DNAzyme-regulated CRISPR/Cas12a based fluorescent biosensor for sensitive detection of alkaline phosphatase activity and inhibition.

Alkaline phosphatase (ALP) is regarded as an emerging biomarker and therapeutic target for various diseases. Herein, we developed a DNAzyme-regulated CRISPR/Cas12a cascade signal amplification strategy for sensitive and selective detection of ALP activity and inhibition. In this assay, the substrate strand of Cu2+-specific DNAzyme was designed as the DNA activator for Cas12a. The substrate strand would be cleaved into two fragments when ALP-catalyzed hydrolysis reaction disabled the complexation between Cu2+ and pyrophosphate (PPi). In this case, the cleaved sequences could not further bind to the Cas12a-crRNA complex to activate the trans-cleavage activity of CRISPR/Cas12a, which would hamper the cleavage of doubly labeled single-stranded DNA (ssDNA) fluorescence reporter, and thus a turn-off fluorescence signal. The cascade signal amplification strategy greatly improved the detection sensitivity for ALP activity, with a detection limit as low as 0.04 U/L. Moreover, the proposed method was successfully applied for ALP activity detection in serum samples and ALP inhibitory capability evaluation. This method overcomes the shortcoming of conventional methods that show unsatisfactory sensitivity and are susceptible to interfering substances, which might hold great promise in clinical diagnosis and biomedical research.