Electron transfer-driven bismuth-gold nanozyme-enhanced aptamer-based lateral flow assay for simultaneous detection of aflatoxin M1 and ochratoxin A in milk.

BACKGROUND: The co-contamination of aflatoxin M1 (AFM1) and ochratoxin A (OTA) poses an increased risk to human health due to their synergistic toxic effects, necessitating the development of sensitive and multiplex mycotoxins analytical methods.

RESULTS: An aptamer-based multiplex lateral flow assay (Apt-MLFA) was developed employing bimetallic bismuth-gold nanoparticles (BiAuNPs) to facilitate the simultaneous detection of AFM1 and OTA. The BiAuNPs were synthesized through in-situ deposition, demonstrating exceptional peroxidase-mimicking activity and storage stability. Specifically, the Bi of valence-rich and Au of high electron transfer efficiency endowed BiAuNPs with high peroxidase-mimicking activity, which oxidized 3,3',5,5'-tetramethylbenzidine through electron transfer. These nanoparticles were further functionalized with AFM1-and OTA-specific aptamers, which served as molecular recognition and signal amplification probes. Complementary DNAs were immobilized as dual test lines to enable a competitive detection system. By combining nanozyme-based signal amplification with smartphone-based analytical device, the assay demonstrated detection limits of detection of 0.11 ng/mL for AFM1 and 0.15 ng/mL for OTA, along with a linear detection range of 0.5-500 ng/mL. Also, this Apt-MLFA was successfully applied to real samples with satisfactory recoveries.

SIGNIFICANCE: This strategy effectively integrates bimetallic nanoparticle signal amplification, aptamer-specific recognition, and spatially separated dual test lines, thereby providing a robust platform for on-site multiplex screening of mycotoxins in food samples.