**3. Conclusions**

To provide stable immunosensors for the detection of mycotoxin ZON, continuous flow OWLS sensor setups were established. To immobilize protein immunoreagents (ZONspecific antibodies or ZON-BSA conjugate), the immunosensor surface was modified by epoxy, amino, and carboxyl functional groups under laboratory conditions by optimised silanisation protocols. Epoxy functional groups allowed direct immobilisation of the proteins under alkaline conditions (pH = 9.5). Amino functional groups allowed direct immobilisation of the proteins with 2.5% GA, or could be converted to carboxylic acid functional groups by 0.2% SA and conjugate to proteins using a 1:1 mixture of 0.1 M NHS and 0.4 M EDC.

In the direct (immobilised antibody) format, immobilisation on epoxy-modified surfaces (GOPS) provided lower binding efficacy and reproducibility than that on amino- (APTS/GA) or carboxyl-modified surfaces (APTS/SA/EDC-NHS). However, detectable ZON concentrations fell in all three cases above 500 ng/mL, not being sufficient for practical purposes. In the competitive (immobilised antigen) format, immobilisation on epoxy-modified surfaces (GOPS) remained improper for use, not providing sigmoid analyte concentration dependence, but amino- or carboxyl-modified surfaces were found of high utility. Both methods (APTS/GA and APTS/SA/EDC-NHS) resulted in similar analytical detection levels (EC<sup>50</sup> values in the range of 0.017–0.083 pg/mL) and linear detection ranges. Higher signal levels—therefore, greater signal decreases by inhibition—were achieved with amino-modified surfaces; however, carboxyl-modified surfaces allowed for

more stable and reproducible results. The optimised competitive immunosensor using 10 µg/mL of ZON-BSA as the surface coating antigen immobilised by the APTS/SA/EDC-NHS method, as well as ZON-specific antibodies at 1:2000 dilution, was found to show excellent sensitivity and specificity to ZON, allowed an LOD of 0.002 pg/mL, and was found to be applicable to the determination of ZON in maize extracts. Detectable analyte concentrations in assay buffer were found to be five orders of magnitude lower by the immunosensor than by the related ELISA method, which, considering the sample preparation requirements, corresponds to a three orders of magnitude improvement for determination of ZON content in maize commodity. Such unique improvements in the analytical sensitivity of the OWLS technique compared to the corresponding ELISA method have previously been evidenced for the detection of other analytes, including a nearly three orders of magnitude enhancement for the endocrine biomarker protein vitellogenin [77] and a six orders of magnitude improvement for a herbicide active ingredient trifluralin [76]. Moreover, the current OWLS immunosensor represents substantial advancements compared to previous immunosensors for ZON, e.g., based on SPR [71], TIRE [73], and electrochemical detection with antibodies immobilised on gold nanoparticles embedded on multi-walled carbon nanotubes [42], to which the LOD of the current competitive OWLS immunosensor represents 30,000-, 5000-, and 75-fold improvements, respectively.
