Gold Nanoparticles/Mo2C/MoO2-Modified Electrodes for Nucleic Acid Detection through CRISPR/Cas12a Photoelectrochemical Assay

Recently, RNA-guided CRISPR/Cas12a systems have been applied in sequence-specific DNA/RNA targeting and detection. However, the CRISPR/Cas12a system mainly relies on fluorescence readout, and the signal conversion depends on the nonspecific cleavage of single-stranded DNA. In this study, a CRISPR/Cas12a assay with site-specific double-stranded DNA (dsDNA) cleavage was developed. A self-powered photoelectrochemical (PEC) detection based on redox cycling with p-aminophenol and Ru(bpy)3Cl2 on a gold nanoparticles/Mo2C/MoO2-modified glassy carbon electrode was used for signal readout. The nucleic acid was detected by an enhancement of the PEC signal produced upon the activation of Cas12a dsDNase activity by dsDNA produced from the recycling of target DNA. dsDNA was cleaved to release alkaline phosphatase to catalyze the generation of p-aminophenol, which can be reused to achieve the PEC signal conversion. Under the optimal conditions, this PEC-CRISPR/Cas12a assay accurately identifies as low as 0.4 fM target HIV nucleic acid, and the dynamic range reaches from 1 to 700 fM. The relative standard deviation was 3.2% at 10 fM. As for the FMS-like tyrosine kinase 3 gene, the limit of detection was 58 aM with a linearity range from 0.2 to 100 fM. This assay enables stable and accurate determination to measure other nucleic acids, indicating important potential in future nucleic acid detection.