Alkaline Phosphatase-RegulatedDNAzyme Cleavage Coupledwith CRISPR/Cas12a for Quantitative Detection of Deoxynivalenol inAgricultural Crops

Sensitive and simplified detection of a mycotoxin such as deoxynivalenol (DON) is crucial for food safety. In recent years, the CRISPR/Cas technology has demonstrated significant potential in detecting non-nucleic acids. Herein, we present a triple enzyme-assisted fluorescence immunoassay (TEFIA) that integrates alkaline phosphatase (ALP)-regulated DNAzyme cleavage with the CRISPR/Cas12a assay for the accurate detection of mycotoxin. By employing this method for detecting DON, we exhibit a low detection limit of 0.05 ng/mL and a satisfactory linear response between 0.1 and 10 ng/mL. This performance exceeds the conventional sensitivity levels found in traditional methods. TEFIA also demonstrates a good correlation with ic-ELISA for testing DON in real samples. Thus, it offers a robust and efficient detection platform for DON in complex matrices. Furthermore, TEFIA can be employed to identify various targets of interest by merely altering the antibody–antigen pairs, indicating its great potential in a wide range of applications.