**3. Small-Molecule–Aptamer Interaction and Characterization**

The secondary structures of aptamers often organize into complex three-dimensional structures to maintain global stability and form functional conformations. Therefore, a stable aptamer can strongly bind to small molecules and shape the aptamers-target complex via weak noncovalent interaction forces, including hydrogen bonding, π-π stacking, van der Waals forces, hydrophobic, and electrostatic interactions. The aptamer-target interactions may either depend on the availability of narrow binding pockets in the three-folded structures of aptamers or the chemical characteristics of targets and the aptamer candidate or the length of the oligonucleotides, for example, removing excess flanking nucleotides outside the binding site (truncated aptamer), which is also a strategy to improve binding affinity and specificity [66,67]. In order to assess the binding strength and specificity of the interaction between an aptamer and its target, binding assays are used to determine the dissociation constant (*K*d) (Table 1), i.e., the binding affinity between an aptamer and its cognate target (and between an aptamer and its non-cognate targets), where the lower the *K*<sup>d</sup> value, the stronger the interaction. Several factors may influence the *K*<sup>d</sup> value, such as temperature, pH value, ionic concentrations, and hydrophobicity of the solution [66]. Below, we discuss the six standard assays used for characterizing the interactions between small-molecule contaminants and aptamers generated using DNA/RNA Capture-SELEX (Figure 2). In contrast with traditional methods, such as HPLC and LC-MS, these assays are highly sensitive, non-toxic, and inexpensive.

**Table 1.** A list of small molecule contaminant-specific aptamers developed by Capture-SELEX method as of to date.


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184

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187


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#### *3.1. SYBR Green I (SGI) Assay*

The binding affinity of Capture-SELEX-generated aptamers for small molecules can be identified by a novel label-free fluorescence SGI assay [24,68,70,73,83]. SGI is a fluorescent nucleic-acid-intercalating dye that was introduced in the 1990s and is widely applied in real-time PCR [92–94], fluorescent gel imaging [95,96], and flow cytometry [97,98]. It intercalates into the minor groove of DNA base pairs or adenine–thymine-rich stem-loop sites to form a fluorescent complex with DNA [99]. The fluorescence intensity of SGI–DNA complexes is measured in the range of 505 to 650 nm, with an excitation wavelength of 495 nm and an emission wavelength of 525 nm, using a spectrophotometer or a microplate reader; thus, an SGI assay identifies the binding affinity of aptamers for small-molecule targets by detecting the change in the fluorescent signal induced by the dissociation of SGI from the aptamers upon their binding with these small-molecule targets, and therefore the fluorescence intensity decreases (Figure 2A). The fluorescence intensity will be recovered as the concentration of the aptamers' small-molecule targets increases.

**Figure 2.** A schematic illustration of six commonly used methods for detecting interactions between small molecule and aptamer. (**A**) SYBR green I (SGI) assay. (**B**) Carbon nanoparticles (CNPs) fluorescence quenching assay. (**C**) Gold nanoparticles (AuNPs) colorimetric assay. (**D**) Microscale thermophoresis (MST) assay. (**E**) Graphene oxide (GO)-based fluorescent assay. (**F**) Isothermal titration calorimetry (ITC) assay.

#### *3.2. Carbon Nanoparticle (CNP) Fluorescence Quenching Assay*

CNPs can be readily synthesized [45] in research laboratories, have low toxicity, and are biocompatible [100]. Due to the strong fluorescence-quenching ability of CNPs' sp2 and π-rich structures, they are useful for characterizing the binding affinity of aptamers for small molecules. This means that when an aptamer is adsorbed onto the surface of CNPs, ππ stacking interactions that form between the nucleobases and nucleosides of the aptamer and the CNPs result in fluorescence quenching. Therefore, in the absence of aptamers' small-molecule targets, aptamers are bound to CNPs, resulting in fluorescence quenching, whereas in the presence of aptamers' small-molecule targets, aptamers are bound to these targets rather than CNPs, and thus fluorescence is not quenched (Figure 2B) [101].

## *3.3. Gold Nanoparticle (AuNP) Colorimetric Assay*

The AuNP colorimetric assay is widely used for characterizing the binding affinity of an aptamer for its target [24,45,70,71,76,80,83,84]. It is a label-free assay as AuNPs allow the identification of a target reaction based on colorimetric signals. AuNPs can be synthesized by reducing chloroauric acid with citric acid and ascorbic acid (the Turkevich method) [102] or with sodium citrate [103]. The Turkevich method affords AuNPs coated with negatively charged citrate ions, and thus the AuNPs are treated with sodium chloride to neutralize their surface charge and thereby induce their aggregation (Figure 2C) [104]. Owing to the electrostatic screening effect, the surface plasma resonance absorption peak of AuNPs undergoes a red shift from 520 nm to 650 nm, which is measured using a microplate reader and reflects the binding affinity of aptamers to their small-molecule targets. In the absence of their small-molecule target, aptamers are adsorbed onto the surface of AuNPs, increasing their colloidal stability of AuNPs and reducing their salt-induced aggregation [105]. However, in the presence of their small-molecule targets, aptamers binds to their targets rather than AuNPs, which decreases the colloidal stability of AuNPs and thereby increases their salt-induced aggregation. This results in a solution containing AuNPs, aptamers, and their small-molecule targets changing color from red to pink to purple or even to blue or gray [70].

#### *3.4. Microscale Thermophoresis (MST) Assay*

The MST assay is a powerful tool for testing the binding affinity of aptamers for their small-molecule targets as it only requires a small amount of sample (up to 4 μL), involves a simple preparation process, and rapidly provides accurate results (within 15 min) [75,76,80]. It is a fluorophore-labeled and immobilization-free assay that is carried out using a Monolith NT.115 instrument. As shown in Figure 2D, the capillary tray of this instrument can accommodate up to 16 thin glass capillaries for operation. An infrared laser heats a specific capillaries area, creating a microscopic temperature gradient on them. Thus, when an aptamer binds to its small-molecule target, there is a significant change in fluorescence intensity due to thermophoresis. An MST binding curve (dose-response curve) is plotted using Affinity Analysis Software to represent specific binding that is specific to its small-molecule target, which automatically calculates the *K*<sup>d</sup> of an aptamer for its small-molecule target.
