Title Details

Yiyang Dong, PhD, is Professor and Director of the Food Safety & Risk Assessment Laboratory of Beijing University of Chemical Technology (BUCT) in Beijing, China.

About the Author xv

Foreword xvii

Preface xix

1 Introduction of SELEX and Important SELEX Variants 1
Yiyang Dong, ZhuoWang, SaiWang, YehuiWu, YufanMa, and Jiahui Liu

1.1 SELEX 1

1.2 Negative SELEX and Its Analogs 3

1.3 One-Round SELEX 5

1.4 CE-SELEX 6

1.5 Microfluidic SELEX 8

1.6 Cell-SELEX 10

1.7 In Silico-SELEX 12

1.8 Post-SELEX and In Chemico-SELEX 14

1.9 Auto-SELEX 17

1.10 Primer-Free SELEX 17

1.11 Genomic SELEX 18

1.12 Photo-SELEX 19

1.13 qPCR-SELEX 19

1.14 Perspectives 20

References 21

2 In Chemico Modification of Nucleotides for Better Recognition 27
Przemyslaw Jurek, MartaMatusiewicz,MaciejMazurek, and Filip Jelen

2.1 Introduction 27

2.1.1 Beyond ATGC 27

2.1.2 The Scope of This Chapter 29

2.2 Modified Functional Nucleic Acids 30

2.2.1 The “Hows” 30

2.2.1.1 Post-SELEX Optimization 30

2.2.1.2 In-line Modifications 30

2.2.2 The “Whys” 31

2.2.2.1 The Hurdles 31

2.2.2.2 The Gains 32

2.2.3 The “Ifs” 33

2.3 Backbone Modifications 35

2.3.1 2′-OH Modifications 36

2.3.2 Phosphodiester Bond Modifications 36

2.3.3 Xeno Nucleic Acids 38

2.3.3.1 TNA 39

2.3.3.2 FANAs 39

2.3.3.3 HNA, CeNA, LNA, ANA 39

2.3.3.4 Other Modifications 40

2.4 Nucleobase Modifications 40

2.4.1 General Information 40

2.4.2 Modified Aptamers and Catalysts 42

2.4.2.1 Introduction of Cationic Moieties 42

2.4.2.2 Catalysts with Protein-like Sidechains 43

2.4.2.3 Nucleobase-linked Nucleobases 44

2.4.2.4 Glycans Targeting with Boronic Acids 44

2.4.2.5 “Click Chemistry”–Based Versatile Approach 45

2.4.2.6 Nonenzymatic Selection – X-aptamers 45

2.4.2.7 Slow Off-rate Modified Aptamers 46

2.5 Aptamers with Expanded Genetic Alphabet 48

2.5.1 GACTZP Aptamers 48

2.5.2 Aptamers with a Hydrophobic Fifth Base 50

2.6 Summary 52

2.A Appendix 52

References 68

3 Immobilization of Aptamers on Substrates 85
Annalisa De Girolamo, Maureen McKeague,Michelangelo Pascale, Marina Cortese, andMaria C. DeRosa

3.1 Introduction 85

3.2 Methods for Immobilization of Aptamers 87

3.2.1 Physical Adsorption 87

3.2.2 Covalent Binding 88

3.2.2.1 Covalent Immobilization of Activated Aptamers on a Functionalized Surface 88

3.2.2.2 Covalent Immobilization of Modified Aptamers on Activated Surfaces 92

3.2.2.3 Covalent Immobilization by Entrapment 95

3.2.2.4 Covalent Immobilization by Electrografting 97

3.2.3 Self-assembled Monolayers 98

3.2.4 Avidin–Biotin Binding (Affinity Coupling) 100

3.2.5 Electrochemical Adsorption 101

3.2.6 Hybridization 101

3.3 Immobilization of Aptamers on Substrates for Diagnostic Applications 102

3.3.1 Flat Gold 102

3.3.1.1 Surface Plasmon Resonance Detection 109

3.3.1.2 Electrochemical Detection 109

3.3.2 Solid Phase 111

3.3.2.1 Optical Detection 112

3.3.2.2 Sample Cleanup 114

3.3.3 Nanomaterials 115

3.4 Future Perspectives on New Substrates and New Immobilization Chemistries 116

3.5 Conclusions 117

References 119

4 Characterization of Aptamer–Ligand Complexes 127
RebecaMiranda-Castro, Noemí de-los-Santos-Álvarez, and María J. Lobo-Castañón

4.1 Introduction 127

4.2 Equilibrium Characterization:Thermodynamics 128

4.2.1 Basic Principles 128

4.2.2 Separation-Based Methods 133

4.2.2.1 Equilibrium Dialysis and Related Techniques 133

4.2.2.2 High-Performance Liquid Chromatography 135

4.2.2.3 Electrophoresis 136

4.2.3 Direct Methods 137

4.2.3.1 Isothermal Titration Calorimetry 138

4.2.3.2 Fluorescence-Based Methods 140

4.3 Kinetic Characterization 146

4.3.1 Heterogeneous Methods 148

4.3.1.1 Surface Plasmon Resonance 148

4.3.1.2 Electrochemical Impedance Spectroscopy 152

4.3.2 Homogeneous Methods 154

4.3.2.1 Rotating Droplet Electrochemistry 154

4.3.2.2 Capillary Electrophoresis 157

4.3.2.3 Nanopore-Based Studies 159

4.4 Concluding Remarks 162

Acknowledgments 163

References 164

5 Utilization of Aptamers for Sample Preparation in Analytical Methods 173
Zhiyong Yan and Yang Liu

5.1 Introduction 173

5.2 Substrate Materials Developed for Immobilization of Aptamers 175

5.3 Utilization of Aptamers for Sample Preparation in SPE 177

5.3.1 Aptamers Utilized in Affinity Column for SPE 181

5.3.2 Aptamers Utilized in Other SPE 182

5.4 Aptamers Utilized in SPME 182

5.4.1 Aptamers Utilized in Fiber SPME 183

5.4.2 Aptamers Utilized in SBSE 184

5.4.3 Aptamers Utilized in Other Formats of SPME 185

5.5 Aptamers Utilized in Other Affinity Chromatography 185

5.6 Aptamers Utilized in Microfluidic Separation System 187

5.7 Aptamers Utilized in Magnetic Separation System 189

5.7.1 Aptamers Utilized in Magnetic Solid-Phase Extraction (MSPE) 190

5.7.2 Aptamers Utilized in Other Magnetic Separation Formats 190

5.8 Aptamers Utilized in CE 191

5.9 Aptamers Utilized in Other Sample SeparationMethods 192

5.10 Conclusion and Outlook 192

References 192

6 Development of Aptamer-Based Colorimetric Analytical Methods 205
Subash C.B. Gopinath, Thangavel Lakshmipriya,M.K.Md Arshad, and Chun Hong Voon

6.1 Introduction 205

6.2 Aptamer Generation for Colorimetric Assay 206

6.3 Aptasensor 206

6.4 Aptamer-AuNP-Based Colorimetric Assays 207

6.5 Applications of AuNP-Aptamer-Based Colorimetric Assays 211

6.6 Conclusions 213

References 213

7 Enzyme-Linked Aptamer Assay (ELAA) 219
Yiyang Dong and SaiWang

7.1 Introduction 219

7.2 Enzyme-Linked Immunosorbent Assay 219

7.3 AnalyticalMerits of Aptamer vs Antibody 221

7.4 Enzyme-Linked Aptamer Assay (ELAA) 223

7.5 Comparison of Direct-Competitive ELAA (dc-ELAA), Indirect-Competitive ELAA (ic-ELAA), and ELISA 225

7.6 Conclusion 226

References 227

8 Development of Aptamer-Based Fluorescence Sensors 229
SeyedM. Taghdisi, Rezvan Yazdian-Robati, Mona Alibolandi, Mohammad Ramezani, and Khalil Abnous

8.1 Introduction 229

8.2 Fluorescent-Dye-Based Aptasensors 230

8.3 Nanoparticle-Based Aptasensors 231

8.3.1 Fluorescent Aptasensors Based on Gold Nanoparticles 231

8.3.2 Fluorescent Aptasensors Based on Carbon Nanomaterials 234

8.3.3 Fluorescent Aptasensors Based on Silica Nanoparticles 236

8.3.4 Fluorescent Aptasensors Based on Silver Nanoparticles 238

8.3.5 Fluorescent Aptasensors Based on DNA Structures 239

8.3.5.1 Fluorescent Aptasensors Based on DNA Nanostructures 239

8.3.5.2 Fluorescent Aptasensors Based on Triple-Helix Molecular Switch (THMS) 240

8.4 Conclusion 241

Acknowledgment 241

SuggestedWebsites 242

References 242

9 Development of Aptamer-Based Electrochemical Methods 247
Jian-guo Xu, Li Yao, Lin Cheng, Chao Yan, andWei Chen

9.1 Introduction 247

9.2 Classification of Electrochemical Aptasensors 247

9.3 Amperometric Aptasensors 248

9.3.1 Covalent Labels 248

9.3.1.1 Enzyme Labels 248

9.3.1.2 Other Covalently Linked Redox Species 250

9.3.2 Non-covalent Labels 256

9.3.2.1 Intercalated Redox Species 256

9.3.2.2 Cationic Redox Species 260

9.3.3 Label-Free Aptasensors 263

9.4 Potentiometric Aptasensors 265

9.5 Impedimetric Aptasensors 266

9.6 Electrochemiluminescence Aptasensors 268

9.7 Conclusion 268

References 269

10 Development of Aptamer-Based Lateral Flow Assay Methods 273
Miriam Jauset-Rubio, Mohammad S. El-Shahawi, Abdulaziz S. Bashammakh, Abdulrahman O. Alyoubi, and Ciara K. O’Sullivan

10.1 Introduction 273

10.2 Development of Aptamer-Based Lateral Flow Assay – Strategy 275

10.2.1 Analogies and Differences Compared to Lateral flow Immunoassays (LFIAs) 275

10.2.2 Fundamental Assay Considerations 276

10.2.3 Fundamental Analytical Considerations 277

10.3 Lateral Flow Aptamer Assays 278

10.3.1 Sandwich Assay 278

10.3.2 Competitive Assay 281

10.3.3 Signal Amplification 283

10.4 Summary and Perspectives 291

References 294

11 Development of Aptamer-Based Non-labeling Methods 301
Huajie Gu, Liling Hao, and ZhoupingWang

11.1 Introduction 301

11.2 Surface Plasmon Resonance (SPR)-Based Aptasensor 302

11.2.1 Introduction 302

11.2.2 The Principle of SPR Technique 302

11.2.3 The Classification of SPR Biosensors 303

11.2.3.1 SPR Biosensors Based on Angular Modulation 303

11.2.3.2 SPR Biosensors Based onWavelength Modulation 304

11.2.3.3 SPR Biosensors Based on Amplitude Modulation 304

11.2.3.4 SPR Biosensors Based on Phase Modulation 304

11.2.4 The Application of Aptamer-Based SPR Technique 304

11.2.4.1 Determination of the Affinity of Aptamers 305

11.2.4.2 Detection Analyte Concentrations 305

11.2.5 Summary and Prospects of SPR Aptasensors 310

11.3 Quartz Crystal Microbalance (QCM)-Based Aptasensor 311

11.3.1 Introduction 311

11.3.2 The Principle of QCM Technique 311

11.3.3 The Application of Aptamer-Based QCM Technique 312

11.3.3.1 Determination of the Affinity of Aptamers 312

11.3.3.2 Detection of Analyte Concentrations 313

11.3.4 Summary and Prospect of QCM Aptasensors 318

11.4 Isothermal Titration Calorimetry (ITC) 319

11.4.1 Introduction 319

11.4.2 The Principle of ITC Technique 319

11.4.3 Thermodynamic Parameters Obtained from ITC Experiment 320

11.4.4 Application of ITC in Association Between Aptamer and Target 322

11.4.4.1 Interaction Between the Aptamer Domain of the Purine Riboswitch and Ligands 322

11.4.4.2 Interaction Between the Cocaine-Binding Aptamer and Quinine 324

11.4.4.3 Affinity Test by ITC After Systemic Evolution of Ligands by EXponential Enrichment (SELEX) 327

11.4.5 Summary 329

11.5 MicroScaleThermophoresis (MST) 329

11.5.1 Introduction 329

11.5.2 The Principle of MST Technique 330

11.5.3 Application of MST in Association Between Aptamer and Target 332

11.5.3.1 Interaction Between Steroid Hormones and Aptamers 332

11.5.3.2 Affinity Test by MST After Systemic Evolution of Ligands by EXponential Enrichment (SELEX) 333

11.5.4 Summary 335

References 335

12 Challenges of SELEX and Demerits of Aptamer-Based Methods 345
Haiyun Liu and Jinghua Yu

12.1 Introduction 345

12.2 Challenges of SELEX 347

12.2.1 Aptamer Degradation 347

12.2.2 Purification 348

12.2.3 Binding Affinity (Kd) 348

12.2.4 Target Immobilization 349

12.2.5 Cross-Reactivity 350

12.2.6 Time and Cost 350

12.2.7 Interaction of Aptamers with Intracellular Targets 351

12.2.8 Bioinformatics Tools 352

12.3 Demerits of Aptamer-Based Methods 352

12.3.1 Sensitivity 352

12.3.2 Selectivity and Specificity 354

12.3.3 Reproducibility 355

12.3.4 Calibration and Uncertainty 355

12.3.5 Regeneration 355

12.3.6 Immobilization of Aptamers 356

12.4 Summary and Perspectives 356

References 357

13 State of the Art and Emerging Applications 365
Lin-Chi Chen, Jui-HongWeng, and Pei-Wei Lee

13.1 Introduction 365

13.2 Frontiers of Analytical Aptamer Selection and Probe Design 368

13.2.1 Biochip-Based Aptamer Selection 368

13.2.2 SELEX with Next-Generation Sequencing (NGS) 372

13.2.3 Aptamer Optimization and Specialized Selection 373

13.2.4 In Silico Aptamer Design 376

13.3 Novel Aptasensing Platforms – From Assays and Sensors to Instrumental Analyses 378

13.3.1 Aptamer Assays 378

13.3.2 Aptasensors 380

13.3.3 Aptamer Chips 382

13.3.4 Cell-Based Aptasensing 384

13.4 Emerging Applications of Aptamer Diagnostics 385

13.4.1 Human Disease Diagnosis 386

13.4.2 Food/EnvironmentalMonitoring – Mycotoxins, Pesticides, Heavy Metal Ions 387

13.4.3 Therapeutic Drug Assessment – Organ-on-a-Chip 387

13.4.4 New Molecular Biology Applications – CRISPR/Cas9, Stem Cells, IHC 388

13.5 Concluding Remarks – Frontiers of Frontiers 389

Acknowledgments 389

References 390

Index 397