Introduction to Fluorescence Sensing: Volume 2: Target Recognition and Imaging [3 ed.] 3031190882, 9783031190889

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Alexander P. Demchenko

Introduction to Fluorescence Sensing Volume 2: Target Recognition and Imaging Third Edition

Introduction to Fluorescence Sensing

Alexander P. Demchenko

Introduction to Fluorescence Sensing Volume 2: Target Recognition and Imaging Third Edition

Alexander P. Demchenko National Academy of Sciences of Ukraine Palladin Institute of Biochemistry Kyiv, Ukraine Yuriy Fedkovych National University Chernivtsi, Ukraine

ISBN 978-3-031-19088-9 ISBN 978-3-031-19089-6 (eBook) https://doi.org/10.1007/978-3-031-19089-6 1st edition: © Springer Science + Business Media B.V. 2009 2nd edition: © Springer International Publishing Switzerland 2015 3rd edition: © Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Dedicated to the memory of my wife Tetiana Ivanchenko

Preface

This book is the second part of third edition on fluorescence sensing that was deeply revised with many additions and now appeared in two volumes. It was written with an intension to combine a textbook, a reference book and a critical review of recent achievements with the vision of future developments. Observing vast literature and realizing how many researchers, their groups and institutions are involved in this research and how strong the public request is for the results of their activities provides a strong motivation for its writing. Indeed, among other sensing and imaging technologies that develop in parallel, the technologies based on fluorescence are unique. They are the most sensitive; their sensitivity reached the absolute limit of single molecules. They offer very high spatial resolution, so that with overcoming the light diffraction limit has reached a molecular scale. They are also the fastest; their response develops on the scale of fluorescence lifetime and can be as short as 10−8 –10−10 s. But their greatest advantage is versatility. Fluorescence sensing can be provided in solid, liquid and gas media and at all kinds of interfaces between these phases. It is because the fluorescence reporter and the detecting instrument are connected via light emission, fluorescence detection can be made non-invasive and equally well suited for remote industrial control and for sensing different targets within the living cells. All these features explain the high popularity of fluorescence sensing, many aspects are explored now and many remain for the future. The field of chemical and biological sensing is immense. It is nearly the whole world of natural and synthetic compounds that have to be analyzed in a broad variety of conditions and for a broad variety of purposes. In the human body, we need to detect and quantify virtually all the genes (genomics) and the products of these genes (proteomics). We need to know how they interact performing their functions (interactomics). In our surrounding, there is a need to analyze a huge number of compounds including millions of newly synthesized products. Among them, we have to select potentially useful compounds (e.g. drugs) and discriminate those that are inefficient and harmful. No less important is to control agricultural production and food processing. There is also a practical necessity to provide control in industrial product technologies, especially in those that produce pollution. Permanent monitoring is needed to maintain safety of our environment. Protection from harmful vii

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microbes, clinical diagnostics and control of patient treatment are the key issues of modern medicine. New problems and challenges appear with the advancement of human society in the twenty-first century. We have to be ready to meet them. Fluorescence sensing technology is the field of research and development in which basic science and technical developments successfully meet. Enrichment of each other and revealing of unexplored areas is an indicator of its rapid growth. Many directions in the present accomplishments and future efforts are overviewed in this book. After the publication of its previous editions, tremendous progress is observed in all fields of research, development and application related to its subject. In order to follow this progress, in the new third edition a number of changes and additions have been made. The present edition is separated into two volumes, V.1 “Materials and Devices” and V.2 “Target Recognition and Imaging”. In V.1, the reader may find critical analysis of methodology in fluorescence sensing and of fluorescence and related methods that can be applied. The properties of different types of fluorescent materials that are molecules and nanoparticles, organic, inorganic or their mixed compositions are analyzed. The present Volume 2 is focused on the practical realization of molecular recognition that is in the background of sensor-target interactions. Different objects of the inorganic and organic world can be used as targets. Of particular interest is the detection of ions, analysis of biological macromolecules, and sensing within living cells and tissues. The fascinating field of fluorescence sensing needs fresh brains. Therefore, the book is primarily addressed to students and young scientists. Together with basic knowledge, they will get an overview of different ideas in research and technology and guide to their own creative activity. Providing a link between basic sciences needed to understand the sensor performance and frontier of research, where new ideas are explored and new products developed, the book will make a strong link between research and education. For the active researcher, it will also be a source of useful information in nearly all areas where fluorescence sensing and imaging is used. Accordingly, this book aims at helping scientists in finding the most appropriate and up-to-date methods to study their molecular recognition systems and to develop highly needed devices. Thus, this book is organized with the aim to satisfy both curious students and busy researchers. This book looks like light reading but it is not. The chapters are selected and organized in such a way as to provide a valuable reference and a transparent learning tool. The list of references in each chapter is an additional resource of information, and the high-quality figures increase clarity and reading pleasure. At the end of every chapter, the reader may find the section “Sensing and Thinking”, in which the most actual and often unresolved issues are raised for the discussion. The series of questions that follows is formulated with the aim to help the reader to check their acquired knowledge.

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This book could not be written without the involvement of my colleagues, friends and students. My special thanks to Sergiy Bobrovnik, Vasyl Pivovarenko, Mykhailo Dvoynenko, Kyrylo Pyrshev, Benoit Michel, Dmytro Dziuba, Sergiy Avilov, Semen Yesylevskyy and Volodymyr Nazarenko. Enjoy your reading. Kyiv, Ukraine December 2021

Alexander P. Demchenko

Contents

1

2

Principles Governing Molecular Recognition . . . . . . . . . . . . . . . . . . . . 1.1 Multivalency: The Principle of Molecular Recognition . . . . . . . . . 1.1.1 Multivalent Pattern of Molecular Interactions . . . . . . . . . 1.1.2 Energetics and Kinetics in Molecular Recognition . . . . . 1.1.3 Reversibility in Molecular Interactions and Mass Action Law . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Lock-And-Key, Induced Fit, Conformation Selection and Induced-Assisted Folding Models . . . . . . . . . . . . . . . . . . . . . . . 1.3 Realization of Principles of Molecular Recognition in Fluorescence Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 The Output Parameters Used in Fluorescence Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.2 Different Strategies in Fluorescence Sensing . . . . . . . . . . 1.4 Molecular Recognition of Different Strength and Specificity . . . . 1.4.1 Sensors Providing Strong Highly Specific Binding . . . . . 1.4.2 Sensors Based on Competitive Target Binding . . . . . . . . 1.4.3 Sensors Based on Reversible Specific Binding and Operating in a Large Volume . . . . . . . . . . . . . . . . . . . 1.5 Direct Reagent-Independent Sensing . . . . . . . . . . . . . . . . . . . . . . . . 1.6 Simultaneous Analysis of Multiple Analytes . . . . . . . . . . . . . . . . . 1.6.1 Systems for Detection of Multiple Analytes . . . . . . . . . . 1.6.2 Specific Target Recognition Versus Pattern Recognition Sensor Arrays . . . . . . . . . . . . . . . . . . . . . . . . . 1.7 Sensing and Thinking. Current Trends that Should Be Highlighted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Basic Theoretical Description of Sensor-Target Binding . . . . . . . . . . . 2.1 Parameters that Need to Be Optimized in Every Sensor . . . . . . . . 2.1.1 The Limit of Detection and Sensitivity . . . . . . . . . . . . . . .

1 3 4 6 10 12 16 16 17 20 20 21 22 22 26 27 28 30 32 37 38 40

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2.1.2

Dynamic Range of Detectable Target Concentrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3 The Sensor Selectivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.4 Multivalent Binding and Cooperativity . . . . . . . . . . . . . . . 2.2 Determination of Binding Constants . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Dynamic Association-Dissociation Equilibrium . . . . . . . 2.2.2 Determination of K b by Titration . . . . . . . . . . . . . . . . . . . . 2.2.3 Determination of K b by Serial Dilutions . . . . . . . . . . . . . . 2.3 Modeling the Analyte Binding Isotherms . . . . . . . . . . . . . . . . . . . . 2.3.1 Receptors Free in Solution or Immobilized to a Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 Bivalent and Polyvalent Reversible Target Binding . . . . 2.3.3 Reversible Binding of Analyte and Competitor . . . . . . . . 2.3.4 Reversible Interactions in a Small Volume . . . . . . . . . . . . 2.4 Kinetics of Target Binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Formats for Fluorescence Detection . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1 Linear Response Format . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.2 Intensity-Weighted Format . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Sensing and Thinking. How to Provide the Optimal Quantitative Measure of Target Binding? . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

4

41 44 46 47 47 49 51 52 53 53 55 57 59 61 61 64 66 69

Recognition Units Built of Small Macrocyclic Molecules . . . . . . . . . . 3.1 Crown Ethers and Cryptands: Macrocyclic Hosts for Ions . . . . . . 3.2 Cavity-Forming Compounds. Structures and Properties . . . . . . . . 3.2.1 Cyclodextrins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2 Calix[n]arenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3 Cucurbit[n]urils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4 Pillar[n]arenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.5 Comparison of Properties and Prospects of Supramolecular Macrocycles . . . . . . . . . . . . . . . . . . . . . 3.3 Porphyrins and Porphyrinoids. Unique Coupling of Recognition and Reporting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Sensing and Thinking. The Recognition Properties of Parent Binders and of Their Derivatives . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

73 74 77 77 81 85 86

Sensors Based on Peptides and Proteins as Recognition Units . . . . . . 4.1 Designed and Randomly Synthesized Peptides . . . . . . . . . . . . . . . . 4.1.1 The Development of Peptide Sensors . . . . . . . . . . . . . . . . 4.1.2 Randomly Synthesized Peptides, Why They Do Not Fold? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.3 Template-Based Approach . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.4 The Exploration of ‘Mini-Protein’ Concept . . . . . . . . . . . 4.1.5 Molecular Display Including Phage Display . . . . . . . . . .

103 104 105

87 90 96 97

106 106 107 109

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4.1.6

Peptide Binders for Protein Targets and the Prospects of Peptide Sensor Arrays . . . . . . . . . . . 4.1.7 Antimicrobial Peptides and Their Analogs . . . . . . . . . . . . 4.1.8 Advantages of Peptide Technologies and Prospects for Their Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Sensors Based on Protein-Based Display Scaffolds . . . . . . . . . . . . 4.2.1 Engineering the Binding Sites by Mutations . . . . . . . . . . 4.2.2 Scaffolds Employing Proteins of Lipocalin Family . . . . . 4.2.3 Other Protein Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Natural Ligand-Binding Proteins and Their Modifications . . . . . . 4.3.1 Bacterial Periplasmic Binding Protein (PBP) Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Engineering PBPs Binding Sites and Response of Environment-Sensitive Dyes . . . . . . . . . . . . . . . . . . . . . 4.3.3 Serum Albumins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Antibodies and Their Recombinant Fragments . . . . . . . . . . . . . . . . 4.4.1 Assay Formats Used for Immunosensing . . . . . . . . . . . . . 4.4.2 The Types of Antibodies and Their Fragments Used in Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.3 Prospects for Antibody Technologies . . . . . . . . . . . . . . . . 4.5 Sensing and Thinking. The Application Range and Benefit from Peptide and Protein Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Nucleic Acids as Scaffolds and Recognition Units . . . . . . . . . . . . . . . . . 5.1 DNA and RNA Fragments in Hybridization-Based Sensing . . . . . 5.1.1 The Types of Nucleic Acid Recognition Units . . . . . . . . . 5.1.2 Fluorescence Reporting in Hybridization Assays . . . . . . 5.2 Nucleic Acid Aptamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2.1 Selection and Production of Aptamers . . . . . . . . . . . . . . . 5.2.2 Integration with Fluorescence-Responding Units . . . . . . 5.2.3 Aptamer Applications and Comparison with Other Binders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3 G-quadruplex-Based Analytical Sensing Platforms . . . . . . . . . . . . 5.3.1 Production and Properties of G-quadruplexes . . . . . . . . . 5.3.2 Fluorescence Reporters for G-quadruplex Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3 Applications of G-quadruplex Sensing Technology . . . . 5.4 The DNA i-motif in Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Sensing and Thinking: The Versatile Recognizing Power of Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

110 111 112 113 113 116 117 119 119 121 122 124 125 127 129 130 131 139 141 141 143 147 147 149 150 153 154 155 156 157 159 160

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7

Contents

Self-assembled, Porous and Molecularly Imprinted Supramolecular Structures in Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Molecular Recognition on Supramolecular Scale . . . . . . . . . . . . . . 6.1.1 Assembly of Organic and Inorganic Functionalities . . . . 6.1.2 The Major Building Blocks . . . . . . . . . . . . . . . . . . . . . . . . 6.1.3 Realization of Multiple Recognition Sites in Self-assembled Structures . . . . . . . . . . . . . . . . . . . . . . . . 6.2 Formation and Operation of Supramolecular Fluorescent Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3 Fluorescence Sensing with Nanoporous and Mesoporous Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.1 Sensing Designed on the Basis of Mesoporous Silica . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.2 The Hydrogel Layers in Sensor Technologies . . . . . . . . . 6.3.3 Porous Structures Formed of Organic Polymers . . . . . . . 6.3.4 Metal–Organic Frameworks . . . . . . . . . . . . . . . . . . . . . . . . 6.4 Molecularly Imprinting in the Polymer Volume . . . . . . . . . . . . . . . 6.4.1 The Principle of Formation of Imprinted Polymers . . . . . 6.4.2 The Coupling of Molecular Recognition with Reporting Functionality . . . . . . . . . . . . . . . . . . . . . . . 6.4.3 Imprinted Polymers in the Form of Nanoparticles and Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.4 Exploration of Collective Properties of Fluorescent Dye Aggregates and Conjugated Polymers . . . . . . . . . . . 6.4.5 Nanomaterials with Molecularly Imprinted Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4.6 Formation of Nanocomposites with Molecular Imprinting Functionalities . . . . . . . . . . . . . . . . . . . . . . . . . . 6.5 Sensing and Thinking: Extending the Fluorescence Sensing Possibilities with Designed and Spontaneously Formed Nano-ensembles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fluorescence Sensing Operating at Interfaces . . . . . . . . . . . . . . . . . . . . 7.1 The Structural and Dynamic Properties of Surfaces and Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.1 Gas–Liquid Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.2 Liquid–Liquid Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.3 Solid–Liquid Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.4 Solid–Solid Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2 The Self-assembled Functional Surfaces . . . . . . . . . . . . . . . . . . . . . 7.2.1 Formation of Functional Surfaces . . . . . . . . . . . . . . . . . . . 7.2.2 The Active Surfaces in Active Use . . . . . . . . . . . . . . . . . . 7.2.3 Organic Dyes Forming Active Surfaces . . . . . . . . . . . . . . 7.2.4 Supported Layers of Conjugated Polymers . . . . . . . . . . .

165 166 167 168 171 173 175 176 177 180 183 184 185 187 189 192 194 195

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Preferential Location of Solutes in the Systems of Structural Heterogeneity and on Active Surfaces . . . . . . . . . . . . 7.4 Binding Affinity at Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5 Surface-Imprinted Sensors and Biosensors . . . . . . . . . . . . . . . . . . . 7.5.1 Surface Imprinting on Support . . . . . . . . . . . . . . . . . . . . . . 7.5.2 Nanoparticle-Based Surface Imprinting . . . . . . . . . . . . . . 7.6 Sensing and Thinking. The Strong Contribution of Surfaces and Interfaces to Sensor Technologies . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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8

Fluorescence Sensing of Physical Parameters and Chemical Composition in Gases and Condensed Media . . . . . . . . . . . . . . . . . . . . 8.1 Sensing the Physical Parameters of Environment: Temperature and Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.1 Molecular Thermometry . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.2 Luminescence for Pressure Measurement . . . . . . . . . . . . . 8.2 Fluorescence Studies in a Gas Phase . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1 Optimal Receptors for the Gas State Molecules . . . . . . . 8.2.2 Determining the Natural Gas Phase Composition . . . . . . 8.2.3 Detection of Hydrocarbon Gasses . . . . . . . . . . . . . . . . . . . 8.2.4 Dangerous Compounds and Explosives . . . . . . . . . . . . . . 8.3 Characterization of Solvents and Their Intermolecular Interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.1 Solvent Polarity Scaling . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.2 Physical Modeling of Solvent Polarity Effects . . . . . . . . . 8.3.3 Wavelength-Ratiometric Response to Solvent Polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.4 Solvent Polarity and Hydrogen Bonding . . . . . . . . . . . . . 8.3.5 Preferential Solvation in Mixed Solvents . . . . . . . . . . . . . 8.4 Fluorescence Probing of Molecular Dynamics in Liquid State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1 Rotating Sphere Approach . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.2 Segmental Probe Rotations and Their Application . . . . . 8.4.3 Molecular Rotors Relaxing to TICT State . . . . . . . . . . . . 8.4.4 Dyes Exhibiting the Excited-State Planarization . . . . . . . 8.5 Dynamics of Solvent Relaxations . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.1 Solvation Dynamics Studied by Time-Resolved Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.5.2 Site-Selective Dynamics in Molecular Ensembles . . . . . 8.6 Detection of Traces of Water in Low-Polar Liquids . . . . . . . . . . . . 8.7 Condensed-Phase Media of Special Interest: Supercritical Liquids, Ionic Liquids and Liquid Crystals . . . . . . . . . . . . . . . . . . . 8.7.1 Molecular Structure and Dynamics in Supercritical Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.7.2 The Properties of Ionic Liquids . . . . . . . . . . . . . . . . . . . . . 8.7.3 Liquid Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

221 223 224 224 228 229 231 237 238 238 242 243 244 245 248 249 251 252 253 256 258 260 262 262 263 264 266 267 268 270 271 273 273 274 276

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The Structure and Dynamics in Polymers . . . . . . . . . . . . . . . . . . . . 8.8.1 Monitoring the Polymerization Process . . . . . . . . . . . . . . 8.8.2 Structures and Structural Transitions in Polymers . . . . . . 8.9 Sensing and Thinking. The Value of Information on Correlation of Macroscopic and Microscopic Variables . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

277 277 278

Quantitative Fluorescent Detection of Ions . . . . . . . . . . . . . . . . . . . . . . . 9.1 Fluorophore-Based Determination of pH . . . . . . . . . . . . . . . . . . . . . 9.2 Determination of Concentration of Cations . . . . . . . . . . . . . . . . . . . 9.2.1 Fluorescent Sensors for Alkali and Alkaline Earth Metal Cations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.2.2 Sensing the Transition Metal Ions . . . . . . . . . . . . . . . . . . . 9.2.3 Detection of Heavy Metal Ions . . . . . . . . . . . . . . . . . . . . . . 9.2.4 Potential for λ-Ratiometric Sensing Based on Excited-State Intramolecular Proton Transfer . . . . . . . 9.3 Sensing the Anions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4 Sensing and Thinking. Selecting the Ways to Apply the Principle of Wavelength-Ratiometry to Sensing Ions . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

295 298 303

10 Detection and Imaging of Small Molecules of Biological Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1 Gaseous Molecules of Physiological Signaling—Gasotransmitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.1 Carbon Monoxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.2 Nitric Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.3 Hydrogen Sulfide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2 Oxygen and Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . 10.2.1 Determination of Oxygen Concentration . . . . . . . . . . . . . 10.2.2 Hydrogen Peroxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.3 Hypochlorous Acid/Hypochlorite . . . . . . . . . . . . . . . . . . . 10.3 Detection of Biothiols (Cysteine, Homocysteine and Glutathione) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.4 Biologically Relevant Phosphate Anions . . . . . . . . . . . . . . . . . . . . . 10.5 Adenosine and Guanosine Triphosphates . . . . . . . . . . . . . . . . . . . . 10.6 Redox Cofactors NADH/NAD+ and NAD(P)H/ NAD(P)+ . . . . . . 10.7 Sensing and Thinking. The Problem of Simultaneous Sensing and Imaging of Many Analytes . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Detection, Structure and Polymorphism of Nucleic Acids . . . . . . . . . 11.1 DNA Detection and Analysis of Its Conformation . . . . . . . . . . . . . 11.1.1 Double-Stranded DNA Structures . . . . . . . . . . . . . . . . . . . 11.1.2 Analysis of Single-Stranded DNA . . . . . . . . . . . . . . . . . . . 11.1.3 Identification of Non-canonical DNA Forms . . . . . . . . . .

280 282

304 307 309 313 315 319 322 329 332 332 334 334 337 338 339 340 342 347 351 354 355 358 367 369 371 373 375

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11.2 Recognition of Specific DNA Sequences by Hybridization . . . . . 11.2.1 The Microarray ‘DNA Chip’ Hybridization Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.2 Sandwich Assays in DNA Hybridization . . . . . . . . . . . . . 11.2.3 Molecular Beacon Technique . . . . . . . . . . . . . . . . . . . . . . . 11.2.4 Specific DNA Sensing with the Aid of Conjugated Polymer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.5 DNA Structure Recognition with Peptide Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2.6 The Use of Nanomaterials in DNA Hybridization . . . . . . 11.3 Probing on the Level of Single Nucleic Acid Bases . . . . . . . . . . . . 11.3.1 Design of Local Site Responsive Sensors . . . . . . . . . . . . . 11.3.2 Operation with Parameters of Fluorescence Emission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.3 Probing the Single-Nucleotide Polymorphism . . . . . . . . . 11.4 RNA Detection, Analysis and Imaging . . . . . . . . . . . . . . . . . . . . . . 11.4.1 RNA Detection in Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.2 RNA G-quadruplexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.5 Sensing and Thinking. Increase of Sensitivity: Amplify the Target or the Detection System? . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

379

12 Fluorescence Detection of Peptides, Proteins, Glycans . . . . . . . . . . . . 12.1 Targeting Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2 Detection of Protein Targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.1 Determination of Total Protein Content . . . . . . . . . . . . . . 12.2.2 Labeling the Surface of Native Proteins . . . . . . . . . . . . . . 12.2.3 The Recognition of Protein Surface by Small Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.4 Protein Sensing with Peptide, Protein and Nucleic Acid Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.5 Molecularly Imprinted Polymers in Protein Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.6 Sensor Arrays and Machine Learning Algorithms . . . . . 12.3 Analysing Pathological β-Aggregated Forms of Proteins . . . . . . . 12.3.1 Organic Dyes as the Sensors for β-Sheets . . . . . . . . . . . . 12.3.2 Following the Kinetics of Amyloid Formation . . . . . . . . 12.4 Polysaccharides and Glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . 12.5 Sensing and Thinking. Precise Affinity Sensors or Chemical Noses? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

417 418 421 422 423

380 383 384 385 386 388 391 392 393 400 401 402 403 404 407

425 428 430 432 436 437 438 441 443 445

13 Detection of Harmful Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453 13.1 Detection and Identification of Vegetative Bacteria . . . . . . . . . . . . 455 13.1.1 The Whole-Cell Detection . . . . . . . . . . . . . . . . . . . . . . . . . 456

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13.1.2 Detection by Characteristic Features of Cell Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.1.3 Detection Based on Bacterial Genome Analysis . . . . . . . 13.2 Discovery and Recognition of Bacterial Spores . . . . . . . . . . . . . . . 13.3 Identification and Analysis of Biofilms . . . . . . . . . . . . . . . . . . . . . . 13.4 Detection of Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5 Sensors for Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.1 Nucleic Acid Based Detection . . . . . . . . . . . . . . . . . . . . . . 13.5.2 Recognition of Viruses by Antibodies and Aptamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.6 Sensing and Thinking. Future Trends in Pathogen Detection: Single-Particle Sensitivity Versus Signal Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Clinical Diagnostics Ex-Vivo Based on Fluorescence . . . . . . . . . . . . . . 14.1 Biological Fluids Available for Sensing . . . . . . . . . . . . . . . . . . . . . . 14.2 Detection of Disease Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.1 Diagnostics of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.2 Diagnostics with Cardiac Biomarkers . . . . . . . . . . . . . . . . 14.2.3 The Markers of Autoimmune Disorders . . . . . . . . . . . . . . 14.2.4 Kidney-Related Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . 14.2.5 Neurodegenerative Diseases . . . . . . . . . . . . . . . . . . . . . . . . 14.3 Glucose Sensing in Diagnosis and Treatment of Diabetes . . . . . . 14.4 Uric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5 Cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.6 Sensing and Thinking. The Era of Digital Health is Approaching? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Imaging and Sensing Inside the Living Cells. From Seeing to Believing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.1 Modern Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . 15.1.1 Epi-Fluorescence Microscopy . . . . . . . . . . . . . . . . . . . . . . 15.1.2 Total Internal Reflection Fluorescence Microscopy (TIRF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.1.3 Confocal Fluorescence Microscopy . . . . . . . . . . . . . . . . . . 15.1.4 Programmable Array Microscope . . . . . . . . . . . . . . . . . . . 15.1.5 Two-Photon and Three-Photon Microscopy . . . . . . . . . . . 15.1.6 Time-Resolved and Time-Gated Imaging . . . . . . . . . . . . . 15.1.7 Wavelength-Ratiometric Imaging . . . . . . . . . . . . . . . . . . . 15.1.8 Traditional Far-Field Fluorescence Microscopy: Advances and Limitations . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2 Far-Field Super-Resolution Microscopy . . . . . . . . . . . . . . . . . . . . . 15.2.1 Breaking the Diffraction Limit . . . . . . . . . . . . . . . . . . . . . .

457 461 464 465 467 471 473 474

478 482 493 495 499 500 503 504 505 507 509 512 514 517 518 529 530 532 533 533 535 536 538 539 540 541 541

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15.2.2 Stimulated Emission Depletion (STED) Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2.3 Single Molecule Localization Microcopy . . . . . . . . . . . . . 15.2.4 Structured Illumination Microscopy (SIM) . . . . . . . . . . . 15.2.5 Correlative Light and Electron Microscopy . . . . . . . . . . . 15.3 Sensing and Imaging on a Single Molecule Level . . . . . . . . . . . . . 15.3.1 The Reason to Study Single Molecules . . . . . . . . . . . . . . . 15.3.2 Single-Molecular Studies in Solutions . . . . . . . . . . . . . . . 15.3.3 The Studies of Molecular Motions and Interactions . . . . 15.3.4 Single Molecules Inside the Living Cells . . . . . . . . . . . . . 15.4 Site-Specific Intracellular Labeling and Genetic Encoding . . . . . . 15.4.1 Attachment of Fluorescent Reporter to Any Cellular Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4.2 Genetically Engineered Protein Labels . . . . . . . . . . . . . . . 15.4.3 Co-synthetic Incorporation of Fluorescence Dyes . . . . . . 15.5 Advanced Nanosensors Inside the Cells . . . . . . . . . . . . . . . . . . . . . 15.5.1 Fluorescent Dye-Doped Nanoparticles . . . . . . . . . . . . . . . 15.5.2 The Quantum Dots Applications in Imaging . . . . . . . . . . 15.5.3 Carbon Nanoparticles in Cell Research . . . . . . . . . . . . . . . 15.6 The Studies of Intracellular Motions . . . . . . . . . . . . . . . . . . . . . . . . 15.6.1 Single-Particle Tracking . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.6.2 Viscosimetry Inside the Living Cell . . . . . . . . . . . . . . . . . 15.7 Sensing Within the Cell Membrane . . . . . . . . . . . . . . . . . . . . . . . . . 15.7.1 Membrane Structure and Dynamics . . . . . . . . . . . . . . . . . 15.7.2 Lipid Asymmetry and Apoptosis . . . . . . . . . . . . . . . . . . . . 15.7.3 Sensing the Membrane Potential . . . . . . . . . . . . . . . . . . . . 15.7.4 Visualizing Membrane Receptors . . . . . . . . . . . . . . . . . . . 15.8 Sensing and Thinking. Intellectual and Technical Means to Go Deeper into Cellular Functions . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Fluorescent Imaging In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.1 Optical Properties of Biological Tissues . . . . . . . . . . . . . . . . . . . . . 16.1.1 Light Propagation Through Tissues . . . . . . . . . . . . . . . . . . 16.1.2 Optical Windows in Near-Infrared . . . . . . . . . . . . . . . . . . . 16.2 Fluorescence Contrast Agents and Reporters . . . . . . . . . . . . . . . . . 16.2.1 Organic Dyes and Their Nanocomposites . . . . . . . . . . . . . 16.2.2 Nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3 Optimal Imaging Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.3.1 Imaging and Microscopy in NIR-I Window . . . . . . . . . . . 16.3.2 Instrumentation for NIR-II Range . . . . . . . . . . . . . . . . . . . 16.4 The Studies on the Level of Tissue Imaging . . . . . . . . . . . . . . . . . . 16.4.1 Contrasting the Blood Vessels and Lymph Nodes . . . . . . 16.4.2 Monitoring Inflammatory Diseases and Response to Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.4.3 Imaging Cancer Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . .

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543 545 549 550 552 553 554 557 559 560 561 564 565 567 569 569 571 571 572 573 577 577 579 580 580 583 585 597 599 599 601 601 602 608 612 612 614 618 618 621 623

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16.5 Fluorescence Image-Guided Surgery . . . . . . . . . . . . . . . . . . . . . . . . 16.6 Cell Tracking Inside the Living Body . . . . . . . . . . . . . . . . . . . . . . . 16.6.1 The Procedures for Cell Labeling . . . . . . . . . . . . . . . . . . . 16.6.2 Tracking Hematopoietic and Cancer Cells . . . . . . . . . . . . 16.6.3 Tracing the Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.7 Combination of Fluorescence with Photoacoustic Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.8 Sensing and Thinking. Towards the Progress in Functional Bioimaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

625 628 629 630 632

17 Phototheranostics: Combining Targeting, Imaging, Therapy . . . . . . 17.1 Light in Theranostics Technologies . . . . . . . . . . . . . . . . . . . . . . . . . 17.2 Photothermal Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.2.1 The Choice of Wavelengths . . . . . . . . . . . . . . . . . . . . . . . . 17.2.2 The Choice of Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3 Photodynamic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3.1 The Factors Needed for Realizing Photodynamic Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.3.2 The Mechanisms of Tumor Destruction . . . . . . . . . . . . . . 17.4 Combining All Power of Phototheranostics . . . . . . . . . . . . . . . . . . 17.4.1 Photoactivation of Prodrugs and Controlling the Drug Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.4.2 Photoimmunotherapy with Near-Infrared Light . . . . . . . . 17.4.3 Non-oncological Clinical Applications . . . . . . . . . . . . . . . 17.4.4 Photothermal and Photodynamic Inactivation of Harmful Microbes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17.5 Sensing and Thinking. The Strategy of Controlling the Diagnostics and Treatment by Light . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

649 652 657 659 659 662

18 Fluorescent Light Opening New Horizons . . . . . . . . . . . . . . . . . . . . . . . 18.1 Genomics, Proteomics and Other ‘Omics’ . . . . . . . . . . . . . . . . . . . 18.1.1 Genomic and Gene Expression Analysis . . . . . . . . . . . . . 18.1.2 The Analysis of Proteome . . . . . . . . . . . . . . . . . . . . . . . . . . 18.1.3 Addressing Interactome . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.1.4 Outlook. Analysis on a Single-Cellular Level . . . . . . . . . 18.2 Unprecedented Scale of Complexity, How to Deal With It? . . . . . 18.2.1 Combinatorial Synthetic Approach on a New Level . . . . 18.2.2 Advanced Sensors in Discovery of New Products . . . . . . 18.2.3 Electronic (Photonic) Noses and Tongues . . . . . . . . . . . . 18.2.4 Realizing the Pattern Recognition Principle . . . . . . . . . . . 18.2.5 Navigating Massive Datasets: Transforming Information into Knowledge . . . . . . . . . . . . . . . . . . . . . . . .

693 694 696 698 700 703 705 705 706 708 710

633 635 637

663 667 669 670 673 674 676 680 682

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18.3 New Level of Clinical Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3.1 The Progressing Sensor Developments . . . . . . . . . . . . . . . 18.3.2 The Sensing in Whole Blood . . . . . . . . . . . . . . . . . . . . . . . 18.3.3 Gene-Based Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.3.4 Confronting the Global Virus Pandemic . . . . . . . . . . . . . . 18.4 Sensors Promising to Change the Society . . . . . . . . . . . . . . . . . . . . 18.4.1 Industrial Challenges and Safe Workplaces . . . . . . . . . . . 18.4.2 Biosensor-Based Lifestyle Management . . . . . . . . . . . . . . 18.4.3 Wearable, Implantable and Digestible Miniature Sensors Are a Reality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.4.4 Living in a Safe Environment and Eating Safe Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18.5 Sensing and Thinking. Where Do We Stand and Where Should We Go? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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718 718 721 723 724 725 726 727 729 731 732 735

Epilogue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 747 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751

Chapter 1

Principles Governing Molecular Recognition

No interaction—no information. This principle governs the science of molecular sensing, with the limitation that the interaction should be highly specific, a molecular recognition. This can be achieved with multivalent pattern of molecular interactions that can be formed with sensor-target conformational adaptations. Different strategies are involved in coupling the recognition event with the response of fluorescence reporter and for obtaining greater information on multiple targets from single experiment by constructing sensor arrays. Addressing new demands, fluorescence sensing technologies become more and more complicated. For better performance of receptor and reporter units and also of transduction and support elements, they use many new materials. As a result, we observe dramatic improvements in heterogeneous and homogeneous sensing assays and of cellular imaging that involves the sensing inside living cells and tissues. An important step to perfection of fluorescent sensors was made by their self-assembly into supramolecular structures and by their attachment to flat or porous surfaces and to different types of particles. Scaling up from small single molecules to larger molecular ensembles aims at achievement of two important goals: improvement of molecular recognition, especially with the targets of large size and complexity, and exploration of collective properties of fluorescence reporters that offer enhanced sensitivity. On this step, a substantial enhancement in performance can be attained and new important functionalities added. The heart of any chemical sensor or biosensor is its recognition unit (binder or receptor), see Fig. 1.1. It is constructed for providing selective target binding from a mixture of different and sometimes closely related compounds. Molecular complementarity is achieved through steric complementarity between interacting structures and through noncovalent bonding such as hydrogen bonding, metal coordination, hydrophobic forces, van der Waals forces, π–π interactions, and/or electrostatic effects. Most versatile and efficient of receptors for applications in sensor technologies are the antibodies, their short fragments and nucleic acid aptamers. Different ligand binding proteins are efficient addressing specific targets. Nucleic acids and artificial peptide nucleic acids became the basis of new technologies of DNA and © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_1

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2

1 Principles Governing Molecular Recognition

Fig. 1.1 Schematic illustration of sensor operation based on molecular recognition exploring fluorescence reporter functionally coupled with the recognition unit (receptor) via some transduction mechanism. Molecular recognition is seen as the highly specific interaction between two or more molecules or supramolecular structures, which display steric and energetic complementarity

RNA detection. Small in size synthetic chelators have become important binders for a great variety of targets. The high specificity and affinity of these molecular units is achieved by their appropriate structures allowing multi-point non-covalent interactions with the target (Chatterji 2016). Molecular recognition is a vast area encompassing every aspect of biology. It provides the way for understanding the mode of interactions in biological systems, biological transport and assembly, biological catalysis. In a biology-inspired way, it expands to molecular and supramolecular sensing in other practically important areas, from industrial technology and environment protection to control of food safety and medical diagnostics. In sensing technologies, multi-point non-covalent interactions with the target of a sensor with the desired affinity is the basis of quantitative analysis. Recognition units are the key functional units of molecular sensors that are responsible for selective target binding in the proper range of target concentrations. Different recognition units can be applied and the principles of their design, construction and performance realized. They range from small coordination compounds targeting small molecules and ions to macromolecules such as enzyme substrates, proteins, nucleic acids, macromolecular assemblies or even the living cells. They can be also molecular structures and nanoparticles representing inorganic world. In order to accommodate the target and to saturate all noncovalent interactions needed for molecular recognition, some extent of molecular flexibility may be required. Being induced by the target binding, in some cases they can even perform the unfolding-folding reaction, which in the best way can be realized with flexible peptides, conjugated polymers and nucleic acid aptamers. We will discuss three principles of realizing molecular recognition that can be applied not only for sensing a particular compound but of their great number, which is often needed for better characterization of the studied system: (a) Strong highly specific binding. This allows after the target-sensor complex is formed, providing manipulation with it, such as reagent addition and washing. DNA hybridization assays and immunoassays use these possibilities by collecting a small sample and determining all the targets therein,

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3

since all of them will be bound. Microarrays can be fabricated, in which every spot corresponds to different target. When the strong covalent bonds are formed, then instead of molecular recognition the principle of reactivity sensing (chemodosimetry) must be applied. (b) Specific binding of variable strength. The sensor-target interactions are reversible and, according to mass action law, they allow quantitative target detection, but only within a limited concentration range. Formation of arrays is possible and frequently provided. Extraction of small samples from large tested volume is not needed; the sensors can be incorporated into large volumes and allow continuous monitoring of production/consumption of targets in them. (c) The binding to multiple targets of variable specificity and strength. This is the field of operation of artificial electronic (photonic) noses and tongues. Here the sensors may have different binding characteristics, none of which are necessarily specific or even very selective. This approach requires that an array of sensors must be created and the composite signal evaluated and interpreted by pattern recognition protocols. All of these receptors or recognition units must be transformed into fluorescence (luminescence) sensors by coupling a dye or nanoparticle generating informative signal. They have to become responding to the presence of the target, without affecting the binding affinity. Therefore our goal is to achieve optimal binding and efficient labeling of the binder but still to maintain the target binding properties intact while adding the reporter function. Different strategies for obtaining the quantitative information on sensor-target interaction will be analyzed. Among those that are in active use, direct reagentindependent sensing is the most advanced strategy that deserves future development. The concept of multiplexing, where multiple analytes can be detected in a single sample, can be tackled based on exact sensing and distributed array approaches. Designing the sensors, we have to keep in mind that we will have to incorporate them into useful sensor devices together with all optical and electronic elements. Many of them should be applicable for point-of-care testing and even for personal use. Nonetheless, the testing should be precise, accurate, cost-effective and highly sensitive. It should not replace in full the huge capacities of centralized laboratory testing but will use them, being connected with smartphones in complicated cases. But everything starts from elementary steps of molecular recognition.

1.1 Multivalency: The Principle of Molecular Recognition The term molecular recognition is commonly used to indicate noncovalent specific interactions between molecules of chemical or biological origin (Lehn 1994; Persch et al. 2015). Such interactions could be hydrogen bonding, metal coordination forces, van der Waals interactions, π–π stacking, etc. (Bishop et al. 2009). For providing stable intermolecular complex formation, each of these forces should be

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1 Principles Governing Molecular Recognition

reversible and not strong enough, allowing to compete with solute–solvent interactions, compensate entropy loss on binding or to withstand thermal fluctuations. Multiplicity of these interactions allows providing sufficient stability of formed intermolecular assemblies (Choi and Jung 2018). These key issues are well elaborated in the literature (Chatterji 2016; Huskens et al. 2018). Multivalent interactions using multiple weak binding sites and many low-affinity binding events occurring simultaneously enhance the overall binding affinity much more than the sum of the constituent monovalent binding interactions (Mammen et al. 1998).

1.1.1 Multivalent Pattern of Molecular Interactions Multivalency is a key principle in realizing molecular recognition for achieving strong, specific, yet reversible interactions operating with rather weak physical forces. Being applicable to both biological (Mammen et al. 1998; Meyer and Knapp 2014) and synthetic (Badjic et al. 2005) systems, it states that molecular recognition can be realized only on formation of multiple, interconnected supramolecular binding modules. The difference between monovalency and multivalency is illustrated in Fig. 1.2. Individually weak interactions produce cumulative effect, so that multipoint binding between interacting structures produces generalized action that differs from that obtained by summation of individual interactions. This principle is the guideline for design and selection of recognition units in fluorescence sensing, since the sensing requires the target binding in complex multi-component systems with components existing in different concentrations and possessing different binding affinities. In contrast to univalent interacting molecules and particles, the multivalent binding nanoparticles that can bind to a larger number of ligands simultaneously (Varner et al.

Fig. 1.2 Illustration of multivalency in intermolecular interactions. a Single-valency (left) and multivalency (right) binding. b Interaction of functional nanoparticles with receptors on cell surface. Left—small number of receptors. Right—large number of receptors and their maximal saturation. c The effect of supersensitivity. The multivalent nanoparticles binding to a large number of ligands simultaneously. The binding curve may change dramatically becoming much sharper

1.1 Multivalency: The Principle of Molecular Recognition

5

2015), display regimes of “superselectivity” (Fig. 1.2c), where the fraction of bound particles varies sharply with the receptor concentration. Direct comparison between monovalent and multivalent binding in a model of interacting nanoparticles with cell surface shows that the fraction of bound particles varies sharply (in a highly nonlinear way) with the receptor concentration (Martinez-Veracoechea and Frenkel 2011). Therefore, such sharp discrimination between surfaces with high and low receptor coverage should be accounted in the design of sensor technologies. It was derived that the superselectivity is due to the fact that the number of distinct ligand-receptor binding arrangements increases in a highly nonlinear manner. To characterize a multivalent binding effect, one can use an enhancement factor b (Mammen et al. 1998), introduced as the ratio of the binding constant for the multivalent binding (K b multi ) of a multivalent ligand to a multivalent receptor with the binding constant for the monovalent binding (K b mono ) of a monovalent ligand to a multivalent receptor, b = (K b multi )/(K b mono ). An advantage of this enhancement factor is that it can be used even if the multiplicity of effective binding interactions is unknown; it can occupy several orders of magnitude. It was frequently observed that the binding between two multivalent (i.e. having more than one binding site) entities involving n (n > 1) binding events occurs with an affinity higher than the sum of n individual monovalent interactions (Mammen et al. 1998). The classical example is the lectin binding with saccharides (Fasting et al. 2012). In the case of monosaccharide molecule, as a result of combination of noncovalent interactions such as hydrogen bonds, hydrophobic interactions and van der Waals interactions, the binding constants K b are typically in the range of 103 –104 M−1 , so the binding is relatively weak and low specific compared to that in other biological recognition events. It becomes much stronger in oligosaccharides, K b up to 106 M−1 , as a result of spatial extension of the binding sites. It can increase further, to 109 M−1 , upon association of lectin molecules. The formation of lectin tetramer is a prototypical illustration of the multivalency generated by self-assembly leading to defined arrays of functional groups and binding patterns (Chmielewski et al. 2014). Essentially, the effects of multiplicity appear when two or more structurally linked sites on one of the partners (receptor) interacts with two or more linked sites of the other partner (ligand). If one of the partners is monovalent and binds to several sites of the second partner, then no multivalency effects will be observed. This effect can be seen without cooperativity (the influence of binding at one site on the binding affinity of other sites). Cooperativity can be rigorously defined for consecutive monovalent interactions at a multivalent platform (Mulder et al. 2004). In general, it does not require multivalency (for instance, binding of oxygen to hemoglobin is cooperative but not multivalent). However, the positive cooperativity, if present, may enhance the effect. Hierarchical molecular recognition may be realized in different sensing technologies (Ariga et al. 2012), inspired by natural examples from nucleic acid (Deng and Walther 2020) world. It may lead to novel nanomaterials (Gong et al. 2019) and even to assembly of macroscopic structures (Harada et al. 2011). Hierarchical

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1 Principles Governing Molecular Recognition

Fig. 1.3 Schematics of hierarchical molecular recognition of bacterial cell by immunoglobulin M (Ig M). Level 1, recognition of exposed antigenic determinants by antibody recognition sites (Fab fragments). Level 2. Involvement of dual binding by two sets of Fab fragments on IgM chains. Level 3. Involvement of greater number out of 10 of antigen recognition sites formed by 5 sets of protein chains

levels of recognition can be envisioned with schematically presented interaction of immunoglobulin M with cell (Fig. 1.3).

1.1.2 Energetics and Kinetics in Molecular Recognition Molecular sensing needs to be provided both in gas and liquid media, and the following chapters demonstrate the obtained achievements. The contribution of different factors to the origin of multiplicity is not easy to evaluate since they reside on the changes of both interaction energy and entropy. The entropy effects at the first site in a sequence of binding events can be complemented by that on mutual steric adaptation and conformational changes in both interacting partners. The effect will depend substantially on whether the interacting sites are connected by rigid or flexible linkers. Different forces combined in steric arrangement of target-receptor interacting partners provide the recognition. The most difficult regarding the sensor design is the finding of proper selective receptors for hydrophilic guests in water (Escobar and Ballester 2021; Ferguson Johns et al. 2021), which is needed for both environment protection and clinical diagnostics. Here we meet both high affinity of our polar analytes to water molecules and their high desolvation cost in water. This makes

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7

the binding event in water less favorable, thus diminishing the contribution of polar interactions for molecular recognition. The hydrophobic effect is commonly invoked as a key component to molecular recognition in water and is a part of the sensor design strategy. The most common way to explain the basis of the hydrophobic effect is to consider water that solvates hydrophobic solutes as being highly ordered, static, and ice-like (Daze and Hof 2016). Indeed, its entropy-driven component can be very important, especially for the compounds with poor water solubility (Rekharsky and Inoue 1998). However, hydrophobic interactions lack the needed selectivity, and in sensor design they have to be combined with other forces. Steric complementarity plays the leading role in this design (Wan et al. 2021). Hydrogen bonding is a key governing force in molecular recognition, notably in biological systems (Baldini et al. 2007; Dong and Davis 2021). Hydrogen (H) bonds are the short-range, unidirectional and specific. Individual bonds being in energy of the order of 10–15 kJ/mol are rather week, but their collective action can result in a much stronger effect. Enough to mention, that these bonds provide stability to α-helix in proteins and to double helix in DNA. Formation of H-bonds of acceptor–acceptor– donor–donor (AADD) type between rather simple organic molecules reveals an unprecedented stability of dimers based on their AADD self-complementary with the dimerization constant K dimer > 5 × 108 M−1 (Corbin et al. 2002). Linking covalently two such units together with a semi-rigid spacer, a number of stable dendritic structures can be obtained that can be a powerful strategy for creating nanocomposites. Collective effects in H-bond formation allows to form the complexes in water solutions and to withstand destructive competition for these bonds in water (Dong and Davis 2021; Escobar and Ballester 2021). Hydrogen bonding complements and adds specificity to molecular recognition in water (Yao et al. 2018). Electrostatic interactions, defined as favorable interactions between oppositely charged species, can be stronger than interactions involved in intermolecular hydrophobic association and extend to larger distances. Electrostatic complementarity has a strong recognition power for charged compounds, such as organic anions, nucleotides, amino acids and peptides (Ferguson Johns et al. 2021). The exact influence of these interactions on molecular recognition depends on charge, polarizability, distance, geometry and dielectric constant of solvent in ways that can be easily derived from simple physical principles (Daze and Hof 2016). Interactions between dipoles have become a central theme of molecular recognition (Persch et al. 2015). Scientists identify other types of small intermolecular interactions, such as π–π stacking, cation-π interactions and halogen bonding (Daze and Hof 2016; Mahadevi and Sastry 2013). In general, the bonds formed of aromatic rings play important role in chemical and biological recognition (Salonen et al. 2011). Reversible covalent binding of target to receptor can be efficient in molecular recognition due to its highly specific nature. Over the past decade a new field, “dynamic covalent chemistry”, has been growing rapidly, in which the formation and exchange of reversible covalent bonds are employed to construct assemblies (You et al. 2015). Among such reversible reactions with low activation threshold and dynamic behavior are imine formation and exchange, acylhydrazone formation and

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1 Principles Governing Molecular Recognition

exchange, disulfide exchange, olefin metathesis, Diels–Alder reactions, acetal and hemiacetal formation and exchange, etc. The disulfide exchange is applied as ligand exchange in different sensor constructions, and the reversible formation of cyclic boronate esters is essential for glucose and other carbohydrate sensors. It should be stressed that the recognition events in nature typically occur in aqueous medium, and the attempts to understand remarkable properties exhibited by proteins, such as high binding affinity, superior binding selectivity, and extreme catalytic performance, have stimulated dramatically the work on design of synthetic receptors. This knowledge is highly needed not only for sensor technologies but also in such areas as self-assembly, drug discovery, and supramolecular catalysis. The design and development of effective water-soluble synthetic receptors based on cyclic, oligocyclic, and concave-shaped architectures (Fig. 1.4) has reached substantial progress (Escobar and Ballester 2021). Evaluation of energetics and kinetics in target-receptor interactions and their prediction based on known 3D structures is a very difficult task because of really existing distributions of their conformations and need inclusion of entropic contributions and solvent effects. Different kinetic steps can be derived from molecular dynamics simulations (Fig. 1.5). The figure presents three kinetic steps found in the process of protein recognition, of diffusion, free conformer selection, and refolding.

Fig. 1.4 Examples of receptors of different nature interacting with their targets in aqueous media (Escobar and Ballester 2021)

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Fig. 1.5 Working model for flexible protein recognition (Grünberg et al. 2004). Protein–protein association may be governed by diffusion, selection of matching conformers, and refolding. Rf and Lf are the free structure ensembles of receptor and ligand, respectively. R*f and L*f are subsets of the free receptor and ligand ensembles (recognition conformers). The middle and lower sections of the figure suggest, schematically, the forces involved at the different stages and the resulting free energy profile. The widths and barrier heights are not meant to reflect real proportions

The factors contributing to free energy and entropy of binding are outlined on each step. Thus, overcoming many difficulties in analysis of particular systems, we have got a strong concern that the 3D complementarity between partner surfaces that involves both geometrical fitness and saturation with weak interactions is the basic structural aspect of macromolecular recognition. Regarding the types of bonds that participate in the recognition and the functional groups that are capable of forming these bonds, the answers are often less definite because no single molecular recognition event in complex systems can or should be reduced to a single interaction. The orchestrated cooperation of many weak interactions is needed to achieve strong and selective molecular recognition.

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1 Principles Governing Molecular Recognition

1.1.3 Reversibility in Molecular Interactions and Mass Action Law Imagine reader that you are at the sea shore and you need to measure the concentration of any compound in sea water. You can take a sample in a fixed volume and apply a colored reaction known from analytical chemistry or, in a more advanced way, to use the sensor molecules or nanoparticles that can bind that entire compound to generate analytical response and then recalculate the concentration in the whole sea. You can do that in a different way, without taking the sample. You can immerse in sea water the sensor that interacts reversibly with the dissolved compound and measures its concentration directly (Fig. 1.6). What is the difference? In the first case you need very strong irreversible binding of all analyte in the sample and in the second case the binding is relatively weak and reversible, in equilibrium with this compound in a large volume of water in the sea. Reversibility (or irreversibility) in target-receptor binding is very important for different sensing technologies. It has to be clearly understood that the sensor detects the bound target, whereas the information is always needed on the total concentration of target present in the system, both bound and unbound. Constellation of weak intermolecular interactions and their optimal arrangement allow achieving high affinity and selectivity even with relatively small molecules as receptors. Multivalency is the means to modulate the strength of these interactions and to achieve their reversibility. Irreversible binding is used in different types of sensors, often with the formation of target-receptor covalent bonds. When interacting with target, the fluorescence

Fig. 1.6 Detection possibilities operating with large and small volumes using the spotted sensor arrays. a Sample of controlled small volume is detached from any large volume and deposited on a sensor plate. All analyte is immobilized on a sensor plate providing the information on its concentration in a large volume. Analyte is immobilized strongly, which allows reagent addition and washing. b The sensor plate is inserted into a large volume. Negligible amount of analyte is bound to a plate in a reversible manner. The strength of binding is selected by the sensor design to indicate the analyte concentration in a large volume

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probe may change its chemical structure resulting in detectable change of its emission, often generating a new band. Fluorescence probes operating in this way are called the reactivity-based probes or chemodosimeters. An example is testing of volatile compounds that provide changes in chemical structures of fluorescent dyes as the probes (see Sect. 8.2). In such cases, methodology is based on the detection of all analyte present in the studied (sub)system. From a large volume of tested sample, a fixed small volume is isolated (e.g. a sample of blood from a patient). The testing is provided within this small volume for the detection of all present therein analyte species that become bound to receptor. Then an analytical result is extended to a large volume (e.g. to a whole patient body). Efficient in this case is a comparative test between two samples, the analyzed and the reference. The higher is the number of analyte molecules, the larger number of receptor sites will be occupied with them and the fewer number occupied with reference molecules that are commonly labeled with the dye of different color. This is the way of operation of different spotted microarrays, such as typical DNA chips (see Sect. 15.4 of Volume 1). Reagent additions (e.g. for enzyme amplification) and washing-out the unreacted species are possible and frequently provided. Though attractively simple, this approach has many disadvantages. The chips are one-use, it is hard to realize with them the pattern recognition techniques, and they do not allow the time-dependent monitoring. Reversible binding provides much larger possibilities but has important limitations regarding the concentration range for detection, depending on the strength of binding, that is dictated by the mass action law (Voit et al. 2015). This law is known as the direct basis for computational enzyme kinetics, ecological systems models, and models for the spread of diseases. Describing molecular sensing, when the binding is reversible and an equilibrium between the concentrations of unbound ligand [L] and this ligand in complex [LR] with receptor [R] is established, it can be used in the simplest form, Eq. (1.1): L + R ⇔ LR

(1.1)

In the conditions of dynamic equilibrium, a rapid binding-dissociation occurs all the time but the concentrations [R], [L] and [LR] do not change over time. In this state the forward reaction of analyte binding proceeds with the same rate as the reverse reaction of its dissociation. Thus, this law establishes relation between concentrations of free ligand, receptor and of their complex. According to mass action law, the equilibrium concentrations of the analyte ligand, [L], of receptor, [R] and of their complex [LR] are related by the binding constant K b : Kb =

k1 [L R] = k2 [L] × [R]

(1.2)

Since the dynamics is at equilibrium, it can be expressed by a rate constant k 1 for the process of binding and by rate constant k 2 for dissociation of the complex. Then, the equilibrium can be characterized by a ratio of these constants.

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1 Principles Governing Molecular Recognition

That can be either the binding constant K b = k 1 /k 2 or its reverse function, the dissociation constant K d = k 2 /k 1 . K b is often called also the stability or affinity constant, it is expressed in reverse molarity units (M−1 ), whereas K d = 1/K b is expressed in molar (M) units. It is essential that the range of ligand (analyte) concentrations is limited to two orders of magnitude, one below and one above the dissociation constant K d (see Chap. 2). Approximately, it is 81-fold change between 10% occupancy and 90% occupancy. This puts additional requirement on sensor operation, which should fit to this rather narrow range of required target concentrations. Too strong binding is often as useless as too weak binding. As it is shown in Chap. 2, different graphs can be plotted based on the correlations between the total analyte concentrations (that are the subject of our analysis) and the concentrations of bound analyte (that should provide the recorded signal). Usually the analyte binding isotherms are constructed, which are the graphs of sigmoid dependence of fractional receptor saturation on logarithm of ligand concentration. Essentially, they belong to stationary regime with no time-dependent changes involved.

1.2 Lock-And-Key, Induced Fit, Conformation Selection and Induced-Assisted Folding Models Molecular recognition proceeds as a dynamic process with a great number of structure making-breaking steps until the optimal interaction is realized. This process of search for complementarity between interacting partners may occur with or without their dynamic conformational adaptation. The theories explaining the selectivity of receptors for target analysis in molecular recognition events were primarily developed for explaining the catalytic power and high specificity of enzymes. Then they were extended to all manifestations of molecular recognition, such as molecular docking in drug discovery, and, finally, for sensing. They must account steric, molecular dynamic and energetic factors (Ciferri 2021). Lock-and-key model (Fischer 1894) postulates that complementarity between partner surfaces is imprinted between their three-dimensional structures. Fitting between them means geometrical fitting, much as the fitting of a key to a lock, Fig. 1.7. Optimal geometrical fitting together with optimal saturation of multiple noncovalent interactions results in tighter binding of specific ligand discriminating all other species that can be bound much less specifically (Chen et al. 2002). Molecular docking algorithms useful in drug design are based on this model, postulating the complete fitting (Brooijmans and Kuntz 2003). This model is actively used for design of molecular sensors. Particularly, it explains the ability of imprinted polymers to serve as receptors for not only of small molecules but also for large macromolecular structures (Bergmann and Peppas 2008), see Sect. 6.4.

1.2 Lock-And-Key, Induced Fit, Conformation Selection …

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Fig. 1.7 Illustration of models explaining specific target-receptor interactions. All of them postulate high degree of precise binding with 3D fitting but account for different levels of initial order and molecular dynamics in interacting partners

Induced fit model (Koshland Jr 1958) extends the lock-and-key principle. It allows conformational adaptation of receptor to the target binding. When an enzyme binds to the appropriate substrate, subtle changes in the active site conformation may occur. As a glove changes shape when a hand slips into it, so an enzyme changes its conformation on binding a ligand (Koshland Jr 1995). Thus, intermolecular interactions between complementary functional groups can be achieved in a dynamic process (Fig. 1.7). The reversible changes in shape of the recognition site to accommodate the target may occur due to local dynamics of participating groups of atoms without global transformation of the receptor. This model can be operative with many types of molecular and nanoscale compositions. Core of nanoparticles, such as semiconductor nanocrystals (quantum dots) or carbon dots, are quite rigid, but appended recognition groups allow conformational adaptation. Conformational selection as the mechanism of molecular recognition and selectivity in enzyme reactions was also described in the literature (Boehr et al. 2009; Hammes et al. 2009). With regard to molecular recognition, an important distinction between it and induced-fit is the step in the binding kinetics, at which the conformational change in the receptor occurs (Vogt and Di Cera 2012). The initial contact with the target may drive the fit induction but also provide the selection among the existing conformational isomers (Pang and Zhou 2017). In proteins, the conformational shift is connected directly with the binding affinity (Michielssens et al. 2015), suggesting one more strategy in sensor design. Following the concept of conformational selection, by providing alternative receptor conformers, the single sensors addressing several targets can be designed. As the receptors, not only the proteins but also the polymers with random substitution of functional groups may serve as the sensors (Jozefowicz and Jozefonvicz 1997). Induced-assisted folding model (Demchenko 2001) deals with the cases, when molecular recognition occurs between flexible molecules that are able to adopt many

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conformations (Fig. 1.7). Single-chain DNA and RNA, peptides with random conformation and linear polysaccharides are among these structures. Moreover, there are naturally unfolded proteins lacking regular conformation (Tompa and Fersht 2009). Without determined structure are synthetic polyelectrolytes. In many reactions of molecular recognition all of them behave in a surprising way. When they interact with the target, they fold into 3D structures maximizing noncovalent contacts both intrinsically and with the target, demonstrating the affinity and selectivity on a high level, similar to the “lock-and-key” receptors. In protein world, many examples demonstrate that folding and binding are coupled; the disorder-to-order transition occurs upon complex formation and can be localized to binding interfaces (Fong et al. 2009; Hegyi et al. 2007; Wright and Dyson 2009). Both the target and the receptor can exist as highly flexible molecular structures (Demchenko 2001). The formation of a specific complex between them is a dynamic process that can occur through sequential steps of mutual conformational adaptation (see Fig. 1.5). The potential receptor can be selected from the library of species without regular structure and become a very specific binder by a strong coupling of the chain folding and target recognition. Here we deal with the ensemble nature of molecular conformations that reduce to a single target-bound form, so that the gain in energy should overcome the strong entropy loss (Tzeng and Kalodimos 2012). This allows modulation of specificity and affinity of interaction in extremely broad ranges. The interacting partners can form a complex with entirely new properties and also produce conformational signal transduction at substantial distance (Fernández 2016). It was shown that rational genetic engineering can destabilize the protein structure in such a way that it becomes naturally unfolded, so that the target-induced folding can be realized (Kohn and Plaxco 2005). Binding the target should make the folded state thermodynamically more stable (Cissell et al. 2008) and subjective to induce efficient fluorescence response. Calmodulin can serve as an example of flexible structures in molecular recognition. It is the intracellular protein binder of Ca2+ ions. Its remarkable structural flexibility was demonstrated not only on binding these ions but also on interaction with different proteins. This molecule consists of two globular domains separated by a linker. Because of flexibility of the linker, calmodulin can accept a large variety of conformations and upon binding it adopts its conformation to the interacting molecules. The structure of its interaction complexes can be predicted using flexible docking algorithms (Schneidman-Duhovny et al. 2005). By appending two fluorescent proteins with conformation-dependent excited-state energy-transfer between them, the sensor for calcium ions was designed (Miyawaki et al. 1997). The binding of Ca2+ ions generates ratiometric fluorescence signal that can be calibrated as a function of their concentration (Fig. 1.8). Thus, different extent of conformational flexibility can be observed in specifically interacting molecular structures, from very rigid, conforming to lock-and-key model, to that with the absence of structural order. In drug design, the structural adaptation upon ligand binding resulting in induced folding is usually very difficult to predict (Fernández 2016). For applications in fluorescence sensing, the stronger change in molecular order is the better, since it gives more possibilities to generate on binding

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Fig. 1.8 Calmodulin molecule demonstrating high conformational variability on binding calcium ions and interacting with proteins. a Calmodulin in open ligand-free conformation (yellow) and closed ligand-bound conformation (semitransparent blue) are aligned by the first domain. The bound Ca2+ ions are shown as red balls. The motion of the second domain induced by the ion binding is indicated by arrow. b The model of structure of the complex of calmodulin with myosin kinase peptide. The conformation of the calmodulin in the bound complex is in red. The myosin kinase peptide is shown in green. c The “chameleon” that changes the fluorescence spectrum in response to Ca2+ ions binding. The sensor is composed of calmodulin (responding to ion binding by conformational change) and green (GFP) and yellow (YFP) fluorescence proteins that are fused to N and C terminals of calmodulin via calmodulin-binding peptide M13 (Miyawaki et al. 1997). The binding of Ca2+ ions induces the conformational change that brings together the two domains. d Resulting switching from the donor to acceptor emission as seen in fluorescence spectra (Truong et al. 2007). e The ratiometric fluorescence response curve built on increasing concentration of calcium ions

the fluorescence response (Cerminara et al. 2012; Nagpal et al. 2020; Plaxco and Soh 2011). Operating with different fluorescence parameters and in broader ranges becomes possible, so that the ability to derive information on target-receptor specific binding by probing the state of receptor conformation can be rather easily realized (Sect. 4.4 of Volume 1).

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1.3 Realization of Principles of Molecular Recognition in Fluorescence Sensing Fluorescence-based sensing technologies develop rapidly in the directions of finding and application of new materials and the extension to new areas that become available for the analysis. All these issues are presented in details in the following chapters. Here we discuss different concepts based on the coupling of molecular recognition with fluorescence reporting that are in the background of these applications.

1.3.1 The Output Parameters Used in Fluorescence Sensors There are limited possibilities for modulation of fluorescence signal providing the sensor response. Fluorescence techniques can suggest only several parameters for reporting, and for their proper use in quantitative analysis they need referencing and calibration. These issues are elaborated in detail in Chap. 3 of Volume 1. Here I highlight the most important points. Due to spectral overlap, only a small number of reporter dye types covering the visible wavelength range can be used simultaneously, and only several types of ground-state or excited-state transformations provide the change of their emission color, anisotropy and lifetime that can deliver a calibrated signal. Fluorescence intensity as a parameter characterizing the sensor response depends on many other instrumental and sample-dependent factors. Commonly the fluorescence intensity F is measured in relative units that are proportional to the number of emitted quanta and have no absolute meaning if they are not compared with some standard measurement. Therefore, it needs calibration. Calibration means the operation, as a result of which at every sensing element (molecule, nanoparticle, etc.) or at every site of the image the fluorescence signal becomes independent of any other factor except the reported concentration of bound target. Hopefully, calibration can be realized on molecular level. Figure 1.9 illustrates the possibilities for obtaining a calibrated signal. The origin of the background mechanisms in self-referencing in four cases shown above is dissimilar (Demchenko 2010b; Demchenko 2014). If the reporting signal is recorded to be different at two wavelengths, these intensities can be considered as being provided by two information channels, and their ratio provides a calibrated signal (Demchenko 2005a; b). This method known as λ-ratiometry is commonly used not only in sensing, but also in imaging, flow cytometry, etc. (Demchenko 2006). Fluorescence anisotropy (polarization) is already self-calibrated since it involves calculating the ratio of intensities at two vertical and horizontal polarizations. Fluorescent decay in time is intrinsically independent on dye concentration and instrumental factors (Wei et al. 2017). In Chap. 2 the reader will learn more about the use of these parameters in sensing.

1.3 Realization of Principles of Molecular Recognition in Fluorescence …

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Fig. 1.9 Possibilities for obtaining the self-calibrated signal in fluorescence sensing. Intensity sensing with reference can be realized with two dyes, one reporting and one referencing, when they are coupled together or included into the same nanoparticle. Switching between two wavelengths of fluorescence emission in a reporting dye can be achieved due to its switching between ground-state forms (conformational changes) or in reactions of charge transfer or proton transfer in the excited state if these reactions are not associated with strong quenching. With two coupled dyes, the excited-state energy transfer can be modulated. Suppression of rotational motion of targetsensor complex can be seen as the increase of anisotropy. The dynamic quenching-dequenching of fluorescence modulates the fluorescence decay, changing the lifetime

1.3.2 Different Strategies in Fluorescence Sensing Here we focus on different strategies for extracting the information on sensor-target interaction and on the possibilities for its transformation into quantitative target assay. If neither sensor receptor nor the target emits fluorescence or if their fluorescence is insensitive to the binding, how we can get information from the appended fluorescence probe? Several working strategies can be realized. I will analyze four of them that are quite characteristic. They are the labeling of all pool of potential targets (as it is commonly used in DNA hybridization assays), sandwich assays, the most developed of which are the immunoassays, competitive binding assays that can be applied to a broader range of sensor-target affinities, and direct reagent-independent sensing, which is the most advanced strategy (Fig. 1.10). The detailed characteristics of these strategies can be found in Chap. 2 of Volume 1. Here their basic features are outlined that are quite variable.

18 Fig. 1.10 The strategies in fluorescence sensing of different analytes. The notations are: (R)—receptor, (T)—target, (I)—labeled indicator and (C)—labeled competitor

1 Principles Governing Molecular Recognition

Labeling the pool of targets

+

R

R

Sandwich assay

+

+

R

R

Competitive binding assay

+

+

R

R

Direct assay R

+

R

In a system, in which the target has to be analyzed, the dye is introduced providing strong, usually covalent, binding of target together with similar compounds. Then, after possible enrichment, the sample is introduced into the device providing strong binding to sensor receptors. If spotted arrays are used, the unbound or weakly bound non-target compounds are washed-out and the sensor plates are analyzed. If suspended arrays are used, the complex with labeled target can be isolated by chromatography or analyzed in flow cytometry. Sandwich assays, which are commonly used with application of antibodies or aptamers, behave differently. Here two recognition steps are needed. First is between target and receptor that are unlabeled and then this complexation is visualized by recognition with the second specific binder. The latter is labeled or even bound with enzyme producing light absorbing or fluorescent reaction product enhancing the assay sensitivity. This is the mode of operation of enzyme-linked immunosorbent assay (ELISA) that is performed with antibodies for sensing either the antigens or the diagnostic antibodies. In competitive binding assays, neither receptor nor target may contain a fluorescence label and for visualization of target binding, a fluorescent competitor is introduced. Usually a competitor is the labeled target analog that binds to the same site on the receptor as the target. After this binding, a mixture of different compounds that contains the target is applied. Due to its specific interaction (molecular recognition) with the receptor, the target displaces the competitor from the binding site. Realizing specific molecular recognition and quantitative analysis of the target can be achieved because it is the only species that can displace the competitor. The competitor reports about its displacement by changing the parameters of its fluorescence emission. With labeling of both receptor and competitor the excited-state energy transfer can be used in sensing, generating two-band ratiometric emission.

1.3 Realization of Principles of Molecular Recognition in Fluorescence …

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Direct sensing with immediate ‘mix-and-read’ fluorescence response to sensortarget interactions could be very attractive technology. But how to realize it? The only possibility for sensing with the dye that stays as ‘the label’ is the use of its response to the change of molecular rotation rate on the target-sensor complexation. In other cases, we need ‘smart dyes’ incorporated into specific locations for becoming sensitive to the change of their intermolecular interactions in the sensor in response to target binding (Altschuh et al. 2006; Arugula and Simonian 2014; Banala et al. 2013). In more detail, direct sensing and selected examples of its application will be discussed below (Sect. 1.5). In all discussed above fluorescence sensing technologies, the reporter dye of organic or inorganic origin, of molecular or nanoscale size, plays very important role and may be the major factor determining the sensitivity of assays. Are there any possibilities for increasing it? There are several of them (Scrimin and Prins 2011), operating on the level of different participants of the sensing process (Fig. 1.11). A great range of possibilities appear when organic dyes are incorporated into nanoparticles formed of organic or inorganic (silica) polymers or form nanoparticles themselves (see Chap. 8 of Volume 1). As well as fluorescent conjugated polymers, these nanoparticles demonstrate superquenching-superenhancement effect (Achyuthan et al. 2005) that allows increasing dramatically the dye sensitivity. Also, the brightness of fluorescence reporters can be enhanced with plasmonic nanoparticles (Badshah et al. 2020), see Chap. 13 of Volume 1. The response of indicators in sandwich-type assays is usually enhanced by attachment of enzymes generating a product that can be detected by light absorption or emission (catalytic amplification), and the popularity of these assays is due to their apparent efficiency (Scrimin and Prins 2011).

R Receptor Modulation of its density

+

+ Target Enrichment of the pool PCR-based enrichment (for nucleic acids)

R

Indicator

Output signal

Assembly into nanoparticles Superquenching Plasmonic enhancement

Catalytic amplification Engineering medium conditions

Fig. 1.11 Possibilities for the enhancement of sensitivity (on an example of sandwich assays)

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1 Principles Governing Molecular Recognition

As with any measurement, fluorescence sensing involves some errors (Ortiz et al. 2003). The error level of optical measurements is usually minimal in comparison with the errors that come from the samples. They can be both random (e.g. due the target or sensor concentrations deviated from average) and systematic. The latter are called false-positive when some part of non-target species happens to be bound, generating increased signal level. When some part of target is missed in the sensing process, this leads to false-negative result.

1.4 Molecular Recognition of Different Strength and Specificity Playing with the strength of target-receptor binding, we can realize three typical possibilities: strong irreversible binding in a small volume extracted from studied system, reversible but specific binding that can be realized in large volume and the binding with relatively low specificity realized in artificial noses and tongues. All of them can be realized for multiplex sensing in arrays but with different procedures in providing the studies and analysis of results.

1.4.1 Sensors Providing Strong Highly Specific Binding The sensor can report on the quantity of bound target to its receptor, which may be only a small part of all targets in the studied system. One of the possibilities to measure the concentration of all the targets in a large system containing much higher amount of free target is to extract the small portion of material from it and to provide its examination, as it is shown in Fig. 1.6a. Then the detection of needed compound can be performed in this small volume in the conditions that the entire target is bound to sensor receptors. The concentration of bound target is determined and, with the knowledge of sample volume, it can be recalculated and assumed to be the concentration in a large volume. One has to be sure that the binding is strong enough for such substantial shifting the equilibrium to the bound form. Also, all these binding reactions should be kinetically realized. The advantage of this conceptually simple approach is apparent. When the binding is achieved, many different manipulations with the sample become possible. If the binding is performed in solution (homogeneous assays), then the analysis with flow cytometry or chromatography, or just counting the microscopic beads under microscope, can be realized. The sensors can be also immobilized on spotted arrays (heterogeneous assays). In this case any their treatments can be performed, including washing-out the unreacted material or the performance of secondary reactions with added reagents. The latter can be used for enzyme-based amplification of output signal.

1.4 Molecular Recognition of Different Strength and Specificity

21

DNA hybridization assays and immunoassays use these possibilities by collecting a small sample and determining multitude of targets therein, since all of the target species will be bound. Microarrays can be fabricated, in which every spot corresponds to different target. DNA hybridization assays and sandwich immunoassays, such as ELISA, frequently use such approach that does not provide immediate result and needs after-treatment before readout of fluorescence data.

1.4.2 Sensors Based on Competitive Target Binding Very good but still limited in application are the sensing technologies that are based on competition between the target and its fluorescent analog for the receptor binding sites (Sect. 2.3 of Volume 1). This allows avoiding the introduction of fluorescence label into analyzed samples, so that neither receptor nor target may contain a fluorescence label (Nguyen and Anslyn 2006; Wu et al. 2013). Instead of labeled indicator (as in sandwich assays), an additional player in the target recognition mechanism is introduced, the fluorescent competitor. Usually a competitor binds to the same site on the receptor as the target. Exposing the sensor to sample that contains the target results in displacement of the competitor from the receptor due to its specific interaction (molecular recognition), competing for the binding site (Fig. 1.10). The target can be analyzed quantitatively because it is the only species that can displace the competitor. This sensor can operate efficiently if it is based on two conditions. First, there should be close correspondence of affinities to receptor of target and competitor. Second, competitor has to provide signaling by recorded difference in its fluorescence parameters, whether it is in the free or receptor-bound form. These changes can be calibrated as a function of target concentration. This technique offers only limited possibilities for fluorescence reporting. Increasing its rotational mobility, the competitor release from the binding with receptor can be detected as the drop of anisotropy. Alternatively, the dye can be reportive being photochemically active, protonation-deprotonation sensitive, environment-sensitive, etc. If the sensor is also labeled with proper dye, then the rupture of excited-state energy transfer to/from the competitor may provide the analytical signal. In any case, this very attractive method is hardly prospective for universal applications. In order to achieve desirable sensitivity, the binding constant of the analyte to the receptor should be comparable to that of the indicator to the receptor. This is not easy to achieve with sensor arrays operating with many analytes and the broad ranges of their analyzed concentrations.

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1.4.3 Sensors Based on Reversible Specific Binding and Operating in a Large Volume For operation in a large volume and continuous monitoring in it of target concentration, the target binding should be reversible and the dynamic equilibrium should be established between free and bound target (Fig. 1.6b). Since only negligible amount of analyte is bound to sensor reversibly, the sensor can indicate the analyte concentration in a large volume. The receptor affinity should be chosen in such a way that the binding sites should be only partially occupied covering the range of less than between 0.1 K d and 10 K d , in accordance to mass action law (see above). A special sensor design only can make this range broader or shorter, as it is shown in Chap. 2. Thus, the sensor-target interactions are reversible and they allow quantitative target detection, but only within a limited concentration range. Collection of small samples is not needed; the sensors can be incorporated into large volumes and allow continuous monitoring of production/consumption of targets in them. Formation of arrays is possible and they can be of multiple use. They can be designed based on the principle of exact sensing (each element of array recognizes particular sensor with highest affinity) and also on the principle of distributed sensing (forming pattern of variable interactions with different sensors). But for exploration in full of this approach, one has to find solution to the problem of introducing the fluorescence response. This problem does not have a general solution, and the existing possibilities based on application of environment-sensitive dyes will be discussed below (Sect. 1.5).

1.5 Direct Reagent-Independent Sensing Direct reagent-independent sensors are the sensors of our future. These are the sensors with target binding that could be fully reversible and without need for physical or chemical manipulation (such as reagent addition, long incubation or washing) in the essay procedure (Sect. 2.4 of Volume 1), see (Altschuh et al. 2006; Miranda et al. 2011). Producing immediate result, they are the most attractive for personal use with smartphone readout, with the possibilities for wireless communication and remote analysis based on machine learning (see Chap. 18). However, the possibilities for signal transduction to generate fluorescence response are rather narrow. The fluorophore must be a part of receptor or, in the sensor design, has to be connected with it using some signal transduction mechanism. Thus, it should be sensitive to the target binding directly, even if the target is not the fluorophore or fluorescence quencher. Response to target binding should be seen in a calibrated manner by the λratiometric changes in intensity (Demchenko 2023a, b) or the changes in anisotropy or lifetime (Demchenko 2010a), see Fig. 1.9. To generate informative change in emission, the fluorophore should be located in proper place. It can participate in molecular recognition site with the target.

1.5 Direct Reagent-Independent Sensing

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Contacting with it should result in the change of its emission, which is not easy to provide without influencing the binding. Rotational diffusion of sensor-analyte complex can be larger than of the sensor alone, suggesting the sensing in anisotropy. However, such method needs tough correlation of fluorescence lifetime τF and rotational correlation times. The τF ~1 ns may be too short. Among all these possibilities, conformational changes in sensor molecules coupled with target response are the most attractive because of broad possibilities for realization. Three examples presented below refer to three flexible structures that can be efficient for molecular sensor production: peptides, conjugated polymers and aptamers. Peptides are flexible structures that can adopt rigid conformation interacting with multiple bonds with the target. Classical example is zinc finger, a domain of 25– 30 residues that folds on coordination of Zn ion. Labeling with fluorescent dye transformed it into Zn sensor (Walkup and Imperiali 1996). A much shorter peptide was applied recently as the sensor for silver ions (Yu et al. 2021). Upon interaction with this ion, the dansyl group of Dansyl-Glu-Cys-Glu-Glu-Trp-NH2 peptide starts to interact with Trp residue becoming the energy transfer acceptor. As a result, switching of emission from that of Trp at 355 nm to that of dansyl at 510 nm occurs as a function of concentration of silver ions, indicating high binding constant 6.4 × 10−9 M obtained in wavelength-ratiometric detection (Fig. 1.7). The conjugated polymers, displaying their conformation-dependent fluorescence (Barbara et al. 2005; Wang et al. 2016, 2017) are prospective for designing the direct sensors. There is an interesting example of obtaining highly sensitive sensor for DNA by assembling its disordered ssDNA structure with the disordered polymer (Doré et al. 2004). The single-stranded capture DNA is combined with conjugated polymer polythiophene possessing appended cationic groups (see Fig. 1.10). Interacting electrostatically with negatively charged DNA, it forms an assembled rigid structure that can specifically bind target DNA. It is known that the chain conformation produces dramatic impact on the properties of π-conjugated system that influences the fluorescence, since it determines the conjugation length (Pecher and Mecking 2010). In this case, the reporting fluorescence emission demonstrates a yellow-to-red color change due to the formation of a planar highly conjugated form of the polythiophene backbone. The addition of complementary DNA target strand leads to superstructure, in which the polythiophene is wrapped in a helical fashion around the DNA duplex, resulting in a blue shift and increase in fluorescence (Fig. 1.12). This fluorescent polythiophene biosensor system allows specific detection of a few hundred molecules of genetic material (Doré et al. 2004). Such recognition is based on simple electrostatic interactions between a cationic polymeric optical transducer and the negatively charged nucleic acid target. This method is versatile enough to detect nucleic acids of various lengths; it can be done simply and affordably, without any chemical reaction or PCR amplification. For further increase of sensitivity, the excited-state energy from polythiophene moiety serving as the donor can be transferred to fluorescent dye serving as emissive acceptor (Ho et al. 2005). This could allow further increase of sensitivity that without PCR could detect only several DNA molecules and recognize the single nucleotide polymorphism.

1 Principles Governing Molecular Recognition

Fluorescence intensity

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Ag+

Wavelength (nm)

Fig. 1.12 Peptide-based direct fluorescence sensor for Ag+ ions operating on ratio detection mode (Yu et al. 2021). Peptide folds around silver ion, demonstrating the change of fluorescence emission from 355 to 510 nm due to energy transfer from Trp to dansyl group that become proximal due to peptide folding

Aptamers are relatively small molecules that behave very efficiently in different fluorescence sensing technologies, substituting and outperforming the antibodies (Sect. 5.2). Their disordered structure may transform to strong ordering on binding target proteins, and due to strong increase of molecular mass on forming their complex, this formation can be easily detected by increase of polarization of emission of appended fluorophore (Baldrich 2010), see Fig. 1.13a. Such result was demonstrated on binding of thrombin (Potyrailo et al. 1998). A detectable change in the fluorophore rotational diffusion rate induced by target binding was measured as the change in the evanescent wave-induced fluorescence anisotropy. Another possibility to use reversible folding-unfolding reaction of aptamer on recognition of proteins can be realized with response in fluorescence quenching (Fig. 1.13b). In both cases, the design was made in such a way that the unfolded structure is transformed into highly ordered and stable G-quadruplex (see Sect. 5.3). The opposite effect can be also realized, so that in free aptamer the fluorophore and the quencher are in close location but on protein binding they move apart giving rise to strong fluorescence (Yamamoto and Kumar 2000). Direct reagent-independent sensing is the area of application of environmentsensitive dyes. When located at proper sites, they are efficient in demonstrating the conformational changes associated with molecular recognition of the target, even if these changes are not very large. Definitely, they are the best performers when the target binding fits the induced-assisted folding model (see Sect. 1.2). In my experience, the best in this respect are the dyes exhibiting the excited-state intramolecular proton transfer (ESIPT) reaction that may change dramatically the color of fluorescence emission showing interplay between initially excited N* band and ESIPT product T* band that can be dramatically shifted to longer wavelengths (Fig. 1.14). Many examples of successful applications of such color-changing approach can be found in the literature (Demchenko et al. 2013) and the advantages of its applications in sensing and imaging were frequently discussed (Altschuh et al. 2006; Demchenko

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Fig. 1.13 Examples of sensing with direct fluorescence response using aptamers demonstrating the conformation arrangement on target proteins binding (Baldrich 2010). a Mechanism of action of a fluorescent aptamer based in the changes in the evanescence wave-induced fluorescence anisotropy generated by target binding. The increase in size due to capture of a relatively large target limits the aptamer rotational diffusion and thus affects the emission of the fluorescent label. The increased polarized emission provides the measure of target concentration. b The flexible aptamer molecule labeled with fluorophore and quencher at two its terminals is disordered, adopting many conformations with no interaction between fluorophore and quencher. On specific interaction with proteins, it folds to ordered structure, so that fluorophore and quencher get together. The direct response to target binding is generated on quenching. The target concentration is measured as a proportional decrease of fluorescence intensity

2006; Klymchenko and Mely 2013). They range from characterizing solvent polarity (Ercelen et al. 2002; Klymchenko and Demchenko 2003), electrostatic interactions (Klymchenko and Demchenko 2002a) and proticity (Shynkar et al. 2004) to the structures of micelles (Klymchenko and Demchenko 2002b), phospholipid bilayers (Klymchenko et al. 2003; Klymchenko et al. 2004) and membranes of living cells (Oncul et al. 2010) including their pathological states (Shynkar et al. 2007). They were successfully applied in the studies of nucleic acid interactions (Michel et al. 2020). Molecular recognition of antibodies by labeled antigenic peptide was also demonstrated (Enander et al. 2008). Regarding protein–protein recognition, the prototype of sensors based on protein molecules exhibiting conformational changes was designed, demonstrating strong λ-ratiometric response in enzyme inhibitor α1 antitrypsin interacting with elastase (Boudier et al. 2009). The 3-hydroxychromone

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Fig. 1.14 The principle of two-band ratiometric fluorescence sensing with functional 3hydroxychromone (3HC) dyes (Demchenko 2006). On excitation of any labeled molecular or supramolecular structure (sensor array, microfluidic device (MFD) or living cell) the fluorescence intensity is distributed between two bands, N∗ and T∗ , centered in green and orange-red regions of the spectrum (a) due to establishment of dynamic equilibrium between two excited-state forms (b). The sensing signal is produced by the change of relative intensities of these two bands (λ-ratiometry)

(3HC) dye was attached to a residue located distantly from the binding site but sensitive to conformation change of enzyme on binding the inhibitor. These and many other examples demonstrating visualization of immediate easily detected and self-calibrated response to different types of intermolecular interactions show promise for efficient implementation of λ-ratiometry into different types of sensing technologies. The observed examples demonstrate that they are not limited to ESIPT but can involve also charge transfer and energy transfer reactions (Pei et al. 2021). Such reactions, however, are often associated with quenching, and it is hard to find the dyes and conditions that could allow to keep comparable level of two intensities (Demchenko 2014).

1.6 Simultaneous Analysis of Multiple Analytes Single analyte sensing is a classical approach for analytical chemistry. It is dominant also in different imaging techniques, where the distribution of a single compound is commonly of the major interest and its highest selectivity is required. It becomes impractical, however, in the analysis of complex mixtures, where many compounds contribute to integrated function and many of these components are with subtle structural differences. Analysis of honey, perfume or vine and of other important objects cannot be complete with this type of sensing. Multiplex sensing means the detection of multiple analytes in a single sample (He et al. 2017; Jarockyte et al. 2020), and it can be provided with the array-based

1.6 Simultaneous Analysis of Multiple Analytes

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Fig. 1.15 Differences between traditional and multiplexed diagnostics (Jarockyte et al. 2020). The main advantage of multiplexed analysis is the capability to detect multiple analytes qualitatively and quantitatively in a single sample

technologies. There is an increasing need to quantify a large number of species from minute sample volumes. Simultaneous detection of multiple analytes is especially important in clinical diagnostics, where it allows more accurate assessment of changes in biomarker expression at the earliest stages. The results are obtained based on the same sample and in the same conditions, saving a lot of time, manpower and materials (Fig. 1.15). Their analysis allows consistent decision making, avoiding many errors. Since many compounds are analyzed simultaneously and in the same conditions, this allows detecting very small differences in their structure, reactivity and other properties. Arrays containing sensors with various affinities to the same target allow them to be measured in a broad range of their concentrations.

1.6.1 Systems for Detection of Multiple Analytes Multiplex sensing gives researchers a possibility to perform thousands of simultaneous assays. The specific interactions between the sensors and their targets can proceed in solutions and also when one of the partners is immobilized on solid support. They can be extended to a scale of assembled multiple sensors known as arrays. Sensor arrays (ensemble-based sensors) are assembled of many sensor units containing recognition and reporting elements. They can operate in solutions (or in suspensions of particles) and also in immobilized conditions on flat surfaces (Fig. 1.16).

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1 Principles Governing Molecular Recognition Excitation

a

●

● ●

Emission

b

● ●

● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ●

Suspension arrays

Spotted arrays

Fig. 1.16 Different principles of formation arrays for sensing multiple analytes. a Suspension arrays performed in solutions. Each suspended sensor contains, in addition to analyte recognition and reporting functionality, a ‘barcode’ recognizing the sensor in flow cytometry or chromatography detection. b Spotted arrays, in which the sensors addressing different targets are deposited on the surface. A scanning fluorimeter (array detector), provides 2D scanning the array, generating fluorescence response from every spot

Both suspension and spotted array technologies develop rapidly. For suspension arrays we need to provide optically programmed identity in the particles themselves, generally by means of a fluorescent signal. Thus, barcodes being ubiquitous in our daily lives and serving for reducing complex information to a simple pattern have found important applications in sensing. Smart nanocomposites and microspheres (Tang et al. 2020) allow designing the suspension array elements that contain ‘barcodes’ recognized by detection devices. Variations of emission colors and intensities can be used as codes to identify targets. Size-dependent light emissive quantum dots (Wu et al. 2020) and multicolored dye-doped nanoparticles (Visaveliya and Köhler 2021) allow many possibilities for barcoding. DNA nanostructures in particular are promising for molecular barcodes, as they can be designed to reconfigure in the presence of molecular biomarkers such as proteins, antibodies and nucleic acids (Chandrasekaran et al. 2021). In spotted arrays, the identification of sensor to particular analyte is made by its location that is provided by 2D scanning (Epstein et al. 2002; Sassolas et al. 2008).

1.6.2 Specific Target Recognition Versus Pattern Recognition Sensor Arrays Arrays can be constructed in such a way that each of its elements specifically senses single analyte. They use the analogy with classical lock-and-key concept to explain and predict the high affinity of every sensor element to its target with an attempt to avoid cross-reactivity. Such arrays can be called specific target recognition arrays or exact sensor arrays. Despite tremendous successes, single analyte sensing arrays demonstrate several drawbacks that limit their applications. Thus, it remains very challenging to design

1.6 Simultaneous Analysis of Multiple Analytes

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Fig. 1.17 Transformation from exact to pattern recognition sensing, and the corresponding outputs that are generated by sensor designs (Peveler et al. 2016). a An antibody gives specific information about a single analyte, due to its high specificity. b Multiple analytes require different antibodies, leading to the creation of an antibody array in which each array component is only specific to a single analyte with no cross-reactivity. c Selective sensor arrays create unique patterns (fingerprints) for each analyte due to the cross-reactivity of the array components. d Hypothesis-free, crossreactive arrays enable the differentiation of multiple analytes, even those of different types (organic, inorganic, etc.), with no previous knowledge needed. In all three of the multisensor platforms patterns are generated that can be analyzed to match against a known pattern using statistical methods

selective receptors for structurally similar analytes and to overcome the crossreactivity that remains a significant problem. For instance, the only difference between glutamate and aspartate is a methylene group in the side chain, and it is highly likely that the receptor designed for one would interact with the other. The arrays made for characterizing complex mixtures cannot recognize them as whole systems, especially if the structure of some ingredients is not known. Can we benefit from these disadvantages? The idea was borrowed from Nature. For the sense of smell and taste, the mammalian nose and tongue operate according to the principle of distributed sensor arrays. Rather than sensing an analyte by its strong affinity for one particular receptor, the recognition is achieved by the composite response of the analyte to the entire array of receptors. The difference between two concepts is illustrated in Fig. 1.17. Interacting with variable affinity but with many different receptors, the analyte studied in a single experimental run generates the pattern of sensor responses that is seen as multiple outputs examined and treated as multidimensional information. A different analyte forms a different pattern. In this way, the whole system is characterized. It may contain the analytes of unknown structure. This is the working principle of artificial electronic (photonic) noses and tongues (see also Sect. 18.2) that is also called differential sensing (Lavigne and Anslyn 2001). Thus, the array-deposited sensors may have different binding characteristics, none of which are necessarily specific or even very selective. It is important that molecular

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recognition occurs with at least several different sensors to generate a pattern. As a consequence, a collection of fingerprint responses are generated, which can lead to a distinct pattern for single analytes or composite mixtures comprising multi-analytes. The composite signal is evaluated and interpreted by pattern recognition protocols (Chen et al. 2020; Li et al. 2018; Wong and Khor 2019).

1.7 Sensing and Thinking. Current Trends that Should Be Highlighted From the time of Emil Fischer 100 years ago when the lock-and-key model was suggested, we started much better understand the mechanisms of formation of strong and selective complexes between molecules, molecular recognition. Both steric complementarity and sterically determined formation of multiple bonds, individually weak but producing strong collective effect, determine the recognition. Meantime, the cases when the interactions occur between rigid structures are rare. The conformational adaptation of interacting partners is more typical. Its extent can be different, from motions of small groups of atoms to rotations of protein domains and folding of DNA aptamers. These processes are activated and they put forward the kinetic aspects of molecular recognition. It is the characteristic feature of fluorescence sensing that we need fluorophore for providing the output signal. Fluorescent targets are very rare, and commonly we need to introduce fluorophore into the test system and to make this fluorophore responsive to target-sensor binding. Which fluorophore to take and how to introduce it? There are two basic possibilities. One is to use any fluorophore that is efficient for labeling or even enzyme generating fluorescent product. Two things can be done with that. One is to label the target and the other is to use the labeled species interacting with the target but not interfering with target-sensor interaction (see Fig. 1.10). To quantify the target, one has to isolate the target-sensor complex containing fluorophore from contaminating species also containing fluorophore by washing, chromatography, etc. The other possibilities are demanding towards fluorophore. The fluorophore should be able to change its emission properties. This change has to provide the signal if the labeled competitor is bound or it is released from the binding site. It can be the part of sensor and has to report if the sensor is free or bound with the target. These smart fluorophores are commonly organic dyes that are performing the change of emission due to their excited-state reactions (Chap. 5 of Volume 1). Then we come up to the most difficult decisions. How to introduce such smart dye into the sensor without perturbation of target binding but with high sensitivity to this binding? And we try to get use from conformational changes in sensor on target binding. These changes should change the environment and interactions of the bound dye, generating the needed fluorescence reporting signal. Different sensing formats are in development and realization in practice and the reader has to evaluate their advantages and disadvantages when applied to particular targets and particular conditions with the account of different situations of sensor

1.7 Sensing and Thinking. Current Trends that Should Be Highlighted

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use. Answering the questions listed below will help the reader to move from passive reading to thinking, analysis and creativity. 1.

Explain the term ‘molecular recognition’. What is the difference from just ‘molecular interaction’? Why molecular recognition should be optimized and what are the criteria for this optimization? 2. Explain collective effect that appears on multivalent binding. How the multivalent binding increases the sensor selectivity? Provide the estimates for increasing the sensor affinity. 3. Explain the source of entropy change on hydrophobic interactions. What other noncovalent forces do you know? Why they lead to reversibility in target-receptor binding? 4. Can covalent bonding be reversible? What are kinetic steps in this binding? 5. What is the mass action law? Why reversible binding always occurs within a limited range of target concentrations? Estimate this range in the simplest case and explain how it can be modulated. 6. Explain the basic difference between reversible and irreversible target-receptor binding. How they are realized in designing the sensors operating in small and large volumes? 7. Explain the difference between lock-and-key, induced fit, conformation selection and induced-assisted folding models. What is essential in terms of conformational changes in the sensor on target binding? What is more attractive regarding the fluorescence sensor design? 8. Why the sensor output in fluorescence intensity needs calibration? How to provide this calibration on molecular level? What is special with fluorescence anisotropy and lifetime sensing? 9. Explain different strategies in fluorescence sensing. Why some of them need adding third component beside target and sensor? Should this component be always fluorescent? 10. Explain the operation of direct sensor. Why the addition of any third component and washing the unreacted species is not needed here? Explain special requirements that should be realized in such cases. 11. Can the two information channels needed for the self-calibrated output fluorescence signal be the intensities at two wavelengths of a single dye? What are the requirements for that? 12. Explain the two strategies that are followed on fluorescence sensing of many targets in one sample (multiplexing)—specific recognition sensor arrays and those based on pattern recognition (electronic noses and tongues). Why the request for determination of a target and any combination of targets can, in principle, be realized following the pattern recognition strategy?

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Shynkar VV, Klymchenko AS, Piemont E, Demchenko AP, Mely Y (2004) Dynamics of intermolecular hydrogen bonds in the excited states of 4‘-dialkylamino-3-hydroxyflavones. On the pathway to an ideal fluorescent hydrogen bonding sensor. J Phys Chem A 108:8151–8159 Shynkar VV, Klymchenko AS, Kunzelmann C, Duportail G, Muller CD, Demchenko AP, Freyssinet J-M, Mely Y (2007) Fluorescent biomembrane probe for ratiometric detection of apoptosis. J Am Chem Soc 129:2187–2193 Tang G, Chen L, Wang Z, Gao S, Qu Q, Xiong R, Braeckmans K, De Smedt SC, Zhang YS, Huang C (2020) Faithful fabrication of biocompatible multicompartmental memomicrospheres for digitally color-tunable barcoding. Small 16:1907586 Tompa P, Fersht A (2009) Structure and Function of Intrinsically Disordered Proteins. CRC Press Truong K, Sawano A, Miyawaki A, Ikura M (2007) Calcium indicators based on calmodulinfluorescent protein fusions. In: Protein Engineering Protocols. Springer, pp 71–82 Tzeng S-R, Kalodimos CG (2012) Protein activity regulation by conformational entropy. Nature 488:236–240 Varner CT, Rosen T, Martin JT, Kane RS (2015) Recent advances in engineering polyvalent biological interactions. Biomacromol 16:43–55 Visaveliya NR, Köhler JM (2021) Softness meets with brightness: dye-doped multifunctional fluorescent polymer particles via microfluidics for labeling. Adv Opt Mater: 2002219 Vogt AD, Di Cera E (2012) Conformational selection or induced fit? A critical appraisal of the kinetic mechanism. Biochemistry 51:5894–5902 Voit EO, Martens HA, Omholt SW (2015) 150 years of the mass action law. PLoS Comput Biol 11:e1004012 Walkup GK, Imperiali B (1996) Design and evaluation of a peptidyl fluorescent chemosensor for divalent zinc. J Am Chem Soc 118:3053–3054 Wan Y-H, Zhu Y-J, Rebek J, Yu Y (2021) Recognition of hydrophilic cyclic compounds by a water-soluble cavitand. Molecules 26:1922 Wang P, Zhao L, Shou H, Wang J, Pan L, Jia K, Liu X (2016) Chain conformation dependent fluorescence of blue-emitting poly (arylene ether nitrile). J Lumin 179:622–628 Wang X, He G, Li Y, Kuang Z, Guo Q, Wang J-L, Pei J, Xia A (2017) Odd–even effect of thiophene chain lengths on excited state properties in Oligo (thienyl ethynylene)-cored chromophores. J Phys Chem C 121:7659–7666 Wei L, Yan W, Ho D (2017) Recent advances in fluorescence lifetime analytical microsystems: contact optics and CMOS time-resolved electronics. Sensors 17:2800 Wong S-F, Khor SM (2019) State-of-the-art of differential sensing techniques in analytical sciences. TrAC, Trends Anal Chem 114:108–125 Wright PE, Dyson HJ (2009) Linking folding and binding. Curr Opin Struct Biol 19:31–38 Wu P, Xu C, Hou X (2013) Exploration of displacement reaction/sorption strategies in spectrometric analysis. Appl Spectrosc Rev 48:629–653 Wu W, Yu X, Gao M, Gull S, Shen L, Wang W, Li L, Yin Y, Li W (2020) Precisely encoded barcodes using tetrapod CdSe/CdS quantum dots with a large stokes shift for multiplexed detection. Adv Func Mater 30:1906707 Yamamoto R, Kumar PK (2000) Molecular beacon aptamer fluoresces in the presence of Tat protein of HIV-1. Genes Cells 5:389–396 Yao H, Ke H, Zhang X, Pan S-J, Li M-S, Yang L-P, Schreckenbach G, Jiang W (2018) Molecular recognition of hydrophilic molecules in water by combining the hydrophobic effect with hydrogen bonding. J Am Chem Soc 140:13466–13477 You L, Zha D, Anslyn EV (2015) Recent advances in supramolecular analytical chemistry using optical sensing. Chem Rev 115:7840–7892 Yu S, Wang Z, Gao L, Zhang B, Wang L, Kong J, Li L (2021) A highly selective and sensitive peptide-based fluorescent ratio sensor for Ag+. J Fluoresc 31:237–246

Chapter 2

Basic Theoretical Description of Sensor-Target Binding

Quantitative measures are highly needed in fluorescence sensing. In this Chapter the reader finds discussion on parameters that have to be optimized in every sensor, such as selectivity, sensitivity and limit of detection. Concentrating on reversible binding, in which the mass action law is observed, the methods of determining the binding constants and modeling the ligand binding isotherms are presented. Kinetics of target binding and its influence on the results of analyte determination are discussed. Focusing on fluorescence sensing, linear and intensity-weighted formats are distinguished and analyzed. The sensing always involves an interaction between the target and the system that is able to detect it (the sensor). The essence of sensing methodologies is the ability to tell about the presence and quantity of target specie in a tested sample from a signal produced by its fraction bound to the sensor. The amount of target binding sites (receptors) composing the sensors is commonly smaller by many orders of magnitude than the number of targets (analytes) in the tested system. For correct detection of this number, different strategies can be applied. One of these strategies is to take out from a tested system a small aliquot of sample, expose it for strong or even irreversible binding by the sensor receptor and then, after calculating the number of bound targets, extrapolate this result to a whole tested system. The reporter signal of the sensor may appear as a result of irreversible change in its chemical structure on interaction with the target. Fluorescence probes operating in this way are called the reactivity-based probes or chemodosimeters (Wu et al. 2017). The other is to design the sensor in such a way that the target-receptor binding is reversible. Upon exposure to tested sample, only a part (often, a small number) of the target species binds to a sensor, and this binding can be sufficient to tell us about the true target concentration. In this case, the testing is not limited to size of the sample. It can be even the whole sea, to which the sensor can be immersed for continuous monitoring its pollution. In this case, we must describe and use the regularities existing between the bound and unbound species for determining their

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whole number, keeping in mind that only the bound species produce the sensor response signal. The interactions on every structural level involve the dimension of time. The target and receptor have to approach and ‘recognize’ each other by many translational and rotational diffusion motions. A ‘conformational adaptation’ can be involved in these steps with time-dependent strengthening of these interactions with the true target and discrimination of target analogs if they are present in the system. Thus, the target-receptor binding involves two important aspects. (a) Thermodynamic, describing the equilibrium between bound and unbound target. (b) Kinetic. The target-receptor binding needs time for their mutual diffusion and optimizing their interactions. The true equilibrium may not be reached in the course of testing, which means that the kinetic variables may influence significantly the readout signal. We observe that there are basic limitations to creation of a sensor that will be useful for target determination in all possible ranges of its concentrations and in all possible ranges of concentrations of low-specific target analogues that may compete with the assay. The regularities describing these systems and that are required for optimization the sensing will be analyzed below. Since these regularities are valid for any intermolecular interactions not always related to sensing, in our discussion below we will use rather general terms—the receptor that stands for sensor element responsible for target binding function and the analyte that can be the target analyte. Not only molecules can be the receptors or analytes but also the particles of different size and even the whole living cells. In this Chapter, after introducing the general parameters that characterize the sensor, we concentrate on these issues.

2.1 Parameters that Need to Be Optimized in Every Sensor In this and following sections we will see that in order to design optimal sensor we need to observe several important parameters that include not only the absolute sensitivity of responding unit but also selectivity against non-specific target binding and reversibility of the binding. These parameters depend strongly on whether the sensor and target form monovalent or multivalent contacts and whether this interaction occurs in solution or in the conditions of immobilized sensor. Kinetic and equilibrium constants characterize these properties. Fluorescence response can proceed in linear and non-linear manner (Fig. 2.1). The concentration ranges that are needed to be analyzed in solutions are tremendous—starting from molar ones down to single molecules (Table 2.1). This puts stringent demands on many sensor parameters. Primarily it is the absolute sensitivity, which is the ability to detect the smallest amounts of target in the tested system. Next is the dynamic range of target concentrations that can be detected. The sensor developed for detecting the picomolar target concentrations may not allow to detect variations of these concentrations on millimolar levels. Specificity can be defined as the ability to discriminate between molecular recognition from other low-affinity binding. It is achieved by optimal spatial location of atoms

2.1 Parameters that Need to Be Optimized in Every Sensor

39

Fig. 2.1 The parameters on which the operation of optimal sensor depends. See text for details Table 2.1 The scale of concentrations used in molecular sensing Notation

Concentration, mol/l

Molecules per 1 μl of solution

Examples

Molar (M)

1

~6 × 1017

Saturated salt (NaCl, KCl) solutions

Millimolar (mM)

10–3

~6 × 1014

Normal concentration of glucose in blood (~3–6 mM)

Micromolar (μM)

10–6

~6 × 1011

Intracellular concentration of NADH

Nanomolar (nM)

10–9

~6 × 108

Intracellular concentration of cyclic AMP

Picomolar (pM)

10–12

~6 × 105

Single molecule in a volume ~1.7 × 10–12 l

Femtomolar (fM)

10–15

~6 × 102

Single molecule in a volume ~1.7 × 10–9 l

Attomolar (aM)

10–18

~0.6

Single molecule in a volume ~1.7 × 10–6 l (~1.7 μl)

Zeptomolar (zM)

10–21

~0.0006

Single molecule in a volume ~1.7 × 10–3 l (~1.7 ml)

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2 Basic Theoretical Description of Sensor-Target Binding

and their groups participating in these interactions, and this determines highly selective complexation of interacting partners. It is often substituted by more precise term selectivity, which is the ability to discriminate in detection the target from its close analogs that can be also present in the tested system (Morales and Halpern 2018). Deviations from ‘ideal’ selectivity can be both false-positive and false-negative. They are false-positive when, in addition to high-specific binding, there occurs the lowspecific binding of non-target species. False-negative result is also possible, when the target binding is less than optimal to show its true concentration.

2.1.1 The Limit of Detection and Sensitivity The sensor can be characterized by the limit of detection (LOD). It is the lowest concentration of an analyte that the analytical process can reliably detect (Kellner et al. 2004). This concentration, C LOD , has to produce a detectable signal, X LOD that must be statistically distinguishable from the noise or background signal. The sensitivity, S, can be defined as the slope of the calibration curve (the dependence of analytical signal, X, on analyte concentration, C): S = dX/dC ≈ ΔX/ΔC.

(2.1)

The sensitivity S and the limit of detection are connected. The higher is the sensitivity the lower is the limit of detection. It is usually accepted that C LOD can be expressed as a function of S and S B , as: CLOD = 3SB /S

(2.2)

Here S B is a standard deviation of a set of blank signals obtained in serial measurements without analyte. The limit of quantitation (LOC) determines the analytical significance of the apparent analyte concentration. It is definitely above the limit of detection, and it is recommended to estimate it on the level of 10S B /S (MacDougall and Crummett 1980). The limit of detection is determined by two very different factors, the response of detection system and the target-receptor affinity. Let us consider these factors in more detail. (a) The limit imposed by the mechanism of detection and the response of detection system. Each detection system records the meaningful signal on the background of some noise, which is the statistical fluctuation of the measured parameter. The noise cannot be eliminated totally, but it can be suppressed by increasing the intensity of useful signal. Thus, the limit appears as an inability to resolve the useful signal on the background of the noise level. The signal-to-noise ratio is an important characteristic of the sensor system. In addition, the interfering signal

2.1 Parameters that Need to Be Optimized in Every Sensor

41

can appear as the background. In fluorescence sensing, this can be the signal from different fluorescent or light-scattering species that cannot be eliminated by instrumental means. If this background is variable, this may be an additional source of error. (b) The limit imposed by target-receptor affinity. Imagine the case when the sensor is able to detect picomolar amounts of target, the target is present in picomplar concentrations but the sensor response is zero. This happens because the sensor responds to the amount of bound target, and the target-receptor affinity is so low that no target is bound. In these cases, for shifting the target-receptor equilibrium towards binding, the affinity should be dramatically increased. This cannot always be done; therefore this limit of detection commonly exists.

2.1.2 Dynamic Range of Detectable Target Concentrations No sensor based on simple reversible target binding can be operative throughout the whole concentration range from moles to single molecules. What happens when at fixed receptor concentration we start increasing gradually the concentration of analyte? When in the system that contains a fixed amount of receptors the analyte concentration is gradually increased, we obtain the dependence of concentration of bound analyte on total analyte concentration (the dose–response curve) as the curve of hyperbolic shape. At the beginning, when there are few analyte molecules, all of them become bound because of plenty of the binding sites. With the increase of analyte concentration and of the occupancy of the binding sites, the equilibrium becomes more and more shifted towards free ligand until all binding sites become occupied and all added analyte remains unbound. It can be seen that this curve tends to saturation. Graph of this function of saturation of binding sites as a function of analyte concentration is called the binding isotherm. This function becomes steeper and shifted to lower concentrations on the increase of analyte-receptor affinity (Fig. 2.2a). The dose–response curves can be transformed into semi-logarithmic plots that are known as the analyte-receptor binding isotherms (Fig. 2.2b). From this plot we derive a strong dependence of the useful for analysis concentration range on binding affinity. Also important is the fact that the range important for analysis, where the graphs demonstrate significant changes, is rather narrow. In line with the mass action law (see below), the dynamic range covers only two orders of magnitude of target concentrations. More specifically, the useful linear dynamic range of singlesite binding spans only approximately two orders of magnitude (specifically it is 81-fold change) between 10% occupancy and 90% occupancy. This means that the sensor affinity must be strongly adapted to the required concentration range. Thus, we obtain the operative range for analyte concentrations and two limits from low and high concentration sides. On a low concentration limit, when all the analytes are bound, we can estimate a number of analyte molecules in a closed volume if this volume is small (and this possibility is used in some sensing technologies), but we can say nothing about analyte concentration when the volume is large and when most of

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2 Basic Theoretical Description of Sensor-Target Binding

Fig. 2.2 Isotherms of analyte-receptor binding obtained by sequential addition of analyte concentrations (titration curves) in linear (a) and semi-logarithmic (b) coordinates. Plots of fractional saturation, f , as a function of total analyte concentration, l, were obtained with variation of l from 1.0 × 10–11 to 6.55 × 10–7 M for the receptor concentration 1 × 10–10 M and the following K b values: 2 × 107 M−1 (1); 2 × 108 M−1 (2); 2 × 109 M−1 (3)

these molecules are unbound. On the side of high analyte concentrations, the sensor will respond to its increasing concentrations up to the point, where all the sensor receptor sites become saturated. Since further increase will not cause additional binding, there will be no response of the sensor. This is the high concentration limit. A very important consequence follows from this simple analysis. There is a fundamental restriction on the concentration range of sensor response. Irrespective of sensor and sample geometry this range is limited roughly to two orders of magnitude, one below and one above K d (~0.1 K d –~10 K d ). Outside this range the sensor becomes insensitive, and determination of target concentration not possible. Extending these affinity-based limits to quantification of broader-ranging concentrations is possible but only based on special sensor design. If an extended range (covering several orders of magnitude) of target concentrations needs to be detected, a series of sensors with different affinities have to be applied for binding the same analyte (Andersson et al. 2009; Drabovich et al. 2007). In Sect. 9.1 we present examples of sensors with broadly extended range of pH detection by including into a single dye of titratable groups with different pK values, see also (Frankær et al. 2019). The extension of sensor response range was done with a series of mutants of maltose binding protein as saccharide receptors (Marvin et al. 1997). Target detection across an extraordinarily broad dynamic range also can be achieved by integrating two different readout mechanisms into a single-molecule construct (Kang et al. 2019). The suggested dual-mode readout DNA biosensor combines an aptamer and a DNAzyme to quantify adenosine triphosphate (ATP) at both low (micromolar) and high (millimolar) concentrations by generating distinct readouts based on changes in fluorescence and absorbance, respectively.

2.1 Parameters that Need to Be Optimized in Every Sensor

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Fig. 2.3 The dynamic range of affinity-based molecular sensor and possibilities for its modulation (Vallée-Bélisle et al. 2012). a The dynamic range of sensors with a single-site receptor spans an 81-fold range of target concentration over which the sensor response transits from 10 to 90% of its signal output. b This useful dynamic range can be extended by combining multiple receptors differing in their affinity for the same target. c The dynamic range can be narrowed, producing a very steep, “ultrasensitive” dose–response curve via a sequestration mechanism that employs a high-affinity inactive depletant receptor. The depletant (white) serves as a “sink” that sequesters free target molecules until it is saturated. With a concentration of the depletant above the dissociation constant of the receptor, KD , an increase in target concentration above this depletant concentration will generate a threshold response, leading to an ultrasensitive response

The result that has to be achieved is illustrated in Fig. 2.3b. It shows that application of two (or more) receptors with different dissociation constants (K d ) instead of single receptor (Fig. 2.3a) extends the concentration range for analysis. It was shown that broad-range editing of the useful dynamic range of an artificial biosensor can be done by engineering a structure-switching mechanism (Ricci et al. 2016; Vallée-Bélisle et al. 2012). Following that approach, a set of receptor variants displaying similar specificities but different target affinities were generated. It was shown that using

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2 Basic Theoretical Description of Sensor-Target Binding

combinations of these receptor variants, the dynamic range was rationally extended (to 900,000-fold). Narrowing the affinity-based limit (Fig. 2.3c) is also possible. It is desirable when a great sensitivity (a steeper relationship between target concentration and output signal) has to be achieved. In this case, a high-affinity conformational switching nonsignaling receptor was suggested. It prevents the accumulation of free target until the total target concentration surpasses the concentration of this “depletant” (the sink is saturated). This allows the fivefold narrowing the dynamic range of synthetic biosensors.

2.1.3 The Sensor Selectivity Selectivity is the basic characteristics of any analytical method that determines the accuracy of results. It is defined as the ability of the method to produce signals that are exclusively dependent on the analyte in the sample independently on the presence of other related species (Kellner et al. 2004). Selectivity is essential for the development of robust point-of-care biosensors especially because biological samples are complex and comprised of numerous competing analytes. In some rare cases, the target is so different from non-target species present in analyzed system that there is no competition from these species to the target binding to the sensor. In a more general case, such interference can exist, especially if the interfering species are present at much higher concentrations than the analyte. The situation with the presence of specific and nonspecific binding is illustrated in Fig. 2.4. When their dissociation constants are separated by two orders of magnitude and more and the response output are similar, the non-specific interference is negligible. It may happen that they are closer or, occasionally, the output from nonspecific binding is much higher. Then the perturbation of analytical signal must be considered. Thus, the sensors operating in the conditions of equilibrium binding can be very selective if they display higher affinity towards specific target. And accordingly, for sensing specific targets in broad range of concentrations, one has to use a series of sensors (receptors) with different affinities. The selectivity can be viewed as interplay of affinities weighted by the concentrations of the target and interfering species. In many cases, the ability of achieving the highest selectivity is vital for sensor applications. For instance, inside the living cells, the calcium ions have to be determined in the presence of much higher concentrations of magnesium ions, and the concentrations of sodium ions are much lower from that of potassium. These ion pairs have the same charge, and the difference in size determine their selectivity. Therefore the usefulness of the sensors that have to be applied depends strongly on the selectivity factor. The term specificity is frequently used in biochemical literature. The binding is often called nonspecific, when the analyte-receptor binding isotherm does not show the effect of saturation in the studied concentration range. Mechanistically, this means that the interaction is of low affinity, so that such saturation must exist but is shifted to

2.1 Parameters that Need to Be Optimized in Every Sensor

45

Fig. 2.4 Explanation of selectivity issue based on two isotherms, for analyte-receptor binding with dissociation constant K D 1 and for nonspecific binding characterized by K D2

much higher concentrations. For describing the ability of the sensor to discriminate between true target analyte and other compounds close in properties, some authors use the term ‘cross-reactivity’. Estimation of cross-reactivity is commonly reduced to a comparison of dissociation constants between two or more species. Specificity usually strongly correlates with the affinity. The stronger is the analytereceptor binding, the more significant can be discrimination between the binding of specific and non-specific analytes (Eaton et al. 1995). This correlation is easy to understand. Specific binding involves multi-point contacts between analyte and receptor. The noncovalent interactions providing these contacts being individually weak produce collective effect increasing both affinity and specificity. The reader must not to be confused by the fact that sometimes the weaker interaction allows revealing more specific changes in target structure. Thus, the application of highaffinity probes for the detection of mismatched DNA and RNA sites shows that the increase of affinity in hybridization of complementary chains may lead to decrease in the ability of detecting single mismatches in the sequence (Demidov and FrankKamenetskii 2004). This is because on the background of lower affinity it is easier to detect its variations associated with the changes of a target structure. Also interesting is the situation with polyreactive antibodies that despite their low specificity may demonstrate high affinity in analyte binding (Bobrovnik 2014). Affinity is directly related to thermodynamic parameters characterizing the binding at equilibrium: the binding free energy ΔG and the changes of enthalpy ΔH and entropy ΔS on binding: ΔG = −RT · ln(K b ) = ΔH − T ΔS

(2.3)

Here R is the gas constant and T is the temperature. K b is the binding constant, which is the quantitative measure of affinity (that will be defined below). Both enthalpy and entropy contributions are important for target binding. ΔH reflects the bond formation energy and is responsible for heat effects. The entropy term determines the change of molecular order and therefore it depends strongly on the temperature.

46

2 Basic Theoretical Description of Sensor-Target Binding

The thermodynamic approach can allow providing some estimates of affinities. For instance, in the case of involvement of electrostatic interactions the affinity will be decreased at higher electrolyte concentrations (ionic screening), and introduction into the sensor of mobile groups that lose their mobility on binding decrease the affinity by influencing the entropy term.

2.1.4 Multivalent Binding and Cooperativity In multivalent binding, there is a possibility for cooperativity of binding among the multiple complexes, as binding of one analyte on the nanoparticle will localize neighboring analytes closer to other receptors, facilitating or hampering further binding events (Mahadevi and Sastry 2016). Binding can be considered cooperative if the binding constant is not a constant but a parameter changing with the extent of binding. In other words, the binding to multivalent sites do not constitute mutually independent events. Cooperativity can be positive or negative. A classic example of positive cooperativity is hemoglobin. Its binding oxygen deviates from typical isotherm in linear coordinates (Fig. 2.2a): instead of steep initial rise with further decline to saturation, there is a very small initial rise that becomes very steep at increasing ligand concentration. Cooperativity is observed for many molecular interactions in biology (Whitty 2008), for instance in binding of Ca2+ ions to calmodulin. Multivalent binding has a number of characteristics that monovalent interactions do not (Mammen et al. 1998; Varner et al. 2015). In particular, multivalent interactions can be collectively much stronger than corresponding monovalent interactions, and this enhancement in strength of binding, called superselectivity (MartinezVeracoechea and Frenkel 2011), can be of several orders of magnitude (see also Sect. 1.1). Bivalent antibodies are the important illustrations for that. There are many examples of multiple interactions determining molecular recognition in biological systems, such as cell–ligand and cell–cell interactions. Avidity is the other name for multivalent binding affinity. It is dependent on both the monovalent binding affinity and the number of analyte-receptor binding pairs (valency) (Mammen et al. 1998): β N  K dmulti = K dmono

(2.4)

Here K d multi is the observed dissociation constant including multivalent effects, K d mono is the dissociation constant of a single analyte-receptor pair, β is the degree of cooperativity (β < 1, negative cooperativity; β = 1, no cooperativity; β > 1, positive cooperativity), and N is the number of affinity analytes bound to receptors. The positive cooperativity of the binding of multiple analytes can dramatically enhance the observed dissociation constant from that of a single analyte-receptor complex. For instance, if the folate receptor ligands are assembled in nanoparticles, their affinity to correspondent receptors (folate binding proteins) in cells is increased

2.2 Determination of Binding Constants

47

2,500–170,000-fold (Hong et al. 2007a). Cooperativity increases targeting efficiency of drugs (Dubacheva et al. 2014, 2015) and also the sensing (Choi and Jung 2018) and imaging ability in vivo (Smith and Gambhir 2017). However, there is a limit, exceeding which a negative cooperativity may appear with an increasing number of analytes per particle. It can be observed on interaction of nanoparticles with cells if these particles are overloaded with additional analytes. This is a result of steric crowding, for which each additional surface analyte will lower the particle affinity by limiting the efficiency of binding (Christy et al. 2021). The same problem may appear with microarrays (Dudley et al. 2002).

2.2 Determination of Binding Constants The solution of many problems in sensor technologies needs correct determination of analyte-receptor affinities (Dsouza et al. 2011; Houk et al. 2003; Whitesides and Krishnamurthy 2005). This can be done on the conditions of application of mass action law. In this case, a titration experiment allows changing gradually the concentration ratio between analyte and receptor, usually by variation of analyte concentration. In the case of reversible binding, the dissociation constant K d value determines the range of target concentrations that can be detected by the sensor. There is always a limit on the lowest and highest of these concentrations. As we observed above, for a monovalent reversible analyte binding the analytically useful concentration range is typically restricted to one order of magnitude below and one order above the K d value, which is approximately the range between 10 and 90% of a sensor fractional saturation. As it will be explained below, this statement follows directly from the mass action law when it is applied to interaction between two species at equilibrium. Heterogeneity of binding usually extends this range, but this may happen only to a small extent. If the dynamic range of measured target concentrations is needed to be broader, one has to use a combination of sensors with similar specificity but of different affinities towards the target (Vallée-Bélisle et al. 2012).

2.2.1 Dynamic Association-Dissociation Equilibrium The simplest example of receptor-analyte interactions is when a single analyte (L) interacts with single receptor (R) to form a single complex (LR) in the conditions of dynamic equilibrium: L + R ⇔ LR

(2.5)

48

2 Basic Theoretical Description of Sensor-Target Binding

In the conditions of equilibrium, a rapid binding-dissociation occurs all the time but the concentrations [R], [L] and [LR] do not change over time. In this state the forward reaction of analyte binding proceeds with the same rate as the reverse reaction of its dissociation. That is why such equilibrium is called dynamic. It allows simple description based on mass action law. Let the kinetics of analyte binding be expressed by a rate constant k 1 and that of dissociation of the complex by rate constant k 2 . Then the equilibrium can be characterized by a ratio of these constants. This can be either the binding constant K b = k 1 /k 2 or its reverse function, the dissociation constant K d = k 2 / k 1 . K b is often called also the stability or affinity constant, it is expressed in reverse molarity units (M−1 ), whereas K d = 1/K b is expressed in molar (M) units. Table 2.1 presents the whole scale of concentrations used in molecular sensing. It has to be recollected that molarity (M) denotes the number of moles of a given substance per liter volume. The mole (mol), in contrast, is the unit that measures an amount of substance. One mole contains the Avogadro’s number (approximately 6.022 × 1023 ) of entities. The molarity dimension of K d corresponds to the analyte molar concentration [L], at which one half of receptor binding sites are occupied. Half of the analytes in this case exist in the bound and another half in the free form. Table 2.2 provides an estimate of the typical ranges of the binding constants that can be useful for sensing. According to mass action law, the equilibrium concentrations of the analyte, [L], of receptor, [R] and of their complex [LR] are related by the binding constant K b : Kb =

[L R] k1 = k2 [L] × [R]

(2.6)

If the total concentrations of receptor and analyte in the system are constant, then only one concentration of the three species ([L], [R] and [LR]) is independent. Therefore if [R] is well determined (as it usually happens in sensing), Eq. (2.6) connects [L] and [LR]. Table 2.2 Examples of intermolecular interactions with different binding constants K b Type of interaction K b (M−1 )

Examples 105

Weak

Less than

Medium

105 –107

Common enzyme–substrate and enzyme-inhibitor interactions

Strong

107 –1010

Interactions of phospholipids in biomembranes; of steroids with their cell receptors; of pharmaceutically useful drugs with their targets; of ligands binding to DNA

Very strong

More than 1010 Interactions of antibodies with highly specific antigens; of some ‘suicide’ enzyme inhibitors with their targets; of avidin with biotin (1015 M−1 )

Interactions of detergents in aqueous micelles; monovalent ions binding to proteins or DNA

2.2 Determination of Binding Constants

49

2.2.2 Determination of Kb by Titration The half-saturation point on this curve corresponds to dissociation constant K d . Thus, K d expressed in concentration units is a convenient characteristic of a sensor, characterizing the dynamic range of target concentrations available for sensing. Meantime, since complete saturation is commonly not reached, such determination of K d is not precise. The precision can be increased by taking into account all the points in titration experiment. This can be done by using nonlinear regression analysis or, alternatively, by transformation of the curve to a linear form, in which all the data points could be efficiently used for building a straight line. The least square method allows fitting the experimental data to a straight line in an optimal way, which allows calculation of precise values of the slope and axis intercepts that are connected with the values of K b or K d . Two types of linearized binding curves, associated with the names of Klotz and Scatchard are presently popular, and they will be considered below. For convenience, Eq. (2.6) can be re-written using the following notations. Let l be the total concentration of analyte, and r 0 —the total concentration of receptors. c = [LR] is the concentration of the analyte-receptor complex. If every acceptor is of valence m (has m binding sites for the analyte), then mr 0 is the total concentration of receptor binding sites. Then the concentration of unbound analyte is l-c, and concentration of free binding sites is mr 0 − c. Thus we have: Kb =

c (l − c) × (mr0 − c)

(2.7)

It is convenient to introduce the term fractional saturation, f , which is the fraction of bound analyte (or of sensor binding sites that are occupied by the analyte). If l − c = f , then Kb =

c f (m × r0 − c)

(2.8)

The Klotz approaches are based on simple algebraic transformation of Eq. (2.7) into linear relationship either between the reverse concentration of bound analyte and reverse concentration of unbound analyte, or between the ratio of unbound analyte to bound, and the concentration of unbound analyte, respectively. Linear dependence of 1/c on 1/f is presented in Eq. (2.9) 1 1 1 1 = × + c mr0 K b f mr0

(2.9)

The graph of this dependence (the Klotz graph) is the straight line with the slope 1/K b that crosses the ordinate axis at the point 1/mr 0 . Thus, with the knowledge of r 0 and using the values of 1/c and 1/f obtained in experiment one can construct the linear dependence similar to that in Fig. 2.5a and determine from its slope and

50

2 Basic Theoretical Description of Sensor-Target Binding

Fig. 2.5 The linearized transformations of the analyte binding isotherms. a The graph of Klotz, the dependence of 1/c on 1/f . The tangent of the slope of straight line equals to 1/mr 0 K b . b The Scatchard graph, the dependence of c/f on c. The tangent of the slope of straight line equals to K b . The graphs are calculated for: m = 4, r 0 = 1.0 × 10–8 M, K b = 1.0 × 108 M−1 . Analyte concentration changes from l 1 = 2.5 × 10–9 to l i = 2.05 × 10–5 M

from its intercept with ordinate the affinity of interaction (expressed by K b ) and the valence of receptor, m. According to Scatchard, the linear relationship is observed between the ratio of bound to unbound analyte, c/f , and the concentration of bound analyte, c: c = K b (mr0 − c) f

(2.10)

Its graph is a declining linear function (Fig. 2.5b) with the slope equal to K b . It crosses the ordinate axis at the point K b mr 0 and abscissa axis at the point mr 0 . It may be noted that in many cases the experiments on determination of K b are made with the excess of analyte (or receptor). In this case, the total concentration of the component that is in excess exceeds substantially the concentration of the formed complex, l ≫ c. This allows approximating the free concentration of this component equal to its total concentration (f ≈ l), which avoids the necessity of its measurement. In the case of two types of binding sites with different affinities the Klotz and Scatchard graph may be represented by two resolvable segments. The Scatchard graph is more suitable for obtaining K b data in the case of two types of analyte binding sites of different affinities. In this case the system of four equations should be solved with four unknowns, K b1 , K b2 , m1 and m2 . Their solution is described in the literature (Klotz and Hunston 1971). When two linear segments are resolved, an approximate graphical determination of these parameters using Scatchard graph is possible.

2.2 Determination of Binding Constants

51

It should be stressed that linear graphs in Klotz or Scatchard coordinates are observed only if the following conditions are satisfied: (a) The receptor has identical binding sites for the analyte; (b) The binding of one (or several) analyte(s) by one (or several) site(s) of the receptor does not change the affinity of its other binding sites, i.e., binding occur without cooperative (positive or negative) effect; (c) The analyte-receptor interaction obeys the law of mass action, which implies that all measurements have been done at the state of equilibrium. Several other methods of linearization of the same binding curves can be found in the literature (e.g. Benesi-Hildebrand plots). They are based on the same Eq. (2.7) and are of similar value (Klotz 1983). These linear regression methods are still in use (Hutterer 2017). They were frequently criticized in literature with indication that as in any linear transformations, such operations provide different statistical values to different data points along the binding curve, and this may cause significant errors in extrapolated values. Therefore it was strongly recommended to use modern computer-based non-linear regression methods for direct fitting of primary data into Eq. (2.7) that may lead to more precise values (Stootman et al. 2006; Tetin and Hazlett 2000; Thordarson 2011).

2.2.3 Determination of Kb by Serial Dilutions In some practical cases, the methodology based on analyte titration is inconvenient or even inapplicable, since the analyte and the receptor cannot always be obtained in purified form but their interaction can be studied in homogeneous solutions. In these cases, an approach based on serial dilutions with the solvent of the sample that contains both receptor and analyte can be applied (Bobrovnik 2003, 2005). With the dilution, the dynamic equilibrium will be shifted to dissociated forms. This shift depends on K b , and the determination of concentrations of bound analyte in the course of serial dilutions allows obtaining this value. After dilution of the system by d i times, a new equilibrium is established and we obtain: Kb = 

l di

− ci

ci 

mr0 di

− ci



(2.11)

Thus, in principle, equilibrium constant can be obtained from re-distribution of analyte between free and bound states as a function of dilution factor d i . In a practically useful case when l/d i ≫ ci , Eq. (2.11) can be reduced to the form: Kb =

l di



ci mr0 di

− ci



(2.12)

52

2 Basic Theoretical Description of Sensor-Target Binding

Transforming Eq. (2.12), one can obtain the dependence of ci on d i . ci =

mr0 l K b di (di + l K b )

(2.13)

Several possibilities exist for linearization of 1/ci d i and l/ci d i functions of dilution factor d i (Bobrovnik 2005). This method has definite advantages over the titration methods discussed above. It can be successfully used when an analyte-receptor mixture already exists and obtaining a series of samples with a constant concentration of receptors and various concentrations of analyte is technically difficult or even impossible. This approach can be especially useful for studying interactions between highly-labile biological receptors and corresponding analytes as found in vivo (Bobrovnik 2008). The methods discussed above are applicable with the use of common techniques measuring the changes of fluorescence intensities (see below). Meantime, in the case of very strong binding the very high dilutions are needed to achieve the presence of analyte-dissociated form together with the bound form. With analytes immobilized on the surface and bivalent or multivalent targets (such as antibodies) in common conditions, the equilibrium can be so strongly shifted that the binding becomes irreversible (Winzor 2011). Then some information can be derived from kinetics of binding/dissociation. More precise information can be obtained by applying fluorescence correlation spectroscopy (FCS) (see Sect. 16.5 of Volume 1) that allows operating with very high sample dilutions. By focusing the laser beam, the volume of several femtoliters (10–13 l) can be illuminated. Based on detection of single molecules entering and leaving the illuminated volume, this method allows providing studying the binding affinities, by switching to the range of very low target and sensor concentrations (Sanchez and Gratton 2005; Tetin and Hazlett 2000).

2.3 Modeling the Analyte Binding Isotherms The sensor response function (often called sensogram) is the function describing the correlation between the target analyte concentration and response of analytical system. Since different parameters that are used for fluorescence response (Chap. 3 of Volume 1) are commonly proportional (or proportional with weighting by light intensity) to the amount of bound analyte, the major task here is to establish correlation between the amount (concentration) of the analyte in the tested system l and its fraction bound by the sensor (fractional saturation) f . This function is called the analyte binding isotherm. It has to be determined for particular sensor operation conditions. The receptor functional elements of sensors can be composed of molecules or particles distributed in a defined volume, or they can be attached to a surface.

2.3 Modeling the Analyte Binding Isotherms

53

2.3.1 Receptors Free in Solution or Immobilized to a Surface Let us consider the case when the sensors are attached to a surface S that accommodates their receptor units with the density σ. Then the total amount of receptors on this sensor surface will be N = σ × S. The sensors interact with the analyte solution with concentration l in volume V and their affinity is characterized by binding constant K b . Let l and K b be known and our aim is to determine the part of receptors that forms the complexes with the analyte and the part that remains unoccupied. When the solution volume is large enough (l × V ≫ N), so that the analyte-receptor binding does not influence the analyte concentration in solution, the approximation l ≈ const is acceptable. Let n be the number of receptors forming a complex with analyte, then the number of free receptors is N–n. Then apparent concentration of the binding sites will be N/V. Let the equilibrium condition of binding be reached, and let n receptors (out of N) bind the analyte and N–n remain free. Then their apparent concentrations will be n / V and (N–n)/V. Applying the mass action law and providing simple transformations we get: f =

l Kb n = N 1 + l Kb

(2.14)

Equation (2.14) represents the analyte binding isotherm by the sensor, which with the account of response of the sensor system can be transformed into sensogram. The graphical forms of the function f (l) in linear and logarithmic coordinates are presented in Fig. 2.2. We observe that the shape of this function does not change by any form of immobilization of receptors. It is solely determined by K b value.

2.3.2 Bivalent and Polyvalent Reversible Target Binding A dramatically increased affinity of receptors present as dimers compared to monomers is explored in the design of fluorescent dyes. Thus, the best fluorescent nucleic acid binders are the dimers of intercalating dyes. The intrinsic DNA binding affinity constants of typical intercalator dye ethidium bromide and its homo-dimer are reported to be 1.5 × 105 and 2 × 108 M−1 correspondingly (Gaugain et al. 1978). Thus, dimerization increased the affinity by three orders of magnitude. The other example is the dramatic (2,500- to 170,000-fold) increase of binding constants between the nanodevices and folate binding protein through multivalency (Hong et al. 2007b). The affinity enhancement effect produced by multivalency can be explained and to some extent predicted on a thermodynamic basis (Kitov and Bundle 2003). The effect of bivalent binding can be analyzed based on formally applied concept that the local target (or receptor) concentration is increased on the formation of primary

54

2 Basic Theoretical Description of Sensor-Target Binding

complex. In this case there is a difference, if the linker between binding sites is rigid or flexible (Bobrovnik 2007). Polyvalent binding at equilibrium means establishing the dynamic equilibrium between unbound form of receptor and its bound forms with saturation of different valences m: L + Rm ⇔ L R1 + 2L R2 + . . . + m L Rm

(2.15)

Instead of a single K b value, this system will be characterized by a matrix of K ij values describing the equilibrium in interaction of each type of the complex with all the rest. In view of experimental data cited above, we can consider the strength of the complex expressed by K ij values to increase with the analyte saturation, reaching the maximum value for mLRm complex. This cannot be true in a general case, but we always can find the complex xLRx , for which affinity is the highest. In order to understand better the receptor response in such complex system, we can consider the limiting cases: (a) The sensor responds to binding of the first target with the formation of LR1 complex. Since this complex is commonly weak, it can be formed only at very high concentrations. But at these concentrations the complex xLRx should be formed already with all population of receptor molecules due to high affinity. Therefore, the sensor response will be seen around some efficient K b value describing equilibrium in interactions of xLRx complex with free analyte and all other complexes. (b) The sensor responds only to formation of complex mLRm , i.e. when all its valences are saturated by the analyte binding. When it is the case of a strongest complex, then we will have the situation similar to case (a) for xLRx with the exception that intermediate complexes are not observed in response. (c) The sensor responds in a well-resolvable manner to formation of each of these complexes. For instance, ketocyanine dye responds to formation of high-affinity hydrogen bond complexes with protic co-solvent molecules at their low concentrations and then a different change in spectra occurs on formation of such complexes of lower affinity observed at higher co-solvent concentrations (see Sect. 5.3 of Volume 1). In this case two K b values can be obtained in titration experiment (Pivovarenko et al. 2000). The range for determining the protic cosolvent concentrations is increased to two areas around these K b values. The other example is the design of a sensor for extended range of pH. In fact, it is the sensor for proton binding-release to the sensor titrating groups. In the case of distinguishable or additive response of sensor groups titrating at different pH we get the sensor for extended pH range (Li et al. 2006; Valuk et al. 2005). Consider now in more detail the simplest but practically important case when there are two analytes in the system that bind to the same receptors with different affinities. Let one (more specific) analyte being in concentration l1 binds receptor with the higher binding constant Kb1 and the other (less specific) in concentration l2 possesses the binding constant Kb2 . Then in the conditions of equilibrium the number

2.3 Modeling the Analyte Binding Isotherms

55

of sensor receptors with the bound first analyte will be n1 , and their number with the bound second analyte—n2 . Based on the mass action law we get two equations representing this case: n1

K b1 =  N −n 1 −n 2 V l1 − V

n1 V

=

n1 V − n 2 )(l1 V − n 1 ) − n (N 1

(2.16)

=

n2 V (N − n 1 − n 2 )(l2 V − n 2 )

(2.17)

n2

K b2 =  N −n 1 −n 2 V l2 − V

n2 V

Attempts to finding analytical solutions for two unknowns n1 and n2 lead to necessity of solving complex cubic equations. Meantime, numerical solution of Eqs. (2.16), (2.17) allows easy determination of n1 and n2 for known Kb1 , Kb2 , l 1 , and l2 . Alternatively, one may find l1 and l2 , by assigning the values of Kb1 , Kb2 , n1 and n2 . Consequently, one can calculate the analyte binding isotherm for this particular case.

2.3.3 Reversible Binding of Analyte and Competitor Now we consider the case of two different analytes, in which the binding of the testing analyte (e.g. fluorescent competitor) can provide informative signal for the binding of target analyte. For testing analyte, the concentration and affinity of binding are known; they can be measured in a preliminary test. This analyte can be a key player as a competitor in competition assays (Sect. 2.3). Is there any possibility of finding in such assay the target concentration in the case when its affinity is unknown? We will try to resolve this issue. Let the unknown concentration of the target analyte be lx , and its unknown binding constant K bx . The binding constant of the testing analyte is known, it is Kb1 . In our experiment we can add to the system any desired concentration of this analyte, l1 . These two analytes can bind with the sensor competing with each other so that the quantity of the bound testing analyte n1 can be measured (for instance, by the response of fluorescence reporter). So, all three of its important parameters, Kb1 , l 1 and n1 are known. In contrast, the amount of target analyte, nx1 , cannot be measured directly, since it does not provide any response. If we measure the bound testing analyte at a different concentration, l2 , then the sensor will bind a different amount of this analyte, n2 , which can also be measured. The amount of analyte analyte, nx2 , remains unknown. But now we are able to compose four linearly independent equations with four unknowns, lx , K bx , nx1 and nx2 . Their solutions will provide numerical values of these unknowns. Based on the mass action law we obtain the equations describing correlations between equilibrium constants K b1 , or K bx , and the concentrations of free analytes or of their complexes.

56

2 Basic Theoretical Description of Sensor-Target Binding

n1 V (N − n 1 − n x1 )(l1 V n2 V = (N − n 2 − n x2 )(l2 V n x1 V = (N − n 1 − n x1 )(l1 V n x2 V = (N − n 2 − n x2 )(l2 V

K b1 = K b1 K bx K bx

− n1) − n2)

(2.18)

− n x1 ) − n x2 )

Numerical solution of Eqs. (2.18) can yield l x , K bx , nx1 and nx2 . Practical examples of these calculations will be presented at the end of this Chapter. When the concentrations of both these analytes are relatively high and their binding constants low, the fractional concentrations of bound analytes will be small compared to their total concentrations. So the changes of free analyte concentrations on binding can be ignored, which simplifies the analysis. We can apply two linearly independent equations based on the mass action law: K b1 =

c1 (C − c1 − cx )l1

(2.19)

K bx =

cx (C − c1 − cx )l x

(2.20)

Here l 1 and l x are the concentrations of two analytes in solution. They bind to the sensor with affinity constants Kb1 and Kbx . C is the efficient concentration of receptors, which can be calculated knowing their amount, N, and solution volume V by formulae: C = N/V × N a . Here N a is the Avogadro’s number and c1 and cx are the efficient concentrations of the bound testing and target analytes correspondingly. After simple algebraic transformations, Eqs. (2.19) and (2.20) can be presented as: c1 =

l1 K b1 C 1 + l1 K b1 + l x K bx

(2.21)

cx =

l x K bx C 1 + l1 K b1 + l x K bx

(2.22)

c1 l1 K b1 = cx l x K bx

(2.23)

It follows that

The number of receptors that have bound first or second analyte will be: l1 K b1 c1 = C 1 + l1 K b1 + l x K bx

(2.24)

2.3 Modeling the Analyte Binding Isotherms

57

Fig. 2.6 Concentrations of the bound competitor and target analyte (c1 and cx ) as a function of total analyte concentration l x at constant competitor concentration l 1 in semi-logarithmic (a) and double-logarithmic (b) coordinates. The graphs were composed for the following parameters: N = 6.0 × 109 , K b1 = 1.0 × 108 M−1 , K bx = 1.0 × 107 M−1 , l 1 = const = 1.0 × 10–8 M, and l x changing from 1.0 × 10–10 M to 8.4 × 10–4 M

l x K bx cx = C 1 + l1 K b1 + l x K bx

(2.25)

If the concentration of one of the analytes is set constant and of the other varies, then we will get the following graph of the dependence of the concentration of either analyte that is bound by the receptor (c1 or cx .) on total analyte concentration lx at constant concentration of the testing analyte. This is the target analyte binding isotherm (Fig. 2.6). This graph shows that when the labeled competitor binds stronger than the target to the receptor, there is a range of sensor insensitivity at low target concentrations because the target cannot replace the competitor from the complex. The substitution occurs at higher concentrations, and if the competitor changes the parameters of its fluorescence in a binding-release process, this allows determining concentration of the target. When the target concentration becomes too high, all the receptor sites become occupied with the target and all the competitors released, so the sensor becomes insensitive again. Note that as in the case of sensors based on direct response to target binding, the range of target concentrations that can be detected is within the same two orders of magnitude.

2.3.4 Reversible Interactions in a Small Volume Now we consider the case when the volume V, in which the detection is made, is so small that on binding the analytes to receptors their concentration in this volume essentially decreases (S. A. Bobrovnik (unpublished)). As we observe, the present tendency in sensor design is the dramatic decrease of the testing volume, which

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was realized in numerous applications. Sensing inside the living cells and using the sensing devices reaching picoliter (10–12 l) detection volumes may present such cases. As in the case discussed above, we consider the case of N receptors immobilized on planar support, whereas the tested target remains in solution. In the absence of analyte, all receptors are unoccupied, and their efficient concentration is N/V, where V is the sample volume. Consider the case of equimolar (1:1) reversible binding, so that at equilibrium n receptors are occupied; they bind n analyte molecules. Then the number of free receptors will be N–n and their apparent concentration in solution will be (N–n)/V. An apparent concentration of analyte-receptor complex will be n/V. The free analyte concentration being initially l/V (l is the total amount of analyte molecules) changes on binding to receptor and becomes l − n/V. The mass action law can be applied for this case in the following form: n

K b =  N −n V l− V

n V

=

nV (N − n)(lV − n)

(2.26)

Equation (2.26) can be rewritten as an expression for f as a function of l (f = n/N)

f =

N K b + lV K b + V −

√

(N K b + lV K b + V )2 − 4NlV K b2 2K b N

(2.27)

Equation (2.27) represents the analyte binding isotherm. Its graph is a sigmoid dependence similar to that in Fig. 2.2. It may be interesting to analyze, how the isotherm of analyte binding will change with the decrease of detection volume. The results are presented in Fig. 2.7. We observe that with the decrease of solution volume the analyte binding isotherm transforms significantly. It becomes narrower and shifts to higher concentrations. The origin of this effect is in analyte redistribution between its free and bound forms. When the sample volume becomes smaller, a higher analyte concentration is needed Fig. 2.7 The influence of solution volume on analyte binding isotherm (the f (l) function). The graphs were composed for the following parameters: N = 1 × 10–10 M, K = 1 × 108 M−1 and the solution volumes V1 = 0.1 ml, V2 = 1 ml and V3 = 10 ml

2.4 Kinetics of Target Binding

59

to occupy the same amount of binding sites as in a large volume. Miniaturization of sensor technologies requires accounting these effects.

2.4 Kinetics of Target Binding The sensing commonly starts from the analyte and receptor physically separated in space, and in order to interact they first have to approach each other. It can be the motion of one partner, analyte, if the receptor is immobilized on the surface of spotted array or of both partners if the receptor molecules are free to diffuse in analyzed solution. In sensor technologies, the knowledge of analyte-receptor association-dissociation kinetics is important in several aspects. For correct applications of conditions of equilibrium binding we always need to know, when this equilibrium is established, and this determines the necessary incubation time. If the binding is irreversible we need to wait until all the analyte is bound. Some sensing technologies allow target determination in kinetic regime. In kinetic analysis of sensor performance, the problem is to find the amount of the analyte bound to the receptor as a function of time t after initial application of sample containing the analyte to the sensor system. The rates of analyte binding reactions are described by differential equations that in the simplest case (1:1 stoichiometry) can be written in the form: dB(t)/dt = k1 [N − B(t)]c L (t) − k2 B(t),

(2.28)

where B(t) is the number of bound analyte molecules and N is the total number of receptor molecules. Here cL (t) is the local concentration of the analyte solution at the reaction surface. It is the function of B(t), and this relation is the subject of modeling (Klenin et al. 2005). In general, the reaction rate as a function of time can be obtained numerically as a solution of a nonlinear integral equation, and its analytical solutions are available only for some special cases. Various sensor geometries of practical interest can be considered, but the practically important result is experimental. The output signal should reach a steady-state value as a function of time. Depending on the receptor-target pair and on immobilization of receptors (their presence in solutions, attachment to nanoparticles or immobilization on flat or porous surface) the rate-limiting step in formation of their complexes can be different. The target-receptor mutual diffusion imposes an upper limit to the rates of all these reactions. Meantime some intermolecular interactions can be coupled with conformational isomerizations in analyte or target molecules, and their rates can be slower than the diffusion. This can be a multistep process of search in a conformational space. In the case if one of interacting components (usually the receptor) is immobilized on the flat surface, the kinetics of analyte binding will become slower not only because only one component has the freedom to diffuse but also because the kinetics of analyte binding becomes time-dependent. This fact can be explained by the following. At

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2 Basic Theoretical Description of Sensor-Target Binding

time zero, t = 0, all receptor binding sites are empty and every interaction with them may lead to binding. However, at t > 0, some of the sites become occupied, and the search for smaller number of unoccupied sites slows down the process. The analyte migration along the receptor sites is usually much slower than free diffusion in solution because the desorption-sorption process at each step occurs with overcoming higher energy barriers (Sadana and Madugula 1993). The observed strong tendency towards miniaturization of sensor systems raises new challenges. Due to small binding area and high target concentration that is usually applied in a small volume, the kinetics of target binding depends on this concentration (Lynn et al. 2014). For micro-flow channel devices, the transport equations have to be solved and the diffusion processes simulated to guarantee perfect mixing (Ward and Fan 2015; Zizzari et al. 2020). The importance of reaching the conditions of thermodynamic equilibrium in target binding was discussed by many authors (Hooyberghs et al. 2010; Walter et al. 2011). They attributed the loss of reproducibility and many systematic errors in assays to the fact that these conditions were not satisfied. Both calculations and experiments show that for sensors immobilized on the surface of planar arrays the target binding occurs by two to three orders of magnitude slower than on binding in solutions. Meantime most of immunosensors and DNA hybridization arrays are made by immobilization on solid surfaces, which increases the time of reaching the equilibrium to many hours and even days (Carletti et al. 2006). Smaller decrease of the rate of binding can be achieved if the sensors are immobilized on microspheres (Sekar et al. 2005). Both array formats are in use and are discussed in this book. The characteristic rate of binding reaction depends on the rates of two processes: (a) the transport of analyte from the bulk compartment to the reaction area (reaction compartment) and (b) the subsequent binding process. In immunosensors the mass transport plays the major role. In contrast, in DNA arrays, the process of recognition of correct sequence, in which a huge amount of target-receptor sequences hybridize simultaneously, plays the major role. Actually, the sensor response time is in general not a fixed quantity but depends on the working condition (e.g., analyte flow and sensor cell volume) and also on the concentration of the analyte. Different strategies for decreasing the assay time can be applied knowing the origin of rate-limiting step: (a) decreasing the volume of tested system, (b) using inert polymers or nanoparticles to provide ‘molecular crowding’ effect, (c) the application of microwaves for local heating. Still the issue of binding kinetics remains very actual. With the knowledge of the origin of such retardation, the binding kinetics can be optimized with acceleration of target binding by several orders of magnitude (Kusnezow et al. 2003). This is especially important for microarray analysis of complex biological samples and achieving reversibility in pattern-generating devices, such as electronic noses (Pode et al. 2017; Röck et al. 2008).

2.5 Formats for Fluorescence Detection

61

2.5 Formats for Fluorescence Detection Working on design of fluorescence sensors for particular analytes and estimating their binding affinities, one should not forget the peculiarities of this method. Fluorescent sensing that relies on reversible target-receptor interactions is based on the measurement of the amount of bound target, and the required results on the amount of total target in the tested system should be derived. The method allows simultaneous detection of a number of targets in a single set (He et al. 2017). The very high absolute sensitivity is characteristic of fluorescence that in common detection schemes occupies the nanomolar and even the picomolar concentration range. This allows easy determination of K D values in 10–9 –10–8 M and above, which means the sensor use in the case of these very strong interactions, when K D is located within this range. This possibility is especially important in drug design, where specific binding affinity is the decisive factor (Smith et al. 2009). The problems in cuvette-type analysis may appear at high fluorophore concentrations, 10–5 –10–6 M and above due to the effect of increased absorbance of excitation light passing through the cuvette. This leads to inner filter effect causing the deviations from linearity (see Sect. 15.1.5 of Volume 1). In such cases, working at low concentrations is recommended, checking that the absorbance at the excitation wavelength used should be less than 0.05, which can also be achieved with special thin cuvettes. The detailed description of fluorescence parameters that are used in fluorescence detection techniques and the analysis of their various uses are presented in Chap. 3 of Volume 1. For the present discussion, we indicate that on target-receptor interaction they have to demonstrate detectable changes. They can be derived from only several types of measurements. Usually at fixed wavelengths, the intensity (in one response channel), intensity ratio (in two channels), anisotropy (or polarization) and the lifetime are recorded. The cases, in which a fluorescence parameter provides the full-scale response, changing from zero to a very high value, are rare. In all practically important cases, we deal with overlap of at least two signals generated by fluorescence reporters indicating the receptor sites with either free or bound analytes. The simplest intensity sensing provides a highly linear response. The applications of anisotropy and lifetime sensing are different; they result in interesting non-linear effects.

2.5.1 Linear Response Format In fluorescence sensing operating in reversible sensing mode, the dependence f (l), where l is the total concentration of analyte, has to be transformed into the dependence of fluorescence response function on analyte concentration that can be used as a calibration curve for a sensor device. The simplest way is to apply the easily recorded single parameter—the fluorescence intensity. Reversible (but

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preferably static) fluorescence quenching or enhancement should be recorded on receptor-analyte binding. Let the fluorescence signal F be proportional to the number of bound analyte molecules n (which is the same as the number of occupied receptors). Its value will be F = φ n, where φ—is the proportionality factor. At saturation, when N = n, we have F = F max . Since f = n/N = F/F max and the function f (l) can be determined by calibration, the values of ligand (analyte) concentration, l, in the tested system can be obtained from the linear fluorescence response by the sensor. In the case if the background fluorescence and/or the fluorescence of the analytefree sensor with the intensity F min contributes to fluorescence signal, this parameter has to be accounted in determination of the analyte concentration, l. It can be presented as:  l = Kd

F − Fmin Fmax − F

 (2.29)

In the design of fluorescence sensors, one has first to estimate the needed range of variation of target concentrations and then try to achieve the K d values that could allow covering this range by variation of sensor response below and above these K d values. The formulas presented above can be of use, so that the response of measured fluorescence parameter can vary between F min for free sensor and F max for this sensor saturated with the target (or vice versa). If the latter values can be reached in experiment, one can obtain K d by nonlinear fitting of the steady-state fluorescence intensity F recorded as a function of free target concentration (Boens et al. 2012). The plots of F as a function of l (direct plot) or F against -log l (semi-logarithmic plot) are quite complicated in the cases of multiple binding and the presence of excited-state reaction (Kowalczyk et al. 1994). Therefore, 1:1 complexation is usually considered and the measurements are provided at isoemissive point. The results on direct fluorometric titration as a function of l using the fluorescence intensities are obtained by nonlinear fitting to equation: F=

Fmin K d + Fmaxl Kd + l

(2.30)

The non-linear fitting is recommended (Thordarson 2011). Meantime, the outdated linearization approach using Scatchard plot or another linear transform, the modified Benesi–Hildebrand equation (Benesi and Hildebrand 1949) is still frequently used (Hutterer 2017): 1 1 1 1 = + ΔF ΔFmax K b ΔFmax l

(2.31)

where ΔF = F − F min and ΔF max = F max − F min . A double-reciprocal plot of 1/ΔF against 1/l gives a straight line, if there exists a one-to-one interaction between the target and receptor.

2.5 Formats for Fluorescence Detection

63

A popular method in intensity sensing that allows compensating for the variation of sensor concentration is the introduction of a reference channel with an analyteindependent intensity F ref (see Sect. 3.2 of Volume 1). Many fluorescent dyes and nanocomposites can provide such two-channel ratiometric response (Demchenko 2010, 2014; Park et al. 2020). Dividing the numerator and denominator of Eq. (2.29) by F ref we get:  l = Kd

R − Rmin Rmax − R

 (2.32)

In contrast to Eq. (2.29), Eq. (2.32) contains only the intensity ratios that are independent of sensor concentration. R = F/F ref , Rmin = F min /F ref and Rmax = F max /F ref . Because of recording the ratios of intensities at different wavelengths λ, such measurements are called λ-ratiometric. The most convenient in fluorescence detection is the application of the ratios of fluorescence intensities at two excitation or two emission wavelengths (Demchenko 2023a, b). In sensor technologies there are the cases when the two sensor forms, analyte-free and analyte-bound, are highly emissive and they differ in positions of excitation or emission spectra. Thus, the increase of intensity at one wavelength is coupled with its decrease at the other wavelength. The spectra of these forms are usually overlapped, and in these cases the account of such variation can be avoided if we select the wavelength for measuring the response to binding, F, at the wavelength of maximal variation of intensity and the reference intensity, F ref , at the crossing point of two spectra (isoemissive point) where this intensity does not change. Then we can easily use Eq. (2.32). It can be more practical to use a broader scale of intensity ratio variations by measuring the intensities at two points of its maximal change, usually at the band maxima corresponding to analyte-free and analyte-bound forms of the sensor. We can again consider the intensity at one wavelength as a ‘sensor signal’ and at the other wavelength as the ‘reference’ but we have to introduce the factor that accounts for intensity redistribution between the two bands. This is the ratio of intensities of free and bound forms, F F /F B , at a ‘reference’ wavelength λ2 :  l = Kd

R − Rmin Rmax − R



FF (λ2 ) FB (λ2 )

 (2.33)

In the case of multivalent receptors these equations become more complicated, and the reader may find them in the literature (Yang et al. 2003). If the sensing is based on the detection of changes in excitation spectra, then the F F /F B ratio has to be substituted by the ratio of ‘brightness values’ at the reference wavelength (εF ϕF /εB ϕB ), which is the ratio of molar absorbances, ε, multiplied by correspondent fluorescence quantum yields, ϕ (Lakowicz 2007). There are important advantages of wavelength-ratiometric detection (Demchenko 2023a, b), observed also on different examples in other chapters of this book. The λratiometric response is the property of fluorophore reporter in particular environment

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Fig. 2.8 The results of typical titration experiment with variation of bovine serum albumin (BSA) concentration at constant concentration of λ-ratiometric 3-hydroxyflavone FA dye 5·10–7 M. a Fluorescence intensity at N* (at 537 nm) and T* (at 617 nm) band maxima. The fits were obtained from the data based on the calculated binding constant and single binding site. b The correspondent Scatchard plot. This change could be detected as the variation of intensities of these bands, as illustrated in the inset above

and participating in particular interactions. It does not depend on its concentration and different instrumental factors, making for particular sensor and particular analyte Rmax and Rmin the well-established values. Moreover, in the case of fluorescence titration, one can follow if the change of molecular interactions occurs in the course of the receptor loading. An example presented in Fig. 2.8 demonstrates that the binding of 3-hydroxyflavone dye FA to serum albumin, that is known by the presence of several ligand binding sites, occurs at only one specific site (Ercelen et al. 2003).

2.5.2 Intensity-Weighted Format There are the methods for which the fluorescence response function in a general case is not linear. They are the methods of anisotropy and lifetime sensing. When measured in a homogeneous population of dyes, these parameters are concentrationindependent. The two states of the sensor, analyte-bound and analyte-free, may differ significantly in these parameters, and based on this difference the analyte concentration has to be determined. In the applications of these methods, an essential nonlinearity in response function appears. This is because each state (bound or unbound)

2.5 Formats for Fluorescence Detection

65

displays its own polarization-resolved emission intensity and intensity decay function. Their fractional contributions depend on the relative intensities of correspondent forms, whereas the additive law is valid only for the intensities. Therefore the parameters derived in anisotropy and lifetime sensing appear to be weighted by fractional intensities. Fluorescence anisotropy (Sect. 3.3 of Volume 1) is extremely sensitive to those intermolecular interactions, in which a small rotating unit becomes a rigidly coupled associate of a much larger size. The bound and free analytes are detected because they possess different values of anisotropy r, of free, r f , and of analyte-bound, r b , receptor. The measured anisotropy is obtained as being formed by contributions of these two forms weighted by fluorescence intensities of these forms: r = F f r f + Fb rb

(2.34)

This means that if the intensity of one of the forms is zero, such anisotropy sensor is useless since it will always show anisotropy of only one of the forms. Meantime, if the test conditions are selected so that fluorescence intensity is not changed on analyte binding, F f = F b , then the fraction of bound analyte can be estimated in a simple way: f =

r −rf rb − r f

(2.35)

The account of fractional intensity factor R = F b /F f (the ratio of intensities of bound and free forms) leads to a more complicated function: r −rf  f = r − r f + R(rb − r )

(2.36)

In different polarization assays the measured steady-state anisotropy is usually a weighted average of the low r values of free analyte and the large anisotropy of high molecular volume analyte–receptor complexes, and the fluorescence intensities as the weighting factors that can vary in both directions. The extent of nonlinearity that can appear can be illustrated in the work on fluorescein binding by antifluorescein antibody (Baker et al. 2000), in which the intensity changes on analyte binding are linear but a strong non-linearity is observed in anisotropy. Anisotropy titration analysis is usually used when the receptor is a small probe that demonstrates bright fluorescence in both free and bound forms but changes dramatically its polarization on binding (Mocz et al. 1998). The method is quite applicable to aptamers (Zhao et al. 2020). It can also be applied efficiently in competition assays (Banco et al. 2018; Samokhvalov et al. 2018). Similar situation is observed in lifetime sensing (that was discussed in Sect. 3.4 of Volume 1). Fluorescence decays as a function of time (in an ideal case, exponentially), and this decay can be described by initial amplitude α and lifetime τF for each of the two free (with index F) and bound (with index B) forms. If both of these forms are

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present in emission, we observe the result of additive contributions of two decays:     F(t) = αF exp −t/τFF + αB exp −t/τFB

(2.37)

The pre-exponential factors αF and αB are related to concentrations of these forms. If for one of the forms α = 0, no sensing will be possible, since irrespective of target concentration we will observe the response of only one form, which is emissive. Meantime, if αF = αB , the sensor response will be determined by the ratio of τF F and τF B values. In a general case, the ratio of concentrations of free and occupied receptors will be determined not only by αF and αB values and correspondent lifetimes τF F and τF F . They have to be weighted by correspondent brightnesses, which are the products of molar absorbances εF or εB and quantum yields ϕF or ϕB (Lakowicz 1999): αB ε B ϕ B τ FF [L R] = αF ε F ϕ F τ FB [L]

(2.38)

It follows from Eq. (2.35) that the ratio of pre-exponential factors (αB /αF ) represents the ratio of concentrations of analyte-bound and free forms ([LR]/[L]) only if there is no change in absorbance at excitation wavelength (εB = εF ) or of the product of quantum yield and lifetime. Fluorescence (luminescence) sensing and imaging (Szmacinski and Lakowicz 1995) is in the best way realized with long-lifetime phosphorescent molecules or lanthanide complexes, where a simple lifetime discrimination technique (Sect. 3.4 of Volume 1) can be applied. Thus, the anisotropy and fluorescence decay functions change in a complex way as a function of target concentration. Species that fluoresce more intensely contribute disproportionally stronger to the measured parameters. Simultaneous measurements of intensities allow accounting this effect. It is also essential to note that neither the choice of reference signal in intensity-ratiometric measurements nor the choice of reference levels in anisotropy or lifetime sensing could change the range of target concentrations to which the sensor can respond. In the state of equilibrium it is determined by the target-receptor affinity.

2.6 Sensing and Thinking. How to Provide the Optimal Quantitative Measure of Target Binding? The first lesson that should be obtained from the presented above considerations is that the range of potential target concentrations is extremely large and covers in molar units 19–20 orders of magnitude. The second lesson is that no single sensor can cover such a vast range in target detection, and even no single strategy can be used for that. There may be a possibility to fit the range of sensor sensitivity by the dilution or

2.6 Sensing and Thinking. How to Provide the Optimal Quantitative …

67

concentration of the sample, but this cannot be done in many practical cases. In all these cases the strict limitations exist. They are determined by the target-receptor affinity. Only in the case of very strong binding, the binding can be considered as irreversible. For determination of such targets, the concentration of receptors should be higher than the upper range of target concentrations in a usually extracted from the tested system small sample volume. Then, an irreversible binding will provide the measure of target concentration. Irreversibility means that the sensor is unable to measure the target continuously. It cannot be re-used or can be used only after regeneration. Reversible binding in some of these cases is also possible but at very high dilutions. If the affinity is lower, then the major operation principle becomes the reversible binding. Thermodynamic analysis and the application of mass action law allows obtaining the optimal sensor parameters, such as the affinity and concentration (or density) of receptors. The determination of target can be provided in the desired concentration range. This range is very narrow and covers only two orders of magnitude, roughly from 0.1 K d to 10 K d . It can be extended by application of an array of sensors with receptors possessing different K d values. The gradual K d increments should cover the whole range of potential target concentrations. In order to achieve the broadest dynamic range of measurement, the receptor concentration should be lower than the expected target concentrations. This is the first reason to use instead of a single type of receptor, an array of sensors with different receptors. The other is the cross-reactivity that is commonly present in real situations and that cannot be easily eliminated by better sensor design. This is the case, for instance, when the non-specific analyte when present in the concentrations much higher than the specific target, produces the same effect of binding. Designing a variety of receptors that bind to analytes with varying affinities allows providing the ‘pattern recognition’ (Geng et al. 2019; Pode et al. 2017; Sandanaraj et al. 2007), and a variety of electronic (photonic) noses and tongues were devised based on this principle (see Sect. 18.2). And the third reason for application the series of receptors derives from the fact that by detecting one compound one never is able to characterize a system of even of medium complexity. The challenges that are put forward are the whole genome and whole proteome requiring analysis of many thousands of different targets. The questions and problems addressed to the reader: 1.

2.

Respond quickly, how many zeptomoles are in 100 pmol? How many attomoles are in 10 nmol? How much should you dilute 50 μM concentration to obtain the concentration of 10 aM? 100 zM? What is the dimension of a cube filled with 1 fL of water? What volumes will you get by separating 5 pL into 100 equal portions? Exercise in transferring the number of molecules (denoted as N or n) into moles. These values should be divided by solution volume, V, and then divided by Avogadro’s number 6 × 1023 . For example, if the sensor is composed of

68

3.

4.

5.

6.

7.

8.

9.

2 Basic Theoretical Description of Sensor-Target Binding

6 × 1010 receptors and it is immersed into the volume of 1 ml, then the number of moles of receptors will be N = 6 × 1010 /10–3 × 6 × 1023 = 1 × 10–10 . Explain why the operational range of target determination and the sensor-target affinity must be strongly correlated. Let the sensor be based on receptors with K d = 100 μM. Can we measure at equilibrium the target concentrations of 1 μM? 100 μM? 100 nM? The system contains the analyte displaying specific and relatively strong (K d = 10 nM) binding. Its concentration varies in the range of 1–100 nM. A nonspecific analyte is also present in this system in unspecified concentrations ranging between 1 and 100 μM. Can the analyte concentration be accurately determined if for this analyte K d is 5 μM? 100 nM? What is the dynamic range of a pH sensor with a single titrating group? How to increase this range by incorporating into the sensor the groups with variable titration properties? For providing a smooth scale of pH sensitivity in the pH range 2–10, how many of these groups are needed? Why weak binding is commonly associated with low selectivity? Provide the explanation based on thermodynamic considerations. Can there be a strong but low-selective binding? Ca2+ ions are present in resting cells on the level of 100 nM, and Mg2+ ions on the level of 1 mM. Imagine that your sensor is based on a receptor that does not exhibit selectivity between these ions but you can vary K d in broad ranges. Can you measure Mg2+ in the presence of Ca2+ or Ca2+ in the presence of Mg2+ ? If yes, select the sensors with optimal K d and estimate the selectivity of such measurement. Analyze the practical example of calculating the occupancies of the sensor binding sites by two analytes in the case of their simultaneous presence in the tested system. Assume that for two analytes dissolved in a defined volume V we know the concentrations and the binding constants. Let l1 = 2 × 10–8 M, K b1 = 1 × 108 M−1 , l x = 5 × 10–8 M, K bx = 3 × 10–8 M−1 , the number of sensor binding sites 6 × 1012 , and the volume, V, is 1 × 10–3 l (i.e. 1 ml). Then the efficient concentration of the analyte binding sites is 6 × 1012 /1 × 10–3 × 6 × 1023 = 1 × 10–8 M. From Eqs. (2.15) and (2.16) we find the number of those sites that bind first analyte: n1 = 4 × 10–9 × 10–3 × 6 × 1023 = 24 × 1011 . The number of receptors that bind second analyte will be nx1 = 3.49 × 10–9 × 10–3 × 6 × 1023 = 20.94 × 1011 . Consider the previous case in which we change the concentration of first analyte (increase it to l 2 = 3 × 10–8 M), whereas the concentration of the second analyte is left unchanged. Then the number of receptors that have bound first analyte will be n2 = 5.08 × 10–9 × 10–3 × 6 × 1023 = 30.54 × 1011 , and the number of receptors that bind second analyte will be nx2 = 2.88 × 10–9 × 10–3 × 6 × 1023 = 17.28 × 1011 . The known values of nx1 and nx2 , can substituted together with the known values l1 = 2 × 10–8 M, Kb1 = 1 × 108 M−1 , l 2 = 3 × 10–8 M into the system of Eqs. (3.17). Their numerical solution yields: lx = 4.9997 × 10–8 M, Kbx = 3.00016 × 10–8 M−1 . Thus we found the values lx and Kbx , which differ

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10. 11. 12. 13. 14.

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very little from those values that were taken for the calculation of the number of analyte-receptor complexes, i.e. lx = 5 × 10–8 M, Kx = 3 × 10–8 M−1 . Compare the effects of K b in competitive and noncompetitive assays. What is the effect of the receptor total concentration on the response in competitive and noncompetitive assay? What is faster, the binding to receptor in solution or the binding to the same receptor immobilized on a surface? By how much? Why the kinetics of target binding to a receptor immobilized on solid surface becomes dependent on its concentration? In what sensor technologies and due to what reason the response is ‘weighted’? How there appear the weighting parameters.

References Andersson O, Nikkinen H, Kanmert D, Enander K (2009) A multiple-ligand approach to extending the dynamic range of analyte quantification in protein microarrays. Biosens Bioelectron 24:2458– 2464 Baker GA, Pandey S, Bright FV (2000) Extending the reach of immunoassays to optically dense specimens by using two-photon excited fluorescence polarization. Anal Chem 72:5748–5752 Banco MT, Mishra V, Greeley SC, Ronning DR (2018) Direct detection of products from Sadenosylmethionine-dependent enzymes using a competitive fluorescence polarization assay. Anal Chem 90:1740–1747 Benesi HA, Hildebrand J (1949) A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons. J Am Chem Soc 71:2703–2707 Bobrovnik S (2003) Ligand–receptor interactions: a new method for determining the binding parameters. J Biochem Biophys Methods 55:71–86 Bobrovnik S (2005) New capabilities in determining the binding parameters for ligand-receptor interaction. J Biochem Biophys Methods 65:30–44 Bobrovnik SA (2007) The influence of rigid or flexible linkage between two ligands on the effective affinity and avidity for reversible interactions with bivalent receptors. J Mol Recognit 20:253–262 Bobrovnik S (2008) A simple and convenient approach for evaluation of the parameters of ligand– receptor interaction. Receptor blocking index and its application. J Mol Recognit 21:96–102 Bobrovnik SA (2014) Avidity of polyreactive immunoglobulins. The Ukrainian Biochemical Journal 86:183–189 Boens N, Leen V, Dehaen W (2012) Fluorescent indicators based on BODIPY. Chem Soc Rev 41:1130–1172 Carletti E, Guerra E, Alberti S (2006) The forgotten variables of DNA array hybridization. Trends Biotechnol 24:443–448 Choi H, Jung Y (2018) Applying multivalent biomolecular interactions for biosensors. Chemistry–A Eur J 24:19103–19109 Christy AT, Kusumaatmaja H, Miller MA (2021) Control of superselectivity by crowding in threedimensional hosts. Phys Rev Lett 126:028002 Demchenko AP (2010) The concept of lambda-ratiometry in fluorescence sensing and imaging. J Fluoresc 20:1099–1128 Demchenko AP (2014) Practical aspects of wavelength ratiometry in the studies of intermolecular interactions. J Mol Struct 1077:51–67

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Demchenko AP (2023a) Tutorial Dual emission and its λ-ratiometric detection in analytical fluorimetry. Pt. I. Basic mechanisms of generating the reporter signal. Methods Appl Fluoresc 11:101095 Demchenko AP (2023b) Tutorial Dual emission and its λ-ratiometric detection in analytical fluorimetry. Pt. II. Exploration in sensing and imaging. Methods Appl Fluoresc 11:101096 Demidov VV, Frank- MD (2004) Two sides of the coin: affinity and specificity of nucleic acid interactions. Trends Biochem Sci 29:62–71 Drabovich AP, Okhonin V, Berezovski M, Krylov SN (2007) Smart aptamers facilitate multi-probe affinity analysis of proteins with ultra-wide dynamic range of measured concentrations. J Am Chem Soc 129:7260–7261 Dsouza RN, Pischel U, Nau WM (2011) Fluorescent dyes and their supramolecular host/guest complexes with macrocycles in aqueous solution. Chem Rev 111:7941–7980 Dubacheva GV, Curk T, Mognetti BM, Auzély-Velty R, Frenkel D, Richter RP (2014) Superselective targeting using multivalent polymers. J Am Chem Soc 136:1722–1725 Dubacheva GV, Curk T, Auzély-Velty R, Frenkel D, Richter RP (2015) Designing multivalent probes for tunable superselective targeting. Proc Natl Acad Sci 112:5579–5584 Dudley AM, Aach J, Steffen MA, Church GM (2002) Measuring absolute expression with microarrays with a calibrated reference sample and an extended signal intensity range. Proc Natl Acad Sci 99:7554–7559 Eaton BE, Gold L, Zichi DA (1995) Let’s get specific: the relationship between specificity and affinity. Chem Biol 2:633–638 Ercelen S, Klymchenko AS, Demchenko AP (2003) Novel two-color fluorescence probe with extreme specificity to bovine serum albumin. FEBS Lett 538:25–28 Frankær CG, Rosenberg M, Santella M, Hussain KJ, Laursen BW, Sørensen TJ (2019) Tuning the pKa of a pH responsive fluorophore and the consequences for calibration of optical sensors based on a single fluorophore but multiple receptors. ACS Sensors 4:764–773 Gaugain B, Barbet J, Capelle N, Roques BP, Le Pecq JB (1978) DNA bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer. Biochemistry 17:5078–5088 Geng Y, Peveler WJ, Rotello VM (2019) Array-based “Chemical Nose” sensing in diagnostics and drug discovery. Angew Chem Int Ed 58:5190–5200 He X-P, Hu X-L, James TD, Yoon J, Tian H (2017) Multiplexed photoluminescent sensors: towards improved disease diagnostics. Chem Soc Rev 46:6687–6696 Hong S, Leroueil PR, Majoros IJ, Orr BG, Baker JR Jr, Holl MMB (2007a) The binding avidity of a nanoparticle-based multivalent targeted drug delivery platform. Chem Biol 14:107–115 Hong S, Leroueil PR, Majoros IJ, Orr BG, Baker JR Jr, Banaszak MM (2007b) The binding avidity of a nanoparticle-based multivalent targeted drug delivery platform. Chem Biol 14:107–115 Hooyberghs J, Baiesi M, Ferrantini A, Carlon E (2010) Breakdown of thermodynamic equilibrium for DNA hybridization in microarrays. Phys Rev E 81:012901 Houk K, Leach AG, Kim SP, Zhang X (2003) Binding affinities of host–guest, protein–ligand, and protein–transition-state complexes. Angew Chem Int Ed 42:4872–4897 Hutterer R (2017) Inclusion of fluorophores in cyclodextrins: a closer look at the fluorometric determination of association constants by linear and nonlinear fitting procedures. Methods Appl Fluoresc 6:013001 Kang B, Park SV, Soh HT, Oh SS (2019) A dual-sensing DNA nanostructure with an ultrabroad detection range. ACS SensS 4:2802–2808 Kellner R, Mermet J-M, Otto M, Valcarcei M, Widmer HM (2004) Analytical Chemistry. WileyVCH, New York Kitov PI, Bundle DR (2003) On the nature of the multivalency effect: a thermodynamic model. J Am Chem Soc 125:16271–16284 Klenin KV, Kusnezow W, Langowski J (2005) Kinetics of protein binding in solid-phase immunoassays: theory. J Chem Phys 122, 214715

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Sandanaraj BS, Demont R, Thayumanavan S (2007) Generating patterns for sensing using a single receptor scaffold. J Am Chem Soc 129:3506 Sekar MM, Bloch W, St John PM (2005) Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres. Nucleic Acids Res 33:366–375 Smith BR, Gambhir SS (2017) Nanomaterials for in vivo imaging. Chem Rev 117:901–986 Smith AJ, Zhang X, Leach AG, Houk K (2009) Beyond picomolar affinities: quantitative aspects of noncovalent and covalent binding of drugs to proteins. J Med Chem 52:225–233 Stootman FH, Fisher DM, Rodger A, Aldrich-Wright JR (2006) Improved curve fitting procedures to determine equilibrium binding constants. Analyst 131:1145–1151 Szmacinski H, Lakowicz JR (1995) Fluorescence lifetime-based sensing and imaging. Sens Actuators, B Chem 29:16–24 Tetin SY, Hazlett TL (2000) Optical spectroscopy in studies of antibody-hapten interactions. Methods 20:341–361 Thordarson P (2011) Determining association constants from titration experiments in supramolecular chemistry. Chem Soc Rev 40:1305–1323 Vallée-Bélisle A, Ricci F, Plaxco KW (2012) Engineering biosensors with extended, narrowed, or arbitrarily edited dynamic range. J Am Chem Soc 134:2876–2879 Valuk VR, Duportail G, Pivovarenko VG (2005) A wide-range fluorescent pH-indicator based on 3-hydroxyflavone structure. J Photochem Photobiol A-Chem 175:226–231 Varner CT, Rosen T, Martin JT, Kane RS (2015) Recent advances in engineering polyvalent biological interactions. Biomacromol 16:43–55 Walter J-C, Kroll KM, Hooyberghs J, Carlon E (2011) Nonequilibrium effects in DNA microarrays: a multiplatform study. J Phys Chem B 115:6732–6739 Ward K, Fan ZH (2015) Mixing in microfluidic devices and enhancement methods. J Micromech Microeng 25:094001 Whitesides GM, Krishnamurthy VM (2005) Designing ligands to bind proteins. Q Rev Biophys 38:385–395 Whitty A (2008) Cooperativity and biological complexity. Nat Chem Biol 4:435–439 Winzor DJ (2011) Allowance for antibody bivalence in the characterization of interactions by ELISA. J Mol Recognit 24:139–148 Wu D, Sedgwick AC, Gunnlaugsson T, Akkaya EU, Yoon J, James TD (2017) Fluorescent chemosensors: the past, present and future. Chem Soc Rev 46:7105–7123 Yang RH, Li KA, Wang KM, Zhao FL, Li N, Liu F (2003) Porphyrin assembly on beta-cyclodextrin for selective sensing and detection of a zinc ion based on the dual emission fluorescence ratio. Anal Chem 75:612–621 Zhao Q, Tao J, Feng W, Uppal JS, Peng H, Le XC (2020) Aptamer binding assays and molecular interaction studies using fluorescence anisotropy-a review. Anal Chim Acta 1125:267–278 Zizzari A, Cesaria M, Bianco M, del Mercato LL, Carraro M, Bonchio M, Rella R, Arima V (2020) Mixing enhancement induced by viscoelastic micromotors in microfluidic platforms. Chem Eng J 391:123572

Chapter 3

Recognition Units Built of Small Macrocyclic Molecules

The possibilities of organic chemistry to synthesize new molecules with increasing complexity and with more and more specialized functional behavior are tremendous. Organic molecules can form cyclic structures with special design that are able to specifically bind small molecules such as ions, monosaccharides, amino acids and short peptides. Whereas small heterocycles (crown ethers, cryptands) are ideal for recognizing ions, the macrocyclic cavity formers, such as cyclodextrins, calix[n]arenes, cucurbit[n]urils and pillar[n]arenes, possess much broader range of possibilities, recognizing different low-polar compounds. Smart porphyrin derivatives recognize particular motives on the surfaces of protein molecules. Such host molecules serve as very efficient receptors when incorporated into supramolecular sensors. Cyclic molecular structures demonstrate many advantages as sensor recognition units (Liu et al. 2017; Mako et al. 2018). They may lead to the generation of architecturally simple but effective receptors that combine in their sensing ability the factor of steric correspondence to target with the most efficient formation of noncovalent bonds. These host molecules interact with diverse guests (e.g., cations, anions, and neutral molecules), and these host–guest interactions play significant roles in the design and construction of powerful receptors having exceptional applications in sensing. Due to their structural diversity and the ability to adopt unique conformations, their molecular recognition properties are really unique. Already with these small binders, we observe the operation of principle of multivalency: the space-filling binding with saturation of all possible weak intermolecular interactions. With cycled rigid structures, multivalency (Fasting et al. 2012) is the most efficiently realized (Baldini et al. 2007) and in this realization attains new features. One is the macrocyclic effect (Melson 2012). The stability in a complex of cyclic molecules with their guests is usually enhanced over a linear multivalent counterpart because the interaction sites are prefixed into a preferred conformation and there is less configurational entropy for macrocyclic receptors to lose on binding. The other is chelate effect (Breslow et al. 2000) that refers to the fact that the formation of first in many noncovalent bonds stabilizing the structure increases dramatically © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_3

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Fig. 3.1 Illustration of three principles in molecular recognition that can be realized with small macrocyclic molecules. a Cyclic structures represented by crown, azacrown and azathiocrown ethers and cryptands with different activities and selectivities towards ions. The fluorophore is appended to cyclic structure. b Cavity-forming molecular structures that can accommodate in their inner volume different types of target (T) molecules, mostly of those that are low soluble in water. The bound fluorophore reports on target binding. c Surface-recognizing molecules that can be functionally modified porphyrins and their analogs that can provide their own fluorescence response

the formation of other bonds just due to proximity that can be thought as an increase of local concentration. Thus, both reduction of flexibility and complementarity in dimension, topology and saturation with noncovalent bonds—all of that matters for the creation of highly stable and selective molecular recognition events. Meantime, fine tuning of these interactions is quite possible (Zhang et al. 2015a, b), which results in high efficiency in molecular recognition between structurally related compounds. Many interesting ideas were realized in very broad areas of applications of sensors with macrocyclic molecular receptors. In order to provide their systematic analysis, I suggest to distinguish their three categories (see Fig. 3.1). One is the focusing on coordination effect of ions with the most efficient application of crown ethers and cryptands and their numerous structural variations (Li et al. 2017). In this case, fluorescence reporter is integrated into cyclic structure or is perturbed by electrostatic effect of ion binding (Valeur and Leray 2000). The others explore the cavityforming structures of cyclodextrins, calix[n]arenes, cucurbit[n]urils, pillar[n]arenes and related molecules for hosting low-polar and amphiphilic compounds (Mako et al. 2018). In this case the fluorophore can be either covalently attached or noncovalently bound and respond to binding either in competition of direct assays. The third case is probing the artificial or natural (protein, biomembrane) surfaces. In this case the macrocycle may respond being fluorescent itself (as in porphyrins), or appended as in functional calixarenes.

3.1 Crown Ethers and Cryptands: Macrocyclic Hosts for Ions Crown ethers are cyclic chemical compounds that contain a ring composed of several ether groups (Fig. 3.2). They are defined as the macrocyclic oligomers of ethylene oxide, forming the cycle (–CH2 CH2 O–)n with n ≥ 4. The oxygen atoms in crown ethers are oriented inside the cycle that allows them to coordinate with cations located

3.1 Crown Ethers and Cryptands: Macrocyclic Hosts for Ions

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Fig. 3.2 Basic macrocycles that are used for recognition of ions. a Crown ether. b Azacrown. c Azathiocrown. d Cryptand

in the interior of the macrocyclic structure, whereas the exterior of the macrocycles are hydrophobic (Gokel et al. 2004; Liu et al. 2017). Oxygens in crown ethers can be replaced by nitrogen and sulfur. Together with nitrogen containing azacrown ethers and those incorporating sulfur (azathiocrown ethers) they can be strong binders of small molecules, particularly cations (Li et al. 2017). Due to the high affinity of sulfur for transition metals such as copper and mercury, sulfur-containing crowns are ideal detection platforms for such ions. Cryptands (Valeur and Leray 2000) are the advanced derivatives of crown ethers that possess three-dimensional spatial structures and higher association constants compared with crown ethers. The introduction of additional arms makes the cryptandbased host–guest systems responsive to more stimuli, which is crucial for the construction of adaptive or smart materials (Zhang et al. 2014). Both crown ethers and cryptands can be fused with organic dyes and in different ways (Li et al. 2017). In this case the dyes, incorporating the target-binding groups, attain new properties. The target binding provides a perturbation of the dye electronic structure, and such perturbation may change dramatically when the target binds, generating a strong spectroscopic response. The targets in these cases are of small size, usually the ions that produce the strongest perturbation. The coupling of these cyclic recognition units with fluorescence reporters can be versatile. The literature contains a great number of dyes and their modifications with incorporation of recognition units (de Silva et al. 1997; Lakowicz 2007; Valeur 2002), and many of them are discussed throughout this book. Organic dyes and conjugated polymers allow combining in one molecule the efficiently of recognition and reporting properties. Several randomly selected examples demonstrating such versatility are given below (Fig. 3.3). We can observe that aromatic group or segment of conjugated polymer can be incorporated directly into crown ether macrocycle. The other possibility to use incorporated nitrogen by modulating its electron-donating properties to appended aromatic fluorophore. Rigidization of structure due to target binding may lead to dramatic enhancement of fluorescence intensity. Thus, various mechanisms of signal transduction and response can be employed here. They include modulation of reactions, in which the electrons participate in the excited states—the electron, charge and energy transfers (PET, ICT and FRET reactions correspondingly). The first two of these reactions can exhibit influences by

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Fig. 3.3 Examples of fluorescent organic molecules that can bind and detect ions. a Conjugated polymer with incorporated crown ether groups. The ions when are bound to some of crown ether groups produce fluorescence quenching of the whole chain (McQuade et al. 2000). b The dye containing azacrown ether group and two carbonyls that provides selective binding of Pb2+ ions with fluorescence enhancement response (Chen and Huang 2002). c Azacrown ether derivative of 3-hydroxyflavone that has two centers of binding bivalent cations, Ma2+ and Ba2+ . The ions bound at azacrown site are ejected upon excitation due to interaction with positive charge that appears on amino group. This leads to different effects of binding in excitation and emission spectra (Roshal et al. 1999). d A laterally nonsymmetric azacryptand derivatized with one 7-nitrobenz-2oxa-1,3-diazole (1) and one/two anthracenes (2). These compounds give a large enhancement on binding of Cu2+ , Ag+ , and proton. The enhancement is observed in the diazole moiety even when the anthracene fluorophore is excited because of substantial fluorescence resonance energy transfer from anthracene to the diazole moiety (Sadhu et al. 2007)

the neighboring charges by electrostatic interactions and therefore these effects are the most applicable for detection of charged compounds and ions. Modulation of EET can be used due to the change of overlap integral on target binding and conformational change leading to the change in electronic conjugation between fragments of the dye molecule. The mechanisms of these reactions are discussed in Chap. 4 of Volume 1. The target selectivity is determined by the size of macrocycle and the composition of constituting atoms (Li et al. 2017). A good example is the sensor for Hg2+ ions that has a strong discriminative power over other cations, which allowed to use it for tracing these ions inside the living cells (Zhang et al. 2013). A BODIPY probe appended to azathiocrown ether recognition unit (see Fig. 3.4) allows achieving a

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Fig. 3.4 Recognition of Hg2+ ions by their selective binding to azathiocrown ether with strong fluorescence electronic charge transfer response of a BODIPY derivative

low nanomolar detection by enhancement of the emission intensity associated with a blue shift of the emission maximum from about 660 to 617 nm. Macrocyclic hosts can serve for specific recognition of different type of guest molecules, such as amines (Li et al. 2017). Moreover, with a special design their binding can be enantioselective, i.e. dependent on the chiral properties of guest molecules, such as amino acids (Móczár and Huszthy 2019). They can be incorporated into different polymeric structures and nanocomposites, for instance, into PAMAM dendrimers becoming very stable indicators of sodium ions in vivo (Lamy et al. 2012).

3.2 Cavity-Forming Compounds. Structures and Properties There are cyclic molecular cavity-forming compounds, such as α-γ-cyclodextrins, calixarenes. cucurbiturils, and pillararenes. They form their hydrophobic cavities for efficient guest complexation allowing their highly selective binding based on fitting to cavity size and the multitude of their noncovalent interactions. Whereas the hydrophobic force is important for their binding within the cavity, the polar and charged group of atoms at periphery of these structures can determine the selectivity and are responsible for solubility in water. The relative rigidity of these structures compared to acyclic molecules promotes highly selective guest binding, although their different classes have widely disparate rigidities, binding strengths, and selectivities (Mako et al. 2018). These molecules are typically basket- or container-shaped, either comprised of a wide upper rim and a narrower lower rim, like cyclodextrins and calixarenes, or with a symmetrical inner cavity, like cucurbiturils and pillararenes. These cyclic structures are extensively used in sensor applications.

3.2.1 Cyclodextrins Cyclodextrins make up a family of cyclic oligosaccharides containing six (α-cyclodextrin), seven (β-cyclodextrin) and eight (γ-cyclodextrin) α-D-glucose

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subunits in a ring, creating a conical shape. Their structures are well described in the literature (Wenz et al. 2006; Szejtli 1998). Different ring sizes and inner cavities are presented by α, β and γ cyclodextrins (Fig. 3.5). In these structures, the primary hydroxyl groups are directed to the narrow side, and the secondary ones are located on the wide side of the torus. With hydroxyl groups facing the outer space, these molecules are highly soluble in water and can serve as the sites for different substitutions, whereas the inner space can accommodate different low-polar molecules, such as cholesterol. Their binding affinity is determined by the cavity size. It increases from α-cyclodextrin (α-CD) to β-cyclodextrin (β-CD) and to γ-cyclodextrin (γ-CD), see Table 3.1. Inclusion complexes are known as the entities comprising two or more molecules, in which one of them, serving as the ‘host’ incorporates a ‘guest’ molecule only by physical forces, i.e. without covalent bonding. Cyclodextrins are typical ‘host’ molecules that can include a great variety of molecules that have the size

Fig. 3.5 Schematic drawing (above) and the chemical structures (below) of α, β and γ cyclodextrins

Table 3.1 Structural parameters of cyclodextrins (CDs). Minimum internal diameters d min , crosssectional areas Amin and inner volume V are presented Cyclodextrin

d min (Å)

Amin (Å2 )

V (Å3 )

α-CD β-CD γ-CD

4.4 5.8 7.4

15 26 43

174 262 427

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conforming to the size of cavity (Szejtli 1998). Thus, α-cyclodextrin can form inclusion complex with one, β-cyclodextrin—with two and γ-cyclodextrin—with three pyrene molecules. Cyclodextrins can be produced in large quantities from starch by simple and cheap enzymatic conversion, which adds its popularity as a carrier of drugs, in cosmetic industry and, of course, in sensing technologies (Szejtli 1998). The presence of a number of exposed hydroxyl groups (18 in α-cyclodextrin, 21 in β-cyclodextrin and 24 in γ-cyclodextrin) allows a practically unlimited number of chemical modifications, and many of them can modify the selectivity in formation of inclusion complexes with different molecular guests. The most popular are methylated and hydroxyalkylated derivatives that increase affinity to low-polar guests. Dimeric and oligomeric forms of cyclodextrins can be easily obtained. They may show increased affinity to large molecules, even to proteins, incorporating their segments (Aachmann et al. 2003). It was observed that such covalently linked dimeric structures could even disrupt the interactions between protein subunits in multimeric proteins by competition for exposed low-polar residues and thereby inhibit protein aggregation. Cyclodextrins have received much attention as the very specific binders in molecular and supramolecular studies, for providing the nanoscale media for chemical reactions (Oshovsky et al. 2007), even for modeling biomolecular catalysis (Breslow and Dong 1998). It was demonstrated that with proper modification, the sensing with cyclodextrins can be enantioselective, so that clear discrimination between D- or L-amino acids can be achieved (Pagliari et al. 2004). Many organic dyes form inclusion complexes with cyclodextrins (Al-Hassan and Khanfer 1998). Decrease of polarity, dehydration effects and immobilization effects were detected in fluorescence response of these dyes. Comparative studies on binding of different neutral, anionic and cationic dyes (Balabai et al. 1998) demonstrated that the binding is mainly hydrophobic and is only insignificantly affected by the charge of guest molecule. In all studied cases the tight complexes are formed. This is manifested by a decrease of anisotropy decay, which detects rotation of the whole complex. Meantime, ultrafast dynamics of guest molecules exists, and it was characterized by time-resolved methods (Douhal 2004). Inclusion into cyclodextrin cavity changes the photophysical and spectroscopic properties of organic dye molecules. Thus, for solvatochromic dye Nile Red a remarkable decrease of irradiative decay of ICT state was observed on inclusion into βcyclodextrin cavity (Hazra et al. 2004). The influence on protonation equilibrium (Mohanty et al. 2006) and excited-state proton transfer from pyranine to acetate in γ-cyclodextrin and hydroxypropyl γ-cyclodextrin (Mondal et al. 2006) was also reported. Different excited-state reactions, such as ESIPT (Organero et al. 2007), are also affected by binding in nanocavity. The fluorescence reporting signal can be generated in a simple but efficient way by displacement by the target molecule of the dye bound in cyclodextrin cavity. If the dye is not attached covalently to cyclodextrin host, the competitor displacement assay can be applied (as explained in Chap. 2 of Volume 1). However, if the dye is covalently bound by a flexible link, we can get the direct sensor. The target binding properties can be modulated by chemical substitutions.

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An increase of selectivity and affinity of cyclodextrin to its target is achieved when the sensor is made ‘bivalent’ so that the linker connects two cyclodextrin molecules (Yang et al. 2003b). Possessing two β-cyclodextrin cavities in close vicinity and a functional linker with good structural variety in a single molecule, these bridged bis(β-cyclodextrin) molecules can significantly enhance the original binding ability and molecular selectivity. In this case the possibility of multipoint interaction with the target increases and recognition of molecules as large as proteins can be achieved by binding the dimer at two exposed sites. It was shown that biquinolino-modified β-cyclodextrin dimers (Fig. 3.6) and their metal complexes may serve as efficient fluorescent sensors for the molecular recognition of steroids (Liu et al. 2004), as shown in Fig. 3.6a. In such cases the dye changing its molecular environment reports by spectral shift or by quenching/dequenching (Ikeda 2020). The length of the linker between two monomers is an important variable that can significantly modify steroid binding. The application of two dyes forming the EET pair sensing (Ogoshi and Harada 2008). Cyclodextrin plus two dyes (donor and acceptor in EET) are assembled on a peptide scaffold. The target molecule (guest) substituting the dye located within the cavity and disrupting EET generates fluorescence output (Fig. 3.6b). It was shown that a γ-cyclodextrin dimer exhibits strong molecular recognition ability for bile acids and endocrine disruptors (Makabe et al. 2002). Attached pyrene moieties allow detecting the binding due to generating a signal as the ratio of their monomer and excimer fluorescence intensities. It was indicated that pyrene residues participate in guest binding and serve as hydrophobic cups for the cavities. Conjugation with peptides expands the possibilities of cyclodextrins for detecting steroid molecules (Hossain et al. 2003). As we observed in Fig. 3.6b, the double

Fig. 3.6 Examples of cyclodextrin conjugates with fluorescence dyes. a From left to right: quinoline dye attached through the spacer (n = 1 − 2); biquinoline dye attached to a monomer; biquinoline dye forming a link between two cyclodextrin molecules. b Illustration of application of cyclodextrins in EET-based sensing. The target by replacing the dye donor D in cyclodextrin cavity disrupts the energy transfer to the appended acceptor A resulting in recordable fluorescence switching

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labeling with EET detection can be a method of choice in fluorescence reporting. Construction of more sophisticated conjugates with proteins was also reported, particularly, for sensing maltose based on E. coli maltose binding protein (MBP) (Medintz et al. 2003). Thus, cyclodextrins being naturally occurring cyclic oligosaccharides bearing a basket-shaped topology can form the basis of different modifications providing highly specific target binding. This binding is based on steric fitting and an “inner– outer” amphiphilic character of the basic structure strengthened by additional noncovalent interactions. Multiple targeting ligands and imaging elements can be attached due to the abundance of hydroxyl groups. This allows realizing different sensing formats in solutions with extensions to molecular imaging. The number of targets, for which cyclodextrin derivatives can be applied, is broad and includes mostly low-polar organic molecules, such as steroids, but also amphiphilic compounds and coordinated ions (Zhang et al. 2019). They are actively used in molecular imaging, and these versatile possibilities are well described in the literature (Lai et al. 2017).

3.2.2 Calix[n]arenes Calix[N]arenes are a class of polycyclic compounds that can allow (by proper chemical substitutions) achieving high-affinity binding of many small molecules (Baldini et al. 2020; Kumar et al. 2019). Calix[n]arenes are cyclic oligomers composed of n = 4–6 phenyl groups connected by methylene bridges that are obtained by phenol– formaldehyde condensation. They exist in a ‘cup-like’ shape with a defined upper and lower rim and a central annulus. Their rigid conformation of skeleton forming a cavity together with some flexibility of side groups (allowing their flip-flop) enables them to act as host molecules for different small molecules and ions. Their basic structure resembling a vase (calix = vase) together with modifications with short peptides in its upper rim (Peczuh and Hamilton 2000) is presented in Fig. 3.7. This beautiful structure resembles a basket of flowers. Based on calixarene scaffold, a broad variety of structures can be realized by modifications of both upper (Sliwa and Deska 2011) and lower (Joseph and Rao 2011) rims (Rodik et al. 2016). Examples of such derivatives are presented in Fig. 3.8. By functionally modifying these sites it is possible to prepare various derivatives with differing selectivity for various guest molecules. Mostly they are small molecules and ions, but with proper arrangement of attached groups they can target the structural elements of large molecules, such as proteins (Peczuh and Hamilton 2000), see Fig. 3.7. At these sites fluorescent labelling can be achieved. Calix[4]arene structure allows many possibilities for targeting proteins. Their upper rim displays four positions that can be used for covalent attachments. Moreover, the nature and symmetry of such recognition elements may be varied to selectively target the highly irregular surfaces, comprised of charged, polar and hydrophobic sites.

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Fig. 3.7 Calix[4]arene derivatives that mimic the properties of antibodies to recognize protein targets. R1 –R2 –R3 –R4 could be H, CH2 COOH; (CH2 )4 NH2 . This arrangement provides the recognition pattern of negative and positive charges

Fig. 3.8 Parent calix[4]arene in a cone conformation. a The sites of substitutions with covalent attachment of recognition units and fluorescent dyes in the upper rim are marked X. In the lower rim, the substitution sites are denoted as R1 and R2 . b Calix[4]arenes with amino groups at the upper rim. They can be used for convenient chemical modifications. c and d Typical calix[4]arenes with substitutions at the upper and lower rims correspondingly

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A series of synthetic receptors was prepared, in which four peptide loop domains are attached to a central calix[4]arene scaffold (Peczuh and Hamilton 2000). Each peptide loop is based on a cyclic hexapeptide, in which two residues have been replaced by a 3-aminomethylbenzoate dipeptide mimetic, which also contains a 5amino substituent for anchoring the peptide to the scaffold. Through the attachment of various peptide loops, the authors made a series of calix[4]arenes expressing negatively and positively charged regions as well as hydrophobic regions and thus achieved binding to complementary regions of several proteins. They demonstrated the binding to platelet-derived growth factor (PDGF) and inhibition of its interaction with its cell surface receptor PDGFR. A similar structure interacts specifically with cytochrome c (see Fig. 3.7). Fluorescence reporting on the binding of different targets to calix[4]arene structures is commonly introduced in two ways. (a) Using the property of these molecules to bind different dyes at the site within the cavity, where there occurs the binding of many targets. Then the binding of target molecule can displace the fluorescent dye. When the dye dissociates, it changes its fluorescence. The chelating properties of calix[4]arenes towards different dyes were reported by many researchers (Kubinyi et al. 2005). In all these cases, formation of the complex leads to dramatic fluorescence quenching, and this result suggests a convenient possibility for transforming the receptors into efficient sensors. This fact suggests using calix[4]arene derivatives in competitive indicator displacement assays (You et al. 2015), see also Chap. 2 of Volume 1. Ample possibilities for modulating the target affinities by covalent modifications and a broad choice of responsive dyes with different affinities promise easy development of simple and efficient assays for many targets. (b) Covalent conjugation of calix[4]arene side groups with fluorescent dyes (Nanduri et al. 2006). In this case, several mechanisms of fluorescence response can be realized. One can be easily applied in ion sensing and is based on induction/perturbation of photo-induced intramolecular charge transfer (ICT). Thus, the tert-butylcalix[4]arene was synthesized either with one appended fluorophore and three ester groups or with four appended fluorescent reporters (Leray et al. 2001). The dyes were 6-acyl-2-methoxynaphthalene derivatives, which contained an electron-donating substituent (methoxy group) conjugated to an electron-withdrawing substituent (carbonyl group). This is a typical arrangement for fluorescent reporters operating on the basis of ICT principle (see Chap. 4 of Volume 1).

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It was shown, that such molecules respond to binding the ions not only by expected red shifts of the absorption and emission spectra but also by a drastic enhancement of the fluorescence quantum yield Φ. Thus, the compound with one substituent was synthesized, for which on binding the Ca2+ ions Φ increases from 0.001 to 0.68. In the case of four substitutions, the emission was strongly depolarized due to the energy transfer (homo-EET). Regarding the complex-forming ability, a high selectivity for Na+ over K+ , Li+ , Ca2+ and Mg2+ was observed in this case. This selectivity (Na+ /other cations) expressed as the ratio of the stability constants was found to be more than 400. This result certifies the abilities of calixarenes as useful building platforms in the design of complex systems, in which not only ICT but also other excited-state phenomena (electron, and proton transfer, excimer formation and resonance energy transfer) are controlled by ions. These applications mainly concern ion sensing with high selectivity (Valeur and Leray 2007). The sensors based on calixarene platform with covalent attachment of reporter dye can be very efficient. A variety of environment-sensitive dyes were used for attachment, particularly, to an upper rim of their structures. In one of the studies, such modification was made with cyanine dyes (Kachkovskiy et al. 2006), which opens the pathway for extending the sensing techniques to a near-IR range. In a number of reports, the dye was constructed as a part of recognition mechanism, and the changes in its interactions with environment induced by target binding produced the necessary reporter signal. Enantioselective (distinguishing stereoisomers) molecular sensing of aromatic amines was achieved using their quenching on interaction with the chiral host tetra-(S)-di-2-naphthylprolinol calix[4]arene (Jennings and Diamond 2001). In addition to the role of recognition elements of small molecular sensors, calix[4]arenes play an important role in construction of fluorescent functional nanoparticles (Shulov et al. 2016). They are used for coating quantum dots (Jin et al. 2006), which allows achieving their bright emission and high stability in aqueous solutions. A fluorescent conjugated polymer poly(phenylene ethynylene) containing calix[4]arene-based recognition units displays a sensitivity to be quenched by the N-methylquinolinium ion. This effect is over three times larger than that seen in a control polymer lacking calix[4]arenes (Wosnick and Swager 2004). Very attractive is the ability of calix[4]arenes to host rare earth cations (Karavan et al. 2010). These ions are highly luminescent only in the ligand-bound forms (Chap. 6 of Volume 1), and calix[4]arenes may serve as such ligands. The water soluble calix[4]arene derivatives can form luminescent complexes with europium(III) and terbium(III) ions (Yang et al. 2003a, b). This makes the whole complex the luminophore with long-duration emission and allows many new possibilities for sensor developments. A broad range of analytes can be determined using fluorescently labeled calixarenes with different functional groups. A comprehensive review (Kumar et al. 2019) may help the interesting reader.

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3.2.3 Cucurbit[n]urils Cucurbit[N]urils are the macrocycles, which, like cyclodextrins and calixarenes, exist in different sizes (n = 5 − 8, 10). They are composed of a different number of glycoluril units joined by pairs of methylene bridges resulting in highly symmetrical structures (Assaf and Nau 2015; Barrow et al. 2015). Their inner volume can accommodate different organic molecules, including fluorophores (Fig. 3.9). In these structures one may pay attention to two identical dipolar portal ends, composed of carbonyl functional groups that may participate in hydrogen bonding and ion–dipole interactions. These compounds are obtained by acid-catalyzed condensation of glycoluril with formaldehyde under carefully controlled conditions. The size of their inner cavity ranges from 68 to 691 Å3 (for n between 5 and 10) allowing different guest molecules of largely variable sizes to be included within the cavity with remarkable selectivity. Similarly to cyclodextrins and calixarenes, cucurbiturils have a nonpolar inner cavity, which is prone to bind organic residues or guests by hydrophobic interactions (Assaf and Nau 2015; Nau et al. 2011). Meantime, there is a remarkable difference from calixarenes, and not only in high rigidity of their structures. In calixarenes the inner surface is electronically polarizable due to the presence of phenyl groups, and in cucurbiturils the cavity is extremely non-polarizable. This class of macrocycles display a relatively low water-solubility and strong complexation with many fluorescent dyes (Dsouza et al. 2011). Their host/guest

Fig. 3.9 The structure of cucurbiturils. a Molecular structures of three smallest homologues. These molecules are composed of a different number of glycoluril units joined by pairs of methylene bridges. b Their inner volume allows hosting different low-polar molecules, including fluorescent dyes. The interaction of curbit[7]uril with coumarin dye forming 1:1 host–guest complex by dye inclusion into the host cavity is shown

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complexes are much stronger than that of cyclodextrins, with binding constants typically ranging from 104 M−1 to much higher values (up to 109 M−1 ). Cucurbiturils are known as cation receptors due to their two identical carbonyl rim portals. Fluorescent dyes may change dramatically their photophysical and spectroscopic properties when they are incorporated into macrocyclic hosts due to rigidization of their structures and screening from water (Koner and Nau 2007). Some of them that are low fluorescent in solutions but become strongly fluorescent in cucurbituril cavities are used in competitor displacement assays for determining different analytes (Elbashir and Aboul-Enein 2015). Covalent binding of fluorescent dye may extend significantly the dynamic range of assays (Bockus et al. 2016). Current literature contains many examples of successful applications of cucurbiturils as macrocyclic hosts for determination of nicotine, pesticides, herbicides, amino acids, various drugs, etc. (Cicolani et al. 2020; Sinn and Biedermann 2018).

3.2.4 Pillar[n]arenes Pillar[N]arenes are a relatively new class of synthetic supramolecular macrocycles (Ogoshi et al. 2016). They possess a particular pillar-shaped architecture consisting of an electron-rich cavity and two fine-tunable rims (Fig. 3.10). Their beautiful and very simple pillar-shaped and regular polygonal structures stimulate many efforts for the construction of versatile functional derivatives. The ease and diversity of the functionalization of the two rims open many possibilities for the design of new architectures, topological isomers, and scaffolds.

Fig. 3.10 Schematics, chemical structures and molecular models of per-hydroxylated pillar[5]arene (a) and pillar[6]arene (b)

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There are interesting distinguishing features of pillar[n]arenes. They are composed of electron-donating dialkoxybenzene units and, therefore, can form host– guest complexes with electron-accepting molecules (Ogoshi et al. 2016). With the charged guest compounds, they can display cation/π interactions that are physical interactions between an electron-rich π-electronic system and a cation. The cavity of pillar[n]arenes is shallow but is hydrophobic because it is composed of hydrophobic benzene moieties. Therefore, small hydrophobic guests in aqueous media are encapsulated in the cavity of pillar[n]arenes to escape the hydrophilic environment. The introduction to the rims of pillar[n]arenes of hydrophilic substituents, such as cationic, anionic, or nonionic moieties, makes them much more soluble in water. Being the relatively new members of a family of macrocyclic receptors, pillar[n]arenes have already shown their potential for the anions/cations sensing, small molecules recognition, biomolecules detection and biomedical imaging (Chen et al. 2017). With the inclusion of organic dyes, they easily form fluorescent supramolecular aggregates, which extends the range of their applications.

3.2.5 Comparison of Properties and Prospects of Supramolecular Macrocycles Summarizing, it may be useful for future developers of sensing technologies to provide comparison of properties of presented above macrocyclic binders. Cyclodextrins are cyclic compounds consisting of glucose units derived from natural predecessors. Therefore, they are non-toxic and biocompatible. They are highly soluble in aqueous systems and cannot pass through cell membranes. Regarding the sensing of small hydrophobic molecules, such as steroids, cyclodextrins are the most attractive. This is because cyclodextrin cavities fit to their sizes; the binding can be selective and involve target penetration into the cavity and forming strong inclusion complexes. Their absence of solubility in many organic solvents may present the problems in synthesis of new derivatives (Lai et al. 2017). They are commercially available and inexpensive. The choice of sizes and shapes of the basic structures is small but fits to common organic dyes that can form the reversible host–guest complexes. Calix[n]arenes are macrocyclic oligomers made up of benzene units. Basic compounds are poorly soluble in water that can be increased by proper modifications. They are well-dispersed in some nonpolar organic solvents. They are chemically stable and in solutions demonstrate segmental flexibility. They are biocompatible and allow great variety of modifications generating different functionalities (Rodik et al. 2016). Their molecular recognition may involve the binding in the cavity (using the electron-donating power of constituting phenols), but mostly involves the designed structures with chemical substitutions in upper and lower rims (Kim and Quang 2007).

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Cucurbit[n]urils are macrocyclic glycolurils having two portals lined with ureidocarbonyl groups. They are biocompatible and provide reversible host–guest complex formation. Their complex-forming ability is probably the strongest among all macrocycles, especially when the guest is hydrophobic but with a positively charged group (Sinn and Biedermann 2018). Their problem is in poor aqueous solubility. They are also insoluble in practically all organic solvents, making chemical manipulation highly challenging (Mutihac et al. 2020). Meantime, being properly functionalized they are able to serve the sensors for zwitterionic compounds, such as amino acids (Bai et al. 2017). Pillar[n]arenes are pillar-shaped macrocyclic compounds made up of hydroquinone units linked by methylene bridges. They possess conformationally stable rigid and symmetrical structures that are easy to functionalize with different substituents (Ogoshi et al. 2016). They are poor in aqueous solubility but soluble in many organic solvents, making functionalization technically feasible. These macrocycles are easily functionalized with different dyes providing versatile fluorescence response (Cao and Meier 2019). For being fluorescence sensors, the macrocyclic compounds have to combine their target-recognition power with fluorescence response. Several possibilities exist for that (Fig. 3.11). The common way for that is the use of competitive binding assays or indicator displacement assays (see Volume 1, Chap. 2). Here the target analyte competitively displaces a fluorescent guest, or vice versa, and results in a change in parameters of fluorescence emission. These methods are the simplest in applications since they do not require the covalent modification of host or guest, thus preserving the host binding properties and obviating the need for additional chemical synthesis (Silva et al. 1992). The noncovalent association of host and indicator dye, however, necessitates the fitting of their binding affinity and working concentrations in order to ensure competitive binding conditions (You et al. 2015). Direct assays are those, in which fluorescent dye can respond to target binding/release by the changes of its fluorescence parameters without its physical displacement (Altschuh et al. 2006; You et al. 2015), see Chap. 2 of Volume 1. Covalent conjugation of a host to an indicator removes the dependence of their association on concentration. Careful design of a conjugate can yield single-component, direct sensors capable of detecting guest binding over a wide range of concentrations (Kim and Quang 2007; Ueno et al. 1992). Excitation energy transfer (EET) requires the presence of two dyes (lightabsorbing donor and fluorescent or quenched acceptor), see Volume 1, Chap. 4. In supramolecular or nanoscale systems in solutions, both of them (or one, at least) must be covalently bound. This increases the difficulties in their synthesis. However, such systems exist (Lou et al. 2017), as illustrated in Fig. 3.6b. From practical considerations, the most attractive are the macrocyclic sensors demonstrating direct fluorescence response that can explore the wavelengthratiometry (Demchenko 2010, 2014) that does need, as indicator the displacement assay and the associated with that fitting of the binding constants and concentration ranges of target and dye indicator. It also has the advantage over EET systems, since there is no need to introduce into the system of additional dye (Demchenko 2023a, b).

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A Fig. 3.11 Different possibilities to use fluorescence reporters for obtaining the responsive signal from target binding in the cavities of macrocyclic hosts. a Indicator displacement assay. The target (T) competes with fluorescent dye (F) for the binding in cavity, and the dye when removed from the cavity changes its fluorescence. b The dye is covalently bound to macrocycle and changes its position on target binding. It reports on target binding by changing the parameters of its fluorescence emission that can be realized in direct assay. c If the size of cavity permits accommodation of both the target and the dye, the dye firmly bound within the cavity responds to target binding by changing on interaction with the target its local environment, generating fluorescence response. Realizing direct assay is also possible here. d The excitation energy transfer (EET) acceptor (A) is introduced that can be bound outside the cavity closely to fluorescent dye within the cavity. The target replaces the dye from the cavity. The dye moves to a distance from the acceptor making EET not possible. The dye emission is restored generating the fluorescence signal

An important point is the behavior of organic fluorophores themselves in macrocyclic hosts. There are many observations indicating that the binding of different dyes in the host cavities may improve dramatically their fluorescence properties due to increase of their rigidity and screening from quenching effects of water (Nau and Mohanty 2005; Patsenker et al. 2010; Sayed and Pal 2016). Moreover, some dyes may start to display the room-temperature phosphorescence. The inclusion of dyes into chelating complexes protects their most reactive groups from undesired chemical transformations (Dsouza et al. 2011). The remarkable success of this approach was demonstrated in the cases of formation of rotaxane-type encapsulation of cyanine (Buston et al. 2000; Yau et al. 2008) and squarene (Arunkumar et al. 2005; Patsenker et al. 2010) dyes into the cyclic saccharide α-cyclodextrin cavity. In this mechanically interlocked molecular architecture, the cyclodextrin is tightly threaded round the central part of the dye. Another type of partners in the dye photoprotection are cucurbiturils (Koner and Nau 2007). The strong stabilizing effect of these host molecules is also due to low polarity of their inner cavity and efficient screening of the dye reactive sites from solvent water (Nau and Mohanty 2005). Thus, a strong improvement was reported for BODIPY complex formation with the macrocyclic host cucurbit[7]uril (Gupta et al. 2017). Macrocyclic compounds are able to associate and they can be connected by covalent linkers (Dubacheva et al. 2014). This allows targeting more than one recognition cites of the target (Xu et al. 2019). In Chap. 12 we will present bright examples how this concept is applied for selective recognition of peptides and proteins.

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3.3 Porphyrins and Porphyrinoids. Unique Coupling of Recognition and Reporting Porphyrins are natural compounds with key role in different biological processes and also with a rich history in synthetic and materials chemistry (Kadish et al. 2000; Kadish 2010). They have unique photochemical and spectroscopic properties. They can coordinate a wide variety of metal ions with the added ability to function as receptors for different types of analytes. Also, being electron-rich, porphyrins easily bind the electron-deficient analytes, such as nitroaromatic compounds, with perturbation of their π-electronic system. This is the basis for the fluorescence detection of nitroaromatic explosives (Kielmann et al. 2018; Tao et al. 2006). Natural porphyrin molecule is a heterocyclic macrocycle derived from four pyrole-like subunits interconnected by means of α carbon atoms via methine bridges (=CH–). This macrocycle is planar and with high electronic conjugation, it contains 26 π-electrons (Fig. 3.12). Porphyrins contain various points (meso-positions, β-positions, and pyrrolic NH), at which they can be synthetically modified by introduction of various functional groups or by heteroatom substitution, allowing for development of numerous porphyrin derivatives (so-called porphyrinoids), which often exhibit characteristic properties. Porphyrins can coordinate with most transition metals and some other cations (M) in a square-planar configuration, leading to metalloporphyrin complexes. The metal cation contained in the porphyrin complex may be further coordinated to by a 5th and/or 6th ligand in the axial positions above and below the plane of the porphyrin macrocycle (shown as L in Fig. 3.12), which can be important for sensing analytes (Ishihara et al. 2014). Typical 5th and 6th ligand molecules are electron-donating heterocycles such as pyridine, imidazole or alcohols, or even smaller molecules such as O2 , CO, NH3 , H2 O, and H2 O2 . Versatility of structural substitutions in the porphyrin skeleton, such as shown in Fig. 3.13a, allows designing receptors for different targets.

Fig. 3.12 The basic structure of synthetic porphyrins. a Metal-free form. b Metal-coordinated form. It is shown that the central core of the porphyrin can be occupied by two hydrogen atoms (free base porphyrin) or by a metal ion (metalloporphyrin). The positions are shown of various substitutions that can be selected for the design of high-affinity target binding

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Fig. 3.13 The porphyrin derivatives and analogs. a The α,α,α,α-atropisomer of 5,10,15,20-mesotetrakis(oaminophenyl) porphyrin. This is a widely used platform that forms cavities for bindingspecific guests, depending on the substituents (Sessler et al. 2012). b The structures of representative porphyrin analogs forming the frameworks for ion sensing (Ding et al. 2017). A contracted porphyrin analog corrole exhibits smaller macrocyclic core size and prefers to coordinate as trianionic ligands rather than in the dianionic form, and thus it shows unique metal ion coordination capabilities. Sapphyrin is an expanded porphyrin analogue that is known to selectively bind F− anion in its protonated core. Porphycene is a porphyrin isomer with planar and aromatic structure. N-confused porphyrins, in which the NH-group extends to periphery, are strong anion binders. Calix[4]pyrrole is a nonconjugated porphyrin analogue that is used for recognition of ion pairs

The family of synthetic porphyrin macrocycles has been expanded by synthetic porphyrin analogues (Costa et al. 2016; Ding et al. 2017; Ishihara et al. 2014), see Fig. 3.13b. Compared to natural porphyrins, the porphyrin analogues show greater structural diversity, unprecedented coordination properties, light absorption/emission, aromaticity, etc. Diverse structures contributing to this large family may be roughly divided into ring-expanded porphyrins, ring-contracted porphyrins, porphyrin isomers, and heteroporphyrins, according to the numbers of pyrrolic units in the molecules or the presence of hetero-donor atoms, such as O, S, Se, and Te instead of pyrrolic N within the porphyrin core. They determine the binding ability to different targets (Beletskaya et al. 2009; Ding et al. 2017). Some expanded porphyrin molecules (that are composed of more than four pyrole groups), see Fig. 3.14, can form supramolecular assemblies with the inclusion of dianions serving as the linker units (Zhang et al. 2015a, b). These systems display unique environmentally responsive behavior. Addition of polar organic solvents or common anions to the ensembles leads to either a visible color change, a change in the fluorescence emission features, or differences in solubility. In this way, they can serve as sensors distinguishing between F− , Cl− , Br− , NO3 − , and SO4 2− anions. These structures demonstrate that expanded porphyrins can attain new very interesting photophysical and target-binding features. The compound shown in Fig. 3.14b binds selectively the Hg2+ cations (Zhu et al. 2006). The binding results in strong fluorescence quenching. Despite very small difference, the species with the structure presented in Fig. 3.14c does not possess appreciable affinity to Hg2+ ions but binds

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Fig. 3.14 Examples of expanded porphyrin analogs containing more than four pyrrole groups. a The type of molecules that can associate in chains on binding of bi-hydroxylic acids. The porphyrins forming such supramolecular structures demonstrate strong response to binding of anions. b Chemical structure of expanded porphyrin chemosensor with very high affinity for and discriminating ability against other ions. c The porphyrin analog selectively binding the Ag+ cation with discrimination of Hg2+ ion

the Ag+ cations with high affinity and selectivity (Zhu et al. 2008). In both cases, the sensitivity of ion detection is about 10–7 M. Numerous experiments demonstrate that the targeted sensing behavior towards ionic species of these structurally diverse porphyrins can be easily modulated in the following ways (Ding et al. 2017): (1) modulation of conformations through structural variation, so that pyrrolic NH moieties are properly arranged for selective anion binding through cooperative hydrogen-bonding interactions; (2) multiple pyrrolic N atoms can be employed to selectively chelate metal ions, including by the replacement of pyrrolic N with other heteroatoms, leading to unique metal ion binding affinity and selectivity; (3) high selectivity and sensitivity toward target anions and metal ions could be further achieved by the modification of binding affinities through variation of the macrocycle size. Many substitutions can be made at the periphery of porphyrin ring, demonstrating the pronounced dependence of porphyrin properties on their structures (Senge et al. 2015). Additional advantages can be achieved on fusion of porphyrins or their analogs with other macrocyclic compounds. Porphyrins are the macrocycles that successfully combine sensing and reporting properties, since they possess their own light-absorption and fluorescence spectra. Their strong light absorption at Soret and Q bands is the basis of design of diverse color-changing probes. Their fluorescence depends on the presence of coordinated metal cation in the center of their structure (Mn+ in Fig. 3.12). In most cases, the emission spectra of free base porphyrins and of porphyrins with coordinated cation (metalloporphyrins) are different from each other. Since the free and bound forms can be easily distinguished, this suggests the possibility of making sensors for these ions (Purrello et al. 1999). It is hard to do that with natural porphyrins, in which the fluorescence intensity of free base in water is very low and the selectivity of binding is not sufficient to discriminate between different ions in their mixture. Hopefully, the porphyrin scaffold allows for many functional substitutions and complexations that are able to improve, if necessary, their spectroscopic as well as their solubility and binding properties (Beyene et al. 2020; Yu et al. 2020).

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The fact that fluorescence properties of porphyrins can be dramatically improved by modifications was demonstrated in numerous studies. Thus, their fluorination allows obtaining emission in the deep red range of spectrum with a two-fold increase of quantum yield (Barker et al. 2007). Additionally, porphyrin analogues such as expanded porphyrins may undergo fluorescence emission at wavelengths longer than 600 nm, even reaching into the near-IR region, making them more suitable for applications involving biological samples. Extension of π-conjugation network in expanded porphyrin analogs is responsible for their near-IR emission. Thus, the intense emission of compound with the structure presented in Fig. 3.14,c is observed at 1030 nm (Zhu et al. 2008). In platinum-coordinated porphyrins, the near-IR phosphorescence was detected, and these spectra were further shifted to infrared region on fusion with aromatic hydrocarbons (Sommer et al. 2011). Whereas porphyrins can act as stand-alone receptors, their integration and functionalization with specific binding moieties is typically required in order to obtain selectivity for a chosen target. Such improvements in ion binding and sensitivity of response can be achieved upon complex formation with cyclodextrins. These complexes can be particularly useful for determination of zinc ion. The zinc binding changes the fluorescence spectrum of porphyrin (Fig. 3.15), which allows the λ-ratiometric recording of zinc concentration (Yang et al. 2003b). Upon binding the zinc ion, the fluorescence emission of tetraphenyl porphyrin at 656 nm band decreases while that at 606 nm increases, which allows the wavelength-ratiometric detection. Fluorescent chemical sensing for aromatic compounds can be achieved by forming a supramolecular complex composed of tin(iv) porphyrin, viologen, and cucurbit[8]uril (KumaráShee and KyoungáKim, 2019). The detection is successfully achieved by the inclusion of an aromatic analyte through the charge-transfer

Fig. 3.15 Ratiometric detection of zinc by fluorescence response in porphyrins-cyclodextrin complex (Yang et al. 2003b). a Possible structure of 2:1:1 complex of cyclodextrin with porphyrin and zinc ion. b Effects of zinc ion concentration on the fluorescence emission spectra (spectra 1–8 correspond to the increasing zinc concentrations from 0 to 7.2 × 10–5 M). c Relative two-band fluorescence ratio, α, as a function of log[Zn2+ ] at pH 8.0

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Fig. 3.16 Fluorescence sensing of metal cations based on tetrapyridyl-substituted porphyrin (left) coordinating surface atoms of the CdSe quantum dots (QDs) (Frasco et al. 2010). The ion binding generates the enhancement of QDs emission (right)

interaction with the viologen unit in the cavity of cucurbit[8]uril, in which the strong charge-transfer interaction interrupts the photo-induced electron transfer (PET) from the tin(IV) porphyrin core to the viologen ligands leading to the efficient fluorescence emission from the porphyrin ring. Porphyrins also find the application in the formation of functional composites with fluorescent nanoparticles, such as quantum dots (QDs). In one example (Fig. 3.16), a hybrid structure that directly senses metal ions was prepared based on the use of quantum dots functionalized with tetrapyridyl-substituted porphyrin serving as an organic ionophore (Frasco et al. 2010). In this construction, the porphyrin derivatives with four pyridyl rings bind efficiently to the QDs surface, while at the same time they preserve the zinc recognition capabilities of the porphyrin. Upon coordination with zinc ions, this porphyrin capping is shown to strongly contribute to the increase in the fluorescence efficiency of CdSe, via an activating interaction with the QD surface. Thus, both the optical properties of the QDs and the ion recognition properties of the porphyrin can operate in a synergic manner. The specific binding of synthetic porphyrins to proteins can be realized by selecting the “hot spots” on the surfaces of these molecules and designing such their derivatives that provide the most efficient saturation of noncovalent interactions with these hot spots. Here electrostatically driven complementarity with contribution of hydrophobic interactions can be realized, and porphyrin analogues simultaneously act as the recognition and reporting units. Such possibility was demonstrated for cytochrome c, for which the site of interaction with its redox partner cytochrome c oxidase was modeled by a porphyrin derivative containing distributed carboxylic groups (Fig. 3.17a). After enhancing the hydrophobicity of the porphyrin core and by increasing the number of peripheral carboxylic acids from 8 to 16, the binding to cytochrome c with sub-nanomolar dissociation constant was achieved. With K d = 0.67 nM, this is probably one of the most potent synthetic protein receptors ever designed (Fletcher and Hamilton 2006). Regarding homotetrameric human Kv1.3 potassium channel, the smart cationic porphyrins interact with them strongly, in the nanomolar range, and, as determined by electrophysiological measurements, significantly reduce the current through the channel (Gradl et al. 2003).

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Fig. 3.17 The structure of tetracarboxyphenylporphyrin derivatives that demonstrated subnanomolar affinity for the binding to the protein surface. a Probe for cytochrome c demonstrating dissociation constant Kd = 0.67 nM (Aya and Hamilton 2003). b Probe for the human Kv1.3 potassium channel (Gradl et al. 2003) providing strong blocking of ion conduction

Arrays of fluorescent surface receptors based on porphyrins were presented for the recognition of different proteins in their mixtures (Baldini et al. 2004). It was reported that in a ‘one-pot’ synthesis, meso-tetracarboxyphenylporphyrin was derivatized with two different amino acids or amino acid derivatives, giving a mixture of six unique products. Iteration of this procedure with further amino acids and/or derivatives led to a library of 35 unique fluorophores, exhibiting peripheral charges from −8 to +8, and with four to eight hydrophobic groups. When incubated with different proteins, an array of eight of the porphyrins interacted differently with various proteins, thereby generating their characteristic fingerprints. Such strategies for protein surface recognition offer a new use for porphyrins as molecular scaffolds (Tsou et al. 2004). In present times the research in this domain is less active. But it is actual, since it is related with molecular recognition patterns on protein surfaces and with the efforts to substitute the antibodies with the binders of simpler design. In more detail, these issues will be discussed in Chap. 4. Porphyrins with special design and also in the form of nanostructures can specifically bind to DNA (Pratviel 2016). One of the methods of detecting the double strand DNA (dsDNA) was based on groove binding along the dsDNA surface of porphyrins decorating gold nanoparticles. A Z-DNA recognition can be performed with cationic zinc porphyrins (D’Urso et al. 2011). Functionally important G-quadruplexes can be recognized by smart porphyrin derivatives (Pratviel 2016). A further opportunity to enhance porphyrin sensing properties is related to their supramolecular organization in the solid state, discussed in Chap. 9. It should be mentioned that porphyrins, interacting between themselves can be engineered to promote the free assembly of very different shapes, such as nanotubes or regular scaffolds (Ishihara et al. 2014; Wang et al. 2013). Such three-dimensional arrangement can be extremely sensitive to interaction with guest analytes, suggesting new possibilities to modulate sensors’ sensitivity. There are many possibilities for designing an important family of sensors in the field of nanostructured devices that are based on hybrid materials formed by

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porphyrins and inorganic materials. Porphyrinoids are the perfect arena for this activity. Their versatility that involves sharing the same macrocycle but with a different metal ion coordinated in the core, possibility to operate the changes in the molecular skeleton using porphyrin analogs and innumerable possibilities of their modifications makes these receptors (and also fluorophores!) adjustable to the many micro- and nanotechnological routes existing for the development of devices.

3.4 Sensing and Thinking. The Recognition Properties of Parent Binders and of Their Derivatives Molecular structures discussed in this chapter can be the most efficiently used for specific recognition and quantitative assays of three classes of molecules: ions, small organic molecules and macromolecules. Organic heterocyclic compounds are better suited for the detection of ions, because the ions can be easily coordinated into heterocycles and because the ion binding can be easily electronically coupled with the response of appended dyes or, as in porphyrins, to provide perturbation of their own electronic systems. The well-developed surface of cyclodextrins, calixarenes and cucurbiturils allows, in addition to specific ion binding, to realize selective binding of small neutral molecules fitting into their inner volume. But in this case, a clever manipulation with external fluorescent dyes is needed to provide reporting. Thus, we have to think how to operate with such a large pool of macrocyclic receptors available that offer unparalleled opportunities for chemical and biochemical sensing. Macrocyclic receptors can be designed and prepared according to the analytical problem to be solved, thanks to the degree of sophistication reached in the fine tuning of weak interactions responsible for molecular recognition. In each case, the transduction platforms to turn the molecular recognition events into a readable signal can be different, as well as their integration into solution-based assays and solid-state devices. The reader has to check that most of the presented above chemical sensors have been applied in organic media, whereas the real-world manipulations with biological samples are commonly needed in aqueous media. Thus, further research on improving water solubility and sensing behavior in aqueous systems is required. For checking the knowledge acquired in this chapter, the reader is encouraged to respond to the following questions: 1.

2. 3.

Crown ethers are cyclic compounds. How do they incorporate the dyes for obtaining fluorescence response to ion binding at their center? In this respect, what is special with azacrowns? How the act of ion binding to crown ethers can be transduced to electronic charge transfer or proton transfer effects in fluorescence response? Why being highly water-soluble, cyclodextrins bind cholesterol and its derivatives and even can extract cholesterol from biological membranes? What are the requirements for such binding?

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4.

What is the difference in target binding to cyclodextrin monomers and dimers in terms of selectivity and affinity? 5. How can the calixarene selectivity be tuned by chemical modifications in order to recognize a particular analyte? 6. How calixarenes and porphyrins interact between each other? Based on these molecules the smart receptors for different targets can be designed. What principles can be implied in this design? 7. How you would modify the protein sensor based on calixarene in order to incorporate the fluorescent dye? 8. What are the attractive features and what are the problems in applications of cucurbit[n]urils in chemical sensing? 9. Pillar[n]arenes are symmetric barrels open from both sides. What kind of targets can they recognize and how to locate the fluorescence responsive unit? 10. Show the sites of ligand binding and chemical modifications in porphyrin structure. 11. Why the expanded porphyrin analogs shift their absorption and emission spectra to near-IR region? 12. Explain the principle of specific protein recognition by modified porphyrin analogs. What kind of modifications is needed for that?

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Chapter 4

Sensors Based on Peptides and Proteins as Recognition Units

Peptides and proteins are the major actors in biological world participating on different steps of biochemical reactions and their regulation. Modern chemistry exploring the principle of molecular recognition has transformed them into powerful recognition elements of the sensors. The range of these receptors is extremely broad, from short peptides of only several amino acids to large proteins and their ensembles. Whereas peptide binders can be found by searching the libraries, the protein sensors use the elements of structure derived from natural proteins. The strong tendency is observed to make the molecular sensors simpler, cheaper, more stable and accessible to techniques of chemical synthesis. An extremely broad range of chemical and biochemical sensors is based on natural or unnatural amino acids connected with peptide bonds. They form the structures of quite different size and spatial arrangement. The assembly of natural amino acid residues can form the recognition pattern of differently distributed charge, polarity, ability to form hydrogen bonds and other types of noncovalent interactions. Additional chemical modifications, including the insertion of fluorescent dyes, can be made using the exposed amino, carboxy and sulfhydryl groups. The functional recognition structures can be both rigid and flexible. They address the targets of different size, arrangement and flexibility. Maximum knowledge is being taken from nature in the design of these smart sensor molecules. They can be those that perform the same or similar function in vivo but also those that contribute to sensor design only the principle of their performance or some element of their structure. In some important cases this allows obtaining artificial miniaturized receptors from the reduction of the known sequence of a natural receptor down to a synthesizable level. In our discussion we start from synthetic peptides that can be rationally designed or randomly selected, discuss the possibilities of their structural arrangement and inducing their functionality, then we proceed with natural and designed protein and protein-like receptors and finish with antibodies and their reduced forms. Peptide nucleic acids that are designed for recognition of nucleic acids will be discussed in Chap. 11.

© Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_4

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4.1 Designed and Randomly Synthesized Peptides Peptides are the oligomers of amino acids connected with peptide bonds. Their ability to form 3D structures and participate in intermolecular interactions (including those that are valuable for sensor recognition function) is determined by hydrophobicity of constituting amino acid residues (side groups) and their ability to participate in intermolecular interactions. For being efficient in addressing particular target, the peptide should form the pattern of these interactions. For that, it should be designed in a rational way or selected from the randomly distributed species in a large library (Fig. 4.1). Peptides with high affinity to targeted analytes have to be either chosen by screening the peptide libraries or have to be known as natural ligands to the target molecule. Additionally, those peptides can be designed to form the recognition sites of proper proteins based on computer modeling. The production of the specific sequences is commonly accomplished by solid-phase synthesis (Behrendt et al. 2016), and modifications for immobilization and labeling may be included in this process. Possessing the possibility of multiple interactions with particular target, they can demonstrate a high affinity to this target. Moreover, the affinity can be further enhanced by easy modifications of the peptides (Pavan and Berti 2012). The peptides can attain the necessary properties as recognition units: flexibility to adapt sterically to any target, rigidity in secondary structure to conserve the binding aptitude and the propensity to form different kinds of non-covalent bonds with potential target. Due to such exceptional versatility, peptide-based sensors have found many applications in analysis, starting from small ions to proteins, DNA and the whole cells (Liu et al. 2015; Pazos et al. 2009). Of special interest is their ability of sensing antibodies when they include specific antigenic determinants (Enander et al. 2008) and to decorate different types of nanoparticles making them functional (Jeong et al. 2018). Operating with synthetic peptides has many technical advantages. Peptides offer functional robustness superior to that of most proteins and are well suited for the longterm storage in dry, dissolved or immobilized forms (Pearlman and Wang 2013).

Fig. 4.1 A cartoon showing a peptide formed of 20 natural amino acids that here are presented in one-letter code and classified according to their polarity. Two possibilities that are basic for the design and selecting of functional peptides are outlined

4.1 Designed and Randomly Synthesized Peptides

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Importantly, they allow full control of the labeling process, including a complete labeling at a single site. Such possibility is extremely important for fluorescence sensing, which allows using full arsenal of fluorescence reporters with their response in electron, proton and excitation energy transfers, formation of excimers, incorporation into supramolecular structures or coupling to plasmonic enhancers (the techniques described in Volume 1 of this book). An oriented immobilization in a chip formats can be readily obtained.

4.1.1 The Development of Peptide Sensors The development of peptide-based sensors proceeds according to different strategies, the major of which are those: (a) Rational design based on already known folded motifs. This means that if this motif is known being observed in a native protein, then it is highly probable that it will fold in the same way when produced as the structure of minimal size by synthetic means. By selecting this fold motif as a scaffold structure, one can introduce different modifications inducing or modifying the target recognition and fluorescence response properties. This strategy is commonly known as the template-based approach (Singh et al. 2006). Synthetic peptides have been designed on the basis of known interactions with the targets. Attention is paid to the presence of those motifs that are known to allow intermolecular selforganization of the sensing peptides over the sensor surface (Pavan and Berti 2012). Sophisticated highly sensitive and selective sensors have been obtained in this way. (b) Selection from a large combinatorial library. In this case, making the combinatorial peptide library and establishing the selection criteria are needed. The efficient binding to the target could be the major selection criterion, so that the affinity chromatography and related methods can provide the selection. Short peptides from random phage display can be selected in a random way from large, unfocussed, and often preexisting and commercially available libraries. To be successful in generation of designed molecules as recognition units, this peptide-based approach has to rely strongly on the ability to create and operate with large libraries together with powerful library selection technologies. The advances in protein engineering, selection and evolution technologies can be actively used (Pavan and Berti 2012; Schröder and Lübke 2014). Such peptides often perform better than antibodies, and the problems appear when the target is a small molecule.

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4.1.2 Randomly Synthesized Peptides, Why They Do Not Fold? Synthetic peptides with random location of amino acid residues are commonly unfolded. The formation of their native-like structures is a low-probable event that can be observed for very rare sequences. This is because of strong connection between the amino acid sequence and the three-dimensional organization, known as the folding code (Demchenko and Chinarov 1999). The folding code is highly degenerate and includes both steric and energetic factors. This means that in relatively large proteins some substitutions (even many of them) still lead to correct folding. Even larger substitutions are allowed if they are compensated in structural or thermodynamic sense by other substitutions. The folding code has distributed character, which means that it is not reduced to interactions between particular residues but involves extended elements of structure. In addition, kinetic variables on different hierarchical levels may not only determine the pathway of folding but also the resultant folded structure (Demchenko 2001a; Yesylevskyy et al. 2005). These possibilities cannot be realized for relatively small peptides, where there are much less possibilities for intramolecular interactions. Because of that, the prediction of folded structures is difficult. Moreover, the folding can be strongly influenced by variations of weak intermolecular interactions (Demchenko 2001b). All these factors complicate the design of protein and peptide 3D structures ab initio. No less difficult is to design the interaction with the target molecule, even if the structure is well known.

4.1.3 Template-Based Approach The well-defined secondary and tertiary structures of peptides and proteins are formed and stabilized by interactions in the main chain and by contacts between side groups. Despite of huge conformational space that allows astronomical number of peptide conformations, the number of folding motifs is very limited (Schulz and Schirmer 1979). The selection of topological peptide template can be made based on the known 3D structures of proteins. The recent advances in the chemistry of coupling reagents, protecting groups, and solid-phase synthesis have made the chemical synthesis of peptides with conformationally controlled and complex structures feasible (Singh et al. 2006). These peptide templates can be used to construct novel structures with tailormade functions. Such peptide-template-based approach demonstrates the utility in achieving molecular recognition of different targets. A statistical picture of amino acids found at protein–protein interaction sites indicates that the proteins recognize and interact with one another mostly through the restricted set of specialized interface amino acid residues, Pro, Ile, Tyr, Trp, Asp and Arg (Sillerud and Larson 2005). They represent the three classes of amino acids: hydrophobic, aromatic and charged

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(one anionic and one cationic). The same residues are the most frequently used for constructing the peptide recognition sites. The automatic solid phase peptide synthesis is the key technology of producing the template-designed peptide sensors (Behrendt et al. 2016). The standard procedures allow obtaining peptides as long as of 20–30 residues, which can be sufficient for making the designed folds. These techniques allow parallel synthesis of several species on solid support. Combining the surface chemistry with the recent technology of microelectronic semiconductor fabrication, the spatially addressable peptide microarrays can be obtained (Hamada et al. 2005). One can choose between two synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate or peptide synthesis in situ.

4.1.4 The Exploration of ‘Mini-Protein’ Concept It was interesting to note that a peptide as small as 20 residues can possess a cooperatively folded tertiary structure (Gellman and Woolfson 2002). Such mini-proteins (Fig. 4.2) are numerous (Škrlec et al. 2015). They serve as a fruitful platform for protein design by positioning all amino acids necessary for biomolecular recognition.

Fig. 4.2 Presentation of typical ‘minimal’ protein scaffolds in ribbon-based graphics derived from protein database. The α-helices are purple and β-sheets are yellow

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Particularly, they can display a high DNA binding affinity and specificity (Pflum 2004). A 38-amino acid peptide was described as an α-helical hairpin stabilized by two disulfide bridges and presented as a scaffold for future sensor developments (Barthe et al. 2004). It was proposed that a synthetic 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles could form a scaffold for molecular sensors (Enander et al. 2002). Different functional groups can be incorporated covalently onto this scaffold in a site-selective manner. Incorporation of a receptor group interacting with the binding site of a protein together with an environment-sensitive dye allows obtaining a sensor for this protein (Fig. 4.3). This fully designed sensor with a group known as an inhibitor of carbonic anhydrase being a recognition unit and a reporting fluorescent dye can detect the binding of carbonic anhydrase (Enander et al. 2002). Essentially, the linker structure and length modulate dissociation constant in a range of two orders of magnitude. The

Fig. 4.3 Schematic representation of the helix-loop-helix polypeptide scaffold forming on dimerization a four-helix-bundle and proposed as a versatile sensing platform (communicated by K. Enander). The scaffold is shown with a dansyl probe and benzenesulfonamide attached at amino acid positions 15 and 34, respectively. Attachment of a bifunctional spacer of different length allows linking covalently the benzenesulfonamide residue to a lysine residue side chain in the polypeptide scaffold. This construct was used for the detection of human carbonic anhydrase II. The graph representing fluorescence response to target binding as a function of a spacer length used for benzenesulfonamide attachment

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binding results in a significant increase of fluorescence intensity, which arises from a disruption of the homodimer in the presence of the target (Enander et al. 2004a). This concept can be applicable to numerous targets, for which the ligand is a small compound that can be grafted on the peptide scaffold. The potential of this approach for microarray applications was demonstrated by designing an affinity array (Enander et al. 2004b).

4.1.5 Molecular Display Including Phage Display Phage display is a powerful method for the discovery of peptide ligands that are used for analytical tools, drug discovery, and target validations (Uchiyama et al. 2005). Phage display technology can produce a great variety of peptides and generate novel peptide ligands. Phage display cycle (Mimmi et al. 2019) includes several steps involving fusion proteins for a viral coat protein and the genes to be expressed in bacteriophage. They infect bacteria, and the genes encoding the higher-affinity binders are isolated. With the introduced random mutations, they are used to perform new rounds of evolution (selection and amplification steps) ending with isolation of binders with the highest affinity. A number of other display platforms include bacterial and yeast display, ribosome display, and mRNA display (Levin and Weiss 2006). Ribosome display (Zahnd et al. 2007) is an in vitro selection and evolution technology for proteins and peptides from large libraries. The diversity of the library is not limited by the transformation efficiency of bacterial cells. The random mutations can be introduced easily after each selection round. This allows facile directed evolution of binding proteins over several generations. Affinity selection of peptides displayed on phage particles can be used for mapping molecular contacts between small molecule ligands and their protein targets (Rodi et al. 2001). Important observations were made in these studies that the binding properties of peptides displayed on the surface of phage particles could mimic the binding properties of peptide segments in naturally occurring proteins. Conformation of these segments can be relatively unimportant for determining the binding properties of these disordered peptides because they adopt ‘induced’ conformation upon binding to the target. Such ‘induced fitting’ to the target (Demchenko 2001b) can be the basis of its molecular recognition behavior. The selection of peptide binders from the library does not need to know the 3D structures of interacting partners. Moreover, these structures can be flexible. This allows a rapid large-scale identification of potential ligand-binding sites and also an easy introduction of fluorescence reporter responsive to target binding in a number of parameters (wavelength ratiometry, lifetime, anisotropy). Dynamic formation of optimal non-covalent bonds compensates the entropy loss associated with the loss of conformation mobility. If in vitro selection techniques could produce short polypeptides that tightly and selectively bind to any of a wide range of macromolecular targets, the possibilities for

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sensor developments would be broad. Some applications have already been demonstrated. One of them is to construct the ‘peptide beacons’ (Oh et al. 2007). It is similar to DNA beacons and is based on the change of relative distance between fluorescent dye and the quencher on target binding. Based on this principle, a robust optical sensor was developed for anti-HIV antibodies (Oh et al. 2007) and the antibodies to p53 protein that are important biomarkers for cancer (Neuweiler et al. 2002).

4.1.6 Peptide Binders for Protein Targets and the Prospects of Peptide Sensor Arrays Labeled peptides with their flexible structures in solutions are very attractive as analytical tools for detecting different protein targets, particularly the antibodies. A prototype of self-referenced protein sensor based on peptide labeled with λratiometric 3-hydroxychromone dye was developed (Enander et al. 2008). Using a model of high-affinity interaction between an 18-aminoacid antigenic peptide derived from tobacco mosaic virus coat protein and a recombinant antibody fragment, Fab 57P, a dramatic change of fluorescence spectrum was observed indicating their interaction by changing the relative intensities of two bands. The non-specific binding does not produce this effect (Fig. 4.4). It was observed that the ratio of intensities of two bands (IN* /IT* ) in the dye fluorescence spectrum changed dramatically (from 0.92–0.94 to 0.60) on interaction with specific and remained unchanged in the presence of nonspecific antibody fragments. Essentially, the labeling was produced outside the antibody binding site and did not influence the binding, so that it is the interaction at the periphery of the binding site that generated this dramatic change. This result shows that the response of fluorescence dye in such constructs is strongly position-dependent and that a rational design should be complemented by comparative studies of systems with different dye locations. More recently, in the studies of a different peptide-antibody system it was shown that on interaction with the antibody fragment, the peptide sensor labeled at Nterminal showed up to 47% change in the ratio of its two emission bands. Competition with two unlabeled peptides of different lengths has led to a dynamic displacement of the construct governed by their relative binding constants (Choulier et al. 2010). In our view, fluorescent indicators based on synthetic peptides are very interesting alternatives to protein-based sensors. They can be synthesized chemically, are stable, and can be easily modified in a site-specific manner for the fluorophore coupling at desired sites and for the immobilization on solid supports (Choulier and Enander 2010). The key issue is the generation of reporting signal to target binding, which can be provided by their labeling by λ-ratiometric dyes and without necessity of target labeling (Demchenko 2023a, b). The peptide based sensors are potentially useful for diagnosis of viral, bacterial, parasitic and autoimmune diseases by detecting the correspondent antibodies

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Fig. 4.4 Synthetic peptide for detecting protein target (Enander et al. 2008). The peptide representing the antigenic segment 134–151 of tobacco mosaic virus protein (shown in pink in peptide sequence) interacts with the specific antibody fragment Fab57P as a model target. The segment of protein structure and the corresponding peptide sequence are shown in the same color code. The pink part of the sequence corresponds to the antigenic binding site for Fab57P. Below are the fluorescence spectra of the λ-ratiometric 3-hydroxychromone dye (FC) covalently attached to indicated sites in the absence (red) and at the presence (blue) of saturating amounts of specific antibody fragment Fab57P. (a) The mutant V151C-FC. The spectra change dramatically on target binding with the decrease of fluorescence ratio between the two bands (IN*/IT*) from 0.94 to 0.60. (b) The mutant S146C-FC. The ratio of fluorescence intensities does not change. The F(ab’)2 fragment with unrelated specificity (green) was used as a negative control

(Gomara and Haro 2007). Synthetic peptides can provide uniform, chemically welldefined antigens for antibody analysis, reducing inter- and intra-assay variation. The success of this approach depends on the extent to which synthetic peptides are able to mimic the antigenic determinants (Timmerman et al. 2005).

4.1.7 Antimicrobial Peptides and Their Analogs Some natural peptides possess functionally important recognition properties and it is straightforward to use them as well as their analogs as the binders. Numerous bacteria, plants, and higher organisms produce antimicrobial peptides as a part of

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their innate immune system, providing a chemical defense mechanism against microbial invasion. Many of these peptides exert their antimicrobial activity by binding to components of the microbe’s surface and disrupting the membrane. They can be incorporated into screening assays for detection of pathogenic species (HoyosNogués et al. 2018; Ngundi et al. 2006). It was shown that the surface-immobilized peptides, such as polymyxins B and E, can be used to detect pathogenic bacteria in two assay formats: sandwich and direct. Different mutant forms of short antimicrobial peptides have been obtained and selected. They are promising as remedies to fight against the antibiotic-resistant microbes. Using antimicrobial peptides as recognition elements in an array biosensor, detection of these microbes can be achieved (Kulagina et al. 2006). Thus, antimicrobial peptides represent a lucky case of receptors that are naturally produced and can be applied to sense their own targets, in addition to much potentially broader scope of use. They can provide antigenic peptide sequences for antibody monitoring, and peptide substrates for enzyme detection.

4.1.8 Advantages of Peptide Technologies and Prospects for Their Development Peptides as chemical products possess many advantages over protein sensors based on ligand-binding proteins, enzymes or antibodies that can be discussed below. They are readily obtained in large amounts by chemical synthesis. The introduction of fluorescent dye can be a part of this synthesis, but not only as the modification at targeted sites, as in proteins (Pazos et al. 2009). The main advantage of the peptidebased approach is the possibility of obtaining the sensing molecules of the minimum possible size. This allows a large-scale application of standard solid-phase techniques of peptide synthesis, which is a rather simple standard technology. We observe that two methods of functional peptide design and production, chemical synthesis and phage display, enrich each other (Uchiyama et al. 2005). The best binders selected from combinatorial libraries can be produced by chemical synthesis on a large-scale level. The synthesis can be robotic. It can be provided directly on arrays to yield the product with highly reproducible properties. In comparison, the large-scale production of mutant recombinant proteins and their subsequent modification are tedious and very expensive. With peptides, all the problems of poor expression levels of the mutated proteins, deleterious effect on binding affinity of the mutation or of fluorophore coupling (or low yield of this coupling) are avoided. Synthetic peptides possess a better thermal and chemical stability than the proteins. They can be easier integrated into nanoparticles, porous materials and polymer gels (Jeong et al. 2018), or deposited in array format for the simultaneous detection of many targets (Kodadek 2002). These technologies are expected to combine the low cost, speed and convenience, with a wide range of applications in diagnosis and the environment protection (Liu et al. 2015). Being located on the

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surface of fluorescent nanoparticles, they are actively used in fluorescence imaging (Jeong et al. 2018; Zhang et al. 2018).

4.2 Sensors Based on Protein-Based Display Scaffolds Now we address a broad class of proteins possessing important functions of binding and transport of different substances along their metabolic routes. Their structures are very diverse. Many protein–protein complexes with multivalent interactions can be found in protein database (Meyer and Knapp 2014). Some of them are almost ready biosensors, and only the fluorescence response units are needed for incorporation into them. Some others can be used as the scaffolds for generation of sensors with new functions. An increased understanding of structure and function of natural ligandbinding proteins together with the advances in protein engineering has also triggered the exploration of various alternative protein architectures. With these tools, valuable protein-binding molecular scaffolds have been obtained. They represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications (Banta et al. 2013; Binz and Pluckthun 2005). Regarding sensor applications, their strong competition with antibodies is expected in the nearest future.

4.2.1 Engineering the Binding Sites by Mutations There are several techniques that can be efficiently applied for generation of new proteins with desired ligand-binding properties. The most efficient of them start from well-known and almost-optimal protein structures that can be taken as scaffolds. Then sequential steps of random mutations and product selection are taken. Mutations are induced in specific regions of protein structure, usually at the ligand-binding pockets. The frequently applied procedure involves display the mutated proteins on the surface of filamentous bacteriophage, the virus that can infect only the bacteria (Uchiyama et al. 2005). Mutations can be induced by standard molecular biology procedures. The phage display technique allows for in vitro mutagenesis-selection. The multiple steps of growth phage-infected bacteria are used to amplify the promising binders and to provide their further selection. This process is often described as ‘in vitro evolution’. It can be especially good for finding the affinity binders for relatively small molecules of metabolites and drugs. These modern methods of molecular biology allow creating huge combinatorial libraries containing millions of structurally diverse species. Usually these libraries are so constructed that they consist of underlying constant scaffold and randomized variable regions that differ from each other. Why then not to synthesize the library of binding proteins de novo, since there are all necessary means for that? The reason is that fully synthetic proteins usually do not fold to compact native-like structures

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(see above) and, if they fold, they possess insufficiently low stability. Therefore it is reasonable to follow the practical trend—to select the structure of a known protein as scaffold and to provide targeted or random mutations only at particular sites (Glasner et al. 2007). The scaffold is usually based on the most rigid elements of protein structure formed by α-helices and β-sheets. These basic structures were assembled from protein data bank (PDB), see Fig. 4.5. According to (Hosse et al. 2006), scaffold proteins can be assigned to one of three groups based on architecture of their backbone: (1) Scaffolds consisting of α-helices (images a–d); (2) Small scaffolds with few secondary structures or an irregular architecture of α-helices and β—sheets (image e), and (3) Predominantly β—sheet scaffolds, representing the majority of proteins used for library display (images f–i).

Fig. 4.5 Representative protein display scaffolds that can be selected for grafting the functional recognition units and library construction of specific molecular recognition binders. Scaffold proteins in a–d consist of α-coils, the small kunitz domain inhibitor (in e) shows an irregular α-coil and β-sheet architecture, whereas f–i show scaffolds predominantly consisting of β-sheet frameworks. α-Helices are depicted in red; β-sheets, in blue; disulfide bonds, in orange; and positions subjected to random or restricted substitutions, in yellow. The PDB IDs used to generate this figure are given in parentheses: a Affibody: Z-domain of protein A (1Q2N), b immunity protein: ImmE7 (1CEI), c cytochrome b562 (1M6T), d repeat-motif protein: ankyrin repeat protein (1SVX), e kunitzdomain inhibitor: Alzheimer’s amyloid b-protein precursor inhibitor (1AAP), f 10th fibronectin type III domain (1FNA), g knottin: cellulose binding domain from cellobiohydrolase Cel7A (1CBH), h carbohydrate binding module CBM4-2 (1K45); and i anticalin FluA: bilin-binding protein (1T0V) with cavity randomization for fluorescein binding

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The scaffolds differ in the presence or the absence of stabilizing disulfide bonds linking spatially separated strands of the protein, a distinction that has consequences for the choice of expression system (Hosse et al. 2006). Various reactive groups extending to the surface can be selected for inducing or modifying particular function, e.g. targeting particular ligand or providing the fluorescence reporter signal. Because of this enormous variety achieved by either rational or combinatorial protein engineering, it is possible to isolate library members binding strongly and specifically virtually to any target. The scaffold principle was in fact borrowed from Nature. Nature implemented it in construction of antibodies (see Sect. 4.4 below). The bodies of these molecules are well-determined and highly homologous, except the so-called variable domains. The latter contain 6 hypervariable loops each, and it is their variability that is responsible for a population of about 108 antibodies of different specificity circulating normally in human body. Therefore, popular among researchers are the β-sandwich and βbarrel scaffolds that resemble the antigen-binding variable domains of antibodies (Binz et al. 2005; Binz and Pluckthun 2005). Remarkable are the properties of knottins (image g in Fig. 4.5). They are small (of 30 to 50 amino acids) proteins with a common folding motif containing cystine knot, in which the formed disulfide bond imparts a high degree of thermal and proteolytic stability (Kintzing and Cochran 2016). Using this scaffold as synthetic products, very stable fluorescent materials can be produced for sensing and imaging. In contrast, the ligand-binding proteins (such as discussed in Sect. 4.3) are very conservative by themselves, and for modulation of their binding properties, the genetic manipulations are required from the researcher. Protein engineering allows insertion of structural elements such as folds or loops and, even more, manipulation with whole domains (Binz et al. 2005), see Fig. 4.6. Thus, proteins with new ligandbinding functions can be engineered through a combinatorial process called random domain insertion (Guntas and Ostermeier 2004). The gene coding domain of one protein can be randomly inserted into the gene sequence of another protein, and this hybrid can show novel not only ligand-binding but also allosteric properties. Design of sensor molecules by random mutations at particular sites has shown its efficiency when applied to proteins with natural binding and transport functions. The results can be compared and even coupled with those obtained by site-directed mutagenesis—amino acid substitutions at pre-defined positions. The latter is the common way of inserting Cys residues that are often needed for labeling with fluorescence reporter dyes that are reactive with –SH groups. Design of such molecular constructs could take lots of skills, especially working with mutant proteins, in which the protein scaffold is stabilized by disulfide bonds. Combination of designed and random-selected protein structures opens far-fetched prospects. Computer design has started to make important contributions to these efforts (Tinberg and Khare 2017).

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Fig. 4.6 Binding-site engineering strategies used with different alternative scaffolds (Binz et al. 2005). In combinatorial engineering approaches, sequences of a scaffold can be diversified at specified positions by means of defined randomized codons (e.g., in loops (a), flat surfaces (b), combinations of loops and helices (c), or cavities (d)), or a random peptide sequence is inserted into the scaffold (e), usually at a loop, or the scaffold sequence is randomized at undefined positions by error-prone PCR (f). Target-binding variants of the resulting libraries are subsequently isolated using selection or screening technologies. In rational engineering approaches, preexisting binding sequences (e.g. loops) have been grafted onto a novel scaffold (g), or binding sites have been engineered de novo into a suitable scaffold (h). The different engineering possibilities are illustrated by alternative binding molecules where the engineering in question has been applied: loop randomization (fibronectin), flat surface randomization (protein Z), loop and helix randomization (ankyrin repeat protein), cavity randomization (lipocalin) random peptide insertion (thioredoxin), error-prone PCR (PDZ domain), loop grafting (neocarzinostatin) and rational design (ribose-binding protein). Many other permutations of randomization strategies and scaffolds are conceivable

4.2.2 Scaffolds Employing Proteins of Lipocalin Family Another class of protein ligand binders, the ‘anticalins’, are the artificial products constructed by introducing structural diversity into the binding site of lipocalins (Skerra 2000), a family of small monomeric ligand-transporting proteins (Weiss and Lowman 2000). Lipocalins are the molecules of 160–180 amino acid residues that are involved in storage of hydrophobic and/or chemically sensitive organic compounds (Flower et al. 2000). They consist of β-barrel formed of eight anti-parallel strands, which is the central folding unit forming a conical cavity. The cavity is relatively deep and largely nonpolar. The entrance to this cavity is formed by four loops that can be randomly mutated for generating of molecular pockets with a diversity of shapes and providing the binding sites to different ligands (Fig. 4.7). The first anticalins were derived from insect origin. They allowed obtaining sensors for small molecules such as steroids (Korndorfer et al. 2003). In addition

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Fig. 4.7 Anticalins. a The general structure. Like immunoglobulins they comprise a structurally conserved framework (grey) formed of a robust β-barrel fold supporting a set of hypervariable loops (colored) that form a cup-like binding site accounting for target recognition. b The location of small hydrophobic target in a lipocalin binding pocket

to low molecular weight compounds, they can be targeted towards proteins. This was shown for a member of the lipocalin family of human origin, apolipoprotein D. Its function is to transport arachidonic acid and progesterone in various body fluids. After randomization of 24 amino acids located within the loop region, a mutant was selected that started to bind hemoglobin (Vogt and Skerra 2004). Anticalins as the binders, offer some advantages over traditional antibodies (Richter et al. 2014; Schiefner and Skerra, 2015). They are especially important in molecular recognition between receptors and small molecule ligands (Weiss and Lowman 2000). As molecular sensors, their application presently limited, though some interesting results have been presented. Their decoration with boronic acid derivatives makes them efficient sensors for sugars (Sommer et al. 2020) and their fused forms with fluorescent protein for sensing tumor biomarkers (Eggenstein et al. 2019). Still, the proper large-scale applications in fluorescence sensing technologies are still in prospect. These technologies should be adapted to small size of these proteins, to the rather rigid conformation of their binding sites and to the absence of global conformational changes upon ligand binding.

4.2.3 Other Protein Scaffolds In addition to scaffolds based on fragments of antibodies, periplasmic ligand-binding proteins and lipocalins, other protein scaffolds were suggested to create the libraries of different binding proteins (Boersma and Plückthun 2011; Skerra 2007). They were developed with different purposes and focused on mostly pharmaceutical applications. But some of them may be attractive as potential sensors. It can be observed, that if the scaffold is based on the protein that binds small ligands, then it is easier

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to achieve specific binding of such ligands. In other cases, the scaffolds are better adapted to large ligands such as proteins (Hosse et al. 2006). If the three-dimensional structure of a target and a scaffold are known, the mutation sites can be assigned based on computational design (Wiederstein and Sippl 2005). A variety of protein scaffolds can be analyzed in silico by generation of computer images and the results of this analysis fit the experimental data satisfactorily. Among the prospective sensors are the ‘affibodies’ (Justino et al. 2015). Derived from bacterial cell surface receptors, they represent an engineered version (Z domain) of one of the five stable three α-helix bundle domains from antibody-binding region of staphylococcal protein A (Eklund et al. 2002; Renberg et al. 2005). They are small (6 kDa and 58 residues only), highly soluble, do not contain S–S bonds and can be fused on a genetic level with other proteins (Ronnmark et al. 2003). Their small size and the ability of spontaneous folding allowed providing their complete chemical synthesis and assembly on automated peptide synthesizer. Moreover, during this synthetic process the fluorescent dyes and reactive groups for attaching protein to the surface can be incorporated in the desired positions (Engfeldt et al. 2005). Also, a biotin moiety can be introduced in the same way. This allows providing not only the reporting function but also the binding to a required site. Because affibodies inherently bind protein targets, they have found application in constructing protein microarrays (Renberg et al. 2007), but the presently suggested technologies still rely on target labeling or on sandwich format. Meantime, being labeled with fluorescent dyes (Miao et al. 2010) or attached to nanoparticles (Pu et al. 2011), the affibodies have found application for tumor imaging. Thus, we observe that the concept of ‘minimal’ protein scaffolds together with the idea of artificial target recognition sites led to many successful developments. Members of several protein families represent promising model systems in this respect. Other examples of ‘minimal’ protein scaffolds include mutated forms of cytochrome b562, ankyrin repeat domains (images c and d in Fig. 4.5, correspondingly), leucine-rich repeat proteins, insect defensin A, protease inhibitors and scorpion toxins (Binz and Pluckthun 2005; Hosse et al. 2006). Small size of these proteins allows an efficient use of computer design and point mutation approach for improving stability and functionality. For scaffold derived from defensin A containing 29 amino acids, a library of such mutations was produced (Yang et al. 2003). Twenty different types of non-immunoglobulin scaffolds were counted already (Škrlec et al. 2015), and their number steadily increases. In order to find scaffolds for new sensors, the scientists started active studies of the proteins that provide immune response in primitive organism (Binz et al. 2005). It is known that not all adaptive immune systems use the immunoglobulin fold as the basis for specific recognition molecules. Sea lampreys, for example, have evolved an adaptive immune system that is based on leucine-rich repeat proteins. Many other proteins, not necessarily involved in adaptive immunity, mediate specific high-affinity interactions. Their transformation into operative fluorescence sensors is the task for future research. Summarizing, we observe that different protein families unrelated to immunoglobulins can provide the basis for specific recognition molecules. The fact that they can

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compete in many valuable properties has stimulated active research. For adaptation to particular target, an approach based on using the protein structure or its basic element as a scaffold and applying the point and random mutations to provide the changes at particular sites can be efficient. Meantime, the examples presented above show that unlimited possibilities offered by the techniques of random and directed mutations are not enough for making optimal binders, and the knowledge of molecular interactions and of their dynamics is needed. A rigid scaffold should provide an extraordinary stable protein architecture tolerating multiple substitutions or insertions at the primary structural level. These substitutions should determine affinity and specificity in target binding. Their prospect will depend strongly on other mechanisms of signal transduction involving those based on direct contact of the target with reporter dye.

4.3 Natural Ligand-Binding Proteins and Their Modifications The proteins, the normal function of which is the binding and transporting of specific ligands, can be seen as the almost ready sensors for these ligands. Such proteins were found primarily among the periplasmic ligand-binding proteins in bacteria. By generation of their mutated forms and providing chemical modifications, their selectivity to particular targets can be modulated (Badilla et al. 2018; Edwards et al. 2016; Ko et al. 2017). We will also discuss the potential of animal ligand-transporting proteins, the serum albumins.

4.3.1 Bacterial Periplasmic Binding Protein (PBP) Scaffolds In line with their function, the transport proteins are highly specific towards their ligands, allowing applications for sensing these ligands as the targets. The periplasmic binding proteins (PBPs) of bacteria are the leaders in this sensing strategy. These structurally diverse transport proteins demonstrate high affinity in binding of their specific ligands, such as maltose, glucose, glutamine, histidine, phosphate, etc. High-resolution crystallographic structures of the ligand-free and ligandbound forms showed that PBPs are formed of two domains linked by a hinge and that a hinge-bending motion occurs upon ligand binding (Quiocho and Ledvina 1996), see Fig. 4.8. The diversity of biological function, ligand binding, conformational changes and structural adaptability of these proteins have been exploited to engineer biosensors, allosteric control elements, biologically active receptors and enzymes using a combination of techniques, including computational design. They demonstrate different mechanisms of coupling the conformational changes with ligand binding. Thus, the histidine binding protein adopts the conformational selection mechanism,

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in which a pre-equilibrium is established between the open and the closed states with the ligand binding to the closed state. Despite being structurally similar, the maltose binding protein adopts the induced fit mechanism, in which the ligand binds to the open state and induces a transition to the closed state (Jayanthi et al. 2020). PBPs and their ligands have been used as models systems to develop fluorescent sensors based on various signal transduction principles (Dwyer and Hellinga 2004). Since the ligand binding site and the fluorescence reporter group may be located in spatially distant areas, these proteins are appropriate for designing the sensors with new ligand-binding specificities but sharing similar reporting functions (de Lorimier et al. 2002; Marvin and Hellinga 2001b). Computational methods for redesigning specific ligand binding of proteins develop actively, so that based on these findings the mutant binding proteins can be constructed by protein engineering methods (Looger et al. 2003; Tinberg and Khare 2017). With an environmentally sensitive fluorophore inserted in the hinge between the two domains, they demonstrate a specific response to the bound targets. Maltose binding protein of E. coli is so far the most efficiently used protein of this family (Medintz and Deschamps 2006). It allows combining two important biosensor properties: specificity of recognition and conformational change. The protein molecule of ~3 × 4 × 6.5 nm in dimension consists of two domains of almost equal size (see Fig. 4.8a). In open form, the binding pocket is exposed to the solvent. Upon binding maltose, this pocket closes by rotation of domains by ~35° and lateral twist by ~8° relative to each other. This brings amino- and carboxy-termini closer by

Fig. 4.8 Schematic illustrations showing the response of PBPs on ligand binding by conformation change and the principle of design of fluorescent biosensors. a Maltose binding protein (Marvin et al. 1997; Marvin and Hellinga 1998). b Phosphate binding protein (Brune et al. 1994) change from an apo state (left) to a liganded state (right) by a bending-twisting motion of the N domain (green) and C domain (blue) about the hinge region (cyan). The fluorescent reporter group (orange) monitors the ligand binding by changing the parameters of its fluorescence emission

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~0.7 nm. The intra-domain conformation also exhibits some changes. This allows exploring both contact and remote response to target binding (Dwyer and Hellinga 2004).

4.3.2 Engineering PBPs Binding Sites and Response of Environment-Sensitive Dyes There were many successful attempts to transform PBPs into sensors by binding the environmentally sensitive dyes (Fonin et al. 2014; Khan and Pickup 2013; Pickup et al. 2013). Initially, in the maltose binding protein a position close to the cleft, where the ligand binds but is not involved in binding itself, was visually selected (Gilardi et al. 1994). It was shown that on the binding of maltose, the fluorescence intensity of acrylodan and IANBD dyes increased dramatically. These changes were accompanied by blue shifts of emission spectra. They can be explained by re-location of the dye from highly polar and exposed to solvent water environment to an environment that is low-polar and screened from the contact with water. Similar effect was observed in phosphate binding protein that was suggested as a sensor for inorganic phosphate (Pi ) (Brune et al. 1994). Upon Pi binding the fluorescence spectrum of this label shifts to the blue and undergoes a 5.2-fold increase of emission intensity. The response is very fast, on the time scale of 50 ms. The possibility to locate the environment-sensitive fluorescence reporter in a position remote from the ligand binding site was thoroughly exploited. Such locations that show the largest structural differences in the ligand-free and ligand-bound forms were identified by comparing the inter-atomic distances in the two forms (Marvin et al. 1997). Spatial separation of the binding site and reporter groups allows their intrinsic properties to be manipulated independently. In the cited research, three different dyes were coupled at six positions, yielding 18 different constructs. Only three out of 18 constructs showed a larger than twofold increase in fluorescence intensity. Provided allosteric linkage is maintained, the ligand binding can therefore be altered without affecting fluorescence reporting. To demonstrate applicability to biosensor technology, the authors introduced a series of point mutations in the maltose-binding site that lower the affinity of the protein for its ligand. These mutant proteins were combined in a composite biosensor capable of measuring the substrate concentration within 5% accuracy over a concentration range spanning five orders of magnitude. A successful attempt to modulate the binding affinity of maltose binding protein that does not involve the binding site was demonstrated. This can be done by introducing mutations located at some distance from the ligand binding pocket that sterically affect the equilibrium between an open, apo-state and a closed, ligand-bound state (Marvin and Hellinga 2001b). The possibility to radically change the specificity of this protein was demonstrated by converting it into a zinc sensor using a targeted design approach (Marvin and Hellinga 2001a). In this new molecular sensor,

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zinc binding is detected in the form of fluorescence signal by use of an engineered conformational coupling mechanism linking the ligand binding to reporter group response. Glucose binding protein was also the subject of extensive studies. The ‘nonallosteric’ and ‘allosteric’ locations of amino acid residues were selected based on the known protein structures in the open and closed forms. These sites were substituted with cysteines for attachment of environment-sensitive acrylodan and NBD dyes. Allosterically located dyes behaved efficiently, demonstrating several-fold change of fluorescence signal intensity (Marvin and Hellinga 1998). Strong changes in fluorescence intensity of ‘allosterically’ located acrylodan dye were also observed in glutamate binding protein (Tolosa et al. 2003). In an extended study (de Lorimier et al. 2002), different environmentally sensitive fluorophores have been conjugated at various positions in eleven members of the PBP family. They selected the positions that either directly contact with the ligand, are located near the ligand-binding site, or are located away from the binding site, in a region that changes conformation on ligand binding. Approximately a quarter of the 320 conjugates gave a satisfactory sensor response. Binding affinities were mostly affected in the group of constructs where the fluorophores contacted directly with the ligand. Wavelength shifts together with the changes of intensity were observed for the environment sensitive dyes, such as acrylodan and NBD. Meantime, for some of labeled PBPs these changes were very little or even undetected, showing that the dye response is strongly position-dependent. The problem of coupling with fluorescence reporter response should be properly addressed and resolved. Protein–ligand binding events usually involve in different proportions the combination of binding pocket solvent exclusion (Liu et al. 2005) and conformational change (Flores et al. 2006). Therefore, the coupling of binding events with conformational changes cannot be a general mechanism of these sensors’ operation.

4.3.3 Serum Albumins Serum albumin is the principal extracellular protein of the circulatory system of animals. It accounts for about 60% of the total plasma proteins and is mostly responsive for the colloid osmotic pressure of blood. Human serum albumin (HSA) is the most studied serum albumin because of its easy availability and high importance for pharmacology. It is known as the fatty acid carrier and is functioning as the transporter, modulator, inactivator of different metabolites and drugs. It also acts as a protective device in binding and inactivation of potentially toxic compounds, to which the body is exposed. Its amino acid sequence consisting of 585 amino acid residues is well known, and its tertiary structure has been determined by X-ray crystallography. It is a single-chain, non-glycosylated globular protein, with 17 disulfide bridges that assist in maintaining its familiar heart-like shape (Fig. 4.9).

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Fig. 4.9 Summary of the ligand binding capacity of HSA as defined by crystallographic studies (Ghuman et al. 2005). Ligands are depicted in space-filling representation; oxygen atoms are coloured red; all other atoms in fatty acids (myristic acid), other endogenous ligands (hemin, thyroxin) and drugs are coloured dark-grey, light grey and orange, respectively

Crystallographic data show that HSA contains three homologous a-helical domains (I, II, and III), each of which includes 10 helices that are divided into sixhelix and four-helix subdomains (He and Carter 1992). A multitude of ligand-binding sites are scattered over the entire protein (Ghuman et al. 2005). These multiple sites underline an exceptional ability of HSA to act as a major depot and transport protein, capable of binding, transporting in the bloodstream and delivering to the target organs an extraordinarily diverse range of endogenous and exogenous compounds. Many of them are low-polar compounds containing also negative charge, but not only them, as it is seen in Fig. 4.9. HSA readily binds different fluorescent dyes, demonstrating high affinity (Kd ~10–6 –10–7 M) and often substituting the drugs, such as warfarin or ibuprofen, from their binding sites (see Fig. 2.8). Their binding sites are rigid and hydrophobic (Ercelen et al. 2003). These properties are variable among serum albumins obtained from different species (Ercelen et al. 2005). The dyes are actively used for determining albumin in different physiological fluids, which is of diagnostical value (see

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Chap. 12). However, in sensor applications the albumin is not used extensively, just because of its promiscuity, the ability to bind different targets and in different sites. It turns-out, however, that the knowledge on HSA binding of different drugs is of utmost importance as it affects their bioavailability and pharmacokinetic behavior. This fact stimulates developing high-throughput screening methods, and it is thought that the fluorescent probe displacement assays because they are highly flexible and amenable to high-throughput screening, are easily scaling up to handle large compound libraries. Recent developments in fluorescence technologies, such as homogenous time-resolved fluorescence, have improved the sensitivity of these assays (Ronzetti et al. 2018). The strong efforts for synthesis of fluorescent dyes mapping the specific binding sites on albumin molecule with subsequent use of probe displacement assays for drugs (Er et al. 2013) are observed.

4.4 Antibodies and Their Recombinant Fragments The antibodies (Abs), the leading players in sensing technologies, are borrowed from nature, where they form the mechanisms of defense of the bodies against the strange and dangerous compounds and cells. They are the powerful recognition tools used in many sensing assays, called immunoassays. They can be developed to bind the target compounds (antigens) of any size, starting from steroids, oligosaccharides and oligopeptides. Very selectively, they target different cells. Usually in sensing applications, the antibodies of immunoglobulin G (IgG) type are used. They are globular glycoproteins with molecular mass of 150,000– 160,000 Da formed of two light (L) chains and two heavy (H) chains joined to form a Y-shaped molecule (Gopinath et al. 2014). This molecule is composed of β-structured domains stabilized by disulfide bonds (see Fig. 4.8). Their antigen (Ag) binding sites are formed of six loops contributed by two different so-called variable domains (called VL and VH ). Because the IgG antibodies contain two VL –VH pairs, they are bivalent: they can react with two antigenic binding sites simultaneously. Specificity to any target is determined by the amino acid composition of these variable domains. Variability of this composition is enormous, it is responsible for the diversity of the antibody repertoire (Fig. 4.10). Antibodies are used extensively as diagnostic tools in a wide array of different analyses (Gopinath et al. 2014). Because of their unique diversity, they provide a never-ending source of molecules with unlimited possibilities for target detection. Antibodies are very efficient in detecting proteins. They recognize regions of the protein surface called antigenic determinants or epitopes. The contact area between protein and bound antibody is relatively large (600–950 Å2 ). The contact surfaces exhibit a high level of steric and physico-chemical complementarity.

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Fig. 4.10 The schematic structure of an IgG type of antibody molecule. The heavy chains (blue) and light chains (pink) form Fab and Fc regions composed of β-structured domains. The VL and VH domains that are present in duplicate are responsible for target antigen recognition. The domains composing the Fc region are usually the sites for covalent labeling with different fluorophores reacting with exposed amino groups

4.4.1 Assay Formats Used for Immunosensing The term ‘immunosensor’ has been applied to various direct or indirect detection assays involving antibodies (immunoassays). Outstanding recognition properties of antibodies allowed realizing these assays in several formats (see Chaps. 1 and 2 of Volume 1). Sandwich immunoassays are the most popular and convenient types of assays that are based on heterogeneous assay principle. Immobilization of receptor molecules (antibodies or their antigens) on solid support, application of the sample and washing steps are required before target detection. The antigen–antibody complex is routinely detected using a secondary antibody specific for the antigen or for the constant domain of antibodies. In enzyme-linked immunosorbent assay (ELISA), the secondary antibody is coupled to an enzyme, allowing to achieve high detection sensitivity due to enzymatic amplification of light absorption or fluorescence signal. Fluorescence polarization immunoassays. Detection in the change of polarization (or anisotropy) of fluorescence emission is one of the most frequently used procedures in sensing with antibodies and is the basis of many commercially available test systems. If the target molecules are not fluorescent, then a competitive assay format is applied. Here the target molecules compete for the Ab binding sites with target analogs coupled with fluorescent dyes and the dye senses the rotational mobility.

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The testing can be made when the antibody is free in solution, but it can be also immobilized on solid support. Time-resolved and time-gated immunoassays. These assays and sensors usually use labeled antibodies or antigen analogs (for competitive assays) with chelating complexes of europium(III) or terbium(III) ions possessing lifetimes typically from 0.01 to 1 s. This allows increasing dramatically the sensitivity, suppressing background emission and avoiding the problems with light-scattering. Excitation energy transfer immunoassays. They are commonly performed when the antibody is labeled with EET donor and the target or competitor is the EET acceptor (quencher). The physical principles and experimental techniques that are in background of these assays are outlined in Chap. 3 of Volume 1 of this book. It may be often attractive to construct a direct sensor by obtaining the response signal from primary interaction of antibody and target antigen in a way that avoids double labeling as in FRET or complicated instrumentation as in polarization or time-resolved immunoassays. But this is not easy in view that the antigen binding is not associated with large conformational changes, so the signal transduction via conformational change for providing the reporter signal may not be efficient. Therefore, a contact sensing using environmentally sensitive covalently attached reporters located in proximity to the antigen-binding site must be used. This can be achieved by introducing at the desired Ab location a reactive group, such as –SH groups of Cys residues, for coupling the reporter dye, which can be done by recombinant techniques. The difficulty lies in the choice of a coupling site. In order to find the best solution for this problem the scientists follow two general strategies. (a) The reporter dye should be located at the periphery of the binding site. It should not interfere with the interaction between the receptor and the target, but its micro-environment should change in response to Ab-target complex formation (Renard et al. 2002). (b) The reporter group should be located within the binding site in such way that it becomes one of the key participants in antigen binding (Jespers et al. 2004). In a latter case, the reporter molecule can be chemically linked to a hypervariable loop of an antibody repertoire displayed on phage, and this repertoire can be selected for antigen binding. The fluorescence of the probe has to respond quantitatively to antigen binding. Recently, the prototypes of “quenchbodies” have been suggested (Ueda and Dong 2014). They work on the principle of fluorescence quenching of attached dye and its antigen-dependent release. Because of lack of sufficient data, it is too early to provide comparative analysis of these two strategies. In experiments when the dye was coupled to an antibody fragment (single chain variable fragment, scFv), seventeen sites were selected for the coupling of responsive dye (Renard et al. 2002). Being located at the periphery of binding site, only few of them provided fluorescence response to the presence of antigen (lysozyme). A much stronger response as a substantial decrease of fluorescence intensity was observed by applying the second strategy (Jespers et al. 2004).

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Thus, the possibility for designing semi-synthetic antibody-based sensors with fully integrated reporter dyes exists and it should be explored in future research.

4.4.2 The Types of Antibodies and Their Fragments Used in Sensing Several types of antibodies and antibody fragments can be used in sensing technologies (Goodchild et al. 2006). Polyclonal antibodies are purified from sera of immunized (exposed to antigens) animals, so that their specificity is developed in the animal bodies in response to compounds that are used for their immunization. Since the blood serum contains also circulating antibodies with varying specificities and affinities, for obtaining the high-affinity antibodies specific for a given target, the purification and fractionation steps are needed. Structurally homogeneous monoclonal antibodies (mAbs) (Sharma et al. 2016) are produced by hybridoma cell line clones. Here the problem of achieving high affinity and selectivity is solved by exposing the animals (usually rabbits or mice) to antigens. Then the mAb-producing cells that are raised in their bodies in response to that treatment are selected. Individual mAb-producing animal cells are known to synthesize antibodies of uniform structure, so when they are fused with myeloma cells that are capable of continuous propagation, they produce identical antibody molecules. These hybrid cells (hybridomas) can be grown for months and years. Their cultivation is already a part of industry allowing large-scale antibody production. Recombinant antibody fragments are attractive for sensing technologies because they allow to decrease the size of sensor molecule and also to include substitutions in their primary structures that can facilitate their attachment to surfaces or carriers (including nanoparticles). These smaller fragments can be of different size, retaining the ability to recognize the target. They can be classic monovalent antibody fragments (Fab, scFv) and also the engineered variants (diabodies, triabodies, minibodies, single-domain antibodies, etc.), see Fig. 4.1. Retaining the targeting specificity of whole mAbs, they can be produced more economically and possess other unique and superior properties for a range of diagnostic and therapeutic applications (Bates and Power 2019; Bruce et al. 2016; Holliger and Hudson 2005). Antibody fragments with desired binding specificity can be selected from libraries constructed from repertoires of antibody V genes, bypassing hybridoma technology and even immunization. The V gene repertoires are harvested from populations of lymphocytes, or are assembled in vitro. They are cloned for display the fragment on the surface of filamentous bacteriophage. Phages that carry the fragments with desired specificity are selected from the repertoire by panning on antigen; soluble antibody fragments are expressed from infected bacteria; and the affinity of binding selected antibodies is improved by mutation. The process mimics immune selection, and antibody fragments with many different binding specificities have been isolated from the same phage repertoire. The probability of identifying a high affinity binder increases

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with library size (Hust and Dubel 2004), which for phage-displayed libraries ranges between 107 and 1010 clones (Azzazy and Highsmith 2002). Short heavy-chain fragments, “domain antibodies” are special. From Fig. 4.11 attentive reader may have noticed that the camel Ig contains only heavy chains. Occurring naturally in camels, the ‘heavy chain’ antibodies are now produced in fully humanized form. Domain antibodies (dAbs) shown as VhH domains are the smallest known antigen-binding fragments of antibodies, ranging from 11 to 15 kDa and being 2–4 nm in size. Because of such small dimensions, they got the name nanobodies (Salema and Fernández 2017). When they are expressed in microbial cell culture, they show favorable biophysical properties including solubility and temperature stability. They are well suited for selection and affinity maturation by in vitro selection systems such as phage display. The chemical synthesis can be employed for their production (Haußner et al. 2017). dAbs are the monomers and, owing to their small size and inherent stability, they can be formatted into larger molecules to create drugs with prolonged serum half-lives or other pharmacological activities. They bind to targets with high affinity and specificity (Bruce and McNaughton 2017; Muyldermans 2001). Groups that can be used for coupling of fluorescence reporters (particularly, –SH groups) can also be introduced on the synthetic steps. Commonly, the antibody fragments retain their specific activity on these modifications. Additional advantages of nanobodies over classical antibodies for designing materials for sensing and imaging are obvious. They have a single binding site and therefore they cannot produce cross-linking and artificial clustering (Sograte-Idrissi et al. 2020). Whereas the stoichiometric labeling of full-length antibodies is challenging, the fluorescent labeling of a nanobody is regularly achieved stoichiometrically at well-determined sites (Grußmayer et al. 2014; Massa et al. 2016).

Fig. 4.11 Schematic representation of different antibody formats, showing intact ‘classic’ immunoglobulins IgG (highlighted) alongside camelid VhH-Ig and shark Ig-NAR molecules (Holliger and Hudson 2005). Camelid VhH-Ig and shark Ig-NARs are unusual immunoglobulin-like structures comprising a homodimeric pair of two chains of V-like and C-like domains (neither has a light chain), in which the displayed V domains bind target independently. Shark Ig-NARs comprise a homodimer of one variable domain (V-NAR) and five C-like constant domains (C-NAR). A variety of antibody fragments are depicted, including Fab, scFv, single-domain VH, VhH and V-NAR and multimeric formats, such as minibodies, bis-scFv, diabodies, triabodies, tetrabodies and chemically conjugated Fab multimers (sizes given in kilodaltons are approximate)

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4.4.3 Prospects for Antibody Technologies Analyzing the application of antibodies as recognition elements of sensors, we obtained very important lessons taught us by Nature. In living body, the generation and synthesis of these high-affinity binders occur via selection-amplification steps starting from genetically available potentially innumerous library. The random mutation-selection principle can also be applied in vitro for creation of libraries of engineered antibodies and their fragments. This allows producing the recognition units to non-immunogenic or toxic molecules. Despite significant technical difficulties, academic and commercial laboratories have now developed large (more than 1010 variants) phage display antibody libraries, from which diverse sub-nanomolar affinity antibody clones can be isolated (Edwards et al. 2003). Possessing the tools for selection and amplified synthesis of optimal binders, one can solve the problem of coupling the binding with fluorescence reporter and try to choose proper sensor format. Despite several promising attempts, direct sensors with the properly attached environment-sensitive dyes still did not find their proper application (Ueda and Dong 2014). With solution of this problem, antibodies will be able to establish themselves as preferred recognition units in all biosensor technologies, including highly needed microarrays for the detection of carbohydrates and proteins, extending to a proteome scale (Sauer 2017). Depicted in Fig. 4.12 is the “old-fashioned” sandwich assay technology. After molecular recognition of the target molecule, such microarrays need a) an additional detection step (e.g., adding labeled detection antibodies) and often b) a separation step (washing off unbound material). The Ig fragments, such as nanobodies, with properly located appended dye responding directly may become a future-directed solution. A great potential for the development in cellular research is expected from presently almost unexplored possibility of fusing antibodies to different marker proteins (Casadei et al. 1990) and to fluorescent proteins (Ries et al. 2012). Obtaining such molecular hybrids may allow combining two important functions—targeted binding at particular sites and sensing different analytes at these sites. Exploring the concept of “on-chip immunoassays”, so that every spot in a microarray can be seen as a reaction chamber for a biosensor, should get much broader vision and application. Regarding the replacement of antibodies by artificial affinity molecules, it is a clearly observed trend. The multiplexed suspension microarrays based on optically encoded microbeads (Vafajoo et al. 2018) is getting new stimulus for development. Addressing this demand and also the request for cellular recognition, imaging and drug cargo delivery, the functional nanoparticles become efficiently decorated with peptide and protein recognition units (Spicer et al. 2018). Here the antibody fragments are expected to play a leading role (Richards et al. 2017).

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Fig. 4.12. Components of protein microarray production and processing. Work flow from left to right: Preparation of the immobilization surface, probe preparation, printing, sample preparation, assay design, instrumentation, read-out and data analysis (Sauer 2017)

4.5 Sensing and Thinking. The Application Range and Benefit from Peptide and Protein Sensors The immense possibilities for proteins and peptides to form their molecular recognition sites are based on two important factors. One is exploring the combination of 20 different types of amino acids plus their modified forms to generate in the recognition sites the optimal patterns of charge distribution and hydrophobicity. The other is the ability to produce and to support this pattern by conformational variables creating proper configurations of polypeptide chains in the folded structures. The researchers follow several strategies for their design. (1) For particular target, we design the sensor searching and using the structural information obtained. The skills in protein engineering are often needed to optimize the fitting with the target. (2) We hook-out the complexes of target or target analogs from the library of peptide or protein binders existing in nature or reproduced in natural source. (3) We create the library of potential binders artificially using the property of a chosen biological system to create it with millions of potential candidates for the most efficient binders and then provide the selection. In the sensor development, the applications of two principles, rational design and combinatorial library selection, that nay involve conformational plasticity, complement each other. Combination of rigid scaffolds with ‘rationally’ located flexible

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recognition units and fluorescence reporters allows achieving highly selective binding and efficient reporting together with the possibility of fine-tuning the affinity to the desired range of target concentrations. In this respect, biopolymers such as proteins, peptides and nucleic acids often demonstrate the necessary flexibility for forming the interaction sites with a huge number of potential targets. In order to check the acquired knowledge, the reader can respond to the following questions: 1.

Explain the two methods of obtaining the high-affinity binders in the design and production of smart peptides. What is the disadvantage in comparison with oligonucleotide binders (Chap. 5)? 2. What are the advantages of synthetic peptides over proteins in sensing technologies? 3. Why self-assembly of peptides into rigid scaffolds may become needed? 4. Compare the affinities to a hypothetical rigid target of a flexible tetrapeptide Leu-Leu-Leu-Asp and of the same sequence incorporated into a rigid structure. Use formulas from Chap. 2 and the rough estimates of free energy change of 2– 3 kJ/mol for the formation of salt bridge, 10 kJ/mol for hydrophobic interaction between amino acids and 3–4 kJ/mol as an entropy penalty for suppression of rotation around a single bond. 5. In which way the fluorescence reporter can be incorporated into peptide sensor? What could be the mechanism of its response? 6. How antimicrobial peptides can be used in sensing? 7. How the protein display scaffolds are formed? Why the scaffolds derived from real functional proteins are the most efficient? 8. Outline the range of applications of sensors based on natural ligand-binding proteins. How the principle of combined flexibility and rigidity is realized in ligand-binding proteins? How the fluorescence reporter can be incorporated in this case? 9. Explain the structure of a typical IgG antibody. How flexibility and rigidity of its structure co-participate in realizing the binding function? 10. Provide comparison of several types of immunoassays noted above in terms of simplicity of design and applicability in microarray technologies. 11. Why antibody fragments become needed? Explain how they can be obtained.

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Haußner C, Lach J, Eichler J (2017) Synthetic antibody mimics for the inhibition of protein–ligand interactions. Curr Opin Chem Biol 40:72–77 He XM, Carter DC (1992) Atomic structure and chemistry of human serum albumin. Nature 358:209–215 Holliger P, Hudson PJ (2005) Engineered antibody fragments and the rise of single domains. Nat Biotechnol 23:1126–1136 Hosse RJ, Rothe A, Power BE (2006) A new generation of protein display scaffolds for molecular recognition. Protein Sci 15:14–27 Hoyos-Nogués M, Gil F, Mas-Moruno C (2018) Antimicrobial peptides: powerful biorecognition elements to detect bacteria in biosensing technologies. Molecules 23:1683 Hust M, Dubel S (2004) Mating antibody phage display with proteomics. Trends Biotechnol 22:8–14 Jayanthi LP, Mascarenhas NM, Gosavi S (2020) Structure dictates the mechanism of ligand recognition in the histidine and maltose binding proteins. Curr Res Struct Biol 2:180–190 Jeong W-j, Bu J, Kubiatowicz LJ, Chen SS, Kim Y, Hong S (2018) Peptide–nanoparticle conjugates: a next generation of diagnostic and therapeutic platforms? Nano Converg 5:38 Jespers L, Bonnert TP, Winter G (2004) Selection of optical biosensors from chemisynthetic antibody libraries. Protein Eng Des Sel 17:709–713 Justino CI, Duarte AC, Rocha-Santos TA (2015) Analytical applications of affibodies. TrAC, Trends Anal Chem 65:73–82 Khan F, Pickup JC (2013) Near-infrared fluorescence glucose sensing based on glucose/galactosebinding protein coupled to 651-Blue Oxazine. Biochem Biophys Res Commun 438:488–492 Kintzing JR, Cochran JR (2016) Engineered knottin peptides as diagnostics, therapeutics, and drug delivery vehicles. Curr Opin Chem Biol 34:143–150 Ko W, Kim S, Lee HS (2017) Engineering a periplasmic binding protein for amino acid sensors with improved binding properties. Org Biomol Chem 15:8761–8769 Kodadek T (2002) Development of protein-detecting microarrays and related devices. Trends Biochem Sci 27:295–300 Korndorfer IP, Schlehuber S, Skerra A (2003) Structural mechanism of specific ligand recognition by a lipocalin tailored for the complexation of digoxigenin. J Mol Biol 330:385–396 Kulagina NV, Shaffer KM, Anderson GP, Ligler FS, Taitt CR (2006) Antimicrobial peptide-based array for Escherichia coli and Salmonella screening. Anal Chim Acta 575:9–15 Levin AM, Weiss GA (2006) Optimizing the affinity and specificity of proteins with molecular display. Mol BioSyst 2:49–57 Liu Y, Liang P, Chen Y, Zhao YL, Ding F, Yu A (2005) Spectrophotometric study of fluorescence sensing and selective binding of biochemical substrates by 2,2’-bridged biso(beta-cyclodextrin) and its water-soluble fullerene conjugate. J Phys Chem B 109:23739–23744 Liu Q, Wang J, Boyd BJ (2015) Peptide-based biosensors. Talanta 136:114–127 Looger LL, Dwyer MA, Smith JJ, Hellinga HW (2003) Computational design of receptor and sensor proteins with novel functions. Nature 423:185–190 Marvin JS, Hellinga HW (1998) Engineering biosensors by introducing fluorescent allosteric signal transducers: construction of a novel glucose sensor. J Am Chem Soc 120:7–11 Marvin JS, Hellinga HW (2001a) Conversion of a maltose receptor into a zinc biosensor by computational design. Proc Natl Acad Sci U S A 98:4955–4960 Marvin JS, Hellinga HW (2001b) Manipulation of ligand binding affinity by exploitation of conformational coupling. Nat Struct Biol 8:795–798 Marvin JS, Corcoran EE, Hattangadi NA, Zhang JV, Gere SA, Hellinga HW (1997) The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors. Proc Natl Acad Sci USA 94:4366–4371 Massa S, Vikani N, Betti C, Ballet S, Vanderhaegen S, Steyaert J, Descamps B, Vanhove C, Bunschoten A, van Leeuwen FW (2016) Sortase A-mediated site-specific labeling of camelid single-domain antibody-fragments: a versatile strategy for multiple molecular imaging modalities. Contrast Media Mol Imaging 11:328–339

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Chapter 5

Nucleic Acids as Scaffolds and Recognition Units

Nucleic acids, beside their crucial role in all manifestations of life, demonstrate high and rapidly increasing importance in man-made technologies. Molecular sensing is not an exception, and here we comparatively discuss two conceptually different approaches that can be realized with nucleic acids in sensing. One is the molecular recognition based on the base pairing that is realized in hybridization assays for nucleic acids and the other is the ability of DNA and RNA aptamers selected from random libraries to recognize a very broad range of analytes and to be in this function even more efficient than the antibodies. Nucleic acids—DNAs and RNAs—have long been known to be very important molecules in cells: DNA carries genetic information and RNA actively participates in many essential cellular processes, such as protein synthesis and gene regulation. The complementary pairing of nucleic acid bases (nucleobases), which are G-C and A-T in DNAs (or A-U in RNAs), resulting in a formation of a double-strand association complex (hybridization) between the formed nucleotide sequences (Fig. 5.1), was and remains to be in the basis of recognition of nucleic acids, their fragments, nucleotides and their analogs. These statements are very easy to accept by every student familiar with the properties of nucleic acids and in this way to understand the basis of the recognition process in nucleic acid sensors. The highly needed in human genetics detection of single base substitutions is achievable in modern hybridization assays. Here, together with these classical issues, the reader will learn also about new well-unexpected properties of nucleic acids. Being designed and selected by their affinities, they can interact strongly and with high selectivity with a broad variety of structurally unrelated molecular targets. Moreover, these DNA and RNA structures can demonstrate catalytic behavior. There emerged the whole field of functional nucleic acids, i.e. those performing these new functions. The new functional players include sensing aptamers (ligand-binding ssDNAs or RNAs), ribozymes (RNA-based enzymes), DNAzymes or deoxyribozymes (ssDNA-based enzymes), and allosteric nucleic acid enzymes or simply aptazymes (ssDNAs or RNAs that contain both ligand-binding

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Fig. 5.1 Recognition between complementary nucleic acid bases as the basis of detection of detection of DNA and RNA and of their fragments (oligonucleotides), of individual bases and their derivatives (from Wikipedia). The sensor and the target form the Watson–Crick base pairs, where the H-bonds between the bases (shown as the dashed lines) play the critical role in their formation

and catalytic elements). The latter can be arranged in such a way that the catalytic activity is controlled by the target binding. The sensing aptamers together with hybridization assays will be in the major focus of our discussion. They are the single-stranded DNA (ssDNA) or RNA fragments that are efficient molecular recognition elements for a variety of molecular targets. They can be tailored to have high specificity and affinity for a broad range of them, including not only nucleic acids, but also proteins, small molecules and ions. It was recognized that, possessing these properties, they can compete in efficiency with antibodies and other smart protein or peptide binders (discussed in Chap. 4). How can they do that, having only four neutral elements of structure (the DNA-RNA bases) against the compositions of 20 amino acids that allow so broad variation of hydrophobicity and charge? One can divide oligonucleotide-based sensors into two main categories: (a) hybridization probes that are based on the formation of complementary base-pairs, and (b) the aptamer sensors (sometimes called aptasensors) that exploit selective recognition of non-nucleic acid analytes and in this function may be compared with immunosensors. Fluorescence reporting function can be common and based on covalent labeling or noncovalent attachment of reporter dyes, but their application range is quite different. The objective of this chapter is to introduce the oligonucleotidebased sensors with fluorescence reporting as versatile molecular recognition tools to detect, quantify or monitor various important chemical and biological targets.

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5.1 DNA and RNA Fragments in Hybridization-Based Sensing The use of fluorescently-labeled oligonucleotide probes complementary to short regions of full-size nucleic acid targets is probably the most reliable method for the detection and study of the structural features and functions of these regions, their quantification in vitro and visualization in cells. The detection of single nucleotide polymorphisms (SNPs), which are the substitutions of single bases, is extremely important, since they are coupled with different diseases (Kwok and Chen 2003; Syvänen 2001). The proper method of revealing them is the probing of chain fragments for their correct double-helix forming complementarity. In many practical fields that require the detection or monitoring of nucleic acids, the sensing based on hybridization is widely used with the application of a variety of methods. They include the DNA microarrays (chips), detection of gene translocation, monitoring the intracellular mRNA and amplification progress in real-time PCR. Fluorescence in-situ hybridization (FISH) assays allow hybridization on the level of chromosomes (Halling and Kipp 2007; Volpi and Bridger 2008). In all these techniques, the molecular recognition is based on complementary between target and probe bases in their double-helical structures. Rapid progress in genomics, transcriptomics, and epigenomics are driving a strong demand for reliable fluorescencebased tools for studying the structural and conformational polymorphisms of nucleic acids, their variability and internal dynamics. Also important are their interactions with proteins, metabolites, DNA-RNA targeting drugs, water molecules, and ions at sub-molecular and atomic levels (Sinkeldam et al. 2010). Such tools should allow direct molecular recognition between tested nucleic acids and probes that produces an easily recordable and interpretable output signal. A diversity of methods for detection of point mutations is based on the hybridization of fluorescent oligonucleotide probes to its complementary nucleic acid target. They have to generate different fluorescent signals depending on the nature of opposite nucleotides (Michel et al. 2020). In general, the probes for revealing the point mutations are designed to display significant hybridization-induced changes in fluorescence emission when targeted to a fully-matched DNA target and moderate changes or complete quenching of fluorescence upon binding with a mismatched target. Thus, the type and the position of the probes have to provide their different binding modes in nucleic acid duplexes depending on the nature of opposite and neighboring nucleic acid bases.

5.1.1 The Types of Nucleic Acid Recognition Units At the moment, different types of fluorescent dye-labeled oligonucleotides or of those incorporating fluorescent base substitutions, have been reported (Michel et al.

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2020). They display strong hybridization-induced changes and a high degree of selectivity of probes relative to the type of nucleic acid target. Fluorescence responding units are the elements forming the integral structures capable of recognition and reporting functioning. There are several types of target hybridizing materials used in fluorescence sensing and imaging technologies. Single-stranded DNA (ssDNA) fragments are the most popular. They behave as negatively charged polyelectrolytes. They can bind to cationic organic dyes and conjugated polymers (Guo et al. 2016). This binding is nonspecific. Specificity appears only if their bases are used in the recognition. Here two different cases can be realized: the binding of selective sequences to complementary bases (hybridization) and those after selection of binders (aptamers) from their huge libraries. Double-stranded DNA (dsDNA) fragments possess much smaller recognition power and they often play a role in supporting the structures of signaling aptamers (see below). It is interesting that on their basis, the DNA triplex structures can be formed when the third strand recognizes and occupies the major groove of the dsDNA (Hu et al. 2017). Molecular recognition in triplexes is due to the formation of extra hydrogen bonds (Guo et al. 2016). Peptide nucleic acids are usually used for recognition of dsDNA segments (see Chap. 11). The RNA fragments demonstrate more limited application, mostly in the construction of signaling aptamers (Gotrik et al. 2018). The RNA-based nanostructures constitute another interesting field of their exploration as scaffolds and building modules (Jasinski et al. 2017). Peptide nucleic acids (PNAs). These oligomeric structures are the mimics of DNA strands, in which a peptide-like repeat of the (2-aminoethyl)glycine unit replaces the sugar–phosphate backbone (Egholm et al. 1992; Ganesh and Nielsen 2000). PNAs recognize and bind to the complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding DNA fragments. They are neutral, therefore the electrostatic repulsion between complementary DNA chains does not exist. More about PNAs and their application in sensing will be discussed in Chap. 11. Locked nucleic acids (LNAs) are a new class of bicyclic high-affinity DNA analogues. The locked nucleic acids are defined as oligonucleotides containing at least one LNA monomer (Kaur et al. 2007; Singh et al. 1998). The LNA monomer incorporates a natural phosphodiester linkage, but its sugar is conformationally locked by an O2' to C4' methylene linkage. LNAs are capable of recognizing complementary DNA and RNA segments with increased mismatch discrimination compared with natural nucleic acids and, therefore, they have found widespread applications in bioanalysis as effective detection probes. Non-canonical DNA structures include the G-quadruplexes and i-motif DNA folds. They demonstrate increasing popularity in sensing technologies. Formation of these structures is realized in the design of very efficient signaling aptamers (see Sect. 5.3). Molecular beacons are the structures that can be made on the basis of both DNA and RNA platforms. They combine molecular recognition of a complementary base with conformational change in the sensing nucleic acid (Zheng et al. 2015). Their structures resemble hairpins and are also known under this name. They contain a

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segment a double-stranded nucleic acid (stem) that is composed of 5–8 hybridized nucleotides that unfolds upon hybridization with the target. The recognition part is the bent structure (loop) that consists of 15–30 nucleotides. It interacts with the target nucleic acid sequence only on such unfolding. It is not the hybridization but unfolding that generates the fluorescence signal. See Chap. 11 to learn about the structures, dynamics and operation of these very popular molecular sensors.

5.1.2 Fluorescence Reporting in Hybridization Assays The demands for fluorescence reporters in hybridization assays are very stringent. They have to provide precise information on the interaction between tested and sensing sequences on the level of single nucleic acid bases. This information should be well recordable and free from interferences. The base analogue 2-aminopurine is the only one intrinsic fluorophore with emission in the visible (Neely and Jones 2015). However, it does not satisfy many needed requirements, simply because of its severe quenching on interaction with other bases. Therefore, the progress in this area will ultimately depend on the ability of synthetic chemists. They have to rationally design fluorophores with the parameters that are optimal for the detection methods and, at the same time, possess the precisely matching recognition units. In principle, to make DNA or RNA fluorescent, the fluorophores can be introduced in two distinct ways: by either non-covalent attachment or covalent labeling (Guo et al. 2016). The non-covalent attachment uses organic dyes of low molecular weight that bind to the DNA minor grooves (such as Hoechst dyes) or intercalate into the helices (such as ethidium bromide) and respond by increasing of fluorescence emission. Cationic conjugated polymers can also be used in this application. The problem is that the vast majority of these dyes are not sequence-specific (or low specific). When they are non-covalently attached to nucleic acids, they are sensitive to conformational changes (e.g. folding-unfolding) but do not exhibit specific base recognition. Therefore, such fluorophores are mainly used for the visualization of nucleic acids in experimental biology procedures (see Chap. 11). As we will see below, they are efficient reporters in aptamer technologies, including those based on G-quadruplexes. In contrast, covalent labeling allows exact positioning of substituted or modified base. Though technically more difficult, it can result in high specificity of molecular recognition and, therefore, has a broad range of applications in hybridization assays (Davies et al. 2000). The chemical structure of nucleic acids allows for multiple ways of attaching exogenous fluorophores, and numerous and diverse examples of this type of labeling have been described (Michel et al. 2020; Xu et al. 2017). The label can be attached to certain position of the chain via a linker or may substitute one nucleobase (Fig. 5.2). External labeling with classical fluorophores, such as rhodamine, cyanine, and fluorescein dyes, linked to the nucleoside via a flexible spacer arm, is very common. The applied dyes are brightly emitting and they are mainly used for fluorescent nucleic acid detection, for example, in DNA sequencing and, in sensors, as

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Fig. 5.2 Main strategies for covalent fluorescence labeling of nucleic acids: via a flexible tether either at the 3' /5' -end (a), or at an internal position (b) illustrated here by amidite building blocks bearing a fluorescein (Michel et al. 2020)

binary probes when combined with a second partner for Excitation Energy Transfer (EET). This type of labeling of DNA fragments is also used for real time PCR detection, single-nucleotide mutation screening, studying the conformational changes in nucleic acids and their interactions with target proteins. For obtaining productive chromophore pairs, EET requires a complicated, time consuming and expensive double labeling. Therefore, every possibility is used for the introduction of a single dye that can be easier realized. But that dye could provide the reporting function generating the changes in fluorescence. The choice of the proper dye for covalent labeling should be based on the ability to attach it in a proper site not only to a specific nucleic acid sequence, but also to locate it within the structure in a non-perturbing manner. In this way, it is possible to probe both major and minor grooves in the double helices. The fluorescent chemical entities, which are incorporated into the DNA, are usually referred as fluorescent nucleoside analogs (FNAs) (Sinkeldam et al. 2010). FNAs are a group of chemically diverse compounds that often (but not always) share partial deoxyribose moieties with natural nucleosides. A few hundred different FNAs have been described to date (Ivancová and Hocek 2019; Saito and Hudson 2018; Xu et al. 2017). The rapid progress is observed towards the technologies designed to address highly specific interactions in nucleic acid world. From the structural point of view, the applied technologies can be divided into several categories. The dye can be grafted with a short linker to one of the nucleic acid bases (Fig. 5.3a) or attached directly to the sugar-phosphate. Alternatively, the fluorophore can replace one of the natural bases, acting as a nucleobase mimic (Fig. 5.3b). Depending on their chemical composition, fluorescent nucleobase mimics can be divided into isomorphic, expanded or extended base analogs that either maintain or not the Watson–Crick base-pairing. They can be the aromatic fluorophores that lack the H-bonding interactions between

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Fig. 5.3 Illustration of strategies aiming to design the dyes for occupying specific positions on hybridization (Michel et al. 2020). a Through a short and rather rigid linker for extra-helical probing (depicted by pyrene), an aromatic polycyclic dye connected to the pyrimidine C5 and purine C8 positions is used as a nucleobase surrogate for intra-helical probing. b Selected isomorphic nucleobase mimics, so that the bases are made emissive by the base modifications or the extension of their conjugation. Excitation and emission wavelengths are given in nanometers

the complementary bases (Sinkeldam et al. 2010). Due to their well-defined positions, FNAs allow site-selective monitoring of conformational or constitutional changes in nucleic acid targets and are therefore the potent signal transducers. Analyzing this vast literature, the reader may notice that at the present date the probes with intensity-based response are over-represented in comparison with FNAs that exploit other reporting principles. The measurement of fluorescence intensity is, definitely, the simplest. But for the quantitative presentation of results, it needs calibration against some reference, which is often a difficult task for the applications in sensing devices (Demchenko 2005a, b) and which is almost impossible to achieve for sensing in biological media. Studying the dye–nucleotide interactions, one must account additional problems that appear because of quite typical strong quenching effects produced by the bases (Nazarenko et al. 2002) that may have both static and dynamic contributions. Crucially important here is the photoinduced electron transfer between a dye and guanosine base (Sauer et al. 1998). One must account that the more advanced fluorescence techniques, that are the polarization or anisotropy and fluorescence lifetime, are sensitive to dynamic quenching (Chap. 3 of Volume 1). In author’s view, the most productive could be using the nucleic base replacement or modification with single but advanced fluorescent dyes that respond to

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hybridization on a single nucleotide level by the changes of fluorescence parameters sensing their local environment (altered polarity, hydration, flipping dynamics, and formation/breaking of hydrogen bonds) (Michel et al. 2020). The most advantageous of them are the dyes generating interplay of two bands in fluorescence emission that provide the intrinsically calibrated λ-ratiometric response (Demchenko 2010, 2014). The most efficient background mechanism generating two spectrally separated and environment-sensitive bands in fluorescence emission is the excited-state intramolecular proton transfer (ESIPT), see Chap. 4 of Volume 1. Realizing these ideas, a series of fluorescent nucleoside analogues were synthetized operating by ESIPT mechanism (Barthes et al. 2015; Barthes et al. 2016; Dziuba et al. 2014). The dyes of 3-hydroxychromone (3HC) family are known for the most efficient demonstration of their two-band switching responding to the changes of environment polarity and hydrogen bonding potential (Klymchenko and Demchenko 2003). Carbonyl connected by H-bonding with 3-hydroxy group serves as intermolecular H-bond sensor modulating the ESIPT reaction and thus providing the changes of intensity between the two emission bands (Shynkar et al. 2004). Based on these findings, the new conjugated nucleobase–3HC fluorophores were synthesized and their strong sensitivity to hydration was shown (Barthes et al. 2015). The DNA strands incorporating the emissive deoxyuridine analog were synthesized and studied (Fig. 5.4). They demonstrated their unique sensitivity, allowing discrimination between the matched and mismatched DNA duplexes, and different B-DNA/DNA and A-DNA/RNA forms (Barthes et al. 2016). More details about the results of these studies are presented in corresponding sections of Chap. 11.

Fig. 5.4 Fluorescent nucleoside analogues incorporating 3-hydroxychromone fluorophore as a nucleic acid base surrogate 1 and natural base modifier 2–4 (Barthes et al. 2015). The marked carbonyl group serves as both intramolecular charge transfer (ICT) acceptor and intermolecular hydrogen bond acceptor influencing the ESIPT reaction and generating the switching between two bands of fluorescence emission

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5.2 Nucleic Acid Aptamers Aptamers are the single-stranded DNA or RNA oligonucleotide sequences that possess the ability of recognizing various molecular targets, including peptides and proteins (Hamula et al. 2006; Tombelli et al. 2005). Commonly, these properties of oligonucleotides are not observed or are very weak, but after the appearance of selection-amplification techniques operating with large libraries, a number of aptamers with very strong and extremely selective binding abilities increased tremendously. Much like in peptide selection, for finding the best binders one has to create a large pool of similar compounds, a combinatorial library, and to screen this library. Critical point is the selection of the best binders within a library of a huge number of species, and the invention of the “in vitro selection” or SELEX (which stands for “Systematic Evolution of Ligands by EXponential Enrichment”) technique (Bayat et al. 2018). Since this process was discovered in the 1990s, it has led to the development of many artificial functional nucleic acids. The efficient aptamers have been selectively identified for a broad range of ions, small molecules, DNA/RNA sequences, proteins, and even cells (Sun and Zu 2015). Aptamers that are able to bind desired target molecules selectively are generated from a random library of 1015 –1018 candidates. The selected aptamer sequences may vary freely and depend on the target analytes with no relation to the genomic DNA or mRNA. Aptamers are generally more robust and stable under different solution environments and are also cheaper and easier to synthesize and modify than the protein antibodies (Bauer et al. 2019; Zhang et al. 2019).

5.2.1 Selection and Production of Aptamers Selection in vitro is an experimental process to derive nucleic acid sequences with binding and/or catalytic activity from a man-made DNA or RNA library (Hamula et al. 2006; Song et al. 2008; Zheng et al. 2006). A typical DNA library consists of 1013 –1016 sequences produced by chemical synthesis; an RNA library is further made from a DNA library by in vitro transcription. The library is subjected to repetitive cycles of selection and amplification: the selection step is to fractionate active sequences from inactive ones. The enriched fraction is then amplified by the polymerase chain reaction, PCR (for DNA-based selection) or RT-PCR (for RNA-based selection). The amplified pool is used for the next round of selection and amplification (see Fig. 5.5). This process is repeated many times until the selected population exhibits a desired activity (Bayat et al. 2018). Thus, individual functional nucleic acids in the pool are identified by cloning and sequencing. By in vitro selection, they are allowed to compete with each other for survival, so that only the most competent species are progressively enriched and dominate at the end. Because the selection and amplification processes are conducted entirely in a test tube, a researcher can set up experimental conditions to derive the species that could meet a defined need. The

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Fig. 5.5 The scheme for SELEX enrichment process (Song et al. 2008). A random nucleic acid library is incubated with a target molecule, and unbound molecules are separated from bound molecules. Bound nucleic acids are eluted, amplified by PCR (polymerase chain reaction) and serve as an enriched library for the next cycle. For making the aptamer highly specific to a particular target, 6–12 consecutive cycles are performed and the final enriched library is cloned and sequenced

ability to simultaneously screen as many as 1016 different molecules brings about a high probability of success. The size of aptamers could vary from tens to thousands of nucleotides. Typically, it is smaller than 200 bases, and this size is sufficient for their optimal performance that often requires very strong conformational changes. Being single-chain molecules, they can fold to make segments of double-helical structure separated by loops (O’Sullivan et al. 2002). Polyanionic nature of nucleic acids does not deprive them from formation of stable three-dimensional structures. These structures are stabilized by intramolecular formation of short double-helical segments composed of complementary nucleic acid bases. They may exhibit an ideal combination of rigidity and flexibility in intermolecular interactions being formed and destroyed in the target binding event. The development of in vitro selection and amplification techniques has allowed the identification of specific aptamers, which bind to the target molecules with very high affinities. These affinities are frequently comparable with those of monoclonal antibodies, so that their dissociation constants can be observed even in nanomolar to picomolar range. Compared to antibodies, the cross-reactivity of aptamers in binding protein targets is typically minimal (Hicke et al. 2001). Thus, they can discriminate protein targets containing only several amino acid substitutions. The methods for further increase of their binding affinities have been suggested (Hasegawa et al. 2016). Due to the fact that they can also be potent pharmacological agents (their specific binding reduces protein activity), the amount of selected and identified aptamers has

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grown tremendously. Aptamer databases were created to help their systematization (Thodima et al. 2006). It is also easy to fuse the selected aptamer serving as a target recognition module, with a conformation-changing reporting module.

5.2.2 Integration with Fluorescence-Responding Units Due to the facts that aptamers are relatively small and flexible molecules and that they may change significantly their conformation on target binding, the generation of sensing response is relatively easy. These conformational changes almost always change the interactions between the nucleotides of the aptamer and the fluorophore, causing a change in the fluorescent output. Therefore, all the arsenals of fluorescence reporting methods described in Chaps. 3 and 4 of Volume 1 can be applied to aptamer sensing. Very often the aptamers are largely unstructured in solutions and they fold into the well-defined three-dimensional structures upon binding their targets (Hermann and Patel 2000; Stojanovic and Kolpashchikov 2004). Another point that facilitates the sensor design is the fact that in aptamers the fluorophore does not participate directly in molecular recognition as the base analog or its derivative. The fluorophore response to conformational changes can occur at the periphery, therefore there is much broader choice of them and of the mode of their response. If the covalent labeling of dyes is selected in the aptamer design, then the methods based on double labeling, such as the donor–acceptor switching in EET or the monomer-excimer switching, can be used. More reports appear on the introduction of λ-ratiometric sensing, for instance, in application of pyrene excimers (Krasheninina et al. 2017). In fact, the double labeling is not a great problem if the assay is made in solution and both 5’-end and 3’-end are available for labeling. For providing the necessary response, the fluorophore and the quencher in one of the forms (with unbound or bound target) should be close together and brake apart in another form. The double labeling is needed for exploring the concept of aptamer beacons that was borrowed from the beacon technology in DNA hybridization but developed to a level applicable to molecular recognition on a much larger scale (Moutsiopoulou et al. 2019). On the target binding, the two dyes, initially located closely at the ends of stem duplex structure, go apart due to conformational change. The reverse is also possible, and an example of that is the sensor for thrombin (Li et al. 2002). Folding of DNA chain around the target molecule brings the 5’-end and 3’-end together, which results in quenching. The same authors showed that the incorporation of emissive EET pair at the same sites allows obtaining the wavelength-ratiometric response to target binding. Structure-switching signaling aptamers are often designed so that they use duplex-complex transition in the conditions of homogeneous assay (Nutiu and Li 2004, 2005a). Duplex in this case is represented by a double-helical structure labeled with fluorescent dye. Using single labeling, which is easier to realize, one may choose the lifetime or polarization sensing. If it is not essential that the response signal is not wellcalibrated, the in vitro assays use the intensity-based recording (Gao et al. 2020). For

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labeling aptamers, all classical methods of nucleic acids post-translational modification can be applied, as well as an incorporation of base-substituting dyes and naturally emitting 2-aminopurine (Katilius et al. 2006). The aptamers for thrombin based on different fluorescence reporting principles were compared (Juskowiak 2011). The absence of strict requirement on covalent attachment of fluorophores stimulated an extended use of both organic and inorganic luminophores for their noncovalent binding (Ma et al. 2013). Simple, called label-free fluorescent detection methods utilize the DNA intercalating dyes or cationic conjugated polymers to probe the changes in DNA conformation (Lee et al. 2011). Certain organic dyes, such as TOTO are able to intercalate into double-stranded DNA, and the intercalated dyes often exhibit significantly different fluorescence properties. Thus, organic dyes can be used to monitor the formation or dissociation of doublehelical elements of an aptamer as a result of ligand binding. The transition metal complexes, in addition to organic dyes, have started to be extensively used (Ma et al. 2013). In this case, the selection of fluorophore for optimal binding can be provided using the same SELEX procedure (Jhaveri et al. 2000). Nucleotides bearing fluorescent labels can constitute a random pool of sequences, from which the ligandbinding species are selected. Then the binding species are screened for aptamers that signal the presence of cognate ligands. The examples of constructions of signaling aptamers with noncovalently incorporated and covalently bound dyes are presented in Fig. 5.6.

5.2.3 Aptamer Applications and Comparison with Other Binders Aptamers are often compared with antibodies (discussed in Chap. 4), since in both of these molecular families the high recognition power to targets is achieved by selection within huge primary libraries. The difference is that in the case of antibodies the selection occurs as a biological process in the living bodies, and the selection of aptamers occurs under the full manual control. The advantages of aptamers are clearly seen in applications. They can be smaller in size, much more stable and, importantly, demonstrate much larger structure-switching capabilities in the sensing event. The small molecule sensing is performed more efficiently with aptamers than with peptides. This can be illustrated by successful isolation from a population of random RNA sequences the subpopulations that bind specifically to a variety of organic dyes. According to rough estimates, one in 1010 RNA molecules folds in such a way as to create a specific binding site for small ligands, such as amino acid derivatives, cocaine or ATP (Nutiu and Li 2005b). Aptamers show superiority to antibodies in specific protein detection (Stadtherr et al. 2005). Moreover, being selected for tripeptide sequences they can recognize these sequences in a large protein, which makes possible a simultaneous multi-site

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Fig. 5.6 The examples of two sensors responding to the binding of cocaine (Stojanovic et al. 2001; Stojanovic and Landry 2002). a Competitive assay based on displacement by cocaine (1) of a cyanine dye (2). The dye changes its absorbance and it aggregates. b The assay is based on inducing by cocaine binding of conformational change that approximates 5’and 3’ends. This results in quenching of fluorescence of fluorescein (F) emission by dabcyl dye (D)

protein recognition (Niu et al. 2007). They are used for detection of a variety of protein targets, including cytokines. Specifically, for the determination of cytokines and growth factors (Guthrie et al. 2006), several assays making use of aptamers have been developed, including the aptamer-based analogs of ELISA, antibodylinked oligonucleotide assay and fluorescence assays based on anisotropy and EET (Hamula et al. 2006). Specific detection and quantitation of cancer-associated proteins (inosine monophosphate dehydrogenase II, vascular endothelial growth factor, basic fibroblast growth factor) in the context of human serum and in cellular extracts has been realized with aptamers. It is expected that this technology will improve diagnosis of

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cancer by enabling direct detection of the expression and modification of proteins closely correlated with the disease (McCauley et al. 2003). Aptamers are useful in the studies of protein–protein interactions, where a competitive assay format can be applied, in which the aptamers are displaced from protein–protein contact areas (Yeldell and Seitz 2020). Aptamer microarrays are becoming one of the most efficient sensing technologies for multiplex analysis of numerous proteins in parallel, furthering the notion that such arrays may be useful in proteomics. Their fabrication is developed to a great detail (Collett et al. 2005). Fluorescence polarization anisotropy can be used for the measurements of target protein binding both in solutions and on solid support (McCauley et al. 2003). The solid-phase aptamer-protein interactions are similar to binding interactions seen in solutions. Usually when the aptamers are used in heterogeneous sensing platforms, they can be the nucleic acid sequences of different lengths, with one of the ends (either 3’ or 5’-end) being normally used for the binding of the aptamer to the solid support. The other end can be used for carrying a dye. Biotin can be attached to one of the ends, so the aptamers can be spotted on streptavidin-coated slides, benefiting from self-assembly based on a very strong streptavidin–biotin interaction (Collett et al. 2005). In addition to sensor technologies, selected oligonucleotides can be used as ‘aptazymes’, the species that possess biocatalytic properties and allow the direct transduction of molecular recognition into catalysis. Together with aptamers they can be used in different bioassays for the detection and quantitation of a wide range of molecular targets (Hesselberth et al. 2000). The aptamers have proved to be useful in imaging, including cancer cell tracking (Hwang et al. 2016) and for providing super-resolution images of membranes in living cells (Wang et al. 2020). Thus, only three decades have passed between the introduction of aptamers technology and its development into one of the most successful fields of molecular sensing. There is no restriction in developing the aptamers as the sensors to any target. Selected aptamers can bind to their targets with high affinity and discriminate between closely related target molecules. Aptamers can thus be considered as a valid alternative to antibodies as well as to any peptide or protein bio-mimetic receptors. The unique binding properties of nucleic acids, which are amenable to various modifications, make aptamers perfectly suitable for not only sensing, but also for other uses in biotechnology, including pharmaceutical applications (Proske et al. 2005). Such combination of already realized and potential applications as analytical, diagnostic and therapeutic tools (Tombelli et al. 2005) still further stimulates their development. All these advantages are not always clearly seen if the aptamers are accommodated into traditional analytical techniques developed for antibodies, such as ELISA. The new techniques exploring their advantages in full are being developed (MacKay et al. 2014). They include microarray and microfluidic technologies. It is expected that they will be highly competitive and demonstrate a full potential for successful realization of extremely valuable properties of aptamers.

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5.3 G-quadruplex-Based Analytical Sensing Platforms G-quadruplexes (G4s) are noncanonical four-stranded nucleic acid structures formed in guanine-rich DNA and RNA sequences (Kwok and Merrick 2017; Spiegel et al. 2020; Yang, 2019). In a plane, four G bases are organized into the planar structure stabilized by hydrogen bonds. Stacking several such structures allows obtaining globally folded compositions, usually consisting of three-four members. These stacking can be inter- and intramolecular (Fig. 5.7).

Fig. 5.7 Schematic illustration of structures formed by DNA quadruplexes (Yang, 2019). a A Gtetrad, which is formed by four guanine bases arranged in a square plane interlinked with hydrogen bonding. Monovalent cations (K+ or Na+ , shown as blue spheres) are required to stabilize Gquadruplexes by coordinating with the O6 atoms of the adjacent G-tetrad planes. b A schematic intermolecular (tetrameric) G-quadruplex with three G-tetrads and examples of intramolecular G-quadruplexes with different folding structures and loop conformations. The experimentally determined molecular structures are shown as examples for parallel, hybrid, and basket-like G-quadruplexes

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It was known for a long time that G-tetrads play a key role in genome structure and its regulation (Spiegel et al. 2020). They have been found to form specific human guanine-rich sequences with functional significance, such as telomeres and oncogene-promoter regions. The methods of their determination and visualization will be overviewed in Chap. 11. Here we are interested in evaluating their possibilities for serving in broad-scale chemical sensors and biosensors. In this respect, they can be considered as advanced signaling aptamers.

5.3.1 Production and Properties of G-quadruplexes The G-quadruplexes can be identified from combinatorial libraries of DNA or RNA oligonucleotides by SELEX technology, an in vitro iterative selection procedure consisting of binding (capture), partitioning and amplification steps (see Sect. 5.2 above). Remarkably, many of the aptamers selected against biologically relevant protein targets are the G-rich sequences that can fold into stable G-quadruplexes. These aptamers may exist in G4 conformation but may also adopt the G-quadruplex conformations upon direct or indirect binding to their target analytes. The difference of G4s from common aptamers is the following. Whereas the aptamers are rather flexible structures that may be stabilized by only the fragments of double helices, the G-quadruplexes are more stable in retaining their conformation under different environment conditions, such as variation of temperature or application of detergents. They are stabilized by not only the π-stacking and network of H-bonds but also by central cations interacting with polarizable electrons of oxygen (see Fig. 5.7a). The metal cations can also mediate the structure formation of a specific G-quadruplex, and this adds diversity to these structures. For formation of these structures, there must be the restrictions in topology. When formed intramolecularly, the G-quadruplexes fold quickly and exhibit great conformational diversity in topology, loop conformation, and capping structures (Neidle and Parkinson 2003). Based on G-strand directionality, a G-quadruplex can be parallel with all four G-strands in the same direction, hybrid/mixed with both parallel and antiparallel strands, and also antiparallel with all adjacent G-strands antiparallel to each other. G-strands in intramolecular G-quadruplexes are connected by different types of loops, such as propeller for connecting parallel strands, lateral for connecting adjacent antiparallel strands, and diagonal for connecting antiparallel strands across the G-tetrad core. The interesting feature of the quadruplex structure is the presence of a central channel formed by stacking of guanine tetrads. Due to the orientation of the carbonyl groups from each guanine base towards the center of the G-tetrad, the central channel is negatively polarized, and because of that it can accommodate cations. Usually, these are the K+ or Na+ ions. The precise coordination site of such cation occurs to be a cavity between two G-quartets that is flanked with eight O-6 guanine atoms. The cation binding contributes significantly to the stability of G-quadruplex structures

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Fig. 5.8 Folding of G-quadruplex on binding potassium ions (Dembska et al., 2020). a Scheme of using the double labeling for generating a λ-ratiometric fluorescent signal based on EET mechanism. b The response to potassium binding plotted as a function fluorescence intensity ratio (F583 /F520 ) plotted against K+ concentration in the presence 150 mM Na+ ions at 25.0 °C (circles) and 36.6 °C (triangles)

and is one of determinants of its folding. The unfolded functionalized DNA fragment can fold into G-quadruplex as a function of concentration of potassium ions (Fig. 5.8).

5.3.2 Fluorescence Reporters for G-quadruplex Structures A large number of small aromatic molecules, both classical and those synthesized in recent years, display an ability to selectively target, stabilize and label the Gquadruplexes with reporting function (Ma et al. 2012; Vummidi et al. 2013). Organic heterocyclic compounds can interact with G4s in different ways by optimizing electrostatic interactions, providing end stacking, intercalation, and groove binding. Therefore, we have to account that many dyes labeling efficiently the double-stranded DNA (such as acridine dyes or ethidium bromide) will also interact with G4. For different sensing technologies that are based on G4, it is not the specific binding to G4 but the binding producing the high level of response to target recognition that has to be optimized, which can be achieved with noncovalently bound dyes. It is a good story that many organic molecules have been reported as such G-quadruplex probes. They include crystal violet, thiazole orange, protoporphyrin IX, thioflavin T and their analogues. These probes exhibit high fluorescence enhancement with addition of Gquadruplex DNA over other forms of DNA, highlighting the dsDNA to G4 transition (Bhasikuttan and Mohanty 2015; Kataoka et al. 2014). The extended range of these possibilities is a positive feature, which allows avoiding complications with covalent labeling, whereas selecting within a number of quadruplex-selective luminophores for their noncovalent attachment is a simple procedure (Ma et al. 2012). The request is different for fluorescence probes that have to be used for G4 detection in biological media. In that case, the selectivity in visualizing G-quadruplexes in the presence of large number of double helices should be the major criterion (see Chap. 11).

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Fig. 5.9 Schematic illustration of the sensing mechanism of the G-quadruplex-based detection platform for insulin by using a G-quadruplex-selective iridium(III) complex (Wang et al. 2016). Coupled with insulin binding, release of iridium(III) complex provides the sensor signal

Among these G-quadruplex-selective probes, the luminescent transition metal complexes (Georgiades et al. 2010), particularly of platinum Pt(II) (Wang et al. 2010) and iridium Ir(III) (Leung et al. 2015), are popular. These complexes coordinating metal cation generally emit phosphorescence in the visible region with a long lifetime, which allows using the time-resolved or time-gating detection to cut-off the fluorescent background. These probes demonstrate strong changes of emission intensity in response to subtle changes of molecular environment. The sensor for insulin (Wang et al. 2016) may serve as an example (Fig. 5.9). Binding of insulin to designed luminescence sensor, generates the folding of G-quadruplex, and the change of location of Ir(III) complex reports on this event by luminescence signal.

5.3.3 Applications of G-quadruplex Sensing Technology The G-quadruplex-based sensing assays can be realized in two ways. One is when starting from unfolded ssDNA or DNA duplexes the folding into G4 structure occurs being induced by the target, and the target presence and concentration is determined by response of the dye emission (Ma et al. 2017a, b). The other is using the G4 scaffold as a robust structure in design that does not change in sensing event. The first approach was found to be efficient when applied to the detection of proteins, nucleotides, different drugs and toxic ions (Ma et al. 2013; Ma et al. 2017a, b). It was shown that both organic substances and ions can generate the appearance of G4 formation on their binding. In a typical G-quadruplex-based sensing platform, the addition of an analyte triggers the conformational switching of a designed DNA sequence, usually in ssDNA or dsDNA form, into a G-quadruplex structure. A Gquadruplex fluorescence probe can be used to transduce this conformational change into a luminescent response. Figure 5.9 is an illustration of this approach (Wang et al. 2016). A sensitive probe was suggested for the detection of insulin. The insulin aptamer (green line)containing DNA ON1 (5' -AG3 AG3 CGCTG3 C6 G3 G2 TG2 TG8 T2 G2 TAG3 TGTCT2

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Fig. 5.10 Schematic diagram for detection of thrombin using the 27-meric aptamer with pyrene molecules labeled at both ends of the aptamer (Zhao and Cheng 2013). The binding of thrombin induces the conformation change of the aptamer, causing the pyrenes to form an excimer. The emission spectra of the aptamer probe in the absence of thrombin (a) and in the presence of thrombin (b) are shown. The excimer gives fluorescence emission at about 485 nm with an excitation at 349 nm, allowing the detection of thrombin

C-3' ), which includes a G-quadruplex-forming sequence (black line) as well at the 5' -end of DNA, is firstly hybridized with a partially antisense DNA strand (ON2, 5' -AC8 AC2 AC5 G6 C3 AGC-3' , blue line), to form a double substrate. Upon the addition of insulin into the ON1-ON2 duplex substrate, it leads to form an insulinaptamer complex, causing the dissociation of ON1 from ON2. After that, due to the stabilizing effect of K+ ions, the released 5' -terminus sequence of ON1 undergoes conformational change into a G-quadruplex motif. The induced G-quadruplex structure is then identified by the G-quadruplex-selective Ir(III)-based luminophore. The case, when G-quadruplex is used as a scaffold and conformational changes on target binding occur on its periphery, is illustrated by aptamer thrombin sensor (Fig. 5.10). A small 27-mer aptamer (5’-GT CCG TGG TAG GGC AGG TTG GGG TGA C-3’) was selected and pyrene groups were introduced at both its terminals (Zhao and Cheng 2013). In the absence of thrombin, the 5’-end and the 3’-end of the 27-mer aptamer were spatially separated, and upon thrombin binding, the aptamer brought the two ends close to form an excimer of pyrene, giving the change of fluorescence that can be recorded by λ-ratiometry. In recent years, the G-quadruplex based biosensors have been intensively studied and applied for detecting different analytes. They include not only molecular targets, such as proteins (Riccardi et al. 2020), but also viruses and bacteria (Roxo et al. 2019; Xi et al. 2020). Their main advantage is an optimal combination of structural stability and its variability, up to folding-unfolding conformational changes that can be adjusted in very broad ranges and allows application of many fluorescence reporting modalities.

5.4 The DNA i-motif in Sensing The i-motifs share with G-quadruplexes a common feature, namely that they are formed from four stretches of identical bases (G or C). Whereas G-quadruplex based biosensors have attracted more research interest than those based on i-motif quadruplex DNA, the interests towards i-motifs are rising only in recent times. The problem

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is in limited stability at neutral pH values that also depends on the temperature. In contrast to G-quadruplexes, for which both DNA and RNA can form stable G4 structures, the RNA i-motifs are less stable than the corresponding DNA i-motif, and application of i-motifs to nanotechnology has so far been limited to DNA oligomers (Mergny and Sen 2019). The basic building block of i-motif structure is a base pair involving one neutral (deprotonated) cytosine and one positively-charged (protonated) cytosine at the N3 position. The resulting C·C+ base pair, which is stable because of the formation of three hydrogen bonds, allows the formation of the parallel duplexes. Therefore, pH plays a crucial role in their formation. The pH range of structural transformation is much narrower than the range of proton dissociation equilibrium in common sensors, see Fig. 5.11. Based on this property, highly responsive pH sensors based on a DNA i-motif were developed (Nesterova and Nesterov 2014). By rational manipulations of i-motif structure as well as by incorporation of allosteric control elements, both response sensitivity and transition midpoint were tuned with high precision over the physiologically relevant pH interval. This strategy delivers molecular sensing systems with a transition midpoint tunable with 0.1 pH units precision and with a total response range as narrow as 0.2 pH units, which can be adjusted to a variety of outputs, including fluorescent readout. Thus, the limited stability at physiological pH is not necessarily a disadvantage for nanotechnology applications. The extreme pH dependence of these structures based on reversible transition between i-DNA and conventional

Fig. 5.11 The pH-induced i-motif folding monitored by color change of fluorescence (Michel et al. 2020) (a) and comparison of probed pH ranges of conventional sensor based on titration of acidic groups and the i-motif based sensor (Nesterova and Nesterov 2014) (b)

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double helices can actually be an asset for pH-responsive devices (Alba et al. 2016; Dembska et al. 2017). Fluorescent base analogs are used for providing the sensor response (Dembska et al. 2020).

5.5 Sensing and Thinking: The Versatile Recognizing Power of Nucleic Acids The more we know about the recognition power of nucleic acids, the more surprise we get. The Watson–Crick pairing is highly selective to composition of bases, and even the changes on the level of single base may result in important functional consequences. Therefore, it is natural to use the sequences producing the base pairing as the sensors recognizing the target sequences and defects in them. The sensors are usually composed of single-strand DNA, which can hybridize with the complementary strand. The fluorescence reporter should be located precisely at the matchingmismatching recognition site. The best performers here are the covalently bound organic dyes. They should possess structure-selective and interaction-stimulated emission characteristics. One may expect that the nucleic acid segments recognize nucleic acids only, and the hybridization is the only mechanism of molecular recognition. But the reality is different and much more interesting. The best DNA and RNA binders selected from huge artificially created libraries complemented with fluorescent reporters demonstrate the ability of being the ideal sensors for extremely broad range of applications. Out of all macromolecular binders, only the antibodies can presently compete with the library-selected aptamers in versatility of adaptation to any particular target. Regarding other aspects, such as production and application, the aptamers possess many important advantages. They demonstrate higher stability to environmental factors, which allows their long-term storage without loss of functional properties. Co-synthetic structural modifications including introduction of fluorescent groups can be achieved much easier. Comparison with library-selected peptide binders is also in favor of nucleic acid aptamers. Automated selection procedures allow rapid identification of DNA and RNA sequences that can target a broad range of species with nanomolar affinities and high specificities. The larger library offers more molecular diversity and higher probability of finding the optimal binders. Thus, with nucleic acids we observe two different trends. One is based on principle of rational design and requires precise location of reporting dyes. The other explores the principle of combinatorial library selection. Here the response to different targets can be mediated by conformational change in the sensing unit and allows for reporting the application of different types of molecular and nanoscale fluorophores. Respond to following questions finding answers in the text: 1.

What is the mechanism of molecular recognition in hybridization-based sensors and how the fluorescence response can be generated? What are the sites

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of attachment of fluorescence dyes? How to achieve specific positions of fluorescent dyes in these sensors? 2. What are the main distinguishing features between fluorescent aptamers and hybridization-based sensors? 3. Why the aptamer sensor can be found to almost any target? Explain the formation of aptamer combinatorial library and the SELEX procedure. 4. Explain the realization of different technologies for providing the fluorescence response in the cases: (a) aptamers in solution; (b) aptamers attached with one of the terminals to solid support. 5. Analyze the difference in requirements to fluorescence reporters in hybridization techniques and in aptamer sensors. 6. What is the mechanism behind the use of intercalating dyes for obtaining the response to aptamer-target binding? What are the disadvantages of this approach? 7. How to introduce the λ-ratiometric detection into the aptamer response mechanism? 8. Can fluorescence nanoparticles be used with aptamers as response units? Can the plasmonic enhancement of fluorescence signal be applied? 9. What are the G-quadruplexes? Explain two different roles that they can play in sensing. How the fluorescence response can be introduced in sensing? 10. What are the i-motifs and what is their role in sensing? What is special in their sensitivity to pH and what is its origin.

References Alba JJ, Sadurní A, Gargallo R (2016) Nucleic acid i-motif structures in analytical chemistry. Crit Rev Anal Chem 46:443–454 Barthes N, Karpenko I, Dziuba D, Spadafora M, Auffret J, Demchenko A, Mély Y, Benhida R, Michel B, Burger A (2015) Development of environmentally sensitive fluorescent and dual emissive deoxyuridine analogues. RSC Adv 5:33536–33545 Barthes NP, Gavvala K, Dziuba D, Bonhomme D, Karpenko IA, Dabert-Gay AS, Debayle D, Demchenko AP, Benhida R, Michel BY (2016) Dual emissive analogue of deoxyuridine as a sensitive hydration-reporting probe for discriminating mismatched from matched DNA and DNA/DNA from DNA/RNA duplexes. J Mater Chem C 4:3010–3017 Bauer M, Strom M, Hammond DS, Shigdar S (2019) Anything you can do, I can do better: can aptamers replace antibodies in clinical diagnostic applications? Molecules 24:4377 Bayat P, Nosrati R, Alibolandi M, Rafatpanah H, Abnous K, Khedri M, Ramezani M (2018) SELEX methods on the road to protein targeting with nucleic acid aptamers. Biochimie 154:132–155 Bhasikuttan AC, Mohanty J (2015) Targeting G-quadruplex structures with extrinsic fluorogenic dyes: promising fluorescence sensors. Chem Commun 51:7581–7597 Collett JR, Cho EJ, Ellington AD (2005) Production and processing of aptamer microarrays. Methods 37:4–15 Davies MJ, Shah A, Bruce IJ (2000) Synthesis of fluorescently labelled oligonucleotides and nucleic acids. Chem Soc Rev 29:97–107 Dembska A, Bielecka P, Juskowiak B (2017) pH-Sensing fluorescence oligonucleotide probes based on an i-motif scaffold: a review. Anal Methods 9:6092–6106

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Chapter 6

Self-assembled, Porous and Molecularly Imprinted Supramolecular Structures in Sensing

Creation of more and more sophisticated sensors can be easier performed by selfassembly of already formed units. These surfaces can be either flat or belong to nanoparticles, but they can also possess high porosity, and, together with their hydrophobicity and charge, these properties can contribute to molecular recognition. Moreover, formation of sites of strong target binding by molecular imprinting can be made in polymer volume. Upon target removal, it leaves behind the spatially arranged functional groups that act as recognition sites. Great diversity of possibilities and still poor involvement of fluorescence detection methods characterizes this area and must stimulate new research activities. On the road to better performance, the development of sensor technologies proceeds in several directions. The scientists try to achieve high durability of sensor instruments with enhanced chemical tolerance and resistance of their receptor and reporter elements in biological milieu, their convenience, reduced size and low cost. To satisfy these strong demands, the organic molecular binders, discussed in the previous chapters, may not be sufficient. In this chapter we concentrate on artificial recognition elements that can be formed by molecular assemblies in supramolecular structures with or without solid support. The solid part can be flat but can also be presented as nanoscale and larger (mesoscale) objects. On this level of complexity in organization of structures, the common fullscale organic synthesis is not efficient, and the self-assembly of functional sensors from the building blocks becomes a method of choice. The solid support can be functionally important and exist in the form of flat solid bodies, of nanoparticles or microsphere structures. Moreover, it can attain porosity, and in this case the molecular recognition can be achieved within the volume of solid system and must involve strict geometrical variables. Such materials are extensively used in many separation techniques, such as thin-layer chromatography, highperformance liquid chromatography and solid-phase extraction. In a new role of sensors, they can offer sufficient affinity and selectivity. Among various artificial recognition elements operating in the solid-state volume are the molecularly imprinted polymers (MIPs). Such materials serving as the target © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_6

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host systems can be efficiently prepared by ‘imprinting’ into the bodies of polymers or inorganic materials of the template molecules, which, upon removal, leave behind the spatially arranged functional groups that act as the recognition sites. After removing the template, an imprint, containing functional groups capable of chemical interaction, remains in the polymer matrix. The imprinted polymer matrix attains the memory for a high-affinity binding to the same template and can be used for its molecular recognition. These new materials potentially offer the specificity and selectivity of the biological receptors with the explicit advantages of durability with respect to the environment. As a rule, they do not require special storage conditions and can be applied over a wide temperature range. Also, their price is estimated to be about two orders of magnitude lower than of those made with the antibodies. In all these cases, the target-receptor molecular recognition is based on multivalency—the ability to form multiple intermolecular bonds (Ariga 2012a, b; Huskens et al. 2018). We observe that there are more chances for the molecular system to be highly specific if it is relatively large and if it can incorporate the species able to form greater number of intermolecular bonds. Versatile building blocks can be used for creation of supramolecular sensors (Fig. 6.1). A rational integration of multivalent and heterotopic interactions generates the hetero-multivalency that involves a combination of ligand–receptor pairs, in which more than one type of ligand interacts with more than one type of receptor simultaneously. This results in greater strength and specificity of binding than the equivalent monovalent interactions or the homo-multivalent counterparts (Xu et al. 2019).

6.1 Molecular Recognition on Supramolecular Scale The increase of size and complexity is the current trend in the design of recognition units in fluorescence sensors for better design enhancing sensitivity and specificity. However, with this increase, the preparation of molecular units by sequential covalent synthesis becomes a very difficult, time-consuming and even unrealizable choice. In contrast, supramolecular chemistry (Atwood and Steed 2004; Schalley 2012), is based on self-assembly of smaller units. It may offer an easier creation of nanoscale systems. Supramolecules are the molecular assemblies that are formed and held together by intermolecular forces rather than by covalent bonds. The term “supramolecular chemistry” was introduced in the middle of 1980s (Lehn 1985), and since then it refers to the chemistry of molecular assemblies that are “beyond the molecule”. Such supramolecules can be obtained by self-assembly from molecular units of different complexity, which avoids lengthy synthetic processes and facilitates rapid sensor development. Without the need for covalent bond formation on the step of assembly, supramolecules are able to form highly ordered and complex systems. Their stability is quite sufficient for considering them as sensor units or even as assembled sensors (Wang et al. 2016b), and their further stabilization can be performed by covalent

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Fig. 6.1 Formation of supramolecular fluorescence sensor systems from different building blocks and obtaining at the outcome the technologies for homogeneous and heterogeneous sensing and imaging with strongly improved performance

linking. Meantime, they can be flexible and adaptable enough for realizing different sensing mechanisms. Combining different receptor and reporter units with different transduction units and support structures can be provided on nanoscale, leading to sensors of extreme efficiency. The range of applications of such supramolecular self-assembled systems can be extremely broad (see Fig. 6.1).

6.1.1 Assembly of Organic and Inorganic Functionalities Different organic and inorganic materials and also organic–inorganic compositions can be used for constructing supramolecular structures that can be used in sensing. As functional nanoscale nanoparticles and nanocomposites with the focus on fluorescence emission, they were overviewed in Volume 1. Here we present them as building blocks for supramolecular structures with the ability of molecular recognition. The supramolecular structures are seen as self-organized complexes between

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or with designed partners, in which the recognition and reporting can be coupled in a most efficient way. In an effort for developing better sensors, an important role is given to nanoscale support materials. They include different types of molecules and particles representing inorganic, organic and biological worlds (Oshovsky et al. 2007). In assembled structures, the role of different organic and inorganic elements is quite variable. Thus, carbon nanoparticles (graphenes, fullerenes, nanotubes and Cdots) can play additional role as fluorescence quenchers (Chap. 9 of Volume 1). Noble metal nanoparticles may serve as fluorescence enhancers based on plasmonic effects, but strong light scatterers and quenchers as well (Chap. 13 of Volume 1), whereas silica particles are quite inert carriers. But, being of controlled porosity, it can participate in target recognition. More possibilities appear for integration and functional improvement of different inorganic and organic dye compositions featuring structurally enhanced and aggregation-induced emissions. Synthetic polymers being polyelectrolytes or possessing alternating hydrophilic-hydrophobic domains can form a variety of self-assembled structures ready for targeted functionalization by covalent linkage. For providing the self-assembly on supramolecular level, one may need to follow definite rules: (1) The interacting particles or molecules should realize the maximal level of noncovalent intermolecular interactions. The most important of them are: (a) electrostatic interactions based on complementarity of charged groups, (b) hydrogen bonding that involves complementarity of proton donor and acceptor groups and (c) hydrophobic interactions that provide stabilization of contacts between low-polar groups in polar media. Individually, these bonds are weak, but their collective action provides significant effects of stabilization of intermolecular complexes. (2) If the partners that are selected for self-assembly are dissolved in non-mixing solvents of different polarities, then the phase transfer steps may become needed. Three strategies can be selected for that: ligand exchange, ligand modification and additional layers of molecules that stabilize the particles in the desired phase (Sperling and Parak 2010). Being a part of these approaches, silanization is used for surface modification and phase transfer. Organic polymers are often applied for formation of coating layers. (3) The resulting composites should allow further steps of functionalization, such as the covalent attachment of proteins, nucleic acids, etc. Such smart functionalities can be added at desired positions in structure (see also Sect. 11.6 of Volume 1).

6.1.2 The Major Building Blocks Carbon-based nanostructures of a very small size (5–20 nm) are known as very strong light absorbers, light emitters and light quenchers (Chap. 9 of Volume 1). Meantime, the role of carbon materials as the building blocks is much broader

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(Maji et al. 2020), especially when it is associated with quenching that can give an output signal in sensing (Demchenko and Dekaliuk 2013). The basic platform of nanoscale carbon is graphene. The carbon atoms of graphene form a densely packed regular two-dimensional pattern of atomic width. The sp2 bonding allows delocalization of electrons that are responsible for unique electron conduction. The familiar graphite can be viewed as the structure composed of many layers of such graphene sheets. Graphene oxide (GO) and reduced graphene oxide (rGO) possess in their graphene sheets the polar groups that break up the regular graphene structure into connected islands. In addition to increasing the solubility, there appear additional sites of covalent binding and self-assembly. Graphene sheet is an ideal absorber of different types of molecules, especially of aromatic nature, of biological macromolecules and nanoparticles, and the association/dissociation of different types of molecules can generate the sensor response. Different aromatic compounds demonstrate increased affinity to graphene, being stabilized by π-π stacking interactions (Mann and Dichtel 2013). Graphene and GO interact strongly with polymers (Salavagione et al. 2011), proteins (Zhang et al. 2013) and, notably, with single-stranded nucleic acids, but not with their double helices. Based on this feature, the methods of the DNA hybridization assays were developed (Deng et al. 2014; Lu et al. 2010). Hiding sticky ends, graphene sheet can be rolled up into a cylinder forming carbon nanotube, and with the inclusion of pentagon elements it can form fullerenes, the structures in the form of hollow spheres. Tubular structures are the rigid mechanically and functionally versatile modules that can be used as nanoscale support materials. Their near-infrared emission at 1200–1400 nm (Choi and Strano 2007) can be quenched by appropriate redox-active dyes, and with proper construction this effect can be used in sensing (Satishkumar et al. 2007). The techniques of integration of nanotubes into various devices and designing the integrated sensing elements were developed (Mahar et al. 2007) exploring the signal transduction by fluorescence quenching and charge transfer (Barone et al. 2005). Fullerenes are known as very potent electron acceptors, and this property is used already in different optoelectronic devices (Ito and D’Souza 2012; Wróbel and Graja 2011). Though, there are practical difficulties due to low solubility of fullerenes and their tendency to aggregate. DNA templates were discussed at length in Sect. 11.3 of Volume 1. Their role is not limited to nucleic acid hybridization and may allow extremely broad range of applications in sensors operating on supramolecular level (Ranallo et al. 2018; Wilner and Willner 2012). Their great advantage is the possibility of strictly designed location in the sequence of nucleic acid bases of different building blocks that can provide programmable arrangement of rigid structure-forming strands that can serve as scaffolds for further modifications (Fischler et al. 2008). Constructing the DNAbased templates can be modulated by temperature (the ‘thermal annealing’ uses controlled high-temperature DNA melting) and also by attached macromolecules and ions. DNA origamis are the structures formed of DNA with very complex topology. Design of specific arrangement of DNA chains allows programmable fabrication of

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non-periodic complex structures. The DNA origami is able to bind at specific locations different molecules and to position peptides, proteins or other supramolecular sensor components. Conjugation with peptide nucleic acids (see Sect. 11.2) can be explored on this new level of structure formation. The examples of these studies are the formations of porphyrin arrays on DNA template (Fendt et al. 2007). The designed pattern and precise spacing of their location adds unlimited possibilities in a bottom-up approach toward self-assembly. Peptide scaffolds can be efficient platforms for self-assembly (see also Sect. 4.1). They are able to form three-dimensional structures of various complexity and of various functional use, not only for the construction of sensor units but also for making the scaffolds for supramolecular sensors. Peptides forming the self-assembled tubular structures can serve as ideal scaffolds for supramolecular sensors (Gao and Matsui 2005). The self-assembled peptide nanostructures can be further organized to form nanowires, nanoparticles and even ‘nanoforests’ (on solid support). Cyclic peptides formed as planar rings by alternating L- and D-amino acids self-assemble via hydrogen bonding to tubular open-ended and hollow structures (Brea et al. 2007). The other types of peptide-based scaffolds are their associates based on the principle of amphiphilicity (the separation of polar and unpolar sites). Their polar sites can be represented by 1–2 charged amino acids and hydrophobic part—by four or more sequential hydrophobic amino acids. They form ordered nanostructures similar to that formed by lipids (Jiang et al. 2007). There are many opportunities for hierarchical formation of supramolecular assemblies of larger complexity based on these peptide assemblies. The principle of formation of the third type of these scaffolds is borrowed from the known examples of amyloid fibrils that may appear in vivo and are associated with a number of human diseases. Application of peptides forming amyloid fibrils as scaffolds was suggested based on the facts of formation by them of very rigid β-sheeted structures (del Mercato et al. 2007). Inorganic colloidal scaffolds, being made of conducting, insulating or semiconducting materials and possessing different optical properties, have something in common: they cannot be used ‘naked’ and have to be ‘decorated’ (Kargozar et al. 2020; Zhou et al. 2017). In such already modified state they can be functionalized or assembled into nanocomposites. For performing such manipulations with them, they have to be present in dispersed forms and stabilized against aggregation. Details of such stabilization reactions can be found in the literature (Sperling and Parak 2010). Different reagents can be used for this purpose, but the general principle is the following. The stabilizing agent should be bi-functional. It has to bind strongly to the particle surface and, simultaneously, prevent the aggregation. The stabilizing agent that is present in the medium of nanoparticle synthesis and forms the surface layer may not be optimal for controlled assembly, the introduction of receptors, fluorescence reporters or other functionalities. Therefore, the ligand exchange may be needed. A common example is the treatment of Au nanoparticles that in an aqueous solution are synthesized by citrate reduction (Saha et al. 2012; Woehrle et al. 2002). They can be replaced by sulfonated phosphines or mercaptocarboxylic acids.

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Amphiphilic molecules, comprising hydrophobic and hydrophilic moieties possess the intrinsic propensity to self-assemble in aqueous environment. Macrocyclic amphiphiles are the amphiphilic systems constructed from the frameworks of a macrocyclic host (Chen et al. 2018). Cyclodextrins, calixarenes, cucurbiturils, and pillararenes (see Chap. 3) can serve as these hosts. They are capable of self-assembling into multidimensional assemblies with defined nanostructures (Li et al. 2020; Tan and Yang 2015; Zhu et al. 2018). By involving host–guest recognition, these amphiphiles can be tailored to fabricate new topological structures and fulfill multiple applications. The introduction of host–guest interactions facilitates the design, synthesis and controllability of these amphiphilic systems. Moreover, host–guest interactions usually possess stimuli-responsive properties. Different combinations with fluorophores are possible with prospect in sensing (Chen et al. 2020b). The application of such strategy of self-assembly of the sensor building blocks use simple production methods, it is robust, practical and affordable.

6.1.3 Realization of Multiple Recognition Sites in Self-assembled Structures Let me draw the reader’s attention to the result that does not deal with fluorescence directly but is informative and instructive. A hetero-multivalent system formed by co-assembly of amphiphilic cyclodextrin (CD) and calixarene (CA) units modified by a hydrophilic head and a hydrophobic tail was arranged into vesicles (Xu et al. 2019). If CD and CA were to assemble into vesicles individually, they showed good but different binding efficiency to different amino acid residues (Tyr and Lys in this case). But the hetero-multivalent recognition platform spontaneously formed by co-assembly of CD and CA units demonstrated dramatically improved binding performance in terms of both affinity and selectivity towards peptides that contain these amino acids (Fig. 6.2). This result shows how the assembling of individual receptors into supramolecular composite can result in substantial improvement of molecular recognition power. Such self-assembled multivalency demonstrates some significant advantages over the use of single multivalent receptors: (1) spontaneous assembly, (2) easily tunable ligands and (3) the ability to assemble different active components into a single nanostructure. Integrating two (or more) different macrocyclic receptors into one ensemble to achieve hetero-multivalent molecular recognition has to drastically improve the binding efficiency and selectivity, especially to biologically relevant heterotopic species, such as proteins. Moreover, the dynamic feature of self-assembly endows the platform with better self-adaptability to recognize multivalent targets. Another example refers to building blocks of one type but already with strong and distinctive fluorescence response to two types of targets. A multi-responsive pyreneappended β-cyclodextrin probe self-assembling into nanoaggregates (150–164 nm)

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Fig. 6.2 Illustration of the hetero-multivalent peptide recognition by the co-assembly of amphiphilic cyclodextrin (CD) and calixarene (CA) units (Xu et al. 2019). Individual CD forms the vesicular assembly affording homo-multivalency, and so does individual CA. The operating principle of constructing the hetero-multivalent platform is the co-assembly of CD and CA amphiphiles. As a result, two types of host–guest recognition site are simultaneously distributed on the surface and can be occupied by the target. Peptide chains bearing two different types of amino acid (represented as rods and spheres) that have high affinities with either CA or CD are shown in purple. The structures and schematic representations of CD and CA are shown (bottom right)

in water was developed (Champagne et al. 2019). The aggregate formation was driven by hydrophobic π–π interactions. The formed fluorescent nanosensors (Fig. 6.3) were found to exhibit an efficient and selective ratiometric detection of pirimicarb, a potent toxic carbamate pesticide, with significant enhancement (85 times) in the monomer to excimer ratio of pyrene emission. Further, among the trinitroaromatic explosives, the formed nanoaggregates displayed discriminative fluorogenic detection of 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB) and picric acid (PA) that were associated with the change of molecular packing of pyrene units. Great advantage of self-assembled supramolecular sensors is an easy incorporation of multiple receptors and multiple reporters into sensors and the possibility of interplay between them for improving their function (Sasaki et al. 2020). As the building blocks of supramolecular structures, performing recognition function, the modified macrocyclic compounds (see Chap. 3) have started to occupy leading positions (Zhu et al. 2018). On the level of monomers they carry the recognition properties and their modification may result in efficient self-assembly into micelles and vesicles.

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Fig. 6.3 Pyrene-appended β-cyclodextrin based multi-responsive self-assembled nanoaggregates that allowed guest specific conformational changes in pyrene arms (a). It allows distinctive fluorescence detection of pirimicarb (PC) (b), TNT (c), and also of TNB and picric acid (Champagne et al. 2019)

6.2 Formation and Operation of Supramolecular Fluorescent Sensors Supramolecular structures with the ability to target binding can be obtained in different ways. The most typical are the self-assembly in solutions and also with the aid of different supports. (a) Self-assembly. The central theme in self-assembly is to direct an outcome of an otherwise spontaneous reaction and to exert control over all its steps, aiming at obtaining the desired structure. For controlling and directing the assembly, a combination of physical and chemical means is often required. The most important is the fitting of complementary molecular interactions in the assembling structures. (b) Template-assisted assembly. Serving as support media and templates, DNA, proteins and lipid bilayers allow precise positioning of different functional

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units, and, based on biospecific interaction, precise self-assembly can be realized. The assembled structures can be deposited on flat surfaces with the formation of self-assembled monolayers or Langmuir–Blodgett films (see Chap. 11 of Volume 1). With the application of supramolecular structures, in addition to realizing the sensor mechanisms described in Volume 1 of this book, the new possibilities appear for the sensor response. (a) They can generate the sensing effect by assembly-disassembly of subunits, directly or indirectly induced by the presence of target (Wang et al. 2016b; Zhang et al. 2017). This can be realized by operating with different molecular and nanoscale structures. The implied mechanisms of fluorescence response could be, for instance, an induction-interruption of electron or energy transfer between constituting units, a formation-disruption of H- and J-aggregates formed of organic dyes or association-dissociation of light scattering gold nanoparticles, as described in Volume 1. As an example, the self-assembly of pillar[6]arenebased structures into supramolecular polymer system generating spherical-like supramolecular nanoparticles was found to be induced by binding Hg2+ ions (Dai et al. 2019). This results in activating the aggregation-induced emission of appended organic dye. (b) These mechanisms can be adaptive in their molecular arrangement. The relatively weak nature of the intermolecular interactions that underlie supramolecular assemblies allows changing their configurations in response to a variety of external stimuli. Such stimuli can be produced by interaction with the target analyte. Thus, the principle of “induced and assisted assembly” (Demchenko 2001) is one of the possibilities that can be realized. (c) The molecular memory can be generated that can be an imprinted configuration of atoms and their groups that can persist for a long time. Such memory can be the most clearly observed with the designing of imprinted polymers, as we will see below (Sects. 6.4.5 and 6.4.6). (d) Molecular assemblies may attain new features, such as cooperativity. Cooperativity involves some interaction between molecular recognition sites, so that multiple interactions can either increase or decrease the affinity of particular site (Lehn 1995), see Chap. 1. Cooperativity is possible also with single sites when the binding of a ligand changes somehow the binding site, so that the binding of another ligand becomes more (or less) favorable (Hancock and Martell 1988). The structural and dynamic changes in the self-assembled structures can be coupled with the measurable changes in an optical signal, including the changes of parameters of fluorescence. Moreover, the collective properties of fluorescence reporters, as discussed in Chap. 8 of Volume 1, can be optimized on the level of supramolecular structures. There are different organic dyes that are able to emit fluorescence in the solid state (Anthony 2012; Shimizu and Hiyama 2010), and it is possible to use them for constructing a solid support. Porphyrins have been used frequently to construct supramolecular assemblies. The expanded porphyrins were found that can form supramolecular polymers with

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Fig. 6.4 The supramolecular constructions based on cyclo[m]pyridine[n]pyrroles (PmPn) and diacids as the building blocks (Zhang et al. 2015). Such assemblies are not formed with monoacids. When exposed to polar solvents and Lewis basic anions these and the related 1:2 ensembles undergo a change in structure, demonstrating the change of color and fluorescence emission that may be easily monitored by the unaided eye

several diacids (Fig. 6.4), and in this form they can be used as chemical sensors for both anions and organic solvents (Zhang et al. 2015). The solubility, color, and fluorescence of the assemblies changes dramatically when they are treated with Lewis basic anions or polar solvents, which decrease the extent of aggregation and the correspondent change in delocalization of π-electrons. The authors have demonstrated that under the conditions of simple mixing this system can be used as a sensor for identifying certain salts and various solvents by solubility, fluorescence or visible color change.

6.3 Fluorescence Sensing with Nanoporous and Mesoporous Materials Materials possessing nano-microscale porosity are quite common in nature, and porous carbon is a good example (Ariga et al. 2012a, b). Porous structures can be also formed by different metal oxides and different organic materials (Moritz and Geszke-Moritz 2015). Cellulose-based nanoporous materials are attractive due to their cheap price and broad range of applications (Fan et al. 2020). Of special interest

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is the mesoporous silica. A material containing pores with diameters between 2 and 50 nm is usually called a mesoporous material. Often “meso-” is added to different words indicating something in the middle. Regarding mesoporous materials, this means their pore sizes larger than several nanometers, but smaller than microns. Sensing here relies not only on specific interaction with the target but also on the limiting of site accessibility due to controlled pore sizes (check, that most proteins fit the 2–50 size range). The limited accessibility can help to shield the sensor receptors from interfering species, and the huge inner surface favors more specific molecular recognition.

6.3.1 Sensing Designed on the Basis of Mesoporous Silica The porous silica-based systems have a specific structure: they possess a 2D hexagonal arrangement of cylindrical pores (Fig. 6.5). This material exhibits a colossal specific surface area, of the order of 1000 m2 /g, and a homogeneous distribution of large volume (5–30 nm in diameter) of pores (Zhao et al. 1998a, b). The structural parameters, such as the diameter of pores and the thickness of walls, can be modified by tuning the parameters of the synthesis (Thielemann et al. 2011). The bulk of ideal silica is non-polar, but the walls of these structures are made of amorphous silica gel exposing the hydroxyls. Abundance of these silanol groups (Si– OH) on the inner surface of mesoporous silica materials allows different types of functionalization. Effective positioning of amine functional groups onto a surface with subsequent coupling of a variety of functional groups by well-developed synthetic means becomes possible (Fayed et al. 2014). A variety of other functionalization methods have been developed. In this way, different target receptor and reporter groups can be covalently attached in the pore volume, to make the sensors.

Fig. 6.5 The model of structure of SBA-15 type mesoporous silica (a) and a schematic illustration of the silica structure with surface hydroxyl units (b). The structure in (b) is simplified and presented in a 2D plane (Laskowski et al. 2019)

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The combination of the properties of organic and inorganic building blocks within a single material represents an example of hybrid organic–inorganic nanoporous materials, which is a current trend in material design. It is particularly attractive because of the possibility to combine the enormous functional variation of species offered by organic chemistry with the advantages of thermally stable and robust inorganic substrates. The common strategy here is to use porous inorganic materials as the templates and their organic derivatives as a flexible decoration (Fayed et al. 2014). Mesoporous silica thin films (Giménez et al. 2020; Innocenzi and Malfatti 2013; Zhao et al. 1998a, b) can be prepared by means of spin coating, and different methods exist for making microspheres of different size and porosity (Rama Rao et al. 2002). The porosity is engineered by manipulating with the size of cylindrical detergent micelles used as templates in the course of synthesis and washed-out after it (Fayed et al. 2014). Different compounds, such as drugs (Doadrio et al. 2015), can be hosted within the pores. Conjugated polymers, such as polypyrrole (Cheng et al. 2006) and polyaniline (Zeng et al. 2008) can be incorporated into the pores in order to achieve the electronic conductance on molecular level. Molecular fluorophores serving for reporting on binding ions were incorporated. Thus, N-pyrene-1-yl-succinamic acid and 4-(pyrene1-yl-carbomoyl) butyric acid were the reporters on Cu2+ sensing in multicomponent mixtures of other metal cations (Kledzik et al. 2007). Sensors for other metal cations have been suggested based on fluorescence quenching of incorporated organic dyes (Melde et al. 2008; Song et al. 2010). The incorporation of calixarenes labeled with fluorophores as specific recognition sites for ions have been described (Métivier et al. 2005). Many other mesoporous silica-based materials have been developed for sensing, recording the changes in relative humidity and in pH. They are able to detect toxic industrial compounds, volatile organic species, small molecules and ions and biologically relevant molecules (Melde et al. 2008). Looking from a biological perspective for sensing and diagnostic applications, miniaturization in the form of microarray platforms is achievable (Maciel et al. 2020). For near-IR imaging, the incorporation of J-aggregates of cyanine dye into silica nanoparticles was achieved (Chen et al. 2019). Still, there remains a notion that the sensor materials based on mesoporous silica are more preferable for electroanalytical rather than for optical detection (Rao et al. 2013; Walcarius 2015).

6.3.2 The Hydrogel Layers in Sensor Technologies The hydrogel layers allow high density of sensor immobilization. Hydrogels are the three-dimensional (3D) polymer networks that swell, but do not dissolve in water. They can be made optically transparent and fluorescent-free, being formed of different hydrophilic polymers, such as polyacrylamide and polyethylene glycol

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(Svedhem et al. 2001). Their development into 3D microarrays for DNA hybridization and protein detection (Rubina et al. 2003; Rubina et al. 2004) allowed increasing substantially the density of sensor locations. Since there are many possibilities for inclusion of fluorophores as the building blocks, the hydrogels are interesting materials for fluorescence sensing (Du et al. 2015). Different materials that can be made for serving as supporting media (Tavakoli and Tang 2017), transducers and even imprinted recognition elements, can be made in a sol–gel process. Both inorganic and organic/inorganic composite materials can be obtained by this procedure (Ahmed 2015). Among the hydrogel formers, polylacticco-glycolic acid (PLGA) and polyethylene glycol (PEG) are actively used, as well as natural polymers, such as polysaccharides, proteins, and DNA. The obtained transparent glass-like structures can attain different shapes and may be obtained with high porosity. The pore size distribution can be controlled by chemical composition and by the reaction conditions. Importantly, fluorescent dyes can be incorporated into these structures in several ways: by adsorption within the pores, by incorporation into reaction mixture (due to mild conditions of synthesis there is no decomposition of dyes), and also by covalent binding to finally obtained material. Covalent attachment should be preferred for obtaining the sensors of high stability. One of the proposed procedures for activation of hydrogels (Burnham et al. 2006) looks in the following way. Disulfide-crosslinked derivative of hydrogel is deposited on the surface of quartz or silicon. Then the application of reducing agent provides the generation of reactive SH groups throughout the hydrogel, leading to ‘activated hydrogel’. These SH groups can be readily modified with the attachment of proteins or other molecules, resulting in functional hydrogel. Affinity coupling in hydrogels allows important possibility. For providing efficient affinity coupling with the use of biotin-streptavidin pair (Xu and Wegner 2020), the SH-reactive biotin derivative is applied. It yields biotinylated hydrogel, which is ready for binding streptavidin. The latter one is particularly useful for immobilization of biotinylated aptamers (Schaferling et al. 2003). Since biotinylated proteins are deposited from solution, such system for immobilization is advantageous for deposition of proteins that need delicate conditions of treatment. Recently, the host– guest interaction with cucurbit[n]urils (Correia et al. 2019) and pillarenes (Wu and Yang 2019) has got popularity. Generally, in addition to increased binding capacity, an immobilization within three-dimensional hydrogels offers many advantages over binding to flat two-dimensional surfaces, such as providing a ‘wet’ and friendly polar environment with stabilizing effect of the gel matrix. A disadvantage is a slow rate of establishing the target-binding equilibrium that is limited by target diffusion in the gel. Different hydrogels are “stimulus-responsive”. When a hydrogel is used in sensing, it is usually one of the three main characteristics that is exploited on the integral level (Buenger et al. 2012): (i) their semi-wet and inert structure that predetermines hydrogels as host-network, (ii) the amplification effect of their sensitivity on a molecular level that is translated into macroscopic effects, such as a change in swelling degree (Fig. 6.6), and (iii) the ability to control the diffusion behavior of molecules through the polymer matrix. They may respond to changes of the

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Fig. 6.6 Swelling of hydrogel responding to analyte binding

environment conditions, such as pH, temperature, pressure, ion concentrations, by their swelling-shrinking behavior (Richter et al. 2008), which allows recording these changes by variation of intensity of appended dyes (McCurley 1994). The smart hydrogels based on DNA molecules have attracted substantial consideration recently due to their efficient response to chemical or physical stimuli (Khajouei et al. 2020). Moreover, various functional motifs, like i-motif structures, G-quadruples and aptamers (discussed in Chap. 5) can be inserted into the polymer network to offer a molecular recognition capability to the complex (Li et al. 2017). For sensing applications, DNA based hydrogels can be fabricated through either chemical linkage of DNA molecules or physical entanglement between DNA chains. Many applications have been reported (Wang et al. 2018a, b, c), including production of sensors for environment monitoring and clinical diagnostics. It was shown also that incorporation of dye-based fluorophores into a porous organic polymer skeleton may prevent the aggregation-caused quenching effect, because porosity allows the spatial isolation of fluorophores to maintain their emission (Li et al. 2015). Detectable changes in this emission allow easy determination of electron-deficient or electron-rich analytes penetrating into the polymer matrix. Due to large amount of void volume filled with water (up to 99% water of their dry weight) and possibility of broad-range variation of the extent of three-dimensional cross-linking, hydrogels are ideal for the construction of biosensors (Buenger et al. 2012). For different applications, they can be used in the form of thin films (White et al. 2013) and microspheres (Le Goff et al. 2015). Hydrogel provides a protection and coating function to sensor parts, preventing undesired interaction with biological molecules or cells. Their open porous structure and hydrophilic environment allows diffusion of analytes through the hydrogel matrix. Beyond simple protection, hydrogels can be used as immobilization matrix for the biosensing elements. Their sensing capability extends to DNA detection, immunosensing and application of aptamers (Tavakoli and Tang 2017).

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6.3.3 Porous Structures Formed of Organic Polymers Organic polymers, on their assembly, can form the structures with high and controlled porosity, usually on solid support. Porous organic polymers (POPs) (Zhang et al. 2020a, b) have attracted considerable attention due to their ability to form pores of several nanometers in dimensions, leading to the very high apparent surface areas (>1000 m2 /g) (Dawson et al. 2012). They demonstrate very high absorption power of small molecules and ions, and, with special design, to proteins. Their broadly variable chemical structure allows molecular recognition based on target size and affinity. The choice of material for porous sensor design can be made between conjugated polymers, conjugated-nonconjugated block copolymers and nonconjugated polymers (Mako et al. 2018). With this choice, one may select between fluorescence response generated over substantial peace of the structure (due to propagating excitons), more local generation of fluorescence signal (due to restricted exciton length) or the absence of light emission from the polymer. These polymers allow incorporating extra fluorescence reporters with the desired position and response properties. Conjugated polymers can imply their sensing properties directly. They are fluorophores possessing extended π-conjugation, resulting in excitonic effects that allow them to serve as the most efficient light harvesters and superquenchers (Volume 1, Sect. 8.5). When they form porous structures, their features are seen as being unique. They are not achievable in other porous materials, which are typically not πconjugated, or in conventional conjugated polymers, which are nonporous (Xu et al. 2013b). Because there is less limitation on size, geometry and functional groups, they offer high flexibility for the molecular design of conjugated skeletons and nanoscale pores (Lee and Cooper 2020; Taylor et al. 2020). They can systematically tune their π-conjugated porous architectures and allow for the optimization of the skeleton. Specific sites that interact with target compounds can be introduced into the conjugated chains to enhance the interaction interface, provide the selective target binding and improve the signaling activity. In view of these unique and very attractive features, we can state that the role of these materials in molecular sensing is definitely underestimated. The sensing is mostly limited to nitroaromatic explosive compounds that are strong fluorescence quenchers (Novotney and Dichtel 2013; Pan et al. 2019; Sang et al. 2015). Other applications are rare. They include sensing of some metal ions (Gao et al. 2020), dopamine (Gu et al. 2014) and aminoglycoside antibiotics in water (Bhunia et al. 2018). The abilities of conjugated polymers to demonstrate the fluorescence quenching by both electron-rich and electron-poor compounds (Lan et al. 2009), and to display high and versatile target-recognition capacity, must find broad range of applications. The polymer network can be composed of conjugated oligomers with precisely determined structures incorporated into general non-conjugated polymer network (Krywko-Cendrowska et al. 2020). This provides many new possibilities in constructing the species with optimal sensor abilities.

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The carefully designed monomers produce extended π-conjugation in the crosslinking network, which allows very high absorbance, excitation energy flow and superquenching. Essentially, these properties are restored in polymer network with high porosity (Chen et al. 2010). When conjugated polymers do not form the whole polymer network but only represented as oligomeric fragments incorporated into the structures formed by other polymers, their unique optical properties are also valid. More importantly, the combination of fluorescence and porosity would provide more space to transduce the molecular interaction between the adsorbed analytes and fluorophores to the detectable changes in light emission. Importantly, the conjugation polymer chains can be synthetically combined with organic dyes. This allows new possibilities for fluorescence response. Interesting in this respect is Nile Red (Zhang et al. 2014), the dye with very strong solvatochromism and solvatofluorochromism. It can serve the acceptor of excitation energy from the polymer and as the color-changing fluorescence reporter. BODIPY derivatives (Liras et al. 2016) and other dyes were also applied for this purpose. Thus, the structural characteristics and porosity of POPs can be easily tuned due to versatile building blocks and polymeric strategies for optimizing the sensors. A wide scope still remains in searching for distinct strategies to develop various fluorescence sensors built on their basis. Nonconjugated fluorescent polymers include the polymers that have fluorescent moieties appended to the nonconjugated polymer backbone as well as those that are composed of fluorescent backbone segments linked by nonconjugated, nonfluorescent linkers (Mako et al. 2018). Among their advantages are more synthetic pathways available to access the broader variety of non-conjugated polymer backbones, including free radical polymerization, step-growth polymerization, and ringopening metathesis polymerization. Also, involvement in the polymer assembly of macrocyclic hosts, such as rotaxanes, allows making them in a very precise configuration (Correia et al. 2019). Disadvantages of their networks, in comparison with of conjugated polymers, include the lack of specific excitonic effects and the resultant lower sensitivity of response. An example of using a nonconjugated fluorescent polymer was reported for the detection of calcium ions in extracellular environments (Ishiwari et al. 2016), see Fig. 6.7. Here, tetraphenylethene (TPE), which is known to become emissive on immobilization and on aggregation, was attached to an acrylate monomer, which then underwent free radical polymerization to yield a poly(acrylic acid)-derived gel. Exposition to the medium containing Ca2+ ions resulted in significant increases in fluorescence intensity, occurring linearly with Ca2+ concentration in a range 0.1– 10 mM. This response could be reversed upon addition of EDTA to sequester the Ca2+ cations. A good selectivity for these ions in the presence of other physiologically relevant cationic analytes was observed. Concluding this section, we can state that a huge amount of effort was made to design 3D structures based on inorganic and organic polymers. Different possibilities are realized to make them to serve as molecular receptors. A comprehensive review (Xia et al. 2020) allows seeing the state of this progress. All types of macrocycles

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Fig. 6.7 Design of Ca2+ sensors based on a polyacrylic acid (PAA) nonconjugated polymer with tetraphenylethene (TPE)-appended units (Ishiwari et al. 2016). a Chemical structures of PAA-TPEx and g-PAA-TPEx, where x, y and z indicate the molar ratios (contents) of TPE, PAA and cross-linker, respectively, and ran means that the monomer sequence is random, i.e., random copolymer. b The graph showing the relationship between Ca2+ concentrations in biological systems and applicable concentration ranges of typical Ca2+ indicators (Fura-2, X-Rhod-5N, YC-2.60 and G-CEPIA1er). c Schematic illustration of the proposed mechanism by which Ca2+ ions induce fluorescence enhancement

that are able to form host–guest interactions (see Chap. 3) can be incorporated into polymer networks that allow these interactions. Covalent organic frameworks (COFs) are a new class of crystalline organic polymers, in which the porosity is structurally predesignable, synthetically controllable, and functionally manageable (Geng et al. 2020; Jiang 2020). When formed of aromatic compounds, they demonstrate unique photophysical properties, including excitonic excited states and proton conductance. Interactions with analyte may provide perturbation of these properties, providing analytical signal (Liu et al. 2019; Qian et al. 2017). COFs can form versatile functional composites with metal organic frameworks (MOFs) that we will discuss below. Their excellent synergistic properties (Wu et al.) are encouraging in achieving tremendous versatility in sensor design.

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6.3.4 Metal–Organic Frameworks Metal–organic frameworks (MOFs) are ordered nanoporous inorganic–organic hybrid materials that can be simply self-assembled from their corresponding inorganic metal ions/clusters with organic linkers. Coupling of single metal ions or metal ion clusters linked together by organic ligand molecules allows formation of an extended, crystalline framework with nanoscale porosity (Fig. 6.8). Due to their inorganic–organic hybrid nature, MOFs can combine the inherent physical and chemical properties of both inorganic and organic photonic units that allow their potential applications in luminescence-based sensing and imaging (Cui et al. 2012; Hu et al. 2014; Lustig et al. 2017). There are diverse emitting mechanisms for tunable dual-emission that can be realized in MOFs (Chen et al. 2020a). They include the metal-centered (MC) emission (e.g. ligand-to-metal charge transfer (LMCT)) and ligands-centered (LC) emission (e.g. metal-to-ligand charge transfer (MLCT)), as it is seen in Fig. 6.8. LMCT and MLCT are mainly associated with relative height of the lowest excited state energy levels in MOFs. If the energy of the lowest excited state level of the organic ligand is lower than that of the metal ions, then the charge will transfer from the metal ions to the organic ligands to emit light, i.e. the process of LMCT. Conversely, the charge will transfer from the ligands to the metal ions, i.e. the process of MLCT. Usually, these processes of charge transfer are complete, and we have to observe only one band in emission. However, a single emission can become dual-emission after adding a characteristic analyst that can disrupt the charge to transfer, which may allow using the whole advantage of λ-ratiometry. The most efficient compositions producing the color-changing emission on sensing are those incorporating lanthanide ions and organic dyes. In this case, the λ-ratiometric response to target binding can be generated, and several possibilities

Fig. 6.8 Formation and operation of typical metal–organic frameworks (Chen et al. 2020a). a Metal ions can be assembled into framework in solutions. Metal nodes are shown as green spheres and organic linkers are yellow cylinders. b Illustration of the photoluminescence emission possibilities. The metal to ligand charge transfer (MLCT) occurs on metal (chelated) ion excitation and ligand that can be an organic linker. In ligand to metal charge transfer (LMCT), the light absorber is the ligand that can transfer its triplet state energy to the metal, resulting in its emission. The framework can also incorporate the ligand (shown as red sphere), demonstrating its own emission

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exist for that (Chen et al. 2020a). Benefitting from the fruitful energy/charge transfer process within the network and with analytes, the luminescence properties of MOFs are very sensitive to their structural characteristics, coordination environment, nature of the pore surfaces, and their interactions with guest species through coordination bonds, π–π interactions, hydrogen bonding, etc., thus providing the solid rationale to develop luminescent MOF sensors (Cui et al. 2018). Meantime, the road for the applications of MOFs in sensing and imaging technologies is not simple; one may find their both positive and negative features. Negative is the low stability of these structures in water solutions. This happens because the electrostatic interactions mainly stabilize the crystalline framework, and surrounding water dipoles decrease this stabilization energy. Recently the developed technologies using incorporation of zirconium (IV) into the MOF construction allowed achieving stable and efficient molecular sensors in water (Wang et al. 2016a). MOFs are known for high sensitivity of their luminescence parameters to both specific and nonspecific interactions with different compounds penetrating into their nanoporous network. The luminescence is sensitive to solvent, temperature, pressure. Moreover, these structures can breathe under the influence of external conditions, changing the properties of their pores. This factor can be both negative and positive, and, depending on the skill of researcher, allows designing highly selective sensors. Positive is the ability of both inorganic and organic components of structure to participate in emission and, due to independence and variability of these sensitive and insensitive contributions, the λ-ratiometric sensing response can be provided. The MOFs building blocks can be the dyes exhibiting the excited-state reactions, which extends the possibilities of reporting. Thus, the sensors for vapor of water was constructed based on the dye demonstrating intramolecular proton transfer (Chen et al. 2017). Moreover, fluorescent nanoparticles, such as carbon dots, can be incorporated as the guests in the network structure (Gu et al. 2017). Benefiting from these possibilities, the MOFs applications occupy a broad range of sensing small molecules in liquid and gas media, particularly of volatile organic molecules (Dong et al. 2014; Wang et al. 2018a, b, c). The methods of their detection in gas phase operate efficiently (see Sect. 8.2). In Chap. 9, their great potential in sensing the ionic species will be demonstrated. It can be realized by generating, instead of single-wavelength (monochromatic) fluorescent response, a switching dual emission based on interplay of intrinsic fluorescent sites and also of included molecular fluorophores and fluorescent nanoparticles.

6.4 Molecularly Imprinting in the Polymer Volume The multi-point noncovalent binding that is necessary for selective detection of analyte in a mixture with structurally close molecules can be achieved not only with the binding sites formed by natural or synthetic molecules or designed porous materials (Chen et al. 2016a). The recognition element of a sensor can also be a hole accommodating precisely the target molecule formed in the structure of synthetic

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polymer. Such accommodation can be made by providing a number of sterically fitting noncovalent interactions of target with the polymer matrix, and the polymer will get the “molecular memory” on this binding. The molecular memory introduced into the polymer allows selective rebinding for many times of the same target, i.e. realizing molecular recognition. This may allow realizing sufficient affinity for the binding of target in analytic procedure and selectivity against the binding of nontarget molecules (Alexander et al. 2006; Sellergren and Andersson 2000). As such, these sites operate by a “lock and key” mechanism to selectively bind the molecule with which they were templated during the formation of polymer structure. In a strict sense, the imprinted polymers are not the ‘molecular sensors’, they are the organized macroscopic bodies assembled in nanoparticles or on solid support, in which many recognition sites for the same target can be formed by one or several polymer molecules (Haupt and Mosbach 2000; Uzun and Turner 2016). This simple idea turned-out to be very profitable; the recognition units produced in polymers in this way compete in efficiency with their natural counterparts, such as the ligandbinding proteins and antibodies. It is frequently explored in both chromatographic separation technology and in drug delivery. In sensor technologies, it offers very interesting prospects that are not limited to molecular targets; it can be applied for recognition of bacteria (Cohen et al. 2010) and viruses (Bolisay et al. 2006; Pan et al. 2018).

6.4.1 The Principle of Formation of Imprinted Polymers Imprinted polymers are produced by assembling and co-polymerizing of synthetic monomers and oligomers to form a polymeric network in the presence of target molecule (the analyte itself or a molecule with a similar structure). This process is often called a ‘template-directed polymerization’. The target serves as a molecular template for creating a cavity in polymer matrix. The formed cavity is complementary to the template. In addition to steric fitting, this allows formation of many noncovalent interactions with the target that become fixed during the polymerization step (Haupt and Mosbach 2000). Moreover, the polymer composition can be chosen in broad ranges to satisfy the optimal multipoint target binding (Chen et al. 2016a). More than 4000 polymerizable compounds can be applied for template formation (Piletsky and Turner 2002). In recent years, organically modified silica was used for synthesis in aqueous media, and 3-aminopropyltrimethoxysilane (APTS) and tetraethyl orthosilicate (TEOS) were chosen as functional monomer and cross-linker, respectively (Ma et al. 2015). After the polymer matrix is formed, the target is washed-out, leaving the ‘imprinted’ binding sites that are complementary in size and shape to the analyte (Fig. 6.9). The progress in polymerization techniques, in formation of molecular imprints and in the techniques of analytes trapping/detrapping is described in many publications (Chen et al. 2016a; Haupt and Mosbach 2000; Mosbach and Haupt 1998). The polymer composition can be selected by the choice of monomers and oligomers in

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Fig. 6.9 The steps of recognition sites formation in imprinted polymer. On the first step, the monomeric or oligomeric units are incubated with the target, so that their noncovalent bonds are formed. Then, polymerization fixes this arrangement, and after that the target is washed-out leaving the hole that can be occupied by the target in a highly selective way

the reaction of polymerization. In a process of synthesis, they can incorporate smaller recognition units, such as cyclodextrins (Komiyama et al. 2018), see Fig. 6.10. In addition, the polymer can be subjected to different post-synthetic modifications. So broad possibilities of variation of structure can make, if necessary, these polymers photo-responsive and responsive to pH and temperature (Komiyama et al. 2018). Variation of these parameters may be needed on the step of detrapping (Chen et al. 2016a). Imprinted polymers can be used in different formats, not only as films, but also as microspheres (Orowitz et al. 2020) and nanoparticles (Wackerlig and Lieberzeit 2015; Yoshimatsu et al. 2007); they can form thin films on supports and coat the supportive nanoparticle surfaces (Komiyama et al. 2018). They can be the parts of different composites including optical elements, such as photonic crystals (Chen et al. 2016b), optical fibers (Wren et al. 2014) and optical waveguides (Chiappini et al. 2020).

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Fig. 6.10 Preparation of molecular imprinted β-cyclodextrin (β-CyD) polymers recognizing oligopeptide templates. β-CyD bearing an acryl group was polymerized with the use of N,N' methylenebisacrylamide (MBAA) as cross-linker (Komiyama et al. 2018). Each of the hydrophobic groups of the template (e.g., indole and phenol) was bound by a β-CyD molecule, so that the β-CyD assembly as a whole memorized the template in terms of its solution conformation. Thus, subtle difference in the primary structure of oligopeptide was clearly distinguished

6.4.2 The Coupling of Molecular Recognition with Reporting Functionality In this section we have to address the key questions, why molecular imprinting, being such a clever idea applicable for sensing (Chen et al. 2016a), has resulted in multitude of efficient sensing technologies based on other modalities (e.g. electrochemistry or quartz crystal microbalance BelBruno 2018; Piletsky and Turner 2002), but regarding the fluorescence reporting the success is rather modest (Gui and Jin 2019; Yang et al. 2018). Based on already achieved results, we have to attain a clear vision, what are the problems and what is the perspective. The major problem dealing with nonfluorescent target is in the mode and efficiency of introduction of reporting functionality, which is not trivial in this case (Henry et al. 2005). In some lucky cases when the target itself contributes to reporter function (being a fluorescent dye, quencher or EET acceptor), the sensor performance is easy

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Fig. 6.11 The popular ways to introduce fluorescence reporting into molecularly imprinted polymer sensing. a Realization of competition assay. The target (T) displaces the fluorescently labeled competitor (C) changing the parameters of its fluorescence. b The fluorescence reporter is a part of the polymer matrix. Interacting with the target, it changes parameters of its fluorescence

(Gui and Jin 2019). For instance, the traces of cancerogenous benzo[a]pyrene in water can be detected by the imprinted polymer binding, since this binding results in enhancement of intrinsic benzo[a]pyrene phosphorescence mediated by heavy atom incorporated into the polymer (Traviesa-Alvarez et al. 2007). Relatively simple is the realization of competitor displacement assays by applying the fluorescent competitor (Navarro-Villoslada et al. 2007), Fig. 6.11a. In this case, the competitor, which is the target analog with attached fluorophore, being replaced from the polymer binding site, changes the parameters of its fluorescence. It can also exchange energy with the EET donor or acceptor incorporated into polymer matrix (Descalzo et al. 2013). On a general scale, with a ‘silent’ target and the absence of competitor, the polymer itself should carry the reporting function by the detectable change of fluorescence emission (Rathbone and Bains 2005; Stephenson and Shimizu 2007). Thus, the two problems are coupled: the dye incorporation into polymer and obtaining the reporter signal from incorporated dye. The cavity becomes fluorescently responsive if the dye is inserted close to the bound target and perturbs its fluorescence, see Fig. 6.11b. On an early stage, many researchers attempted the incorporation of organic dyes into the polymer matrix on the step of polymerization process (Rouhani and Nahavandifard 2014; Xu et al. 2016), making them a part of polymer network (Inoue et al. 2013). The important problem appears when the fluorescent reporter appears to be located outside the imprinting cavity or encapsulated randomly into the material; then some or even considerable number of reporters will not participate in the target recognition event. Therefore, the imprinted polymers prepared with conventional dyes as fluorescent monomers commonly suffer from considerably high background fluorescence and signal heterogeneity, see (Deng et al. 2013) as example. More efficient is the specific incorporation of dye into the target-binding cavity. This can be achieved by dye conjugation with the target analog and its use on the step of polymerization and formation of cavities. In order to introduce a fluorescent reporter molecule exactly into the molecularly imprinted cavity, the technique of post-imprinting modification was developed, resulting in some lucky cases in efficient imprinted fluorescent sensor. Its formation can be achieved by post-imprinting treatment after removal of cavity-forming

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target (Sunayama et al. 2010). It was suggested to use a protein covalently conjugated to cleavable functional monomer that after template removal, allows for the introduction of the fluorophore only inside the cavity (Suga et al. 2013). To ensure proper functionality and fixation in the matrix, the dye molecules have to be covalently integrated into the polymer. This can also be done on the polymerization step by equipping a fluorescent dye with a polymerizable functional group. Then the dye can participate in formation of cavity being incorporated into polymer but containing cleavable bond(s) with the cavity-forming target (Wan et al. 2013; Wan et al. 2016). For achieving the transduction of binding event into fluorescence signal of matrixincorporated dye, a number of methods have been suggested (Alexander et al. 2006). This turned-out to be a difficult task, so that no general satisfactory solution of existing problems have been found (Stephenson and Shimizu 2007). It is hard, for instance, to explore the anisotropy sensing (see Volume 1, Sect. 3.3), because no free segmental rotation could be allowed in the polymer. Meantime, in view of limitations in other possibilities, such attempts have been made (Hunt and Ansell 2006). Due to existing difficulties, the application of lifetime or wavelength-shifting response (see Chap. 3 of Volume 1) is very rare and the primitive spectroscopic response providing the change of light intensity (Volume 1, Sect. 3.2) still dominates in this area. The sensors based on a single-wavelength emission do not need more than the single type of fluorescent material. Its typical realization is by photoinduced electron transfer (PET) mechanism (Volume 1, Sect. 4.1). Many organic fluorophores can “light up” upon binding to the analyte (Ivanova-Mitseva et al. 2012; Wan et al. 2013) by their screening from contacts with solvent water. The realization of twowavelength ratiometric response (Demchenko 2014) with the application of single reporter dye sensing by charge-transfer shifts, the dye protonation, intramolecular proton transfer or H-bonding is not very successful by now. At present, in the best way this can be achieved with two fluorophores, which is commonly realized in nanocomposites (see below).

6.4.3 Imprinted Polymers in the Form of Nanoparticles and Microspheres The use of imprinted polymers in the form of nanoparticles or microspheres offers a number of advantages. Particularly, it is a faster diffusion of molecules and particles and the easy possibility of mixing the sensors specific for different targets for multiplex sensing. Formation of these structures usually follows the core–shell principle. Many such hybrid structures can be constructed on nanoscale that can allow the recognition in suspension volume (Rico-Yuste and Carrasco 2019). The literature describes the application of fluorescent gold nanoclusters (Volume 1, Sect. 6.1), (Wu et al. 2015), lanthanide metal complexes (Volume 1, Sect. 6.3), (Özgür et al. 2020), and carbon dots (see Volume 1, Sect. 9.4), (Dai et al. 2017; Qin et al. 2020). It has

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become popular to introduce into the conjugated polymer, as fluorescence emitters, the nanoparticles (Zhao et al. 2012), such as quantum dots (QDs, see Volume 1, Sect. 7.2). High stability and brightness is their attractive feature, and variation of light intensity (target-induced quenching) is their typical mode of response. An example of successful design of molecularly imprinted sensor for 2,4,6trinitrotoluene (TNT) can be cited (Xu et al. 2013a). The sensor (Fig. 6.12) was constructed through a sol−gel seed-growth approach, using functionalized siloxanes as monomers and trinitrophenol as a dummy template, and then capped with CdTe quantum dots as fluorophores. The sensor shows selective binding affinity to TNT against other possibly competing explosive molecules with distinct fluorescence quenching occurring due to electron transfer. With the presence and increase in quantity of TNT in sample solutions, a complex formation occurs between TNT and the primary amino groups on the surface of the QDs, resulting in quenching of its emission. As well as in other imprinted polymer constructions, the introduction of wavelength-shifting and two-band ratiometric reporting functionalities (described in Sects. 3.5–3.6 of Volume 1) is in great demand (Demchenko, 2023a, b). It is technically easy by combination of two different fluorophore types to assemble the structures generating the self-calibrating λ-ratiometric fluorescence response (Demchenko 2010, 2014). In the imprinted-polymer sensors, the wavelengthratiometric sensing response was realized with the technologies using two molecular or nanoparticle fluorophores serving as the reporting probe-reference pair (Wang

Fig. 6.12 Schematic representation of CdTe quantum dots (CDs) encapsulated as reporters into molecularly imprinted sensor based on a sol–gel seed-growth method that provides the fluorescence intensity quenching response to trinitrotoluene (TNT), (Xu et al. 2013a). On TNT removal, the fluorescence is restored. TEOS is tetraethyl orthosilicate

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et al. 2018a, b, c, 2020; Xu and Lu 2015) or the donor–acceptor pair in EET (Wang and Yu et al. 2016c; Xu et al. 2017). Figure 6.13 illustrates the underlying mechanisms. In the case of probe-reference pairs forming the λ-ratiometric response, the mechanism that was described in Sect. 3.6 of Volume 1), the reference, that needs to be protected from interaction with the target, is usually located in the particle core, and the probes reporting by the change of intensity of corresponding band—in the surrounding polymer matrix. In the case of EET (described in Volume 1, Sect. 4.3), the responsive element should be the donor, providing the energy transfer to acceptor in modulated extent. It is important to note, that the requirements for optimal performance of participating fluorophores in these formats are mutually exclusive. The reporting and reference dyes should be excited at the same wavelength and provide comparative in intensities fluorescence spectra at different wavelengths. In order to emit independently, they should be structurally separated. In contrast, the acceptor dye in EET should not be excited at the excitation wavelength of the donor, since its own excitation reduces the light intensity variation in sensing (Demchenko, 2023a, b). It was, for instance, suggested to achieve the two-wavelength ratiometric recording by interplay of emission between the embedded blue-emitting carbon dots located in silica core and the green-emitting CdTe/CdS quantum dots (QDs) encapsulated

Fig. 6.13 Generation of wavelength-ratiometric fluorescence response to target (T) binding in molecularly imprinted polymer sensor based on interplay of emission spectra of two types of molecular or nanoscale fluorophores. a Reporter with reference format. Reporter (blue) emitting at the wavelength λ1 demonstrates the quenching (or enhancement) of fluorescence. The reference (red) emitting at λ2 does not change its emission. b Excitation energy transfer format. The donor (blue) emitting at λ1 provides the transfer of energy to acceptor (red) emitting at λ2 . The extent of transfer is determined by target binding. Dashed arrows indicate the emission of variable intensity

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into the mesoporous silica pores. They act as the reference and the response signal label, respectively, providing the λ-ratiometric fluorescence detection of diniconazole (Amjadi and Jalili 2017). A novel ternary-emission fluorescence sensor was proposed by post-imprinting mixing blue-/green-/red-emission bovine hemoglobin (BHb) imprinted polymers (b-MIPs, g-MIPs, and r-MIPs) at a proper ratio and realized the multiplexed and visual detection of BHb (Yang et al. 2019). The three molecularly imprinted proteins were individually embedded with blue-emission 7hydroxycoumarin, green-emission CdTe quantum dots, and red-emission CdTe/ZnS QDs. Upon interaction with BHb, the fluorescence of CdTe and CdTe/ZnS QDs were simultaneously turned off, whereas the 7-hydroxycoumarin turned on the fluorescence intensity. In comparison with common dual- or single-emission sensors, the ternary-emission fluorescence sensor provides a wider color variation covering the green−red−blue window for accurate naked-eye determination of BHb, as well as a lower detection limit. Considering sensor technologies involving quantum dots incorporated into polymer matrices, one has to account their relatively large size for being the cavity formers. This produces difficulties of their location at the required close distance to the bound target for generating the PET quenching, in view that this reaction is extremely distance-dependent (Volume 1, Sect. 4.1). The same refers to other nanoscale reporters.

6.4.4 Exploration of Collective Properties of Fluorescent Dye Aggregates and Conjugated Polymers Being in a special arrangement of H- and J-aggregates, the organic fluorescence dyes, as well as conjugated polymers possessing ordered monomer groups, attain unusual excitonic properties (see Volume 1, Chap. 8). They are poorly known and explored as fluorescence reporters in sensor technologies based on molecularly imprinted polymers. However, they are very prospective for application. Among their general properties is the ability of superquenching—the quenching of the whole ensemble of fluorophores if one of its members is quenched. Most prominently this effect is observed for J-aggregates of organic dyes (Bricks et al. 2017), for H-aggregates of these dyes and, possibly, for carbonic nanostructures (Demchenko 2019). They are well described for conjugated polymers (Zhang et al. 2020a, b). The possible application of this effect is illustrated in Fig. 6.14. In the case of excitonic H- and J-aggregates of organic dyes, even on the level of their dimers, the absorbance is greatly enhanced, but the quenching of one molecule quenches the whole aggregate providing dramatic quenching effect on target binding. The aggregates are self-assembled and can be incorporated into polymer. The researchers may be detracted by low stability of these aggregates, but the methods of their stabilization already exist (Bricks et al. 2017). Such sensing technology is waiting for its exploration.

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Fig. 6.14 General idea on application of superquenching effect in the design of imprinted polymers. a The common case. The dyes are located in polymer matrix and only those contacting with a target in the cavity produce the reporting signal. b The dyes are electronically connected forming excitons (marked with solid arrows). The exciton propagation allows on the quenching of one dye exposed to ligand-recognition cavity to quench the whole coupled molecular ensemble. Other ensembles and individual dye molecules being connected by exciton diffusion (dashed arrows) may also be quenched. Exciton formation increases the brightness and the magnitude of response manifold: the quenching of one dye unit results in superquenching

Conjugated polymers (see Volume 1, Sect. 8.5) were very successful in applications in fluorescence sensing technologies (McQuade et al. 2000; Thomas et al. 2007), and substantial progress, still not realized in full, is expected from the combination of imprinting methodology with their responsive properties (Anantha-Iyengar et al. 2019). They can form polymer layers and nanoparticles with many useful and interesting properties, including very high fluorescence brightness, light-harvesting, excellent photostability, and sensing capabilities (Jiang and McNeill 2017). On interaction of quencher with a single unit, as much as hundreds of polymer chromophore units may result in superquenching and supersensitivity. Despite their unique obvious advantages, conjugated polymers and their easily formed nanoparticles did not receive proper attention in imprinted polymer design, and only sporadic publications appear. Thus, it was reported (Li et al. 2007) on success in detection of 2,4,6-trinitrotoluene (TNT) and related nitroaromatic compounds that are dangerous explosives and are known to be the most popular analytes for conventional conjugated polymer sensors. Figure 6.15 illustrates the performance of this sensor and the graph of its application in detecting the TNT vapor in kinetic regime. Here, TNT was imprinted via a covalently attached “dummy” template, which was removed through cleavage after polymer formation. The sensing response then showed the typical TNT interaction features (i.e. strong fluorescence quenching). The described polymeric sensor shows remarkable air stability and photostability, high fluorescence quantum yield, and reversible analyte binding. The reason for so low popularity of excitonic effects in designing imprinted polymers can be explained by their insufficient knowledge of their excitonic nature. A precaution exists, that the dye aggregate or conjugated polymer should always adapt as strong as possible to the specific geometrical requirements of a template, but this cannot always be done since an aggregate should be highly ordered, and a conjugated

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Fig. 6.15 Schematic design of trinitrotoluene (TNT) sensor based on fluorescent conjugated polymer (a) and the quenching effect of TNT binding on fluorescence spectra as a function of exposition time on its vapors at room temperature (b). Redrawn from Li et al. (2007)

polymer will at best be as rigid and planar as possible to guarantee the optimum delocalization length within the conjugated chain. In reality, some degree of flexibility is allowed in molecular excitonic structures, and such structure does not need to “wrap” the template. Its “gentle touching” the template may be sufficient for providing the superquenching effect.

6.4.5 Nanomaterials with Molecularly Imprinted Sensing Some authors have started to call the imprinted polymers the “artificial antibodies” or “plastic antibodies” (Hoshino et al. 2008) pointing to their high efficiency as target binding units. However, the two features distinguish them from real antibodies, their fragments and other peptide and protein sensors, discussed in Chap. 4. There are the size and valency of these materials. Can these polymers be designed in the form of small nanoparticles with single imprinted recognition sites? Success in such design may help in resolving their other weak points, achieving better water solubility and faster target binding kinetics. The introduction of novel nanotechnologies and surface chemistry into molecular imprinting strategy has opened this possibility (Awino and

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Zhao 2013; Guan et al. 2008; Xiao et al. 2020). The molecular imprinting nanotechnologies are expected to greatly enhance the molecular affinity of these materials, and thus provide a wider range of applications, approaching the biological receptors. Among many attempts to create the chemosensing imprinted polymer nanoparticles, two types of developments has attracted attention of the present author. One is based on dendrimers, in which the central core molecule (in original case, the porphyrin derivative) serves as a template (Zimmerman et al. 2003). Hydrolytic removal of the core afforded imprinted hosts for target recognition. The other case is the creation of small nanoparticle library formed by polymerization of monomers in the presence of peptide melittin (Hoshino et al. 2008). After melittin is removed by dialysis, the best melittin binders could be selected. More complicated is the application of cross-linked detergent micelles (Zhang and Zhao 2019) that will be discussed in Sect. 7.5. These nanoparticle forms of imprinted structures are expected to greatly improve the binding capacity and achieve higher site accessibility together with faster kinetics of target materials. A more homogeneous distribution of recognition sites can also be achieved. Nanostructured, imprinted materials have a small dimension with extremely high surface-to-volume ratio, so that most of template molecules can be located in the proximity of particle surface, which should accelerate the target diffusion. However, non-specific binding may also be increased. In Sect. 7.5 we discuss the possibilities for nanoparticle-based surface imprinting. An interesting possibility is to provide the fluorescence reporting function by applying as the particle core the carbon dots, semiconductor quantum dots or nanoparticles composed of organic dyes (Xiao et al. 2020). In this case, the target bound to particle shell formed of imprinted polymer could provide the recordable perturbation of fluorescence signal.

6.4.6 Formation of Nanocomposites with Molecular Imprinting Functionalities Based on molecularly imprinted polymers as recognition elements, a multitude of architectures are conceivable, combining, for instance, a magnetically functionalized core with a thin polymer shell or a polymer shell surrounding a dye-labeled core. In principle, any combination of polymer and silica, and also the designs integrating any nanoparticles, organic dyes and clusters of noble metals, can be imagined for constructing such composite systems. Therefore, it is quite natural to design multifunctional and multimodal composites that combine different, not only molecular recognition, functions (Ma et al. 2015). The results on obtaining the nanocomposites combining molecular recognition and sensing with magnetic platform for detection of fungicide λ-cyhalothrin has been developed on the basis of paramagnetic Fe3 O4 nanoparticle serving as the nanocomposite core (Gao et al. 2014), Fig. 6.16. Thin layer of 3-(methacryloxyl)

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Fig. 6.16 Schematic illustration for the preparation of fluorescent core–shell magnetic molecularly imprinted polymer nanoparticles, Fe3O4/SiO2-MPS/MIPs (Gao et al. 2014). Here MPS = 3-(methacryloxyl) propyl trimethoxysilane, MIPs = molecularly imprinted polymers, Fe3 O4 /SiO2 MPS serve as core, MIPs as shell. TEOS is tetraethyl orthosilicate. A surface molecular imprinting technique was used for optical detection of traces of λ-cyhalothrin. To form the recognition sites, the shell was first prepared by copolymerization of acrylamide with a small quantity of allyl fluorescein in the presence of λ-cyhalothrin. The magnetic Fe3 O4 /SiO2 -MPS/MIPs exhibited paramagnetism, high fluorescence intensity, and highly selective recognition

propyl trimethoxysilane protects the fluorescein derivative incorporated into polymer from its fluorescence quenching and serves as reporter to target binding, responding by variation of its emission. The trace amounts (in a concentration range of 0–50 nM) of λ-cyhalothrin in food samples can be detected in this way. In addition, magnetic separation can be applied for capture of bound fungicides after the detection. To say in conclusion, it is amazing that there is virtually no limit to size and chemical nature of analyzed compounds that can be detected with molecularly imprinted polymers. They have been developed for small organic molecules, such as steroids, for amino acids, sugars, drugs, pesticides, and also for proteins and even the cells (BelBruno 2018; Haupt and Mosbach 1999; Saylan et al. 2017). In contrast to sensors based on biological macromolecules, these polymeric binders are resistant to adverse environmental conditions such as heat and extremes of pH, they are durable and inexpensive. Their affinity and selectivity can approach that observed for biospecific molecular recognition (Hillberg et al. 2005). They offer good potential for applications in sensor array technologies (Shimizu and Stephenson 2010), optical waveguides (Ton et al. 2015) and even for live cell imaging (Vaneckova et al. 2020). Molecular recognition is achieved through hydrophobic, hydrophilic and electrostatic interactions towards specific target sites (Zeng et al. 2010), so they may be

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termed “plastic antibodies” (Takeuchi and Sunayama 2018). Still, the devices based on this technology did not become true competitors of sensors based on recognition by biological structures, such as aptamers or antibodies. There are several reasons for that. It is hard to avoid broader structural heterogeneity of recognition sites, slower binding-dissociation kinetics of the targets, poor solubility, etc. Rather difficult problems are in building-in signaling elements for fluorescence reporting and amplification of fluorescence signal (Gui and Jin 2019; Xiao et al. 2020). Thus, these techniques need further improvement.

6.5 Sensing and Thinking: Extending the Fluorescence Sensing Possibilities with Designed and Spontaneously Formed Nano-ensembles Scaling up from small single molecules to larger molecular ensembles aims at achievement of two important goals: improvement of molecular recognition, especially with the targets of large size and complexity, and exploitation of collective properties of fluorescence reporters that offer enhanced sensitivity. Reading this chapter, we imagine a myriad of opportunities for manipulating with interactions of molecules, nanoparticles and surfaces. Since there is no limit regarding the sensor construct, the choice of optimal solution can be made on a very broad scale. This may involve not only molecular complexes and responding nanoparticles, but also the self-assembled supramolecular structures, composite nanoparticles (that combine recognition and response properties) and various types of surfaces and interfaces decorated with functional units. Are these possibilities used in full? The optimized libraries of sensor building blocks still are seen only in perspective, promising such a broad choice. Synthetic strategies for obtaining supramolecular structures based on covalent cross-linking, affinity coupling, self-assembly and coupling to surface, offer many advantages. They include (i) the minimization of synthetic work, (ii) the ease of modification and optimization of the sensor, (iii) the possibility to tune its properties by a simple adjustment of the ratio of components. Self-assembly of composite sensor units is based on the same principles as the sensing itself—on complementarity between interacting partners and formation of non-covalent interactions between them, with the possibilities of stabilizing covalent cross-links. These structures can be highly stable, with or without support. Broad room for optimization is available here. Modular design of sensor elements tend not only to combine the target binding and the fluorescence response but also to improve this response by creating optimal supramolecular ensemble, allowing activating optimal communication and signal transduction between these elements. The self-assembled systems allow great possibilities for introduction of fluorescence response functionality and thus for providing additional important properties. In the assembly, the receptor and the reporter units

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may not interact directly, and the signal on target binding can be transmitted due to their spatial closeness ensured by the template and even indirectly via the change of integral property of nanoscale system. The weak point of all these techniques is the absence of efficient generally applicable method(s) of providing the fluorescence response with the broad-scale selection of fluorophores and of their response mechanisms, such as seen over the whole Volume 1 of this book. Among the methods of fluorescence detection (described in Chap. 3 of Volume 1), the simplest recording of light intensity dominates, and efficient implementation of other methods remains for the future. A room for thinking and creativity exists regarding every subject presented in this chapter. For instance, can the molecular imprinting concept be applied for metal– organic frameworks? Or with covalent organic frameworks? Clearly, this relatively new field of development of fluorescence sensors is far from being mature. Many of the results discussed above were reported in preliminary or proof-of-concept stages with minimal testing for interference, recoverability, and long term stability. These features should improve with time, and the formation of more interdisciplinary collaborations aimed at adaptation of new materials to applications involving environmental and biological sensing is expected. On a route to success, a higher creativity is required from the researcher. Questions and problems addressed to the reader are the following: 1.

Explain the principle of multivalency and the potentials of its realization in assembled supramolecular structures. 2. Evaluate the possibilities of self-assembling the supramolecular structures in terms of production and performance. Explain their potential advantages and disadvantages over molecular-scale sensors. 3. How the supramolecular structures are formed? Is molecular flexibility of the partners needed? 4. Count the most efficient support materials. Can they perform as optical signal transducers? As the EET donors or acceptors with organic dyes? 5. Thin layers of gold as support material, why they are attractive and what is their role? 6. Why the self-assembled nanoscale materials with core–shell composition are so attractive? How they can combine recognition and response properties? 7. Compare the advantage of DNA scaffold and that made of synthetic polymers. How does the DNA structure allow incorporation of bulky substituents? Provide the modeling and compare your predicted structures with the results on porphyrins-DNA composites described in ref. (Fendt et al. 2007). 8. How the macrocyclic compounds, their host–guest complexes and substituents of different polarity are used for the creation of self-organized network? 9. Is it possible to design the partner for self-assembly based on the known threedimensional structure of the other partner? On what principles this design should be based? 10. What are the hydrogels? What is the size range of their pores? How the sensing is provided with them and what are the possibilities for reporting?

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11. How to generate the λ-ratiometric response to target binding in metal–organic frameworks? 12. In the case of imprinted polymer, what are the possibilities to provide fluorescence response in the following cases: (a) if the polymer does not contain fluorescence reporter? (b) if it contains the reporter that is not contacting with the bound target? (c) if fluorescence reporter is located within the target-binding cavity? 13. Suggest the possibilities to locate the reporter in the imprinted polymer cavities available for direct contact with the target. What detection techniques could you suggest to use in this case?

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Chapter 7

Fluorescence Sensing Operating at Interfaces

Determination of composition, structural arrangement and dynamics of molecules and their groups of atoms at the air–liquid, liquid–liquid and liquid–solid interfaces are extremely important for understanding various phenomena related to molecular recognition in these systems and to the design of efficient sensors. Different technologies that allow formation of functional surfaces are discussed here. The sensor receptors and fluorescent reporters can be self-organized on a surface acting as a template. The sensing technologies should be simple and convenient in applications. They include formation of self-assembled monolayers and Langmuir–Blodgett films exposing their functional groups. Monolayers of conjugated polymers play a special role of not only as target binders but also as of extremely efficient fluorescence reporters. Porous materials can be of different kind—from nanoporous silica to metal–organic frameworks. They allow dramatic increase of surface areas and impose the size-dependent selection of potential targets. The surfaces and interfaces may influence dramatically the binding affinity of different ligands (usually, increasing it), which should be accounted in sensor design. Finally, the surface imprinting technology is discussed. It can be realized both on flat surfaces and in nanoparticle format. When sensing the impurities in air and different gas media, the most convenient could be the sensors exposed on solid surface, but not dispersed in liquids. Solid surface can be made nanostructured or highly porous, dramatically increasing the surface. Naturally formed porous materials synthesized on solid supports demonstrate their increasing importance for sensing technologies. When the target is sensed in liquid phase, the interface with solid can also be of great use. It allows designing a strict pattern of interactions that is important for recognition of large molecules, such as proteins. Such pattern can be imprinted and allow without change of structure the multiple binding-release steps for the target. Designing the sensors, we have to account that close to the surfaces, the solvents change their properties, and the pre-surface areas become enriched or dispossessed of solutes. Similar effects are observed at interfaces of immiscible liquids. Solute © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_7

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concentrations, distribution of target and non-target species, may change dramatically in thin layers at interfaces (Benjamin 2006). The formation of solute coronas (wellknown in protein research) is the limiting case of these distributions. The active surfaces allow not only dense assembly of recognition units but also of fluorescence/luminescence reporters. We need to know their spectroscopic behavior if they interact being located at close distances. Attractive in this respect are the Hand J-aggregates that can be formed on surfaces from organic dyes, generating the collective effect of fluorescence enhancement. The other systems with such collective excitonic properties are the conjugated polymers. Sensors based on imprinted polymers, discussed in previous Chapter, are in active development. The surface imprinting that will be discussed here allows avoiding some disadvantages of imprinting in polymer volume, particularly the slow target bindingrelease rates. The surface-imprinted sensors can be made both on flat surfaces and on nanoparticles, which opens a broad range of new possibilities.

7.1 The Structural and Dynamic Properties of Surfaces and Interfaces When we design the sensors, it is important to understand the structure, dynamics and interactions at liquid–liquid, liquid–solid and solid–solid interfaces and at the liquid and solid surfaces exposed to air. Such surfaces are able to exhibit the interactions that are not present in constituting bulk media and that must influence on both molecular recognition and fluorescence reporting. The sensors (or their components) may demonstrate not only preferential location in one of interfacing phases but also a strong specific affinity to interfacial region. The small-size organic dye molecules are commonly used to characterize the interfacial polarity, fluidity, solvation dynamics, statics and dynamics of translational and orientation diffusion of solute molecules. The surfaces and interfaces that will be discussed in this Section are depicted in Fig. 7.1.

7.1.1 Gas–Liquid Interfaces The polar liquid surfaces contacting with air or other gasses are probably the simplest cases, in which the fluorescence probes can successfully combine surfactant and reporting properties (Rüttinger et al. 2018). The experiments demonstrate the wellexpected results: they often show the properties intermediate between vacuum and bulk solvent, conforming to the ‘half hydration’ concept (Watanabe et al. 2010). Meantime, it is obvious that the obtained estimates of interfacial polarity depend strongly on the probing dye and on its interactions at the interface.

7.1 The Structural and Dynamic Properties of Surfaces and Interfaces

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Fig. 7.1 Schematic representation of fluorescence reporter dye located at interfaces. a The liquid surface exposed to air or any gas. The species adsorbed on the surface are oriented with their stronger interacting part to the liquid phase. So does the fluorescence probe that has affinity to the surface. b The interface between non-mixable liquids (low-polar above and highly polar below). The solutes are distributed between the phases according to their polarities. The amphiphilic probe has high affinity to interface. c The liquid–solid interface. The probe can be a part of solid structure or can be adsorbed on the solid surface. d The solid–solid interface. The charges may be adsorbed at an interface. The probe may be a part of structure of one of the solids or incorporated at the interface

The coupling of probe response to such properties as the surface tension and surface diffusion is probably more important than the determination of ‘polarity’. The application of Malachite Green dye as viscosity indicator has shown higher local viscosity at the interface than in bulk water demonstrating a substantially retarded solvation dynamics (Pal et al. 2010). It was shown that the rotational dynamics of the probes at the air/water interface is influenced by the surface-active agents, especially by such strongly charged surfactants as sodium dodecyl sulfate (SDS). The rotational dynamics of the Coumarin 314 probe is three times slower in the presence of SDS, although the dependence of this effect on the surfactant charge density is rather weak (Nguyen et al. 2006). Molecular dynamics (MD) simulations revealed atomic details of the interface organization and of the localization of the probe at different surfactant coverage. The probe can be lying adjacent to two-dimensional cluster of the surfactant molecules or be embedded within the compact surfactant domain. There is a gradual transition from parallel to perpendicular dipolar alignment of the probe with respect to the interface as the concentration of surfactant increases (Pantano et al. 2005). The role of the hydrogen bonds in the dynamic behavior of the vapor-water interface was studied by means of the femtosecond pump-probe spectroscopy. It was shown that different solvation dynamics at the surfactant-modified interface is

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attributed to rearrangement of the hydrogen bonding network of water molecules near the interface (Benderskii and Eisenthal 2000). The atomistic details of the interfacial dynamics obtained in MD simulations have shown that at interfaces the relaxation of hydrogen bonds is substantially slower than in bulk water (Liu et al. 2005). The MD studies of the liquid–vapor system are not limited to pure water. The studies of the trimethylamine-N-oxide (Paul 2009), nitrobenzene (Johnson et al. 2009) and ammonia (Paul and Chandra 2005) mixed with the water–vapor systems were also reported. In the case of nitrobenzene the surface rotation is much faster than in the bulk liquid, but it slows down and approaches the bulk behavior as the solute becomes more polar. The reorientation dynamics of the nitrobenzene is quite anisotropic, with out-of-plane rotation faster than the in-plane rotation (Johnson et al. 2009). Experimental data (Sovago et al. 2009) demonstrate that at the water–air interface the hydrogen-bonded region resembles the bulk water. In contrast, for silica–water and lipid–water interfaces, interfacial hydrogen bonding is substantially stronger. It is rearranged with a larger degree of heterogeneity (Fayer and Levinger 2010).

7.1.2 Liquid–Liquid Interfaces These interfaces are formed between contacting liquids that are immiscible and differ in many properties, such as polarity, electronic polarizability and association via Hbonds. The strongly amphiphilic dye molecules (containing both apolar and polar sites) can act as surfactants concentrating at the interface between these liquids and reporting about the properties of the interface (Perera and Stevens 2009). Formation of supramolecular structures and aggregation of nanoparticles occurs at interfaces (Binder 2005). Many questions can be addressed in the studies with fluorescent dyes possessing such properties. Are these interfaces sharp or there is a continuous transition from one phase to another? If such transition area exists, what is its width? How this width depends on the ability of contacting solvents to associate? If one of the solvents is a strong H-bond former (e.g. water), what happens with its unsaturated H-bonds at the interface? It is not easy to resolve these questions in view of the size of organic dye probes. Some dyes sow the distribution between two types of solvation sites (Bessho et al. 1997; Pantano et al. 2005), and some are located exactly at the interface (Ishizaka et al. 2001). Moreover, the dyes with some difference in structure may demonstrate different location and also the excited-state dynamics, as it was shown in the studies on phospholipid membranes by the present author many years ago (Demchenko and Shcherbatska 1985; Gakamsky et al. 1992). The relaxations involve multiple time scales corresponding to contributions from both solvents and from the unique structural and dynamic properties of the interface. The dyes consisting of charged sulfate group and small hydrophobic dye separated by linker of different length and located at the interface between different alcohols and water demonstrated gradual polarity-dependent shifts of spectra as a function of

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linker length (Steel et al. 2004). This and similar results (Leich and Richmond 2005) suggest (but still do not prove) the existence of transition zone between contacting liquids. Meantime, several experiments and theoretical studies suggested that the liquid/liquid interfaces are molecularly sharp and therefore can be modeled by a simple ideal plane (Neugebauer et al. 2005). Whatever is the width of transition zone at the liquid–liquid interfaces, the reader should mark three important points that are derived from these studies. (i) The interface between two immiscible solvents forms very strong gradients of polarity. This means that for very different solutes (such as our targets) their affinity to interface may be much stronger than the solubility in contacting solvents. (ii) The rates of two-dimensional diffusion of solutes at interfaces are different from that in volume and may be strongly retarded. The two-dimensional “viscosity” is not a direct function of viscosities of contacting solvents. (iii) There may be specific effects at the interface due to uncompensated H-bonding and the appearance of interfacial charges. (iv) The local concentration of different (and, particularly, amphiphilic) solutes can be much higher than in bulk phases, leading to self-aggregation and formation of nanostructures (Molina-Osorio et al. 2020; Yamamoto et al. 2017).

7.1.3 Solid–Liquid Interfaces In solid–liquid interfaces, some interactions that are saturated in bulk liquid appear unsaturated with some new interactions formed (Zaera 2012). Stability of suspensions of nanoparticles formed by these solids is determined by these interactions, as well as the efficiency of catalyzed reactions at an interface. Meantime, the researcher should be extremely cautious in application of fluorescence probes, since being immobilized on solid surface the dyes may change dramatically their photophysical properties, up to generation of new bands (El-Rayyes et al. 2005). The method of resonance-enhanced second harmonic generation (SHG) could be used with great success to study solvation of such molecules as p-nitroanisole or indoline at different solid/liquid interfaces (Brindza and Walker 2009). SHG spectra of these compounds adsorbed to silica and solvated by organic solvents interfaces allow to probe either interfacial polarity or interfacial hydrogen bond donating/accepting abilities. SHG results show that the interfacial polarity probed by p-nitroanisole depends strongly on solvent structure, while the hydrogen bonding interactions probed by indoline are insensitive to solvent identity but depend on the hydrogen bond donating properties of the polar silica substrate.

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7.1.4 Solid–Solid Interfaces Intermolecular interactions at solid–solid interfaces are critical for the function of different composite materials, since the interfacial regions determine many properties, such as their strength, thermal stability and durability. Their importance is well recognized, particularly for the design of polymer-based sensors but the possibilities for their studies are still very limited and are frequently reduced to determination of the glass transition temperature (T g). Above T g, the material behaves like a supercooled liquid. Lowering the temperature brings rigidity to the polymer matrix. Due to the low weight fraction of the interface region, the conventional techniques of thermal analysis are not adequate to characterize such thermal transitions. At polymer interfaces, the T g change and its range broadens indicating the change of molecular organization and dynamics (Lenhart et al. 2001). A number of experimental and theoretical studies describe a spatial heterogeneity at the glass transition that can be significantly perturbed by the contact with odd surface. The glass-polymer composites are technologically important for many sensor designs. They allow chemical grafting a fluorescent probe onto an organosilanemodified glass surface with subsequent formation of polymer on this surface (Lenhart et al. 2000). This suggests an efficient way to monitor the chemical and physical properties of the buried polymer/glass interfacial region by obtaining the signal only from the dye located at interface. Meantime the level of application and analysis of spectroscopic data is rather poor and commonly does not go beyond empirical ‘polarity’ and ‘viscosity’ correlations. Ultrathin polymer films of several nanometers in the form of flat layers or as monolayer and multilayer shells of nanoparticles have found a variety of applications. Fluorescence studies have shown that in these films incorporated into fibers the surface-induced perturbations can propagate away from a fiber surface to induce the formation of a three-dimensional zone with the properties different from those of the bulk polymer (Ellison and Torkelson 2002). The properties of these films strongly depend on interaction with substrates (Sulpizi et al. 2005). Such findings indicate that the dynamics of nanoscale layers can be greatly influenced by adjacent domains. The fluorescence probing of the interfaces between glass fibers or silica particles coated with silanes and epoxy polymer (Olmos et al. 2003, 2005) demonstrated that the curing reaction at the interface proceeded faster than in the polymer bulk. There is a special interest to interfaces formed by water (Björneholm et al. 2016). The interface weakens the interactions between adjacent water molecules that cannot be substituted by bonding with organic molecules. This results in significant interfacial structuring and molecular orientation on both sides of the interface. Upon solute addition, the H-bonding between neighboring water molecules must change to adjust to its new solid, liquid, or gaseous neighbor. Concluding this section, we must stress the following. Surfaces and interfaces present the strongest molecular-scale gradients of many physical properties. Located at the interface, organic dye molecules experience strong anisotropy in these interactions, and being structurally anisotropic themselves, they adopt preferred location

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and orientation together with some distribution around these preferred values. This means that any molecules that are devised for molecular recognition may be preferentially oriented and demonstrate the changed intermolecular and intramolecular dynamics. Regarding fluorescence reporters, due to very steep gradients at an interface, a fine change of their position and orientation may produce a dramatic change of their response.

7.2 The Self-assembled Functional Surfaces Many sensor devices for locating the receptor and reporter units use solid support in the form of plain surfaces, microspheres and nanoparticles. Sensor events occurring at interfaces have relevance to biological systems, such as biomembrane and organelle surfaces, where the molecular recognition occurs mostly within the interfacial environments. As it was shown by modeling, the binding constants here may be much higher, than in neat solvents (Ariga et al. 2007), see Sect. 7.4. Due to the presence of strong gradients of local electric fields, of polarity, and water accessibility, the reporter dyes may demonstrate much broader range of response (Demchenko and Yesylevskyy 2011). Surfaces are active participants in sensor design. We observe the broadening of the concept of molecular imprinting, which now involves, together with recognition in the volumes of polymer structures (Sect. 6.4), also the surface imprinting of macromolecules, viruses and cells based on the surface-assembled monolayers (Wang et al. 2018c). The latter requires the formation of “smart” molecular layers with the aid of reactive surface-exposed groups. These layers can be easily decorated to produce unique structures.

7.2.1 Formation of Functional Surfaces Formation of an organized assembly can be achieved by self-organization of receptors and fluorescent reporters on a surface acting as a template (Arduini et al. 2007; Guan et al. 2015). In this way, one can form a construction, in which the target binding and reporting units do not interact directly and the communication between these two units is determined only by their spatial closeness ensured by the template. Already in this simple construction, there are many possibilities for modification and optimization of the sensor by simple adjustment of the ratio of components. With the combination of covalent binding and self-organizing methodologies, the more complicated multifunctional sensors can be constructed. In this Section we discuss different technologies for formation of surfaces with active and passive role in sensing. In Volume 1, Chap. 11, we discussed the formation of different surfaces, focusing on them as on passive supports and as on fluorescence reporter carriers. Here we

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overview the abilities of self-assembled surfaces to carry the recognition functions and to provide these functions in sensor design. Commonly, small molecules forming functional unimolecular layers carry different functional groups on their opposite ends. One is for binding supportive template and the other for performing recognition function or binding the recognition unit. There are different possibilities for forming the active surfaces based on such molecules (Ariga et al. 2015): (a) The surface-assembled monolayers (SAMs). They are well-known structures consisting of a single layer of organic molecules chemically grafted on a surface (Volume 1, Sect. 11.1). SAMs typically behave as robust conformal coatings and dense packing of a uniform thickness (1–3 nm), allowing the molecules to function as ultrathin coatings with predetermined surface chemistries. Further chemical modifications can substantially broaden the range of possible functional groups at the surface. Strong affinity to support surface is needed, and therefore alkanethiols are usually used for gold surfaces (Srisombat et al. 2011) and alkylsilanes for inorganic oxides (Pujari et al. 2014). (b) Langmuir–Blodgett films (Acharaya et al. 2009; Ariga 2020; Roberts 2013) are based on the effects of polarity and layer-by-layer arrangements that are formed by electrostatic forces with the charged surface (Chapel and Berret 2012). Multilayers can be organized by alternating layers of the different polarity or opposite charge. If these layers are formed by polyelectrolytes, they are stable enough, and their chemical bonding with support is not required. (c) Polymer brushes are the type of grafted surfaces that are formed by polymer chains extending from the surface (Nguyen et al. 2012; Park et al. 2016). Uniform polymer brush films can be tailored to precise thicknesses with appropriate polymerization methods. Many types of copolymers can form brush layers and are suitable for grafting, allowing a large degree of customization. Grafting is a method, in which existing polymer chains are covalently joined to a support surface that has been modified with an assembled layer of linker groups. Or, instead, the polymer brush can be formed after the linking, by providing the chain growth. When the sensor molecules, nanoparticles or supramolecular structures are immobilized on the surface, they cannot diffuse freely in solution and mix with other sensors. Many attractive analytical advantages can be derived from sensor attachment to the surface. We indicate several of them offering new possibilities. (a) The sensors composed in this way allow their use for many times with simple sample addition-washing protocols or providing continuous monitoring in the flow. (b) In such many-use systems, the reagent additions, if needed, can be easily applied. This can be done, for instance, by the addition of fluorescent competitor. (c) Due to the fixed spatial separation between sensor molecules, the sensing of different analytes (multiplex sensing) can be made on the same plate simultaneously. For such multiplex sensing, the microarrays (sometimes called biochips) can be developed for simultaneous sensing of hundreds or even thousands of targets (see Chap. 15, Volume 1).

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The choice of methods of surface immobilization depends on the chemical structures of the sensor and the surface; these methods are well described in original literature and reviews (Sobek et al. 2006). They do not differ much from those that are used in other sensor technologies, such as surface plasmon resonance (SPR), where the immobilization on the surface is also needed. Meantime, each sensor and each application may demand its own solution; the demands on applied solid support are thereby as manifold as the applications themselves. Surface immobilization should satisfy many requirements, particularly, it should involve proper orientation of sensors and availability of receptor sites for target binding. The example of porphyrin binding to gold surface (Otte et al. 2014) demonstrates that proper distance and proper orientation between porphyrin units is achievable (Fig. 7.2). It influences the electronic properties of the system that can be used for efficient light harvesting.

Fig. 7.2 The surface assembled monolayer (SAM) structures with porphyrins upright on a molecular platform (Ariga et al. 2015). Such platform approach enables controlling both the distance between porphyrin chromophore units and their orientation relative to the surface. In the array, the free-standing porphyrin units can be aligned vertically through interaction involving dispersion forces between phenyl substituents of neighboring molecules. Ethynyl unit as a spacer and pivot joint provides almost free azimuthal rotation of the unsubstituted porphin. However, rotation of the larger triphenylporphyrin unit is sterically restricted, which leads to an alignment of the azimuthal rotators (Otte et al. 2014)

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7.2.2 The Active Surfaces in Active Use The ability of functionally modified surface to provide recognition on the level of the whole living cells is illustrated by presenting the platform to anchor cancer cells expressing folate receptors (FR) in Fig. 7.3 (Chen et al. 2015). A great number of folic acid receptors on their surface is a characteristic feature of these cells. The monolayer of PLL-g-PMOXA-c-FA was self-assembled at the glass interface, expressing folic acid on the surface of the formed layer. After one hour of incubation, the FR-negative cells could be removed by washing because of a lack of anchoring sites, and FRpositive cancer cells, due to the multivalent binding, remain on the surface and can be analyzed.

7.2.3 Organic Dyes Forming Active Surfaces Thin monomolecular layers can be formed not only of functional binding molecules with the addition of organic dye reporters, but also of organic dyes only. Forming the layers, these dyes can be decorated with different surface-binding groups. They can interact between themselves being organized into J-aggregates (Hada et al. 1985; Ishimoto et al. 1986; Kuroda 2004) and H-aggregates (Saito 2001). These ordered dye structures that demonstrate dramatic spectroscopic differences, were described at length in Chap. 8 of Volume 1.

Fig. 7.3 Illustration of the use of bioactive copolymer poly(l-lysine)-graft-folic acid-coupled poly(2-methyl-2-oxazoline) (PLL-g-PMOXA-c-FA), immobilized on glass substrate (Chen et al. 2015). Due to the presence of folate groups, it has a specific interaction with folate receptor of the (FR)-positive HeLa cancer cells. These cells only remain immobilized on surface and can be analyzed, whereas the normal cells are easily removed

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The formation-destruction of J-aggregates (typical for cyanine and merocyanine dyes) and the transitions between H- and J-forms (see Fig. 7.4), can be used for the formation of sensors responding as the spectral shifts in absorption or fluorescence spectra (Bricks et al. 2017). Thus, in early studies (Ariga et al. 1996) it was suggested to use the porphyrin dye forming J-aggregates assembling in monolayers. The porphyrins display spectral shifts upon forming these aggregates, and that can be the result of binding the iodide ions. The H-aggregates were formed under the influence of other halide ions (fluoride, chloride, and bromide) and in the absence of anions. Stabilization of the J-aggregate structure may be due to the larger size of the bound iodide ions, probably resulting in stable binding of these ions to the film. In H- and J-aggregates, the dyes attain collective behavior that can be realized in thin films (Roodenko and Thissen 2018). Their properties are described by the theory of molecular excitons (Chap. 8, Volume 1). This allows using the most important for sensing property of molecular excitons, the effect of enhanced quenching, often called superquenching (the quenching of fluorescence of the whole aggregate on interaction of quencher with one of its members). Such dramatic change of light intensity should provide dramatic enhancement of sensitivity of any analytic measurement. Ordering of surface-attached dyes on different surfaces with the observation of superquenching was reported (Bujdák and Iyi 2008; Lu et al. 2002; Sato et al. 2015).

Fig. 7.4 Formation of H- and J-aggregates by organic dyes attached to the surface and the transformation of their spectra. The absorption (blue) and fluorescence (red) spectra of dye monomer are shown in the center. On the formation of H-aggregates (left) the dyes are assembled into side-toside configuration, and, compared to that of monomers (shown as shadows), the absorption spectra move in the blue, and the fluorescence spectra move in the red directions. On the formation of Jaggregates, which results in declined and shifted configuration of dyes (right), both absorption and emission spectra become very narrow and shift to the red. The transition between H- and J-aggregate is possible, resulting in dramatic transformation of spectra

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Summarizing, the functional surfaces can be prepared by many easy ways by assembly and ordering of small molecules. But the capacity of such sensors and resulting sensitivity may be weak. Also, for increasing the recognition power it is rather difficult to provide surface patterning, to “imprint” the specific pattern of a variety of functional groups together with fluorescence reporters in their desired configuration (Movilli and Huskens 2020).

7.2.4 Supported Layers of Conjugated Polymers If a reader is interested to make a sensor based on thin polymer layer, the most probably, the layer of conjugated polymer will be selected. The conjugation unites the chain of molecular fluorophores, making it fluorescent in a special way—by the propagating exciton-hole recombination (see Chap. 8 of Volume 1). In such a molecular wire, excitons generated at any point along the polymer chain propagate along the chain, and the fluorescence of the whole chain along the exciton propagation can be quenched (Rochat and Swager 2013). Thus, the fluorescence of the entire polymer chain can be turned on or off by single intermolecular interaction. In this way, the problem of sensor response can be basically resolved. The response signal can be modified, enhanced or made more specific based on the properties of target. The brightly emissive polymer itself possesses the property of superquenching that can be realized on interaction with electron-rich or electron-poor target. The exposingscreening from water can also provide such signal (Joly et al. 2006). Fluorescent polymers allow many possibilities for variation the structure. They can be π-conjugated in the backbone, but the conjugation can also be made by fluorescent moieties appended around the main polymer chain (Swager 2017). The polymer backbone is commonly low-polar, and for the appended side groups, it may be necessary to increase the polarity. They can be charged, making the polymer polyelectrolyte. Coupled to target binding, the specific quenchers (usually, operating by electron transfer) may be a part of response, and the dyes of different origin, being the energy transfer donors/acceptors, can mediate and enhance the effect of reporting. The polymer can be fabricated into a spin-coated thin film or coated in the interior of a capillary tube. The fluorescent platform of conjugated polymer film allows constructing a huge variety of different types of molecular sensors (Rochat and Swager 2013; Wang et al. 2018b). The most typical is the exposition of Langmuir–Blodgett monolayer films of low-polar polymer on solid support for target detection in water or in gas phase (Joly et al. 2006). Regarding sensing in the gas phase, probably the most important are the sensors for explosive nitroaromatic compounds (Fu et al. 2016). The electron-deficient conjugated polymers provide the effective sensing platform for different electron-rich compounds, also in solutions. They include aromatic amino acids, neurotransmitters, naturally occurring polyamines spermine, spermidine, and putrescine, and also different drugs. The use of highly fluorinated conjugated polymers enhances their performance. Different recognition units can be appended, such as urea derivatives

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for recognition of anions (Sakai 2016). These polymer-based films became valuable tools in disease diagnostics (Lv et al. 2016; Wu et al. 2017). Electrostatically driven complementarity is in the background of detecting of oligonucleotides, and proteins. The pattern of charges on polymer surface allows recognizing normal and cancer cells (Bajaj et al. 2010).

7.3 Preferential Location of Solutes in the Systems of Structural Heterogeneity and on Active Surfaces It was interesting to compare the location and fluorescence response of dyes that are very similar in structure but located in different sites of complex molecular system that differ in polarity and local mobility. This was elegantly done with three coumarin dyes that differ only in small substituents in the studies of triblock-copolymers of poly(ethylene oxide)-poly-(propylene oxide)-poly(ethylene oxide) in aqueous solutions (Grant et al. 2005). By variation of temperature, the copolymers can be studied in one of three microphases: unimers, micelles, or hydrogels formed from body centered cubic aggregates of micelles. These dyes were found in different aggregate regions (Fig. 7.5) and could be probed independently on the difference of molecular dynamics at the sites of their location.

Fig. 7.5 Three 7-aminocoumarin probes C153, C102, and C343 (above) share very similar molecular geometries, volumes, and spectroscopic characteristics but they have greatly varying hydrophobicities (and solubilities) due to small substitutions (Grant et al. 2005). By selecting coumarin fluorescence probe molecules with different solubility, their different locations can be achieved in the triblock-copolymer micelles, which allows reporting on different polarities and molecular mobilities of their surrounding

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Numerous studies on micellar, dendritic and vesicular systems provide definite evidence that very small variations of probing dye structure may result in its different location in structurally heterogeneous systems. In these systems one can distinguish the more densely packed core and loosely packed periphery regions. Together with adsorbed molecules, a special phase of nanometer dimensions, that is often referred as “corona” (Kumbhakar et al. 2006; Shiraishi et al. 2010), is formed. The property of forming coronas in mixed solvents (Cooksey et al. 2017) is related to preferential solvation that will be discussed in Chap. 8. Regarding the chemical sensing, formation of such coronas increases the local concentrations of nonspecific ligands, which may compete with the determinations of targets in the same system. Here “coronas” can be considered as the clouds of low-specificity binders around the recognition sites that may increase dramatically their local concentration (Schroffenegger et al. 2020; Wang et al. 2019). Many factors influence molecular recognition at the interface differing in molecular interactions (Rodriguez-Quijada et al. 2018), including the appearance of coronas. The formation of a protein corona is very common when the analysis is performed in biological media (Li and Lee 2020; Pareek et al. 2018). If it is formed around performed nanoparticles, it is seen as the increase of their actual size and decrease of their colloidal stability. The common procedure for suppressing the corona formation is by covering the surfaces with the shell of inert hydrophilic polymer (Ke et al. 2017). The suggestions to form thick, highly hydrated, and densely grafted polymer shells, the so-called “polymer brush” shells were quite productive (Ke et al. 2017). Avoiding corona formation is the means to avoid the aggregation of sensing nanoparticles (see Fig. 7.9). However, such grafting of hydrophilic polymers can make problems in the availability of molecular recognition sites and in incorporation of fluorescence reporters.

Fig. 7.6 Difference in protection efficiency against formation of human serum albumin (HSA) corona around Fex Oy nanoparticles by shells formed of linear (a) and cyclic (b) poly(2-ethyl-2oxazoline) (PEOXA) polymers (Schroffenegger et al. 2020). Cyclic C-PEOXA shells quantitatively prevent the formation of a protein corona. The interaction of proteins with nanoparticles grafted with linear brush L-PEOXA shells cannot be entirely prevented

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It was shown that linear and cyclic poly(2-ethyl-2-oxazoline) (PEOXA) shells provide excellent colloidal stability to superparamagnetic iron oxide (Fex Oy ) nanoparticles incorporated into protein-rich media (Schroffenegger et al. 2020). However, dense shells of linear L-PEOXA brushes cannot prevent weak but significant attractive interactions with human serum albumin. In contrast, their cyclic CPEOXA counterparts quantitatively hinder protein adsorption (Fig. 7.6). It is evident that the cyclic PEOXA brushes generate the shells that are denser and more compact than their linear counterparts, entirely preventing the formation of a protein corona.

7.4 Binding Affinity at Interfaces A strong enhancement in binding constants of different ligands when absorbed on the surfaces compared to those in solutions was reported for different cases (Ariga 2020; Ariga et al. 2015). The system comprising the fixed recognition pairs of the phosphate-guanidinium system was studied in most detail (Onda et al. 1996). This pair is known to interact through both electrostatic interaction and hydrogen bonding. It was shown that at different types of interfaces the molecular recognition behavior may be dramatically different (Fig. 7.7). Although in water the binding constant between molecularly dispersed guanidinium and phosphate is quite low (1.4 M−1 ), the binding constants for adenosine monophosphate (AMP) and adenosine triphosphate (ATP) at the air–water interface are enhanced dramatically, to 3.2·106 and 1.7·107 M−1 , respectively. The binding constants of AMP to guanidinium functionality in aqueous aggregates, such as micelles and bilayer vesicles, have been evaluated to be 102 –104 M−1 . These results suggest that the interfacial media enhance significantly the molecular recognition efficiency. The increase of the binding constants depends on the size and type of interfaces.

Fig. 7.7 Comparison of binding efficiency between guanidinium and phosphate in three different environments. Typical binding constants are shown for typical cases: a in aqueous solution; b on the surface of micelles and bilayers exposed to water; c at the air–water interface (Onda et al. 1996)

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It was stated that the air–water interface may possess the advantage of stronger binding, since it is much larger and smoother than the surfaces of aqueous micelles and bilayers. Interfaces of micelles and lipid bilayers are also more dynamic than those available at the air–water interface where water penetration and structural fluctuation at the interfaces may lower binding constants. Unexpectedly large differences in protonation and deprotonation of amino acid side chains were observed when they were inserted into Langmuir monolayers or aqueous vesicles (Ariga et al. 2005). This fact indicates that molecular interactions related to the molecular recognition are significantly influenced at interfaces and depend on the membrane curvature. These results indicate clearly that molecular recognition can be achieved much more efficiently at an appropriate interface. They provide a link to understanding the fact how the biological systems adopted interfacial environments for molecular recognition.

7.5 Surface-Imprinted Sensors and Biosensors The surface imprinting technologies, though using the same principle as imprinting in polymer volume (Sect. 6.4), are basically different from them. They are able to overcome the weak points of traditional 3D molecular imprinting, such as a relatively slow response time, commonly extending to many minutes. This is because the recognition sites buried inside of the matrix are not subject to immediate access by the target molecules. Therefore, this method is hard to be applied if the analyte is too large to diffuse to or from the cured polymer. Also, the majority of the imprinting procedures are conducted in apolar organic solvents aiming to maximize electrostatic interactions, which is not always applicable for biological macromolecules. In order to overcome these difficulties, the methods to generate the imprinted binding sites on a surface were developed (Turner et al. 2006).

7.5.1 Surface Imprinting on Support The surface imprinting uses specially prepared composition of groups of atoms forming a recognition pattern at the surface. Extremely small thickness of the sensor layer, which offers the benefit of faster response, can be achieved by forming not only the molecular layer of polymer, but also by organizing the self-assembled monolayers (SAMs). The latter are molecular assemblies formed by the adsorption of small molecules by their active groups on a solid surface (Ulman 1996). The molecules forming the layer are self-organized into stable order by interplay of non-covalent or covalent interactions (Sect. 11.1 of Volume 1). In the case of SAMs, the target recognition pattern has to be formed in a small surface layer of sub-nanometer thickness. Thin films can be grafted onto a variety

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of different substrates, most of all, the glass slides or optical fibers. The surfaceimprinted sensors using the organic self-assembled monolayers have the advantages of high contact areas, fast response, easy construction, as well as integration of the recognition element with the transducer, all of which can lead to high sensitivity (Shin and Hong 2011). They are compatible with many commercial instruments, and especially the thin-film-coated optical fibers that can be used for remote sensing. Essential feature of SAMs is their ability to be patterned, so that specific selfassembling components have a deliberate spatial distribution on the surface (Smith et al. 2004). Such fabricated sophisticated 2-D nanoscale architectures can be formed by different means, and they can provide the well-characterized supports for molecular recognition. It is possible to introduce patterned features into both SAMs and the substrates that support them and in this way to make sensor arrays. One can manipulate the monolayers both during and after their formation by means of thermal, chemical, and electrochemical processing. The SAM-forming molecules can be found bi-functional, so that one their terminal group can be used for binding the surface and other groups participate in formation of the target-binding cage. The possibility of using SAM molecules with different tail groups offers the flexibility of inducing and changing the affinity of the sensor to their targets. These tail groups of the self-assembling molecules can render the surfaces a wide variety of physical/chemical properties, such as a controlled hydrophilicity/hydrophobicity and electrostatic charge. Different categories of SAMs (Ulman 1996) can be used in surface imprinting. They can be monolayers of long-chain alkanoic acids on metal oxide surface. The driving force of the interaction is the formation of a surface salt between the carboxylate anion and the surface metal cation. There can be also monolayers of organosilicon derivatives. This type of SAM is formed by alkylchlorosilanes, alkyloxysilanes, alkylaminosilanes on hydroxylated substrates (Pawlenko 2011). The driving force of the SAM formation is the in-situ assembly of polysiloxane that is connected to the substrate silanol groups (-SiOH) via Si–O–Si bond. The most popular, however, are the organosulfur adsorbates on metal (preferably, gold) surfaces. The alkanethiol SAMs (Guo and Li 2014) are easily prepared by immersing the pre-cleaned gold substrate in dilute thiol solutions. Also, sulfur and selenium compounds have a strong affinity to transition metal surfaces (Chand et al. 2020), which can be used for the formation of single-molecular shells around semiconductor nanoparticles. Early studies have demonstrated the ability of SAMs formed of hexadecyl mercaptan on gold surfaces for sensing cholesterol (Piletsky et al. 1999). In recent decades, the application of surface imprinting in the detection of a variety of small molecules has achieved success (Shin et al. 2018; Wang et al. 2018a). The fabrication of the surface-imprinted sensors for proteins may be encountered with technical difficulties (Turner et al. 2006; Zhang et al. 2013). These molecules are large in size, compared to the thickness of the SAMs, which makes the imprinting process less feasible. Also, they may display structural liability depending on the environment, which may influence primarily the formation of SAMs, especially if it is carried out in organic solvents. Overcoming these difficulties, the SAMs-based

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protein detection demonstrates very rapid progress (Ansari and Masoum 2019). This field shows many achievements in the development of the surface-imprinted biosensors for proteins, cancer markers and viruses, mostly based on potentiometric responses (Wang et al. 2018c). Recently developed technologies allow immobilization of protein template in a well-determined conformation and orientation (Kalecki et al. 2020). Biocompatible imprinted cavities can be formed from functional thiols. This allows formation of protein-imprinted self-assembled monolayers (SAMs) with multiple binding sites on gold support (Zhang et al. 2013). An example of formation of such recognition site for specific detection of proteins is presented in Fig. 7.8. It can be seen that a variety of functional groups were introduced into compounds that bind to the template protein complex forming the proper environment saturated with noncovalent bonds. Interacting with gold surface with their terminal sulfo groups, these complexes are self-assembled into SAM. The created protein-imprinted SAMs exhibited the excellent ability of specific binding of target proteins. They are determined by multiple binding sites and imprinted cavities. The strategy generates the tailor-made monolayer surfaces with specific protein binding, which opens the possibility of controlled assembly of intellectual biomaterials and preparation of biosensors.

Fig. 7.8 Protein recognition based on molecular imprinting exploring the surface-assembled monolayer forming the recognition site on a gold surface (Zhang et al. 2013). Selected thiols (shown above) assemble with protein in solution in such a way that their sulfo groups remain exposed. They are used for the attachment of the formed complex to the gold surface. Then the protein is washed-out and the remaining configuration of bound thiols serves as the recognition site for specific binding and quantitative analysis of this protein

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The usefulness of Langmuir–Blodgett films (see Volume 1, Sect. 11.1) formed by phospholipids at an air–water interface was demonstrated in the design of sensors for anionic protein ferritin (Turner et al. 2007). In order to explore the electrostatic interactions with protein surface, monolayers consisting of cationic dioctadecyldimethylammonium bromide, non-ionic methyl stearate, and poly(ethylene glycol) bearing phospholipids were imprinted with ferritin at the air/water interface and transferred hydrated to hydrophobic substrates. In such monolayers, the in-plane diffusion of both proteins and lipids was strictly suppressed. The rebinding studies demonstrated up to a six-fold increase in ferritin adsorption to imprinted versus control monolayers. The surface imprinting based on polymer materials also develops rapidly (Abo Dena et al. 2020). The ‘molecular brush’ technology that uses thin polymer films was applied successfully for recognition of protein molecules (Zdyrko et al. 2009). It consists in the following. Protein molecules are first chemically bound to an ultrathin (1–3 nm) polyglycidyl methacrylate reactive polymer layer and later removed by protease treatment. Residual amino acids became grafted to the surface. To a certain extent they imitate the surface chemical composition and shape of the template molecule. The space surrounding the adsorbed biomolecules can be modified with grafted polyethylene glycol layer. This leads to formation of islands of spatial nanosized pockets complementary to the protein shape. The adsorbing protein recognizes the surface imprinted and is anchored to the substrate. In general, using the thin film technologies, one can obtain a reproducible and uniform film thickness and prevent its irregularities. The technique of thin film imprinting on flat support is well developed and can be efficiently used (Schirhagl et al. 2012). Formation of thin molecular shells around nanoparticles of organic and inorganic origin can be also provided with a variety of post-synthetic modifications (Komiyama et al. 2018). With proper grafting onto supporting substrate, any harsh subsequent mechanical influences can be avoided. A rapid detection rate, reusable performance and excellent selectivity over several target competitors can be achieved with the surface imprinting. This method is well applicable for microfluidic devices (Schirhagl et al. 2012). Furthermore, the fluorescence sensor could efficiently perform in real biological samples. These results demonstrated clearly the potential value of this smart sensor nanomaterial in environmental monitoring and biological diagnosis (Piletsky et al. 2020). However, the limiting factor is the application and efficiency of fluorescence reporters. Future investigators should pay attention to the possibility of applying the technologies that were developed for the studies of biological membranes and their phospholipid models (Demchenko 2006; Demchenko et al. 2015, 2009). Let us sum-up, addressing fluorescence reporting techniques that are applicable for imprinted-polymer sensing. The simplest could be the photoinduced electron transfer (PET) quenching to or from electron-abundant or deficient target. This effect that results in modulation of light intensity is easily achieved. If the target or its analog is fluorescent, then the quenching will result in one-wavelength recording, and the fluorescence reference for λ-ratiometric recording can be easily introduced with the surface emission channel coming from the template. With the two types of emitters in the polymer matrix, the excimer formation and exchange of excitation energy (EET)

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between them provide this possibility. Meantime, the most powerful λ-ratiometry from a single dye (Demchenko 2010, 2014) remains “in reserve”. The same can be said about anisotropy and lifetime sensing (see Chap. 3 of Volume 1).

7.5.2 Nanoparticle-Based Surface Imprinting Creation of synthetic analogs of such natural receptors as antibodies and their fragments (Chap. 4) or aptamers (Chap. 5) that could be superior in efficiency, stability and price, are in the minds of many researchers. However, the attempts to produce “plastic antibodies” by formation of small nanoparticles based on polymer imprinting technology (Sect. 6.4) meets many difficulties (Haupt 2010). One of them is related to the necessity of processing the particles in nonaqueous media, including the operation with templates. Both the particle solubility and the molecular recognition ability in aqueous systems may create problems, which is not acceptable for biotechnological applications. For overcoming them, a different technology was developed. It is based on formation of surfactant micelles in the presence of target templates, stabilizing these micelles by cross-linking and, by removing the template, making them as the ready for use receptors (Fig. 7.9). Micelles are very flexible structures. Therefore in this technology, for preparing the molecularly imprinted nanoparticles, the surface crosslinking is applied (Zhang and Zhao 2019). It was suggested to use surfactant that has a tripropargylammonium head group cross-linkable on the surface by the click reaction. For making a rigid surrounding around a hydrophobic template, additional cross-linking can be made

Fig. 7.9 Molecular sensors based on imprinting in cross-linked micelles. a The steps of formation of sensor nanoparticles. (1) Surfactant molecules form micelles with the inclusion of template, which is the target analog with appended polar group. (2) The micelle is stabilized by cross-linking between head groups of surfactant molecules, and additional polar groups may be added to prevent micelle aggregation. Cross-linking within hydrophobic chains can also be made. (3) Template is removed by washing, exposing the target binding site. b The sensor operation. The equilibrium is established between free and bound target

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in the particle core by introducing methacrylate group at the hydrophobic tail. The template is included into micelle on the step of its formation in water. For that, it should have a charged group that could be extended to micelle surface. It was shown that selective binding pockets with prescribed shapes could be created in such a way for the recognition of bile salts (Awino and Zhao 2013). Without specific binding groups in the core, the pockets in such surface–core doubly cross-linking surfactant micelles recognize guests primarily based on their size and shape. Therefore, the groups providing more specific recognition should be included into the core structure (Arifuzzaman and Zhao 2016). In this way, the specific receptors for peptides (Zhao 2018), glycans (Zangiabadi and Zhao 2020) and alkaloids (Duan and Zhao 2019) were developed. The possibilities for fluorescence reporting on target binding have also to be found. The target can provide signaling by itself if it is fluorescent and if its fluorescent signal changes on the binding. If it is fluorescent but nonresponsive, fluorescence resonance transfer can be achieved to/from fluorophore located in the micelle (Awino and Zhao 2014). This can be achieved in view of short distances due to small micelle size (~1.5 nm for hydrophobic core and ~2.5 nm including the surface ligands), which is of the order of typical small protein. Micelle has to incorporate the responsive fluorescent dyes in other cases. The innovations in chemistry and material science in the last decades have led to the rapid development of micelle-based artificial antibodies, and their molecular affinity has started to approach that of natural antibodies. In the case of such nanosized particles, most of imprinted sites are situated at the surface or in the proximity of surface. Compared with the imprinted films and surface-imprinted materials, they are monovalent, with a more homogeneous distribution of recognition sites between the particles and improved site accessibility of imprinted materials. Therefore, they offer strong potential to substitute or even to transcend the performance of natural antibodies (Xu et al. 2020).

7.6 Sensing and Thinking. The Strong Contribution of Surfaces and Interfaces to Sensor Technologies It is amazing story that different functional materials can be self-assembled in one step without active participation of researcher. The researcher’s activity is driven to other issues. It is the understanding the properties of starting materials and of the formed final products, the choice of these components and optimizing the external conditions. The solid templates, flat or particulate, allow regular patterning of sensors at the interface and realizing all the advantages of heterogeneous format in sensing. Enrichment or deprivation of interfacial layers with molecules of different affinity is a very common phenomenon. Our task is to transform it into very efficient sensor performance.

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Several trends are of special concern and with good prospects for further development. First of them are the applications of nanoporous materials that can be formed on support, particularly nanoporous silica and metal–organic frameworks. In addition to providing greatly increased surface areas and possibility of great increase of density of incorporated sensors, they demonstrate high efficiency of detecting small molecules, especially in gas phase. The other trend is the extended development and application of surface imprinting that can be provided on the basis of microarray technology. This allows simultaneous sensing many targets that can be as large as the living cells. Regarding fluorescence reporting, three important developments should be outlined. First is the application of conjugated polymers with their ability to provide greatly enhanced reporting signal. Second is the exploration of H- and J-aggregates of organic dyes self-assembled at the surface that can also provide enhancement in their reporting. The third is building the sensors based on metal–organic frameworks that have emerged as particularly exciting inorganic–organic hybrid porous materials (see Sect. 6.3.4). They can combine the inherent physical, chemical and light-emissive properties of both inorganic and organic photonic units due to their inorganic–organic hybrid nature. In order to verify the acquired knowledge, the reader is asked to respond to the following questions: 1.

What is the common feature of different interfaces regarding concentrating of different analytes and contaminating compounds? 2. Explain the difference in affinities to targets between the sensors in the volume of liquid phase and at interfaces. 3. How to create an active surface on a support? Evaluate different possibilities that can be realized with species of different polarity and affinity to support, their ability to form with support the electrostatic interactions and chemical bonds. 4. How to introduce fluorescence dyes into the interface layer? What should be their properties? 5. What are the advantages (or disadvantages) of using the surface-appended Jand H-aggregates, instead of individual dyes? 6. What are the properties of conjugated polymers? What is their special role in sensor design? 7. Provide examples how interfacial phenomena help sensing the water-soluble analytes with water-insoluble sensors. 8. What are the advantages of porous materials in applications of sensor technologies? Explain their formation and practical use with incorporation of sensor receptors and reporters. 9. What are the surface imprinted sensors? How they can be formed and how they operate? 10. Explain the design and operation of surface-imprinted nanoparticles as the sensors.

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Park CS, Lee HJ, Jamison AC, Lee TR (2016) Robust thick polymer brushes grafted from gold surfaces using bidentate thiol-based atom-transfer radical polymerization initiators. ACS Appl Mater Interfaces 8:5586–5594 Paul S (2009) Liquid–vapour interfaces of aqueous trimethylamine-N-oxide solutions: a molecular dynamics simulation study. Chem Phys 368:7–13 Paul S, Chandra A (2005) Liquid-vapor interfacial properties of water-ammonia mixtures: dependence on ammonia concentration. J Chem Phys 123:174712 Pawlenko S (2011) Organosilicon chemistry. Walter de Gruyter Perera JM, Stevens GW (2009) Spectroscopic studies of molecular interaction at the liquid–liquid interface. Anal Bioanal Chem 395:1019–1032 Piletsky S, Canfarotta F, Poma A, Bossi AM, Piletsky S (2020) Molecularly imprinted polymers for cell recognition. Trends Biotechnol 38:368–387 Piletsky S, Piletskaya E, Sergeyeva T, Panasyuk T, El’Skaya A (1999) Molecularly imprinted self-assembled films with specificity to cholesterol. Sens Actuators B Chem 60:216–220 Pujari SP, Scheres L, Marcelis AT, Zuilhof H (2014) Covalent surface modification of oxide surfaces. Angew Chem Int Ed 53:6322–6356 Roberts G (2013) Langmuir-blodgett films. Springer Science and Business Media Rochat S, Swager TM (2013) Conjugated amplifying polymers for optical sensing applications. ACS Appl Mater Interfaces 5: 4488-4502 Rodriguez-Quijada C, Sánchez-Purrà M, de Puig H, Hamad-Schifferli K (2018) Physical properties of biomolecules at the nanomaterial interface. J Phys Chem B 122:2827–2840 Roodenko K, Thissen P (2018) Optical dielectric properties of thin films formed by organic dye aggregates. In: Ellipsometry of functional organic surfaces and films. Springer, pp 319–333 Rüttinger S, Spille C, Hoffmann M, Schlüter M (2018) Laser-induced fluorescence in multiphase systems. ChemBioEng Reviews 5:253–269 Saito K (2001) H-aggregate formation in squarylium Langmuir−Blodgett films. J Phys Chem B 105:4235–4238 Sakai R (2016) Conjugated polymers applicable to colorimetric and fluorescent anion detection. Polym J 48:59–65 Sato N, Fujimura T, Shimada T, Tani T, Takagi S (2015) J-aggregate formation behavior of a cationic cyanine dye on inorganic layered material. Tetrahedron Lett 56:2902–2905 Schirhagl R, Ren K, Zare RN (2012) Surface-imprinted polymers in microfluidic devices. Sci China Chem 55:469–483 Schroffenegger M, Leitner NS, Morgese G, Ramakrishna SN, Willinger M, Benetti EM, Reimhult E (2020) Polymer topology determines the formation of protein corona on core-shell nanoparticles. ACS Nano 14:12708–12718 Shin MJ, Hong WH (2011) Sensing capability of molecularly imprinted self-assembled monolayer. Biochem Eng J 54:57–61 Shin MJ, Shin YJ, Shin JS (2018) Cholesterol recognition system by molecular imprinting on self-assembled monolayer. Colloids Surf A 559:365–371 Shiraishi Y, Inoue T, Hirai T (2010) Local viscosity analysis of triblock copolymer micelle with cyanine dyes as a fluorescent probe. Langmuir 26:17505–17512 Smith RK, Lewis PA, Weiss PS (2004) Patterning self-assembled monolayers. Prog Surf Sci 75:1–68 Sobek J, Bartscherer K, Jacob A, Hoheisel JD, Angenendt P (2006) Microarray technology as a universal tool for high-throughput analysis of biological systems. Comb Chem High Throughput Screening 9:365–380 Sovago M, Campen RK, Bakker HJ, Bonn M (2009) Hydrogen bonding strength of interfacial water determined with surface sum-frequency generation. Chem Phys Lett 470:7–12 Srisombat L, Jamison AC, Lee TR (2011) Stability: A key issue for self-assembled monolayers on gold as thin-film coatings and nanoparticle protectants. Colloids Surf A 390:1–19 Steel WH, Beildeck CL, Walker RA (2004) Solvent polarity across strongly associating interfaces. J Phys Chem 108:16107–16116

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Chapter 8

Fluorescence Sensing of Physical Parameters and Chemical Composition in Gases and Condensed Media

Versatility of fluorescence sensing extends to different gas, liquid and solid media. Probing these media of different chemical composition and external conditions involves obtaining quantitative fluorescence response to temperature, pressure, polarity, viscosity, and the rates of dielectric relaxation and of molecular diffusion. Structural transformations in these media can be probed on molecular level. Different analytes can be detected in a more and more sophisticated manner. This chapter demonstrates the broad range of these possibilities that can be realized with fluorescent dyes. There are many methods to measure different characteristics of and within different states of matter, their compositions and impurities in them. We prefer these methods to be simple, convenient and reliable. Fluorescence sensing occupies a strong niche in all the cases, when the response should be obtained on a molecular level: in microscopic volumes, in complex environments, at interfaces. They are of preference if we need to create an image in fluorescence response parameters, if there is a necessity to make the measurements distantly or with high resolution in time. Analyzing different applications of fluorescence sensors and probes, we witness the interesting crossing points of microscopic and macroscopic concepts, as it will be illustrated in this chapter. Molecules that respond to thermal collisions with their neighboring molecules allow detecting the temperature and pressure gradients on the surface of airplanes, and the ion sensors are used for detecting pollution of large pieces of land. Next we observed active use on molecular level of description based on macroscopic polarity and viscosity by introducing “micropolarity” and “microviscosity”. Applying such description of the tested objects as continuous (on molecular scale) media, we can achieve their photophysical description. Such description is useful in the studies of solvent mixtures, interfaces and microheterogeneous systems (Yesylevskyy and Demchenko 2011). The photophysical processes in dye molecules may differ dramatically in these media (Kalyanasundaram 1987), which allows generating informative fluorescence response. Our macroscopic world and the world of atoms and small molecules are separated by length scales differing by seven or more orders of magnitude. The same difference © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_8

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exists between the time scales of molecular motions and of our visually observed processes. We have to find the best solutions of their coupling by building molecular models and not forgetting the limitations of our techniques. This chapter starts with fluorescence probing of temperature and pressure in condensed media and then immediately comes to overview the probing in gas phase. In focus will be the gas composition and the presence of harmful additives. The description of liquid structure and dynamics is viewed from a general aspect of continuous approximation. This allows introducing and exploration of the concepts of micropolarity and microviscosity and discussing the methods of their quantitative evaluation in different solvent compositions and microheterogeneous systems. Spectroscopy of molecular relaxations offers complementary approach for characterizing molecular dynamics with time-resolved and wavelength-selective techniques. Possessing these tools, we are able to resolve the unusual properties of supercritical fluids, ionic liquids and liquid crystals. We finish the story discussing the formation of polymers and the transitions in polymer structures.

8.1 Sensing the Physical Parameters of Environment: Temperature and Pressure In both cases we deal with modulation of internal energy in the probe-environment systems. Its changes are visualized as the changes in the parameters of probe emission.

8.1.1 Molecular Thermometry The measurement of temperature is ubiquitous in common life, as well in all fields of science, engineering, medicine. Nowadays most of temperature measurements are based on the temperature-dependent electrical signals and the use of thermocouples, thermistors or resistance thermometers. For remote measurements, the detection of IR radiation (pyrometry) is frequently used. These methods have essential limitations. For instance, accurate measurement of temperature distribution, ‘thermal image’, cannot be easily obtained with thermo-electrical devices, and it is hard to measure IR radiation at low temperatures (due to low signal) and in water or in humid media (because of strong absorbance of water molecules in the whole IR region). In all these conditions, the methods based on luminescence demonstrate their advantage (Bradac et al. 2020). They allow the sensing response (a) to be generated on a molecular level, (b) to be detected remotely and (c) to achieve very high spatial and temporal resolution (Mazza and Raymo 2019). Since the thermal quenching of all kind of luminescence is a very common phenomenon, both fluorescent and phosphorescent dyes, as well as luminescent

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metal–ligand complexes, can be used for sensing the temperature (Wang et al. 2013b). Thermal quenching is a dynamic process that reduces the intensity and lifetime in a coupled manner, so both these parameters can be measured and calibrated on a temperature scale. The advantage of intensity measurement is the simplicity and of lifetime detection—the independence of response on instrumental and photo-bleaching effects. The calibration of probe response is needed if a single-channel reporting of changing the emission intensity is chosen as the response signal. It could be natural to make the response signal λ-ratiometric (this problem was discussed in Chap. 6, see Fig. 6.13). The two possibilities can be realized: (a) the intensity measurements with the application of insensitive reference and (b) the redistribution of intensity between emission bands (Demchenko 2023a, b). The latter case can be achieved in photophysical or photochemical reactions. Different means to attain these possibilities are discussed in the literature (Li et al. 2020a, b). There can be molecular dyads containing two fluorophores, hetero-dimetallic clusters, mixed-metal MOFs, and energy donor–acceptor assemblies. They are efficient not only in color-changing temperature sensing but also in multicolor biological imaging. The sensors can be constructed using the temperature-dependent structural properties of self-assembled or polymeric materials, into which the environment-sensitive dyes can be embedded. This idea was realized with a series of thermally responsive polymers (Ellison and Torkelson 2002). The dyes exhibiting fluorescence and phosphorescence simultaneously or the systems allowing detection of both fluorescence and long-living luminescence of metal complexes deserve special attention. They demonstrate the difference in thermal quenching between the two types of these emissions; the emission with longer lifetime is quenched stronger. Because of that, the ratio of two light intensities is temperature-dependent and can be calibrated as an empirical ratiometric parameter in temperature units. Since the phenomenon of thermal quenching is very general, many possibilities exist in optimal design of such temperature sensors. The complexes of rare earth ions incorporated into different glasses and crystals were reported to be functional thermometers that operate based on this principle. They can be efficient at the temperatures up to 600 °C with the resolution of the order of 1 °C (Wade et al. 2003). The monomer-exciplex equilibrium between perylene and N-allyl-Nmethylaniline embedded into polymer matrix is temperature-dependent. The monomer emits blue light and the exciplex green light. On the temperature increase, this equilibrium is shifted towards the monomer. The linear temperature dependence of the ratio of these emission intensities can be used for reading the temperature (Chandrasekharan and Kelly 2001). Other excited-state reactions, such as the excited-state intramolecular proton transfer (ESIPT), are sensitive to temperature, which allows interplay of initial and proton-transfer emissions. The aggregates of 2-(2' -hydroxyphenyl) benzoxazole (HBO) show a fluorescent λ-ratiometric change in a range of temperatures from 15 to 60 °C. The reversibility and robustness as well as the stability of the aqueous

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dispersion of aggregates demonstrate very good performance, which may be useful in the applications of molecular thermometers (Huang et al. 2006a). Conformational change in polymers and biopolymers occurring in the narrow temperature interval can be used as a responding mechanism reporting on the change of temperature within this interval. The application of such temperature probes is especially important when working with living systems at physiological conditions and, particularly, for detecting subtle local changes of temperature and their display in cellular images. For reporting on such temperature-dependent changes, the studies of excitation energy transfer between two appended dyes as the donor and acceptor of energy are efficient. The changes of distance between them produce the interplay of intensities of their emissions, generating the λ-ratiometric signal. An example of such temperature sensor is presented in Fig. 8.1. A DNA chain forming molecular beacon (see Volume 1, Sect. 4.4) is labeled by two dyes at its two ends, and the temperature-dependent change of distance between them is transformed into recorded fluorescence signal. The reader may expect that the thermometers using molecular mechanism of response should, in principle, achieve a molecular-scale spatial resolution. Such molecular thermometers have the potential of measuring the temperature gradients

a A

A

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Intensity Ratio

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600

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Wavelength (nm) Fig. 8.1 Molecular-beacon temperature sensor based on nucleic acid folding-unfolding (Barilero et al. 2009). a As temperature increases, the DNA hairpin unfolds and donor/acceptor energy transfer (FRET) becomes less efficient. b Fluorescence spectra with fluorescein as the FRET donor and Texas Red as the acceptor, measured from 7.3 to 36.5 °C. With increasing the temperature, the fluorescein emission (518 nm) increases and the Texas Red emission (610 nm) decreases. c Ratio of intensities at 518 and 610 versus temperature, normalized at room temperature, shows the dynamic range of these measurements

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inside the living cells (Gota et al. 2008; Mazza and Raymo 2019). In fact, there are two possibilities for such measurements. One is to use a protein or a nucleic acid with the defined range of thermal transition and to label it with an environment-sensitive fluorescent dye or an EET pair (Ke et al. 2012), as it is observed in Fig. 8.1. Such construction can be a sensitive molecular thermometer in the range of this transition, on a condition that no uncontrollable factors such as ligand binding, digestion by hydrolases or local pH could influence it (del Rosal et al. 2017; Meng et al. 2020). The other possibility is to incorporate the temperature-responsive fluorescent nanoparticles calibrated in wavelength shift, lifetime or two-wavelength ratiometric ratio (Jaque et al. 2014; McLaurin et al. 2013; Wang et al. 2013b). Such nanoscale thermometers are particularly needed for sensing on a tissue level. They can provide control of clinical treatment of tumors by hyperthermia. It is known that the tumors are commonly more sensitive to high temperatures than the normal cells. Because of that, they can be selectively destroyed by thermal treatment. In order to avoid destroying the normal cells, the temperature has to be kept locally at elevated but narrow ranges (at about 42.5 °C). The dyes and nanoparticles with near-IR emission that fits the wavelength range of transparency of human skin and tissues can be used for such monitoring. It was shown that the nanoscale gold thermometers can effectively determine the local temperatures inside or around the targeted cancer cell (El-Sayed et al. 2006; Huang et al. 2006b). For rapid and sensitive temperature detection in small volume samples ( τF ). When (τR < τF ), which is the condition realized in low-viscous liquid solvents, this effect is hard to observe because of rapid averaging of species within the distribution. When τR ≈ τF , with the knowledge of τF we get a tool for estimating τR from the shifts of steady-state spectra by using external perturbations that modulate τR (temperature or pressure) or τF (dynamic quenchers). The method for obtaining the relaxation rates from the steady-state data using τF as the time marker is described in detail elsewhere (Demchenko 1986, 1988). edge Essentially, when the Red Edge effect in the limit of slow relaxations vt=0 − vt=0 is known, then τ R can be estimated from this effect in relaxation zone:  edge v − vedge = vt=0 − vt=0 τ R /(τ R + τ F ) (8.10) The time-resolved spectroscopy allows observing the dynamics of molecular relaxations of Red-Edge selected species and comparing them with the relaxations of mean in ensemble (Rubinov et al. 1982; Vincent et al. 1995). These experiments showed the appearance of the Red Edge effect in polar solvent with the increase of solvent viscosity by lowering the temperature. The problem here is that the high and

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wavelength-selective temporal resolution is needed in the conditions when the dielectric relaxation rates match the measured time scale of fluorescence lifetimes. For lowviscosity liquids relaxing on a scale of several picoseconds this scale is inconvenient for steady-state measurements. However, these methods can be efficiently used in highly viscous media (in which the relaxations are nanosecond or longer) and allow characterizing the nanoscale systems, such as biological membranes and protein molecules (Demchenko 2008a). In fluid aqueous media, even when the macroscopic viscosity of studied systems is low, these objects of tens or even several nanometers in dimension behave as nanoscopic solids. There is an attractive but still rarely explored possibility to amplify the Red Edge effects by observing the site-photoselection-dependent excited-state reactions, such as intramolecular electron transfer (Demchenko and Sytnik 1991a, b), intramolecular proton transfer (Demchenko et al. 2002; Nemkovich et al. 2001) and intermolecular resonance energy transfer (Demchenko and Sytnik 1991a; Nemkovich et al. 1982). Those are the reactions that are controlled by the dynamics of their solvent environments. Coupled with dielectric relaxations, these reactions produce new spectral bands that signal on the polarity and dynamics of dielectric relaxations on picosecond-nanosecond time scale. Thus, in this section we observed that fluorescence probing allowed obtaining polarity and viscosity scales of common liquids. These scales can be used for evaluation and selection of different liquid compositions and are efficient in application to small nanoscale objects. The studies of dielectric relaxations in time domain or with the Red Edge effect are applicable in the best way to the systems with retarded molecular flexibility. These tools are efficient for characterization of heterogeneous media and even, as we will see below, the condensed media with poorly understood molecular properties, such as ionic liquids and supercritical liquids.

8.6 Detection of Traces of Water in Low-Polar Liquids Determining traces of water in organic solvents and traces of organic impurities in water are highly needed in industry and common life (Kumar et al. 2021). Fluorescent organic probes are frequently used for this determination. The most attractive are those that respond to the presence of water molecules by wavelength shifts and changes of fluorescence intensity that allow λ-ratiometric recording, exploring the mechanisms of photoinduced charge transfer (Uahengo and Naimhwaka 2021; Wang et al. 2018a, b; Yoon et al. 2019), EET (Jinbo et al. 2020; Pal et al. 2014) and excited-state intramolecular proton transfer (Wang et al. 2020a, b, c). In some cases, the concentration of water molecules is so low, that for the analytical determination, their efficient but easy concentrating should be provided. This can be done directly in solutions with the addition of surfactant that forms reverse micelles with polar heads assembled in the center and low-polar chains extended to solvent. In pure solvent, this central part is “empty”, and if the water molecules are present, they can be solubilized there. It was shown that sodium bis(2-ethylhexyl) sulfosuccinate

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(AOT) reverse micelles can serve to this purpose (Klymchenko and Demchenko 2002). The 3-hydroxychromone fluorescent probes FE and FA (Fig. 8.18) were applied in this research. When incorporated into empty micelles, these two-wavelength ratiometric dyes, demonstrating ESIPT reaction, showed bright emission with predominance of short-wavelength N* band. When individual water molecules are captured by the micelle, there is a change in micelle organization, which is associated with the change of relative intensity of two bands in the dye spectra. Thus, the principle “concentration without separation” in this case works perfectly. The incorporation of several water molecules provides an almost total switch of emission color from blue-green to orange-red! Development and introduction of metal–organic frameworks (MOFs) (see Sect. 6.3) has opened a new story in these analytical procedures (Yin et al. 2017). The proper MOFs design with inclusion of lanthanide ions (Yan 2021; Zhao and Li 2020) allows achieving the two-band ratiometric detection of water. The two-color light-emitting nanohybrids (Eu-MOFs/N,S-CDs) were constructed by encapsulating the blue emitting nitrogen and sulfur co-doped carbon dots (N,SCDs) into europium metal–organic frameworks (Eu-MOFs) with red emission

Fig. 8.18 Detection of traces of water in hexane by forming AOT reverse micelles with incorporation of fluorescent dyes 4’-Diethylamino-3-hydroxyflavone (FE) (a), and 2-(6Diethylaminobenzo[b]furan-2-yl)-3-hydroxychromone (FA) (b) (Klymchenko and Demchenko 2002). Fluorescence spectra of these λ-ratiometric probes are presented for 0.1 M AOT in hexane at different W 0 values, which are the [water]/[AOT] relative molar ratios. Solubilization of first water molecules produces the most spectacular effect, and binding of 30 molecules results in almost total loss of intensity of short-wavelength N* band

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Fig. 8.19 Synthesis of two-color light-emitting Eu-MOFs/N,S-CDs nanohybrids of MOFs and carbon dots (CDs) for the detection of water in organic solvents (Dong et al. 2016). They generate red light emission (623 nm) of Eu-MOFs and blue light emission (420 nm) of N,S-CDs. The redlight emission can be quenched on interaction with water while the blue light was enhanced due to the release of N,S-CDs. Thus, the increase of water content linearly increases the light intensity ratio (I420 /I623 ), providing its quantitative determination

(Fig. 8.19). When water molecules interact with this construction, the red Eu3+ emission becomes quenched, and the released N,S-CDs produce enhanced blue emission. The λ-ratiometric signal changes linearly with water content in the range from 0.05 to 4 v% (Dong et al. 2016).

8.7 Condensed-Phase Media of Special Interest: Supercritical Liquids, Ionic Liquids and Liquid Crystals 8.7.1 Molecular Structure and Dynamics in Supercritical Fluids Supercritical fluid is a unique state of condensed matter, which can be reached in liquids by simultaneous increase of temperature (T ) and pressure (P) above their critical points T c and Pc . In these conditions, the liquid and gas phases coalesce into a single phase. Its properties, such as density, viscosity and the diffusion rate, can be tuned continuously in broad ranges by simply adjusting T and P values without changing the solvent composition. Combination of liquid-like but tunable solvation properties with gas-like rates of molecular diffusion makes supercritical fluids very attractive solvent media for chemical reactions (Knez et al. 2014). In this respect, supercritical CO2 , being both efficient and non-toxic, presents an attractive example

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of the medium for the future ‘green’ chemical synthetic technologies (Knez et al. 2019; Nikolai et al. 2019). A high level of fluctuations of physical properties is characteristic for critical phenomena (Wang et al. 2020a, b, c; Wei et al. 2020). Dynamic molecular clusters are forming and breaking in these systems. Such clusters have also to surround the solute molecules that can be the fluorescent dyes. As in liquid solvents, polarity may serve as an important characteristic predicting the solubility and reactivity of different compounds. The studies with solvent-sensitive fluorescent dyes have shown that close to the critical temperature and pressure, the local solvent density around a solute molecule is higher than the actual bulk value. Such enhanced solvent density was explained as the witness of such ‘clustering’ or ‘local density augmentation’ (Abbott et al. 2007). An interesting effect was observed in the pressure dependence of fluorescence of wavelength-ratiometric 3-hydroxychromone dye in supercritical CO2 (Barroso et al. 2006). The dye exhibiting the ESIPT reaction, is similar to FE dye presented in Figs. 8.12 and 8.18. It was found that with increase in the pressure its fluorescence spectra, normalized at the normal emission maximum (422–428 nm) nm, demonstrate a dramatic increase in the tautomer band (535–538 nm) intensity. As the density exceeds 0.7 g/mL, the relative intensity of the two bands tends to a constant value, indicating the polarity similar to that of apolar organic solvents, such as toluene and di-n-butyl ether. At lower densities, the substantial decrease of the total fluorescence intensity (a 600-fold decrease as the pressure decreases from 100 to 80 bar) is accompanied by an even more accentuated decrease of the tautomer fluorescence. If the intensity ratio is considered only, such behavior could mean a dramatic increase of polarity. Meantime, the true explanation is different. An impressive dynamic fluorescence quenching at low pressures makes the fluorescence rates comparable or even shorter than the rates of ESIPT reaction, so the excited species simply have no time for transition to a tautomer state exhibiting the long-wavelength band in emission. The origin of such dramatic quenching at low pressures is probably related to a more ‘naked’ environment of fluorescent dye and its greater exposure to collisions with CO2 molecules possessing high kinetic energies.

8.7.2 The Properties of Ionic Liquids Room-temperature ionic liquids have recently acquired a lot of interest in view of their attractive properties as the media for chemical reactions involving synthesis, catalysis and extraction (Hayes et al. 2015; Samanta 2011). They are usually composed of organic cation (alkylammonium, alkylphosphonium, imidazolium, pyridinium, isoquinolinium) and an inorganic anion (Fig. 8.20). They remain liquid over a wide temperature range, including room temperature, and in normal conditions they behave as molten salts. Many ionic liquids are colorless and optically transparent over a wide spectral range, from UV to near-IR, which does not create any problems for their optical studies.

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Fig. 8.20 The structures of typical cations and anions forming ionic liquids. Molecular dynamics in ionic liquids is quite different from that observed in common liquid. When it is probed by ICT forming fluorescent dye, the relaxation of surrounding structure occurs not by reorientation of dipoles but by translational motion of cations and anions induced by the changed electrostatic field produced by the dye

High viscosity (Jiang et al. 2019) and the absence of sharp freezing point suggest a ‘supercooled’ nature of ionic liquid media and their nano-structuring. Therefore, the study of their molecular dynamics with fluorescence probes attracted much attention. The time-resolved spectroscopy (Nagasawa and Miyasaka 2014) with excitationwavelength selection (Funston et al. 2007) allowed to show that in the origin of high viscosity of these solvents is the very slow rate of dielectric relaxations, which are in the range of hundreds of nanoseconds. This is by a factor of 104 –105 slower than in common liquids (Huang et al. 2006b). Meantime, the mechanism of these relaxations is different to that observed in common polar solvents, and the difference is in formation and reorganization of solvent shells (Samanta 2011), which occurs as the motion of charges (see Fig. 8.20). This property may significantly affect chemical reactions accompanying charge redistribution. It was shown, that ionic liquids exhibit both static and dynamic disorder (Clark et al. 2020). Static disorder is the characteristics of the system when on a relatively long time scale each molecule reveals the peculiar local and stable characteristic of its environment that may be different from that of another molecule situated within this system. A dynamic disorder appears when the probe molecules are located in different environments but these environments interchange dynamically on a time scale of emission (Nemkovich et al. 2002). Static disorder is characteristic of solid systems (e.g. glasses) and it is characteristic for the polymers in glassy states, whereas dynamic disorder is observed in molten polymer states. They can be easily distinguished and characterized with fluorescent dyes using the Red Edge wavelength-selective fluorescence spectroscopy (Demchenko 2002).

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Interesting features of the excited-state reactivity in ionic liquid were revealed with 3-hydroxyflavone probe (Kimura et al. 2010). It was confirmed that solvation dynamics corresponds to that in high viscosity solvents and is much slower than the fluorescence emission. Meantime, the excited-state proton transfer reaction proceeds on a much faster scale, in the absence of dynamic equilibrium with the solvent. The fact that reaction dynamics can be decoupled from solvation dynamics may be an important feature of chemical reactivity in ionic liquids as the solvent media.

8.7.3 Liquid Crystals There are materials with a strong structural anisotropy that are very important for basic science and practical applications. Liquid crystals are highly anisotropic materials, in which elongated or disk-like organic molecules have no positional order, but they self-align to attain the long-range directional order with their roughly parallel axes. Organic dyes serve not only for probing their structure, dynamic and transformations (Gayvoronsky et al. 2005; Lisetski et al. 2009). They demonstrate unique optical properties (Gayvoronsky et al. 2006) and are a part of efficient applications in liquid crystal displays (Lagerwall and Scalia 2012), miniature optical waveguides (Zografopoulos et al. 2012) and lasers (Coles and Morris 2010). There are several types of liquid crystals, which differ by the types of molecules and the characteristics of their ordering. The molecules could be ordered in one (nematics) or two (smectics) directions or be organized into chiral spirals (cholesterics). All liquid crystals possess large molecular anisotropy and intermolecular ordering described by the preferred orientation vector (so-called director). The centers of masses of the molecules are randomly distributed as in a liquid but still maintain long-range directional order as in crystal, retaining the freedom to flow (Fig. 8.21). The interfacial ordering and induction of structural anisotropy may influence substantially the fluorescence spectra of incorporated ICT dyes (Tajalli et al. 2008). Anisotropy in molecular interactions and the dynamics that includes strong electric field gradients can be sensed by probing dyes displaying the property of electrochromism (Yesylevskyy and Demchenko 2011). This is in contrast to isotropic media, in which spatial averaging of local interactions can justify their description in terms of “polarity” and “viscosity”. Among new properties of liquid crystals discovered with fluorescent dyes is their slow anisotropic diffusion (Pumpa and Cichos 2012; Turiv et al. 2013) and anisotropic behavior in electron-transfer reactions (Kapko and Matyushov 2006; Pasitsuparoad and Angulo 2021). The stronger solvation energy in one direction and unusual character of relaxations suppresses these reactions. It is quite natural to observe that the host–guest interactions in liquid crystals are depressed.

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SMECTIC

CHOLESTERIC

Fig. 8.21 Three major classes of liquid crystal mesophases depending on their molecular symmetry and lattice order: nematic, smectic and cholesteric. Nematic liquid crystals tend to align themselves parallel to one another along principal axis (molecular director). In smectic phase, the molecules align parallel to one another within a layer and their inter-layer translational movements are restricted. In cholesteric phase the twisting of the nematic director between consecutive layers is allowed

8.8 The Structure and Dynamics in Polymers Polymers are the well-known systems in which estimation of molecular parameters characterizing segmental dynamics and interactions is important for understanding their macroscopic properties. This can be done by fluorescence probe techniques. Their application allows to study the dynamic processes of interest, such as polymerization kinetics and mechanisms, thermal transitions, photodegradation, swelling in solvents, and so forth (Bosch et al. 2005; Raja and Brouwer 2011). While most polymeric materials are not fluorescent, they can be investigated by embedding fluorescent probe molecules that can be used to detect properties of their immediate surroundings, and dynamical processes of its change on a variety of timescales, from picoseconds (solvent relaxation) up to months or years (physical aging). The combination of fluorescence techniques, such as steady-state, timeresolved fluorescence, and fluorescence microscopy, provides excellent opportunities to polymer scientist to investigate structure and dynamics in polymers. Fluorescencebased imaging gives access to 3D-stucture and morphology information with a potential resolution down to a few tens of nanometers. These methods have great potential for further development in application to polymer science.

8.8.1 Monitoring the Polymerization Process The polymerization process is a reaction that starts with monomers, which are often low molecular weight and low viscosity liquids that are converted into high molecular weight polymers that become solid. Polymer curing produces the toughening or hardening of a polymer material by cross-linking of polymer chains. There may be

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a high need to follow this process and its steps on molecular level (Fonseca et al. 2009), which can be realized on dye incorporation before polymerization or building the polymer from labeled oligomers (Breul et al. 2013). Polymerization produces a great change in the environment of incorporated probes (Raja and Brouwer 2011) that can be followed by a variety of detection methods described in Sect. 8.4. Thus, restriction of molecular motion leads to increase of emission anisotropy when the molecular rotation of the probe molecule is hindered on the timescale of the excited-state lifetime. Enhancement of intensity and increase of fluorescence lifetime is observed for molecular rotors (Haidekker et al. 2006). During polymerization, the mobility of molecular rotors is progressively hindered as more rigid polymeric structures are formed from monomers. Polarity-sensitive probes (Sect. 8.5) report on polymerization process being sensitive to the change of structure and dynamics in their environment. Their emission becomes shifted toward shorter wavelengths because the dipolar excited states of the probe molecules are less stabilized in polymeric networks than in corresponding monomeric phases (Józefowicz et al. 2013; van den Berg et al. 2004). The blue shift increases when the monomers with shorter spacers were used, because this leads to a denser polymer network (Jager et al. 1995). Fluorescent dyes can be ideal monitors of free-radical polymerization process, and the dyes were designed for its λ-ratiometric detection (Topa et al. 2020). Other phenomena that can be used to probe polymerization via the viscosity of the medium are those based on excited-state interactions of fluorophores with other molecules. Diffusive quenching processes can be used to probe the restriction on polymerization of translational diffusion. Excimer formation, energy transfer, and any quenching mechanism can be of use (Raja and Brouwer 2011). Ratiometric detection of pyrene excimer formation followed excellently the polymerization process (Valdes-Aguilera et al. 1990). The rate of consumption of monomeric species that are the fluorescence quenchers can be followed (Valdes-Aguilera et al. 1990). Organic dyes can serve as photoinitiators of polymerization process that can be activated under visible light and low intensity, and in this role they are advantageous over UV-photoinitiators (Dumur 2020). Thus, they may achieve a unique role as initiators of polymerization and also as the monitors of its development (Ortyl et al. 2019). Moreover, they can be simultaneously used for elucidating the mechanisms of photopolymerization (Shanmugam et al. 2017).

8.8.2 Structures and Structural Transitions in Polymers Upon cooling, the polymer melts exhibit a tremendous reduction in molecular mobility, so that the structural relaxation time becomes longer than the time allowed for equilibration. This brings the system out of equilibrium, and the liquid becomes a glass—a solid lacking long-range order. The glass transition temperature Tg is one of the most important physical characteristics of different polymers. The glass state is the state of low order but high rigidity of structure. Above the glass transition,

a

b Fluorescence intensity

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Internal energy. Dynamics

8.8 The Structure and Dynamics in Polymers

Temperature

Tg

Tm

Tg Temperature (K)

Fig. 8.22 Schematic of the glass transition (shown in red) and crystal melting (shown in blue) plotted as internal energy or molecular dynamics versus temperature (a). Tg is the temperature at which a solid glassy polymer existing out of equilibrium is transformed into more relaxed and mobile elastic or highly viscous liquid state. In contrast, ideal crystal melts at a single temperature Tm . In real semicrystalline polymers, a number of low-temperature transitions are revealed (see below). b Fluorescence intensity as a function of temperature monitored at emission wavelengths of 395 nm (circles) and 384 nm (triangles) for pyrene-doped polystyrene film (Ellison et al. 2002). The Tg position is marked

the polymer has a larger free volume, the polymer chains are more mobile, and the material is softer than below Tg . For many polymers it is the rubbery state, for others it is the transition to highly viscous fluid (see Fig. 8.22). Probing with different organic dyes allows detecting these structural and dynamics changes (Raja and Brouwer 2011). The glass transitions are usually found by observing nonlinearity in temperature dependence of fluorescence intensity (Ellison and Torkelson 2002). The local dynamics of a polymer matrix at the onset of the glass transition is one of the most interesting subjects (Deschenes and Vanden Bout 2001). Fluorescence probing allowed studying the dependence of Tg on different structural variables (Napolitano et al. 2017; Shavit and Riggleman 2013). Thus, fluorescence polarization allows characterizing polymer dynamics in the area of glass transition (Choi et al. 2019) and above it (Nguyen et al. 2018), and also in different films or solid-contacting layers (Paeng et al. 2012). It was used to follow a slow rotational diffusion of individual molecules over several hours. The studies of polymer crystallinity and structural transformations in semicrystalline state can be performed with fluorescent probes (Corrales et al. 2004). The relaxation range is composed basically of three relaxation processes, known as γ, β and α, in order of increasing temperature (Menard and Menard 2002). The recognized with different methods characteristic relaxation temperatures Tγ , Tβ and Tα indicate the sequential activation on temperature increase of bend-and-stretch motions and mobility of side groups, some main chain dynamics. Tα corresponding to Tg in Fig. 8.22 may not be observed, and the main transition is melting at temperature Tm . Associated with these relaxation processes are variations in the thermal, mechanical and dielectric properties of the polymer sample.

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Some polymers can exist in highly elastic state. Being macroscopically solid, they differ substantially in dynamic properties from that in glassy state. Application of polymethine dye as a probe and investigation of static and time-resolved spectra allowed to reveal and characterize the relaxation processes in one of them, polyurethane acrylate, on the nanosecond time scale (Przhonska et al. 1999). The dye developed and used in these studies has a unique property of ICT-dependent change in the conjugation length in the excited state, which leads to strong Stokes shifts, even in rigid polymer media. Its application allowed revealing the important features of molecular dynamics in elastic polymers that could not be detected with common dyes. The studies on single molecular level have shed a new light on polymer states demonstrating both static and dynamic disorder (Flier et al. 2012; Uji-i et al. 2006). The disorder is highly pronounced in the range of glass transition. An important application of fluorescent probes is in optimization of polymer properties by rapid screening of many samples varying in composition, technology of preparation, etc., so that the combinatorial discovery cycles can be developed (Potyrailo 2006). Selection of fluorescence dyes for this purpose depends upon the desired properties to be optimized.

8.9 Sensing and Thinking. The Value of Information on Correlation of Macroscopic and Microscopic Variables There are many analytical methods to study the composition of gas, liquid and solid media, and the apparent advantage of fluorescence methods is the absence of necessity of preliminary sample preparation, extraction or separation of its components. The sample size can be very small, and the response can be obtained on the level of molecular interactions, rapidly and in a form that is easy to analyze. The fluorescence methods allow recording different kinetic processes in real time, such as mixing of solvents and formation of polymer structures. Dynamics of intermolecular interactions occurring on ultrafast time scales (10–8 –10–12 s) is the field, where the fluorescence keeps its top priority. The readers are invited to discuss the weak points of fluorescence method. One of them is the request that the samples should be optically transparent. Please, suggest the possibilities to overcome that by manipulating with the probe properties and the mode of illumination and observation. The other weak point is conceptually more important. One may argue that with fluorescence methods we can measure only what we want. The sensing and probing cannot characterize the whole system under study. The probes are selected to respond with maximal efficiency to the parameter, in which we are interested, and we often

8.9 Sensing and Thinking. The Value of Information on Correlation …

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consider as undesired the sensitivity to something else. In a sample of unknown composition we try to find particular target and develop the sensor with maximal efficiency to this target, ignoring other, may be also important, constituents. Sensor arrays, optoelectronic noses and tongs that are demonstrated in different chapters of this book, are the tools to, at least partially, overcoming this limitation. How to extend these possibilities? Fluorescence analysis depends strongly on physical modeling leading to simplification of molecular systems or on empirical correlations relating spectroscopic parameters and intermolecular interactions (Reichardt 1990). Both approaches lead to quasi-continuous characterization of reporter surrounding with the exploration of such terms as micropolarity, microviscosity or proticity as the nanoscale analogs of parameters characterizing macroscopic condensed media. Such correlation of parameters that refer to macroscopic scale and molecular scale is not easy and requires different assumptions and approximations (Yesylevskyy and Demchenko 2011) that are rarely accounted in original works. They should be discussed. The reader is asked to find the answers to several questions related to the subject of this chapter. 1.

2. 3.

4. 5. 6.

7.

8.

What are the advantages and disadvantages in the application of luminescence to temperature and pressure sensing compared to other popular methods? Which method would you select for sensing temperature inside the oven? Inside the cryogenic device? Inside the water pipe? Inside the targeted cells in a living body? What are the problems in detecting the targets in gas phase? What mechanisms in molecular recognition can be used to overcome them? Explain the difference between different definitions of the term ‘polarity’. What are the mechanisms behind fluorescence sensing of solvent polarity? What fluorescence probes and what their properties are needed for that? Should the polarity of the probe molecule itself correspond to the polarity of studied medium? How the sensor response to polarity and viscosity can be made ratiometric? Analyze the presented examples. How to probe the solvent proton-donating property in H-bonding (solvent proticity)? What is the static and what is the dynamic disorder in fluid systems? Does dynamic disorder depend on observation time window? How the fluorescence signal manifesting the disorder can be transformed into analytical signal characterizing molecular dynamics? Why the fluorescent nanoporous materials are so efficient in sensing the rare components both in gas and liquid media? Suggest different sensing mechanisms that can be realized with them. Analyze possible applications of “concentration without separation” principle that can be realized with micellar structures for discovery and analysis of minor components in solutions.

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9.

What is the major effect discovered with the wavelength-ratiometric probe in supercritical CO2 ? (a). Change of polarity? (b). Change of solvent reactivity? (c). Change of size and dynamics of solvent clusters? 10. What probes and what measurements can you suggest for studying the dynamic properties in polymers?

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Chapter 9

Quantitative Fluorescent Detection of Ions

This Chapter covers the basic knowledge and recent developments on quantitative detection of small ionic species. The broad diversity of approaches exists regarding selection and operation of recognition units. Meantime, three major principles in fluorescence reporting dominate in ion sensing. They are the ionization behavior of coupled to fluorophore charged groups, the modulation of photophysical processes in fluorophore (such as electron, proton, charge and energy transfers) and chemodosimetry (chemical transformations in fluorophore changing its fluorescence). The possibilities for obtaining the wavelength-ratiometric reporting signal in these cases are highlighted. The sensing of ionic species is of great importance because of their widespread distribution in environmental and biological systems and of their important role in modulating different industrial and biological processes. Discussion provided below is divided into three sections because of three different classes of species considered and because of different principles of sensing and reporting applied for their detection. First, it is the determination of pH. It should be the quantitative assay of free protons in aqueous solutions, but in reality it is the determination of a “hydrated proton”, hydronium ion H3 O+ . The major principle in this case is the direct coupling or dissociation of proton from ionizable group with generation of the response of fluorescence reporter. In the sensing of metal cations, the major principle of sensing is different. A great variety of chelating groups was developed for ion binding in their required concentration range. They create a strong local electric field influencing directly or with the aid of conformational changes on the electronic structure of fluorescence reporter. The reporter, being excited, responds by inducing the electron transfer, intramolecular charge transfer or proton transfer reactions. These reversible changes of fluorescence parameters on the ion binding are recorded. Some reactive ions may also produce irreversible changes in fluorophore structure. Though they are analytically useful, they are not considered here. In anion sensing, the principle of ion chelation is used, but it is not sufficient. Many anions are of larger size and with stronger hydration. Therefore, the more © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_9

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Absorption

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a Photoinduced electron transfer (PET) λ

b

c

λ

Ground-state charge transfer, proton transfer Excited-state charge transfer, proton transfer

λ

λ

λ

λ

Fig. 9.1 The possibilities of fluorescence response in sensing of ions and charged molecules. a The target charge is a strong modulator of photoinduced electron transfer (PET). Inducing or suppressing this reaction by target binding, one may only get information based on quenchingdequenching changes in fluorescence intensity without transformations in fluorescence spectra. b The target produces the switch between two ground-state forms. If these forms absorb and emit light independently, one may observe interplay of two band intensities not only in absorption (and excitation) spectra, but also in fluorescence spectra. In the excited state two possibilities can be realized. One is for two ground-state forms emit independently with generation of two bands. The other is a switch to one emissive form, the intensity of which changes proportionally to absorbance. c The absorption spectrum is of single ground-state species, but in the excited state the reversible charge or proton transfer occurs being modulated by target binding. This results in interplay of two excited-state forms. The ratio of their fluorescence intensities results in analytical signal

sophisticated caging compounds are devised, and fluorescence reporting relies to a more significant extent on conformational changes in the sensor, on dye substitutions in the binding sites and on other special tricks. Meantime, the most efficient is the chemodosimetry that is based on chemical transformations produced by the target. The target reactivity is transformed into quantitative assays by providing irreversible changes in the probe structures. In these cases we lose the reversibility of target effect but gain on specificity of reaction produced by particular target. In all these cases, depending on the fluorophore properties, we can explore several types of analytical signal displayed in steady-state fluorescence spectra (Fig. 9.1). The simplest way to obtain the target concentration from fluorescence spectra is to measure the changes of fluorescence intensity (a). It is typically realized in photoinduced electron transfer (PET) reactions, since their products are commonly nonfluorescent. Regarding signal transduction and reporting mechanism, the electron transfer is also easiest to realize. For instance, removal of PET by target binding produces fluorescence enhancement. But here arises the problem of calibration. For a single-site reversible target binding, the target concentration can be determined: [Target]/K d = (F − Fmin )/(Fmax − F)

(9.1)

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Thus, we need to measure the fluorescence F and know the dissociation constant Kd . We have also to know the fluorescence intensity in the absence of target (F min ) and the fluorescence intensity of the same solution under target-saturating conditions (F max ). For getting the precise result, F, F min , F max and the Kd must be determined at the same dye concentration, ionic strength, temperature, optical path-length and instrument gain and sensitivity. Of some help is the determination of dynamic range (DR = F max /F min ) that still depends on all above mentioned parameters (Maravall et al. 2000). Wavelength ratiometry (Demchenko 2023a, b) allows analysis of two signals that change in the same manner under all these conditions. Their different response to target binding allows obtaining the calibrated response in target concentration (Demchenko 2010, 2014). In the case when two emissions interconvert, one band decrease, and the other—increase as a result of this reaction, we observe interplay of intensities at two selected wavelengths, λ1 and λ2 , with their change in converse manner and the generation of isobestic and isoemissive points (they indicate that the sensing reaction is a single transformation). The target concentration can be determined in the following way (Grynkiewicz et al. 1985): [ ] Target /K d = (R − Rmin )/(Rmax − R) · Fmin (λ2 )/Fmax (λ2 )

(9.2)

Here R = F(λ1 )/F(λ2 ) is the ratio of intensities at two bands in excitation or emission spectra. R is measured, and the same ratios for free and target-saturated sensor, Rmin and Rmax , as well as the K d value must be known. The factor F min (λ2 )/F max (λ2 ) is the ratio of intensities of free and bound forms at wavelength λ2 . It is needed to provide compensation for the change of intensity only if the detection wavelength is not an isobestic or isoemissive point. The λ-ratiometric sensors provide direct information about the concentration of the target analyte without the need for calibration. The band switching on target binding can be achieved in absorption (and excitation) spectra (case b in Fig. 9.1) with response in fluorescence spectra by changing the intensity or even by generation of new band. There can be also the target-dependent excited-state reaction generating new species existing only in the excited states with new fluorescence bands (case c in Fig. 9.1). The intramolecular reactions in fluorophore sites can be complemented by conformational changes, or involvement of two or more dyes that may form exciplexes or excitation energy transfer pairs. In all these sensing technologies, the smart organic molecules dominate. They suggest a simple way of forming the binding sites and of switching between different ground and excited-state forms, generating the reporter signal. The increasing contribution of metal–organic frameworks (Chen et al. 2020; Pamei and Puzari 2019) and different nanocomposites (Huang et al. 2018) is also observed. The examples that will be presented below demonstrate that ion sensing is a highly active area, and that its achievements still do not satisfy the existing demands. A general tendency of transition from “monochromating” to color-switching sensor response requires new efforts for its realization. One of important problems is the water solubility of molecular or nanoscale sensors and reaching their proper sites

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of incorporation in biological structures. Achieving multiple signals reporting on different targets simultaneously has to be one of important directions in future developments.

9.1 Fluorophore-Based Determination of pH Proton concentration in the studied medium is measured in pH units (Steinegger et al. 2020; Wencel et al. 2014). In reality, free protons (hydrogen ions) do not exist in aqueous solutions; they associate with water molecules and should be described as hydronium ions H3 O+ . These ions are formed by very strong association because hydrogen ion is very small, very reactive and possessing very high charge density. By definition, pH of a solution is the characteristics of a proton or hydrogen ion H+ (more correctly hydronium ions, H3 O+ ) activity in the form: pH = − log aH+

(9.3)

where aH+ is the activity of the hydrogen ions. Activity and concentration are linked by the activity coefficient f H+ , so that aH+ = f H+ ·[H+ ]. The negative sign in Eq. (9.1) assures that pH of most solutions is always positive. Primarily, pH is a measure of acidity/basicity of water, with pH 7 being neutral. pH of less than 7 indicates acidity, whereas if it is greater than 7 indicates a base. It is also an indicator of ionization state of many compounds dissolved in water and modulator of their reactions. It is also an essential parameter in cell physiology determining the rates of many biochemical reactions that operate within a narrow pH range (Han and Burgess 2010). Therefore, many fluorescent pH indicators and sensors are developed and actively used. Fluorescence sensing of pH is based on the fact, that in some fluorescent dyes the ionization of attached groups changes dramatically the spectroscopic properties leading to the appearance of new bands in absorption and in emission spectra. Usually, the titration ranges in absorption and in emission do not correspond (Fig. 9.2). Because the acidity of the groups accepting proton is commonly much higher in the excited than in the ground state, the pH range of sensitivity in emission becomes often strongly shifted to lower pH, making photoacids. The equilibrium between these forms can be established on a very fast time scale (Davenport et al. 1986, Laws and Brand 1979). If the two protonated and deprotonated forms are fluorescent, one can obtain with a single-band excitation the two emission bands that belong to neutral and proton-dissociated forms. During gradual pH change with fluorescence detection, one of the forms disappears and the other one appears in a coupled manner. This allows λ-ratiometric pH recording (Demchenko 2014). The optical pH sensors are typically weak organic acids or bases with distinct optical properties associated with their protonated (acidic) and deprotonated (basic) forms. Among the dyes suggested as fluorescent indicators of pH, benzo[c]xanthene derivatives such as C-SNAFL-1 (Mordon et al. 1995; Whitaker et al. 1991) were

9.1 Fluorophore-Based Determination of pH

Absorption

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+

+

+ + λ

λ

pH

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+

+ +

+ +

λ

λ

Fig. 9.2 Illustration of proton dissociation equilibrium leading to pH sensing in absorption and fluorescence spectra. At higher pH values one may observe the interplay of two absorption bands due to simultaneous presence of proton-bound and proton-dissociated forms. In the excited state, the equilibrium at this pH is commonly shifted to proton-dissociated form, and we can observe one band in fluorescence emission. At lower pH, the ground-state equilibrium is shifted to proton-bound form, and the absorption spectrum is represented by a single band. In the excited state at this pH, one may reach the conditions of equilibrium between two forms (proton-bound and dissociated), that are represented by correspondent fluorescence bands. This will make the λ-ratiometric fluorescence probe for this pH range

the first to become in extensive use. Their excitation spectra reflect the ground-state proton dissociation, and in emission spectra the pH-dependent appearance of strongly red-shifted second band is due to deprotonation of hydroxy group in the excited state. Further developments in pH-sensitive dyes included stronger band separation, efficient two-photon absorbance and adaptation for the requests for cellular imaging (Yao et al. 2007). Dissociation of substituent groups participating as proton donors in the excited-state intramolecular proton transfer (ESIPT) reactions interrupts these reactions with the appearance of new fluorescence bands, as it was shown for 2(2' -arylsulfonamidophenyl)-benzimidazole (Henary et al. 2007), hydroxythiopheneconjugated benzothiazole (Hong et al. 2019) and 3-hydroxychromone (Klymchenko and Demchenko 2004) derivatives. Such effects open new possibilities for the design of ratiometric pH sensors. The range of detectable titration of ionizable groups obeys to the rule of equilibrium between two (free and bound) states giving the small range of two orders of magnitude for efficient recording of ligand binding (see Chap. 2). Therefore, the range of pH sensitivity (as a logarithmic function) in titration of a single group is limited to two sequential digits of pH. Examples of several recently reported dyes producing λ-ratiometric response in different pH ranges and prospective for different applications are presented below. A dye 6-(2-(benzothiazol-2-yl)vinyl)naphthalen-2-ol (BTNO), see Fig. 9.3, is remarkable by its high quantum yield (0.61 in water) and by its use for λ-ratiometric recording (F 456 /F 526 ) of pH in the range 9.50–7.00 with a pK a value of 7.91 (Lin et al. 2020). With these properties, it is prospective for measuring and monitoring the cytoplasmic pH fluctuations in biomedical research. For weakly acidic pH, a novel λ-ratiometric emission NIR-fluorescence probe N,N-dimethyl-4-((1E,3E)-4-(3,3-dimethyl-3H-indol-2-yl)buta-1,3dienyl)benzenamine (DIDBA) was reported (Niu et al. 2018). The probe demonstrates a remarkable NIR ratiometric fluorescence emission (F 618nm /F 697nm )

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pH

+ H+

- H+

Fluorescence intensity

BTNO

Wavelength (nm) Fig. 9.3 The dye BTNO for neutral and weakly alkaline pH range (Lin et al. 2020). Changes of the fluorescence spectra at excitation wavelength 349 nm with decreasing pH from 11.50 to 4.00 result in the change of fluorescence color from green to aquamarine. The pK a value is acquired fitting the pH dependence of ratiometric fluorescence intensity (F 456 /F 526 ) versus pH (see insert)

Fig. 9.4 The λ-ratiometric sensor for low pH region (Niu et al. 2018). The dye DIDBA emits light demonstrating two bands in the red and near-IR range of spectrum. Excitation was at the isosbestic point 495 nm. The pK a value was calculated from the pH dependence of λ-ratiometric fluorescence intensity (F 618nm /F 697nm ). Sigmoidal fitting yields pK a = 4.5 (insert)

characteristic with pK a 4.5 (Fig. 9.4). When the pH value is higher than 5.5, the DIDBA solution exhibits an intense emission band centered at 618 nm with the fluorescence quantum yield of 0.18. As the pH decreases from 5.5 to 2.4, the fluorescence intensity at 618 nm reduces gradually and, concomitantly, a new peak at 697 nm appears and increases dramatically with the fluorescence quantum yield of

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Fluorescence intensity

0.21. The emission ratio (F 618nm /F 697nm ) changes from 2.79 at pH 7.00 to 0.15 at pH 1.6. It was reported that the dye has low cytotoxicity and excellent cell membrane permeability, together with fine photostability. Due to red-NIR emission, it can be applied successfully to visualize intracellular pH fluctuations in live cells without influence of autofluorescence and native cellular species in biological systems. Regarding fluorescence measurements at extremely low pH, an interesting example is the dye 4-((1,3)-4-(benzo[d]thiazol-2-yl)buta-1,3-dienyl)-N, Ndimethylbenzenamine (BTDB) (Zhang et al. 2016b) that displays a switching between two strongly separated fluorescence bands (Fig. 9.5). This makes the twoband ratiometric pH measurements very efficient. With pKa = 2.34, the probe operates in strongly acidic environments. The probe displayed excellent cell membrane permeability and can be applied for lysosome targeting. One may notice that operational potential of fluorescence pH probes with single titrating group, such as those discussed above, is about two orders of magnitude in proton concentrations, which is two pH units. For increasing this range, several ionisable groups ranging on a pH scale can be attached to a dye. Based on this principle, an optode-based sensor showing an almost linear pH response in the range from 2 to 10 was constructed (Li et al. 2006). It uses amino-functionalized corrole immobilized in a sol–gel glass matrix. Other researchers (Niu et al. 2005) constructed a ratiometric fluorescence sensor with broad dynamic range based on two fluorescent dyes that were sensitive in different pH-ranges. The dyes were co-polymerized with acrylamide, hydroxyethyl methacrylate and triethylene glycol dimethacrylate on the silanized glass surface. The sensor covers a broad dynamic range of pH 1.5–9.0.

400

500

600

700

800

Wavelength (nm)

Fig. 9.5 The λ-ratiometric probe for strongly acidic pH (Zhang et al. 2016b). The change of fluorescence spectra of BTDB occurs with decreasing pH from 7.0 to1.2 (λex = 383 nm). With decreasing pH, the fluorescent color of the solution changed from yellow–red to blue providing the band separation of 140 nm. Insert: Sigmoidal fitting of the pH-dependent emission (F425nm /F595nm ) yielding pKa = 2.34

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Fig. 9.6 Excitation spectra of 3,4’-dihydroxy-3’,5’-bis(dimethylaminomethyl)flavone (dye FAM345), 5 μM, in phosphate-citrate-borate buffer in the range of pH from 2.5 to 11.8 (Valuk et al. 2005). Chemical structure of FAM345 is presented at pH 2.5 together with the numbers indicating a sequence of deprotonation proposed by the changes in absorption and fluorescence spectra. Right panel: fluorescence intensity ratios F 385 /F 350 vs. pH. Emission wavelength was 515 nm in all cases

Meantime, obtaining the color-changing λ-ratiometric response in a broad pH range is a difficult task. The new water-soluble 3-hydroxyflavone dye was proposed for ratiometric measurement of pH of aqueous solutions in its range from 2.5 to 11.5 (Valuk et al. 2005). The proposed dye, 3,4’-dihydroxy-3’,5’-bis(dimethylaminomethyl)flavone, possesses four acidic centers, two of which are conjugated with molecular fluorophore (Fig. 9.6). Sequential deprotonation of the centers generates the spectral changes in absorption, fluorescence excitation and emission spectra and allows several regimes of ratiometry. The most suitable for application in the widest range of pH is the intensity ratio F385 /F350 in fluorescence excitation spectra. In the past decades, there has been growing interest in the use of nanoparticles for pH measurement, especially for intracellular pH sensing and imaging (Shamsipur et al. 2019). The nanoparticles are versatile. They may respond to pH themselves, directly or indirectly. They may be nonfluorescent but serve as scaffolds and carriers for pH-sensitive fluorescent dyes. There may be also nonfluorescent particles, whose pH-responsive structural change is converted into the fluorescence signal of their conjugated dyes. The described nanostructures include semiconductor QDs, carbonbased dots, polymer dots, upconversion nanoparticles, metallic and silica nanoparticles, fluorescent metal–organic frameworks, etc. Also, macrocyclic Eu3+ complexes, such that incorporate an N-methylsulfonamide moiety (Pal and Parker 2007), can provide λ-ratiometric pH measurements in the neutral pH range. A number of other pH sensors based on emission of lanthanide chelates have been suggested (Bunzli and Piguet 2005). They use the change of Eu3+ fluorescence due to pH-dependent change in ionization state of chelating units.

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Many processes in the living cell depend on pH, and therefore its values (pHi ) are strongly regulated and kept constant near the neutral range (exceptions are certain organelles such as lysosomes, in which pH is acidic). This regulation fails in some pathological conditions leading to acidosis or alkalosis. That is why an effort has been made to synthesize the cell-permeable fluorescence probes (Han and Burgess 2010). To address the need of researchers, the pH-sensitive fluorescent proteins were developed and they are applicable for λ-ratiometric detection (Hanson et al. 2002; McAnaney et al. 2005). The most popular version of red fluorescent protein operates in the neutral pH range as a dual excitation dye at 440 and 585 nm with recording emission at 610 nm (Tantama et al. 2011). The role of optical pH sensors is not limited to detection of proton (in fact, H3 O+ ) concentrations. In a broader sense they should be considered as acid–base indicators. Based on response in pH, the λ-ratiometric fluorescence reporters can be used as transducers in the sensors for different analytes. The pH-dependent change in ionization of the dye functional groups is local and can respond to the shift in association-dissociation equilibrium of nearby groups in macromolecules without the change of medium pH. This property can be efficiently used in different sensing technologies, such as determining urea, penicillin, acetylcholine and organophosphorous pesticides (Borisov and Wolfbeis 2008), metal cations (Xu et al. 2005). If such coupling of target binding-release with protonation-deprotonation of the dye is realized, then the yield could be a dramatic change of emission color.

9.2 Determination of Concentration of Cations There are a number of metal ions that play a vital role in biological functioning. Control of their level is important for our daily physiological life, and deviations may be associated with pathology. These include sodium (Na+ ) potassium (K+ ), calcium (Ca2+ ), magnesium (Mg2+ ), copper (Cu+ and Cu2+ ) and zinc (Zn2+ ). Health and environmental problems may be caused by other toxic metal ions, such as lead (Pb2+ ), cadmium (Cd2+ ) and mercury (Hg2+ ). These ions differ in many properties, and the range of their concentrations to be detected is great, starting from picomoles for toxic compounds and 10–6 -10–7 M for Ca2+ in cells to 0.15 M of Na+ in blood. The ion-chelating compounds (ionophores) that are the most frequently used for ion recognition can be chelators, open-chain structures (podands), macrocycles (coronands, e.g. crown ethers), macrobicycles (cryptands), etc. Some of these structures were described in Chap. 3. Such a great choice of receptors allows the proper fitting of their affinities to the desired range of target concentrations. The reporting dyes can also be versatile. Being in close interaction with the donor or the acceptor moiety of reporting dye, the ion will change its photophysical properties, generating the response, as described in Sect. 4.1 of Volume 1. Observing it as a single-parameter quenching-dequenching is easy due to PET mechanism. Generation of two-band ratiometry is more difficult. It can be realized by producing the ICT reaction in the ground or excited state (see Fig. 9.1).

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Location of bound ion with respect to fluorophore dipole is also important (Valeur and Berberan-Santos 2012). In the excited state, the cation binding at the electron acceptor site becomes stronger and at the donor site weaker. The ionophore may be linked to the fluorophore via a spacer, but in many cases some atoms or groups participating in the complexation may belong to the fluorophore. Therefore, the selectivity of binding often results from the whole structure involving both signaling and recognition moieties.

9.2.1 Fluorescent Sensors for Alkali and Alkaline Earth Metal Cations Sensing Na+ and K+ ions is important just because every living cell forms a concentration gradient of these ions between the intracellular space and the extracellular space. A Na+ gradient is between ∼5–15 mM inside and ∼100–150 mM outside the cells. This Na+ gradient is maintained by membrane transport protein Na+ /K+ ATPase; it is important for a variety of functions, and its misbalance is associated with different pathologies. Selectivity between these ions (and also of Li+ ions on their background) is needed in sensing, and this is achieved by using crown ethers, coronands and cryptands, fitting their ligand cavity to ion diameter. Lithium ion (Li+ ) monitoring is needed for health and environment protection (due to presently widespread application of Li-ion batteries). Different crown ethers (e.g. 14 crown 4) and cryptands were suggested as Li+ recognition units (Kamenica et al. 2017; Villemin and Raccurt 2021), however, the coupling with reporting function is still not efficient. Thus, the attached BODIPY derivatives provided very small ICT-based ratiometric signal (Ando et al. 2009). Sodium ion (Na+ ) sensing is often provided with a 15-crown-5 ether and azacrowns of similar size. The Na+ binding site is linked to the fluorophore, such as fluorescein and benzofuran derivatives, providing the PET-based quenching. Such are commercial dyes CoroNa Green and Sodium Green (Haugland 2003). Sodium Green displays improved optical properties with excitation in the visible spectral range, but it is a non-ratiometric dye. It comprises two 2,7 –dichlorofluorescein dyes that are linked to two nitrogen atoms of a crown ether that serves as an ion recognition unit with K d of 6.0 mM. Such dyes demonstrate fluorescence quenching/dequenching without spectral shifts (Fig. 9.7), therefore the problem of their calibration must exist. It was suggested to resolve it by application of time-resolved spectroscopy in view that the fluorescence decay rates are the dye concentration-independent (Szmacinski and Lakowicz 1997). This approach has got further development in fluorescence lifetime imaging microscopy (Despa et al. 2000). Different possibilities are being realized to develop efficient molecular sensors for Na+ ions (Schwarze et al. 2019; Taki et al. 2015). The λ-ratiometric sensitivity in Na+ and K+ detection with π-conjugated systems has been achieved (Parr and

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Fig. 9.7 The sodium ion sensor Sodium Green (Szmacinski and Lakowicz 1997). a Absorption spectra of free (solid line) and Na+ -bound (dashed line) forms. b Emission spectra at various NaCl concentrations. Inset is the Na+ -dependent intensity at the emission maximum (530 nm)

Nielsen 2020). We can expect for the appearance of efficient sensors based on this principle in near future. Potassium ions (K+ ) play essential role in living organisms. In mammals, the concentration of K+ inside cells is about 150 mM, which is nearly 30 times higher than that in the extracellular environment. A variety of fluorescence sensors for K+ ions is based on fluorescence enhancement of appended fluorophore due to reduction of PET upon complexation of these ions with crown or azacrown ethers (Müller et al. 2016). There were many attempts to develop the potassium probe displaying ratiometry. One of them was based on the use of dyes forming-disrupting the exciplexes (Sinn et al. 2016) or providing dual emission with K+ -responsive and unresponsive dyes (Wang et al. 2021). Calcium ions (Ca2+ ) are unique modulators of intracellular activities, including muscle contraction. The 1,2-bis(2-aminophenoxy) ethane-N,N,N0,N0-tetraacetic acid (BAPTA) was applied as recognition moiety in the design of Ca2+ probes for biological research (Grynkiewicz et al. 1985). In suggested compounds, the fluorophore is a donor–acceptor molecule with an amino group as the electron-donating site that participates in the complexation, and the BAPTA site is an ion chelating group (Fig. 9.8). Binding of Ca2+ in water, shifts the absorption spectrum of FURA2 to blue, whereas there is almost no shift of the fluorescence spectrum. This is due to the reduction on excitation of the electron density of the nitrogen atom conjugated with the electron-withdrawing group of the fluorophore. This causes disruption of the

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Fig. 9.8 The structures and the spectroscopic response to Ca2+ ion binding in fluorescence probes FURA 2 and INDO-1 (Grynkiewicz et al. 1985). Both probes employ BAPTA recognition site and an appended fluorophore oriented by electron-donor part to ion binding site. For FURA-2, the strong ratiometric response is observed in excitation spectra, whereas the emission spectra are unchanged. For INDO 1, the two-band ratiometry is also seen in fluorescence emission spectra

interaction between this nitrogen atom and a bound cation. Consequently, fluorescence emission closely resembles that of the free probe. In contrast to FURA-2, the photoinduced charge transfer in INDO-1 may not be sufficient to cause the nitrogen– Ca2+ bond breaking. Detailed analysis of this mechanism can be found in ref. (Valeur and Berberan-Santos 2012). Though FURA-2 and INDO-1 are very popular in use for decades, there were many attempts to overcome their weak points, such as excitation in the UV and low photostability. Thus, a two-photon excited Ca2+ sensor was synthesized that demonstrated the ratiometric behavior (Kim et al. 2017). However, the implementation of two coupled dyes was needed for that. They operate with two output windows with eliminated EET interference: a Ca2+ -sensing window and an internal reference window. The ratiometric two-photon microscopy images revealed that this probe could directly and quantitatively estimate Ca2+ in live neurons and various tissues. Magnesium ion (Mg2+ ) is also a cation of life having a number of critical roles in cells. Till present, the most popular sensors for Mg2+ are the “turn-on” sensors based on suppression of PET (Orrego-Hernández et al. 2016), however, different other possibilities are explored (Liu et al. 2018a). As a recognition unit, the o-aminophenolN,N,O-triacetic acid (APTRA) structure was suggested, which is similar to BAPTA but forms smaller cavity (Csernoch et al. 1998). This resulted in the appearance

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of dyes Mag-INDO 1 and Mag-FURA 2 that provided the possibility of internally calibrated λ-ratiometric signal. Obtaining their fluorinated derivatives, shifting the excitation from UV to visible range, synthesis of two-photonic dyes were the subject of many further developments (Liu et al. 2018a; Suzuki and Yokoyama 2015). In sensing Mg2+ ions, the ESIPT-based probes were applied, but their potential to generate new bands on ion binding was not realized in full. Thus, fluorescence enhancement only was demonstrated on the ion binding to the form that already exhibited the proton-transfer reaction (Singh et al. 2008).

9.2.2 Sensing the Transition Metal Ions In contrast to the above-described fluorescent sensors for alkali and alkaline earth metal ions, which are based on coordination interaction, for transition metal ions some chemical reaction-based fluorescent chemical sensors have been developed. Their potential differs. Copper ions Cu2+ are known as very efficient quenchers of fluorescence arising from a photoinduced electron transfer (PET) from the fluorophore to the metal center. For other transition metal ions like Mn2+ , Fe2+ , Co2+ and Ni2+ this effect is negligible, but the single-channel intensity sensing based on PET mechanism is frequently used in their sensing and imaging. Copper exists in stable oxidized Cu2+ and reduced Cu+ states, and both of them are present in living bodies, being involved in various physiological and pathological processes. Copper serves mainly as a redox-active catalytic center in enzymes cycling between Cu+ and Cu2+ . The main oxidation state in the cell is Cu+ , whereas Cu2+ is mainly found in extracellular fluids (Falcone et al. 2021). Detection of Cu+ is just as important as the detection of Cu2+ , however, many fewer fluorescent sensors have been developed for Cu+ than for Cu2+ . Reliable sensors for simultaneous determination of both of these forms are highly needed, and at present they are lacking. There are many publications on sensors for Cu2+ ions and their applications to studies in living cells (Liu et al. 2017; Saleem et al. 2018). They can easily operate in “turn-on” or “switch-off” modes based on paramagnetic properties of these ions that can serve as potent PET active agents (Rorabacher 2004) and fluorescence quenchers (Udhayakumari et al. 2014). As the reporters, very simple derivatives of organic dyes can be used, such as coumarin (Warrier and Kharkar 2018) or 1,3,4-oxadiazole (Wang et al. 2018). Due to the same reason, creation of efficient λ-ratiometric sensors becomes a great problem. Cobalt (Co2+ ) is an essential trace element that is usually tightly bound with proteins performing different biological functions. However, its high free redox activity makes it highly toxic to living cells. This requires the development of fluorescent probes for the detection of relatively low concentrations of free cobalt species (Okamoto and Eltis 2011). Due to high reactivity of Co2+ ions, the current methods of their detection are based on measuring the enhanced fluorescence intensity of reaction products (Au-Yeung et al. 2012; Maity et al. 2012).

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Zinc ions (Zn2+ ) are very important players in biological systems, and their determination is of diagnostic value. Most of the methods for their visualization and analysis are based on single-channel recording of fluorescence enhancement (Wu et al. 2020), and the two-channel ratiometric sensing becomes increasingly important. One of the most efficient is the cyanine-based fluorescent probe, CTMPA (Guo et al. 2014). The low-intensive emission maximum of CTMPA is at 730 nm, whereas that of the CTMPA-Zn2+ complex, demonstrating strong emission, is around 590 nm, which enables successful λ-ratiometric measurement of Zn2+ concentrations in living cells. Ratiometric colorimetric and fluorescent boron-dipyrromethene (BODIPY) based sensor was described for the detection of zinc ions in solution and living cells (Xia et al. 2018). Zinc ions could be trapped into the tetra-coordinating complex that enables quantitative analysis of its concentrations. Due to ICT transformation, the BNDP dye possessing absorption and fluorescence band maxima at 525 nm and 540 nm, correspondingly, are dramatically transformed and demonstrate absorption spectra at 552 nm and fluorescence bands at 580 nm (Fig. 9.9). Such significant changes in spectroscopic properties of BNDP, resulting in red-shifts between unbound and bound forms for 27 nm in absorption and 40 nm in emission spectra, allow easy λ-ratiometric detection of Zn2+ ions in low nanomolar range.

Fig. 9.9 The wavelength-ratiometric molecular sensor BNDP for Zn2+ ions based on BODIPY fluorophore (Xia et al. 2018). a The changes in absorption spectra of BNDP (5 μM) with various amounts of Zn2+ . Insert: The absorption ratio A552nm /A525nm change of BNDP with various equiv. of Zn2+ ions. b The changes in fluorescence spectra of BNDP (5 μM) with various amounts of Zn2+ ions (0 → 2.0 eq.). Insert: The fluorescence ratio change(F580nm /F540nm ) of BNDP with various equiv. of Zn2 + . The excitation was 535 nm; the samples were in CH3 CN/0.02 M HEPES buffer (1:1, v/v, pH 7.0)

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Supramolecular constructions, such as porphyrin-cyclodextrin complexes (Yang et al. 2003), were also applied for λ-ratiometric detection of zinc ions. The promising prospect for wavelength-ratiometric sensing and imaging of Zn2+ ions is expected from new fluorescent nanomaterials, such as carbon dots (Bu et al. 2020).

9.2.3 Detection of Heavy Metal Ions Industrial pollution raises the necessity of determining the concentrations of heavy metal ions on a very low level, down to nanomoles, since even in such concentrations these ions can accumulate and display a negative effect. The most needed ions to be determined are Hg2+ , Cd2+ , Co2+ , Pb2+ (Aragay et al. 2011). The heavy metal ions are known as potent fluorescence quenchers (the quenching mechanism is the induction of intersystem crossing, the Kasha effect), and therefore the first-generation sensors were based on this effect. Further developments suggested the ‘turn-on’ sensors with the increase of intensity induced by ion binding (Liu et al. 2013). Such sensors were developed for copper (Konishi and Hiratani 2006), mercury (Huang and Chang 2006) and many other ions (Dutta and Das 2012) in aqueous solutions. Metal nanoparticles, quantum dots and dendrimers (Bergamini et al. 2010; Kumar et al. 2017) are becoming involved in the construction of nanosensors, and they offer an increase of sensitivity in detection. Meantime, the sensors that could fully satisfy practical needs have still to appear. An accent on color-changing ratiometric sensors is made here. Mercury ions (Hg2+ ) belong to the most prevalent deadly toxins on earth. The danger arises from many sources such as gold production, coal plants, thermometers, barometers and mercury lamps. In the past several decades, a huge number of fluorescent sensors have been developed for the detection of Hg2+ ions (Saleem et al. 2017). The PET reaction realized with different dyes was in the basis of operation of many Hg2+ sensors (Chen et al. 2015; Shanmugapriya et al. 2018). They explored strong fluorescence quenching ability of this ion, and the major effort was made on making it the most selective. A visible to near-IR sensor (MCy-1) for mercury ions was devised and characterized (Zhu et al. 2008). A large red-shift (122 nm) of the absorption maximum of MCy-1 on Hg2+ ions binding together with high selectivity towards mercury ions over the other competitive species was observed (Fig. 9.10), making the “nakedeye” detection of mercury ions possible. The sensor uses heptamethine cyanine as the fluorophore and di-thia-dioxa-monoazacrown ether moiety as the receptor. Blue-shifted absorption of Mcy-1 relative to the parent dye can be attributed to an efficient excited-state ICT process from the donor nitrogen atom on the di-thiadioxa-monoaza macrocycles to the acceptor tricarbocyanine group. The absorption at 695 nm decreases sharply with the gradual addition of Hg2+ to the solution of Mcy-1. At the same time, a new band at 817 nm increases prominently, with an isosbestic point at 740 nm. A possible explanation for the red-shift is that the coordination of the Hg2+ ions reduces the electron donating ability of the nitrogen atom

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Fig. 9.10 A near-IR sensor (MCy-1) for mercury ions (Zhu et al. 2008) a Changes in the absorption spectra of Mcy-1 upon titration by Hg2+ ions. b The changes of absorption spectra of Mcy-1 upon addition of different cations (10 equiv). c The demonstration of sensor selectivity. The bars represent the percentage of fluorescence quenched

at the macrocycle. Thus, the ICT process becomes not possible, and the blue-shift in absorption spectra becomes suppressed. A ratiometric fluorescent probe synthesized for detecting mercury ions (Yu et al. 2021) used mercaptoethanol moiety as the recognition receptor (Fig. 9.11). The ICT-generating coumarin dye served as a reporter. The fluorescence intensity ratio (F625 nm /F495 nm ) demonstrates linear dependence on the concentration of Hg2+ ions in the range 0–1.6 μM, which allows achieving the detection limit 7.6 nM. This probe was found to be useful in biological imaging. The other attempts to achieve ratiometry in detecting Hg2+ ions were based on the C = N bond cleavage between two dyes (Jiao et al. 2017) and on phototautomerism of 7-hydroxycoumarin in aqueous solutions (Ngororabanga et al. 2019). Ratiometric sensor for two-photon excited Hg2+ imaging was also reported (Chen et al. 2019). Aluminum is a very useful metal for various industries and in our common life. Meantime, the toxic effects of Al3+ ions affect plants and aquatic ecosystems, and they also affect humans. Long-term exposure to aluminum is known to cause harm to human bodies and may be associated with cancer. The commonly applied sensors for aluminum ions are based on organic dyes demonstrating fluorescence emission enhancement (Gupta and Kumar 2016, Huang et al 2016). The attempts to construct the λ-ratiometric sensor based on enhancement of excitation energy transfer between two attached dyes was made, though with UV excitation (Zhu et al. 2016). Dimerization of pyrene-based sensors with reporting based on excimer formation was also used for generating the λ-ratiometric response (Hwang et al. 2018). A small benzothiazole derivative BHM was recently applied for sensing Al3+ ions (Tian et al. 2019). The ion binding results in formation of ICT complex generating the absorption band dramatically shifted to shorter wavelengths. The fluorescence spectra are

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shifted accordingly (Fig. 9.12), which allows the λ-ratiometric measurements of the concentrations of Al3+ ions. Cadmium ions (Cd2+ ) represent highly toxic industrial and environmental pollutants, and they are classified as a human carcinogen. There are many attempts to

Fig. 9.11 The color-changing ratiometric probe CMER for detecting Hg2+ ions based on mercaptoethanol moiety as the recognition unit and coumarin as fluorescence reporter (Yu et al. 2021). a Absorption spectra of CMER (5 μM) in the absence/presence of Hg2+ (3 μM) in aqueous HEPES (5 mM, pH = 7.4) solution. b Fluorescence spectra of CMER (5 μM) in the presence of various concentrations of Hg2+ ions (from 0 to 3 μM) in HEPES (5 mM, pH = 7.4) buffer solution. λex = 450 nm

Fig. 9.12 The benzothiazole-based BHM dye as the sensor for aluminum ions (Tian et al. 2019). a UV–Vis absorption spectra of BHM (10 μM) in the absence and presence of tested Al3+ ions in increasing concentrations demonstrating the redistribution of intensity between two bands. b Fluorescence spectra showing the interplay of two bands in emission. The solvent was DMF/H2 O (1/1, v/v)

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develop fluorescent sensors with high sensitivity and selectivity and to explore the ratiometric sensing capability. One of the recent examples is the sensor for cadmium ions based on BOPHY fluorophore (Cheng et al. 2017). The dye tetramethyl substituted bis(difluoroboron)-1,2-bis[(1H-pyrrol-2-yl)methylene]hydrazine (Me4 BOPHY) was modified with an electron donor moiety of N,N-bis(pyridin-2ylmethyl)benzenamine (Fig. 9.13). In a free form, this sensor demonstrates significantly quenched ICT fluorescence with a very strong red shift to 675 nm. On Cd2+ binding, the ICT is suppressed, increasing the probability of the emissive π-π transition. As a result, the fluorescence is greatly enhanced and observed at short wavelength, 550 nm. The ratio of fluorescence intensities measured at 570 nm and 730 nm, (F570 /F730 ), demonstrates linear dependence as a function of the Cd2+ concentration. An excellent limit of detection (LOD) of 0.77 ppb was achieved. Indium ions (In3+ ) are the newborn toxic contaminants. Their appearance is associated with the increased use for manufacturing various electronic devices including notebooks, mobile phones, and PC monitors. Wavelength-ratiometric sensors for In3+ detection were recently developed based on fluorescent peptidyl probe and metal chelating agent (Park et al. 2020a). They can be designed based on dimerizing pyrene excimer reporters (Neupane et al. 2018).

Fig. 9.13 The sensor for cadmium ions based on the mechanism of suppression of ICT (Cheng et al. 2017). The ground-state charge transfer results in weak fluorescence strongly shifted to longer wavelengths (to 675 nm at excitation 460 nm). Binding of Cd2+ ions suppresses the ICT reaction generating a short-wavelength fluorescence band (at 570 nm) with strongly increased intensity. a UV–Vis absorption spectral changes and b fluorescence spectral changes recorded for an acetonitrile sensor solution (2 μM) upon the titration of Cd2+ ions

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Different types of nanocomposites (Zhang et al. 2016a) and metal organic frameworks (Razavi and Morsali 2020) have been presented recently for sensing metal ions. DNA-aptamers also demonstrate their efficiency (Zhou et al. 2017). Future will show if they continue to stand in competition with smart organic dyes.

9.2.4 Potential for λ-Ratiometric Sensing Based on Excited-State Intramolecular Proton Transfer This potential is great, though it is mostly unrealized (Sedgwick et al. 2018). Very significant (up to ~100 nm and more) shifts of fluorescence spectra between normal N* (reactant) and tautomer T* (reaction product) forms can be achieved, and the ability to perform this reaction can be modulated by switching between different ground-state forms. Thus, the excited-state intramolecular proton transfer (ESIPT) reaction offers an ideal reporting mechanism providing dramatic change of emission color (see Sects. 4.1 and 5.3 of Volume 1), particularly in sensing of ions. In the ESIPT performing dyes, the reaction site is also the site that can be used for strong ion binding, which can modulate the ESIPT reaction generating the fluorescence response. There are different types of such donors and acceptors participating in ESIPT, including amino proton donors (Chen et al. 2018), but typically the hydroxyl groups serve as the donors and carbonyls as the acceptors (Hsieh et al. 2010). They must be located closely together and connected by hydrogen bond. The other site for ion binding can be the attached ion-chelating group that modulates the donor acidity or acceptor basicity of ESIPT reaction via the intramolecular charge transfer (ICT). Such ESIPT-ICT coupling is well studied and described in vast literature (Demchenko et al. 2013). It can be realized by attaching the ion-chelating azacrown, in which the nitrogen can serve as the electron donor. The interesting fact was described in early studies on cation binding to 3hydroxychromone derivative 4’-(Aza-15-crown-5)-flavonol, in which these two types of binding sites were present (Roshal et al. 1998, 1999). In acetonitrile it forms two types of complexes with Mg2+ and Ba2+ cations with cation:flavonol stoichiometry of 1:1 or 1:2, but the sequence of steps in complex formation is different. The Mg2+ ion is primarily bound in a chelating site formed by groups 3-OH and 4-C=O (the site of ESIPT reaction) and then is bound by the crown cycle (Fig. 9.14a). In contrast, the Ba2+ ion is bound first with the crown cycle and then forms the complex with the oxygen of carbonyl 4-C=O. The ejection of the cation from the crown complexes in the excited state is observed. The opposite sequence of interaction of the coordination centers of crown flavonol with Mg2+ and Ba2+ ions at complex formation leads to different changes in absorption and fluorescence spectra of this dye. In the cases discussed above, the ions interact directly either with the site of ESIPT reaction or by perturbing by ICT the distribution of electronic charge, perturbing this reaction. There is a different possibility for fluorophore to sense the ion binding. As a source of electric field, the proximally located ion influences the energies of

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Fig. 9.14 Examples of potential application of excited-state intramolecular proton transfer (ESIPT) reaction for sensing cationic species. a The azacrown-flavonol possessing two metal binding sites demonstrate different properties in the ground and excited states (Roshal et al. 1998, 1999). The azacrown-formed complex is more stable in the ground state but can eject the ion in the excited state due to interaction with positive charge on nitrogen serving as electron donor. The binding at the site of ESIPT reaction blocks this reaction resulting in normal emission. b The use of electrofluorochromism to modulate the ESIPT reaction (Klymchenko and Demchenko 2002). The electronic transition energy changes under the influence of electric field created by nearby charge electronically unconnected with the fluorophore. Depending on the positive or negative charge and its position, the absorption and excitation spectra shift on the wavelength scale. In ESIPT-performing dye, this reaction will be modulated with the observation of normal (N*) and tautomer (T*) bands in emission. c The fluorescent probe for Hg2+ ions based on the principle of chemodosimetry (Santra et al. 2011). The chemical reaction produced by this ion releases the O–H group able to perform ESIPT. As a result, strongly wavelength-shifted fluorescence emission band can be observed. The probing is highly specific because other ions do not generate this reaction. In all presented cases, the ESIPT site is circled

electronic transitions giving rise to the shifts in absorption spectra (solvatochromism) and fluorescence spectra (solvatofluorochromism). If the dye demonstrates ESIPT, the electrochromic modulation of ESIPT can be observed resulting in interplay of two emission bands (Klymchenko and Demchenko 2002). These effects are in the background of operation of membrane potential-sensitive probes (Volume 1, Section 5.3). Fig. 9.14b shows that an attached charge (even with a spacer!) shifting the excitation spectrum changing the relative intensities of two N* and T* fluorescence bands.

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The principle of chemodosimetry (Yang et al. 2013) can be realized on the basis of ESIPT reaction for sensing ions. In this case the target ion interacting with the probe should provide the irreversible transformation leading to the change in emission. Though the most popular range of application of chemodosimetry is in sensing chemically reactive but uncharged small molecules (see Chap. 10), there are the examples of sensing metal ions. Thus, a ratiometric fluorescent probe was developed for the detection of mercury species (Santra et al. 2011). The mercury ion promoted deprotection of a vinyl ether group in compound shown in Fig. 9.14c to afford its corresponding phenol (Scheme 17). The released hydroxyl group becomes able to participate in ESIPT reaction, generating a new band in emission. Such probing demonstrates an excellent selectively in detecting mercury species over other metal ions.

9.3 Sensing the Anions The technologies for sensing the anions are driven by the important role they play in biological and industrial processes. Their role is versatile; many of them act as nucleophiles, bases, redox agents or phase transfer catalysts. However, they may behave as pollutants in the environment. One must recognize that the development of methods for their detection has received much less attention than for molecular sensors for cations. The reason is that a strong hydration (Kubik 2017) or large size of some anions (Szumna and Jurczak 2001) does not allow strong electrostatic effects and easy generation of reporter signal based on electron, charge or proton transfer to be provided. Many organic motifs, such as ureas, amides, ammoniums, guanidiniums, hydrazones, pyrroles, imidazoliums, triazoles, and electron-deficient arenes have been incorporated into anion receptors. The recognition sites for ions are commonly made of dyes possessing either H-bond forming groups or incorporated metal cations (usually, zinc (O’Neil and Smith 2006)) in order to provide complementarity of electrostatic interactions (Zhao et al. 2019a, b). If we start designing based on geometry, we have to account various geometries: spherical (e.g., F− , Cl− , Br− , I− ), linear (e.g., CN− ), trigonal planar (e.g., NO3− ), and tetrahedral (e.g., PO4 3− ). As a result, we observe a broad diversity of solutions in design of anion sensors (Busschaert et al. 2015; Gale and Caltagirone 2015). The chemical reactivity of anions (F− and CN− and some others) allows using for the detection of these ions the chemodosimeters – the reporting molecules, in which the targets produce the changes in their structures (Ashton et al. 2015; MartínezMáñez and Sancenón 2005; Yang et al. 2013). Chemodosimeters provide the significantly higher selectivity due to specificity of their reactions and also better opportunities for analysis in aqueous environment, as they are mostly independent of hydrogen bonding interactions in comparison to those of chemical sensors. These changes are irreversible, and if they produce the response in absorption (excitation) or fluorescence spectra, they can be used for quantitative determination of ion concentrations. A variety of such chemodosimeters have been suggested for detection of fluoride

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a

401-406 nm

510-512 nm

b

688 nm

519 nm

Fig. 9.15 Two characteristic types of chemodosimeters for anions. a Example for chemodosimeters for fluoride ions based on a F− -induced breaking of Si–O bond. Providing the change in the dye structure, it induces the appearance of new fluorescence band (Wu et al. 2019). This test operates in aqueous media and can be used in paper strip format. b The cyanide probe providing the λratiometric signal by switching the emission from 688 to 519 nm on CN− binding (Long et al. 2020). Treating with AgNO3 dissociates this complex

(Dhiman et al. 2020; Zhou et al. 2014) and cyanide (Xu et al. 2010) ions. Figure 9.15 illustrates the application of chemodosimeter concept to sensing these ions. Fluoride ion (F− ) is the smallest anion (ionic radius 1.19 Å) and, therefore, it can be accommodated within the smallest cavities. Because of the highest charge density, it can influence the fluorophore response by strong H-bonding perturbation (Liu et al. 2018b; Renuga et al. 2012) and generating the electron transfer, charge transfer (Bozdemir et al. 2010) or proton transfer (Liu et al. 2020; Mahapatra et al. 2016; Peng et al. 2005) reactions. Its ability to provide cleavage of the O–Si bond (Han et al. 2019) may ‘turn on’ these reactions. In successful cases this generates the λratiometric response (Hu et al. 2010). The case presented in Fig. 9.15a (Wu et al. 2019) is taken as one of many examples. The two probes were prepared with naphthalene−b enzothiazole as the reporting fluorophore and the splitting Si−O bond as the reaction site. These probes could be used for the quantitative detection of F− in drinking water, so that even the test paper format can be applied. In general, chemodosimeters with a F− -induced bond-breaking mechanism are highly selective and sensitive. They can be λ-ratiometric, which can reduce an interference from environmental conditions, instrumental efficiency, excitation intensity, and concentration. The efficiency of metal–organic frameworks (MOFs) application for fluoride sensing was also demonstrated. In one of such cases (Yang et al. 2017), MOF was synthesized with incorporated Eu3+ ions and the high-affinity boric group was used for fluoride binding. In many cases, as with other anion sensors, in sensing of fluoride the constructions based on organic dyes or MOFs actively use the incorporated cations for introducing the interaction of charges into sensing process. Cyanide (CN− ) is a well-known toxic anion that can lead to the death of human beings in a very small dosage due to tight binding to cytochrome oxidase, resulting in the paralysis of cellular respiration. Nonetheless, it is extensively used in a number of

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Spacers:

Fluorescence intensity

industrial processes, such as metallurgy, gold mining, electroplating, plastics manufacturing, etc. Environment pollution and the fact that different food plants could produce endogenous cyanide require the methods for its monitoring. An example of ratiometric near-infrared fluorescent probe for sensing cyanide in food samples (Long et al. 2020) is presented in Fig. 9.15b. The switching of fluorescent intensities to 519 from 688 nm occurs on cyanide binding, and their ratio was found to be linear with added cyanide concentrations from 0 to 80 μM. This probe has been successfully applied for monitoring cyanide in various food samples, such as almonds, sprouting potatoes, and bamboo shoots. Several types of organic dye sensors have been developed based on the principle of blocking the ICT reaction (Kang et al. 2019; Sun et al. 2016), similarly to the case described above. A new fluorescence emission band appears at shorter wavelengths with impressive band separation and ideal possibility for ratiometric cyanide detection. Description of many other cyanide sensors can be found in the literature (Xu et al. 2010). Chromium (Cr6+ ) anions are in the focus of new developments. The sensors are based on MOFs with the inclusion of cations as a part of recognition system (Jin et al. 2018; Mukherjee et al. 2019). Thus, being as CrO4 2− and Cr2 O7 2− species, they exhibit sensitive and selective PET-based luminescence quenching response in aqueous solution (Adotey et al. 2020). Becoming efficient sensors of chromium ions in aqueous solutions (Yao et al. 2018), MOFs attain the ability of two-band ratiometric response (Jin et al. 2018). Chloride ion is the most abundant anion in the living body and it plays crucial roles in physiology across diverse cell types. As such, dysfunctional chloride homeostasis leads to a number of serious diseases. Correct chloride flux is maintained by diverse and often tissue-specific families of chloride channels that are of low selectivity among other biological anions. The ideal chloride sensor that could satisfy the demands of both chemists and biologists by now was not found (Zajac et al. 2020), and I refer to a classical example (Jayaraman et al. 1999) exploring for λ-ratiometric response a pair of responsive and irresponsive dyes, see Fig. 9.16.

Wavelength (nm) Fig. 9.16 Dual-wavelength chloride ion sensors (Jayaraman et al. 1999). The sensor structures with different spacer groups. Fluorescence emission spectra (20 μM) for 365-nm (solid lines) and 410-nm (dashed line) excitation in the presence of specified NaCl concentrations in 5 mM sodium phosphate at pH 7.2

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In a series of chloride sensors presented in Fig. 9.16, the fluorescence of 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) generating blue fluorescence is quenched by Cl− by a collisional mechanism without change in spectral shape. An appended N-substituted 6-aminoquinolinium (AQ) was chosen as the Cl− -insensitive moiety because of its different spectral characteristics (green fluorescence), positive charge (to minimize quenching by chromophore stacking/electron transfer), and reducibility (for noninvasive cell loading). The dual-wavelength indicators were stable and nontoxic in cells and were distributed uniformly in cytoplasm, with occasional staining of the nucleus. Changes in Cl− concentration in response to Cl− /nitrate exchange could be recorded by emission ratio imaging (450/565 nm) at 365-nm excitation wavelength. Other λ-ratiometric probes were also synthesized by fusion of Cl− -responsive and irresponsive dyes and applied for cellular imaging (Li et al. 2014). Bisulfite (HSO3 − ) and sulfite anions (SO3 2− ) are important for their detecting in view of their importance for cell biology and medicine. Most of the ratiometric fluorescent probes for HSO3 − /SO3 2− anions have been developed based on nucleophilic addition reactions that disrupt the π-conjugate systems suppressing ICT between their aromatic fragments. This causes the appearance of a new strong blue-shifted band in fluorescence spectra. Such dramatic changes of spectra are easy to record and to quantify. A number of such examples using the principle of chemodosimeter can be found in the literature (Park et al. 2020b). Carboxylate ions can be recognized and sensed by a complex of an anthrylamine (analogous to A-8) with Zn2+ cation (Fig. 10.35). The resulting four-coordinate metal center (A-17) has a vacant site for coordination of an anion to give a trigonalbipyramidal arrangement. Affinity towards anions bearing a carboxylate group is strong. Recognition is signaled via fluorescence quenching of the appended fluorophore as a result of intramolecular electron transfer, e.g. from a bound 4-N,Ndimethylaminobenzoate to the excited anthracene moiety. Such a transfer is favored by the stacking of benzoate and anthracene. The selectivity is essentially determined by the energy of the metal–anion coordinative interaction; moreover, only the interactions with anions displaying distinctive electron donor or electron acceptor tendencies cause fluorescence quenching of anthracene. For instance, NO3 − and SCN− do not affect the anthracene emission and do not compete with dimethylaminobenzoate for the binding to the metal center, whereas Cl− causes an intensity decrease of less than 5% and competes for binding. The acetate ion behaves in a similar way to Cl− . Summarizing, fluorescence sensing of ions offers many opportunities. It is the result of rapid growth that some of the results are seen as the research tools only and only their limited amount are prospective for clinical settings, cellular imaging and other applications. Our discussion on biologically relevant anions (e.g. phosphates) is extended to the next Chap. 10.

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9.4 Sensing and Thinking. Selecting the Ways to Apply the Principle of Wavelength-Ratiometry to Sensing Ions It could be expected that the quantitative assay of ions is the easiest task for fluorescence sensing technologies, because of their small size and the possibility to use in their recognition the strong electrostatic interactions. It may be so if we select proper ion receptors out of many available possibilities and are satisfied by response in monochromatic change of fluorescence intensity, producing on target binding the quenching or enhancing effect. In the case if the fluorescence quenching is dynamic, one can also use the response in anisotropy or lifetime (see (Lakowicz 2007) and Chap. 3 of Volume 1). This is not possible in the case of PET, since the electron transfer usually results in complete quenching, excluding the variation of these parameters. To overcome the limitations of intensity-based probes, we can use the technique of λ-ratiometry that relies on changes in the intensity of two or more emission bands (induced by the target ion), resulting in an effective internal referencing (Demchenki 2023a, b). The self-calibration makes the ratiometric fluorescent sensors more sensitive and reliable, resulting in more precise detection. In the simplest cases, the λratiometry can be achieved with the introduction of another dye emitting light in a different range of spectrum and serving as a reference (Fig. 9.17a). If we wish to achieve the λ-ratiometric reporting from a single dye molecule, this molecule should be able to change its structure or electronic distribution in the ground state (changing the excitation spectra) or the excited state (changing the fluorescence emission spectra), see Fig. 9.1. The easiest way of achieving that is in the case of pH sensors, where the proton-dissociating group is a part of fluorophore system and titration of these ionizable groups can generate new bands in both excitation and emission spectra. In sensors for other ions, the coupling with ICT and ESIPT reactions should be provided. In general, ICT and ESIPT are the intramolecular reactions that generate new fluorescence bands, and they should be coupled with the receptor performing the target binding-release. On target binding, the two (reactant and reaction product) bands change their intensities in opposite manner, and the ideal case should be if these bands are on a comparable level of intensities (Fig. 9.17b). From the results presented in this Chapter, it can be seen that the ratiometric response can be also generated by operation with two linked dyes. They should form EET pairs or exciplexes. Binding the ion either puts the dyes together or disrupts their linkage. Since the interacting and disrupted forms possess different spectra, this allows the ratiometric detection of a particular ion (Fig. 9.17c). The chemical reactivity of ions is in the background of “chemodosimeter” approach (Ashton et al. 2015). These anion-generated reactions are usually highly specific. When an anionic substrate reacts with a chemodosimeter, it can remain covalently bound to it. It can also catalyze a chemical reaction, often by splitting a molecular fragment or making it optically inactive (Park et al. 2020b). In both cases the product formed is different from the starting material. The changes in its

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The area of λ-ratiometric fluorescence sensing a

b

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Ratiometry with two active participants. Excimers and EET.

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Chemodosimetry. Decaging ground states allows excited-state reactions.

Fig. 9.17 Different cases of wavelength-ratiometric sensing that can be realized in application to ions. (a) The uncoupled pair of fluorescent dyes, one is quenched on the ion binding/release due to photoinduced proton transfer (PET), molecular collisions, etc., and the other, insensitive, serves as the reference. (b) The ion binding/release influences the reactions of intramolecular charge transfer (ICT) or the excited-state intramolecular proton transfer (ESIPT) and provides the switching between two separate reactant and reaction product emission bands. (c) The switching between spectrally different emissive forms is achieved by changing the interaction between two dyes as a result of ion binding/release. The interactions may generate the excitation energy transfer (EET) or excimer formation. (d) The interaction of target ion results in irreversible reaction of analytical value (chemodosimetry). To observe this reaction and quantify in ratiometric manner it in target ion concentration, both its reactant and product should be emissive at different wavelengths or the product should display the excited-state reaction, generating the emissive state

optical properties generate new fluorescence bands allowing the anion to be detected (Fig. 9.17d). The reader must evaluate the chemodosimeter approach in comparison to common sensing technologies that require reversible target binding in equilibrium conditions. There can be several points in this comparison. High specificity in ion chemical reactivity towards particular receptors allows achieving higher selectivity in ion recognition. However, the concentration range in such testing has to be different, and, in order to provide response corresponding to real target concentration, the reaction developing in time may require the time-dependent observation (Ashton et al. 2015). Regarding the fluorescence response, different possibilities may exist. One of them is when the probe emits light at long wavelength and the target disrupts electronic conjugation between molecular fragments, resulting in a short-wavelength emissive state (Park et al. 2020b). The other is when the ion-induced reaction starts the excitedstate process (ICT, ESIPT), and in this way the second band is generated at longer wavelengths.

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The other important point can be raised. Are the organic dyes the only materials that can display the λ-ratiometry in ion sensing? This book describes many different types of fluorescence emitters of both organic and inorganic origin, their composites and nanoscale formations. Can the ion sensing technology be based on them? Indeed, the metal–organic frameworks (MOFs) as an important class of inorganic–organic hybrid crystals with applications in various fields (see Sect. 6.3) can demonstrate the λ-ratiometry (Chen et al. 2020; Sun Gao et al. 2020). They allow different constructions for ion sensing: MOFs’ intrinsic dual-emission, single emissive MOFs with a fluorophore and non-emissive MOFs with two fluorophores. They allow providing response to target binding as the ratio of their band intensities. Fragmentary data exist on other fluorophore classes (Zhao et al. 2019a, b), their assemblies and nanocomposites (Bigdeli et al. 2019; Han et al. 2020; Li et al. 2015; Lv et al. 2020). Are there any alternatives to λ-ratiometry as a general methodology in futureoriented fluorescent ion sensing? Here the simplicity of generating and recording the sensor signal is attractive, but not only that. In comparison, the lifetime and anisotropy sensing as a possible alternative is not easy to realize. This is not only because of technical difficulties, but also due to sensitivity of results obtained with the latter methods to variations of temperature, collisional quenching and (in the case of anisotropy) to light scattering. More significant is the problem of constructing fluorescence sensors that could satisfy the demands of these methods regarding the molecular recognition of ions. The reader is asked to check the knowledge obtained after reading this Chapter by answering the following questions: 1. Explain why the λ-ratiometric sensing is needed. What is its advantage over the methods based on simple variations of fluorescence intensity? What are the requirements for its observation in excitation spectra? In emission spectra? 2. In fluorophores used for pH sensing, the reporting effect is realized due to switching between two bands in excitation or in emission spectra as a function of dissociation/association of proton. The pH ranges of this switching may correspond but may be quite different in excitation and emission spectra. Explain that. 3. What factor determines the range of sensitivity of pH indicators? How can this range be expanded? 4. Comment on the structure of a molecular ion sensor and the role of each distinct molecular module in the sensing process. 5. Explain how the fluorophore intensity as a result of ion binding can be switchedon and how switched-off. 6. How the new bands in fluorescence spectra can be generated in ion binding? What are the differences between binding cations and anions? 7. Can the interactions between two dyes be used as reporting mechanism in ion sensing? What properties these two dyes should possess? 8. What are chemodosimeters? What is special in their application to sensing ions? Why commonly for anions but not cations?

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9. Can the fluorescent materials other than organic dyes be applied for ion sensing? Can the λ-ratiometric response be engineered with them? Find several examples in the current literature.

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Chapter 10

Detection and Imaging of Small Molecules of Biological Significance

Small biology-related molecules that are neutral or without a high charge density require special methods for their detection that will be highlighted in the present chapter. Due to small strength and number of potential contacts with the sensor, molecular recognition may not be very efficient, and their detection can be based on chemodosimetry concept. Many of these compounds possess specific reactivity that is the basis of their function, and this reactivity can be used to chemically modify the sensor for providing analytical signal. Here, the basic principles in the design of such sensors and the construction of smart molecular sensors and nanosensors that are able to detect multiple analytes is discussed. Recognition of small neutral or charge-distributed organic molecules in solutions and, especially, in biological media is a much greater challenge than the recognition of ionic species because the pattern of involved forces (electrostatic and Van der Waals interactions, hydrogen bonds) is much smaller and these interactions are commonly weaker. This creates problems in application of affinity sensing based on molecular recognition concept. However, in many cases one may design the sensors based on specific irreversible chemical reaction with their targets producing the analytical signal. This is the essence of chemodosimetry concept (Hao et al. 2019; Wu et al. 2017a). Here, the specific reactivity of analyte compounds that can be related to their function is used for providing analytical signal by irreversible chemical modification of the sensor. A large range of both types of fluorescent sensors have been established for the detection of biologically and/or environmentally important species. Though molecular recognition and chemodosimetry are conceptually different in kinetic regimes and the ranges of acquired target concentrations, the range of possibilities of reporter response is the same. Figure 10.1 demonstrates different mechanisms of coupling the primary events in sensing with reporting generating different signals in fluorescence emission. There can be a single enhancing-quenching (“on–off”) coordinate of change in fluorescence intensity. The other possibility is the wavelength shifting and generation of new bands that can be recorded as the λ-ratiometric signal (Demchenko 2023a, b). This scheme is of general value, but it © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_10

329

330

10 Detection and Imaging of Small Molecules of Biological Significance Primary mechanism

Chemodosimetry (irreversible reaction)

Molecular recognition (reversible binding)

Reporting mechanism

Spectroscopic result In absorption/excitation In emission

Photoinduced electron transfer (PET)

No change

Quenching

Paramagnetic or heavy atom effect

No change

Quenching

Excited-state energy transfer (EET)

No change

Quenchingg o or new band generation

Excimer formation

No change

New band generation

Excited-state intramolecular proton transfer (ESIPT)

No change

New band generation

Intramolecular charge Spectral shift or transfer (ICT) new band generation

Spectral shift or new band generation

Fig. 10.1 The scheme illustrating the coupling between molecular interaction of target with the sensor and the generation of fluorescence signal in intensity change at a single wavelength and in λ-ratiometry

is of special importance for the detection of small molecules that are neutral or do not possess a high charge density. Thus, the sensing can be realized by two means. One, the chemodosimetry approach (Hao et al. 2019; Wu et al. 2017a), relies on the chemical reactivity of small molecular targets. For every of these target analytes, a highly selective reaction has to be found and realized in molecular structures that allows coupling with the reporting mechanisms. As we saw in Chap. 9, such approach worked well with anions F− , CN− and Cl− . With this approach, we deal with kinetically controlled reactions but lose important property of reversibility in sensor-target interactions. Moreover, the irreversible binding may change internal balance of target concentrations when the small volumes or living cells and tissues are studied. But, as we will discuss below, this is the only means to provide detection and quantitative analysis of gasotransmitters CO, NO and H2 S and also of reactive oxygen species (ROS). In contrast, the concept of molecular recognition is very general, and the term “affinity sensing” is often used to describe the operation of such sensors (see Chap. 1). It is based on a reversible target binding to the sensor in the conditions of thermodynamic equilibrium. The coupling of several weak molecular forces is needed to produce the necessary collective effect, which is not easy to realize with very small or neutral molecules. The target binding should be translated into the response of fluorescence reporter. The simplest way of response is the change of intensity of a single fluorescence band. This can be realized by the mechanism of PET (see Chap. 4 of Volume 1). The electron donor or acceptor power of a target should drive the PET reaction to or from the fluorescent reporter. The dye that obtained excess or deficiency of electron

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commonly demonstrates a non-emissive decay to the ground state. The target binding can also decouple the performed PET chain, providing a fluorescence enhancement. Quenching of fluorescence by coordinated ion can be realized in metal–ligand complexes. In designed probes, the paramagnetic or heavy atom effects of transition metal ions (such as Cu2+ , Pt2+ and Pl2+ ) can quench the fluorescence signal (Alday et al. 2020). After the fluorophores react with these targets, the reduction and/or release from the bound state of the transition metal may prevent the fluorescence quenching, thereby providing the fluorescence emission enhancement of the probes. Here also, only the single-channel response in intensity is possible. Excited-state energy transfer (EET) needs two dyes, the donor and the acceptor. Their incorporation into a single designed structure may need a significant synthetic effort. The sensing can be realized at a distance as the Förster-type resonance event, and in this case it is called the Förster resonance energy transfer (FRET). The target binding can intervene into EET, coupling or decoupling this process (Zhang et al. 2016a, b). When the acceptor is nonfluorescent, only the fluorescence intensity of the donor may change, providing a single-wavelength recording. Such probes may have advantage if it is needed to exclude the direct excitation of the acceptor. But when both donor and acceptor are emissive, the switching between their emissions on target binding may allow the λ-ratiometric recording. Excimers are the molecular complexes formed between two identical dyes (usually pyrenes) upon excitation of one of them. Since the fluorescence spectra of monomer and excimer differ dramatically and the target may influence the excimer formation, the ratiometric recording of target binding can be provided. The target concentration can be derived from these data. The ESIPT reaction can result in dramatic red-shifted fluorescence emission. The influence of target binding can be provided in different ways, such as by coupled ICT switching (Demchenko et al. 2013) or by bond-breaking chemodosimetry (Sedgwick et al. 2018). The two-band ratiometry can be achieved based on the target effect on this reaction, and corresponded dependences can be plotted in terms of target concentrations. The ICT process can occur as a dramatic shift in electron density within the same π-electronic structure, commonly with the formation of new bands. This change can occur both in the ground and the excited states by attaching-detaching the electron donor or acceptor groups, by the proximal charges, etc. If this occurs in the ground states, the new bands can be seen in absorption and excitation spectra and in fluorescence spectra at proper excitation wavelengths. If the ICT occurs in the excited state, the two-band ratiometry can be achieved in fluorescence emission spectra with the possibility to couple these changes with the target binding. There are other possibilities to generate the wavelength-ratiometry (Demchenko 2010; Gui et al. 2019). Some dyes demonstrate in common conditions both fluorescence and phosphorescence, and the switching between their intensities can be realized in sensing. Always remains the possibility to attach to the sensor the “irresponsive” dye that can be used as the reference for obtaining the ratiometric signal.

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Essentially, like in affinity sensing, all these possibilities, including the sensor-withreference concept, can be realized with the sensors operating according to the principle of chemodosimetry (Yang et al. 2020b). In addition to systems based on organic dyes, the active development of composite nanoprobes (Huang et al. 2018) and MOFs (Chen et al. 2020; Wu et al. 2020a, b) is observed.

10.1 Gaseous Molecules of Physiological Signaling—Gasotransmitters Carbon monoxide (CO), nitric oxide (NO), and hydrogen sulfide (H2 S) are well known for their toxic effects and environmental hazards. Paradoxically, in due locations and concentrations, they are extremely valuable intracellular regulators. Participating in various signaling pathways in biological systems, they are called gaseous signaling molecules or gasotransmitters (Hermann et al. 2012; Lee et al. 2018). Deviations of their concentrations from strictly determined normal values are associated with various diseases (Ali et al. 2020; Lee et al. 2018; Shefa et al. 2017). They are potential therapeutic agents in clinical applications (Lee et al. 2018; Qian and Matson 2017). Considering significant physiological roles and promising therapeutic actions of these molecules, it is essential to develop highly efficient techniques for their quantitative assay and for tracking them in biological systems. Since the gaseous signaling molecules are the neutral molecules of very small size, it is hard to apply here the molecular recognition principle. Hopefully, they are highly reactive species, and their high and specific reactivity can be in the background of their detection. Thus, the principle of chemodosimetry was found to be the most efficient (Wu et al. 2017a). Based on this principle, numerous fluorescent probes have been constructed for their analysis (Strianese and Pellecchia 2016; Yang et al. 2020b). It becomes a real piece of art to develop the receptors providing fast and highly specific chemical reactivity. They should generate the optical signal, employing one of photophysical mechanisms leading to fluorescence output (see Fig. 10.1).

10.1.1 Carbon Monoxide Carbon monoxide (CO), as a crucial gasotransmitter, is endogenously produced by the degradation of heme. It plays a critical role in regulating various physiological and pathophysiological processes, such as oxidative stress. Exogenous CO delivered at low concentrations has shown therapeutic potential as an anti-inflammatory agent (Motterlini and Otterbein 2010). Thus, it is necessary to develop highly effective and sensitive fluorescent probes for detecting CO in biological samples. Simple and efficient chemodosimeters with light-up CO detection have been developed (Das et al. 2018; Madea et al. 2020; Mukhopadhyay et al. 2020; Yuan

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et al. 2013). Because CO has a strong reducing ability, it can reduce the nitro group to an amino group under certain conditions, and this property is used in many sensor constructions. They include the probes demonstrating very strong Stokes shifts extending to the near-IR range of spectrum (Tian et al. 2021; Zhou et al. 2020). Regarding the λ-ratiometric probes for CO, the first such probe was synthesized based on attachment of a CO-specific carbamate derivative to the amino unit of naphthalimide (Feng et al. 2017). In this case, ICT is restricted and the emission wavelength is blue-shifted, at 472 nm. When CO was added, the allyl acetate group was eliminated and amino-naphthalimide was released simultaneously through the Pd(0)-initiated Tsuji–Trost reaction. The ICT process was restored and the emission wavelength was red-shifted to 545 nm (Fig. 10.2). This probe system shows excellent sensing properties for CO including rapid response, high selectivity and sensitivity with a low detection limit (58 nM), and capable of giving distinct colorimetric and λ-ratiometric fluorescent signal changes for CO in aqueous solution under mild conditions. A ratiometric fluorescent probe (Mito-NIB-CO) was developed for imaging of CO in mitochondria (Du et al. 2021). The mitochondria-targeting unit (triphenylphosphonium moiety) and CO-responsive unit (allyl ether moiety) are covalently linking into the single molecule. Treating with CO resulted in the cleavage of allyl ether element in the presence of PdCl2 , leading to the structural and spectral changes. The appearance of new band in emission allow ratiometric measurements. What’s more, profiting

Fig. 10.2 Chemodosimeter for fluorescent λ-ratiometric detection of CO based on Pd2+ -assisted structural change (Feng et al. 2017). a The fluorescence spectra showing the selectivity of CO sensor over many other related compounds (reactive forms of sulfur and oxygen, acids, etc.). b The spectroscopic transformations occurring on titration with CO concentrations in the range 0–0.35 μM

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from triphenylphosphonium moiety, the probe Mito-NIB-CO can specifically target the mitochondria and realize quantitative detection of exogenous/endogenous CO.

10.1.2 Nitric Oxide Nitric oxide (NO) is a small signaling molecule that is known to mediate many physiological processes, such as neurotransmission, blood pressure regulation, smooth muscle relaxation, and immune regulation (Adach and Olas 2019; Hsu and Tain 2019). However, uncontrolled NO production can lead to nitrosative stress, which is implicated in neurodegenerative diseases and ischemic-reperfusion injury (Schiattarella et al. 2019). Therefore, the development of fluorescence-based probes for NO could provide useful tools allowing for its direct detection and real-time monitoring in biological environments. Here also, the necessary tools can be found based on the principle of chemodosimetry. The sensors for NO can use the NO ability as a reductant reacting with Cu2+ to generate Cu+ and NO+ , and also with palladium and rhodium ions that can coordinate with various ligands, such as fluorophores). Using their quenching ability, one may achieve only the change of intensity. The change of electronic structure of fluorophore is needed for λ-ratiometry (see Fig. 10.1), which can be realized in organic dye molecules. A ratiometric fluorescence probe for NO (Zhu et al. 2017a, b) was based on a naphthalimide fluorophore. Through the Suzuki reaction, 3-dimethylaminobiphenyl was incorporated into the naphthalimide scaffold as a NO-recognizing group. It can successfully detect the NO level in living cells, tissues and an inflammatory mouse model without interference from other active oxygen and nitrogen species. For the selective and ratiometric detection of NO, a rather simple fluorescent probe was developed (Chen et al. 2018). Initially, it exhibited a fluorescence emission peak at 470 nm. Interaction with NO initiated the ESIPT process leading to the formation of a new band at longer wavelengths. This spectral transformation can be plotted as a ratiometric change in fluorescence intensity (F 560 /F 470 ) as a function of NO concentration (Fig. 10.3). The probe is cell-permeable and demonstrates good selectivity and sensitivity (LOD = 17 nM) for NO over other relevant analytes.

10.1.3 Hydrogen Sulfide Hydrogen sulfide (H2 S), along with NO and CO, is considered to be the third gaseous signaling agent, or gasotransmitter (Nagy et al. 2014). It regulates several physiological and pathological processes, including those involved in neurotransmission, vasodilation, inflammation, atherosclerosis, oxidative stress, and inhibition of insulin signaling (Dilek et al. 2020, Giuffrè et al. 2020). Considering the pKa values for H2 S are 7.05 (at 25 °C and pH 7.4), the ratio of its natural and proton-dissociated forms

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Fig. 10.3 The dye performing the ESIPT transformation after the reaction with NO (Chen et al. 2018). Fluorescence spectra of probe (10 μM) upon the addition of DEA·NONOate probe (0– 60 μM) in PBS buffer (1.0 mM, pH = 7.4). Inset. Changes in the fluorescence intensity ratio, F560 /F470 , in response to increasing concentrations of DEA·NONOate (0–40 μM). Spectra were recorded after incubation with different concentrations of nitric oxide for 30 min. λex = 420 nm, λem1 = 470 nm, λem2 = 560 nm

may differ in different cell compartments. Therefore it is important to measure both its neutral and anionic forms. Detection of HS− is easier, since it is reactive and allows applying chemodosimeter approach. Different “light-up” intensity sensing, some with strong intensity change and Stokes shifts and operating in near-IR region, have been reported based on splitting of dinitrophenyl ester group that is active in PET quenching, see e.g. (Jose et al. 2020; Zhong et al. 2018) or producing other irreversible chemical events (Guo et al. 2015; Lin et al. 2015). The same approach was used in operation of sensors performing the λ-ratiometry by activating the changed ground-state form or the excited-state reaction with the appearance of new fluorescence emission band (Maity et al. 2014). Based on this design strategy of λ-ratiometric probes, the probes for two-photon excitation microscopy were synthesized (Liu et al. 2014). They using 4-amino-1,8naphthalimide as the fluorophore and 4-azidobenzyl carbamate as a H2 S response site (Fig. 10.4). As the azide is reduced to amine, the probes may undergo a cleavage of the carbamate and release of the amino group. Through introducing an electronwithdrawing carbamate group to convert the 4-amino donor into a weak donor, the ICT effect of the fluorophore is weakened to result in a blue shift of fluorescence. The time-dependent fluorescent response to H2 S was also observed. Consequently, the fluorescence emission intensity in the short wavelength region gradually decreases, accompanying appearance of a new emission band (475−650 nm) with a peak at 530 nm, exhibiting a ratiometric change with a large red-shift over 62 nm.

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Fig. 10.4 The two-photon excited ratiometric fluorescence probes AcSH (Liu et al. 2014). The reaction scheme above shows how the H2 S sensing is coupled with fragment splitting and ICT reaction. a Time-dependent UV/vis absorption and b fluorescence spectra of 10 μm AcHS-1 probe in EtOH/PBS (v/v1:4, pH 7.0) response toward Na2 S (1 mM), respectively, excitation at 410 nm. Insets in (a) and (b): photograph for the solution color and the fluorescent changes of AcHS-1 without and with Na2 S in the buffer solution, respectively

These probes exhibit high selectivity toward H2S over biothiols and other reactive species, low detection limits of 50−85 nM, low cytotoxicity, and high stability under physiological conditions. A successful example is a novel probe BPO-N3 that uses phenoxazine as the fluorescent matrix (Lv et al. 2021). The introduced electron-withdrawing azide group into the phenoxazine matrix allowed obtaining a ratiometric signal when the azide group is effectively reduced to an amino group by H2 S. On the phenoxazine fluorescent framework, the electron-withdrawing group is converted into an electron donor group, which leads to the change of fluorescence spectrum. This mechanism was realized in a ratiometric tool that can be used for detecting H2 S in vivo (Fig. 10.5). The sensors operating in near-IR range of spectra are especially valuable for H2 S studies in vivo (Jose et al. 2020). Co-assembly of H2 S responsive and inert fluorescence moieties can also be provided. Semiconducting polymer nanoparticle can provide a strong reference signal, on the background of which the fluorescence enhancement produced by interacting with H2 S responsive dye can be ratiometrically recorded (Wu et al. 2020a, b). The ratiometric sensors based on MOF platforms deserve mentioning (Chen et al. 2020). The efficiency of many of them, e.g. (Zhang et al. 2016a, b), is based on reporting signal from Tb3+ and Eu3+ ions and Cu2+ ions as the signal modulators.

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Fig. 10.5 Fluorescence probe BPO-N3 for detection of hydrogen sulfide based on reduction of azide group to an amino group (Lv et al. 2021). a Absorption spectra of BPO-N3 (20 μM) before and after reacting with H2 S (200 μM), and of nonreactive BPO-NH2 (10 μM). Inset is the color change. b Fluorescence spectra of BPO-N3 (10 μM) in the presence of 0–20 μM H2 S. Inset: Linear relationship between fluorescence intensity ratio (F625 /F560 ) and concentration of H2 S (0.1–20 μM)

10.2 Oxygen and Reactive Oxygen Species Oxygen is a synonym of life. Its quantitative detection is highly needed in different areas of biology (Dmitriev and Papkovsky 2015; Papkovsky and Dmitriev 2013), as well as in many other fields of research and industry (Wang and Wolfbeis 2014). Regarding the reactive oxygen species (ROS), dramatic transformation of their role occurred in last decades. At the beginning, they were considered as only nonspecific harmful metabolic products. The establishment of their very important modulatory role in normal cells and tissues (Dickinson and Chang 2011) has recently got a lot of support after the discovery of their various signaling functions (Finkel 2011; Holmström and Finkel 2014). The mechanism of ROS-dependent signaling involves the reversible oxidation and reduction of specific amino acids, with the reactive Cys residues within the redox-sensitive proteins being the most frequent targets. The ROS overproduction is harmful, and there is a high need to study their concentration level and its intracellular distribution. The analysis of fluorescence detection technologies demonstrates the dominance of two different approaches. For oxygen, it is its effect as luminescence quencher, whereas high chemical reactivity of ROS allows exploration of the concept of chemodosimetry. However, reversibility of interaction with the sensors may become needed to trace rapid fluctuations in concentration of these species (Wen et al. 2021).

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10.2.1 Determination of Oxygen Concentration Determination of oxygen is commonly based on the ability of its molecules to very efficiently quench the long-living luminescence emission, close to a diffusioncontrolled rate. Oxygen is known as a very potent collisional quencher of luminescence, so if the lifetime of sensor emission is sufficiently long on the scale of oxygen diffusion, the quenching rate is proportional to its concentration in the studied medium. The process involves dynamic collision between molecular (triplet) oxygen and the probe excited electronic state, and, since the quenching is a stochastic process, the lifetime decreases. This excited-state process is reversible that does not alter the optical probe and does not change its absorption spectrum. The relationship between emission intensity F (or decay time τ) and the concentration of oxygen [O2 ] can be presented by the Stern–Volmer equation which, in its most simple form, reads as F0 /F = τ/τ0 = 1 + K SV · [O2 ]

(10.1)

where F 0 and F, respectively, are the luminescence intensities of a probe in the absence and presence of oxygen, KSV is the Stern–Volmer constant, which depends on the oxygen diffusion rate in the studied sample, and [O2 ] is the concentration of oxygen to be determined. The term luminescence is used here in order to include the long-lifetime emission. The fluorescence with sufficiently long lifetime is rare, and it is mostly represented by pyrene derivatives (Wang and Wolfbeis 2014). Therefore, the probes displaying phosphorescence are commonly used. The useful dyes emitting phosphorescence emission at normal conditions are also not very common. Their phosphorescence involves transition to the triplet state (see Sect. 5.4 of Volume 1). Metal–ligand luminescent complexes are also popular in oxygen assays. The Pt or Pd incorporated porphyrin derivatives are widely employed, since their phosphorescence emission possessing long τ0 in the range of microseconds, can be efficiently quenched by molecular oxygen (Paolesse et al. 2017). Polypyridyl complexes of ruthenium, rhenium and osmium are also very efficient in detection of oxygen (Wang and Wolfbeis 2014). Thus, the decrease of quantum yield and lifetime is used as the major principle applied in oxygen sensing (Nagl et al. 2007; Quaranta et al. 2012). The most typical are the lifetime-based methods (Ding et al. 2019), since they allow avoiding the problem of self-calibration of an output signal (Sect. 3.4 of Volume 1). For the purpose of spectroscopic analysis of oxygen, the probes exhibiting both fluorescence and phosphorescence on the same intensity scale are very attractive. The short-living fluorescence is not affected by the presence of oxygen and may serve as the reference, whereas the long-living phosphorescence can provide the necessary response in quenching. Since phosphorescence band is strongly shifted to longer wavelengths, a very precise, convenient and self-calibrating detection can be achieved in a steady-state recording at two emission wavelengths. This ratiometric

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approach makes alternative to lifetime measurements and may offer similar precision but allows using a simpler instrumentation. The λ-ratiometric imaging based on this principle (Hochreiner et al. 2005) can be realized both in industrial systems and in living cells (Feng et al. 2012; Sun et al. 2015). In addition to fluorescentphosphorescent dyes, the designed MOFs (Xu et al. 2016a, b) and polymer-based nanoscale composites (Dmitriev et al. 2015), can operate in λ-ratiometric manner. Equation (10.1) can be used, in which F and F 0 can be substituted by R and R0 , the ratios of intensities of quenched and unquenched bands. Of course, if the two dyes, one phosphorescent and the other fluorescent, are incorporated into one nanoparticle, they can also be used for wavelength-ratiometric sensing of oxygen. A disadvantage in this case could be their different photostabilities. The photodegradation of dyes can be strongly oxygen-dependent (Demchenko 2020). If one of the dyes is photobleached, this will change the ratio of two signals in emission unrelated to oxygen sensing. Different detection formats can be realized in oxygen sensing. Thus, for application in cell culture bioreactor, an external fiber-optical probe was developed (O’Neal et al. 2004). The suggested probe takes advantage of the oxygen-stimulated luminescence quenching of dichloro(tris-1,10-phenanthroline) ruthenium (II) hydrate. This probe was immobilized in a photo-polymerized hydrogel made of poly(ethylene glycol) diacrylate. The sensor showed a high degree of reproducibility across a range of oxygen concentrations that are typical for cell culture experiments. The methods of oxygen determination in microreactors and microfluidic systems have been developed (Lasave et al. 2015; Sun et al. 2015).

10.2.2 Hydrogen Peroxide Hydrogen peroxide (H2 O2 ) with an oxygen–oxygen single bond is a strong oxidizer that is used in different industrial technologies. In trace amounts, it is naturally produced in living bodies and serves as a second messenger in cellular signal transduction. It can also be a marker for oxidative stress. Overproduction of H2 O2 is implicated with various diseases including cancer, diabetes, and cardiovascular and neurodegenerative disorders (Lippert et al. 2011). Therefore, quantitative detection of H2 O2 (Zheng et al. 2019) and of other reactive oxygen species (ROS) is important in various areas of human life. Figure 10.6 represents the mode of operation of fluorescence probe for λratiometric imaging of hydrogen peroxide in mitochondria (Tang et al. 2018). Oxidative hydrolysis and elimination reaction of fluorescent probe HBTPB results in nonfluorescent ground-state species. But this reaction leads to releasing the phenolic OH group, which can participate in the ESIPT reaction. This reaction leads to dramatically Stokes-shifted emission, up to the near-IR range. Thus, in sensing H2 O2 we can observe interplay of two emissions, without treatment and after treatment with H2 O2 . Probe HBTPB displays high selectivity toward H2 O2 over various ROS, biological thiols and common anions. Importantly, this probe is mitochondria targetable and

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Fig. 10.6 Combination of irreversible chemical process (oxidative hydrolysis) and reversible photophysical process (excited-state intramolecular proton transfer, ESIPT) for the detection of hydrogen peroxide (Tang et al. 2018). a Proposed sensing mechanism of probe HBTPB to H2 O2 . b Fluorescence spectra of HBTPB (10 μM) in CH3 CN/H2 O (1/1, v/v, HEPES 10 mM, pH = 7.4) solution upon the incremental addition of H2 O2 (0–80.0 equiv.). λex = 373 nm. Fluorescence spectra were recorded after 90 min of H2 O2 addition

has been applied to provide their imaging with a near-IR emission in a ratiometric manner. Many other organelle-targetable probes for hydrogen peroxide have been synthesized based on chemical transformation that allows starting the excited-state process generating new fluorescence bands (Biswas et al. 2017; Sedgwick et al. 2018; Wen et al. 2019). This approach was efficient for providing λ-ratiometric detection and color-changing in imaging.

10.2.3 Hypochlorous Acid/Hypochlorite Hypochlorous acid (HClO/ClO− ) is commonly known as universal disinfectant that is often used in sanitary and even in disinfection of drinking water (Manna and Goswami 2015). Meantime, in biological systems it is considered as a reactive oxygen species (ROS) that plays an important role in many pathological and physiological processes. HClO could kill invading pathogens and bacteria in the immune system (Zhang et al. 2018). However, its excessive concentrations may cause different diseases. The fluorescence methods of its detection are based on their specifically applied oxidative power, which allows realizing actively the principle of chemodosimetry (Wu et al. 2019). Several examples of its application are presented below. A wavelength-ratiometric fluorescence probe specific in ClO− response and with selective location in mitochondria was proposed (Xu et al. 2016a, b). It was based on 7-diethylaminocoumarin that was conjugated with benzo[e]indolium by an ethylene group that increased the water-solubility and helped the probe to accumulate selectively in mitochondria of live cells. Moreover, in such construction, benzo[e]indolium

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extended the π-conjugation and provides strong intramolecular charge transfer (ICT). Such an extended “push–pull” construction allows shifting the absorption and fluorescence spectra to longer wavelengths, demonstrating the near-IR emission band. When the connecting bond is split by ClO− , the π-conjugation is disrupted and there appears the short-wavelength (blue) band from coumarin moiety. Therefore, such probe exhibits a colorimetric and ratiometric fluorescence dual response towards ClO− . The fluorescent OCl− detecting probe NIB was prepared (Pak et al. 2018), in which an imidazoline-2-borane group is fused to a naphthalene fluorophore (Fig. 10.7). It is shown to be selective for HOCl over other reactive oxygen species being distinguished by its electrophilic oxidation mechanism involving B–H bond cleavage. In response to OCl− , the ratiometric changes taking place in going from NIB to the corresponding oxidation product originate from differences in their charge-transfer properties. NIB was successfully applied in two-photon microscopy experiments in living cells and tissues to imaging exogenous and endogenous OCl− . Interestingly, NIB has a high endoplasmic reticulum (ER)-targeting capability and can be applied for monitoring of endogenous HOCl generation and changes in HOCl concentrations during oxidative stress situations. Mitochondria-selective HOCl targeting dyes are also known (Hu et al. 2018). Sensing and imaging of ROS has become very important in evaluating the toxicity produced by poisonous materials, the effects of different drugs and of other species that intervene into cell metabolism. Presented examples have shown that the wavelength-ratiometric detection (that can be transferred to imaging) can be achieved by exploiting high chemical reactivity of these species (Andina et al. 2017). A high specificity of their reactions with molecular sensors can be achieved due to specific sensor constructions. The major ways for ROS to generate new bands in fluorescence

Fig. 10.7 The naphthoimidazolium borane (NIB) probe for detection of OCl− (Pak et al. 2018). a The structure of NIB and proposed mechanism of its change on interaction with NIB. b Fluorescence spectra of NIB (10 μM) with titration of OCl− . Inset: photographs of NIB in the absence (left) and presence (right) of 100 μM of OCl− under UV irradiation

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emission are to break the luminophore integrity by disrupting its π-conjugation and also by inducing/disrupting the ICT and ESIPT reactions. These possibilities can be largely extended by using nanocomposites incorporating multiple emitters and of metal–organic networks (Kwon et al. 2021, Sun et al. 2021b).

10.3 Detection of Biothiols (Cysteine, Homocysteine and Glutathione) Intracellular thiols such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play key roles in biological systems. They regulate oxidation/reduction potential in cells and serve as potent antioxidants (Baba and Bhatnagar 2018). Amino acid Cys is present in many proteins either stabilizing their structures by forming S–S bonds or, in reduced form, is present in their active sites. Glutathione is a tripeptide with internal Cys residue. It is present at very high levels (0.1–10 mM) in the cell (comprising about 90% of non-protein sulfur) and protects the cells against oxidative stress. Abnormal levels of these molecules have been linked to a number of diseases, such as liver damage, leucocyte loss, psoriasis, cancer and AIDS. Accordingly, the detection of these thiol-containing biomolecules in biological samples and their imaging on cellular level is very important (Dai et al. 2020; Jung et al. 2013; Xia et al. 2019; Yan et al. 2018). Differentiating cysteine from GSH and Hcy by applying common sensor technologies is difficult because these compounds contain the same sulfhydryl groups that demonstrate similar binding ability and reactivity. Therefore, the reaction-based probing (chemodosimetry) becomes of absolute necessity in these cases (Jung et al. 2013; Niu et al. 2015). Probes bearing an electrophilic center may easily undergo reaction with the sulfhydryl group. Thiol detection methods include probes and labeling agents based on nucleophilic addition and substitution, Michael addition, disulfide bond or Se-N bond cleavage, metal-sulfur interactions and more (Peng et al. 2012). However, due mainly to the similarity in nucleophilicity, the discrimination of one biothiol over another still remains a great challenge (Niu et al. 2015). The solution can be found within chemodosimetry concept. Different reactive groups recognizing biothiols selectively can be found (engineered) even within a single probe molecule (Yin et al. 2018). An enhanced doubly-activated (dual excitation) and dual emission system was developed based on fluorescent coumarin-based sensing technology (Mulay et al. 2018). The double optical channel was designed based on a single dye (DACP1 and the closely related DACP-2) that, initially, does not emit fluorescence. It starts emitting after interaction with analyte and this emission is different because different products are obtained on interaction with Cys/Hcy and GHS (Fig. 10.8). The phenylselenide group at the 4-position of the coumarin was flanked by two proximal carbonyl moieties; outstanding leaving-group action upon nucleophilic attack was

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Fig. 10.8 The structures of cysteine, homocysteine and glutathione (a) and a successful result in attempt to distinguish them in molecular probing (b). The engineered dye is able to selectively detect glutathione (GSH) through the use of one optical channel (excitation at 550 nm and emission at 590 nm), and the detection of cysteine (Cys) by another channel (excitation at 410 nm and emission at 510 nm) (Mulay et al. 2018)

enabled. The same chemical group aids in quenching the fluorescence via photoinduced electron transfer (PET) and induces the chromophore non-fluorescence. The selectivity for GSH is observed in the red region and for Cys/Hcy in the green region. When treated with GSH, the probe showed strong fluorescence enhancement compared to that for closely related species. Such selective detection was confirmed in cellular studies. Thus, we have an example that the dark probe responds to Cys/Hcy and GHS by light-up emission with different colors. If such situations happen on a general scale, what results of analytical value can we obtain from ratiometric measurements of emission intensities at two bands? These two intensity channels are independent in conditions when the concentration of sensor itself does not influence this intensity distribution. Then, after proper calibration, we can obtain the distribution of these analyte concentrations, but not their absolute values. Let us consider different cases, in which the ratiometric signal can be obtained between fluorescence spectra before and after the interaction with target. In order to obtain new bands in absorption and/or in emission, some changes should occur in the ground or excited states (see Fig. 9.1). The reaction with biothiol can produce such changes in the probe ground state that influence the intramolecular charge transfer (ICT) and, therefore, change the absorption and excitation spectrum. Then, when both sensor forms, unmodified and modified by analyte, are fluorescent, then we will have two bands in emission excited at two different wavelengths (Zhang et al. 2017; Zhu et al. 2014). Establishing ratiometric

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analytical signal is difficult but possible if we perform excitation at the wavelength, where both forms could be excited. One of good ideas was to manipulate by targeted chemical transformation with the π-conjugation length of the probe. In one of the reference papers (Fu et al. 2017), fluorescein and coumarin platforms were combined for achieving the dual-mode and ratiometric response to Cys over Hcy/GSH, see Fig. 10.9a. Coumarin shares benzene ring with fluorescein, which allows extending the π-conjugation between two fluorophores. Because of the closed-loop fluorescein, the probe only displays the fluorescence (λex /λem = 332 nm/472 nm) of coumarin. In the presence of Cys, the addition-cyclization reaction converts the probe from enol to keto and fluorescein ring-open, which forms a larger conjugate structure resulting from xanthene and coumarin, so the probe exhibits another fluorescence band excited at the longwavelength shifted absorption band (λex /λem = 450 nm/540 nm). This transformation is fast, within 10 min. A good linear correlation was established between fluorescence intensity (λem = 540 nm) and concentration of Cys (0–10 μM). Essentially, decrease in number of blue emitting unreacted sensors is accompanied by an increase in number of green emitting sensors after the reaction with analyte, which informs on analyte concentration. The result presented in Fig. 10.9b demonstrates quite opposite effect (Zhang et al. 2017). The extended π-conjugated electronic system in an unreacted probe allowed observing upon excitation with red light (610 nm) the near-IR emission peaked at 707 nm. These characteristics are attributed to the specific acceptor–donor-acceptor structure of the probe, providing two different ICT pathways. The nucleophilic addition reaction of the double bond and Cys results in shrinking the π-conjugation, abolishing the strong ICT process. Because of that, the long-wavelength emission band disappears. At the same time, the weak ICT process starts to be present, generating the absorption band at 465 nm and causing the short-wavelength emission (λem = 588 nm). The reaction can be completed in 2 min. It shows a good selectivity and sensitivity for Cys. The ratiometric fluorescent change (F 588 /F 707 ) exhibits good linear relationship with the low concentration of Cys (0–45 μM), and the LOD was calculated to be 74 nM. This case shows that though the spectroscopic result is opposite to that discussed above, the analytical result is quite similar—the analyte concentration is proportional to the relative intensity of the band signaling on the reaction product. Changing the excited-state reactivity as a result of reaction with the analyte is one more possibility that can be used in sensing biothiols. In this case, the sensor can be emissive but unable to excited-state transformations. Reaction with analyte removes this restriction and allows the excited-state reaction (ICT, ESIPT, EET) to proceed. What is the benefit? As a result of these reactions, the fluorescence spectra may become strongly shifted to longer wavelengths, which allows achieving strong band separation with the spectrum of unreacted target and efficient ratiometric recording. An example of λ-ratiometric fluorescent probe based on ESIPT reaction designed for the detection of Cys is presented in Fig. 10.10. The probe BTP-Cys uses the acrylates moiety as a recognition site. This site also serves as the protection unit preventing the ESIPT reaction, so that this reaction is activated on this bond splitting.

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Fig. 10.9 The sensors used for specific detection and visualization of cysteine based on reactions changing the dye π-conjugation length. a Composite fluorescent probe, in which coumarin shares benzene ring with fluorescein. This allows extending the π-conjugation between two fluorophores in a Cys-based reaction (Fu et al. 2017). Initially, the probe displays the absorption (at 332 nm) and fluorescence (at 472 nm) bands of coumarin because of the absence of extended conjugation. Increase of conjugation length in specific reaction with Cys results in the appearance of new absorption (at 450 nm) and fluorescence (at 540 nm) bands. b The unreacted probe demonstrates the long-wavelength absorption (at 610 nm) and emission (at 707 nm) bands due to extended π-conjugation length of fluorophore (Zhang et al. 2017). Reaction with Cys results in smaller π-conjugation leading to disappearance of these bands. The reduced fluorophore generates the absorption and emission bands at much shorter wavelengths, 465 and 588 nm, correspondingly

When unreacted with Cys, the probe emits blue light. The ESIPT reaction is known to shift the fluorescence emission band very strongly to red (see Sect. 4.1 of Volume 1). In the present case, this emission shift is very large (113 nm). This allows the decrease of intensity of blue (410 nm) band and an increase of green (523 nm) band to use for the detection of Cys in the range 0–250 μM, so that a dramatic fluorescent intensity ratios enhancement (from 0.03 to 18.3) can be recorded. Another example of using the excited-state reaction for biothiol sensing is presented in Fig. 10.11. This time it is for sensing glutathione, and the excitedstate process is the excitation energy transfer (EET) between two fragments of the sensor molecule (Umezawa et al. 2017). In this case, the sensor is able to perform the EET reaction between two coupled rhodamine derivatives, and a strong Stokesshifted emission band is observed. The reaction with analyte destroys the light absorbance of EET acceptor, and the donor starts emitting light itself, providing good possibilities for ratiometric analyte detection. The synthesized probe exhibits the GSH concentration-dependent, reversible and rapid absorption/fluorescence changes

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Fig. 10.10 The probe selective for cysteine generating the two-band ratiometric response based on excited-state intramolecular proton transfer (ESIPT) reaction (Li et al. 2019a). a Proposed mechanism of response to Cys of probe BTP-Cys. b Fluorescence spectra of (1 μM) in pH 7.4 HEPES-CH3CN (3:2, v/v) in the absence or presence of Cys (0–400 μM). Inset: fluorescence intensity ratio (F 523 /F 410 ) changes of BTP-Cys (1 μM) with the amount of Cys, and the fluorescence color change of probe BTP-Cys before and after addition of Cys under a 365 nm UV lamp. The spectra were recorded after incubation of the probe with Cys for 25 min. Excitation at 350 nm

(t1/2 = 620 ms at [GSH] = 1 mM), as well as appropriate Kd values within the range of intracellular GSH concentrations (1–10 mM). Such probes are useful for quantifying GSH concentrations in various cell types and also for real-time live-cell imaging of GSH dynamics with temporal resolution of seconds. With this and similar probes based on EET we observe the possibility to achieve the ratiometric response by manipulating with the reaction between the aromatic donor and a dye whose properties as an acceptor change on interaction with GSH. Transformation of the acceptor occurs in the ground state, which is seen as the decrease of its absorption band. When the excitation at the donor band is provided, the acceptor band in emission spectrum is decreased accordingly. This allows plotting the GSH concentration as a function of intensity ratio of two bands, as is seen in Fig. 10.11c. The use of intramolecular rearrangement of Cys/Hcy with chemosensors is a typical strategy for the design of fluorescent sensors to discriminate Cys/Hcy and GSH. Concluding this section it is worth noting that selective ratiometric detection and imaging of biothiols can be achieved also with upconversion nanoparticles (Guan et al. 2016), which provides great advantages in fluorescence microscopic studies.

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Fig. 10.11 Example of glutathione probe based on suppression of excitation energy transfer (EET) between two coupled rhodamine dyes (Umezawa et al. 2017). Reversible reaction with GSH destroys the EET receptor and activates the short-wavelength emission of the donor. a Absorption spectrum of the probe (1 μM) showing the decrease of intensity of acceptor band upon addition of GSH (0–20 mM). b Fluorescence spectrum (excitation at 550 nm) demonstrating redistribution of intensity from long-wavelength band of the acceptor to shortwavelength band of the donor. c The dose–response curve plotted in two-wavelength F 593 /F 632 ratio units as a function of GSH concentration. The blue region indicates the reported range of general intracellular GSH concentrations

10.4 Biologically Relevant Phosphate Anions Phosphates have attracted much attention because of their biological significance (Li et al. 2019b). The phosphorylation (binding of phosphate) is a key reaction in metabolism of sugars, and the addition and removal of phosphate groups modulate the activity of a great number of enzymes, participate in formation of bones, etc. Phosphorylation/dephosphorylation in nucleic acid residues ATP and GTP is coupled with many biosynthetic reactions. The orthophosphate (or simply, phosphate) ion [PO4 ]3− , that is derived from phosphoric acid by the removal of three protons, and pyrophosphate [P2 O7 ]4− ion are the most abundant phosphate forms. Intensity-based sensing with the reference (Sect. 3.2 of Volume 1), being conceptually simple and typical for detecting different analytes, has found application in sensing of phosphate ions. The very low fluorescence of the compounds presented in Fig. 10.12 is due to photoinduced electron transfer (PET) from the unprotonated amino group to anthracene fluorophore. These cationic groups can bind a complementary structure, which can be the phosphate and pyrophosphate anions. The remaining phosphate OH groups are in a favorable position to undergo an intra-complex

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Fig. 10.12 Sensors for phosphate groups based on protonated polyamines. a Phosphate anions (Huston et al. 1989). b Pyrophosphate anions (Vance and Czarnik 1994). In both cases the PET quenching was removed resulting in the appearance of bright emission

proton transfer to the unprotonated amino group, which eliminates intramolecular quenching. Then, the binding is accompanied by a drastic enhancement of fluorescence. Still, the stability of such complexes is low. As an example of polymeric sensor, the positively charged surface of polyethylenimine was used for binding the responsive dye 8-hydroxyquinoline-5-sulfonate through electrostatic interaction. The reference was provided by gold nanoclusters covered by the polymer (Zhang et al. 2019). Ratiometric response based on such introduction of the reference allowed quantitative detection of phosphate in human serum samples. Metal–organic frameworks (MOFs) have started to be used for phosphate sensing (Gao et al. 2018). A complicated dual lanthanide MOF showing emission at 375 nm was synthesized via hydrothermal reaction based on Tb3+ and Ce3+ as the center metal ions and terephthalic acid as the organic ligand. In the presence of PO4 3− , the fluorescence intensity at 500 nm and 550 nm increased, while the intensity at 375 nm was reduced. Hence, the λ-ratiometric fluorescence detecting of PO4 3− can be achieved by measuring the ratio of fluorescence intensities at 550 nm to 375 nm (Li et al. 2021). In a stable Zr-based MOF, the ligand-to-metal charge transfer (LMCT) was established that was weakened due to the strong binding of phosphate ion, providing the reporter signal, and rhodamine B dye was used as the reference (Gao et al. 2018). In both cases the micromolar range of sensitivity was adapted for measuring the phosphate concentration in blood serum.

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Pyrophosphate (P2 O7 4− , PPi) anion is central to a number of biological processes, including ATP hydrolysis, DNA and RNA polymerization, and a number of enzymatic reactions. Moreover, its abnormal production, degradation, and transport are associated with a range of diseases. The popular methods of its determination are those using the recognition (affinity sensing) of its bivalent charged structure. This strategy when applied to the recognition of PPi allow the sensor with the structure presented in Fig. 10.12b to bind these ions over 2000 times more tightly than phosphate ions, permitting the real-time monitoring of reaction of pyrophosphate hydrolysis. Pyrophosphate ion can be determined with the use of a pyrene-functionalized guanidinium receptor in MeOH (Nishizawa et al. 1999). The receptor (Fig. 10.13) was found to self-assemble forming a 2:1 (host:guest) complex due to the electrostatic interaction. Formation of the self-assembled structure results in a remarkable change in the ratio of fluorescence emission intensities of excimer to monomer in pyrene moiety that demonstrate characteristic spectra at different wavelengths (see Volume 1, Sect. 3.6). Promoting the formation of the self-assembly of two reporting units with pyrene derivatives, this system shows high selectivity for PPi. The application of excimer formation as a reporter signal was also used in other publications. Thus, a PPi reporting system as a 2:2 complex assembled in water solutions was developed (Lee et al. 2007). The other numerous possibilities include the complex formations incorporating metal cations (Jang et al. 2005) that due to ICT perturbation of fluorophore may allow ratiometric measurements. The structures as presented in Fig. 10.13 require molecular diffusion of pyrene sensors and their assembly induced by the target. For providing the ratiometric response in an intramolecular way, there should be the construction created

Fig. 10.13 The application of pyrene excimer formation in sensing pyrophosphate (Nishizawa et al. 1999). a Guanidinium receptor with appended pyrenyl group binds PPi at two phosphate sites. The pyrenes become in contact forming an exciplex. b Fluorescence spectra upon addition of PPi (0, 140, 270, 410, 540 μM) as [K + [18]crown-6] salt to 8.0 × 10–4 M sensor in MeOH. Excitation wavelength was 312 nm. Inset: Dependence of two-band intensity ratio F 476 /F376 on the concentrations of a PPi and b H2 PO4 , demonstrating high selectivity for PPi

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for coupling the selective bivalent target binding with the process generating new emission bands intramolecularly. It was shown that this process can be the excited-state intramolecular proton transfer (ESIPT) that can be realized with 2(2-hydroxyphenyl)-1,3-benzoxazole (HBO) fluorophore. In molecular construction incorporating Zn2+ ions for molecular recognition, the conformational change occurring on PPi binding makes ESIPT possible (Chen et al. 2011). This well-defined chemical event in the molecular design leads to a highly selective probe for PPi. The PPi binding provides dramatic transformation of spectra (Fig. 10.14). The shortwavelength (blue) normal band decreases and the long-wavelength ESIPT band (green) shifted by ~100 nm increases accordingly. This result shows a high efficiency of the ESIPT as a switching mechanism in anion detection (Sedgwick et al. 2018). The use of a Zn2+ complex as a binding site for PPi has been found to be a particularly successful strategy due to the strong binding affinity between Zn2+ and PPi. As we will see below, it will be efficiently used for the ATP and GTP recognition.

Fig. 10.14 Fluorescence sensor for pyrophosphate based on generation of new fluorescence band due to activation of excited-state intramolecular proton transfer (ESIPT) reaction (Chen et al. 2011). The sensor molecule coordinates two Zn2+ ions that change their interactions on PPi binding making ESIPT possible. The sensor responds by the changes in distribution of fluorescence emission between two bands that allows wavelength-ratiometric recording. Presented is the change in fluorescence emission of the sensor (12 μM) upon addition of PPi (sodium salt) in an aqueous solution at pH 7.4

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From the above discussion we derive that the design of PPi sensors requires the following: an understanding of the molecular recognition between PPi and the binding sites, the desired solubility in aqueous solutions, the communicating and signaling mechanism, and most importantly, selectivity for PPi over other anions such as AMP and ADP, and particularly phosphate and ATP.

10.5 Adenosine and Guanosine Triphosphates Recognition and sensing of nucleotides, and of ATP and GTP in particular (Fig. 10.15), has been an especially active area of research due to their biological significance described in many biochemistry and cell biology textbooks. In different biological processes, ATP is not only a universal energy source but also an extracellular signaling mediator (Khakh and Burnstock 2009), and GTP is involved in RNA synthesis, citric acid cycle, and acts as an energy source for protein synthesis. Adenosine monophosphate (AMP), adenosine diphosphate (ADP) and guanosine monophosphate (GMP) also play pivotal roles in various physiological events. The high energy stored in ATP terminal phosphate group being the main source of energy for intracellular processes, is released upon hydrolysis to form ADP, and this process has to be followed with space and time resolution. Cytosine and uracil based compounds also play important roles in several metabolic processes. Analyzing biological systems, ATP and GTP should be distinguished from other nucleoside polyphosphates containing these and other bases. Two types of recognition sites can be used for efficient sensing of nucleotides. The first one is the phosphate moiety that in normal conditions is negatively charged (Butler and Jolliffe 2020). It interacts readily with imidazole groups (Zhu et al. 2017a, b) or quaternary ammonium groups. These elements of structure have to be coupled with fluorescence reporters (Zhou et al. 2011). Thus, the rather simple water-soluble imidazolium anthracene derivative differentiates two structurally similar compounds GTP and ATP and may act as their potential fluorescent chemosensor (Kwon et al. 2004). The other option is to use electrostatic interactions with zinc ions incorporated into organic structures that may serve as efficient recognition units (BazanyRodríguez et al. 2020). Both possibilities allow selective recognition of triphosphates out of other phosphate derivatives (Agafontsev et al. 2019; Li et al. 2019b). The hydrogen bonding-based receptors can be made of thiourea that can be coupled with fluorescent reporter (Swamy et al. 2019). The other sites for molecular recognition are the purine bases. Here the π-π stacking interactions together with H-bonding and steric effects can be very efficient. In this respect, a classical example of ATP sensor based on disruption of pyrene excimer can be presented (Xu et al. 2009). In this smart construction, the cluster of imidazolium groups served as the binding site for ATP phosphate groups, and pyrene dimer was used for selective binding the adenine base and for providing the reporter signal by disrupting the excimer (Fig. 10.15). Symmetric pendants were bridged by one benzene group with flexible

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Fig. 10.15 Molecular sensor designed for the detection of ATP providing wavelength-ratiometric response and strong discrimination against GTP (Xu et al. 2009). a The structures of GTP and ATP and of correspondent monophosphate and biphosphate forms. b The structure of the sensor and illustration of differences in its interaction with GTP and ATP. c The spectroscopic response to ATP by disruption of excimers. Fluorescent titrations of probe (10 μM) upon addition of sodium salt of ATP at pH 7.4 (20 mM HEPES). d Fluorescent emission changes of the probe (10 μM) upon addition of sodium salt of AMP, ADP, and ATP (20 equiv) at pH 7.4 (20 mM HEPES). Inset: Ratiometric calibration curve for the intensity ratio F 375 /F 478 as a function of the concentration of AMP, ADP, or ATP. Excitation at 345 nm

methylene, and the short distance between two pendants was made possible for the initial π-stacking between pyrene units at each side and the resulted excimer emission (see central part in Fig. 10.15b). The ATP binding occurs by insertion of its base between these units. It gets inserted in a π-stacked pyrene-adenine-pyrene sandwich pattern. The extended distance between pyrene units does not make possible the excimer emission, and the emission of monomers starts to be observed (Fig. 10.15c).

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The ratio of intensities of monomer (at 375 nm) and excimer (at 478 nm) emissions was applied for ATP determination. As can be seen in Fig. 10.15b, this construction allows discrimination of ATP against GTP. Sharing similar structural skeleton with ATP, the guanine group of GTP does not fit into the sandwich formed by two pyrene groups. It could only bind with this sensor from the outside, hardly making damage to the stable pyrene dimer. Since the pyrene ability to form exciplex is not disrupted, only the long-wavelength structureless exciplex emission is observed. This happens also with other nucleoside triphosphates (CTP, UTP and TTP), and this determines the high selectivity of ATP binding. Providing the F 375 /F 478 ratiometric reporting, this probe can be used for large-scale ATP sensing and imaging in the neutral pH range. Naphthalimide–rhodamine coupling is one of the most efficient ways for constructing the sensors based on excited-state energy transfer. A ratiometric fluorescent probe for ATP detection built on this mechanism was reported (Tang et al. 2014). It shows an unexpected high selectivity for ATP over other anions, especially organic phosphate anions, due to simultaneous interactions of two recognition sites, which benefits the fluorescence imaging in living cells. Among many other ATP sensors (Agafontsev et al. 2019; Wu et al. 2017b) operating in intensity or more attractively, in λ-ratiometric mode, the sensors based on 3hydroxychromone (3HC) scaffolds (Pivovarenko et al. 2017) appear very interesting. Surprisingly, a smart single dye can substitute a complicated dimer. Whereas typically for providing ratiometric signal the reporter functionality requires two coupled fluorophores for operation based on excimer formation or excitation energy transfer between them (Tang et al. 2014), the dye demonstrating the excited-state intramolecular proton transfer (ESIPT) may serve efficiently to this purpose. On interaction with ATP, it exhibits new bands in its fluorescence excitation and emission spectra that are strongly shifted to longer wavelengths (Yushchenko et al. 2007). This suggests intensity-based and λ-ratiometric measurements of ATP concentration. In a recent study on a broad series of 3HC and related dyes (Pivovarenko et al. 2017), it was shown that these dyes may differ by the total charge, the size and number of their aromatic units, as well as by the position or electron donating ability of their substituents, but display similar properties. All of them were suggested to form complexes with ATP of 1:1 and 1:2 stoichiometry at neutral pH, which can be evidenced by their 3000–6000 cm−1 red-shifted excitation band and their bright fluorescence. Regarding the mechanism of binding, the comparative study shows a surprising fact that electrostatic interactions play only a minor role, so that the majority of effect is due to the H-bonding and stacking interactions between the chromone unit of the dye and the adenine base of the ATP molecule, discriminating ATP from other nucleotides. The stability constants of the formed complexes depend on the dye structure and lie in the range of 0.3–3 × 103 M−1 . The dyes penetrate easily into the cells and their mitochondria. Therefore they are prospective for ATP imaging. Sensing of GTP did not receive so much attention as that of ATP (Li et al. 2019b). Usually in biological systems GTP is present in much lower concentrations than ATP. If their affinities for the receptor sites are not strongly different, this can make

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problems for its competing with ATP for these sites. Genetically encoded sensor for intracellular GTP detection has been recently developed based on genetically encoded protein (Bianchi-Smiraglia et al. 2017; Zhang et al. 2020). Summarizing, it should be stated that affinity sensing based on molecular recognition is the major principle behind the design of fluorescence probes for purines and pyrimidines. However, the efficiency of most of them does not extend to sensing on the level of several micromoles or lower (Bazany-Rodríguez et al. 2020), which is their typical concentration in extracellular fluids, such as human blood (Traut 1994).

10.6 Redox Cofactors NADH/NAD+ and NAD(P)H/ NAD(P)+ Reduced nicotinamide adenine dinucleotide NADH and its phosphate ester NAD(P)H are ubiquitous molecules, which are involved in numerous metabolic events in living cells as electron carriers. Providing transfer between their reduced and oxidized forms, NADH/NAD+ and NAD(P)H/ NAD(P)+ are key electron carriers in living cells, coupling the transfer of hydrogen atoms and electrons from one metabolite to another in many cellular redox reactions. Serving as coenzymes, they participate in energy metabolism, mitochondrial functions, immune function, biosynthesis, gene expression and cell death (Ying 2006). It is important to monitor in real-time their levels in living cells and in vivo (Sun et al. 2021a). Direct sensing and visualization of NADH/NAD+ and NAD(P)H/ NAD(P)+ using their own fluorescence is complicated. NADH can be excited at ~340 nm and its fluorescence can be observed as a broad band at ~450 nm, with lifetime 0.41.0 ns (Lakowicz et al. 1992), but NAD+ is nonfluorescent. NADH and NAD(P)H have similar fluorescence properties though their functions in the cell are different. Therefore, their distinction should be provided in sensing and cell imaging. Affinity sensing technology has been applied for NADH and NAD(P)H analysis using the benefits of their extended structures, charges and the possibilities of their multipoint binding with the sensor. The techniques based on genetically encoded fluorescence proteins (Tao et al. 2017; Zhao et al. 2011) were applied that allow their differentiating in cell imaging (Bilan et al. 2014). A number of NADH and NAD(P)H-directed organic dye indicators that demonstrate fluorescence intensitybased response have been also suggested (Fomin et al. 2016; Pan et al. 2019; Podder et al. 2020). Directed to recognition of the negatively charged phosphate groups based on the electrostatic attraction with the positively charged imidazolium heads, an imidazolium functionalized polydiacetylenes sensing system was reported (Jiang et al. 2020). Detecting selectively NADPH and NADP+ , this system does not respond to the less negatively charged NADH and NAD+ in the tested concentration range. Owing to the positively charged nicotinamide of NADP+ , its detection sensitivity

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towards NADP+ is much lower than that towards NADPH. Successfully differentiating NADPH from the rest three cofactors, it can be used for practical applications in NADPH detection. The application of principle of chemodosimetry (see above) turned out to be quite efficient in quantitative detection of redox cofactors because, as in other relevant cases, these targets easily produce the chemical transformation in the sensor. The fact of their very high reactivity in redox reactions suggested to use this property for their detection based on switching the sensor between its oxidized and reduced forms. The redox transformation with NADH and NAD(P)H-dependent enzymes is frequently used in vitro and can be recorded with different analytical techniques (Heikal 2010). This transformation is selective and can provide the recorded signal. The enzyme-linked reactions are convenient for applications in vitro, but they are hardly applicable for cellular imaging. Therefore, the redox reactivity was used for chemical transformations in the designed fluorescence sensors (Park et al. 2021). Both NADH and NADPH coenzymes can reduce many electron-deficient heterocyclic compounds that can serve as different recognition units in fluorescent probes. They can be coupled with PET, ICT and spirolactone ring-opening reactions providing modulation of fluorescence response (Sun et al. 2021a). Among these compounds are quinone, resazurin, quinolinium and pyridine.

10.7 Sensing and Thinking. The Problem of Simultaneous Sensing and Imaging of Many Analytes In this chapter, by selecting the examples of the most essential and diagnostically valuable small molecular analytes we demonstrate the very rapid development of this field of research and analysis. Now we have to evaluate what are the arising problems and what are the natural limits in this development. It is especially important to do that every time, since most of the reported probes have been used only for in vitro spectroscopic detection, their detection limits and response concentration ranges often do not match intracellular or environment-valuable analyte concentrations. For those that match, the problems of solubility and access to targets in structured and, especially, biological media may be associated with problems that have to be properly resolved. We observe also that the general principle of affinity sensing cannot be simply applied to considered type of molecules. An alternative approach, chemodosimetry, based on sensor-target reactivity, is needed for active use in many cases. These principles are often not complementary in application to particular targets, since when molecules are small and neutral, their contacts with the designed sensor will not be sufficient for their selective recognition, but their chemical reactivity may be specific and strong. Enzyme sensors may be considered as an extension of this approach (Karube and Nomura 2000; Wilson and Hu 2000); they are not considered here.

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Determination of biologically relevant compounds individually, of their selected examples presented above or more extended range of them existing in nature, cannot satisfy the researchers. Each of these species participates in the network of metabolic pathways demonstrating a range of time-dependent transformations (Papin et al. 2003). Signaling molecules work together (Mukherjee and Corpas 2020; Xuan et al. 2020). For multiple target detection (Qian et al. 2019), one may use the sensor arrays (see Sect. 15.4 of Volume 1) after destroying the cells and tissues and extracting the needed fractions. The array format cannot be fitted to living cell interior directly, and the extraction-based approach can be misleading in view of complexity of studied systems and possibilities of functionally important mutual transformations of the targets. The microarrays that can be formed of self-assembled molecular structures can allow detection of several analytes based on their “scores” (Sasaki et al. 2020), which is still an in vitro assay. So, to what extent the fluorescence probing allows simultaneous detection of certain number of targets within the cells and in biological tissues? What was done and what are the prospects? “Lab-on-a-molecule” concept, which could incorporate the whole sensing ensemble responding simultaneously to a number of analytes (Scerri et al. 2019), was formulated. Some attempts in this direction have been made already (Huang et al. 2020). Thus, by exciting and/or emitting at different wavelengths one can measure with a single sensor the water content and hypochlorite concentration (Song et al. 2019), intracellular and intra-tissue ClO− and H2 O2 (Du et al. 2020), H2 O2 and NO (Yuan et al. 2012), bisulfite and hydrogen peroxide (Zhou et al. 2021). Porphyrinbased pH indicators can be further developed to sense different targets (Dini et al. 2015). Fluorescent probes to simultaneously detect GSH, Cys, and SO2 derivatives were developed (Jia et al. 2020). Simultaneous detection of biothiols Cys, Hcy and GSH was realized in separate output channels (Xu et al. 2019). This can be achieved by designing multiple binding sites in small organic receptor molecules (Yue et al. 2019). Regarding materials, metal–organic frameworks (MOFs), in addition to organic dyes, are becoming more frequently used for constructing the multi-analyte sensors (Wang et al. 2018; Yang et al. 2020a; Yin and Yin 2020). Logical operations can be performed with input signals coming from binding several targets, and such procedure has got the name “molecular computing” (see Sect. 4.5 of Volume 1). Now we concentrate on the problems of analysis of multiple output signals that have relation to the problem of optical barcoding (Sect. 15.5 of Volume 1). In essence, it is related to the ability of pattern recognition in spectroscopic analysis. We outline three problems (Fig. 10.16). Problem one is the limitation in color window. The possibilities of spectroscopic recognition are limited due to commonly broad spectra of emitters and their overlap on the wavelength scale. The complicating factors are the gradual (but not discrete) changes of band intensities for each component and the coupled changes for two bands in the case of λ-ratiometry. Problem two is the interplay between output signals. Excited-state interactions leading to quenching (PET, excimer and exciplex formations) operate at short

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Fig. 10.16 The multi-analyte multi-responsive fluorescence sensors and the problems arising in recording and analysis of their emission

distances, and the energy transfer (flow) between the emitters shifting the spectra to longer wavelengths (see Sect. 4.1 of Volume 1) may operate on a longer scale. These effects are similar to that observed for aggregates and nanostructures made of organic dyes (Chap. 8 of Volume 1). The problem of chromophore communication exists in MOFs (Martin et al. 2020). Generally, this results in loss of individuality in output from individual emitters. Problem three is related to differences in photobleaching. . Organic dyes lose their fluorophore properties in time on exposure to light. The ability to withstand such photoinduced transformation depends on the dye structure and may vary over several orders of magnitude (Sect. 5.1 of Volume 1), see also (Demchenko 2020). This means that as a result of photobleaching the spectrum formed of dissimilar fluorophores may change as a function of illumination time. The reader is encouraged to take an example from the literature and estimate, to what extent these factors may influence the result of analysis of two or more analytes with a single probe. In addition, the reader may check the effect of reading this chapter by responding the following questions: 1.

2. 3. 4. 5.

6. 7.

Explain the chemodosimetry concept. Why the affinity sensing and chemodosimetry being so different may rely on the same photophysical reactions leading to fluorescence output? List the photophysical mechanisms that can allow generating the λ-ratiometric response and that can operate only in “on–off” mode. What is common between carbon monoxide, nitric oxide and hydrogen sulfide and what is their functional role? Why it is difficult to develop for CO, NO, and H2 S the affinity sensors? How in these cases the chemodosimetry approach is realized? List different possibilities to detect molecular oxygen. How to achieve the wavelength-ratiometric oxygen sensing in solutions? Can this approach be applied to other dissolved gasses? What are the reactive oxygen species and how their oxidation/reduction potential is used in fluorescence sensing? What are biothiols and how to provide their differential sensing? How the λratiometry can be achieved in their detection? Explain the use in this case of

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ESIPT reaction in the fluorophore. EET can also be used. What is the difference between them in generation of reporting signal? 8. Why for sensing the phosphate [PO4 ]3− and pyrophosphate [P2 O7 ]4− ions the affinity sensing can be realized? Explain different possibilities for this realization based on the principle of charge complementarity. 9. How to differentially recognize [PO4 ]3− and [P2 O7 ]4− ions in fluorescence sensing? How the λ-ratiometric signal can be generated in these cases? 10. ATP and GTP are very similar molecules. How to analyze them separately? What ground-state and excited-state transformations may be used to generate their λ-ratiometric signal? 11. Redox cofactors NADH/NAD+ and NAD(P)H/ NAD(P)+ can exist in both reduced and oxidized forms. Their detection can be based on both affinity sensing and on chemodosimetry. Analyze strong and weak points of these approaches. 12. Why the problem of simultaneous sensing and imaging of many analytes is important, particularly regarding the small biology-relevant molecules studied in this chapter? Explain the problems associated with the generation of multicolor response.

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Chapter 11

Detection, Structure and Polymorphism of Nucleic Acids

Detection and characterization of DNA and RNA is needed in many areas, which include all branches of biology, medicine, environmental control and forensic detection. Such extremely broad range of applications requires the search of diverse methodologies that should be optimal for every particular task. Fluorescence techniques offer a broad range of possibilities, from quantitative nucleic acid detection and visualization in cells to characterizing their structural and dynamic variations on the level of single nucleic acid bases. The DNA analytic techniques have led to revolutionary changes in modern biology and related areas. The applications range from simple qualitative detection of a single nucleic acid target to quantitative whole-cell gene expression profiling. They brought unique possibilities for identification of personality, the characteristics of single nucleotide polymorphism (SNP), recognition of cancer cells and tissues and detection of pathogenic microorganisms. They allow comparison of closely related species in plant and animal worlds. This huge amount of tasks requires development of different competitive technologies, what is actually observed. In order to understand the functions of nucleic acids, it is important to study their molecular mechanisms, dynamics, conformational changes and interactions with cellular partners. However, accessing this information is challenging due to dynamic nature of these molecules, the instability of their structures, and the transitory nature of the associated intermediates. Nucleic acids are highly polymorphic, as can be seen from the multitude of canonical and non-canonical structures (B-, A-, and Z-DNA duplexes, triplexes, quadruplex, etc.). RNA is also well-known to form functionally important folded structures, as is seen in ribosomes, tRNAs, ribozymes, riboswitches, and other non-coding RNAs. Quite a different task is to follow a cell’s developmental process at the nuclear level (Weiss et al. 2018). Nuclear probes are extremely significant, as they allow for visualizing nuclei and chromosomes, and they also are very useful in the analysis of chromosome banding patterns, as well as in their division. Furthermore, as the targeted genetic material can be of different forms, i.e., most often double-stranded DNA (genomic DNA in cellular organisms) but sometimes single- or double-stranded © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_11

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RNA (viruses), biosensors must ideally be versatile enough to be able to detect all these different targets. Fluorescence methods are the most suited to satisfy these demands operating on different levels, from chromosomes to single base substitutions and from cell and tissue imaging to characterization of single molecules. They are able to operate at the level of single bases/base pairs and to probe the interactions between nucleic acids with proteins, metabolites, gene-targeting drugs, water molecules, and ions at sub-molecular and atomic levels (Wilhelmsson and Tor 2016). Hybridization sensors can be used to reveal mutant genes associated with genetically determined diseases. Such genetic mutations can be revealed in mismatching in the hybridization process. Another application of hybridization consists of identifying pathogenic micro-organisms in water supplies, foodstuffs and biological samples by their specific nucleic acids, DNA or RNA. Similarly, genetically close and modified organisms can be detected by means of hybridization. Different strategies of introducing fluorescence reporters can be realized. If the DNA detection and analysis does not require achieving high nucleic base specificity, the probes demonstrating strong noncovalent binding can be applied. In cellular research the desired penetration or no-penetration into cell or its nucleus can be selected. In contrast, if the local change of structure, such as in single-nucleotide polymorphism (SNP), has to be studied, the dyes representing the single base modifications or substitutions inserted covalently into the probing chain structure may be applied. The applied fluorescent reporters may be of different origin. There may be organic dyes and all types of fluorescent nanomaterials, including those that were introduced quite recently (such as metal–organic frameworks). As in other areas of application of fluorescence sensing, the most popular here is the simplest type of response, by the change of intensity. This may not be sufficient, and, in addition to lifetime sensing, different forms of λ-ratiometric sensing were suggested (Demchenko 2023a, b). They can be of two types, involving two fluorophores or implemented in a singlefluorophore format. Regarding two fluorophores, the response on interaction between them based on mechanisms of excimer formation and excitation energy transfer can be realized. The more progressive and simple in realization response can be realized with a single molecule according to mechanisms of intramolecular charge transfer (ICT) and excited-state intramolecular proton transfer (ESIPT). Ratiometric fluorescent imaging is widely used for visualization and quantitative analysis of the microenvironment in living cells because of its self-calibration and anti-interference ability. Compared to fluorescence intensity-based imaging, ratiometric imaging has more advantages, such as reducing background and microenvironment interference, being independent of probe concentration, and being able to perform quantitative analysis. A high-throughput screening is often needed in molecular diagnostics, genotyping and gene expression studies. The limitations in operation of this technology and insufficient reproducibility of its data required searching for new solutions that can be found in application of much brighter and more informative organic dyes and inorganic/organic nanocomposites.

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The problem of increasing the sensitivity of detection is critical for the development of DNA and RNA sensing technologies. Since many genetic diseases are caused by single mutations in the patient genome, the selectivity of assay must be sufficiently high to accurately distinguish the mutated disease-related alleles. Also, the majority of infectious diseases need to be diagnosed promptly based on only a few copies of the pathogen that are usually present in the blood or other body fluids at the onset of an infection. Thus, DNA detection techniques need to be ultrasensitive to the point of being able to detect as little as a few copies of the target DNA sequence. This requires the signal amplification. The amplified sensor response can be achieved by two means. One is the increase of target copy number, which is the reason of use the polymerase chain reaction (PCR) or related nucleic acid amplification techniques. This needs thermal cycling and enzyme application, which is expensive and time-consuming. The other is the improvement of output signal based on available amount of sample material; let it be only several molecules and a single mutated site in them. Thus, to reach this level of sensitivity, researchers have investigated several amplification strategies, either enzymatic amplification of the target DNA material or amplification of the signal generated by each detection event. Discussion in the present chapter will be focused on the amount of latter possibilities that require new detection schemes, proper sensor design and smart fluorescence reporters. The progress in the field suggests that new exciting technologies for amplified DNA and RNA detection will emerge.

11.1 DNA Detection and Analysis of Its Conformation There is a high need to determine rapidly and reliably the nucleic acids in different analytical procedures. Quantification in electrophoretic gels, blots, arrays, separation of live and dead cells in flow cytometry, real-time PCR analysis and many other related methods require DNA staining without the need of resolving its nucleic acid base sequence. Likewise, live cell staining of the chromosomal DNA do not require obtaining such detailed information (Martin et al. 2005). In all these cases the dye molecules with a high affinity for DNA and no-to-little sequence preference can be efficiently used. A huge variety of such dyes can be found in the literature; they are very popular and many of them are commercially available (Haugland 2005; Spence and Johnson 2010). The researcher may choose the dyes that are cell-permeable or not, those targeting particular organelles or absorbing and emitting light in particular wavelength region. Efficient in applications are the dyes that are not (or low) fluorescent producing low background in the unbound state but becoming strongly emissive on target binding. Among small molecular fluorescent probes, Hoechst (bis-benzimidazole probes), 6-diamidino-2-phenylindole (DAPI), ethidium bromide (EtBr), propidium iodide (PI), SYBR green I, PicoGreen and thiazole orange (TO) are the most popular ones. Particularly, acridine dyes and ethidium bromide possess

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the property of displaying almost no fluorescence in water but a very strong emission when they are bound with high affinity to DNA (with enhancement by a factor of ~103 and higher), see Sect. 5.1 of Volume 1. Recent years are marked by rapid development of far-red, near-infrared (Suseela et al. 2018; Yuan et al. 2013) and two-photonic (Gao et al. 2019) dyes for cellular and in vivo imaging. The majority of these dyes are based on reversible non-covalent attachment (binding) to nucleic acids and, therefore, they are very simple in application. They do not exhibit specific base recognition but are sensitive to the state and changes of molecular conformation (Ma et al. 2013; Tatikolov 2012). They are mainly used for the visualization of DNA in experimental biology procedures (Kapuscinski 1995; Narayanaswamy et al. 2015) and can be applied to monitor the folding and interactions of nucleic acids with their targets but are not able to provide the site-specific data. In a living cell, genomic DNA typically adopts a right-handed, double-helix structure supported by A·T (adenine·thymine) and G·C (guanine·cytosine) base pairing. These standard double-stranded structures (dsDNA), termed canonical structures, are supported by Watson–Crick hydrogen bonding (Fig. 11.1). Beyond the well-known B-DNA form, the DNA can fold into a Z-duplex, triplex, G-quadruplex or i-motif. It forms bulges, loops, holiday junctions, and pseudoknots, etc. (Choi and Majima 2011; Doluca et al. 2013). These non-canonical motifs are believed to have an important influence on major cellular processes. As we will see below, the visualization and characterization of non-canonical structures have attracted much attention along with discoveries of their important functional role. Fig. 11.1 Double-stranded helical DNA structure (from Wikipedia). The adenine–thymine (A-T) and guanine-cytosine (G-C) base pairing stabilized by hydrogen bonds results in formation of double-helix right-handed conformation of B-DNA

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11.1.1 Double-Stranded DNA Structures Double-stranded DNA can exist in several forms (A-DNA, B-DNA, Z-DNA) which differ by their structural and conformational parameters (Belmont et al. 2001). BDNA is the most abundant form under near-physiological conditions. It forms major and minor grooves exposed to the environment. Both grooves have moderate depths and are well solvated by water molecules. The minor groove is broad and shallow, and the major groove is deep and narrow. There are three classes of dyes regarding their binding to different locations of the double-helix. They are intercalating dyes (Lyles and Cameron 2002), minor groove binders (Reddy et al. 1999; Sarwar et al. 2015) and other nucleic acid stains (Spence and Johnson 2010), such as major groove binders (Kim and Nordén 1993) and external binders. The latter are usually associate with the phosphate backbone (Rehman et al. 2015). The different binding modes are schematically shown in Fig. 11.2. One mode is the intercalation. It is the insertion and stacking of planar smallsize molecules between the base pairs of the DNA double helix due to hydrophobic and van der Waals interaction. In the double helix, the bases form planar couples connected by hydrogen bonds. Intercalation is a noncovalent binding, in which the molecule of the same size as the base pair accommodates between the neighboring base pairs. It is held perpendicular to the helix axis. This type of binding is characteristic for planar acridine, ethidium and thiazole dyes and their dimers. The major effect of fluorescence enhancement of these dyes is their screening from the quenching effect of water. Affinity of these dyes towards DNA can be dramatically (by 103 –104 times) increased upon the use of their dimers instead of previously applied monomers. In the cases of the dye dimers, the two heterocyclic parts intercalate between the base pairs, and the linker accommodates to the minor groove. These dimers, such as YOYO,

Fig. 11.2 Several modes of binding fluorescent organic dyes to dsDNA and an example of minor groove binder, Hoechst 33342 (Spence and Johnson 2010)

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YOYO

b

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YOYO

Wavelength (nm)

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a

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Fig. 11.3 The YOYO-type dyes that are composed of two organic heterocycles connected with a positively charged linker. a Fluorescence excitation (solid) and emission (dashed) spectra of YOYO1 and the model of its intercalation into double-helix DNA structure (inset). b The assortment of similar to YOYO dimeric dyes for incorporation into dsDNA covering the whole visible range of spectrum (Spence and Johnson 2010)

EthD and TOTO (Thiazole Orange dimer), carry four positive charges allowing strong electrostatic interaction with the negatively charged DNA phosphates (Fig. 11.3). The binding of these dyes is so strong, that electrophoresis of DNA labeled with them in nanomolar quantities can be performed without the dye loss (Glazer et al. 1990). The affinity towards DNA and the brightness of fluorescence response are sufficient for the single-molecular detection. YOYO belongs to the family of monomethine cyanine dyes and is a tetracationic homodimer of Oxazole Yellow (YO), which is the origin of the name YOYO. Free YOYO dye in aqueous buffer has absorption maximum at λmax 458 nm and very low intensity of fluorescence emission at λmax 564 nm. As for other intercalating dyes, its intensity increases (by ~3200 times) upon binding to double-stranded DNA with some spectral changes (absorption: λmax = 489 nm, emission: λmax = 509 nm). The mode of incorporation (intercalation) into dsDNA is shown in the insert of Fig. 11.3a. The minor groove binders represent the other type of DNA-staining dyes. They are the dyes that bind spontaneously directly to the minor groove of the double helix. In this case, the binding of dye molecules is mostly electrostatic, and great enhancement of their fluorescence upon the binding can be due to both displacement of water from their contact areas with DNA and to the suppression of their internal segmental mobility. The most known dye of this type is blue-emissive 4’,6-diamidino2-phenylindole (DAPI), which is extensively used in fluorescence microscopy to stain DNA in cell nucleus (Kapuscinski, 1995). Other examples are the dyes known as Hoechst dyes (e.g. Hoechst 33258 or Hoechst 33342). They possess three positive charges per molecule (see Fig. 11.2), which determines the mode of their binding (Pjura et al. 1987). They are cell membrane permeable, and their positive feature

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is the strong Stokes shifts that allow exciting fluorescence at about 350 nm and observing it in the 500 nm region. There are different cyanine dyes (Sect. 5.2 of Volume 1) that are frequently used for DNA detection (Tatikolov, 2012). Their advantage is the presence of low-polar groups together with the positive charge that provides specific affinity towards nucleic acids together with high brightness necessary for ultrasensitive detection. Fluorescence enhancement on their binding is due to dye fixation in trans-conformation. They demonstrate remarkable diversity of the binding patterns. The short-chain monomethine dyes act as intercalators, while the increase in polymethine chain length leads to increasing the potency for groove binding (Kovalska et al. 2006; Yashchuk et al. 2007). Their sensitivity can be even further increased by formation on this interaction of cyanine dye aggregates (Ogul’chansky et al. 2000; Yarmoluk et al. 2002). When applied in living systems, the dye probes should not interfere into cellular processes. Thus, it was shown that YOYO-1 dye (shown in Fig. 11.3) provides a simple and reliable green-fluorescent stains for FISH (fluorescent in situ hybridization) analysis, usually on fixed cells. This technique is used to diagnose chromosome abnormalities going through cell divisions.

11.1.2 Analysis of Single-Stranded DNA Although most DNA exists as double-stranded DNA (dsDNA), the single-stranded (ssDNA) forms are important intermediates in the processes of DNA replication, transcription, repair, and recombination (Técher et al. 2017). Besides that, ssDNA is the genomic material of a subgroup of viruses. The linear DNA chains can create a variety of three-dimensional motifs, which define structural polymorphisms. Many fluorescent dyes bind not only to dsDNA but also to single-stranded DNA and RNA. Single-stranded DNA harbors a polyanion skeleton assembled by multiple negatively charged phosphate groups. This skeleton is the site of binding of positively charged fluorescent dyes. Moreover, these dyes can aggregate along ssDNA through electrostatic interaction. Acridine orange is one of the earliest examples, for which the binding to both dsDNA and ssDNA was described. The mechanisms of binding to double and singlestranded nucleic acids are so different, that there is a difference in emission color. When this dye binds to dsDNA, it exhibits green fluorescence, whereas bound to RNA its fluorescence is red. Thus, the DNA in the nucleus stains green, while the RNA in cytoplasm - red. These observations were extended by an application of a number of acridinium derivatives, and those of them were found that bind to single strands of DNA and RNA with negligible binding to the double-strand DNA (Kuruvilla and Ramaiah 2007). Due to the presence of four positive charges, the ethidium dimer and also YOYO and TOTO dyes can also bind to ssDNA and RNA but with lesser affinity. To overcome these limitations, new fluorescent dyes were developed, which can discriminate between ssDNA, dsDNA and RNA. Advanced variants of cyanine dyes marked by

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ThermoFisher under names PicoGreen®, OliGreen® and RiboGreen® can be used to selective quantitative analysis of dsDNA, ssDNA and RNA in solutions, respectively (Spence and Johnson 2010). Many new possibilities appear in the studies of structures and interactions of nucleic acids using neutral 3-hydorxychromone (3HC) derivatives as color-changing λ-ratiometric dyes when they are attached to poycations. Using the labeled spermine as a positively charged targeting moiety, the binding to single-stranded or doublestranded DNA is easily distinguished by dramatic difference in intensities of their double-band spectra (Klymchenko et al. 2008). As shown in Fig. 11.4, the response of 2-furyl-3HC dye as the label demonstrates the dramatically increased emission of the long-wavelength T* band, so that the ratio of the two N* and T* emission bands change up to 16-fold. This suggests an efficient screening of the 3HC fluorophore from water molecules in the complex with DNA. In sharp contrast, only moderate changes in the dual emission on binding to a single-stranded DNA are observed, indicating a much higher fluorophore exposure to water at the binding site. Thus, the fluorophore being conjugated to spermine recognizes easily the binding of this polycation to dsDNA from that to ssDNA. Cationic polyelectrolytes are frequently used as the ssDNA binders. Cationic polythiophene derivatives (CPD) deserve special attention as water-soluble polymers with adverse fluorescence in monodisperse status and highly aggregated form respectively. As we will see below, these polymers have become valuable tools in DNA hybridization techniques (Ho et al. 2002, 2005).

...

ESIPT

Fluorescence intensity

Spermine – 3HC

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Fig. 11.4 Fluorescence spectra of spermine derivative labeled with 2-furyl-3HC dye interacting with single-strand (blue) and double-strand (red) DNAs (Klymchenko, Shvadchak et al. 2008). Dramatic increase of relative intensity of long-wavelength product T* band relative to initially excited N* band intensity in ESIPT reaction indicates incorporation of the fluorophore into the double-helical structure

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11.1.3 Identification of Non-canonical DNA Forms The properties of DNA as a polymeric structure allow both rigidity and flexibility of its conformation. Under special physiological conditions, DNA molecules adopt distinct spatial organizations, giving rise to non-canonical conformations different from classical right-handed double helices and supported by hydrogen bonding other than the Watson–Crick type. Such Hoogsteen and Wobble base pairing depends not only on external environmental conditions but primarily on the sequence of nucleic acid bases (Choi and Majima 2011; Doluca et al. 2013). G-quadruplex (G4) DNA is a fully stable non-canonical secondary nucleic acid structure formed by guanine-rich genomic sequences (Varshney et al. 2020). Quadruplex structures are not exotic forms (in contrast to triplex DNA), they are fully natural, existing e.g. in telomeres and promoter regions of numerous genes. The biological roles of DNA G4s are still poorly understood, but there are many evidences on their involvement into the regulation of gene expression at the transcriptional level (Hänsel-Hertsch et al. 2016; Wang and Vasquez 2014). Moreover, they are involved in pathological transformations, such as oncogene overexpression, alterations in DNA replication and telomere stability. Since G4-specific DNA-binding ligands are the telomerase inhibitors, it is thought that having definite role in controlling gene expression, such non-canonical structures can serve as novel diagnostic and therapeutic targets (Balasubramanian et al. 2011; Shahsavar et al. 2021) being prospective for anticancer therapy. The planar guanine quartets (G-quartets) that are composed of four guanine bases are stabilized via the Hoogsteen pairing. The central cavity of G-quadruplexes is occupied by monovalent cations, which neutralize the electrostatic repulsion between guanine oxygen atoms, and thus stabilize the overall structure shown in Fig. 5.7. Looking from biotechnological side, the DNA quadruplexes represent valuable tools for constructing the fluorescence sensing devices (Sect. 5.3), see also (Shahsavar et al. 2020; Umar et al. 2019). Indeed, as we discussed in Sect. 5.3, the possibilities exist to use these structures and their transformations for forming efficient molecular sensors. Here we focus on G4 as the targets themselves trying to understand their properties in vitro and in vivo. Fluorescence probing is the key method to acquire this information. Several tasks can be formulated (Bhasikuttan and Mohanty 2015; Vummidi et al. 2013) that have to be realized. The first task is manipulating with DNA of variable sequence in different conditions in vitro in order to understand, what structures are realizable and what factors influence on that (Lightfoot et al. 2019). Here a variety of other methods of G4 structure determination can be used, and we can learn more on their interactions with fluorescence probes (Asamitsu et al. 2019). The most promising probes are then can be selected for cellular studies. Their mode of binding can be diverse (Fig. 11.5). They can interact with G-quadruplex DNA through different binding strategies, such as electrostatic interactions, end stacking, intercalation, and groove binding (Asamitsu et al. 2019; Ma et al. 2015; Ruttkay-Nedecky et al. 2013).

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K+

K+

K+

K+

End stacking

Groove binding

K+

Intercalation

Fig. 11.5 Three modes of binding of fluorescence probe to G-quadruplex structure (the antiparallel G4 folded structure is shown)

The second task is to introduce fluorescence probes for the G4 labeling and tracing their transformations within the cells. Here the ability of probe to penetrate into the cell, into its nucleus and nucleolus is the necessary condition, and of value is their selective binding and efficient reporting. Additional requirements are imposed by fluorescence microscopy technique, such as the high brightness and convenience of excitation and emission wavelengths. Numerous small molecular fluorescent probes with emission in the far red to near-IR wavelength region have been reported for targeting these DNA polymorphic structures (Suseela et al. 2018; Umar et al. 2019; Wang et al. 2021). They are able to light-up the G-quadruplex structures providing the “OFF–ON” emission switching on binding. The third task is the finding of drugs as the ligands that selectively bind to G-quadruplex structures without binding to duplex DNA, which is an important and challenging goal of drug discovery (Chilka et al. 2019). Since such drugs and the G4-specific fluorescence probes may compete for the same binding sites, such competition can be used by performing the high-throughput fluorescent indicator displacement assays (del Villar-Guerra et al. 2018; Xie et al. 2019). For performing these tasks, a large number of fluorescence probes were tested, from very simple to highly sophisticated. Thus, a water-soluble fluorogenic dye, Thioflavin T (ThT), which is known for its frequent use in staining fibrillar protein aggregates, has shown selective ability to bind to G4 sites (Fig. 11.6). This dye induces the quadruplex folding in the 22AG human telomeric DNA, both in the presence and absence of Tris buffer/salt, and senses the formed G4 through its fluorescence light-up. The emission enhancement of the order of ~1700–2100-fold in the visible region can be observed (Mohanty et al. 2013). Such a large increase in ThT fluorescence emission contrasts with the results on control DNA duplexes and single strands that had a much smaller effect on emission. This differential behavior suggests designing a high-throughput assay to detect the G4 formation. Hundreds of different oligonucleotides may be tested in parallel for G4 formation with a simple fluorescence plate reader (Renaud de la Faverie et al. 2014). The large aromatic π-surfaces formed of planar G-quartets were suggested using the π–π-stacking interactions for their recognition with fluorescent probes (RuttkayNedecky et al. 2013), and porphyrins have become the subjects of active research

Fluorescence intensity

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Absorbance

a

Wavelength (nm)

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Fig. 11.6 The application of Thioflavin T (ThT) for induction and detection of formation of Gquadruplex structures (Mohanty et al. 2013). Absorption (a) and fluorescence (b) spectra of ThT with 22AG DNA at different concentrations. ThT solution (3.5 μM) contained 5 mM Tris (pH 7.2) with [22AG]/μM: (1) 0, (2) 0.12, (3) 0.25, (4) 0.75, (5) 1.5, (6) 2.5, (7) 4.0, (8) 5.0

and applications (Norvaiša et al. 2020; Zhang et al. 2019). For example, studies on cationic porphyrins showed the ability to specifically recognize nucleic acid G4 over dsDNA and single-stranded DNA (ssDNA) (Huo et al. 2017). The cyanine dyes can bind to G-quadruplex structures without targeting duplex DNA (Owens et al. 2019). Due to their high amenability to structural modifications, the asymmetric trimethine cyanines are attractive not only as fluorophores but also as drug candidates. In an attempt to provide high affinity and selectivity for G4s and also to generate fluorescence turn-on properties based on suppression of photoinduced electron transfer (PET) quenching, a “twice-as-smart” probe was developed (Laguerre et al. 2016). This was achieved by quartet–quartet recognition on the self-assembly of guanine quartets, one from the quadruplex and one from the probe (Fig. 11.7) offering selectivity for particular G4s over other nucleic acid conformations. When free in solution, N-TASQ exists in its open conformation, and the fluorescence of its naphthalene template is quenched by the four surrounding guanines via intramolecular photoinduced electron transfer. However, upon interaction with N-TASQ, the intramolecular G-quartet formation leads to the redistribution of the guanine electrons onto the four guanines, restoring fluorescence. The probe allows tracking quadruplexes in live cells with two-photon microscopy. The noncanonical DNA i-motifs are flexible (Dai et al. 2010) and strongly pHdependent (Choi et al. 2011) structures (see Sect. 5.4). Therefore their detection and characterization in cells, being very important (Abou Assi et al. 2018), presents a great problem. The basic building block of the i-motif structure is a base pair involving one neutral (deprotonated) cytosine and one positively-charged (protonated) cytosine at the N3 position. The resulting C·C+ base pair, which is stable because of the formation of three hydrogen bonds, allows the formation of the parallel duplexes. The base

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Fig. 11.7 Recognition of G-quadruplexes by synthetic G-quartet probes (Laguerre et al. 2016). The structures of a template-assembled synthetic G-quartet (N-TASQ, left) and of a G-quartet (right) represent schematically the quadruplex-forming sequences in their unfolded and folded states. The bioinspired interaction between N-TASQ and quadruplexes takes place through quartetquartet recognition, stabilized by π-stacking interactions (grey arrows) and cation chelation (yellow dashed lines), along with electrostatic interactions (pink arrows). The ordered hydration shell of the N-TASQ/quadruplex assembly is schematically represented as a pale blue rectangle

protonation plays important role in formation and stability of these structures. Like for G4, modulation of formation and properties of i-motifs is one of key points of cell biology (Masoud and Nagasawa 2018). The i-motif-binding ligands are used as tools to investigate the i-motif structure and biological functions and also as potential drug candidates (Brown and Kendrick 2021). It is relatively easy to study the i-motifs formation and reorganization in vitro and the influence of different ligands on this process. A fluorescent intercalator displacement assay can be applied for identification of new DNA i-motif binding ligands (Sheng et al. 2017). Thiazole orange was used as the dye interacting with imotif DNA and used in this assay. Using the fluorescence labeling of DNA fragment at its terminal group, the structural transitions in i-motif sequences can be studied by fluorescence anisotropy (Huang et al. 2015). Regarding a visualization of i-motif on cellular level, it is a very difficult task, but for finding its solution, simple probes have started to be found. Thus, a near-infrared fluorescent probe called Quinaldine Red, the same as used to lights up the β-sheet structure of amyloid fibrils (Wang et al. 2020), was reported to be applied efficiently (Jiang et al. 2017). Other dyes have also been tested (Chen et al. 2021), and essential progress is expected in near future. Concluding this section, we can state that intensity-based light-up recording of fluorescence dominates in the studies of non-canonical DNA forms (Umar et al. 2019) and more sophisticated dyes reporting in time-resolved and λ-ratiometric manner must substitute them in near future.

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11.2 Recognition of Specific DNA Sequences by Hybridization Nothing is more specific than the recognition between specific DNA or RNA sequences that leads to the formation of double-helical structures. Such recognition is always in the background of hybridization assays. The two strands recognize each other by formation of very selective hydrogen bonds between the complementary nucleic acid bases (see Fig. 11.1). Three such bonds are formed between guanine (G) and cytosine (C) base pairs, and two bonds are formed in complementary pairs of adenine (A) and thymine (T), for DNA, or adenine and uracyl (U), for RNA. Due to collective effect produced on the affinity of interaction by multipoint binding, the formation of the first three-four base pairs is sufficient to trigger the process of hybridization. The formed hybrid double helices are highly specific and stable. In order to possess the detection method that could satisfy many demands (such as in genomic analysis and/or clinical gene expression profiling), we have to resolve three technological problems. First, we need the adequate transduction method to generate a physically measurable signal from the hybridization event. Second, this method should be extremely sensitive to allow the detection of formed hybrids on a picomolar level of concentrations and below. Third, this method should allow the application in microarrays to apply essentially the same way of detection for all the spotted (or otherwise marked) sensor sequences. Oligonucleotides (ONs) are commonly used for the detection of specific nucleic acid sequences in hybridization assays. ONs are oligomers of desired sequences, of their nucleotide order and lengths, obtained by chemical synthesis. Their minimum size for recognition a single specific sequence in the whole human genome (3 × 109 base-pairs) is on the level of 15–19 nucleotides. The detection limit in standard hybridization procedures should be about 104 –105 genome copies. It can be dramatically reduced when working with microscopy on a single-cell level. It is important to stress that the selectivity provided by DNA hybridization technologies is of two sides. It is quite positive that only the genes, for which the capture sequences are present in the system (e.g. in an array), can be captured and analyzed with the desired absence of interference of sequences representing all other genes. The other side is our inability to detect any sequence present in the applied sample, for which we did not prepare complimentary capture sequence. This means that the genes not yet been annotated in a genome will not be represented on the arrays and will avoid the analysis. Thus, we can only detect sequences that were designed to be detected (Bolón-Canedo et al. 2019).

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11.2.1 The Microarray ‘DNA Chip’ Hybridization Techniques With the prospective of recognition of particular DNA sequences coding particular genes, we can critically analyze the spotted microarray ‘DNA chip’ technology that presently dominates in biomedical research and clinical diagnostics. It is not excluded, however, that these chips may be slowly substituted by nextgeneration sequencing (NGS) based methods (McCombie et al. 2019), since the cost of sequencing has dropped dramatically during the last decade and this tendency continues. MNG can be applied on the level of single cells and allows to capture all sequences, not only those programmed in the microarray. It is particularly prospective for diagnosis of virus diseases (Beerenwinkel et al. 2012). A spotted DNA microarray consists of a set of different probes, each of them being specific to a particular gene and placed at a particular site on a small plate. In standard microarrays, the probes are attached by covalent linking to a solid support made of glass, polymers or silicon. This allows parallel and simultaneous detection of a great number of target genes. The use of such microarrays is versatile. They are widely applied for diagnostic purposes for gene expression analysis or for developing drugs against gene expression related diseases, such as cancer. Also, DNA microarrays are employed for detecting mutations or polymorphisms in a gene sequence. The array throughput is determined by the spot density, and great success is achieved in DNA chip miniaturization. Meantime, the sensitivity of analysis still relies on amplification by polymerase chain reaction (PCR) or reverse transcription polymerase chain reaction (RT-PCR) of the whole pool of tested sequences. Organic dyes are applied as the labels for the amplified products in this pool. The common DNA chip technology that provides comparative analysis of two DNA samples is well described (Sassolas et al. 2008). It consists of three essential steps (Fig. 11.8): (a) The array fabrication. The sensing ssDNA sequences (oligos) possessing no label are immobilized or directly synthesized on the surface (slices or membranes) in multiple copies, forming the microarray. Each type of ssDNA sequence occupies the separate spot. (b) The preparation of the target and the reference. A comparative study is commonly made (competitive hybridization). The pool of DNA or RNA fragments that contain potential targets are amplified by PCR or RT-PCR and are covalently labeled with a fluorescent dye. The same is done with the reference sample. In this case, the labeling is made with the dye emitting with a different color. Then both labeled samples are mixed and hybridized to an array. The target sequences compete for the binding sites and those of them that are present in greater quantities in solution are predominantly bound. (c) The data acquisition and analysis. It is provided by a special reader, recording in two channels the spectrally resolved fluorescence emission. The formed image is analyzed with the aid of special computer software.

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Fig. 11.8 Schematic showing the steps involved in fabrication (a), hybridization (b), and analysis (c) of a spotted DNA microarray. The standard DNA microarray consists of a collection of microscopic DNA-probe spots attached to a solid surface. Each spot identifies by hybridization a particular target in a sample. Hybridization is performed with target sequences from a test sample and a reference sample that have been labeled with different fluorophores. They are mixed and hybridized to the same array. Signal reading is carried out by means of an optical scanner that includes two laser excitation sources and very sensitive light detectors

Thus, the pattern of spots location allows finding each DNA sequence at a unique site. The elementary units of DNA flat microarrays (biochips) are the spots containing numerous ssDNA chains of identical sequence terminally anchored to the support surface. These chains hybridize selectively with free fluorescently labeled ssDNA chains having a complementary sequence (see Fig. 11.9a). The presence of specific sequences (targets) is signaled by hybridization on the corresponding spot as monitored by correlating the strength of the label signal with the position of the spot (Dharmadi and Gonzalez 2004). Based on this principle, the functional and convenient DNA biochips have been applied to analyze genomes, to detect inherited and acquired diseases, such as cancer, to detect cellular responses to different stimuli, such as the effects of drugs on a whole-transcriptome level. They have proved to be very useful for rapid detection of mutations, single nucleotide polymorphism (SNP) and also for gene discovery and expression monitoring.

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a

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Indicator

Fig. 11.9 Different principles that are followed in the design of DNA sensor arrays. a Target pool labeling. The sensor chain is unlabeled. b The sandwich assay. Two sites on the target are recognized. Neither target pool nor sensor chain is labeled. The indicator chain brings the label. c Both sensor and indicator sequences are labeled. Binding the target brings two labels together and allows realizing EET, providing switching of emission color. The sensor and the indicator can be connected by flexible linker. d Molecular beacon. The target binding to complementary loop structure disrupts the stem and removes the label from quenching by the neighboring groups or by the surface

Modern technology allows depositing as much as 105 of different oligonucleotides on a square centimeter chip, which shows the capability of extension to the whole transcriptome profiling. Arrays containing many thousands of unique probe sequences have been constructed, and sophisticated algorithms were developed for fluorescence readout data analysis. With the increase of microarray size, this analysis becomes less trivial and more demanding. Deep learning methods are rapidly developing for such analysis (Bolón-Canedo et al. 2019). This technique is still far from being ideal, and there exist at least two important critical steps that have to be improved. One is the use of PCR, and the other—the target labeling. Both refer to manipulations with the studied sample that need to be avoided in all cases when the time of analysis and the costs of reagents and labor are the critical points. The PCR technique allows producing many nucleic acid sequences using the target nucleic acid fragment as a template. With its aid, the detection limit reaches the subpicomolar range. Meantime, this reaction is time-consuming; it requires temperature cycling and consumes expensive materials (enzymes and labeled nucleotide derivatives). Furthermore, the selective and nonlinear target amplification in PCR may distort the results. Portability and miniaturization of this reaction remains a problem. Avoiding the PCR reaction can be possible only when the necessary level of sensitivity is achieved with alternative procedures. We have to admit also that the target pool labeling used here is the most primitive concept in sensing. Its success in DNA hybridization assays is due to a lucky fact that the same chemistry can be used for labeling all nucleic acids present in the

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pool. There are attempts for avoiding the target labeling based on the competitive DNA hybridization approach (Peterson et al. 2016). In this method, a labeled analyte analog is initially hybridized to the capture probe so as to achieve saturation. Next, the saturated receptor layer is allowed to interact with the sample. On this stage of the process, the sample analyte displaces a fraction of the labeled analog, leading to a decrease in the fluorescence. The attempts to substitute the target pool labeling with the techniques based on more advanced concepts (as described in Chap. 1 of Volume 1) are discussed below. Still, the simple ‘DNA chip’ technology has opened exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. The multiplexed microarray platforms provide parallel detection capabilities enabling the measurements of many thousands of simultaneous responses. The DNA microarrays are especially attractive due to achieved combination of high throughput, parallelism, miniaturization, speed, and automation. Inclusion of the expression profile of the whole genome eliminates the bias related to any preliminary selection. Moreover, the global nature of the DNA microarray technique holds tremendous promise for the discovery of complex genetic and metabolic networks. In this rapidly developing technology, the measurement is ahead of analysis and the sensitivity is ahead of accuracy. Overcoming these difficulties is expected soon.

11.2.2 Sandwich Assays in DNA Hybridization DNA hybridization can be carried out in the sandwich format (see Sect. 2.2 of Volume 1). In this approach, the probe is designed so that after hybridization with the target, an overhanging target fragment remains in the single-strand form. This fragment is then used to bind by hybridization a signaling probe tagged with a fluorescent label. Sandwich assay avoids the necessity of pool labeling but requires recognition of the target at two different sites. In nucleic acid detection one of the DNA terminal sequences can be recognized by the capture sequence (that is usually attached to support), and the remaining single-chain segment exposed to the solvent can be hybridized with the indicator sequence that contains fluorescence label. Illustration of this principle is presented in Fig. 11.9b. As in the case of immunoassays, this label can be not only the fluorescent dye but also a fluorescent nanoparticle that can provide increased brightness. The dye-silica doped nanoparticles were successfully used for DNA detection in this assay (Zhao et al. 2003). An impressive detection limit of 0.8 fm (femtomoles) and selectivity ratio of 14:1 against one-base mismatches have been achieved. This approach can allow to make one more step forward and to develop the wavelength-ratiometric fluorescence nanosensor (Demchenko 2023a, b) based on double labeling (Fig. 11.9c). Indicator DNA is modified with the attachment of fluorescent dye that serves as EET acceptor. The sensor sequences can be attached to a plain support but also can be bound to fluorescent nanoparticles that serve as

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EET donors. When both of them are present in test medium without target DNA, they do not interact and we observe emission of the donor. In the presence of target DNA sequence, it hybridizes with both capture and indicator sequences, the EET donor and acceptor approach each other and we observe the emission of the acceptor (Zhang et al. 2005). Many constructions like that can be devised, but their extension to high throughput screening in suspension array format needs precise identification of nanoparticles carrying specific sensors, the ‘barcoding’.

11.2.3 Molecular Beacon Technique Quite different methodology is behind the molecular beacon techniques (Fig. 11.9d). These “beacons” operate according to the principle of direct sensing, in which the recognizing sequence possesses also the reporting function. Reporting is coupled to conformational change in this sequence. In Sect. 4.4 of Volume 1 the reader can find the detailed description of this mechanism, and below we concentrate on different possibilities of its realization and on some of obtained results. Molecular beacons are the nucleic acids that contain two structural components, a loop and a stem forming a ‘molecular hairpin’ (see Fig. 4.15 in Volume 1). The loop is the recognition element for complementary nucleic acid sequences of the target. The stem is formed of two complementary sequences that flank the loop. It is a 4–7 base pair sequence at each of the 5' and 3' ends that is self-hybridizing and stabilizes the overall hairpin shape of the closed beacon. In an inactive, unbound conformation, the molecular beacon resembles a short hairpin brought together by binding between the two stem nucleotide sequences. In its initial presentation, it was end-labeled with a reporter dye and a quencher (Tyagi and Kramer 1996; Zheng et al. 2015). Other double labeling techniques were also reported (Tan et al. 2004). The process of recognition in molecular beacons is associated with the disruption of intramolecular contacts between DNA bases in the stem in favor of intermolecular contacts with the target formed by the loop resulting in proper fluorescence response. Because of such competition, this method is much more sensitive to single nucleotide substitutions than the simpler hybridization assays (Demidov and Frank-Kamenetskii 2004). Hybridization of the target nucleotide sequence to the loop opens the stem-loop, inducing a separation between them removing the interaction between two labels or label and quencher (see Fig. 13.12d). Different signal transduction mechanisms, in addition to fluorescence de-quenching, were suggested to provide the response to analyte ssDNA or RNA binding (Guo et al. 2012; Huang et al. 2014). Moreover, the hairpin is the simplest but not the only possible structure of a molecular DNA sensor that combines the target binding and reporting. Hybrid structures were proposed that contain DNA segments connected by a flexible polymer linker (Yang et al. 2006). Upon the hybridization, the labeled DNA segments appear in close proximity, so that the response in EET is generated.

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A great advantage of molecular beacons is the possibility of their use for intracellular studies (Wang et al. 2013). The labeled hairpins are the molecular instruments that do not need any support and contain everything for both recognition and reporting. There is a problem, however, that any nonspecific disruption of stem-loop structure would give a false positive result. This can happen due to degradation by nucleases, protein binding or other conditions making the stem structure unstable.

11.2.4 Specific DNA Sensing with the Aid of Conjugated Polymer Cationic conjugated polymers exhibit strong electrostatic binding to nucleic acids with the change of their fluorescence properties. An example is polythiophene polymer which was used for colorimetric and fluorometric detection of nucleic acids (Ho et al. 2002). They can be strongly fluorescent in their native random coil conformation, but fluorescence is quenched when they couple with a single-stranded DNA chain to adopt a highly conjugated, planar conformation. The fluorescence (λexc = 420 nm, λem = 525 nm) appears again when a complementary oligonucleotide target is added to this molecular system, because with the double-stranded DNA such interaction is different. In this case, the polymer binds in a helical and non-planar configuration (a ‘triplex’) with the negatively charged phosphate backbone of the double-stranded DNA (Fig. 11.10).

DUPLEX

Positively charged Polythiophene

Single-stranded DNA probe

TRIPLEX

Fig. 11.10 Schematic description of the formation in solution of single-stranded nucleic acid duplex with cationic polythiophene derivative and polythiophene/hybridized nucleic acid triplex (Doré et al. 2009). Addition to cationic conjugated polymer of a capture DNA strand induces its planar, highly conjugated conformation. This change in conformation is accompanied with a yellow to red color change and a complete fluorescence quenching. Upon addition of the complementary target DNA strand, a triplex structure is formed, in which the polymer is wrapped in a helical fashion around the DNA duplex, resulting in a blue-shifted absorption band and the appearance of strong fluorescence

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Thus, the conjugated polymer can serve as a very sensitive indicator of hybridization. Moreover, since it exhibits a remarkable property of antenna effect in EET and superquenching (Sect. 8.5 of Volume 1), the sensitivity of response can be dramatically increased with the inclusion into this system of additional organic dye (Liu et al. 2011). It was shown, that tagging the sensor chain with fluorescent dye serving as EET acceptor that emits with substantial wavelength shift allows detecting as much as only five copies of dsDNA in 3 ml in only 5 min in the presence of the entire human genome (Ho et al. 2005). This approach is suitable for the rapid assessment of the identity of single nucleotide polymorphisms (SNPs) of different genes, and of pathogens without the need for nucleic acid amplification (Dore et al. 2006).

11.2.5 DNA Structure Recognition with Peptide Nucleic Acids All current techniques for quantifying specific DNA or RNA exploit the base pair fitting between target polynucleotide and a complementary nucleic acid sequence serving as the sensor recognition unit. The major effort of researchers is now directed towards developing the techniques that could dramatically increase the sensitivity of response to this primary interaction. This search was extended to synthetic polymers. It was found that the neutral polymer called ‘peptide nucleic acid’ demonstrates superior to DNA or RNA binding properties (Li et al. 2016). Peptide nucleic acid (PNA) is not an acid! It is a neutral oligomer, in which the entire negatively charged backbone is replaced by an uncharged N-(2-aminoethyl) glycine scaffold. It can contain the same side groups (bases) as DNA and RNA and is capable of forming the highly stable complexes with complementary sequences of nucleic acids. The nucleotide nitrogenous bases are attached to its backbone via a methylene carbonyl linker. Therefore, PNA can be regarded as DNA with a neutral peptide backbone instead of a negatively charged sugar-phosphate backbone of natural nucleic acids (Shakeel et al. 2006). The size and shape of this polymer allows ideal hybridization based on the base–base interaction with DNA and RNA (Fig. 11.11). Because of the absence of negative charge, peptide nucleic acids exhibit superior hybridization properties than the natural nucleic acids. PNA is chemically stable and resistant to enzymatic cleavage, which provides its stability towards degradation in a living cell. PNA is capable of recognizing specific sequences of DNA and RNA obeying the Watson–Crick hydrogen bonding scheme, and the hybrid complexes exhibit extraordinary thermal stability and unique ionic strength effects (Shakeel et al. 2006). The great interest to PNA as a recognition functionality for DNA or RNA detection appeared after it was demonstrated that both ssDNA and DNA-PNA hybrids bind cationic conjugated polymer (e.g. polythiophene derivative) with a dramatic change of its fluorescence (see subsection above). When the neutral peptide nucleic

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Fig. 11.11 Chemical and cartoon representations of L Kγ-PNA assembled onto DNA (Englund et al. 2012). a Chemical structure of L Kγ-PNA (Lys-bound PNA) bound to DNA. b, c Ribbon and cartoon diagrams of four L Kγ-PNAs (each bearing one ligand) bound to a linear DNA. Chemical and cartoon representations of L Kγ-PNA assembled onto DNA. Lysine-substituted PNA enabled the attachment of a wide variety of ligands via the reactive amine handle (including fluorescent dyes). Below: Comparison of PNA and DNA structures showing structural similarity between them. Similar size of repeat units allows hybridization between the chains in the case of complementarity of side groups (nucleic acid bases). Because of absence of electrostatic repulsion, PNA binds to DNA stronger than a complementary DNA chain

acid is immobilized as the recognition unit on the surface of planar array, it does not interact with the cationic polymer. The triplex formation similar to that described in Fig. 11.10 occurs on hybridization with the target nucleic acid. A combination of complementary functions of specific base-to-base PNA recognition with the extraordinary reporting function of conjugated polymer suggests a revolutionary change in DNA detection methodology. For detecting and identifying the unlabeled target nucleic acid on microarrays, a combination of surface-bound PNA probes and soluble fluorescent cationic conjugated polymers was suggested (Raymond et al. 2005). Application of PNA instead of DNA on supported arrays has the advantage in the absence of interaction between PNA and cationic polymer. Only in the target DNA presence such interaction appears in the form of ‘triplex’. In addition to active role in DNA recognition and passive role in reporting, PNA can play an active role when labeled with the dye that can serve the EET acceptor. Then in combination with the conjugated polymer that serves an antenna and an

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energy donor, a superquenching effect (Sect. 8.1 of Volume 1) can be achieved with substantial increase of response sensitivity (Liu and Bazan 2005). Interaction of PNA with the double-stranded DNA is the prospective trend in development of DNA sensing technologies. PNA is able to form specific higherorder (i.e. three- and four-stranded) complexes with DNA. This makes it an ideal structural probe for designing the strand-specific dsDNA biosensors (Baker et al. 2006). The formation of higher-order complexes can be detected by the dye-labeling of PNA. Addition to this system of cationic conjugated polymer allows providing the amplification of reporting effect. This finding has important consequences. Commonly, the DNA hybridization assays require thermal denaturation producing the dissociation of the two strands, which is necessary for subsequent hybridization. Introduction into a sensing technology of a detection method for dsDNA that eliminates the need for thermal denaturing steps must have good prospects. The single nucleotide polymorphism (SNP) is the major type of variation in the human genome that can characterize the identity of a person on a genetic level (see below). The strategy employing a combination of PNA recognition units, an optically amplifying conjugated polymer detectors, and S1 nuclease enzyme is capable of detecting SNPs in a simple, rapid, and sensitive manner (Baker et al. 2006). The recognition is accomplished by the sequence-specific hybridization between the uncharged fluorescein-labeled PNA probe and the DNA sequence of interest. After subsequent treatment with S1 nuclease, the cationic polymer associates with the remaining anionic PNA/DNA complex, leading to sensitized emission of the dye-labeled PNA probe via EET mechanism. An improvement of this assay can be provided by additional application of nonionic detergent (Al Attar et al. 2008). The techniques addressing detection of single mismatches in hybridization assays can benefit from the possibility of using PNA segments in which one base is substituted with a fluorescent dye. This dye serves as a replacement of a canonical nucleic acid base with the ability of performing a reporting function (Socher et al. 2008). If a complementary DNA molecule (except at the site of the dye) hybridizes to the probe, the dye exhibits intense fluorescence emission because the stacking in the duplexes enforces its coplanar arrangement. However, a base mismatch at either position immediately adjacent to the dye dramatically decreases fluorescence, presumably because the dye becomes allowed to undergo torsional motions that lead to rapid depletion of the excited state. The number of dyes tested for this functional response (called ‘forced intercallation’) increases (Bethge et al. 2008).

11.2.6 The Use of Nanomaterials in DNA Hybridization An ultimate goal of achieving the sensitivity of DNA assays to a level that could allow avoiding the target multiplication procedures (e.g. PCR) can be reached when the commonly applied organic dyes are substituted by much brighter fluorescent nanoparticles. Nanoparticles can play also different roles as support materials and

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also as plasmonic enhancers and efficient quenchers (Vikrant et al. 2019). The detection strategy can be based on the differences in affinities of the labeled single-stranded and double-stranded DNA oligonucleotides towards spontaneous self-assembly with different types of nanoparticles. Two examples of using the nanoparticles in modulation of fluorescence response in hybridization assays that allow using the homogeneous format are presented in Fig. 11.12. It is known that different carbonic structures (see Chap. 9 of Volume 1) are among the strongest fluorescence quenchers and also that nucleic acids can be readily adsorbed on their surface as single chains but not as double-helical structures. These facts suggested providing the hybridization assay, in which the labeled probing sequence is attached and its fluorescence is quenched. Dissociation coupled with the target binding results in dequenching. Such possibility has been demonstrated with graphene oxide nanoparticles (Liu et al. 2014), carbon nanotubes (Li et al. 2013) and carbon dots (Loo et al. 2016). Metal–organic frameworks (MOFs) demonstrating differential binding ability to single-stranded and double-stranded DNA molecules can also be used for exploitation of this idea (Zhang et al. 2018). High binding capacity of MOFs allows operating with several capture probes labeled with dyes of different color (Wu et al. 2018). Plasmonic enhancement of fluorescence (see Chap. 13 of Volume 1) can be efficiently used with gold nanoparticles in application to DNA assays (Peng and Miller 2011). Dissociation from plasmonic nanoparticles results in fluorescence loss. An optimal combination of plasmonic enhancement and quenching allows achieving

Fig. 11.12 Different possibilities to use nanostructures in modulation of fluorescence response in nucleic acid hybridization assays. (a) Carbon nanoparticles strongly quench fluorescence of attached single-chain labeled sequences. Hybridization with the target sequence results in its detachment from the nanoparticle surface resulting in fluorescence enhancement. (b) Plasmonic nanoparticle provides strongly enhanced fluorescence of attached single-chain probe. Hybridization with target sequence results in its detachment that removes plasmonic enhancement retaining a weak molecular fluorescence

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strong increase in sensitivity of hybridization assays (Vietz et al. 2017; Zhu et al. 2018). In the efforts to avoid tedious and expensive DNA target amplification (generation of its multiple copies), various methods for enhancing the fluorescence signal have been advanced (Fozooni et al. 2017; Willner et al. 2009). Trivial but efficient is the substitution in conventional schemes of organic dyes by brighter emitters, such as fluorescent nanoparticles. Conjugation of nucleic acids with properly functionalized metal nanoparticles, semiconductor nanocrystals (quantum dots), carbon dots and dye-incorporated polymeric nanostructures (see Volume 1, Chaps. 7, 8, 9, and 10) have frequently been reported. Signaling oligonucleotides are usually linked covalently to nanoparticles and employed in sandwich, the EET-based or molecular beacon formats (Fig. 11.9). Optimization of fluorescent nanoparticles can be made in different ways, and one of them is the increase of nanoparticle brightness (Melnychuk and Klymchenko, 2018). Thus, extremely high brightness with low background can be achieved in nanocomposites encapsulating ~1000 strongly coupled and highly emissive rhodamine dyes into a 20-nm light-harvesting polymeric particle that can transfer the excitation energy to a single acceptor. The obtained nanoprobes enable singlemolecule detection of short DNA and RNA, encoding a cancer marker (survivin), and imaging the single hybridization events by an epifluorescence microscope (Melnychuk et al. 2020). The remark should be made concerning the comparison of in-vitro and in-vivo hybridization assays. Working in cell-free settings, it is easy to optimize a variety of conditions (such as temperature, pH, molecular crowding, and density of targets) and to make them to be precisely controlled. There is no trouble regarding the reagent delivery and accessibility of the specific sequence on the target, and a risk of its degradation by nucleases. In contrast, the in vivo assays, such as fluorescence in situ hybridization (FISH), face important problems, such as the delivery and distribution of exogenous probes to the cell. Because of that, FISH is commonly used only with fixed cells. They may not be always accessible as target nucleic acids due to the presence of their complex secondary structures (Liehr 2017). The ability to detect, localize, quantify and monitor the expression of specific genes in living cells in real time without altering the cell morphology or the integrity of its various compartments is very demanding in realization. No less demanding is the requirement for providing the fluorescence response. In living cells, the detection of formed hybrid requires a change of the fluorescent signal upon hybridization that cannot be only the switchon of intensity. A strong demand exists towards the time-resolved and wavelengthratiometric probing.

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11.3 Probing on the Level of Single Nucleic Acid Bases In previous sections we discussed probing the structures of nucleic acids and their hybridization with complementary chain sequences that did not require precise localization of reporting fluorophores. These dyes did not demonstrate selective affinity to particular bases, and this feature was not actually needed. Thus, the reporters for double helices, G-quadruplexes and i-motifs responded to conformations with particular folding, but not to the bases that participated in their formation. Likewise, in hybridization assays the reporter had to provide signal on the hybrid formation regardless of its own location. Thus, in these cases the role of fluorophores was rather passive and precision of their location was not strongly requested. The situation becomes quite different when we study point mutations in nucleic acid sequences. These changes may not alter significantly the affinity between hybridized chains, which makes problematic their detection in standard hybridization assays. Instead, the dyes are needed to be precisely located at the point mutation site; they should not perturb significantly the structure and interaction energies at this site but have to provide reliable information on the presence of this mutation. To achieve the needed precision of location, the reporting dyes must be covalently linked to the probing sequence. The chemical structure of nucleic acids allows for multiple ways of attaching exogenous fluorophores with high specificity. The label can be attached to certain position of the structure via a linker or may substitute one nucleic acid base. The examples of such linkages are presented in Fig. 11.13. The possibility exists to attach the probe not only to a specific sequence, but also to a desired position in the structure in non-perturbing manner. Moreover, it is possible to locate the probe both in major and minor grooves in DNA double helices. Examples of realization of these possibilities regarding pyrene groups can be found in references (Preuß et al. 1997), (Ono et al. 2012), (Okamoto et al. 2004), and (Hrdlicka et al. 2005). The data on the studies of 3-hydroxychromone dyes incorporated in different manner (see Fig. 5.26 of Volume 1) will be discussed below. In these applications, the ultra-high sensitivity in light intensity is not enough; the fluorescence reporting signal must be informative and perceptible to even weak intermolecular interactions at the sites of probe location. On a fluorophore level, it can be expressed as the effects of π-π stacking, screening from water, polarity and H-bonding. The informative signal should be provided by properly located single base substitute (surrogate) reporting by recording one of fluorescence parameters: intensity, lifetime, optical anisotropy, wavelength shift or the appearance of new bands in emission (see Chap. 3 of Volume 1). The possibility of applying really smart base surrogates demonstrating unique response as the wavelength-shifting and new band generation of their fluorescence emission will be demonstrated below. We will show that such important developments are a reality (Michel et al. 2020) and will analyze different already realized and prospective pathways leading to this ambitious goal.

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Fig. 11.13 Illustration of multiple ways for attaching exogenous fluorophores to double helical DNA structures using pyrene (depicted in blue) as example. a Fluorophore attached directly to the terminal phosphate via a long flexible linker; b Fluorophore replaces the base, being a base mimic bonded to the anomeric carbon of deoxyribose; c Fluorophore attached to a natural nucleoside either at the base or sugar parts via a short linker

11.3.1 Design of Local Site Responsive Sensors The fluorescent chemical entities, which are incorporated into the DNA, are usually called fluorescent nucleoside analogs (FNAs). The fluorophore can replace one of the natural nucleic acid bases, acting as a nucleobase mimic. Depending on their chemical composition, fluorescent nucleobase mimics can be isomorphic, expanded or extended base analogs that maintain or not the Watson–Crick base-pairing. They can be aromatic fluorophores that lack the H-bonding groups, the so-called chromophoric FNAs (Sinkeldam et al. 2010). Due to their well-defined position, FNAs allow the site-selective monitoring of conformational or constitutional changes of nucleic acids and are therefore the potent signal transducers. FNAs are a group of chemically diverse compounds that often (but not always) share partial deoxyribose moieties with natural nucleosides, and several hundred such compounds are available to researchers. The following comprehensive reviews are especially recommended to the reader (Saito and Hudson 2018; Sinkeldam et al. 2010; Wilhelmsson 2010; Xu et al. 2017) for making their proper choice. In order to minimize disturbance in the labeled nucleic acid sequences, the FNA fluorophoric groups should resemble the natural nucleobases in size and hydrogenbonding patterns. Isomorphic and expanded FNAs are the two groups that best

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meet the criteria of non-perturbing fluorescent labels for DNA and RNA. Significant progress has been made over the past decades in understanding the fundamental photophysics that governs the spectroscopic and environmentally sensitive properties of these FNAs (Dziuba et al. 2021). Ideally, the fluorophore moiety should of similar size as the natural nucleobases, but this is rarely possible. They need to be larger for providing the necessary signal in the necessary spectral range because the size increase in π-electronic system is usually connected with the needed increase in molar absorbance and shifts of the spectra to longer wavelengths. Both shape complementarity and formation of Hbonds with complementary base are needed for the formation of stable double helices. Therefore, the skill of researchers should be applied to prepare the derivatives of bases demonstrating their noncovalent interactions as close as the natural bases with the retention or even enhancement of their spectroscopic properties. In order to induce a large signal change, such reporter should be located close enough to the sites of interest, particularly the sites of insertion, deletion or substitution in mutating genes. Also, it may be of particular interest to probe the near environment of the bases, which can be achieved by probe location in the major and minor grooves formed in double helices (see Fig. 11.2). These problems have to be resolved by the means of organic synthesis. Labeling, which in this case is the covalent incorporation of dye into the nucleic acid chain, can be accomplished according to different strategies that are welldescribed in bioorganic chemistry. FNAs can be introduced using either solid-phase phosphoramidite chemical synthesis (Hogrefe et al. 2013) or by enzymatic (polymerase) incorporation (Hocek, 2019). The polymerase incorporation method with the base-modified (deoxy)nucleoside triphosphates (d)NTPs complements the phosphoramidite approach, overcoming many of its limitations. Several DNA and RNA polymerases tolerate diverse variations in the chemical structure of substrates, which opens a possibility for the enzymatic constructions of base-modified nucleic acids (Hocek and Fojta 2008; Hollenstein, 2012), including those bearing FNAs. The polymerase method is suitable for the preparation of medium-size (20–100 bp) and long (100–1000 bp) modified DNA. The disadvantages of the method are the high cost of nucleic acid polymerases, as well as difficulties with programmable site-selective labeling. All these approaches can be applicable to double and even multiple labeling.

11.3.2 Operation with Parameters of Fluorescence Emission The choice of detection method should be dictated by the aim of considered research project or by the new technology to be developed. Whereas the double-helical conformation can be detected in a simple way by increasing the intensity of intercalating dyes, the studies on a single-base level require the involvement of more sophisticated tools. For the detection of atomic-scale variations of structure (hydration), the sensing based on lifetime, anisotropy, and ratiometric sensing with internal or external standard are needed. They give an analytical signal, which does not depend

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on probe concentration or instrumental setting (Demchenko 2005, 2014; Gryczynski et al. 2003). Fluorescence, after initial electronic excitation, develops in time in subnanosecond-nanosecond time range. If molecules emit light independently, the decay function of their fluorescence emission is essentially concentrationindependent and, therefore, no reference is needed. Fluorescence anisotropy is internally calibrated but depends strongly on fluorescence lifetime. Environment-sensitive probes responding in spectral shifts and generation of new bands offer a lot of promise. Here we critically discuss in short the most popular fluorophores, addressing the reader to find more detailed information in ref. (Michel et al. 2020). In fluorescence sensing, the output signal is always the number of emitted light quanta that can be integrated and frequently presented as fluorescence intensity (Sect. 3.2 of Volume 1). Many known FNAs have been shown to change their emission intensity upon different molecular events, such as stacking with the flanking nucleobases, forming hydrogen bonds with a complementary nucleobase, etc. Since this parameter depends on fluorophore concentration and many instrumental factors (such as light source brightness, detector sensitivity and illuminated volume), its quantitative determination needs a reference. Within a single experiment series, all parameters related to the instrumental settings are typically kept constant and thus, all changes in fluorescence emission can be attributed to the changes in quantum yield (.). The switching between emissive and non-emissive decay from the excited to the ground states that can be very efficient with the involvement of photoinduced electron transfer (PET). The 2-aminopurine (2AP) probe is an environment-sensitive isomorphic FNA that absorbs and fluoresces in the ultraviolet region of the spectrum (Dziuba et al. 2021; Jones and Neely 2015). It is a constitutional isomer of adenine demonstrating high degree of structural similarity with the natural purine bases. 2AP can form stable hydrogen-bonded base pairs with T and C. Incorporation of 2AP introduces minimal, although notable, perturbations to the dynamic and thermodynamic stability of double-stranded DNA (Dallmann et al. 2010). In a free form, 2AP is brightly fluorescent in water (. = 0.68). Meanwhile, the fluorescence of 2AP is severely quenched upon incorporation into dsDNA (Ward et al. 1969). The π–π stacking interactions are crucial for quenching and 2AP is quenched by all four natural bases with G being the most efficient quencher (Somsen et al. 2005). It was established that the excited 2AP oxidizes G and A, but reduces T. The base C can be either reduced or oxidized (Narayanan et al. 2010b). 8-Vinyl-deoxyadenosine (8VdA) and 8-vinyl-deoxyguanosine appear to be useful fluorescent mimics of the natural purine bases (Gaied et al. 2005; Nadler et al. 2011), demonstrating similar quantum yields but higher molar absorbance compared to 2AP. A sequence-dependent quenching was observed for 8VdA within DNA. Stacking with neighboring nucleobases causes quenching of fluorescent emission that depends on the nature and relative orientation of the flanking bases (Kenfack et al. 2006). FNAs bearing aromatic hydrocarbons as a nucleobase mimic can also be used as a reporting group with its fluorescence quenched by natural nucleobases. Among aromatic hydrocarbons, pyrene shows the most sufficient degree of quenching upon

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interaction with flanking pyrimidines, with T being the strongest quencher (Wilson et al. 2008). Another important group of FNAs quenched upon incorporation in DNA are pteridine nucleoside analogs (Hawkins 2001; Hawkins et al. 2008). Several members of this family are 6MI, 3MI, 6MAP and DMAP. These compounds are structurally similar to purines. They can be incorporated into DNA using the phosphoramidite chemistry to provide constructs of similar stability comparing to unmodified DNA. Compared to 2AP, pteridines exhibit a red-shifted absorption band thus allowing selective excitation without interfering with absorption of natural nucleobases and amino acids. Like 2AP, free pteridines fluoresce brightly in water, and the fluorescence is significantly quenched when they are incorporated into DNA (Moreno et al. 2012; Wojtuszewski Poulin et al. 2009). Experimental results prove that pteridines can be quenched by PET with the natural nucleobases (Narayanan et al. 2010a; Wojtuszewski Poulin et al. 2009). Pyrrolo-dC is a class of emissive artificial bases, preserving the H-bonding pattern of natural cytosine and the ability to selectively base-pair with G.. Their fluorescence quantum yield is significantly decreased in perfectly matched duplexes (Berry et al. 2004). As a consequence, pyrrolo-dC family is attractive as base-discriminating fluorescent (BDF) nucleosides, i.e. as probes able to distinguish single-nucleotide polymorphisms (SNPs) (Dodd and Hudson 2009). The fluorescence lifetime may become the source of valuable information from fluorescent nucleic acid analogs on a condition that very short lifetimes are detected (Sect. 3.4 of Volume 1). For instance, 2AP exhibits monoexponential decay in water, whereas on incorporation into DNA the decay becoming short is resolved as a sum of four exponential components with typical lifetimes ranging from 30–50 ps to 10 ns (Jones and Neely 2015). These lifetime components have been previously attributed to conformations with the fluorophore having different degrees of stacking with the adjusted nucleobases. The major shortest lifetime component was typically referred to a stacked sufficiently quenched 2AP conformation, whereas the other species represent small populations of unstacked and partially stacked 2AP conformations. The longest component was attributed to an unstacked conformation, in which 2AP is protruded from the contact with nucleobases and was highly emissive. Later studies (Remington et al. 2016) showed that the fully stacked conformation contributes negligibly to the emission signals detected more than 50 ps after excitation, and all the four lifetime components originate from the unstacked conformations. Moreover, the same study showed that the decay of 2AP can be modeled using a continuous lifetime distribution originated in a multitude (> 4) of different conformations. Optical anisotropy is sensitive to rotational motion of the dye inside the molecular construct to which it is incorporated (Sect. 3.3 of Volume 1). It is essential that anisotropy sensing allows direct response to intermolecular interaction that is independent on absolute fluorescence intensity and, therefore, on fluorophore concentration. This method allows determining single-base mismatches. That was shown with molecular rotor, trans-stilbene annulated uracil derivative (Karimi et al. 2020). Serving as a “smart” thymidine analog, it exhibited 28-fold brighter fluorescence intensity when base paired with A as compared to T or C and is highly sensitive to

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local mechanical motions in duplex DNA, reporting in anisotropy decay. The brightly emissive cytidine analogs tC and tCo were developed (Lin et al. 1995; Teppang et al. 2019; Wilhelmsson et al. 2001). They incorporate nucleobase surrogates 1,3-diaza-2-oxophenothiazine and 1,3-diaza-2-oxophenoxazine, respectively. The tricyclic nucleobase tC exhibits interesting properties for anisotropy-based sensing. The tC base pair with G, shows well-defined position and geometry within the DNA helix and exhibits bright fluorescence with negligible sensitivity to the surrounding bases (Sandin et al. 2005). These fluorescent base analogs have a base-flipping rate that will not interfere with the signal measured in fluorescence anisotropy. Pteridine nucleobases can also be employed in anisotropy sensing (Hawkins et al. 2008). Polarity-sensitive dyes respond to changes of their local environment by spectral shifts and generation of new bands. Commonly, they red-shift their emission spectra when placed into a medium of increased polarity. The general principles governing the performance of these dyes are well understood (see Sect. 5.3 of Volume 1). They should contain the functional groups attached to aromatic rings that could become in the excited state strong electron donors and electron acceptors. Being appended to opposite sides of π-electronic structure they generate strong dipole moment in a push–pull manner that, interacting with local molecular environment, results in modulation of energy of electronic transition. This generates shift to longer wavelengths (red shift) that is the stronger the higher is the polarity of environment. Such shift can be also modulated by intermolecular H-bond of one of these groups in polar environment. Several approaches for the construction of polarity-sensitive nucleosides were realized, see ref. (Michel et al. 2020). Within the first approach, the dye was grafted to deoxyribose, typically via C-glycoside bond, to be further intercalated between nucleobases within DNA. Second approach employs fluorescent dye to be conjugated to the natural nucleobases, typically to the position 5 of pyrimidines. Third approach features minor decoration of the natural nucleoside with additional chemical functionalities. The later seems to have a distinct advantage, since in this way minimal perturbations to the structure of DNA are introduced. An example is 5furan-uracil that was designed for the detection of DNA abasic sites (Greco and Tor 2005). Solvent-dependent emission sensitivity was also observed for structurally related compound containing thiophene, namely for 6-aza uridines (Sinkeldam et al. 2012), nucleoside analogs based on thieno-fused pyrrolo cytosines (Noé et al. 2012) and thieno[3,4-d]-pyrimidine scaffold (Shin et al. 2011). However, being incorporated into polynucleotides, these compounds show only minor shifts in their emission bands, being more useful for the construction of intensity-based probes. A new fluorescent 2' -deoxycytidine nucleoside linked to a strongly wavelength-switching fluorene derivative through a non-conjugated tether was synthesized (Dziuba et al. 2016). The nucleoside was converted to its triphosphate variant, which was found to be a good substrate for DNA polymerases suitable for the enzymatic synthesis of oligonucleotide or DNA probes and for probing of DNA interactions. The sensitivity of fluorescence reporters to the change of intermolecular interactions can be greatly increased if the sensing signal is the change between two spectrally resolved forms. This is because the wavelength-ratiometric signal will

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be recorded as a ratio of intensities of the band maxima providing comparison of fluorescence intensities on a higher level and achieving higher wavelength precision (Demchenko 2010, 2014). In order to achieve that, the researcher has to select the fluorophore that can exist in two ground-state or two excited-state forms (see Sect. 8.3). Switching between the ground-state forms allows obtaining the effects in fluorescence excitation spectra. In order to get interplay of intensities in fluorescence emission spectra, the dye has to be present in two excited-state forms with comparable fluorescence intensities (Demchenko 2023a, b). One of the excited-state reactions that can be exploited is the intramolecular charge transfer (ICT) that has to be stabilized by isomerization (twisting), see Sect. 4.1 of Volume 1. Such twisted intramolecular charge transfer (TICT) can modulate drastically the emission spectra depending on the environment, since only in the conditions of high polarity the charge-transfer form can be stabilized. Since the two isomers that appear in the excited state emit light quanta of different energies, they show two well-resolved emission bands. The very large Stokes shift of the long wavelength band due to the emission of the generated excited state isomer is characteristic for this type of dual probe (Fig. 11.14). The probe presented in Fig. 11.14 is a pyrene derivative coupled to a dimethylamino donor and an amide acceptor (Okamoto et al. 2005b). It was tethered to deoxuridine for incorporation in DNA and hybridization with DNA and RNA. While the nucleoside conjugate showed a single band TICT emission at 540 nm, the single-stranded and double-stranded oligonucleotides showed dual emission with the appearance of a new LE band centered at 440 nm. The equilibrium between the LE and TICT states, and thus the intensity ratio of these two bands, was due to restricted twisting motion of the dsDNA environment. In this case, the steric hindrance and a narrow free space in the duplex acted as a barrier to the internal rotation of the fluorophore. More recent publications underlined the effort to find new nucleoside b LE

TICT

Fluorescence intensity

a

LE

TICT

Twisting

400

500 600 Wavelength (nm)

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Fig. 11.14 Fluorescent nucleoside analogue containing fluorophore undergoing twisted intramolecular charge transfer (TICT) that generates a new band in emission. a Schematic representation of geometrical arrangements of normal locally excited (LE) and twisted intramolecular charge transfer (TICT) excited states (Rettig 1986) and the structure of nucleoside analog displaying TICT (Okamoto et al. 2005b). b The change in fluorescence spectrum of this analog incorporated into ssDNA (red) and dsDNA (blue). Excitation was at 380 nm

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analogues with TICT properties and to extend this new approach for DNA labeling and sensing (Schweigert et al. 2018; Suzuki et al. 2014). The other reaction generating two bands in emission spectra is the excited state intramolecular proton transfer (ESIPT), see Sect. 4.1 of Volume 1, that proceeds between proton donor and acceptor groups closely located in the dye structure and commonly connected with intramolecular H-bond (Demchenko et al. 2013). This reaction is subjective to perturbation by the change of polarity in the environment and to formation of additional, intermolecular, H-bonds (Klymchenko and Demchenko 2003; Shynkar et al. 2004). ESIPT reaction is observed only in those aromatic compounds having proton donor and a proton acceptor groups in close proximity connected with intramolecular hydrogen bonding. Common proton donors and acceptors are the hydroxyl or amino groups and the carbonyl oxygen or imino nitrogen, respectively. After photon absorption, the electronic charges are redistributed, making the proton donor more acidic and the acceptor more basic favoring the intramolecular proton transfer in the excited state (Demchenko et al. 2013; Zhao et al. 2012). 3-Hydroxychromones (3HCs) are typical representatives of ESIPT dyes; they are among the most useful probes for practical applications. 3HCs are heterocyclic compounds bearing hydroxyl and carbonyl groups involved in a 5-member H-bonding ring system (Fig. 11.15). Due to ESIPT, the 3HC fluorophores exhibit two excited states, which generates two well-resolved emission bands (Demchenko 2006; Demchenko et al. 2013). The short-wavelength band is due to the emission from the normal (N*) excited form, whereas the long-wavelength band is produced by the tautomer form (T*). In the excited normal state (N*), the oxygen carbonyl is more basic making this group sensitive to the donating ability of hydrogen bond of protic solvents and hydration (Shynkar et al. 2004). As a consequence, an increase of the solvent acidity or of the hydration rate favors H-bonding to the carbonyl oxygen resulting in decrease of the relative intensity of the T* form due to slower ESIPT reaction. Another consequence of the electronic charge distribution for the N* state is related to its strongly enhanced dipole moment. The magnitude of the dipole moment of N* can be finely tuned by appropriate chemical modifications, while the dipole moment of the T* state is much less affected (Ercelen et al. 2002). For instance, the electron-rich aryl groups at position 2 (e.g. 4-dimethylaminophenyl) increase dramatically the magnitude of the dipole moment of the N* state due to internal charge transfer (ICT) rendering the 3HCs sensitive to polarity change in aprotic solvents. In polar solvents, strong and favorable dipole–dipole interactions between solvent molecules and the dye stabilize the N* state much more than the T* state resulting in increased emission of the N*. The N*/T* ratio is therefore a robust analytical signal reporting directly the properties of the microenvironment of 3HC (Klymchenko and Demchenko 2003). These properties have been used extensively for the construction of environment-sensitive fluorescent dyes to probe proteins and lipid membranes (Demchenko 2006). Recently ratiometric fluorescent nucleoside analogs based on 3HC were introduced and investigated (Barthes et al. 2014, 2015,

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Fig. 11.15 Fluorescent nucleoside analogs based on 3-hydroxychromone (3HC) dyes demonstrating λ-ratiometric response. a Comparison of sizes of furyl-3HC and natural base pair dA-dT. b Base-substituting and base-extending 3HC derivatives described by (Dziuba et al. 2012) and (Barthes et al. 2015). c Dual-emissive dA analogs that allow the two-band variability of response depending on polarity of their environment (Le et al. 2019). This variability is determined by the differences in the dipole moments of excited and ground states—given in Debye (D) units. Donor and acceptor groups are depicted in blue and red, respectively. Gradient colored arrows represent the D–π–A push–pull system

2016; Dziuba et al. 2012; Le et al. 2019). Extended analysis of this approach was presented recently (Michel et al. 2020). 3-Hydroxychromones bearing a thienyl or furyl ring at position 2 are known to be extremely sensitive to hydration. The emission switches from the dominating N* band in water to T* emission in aprotic media. 2-Thieno-3-hydroxychromone was therefore selected for DNA labeling. It was formulated as a deoxyribose derivative and incorporated into DNA via standard phosphoramidite method. The dye showed a dominant tautomer emission in the single-stranded form. A further rise of T* band was typically observed upon hybridization to a complementary strand. Most importantly, the emission of single-stranded DNA was highly sensitive to the formation of the complex with HIV-1 nucleocapsid protein. An emphatic increase of the normal emission was observed, indicating that the 3HC nucleoside can be a good sensor for DNA–protein interactions (Dziuba et al. 2012). In a comparative study with commercially available fluorescent nucleosides, the fluorescent label was exploited to investigate the mechanism of the DNA repair enzyme, endonuclease WIII, in interactions with damaged DNA. The results of this study showed that the 3HC label exhibits higher sensitivity and is more informative about the conformational changes of DNA

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binding and processing. Using 3HC-based molecular probes, the kinetic mechanism of endonuclease VIII action was specified (Kuznetsova et al. 2014). The possibility to construct extended FNAs based on 3HC for internal labeling of DNA has been also explored (Dziuba et al. 2014). New conjugated nucleobase-3HC fluorophore demonstrated high sensitivity to solvent polarity and hydration (Barthes et al. 2015; Le et al. 2019).

11.3.3 Probing the Single-Nucleotide Polymorphism Oligonucleotides incorporating stacking-sensitive fluorescent nucleosides can be used as single-label probes for direct probing of single-nucleotide polymorphism (SNP). It is known that within the entire human genome, which comprises approximately 3.2 billion base pairs, individual human beings differ in approximately 0.1% of their nucleotide sequence. The most common of these three million genetic differences are the variations of single-base pairs within an otherwise unchanged genetic context. Some of these genetic variations are directly linked to diseases and also to different effects of drugs on different patients (Price et al. 2015). Therefore, the development of personalized medicine depends strongly on our ability to detect single base substitutions located either within or outside a gene. In the case of SNPs, the thermodynamic differences between the fully matched and the mishybridized duplexes are often too small to be detectable, and only locally positioned fluorescent probes can detect them. To identify SNPs, the emissive oligonucleotides, complementary to the domain of interest, are hybridized to their target DNA. They are typically placed across from the base of interest, yielding, under ideal conditions, markedly different signals depending upon their pairing partner (Fig. 11.16). Pyrrolo-cytosines (pC) are representatives for this approach (Fig. 11.16a). The fluorophore is positioned in the probe strand opposite to the SNP site. Hybridization to a perfectly matched target causes the fluorophore to stack between the surrounding nucleobases. A light-down response (ON–OFF) is observed in this case. However, in the case of mismatch, a perfectly stacked conformation cannot be achieved and a light-up response (ON) is observed (Hudson and Ghorbani-Choghamarani 2007). Numerous SNP-detecting probes showing such response upon mismatch detection are known (Dodd and Hudson 2009; Okamoto et al. 2005a), and their number increases. Among light-up base discriminating probes are the anthracene derivatives (Duprey et al. 2018). Different strategy to distinguish a fully complementary strand from a mismatched target is illustrated in Fig. 11.16b. For this purpose, a deoxyuridine derivative connected to fluorene at position 5 (UFL ) was used as fluorescent probe and incorporated into the loop region of a hairpin (Hwang et al. 2004; Ryu et al. 2007). The beacon signal exhibited a twofold increase and a sixfold decrease upon hybridization with fully complementary and single-base mismatched sequences, respectively.

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Fig. 11.16 Single-dye hybridization probes for the detection of SNPs (Michel et al. 2020). a Pyrrolocytosine nucleobase allowing mismatch discrimination by a fluorescent turn-on response; and b quencher-free molecular beacon approach revealing match cases by a light-up signal

The techniques are progressing aiming at seeing genetic information directly in single cells (Zhang et al. 2020). With recent developments, the single-nucleotide variation (SNV) imaging methods visualizing the subtlest sequence alterations have opened a new door for studying the heterogeneous and stochastic genetic information in individual cells.

11.4 RNA Detection, Analysis and Imaging The central dogma ‘DNA encodes RNA, RNA encodes protein’ states that RNA is an essential molecule in the flow of genetic information in cells. Beyond serving as a bridge between DNA and protein, RNA is critical in a cell to regulate this information flow, both qualitatively and quantitatively, thus constituting an essential control point for cellular processes (Dumas et al. 2020). In contrast to DNA, RNAs demonstrate great versatility of structures and performing functions. The classical examples are the transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), the central components of the cellular protein biosynthesis machinery. Micro-RNAs (miRNAs) and long non-coding RNAs (lncRNAs) are involved into post-transcriptional regulation of gene expression, shaping and

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maintaining of the chromatin landscape, and other key biological processes. In the nucleus, RNA is concentrated mostly in the nucleolus (Hernandez-Verdun et al. 2010; Olson et al. 2002; Raška et al. 2006), which is the key site for ribosomal RNA (rRNA) transcription, processing and assembly. In cytoplasm, it is also found in the form of small (tRNAs and microRNAs) and large messenger RNAs (mRNAs) and also ribosomal RNAs. These molecules penetrate from the nucleus through nuclear pore complexes for gene expression and its regulation/silencing. The RNA-type viruses bring us different pathologies (Hiscox 2007). Like DNA, the RNA molecule contains A, G, and C nucleobases, but thymine is replaced by uracil (U). Such difference from DNA results in altered structural and functional properties. RNA exhibits a superior degree of structural complexity and plasticity, and because of that it can adopt diverse three-dimensional structural forms responsible for intermolecular interactions and catalytic activity. Due to their flexible secondary and tertiary structural conformations, designing the RNA-specific fluorescent probes is a challenging task.

11.4.1 RNA Detection in Cells In recent times, dedicated efforts have been directed at visualizing cellular RNAs and understanding their role in various physiological processes using several techniques established for RNA detection in living cells. They include fluorescence-labeled microinjection of tRNA beacons for mRNA imaging in the cell cytoplasm (Mhlanga et al. 2005) and fluorescence in situ hybridization (FISH) technique (Huber et al. 2018; Liehr 2017). The latter allows single-cell analysis of RNA expression (and localization), whereby in fixed and permeabilized cells the labeled linear oligonucleotide (ODN) probes are used to probe the intracellular RNA (Guo et al. 2012). For reducing background and achieving specificity, the unbound probes are removed by washing. There exists a unique possibility for using designed fluorescent dyes that enter the cells and their organelles without membrane disruption occurring on cell fixing. Moreover, it is possible to study the nucleolar dynamics in prefixed cells and, therefore, it is extremely beneficial to have RNA-selective fluorescent probes to monitor the RNA content and distribution in comparison with the DNA organization inside the nucleus (Dupuis-Sandoval et al. 2015). However, RNA-staining small molecular probes are rare due to difficulty in designing probes with differential selectivity for RNA over DNA and poor nuclear membrane permeability. Visualization of gene expression and regulation can be implemented by imaging various RNAs, mostly messenger RNAs (mRNAs) and micro-RNAs (miRNAs). Imaging of mRNAs and miRNAs not only allows us to learn the formation and transcription of mRNAs and the biogenesis of miRNAs involved in various life processes, but also helps in detecting cancer (Xia et al. 2017). Similarly to DNA detection, molecular beacons can be used for RNA (Bao et al. 2009).

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Messenger RNAs (mRNAs) convey genetic information from the DNA genome to proteins and thus lie at the heart of gene expression and regulation of all cellular activities. Live cell single molecule tracking tools are needed for the investigation of mRNA trafficking, translation and degradation within the complex environment of the cell and in real time (Schmidt et al. 2020). Fluorescent protein technology is actively used for its detection and cell trafficking (Wu and Jaffrey 2020). The advent of CRISPR-Cas9 in combination with fluorescent proteins made it possible to track individual RNAs in living cells without perturbing their biological functions (Gleditzsch et al. 2019). Micro-RNA (miRNA) are short (19–25 base long), single-stranded, noncoding RNAs that regulate an array of cellular functions through the degradation and translational repression of mRNA targets. Tissue levels of specific miRNAs correlate with pathological development of diseases, therefore rapid and efficient methods of assessing miRNA expression are needed for diagnosing diseases and identifying novel therapeutic targets. Molecular beacon hybridization assays have been efficiently used for their quantitative analysis in vitro (Baker et al. 2013; D’Agata and Spoto 2019). They can be detected in cell cultures by graphene oxide specific fluorescence quenching (Nitu et al. 2021). Different nanomaterials were suggested for constructing the RNA imaging probes (Xia et al. 2017).

11.4.2 RNA G-quadruplexes G-quadruplexes (G4s) are the four-stranded nucleic acid structures that arise from the stacking of G-quartets, cyclic arrangements of four guanines engaged in Hoogsteen base-pairing (Banco and Ferré-D’Amaré 2021). These structural elements formed in RNA of the same G nucleobases are similar to the DNA G-quadruplexes (see Sect. 11.1.3). The placement of four guanine carbonyl oxygens near the four-fold axis of the G-quartet generates an electronegative central pore, which is stabilized by coordination of monovalent cations. This cation is most commonly the potassium ion. Meantime, instead of expected relatively simple RNA G4 structures, recent crystallographic and solution NMR structure determinations of a number of in vitro selected RNA aptamers have revealed the RNA G4s of unprecedented complexity (Banco and Ferré-D’Amaré 2021). Their transcriptome-wide presence (Yang et al. 2018) suggests their possible roles in transcription regulation and chromatin organization (Varshney et al. 2020; Yang et al. 2018). Their involvement in the mechanisms (Cammas and Millevoi 2017) and treatment (Neidle 2017) of disease have been found. Likewise with DNA, the RNA quadruplexes can be visualized by synthetic Gquartet probes similar to that shown in Fig. 11.7 (Laguerre et al. 2016). These structures form a template-assembled synthetic G-quartet (N-TASQ) themselves, which allows efficient target recognition. The dyes targeting the G-quartet and phosphate backbone (Yu et al. 2020) are among the best candidates with the strongest G-quadruplex binding affinity and specificity.

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Increased flexibility of RNA compared to DNA formed structures is realized in full in the dynamics of their G-quadruplexes (Banco and Ferré-D’Amaré 2021). The latest breakthroughs support the notion that the G4 transient structures fluctuate dynamically in cellulo and that their binding proteins are determinants of their transient conformation and effectors of their biological functions (Dumas et al. 2020). These peculiarities determine the strategy for their visualization (Pandith et al. 2019). Among different tools to study G-quadruplexes, the application of J-aggregate forming-dissociating fluorescent dye CyT (Xu et al. 2015) has attracted my attention. Selective recognition of RNA G4s results from the disassembly of J-aggregates and binding of the monomer to RNA GQs, which result in restricting the molecular rotation and prevention of non-radiating transition. CyT exhibited negligible fluorescence in the unbound state. Fluorescence enhancement (λem = 595 nm at λex = 532 nm) of over 1000-fold was observed in the presence of an RNA G4 compared to a mere 25-fold increase with non-G4 RNAs.

11.5 Sensing and Thinking. Increase of Sensitivity: Amplify the Target or the Detection System? It is a dream of researchers to provide genetic analysis on the level of single DNA/RNA molecules without pull labeling of tested genes, without immobilization and washing, without gene amplification (temperature cycling, PCR)—just in a “mix and read” manner, as described in Sect. 2.4 of Volume 1. Such techniques must be based on direct molecular recognition between tested and probing nucleic acid sequences providing easily recordable and comprehensive output signal. It must be applicable in different heterogeneous media, including living cells and tissues. In order to achieve this goal, the scientists follow two strategies that are schematically presented in Fig. 11.17. One is the amplification of target copies, which is the direct increase of the copy number of target molecules. The other is the amplification of output signal. It has to use highly sensitive reporter molecules or probes to detect the target without increasing the amount of the target molecules, just by increasing the sensitivity of detection system. The existing reality of very weak reporting signal that is proportional to a number of target copies stimulated the development of methods that provide increase of this number. The result was successful, but, being based on enzyme-assisted synthesis of a large number of target replicas, is not so simple and cheap. Different variants of polymerase chain reaction (PCR) are commonly used in practice because of their high efficiency, as they allow synthesizing up to 109 copies (amplicons) of the analyzed sequence (Ikbal et al. 2015; Watson 2012). However, there are a number of drawbacks, e.g., possible nonspecific hybridization resulting in the accumulation of undesired products. The melting of the DNA duplex in the course of reaction is achieved by reversible sample heating (temperature cycling), which cannot be applied in analysis of living cells and tissues. The development of various platforms

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enhancement sensitivity enhancem ement of sens e it i ivity in dna/rna dna/ a/rna assays Amplification of target copies

Amplification of reporter signal

High efficiency, as it allows synthesizing up to 109 copies

Efficiency depends on the sensitivity of reporter system

Amplification factor is determined by the number of reaction cycles

Amplification factor is determined by brightness of material

The procedure is labor demanding and time consuming

The procedure is immediate, simple and robust

Improvements are limited. They lead to higher complexity and cost

Improvements are unlimited due to unlimited number of fluorescent materials

Fig. 11.17 Comparison of two strategies for achieving dramatic enhancement of sensitivity in DNA assays

for the isothermal DNA/RNA detection, still at elevated temperature and with additional enzyme treatment (Bodulev and Sakharov 2020; Yan et al. 2014), is seen in recent years. However, it does not satisfy researchers and practical users. Fluorescence detection techniques are also booming (Fozooni et al. 2017). The brighter is the response signal, the smaller amount of sample is needed for the analyses, and the success in single-molecular spectroscopy adds enthusiasm to the researchers. In organic dyes, there is a limit of brightness that is determined by the light absorption cross-section (that is connected with geometrical dimensions) and the fluorescence quantum yield that cannot be higher than 100%. Similar limitations exist in the world of fluorescent nanoparticles. The obvious solution here is the increase in the number of fluorophores in every detection unit and the induction of their collective effects, such light-harvesting, superenhancement and superquenching (see Chaps. 8 and 10 of Volume 1). It is hard to expect that on this pathway the sensitivity will increase more than several hundred times. But it may be foreseen that this increase will reach a level that does not require any target amplification, purification or the removal of unhybridized probes. Fluorescent conjugated polymers are very advantageous in performing these tasks, since their collective system response is in itself a form of signal amplification, i.e., each repeating unit is a fluorescent entity but with the entire polymer chain acting as one signaling probe (Knoops et al. 2018; Wu et al. 2017). Possessing positive charge and with the emission properties changing upon formation of the double helix, cationic fluorescent conjugated polymers can be used as direct indicators of DNA hybridization. New efforts are needed to make them sensitive to the changes on the level of single bases on an enhanced level of response (Yucel et al. 2021).

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Fluorescent nanoparticles and nanocomposites of different origin and with different functions (ultra-high brightness, light-harvesting, plasmonic enhancement) offer the solution from the side of reporting, but the problem is their coupling with molecular recognition event. Thus, with the dye-doped nanoparticles the record brightness can be achieved (Melnychuk et al. 2020) and the base surrogates used in recognition may be connected via excited-state process with such structures. Such technologies may be extended to live cell imaging (Fozooni et al. 2017). The methods that offer avoiding the PCR amplification and labeling of the target pool are expected to complement and, in many important areas, to substitute the standard ‘DNA chip’ approach. These methods are expected to make the DNA tests considerably simpler, cheaper, and quicker and therefore more applicable for large-scale applications. This goal can be achieved on one condition: they have to demonstrate superior sensitivity. This condition can be realized when the ultrabright nanoparticles will substitute or complement organic dyes and when enzymatic amplification mechanisms will be substituted by photophysical ones. The dyedoped nanoparticles (Zhao et al. 2003), Quantum Dots as FRET donors (Zhang et al. 2005), conjugated polymers as self-amplified systems (Dore et al. 2006; Liu and Bazan 2005) the metal-enhancement support (Aslan et al. 2006), as well as enhancing support of porous semiconductor materials (Dorfman et al. 2006) are the trends pointed forward by pioneering research. This is indeed a largely unexplored area waiting to be conquered by designing very selective, sensitive and biologically benign probes. In order to check the efficiency of reading, the readers are asked to respond to the following questions: 1. 2.

3.

4. 5.

6.

7.

What is the mechanism of single-strand association in canonical double helices and in noncanonical structures? Explain the formation of G-quadruplexes and i-motifs. What is their conformation and what forces stabilize them? How can they be detected by fluorescence probing? Why the methods of probing the single-strand and double-strand DNA differ? Present an example of different two-band ratiometric response of the same probe. What are the differences in affinity, selectivity and the mechanisms of binding to ssDNA and dsDNA between monomers and dimers of acridine dyes? Explain advantages and disadvantages in application of planar arrays with target pool labeling (DNA chips). Why it is hard to apply the same methodology to make protein arrays? Provide the comparative review of the mechanism of nucleic acid hybridization in detection of specific sequences with regard to generating the sensor response. Explain advantages and disadvantages of the whole pool labeling and the sandwich assays. How to use the double labeling for generating the two-band ratiometric response? Explain the performance of molecular beacons. Can they be used in homogeneous formats?

References

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10. 11. 12. 13. 14. 15. 16. 17.

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What are the advantages of covalent immobilization of capture sequences on solid support? Review the properties of solid surfaces used for covalent immobilization of nucleic acids. Can these supports be functional serving as fluorescence enhancers or quenchers? How nucleic acids associate with cationic conjugated polymers? What is the difference in their binding between single chains and double helices? How and why their fluorescence response may change? What is the structural and energetic basis for DNA hybridization with peptide nucleic acids? How to use the ability of different materials to adsorb on their surface the DNA molecules as single chains but not in their double helical form? What are the possibilities for incorporation of fluorescent dyes into nucleic acid structures by covalent labeling? What type of fluorescence response is the most popular and what is the most efficient for reporting in the DNA SNP detection field? What is the single nucleotide polymorphism and what are the surrogates of DNA bases to recognize it? What forms of RNA exist in cells and how they can be detected? Can RNA form G-quadruplexes? What are their specific features? Provide comparative characteristics of two strategies that can be followed in order to increase dramatically the sensitivity in nucleic acid specific recognition, the amplification of target copies and the amplification of reporter signal.

References Abou Assi H, Garavís M, González C, Damha MJ (2018) i-Motif DNA: structural features and significance to cell biology. Nucleic Acids Res 46:8038–8056 Al Attar HA, Norden J, O’Brien S, Monkman AP (2008) Improved single nucleotide polymorphisms detection using conjugated polymer/surfactant system and peptide nucleic acid. Biosens Bioelectron 23:1466–1472 Asamitsu S, Bando T, Sugiyama H (2019) Ligand Design to Acquire Specificity to Intended GQuadruplex Structures. Chemistry–A Eur J 25:417–430 Aslan K, Huang J, Wilson GM, Geddes CD (2006) Metal-enhanced fluorescence-based RNA sensing. J Am Chem Soc 128:4206–4207 Baker ES, Hong JW, Gaylord BS, Bazan GC, Bowers MT (2006) PNA/dsDNA complexes: site specific binding and dsDNA biosensor applications. J Am Chem Soc 128:8484–8492 Baker MB, Bao G, Searles CD (2013) The use of molecular beacons to detect and quantify microRNA. In: Nucleic acid detection. Springer, pp 279–287 Balasubramanian S, Hurley LH, Neidle S (2011) Targeting G-quadruplexes in gene promoters: a novel anticancer strategy? Nat Rev Drug Discovery 10:261–275 Banco MT, Ferré-D’Amaré AR (2021) The emerging structural complexity of G-quadruplex RNAs. RNA 27:390–402 Bao G, Rhee WJ, Tsourkas A (2009) Fluorescent probes for live-cell RNA detection. Annu Rev Biomed Eng 11:25–47 Barthes NP, Gavvala K, Dziuba D, Bonhomme D, Karpenko IA, Dabert-Gay AS, Debayle D, Demchenko AP, Benhida R, Michel BY (2016) Dual emissive analogue of deoxyuridine as

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Chapter 12

Fluorescence Detection of Peptides, Proteins, Glycans

Peptides, proteins and glycans are major constituents of the living cells and their detection is very important for basic research and clinical diagnostics. Their largely variable molecular weights, complex structures and high functional diversity create great problems for their specific detection. In this chapter we analyze these problems and describe their solutions. Two major trends can be outlined—detection of individual species with highest possible affinity and simultaneous measurement of multiple species using microarrays with the aid of recently introduced machine learning algorithms. In previous chapter we observed that the use of nucleic acid base complementarity as the tool for their molecular recognition allows providing their highly specific detection both on the whole sequence level of gene and on the level of individual bases. For other biological molecules, such possibility does not exist, and the problem of molecular sensing is more complicated. Specific feature of peptides is their large conformational flexibility, which often complicates their multi-point molecular recognition. For recognition of individual amino acids and of several neighbors in their structures, the macrocyclic caging compounds can mostly be used. The same flexibility allows assembling these macrocycles in linear fashion or on nanoparticle surface for the increase of their selective binding at multiple sites. To some extent, this approach is applicable to proteins. Some patches of their structures, “hot spots”, can be recognized with designed macrocycles and, more efficiently, with a variety of modified porphyrins. More proficient, however, are the antibodies and nucleic acid aptamers, which can offer very high specificity of interaction that is especially needed if the target protein is in ultralow concentration. The substitution of these valuable sensors by synthetic analogs requires generating the receptors in a combinatorial fashion and characterizing their properties by highthroughput screening to identify specific binders for given target proteins. A rational approach could involve receptors with large surface areas and the integration of various weak interactions to overcome the highly solvated character of the protein surface. © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_12

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The challenges that exist with protein detection methods can be counted (Cohen and Walt 2018). First, a sample may contain many interfering molecules that make it difficult to detect a specific protein. Thus, the high-affinity sensors must be applied. Second, many proteins may be present at low concentrations in a biological sample, and the sensors with ultra-high sensitivity may be requested. Third, the concentrations of different proteins can vary by many orders of magnitude, and the sensor arrays covering a very broad concentration range are requested (Corthals et al. 2000). Finally, protein processing, such as post-translational modifications and spliced variants that result in different isoforms, can make it difficult or complicated to detect the specific protein of interest. It is often necessary to detect more than one protein in a single sample. Many proteins work in networks to exert their function. Some proteins are overexpressed, while others are under-expressed. A limited ability to simultaneously measure multiple proteins in a sample has constrained our ability to fully study the proteome. Protein measurements on the omics scale may offer many new possibilities. These possibilities have started to be realized with the use of array-based ‘chemical nose/tongue’ platforms and with the development and application of machine learning algorithms that allow recovering from mosaic pattern of sensor responses at every spot of microarray the true distribution of target concentrations in the studied sample. Recent discoveries witness that the appearance of pathologically folded protein forms that are β-sheeted amyloid aggregates is not a rare event. It is observed for a larger amount of proteins, and their quantitative analysis is needed in many areas, including clinical pathology. Simultaneous measurement and analysis of multiple samples is the demand from clinical diagnostics. Here different peptides, proteins and glycans can serve as disease markers, but only consistent analysis of several of them may commonly lead to correct diagnosis. Detection and analysis of disease-related proteins as biomarkers will be provided in Chap. 14.

12.1 Targeting Peptides The sequence-selective molecular recognition of peptides in water was and remains to be one of the most important problems in chemistry and biology (Peczuh and Hamilton 2000; Sewald and Jakubke 2015). Their role in biological systems is great. In many organisms, oligopeptides act as neurotransmitters, neuromodulators, and hormones, and their interactions with protein receptors influence cell–cell communications, metabolism, and immune response (Sewald and Jakubke 2015). In addition to common ribosomal synthesis, the non-ribosomal mechanisms of their production exist that allows, in addition to common 20 amino acids, to incorporate a number of new components (Sieber and Marahiel 2005). This added to complexity of structural and functional analysis but also opened the ways to design new antibiotics (Pasupuleti et al. 2012). The post-synthetic modifications, such as methylation and

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phosphorylation (Ojida et al. 2002), play an important functional role. Whereas peptide chemistry is well-developed (Liskamp et al. 2011), their sensing is an arena of active efforts of enthusiastic researchers. As it was stated long ago (Hossain and Schneider 1998), reading and transmission of information in peptides relies exclusively on interactions with their different amino acid side chains, in contrast to the mechanism in nucleic acids, where the recognition via hydrogen bonding provides the corresponding codes. Recognition of amino acid sequences, in particular by synthetic receptors, is furthermore hampered by the lack of conformational order, again in contrast to that in the conformationally more stable nucleic acids. In principle, unique composition of every peptide allows unique possibilities for their specific multipoint molecular recognition (Smith 2015). The 20 classical amino acids differ dramatically in charge and hydrophobicity. Unfortunately, some of the side chains are quite similar. Leucine (L) and isoleucine (I), for example, differ by the position of a single methyl group. Glutamic acid (E) has one extra methylene than aspartic acid (D), and tyrosine (Y) has one extra hydroxyl in comparison to phenylalanine (F). When operating in aqueous solutions, hydration of these residues is very different and has to be accounted for molecular recognition in water (Schmuck and Wich 2006) because the solvent molecule itself is an excellent hydrogen-bond donor and acceptor (Awino et al. 2017). Intrinsic conformational flexibility of many peptides is an important factor. Their immobilization on receptors needs energy against the entropy factor. In view of these problems, the design of synthetic receptors for peptides with the ability of providing fluorescence response is not a simple task. The multi-point structural and energetic complementarity with the receptor has to be realized. The macrocyclic hosts may be efficiently used for recognition of amino acids (Martins et al. 2021), but they are too small for hosting peptides. The application of antibodies (such as discussed in Sect. 4.4) can be used if the recognition of 3–4 residues in sequence is sufficient for specific peptide detection. Thus, for relatively large peptides, of 10–20 amino acids, either these small recognition sites have to be used, or more complicated receptors have to be devised. The use of macrocycles for developing of methods of peptide detection has started several decades ago (Hong et al. 1991). Many different materials have been used to construct peptide receptors for smallest peptides since then (Schmuck and Wich 2006; Wright et al. 2005). They included functionalized cyclodextrins (Yamamura et al. 2004) and their dimers (Breslow et al. 1998), and also cucurbit[n]urils (Smith et al. 2015). For providing recognition at the sites of multiple amino acid residues, these receptors could include different combinations of assembled caged compounds (Martins et al. 2021). The reader may refer to Fig. 6.2 to see how the combination of different chelating compounds can be organized. Cyclodextrin and calixarene amphiphiles form two types of host–guest recognition sites that are simultaneously distributed on the surface of the co-assembly. Peptide chains bearing two different types of amino acid in peptide sequence demonstrate high affinities with either of these chelators (Xu et al. 2019). It was shown that such construction designed according to the principle of

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heteromultivalent peptide recognition demonstrates a supreme binding affinity to heterotopic amyloid β-peptide. The selected case (Fig. 12.1a) demonstrates a biomimetic approach to the construction of discrete, modular, multivalent receptors via molecular self-assembly in aqueous solution (Reczek et al. 2009). In this construction that can detect specifically located Trp residues in peptide sequence, the scaffolds presenting 1–3 viologen groups recruit a respective 1–3 copies of the synthetic host, cucurbit[8]uril. The assembled mono-, di-, and trivalent receptors bind to their cognate target peptides containing 1–3 Trp residues in predetermined mono- or multivalent binding modes with 31–280-fold enhancements in affinity and additive enthalpies due to multivalency. The extent of valency was determined directly by measuring the visible charge transfer absorbance due to the viologen-indole pair. The predictable behavior of this system and its ease of synthesis and analysis make it well suited to serve as a model for multivalent recognition of peptides by design. Other strategies that are based on templating methods use the principle of molecular imprinting (see Sect. 6.4). In this case, the difference in size and shape of amino acid can provide contribution to the peptide recognition pattern. Peptide serves as the template for the formation and stabilization of structure around it, and after its removal by washing, the empty space contains the “memory” that is able to selectively bind the target peptide with high affinity (Chen et al. 2011; Janiak and Kofinas 2007). In one of the early reports, the combinations of functional monomers were

Fig. 12.1 Typical composite receptors developed for peptide recognition (left) and their building blocks (right). a The use of polymer scaffold for assembling cucurbit[8]urils (Q8) on their methyl viologen (MV) side groups (Reczek et al. 2009). Binding of target peptide containing Trp residues occurs by Trp insertion into this structure. b Peptide-binding nanoparticle formed by molecular imprinting in double-crosslinked micelles (Awino et al. 2017). Peptide recognition is provided by a pattern of hydrophobic sites formed in these micelles

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polymerized in the presence of the imprinting peptide melittin in aqueous solution at room temperature (Hoshino et al. 2008). Recent studies showed applicability of this approach. Hydrophobic pocket can be formed complementary to the hydrophobic side chains of peptides. A layer of hydrogen bonds at the surfactant/water interface was found to enhance the molecular recognition of peptides in water, particularly hydrophilic ones rich in polar residues (Zangiabadi and Zhao 2020). Presented in Fig. 12.1b, is one of the examples using smartly designed nanoparticles. To recognize peptides by the location, number, and nature of their hydrophobic side chains, the water-soluble surface–core doubly cross-linked micelles were synthetized (Awino et al. 2017). After the template is removed by repeated solvent washing, a hydrophobic binding site complementary to the template in size and shape is left on the surface of the resulting molecularly imprinted nanoparticle. In this way, an array of “hydrophobically coded dimples” on the surface of nanoscale receptors was created. Minute differences in the side chains could be distinguished, and affinities up to 20 nM were obtained for biologically active oligopeptides in water. Further development of this trend allows including electrostatic complementarity into molecular recognition (Fa and Zhao 2019). This allowed achieving highly specific detection of amyloid peptides. From these examples, one may derive, how difficult is to develop the highly specific sensors for peptides. Therefore, quite natural was the appearance of new trend involving the sensor arrays (Rochat et al. 2010). Each element of the array does not demonstrate unique specificity to target peptide, but together they form the background for the required pattern-based peptide recognition (Rochat et al. 2010). The array format (Sect. 15.5 of Volume 1) allows the inclusion of reporting fluorophores that may remove this general problem in the design of peptide sensors.

12.2 Detection of Protein Targets Proteins are biological macromolecules that are the most essential constituents of cells. They are the building blocks of all their organelles and the catalysts of all biochemical reactions. Proteins are complex polymer structures formed by linear chains of amino acids that can be cross-linked and altered by covalent post-translational modifications including phosphorylation, acetylation, methylation, acylation, glycosylation, ubiquination, deamidation, oxidation, sulfation, and nitration. They can contain different prosthetic groups and bind ions. Thus, sensing is not only important to detect specific proteins but also to detect specific modifications on certain proteins (Cohen and Walt 2018). In different conditions and for different purposes there is a need to determine (a) the total protein content, (b) the specific proteins, such as particular enzyme or antibody, (c) all protein composition in a given system and (d) all human proteome. Pathological protein forms and disease markers are very important targets. We will start with the simplest tasks and proceed with the more complicated ones.

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12.2.1 Determination of Total Protein Content Direct detection and quantification of total protein content in the studied sample (Noble and Bailey 2009) is important for many applications, starting from visualization of results of separation techniques to control of pharmaceutical formulations (Hovgaard et al. 2012) and analysis of proteome (Wi´sniewski and Gaugaz 2015). Classical protein assays known from the textbooks on biochemistry, such as the Biuret, Lowry and Bradford methods based on the light absorbance of the bound dye or intrinsic absorbance of aromatic amino acids (Noble and Bailey 2009), have a strong tendency to be substituted by rapid, sensitive and precise methods employing the fluorescence. The problems with both absorption and fluorescence assays based on intrinsic spectroscopic properties of proteins are in broadly heterogeneous distribution of their tyrosine and tryptophan residues absorbing in the UV (280–290 nm) and tryptophan emitting in 330–350 wavelength range (Demchenko 2013). Heterogeneity of binding sites is the problem with the application of different dyes. The popular dye Coomassie binds selectively to certain amino acids and tertiary protein structures (Noble and Bailey 2009). Thus, a great diversity and even uniqueness of every protein complicates its recognition ‘as a protein’. Nevertheless, different fluorescent dyes are used for protein assays (Harvey et al. 2001; Jones et al. 2003; Suzuki and Yokoyama 2005). Modified cyclodextrins combined with attached reporting dyes were also suggested (Zhu et al. 2007). Sufficiently precise results can be achieved for a known protein based on previous calibration. In other cases some uncertainty is unavoidable. Protein assay in denaturation conditions allows equalizing to some extent the reactivity of protein groups with the dyes, which can be achieved by thermal treatment of the studied sample with the dye in the presence of detergent. Such treatment was recommended for popular protein label merocyanine NanoOrange (Jones et al. 2003). The NanoOrange assay allowed for the detection of 10 ng/mL to 10 μg/mL protein with a standard fluorimeter. It offers a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The advantage of such dyes is also in their convenience. Reacting with proteins, they increase manifold their fluorescence emission that can make the step of washing-out of unreacted dye unnecessary. The methods described above vary in their ability to detect and quantify different proteins (Cohen and Walt 2018). They are subject to interference from other molecules in the sample that may contain the same reactive groups. Careful calibration is recommended in all cases. Therefore, the particular choice of protein assay is highly dependent on the sample type. Nevertheless, these methods are efficient for direct and simple detection and quantification of total protein and are widely in use.

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12.2.2 Labeling the Surface of Native Proteins Different biotechnologies related to proteins require proper protein modifications. So do fluorescence sensing technologies (Holmes and Lantz 2001). Such modifications include binding not only fluorescent dyes, but also chemical cross-linkers, pharmacologically active compounds and different other genetically non-encodable synthetic molecules (Wong 1991). The labeling of proteins with synthetic molecules provides exciting new functions. Their labeling in vitro needed for biotechnology will be discussed here. Protein labeling for their visualization and sensing within the living cells will be analyzed in Sect. 12.4. Each globular protein contains charged and polar groups on its surface that determines its solubility and, to significant extent, the pattern of interactions with other molecules. Protein–protein contacts that can be used in protein sensing may involve large areas of interactions between these groups. Location of responsive fluorescent dyes within these contact areas or close to them allows obtaining direct sensor response (Altschuh et al. 2006). Techniques of chemical modifications of proteins are well described elsewhere, and the reader is advised to consult classical monographs (Hermanson 1995) and reviews (Brinkley 1992; Brunner 1993), in which these modifications are described in sufficient detail. Amino groups and SH-groups are the usual targets for chemical modifications of proteins, and their presence at the sites of desired modification with the absence in other sites may guarantee the necessary precision (Brinkley 1992). SH-targeting is more actively used with peptides and proteins because these groups present as Cys residues are less abundant than the amino groups (N-terminal and that of Lys residues). Solid phase synthesis of peptides and site-directed mutagenesis of large proteins are used for incorporation of SH- groups into desired sites of the sequence (Gilardi et al. 1994; Sloan and Hellinga 1998) for subsequent attachment of fluorescent dyes according to well-known chemistry. Usually the fluorescence dyes are synthesized with the inclusion of reactive group for labeling. The three-dimensional structures of protein molecules, if they are known, are actively used for determination of the optimal labeling sites. Comparative studies of sensor proteins, in which the engineered Cys locations were rationally designed based on the strongest differences of environments of these sites in unbound and bound forms derived from X-ray structures (Marvin et al. 1997) witness for usefulness of this approach. The retention of native protein structure during SH labeling is especially important for proteins that contain significant amount of S–S bonds that are necessary for supporting this structure, and, particularly, for antibodies. Thus, the skill is to select the location of introduced SH-group in such a way that could allow correct protein folding with correct formation of S–S bonds that are essential for structure and, simultaneously, to keep the introduced SH-group free and available for chemical modification. It was found (Renard et al. 2002) that a mild reducing treatment is necessary to reactivate mutant Cys residues before coupling with SHreactive dye. The reaction of coupling in these conditions allows preserving the essential disulfide bonds of the antibody variable domains. In the same study, the

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structural and energetic criteria were applied to locate the sites of introduction of reactive Cys residues by mutagenesis. The authors selected the target residues as that belonging to topological neighborhood of the antigen in the structure of complex between antibody and antigen, its absence of functional importance for binding the antigen and its solvent accessibility. The feasibility of this rule was shown in experiment. Thus the amino and sulfhydryl groups on protein surface are the major sites of labeling, and the question arises if these sites of labeling can be distinguished by spectroscopic means. The effect of electrochromic modulation of intramolecular proton transfer (Klymchenko and Demchenko 2002) that we discovered working with 3HC dyes possessing substitutions of different charge (see Fig. 5.19 of Volume 1) has found unexpected application in the dye labeling of proteins (Klymchenko et al. 2004). In this work (Klymchenko et al. 2004), a reactive derivative of 3' hydroxychromone, 6-bromomethyl-4 -diethylamino-3-hydroxyflavone, was applied to label covalently bovine lens α-crystallin. The labeling of SH and NH 2 groups are clearly distinguished by spectroscopic criteria (Fig. 12.2). We observe that the NH2 labeling creates the positive charge in the proximity to fluorophore, which results in strong internal Stark effect producing the shift in excitation spectrum by ca. 15 nm. Analysis of excitation-dependent fluorescence spectra allows separation of the emission profiles of these SH- and NH2 -labeled species. The novel label due to its two-wavelength ratiometric response and high sensitivity to the type of labeling may offer new possibilities in the studies of structure, dynamics, and interactions of proteins by probing their SH- and NH2 -labeling sites.

Fig. 12.2 The dye displaying different fluorescence spectra on protein binding depending on conjugation with SH or NH2 groups (Klymchenko et al. 2004). a Structure of 3-hydroxychromone label BMFE and its reaction with the SH and NH2 groups of proteins. b Fluorescence spectra of αcrystallin labeled with BMFE dye (pH 7.3) and the corresponding spectra of NH2 (dotted line) and SH (dashed line) labeled species

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12.2.3 The Recognition of Protein Surface by Small Molecules When compared to great progress in nucleic acid recognition, the techniques for molecular recognition of specific proteins are seen as insufficiently well developed, and the general mechanisms that could be suggested for their detection are not existent. This is because each type of protein presents its own structure and own chemistry and unique topological distribution of charged, polar and hydrophobic residues on its surface. Generally in proteins, unlike the well-defined active sites of enzymes that can readily accommodate synthetic binders, the surfaces are large and non-contiguous; they lack well-defined grooves and pockets that can serve as templates for designing a specific binding partner. The most general way that may be suggested is to look for a unique mosaic distribution of hydrophobic, H-bonding and charged groups of atoms (Crowley et al. 2008; Wodak and Janin 2002). When probing this mosaic area, we can attempt to provide saturation in weak intermolecular interactions within a particular site, a “hot spot” of the surface of target protein. The size of such spot can be of several square nanometers only, which is comparable to that for the recognition by antibodies and their fragments. In immunochemistry, these recognition sites are called epitopes, and often this term in application to proteins is used in a broader sense, as a synonym of hot spots (Yang et al. 2014). The challenge with the design of artificial protein binders is, therefore, to encode a small molecule with sufficient recognition information to bind to the specific site on protein surface (Kubota and Hamachi 2015). Polar and charged side chains take up the periphery of the binding site, and frequently there is a juxtaposition of complementary charged groups across the interface (Janin et al. 2007). Macrocyclic receptors (see Chap. 3) can be designed and prepared according to the analytical problem to be solved, thanks to the degree of sophistication reached in the fine tuning of weak interactions responsible for molecular recognition (van Dun et al. 2017). Interacting with proteins, they can find on their surface the groups of atoms that are extending from the structure and can be incorporated in the inner volume of macrocycles (Fig. 12.3). By the combination of crystallography data with binding data obtained in solution from NMR titration experiments, the dynamic peripheral superficial binding of calixarene bsclx4 with cytochrome c was explored (McGovern et al. 2012). The crystal structure shows three different binding sites, all involving a Lys side chain (Fig. 12.3a). All three Lys side chains adopt a bent conformation, thereby enabling the formation of salt bridges with the sulfonate substituents and displacement of water from the hydrophobic calixarene interior. Dependent upon the position of the Lys, additional polar contacts were made by sclx4 with neighboring polar residues or the backbone amides. Sclx4 binds selectively to methylated over regular Lys, as was shown for dimethyllysine lysozyme variant for specific calixarene recognition using crystallography and NMR spectroscopy (McGovern et al. 2015), see Fig. 12.3b. Of the six available LysMe2 residues present on the lysozyme surface, only Lys116 Me2 was buried in the calixarene cavity as a result of its steric accessibility and an advantageous local charge environment.

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Fig. 12.3 Molecular recognition of proteins by functionalized calixarenes (PDB data), see (van Dun et al. 2017). a Crystal structure of sclx4 bound at three positions to an asymmetric cytochrome c dimer with a zoom in on the calixarene binding site with Lys89 (PDB entry 3TYI). b Crystal structure of the asymmetric methylated lysine-lysozyme dimer bound to sclx4 with a zoom in on the calixarene binding site with Lys116-Me2 (PDB 4N0J)

The gained structural information casts light on the interactions that are important for the development of (small) supramolecular host molecules for protein − surface recognition. Charge complementarity is seen on interaction of negatively charged calixarene with rich in Lys residues positively charged surface of cytochrome c (Engilberge et al. 2019). When in a series of sulfonatocalix[4]arenes interacting with cytochrome c a single sulfonate is substituted with either a bromo or a phenyl substituent, this resulted in altered recognition of cytochrome c (Doolan et al. 2018). Unsymmetrical calixarene analogues, with larger cavities and functionalized peripheries promise to increase the specificity of these host molecules. The cucurbit[n]uril class of supramolecular host molecules has been shown to provide an attractive playing field for application of protein recognition (de Vink and Brunsveld 2019). The hydrophobic cavity of these host molecules typically provides for a binding affinity to specific protein surface patterns via a substantial hydrophobic component. The large cavity of cucurbit[8]urils allows single amino acid and co-guest binding (check with Fig. 12.1a). This interaction can be further modulated by polar non-covalent interactions. There were many reported attempts to apply porphyrins (Baldini et al. 2004), coordinated ruthenium complexes (Hewitt and Wilson 2017), pyrene derivatives (Fan et al. 2015) and other macrocyclic compounds to recognize the surface of specific proteins (Kubota and Hamachi 2015). However, the fact that the same structural motifs, such as glycans, phosphates, and amino acid side chains are manifested on the surfaces of various proteins in the mixture is an additional factor that makes the design of specific protein surface sensors extremely difficult (Margulies et al. 2016).

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Different proteins, such as enzymes or ligand carriers, possess specific binding sites for small molecules represented as deep cavities. The design of probe, in which specific receptor is linked to low-specific binder probing a desired site of protein surface can recognize such site (Fig. 12.4). Attached fluorescence dye may serve as reporter for low-affinity binding at this site. In this way, one may distinguish protein isoforms and study conformational changes involving the selected site (Nissinkorn et al. 2015). Among recent trends to obtain the excellent binders (sensors) is to use a combinatorial library and a selection system, just like proposed for synthetic peptide binders (see Sect. 12.2.6). This is not easy, highly challenging but also highly promising. Unlike antibody arrays, this methodology does not require the expression of expensive proteins or stepwise protocols and therefore, it can provide high-throughput detection, where patterns are obtained in a single step (Geng et al. 2019). The designed library can be used for forming the protein-detecting array based on one type of fluorescent receptors with a magnitude of modifications. Such are porphyrins containing peripheral amino acids acting as protein surface receptors (Zhou et al. 2006). The array of porphyrin receptors showed a unique pattern of fluorescence change upon interaction with certain protein samples. Both metal and nonmetal-containing proteins and mixtures of proteins provide distinct patterns, allowing their unambiguous identification.

Fig. 12.4 Schematic illustration of a targeted, protein surface sensor that can interact with a specific site on the surface of the protein target and consequently, respond to a specific structural modification involving this site (Margulies et al. 2016). A relatively non-specific synthetic receptor is attached to a highly specific protein binder. In this way, the specific binder brings the synthetic receptor in the vicinity of the protein of interest, enhances its effective molarity and consequently, its affinity toward the surface of the target protein. Decorating this receptor with a suitable fluorescent reporter and supramolecular recognition elements allows the sensing specific structural motifs on protein surfaces. The same sensor can interact with different isoforms (e.g., state I and state II) and the favorable interaction of the non-specific receptor with the surface of one isoform provides this sensor with a selective fluorescence response

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The cross-reactive sensor arrays allow overcoming the necessity to design a specific receptor for each protein (Cohen and Walt 2018). Selective protein recognition can be achieved when the global response of different receptors generates a unique pattern that can be regarded as a fingerprint of the analyte(s). Each receptor interacts with the protein in a slightly different manner (or not at all), which is signaled by a characteristic optical readout. The whole plate thus generates a distinct pattern, which can be analyzed by chemometric methods such as principal component analysis (PCA) or linear discriminant analysis (LDA). On a simple scheme, the arrays allow realizing a discriminating search among different proteins in their mixtures and resolving the proteins belonging to the same class. Summarizing, we indicate that, although a variety of synthetic binders to the protein surface were synthesized, it is still difficult to rationally design and adapt to particular target the binders with high selectivity and affinity. Synthetic molecules possess advantages compared with large biological protein binders (antibodies and aptamers) being smaller, often much easier available and stable under physiological conditions. However, combining in their use the principles of fluorescent molecular probe design and concepts of host–guest chemistry, such as multivalency and binding cooperativity, is attractive as a general idea but difficult for realizing in practice.

12.2.4 Protein Sensing with Peptide, Protein and Nucleic Acid Receptors The most commonly used affinity reagents (the binders with high affinity) for proteins are antibodies and aptamers. They satisfy the best characteristics of protein affinity reagents demonstrating together with high affinity (low dissociation constants) a high specificity (ability to recognize the target protein in a sample containing many other potentially interfering molecules). They can operate in the presence of many interfering molecules allowing to detect a specific protein. In addition, different designed binding proteins and peptides demonstrate their increased utility. Because of dramatic variations of protein structures, of their surface topologies and of their surface modifications there is a great difficulty in unifying the strategies for proteinreceptor interactions. Often a special recipe is needed to target a particular protein. Antibodies, their properties and the principles of their interaction with the targets are overviewed in Sect. 4.4. Being the major components of the vertebrate immune system, they bind to foreign molecules known as antigens. An antibody binds specifically to a structure on the antigen called an epitope. Antibodies recognize linear segments or conformationally unified clusters of 3–5 amino acids on the protein surface (Kusnezow et al. 2003). Most antigens are proteins. Moreover, each protein molecule may contain several epitopes, which allows realizing sandwich assays, in which two different antibodies interact with the protein at two different sites (Sect. 2.2 of Volume 1).

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Antibody−antigen interactions are particularly strong and have very high affinities in the nanomolar to picomolar range, but a directed evolution of antibodies with affinities in the femtomolar range have been achieved (Peng et al. 2014). Overall, due to their high affinity and specificity, antibodies are at present the key reagents for protein detection. Antibodies of immunoglobulin G (IgG) type are relatively large (~150 kDa) Yshaped protein molecules. They consist of two identical heavy and light polypeptide chains that are connected to each other by disulfide bonds. They can be easily integrated into different protein assays. Their multiple reactive side groups can serve as attachment sites for conjugation to solid supports (such as magnetic beads or nanoparticles) or labels that act as signal transducers. Antibodies are usually covalently attached to plain surfaces or nanoparticles. The techniques of spotting antibodies to different surfaces are well developed. Obtaining the recombinant fragments of antibodies possessing similar binding properties, the minibodies and nanobodies (Doshi et al. 2014; Gonzalez-Sapienza et al. 2017) demonstrates a great progress (see Fig. 4.11). Aptamers are single-stranded DNA or RNA oligonucleotides that can bind to proteins with high affinity and specificity (see Sect. 5.2). They are created by an in vitro process known as SELEX, systemic evolution of ligands by exponential enrichment. The SELEX process (see Fig. 5.5) starts with a large combinatorial library of DNA or RNA oligonucleotides of random sequences. These sequences are allowed to interact with the target protein that is usually attached to a solid support. Those species that bind strongly are selected and amplified. They are put to undergo another round of selection. This process is repeated, typically between 8 and 15 rounds, with sequential selection of stronger binders. Once an optimal sequence has been determined, it can be easily and reproducibly generated by chemical synthesis. Aptamers can be easily integrated into many protein assay formats by substituting for antibodies (Fang et al. 2003). They are easily labeled and modified with various molecules and functional groups. Aptamers are highly stable and can also be regenerated for multiple uses. The advantages of aptamers for use as the protein affinity reagents are their low cost, high stability, and high specificity, with dissociation constants achievable in the femtomolar to picomolar range. In contrast to monoclonal antibodies, they can be generated against almost any protein, even toxic proteins and those that are not immunogenic. Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and clinical diagnostics (Sect. 4.2). The advantages include smaller size and a more robust, singledomain structural framework with a defined binding surface amenable to mutation. Therefore, there were many attempts to develop affinity binders structurally unrelated to antibodies and aptamers (Banta et al. 2013; Pham et al. 2021; Skerra 2007; Yu et al. 2017). Some of them, often called as antibody mimetics, were discussed in Chap. 4. In general, a good small protein scaffold should display a high stability, together with flexibility of binding. Chemical (of aptamers) or biological (of antibodies) selection allows obtaining very efficient binding to almost unlimited number of protein species. Other protein

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binders lack such efficient mechanisms of amplification and selection. Therefore, efficient sensor receptors are found in a different way, by selecting protein or peptide scaffolds and making libraries composed of mutated forms. In this way, the sensors to particular important targets are constructed. Peptide sensors (Sect. 4.1) are less popular than proteins and nucleic acids in the studies of proteins. They are efficient in detecting the antibodies, and when they are labeled with λ-ratiometric dye, they can report on their binding by the change of fluorescence color (Enander et al. 2008), see Fig. 4.4.

12.2.5 Molecularly Imprinted Polymers in Protein Sensing The sensing based on the principle of molecular imprinting involves the formation of structure in the presence of analyte molecule or its analog that contains the analyte recognition sites, so that this structure remains intact after removal of the guest compound and then can serve as the analyte receptor on its detection in tested media. Molecular imprinting can be of two types. One is the imprinting in the polymer volume, where the polymerization step is necessary to form rigid 3D structure (see Sect. 6.4) and the other is the imprinting on the performed functional surface, where imprinting is achieved by attachment of small molecules to solid surface (see Sect. 7.5). The mechanisms of their formation are different. Meantime, both of them are efficient in recognition of proteins (Ansari and Masoum 2019; Dabrowski et al. 2018; Yang et al. 2012), since the broad selection of available monomers and oligomers allows fine adaptation to the size of analyte protein molecule together with the establishment of multiple interactions with the formed matrix. Despite these apparent advantages, the implementation of this method into protein sensing research is slow and meets essential difficulties (Yang et al. 2012), which are not only in large size, variable configuration, unstable physical and chemical properties of target proteins (Ansari and Masoum 2019). More successful are the applications to chromatographic separation and detection of captured molecules. Let us critically analyze the existing problems. First, heterogeneity of target binding leading to the loss of selectivity is usually observed. It may appear as a result of non-ideal fitting to template structure on polymer formation. The pre-polymerization step is not a well-defined process; it results in the formation of complexes with different ratios of template to monomer that are fixed on polymerization (Chen et al. 2011). Nonspecific binding to the matrix is possible (Zhang et al. 2021). It increases the background signal and reduces the sensitivity of the assay. Second, it usually takes long time for proteins to reach their recognition sites because of the surface structure of the imprinted materials (Yang et al. 2012). This means slow establishment of equilibrium between free and bound target protein, especially in the presence of nonspecific binders. This effect is reduced substantially with the use instead of flat support of polymer nanoparticles (Chen et al. 2016).

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Third, the detection mechanisms do not readily enable effective signal conversion. The fluorophore should be located within the system and report on target binding in the cavity. The solution for providing satisfactory signal conversion for obtaining fluorescence reporting is not easy and has to be found in every particular case (see Sect. 6.4). There are two tendencies in the development of protein detection techniques based on molecular imprinting. First is increasing the selectivity of interaction with the target by introduction into the template of functional groups (Takeuchi and Sunayama 2018). Thus, introduction of boronic acid derivatives into imprinted cavities functionalized for molecular recognition of glycoproteins allows achieving desired effect (Zhang and Du 2020). Boronic acid is able to interact strongly but reversibly with carbohydrate moiety of glycoproteins, and in this way a strong increase of selectivity is achieved. The second tendency is the use on the step of template formation instead of whole target protein of short peptides or other smaller molecules carrying the target protein recognition sites (epitopes). Epitope molecularly imprinted polymers (EMIPs) emerged as novel imprinted materials using as templates the short characteristic peptides rather than entire proteins (Wang et al. 2021; Yang et al. 2019). For providing this specific effect, the amino acid sequence of the template peptide can be the same as an exposed N- or C-terminus of a target protein, or its amino acid composition and sequence replicate a similar conformational arrangement as the same amino acid residues on the surface of the target protein (Fig. 12.5).

Fig. 12.5 The epitopes arising from a continuous amino acid sequence that are the most promising to imprint (Pasquardini and Bossi 2021). a Epitopes located at the N- or C-terminus of the protein, or epitopes placed within the amino acid sequence, thus internal. b Some epitopes that are characterized by a secondary structure, such as α-helix or β-strand, and can be defined as structured epitopes. c Most of the epitopes that lack a properly defined secondary structure and are therefore loose terminal stretches, or flexible loops of the protein, characterized by structural flexibility. Nevertheless, the exposure to the solvent of both the terminal stretches and the loops and their accessibility to binding partners come with a defined orientation and with inherent directional constrains. In the examples, the following structures are shown: a human serum albumin (HSA); b co-crystal of HSA with shark IgNAR variable domain; c co-crystal of Fab fragment with human serum kallikrein

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Some epitopes are portions of the protein’s sequence characterized by a defined secondary structure, such as α-helix or β-strand. The epitope exposed at the protein’s surface, whether it is located terminally or internally, is characterized by a directional exposure and accessibility both to the solvent and to its possible interacting partners. Thus, the orientation of the epitope during the imprinting process can also be provided by exploiting inhibitors, or more generally, by coupling the epitope to a solid support in a defined direction (Pasquardini and Bossi 2021). Several advantages were found in this approach, mainly in simplifying the process of imprinting. Still, as it was indicated (Wang et al. 2021), some challenges remain to be solved. In order to increase the versatility of the methods for facile and efficient epitope immobilization, further developments are needed (Xing et al. 2019). Poorly realized are the possibilities for post-imprinting modification introducing new functions into the molecular imprinting cavity in a site-directed manner. Attractive are the nanostructured imprinted polymer systems (Dabrowski et al. 2018; Zhang 2020), which allows using them in the studies of living cells and tissues. Comparison of ‘synthetic antibodies’, which are the imprinted polymer sensors, with natural antibodies and aptamers shows their apparent advantages. They are less expensive, more durable and able to withstand harsher environmental conditions (Luan et al. 2018; Xu et al. 2020). Regarding proteins, their recognition power involves both the factors of size and shape and also the sites on molecular surface that can be large or presented by small patches (epitopes). The idea of coupling with fluorescence reporting is expected to find many good solutions, particularly on nanoscale level (Wang et al. 2020).

12.2.6 Sensor Arrays and Machine Learning Algorithms Strong demand for high-throughput analysis on proteomic level stimulates rapid development of protein arrays for simultaneous detection of many proteins. Due to their high throughput, they must obtain great potential to reduce the cycle time of drug discovery and to improve the efficiency of medical diagnostics, providing simultaneous detection of a number of disease markers (see also Chap. 14). For realworld use, a protein array have to become simple and inexpensive to manufacture, its fabrication should be amendable to automation, the size and shape of its spots must be controlled, it must be durable and reproducible in application. The sensitivity of these arrays should be on a competing level with that of ELISA, which means a picomolar detection limit. These facts explain why despite the offer of different ligand-binding proteins, peptides and aptamers, the antibodies are still very much in vogue, as well as the recombinant antibody fragments (Pavlickovan et al. 2004). Many problems with the design and production of microarrays based on them have been resolved, including their immobilization in 3D hydrogels (Rubina et al. 2003).

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The protein microarrays based on immobilized DNA and RNA aptamers, selected against many different protein targets, allow simultaneously detecting and quantifying the levels of individual proteins in complex biological mixtures (Zhou and Zhang 2021). Each aptamer can be fluorescently labeled and immobilized on a glass or plasmonic metal (Hou et al. 2014) substrate. Different methods including fluorescence polarization (anisotropy) can be used for detecting the target protein binding. The latter detection method can operate both in solution and on solid support. Attractive is the possibility to achieve the λ-ratiometric response in the near-IR region (Demchenko 2023a, b; Wang et al. 2019). Water-soluble conjugated polymers with pendant-charged residues provide an excellent scaffold for the design of sensor arrays (Miranda et al. 2007), since they combine the binding and responsive properties (Freudenberg et al. 2017; Thomas et al. 2007). The introduced side groups can provide specific protein binding through multivalent interactions, and their optical properties are sensitive to minor changes in their conformation with the possibility of fluorescence superquenching (see Sect. 8.5 of Volume 1). Complementary to the arrays based on highly specific recognition described above is the strategy of cross-responsive arrays that are inspired by the mammalian olfactory system (Sect. 18.2). This led to development of array-based ‘chemical nose/tongue’ platforms. Multiple sensor elements in these devices interact selectively with target analytes, producing a distinct pattern of response and enabling analyte identification (Geng et al. 2019). This approach offers unique opportunities relative to ‘traditional’ highly specific sensor elements such as antibodies. A high level of cross-reactivity still allows precise determination of particular target. This is because each target generates a unique composite pattern of interactions with olfactory receptors. In analogy, an array of nonspecific or weakly interacting agents would give rise to distinctive fingerprints in response to a target. This strategy can be the basis for identification of “hot spots” on protein surfaces. Following it, a smart sensor array (chemical nose) was developed for sensing specific proteins (You et al. 2007). It is based on reversible binding of anionic poly(paraphenyleneethynylene) (PPE) polymer with cationic gold nanoparticles (Fig. 12.6). The fluorescence in these complexes is initially quenched, but in the presence of proteins they become disrupted giving a turn-on response. The varied affinities of different proteins to gold particles have led to different scales of signal response, so that the fluorescence response generates fingerprint pattern for individual proteins. These affinities are determined by their respective structural features, allowing charged, hydrophobic, hydrophilic, and hydrogen-bonding interactions. An interesting trend was recently presented—to combine in a single synthetic molecule several recognition and several reporting functions in order to realize the chemical nose/tongue concept on molecular level (Pode et al. 2017; Rout et al. 2012). Such unimolecular pattern-generating device (Fig. 12.7) stands between conventional small-molecule fluorescent probes and larger analytical systems that can provide multiplexed protein detection. The covalent integration of the various signaling and recognition elements enables the probe to interact reversibly with its

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Fig. 12.6 Experiment on specific protein detection based on chemical nose concept (You et al. 2007). a The protein analyte releases fluorescent polymer from its quenched complex with gold nanoparticle with concomitant restoration of fluorescence. b Pattern generation through differential release of fluorescent polymers from gold nanoparticles. The combination of an array of sensors generates the fingerprint response patterns for individual proteins

protein targets, switch between multiple structural states and operate within confined microscopic environments. In contrast to common nose/tongue technologies that use spotted microarrays, this molecular device can be easily used in confined biological compartments, identifying the groups of similar proteins and following their transformations, which are beyond the reach of current electronic or optical devices that can produce identification patterns. Pattern recognition is central to array-based sensing. Each sensing event is a point in a multidimensional space where the dimensionality is the number of sensorarray elements used. Traditionally, linear discriminant analysis (LDA) has been widely used because of its easy interpretability. Recently, it has been shown that pattern recognition based on nonlinear machine learning (ML) may substantially improve detection efficiency (Ha et al. 2020). Currently emerging, compelling pattern-recognition method based on machine learning (ML) enables a machine to “learn” a pattern by training without having the recognition method explicitly programmed into it. Thus, ML has an enormous potential to analyze the sensor output data better than the widely used statistical pattern-recognition methods. Nowadays, machine learning as a branch of artificial intelligence involving computer programs that are able to improve their own performance through experience (training) has achieved impressive advances (Jones 2019; Joshi 2020). Novel advanced ML methods, especially deep learning, which were successful in image analysis, facial recognition, and speech recognition, have started to be actively applied for sensor array technologies (Cui et al. 2020). The utility of using machine learning algorithms in pattern recognition of fluorescence signals from the array has been demonstrated. Thus, an array-based sensor using easy-to-synthesize carbon dots with varied surface functionality is reported (Pandit et al. 2019). It can differentiate between eight different proteins at 100 nM concentration. Another example is an array of differently functionalized MoS2

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Fig. 12.7 Pattern-based recognition of specific protein populations in biological mixtures by a unimolecular probe (Pode et al. 2017). a The probe 1 integrating four fluorophores with binders for three different protein groups (blue, green and purple marked proteins). The probe can discriminate among different combinations of proteins that belong to different families (pattern 1 versus pattern 2), as well as among combinations with different isoforms of the same family (pattern 1 versus pattern 3, green isoforms). b Chemical structure of probe 1 that consists of a cis-amino proline scaffold appended with four fluorophores—NBD, NR, Cy5.5 and Cy7—and three protein binders—bis-EA, MT and an Apt—that are known to bind glutathione-S-transferases (GSTs), matrix metalloproteases (MMPs) and platelet-derived growth factors (PDGFs) in complex mixtures, such as human urine. The excitation (ex) and emission (em) wavelengths (nm) of each dye are denoted in brackets (ex/em)

sheets, in which the dequenching of fluorescence of green fluorescent protein occurs differently on interaction with different target proteins (Fig. 12.8). It can be predicted that implementation of machine learning algorithms will provide stimulus to further development of molecular computing (Shani et al. 2019) that becomes an established technique for providing logical operations on molecular level (Sect. 4.5 of Volume 1). The idea in molecular computing applied to sensing is providing on molecular level of decisive output signal on the presence of particular target(s) on the condition of absence or presence of other target(s) (De Silva 2016; Erbas-Cakmak et al. 2018). Clinical diagnostics often requires simultaneous analysis of several protein markers, so we may expect that artificial intelligence on molecular level (Butler et al. 2018) will soon complement the intelligence of our doctors.

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Fig. 12.8 Schematic representation of sensing system that uses machine learning (Behera et al. 2021). The sensor array is composed of green fluorescent protein (GFP) deposited on cationic MoS2 nanosheets, on which the fluorescence is quenched. The nanosheets serve as multiple crossreactivity receptors forming their array. Interaction with analyte proteins results in displacement of GFP from the cationic MoS2 surface followed by intense fluorescence, which allows building the fluorescence response pattern. Machine learning algorithm allows processed on the basis of smart model recovery of true data for every protein analyte

12.3 Analysing Pathological β-Aggregated Forms of Proteins When we discussed the sensors for nucleic acids we had to recognize the usefulness of their ability to adopt the canonical double-helical structure that allowed providing their efficient molecular recognition both on the level of their overall structure representing the gene and also on the level of single nucleobases. Proteins also have regular elements of structure—λ-helices and β-strands. They occupy very definite positions in the structures and their content differs in very broad ranges among proteins and may, in principle, serve as the elements for their recognition in fluorescence sensing. This does not happen because of lacking the proper tools. The only case, in which fluorescence sensing is efficient is the discovery and analysis of pathological protein folding leading to formation of their β-structured aggregates (Chiti and Dobson 2017). Those are amyloid fibrils detected in certain nerve cells on Parkinson and Alzheimer diseases (Soto and Pritzkow 2018). Such structures are also characteristic for prion (mad cow) disease (Ritchie and Barria 2021). Formation of such highly ordered βstructured fibrils can be reproduced in experiment with purified proteins. Moreover, a larger number or proteins, such as myoglobin (Fändrich et al. 2001), lysozyme (Tokunaga et al. 2013) and insulin (Iannuzzi et al. 2017) are able to form them under some special conditions. Clinically important pathological forms of fibrinogen and fibrin were also found (Pretorius et al. 2017). Moreover, some amyloid protein forms can be important for normal functioning (Rubel et al. 2020). Amyloid-type fibrils

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of proteins are the basis of bacterial biofilms (Shanmugam et al. 2019); they were found in food products (Cao and Mezzenga 2019). The reader may follow the papers showing the development in our understanding of this unusual phenomenon (Chatani et al. 2021; Kell and Pretorius 2017; Makin and Serpell 2005; Rambaran and Serpell 2008; Tycko 2004).

12.3.1 Organic Dyes as the Sensors for β-Sheets Many mysteries still are hidden in the phenomenon of pathological protein folding. One of them is the fact that a number of small organic dyes can serve as efficient sensors of large and regular cross-β-sheeted structures (Zhang and Ran 2013). The most popular dye to detect them is Thioflavin T (ThT) (Naiki et al. 1989), see Fig. 12.9. The torsion angle between the benzothiazole ring and the dimethylaminobenzene ring can be variable (Wolfe et al. 2010), and immobilization of this structure on binding leads to fluorescence increase. Many other dyes were found to be able to bind with β-sheeted fibrils specifically, in nanomolar concentrations. Their list grows (Dzyuba 2020; Lee et al. 2019; Bertoncini and Soledad Celej 2011), and it is hard to find structural similarity between them. Regarding the best studied and the most frequently used Thioflavin T, its fluorescence is quenched in solutions (even at high viscosities) but is enhanced on binding to protein fibrils (Groenning 2010). This means that not only the loss of intramolecular mobility but immobilization in some favorable conformation is needed for its bright fluorescence. It is thought that it binds to the hydrophobic grooves along the pleat surface that runs parallel to the peptide chains (Biancalana and Koide 2010). Due to its positive charge, the closeness of anionic side groups may contribute to its strong binding. Surprisingly, ThT does not bind to β-pleated sheeted polypeptides or to

Fig. 12.9 The structure and spectra of Thioflavin T. a The torsion angle between the benzothiazole ring and the dimethylaminobenzene ring can be variable. b The typical excitation and emission spectra on binding to β-structured protein aggregated forms

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native proteins that contain a high amount of β-structure. These facts suggest that in pathologically folded fibrils the fluorescent form of ThT is not simply immobilized; it should be immobilized in particular conformation favorable for fluorescence. Since there are so many dyes that become fluorescent on binding to amyloid fibrils, it becomes natural to search for a fluorophore that can provide the most informative response. Those are the dyes of 3-hydroychromone (3HC) family that provide multiparametric spectral response to polarity and hydration of their environment (Klymchenko and Demchenko 2003; Shynkar et al. 2004); their versatile applications were presented in different chapters of this book. These dyes can be present in two ground-state forms, normal N and hydrogen-bonded to water molecule, H-N. They can be distinguished in excitation spectra, and the ratio of emissions of their excited-state forms, H-N*/N*, can be used as characteristics of probe environment. In addition, the N*/T* ratio is a very sensitive characteristics of polarity. Special software were developed for acquiring these parameters from spectroscopic information (Caarls et al. 2010). The key protein that exhibits pathogenic folding leading to Parkinson disease is α-synuclein, a 15 kDa single-chain protein. The results have shown that 3hydroxychromone dye 4-(diethylamino)-3-hydroxyflavone can bind to amyloid fibrils formed of α-synuclein with strongly enhanced fluorescence. Deconvolution of its two-dimensional excitation-emission spectra allows characterizing simultaneously the polarity and hydration (proticity) of its environment by observing the interplay of N*, T* and H–N* forms in their emission (Celej et al. 2009). Moreover, it can distinguish between polymorph forms derived from naturally occurring point mutations in α-synuclein structure (Fig. 12.10). The sensitivity of such a simple external probe to conformational alterations induced by point mutations is unprecedented and provides new insight into key phenomena related to amyloid fibrils: plasticity, polymorphism, propagation of structural features, and structure–function relationships underlying toxicity. Polymorphism of pathological protein aggregates is observed not only in the case of point mutations but also on their formation under different experimental conditions (Chatani et al. 2021), e.g. on variation of temperature (Ziaunys et al. 2021). It is essential that the morphology of a single fibril does not change along the length of the fibril (Tycko 2014). Thus, morphological features such as fibril width and twist period are self-propagating. Moreover, when a morphologically homogeneous batch of “parent” fibrils is used as the source of “seeds” for the growth of subsequent batches of “daughter” fibrils, the morphology passes from parent to daughter and from daughter to subsequent generations.

12.3.2 Following the Kinetics of Amyloid Formation There are many evidences allowing to say that it is not the finally formed fibrils or filaments but the intermediates in their formation (soluble oligomers) are the primary toxic agents (Chatani et al. 2021). These intermediate oligomer species are

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Fig. 12.10 The structure of α-synuclein and of its disease-related mutants and the multiparametric response of the bound fluorescent dye (Celej et al. 2009). a The presentation of α-synuclein structure, in which the charged and Pro residues are marked with different colors. Three structural regions are indicated. Three point mutations (A30P, E46K, A53T) are found in patients suffering from early onset of Parkinson’s disease and their positions are marked with arrows. b The 3D representation and contour plots of correlated excitation-emission spectra of 3-hydroxychromone probe 4-(diethylamino)-3-hydroxyflavone showing the difference between these mutants. c Deconvolution of fluorescence spectra into three individual components, N*, H–N* and T*. d The ratio of these individual components characterizing polarity (N*/T*) and hydration (H-N*/N*) of the probe environment. Ratios of the integrated areas of the N* and T* bands (violet bars) and of the H–N* and N* bands (orange bars)

highly toxic, affecting mitochondrial function, endoplasmic reticulum–Golgi trafficking, protein degradation and/or synaptic transmission that may induce neurodegeneration. The scheme depicting one of suggested pathways of their formation and transformation (Lashuel et al. 2013) is presented in Fig. 12.11. Most probes that are used to follow the kinetics of aggregate formation are insensitive to the early oligomeric states of aggregation and detect only the terminal fibrillar species, so that the observed kinetics is actually the recording of their growth. Thus,

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Fig. 12.11 Insight into kinetics of formation of α-synuclein fibrils. a Schematic presentation of aggregation pathways (Lashuel et al. 2013). Unfolded monomers interact to form two types of dimers: anti-parallel dimers, which do not propagate (not shown), and parallel dimers, which do propagate. A dynamic equilibrium is established between unfolded monomers and both forms of dimers that can take place either in the cytoplasm or on association with the cellular membrane. Propagating dimers can grow by the addition of unfolded monomers and generate ring-like oligomers. Ring-like oligomers interact with the cytoplasmic membrane and form trans-membrane pores, inducing abnormal intracellular calcium influx. Cytoplasmic oligomers grow by the addition of soluble monomers, forming small amyloid fibrils and then longer fibrils. b A thiol-reactive (maleimide) probe MFC based on 2-(2-furyl)-3-hydroxychromone and the results of monitoring the kinetics of aggregation of α-synuclein covalently labeled with MFC in comparison with ThT probing (Yushchenko et al. 2010)

the black curve in Fig. 12.11b presents the kinetics of increasing the ThT fluorescence intensity that roughly corresponds to increase of light scattering and indicates the formation of terminal large aggregates. The attempts to catch the first steps that include the formation of early intermediates were first performed with fluorescent covalent labeling of initial α-synuclein monomers (Thirunavukkuarasu et al. 2008). This approach involves tagging functionally neutral Ala-to-Cys variants located at the desired sites in protein sequence with different dyes, such as the longlifetime fluorophore pyrene. The entire family of descriptors of pyrene emission was shown to change dramatically during the early stages of folding, in which oligomeric intermediates form and evolve. In development of these studies, the Cys labeling of α-synuclein monomers was performed with a thiol-reactive 2-(2-furyl)-3-hydroxychromone derivative MFC

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(Yushchenko et al. 2010), see Fig. 12.11b. In contrast to ThT probing, the results of monitoring the kinetics of aggregation of α-synuclein covalently labeled with MFC, allowed providing the information on early and intermediate stages of the overall reaction. The most notable spectroscopic feature was a dramatic (15-fold) increase in the T* band intensity, with a smaller, twofold increase of the N* band. The net effect was a 9.5-fold rise in the T*/N* ratio to a peak value of 8.8 at 20 h, decreasing to 7.6 at the end of the reaction (70 h). The large increase in the T*/N* ratio as well as the red-shifted T* band upon aggregation reflect a transfer of the probe to a significantly less protic (H-bonding) and moderately less polar environment. These values evidence unambiguously that the reaction intermediates differ significantly from monomeric and also from the fibrillar amyloid forms of α-synuclein. They witness (indirectly) that β-sheets are not formed on early steps of aggregation. A range of functionally and structurally unrelated proteins form amyloid structures (Chatani et al. 2021). Moreover, the same protein can form different highly structured, densely packed protein aggregates (Ziaunys et al. 2020). Thus, it can be thought that the amyloid structure is an alternative stable structure for peptides and proteins accessible to many of them. The existence of distinct conformers and interchange among them present heterogeneity and complexity, hindering the development of effective detection approaches. Protein engineering under the control of fluorescence that involves formation and transformation of amyloid fibrils is becoming one of important trends in future developments.

12.4 Polysaccharides and Glycoproteins Known under the common name of glycans, polysaccharides, and carbohydrate portions of glycolipids and glycoproteins constitute a large family of compounds that involve sugar elements (Jelinek and Kolusheva 2004). These molecules are present both inside and on the surface of cells and are integral constituents of a vast number of proteins that are involved in a myriad of cellular events. Carbohydrates and glycoconjugates of proteins and lipids participate in such processes as the normal and pathogenic cell adhesion, inflammation, signal transduction, etc. Therefore, a new field of glycomics (Rudd et al. 2017; West et al. 2021) has to follow in development the more advanced fields of genomics and proteomics with a strong extension into clinical diagnostics (Miyoshi et al. 2020; Peng et al. 2018; Svarovsky and Joshi 2014). In addition, control on carbohydrate level is needed in industrial biotechnologies and in many other industrial processes such as food processing and storage. Similarly to antibodies recognizing the surface antigens, the glycan sensors can recognize carbohydrate pattern on the surfaces of pathogenic microbes. It is known that a large number of bacterial toxins, viruses and bacteria possess carbohydrate derivatives on the cell surface to attach and gain entry into the cell, and glycan sensors can detect them (Ngundi et al. 2006). Because of that, they find important application for the determination of microbial contaminants in food.

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Many suggested sensors for glycans and glycoproteins rely on recognition power of boronic acid and its derivatives (Qin et al. 2020). Detailed mechanism of their interaction with saccharides will be explained in Chap. 14 on an example of sensing glucose. In glycan detection, the advantages are the pH-controlled binding/release, reversibility of the reaction, high specificity, and high selectivity, showing their wide application prospects. Boronic acid can serve as electron acceptor from different luminophores, including quantum dots, and the latter can serve as the base for nanosensors for glycoprotein recognition. Lectins are the proteins that specifically bind to carbohydrates (Hendrickson and Zherdev 2018; Surya, Deepti et al., 2020). Being made on the basis of lectins, the glycan sensors provide the profiling of carbohydrate expression on the surface of human cells (Zheng et al. 2005). Sensing the glycan markers of diseases is an important approach in clinical diagnostics (Dai et al. 2006). In this respect, glycan microarrays technology becomes promising in high-throughput identification of glycanbinding proteins and pathogenic microorganisms and even for decoding of the whole glycome (Rillahan and Paulson 2011). Glycan microarrays have played important roles in detection and specificity assignment of glycan-recognition by proteins. However, the size and diversity of glycan libraries in current microarray systems are small compared to estimated glycomes, and these may lead to missed detection or incomplete assignment. Before such highly needed arrays come into practice, many technical problems have to be resolved (Hyun et al. 2017; Li et al. 2021; Shin et al. 2005; Temme et al. 2019). These problems are much more difficult than with DNA and protein arrays. They can be outlined as follows: (a) Glycans are rather diverse chemical structures, and there are no unified methods for their synthesis, modification and immobilization on the surface. Moreover, the designed sensors must be able to probe the protein-carbohydrate, carbohydrate-carbohydrate, nucleic acid-carbohydrate and intact cell-carbohydrate interactions. This makes difficult to develop a unified strategy of sensor design. The questions to be resolved involve the proper surface preparation, the proper linking chemistry, and immobilization with proper orientation. (b) The glycan interactions display diverse affinities that often are rather weak (K d ~ μM to mM). This is because their molecular recognition is hampered by their strong solvation in water and subtle structural differences among analogues. Definitely, such strength of sensor-target interactions does not allow using in full the sandwich techniques that require washing steps. In addition, the researcher should often address the problem of nonspecific adsorption effects and false-positive signals. The application of competition assays can also be problematic. Thus, lectin–glycan interactions possess high discriminating power for lectins but within this family a high “promiscuity” may be observed (Kolarich et al. 2012) that needs to prepare more specific binders, such as recombinant lectins or DNA and peptide aptamers (Alley et al. 2013; Oliveira et al. 2013).

12.5 Sensing and Thinking. Precise Affinity Sensors or Chemical Noses?

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(c) There is no unified procedure for fluorescent labeling of lectins, and existing methods of introduction of reactive groups are not enough specific. Also, close fluorescent analogs of carbohydrates that could be used in sensing do not exist, and conjugation of fluorescent dyes may interfere strongly with the target recognition. These difficulties do not detract the researchers, and with the implementation of new ideas and new technologies they have to be overcome. Several examples are presented below. It was shown that with the aid of combinatorial methods of sensor design, a library can be constructed for specific binding of lectins for specific carbohydrate binding. A 15,625-membered short peptide-dendrimer combinatorial library was produced and acylated with an alpha-C-fucosyl residue at its four N-termini and screened for binding to fucose-specific lectins (Kolomiets et al. 2007). In this way, the targets on a sub-micromolar level can be identified. The principle of molecular imprinting was applied for glycoprotein sensing (Zhang and Du 2020). The glycoprotein imprinted self-assembled monolayers incorporating boronic acid recognition sites were developed for their detection. The other possibilities to use cross-linked micelles as synthetic lectin analogues was also realized (Duan et al. 2020). Functionalized with boroxole groups in the binding sites, these water-soluble synthetic lectins are able to bind to carbohydrate components of several proteins selectively in water with an association constant of Ka = 104 –105 M−1 .

12.5 Sensing and Thinking. Precise Affinity Sensors or Chemical Noses? Great demand exists for peptide, protein and glycan sensors. We observe that it is not easy to satisfy it due to great diversity of these targets leading to great complexity of existing problems. One of them to mention, is in a difficulty to achieve efficient molecular recognition due to the effect of solvent water: dielectric screening of complementary charges and saturation with hydrogen bonds of interacting surfaces. Therefore there appeared a great variety of suggested and exploited methods. We will discuss two tendencies in the development of these methods. On one hand, the focus is on particular molecular targets and on the ability of their quantitative evaluation in broad concentration ranges and in the presence of other contaminating species. The multivalent molecular recognition scheme must be realized, and the best performers here are the antibodies (or their small recombinant fragments) and nucleic acid aptamers. They can achieve a high specificity of binding and low level of side effects. The techniques of their selection may encompass a broad range of affinities fitting the optimal target concentration range. The sensor arrays focusing on a number of targets should display a minimum level of cross-reactivity. The templating methods that use the principle of molecular imprinting, being highly prospective, still have to deserve the name “artificial antibodies”, because of slow

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target binding/release rates and difficulties in achieving the required selectivity. Though, they have an advantage over real antibodies due to additional possibility to recognize the targets according to their size and shape. Search for high-affinity binders among enzyme analogs of inhibitors or ligands follows the same line as the search for optimal drugs that may involve searching great libraries of related molecules. On the other hand is the focusing on characterizing the whole studied sample or a number of its most interesting components without attempt to achieve high binding affinities to each of them. Here the array-based ‘chemical nose/tongue’ platforms are efficient. Such array-based sensing systems utilizing combined recognition of multiple specific targets with a selectivity-based modality have been developed and represent convenient and rapid methods that do not require high degrees of specificity between the recognition element and the target. To develop a successful array-based sensing system, the recognition elements and the targets should possess distinct selectivities that result in different responses. Therefore, the cross-reactive recognition elements are not harmful. Pattern-based recognition can be provided with the aid of machine learning algorithms. It should be recalled that many drugs, especially the drugs derived from plant origin, interact with multiple targets producing multiple pharmacological effects. The competition between arrays with individual response of each sensor element and arrays based on response patterns continues. In order to verify the efficiency of reading (and thinking), the reader is asked to respond to the following questions: 1. 2. 3. 4.

5.

6. 7. 8. 9.

What are the recognition elements in peptide sensing? Can they be recognized by antibodies? What synthetic compounds can be used as their specific binders? What is the difference in molecular recognition of small and large peptides? How the large peptides can be specifically detected? Explain on an example the principle of application of molecular imprinting to peptide sensing. Explain the application of fluorescence for determining total protein content. What are the problems with that? What alternative methods do you know? Critically evaluate them. What reactive groups on protein surface are the best fitting for fluorescent labeling? How to provide the protein labeling with λ-ratiometric fluorescent dye? What are the ‘hot spots’ on protein surfaces and how they can be used for protein recognition? Macrocyclic receptors, do the charge-charge interactions play a role in their molecular recognition of proteins? What protein-targeting affinity reagents do you know? Characterize them. How molecularly imprinted polymers are used in protein sensing? Provide critical analysis of application of this technology.

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10. Explain different possibilities to construct the protein sensor arrays. Explain the operation of array-based ‘chemical nose/tongue’ platforms. What is the need to apply the machine learning algorithms for their analysis? 11. What structures are formed on pathological protein folding? In what cases they are formed in human body? How they can be analyzed? 12. What techniques and what molecular receptors are used for specific recognition of glycans?

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Smith BD (2015) Synthetic receptors for biomolecules: design principles and applications. Royal Society of Chemistry Smith LC, Leach DG, Blaylock BE, Ali OA, Urbach AR (2015) Sequence-specific, nanomolar peptide binding via cucurbit [8] uril-induced folding and inclusion of neighboring side chains. J Am Chem Soc 137:3663–3669 Soto C, Pritzkow S (2018) Protein misfolding, aggregation, and conformational strains in neurodegenerative diseases. Nat Neurosci 21:1332–1340 Surya P, Deepti M, Elyas K (2020) Plant lectins: sugar-binding properties and biotechnological applications. In Plant metabolites: methods, applications and prospects, pp 401–439. Springer Suzuki Y, Yokoyama K (2005) Design and synthesis of intramolecular charge transfer-based fluorescent reagents for the highly-sensitive detection of proteins. J Am Chem Soc 127:17799–17802 Svarovsky SA, Joshi L (2014) Cancer glycan biomarkers and their detection–past, present and future. Anal Methods 6:3918–3936 Takeuchi T, Sunayama H (2018) Beyond natural antibodies–a new generation of synthetic antibodies created by post-imprinting modification of molecularly imprinted polymers. Chem Commun 54:6243–6251 Temme JS, Campbell CT, Gildersleeve JC (2019) Factors contributing to variability of glycan microarray binding profiles. Faraday Discuss 219:90–111 Thirunavukkuarasu S, Jares-Erijman EA, Jovin TM (2008) Multiparametric fluorescence detection of early stages in the amyloid protein aggregation of pyrene-labeled α-synuclein. J Mol Biol 378:1064–1073 Thomas SW 3rd, Joly GD, Swager TM (2007) Chemical sensors based on amplifying fluorescent conjugated polymers. Chem Rev 107:1339–1386 Tokunaga Y, Sakakibara Y, Kamada Y, Watanabe K-i, Sugimoto Y (2013) Analysis of core region from egg white lysozyme forming amyloid fibrils. Int J Biol Sci 9:219 Tycko R (2004) Progress towards a molecular-level structural understanding of amyloid fibrils. Curr Opin Struct Biol 14:96–103 Tycko R (2014) Physical and structural basis for polymorphism in amyloid fibrils. Protein Sci 23:1528–1539 van Dun S, Ottmann C, Milroy L-G, Brunsveld L (2017) Supramolecular chemistry targeting proteins. J Am Chem Soc 139:13960–13968 W Bertoncini C, Soledad Celej M, (2011) Small molecule fluorescent probes for the detection of amyloid self-assembly in vitro and in vivo. Curr Protein Pept Sci 12:206–220 Wang X, Chen G, Zhang P, Jia Q (2021) Advances in epitope molecularly imprinted polymers for protein detection: a review. Anal Methods 13:1660–1671 Wang X, Zhao X, Zheng K, Guo X, Yan Y, Xu Y (2019) Ratiometric nanoparticle array-based near-infrared fluorescent probes for quantitative protein sensing. Langmuir 35:5599–5607 Wang Y-f, Pan M-m, Yu X, Xu L (2020) The recent advances of fluorescent sensors based on molecularly imprinted fluorescent nanoparticles for pharmaceutical analysis. Curr Med Sci 40:407–421 West CM, Malzl D, Hykollari A, Wilson IB (2021) Glycomics, glycoproteomics and glycogenomics: an inter-taxa evolutionary perspective. Mol Cell Proteomics 20:100024 Wi´sniewski JR, Gaugaz FZ (2015) Fast and sensitive total protein and Peptide assays for proteomic analysis. Anal Chem 87:4110–4116 Wodak SJ, Janin J (2002) Structural basis of macromolecular recognition. Adv Protein Chem 61:9–73 Wolfe LS, Calabrese MF, Nath A, Blaho DV, Miranker AD, Xiong Y (2010) Protein-induced photophysical changes to the amyloid indicator dye thioflavin T. Proc Natl Acad Sci 107:16863– 16868 Wong SS (1991) Chemistry of protein conjugation and cross-linking. CRC Press Wright AT, Anslyn EV, McDevitt JT (2005) A differential array of metalated synthetic receptors for the analysis of tripeptide mixtures. J Am Chem Soc 127:17405–17411

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Chapter 13

Detection of Harmful Microbes

Rapid, sensitive, and selective detection of pathogenic bacteria and viruses is extremely important for proper prevention, diagnosis and treatment of diseases. Strong move from culture-cultivation methods to direct sensing that could avoid enrichment and purification processes is a goal that must be reached in near future. Fluorescence-based sensing methods are highly attractive in this respect owing to their real-time, economic, highly selective, and sensitive features that allow their miniaturization and use in point-of-care formats. Sensitively and selectively in detecting harmful microbes is the problem of ultimate significance. The consumption of water and food contaminated by pathogens is a major cause of numerous diseases and deaths globally (Ramírez-Castillo et al. 2015). Global biosecurity threats such as the spread of emerging infectious diseases, i.e. Ebola, avian influenza and recently Covid-19 (Janik et al. 2020), and bioterrorist attacks (Pohanka 2019) have dramatically increased the importance of this problem (Rajapaksha et al. 2019). The control for microbial contamination is not only a medical problem. It plays an important role in pharmaceutical clean room production, in veterinary and in food processing technology. To control pathogen contamination and reduce the risk of illness, quick detection and monitoring target pathogens are highly needed. What does the future hold and which biosensor technology platform is suitable for the real-time detection of infectious microbes? The ideal detection method for pathogenic microorganisms must be rapid (capable of delivering results within minutes but not days), reliable, and cost-effective. It should be simple, with no requirement for expensive instrumentation or skilled personnel. The results should be easily interpreted. The devices could be the portable, miniaturized and multi-targeting; they can be used directly in the field or at the point of care. In reality, we deal with microbes as very complicated objects with often minor recognition features between them. They can be present in very low quantities and can demonstrate large variability. Thus, we deal with challenges that are not found in the detection of small molecules, anions, or cations, or even macromolecules. Conventional diagnostic techniques are frequently time-consuming, labor-intensive © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_13

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and require to be performed on sophisticated equipment by trained professionals (Fauci and Morens 2012; Pulido et al. 2013). The prospective fluorescence sensors should perform much, much better (Miller et al. 2019; Sharafeldin and Davis 2020). There are several strategies developed that will be analyzed here. • Pathogen is detected as a whole avoiding sample preparation or colony growth steps. • Pathogen is recognized on molecular level targeting some characteristic site on its surface. These can be the sites of recognition by antibodies or aptamers. • Pathogen is identified by genetic information it contains. Among presented detection strategies, the nucleic acid-based biosensors appear being highly suitable for swift and sensitive testing. Their limitation lays in sample preparation steps that include nucleic acids extraction and, commonly, the amplification. In all these cases, the sensing elements should be associated with the transducing elements leading to a direct signal when the target is recognized. In many cases, this signal is fluorescence. Control on microbial contamination and on their inactivation is highly needed. Identification of the causative bacterial agent should be followed by studying its susceptibility to anti-bacterial agents (Vasala et al. 2020). Providing rapid sensor response would have significant advantages in terms of directed (rather than empirical) antibacterial therapy. The presently developed detection methods allow the readout of bacterial viability at the single-cell level (Shanmugakani et al. 2020). For removing of undesired microbes, different agents are used, from antibiotics to disinfectants. Disinfectants are chemical agents used on inanimate objects to inactivate virtually all recognized pathogenic microorganisms. Unlike antibiotics, which are chemotherapeutic drugs mostly used internally to control infections and which interact with specific structures or metabolic processes in microbial cells, disinfectants act non-specifically against multiple targets (Meyer and Cookson 2010). Resistance to their action may be a great problem (Russell 1999). In general, the microbial pathogens come in a variety of formats that include vegetative cells, dormant spores and the biofilm forms (Fig. 13.1). These different formats have their own characteristics that must be considered in the design of detection methods. The microbes have evolved to be dormant until they find a host to reproduce, which complicates their detection. Therefore, current rapid tests should allow detecting the vegetative forms of bacteria. Microbes are constantly mutating organisms, which further complicates the development of sensors. In each class of infectious agents, there are numerous strains, each with its own transmissibility and killing efficiency (virulence). Thus, detecting pathogenic bacteria face great scientific challenges and practical problems, much greater than the development of sensors to any chemical target. Going deeper into the problem of pathological pathogen detection, we may find quite reliable methods. Typically, a culture-based method that involves colony growth is the gold standard for the diagnosis of pathogens in hospitals, but it is often laborious and time-consuming (could be of several days), and not suitable for culture-negative

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Bacterial spore, ~100 nm Vegetative bacterial cell, ~2-10 μm

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Metabolically inactive

Metabolically active

Spike glycoprotein

d

Hemaggluteninesterase dimer (HE) M-Protein Ale

E-Protein Envelope RNA and N protein

Biofilm formed of bacterial cells

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Fig. 13.1 The most typical microbial objects. a Prokaryotic vegetative bacterial cell. b Bacterial spore which is a dormant form of pathogen. c Biofilm formed by bacteria on surfaces. d Virus, a particle of sub-micron size but with strong structural organization

bacteria or viruses. The problems with culture-independent methods, such as enzymelinked immunosorbent assay (ELISA) and polymerase chain reaction (PCR), are the need for trained and skilled personnel and sophisticated diagnostic instruments. The environmental, clinical and foodstuff monitoring or the detection of bioterrorist agents requires novel techniques that have to be very sensitive but also fast and easy in performance. We will analyze these possibilities.

13.1 Detection and Identification of Vegetative Bacteria As can be seen in Fig. 13.1, bacteria are the living bodies of complicated structure and well-specialized distribution of functions. Nucleoid is the region of the cytoplasm where the DNA is located in stranded form. Plasmids are autonomous circular DNA molecules that may be transferred between bacteria (horizontal gene transfer). Ribosomes are the complexes of RNA and protein that are responsible for polypeptide synthesis. Some bacteria possess both cell membrane (semi-permeable and selective barrier surrounding the cell) and cell wall (the rigid outer covering made of peptidoglycan, maintains shape and prevents bursting (lysis). Slime capsule is a thick polysaccharide layer used for protection against dessication (drying out) and phagocytosis. Pili are hair-like extensions that enable adherence to surfaces

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(attachment pili) or mediate bacterial conjugation (sex pili). Movement of bacteria is provided by flagella—the long, slender projections containing a motor protein. Observation and functional description of such complicated structures is needed for development of the most efficient methods for their detection. These methods can be roughly classified into phenotypic (studying the cell morphology), enzyme-specific (based on biochemical markers), surface-recognizing (using molecular receptors) and genotypic (based on manipulation with its nucleic acid sequence).

13.1.1 The Whole-Cell Detection The role that the sensors must play in analysis of microbes is primarily in simplification and speeding up the assays that presently are quite unsatisfactory. The major problems are the duration and cost of analysis, involvement of costly equipment and of highly professional personnel (Fournier et al. 2013; Van Belkum et al. 2013). Colony growth in culture media remains the “gold standard” for the identification of the presence of bacteria. Existing microbiology-based methods for bacterial identification and antibiotic resistance testing rely on monitoring cultures that contain high numbers of bacterial cells and require the growth of the culture to progress until sufficient numbers of cells can be achieved for analysis. Conventional microbiological classification tests usually involve visual microscopy after staining with dyes. This is a very accurate procedure but with many drawbacks. First, it is time-consuming requiring tedious cultivation for several (2 to 7) days before the results are obtained, which cannot be tolerated. Second, the nature of the method does not lend itself to integration and miniaturization into high-throughput microanalytical systems. Third, it requires trained personnel to interpret the results for providing visual analysis of only one bacterial pathogen at a time. The application of digital microscopic cell imaging is possible, allowing automatic identification of cells according to their morphological characteristics (Hiremath et al. 2013; Kshikhundo and Itumhelo 2016). It suggests only partial solution of existing problems. Single-cell methods have found their way to bacterial analysis (Kelley 2017). Recent advances in single-cell monitoring may provide new solutions that can speed bacterial analysis by monitoring changes in growth on a cell-by-cell level (Li et al. 2017). Most of recent advances are based on microfluidic platforms (Kaprou et al. 2021). Enzyme activity in fluorogenic media is the method used in many clinical laboratories (Váradi et al. 2017). Fluorogenic enzyme substrate is the substrate fused to a dye that is not fluorescent in the bound form. Its fluorescence appears after the substrate is hydrolyzed by unique enzyme that is expressed by the bacterium of interest. Thus, the appearance of fluorescence indicates the presence of bacteria in the sample under investigation. Phage-mediated cell lysis can be used for the release of target enzyme (Burnham et al. 2014). A major drawback with this method is the need for incubation time for producing sufficient bacterial colonies (and thus the enzyme) for the development of color. Positive sign is the possibility to use functional nanoparticles

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Fig. 13.2 Principles for the detection of E. coli bacterial cells using a paper-based handheld culture device (Kumar et al. 2019). The detection is based on release of enzyme β-galactosidase induced by T 4 bacteriophage cell lysis and the detection of fluorescence of its reaction product

that enhance the fluorescence signal to capture and analyze the bacteria (Santopolo et al. 2019). A simple device based on this principle (Burnham et al. 2014) making the assay suitable for on-site analysis is illustrated in Fig. 13.2. Flow cytometry allows studying cells one by one when they pass through a very thin capillary. It can measure several parameters simultaneously with a precision of a few per cent and at a rate of 1000 cells per second (Steen 2000). Usually the fluorescence—light scattering histograms are obtained, in which the cell identity depends on specificity of cell labeling (Müller and Nebe-von-Caron 2010). The method can easily discriminate live and dead bacteria (Ou et al. 2019). A broad variety of fluorescent materials are available to researches, and some of them are specific to studied target cells, including labeled antibodies and cell membrane reactive dyes (Adan et al. 2017; McKinnon 2018; Veal et al. 2000). Complicated equipment and skillful personnel are needed here. Surface imprinting is the technique based on recognition of cellular surface by specially performed configuration of recognition molecules assembled on a support imprinted by a target cell. Such structures are able to preserve molecular memory (see Sect. 7.5). Different methods of cell detection based on the principle of imprinting are developed. They allow imprinting on plasmonic surface for providing fluorescence enhancement and also the use of nanostructures (Mako et al. 2018). The above listed methods are suggested for applications using very small amount of cells in microfluidic format and the detection strategies providing ultrasensitive readout mechanisms (Kaprou et al. 2021).

13.1.2 Detection by Characteristic Features of Cell Surface Exposing on its surface different molecular structures with their characteristic properties makes the surface of bacterial cell very characteristic and very recognizable. Even within one cellular subtype, different cells often display slightly different exterior

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surfaces; therefore, the sensors with flexibility to recognize this degree of variability may be of diagnostic value. For our purpose, it is important that these features are resolved by different tools that are used for recognition of much smaller molecular targets (see previous chapters). Cell membrane and/or cell wall that are exposed to outer environment can serve as the field for different target. In view of its large dimensions on molecular scale, the recognition sites can be rather small, just fitting the capacities of aptamers (Sect. 5.2) and antibodies (Sect. 4.4). Therefore, many assays, mostly immunoassays, were suggested for practical use. In this way, we can detect the presence of specific proteins and peptidoglycans displayed on or near the cell surface (Bertsche et al. 2015; Hernández and Cava 2021; Irazoki et al. 2019; Tosoni et al. 2019). Antibodies against surface cell receptors are the traditional tools for bacterial cell recognition. The sensing is mostly based on well-established sandwich assays and sensor arrays. Figure 13.3 illustrates the strategy to identify the surface antigen that can be protein or glycan. Then, different types of antibodies or their recombinant functional fragments are synthesized (see Sect. 4.4), for which the optimal sensing platform and assay format can be selected. Critical analysis of these assays can be found elsewhere (Byrne et al. 2009; Sapsford et al. 2006; Skottrup et al. 2008). Nucleic acid aptamers described in Sect. 5.2 are short (20–60 nucleotides in length) single-stranded RNA or DNA sequences that are very efficient as molecular receptors. They achieve their increased efficiency by a procedure that involved alternate cycles of their selection from a pool of random sequences and an amplification of the bound species. This process is termed as systematic evolution of ligands by exponential enrichment (SELEX). The aptamers can bind with various targets, ranging from inorganic molecules to protein complexes, but also with whole cells. Aptamers are often called the nucleotide analogues of antibodies because they can be used in the same manner as antibodies (Bauer et al. 2019; Zhang et al. 2019b). Immunosensor-based analysis

Proteins Glycans

Polyclonal Monoclonal Recombinant

Flat arrays Nanocomposites

Antigencapture Sandwich

Fig. 13.3 The common strategy in immunosensors-based analysis of bacteria. For sensing the surface-exposed proteins and glycans the fluorescence-based platforms are developed for the monitoring of bacterial pathogens by incorporating monoclonal, polyclonal or recombinant antibodies in a variety of assay formats

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Fig. 13.4 Schematic illustration of the bacterial whole cell SELEX process in combination with qPCR-based and NGS tools (Kolm et al. 2020). Eleven SELEX rounds of positive selection with E. faecalis cells as target were performed. In rounds R03, R04, R05, R06 and R09, subtractive counter-selections were carried out with mixtures of enterococcal and non-enterococcal species, respectively, which were incubated with the enriched single-stranded ssDNA pool prior to positive selection

Fig. 13.5 Tethering antimicrobial peptides (AMPs) can prevent the triggering of killing mechanisms of the bacteria (Pardoux et al. 2020). a Free floating peptides can self-organize onto the bacterial membrane and subsequently disrupt it or insert themselves inside the cell in order to kill it. b Tethered peptides can no longer self-organize at the surface of the bacterial membrane, thus inhibiting their bactericidal activity

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Their popularity in pathogen detection increases rapidly (Vishwakarma et al. 2021) (Fig. 13.4). The reason for the increasing popularity of aptamers in the construction of aptamer-based sensors (aptasensors) is because they are economical to manufacture, thermally stable, and non-immunogenic (Bauer et al. 2019). They can be easily modified by conjugation with various molecules serving as sensor transducers and reporters (Odeh et al. 2020) to detect potential pathogens in water. Therefore they are prospective for use in point-of-care and point-in-field devices (Yoo et al. 2020). Comparison of properties of antibodies, aptamers and imprinted polymers that are valuable for environmental monitoring can be found in ref. (Naseri et al. 2020). Antimicrobial peptides are different types of recognition units (Hoyos-Nogués et al. 2018; Pardoux et al. 2020). They can be found in bacteria, plants, and higher and lower animals and exert their antimicrobial activity by binding to the microbe surface and disrupting its membrane. Their selectivity is not due to interaction with specific receptors but is determined by the difference in lipid compositions of the membranes and by their cationic and amphipathic character (Bobone and Stella 2019). Their recognition power to detect E. coli and Salmonella was demonstrated (Ngundi et al. 2006) (Fig. 13.5). An interesting approach for the detection of bacteria is the use of carbohydratefunctionalized fluorescent polymers (Disney et al. 2004), which allows exploring multiple interactions. Fluorescent polymers (Sect. 8.5 of Volume 1) can be modified with different functional substituents, which displays many carbohydrate ligands on a single polymer chain and allows observing brightly fluorescent aggregates of bacteria. Combination of fluorescent polymer and antimicrobial peptides allows using quenching effect of the latter for discriminative sensing of bacteria (Han et al. 2017). New nanoscale materials such as carbon dots (Chap. 9 of Volume 1) allow achieving with a very simple synthetic procedure a high sensitivity together with discriminating ability in sensing microbes, providing strong modulation of their fluorescence (Yang et al. 2019). For instance, they can easy identify the Gram-type of bacteria (Yang et al. 2019). They can not only discover but efficiently kill them (Dong et al. 2020; Lin et al. 2019). Summarizing, the peculiarities of bacterial cell surface really exist, they are sufficient for recognition of not only the type of bacteria. Even within one cellular subtype, different cells often display slightly different exterior surfaces. Therefore, a sensor with flexibility to recognize this degree of variability is possible. The new materials, concepts and ideas that are exploited in other areas of sensing science and technology are actively used in sensing bacteria (Liu et al. 2020c) with increasing implication of nanoscience (Bhardwaj et al. 2019; Shen et al. 2021). In addition to fluorescent polymers and carbon dots, plasmonic nanoparticles are applied for enhancement of emission intensity (Fothergill et al. 2018). The composite nanoparticles with λratiometric fluorescence response to bacterial target binding allowed achieving their detection on a single-cell level (Shen et al. 2020). Also applicable are the sensor arrays based on optical nose/tong concept (Mungkarndee et al. 2015). These arrays are cross-reactive and not intrinsically selective, but they are often based on stable

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small molecules and provide more flexibility. Thus, the simultaneous detection of multiple pathogenic bacteria (Shen et al. 2021) becomes a reality.

13.1.3 Detection Based on Bacterial Genome Analysis Now we switch from phenotypic to genotypic determination of bacteria. Analysis of bacterial genome becomes more and more simple, and the amount of sample with respect to sensitivity of the method becomes a key factor. Signal amplification on a sample level can be achieved with the application of PCR and related techniques. Polymerase chain reaction (PCR) is frequently used for identification and quantitative evaluation of bacteria (Garibyan and Avashia 2013). PCR is a nucleic acid amplification technique that is based on the isolation, amplification and quantification of a short DNA sequence. Among the developed PCR methods applicable to analyze genetic material of the target bacteria are: real-time PCR (RT-PCR), multi˙ 2021). PCR is much plex PCR and reverse transcriptase PCR (RT-PCR) (Zukowska less time consuming than other conventional techniques utilizing culture and plating. It takes about 5 to 24 h to produce a result, but this depends on the specific version of PCR. PCR technique allows amplifying a specific DNA fragment from a complex pool of DNA, and this enriched material becomes available to different types of analysis. PCR assay requires template DNA, primers, nucleotides and thermostable DNA polymerase (Taq polymerase) enzyme. PCR proceeds through three main phases; denaturation, primer annealing and primer extension (Fig. 13.6). Different synthetic cycles are performed, in which the temperature cycling with the increase above melting point of DNA is applied to denature the targeted double-stranded DNA sample (Garibyan and Avashia 2013). The DNA is then purified followed by an extension phase using primers and a thermostable polymerase. Exponential amplification is attained in subsequent steps, so that each new double-stranded DNA acts as a target for a subsequent cycle (Rebrikov and Trofimov 2006). The primers are extended from 5' to 3' direction by polymerase enzyme to overlap the copies of original template. Subsequent detection of the amplified sequence is performed using gel electrophoresis or chromatography. Recent advance in PCR technology is the real-time PCR, in which faster results can be obtained through the detection of fluorescent emission (Garibyan and Avashia 2013). Visualizing the PCR products can be performed by staining of the amplified DNA product with a chemical dye such as ethidium bromide, which intercalates between the two strands of the duplex. The labeling of PCR primers or nucleotides with fluorescent dyes prior to PCR amplification is also possible. Fluorescence intensity is directly proportional to the amount of amplified product and, therefore, it is possible to follow this response in real time eliminating laborious post-amplification processes. One must recognize the limitations of PCR techniques (Garibyan and Avashia 2013) that may lead to false-positive results (Derveaux et al. 2010). They are the following. (a) The DNA polymerase used in the PCR reaction is prone to errors and

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Fig. 13.6 Schematic of the polymerase chain reaction (PCR) used to amplify nucleic acids that are present in an isolated sample

can lead to mutations in the fragment that is synthetized. (b) The specificity of the generated PCR product may be altered by nonspecific binding of the primers to other similar sequences on the template DNA. (c) In order to design primers to generate a PCR product, some prior information on the sequence is usually needed. Since the multi-step reagent addition and temperature cycling are involved, there is a need for rather sophisticated instruments, being not suitable for in-field analysis (Váradi et al. 2017). Isothermal DNA amplification, eliminating the need for temperature cycling, simplifies the technique and reduces the time of analysis (Glökler et al. 2021; Váradi et al. 2017). Several such methods have been developed, such as strand displacement amplification (SDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification (HDA) (Craw and Balachandran 2012). These methods have paved the way for the implementation of rapid, nextgeneration molecular diagnostics (Becherer et al. 2020; Martzy et al. 2019). The complex reaction mechanisms and the need of several primers or several enzymatic steps are the weak points of these techniques. Although LAMP needs a complex primer design, it is the most popular among these methods. However, in contrast to techniques based on thermal cycling, the methods based on isothermal DNA amplification can be used in microfluidics (Zanoli and Spoto 2013); they allow constructing simplified biosensor devices (Leonardo et al. 2021). Multiplexed isothermal amplification and detection (Mayboroda et al. 2018) is a new step in development. Whilst there are several very elegant isothermal amplification approaches, multiplexed amplification remains a challenge, requiring careful experimental design and optimization, from judicious primer design in order to avoid the formation of primer dimers and non-specific amplification. Still, the recent data show that parallel multiplexing can be achieved. The DNA microarray technique is a method possessing high potential in identifying particular pathogen but without strong attempt to achieve its quantitative

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measure. This approach relies on the detection of the presence or absence of genes in a target organism when compared to a reference strain or genome. In DNA microarrays, numerous specific DNA probes relying on reference genes present in a characterized strain and comparative genomic hybridizations are performed followed by the analysis of the hybridization results. In this way, DNA microarrays offer the potential for simultaneous detection of many pathogens (see Sect. 5.1). DNA microarrays can be coupled with multiplex PCR amplification for production of more copies of the target and in this way to increase the sensitivity of testing (Call 2005), but the challenge is to avoid it and to obtain the result based on direct sensing. Direct hybridization of DNA or RNA provides the least bias in gene detection but also the lowest level of analytic sensitivity that may depend on fluorescence response. DNA microarrays are interesting in view of prospective applications as point-of-care testing systems. DNAzymes can demonstrate together with molecular recognition function (RNAcleaving DNAzyme) the signaling mechanisms, such as peroxidase mimicking DNAzyme, that are simple and efficient for pathogenic bacteria detection, including fluorescence and color recording (Ma et al. 2021). DNAzymes are compatible with isothermal amplification technology for pathogenic bacteria detection. Enhancement of fluorescence by plasmonic nanoparticles can be used to amplify the output signal (Yu et al. 2017). Presently the genes of virtually all clinically relevant common pathogens have been fully sequenced, and the design of specific probe sequences to target antibiotic resistance genes and subtyping of bacteria can be performed rapidly. The detection system can be indirect, modulating the DNA polymerase activity (Fig. 13.7). The complete detection system (Park et al. 2016) is based on measuring the changes in fluorescence anisotropy when detection probes recognize target bacterial nucleic acids. Whereas nucleic-acid-based rapid diagnostic tests are definitely more sensitive and specific than antibody-based detection, PCR requires the use of appropriate primers and a relatively clean sample. These detection tests cannot determine whether a bacterium is alive or dead and since it is a destructive technique, archiving the organism for subsequent studies is often not possible. Summarizing, it can be stated that the current tendency is to achieve the integrated and automated platforms based on obtaining genetic information that is unique for every bacteria. The attempts are to do that in the most practical way, approaching a broad range of applications by integration and automation of processes, such as nucleic acid extraction, purification, amplification, and detection, coupled with sophisticated data analysis software. Since PCR-based methods have limited multiplexing ability and therefore are difficult to apply to large families of sequences, there is an additional motivation in development of ultrasensitive amplification-free methods.

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Fig. 13.7 Polarization anisotropy diagnostics (PAD) system based on target-modulated DNA polymerase activity (Park et al. 2016). a Assay procedure. Bacteria are lysed, and total RNA is extracted. Following the RT-PCR amplification, samples containing amplicons and DNA polymerase are incubated with an all-in-one master mix that has the detection key and the reporter. The resulting fluorescence anisotropy of the sample is then measured. b Photograph of a disposable RNA extraction cartridge made in plastic. The device has an RNA extraction chamber packed with glass beads (inset). c Photograph of a portable system for fluorescence anisotropy detection. Four separate optical cubes can be plugged into an electronic base station. d PAD measurement is controlled through a custom-designed application in a smartphone. The PAD device and the smartphone communicate via Bluetooth

13.2 Discovery and Recognition of Bacterial Spores Many harmful microbes form protective spores (endospores) that are highly resistant to heat, salinity, acidity, radiation, oxygen, water depletion, and so on (Setlow and Johnson 2019). This remarkable feature permits spores to remain viable for a long time. The resistance properties of bacterial spores lie at the heart of their widespread occurrence in food ingredients (Setlow and Johnson 2019). They survive during extremely long periods of time under even extreme conditions and transform into active microbes (the process called germination) when the conditions become favorable for their living and population growth (Christie and Setlow 2020). Such metabolically dormant forms can be very dangerous. The spores are composed of DNA, the DNA-stabilizing agents (such as calcium dipicolinate) and the protecting shell (Fig. 13.1b). This shell can be recognized by fluorescence sensors. The spores of various Bacillus and Clostridium species are among the most resistant life forms known (Setlow 2014). Since the spores of some species are causative agents of much food spoilage, food poisoning, and human disease, and there were attempts to use the spores of Bacillus anthracis as a biological weapon, there is much interest in the mechanisms of spore resistance and how these spores can be inactivated. Despite their extreme resistance, spores can be inactivated by damaging the

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DNA, crucial spore proteins, the spore’s inner membrane, and one or more components of the spore germination apparatus (Cortesão et al. 2019; Ulrich et al. 2018; Wells-Bennik et al. 2016). An example of fluorescence assay for the detection of Bacillus thuringiensis (BT) spores is based on aptamer-quantum dot binding (Ikanovic et al. 2007). The SELEX technique was used for selecting and identifying the DNA aptamer sequence specific for BT. The 60-base aptamer was then coupled to fluorescent zinc sulfide-capped CdSe quantum dots. The assay is semi-quantitative, specific and can detect BT at concentrations of about 1,000 colony-forming units/ml. Detection of Bacillus anthracis spores (anthrax) has received much attention due to their high potential danger and the attempts to use them as biological weapon. Here a very high sensitivity can be achieved due to application of nanocomposites with dual fluorescence, in which the lanthanide binding to spore biomarker indicates the spore presence, as it is seen in examples discussed below. Dipicolinic acid (DPA) is commonly used as an anthrax spore biomarker, representing 5–15% of its content. Different fluorescence nanocomposites were developed for providing its detection. A dual-emissive metal–organic framework (MOF) hybrid, which is formed by encapsulation of Tb3+ cations into an anionic MOF, was used for detecting DPA in a λ-ratiometric manner (Zhang et al. 2018). The wavelength ratiometry allowed providing self-calibrating and background-free determination of DPA. Other λ-ratiometric fluorescence sensors used europium (Eu3+ )-doped silicon nanoparticles with the sensitivity that allowed using the test paper (Na et al. 2020). The fluorescent color-changing test paper was also devised based on Eu3+ -doped carbon dots (Wang et al. 2020). In these cases, upon addition of DPA, the intrinsic luminescence of lanthanide ions can be sensitized due to coordination with DPA chromophore. The fluorescence of MOF or carbon dots served as the reference, the fluorescence of which remained essentially constant. A smartphone-integrated detection scheme was realized on competition for Eu3+ binding with its chelating dye and DPA (Xu et al. 2019) and also when using the Eu3+ -MOF system (Jia et al. 2021). Figure 13.8 demonstrates one of the results on application of carbon dot nanoparticles coupled with Tb3+ ions for the determination of DPA (Zhang et al. 2019a).

13.3 Identification and Analysis of Biofilms A biofilm can be defined as a community of microorganisms adhering to a surface and surrounded by a complex matrix of extracellular polymeric substances (EPS) (Lewandowski and Beyenal 2019), see Fig. 13.1c. Biofilms are widespread in nature and constitute an important strategy implemented by microorganisms to survive in sometimes harsh environmental conditions (Azeredo et al. 2017). It is now generally accepted that the biofilm growth mode induces microbial resistance to disinfection that can lead to substantial economic and health concerns. Although the precise origin of such resistance remains unclear, different studies have shown that it is a

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b

c F545/F445 ratio

Fluorescence intensity

a

Wavelength (nm)

Concentration of DPA (μM)

Fig. 13.8 The application of carbon dots incorporating Tb3+ ions for the determination of dipicolinic acid (DPA) as the anthrax biomarker (Zhang et al. 2019a). a Schematic diagram demonstrating the nanoprobe for DPA recognition. b Fluorescence response of nanoprobe upon the addition of DPA (λex = 270 nm). c Ratiometric calibration plot of F 545 /F 445 fluorescence intensity ratio on DPA concentration

multifactorial process involving the spatial organization of the biofilm (Bridier et al. 2011). In most wet environments, microorganisms are able to adhere to a surface, producing a matrix of protecting extracellular polymeric substances (Saxena et al. 2019). A sticky and strong EPS matrix protects them (also from antibiotic treatment). It mainly comprises polysaccharides, proteins, lipids, and DNA (Flemming et al. 2007; Karygianni et al. 2020). Functionally active bacteria can be released from biofilms (Fig. 13.9). Fig. 13.9 Biofilm formation and development stages (Carrascosa et al. 2021)

Planctonic bacteria

Maturation and dispersal Attachment

Microcolony formation

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Biofilm-associated infections constitute a great problem for medicine. Many untreatable chronic diseases affecting millions of people are biofilm-related (Bjarnsholt 2013). Dental plaque has the properties of a biofilm, similar to other biofilms found in the body and the environment (Jacob 2006). Biofilms are largely responsible for food spoilage and outbreaks (Carrascosa et al. 2021). They damage industrial facilities by corroding pipes, blocking filtration membranes, and fouling marine surfaces. However, there were attempts to use biofilms in a positive manner by forming protective layers on some industrial products when they are exposed to extreme environments (Yin et al. 2019). Therefore, efficient tools are needed to detect biofilms, to control over their development and to test the factors that could destroy them. Sensing and imaging the biofilm-encased microorganisms is challenging because of their shielding by a sticky and strong extracellular polymeric substance matrix that blocks to a significant extent the penetration of dyes or other molecular sensors. Microbiological culture-based studies of replicas from biofilm surface (Branda et al. 2005) and enzyme-linked generation of color (Ripolles-Avila et al. 2018) are the major methods presently used for biofilm detection. Confocal microscopy (see Sect. 15.1) is presently the major method to study the structures of biofilm based on assortment of fluorescent dyes that allow their staining. These studies address the problems of biofilm formation, the matrix properties, the composition of living and dead resident cells and resistance to disinfectant actions (Bridier et al. 2011; Reichhardt and Parsek 2019). The studies of matrix composition and of its properties (such as their localization and interaction between them) is of special concern (Reichhardt and Parsek 2019; Schlafer and Meyer 2017). DNA is an important constituent of the matrix participating in adhesion, aggregation and penetration reduction, and its staining in the matrix can be achieved (Boháˇcová et al. 2019; Svarcova et al. 2021). Regarding the studies of biofilm specificity and sensing within biofilm structures, only a modest success was achieved. Such situation is related to the problem of sensor penetration into biofilm structure. In the recent study, a collection of fluorescently labeled peptides was assembled to specifically explore their biofilm targeting properties (Locke et al. 2020).

13.4 Detection of Toxins Harmful bacteria can be detected by the products that they synthesize. These products are the toxins. Toxins are the poisons that are produced biologically and originate from diverse sources including animals, plants, and microbes. This refers not only to the most dangerous infections (e.g. cholera). For instance, staphylococcal enterotoxins are a major cause of food poisoning (Witczak and Sikorski 2017). Other toxins are potential biological weapons, and in hands of criminal can be used for bioterrorist attacks (Anderson 2012; Janik et al. 2019).

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Fig. 13.10 Chemical structures of selected low-molecular toxins: A trichothecene mycotoxin, B tetrodotoxin, C saxitoxin, and D brevetoxin B, the ladder-shaped polyether of 11 rings (Walper et al. 2018)

Toxins are chemically diverse. They can be proteins, such as botulinum toxin, staphylococcal enterotoxins, Clostridium perfringens toxins, ricin and abrin, are proteins (Janik et al. 2020), whereas others are small-molecular species (Walper et al. 2018), see Fig. 13.10. Depicted in Fig. 13.10 mycotoxin represents a class of toxins produced by fungi. Tetrodotoxin (TTX) is a deadly toxin found in fugu fish, which is popular in traditional Japanese cuisine. Contrary to popular belief, TTX is not endogenously produced in the fish but rather by the presence of either symbiotic or infective bacteria. The lethal amount of this substance found in those fish, requires very specialized licensed chefs to prepare it for consumption. Saxitoxin was first isolated from the Saxidomus butter clam but actually constitutes more than structurally related compounds typically found in shellfish that have been contaminated by toxic algal blooms. Brevetotoxin also derives from shellfish. Harvesting and consumption of infected mollusks provide its entry point into the food chain. Here the role of sensor technologies is not only to provide rapid and sensitive tests but also to avoid extensive pretreatment or concentration of the sample prior to analysis. Though not fully conforming to these needs, immunosensor arrays based on immobilized antibodies have become the common approach for the detection of toxins (Cunningham et al. 2022; Rucker et al. 2005), and the most popular for detection of small-molecular toxins is the competition format (Ngundi et al. 2006). Immunosensor arrays can be assembled on waveguides where the antibody molecules are first immobilized in specific locations on the waveguide and the resultant patterned array was used to interrogate different samples for the presence of multiple different analytes (Ligler et al. 2003). Upon binding of a fluorescent analyte or fluorescent immunocomplex, the pattern of fluorescent spots can be detected using a CCD camera.

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The array formats can also be realized with the application of signaling aptamers (Guo et al. 2020; Zhang et al. 2020b) and these formats can become quite sophisticated. Simple design, easy operation, fast response, low cost, and analytical performance can be realized in this way. One has to account, however, that only those toxins can be found and analyzed, for which the proper binders are displayed on microarrays. Among the toxins that are the major pathogenicity factors involved in severe diseases in humans and animals and produced by numerous bacteria are the proteins. In addition to botulin neurotoxins there are abrin, staphylococcal enterotoxin B and other very dangerous species (Bozza et al. 2015; Bradberry 2016; Janik et al. 2019). Classical way for their detection is the mouse lethality assay based on survival of mice after target addition. Cellular-based toxicity assays are also applied (Boldt et al. 2006). To be useful, analytical tools should perform on a similar level of sensitivity, or better. Botulin neurotoxins with a median lethal dose (LD50 ) of 1–5 ng/kg are the most poisonous substances known (Montecucco and Molgó 2005). They are proteins with a single polypeptide chain of ~ 150 kDa (Fig. 13.11) that are synthesized as proproteins by the Clostridium botulinum, which are the gram-positive, spore forming and anaerobic bacteria. Their active form is composed of a heavy chain linked to a light chain via disulfide bond. The protein binds selectively and irreversibly to nerve terminals and thus prevents neurotransmission by inhibiting the release of acetylcholine. Foodborne botulism is a life-threatening disease caused by the ingestion of food containing preformed botulin neurotoxins; it is considered a public health emergency (Scalfaro et al. 2019) and potential bioterrorist threat (Janik et al. 2019). The most common format for the detection of these toxins is the exploitation of a fluorescently labeled antibody with recognition specificity towards a given toxin when they are applied in some type of immunoassay, be it direct, indirect, sandwich, ˇ etc. The most popular here are the sandwich assays (Capek and Dickerson 2010; Hobbs et al. 2019) with different variations, including the substitution of capture antibodies by fluorescent peptide binders (Ma et al. 2006). Fig. 13.11 Structure of botulin neurotoxin, type A. The crystal structure of protein (PDB: 3BTA) (Lacy et al. 1998) taken from the RCSB PDB databank and reproduced in ref. (Janik et al. 2019)

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Fluorescent sandwich immunoassays have been also performed on beads with flow ˇ cytometry instrumentation used to detect and quantify toxins (Capek and Dickerson 2010). This can be done in multiplex with the detection of other toxins (e.g., cholera toxin, ricin and staphylococcal enterotoxin B (SEB)) using different color-coded beads. The use of magnetic beads in flow cytometry was shown to have several advantages including the possibility of involvement of a pre-concentration step, resulting in increased sensitivity. The analysis of turbid or heterogeneous samples becomes possible as beads can be easily separated from the matrix. An automatic fluidic system format can be applied. Before the flow cytometric analysis, the beads with a capture antibody were trapped in a flow cell, where the capture, washing, and binding of detection antibody were performed under the control of a system. The benefits of flow cytometry assays over common ELISA sandwich immunoassay are the easier to automate and to interrogate the samples for the presence of multiple toxins or serotypes in one test tube; there are better capture kinetics and enhanced possibility to concentrate analyte. The detection of often very small concentrations of target may require substantial increase of test sensitivity. The amplification of signal sensitivity in sandwich assays occurs on the level of capture (indicator) antibody. In ELISA-type techniques the attached enzyme can be used that produces accumulating light-absorbing or fluorescent product. Several additional possibilities were suggested (they will be discussed in Sect. 13.6). One of them is immuno-polymerase chain reaction (Immuno-PCR) that is an ELISA-type immunological test using PCR to increase amplification of the ELISA signal. The detection methodology is like in common ELISA relying on forming the complexes of antigen and antibody. But instead of the normally used enzyme format, the capture antibody binds to known DNA molecules. Once the binding has occurred, the amplification of the DNA fragments can be achieved, like in common real-time quantitative PCR (qPCR) (Niemeyer et al. 2005). This approach was applied for diagnosis of different bacterial and viral infections (Barkova et al. 2019), particularly for ultrasensitive detection of botulin neurotoxins (Hobbs et al. 2019). The other is the liposome-PCR test. It is an ultrasensitive immunoassay for detecting toxins that uses a liposome with encapsulated DNA reporters, and gangliosides embedded in the bilayer, as the detection reagent (Mason et al. 2006a). After immobilization of the target toxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the DNA reporters, which are amplified and quantified by the real-time PCR. Finally, an intrinsic metalloprotease activity of botulin neurotoxins can be measured with a fluorogenic substrate after enrichment on a large immunosorbent surface area (Bagramyan et al. 2008). It was reported that this method has high specificity for the targeted type A toxin and reaches attomolar sensitivities in serum, milk, carrot juice, and in diluent fluid used in the mouse assay. Further increase of sensitivity can be achieved by concentrating the fluorescent reaction product on silica beads by evaporation in microfluidic platform and detecting using microscopy (Frisk et al. 2008).

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Nanocomposites of different origin are actively used for detection of toxins (Bhardwaj et al. 2019, Burris and Stewart Jr 2012). Thus, quantum dots are useful for multiplex detection (Ganesan and Nagaraaj 2020), since they emit fluorescence of different colors depending on their size and the same procedure can be used for immobilization of antibodies. Lanthanide-doped nanoparticles were suggested as fluorescent probes (Tang et al. 2009) with the use of specific aptamers against the target toxins (Huang et al. 2015). Gold nanoparticles can be used as reporters in two ways, by their aggregation-dependent change of color (Verma et al. 2015) or as plasmonic enhancers of emission of reporting fluorophores (Yue et al. 2016).

13.5 Sensors for Viruses Viruses are the sub-microscopic infectious agents that are able to grow or reproduce only inside a host cell. Each viral particle consists of genetic material in the form of DNA or RNA and a protective coat made of proteins, called a capsid. Both these nucleic acids and capsid proteins can be the targets of viral infection. Of diagnostic importance are also the antibodies raised in the organism in response to virus infection. Influenza viruses are among the most common causes of respiratory tract infections in humans (Barik 2012; Medina and García-Sastre 2011). They cause seasonal epidemics in the winter months and global pandemics resulting in high morbidity. All influenza viruses are enveloped, negative-sense single-strand RNA (ssRNA) viruses. They demonstrate magnitude of mutated forms, one of which is the introduction of a glycosylation site on the antigenic globular head of influenza hemagglutinin (Medina et al. 2013). The human immunodeficiency virus (HIV) is a positive-sense ssRNA virus. It is the primary cause of acquired immune deficiency syndrome (AIDS), which causes significant morbidity and mortality with a significant consequent decrease in the quality of patient’s lives. The absence of its efficient treatment is a great medical and social problem. Its early detection is an extremely important problem in all efforts to prevent epidemic propagation of AIDS disease. Ebola viruses are responsible for the large outbreaks of disease in Africa (Goeijenbier et al. 2014; Osterholm et al. 2015). Their RNA are linear, non-segmented, negative stranded structures. The enveloped EBOV virion consists of a nucleocapsid complex, surrounding matrix and coating envelope (Kost 2018). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the coronavirus disease COVID-19, which is an ongoing worldwide pandemic and a global health threat. Pandemic spread of COVID-19 provided a great stimulus for the development of new methods of virus molecular diagnostics, particularly for point-of-care detection (Choi 2020; Etienne et al. 2021; Gowri et al. 2021). Being similar to previously identified SARS-CoV and the Middle East Respiratory Syndrome coronavirus (MERS-CoV). SARS-CoV-2 is an enveloped, non-segmented, positive sense RNA β-coronavirus. Its S-protein is the major target for antibody-based virus detection.

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Distinct fluorescence spots derived from single virus particles can be observed with the mobile imaging platform, which allowed digital virus counting (Minagawa et al. 2019). The number of detected fluorescence spots showed good linearity against the influenza virus titer, suggesting that high sensitivity and quantification were achieved. Fluorescence imaging allows tracking of the infection pathway in living cells (Liu et al. 2020b). Based on single-molecule imaging, the real-time visualization of single viruses, each labeled with only one fluorescent dye molecule, can be tracked in cells (Seisenberger et al. 2001). Diffusion trajectories with high spatial and time resolution show various modes of their motion during their infection pathway into cells (Fig. 13.12). This process is surprisingly very fast. Fighting for healthy life is to a significant extent the fighting with pathogenic viruses. In biological fluids, viruses can be identified in two ways. One is based on recognition of nucleic acids that represent their genome and the other relies on their behavior as antigens exposing the sites that can be recognized by specific receptors, such as antibodies. Both approaches are used in the analysis of clinical samples. One of examples signifying rapid progress in the field of detection of viruses is the application of fluorescent silver nanoparticles and DNA templates (Fig. 13.12). Further developments may involve exploitation of labeled DNA aptamers as recognition and detection units.

Fig. 13.12 Potential platform for detection of various viruses using the sensors based on silver nanoclusters (AgNCs) and the generation of different fluorescent readouts, which rely on the fluorescence enhancement (turn on), quenching (turn off), or shift (color change) (Li et al. 2021a). SARS-CoV-2 and influenza virus images were originally produced by the Centers for Disease Control (CDC), USA. bAgNCs, gAgNCs, and rAgNCs indicate blue, green, and red fluorescent AgNCs, respectively. The various readouts (filled with blue, green, and red colors) indicate the sensing signals are obtained from different fluorescent AgNCs

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13.5.1 Nucleic Acid Based Detection Nucleic acid based tests target directly the signature regions of RNA viruses and, in principle, provide their molecular diagnosis with the highest accuracy and sensitivity (Carter et al. 2020; Habibzadeh et al. 2021). Very low virus quantities that are usually seen in tested samples often require the amplification. It can be made using reverse transcription quantitative PCR (RT-qPCR) with the fluorescence-based detection (Arya et al. 2005), which is considered today as the gold standard for accurate, sensitive and fast measurement of gene expression. In a typical nucleic acid test, target viral RNAs are transferred to cDNA via the reverse-transcription process, followed by either PCR or isothermal amplification to obtain abundant copies of target sequences. Providing high sensitivity, nucleic acid amplification methods also allow achieving high sequence signature selectivity and discriminating between nucleic acid strands with only minor differences. Essentially, these methods allow a direct detection of virus on an early step of infection, when the detection based on immune response of the host body is still absent. It is not a proper place to analyze here a number of suggested (and FDA approved) in vitro DNA and RNA pathogen diagnostic tests. One may check the site https://www.fda.gov/medical-devices/in-vitro-diagnostics/nucleicacid-based-tests#microbial, see also (Falzone et al. 2021; Ghaffari et al. 2021; Guglielmi 2020). Many of these tests have been commercialized. They assay very low (down to femtomolar) levels of pathogens in a range of biological samples, including sputum, nasal swaps, serum, and whole blood. Remarkable are those technical solutions that offer portable devices approaching the need for massive screening and point-of-care performance. The requirement for temperature cycling in RT-PCR is a great limitation in reducing the size and cost of devices, but it can be overcome by applying thermoplasmonics, a method for fast light-to-heat conversion mediated by light-illuminated plasmonic nanoparticles (Cheong et al. 2020). Thus, thermal cycling can be provided on nanoscale level, bringing to life new generation of miniaturized devices (Mohammadyousef et al. 2021; Wu et al. 2021). There are many attempts to find alternatives to PCR-based assays (Li et al. 2021b). An important step in development of sensitive, simple and fast nucleic acid diagnostic tests for virus diseases was the demonstration of assay based on a sustained isothermal reaction cascade producing an RNA aptamer that binds to a fluorogenic dye (Woo et al. 2020). A DNA ligase was used to form the ligation product of a promoter DNA probe and a reporter DNA probe that hybridize with the target single-stranded RNA sequence (Fig. 13.13). The assay was termed sensitive splintbased one-pot isothermal RNA detection (SENSR). Thus, SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR DNA ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that binds to a fluorogenic dye and produces fluorescence only when the target RNA exists in a sample. It was reported that the assay can be performed within 30–50 min of incubation time and can reach a limit of detection of 0.1-attomolar RNA concentration.

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Fig. 13.13 Schematic of SENSR, a one-pot isothermal reaction cascade for the rapid detection of RNA (Woo et al. 2020). The one-pot reaction is composed of four main components: a set of probes, SplintR ligase, T7 RNA polymerase and a fluorogenic dye. In the presence of target RNA, hybridization, ligation, transcription and aptamer-dye binding reactions occur sequentially in a single reaction tube at a constant temperature

Meantime, it has to be noted that in real samples the false-negative results appear with some probability (Kucirka et al. 2020; Mushtaq et al. 2021) because of the errors produced by involved nucleic acid synthetizing enzymes (Deng et al. 2017). In addition, the nucleic acid-based pathogen diagnostics fundamentally fail to differentiate between active and degraded virus species; they can remain in the environment for several days or weeks.

13.5.2 Recognition of Viruses by Antibodies and Aptamers Viruses, like bacteria and their spores, present unique spatial patterns of antigens (epitopes) on their surfaces. They raise immune response in the bodies and can be the targets for their recognition with antibodies and aptamers. In the design of

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immunosensors, such epitopes are recognized as the protein markers and primary sites for sensor design. This orientation on recognition sites is clearly seen in the design of sensors for SARS-CoV-2 virus, where these sites on S- and N-proteins were found and explored (Forni et al. 2021). It is generally thought that compared to nucleic acid tests, antigen-detection rapid diagnostic tests (Ag-RDTs) are more accessible and better fit to on-site detection, but typically have lower sensitivity and specificity (Ebrahimi et al. 2022; Ricks et al. 2021). Different versions of sandwich ELISA-type technique are commonly applied in laboratories and are the basis for different rapid tests. Methods relying on viral antigen detection are expected to experience revolutionary changes with the application of novel fluorescence sensing technologies. They proceed along several lines, one of which is the application of nanotechnologies. Nanoparticles demonstrate several advantages that can be realized in the diagnosis of viruses. The large surface area of nanoparticles is suitable for versatile bioconjugation; it offers multivalency that allows pre-concentration and enrichment of the low virus load. The surface can be modified to ensure high capacity of loading. They can work as fluorescent reporters and enhancers of fluorescence emission. Magnetic nanocomposites allow easy purification and separation. Thus, a digital single virus immunoassay was realized for multiplex virus detection by using fluorescent magnetic multifunctional nanospheres as both capture carriers and signal labels (Wu et al. 2019). The strong magnetic response ability of nanospheres realized efficient capture and separation of targets without sample pretreatment. Due to their distinguishable fluorescence imaging and photostability, the nanospheres enable single-particle counting for ultrasensitive multiplexed detection. Based on multifunctional nanospheres and digital analysis, a digital single-virus immunoassay was proposed for simultaneous detection of H9N2, H1N1, and H7N9 avian influenza virus without complex signal amplification. In fluorescence reporting, nanoparticles and nanocomposites increase the brightness and suppress the background signal; they allow time-resolved and singlemolecular detection. Thus, the application of time-resolved detection of Eu3+ doped luminescence nanoparticles with their extremely long luminescence lifetimes (Vuojola and Soukka 2014) was shown to improve dramatically the sensitivity of adenovirus detection (Valanne et al. 2005). Many developments occurred thereafter, starting from simple antibody labeling to lanthanide emission enhancement coupled with ELISA enzyme product generation (Hagan and Zuchner 2011). Enhanced fluorescence response in immunosensors avoiding enzyme amplification can be achieved by using localized surface plasmon resonance (LSPR) fluorescence enhancement (Hassan et al. 2020; Shrivastav et al. 2021; Takemura et al. 2019). In such immunosensor (Takemura et al. 2017), gold nanoparticles were used to provide fluorescence enhancement of fluorescent quantum dots serving as reporters (Fig. 13.14). They were decorated with different anti-virus antibodies and allowed to interact on virus surface. In this way they generate very strong reporting signal that changes in proportion to the concentration of the target virus. This resulted in ultrasensitive, rapid and specific immunofluorescence sensor for the influenza virus.

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Fig. 13.14 Schematic representation of detection principle for the influenza virus using the localized surface plasmon resonance induced fluorescence nanobiosensor (Takemura et al. 2017). Both quantum dots (QDs) and gold nanoparticles (AuNPs) are derivatized with antibodies to virus surface antigens and can assemble on virus surface. This results in plasmonic enhancement of QDs fluorescence

The receptor functionality demonstrates rapid development. To overcome some of the problems associated with the antibody-based recognition, several other recognition elements based on recombinant antibody fragments (Sect. 4.4) have been proposed for virus detection (Chitray et al. 2020; Torrance et al. 2006). These recognition molecules demonstrate increased stability together with high affinity to the target where they can respond by fluorescence quenching (Dong and Ueda 2021). More efforts are needed to transform them into efficient fluorescence sensors, and promising trend is to incorporate into their structure the wavelength-shifting λ-ratiometric fluorescence dyes that could allow providing the direct sensing technologies (Islam et al. 2019). It is anticipated that some of these technologies will become viable in the future (Demchenko 2023a, b). Aptamers can be raised by SELEX technique (see Sect. 5.2) and demonstrate very high affinity to virus spike proteins (González et al. 2016; Song et al. 2020). They are easier, cheaper and more stable in production and applications than antibodies and therefore they have started to be used as their substitutions in ELISA-type sandwich assays (Zhang et al. 2020a; Zou et al. 2019). They can serve as primary receptors or the labeled detectors instead of secondary antibodies. A manifold increase of efficiency in virus detection on this substitution was reported (Lee and Zeng 2017). A proximity ligation assay can be realized with aptamers. It is based on binding two aptamer probes to the same protein target that brings the ligation DNA region into close proximity, thereby initiating ligation-dependent qPCR amplification. In

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this way, an aptamer-based protein recognition is converted into a detectable qPCR signal using a simple, homogeneous and fast detection workflow in ∼2 h (Liu et al. 2020a). Fluorescent aptasensors are more than aptamers. They are the aptamers that respond to target binding directly by modulation of response of their fluorescence reporting units (W˛edrowska et al. 2020). Such possibility can be realized in several ways exploiting the flexibility of aptamer molecules—their ability to change the conformation on target binding (see Sect. 5.2). This property can be explored in reporting as the change of interaction between fluorophore and quencher, the effects of superquenching, the charge-transfer or proton-transfer generated change of fluorescence emission color. Dynamic quenching can be recorded as the change of time-resolved fluorescence (luminescence) emission, and the molecular or segmental mobility reflected as the change of emission anisotropy. Detailed description of all these possibilities can be found in Volume 1 of this book. Here several examples of their realization are presented regarding the detection of viruses. For the detection of H5N1 avian influenza virus hemagglutinin in human plasma, the aptamers rich in guanine residues were synthesized by SELEX technique and immobilized on the surface of the Ag@SiO2 nanoparticles, which performed as a metal-enhanced plasmonic fluorescence sensing platform (Pang et al. 2015). In the absence of target protein, the dye was free in solution with almost no fluorescence emission. Interacting with the target, these aptamers underwent conformational change, resulting in the formation of G-quadruplex structure. This change allows binding of the dye with fluorescence strongly enhanced by plasmonic effect. This system does not require covalent labeling with fluorophores to the aptamer and the background noise is very low. It looks suitable as a self-contained diagnostic kit for H5N1 influenza virus point-of-care diagnostic, detecting the viral protein in human plasma in a one-step process, as early as 30 min and at a concentration of only 3.5 ng/mL. The aptamers can be integrated with a relatively small microfluidic system. It was proposed to detect three different types of influenza virus, including two type A— H1N1, H3N2—and influenza B virus type with one fluorescent aptamer responding differently to ion concentrations modulating their virus selectivity (Wang et al. 2016). An example of application of unlabeled aptamers to virus protein detection is presented in Fig. 13.15 (Feng et al. 2015). Single-stranded DNA aptamer specific to the hemagglutinin (HA) protein of avian influenza virus was obtained by SELEX and applied to an affinity bioassay. It forms hairpin consisting of two DNA regions, viz. (a) the aptamer for the HA protein and (b) an oligonucleotide designed to form a stem-loop structure. In the absence of target, it maintains hairpin structure and is adsorbed to graphene oxide sheet, so that the fluorescence of SYBR Green I dye is almost quenched. Interaction with the target protein results in unfolding and detachment from the sheet. By applying a polymerase elongation reaction, a long dsDNA product is generated. As a result, the dye interacts with the dsDNA product to produce a strong enhancement of fluorescence response.

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Fig. 13.15 An aptamer-based label-free fluorescence assay for the detection of avian influenza virus by targeting its hemagglutinin protein (Feng et al. 2015), see (Park 2018). This sensing event involves the detachment of aptamer from graphene oxide sheet and action of DNA polymerase. The synthetized dsDNA binds the dye SYBER green I with great enhancement of its fluorescence

Fluorescent nanomaterials (described in Vol. 1 of this book) offer substantial improvement in sensor technologies that also focused on detecting harmful species (Li et al. 2022; Pirzada and Altintas 2022).

13.6 Sensing and Thinking. Future Trends in Pathogen Detection: Single-Particle Sensitivity Versus Signal Amplification Advancement in sensing pathogens and their dangerous products that could allow their easy and efficient in-field and point-of care detection requires finding solutions to several important problems (Kumar et al. 2019). One of them is difficulty in operation with their very low concentrations in the test systems. Microbiological methods allow increasing the bacterial concentration by using culturing in a liquid medium and then proceeding to the analysis of the enriched samples. This cannot be done with toxins or viral samples. The methods based on molecular sensing are more general, but in direct detection the sensitivity may not be sufficient and often needs to be increased by several orders of magnitude by some enrichment procedure. Figure 13.16 illustrates the possibilities to achieve the signal amplifications in sensing that are applicable for analysis of microbes or are prospective for that. The pathogen identification by its genome needs the extraction of nucleic acid and the amplification performed using one of the versions of PCR technique. Beside DNA or RNA, this procedure cannot be applied to any other molecular marker of pathogen. Though it is time-consuming, quite expensive, and requiring a high level of skills, they are the best what presently can be suggested to clinics. They do not fit for testing with immediate response implemented in portable devices. Immunochemistry offers the methods that are much simpler in realization and that can be provided even in microfluidic format (Zhang et al. 2021). The most popular are the ELISA-type sandwich assays that can operate even at very low initial loads due to the possibility of amplification on a detection step. In classical ELISA the

13.6 Sensing and Thinking. Future Trends in Pathogen Detection ...

a

Amplification of target

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Amplification of reporting signal Capture antibody

PCR

Enzyme amplification Immuno-PCR

Detection antibody

b

Liposome-PCR

Amplification by increasing brightness of fluorescence reporter H Q

Q

Superenhancement - superquenching

J Formation of Hand J-aggregates

Localized plasmons

Propagating plasmons

Au

Au

Plasmonic enhancement

Amplified stimulated emission

Fig. 13.16 The possibilities of signal amplification in sensing microbes. a The techniques currently in active use. b The possibilities of enhancement of the output fluorescence signal

amplification of reporting signal is realized by attaching enzyme that produces lightabsorbing or fluorescent product. Other versions suggesting the improvement are immune-PCR and liposome-PCR. Immuno-PCR is an ELISA-type immunological test that uses polymerase chain reaction (PCR) to increase the ELISA signal instead of the normally used enzyme format (Abud et al. 2019; Dahiya and Mehta 2019). The detection antibody is bound to known DNA molecules, and once the binding has occurred, the amplification of the attached DNA fragment is provided by real-time quantitative PCR with fluorescent dye labeling of the formed PCR product. There are many interesting applications of this method in detection of microbial targets (Barkova et al. 2019) and toxins (Chao et al. 2004). The liposome-PCR assay uses liposomes with encapsulated DNA reporters, and (in the case of botulin toxin) ganglioside receptors embedded in the bilayer, as a detection reagent (Mason et al. 2006b). After immobilization of the target toxin by a capture antibody and co-binding of the detection reagent, the liposomes are ruptured to release the reporter DNAs, which are quantified by real-time PCR. The two latter methods require checking for the presence of external contaminating DNA in test medium. They also share disadvantages of common ELISA, such as the multi-step complexity of testing and the need of incubation period, since the reactions leading to amplification develop in time. Only partial improvement can be offered with the introduction of nanoparticles into the ELISA-type tests. Being loaded with primary antibodies, they offer more space for attachment of secondary (detection) antibodies (Billingsley et al. 2017). Providing output signal, they can serve as nano-enzymes and nano-reporters (Gao et al. 2019; Tabatabaei et al. 2020; Zhao et al. 2020). In direct sensing techniques eliminating ELISA, we lose the essential feature of ELISA—the possibility of amplification, since fluorescent nanoparticles cannot

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reproduce themselves. But the strongly increased brightness may compensate that (Drobysh et al. 2021; Gao et al. 2019). The attachment of quantum dots of different sizes (Taranova et al. 2015) allows realizing multiplex formats with multicolor fluorescence detection. Moreover, the introduction into sandwich-type assay of upconversion nanoparticles as reporters was shown to be very efficient, since the result was the 400 times higher sensitivity than in common ELISA (Poláchová et al. 2019). A lot of effort has been put to design the portable ELISA-type devices (Dong and Ueda 2017; Hosseini et al. 2018; Zhdanov et al. 2018). But since the desired sensitivity can be achieved in such a simple way, why do we need any type of amplification? There are many possibilities to increase the sensitivity of different fluorescence assays by enhancing the brightness of applied fluorescence reporters (Fig. 13.16b). Collective electronic effects in fluorescence emitters can be realized on nanoscale level (see Sect. 8.1 of Volume 1). Organic dyes are known to be very potent light absorbers and when they form molecular assemblies, the absorbance of formed structures proportionally increases, and this increase may be by 2–3 orders of magnitude. However, in fluorescence they often exhibit the self-quenching, in some of these assembled nanostructures the self-quenching can be avoided, and they demonstrate extreme brightness. The electronic interactions in these structures between the dyes exist, which allows exchange of excitation energy between them and realizing the effect of superquenching. Interacting with any quencher in sensor response, the very bright fluorescence emission can be quenched in one event. Such outstanding properties can be realized both in dye-doped particles and in fluorescent polymers (Chap. 8 of Volume 1). Upon association, some dyes can form the assemblies of well-defined geometry, H- and J-aggregates. They can be recognized by dramatically changed absorption and emission spectra in comparison with their monomeric forms. Remarkably, in J-aggregates the absorption and emission are concentrated in very narrow red-shifted bands that may demonstrate dramatically increased brightness and the valuable for sensing superquenching effect. Plasmonic enhancement can be seen due to the effect of local electric field gradient on fluorophore, changing its absorption and emission properties. Usually, this change is quite positive and is frequently used in different sensing technologies (Sect. 13.2 of Volume 1). Finally, the amplified stimulated emission is commonly observed when the fluorescent dyes are excited by highly intensive focused light beam. In this case, the majority of emitters appear in the excited state, demonstrating narrow red-shifted emission spectrum. The sensing response can be provided by shifting between normal and stimulated emissions, together with other possibilities (see Sect. 16.1 of Volume 1). These examples suggest that the nanostructures exhibiting extraordinary intrinsic light-emitting properties can be efficiently used in ultrasensitive immunosensor devices as signal transducers and reporters, avoiding signal amplification (Drobysh et al. 2021). Moreover, they may also allow providing pathogen detection based on DNA hybridization assays (see Sect. 5.1) without the need of amplification by PCR with single-molecular sensitivity (Furth et al. 2021). Implementation of these potentials into routine sensor technologies must result in remarkable achievements:

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• Simplicity in assays resulting in broader possibilities for miniaturization and integration into the chips (Chircov et al. 2020) and microfluidic devices (Loo et al. 2019; Niculescu et al. 2021), substituting more complicated signal transduction modules. • Easy realization of multiplex platform for simultaneous detection of multiple analytes by operation with nanoparticles generating different colors of fluorescence emission with on–off and wavelength-shifting possibilities. Such systems will represent smart combination of a high-performing device with a low system complexity, see (Dincer et al. 2017) and (Kim et al. 2021), for comparison. • Immediate obtaining of result in direct sensing with minimal operator requirements, avoiding washing and/or changing the reagents, see Sect. 2.4 of Volume 1. • Processing, storage and telecommunication of results using smartphone capabilities integrated into the process of sensing (Chen et al. 2020; Quesada-González and Merkoçi 2017). Moving towards in-field, point-of-care and self-sensing of complex and relevant samples such as foodstuff, urine or blood (Rani et al. 2021; Wang et al. 2021a), we benefit from recent technological developments (Ligler and Gooding 2019). The integration of narrow spectrum LED excitation sources, fiber optics, and miniature, highly-sensitive optoelectronic sensors, such as those routinely integrated into smartphones, provide a mobile fluorescence imaging platform that is very different from benchtop fluorescence instruments (Shrivastava et al. 2020; Wang et al. 2021b). Naked eye detectable fluorescence detection, can they be realized with bacteria and viruses? Yes, such possibility exists. For instance, distinct fluorescent spots derived from single influenza virus particles can be observed based on a fluorogenic assay of neuraminidase activity of the virus (Minagawa et al. 2019). Many possibilities can be realized with the application of biomembrane-incorporating and double stranded DNA-intercalating dyes. The prospect of reaching the ultimate limit of detection has stimulated the development of single-molecule bioaffinity assays (Farka et al. 2020). This novel and highly promising area regarding fluorescence sensing bacterial cells and viruses requires functional nanomaterials with excellent performance (Liu et al. 2021). They can be found among quantum dots, gold nanoparticles, upconversion nanoparticles, fluorescent conjugated polymer nanoparticles, nanosheets, and magnetic nanoparticles and realized in single-cell and single-virus assay formats (Wu et al. 2019). Surveying the pathway from past and present to future developments (Fig. 13.17), we observe revolutionary changes. The methods for detection, quantification and analysis of harmful microbes become more and more efficient and friendly to every user. For controlling the efficiency of reading this chapter, the reader is asked to respond to the following questions. 1. 2.

Explain three commonly realized strategies in pathogen detection. How they complement each other? Provide examples. What is the difference between antibiotics and disinfectants?

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Fig. 13.17 Past and future of routine pathogen detection. Conventional methods of microbiology using microscopic study of growing pathogen colonies (left). Smartphone-based fluorescence imaging with possibility of processing and telecommunicating of results (right)

3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Explain the culture-based methods used in microbiological laboratories. Why they are not satisfactory? Provide comparison with molecular-based methods. Can the bacteria be recognized by their release of active enzyme? For the fluorescence-based detection of bacteria, what in your view are the best recognition units (choose between antibodies, antimicrobial peptides, aptamers, etc.) and reporting units (choose between the dye-doped nanoparticles, conjugated polymers, the systems exhibiting FRET, etc.). Suggest the optimal composition of receptors and reporters. How to use chromogenic and fluorogenic enzyme substrates for bacterial detection? Why aptamers are preferable in comparison to antibodies regarding fluorescence reporting possibilities? How antimicrobial peptides are used in sensing bacteria? Explain the principle of operation of PCR technique. Why thermal cycling is needed and what are the possibilities to substitute it? What are bacterial spores and how they are recognized by their biomarkers? How biofilms are formed and what is the need to analyze them? How to recognize viruses determining their nucleic acid content? Explain the principle of recognition of viruses based on their surface antigens. Compare the principles of amplification in detection of pathogens. What are novel developments that allow obtaining the sensor response directly avoiding amplification steps? What is achieved?

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Li S, Huang J, Ren L, Jiang W, Wang M, et al (2021b) A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): application for the diagnosis of COVID-19. Talanta:122591 Li Y, Yang X, Zhao W (2017) Emerging microtechnologies and automated systems for rapid bacterial identification and antibiotic susceptibility testing. SLAS TECHNOLOGY: Transl Life Sci Innov 22:585–608 Ligler FS, Gooding JJ (2019) Lighting up biosensors: now and the decade to come. Anal Chem 91:8732–8738 Ligler FS, Taitt CR, Shriver-Lake LC, Sapsford KE, Shubin Y, Golden JP (2003) Array biosensor for detection of toxins. Anal Bioanal Chem 377:469–477 Lin F, Bao Y-W, Wu F-G (2019) Carbon dots for sensing and killing microorganisms. C 5:33 Liu M, Qiu JG, Ma F, Zhang CY (2021) Advances in single-molecule fluorescent nanosensors. Wiley Interdiscip Rev: Nanomed Nanobiotechnol:e1716 Liu R, He L, Hu Y, Luo Z, Zhang J (2020a) A serological aptamer-assisted proximity ligation assay for COVID-19 diagnosis and seeking neutralizing aptamers. Chem Sci 11:12157–12164 Liu S-L, Wang Z-G, Xie H-Y, Liu A-A, Lamb DC, Pang D-W (2020b) Single-virus tracking: from imaging methodologies to virological applications. Chem Rev 120:1936–1979 Liu W, Miao L, Li X, Xu Z (2020c) Development of fluorescent probes targeting the cell wall of pathogenic bacteria. Coord Chem Rev:213646 Locke LW, Shankaran K, Gong L, Stoodley P, Vozar SL et al (2020) Evaluation of peptidebased probes toward in vivo diagnostic imaging of bacterial biofilm-associated infections. ACS Infectious Diseases 6:2086–2098 Loo JF, Ho AH, Turner AP, Mak WC (2019) Integrated printed microfluidic biosensors. Trends Biotechnol 37:1104–1120 Ma H, Zhou B, Kim Y, Janda KD (2006) A cyclic peptide–polymer probe for the detection of Clostridium botulinum neurotoxin serotype A. Toxicon 47:901–908 Ma X, Ding W, Wang C, Wu H, Tian X, et al (2021) DNAzyme biosensors for the detection of pathogenic bacteria. Sens Actuators B: Chem 331:129422 Mako TL, Racicot JM, Levine M (2018) Supramolecular luminescent sensors. Chem Rev 119:322– 477 Martzy R, Kolm C, Krska R, Mach RL, Farnleitner AH, Reischer GH (2019) Challenges and perspectives in the application of isothermal DNA amplification methods for food and water analysis. Anal Bioanal Chem 411:1695–1702 Mason JT, Xu L, Sheng Z-m, He J, O’leary TJ (2006a) Liposome polymerase chain reaction assay for the sub-attomolar detection of cholera toxin and botulinum neurotoxin type A. Nat Protoc 1:2003–2011 Mason JT, Xu L, Sheng Z-m, O’Leary TJ (2006b) A liposome-PCR assay for the ultrasensitive detection of biological toxins. Nat Biotechnol 24:555–557 Mayboroda O, Katakis I, O’Sullivan CK (2018) Multiplexed isothermal nucleic acid amplification. Anal Biochem 545:20–30 McKinnon KM (2018) Flow cytometry: an overview. Curr Protoc Immunol 120:5.1.1–5.1.11 Medina RA, García-Sastre A (2011) Influenza A viruses: new research developments. Nat Rev Microbiol 9:590–603 Medina RA, Stertz S, Manicassamy B, Zimmermann P, Sun X, et al (2013) Glycosylations in the globular head of the hemagglutinin protein modulate the virulence and antigenic properties of the H1N1 influenza viruses. Sci Transl Med 5:187ra70–87ra70 Meyer B, Cookson B (2010) Does microbial resistance or adaptation to biocides create a hazard in infection prevention and control? J Hosp Infect 76:200–205 Miller MB, Atrzadeh F, Burnham C-AD, Cavalieri S, Dunn J et al (2019) Clinical utility of advanced microbiology testing tools. J Clin Microbiol 57:e00495-e519 Minagawa Y, Ueno H, Tabata KV, Noji H (2019) Mobile imaging platform for digital influenza virus counting. Lab Chip 19:2678–2687 Mohammadyousef P, Paliouras M, Trifiro M, Kirk A (2021) Plasmonic and label-free real-time quantitative PCR for point-of-care diagnostics. Analyst 146:5619–5630

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Chapter 14

Clinical Diagnostics Ex-Vivo Based on Fluorescence

Fluorescence (luminescence) is the only method with broad versatility that in target detection can combine multi-analyte ex vivo and in vivo studies. It offers the testing that is simple in performance, low invasive and applicable to point-of-care conditions. The progress in nanotechnology brings to biosensors the possibility for constructing the microarrays for high throughput parallel measurements of many analytes and for integration of biosensors with microfluidics into lab-on-a-chip devices. In recent years, clinical diagnostics has made dramatic steps from exploring empirical correlations between the appearance and severity of disease based on its visual or tested characteristic features towards understanding the basis of these features on the background of cell physiology. Many clinical tests that actively explore fluorescence techniques were developed. The studies on molecular level allow detecting the changes in metabolic biomarkers that can be abnormal levels of common metabolites or highly disease-specific biomarkers and pathology-originated mutations on molecular level (Fig. 14.1). The ‘in vivo’ methods are more limited and involve the whole body tissue imaging and contrasting of blood vessels. Attempts are being made to develop efficient fluorescence tomography and to control by fluorescent imaging the surgical operations, which will be described in Chap. 16. Biomarker is a term defined by a National Institutes of Health (NIH) working group as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.” (McMahon and Waikar 2013). Thus, biomarker is a biochemical indicator detecting the changes in molecular system, which can be applied in disease diagnosis. The use of biomarkers in basic and clinical research has become routine in many areas (Wang and Witzmann 2016). Operating with biomarkers, the doctor needs to obtain the valuable information in the simplest and fastest possible ways, which can be realized with new types of analytical devices called molecular sensors or biosensors. These instruments are able to provide more and more accurate detection of chemical or biochemical compounds that have to show, how the occurrence of given pathologies are being developed and what clinical decision should be taken in a particular case. © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_14

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Pathology diagnosis Ex-vivo Cells

Molecular pathology

Molecules

In-vivo Histology

Metabolic biomarkers

Disease specific biomarkers

Cellular dynamics

Tissue imaging

Contrasting blood vessels

Fig. 14.1 Pathology diagnosis techniques in which fluorescence is implied

Blood and urine are traditionally the major sources of information obtained in clinical laboratories. A strong demand in sensor devices operating at the bedside or even by patients at home brought into practice such unusual media as saliva, sweat and tears. They allow integrated proteome-based body fluid analysis (Corrie et al. 2015). Taken together they have to predict clinical outcome. Two types of biomarkers are usually considered. Of diagnostic value is the decrease or enhancement in biological fluids of concentrations of different metabolites, such as glucose. There are also tissue-specific makers that are not detected normally but appear as a result of pathological process in particular tissue. Point-of-care (POC) is the place of patient care or bedside, where the diagnosis should be made and where the information from biosensors should be realized in clinical decisions (Liu et al. 2020). To date, there are still great challenges in the development of simple, quick, affordable, yet highly effective and selective biosensors for POC testing (Kaushik and Mujawar 2018). To meet the increasing demands, researchers need to consider biosensor miniaturization. The miniaturization strategies include handheld instruments and microfluidics-assisted miniaturized biosensing systems. Typical types of instrumentation in clinical laboratories are those performing solid-phase extraction followed by analyses including high-performance separation techniques coupled to mass spectrometry detection (Wu and French 2013). Although these techniques are the most sensitive and accurate, they require sophisticated/expensive instruments and time-consuming manipulations by well-trained personnel. The other trend is the integration and automation of different immunoassays (Prabhu and Urban 2017), and these instruments is hard to apply at bedside or for personal use. Thus, new techniques are needed for clinical tests that satisfy new demands: – to reduce to a minimum the size and cost of instrumentation and complexity of its operation; – to reduce to a minimum or, better, completely avoid the manipulations with clinical samples before or during the test; – to make the result of analysis available as fast as possible;

14.1 Biological Fluids Available for Sensing

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– to provide the possibility of easy analysis of results with the use of databases, in comparison with previous data, etc. Biosensors appeared for satisfying these demands (Chen et al. 2020; Miranda et al. 2021). They assemble versatile new technologies. Aptamers, recombinant fragments of antibodies together with imprinted polymers make revolution in molecular recognition of analytes, step-by-step substituting enzyme-based technologies and allowing the screening of a wide variety of biomolecules with high specificity, low limits of detection, and great sensitivity. On the level of signal generation and transduction, various types of nanostructures have become of use (Welch et al. 2021). Miniaturization reached every optical element, from laser diodes to smartphone matrices. Machine learning algorithms are ready to analyze this information (Kim et al. 2020; Zhang et al. 2021). The era of digital health is approaching (Tu et al. 2020). In this rapidly developing area it is hard to draw a separation line between desirable and already achievable, between existing only on the table of researcher and already applicable in large-scale practice. This line can be provided by USA Food and Drug Administration (FDA) by approving the tested and useful products. One may study the web site: https://www.fda.gov/medical-devices/products-and-medical-pro cedures/in-vitro-diagnostics. The news on important developments appears every day. Below we will concentrate on general tendencies and illustrate them by selected examples.

14.1 Biological Fluids Available for Sensing Everyone is used to clinical tests of blood and urine, from which many important diagnostic variables are derived. Less common but increasingly popular are the tests with the use of saliva, sweat, and tears. These samples can be collected in a simple non-invasive manner, and the results with the application of clinically relevant protein biosensors become valuable for integrated body fluid analysis (Corrie et al. 2015). All these body fluids are highly complex mixtures that contain a variable concentration of cells, proteins, macromolecules, metabolites, small molecules and ions. The studies of their constitution can be used in the prediction and diagnosis of various diseases (Mischak et al. 2010). Blood is the most commonly collected biological fluid of human beings for clinical diagnostics (Fig. 14.2). In addition to cells, proteins, small molecules and ions forming the natural blood composition, it may contain the disease markers released by cells of the body deviating from normal functioning. In addition to standard blood panel of metabolites and electrolytes, over 200 proteins are used in clinically approved tests in the USA as the markers (Anderson 2010). The blood fractions that do not contain cells are called blood plasma and blood serum. Plasma contains blood clotting protein fibrinogen and serum—not. Blood serum, contains a total protein content of 60–80 g per liter, mainly consisting of albumin and globular

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Human blood

Cells

Cellular components

Molecular biomarkers Fig. 14.2 Peripheral blood as the source of samples for clinical analysis (Herbáth et al. 2014). The white blood cells originate from the bone marrow, lymph nodes, spleen and other lymphoid tissues. The serum component of blood carries proteins from all over the body and some of them are of diagnostic value. The analysis includes screening of plasma membrane constituent of intact cells as well as the measurement of cellular components after cell lysis or by using specific extraction methods

proteins. Many clinically relevant biomarkers are present at concentrations ranging from several nanomolars to hundreds of micromolars (Anderson and Anderson 2002). Serological tests are the analyses provided in blood usually in diagnostics of viral diseases. They are reliable to study the immune response and can be used to identify the recovery or post-infected people by detection of circulating antivirus antibodies (Elledge et al. 2021; Galili 2020), but it is not the way for direct detection of virus. A fluorescently labeled peptide derived from the antigenic virus protein can be used as the test antigen, and the response to their binding can be obtained as the change in color of fluorescence emission, as it was demonstrated in model experiments (Choulier et al. 2010; Enander et al. 2008). A great variety of synthetic and phage-displayed peptides have been presently collected as test antigens (Palacios-Rodriguez et al. 2007). As for serological tests, the additions of photonic ring immunoassays and bead-based digital ELISA show promising results in the ability to simultaneously measure the level of multiple antibodies against multiple antigens (Giri et al. 2021), which allows ultrasensitive high-resolution profiling of early transformation of immune response in patients with COVID-19 (Norman et al. 2020), see Fig. 14.3. Urine is biological fluid of important diagnostic value (Barratt and Topham 2007, Decramer et al. 2008), particularly in clinical cases of kidney injuries (Su et al. 2011) and diabetes (Rao et al. 2007). Urine is a desirable material for the diagnosis and classification of diseases because of the convenience of its collection in large amounts. Urine biosensors applied to human sampling have typically focused on small molecule analytes, that may be indicative of renal tract pathology, such as

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Fig. 14.3 Schematic of the developed ultra-sensitive, multiplexed single molecule array (Simoa) for immunoglobulin isotypes against immunogenic SARS-CoV-2 proteins (Norman et al. 2020). Plasma is incubated with four types of dye-encoded beads that are each coupled to one of four viral targets (spike, S1, RBD and nucleocapsid). IgG, IgA and IgM antibodies specific to the SARS-CoV2 targets bind to the viral antigen-coated beads. After washing, beads are introduced to biotinylated anti-human immunoglobulin antibodies to label either IgG, IgM or IgA in the different reactions. After additional washes, the enzyme streptavidin-β-galactosidase (SβG) is introduced. The beads are washed, resuspended in fluorogenic resorufin β-d-galactopyranoside (RGP) and loaded into a 216,000-microwell array for multicolor imaging

oxalate, glucose, uric acid, but also proteins (Corrie et al. 2015). The human urinary proteome contains more than 1500 proteins, including a large portion of membrane proteins (Adachi et al. 2006; Rodríguez-Suárez et al. 2014). The presence of serum albumin in urine clinical samples is a clear indication of type 2 diabetes (Rowe et al. 1990). Saliva contains a varied range of composites, organic and inorganic. There are proteins, carbohydrates, and lipids, which justifies its use as a diagnostic material. Moreover, drugs and their metabolites are also secreted into saliva. Saliva collection is noninvasive, and self-collection is possible. It offers the advantages of easy accessing and volume, but with a major disadvantage of large range of variability in components and their concentrations, depending on the extent of oral cleanliness, diet and liquid uptake (Schulz et al. 2013). Human saliva has been successfully used in the diagnosis of many systemic diseases like cancers, autoimmune diseases, infectious diseases (HIV, hepatitis, and malaria), and endocrine diseases, as well as diseases of the gastrointestinal tract (Gug et al. 2019; Yoshizawa et al. 2013). It can serve as important indicator of immunity (Riis et al. 2020). Particularly, detection of cytokines helps in diagnosis of inflammation (Belstrøm et al. 2017). Also, it is used in toxicological diagnostics, drug monitoring and forensic medicine (Beatty et al. 2019). The usefulness of saliva as a biological marker has also been extended to psychiatry (Klimiuk et al. 2019; Kułak-Bejda et al. 2019).

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One such biomarker, whose measurement in saliva could potentially contribute in a significant manner to the understanding and diagnosis of disease, is the Creactive protein. This is an important inflammation marker and cardiovascular disease predictor that is derived from the liver and whose production is regulated by cytokines. It was found to be a stronger predictor of coronary heart disease. Microsystem-based diagnostics of this protein with the use of saliva deserves active development and applications (Christodoulides et al. 2005; Pay and Shaw 2019). Currently, tear-based sensors have focused primarily on glucose monitoring and demonstrate correlation with glucose level observed in blood (Lee et al. 2021). They show considerable promise for detecting other physiologically important biomarkers (Yu et al. 2019). The scope of new tear analytes can be expanded to include additional metabolites, for example, catecholamines (Sharma et al. 2019). Since tear fluid contains thousands of proteins, the most abundant of which are lysozyme (Ballard et al. 2020), lactoferrin, and albumin, noninvasive tear monitoring could also be used to detect protein biomarkers correlated with a variety of diseases. Various putative tear fluid biomarkers have been identified to date, ranging from ocular surface disease and retinopathies to cancer and multiple sclerosis (Hagan et al. 2016). Putative tear fluid biomarkers of ocular disorders, as well as the more recent field of systemic disease biomarkers, will be shown. Proteomic analysis of tear samples may be an approach to identify biomarkers linked to ocular diseases (Azkargorta et al. 2017). Contact lens biosensors were suggested as the sensor instruments, and they demonstrate steady improvement (Tseng et al. 2018). A very elegant solution was found, in contrast to common thinking focused on implanting glucose sensor protected by membrane from interference of blood cells and proteins. Since the increased glucose level can be also detected in tears, application of smart disposable and colorless contact lenses was suggested (Fig. 14.4). These contact lenses can be worn by diabetics patients who can see the changes in their contact lens color or other fluorescence-based properties, giving an indication of tear and blood glucose levels (Badugu et al. 2004). Passive eccrine sweat is a functional biological fluid that is valuable for diagnosis and monitoring of disease states (Ghaffari et al. 2021a; Katchman et al. 2018). The rich composition of electrolytes, metabolites, hormones, proteins, nucleic acids can be found in sweat. It is applicable for proteome and metabolome analysis (Serag et al. 2021). It provides response to inflammation (Hladek et al. 2018; Jagannath et al. 2021). Cytokines are biomarkers that orchestrate the manifestation and progression of an infection/inflammatory event. Hence, noninvasive, real-time monitoring of cytokines can be pivotal in assessing the progression of infection/inflammatory event, which may be feasible through monitoring of host immune markers in eccrine sweat. For performing sweat analysis, different wearable biosensor designs have been suggested (Ghaffari et al. 2021a; Koralli and Mouzakis 2021; Moonen et al. 2020). They allow prolonged semi-continuous health monitoring. Exhaled breath biomarkers can be of interest for diagnostics (Das and Pal 2020; Vasilescu et al. 2021). Compared to clinical blood and urine tests, this method of sample collection is noninvasive, easily repeatable, has a very low infection risk, and is convenient for long-term clinical monitoring. Biological samples for diagnostic analysis can be collected spontaneously and in large quantities. The oxygen/carbon

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Fig. 14.4 The non-invasive tear-analyzing glucose sensor (Badugu et al. 2004). a Schematic of a device. The hand-held device works by flashing a light into the eye and measuring the emission intensity. b Contact lens doped with optical probe for glucose with sensor spots on the surface of the lens that allow providing the excitation (Ex) and monitoring the emission (Em). Other analytes, in addition to glucose, such as chloride or oxygen, can be measured, allowing for ratiometric, lifetime or polarization based fluorescent sensing. c Variation of fluorescence intensity as a function of glucose concentration

dioxide respiratory cycle is the pulmonary gas exchange. Except for the gases, the exhaled breath contains water vapors and hundreds of different trace compounds of exogenous and endogenous origin in ppb to ppm levels (Kim et al. 2012; Wang and Sahay 2009). Though slowly, breath analysis becomes a new field of diagnostic medicine. Above we listed the information channels that could allow extracting the versatile and important data on the life of human body on molecular and cellular levels. They can be analyzed by many technical means, often after sample purification and pre-treatment that can be done with integrated in vitro diagnostic devices (Broza et al. 2019). We need better integration between body fluid sampling and these devices. For better diagnostics, we may require to tackle immense challenges in detecting low abundance analytes in complex fluids, including protein analytes in ultra-low concentrations. But the reward is the implementation into clinical practice. Non-invasive sample collection allows body-interfacing continuous monitoring of analytes (Bhide et al. 2021; Zhao et al. 2019). The comparison between diagnostic patterns of analytes between this broadening range of body fluids with their different small-molecular compositions and proteome sub-sets should yield new insights into biosensor development and enlarge diagnostic possibilities.

14.2 Detection of Disease Biomarkers To make the diagnosis most precise, a number of biomarkers (the output parameters of diagnostic value) have to be analyzed in parallel, and therefore several channels of fluorescence response have to be activated on a cellular and tissue level. Novel biosensor technologies address cancer biomarkers, cardiac biomarkers

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as well as biomarkers for neurodegenerative and autoimmune diseases, and also infectious diseases. Age-related mental disorders should be recognized as early as possible. These biomarkers can be produced either by the malignant tissues and cells themselves or by the body in response to the presence of disease.

14.2.1 Diagnostics of Cancer Early diagnosis of cancer is an urgently needed strategy to reduce the incidence of the disease and improve significantly the survival rate. Still now, the commonly used cancer diagnostic and prognostic indicators are the morphological and histological characteristics of tumors. Cancer emerges from our own tissues, complicating both detection and treatment methods due to the similarities between the diseased and healthy tissues. Meantime, biomarkers can recognize them and they have started to play their important role as molecular signatures of the disease. They can be of two types: metabolic and specific protein-based. Differential metabolic profile between cancerous cells and normal cells was observed in many types of cancer, resulting in discovery of metabolic biomarkers specific to cancerous cells and, most importantly, to different cancer types. As an example, the metabolic biomarkers indicating prostate cancer are citrate and spermine (Giskeødegård et al. 2013). Detection of abnormal concentrations of small molecules is of diagnostic value. Aberrant activity of many enzymes leads to abnormal levels of their products thereby affecting cellular homeostasis linked to cancer formation and progression. Certain enzymes are “overexpressed” or “underexpressed”, and their products are the targets for diagnostics (Sharma et al. 2010). Fluorescence probing of different enzyme activities have shown important cancer-related changes (Singh et al. 2018). Single specific protein biomarkers, such as prostate specific antigen (PSA) have already found their broad use in clinical practice (Barry 2001). Biomarkers for other types of malignancy, such as breast cancer (Cheung et al. 2000) and colorectal cancer (You et al. 2018) were also identified. The protein with clinical evidence for breast cancer is the human epidermal growth factor (HER2) (Loibl and Gianni 2017). Meantime, since tumor development involves many biological changes, the signatures of this disease can be very complex and characteristic for particular type of tumor (Jain 2007; McShane et al. 2005). Therefore, multiple targets have to be determined simultaneously. An example of a sensor determining three different targets providing multicolor response is presented below (Fig. 14.5). Presented in Fig. 14.5 is an illustration of a method for simultaneous recording of three breast cancer biomarkers based on single-band upconversion nanoparticles (sbUCNPs) emitting light with different colors (Zhou et al. 2015). The expression levels of three biomarkers in breast cancer cells were determined with sb-UCNPs coated with a screen layer containing an organic dye with a high molar absorption coefficient. As a result of the efficient reabsorption of the organic dye, remarkably pure singleband upconversion emissions can be generated in the blue, green and red regions. Conjugation with antibody allowed applying a molecular profiling technology for

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Fig. 14.5 Schematic diagram of single-band upconversion nanoparticles (sb-UCNPs) fabrication for multiplexed detection (Zhou et al. 2015). Surface amino modifications of the multi-layer structure of green, blue and red sb-UCNPs were made. They were conjugated with the antibodies to the breast cancer biomarkers PR, ER and HER2, respectively, for multiplexed in situ molecular mapping of these biomarkers

achieving the multiplexed simultaneous in-situ detection of biomarkers in breast cancer cells and tissue specimens. Proteomics approaches based on comparative detection of increased levels of several among many thousands of proteins have been used to generate differential protein expression maps of the normal cells (Ray et al. 2011). They can be compared to that of cancer cells with the detection of proteins, whose levels change significantly (Kuramitsu and Nakamura 2006). The microarrays with fluorescence readout (Zajac et al. 2007) and microfluidic devices (Kartalov et al. 2006) must be of efficient use for their detection. Exosomes are naturally occurring, nanoscale (30–100 nm) vesicles that transfer biomolecules (proteins and nucleic acids) to facilitate cell-to-cell communication (Sato and Weaver 2018). They participate in spreading of cancer cells and can serve as their important biomarkers carrying the information that reflects the origins of the cancer cells (Makler and Asghar 2020). Aptamer-based fluorescence assays are efficient for their recognition and analysis (Hassan and DeRosa 2020; Kordasht and Hasanzadeh 2020; Zhang et al. 2019). Genome derangement is frequently involved in carcinogenesis and may contribute to abnormalities in genes encoding proteins that have a critical role in key pathways

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related to cell growth and survival, leading to cancer development. Therefore, identification of potential molecular biomarkers with high sensitivity and specificity would be beneficial for cancer diagnosis and patient prognosis as well as targeting therapeutics (Seeree et al. 2015). Current research tools have permitted the identification of these genetic alterations benefiting from recent advances in “omics” technologies (see Fig. 14.6). The studies of cancer-related biomarkers follow in line with direct identification of cancer cells. The aptamers specifically reacting with cell surface can distinguish the cancer type (Mehmood et al. 2019; Safarpour et al. 2020; Zahra et al. 2021). The array sensors based on fluorescent nanoparticles formed of conjugated polymers have also shown their efficiency (Le et al. 2014). Serving as signal amplification carriers or direct signal generating elements, nanomaterials can form the basis of direct no-wash biosensors (Huang et al. 2017), as it is discussed in Sect. 13.6.

Fig. 14.6 An overview of molecular biomarkers for cholangiocarcinoma (CCA), a highly malignant form of cancer, based on their potential use in early diagnostics, prognostics, and therapeutics. “Omics” approach allows simultaneous detection of magnitude of biomarkers on different levels of cell organization. It is based on universal detection of genes (genomics), mRNA (transcriptomics), proteins (proteomics), and metabolites (metabolomics).These techniques are useful for retrieving cancer biomarkers as they simultaneously investigate multiple molecules. For detailed information on presented biomarkers see original publication (Seeree et al. 2015)

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Cancer is a life-threatening concern worldwide, and its early-stage diagnostics means saving many lives. To efficiently combat this menace, sensitive and informative diagnostics of different cancer types can make it possible for patients to get through the best available treatment options. The biomarker-based prediction of patient’s cancer resistance to drug treatment is the pathway to personalized medicine (Housman et al. 2014).

14.2.2 Diagnostics with Cardiac Biomarkers Many current activities are directed towards the use of biosensors for achieving sensitive detection of cardiac biomarkers for early diagnosis of acute myocardial infarction. Identification of biomarkers provides a powerful approach for screening, diagnosis, prognosis and therapeutic monitoring especially in the emergency room, which is crucial to design an appropriate patient care strategy (Christenson et al. 2014; Vasan 2006). Addressing and improving the detection of early-stage disease permits early intervention with more efficient disease management and a significant reduction in premature mortality. This condition is associated with increase in the blood of several key protein-based biomarkers (Ho et al. 2018; Yang and Zhou 2006). These markers indicate different features of cardiac disease. C-reactive protein reports on inflammation that is associated with cardiovascular damage, the D-dimer fragment of fibrinogen is the risk factor of thrombosis, troponin I and troponin T levels in blood indicate the damage of muscle cells. (Maznyczka et al. 2015). Therefore simultaneous measurement of several biomarkers not only reduces the errors but allows to characterize the disease in most detail (Wang et al. 2006; Wu et al. 2020). Of diagnostic value are lactate dehydrogenase1 (H4), aspartate aminotransferase and creatine phosphokinase (Batta et al. 2012). Figure 14.7 illustrates the steps that are required to pass for achieving the results in point-of-care testing. The applied techniques are on the steps of active improvement.

Fig. 14.7 Point-of-care testing (POCT) utilizes traditional receptors, such as antibodies, for cardiac biomarker measurements (Regan et al. 2019). The receptors are integral in the detection of released cardiac biomarkers for POCT. Myo—Myoglobin, BNP—Brain natriuretic peptide, H-FABP—Heart type fatty acid binding protein

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It was noted however, that the use of current biomarkers that are determined in popular sandwich immunoassays improves standard assessment only moderately, even in a multiplexing approach. Therefore, there is an urgent need for technological advances that could allow not only to determine these protein biomarkers quantitatively above their background levels, but also to do this in a most rapid procedure to provide correct clinical decision timely. The progress must be associated with fluorophore-mediated multi-analyte sensing systems (Tang et al. 2006). The problem is in extremely low concentration level of some of these biomarkers (in the pM to nM range) not only in norm but also in disease and the presence of other structurally similar proteins. The sandwich immunoassays are of sufficient sensitivity, but they are time-consuming, expensive and technically complicated. Therefore, they cannot satisfy practical medicine that needs rapid decisions. Direct and competitive assays (see Chap. 2 of Volume 1) are still on initial steps of their development, but offer promise. The recently reported application of competitive anisotropy assay allowed achieving in the detection of human cardiac troponins I and T the limit of 15 pM concentration rapidly in homogeneous conditions (Qiao et al. 2011). Myoglobin, while not cardiac specific, is one of the very early markers that increases its concentration in blood directly after an acute myocardial infarction (Govindan et al. 2013). Therefore it can be used as a suitable marker for rapid diagnosis using the new microwave-accelerated metal-enhanced fluorescence approach (Aslan and Geddes 2006). Presently it is developed for immunoassay and offers an ultra-rapid and sensitive platform. For the diagnosis of ischemic stroke, in addition to cardiac troponins, the brain natriuretic peptide (BNP), and neuron-specific enolase (NSE) are the critically important biomarkers that are specific to brain injuries (Dolati et al. 2021; Katan and Elkind 2018). Rapid implementation of biosensors as rapid diagnostic tests has to overcome important limitations in the evaluation and treatment of this disease. They allow providing differentiated diagnosis that may determine strategy of treatment (Harpaz et al. 2020). Screening cholesterol, triglycerides, and other plasma lipids, indicating biomembrane damage of cardiac cells, is also an important component in the management of cardiovascular disease. Stroke and diabetes are also linked to high cholesterol level, strengthening its importance as a diagnostic target. Meantime, efficient express methods for their determination in blood are still lacking (see Sect. 14.5).

14.2.3 The Markers of Autoimmune Disorders Autoimmune diseases are the disorders characterized by the presence of autoantibodies, which bind the patient’s own proteins, peptides or other structural compounds as target antigens (Wang et al. 2015). Autoantibodies that can be found in blood serum accompany or even determine many diseases. They can be manifested in a single organ or tissue (localized or organ-specific autoimmune diseases), such as type

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1 diabetes, or affect multiple organs and tissues (systemic autoimmune diseases), such as systemic sclerosis. They are usually disease-specific and have to be analyzed together with other disease markers in order to identify disease type and state. Several examples are presented below. Cancer autoantibodies are circulating antibodies to cancer-related antigens; they appear in early stages of disease and are recognized early as biomarkers in early cancer detection (Tan et al. 2009). Presently, these autoantibodies are increasingly investigated as cancer biomarkers (Yadav, Kashaninejad et al. 2019) and demonstrate signatures for common cancers (Kobayashi et al. 2020a), particularly to breast cancer (Jeong et al. 2020; Qiu et al. 2018). Proteome profiling of autoimmune response to ovarian cancer pathogenesis was identified (Kobayashi et al. 2020b). Rheumatoid arthritis (RA) is characterized by chronic inflammation of the joints, sometimes systemic complications may be involved. Currently, antibodies against citrulline-containing peptides are recognized as the most specific serological markers for diagnosing RA (de Gracia Villa et al. 2011). Carbon nanotubes with immobilization of these peptides were used to detect those antibodies. Serum samples obtained from RA patients and from healthy individuals could be distinguished. In type 1 diabetes, the destroying autoimmune response is directed against the insulin-producing β-cells of the pancreas (Seissler and Scherbaum 2006). Diabetesrelated autoantibodies include antibodies reactive with insulin (Church et al. 2018) and antibodies to tyrosine phosphatase-like protein IA-2 (Hawa et al. 1997). Knowledge about immunological disorders is likely to be further expanded as more antibody targets in disease are discovered. There is a lot of choice to detect autoantibodies in different biosensor formats (Ghorbani et al. 2019; Zhang et al. 2017). The identification of relevant target proteins requires the development of new strategies to handle and process the vast quantities of data, so that these data must be evaluated and correlated with relevant clinical issues, such as disease progression, clinical manifestations and prognostic factors (Abel et al. 2014). Therefore, protein microarrays may become an established tool for routine diagnostic procedures in the future. Though the advantages of multiplexed analyses of autoantibodies by the use of microarrays were emphasized (Schlichtiger et al. 2012), the most popular still remain the single-analyte sandwich-type biosensors.

14.2.4 Kidney-Related Diseases In kidney-related diseases, urine is a primary source for biomarkers to be analyzed (Edelstein 2016). There are several traditional disease-related biomarkers, and serum albumin is one of them. Microalbuminuria, which is the presence of very small amounts of serum albumin (SA) in urea (30–300 mg/day), is the diagnostic test for kidney diseases (Gansevoort and de Jong 2009) and also for type 2 diabetes (Hellemons et al. 2012) and as a cardiovascular risk predictor (Cerasola et al. 2010). The test for quantification of urinary albumin excretion in terms of albumin to creatinine ratio is used to predict

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albuminuria accurately and becomes increasingly relevant in assessing the prognosis and treatment of kidney diseases (Abdelmalek et al. 2014). Serum albumin is the protein, the function of which is to carry in the blood the molecules that are low-soluble. They can be adsorbed at specific sites on this protein surface. Those are different fluorescent dyes and, since the albumin is excreted in the native form with retention of its binding sites, it can bind the dyes with high selectivity and affinity. One of these dyes, Albumin Blue 580, is used already for many years for the detection of microalbuminurea in clinics (Kessler et al. 1997). This dye is almost non-emissive, and its strong fluorescence appears on albumin binding. The albumin concentration can be calculated from emission intensity at 616 nm (excitation at 590 nm) based on preliminarily obtained calibration curve. The albumin concentration was calculated from emission intensity at 616 nm (excitation at 590 nm) and a calibration curve. This method, though not new, demonstrates important advantages over albumin detection with antibodies and can be realized in microplate format (Laiwattanapaisal et al. 2008). Different other dyes with fluorescence enhancement on albumin binding have been suggested. The mechanism of their fluorescence enhancement is simple: suppression of intramolecular mobility and screening from quenching by water (Harvey et al. 2001; Wu, Yu et al. 2014). Among recent developments is the albumin test based on NIR fluorescence modulation of carbon nanotubes (Budhathoki-Uprety et al. 2019). A nanosensor paint was developed for the detection of microalbuminuria, in which a suite of polycarbodiimide polymers to concomitantly encapsulate carbon nanotubes and interact with specific features of albumin (Fig. 14.8). Probably due to mimicry of the head group of albumin-binding fatty acids at its specific site, the polymer exhibited specific photoluminescence responses to albumin in the microalbuminuria range via both intensity enhancement and a blue shift of spectrum. Recently, there has been an explosive growth in the search for more sensitive, specific, and prognostically accurate biomarkers to assist in the care of patients with or at risk of kidney disease. It has been shown that identification of urinary polypeptides as the biomarkers of kidney-related diseases allows to diagnose the severity of the disease several months before the manifestation of pathology (Devarajan 2007). They

Fig. 14.8 Optical nanosensor for albumin detection based on recognizing feature of hydrophobic polymer and fluorescence response of carbon nanotube in in near-infrared region (BudhathokiUprety et al. 2019)

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can be used in diagnosis of acute kidney injury (AKI), chronic kidney disease (CKD), nephrotoxin exposure, and glomerulonephritis (McMahon and Waikar 2013). Examination of blood urea nitrogen concentration and the serum creatinine concentration are used to urea studies. In addition to specific peptide and protein biomarkers, metabolic biomarkers, such as creatinine, ammonia and urea are also of diagnostic value. Creatinine, a byproduct of kidney function, is produced at an almost constant rate in healthy individuals; its level is diagnostic for defective renal function through estimation of the glomerular filtration rate (Shephard et al. 2010). Renal dysfunction, liver disease, and asthma can often be detected through the measurement of urea and ammonia levels. Optical array biosensors for simultaneous measurement of markers for renal disease (urea, creatinine, uric acid, glucose) were produced by immobilizing the appropriate enzymes and fluorescent dyes in a sol–gel matrix. For urea and creatinine, the actions of hydrolase enzymes urease and creatinine deiminase produced hydroxide ions, which were detected via an immobilized pH-sensitive fluorescent indicator. For glucose and uric acid, the actions of oxidase enzymes glucose oxidase and uricase produced hydrogen peroxide, which was consumed by co-immobilized horseradish peroxidase to give a fluorescent signal at 590 nm as a result of reduction of Amplex red to resorufin (Summers et al. 2013). Such complicated assays that rely on enzyme activities must be substituted by more convenient direct sensor technologies.

14.2.5 Neurodegenerative Diseases Multiple neurodegenerative disorders, such as Alzheimer’s disease and Parkinson’s disease share common pathogenic mechanisms leading to accumulation of misfolded protein species that provide irreversible damage of nerve cells. Common among them is the absence of full understanding of their origin and the absence of specific functionally oriented treatment. There are no specific tests for them; their diagnostic on molecular and cellular level is complicated, so that a series of different biomarkers can be used only. Alzheimer’s disease is an age-related neurodegenerative disorder causing severe disability and important socio-economic burden. It is associated with gradual failure of synaptic integrity and loss of cognitive functions, and there is no cure available to date (Kumar and Singh 2015). Abnormal processing and accumulation of amyloid-β peptide (Aβ) has been considered the main cause and trigger factor of the disease (Bruni et al. 2020). The increase in amount of Aβ in body fluids can be used for diagnostics. Also, tau protein and microRNA, are the most important biomarkers that are detected using biosensors (Mobed and Hasanzadeh 2020). They can be found in body fluids, including blood, saliva, urine and tears (B˘alas, a et al. 2020; Pawlik and Błochowiak 2021). Tear fluid was shown as a novel source of diseasespecific protein and microRNA-based biomarkers for the development of this disease (Kenny, Jiménez-Mateos et al. 2019). Within rich tear protein content, an elongation

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initiation factor 4E (eIF4E) was identified as a unique protein present, and among individual microRNAs, microRNA-200b-5p was identified as a potential biomarker. Parkinson’s disease is a progressive neurodegenerative disease, one of the most critical disorders of the elderly. It is strongly associated with increased disability and reduced quality of life (Raza and Anjum 2019). This disease is characterized by the progressive degeneration of dopaminergic neurons, with consequent dopamine depletion, and the accumulation of misfolded small protein α-synuclein aggregates. Its diagnosis based on evaluation of clinical manifestations as well as on evaluation of movement disorders by a neurologist has several disadvantages and limitations. Biosensors technology opens up new diagnostic approach that allows reliable, repeatable, and multidimensional identification to be made with minimal problem and discomfort for patients (Mobed et al. 2021). One of the most promising sources of biomarkers for Parkinson’s disease is represented by extracellular vesicles (EVs), a heterogeneous population of nanoparticles, released by all cells in the microenvironment (Leggio et al. 2021). Brain-derived EVs are able to cross the blood–brain barrier, protecting their payload from enzymatic degradation, and are easily recovered from biological fluids. Detection of decrease of dopamine concentration can be made by a biosensor based on fluorescence quenching of quantum dots in the presence of ascorbic acid (Ankireddy and Kim 2015) and also using fluorescent gold nanoclusters (Govindaraju et al. 2017) and carbon dots in conjugates with gold clusters (He et al. 2018). There are other diseases associated with improper functioning of nervous system. For their proper diagnostics, a number of neurotransmitters have to be determined (Nawrot et al. 2018). Among them are dopamine, epinephrine (adrenaline) and norepinephrine (noradrenaline), which belongs to the catecholamines, and serotonin. Their level can be measured in different biological fluids, such as plasma, saliva, urine, or blood serum (Marc et al. 2011). For their recognition, two types of receptors are applied and implemented in biosensors. One involves the antibodies and the other incorporates modified calixarene, cucurbiturils (Saluja and Sekhon 2013) and pillar[6]arenes (Paudics et al. 2019), usually using fluorescent indicator displacement assays (Rather and Ali 2021). The understanding of the role of neurotransmitters in different pathologies, especially in the field of central nervous system disorders, has dramatically improved in recent years mainly due to novel methods of screening the antigens with the aid of microarray techniques, Identification of antibodies directed against ion channels, receptors and other synaptic proteins helped in assigning their causative roles in different disorders. Diagnostics of neurodegenerative disorders is an active area of application of artificial intelligence techniques like machine learning algorithms (Chang et al. 2021). With novel biomarkers and multiple variables these tools may increase substantially the sensitivity and specificity of analysis (Gao et al. 2021).

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14.3 Glucose Sensing in Diagnosis and Treatment of Diabetes Glucose (Fig. 14.9a) is one of the most important molecules in the body and the main source of energy in cellular metabolism. Deviations from normal glucose levels can cause many serious diseases such as diabetes or hypoglycemia. Dramatic increase and oscillation of blood glucose level in blood over its normal value of 3.3–5.5 mM is the characteristic feature of diabetes. Close monitoring of glucose is needed for diabetes patients, so that its increase requires immediate treatment. The measurement should be frequent and, ideally, continuous. The aim of producing the sensors that conform to these requests is still not achieved, despite the broad market that is estimated in tens of billions euros (Steiner et al. 2011; Vashist 2012). Such situation stimulates very tough competition between different technologies, one of which is based on fluorescence. The conventional approach to glucose level monitoring requires invasive treatment to draw the drop of blood and to manipulate with it in glucometers or paper-strip devices (Vashist et al. 2011). Hence the strong demand exists towards non-invasive glucose monitoring devices (Tang et al. 2020; Vashist 2012; Villena Gonzales et al. 2019). The suggested solutions start from glucose monitoring in sweat (Lee et al. 2017) and tears (Bruen et al. 2017; Kownacka et al. 2018) to application of tattoo to the patient’s skin (Bandodkar et al. 2015). Different glucose recognition units were suggested. They include small-molecular organic boronic acid, concanavalin A and bacterial periplasmic saccharide binding proteins, as well as glucose transforming enzymes (Steiner et al. 2011). The majority of commercial self-test glucose measurement systems are now based on redoxcouple-mediated enzymatic oxidation of glucose by either glucose oxidase or glucose dehydrogenase with electrochemical detection. Their application is not easy or reliable, since enzyme reactions depend on many uncontrolled factors and require

Fig. 14.9 Glucose structure and its space-filling model (a) and interaction of boronic acid with diols, reaction frequently used for determination of glucose (b)

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frequent calibration. Thus, the ideal glucose sensor should avoid sample manipulations and reagent additions, the response should be direct and reversible allowing continuous monitoring (Van Enter and Von Hauff 2018). With this aim in mind, we consider the possibilities offered by fluorescent boronic acid derivatives. Boronic acids and their derivatives are valuable as molecular recognition units due to their distinguished features to recognize diol motifs through boronic ester formation (Nishiyabu et al. 2012; Wu et al. 2013). They can reversibly interact with 1,2-diols or 1,3-diols in aqueous solution to form 5- or 6-membered ring cyclic esters (Fig. 14.9b). This reaction results in formation of covalent bonds, but still it is reversible. Since it occurs in aqueous media at neutral pH values, it has become very attractive as the basis for an important sensing scheme in biologically relevant conditions for saccharides, including glucose (Fang et al. 2018; Williams et al. 2021). The small-molecule sensors are represented by boronic acid derivatives (which bind the diols of sugars, see Sect. 8.2) coupled to organic fluorescence dyes. The boronic acid group is an electron-deficient Lewis acid having an sp2-hybridized boron atom with a trigonal planar conformation. The anionic form of the boronic acid that is formed in the presence of glucose is characterized by a more electron-rich sp3-hybridized boron atom with a tetrahedral geometry. The change in the electronic properties and the geometry at the boron atom induces the fluorescence spectral changes of the attached aromatic groups. Upon addition of glucose, the electron density on the boron atom is increased, facilitating the partial neutralization of the positively charged quaternary nitrogen of the quinolinium moiety. This change can be transmitted to appended fluorescent dye changing its electronic properties. In this case, the direct mechanisms of their response based on PET or ICT (see Sect. 4.1 of Volume 1) can be used with the detection in intensity, spectral shift or lifetime. Boronic acid derivatives show good examples of multivalency (Williams et al. 2021). Binding of diols is already bivalent. In order to increase affinity to multihydroxylic compounds, a number of dimeric derivatives of boronic acid were synthesized (Fig. 14.10). They became ideal for highly selective binding of saccharides interacting with their linked arrays of hydroxyl groups. Close location of two boronic structures can be realized on interaction with saccharides, particularly with glucose. In this case, appended dye molecules may interact providing fluorescent signal on saccharide binding. It can be pyrenes forming the excimers, as depicted in Fig. 14.11. Their monomer emission at ~390 nm is transformed into emission at ~500 nm, which allows ratiometric detection of glucose. The change in molecular sensor construction allowed to discriminate glucose from fructose and other saccharides (Wu et al. 2013). The sensors using the glucose binding proteins can benefit from the property of these proteins to exhibit extended conformational changes on ligand binding (see Sect. 4.3). Bioanalytical recognition units include the lectin concanavalin A (Con A), enzymes such as glucose oxidase, glucose dehydrogenase and hexokinase/glucokinase, bacterial glucose-binding protein (Lee et al. 2018). In these cases the environment-sensitive fluorescence probes and the pairs of dyes with the modulation of EET between them can be used for sensing (Pickup et al. 2013). The popular mechanism of response is the EET between a fluorescent donor and an acceptor within

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Fig. 14.10 The strategies in applications of boronic acid derivatives for selective saccharide sensing via multivalent boronic acid–saccharide interactions (Wu et al. 2013). a Typical synthetic diboronic acids that form 1: 1 cyclic boronate esters with glucose. b Cartoon representation of diboronic acid binding to glucose; c aggregation of simple boronic acids via noncovalent interactions allowing multivalent glucose binding; d boronic acid-containing polymers or nanomaterials that bind glucose with two of pendant boronic acid moieties

Fig. 14.11 Selective glucose sensing utilizing complexation with fluorescent boronic acid on polycation (Kanekiyo and Tao 2005). Excimer formation between attached pyrenes is used for reporting. Before addition of glucose: monomer emission. After addition of glucose: 1: 2 complex formation between glucose and fluorescent boronic acid in the presence of polycation poly(diallyl dimethylammonium) chloride results in excimer emission

a protein that undergoes glucose-induced changes in conformation or re-arrangement of subunits. Competitive displacement assays were also developed with application of saccharide binding proteins. They use fluorescently labeled dextran as a competitor in glucose binding to concanavalin A (Ballerstadt et al. 2004) or apo-glucose oxidase

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(Chinnayelka and McShane 2004). Plasmonic amplification of such assay based on gold colloid particles was reported (Aslan et al. 2004). The patients prefer non-invasive or minimally invasive methods for monitoring the glucose level that should be always at hand (Villena Gonzales et al. 2019). The use of optical and portable biosensor strips and devices becomes increasingly important in the remarkable development of sensitive visualization (i.e. visible inspection by the human eye) assays, low-cost analyses and personalized home testing of patients with diabetes (Reda et al. 2021). A special story is the quantitative determination of insulin (Luong et al. 2021; Sabu et al. 2019; Schirhagl et al. 2012). Disorders in the amount of glucose that are the cause of diabetes are the results of improper insulin action. Good glucose management through an insulin dose regime based on the metabolism of glucose helps millions of people worldwide managing their diabetes. Insulin is a large (5.8 kDa) peptide hormone produced by beta cells of the pancreatic islets. It can be detected in human plasma or serum with specific aptamers, antibodies or molecularly imprinted polymers (Luong et al. 2021) using typical for protein and large peptides methods of generating fluorescence response. Its direct point-of-care detection is still a significant problem (Soffe et al. 2018) that has to be resolved with novel nanomaterials (Shafiei-Irannejad et al. 2019) and devices (Singh 2020). Glucose and insulin are the main indicators in the monitoring and control of diabetes, a chronic metabolic disorder lasting for the lifetime of a person. An uncountable number of biosensors have been developed based on various mechanisms of operation, which allow glucose as well as insulin monitoring (Sabu et al. 2019; Villena Gonzales et al. 2019). However, the general strategy is absent. If we assume that monitoring of diabetes biomarkers is needed along the person’s lifetime, then implantable receptor units may be required with long-term service. They should respond as reporters reversibly and without any manipulation. Present fluorescencebased technologies offer such possibility. External instrumentation may be used for fluorescence excitation and reading the information.

14.4 Uric Acid Uric acid (UA, 2,6,8-trihydroxypurine) is the main terminal product of purine nucleotide metabolism that is commonly detected in urine and blood serum (Fig. 14.12). It is the metabolite of high diagnostic value. In a healthy person, its concentration varies between 0.13 and 0.46 mM (2.18–7.7 mg/dL) in plasma and between 1.49 and 4.46 mM (25–74 mg/dL) in urine. Altered serum uric acid concentrations, both above and below normal levels, have been linked to a number of disease states (El Ridi and Tallima 2017; Kutzing and Firestein 2008). An abnormally high uric acid level has been correlated with gout, hypertension, cardiovascular disease, and renal disease, whereas reduced concentrations have been linked to multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, and optic neuritis. Uric acid is not only a marker of these disease states; it may actually play a role in their

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513

Fig. 14.12 Uric acid and its enzymatic transformation by uricase

development and progression. In contrast, at normal concentrations it is very useful, demonstrating antioxidant properties. Therefore, it is necessary to find easy, fast and sensitive methods for determining this compound. For the purpose of uric acid investigation, various analytical techniques are used. They are directed at determining this compound directly and also by the analysis of its oxidation products. The detection methods exploit both enzymatic and nonenzymatic strategies (Wang et al. 2020). For direct determinations, the methods of analytical chemistry, such as liquid chromatography, are frequently applied (Kim et al. 2009; Perelló et al. 2005). However, for practical applications, the use of enzymes with the detection of reaction products was found to be more convenient. The methods of indirect detection of uric acid are based on enzyme coupling. The procedures start from application of uricase that converts this substrate in a coupled reaction with oxygen, releasing hydrogen peroxide (see Fig. 14.12). As an illustration, Fig. 14.13 presents the reaction scheme for uric acid determination using dual enzymes with fluorescent quantum dots as reporting units (Azmi et al. 2015; Jin et al. 2016). In a similar approach, graphene quantum dots emission was quenched when uric acid was first oxidized using uricase, hydrogen peroxide produced and the latter oxidized phenol to benzoquinone in the presence of horseradish peroxidase as the catalyst (Jiao et al. 2021). In order to increase the precision of enzyme-coupled assays, the sensors were suggested that generate the two-band ratiometric fluorescence output (Demchenko 2023a, b). For that, it must be needed that two or more emission bands appear and switching the emission intensity between them occurs under the action of generated H2 O2 . Thus, a certain fluorescence emission peak should be quenched, and the other should not be affected. This idea was realized by coupling of the generation of H2 O2 by uricase with fluorescence quenching of upconversion fluorescent nanoparticles (Fang et al. 2016). By observing differential effect of quenching of their visible fluorescence excited by near-IR light, the λ-ratiometric detection was achieved. Further development of this trend resulted in application of gold/silver nanoclusters (Wang et al. 2019). The use of Cu nanoclusters incorporated into carbon dots allowed achieving the switching of dual emission at 460 and 620 nm by H2 O2 (Ma et al. 2021). In order to avoid application of natural enzymes, researchers have developed different strategies in fluorescence-based methods. One of them is to use the own reducing power of uric acid. For this purpose, carbon dots-MnO2 nanosheets were used as effective fluorescent probes (Amjadi et al. 2018). However, the problem

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Fig. 14.13 Illustration of uric acid detection using the uricase/HRP (horseradish peroxidase) based on H2 O2 -sensitive quantum dots (QDs) (Azmi et al. 2015). In the presence of uricase, uric acid is oxidized to allaintoin and H2 O2 is produced. Based on the quenching effect of H2 O2 on the QDs fluorescence, the uric acid is detected via monitoring the effect of quenching

with this approach is the presence in real biological fluids of other reducers, such as ascorbic acid, which complicates the analysis. The attempts to establish the direct sensing techniques that could provide response just by uric acid binding are rare. A heterocyclic substitute of Bodipy dye on binding with uric acid produces enhancement of dye emission (Pradhan et al. 2015). When the surface of CdTe quantum dots was modified with 2-mercaptoethylamine, their formation of hydrogen bonds with uric acid results in quenching of their fluorescence (Zhang et al. 2011). A designed chelating complex of Tb ion demonstrated quenching of its emission on uric acid binding (Yang et al. 2018). It was reported on successful use of a fluorescent platform with Ag doped carbon dots (Zhuo et al. 2019). Based on the strong affinity of Ag to the nitrogen-containing groups in uric acid, the stable complexes can be formed, in which due to electron and/or energy transfer the fluorescence is quenched. According to reports, the proposed sensing systems were successfully demonstrated for use as the assays of uric acid in human serum or urine samples. Thus, development of uric acid sensors proceeds in several directions. The enzyme-based sensors are expensive and difficult to use, the urease-free reducing sensors are not specific enough in biological media and the promising direct sensors are underdeveloped. There is a lot of room for future progress.

14.5 Cholesterol Cholesterol is an important lipid, being one of the main components of the cell membrane that helps to keep membrane permeability and fluidity (Cebecauer et al. 2018). There exists a strong correlation between total cholesterol and its bound

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forms with lipoproteins in human blood serum with the diseases of heart and blood vessels (Maxfield and Tabas 2005). A large number of clinical disorders are related to arteriosclerosis, in which the role of cholesterol is very important. In blood, there are two forms for cholesterol, 30% in free and 70% in esterified form (Luo et al. 2020). The coupling of enzymes cholesterol esterase, cholesterol oxidase and peroxidase is the most frequently used method to determine total cholesterol in clinical samples, and the detection of hydrogen peroxide (H2 O2 ), a final product of these reactions is usually provided. Nanomaterials are actively used for immobilization of these enzymes (Solanki et al. 2009) and for providing fluorescence response (Barua et al. 2018). There were many attempts to design a sensor that avoids the action of enzymes. A sensitive and selective fluorescent method was based on β-cyclodextrin functionalized carbon quantum dots (β-CD-CQD) using the principle of competitive host–guest recognition (Sun et al. 2017). The bright fluorescence of β-CD-CQD nanoprobe can be effectively quenched by the introduction of a very small amount of p-nitrophenol forming the host–guest interaction. Cholesterol, as a more suited guest molecule of βcyclodextrin, forms an inclusion complex with a much greater binding constant than p-nitrophenol and causes a replacement of guest molecule of β-CD moiety inducing significant fluorescence enhancement. Under the optimized detection conditions, this method shows good analytical performance and enables its use in practical serum samples. The attempts to establish the direct sensing methods that avoid manipulation with enzymes include the application of cyclodextrin dimers labeled with fluorescent reporter dye (Liu et al. 2004) and europium-tetracycline complex (Silva et al. 2008). Later, direct sensing of cholesterol was realized with gold–carbon dot nanoconjugates (Priyadarshini and Rawat 2017). The Au particles perform dual function of displaying colorimetric sensing and fluorescence quenching in response to cholesterol in the concentration range of 10–100 ppm (0.208–2.08 mM), wherein the carbon dots act as the fluorescent entity. The induced by cholesterol precipitation of these particles was quite specific resulting in a visible change in color. It was accompanied with the quenching of carbon dots fluorescence. The natural function of cholesterol is its participation in formation of biological membranes (Cebecauer et al. 2018). Such incorporation into phospholipid structures (see Fig. 14.14) changes dramatically the biomembrane properties and results in formation of very rigid complexes (called ‘rafts’) when this lipid is sphingomyelin. An idea to use the phospholipid bilayer as a direct cholesterol sensor with fluorescence response of environment-sensitive dye has led to the observation of its dramatic λ-ratiometric response to the binding of cholesterol (Turkmen et al. 2005). Other attempts in this direction included the formation of Langmuir–Blodgett sensor surfaces formed of phospholipids (Roy et al. 2018). Molecularly imprinted self-assembled films with specificity to cholesterol have been constructed by the formation of surface-assembled polymer monolayers (Piletsky et al. 1999). The imprinted structures could be formed on gold electrode (Shin and Shin 2020). However, this type of technique was not developed up to the application of fluorescence.

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a

Cholesterol

Hydrocarbon side chain

Steroid nucleus Hydroxyl group

b

Emission

SM SM + Cholesterol DOPC + Cholesterol DOPC + SM + Cholesterol

Fig. 14.14 The structure of cholesterol and the mode of its incorporation into phospholipid bilayer. a Its structure is composed of three parts: the polar part that allows orientation of molecule to lipid heads is represented by hydroxyl group, the central steroid part and the non-polar (hydrophobic) part that extends to the center of bilayer. b Molecular hybrid of steroid with fluorescence reporter transforms phospholipid vesicles into ratiometric cholesterol sensor. The arrow demonstrates dramatic effect on spectra of cholesterol incorporation into sphyngomielin (SM) vesicles and the absence of any effect on DOPC vesicles (Turkmen et al. 2005)

Cholesterol does not float freely in the blood—it must be carried by lipoproteins, the particles formed in the liver that are made of proteins and phospholipids. There are several their types, including high-density lipoprotein (HDL), which helps removing cholesterol from the arteries and prevent fatty buildup, and low density lipoprotein (LDH), which in excess is linked to artery damage, heart disease and stroke. The same cholesterol molecules may be “good cholesterol” when they are incorporated into HDL and “bad cholesterol” when they appear in LDH. These proteins can be isolated and identified by centrifugation. In order to avoid it, a simple blood test called a lipid panel is used to measure total cholesterol, HDL cholesterol and triglycerides. Thus, the LDL cholesterol is estimated rather than directly measured. To calculate LDL, the clinical labs have traditionally used a formula known as the Friedewald equation, according to which the LDL cholesterol is the total cholesterol minus HDL cholesterol minus triglycerides divided by five. This approach is frequently criticized but still used in clinical labs (DeLong et al. 1986; Martin et al. 2013; Song et al. 2021). Cholesterol testing on a smartphone is in high need for personal use (Oncescu et al. 2014). But only the sensing based on paper strips with response based on changing the color of absorbed light can be presented now.

14.6 Sensing and Thinking. The Era of Digital Health is Approaching?

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14.6 Sensing and Thinking. The Era of Digital Health is Approaching? One of the key aims for the future development is achieving the point-of-care settings in clinical analyses without stationary laboratory support (Tu et al. 2020). It is expected that quantitative performance of future devices will soon become comparable to laboratory methods with the advantages of convenience, low cost and high speed. The diabetes patients, the patients in critical conditions in rural locations and the population under the risk of infectious diseases in developing countries have to get the greatest benefit from these developments (Jiang et al. 2021; Syedmoradi et al. 2020; Vashistha et al. 2018). They will help withstanding virus pandemic (Merkoçi et al. 2021). However, addressing this aim, we observe that the progress is unexpectedly slow. Essentially, the limiting factors are not in instrumentation, which is able to support current developments on a sufficient level (O’Sullivan et al. 2019; Romao et al. 2017) but in construction of sensor units. The development proceeds along several directions, some of them are very promising and some—not. Major progress is expected from substituting the procedures requiring manipulation steps with the sample (reagent addition, washing, temperature cycling) by direct ‘mix-and-read’ tests. Microfluidics and paper-based assays systems have limitations (Bhide et al. 2021), and only the direct sensors allow continuous monitoring of required analytes. Presently, the sample and reagent manipulation steps are unavoidable if the amplification of output signal is needed for increasing the copies of target molecules or of fluorescent product. Therefore, we have to avoid such amplification by increasing dramatically the efficiency of fluorescence (luminescence) reporting. Possibilities for that exist, as can be seen in other chapters in this book. They are in using novel materials and forming their nanocomposites, in optimizing the photophysical mechanisms of their response. The other direction of future progress is in making our tests non-invasive or lowinvasive for the patient. Blood in systemic circulation reflects the state of health or disease of most organs providing a sensitive assessment of health and disease. Its analysis will continue as well as the use of urine as the traditional test material (Lei et al. 2020). Meantime, the possibility of dramatic decrease of sample volume and of strong increase of sensitivity will result in broader application of other biological fluids, such as sweat, saliva or tears (Ates et al. 2021; Ghaffari et al. 2021b). The third direction that determines the progress is the achieving of multiplicity of analyzed data attributed to particular person. Parallel sensing of many targets including specific and metabolite biomarkers should be analyzed with algorithms clearly understood to doctors making clinical decisions. Only on this condition, the attempts of establishing personalized medicine will make sense. It is expected that the future sensors will possess integrated decision-making units based on artificial intelligence (Haick and Tang 2021; Jin et al. 2020), connection to global databases and Internet communication abilities with health expert and medical center (Jain et al. 2021). It was stated that “nearly every person will experience diagnostic error in their lifetime” (Haick and Tang 2021), and integrated human

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knowledge must be mobilized to avoid that. We enter the era of digital health (Tu et al. 2020). The following questions and problems are addressed to the readers: 1. What is a conceptual difference between ex-vivo and in-vivo diagnostics? On what levels of organization of living matter they are directed? 2. What are the metabolic and what are specific protein-based biomarkers? Present examples. 3. Analyze the present state of biomarker tests in clinical laboratories that you know. Formulate, to what requirements should satisfy the future sensor devices. 4. Provide critical analysis of clinical tests involving different biological media: blood, serum, saliva, etc. What specific demands they pose to fluorescent sensors? 5. List the methods of glucose determining and explain their basic recognition and reporting mechanisms. 6. Explain the properties of boronic acid. Why it is so attractive for the design of fluorescence glucose sensors? What are the principles of construction of these sensors? 7. There are two general trends in construction of sensors for uric acid. What are they? Estimate their advantages and disadvantages. 8. What is total cholesterol and how it can be determined? 9. How to determine lipoproteins and their fractions?

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Chapter 15

Imaging and Sensing Inside the Living Cells. From Seeing to Believing

The full power of fluorescence technique can be demonstrated in imaging and sensing within living cells. Its most important advantage that will probably be never competed by any other technique is a high spatial resolution together with ultimate contrast and sensitivity obtained in a noninvasive or low invasive manner. Exploration of these possibilities has led to development of powerful methods of fluorescence microscopy, such as confocal, two-photon and evanescent-wave microscopy. Resolution in time and anisotropy were successfully applied to microscopy. Recent advances allowed achieving superresolution images and observing individual molecules. Humans have an uncanny visual capability to recognize patterns, making microscopy a powerful analysis tool that develops rapidly since the first microscopes were produced at the end of the 16th century. The present-date microscopy in fluorescent light is quite different from common light transmission microscopy. The optical contrast needed in transmission imaging is not important here and there is no need to enhance it by dye staining. Instead, the dyes can be applied in much lower concentrations so that they do not interfere with cellular life. They can form fluorescent image contrasting different structures and organelles, demonstrating distributions of different types of molecules and their dynamics. High resolution in images is achieved due to focusing the exciting light beam to particular sites forming focal plane or focal volume, and this allows suppression of out-of-site emission. The illuminated volume can be dramatically reduced, which leads to detection of single molecules. In order to achieve localization of macromolecules and formation of image with molecular resolution, the fundamental restriction known as diffraction limit has been overcome by recently developed techniques. All these achievements become very useful when they provide addressing fundamental problems of cellular biology and also when they allow characterizing the species inside the cell that are of diagnostic and prognostic value. Extremely important are the possibilities of imaging the interactions between macromolecules and their biocatalytic transformations. Manipulation with fluorescent dyes and nanoparticles allows many possibilities for their use as tags, probes and sensors (Schäferling 2012). The applied species can be intrinsic or extrinsic to the cell; they can be not only © Springer Nature Switzerland AG 2023 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-031-19089-6_15

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silent observers but also participants, modulators or disruptors of specific activities that form biological functions.

15.1 Modern Fluorescence Microscopy The basic function of a fluorescent microscope is to provide the image of studied object in fluorescent light with the full rejection of incident light. Advancement in such instrumentation stimulated a great progress in imaging. For forming the image that is informative for sensing in fluorescent light, a variety of different dyes were suggested as the labels, tags, probes and sensors. Discussion of different practical aspects of fluorescence microscopy can be found in books (Chiarini-Garcia and Melo 2011, Markaki et al. 2017) and reviews (Chen and Lukinaviˇcius 2018; Galas et al. 2018). There is a choice to compose an image based on selected parameter characterizing their emission (Ishikawa-Ankerhold et al. 2012). Commonly it is the light intensity, but it can be also anisotropy, lifetime and ratio of intensities at selected wavelengths (λ-ratiometry) (Demchenko 2023a, b). Microscopes allow the selection of excitation wavelengths (often, in a limited range) and detection at different emission wavelengths that are usually selected in a broader range by the filters. Thus, the studied object is illuminated by light of an (almost) desired wavelength, and then the much weaker emitted fluorescence is separated from the excitation light, an image is formed. This image can be recorded with a high contrast against the background. There are two sources of this background, and the instrument constructors and researchers combine their efforts to eliminate (or, at least, to reduce) them. One is the intrinsic fluorescence of cellular components (autofluorescence). It is caused by such pigments, as reduced nicotinamide dinucleotide (NADH) and flavine adenine dinucleotide (FAD) that are always present in a living cell. Such background signal can be reduced by proper selection of excitation and emission wavelengths (usually, by shifting from near-UV and violet to green and longer wavelengths). Of course, the applied fluorescence reporter should have the highest brightness in these conditions. The other source of background is fundamentally unavoidable but can be dramatically reduced by optical means. It is the excitation of emitting species by out of focus light. If close and distant objects are in focus, the image looks flat. This happens in epi-fluorescence microscopy. But if the researcher wants obtaining a focused image at particular depth of studied object, the out-of-focused light should be rejected. Actually, the aim here is the opposite of that in common photography, where a photographer may benefit from keeping both close and distant objects in focus. Here various optical principles and instrument constructions are employed to restrict the excitation and detection of fluorophores to a thin region of the sample (focal plane) and to make the fluorescence from this region much stronger than that coming from the regions outside it. Below, we will overview three technologies that provide dramatically increased response from the dyes located within the focal plane. These are the total internal reflection microscopy, the confocal microscopy and the

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two-photon microscopy. Elimination of background fluorescence from outside the focal plane can dramatically improve the signal-to-noise ratio, and consequently, the contrast of obtained images. With all these advancements, the spatial resolution remains the most important factor to be considered. The fundamental limit is given by the length of light wave and is known as the diffraction limit. It is associated with the name of Ernst Abbe, who in 1873 formulated this limit as the ability of a lens-based optical microscope to discern only those details that are larger than a half of the wavelength of light. The diagram presented below (Fig. 15.1) may help the reader in orienting in dimensions of the cellular components and the whole cells in comparison with the wavelength of light. The objects with dimensions smaller than 200–400 nm (presented to the left of grey vertical band) are not resolvable by conventional image-making microscopy. Thus, two or more orders of magnitude separate the best resolution that can be obtained by conventional diffraction-limited optical techniques and the molecular level. However, there are two possibilities to overcome this limit in producing the fluorescence images. One is to make a waveguide with a pointed edge of nanometer dimension and to provide scanning with a ‘nano-beam’; this is the near-field fluorescence microscopy. Recently, the other possibility to break the diffraction limit was suggested and realized in both scanning and wide-field optical nanoscopy.

Fig. 15.1 The sizes of cells and of their components with respect of wavelength of visible light

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15.1.1 Epi-Fluorescence Microscopy The wide field epi-fluorescence microscopy is the most popular fluorescence method in cellular research. In this configuration the excitation and observation of the fluorescence are from above (epi) the specimen, in contrast to common microscopes that collect transmitted light. This type of microscopy has become an important tool in the field of biology, opening the doors for more advanced technical designs. The construction of epi-fluorescence microscope (Fig. 15.2a) allows very efficient collection of fluorescent light that forms the image. The epi-configuration allows the microscope letting the excitation light illuminate the specimen and then sort out the much weaker emitted light from the scattered excitation light to form the image. For that, the microscope has a filter that allows passing only the radiation with the desired wavelength that matches the fluorescing material. The emitted light is separated from the much brighter excitation light with a second filter. Here, the fact that the emitted light has a spectrum at longer wavelengths (being Stokes-shifted) is used. The bright fluorescing areas can be observed in the microscope against a dark background with a high contrast.

Fig. 15.2 Schematic representation of a simplest epi-fluorescence microscope (a) and total internal reflection fluorescence microscope (b). In epi-fluorescence microscope the incident excitation light, after passing the optical filter, is focused on the specimen from above. Fluorescent light is directed to the detector after passing the filter that rejects the reflected and scattered excitation light. The total internal reflection microscope (the scheme is shown with illumination ‘from below’ and detection ‘from above’) is different. Here the incident beam does not penetrate into the medium, where the specimen is located, and excites its fluorescence by producing evanescent field in a narrow pre-surface area

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The major disadvantage of this microscope is that it allows collecting the image of a specimen with the three-dimensional distribution of fluorescent species as a twodimensional picture formed by both in-focus and out-of-focus located fluorescence emitters. It produces ‘photographic’ imaging of the object, in which sometimes it is hard to judge, what is in front and what is behind. Thus, a significant part of spatial information can be lost.

15.1.2 Total Internal Reflection Fluorescence Microscopy (TIRF) The total internal reflection microscopy takes advantage of the evanescent wave that exists when the light is totally internally reflected at the interface between two media having dissimilar refractive indices (see Sect. 16.3 of Volume 1 for discussing the mechanism of evanescent wave generation). In this technique, a prism of sapphire or special glass with high refractive index is used to illuminate the sample that is located on top of it. If the light is directed into the prism at an angle higher than the critical angle for the interface of two media, the beam will not enter the studied specimen in low-refractive aqueous environment and will be totally internally reflected at the interface. Meantime, some of the light energy can propagate at a short distance and excite fluorescent dyes close to the interface. The reflection phenomenon develops at the interface a so-called evanescent wave that permeates about 100–200 nm outside the interface. The light intensity of evanescent wave is sufficiently high to excite the dyes, but remains located within this short distance. Because of such shallow penetration depth of excitation energy, the x–y plane close to the interface becomes in fact the focal plane (Fig. 15.2). Depicted in Fig. 15.2b is the simplest configuration of microscope based on this principle; it allows many modifications. They are well described in the literature and in information materials provided by the instrument manufacturers. Because the excitation of fluorophores in the bulk of the object is avoided and the fluorescence emission is confined to a very thin region, a much higher signal-to-noise ratio is achieved compared to conventional epi-fluorescence imaging. Some instruments allow varying the illumination incidence angle and, consequently, the penetration depth of the evanescent wave. This allows increasing the resolution along z axis and distinguishing the depths of dye location on a nanometer scale. Meantime, these variations are limited to the pre-surface area and do not allow seeing the whole cells.

15.1.3 Confocal Fluorescence Microscopy Confocal fluorescence microscopy offers a different principle of forming sharp images. The conventional focusing system is applied here, in which the excitation

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Fig. 15.3 Schematic representation of a typical confocal microscope showing the beam geometry that allows rejection of photons emitted from outside the focal plane

of out-of-focus located dyes by incident beam is not avoided, but an optical configuration of the instrument allows rejecting their emission. This is achieved with the application of scanning the focal plane with the use of confocal pinhole that provides geometric restriction to passing the out-of-focus emission (Fig. 15.3). Application of this principle allows achieving very sharp fluorescence images due to dramatically improved signal-to-noise ratio. In confocal microscopy, the resolution along z axis is on the level of 1 μm, which is by one order of magnitude less than in total internal reflection microscopy. But the great advantage here is the possibility to move the focal plane providing scanning the object at different z levels, so that the cross-sections at various depths can be obtained. If necessary, by computational means these cross-sections can be transformed into the three-dimensional image of the object. A remarkable explosion in the popularity of confocal microscopy in recent years is due in part to the relative ease, with which the high-quality crosssectional images can be obtained from the samples prepared for conventional optical microscopy. One weak point of “classical” confocal microscopy is the requirement to use the high-intensity lasers for excitation and to illuminate with them a large volume, not only within the focal plane. It generates not only fluorescence but also produces

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photobleaching and phototoxicity in the whole illuminated volume throughout the object. Involvement of these factors depends strongly on the properties of used dyes. An attempt to overcome this limitation is to form an image by simultaneous illumination of many pinholes, as in the “spinning disk” microscopes. Confocal imaging here is provided by synchronous rotation of two discs (called Nipkow disks). One of these disks contains about 20,000 microlenses, and the other is placed with the same number of pinholes (Straub et al. 2000). Passing through the array of microlenses and focusing upon correspondent pinholes, the excitation laser beam can create about 1000 independent focal volume elements. By rotating the disk pair, a full high-resolution confocal image can be acquired within 50 ms. The disadvantage of rotating disks is that the pattern of illumination and detection cannot be adjusted at will for particular purpose.

15.1.4 Programmable Array Microscope The optical sectioning ‘intelligent’ Programmable Array Microscope (iPAM) can serve as an add-on module for conventional commercial wide-field fluorescence microscope, converting it into a high-speed optical sectioning (“confocal”) instrument (De Beule et al. 2011; de Vries et al. 2015). It is unique in that it combines both wide field of view and high speed, without compromising the imaging performance. In iPAM the structured illumination and structured detection operate in synchrony. A single digital micro-mirror device (DMD) acts as a spatial light modulator defining a pattern in both the fluorescence excitation and emission paths. A sequence of binary patterns of excitation light is projected into the focal plane of the microscope. The resulting fluorescent emission is captured as two distinct signals: conjugate, c (“onfocus”) consisting of light impinging on and deviated from the “on” elements of the DMD, and the non-conjugate, nc (“out-of-focus”) light falling on and deviated from the “off” elements (Fig. 15.4). Thus, the c image comprises the in-focus information and part of the out-of-focus background, while the nc image receives the rest of the out-of-focus background. The two distinct, deflected beams are optically filtered and detected either by two individual cameras or captured as adjacent images on a single camera after traversing an image combiner. The sectioned image is gained from a subtraction of the nc image from the c image, weighted in accordance with the pattern(s) used for illumination and detection and the relative exposure times of the cameras. The wide-field image is given by the sum of the c and nc images. This system can function at very high acquisition speed with low light intensities, preventing saturation and minimizing photobleaching of sensitive fluorophores. The programmable array allows optimization of the patterns (duty cycle, feature size and distribution), thus enabling a wide range of applications, from patterned photobleaching to superresolution microscopy.

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Fig. 15.4 Operation principle of programmable array microscope (above) and comparison of the conjugate, non-conjugate and the resulting sectioned images (below). The conjugate image reveals the extra in-focus detail in the image that is missing in the non-conjugate image. It is this in-focus component in the conjugate image which is left after (weighted) subtraction of the non-conjugate

15.1.5 Two-Photon and Three-Photon Microscopy In Volume 1, Sect. 16.1 of this book, we described the physical background of a phenomenon that brings the electronic system to an excited state by absorption of two photons simultaneously. Two photon excitation occurs only when the photon density is very high, which can be realized by confining the light flux on a spatial and temporal scale. Very short pulses of a well-focused laser can do this. In this case, the laser focal point is the only location along the optical path where the photon density is high enough to generate a significant occurrence of two-photon excited species. The generation of two-photon excitation in a fluorophore-containing specimen at the microscope focal point on a focal plane is illustrated in Fig. 15.5. Above and below the focal point, the photon density is not sufficiently high for two photons to pass at the same instant within the absorption cross section of a single fluorophore. Only at the focal point, this density can be so high that the two photons will be absorbed simultaneously with sufficient probability. Thus, if we want to express in two words the key difference between confocal and two-photon microscopy, we must say: in confocal microscopy the out-of-focus background is rejected, but in two-photon microscopy it does not appear!

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Fig. 15.5 Illustration of the principle of two-photon and three-photon microscopy. The focused light provides the high photon density only at the focus plane, so that it can be absorbed by only in-plane dyes, thus forming a sharp image. The near-IR laser can excite ‘blue-green’ and ‘near-UV’ emissions by two-photon and three-photon absorption, respectively

This phenomenon has found a very important application in microscopy because it brings many benefits. (a) In contrast to confocal microscopy, the two-photon microscopy allows concentrating all the excitation in a narrow focal plane. Outside this plane, the dyes are not excited simply because the photon flux is insufficient. This brings not only a sharp image but also the absence of photobleaching and phototoxicity outside the focal plane. (b) In two-photon microscopy, the wavelengths of excitation and emission differ dramatically (for instance, the dye with normal absorption band maximum at 520 nm can be excited by two photons of 1050 nm laser, and an emission is detected at 560 nm). Therefore, the scattered excitation light is easily rejected. (c) The cells and tissues are relatively transparent in the near-IR, so for exciting with two photons an intensive light can penetrate without essential losses at substantial depths into the living tissue or even inside intact animal specimens. Additionally, because there is no absorption in out-of-focus specimen areas, more of the excitation light penetrates through the specimen to the plane of focus. Together with the formation of focal planes at substantial and modulated depths this method provides unique prospects in imaging. The three-photon excitation occurs in much the same way as the two-photon process. One of its benefits is the possibility to provide an excitation of two dyes absorbing light at two quite different wavelengths with the same laser. With the 1050 nm laser the blue-green dye can be excited by two photons at about 520 nm whereas a near-UV absorbing (at about 350 nm) dye can be excited simultaneously by three photons. Fluorescence of these two dyes can form a two-color image. New possibilities appear with intracellular applications of reporting dyes operating by variation of lifetime with two-photon excitation microscopy that utilizes pulsed lasers. Detection of various analytes and establishing proximity relations between molecules often use the excitation energy transfer (EET) methodology

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(Sect. 4.3 of Volume 1), and the best applicable to cellular research EET detection is the lifetime imaging. Different two-photon excited probes were developed for visualization of sub-cellular structures and measuring local pH and concentrations of analytes (Lim 2013; Zhang et al. 2013).

15.1.6 Time-Resolved and Time-Gated Imaging The fluorescence lifetime imaging (FLIM) has become one of the most powerful techniques in cellular research (Berezin and Achilefu 2010; Datta et al. 2020; Kumar 2011). Lifetime is the fluorescence parameter that is extremely sensitive to intermolecular interactions. In sensing applications it can be used in different aspects, mostly in configurations that allow dynamic fluorescence quenching and EET. Lifetime is the dye concentration-independent parameter but it requires clever operation, since totally quenched species cannot be detected (Sect. 3.4 of Volume 1). The purpose of FLIM is by measuring the lifetime at each point of the image to detect the variations of intermolecular interactions (Marriott et al. 1991). These variations can be correlated with the presence of target species and their interactions. A number of fluorescence reporters have been suggested for lifetime imaging (Berezin and Achilefu 2010). This technique is especially applicable for working with the visible fluorescent proteins, such as GFP (Sect. 5.5 of Volume 1), since these proteins, being EET donors, easily exhibit the dynamic quenching effects but do not respond so easily by spectral changes. The two-photon lifetime imaging develops with the use of novel fluorescent dyes (Li et al. 2018). The time-resolved microscopy is implemented in two different ways. Both of them came from time-resolved spectroscopy. One uses the time-domain measurement and the detection with single-photon counting technique (Suhling et al. 2019). Its simplified version is the time-gated technique that allows collecting the quanta within a selected time window (see Sect. 3.4 of Volume 1). Close in methodology and simpler in performance is the stroboscopic technique, which is based on formation of the time window by opening and closing the detector voltage by synchronized pulses (strobs). The frequency domain measurement uses a different principle. It is based on excitation by frequency-modulated light, so that the lifetime information can be obtained from phase shift and demodulation of excitation light (Lakowicz 2007). The application of time-gated microscopy is reasonable when there is a need to distinguish between the emissions possessing well separated lifetimes, and this necessitates acquiring and analysis of two sets of images (at short and long lifetimes). This technique is the best suited for the images formed by lanthanide chelates and metal–ligand complexes, possessing long lifetimes (Maury et al. 2014). The timeresolved imaging allows more flexibility, and a combination of intensity and lifetime imaging allows improving the application of EET (Hanley et al. 2002), which is a key approach to elucidating the proximity relations and dynamics inside the cell.

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15.1.7 Wavelength-Ratiometric Imaging In the studies of living cells and other heterogeneous microscopic objects, the great advantages of λ-ratiometry (Sects. 3.5–3.6 of Volume 1) can be efficiently realized providing imaging in different colors of targets distributions and their transformations (Demchenko 2010, 2023a, b). This technique was successfully stimulated by introduction of the first Ca2+ probes (Grynkiewicz et al. 1985) and continues its development and exploration, addressing different targets and employing different mechanisms of wavelength-switching, such as ESIPT and EET (see Chap. 4 of Volume 1). One of such techniques was applied for detecting and characterizing the cell membrane transformations in the course of apoptosis with the use of the two-band electrochromic dye (Shynkar et al. 2007). In this work the two images in different colors are taken in confocal microscope using different photodetectors and then the ratio of these images was calculated (Fig. 15.6). In this case both intensity and color are informative: the intensity demonstrates the distribution of emissive fluorophores, while the color shows the ratio values. The technique of obtaining multicolor λ-ratiometric images can be simplified by avoiding the use of expensive CCD cameras, substituting them by PMT. Also, the CMOS matrices that are used in every smartphone are quite applicable for that (see

Fig. 15.6 One of the possibilities to construct ratiometric image in microscopy (Shynkar 2005). The fluorescence spectrum of 3-hydroxychromone biomembrane fluorescence probe F2N12S is presented above. The green and red parts of this spectrum are selected with filters and recorded over the image with different detectors. Then the ratio of two images is calculated. Presented in pseudocolor, it does not depend on the distribution of dye molecules but only on the redistribution of intensity in their spectra between two channels

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Sect. 15.2 in Volume 1). Importantly, the multicolor λ-ratiometric images can be easily combined with polarization (Jameson and Ross 2010) and lifetime imaging (Peng et al. 2011).

15.1.8 Traditional Far-Field Fluorescence Microscopy: Advances and Limitations Several trends of traditional far-field (lens-based) optical imaging demonstrate its usefulness, but approach the limit of perfectness in their development. All possibilities of fluorescence spectroscopy (spectral resolution, anisotropy, and lifetime) can be realized in microscopy, the simple epi-fluorescent microscope remains basic in many studies competing with more advanced techniques. Out-of-focus emission spoils the image and therefore different microscopic techniques were devised, offering optical sectioning, i.e. the ways to reject the out-of-focus light and maintain only in-focus information in the final image. In a confocal microscope, the fluorescence is excited through the illuminated volume of the specimen, but only the signal originating in the focal plane passes through the confocal pinhole, allowing collecting the background-free data, but this greatly reduces the number of quanta reaching the detector. By contrast, the twophoton excitation generates the fluorescence only at the focal plane, and, since no background fluorescence is produced, a pinhole in front of the detector is not required. In both methods, the achievable spatial resolution along z axis is similar, it is about 500–600 nm. In this aspect, they are behind the total internal reflection microscopy, in which this resolution is about 100 nm, but without the possibility to move the focal plane located at the interface. All three methods are adaptable for time-resolved measurements. The timeresolved imaging, with or without using of EET, allows making important steps towards real intracellular sensing of many analytes. It is an advantageous version because the fluorescence lifetime is concentration-independent, and a single emission wavelength is sufficient to be used for detecting the motions that cause dynamic quenching (see Sect. 3.4 of Volume 1). Its disadvantages, however, are in a more complicated microscope construction and the low number of photons that are usually collected per pixel for scanning the images. Also, the presence of any dynamic quencher in the studied system would interfere with the results. The in-plane resolution in conventional fluorescence microscopy has reached the values of 200–300 nm and approached the diffraction limit. This does not allow resolving molecules and molecular structures having smaller dimensions, as seen in Fig. 15.1. Other limiting factors remain also important. The scanning confocal microscopy requires high power of excitation light, which is not tolerable by many organic dyes (Demchenko 2020), and photostable inorganic nanocrystals are often larger in size than their macromolecular targets. Slow speed of collecting

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the images makes working with live cells in their natural environment complicated. Programmable array microscopy offers the possibility of overcoming these limitations. The information content of obtained images becomes the key factor. It is not enough to localize the cell organelles and determine their number and sizes. An amount and localization of at least some out of many thousand types of molecules is of major interest, and this constitutes the intracellular sensing.

15.2 Far-Field Super-Resolution Microscopy Super-resolution microscopy demonstrates dramatic development and broad range of applications in cell biology (Schermelleh et al. 2019). The spatial resolution of standard optical microscopy techniques is limited to roughly half the wavelength of light. As a result of diffraction, the image of an arbitrarily small source of light imaged using a lens-based microscope is not a point but a point spread function (PSF), with a central peak approximately ~200–300 nm in width, resulting in a blurring of structures below this spatial scale (Lelek et al. 2021). Super-resolution methods break this limit (Vangindertael et al. 2018). They are generally classified into three main concepts: stimulated emission depletion (STED), single molecule localization microscopy (SMLM) and structured illumination microscopy (SIM) (Reymond et al. 2020).

15.2.1 Breaking the Diffraction Limit Improving the spatial resolution is a strong demand from the side of researchers and practical users of intracellular sensing. Resolution is the power to resolve objects from each other, and in optical imaging it is limited by the diffraction of light wave. Therefore, the lens-based amplification in light microscopy has its limit. The mathematical expression for the diffraction limit was formulated in 1863 by Ernst Abbe: the two spots in the microscope image (Fig. 15.7) cannot be resolved if their separation distance is smaller than the d value that equals to λ/2nsinα in the focal plane (xy) and 2λ/nsin2α along the optical axis (z). Here λ is the wavelength of light and nsinα is the numerical aperture of the lens. As most lenses have a numerical aperture of