A magnetic separation-assisted cascade hybridization chain reaction amplification strategy for sensitive detection of flap endonuclease 1

Abstract

Sensitive and accurate detection of flap endonuclease 1 (FEN1) is essential to understand its roles in DNA replication and repair and explore its functions in tumor diagnosis and prognosis. Here, we designed a magnetic separation-assisted cascade hybridization chain reaction (HCR) amplification strategy for enzyme-free and sensitive detection of FEN1. Firstly, the forked dsDNAs contained the recognition site (5′ overhanging DNA flap) of FEN1 were modified on magbeads surface to form magnetic recognition probes. By the recognition and catalytic cleavage of FEN1, the 5′ flaps were removed into solution and then purified by magnetic separation. Then, the obtained flaps served as triggers to activate the linear-assembly of hairpin probes H1 and H2 (primary HCR), forming nicked double helices with split fragments. Finally, the fragments acted as new triggers to activate the branched-assembly of hairpin probes H3 and H4 (secondary HCR), causing fluorophore/quencher labeled on H3 far away and fluorescence recovery. Benefiting from the purification of triggers by magnetic separation and the signal amplification effect of cascade HCR, this strategy achieved a low detection limit of 3.6 × 10−5 U μL−1 for FEN1, and enabled the assay of it in actual samples. The results demonstrate that our strategy will provide a potent approach for sensitive detection of FEN1 in early tumor diagnosis and prognosis.