An ultrasensitive biosensing platform for FEN1 activity detection based on target-induced primer extension to trigger the collateral cleavage of CRISPR/Cas12a

Abstract

Flap endonuclease 1 (FEN1), a structure-selective endonuclease essential for DNA replication and repair, has been considered as a new promising marker for early cancer diagnosis. However, reliable, sensitive and convenient biosensors for FEN1 detection are still technically challenging. Herein, a fluorometric biosensor based on target-induced primer extension to initiate the collateral cleavage of CRISPR/Cas12a has been established for ultrasensitive and specific detection of FEN1 activity. Using branched DNA to probe FEN1 activity, the cleaved 5′ flap initiated DNA polymerase-mediated primer extension to produce plenty of DNA duplexes containing protospacer adjacent motif (PAM) which act as activators to initiate the collateral cleavage activity of Cas12a protein, producing an significantly amplified fluorescence response for ultrasensitive determination of FEN1 activity. The developed biosensing platform displays excellent analytical performance, with a limit of detection (LOD) down to 8.9 × 10−5 U μL−1, and a wide linear range from 1.0 × 10−4 to 5.0 × 10−1 U μL−1. Moreover, the proposed strategy was successfully used for FEN1 detection in serums and cell lysates and suggests potential clinical applications, which may provide a reliable approach for FEN1 that will allow effective diagnosis in the early stages of related cancer.