4.4.3. Immunoassay

Plates were coated with 1 µg/mL ZON-60 -carboxymethyloxime–BSA conjugate (ZON– BSA) in carbonate buffer (15 mM Na2CO3, 35 mM NaHCO3, pH = 9.6) overnight (~8 h) at 4 ◦C. The unbound conjugate was washed 4 times with PBS with 2% Tween20 (137 mM NaCl, 2.7 KCl, 10 mM Na2HPO<sup>4</sup> × 2H2O, pH = 7.4). Blocking was carried out with 150 µL/well 1% gelatin in PBS, for 1.5 h of incubation at 37 ◦C. After washing, competition was initiated by adding 50 µL/well of both ZON analytical standard and purified rabbit anti-ZON serum (in PBS buffer with 5% Tween20, dilution 1:1000). An analytical ZON standard stock solution (1 mg/mL ZON in MeOH) was used in serial dilution (0.004 pg/mL– 2 µg/mL). Thus, MeOH content in the actual ELFIA measurements was 0.2% or below. After 1 h of incubation at 37 ◦C and four washes, 100 µL/well goat antirabbit IgG–HRP (horseradish peroxidase, dilution 1:7500) conjugate was added as secondary antibody and incubated for 1 h at 37 ◦C. Unbound secondary antibodies were washed out 4 times with PBS and 100 µL/well working solution of QuantaRed Enhanced Chemifluorescent HRP Substrate Kit were added (content of the working solution: QuantaRed ADHP concentrate, QuantaRed Enhancer Solution, and QuantaRed stable Peroxide in 1:50:50 (*v*/*v*) proportion). The kit contains 10-acetyl-3,7-dihydroxyphenoxazine (ADHP), a nonfluorescent compound that is dehydrogenated (oxidized) by HRP to resorufin, a highly fluorescent reaction product, which can also be measured in a colorimetric plate reader. After 5 min of incubation, the enzymatic activity was stopped by 10 µL of QuantaRed Stop Solution. Both absorbance and fluorescence were measured at 576 and at 593 nm wavelengths, respectively. After the colorimetric assay, the liquid phase was transferred with an 8-channel pipette into a white, low profile 96-well microplate where fluorescence was determined. Absorbances were read by SpectraMax iD3 Multi-Mode Microplate Reader (Molecular Devices, San Jose, CA, USA) at 576 nm wavelength, while relative fluorescence signals were determined by the prototype fluorimeter developed in project Aquafluosense [69,74] (see Section 4.2). Standard curves were obtained in different surface water samples as well, for evaluation of matrix effects in ZON determination from natural water bodies. Statistical analysis

of standard curves was performed by comparison of IC<sup>50</sup> values by one-way analysis of variance followed by post hoc Tukey test at a significance level of 0.05.

The immunoassay developed in this study can be performed in situ by the instrument developed and built in a laboratory motor vehicle. Coating the assay microplates by the ZON–BSA conjugate can be carried out under laboratory conditions (method in Section 4.4.3) prior the measurement, and in situ determination can be performed on precoated microplates in the mobile equipment. Thus, the total immunoassay performance time is 2.5 h. Since the immunofluorescence instrument is equipped with stepping motor units, fluorescence determination on an entire plate requires approximately 2 min. A possibility of further shortening the procedure is potentially through performing both the coating and blocking steps in the laboratory a day before the in situ determination. In this case, the coating step can be applied at room temperature for 1 h and the blocking step with 1% gelatin in PBS for 1.5 h. Coated and blocked microplates can be stored at 4 ◦C until measurement; however, it is necessary to prevent evaporation in the wells by covering the microplate with parafilm.
