Photonic Microbead Array Digital Time-Resolved Fluorescence Ultrasensitive Platform for Simultaneous Detection of Multiple Mycotoxins

Limitations in the sensitivity, linear detection range, and cross-reaction of lateral flow immunoassays mainly hamper their application in rapid screening for multiple targets. In this work, we designed a new time-resolved fluorescence immunoassay (TRFIA) platform to overcome these limitations. This platform uses europium chelate polystyrene (PS@Eu) nanoparticles conjugated with monoclonal antibodies to sense multiple mycotoxins. We employed a competitive TRFIA protocol in which the conjugated PS@Eu was used on the surfaces of photonic microbead arrays (PMAs). The TRFIA signal of PMAs on the pad was recorded with the digital time-resolved fluorescence reader. The developed TRFIA shows wide detection linear ranges (0.01-1000 ng/mL for DON, 0.1-100 ng/mL for OTA, and 0.01-100 ng/mL for AFB₁), low limits of detection (LODs) (7.9 pg/mL for DON, 18 pg/mL for OTA, and 7.7 pg/mL for AFB₁), good specificity, good recovery ratios (76.68-117.26%), and good reproducibility in grain samples. The simulated fluorescence enhancement effect of PMA indicated that the electric field distribution on the surface of PS@Eu on PMA is twice higher than that on the surface of PS@Eu. The new TRFIA for three kinds of mycotoxins was 1000-fold more sensitive than the classical TRFIA, and it has great potential application in rapid screening for multiple targets.