Dual cascade signal amplification lights up G4 dimers for fluorescent detection of DNA repair enzyme FEN1

DNA damage resulted from various endogenous and environmental factors would induce various diseases by DNA mutations and replication errors. Flap endonuclease 1 (FEN1) is a structure-specific nuclease and it coordinates genomic stability via the specific excision of 5′ flaps in base excision repair and DNA replication. Herein, we develop a dual-cascade signal amplification fluorescent strategy for unlabeled and ultra-sensitive detection of FEN1. The assay comprises four consecutive steps: (1) FEN1-catalyzed cleavage of the 5′ flap from a branched double-stranded DNA substrate, (2) cyclization of the released flap into a dumbbell DNA structure, (3) generation of numerous G-quadruplex (G4) dimers via the synergistic integration of strand displacement amplification and rolling circle amplification, and (4) lighting up thioflavin T (ThT) by the G4 dimers to produce a significant fluorescence signal. This method demonstrate excellent sensitivity and achieve a detection limit of 2.33 × 10⁻⁵ U/μL, significantly enhanced sensitivity (by 1459-fold) compared to conventional ELISA kits. It enables high-throughput screening of FEN1 inhibitors, single-cell-resolution quantification of intracellular FEN1 activity, and diagnostic discrimination between malignant and normal cells based on their differential enzymatic activity profiles. Notably, this method enables high probe utilization, resulting in virtually no probe wastage, and the introduction of G4 dimer/ThT complex enables label-free fluorescence output without the need for cumbersome chemical labeling. It has great potential in clinical diagnosis and biomedical research.