Programming DNA walker and DNA tetrahedral nanostructures mediated CRISPR/Cas12a electrochemiluminescence biosystem and its biosensing application

A CRISPR/Cas12a electrochemiluminescence (ECL) biosensor based on the multiple sites DNA walker with controlled swing arm position, integrating Tribulus terrestris-like AuCu nanoterrestris (AuCu NTs) and DNA tetrahedral nanostructures (DTNs) is constructed for ultrasensitive detection of miRNA-21. The AuCu NTs was served as a co-reaction accelerant to promote the reduction of S2O82- at the cathode, which can be rapidly converted with S2O83•- and significantly enhance the ECL signal from graphitic carbon nitride nanosheets. Meanwhile, by virtue of the excellent stability of DNA tetrahedral nanostructure, the binding efficiency between single strand DNA and luminescent materials was improved. Afterward, DNA walker signal amplifier converted and amplified the target miRNA-21 into transitional DNA under the activity of Nt.BsmAI nicking endonuclease, thereby activating trans-cleavage ability of CRISPR/Cas12a and achieving the efficient annihilation of ECL signals was employed. As a proof of concept, the as-constructed biosensor demonstrated well specificity and sensitivity towards miRNA-21 with the range of 1.0 fM to 10 nM and the detection limit was 0.339 fM (S/N = 3). Therefore, the proposed strategy expanded the scope of application of CRISPR/Cas12a and demonstrated potential of diagnostic analysis.