Aptamers in Biotechnology [1st ed.] 9783030540609, 9783030540616

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Advances in Biochemical Engineering/Biotechnology  174 Series Editor: T. Scheper

Katharina Urmann Johanna-Gabriela Walter   Editors

Aptamers in Biotechnology

174 Advances in Biochemical Engineering/Biotechnology Series Editor T. Scheper, Hannover, Germany Editorial Board S. Belkin, Jerusalem, Israel T. Bley, Dresden, Germany J. Bohlmann, Vancouver, Canada M.B. Gu, Seoul, Korea (Republic of) W.-S. Hu, Minneapolis, USA B. Mattiasson, Lund, Sweden H. Seitz, Potsdam, Germany R. Ulber, Kaiserslautern, Germany A.-P. Zeng, Hamburg, Germany J.-J. Zhong, Shanghai, China W. Zhou, Shanghai, China

Aims and Scope This book series reviews current trends in modern biotechnology and biochemical engineering. Its aim is to cover all aspects of these interdisciplinary disciplines, where knowledge, methods and expertise are required from chemistry, biochemistry, microbiology, molecular biology, chemical engineering and computer science. Volumes are organized topically and provide a comprehensive discussion of developments in the field over the past 3–5 years. The series also discusses new discoveries and applications. Special volumes are dedicated to selected topics which focus on new biotechnological products and new processes for their synthesis and purification. In general, volumes are edited by well-known guest editors. The series editor and publisher will, however, always be pleased to receive suggestions and supplementary information. Manuscripts are accepted in English. In references, Advances in Biochemical Engineering/Biotechnology is abbreviated as Adv. Biochem. Engin./Biotechnol. and cited as a journal.

More information about this series at http://www.springer.com/series/10

Katharina Urmann • Johanna-Gabriela Walter Editors

Aptamers in Biotechnology With contributions by J. Bahnemann
N. Bahner
E. Boschetti
J. G. Bruno
V. Calzada
B. Chaterjee
A. Eilers
H. Kaur
G. Perret
M. Plach
J.-A. Preuß
P. Reich
T. Schubert
T. K. Sharma
J. Walter
S. Witt

Editors Katharina Urmann Basel, Switzerland

Johanna-Gabriela Walter University Hannover Gottfried-Wilhelm Leibniz Hannover, Niedersachsen, Germany

ISSN 0724-6145 ISSN 1616-8542 (electronic) Advances in Biochemical Engineering/Biotechnology ISBN 978-3-030-54060-9 ISBN 978-3-030-54061-6 (eBook) https://doi.org/10.1007/978-3-030-54061-6 © Springer Nature Switzerland AG 2020 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

Preface

We are very pleased to present the current volume of the series Advances in Biochemical Engineering and Biotechnology entitled “Aptamers in Biotechnology.” In 1990, Professors Larry Gold and Craig Tuerk developed the SELEX process and discovered that nucleic acids could be used as ligands specifically binding to proteins. Simultaneously and independently, these protein binding nucleic acids were discovered by Andy Ellington and Jack Szostak, who termed this new class of oligonucleotides “aptamers.” In the early years of aptamer research, most efforts were focused on the exploitation of aptamers in the medical field. In 2004, this resulted in Macugen, the first FDA-approved aptamer-based drug, for the treatment of macular degeneration. Nonetheless, the favorable properties of aptamers, such as simple and economic large-scale production, high stability, and switchable structural changes made them also ideal candidates for applications outside of therapeutics. Today, 30 years after the discovery of SELEX, aptamers have penetrated almost all areas of biotechnological research, ranging from classical biotechnological approaches such as biosensing, to modern red biotechnology, where aptamers can be used for the development of targeted treatment strategies. This book attempts to cover this broad range of applications. Since the selection of aptamers is intensively reviewed elsewhere, this book starts with a chapter on the characterization of aptamers, as a thorough characterization is the basis for each successful application. Within the next chapters, the use of aptamers in biosensing is described exemplarily for impedimetric aptamer-based biosensors, where the underlying principles are described and the broad variety of possible applications are elaborated. Besides biosensing, the affinity separation of proteins is another classical field of biotechnology that can profit from the use of aptamers and is thus represented as a chapter in this book. Finally, diverse medical applications are summarized, including the use of aptamers in diagnostics and targeted therapies.

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Preface

We hope that this collection of chapters and the wide variety of included topics will be useful, not only for experts in the field but also for researchers starting their work with aptamers. The chapters cover important current aspects, as well as recent developments and will hopefully inspire future research work. Last but not least, we would like to thank all the authors for their contributions, as well as Springer for the implementation and support of this project. We would like to thank Ms. Alamelu Damodharan and Prof. Thomas Scheper for their excellent support during the preparation of this volume. Basel, Switzerland Hannover, Niedersachsen, Germany

Katharina Urmann Johanna-Gabriela Walter

Contents

Biophysical Characterization of Aptamer-Target Interactions . . . . . . . . Maximilian Plach and Thomas Schubert

1

Impedimetric Aptamer-Based Biosensors: Principles and Techniques . . Peggy Reich, John-Alexander Preuß, Nicole Bahner, and Janina Bahnemann

17

Impedimetric Aptamer-Based Biosensors: Applications . . . . . . . . . . . . . John-Alexander Preuß, Peggy Reich, Nicole Bahner, and Janina Bahnemann

43

Aptamer-Based Affinity Chromatography for Protein Extraction and Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Perret and E. Boschetti

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Aptamers in Diagnostic and Molecular Imaging Applications . . . . . . . . 141 Victoria Calzada Aptamer-Modified Nanoparticles in Medical Applications . . . . . . . . . . . 161 Alina Eilers, Sandra Witt, and Johanna Walter Defining Target Product Profiles (TPPs) for Aptamer-Based Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 Harleen Kaur, Bandhan Chaterjee, John G. Bruno, and Tarun Kumar Sharma Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211

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Adv Biochem Eng Biotechnol (2020) 174: 1–16 DOI: 10.1007/10_2019_103 © Springer Nature Switzerland AG 2019 Published online: 3 August 2019

Biophysical Characterization of Aptamer-Target Interactions Maximilian Plach and Thomas Schubert

Contents 1 Basic Binding Parameters in Aptamer Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Biophysical Techniques to Study Basic Binding Parameters of Aptamers and Their Target Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Biophysical Principles and Readouts of Selected Techniques . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Sample Material Requirements of the Selected Biophysical Methods . . . . . . . . . . . . . . . . 2.3 Application Range of the Selected Biophysical Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . 3 The Characterization Strategy: Aspects to Consider . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Affinity Constant: Estimated by Biophysical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Summary and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 3 4 8 10 11 11 13 14

Abstract Aptamers are single-stranded nucleic acid molecules forming welldefined 3D structures. Aptamers typically bind to their ligands with high affinity and specificity. They are capable of interacting with various kinds of ligands: ions, small molecules, peptides, proteins, viruses, bacteria, and even cells. Therefore, aptamers are in widespread use as sensor molecules or as targeting agents in diagnostics and pharmaceutics. As a prerequisite for their use in these economic high-value areas, aptamers must be studied in detail with respect to different biophysical characteristics. Of central importance are basic binding parameters of the aptamer-target interaction, such as binding affinity and kinetics. Numerous biophysical methods with different features, characteristics, and capabilities are used in the field today for this purpose. This chapter provides an overview of the current state-of-the-art technologies for studying interactions between aptamers and targets and discusses their advantages as well as drawbacks. Furthermore, essential aspects influencing any aptamer M. Plach and T. Schubert (*) 2bind GmbH, Regensburg, Germany e-mail: [email protected]

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characterization strategy will be presented. Finally, issues of comparability of binding data between different aptamer characterization technologies will be discussed. Graphical Abstract

Keywords Affinity, Binding parameters, Biolayer Interferometry, Biophysical characterization, EMSA, Filter-Binding Assay, Flow Cytometry, Fluorescence Polarization, Isothermal Titration Calorimetry, Kinetics, MicroScale Thermophoresis, Surface Plasmon Resonance, Thermodynamics, SwitchSense

1 Basic Binding Parameters in Aptamer Development Basic biophysical binding parameters such as affinity, kinetics, or thermodynamics are key aspects in the development of aptamers for pharmaceutical and diagnostic use. Binding affinity is a measure of binding strength between aptamer and target and is usually reported as an equilibrium dissociation constant (KD). The lower this constant, the higher the binding strength between aptamer and target. In the course of aptamer development, affinity is often used to initially rank a pool of aptamers to select relevant candidates. In addition, affinity enables to express selectivity of

Biophysical Characterization of Aptamer-Target Interactions

3

different aptamers to one target or the specificity of an aptamer to one or few of multiple targets. Binding kinetics describes the time-dependent, dynamic component of the binding event between aptamer and target. The association rate constant ka (kon, in M 1 s 1) describes the association of aptamer and target to the binary (or higher order) complex over time. The dissociation rate constant kd (koff, in s 1) describes the rate of dissociation of aptamer and target and is hence a measure of temporal stability of the aptamer-target complex. In order to ensure proper functionality of an aptamer in its final application (e.g., in diagnostic use), aptamers with desired binding kinetics may be chosen during the development phase. Binding thermodynamics describe the enthalpic (ΔH) and entropic ( TΔS) parameters of the interaction between aptamer and target, which will only occur spontaneously when the Gibbs free energy (ΔG) of the interaction is negative (either enthalpy or entropy driven). The enthalpic parameter ΔH is the energy change resulting from the formation of non-covalent interactions between aptamer and target and the changes of hydrogen bond and van der Waals interactions between aptamer, target, and the solvent. The entropic counterpart ΔS represents the global thermodynamic property of the system, hence the degree of freedom of the system. Thermodynamic parameters are thus helpful to understand the molecular principles of aptamer-target interactions and to optimize aptamers with respect to certain thermodynamic characteristics. In the following, different biophysical methods to characterize aptamer-target interactions are described. Besides the seven well-established and broadly applied methods, Surface Plasmon Resonance (SPR) [1–5], Biolayer Interferometry (BLI) [6, 7], Isothermal Titration Calorimetry (ITC) [2, 8], Fluorescence Polarization (FP or FA) [9, 10], Flow Cytometry [11, 12], Filter-Binding Assay (FB)/Filter Retention Assay [4, 13], and Electromobility Shift Assay (EMSA) [14, 15], two emerging technologies SwitchSENSE (SwS) [16] and MicroScale Thermophoresis (MST) [3, 13, 17] are discussed.

2 Biophysical Techniques to Study Basic Binding Parameters of Aptamers and Their Target Molecules Numerous physical and biophysical methods and technologies are available today for determining the aforementioned basic binding parameters of aptamer-target interactions. The methods differ with respect to the type of physical readout and their information content. Some determine binding parameters in an indirect manner, others by direct readout; some work in solution, whereas others require immobilization of either target or aptamer to a solid phase. Modification-free, label-free, or fluorescent technologies are available. Some methods characterize interactions in a steady-state equilibrium, whereas others analyze dynamic binding kinetics. Some techniques are capable of studying interactions between aptamers and whole cells,

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and others can detect binding of the smallest ions to an aptamer. Furthermore, all available technologies have different prerequisites and requirements with respect to the sample material. Development of an aptamer for later use in diagnostics or as a therapeutic agent thus needs careful selection of the right tools and methods at the right time and for the right application.

2.1

Biophysical Principles and Readouts of Selected Techniques

Typically, a complex of two interaction partners differs from the respective individual molecules in many molecular parameters, such as size, structure, shape, energetic state, charge, or hydration shell. Biophysical technologies either directly read out these changes in order to obtain basic binding parameters or they indirectly monitor effects correlated to these changes. Table 1 summarizes the readout principles of the biophysical methods described below, as well as their key information content. Furthermore, the key advantage of each respective technique is indicated.

2.1.1

Surface Plasmon Resonance (SPR) [1–5]

SPR appears when a polarized light beam hits a metal layer (commonly a gold film) at the interface of two media with different refractive indices. Monitoring changes in refractive index upon binding of an interaction partner (analyte) to an immobilized partner (ligand) on the metal layer enables to calculate kinetic parameters (kon and koff) and steady-state affinity (KD). Furthermore, thermodynamic parameters can be estimated from experimental repeats at different constant temperatures [19]. In a typical SPR experiment, one of the interaction partners is immobilized on the surface of an SPR sensor chip, whereas the other interaction partner is supplied in different concentrations via a microfluidic system. Numerous different immobilization strategies and coupling chemistries are available for both nucleic acids, proteins, peptides, and larger particles so that aptamers can be used both as the immobilized binding partner and the partner being free in solution.

2.1.2

Biolayer Interferometry (BLI) [6, 7]

BLI analyzes interference patterns of white light that is reflected from two optical layers of a sensor tip. One internal reference layer is located inside the tip and one layer at the interface between the tip and the surrounding liquid phase. Each reflection generates constructive and destructive interferences that vary with the wavelength of the incident light. Any change at the outer layer of the tip (a biocompatible surface with one interaction partner immobilized on it), for

Biophysical Characterization of Aptamer-Target Interactions

5

Table 1 Readout, information content, and key advantages of selected biophysical methods Technique SPR

Principle Changes in refractive index

BLI

Changes in biolayer thickness

SwitchSENSE

Changes in movement of DNA nano-levers

MST

ITC

Changes in thermophoretic mobility and fluorophore microenvironment Heat changes

Flow cytometry

Fluorescence changes

Fluorescence polarization EMSA

Changes of polarization Changes in molecular size and hence changes in electrophoretic mobility Fluorescence changes

Filter-binding assay

Information obtained Affinity (steady-state): KD from 1 nM to 500 μM. Kinetics: Association rate (kon) down to 1E 5 s 1 and dissociation rate (koff) up to 1E7 M 1 s 1. Thermodynamics: Binding enthalpy ΔH by Vant-Hoff (25–40 C), derived ΔG Affinity (steady-state): KD from 1 nM to 500 μM. Kinetics: Association rate (kon) down to 1E 5 s 1 and dissociation rate (koff) up to 1E7 M 1 s 1 Thermodynamics: Binding enthalpy ΔH by Vant-Hoff (25–40 C), derived ΔG Affinity (steady-state): KD from 1 nM to 1 mM. Kinetics: Association rate (kon) down to 1E 5 s 1 and dissociation rate (koff) up to 1E7 M 1 s 1. Thermodynamics: Binding enthalpy ΔH by Vant-Hoff (25–40 C), derived ΔG. Hydrodynamic radius Affinity (steady-state): KD from 10 pM to 10 mM. Thermodynamics: Binding enthalpy ΔH by Vant-Hoff (22–45 C), derived ΔG Affinity (steady-state): KD from 1 nM to 500 μM. Thermodynamics: Directly binding enthalpy ΔH, derived free enthalpy of binding ΔG and binding entropy ΔS. Stoichiometry: Directly determinable. Kinetics [18] Affinity (steady-state): KD from 10 nM to 1 mM

Affinity (steady-state): KD from 1 nM to 1 mM Affinity (steady-state): KD from 10 nM to 1 mM

Affinity (steady-state): KD from 1 nM to 1 mM

Key advantages Well-established and well-characterized technique

Offers kinetics without drawbacks of microfluidics

Fast and efficient immobilization of aptamers on the DNA nano-lever by simple sequence extension

Ultra-low sample consumption, largest application range, and possibility to work in bioliquids Highest content of information. Thermodynamics directly accessible

Only method allowing to study aptamer-cell interactions Highest throughput Cost-efficient technique with low lab requirements

Rapid and costefficient technique

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example, due to binding of a ligand, leads to different interference patterns at this reflective layer. This, in turn, causes a shift of the interference spectrum to different wavelengths. From the time-resolved monitoring of this shift, it is possible to derive real-time association (kon) and dissociation rates (koff) of an aptamer-target interaction. The steady-state affinity (KD) can be extracted from equilibrium titrations. As for SPR analyses, repeat of BLI experiments at different temperatures allows for determination of thermodynamic parameters. In a typical BLI experiment, one of the interaction partners is immobilized to the sensor tip, whereas the other partner is supplied in different concentrations in a microwell plate. As for SPR, numerous coupling methods exist that allow to analyze aptamers both as the immobilized and the in-solution interaction partner.

2.1.3

SwitchSENSE (SwS) [16]

The SwitchSENSE technology monitors voltage-driven movement of DNA nanolevers attached to a sensor surface. Usually, such a nano-lever carries one of the interaction partners by direct, covalent attachment. Binding of the other partner affects the hydrodynamic friction of the nano-lever and hence its movement on the sensor surface, which can be monitored through time-resolved single-photon counting. Kinetic parameters (kon and koff) and steady-state affinity (KD) can be extracted. Furthermore, thermodynamic parameters can be estimated by analyses at different temperatures. In a typical SwitchSENSE experiment, one interaction partner is immobilized to the nano-levers on the sensor surface, whereas the other interaction partner is titrated in different concentrations. Aptamers can often be coupled directly to the nucleic acid-based nano-levers by base-pairing.

2.1.4

MicroScale Thermophoresis (MST) [3, 13, 17]

The optical method MST is based on the combined effect of Temperature-Related Intensity Change of fluorescent molecules (TRIC) and their directed movement along temperature gradients (thermophoresis). Both the TRIC effect and the thermophoretic component of the MST signal vary with three key molecular features that change upon binding between an aptamer and its target: molecular size, molecular charge, as well as the hydration shell of the molecules. Information on steady-state binding affinity (KD) can be directly obtained from a ligand titration. By variation of assay temperatures, also thermodynamics can be determined. In a typical MST experiment, one binding partner is held at a constant concentration and is monitored for its TRIC effect and thermophoretic movement by its intrinsic fluorescence or by a coupled fluorescent dye. The other binding partner is titrated usually in 16 dilution steps in order to sample a very large ligand concentration range. Aptamers can be used in MST very straightforward as the constant, fluorescent interaction partner, because they can be easily obtained with all kinds of

Biophysical Characterization of Aptamer-Target Interactions

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fluorescent dyes attached. Alternatively, proteins can be labeled with fluorescent dyes for MST, and an aptamer can be used as the non-fluorescent, titrated interaction partner.

2.1.5

Isothermal Titration Calorimetry (ITC) [2, 8]

The calorimetric method ITC directly measures the heat released or consumed in the course of a molecular binding event. The technology offers high information content. Besides thermodynamic parameters such as ΔH, ΔS, and ΔG, the equilibrium-binding affinity (KD) and interaction stoichiometry can be determined from the same experiment. Recent developments even allow to study kinetics by ITC [18]. Hence ITC offers the highest information content. In a typical ITC experiment, one interaction partner is put into a reaction cell at a constant volume and concentration, whereas the other partner is titrated into the reaction cell via a rotating syringe. ITC allows to study aptamer-target interactions without modification of the molecules.

2.1.6

Fluorescence Polarization or Fluorescence Anisotropy (FP or FA) [9, 10]

FP (often called FA) is based on the phenomenon that the polarization plane of emitted light of a small fluorescent molecule (excited with plane-polarized light) changes upon binding of an interaction partner. FP enables to calculate the affinity (KD) of the aptamer-target interaction. In a typical FP or FA experiment, one binding partner (the smaller one) is monitored via an attached fluorescent dye and held at constant concentration during the experiment, whereas the other binding partner is titrated across a certain concentration range.

2.1.7

Flow Cytometry [11, 12]

This optical method is commonly used to quantify the interaction strength between aptamers and whole cells, by sorting populations of cells that show interaction to fluorescently labeled aptamers, combined by quantification of the fluorescence signal coming from the aptamers. The steady-state affinity (KD) can be derived from flow cytometry assays, in which the target cells are incubated with increasing concentrations of fluorescently labeled aptamers. In a typical flow cytometry experiment, cells are incubated with fluorescent aptamers.

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M. Plach and T. Schubert

Filter-Binding Assay/Filter Retention Assay [4, 13]

Filter-binding assays quantify the signal of a fluorescently or radioactively labeled aptamer binding to a target that is immobilized on a filter membrane. Reading out different concentration steps of fluorescent aptamers allows for determining the equilibrium-binding affinity (KD). In a typical filter-binding assay, one interaction partner (usually the aptamer target) is immobilized on a membrane, whereas the second interaction partner (usually the aptamer) is titrated across a certain concentration range.

2.1.9

Electromobility Shift Assay (EMSA) [14, 15]

EMSA monitors mobility differences between complexed and unbound molecules in net-like matrices or gels, in order to determine the equilibrium-binding affinity (KD) between the interaction partners. In a typical EMSA experiment, a constant concentration of a labeled aptamer is incubated with increasing concentrations of its target. The mixed samples are loaded on a gel matrix in order to separate complexes from unbound aptamers by size. A fluorescent or radioactive readout allows for quantifying the interaction strength.

2.2

Sample Material Requirements of the Selected Biophysical Methods

Besides the fact that the data quality of any biophysical method is enhanced with increased purity, homogeneity, stability, solubility, and reduced aggregation tendency of the sample material, every method has its specific prerequisites and requirements to the sample material due to its specific principle and technical setup. Surface-based methods, such as SPR, BLI, and SwitchSENSE, require immobilization of one interaction partner to the sensor surface. In SPR and BLI, various immobilization strategies are available using direct immobilization via capturing of epitopes already available on the target (e.g., via a hexahistidine tag on a protein). Alternatively, chemical processes are available for linking the target either directly (via amino acid side chains) on the sensor surface or to adaptors (such as biotin), which are then captured on a pre-coated surface. In SwitchSENSE, a target molecule needs to be modified with a DNA strand, which is then hybridized to a counterpart on the sensor surface. Aptamers may directly be hybridized via extended sequences that bind to the DNA nano-lever on the sensor surface. In MST, flow cytometry, EMSA, FP, and filter-binding assays, one interaction partner must be fluorescent. MST can either work with intrinsic molecule fluorescence (tryptophan fluorescence in proteins or peptides, label-free MST) or relies on labeling one of the interaction partners with a fluorophore. In flow cytometry, the

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Table 2 Consumption, throughput, and sample pretreatment of selected biophysical methods

Technique

Consumption

Throughput Sample pretreatment

SPR

Immobilization via chemical modification or direct capturing of target epitopes

BLI

Immobilization via chemical modification or direct capturing of target epitopes

SwitchSENSE

Immobilization via DNA adaptor sequence on target

MST

Fluorescent labeling via chemical modification or monitoring of target-intrinsic tryptophan fluorescence

ITC

Extensive dialysis of target and ligand into the exact same buffer

Flow cytometry

Fluorescent labeling of the aptamer

Fluorescence polarization

Fluorescent labelling of the smaller partner

EMSA

Fluorescent or radioactive labeling of the aptamer

Filter binding assay

Fluorescent or radioactive labeling of the aptamer

Green indicates low consumption or high throughput, orange medium consumption or throughput, and red high sample consumption or low throughput

aptamer needs to be fluorescent. EMSA and filter-binding assays can be performed either with fluorescent or radioactive signal readout. Only label-free MST and ITC do not need any fluorescent modification of the interaction partners. The following table (Table 2) summarizes sample consumption, throughput, and necessary sample pretreatment of the selected biophysical techniques.

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Application Range of the Selected Biophysical Techniques

Different in vitro selection processes allow for the selection of aptamers for various target classes, starting at aptamers against the smallest molecules, such as ions. Other target classes for aptamer selection are small chemical molecules, peptides, nucleic acids, proteins, high-molecular-weight protein complexes, particles, viruses, bacteria, and even whole cells. Due to the enormous differences between target classes in size, charge, shape, and structure, there is currently no universally applicable biophysical method for studying all possible aptamer-target combinations. The limit of detection of the available biophysical methods is considerably influenced by the rather small size of aptamers (low to middle kDa range) and the size of the interaction partner (greatly varying from few Da as for ions to several MDa and more for whole cells). Consequently, different biophysical methods have to be applied for studying the different classes of aptamer interaction partners, as indicated in Fig. 1. Given the small molecular sizes of aptamers, most biophysical technologies have their analytical optimum (dark blue areas in Fig. 1) in the size range of 1–500 kDa (of the aptamer’s interaction partner). Target sizes 1 kHz, whereas at the nano-gap electrode a relative permittivity of ~80 was reached in the frequency range of 10–1,000 Hz and at higher frequencies decreased down to a value of ~3. Thus, the influence of electrode polarization was diminished. Upon aptamer immobilization the relative permittivity was reduced, and upon addition of thrombin (0.2 μL), the value decreased another ~20%, while in controls with lysozyme or random oligonucleotide, no changes were observed. In summary, the limited amount of research conducted on non-faradaic aptasensors is mainly based on big targets (molecular weight, >20 kDa) for clinical application, but also smaller targets (Rev peptides, 2.4 kDa) were detectable [50]. Until now, reached sensitivities are not as good as in faradaic impedance sensors, but the main drawback of low selectivity was resolved by the use of aptamers, and additionally it was shown that direct detection in diluted serum is possible [39–41, 45]. Although further development and research is needed, non-faradaic aptasensors offer a promising tool for point-of-care diagnostics, screenings, and online monitoring as a truly label-free technique.

4 Criteria for the Comparison of Impedimetric Aptamer-Based Biosensors Due to the almost unlimited variety of impedimetric aptasensors based on the countless possibilities to choose electrode design, surface modification, immobilization method, detection strategy, amplification strategy, and furthermore assay design (i.e., microfluidic or batch, real time or endpoint), it is difficult to compare the performance of different biosensors. Thus, guidelines for their characterization are needed as Thévenot et al. [52] already recommended for electrochemical biosensors based on enzymes. Based on their publication, recommendations for the characterization of impedimetric aptasensors are given in this paragraph. Further suggestions are included and open for discussion. A biosensor is defined by the integration of a biological recognition element with a transducer that converts the binding of the analyte into an electrical signal. By immobilizing the biological recognition element on the transducer, a close spatial coupling is achieved. The direct coupling enables integrated devices which distinguish biosensors from bioassays. Therefore, we recommend that a biosensor should be classified by the measurement method of the transducer and its biological recognition element in the form “receptor-based methodic biosensor” (e.g., aptamer-based impedimetric biosensor) with the addition of how the direct coupling was achieved. These key points should be stated in the abstract of a publication as well as the information if the detection strategy is direct or indirect.

Impedimetric Aptamer-Based Biosensors: Principles and Techniques

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As this chapter is reviewing impedimetric aptasensors, the following recommendations refer to this class of biosensors, although most criteria are common to all classes. Aptasensors belong to the affinity sensors, and the biological signal is generated by the interaction of the aptamer Apt and its target T with n binding sites for Apt: ka

nApt þ T Ð Aptn T kd

ð2Þ

After some time, the interaction will reach an equilibrium as the ratio of the dissociation rate constant kd and the association rate constant ka will be constant. In comparison to enzyme-based biosensors, aptasensors are not suitable for continuous monitoring, but the aptamer-analyte complex can be regenerated [53], and thus aptasensors are not necessarily single-use biosensors. In impedimetric aptasensors the biological signal is a change in the electrical behavior of the measurement cell that is detected by electrodes, and the direct coupling is achieved by immobilization of the aptamer on the electrodes. As proven by many publications, this class of biosensors was successfully applied to biological matrices like urine, blood serum, and food extracts [54–58]. The success depends strongly on the inertness of the biosensor surface, referring to both directions. In other words, the effect of the biosensor surface on the sample and the influence of the sample on the biosensor surface should be neglectable. Aptamers are highly biocompatible and have no degrading effect on the sample, but the sample might contain enzymes leading to the degradation of the aptamer or the electrode modification. Thus, characterization of the biosensor response in different situations is very important for its optimization and transfer into industry. The fast-growing field of biosensors still lacks standard procedures for the characterization of a biosensor. The IUPAC (International Union of Pure and Applied Chemistry) established some standard protocols, but these need revision and adaptation to the newly developed recognition elements and transducer. Guidelines for the evaluation of analytical methods can be found but are meant for the usage of a method not for its development and are application-specific, like the “Guidelines for performance criteria and validation procedures of analytical methods used in controls of food contact materials” (EUR 24105 EN) by the Joint Research Centre of the European Commission [59] or the “Guideline on bioanalytical method validation” by the European Medicines Agency [60] that is for pharmacokinetic and toxicokinetic parameter determination. Adapted from these guidelines and Thévenot et al., we recommend the following criteria for the characterization of a biosensor in development with the aim for its optimization and comparison: • Immobilization density and capture capacity • Response time • Calibration curve including: – Apparent KD – Linear and working range

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– Sensitivity – Detection and quantitative limits • • • • •

Selectivity/specificity Reliability Regeneration Repeatability/reproducibility Stability

In the following, every criterion will be explained in more detail. For the development of a biosensor, the immobilization of the recognition element has to be validated. During this validation, it would be useful to determine the number of immobilized recognition elements and if possible the capture capacity which is the ratio of active to immobilized recognition elements. The number of immobilized recognition elements should be normalized to the unit of area, e.g., molecules per cm2, whereas the capture capacity should be expressed in percentage. With these values, it is possible to evaluate the efficiency of the immobilization procedure and to determine which concentration range should be used for the calibration curve. The time passed until the signal reached 90% of the maximal response Rmax is called the steady-state response time. This parameter depends mainly on the diffusion of the analyte, the affinity of the aptamer, and if the sample is stirred. The calibration curve should be obtained from steady-state responses close to equilibrium, because these are unaffected by diffusion rates and analyte reassociation. The measurement time should be kept constant for every of the minimum of six freshly prepared analyte concentrations used, and each concentration should be repeated at least three times. The sample matrix should be adjusted to the final application of the developed biosensor. And most important is that the authors clearly specify the measurement procedure including the washing steps, the composition of the samples used, and how the reproducibility was determined. The calibration curve should be displayed as scatter plot of the signal versus logarithm of the analyte concentration including the standard deviation of the repeated measurements for every concentration. For better comparison of electrochemical biosensors, we suggest that the signal measured is normalized to the signal of a blank sample and to the electroactive electrode surface area. At equilibrium, the dissociation constant KD is the ratio of dissociation rate constant kd and association rate constant ka according to the law of mass action. KD describes the ratio of unbound molecules to bound molecules as demonstrated for the affinity reaction in Eq. (2): KD ¼

n m k d ½Apt ½T  ¼ ka ½Aptn T m 

ð3Þ

The smaller the KD, the higher is the affinity of the aptamer to its target. The apparent KD is often used to compare different biosensors, but this constant

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is strongly dependent on the measurement method and measurement conditions like temperature, pH, ionic strength, flow rate, etc. Therefore, we recommend that it should only be used to compare different molecules measured with the same method under the same conditions or to compare the same molecule measured with different methods or different conditions. Besides, the conditions of the measurement should be considered carefully and stated clearly, because limitations to the binding of aptamer and target will lead to inaccurate affinity rate constants. For example, if the diffusion rate is slower than the association rate, then the limiting mass transfer will result in a decrease of the rate constants. KD also depends strongly on the number of immobilized and/or active recognition elements; thus this should be stated. From the obtained calibration curve, many parameters can be determined. KD corresponds to the concentration that is equivalent to a response that is 50% of the maximum response Rmax. It can be determined by fitting the curve with a binding site model. As differences appear due to the application of different models, we recommend for all sigmoidal calibration curves to use the four-parameter logistic function as a standard model: R¼

Rmax  Rbl  S þ Rbl 1 þ KxD

ð4Þ

S is the slope in the middle of the linear range and represents the sensitivity of the biosensor response. In general, a high sensitivity and a wide linear range are desired, but as seen from Eq. (4), a wider linear range results in a lower sensitivity; thus compromises are required during optimization. By measuring the response of a blank sample Rbl at least six times in repetition according to the measurement procedure, the mean xbl and standard deviation σ bl of the measurement method are determined. According to IUPAC, the limit of detection (LoD) is defined as the concentration corresponding to a signal of xbl þ 3σ bl which refers to a signal-to-noise ratio of 3, and the limit of quantification (LoQ) is determined from xbl þ 5σ bl . The working concentration range is determined by the lower and upper limits of quantification. For aptasensors, the selectivity or specificity should be already examined during or after its selection. Thus, it is more important to test for unspecific binding of the biosensor surface, which is performed using random oligonucleotides of the same length as the aptamer. Besides, to test for interferences, a sample with an analyte concentration close to KD should be spiked with the interfering substance, and the percentage change of the signal compared to a sample with analyte alone should be reported. To test the reliability of the biosensor response, the concentration of the interfering substance should be changed while keeping the analyte concentration constant. The fluctuation of the signal describes the reliability of the biosensor and can be expressed in percentage. If the biosensor is regenerable, it is important to state the recovery time needed to return to the baseline and the regeneration repeatability defining the number of cycles performed until the signal response changed about 10%.

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Reproducibility is another important parameter for evaluating the quality of a biosensor, but the expression is often misused as testing the reproducibility would require to measure the same concentrations on a different instrument and by a different operator under reproducible conditions which is easiest performed in another laboratory. What is most often measured is the repeatability, which is defined as the variation of the signal measured of an analyte concentration in the linear range on different time points. In most publications about biosensor development, the calibration curve is obtained from analyte dilution series in buffer solutions, although it would be preferable to use spiked artificial or natural sample matrices. However, the performance of the biosensor on a real sample has to be evaluated. Therefore, measurements of a natural sample should be performed with the biosensor and a reference method. If reference methods are not available, spiked natural sample matrices may be used. The recovery is expressed in percentage. As the last criteria, stability should be mentioned which refers to the influence of every external change on the performance of the biosensor. Mostly, the storage stability (also called lifetime) is the most important external influence. To examine a biosensor’s lifetime, a batch of biosensors should be produced and used after different storage times, while the change of the response signal under identical conditions is stated in percentage. In conclusion, a thorough characterization of a biosensor is extensive, and a publication including all criteria is seldom. What has already become established among publications is the indication of linear range and detection limit of a biosensor, presumably caused by the pursuit for single-molecule detection. But most of the published detection limits fulfill the requirements for analytical diagnostics, but lack real sample validation. Thus, especially for impedimetric aptasensor, it would be desirable to present the calibration curve from measurements in spiked natural or artificial sample matrices and perform validation with natural samples. Besides, normalization of the response to biosensor-specific characteristics like electrode area, number of recognition elements, and blank signals would be helpful for proper comparison of different impedimetric aptasensors.

5 Summary and Outlook Most publications on impedimetric aptasensors are based on the sequential measurement of frequencies in a small range with measurement times of 1–3 min. As most biological systems are changing over time, more applications using multisine and potential step techniques are needed to avoid errors of non-stationarity and enable kinetic measurements. To increase sensitivity and reduce non-specific binding, a special design of the electrode surface is needed. DNA origami and nanocomposites are promising tools for application-specific surface designs. However, the aim should be to keep it as simple as possible to enable thorough characterization of the surface and

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mathematical modeling of the sensor response. There is a lack of models for impedimetric aptasensors, which is probably one reason for the low transfer to commercial products. In general, impedimetric biosensors are favored due to the possibility of label-free measurements. But only non-faradaic measurement techniques are truly label-free, and recent applications of non-faradaic impedimetric aptasensors show significant lower sensitivities than faradaic impedimetric aptasensors. Thus, more research in non-faradaic impedance sensors is needed. Despite the doubt that aptamers are not suitable for the application in real samples, their excellent performance in human serum and blood samples down to the pM range has been shown by several researchers. However, the majority of publications lack validation with real samples. As the work with real samples might often not be possible, we recommend that calibration curves are taken in spiked artificial matrices instead of buffer solution. Furthermore, we recommend that instead of absolute signal values, relative values are plotted and normalized to the electrode area or the number of aptamers to enable comparison of sensor performance. The full potential of impedimetric aptasensors has not yet been exploited. However, the success of impedimetric aptasensors depends equally on the advances in aptamer selection and synthesis, in impedimetric measurement techniques, and in surface modifications and assay design. Besides other transducer types, the potential of electrochemical biosensors is outstanding for the application in miniaturized point-of-care devices. Acknowledgments We thank SciGraphics ([email protected]) for contributing the illustrations for this chapter.

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Adv Biochem Eng Biotechnol (2020) 174: 43–92 DOI: 10.1007/10_2020_125 © Springer Nature Switzerland AG 2020 Published online: 21 April 2020

Impedimetric Aptamer-Based Biosensors: Applications John-Alexander Preuß, Peggy Reich, Nicole Bahner, and Janina Bahnemann

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Impedimetric Aptasensors for Health Care Applications (Red Biotechnology) . . . . . . . . . . . . 2.1 Diagnosis of Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Cancer Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Other Use Cases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Impedimetric Aptasensors for Environmental Application (Gray Biotechnology) . . . . . . . . . 3.1 Drug Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 An Outlook on Environmental Application of Impedimetric Biosensors . . . . . . . . . . . . . 4 Impedimetric Aptasensors for Food Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Mycotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Benefits and Challenges of Impedimetric Aptasensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

J.-A. Preuß, P. Reich (*), and J. Bahnemann Institute of Technical Chemistry, Leibniz Universität Hannover, Hannover, Germany e-mail: [email protected]; [email protected]; [email protected] N. Bahner Institut für Biologische Verfahrenstechnik, Hochschule Mannheim, Mannheim, Germany e-mail: [email protected]

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Abstract Impedimetric aptamer-based biosensors show high potential for handheld devices and point-of-care tests. In this review, we report on recent advances in aptamer-based impedimetric biosensors for applications in biotechnology. We detail on analytes relevant in medical and environmental biotechnology as well as food control, for which aptamer-based impedimetric biosensors were developed. The reviewed biosensors are examined for their performance, including sensitivity, selectivity, response time, and real sample validation. Additionally, the benefits and challenges of impedimetric aptasensors are summarized. Graphical Abstract

Keywords Aptamer, Aptasensor, Bacteria, Biomarker, Biosensor, Cancer, Contaminants, Diagnostics, Disease, Drug, Environmental, Food industry, Health care, Impedance, Infection, Monitoring, Pathogen, Point-of-care, Serum, Therapy, Toxins, Tumor marker, Virus

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1 Introduction The main focus of this book chapter is on the review of impedimetric aptasensors since 2005 for their possible applications in biotechnology, in particular in the fields of health care, food inspection, and environmental protection (Sects. 2–4) with emphasis on target type, limit of detection, and real sample testing. Their performance will be compared to other biosensors to discuss, which benefits they offer and which challenges still have to be taken for their introduction into market (Sect. 5). Although several academic databases and search engines were sifted, some articles might have been overlooked.

2 Impedimetric Aptasensors for Health Care Applications (Red Biotechnology) Red biotechnology aims to advance the application of biotechnological tools to benefit (bio)medical purposes. Besides developing and providing drugs, a major purpose of red biotechnology addresses the field of medical diagnostics. Instrumental diagnostic procedures are necessary in order to derive reliable diagnoses and thereby specific therapy decisions. With regard to red biotechnology, the analysis of usually complex samples (e.g. whole blood) often requires multiple processing steps in standard laboratories which demand time, highly trained personnel, and money. An ideal sensor offers cost-efficient, selective, and rapid measurements of non-processed as well as low-volume samples. Point-of-care tests point toward such specifications and enable rapid detection of analytes on-site, which can be crucial depending on the specific disease as well as the medical supply available to the patient. Readouts of point-of-care tests by smartphone applications might be the highlight of the development of simplified analysis and exchange of information with even distant professionals. The crucial question is if impedimetric aptasensors match these requirements. Impedimetric aptasensors combine the advantages of aptamers with the advantages of EIS (electrochemical impedance spectroscopy). EIS-based sensors are promising with regard to simplified diagnostics due to rapid and label-free application without complex sample preparation. Based on the sensitivity of impedance changes at the interface of electrode and electrolyte, even the detection of analytes in the pM range is feasible. The selectivity of the sensor is ensured by aptamers which are short, stable and cost-efficient molecules [1–3]. This paragraph reviews recent publications in the field of aptamer-based impedimetric biosensors targeting medically relevant analytes, grouped by their application in the diagnosis of diseases, cancer detection, and other use cases. This review shows the potential of impedimetric aptasensors for detection and analysis of a variety of targets (e.g. proteins, cells, small molecules) from even complex origin (e.g. serum, plasma, urine).

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Diagnosis of Diseases

The body’s health status can be portrayed by the quantitative determination of different biomarkers which include hormones or infection-related components. Depending on the application, the diagnostic tool can facilitate a rapid alert system by the determination of general biomarkers such as the inflammatory marker CRP (C-reactive protein) or specific biomarkers for a virus type during the early stage of disease progression (see Fig. 1). Thereby, such sensitive and rapid diagnostic tools may support initial clinical examination as well as long-term monitoring of a condition, with regard to progression, recession, or recurrence. C-reactive protein (CRP) is an unspecific biomarker that detects the risk for cardiac disease or inflammation at an early stage. Thereby, concentrations below 8 nM CRP in blood point to a low risk while concentrations above 25 nM CRP indicate a high risk [5]. So far, only non-faradaic impedimetric aptasensors have been developed for the detection of CRP but have not yet been applied in a clinically relevant matrix (serum, blood, plasma, urine, saliva). An overview of affinity-based detection methods is presented in Table 1. Qureshi et al. presented an RNA-aptamer-based sensor with gold-interdigitated capacitor arrays for the purpose of surface maximization [6]. A linear range of 4–20 pM was achieved. In the process of sensor optimization, the authors showed the influence of AC frequency on the aptamer-CRP-complex

Fig. 1 The progression of a disease. A rapid increase of severity after appearance of symptoms requires more sensitive diagnostic tools facilitating detection on the very beginning of biomarker genesis. (Adapted from [4]; copyright John Wiley & Sons, Inc.)

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Table 1 Biosensors for CRP determination using different methods Method EIS EIS SPR EIS EIS SPR LMR

Biorecognition element Aptamer Aptamer Antibody Antibody Antibody Aptamer-antibodySandwich Aptamer

Sample Buffer Buffer Serum Serum Serum Diluted serum Buffer

LoD 4 pM 1 pM 0.36 nM 176 pM 176 pM 10 pM 2.5 nM

Linear range 4–20 pM 101–104 pM 0.24–2,800 nM 0.5–50 nM 0.5–70 nM 10 pM to 100 nM 2.5–40 nM

Reference [6] [7] [8] [9] [10] [11] [12]

SPR surface plasmon resonance, EIS electrochemical impedance spectroscopy, LMR lossy mode resonance

formation. Depending on the AC frequency, the dissociation constant changed in a non-linear manner. Hence, the impact of an electric field on binding events of aptamer and target should be considered during the experiments. In 2018, Piccoli et al. presented another aptasensor based on capacity measurements with a linear range of 10–104 pM and no interference with HSA [7]. Lysozyme, also called the body’s own antibiotic, plays a key role in the innate human immune system [13]. It protects the organism from infections by grampositive bacteria by hydrolyzing the murein in the cell walls. Muramidase lysozymes are especially present in secretions such as tears or saliva. Lysozymes can serve as medical biomarkers with regard to several infections such as AIDS (acquired immune deficiency syndrome), malaria, and bacterial meningitis as well as autoimmune Alzheimer’s disease or rheumatoid arthritis [13]. Lysozyme concentration in the saliva and serum of healthy patients ranges from 32 to 207 nM, while an increase points to different disorders. Herein, the potential of impedimetric aptasensors for lysozyme quantification will be discussed and compared to other biosensors (see Table 2). Similar strategies have been published by Peng et al. and Xia et al. enabling lysozyme detection [14, 15]. The aptamer is hybridized on an immobilized, complementary strand. By addition of 100 μl sample containing lysozyme, the aptamer is released (target induced dissociation). The regeneration requires rehybridization of an aptamer sequence after formamide treatment. Mentionable features of the Table 2 Biosensors for lysozyme determination using different methods Method EIS EIS EIS EIS EIS ECL

Biorecognition element Aptamer Aptamer Aptamer Aptamer Aptamer Aptamer

Sample Buffer Buffer Buffer Buffer Buffer Spiked serum

LoD 0.2 nM 0.07 nM 1.04 pM 10 fM 6.3 nM 0.4 fM

Linear range 0.2–100 nM 0.2–4 nM 3.5–70 pM 0.1–500 pM 6.3–70 nM 3.5 fM to 350 pM

Reference [14] [15] [16] [17] [18] [19]

EIS electrochemical impedance spectroscopy, ECL electrogenerated chemiluminescence

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approach by Peng et al. are a bulge of the cDNA during hybridization which increases the charge-transfer resistance as well as a covalently attached ferrocene. Their design results in a linear range of 0.2–100 nM in a buffer system, while Xia et al. enabled a linear range of 0.2–4 nM and a limit of detection (LoD) of 70 pM [14, 15]. Additionally, Xia et al. have shown the impact of incubation time. Maximal charge-transfer resistance was achieved after 80 min of incubation [15]. In order to achieve improved fabrication of the functionalized electrode with regard to higher reproducibility and automation, Khan et al. deposited carbonnanotube-aptamer complex by inkjet printing (see Fig. 2). The anionic character of DNA results in a high charge-transfer resistance and a binding event decreases this resistance. A detection limit of 6.3 nM could be measured [18]. Hence, the approach is a seldom example of lowered impedance due to a binding event. By immobilizing the aptamer on gold nanoparticles deposited on a gold electrode, a linear range of 0.1–500 pM and a LoD of 10 fM has been achieved [17].

Fig. 2 Instead of a direct immobilization of the aptamer, a carbon-nanotube-aptamer-complex (CNT-Aptamer) is inkjet-printed on the working electrode. The complex is based on the high affinity between single-stranded DNA and carbon nanotubes. The anionic character of DNA results in a high charge-transfer resistance and binding of the positively charged analyte decreases this resistance. (Adapted from [18] under the CC BY license (http://creativecommons.org/licenses/by/ 4.0/), licensed under CC BY by SciGraphics)

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In conclusion, several groups have reported aptamer-based impedimetric biosensors for lysozyme detection at picomolar concentrations (see Table 1). By using electrochemical chemiluminescence and aptamers as recognition element Dong et al. showed an astonishing detection limit of 0.4 fM in spiked serum [19]. Due to the high sensitivity of EIS aptasensors presented by Chen et al. and Zhang et al. which is magnitudes lower than the physiological concentrations, sample dilution in an appropriate buffer might be a promising strategy. Nonetheless, a proof of concept in a diluted clinical sample still needs to be provided. Thrombin, as coagulation agent in human blood, plays a central role in human physiology. Usually, thrombin levels are low and additionally inhibited by anti-thrombin agents in order to prevent non-functional blood clogging (thrombosis). Moreover, thrombin is involved in different neurodegenerative diseases such as Alzheimer’s disease or Parkinson [20]. To increase sensitivity, signal amplification is a possible strategy. For instance, a strategy presented by Deng et al. utilized the two binding sites of thrombin and consists of a multiple step detection enhancement [21]. First, thrombin is captured by immobilized aptamers on a gold surface. Second, gold nanoparticles coupled with aptamers build a sandwich with captured thrombin. Third, the gold nanoparticles serve as seed for further nanoparticle growth and thus increased steric hindrance (reduction of HAuCl4). Fourth, SDS (sodium dodecyl sulfate) builds a selfassembled monolayer on the gold nanoparticles. Their negative charge further enhances the charge-transfer resistance resulting in a linear range from 50 pM up to 35 nM (LoD 100 fM) and showing fair recoveries in spiked serum. Lu et al. developed a design suitable for EIS as well as ECL determination with the same aptamer sequence as Deng et al. The aptamer is immobilized on a gold electrode while a complementary strand coupled to a Quantum Dot is supposed to be released in the presence of thrombin (target induced dissociation). Thus, the impedance increases while the ECL signal decreases. The limit of detection of both measurement strategies is 2.7 aM. Both measurement methods result in wide linear ranges from 2.7 aM up to 2.7 μM for EIS and 2.7 aM up to 27 nM for ECL [22]. Although there is no investigation in serum, it might work since the same sequence was applied by Deng et al. for biosensor experiments in human serum. An aptasensor based on another sequence was reported by Heydari-Bafrooei et al. in 2016 [23]. Based on a nanocomposite consisting of TiO2, MWCNT (multiwalled carbon nanotube), chitosan, and a Schiff base applied on a glassy carbon electrode, a LoD of 1 fM was achieved due to increased surface (linear range from 50 fM up to 10 nM). The aptamer is immobilized by simple π-π stacking. Compared to an ELISA kit on healthy serum and serum of patients with different diseases (e.g. Parkinson, Epilepsy), similar results were obtained showing the applicability of the design even in complex matrices. Interleukins, member of the cytokines, are peptide hormones that regulate immune cells. The analysis of different interleukin levels in human blood allows inferences of cell state and cell-to-cell communications. For instance, interleukin 17A (IL17A) is a biomarker for different autoimmune diseases like arthritis or multiple sclerosis [1]. The synthesis of IL17A strictly

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correlates to the synthesis of its corresponding receptor interleukin 17RA [24] and therefore is a promising target. Jo et al. presented a simple impedimetric aptasensor which is based on the coupling of thiol-modified aptamers on the gold electrode with deposited gold nanoparticles that are supposed to increase the surface for enhanced sensitivity. The approach enabled selective target recognition with regard to other interleukin proteins and a linear range of approx. 10–40 fM [1]. The pro-inflammatory Interleukin 6 is thought to play a role in the occurrence of major depression as it influences the neurotransmitter metabolism and neural plasticity [25]. Tertiş et al. applied a screen-printed carbon electrode with a film of polypyrrole and gold nanoparticles. A detection limit of 14 fM and a linear range from 42 fM to 633 nM cover the essential range [25]. Troponin I gained due to its high specificity for heart damages high relevance as a biomarker for early detection of, e.g., acute myocardial infarction. Antibody-based tests, usually ELISA and radioimmunoassay, generally lack thermal stability, require a complex sample preparation, and are cost-inefficient, albeit being sufficiently specific and sensitive [26]. Troponin I concentrations below 24.9 pM represent normal level while high concentrations above 70 pM are indicative for cardiac tissue damage [27]. In 2015, Jo et al. presented an electrochemical aptasensor that is able to quantify Troponin I with a linear range of 2 nM to 2 μM in human serum [26]. Wang et al. introduced a 13-mer peptide aptamer, immobilizing Troponin I on deposited gold nanoparticles by using the thiol-functionality of the terminal cysteine [28]. This approach achieved a linear range of 0.7–700 pM. Troponin I detection from clinical samples showed fair recoveries of 91–105%. Higher sensitivity was achieved by Akter et al. in 2017 by an antibody-based EIS sensor [29]. Dendrimers between the electrode and the aptamer enhanced the performance resulting in even lower detection limits of 11.7 fM and a linear range of 46 fM to 46 nM. The lowest concentration measured in spiked serum was 460 fM. Malaria results from an infection caused by parasites belonging to the Plasmodium family. P. falciparum and P. vivax are the main species which are transferred by the Anopheles mosquito. In 2016, 216 million individuals were affected by malaria and 445,000 died from it (see WHO malaria report 2017). Recommended by the WHO as state-of-the-art blood diagnostics are microscopy and rapid diagnostic testing (RDTs) [30]. However, both methods show significant disadvantages: While microscopy requires time and well-trained experts, RDTs based on antibodies against Plasmodium lactate dehydrogenase (LDH) show limitations with regard to costs, sensitivity at low concentrations, specificity, and thermal stability [31]. Nonetheless, since Plasmodium LDH is expressed in the sexual as well as the asexual stage of the parasite, it is a reliable target for affinity-based sensors. Lee et al. and Figueroa-Miranda et al. presented impedimetric aptasensors for Plasmodium LDH determination from patient’s blood and diluted human serum [31, 32]. In this way, they were able to distinguish between infected and uninfected blood. A simple design based on co-immobilization of 6-mercapto-1-hexanol and thiolated aptamer on a gold electrode resulted in a linear detection range of 1–1,000 pM [31]. Figueroa-Miranda et al. reported a linear range of 1 pM to 10 nM of the

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Plasmodium LDH covering the necessary range from 13 to 206 pM. Depending on the pH, the required linear range can be adjusted. Moreover, the regeneration of the sensor was demonstrated [32]. For influenza virus H1N1, Kiilerich-Pedersen et al. presented an impedimetric aptasensor that is potentially applicable as a point-of-care test [33]. Standard methods include RT-PCR, antigen staining, and virus cultivation. Especially virus cultivation requires several days and is therefore not suitable for rapid testing. However, by way of example, an influenza virus that is responsible for a potentially pandemic flu, as arisen in 2009, needs to be determined in a rapid and reliable manner. In a buffer system, a linear range of 101–106 Plaque-forming units (PFU) per ml was achieved. Briefly, a suitable DNA-aptamer was immobilized on a conductive polymer in a microchannel. A saliva sample spiked with 103 PFU/ml (100-fold lower than in real samples) caused a signal within 15 min, which was not induced by a H1N1 virus-free saliva sample. Cyclic adenosine monophosphate (cAMP) is a secondary messenger with various functions in different physiological contexts [34]. cAMP levels are usually low and stable, while an increase in urine or plasma points to different diseases. The detection of cAMP is an example of detecting relatively small molecules by means of impedance increase. Zhao and coworkers applied a RNA aptamer in a simple approach that uses gold electrodes [35]. These gold electrodes were enhanced by gold nanoparticles that allowed for the detection of cAMP in buffer and spiked diluted serum. In spiked serum, a linear range of 50 nM to 1 μM was achieved. Three orders of magnitude lower, in a linear range of 50–250 pM, were detectable in a buffer system. Selectivity was proven, as the sensor was not triggered by ATP, AMP, or c-diGMP. For the detection of adenosine, Wang et al. used a methodically similar design [36]. The study demonstrates the influence of backfiller such as DTT and MCH which are added to functionalized aptamer solutions (see Fig. 3). Depending on the backfiller composition, linear ranges of 0.1–61 nM for pure MCH backfilling (LoD 0.03 nM), 0.5–27 pM for pure DTT backfilling (LoD 0.2 pM), and 0.05–17 pM for a mixture of DTT and MCH (LoD 0.02 pM) were achieved. For testing clinical suitability, diluted clinical samples were analyzed, resulting in recoveries of 95–101% (lowest sample concentration 0.066 pM).

2.2

Cancer Detection

The uncontrolled proliferation of the body’s owns cells and the inhibition of natural cell death (apoptosis) as present in cancer require early diagnosis in order to prevent the formation of metastases. Since aptamer sequences with a high and specific affinity to different tumor-related markers and cancer cells have been selected lately, the development of impedimetric aptasensors for the detection of low level tumor markers at an early point is pursued [37, 38]. Beside the benefits of specific

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Fig. 3 Gold electrodes are often functionalized by well-known gold sulfur chemistry. In order to achieve a self-assembled monolayer (SAM) and to reduce non-specific binding, the application of short backfillers is a common strategy besides co-immobilization or post-functionalization of SAM. The impact of backfiller composition should be taken into account. (Adapted from [36], with permission from Elsevier)

diagnostics, this chapter deals with the potential of impedimetric aptasensors for aptamer-based cancer treatment by oncolytic virus.

2.2.1

Cancer Cells

The state-of-the-art diagnostics of cancer types that are based on imaging methods are limited since they mainly analyze morphology. However, morphology-based classification does not allow for early-stage-diagnosis as morphologic changes do not occur right away. As an alternative, impedimetric aptasensors are an opportunity of rapid and label-free detection of cancer cells themselves. For instance, CT26 (colorectal cancer) were detected by an impedimetric aptasensor as a rapid and labelfree approach [39]. The proposed sensor enabled LoD of 2 cells/ml and a linear range of 1
105–6
106 cells/ml. Diagnosis of hepatocellular carcinoma (HCC) goes along with poor prognosis for survival the next 5 years due to the lack of effective treatment strategies. So far, the

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majority of patients are diagnosed at a late stage, because α-fetoprotein, considered to be the most useful HCC marker in human serum, lacks sensitivity and specificity as increased concentrations might be due to other liver related diseases [41]. A more specific and reliable tool for diagnostics might be the direct detection of cancer cells as published by Kashefi-Kheyrabdi et al., who proposed an impedimetric aptasensor for quantification of HepG2 cells which are a hepatocellular carcinoma cell line [38]. A LoD of 2 cells/ml and a linear range of 102–106 cells/ml were achieved based on the covalent immobilization of aptamer molecules on a gold electrode. Since the blood of tumor patients contains low levels of circulating tumor cells (CTCs), screening of isolated CTCs enables diagnosis at early stage. Shen et al. [40] presented an approach that focuses on the reuse of the biosensor surface and the collection of viable cells after detection [40]. The authors used an aptamer that targets an epithelial adhesion molecule that is specifically overexpressed in breast cancer cells. The sensor design consisted of an immobilized capture probe complementary to the aptamer. The hybridized aptamers captured the CTCs and the impedance is measured. A uracil excision enzyme specifically digested the aptamer, leaving the DNA capture probe intact for further experiments and the isolated cells are released for further cultivation (see Fig. 4). With regard to the CTC, a LoD of 10 cells/ml and a high linear range of 30–106 cells/ml were achieved.

2.2.2

Soluble Tumor Marker

The carcinoembryonic antigen (CEA) is an example of a protein tumor marker that is quantifiable by impedimetric aptasensors. Despite its unspecificity, CEA plays an important role in diagnostics and monitoring. CEA is expressed on the tumor membrane and additionally secreted into the blood. Normal concentrations of CEA in the blood range from 3 to 5 ng/ml; increased CEA levels indicate tumor progression or recurrence [42, 43]. Different biosensors for CEA detection have recently been published as summarized in Table 3. Shekari et al. developed a selective aptasensor with a linear range of 1 pg/ml to 100 ng/ml [43]. An even lower linear range of 0.1 fg/ml to 1 pg/ml was achieved by Wang et al. in 2015 by excluding the usual need of covalent immobilization [44]. The required sample volume was 50 μl. The working principle engages the differences of adsorption from ssDNA and dsDNA on graphene surfaces when target binding to the single-stranded aptamer induces dsDNA formation. The lowered charge-transfer resistance results from dsDNA desorption. Since the linear range falls far below the diagnostic range, the required dilution of serum is not limiting. In 2017, Guo et al. reported about a design based on silver nanoclusters and aptamers embedded in zirconium metal-organic clusters [45]. Achieving a linear range of 10 pg/ml up to 10 ng/ml (LoD: 5 pg/ml) in spiked serum, the approach potentially enables appropriate diagnostics. Good repeatabilities (n ¼ 5), selectivity, lifetime (12 days), and possible regeneration (n ¼ 8) were achieved. Just the sample incubation time of 3 h might be limiting with regard to rapid diagnostics.

Fig. 4 Design for detection and capture of CTCs by an impedimetric aptasensor: Aptamers are hybridized to complementary DNA capture probes on the sensor surface. Then, CTCs from the sample bind to these aptamers and an increase in impedance is measured. Cells are released by the application of a uracil excision enzyme, specifically targeting the aptamer due to its uracil bases. Recovered CTCs can be used for further experiments and the recovered sensor can be reused. (Adapted from [40], with permission from Elsevier)

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Table 3 Different biosensors for CEA detection Method EIS

Biorecognition element Aptamer

EIS

Aptamer

EIS DPV

Aptamer Aptamer

ELISA

Antibody

Sample Diluted serum Diluted serum Spiked serum Human serum Spiked plasma

LoD 1 pg/ml

Linear range in g/ml 1 p to 100 n

Reference [43]

0.1 fg/ml

0.1 f to 1 p

[44]

5 pg/ml 3.4 ng/ ml 2 ng/ml

0.01–10 n 5–40 n

[45] [46]

2–64 n

[47]

DPV differential pulse voltammetry, ELISA enzyme-linked immunosorbent assay, EIS electrochemical impedance spectroscopy

The vascular endothelial growth factor (VEGF) is an important biomarker for cancer diagnostics as well as for other diseases [48, 49]. Secreted into the blood for the purpose of angiogenesis, healthy individuals have serum concentrations of 1–177 pg/ml VEGF while individuals with cancer show a serum level of 18–328 pg/ml VEGF due to increased tissue formation [50]. Therefore, highly sensitive detection methods are required in order to detect the fine differences between healthy and cancerous VEGF blood concentrations [51]. In 2015, two studies were published presenting impedimetric aptasensors for VEGF detection in human serum. Tabrizi et al. reached a linear range of 10–300 pg/ ml and a detection limit of 1 pg/ml by using mesoporous gold nanocomposites on a screen-printed electrode [49]. For the immobilization, the affinity of gold toward thiolated aptamer molecules was used. As proof for a potential application, diluted serum of a patient with lung cancer was tested. The value measured with a standard ELISA at the local hospital could be recovered to 97% by the developed biosensor. A more exotic approach was proposed by Qureshi et al. who designed a non-faradaic aptasensor. Instead of impedance change, this sensor used changes in capacitance [48]. A linear detection range of 400 pg/ml up to 1 ng/ml VEGF in spiked serum was obtained for a low volume sample of 5 μl (LoD: 5 pg/ml). The authors showed the increased capacitance change by a sandwich assay design compared to a simple aptamer immobilized on a gold surface. The sandwich assay additionally included an antibody linked to a magnetic bead (see Fig. 5). The approach is promising with regard to potential parallelization and the need of low volumes. However, the complexity of the approach might be limiting due to the need of an additional element such as an eventually expensive antibody. Moreover, the antibody must not bind the same epitope as the aptamer. The approaches presented by Tabrizi et al. and Qureshi et al. are close to the medically relevant range. Compared to other VEGF-biosensors, the presented impedimetric biosensors stand out due to its high sensitivity even in a complex biological matrix (see Table 4). Da et al. presented an aptasensor based on photoelectrochemical detection

Fig. 5 An aptamer immobilized on a gold electrode binds VEGF. An anti-VEGF antibody coupled with magnetic beads used to build a sandwich and to further increase the capacitance. (Adapted from [48], with permission from Elsevier)

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Table 4 Different biosensors for VEGF determination Method EIS

Biorecognition element Aptamer

EIS EIS

Aptamer-antibody VEGF receptor 1

DPV

Antibody

PECa

Aptamer

Sample Diluted serum Spiked serum Artificial serum Human serum Buffer

LoD in pg/ml 1

Linear range in g/ml 10–300 p

Reference [49]

5 100

400 p to 1 n 100–700 p

[48] [52]

50

50 p to 100 n

[53]

1.16

3.86 p to 386 n

[54]

EIS electrochemical impedance spectroscopy, DPV differential pulse voltammetry, PEC photoelectrochemical a To calculate concentration, VEGF was assumed to form a dimer

with a dynamic range of approx. 4 pg/ml to 386 ng/ml VEGF, but a proof of concept in serum is missing [54].

2.2.3

Oncolytic Virus

Biosensors are useful not only to detect tumor marker or cancer cells, but also to monitor cancer treatment or to test new treatment strategies. Therapies based on oncolytic viruses might be a game changer for modern cancer treatment. With a special focus on cancer cells, the infection with oncolytic virus causes tumor lysis and supports the development of tumor-specific immunity. Especially promising is this treatment strategy’s versatility [55]. However, the immune response might release neutralizing antibodies, shortening the circulation time of oncolytic virus. As a consequence, due to the immunological memory, the application of the oncolytic virus at a later time point is then limited. Impedimetric aptasensors might contribute to overcome this limitation since aptamers as nucleic acids do not activate an immune response. Therefore, aptamers protecting the epitopes can be utilized in order to extend circulation time. Labib et al. proposed an impedimetric aptasensor for testing the degree of protection of an oncolytic virus by different anti-vesicular stomatitis virus (VSV) aptamers [56]. In this study, the oncolytic virus VSV was captured by an aptamer immobilized on an electrode surface. After incubation with polyclonal antibodies, the change of impedance determines the degree of virus protection (compare with Fig. 6). The presented aptasensor enabled a linear range of 40,000–110,000 plague forming units (PFU) per ml and a LoD of 30,000 PFU/ml.

Fig. 6 Impedimetric aptasensor to determine the degree of protection of an oncolytic virus by a specific aptamer with regard to polyclonal antibodies. The approach is proposed as a screening for an aptamer sequence for cancer treatment based on aptamer shielded oncolytic virus injection. (Adapted with permission from [56]. Copyright 2012 American Chemical Society)

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Table 5 Different biosensors for endotoxin detection Method EIS EIS EIS LSPR PCTIR

Biorecognition element Aptamer Aptamer Aptamer Polymyxin B LAL

Sample Diluted insulin Buffer Buffer Buffer LAL reagent water

LoD 7.94 zMa

Linear range 0.01 aM to 1 pM

Reference [59]

5 pg/mlb 0.01 ng/mlc 340 pg/mld 2.5
106 EU/ mle

0.01–10.24 ng/ml 0.01–1 ng/ml 106–10 μg/ml 2.5
106 – 5
102 EU/ml

[58] [60] [61] [57]

LSPR localized surface plasmon resonance, EIS electrochemical impedance spectroscopy, PC-TIR photonic-crystal total-internal-reflection. For endotoxin, the LoD depends not only on the concentration, but also on the pyrogenicity of the endotoxin that is defined by the endotoxin unit (EU), which varies depending on the source of the endotoxin. Calculations of the endotoxin unit (EU) were based on information about EU and concentrations specified by the author a 1 EU/kg body weight ¼ 100 pM b 5 pg/ml ¼ 2.5
103 EU/ml c 2 μM ¼ 2
107 EU/ml ¼ 4 mg/ml d Approximately 340 pg/ml ¼ 3 EU/ml e 1 EU/ml ¼ 0.1–0.2 ng/ml

2.3

Other Use Cases

This paragraph gives a short overview about targets which points beyond the classic diagnostics as a major field of red biotechnology. Since red biotechnology deals with biotechnology in health care related applications in general, the analytics for diverse non-protein or non-cell targets such as drugs are required in order to ensure safe production of pharmaceuticals, drug monitoring, or forensic analytics. The pyrogenic endotoxin, also called lipopolysaccharide (LPS), causes an immune response including fever or septic shock. Therefore, the removal of endotoxins is of great interest for the drug industry concerning potential contamination of pharmaceutical products. Endotoxins originate from the outer membrane of gram-negative bacteria and are released after cell death and are chemically stable even with regard to sterilization [57, 58]. Thus, the drug industry needs to meet legal limits with regard to endotoxin concentration which lies at approximately 20 pM for drug distribution [59]. The most popular endotoxin detection method is the Limulus amebocyte lysate (LAL) test which utilizes the unique coagulation based on an endotoxin induced enzyme cascade in the Limulus blood. Chromogenic and turbidimetric assays are available [57]. The need for more rapid testing and the increased need for pharmaceutical contamination detection require more sustainable alternatives than the horseshoe crab based LAL test. Several impedimetric aptasensors have been developed for the detection of endotoxin. As an example, Posha et al. quantified LPS in diluted, spiked urine with excellent recoveries and a wide linear range from 1 aM to 1 pM, while the lower limit of the range in a PBS buffer system was even 100-fold lower [59]. The surface for aptamer immobilization was increased by gold atomic clusters. In Table 5

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biosensors for endotoxin determination are listed, but since EU (endotoxin unit) is a measurement for pyrogenic activity, comparability of different sensor methods is restricted. In all conscience of the announced information by the authors, endotoxin detection limits and ranges are summarized. Therapeutic drug monitoring (TDM) aims at determining drug concentrations in blood or blood-related samples. Many drugs can be classified as small organic molecules. For the field of affinity-based detection in general, antibodies are the most important strategy. However, the need of immunogenicity usually limits the application of antibodies for small molecule detection [62]. Additionally, antibodies’ high molecular mass might reduce its feasibility in many sensor designs with regard to low mass targets. Alternatives such as GC-MS or HPLC require highly trained personnel [63]. Thus, aptamers are considered as an opportunity for drug monitoring and testing. Medication is sought to be improved by TDM for drugs demanding narrow concentration ranges. Since many drugs are small molecules, the sensors need to be sufficiently sensitive on a small scale. For instance, Roushani and Shahdost-fard presented a sensor for the determination of ibuprofen [64], as the long-term medication risk of ibuprofen with regard to heart attacks is especially striking. Common approaches for its detection are GC-MS or HPLC reaching a LoD of 1 – 8 μM [65, 66]. Roushani and Shahdost-fard reported an ibuprofen detection down to 16 pM with a wide linear range of 50 pM up to 20 μM with high recovery rates in spiked blood. This approach is based on functionalized Quantum dots on an electrode surface. The Quantum dots are supposed to increase the surface and therefore the sensitivity for small molecule determination. The authors immobilized a capture DNA probe on the Quantum dots. The aptamer contains a single-stranded target binding site, while the rest is hybridized on the capture probe for immobilization purposes (see Fig. 7). By a conformational change due to complex formation, an increase of impedance was measured.

2.4

Conclusions

Aptamer-based impedimetric biosensors have been applied to a versatile range of targets underlying the universal qualification as biotechnological tool while the numbers of faradaic EIS applications dominate the non-faradaic ones. From cells to small molecules, the targets of interest generally seem unlimited. Nonetheless, aptamers for further medically relevant targets are required in order to cover the variety of biomarker diagnostics. As a possible alternative to standard techniques, impedimetric aptasensors have been proven to be an easy and rapid diagnostic tool even without elaborate sample preparation due to the label-free application. In many reports, low sample volumes of 5–100 μl were sufficient. A range of authors reported on picomolar detection levels for small molecule and protein targets, while some even reported (sub-)femtomolar LoD levels. Thereby, the success of aptamers as a sensitive recognition element might root in high immobilization densities (approx.

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Fig. 7 Ibuprofen (IBP) causes a conformational change of the aptamer resulting in an increase of impedance. A capture probe is immobilized on Quantum dots in order to immobilize the aptamer by hybridization. (Adapted from [64], with permission from Elsevier)

1013 aptamer molecules per cm2) due to its lower molecular mass compared to antibodies [62]. However, for some targets (e.g. lysozyme) no measurement in a medically relevant matrix is reported. With regard to point-of-care tests, EIS might not be the method of choice since high-priced potentiostats are required and are not suitable for in-field diagnostics (malaria or virus detection). Nonetheless, organizations, which are able to cover the investment costs (e.g., cancer medicine or food industry), might benefit from it by reducing current costs and time. Thus, the important aspects to be clarified are costs per unit, proof of standardized mass production, and tests of stability. The common strategy of self-assembled monolayer formation which requires several hours might be a limiting factor of fabrication and commercialization. The increase of charge-transfer resistance by a binding event is the most dominant design strategy. An advantage by aptasensors is the relatively low molecular weight of an aptamer compared to an antibody. Therefore, a binding event and the subsequent conformational change might have a higher impact on the charge-transfer. Another aspect of red biotechnology is the monitoring in biopharmaceutical processes and has not been a topic in literature. To overcome limitations with regard to manual handling which is needed for sample dilution or continuous monitoring, the combination of EIS aptasensors with microfluidic tools might be promising.

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3 Impedimetric Aptasensors for Environmental Application (Gray Biotechnology) In gray biotechnology, impedimetric aptasensors attracted attention in recent years due to Guideline 2008/105/EG of the European Parliament and the Council of 16 December 2008. Gray biotechnology covers biotechnological applications in the environment such as monitoring of drinking water quality and efficiency of wastewater treatment and is therefore also called environmental biotechnology. Impedimetric aptasensors were developed for various purposes in environmental biotechnological applications with the main focus on the detection of pollutants in water sources such as drug residues, bacteria, toxins, and pesticides. The sources of pollution are versatile, as drug residues are introduced by medical treatments while micropollutants are released into the environment through plastic production. Toxins produced by microorganisms enter the environment during cell death or through their metabolism and accumulate, for example, in shellfish. With impedimetric aptasensors due to the in vitro synthesis of aptamers, it is possible to detect not only proteins, but also toxins, whole cells, and small molecules such as ethanolamine, which has a molecular weight of 61 g/mol and is released into the environment by the textile, chemical, and pharmaceutical industries. In 2016, Liang et al. developed an impedimetric aptasensor for ethanolamine with a detection limit of 0.08 nM, thus more sensitive than currently used chromatographic methods (0.4 μM) [67]. This chapter is intended to give an overview of environmental application of impedimetric biosensors and compares them with classical analytical methods.

3.1

Drug Residues

As mentioned above, drugs are important for medical treatment, but they have significant side effects when they are released into the environment or accumulate in animal products (like milk). The residues of drugs are continuously released into the environment, especially into the water. They can intervene in the regulatory mechanism of the organisms present there. They can be detected there unchanged, since they are often difficult to degrade and mobile. In sewage treatment plants, these can only be retained to a limited extent. Due to the low concentration, many drug residues cannot be detected with the previous methods; therefore, numerous impedimetric aptasensors have been developed for the detection of antibiotics such as tetracycline and chloramphenicol, chemotherapeutics like doxorubicin and hormones such as 17ß-estradiol and progesterone (see Table 6). These drugs are used in cancer therapy, hormone therapy, and animal breeding. With these sensors a faster, simpler, and more sensitive measurement is possible.

Impedimetric Aptamer-Based Biosensors: Applications Table 6 List of impedimetric aptasensors for the detection of drug residues

Drug residue Chloramphenicol Doxorubicin 17ß-estradiol Progesterone

3.1.1

63 Detection limit (M) 1.76
109 2.80
108 1.28
107 1.00
1015 2.00
1012 3.00
109

Reference [68] [69] [70] [71] [72] [73]

Antibiotics

Chloramphenicol is an effective broad-spectrum antibiotic used in the treatment of infectious diseases. Due to its potential lethal side effects it was defined that chloramphenicol in any concentration is a health risk. To ensure food safety, the European Commission set a minimum required performance limit (MRPL) value, defining the lowest detectable concentration of the analysis method [74]. Currently, the detection of chloramphenicol is based on gas chromatography, high performance liquid chromatography, and mass spectrometry, which are very sensitive, but are expensive, time-consuming, and require highly trained personnel. Pilehvar et al. developed a simple low-cost aptamer-based impedimetric biosensor with an approximate assay time of 5 h and a detection limit of 1.76 nM (0.6 μg/l), which approaches the MRPL value of 0.3 μg/kg [68].

3.1.2

Anthracycline

Doxorubicin belongs to the group of anthracycline antibiotics and is used as chemotherapeutics in cancer therapy. By incomplete degradation in wastewater, this drug residue reaches our tap water. Due to its cardiotoxicity, it contaminates nature and threatens humans and the environment. An aptamer-based biosensor for detection of doxorubicin by electrochemical impedance spectroscopy was developed by Bahner et al. [69]. The developed sensor was able to reach a detection limit of 28 nM in buffer and thus a lower detection limit as previously reported by Erdem et al. (128 nM) [70].

3.1.3

Hormones

Hormones can also be detected with aptasensors. The hormone estradiol is one of the most important active ingredients for contraception and hormone treatment. When ingested by humans, it can enter the sewage system. Zhu et al. developed a sensitive impedimetric aptasensor for the detection of 17ß-estradiol with a detection limit of 1 fM [71]. They successfully detected estradiol in spiked biologically complex human urine samples as well as spiked untreated tap water samples and obtained

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Fig. 8 Principle of an aptamer-based impedimetric biosensor for the detection of progesterone. (Adapted with permission from [73]. Copyright 2015 American Chemical Society)

similar results to estradiol in buffer solution. Also Lin et al. developed in 2012 an aptasensor for the detection of 17ß-estradiol with a detection limit of 2 pM [72]. With the sensor, it was possible to detect 17ß-estradiol in urine, which was added with 17ß-estradiol. Average recovery rates range from 92% to 101% with relative standard deviations below 4.4%. The sensor can therefore be used to determine 17ß-estradiol in urine samples. Another hormone is progesterone, which is used in hormone therapy. For detection of progesterone in water an aptasensor with a detection limit of 3 nM was developed by Contreras Jimenez et al. (see Fig. 8) [73]. This sensor shows a higher sensitivity than instrumental analysis methods such as HPLC, LC/MS, or GC/MS, because they can detect progesterone concentrations as low as 32 μM.

3.2

Toxins

Toxins produced by bacteria enter the environment during cell death or according to their metabolism. Also the use of insecticides in modern agriculture contaminates the natural environment and through plastic production bisphenol A is released into the

Impedimetric Aptamer-Based Biosensors: Applications Table 7 List of impedimetric aptasensors for the detection of bacteria and toxins

Analyte E. coli O157:H7 Brevetoxin-2 Cylindrospermopsin Microcystin-LR Atrazine Bisphenol A

65 Detection limit 100 CFU/ml 0.12 nM 0.3 nM 18 pM 10 pM 45 fM 10 fM 7.2 fM

Reference [75] [76] [77] [78] [79] [80] [81] [82]

environment. Therefore, impedimetric aptasensors have been developed for the detection of toxins as the examples shown in Table 7.

3.2.1

Escherichia coli and Toxins Produced by Bacteria

One of the main causes of human infectious diseases are pathogenic bacteria such as Escherichia coli (E. coli). The presence of E. coli bacteria in water usually indicates fecal contamination; therefore, a detection of E. coli in tap water is required. For routine measurements, a method based on colony forming units is used, which is very sensitive but takes up to 24 h. Thus, biosensors offer a valuable alternative for rapid detection of E. coli. For example, the impedimetric aptasensor developed by Brosel-Oliu et al. detects E. coli O157:H7 in tap water within 30 min and reaches a detection limit of 100 CFU/ml [75]. Although this sensor is not as sensitive as cultivation methods, it is more sensitive compared to other previously reported biosensors. Furthermore, microorganisms produce toxins that enter the environment during cell death or through their metabolism. One associated toxin is Brevetoxin-2 that is produced by dinoflagellates Karenia brevis and accumulates in shellfish. It has no toxic effect on shellfish, but it is toxic to the sea, mammals, birds, fish, and humans [83]. Eissa et al. developed in 2015 an impedimetric aptasensor against Brevetoxin-2 in shellfish with a detection limit of 0.12 nM [76], which is comparable with previously reported electrochemical immunosensor (0.12 nM) [84]. Cylindrospermopsin, produced by cyanobacteria, is also a kind of widespread toxin in water sources and exhibits strong cytotoxicity and carcinogenic activity [85]. The aptasensor for detection of cylindrospermopsin in lake water has a detection limit of 0.3 nM (0.12 ng/mL) [77] and thus shows a higher sensitivity than instrumental analysis methods such as HPLC or LC/MS that detect cylindrospermopsin up to 1 and 0.5 ng/L [86, 87]. Another toxin produced by cyanobacteria is microcystin-LR (MC-LR), which is released in situation of cell rupture into the water. Due to the toxicity of microcystinLR, it causes health and environmental problems, e.g. it leads to liver damage and possible death when exposed to humans and animals. Lin et al. developed a sensor

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against microcystin-LR and reached a detection limit of 18 pM [78]. Many other methods for MC-LR detection have been reported and showed good detection limits, but the impedimetric aptasensor tops with its simplicity, low-cost, and rapid detection.

3.2.2

Pesticides and Bisphenol A

Pesticides are chemicals that kill fungi, bacteria, insects, snails, slugs, or weed to avoid plant diseases, plant damage, or suppressed growth. Subgroups of pesticides are insecticides, which are substances that are used to kill, harm, repel, or mitigate a specific species of insects. Insecticides are often used to increase crop yields and improve the quality of agricultural products. The use of insecticides in modern agriculture contaminates the natural environment, which can pose a potential health risk to humans. One of the most extensively employed insecticides is acetamiprid that belongs to the class of systemic broad-spectrum insecticides and is used for controlling insects such as Lepidoptera and Thysanoptera [88]. Aptamer-based biosensors for the detection of acetamiprid are reviewed in Verdian [89] including optical and electrochemical transducers. An impedimetric aptasensor for detection of acetamiprid residues in wastewater and tomatoes was developed by Fan et al. who achieved a detection limit of 1 nM, which is comparable or even lower than conventional instrumental methods (see Table 8) [95]. Fan et al. also examined the application of the sensor for real samples. For this purpose, wastewater samples were added with three different concentrations of acetamiprid and the concentration was determined by the sensor. Average recovery rates range from 94% to 103% with relative standard deviations of 4.9%. The sensor can therefore be used to determine acetamiprid residues in the environment. Furthermore, an aptamer-based impedimetric biosensor for detection of acetamiprid and atrazine in water was developed [79]. Atrazine is one of the most commonly used herbicides, a subgroup of pesticides used to kill undesirable plants. The developed sensor by Medianos et al. reached a detection limit of 1 pM for acetamiprid and 10 pM for atrazine. In this case, it can be seen that aptasensors allow the detection of molecules in the picomolar concentration range. As far as the Table 8 Comparison of techniques for the determination of acetamiprid

Method HPLC GC ELISA Aptasensor

Detection limit 2.69 nM 0.157 nM 4.49 nM 4.49 nM 4.49 nM 0.898 nM 17 fM 33 fM 0.9 pM

Reference [90] [91] [92] [93] [94] [95] [96] [97] [79]

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detection of acetamiprid is concerned, the proposed impedimetric biosensor is more sensitive than conventional instrumental methods and the aptasensor reported by Fan et al. but lower detection limits of 17–33 fM are reached by other impedimetric biosensors [96, 97]. The developed aptasensor reported the lowest detection limit for atrazine at this time. Bisphenol A is an organic monomer and is widely used in the chemical industry for manufacturing polycarbonate plastics. In food packaging, it was used, for example, in beverage bottles, baby bottles, and medical devices [98]. Due to its toxicity and harmful endocrine disrupting activity, bisphenol A was banned in baby bottles since 2010 [99]. In recent years, several impedimetric aptasensors against bisphenol A were developed, which allow quick and easy measurement. These sensors reached detection limits of 45 fM [80], 10 fM [81], and 7.2 fM [82], which are the lowest compared to other biosensors.

3.3

An Outlook on Environmental Application of Impedimetric Biosensors

The detection and quantification of pathogens and chemical agents are of great importance for water and environmental analysis. The classical methods such as HPLC and GC/MS are usually time-consuming and require highly qualified personnel and highly developed equipment. Therefore, new technologies are constantly being developed. Aptasensors are real-time measurements of different targets in the environment with high sensitivity and specificity (see Fig. 9). The aptasensors presented here were able to withstand the interference of complex matrices and can therefore be used for determination in environmental samples. In addition, the detection process with these aptasensors usually only takes 3 h. To improve detection, the aptamers used should be optimized by shortening and stabilizing so that cross sensitivities are reduced. Furthermore, the reproducibility and also the possibility of regeneration must be further investigated.

4 Impedimetric Aptasensors for Food Control White biotechnology is descriptive for industrial processes to generate products useful for us humans such as chemicals and food. The role of biotechnology in the food industry is copious, ranging from increasing productivity in food manufacturing and handling to fabrication of food additives and preservatives [100]. During growth, fermentation, and processing, the food can be contaminated with microorganisms or toxins. The World Health Organization (WHO) estimated that there are 600 million cases of foodborne diseases every year worldwide. The economic burden of foodborne diseases in Europe is about 171 million US$ per year

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Fig. 9 Overview of the applications of aptasensors in gray biotechnology

[101]. During transportation and storage, the food is densely packed, often still moist and not cooled, which is why microorganisms grow and produce toxins (see Fig. 10). Thus, food control upon arrival at the factory as well as before the delivery to the consumer is essential. Especially small manufacturers have a high demand in compact devices with easy handling for rapid on-site detection. Beside ELISAs and microarrays, biosensors are an alternative for rapid detection of contaminants. An important step in food control is the extraction of the analytes from the food matrix which might be reduced in biosensor setups by the use of stable and biocompatible recognition elements, such as aptamers. In this chapter, we will review the advances in aptamer-based impedimetric biosensors for the detection of mycotoxins and microorganisms in food.

Fig. 10 Role of food control in white biotechnology – it is essential to control food before processing at the factory – methods for fast detection in situ are desired, as e.g. biosensors

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Mycotoxins

Mycotoxins are metabolites produced by fungi that are toxic for humans and animals. Fungi grow best in moisture and warm environments and thus, mycotoxins are found in products grown in hot climates or stored and shipped for long periods such as coffee beans or peanuts. The toxins withstand high temperatures and thus remain active after heat treatment. Furthermore, mycotoxins do not induce an immune response and thus are bypassing our immune system. To produce antibodies against a mycotoxin, it needs to be bound to an immunogenic protein carrier which is cumbersome. Therefore, especially for small molecules like mycotoxins, aptamers are a perfect alternative recognition element in biosensors. The major groups of mycotoxins are aflatoxin, ochratoxin, citrinin, ergot alkaloids, patulin, and fusarium [102]. Vidal et al. published a review discussing electrochemical biosensors for the detection of mycotoxins in food and they conclude that label-free and direct binding properties, as impedimetric biosensors have, are beneficial in quality control in food processing [103]. A list of aptamer-based impedimetric biosensors for the detection of mycotoxins is presented in Table 9. Aflatoxins are produced mainly by Aspergillus flavus, A. parasiticus, and A. nomius, contaminants found in nuts and grains. Not only concentrations of ~10 μg/kg body weight have an acute hepatotoxic effect, but also concentrations below are carcinogenic [102]. From 20 known types of aflatoxins, aflatoxin B1 (AFB1) is considered the most prevalent, followed by aflatoxin B2, G1, and G2. The European Commission set the limit for AFB1 in food products containing nuts and grains depending on the kind and handling to 2–12 ppb (equivalent to 2–12 ng/g or 6.4–38.4 nM) and for processed food intended for babies or children to 0.1 ng/g (equivalent to 0.32 nM) [116]. Castillo et al. developed an aptamer-based impedimetric biosensor for the detection of AFB1 by using PAMAM G4 dendrimers on gold electrodes to increase the Table 9 Impedimetric aptasensors for the detection of mycotoxins Toxin OTA OTA OTA OTA OTA OTA OTA OTA AFB1 AFB1 AFM1 FB1

Conc. range in g/ml 100 p to 25 m 40 p to 40 n 120 p to 12 n 1 p to 0.5 n 4–40 p 2 p to 6 n 0.01 f to 0.1 n 0.1–10 p 31 p to 2 n 1 f to 1 n 20 p to 1 n 72 p to 72 m

LoD in pg/ml 100 50 20 0.2 6 2 105 0.05 100 0.4
103 1 1

Real sample – Coffee, flour, wine Beer Beer White wine Wine Soybean Grape juice Peanuts Peanuts Milk Maize

Response time 15 min 30 min 30 min 1h 90 min 50 min 9 min 5–7 min 40 min 3h 30 min 30 min

Reference [104] [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115]

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active surface ([112], see Fig. 11). As the pK of AFB1 is 5.84, the impedance increased upon its binding to the immobilized aptamers. An affinity constant Kd of 0.59  0.33 nM and a limit of detection (LoD) of 0.4  0.03 nM in buffer were measured, proving high affinity and sensitivity. Slight interferences with aflatoxin B2 were observed, but no interference with ochratoxin A. Measurements of certified contaminated peanut extracts resulted in recovery rates of 96–120%, but in spiked peanut samples matrix effects were discovered. The developed biosensor has an assay time of 30 min, is regenerable with 0.2 mM glycine, and is stable for 60 h when stored at 4 C. As a rule of thumb, the detection limit should be ~5 times below the legislative limit, thus the detection range is sufficient for the food control of nuts and grains for the direct human consumption but not for baby food. As comparison, the LoD of other developed biosensors were in the range from 0.1 to 6.4 nM, but some researchers claimed lower limits, e.g. Li et al. who achieved a LoD of 1.3 fM by aptamer-based surface enhanced Raman spectroscopy (SERS) and signal enhancement by a recycling mechanism [113]. However, the impedimetric aptasensor developed by Castillo et al. is 4–5 times faster and thus offers a cost-effective alternative to ELISAs for small food manufacturers. The presence of AFB1 in feed leads to its digestion in lactating animals producing its hydroxylated metabolites aflatoxin M1 (AFM1) and M2. Thus, the EU regulated the concentration of AFM1 in raw milk to 0.05 μg/kg (equivalent to 152 nM) and in milk-based products intended for infants to 0.025 μg/kg [116]. Istamboulié et al. developed a faradaic impedance biosensor using screen-printed carbon electrodes

Fig. 11 Modification of the surface with the dendrimer PAMAM G4 increases the active surface and thus enables signal enhancement. (Adapted from [112], with permission from Elsevier)

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(SPCE) covered with 4-carboxyphenyl diazonium salt (4-CP) and 50 -aminohexaethyleneglycol modified aptamers against AFM1 [114]. They reached in buffer a LoD of 1.15 ng/l and in milk a linear range of 20–1,000 ng/kg. Measurements of raw milk and pasteurized milk spiked with 500 ng/kg AFM1 resulted in recovery rates of 99–111%, whereas measurements with a standard immunoassay resulted in similar recoveries of 98–114%. The comparison to a standard method proves its functionality, although interferences with other substances should be tested. Hianik’s research group immobilized aptamers against AFM1 on the PAMAM G4 modified electrodes and compared them to biotin-avidin surfaces [117]. The results demonstrate that both immobilization methods show the same characteristics with a dynamic range of 15–120 ng/l and a LoD of 8.5 ng/l in milk which is sufficient for the legislative limits. Ochratoxin is produced by fungi of the genera Aspergillus and Penicillium. It can be found in cereals, coffee, dried fruits, cocoa, grapes, and spices as well as in processed food like red wine, bread, beverages and it can be accumulated in animals, e.g. pork meat. Ochratoxin A (OTA) is the most prevalent and relevant representative of the ochratoxins and has several toxicological effects such as neurotoxicity, nephropathy, immunosuppression, and carcinogenicity [118]. The European Commission set regulatory limits of OTA in foodstuff depending on the process level from 2 to 20 ng/g and for foodstuff intended for babies or infants to 0.5 ng/g [116]. Castillo et al. from Hianik’s research group developed an aptamer-based impedimetric biosensor for the detection of OTA by simple chemisorption of the aptamers on a gold electrode and measuring the impedance in buffer with 1 mM of the redox mediator ferri-/ferrocyanide [Fe(CN)6]3/4 [105]. They tested different modifications of the aptamer for immobilization; interestingly, the simplest modification with a thiol on the 50 end showed the best results reaching a LoD of 0.12 nM (¼0.05 ng/ml) and they notified that Ca2+ ions were essential for OTA binding. They also measured the surface density of aptamers and obtained 1.85
1013 aptamers/ cm2. Furthermore, OTA showed a six times higher signal than OTB and N-Acetyl-Lphenylalanine. The recoveries in coffee, flour, and wine spiked with 1, 5, and 10 ng/ g OTA were 78–108%. The sensor could also be regenerated by immersion into 1 mM HCl and no significant decrease of the signal was observed within 10 cycles of regeneration. Evtugyn et al. from the same research group used silver nanoparticles to enhance the signal and obtained a LoD of 0.05 nM (¼0.02 ng/ml) for OTA in buffer and a concentration range of 0.3–30 nM [106], although they observed instability of the coating and used significant higher concentrations of [Fe(CN)6]3/4 (0.1 M instead of 1 mM). For the direct detection in situ, biosensors with a wide concentration range are favored. Hayat et al. developed an aptamer-based impedimetric biosensor for the detection of OTA with a concentration range of 1.25–500 ng/l and a LoD of 0.25 ng/l [107]. This low sensitivity was achieved by the modification of a SPCE with diazonium salts and the immobilization of the aptamer via click chemistry. The surface modification led to high impedances of about 150 kΩ and thus low frequencies of 10 mHz were required to measure the charge-transfer resistance in a buffer

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solution with 1 mM [Fe(CN)6]3/4. Although the author enabled a wide concentration range, it is two magnitudes below the legislative limits and thus a dilution step is required. The concentration range achieved by Castillo et al. of 0.04–40 ng/ml is better suited for direct detection [105]. However, dilution of the sample can increase the signal-to-noise ratio as the matrix is diluted as well. The authors were able to regenerate the aptasensor up to 10 times without significant signal loss. A wide detection range can be obtained by increasing the active-surface-tovolume ratio as Mejri-Omrani et al. achieved by modifying a gold electrode with a conductive polypyrrole layer (PPy) and above a layer of dendrimers (PAMAM G4) containing a high number of immobilization sites for the aptamer (see Fig. 12, [109]). They measured a detection range from 2 pg/ml to 6 ng/ml, which is similar to the ranges measured with high pressure liquid chromatography (HPLC), but shifted to a lower detection limit [119]. The lower concentrations reached by Mejri-Omrani et al. compared to Castillo et al. for AFB1 [112] may be grounded in a higher affinity of the aptamer, the higher concentration of the redox mediator [Fe (CN)6]3/4 (10 mM) or it may be due to the addition of the PPy layer. However, the median concentration of OTA in wine is 50 pg/g and thus the demonstrated aptasensor is a promising alternative for OTA detection in wine. An astonishing wide concentration range and detection limit of 0.01 fg/ml to 0.1 ng/ml was achieved by Wei et al. by using a nanocomposite of porous carbon structures with incorporated gold nanoparticles which significantly increases the

Fig. 12 Signal enhancement by modification of the surface with the dendrimer PAMAM G4 and the conductive polymer polypyrrole (PPy). (Adapted from [109], with permission from Elsevier)

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active surface [110]. The achieved detection limit is even lower than the LoD obtained with real-time PCR (1 fg/ml), although such sensitivities are not necessary for the application in food control. The developed aptasensor was tested on soybean samples and recoveries of 95–108% were obtained. Compared to the simple sensor surface of Castillo’s aptasensor [105], the signal ratio of OTA to AFB1 is only ~2.5. It is reasonable to presume that by increasing the active surface, the unspecific binding is increased as well. In conclusion, impedimetric aptasensors for mycotoxin detection are welldeveloped, providing detection limits and ranges comparable with HPLC and sufficient for the legislative limits set by the EC. They were validated with real samples and complex food matrices with short measurement times of 5–90 min and have mechanisms for regeneration. For the transfer of these biosensors into commercial products, handheld impedance analyzers with microfluidic cartridges are needed.

4.2

Microorganisms

Besides mycotoxins from fungi, another major cause of foodborne diseases are the toxins produced by bacteria like Salmonella, Escherichia coli, Staphylococcus aureus, and Bacillus cereus. Although it is more accurate to directly determine the toxins, often in food control the microorganisms are detected. Optical biosensors for the detection of pathogenic bacteria are reviewed in Yoo and Lee [120], whereas electrochemical biosensors are reviewed by Amiri et al. [121] who concluded that aptamers, imprinted polymers, and bacteriophages are promising alternatives to antibodies and that improvements are needed in reproducibility and reliability as well as in minimizing non-specific binding. In comparison with clinical applications that require spotting of a single bacterium, in food control higher concentrations are sufficient, as for example 105 colony forming units (CFU) per ml of Staphylococcus aureus (S. aureus) are producing toxins in a quantity that can cause food poisoning. A general review of biosensors for the detection of foodborne pathogens is given by Arora et al. [122], whereas Teng et al. [123] reviewed aptamer-based biosensors and Kant et al. [124] concentrate on microfluidic devices. In this chapter, we will review aptamer-based impedimetric biosensors for the detection of food borne pathogens (see Table 10). Salmonella are gram-negative, flagellated, and facultative anaerobic bacteria from the family Enterobacteriaceae. More than 2,600 serotypes are distinguished by the existence of different antigens, like the somatic O-antigen, also known as lipopolysaccharide or endotoxin (see Sect. 2.3) and the flagellar H antigen. However, the most common serovar found in humans and animals are S. typhimurium and S. enteritidis that also the major pathogens in gastrointestinal infections. Salmonella food poisoning is mainly caused not only by contaminated poultry and eggs, but also by the feces of infected humans and animals, insufficient hygiene in food handling, or contaminated surface water. Thus, the EC regulation No. 2073/2005 demands the

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Table 10 Impedimetric aptasensors for the detection of microorganisms and their toxins in food Analyte S. typhimurium S. typhimurium

Method EIS EIS

LoD in CFU/ml 600 25

S. typhimurium S. typhimurium S. typhimurium S. typhimurium S. typhimurium

EIS EIS EIS EIS CA

6 3 3 1 20

S. typh. and E. coli S. tnteritidis E. coli E. coli E. coli Listeria L. monocytogenes S. aureus

RTPCR EIS EIS EIS EIS EIS EIS EIS

78 600 2
104 4 12 100 5 10

S. aureus SEB

EIS EIS

10 6 pM

Real sample – Chicken meat Apple juice Apple juice Pork meat Egg Chicken meat Milk – – – Milk – – Fish and water – Milk

Incubation time in min 60 60

Reference [125] [126]

60 45 35 40 120

[127] [128] [129] [130] [131]

120

[132]

60 60 12 120 1015 random oligonucleotides) and followed by iterative rounds of purification and amplification, it is possible to obtain aptamers with desirable selectivities and Kd values [3]. The interaction between the target and aptamers is a consequence of electrostatic forces and conformational motifs, principally. Moreover, Van der Waals forces, charge interactions, and hydrogen bonding are responsible for binding. Aptamers also display a variety of secondary and tertiary structures, like loops, hairpins, and

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bugles, which drive more interaction through tridimensional contacts with the target [4]. Thus, a variety of targets, such as proteins, peptides, lipids, and toxins, are able to be recognized by aptamers. The oligonucleotide nature confers a wide range of pH and temperature stability, compared with their protein analogues, the antibodies. Aptamer size, charge, and flexibility also enable binding to hidden epitopes, as well as high tissue penetration [5, 6]. This feature also allows easy chemical modifications and thus diverse applications. Additionally, non-immunogenic and nontoxic properties provide a biological safety profile for pharmaceutical approaches [7]. For many years, antibodies were the primary molecule used to specifically detect an expressed molecule in a pathology sample. However, aptamers can achieve similar affinities as antibodies, resulting in binding specific and diverse targets with several advantages [6]. Fast and easy identification methodologies with a stable and reproducible product are critical features at this time. Because of their physicochemical and biological advantages and the critical situation of the high-cost market place, aptamers have emerged as a powerful tool [8].

2 Aptamers-Based Diagnostic It is well-known that an early diagnosis has a direct impact on the prognosis of a disease. Even when age and other risk factors impact treatment selection, an early and accurate diagnosis is the key to successful treatment. In this sense, it is imperative to detect the disease in at the moment of appearance, avoiding its advancement. Early diagnosis is possible with sensitive methods involving acute detecting agents. As the amount of target to be detected becomes smaller, the more sensitive the diagnostic method must be [9]. There are two conditions necessary to perform a successful diagnosis: recognition of abnormal characteristics and the accurate detection. Because effective strategies to identify and recognize targets are crucial, recent efforts have emphasized the use of aptamers. As a targeting component, aptamers are ideal and can be easily functionalized with the attachment of a signal agent, which allows for detection with measurement instruments. In most cases, these additional components must be considered with the aim to detect the binding and amplification of the signal. Aptamer modifications can be achieved without impacting their binding capacity, based on the chemical composition, size, charge, and the wide range of pH and temperature stability. Hence, aptamers and functionalization methodologies are the fundamental parameters to creating a sensitive diagnostic technique, which includes big efforts in the conception of strategies and the design of the probe.

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Probe Design for Diagnostics

Effective tools to identify biomarkers are in high demand in medicine. Probe development is one of the most frequently used strategies for diagnostics. Here, a probe is considered using two components: the uptake component and a signal agent to facilitate the measurement. In fact, a variety of diagnostic probes can be developed using aptamers labeled via physical or chemical conjugation of both components, in order to confer diverse functions. There are several strategies to consider when creating a probe with high affinity and specificity to the target [10]. Additional probe considerations relate to their sensitivity, specific activity, signaling agent, and stability. The following describes each consideration: • High affinity to target. High uptake of the target usually requires nM-range Kd values or less. This principally depends on the targeting component, but it is very important that the recognition will not be affected by subsequent modifications. Considering small molecules, any incorporations can affect charge and other binding features. • High specificity. This parameter permits the discrimination between the correct target and others, because small molecular variations in specificity measure the degree to which the probe differentiates between targets, isotypes, similar domains, and conformational stages. • Specific activity. This refers to the amount of signal component per mass unit. This means how much signal component is available to be attached to the molecular component and improve the detection. • Signaling agent. For a probe to be used in quantification or qualification assays, it needs a signaling agent, which can be a complementary dye, fluorophore, radionuclide, or a more complex system. Oligonucleotide composition exposes a variety of chemical groups to attach signal agents to. Here, we must be very careful in choosing the desired signal range which is directly dependent on the application. • Purity. The purity of the probe has direct consequences on the results. Free signal agent increases the background noise affecting the real measurement. Free target component and others decrease the site-specific signal. • Stability. This is the most controversial point principally due to degradation of biomolecule components. However, proper targeting and signal components must be stable for as long as the assay requires. The pharmacokinetic parameters should be also considered. Additionally, robust binding between the target and signal agent is fundamentally related to the stability of the probe as a whole. A covalent bond between both components is usually preferred. Extra links can be added, but it is well documented that the addition has a profound impact on the final physicochemical and biological characteristics [11]. • High sensitivity. Especially with small amounts of target, the probe must be highly sensitive. The sensitivity is closely related to most of the features mentioned above but also is dependent on the detection equipment.

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• Reproducibility. The methodology must be reproducible with no significant result variations. In this context, the design of the aptamer-based probes begins with the correct aptamer selection. First, systematic evolution of ligands by exponential (SELEX) enrichment is the most established methodology of aptamer obtention and initially restricts the affinity and specificity parameters [1, 2]. At this moment, it is important to decide if DNA or RNA will be used [12]. Both have no trivial differences in stability and chemical composition. While the use of unnatural nucleic acids [13, 14] has emerged as an interesting tool, DNA and RNA native libraries are used in most SELEX procedures. Since RNA plays an important role in gene regulation, it represents the majority of the libraries used in genomic SELEX [15]. Additionally, some authors suggest RNAs have more conformational motifs, which result in high affinity binders [16]. However, DNA has clear advantages, since it is reportedly more chemically and biologically stable [17]. In fact, most of the commercial-developed aptamers are from DNA [18]. Typically, the SELEX library includes a randomized region between two constant primer binding sequences on the ends. Consequently, the binding depends on the constant and random regions, as well as their length and GC percentage. The library design comprises variations [19], and the introduction of artificial nucleotides, polymers, and functional groups into the library results in a challenge to find a good starting point [12]. The molecular diversity can be improved using an extend alphabet of SELEX starting libraries. This expanded alphabet includes unnatural hydrophobic bases and functional groups that mimic amino acid side chains [20]. The probability to select more affine aptamers is higher due to the increased number of possible interactions between aptamers and target. Additionally, stabilization of aptamer structures with a 2’OH group of RNA [21] or locked nucleic acids (LNA) may improve their target binding affinities [22]. Nonetheless, the development of modified SELEX libraries requires the adaptation of compatible steps of SELEX, PCR amplification, and cloning [23]. Second, target presentation could be determinant. Correct antigenic fragments can be exposed to the library in a soluble resin, in target fragments, and by tissues or whole cells [24]. The assays’ settings must be approximate to the real conditions. At this point, the correct folding of the oligonucleotides is fundamental in the presentation of the target. Ionic force, salts, pH, and temperature are some parameters to be considered in order to properly reproduce the binding conditions. In order to control the media conditions, the buffer is usually enriched with magnesium. Addition of magnesium chloride to the aptamer enhances G-quadruplex folding [25]. Successful SELEX also requires efficient separation of bound and unbound sequences, allowing increase of target bound/unbound ratio and progress in the enrichment. There are diverse variants of the methodology, which showed to improve the success rate [26, 27].

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After the selection procedure, sequencing is performed, and the aptamers are synthetized [28]. Based on multiple alignment and structural simulation software, it is possible to predict different characteristics such as structural motifs, G-quadruplex, and conserved sequences. There are multiple post-SELEX reactions described, and fortunately, aptamers are versatile enough to undergo diverse modifications [29]. For example, nowadays, it is possible to introduce new chemical modifications to protect aptamers against degradation or confer new functionalities. Oligonucleotide composition exposes a variety of chemical groups and thus enables the attachment of signaling agents. Likewise, signaling agents can enable diverse chemical reactions or physical interaction with aptamers. Most frequently, functionalities consist of the covalent incorporation of 50 or 30 groups, such as amines and thiols, by phosphor chemistry reactions [30]. However, it must be verified that post-SELEX modifications do not affect the initial characteristics of the oligonucleotide sequence. Finally, the purification of the probe is very important. The molecular weight and physicochemical properties of aptamers allow for the use of molecular exclusion techniques or similar, which enable a complete isolation of the probe.

2.2

Aptamer In Vitro Applications

Aptamer characteristics, as summarized above, make them ideal diagnostic reagents. Accordingly, the number of applications continues to grow and is expected to make great contributions to the biotechnology industry in the near future [31]. To date, a high number of in vitro aptamer diagnostic applications have been reported [32]. The most developed application of diagnostic aptamers is in the field of biosensors (“aptasensors”), wherein the aptamers’ versatility allows for a wide array of sensor formats. Aptamers often recognize their targets by a mechanism in which they undergo structural rearrangements [33] which can be used to transduce the binding event into a measurable signal [34]. Surface plasmon resonance (SPR) is a modern analytical technique based on the measured signal being caused by refractive index changes, due to the mass of a compound in solution that interacts with a target immobilized at a liquid–solid interface, in an evanescent wave field [35]. Recently, a SPR aptamer biosensor was explored for the detection of a label-free toxin. These strategies have great advantages in terms of rapidity and real-time sensing as well as sensitivity [36]. Otherwise, a variety of aptamer probes are used for biosensor development, such as electrochemical sensors, surface-enhanced Raman spectroscopy (SERS), and optical formats. The SERS technology is based on the frequency shifts of Raman scattering and can provide “fingerprint” information of the analytes’ chemical structure [37]. In this sense, an aptamer sandwich methodology was developed for influenza virus

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detection. Primary aptamers were attached to metal particles of a SERS substrate, and influenza viruses were captured and bound with secondary aptamers labeled with Raman-active molecules. Aptamer-based sensors provided recognition of various influenza viral strains, including H1, H3, and H5 hemagglutinin subtypes [38] (see Fig. 1). SERS-based biosensors have been used increasingly over the past few years, including an extremely highly sensitive methods [39].

Fig. 1 Design of aptasensor for influenza virus. (a) Scheme of sandwich-like aptasensor for IV detection: (1) primary aptamer is immobilized onto Ag nanoparticles, (2) IV is captured with primary aptamers, and (3) secondary aptamers interact with IV, providing the SERS signal. (b) SEM image of the empty substrate. (c) AFM image of the control zone with adsorbed components of allantoic fluid. (d) AFM image of the experimental zone with a dense layer of virus particles adsorbed from the sample with 0.64 HAU/mL concentration (Figure from [38])

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Electrochemical aptasensors provide a highly sensitive and selective methodology. Since only a few target analytes exhibit redox properties, an aptamer probe is usually created by attachment of electroactive labels, such as methylene blue, ferrocene, or similar [40]. Additionally, molecular beacons consist of a dual-label molecule which adopts two or more conformations [41]. After target recognition, molecular beacons change their conformation producing a signal change which is detectable. In fact, electrochemical beacons (e-beacons) have been explored by Radi et al. Therein, a bifunctional derivative of the thrombin-binding aptamer is used with a redox-active moiety. The ferrocene-labeled aptamer with thiol functionality was bound to a polycrystalline gold electrode surface. The results showed a linear response of the ferrocene oxidation signal to the increase in the thrombin concentration [42]. Nonetheless, optical devices are most widely developed and available in the market place [8]. A molecular beacon fluorescence resonance energy transfer (FRET) phenomenon is the most commonly used strategy and will be described in Sect. 3.1. Aptamer-linked immobilized sorbent assay (ALISA), flow cytometry, microfluidics, precipitation, and magnetic nanomaterials are technologies that have also been adapted to aptasensors [43]. In brief, the use of aptamers as a diagnostic tool is developed by diverse methodologies, applicable to diverse targets, with growing market place products and sensitive, specific, robust, and affordable results.

2.3

In Vivo Aptamer Diagnostics

The successful use of probes for in vivo applications requires additional characteristics, to assure the effectiveness and a safety profile. A molecular imaging probe needs to reduce the pharmacological effects to biological systems and processes to a minimal level [10]. Principally, assurance profiles and overcoming biological barriers present a challenge [44]. Thus, a probe with clinical translation potential is expected to exhibit the following properties. Non-immunogenic and Nontoxic Both aspects provide the biological assurance profile for an in vivo probe. Probes for in vivo applications are treated as a particular class of pharmaceutical, and world pharmaceutical regulations are applied. Although a diagnostic probe allows the observation of pathology, it has no pharmacological effect and is administered in a very small amount. In addition, the absence of immunogenicity and toxicity of aptamers results in the biological assurance profile for the development of an in vivo probe [7]. High In Vivo Stability This is a huge challenge due to numerous enzymes in serum and tissue, which may degrade the probe. Probes have to overcome this first biological barrier to reach the target at a sufficient concentration. Since endo- and exonucleases are abundant in biological fluids, several modifications of nucleotides

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are introduced to protect the vulnerable 20 -position of the ribose-phosphate backbone against endonucleases (20 -fluoro, 20 -amino, 20 -O-methyl) [45]. Terminal nucleotides are also attached to prevent exonuclease susceptibility [46, 47]. A more extensive strategy is the use of mirror-image configuration oligonucleotides. These aptamers, built from nonnatural L-nucleotides, are known as a Spiegelmers® and are not recognized by the ubiquitous plasma nucleases. Thus, the mirror-image configuration confers plasma stability in vitro and immunological passivity. Their selection process consists on the use of D-configured oligonucleotide libraries against the mirror-image of the biological target of interest. Identified sequences are finally synthesized using enantiomeric (L-)ribonucleotides [48]. High Contrast Ratio High image quality requires high contrast which is a consequence of a high target-to-background signal ratio. First, the target must uptake and then retain the probe long enough. High target uptake is achieved by overcoming the biological barriers and through effective tissue penetration. For tumoral and intracellular targets, additional barriers must be considered. Aberrant blood vessels, heterogeneous microvascular perfusion, interstitial pressure, and pH are relevant in pathologies, such as cancer [49]. The aptamers’ size, charge, and composition are important characteristics to obtain tissue penetration and significant target uptake. However, low signal on normal tissue is very important to decrease the background on images and depends on systemic clearance. Probe decay within the biological environment is dependent on its pharmacokinetic properties. Route of administration and physicochemical properties, such as pKa, molecular weight, and logP, directly affect probe pharmacokinetics, including rates of adsorption, distribution, metabolism, and excretion. The small size of aptamers, between 25 and 40 nucleotides, results in a molecular weight of ~15,000 Da, which is excreted by the kidneys in minutes [50]. In most of cases, this is a desirable characteristic for in vivo probes. Scale Production and Economic Feasibility In vivo applications need to be scaled up, which can directly affect their application. Low manufacturing costs, shorter generation time, and no batch-to-batch variability are fundamental aspects for translation to clinic. Aptamers’ versatility enables the combination of all these attributes in a unique probe for in vivo application [12]. Additional characteristics conferred by attachment of nanoparticles or multivalent structures might significantly improve the probes’ bioavailability. Simple building blocks of the same aptamer motif can significantly improve the avidity due to multiple target binding sites. The combination of different aptamer motifs offers opportunities to build multifunctional molecules that can serve as a basis for diverse approaches [51]. Lei and colleagues designed a novel strategy of nanotriangle-scaffolded multivalent split activatable aptamer probes, which combine advantages of programmable self-assembly, multivalent effect, and targetactivatable architecture [52, 53]. The in vivo diagnostic approaches join into the imaging development, and the contribution of aptamers includes molecular target strategies in this field.

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3 Molecular Imaging Image-based detection platforms are used extensively in medicine to detect, localize, and characterize pathologies. Also, imaging has become an indispensable tool in research, clinical trials, and diverse disciplines. Imaging can reveal complex structures and dynamic interactive processes located deep in the sample that are otherwise difficult to decipher [54]. While conventional images provide anatomical and physiological information by an external radiation field applied to the patient, new imaging strategies are used as diagnostic tools. Molecular imaging involves in vivo characterization and measurement of biologic processes at the cellular and molecular level [55]. Through noninvasive methodologies, molecular imaging is being increasingly used for in vivo diagnosis as it provides additional information to the conventional imaging strategies. Among them, in vivo gene expression, antigen presence, metabolic alterations, cell pathways, and molecular localization can be identified. Generally the methodology consists in probe administration to the patient, and after biodistribution, the probe accumulates on the target as a result of the probe transport, binding to receptors and antigens, enzymatic reactions, biochemical or mechanism trapping, phagocytosis, or combinations of these [56]. Finally, the signal of the probe must be transferred throughout the tissue to be detected and to create the image. In fact, molecular imaging provides an alternative or complementary diagnostic tool. Obtaining molecular information from a temporary context and in the environment of interest is the goal of this methodology. Real-time measurements make it possible to detect changes produced by stimuli and in a wide range of unique studies. Additionally, molecular imaging has the potential to play a leading role in the routine use of therapeutics, particularly in oncology, where drug resistance develops over time and targeted therapies can be extremely expensive [54]. For successful in vivo imaging, localization of the target, reasonable distribution time, signal depth penetration, and energy and detection equipment are also features to be considered to the design of the methodology. However, the molecular imaging probe is most crucial, as it has both a targeting and an imaging component, and is able to be introduced into the patient. However, the ratio between pathologic and normal tissue must be high, and probe should not reside long in blood or in the organs of metabolism [57]. Aptamers as targeting components are biomolecules with strong potential for the development of molecular imaging agents. The small size and polyanionic nature of aptamers may lead to rapid blood clearance and tissue uptake and may minimize the residence time in the liver and kidneys, providing potentially useful features for imaging [57]. Each imaging modality has advantages and disadvantages, and there are increasing numbers of hybrid instruments that offer the possibility of combining them [58]. Furthermore, the aptamer’s ability to bind a wide range of targets allows extraordinary applications of aptamer-imaging probes [52, 53].

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Aptamers have already been included into a variety of molecular imaging modalities such as optical images, nuclear imaging, computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound. They differ in type, spatial resolution, sensitivity, imaging time, and cost of currently available common systems. Details are discussed below.

3.1

Optical Imaging

Optical imaging is one of the molecular imaging modalities that has been more studied, including both in vitro and in vivo approaches. It is based on detecting the transmission of light (photons) [59]. This includes photon emission in the range between ultraviolet and infrared wavelengths. Luminescence comprises the emission of photons produced by different phenomena, i.e., chemical or biological reactions, in the visible and near-infrared spectra (400–1,700 nm). Fluorescence is more frequently applied and based on photons that are emitted after light absorption from an external source. For fluorescence imaging acquisition, the fluorophore is excited by a laser, and the light is detected by an appropriate camera. In the near-infrared (NIR) range wavelength (650–1,000 nm), background interference is minimum because of considerably lower tissue absorption coefficient in the region [60]. Thus, near-infrared (NIR) optical imaging is widely used for in vivo applications with adequate fluorophores [61], providing a micron-scale resolution at 0.2 mm depth [62, 63]. These imaging modalities can principally be used for imaging of surface pathologies and surgically exposed organs or for intraoperative imaging but are limited in their imaging depth (cm) and resolution [64]. Promising applications in the clinic include fluorescence endoscopy, optical coherence tomography, and confocal microendoscopy. These methods, collectively termed “optical biopsy,” are nondestructive in situ assays of mucosal histopathologic states using light that can provide instantaneous tissue assessment, alternative to conventional biopsy [65, 66]. Recent technological advances in fiber optics, light sources, and detectors have stimulated the development of numerous optical methods that promise to significantly improve our ability to visualize and evaluate the human epithelium in vivo. To create these imaging probes, direct conjugation by covalent attachment of fluorophores to aptamers is widely used. Either a standard chemical reaction at the 50 or 30 prime ends [67] or fluorescent-labeled bases [68] are commonly used (see Fig. 2). Many of such produced aptamers are promising imaging probes for in vivo studies [67–71]. A very useful strategy for molecular imaging consists of designing the probe with a fluorophore and an acceptor included in the aptamer system. This fluorescence resonance energy transfer (FRET) can be detected as a consequence of

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Fig. 2 In vivo imaging of β55 positive amyloid plaques. In vivo 2-photon microscopy images from an 18-month-old APP/PS1 transgenic mouse obtained 1 h after topical application of fluoresceinlabeled β55 (a, b). Texas Red labeled dextran was intravenously injected for visualization of blood vessels. β55 positive plaques and cerebral amyloid angiopathy are clearly visible in the cortex (a) and vasculature (b), respectively (scale bars: 20 μm) (figure from [68])

conformational change or molecular interaction between the fluorophore and the acceptor (a quencher or a second fluorophore), depending on the distance. Target recognition can be the switch to produce this activation and the final emission [70– 73]. Some authors reported the use of conformational changes associated with aptamer/target binding to generate activatable aptamer probes (AAP) [72, 74]. When the probe binds to a target, conformational changes separate the fluorophore from a quencher, resulting in active fluorescence [75]. As an innovative alternative, aptamers are conjugated to quantum dots (QDs), nanocrystals of semiconductor materials, which have interesting emission properties [76]. Quantum dots are also used for in vivo imaging. Their narrow and symmetric emission peaks, obtained by excitation with broad ranges of wavelengths, allow for multicolored single QDs that enable multimodal imaging [77]. Compared with conventional fluorescent materials, QDs exhibit more advantages, including water solubility, high quantum fluorescence yield, low photobleaching, and chemical stability [78]. Aptamer-QDs are reported to have strong fluorescence and excellent photostability and have been successfully applied in active tumor-targeted in vivo imaging [79]. Recently, a nanoprobe constructed with a QD-labeled aptamer was reported to be capable of binding to the epidermal growth factor receptor variant III (EGFRvIII) spacially distributed on the surface of glioma cells. The fluorescence imaging in vivo, using glioma model mice, showed the probe could penetrate the blood-brain barrier and generate a strong fluorescence on tumors, which contributed to clearly visualizing the glioma margins [76].

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Aptamer-Based CT and MR Imaging

Computed tomography (CT) and magnetic resonance imaging (MRI) are conventional imaging techniques commonly used in clinics, known as anatomical imaging modalities. Both usually use contrasting agents, which alter image contrast to help distinguish between normal and abnormal conditions. However, using a proper probe, these modalities can provide more information. Using aptamers as targeting components, these modalities can be part of the molecular imaging field. In CT, the patient is exposed to an external source of X-rays, and the image is based on X-ray attenuation by tissue. Analogous to the 2D X-rays, CT includes a rotating detector producing a 3D image. Results include anatomical information with high resolution, and there is no limitation on tissue depth. Soft tissues have low X-ray attenuation, and CT results are poor in contrast. Elements with high atomic weight, such as iodine, are administrated to increase the attenuation but are generally not specific and require considerable quantities. To overcome these hurdles, molecular imaging approaches have been developed using contrast agents attached to aptamers for CT molecular imaging [80, 81]. Magnetic resonance imaging (MRI) offers a primarily diagnostic method, given its high soft tissue contrast, spatial resolution, low risk, availability, and relatively low cost [82]. For anatomical and physiological information, MRI is an excellent technique. Images are obtained by exposing endogenous nuclei (most commonly 1H in H2O) to a static magnetic field, perturbing a steady-state equilibrium with time and space varying magnetic fields. After perturbation, all nuclei relax by two unique and co-dependent relaxation mechanisms: T1 (spin-lattice relaxation) and T2 (spin– spin relaxation) [64]. Images depend on different levels of magnetic nuclei relaxation in tissues. MRI also provides micrometer resolution, and contrast agents are frequently used. Commonly, these agents include the injection of small complexes based on gadolinium (Gd3+) or include biocompatible superparamagnetic iron oxide nanoparticles (SPIONs) [83, 84], which increase the relaxation times. The development of MRI has advanced to create contrast agents based on specific targeting strategies. Aptamers have been complexed with contrast agents to develop MRI-based molecular imaging [85]. Some authors indicate SPION disrupted conformational changes due to aptamer-target interactions displaying larger relaxation values [86, 87]. Recently, preclinical studies showed a target aptamer conjugated to the gadolinium on nanocarriers with promising results [88]. A multimodal nanoprobe was designed against a type I membrane glycoprotein, endoglin (CD105), which is expressed abundantly in the tumor periphery of hepatocellular carcinoma. The nanoprobe included a single-stranded DNA aptamer conjugated with gadolinium in a G5 dendrimer (aptamer/DTPA/Gd/PEG/dendrimer). The invasive tumor margins were clearly delineated in a model mouse tumor [89].

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Ultrasound Imaging with Aptamers

Ultrasound is one of the most widely used conventional imaging modalities in the world because it is the most accessible (transportable) and safe and incurs a lower cost. Here, high frequency sound waves are transmitted to the tissue and scattered by differences in density and compressibility. Micrometer resolution and real-time images demonstrate the utility of this anatomical imaging modality. However, the visuality and veracity of ultrasound imaging for the diagnostic and prognostic prediction of some pathologies are controversial [90]. Limitations include depth and inability to discern abnormalities from surrounding tissue, owing to the similar physical properties of soft tissues within the body [91]. New ultrasound probes have been designed to include this modality into molecular imaging using aptamers [92]. This includes modified contrast agents as probes for target recognition. Multi-walled carbon nanotubes have been reported as promising ultrasound contrast material due to their particular structure and properties. Recently, these nanostructures with pegylated aptamers were studied as ultrasound contrast agent [93]. In this case, an aptamer anti-PSMA was attached to the surface of multi-walled carbon nanotubes to further enhance their targeting ability and biocompatibility. The nanotubes were modified with a bifunctional polyethylene glycol (by amino and carboxyl group), and the aptamer was attached. The intravenous injection of the probe was performed in BALB/c xenograft nude mice models of a prostate cancer. The results showed a better visuality and veracity of the images as compared with the traditional contrast agent [93]. This exemplary work could have a significant impact on the improvement and adaptation of ultrasound contrast agents, available for new developments, but it still remains in the preclinical phase.

3.4

PET and SPECT Imaging

Positron emission tomography (PET) and single photon emission computed tomography (SPECT) are both medical imaging modalities. These diagnostic techniques include the use of radioactivity for high tissue penetration and are well-known nuclear imaging or emission tomography imaging techniques. The basis of these modalities consists of detecting emitted high energy photons prevenient of a radioactive decay. They include the administration of a radioactive probe to the patient, known as radiopharmaceutical formulation, generally by intravenous injection. After distribution, the probe reaches the pathology area, and its retention there is a result of transport, binding to receptors and antigens, enzymatic reactions, biochemical or mechanistic trapping, phagocytosis, or combinations of the aforementioned [56]. Finally, the probe emission is measured by an external detector. Thus, essentially PET and SPECT are molecular imaging techniques, with poor morphologic information. Despite millimeter spatial resolution, both modalities

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provide high molecular sensitivity (1011–1012 mol/L) and independent location depth of the emission source [56]. Moreover, image fusion techniques have been developed, and the combination of CT and MRI with PET or SPECT modalities is now possible, offering a more complete and accurate assessment of disease [94]. PET and SPECT substantially differ in the type of radionuclides used. PET is based on the use of probes labeled with a radionuclide that decays by positron emission. When the positron is combined with an electron, both are annihilated, and their mass is converted. Each annihilation produces simultaneously two 511 keV photons in opposite directions at an angle of 180 . SPECT radionuclides, such as 99m Tc, 111In, and 67Ga, decay with single photon emission. The use of both is principally dependent on the radionuclide availability and imaging devices. To create the radioactive probe, conjugation to radionuclides usually requires labeling by isotopic or coordination reactions. This attachment creates the probe and includes strict reaction time considerations due to radioactive decay and a very high necessary final purity. Aptamers could be exposed to high-temperature reactions and enable fast conjugation and high purity yields without altering their binding properties [11], a characteristic highly desired. Both PET and SPECT are the most advanced molecular imaging modalities, currently available in clinics. Besides their in vivo advantages, in addition to their affinity and specificity, aptamers have great potential for this image modality in which pharmacokinetics and tissue penetration play a central role [95]. The good tissue penetration and fast clearance of aptamers lead to image acquisition in a considerable time. Radioactive aptamer-based probes have been reported since 1997 [96]. Preclinical studies with aptamers in PET include the use of a positron emitter, such as fluorine-18, gallium-68, and copper-64 (see Fig. 3) [97, 98]. Additionally, a metabolic profile study of [18F]-L-RNA Spiegelmer after intravenous

Fig. 3 Representative images of in vivo 18F-labeled HER2-aptamer PET in HER2-positive and HER2-negative tumor-bearing mice. (a) HER2 overexpressing BT474 tumor shows increased uptake, compared to the (b) HER2-negative MB-MDA231 tumor. (c) %ID/g of tumor calculated from 18F-labeled HER2 aptamer. The %ID/g of aptamer in the BT474 tumor was significantly higher than those in the MDA-MD231 tumor (Figure from [97])

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administration in animals was successfully performed, using whole-body PET imaging modality [99]. Single gamma emitters, such as technetium-99, gallium-67, and indium-111 radiolabeled aptamers, have been investigated for SPECT imaging [69, 100, 101]. Hence, PET and SPECT aptamer probes have shown promising results, whereas experience in this imaging modalities is a powerful base to enhance their development. Aptamers constitute molecules versatile enough for this kind of imaging modality, and their in vivo properties are perceived with high expectations in this field.

4 Summary Precision diagnostics and personalized medicine are the goals of the new century. The benefits of diagnostics with aptamers include directly measuring the activity of target molecules for the early detection of pathology, changes in therapy responses, and detecting metastases in cancer. Indeed, market demand of biotechnological advancements in diagnostics provides the basis for aptamer developments. Consequently, the significant increase in aptamer development is clearly visible over the past decades, with strong research and patents. However, the successful use of aptamers is still a challenge while overcoming antibodies in the marketplace, which are their main competitor. The expiration of the SELEX patent will be allowing the advance of aptamers as well. Currently, the industry is more concentrated on aptasensor development, which is expected due to a more direct translation from the use of antibodies. For molecular imaging, aptamers have unique properties and are ideal to create a wide range of probes. In vivo diagnostic approaches need more efforts comparable to therapeutics for biological effectiveness and assurance. Nevertheless, aptamers are molecules with remarkable potential and versatility, and their future success is only a matter of time.

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Adv Biochem Eng Biotechnol (2020) 174: 161–194 DOI: 10.1007/10_2020_124 © Springer Nature Switzerland AG 2020 Published online: 11 March 2020

Aptamer-Modified Nanoparticles in Medical Applications Alina Eilers, Sandra Witt, and Johanna Walter

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Coupling of Aptamers to Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Aptamer-Modified Nanoparticles and Multivalent Binding . . . . . . . . . . . . . . . . . . . . . . . . . 2 Assays with Aptamer-Modified Nanoparticles in Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Assays Using Aptamer-Modified Gold Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Biosensors Using Aptamer-Modified Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Cell Detection with Aptamer-Modified Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Aptamer-Modified Nanoparticles for Therapeutic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Drug Delivery with Aptamer-Modified Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Photothermal and Photodynamic Therapy with Aptamer-Modified Nanoparticles . 3.3 Other Therapeutic Applications of Aptamer-Modified Nanoparticles . . . . . . . . . . . . . . . 4 Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Since aptamers have been selected against a broad range of target structures of medical interest and nanoparticles are available with diverse properties, aptamer-modified nanoparticles can be used in various diagnostic and therapeutic applications. While the aptamer is responsible for specificity and affinity of the conjugate, the nanoparticles’ function varies from signal generation in diagnostic approaches to drug loading in drug delivery systems. Within this chapter different medical applications of aptamer-modified nanoparticles will be summarized and underlying principles will be described.

Alina Eilers and Sandra Witt contributed equally to this work. A. Eilers, S. Witt, and J. Walter (*) Institut für Technische Chemie, Hannover, Germany e-mail: [email protected]

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Graphical Abstract

Keywords Aptamer, Biosensor, Diagnostics, Drug delivery, Nanoparticle, Therapy

1 Introduction Aptamers were first described in 1990 [1]. Since then many aptamers against a wide variety of target molecules have been selected [2]. At the same time, nanotechnology has advanced, thus providing the use of aptamer-coupled nanoparticles for many applications in therapeutic applications, for biosensing or cellular imaging [2, 3]. In contrast to free drug molecules, it was shown that targeted nanoparticles can accumulate in cancer tissue. So systemic side effects of drugs can be prevented in therapeutic applications, and in vivo imaging of target tissue or biomarkers can be enabled in diagnostics [3]. Also ex vivo applications for biomarker detection like lateral flow assays can be performed with aptamer-modified nanoparticles, where the nanoparticles are used for imaging and the aptamers to achieve specificity [4]. Nanoparticles are structures with a size smaller than 100 nm. They can be composed of different materials, resulting in many different types of nanoparticles such as gold nanoparticles, quantum dots, iron oxide nanoparticles, polymer nanoparticles, dendrimers, liposomes, and carbon nanotubes. Although derived from different materials, nanoparticles commonly display a high surface area to

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volume ratio and offer the opportunity to modify this surface with different molecules. For example, targeting ligands such as aptamers can be used for nanoparticle modification [5]. Important aspects of aptamer coupling to nanoparticles are highlighted in the section below. This chapter deals with the application of aptamer-modified nanoparticles with a focus on both therapeutic (Sect. 3) and diagnostic (Sect. 2) applications.

1.1

Coupling of Aptamers to Nanoparticles

Aptamers can either be immobilized to nanoparticles by covalent coupling or in a non-covalent way [6–9]. Most immobilization strategies require a site-specific modification of the aptamer; these modifications are commonly introduced during the aptamer synthesis [10]. Covalent coupling can, e.g., be performed via carbodiimide coupling chemistry [6]. Non-covalent coupling can be performed by utilizing affinity ligands (e.g., streptavidin and biotin) [9]; physisorption, e.g., between thiol-modified aptamers and gold nanoparticles [7]; or π-stacking between nucleotide bases of the aptamer and the side walls of single-walled carbon nanotubes (SWNTs) [8]. To ensure the functionality of nanoparticle-coupled aptamers, some parameters have to be considered. Aptamers depend on their three-dimensional structure for target recognition and binding; disrupting the native structure of an aptamer may lead to a loss of affinity [11]. Therefore the density of immobilized aptamers must be optimized carefully. Too high densities can result in non-correct folding of aptamers [12], while too low aptamer density can reduce the binding capacity of the functionalized nanoparticle. Furthermore the surface charge of the nanoparticle can affect aptamer folding, e.g., a positive-charged surface can interact electrostatically with the negative-charged aptamers and lead to aptamer unfolding. In some cases aptamers that are bound directly to a surface cannot adapt their correct folding because of their proximity to the surface; in these cases spacers (e.g., poly T spacers) can be used to increase the distance of the aptamers to the surface and therefore allow correct folding. The orientation of aptamer immobilization can also affect aptamer functionality; in this case both 30 and 50 terminal modifications should be investigated to optimize functionality of the aptamer orientation [13]. If these aspects are taken into account, aptamer-modified nanoparticles can be a promising tool for medical applications including diagnostics and therapy.

1.2

Aptamer-Modified Nanoparticles and Multivalent Binding

Nanoparticles hold the potential to carry more than one aptamer, thereby facilitating multivalent binding with dramatically increased affinity. Here either several copies

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of one aptamer or different aptamers directed against the same target can be immobilized on a nanoparticle. This can dramatically increase the affinity of the system due to avidity effects [14]. While the term affinity describes the interaction between one ligand and its target structure, avidity describes the overall affinity of multiple binding events between the multimeric ligand construct and the target [15]. Avidity effects are possible when either different aptamers binding to distinct sites of a target molecule are available or in case of multivalent target structures, such as multimeric proteins or targets expressed in high density on a cell surface [15]. Multivalent aptamer-modified nanoparticles have already been proposed for therapeutic applications. They were used to develop theragnostic agents for cancer cell therapy [16] and drug delivery [17]. Moreover aptamer-modified nanoparticles were used to develop a system to control thrombin activity [18, 19]; one of these systems is described in Sect. 3.3 of this chapter in detail.

2 Assays with Aptamer-Modified Nanoparticles in Diagnostics Common types of diagnostic applications are immunoassays using antibodies or analytical methods like liquid chromatography (LC) [20–23]. Because of their high costs and some other disadvantages, efforts are made to replace antibodies by aptamers [4, 6, 12, 24]. Due to their low cost production and the possibility to target even small and toxic analytes, aptamers provide the possibility for new diagnostic assays and applications. For generation of a signal, the aptamers often get conjugated to nanoparticles [12, 23]. The nanoparticles can produce a visible signal, either based on their color like gold nanoparticles or, e.g., an electrochemical signal [25]. Different assays exploiting aptamer-modified nanoparticles for diagnostic applications are discussed in the following sections.

2.1

Assays Using Aptamer-Modified Gold Nanoparticles

In assays with gold nanoparticles (AuNPs), the optical properties of AuNPs are utilized. Color shifts due to the size and distance-dependent light absorption can be analyzed by the naked eye. Colloidal gold nanoparticles are red; agglomerated particles show a color shift over purple to blue and black eventually. The AuNPs are mostly either produced by reduction of tetrachloroauric acid (HAuCl4) by sodium citrate or by laser ablation of gold [26]. AuNPs can easily be modified with ligands, such as aptamers but also proteins like antibodies or other molecules [27, 28]. Often the high affinity of thiol toward gold is exploited, e.g., to immobilize thiolmodified aptamers on the AuNP surface, resulting in a strong semi-covalent

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physisorption [29, 30]. Coating gold nanoparticles with a silica layer leads to free hydroxyl groups on the surface, which can further be functionalized with aptamers [31, 32]. Additionally “click” reactions of terminal alkynes and azides can be performed. These “click” reactions utilize the Au-thiol bond, which can further be used for modifying AuNPs with Polyethylene glycol (PEG) ligands [33]. Non-modified aptamers get adsorbed as well, just with a weaker, electrostatic interactions [34]. The assays use the ability of aptamers to specifically bind their target. Some assays need an additional interaction partner, to generate signals. In case of small molecule detection, oligonucleotides complementary to the aptamers are frequently used. The assay setup can be a sandwich or a competitive assay; properties like size and binding mechanism of the aptamer, target, and oligonucleotide are crucial for the selected setup. In this chapter, the most common assays using aptamer-modified AuNPs are presented.

2.1.1

Lateral Flow Assays

Lateral flow assays (LFAs) are paper-based platforms for detection and sometimes even quantification of an analyte. Many LFAs work with complex media, such as blood, urine, saliva, or serum [35]. The most popular LFA is the pregnancy test, invented in the 1970s [36], using urine or blood as sample probe. But there are numerous LFAs for different analytes, some of them shown in Table 1. They all have in common that the visible signal determining between positive or negative is derived by ligand-modified AuNPs. In the case of the pregnancy test the ligands are antibodies [37], but more recently also aptamers have been used. The aptamers are immobilized to the nanoparticles and bind their target based on their threedimensional structure. In case of aptamer-based LFAs, two different setups are most frequently used, competitive LFAs with a complementary oligonucleotide (cOligo) or sandwich format with two different aptamers binding the analyte simultaneously. Depending on the setup, the presence of the targeted analyte results in a colored or uncolored test zone. Table 1 Assays with aptamer-modified gold nanoparticles Setup Lateral flow assay Lateral flow assay Lateral flow assay Colorimetric assay Colorimetric assay Colorimetric assay Colorimetric assay

Target Cholera toxin Kanamycin Zearalenone Cholic acid 17 β-estradiol Human insulin Serotonin

Limit of detection 2–10 ng mL1 35 nM 5–200 ng mL1 1 μM 0.1 ng mL1 0.0156 ng mL1 52 ng mL1

Sample Spiked buffer Food samples Spiked corn samples Spiked buffer Spiked buffer Serum Spiked buffer

Reference [45] [46] [47] [48] [49] [50] [51]

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Figure 1 shows the scheme of a LFA; it consists of three to four different parts: sample pad, where the liquid sample is applied; conjugate pad (CP) which possesses the dried AuNP conjugates; membrane (mostly nitrocellulose) which has one test and one control zone; and absorbent pad (AP), which soaks up the test liquid. The LFA uses the capillary force; therefore the sample flows through all the pads while collecting the AuNPs of the CP, interacting on the membrane and finally reaching the AP [35, 38]. The analyte concentration in the sample can affect the assay and result in a concentration-dependent intensity of the test zone, thereby allowing quantification. For reliable results LFAs have a readout window after running the test; afterward the signals can change due to detachment of the particles or analytes. In general they are point-of-care devices that deliver a fast result and are easy to use, even for untrained personnel [38] and exist for various medical targets, as shown in Table 1. The listed LFAs are currently still restricted to research applications, but the usage in complex samples for diagnostic analysis is assumed to allow clinical use.

2.1.2

Colorimetric Assays

In contrast to LFAs, which use a membrane as a solid phase, AuNPs can also be used in liquid-phase assays. Colorimetric assays with gold nanoparticles use the color shifting effect from red to purple/blue, when AuNPs aggregate or assemble [39]. There are two different setups, one with aptamers adsorbed on the particles and the other with stable aptamer-AuNP conjugates. Colloidal AuNPs have a special localized surface plasmon resonance (LSPR), resulting in their optical properties. The LSPR is sensitive to the size of the AuNPs and the local refractive index near their surface. Changes in the LSPR are visible due to the color change of the AuNPs [40]. AuNPs synthesized with citrate are stable in solution because the citrate ions form an adsorbed protective layer around the AuNPs, and the electrostatic repulsion from these anions keeps the AuNPs separated [41]. When salt (e.g., NaCl) is added, the AuNPs aggregate, because the salt ions shield the negative charge on the AuNPs and the interparticle distance decreases, thereby the LSPR changes and the AuNPs solution turns purple [42, 43]. Ligands like aptamers can be bound to the AuNPs and stabilize the AuNPs at higher salt concentrations [44]. Aptamers can form electrostatic interactions with AuNPs and adsorb reversibly on their surface or they can be conjugated covalently or covalently like, as described in Sect. 1.1. The negatively charged aptamers provide stabilization of AuNPs by electrostatic repulsion and steric stabilization, so that the AuNP solution retains it red color. If an analyte is added and two aptamers bind in a sandwich setup simultaneously, the AuNPs assemble and the interparticle distance gets reduced; therefore the LSPR shifts and the visible color changes to purple [39]. The different colorimetric assay setups are shown in Fig. 2. Salt-induced aggregation assays with adsorbed aptamers are shown in Fig. 2a, and directed assembly of conjugates is shown in Fig. 2b.

Fig. 1 Lateral flow assay with aptamer-modified gold nanoparticles. (a) Competitive assay format, cOligo on test zone, control oligo on control zone (binds, e.g., part of aptamer on spacer sequence, which doesn’t interact with target/is free to bind even when target binds). (b) Sandwich format, aptamer on test zone which binds target, conjugated AuNPs bind target as well. AuNP sizes from CP/AP and NZ differ for better visualization

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Fig. 2 Colorimetric assay with aptamer-modified gold nanoparticles, (a) adsorbed aptamers detach from AuNP to bind their target, AuNPs aggregate due to salt addition. (b) AuNPs are modified with aptamer 1 or 2, which can bind in a sandwich format to the added analyte and AuNPs build an assembly

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By comparing different samples, the samples’ color shift gives information about the analyte concentration, and therefore the assay can be quantitative. An overview of various assays with aptamer-modified gold nanoparticles is given in Table 1.

2.2

Biosensors Using Aptamer-Modified Nanoparticles

Besides colorimetric applications, aptamer-modified nanoparticles can be used in various types of biosensors. Depending on the nanoparticle composition, fluorescence signals, UV-Vis analysis, or electrochemical signals can be used for biosensors.

2.2.1

Fluorescence Biosensors

Fluorescence sensors benefit from the property of metal nanoparticles to alter the fluorescence of fluorophores. Dependent on the nanoparticles size and the distance of the fluorophore to the particle, the fluorescence can be enhanced or more likely be reduced; hence most particles quench the fluorescence signal. Crucial for the fluorescence change is the plasmon field around the particle, which is generated by incident light and the dipole energy around the particle [27, 52]. Many sensors are FRET (Förster resonance energy transfer)-based sensors [53]. For FRET sensors the aptamer or cOligo is modified with a fluorophore. In one setup fluorescence-modified aptamers get adsorbed onto nanoparticles and due to quenching generate a low fluorescence signal. When the analyte is present, aptamers detach from the nanoparticles to bind their target; thus the fluorescence increases. This principle is called “signal-on.” Depending on the nanoparticles and fluorophore properties, the opposite “signal-off” can be used as the sensor setup. For the “signal-off” sensor, the aptamer is covalently or semi-covalently bound to the nanoparticle, with the fluorophore having the furthest distance from the nanoparticle as possible. Once the target is present, the aptamers’ structure changes, and therefore the distance of the fluorophore to the nanoparticle decreases, leading to a higher quenching efficiency and lower signal intensity [52, 54]. Another setup has aptamers immobilized to the nanoparticle. When the cOligo is fluorescence-modified and is competing with the target to bind the aptamer conjugated to the AuNP, the cOligo fluorophore quenching evaluates the assay. A sandwich assay would work similarly. Another fluorescence sensor setup uses the aptamers’ structural changes, operating in a fluorophore distance change [53]. Fluorescent-modified aptamers that bound to nanoparticles generate different signals depending whether the target is bound or not bound to the aptamer. Crucial for this signal change is the fluorophore distance to the particle, which has to change distinctly with the aptamer structural change upon

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Table 2 Fluorescence biosensors using aptamer-modified nanoparticles Setup FRET FRET

Target Cocaine Human cardiac troponin I

FRET FRET

Tumor marker Mucin 1 VEGF165

Fluorescence detection Fluorescent enhancement

Theophylline Protein of H5N1 influenza virus

Nanoparticle Quantum dots Gold NPs, CdSeS/ZnS quantum dots CdSe quantum dots Silver NPs, Mn-ZnS quantum dots Gold NPs

Reference [55] [56]

[59]

Silica-coated silver NPs

[60]

[57] [58]

target binding. Thus this setup only works for aptamers which exhibit a large conformational change upon target binding [52]. The methods for fluorescence biosensors listed above consist of labeled aptamers, but there are some label-free methods as well. A competitive assay setup uses aptamer-modified nanoparticles with free fluorophores intercalated to the aptamers. Upon addition of the target, the fluorophores detach from the aptamer, and the fluorescence signal increases, resulting in a signal-on sensor [53]. An overview of different fluorescence biosensors is listed in Table 2.

2.2.2

Other Biosensors

Besides the fluorescence sensors, there are various other applications using aptamermodified nanoparticles. For once there are UV-Vis sensors using absorption properties of nanoparticles. For example, most AuNPs have an absorption peak around 520 nm, which can be influenced by different factors [61]. It shifts to higher wavelength with the particles’ aggregation, or the absorption intensity can change upon binding of analytes. The aggregation shift can easily be monitored by a colorimetric assay due to the sample color shift. When aptamer-modified AuNPs bind the aptamer target, such an intensity shift can occur; the intensity changes proportional to the analyte concentration. The intensity can be enhanced or decreased, depending on assay components [62]. Some electrochemical sensors measure the change in proton relaxation times (ΔT2). Magnetic nanoparticles (MNPs) can enhance the magnetic resonance signal of protons from the surrounding water molecules [63]. Whether aptamer-modified MNPs aggregate or disassemble their cluster due to target binding results in a signal change, measurable by NMR (nuclear magnetic resonance), MRI (magnetic resonance imaging), or relaxometry [64].

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Measuring voltammetry for aptamer-based sensors is a further application. These sensors use various materials as the sensors base, like graphene nanocomposites or carbon electrodes or other nanohybrids, but often use a layer of AuNPs on top for aptamer conjugation. Due to target binding to the aptamer, the electrodes measure changes in cyclic voltammetry, differential pulse voltammetry, or square wave voltammetry [65–67].

2.3

Cell Detection with Aptamer-Modified Nanoparticles

The assays and biosensors described in the previous sections allow the detection of small molecules or proteins. But detection of whole cells is also possible. Specific ligands on the cell membrane can be targeted by the aptamers [68– 76]. Mainly assays targeting bacterial cells or human cancer cells have been described so far. Table 3 gives an overview of different methods for cell detection with aptamermodified nanoparticles.

3 Aptamer-Modified Nanoparticles for Therapeutic Applications Besides analytical and diagnostic applications, aptamer-modified nanoparticles are also a promising approach for treatment of different diseases like cancer, Alzheimer’s disease, or blood-clotting disorders [19, 79, 80]. Due to their size, nanoparticles cannot penetrate the vasculature of healthy tissues. Thus nanoparticles Table 3 Cell detection methods with aptamer-modified nanoparticles Setup Electrochemical, H2O2 reduction Fluorescence and magnetic separation Light scattering with plasma mass spectrometry Paper-based electrochemiluminescence electrode Electroluminescence Fluorescence – flow cytometry Dual-recognition units based on FRET Magnetically assisted surfaceenhanced Raman scattering Electrochemical impedance spectroscopy

Target A549 lung cancer cells HER2 and MUC1 breast cancer cells Hs578T breast cancer cells MCF-7 breast cancer cells HL-60 (cancer) cells Leukemia cells Staphylococcus aureus Staphylococcus aureus Salmonella typhimurium

Nanoparticle Gold NPs

Reference [68]

Silica NPs

[77]

Gold NPs

[70]

Gold NPs

[71]

Gold NPs Fluorescent silica NPs Gold NPs

[78] [73] [74]

Superparamagnetic ferrite NPs, gold NPs Gold NPs

[75] [76]

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accumulate in tissue with leaky vasculature such as inflamed tissue or tumors [79]. This effect, called enhanced permeability and retention effect (EPR effect), alters the biodistribution and pharmacokinetics of a drug, so it can reduce the systemic side effects of nanoparticle-bound drugs and increase drug concentrations in target tissues [5, 79]. The addition of a targeting ligand such as an aptamer further increases the specificity of the system and can promote target binding and cellular uptake of nanoparticles (e.g., via receptor-mediated endocytosis) [5, 79]. This results in higher cytotoxicity of aptamer-modified nanoparticle systems and therefore can reduce the amount of the drug that is needed for therapy [81, 82]. There are several possibilities to use aptamer-modified nanoparticles for therapeutic applications. A frequently used approach is the use of nanoparticles for drug delivery (see Sect. 3.1). Another therapeutic application using aptamer-modified nanoparticles is the photothermal and photodynamic therapy, which uses the characteristics of the nanoparticle material to create damaging effects to cells upon irradiation with certain wavelengths (see Sect. 3.2). In Sect. 3.3 some less frequently used applications of aptamer-modified nanoparticles for therapeutic applications are shown.

3.1

Drug Delivery with Aptamer-Modified Nanoparticles

The most common approach of using aptamer-modified nanoparticles for therapeutic applications is the delivery of drugs to the target tissue with nanoparticles as carrier materials. By the modification with aptamers, these drug-nanoparticle conjugates obtain their specificity for the target cells. Several different materials can be used as carrier materials. Their specific advantages and disadvantages will be discussed below. In Table 4 a summary of different aptamer-based targeted drug delivery systems (TDDS) is shown.

3.1.1

Gold Nanoparticles

Gold nanoparticles (AuNPs) can be produced with different methods; commonly citrate-based methods resulting in citrate-functionalized AuNPs or the BrustSchiffrin method resulting in alkanethiolate-modified AuNPs are used. Preparation methods usually offer high yields and result in nanoparticles with characteristic sizes and shapes (e.g., spherical or nonspherical). The sizes of AuNPs can be between a few nanometer and over 200 nm. The cytotoxicity of AuNPs is dependent on their size and surface modification and has to be investigated for each system individually. The surface area to volume ratio of AuNPs is usually high, and the surface can be modified in many different ways (e.g., with thiols or amines) [83]. Targeting ligands can easily be added to AuNPs, e.g., by physisorption of thiol-modified aptamers or by coupling to functional groups on the nanoparticle surface [7, 83]. For targeted

Aptamer sgc8

sgc8 and AS1411 sgc8

AS1411

S15 and S6 AS1411 A10

A10

A10

A10

AS1411 A10

FB4 AS1411 S2.2 CD 30 aptamer

AS1411

Nanomaterial Au

Au Liposome

Liposome

PEG-PCL PEG-PCL PLGA-b-PEG

PLGA-b-PEG

PLGA-b-PEG

PLGA-PEG

CA-PLGA-b-TPGS PLA-PEG-COOH

PEG-PLA PLGA PLGA PEI-citrate

Mesoporous silica nanoparticle Nucleolin

A549 cells Nucleolin Prostate-specific membrane antigen (PSMA) Prostate-specific membrane antigen (PSMA) Prostate-specific membrane antigen (PSMA) Prostate-specific membrane antigen (PSMA) Nucleolin Prostate-specific membrane antigen (PSMA) bEND5 cells Nucleolin MUC1 CD 30

Target Protein tyrosine kinase 7 (PTK 7) PTK 7 and nucleolin Protein tyrosine kinase 7 (PTK 7) Nucleolin

Table 4 Examples of aptamer-based targeted drug delivery systems

Doxorubicin

Docetaxel Rhodamine-dextran (model) Flurbiprofen Paclitaxel Paclitaxel siRNA

Cisplatin + docetaxel

Doxorubicin + docetaxel

Cisplatin

Paclitaxel Docetaxel Docetaxel

Daunorubicin FITC-dextran (model drug) siRNA (anti BRAF)

Drug Doxorubicin

Alzheimer’s disease treatment Glial cancer treatment Breast cancer treatment Anaplastic large-cell lymphoma (ALCL) treatment Breast cancer treatment

Breast cancer treatment Prostate cancer treatment

Prostate cancer treatment

Prostate cancer treatment

Prostate cancer treatment

Treatment of malignant melanoma Lung cancer treatment Brain glioma treatment Prostate cancer treatment

Leukemia treatment Leukemia treatment

Possible application Leukemia treatment

(continued)

[104]

[80] [100] [101] [102]

[98] [108]

[97]

[84]

[82]

[6] [95] [81]

[92]

[86] [91]

Reference [7]

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Nanomaterial Mesoporous silica nanoparticle Mesoporous silica nanoparticle Mesoporous silica nanoparticle Dendrigraft poly-Llysine (DGL) MS2 bacteriophage capsid

Table 4 (continued) Target EpCAM ATP ATP Nucleolin; cytochrome C and ATP MCF-7 cells

Aptamer EpCAM aptamer

ATP aptamer

ATP aptamer

AS1411; cytochrome c aptamer; ATP aptamer G-quadruplex targeting aptamer Porphyrins for PDT

Doxorubicin

Fluorescein (model)

Dye (model)

Drug Doxorubicin ATP-responsive controlled release of drugs ATP-responsive controlled release of drugs Treatment of multidrugresistant cancer cells Breast cancer treatment

Possible application Colon cancer treatment

[107]

[106]

[105]

[103]

Reference [109]

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drug delivery based on AuNPs, drugs can be integrated into the system by three commonly used ways that are described below (see Fig. 3). The first way is the intercalation of drugs into hairpin regions of DNA (see Fig. 3a). For example, the drug doxorubicin is able to intercalate into DNA hairpins. As specificity-mediating ligands, aptamers can be bound to the surface in addition to the hairpin DNA [7]. The second way is the direct intercalation of drugs into aptamers (see Fig. 3b). For example, the aptamer A10 has a GC-rich stem region, where the drug doxorubicin can intercalate [84]. This intercalation often takes place at GC-rich parts of oligonucleotides, e.g., in G-quadruplex structures [85]. Thirdly the drug can be loaded to the surface of AuNPs (see Fig. 3c). For example, Taghdisi et al. loaded gold nanoparticles with the drug daunorubicin [86]. Figure 3 shows the components of different AuNP-based targeted drug delivery systems. Additionally AuNPs offer the opportunity of photodynamic therapy which is discussed in Sect. 3.2.1. One disadvantage of AuNPs in drug delivery is that only the surface of the AuNPs or the surface-bound ligands can be used as a drug carrier. Some other nanoparticle types do also allow the utilization of the nanoparticle volume to enhance loading capacity, as discussed in the next paragraphs.

3.1.2

Liposomes and Micelles

Liposomes usually consist of a bilayer of natural or synthetic phospholipids (e.g., phosphatidylcholine or phosphatidylethanolamine (see Fig. 4) [85, 87]. The bilayer structure enables encapsulation of hydrophilic drugs inside of the core and hydrophobic drugs inside of the lipid bilayer [85]. At body temperature the bilayers are in a fluid state, making them leaky for encapsulated drugs. If cholesterol is added, the structure of the liposomes gets more stable, and unintended drug release can be prevented [87]. Liposomes can be prepared in different ways; the first described method was the rehydration of a thin film of lipids with aqueous solvents, which was generated by organic solvent evaporation in a round bottom flask. The size can further be reduced by sonication or extrusions through a polycarbonate membrane. One limitation of this method is the rather low encapsulation efficiency [87, 88]. Another method is the addition of lipids dissolved in an organic solvent into an aqueous drug solution (solvent injection technique) and subsequent removal of the organic solvent [87, 89]. However, removal of the organic solvent can be difficult. By using microfluidics, liposomes with a defined size can be produced continuously, thereby offering the opportunity for an industrial preparation method in a large scale [87, 90]. One drawback of liposomes is their short half-life in vivo. However, this can be improved by surface modification with polyethylene glycol (PEG). PEG can prevent plasma protein binding and therefore increase the half-life of liposomes (“stealth liposome”) [87]. Targeting ligands like aptamers can be attached, e.g., via coupling to maleimide or N-hydroxysuccinimide-activated PEG-modified phospholipids [91, 92]. A

Fig. 3 Different designs of targeted drug delivery systems containing aptamer-modified gold nanoparticles. Drugs can either be intercalated into hairpin DNA (a), directly into some aptamers (b), or loaded to the surface of the particles (c) (according to [85])

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Fig. 4 A possible composition of a liposome for targeted drug delivery (according to [85])

PEG-modified liposome with encapsulated doxorubicin was the first FDA-approved nanodrug (Doxil®), but this system was still without a targeting ligand [93]. The results of Kang et al. showed that aptamers increase the binding of the system to target cells, which could be a promising advancement for future applications [91]. Figure 4 shows the components of different liposome-based targeted drug delivery systems.

3.1.3

Polymer Nanoparticles

Polymer nanoparticles are commonly used nanoparticles for drug delivery. They can be prepared out of preformed polymers or directly out of monomers by different methods. Polymer nanoparticles can be organized in a complete solid structure (nanospheres), or they can contain a fluid core (nanocapsules) [94]. In the literature many examples are described, in which block copolymers are used, composed of a hydrophilic and a hydrophobic part [6, 80, 82, 84, 95–98]. These block copolymers usually build nanoparticles through spontaneous self-assembly [99]. The resulting polymer nanoparticles usually have a hydrophobic core and hydrophilic shell (see Fig. 5a). This hydrophobic core enables to encapsulate hydrophobic drugs, whereas targeting ligands, such as aptamers, can conveniently be coupled to the hydrophilic shell compounds [85]. PEG is often used as a shell compound, which decelerates

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Fig. 5 Design of aptamer-modified polymer nanoparticles for drug delivery. Nanoparticles can be produced out of block copolymers that have a hydrophobic and a hydrophilic part and therefore form a hydrophobic core in aqueous solution. In this hydrophobic core, hydrophobic drugs can be encapsulated (a). If nanoparticles are produced only out of a hydrophobic polymer, there is no coreshell structure, but also hydrophobic drugs can be encapsulated (b). Aptamers are usually coupled covalently (according to [85])

systemic clearance by preventing the binding of plasma proteins and thereby preventing recognition by the mononuclear phagocyte system [84, 87]. Despite the popularity of block copolymers, other nanoparticles are composed of only a single type of polymer, e.g., poly(lactic-co-glycolic acid) (PLGA), and therefore do not form a core-shell structure but a nanosphere (see Fig. 5b) [100, 101]. The aptamers serve as affinity ligands for target cell binding and thereby enable specificity, and in some cases they can also promote the internalization of nanoparticles into target cells, e.g., via receptor-mediated endocytosis [80, 81, 96, 98, 100–102]. Figure 5 shows the components of different polymer nanoparticlebased targeted drug delivery systems.

3.1.4

Mesoporous Silica

Mesoporous silica nanoparticles (MSNs) are used for drug delivery because of their good biocompatibility, their high surface area that allows high drug loading, and the possibility of easy functionalization [103]. Furthermore there are several applications to control the drug release from the MSN, including pH-dependent drug release or target-controlled drug release (see Fig. 6) [103–105]. One possibility for controlled drug release is locking the drug-containing pores with DNA hybrids formed by aptamers and oligonucleotides complementary to

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portions of the aptamer sequence. Here drugs cannot escape the pores until the target molecule binds to the aptamer resulting in dissociation of the aptamer from the oligonucleotide (see Fig. 6a). Upon target binding, the aptamer dissociates from the MSN, and due to the flexibility of the complementary DNAs the MSNs pores are open and facilitate the release of the drug [103]. Alternatively, pores can be blocked by aptamer-modified gold nanoparticles (see Fig. 6b). Therefore molecules have to be coupled to the MSN, to which the used aptamer also binds, but the binding strength has to be weaker than to the target molecule (e.g., adenosine also binds to ATP-targeting aptamers, but binding strength of the aptamer is stronger for ATP, so adenosine is released from the ATP aptamer in the presence of ATP). If there is no target, the aptamer-modified gold nanoparticles bind to the surface of the MSN and prevent drug release. In the presence of the target, it will bind to the aptamers, thereby releasing the aptamer-modified AuNPs from the MSN, the pores aren’t blocked anymore, and the drug is released [105]. For example, Zhu et al. and He et al. developed this controlled drug release system using ATP as target molecule to trigger drug release [103, 105]. The surface of MSN can easily be functionalized with different molecules. If drugs are bound to MSN by electrostatic interactions with surface molecules, drugs can be released due to pH shift. For example, Li et al. developed a MSN system with phosphate-modified inner channels that can bind positively charged doxorubicin via electrostatic interactions at physiological pH. If the pH decreases, like in tumor tissue, the charge of the phosphate gets positive, and doxorubicin is released from the pores. Aptamers were bound to the surface of the MSN for targeted cell binding and receptor-mediated endocytosis [104]. Figure 6 shows the components of different MSN-based targeted drug delivery systems.

3.1.5

Others

There are some nanomaterials that are less frequently used for drug delivery but also worth mentioning. These are explained in more detail in the following sections; a short summary of the systems can be found in Table 4. Dendrimers Dendrimers are branched polymers that contain a central inner core. To this core several repeating groups of the polymer are bound, which themselves can bind several repeating groups, so they build a branched outer layer [85]. The surface can be modified with several functional groups, thus providing a wide range of modifications, e.g., with targeting ligands such as aptamers [106]. Drugs can be bound inside the dendrimers. Chen et al. developed a sophisticated dendrimer-based drug delivery system containing three different aptamers for mitochondrial targeting and to circumvent multidrug resistance (MDR) of tumor cells. The aptamer AS1411 was used for targeting and internalization into nucleolin presenting cells. Furthermore, a cytochrome c targeting aptamer was used for internalization of the dendrimers into the mitochondria, and an ATP aptamer was used for selective

Fig. 6 Different designs of aptamer-modified mesoporous silica nanoparticles. Drug release can be prevented by blocking of drug-containing pores with complementary DNA and aptamer upon target binding (a), or with aptamer-modified gold nanoparticles, that bind on targets with lower binding strength next to the drug-containing pores upon competitive displacement by the targets (b). By using electrostatic interactions for drug binding inside the pores, drug can be released by pH shift (c) (according to [103–105])

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drug release. The ATP aptamer was hybridized with a complementary DNA, and the drug doxorubicin was intercalated in this DNA duplex. At high concentrations of ATP, the ATP aptamer binds ATP and therefore dissociates from the duplex structure resulting in drug release. This system ensures that drugs are only released within the mitochondria, where the ATP level is high. Using these multifunctional nanoparticles, Chen et al. could show that this drug delivery system is even effective in multidrug-resistant cancer cells (MCF-7/ADR cells) [106]. Virus Capsid Virus capsids are protein-based nanoparticles that can be used to encapsulate different molecules; the surface can also be modified with different molecules, such as aptamers [85]. Cohen et al. developed a virus capsid based on the MS2 bacteriophage and loaded it with cationic porphyrins, which can be used for photodynamic therapy. The surface was modified with an aptamer that binds to MCF-7 cancer cells. This system showed cytotoxicity to MCF-7 cells, but no cytotoxicity to non-targeted cells; furthermore no cytotoxicity was observed when a non MCF-7 binding aptamer was used [107].

3.2

Photothermal and Photodynamic Therapy with Aptamer-Modified Nanoparticles

Apart from nanoparticles as delivery vehicles for drugs, nanoparticles themselves can be the therapeutic part of an aptamer-modified nanoparticle system. Some materials have the properties to cause photothermal or photodynamic effects [110]. In photodynamic therapy (PDT) usually a nontoxic photosensitizer is activated by irradiation of the targeted tissue. Thereby, singlet oxygen (1O2) is generated by the photosensitizer using the energy from the radiation. Singlet oxygen is highly reactive and causes severe reactions with cellular molecules which finally lead to cell death. In PDT it is possible that the nanoparticle itself is the photosensitizer or the photosensitizers are conjugated to nanoparticles via aptamers. In the latter case, the nanoparticles quench the cytotoxic effects if the photosensitizers are close to the nanoparticles in the absence of the target. By conformational changes upon target binding of the aptamer, the photosensitizer is not quenched any longer, and cytotoxic effects can occur after illumination (see Fig. 7) [8]. In photothermal therapy (PTT) nanoparticles prepared of a suitable material (e.g., gold) are irradiated. The resulting photon energy is transformed into heat which then causes cell damage and finally cell death (see Fig. 8a) [7]. Table 5 shows the components of aptamer-modified nanoparticles for PTT and PDT.

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Fig. 7 Design of aptamer-modified gold nanorods for PDT. The aptamer is elongated with a poly T spacer and a DNA sequence that is complementary to the terminus of the aptamer that is conjugated with the photosensitizer Ce6. Without target binding the photosensitizer is close to the gold nanorod, which quenches the Ce6 activity. Upon target binding the Ce6 isn’t close to the gold nanorod anymore and light irradiation leads to formation of singlet oxygen (1O2) that causes cell damage (according to [8])

3.2.1

Gold Nanoparticles for Photodynamic and Photothermal Therapy

Gold nanoparticles have a high plasmon resonance and low quantum yield; therefore photon energy which is absorbed by AuNPs after irradiation is nearly completely transformed into heat. This offers the opportunity for their usage for photothermal therapy. Additionally the generated heat can lead to a controlled release of drugs if the AuNPs are used for drug delivery (see Fig. 8b) [7]. Nevertheless, not all tissues can be reached by direct laser irradiation. For example, a laser with a wavelength of 532 nm, which was used by Luo et al. for controlled release of drugs from AuNPs, can only penetrate the skin to a depth of 0.3 to 0.5 mm [7, 111]. However, by the use of fiber optics, irradiation can reach nearly all targeted tissues in a minimal invasive way [112].

3.2.2

Single-Walled Carbon Nanotubes (SWNT)

Besides gold nanoparticles also single-walled carbon nanotubes (SWNTs) can be used for photodynamic therapy [8]. They consist of a cylinder out of a single

Fig. 8 Different designs of aptamer-modified gold nanoparticles for PTT. Illumination of the AuNPs leads to generation of heat, which causes cell damage (a), or drugs that are intercalated into hairpin DNA are released after irradiation due to light-induced heat development (b)

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Table 5 Examples of aptamer-modified nanoparticles for PTT and PDT Nanomaterial Gold nanorods (AuNR) Au

Aptamer sgc8

SWNT

Tmb aptamer

sgc8

Target Protein tyrosine kinase 7 (PTK 7) Protein tyrosine kinase 7 (PTK 7) Human α-thrombin

Drug –

Possible application Leukemia treatment

Reference [110]

Doxorubicin

Leukemia treatment

[7]

–

Treatment of diseases related to blood-clotting disorders

[8]

graphene layer with a diameter of around 1 nm and a length from 1 to 100 μm. There are three common ways to prepare SWNT, the carbon arc-discharge technique, the laser-ablation technique, and the chemical vapor deposition technique [113]. SWNTs are known to be good quenchers of fluorescence. Furthermore they can quench the activity of photosensitizers. This characteristic enables SWNTs for the following example application. Aptamer-coupled photosensitizers can be bound to SWNTs non-covalently by π-stacking interactions between the aptamer and the surface of the SWNTs when no target is bound by the aptamer (see Fig. 9). The close proximity of the SWNT quenches the cytotoxic effects of the photosensitizer. Upon target binding, the structure of the aptamer changes and the aptamer is released from the SWNT. Therefore the photosensitizer is no longer quenched by the SWNT, and singlet oxygen is produced upon irradiation [8].

3.3

Other Therapeutic Applications of Aptamer-Modified Nanoparticles

The following section presents selected special applications of aptamer-modified nanoparticles that can be used for therapeutic applications. Aptamer-Modified Superparamagnetic Nanoparticles as Nanosurgeons Superparamagnetic nanoparticles are based on magnetite (Fe3O4) and therefore can be controlled by external magnetic fields [114, 115]. They have sizes between a few nanometers and 180 nm. Their half-life in the blood circulation is dependent on size and modification [115]. Some formulations of superparamagnetic nanoparticles are approved for magnetic resonance imaging, but they are moreover a promising tool for usage in nanosurgery [114, 115]. They can be transported to the target tissue by usage of a three-dimensional magnetic field generator [114, 116]. The surface of these magnetic nanoparticles can easily be modified and aptamers can be coupled. If aptamers are chosen which bind, e.g., to cancer cells, they selectively can be separated from healthy cells (see Fig. 10). For example, Nair et al. were able to separate targeted cells from non-targeted cells; furthermore a large extend of the

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Fig. 9 Design of a single-walled carbon nanotube-aptamer system for photodynamic therapy. In absence of the target of the used aptamer, the activity of the photosensitizer is quenched by the SWNT. Upon target binding the aptamer dissociates from the SWNT and singlet oxygen can be produced by the photosensitizer upon irradiation (according to [8])

targeted cells could be detached from a cell culture surface by rapid changes of the direction of the magnetic field. This method might also be useable in the treatment of solid tumors. Additionally the target cells can be destroyed by the nanosurgeons presumably caused by mechanical strain-induced apoptosis or necrosis. Therefore aptamer-modified superparamagnetic nanoparticles are a promising tool for surgical actions on cellular level [114]. Table 6 shows the compounds of the nanosurgeon system developed by Nair et al. Aptamer-Modified Gold Nanoparticles for Reversible Treatment of Blood-Clotting Disorders Another extraordinary method that uses an aptamer-modified nanoparticle system was developed by Huang et al. They used modified aptamers as drugs in combination with gold nanoparticles. These triblock aptamers contained a poly A sequence for self-assembly on the surface of the gold nanoparticles, a short sequence that can be used for hybridization and a thrombin binding block. Two different triblock aptamers were used which contain two thrombin binding aptamers that bind to different parts of thrombin. For drug preparation both triblock aptamers were hybridized and then immobilized to gold nanoparticles (they build a self-assembled monolayer on the nanoparticle surface). Due to the spatial proximity of the two aptamers, they can bind thrombin effectively and thus prevent the conversion of fibrin into fibrinogen and further prevent blood clotting. In emergencies, where blood clotting is required, the aptamers can be detached from the gold nanoparticles

Fig. 10 A possible application of aptamer-modified superparamagnetic nanoparticles for nanosurgeon. Aptamer-modified nanoparticles can be transported to the target tissue by an external magnetic field, where they could bind to their target cells. Subsequently the position of the external magnetic field could be changed and nanoparticle-bound cells can be separated from non-target cells

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Table 6 Compounds of unusual aptamer-modified nanoparticle systems Nanomaterial Superparamagnetic NP

Aptamer GB-10

Au

Triblock aptamer for thrombin binding

Target Tenascin C receptor Thrombin

Drug –

Possible application Glioblastoma treatment

Reference [114]

–

Treatment of diseases related to bloodclotting disorders

[19]

by irradiating with 532 nm light. The spatial proximity of the two aptamers is lost, and thrombin cannot be effectively bound any longer, so the blood can clot again [19]. However, light with a wavelength of 532 nm can only penetrate the skin to a depth of 0.3 to 0.5 mm [111]. Table 6 shows the compounds of the above described system. Aptamer-modified nanoparticles can be used for various targets in therapeutic applications. Thereby the nanomaterial plays an important role for the possible applications. As of now there are some nanodrugs or aptamers as drugs in clinical use or trials, but the combination of both is still not in clinical stages [10, 117]. Nonetheless, this combination might be an advantageous tool for targeted treatment of diseases in future.

4 Summary and Conclusions Aptamer-modified nanoparticles for medical applications are in the focus of current research efforts. Currently there are many diagnostic applications for small molecule detection but also for whole cancer cell detection described in the literature. Their common usage in, e.g., hospitals or for consumers at home is often not yet established, but the setup principle and components are well described. For therapeutic applications in vivo, different clinical stages have to be finished, before their deployment in routinely usage can be proven. For some cancer types, the combination of diagnostics and therapeutics has already been efficiently shown. For imaging purposes in computed tomography (CT) and magnetic resonance imaging (MRI) with combination of drug delivery for therapy, different aptamer-modified nanoparticles have been utilized [117– 121]. Therefore the deployment of aptamer-modified nanoparticles in diagnostics and therapeutics, or the combination of both, provides various application possibilities and research areas in future. Acknowledgments This work was funded by the Ministry of Science and Culture of Lower Saxony (MWK Niedersachsen).

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Adv Biochem Eng Biotechnol (2020) 174: 195– 210 DOI: 10.1007/10_2019_104 © Springer Nature Switzerland AG 2019 Published online: 23 July 2019

Defining Target Product Profiles (TPPs) for Aptamer-Based Diagnostics Harleen Kaur, Bandhan Chaterjee, John G. Bruno, and Tarun Kumar Sharma

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Designing of TPP, Benefits, and Its Key Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Benefits of Designing a TPP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Features of a TPP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Applications of Defining TPPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 TPP for Tuberculosis (TB) Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 TPP for Point-Of-Care (POC) Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Diagnostic Detection of Bacterial Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Other Aptamer-Specific TPPs and Diagnostic Applications . . . . . . . . . . . . . . . . . . . . . . . . . 4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract Defining target product profiles (TPPs) for aptamer-based diagnostics is crucial to the success or failure of aptamer businesses or products. A well-conceived TPP will place the aptamer in an assay for a target against which antibodies are ill-suited or have difficulty detecting the analyte, such as some highly related proteins or poorly immunogenic small molecule haptens. Strong TPPs can also take advantage of the unique nucleic acid nature of aptamers, to produce assays with

Harleen Kaur and Bandhan Chaterjee contributed equally to this work. H. Kaur Aurobindo Biologics, Hyderabad, India B. Chaterjee and T. K. Sharma (*) Centre for Biodesign and Diagnostics, Translational Health Science and Technology Institute (THSTI), Faridabad, Haryana, India e-mail: [email protected] J. G. Bruno Nanohmics Inc., Austin, TX, USA

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longer shelf life or special chemical properties and ability to be modified versus protein-based antibodies. The following chapter reviews the essence of wellconceived TPPs especially with respect to aptamer targets for diagnostics and illustrates several examples of commercial aptamer diagnostic success. Graphical Abstract

Keywords Aptamer, Diagnostics, Health, TPPs

1 Introduction With two simultaneous seminal works by Ellington and Szostak [1] and Tuerk and Gold [2] in 1990, the genesis of a new era started. What was discovered was going to be a potential panacea to diagnostic and therapeutic challenges. They realized what was already known from studies on HIV and adenovirus in the 1980s, which established that viruses code structured RNAs that then bind proteins with high affinity and specificity [3], and that the capacity of single-stranded nucleic acids to form limited, but sophisticated, 3D structures, conferred their ability to bind ligands specifically. However, the moment of epiphany was that random single-stranded nucleic acid sequences can be screened for candidates that will be binding to a specific analyte with affinity and specificity at par with antibodies, hence the discovery of nucleic acid “antibodies.” Both the groups independently devised an iterative selection process known as SELEX (Systematic Evolution of Ligands by EXponential enrichment) for screening of chemically synthesized random nucleic

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acid sequences against a target, and the resultant high-affinity sequences were termed as “aptamers” (a sobriquet coalescing the Latin word aptus (“to fit”) and the Greek word meros (“part”)) [4, 5]. The development of aptamers includes designing a library of random nucleic acid sequences, typically 60–90 bp long, forming secondary structures (these structures may range from or include stem, loop, bugle, pseudoknot, G-quadruplex, and kissing hairpin) [6, 7] which in turn is functionally analogous to the binding site of antibodies (Fab fragment). Sequential cycles of repetitive selection against a target and PCR-assisted enrichment follow. The final product of SELEX is aptamers which can bind their cognate targets with high affinity and specificity. Consider the following points to understand how aptamers are at least an ideal surrogate, if not better, for antibodies: • First of all, antibodies are generated in biological systems, typically in horses or sheep. This poses a limitation, as antibodies cannot be generated against toxins that cannot be tolerated by the animal system. Further, antibodies cannot be generated against non-immunogenic (targets that do not illicit immune response in the host systems) entities. Conversely, as aptamers are generated chemically and the selection is in vitro, the intended number of targets can be theoretically endless. • The synthesis procedure of aptamers is rapid and cheap and suffers only minimally from batch to batch variations. Antibody synthesis is opposite to it on every account. Furthermore, the selection process for monoclonal antibodies is far more time-consuming and costly than aptamer selection. • Both offer comparable range of affinity (low nanomolar to picomolar range, though aptamers have gone to zeptomolar (10 21 M) [8]) and selectiveness. Because aptamers are chemically synthesized and selected in vitro, aptamers offer greater room for modifications than antibodies. Features like the ability to be chemically modified as per requisite, selection of target epitope, even pharmacokinetic (PK) parameters can be tailored according to needs, and simply do not exist with antibodies. • Aptamers have the ability to refold into their functionally active native state after high-temperature exposures and thus have less stringent storage conditions than antibodies which only remain functional when stored in refrigerated conditions. Because of such technical superiority aptamers were destined for the diagnostics and therapeutics market. Despite being only discovered in 1990, the industry’s worth estimate runs as high as $2.1bn by 2018 [9] and is poised to grow at unprecedented rate in the coming years. With such optimism and opportunities, a profound understanding of the requirements from end users and the aptamer’s intended use becomes of paramount importance. Thoughtful consideration of facts, such as (1) what is the specific use of the espoused product and if it aligns properly with the needs of the end users or the target populations and (2) what features to be incorporated so that the product can compete with the contemporary gold standards, facts that concern the investors and the remaining stakeholders, is indispensable for a viable market product. Luckily such motley but germane questions can be conjoined in a document

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that will be used as a tool to guide the entire process of the product development, known as target product profile (TPP). A TPP is nothing but an elaborate and welldeliberated plan, spanning all the relevant sections from product development to marketing [10]. A well-designed TPP ensures that the R&D is well directed to develop products that satisfy the context and need of the end users properly. Organizations like the Foundation for Innovative New Diagnostics (FIND) further simplify the process by convening the various stakeholders like researchers, clinicians, and end users to gather their views on the product, to develop a better TPP. While most target product profiles (TPP) for aptamers are similar to almost any other business or product development plan to sell a “widget” which targets a specific need, aptamers present some unique opportunities and challenges when compared to their antibody or immunodiagnostic competitors which have been quite commercially successful. Thus, planning of specific aptamer development for particular targets (TPPs) is extremely important to the eventual success or failure of aptamer-based diagnostics. We begin with a general discussion of TPPs with emphasis on aptamer diagnostic products below.

2 Designing of TPP, Benefits, and Its Key Features A TPP document can be used as a strategy planning tool in the development of aptamers for diagnostic assays and also other biomolecules. The development of the TPP and its execution is a joint effort between various stakeholders, such as the team of scientists (or technical team), regulatory authorities, investors, supply chain, and senior management, and can often be used as a reference document for post development discussions.

2.1

Benefits of Designing a TPP

Defining TPP as per its intended use from the beginning of the execution of a project increases the chance of success many folds, as it allows the researchers to design their aptamer as per the requirement of regulatory bodies. The other main advantage of a TPP is that it can serve as an excellent tool for designing, communicating, and tracking the progress and modifications during the aptamer-based diagnostics development. In addition, as it is not limited to aptamer diagnostics, TPPs could also be employed for drug discovery research as well as drug development and have already been utilized for in vitro diagnostics (IVD)-based products for several companies [11]. Also, this kind of exercise could gather a lot of information and feedback from a wide variety of stakeholders from different disciplines, background, and geographies which helps to make well-informed decisions and provide value to the end users, investors, and sponsors.

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Features of a TPP

In order to develop the business strategy for TPPs and quantify the potential market value, five key differentiating features must be put in place for product positioning, raising capital, and discussions with investors and key stakeholders. The key features are discussed in detail below: 1. Statement of intended use: This section of the TPP provides a brief description of the assay or product under development. The following questions need to be addressed by the stakeholders during this phase of product development: • What is the purpose of the assay? Is it for screening, monitoring, or diagnosis? • What is the target population? Is it limited to a particular geography or demographics? • Is it a quantitative or qualitative diagnostic assay? How can the end result(s) be expected, and how can it assist the physician to understand the entire clinical picture and take a well-informed decision related to the treatment? • What is the analyte or marker of interest? Is it well characterized or validated for the assay development? • What instruments or additional diagnostics tests are required to execute the assay? • What is the “gold standard” against which performance of your test will be compared? 2. Brief explanation of test: A concise summary about the diagnostic test along with the usefulness to the patients is helpful to complete this section of the TPP. Questions raised during this section could be as follows: • What is the intent of the test? • What kind of test will it be? Is it a point-of-care test, laboratory test, or use at home test? • What analytes can be detected from the test? • Does the test and target of interest need to be validated per the regulatory guidelines? • What could be accomplished from the test? • When should the test not be used for detection? 3. Summary of test procedure: A good understanding about the simplicity or complexity of the diagnostic test is useful to determine whether or not the target location is ideal for performing the test. For simple and easy-to-use diagnostic kits, such as glucose level monitoring or pregnancy detection, robustness and reliability would be important. On the other hand, complex laboratory tests, such as pathogen detection or tumor biopsy in a clinical laboratory, must be carried out under the supervision of trained and qualified laboratory personnel. The following questions need to be addressed in order to complete this section:

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• What is the layout of the entire test starting from sample collection, sample analysis to final results? • What equipment (or instruments), reagents, and assistance are required to perform the test? • Is the diagnostic assay kit easily accessible in different geographies? • In case of complex testing, what is the turnaround time from sample collection to availability of results? 4. Interpretation of results: The results of a clinical test could be interpreted by visual observation and manual calculation or could be determined through computer software. Regardless of how the results are obtained and reported, this key feature should include how the results are calculated and interpreted. In order to do so, the following questions must be answered and taken into consideration. • How will the results be calculated? • What are the acceptable criteria for a positive or negative/clinically insignificant test result? • What format should be used for reporting the result? • What factors can impact the test results? For example, improper handling of samples or reagents, calibration of equipment, and storage of samples or reagents. 5. Performance characteristics: The last key step to complete the TPP for a particular diagnostic assay would be to summarize the critical performance characteristics that would be used as a standard to build the road map of the studies required to generate data to test and validate the specifications. A checklist of performance characteristics for a diagnostic test is as follows. • Robustness: Robustness is a measure of a diagnostic test capacity to remain unaffected by small but deliberate variations in test parameters and, thus, provides an indication of its reliability during normal usage. • Specificity: Specificity is the ability of a diagnostic test to assess unequivocally the analyte in the presence of components which may be expected to be present such as impurities and degradants. • Sensitivity: Sensitivity is the ability of a diagnostic test to detect the minimum concentration of an analyte in a sample. • Reproducibility: The reproducibility of a diagnostic test is the ability of the diagnostic test to measure the sample and generate data within acceptable range under the same operating conditions. • Limit of quantification (LOQ) and limit of detection (LOD): The quantitation limit of a diagnostic test is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. The detection limit of a diagnostic test is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value. To understand TPPs better, consider this example. In 2017, the WHO outlined an overarching TPP for developing a test for predicting the advancement of tuberculosis

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infection to active disease. For this, the WHO conducted two surveys, first in May 2016 and second in January 2017. The latter had more specific participation, engaging all stakeholders including academia, multilateral and international agencies, NGOs, civil society and community representatives, endemic countries, test developers, and members of the New Diagnostics Working Group (NDWG) to arrive at a consensus TPP. The consensus meeting report outlines TPPs for optimal features of the test and can be summarized as follows [12]: Intended Use • Goal of test – To predict and quantitatively correlate the progression of infection to active disease within the next 2 years, to reflect the effect of treatment • Type of specimen – Invasive specimen like capillary whole blood (finger-prick sample) or noninvasive specimens like saliva, urine, stool, or breath • Target population – Persons who highly likely came in contact with affected persons, individuals showing early symptoms marking the progression of the disease • Target user – Health workers with minimal lab training • Setting (lowest level of implementation in healthcare system) – Health post or tertiary level Performance Characteristics • Sensitivity – 90% sensitivity • Specificity – 90% specificity • Reproducibility – Inter-assay CV 10.0% at high and low extremes of the assay Operational Characteristics • • • • • • • • • • • • • •

No. of steps –