DNA Engineered Noble Metal Nanoparticles

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DNA Engineered Noble Metal Nanoparticles

Ignác Capek

Fundamentals and State-of-the-art-of Nanobiotechnology

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ISBN 978-1-118-07214-1

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10 9 8 7 6 5 4 3 2 1

Dedicated to my grandchildren Andrej, Juraj and Michaela

vii

Contents

Acknowledgement ixPreface xi

1 Introduction 1

2 Nucleic Acids 292.1 DNA/RNA Basics 292.2 Aptamers, Telomers and Oligonucleotides 502.3 Techniques and Approaches 632.4 DNA-Based Molecular Nanomachines 902.5 Peptide Nucleic Acid 1042.6 Nanobiotechnology 112

3 Noble Metal Nanoparticles 1213.1 Preparation and Modifi cation 1213.2 Optical and Physical Properties 1373.3 Conjugates 144

4 DNA-Based Conjugates 1494.1 General 1494.2 Condensation 1524.3 Conjugates 170

5 DNA-Noble Metal Nanoparticle Conjugates 1735.1 General Approaches 1735.2 DNA Monomers and Oligomers 178

5.2.1 Gold Nanoparticles 1785.2.2 Silver Nanoparticles 207

5.3 Hybridization and Denaturation 2245.3.1 General Background 2245.3.2 Linkers and Probes 2285.3.3 Particle Size and Shape 2385.3.4 Th ermodynamics 2525.3.5 Salt Eff ect 2625.3.6 Approaches 267

5.4 DNA Biotemplates 273

viii Contents

6 DNA-Gold Nanoparticle Conjugates 2816.1 DNA-Gold Zero-Dimensional Nanoparticle Conjugates 2816.2 DNA One-Dimensional Gold Nanoparticle Conjugates 295

7 PNA-Noble Metal Nanoparticles Conjugates 3157.1 PNA-Gold Nanoparticle Conjugates 3157.2 PNA-Silver Nanoparticle Conjugates 328

8 DNA-Silver Nanoparticles Conjugates 335

9 Th e Structure of DNA-Noble Metal Nanoparticles Conjugates 3459.1 Confi guration of DNA-Noble Metal

Nanoparticles Conjugates 3459.2 Stabilization of DNA Conjugates 3589.3 Nanostructures and Nanoconstructs 3699.4 Colorimetric and Sensing Assays 385

10 Photochemical and Photophysical Events 40310.1 Noble Metal Nanoparticles 40310.2 DNA Nucleobases 40610.3 DNA/PNA 41710.4 DNA-Dyes Conjugates 42910.5 DNA-AuNP-Dye Conjugates 44310.6 DNA-Gold Nanoparticle Conjugates 45510.7 DNA-AgNPs 46510.8 Hot Gold Nanoparticles 475

11 Nanoparticle Th erapeutics 48111.1 Biodecorated Nanoparticle-Based Th erapies 48111.2 Photothermal Th erapy 48911.3 Cells 49511.4 Gene Th erapy 49911.5 Blood Fluid Eff ect 50511.6 Other Application Approaches 507

12 Conclusion 515Nomenclature 533DNA Entities 545Vocabulary and Defi nitions 549References 564

Index 637

ix

Acknowledgement

Th is work is supported by the VEGA and APVV research projects at the Polymer Institute, SAS, Bratislava as well as the Faculty of Industrial Technologies, TnUni, Púchov, Slovakia. Th e author thanks Mrs. Katke Cinovej for the drawing the fi gures and schemes.

xi

Preface

A discussion on the extensive topic of DNA-noble metal nanostruc-tures is presented in the current volume, DNA Engineered Noble Metal Nanoparticles: Fundamentals and State of the Art of Nanobiotechnology, which will be continued in the complementary forthcoming volume on self-assembling phenomena and DNA biosensors. Th is volume summa-rizes the basic knowledge about nucleic acid and noble metal nanopar-ticle conjugates. Recent advances in the preparation, characterization and applications of noble metal nanoparticles that are conjugated with DNA are discussed, along with their aptamers and oligomers. Highlighted in the book are the advantages and disadvantages of biodecorated nanoparticles through various detection modes; and the great potential in biosensing shown by functionalized noble metal nanoparticles that are selective and sensitive for the analytes. Furthermore, reviews are also presented of recent progress in the area of DNA-noble metal nanoparticles-based artifi cial nanostructures, that is, the preparation, collective properties, and applica-tions of various DNA-based nanostructures.

Th e book is organized into twelve chapters and their subchapters. In the introduction, general characteristics and recent advances in nucleic acids and inorganic nanoparticles (NPs) and their conjugates are described, with an emphasis on gold and silver nanoparticles. Th e second chapter, “Nucleic Acids,” consists of six subchapters. Th e fi rst one covers basic knowledge about the structure of DNA/RNA, the Watson-Crick (WC) base pairs approach, the framework of a regular double helix, the basic components, or building blocks, of DNA or RNA, the double-helical structure of DNA, covalent and noncovalent bonds and interactions among the DNA build-ing blocks. Th e second subchapter deals with aptamers, telomers and oligo-nucleotides. A number of molecular imaging techniques are summarized in the third subchapter, and the fourth subchapter deals with molecular machines based on DNA’s ability to perform tasks on the nanometer scale. Th e fi ft h subchapter describes the bionanomaterials of peptide nucleic

xii Preface

acid (PNA) analogues of DNA and the last one deals with nanotechnology, nanobiotechnology and nanomaterials. Materials in nanostructured form are excellent candidates as probes because they can achieve high response to very small targets in practical conditions. Th e third chapter, “Noble Metal Nanoparticles,” consists of three subchapters. Th e fi rst one discusses various approaches for the synthesis of water-soluble noble metal nanopar-ticles. Th e second subchapter describes the optical and physical properties of noble metal nanoparticles and the third one introduces metal nanopar-ticles as a modifi er of carbon nanomaterials, as well as their electronic structure. Th e fourth chapter, “DNA-Based Conjugates,” consists of two subchapters. Th e fi rst one covers surface-immobilized deoxyribonucleic acid (DNA), which can store hereditary information and regulate gene expression. Th e second subchapter describes DNA condensation induced by a variety of processes and agents such as multivalent ions, inorganic nanoparticles, solvents with low dielectric constants, surfactants, small organic compounds and polymers.

Th e fi ft h chapter, “DNA-Noble Metal Nanoparticle Conjugates,” consists of four subchapters. Th e fi rst one presents general approaches used in the conjugation of thiolated DNA strands on the surface of gold nanoparticles performed by ligand exchange steps. Th ere are several methods for conju-gating oligonucleotides to gold nanoparticles in which thiol-modifi ed and disulfi de-modifi ed oligonucleotides spontaneously bind to gold nanopar-ticle surfaces. Th e second subchapter deals with the bioconjugation of gold nanoparticles with (oligo)nucleotides and the third deals with the agglomeration (hybridization) of nanoparticles or scission (dehybridiza-tion or denaturation) of nanoparticle assemblies (agglomerates). Th e fi nal subchapter covers DNA biotemplates.

Th e sixth chapter, “DNA-Gold Nanoparticle Conjugates,” consists of two subchapters which describe the formation and properties of DNA zero- and one-dimensional gold nanoparticle conjugates. Th e seventh chapter, “PNA-Noble Metal Nanoparticle Conjugates,” consists of two subchapters which both discuss peptide nucleic acid (PNA) DNA analogues. Th e eighth chapter, “DNA-Silver Nanoparticle Conjugates,” describes silver nanopar-ticle conjugates. Th e ninth chapter, “Structure and Stabilization of DNA Conjugates,” consists of four subchapters. Th e fi rst one discusses the con-fi guration of DNA-particle conjugates and the second one deals with the colloidal stability of DNA nanoconjugates. Th e third subchapter describes DNA-AuNPs nanostructures and nanoconstructs and the fourth describes the sensing ability of DNA nanoconjugates. Colorimetric DNA detection was performed by DNA (shell)-functionalized gold nanoparticle(core) (AuNP@DNAs).

Preface xiii

Th e tenth chapter, “Photochemical and Photophysical Events,” consists of eight subchapters. Th e fi rst subchapter describes plasmonics as a branch of nanophotonics that examines the properties of the collective electronic excitations in noble metal nanoparticles. Th e second subchapter describes electronic excitation of DNA by solar ultraviolet (UV) light which initiates photochemical and photophysical processes, leading to photolesions and some harmful photoproducts as well. Th e third subchapter describes the excited states of DNA, PNA and DNA/PNA complexes. Th e lesion distri-bution depends on the sequence around the hotspots, suggesting coop-erativity between bases. Th e fourth and fi ft h subchapters deal with the excited states of DNA-dyes-noble metal nanoparticle nanostructures. An important step into the genomic era was enabled by the development of the YOYO and TOTO dye families since they allowed the detection of DNA at a sensitivity comparable to that of radioactive probes, but without the danger inherent in radioactivity. Th e ability of these dyes to interact with DNA is addressed through a variety of spectroscopic studies. Th e sixth subchapter describes the photochemistry of exited DNA-gold nanoparticle conjugates. In correlation with surface or interfacial phenomena, the predominant role of nonequilibrium electrons in driving the most basic reactions, such as desorption, dissociation, or motion of molecules on metal surfaces, has been established. Th e seventh subchapter deals with the excited states of DNA-silver nanoparticle nanoconjugates and the eighth describes particle heating via light absorption. Th e eleventh chapter, “Nanoparticle Cancer Th eraupeutics,” consists of six subchapters. Th e fi rst subchapter describes composite nanoparticles tailored to simultaneously carry both drugs and imaging probes and specifi cally designed to target molecules of diseased tissues. Th e second subchapter deals with photothermal treatments using immunotargeted gold nanoparticles that have a demonstrated ability to selectively induce cancer cell damage via hyperthermia while minimally aff ecting nontargeted cells. In the third subchapter, biodecorated metal nanoparticle–cell surface interactions are covered, which can play a vital role in the ultimate location of the nanoparticle. Th e fourth subchapter describes the inserting or altering of genes in cells, resulting in therapeutic benefi ts for specifi c diseases. Th e fi ft h subchapter is on blood, a highly com-plex fl uid composed of salts, sugars, proteins, enzymes, and amino acids that can destabilize noble metal nanoparticles (or their conjugates), caus-ing aggregation and embolism. Th e sixth subchapter describes the various applications of DNA-noble metal nanoparticle conjugates. Th e concluding twelft h chapter is a further discussion of some of the data presented above.

Th e fi rst two or three chapters of this book are convenient for beginners, who might also enjoy further chapters even if they lack in-depth knowledge

xiv Preface

of topics related to DNA-noble metal nanostructures. Th is book serves as a general introduction to those just entering the fi eld and for the expert seeking more information in various subfi elds. Th e intention is for it to be a mostly comprehensive review. Since it is impossible for a book to cover all the aspects of DNA entities, noble metal nanoparticles and DNA-noble metal nanoparticle conjugates, those interested in a deeper insight into, and analysis of, the topic will fi nd representive references to original books and journal publications.

Th e goal of this book is to provide a platform for researchers working in the fi eld of nanobiotechnology to discuss recent developments on various topics in the exciting area of DNA-noble metal nanoparticles conjugates. Th e present book might only partly fi ll in the gaps in the literature dealing with DNA-noble metal nanostructures. Th e discussed topic is so extensive today that full treatment of a single aspect would require a scope much greater than can be provided in this book. Th e same is true for all discussed topics. Taking this fact into account, the aim of this book is to provide a brief but, as far as possible, comprehensive insight into the problems of preparing biodecorated noble metal nanoparticles or nanostructures. Th e necessary selection of the materials, topics and approaches, and the extent of their analysis, refl ect the personal approach of the author. Despite this, it is believed that the optimum alternative has been chosen, which is to characterize the biocolloids in an objective and comprehensive way. It is up to the reader to judge to what extent the author has succeeded in his aim.

I. CapekBratislava

January 2015

1

Although the detailed structure of deoxyribonucleic acid (DNA) was revealed by Watson and Crick [1] back in 1953, stunning and useful new structural modes are still being discovered even today for this versatile macromolecule. Taking lessons from its in vivo role and aided by techno-logical advances, nanoengineers have begun to explore novel and creative uses for DNA including: molecular detection [2], therapeutic regimens [3], complex nanodevices [4], nanomechanical actuators and motors [5], directed organic synthesis [6], and molecular computation [6]. Owing to its unique Watson-Crick hydrogen-bonding nature, DNA ensures the specifi city and precision required by biosensors and programmable nano-assemblies [7]. Nucleic acid has been recognized as an attractive scaff old-ing material because of its very long linear structure and its mechanical rigidity over short distances [8].

Th e synthesis of nanomaterials using DNA templates is attracting sub-stantial interest in current nanoscience research due to their enormous potential for applications in industrial and medical fi elds. Utilization of the biochemical functionalities of DNA has been exploited to fabricate and organize nanomaterials. Th e DNA-based approaches have several

1Introduction

2 DNA Engineered Noble Metal Nanoparticles

advantages over conventional chemical methods when preparing struc-tured nanoscaled materials. Th ese benefi ts derive from the unique function-alities of biological substances. Th e numerous active functional moieties of DNA can be conjugated with other organic and inorganic substances. Th e charged and chemically reactive moieties on the biomolecules, such as amine and carboxyl groups, can be exploited to attract and react with other chemical molecules. Furthermore, their natural substrate-specifi c affi nity makes it possible to assemble and align the biomolecule in a specifi c pat-tern. For example, the specifi c affi nities of base pairs of nucleotides have been used to assemble substances in a programmed position, to align small structured materials in a designer pattern, and to conjugate biomolecular substances with each other.

A variety of biomolecules possessing single or multiple functionalities have been used for the preparation of nanoscaled materials. Nucleotides are biomolecules which are commonly used in bionanotechnology due to their hybridizing functions and ease of preparation in the laboratory [9–11]. Moreover, their topological structures are tunable with proper sequence design. Reconfi gurable structures of the ribbon, supercoil ring, or triangle can be made from the designed DNA strands [12,13].

Noble metal nanoparticles are fascinating materials with great nano-technological potential due to their unique and strongly size-dependent electronic, optical, physical, and chemical properties [14,15]. In general, the particulate matter is categorized into particles ≤ 10 μm, ≤ 2.5 μm, and < 0.1 μm in diameter. Th e latter ones are also referred to as ultrafi ne par-ticles or nanoparticles (NPs). Th ey are classifi ed into metal-based (e.g., metal and metal oxides, quantum dots [QDs]), carbon-based (e.g., single- and multiwalled carbon nanotubes [SWCNT and MWCNTs], fullerenes), polymer-based and lipid-based (e.g., liposomes) subgroups. Depending on their basic material, nanoparticles are expected to aff ect biological systems in diff erent ways. However, they share the common characteristic that they exhibit a large surface-to-mass ratio and, therefore, are considered to be biologically more reactive than larger particles of identical material and form. Additionally, the surfaces of nanoparticles can be easily functional-ized with various organic and biomolecular ligands, among which the mol-ecules with a sulfur headgroup have been attracting considerable interest [16]. Simple thiol chemistry or electrostatic attachment can bind DNA to gold nanostructures. When attached to gold nanostructures DNA has an increased half-life from minutes to hours [17] against attack by large nucle-ases due to the increased steric hindrance caused by attachment to the gold surface [17]. Additionally, polyvalent cations near the gold nanoparticle surface electrostatically repel dications located within the nucleases, also

Introduction 3

increasing oligonucleotide stability [18]. Th e strong affi nity of sulfur to gold has been exploited to form molecular contacts, to link other species to the gold surface, or to form well-ordered self-assembled monolayers (SAMs) [19] for applications like surface patterning [20] and molecular electronics [21]. Several strategies [22] have employed alkanethiol-capped DNA oligonucleotides to link gold nanoparticle building blocks to form periodic functional assemblies, in addition to serving as effi cient DNA detection schemes.

DNA represents an ideal scaff old for the generation of ordered nano-structures with noble metal nanoparticles. Over the past decade, researchers have developed many uses for oligonucleotides-noble metal nanoparticle(s) (DNAs-gold nanoparticle(s)) conjugates [23]. Th ese nanostructures, which consist of a nanoparticle core (typically 2–200 nm in size) and many oligo-nucleotide strands covalently attached to their surface [24], exhibit several unusual properties that make them attractive for nanoconjugate applica-tions and particle stabilizations [25]. Th ese properties include coopera-tive binding and enhanced affi nities for complementary nucleic acids [26] that can be used for signal amplifi cation [27], unusual distance-dependent plasmonic properties [28], and the ability to enter cells without the use of auxiliary transfection agents [29]. Th ey also exhibit an extraordinary intracellular stability that makes them useful for antisense studies, drug delivery, and intracellular molecular diagnostics [30]. Indeed, nucleic acid stability is a key property of any system that aims to use such structures for intracellular regulatory or diagnostic events. Th e problem is that Nature has evolved an arsenal of enzymes, known as nucleases, to degrade foreign nucleic acids that enter cells [31]. DNAs-gold nanoparticle(s) conjugate might suppress the enzymatic degradation of DNA.

Th e colloidal stability of gold nanoparticles and their bioconjugates is a complex function of amphiphilic molecules, ligands and biomolecules. Amphiphilic molecules are very popular in nanotechnology due to their self-assembly properties. Most common surface active compounds do not carry a strong charge in the polar headgroup and, therefore, do not interact strongly enough to induce the compaction of negative DNA or its oligo-mers. In fact, it is the surface active agent self-assembly process itself that, while facilitated by the presence of the DNA molecule, induces compac-tion of the DNA. Since the self-assembly of the surfactants is relatively easy to control, it is in principle possible to control the compaction of DNA. In fact, this concept has been used with other positively charged agents to improve their effi ciency and control. Th is self-assembly of the surface active compounds and gold nanoparticles was also ascribed to the hydro-phobic interactions [32].

4 DNA Engineered Noble Metal Nanoparticles

Th e conjugation of a limited number of thiolated DNA strands on the sur-face of gold nanoparticles is performed following several ligand exchange steps [33]. In order to minimize nonspecifi c interactions between the neg-atively charged DNA strands and the metal surface and to optimize col-loidal stability, gold nanoparticles are prepared with a negatively charged shell. Th is labile ligand can be displaced by thiolated DNA strands in the presence of charge screening cations (typically Na+). However, colloidal stability of larger stabilized gold nanoparticles (AuNPs) (> 30 nm in diam-eter) is only optimum for lower NaCl concentrations lower (< 50 mM). In order to further stabilize the AuNP-DNA conjugates, the gold surface can be passivated by adding a large excess of short thiolated poly(ethylene glycol) (PEG) oligomers [34].

A bottom-up strategy has been adopted for the hierarchical structur-ing of atoms or molecules to nanometer-scale bioconjugates. Over the past decade, by using the self-assembling nature of artifi cially designed mole-cules, chemists have succeeded in constructing many kinds of nanometer-scale molecular assemblies, e.g., molecular recognition-directed molecular assemblies [35], surfactant bilayer membranes [36–38], self-assembled monolayers [39] and alternatively deposited polyelectrolyte multilayers [40]. Nanometer-scale molecular self-assembling is the fi rst step of the biomimetic approach of the bottom-up strategy for materials fabrica-tion. Th e second step of the bottom-up strategy is to organize the nano-meter-scale molecular assemblies into larger supramolecular systems in the mesoscopic scale of nanometer [41] to the submicrometer range [42]. Self-assembly is one of the few practical strategies for making ensembles of nanostructures and will therefore be an essential part of nanotechnol-ogy [43]. In order to generate complex structures through self-assembly, it is essential to develop methods by which diff erent components in solu-tion can come together in an ordered fashion. One approach to achieve ordered self-assembly on the nanoscale is to use biomolecules as scaff olds for directed assembly because of the specifi city and versatility they provide [44]. Th e nanoparticle networks or superstructures assembled on various DNA substrates are expected to produce systems with interesting elec-tronic and optical properties [45].

DNA-functionalized colloidal gold nanoparticles (AuNP@DNAs) hold promise for applications in bionanotechnology [46]. Following the pioneer-ing work of Mirkin and coworkers, these modifi ed nanoparticles can act as useful building blocks to form spatially well-defi ned superstructures, including nanocrystals [47], binary [48] and multilayered [49] nanoparticle assemblies, and well-ordered three-dimensional nanoclusters [23]. More importantly, DNA-functionalized gold colloids have been widely used to

Introduction 5

develop highly sensitive biosensors. Mirkin and coworkers have reported selective colorimetric detection of DNA up to one single mismatch [50] and an ultrasensitive scanometric DNA array detection [27]. Maeda et al. later reported that aggregation can be triggered by non-crosslinking DNA hybridization, and that the technique is also capable of single mismatch dis-crimination [51]. Increasing research eff orts in DNA-AuNP-based biosen-sors have expanded the application of AuNP@DNAs to other DNA detection techniques, such as the quartz crystal microbalance [52] and surface plas-mon resonance [53], as well as to the detection of other biomolecules [25].

Gold and silver nanoparticles, with desirable nanoscaled sizes and unique physical properties (particularly the colors associated with their surface plasmon resonance), are highly suitable signal transducers for bio-sensors and building blocks in nanoassemblies [54]. Th e combination of gold nanoparticles and biomolecules has enabled considerable advances in diagnostic and therapeutic nanomedicine [55]. Gold nanoparticles have been extensively studied as photothermal [56] and optical contrast agents [57] thanks to their large absorption and scattering cross sections at visible plasmon resonance frequencies. In addition to biocompatibility and ease of fabrication and functionalization, the optical properties of certain noble metal nanoparticles are ideal for biomedical applications. Th e interaction of light with noble metal nanoparticles results in collective oscillations of the free electrons in the metal known as localized surface plasmons. On resonance, a metallic nanoparticle interacts strongly with incident light, possessing an extinction cross section nominally fi ve times its physical cross section. Resonant illumination can result in strong light scattering (useful in biological sensing and imaging) and strong absorption, with rel-ative magnitudes depending upon absolute nanoparticle size. As absorbers, plasmon-resonant nanoparticles are unparalleled light-to-heat converters, dissipating energy via their lattice phonons [58].

Biosensing assays can take advantage of gold nanoparticle aggre-gation induced by the loss (or screening) of surface charges [59]. Nanobioconjugates that consist of various functional nanoparticles linked to biological molecules have been used in many areas such as diagnostics, therapeutics, sensors, and bioengineering. DNA-modifi ed gold nanopar-ticles, for example, can be associated into aggregates in the presence of complementary DNA strands; the aggregation of gold nanoparticles is accompanied by a red-to-purple (or blue) color change [60]. Th e redis-persion of DNA-crosslinked gold nanoparticle aggregates, associated with the inverse purple-to-red color transition, has also been developed for the detection of metal ions and small molecules [61]. Detection methods based on the AuNPs/DNA nanobioconjugates show increased selectivity

6 DNA Engineered Noble Metal Nanoparticles

and sensitivity as compared with many conventional assays that rely on molecular probes [23,59,62].

Th is book reviews work that was performed dealing with DNA structure and functionalities, interactions between DNA, noble metal nanoparticles, surface active agents, solvents and some selected additives [45]. Particular attention is given to how the DNA’s chain length and conformation aff ect the interaction and structure of the nanoconjugates and nanostructures that are formed. Also discussed are recent advances in the preparation, characterization, and applications of noble metal nanoparticles that are conjugated with DNA and their aptamers and oligomers. Highlighted are the advantages and disadvantages of functionalized nanoparticles through various detection modes, including colorimetry, fl uorescence, electro-chemistry, surface plasmon resonance (SPR), and mass spectrometry for the detection of small molecules and biomolecules. Th e functionalized noble metal nanoparticles are selective and sensitive for the analytes, show-ing their great potential in biosensing. Furthermore, this book reviews recent progress in the area of DNA-noble metal nanoparticles-based arti-fi cial nanostructures, that is, the preparation, collective properties, and applications of various DNA-based nanostructures are also described. Th e goal of this book is to provide a platform for researchers working in the fi elds of nanobiotechnology to discuss the recent developments on various topics in this exciting area.

Th is book is organized in the following twelve chapters and subchapters: In the Introduction, general characteristics and recent advances in

nucleic acids and inorganic nanoparticles (NPs) and their conjugates are described, with an emphasis on gold and silver nanoparticles.

Th e second chapter, “Nucleic Acids,” consists of six subchapters. Th e fi rst one describes basic knowledge about the structure of DNA/RNA, the Watson–Crick (WC) base pairs approach, the framework of a regular double helix, the basic components or building blocks of DNA or RNA; the double-helical structure of DNA; covalent and noncovalent bonds and interactions among the DNA building blocks; the eff ect of temper-ature, salt and solute types on the structure, rigidity or stability of DNA macromolecules and the helix bundle type. Th e second subchapter deals with aptamers, telomers and oligonucleotides. In addition to hybridization with its complementary nucleic acid strand via Watson-Crick hydrogen bonding and base stacking, DNA or RNA aptamers can also specifi cally recognize non-nucleic acid targets. Aptamers (DNA or RNA) have been used to bind from small solutes to peptides, proteins, cells, viruses, or para-sites, with high affi nity. Th ese functional nucleic acids can fold into well-defi ned three-dimensional structures to form binding pockets and cleft s

Introduction 7

for the specifi c recognition and tight binding of any given molecular target. Unlike traditional polyelectrolytes, DNA aptamers can fold into compact tertiary structures in the presence of their cognate targets. Telomers are nucleic acids of constant repeat sequences tethered to the ends of the chro-mosomes. During cell proliferation, telomers are eroded, and this provides a cellular signal for the termination of the cell cycle. Th e conjugation of oligonucleotides to other molecules (target) and drugs provides an alter-native approach to modulate oligonucleotide properties. Th e third sub-chapter summarizes a number of molecular imaging techniques such as polymerase chain reaction (PCR), rolling circle amplifi cation (RCA), opti-cal imaging (OI), magnetic resonance imaging (MRI), ultrasound imag-ing (USI), positron emission tomography (PET), electrophoretic mobility shift assay (EMSA), electrospray ionization mass spectrometry (ESI-MS), and so on. Th e fourth subchapter deals with molecular machines based on DNA that have the ability to perform tasks on the nanometer scale. In addition to movements such as stretching and rotation, these nanodevices can execute useful functions such as grabbing and releasing a single pro-tein and walking a defi ned distance along a circular or linear track. Th ese nanodevices can be incorporated within living organisms in order to artifi -cially control processes on the molecular scale. DNA nanomachines oper-ate through hybridization of the machine with manually added ssDNA signals. Th e integration of the instructions for nanomachine operation into a DNA gene and the genetic regulation of the expression of these instruc-tions can enable these nanodevices to function independently and respond to environmental stimuli. Th e fi ft h subchapter describes bionanomaterials of peptide nucleic acids (PNAs) analogues of DNA. A key feature of PNA is the absence of negatively charged phosphate groups, which eliminates the Coulombic repulsion that occurs in natural nucleic acid hybridization. As such, DNA and RNA tend to bind to PNA strands more tightly than to each other, and more readily form higher-order PNA/dsDNA complexes. Th e PNA has also been found to be stable toward nuclease, protease, and peptidase activity, indicating that it is more robust in cells than DNA, RNA, and proteins. One interesting modifi cation is the incorporation of peptide nucleic acid units into the canonic nucleic acid backbone leading to PNA-DNA hybrids, the so-called PNA-DNA chimeras. Furthermore, PNA-DNA chimeras possess interesting biological properties as antisense agents and also as decoys against some transcription factors. Th e last subchapter deals with nanotechnology, nanobiotechnology and nanomaterials. Th e mate-rials in nanostructured form are excellent candidates as probes because they can achieve high response to very small targets in practical condi-tions. Nanomaterials have been explored in many biomedical applications

8 DNA Engineered Noble Metal Nanoparticles

because their novel properties and functions diff er drastically from the bulk counterparts. Particularly, their high volume/surface ratio, surface tailorability, and multifunctionality open many new possibilities for bio-medicine. Molecular nanotechnology especially aims at the generation of nanometer-sized structural and functional elements by means of the “bottom–up” approach. Currently, there has been an increasing interest in the use of DNA as a construction material for the biomimetic synthesis of nanostructured materials. Th e organization of nanoparticles might be of considerable interest with respect to future applications in microelectronic devices. Among the spectacular developments of nanotechnology, a new exciting fi eld that combines nanotechnology and biotechnology—nano-biotechnology—is receiving broad attention. Upon the comprehensive and emerging clinical needs, the probes for in vitro diagnostics are needed to be effi ciently produced, highly sensitive, quantitative, rapid, handy, and even multiplexed to detect and monitor the biomolecules or bioentities from small amounts of diverse clinical samples.

Th e third chapter, “Noble Metal Nanoparticles,” consists of three sub-chapters. Th e fi rst one describes and discusses various approaches for the synthesis of water-soluble noble metal nanoparticles. Th ese nanoparticles are indispensable for various biomedical applications and they can be produced by reducing chloroauric acid in aqueous phase. Furthermore, the coating helps to convert well-defi ned hydrophobic nanoparticles into hydrophilic nanoparticles and introduce chemical functionality onto the particle surface so that diff erent chemicals and biomolecules can be cova-lently attached. In addition, various approaches of thiol-based methods to make a stable coating, which involves the use of ligands with either multiple thiols, thiolated dendrimers, dendrons, or the crosslinking of surface ligands, are mentioned. Th e further approach involves the inter-digited bilayer formation between amphiphilic molecules/polymers and the passivating surfactant layer surrounding nanoparticles. Surface modi-fi cation is usually required for improving their aqueous dispersibility and biocompatibility, and obtaining appropriate surface functional groups for bioconjugation purposes. Biomolecules can be attached to nanoparticles through direct linkage by either physical adsorption or covalent coupling. In physical adsorption, hydrophobic and electrostatic interactions between biomolecules and the nanoparticles dominate over the interaction among nanoparticles. Covalent binding of ligands with gold nanoparticles off ers high stability and is demonstrated to be quite robust: they can withstand a very high salt concentration; they are extremely stable under thermal conditions; and they can also resist, to some extent, attack by molecules bearing SH, phosphine, and NH2 groups. Most of the techniques reported

Introduction 9

for immobilizing ligands to gold nanoparticles surfaces are based on Au–S covalent bond formation between the ligands and the gold atoms on the particle surfaces. Place-exchange reactions were used to remove loosely bound molecules to modify particle surface by mercapto alkanes. Th e sec-ond subchapter describes the optical and physical properties of the noble metal nanoparticles. Surface plasmon resonance results show that gold nanoparticles exhibit strong extinction properties at the visible-to-near-infrared region, depending on their sizes and shapes. Because nanopar-ticles have a high surface area to volume ratio, the plasmon frequency is exquisitely sensitive to the dielectric (refractive index) nature of its inter-face with the local medium. Any changes to the environment of these nanoparticles leads to colorimetric changes of the dispersions. Due to coupling of the plasmons, assemblies (or aggregations) of gold nanopar-ticles are oft en accompanied by distinct color changes. Not only is light strongly absorbed by the plasmons, it is also Rayleigh (elastically) scattered by them, and as the particle gets larger, a larger proportion of the outgoing light is scattered, compared with that absorbed. Besides nanorods, other examples of gold-based nanoparticles of interest for biomedical applica-tions include nanoshells, nanocages, and other geometries that allow the plasmon resonance to be shift ed from the visible into the physiological “water window” in the near-infrared region of the spectrum. Illumination at their plasmon resonant frequency results in light absorption, where the absorbed energy is effi ciently converted to heat and can be exploited for hyperthermal cancer therapy or photothermal drug delivery. An addi-tional property of the plasmon resonance of nanoparticles, frequently overlooked, is the generation of nonequilibrium “hot” electron-hole pairs, a dominant mechanism for plasmon decay. In addition to damping the plasmon resonance, hot electrons can react with molecules at the surface of the noble metal nanoparticle, resulting in enhanced photoinduced charge transfer reactions. Th e third subchapter introduces metal nanoparticles as modifi ers of carbon nanomaterials as well as their electronic structure. One can control the electronic properties of carbon nanotubes by changing the size and concentration of the metal nanoparticles incorporated. Th is new type of nanocomposite is important not only for fundamental studies of the interactions between the matrix and the metallic nanoparticles, but also for diff erent applications such as magnetic devices and nanosensors. Doping carbon nanotubes with foreign atoms yields sites that are more easily chemically modifi ed than the carbon atoms of the regular sidewall structure. Also, sidewall substituents have been formed by applying pho-tochemical and electrochemical methods to generate active species for the reactions with carbon nanotubes. Using direct electrodeposition, it

10 DNA Engineered Noble Metal Nanoparticles

was possible to control the density and size of metal nanoparticles via the applied potential and time, while the corresponding amperometric signal provided information on the underlying growth mechanism.

Th e fourth chapter, “DNA-Based Conjugates,” consists of two subchap-ters. Th e fi rst one describes the surface immobilized deoxyribonucleic acid (DNA), which can store hereditary information and regulate gene expres-sion. Th e development of effi cient surface chemistries for the manufac-ture of spatially resolvable microscale DNA arrays onto solid supports has become essential for the realization of DNA chip technology. Parameters that govern the development of useful and reliable chemistries for DNA microarray fabrication include the accessibility and functionality of sur-face-bound DNA strands, density of attachment, and the stability of the array. Two general generic approaches have been evolved for making DNA arrays: fi rst, base-by-base attachment to build diff erent DNA strands at diff erent sites in the array, or, alternatively, the attachment of diff erent complete strands to individual array sites. Attachment of complete DNA strands to a surface off ers a number of distinct advantages, of which the most important is the fact that the oligonucleotides can be rigorously puri-fi ed prior to surface immobilization. Several methods exist for immobi-lizing single-stranded DNA (ssDNA) oligonucleotides onto solid surfaces. Th e two most promising approaches have been direct assembly of thiol-terminated ssDNA molecules onto gold, and linking of thiol-DNA via the heterobifunctional linker to shell bilayer. Additionally, carbon nanotubes have been functionalized directly with DNA, allowing the nanotubes to be directed into a nanoassembly by DNA-DNA interactions. Th e physi-cochemical interactions between cationically functionalized carbon nano-tubes and DNA build the novel constructions, carbon nanotube-based gene-transfer vector systems.

Th e second subchapter describes DNA condensation induced by a vari-ety of processes and agents such as multivalent ions, inorganic nanopar-ticles, solvents with low dielectric constants, surfactants, small organic compounds and polymers. Th e binding of charged surfactants, micelles and multivalent polyamines is known to condense large DNA coils result-ing in (associative) phase separation in suffi ciently concentrated solu-tions. To incorporate DNA into various organized systems and perform an assortment of functions, cationic surfactants are usually applied to modify its surface properties by interacting with counterions present in the DNA molecule. DNA-surfactant conjugates present a new type of material capa-ble of demonstrating several advanced functions. As expected, the asso-ciation increases markedly in strength with the number of charges of the cosolute as well as its charge density; on the other hand, the association is

Introduction 11

weakened in the presence of a screening electrolyte. Surfactant aggregates induced by the polymer act as its counterions, thereby reducing the charge of the complex and the entropic driving force for mixing as well as inter-polymer repulsions. Th e extent of phase separation increases strongly with the surfactant’s alkyl chain length. Th e observation that negatively charged surfactant monomers can aff ect the rate of dissociation of DNA ligands is remarkable, since it implies that they have to bind or at least get very close to the strongly negatively charged DNA polyelectrolyte. Th e large aromatic ring systems common to the large DNA ligands in combination with their positive charges, however, may provide an attractive environment for the association of surfactant molecules with their negative headgroup and hydrophobic tail, and this could make it easier for the surfactant mono-mers to get close to the DNA. Th e eff ects are also expected to be larger with increasing charge on the ligand, because a high charge would neutralize the negative charge on DNA more, and thereby facilitate the approach of the negatively charged surfactant monomers. Information about the con-formation of the DNA molecules in the gels was obtained by fl uorescence measurements with dyes. Th us the ability to reversibly condense DNA is a prerequisite for being an eff ective vector, that is, good condensing agents should not only have the ability to condense DNA, but also should release DNA from the condensates under proper conditions.

Th e fi ft h chapter, “DNA-Noble Metal Nanoparticles Conjugates,” consists of four subchapters. Th e fi rst one describes general approaches used in the conjugation of thiolated DNA strands on the surface of gold nanoparticles performed by ligand exchange steps. Th ere are several meth-ods for conjugating oligonucleotides to gold nanoparticles in which thiol-modifi ed and disulfi de-modifi ed oligonucleotides spontaneously bind to gold nanoparticle surfaces. Asymmetric disulfi de modifi cation adds an additional mercaptoalcohol ligand to the gold surface, but the density of oligonucleotides formed on the nanoparticle surface is the same as for thiol-terminal oligonucleotides. Di- and trisulfi de-modifi ed conjugates and oligothiol-nanoparticle conjugates are formed. Although four thiol connections are shown, any number are possible via sequential addition of a commercial dithiane phosphoramidite during solid-phase oligonucle-otide synthesis. Attempts to attach oligonucleotides to nanoparticles made from materials other than gold (such as silver, palladium and platinum) have been less successful. Th e second subchapter deals with the bioconju-gation of gold nanoparticles with (oligo)nucleotides. Gold nanoparticles conjugated with nucleobases have emerged as promising materials for bio-logical sensing as well as for bottom-up nanotechnologies based on the Watson-Crick base pairing. Th e capping of the gold nanoparticles with the

12 DNA Engineered Noble Metal Nanoparticles

nucleobases and peptide nucleic acid (PNA) base monomers was achieved by mixing of gold nanoparticles with DNA and PNA monomers and their oligonucleotides. Th e formation of DNA-silver aggregates manifested itself mainly in the substantial decrease of intensity of surface plasmon peak and a small shift of its maximum. In the case of hybridized gold nanoparticles, on the other hand, the shift of surface plasmon peak maximum is clearly dominant over the decrease of peak intensity. Th e spectrum of hybridized silver and gold nanostructures combines both types of spectral changes; a signifi cant dampening of the silver surface plasmon resonance (SPR) peak and a shift of the gold surface plasmon resonance peak mark the process of particle aggregation. Th e melting curves of all types of studied DNA-linked aggregates exhibited relatively sharp slopes and high melting tempera-ture characteristic for oligonucleotide conjugated metallic nanoparticles. Gold nanostructures can be easily tracked with infrared (IR) absorption, and they were proved to have characteristics adaptable to photothermal therapy, making them ideal for testing biomedical applications. Binding of nanoparticle probes to a complementary target sequence modifi ed with a molecular fl uorophore resulted in quenching and decreased fl uorescence intensity. From the overall bathochromic shift of λmax in the absorption spectra, the strength of interaction of the nucleobases with silver nanopar-ticles can be estimated. Th e diff erence in strength of interaction is likely due to the varying ability of the bases to coordinate the nanoparticle surface as a result of the diff erent types of possible surface binding moieties, and these possible nonspecifi c chemical interactions result in diff erent aggrega-tion mechanisms for diff erent nucleobases. In the presence of nucleosides, absorbance at plasmon resonance absorption (PRA) increases. Th is may be due to the strong adsorption of nucleosides at the surface. Th e surface-enhanced fl uorescence (SEF) became stronger with an increase of particle size.

Th e third subchapter deals with the agglomeration (hybridization) of nanoparticles or scission (dehybridization or denaturation) of nanopar-ticle assemblies (agglomerates). Oligonucleotide-derivatized nanoparticles have been extensively employed for detecting DNA hybridization in aque-ous dispersions and on surfaces. Th e simplest choice is the exploitation of gold nanoparticles whose LSPR absorption (or scattering) maximum is a function of size and composition of core@shell gold@DNA oligonucleotide nanoparticle conjugates. For multiple assays, however, it is highly desirable to have two or more markers of diff erent color, that is, the fi rst marker is a gold nanoparticle and a second marker is a silver nanoparticle. Th e sim-plest choice is the exploitation of silver nanoparticles whose LSPR absorp-tion (or scattering) maximum occurs at a wavelength diff erent from that

Introduction 13

of the gold nanoparticles. Under comparable experimental conditions, the melting analyses show that the melting temperature (Tm) increases with the length of spacer. As with native DNA, Tm of these DNA-linked nanopar-ticle structures increases with increasing salt concentration. DNA-gold nanoparticle conjugates were suggested that when hybridized to comple-mentary particles of the same diameter, exhibit Tms that are dependent upon particle size. As the particle size increases, there is a signifi cant and measurable increase in Tm for all particle sizes. Because of these Tm diff er-ences and the sharp, highly cooperative melting transitions, which are char-acteristic of aggregates formed from these nanoparticle-DNA conjugates, it was found that mixtures of particles of diff erent sizes can be separated by size-selective hybridization at a specifi c temperature that is between the Tms of each homoparticle aggregate. It was concluded that electrostatic interac-tions (particle-particle repulsion), which would be expected to be sensitive to the interparticle distance, are the dominant factors which aff ect the Tm. However, binding double-stranded (ds)DNA to nanoparticle surfaces for either thermal or light-induced release introduces a new local environment for the DNA molecules, which dramatically modifi es solution-phase Tm val-ues. We can compare the amount of ssDNA released by light-controlled and thermal dehybridization (from spherical and nanorod conjugates) to obtain the eff ective DNA-ambient melting temperature for both processes. As the salt concentration was increased, while keeping the nanoparticle and tar-get concentration constant, the Tm also increased. Normal DNA of identical sequence exhibits a similar salt concentration dependence but with lower absolute Tm values. Th is is consistent with the conclusion that the increased dielectric created by the nanoparticle probes stabilizes the duplex DNA interconnects.

Th e sixth chapter, “DNA-Gold Nanoparticles Conjugates,” consists of two subchapters. Th e fi rst one describes the formation and properties of DNA-zero-dimensional gold nanoparticles conjugates. Nucleic acids serve as templates that bind DNA-functionalized nanoparticles at complemen-tary segments. When DNA templates are fi xed at a surface of a solid sup-port, the resulting assemblies of nanoparticles can yield a pattern that is dependent on either the shape produced by the DNA template itself or on the pattern produced upon its immobilization. Gold nanoparticles capped with positively charged ligand were deposited onto a surface that was coated with a thick, negatively charged DNA template. Multilayers of nanoparticles can be assembled on solid supports by utilizing DNA com-plementarity. Current protocols for nonspecifi c silver deposition of DNA strands involve either photoreduction silver ions complexed to DNA or chemical reduction of silver ions by glutaraldehyde-modifi ed DNA.

14 DNA Engineered Noble Metal Nanoparticles

Th e second subchapter describes DNA-functionalized anisotropic nanoparticles conjugates. Nonspherical nanostructures provide advantages in understanding the collective properties of ligand shells. Ligand-coated gold nanorods (AuNRs) were used to detect nucleic acid hybridization using a controlled removal of the stabilization force. Particularly, an anionic substance was used to aggregate the particles in the presence of a single-stranded DNA probe before and aft er its hybridization with its target DNA. Th e negatively charged substances can aggregate the ligand+-AuNRs, and the inhibition of these coagulation eff ects can be used to design colori-metric assays. TEM images taken for AuNRs samples confi rm the diff er-ential protection eff ects; i.e., the dsDNA-containing AuNRs remain well dispersed, whereas ssDNA-containing ones underwent a certain degree of aggregation, but were less intensive than those without DNA. It is the adsorption of negatively charged DNA to CTA+-coated AuNRs (through electrostatic attraction that provides charge repulsion) and a steric barrier to prevent the citrate anions from coming closer to neutralize the surface charges. Gold triangular nanoprisms and spherical gold nanoparticles were synthesized and functionalized with oligonucleotides containing a termi-nal alkylthiol moiety. Th ese linkers consist of a recognition sequence that binds to the strands anchored on the particle surface and present a short, terminal, self-complementary “sticky end,” which facilitates hybridiza-tion between DNA-AuNP conjugates. By selecting particle sizes such that each shape has the same number of oligonucleotides, one can ensure that prisms and spheres have the same opportunity for binding and that any diff erences observed are due to anisotropy eff ects. At high temperatures, both the nanoprisms and spherical particles are discrete and dispersed. As the temperature is decreased, a sharp drop in the extinction is observed, indicating oligonucleotide-mediated aggregation. Th e higher temperature at which the prisms hybridize demonstrates that they are able to stabilize interparticle interactions more readily than spheres. As in the case of DNA, interparticle association between prisms occurs selectively compared to spheres, as fewer protonated carboxylates are required to facilitate prism-prism interactions because of the increased contact area and elevated local concentration of terminal functional groups induced by the particle shape. Research eff orts have also been focused on developing inducible systems to control the biological activities of bionanomaterial conjugates in cultured mammalian cells by applied electronic, optical, ultrasonic, or magnetic signals. Specifi cally, gold nanorods have been found to exhibit strong sur-face plasma absorption in the near infrared (NIR) to IR region depending on their aspect ratios. In addition, the chemical modifi cation on the gold nanorod surface can be easily achieved compared to the surfaces of metal