A Time-Resolved Fluorescent Lateral Flow Immunoassay for the Rapid and Ultra-Sensitive Detection of AFB1 in Peanuts and Maize

Aflatoxin B₁ (AFB₁), a highly toxic and carcinogenic mycotoxin, poses significant public health risks due to its widespread contamination of staple food crops such as peanuts and maize. Although conventional lateral flow immunoassays (LFIAs) are widely employed for rapid on-site screening, their limited sensitivity frequently compromises accurate quantification at trace levels. To improve the analytical performance of LFIAs, we developed a novel time-resolved fluorescence-based lateral flow immunoassay (TRFN-LFIA) by integrating reverse artificial antigen labeling with time-resolved fluorescence signal amplification. This method enhances detection sensitivity and enables rapid, ultra-sensitive, visible, and quantitative determination of AFB₁ in peanut and maize samples. Under optimized conditions, the TRFN-LFIA achieved a visible limit of detection (vLOD) of 0.30 ng/mL (2.22 µg/kg), a quantitative limit of detection (qLOD) of 0.04 ng/mL (0.30 μg/kg), and a half-maximal inhibitory concentration (IC₅₀) of 0.09 ng/mL. Recoveries from spiked peanut and maize samples ranged from 81.33% to 117.86%, with coefficients of variation (CVs) below 13.04%. Analysis of 21 real samples (13 maize and 8 peanut samples) yielded results highly consistent with those obtained by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Moreover, the method demonstrates significant advantages in terms of detection speed, cost-effectiveness, and operational convenience. Therefore, the results established the TRFN-LFIA method as a reliable and practical tool for on-site rapid detection of AFB₁ in contaminated food matrices, providing both a rapid and accurate approach for trace-level quantification and a novel strategy for enhancing the sensitivity of lateral flow immunoassays.