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ELECTROCHEMICAL
DNA
BIOSENSORS
Edited by
Mehmet Ozsoz

ELECTROCHEMICAL
DNA
BIOSENSORS

ELECTROCHEMICAL
DNA
BIOSENSORS
Edited by
Mehmet Ozsoz

Published by
Pan Stanford Publishing Pte. Ltd.
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8 Temasek Boulevard
Singapore 038988
Email: editorial@panstanford.com
Web: www.panstanford.com
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A catalogue record for this book is available from the British Library.
Electrochemical DNA Biosensors
Copyright c○ 2012 Pan Stanford Publishing Pte. Ltd.
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ISBN 978-981-4241-77-9 (Hardcover)
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PrintedintheUSA

Contents
Preface
xvii
1 Terminology Related to Electrochemical DNA-Based
Biosensors 1
Jan Labuda
1.1 Introduction 1
1.2 Detection Features of DNA-Based Biosensors 3
1.3 Detection of Specific DNA Interactions 7
1.3.1 DNA Hybridization Biosensors 7
1.3.2 DNA Damage 9
1.3.3 DNA Association Interactions 13
1.3.3.1 Binding of low molecular mass
compounds 13
1.3.3.2 Binding of proteins 14
1.4 Conclusions 15
2 Electrochemical Aptamer-Based Biosensors 29
S. Centi, S. Tombelli, and M. Mascini
2.1 Introduction 29
2.2 Electrochemical Detection Strategies
Based on Labeling 31
2.3 Electrochemical Aptasensors Based on
a Sandwich Assay 32
2.4 Electrochemical Aptasensors Based on
a Competitive Assay 34
2.5 Electrochemical Aptasensors Based on a Direct
Assay 37
2.6 Electrochemical Metal Nanoparticle-Labeled
Aptasensors 39

vi
Contents
2.7 Electrochemical Aptasensors Based on Noncovalent
Redox Species Label 43
2.8 Electrochemical Aptasensors Based on the Aptamer
Conformational Changes 46
2.9 Electrochemical Aptasensors Based on
Target-Induced Aptamer Displacement 49
2.10 Conclusions 52
3 Carbon-Polymer Bio-Nano-Composite Electrodes for
Electrochemical Genosensing 57
María Isabel Pividori and Salvador Alegret
3.1 Introduction 57
3.2 Composites Materials: Main Features and
Classification 61
3.3 Carbon Composites 65
3.3.1 Carbon-Based Materials as Conductive
Fillers in Composites 65
3.3.2 Rigid Carbon-Polymer Composite 69
3.3.3 Graphite-Epoxy Composites 71
3.4 Electrochemical Genosensing Based on
Graphite-Epoxy Composite 73
3.4.1 Electrochemical Genosensing Based on DNA
Dry Adsorption on GEC as Electrochemical
Transducer 73
3.4.2 Electrochemical Genosensing Based on DNA
Wet Adsorption on GEC as Electrochemical
Transducer 77
3.4.3 Electrochemical Genosensing Based on
Graphite-Epoxy Biocomposite Modified with
Avidin (Av-GEB) as Electrochemical
Transducer 78
3.4.4 Electrochemical Genosensing Based on
Magnetic Beads and m-GEC Electrochemical
Transducer 81
3.4.5 Electrochemical Genosensing Based on
Graphite-Epoxy Composite Modified with
Gold Nanoparticles (NanoAu-GEC) as
Electrochemical Transducer 87
3.5 Final Remarks 93

Contents
vii
4 Gold Nanoparticle-Based Electrochemical DNA
Biosensors 103
María Pedrero, Paloma Yáñez-Sedeño,
and JoséM.Pingarrón
4.1 Introduction 103
4.2 Configurations Used for DNA Immobilization 107
4.2.1 Au-Thiol Binding 108
4.2.2 Gold Nanoparticles: Metallic Oxide
Composites 110
4.2.3 Carbon Nanotube–Gold Nanoparticle
Hybrids 111
4.2.4 Polymer–Gold Nanoparticle Hybrids 111
4.2.5 Avidin–Biotin Affinity Reactions 113
4.3 Signal Transduction and Amplification Strategies 114
4.3.1 Detection Strategies Not Involving Direct
Participation of Au-NPs in the Generation
of the Electrochemical Signal 114
4.3.1.1 Direct detection of redox markers 115
4.3.1.2 Detection based on enzymatic labels 116
4.3.1.3 Detection based on electrochemical
labels intercalated within dsDNA 118
4.3.1.4 Detection involving the use of
Au-NPs as carriers 120
4.3.2 Detection Strategies Involving Direct
Participation of Au-NPs in the Generation of
the Electrochemical Signal 124
4.3.2.1 Detection based on Au-NPs’ acidic or
electrochemical dissolving 124
4.3.2.2 Label-free electrical detection 127
4.3.2.3 Signal enhancement methods 129
4.4 Conclusions and Outlook 136
5 Nanoparticle-Induced Catalysis for Electrochemical
DNA Biosensors 141
Marisa Maltez-da Costa, Alfredo de la Escosura-Muñiz, and
Arben Merkoçi
5.1 Introduction 142
5.2 Catalysis Induced by Gold Nanoparticles 145

viii
Contents
5.2.1 Electrocatalytic Activity of Gold Nanoparticle
Labels on Silver Deposition 145
5.2.2 Electrocatalytic Activity of Gold Nanoparticle
Labels on Other Reactions 146
5.2.3 Electrocatalytic Activity of Gold
Nanoparticles Used as Modifiers of
Electrotransducer Surfaces 149
5.3 Catalysis Induced by Platinum and
Palladium Nanoparticles 149
5.3.1 Electrocatalytic Activity of Platinum
Nanoparticle Labels 149
5.3.2 Electrocatalytic Activity of Palladium
Nanoparticle Labels 151
5.4 Catalysis Induced by Other Nanoparticles 152
5.4.1 Electrocatalytic Activity of Titanium Dioxide
Nanoparticle Labels 152
5.4.2 Electrocatalytic Activity of Osmium Oxide
Nanoparticle Labels 155
5.4.3 Electrocatalytic Activity of Other
Nanoparticles 156
5.5 Conclusions 157
6 Application of Field-Effect Transistors to Label-Free
Electrical DNA Biosensor Arrays 163
Peng Li, Piero Migliorato, and Pedro Estrela
6.1 Introduction 163
6.2 Field-Effect Transistors 165
6.2.1 Field-Effect Transistor Technologies 168
6.2.1.1 Single crystalline silicon and CMOS 168
6.2.1.2 Thin-film transistors 170
6.2.2 Field-Effect Transistor Arrays 173
6.3 Field-Effect DNA Sensing 174
6.3.1 Physical Mechanisms of Detection 176
6.3.1.1 Description of the electrochemical
system 177
6.3.1.2 DNA charge fraction 178
6.3.1.3 Quantitation of the field-effect device
signal 180

Contents
ix
6.3.1.4 Equivalent electrical circuit model of
functionalized FET 183
6.3.2 Differential OCP Measurement 184
6.4 Electrochemical Impedance Spectroscopy 185
6.4.1 PNA-Based Sensing 188
6.4.2 Modeling of the Signal 189
6.5 Application of FETs on Biosensor Arrays 192
6.5.1 FET-Addressed Biosensor Arrays 192
6.5.2 Specifications of the Biosensor Arrays 194
6.5.3 Development of Biosensor Arrays Based on
FETs 197
6.5.4 Fabrication Technologies and Future
Trends 198
6.6 Conclusions 201
7 Electrochemical Detection of Basepair Mismatches in
DNA Films 205
Piotr Michal Diakowski, Mohtashim Shamsi,
and Heinz-Bernhard Kraatz
7.1 Introduction 206
7.2 Surface Immobilization 207
7.2.1 Covalent Attachment 208
7.2.2 Adsorption 209
7.2.3 Affinity Binding 210
7.3 Detection Strategies 210
7.3.1 Direct DNA Electrochemistry 211
7.3.2 Charge Transduction Through DNA 212
7.3.3 Hybridization Indicators, Intercalators and
Groove Binders 216
7.3.4 Peptide Nucleic Acids (PNA) 221
7.3.5 Protein Mediated DNA Biosensors 225
7.3.6 DNA Stem-Loops 227
7.3.6.1 Enzyme-mediated sensors 228
7.3.7 Nanoparticle-Based Sensors 231
7.3.8 Metal-Ion Amplified Sensor 233
7.3.9 Miscellaneous Methods 236
7.4 Conclusion 239

x
Contents
8 Electrochemical Detection of DNA Hybridization: Use
of Latex to Construct Metal-Nanoparticle Labels 245
Mithran Somasundrum and Werasak Surareungchai
8.1 Introduction 245
8.2 Synthesis of Metal Nanoparticles 246
8.3 Use of Metal Nanoparticles as Electrochemical
Labels 249
8.4 Voltammetric Detection of Metal-Nanoparticle
Labels 254
8.4.1 Principles of Analytical Voltammetry 254
8.4.2 Anodic Stripping Voltammetry (ASV) 256
8.4.3 Quantification 258
8.4.3.1 Linear sweep voltammetry 258
8.4.3.2 Differential pulse voltammetry 260
8.4.3.3 Potentiometric stripping analysis 262
8.5 Latex as a Label Support 262
8.5.1 Introduction 262
8.5.2 Latex Synthesis 263
8.5.3 Latex Solution Properties 264
8.5.4 Layer-by-Layer Deposition: Theory 265
8.5.5 Layer-by-Layer Modification of Latex 267
8.5.5.1 Latex surface charge excess 267
8.6 DNA Measurement 278
8.6.1 DNA Immobilization 278
8.6.2 Probe Attachment 280
8.6.3 Detection Sequence 280
8.7 Areas for Further Research 284
9 Screen-Printed Electrodes for Electrochemical DNA
Detection 291
Graciela Martínez-Paredes, María Begoña González-García,
and Agustín Costa-García
9.1 Introduction 292
9.2 Fabrication of Screen-Printed Electrodes 292
9.2.1 Types of Screen-Printed Electrodes 293
9.3 Genosensors on Screen-Printed Electrodes 294
9.3.1 Electrochemical Detection of Hybridization
Reaction 295

Contents
xi
9.3.1.1 Direct transduction methods 295
9.3.1.2 Indirect transduction methods 296
9.3.2 Strategies for Immobilization of ssDNA over
SPEs 298
9.3.2.1 Immobilization of ssDNA over
carbon electrodes 300
9.3.2.2 Immobilization of ssDNA over gold
electrodes 302
9.4 Applications 303
9.4.1 Enzymatic Genosensors on
Streptavidin-Modified Screen-Printed Carbon
Electrode 304
9.4.1.1 Genosensor design 305
9.4.1.2 Analytical signal recording 306
9.4.2 Alkaline Phosphatase-Catalyzed Silver
Deposition for Electrochemical Detection 310
9.4.2.1 Genosensor design 311
9.4.2.2 Results 312
9.4.3 Genosensor for SARS Virus Detection Based
on Gold Nanostructured Screen-Printed
Carbon Electrode 314
9.4.3.1 Gold nanostructuration of
screen-printed carbon electrodes 315
9.4.3.2 Genosensor design 315
9.4.3.3 Results 315
9.4.4 Simultaneous Detection of Streptococcus and
Mycoplasma Pneumoniae Using
Gold-Modified SPCEs 318
9.4.4.1 Genosensor design 319
9.4.4.2 Results 320
9.5 Conclusion 321
10 Synthetic Polymers for Electrochemical DNA
Biosensors 329
Adriana Ferancová and Katarína Beníková
10.1 Introduction 329
10.2 Modification of Electrode Surface with Polymers 330
10.2.1 Solvent Casting 330

xii
Contents
10.2.2 Spin Coating 330
10.2.3 Electropolymerization 331
10.3 Polymer-Assisted DNA Immobilization 332
10.3.1 Immobilization of DNA onto
Polymer-Modified Electrode Surface 332
10.3.2 Immobilization of DNA Within a Polymeric
Matrix by Electropolymerization 334
10.4 Application of Synthetic Polymers in DNA
Biosensors 334
10.4.1 Electronically (Intrinsically) Conducting
Polymers 334
10.4.1.1 Polypyrroles 335
10.4.1.2 Polyaniline 339
10.4.1.3 Polythiophene and its
derivatives 341
10.4.2 Redox Polymers 342
10.4.2.1 Quinone-containing polymers 342
10.4.2.2 Redox-active polymers
containing organometalic
redox center 343
10.4.3 Nonconducting Polymers 344
10.5 Conclusions 346
11 Electrochemical Transducer for Oligonucleotide
Biosensor Based on the Elimination and Adsorptive
Transfer Techniques 355
Libuse Trnkova, Frantisek Jelen, and Mehmet Ozsoz
11.1 Introduction 355
11.2 Theoretical Fundamentals of Elimination
Voltammetry with Linear Scan (EVLS) 356
11.2.1 Elimination Functions 356
11.2.2 EVLS of Adsorbed Species 359
11.2.3 Single and Double Mode of EVLS 360
11.3 EVLS Increasing the Transducer Potential
Range 362
11.4 EVLS in Connection with Adsorptive Stripping
Technique 362
11.4.1 AdS EVLS of Homo- and
Hetero-oligonucleotides 364

Contents
xiii
11.4.2 AdS EVLS of Hairpins 366
11.5 EVLS of Nucleobases and Oligonucleotides in the
Presence of Copper Ions 368
11.5.1 Mercury and Mercury-Modified
Electrodes 368
11.5.2 Solid Electrodes 371
11.6 Conclusions 373
12 Electrochemical DNA Biosensors for Detection of
Compound-DNA Interactions 379
D. Ozkan-Ariksoysal, P. Kara, and M. Ozsoz
12.1 Introduction 380
12.1.1 Aim of Electrochemical DNA Biosensors 380
12.2 The Structure of DNA 380
12.3 Natural Electronalytical Characterictics of DNA 383
12.4 Types of DNA Immobilization Methodologies onto
Sensor Surfaces 385
12.4.1 Adsorption (Wet Adsorption/Electrostatic
Accumulation) 386
12.4.2 Covalent Binding to Activated/
Nonactivated Surfaces 386
12.4.3 DNA İmmobilization onto Transducer
Surfaces Via Avidin-Biotin İnteraction 387
12.5 DNA-Compound Interactions 387
12.5.1 Types of Molecular Binding to DNA 388
12.5.1.1 Electrostatic interactions 388
12.5.1.2 Groove binding interactions 388
12.5.1.3 Intercalation mode 389
12.5.1.4 Specific binding for
single-stranded DNA 390
12.5.2 Detection Techniques for Compound-DNA
Binding Reactions Using Electrochemical
DNA Biosensors 390
12.5.2.1 Label-free detection based on
intrinsic DNA signals (direct
detection) 390
12.5.2.2 Compound-based detection
(indirect redox indicator-based
detection) 392

xiv
Contents
12.6 Calculations About Compound-DNA Interactions 394
12.7 Conclusions 395
13 Electrochemical Nucleic Acid Biosensors Based on
Hybridization Detection for Clinical Analysis 403
P. Kara, D. Ariksoysal, and M. Ozsoz
13.1 Introduction 403
13.2 Biosensors 404
13.2.1 Nucleic Acid Hybridization Biosensors 405
13.3 Electrochemical Nucleic Acid Biosensors 407
13.3.1 Label-Based Electrochemical Nucleic Acid
Biosensors 408
13.3.1.1 Electrochemical genosensing by
using hybridization indicator 408
13.3.1.2 Electrochemical genosensing
with labeled signaling probe or
labeled target DNA 414
13.3.2 Label-Free Electrochemical
Genosensing 415
13.4 Conclusion 420
14 Nanomaterial-Based Electrochemical DNA Detection 427
Ronen Polsky, Jason C. Harper, and Susan M. Brozik
14.1 Introduction 427
14.2 Nanoparticle-Based Electrochemical DNA
Detection 429
14.2.1 Nanoparticle Modification of Electrodes
and Their Use as Supports for DNA
Immobilization 429
14.2.2 Gold Nanoparticle Supports 430
14.2.3 Magnetic Particles 432
14.2.4 Layer-by-Layer Immobilization Techniques 434
14.2.5 Metal Nanoparticle Labels for DNA
Hybridization Detection 435
14.2.5.1 Direct detection of the
nanoparticle label 435
14.2.5.2 Non-stripping-based
nanoparticle electrochemical
DNA detection methods 440

Contents
xv
14.3 Nanowires, Nanorods, and Nanofibers 443
14.3.1 Nanorods as Labels 444
14.3.2 Nanowires Interfaced with Electrodes as
an Immobilization Matrix 445
14.3.3 Nanowire Conductance Based DNA
Detection 448
14.3.4 Electrochemical Impedance Spectroscopy
at Nanowires for DNA Detection 451
14.3.5 Dendrimers 452
14.3.6 Apoferritin Nanovehicles 455
14.3.7 Silica Nanoparticles 456
14.3.8 Liposomes 458
14.4 DNA Detection Using Carbon Nanotubes 461
14.4.1 Functionalization of Carbon Nanotubes
with DNA 462
14.4.2 CNTs for Electrochemical DNA Sensing 464
14.4.3 Progress toward CNT-Based Sensors for
DNA Detection 470
14.5 Conclusion 472
15 Electrochemical Genosensor Assay for the Detection
of Bacteria on Screen-Printed Chips 481
Chan Yean Yean, Lee Su Yin, and Manickam Ravichandran
15.1 Introduction 482
15.2 Methods for the Detection and Identification of
Microorganism Utilizing Enzyme-Based
Genosensors on Screen-Printed Chips 482
15.2.1 Electrochemical Genosensors for the
Detection of Bacteria 482
15.2.2 Principles of Enzyme-Based PCR
Amplicons Target DNA Detection
Methods 486
15.2.2.1 Direct method 486
15.2.2.2 Indirect method 488
15.2.2.3 Rapid method 488
15.2.3 Screen-Printed Transducer Surface 490
15.2.3.1 Screen-printed gold chip
genosensors 490

xvi
Contents
15.2.3.2 Screen-printed carbon-chip
genosensors 491
15.3 Advantages of the Enzyme-Based Electrochemical
Genosensors in Detecting Bacteria on
Screen-Printed Carbon Chips 492
15.4 Discussions 493
15.4 Conclusions 493
16 Introduction to Molecular Biology Related to
Electrochemical DNA-Based Biosensors 499
Yalcin Erzurumlu and Petek Ballar
16.1 Introduction 499
16.2 Nucleic Acids 501
16.3 Deoxyribonucleic Acid 506
16.4 DNA in Electrochemical DNA-Based Biosensors 509
16.5 Nucleic Acid Variants Used in Electrochemical
DNA-Based Biosensors 511
16.5.1 Peptide Nucleic Acid (PNA) 511
16.5.2 Locked Nucleic Acid (LNA) 513
Index 517

Preface
The discovery of DNA, the carrier of genetic information in cells,
brought with it many important technological accomplishments
such as the development of various diagnostic tools to unravel the
nature of hereditary diseases, gene expression profiling methods,
and genotyping. Among these, DNA biosensors constitute an
important class of point-of-care diagnostic devices because they
are capable of converting the Watson-Crick base pair recognition
event signal into an interpretable analytical signal in a shorter time
compared with other methods, thereby producing accurate and sensitive
results. Moreover, they are also suitable for miniaturization.
The terms “electrochemical DNA biosensor” and “nucleic acid–based
electrochemical biosensor” are used interchangeably.
By definition, biosensors are devices that fall into the subgroup of
biomedical sensors, combine a biological component with a detector
component, and are composed of three parts: (1) the biorecognition
element, such as an antibody, an enzyme, nucleic acids, or cell
lysates, which serves as a mediator; (2) the detector/transducer
element, which converts a biological signal into a readable output;
and (3) the signal processor, which displays a user-friendly version
of the transformed signal. Biosensors are classified according to
either the detector they are equipped with or the biorecognition
element they include. In general, the term “nucleic acid biosensors”
connotes devices that use single-stranded DNA as a biological
element. However, because of the advances in biosensor design, new
nucleic acid/nucleic acid analog interactions have been described
that are also considered to fall in this category, such as aptamer–
nucleic acid, RNA–DNA, peptide nucleic acid (PNA)–DNA, and locked
nucleic acid (LNA)–DNA. For the transduction of biological signals,
various kinds of detectors are available, but they can be categorized

xviii
Preface
into three main classes: optical, electrochemical, and piezoelectric.
Because electrochemical DNA biosensors are miniaturizable (i.e.,
reducible in size to nanoscale dimensions), fast, accurate, simple,
and low cost, they have played perhaps the greatest role in the fields
of molecular and medical diagnosis, environmental monitoring,
bioterrorism, food analysis, pharmacogenomics, and drug discovery.
The aim of this book is to cover the full scope of electrochemical
nucleic acid biosensors by emphazing on DNA detection. The
material is presented in 16 chapters. Starting with the terminology
related to electrochemical DNA–based biosensors in Chapter 1,
the researchers active in the fields of biosensor design, molecular
biology, and genetics describe types of detection used for analysis
(chapters 6, 9, 11, and 13), types of materials used for biosensor
design (chapters 3, 4, 5, 8, 10, and 14), and types of nucleic acid
interactions detected (chapters 2, 7, 12, and 15).
I hope that this state-of-the-art book will continue to inform and
inspire all levels of scientists for many years. I wish to express my
gratitude to the researchers throughout the world who contributed
to the book by sharing their valuable studies in the field of
biosensors. In their honor, I quote the amazing scientist Albert
Einstein: “Imagination is more important than knowledge.”
I would also like to thank my wife, Ayse, for her love and patience
as well as the editorial group of Pan Stanford Publishing for their
assistance and support.
Mehmet Ozsoz
Izmir, Turkey