Competitive aptamer assay for aflatoxin B1 detection using CRISPR/Cas12a as a signal amplifier

Sensitive and reliable determination of aflatoxin B1 (AFB1) is critical due to its high toxicity and wide distribution. Recently, the clustered regularly interspaced short palindromic repeats and its associated protein systems (CRISPR/Cas) have sparked a research upsurge in biosensing field, and a major challenge for non-nucleic acid molecule detection is to construct universal signal conversion strategy. Here, we proposed a competitive aptamer assay for AFB1 detection using CRISPR/Cas12a as a signal amplifier. In our strategy, a dual functional DNA probe was designed by linking AFB1 aptamer with Cas12a activator, and it was applied to regulate the activity of Cas12a in response to the AFB1 input. The probes are captured by bovine serum albumin (BSA)-AFB1 conjugates immobilized on the microplate by the aptamer-AFB1 binding, and the activator sequence activates Cas12a to indiscriminately scissor single-stranded DNA reporters, generating fluorescence signals. With the increase of AFB1 in the sample solution, the probe binds to free AFB1 instead of being captured by BSA-AFB1 on the microplate, so that the number of captured probes and activated Cas12a gradually decrease, resulting in a significant decline in fluorescence signal. Under optimized conditions, we achieved sensitive detection of AFB1 in the range from 0.15 ng/ml to 78 ng/ml within 110 min, and a low detection limit of 0.15 ng/ml AFB1 and a good linear range were obtained. The assay responded well in complex corn flour and liqueur samples, with satisfactory recovery rates ranging from 91 % to 122 %. The developed assay showed advantages in simple operation, superior generality, high selectivity, and satisfactory performance in the real sample analysis.