Development of an Immunofluorescence Assay Module for Determination of the Mycotoxin Zearalenone in Water

Abstract

Project Aquafluosense is designed to develop prototypes for a fluorescence-based instrumentation setup for in situ measurements of several characteristic parameters of water quality. In the scope of the project an enzyme-linked fluorescent immunoassay (ELFIA) method has been developed for the detection of several environmental xenobiotics, including mycotoxin zearalenone (ZON). ZON, produced by several plant pathogenic Fusarium species, has recently been identified as an emerging pollutant in surface water, presenting a hazard to aquatic ecosystems. Due to its physico-chemical properties, detection of ZON at low concentrations in surface water is a challenging task. The 96-well microplate-based fluorescence instrument is capable of detecting ZON in the concentration range of 0.09-400 ng/mL. The sensitivity and accuracy of the analytical method has been demonstrated by a comparative assessment with detection by high-performance liquid chromatography and by total internal reflection ellipsometry. The limit of detection of the method, 0.09 ng/mL, falls in the low range compared to the other reported immunoassays, but the main advantage of this ELFIA method is its efficacy in combined in situ applications for determination of various important water quality parameters detectable by induced fluorimerty-e.g., total organic carbon content, algal density or the level of other organic micropollutants detectable by immunofluorimetry. In addition, the immunofluorescence module can readily be expanded to other target analytes if proper antibodies are available for detection.

Keywords: competitive immunoassay; fluorescence detection; high-performance liquid chromatography; mycotoxin; total internal reflection ellipsometry; zearalenone.

Figure 1

Results of zearalenone (ZON) quantification by autofluorescence. (a) A fluorescence spectral map of ZON in phosphate buffer saline and the optimized peak (in the range of a red patch, the middle point of cross-hair indicating optimal detection conditions) with 280 and 520 nm wavelengths for excitation (ordinate) and emission (abscissa), respectively. (b) A calibration curve obtained in a concentration range between 175 and 1,000,000 ng/mL of ZON and the chemical structure of ZON (insert).

Figure 2

Analytical characterization of antisera collected from two 3-month old female New-Zealand white rabbits: (a) titer curves of antisera from rabbit 1 (■) and rabbit 2 (■) (dilution factor range of 1:50–1:12,200) using a zearalenone-6′-carboxymethyloxime-bovine serum albumin conjugate as a coating antigen at 5 µg/mL; blocked with 1% gelatin in phosphate buffer saline; (b) checkboard titration of the antiserum from rabbit 1 using the coating antigen at concentrations in the range of 0.3125–2.5 µg/mL and the serum at various dilution factors (solid symbols, solid lines): 1:1000 (■), 1:1500 (■), 1:2250 (■), 1:3375 (■). Titration was also performed under the same conditions with the serum inhibited by 3.2 ng/mL of zearalenone at various dilution factors (hollow symbols, slashed lines): 1:1000 (☐), 1:1500 (☐), 1:2250 (☐), and 1:3375 (☐).

Figure 3

Competitive indirect calibration curves for zearalenone obtained in assay buffer (hollow marker, dashed lines) and in surface water from river Danube (solid marker, solid lines) determined by absorbance (■, □) and fluorescence (●, o), detected at 576 and 593 nm wavelengths, respectively.

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 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 6

The immunofluorescence module developed in project Aquafluosense and appropriate for zearalenone determination (top left). The optical path in the detector head (top right). The modular instrumental setup during on site operation in a laboratory motor vehicle (bottom).