MicroRNAs (miRNAs) have emerged as critical biomarkers for early cancer diagnosis due to their high specificity and stability in biological fluids. In this study, we report a highly sensitive and specific electrochemiluminescence (ECL) biosensor for the detection of miR-31, a miRNA closely associated with non-small cell lung cancer. The sensing platform integrates a PEI-Ru@Ti₃C₂@AuNPs-modified electrode with a ferrocene-labeled DNA probe (DNA1-Fc) to construct a target-responsive signal-on system. Upon recognition of miR-31, an isothermal strand displacement amplification (ISDA) reaction is initiated, producing abundant double-stranded DNA (dsDNA) that activates the CRISPR/Cas12a complex. The trans-cleavage activity of Cas12a then cleaves the DNA1-Fc probes on the electrode surface, removing the quenching ferrocene moiety and restoring the Ru-based ECL signal. Under optimized conditions, the biosensor exhibited a wide dynamic range from 10 aM to 100 pM and a remarkably low detection limit of 1.67 aM. The system also showed excellent specificity against homologous miRNAs, and its applicability was successfully validated in spiked human serum samples, achieving high recovery and reproducibility. The synergistic combination of nanomaterial-enhanced ECL emission, isothermal nucleic acid amplification, and CRISPR-based enzymatic cleavage provides a powerful strategy for ultrasensitive nucleic acid detection. This work offers a promising approach for early cancer diagnosis and has great potential for clinical translation and point-of-care testing.
