A quadruple-label time-resolved fluorescence immunochromatographic assay for simultaneous quantitative determination of three mycotoxins in grains

Abstract

A multiplex quadruple-label time-resolved fluorescence immunochromatographic assay (QL-TRFICA) for simultaneous quantitative detection of aflatoxin B₁ (AFB₁), zearalenone (ZEN) and ochratoxin A (OTA) in grains was established in this paper. This assay was developed based on quadruple-label immunoprobes, one of which was a chicken IgY-specific antibody coupled with time-resolved fluorescent nanobeads (TRFN) for the test C line signal while the other three were specific to AFB₁, ZEN and OTA monoclonal antibodies coupled with TRFN for the test T line signal. Quantitative relationships between the concentrations of the three mycotoxins and T/C ratios were established to determine the analyte concentration through a portable multi-channel fluorescence reader. The limits of detection (LODs) for AFB₁, ZEN and OTA were 0.04 μg kg⁻¹, 0.20 μg kg⁻¹ and 0.10 μg kg⁻¹ in 6 grains (corn, soybean, sorghum, wheat, rice and oats), respectively. Recoveries ranged from 71.60% to 119.98% at fortified concentrations of LOD, 2LOD and 4LOD, with a coefficient of variation <15%. Analysis of field grain samples by QL-TRFICA was in accordance with that of LC-MS/MS. Therefore, this proposed multiplex QL-TRFICA was a promising method for on-site screening of multiple mycotoxins in grains.