Label-free and highly sensitive fluorescent detection of bleomycin based on CRISPR-Cas12a and G-quadruplex-thioflavin T

CRISPR-Cas12a system opens up a new avenue for biosensing due to its flexible programmability and excellent target recognition accuracy. In this work, a novel label-free fluorescent biosensor for highly sensitive detection of bleomycin (BLM) was developed based on BLM-mediated inactivation of CRISPR-Cas12a and thioflavin T (ThT)-induced G-quadruplex formation. We designed a single-stranded DNA (ssDNA) probe containing the scission site of BLM as a DNA activator for Cas12a-crRNA. In the absence of BLM, the DNA activator binds to and activates the Cas12a, the activated CRISPR-Cas12a system trans-cleaves the ThT-induced G-quadruplex formation. Upon the addition of BLM, the DNA activator undergoes an irreversible cleavage event at the scission site via the oxidative effect of BLM with Fe(Ⅱ) as a cofactor. As a result, the trans-cleavage activity of Cas12a cannot be activated so the G-quadruplex-ThT complex remains intact, resulting in a significant increase of fluorescence signal. Attributing to the specific recognition of BLM and the excellent cleavage activity of Cas12a on DNA G-quadruplexes, the fluorescent biosensor exhibits high sensitivity and selectivity for detection of BLM in human serum samples, illustrating a promising tool for BLM assay in biomedical research.