*4.2. Lateral Flow Aptasensors (LFAs)*

LFAs have been developed as portable detection devices with cost-effective, rapid (≤15 min), and easy operation and broad applications, such as in the detection of pregnancy, severe acute respiratory syndrome coronavirus 2 [118,119], and infectious diseases [120]. Owing to the drawbacks of antibodies, LFAs are gradually replacing antibody-based lateral flow biosensors, although the latter remains predominantly used. In addition, LFAs have been used to detect antibody-inaccessible small-molecule contaminants [24,89,121]. Figure 3B depicts the LFA concept [121–123], in which AuNPs are typically used for colorimetric identification of the test line and control line on the strip [124]. The high affinity aptamer generated from Capture-SELEX against the specific small-molecule target that splits into two fragmented aptamers (aptamer 1 and 2; Figure 3B) [123]. A sample solution (a solution of an aptamer's small-molecule target in buffer) is loaded onto the sample pad and flows through the conjugate pad (containing AuNP–aptamer 1 conjugates) by capillary action. When aptamer–small-molecule-target binding occurs, two fragmented aptamers could rejoint the three-dimensional structure without affecting the binding affinity, and thus a red band appears in the test line. A complementary DNA (cDNA) is designed to hybridize with aptamer 1 in the control line and generate a visual signal, which is used to verify whether the aptasensor is working properly based on the appearance of red band in both presence or absence of the aptamer target. The excess sample reagent flows from the test line to the control line and to the absorbent pad eventually. Two slightly different LFAs have also been developed that use aptamers generated using Capture-SELEX. The LFA devised by Du et al. [24] combines lateral flow strips and the recombinase polymerase amplification technique for detecting erythromycin in tap water, whereas that devised by Xia et al. [89] uses aptamer EC1-34 as a recognition probe for detecting ethyl carbamate. The latter LFA differs from the former LFA only in terms of its use of a cationic polymer (such as poly(dimethyldiallylammonium chloride)) instead of streptavidin in the test line.

#### **5. Biosensor Applications**

*5.1. Food Safety Analysis*

#### 5.1.1. Veterinary Drug Residues and Pesticides

Ractopamine (RAC) is a β-adrenergic agonist used illegally as an animal feed additive for increasing skeletal muscle mass, reducing fat deposition, and increasing protein accretion in livestock [125,126]. The accumulation of RAC in animals may increase the risk of food poisoning in humans and have other adverse effects on human health, such as causing headache, tachycardia, and muscle tremors [127]. Therefore, the development of a rapid and cost-effective biosensor for RAC contamination in food is warranted. Duan et al. [82] obtained nine aptamer candidates for RAC through 16 selection rounds of Capture-SELEX. The aptamer RAC-6 showed the highest binding affinity for RAC in a GO-based fluorescent assay, with weak binding affinity for other off-target species (<22%). The researchers further developed a fluorescent aptasensor based on RAC-6 that exhibited a linear detection range of 0.33 to 331.79 nM, a low limit of detection (LOD; 0.13 nM; Table 2), and high recovery rates (82.57–104.65%). They found that RAC-6 was especially useful for detecting RAC contamination in pork samples.

λ-Cyhalothrin is a broad-spectrum pyrethroid insecticide used to control agricultural insect pests, such as Coleoptera, Lepidoptera, and mites, and thus increase agricultural productivity [128]. Compared with older-generation pesticides, the insecticidal effect of λ-cyhalothrin is 10–100 times stronger, and overuse of this insecticide may lead to food contamination [129]. Due to its toxicity, the ingestion of λ-cyhalothrin residues in food may cause serious adverse effects, including mouth ulcers, nausea, abdominal pain, and vomiting [130]. Yang et al. [76] used Capture-SELEX to obtain several candidate aptamers against λ-cyhalothrin. The aptamer LCT-1 showed the strongest affinity and

specificity for λ-cyhalothrin in a colorimetric assay and its binding affinity was further optimized by truncation. The dissociation constant of the truncated aptamer, named LCT-1-39, was improved by approximately 40 nM relative to LCT-1, and similar results were obtained using the MST assay. LCT-1 and LCT-1-39 were thus used to establish colorimetric aptasensors for detecting λ-cyhalothrin. These aptasensors demonstrated low LODs for LCT-1 and LCT-1-39 (43.8 nM and 41.35 nM, respectively; Table 2) and mean λ-cyhalothrin recovery rates of 82.93–95.50%. Compared with traditional quantification methods, these colorimetric aptasensors demonstrated more rapid detection of λ-cyhalothrin in cucumber and pear samples.

#### 5.1.2. Food Additives and Flavoring Agents

Vanillin is the second most popular flavoring agent worldwide and is used as a food additive in sweet foods and beverages, and a masking agent in numerous pharmaceutical formulations [131]. It is a phenolic aldehyde that has demonstrated antioxidant, antimicrobial, and antifungal activities in various food products [132,133]. Hence, a rapid detection biosensor is required to monitor vanillin concentrations during processed food production. Through Capture-SELEX, Kuznetsov et al. [23] obtained six aptamer candidates against vanillin and found that Van\_74 had the highest binding affinity by nondenaturing PAGE. Its specificity for vanillin was also confirmed in the presence of interferents, such as benzaldehyde, guaiacol, furaneol, ethyl guaiacol, and ethyl vanillin. The authors also found that Van\_74 was sensitive to the composition of the selection buffer. Van\_74 was then used in the development of an ion-sensitive field-effect transistor (ISFET)-based biosensor that demonstrated a low LOD (0.155 μM) and a dynamic detection range of 0.155–1 μM (Table 2). This novel aptasensor can be applied for the rapid on-site detection of vanillin contamination in coffee extracts and mixtures.

Another aroma compound, furaneol, is extensively used as an artificial flavoring agent as it imparts fruit flavor to food [134], and thus a rapid detection biosensor is required to monitor furaneol concentrations during processed food production. Komarova et al. [68] obtained eight aptamer candidates against furaneol through 13 selection rounds of Capture-SELEX. These aptamers' binding affinity for furaneol was analyzed by three methods: an exonuclease protection assay, an SGI assay, and an MB-associated elution assay. The results revealed that the aptamer Fur\_14 had the highest binding affinity for furaneol; therefore, Fur\_14 was used to develop an ISFET-based aptasensor. Fur\_14 was further modified with an alkyne label at its 5 -end, and this Fur\_14 derivative exhibited a furaneol detection range of 0.1–10 μM (Table 2).

Spermine, tyramine (TYR), and β-phenethylamine (PHE) are biogenic amines (BAs) that are typically present in foodstuffs. As the consumption of foods containing high concentrations of BAs may cause toxic effects, biosensors are needed for BA detection in foods [135,136]. Tian et al. [44] obtained aptamer candidates against spermine by Capture-SELEX selection and tested them using ITC and fluorescence assays. The aptamer APJ-6 showed the highest affinity and specificity for spermine and was, thus, used to develop a fluorescent aptasensor for spermine detection in pork samples. This aptasensor demonstrated a linear detection range of 0.1–20 nM and a low LOD (0.052 nM). For detecting TYR and PHE, Kuznetsov et al. selected and isolated several aptamers using Capture-SELEX. The selection process was monitored by the melting temperature (Tm) in the screening process, and Tm peaked during the 14th round for both TYR and PHE. The aptamers TYR-2 and PHE-2 were identified to have the strongest binding affinity and specificity for TYR and PHE, respectively, based on a GO-based fluorescent assay. TYR-2 and PHE-2 were then used to develop fluorescent aptasensors for the detection of TYR and PHE in pork and bear meat samples. These aptasensors demonstrated LODs for TYR and PHE of 2.48 and 3.22 nM, respectively (Table 2), with target recovery rates in the range of 95.6–104.2%, suggesting their efficacy in detecting TYR and PHE in foods.

#### *5.2. Aquatic Environment*

#### 5.2.1. Veterinary Drug Residues and Pesticides

Erythromycin is a broad-spectrum macrolide antibiotic used to treat diseases such as diphtheria, pertussis, and bacillary angiomatosis [137]. The natural degradation of erythromycin is prolonged due to its stable structure, leading to increased erythromycin resistance among bacteria [138]. As erythromycin diffuses rapidly into most tissues of the human body, erythromycin pollution of environmental media poses a serious threat to human health, in addition to the ecosystem. Du et al. [24] obtained 10 aptamer candidates against erythromycin through 20 selection rounds of Capture-SELEX. The binding affinity and specificity of the candidates were determined using an SGI fluorometric assay, an AuNP-based colorimetric assay, a quartz crystal microbalance with dissipation assay, and an agarose chasing diffusion assay, resulting in the selection of the aptamer Ery\_06 for the development of a novel LFA. This LFA demonstrated an erythromycin-detection range of 250–500 pM in water samples, with a low LOD (3 pM; Table 2) and rapid detection (within 15 min), suggesting its efficacy for erythromycin detection in water.

Roxithromycin is a macrolide antibiotic that poses a similar risk to the ecosystem and human health as erythromycin, indicating the need to establish a rapid and effective detection device for monitoring roxithromycin residues in environmental media. Jiang et al. selected aptamer candidates against roxithromycin after 16 selection rounds of Capture-SELEX. The aptamer Ap01 demonstrated the highest affinity and specificity for roxithromycin, as indicated by the results of an SGI assay, and was therefore selected for the development of a colorimetric aptasensor for roxithromycin. The developed aptasensor demonstrated a low LOD (0.077 μM) for roxithromycin in water samples (Table 2) and high recovery rates in the range of 90.48–109.39%.

#### 5.2.2. Toxins and Plasticizers

Gymnodimines (GYMs) are fast-acting cyclic imine toxins that are biosynthesized by dinoflagellates and have deleterious effects on the aquatic environment with the accumulation. The contaminated environment can have serious toxic effects on filter feeding shellfish and thereby pose a threat to human health [139]. Zhang et al. [75] used Capture-SELEX to screen and obtain six aptamer candidates against gymnodimine-A (GYM-A). G48 exhibited the highest binding affinity (*K*d: 288 nM) and was therefore chosen for further optimization and investigation. The truncated aptamer G48nop demonstrated an improved *K*<sup>d</sup> value of 34.5 ± 1.72 nM and high specificity for GYM-A. A novel BLI-based aptasensor was established using this aptamer that detected GYM-A in the range of 55–1400 nM (linear range of 55–875 nM) and had a low LOD (6.21 nM; Table 2). This BLI-based aptasensor also demonstrated high recovery rates in the range of 96.65%–109.67%, indicating that is reliable and efficient in detecting and monitoring GYM-A in water samples.

Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer that is widely used as an additive in packaging materials, and its residues are known to accumulate and dissolve in water [140]. DEHP is also a well-known endocrine disruptor that can enter the human body through ingestion of food or water and inhalation with contaminated air that disrupts the immune system. Lu et al. [71] selected aptamer candidates against DEHP through eight rounds of Capture-SELEX. Upon high-throughput sequencing and characterizing the candidate aptamers using an AuNP colorimetric assay and localized surface plasmon resonance, aptamer 31 was revealed to have high affinity and specificity. Aptamer 31 was thus used to develop an ultrasensitive electrochemical impedance spectroscopy aptasensor to detect DEHP in real water samples; this aptasensor demonstrated a low LOD (0.264 pM; Table 2) and a mean recovery rate ranging from 76.07% to 141.32%.

#### *5.3. Other Potential Applications*

Synthetic riboswitches can have several biotechnological applications, such as regulating gene expression, e.g., the construction of genetic circuits [141,142]. Natural riboswitches are mainly found in bacteria, while synthetic riboswitches are artificially generated by

combining aptamer domains (using in vitro SELEX method) with expression platforms to regulate gene expression via small-molecule-RNA interactions [143,144]. However, using the conventional SELEX method, only a ciprofloxacin riboswitch aptamer has been developed, as most aptamers have limitations, such as excellent binding affinity and conformation switching, and require cellular screening after in vitro selection [145]. Subsequently, Boussebayle et al. [146] identified a paromomycin riboswitch aptamer using Capture-SELEX and found it had a high affinity (*K*d: 21 nM) using an ITC assay. Through further in vivo selection, this aptamer was revealed to have riboswitching properties. This work has introduced an efficient protocol for developing synthetic riboswitches and boosted the development of real-time intracellular biosensors for monitoring metabolic flows in living cells.

Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin produced by fungi and is known to contaminate cereal grains and other crops [147]. Due to its high estrogenic activity, long-term intake of ZEN residues adversely affects human health by causing cervical cancer or hyperestrogenic syndrome [147,148]. Zhang et al. [84] obtained aptamer Z100 against ZEN after eight rounds of Capture-SELEX. Z100 was shown to have high affinity and specificity for ZEN in a fluorescence assay, and hence was selected to develop a rapid and on-site AuNP-based label-free aptasensor for detecting ZEN in agricultural produce. The developed aptasensor had a low LOD (12.5 nM), a linear detection range of 12.5–402.1 nM (Table 2), and high recovery rates in corn powder and feed (96.42–99.78% and 95.99–103.73%, respectively). This study revealed the great potential for developing aptamer-based inhibitors for ZEN to enhance animal feed safety.

Fenitrothion (FEN) is a broad-spectrum organophosphorus insecticide mainly used to control insect pests in agriculture [149]. As FEN is available at a low cost, large amounts of FEN are frequently applied in agriculture. High concentrations of FEN residues have been found in foods, which has become a great concern for human health and the environment. Trinh et al. [85] screened aptamer candidates against FEN through Capture-SELEX. In the thioflavin T (ThT) displacement assay, the aptamer FenA2 was identified to exhibit high-affinity FEN binding, as indicated by the loss of fluorescence. FenA2 was further optimized and used to develop a label-free ThT sensor. The developed aptasensor has a G4-quadruplex-like structure and a low LOD (14 nM; Table 2). This aptamer may be further optimized to develop a real-time FEN-detecting aptasensor.

#### **6. Conclusions**

Small-molecule contaminants are ubiquitous in the aquatic environment, agriculture produce, and animal feed due to the overuse of small molecules such as antibiotics and pesticides, and these contaminants pose a serious threat to human health and the environment. Owing to the few functional groups on these small molecules, screening aptamers against them is more challenging than doing so against large-molecule targets. Compared with other SELEX approaches, which involve the immobilization of small-molecule targets, the Capture-SELEX approach is more feasible, as they involve the immobilization of a biotinylated ssDNA/RNA library against which the binding affinity and specificity of small-molecule targets can be screened. To date, fewer than 50 studies have reported using Capture-SELEX to identify novel aptamers against specific small-molecule contaminants, suggesting that this process remains challenging, such as false-positive and lack of diversity. However, this research field has recently been receiving increasing attention from scientists. We hope that this review will encourage further research into the use of Capture-SELEX in generating aptamers against small-molecule contaminants. Six small-molecule aptamer characterization methods are introduced in this review. The high affinity and specificity aptamer work as a biorecognition element in aptasensor to detect specific small-molecule contaminants in environmental media and agricultural produce. This will help to improve food safety, aquatic environments, and agricultural crop production.

**Author Contributions:** Conceptualization, S.Y.L. and C.K.K.; writing—original draft preparation, S.Y.L. and C.K.K.; writing—review and editing, S.Y.L., H.L.L. and C.K.K.; visualization, S.Y.L. and C.K.K.; supervision, C.K.K.; funding acquisition, C.K.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Shenzhen Basic Research Project [JCYJ20180507181642811]; National Natural Science Foundation of China Project [32222089]; Research Grants Council of the Hong Kong SAR, China Projects [CityU 11100222, CityU 11100421, CityU 11101519, CityU 11100218, N\_CityU110/17]; Croucher Foundation Project [9509003]; State Key Laboratory of Marine Pollution Director Discretionary Fund; City University of Hong Kong projects [7005503, 9667222, 9680261] to C.K.K.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
