A highly sensitive homogeneous electrochemiluminescence biosensor for flap endonuclease 1 based on branched hybridization chain reaction amplification and ultrafiltration separation

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)32+ 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 × 103 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.