An ultra-sensitive fluorescence biosensor with rolling circle amplification and CRISPR/Cas12a one-pot system for FEN1 detection

Abstract

Flap endonuclease 1 (FEN1), a structure-specific nuclease, has been reported to be widely involved in the development of cancer, and recognized as a new biomarker for cancer. However, there remains a deficiency in the availability of simple, rapid and reliable biosensors for its detection. We have constructed a cascade signal amplification fluorescence biosensor for ultra-sensitive and rapid detection of FEN1. This biosensor relied on FEN1-induced production of the 5’ flap DNA, and combined rolling circle amplification (RCA) and CRISPR/Cas12a one-pot system (RCOS). By utilizing branched dsDNA substrates to provoke FEN1 activity, the 5’ flap DNA was cleaved and isolated through magnetic separation. Subsequently, these DNA fragments initiated the RCA and CRISPR/Cas12a one-pot exponential amplification reaction, activating the cis and trans-cleavage activity of Cas12a and resulting in a significant fluorescence signal for readout. By combining RCA and CRISPR/Cas12a one-pot cascade signal amplification, the detection signal was remarkable enhanced. The RCOS exhibited excellent sensitivity with a limit of detection (LOD) of 4.1 × 10-7 U/μL, which was more sensitive and expeditious than many other approaches. Furthermore, the biosensor successfully facilitated accurate determination of FEN1 in cell extracts and plasma samples, revealing the potential clinical application and providing a dependable and rapid approach for FEN1 inhibitor screening. Compared with traditional methods, this approach has several benefits including improving the selectivity and sensitivity for FEN1 assay, reducing the complex operation process, and providing a method for the FEN1 inhibition screening.