*2.4. Method Validation in Commodity Matrix*

The optimised competitive immunosensor was applied to determine ZON concentrations in maize commodity. For this purpose, maize samples spiked with ZON at concentration levels of 0–10 µg/kg were extracted in acetonitrile/water (6:4), and were analysed by the competitive OWLS immunosensor and ELISA. These analyses aimed to assess matrix effects by the maize extract on the one hand, and were also targeted to investigate whether the two analytical methods detect the same ZON concentrations, identical to the nominal values, on the other hand. To assess possible matrix effects on immunosensor performance, the aqueous extracts were diluted 1:100 to 1:10,000 in 42 mM 2-amino-2- (hydroxymethyl)-1,3-propanediol (Tris) buffer (pH 7.4). Figure 5 shows analytical standard curves obtained by the optimised competitive immunosensor setup in diluted maize extracts, and demonstrates that matrix effects are diluted out at 1:10,000. ZON concentrations detected by the competitive immunosensor indicated analytical recoveries at initial ZON concentrations between 5 ng/kg and 10 µg/kg, carried out in triplicates, were found to be 84% and 124%, mostly suitable for practical use. It has to be noted, however, that the maximal recovery value fell by 3.3% out of the acceptable recovery range of the European legislation performance criteria for ZON detection set to be 60–120% and 70–120% for ZON concentration at or below 50 µg/kg and above 50 µg/kg, respectively [79].

**Figure 5.** Standard calibration curves for zearalenone (ZON) determination by the competitive optical waveguide lightmode spectroscopy (OWLS) immunosensor format in maize extract at various dilutions. The amino-modified sensor surface with ZON conjugate to bovine serum albumin (ZON-BSA) immobilised at 10 µg/mL on amino-modified sensor surfaces by (3-aminopropyl) triethoxysilane, succinic anhydride and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide with N-hydroxysuccinimide (APTS/SA/EDC-NHS) using ZON-specific serum at 1:2000 dilution. Maize extracts were applied at dilutions of 1:100 (, green line), 1:1000 (, red line), and 1:10,000 (, blue line); ZON calibration curve in 42 mM 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer (pH 7.4) (, black slashed line).

ZON concentrations measured in maize extract by OWLS and ELISA methods were compared to each other as shown in Figure 6. Results indicate that concentrations detected by the two methods well correlated with each other in the 0.1–10 µg/kg range (r<sup>2</sup> = 0.984), and both methods are applicable. However, while the ELISA method required an extract dilution of 1:10 and detected ZON above 0.1 ng/mL, the OWLS immunosensor required an extract dilution of 1:10,000, but detected ZON above 0.01 pg/mL.

**Figure 6.** Determination of zearalenone (ZON) content in maize samples by optical waveguide lightmode spectroscopy (OWLS) immunosensor and enzyme-linked immunosorbent assay (ELISA). The sensing range for OWLS (>0.002 µg/kg) and for ELISA (>0.09 µg/kg) are indicated in blue and red, respectively. (Ordinate values for ZON concentrations below 0.09 µg/kg are virtual for visualisation—indicated by hollow rectangles).
