Catalytic hairpin assembly assists CRISPR/Cas12a-mediated high-sensitivity detection of aflatoxin B1

Aflatoxin B1 (AFB1) is recognized the most toxic and carcinogenic mycotoxin and is widely present in cereals and various foods. Therefore, its precise detection is crucial to safeguard food quality and human health. In this study, we proposed a highly sensitive detection system for AFB1 by combining the catalytic hairpin assembly (CHA) and CRISPR/Cas12a techniques. The Aptamer of Aptamer-Initiator interacts with AFB1 to release the blocked Antisense. As a result, the Initiator of the Aptamer-Initiator becomes free and can act as a toehold to bind with H1, which can initiate the CHA to generate a large amount of double-stranded DNA, which hybridized with the Cas12a-crRNA duplex to form the Cas12a-crRNA-DNA ternary complex, wherein Cas12a subsequently cleaves the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. After optimization, we observed a linear relationship between fluorescence intensity and the AFB1 concentration in the range of 50 pM to 1 nM, with a limit of detection (LOD) of 10 pM. Also, the system was robust and could operate with excellent reliability and accuracy even in complex samples. The recovery values in food samples ranged from 92.23 % to 111.72 %, with relative standard deviation (RSD) below 5.68 %. The system exhibited remarkable advantages, including high sensitivity, strong specificity, and rapid response, thereby showed great potential in the efficient detection of AFB1 contaminants in food.