<p>About the Author xv</p> <p>Foreword xvii</p> <p>Preface xix</p> <p><b>1 Introduction of SELEX and Important SELEX Variants 1<br /></b><i>Yiyang Dong, ZhuoWang, SaiWang, YehuiWu, YufanMa, and Jiahui Liu</i></p> <p>1.1 SELEX 1</p> <p>1.2 Negative SELEX and Its Analogs 3</p> <p>1.3 One-Round SELEX 5</p> <p>1.4 CE-SELEX 6</p> <p>1.5 Microfluidic SELEX 8</p> <p>1.6 Cell-SELEX 10</p> <p>1.7 In Silico-SELEX 12</p> <p>1.8 Post-SELEX and In Chemico-SELEX 14</p> <p>1.9 Auto-SELEX 17</p> <p>1.10 Primer-Free SELEX 17</p> <p>1.11 Genomic SELEX 18</p> <p>1.12 Photo-SELEX 19</p> <p>1.13 qPCR-SELEX 19</p> <p>1.14 Perspectives 20</p> <p>References 21</p> <p><b>2 In Chemico Modification of Nucleotides for Better Recognition 27<br /></b><i>Przemyslaw Jurek, MartaMatusiewicz,MaciejMazurek, and Filip Jelen</i></p> <p>2.1 Introduction 27</p> <p>2.1.1 Beyond ATGC 27</p> <p>2.1.2 The Scope of This Chapter 29</p> <p>2.2 Modified Functional Nucleic Acids 30</p> <p>2.2.1 The “Hows” 30</p> <p>2.2.1.1 Post-SELEX Optimization 30</p> <p>2.2.1.2 In-line Modifications 30</p> <p>2.2.2 The “Whys” 31</p> <p>2.2.2.1 The Hurdles 31</p> <p>2.2.2.2 The Gains 32</p> <p>2.2.3 The “Ifs” 33</p> <p>2.3 Backbone Modifications 35</p> <p>2.3.1 2′-OH Modifications 36</p> <p>2.3.2 Phosphodiester Bond Modifications 36</p> <p>2.3.3 Xeno Nucleic Acids 38</p> <p>2.3.3.1 TNA 39</p> <p>2.3.3.2 FANAs 39</p> <p>2.3.3.3 HNA, CeNA, LNA, ANA 39</p> <p>2.3.3.4 Other Modifications 40</p> <p>2.4 Nucleobase Modifications 40</p> <p>2.4.1 General Information 40</p> <p>2.4.2 Modified Aptamers and Catalysts 42</p> <p>2.4.2.1 Introduction of Cationic Moieties 42</p> <p>2.4.2.2 Catalysts with Protein-like Sidechains 43</p> <p>2.4.2.3 Nucleobase-linked Nucleobases 44</p> <p>2.4.2.4 Glycans Targeting with Boronic Acids 44</p> <p>2.4.2.5 “Click Chemistry”–Based Versatile Approach 45</p> <p>2.4.2.6 Nonenzymatic Selection – X-aptamers 45</p> <p>2.4.2.7 Slow Off-rate Modified Aptamers 46</p> <p>2.5 Aptamers with Expanded Genetic Alphabet 48</p> <p>2.5.1 GACTZP Aptamers 48</p> <p>2.5.2 Aptamers with a Hydrophobic Fifth Base 50</p> <p>2.6 Summary 52</p> <p>2.A Appendix 52</p> <p>References 68</p> <p><b>3 Immobilization of Aptamers on Substrates 85<br /></b><i>Annalisa De Girolamo, Maureen McKeague,Michelangelo Pascale, Marina Cortese, andMaria C. DeRosa</i></p> <p>3.1 Introduction 85</p> <p>3.2 Methods for Immobilization of Aptamers 87</p> <p>3.2.1 Physical Adsorption 87</p> <p>3.2.2 Covalent Binding 88</p> <p>3.2.2.1 Covalent Immobilization of Activated Aptamers on a Functionalized Surface 88</p> <p>3.2.2.2 Covalent Immobilization of Modified Aptamers on Activated Surfaces 92</p> <p>3.2.2.3 Covalent Immobilization by Entrapment 95</p> <p>3.2.2.4 Covalent Immobilization by Electrografting 97</p> <p>3.2.3 Self-assembled Monolayers 98</p> <p>3.2.4 Avidin–Biotin Binding (Affinity Coupling) 100</p> <p>3.2.5 Electrochemical Adsorption 101</p> <p>3.2.6 Hybridization 101</p> <p>3.3 Immobilization of Aptamers on Substrates for Diagnostic Applications 102</p> <p>3.3.1 Flat Gold 102</p> <p>3.3.1.1 Surface Plasmon Resonance Detection 109</p> <p>3.3.1.2 Electrochemical Detection 109</p> <p>3.3.2 Solid Phase 111</p> <p>3.3.2.1 Optical Detection 112</p> <p>3.3.2.2 Sample Cleanup 114</p> <p>3.3.3 Nanomaterials 115</p> <p>3.4 Future Perspectives on New Substrates and New Immobilization Chemistries 116</p> <p>3.5 Conclusions 117</p> <p>References 119</p> <p><b>4 Characterization of Aptamer–Ligand Complexes 127<br /></b><i>RebecaMiranda-Castro, Noemí de-los-Santos-Álvarez, and María J. Lobo-Castañón</i></p> <p>4.1 Introduction 127</p> <p>4.2 Equilibrium Characterization:Thermodynamics 128</p> <p>4.2.1 Basic Principles 128</p> <p>4.2.2 Separation-Based Methods 133</p> <p>4.2.2.1 Equilibrium Dialysis and Related Techniques 133</p> <p>4.2.2.2 High-Performance Liquid Chromatography 135</p> <p>4.2.2.3 Electrophoresis 136</p> <p>4.2.3 Direct Methods 137</p> <p>4.2.3.1 Isothermal Titration Calorimetry 138</p> <p>4.2.3.2 Fluorescence-Based Methods 140</p> <p>4.3 Kinetic Characterization 146</p> <p>4.3.1 Heterogeneous Methods 148</p> <p>4.3.1.1 Surface Plasmon Resonance 148</p> <p>4.3.1.2 Electrochemical Impedance Spectroscopy 152</p> <p>4.3.2 Homogeneous Methods 154</p> <p>4.3.2.1 Rotating Droplet Electrochemistry 154</p> <p>4.3.2.2 Capillary Electrophoresis 157</p> <p>4.3.2.3 Nanopore-Based Studies 159</p> <p>4.4 Concluding Remarks 162</p> <p>Acknowledgments 163</p> <p>References 164</p> <p><b>5 Utilization of Aptamers for Sample Preparation in Analytical Methods 173<br /></b><i>Zhiyong Yan and Yang Liu</i></p> <p>5.1 Introduction 173</p> <p>5.2 Substrate Materials Developed for Immobilization of Aptamers 175</p> <p>5.3 Utilization of Aptamers for Sample Preparation in SPE 177</p> <p>5.3.1 Aptamers Utilized in Affinity Column for SPE 181</p> <p>5.3.2 Aptamers Utilized in Other SPE 182</p> <p>5.4 Aptamers Utilized in SPME 182</p> <p>5.4.1 Aptamers Utilized in Fiber SPME 183</p> <p>5.4.2 Aptamers Utilized in SBSE 184</p> <p>5.4.3 Aptamers Utilized in Other Formats of SPME 185</p> <p>5.5 Aptamers Utilized in Other Affinity Chromatography 185</p> <p>5.6 Aptamers Utilized in Microfluidic Separation System 187</p> <p>5.7 Aptamers Utilized in Magnetic Separation System 189</p> <p>5.7.1 Aptamers Utilized in Magnetic Solid-Phase Extraction (MSPE) 190</p> <p>5.7.2 Aptamers Utilized in Other Magnetic Separation Formats 190</p> <p>5.8 Aptamers Utilized in CE 191</p> <p>5.9 Aptamers Utilized in Other Sample SeparationMethods 192</p> <p>5.10 Conclusion and Outlook 192</p> <p>References 192</p> <p><b>6 Development of Aptamer-Based Colorimetric Analytical Methods 205<br /></b><i>Subash C.B. Gopinath, Thangavel Lakshmipriya,M.K.Md Arshad, and Chun Hong Voon</i></p> <p>6.1 Introduction 205</p> <p>6.2 Aptamer Generation for Colorimetric Assay 206</p> <p>6.3 Aptasensor 206</p> <p>6.4 Aptamer-AuNP-Based Colorimetric Assays 207</p> <p>6.5 Applications of AuNP-Aptamer-Based Colorimetric Assays 211</p> <p>6.6 Conclusions 213</p> <p>References 213</p> <p><b>7 Enzyme-Linked Aptamer Assay (ELAA) 219<br /></b><i>Yiyang Dong and SaiWang</i></p> <p>7.1 Introduction 219</p> <p>7.2 Enzyme-Linked Immunosorbent Assay 219</p> <p>7.3 AnalyticalMerits of Aptamer vs Antibody 221</p> <p>7.4 Enzyme-Linked Aptamer Assay (ELAA) 223</p> <p>7.5 Comparison of Direct-Competitive ELAA (dc-ELAA), Indirect-Competitive ELAA (ic-ELAA), and ELISA 225</p> <p>7.6 Conclusion 226</p> <p>References 227</p> <p><b>8 Development of Aptamer-Based Fluorescence Sensors 229<br /></b><i>SeyedM. Taghdisi, Rezvan Yazdian-Robati, Mona Alibolandi, Mohammad Ramezani, and Khalil Abnous</i></p> <p>8.1 Introduction 229</p> <p>8.2 Fluorescent-Dye-Based Aptasensors 230</p> <p>8.3 Nanoparticle-Based Aptasensors 231</p> <p>8.3.1 Fluorescent Aptasensors Based on Gold Nanoparticles 231</p> <p>8.3.2 Fluorescent Aptasensors Based on Carbon Nanomaterials 234</p> <p>8.3.3 Fluorescent Aptasensors Based on Silica Nanoparticles 236</p> <p>8.3.4 Fluorescent Aptasensors Based on Silver Nanoparticles 238</p> <p>8.3.5 Fluorescent Aptasensors Based on DNA Structures 239</p> <p>8.3.5.1 Fluorescent Aptasensors Based on DNA Nanostructures 239</p> <p>8.3.5.2 Fluorescent Aptasensors Based on Triple-Helix Molecular Switch (THMS) 240</p> <p>8.4 Conclusion 241</p> <p>Acknowledgment 241</p> <p>SuggestedWebsites 242</p> <p>References 242</p> <p><b>9 Development of Aptamer-Based Electrochemical Methods 247<br /></b><i>Jian-guo Xu, Li Yao, Lin Cheng, Chao Yan, andWei Chen</i></p> <p>9.1 Introduction 247</p> <p>9.2 Classification of Electrochemical Aptasensors 247</p> <p>9.3 Amperometric Aptasensors 248</p> <p>9.3.1 Covalent Labels 248</p> <p>9.3.1.1 Enzyme Labels 248</p> <p>9.3.1.2 Other Covalently Linked Redox Species 250</p> <p>9.3.2 Non-covalent Labels 256</p> <p>9.3.2.1 Intercalated Redox Species 256</p> <p>9.3.2.2 Cationic Redox Species 260</p> <p>9.3.3 Label-Free Aptasensors 263</p> <p>9.4 Potentiometric Aptasensors 265</p> <p>9.5 Impedimetric Aptasensors 266</p> <p>9.6 Electrochemiluminescence Aptasensors 268</p> <p>9.7 Conclusion 268</p> <p>References 269</p> <p><b>10 Development of Aptamer-Based Lateral Flow Assay Methods 273<br /></b><i>Miriam Jauset-Rubio, Mohammad S. El-Shahawi, Abdulaziz S. Bashammakh, Abdulrahman O. Alyoubi, and Ciara K. O’Sullivan</i></p> <p>10.1 Introduction 273</p> <p>10.2 Development of Aptamer-Based Lateral Flow Assay – Strategy 275</p> <p>10.2.1 Analogies and Differences Compared to Lateral flow Immunoassays (LFIAs) 275</p> <p>10.2.2 Fundamental Assay Considerations 276</p> <p>10.2.3 Fundamental Analytical Considerations 277</p> <p>10.3 Lateral Flow Aptamer Assays 278</p> <p>10.3.1 Sandwich Assay 278</p> <p>10.3.2 Competitive Assay 281</p> <p>10.3.3 Signal Amplification 283</p> <p>10.4 Summary and Perspectives 291</p> <p>References 294</p> <p><b>11 Development of Aptamer-Based Non-labeling Methods 301<br /></b><i>Huajie Gu, Liling Hao, and ZhoupingWang</i></p> <p>11.1 Introduction 301</p> <p>11.2 Surface Plasmon Resonance (SPR)-Based Aptasensor 302</p> <p>11.2.1 Introduction 302</p> <p>11.2.2 The Principle of SPR Technique 302</p> <p>11.2.3 The Classification of SPR Biosensors 303</p> <p>11.2.3.1 SPR Biosensors Based on Angular Modulation 303</p> <p>11.2.3.2 SPR Biosensors Based onWavelength Modulation 304</p> <p>11.2.3.3 SPR Biosensors Based on Amplitude Modulation 304</p> <p>11.2.3.4 SPR Biosensors Based on Phase Modulation 304</p> <p>11.2.4 The Application of Aptamer-Based SPR Technique 304</p> <p>11.2.4.1 Determination of the Affinity of Aptamers 305</p> <p>11.2.4.2 Detection Analyte Concentrations 305</p> <p>11.2.5 Summary and Prospects of SPR Aptasensors 310</p> <p>11.3 Quartz Crystal Microbalance (QCM)-Based Aptasensor 311</p> <p>11.3.1 Introduction 311</p> <p>11.3.2 The Principle of QCM Technique 311</p> <p>11.3.3 The Application of Aptamer-Based QCM Technique 312</p> <p>11.3.3.1 Determination of the Affinity of Aptamers 312</p> <p>11.3.3.2 Detection of Analyte Concentrations 313</p> <p>11.3.4 Summary and Prospect of QCM Aptasensors 318</p> <p>11.4 Isothermal Titration Calorimetry (ITC) 319</p> <p>11.4.1 Introduction 319</p> <p>11.4.2 The Principle of ITC Technique 319</p> <p>11.4.3 Thermodynamic Parameters Obtained from ITC Experiment 320</p> <p>11.4.4 Application of ITC in Association Between Aptamer and Target 322</p> <p>11.4.4.1 Interaction Between the Aptamer Domain of the Purine Riboswitch and Ligands 322</p> <p>11.4.4.2 Interaction Between the Cocaine-Binding Aptamer and Quinine 324</p> <p>11.4.4.3 Affinity Test by ITC After Systemic Evolution of Ligands by EXponential Enrichment (SELEX) 327</p> <p>11.4.5 Summary 329</p> <p>11.5 MicroScaleThermophoresis (MST) 329</p> <p>11.5.1 Introduction 329</p> <p>11.5.2 The Principle of MST Technique 330</p> <p>11.5.3 Application of MST in Association Between Aptamer and Target 332</p> <p>11.5.3.1 Interaction Between Steroid Hormones and Aptamers 332</p> <p>11.5.3.2 Affinity Test by MST After Systemic Evolution of Ligands by EXponential Enrichment (SELEX) 333</p> <p>11.5.4 Summary 335</p> <p>References 335</p> <p><b>12 Challenges of SELEX and Demerits of Aptamer-Based Methods 345<br /></b><i>Haiyun Liu and Jinghua Yu</i></p> <p>12.1 Introduction 345</p> <p>12.2 Challenges of SELEX 347</p> <p>12.2.1 Aptamer Degradation 347</p> <p>12.2.2 Purification 348</p> <p>12.2.3 Binding Affinity (Kd) 348</p> <p>12.2.4 Target Immobilization 349</p> <p>12.2.5 Cross-Reactivity 350</p> <p>12.2.6 Time and Cost 350</p> <p>12.2.7 Interaction of Aptamers with Intracellular Targets 351</p> <p>12.2.8 Bioinformatics Tools 352</p> <p>12.3 Demerits of Aptamer-Based Methods 352</p> <p>12.3.1 Sensitivity 352</p> <p>12.3.2 Selectivity and Specificity 354</p> <p>12.3.3 Reproducibility 355</p> <p>12.3.4 Calibration and Uncertainty 355</p> <p>12.3.5 Regeneration 355</p> <p>12.3.6 Immobilization of Aptamers 356</p> <p>12.4 Summary and Perspectives 356</p> <p>References 357</p> <p><b>13 State of the Art and Emerging Applications 365<br /></b><i>Lin-Chi Chen, Jui-HongWeng, and Pei-Wei Lee</i></p> <p>13.1 Introduction 365</p> <p>13.2 Frontiers of Analytical Aptamer Selection and Probe Design 368</p> <p>13.2.1 Biochip-Based Aptamer Selection 368</p> <p>13.2.2 SELEX with Next-Generation Sequencing (NGS) 372</p> <p>13.2.3 Aptamer Optimization and Specialized Selection 373</p> <p>13.2.4 In Silico Aptamer Design 376</p> <p>13.3 Novel Aptasensing Platforms – From Assays and Sensors to Instrumental Analyses 378</p> <p>13.3.1 Aptamer Assays 378</p> <p>13.3.2 Aptasensors 380</p> <p>13.3.3 Aptamer Chips 382</p> <p>13.3.4 Cell-Based Aptasensing 384</p> <p>13.4 Emerging Applications of Aptamer Diagnostics 385</p> <p>13.4.1 Human Disease Diagnosis 386</p> <p>13.4.2 Food/EnvironmentalMonitoring – Mycotoxins, Pesticides, Heavy Metal Ions 387</p> <p>13.4.3 Therapeutic Drug Assessment – Organ-on-a-Chip 387</p> <p>13.4.4 New Molecular Biology Applications – CRISPR/Cas9, Stem Cells, IHC 388</p> <p>13.5 Concluding Remarks – Frontiers of Frontiers 389</p> <p>Acknowledgments 389</p> <p>References 390</p> <p>Index 397</p>