*3.5. GO-Based Fluorescent Assay*

GO can be synthesized from graphite via the Hummers and Offeman method [106], which uses potassium permanganate (as the oxidizing agent) and sodium nitrate in a solution of sulfuric acid [107]. GO has an sp2 structure and numerous oxygen-containing functional groups [108], such as carbonyl groups, hydroxyl groups, and carboxylic acid groups, and thus can strongly quench fluorescence. This quenching occurs when a 5 fluorescein-labeled aptamer is adsorbed onto the surface of GO through *π*- *π* stacking interactions and hydrogen bonding (Figure 2E). When 5 -fluorescein labeled aptamer is absorbed onto GO, GO-mediated fluorescence quenching occurs. The fluorescence intensity will be recovered when the labeled aptamer is desorbed from the surface of GO in the presence of small-molecule target. The fluorescence intensity change are measured using a microplate reader, and eventually the *K*<sup>d</sup> value of the aptamer for its small-molecule target is calculated [72,77,109].

#### *3.6. Isothermal Titration Calorimetry (ITC) Assay*

The ITC assay is a label-free and immobilization-free assay for measuring the binding affinity of an aptamer for its small-molecule target [44,83] in an instrument comprising a reference cell and a sample cell (Figure 2F). The reference cell is filled with a buffer and water (without the aptamer or its small-molecule target), while the sample cell is filled with the buffer and the aptamer and is titrated via syringe with up to 20 volumes of the aptamer's small-molecule target [77]. The temperature in the two cell units is maintained at a constant value, and when the small molecule is titrated against the aptamer in the sample cell, an exothermic binding reaction occurs. The feedback system of the ITC instrument then reduces the power supply to the sample cell to prevent an increase in temperature, leading to a difference in the amount of energy supplied to the reference cell and the sample cell. Therefore, during this titration, the power supply to the sample cell continuously decreases until the small-molecule–aptamer binding has been completed. The binding affinity of the aptamer can then be determined from the energy curve generated [110]. The ITC instrument measures the exothermic reaction (heat released) and endothermic reaction (heat absorbed) when the molecules interact. The peak goes downward when the exothermic reaction occurs, whereas an upward peak is measured when an endothermic reaction occurs. The downward peak between the interaction of aptamer and smallmolecule target are measured from the ITC instrument using software such as MicroCal ITC200 and MicroCal PEAQ-ITC.

#### **4. Development of Aptamer-Based Biosensors**

To date, Capture-SELEX has been used for screening and generating a series of aptamers against small-molecule contaminants (Table 1). As aptasensors can be used for low-cost, rapid, and real-time detection of small-molecule contaminants, they have increasingly supplanted conventional antibody-based biosensors. Fluorescent aptasensors are the most popular (Table 2), and their working principle is similar to that of fluorescent-based characterization methods. Therefore, two other types of aptasensor are discussed here (Figure 3).

**Table 2.** Representative biosensors using aptamers against small molecule contaminants that are generated from Capture-SELEX.


**Figure 3.** The schematic illustration of aptasensors for the detection of small molecules using novel aptamer generated from Capture-SELEX. (**A**) Biolayer interferometry (BLI)-based aptasensor. (**B**) A lateral flow aptasensor (LFA) is generally constructed by 4 sections: sample pad, conjugate pad, nitrocellulose membrane with test line and control line, and absorbent pad. The sample flow from left to right laterally (left). When the target is loaded onto the sample pad, positive result (red band for both control and test line) is observed after 15 min (right).

#### *4.1. Biolayer Interferometry (BLI)-Based Aptasensor*

Optical-fiber-based BLI biosensors have been developed for real-time, sensitive, and rapid measurement [112] of interactions between biomolecules, including DNA– protein [113,114], antibody–antigen [115], and DNA–small-molecule-contaminant interactions [116,117]. The sensor tip of an optical fiber consists of two reflective surfaces: a streptavidin-coated biocompatible surface, which is immersed in the sample solution, and an optical layer, which functions as an internal surface (Figure 3A). The biocompatible surface is functionalized to immobilize a biotinylated aptamer and minimize non-specific binding. Inside the sensor, these two surfaces generate an interference pattern by reflecting incident white light. When an aptamer binds to its small-molecule target, a spectral redshift (Δ*λ*) occurs as the bound compound immobilizes on the tip surface and the surface thickness increases. The false-positive results from this sensor can be minimized as the

non-specific and unbound molecules can be differentiated from the molecules with high binding affinity, resulting in high detection accuracy [116].
