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.
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