Structure-Specific Electrochemiluminescent Biosensor for FEN1 Detection via Dumbbell Probe-Mediated Transcription and CRISPR/Cas13a-Induced G-quadruplexes Cleavage

Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5′-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti 3 C 2 /AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10 -9 U μL -1 and a wide dynamic range (1 × 10 -8 to 1 × 10 -5 U μL -1 ), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.