Direct and Competitive Optical Grating Immunosensors for Determination of Fusarium Mycotoxin Zearalenone.

Inna Székács,András Székács,István Szendrő,Nóra Adányi

doi:10.3390/toxins13010043

Abstract

Novel optical waveguide lightmode spectroscopy (OWLS)-based immunosensor formats were developed for label-free detection of Fusarium mycotoxin zearalenone (ZON). To achieve low limits of detection (LODs), both immobilised antibody-based (direct) and immobilised antigen-based (competitive) assay setups were applied. Immunoreagents were immobilised on epoxy-, amino-, and carboxyl-functionalised sensor surfaces, and by optimising the immobilisation methods, standard sigmoid curves were obtained in both sensor formats. An outstanding LOD of 0.002 pg/mL was obtained for ZON in the competitive immunosensor setup with a dynamic detection range between 0.01 and 1 pg/mL ZON concentrations, depending on the covalent immobilisation method applied. This corresponds to a five orders of magnitude improvement in detectability of ZON relative to the previously developed enzyme-linked immonosorbent assay (ELISA) method. The selectivity of the immunosensor for ZON was demonstrated with structural analogues (α-zearalenol, α-zearalanol, and β-zearalanol) and structurally unrelated mycotoxins. The method was found to be applicable in maize extract using acetonitrile as the organic solvent, upon a dilution rate of 1:10,000 in buffer. Thus, the OWLS immunosensor method developed appears to be suitable for the quantitative determination of ZON in aqueous medium. The new technique can widen the range of sensoric detection methods of ZON for surveys in food and environmental safety assessment.

Highlights

optical waveguide lightmode spectroscopy (OWLS) immunosensors were devised both in the direct and competitive-based formats
Immobilisation of the protein reactants has been carried out by several chemical routes utilising hydroxyl groups on the sensor surface converted into epoxy or amino functionalities, further reacted with appropriate chemical reagents for covalent immobilisation of the protein immunoreagents (Figure 1)
Immunoglobulin (IgG) fractions purified from ZON-specific rabbit antisera obtained against a conjugate of ZON to conalbumin as an immunogen were used in an immobilised antibody-based immunosensor format

Summary

Introduction

Zearalenone (ZON) is a mycotoxin produced by several Fusarium species, most frequently by F. graminearum, and is commonly found in maize and in wheat, barley, sorghum, and rye throughout various countries of the world, causing substantial human exposure [1]. ZON and its metabolites have oestrogenic activity in several species [2,3,4,5]. Tolerance levels in grains and grain products have been set in several countries at a concentration range of 20 to 1000 μg/kg [9], e.g., 20 to 200 μg/kg in unprocessed and processed cereal products in the EU [10]. Data evaluation on the most sensitive animal species—swine—and comparing with humans, a tolerable daily intake for ZON has been set as 0.25 μg/kg body weight [11,12]

Methods

Results

Conclusion