*3.1. Solution-Based Assays*

In-solution assays rely on the colloidal interaction between different biomolecules without their immobilization on a substrate. The colorimetric signal is usually generated after cascade additions of reagents in a total volume of some hundreds of microliters where the colorimetric probe, the bioreceptor, and the target can meet. Numerous reports of homogenous solution-based assays for mycotoxins detection have been developed owing to their rapid operation and facile design. Most of these patterns are based on targetinduced/disabled nanoparticles aggregation, enzymes or enzyme-like catalytic activities, or chemical dyes in label-based or label-free formats.

As described earlier, noble nanoparticles are characterized by an intrinsic size- and distance-dependent optical signal. Particularly, AuNPs showed a great success in the design of solution-based colorimetric assays using different aggregation approaches.

As an example, a simple colorimetric assay has been described by Chotchuang et al. for the detection of fumonisin B1 using dispersed cysteamine-functionalized gold nanoparticles (Cys-AuNPs) [42]. The target mycotoxin was first hydrolyzed (HFB1) to then induce NPs aggregation via hydrogen bonding. At an optimal pH of 9, color change from wine-red to blue-gray and absorption spectra from 520 nm to 645 nm can be either observed visually or measured by a UV-vis spectrophotometer. This 3 min sensing approach achieved satisfactory results between 2–8 µg kg−<sup>1</sup> FB1 concentration range and a detection limit of 0.90 µg kg−<sup>1</sup> . Although this method was successfully applied to corn samples, its specificity could be decreased in the presence of interfering molecules that are able to aggregate AuNPs in the absence of target. Therefore, the use of specific bioreceptors such as aptamers is more common in AuNPs-based detection of mycotoxins.

Nucleic acid strands are known to protect AuNPs against salt-induced aggregation because of strong van der Waals interactions between DNA bases and gold [48]. This electrostatic affinity induces aptamer's adsorption which stabilizes the dispersed nanoparticles. Upon mycotoxin recognition, aptamers desorb from the surface of AuNPs to preferentially complex with the target. Subsequently, stable gold aggregates are formed under the action of electrolytes or cationic polymers leading to the solution color changing. According to this aptasensing strategy, label-free AuNPs-based aptasensors were frequently reported for the rapid detection of mycotoxins [49], including ochratoxin A [38,40,50], aflatoxin B1 [27,51], and zearalenone [52].

Interestingly, Liu and collaborators have found that aromatic targets sch as ochratoxin A can also adsorb on the surface of AuNPs after aptamer folding and further inhibit salt-induced aggregation [40]. This limitation renders the assay unreliable at high target concentrations. To expand the detection range, they described a double calibration curve method in which both aggregation mechanisms are combined using two experimental conditions (Figure 3a). Using this system, they claimed that the OTA concentration range could be widened from 10−10.5–10−<sup>8</sup> to 10−10.5–10−6.5 <sup>g</sup>·mL−<sup>1</sup> .

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**Figure 3.** (**a**) Gold nanoparticle-aptamer-based LSPR sensing of ochratoxin a at a widened detection range by double calibration curve method; (**b**) Colorimetric aptasensor for the ochratoxin A (OTA) assay based on the structure-switching of OTA aptamer coupling with alkaline phosphatase (ALP)-MnO<sup>2</sup> cascade catalytic reaction; (**c**) Multicolor colorimetric detection of OTA via structure-switching aptamer and enzyme-induced metallization of gold nanorods; (**d**) pH-Resolved simultaneous detection of four targets based on magnetic separation of two GO platforms with allochroic dyes. Reproduced with permission from [37,40,53,54].

−1 −1 Although the achieved limits of detection are demonstrated to be in compliance with regulatory levels, such colorimetric assays present relatively high LOD values compared to other optical or electrochemical approaches. The lack of sensitivity was explained by the number of NPs required to generate a significant color change. Aiming to overcome this constraint, Xiao et al. [39] described a colorimetric aptasensor based on the disassembly of aggregates of oriented AuNP dimers by target molecules. This AuNPs dimer-based sensor has shown better stability, sensitivity (LOD = 0.02 µg·L −1 ) and OTA detection dynamic range (0.08–100.8 µg·L −1 ). Furthermore, it was noted that the disassembly of AuNPs dimers was faster than that of large aggregates reducing thus the analysis time [41].

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In another option, the colorimetric signal of substrate-free assays can be amplified by catalytic reactions using either enzymes or nanozymes. Harnessing the inherent peroxidaselike activity of AuNPs, Sun et al. [41] developed a rapid apta-assay specific to zearalenone. In this assay, ZEN aptamer inhibits the catalytic activity of AuNPs in the presence of H2O<sup>2</sup> and TMB. The solution remains red until the target binding to the aptamer, which restores the peroxidase-mimicking of nanozymes to oxidize the colorless TMB into blue oxTMB. Quantitative analysis was reported in the ZEN concentration range of 10–250 ng·mL −1 , and the limit of detection is 10 ng·mL −1 . The assay was applied to test ZEN in corn and corn oil samples, but high sensitivity was still challenging.

As an alternative, a combination of enzymatic action and gold nanoparticles aggregation was suggested by He et al. [37]. This colorimetric method was developed to detect OTA indirectly via the activity of alkaline phosphatase (ALP) (Figure 3b). Briefly, aptamermodified magnetic beads (MBs) were conjugated to DNA-linked ALP by hybridization. After OTA recognition, magnetic separation allowed to collect the released quantity of enzyme. The ALP can then hydrolyze ascorbic acid 2-phosphate (AAP) to ascorbic acid (AA), which mediates the reduction of MnO<sup>2</sup> nanosheets to Mn2+. These metal cations allow thereby the aggregation of AuNPs and lead to vivid color changes in the sensing system [37]. The dynamic range extends from 6.25 to 750 nM and an improved LOD of 2 ng·mL−<sup>1</sup> was recorded. This colorimetric method was applied to grape juice and red wine matrix with satisfactory recoveries.

A comparable approach was also described by the same group while replacing MnO<sup>2</sup> nanosheets by gold nanorods (AuNRs) and silver ions [53]. After magnetic separation, generated AA acted as reducing agents that transform Ag<sup>+</sup> to metal silver forming an Ag shell on the surface of AuNRs (Figure 3c). This caused a blue-shift of the longitudinal AuNRs' LSPR and a rainbow-like multicolor change.

Multicolor detection of OTA was also reported by AuNRs etching (diameter ~ 14 nm) mediated by G-quadruplex (AG4-OTA)-hemin DNAzyme and exonuclease I [55]. The product of peroxidase-like activity in acidic solution TMB2+ can etch the AuNRs by oxidizing Au(0) into Au(I).Variation of the optical characteristics of AuNRs arising from the change in interparticle distance and the number of hydrogen bonds has been reported as the key sensing strategy. A linear response range of 10–200 nM OTA was found with a LOD of 30 nM by visual observation and a lower LOD of 10 nM by spectrophotometry. The selectivity towards OTA was tested with the interfering mycotoxins AFB1, ZEN, and OTB. The method was successfully applied to the determination of OTA in spiked beer samples.

Besides enzymes and nanomaterials, commercially available organic dyes have also been used to conceive solution-based colorimetric methods. Interestingly, Wang's research group developed some multiplexed assays for the real-time detection of different mycotoxins based on allochroic dyes [45,54]. For instance, Hao et al. proposed a pH-resolved colorimetric aptasensing method for the simultaneous detection of four targets, including three mycotoxins, ochratoxin A, aflatoxins B1, fumonisin B1, and a marine toxin, microcystin-LR [54]. This assay involves four allochroic dyes—namely, phenolphthalein (PP), malachite green carbinol base (MGCB), thymolphthalein (TP), and methyl violet (MV)—as multiple signal indicators with colors of different wavelengths. Two DNA-GO platforms were prepared; the first was modified with Fe3O<sup>4</sup> for magnetic separation while the second adsorbed the hydrophobic dyes (Figure 3d). Both platforms were linked by partial hybridization to a target-specific aptamer. Upon target recognition, aptamer structure switching disabled hybridization and dissociated GO platforms. The subsequent magnetic separation followed by centrifugation allowed the spectroscopic analysis of supernatant in acidic solution and precipitate in alkaline solution. The absorption of supernatant solutions was directly proportional to AFB1 and MC-LR concentrations because of the MGCB and MV release at pH 3. Whereas the absorption of precipitates containing PP and TP adsorbed dyes was inversely proportional to OTA and FB1 mycotoxins in an alkaline pH of 12. This approach enabled the simultaneous detection of OTA and AFB1 in peanut samples with satisfactory recoveries (97.8–104.3%).

More recently, a derived nanocomposite-based strategy was described by Zhu et al. [45] employing TP dye signaling in acidic conditions for AFB1 detection and AuNPs as nanozymes to detect OTA via TMB catalysis in the alkaline precipitate. Competitive limits of detection as low as 1.5 ng·mL−<sup>1</sup> and 0.15 ng·mL−<sup>1</sup> were thus obtained for AFB1 and OTA, respectively.
