CRISPR/Cas12a-Powered\nElectrochemical Platform for\nDual-miRNA Detection via an AND Logic Circuit

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