Amplified fluorescence detection of pb2+ using pb2+-dependent dnazyme combined with nicking enzyme-mediated enzymatic recycling amplification

A fluorescence sensing system was developed for the detection of Pb2+ with excellent sensitivity and selectivity based on Pb2+-dependent DNAzyme (8-17E DNAzyme) with nicking enzyme (Nt.BbvCI)-assisted signal cascade amplification strategy. In the presence of Pb2+, the 8-17E DNAzyme can catalyze the cleavage of its substrate. And subsequently, the partial substrate strand dissociated from DNAzyme could hybridize with molecular beacon (MB), resulting in the restoration of fluorescence signal as well as the formation of the double-stranded recognition site for nicking endonuclease (Nt.BbvCI). After the Nt.BbvCI mediated the cleavage of MB, the released partial substrate strand could hybridize with another MB probe again and be re-used for the second cycle of cleavage. Eventually, each target-induced partial substrate strand can trigger many cycles of cleavage to achieve the amplified fluorescence detection of Pb2+. This new design avoids the modification on DNAzyme and substrate, and significantly improves the sensitivity with a detection limit down to 1.0 × 10-10 M. Moreover, it also exhibited satisfactory selectivity for Pb2+ detection, even in the presence of 2 times concentrations of Zn2+ and 5 times concentrations of each other interferential metal ions. Furthermore, this proposed method was successfully used for the determination of Pb2+ in river water samples with recoveries from 96.1% to 108%.