An electrochemiluminescence biosensor for miR-21 detection via TMSD-initiated transcription and CRISPR/Cas13a-assisted signal amplification on a Mxene-based electrode

MicroRNA-21 (miR-21) is a critical biomarker involved in various pathological processes, yet its low abundance in biological fluids poses challenges for accurate detection. Herein, we present a highly sensitive electrochemiluminescence (ECL) biosensor for miR-21 detection. The system integrates a multi-stage amplification cascade that begins with toehold-mediated strand displacement (TMSD) and continues through T7 RNA polymerase transcription and CRISPR/Cas13a-mediated collateral cleavage. The sensing interface was constructed by immobilizing ferrocene-labeled DNA probes onto a conductive AuNPs/Ti₃C₂Tx/Ru(II)-PEI nanohybrid-modified electrode. In the absence of miR-21, ferrocene effectively quenched the ECL signal. Upon target recognition, TMSD activates a split T7 promoter/template system, triggering RNA transcription. The resulting single-stranded RNAs activate Cas13a, which cleaves DNA probes and restores the ECL emission. Under optimized conditions, the biosensor exhibited a wide dynamic range from 10 aM to 500 fM and a detection limit as low as 5.23 aM. It also demonstrated excellent specificity, repeatability (RSD = 4.3 %), and recovery (99.93–106.20 %) in spiked serum samples. This work provides a versatile and modular ECL sensing platform for ultrasensitive miRNA detection, offering promising applications in clinical diagnostics and precision medicine.