A dual mode one-pot assay detecting FEN1 activity through magnetic separation-based cascade exponential amplification strategy.

As a structure-specific nuclease with 5’flap endonuclease activity, Flap endonuclease 1 (FEN1) can specifically recognize and cleave the trinucleotide overlapping structure of 5’flap dsDNA, which is closely associated with tumor development and progression. Accordingly, hypersensitive and accurate detection of FEN1 is pivotal for cancer diagnosis and monitoring. Herein, we propose a dual mode one-pot testing platform through magnetic separation-assisted rolling circle amplification-coupled CRISPR/Cas12a (MRCAC) for detecting FEN1 activity. Upon incising bead-coupled 5’flap dsDNA substrate by FEN1, the separated short single-stranded DNA (ssDNA) triggers rolling circle amplification (RCA). We leverage tandemly repeated RCA products to activate Cas12a, which cleaves RCA products into short fragments that initiate new RCA. On the one hand, Cas12a catalyzes trans-cleavage of hairpin reporters, releasing abundant fluorescence signals. This exponential cascade amplification model significantly enhances sensitivity with a detection limit of 3.17 × 10⁻6 U/µL. On the other hand, the Cas12a catalyzed cleavage of linkers between DNA-functionalized gold nanoparticles (DNAs-AuNPs) and unlocks a reversible aggregation to dispersion transition. This shift is visually discernible and quantifiable by a portable colorimeter. Combining MRCAC with AuNPs, a simple and equipment-free POCT was successfully created. Applying to spiked serum and cellular extracts, the MRCAC biosensor exhibits excellent performance in complicated biological samples. Furthermore, it verifies intense potential for screening FEN1 inhibitors, highlighting valuable application prospects in early therapeutic evaluation and anticancer drug development.