An ultrasensitive biosensor with suppressed background signals for FEN1 detection in a homogeneous reaction via cascade primer exchange reaction and CRISPR/Cas12a system

Flap endonuclease 1 (FEN1) is a structure-specific enzyme which plays a crucial role in DNA replication and repair within mammalian cells. FEN1 is also known to be overexpressed in various cancer cells and has emerged as a potential biomarker for cancer diagnosis. However, there have been limited progress in development of sensitively analytical methods toward FEN1 detection. Herein, we have developed an ultrasensitive FEN1 sensing biosensor for FEN1 detection by combining primer exchange reaction (PER) with CRISPR/Cas12a signal amplification (PCSA). This innovative method achieves remarkable sensitivity for FEN1 detection, with a limit of detection (LOD) as low as 3.72×10-7 U/μL. Notably, PCSA effectively suppresses background signals, resulting in a high signal-to-noise ratio exceeding 31-fold due to three specific enzymatic recognition reactions. Consequently, PCSA demonstrates outstanding selectivity and robust anti-interference capabilities. The PCSA was employed to detect the FEN1 in complex serum samples, as well as in normal and tumor cells, achieving an LOD for FEN1 as low as in 10 cancer cells. Additionally, PCSA offers a simple operational process and yields a visually observable signal output, simplifying its use for the point-of-care testing (POCT) in some resource-limited regions. This straightforward approach holds significant promise for broad applications in FEN1 sensing and inhibitor screening.