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

A multiple quadruple-label time-resolved fluorescent immunochromatographic assay (QL-TRFICA) for simultaneous quantitative detecting 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 three mycotoxins concentrations 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 concentration of LOD, 2LOD and 4LOD, with 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.