CRISPR/Cas12a-Powered Electrochemical Platform for Dual-miRNA Detection via an AND Logic Circuit

Abstract

The CRISPR/Cas technology shows great potential in molecular detection and diagnostics. However, it is still challenging to detect multiple targets simultaneously using the CRISPR-Cas system. Herein, we ingeniously leverage the synergistic effect of two short single-stranded DNA activators to construct a CRISPR/Cas12a-driven electrochemical sensing platform based on an AND logic circuit ("AND" LC-CRISPR) for the simultaneous detection of dual miRNAs. Specifically, the exponential amplification reaction products triggered by the dual-specific miRNAs are designed as binary inputs to bind with Cas12a/crRNA, forming an AND logic circuit and activating the trans-cleavage ability of the CRISPR-Cas12a system. Subsequently, the hairpin probe biogate on the surface of the functionalized electrochemical signal probe (MB@HP-Fe-MOF) is cleaved by activated Cas12a, leading to the release of the encapsulated electroactive signal molecule methylene blue, thereby generating a strong electrochemical signal. As a result, this "AND" LC-CRISPR sensing platform, requiring only a single crRNA assembled with Cas12a, achieves simultaneous detection of miRNA-155 and miRNA-21 at concentrations as low as 3.2 fM. Moreover, the platform allows easy adjustment of the AND logic circuit inputs according to different detection targets, allowing it to be easily expanded for the analysis and diagnosis of other multibiomarkers. This approach demonstrates promising potential for future applications in intelligent diagnostic medicine.