*2.4. Total Internal Reflection Ellipsometry*

The immunoassay setup has also been applied in a system based on detection by total internal reflection ellipsometry (TIRE), where two parameters Ψ and ∆ are related, respectively, to the amplitude and phase shift of p- and s-components of the polarized light detected [73]. Since variations in the refractive index and the thickness of the adsorbed layers cause 10 times higher values of ∆ than of Ψ, ∆(λ) spectra were used in the TIRE method as a sensor response. A typical series of ∆(λ) spectra for ZON competitive immunoassays and the respective calibration curve of the assay signal in TIRE (δd corresponding to the shift in the adsorbed layer thickness vs. the concentration of ZON) obtained by sigmoidal fitting are depicted in Figure 5. The response is similar to that shown in Figure 2, where the

highest concentration of ZON yields the lowest response, which is typical for competitive immunoassays. On the basis of the standard calibration curve, a LOD of 0.01 ng/mL for ZON was determined. *Toxins* **2021**, *13*, x FOR PEER REVIEW 10 of 18

*Toxins* **2021**, *13*, x FOR PEER REVIEW 10 of 18

**Figure 4.** High-performance liquid chromatography (HPLC) chromatogram of zearalenone (ZON) at 1 µg/mL concentration dissolved in methanol:phosphate buffer saline (1:1). Linear calibration (average of peak area from three parallel measurements and their SDs) of ZON in a concentration range of 10–2000 ng/mL determined at 236 nm by high-performance liquid chromatography coupled with UV detection (insert). **Figure 4.** High-performance liquid chromatography (HPLC) chromatogram of zearalenone (ZON) at 1 µg/mL concentration dissolved in methanol:phosphate buffer saline (1:1). Linear calibration (average of peak area from three parallel measurements and their SDs) of ZON in a concentration range of 10–2000 ng/mL determined at 236 nm by high-performance liquid chromatography coupled with UV detection (insert). and the respective calibration curve of the assay signal in TIRE (δd corresponding to the shift in the adsorbed layer thickness vs. the concentration of ZON) obtained by sigmoidal fitting are depicted in Figure 5. The response is similar to that shown in Figure 2, where the highest concentration of ZON yields the lowest response, which is typical for competitive immunoassays. On the basis of the standard calibration curve, a LOD of 0.01 ng/mL for ZON was determined.

**Figure 5.** A competitive immunoassay for zearalenone (ZON) carried out by detection via total internal reflection ellipsometry (TIRE). (**a**) A typical set of Δ(λ) spectra measured on bare Au surface (1), polyallylamine hydrochloride (2) ZON–bovine serum albumin conjugate (3), bovine serum albumin (4), Ab-ZON of from preincubated mixtures containing ZON: 100 (5), 10 (6), 1 (7) and 0.1 ng/mL (8). (**b**) Changes in the adsorbed layer thickness versus the concentration of ZON (in the mixture with Ab-ZON) obtained by fitting the TIRE data. **Figure 5.** A competitive immunoassay for zearalenone (ZON) carried out by detection via total internal reflection ellipsometry (TIRE). (**a**) A typical set of ∆(λ) spectra measured on bare Au surface (1), polyallylamine hydrochloride (2) ZON–bovine serum albumin conjugate (3), bovine serum albumin (4), Ab-ZON of from preincubated mixtures containing ZON: 100 (5), 10 (6), 1 (7) and 0.1 ng/mL (8). (**b**) Changes in the adsorbed layer thickness versus the concentration of ZON (in the mixture with Ab-ZON) obtained by fitting the TIRE data.
