Nanotechnology for Bioapplications (Advances in Experimental Medicine and Biology, 1309) 9813361573, 9789813361577

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Advances in Experimental Medicine and Biology 1309

Bong-Hyun Jun  Editor

Nanotechnology for Bioapplications

Advances in Experimental Medicine and Biology Volume 1309 Series Editors Wim E. Crusio, Institut de Neurosciences Cognitives et Intégratives d’Aquitaine, CNRS and University of Bordeaux UMR 5287, Pessac Cedex, France Haidong Dong, Departments of Urology and Immunology, Mayo Clinic, Rochester, MN, USA Heinfried H. Radeke, Institute of Pharmacology & Toxicology, Clinic of the Goethe University Frankfurt Main, Frankfurt am Main, Hessen, Germany Nima Rezaei, Research Center for Immunodeficiencies, Children’s Medical Center, Tehran University of Medical Sciences, Tehran, Iran Ortrud Steinlein, Institute of Human Genetics, LMU University Hospital, Munich, Germany Junjie Xiao, Cardiac Regeneration and Ageing Lab, Institute of Cardiovascular Science, School of Life Science, Shanghai University, Shanghai, China

Advances in Experimental Medicine and Biology provides a platform for scientific contributions in the main disciplines of the biomedicine and the life sciences. This series publishes thematic volumes on contemporary research in the areas of microbiology, immunology, neurosciences, biochemistry, biomedical engineering, genetics, physiology, and cancer research. Covering emerging topics and techniques in basic and clinical science, it brings together clinicians and researchers from various fields. Advances in Experimental Medicine and Biology has been publishing exceptional works in the field for over 40 years, and is indexed in SCOPUS, Medline (PubMed), Journal Citation Reports/Science Edition, Science Citation Index Expanded (SciSearch, Web of Science), EMBASE, BIOSIS, Reaxys, EMBiology, the Chemical Abstracts Service (CAS), and Pathway Studio. 2019 Impact Factor: 2.450 5 Year Impact Factor: 2.324 More information about this series at http://www.springer.com/series/5584

Bong-Hyun Jun Editor

Nanotechnology for Bioapplications

Editor Bong-Hyun Jun Department of Bioscience and Biotechnology Konkuk University Seoul, Korea (Republic of)

ISSN 0065-2598     ISSN 2214-8019 (electronic) Advances in Experimental Medicine and Biology ISBN 978-981-33-6157-7    ISBN 978-981-33-6158-4 (eBook) https://doi.org/10.1007/978-981-33-6158-4 © Springer Nature Singapore Pte Ltd. 2021 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 Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Contents

1 Introduction of Nanobiotechnology����������������������������������������������    1 Kwee-Yum Lee, Xuan-Hung Pham, Won-Yeop Rho, Hyejin Chang, Sang Hun Lee, Jaehi Kim, Eunil Hahm, Jong Hun Lee, Yoon-Sik Lee, and Bong-Hyun Jun 2 General in Colloidal Nanoparticles����������������������������������������������   23 Jaehi Kim, Xuan-Hung Pham, Hyejin Chang, Byung Sung Son, Eunil Hahm, Sang Hun Lee, Won-Yeop Rho, and Bong-Hyun Jun 3 Silica Nanoparticles ����������������������������������������������������������������������   41 Hyejin Chang, Jaehi Kim, Won-Yeop Rho, Xuan-­Hung Pham, Jong Hun Lee, Sang Hun Lee, Dae Hong Jeong, and Bong-­Hyun Jun 4 Luminescent Nanomaterials (I)����������������������������������������������������   67 Hyejin Chang, Michael M. Murata, Won-Yeop Rho, Jaehi Kim, Jong Hun Lee, Sang Hun Lee, Dae Hong Jeong, and Bong-Hyun Jun 5 Luminescent Nanomaterials (II)��������������������������������������������������   97 Hyejin Chang, Jaehi Kim, Sang Hun Lee, Won-­Yeop Rho, Jong Hun Lee, Dae Hong Jeong, and Bong-Hyun Jun 6 Plasmonic Nanoparticles: Basics to Applications (I)������������������  133 Hyejin Chang, Won-Yeop Rho, Byung Sung Son, Jaehi Kim, Sang Hun Lee, Dae Hong Jeong, and Bong-Hyun Jun 7 Plasmonic Nanoparticles: Advanced Researches (II) ����������������  161 Hyejin Chang, Sang Hun Lee, Jaehi Kim, Won-­Yeop Rho, Xuan-Hung Pham, Dae Hong Jeong, and Bong-Hyun Jun 8 Magnetic Nanoparticles����������������������������������������������������������������  191 San Kyeong, Jaehi Kim, Hyejin Chang, Sang Hun Lee, Byung Sung Son, Jong Hun Lee, Won-Yeop Rho, Xuan-Hung Pham, and Bong-Hyun Jun 9 Lithography Technology for Micro- and Nanofabrication��������  217 Dahee Baek, Sang Hun Lee, Bong-Hyun Jun, and Seung Hwan Lee

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10 Bioapplications of Nanomaterials������������������������������������������������  235 Kim-Hung Huynh, Kwee-Yum Lee, Hyejin Chang, Sang Hun Lee, Jaehi Kim, Xuan-Hung Pham, Yoon-Sik Lee, Won-Yeop Rho, and Bong-Hyun Jun 11 Carbon Nanomaterials for Biomedical Application�������������������  257 Sang Hun Lee, Won-Yeop Rho, Hyejin Chang, Jong Hun Lee, Jaehi Kim, Seung Hwan Lee, and Bong-Hyun Jun 12 Optical and Electron Microscopy for Analysis of Nanomaterials������������������������������������������������������  277 Hyoyeon Kim, Michael M. Murata, Hyejin Chang, Sang Hun Lee, Jaehi Kim, Jong Hun Lee, Won-­Yeop Rho, and Bong-­Hyun Jun 13 Conclusion and Perspective����������������������������������������������������������  289 Jaehi Kim, Xuan-Hung Pham, Hyejin Chang, Sang Hun Lee, Won-Yeop Rho, Byung Sung Son, Yoon-Sik Lee, Dae Hong Jeong, and Bong-Hyun Jun

Contents

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Introduction of Nanobiotechnology Kwee-Yum Lee, Xuan-Hung Pham, Won-Yeop Rho, Hyejin Chang, Sang Hun Lee, Jaehi Kim, Eunil Hahm, Jong Hun Lee, Yoon-Sik Lee, and Bong-Hyun Jun

Abstract

Keywords

Nano is a fine metric unit which means “one billionth.” Nanotechnology is attracting attention as a technological basis to lead the fourth industry. By utilizing synergistic properties obtained from controlling the structure or arrangement of materials at the nanoscale, nanotechnology has evolved rapidly over the past half century and is active in a variety of fields such as materials, pharmaceuticals, and biology. This chapter briefly describes the concept and features of nanotechnology, as well as the preparation, analysis, characterization, and application of nanomaterials. Also, the prospects for nanotechnology along with the nanotoxicity are described.

Nanotechnology · Nanomaterials · Size-effect · Nanotoxicology

Kwee-Yum Lee and Xuan-Hung Pham contributed equally to this work.

1.1

Introduction

This book serves to provide an overview of nanotechnology that has been heavily applied to bioscience and medicine. Types of nanomaterials, their characteristics, and characterization methods will be discussed and how these properties are and will be leveraged to find a perfect niche in bioapplications will be presented. The readers of this book are primarily aimed at undergraduate and postgraduate students, as well as researchers and technicians who are studying nanotechnology for bioapplications.

K.-Y. Lee Royal Brisbane and Women’s Hospital, Herston, QLD, Australia X.-H. Pham · J. Kim · E. Hahm · B.-H. Jun (*) Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea e-mail: [email protected] W.-Y. Rho School of International Engineering and Science, Jeonbuk National University, Jeonju, Republic of Korea H. Chang Division of Science Education, Kangwon National University, Chuncheon, Republic of Korea

S. H. Lee Department of Chemical and Biological Engineering, Hanbat National University, Daejeon, Republic of Korea J. H. Lee Department of Food Science and Biotechnology, Gachon University, Seongnam, Republic of Korea Y.-S. Lee School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea

© Springer Nature Singapore Pte Ltd. 2021 B.-H. Jun (ed.), Nanotechnology for Bioapplications, Advances in Experimental Medicine and Biology 1309, https://doi.org/10.1007/978-981-33-6158-4_1

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We also hope that this book will be a guidance to those in the medical field, nanotechnology industries, and relevant fields of chemistry, who are interested in nanobiotechnology. As an introduction to nanobiotechnology, this book focuses on basic principles and broad snapshots of the field without dealing with intricacies of the theoretical explanations or equations. Overlap of themes in bioapplications of nanomaterials across chapters is inevitable, and some examples of nanomaterials and their applications covered in one section are discussed in other section to highlight different aspects and to achieve intended delivery of contents.

1.2

What Is Nanotechnology?

Formal definitions of nanotechnology, including that by the National Nanotechnology Initiative (NNI, a multidisciplinary strategy in US), typically involve the ability to handle materials, device, and systems at the level at which at least one dimension is on the nanometer length scale, that is, a one-billionth of a meter. This ability might be described as capacity to assemble molecules into objects or disassemble objects into molecules, spanning several lengths scales. In fact, sources of this ability can be found from nature, which has slowly evolving biological systems, rearranging matter over a long period of time at the nanoscale by forces such as van der Waals interaction, hydrogen bonds, and various surface forces. These processes can be called “wet” nanotechnology, as opposed to “dry” nanotechnology, which refers to the man-made nanoscale materials. The dry nanotechnology in fact has its origins as old as chemistry itself, from fantastic colors of Chinese silk and medieval stain glass to the Roman’s famous Lycurgus cup that shows red-green dichroism (the property of emitting different colors by reflected or transmitted light) from the surface plasmon phenomena of the gold nanoparticles (AuNPs). The analysis of the Lycurgus cup showed

that it contains silver and gold alloy nanoparticles with a ratio of 7:3 and 10% Cu (Fig.  1.1) (Freestone et al. 2007). Yet, it had not been until the scientific community has acquired enough technical dexterity in handling nanoparticles (NPs) and finer resolution of measuring their mechanisms that the field has achieved the status as one of the research frontiers that underpins many of the current progresses. The field is currently extending the addressable unmet needs of science and technology of the twenty-first century. About six decades ago, the Nobel Laureate Richard Feynman foresaw the enormous potential of nanotechnology and gave a seminal lecture that was aptly titled There’s plenty of room at the bottom, providing an outlook of the future technology wherein working with individual atoms for increasingly smaller machines can be realized. In his lecture, he imagined the whole Encyclopedia Britannica written on the head of a pin! It is worthwhile to watch his lecture, which is available online, providing excellent insights and outlook of nanotechnology to this date. Nanotechnology began, as a separate field, to enter into mainstream science and technology some 30 years ago. The term “nanotechnology” was not used until 1974 and first coined by Noria Taniguchi from University of Tokyo to emphasize the traction behind miniaturization to produce smaller, faster, and complex electronic devices on silicon chips in the electronic industry. This conceptual underpinning of nanoscience and nanotechnology was reemphasized in 1996 by Richard E Smalley, following the Nobel-­ prize-­winning discovery of fullerene (a nanoscale carbon structure composed of 60 carbon atoms arranged in a soccer ball-like architecture also called “buckyball”), when he stated: “There is a growing sense in the scientific and technical community that we are about to enter a golden new era when referring to the potential applications of nanotechnology.” Nanotechnology manifests itself a broad range of materials, and the nanoconstructs are capable

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Fig. 1.1  The Lycurgus cup. The glass appears green when illuminated from the outside (a) and purple-red when illuminated from the inside (b)

Fig. 1.2  Number of publications in nanotechnology and nanoscience in 1970–2018. The analysis was obtained from ISI Web of Science using a search term “nano*”

of combining multiple functionalities into a single nanosystem, affording unprecedented freedom to design and modify nanomaterials. Since year 2000, more than 35 countries have developed own national programs in nanotechnology, and numerous journals have been established including Nature Nano, Nano

letters, ACS Nano, etc. As can be seen in Fig. 1.2, the number of publications on nanomaterials has grown immensely over the past 50 years. The multidisciplinary nature of nanotechnology is exemplified in the analysis of the relevant publication domains in 1.8 million published

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Fig. 1.3  Percentage of nano-related subject domains of science and technology. The analysis was obtained from ISI Web of Science using a search term “nano*”

papers that had NANO* (* signifies a wildcard search terminology) as their topic (Fig. 1.3). Terminology of the subject domains relevant to nanotechnology included CHEM*, PHYSICS, BIO*, ELECTR*, OPTICS, ENERGY, PHARMA*, ENVIRONMENT, and so on, which shows pervasiveness of nanotechnology in varying ranges of technological sectors. A complete list of the potential applications of this field are too vast to describe in detail in this chapter, but without a doubt, one of the greatest beneficiaries from the technology will be in the field of biology including medicine, where immense interests have been devoted over the past decades. This area will be discussed in Chap. 10.

1.2.1 How Small Is Nano? The prefix “nano” in the International System of Units (SI) is derived from a Greek word for “dwarf,” which is 1000−3, the third order of 10−3.

With the rapid progress of nanotechnology, research has been active that spans other smaller molar scales, which include pico- (Spanish meaning “bit” for the factor of 1000−4), femto(Danish “femten” meaning “fifteen” for the factor of 10−15 [1000−5]), atto- (Danish “atten,” meaning “eighteen” for the factor of 10−18 [1000−6]), and even zepta- (Greek [h]epta meaning seven, for the factor of 1000−7) molar scales. NPs are colloidal particles with a size of 10–1000 nm. An atom is smaller than 1 nm (most of them ranging 0.1–0.3 nm), and a width of 10 water molecules is about 1 nm. Nanosized objects are 100–10,000 times smaller than the size of human cells (10,000–20,000 nm in diameter) and similar in size to biological macromolecules such as enzymes and receptors, for example, lipid bilayer of the cell membrane is about 6 nm thick. NPs smaller than 20  nm can pass through the walls of blood vessels, and even the blood–brain barriers (BBB) can be penetrated by NPs. It is hard to gather the extreme “tininess” of an NP,

1  Introduction of Nanobiotechnology

but if we can stretch our imagination, 10  nm is 1000 times smaller than the diameter of a human hair (10–50 μm). To put it differently, if one can imagine 1 m as a width of the earth, 1 nm will be a size of a tennis ball, or a red blood cell (¬5 μm) to an NP is a stadium to a football.

1.2.2 Nanofabrication and Analytical Tools Research into methods for manufacturing and characterizing nanomaterials has a long history in the evolution and is not alien to the premillennial science communities. In 1857, Michael Faraday prepared the colloidal gold, which he called “activated gold,” and described them to be fine “colloids” of gold with “very minute in their dimensions.” The colloidal gold he made was known to be optically active for 100 years before they were discarded during the Second World War. As mentioned earlier, the intense momentum of development in nanotechnology established in recent years are in effect primarily due to the advent of novel ways to refine the control in fabrication and to enhance the resolution of measurements in analysis. In order to explore properties and potential applications of novel NPs, a prerequisite is to have established methods of fabricating NPs and nanostructures. A diverse range of fabrication methods has been proposed, but in general, nanofabrication can be divided by two approaches: top-down (i.e., size reduction from bulk materials) and bottom-up (i.e., material synthesis from atomic level). These fabrication methods will be discussed again in the next chapter. Top-down approach involves physical methods, which can be regarded as an extension of lithography for manufacturing semiconductors. In nanoimprint lithography, for example, a nanoscale mold is pressed on to a resist-coated wafer (resist: a mask that temporarily protects selected areas of the underlying substrate; wafer: a thin slice of semiconductor) with an appropriate force, and following the removal of the mold, further processing is performed to remove the resist (Fig. 1.4) (Yang et al. 2014). This approach allows mass production, but the synthesis of

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uniform-sized nanocrystals and their size-control is onerous. In particular, the route often creates imperfection of the surface structure, which can significantly impact physiochemical properties of the prepared particles. Conversely, bottom-up approach broadly refers to forming a nanostructure by joining small units of particles such as formation of crystals by surface adhesion of atoms or ions, or fabrication of polymers from single molecules. The process is similar to building a toy car from Lego bricks. Elegant examples of this approach can be found in nature where self-assembly of molecules on various substrates can be formed in a perfect manner based on supramolecular chemistry (i.e., noncovalent bonding), which would be extremely cumbersome with the top-down approach. “Biomimetics” is using principles borrowed from nature to create new materials or devices, whereof the controlled self-assembly or self-organization of nanobiomaterials or devices is one type of examples (Fig. 1.5) (Nandiyanto et al. 2009). The bottom-up method inspired from biological models is the most promising assembly method at the present moment and can expand to develop biohybrid products. The creation of organic–inorganic hybrid nanomaterials can function as building blocks, or “molecular Legos,” to manufacture nanodevices. The bottom-up route of nanofabrication uses chemical methods that are capable of synthesizing uniform nanocrystals with a controlled particle size and various-shaped nanocrystals. Most colloidal NPs which will be discussed in the following chapters, such as silica NPs, quantum dots (QDs), and metal NPs, are produced using the bottom-up method. In terms of analytical tools, the invention of electron microscopy by Max Knoll and Ernst Ruska in 1931 has been a major breakthrough, overcoming the limited resolution by visible light, and therefrom its resolution has been continually enhanced over the ensuing years. At the present moment, it is possible to observe a particle whose size is smaller than 0.1  nm. In 1981, Gerd Binnig and Heinrich Rohrer invented (both earned the Nobel Prize in Physics in 1986) scanning tunneling microscopy (STM), which can image surfaces at the atomic level. With

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Fig. 1.4  Scheme of nanoimprint lithography. (Yang et al. 2014)

Fig. 1.5 Examples of nanofabrication using self-­ electron microscope image of a particle. (Reproduced assembly. SEM (a) and TEM (b) images of mesoporous from Nandiyanto et al. © 2009 Elsevier Inc. (Nandiyanto silica nanoparticles. The insert image is a high-resolution et al. 2009))

0.1  nm lateral resolution and 0.01  nm (10  pm) depth resolution, individual atoms can be imaged and manipulated. Analyses of the chemical properties and intermolecular mechanics of a single molecule within a single cell in vivo has been made possible with atomic force microscopy (AFM). Similar to sensing the dents on the vinyl record by a stylus of a phonograph, AFM monitors the interatomic

interactions that occur between a nanoscale probe at a cantilever and the substrate. In contrast to most high-resolution microscopes which require the sample to be subjected to high vacuum, AFM is performed under atmospheric pressure, allowing the sample to be relatively unaltered. With a spatial resolution of a few angstrom, AFM imaging has been used to measure intermolecular binding strength between biomolecules

1  Introduction of Nanobiotechnology

and structures under physiological conditions, contributing to the discovery of small RNA (noncoding RNA molecules of 18–30 nucleotides that function to regulate gene expression).

1.2.3 Exotic Properties of the Nanomaterials On the surface, biological and mechanical components can be engineered by miniaturized particles which provide cost-effective solutions. Less obviously known fact is that the nanomaterials possess fascinating behavior of self-ordering and self-assembly and demonstrate fundamentally new physiochemical properties quite different from macromaterials. As bulk materials are broken into infinitesimally smaller particles, they tend to have radically different physiochemical properties compared to the original matter. For instance, a copper wire is well known for its high conductance, but a copper atom is not. Such changes of fundamental properties with the particle size are known as “size effect,” which can broadly include structural, thermal, electromagnetic, optical, and mechanical properties. In fact, engineering novel materials, particles, or systems by leveraging these unique properties at a nanoscale represents a fundamental strategy in nanotechnology. Structural properties of NPs are different from their bulk materials as the particle surface is exponentially exposed with smaller size. In a spherical particle with 1  μm size, the ratio of surface-to-volume is only 1%, whereas for a particle of 10  nm size, the ratio becomes 25%. The surface-to-volume ratio reaches 100% when the particle size is around 1 nm (i.e., the size of three atomic shells or less). To put it differently, at 4–5 nm size, about half of the molecules are on the surface, and for a single-walled carbon nanotube (CNTs) or graphene, every atom is on the surface. This vastly increased ratio of surface area to volume can maximize the interactions between the surfaces of contacting materials. Greater number of particles per volume can also influence the particle behavior. If a cube with a width of 1 mm (10−3 m) is split into a cube of

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1 nm (10−9 m) width, the total number of particles amplifies to 1018. The advantage of having an increased number of particles per unit volume is that more information or functions can be packed in the same cubic space. Another practical benefit is finer resolution of NP fabrication as can be seen in reduced blemishes in magnetic NPs, which can create extremely strong magnetic fields. Other types of size-effect of NPs include quantum confinement, proximity effects, and interparticle tunneling, which explain the optical properties of QDs and AuNPs, which will be briefly discussed in later sections. The reason behind this exotic size-effect shown at a nanometer length scale is because the size of NPs lies at the point of which a transition from atomic to bulk-like behavior occurs. For instance, a nanoscale crystalline structure has a lower melting/freezing point and higher lattice parameter compared to those of the bulk counterpart. By having relatively greater number of atoms on its surface, the fraction of surface energy contributing to the overall thermal stability increases. For instance, AuNPs have a substantially low melting temperature (300– 400  °C), compared to that of the bulk gold (1064 °C). Likewise, particles that show stability only at high temperature can be stabilized at lower level when they become nanometer-sized, such as zirconia particles in the nanosize range as shown by Chandra et al. (2010). Besides that, a bulk material that show ferroelectricity or ferromagnetism at room temperature can lose those characteristics at the same temperature when it is split into nanoscale size. As the ferroelectricity or ferromagnetism occurs from dipole–dipole and spin–spin interactions of the material, diminishing the size of the particle relatively increases the surface energy, which in turn tends to weaken those interaction forces. Lastly, a bulk material with semiconductor characteristics can be turned into an insulator when it becomes nanoscaled particles. The “quantum size effect” occurs when de Broglie wavelength of the valence electrons is of the same order as the size of the particle, whereby the particles behave as zero-dimensional QDs according to quantum mechanics. Free electrons

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in such particles show a characteristic oscillation frequency of the plasma resonance, so-called plasmon resonance band, which is observed around 520 nm in the 5–20 nm diameter range in AuNPs for example. Furthermore, single-electron transitions from a size-dependent quantization effects at a given temperature can occur in NPs as they are small enough, and, unlike bulk metals, there is a gap between the valence band and the conduction band. Exploiting this feature affords large variations of electrical and optical properties of NPs, which is of great practical importance in applications such as transistors, biosensors, or catalysis. The most valuable features of QDs is their fluorescence spectrum, allowing QDs to be used as fluorophores for bioimaging. The quantum size effects confer QDs to have sharp and symmetrical emission and broad absorption spectra, high quantum yield, tunability of emission wavelength through their size, and chemical and photostability (see “QDs” section for more details).

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Besides, one can broadly categorize nanotechnology by its applications, namely, information, biological, chemical, energy, and environmental technology. An example of nanotechnology applied to information technology is best depicted in the development of integrated circuits (ICs), also known as microelectronic circuits. Gordon E. Moore, co-founder of Intel, stated in 1965 that the number of transistors inside a computer chip is estimated to double every 18 months, which is coined as “Moore’s Law.” The increased number of transistors on a chip with decrease in their sizes allow rapid enhancement of speed and performance of a computer. In the year 2000, a critical dimension was set at 120 nm for a 1 Gbit dynamic random access memory (DRAM). The critical dimension has decreased to 50 nm for a 16 Gbit DRAM around 2005 and to 10 nm for the third generation of an 8 Gbit Double Data Rate 4 (DDR4) DRAM in 2019. Moore’s Law, at some point, might not be possible to predict the rapid downsizing of microelectronics, since below certain critical dimension, finite size-effects have to be taken into account. An example of this is 1.3 Classification superparamagnetism, whereby small ferromagnetic or ferrimagnetic NPs can have of Nanomaterials and Their their magnetization to randomly alter the Application direction depending on the level of temperature. Diverse morphologies of NPs are available, and Overall, the microelectronics has been coevolved examples include nanosheets, nanofibers, with advancement of nanodevices and nanorods, nanowires, nanofilms, and so on nanotechnology. (Fig. 1.6) (Jeevanandam et al. 2018). Given this Rapid expansion of nanoscale applications in diversity, there are many ways to categorize NPs biotechnology can be attested in microelectronic using different schemes (e.g., Gleiter scheme and mechanical system (MEMS), lab-on-a-chip, and dimension-based Scheme [0D, 1D, 2D, and 3D]). microarray in which biological signals and Another way to classify nanomaterials is by information are obtained in real-time with high using the phase (gas, liquid, or solid) of the sensitivity. Particle-based QDs, AuNPs, and growth medium in which they were formed. A magnetic NPs have also been applied to material-based classification system can also be molecular monitoring. Recently, a DNA-bridged used, which divides NPs into four categories: (1) assemblies of gold nanorods have been shown carbon-based nanomaterials; (2) inorganic-based to allow quantitative zeptomolar in vivo imaging nanomaterials (metal and metal oxide NPs); (3) of intracellular nucleotide (Fig.  1.7) (Qu et  al. organic-based nanomaterials (excluding carbon-­ 2019). based NPs); and (4) composite-based Another closely related area that exploits the nanomaterials (NPs are combined with other “size-effect” of NPs is energy technology. Active types of NPs or with larger materials). research has been done on nanostructure-based

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Fig. 1.6 Nanomaterials with different morphologies: (a) nonporous Pd NPs (0D), (b) graphene nanosheets (2D), (c) Ag nanorods (1D), (d) polyethylene oxide nanofibers (1D), (e) urchin-like ZnO nanowires (3D), (f) WO3 nanowire network (3D). (Reproduced from Jeevanandam et al. © 2018 Beilstein-Institute. (Jeevanandam et al. 2018))

fuel cells or batteries to increase the efficiency, and some TiO2 NPs have been applied to solar energy panels. Mesoporous NPs can be used as molecular sieves by their morphologic characters and combined with other nanoconstructs to develop lighter but tougher materials, which are commercially showcased in automotive applications (e.g., timing belts and tires) and sport equipment (e.g., tennis rackets, hockey sticks, tennis balls, and soccer balls). In tennis rackets or hockey sticks, CNTs at lower concentration can reinforce the epoxy matrix of the carbon fiber materials. Developing more efficient biodegradable polymers and

manufacturing biocompatible green materials are other areas of environmental technology where NPs can partake an important role. Food and beverage packaging can be fabricated from biorelevant nanocomposites that are biodegradable in few years as opposed to plastics, which takes around 500–1000  years to decompose. In essence, the scope of application of NPs is immense and encompasses, but not limited to, information, biological, chemical, energy, and environmental technology. As a result, influence of nanotechnology is becoming progressively more pervasive in everyday lives from medications, food packaging, cosmetics to

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Fig. 1.7  Quantitative zeptomolar imaging of miRNA cancer markers with gold nanorod assemblies. (Reproduced from Qu et al.© 2019. (Qu et al. 2019))

condoms that can prevent sexually transmitted diseases. In the following section, we discuss five widely utilized types of NPs—metal (highlighting gold NPs) and magnetic NPs, QDs, carbon, and silica NPs—to illustrate examples of how properties of NPs are applied to various sectors of bioscience.

aspect of metal NPs is tailoring their shape or coordination chemistry to afford desired catalytic properties, as well as optical properties, such as surface plasmon resonance. In terms of applications in catalysis, the need for in-depth characterization of the coordination chemistry has been recognized that is facilitated and controlled by small ligands, including halides, carbon monoxide, amine, and thiolate. 1.3.1 Metal NPs Yet, deeper understanding of the surface coordination is far from complete as Metal NPs are submicron scale (10–100  nm) characterization tools of surface and interfacial materials composed of pure metals or their structure of metal NPs have been lacking. compounds, such as gold, silver, platinum, Different coordination environments are usutitanium, zinc, iron, cerium, and thallium. Most ally identified in different facets of the surface or synthetic methods adopt a solution-based interface of solid metal atoms, conferring various colloidal synthesis, which is a bottom-up activation capacities to reactants. Various metal approach through thermal decomposition or coordination sites can be designated in metal chemical reduction of a metal precursor. Some NPs, including corner, facet, or edge, which are novel top-down methods are also developed related to discrete coordination chemistry via steincluding mechanical compression, exfoliation ric and electronic properties (Fig. 1.8) (Liu et al. technique, and nanolithography. 2017). This surface-­dependent feature of catalyBy virtue of unique properties of metal NPs, sis thus bears diverse “nano effects,” such as parsuch as quantum effects and unusual ticle size, shape, physiochemical properties from high surface-to-­ alloy, and support, which are pronounced in volume aspect ratio, burgeoning areas of metal NP-based catalysts. Cost-effective, enviapplications are being witnessed in optics (e.g., ronmental-friendly catalysts with high yields and SERS, biosensing, and bioimaging), electronics selectivity have been called for in industrial (e.g., solar cells), and catalysis. One pivotal applications, which favors the usage of

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Fig. 1.8  Involvements of coordination chemistry on the surface and interface of metal nanomaterials in different aspects: coordination number (C.N.) of surface metal atoms; coordinating ligand array interfering the reactants’ interaction with metal; electronic effect induced by

surface coordinating ligands; the local coordination environments of metal-support interface. (Reproduced from Liu et al. © 2017 American Chemical Society. (Liu et al. 2017))

heterogeneous metal NP-based catalysts, since additive ligands can tune selectivity through modifying catalytic surface (Jun et  al. 2015; Pham et al. 2018). Gold is one of the most ancient themes of scientific investigation. The extraction of gold is estimated to begin around fifth millennium B.C., and colloidal gold was used to make ruby glass and for coloring ceramics. As mentioned at the beginning of this chapter, the Lycurgus Cup, manufactured in fifth to fourth century B.C., contains gold colloids, which show transmitted light as ruby and reflected light as green in the glass. The term “colloid” is derived from the French, colle (to paste), and was coined by Thomas Grahm in 1861. Michael Faraday reported the deep-red solutions of colloidal gold in which he found the optical properties of thin films that demonstrated reversible color changes with mechanical compression. This phenomenon is due to surface plasmon resonance (SPR), which is characterized as a broad absorption band around the visible region of 520  nm (red wine color) in AuNPs. This is due to the collective oscillation of the electrons of the conduction band at the surface of particles that are coherent to the electromagnetic field of the inci-

dent light. Such oscillation provides strong extinction of scattering and absorption light. The SPR band is dependent on the size, shape, surface characters, and agglomeration status of AuNPs and shows decrease in intensity as particle size diminishes with broadening of the plasmon bandwidth. In fact, the SPR band is absent in AuNPs with a core diameter less than 2 nm. AuNPs are the most stable metal NPs, exhibiting other fascinating features such as assembly of multiple types, size-related electronic, optical, magnetic properties (i.e., quantum size effect), and have been applied as catalysts. All AuNPs need a certain type of stabilizing ligands or polymer (i.e., capping agents) to prevent aggregation. Examples of AuNP assemblies include bimetallic NPs, polymers (e.g., polyvinyl pyrrolidone [PVP] polyethylene glycol [PEG]), and dendrimers), films, silica, and various other materials and shapes (e.g., nanosphere, nanorod, nanopyramid, nanostar, nanocube, and nanocage), and diverse methods for preparation of AuNPs were reported in the twentieth century including the citrate reduction, Brust-Schiffrin method using thiols, stabilizing AuNPs with other sulfur ligands, and so on.

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AuNPs, such as gold nanoshells, which have a gold-based outer shell surrounding a metal or inorganic NPs, can be selectively activated by adjusting the thickness of the shell. Tissue irradiation with near-infrared (NIR) light stimulates the nanoshells to release energy in the form of heat (10–1000 of degrees in Kelvin) and can  cause localized cauterization of the tumor vasculature, enabling their potential use in highly selective and externally or internally triggered therapeutic agents for cancer. Gold nanoshells can be decorated with cancer-­ specific ligands such as monoclonal antibodies and aptamers (i.e., oligonucleotides with high affinity to protein or other molecules) for targeted optical imaging of overexpressed biomarkers in the cancer cells including epidermal growth factor (EGF) receptor, matrix metalloproteases (MMPs—a family of metal-dependent proteins), oncoproteins from human papilloma virus infection. Optically responsive nanoshells in the NIR also confer the potential to image virtually all tissues in the body without needing fiber optics, as NIR can penetrate as deep as 10  cm into the tissue. DNA-driven AuNPs constructs have been designed by Mirkin et al. to monitor DNA modification based on colorimetric technique exploiting the distance-related SPR of AuNPs (Mirkin et  al. 1996). Fluorescence resonance energy transfer (FRET) is exploited in this method, wherein the excitation energy of one dye molecule (the donor) is transferred to the other dye molecule (the acceptor) via a dipole– dipole interaction. The color change is detected with AuNP aggregation corresponding to DNA hybridization and cleavage. DNA transfection (i.e., delivering DNA into the cell) or RNA interference (i.e., cleaving mRNA by delivering small interfering RNA [siRNA] in order to halt processing protein synthesis) can be favorably performed by AuNPs due to their strong affinity to biorelevant molecules. For instance, AuNPs conjugated with thiol-modified oligonucleotides (antisense agent) developed by Rosi et al. are shown to be highly efficacious in stopping a certain translational process without

causing any cytotoxicity to cells (Rosi et  al. 2006). Surface-enhanced Raman scattering (SERS), which is a combined technology of laser spectroscopy and optics of metal NPs, provides an extremely strong Raman signal from Raman active molecules on metal (gold, silver, or copper) nanostructure surfaces. Gold or silver nanostructures have been popular SERS substrate, as the particles are noted to show great enhancement of local electromagnetic field. SERS-based detection of circulating tumor cells (CTCs) in peripheral blood of patients has been demonstrated by Wang et  al. (2011). Yet, their translation to clinical use has not been widely realized with SERS imaging since it requires complex equipment and has several challenges in regulation clearance. Although inert gold metals are one of the most stable metals that resist oxidation, AuNPs supported on metal oxides have high catalytic activity for carbon monoxide (CO) and H2 oxidation as shown by Galvagno and Parravano. Noble metals are very costly but as the catalytic activity occurs on the surface of particles, possessing relatively higher surface area can save substantial cost while enjoying enhanced efficiency of catalytic activity.

1.3.2 Magnetic NPs Magnetic NPs can maneuver a mechanical force inside the cells, which is highly advantageous for monitoring and controlling cell signaling. Most widely used magnetic NPs are the nanoscale zerovalent iron, Fe3O4 (magnetite), and gamma-­ Fe2O3 (maghemite). Different magnetic states are shown during the crystallization of iron (Fig. 1.9) (Mohammed et  al. 2017). For example, paramagnetic crystal shows random direction of magnetic moments that has zero net magnetization, but when it is under external field, a small net crystal magnetization can be formed. Paramagnetic NPs have been used for detection of biomarkers for cancer, viruses, and bacteria. On the other hand, ferri- and antiferromagnetic

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Fig. 1.9  Alignment of the magnetic moment of individual atoms of iron. (Reproduced from Mohammed et al. © 2017 Elsevier. (Mohammed et al. 2017))

crystal has several magnetic domains with uniformly aligned areas separated by nonuniform distribution of magnetization arrays, causing low net magnetization. The magnetic moment and field gradient are two key properties that underlie efficacious bioapplication of magnetic NPs. For instance, single-core superparamagnetic NPs are less favorable to produce magnetic force owing to their small size and magnetic moments, whereas multicore materials can generate sufficiently strong magnetic field to guide magnetic targeting using moderate degree of field intensity and gradient. Utilization of magnetic NPs has been prominent in main areas of bioapplication, namely, magnetic resonance imaging (MRI), magnetic separation of biomolecules for diagnostics, magnetic vectors for drug delivery, and radio frequency controllable magnetic NPs for cancer treatment such as thermal ablation. Besides that, other areas of their applications range from catalysis, water treatment and purification, and toxin and heavy metal chelation and remediation. The scope is possibly limited only by the biocompatibility and toxicity of the particles, which can be overcome by strategic coating of their surfaces. Pure metals have excellent magnetic properties but without surface modification, they are highly toxic and sensitive to oxidation, and the dipole–dipole and dipole–field interactions might lead to considerable aggregates that could potentially block small blood vessels.

Since magnetic NPs can respond to the external magnetic field gradient, they constitute promising methods for targeted drug delivery that enable spatially, temporally, and dosage-­ wise tunable drug release with minimal side effects. Take doxorubicin as an example, which is an anthracycline anticancer agent. Doxorubicin has been encapsulated into apoferritin for targeted cancer therapy with MRI using integrin as high-­ affinity ligands to tumor. In addition, platinum-­ incorporated magnetic NPs can be used as dual contrast agents for MRI and X-ray CT, and porous magnetic NPs allow storage and release of drugs. Other MRI-guided applications of magnetic NPs include cell replacement therapy and cancer-specific gene transfection. The size of the magnetic NPs is a paramount factor in functionality, which affects magnetism and surface area, and thus various size-controlled synthesis methods have been explored, including organic-phase synthesis that produces sub 20 nm particles and a seed-mediated growth which increases the particle size (bottom-up methods). In order to create colloidally stable particles that resist precipitation and agglomeration, the ratio of inert to reactive surface-coating components can be optimized. Physical (top-down) methods of synthesizing magnetic NPs include laser ablation or evaporation, size reduction using a colloidal route, and condensation of the precursors from a liquid or gaseous phase. Lastly,

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microbial methods generate magnetic NPs through a biomineralization process from magnetotactic bacteria, which show great promise as they allow scale-up production and excellent reproducibility with good size control. Added functionality can be achieved by linking the NPs with antibodies and oligonucleotides for active targeting of a tumor or with other nanomaterials, such as QDs, for cellular imaging. The drug conjugation is usually achieved by covalent and noncovalent (e.g., hydrophobic interaction, electrostatic interaction, or coordination chemistry) modification.

1.3.3 Quantum Dots (QDs) QDs are NPs in nanocrystalline form prepared with core, core-shell, or core-multishell formulation from binary, tertiary, alloyed, or from other semiconductors. They are one of the most versatile luminophores, which demonstrate quantum confinement effects that stem from size-­effects of nanodimension. When the particle size nears the bulk exciton Bohr diameter of the material, the band gap increases and becomes discrete with decreasing size. As the effects allow discrete and tunable electronic energy state by the particle size, it becomes possible to transfer energy as a donor or an acceptor (also tunable by the particles size) and to increase the efficiency of photoluminescence (PL). QDs have been widely researched as biolabels and contrast agents for biosensing and bioimaging, as well as solar power and energy harvesting applications (Fig.  1.10) (Algar et al. 2011). The breadth of QD-based bioassay applications is vast and covers optical transduction, electrochemical luminescence, and photoelectrochemical assays, with an added choice of various multiplexing strategies. Despite these unprecedented advantages, potential cytotoxicity of QDs is a major roadblock that limits their clinical translation. The elements of QDs can be from group II–VI (e.g., CdTe or ZnS), III–V (e.g., InP or InAs), or

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group I-III-VI2 (e.g., CuInS2), which constitute classical inorganic QDs. Other types of QDs constitute group IV–VI (e.g., PbSe or PbS) or group IV (e.g., Si or C) elements. The size of the commonly used QDs in bioapplication is around 2–20 nm (3–60 nm with surface coating), which marks one of the smallest spherical NPs. In a single QD core, 200–10,000 atoms can usually be found. Taken together, high contrast in bioimaging is possible with QDs due to sufficient electron density and high molar extinction coefficients, which measure how strongly particles absorb light at a given wavelength per molar concentration. Among all QD materials, cadmium chalcogenide (e.g., CdS, CdSe, and CdTe) QDs are most popular and intensely researched QDs due to their unmatched wide spectral coverage (visible to NIR) with a narrow (1700 °C) and vacuum conditions, which are costly. Now the methods are mostly replaced by chemical vapor deposition method that can be carried out at a much lower temperature (< 800  °C). The chemical vapor deposition is suitable for scale-up production that involves the pyrolysis of hydrocarbons or polymers and CO using metal catalysts such as Ni, Fe, and Co. Relatively simple instrumentation and greater yield rate have facilitated this method to be utilized for mass production of CNTs, whereby the diameter, length, and alignment of the materials can be effectively controlled by adjusting the level of temperature. The widespread adoption of CNTs in terms of manufacturing and applications is expected in near-term future, but the trend raises a significant concern regarding their potential issues on cytotoxicity and

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Fig. 1.13  The mother of all graphitic forms. Graphene is a 2D building material for the carbon materials of all other dimensionalities. It can be wrapped up into 0D buckyballs,

rolled into 1D nanotubes, or stacked into 3D graphite. (Reproduced from Geim & Novoselov © 2007 Nature Publishing Group. (Geim and Novoselov 2007))

electronic waste that can influence human health and environment. Graphene is a monolayer 2D sheet within which each carbon atoms is sp2-hybridized to three others, arranged like a honeycomb or benzene-like structure. It provides a starting material for fabricating other carbon NPs such that when it is rolled, it becomes CNTs, and 3D graphite is constructed when it is stacked (Fig. 1.13) (Geim and Novoselov 2007). Due to its planer morphology, graphene exhibits remarkable optical properties, large surface area, high Young’s modulus, and excellent thermal and electrical conductivity. Through covalent and noncovalent interactions, various biomolecules can be immobilized on its surface without further

functionalization. Such exceptional properties, as well as its biocompatibility, have rendered graphene to be used in a wide array of bioapplications for bioimaging, drug delivery, and photodynamic and photoacoustic therapies. Fullerenes are hollow spheres, consisting of 60 carbon atoms and 30 conjugated carbon double bonds, containing 20 hexagons and 12 pentagons. Owing to their insoluble nature in the aqueous media, surface modification is usually required for them to be utilized in biorelevant applications. A number of water-soluble and functionalized fullerenes have been developed, exploiting their unique morphology as a 3D delivery vehicle for pharmaceutical payloads. Similar to other carbon NPs, their application

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covers bioimaging, biosensing, drug delivery, as well as photodynamic and photoacoustic therapies. Nanodiamonds are less than 5 nm in size and have a shape of truncated octahedron with a crystalline diamond core surrounded by an amorphous layer of functional groups immobilized (by sp2 and sp3 bonds) on their surface to stabilize the particles. Nanodiamonds feature high surface areas, chemical inertness, physical hardness, biocompatibility, optical transparency, and high thermal conductivity. Promising candidates such as detonation nanodiamonds (DNDs) and fluorescent nanodiamonds (FNDs) have been developed for bioapplications to exploit such extraordinary properties of the material. For example, both nanodiamonds can be used as thermal therapy since they can convert incident light into heat energy. DNDs can be utilized as drug carriers to intracellular targets, whereas FNDs are suitable for bioimaging of living cells as they contain high photostability, far infrared emission, and long fluorescence lifetime.

1.4

Nanotoxicology and Future Perspective

Although tremendous hopes have been placed on nanotechnology across the globe, the issue that would likely to backfire, as voiced by the survey of experts, is the misled promise that “nanotechnology can fix everything” (Moghimi 2005). Since there lie challenges and risks to in vivo applications, a lack of detailed understanding of the toxicology of NPs needs to be acknowledged first, maintaining an intellectually humble stance. Applying NPs into a living system will have more than trillions of interactions with the surrounding system that are difficult to monitor. It is impossible to measure all those interactions in real time or in situ as yet, but application of systems biology can guide future endeavors. Nanotoxicology is the investigation of biorelevant effects of nanomaterials and has been a rapidly developing field as it reflects an urgent need to examine the toxic effects of nanomaterials on the living systems. However, it is often

difficult to confirm the toxicity of specific nanomaterials, as most nanotoxicology studies are done on animals or cultured cells, and the synthetic processes of the materials are very specific to individual research group where detailed information on synthesis is hard to obtain. A fundamental task is therefore to establish standard protocols with standardized assays in preparing and evaluating the NPs and to develop databases that can relate toxicological properties to physiochemical information of the particles. Although, at present, there are no agreed international regulation and protocols for evaluating toxicity of NPs, such endeavors are actively sought in USA and EU with their regulatory bodies and guidelines. One of the fundamental attributes of nanotoxicity is reactive oxygen species (ROS), whereby release of ROS by the nanoconstructs, as well as the size and shape of the particles, can affect cell membrane stability. Once the particles are internalized into the cell, cell viability can be influenced by the ROS disrupting subcellular units from DNA damage or lipid peroxidation. The functional groups, surface charge, and dissolution characteristics of the nanomaterials also can undesirably affect their surroundings. The wellstudied acute effects from exposure to NPs include ROS generation, protein denaturation, mitochondrial dysfunction, and disturbance in phagocytic activities (Fig.  1.14). Common chronic effects of NPs toxicity are known to include possible cancer, asthma, and generation of neoantigens that might result in organ dysfunction. When examining toxic effects of specific NPs, although QDs such as CdSe/ZnS NPs are not foreign to bioimaging studies and preclinical trials, they are notorious for high toxicity from their heavy metals. In particular, heavy metals can be retained in the body for up to 10 years and are known to be carcinogenic with long-term exposure. Alarifi et al. have shown that magnetite NPs have potential to damage healthy cells through oxidative stress or by damaging cytoskeletons (Alarifi et  al. 2014). Lastly, carbon NPs, which is known to be nontoxic in cytotoxic assays, have resulted in lipid peroxidation in the brain tissues of fish and inflammation of lungs in rats (Muller and Keck 2004).

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Fig. 1.14 Electron microscope images show how NPs can penetrate and relocate to various sites inside a phagocytic cell line. (a) Untreated phagocytic cell line (RAW 264.7). Cells were treated with (b) ultrafine particles ( R2) are mixed with the solvent, each parti-

2  General in Colloidal Nanoparticles

31 (b) Time

(a) Time

Nucleation & Growth

Growth Only

Final product Sum Sum

Fig. 2.3  Schematic illustration of monodisperse nanoparticle fabrication. Fabrication with single nucleation (a) and complete separation of nucleation and growth (b). The nucleation section is regarded as a condition where

Final product

both nucleation and growth occur, and the growth section is regarded as a section where only growth occurs without nucleation

cle equilibrates with the surrounding solvent. tions. Ostwald ripening often leads to the undeAccording to equation known as Gibbs-­ sirable results when applied to the processes of Thompson relation, the solubility of small parti- many materials. In sintering of polycrystalline cles will be greater than that of large particles materials, Ostwald ripening leads to abnormal because larger curvature of small particles make grain growth, resulting in uneven microstructure dissolve them into the solvent. As a result, there in size and poor mechanical properties of the will be a net diffusion of the solute from the prox- product. As a rule, one or several large grains imity of the small particles to the proximity of the grow at the expense of the surrounding small large particles. To maintain equilibrium, solutes grains, creating uneven microstructures. But can be deposited on the surface of large particles, Ostwald ripening was explored in the synthesis while small particles must continue to dissolve to of nanoparticles. More specifically, Ostwald ripcompensate for the diffused amount of solute. In ening has been used to narrow the size distribuconsequence, larger particles become larger, tion of nanoparticles by removing small particles. meanwhile, smaller particles become smaller and The situation here is very different. Many relaeven disappear when fully dissolved.  This phe- tively large particles grow at the expense of a nomenon is called as Ostwald Ripening. relatively few number of smaller particles. As a Assuming that there are no other changes result, small particles are removed, which narbetween the two different particles, the change in rows the size distribution of the nanoparticles. the chemical potential of the atoms moving to R2 Ostwald ripening can be promoted by changing from the spherical surface of radius R1 is given the temperatures of processes. In the synthesis of by: nanoparticles from solution, after initial nucleation and subsequent growth, the temperature 1 1   2 R2  R1 rises, thus increasing the solubility of solids in This equation should not be confused with the the solvent to promote Ostwald ripening. As a Kelvin equation. Depending on the processes and result, the concentration of solids in the solvent applications, Ostwald ripening can have a posi- falls below the equilibrium solubility of the small tive or negative effect on the produced materials. nanoparticles, and the small particles dissolve in Ostwald ripening can broaden or narrow the size the solvent. As the nanoparticles dissolve, the distribution under the control of process condi- nanoparticles become smaller and have higher





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solubility. Once the nanoparticles begin to dissolve in the solvent, it is clear that the dissolution process is stopped only when the nanoparticles are completely dissolved. On the other hand, since the concentration of solids in the solvent is still higher than the equilibrium solubility of the larger particles, these larger particles will continue to grow. This growth process will be stopped when the concentration of solids in the solvent is equal to the equilibrium solubility of these relatively larger nanoparticles. The reduction in total surface energy is the driving force for surface restructuring, the formation of faceted crystals, sintering, and ­ Ostwald ripening. These kinds are the reduction mechanisms for individual surfaces, individual nanostructures, and the entire system. In addition to sintering and Ostwald ripening, the system can set aside another mechanism to reduce the overall surface energy. This is an agglomeration (and also called as aggregation). When small nanostructures form agglomerates, it is very difficult to disperse them. In nanostructure fabrication and processing, it is very important to overcome the huge total surface energy to produce the desired

nanostructure. It is equally important to prevent the nanostructures from agglomerating. As the dimensions of nanostructured materials decrease, the van der Waals attraction between nanostructured materials becomes increasingly important. If proper stabilization mechanisms are not applied, nanostructured materials are likely and easy to form agglomerates. • Sintering In general, sintering can be regarded as a process of replacing a solid–vapor surface by a solid–solid interface by reshaping the nanostructures such that the individual nanostructures are packed so that there are no gaps between the solid nanostructures. Ostwald ripening takes a radically different process, where two separate nanostructures become a single structure. The big grows at the expense of the small until the latter completely disappears. While the sintered product is a polycrystalline material, Ostwald ripening results in a single uniform structure. Figure 2.4 shows the results of the two processes schematically, but the results of the two processes

Grain boundary

(a)

Pore

Particles

(b)

Particles

Initial growth of droplet

Fig. 2.4  Schematic illustration showing sintering and Oswald ripening processes. (a) Sintering is to combine individual particles to a bulk with solid interfaces to con-

nect each other, (b) Ostwald ripening is to merge smaller particles into a larger particle. Both processes reduce the solid–gas surface area

2  General in Colloidal Nanoparticles

are similar, that is, the reduction of the total surface energy. Macroscopically, the reduction of total surface energy is the driving force of both sintering and Ostwald ripening. Differential surface energy of surfaces with different surface curvatures under a microscopic view is a real driving force for mass transport during sintering or Ostwald ripening. In addition to combining individual nanostructures to form large structures through sintering or Ostwald ripening, aggregation is another way to reduce the overall surface energy. In aggregates, many nanostructures are associated with each other through chemical bonds and physical attraction at the interface. Once formed, the aggregates are very difficult to destroy. The smaller the individual nanostructures, the stronger the bonds to each other and the more difficult it is to separate. For the practical application of nanomaterials, it is necessary to prevent the formation of aggregates.

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droplets coated by surfactant that is dispersed throughout the continuous, other phase. These emulsions are milky or turbid in appearance due to the fact that the droplet sizes range from 0.1 to 1 micron. As a general rule, the type of surfactant used in the system determines which phase is continuous. If the surfactant is hydrophilic, oil will be emulsified in droplets throughout a continuous water phase. The opposite is true for more lipophilic surfactants. Water will be emulsified in droplets that are dispersed throughout a continuous oil phase in this case. Emulsions are kinetically stable, but are ultimately ­thermodynamically unstable, and will begin to separate back into their two phases. The droplets would merge together, while the dispersed phases will sediment. At this point, they degrade back into bulk phases of pure oil and pure water with some of the surfactant dissolved preferentially in one of the two. If a surfactant possessing balanced hydrophilic and lipophilic properties is used in the proper concentration, a different oil and water 2.2.2.3 Microemulsion Method system will be produced. The system remains as (Template-Based Method) emulsion but exhibits some characteristics that Microemulsion method is one of the recent and are different from the milky emulsions discussed ideal techniques for the preparation of inorganic earlier. These new systems are “microemulnanoparticles. Oil and water are immiscible, and sions”. The interfacial tension between phases, they separate into two phases when mixed, each amount of energy required for formation, droplet saturated with traces of the other component. An sizes, and visual appearance are only a few of the attempt to combine the two phases requires differences seen when comparing emulsions to energy input that would establish water–oil asso- microemulsions. Water-in-oil microemulsions ciation replacing the water–water/oil–oil con- are also known as reverse micelles. These systacts. The interfacial tension between bulk oil and tems can solubilize both hydrophilic and hydrowater can be as high as 30–50 dynes/cm. This can phobic substances. be overcome by using surface-active molecules Microemulsions usually exhibit low viscosiknown as surfactants. Surfactants contain water-­ ties and Newtonian flow characteristics. Their friendly (hydrophilic) and oil-friendly (lipo- flow remains constant when subjected to a variety philic) moieties. Owing to this characteristic, of shear rates. Bicontinuous formulations may they tend to adsorb at the water–oil interface. If show some non-Newtonian flow and plasticity. enough surfactant molecules are present, they Microemulsion viscosity is close to that of water, align and create an interface between the water even at high droplet concentrations. The microand the oil by decreasing the interfacial tension. structure constantly changes, making them very An emulsion is formed when a small amount dynamic systems with reversible droplet coalesof an appropriate surfactant is mechanically agi- cence. A variety of techniques are employed to tated with the oil and water resulting in a two-­ characterize different properties of microemulphase dispersion where one phase exists as sions. Light scattering, X-ray diffraction, ultra-

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centrifugation, electrical conductivity, and viscosity measurements have been widely used. Based on the phase equilibrium of microemulsions, they can be further classified into four different types. Winsor (1948) has developed a classification scheme for emulsions (micro- and macro) (Winsor 1948). Oil-in-water (o/w) microemulsions are droplets of oil surrounded by a surfactant (and possibly cosurfactant) film that forms the internal phase distributed in water, which is the continuous phase, Winsor-I. The Winsor-II type is water-­ in-­oil (w/o) microemulsion in equilibrium with excess water phase at the bottom. The o/w type microemulsion has generally a larger interaction volume than the w/o microemulsion. Water-in-oil microemulsions are made up of droplets of water surrounded by an oil continuous phase. These are generally known as “reverse-micelles.” The middle phase bicontinuous microemulsions in equilibrium with excess oil phase at the top and excess water phase at the bottom have been classified as Winsor-III type. These may show non-­ Newtonian flow and plasticity. These properties make them especially useful for topical delivery of drugs or for intravenous administration, where upon dilution with aqueous biological fluids form an o/w microemulsion. Water-in-oil microemulsions are made up of droplets of water surrounded by an oil continuous phase. These are generally known as “reverse-micelles”. The fourth type, basically a macroemulsion, Winsor-IV, may exist in the form of one of three possible different microstructures: oil-in-water (o/w), water-in-oil (w/o), and bicontinuous. Generally, one would assume that whichever phase was in larger volume would be the continuous phase, but this is not always the case. The monolayer of surfactant forms the interfacial film that is oriented in a “positive” curve, where the polar head-groups face the continuous water phase and the lipophilic tails face into the oil droplets.

2.3

Stabilization of Nanocrystals Against Aggregation

Nanoparticles basically try to aggregate each other to lower their surface energy, and electrostatic stabilization and steric stabilization are the two main methods to prevent the aggregation.

2.3.1 Aggregation Particle movements in the solution phase or in the gas phase include a variety of motions, such as Brownian Motion, motion by shear stress (or force), motion caused by fluid flow, up and down motion due to gravity and buoyancy, particle motion due to speed differences, and so on, and thus these movements keep on the particles moving, thereby reducing the span of time that the particles hold together, preventing the particles from aggregating together. Here, the main factor that makes the particles stick together can be called van der Waals attraction. Of course, if the particles are charged, strong repulsive forces can act between the particles. The smaller the particle size, the greater the effect of van der Waals attraction can be. It is understood that particles of about 10 μm show mechanical dispersion even if the van der Waals attraction between particles is strong, and even particles of about 1 μm can be dispersed by applying mechanical force to the colloidal dispersion. But as reaching the 100 nm level, it can be expected that the particles will not be dispersed and will stick together because the van der Waals attraction is far superior to mechanical dispersion. Therefore, in order to maintain the nanostructures to express nanosized properties, additional treatment is required to prevent the structures from aggregating each other. In order to control the aggregation between particles, the surface modification of nanoparticles can reduce van der Waals attraction or introduce electrostatic repulsion or steric hindrance to prevent the aggregation of particles. These are called electrostatic stabilization and steric stabilization, respectively, and they are the most generally used method for preventing aggregation.

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In addition to the above mentioned, there are other applicable methods, that is, method of electrostatic stabilization which simultaneously creates charge and steric hindrance, methods of preventive collisions between particles by dissolving a polymer-like material in a dispersion to increase viscosity, method of masking van der walls forces by dosing together molecules that can screen the attraction between particles, method of providing a dispersing force by enclosing the particles and molecules with water molecules using a polymer material such as poly(N-isopropylacrylamide-co-acrylonitrile) (poly NIPAAm) hydrogel and so on.

2.3.2 Surface Charge The term “surface charge” literally refers to the charges that are generated on the surface of an object. Colloidal particles are charged on their surface, and the surface charge is a major factor that prevents the particles from aggregating. Surface charge also stabilizes the particles due to electrostatic repulsion between the nanoparticles, and this charge on the surface is called primary charge. The reason why the colloidal particles are charged is that the chemically inert substances dispersed in the water are negatively charged by the selective adsorption of anions (especially, hydroxide ions) existing in the medium (water), or in the case of substances such as proteins or microorganisms the surface charge is obtained by ionization of the carboxyl group and the amino group, that is, the active group at the end of the proteins or microorganisms, and clay particles are negatively charged on the surface by ­isomorphous replacement, which polyvalent ions such as Si (IV) ions and Al (III) ions in the clay are replaced by lower valent ions such as Ca, Mg, and metal ions that have smaller charges than the Si (IV) ions and Al (III) ions. Therefore, clay-­ suspended colloids, algae, bacterial cells, and proteins, which are major suspended substances in natural water, and activated sludge, which is generated during sewage treatment, are mostly negatively charged; hence, the surface charge is neutralized to cause aggregation by a positively

Fig. 2.5 Schematic illustration of surface charge of nanoparticles

charged inorganic coagulant or a cationic organic polymer flocculant.

2.3.3 Electrical Double Layer Particles present in the liquid phase have surface charges characterized by “ionization of an acid or basic groups”, “differential solution of cations or anions”, “isomorphic substitution”, “specific adsorption of cationic or anionic surfactant interfaces”, and etc. (Fig. 2.5). Charged particles in solution attract oppositely charged ions or polar molecules near the surface. Thus, the concentration of polar molecules increases near the surface compared to the average concentration in the solution. The potential difference between the particle surface and the solution has a gentle gradient generated by diffusion due to thermal vibrations. A generally known model consists of “a relatively immobile counterion adsorption layer” and

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“a counterion diffuser layer with a concentration gradient reaching a steady-state array”, which is called a diffusion electrical double layer. In electrokinetic experiments, slippage during a flow should occur at a specific surface within the bilayer, and the potential at this specific surface (sliding surface) is called the zeta potential. Normally, it is called a low-charged surface when it has a small value of ±25  mV, and it is classified as a highly charged surface when it has a higher value than ±25 mV.

2.3.4 Van der Waals Attraction Van der Waals attraction is rooted in the pulling interaction between the oscillating electric dipoles in one molecule and the released dipoles in the neighboring molecule, and if present, the pulling interaction with the permanent dipole. It is a relatively weak electric force that attracts molecules each other that are neutral in gases, liquefied, or sublimed gases, and also liquids and solids of most organic compounds. This interaction is called van der Waals force (or attraction), after Dutch physicist Johannes van der Waals, who first assumed such intermolecular force when he was developing a theory explaining the properties of real gases other than ideal gases over 1873. Solids bound by van der Waals forces are softer and melt at lower temperatures than solids made of stronger ionic, covalent, and metal bonds. Van der Waals interaction can come from three sources. First, molecules of some materials can be permanent electric dipoles, even if they are electrically neutral. Some molecules have a structure in which the distribution of charges is uneven and unbalance-fixed, so that either side of the molecule always has some positive charge and the other side always has a slight negative charge. The tendency of these permanent dipoles to align in the same direction creates net attracting force. Second, the presence of molecules that are permanent dipoles temporarily alter the charge distribution of other polar or nonpolar molecules

in the vicinity, leading to greater polarization. Another attraction arises from the interaction between the permanent dipoles and the surrounding dipoles. Third, even if there are no molecules having permanent dipoles (for example, in the case of argon as an inert gas or benzene as a liquid of an organic compound), there may be attraction between molecules, which explains the condensation into the liquid state at sufficiently low temperatures. Quantum mechanics is required to accurately describe the nature of the attraction between molecules, Polish-born physicist Fritz Wolfgang London first discovered this in 1930s while investigating the motion of electrons in a molecule (London 1937). London pointed out that at some point, the center of the negative charge of the electrons and the center of the positive charge of the atomic nucleus tend to be inconsistent. As a result, the oscillations of the electrons cause the molecules to become dipoles that vary over time, which is averaged to zero over the macroscopic period. Thus, dipoles that vary over time—that is, instantaneous dipoles—continue to change, namely, quick direction change, so they cannot be arranged in a form that describes the actual attraction. However, they induce a properly aligned polarization with adjacent molecules, which in turn causes attraction. This particular interaction, which is caused by the oscillations of electrons in the molecule, is called force (known as Rondon force or dispersion force), even between permanently polar molecules, and the interaction is usually the largest out of the three components that contribute to van der Waals interactions. • Hydrogen Bond When atoms having high electronegativity and unshared pairs of electrons such as fluorine, oxygen, and nitrogen are covalently bonded to hydrogen, it refers to the attraction force between hydrogen having a partial positive charge and an atom having a charge in another

2  General in Colloidal Nanoparticles

molecule. Representative materials are HF (hydrogen fluoride), CH3CO2H (acetic acid), H2O (water), and all other materials have the dipole force. • Polarization When a molecule approaches a polar molecule, the electrons of the molecule are biased toward the positive charge of the polar molecule, and the larger the number of electrons, the greater the degree of polarization. • Induced Dipole

37

between van der Waals attraction and the electric double layer is generated. The maximum potential of the electrostatic repulsive force is related to the zeta potential, and the attraction between the two particles shows the maximum potential when the distance is close. At this time, the total potential energy of the particles (UDLVO) can be expressed by the arithmetic sum of van der Waals attraction potential (UVDW) and repulsive potential (UES) in the following equation:

U DLVO  U ES  U VDW

The total potential energy can be a criterion for predicting whether the colloid may be in a When the polarization occurs, the electron-­ stable depending on the value of the total potenbiased side is temporarily charged, and this tial energy or the aggregation occurs. dipole is called the induced dipole. The approximate outline of change in potential depending on the distance of two particles is • Dispersion shown in the Fig. 2.6, creating an energy barrier between the first and second minimums, and it The electrons of nonpolar molecules are not forms the energy barrier between the first and always in a fixed position. Therefore, when elec- second minimums. For two particles to aggretrons are biased to either side, molecules become gate, they must have enough kinetic energy to instantaneously polarized and have induced cross the energy barrier in the event of a collision. dipoles; thus, the force acting between molecules As the Hamaker constant increases, the surface having these induced dipoles is the dispersing potential decreases, and the electrolyte concenforce. One thing to add is that dispersing forces tration increases, the size of the energy barrier work between all molecules, but they have a very decreases, causing aggregating of the two small impact on the properties of matter between particles. molecules other than nonpolar molecules. The fact that the DLVO theory explains the However, since only the dispersing force acts dispersion and aggregation of particles in aquebetween the nonpolar molecules, the properties ous solution, is discussed above, and shows an of the nonpolar molecules are greatly affected by example of a potential curve based on this theory. the dispersing force. In the case of suspensions with surface potentials and electrolyte concentrations of 64.9  mV and 0.6 mM, respectively, the thickness of the electric double layer is calculated to be 12.4 nm. In gen2.3.5 DLVO (Derjaguin, Landau, Verwey, Overbeek) Theory eral, when the height of the energy barrier is more than 15 times over the product of the absoThe interaction of two particles with identically lute temperature and the Blitzmann constant, the charged electric double layers has been investi- particles are dispersed in the aqueous solution, gated by the above four scientists, and the combi- and when less than 10 times, the aggregation nation of their studies is called the DLVO theory occurs. When the particle size is 100 nm or less, (Derjaguin 1993; Derjaguin and Landau 1993; for example, 20  nm or less, the height of the Verwey 1947). When charged particles approach energy barrier becomes 10 kT or less, causing each other and come closer than a certain dis- aggregation, and when the particle size is 100 nm tance, the repulsive force due to the interaction or more and 300 nm, the height of the energy bar-

J. Kim et al.

38

(a)

+ + + + + + + +

Electrostatic effect

(b)

Steric hinderance Fig. 2.7  Stabilization of nanoparticles via (a) charge repulsion and (b) steric repulsion

2.3.6 Steric Stabilization

Fig. 2.6 Energy diagram between the nanoparticles according to the distance

rier becomes 15 kT or more, causing dispersion. In addition, in order to disperse a particle having a size of 20  nm, the surface potential must be increased by hundreds of mV or more. To achieve this high surface potential, special methods and techniques must be implemented. Another thing to consider regarding the dispersion of nanoparticles is the average distance between the particle surfaces. The maximum repulsive force between the particles appears at several nanometers on the surface, and when the average distance of the particle surface is smaller than several nanometers, aggregation is inevitable.

In addition to stabilization of the colloidal particles by the electric double layer, dispersion stability due to steric hindrance of the adsorbed layer can be obtained too (Fig. 2.7). Steric stabilizers are usually amphiphilic blocks or graft polymers, which consist of anchor groups and stabilizing moieties. Steric stabilization is largely explained by two mechanisms. Unlike typical low molecular compounds, polymers have a large difference in entropy (s) due to spatial changes in polymer chains. When the polymer chains are freely moved, if the polymer chains are forced to be pushed into tight spaces, a large decrease in entropy has to be tolerated. If the two polymer chains are of the same kind and affinity with the solvent, there is little change in enthalpy when the polymer chains are stretched and shrunken together. Therefore, since only entropy is reduced, free energy (G) has a positive value so that the two surfaces cannot stick together. In addition to stabilization of colloidal particles by the electric double layer, there is also dispersion by steric hindrance of adsorption layer.

2  General in Colloidal Nanoparticles

If the polymer as the same is attached to the nanoparticles as a surfactant, as the particles are closer to each other and the entropy of the polymer chain is reduced in consequence, therefore the particles are thermodynamically repulsive against each other to prevent the aggregation of particles. However, when the particles are dispersed in a poor solvent, the polymer chains are in a much contracted state. When two particles approach and come into contact with each other, it is advantageous to create degrees of freedom through contact between the chains rather than contraction through contact with a solvent from the viewpoint of the polymer chains, therefore entropy increases as a result, causing aggregation between particles. If the polymer chains are charged, free energy is greatly increased by the electrostatic repulsion when the two chains are approached and become entangled. In this case, an increase in enthalpy, rather than a change in entropy, acts as a major mechanism to maintain dispersion. The matter which mechanism prevails can be distinguished by how the dispersion stability changes with temperature, and enthalpy stabilization is when the aggregation occurs during heating and then has a phase-critical aggregation temperature. In addition to kinetic stabilization, thermodynamically stabilized dispersions such as microemulsions are also possible. That is, when the pH of the precipitate is different from the point of zero charge (PZC) and the ionic strength is sufficiently strong, ripening of the nanoparticles can be avoided. The stabilization conditions defined here as “zero” interfacial tensions are related to chemical and electrostatic saturation at the water-­oxide interface. If adsorption occurs under the condition that the density of the charged surface group reaches its maximum value and also the interfacial tension reaches its minimum, the nanoparticles can be stabilized thermodynamically. Acknowledgments  This work was supported by the KU Research Professor Program of Konkuk University and funded by the Ministry of Science, ICT and Future Planning [NRF-2016M3A9B6918892]. Special thanks to Miye Cho for drawing the figures.

39 Conflict of Interest  The authors declare no conflict of interest.

Bibliography Choi WK, Liew TH, Chew HG, Zheng F, Thompson CV, Wang Y, Hong MH, Wang XD, Li L, Yun J (2008) A combined top-down and bottom-up approach for precise placement of metal nanoparticles on silicon. Small 4(3):330–333. https://doi.org/10.1002/ smll.200700728 Derjaguin B (1993) A theory of interaction of particles in presence of electric double layers and the stability of lyophobe colloids and disperse systems. Prog Surf Sci 43(1–4):1–14 Derjaguin B, Landau L (1993) Theory of the stability of strongly charged lyophobic sols and of the adhesion of strongly charged particles in solutions of electrolytes. Prog Surf Sci 43(1–4):30–59 Dhawan A, Du Y, Batchelor D, Wang H-N, Leonard D, Misra V, Ozturk M, Gerhold MD, Vo-Dinh T (2011) Hybrid top-down and bottom-up fabrication approach for wafer-scale plasmonic nanoplatforms. Small 7(6):727–731. https://doi.org/10.1002/ smll.201002186 Hao F, Nehl CL, Hafner JH, Nordlander P (2007) Plasmon resonances of a gold nanostar. Nano Lett 7(3):729–732 London F (1937) The general theory of molecular forces. Trans Faraday Soc 33:8b-26 Mahmoud MA, Tabor CE, El-Sayed MA, Ding Y, Wang ZL (2008) A new catalytically active colloidal platinum nanocatalyst: the multiarmed nanostar single crystal. J Am Chem Soc 130(14):4590–4591 Majumder DD, Banerjee R, Ulrichs C, Mewis I, Goswami A (2007) Nano-materials: science of bottom-up and top-down. IETE Tech Rev 24(1):9–25 Naik AN, Patra S, Sen D, Goswami A (2019) Evaluating the mechanism of nucleation and growth of silver nanoparticles in a polymer membrane under continuous precursor supply: tuning of multiple to single nucleation pathway. Phys Chem Chem Phys 21(8):4193–4199. https://doi.org/10.1039/ C8CP06202A Verwey EJW (1947) Theory of the stability of lyophobic colloids. J Phys Chem 51(3):631–636 Wang H, Sun M, Ding K, Hill MT, Ning C-Z (2011) A top-down approach to fabrication of high quality vertical heterostructure nanowire arrays. Nano Lett 11(4):1646–1650. https://doi.org/10.1021/nl2001132 Wang H, Zhou S, Gilroy KD, Cai Z, Xia Y (2017) Icosahedral nanocrystals of noble metals: synthesis and applications. Nano Today 15:121–144. https://doi. org/10.1016/j.nantod.2017.06.011 Winsor P (1948) Hydrotropy, solubilisation and related emulsification processes. Trans Faraday Soc 44:376–398

40 Yang Y, Matsubara S, Xiong L, Hayakawa T, Nogami M (2007) Solvothermal synthesis of multiple shapes of silver nanoparticles and their SERS properties. J Phys Chem C 111(26):9095–9104 Zhang J, Li S, Wu J, Schatz GC, Mirkin CA (2009) Plasmon-mediated synthesis of silver triangular

J. Kim et al. bipyramids. Angew Chem Int Ed 48(42):7787–7791. https://doi.org/10.1002/anie.200903380 Zhou Z-Y, Tian N, Huang Z-Z, Chen D-J, Sun S-G (2009) Nanoparticle catalysts with high energy surfaces and enhanced activity synthesized by electrochemical method. Faraday Discuss 140:81–92

3

Silica Nanoparticles Hyejin Chang, Jaehi Kim, Won-Yeop Rho, Xuan-­ Hung Pham, Jong Hun Lee, Sang Hun Lee, Dae Hong Jeong, and Bong-Hyun Jun

Abstract

Keywords

Silica consists of one silicon atom and two oxygen atoms (SiO2) and is commonly used in various aspects of daily life. For example, it has been used as glass, insulator, and so on. Nowadays, silica is used as core reagents for fabricating and encapsulating nanoparticles (NPs). In this chapter, the usage of silica in nanotechnology is described. Synthesis and surface modification of silica nanoparticles (SiNPs), including via the  Stöber method, reverse microemulsion method, and modified sol-gel method,  are  illustrated. Then, various NPs with silica encapsulation are explained. At last, the biological applications of those mentioned NPs are described.

Reverse microemulsion method · Silica nanoparticles · Sol-gel method · Stöber method

Hyejin Chang and Jaehi Kim contributed equally to this work.

H. Chang Division of Science Education, Kangwon National University, Chuncheon, Republic of Korea J. Kim · X.-H. Pham · B.-H. Jun (*) Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea e-mail: [email protected] W.-Y. Rho School of International Engineering and Science, Jeonbuk National University, Jeonju, Republic of Korea

3.1

Introduction

“Silica” is the name given to the mineral group consisting of silicon and oxygen, the two most abundant elements in the earth’s crust, which consists of one silicon atom and two oxygen atoms to form SiO2. Silica in the crystalline state and almost amorphous state is most commonly found naturally in quartz and various organisms. Silica is one of the most complex and abundant materials that exist as compounds and synthetic products of several minerals, and is used as structural materials, microelectronics (electrical insulators), and as components of the food and pharmaceutical industries.

J. H. Lee Department of Food Science and Biotechnology, Gachon University, Seongnam, Republic of Korea S. H. Lee Department of Chemical and Biological Engineering, Hanbat National University, Daejeon, Republic of Korea D. H. Jeong Department of Chemistry Education, Seoul National University, Seoul, Republic of Korea

© Springer Nature Singapore Pte Ltd. 2021 B.-H. Jun (ed.), Nanotechnology for Bioapplications, Advances in Experimental Medicine and Biology 1309, https://doi.org/10.1007/978-981-33-6158-4_3

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H. Chang et al.

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Silica (including silica coated and doped silica materials) obtained through “sol-gel” inorganic polymerization processes is attracting attention as a substance having an impressive range of applications, such as controlled release and protection, including two pillars of practical application in the chemical field, synthesis and analysis. It is chemically and thermally very stable and relatively inert environmentally. Silica nanoparticles (NPs) have technically attractive physical and chemical properties. Silica is optically transparent, free from, or very limited in soil microbes, and can act as an insulator that serves to limit external effects. It also has a variety of uses in catalysts, energy, electronics, sensors, coatings, adsorption, chromatography, separation, biotechnology, environmental improvement, and many other modern technologies. Silica NPs have been intensively studied as a type of colloidal metal oxide NPs. They consist of siloxane (-Si-O-Si-O-) structures, exhibit high concentrations of silanol (Si-OH) groups on the surface, and can usually be produced by Stöber process or microemulsion. They are stable in aqueous solution and are very promising for biological use because of its biocompatibility, low toxicity, thermal stability, easy synthesis route, and large-scale synthetic availability. Particle size, crystallinity, porosity, and shape can be precisely manipulated to enable silica NPs for a variety of applications. Moreover, nanosized silica particles with large surface areas are known as ideal protein hosts, and many available surface modifications of silica NPs allow control of surface chemistry to achieve drug loading, good partitioning, and site-specific targeting. These properties make silica NPs a good platform for biomedical imaging, detection, therapeutic delivery, monitoring, and resection therapy. Silica is also used as a coating for surface modification of various NPs, and with the design of various dopants, surface functional groups and assembly techniques, multifunctional NPs can be developed. Silica NPs can be classified into two main types: solid silica NPs and mesoporous silica NPs. Mesoporous silica NPs, like  Mobil Crystalline Materials (MCM-41), consist of a porous structure of a silica matrix with many hol-

low pores and channels that can be filled with large quantities of various biomolecules and drugs. They have high surface area and large pore volume. Indeed, mesoporous silica, generally 2–50  nm in pore size, can be loaded more frequently with fluorescent dyes, photosensitizers, or diagnostic reagents and thus is used more often as a therapeutic nanoplatform and also has a combined function with fluorescence. Such NPs have low cytotoxicity and good cell imaging ability. Compared to mesoporous silica NPs, soild silica NPs do not exhibit a porous structure. In recent decades, among these nano platforms, silica NP offers many advantages over other similar systems for therapeutics, bio-imaging, bio-­ adhesives, and many other biomedical applications. This chapter provides an overview of recent developments in nonporous and porous silica NPs in the 1–200 nm size range. First, their synthesis method is described, and a method of designing size, shape, surface modification, and defects is presented. Finally, various types of silica-­applied NPs and their biomedical applications such as stimulus-induced drug delivery and accurate cancer diagnosis are described.

3.2

Synthesis of Silica Nanoparticles

The most commonly used synthetic method for silica NPs can be referred to as sol–gel reaction. Since synthesis of silica NPs with a uniform size is well established, the Silica NPs is utilized in various applications. The sol–gel reaction is a polycondensation reaction of an organometallic precursor, which loses water during the polycondensation and polymerizes to form a three-­ dimensional network structure (Fig.  3.1). For example, the Stöber process is an example of a sol–gel process in which molecular precursors (typically tetraethyl orthosilicate; TEOS) first react with water in an alcohol solution and then the resulting molecules combine to form larger structures. Since the bond between metal (M) and carbon (M-C) is not relatively strong, it is difficult to attach large organic molecules to metal atoms.

3  Silica Nanoparticles

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Condensation**

Hydrolysis*

TEOS

(+EtOH)

SiO2 + H2O Silica

(Tetraethyl orthosilicate)

* Hydrolysis : (EtO)3Si-OEt + H2O → (EtO)3Si-OH + EtOH ** Condensation :

(EtO)3Si-OH + EtO-Si(OEt)3 → (EtO)3Si-O-Si(OEt)3 + EtOH (EtO)3Si-OH + HO-Si(OEt)3 → (EtO)3Si-O-Si(OEt)3 + H2O

Fig. 3.1  Explanation of sol–gel reaction. Silica precursors (e.g., TEOS) are changed into silica via hydrolysis and condensation reactions

However, Si-C bonds can be fairly stable because they resist hydrolysis. Basically, the precursor having the structure of R4-nSiXn (n  =  1–4, X  =  OR′, halogen) changes into Si-OH group since Si-X bond is relatively weak to hydrolysis reaction, so the bond is broken to meet the water, halogen forms HX molecules. This causes condensation reaction between −OH again, causing Si-O-Si bonds and release water molecules. At the beginning of this reaction, a short oligomer-­ level condensation polymerization occurs or forms a cyclic structure, where the state is called a sol. As the reaction proceeds further into a complete network, now this state is called a gel. In order to enhance the sol–gel reaction, catalytic action by acid or base is required (Alothman 2012). Acids and bases, however, induce condensation reactions identically, but differ in terms of kinetics. The silica sol–gel reaction is sensitive to the acidity (pH) of the environment showing the slowest reaction at the isoelectric point of silica (pH: 2.5~4.5), and the reaction rate increases as the pH increases. In general, the larger the organic material attached to the metal is, the slower the reaction rate. Similarly, the larger the metal size, the faster the reaction rate. Therefore, Ti-based precursors exhibit much faster reaction rates than Si-based precursors. The pH also greatly affects the structure of the final material. Acid catalysts result in small clusters because they induce formation of  an open network structure, whereas base catalysts are likely to develop into large colloids because they maintain a high degree of cur-

ing from the beginning. Therefore, in general, an acid catalyst is used to make a sol-gel thin film, and a base catalyst is used to make a colloid. Spherical silica NPs are usually made by one of two synthetic routes: Stöber method or reverse microemulsion. The following section describes each of these two synthetic pathways and discusses how to control the size, shape, and porosity of silica coatings and particles based on them.

3.2.1 S  töber Method (Nucleation and Growth) The Stöber process was reported by Werner Stöber and his colleagues in 1968 (Stöber et al. 1968), and now is the most common method for synthesizing colloidal silica-based NPs below 200 nm. Since the reaction proceeds slowly compared to other sol–gel precursors, mass production is possible, and commercial applications are already being made. The Stöber process begins with the hydrolysis of silica alkoxide precursors, typically tetraethyl orthosilicate (Si(OEt)4, TEOS), in a mixed solution of alcohols (generally methanol or ethanol) and water containing a base catalyst (ammonium hydroxide) (Reaction 1). This reaction produces a mixture of ethanol and ethoxysilanol such as Si(OEt)3OH, Si(OEt)2(OH)2, even Si(OH)4. Loss of alcohol or water molecules can cause condensation with TEOS or other silanols. Further hydrolysis and subsequent condensation of ethoxy groups lead

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to cross-linking, and because hydrolysis and condensation occur together in a single reaction vessel, the process is regarded as a single-step process. Silicic acid ([SiOx(OH)4-2x]n) is produced during hydrolysis and if the concentration is higher than solubility in ethanol, it is nucleated homogeneously to form submicron silica NPs. Silica particle size can be controlled from several tens of nanometers to several micrometers by adjusting the reaction time and the concentrations of catalyst and precursor. To understand the reaction rate and mechanism, the effects of various alcohol solvents and tetra alkoxysilanes, as well as the concentration of each component on the particle size, have been intensively and systematically studied (Bogush et al. 1988; Hsu et al. 2008; Van Blaaderen et al. 1992). It was demonstrated that growth proceeds through surface reaction limiting condensation of hydrolyzed monomers or small oligomers. Particle formation through nucleation proceeds through an agglomeration process of siloxane substructures that is greatly affected by the surface potential of the silica and the ionic strength of the reaction medium. Thus, hydrolysis and condensation rate constants as a function of the reaction mixture and the behavior-­ related stability of silica NP in mixtures of alcohol, water, and ammonia play an important role in determining the final size of silica NP. Higher concentrations of water and ammonia result in a wider particle size distribution and larger particles. The initial concentration of TEOS is inversely proportional to the size of the particles produced. On average, higher concentrations of TEOS result in smaller particles due to more nucleation, leading uneven size distribution (Van Helden et al. 1981). If the initial precursor concentration is too high, irregularly shaped particles can be obtained. If the pH is higher than 11.6, the growth of silica is much faster than the rate of nucleation, so that uniform particles are not produced, and the poor particles are made. When the pH is less than 10, the hydrolysis reaction proceeds too slowly and the time required to produce nucleation becomes too long, therefore the uniformity of the particles is reduced. The process is temperature dependent and when it cools (and

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therefore slows the reaction rate), the average particle size monotonically increases, but control over the size distribution cannot be maintained at too low temperature (Bogush et al. 1988; Razink and Schlotter 2007). Particle size can be varied with reaction time. The particle aggregation model provides a high level of control over particle size and distribution and allows fine tuning of the physical properties of the resulting material to suit the intended application. The generated amorphous silica NPs are spherical having a size of 20 nm to several microns and have a monodispersity with a coefficient of variation (CV) of about 10–15%. The surface of the particles is negatively charged, which prevents adhesion of the particles in dispersion and is very hydrophilic. If the particles are smaller than 50 nm, the particles are less spherical, the monodispersity is reduced, and the surface of the particles is less smooth. Seed regrowth strategies have been attempted to improve the size control for larger NPs (Giesche 1994; Kim et  al. 2007). Recently, Hartlen et al. and Yokoi et al. produced monodisperse silica NPs with 12 nm size in diameter through heterogeneous reaction with tetraethoxysilane in the organic layer using lysine or arginine as the basic catalyst in aqueous media (Hartlen et al. 2008; Yokoi et al. 2009).

3.2.2 Reverse Microemulsions The reverse micelle method, also known as a water-in-oil (W/O) microemulsion system, produces a thermodynamically stable amphiphilic surfactant aggregates (Van Helden et  al. 1981). Hydrophilic head regions containing water cores of nanometer sizes are formed, each having a hydrophobic tail extending into the nonpolar continuous phase (Fig. 3.2). Various amphiphilic surfactants are used, and commonly cetyltrimethyl-­ammonium bromide (CTAB) and sodium dioctylsulfosuccinate (IGEPAL CO-520) are used (Ding et al. 2012). TEOS, a silica precursor, can be dissolved in a nonpolar organic solvent, so the alkaline catalyst solution meets the surfactant to form an emulsion. Proper mixing of surfactants with hydrocarbons leads to dis-

3  Silica Nanoparticles

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Water in Oil (W/O)

O C9H19

n~5

OH n

Igepal® CO-520

Water (+NH3)

Oil (+TEOS) Fig. 3.2  Illustration of microemulsion in water in oil (W/O) method by using Igepal® CO-520 as surfactant. Ammonia-contained water phase was surrounded with hydrophilic oligo  (ethylene glycol) moiety

persion formation of reverse micelles that can form reverse microemulsions with an infinite amount of macroscopically isotropic water. A nucleus section made of water acts as a nanocontainer to limit the Stöber process, allowing particularly effectiveness for synthesis of very spherical and monodisperse NPs less than 100 nm in diameter (Arriagada and Osseo-Asare 1992, 1999; Osseo-Asare and Arriagada 1990). As in the Stöber process, silica NPs are formed by hydrolysis of the silane precursor. Since the reaction is limited in the nucleus, the diameter of the particles produced is controlled by the size of the nucleus and the molar ratio of water/surfactant. Compared with the Stöber method, particle synthesis requires a much longer period, that is, about 1–2 days versus several hours. In addition to pure silica particles, the reverse micelle method can be used to prepare dye-doped metal NPs having a magnetic or semiconductor quantum dot core. The use of positively charged hydrophilic dyes is usually appropriate because these dyes easily integrate into the core formed of water of reverse microemulsion and are well pre-

(described as blue), meanwhile hydrophobic alkyl chain (described as red) was stretched toward oil phase (outside of emulsion)

served in the silica medium of the NPs by electrostatic interaction. Polar and water-soluble dye molecules can be easily encapsulated with silica NP by this method due to electrostatic attraction in the negatively charged silica matrix (Santra et  al. 2001; Wang and Tan 2006). Bagwe et  al. and Jin et  al. synthesized dye-doped silica NPs with continuously adjustable sizes in reverse microemulsions and applied them for cell imaging (Bagwe et al. 2004; Jin et al. 2008). With regard to hydrophobic organic dyes, doped silica NPs can be more difficult to manufacture because they are apparently soluble in water nanodroplets. Various trapping methods have been applied (Zhao et  al. 2004), such as introducing hydrophobic silica precursors or synthesizing water-soluble dextran-conjugated dyes under acidic conditions to synthesize NP doped with organic dyes. However, in these cases, the fluorophore is physically attached to the silica matrix and can leach out of the particles over time. In addition, the use of surfactants requires extensive washing to remove the surfactant molecules prior to biological application to avoid

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destruction or dissolution of the biofilm by the surfactant molecules.

3.2.3 M  odified Sol–Gel Method for Silica Coating Core/silica shell type NPs are the most basic type of structure that heterogeneous NPs using silica can have. If functional inorganic NPs are used as the core and silica is coated, concentric spherical NPs can be obtained. Functional NPs having various shapes and properties have been developed; however, the practical application of NPs requires the stability of physical, chemical, electrical, and optical properties in various environments. Therefore, silica has been used to address these problems. In order to make functional particles of hybrid structure using sol–gel reaction of silica, silica is coated on various colloids by taking advantage of the phenomena that heterogeneous reactions accompany thermodynamically favored nucleation more than homogeneous reactions do. The method based on the Stöber process is mainly used for coating silica shells on core NPs dispersed in solution (Liz-Marzan et  al. 1996). As described above, TEOS, a typical silica precursor, is hydrolyzed in the form of silicic acid in the presence of an alkaline catalyst and condensed on the surface of the NPs to be coated. Therefore, the hydrolysis rate of TEOS is affected by the aggregation rate, the size and concentration of the core NPs, the composition, and the ligand type. By adjusting the above conditions, the thickness of the silica shell can be adjusted by several nanometers. The synthetic basis to form core-silica shell NPs is to grow silica on core NPs as seeds. A representative example is silica-coated gold NPs. Low material affinity between crystalline noble metal NPs, including gold NPs, and amorphous silica makes it difficult to grow silica shells directly on gold NP surfaces. Therefore, various methods for coating silica shells directly on the surface of metal NPs have been researched. By pre-coating the surface of the gold NPs with a

H. Chang et al.

specific ligand to reduce the interfacial energy between the silica and the gold NPs, it is possible to thermodynamically reduce the tendency of phase separation between gold and silica. By doing this, it is possible to effectively induce nucleation of silica on the surface of gold NPs and to achieve direct silica shell growth. Methods of growing silica shells using TEOS were widely used by adsorbing 3-­mercaptopropyltrimethoxys ilane (3-MPTMS), a silane-based chemical with thiol functional groups, which is known to have strong affinity and strong binding with gold (Fig.  3.3) (Li et  al. 2012b). In this method, the concentration of TEOS should not  be kept too high, since the homogeneous reactions that lead to the independent silica particles should be suppressed. Thus, continuous injection of precursors can be an effective method. After coating with silica, the surface can be polymerized again, making it a triple structure of Au@silica@polymer (Liu et  al. 2008). The Wang group  put the nanostructures in dilute HF solution ( 200 nm) suitable for colloidal templating and formation of ordered arrays. Langmuir 24(5):1714–1720. https://doi.org/10.1021/la7025285 He XX, Nie HL, Wang KM, Tan WH, Wu X, Zhang PF (2008) In vivo study of biodistribution and urinary excretion of surface-modified silica nanoparticles. Anal Chem 80(24):9597–9603. https://doi. org/10.1021/ac801882g He XX, Wang YS, Wang KM, Chen M, Chen SY (2012) Fluorescence resonance energy transfer mediated large stokes shifting near-infrared fluorescent silica nanoparticles for in vivo small-animal imaging. Anal Chem 84(21):9056–9064. https://doi.org/10.1021/ ac301461s Helle M, Rampazzo E, Monchanin M, Marchal F, Guillemin F, Bonacchi S, Salis F, Prodi L, Bezdetnaya L (2013) Surface chemistry architecture of silica nanoparticles determine the efficiency of in  vivo fluorescence lymph node mapping. ACS Nano 7(10):8645–8657. https://doi.org/10.1021/nn402792a Hsu C-M, Connor ST, Tang MX, Cui Y (2008) Wafer-­ scale silicon nanopillars and nanocones by Langmuir– Blodgett assembly and etching. Appl Phys Lett 93(13):133109. https://doi.org/10.1063/1.2988893 Hu HC, Liu JJ, Yu JQ, Wang XC, Zheng HW, Xu Y, Chen M, Han J, Liu Z, Zhang Q (2017) Synthesis of Janus Au@periodic mesoporous organosilica (PMO) nanostructures with precisely controllable morphology: a seed-shape defined growth mechanism. Nanoscale 9(14):4826–4834. https://doi.org/10.1039/c7nr01047h Jetty R, Bandera YP, Daniele MA, Hanor D, Hung H-I, Ramshesh V, Duperreault MF, Nieminen A-L, Lemasters JJ, Foulger SH (2013) Protein triggered fluorescence switching of near-infrared emitting nanoparticles for contrast-enhanced imaging. J Mater Chem B 1(36):4542–4554. https://doi.org/10.1039/ C3TB20681E Jin YH, Lohstreter S, Pierce DT, Parisien J, Wu M, Hall C, Zhao JXJ (2008) Silica nanoparticles with continuously tunable sizes: synthesis and size effects on cellular contrast imaging. Chem Mater 20(13):4411–4419. https://doi.org/10.1021/cm8007478 Jo H, Her J, Ban C (2015) Dual aptamer-functionalized silica nanoparticles for the highly sensitive detection

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64 Rahman IA, Padavettan V (2012) Synthesis of silica nanoparticles by sol-gel: size-dependent properties, surface modification, and applications in silica-­ polymer nanocomposites—a review. J Nanomater 2012:132424. https://doi.org/10.1155/2012/132424 Rampazzo E, Bonacchi S, Genovese D, Juris R, Sgarzi M, Montalti M, Prodi L, Zaccheroni N, Tomaselli G, Gentile S, Satriano C, Rizzarelli E (2011) A versatile strategy for signal amplification based on core/shell silica nanoparticles. Chem Eur J 17(48):13429–13432. https://doi.org/10.1002/chem.201101851 Razink JJ, Schlotter NE (2007) Correction to “Preparation of monodisperse silica particles: control of size and mass fraction” by G.H.  Bogush, M.A.  Tracy and C.F.  Zukoski IV, Journal of Non-Crystalline Solids 104 (1988) 95–106. J Non-Cryst Solids 353(30):2932–2933. https://doi.org/10.1016/j. jnoncrysol.2007.06.067 Reineck P, Gómez D, Ng SH, Karg M, Bell T, Mulvaney P, Bach U (2013) Distance and wavelength dependent quenching of molecular fluorescence by Au@SiO2 core–shell nanoparticles. ACS Nano 7(8):6636–6648. https://doi.org/10.1021/nn401775e Rose S, Prevoteau A, Elziere P, Hourdet D, Marcellan A, Leibler L (2014) Neanoparticle solutions as adhesives for gels and biological tissues. Nature 505(7483):382– 385. https://doi.org/10.1038/nature12806 Russel WB, Russel W, Saville DA, Schowalter WR (1991) Colloidal dispersions. Cambridge University Press, Cambridge Santra S, Tapec R, Theodoropoulou N, Dobson J, Hebard A, Tan WH (2001) Synthesis and characterization of silica-coated iron oxide nanoparticles in microemulsion: the effect of nonionic surfactants. Langmuir 17(10):2900–2906. https://doi.org/10.1021/la0008636 Sanz-Ortiz MN, Sentosun K, Bals S, Liz-Marzan LM (2015) Templated growth of surface enhanced Raman scattering-active branched gold nanoparticles within radial mesoporous silica shells. ACS Nano 9(10):10489–10497. https://doi.org/10.1021/ acsnano.5b04744 Shen DK, Yang JP, Li XM, Zhou L, Zhang RY, Li W, Chen L, Wang R, Zhang F, Zhao DY (2014) Biphase stratification approach to three-dimensional dendritic biodegradable mesoporous silica nanospheres. Nano Lett 14(2):923–932. https://doi.org/10.1021/nl404316v Slowing II, Vivero-Escoto JL, Wu CW, Lin VSY (2008) Mesoporous silica nanoparticles as controlled release drug delivery and gene transfection carriers. Adv Drug Deliv Rev 60(11):1278–1288. https://doi. org/10.1016/j.addr.2008.03.012 Stöber W, Fink A, Bohn E (1968) Controlled growth of monodisperse silica spheres in the micron size range. J Colloid Interface Sci 26(1):62–69. https://doi. org/10.1016/0021-­9797(68)90272-­5 Suzuki K, Ikari K, Imai H (2004) Synthesis of silica nanoparticles having a well-ordered mesostructure using a double surfactant system. J Am Chem Soc 126(2):462–463. https://doi.org/10.1021/ja038250d

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4

Luminescent Nanomaterials (I) Hyejin Chang, Michael M. Murata, Won-Yeop Rho, Jaehi Kim, Jong Hun Lee, Sang Hun Lee, Dae Hong Jeong, and Bong-Hyun Jun

Abstract

From molecular probes, also known as fluorophores (typically emitting a longer wavelength than the absorbing wavelength), to inorganic nanoparticles, various light-emitting materials have been actively studied and developed for various applications in life science owing to their superior imaging and sensing ability. Especially after the breakthrough development of quantum dots (QDs), studies have pursued the development of the optical properties and biological applications of luminescent inorganic nanoparticles such as upconversion nanoparticles (UCNPs), metal nanoclusters, carbon dots, and so on. In this review, we first Hyejin Chang and Michael M. Murata contributed equally to this work.

provide a brief explanation about the theoretical background and traditional concepts of molecular fluorophores. Then, currently developed luminescent nanoparticles are described as sensing and imaging platforms from general aspects to technical views. Keywords

Fluorophore · Inorganic nanoparticles · Quantum dot (QD) · Upconversion nanoparticles (UCNPs)

4.1

Introduction

Fluorescence has long been used in biological applications because it has several important favorable characteristics for quantitative and ana-

H. Chang Division of Science Education, Kangwon National University, Chuncheon, Republic of Korea M. M. Murata Department of Surgery, Cedars-Sinai Medical Center, Los Angeles, CA, USA W.-Y. Rho School of International Engineering and Science, Jeonbuk National University, Jeonju, Republic of Korea J. Kim · B.-H. Jun (*) Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea e-mail: [email protected]

J. H. Lee Department of Food Science and Biotechnology, Gachon University, Seongnam, Republic of Korea S. H. Lee Department of Chemical and Biological Engineering, Hanbat National University, Daejeon, Republic of Korea D. H. Jeong Department of Chemistry Education, Seoul National University, Seoul, Republic of Korea

© Springer Nature Singapore Pte Ltd. 2021 B.-H. Jun (ed.), Nanotechnology for Bioapplications, Advances in Experimental Medicine and Biology 1309, https://doi.org/10.1007/978-981-33-6158-4_4

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lytical measurements. Over the past several decades, various light-emitting nanoparticles (NPs) have been introduced. These luminescent inorganic NPs, which include quantum dots (QDs), upconversion nanoparticles (UCNPs), metal nanoclusters, and carbon dots, have utility in environmental and life sciences due to several advantages. These include having unique optical properties such as narrow emission bands relative to traditional organic fluorophores. The development and innovation of luminescent NPs with unique optical properties, accompanied by advances in surface chemistry techniques, have provided new directions for in vitro and in vivo labeling in cells, tissues, and organisms, as well as fluorescence imaging and sensing. Luminescent NPs are gradually replacing traditional organic fluorescent chromophores in many biological applications, particularly immunoassays, microarrays, fluorescence imaging, and other sensor platforms. New molecular sensing strategies based on these NPs seek high sensitivity, high efficiency, and multiplexing capabilities. In addition, there is an abundance of more advanced luminescent probes based on phenomena such as fluorescence resonance energy transfer (FRET) and quenching. When coupled with technologies such as microfluidics and fluorescence spectroscopy, the detection limit could be as low as to a single molecule. These approaches will be more widely used in proteomic and genomic studies, disease diagnosis, drug screening, drug delivery, protein purification, therapy, and imaging. This chapter provides a general review of the current development of luminescent NP-based sensing platforms such as fluorescence dye, dye-­ doped NPs, QDs, and UCNPs (Details of carbon-­ based materials such as graphene quantum dots are covered in Chap. 11). We will also provide the theoretical background needed to understand the general and most common processes affecting fluorescence and introduce several techniques and applications for biosensing and imaging based on the introduced materials. Finally, we will briefly explain the major limitations and future directions of NPs in these research fields.

4.2

Basics of Fluorescence

Nanomaterials are expected to be a technological starting point that can revolutionize the field of optics and play an important role with broad applicability. Research in optics is closely related to the field of nanotechnology because both are essentially performed in the nano-sized wavelength range of light. The interaction of light and matter is covered throughout this book, including Chaps. 6 and 7 related to metals, Chap. 10 related to lithography, Chap. 11 related to carbon materials, and so on. This section briefly discusses the general aspects of light and fluorescence processes and then discusses luminescent nanomaterials.

4.2.1 Light and Luminescence In the narrow sense, “light” refers to visible rays. In particular, visible rays are electromagnetic waves with wavelengths between about 400 nm and 700  nm, which are generally detectable by the human eye. In broader terms, the electromagnetic spectrum includes radio waves, microwaves, infrared rays, X-rays, and gamma rays that bookend the visible wavelengths. Electromagnetic radiation is the energy that is transmitted or emitted through space in the form of periodic oscillations of an electric field and a magnetic field. Light travels linearly at a speed of 186,000 miles per second (300,000 km/sec) in a vacuum, and slower in other materials such as air, water, and glass. These periodic oscillations of the electric field and magnetic field are characterized by photons, the quantized unit of electromagnetic radiation. The electric field strength (E) at a given time (t) is expressed by E = E0 cos2πνt, where E0 is the amplitude and ν is the frequency of radiation as defined later. The distance between two points of the same phase in successive waves is called the “wavelength” λ,° which is measured in units of distance such as A (angstrom), nm (nanometer), and cm (centimeter).° The relationships between these units are: 1 A = 10-1 nm = 10−8 cm.

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The frequency of radiation, ν, is the number of waves in the distance light travels in one second and is expressed by

n=



c l

where c is the velocity of light (3 × 1010 cm/s). If λ is in units of centimeters, the dimension of ν is (cm/s)/(cm) = 1/s. This “reciprocal second” unit is also called the “hertz” (Hz). The third parameter, which is most common to ˜vibrational spectroscopy, is the “wavenumber” n , defined by ˜



n=

n 1 = c l

which has the dimension of (l/s)/(cm/s) = 1/cm = cm−1. Various types of electromagnetic radiation all have the same speed (c) but differ in wavelength and frequency. The electromagnetic radiation energy E is directly proportional to the frequency ν of the electromagnetic wave and thus is inversely proportional to the wavelength λ. High frequencies have more energy and shorter wavelengths. The energy of light is closely related to its color in the visible range where red light has lower energy (longer wavelength, lower frequency) than blue or purple light which have higher energy (shorter wavelengths, higher frequencies). The word luminescence comes from Latin (lumen  =  light). In contrast to the phenomenon that occurs in incandescent lamps, which are high temperature lighting, luminescence is often regarded as “cold light.” Luminescence is defined as the spontaneous emission from an electronically or vibrationally excited species that is not in equilibrium with the environment. According to the excitation mode, it is classified into various types such as photoluminescence, radioluminescence, and chemiluminescence. When the external energy supply is due to the absorption of light such as infrared, visible, or ultraviolet radiation, the emitted light is called photoluminescence, which occurs in all fluorescence analyses. The luminescent compound can be selected from the group consisting of (1) aromatic com-

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pounds: aromatic hydrocarbons and derivatives, dyes such as fluorescein, rhodamines, and coumarins; (2) inorganic compounds: uranyl ions (UO2+), lanthanide ions (e.g., Eu3+, Tb3+), semiconductor nanocrystals such as CdSe, metal clusters, and carbon nanodots; or (3) organic metal compounds: porphyrin metal complexes, ruthenium complexes, for example, Ru(bpy)3+2, copper complexes, and so on. In fluorescence and phosphorescence, a chemical species absorbs one or more photons, resulting in electronic excitation. It is de-excited through the spontaneous emission of photons, one of the physical effects caused by the interaction of light and matter. (Fig.  4.1) The Glossary of Terms Used in Photochemistry published by the International Union of Pure and Applied Chemistry (Braslavsky 2007) gives the definition of fluorescence and phosphorescence as follows: • Fluorescence: spontaneous emission of radiation (luminescence) from an excited molecular entity with retention of spin multiplicity. • Phosphorescence: phenomenologically, term used to describe long-lived luminescence. In mechanistic photochemistry, the term designates luminescence involving a change in spin multiplicity, typically from triplet to singlet or vice versa. (Note: e.g., the luminescence from a quartet state to a doublet state is also phosphorescence.) These definitions can be applied to organic molecules. However, other emissive species such as nanocrystal semiconductors (QDs), UCNPs, and metal nanoclusters, which are the main concern of this chapter, are not related to the concept of spin multiplicity. Generally speaking, fluorescence is emitted from an excited state that can be reached by direct light excitation, while phosphorescence is emitted from another excited state with a corresponding forbidden radial transition. Although nanocrystal semiconductors (i.e., QDs) are often regarded as fluorescent species, the term “luminescent QD” is preferred over “fluorescent QD” because the emission process is complex. The term photoluminescence, or sim-

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Fig. 4.1  Position of photoluminescence in the frame of light–matter interactions. (Valeur and Berberan-Santos 2012b)

ply luminescence, can be used whenever the nature of the state involved in the emission process is questioned.

4.2.2 Fluorescence Process and Related Terminologies Let us look further at the concepts that have traditionally been used in relation to the fluorescence of common organic molecules. To illustrate the process that follows light absorption by a molecule, it is convenient to use an energy diagram that represents the electronic state of a molecule with arrows to indicate possible transitions between them. This diagram is called the Jablonski diagram, derived from the name of “Aleksander Jablonski,” a Polish physicist in the 1970s. The Jablonski diagram visualizes simple processes such as photon absorption, internal conversion, fluorescence, intersystem crossing, and phosphorescence. Within each electronic level, there are a number of vibrational levels that can be combined with the electron state, and each of these vibrational levels can be further subdivided into rotational energy levels, but in the typical Jablonski diagram, the rotational level is omitted.

The vertical arrows corresponding to the absorption start at the 0 (lowest) vibrational energy level of S0 because most of the molecules are at this level at room temperature. This is also called the ground state. Once the electrons are excited, there are many ways in which energy can be dissipated, namely non-radiative processes (vibration mitigation and internal conversion) and radiative processes. As a radiative process, fluorescence is one of the passages of molecules that deal with the energy absorbed from a photon via emitting a photon. This process is represented by a straight line along the energy axis between the electron states in the Jablonski diagram. Fluorescence emission is terminated immediately after turning off the excitation source since this process is almost instantaneous (10−8  s). Because of the many vibration levels within an electronic state, most fluorescent molecules exhibit broad and unstructured absorption and emission bands. The 0–0 transition from the lowest vibration level of the bottom electron state to the lowest vibration level of the first excitation state is common to both absorption and emission while all other absorption transitions require more energy than any transition in fluorescence emission do. Therefore, we can expect that the emission spectrum overlaps with the absorption

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spectrum at the wavelength corresponding to the 0–0 transition and the remaining emission spectrum becomes the lower energy or longer wavelength (Fig. 4.2). However, the 0–0 transition of the absorption and emission spectra is hardly coincident, and the difference represents a small energy loss due to the interaction of fluorescent molecules and surrounding solvent molecules. The absorption of energy to produce a first excited state does not significantly disturb the morphology of the molecule, which means that the distribution of vibration levels is very similar

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in both the base and first excited states. The band-­ to-­band energy difference of the emission spectrum will be similar to the energy difference of the absorption spectrum, and often the emission spectrum is similar to the mirror image of the absorption spectrum. The commonly used terms in fluorescence spectroscopy are described as below. Stokes Shift  0–0 transitions are generally the same for absorption and fluorescence. However, due to energy loss in the excited state due to

Fig. 4.2  Perrin-Jablonski diagram of absorption, fluorescence, and phosphorescence spectra (above) and definition of the Stokes shift (below). (Valeur and Berberan-Santos 2012a)

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oscillation relaxation, the energy associated with the fluorescence emission transition is smaller than the absorption energy. Thus, the emitted photons travel at longer wavelengths than the absorbed photons (Fig. 4.2), which is commonly known as the Stokes shift. The main cause of the Stokes shift is that the excited electrons rapidly decrease to the lowest vibration energy level of the S1 excited state. In practice, the Stokes shift is measured as the maximum wavelength difference of the excitation and emission spectrum of a particular fluorescent dye or fluorescent chromophore. The degree of shift depends on the molecular structure but can range from a few nanometers to hundreds of nanometers. The presence of Stokes shift is important for extremely high sensitivity of fluorescence imaging measurements. Mirror Image Rule  In many of the common fluorescent materials, the vibrational energy-­ level spacing is similar for the ground and excited states, resulting in a fluorescence spectrum very similar to the mirror image of the absorption spectrum. This is due to the fact that the same conversion is the most advantageous for both absorption and emission. In solution, the detailed vibrational structure is generally lost and the emission spectrum appears in a broad band.

fluorescent emission or phosphorescence after intersystem crossing. Intramolecular pathways of de-excitation excitation such as electron transfer, proton transfer, energy transfer, and excimer formation can also compete with fluorescence emission if those processes occur on a time scale compared to the average time (lifetime) in which the molecule stays in the excited state. Fluorescence lifetime is a characteristic time that a fluorophore spends in the excited state before returning to the ground state by emitting a photon. The reduction in fluorescence intensity as a function of time in a uniform population of molecules excited by a pulse of short light is described by an exponential function:

I ( t ) = I 0 • e - t /t

where I(t) is the fluorescence intensity measured at time t, I0 is the initial intensity observed immediately after excitation, and τ is the fluorescence lifetime. Formally, the fluorescence lifetime is defined as the time at which the initial fluorescence intensity of the phosphor decreases to 1/e (about 37%) of the initial intensity. This quantity is the reciprocal of the rate constant for fluorescence attenuation from the excited state to the ground state.

Full Width Half Maximum  When analyzing the spectral peak, the full width-half maximum (FWHM) is a useful way of characterizing the shape and overall value of the peak. The FWHM is given by the difference in wavelength between two points on either side at which the intensity is half the maximum of the peak. This measures not only the height of the peak but also its width. The concept is simple, but this is a very important figure because the FWHM is used to define the resolution. For example, if the two peaks have overlapping FWHMs, they are not resolvable.

Quantum Yield  The quantum yield indicates the probability that a given excited molecule emits a photon as described above. More precisely, it is derived from the ratio of the fluorescence rate to all deactivation rates, including intersystem crossing to nonradioactive decay and triplet state. All nonfluorescent processes that compete for deactivation of the excited state electrons are called nonradiative rate constants and can conveniently be represented by a single rate constant denoted by the variable knr (Fig. 4.3). The quantum yield can be expressed as a rate constant given by:

Lifetime  Once a molecule is excited by the absorption of a photon, it could undergo intramolecular charge transfer and structural changes, although it may return to its ground state by

F = emitted photon / absorbed photon = kf / ( kf + knr ) = t f / t o



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Fig. 4.3  Possible de-excitation pathways of excited molecules. (Valeur and Berberan-Santos 2012b)

where kf is the rate constant for fluorescence decay. The reciprocal of the attenuation rate constant (1/ (kf + knr)) is equal to the eigenstate lifetime (τo), and the excitation state is defined as the lifetime of the excitation state in the absence of all processes competing for excitation state deactivation. Indeed, the fluorescence excited state lifetime is shortened by the non-radiative process, which results in a lifetime (τf) measured by the combination of inherent lifetime and competitive non-fluorescence relaxation mechanisms. Since the measured lifetime is always less than the intrinsic lifetime, the quantum yield never exceeds the value of 1. Generally, high quantum yield is desirable in most imaging applications. The quantum yield of a given fluorescent material is sometimes extremely varied depending on environmental factors such as pH, concentration, and solvent polarity. Brightness  The brightness of a fluorophore is defined as two parameters: extinction coefficient and quantum yield. To compare the brightness of different fluorophores, the relative brightness of the fluorophore is calculated by multiplying the extinction coefficient by the quantum yield. The extinction coefficient is a measure of the

probability of the electronic transition in a molecule by absorption of a photon, hence a higher extinction coefficient means higher absorption of light. Once photons are absorbed, the probability of photons emitted through fluorescence is related to the quantum yield. This is the ratio of the number of photons emitted relative to the number absorbed. Molecules with a low deactivation rate relative to fluorescence have high quantum yield. Therefore, molecules with high quantum yield and extinction coefficient are brighter.

Quenching and Photobleaching  Quenching and optical bleaching are phenomena that reduce emissions, and hence should be considered when designing and implementing fluorescence studies. Quenching occurs through a variety of competing intracellular or intermolecular processes that induce nonradiative relaxation of the excited state electrons to a ground state, reducing the excitation lifetime and quantum yield of the affected fluorophores. The excited fluorescent molecule often collides with other (nonfluorescent) molecules in the solution to inactivate the fluorescent substance and return to the ground state.

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Unlike quenching, photobleaching refers to a permanent loss of fluorescence due to photonic-­ induced chemical damage and covalent deformation. The average number of excitation and emission cycles for a particular fluorescent material prior to photobleaching depends on the molecular structure and local environment. While limiting the illuminating power and time can reduce photobleaching, this also reduces measurable fluorescence signals. Photodynamics, an important class of photobleaching events, refers to the reaction between the fluorescent molecules and oxygen which produces a free radical-neutral oxygen species, and this can result in permanent destruction of the fluorescence and chemical transformation of other molecules in living cells. The best protection against photobleaching is to limit the light exposure in combination with the wise use of commercially available anti-fade reagents that can be added to the mounting solution or cell culture medium.

4.2.3 Organic Dyes As Fluorophores Fluorescent dyes have been the most widely used in bioapplications, and these molecules generally have delocalized electrons due to their structures containing conjugated double bonds such as several benzene rings. In these systems, the energy gap between the lowest unoccupied molecular orbital (LUMO) and the highest occupied molecular orbital (HOMO) is generally in the visible light range. Table 4.1 shows popular fluorophores with their absorption and emission features. Fluorescein, one of the most popular fluorophores, has been widely applied in the field of immunofluorescence labeling. Fluorescein isothiocyanate (FITC), an amine-reactive isothiocyanate derivative, is the most widely used variant. The maximum absorption wavelength of this dye is in good agreement with the blue spectral lines of mercury and xenon lamps as well as argon ion and krypton-argon lasers. For fluorescein, the quantum yield is very high, but the emission intensity is strongly influenced by environmental factors (e.g., pH), and its relatively broad emission spectra often overlaps with the fluorescence

Table 4.1  λmax,abs and λmax,em of popular fluorophores Fluorophore Cascade Blue Marina Blue Fluorescein (FITC) Alexa Fluor488 Cy3 Alexa Fluor555 Tetramethylrhodamine (TAMRA, TRITC) Rhodamine Red Texas Red Cy5 Alexa Fluor647

λmax, abs(nm) 400 360 499 488 554 555 552

λmax, em (nm) 418 459 515 519 566 563 575

buffer pH 7.0 8.0 8.0 8.0 7.2 7.2 8.0

573 595 645 647

590 613 664 671

8.0 7.2 7.5

spectra of other fluorescent materials in doubleand triple-labeling experiments. Rhodamine dyes are widely used as fluorescent probes due to their high absorption coefficient, high quantum yield, and photostability in the visible region. Tetramethylrhodamine (TMR) and its isothiocyanate derivatives (TRITC) are often used in multiple-label studies in broadband microscopy due to the efficient excitation by the 546-nanometer spectral line of mercury arc discharge lamps. The fluorescence emission intensity of rhodamine derivatives does not depend strictly on environmental conditions. An example of a breakthrough in advanced fluorescence technologies is the Alexa Fluor dye from Molecular Probes. These sulfonated rhodamine derivatives exhibit higher quantum yields than spectrally similar probes and exhibit some additionally improved characteristics including enhanced photostability, absorption spectra suitable with common laser lines, low pH susceptibility, and high water solubility. Alexa Fluor dyes can be used in a wide range of fluorescence excitation and emission wavelengths ranging from ultraviolet and deep blue to near infrared regions. Cyanine such as Cy3 and Cy5 is a good alternative to conventional dyes such as fluorescent dyes and rhodamine because it produces bright, stable fluorescence. The combination of Cy3 and Cy5 (which emit red and greenish yellow respectively) is commonly used in two-color detection. Cy3 can be detected with a variety of fluores-

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cence detectors, imagers, and microscopes using standard filters for tetramethylrhodamine (TRITC), and is easily detectable visually in electrophoresis gels and solutions due to its high molar extinction coefficient. Cy5 has been widely used as a near-infrared fluorescent dye because it is a chromophore with a high extinction coefficient in the red region where most CCD detectors have maximum sensitivity and biological objects give low background interference. Cyanine dyes generally have a broader absorption spectrum than the Alexa Fluor dyes, which results in a diverse choice of laser excitation sources for confocal microscopy. Sulfo-cyanine dyes have one or two sulfo groups, which improves the water solubility of Cy-series dyes. Dye molecules can be attached to a protein through specific functional groups such as amino (active ester, carboxylate, isothiocyanate, and hydrazine), carboxyl (carbodiimide), and thiol (maleimide, acetyl bromide) groups. They can also be nonspecifically conjugated via glutaraldehyde or physically adsorbed by hydrophobic interaction. Various fluorescent dyes can be used in living cells, but in many cases, their applicability is still limited. Traditional fluorescence organic molecule-­ based bioanalysis has disadvantages such as photobleaching, relatively low brightness, and short tendency of Stokes shift. Time-­ lapse studies are more difficult due photobleaching and this reduction in fluorescence over time can complicate quantitative analyses. The problem of low brightness naturally causes sensitivity issues when working with samples with high background fluorescence. In addition, typically, only a limited number of phosphors can be attached to biomolecules before they significantly interfere with binding specificity. As a result, when targeting a trace amount of an analyte, a signal amplification step is required and the analysis becomes complicated. A new generation of phosphors (such as Alexa dyes) dramatically improves labeling efficiency, but the limitations mentioned are not completely addressed. In particular, there remain several difficulties performing highly multiplexed fluorescence-­ based analyses due to spectral overlap or com-

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plex equipment requirements. The emission spectrum of fluorescent dyes can be as large as 80 nm, which limits studies to only three colors without spectral overlap in the visible light region. Fluorescent organic dyes primarily absorb light with a wavelength slightly shorter than the emission band (Stokes shift of up to 50  nm), which means that if one wants to use three different fluorescent organic dyes with good resolution, three different excitation lasers and filters are required such that the equipment becomes complicated and expensive. Here, we review various materials that have been proposed to compensate for the disadvantages of organic dyes.

4.3

Luminescent Nanoparticles

Many types of emitters (optical labels) used in bioapplications, including internal and external detection and imaging, are classified mainly into three categories: (1) organic- and biologically derived fluorophores, (2) nanocrystals such as QD, and (3) molecular systems with defined structures containing particles from nanometer to micrometer size. This section introduces various colloidal emitters such as silica-based NPs, QDs, and UCNPs loaded with fluorescent dyes (Jun et al. 2012; Kim et al. 2018).

4.3.1 F  luorescence Organic Dye-­ Incorporated Materials Fluorescence organic dye-based materials have been introduced to complement some of the crucial shortcomings of organic dyes. Here, dye-­ doped silica NPs introduced into silica NPs and fluorescence encoded beads in which fluorescence is introduced into micro-sized polymers already commercialized are introduced.

4.3.1.1 Dye-Doped Silica Nanoparticles Silica is used in the development of various hybrid silica nanomaterials with different properties and is suitable for the construction of a hybrid system capable of loading and transporting materials appropriate for various fields of application

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of silica nanomaterials (see Chap. 3). In Chap. 3, we discussed the synthesis, surface modification, and general properties of silica-based materials, so we will briefly focus on the characteristics of fluorescent dye-doped NPs. Molecular tags like the traditional dyes mentioned in the previous section are being replaced by NPs because of their advantages in signal and selectivity. Fluorescent dye-doped silica nanoparticles (FSiNP) have a large number of dye molecules within the silica matrix and hence have intense and stable fluorescence signals without additional amplification steps. This facilitates sensitive analyses and monitoring of biological events that conventional fluorescence labeling techniques cannot detect. In addition, it is advantageous for selective recognition by using various surface modification techniques and for drug loading via the porous surfaces of silica. Thus, FSiNPs are also a promising candidate for use in smart drug delivery and therapy systems. Silica NPs can easily be doped with various organic, metal organic, and metal luminophore (hydrophobic, hydrophilic or ionic) materials and their emission wavelengths can be adjusted widely in the range of 300–1000  nm. Designs that allow longer emission wavelengths (>600  nm) are preferred due to the noise from autofluorescence of living tissue below 500  nm wavelengths. Organic dye-doped silica NPs exhibit absorption and emission spectra similar to or slightly shifted from the native dye molecules. When silica NPs are used as fluorescent tags (e.g., for biomolecule detection), incorporation of the fluorescent dye should occur during formation of silica NP or the fluorescent dye must be bonded to the silica surface after preparation. Typically, two methods are available to produce silica-based NPs, namely the Stöber and reverse micelle processes. The production of NPs doped with hydrophobic dyes is mainly carried out using the Stöber method while hydrophilic dyes are preferably encapsulated using a reverse micelle process. Reverse micelle technology continues today with the Tan group developing fluorescent NPs by physically trapping organometallic dyes in silica NPs in the mid-1990s (Santra et  al. 2001). The

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nanoreactor environment within reverse micelles has been shown to produce highly monodisperse particles and increase the incorporation of nonpolar molecules that are difficult to incorporate into the hydrophilic silica matrix. However, since the dye molecules are not covalently bonded to the silica matrix, they leach out of the NPs over time, resulting in a decrease in brightness of particles and an increase in background signal. Thus, the Tan group has developed a new synthetic method of organic dye (TMR)-doped silica NPs for reducing leakage of dyes via electrostatic attraction to the silica matrix (Zhao et al. 2004). TMR dye is bound to hydrophilic dextran molecules, dissolved in water, and synthesized into particles under acidic conditions. Encapsulation of fluorescent dye molecules in the hollow spheres of silica was also reported to effectively insulate the dye molecule from the effects of the external environment (Makarova et  al. 1999). Nonetheless, reverse micelle synthesis is often of low yield, and the use of surfactants requires extensive washing to remove surfactant molecules prior to biological application to avoid destruction or dissolution of the biological membranes by surfactant molecules. As an alternative, there have been various attempts to modify the Stöber method. Ow et al. reported covalent coupling method of the organophosphorus and the silica matrix using a reactive organosilicon compound (Ow et  al. 2005). The organic dye molecule is first covalently bound to the silica precursor to be concentrated to form a dye-rich core, followed by the addition of a silica sol-gel monomer to form a dense silica network around the core material. The resulting shell enhances the stability by shielding the solvent interaction. In this way, the particles have been validated for a wide variety of phosphors (mainly Alexa phosphors), and their sizes ranged from hundreds of nanometers to a few micrometers. This is useful for many applications such as colloid mechanics and photonic crystal studies, but is generally somewhat on the larger end of the spectrum for use in biological applications. The emission band of FSiNP can be adjusted by changing the ratio of the two dyes or excitation wavelengths while keeping the ratio (Xu

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et  al. 2010). Using this method, multiple phosphors can be loaded into a single particle, so that light absorption and emission characteristics of the particles can be controlled regardless of particle size. This is an important advantage because the optimal or desired particle size for use in vivo depends on the anatomical or cellular characteristics selected for targeting or diagnosis. In order to optimize the optical properties of the above-mentioned FSiNPs, several details need to be considered. For example, the loading density should be optimized to avoid self-­ quenching, which is commonly observed in most organic dyes in high concentrations or agglomerates.

4.3.1.2 Fluorescence-Encoded Beads Fluorescence-encoded beads, one of the most widely used materials for the multiplexed detection of biomolecules, are produced by capturing fluorescent dyes in microbeads composed of polymers such as polystyrene. By changing various dyes and adjusting the concentration, the microbeads can have different kinds of codes. Bead-based multiplexed analysis is the most commonly used form developed by many companies. Here, fluorescence-encoded beads will be explained briefly, since they have different application methods from NPs due to their larger (micrometer) size, although they can be used as templates. A commercially available Luminex protein detection system is a representative example of a fluorescence bead–based assay. The Luminex system uses a polystyrene-based microbead of 5.6 μm size as the carrier. Microbeads coded via the ratio of red and orange fluorophores are excited and measured using a red laser for decoding (target identification). Capture molecules that specifically bind to the target analyte are immobilized on the corresponding native barcode microbeads. By decoding the bead, the identity of the captured analyte can be determined. To quantify the target protein, the green fluorescence intensity is measured using a green laser. The green fluorescence intensity reflects the amount of target because it hybridizes, as in sandwich immunoassay or ELISA, and fluorescence is emitted

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from the secondary probe added after target capture. In the flow cytometry system, these beads can be separated by optical properties and target amounts. The encoded microbead-based techniques have several advantages over chip-based techniques when developing high-throughput systems for protein detection. Micro-bead-based techniques are capable of capturing about 106 times more molecules per bead than a planar chip on a large surface area. Thus, these beads are more flexible and sensitive in order to detect a wide range of target proteins. Additionally, since the interaction between the bead and the target molecule is almost similar to solution phase kinetics, it is very time-efficient. Also, the target molecule can be collected using flow cytometry. Because it is easy to mass-produce and store, it can be customized by selectively mixing antibody-­ conjugated microbeads according to the target protein. However, the wide and overlapping features of the emission bands, the complex optical system requiring multiple excitation laser lines, and the limit of practically available dyes hinder the widespread use of this method.

4.3.2 Quantum Dots (QD) Nanometer-sized crystals of certain semiconductors, such as cadmium selenide (CdSe), have interesting luminescent properties. The emission wavelength depends on the nature of the semiconductor material and the size of the crystal, which is known to originate from quantum confinement. Therefore, these nanocrystals are called “quantum dots (QDs).” If the radius of the nanocrystal is smaller than the exciton Bohr radius, the motion of electrons and holes is restricted in all directions. The quantum effect is felt also in all directions and the energy level of the material becomes discontinuous in all directions. A typical exciton radius of a semiconductor ranges approximately from 2 nm to 10 nm. Therefore, a quantum confinement effect can be realized when the size of the nanomaterial falls within this range. In general, CdS, CdSe, CdTe, ZnSe, InP, and InAs nanocrystals are typically used (Yao

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et al. 2014) resulting in properties between bulk semiconductors and discontinuous molecules. The smaller the QD, the shorter the emission wavelength. For example, the emission color of CdSe QDs changes from purple to red when the size increases from 2 nm to 7 nm. Quantum dots have attracted a great deal of attention in a wide range of fields related to electronics, optics, biosystems, and materials synthesis due to various properties distinct from general fluorescent dyes. The following section describes the structure, properties, and multiplexing performance of QDs.

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Quantum Confinement Effect As mentioned above, QDs have the size and the number of atoms between the atomic molecule level and the bulk material, and the bandgap varies in complex ways depending on a number of factors. In the case of an isolated atom, a sharp and narrow emission peak is observed. NPs com-

posed of approximately 102–104 atoms exhibit a distinct narrow optical line spectrum. This is why quantum dots are described as artificial atoms. When a material is reduced to the nanoscale, a quantum phenomenon occurs in which the energy level becomes discrete due to the limited wave motion of electrons. Figure  4.4 schematically shows the transition from atomic orbitals (s, p, or sp3) through molecular orbitals to QDs and bulk semiconductor energy bands. The energy gap between HOMO (the valence band of the semiconductor) and LUMO (the conduction band) decreases as the number of atoms increases in the transition region between the molecule and the bulk solids. In the case of semiconductor QDs, since the Fermi level exists between the bands, the edges exist in a discontinuous energy level, which greatly affects the optical and electrical properties of the semiconductor. Electrons and holes generated by excitation from the valence band to the conduction band have a bound state due to coulombic attraction, and a specific size of the coupled state is the exciton Bohr radius.

Fig. 4.4  Electronic energy levels depending on the number of bound atoms. By binding more and more atoms together, the discrete energy levels of the atomic orbitals merge into energy bands (here shown for a semiconduct-

ing material) (Alivisatos 1997). Therefore, semiconducting nanocrystals (quantum dots) can be regarded as a hybrid between small molecules and bulk material. (Parak et al. 2010)

4.3.2.1 Fundamentals of QDs

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There are three consequences when the size of the semiconductor nanocrystals becomes smaller than the Bohr radius: (1) the description of the wave function of electrons and holes is influenced by the boundary condition, (2) the energy level is quantized, and (3) the band gap is changed. The Bohr radius is the physical property of the semiconductor determined by the dielectric constant of the semiconductor. For example, it has a Bohr radius of 4.9 nm for CdSe and 46  nm for PbSe (Goesmann and Feldmann 2010). The quantum confinement effect can be understood based on the wave characteristics of the particles. If a particle has a wave characteristic, its behavior can be understood basically as a wave equation. In other words, based on the wave equation presented in classical mechanics, the quantum mechanical properties of the particles can be expressed by using the quantum mechanics operator. This is the Schrödinger equation. The best known of the solutions of the Schrödinger equation is the “particle in the box” model. If this box has a spherical shape with a diameter of d, the Schrödinger equation can be solved by introducing a spherical coordinate system and separating the radial part and the part containing the angular momentum. The lowest energy level is

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density of state (DOS) is defined as the number of available states per unit volume per unit energy per unit energy E. The lower the dimension of materials (3, 2, 1, and 0), the more limited the DOS of the electrons and the greater the quantum confinement effect.

Optical Properties As mentioned previously, the optical response of QDs is controlled by the band gap energy (Eg), particle size, and composition, and surface state. Like bulk semiconducting materials, QDs absorb photons of higher energy than the ­bandgap. When a photon with energy similar to Eg is absorbed, the electrons are excited into the conduction band in the valence band and holes remain in the valence band. Absorbing a photon with a much higher energy than Eg will excite the edge of the conduction band, and these electrons lose excess energy in nonradiative processes. The absorption intensity also varies depending on the occupancy level of the floor state and the number of nonoccupancy levels of the excited state. The absorption coefficient decreases with increasing size of the QD. In addition, as the photon energy increases, the probability of absorption increases because more valence band/conduction band transitions are possible. Compared with organic dyes, QDs have a very æ1ö 2 2 Ewell ,3 d ( sphere) = ç ÷ h / md . broad absorption spectrum, which allows the QD è2ø material to be excited by all light sources having The bulk band gap energy (Eg (bulk)), the con- an energy higher than the emission band. This finement energy of the carriers (Ewell), and the characteristic can reduce the cost and complexity coulomb energy between the electron-hole pairs of multi-wavelength systems in many applica(Ecoul) should be considered for obtaining the tions because QDs of different colors can be minimum energy (Eg (dot)) required to generate an excited with the same light source. Looking at the absorption spectrum, the quantum size effect that electron–hole pair in QD. Therefore, the particle size increases and shifts toward the Eg( dot ) = Eg ( bulk ) + Ewell + Ecoul , high energy band edge (blue shift) are directly h2 1.8e2 observed. The absorption spectra for CdSe QDs = Eg ( bulk ) + 2 at radii 4, 3, 2.7, 2.3, and 2.1  nm shown in pe . 2 d 2 ud 0 Fig. 4.6a clearly show a consistent blue shift as QDs emit different colors as the size changes. the particle size increases. In photoluminescence process of semiconducFigure  4.5 shows the change of emission color according to the size change of CdSe QDs. As tor, carriers (a pair of free electrons and holes) shown in the equation, if the size (d) of the quan- created by the absorption of photons with higher tum dots becomes smaller, the band gap is re-­ energy than the bandgap are relaxed in a two-step standardized, and the value becomes larger. The process. Initially, energy is lost quickly by

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Fig. 4.5  CdSe semiconductor quantum dots as nanoscale luminophores: (a) Surface color of suspensions in toluene in visible light. (b) Schematic diagram of band gap and

emission color as a function of particle size. (c) Light emission of suspensions in toluene when excited with UV light. (Goesmann and Feldmann 2010)

a

b 21 Å diameter Normalized Fluorescence

(B) Optical Density

23 Å

27 Å 30 Å 40 Å

1.8

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

Energy (eV)

400

(A)

500

600

700

800

Wavelength / nm

Fig. 4.6 (a) Optical absorption versus size for CdSe nanocrystals (Mittleman et  al. 1994). (b) The emission of nanoparticles (CdTe QDs) of different sizes. (Li et al. 2006)

phonon emission. In other words, the electron relaxes to the lowest level of the conduction band and the hole relaxes to the highest energy level of the valence band. The carrier can now be relaxed across the bandgap by radiative or nonradiative

recombination. Nonradiative processes compete with the radiative process that results in photon emission and affect quantum efficiency. As with absorption, the emission of QDs depends strongly on energy and electron state,

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and the emission wavelength is a function of size. Therefore, QD emission characteristics are changed by designing its size and shape. The bandgap of nanocrystals increases as the size decreases and the emission wavelength becomes shorter. Figure 4.6b shows that the fluorescence emission spectrum of CdTe QDs can be adjusted from 500 to 700 nm by varying the diameter. In addition to particle size, the chemical composition of QDs plays an important role in the emission wavelength. By changing both the particle size and the chemical composition, the fluorescence emission can be adjusted from the visible to the near infrared region and adjusted over a wide wavelength range of 400–2000  nm. Table  4.2 shows the range of variability of the various QD materials. The manipulation of the photoluminescent emission of QDs is important when used in biological applications such as fluorescent labels for biological macromolecules. In addition, surface states can have a direct impact on the emission characteristics because the QDs have a large surface area-to-volume ratio. When QDs contain impurities, the excitons can be trapped and the photoluminescence emission energy can complicate the photoluminescence spectrum. Quantum Yield and Surface Structures In addition to the absorption and emission characteristics described above, many parameters such as chemical or physical stability, manufacturing cost, reproducibility, and toxicity are required for practical compatibility of the luminescent materials. Quantum efficiency, a key Table 4.2  Photophysical characteristics of representative quantum dots Core (shell) materials CdTe CdTe(CdSe) CdSe(ZnS) CdS ZnS ZnSe InP InAs Yao et al. (2014)

Core diameter size range (nm) 3.2–9.0 4.0–9.4 2.0–8.0 2.8–5.3 0.7–2.1 2.0–5.0 2.6–4.5 3.4–6.0

Typical emission range (nm) 540–750 640–860 480–650 375–475 300–400 325–150 625–720 860–1250

value for high-quality luminophores, is a particularly important consideration. As described in Sect. 2, quantum efficiency is defined as the ratio of emitted photons to absorbed photons (Nem / Nabs) and the maximum possible value is 1.0 except for multi-photon emission. Quantum dots exhibit a higher quantum efficiency than conventional organic fluorescent layers. For example, the fluorescence intensity of ZnS-coated CdSe QDs is 20 times brighter than a single rhodamine 6G molecule and 100 times more stable for photobleaching than the organic fluorophore (Mitchell et al. 1999). Therefore, QDs have relatively good analytical sensitivity. A quantum yield of less than 1 means that some of the electrons in the excited state relax nonradiatively through competitive dissipation processes. In general, the quantum yield of NP luminophores is known to be about 40% which is much lower than the quantum yield of bulk luminescent materials  (over 80%) (Goesmann and Feldmann 2010). This is because the quantum yield is drastically reduced by very small defects of the crystal lattice, and even highly crystallized NPs with high purity inevitably have a definite inherent defect, called surface. High energy oscillation also causes a decrease in quantum yield. Surface functionalization of NPs by organic molecules or polymers have advantages from the standpoint of colloidal stability. However, it can also cause a drastic reduction in quantum yield due to molecular vibrations. Since the volume ratio of the QDs to the surface is high (e.g., 15% of the atoms in the 5 nm CdS QDs are on the surface), the surface state is important for the optical characteristics of the QDs. Their photoluminescent properties such as quantum yield and lifetime are affected by the nature of the shell or surface-capping organic molecule coating, synthesis conditions, changes in surface ligands, and environmental conditions (e.g., temperature and pH). Defects on the surface act as temporary “traps” for electrons, holes, or excitons, thereby suppressing radiative recombination and reducing quantum yield. Therefore, surface-capping or passivation research has been of great interest for the development of highly efficient and photostable QDs. All band edges of

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the QD with fully immobilized surfaces only have an internally quantum-confined state. Different passivation results in new energy levels near the band edge, which can result in different optical properties. The surface modification of the QDs is very tricky and is generally performed by depositing an organic or inorganic capping layer. The fabrication process for monodisperse QDs generally includes introducing organic molecules, which are adsorbed on the surface and act as capping agents. Tri-n-octyl phosphene oxide (TOPO) is the most widely used ligand, and the surface derivatization with thiolates (-SH) has the effect of minimizing surface defects and enhances the possibility of electron–hole recombination and improves the fluorescence intensity of QDs (Noglik and Pietro 1994). The simultaneous introduction of an organic capping layer with the fabrication of QDs is convenient, and also advantageous for biomolecule introduction. However, choosing an organic ligand that binds to the surface atoms of QDs is a tricky problem. The cover of the surface atoms can be sterically hindered by the organic capping molecule due to their distortion on surface and large size relative to surface areas. Another important problem is the simultaneous passivation of anionic and cationic surface sites using a capping agent. Finally, the bond between the organic capping molecule and the surface atom is generally weak, so the organic capped QD is optically unstable. A second approach for passivating the surface of QDs is to use an inorganic layer, especially a material with a larger bandgap. The passivating shell is grown on the core as an epitaxial or nonepitaxial crystal. The quantum yield of these QDs increases with a uniform and nondefect shell coating. The shell material could modulate the lattice parameters of the core during epitaxial growth to change the characteristics of the core/ shell system, such as absorption and emission spectra (Chen et  al. 2003). The quantum yield and emission band of the core/shell QD depends on the thickness of the shell layer. The epitaxial growth is described in detail in the next section.

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4.3.2.2 Synthesis of Quantum Dots Sufficient control during synthesis is required because the properties of QDs are determined by different intrinsic factors such as size, shape, defects, impurities, and crystallinity. Over the last 30  years, various approaches have been developed to synthesize QDs with improved properties. Here, hot injection, heating-up, clustering, and microwave-assisted synthesis ­ methods are discussed. Currently, there are many types of semiconductor NPs that can be synthesized. Chalcogenide compound semiconductors of groups II–IV particularly have been studied for a long time because they are direct bandgap semiconductors with high fluorescence efficiency and relatively easily synthesized. The synthesis of QDs has mainly relied on the thermal decomposition of organometallic precursors. Therefore, unintended synthesis of metal atoms cannot be avoided. QD synthesis is usually carried out in an atmosphere of nitrogen and argon as much as possible because the electrical and optical properties of the QDs are deteriorated by oxygen. In order to prevent the aggregation of particles during the synthesis process, amphiphilic organic coating agents with low molecular weight such as acids and alkyl amines are used. These coating agents play an important role in controlling the size and distribution as well as preventing aggregation. The current approaches to synthesizing monodisperse QDs are based on fast nucleation and controlled growth of nuclei (Fig.  4.7) (Murray et al. 1993; Alivisatos 1996; Peng et al. 1998; Murray et  al. 2000). Alivisatos and Bawendi et  al., introduced a “hot-injection” method of synthesizing monodisperse CdS, CdSe, and CdTe QDs. The hot injection method is the most common method for synthesizing monodisperse nanocrystals, rapidly injecting organometallic reagents into a high-temperature solvent to produce uniform nuclei. Separation of nucleation and growth is effective for particle size and size distribution control. The reaction solution contains surfactant molecules/ligands such as tri-n-­octylphosphine (TOP) and/or tri-n-

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isolated by periodically removing aliquots from the reaction vessel. (b) Representation of the simple synthetic apparatus employed in the preparation of monodisperse nanocrystal samples. (Murray et al. 2000)

octylphosphine oxide (TOPO), oleic acid (OA), and oleamines (OM), to prevent QDs from aggregating. The process initially starts at a high temperature (350 °C), which is lowered (280 °C) after introducing the precursor. This pyrolysis at a high temperature causes nucleation to occur at once throughout large amounts of Cd and Se atom supersaturation. The generated nuclei are induced to grow while suppressing further nucleation. During the uniform diffusion-controlled growth, larger QDs grow more slowly than smaller QDs, resulting in scaling effects. Although larger QDs continue to grow, small QDs dissolve due to higher chemical potentials (Ostwald ripening). This process is called aging, and the size of the QDs can be controlled to be a few nm to 10  nm according to the aging time. After the reaction is completed, the reaction solution is cooled to 60  °C, and then methanol is added to the reaction solution. As a result, TOP and TOPO attached to the surface of the particles become less compatible with methanol, resulting

in aggregation of NPs. After centrifuging the mixture, butanol is added. The pure Cd and Se particles are precipitated, but the CdSe NPs are dispersed and can be easily separated. By washing the NPs two or three times with alcohol, the remaining TOP and TOPO are removed and the synthesis of CdSe NPs is completed. From the thermodynamic point of view of CdSe synthesis, the formation of independent NPs of Cd and Se is not thermodynamically favorable because the CdSe alloy form is lower than the energy of the single component particles of Cd and Se. Of course, it is inevitable that small amounts of Cd and Se NPs will form, but after synthesis, they can be removed using centrifugation and solubility differences. By varying the temperature, concentration of the surfactant, and reaction time, QDs with various sizes and nanocrystal structures can be obtained. In the case of TOP and TOPO, the unpaired electron pair of phosphorus and oxygen atoms coordinate with the transition metal Cd

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and Se atoms to form a self-assembled layer on become supersaturated enough to generate an the QD surface. The presence of such an organic independent nucleus. Further, in order to control layer not only prevents aggregation between par- the thickness of the shell well, it is preferable to ticles but also delays the surface adsorption of Cd keep the temperature low so that the reaction rate and Se atoms participating in the reaction so that is not too fast. the entire reaction can be controlled by diffusion. When synthesizing hybrid semiconductor However, capping molecules that bind too QDs, the stoichiometry between the different strongly to the surface are not suitable for synthe- precursors must be precisely adjusted. Otherwise, sis because they do not allow growth. Weak coor- each single-component metallic NP is highly dination of the molecule results in the formation likely to form, making separation difficult. In of large particles or aggregates. Superior control addition, even if the stoichiometry ratio is of size and shape is possible by controlling the adjusted properly, there is no guarantee that only mixture of surfactant molecules, and they could mixed semiconductors will be fabricated due to be exchanged with other coating molecules hav- the different thermal decomposition rate of the ing different functional groups or polarity after two reactants depending on the reaction condisynthesis if necessary. tions. Recently, in order to solve this problem, The QDs synthesized in this manner have there have been many studies on the synthesis of weak luminescence due to a large number of non-­ NPs by preparing a single precursor having both radiative recombination of excitons on the sur- reactive elements. Since these precursors are face, and the relatively short lifetime due to already in stoichiometric proportions, there is oxidation. As mentioned in the previous section, less concern about the presence of pure single a surface passivation process is required to solve metals. this problem, and the most widely used method is epitaxial growth. A 5% size difference between 4.3.2.3 Surface Modifications the coated and coating crystal lattice surface per- For biological applications of QDs, it should first mits epitaxial growth. Commercially available be stable in water and should be biocompatible, QDs have a core/shell structure such as a CdSe/ especially when applied to biomedical imaging. ZnS. These epitaxy-based coatings are different It is also important for optical sensing to modify from physical coatings such as silica coatings in the surface with various functional groups (-NH2, that they share the same crystal lattice and there- -COOH, -OH, etc.) that allow interaction of QDs fore can also share electronic states. In particular, to enhance selectivity and sensitivity to target materials such as CdSe @ CdS have a lattice mis- analytes. Synthetic methods using surfactants, match of 3.9% between CdS and CdSe, and the such as TOP, TOPO and octadecylamine, which bandgap of CdS is larger than the bandgap of are the most widely used stabilizers, result in CdSe, thereby increasing the luminescence effi- water-insoluble QDs that are not suitable for biociency and increasing the stability of the parti- logical applications. Two approaches are genercles. For achieving epitaxial growth, prior ally available to overcome this surface property: removal of the organic layer on the surface is nec- (1) nanocrystal surface coatings using various essary so imitating the crystal faces exposed on organic materials such as organic ligands or polythe surface is possible. Therefore, CdSe QDs sta- mers and (2) inorganic shell coatings such as bilized by TOPO are dispersed in pyridine and other silica and titanium. It should be noted that TOPOs attached to the surface are dissolved and various surface modification strategies may result replaced with pyridine which have much weaker in changes in optical and chemical properties, bonds. For preventing nucleation and growth dur- including quantum yield, as discussed in the ing Zn, S, and ZnS coating, it is necessary to previous section. Most surface modifications lead gradually and continuously supply a low-­ to a rapid decrease of quantum yield, which concentration precursor solution so as not to ultimately limits their practical application.

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There are many ways to make QDs water-­ soluble with several considerations compromising between complexity, conditional stability, hydrodynamic size, and optical properties. Replacement of the organic material attached to the QD with a biocompatible material with a thiol group, such as an oligonucleotide or protein, could be one such way. The high affinity of thiol ligands for metal surfaces allows the hydrophilic molecules to naturally exchange with the weakly bonded hydrophobic TOPO ligands. Direct attachment of thiol-containing molecules to QDs instead of bulky TOPO or polymer reduces the hydrodynamic size of QDs. This is essential for many applications. For example, particle size can dramatically affect in  vivo biodistribution and pharmacokinetics as well as the efficiency of FRET-based assays that are highly sensitive to donor–acceptor distances. Recently, the development of polymer synthesis has allowed biocompatible coatings with amphiphilic molecules (polymers, small molecules, and lipids) directly to surface of QDs (Tomczak et al. 2013). For example, polyacrylic acid (PAA) having an amine group can be used to control hydrophilicity of TOPO-coated QDs without removing TOPO by using a strong hydrophobic reaction with TOPO (Gomaa et al. 2018). As another example, introduction of polyethylene glycol (PEG) enhances resistance to environmental changes, promotes effective use in bioassay and live cell imaging, and increases circulation time (Ballou et al. 2004; Mei et al. 2009; Papagiannaros et al. 2009). When the polymer is added to a solution of QDs coated with a hydrophobic surfactant (TOP, TOPO), a hydrophobic chain of the polymer is inserted between the surfactant molecules and a hydrophilic group is exposed to the surface to stabilize the QD in an aqueous environment (Shibasaki et al. 2009; Lin et al. 2008; Schmidtke et al. 2013). The polymer on the surface of the QD can be further crosslinked to increase stability of QDs. If the polymer contains an amine group or a carboxylic acid group, it is also convenient to impart additional functionality. A method of directly coating positively charged proteins on negatively charged

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QDs with electrostatic attraction has also been introduced (Guerrini et al. 2018). Layer-by-layer (LBL) technology is also used, which consists of depositing a single layer of polymeric molecules of alternating polarity or charge, and works best with an alternately charged ionic material (Costa and Mano 2014). Although LBL is very viable, it can be problematic if the QD is applied for complex specimens such as serum. As another method of encapsulation, the TOPO on QD surfaces is reacted with silane compounds such as mercaptopropyltri(methyoxy) silane (MPS) to form the silica shell using a sol-­ gel method (Ung et  al. 1998; Erathodiyil and Ying 2011). Silica encapsulation makes QDs ­biologically inactive to avoid toxicity, builds negative charge up on the surface, and most importantly, protects the core from external extinction. When silica is coated, basic catalysts such as ammonia are used to ensure that the degree of supersaturation is not so high as to prevent the generation of independent silica NPs. Additional functionalization can be achieved through new silane precursors containing functional groups (-SH; -NH2; -PO-(O-)CH3) that can be incorporated into the shell. The method of coating QDs with silica is well established and reproducible. In addition to the above approach to solubilization and functionalization of the QD surface, a number of combined approaches including silica and polymer capping have also been proposed (Liu et  al. 2013). This combination of synergies produces QDs with greatly enhanced resistance to aggressive chemicals, including strong acids.

4.3.3 Upconversion Fluorescent Nanoparticles Semiconductor nanocrystals (i.e., QDs), which have attracted much attention as an alternative to organic dyes, have excellent optical properties. However, their use is limited due to toxicity concerns for the human body. Also, the light source in the UV–Vis region, which is generally used to excite both organic dyes and QDs, is in a region

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absorbed by most biological samples. This causes autofluorescence that interferes with the signal to be obtained from the targets to be monitored as well as the possibility of optical damage and mutation due to continuous exposure to ultraviolet light. Therefore, it has become necessary to develop novel substances to overcome the limitations of organic dyes and QDs. Recently, upconversion nanoparticles (UCNPs) have been attracting attention as a substitute for organic dyes and QDs. UCNPs, a NP doped with lanthanide ions, sequentially absorb two or more photons in the near-infrared region, and exhibit anti-Stokes luminescence properties having the shorter emission wavelength (UV-Vis region) than absorption unlike other conventional down-conversion fluorescent materials. Two-­ photon fluorescence microscopy techniques have been developed and used extensively in life sciences even before UCNPs were used for imaging. However, the barrier for typical two-photon fluorescence techniques is high since it requires a high-power pulsed laser. In contrast, in the case of UCNPs, the long excitation lifetime of lanthanide atoms makes it possible to use a low power continuous wave source. UCNPs exhibit strong luminescence intensity, narrow emission band, biocompatibility, and excellent optical stability against photobleaching or blinking. In addition, the use of near-infrared excitation wavelengths generates much less autofluorescence signal and permits excellent tissue penetration characteristics. As a result, studies on biological applications such as biosensing, bioimaging, and phototherapy using UCNPs have been actively conducted.

4.3.3.1 Fundamentals of UCNPs The lanthanides are the elements from atomic number 57 lanthanum (La) to atomic number 71 lutetium (Lu). In general, they are sometimes referred to as rare earth elements, adding yttrium and scandium, due to the similarity of chemical properties. The lanthanide elements belong to the f-zone elements, except for lutetium, and exhibit very similar chemical and physical properties due to the characteristic electronic arrangement of the f elements. This f electron is shielded by

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the external 5p and 6 s orbital, and is less affected by the binding of the elements or the external environment. Therefore, the absorption and emission spectra due to the f electrons have a relatively small influence on the composition change. Lanthanides exhibit interesting inherent absorption and luminescence characteristics and a very narrow spectrum even with changes in the composition of the material and surrounding environment. The absorption and luminescence transitions of the lanthanide cation between the f-level (4f-­ 4f) are both forbidden and thus exhibit a very low molar extinction coefficient. The lifetime of an excited electron is very long from microseconds to milliseconds. These properties can lead to efficient energy transfer between the energy levels of the f electrons when doping one or more lanthanide elements into a NP host made from a few to tens of nanometers. This is because the energy transfer between the elements can be very effective due to the long excited lifetime and the distance between the dopants constrained by the physical size of the NPs. Among the unique optical phenomena that the f-electron of the lanthanum element exhibits, the upconversion phenomenon occurs when NPs absorb photons to generate excited f-electrons with long lifetime, the electrons absorb additional photons, and finally the NPs emit photons of higher energy (Sarkar et al. 2019). Unlike two-­ photon absorption using imaginary energy levels, the two-photon upconversion of a lanthanum element uses the actual energy level of the element and thus exhibits a relatively high efficiency. Upconversion is a nonlinear optical process that continuously absorbs two or more pump photons through a long-lived intermediate energy state and then emits radiation of a shorter wavelength than the pump wavelength (Wang and Liu 2009). The energy upconverter is synthesized by simultaneously doping the light-absorbing element and the light-emitting element in the host. In general, ytterbium (Yb) cations are used as light-absorbing elements. The emission spectra of NaYF4: Yb3+ and Er3+, typical UCNPs, emit green light with wavelengths of 525  nm and 540  nm and red light of 655  nm

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(Fig.  4.8a). Yb3+ has a ground state and one excited state energy level (van der Ende et  al. 2009), which can absorb an infrared photon at about 980  nm and transition the ground state electrons to an excited state (2F5/2). The absorbed energy is transferred to another lanthanide cation, which is used as the emission center (e.g., Erbium (Er), Thulium (Tm)). Then, energy is absorbed from the other Yb3+ and transition to a higher energy state takes place in the emission center. This two-photon transition is very efficient (a few μs–ms) since the excitation lifetime of a luminescent center such as Er3+ or Tm3+ is very long compared to a general fluorescent material (a few fs–ps). When the excited electrons are radiated, visible light emission characteristics can be realized from the absorbed 980  nm infrared rays. Figure  4.8b shows an energy diagram of the upconversion process. Depending on the type of host and the dopant ion used, it is possible to realize upward light emission characteristics such as green and red (Er) or blue and infrared (Tm). The upconversion process is typically divided into three categories: excited state absorption (ESA), energy transfer upconversion (ETU), and photon avalanche (PA) as shown in Fig.  4.9 (Wang and Liu 2009). In addition to this, there is literature categorizing six process types including cooperative sensitization upconversion (CSU) and cross relaxation (CR) (Chen et  al. 2014). However, here we only discuss ESA, ETU, and PA, which are more generally considered. Excited State Absorption (ESA)  ESA, also known as sequential two-photon absorption, was proposed by Bloembergen in 1959 and is the only UC process that occurs at low dopant ­concentrations. Here, in a three-level system, the gap between the ground state (G) and the E1 level (intermediate long excited state) and the gap between the E1 and the E2 level are equal. The ground state absorption (GSA), transition from G to E1, occurs by the excitation light first. Then second absorption from the E1 level to the E2 level occurs before the excited photons at the E1 level relax to the G level, followed by upconversion emission from E2 to G. The lanthanum ions that show this upconversion are

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Er3+, Ho3+, Tm3+, and Nd3+. In this mechanism, the luminescent efficiency is independent of the concentration of the dopant since this process occurs in one ion.

Energy Transfer Upconversion (ETU)  UC through energy transfer was extensively studied in the mid-1960s and is observed in materials with high dopant ion concentrations. ETU is similar to ESA, in that it uses successive absorption to move two photons to metastable levels, but it occurs between two adjacent, but not single, ions. Two neighboring ions each absorb the same energy and are located at the metastable level E1. At this time, energy transfers from ion 1 acting as a sensitizer or donor, to ion 2 acting as an activator or receptor. The transferred energy is used to absorb the pump photons from the E1 level to the E2 level at ion 2. The nonradiative energy transfer process allows one ion to go to a higher emission level, E2, but the other ions go down to the ground level, G. A variety of well-known energy transfer (ET) mechanisms include ET followed by ESA (EFE), successive energy transfer (SET), cross relaxation (CR), cooperative sensitization (CS), and cooperative luminescence (CL). Depending on the distance between the photosensitizer and the activator as well as the dopant concentration, the efficiency of the energy transfer upconversion varies (Chen et  al. 2014). The lanthanum most commonly used as photosensitizers/activators for ETU are Yb3+/Tm3+ and Yb3+/ Er3+ (Zhou et al. 2015).

Photon Avalanche (PA)  PA, one of the most efficient types of UC, was first discovered by Chivian in 1979. The process of generating UC by PA involves intense pumping above a certain threshold, where a very small amount of fluorescence is generated by upconversion below the pump threshold. In this mechanism, the level E1 of ion 2 is initially filled by the nonresonant weak GSA. It is then raised by the resonant ESA to the upper level E2 which has visible range emission (ESA process). Then, a cross-relaxation energy transfer occurs between the excited ion and the

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Fig. 4.9  Principal UC processes for lanthanide-­ doped crystals: (a) excited state absorption, (b) energy transfer upconversion, (c) photon avalanche. The dashed/ dotted, dashed, and full arrows represent photon excitation, energy transfer, and emission processes, respectively. (Wang and Liu 2009)

adjacent ground state ion, so that both ions occupy E1 (E2 (ion 2)  +  G (ion 1)  →  E1 (ion 1) + E1 (ion 2)), and this process is repeated. As a result of repetition, one ion 2 in the metastable state (E1) generates two ion 2 in E1 states, and the ion distribution of E2 increases exponentially by ESA over time generating upconversion emission as an avalanche. High pump strength and long energy irradiation times are required for luminescence through the PA. The PA process is least observed among all UC systems, including ESA of incident light and intrinsic cross-relaxation. The upconversion efficiency of these three processes is significantly different from each other. ESA is the most inefficient upconversion process. PAs with metastable intermediate levels that can serve as pump energy reservoirs are much more efficient than ESAs, but the PA process relies on pump power and the response to excitation is significantly slower (within a few seconds) due to numerous ESA circuit repetition and cross-relaxation processes. In contrast, the ETU is instantaneous and independent of the pump power and hence has been widely used for highly efficient upconversion (twice as large as ESA) processes over the past decade. Control of size, emission, and excitation spectra of UCNPs is still a challenge (Sun et al. 2014). The color of the UCNP emission is independent of the excitation wavelength around 750– 1000  nm. UCNP luminescence exhibits various colors depending on the peak wavelength that is changed by the dopant of lanthanide (Wang and

Liu 2008; Zhou et al. 2015). The wavelength of the emitted light can be controlled by varying the host, sensitizer, activator combination, and their concentrations. The size of UCNPs affects the quantum yield and can be broadly tuned to 10–100 nm. Despite recent advances in improving UCNP efficiency, the insufficient brightness of UCNPs still limits popular use in biological applications. The low extinction coefficient of UCNPs due to the intrinsic nature of the 4f-4f optical transition in the trivalent lanthanum ion is to be overcome by using the antenna effect of dyes, plasmons, QDs, or other doping elements with strong absorption. As a representative example, we observed that the brightness of silver NPs increased by 30 times compared with that of UCNPs without the nucleus of silver NPs by covering the nucleus with UCNPs (Yin et  al. 2014).

4.3.3.2 Synthesis and Surface Modification of UCNPs The development of a simple synthesis strategy for high-quality UCNPs with controlled composition, crystal phase, morphology, and size is important in tuning chemical and optical properties and exploring potential applications in various fields. Here, thermal decomposition and hydro/solvo-thermal synthesis are described among various synthesis processes. Thermal decomposition was first developed by the Yan group by synthesizing monodisperse LaF3 UCNPs (Zhang et al. 2005) and then later

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generalized as a common method of synthesizing NaYF4 UCNPs. The thermal decomposition method generally uses organometallic compounds as precursors that are decomposed at high temperatures with high-boiling organic solvents with the aid of surfactants. Typical precursors used are metal trifluoroacetate salts and the solvent may be 1-octadecene (ODE). Surfactants may be OA, OM, or TOPO, which generally contain functional capping groups that coordinate metal elements and long hydrocarbon chains. For example, the Capobianco group has prepared NaYF4 UCNPs simultaneously doped with Yb/Er or Yb/Tm by thermal decomposition of metal trifluoroacetate precursors in the presence of OA and ODE (Boyer et al. 2006). The noncoordinating ODE with high boiling point (315  °C) was used as the primary solvent, and OA acted as a passivating ligand to prevent aggregation of UCNP and also as a solvent. By carefully varying experimental parameters such as the type of solvent, concentration of the metal precursor, reaction temperature and time, high-quality UCNPs having a narrow size distribution, good crystallinity, and excellent optical properties from thermal decomposition can be easily obtained. On the other hand, this method uses expensive and air-sensitive metal precursors and produces toxic byproducts. The hydro/solvo-thermal synthesis is a method that is developed to synthesize UCNPs which are more size-controlled and well-dispersed in solution compared to those produced by the thermal decomposition method. Hydro/solvo-thermal synthesis uses a solvent at a pressure and temperature above the critical point to increase the solubility of the solids and the reaction rate between the solids. In a typical hydro/solvo-­ thermal synthesis process, a suitable reaction precursor, a solvent, and a surfactant are mixed and then heated in an autoclave. Surfactants such as polyethyleneimine (PEI), ethylenediaminetetraacetic acid (EDTA), cetyltrimethylammonium bromide (CTAB), and OA provide chelating ability with cations to control the reaction concentration, crystalline phase, size, and morphology, as well as the surface functional groups. A “liquid–

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solid-solution” strategy in which reactions, phase transitions, and separations take place at the interface has been reported (Wang et  al. 2005) and using this strategy, NaYF4 (Jiang et al. 2008; Wang et  al. 2012), NaLaF4 (Wang et  al. 2007), YF3 (Li et al. 2008), and LaOF (Fu et al. 2013) UCNPs having well-controlled crystallinity, size, and morphology were synthesized. This method uses low-cost raw materials, does not require post-heat treatment, and provides an effective way to control particle size and shape, but its disadvantages include requiring an autoclave and that it is impossible to observe the growth process of NPs. The surface properties of UCNPs are important for photoluminescence efficiency as with other luminescent NPs because the surface properties are related to deactivation caused by surface impurities, lattice strain, high phonon energy, ligands, and solvents. The light emission of the dopant ions on the surface tends to be quenched because the protection by the host lattice is not effective. Therefore, an undoped inert crystalline shell around the doped NPs traps the dopant ions in the inner core, effectively suppressing energy transfer to the surface of the NPs and increasing the efficiency of UC luminescence. In order to grow the shell layer on the surface of the core NPs, UCNPs as cores are first synthesized by the above-mentioned method. To ensure epitaxial growth in subsequent growth of the shell and to create a homogeneous interface between the core and the outer shell, the host material of the shell should have a low lattice mismatch with the core material. Epitaxial growth is achieved by gradually depositing a precursor of the shell material onto the core NPs or by depositing the shell monomers from the smaller sacrificial NPs with less stability onto the larger (and hence more stable) core NPs through the Ostwald ripening process. The precisely defined concentration of the precursor or sacrificial NP can be used to control the shell thickness and optical properties. As for QDs, the surface modification of UCNPs not only improves the photostability of NPs but can also serve as a platform for attaching

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biological ligands for a variety of biomedical applications. In order to be used in bioimaging applications, UCNPs should not only have high luminescence efficiency but also have hydrophilicity, and hence, it is necessary to functionalize hydrophilic ligands on the particle surface. Two strategies similar to those described in QDs can be used: (1) to replace the originally existing ligand with a polymer molecule having two functional groups or (2) absorption of additional amphiphilic block copolymers through the hydrophobic attraction between the original ligand and the hydrocarbon chain of the polymer.

4.3.4 Other Luminescent Nanomaterials 4.3.4.1 Europium-Based Materials Lanthanide-based NPs exhibit excellent luminescent and magnetic properties and are attracting interest in biomedical applications. Among the luminescent lanthanide NPs, europium (Eu3+)based NPs exhibiting stable luminescence, long luminescent lifetime, sharp emission peaks with narrow band width, and biocompatibility are excellent candidates in the field of immunoassay and imaging. Herein, the trivalent europium ion (Eu3+)-activated inorganic material, which is known as one of the most important red-emitting luminophores, will be briefly described. Among the lanthanides, the europium element, atomic number 63, is a very interesting chemical species, especially in the trivalent oxidation state. The ternary europium ion (Eu3+) is a typical and well-known red emission activator. The unique red emission is observed not only in the Eu3+ complex but also in the inorganic luminophores doped with Eu3+ ions in the lattice. The emission of Eu3+ is characterized by a sharp emission peak set that provides a distinguishable spectral fingerprint for probing the surrounding symmetry (Tu et  al. 2019). The main radiative transition of Eu3+ is shown in Fig.  4.10 (Syamchand and Sony 2015). Currently, the Eu3+

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ion is considered to be one of the most ideal spectroscopic probes in the nondegenerated ground state 7F0 and the excited state 5D0. The transition from 5D0 to the low J value of 7FJ (J = 0, 1, 2, 3, 4) is the most important in the emission spectrum of Eu3+. Since the number of possible crystal field transitions is relatively small, interpretation of the emission spectrum is easy (Tourani et  al. 2015). For example, the 5D0 →  7F0 transition of Eu3+ is allowed only in symmetry site without inverse symmetry such as Cs, C1, Cn, and Cnv (n = 2, 3, 4, 6), providing information about the environment. Previously, photoluminescence spectroscopy of Eu3+ in various bulk phosphors has been well studied, but Eu3+-activated NPs with a particle size of less than 100  nm can exhibit different optical, electronic, and magnetic properties. For example, several new spectroscopic properties have been reported, ­ such as multiple-site luminescence, extended lifetime, improved quantum efficiency, and abnormal heat treatment (Liu et al. 2007). Most of the various europium-based nanoparticles (Eu-NPs) are prepared by incorporating Eu3+ as a dopant that produces stable luminescence. Eu-NP-based luminescent reporters exhibit signal-to-noise ratios advantageous to serve as efficient probes. The large Stokes shift of Eu-NPs helps to avoid self-absorption of the ligand and reduce the background signal. (However, the term ‘Stokes shift’ meaning the emission and absorption gap here can be somewhat confusing. Please refer to a review article about the concept (Tanner 2013). Long-lived luminescence of Eu-NPs can be collected using time-resolved fluorescence measurements and the narrow emission band of Eu-NPs can improve detection sensitivity (Wang et  al. 2014). The Eu-chelates formed by the coordination of various chelating groups with Eu3+ can function as luminescent agents in lanthanide-based immunoassay and bioimaging. Eu-NPs have demonstrated their efficacy in various bioapplications in vitro and in vivo, and those research examples will be discussed in Chap. 5.

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Fig. 4.10 (a) Energy levels of Eu(III) ion with main radiative transitions. (b) The ligand-Eu(III) energy transfer in Eu-chelates. (Syamchand and Sony 2015)

4.3.4.2 Noble Metal Nanoclusters In general, noble metal nanoclusters having a size of 2 nm or less are known to exhibit fluorescence characteristics. In bulk metals, there is no energy band gap between conduction and valence bands, and electron transitions cannot cause fluorescence. In metallic NPs with a size smaller than the electron mean free path (~20–30 nm for silver and gold), electron motion is restricted and localized plasmonic effects with strong absorption and scattering are exhibited (Link and Ei-Sayed

2003). Plasmonic absorption completely disappears for NPs below 2  nm size at which Mie’s theory is no longer applicable (Alvarez et  al. 1997; Hostetler et al. 1998) (Plasmon properties of gold NPs are described in more detail in Chap. 6). However, when the size of a metal is reduced to a few nanometers or less (also known as nanoclusters), a discontinuous discrete energy level is formed. Therefore, the excitation of the electrons and the relaxation to the ground state by the corresponding light irradiation can cause a fluores-

4  Luminescent Nanomaterials (I)

cence with a mechanism similar to the organic dye molecule. Gold and silver are two types of metals that have been reported to form fluorescent nanoclusters. Due to their low toxicity and ultrafine size, they are suited for bioimaging and biolabeling applications. Copper metal also exhibits fluorescence properties and has been used as a detection probe for hydrogen peroxide detection. Luminescence of nanoclusters is often due to size effects, while structural parameters such as surface ligands, valence states of metal atoms, and the crystallinity of NPs also affect spectra and decay times. Recently, as the use of metal-containing compounds in treatment and diagnosis has increased, metal nanoclusters have been developed based on their luminescent properties as an alternative biomedical probe. Please refer to recent reviews for detailed theory, synthesis, properties, and applications of noble metal nanoclusters (Díez and Ras 2011; Jin et al. 2016; Li et al. 2014; Yuan et al. 2011).

4.3.4.3 Other Carbon-Based QDs Carbon-based quantum dots (CQDs) are an organic material-based carbon nanomaterial exhibiting photoluminescence and semiconductor properties similar to inorganic QDs. CQDs contain carbon as its main element and additional elements such as hydrogen, oxygen, and other elements (e.g., nitrogen) depending on synthetic materials. Although there are slight differences depending on the synthetic process, CQDs exhibit generally good water-solubility because they have many hydrophilic functional groups on the surface such as hydroxyl or carboxylic acid groups. Therefore, unlike the conventional carbon nanomaterials such as carbon nanotubes and graphene, there is no problem regarding the dispersion stability of the solvent. In addition, it is known that CQDs show almost no toxicity regardless of the raw materials used. According to some reports, the emission characteristics of CQDs can be up to 90% of the existing QD light-­ emitting efficiency. In addition, the unique emission characteristics are different in mechanism from those of conventional QDs and remarkably, change according to the external environment. Thus, many applications as bioenvironmental

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sensors have been studied. Carbon-based QDs are discussed in detail in Chap. 11. Acknowledgments This work was supported by the National Research Foundation of Korea  (NRF) grant funded by the Korea government  (MSIT) (No. 2019R1G1A1100734). Conflicts of Interest  The authors declare no conflicts of interest.

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5

Luminescent Nanomaterials (II) Hyejin Chang, Jaehi Kim, Sang Hun Lee, Won-­ Yeop Rho, Jong Hun Lee, Dae Hong Jeong, and Bong-Hyun Jun

Abstract

In this review, we focus on sensing techniques and biological applications of various luminescent nanoparticles including quantum dot (QD), up-conversion nanoparticles (UCNPs) following the previous chapter. Fluorescent phenomena can be regulated or shifted by interaction between biological targets and luminescence probes depending on their distance, which is so-called Fӧrster resonance energy transfer (FRET). QD-based FRET technique, which has been widely applied as a bioanalytical tool, is described. We discuss time-resolved fluorescence (TRF) imaging and flow cytometry technique, using photoluminescent nanoparticles with unique properties for effectively improving selectivity and sensitivity. Based on these techniques, bioanalytical and biomedical application, bioimag-

ing with QD, UCNPs, and Euripium-activated luminescent nanoprobes are covered. Combination of optical property of these luminescent nanoparticles with special functions such as drug delivery, photothermal therapy (PTT), and photodynamic therapy (PDT) is also described. Keywords

Quantum dot (QD) · Up-conversion nanoparticles (UCNPs) · Fӧrster resonance energy transfer (FRET) · Time-resolved fluorescence (TRF) · Drug delivery · Photothermal therapy (PTT) · Photodynamic therapy (PDT)

Hyejin Chang and Jaehi Kim contributed equally to this work. H. Chang Division of Science Education, Kangwon National University, Chuncheon, Republic of Korea J. Kim · B.-H. Jun (*) Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea e-mail: [email protected] S. H. Lee Department of Chemical and Biological Engineering, Hanbat National University, Daejeon, Republic of Korea

W.-Y. Rho School of International Engineering and Science, Jeonbuk National University, Jeonju, Republic of Korea J. H. Lee Department of Food Science and Biotechnology, Gachon University, Seongnam, Republic of Korea D. H. Jeong Department of Chemistry Education, Seoul National University, Seoul, Republic of Korea

© Springer Nature Singapore Pte Ltd. 2021 B.-H. Jun (ed.), Nanotechnology for Bioapplications, Advances in Experimental Medicine and Biology 1309, https://doi.org/10.1007/978-981-33-6158-4_5

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5.1

Sensing Mechanisms and Techniques

widely applied in many fields, such as single-­ molecule experiments, molecular motors, biosensors, and DNA mechanical movements. FRET 5.1.1 Förster Resonance Energy is also called a “spectroscopic ruler” because of Transfer (FRET) its inherent convenience. A Jablonski diagram showing the transition Resonance energy transfer (RET) refers to the between donor emission and acceptor absorbance through-space dipolar coupling mechanism of in fluorescence resonance energy transfer is prethe energy transfer (ET) between the excited-­ sented in Fig.  5.1a. As shown in Fig.  5.1a, the state donor (D*) and the ground-state acceptor donor group (D) is excited by a photon and then (A), where the product of the ET involved with- relaxed to the lowest excited singlet state S1. If out photons or physical contact is the ground-­ the suitable acceptor group is not too far away, state donor (D) and the excited-state acceptor the nonradiative energy released when the elec(A*) (Hildebrandt et al. 2017). trons return to the ground state S0 can be used to excite the acceptor group. This process is called   D A  DA “resonance” and does not require collisions and The best-known RET mechanism is Fӧrster does not involve heat generation. Resonance resonance energy transfer (FRET), described by mechanisms are associated with Coulomb interFörster in the 1940s (Förster 1948), and this actions between electrons, only the superposition transfer of the electronic excited state occurs in of the spectrum is necessary. When energy transthe dipole–dipole interaction between the donor fer occurs, the acceptor molecule disappears the and the acceptor’s electronic states. FRET is donor molecular fluorescence, and the excited based on the concept of treating the excited acceptor returns to the ground state, releasing fluorophore as a vibrating dipole capable of increased or sensitized photons unless another energy exchange with a second dipole having a quenching state is present. The resulting sensisimilar resonance frequency. That is, the transfer tized fluorescence emission has properties simioccurs when the vibration of optically induced lar to the emission spectrum of the acceptor. FRET does not require physical contact, but electromagnetic interference on the donor resonates with the electron energy gap of the the donor and acceptor should be close enough to acceptor. Unlike radiant energy transfer, it does interact (typically 10 nm or less). The degree of not include the emission and re-absorption of interaction depends on the magnitude of the transition dipole interaction, which depends on the photons. FRET, which relies heavily on distance, has size of the donor and acceptor transition matrix become a widely used tool for measuring the elements and the alignment and separation of the dynamic activity of biological molecules on the dipoles. In other words, in addition to the proxnanoscale. One common application is to attach imity between the donor and the acceptor, the appropriate donor-acceptor groups to large mol- transition dipole must have a favorable orientaecules to measure the distance between two posi- tion with respect to each other, there must be a tions of interest in large molecules, generally resonant transition between the donor and the biological macromolecules. If the large molecule acceptor, and the ET must compete with other contains only one donor and one acceptor group, paths to relax the donor to ground. These distance the distance between the donor and the acceptor conditions are important design considerations can easily be measured if there are no morpho- for the donor–accceptor configuration. Förster’s logical changes within this process. It is also pos- theory explained the energy transfer efficiency sible to measure the dynamic activity between (ET) for these factors (Boens et al. 2007; Lakowicz two sites on this large molecule, such as protein 2013). Energy transfer efficiency ET is a measure interactions, when the molecule has a large mor- of the fraction of photons absorbed by a donor phological change. Today, this technique is

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Fig. 5.1 (a) Schematic diagram of Förster Resonance Energy Transfer. (b) Absorption (solid lines) and fluorescence (dashed lines) spectra of donor– acceptor pairs exhibiting FRET. For efficient energy transfer, the absorption spectrum of the acceptor and emission spectrum of the donor should overlap (shaded area). This process also depends on the distance between two FRET partners (Yao et al. 2014)

delivered to a acceptor, which can be changed by several factors. ET 

kFRET  kFRET  kDR  kDNR

1 6

 r  1    R0 

r is the distance separating the donor and acceptor chromophores, and R0 is the Förster critical distance. The energy transfer at this distance (R0) between the donor and acceptor is 50% efficient, and R0 typically ranges from 2 to 7 nm for most resonant energy transfers measurements. At this separation radius, half of the donor excitation energy is delivered to the acceptor via resonance energy transfer and the other half is dissipated

through a combination of all other available processes, including fluorescence emission. To improve FRET efficiency, donor groups must be capable of absorbing and emitting photons. That is, the donor group should have high absorption coefficient and high quantum yield. Also, the more overlap between the donor’s emission spectrum and the acceptor’s absorption spectrum, the better the donor can transfer energy to the acceptor (Fig. 5.1b). The resonance energy transfer mechanism is also influenced by the orientation of the release transition dipole of the donor and the absorption dipole of the acceptor. The orientation parameter κ2 provides a quantitative value of the interaction between two dipole moments, which accounts for the relative direction in space between the two transition dipoles.

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κ2 may theoretically be a value from 0 (when the dipoles are perpendicular to each other) to 4 (when the dipoles are on the same line). κ2 equals 1 when these two transition dipoles are parallel. For free-rotating donors and acceptor groups, the mean κ2 is treated as 2/3. Longer R0 can increase FRET efficiency. R0 is a function of the index of refraction n of medium, the quantum yield ΦD of the donor chromophore, the spectral overlap J (λ) of the donor and acceptor, and the factor κ2. In a typical FRET experiment, an organic molecule-based donor or acceptor was used, and the structure was moved from the excited state by the light source to the ground state through the FRET process and did not emit light. A typical example of a classic FRET sensor is single-­ stranded DNA, with molecular beacons containing donor and acceptor molecules at each end (Wang et  al. 2009a). The loop becomes spread out when the target DNA (or protein) exists, which results in fluorescence while the distance between donor and acceptor is farther away. However, since such materials are based on fluorescent organic materials, there may be limitations in utilization due to general disadvantages of the fluorescent organic materials, that is, photobleaching or lack of diversity of fluorescent colors. While replacing this, the luminescent nanoparticles can act as donors by absorbing light energy and can transfer energy to nearby surrounding species or species that interact with the surface of the nanoparticles. In this process, emission of luminescent nanoparticles is lost, while acceptors (e.g., fluorescent species) enhance the emission. The degree of change can be correlated to generate quantitative analytical information of the acceptor species. In FRET application, bioassays and bioprobes using QDs are the most developed and widely used approaches to integrate QDs into bioanalyses. Some groups have attempted to use QD conjugated with biomolecules (e.g., antibodies) in FRET- based assays and biosensors. QD for FRET applications was first reported in 1996, and since then many studies have shown that QD-based FRET can be used in biological systems in which QDs can be energy donors or acceptors. In general, QD acts as an energy donor

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and biomolecules are represented by acceptor dyes. Biometric events on the QD surface are used to induce the association or dissociation of acceptors or to alter the QD–acceptor separation distance. The resulting modulation of the FRET efficiency provides the analytical signal. When detecting the targets, the aptamer’s binding-­ induced conformational changes alter the beacon’s donor–acceptor distance, resulting in a change of FRET. In addition, new materials such as gold nanoparticles were used in the QD-FRET system and many new possibilities opened up for FRET analysis (Yang et al. 2009; Yao et al. 2014). Specific examples are discussed in Sect. 5.2. Similar to FRET, chemiluminescence-­ resonance energy transfer (CRET) and bioluminescence-­ resonance energy transfer (BRET) generate excited-state donors through chemical reactions. This includes nonradiative (dipole-dipole) transitions of energy from chemiluminescent donors to the appropriate acceptor molecules in the CRET (or BRET) system. Non-­radiative energy transfers in CRET and BRET require the donor/acceptor to be in close proximity. The difference between CRET and BRET is that biochemical reactions are the basis of the latter. The advantages of BRET and CRET over FRET include the following: (i) CRET and BRET experiments are very simple. When the luminescent donor transfers energy to the QD non-­radiatively, a self-illuminated QD-conjugate is created without the need for external excitation light. (ii) The background associated with CRET and BRET is very low. Without optical excitation, auto-fluorescence, strong scattering of source light, and spurious acceptor emission from direct excitation can be avoided. (iii) CRET and BRET can eliminate the difficulties resulted from use of QD as an acceptor fluorophore. QDs act as energy acceptors in CRET and BRET due to their strong absorption and excellent spectral separation between donor and acceptor emission. However, CRET has its own limitations that an oxidizing agent (e.g., H2O2) in the CRET system could quench the fluorescence of QD (Ma and Su 2011).

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5.1.2 Time-Resolved Fluorescence (TRF) Time-resolved fluorescence (TRF) can use fluorescence lifetime to obtain information about the molecule or its environment because the fluorescence lifetime is characteristic of each fluorescent molecule and is also affected by the chemical composition of the environment. However, using the term time-resolved fluorescence in the life sciences can be used in two different ways: (1) monitoring technique as a function of time after the fluorescence of the sample is excited by a light pulse; (2) a method of using fluorophores with unique fluorescence lifetimes to avoid interference by molecules with very different fluorescence lifetimes or molecules with other factors (most importantly, excitation light). The former is used to monitor molecular interactions and movements occurring in the picosecond or nanosecond scale time range and is particularly useful for the analysis of biomolecular structure and dynamics. Biological species in condensed phase (solution) are always surrounded by other molecules, such as proteins, water, lipids, etc.; therefore, in addition to the properties of the molecules themselves, there are several more processes that affect the dynamics of the excited state, for example, transfer of excitation energy to neighboring molecules, solvation (regrouping of solvent molecules to accommodate the excited-state electron arrange-

Fig. 5.2  Schematic of fluorescence decay upon pulsed excitation. Fluorophores are excited using a short pulse of light. The emitted fluorescence is measured in a time-­resolved manner, forming the basis for time-domain lifetime measurement (Jain et al. 2009)

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ment of molecules), and between photo-inductive intermolecular reactions such as proton or electron transfer from excited molecules to solvents or surrounding molecules and the like. All of these processes affect the state of the molecule and thus the fluorescence spectrum, so these effects can be studied using time-resolved fluorescence. For biological sensing or imaging applications, there are plenty of examples in the second way. The time-resolved fluorescence (TRF) measurement is very similar to the fluorescence intensity (FI) measurement, the only difference being the timing of the excitation and measuring process. When measuring FI, the excitation and emission processes take place simultaneously, and the light generated from the excitation is measured during the emission of the sample. Although the emission system is very efficient at removing excitation light before it reaches the detector, it always has a fairly high background signal in FI measurements because of the strong excitation light compared to the emission light. TRF provides a solution to this problem. When a mixture of fluorescent compounds is excited with short pulses of light from a laser or flash lamp, the excited molecules emit short- or long-term fluorescence (Fig. 5.2). Both types of fluorescence decay follow the exponential curve, but the short-term fluorescence (background) dissipates to zero at 1100  nm) facilitates micrometer-­ level resolution of both peripheral vascular structures and tumor angiogenesis with respect to cancer growth and metastasis (Dong et al. 2013a; Li et al. 2014). In addition, NIR-II imaging with Ag2S QD significantly improved clarity and penetration depth for visualizing lymphatic vessels and lymph nodes as compared to ICG, the clinical standard contrast medium for induced growth lymph node dissection.

PbS QDs are one of the best NIR-II fluorophores for biological imaging due to their very high quantum yield and long emission wavelengths (Nakane et  al. 2013). Applicability to human patients is very low at present, but various biological coatings of PbS QD have been developed for tissue preclinical animal imaging. Direct encapsulation methods make PbS QD vulnerable to fluorescent quenching when exposed to water. However, the PbS-CdS core-shell ligand coating structure that shields the PbS core maintains a high quantum yield of 17%, resulting in a very bright inorganic NIR-II contrast agent. Changing the QD core diameter shifts the emission wavelength to the NIR-II region in the range of 1100–

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1300  nm (Tsukasaki et  al. 2014b). These PbS-CdS contrast agents can image the blood vascular structures of the brain with high fidelity, and in mouse hind limb lymphatic structures, the SBR ratio increases nearly tenfold while imaging at progressively longer detection wavelengths from 720 to 1500 nm (Tsukasaki et al. 2014a). Rare earth-doped nanoparticles (RE NPs) are another recently developed class of NIR-II fluorophores that have demonstrated excellent emission wavelength control through doping with other RE metal ions (Naczynski et  al. 2013; Rocha et  al. 2014). Adjusting the atomic composition of NPs can produce narrow, sharp emission peaks across the NIR-II region. Similar to other inorganic fluorophores, RE NPs exhibit minimal photobleaching under systemic imaging conditions, indicating the possibility of monitoring heterologous biological processes through long-­ term multicolor imaging. Re NPs with discontinuous emission peaks at 1185 or 1525 nm variable activator dopants allowed for in vivo multicolor NIR-II imaging of xenograft tumors (Naczynski et al. 2013).

5.4

Therapeutics Cooperated with Luminescent Nanoparticles

One of the very interesting trends at the moment is the combination of drug or gene delivery and fluorescence imaging. They rely on the release of diagnostically active species. It also includes fluorescence imaging combined with photodynamic therapy (PDT) or photothermal therapy (PTD), which may or may not be released in NP. These methods will be discussed mainly in terms of nanomaterials in the subsequent sections.

5.4.1 Drug Delivery Drug delivery systems can enhance the efficacy of various pharmaceutical payloads and improve drug solubility, stability, biodistribution, and pharmacokinetics. To build a UCNP-based system for drug delivery, NaYF4:Yb3+/Er3+ UCNPs can create a hydrophobic pocket on the particle surface to partition the anticancer drug molecule

of doxorubicin (DOX). The release of DOX was controlled by changing the pH of the solution, which has a higher rate at decreasing pH.  The results are advantageous for controlled drug release in tumor cells (Naczynski et  al. 2013). Recently, similar methods have been reported in NaYF4:Yb3+/Er3+ and iron oxide nanocomposites, which are simultaneous optical imaging and self-­ targeting drugs (Xu et al. 2011a). The mesopores of the silica shell have a high surface area and a large pore volume to accommodate a large amount of drugs. Therefore, a method of depositing a drug in the pores of a mesoporous silica shell coated on the surface of the UCNP has also been reported. For example, ibuprofen was added to mesoporous silica-coated β-NaYF4: Yb3+/Er3+ UCNP fibers and prepared by an electrospinning process (Hou et al. 2011). Ibuprofen loading capacity was further improved by the same researchers who developed mesoporous silica-­ coated α-NaYF4:Yb3+/ Er3+ nanospheres through a simple two-step solgel modification process. Ibuprofen loading was controlled by varying the thickness of the mesoporous SiO2 layer (Kang et  al. 2011). The Qu groups impressively showed that lanthanide-modified hollow mesoporous nanoparticles can be applied to simultaneous imaging and delivery of genes and drugs (Li et al. 2013). Drugs can also be loaded into hollow UCNPs with mesoporous shells. The hollow structure allows for appropriate levels of drug loading while maintaining UC PL imaging capabilities. In this regard, Lin et al. reported for monodisperse core/shell structured up-­converting Yb(OH)-CO3@YbPO4:Er3+ hollow spheres as a drug carrier for anticancer agents of DOX (Xu et  al. 2011b). DOX were transported into cells by core/shell hollow sphere carriers, released into cells after endocytosis, and the DOX-loaded spheres showed much greater cytotoxicity than free DOX.  Similarly, Y2O3: Yb3+/ Er3+ hollow nanospheres were synthesized to deliver DOX to HeLa cells, which allows highcontrast cell and tissue imaging without damaging by radiation (Dong et al. 2013b). Light-controlled release processes in vitro and in vivo is particularly important in the delivery of various molecules such as proteins, peptides, nucleic acids, amino acids, and drugs to desired cell/tissue/body sites. Photosensitive functional

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Fig. 5.12  Experimental design for uncaging D -luciferin and subsequent bioluminescence through the use of photocaged core-shell upconversion nanoparticles (Yang et al. 2012)

groups are generally involved in the delivery system, which can be cleaved upon UV irradiation. Based on this simple idea, various systems were developed. Zhang et al. have been reported in NIRto-UV-UCNPs for activating optical cage nucleic acids in much deeper tissues compared to conventional systems (Jayakumar et al. 2012). Using optical cage GFP-loaded UCNPs and NIR lasers, drug release process was also activated under the skin of a mouse. Xing et al. reported a system for the controlled uncaging of D-luciferin in D-luciferinconjugated NIR-UV UCNPs (Fig.  5.12) (Yang et  al. 2012). The released D-luciferin effectively provided enhanced fluorescence and bioluminescence signals in vitro and in vivo, resulting in deep light penetration and low cell damage.

5.4.2 Photothermal Therapy Photothermal therapy (PTT) uses light absorbers to induce heat removal of cancer cells to generate heat from light absorption. Recently, PTT has been increasingly recognized as an alternative to classic cancer therapies such as surgery, radiation therapy, and chemotherapy. In this application, various nanomaterials with high absorbance have been very successful. Because of the low absorption coefficient of lanthanide ions, UCNPs generally have limited ability to convert light directly into heat. However, UCNP can be easily bound to plasmonic nanoparticles that has a strong extinction coefficient and are actively used in the treatment of PTT in diseases.

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a

Au seeds IONPs PEG FA

b

c

d

500 nm

500 nm

500 nm

e

f

g

5 nm

100 nm

100 nm

100 nm

Fig. 5.13 MFNP synthesis and characterization. (a) Strategy for MFNP synthesis and functionalization. (b–d) SEM images of the UCNPs (b), UCNP–IONP nanocomposites (c), and UCNP-IONP-Au MFNPs (d). (e–g) TEM

images of UCNPs (e), the inset shows the HRTEM image of a MFNP and the indicated d spacing is 0.52  nm; UCNP–IONP nanocomposites (f), and MFNPs (g) (Cheng et al. 2011)

When excited by a NIR laser, the UCNP is heated. It is not desirable for bioimaging and an excitation wavelength of 915  nm was ­recommended to reduce the laser-induced heating of NaYbF4:Tm3+/Er3+/Ho3+ type UCNPs (Zhan et  al. 2011). However, heating may be desirable because local heating can be used in cancer treatment. In a typical application, oleatecapped UCNPs were first coated with a silica shell and then with a silica layer doped with a blue arbocyanine dye. Silica shell formation allowed also enhanced stability in aqueous solution. The light emission of the UCNP is absorbed by the dye, causing local heating up to 21  °C, which results in cell destruction.

Multifunctional nanoparticles have been reported to combine UC luminescence, magnetic properties, and PTT function (Chen et al. 2014). The ultrasmall superparamagnetic Fe3O4 nanoparticles (IONPs) produced using layered self-assembly were coated on a 200  nm size NaYF4:Yb3+/Er3+ UCNP surface, where a thin gold shell was formed by seed-mediated reduction growth (Fig. 5.13) (Cheng et al. 2011). The IONP layer between UCNP and the gold shell not only provided magnetic properties but also reduced the luminescence quenching effect of gold nanostructures to UCNP. In addition, double targeted PTT ablation of cancer cells was successful at the cell culture level. There was a similar report that the core-shell structured

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silver-coated β-NaYF4:Yb3+/Er3+ nanoparticles were used in PTT experiments under excitation of a continuous 980 nm diode laser (Dong et al. 2011). PTT was applied in vitro to HepG2 cells of human liver cancer and BCap-37 cells of human breast cancer, with optimal mortality approaching 95% with a power density of 1.5 W/ cm2, which was much lower power than that reported for gold nanoshells and gold nanorods.

5.4.3 Photodynamic Therapy Photodynamic therapy (PDT) is a clinical treatment that kills tumors by generating single oxygen (1O2) using photo-triggered chemical drugs (photosensitizers). Typical PDT treatments include three components: photosensitizer (PS), a light source, and oxygen in the tissue at the disease site. At the appropriate photoexcitation (typically within the visible range), the PS can be excited from the bottom singlet state to the excited singlet state, and after going through intersystem crossing to triplet state with a longer lifetime, this excited PS reacts with near oxygen molecules (3O2) to produce highly cytotoxic 1O2. PDT has been used to treat prostate, lung, head and neck, or skin cancer. However, conventional PDTs are limited by the penetration depth of visible light required for activation. NIR range with “optical transparency” (750–1100  nm) of tissue can penetrate deeper into tissue than visible light because of its lower absorbance. Semiconductor QDs have great potential for PDT applications. QDs can have unique optical and emission characteristics that can be precisely adjusted from the UV to the NIR region depending on size (range of 1–6 nm) and configuration. The surface of the QDs can be modified and functionalized to be water-soluble and biocompatible. The large transition dipole moment is also a powerful light absorber ideal for PDT applications. QDs can act as energy donors to transfer energy to cellular molecules such as triplet oxygen, reducing equivalents or pigments, potentially inducing the production of reactive oxygen species (ROS), leading to cell death. Samia et  al. reported that CdSe QD could be a

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potential PS by itself and produce 1O2, and the quantum yield of CdSe-generated 1O2 was rather low (40–60% by traditional PS) (Samia et  al. 2003). However, the authors noted that more efficient (77%) energy transfer through the FRET mechanism can occur between QD and Pc4, a known silicon phthalocyanine PDT photosensitizer, by excitation at 488 nm because of the close proximity between the two structures. Pc4 is excited by CdSe-QD emission at 568  nm to enable the use of excitation wavelengths that the PS alone does not absorb in normal conditions. The insolubility of the conjugate in water has hampered its application in biological systems. Shi et al. synthesized water-soluble QDs electrostatically attached to meso-tetra (4-­sulfunatophenyl) porphyrin (TSPP) (Tsay et al. 2007). Upon excitation at 355  nm which photosensitizer does not absorb, the QD-PS hybrid nanocomposite was found to produce 1O2. However, the main toxicity was the potential toxicity of the synthetic uncapped QDs. Tsay et  al. bound two different PS, Rose Bengal (RB) and Ce6 to peptides, which in turn were attached to CdSe/CdS/ZnS QD (Tsay et al. 2007). Although the QD itself did not generate 1O2 and the quantum yield of 1O2 was relatively low, both in direct excitation of the photosensitizer or indirectly through the FRET mechanism, the advantage of this technique is that multiple PS molecules can be coupled to the QD to improve FRET efficiency. Hsu et al. recently demonstrated that self-­illuminated QDs can serve as a versatile light source for PS activation and overcome the limitations of light penetration into PDT (Hsu et al. 2013). They prepared a Renilla Luciferasefixed QD (QD-RLuc8) conjugate. After addition of coelenterazine (substrate of RLuc8), energy is released from RLuc8 and transferred to QD via BRET, resulting in self-illumination of the QD-RLuc8 conjugate at 655 nm. This bioluminescent photon emitted from the QD-RLuc8 conjugate successfully produced ROS to stimulate Foscan-loaded micelles and to achieve ~50% cell death in vitro and delayed tumor growth in vivo. Importantly, UCNP can efficiently convert deeply penetrating NIR radiation into visible range light, stimulating photosensitizers to produce cytotoxic 1O2. Chatterjee et al. demonstrated

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Fig. 5.14 (a) Schematic diagram for the synthesis of ZnPc-loaded SOC-UCNPs. (b) TEM images of the prepared nanoparticles (ZnPc-SOC-UCNPs). (c) Viability of

MCF-7 cells incubated with SOC-UCNPs and ZnPc-­ SOC-­UCNPs for different irradiation time intervals (0, 5, 10 and 15 min) (Cui et al. 2012)

the ability of ZnPC-loaded PEI-modified NaYF4:Yb3+, Er3+ nanocrystals to disrupt HT29 human colon adenocarcinoma cells (Chatterjee and Yong 2008). In this study, nonpolar ZnPC was physically adsorbed on the highly polarized UCNP core resulting in a high PS encapsulation efficiency of 97%. In addition, the effect of these folic acid conjugated nanostructures was demonstrated in  vitro on the selective targeting and destruction of folate receptor (FR) overexpressing HT29 cells under NIR irradiation at 980 nm for 5  min. Chitosan-capped UCNP loaded with PS has shown promising results as PDT agonists (Cui et  al. 2012; Zhou et  al. 2012). Cui et  al. reported that PS-loaded ZnPC achieved one of the highest PS payloads of 10.8% through hydrophobic interactions (Fig. 5.14) (Cui et al. 2012). After 24  h of incubation, 400  mg/mL chitosan-­ capped ZnPC-loaded UCNPs were used for

in vitro PDT to kill about 80% of MCF-7 breast cancer cells through apoptosis at 980 nm excitation of the output of 0.6 W for 10 min. To realize the need for controllable and stable loading of PS, Zhang group developed a method of uniform surface coating of photocatalyst TiO2 on individual UCNPs to achieve well-defined core-shell structured nanoparticles (Idris et  al. 2014). The photocatalyst TiO2 was stably and uniformly coated on the surface of the silica-­ coated UCNP having a positively charged amino group by using the modified Stӧber method according to the electrostatic induction adhesion of the Ti precursor. This method has the advantage of stable PS attachment with minimal batch variation, which prevents the possibility of PS leaks and ensures consistent PDT results. After 980  nm NIR irradiation, photo-induced TiO2 resulted in the production of one or more types of

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ROS including hydroxyl radicals, peroxide anions, and hydrogen peroxide, and the potential for induction of cell death was successfully demonstrated in  vitro. In subsequent studies, they surface modified core-shell TiO2-UCNP with ­ polyethylene glycol (PEG) to confer stability and highly reproducible PS loading, making them more advantageous for biological applications (Lucky et al. 2015). PEGylated TiO2-UCNP was found to be biocompatible and blood-compatible and demonstrated capacity-dependent ROS production and corresponding cell-killing capacity in vitro. The biggest challenge in the application of UCNP to PDT in vivo was that the particles were delivered and selectively accumulated in the tumor to prevent systemic toxicity. Most of the early studies reported the delivery of UCNP via intratumoral injection. This route of administration was initially chosen as the synthesized particles were agglomerated in a large and physiological environment. In addition, specificity and selectivity of UCNPs for tumors were limited, most of which accumulate in the organs of mononuclear phagocytic system, causing toxicity. Therefore, UCNP was administered intratumorally to increase PDT efficiency and reduce side effects. Wang et  al. demonstrated in  vivo PDT in murine breast tumors using chlorin e6 (Ce6)-loaded PEGylated UCNPs (Ce6-PEG-­ UCNP) (Wang et al. 2011). PEGylation improves stability as well as reduces the toxicity of Ce6-­ PEG-­UCNP, where the hydrophobic Ce6 molecules were simply adsorbed to the oleic acid layer of PEGylated UCNP to achieve very close binding of Ce6. This strategy has the additional advantage of providing higher resonance energy transfer between photon donor and acceptor molecules due to proximity. The particle size (~30  nm) was the smallest compared to all UCNPs previously reported for PDT. In vivo PDT (1 min interval after 1 min of each irradiation) with a 980 nm laser irradiated for 30 min at a power density of 0.5 W cm2 showed that 70% of tumors disappeared without regrowth for 2  weeks. However, the particles were administered to the animal intratumor rather than intrave-

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nously. Moreover, retention of UCNP was enhanced in organs such as the spleen and liver 15  days after injection, indicating an apparent translocation of UCNP from the injection site via lymph circulation. The first study demonstrating systemic administration of UCNP (42  nm) carrying Ce6 was reported by Park et al. (2012). Ce6 was attached through physical adsorption to the phospholipid layer and covalent conjugation to amino groups on the UCNP to achieve the stable and highest PS payload (>1000 Ce6 molecules/UCNP). Due to the presence of the biocompatible polymer PEG, these UCNPs carrying Ce6 had better circulation half-lives than free-Ce6. Particles accumulate mainly in the liver and spleen, but decrease significantly on day 7. Tumors were clearly observed in magnetic resonance images as well as up-­ conversion images, and after in vivo PDT at 980  nm, tumor size decreased sixfold in the treated groups, clearly demonstrating their potential as PDT agonists.

5.5

Conclusions and Outlook

The field of fluorescence or PL-based bioanalysis and imaging has made impressive advances in materials science, spectroscopy, and microscopy. PL imaging plays an important role in biomedical research and is very useful for early detection, screening, and image-guided therapy of life-­ threatening diseases. In particular, luminescent nanomaterials have been widely used due to the development of nanotechnology in this field of analysis and imaging. This chapter introduced recent advances in the use of nanomaterials such as semiconductor QDs and UCNPs in chemical and biodetection. In addition, insights into various detection methods based on nanoparticles can provide valuable information for researchers. Key issues in fluorescence imaging, particularly with regard to design considerations for nanomaterial bioapplication, include autofluorescence, quenching/photobleaching, and penetration depth. QDs have excellent photostability and

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help solve the photobleaching problem, in particular red-shift NIR emission QDs substantially improve penetration problems, at least for small animal imaging. However, autofluorescence and especially penetration depth make fluorescence highly limited in human imaging except in very specific applications such as fluorescence-guided surgery and endoscopy. When considering clinical applications, QD nanomaterial compositions should be carefully considered, as general imaging requires no toxic and bright particles with good cell permeability if necessary. Lanthanide-­ doped UCNP as next-generation fluorophores have attracted considerable interest in the biological field. The inherent advantages over organic fluorophores and QDs make them more likely to be used as fluorescent labels. For example, UCNP has high quantum yield, narrow emission peak, large stoke shift, good chemical stability, and low toxicity. This striking advantage makes UCNP a promising candidate as a donor for FRET-based biological detection. In addition, unlike QD, which requires UV radiation, UCNP can convert NIR (long wave) radiation into visible light (short wave fluorescence) through a nonlinear optical process. All major radiation modalities as imaging techniques including MRI, CT, and PET are expected to provide anatomical, physiological, molecular, and genomic information for accurate disease diagnosis, prediction of therapeutic responses, and development of highly specific and sensitive drugs and imaging agents. However, none of the current imaging methods used in humans provide comprehensive medical imaging. To take advantage of the various imaging methods, multimode imaging has become an attractive strategy for in vivo research. NPs with highly structural precision are required for use in various multimodal imaging. Such NPs exhibiting bright luminescence are expected to be applicable to a second type of imaging, for example, MRI.  For achieving this, additional functions (functional materials) should be incorporated into the core or shell of the NP without compromising other characteristics, including size and cell viability.

NP-based sensing techniques are now expanding to an application for “theranostics” that can be diagnose and treat simultaneously. Those multifunctional nanoparticles are covered with a porous shell that can be loaded with drugs, genes, or photodynamics or photothermal agents. When NP reaches the site of action, it can release drugs and genes. One of the important factors for NPs to be successfully used as sensors is a wide and better understanding and control of the phenomena on NP’s surface and its surroundings. NPs for use in optical imaging in combination with therapeutic regimens also require high sophistication. Appropriate strategies are also needed if the NP surfaces are modified  for utilizing as sensors, since NPs with modified surfaces differ in physical properties from NPs with unmodified surfaces. In addition, there is a need for a full understanding of the toxicity and biocompatibility for nanoparticles. Acknowledgments This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(MSIT) (No. 2019R1G1A1100734). Conflicts of Interest  The authors declare no conflict of interest.

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6

Plasmonic Nanoparticles: Basics to Applications (I) Hyejin Chang, Won-Yeop Rho, Byung Sung Son, Jaehi Kim, Sang Hun Lee, Dae Hong Jeong, and Bong-Hyun Jun

Abstract

This review presents the main characteristics of metal nanoparticles (NPs), especially consisting of  noble metal such as Au and Ag, and brief information on their synthesis methods. The physical and chemical properties of the metal NPs are described, with a particular focus on the optically variable properties (surface plasmon resonance based properties) and surface-enhanced Raman scattering of plasmonic materials. In addition, this chapter covers ways to achieve advances by utilizing their properties in the biological studies and medical fields (such as imaging, diagnostics, and therapeutics). These descriptions will help researchers new to nanomaterials for biomedical diagnosis to understand easily the related knowledge and also will help researchers

Hyejin Chang and Won-Yeop Rho contributed equally to this work.

involved in the biomedical field to learn about the latest research trends. Keywords

Surface plasmon resonance (SPR) · Surface-­ enhanced fluorescence (SEF) · Surface-­ enhanced Raman scattering (SERS) · Plasmon resonance energy transfer (PRET)

6.1

Introduction

Nanoparticles (NPs) are considered to be the most valuable and important functional materials in the field of material science. Noble metal NPs comprise one of the most actively studied fields in nanotechnology, with carbon materials, magnetic materials, and quantum dots. The interesting ability of metal NPs to interact effectively with electromagnetic radiation makes them suitable for many biomedical applications, including

H. Chang Division of Science Education, Kangwon National University, Chuncheon, Republic of Korea W.-Y. Rho School of International Engineering and Science, Jeonbuk National University, Jeonju, Republic of Korea B. S. Son · J. Kim · B.-H. Jun (*) Department of Bioscience and Biotechnology, Konkuk University, Seoul, South Korea e-mail: [email protected]

S. H. Lee Department of Chemical and Biological Engineering, Hanbat National University, Daejeon, Republic of Korea D. H. Jeong Department of Chemistry Education, Seoul National University, Seoul, Republic of Korea

© Springer Nature Singapore Pte Ltd. 2021 B.-H. Jun (ed.), Nanotechnology for Bioapplications, Advances in Experimental Medicine and Biology 1309, https://doi.org/10.1007/978-981-33-6158-4_6

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diagnosis, treatment, and evaluation of disease. The interaction with electromagnetic radiation causes a unique optical phenomenon, called surface plasmon resonance (SPR), usually in the ultraviolet (UV), visible, and near-infrared (NIR) spectrum region. Under the irradiation of light, the electrons in metal NPs collectively oscillate at a resonant frequency relative to the lattice of positive ions. This results in the strong absorption or scattering of light by the metal NPs. The SPR frequency is sensitive to subtle changes in physicochemical conditions such as distance between NPs and particle size and shape. In some cases, the shift of the plasmon is so great that the color change can be observed visually, which provides the great advantage that expensive equipment and complicated equipment are not necessary. Biomedical applications generally utilize gold nanoparticles (Au NPs) and silver nanoparticles (Ag NPs), which exhibit superior plasmonic properties. Also these NPs are widely used because their size and shape can be controlled easily during fabrication. Furthermore, the surface chemistry and modification of Au and Ag NPs are well known, enabling them to be used as a sensor based on their SPR band changes. In addition to colorimetric sensing, the intensive absorption and scattering of light due to SPR can be coupled to interesting applications of the metal NPs: surface-enhanced fluorescence (SEF) and surface-enhanced Raman scattering (SERS). Especially, the SERS technique has received great attention in the past two decades because of its unique virtues such as high sensitivity, multiplexing capacity, and photostability. In addition, SERS can be realized with an optical microscope, and deep tissue penetration and analysis can be performed when the NIR region is used as the excitation light source. The history of utilizing metal NPs is introduced briefly here, and then there is a discussion about representative particle synthesis and the unique properties and applications of NPs. Historically, Au  NPs have been used and studied for a long time. They were used for medicinal purposes in ancient China and Egypt

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(around 2500  BCE) according to the earliest records. After that, several ancient cultures utilized Au-based nanomaterials for the treatment of several diseases. In the Middle Ages in Europe, the alchemist Paracelsus and his contemporaries made soluble “Aurum Potabile” (Latin, Potable Gold) and studied its therapeutic properties. Au NPs were also applied to Chinese ceramic porcelain as red-colored inorganic dye. In the late Bronze Age (1200– 1000  BCE), red glass from Frattesina (Italy) was colored by the unique optical properties of Cu NPs (Angelini et al. 2004). The use of metal NPs for coloring glass spread during the Roman period. Lycurgus Cup (Roman, 400 CE) appears green in reflected light, but it appears (or transmits) red when light is shone from inside (Freestone et al. 2007). This unique property of scattering and absorption is from the Au  NPs embedded in the glass. Purple of Cassius from the seventeenth century was a purple pigment precipitated as a sol by the interaction of gold chloride and a solution of stannic acid and stannous chloride. Au NPs were used also as a pigment of ruby-colored stained glass. The bright red and purple colors of the Rose Window of the Cathedral of Notre Dame are produced by Au NPs (Pérez-Villar et  al. 2008). Actually, from ancient times, stained-­ glass makers have known that they could produce the red and yellow found in stained-glass windows by putting varying, tiny amounts of gold and silver in the glass. Michael Faraday attributed the beautiful color of stained glass to finely divided gold particles and prepared gold colloids in 1857 (Faraday 1857). Many scientists began to focus on the optical properties of metal colloids from the early twentieth century with the Mie theory in 1908 (Mie 1908). In recent years, colloidal metal particles with varying sizes in the nm–μm range have been prepared by various methods, and their morphology and geometric structures have been examined by a variety of microscopic techniques. As described above, the unique characteristics of metal NPs such as gold and silver have been used from ancient times. In this era of nanotech-

6  Plasmonic Nanoparticles: Basics to Applications (I)

nology, Au NPs have been used for the treatment of diseases such as rheumatoid arthritis, and considerable research is proceeding to establish their potential in anticancer, antimicrobial, and diagnostic applications. Also current applications of the various metal NPs are being studied in more extensive fields.

6.2

Synthesis of Metal Nanoparticles

6.2.1 General Information Enormous efforts to develop a synthetic method for well-controlled NPs have continued because the size, shape, and composition of NPs determine their properties. A breakthrough in reliable synthesis and analysis techniques achieved the basis for research based on nanotechnology. Basically, synthetic methods of metal NPs, as with other NPs, can be divided into bottom-up and top-down approaches. Most of them are based on the bottom-up method to take advantage of its relative simplicity and cost-effectiveness. Bottom-up methods generally begin with atoms (ions) and build up to nanostructures, reducing the metal precursor. This reduction method can also be broadly divided into four types: chemical reduction, thermal decomposition, photochemical reduction, and sonochemical reduction. Among these methods, chemical reduction is an effective way to synthesize metal NPs that are well controlled in size and shape. Therefore, chemical reduction is most common and has been variously developed. At present, NPs of almost all metallic species, and even some alloys, can be uniformly synthesized via chemical reduction with controlled size and shape. For noble-metal systems such as those with Au, Ag, Pd, Pt, and Cu, a metal salt precursor is reduced in solution with a reducing agent such as sodium borohydride, ascorbic acid, or trisodium citrate (Toshima and Yonezawa 1998). Commonly, the procedures are carried out in the presence of a stabilizing agent that prevents aggregation or improves the chemical stability of

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the formed NPs. These processes are based on nucleation and growth, the reactions of which are governed by thermodynamic (e.g., temperature, reduction potential) and kinetic (e.g., reactant concentration, diffusion, solubility, reaction rate) parameters (Polte 2015; Thanh et al. 2014). For theoretical interpretation, LaMer and his colleagues first transferred the classical nucleation theory to the nanoparticle synthesis mechanism, conceptually separating homogeneous nucleation and growth (LaMer and Dinegar 1950; Mer 1952). Although the LaMer model fails to describe nanoparticle growth and the formation mechanisms of NPs are still controversial, the LaMer model (including its modifications) is still seen as the basic and commonly accepted model for describing the general NP formation process. The mechanism starts with a rapid increase of the free atoms (monomers) generated by reducing the metal precursors in solution (Fig. 6.1a). At a certain time, the system reaches a certain critical supersaturation level, overcoming the energy barrier, and the monomer undergoes burst nucleation (Fig. 6.1b). After the burst nucleation occurs and significantly reduces the concentration of free monomers and the rate of the nucleation, almost no nucleation exists. Then, particles grow by diffusion of further monomers in the solution. Nuclei larger than a critical size will continue to grow because growth can lower the energy of the entire solution. In order to produce uniform NPs in size, the generation of nuclei must occur in a short period of time, so that no additional nucleation occurs, and the concentration and the temperature must be maintained homogeneously throughout the solution. To ­prevent additional nucleation, the conditions of the reaction solution should be maintained so that the concentration of the metal atoms does not rise above the critical concentration for nucleation after the initial nucleation period. In addition, diffusion-controlled growth conditions must be maintained during the growth of nuclei. Because polymer-based surfactants or low-molecular capping agents are used in most cases, the fabricated nuclei are surrounded by an organic layer and provide this condition. Compared with the syn-

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Fig. 6.1 (a) The principle of NP nucleation due to LaMer’s mechanism of (sulfur) nucleation derived from CNT.  The (theoretical) qualitative curve describes the monomer concentration as a function of time. (b) The dependence of the cluster free energy, ΔG, on the cluster

radius, r according to the classical nucleation theory (CNT). The curve has a maximum free energy ΔG at a critical cluster size, rc, which defines the first stable particles—the nuclei. (c) Summary of growth process and the underlying chemical processes (Polte 2015)

thesis of semiconductor NPs such as quantum dots, the synthesis of metal NPs is rather slower and generally takes longer because the semiconducting material is obtained in thermal decomposition, which requires a very high initial concentration of the reactant at high temperature. In the following section, we will consider more specific methods such as citrate reduction, inverse micelle, and polyol methods as widely used chemical reductions.

6.2.2 Citrate Reduction Method For a long time, the most popular method among the conventional synthesis methods of Au NPs has been that using citrate-mediated reduction of HAuCl4 in water. When first reported by Turkevitch et al. in 1951, it resulted in Au NPs of about 20 nm (Turkevich et al. 1951), and several modified methods have been developed for tuning the particle size and also applying with other metal species (Frens 1973; Watson et  al. 1999; Bastús et al. 2011; Dong et al. 2009; Bastús et al. 2014). In this method, gold(III) anions (Au3+) of

6  Plasmonic Nanoparticles: Basics to Applications (I)

chloroauric acid are reduced to Au by citrate ions, which act both as a reducing agent and as a capping agent. As a specific experimental method, 1 ml of 0.5% trisodium citrate is added to a boiling HAuCl4 solution (20  ml of 2.5 × 10−4 M) to produce highly stable and relatively uniform Au NPs. In a precursor solution, tetrachlorauric acid, which is a strong acid, completely ionizes to H+

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and AuCl4−. On the other hand, the reducing agent of sodium citrate solution is basic because a citrate ion (Ct3−) consumes H+ ions generated by self-ionization of water by transformation into HCt2−, H2Ct–, and H3Ct. During Au NP formation, Ct3− is oxidized to acetone dicarboxylate (ACDC2−), a ligand that complexes Au3+, thus facilitating nanoparticle growth (Leng et  al. 2015).

2 AuCl -4 + 3Ct 3- ® 2 Au + 3ACDC2 + 3CO2 - +8Cl - + 3H +

ACDC2− is known to be rapidly degraded to acetate at a synthesis temperature of 100  °C (Turkevich et al. 1951).

being exchanged by other functional ligands, and (3) thin capping layer ensuring effective enhancement of the Raman signals.

100 C

ACDC2 - + 2H 2 O ® acetone + 2CO2 - +2OH - . Several reports suggested that ACDC2− or its degradation products could contribute in additional redox reactions (Brown et  al. 2000). The system contains several types of carboxylate including 3Ct3−, ACDC2−, and their byproducts by oxidation, reduction, or degradation. Regardless of the exact species involved, the carboxylate moiety is likely interacting or capping with the Au NP surface. With increasing pH value of the reacting system, the Cl− ligands in AuCl4− complexes are exchanged successively by OH−, shifting ratios between the different [AuCl4-xOHx]− species (Ojea-Jiménez and Campanera 2012). Both pH value and temperature, which vary the ratios between the gold species and the ratios between the citrate species, affect the seed particle formation and finally decide the size of the Au NPs (Fig.  6.1c) (Wuithschick et al. 2015). For the case of synthesizing spherical Ag NPs, one of the most common approaches is the Lee– Meisel method that reduces AgNO3 with citrate in an aqueous solution on boiling (Lee and Meisel 1982). In this approach, citrate serves as a reducing agent and as a stabilizer, as it does in the Turkevich method. Citrate reduction has the following advantages compared with other reduction methods: (1) compatibility with biomolecules, (2) ease of

6.2.3 Reverse Micelle Method Reverse micelles are nanometer-sized droplets of aqueous phase, thermodynamically stabilized by organized assemblies of surfactant molecules in an organic phase. Reverse micellar systems have been developed using various organic and aqueous phases and surfactants. Nanometer-sized aqueous systems are used to carry out specific reactions for the development of materials of controlled size and shape. The size of reverse micelles is one of the parameters for controlling the size of nanomaterial during nanomaterial synthesis, and reverse micelles have been successfully used as nanoreactors for the synthesis of various metal NPs. The field has grown considerably from the initial synthesis of spherical metal NPs in the 1980s to the highly complex and multifunctional nanostructures of today. Boutonnet et  al. in 1982 introduced reverse micelles as a template to synthesize metal NPs of platinum, palladium, rhodium, and iridium for the first time (Boutonnet et al. 1982). Pileni et al. synthesized various metal NPs including cobalt, copper, and silver by the reduction of metal salts in reverse micelles (Petit et al. 1990). The parameters (such as the reducing agent concentration/ water content) that affect the final particle size for the metal NPs have been studied (Capek 2004; Naoe et  al. 2008). Detailed investigation

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on metal NPs has elucidated the role of water content, capping agent, and concentration of reducing agent on the shape and size, although there are no simple rules so far for deciding the morphology of the synthesized product. The reverse micelle method offers several advantages such as the ability to control easily the particle size and morphology by adjusting the concerned parameters, for example, the concentration and type of surfactant, the type of continuous phase, the concentration of precursors, and molar ratio of water to surfactant. However, there are several limitations for the synthesis of NPs using reverse micelles: (1) NPs synthesized in reverse micelles are generally poorly crystalline because the reactions are usually performed at low temperature, (2) the yield of the NPs is often very low, and (3) polydispersed NPs are often produced.

6.2.4 Polyol Method Among a number of promising synthetic routes, the polyol process is a versatile liquid-phase method utilizing high boiling and multivalent alcohols to produce NPs. The polyol method was first reported by Fievet et al. in 1989 (Fievet et al. 1989a, b). The polyol method, which can be applied to most metal salts, is a robust strategy for preparation of well-defined metal NPs in terms of size, shape, composition, and crystallinity, as shown in Fig.  6.2 (Lu et  al. 2009; Dong et al. 2015; Fiévet et al. 2018b). Several kinds of polyols such as ethylene glycol (EG), propylene glycol (PG), and up to polyethylene glycol (PEG) play a dual role as a reducing agent and solvent; also they are capable of controlling particle growth. Polyols exhibit high boiling points and viscosities because of the presence of several OH groups. The several OH groups grant reducing and coordinating properties to polyols, which contribute greatly to the synthesis of NPs in terms of controlling their size and shape. At elevated temperatures, polyols are oxidized to various aldehyde and ketone species, enabling the reduction of metal precursors.

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EG is probably the most common polyol in polyol-mediated synthesis, and acetaldehyde is assumed to be the primary oxidant, which is then transformed into diacetyl by oxidative coupling (Blin et al. 1989). In the process that ethylene aldehyde and a carbonyl group form a coordination bond with the metal ion, they lose electrons and bond with oxygen. Through this process, ethylene aldehyde acts as a weak reducing agent to gradually reduce each type of metal. However, each metal and each polyol must be considered as a specific case. In the case of silver, some results also showed that glycolaldehyde is the primary reductant (Blin et al. 1989). Some polyols can decompose to give carbon dots at temperatures well below their boiling points. Generally, the use of the polyol alone is not sufficient to control the size and shape of the particles and several strategies have been used to control size and morphology, including the use of capping agents (long-chain alkylamines such as oleylamine, polymers such as polyvinylpyrrolidone (PVP), carboxylates, or halide anions) (Xia et al. 2009; Zhang et al. 2012; Yin et al. 2008). The addition of capping agents is very often required to tailor NP shape and size by influencing the nucleation and growth steps. Because of the polar nature of polyol media, PVP has been largely used as a capping agent, particularly for noble metal syntheses (Koczkur et al. 2015). PVP enables production of a wide range of metal nanocubes via adsorption on the metal surface through the carbonyl group of the pyrrolidone ring. It tends to preferentially cover certain facets of the growing seeds, namely, (100) facets rather than (111) facets for silver, but the reverse for Au (Fiévet et al. 2018a). Besides its reducing ability, a polyol medium offers several advantages: (1) obtaining well-­ crystallized materials because of its high boiling point that allows reaction at high temperature, (2) minimized coalescence due to its ability to coordinate metal precursors as well as the particle surface, and (3) a diffusion-controlled regime for particle growth because of its high viscosity, which results in controlled structures and morphologies.

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Fig. 6.2  A schematic illustrating the reaction pathways that lead to noble-metal NPs having different shapes. First, a precursor is reduced or decomposed to form nuclei (small clusters). Once the nuclei have grown past a certain size, they become seeds with a single-crystal, singly twinned, or multiply twinned structure. If stacking faults

are introduced, plate-like seeds form. Green, orange, and purple represent the {100}, {111}, and {110} facets, respectively. The parameter R is defined as the ratio between the growth rates along the and directions. Twin planes are delineated in the drawing with magenta lines (Lu et al. 2009)

6.3

have been extensively explored for numerous applications including bio/chemical/environmental sensors, SERS spectroscopy, photothermal therapy, and polymer photovoltaics. Manipulation of the plasmonic characteristics of metal NPs is the most critical issue in such applications. Accordingly, tremendous efforts have been devoted to the control of their size, shape, dielec-

Property of Metal Nanoparticles

6.3.1 General Information The excitation of surface plasmons in metallic NPs induces optical properties hardly achievable in other optical materials. In this regard, they

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tric environment, and spatial arrangement because these parameters explicitly determine their plasmonic characteristics such as the localized surface plasmon resonance (LSPR) profile and SERS efficiency. In particular, the controlled assembly of metal NPs has recently proved to be a promising strategy for constructing functional plasmonic platforms with specific optical properties such as Fano resonance and promoting SERS activity. Here, we describe an overview of the optical properties exhibited due to the surface plasmons in metal NPs and the effect of different parameters governing the optical features. Gold and silver still remain the most popular plasmonic materials because of their optical property as well as high stability, making them attractive to work with particularly in biomedical applications. Therefore, we mainly describe the properties of gold and silver NPs here. Au NPs, as typical noble metal NPs, have a characteristic red color. The origins of the optical properties on the nanoscale metal are different from those of semiconductor NPs. In a semiconductor, the size effect in nanoscale is important because of the quantum confinement of the electrons (Alivisatos 1996). The energy gap between the conduction band and valence band in regular semiconductors is very small from the merging of adjacent energy levels of very large numbers of atoms and molecules. However, as a bulk material is reduced to nanoscale dimensions, the number of overlapping energy levels decreases, causing discrete exciton states. When light is absorbed, an electron become excited from the valence to the conduction band, leaving behind a hole. The electron and the hole can bind to each other to form an exciton. When exciton recombination (the electron transition to the ground state) occurs, the exciton’s energy can be emitted as photoluminescence. The corresponding band gap emission increases with decreases in the particle size below the Bohr radius, shifting through the entire visible region from red to blue. In the case of a bulk metal as a conducting material, there is no energy gap between the conduction band, which is partially filled with electrons, and the valence band, or the conduction band and valence band overlap each other. Thus,

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many electrons from below the Fermi level can shift to higher energy levels above the Fermi level in the conduction band and behave as free electrons by acquiring a little more energy from other sources. The oscillation frequency for noble metal NPs is usually in the visible region, and this leads to strong surface SPR absorption. When metal NPs are enlarged, their optical properties only slightly change compared with those of semiconductor NPs. The SPR peak position of metal NPs depends on the dielectric constant of the environment; thus, a different solvent or ­capping agent results in a slight variation for the SPR band position. In addition, aggregation of metal NPs leads to a color transition from red to purple because of plasmonic coupling between NPs. The plasmons of spherical metal NPs with a size in the 10–100 nm range are well described using Mie theory, a classical electromagnetic approach, which is the exact solution to Maxwell’s electromagnetic field equations for a plane wave interacting with a homogeneous sphere of radius R with the same dielectric constant as bulk metal. However, electronic structures of metal NPs in the diameter range 1–10 nm are governed by quantum mechanical rules. The resulting physical properties are neither those of bulk metal nor those of molecular compounds, depending heavily on the particle size, interparticle distance, nature of the protecting organic shell, and shape of the NPs. The recent development of plasmonic NPs with efficient heat-generating ability under illumination of laser radiation has attracted much attention for utilizing Au NPs for therapeutic applications: so-called photothermal therapy (PTT). The photothermal effect can be referred to as light scattering/absorption and the subsequent heat generation and dissipation in the metallic nanomaterials. When the Au NPs interact with light in the visible and near-infrared region, that is, strong scattering and absorption occurs owing to their LSPR, a large portion of the energy of the incident photons is transferred to the energy of the collective oscillation of excited electrons within the metal. Resistive heat is generated in the gold nanostructure when surface plasmon polaritons (SPPs) are excited around the nano-

6  Plasmonic Nanoparticles: Basics to Applications (I)

structure. The heat generated per volume is proportional to the square of the electric field, and the heat density is localized on the surface of the plasmonic NPs because the surface plasmonic wave decays exponentially from the interface. Once surface plasmons are generated, they transfer their absorbed energy to the metallic lattice and the surrounding medium in the form of thermal energy during the plasmon relaxation process, which is called the photothermal effect. During the treatment of cancer, specifically accumulated Au NPs near the cancerous cell generate localized heating under external light irradiation, resulting in cell death.

6.3.2 L  ocalized Surface Plasmon Resonance Metal NPs have great potential in medical diagnostics because of optical phenomena related to surface plasmon resonance (SPR) that can be associated with the presence or absence of target molecules. Plasmon means a collective oscillation of free electrons within a metal. In the case of metal NPs, plasmons are sometimes referred to as surface plasmons (SPs) because of the local presence of plasmons. In metal NPs, the electric fields from visible to near-infrared band light and the plasmons are paired with each other, resulting in absorption of light and then a vivid color; therefore, this phenomenon is referred to as surface plasmon resonance, and it generates a localized, highly enhanced electric field. This means that light energy is converted to surface plasmons and accumulated on the surface of metal NP, which means that light can be controlled in a region smaller than the diffraction limit of light. For readers who are new to the field, we would like to provide again a brief description of the relevant terms. • Plasmons: a quantum of plasma is oscillation (a type of quasi-particle like a phonon) and described as collective oscillations of the “free electron gas” density, often at optical frequencies. They exist mainly in metals, where electrons are weakly bound to the atoms and free to roam.

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• Surface plasmons: plasmons confined to a surface (interface) and that interact with light resulting in polaritons. • Surface plasmon resonance: light (λ) in resonance with surface plasmon oscillation. • Plasmonics: design, control, and application technology of surface plasmons. Plasmonics relates to localization, guiding, and manipulation and control of nanometer-length electromagnetic waves above the diffraction limit. SPR is caused by the collective movement of an electron cloud on the metal surface when electromagnetic waves reach the metal surface from the outside. This characteristic appears in noble metals (Au, Ag, Pt, Pd) having no oxide film because there is an electron cloud on the top layer of the surface. The same phenomenon can be observed even when a semiconductor has a large conductivity by doping. There are two surface plasmon modes: propagating surface plasmon resonance (PSPR) and localized surface plasmon resonance (LSPR). While PSPR is evanescent electromagnetic waves bounded by flat smooth metal-dielectric interfaces (Fig.  6.3a), LSPR is observed when surface plasmons are confined on periodic, colloidal, or other nanosystems. LSPR usually leads to highly localized electromagnetic fields outside the metal NPs. As the metal becomes smaller in nanometer size, the entire electron cloud of the metal is affected by external electromagnetic waves, which results in a much stronger localized electric field than with PSPR. In addition, as the size of the NPs becomes smaller, the space in which free electrons can oscillate becomes smaller (Fig.  6.3b); and as a result, the energy of the external electromagnetic wave, which can cause resonance as a whole, becomes shorter. At a ­certain excitation frequency (ω), this collective local oscillation resonates and produces stronger oscillation (vibration). This is called LSPR. The exact analysis of surface plasmons, which correspond to an interaction between matter and the electromagnetic field of light, is possible by solving Maxwell’s equations with the appropriate boundary conditions. However, a simplified clas-

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Fig. 6.3  Schematic diagrams illustrating (a) a surface plasmon polariton (or propagating plasmon) and (b) a localized surface plasmon (Willets and Van Duyne 2007)

sical picture can describe better the physical meaning of the SP (García 2011). A metallic NP can be interpreted as a lattice of ionic cores with conduction electrons moving almost freely inside the NP. When illumination comes to the NP, these conduction electrons are forced to move toward the NP surface by the electromagnetic field of the light. This causes accumulations of negative charge on one side and positive charge on the opposite side, generating an electric dipole. This dipole produces an electric field opposite to that of the light inside the NP, driving the return of electrons to the equilibrium position. As the electronic displacement increases, the electric dipole

also becomes larger, resulting in greater resilience. When the electrons deviate from the equilibrium position and the field is later removed, it oscillates at a specific frequency called the plasmon frequency (resonant frequency in general). The electron movement inside the NP shows some degree of damping, hence the system is similar to a linear oscillator with some damping. If the frequency of the external force is equal to the plasmon frequency of the NP, it will be easier to make the electrons oscillate. The more the electrons oscillate, the greater the light extinction by absorption and scattering. The plasmon frequency in metal NPs corresponds typically to

6  Plasmonic Nanoparticles: Basics to Applications (I)

UV–visible light, consequently resulting in the extinction (the sum of absorption and scattering) spectrum of metal NPs in this region (Creighton and Eadon 1991). The extinction cross section (the sum of absorption and scattering cross section) for noble metal NPs can be up to 10 times their geometrical section, which means that the NPs are capable of absorbing and scattering photons that are even away from the physical position of the NPs. The light absorption has an exponential dependence on the absorption cross section. Therefore, a moderate increase in the extinction cross section can lead to a huge enhancement of light absorption. The electric dipole produced by the incident light in metal NPs creates an intense field inside the NPs (the restoring field) as described above, as well as out of the NPs, and 100 ~ 10,000 times more electromagnetic waves can be generated than external electromagnetic waves. If there are factors that can give a spectrum around the NPs, the intensity of the spectra increases by several thousand times because of the electromagnetic waves generated at the surface of the NPs. This property is used for surface-enhanced Raman spectroscopy (SERS), surface-enhanced fluorescence (SEF), surface-enhanced second harmonic, etc. (Ding et  al. 2017; Fothergill et  al. 2018; Pipino et al. 1994). Based on this phenomenon, it became possible to carry out highly sensitive analysis in conjunction with plasmon-enhanced spectroscopic techniques such as SERS. Furthermore, a change in refractive index (RI) was observed with a high sensitivity. Metal nanostructures have been used for antigen–antibody binding studies, for volatile organics, and even for catalytic studies and as contrast agents in medical biomedical imaging and in phototherapy-­ based cancer treatments. Also, research has been carried out on the possibility of metal nanostructures being applied in a wide range of fields such as energy, catalysis, electronics, and optics. In summary, the confinement of a surface plasmon in a NP of size comparable to or smaller than the wavelength of light used to excite plasmons leads to two important effects:

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1. Electric fields near the particle’s surface are greatly enhanced. This enhancement falls off quickly with distance from the surface. 2. The particle’s optical excitation has a maximum at the plasmon resonant frequency. For noble metal NPs, this occurs at visible wavelengths.

6.3.3 E  ffects of Size, Shape, Composition, and Environment 6.3.3.1 Size-Dependent Optical Property The size of NPs has a dramatic effect on the SPR process. The color changes of gold NPs from purple to ruby red are largely geometric changes that can be explained with Mie theory, which provides an exact solution of Maxwell’s electromagnetic field equations for a planewave interacting with a homogeneous sphere of radius R with the same dielectric constant as bulk metal (Mie 1908). When R is much smaller than the wavelength of the radiation (2R