Catalytic hairpin assembly-assisted CRISPR/Cas12a mediated photoelectrochemical biosensor for sensitive detection of miRNA-122

Abstract

Herein, based on CRISPR/Cas12a and catalytic hairpin assembly (CHA) non-enzyme amplification technology, a general photoelectrochemical (PEC) biosensor with high sensitivity and specificity is constructed using Bi/g-C3N4 nanohybrids as photoelectric composite electrode for the detection of miRNA-122. Notably, the single-strand DNA labelled with the sensitizer methylene blue (MB) was combined with the electrode to secondary improved the photocurrent response and effectively reduced the detection background. The miRNA-122 was specifically recognized by the CHA process, and then amplified into a rich signal output, which effectively improved the detection sensitivity. The target DNA complex generated by CHA cycle on the electrode surface hybridized with Cas12a-crRNA duplex to formed the Cas12a-crRNA-target DNA ternary complex, which activated their trans-cleavage ability and repelled MB to leave the electrode surface. The PEC signal was significantly reduced, achieving accurate detection of the miRNA-122. Under the action of multiple amplification mechanisms, the sensor showed prominent sensitivity and selectivity, the universal CRISPR/Cas12a cutting characteristics further expanded its analysis potential and simplified the operation difficulty, which can expand the application in biomedical detection and clinical diagnosis, showing great potential in efficient nucleic acid detection and in vitro diagnosis.