Development of aptamer fluorescent switch assay for aflatoxin B1 by using fluorescein-labeled aptamer and black hole quencher 1-labeled complementary DNA

Aflatoxin B1 (AFB1) is one of the most toxic mycotoxins and draws great concern in health and food safety. A DNA aptamer against AFB1 having a stem-loop structure shows high binding affinity to AFB1 and promise in assay development for AFB1 detection. Based on the structure-switching property of the aptamer, we report an aptamer fluorescence assay for AFB1 detection. Aptamer with fluorescein (FAM) label at 5' end was used as affinity ligand, while its short complementary DNA (cDNA) with BHQ1 (black hole quencher 1) label at 3' end was used as a quencher. In the absence of AFB1, FAM-labeled aptamer hybridized with BHQ1-labeled cDNA, forming a duplex of cDNA and aptamer, resulting in fluorescence quenching of FAM. When AFB1 bound with aptamer, the BHQ1-labeled cDNA was displaced from aptamer, causing fluorescence restoration of FAM. We tested a series of FAM-labeled aptamers and BHQ1-labeled cDNAs with different lengths. The lengths of the aptamer stem and the cDNA, Mg2+ in binding buffer, and temperature had significant influence on the performance of the assay. Under optimized conditions, we achieved sensitive detection of AFB1 by using a 29-mer FAM-labeled aptamer and a 14-mer BHQ1-labeled cDNA, and the detection limit of AFB1 reached 0.2 nM. The maximum fluorescence recovery rate of FAM-labeled aptamer caused by AFB1 was about 69-fold. This method enabled the detection of AFB1 in complex sample matrix, e.g., diluted wine samples and maize flour samples. This aptamer-based fluorescent assay for AFB1 determination shows potential for broad applications.