*2.2. Nanomaterial-Based Probes*

Accelerated by the advances in nanomaterials (NMs), colorimetric methods for the detection of mycotoxins have undergone a rapidly developing stage in the past few years [22]. Their nanometric size (less than 100 nm) and unique physicochemical features, including distinctive optical and catalytic properties, have promoted the extensive use of nanostructured materials in colorimetric methods. Accordingly, researchers handled each nanomaterial differently to adapt it with the desired function in the sensing assay. It is widely reported that NMs are attractive candidates to immobilize bioreceptors, including enzymes, antibodies, and aptamers, thanks to their large size to volume ratio, which provides a high specific active surface. In particular, magnetic nanoparticles (MNPs) from iron-based nanoparticles are widely used in colorimetric bioassays to capture, separate, and enrich target analytes, especially when a low detection limit is required. However, we emphasize in this section the signaling roles of NMs in colorimetric methods dedicated to mycotoxins detection. Glimpsing at the relevant literature, two prominent roles of NMs are depicted. NMs mainly based on metal nanoparticles show color switching tunable properties and are thereby used as direct colorimetric probes. Enzyme-like NMs (or nanozymes) also contribute to the advances in colorimetric assays, particularly through peroxidase and oxidase-like catalysis that generate colored products. Some NMs can be also employed as signal mediators to enhance assay sensitivities in cascade amplification systems.

As optical signal generators, noble metal nanoparticles—including gold NPs, silver NPs, etc.—are majorly used in mycotoxins' (bio)assays due to their unique physicochemical properties. In particular, detection strategies based on changes in the localized surface plasmon resonance (LSPR) signal caused by the aggregation of noble metal NPs have shown suitable sensitivities to detect mycotoxins [23]. In such systems, NPs can be dispersed in colloidal solution via surface anionic repulsion. In the presence of electrolytes containing salt cations or cationic polymers, charges are stabilized, and NPs tend to aggregate. This aggregation alters the LSPR effect, resulting in a red shift of the UV-vis absorption spectrum [24]. Harnessing this property, AuNPs have been extensively tested in the plasmonic sensing of some fungal toxins, owing to their easy synthesis, high extinction coefficients, photostability, and non-toxicity. AuNPs have been considered as ideal signal generating probes because of the visible color change from red to blue through salt-induced nanoparticles assembly, or inversely through their redispersion [23].

Specifically, the advances of nucleic acid manipulation and aptamers selection have powerfully accelerated the progress in plasmonic mycotoxins detection [25]. Nucleic acid strands are more convenient than antibodies for unmodified AuNPs aggregation-based assays, with promising results in the semi-quantitative and quantitative real-sample application [26]. For instance, A label-free optical sensor was reported for the selective detection of AFB1 using a DNA-based aptamer along with unmodified spherical colloidal AuNPs (diameter ~ 13 nm). Recognition of AFB1 was achieved based on the salt-induced AuNPs aggregation. High selectivity was observed against the presence of OTA. Low detectionlimit of 0.025 ng·mL−<sup>1</sup> AFB1 was reported with the linear dynamic determination range of 0.025–100 ng·mL−<sup>1</sup> [27]. More recently, Phanchai et al. [28] have performed in silico studies to investigate the molecular dynamics (MD) of this detection approach using AuNPs aggregation taking as an example anti-OTA aptamer (Figure 2a). This offered new insights into the mechanism of recognition highlighting the effect of the ionic composition of solvent as well as the kinetics behind the interaction between the three molecular partners—i.e., AuNPs, aptamer, and the mycotoxin. The reported MD simulation revealed an insightful analysis of the interaction mechanisms in the AuNP-based aptasensing platforms that can be projected to any other similar pattern.

Another strategy of colorimetric signal generating relies on nanomaterial-based labels like common in lateral flow immunochromatographic assays (LFIAs). A number of NMs was described as antibodies' nano-labels for the visual rapid detection of mycotoxins [29], such as AuNPs [30], graphene oxide (GO) [31], Prussian blue nanoparticles (PBNPs) [32], etc. In such devices, the color of test lines is usually drawn by the labeled antibodies involved in specific immunoreactions (cf. Section 3.3). Typical mycotoxins' LFIAs use AuNPs as convenient nano-labels. Interestingly, Kong et al. [33] described a semi-quantitative and quantitative AuNPs-based LFIA for the simultaneous detection of 20 types of mycotoxins from five classes—including zearalenones, deoxynivalenols, T-2 toxins, aflatoxins, and fumonisins—in cereal food samples (Figure 2b). The whole detection process took 20 min in total and was used for the reliable detection of mycotoxins in cereal samples. The LOD of three mycotoxins (AFB1, ZEN, and OTA) were far below the European maximum residue limits. involved in specific immunoreactions (cf. Section 3.3). Typical mycotoxins' LFIAs use — —

**Figure 2.** (**a**) Simulation of the molecular interactions involved in the aggregation of citratecapped AuNPs for the rapid aptasensing of OTA; (**b**) A gold nanoparticle-based semi-quantitative and quantitative LFIA for the simultaneous detection of 20 mycotoxins; (**c**) Mechanism of MnO<sup>2</sup> nanozyme-based cascade colorimetric aptasensor for OTA detection. Reproduced with permission from [28,33,34].

Nanozymes are unique nanomaterials that have been proven to show catalytic activities in a similar way to biological enzymes with greater stability. This particular feature enables the enhancement of enzymatic response or the development of enzyme-free colorimetric methods. Accordingly, some nanozymes owing peroxidase-like and oxidase-like activities have been used as colorimetric probes for mycotoxins analysis [35]. They are

widely used in different formats to afford rapid colorimetric observation, sensitive response, and cost-effective analysis.

For example, Tian et al. [34] developed a sensitive OTA aptasensor harnessing the oxidase-mimicking activity of MnO<sup>2</sup> nanosheets to catalyze the TMB oxidation (Figure 2c). In this assay, ascorbic acid generated under ALP action reduces MnO<sup>2</sup> nanosheets to Mn2+ ions, and thereby inhibits the catalytic activity of MnO<sup>2</sup> in the presence of TMB. With the increasing amount of OTA, a highly sensitive color change from blue to colorless was obtained. This sensing method enabled competitive LOD (0.07 nM) compared to conventional single enzymatic colorimetric schemes. While in the colorimetric sensing system based on peroxidase-like nanomaterials, the detection of targets is performed through measuring the absorption variation of the TMB-H2O<sup>2</sup> reaction. According to this scheme, analysis of OTA has been demonstrated using a hybrid recognition matrix composed of Fe3O<sup>4</sup> doped with AuNPs, amino-modified capture DNA and anti-OTA aptamer deposited on glass beads (GB-aptamer/cDNA-Au@Fe3O4) [36]. The peroxidaselike activity of Au@Fe3O<sup>4</sup> NPs was effectively enhanced due to the synergistic effect between the AuNPs and Fe3O<sup>4</sup> NPs. Low detection limit of 30 pg mL−<sup>1</sup> OTA was achieved with a linear current response range of 0.05–200 ng·mL−<sup>1</sup> . Selectivity has been proven in the presence of OTB, FB1, and AFB1. Sensor performance for the determination of OTA from real samples has been demonstrated with peanut and corn samples.

It is worth noting that other detection strategies can implicate NMs as signal mediators in combination with other main colorimetric probes for sensitivity enhancement. As an example, MnO<sup>2</sup> nanosheets can be used in new colorimetric methods based on AuNPs aggregation schemes since MnO<sup>2</sup> nanoflakes can produce abundance metal ions Mn2+ after decomposition [37], or combined with enzymes to react with catalysis products [34].

Representative examples of NMs-based assays from the recent literature are summarized in Table 2.

*Toxins* **2021**, *13*, 13

**Strategy Detection Probe Target LOD Linear Range Specificity Sample Advantages/Disadvantages Ref.** Aptamer assay based on AuNPs aggregation by poly diallyldimethyl ammonium chloride polymer (PDDA). AuNPs OTA 0.009 ng·mL−1 0.05–50 ng·mL−1 High Chinese liquor sample Cost-effectiveness, few steps, rapid detection (15 min), good sensitivity/Possible cross-reactivity at high target concentrations. [38] AuNP dimer disassembly by the target-induced release of complementary DNA probes. AuNPs OTA 0.05 nM 0.2–250 nM High Red wine Improved sensitivity, low-cost, short detection time (15 min)/Difficult applicability to colored complex samples [39] Double calibration curve of label free aptasensing assay based on salt-induced aggregation of AuNPs. AuNPs OTA 0.03 ng·mL−1 0.03–316 ng·mL−1 Good Corn Widened detection range, enhanced sensitivity, reliability, rapid detection/low selectivity [40] Peroxidase-like activity of AuNPs in the presence of H2O2 and TMS substrate. AuNPs ZEN 10 ng·mL−1 10–250 ng·mL−1 High Corn and corn oil Simple one-step assay, short detection time/Relatively high detection limit [41] Chemical nano-sensor based on cysteamine-modified AuNPs aggregation via electrostatic interaction with hydrolyzed target. AuNPs FB1 0.90 µg·kg−1 2–8 µg·kg−1 Low Corn Simple one-step assay, Rapid homogenous test (3 min)/Real sample interferences, low sensitivity and selectivity. [42] ALP- induced gold nanoparticle aggregation mediated by MnO2 nanosheets reduction in the presence of generated ascorbic acid. AuNPs, MnO2 nanosheets OTA 5.0 nM 6.25–750 nM High Grape juice & red wine Enzymatic amplification, high selectivity/multi reaction steps, possible cross reactivity in real samples [37] Colorimetric aflatoxins immunoassay by using mesoporous silica nanoparticles decorated with gold nanoparticles. AuNPs@m-SiNPs nanocomposite AFs (AFB1, AFB2, and AFG2) 0.16 ng·mL−1 1–75 ng·mL−1 for AFB1 High Nuts, cornflakes, cornmeal, peanuts, peanut butter and pecan nuts High sensitivity, versatile real matrix applicability, 30 min incubation time/long synthesis and modification of transducer [43] AFB1 hydrolyzed to phenolate anions react with curcumin enol form-Zn red complex to give curcumin enol form-ZnO-Phenol yellow complex. ZnO NPs AFB1 11 µg·kg−1 0–36 µg·kg−1 Good Rice Simple and rapid detection, bioreceptor-free sensor, HPLC validation/Chemical modification of target [44] Cascade aptasensor by double catalytic amplifications using ALP activity combined to the inhibition of the MnO2 oxidase-mimicking activity. MnO2 nanosheets OTA 0.07 nM 1.25–250 nM Excellent Grape juice Amplified colorimetric signal, high sensitivity and selectivity/Many washing and addition steps [34] Simultaneous dual target detection via the combination of two the catalysis of TMB under acidic conditions and the release of TP under alkaline conditions. Fe3O4 -GO nanocomposite and AuNPs AFB1 1.5 ng·mL−1 5–250 ng·mL−1 High Peanuts [45] OTA 0.15 ng·mL−1 0.5–80 ng·mL−1 Multiplexed detection, high sensitivity and selectivity/Tedious probes synthesis, specific pH and temperature conditions, long incubation time (90 min), multiple steps of washing and separation Salt-induced coagulation of iron-modified 2D covalent triazine framework nanosheets (2D Fe-CTFs) that showed strong peroxidase-like activity 2D Fe-CTFs OTA NR 0.2–0.8 µM NR NR Promising proof of concept/limited detection range, analytical performances not reported [46] PAD sensor array based on silver and gold nanoparticles aggregation synthesized by three different capping agents. AgNPs and AuNPs AFB1 2.7 ng·mL−1 3.1 ng·mL−1 –7.8 µg·mL−1 Excellent Mixtures of pistachio, wheat and coffee, milk Very fast colorimetric response (5 s), multiplexed detection of five mycotoxins, low cost, device portability/Optical nanoprobes fabrication [47] AFG1 7.3 ng·mL−1 8.2 ng·mL−1–8.4 µg.mL−1 AFM1 2.1 ng·mL−1 2.5 ng·mL−1–8.2 µg.mL−1 OTA 3.3 ng·mL−1 4.0 ng·mL−1–3.8 µg.mL−1 ZEN 7.0 ng·mL−1 8.0 ng·mL−1–7.9 µg.mL−1

**Table 2.** Representative examples of recent developed nanomaterial-based methods for the colorimetric detection of mycotoxins.
