**1. Introduction**

Accumulating evidence suggests that small-molecule contaminants, such as antibiotics, pesticides, and plasticizers, pose a severe threat to human health and the environment worldwide. Antibiotics have been extensively used for decades to treat bacterial infections, and most are recalcitrant to biodegradation and elimination [1]. Moreover, the global overuse of antibiotics has led to their long-term persistence in the marine environment. More than 700,000 human deaths per year are attributable to bacterial resistance to antimicrobials (including antibiotics), with this number projected to increase to 10 million by 2050 [2]. Pesticides are widely used in agriculture to control pests and disease carriers. According to the World Health Organization, 193,460 unintentional deaths in 2012 were due to pesticide poisoning [3]. Plasticizers are widely applied in plastic products, such as Di(2-ethylhexyl)phthalate (DEHP), contaminating the environment. Toxic plasticizers with their toxicological effects, including endocrine disruption, carcinogenicity, and bioaccumulation potential [4–6], may enter human body via inhalation, ingestion, and dermal contact, reflecting the high risk of harm to human health.

Some traditional techniques for detecting small-molecule contaminants with high accuracy include high-performance liquid chromatography [7,8], enzyme-linked immunosorbent assays [9,10], and liquid chromatography–mass spectrometry [11,12]. However, these techniques are costly and laborious, and can only be performed by professional technicians. Therefore, there is an urgent need for robust aptamer-based biosensors (also called aptasensors), for low-cost, user-friendly, and rapid detection of small-molecule contaminants.

Aptamers are short, single-stranded DNA or RNA oligonucleotides that are able to bind its target with high affinity and specificity. The development of an aptasensor for detecting small-molecule contaminants requires the generation of a novel aptamer. It has gradually replaced traditional antibodies in some applications as they have several advantages over antibodies, such as thermal stability, low cost, amenability to chemical modification, and ease of generation [13,14]. Furthermore, when designed appropriately, the chemical modification of aptamers often does not affect—and sometimes even strengthens—their binding affinity for their targets, whereas the chemical modification of antibodies may remove their affinity for their targets [13]. Moreover, antibodies can only bind to a narrow range of targets, such as immunogenic non-toxic molecules and other molecules that do not cause strong immune responses [13]. In contrast, aptamers can selectively and tightly bind to a broad range of targets, including proteins [15–17], cells [18,19], microorganisms [20], and small-molecule contaminants [21–24], which may be either immunogenic or non-immunogenic. In addition, the in vivo generation of antibodies is a lengthy process (~6 months or longer) and requires a living host, whereas aptamers can be generated through in vitro selection (~2–8 weeks) using a method known as systematic evolution of ligands by exponential enrichment (SELEX) [13].

Three research groups independently developed the first SELEX method and all reported their findings in 1990: Tuerk and Gold [25], Ellington and Szostak [26], and Joyce [27]. Since then, many derivative methods of SELEX have been developed, such as capillary electrophoresis (CE)-SELEX [28–30], graphene oxide (GO)-SELEX [31–33], cell-SELEX [34–36], in vivo SELEX [37,38], and Capture-SELEX [22,23,39]. Capture-SELEX selects DNA or RNA aptamers that bind small-molecule contaminants and is partly based on FluMag-SELEX. Stoltenburg et al. reported FluMag-SELEX in 2005 [40], and it is based on the magnetic bead (MB)-based SELEX, whereby the fluorescein-labeled oligonucleotides is to monitor the enrichment of target-specific aptamers with the quantification in each round, and biotinylated target molecules are immobilized onto the surface of streptavidin-coated magnetic beads (SA-MBs). However, it is more challenging to immobilize biotinylated small-molecule targets than biotinylated large-molecule targets such as proteins [41], as small-molecule targets have fewer functional groups. Thus, Capture-SELEX is an effective alternative to FluMag-SELEX, as Capture-SELEX involves the immobilization of a biotinylated hybridized library on a solid support, which is more feasible than the immobilization of biotinylated targets on a solid support (Figure 1). Due to the differences in the principle, Capture-SELEX also has some deficiencies compared to other SELEX methods [41]. The false-positive may happen in the immobilization process of Capture-SELEX. The complemented library may have a dynamic dissociation equilibrium due to the weak force, and some unbound sequences (non-biotinylated) are also dissociated from the solid support. Moreover, conformational changes of the target are essential for the success of aptamer screening and diversity in the selection process. This review discusses Capture-SELEX-based selection strategies for small-molecule contaminant targets, methods used to identify and characterize small molecule–aptamer interactions, and biosensing applications of aptamers generated using Capture-SELEX.

**Figure 1.** The experimental flowchart of DNA Capture-SELEX and RNA Capture-SELEX. (**A**) DNA Capture-SELEX. Each DNA Capture-SELEX selection round consists of 7 main steps: (1) The designed ssDNA library template is hybridized with biotinylated capture-oligonucleotide. (2) The hybridized product is immobilized on the washed streptavidin-coated magnetic beads. (3–4) Target-bound sequence elution: counter selection is followed by positive selection. (5) PCR amplification was performed on the eluted ssDNA to obtain the amplified dsDNA pool, (6) and regenerate ssDNA for the next DNA Capture-SELEX round (either gel purification or magnetic bead-based method). (7) After DNA Capture-SELEX, aptamer sequences were identified through sequencing. (**B**) RNA Capture-SELEX. The principles and procedures are similar to (**A**), except that RNA Capture-SELEX consists of additional transcription (step 1) to convert DNA to RNA and reverse transcription (step 6) to convert RNA to DNA steps.

#### **2. Principle and General Procedure of Capture-SELEX for Aptamer Selection**
