**1. Introduction**

Zearalenone (ZON) is a mycotoxin produced by several *Fusarium* species, most frequently by *F. graminearum*, and is commonly found in maize and also in wheat, barley, sorghum, and rye throughout various countries of the world, causing substantial human exposure [1]. ZON and its metabolites have oestrogenic activity in several species [2–5] accompanied by hepatotoxicity, haematotoxicity, immunotoxicity, and genotoxicity [6–8]. No uniform regulations have been imposed for this toxin in different countries. Tolerance levels in grains and grain products have been set in several countries at a concentration range of 20 to 1000 µg/kg [9], e.g., 20 to 200 µg/kg in unprocessed and processed cereal products in the EU [10]. Data evaluation on the most sensitive animal species—swine—and

**Citation:** Székács, I.; Adányi, N.; Szendr˝o, I.; Székács, A. Direct and Competitive Optical Grating Immunosensors for Determination of *Fusarium* Mycotoxin Zearalenone. *Toxins* **2021**, *13*, 43. https://doi.org/10.3390/toxins13010043

Received: 1 December 2020 Accepted: 5 January 2021 Published: 8 January 2021

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comparing with humans, a tolerable daily intake for ZON has been set as 0.25 µg/kg body weight [11,12].

Common analytical methods for identifying and quantifying mycotoxins include thin-layer chromatography (TLC) [13,14] or high-pressure TLC [15], laser fluorimetry [16], gas chromatography (GC) [14] often coupled with mass spectrometry (GC-MS) [17,18], high-performance liquid chromatography (HPLC) [19–21] with standardised sample preparation [22,23], ultra-performance liquid chromatography (UPLC) [24], and capillary electrophoresis [25,26]. HPLC methods have become the most widespread for mycotoxin analysis. These methods are sensitive and accurate but require extensive sample preparation steps, well-trained personnel, and expensive instrumentation. Therefore, just as for other mycotoxins, and on the basis of the historical radioimmunoassay method [27], immunochemical methods, e.g., enzyme-linked immunosorbent assays (ELISAs), have been developed and utilised for rapid screening of ZON [14,19,28–33]. These immunoassays were further amplified with fluorescent quantum dots [34–36], magnetic nanoparticles [37], or helical carbon nanotubes [38]. Alternatively, antibodies [24,36,39–44] or molecularly imprinted polymers [45–47] could be applied for affinity chromatography or pre-column sample purification prior to chromatographic analyses (HPLC, UPLC). Similarly, nanoparticleassisted lateral flow immunochromatographic strips [48,49] were devised, occasionally with surface-enhanced Raman scattering detection [50]. Recently, micro- and nanoarray immunoassays were reported in microplate-based [51] or microfluidic sensor-based [52] setups. A cut-off level of 100 µg/kg was established (4 min) for ZON and T2 toxin in a gel-based immunoassay [53]. Fluorescence polarisation immunoassays allowed for a detection range for ZON of 150–1000 µg/kg and a limit of detection (LOD) of 137 µg/kg, and required less than 2 min per sample to carry out [54]. A magnetic nanotag-based immunoassay [55] and a multiplexed quantum dot immunochromatographic assay [56] allowed the parallel detection of ZON in the presence of other mycotoxins. Label-free biosensors on the basis of antibodies [57–60], aptamers [58,61–67], or molecularly imprinted polymers [68–71] as recognition elements have also been developed with various signal amplification and detection routes involved, and the range of sensoric detection techniques is expanding [72]. Thus, a surface plasmon resonance (SPR) biosensor has been developed for the simultaneous detection of four mycotoxins, with an LOD below 0.2 ng/mL for ZON [73], a gold nanoparticle-amplified imaging SPR (iSPR) biosensor allowed an LOD for ZON of 59.2 pg/mL in multiplex mycotoxin determination [74], a method of total internal reflection ellipsometry (TIRE) allowed detection of ZON at concentrations as low as 0.1 ng/mL [75], and electrochemical sensors resulted in LODs of 0.15–0.25 pg/mL [42,60]. The immunosensors developed allow rapid quantitative determination of the target compounds in plant samples and in environmental matrices, mainly in ground water.

Immunosensors based on the technique of optical waveguide lightmode spectroscopy (OWLS) have been applied with success to detect different molecules, and gained importance in environmental and food analysis [59,76,77]. In the current study, an OWLS immunosensor has been developed for the determination of ZON in maize samples. Different chemical methods for functionalisation and accordingly for immobilisation were compared regarding analytical sensitivity and sensor stability. Upon optimisation, the novel immunosensor was used for the detection of ZON contamination in maize and the results were compared to ELISA measurements to demonstrate the outstanding applicability of the method in complex food matrices and assumedly, in environmental samples as well.
