Multiple fluorescence immunoassay for the simultaneous detection of Zearalenone and Ochratoxin A

A sensitive and accurate multiple fluorescence immunoassay for the simultaneous quantitative detection of Zearalenone (ZEN) and Ochratoxin A (OTA) in single spot based on multicolor quantum dots (QDs) labeling was developed for the first time. Two kinds of ZnCdSe/ZnS (core/shell) QDs with maximum emission wavelengths at 520 nm (green) and 610 nm (orange-red) were selected as marking materials, respectively. The anti-ZEN-mAb-QDs and anti-OTA-mAb-QDs were designed as the immune fluorescent probes. Fluorescence was measured at the same excitation wavelength and two different emission wavelengths to determine each target. The procedure for QDs-based multiple fluorescence labeled immunosorbent assay (M-FLISA) was developed. The 50% inhibition concentrations (IC₅₀) of ZEN and OTA were 0.034 and 1.175 ng/mL. Moreover, the limits of detection (LOD) for the simultaneous determination were 0.0239 and 2.339 ng/g for ZEN and OTA in maize, respectively. In addition, the recoveries ranged from 93.15 to 101.90% for ZEN and from 95.29 to 102.43% for OTA, with the coefficient variation (CV) of 2.70-8.86% and 3.51-6.22% respectively. There was good consistency between the M-FLISA and high performance liquid chromatography (HPLC) results, which confirmed that the M-FLISA was suitable for the simultaneous quantitative detection of various mycotoxins.