A multi-level signal conversion architecture for enzyme sensing: Integrating MXene nanoplatforms with CRISPR-driven...

Precise and ultrasensitive detection of flap endonuclease 1 (FEN1), a key DNA repair enzyme implicated in cancer diagnostics, remains challenging due to its subtle structural cleavage activity. Herein, we present a cascade-amplified electrochemiluminescence (ECL) biosensor based on a Ti₃C₂ MXene-supported Ru (bpy)₃²⁺/Au nanocomposite integrated with a CRISPR-Cas13a system and DNA walker circuitry. Upon specific recognition and cleavage of a 5'-flap substrate by FEN1, a nicked DNA product is circularized and transcribed via T7 RNA polymerase, yielding RNA activators that trigger Cas13a-mediated collateral cleavage. This event releases a blocked DNA walker, which reorganizes Fc-labeled DNA on the electrode surface and restores the ECL signal suppressed by resonance energy transfer. The system achieves a detection limit as low as 1.48 fU/mL and exhibits a dynamic range spanning five orders of magnitude. Compared to fluorescence-based CRISPR detection systems, the ECL-based platform offers low background, high signal-to-noise ratios, and operational simplicity using standard electrochemical instrumentation, supporting practical deployment in clinical diagnostics. Furthermore, the platform demonstrates high selectivity against other nucleases and proteins, along with excellent performance in spiked human serum samples. This work presents a robust and modular strategy for accurate enzyme activity profiling with promising applications in early-stage disease diagnostics.