*2.3. Immunosensor Specificity*

Immunosensor specificity was tested on the optimised immunosensor setup (see above) by measuring EC<sup>50</sup> values obtained with ZON derivatives and structurally unrelated mycotoxins, and cross-reactivities (CRs), defined as a percentage ratio between the EC<sup>50</sup> values of ZON and the given compound, were calculated. Among the structurally unrelated compounds tested, aflatoxin B1 and ochratoxin did not cause a decrease in the OWLS sensor signal up to 1000 ng/mL concentration in the diluted standards. Among the compounds tested, only α-zearalenol, α-zearalanol, and β-zearalanol showed significant CRs (Table 1) with ZON in the competitive immunosensor format. These are major reductive metabolites of ZON in mammals, but are also formed to a lesser extent in plants as well [12]; therefore, the potential presence of these metabolites should be also considered upon positive detection of ZON in commodities by the current immunosensor method. These CR values are in good agreement with the corresponding values reported for our ELISA system for ZON [29]; however, the detection sensitivity of the current OWLS immunosensor exceeds that of the ELISA by five orders of magnitude. Such outstanding improvement in the detection range of an OWLS immunosensor compared to the corresponding ELISA has been reported [59,78].

**Table 1.** Percentage of cross-reactivity (CR%) of the competitive OWLS immunosensor and the corresponding ELISA method [29] with zearalenone and its derivatives.


<sup>1</sup> Cross-reactivity defined as the percentage ratio of the 50% inhibitory concentration (IC50) values of zearalenone and of the given derivative.
