reading

Cover
Dedication
About the Author
Foreword
Preface
1 Introduction of SELEX and Important SELEX Variants
1. 1.1 SELEX
2. 1.2 Negative SELEX and Its Analogs
3. 1.3 One‐Round SELEX
4. 1.4 CE‐SELEX
5. 1.5 Microfluidic SELEX
6. 1.6 Cell‐SELEX
7. 1.7 In Silico ‐SELEX
8. 1.8 Post‐SELEX and In Chemico ‐SELEX
9. 1.9 Auto‐SELEX
10. 1.10 Primer‐Free SELEX
11. 1.11 Genomic SELEX
12. 1.12 Photo‐SELEX
13. 1.13 qPCR‐SELEX
14. 1.14 Perspectives
15. References
2 In Chemico Modification of Nucleotides for Better Recognition
1. 2.1 Introduction
2. 2.2 Modified Functional Nucleic Acids
3. 2.3 Backbone Modifications
4. 2.4 Nucleobase Modifications
5. 2.5 Aptamers with Expanded Genetic Alphabet
6. 2.6 Summary
7. Appendix 2.A
8. References
3 Immobilization of Aptamers on Substrates
1. 3.1 Introduction
2. 3.2 Methods for Immobilization of Aptamers
3. 3.3 Immobilization of Aptamers on Substrates for Diagnostic Applications
4. 3.4 Future Perspectives on New Substrates and New Immobilization Chemistries
5. 3.5 Conclusions
6. References
4 Characterization of Aptamer–Ligand Complexes
1. 4.1 Introduction
2. 4.2 Equilibrium Characterization: Thermodynamics
3. 4.3 Kinetic Characterization
4. 4.4 Concluding Remarks
5. Acknowledgments
6. References
5 Utilization of Aptamers for Sample Preparation in Analytical Methods
1. 5.1 Introduction
2. 5.2 Substrate Materials Developed for Immobilization of Aptamers
3. 5.3 Utilization of Aptamers for Sample Preparation in SPE
4. 5.4 Aptamers Utilized in SPME
5. 5.5 Aptamers Utilized in Other Affinity Chromatography
6. 5.6 Aptamers Utilized in Microfluidic Separation System
7. 5.7 Aptamers Utilized in Magnetic Separation System
8. 5.8 Aptamers Utilized in CE
9. 5.9 Aptamers Utilized in Other Sample Separation Methods
10. 5.10 Conclusion and Outlook
11. References
6 Development of Aptamer‐Based Colorimetric Analytical Methods
1. 6.1 Introduction
2. 6.2 Aptamer Generation for Colorimetric Assay
3. 6.3 Aptasensor
4. 6.4 Aptamer‐AuNP‐Based Colorimetric Assays
5. 6.5 Applications of AuNP‐Aptamer‐Based Colorimetric Assays
6. 6.6 Conclusions
7. References
7 Enzyme‐Linked Aptamer Assay (ELAA)
1. 7.1 Introduction
2. 7.2 Enzyme‐Linked Immunosorbent Assay
3. 7.3 Analytical Merits of Aptamer vs Antibody
4. 7.4 Enzyme‐Linked Aptamer Assay (ELAA)
5. 7.5 Comparison of Direct‐Competitive ELAA (dc‐ELAA), Indirect‐Competitive ELAA (ic‐ELAA), and ELISA
6. 7.6 Conclusion
7. References
8 Development of Aptamer‐Based Fluorescence Sensors
1. 8.1 Introduction
2. 8.2 Fluorescent‐Dye‐Based Aptasensors
3. 8.3 Nanoparticle‐Based Aptasensors
4. 8.4 Conclusion
5. Acknowledgment
6. Suggested Websites
7. References
9 Development of Aptamer‐Based Electrochemical Methods
1. 9.1 Introduction
2. 9.2 Classification of Electrochemical Aptasensors
3. 9.3 Amperometric Aptasensors
4. 9.4 Potentiometric Aptasensors
5. 9.5 Impedimetric Aptasensors
6. 9.6 Electrochemiluminescence Aptasensors
7. 9.7 Conclusion
8. References
10 Development of Aptamer‐Based Lateral Flow Assay Methods
1. 10.1 Introduction
2. 10.2 Development of Aptamer‐Based Lateral Flow Assay – Strategy
3. 10.3 Lateral Flow Aptamer Assays
4. 10.4 Summary and Perspectives
5. References
11 Development of Aptamer‐Based Non‐labeling Methods
1. 11.1 Introduction
2. 11.2 Surface Plasmon Resonance (SPR)‐Based Aptasensor
3. 11.3 Quartz Crystal Microbalance (QCM)‐Based Aptasensor
4. 11.4 Isothermal Titration Calorimetry (ITC)
5. 11.5 MicroScale Thermophoresis (MST)
6. References
12 Challenges of SELEX and Demerits of Aptamer‐Based Methods
1. 12.1 Introduction
2. 12.2 Challenges of SELEX
3. 12.3 Demerits of Aptamer‐Based Methods
4. 12.4 Summary and Perspectives
5. References
13 State of the Art and Emerging Applications
1. 13.1 Introduction
2. 13.2 Frontiers of Analytical Aptamer Selection and Probe Design
3. 13.3 Novel Aptasensing Platforms – From Assays and Sensors to Instrumental Analyses
4. 13.4 Emerging Applications of Aptamer Diagnostics
5. 13.5 Concluding Remarks – Frontiers of Frontiers
6. Acknowledgments
7. References
Index
End User License Agreement

List of Tables

Chapter 02
1. Table 2.A.1 Modified nucleotides derived from thymine/uracil nucleobase.
2. Table 2.A.2 Modified nucleotides derived from cytosine nucleobase.
3. Table 2.A.3 Modified nucleotides derived from guanine nucleobase.
4. Table 2.A.4 Modified nucleotides derived from adenine nucleobase.
Chapter 03
1. Table 3.1 Summary of different aptamer immobilizing methods in relation to the surface.
2. Table 3.2 Some examples of flat gold surfaces for immobilization of aptamers.
3. Table 3.3 Selected examples of solid phases for immobilization of aptamers.
4. Table 3.4 Selected examples of nanomaterials for immobilization of aptamers.
Chapter 05
1. Table 5.1 Recent applications of substrate materials developed for immobilization of aptamers.
Chapter 06
1. Table 6.1 Limit of detection of AuNP‐based colorimetric aptasensors.
Chapter 07
1. Table 7.1 Detection by dc‐ELAA, ic‐ELAA, and ELISA.
Chapter 10
1. Table 10.1 Aptamers in lateral flow assays.
Chapter 11
1. Table 11.1 Thermodynamic parameters for the Interaction between quinine and the aptamersa.
2. Table 11.2 17β‐Estradiol aptamer affinity dissociation constants for different steroids determined by MST.
Chapter 13
1. Table 13.1 Summary of post‐SELEX optimization methods for as‐selected aptamers.
2. Table 13.2 Summary of specialized aptamer selection designs.
3. Table 13.3 Emerging aptamer assay applications.
4. Table 13.4 Partial list of emerging aptasensing modes.

List of Illustrations

Chapter 01
1. Figure 1.1 Schematic drawing of SELEX procedures.
2. Figure 1.2 Schematic drawing of the isogenic cell‐SELEX procedure.
3. Figure 1.3 A schematic diagram of one‐round proximity ligation selection (PLS).
4. Figure 1.4 Schematic of CE‐SELEX.
5. Figure 1.5 Overview of a microfluidic‐SELEX process.
6. Figure 1.6 Mechanism of cell‐SELEX [35].
7. Figure 1.7 Analysis of the predicted intermolecular interactions in the selected ERα‐RNA complex. (a) and (b) Numbers of the predicted hydrophobic interactions and H‐bonds in complex of ERα with ERaptR1–ERaptR5. These interactions are predicted using Ligplot and Nucplot. (c) Ribbon view of the HADDOCK predicted ERα (1SJ0)‐ERaptR4 complex, depicting the interacting residues and the spatial arrangement of protein chains in the vicinity of aptamer molecule. (d) H‐bonding residues in the AutoDock Vina generated complex of ERα‐ERaptR4. The dark gray color represents the aptamer bases while the light gray color indicates the amino acids. (e) Surface view of the PatchDock‐generated ERα‐ERaptR4 complex showing the relative orientations of interacting bases and amino acid chain. (f) Structural representation of H‐bond and hydrophobic interactions in the ERα‐ERaptR4 complex as predicted using Ligplot. H‐bonds are represented by dashed lines between H‐bonding atoms, whereas the hydrophobic interactions are shown by an arc with spokes radiating toward the interacting ligand atoms.
8. Figure 1.8 Process of active species enrichment in selection rounds. (a) Capillary electrograms for library E of each round with human thrombin (left graphic). All electrograms recorded fluorescent intensity of 5′‐labeled 6‐FAM vs migration time. Saturation curve of library enrichment for TBA acquisition (right graphic). (b) Enlarged and overlapped view of each round of capillary electrograms with or without human thrombin.
9. Figure 1.9 Relative sensitivity value of family A 66‐mers to degradation by DNase I. Unmodified, phosphoryl duplex (•) and monothiophosphorylated at nonprime dA sites only (□) with 0.0 and 1.0 at time 0 and after 60 minutes, respectively (▪).
10. Figure 1.10 Chemical structures of the six nucleotides and AEGIS‐LIVE (laboratory in vitro evolution). (a) Molecular structures (left) and space‐filling models (right) of C:G, T:A, and Z:P pairs showing their similarity (PDB ID: 4RHD). (b) Engineering hGPC3‐overexpressing cells and AEGIS‐LIVE procedure.
11. Figure 1.11 X‐aptamer selection kit developed by AM Biotech.
Chapter 02
1. Figure 2.1 The natural diversity possible for polynucleotide (a) and polypeptide (b) chains.
2. Figure 2.2 Possible modifications of (a) 2′‐OH functional group in the sugar moieties of oligonucleotide backbone and (b) phosphodiester bond in the oligonucleotide backbone.
3. Figure 2.3 Xeno nucleic acids – building blocks with their ribose or deoxyribose exchanged to different sugar moiety, retaining ability to form double helix.
4. Figure 2.4 Hydrogen Interactions and a spatial organization of nucleotides in the minor and the major groove of double helix.
5. Figure 2.5 An overview of SOMAmers employing protein‐like side chains, developed by SomaLogic research group.
6. Figure 2.6 An overview of unnatural base pairs capable of being replicated by high‐fidelity polymerase.
Chapter 03
1. Figure 3.1 Schematic representation of aptamer immobilization onto sensor surface. The aptamer could be functionalized with biotin, amine, or thiol group. A spacer group (alkyl and/or nucleotides) could also be required for improving the aptamer–ligand binding.
2. Figure 3.2 Electrostatic interactions on charged surfaces.
3. Figure 3.3 DNA activation and covalent coupling on amino‐functionalized surfaces.
4. Figure 3.4 Some organosilanes used for glass modification.
5. Figure 3.5 Typical reaction sequences reported for covalent attachment of aptamers to hydroxyl (OH) and amine (NH₂)‐modified surfaces using carbonyldiimidazole (CDI) or glutaraldehyde, respectively, as bifunctional cross‐linkers.
6. Figure 3.6 Homobifunctional cross‐linker dendrimer: an initiatory core is extended through a number of branching reactions to form a dendrimer.
7. Figure 3.7 Immobilization of aptamers by electrografting using aryl diazonium electroreduction.
8. Figure 3.8 Representative immobilization of thiolated aptamers on gold surface. Large‐scale immobilization of the thiolated aptamers forms a self‐assembled monolayer (SAM).
9. Figure 3.9 Immobilization of biotinylated aptamers to avidin‐coated surfaces.
10. Figure 3.10 Example of electrochemical adsorption between the negatively charged sugar‐phosphate backbone of aptamer and a positively charged electrode.
11. Figure 3.11 Hybridization of aptamers to partially complementary DNA sequences immobilized onto surfaces.
12. Figure 3.12 Illustration of an SPR aptasensor for ochratoxin A (OTA). The immobilization of a streptavidin layer (a) as well as the capture of a biotin‐labeled OTA aptamer (b) can be tracked in real time to prepare a functional sensor that can detect the presence of the OTA in buffer, wine, or peanut oil (c).
13. Figure 3.13 (a) Microelectrode array on a chip. (b) A schematic representation of aptamer‐induced target binding in an electrical double layer at the gate–solution interface. Specific lysozyme detection (c) and thrombin detection (d) using the appropriate aptamer‐immobilized SB SAM on the extended gate‐FET in 10% fetal bovine serum (FBS).
14. Figure 3.14 (a) Mixed SAM of tenofovir‐aptamer and 6‐mercaptohexanol as the sensing surface of biosensor. (b) Dose–response curve of AptaFET biosensor. Specific response (circle) fitted to Hill function with linear range between 1 nM and 100 nM, and EC50 of 5.8 ± 0.5 nM and responses related to non‐specific drugs and non-specific aptamers as negative controls (triangles and dashed line).
15. Figure 3.15 Schematic illustration of the dipstick assay for the simple and rapid detection of AFB₁. A construction of dipstick assay is shown on the upper part. The procedures and results of the dipstick assay for negative (bottom left) and positive tests are presented at the bottom of the schematic illustration.
16. Figure 3.16 SOMAmers are modified with a photocleavable linker and biotin. Following binding, SOMAmers are immobilized on a streptavidin‐substituted solid support. Proteins immobilized through interaction with bound SOMAmers are biotinylated. After washing, the entire SOMAmer population is released into solution via long‐wave ultraviolet light‐catalyzed cleavage of the photocleavable linker. The biotinylated analyte–SOMAmer complexes are then selectively captured on another streptavidin support. Finally, analyte‐bound SOMAmers are eluted by disrupting the affinity interaction and can be quantified by hybridization to microarrays.
17. Figure 3.17 A sample is incubated with an aptamer‐immobilized column material. The target analyte becomes immobilized to the column through the specific interaction with the aptamer. Thus, washing steps permit the efficient removal of interfering compounds. The target analyte (star) can be eluted from the column using denaturing conditions and quantified by a variety of techniques (e.g. LC–MS, fluorescence, etc.).
18. Figure 3.18 Scheme illustrating click immobilization of 5′‐alkyne‐modified ErbB2 aptamer. (1) Carboxy‐functionalization, activation, and azido‐functionalization, (2) 5′‐terminal‐alkyne‐modified DNA aptamer immobilization, and (3) SPR measurement.
Chapter 04
1. Figure 4.1 Simulated binding curves for aptamer–target interaction with different affinity constants plotted on a linear scale (a), and a semi‐logarithmic scale revealing the characteristic sigmoid plot (b).
2. Figure 4.2 Experimental setup used in equilibrium dialysis. Dots represent the target molecules (T) and the folded structure is the aptamer (A).
3. Figure 4.3 (a) Schematic setup of ITC. Sample and reference cells are placed into an adiabatic jacket and set at the same temperature (ΔT < 10⁻⁶ °C). An injection device allows addition of titrant to the sample cell and mixing the solution. (b) Example of a raw ITC thermogram for an exothermic reaction. (c) The binding isotherm from the integrated thermogram fitted to a single‐site binding model.
4. Figure 4.4 (a) Typical fluorescence polarization assay (saturation binding analysis). Under equilibrium conditions, the fluorescent smaller partner has much lower polarization value compared to that of the fluorescent binding complex. (b) Scheme of a spectrofluorometer for FP measurements (L‐format or single channel). The second polarizer, located in the emission light path, is rotated along parallel and orthogonal directions to record I _∥ and I _⊥ , respectively.
5. Figure 4.5 (a) Schematic representation of the experimental setup for MST measurements. (b) Typical MST signal – fluorescence intensity over time – for a single capillary. (c) Binding experiment based on MST measurements. See the main text for details.
6. Figure 4.6 (a) Free energy profile of binding of aptamer, A, to target, T, to form the AT complex. Binding affinity depends on the free energy difference between the unbound and bound states, ΔG _b. The free energy difference between these states and the transition state, and , determines the association and dissociation rate constants. The dashed profile shows modulation leading to an increase in affinity constant by slowing the dissociation process. (b) Simulated kinetic profiles showing that the same binding affinity (iso‐affinity lines) may be achieved with different association and dissociation rates.
7. Figure 4.7 (a) Typical setup for SPR measurements (Kretschmann configuration). (b) Reflectivity vs angle of incidence depicts a drop in reflected light or dip, as a result of SPR. The angular position of the SPR dip is measured over time. (c) Variation of SPR angle as a function of time or sensorgram for a typical SPR experiment involving association, dissociation and regeneration steps.
8. Figure 4.8 (a) Nyquist plot of impedance spectrum for mixed kinetic and charge‐transfer control circuit. Inset: Randles circuit applied to fit the FIS experimental data. (b) Aptamer–target binding kinetics. R _ct values are obtained from impedance spectrum fitting.
9. Figure 4.9 (a) Schematic representation of the rotating droplet system with a detailed view of an individual screen‐printed electrochemical cell. (b) Normalized amperometric kinetic plot recorded at the rotated droplet for the recognition binding between L‐tyrosinamide and its DNA aptamer.
10. Figure 4.10 (a) Scheme showing the initial and boundary conditions in NECEEM, (b) simulated electropherogram, and (c) a typical signal obtained in the NECEEM method for aptamer characterization.
11. Figure 4.11 (a) Schematic representation of the α‐hemolysin (αHL) nanopore. (b) Typical setup for nanopore‐based measurements. (c) Current traces from a single nanopore showing characteristic signature blocks.
Chapter 05
1. Figure 5.1 The schematic representation of SPE with an oligosorbent.
2. Figure 5.2 The schematic representation of SPME with an oligosorbent.
3. Figure 5.3 The general extraction procedure of aptamer‐based fiber SPME.
4. Figure 5.4 The general procedure of an oligosorbent chromatographic column for sample screening.
5. Figure 5.5 The schematic representation of the aptamer‐based microfluidic technique.
6. Figure 5.6 The general extraction procedure of an aptamer‐based magnetic separation system.
Chapter 06
1. Figure 6.1 SELEX strategy. Five major steps involved are indicated. The critical steps are separation and amplification.
2. Figure 6.2 (a) Aptamer‐mediated controlled assembly and disassembly of AuNP to detect oligonucleotide as the target. (b) Schematic representation of the method involved. This strategy is mainly due to the duplex formation between the nucleic acid strands. In the presence of aptamer and NaCl, the gold colloid shows the dispersion. In the presence of the target, it turns into the state of aggregation.
3. Figure 6.3 Aptamer‐mediated controlled assembly and disassembly on AuNP to detect protein as the target. In the presence of aptamer and NaCl, the gold colloid shows the dispersion. In the presence of target, it turns into the state of aggregation. The spectrophotometric measurements display from red‐shift to blue‐shift with increasing target concentrations.
4. Figure 6.4 Aptamer‐mediated controlled assembly and disassembly on AuNP to detect DNA as the target. This strategy is different from the abovementioned strategies, in which AuNP will be added after mixing the probe and the target.
5. Figure 6.5 Aptamer‐mediated controlled assembly and disassembly on AuNP to detect protein as the target. AuNP will be added after mixing the probe and the target.
6. Figure 6.6 Split aptamer‐mediated controlled assembly and disassembly on AuNP. This strategy has a different approach, in which two pieces of aptamer are used.
7. Figure 6.7 Aptamer‐mediated controlled assembly and disassembly on AuNP to detect metal ion as the target. The cationic polymer is also involved.
Chapter 07
1. Figure 7.1 The four‐chain structure of an antibody, or immunoglobulin, molecule. The basic unit is composed of two identical light (L) chains and two identical heavy (H) chains, which are held together by disulfide bonds to form a flexible Y shape. Each chain is composed of a variable (V) region and a constant (C) region.
2. Figure 7.2 ELISA kit of β‐estradiol.
3. Figure 7.3 Typical calibration curve for the hVEGF ELONA; the bottom portion of the curve was expanded in the inset to better display the error. The range of the assay was from 31.25 to 8000 pg ml⁻¹.
4. Figure 7.4 Schematic illustration of the aptasensor based on indirect competitive ELAA for TC detection.
5. Figure 7.5 Schematic illustration of the aptasensor based on direct competitive ELAA for TC detection.
Chapter 08
1. Figure 8.1 Detection of STR using SYBR Gold and Exo III. In the absence of a target, the aptamer bound to its CS and formed a dsDNA. The formed dsDNA was digested by Exo III. So, a low fluorescence intensity was detected. Upon addition of a target, the aptamer bound to STR, the complementary strand was released from the aptamer, and more protection against Exo III was obtained. Upon addition of SYBR Gold, a strong fluorescence intensity was observed.
2. Figure 8.2 AuNPs and FAM‐labeled CS were used to design a fluorescent aptasensor for STR detection. In the absence of STR, aptamer/FAM‐labeled complementary strand (dsDNA) was stable, resulting in the strong emission of fluorescence. By addition of a target, the aptamer bound to its target and FAM‐labeled CS was adsorbed on the surface of AuNPs and the fluorescence of FAM‐labeled CS was efficiently quenched by AuNPs.
3. Figure 8.3 Fluorescent aptasensor to detect kanamycin based on exonuclease III activity (Exo III), gold nanoparticles (AuNPs), and FAM‐labeled CS. In the absence of kanamycin, the aptamer and its CS formed a dsDNA and left the surface of AuNPs. Using Exo III, the aptamer was recycled from dsDNA and the cycle continued, resulting in a very strong fluorescence emission. Addition of kanamycin caused the aptamer to bind to its target and CSs remained on the surface of AuNPs, resulting in a weak fluorescence emission.
4. Figure 8.4 Detection of digoxin based on rapid fluorescence quenching. In the absence of digoxin, aptamers were adsorbed on the surfaces of AuNPs. Therefore, the fluorescence of the labeled aptamer was efficiently quenched by AuNPs. In the presence of digoxin, aptamers bound to digoxin, leading to the turning on of the fluorescence emission.
5. Figure 8.5 Super quenching capability of SWNTs and a labeled aptamer were used for design of this sensor. In the absence of Pb²⁺, the fluorescence of labeled aptamer was quenched by SWNTs. Upon addition of Pb²⁺, G‐quadruplex/Pb²⁺ complex was formed and ATTO 647 N‐aptamer started fluorescence emission.
6. Figure 8.6 Kanamycin aptamer as the molecular recognition probe was immobilized on the surface of the SNPs. In the absence of a target, the SNPs–dsDNA complex was intact and the strong fluorescence emission was obtained. In the presence of kanamycin, the aptamer bound to its target and labeled CS was released from the aptamer and SNPs, so the fluorescence intensity was reduced.
7. Figure 8.7 Biotin‐labeled immobilized sequence (IS) as the complementary strand (CS) was attached on the surface of SNPs. In the absence of Mb, Apt/CS conjugate (dsDNA) was digested by Exo III. In the presence of a target, the aptamer bound to its target and free CS bound to IS. So, upon addition of PG, a very strong fluorescence emission was detected.
8. Figure 8.8 Cocaine detection based on fluorescent assay. In the presence of a target, a very strong fluorescence signal was detected due to the formation of the hairpin structure of CS. In the absence of a target, aptamer‐modified AuNPs/CS‐SNPs‐streptavidin complex remained intact, resulting in a weak fluorescence emission.
9. Figure 8.9 In the presence of tetracycline, the aptamer bound to its target and the THMS complex was disassembled. So, the free STP was folded into a stem‐loop structure, and the fluorescence was quenched. In the absence of a target, the fluorescence was on.
Chapter 09
1. Figure 9.1 Sketch of (a) the preparation of Apt2–AuNPs–HRP conjugates and (b) the formation of AuMNPs–Apt1/thrombin/Apt2–AuNPs–HRP.
2. Figure 9.2 Preparation of (a) the second thrombin aptamer and (b) the sandwich‐type electrochemical aptasensor.
3. Figure 9.3 Schematic representation of the enzyme‐labeled electrochemical aptasensors based on labeled aptamer displacement for thrombin detection.
4. Figure 9.4 Scheme of one‐step electrochemical aptasensor using the thiol‐ and methylene blue (MB)‐dual‐labeled aptamer‐modified gold electrode for determination of ochratoxin A.
5. Figure 9.5 Schematic illustration of the detection of ATP based on electrochemical signal amplification by dual‐hairpin DNA structure in combination with the insertion approach.
6. Figure 9.6 Schematic representation of the TISR E‐AB sensor.
7. Figure 9.7 Mechanism of the structural transition of a G‐rich aptamer from random coil to G‐quadruplex induced by K⁺.
8. Figure 9.8 Mechanism of a reusable “signal‐on” aptasensor based on cocaine‐binding‐induced conformational change of immobilized MB‐labeled aptamers.
9. Figure 9.9 Schematic illustration of the dual‐signaling E‐AB biosensor for ATP analysis.
10. Figure 9.10 Schematic diagram for the fabrication of the dual‐signaling electrochemical aptasensor.
11. Figure 9.11 Schematic representation of biotinylated aptamers immobilized on a gold electrode through avidin and the charge transfer from the electrode to the electrochemical indicator methylene blue (MB).
12. Figure 9.12 Schematic representation of the immobilization of beacon aptamer and detection of its target protein.
13. Figure 9.13 Schematic representation of the rapid and ultrasensitive electrochemical aptasensor for bisphenol A (BPA) determination.
14. Figure 9.14 Schematic diagram of the RCA‐based signal‐amplification electrochemical aptasensor for detection of OTA.
15. Figure 9.15 The schematic process of signal‐amplified electrochemical aptasensor for AFB1 detection: (a) formation of stem‐loop structure of the immobilized probe; (b) immobilization of TS‐primer‐AuNP‐cDNA onto the sensing interface; and (c) telomerase‐based first‐round amplification.
16. Figure 9.16 Schematic representation of the overall detection scheme of lysozyme with anti‐lysozyme aptamers immobilized on gold electrodes via self‐assembly.
17. Figure 9.17 Schematic representation of the chronocoulometric aptamer sensor for AMP.
18. Figure 9.18 Schematic illustration of the stepwise aptasensor fabrication based on exonuclease‐catalyzed target recycling and surface‐initiated enzymatic polymerization for amplification.
19. Figure 9.19 Schematic illustration of triple‐helix molecular‐switch‐induced hybridization chain reaction amplification for developing a universal and sensitive electrochemical aptasensor.
20. Figure 9.20 Schematic illustration of the label‐free electrochemical (EC) aptasensor for ultrasensitive detection of ractopamine (RAC).
21. Figure 9.21 Schematic illustration of the label‐free electrochemical aptasensor based on functionalized multiwall carbon nanotube/gold nanoparticle (f‐MWCNT/AuNP) nanocomposite‐film‐modified gold electrode.
22. Figure 9.22 Schematic representation of the label‐free and reagentless aptasensor allowing direct detection of thrombin without using additional electroactive label and based on gold NP redox properties.
23. Figure 9.23 Schematic representation of the reusable potentiometric screen‐printed sensor and label‐free aptasensor with pseudo‐reference electrode.
24. Figure 9.24 Schematic of the antifouling impedimetric aptasensor for the determination of thrombin.
25. Figure 9.25 Schematic representation of the impedimetric biosensor based on bimetallic AgPt nanoparticle‐decorated carbon nanotubes.
26. Figure 9.26 Schematic illustration of AuNP‐driven ECL aptasensors for FB1 detection.
Chapter 10
1. Figure 10.1 Schematic representation of the architecture of lateral flow assays.
2. Figure 10.2 Schematic representation of a sandwich assay based on a pair of aptamers. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
3. Figure 10.3 Schematic representation of LFA for salivary α‐amylase. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
4. Figure 10.4 Schematic representation of ATP detection by split aptamers. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
5. Figure 10.5 Schematic representation of β‐conglutin detection by competitive assay. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
6. Figure 10.6 Schematic representation of OTA detection by competitive assay. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
7. Figure 10.7 Schematic representation of aflatoxin B1 competitive assay dipstick detection.
8. Figure 10.8 Schematic representation of bacteria detection with digoxigenin‐5′‐reporter aptamer‐3′‐biotin‐streptavidin‐AuNP conjugates. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
9. Figure 10.9 Schematic representation of adenosine–cocaine detection. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
10. Figure 10.10 Schematic illustration of thrombin detection using two kinds of gold nanoparticles conjugates. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
11. Figure 10.11 Schematic representation of thrombin detection based on aptamer‐cleavage reaction. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
12. Figure 10.12 Schematic representation of IgE detection based on aptamer‐phage reporters. (a) Configuration of lateral flow assay; (b) the principle of visual detection in the presence and absence of analyte.
13. Figure 10.13 Schematic representation of ATP detection based on aptamer‐gated silica nanoparticles. (a) Configuration of lateral flow strip; (b) the principle of visual detection in the presence and absence of analyte.
14. Figure 10.14 Schematic representation of bacterium detection based on aptamer‐mediated strand displacement amplification. (a) Configuration of lateral flow assay; (b) Principle of visual detection in the presence and absence of analyte.
15. Figure 10.15 Schematic representation of β‐conglutin detection combining competitive assay with recombinase polymerase amplification and lateral flow. (a) Configuration of lateral flow strip; (b) Principle of visual detection in the presence and absence of analyte.
Chapter 11
1. Figure 11.1 Interrogation modes for commercial surface plasmon resonance (SPR) instruments.
2. Figure 11.2 Design of the aptasensor for lysozyme detection in wines.
3. Figure 11.3 Dual aptamer using the combination of SPR assay and secondary aptamer‐conjugated AuNP. (a) The scheme depicts the principal of SPR assay using secondary aptamer conjugated‐AuNP. (b) The pair of aptamers IF1‐IF12, IF4‐IF22, IF10‐IF12, and IF10‐IF22 were rechecked using aptamer conjugated‐AuNP. (c) The dose‐dependent binding assays of the pair of aptamers IF10‐IF22 using SPR‐AuNP.
4. Figure 11.4 Schematic overview of strategy for TC detection. The TC aptamer was first immobilized on an SPR chip, followed by the covalent attachment of aptamer probes. TC binding was performed in both buffer and Sincheon river samples, followed by the subsequent binding of GNS‐antiTC's for further SPR signal amplification.
5. Figure 11.5 (a) PLP design. The PLP contains target‐complementary sequence (T), general sequence (G), and tag sequence (Tag). Tag sequence is unique for each PLP and complementary to the capture probe immobilized on the chip surface. The general sequence provides the repeats on the RCA products for AuNP binding. “C” means the cutting site recognized by restriction endonuclease to remove the RCA products from the chip surface after the detection is completed. (b) Schematic representation of the proposed detection strategy. (c) Real‐time detection of multiple point mutations by the SPR sensor.
6. Figure 11.6 Schematic illustration of AuNPs and strand displacement cycle signal amplification‐based SPR assay for the detection of adenosine.
7. Figure 11.7 General principle of SPR imaging [48].
8. Figure 11.8 Schematic of different sensor configurations investigated.
9. Figure 11.9 The selection process of DNA aptamers against S. typhimurium using the QCM‐based SELEX.
10. Figure 11.10 (a) Schematic representation of the selective collection and detection of target CCRF‐CEM cells on the magnet–QCM system using aptamer‐conjugated magnetic beads. (b) Schematic diagram of the apparatus for magnet–QCM experiments.
11. Figure 11.11 The schematic representation of the sandwich QCM‐D biosensor for on‐line detection of thrombin. The solid (dashed) curves show the corresponding frequency (dissipation) shift for the abovementioned three steps.
12. Figure 11.12 Schematic representation of the fabrication of the QCM biosensor for analysis of leukemia cells.
13. Figure 11.13 Schematic representation of ATP detection through DNAzyme‐activated two‐cycle amplification strategy based on the strand‐displacement polymerization activity, which released AuNP‐functionalized probes captured on the QCM biosensor.
14. Figure 11.14 Concept of the novel QCM detector based on the phase shift measurements. The piezoelectric sensor is supplied with a constant frequency f _t within its resonant frequency band; the phase shifts resulting from surface interactions are then monitored using the circuit consisting of the highly stable source of interrogation voltage and mixer‐based phase shift detector.
15. Figure 11.15 Schematic of the ITC instrument.
16. Figure 11.16 Raw data (upper panel) generated by an ITC experiment represent the heat released (or adsorbed) during titration (μcal s⁻¹). These raw data are converted into the binding isotherm (lower panel) by integration of each injection producing the thermal energy (ΔH) of each titration step. The signal is reduced until only the background heat of dilution remains. Corrections of such heat are necessary and are usually performed by control experiments. The change in enthalpy (ΔH), the stoichiometry (n), the dissociation constant (K _d), and affinity (1/K _d) can be derived from the binding isotherm. The change in enthalpy is presented by the distance of the two asymptotic lines corresponding to the minimal and maximal heat formation. Stoichiometry is molar ratio at the inflection point of the sigmoidal curve. The slope at the inflection point presents the association constant (affinity).
17. Figure 11.17 (a) A cartoon representation of the mechanism of genetic regulation by the guanine riboswitch. Early in transcription, the aptamer domain of the riboswitch forms, in which the terminal loops (L2/L3) form a tight interaction but the three‐way junction is locally disordered. In the presence of high concentrations of guanine or hypoxanthine, ligand binding stabilizes the riboswitch core and the P1 helix, allowing the mRNA to form the terminator element. Without ligand binding, the 3′‐side of the P1 helix and the 5′‐side of the terminator are used to form an anti‐terminator element, allowing transcription to continue. (b) Secondary structure representation of the aptamer domain, highlighting the ligand‐binding pocket (nucleotides in light gray box in bold alphabet and C₇₄ in square box) in the presence of hypoxanthine (HX in highlighted oval box). Nucleotides highlighted in bold are at least 90% conserved across purine riboswitch phylogeny. (c) Global structure of the guanine riboswitch, with coloring consistent with the secondary structure.
18. Figure 11.18 Representative data of 2,6‐diaminopurine binding the (a) A35U, (b) C61U, and (c) G37A mutants of GRA RNA at 30 °C in a buffer containing 50 mM K⁺‐HEPES, pH 7.5, 100 mM KCl, and 10 mM MgCl₂.
19. Figure 11.19 Sequence of the aptamer and chemical structure of ligands. Compared to MN4, aptamer MN19 has three base pairs removed from the end of stem 1. Aptamer WC contains a change at the three‐way junction in which A21 has been replaced with a C, restoring the Watson–Crick base pair. MN16 is a weak‐binding variant in which T19 has been swapped for an A. Dashes between nucleotides indicate Watson–Crick base pairs, and dots indicate non‐Watson–Crick base pairs.
20. Figure 11.20 Structures of the molecules used in this study. In (a) are cocaine, atropine, and the cocaine metabolites investigated. In (b) are quinine and the different analogs of quinine studied. The chemical groups on quinine that are most important for aptamer binding are in bold and are drawn with thicker bonds.
21. Figure 11.21 (a) Molecular interactions of the T2 aptamer and tebuconazole measured by ITC analysis with 50 mM tebuconazole titrated into 0.025 mM T2 aptamer. (b) Molecular interactions of the T2 aptamer and inabenfide measured by ITC analysis with 50 mM inabenfide titrated into 0.025 mM T2 aptamer.
22. Figure 11.22 MST setup and experiments.
23. Figure 11.23 Affinity and specificity of T5 testosterone aptamer candidate studied by MST.
24. Figure 11.24 (a) The corresponding MST curves of CA solutions incubated with FAM and FAM‐Apt, respectively. (b) Binding of CA to FAM‐labeled aptamers and FAM. The concentration of the FAM‐Apt was kept constant at 0.05 μM, while the concentration of CA was varied from 0.03 to 250 μM. Error bars indicate the SD of three independent experiments.
25. Figure 11.25 Full binding curve obtained with MST for aptamer Tyr_10 and its corresponding secondary structure predicted by Mfold software.
Chapter 12
1. Figure 12.1 DNA‐polymerase activity of Pol κ and Pol β in the presence of aptamer IKL5.
2. Figure 12.2 The detection limit of 1 nM could be readily achieved with the concentrations of ATP in the range from 1 to 20 nM.
Chapter 13
1. Figure 13.1 Overview of the features and innovation models for aptamer diagnostics.
2. Figure 13.2 (a) In‐round size control with Bioanalyzer® for ligand pool amplification to avoid elongated and truncated PCR parasites. (b) An automated SELEX platform.
3. Figure 13.3 (a) Microfluidic and (b) microarray SELEX with an advantage for parallel examination of ligand pool specificity against target and nontarget proteins in selection.
4. Figure 13.4 (a) A pipeline of in silico 3D structure prediction for an aptamer sequence. (b) In silico aptamer‐hemagglutinin protein (H1) docking simulation experiments.
5. Figure 13.5 (a) Bacterial protein expression profiling on an aptamer microarray with fluorescent labels. (b) Simultaneous detection of an E. coli protein (light gray spots, Cy3 signal) and thrombin (dark gray spots, Cy5 signal) on an aptamer microarray.
6. Figure 13.6 (a) Cancer cell surface recognition and fluorescent Dox drug delivery by an aptamer for cancer cell theranostics. (b) Decoy oligodeoxynucleotide (dODN) as an intracellular aptamer targeting an oncogenic transcription factor for cancer inhibition.
7. Figure 13.7 Immunocytochemistry with aptamers: prostate cancer cell lines (a) LNCaP (AMACR(+)) and (b) PC3 (AMACR(−)) stained with Cy3‐labeled AMACR aptamer.

Aptamers for Analytical Applications

Affinity Acquisition and Method Design

Copyright

Dedication

About the Author

Foreword

Preface