Time-Resolved Fluorescence Immunochromatographic Assay Developed Using Two Idiotypic Nanobodies for Rapid, Quantitative, and Simultaneous Detection of Aflatoxin and Zearalenone in Maize and Its Products

Aflatoxins and zearalenone (ZEN) are highly common mycotoxins in maize and maize-based products. This study aimed to report a time-resolved fluorescence immunochromatographic assay (TRFICA) developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin B₁ (AFB₁) and ZEN in maize and its products. A novel Eu/Tb­(III) nanosphere with enhanced fluorescence was prepared as a label and conjugated to anti-idiotypic nanobody (AIdnb) and monoclonal antibody (mAb). On the basis of nanosphere–antibody conjugation, two patterns of competitive time-resolved strip methods (AIdnb–TRFICA and mAb–TRFICA) were established and compared. The half inhibition concentration of AIdnb–TRFICA was 0.46 and 0.86 ng·mL^–1 for AFB₁ and ZEN, which was 18.3- and 20.3-fold more sensitive than that of mAb–TRFICA for AFB₁ and ZEN, respectively. Under optimal conditions, AIdnb–TRFICA for dual mycotoxin was established and provided a quantitative relationship ranging from 0.13 to 4.54 ng·mL^–1 for AFB₁ and 0.20 to 2.77 ng·mL^–1 for ZEN, with a detection limit of 0.05 and 0.07 ng·mL^–1 in the buffer solution, respectively. AIdnb–TRFICA showed good recoveries (72.6%–106.6%) in samples and was applied to detect dual mycotoxin in maize samples with satisfying results. To the best of our knowledge, it is the first report about a time-resolved strip method based on AIdnbs for dual mycotoxin