*2.1. Direct Immunosensor*

Immunoglobulin (IgG) fractions purified from ZON-specific rabbit antisera obtained against a conjugate of ZON to conalbumin (ovotransferrin, CONA) as an immunogen were used in an immobilised antibody-based (direct) immunosensor format. The main characteristics that determined achievable assay signals were the quality and concentration (dilution) of the ZON-specific antibodies. Using a dilution of the IgG purified from the serum of 1:2000 for immobilisation by all three methods, the epoxy-functionalised sensor surface modified with γ-glycidoxypropyl-trimethoxysilane (GOPS), as well as the amino-functionalised sensor surface modified with (3-aminopropyl)triethoxysilane (APTS), and glutaraldehyde (GA) or succinic anhydride with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Nhydroxysuccinimide (SA/EDC-NHS), standard calibration curves were obtained for ZON determination by applying ZON onto the immobilised antibodies on the sensor surface at various concentrations up to 100 µg/mL (Figure 2).

The highest sensor signals were obtained by APTS/GA modification, followed by APTS/SA-EDC/NHS, while immobilisation with GOPS provided the lowest assay signals. Detection sensitivity, characterised with the analyte (ZON) 50% effective concentrations (EC50) corresponding to the half-maximal signal level, indicated EC<sup>50</sup> values of 3.6 ± 0.2, 2.2 ± 0.6, and 1.1 ± 0.1 µg/mL for the GOPS, APTS/GA, and APTS/SA-EDC/NHS modifications, respectively. Signal intensities and statistics indicated that immobilisation on the epoxy-modified surface (GOPS) provided lower binding efficacy and reproducibility than that on amino-modified surfaces (APTS) with homo-bifunctional cross-linking (GA) with further modification to carboxyl groups (SA/EDC-NHS). Nonetheless, the lowest detectable ZON concentrations in these setups were in all three cases above 500 ng/mL, which is not sufficiently sensitive for analysis of real samples.

**Figure 2.** Standard calibration curves for zearalenone (ZON) determination by the direct optical waveguide lightmode spectroscopy (OWLS) immunosensor format. Sensor signals proportional to relative surface mass (ng mm−<sup>2</sup> ) on the OWLS sensor, expressed in arbitrary units (a.u.), as a function of concentration of ZON applied in the calibration standard samples in the sensor format with ZON-specific serum immobilised on amino- and epoxy-modified sensor surfaces using (3-aminopropyl) triethoxysilane and glutaraldehyde (APTS/GA) (, red dashed line), (3-aminopropyl) triethoxysilane, succinic anhydride and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide with N-hydroxysuccinimide (APTS/SA/EDC-NHS) (, blue solid line), and γglycidoxypropyl-trimethoxysilane (GOPS) (N, green dotted line).
