*4.6. Total Internal Reflection Ellipsometry (TIRE)*

Total internal reflection ellipsometry (TIRE) experiments were carried out on an M-2000 automatic spectroscopic ellipsometer (J.A. Woollam Co., Lincoln, NE, USA) operating in the 370–1000 nm range using glass-based sensor chips fabricated in the laboratory by vacuum evaporation. Sensor surfaces were prepared by thermal evaporation of layers of chromium (Cr)—3 nm thick and gold (Au)—25 nm on standard microscopic glass slides (BK-7). The Cr layer improves the adhesion of gold to the glass surface. The Au surface was modified with mercaptoethyl sodium sulfonate to enhance the negative surface charge. The ellipsometer was equipped with a 68◦ trapezoidal prism which allowed coupling the light beam at total internal reflection conditions to the gold film on the glass slide. The 0.2 mL reaction cell with the inlet and outlet tubes was attached underneath to the gold surface, allowing the injection of the required chemicals to perform binding reactions. The ellipsometry spectral scans were performed in a standard Trisma/HCl buffer solution (pH = 7.5) after completing each adsorption (binding) stage. For a competitive immunoassay, a ZON-60 -carboxymethyloxime– BSA conjugate (ZON–BSA) was electrostatically immobilized on the Au surface via a polyallylamine hydrochloride layer. In order to block all the remaining binding sites, an additional adsorption of BSA was carried out. Then a mixture of ZON-specific antiserum and solutions of free ZON (at a concentration range of 0.01 ng/mL–10 µg/mL) were injected into the cell with the intermediate rinsing with buffer. The mixtures were preincubated for 5 min before injection. A series of ∆ spectra were recorded after binding of ZON to the antibodies immobilized on the chip surface.

**Author Contributions:** Conceptualization, A.S., L.K., N.A. and A.N.; methodology, A.B., G.S., M.M., S.L., A.C., S.K. and E.T.; software, A.B., P.G. and L.D.; formal analysis and investigation, B.G., M.M., A.N. and E.T.; data curation, A.S., E.T., A.N. and M.M.; writing—original draft preparation, B.G., A.S. and E.T.; writing—review and editing, A.S., L.K. and A.B.; visualization, B.G., E.T., M.M., A.S. and A.N.; supervision, A.S.; project administration, A.S., L.K., A.B. and N.A.; funding acquisition, A.S., L.K., A.B., A.N. and L.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Hungarian National Research, Development and Innovation Office within the National Competitiveness and Excellence Program, project NVKP\_16-1-2016- 0049 "In situ, complex water quality monitoring by using direct or immunofluorimetry and plasma spectroscopy" (Aquafluosense 2017-2021).

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Research on Animals of the Food Science Research Institute, Institute of Food Sciences, Hungarian University of Agriculture and Life Sciences, Budapest, Hungary (protocol code PE/EA/45-6/2020, last date of approval 21 February 2020).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy reasons.

**Acknowledgments:** The authors express their appreciation to Csilla Magor at the Agro-Environmental Research Institute, National Agricultural Research and Innovation Centre, Budapest, Hungary, and to Ali Madlool Al-Jawdah at the Materials and Engineering Research Institute, Sheffield Hallam University, Sheffield, UK, for their technical contribution in the HPLC determination and the TIRE experiments, respectively.

**Conflicts of Interest:** The authors declare no conflict of interest.
