Bipedal DNA walker- CRISPR/Cas12a coupling with sulfur doped boron nitride-carbon nitride composite biosensing for the detection of miRNA-21

A novel photoelectrochemical (PEC) biosensor based on 2D-2D heterojunction S-BN/CN and dual-orbit threedimensional (3D) bipedal DNA walker-synergized CRISPR/Cas12a signal amplification was fabricated for sensitive detection of miRNA-21. First of all, S-BN/CN, which sulfur was doped into boron nitride (BN) to construct heterojunction of 2D S-BN and 2D g-C3N4, was used as photosensitive materials to provide initial photocurrent, and combined methylene blue (MB) labeled hairpin DNA (MB-HP) as a signal probe. Secondly, after identifying the target miRNA-21, the two swing arms were simultaneously activated on two 3D orbits, cleaving along the orbit to output massive short single strand DNA (O-DNA) under the action of Nt.BsmAl endonuclease. Then trigger the trans-cleavage of CRISPR/Cas12a and cut off the hairpin DNA labeled with MB to achieve signal-off. The result showed that the proposed PEC biosensor had a good sensitivity for the detection of miRNA-21. It was a good linearity in the range of 10 fM - 100 nM, and the limit of detection was 3.98 fM (S/N = 3). The proposed PEC biosensor achieved sensitive detection with high specificity and low limit of detection for miRNA-21, while the rational design could be extended to achieve the detection of other targets.