*3.3. Lateral Flow Assays*

Lateral-flow assays (LFAs), also known as immunochromatographic assays (ICAs), are among the most widely used and popular methods in detecting various analytes such as microorganisms, pesticides, heavy metals, diseases biomarkers, and mycotoxins. In recent years, researchers have paid more attention to screening mycotoxins by LFA. LFA is based on the movement of fluid sample across the membrane by capillary force and binding reaction between antibody-antigen or nucleic acid-target analyte [67]. The standard LFA strip is comprised of four parts including a sample pad (the area where the sample is dropped); a conjugate pad (the area where biorecognition element conjugated with label is immobilized); a reaction nitrocellulose membrane (the area containing test line and control line for target binding to antibody or nucleic acid probe); and absorbent pad (as a wick to reserve additional fluid flow) [68,69]. Sandwich mode (for large analytes) and competition mode (for small analytes) are the two most widely used detection formats. Competitive mode is suitable for mycotoxins with low molecular weight and single epitope.

The optimization of the experimental conditions is crucial to develop a LFA with excellent performance and high sensitivity. High sensitivity, low immunoreagent consumption, and ideal color intensity are major parameters for the construction of LFAs. Utilization of an appropriate label is important for a sensitive analysis. Different colored labels such as colored latex beads, AuNPs, magnetic particles (MPs), carbon nanostructure, and enzymes have been used for developing LFA. In addition to sensitivity, label should not change the features of biorecognition element, and it must create stable conjugation with recognition element. AuNPs have been frequently used colorimetric labels in developing LFA strip due to having all the mentioned features [68–70]. Di Nardo et al. developed a novel LFA using dual color AuNPs and a single Test line for simultaneous determination of AFB1 and type-B fumonisins (FMBs) [71]. In this assay, red (spherical, mean diameter ≈ 30 nm) and blue (desert rose-like, mean diameter ≈ 75 nm) AuNPs were conjugated to anti-aflatoxin and anti-fumonisins antibodies, respectively. The single test line was formed by spraying the mixture of two antigens including AFB1-BSA and FMB-BSA. According to the competitive format, mycotoxin-free samples provided a purple test line due to the combination of the red and blue AuNPs. Contaminated samples with AFB1 or FMBs resulted in the blue and red color Test line, respectively. The simultaneous presence of both mycotoxins provided the usual disappearance of the Test line. (Semi-) quantitative analysis was obtained using a simple smartphone and RGB colorimetric analysis. The use of a single strip to multiplex analysis provided a simple, rapid, low-cost and reagent-saving assay. The developed strips with LOD at 0.5 and 20 ng·mL−<sup>1</sup> for AFB1 and FMB1, respectively, were employed to determine these two mycotoxins in wheat and pasta samples.

Conventional AuNPs-based LFA suffers from a major challenge in measuring target concentration in complex food matrices with dark color due to its poor resistance to the background matrix and color interference. To address this issue, Hao et al. developed a novel LFA using bifunctional magneto-gold nanohybrid (MGNH) label as a hetero-structured nanomaterial for the simultaneous magnetic separation and colorimetric detection of OTA in grape juice [72]. In this assay, MGNH-labeled monoclonal antibodies (mAb) were used for the MGNH-mAb-OTA complex formation and subsequently rapid separation of the complex from sample using an external magnetic field. Then, MGNHmAb-OTA complex was resuspended in buffer and applied on LFA strip for colorimetric detection (Figure 5a). Grape juice with purple color and high concentrations of sugar, pigment, and tannins was used as complex matrix to evaluate the designed method. The novel LFA was highly sensitive with LOD at 0.094 ng·mL−<sup>1</sup> . The assay showed high accuracy, reproducibility, practicability, and short detection time (10 min of magnetic separation and 5 min of immunoreaction).

Most of the multiplex LFAs for mycotoxins analysis have been designed for detection of only two or three kinds of mycotoxins [73], while sometimes more than this occurs in some foods such as cereals. On the other hand, quantitative analysis is a main issue in LFA technology which is often carried out by desktop readers or handheld readers. These

devices are slightly inferior in terms of popularity, portability, and timely data sharing compared to smartphone-based analysis [74]. Therefore, these existing limitations must be overcome to receive a practical LFA for multiplex and on-site detection. For this purpose, Liu et al. developed two kinds of multiplexed LFA strips using AuNPs and time-resolved fluorescence microspheres (TRFMs) as label for the detection of AFB1, zearalenone (ZEN), deoxynivalenol (DON), T-2 toxin (T-2), and fumonisin B1 (FMB1) in cereals (Figure 5b) [30]. Five test lines were sprayed on a single test strip for each mode of detection. Quantitative results were obtained using a smartphone dual detection mode device. The visual LODs of AuNPs-LFA were 10, 2.5, 1, 10, and 0.5 ng·mL −1 for AFB1, ZEN, DON, T-2 and FMB1, respectively. In the TRFMs-LFA format, LODs were 2.5, 0.5, 0.5, 2.5, and 0.5 ng·mL −1 , respectively for the mentioned mycotoxins. Quantitative LODs (qLODs) for these mycotoxins were obtained 0.59, 0.24, 0.32, 0.90, and 0.27 ng·mL −1 (in AuNPs-LFA), and 0.42, 0.10, 0.05, 0.75, and 0.04 ng·mL −1 (in TRFMs-LFA). TRFMs-LFA was more sensitive than AuNPs-LFA due to large surface area and stokes shift of TRFMs. On the other hand, AuNPs was low-cost, more popular, stable and easy to synthesize. The assay was reliable, quantitative and highly sensitive for on-site detection of multiple mycotoxins. However, a main problem of a multiplex LFA is the cross reactivity between Ag-Ab pairs, so that the developed LFA was able to detect 20 mycotoxins from five classes. −1 −1 −1 −1

**Figure 5.** (**a**) Schematic illustration of the detection principle of the MGNH-based LFA strip; (**b**) smartphone-based AuNPs and TRFMs-LFAs for multiplex mycotoxins detection; (**c**) aptamer-based LFA for ZEN detection in the presence and absence of ZEN analyte. Reproduced from [30,72,75], respectively, with permission.

−1 In addition to AuNPs, colloidal carbon can be used as a colored label in LFAs. It is comparatively inexpensive and can be synthesized in a large scale. Furthermore, it shows high chemical stability and recognizable color to develop LFA with high sensitivity. Many colloidal carbon-based-LFA have been developed for detection of different analytes. For example, Yu et al. proposed a LFA using Graphene oxide (GO) and carboxylated GO as labels for AFB1 detection [31]. GO can be easily conjugated with biomolecules without any additional activation due to having a large variety of oxygen-containing chemical groups. Moreover, it shows excellent hydrophilicity and high stability at room temperature. In this study, mA against AFB1 was conjugated with GO. The vLOD and cut-off values for AFB1 were 0.3 and 1 ng·mL −1 , respectively. It was exhibited that GO and carboxylated GO can be used as viable black labels to develop a low-cost LFA compared to the AuNPs labels. The method was successfully applied for AFB1 detection in peanut oil, maize, and rice.

In recent years, many efforts have been made to replace aptamer with antibody in LFA technology due to potential advantages of aptamers (as mentioned earlier). However, aptamer-based assays mostly require laboratory infrastructure, which limits their application. Incorporating the advantages of aptamers and LFA technology is a basic step to complete the user-friendliness of aptamers. In this context, Wu et al. developed a competitive aptamer-based LFA for rapid and sensitive detection of ZEN [75]. The assay was based on competition between the DNA 1 on the test line and ZEN in the for binding to AuNPs-labeled aptamer. In the absence of ZEN, AuNPs-labeled aptamer hybridized with DNA 1 on the test line and DNA 2 on the control line, resulting in two colored lines on the strip while, in the presence of high concentration of ZEN, the test line was colorless (Figure 5c). The proposed aptamer-based LFA with high specificity and sensitivity (vLOD and qLOD of 20 and 5 ng·mL−<sup>1</sup> , respectively) and short detection time (5 min) was applied for ZEN detection in spiked corn samples.

Representative examples of recent developed LFA test strips for the detection of mycotoxins are reported in Table 4.



**Table 4.** Representative examples of recent developed LFA strip tests for the detection of mycotoxins.
