In Situ-Generated Dual-Catalytic DNAzyme Walkers for High-Fidelity Electrochemiluminescence Detection of FEN1 Activity

Flap endonuclease 1 (FEN1), an essential enzyme responsible for 5'-flap excision during DNA repair, serves as a key biomarker for disease diagnostics. However, current DNA probe-based methods for detecting FEN1 activity often suffer from nonspecific activation and signal leakage. Herein, we developed an innovative target-triggered in situ generation strategy that converted the linear DNA precursor into a dual-catalytic circular DNAzyme (DARE) walker, enabling high-fidelity electrochemiluminescence (ECL) detection of FEN1 activity. Unlike conventional preblocked DNA walkers that are susceptible to signal leakage due to unintended activation, the DARE walker is specifically generated from a linear DNA precursor via a target-triggered enzymatic cascade process. This in situ generation strategy minimizes background signals through degradation of unreacted precursors, thereby ensuring high analytical fidelity. Moreover, the DARE walker, characterized by a closed circular architecture with dual catalytic cores, exhibited a 35-fold greater resistance to exonucleases and an 8-fold enhanced stability in cell lysates compared to its linear counterparts, as well as a 6-fold higher cleavage efficiency relative to a single-core configuration. When incorporated into a DNA wireframe track-based ECL biosensor, the DARE walker demonstrated excellent sensitivity and selectivity with a limit of detection of 9.0 × 10⁻¹⁰ U μL⁻¹, offering a robust tool for high-fidelity bioanalysis.