CRISPR/Cas12a-based photoelectrochemical sensing of microRNA on reduced graphene oxide-anchored Bi2WO6 coupling with catalytic hairpin assembly

An innovative and generic CRISPR/Cas12a-driven photoelectrochemical (PEC) biosensing platform was developed for screening of microRNA-21 (miR-21) by coupling with target-triggered catalytic hairpin assembly (CHA) and reduced graphene oxide-anchored Bi2WO6 (rGO-BWO) as the photoactive material. CHA isothermal amplification involved two programmable hairpin DNA modules and miR-21 as an activator. In the presence of miR-21, the products of target-triggered CHA circuit were inserted into the Cas12a-crRNA duplex to initial trans-cleavage capacity of CRISPR/Cas12a nuclease, accompanying the digestion on alkaline phosphatase (ALP)-labeled single-stranded DNA (ssDNA)-encoded magnetic bead (MB) through the activated CRISPR system. The ALPs were detached from magnetic beads and promoted the generation of ascorbic acid (AA), which increased the photocurrent of rGO-BWO-modified electrode. The value of photocurrent was positively proportional to the level of AA, which was also linearly correlated with target concentrations. Under optimum conditions, the CRISPR-based PEC sensing system displayed satisfying photocurrent responses toward miR-21 within the range from 1.0 fM to 1.0 nM with a limit of detection of 0.47 fM. In addition, the biosensor exhibited acceptable stability and excellent selectivity. Impressively, CHA-mediated CRISPR-based PEC biosensing platform provides a universal and sensitive method for clinical cancer diagnostics and biomolecular research.