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A fluorescent labeling system for the determination of endonuclease activity using the hybridization chain reaction (HCR) is reported. Two hairpin probes, H1 and H2, were well-designed in which two split cleavage sequences of endonuclease  Eco RI were respectively integrated into H1 and H2. Black Hole Quencher 1 (BHQ1) and carboxyfluorescein (FAM) were labeled at the 5′ and 3′ ends of H1, respectively. Once trigger DNA was introduced, HCR between H1 and H2 was triggered to form long DNA nanowires containing the intact cleavage sequences of  Eco RI. At the same time, the BHQ1 and FAM were close together and the fluorescence signal was quenched. In the presence of  Eco RI, the cleavage sequences were recognized by  Eco RI and long DNA nanowires were cleaved to short double-stranded DNA (dsDNA) polymers. The FAM was far from BHQ1, resulting in the recovery of fluorescence signal. Under the optimized conditions, the fluorescence intensity was linear with  Eco RI concentration over the range from 0.001 to 0.2 U μL −1  and the detection limit is 1.2 × 10 −4  U µL −1 . In addition, this strategy is versatile and may be used to determine other endonucleases activity by simply changing the specific cleavage sites in HCR system.

Determination of Endonuclease Activity by an Enzyme-Free Fluorescent Biosensor Using the Hybridization Chain Reaction (HCR)