2.2.2. Competitive Immunosensor Setups with Different Surfaces Modifications

The protein-heterologous conjugate of ZON (ZON-BSA) was used as a sensor surface antigen at various concentrations between 2 and 20 µg/mL with the above three immobilisation methods. Upon serum titration, surface coating conditions were optimised for immunosensor sensitivity i.e., analytical standard curves were obtained using concentration series of ZON and recording its inhibitory effect on antibody binding to the treated immunosensor surfaces (Figure 4).

**Figure 4.** Standard calibration curves for zearalenone (ZON) determination by the competitive 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, as a function of concentration of ZON applied in the calibration standard samples in the sensor format with ZON conjugate to bovine serum albumin (ZON-BSA) immobilised on amino- and epoxy-modified sensor surfaces with (3-aminopropyl) triethoxysilane and glutaraldehyde (APTS/GA) using 10 µg/mL ZON-BSA (, blue solid line) and 5 µg/mL ZON-BSA (, blue dashed line); (3-aminopropyl) triethoxysilane, succinic anhydride and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide with Nhydroxysuccinimide (APTS/SA/EDC-NHS) using 10 µg/mL ZON-BSA (, red solid line), 5 µg/mL ZON-BSA (♦, red dashed line), and γ-glycidoxypropyl-trimethoxysilane (GOPS) (N, green dotted line).

The highest and stable sensor signals were obtained when ZON-BSA was at 10 µg/mL concentration, above which further improvement in assay signals could not be obtained, and excess of the antigen even caused less reproducible or deteriorated signals. Similarly to the direct sensor format, immobilisation on the epoxy-modified surface (GOPS) was found to be of limited utility in the competitive sensor format as well. Although addition of ZON resulted in concentration-dependent sensor signals, nonetheless, this means of

immobilisation was improper for analytical purposes due to the lack of proper regression possibility with the four-parameter logistic fitting. Immobilisation on amino- (APTS/GA) or carboxyl-modified surfaces (APTS/SA/EDC-NHS) allowed for better quantitative detection possibilities, with dynamic detection ranges of 0.001–1 pg/mL when the antisera were used in 1:2000 dilution. Immobilisation with GA appeared to be applicable at coating levels with ZON-BSA both at 10 and 5 µg/mL, while the SA/EDC-NHS method resulted in a standard sigmoid curve only at a coating level of 10 µg/mL. In these cases, linear detection ranges were found to be similar with good reproducibility. Antisera obtained from different rabbits (under the same immunisation protocol), although showing somewhat different titration characteristics, provided similar results in the competitive formats, indicating that slight differences in serum composition did not have inhibitory activity. The highest sensor signals were obtained here also by APTS/GA modification. Detection sensitivity showed outstandingly low EC<sup>50</sup> values in the range of 0.017–0.083 pg/mL, corresponding to at least six orders of magnitude improvement in detection range compared to the direct immunosensor. The dynamic detection range of ZON was found in the 0.010–1 pg/mL ZON concentrations, and an LOD for ZON of 0.002 pg/mL was obtained in the APTS/GA modification using the ZON-BSA conjugate as the surface coating antigen at 10 µg/mL. As the competitive immunosensors based on amino- and carboxyl-modified surfaces provided similar ranges of detection, due to the better reproducibility and longer shelf-life of the latter, the sensor setup using 10 µg/mL of ZON-BSA as the surface coating antigen immobilised with the APTS/SA/EDC-NHS method, as well as ZON-specific antibodies at 1:2000 dilution, was chosen to be used for practical purposes.
