A sensitive homogeneous electrochemiluminescence (ECL) biosensor for flap endonuclease 1 (FEN1) detection was developed by combining highly sensitive ECL detection, high efficiency of branched hybridization chain reaction (BHCR) amplification, a convenient homogeneous strategy, and simple ultrafiltration separation. Magnetic beads were first modified with well-designed double flap DNAs containing 5′-flaps. In the presence of FEN1, the 5′-flap can be cleaved, and a large amount of single-stranded DNA can be produced, which can be separated easily from the double-flap DNA-modified beads by a magnet. Then, the cleaved 5′-flap can be used to initiate BHCR amplification to produce a large amount of long-strand dsDNA. Ru(phen) 3 2+ can insert dsDNA to form Ru-dsDNAs, which can be easily separated from the main solution through ultrafiltration. The ECL signal from the separated Ru-dsDNAs has a good linear relationship with the logarithm of the FEN1 concentration ranging from 6.5 × 10 −2 ∼ 6.5 × 10 3 U/L with a detection limit of 2.2 × 10 −2 U/L. The proposed biosensor was used to evaluate FEN1 activity in real samples with satisfactory results.
Flap endonuclease 1 (FEN1) is a nuclease overexpressed in cells with a high replicate rate, which has been recognized as a potential biomarker in the molecular diagnosis of cancers. In this work, we developed a fluorescent biosensor based on exponential amplification reaction (EXPAR) to realize the rapid and sensitive detection of FEN1. A DNA probe specific for FEN1 is used for the generation of reporter DNA (rDNA), and then the rDNA hybridizes with a template, triggering EXPAR to produce substantial rDNA exponentially. However, when no FEN1 is in the system, the EXPAR will not be initiated because of the lack of rDNA. Derived from the high amplification efficiency of EXPAR, the rapid quantification of FEN1 with a limit of detection (LOD) of 0.5 fM was realized. It is also revealed that this biosensor exhibits good selectivity towards FEN1 under the challenge from real samples including extracts of cells and solid tumors. Furthermore, this sensor was reengineered for the analysis of FEN1 inhibitors, which shows promise in the screening and evaluation of potential drugs targeting FEN1. This work integrates EXPAR into the biosensing of FEN1 for the first time, which provides a promising tool for FEN1-related biomedical investigations.
危险品化学品经营许可证（带存储）