基于核酸切割酶与脱氧核酶的荧光循环放大系统检测铅(Ⅱ)

利用核酸切割酶(Nicking endonuclease)识别特定DNA双链并切割其中某条单链的性质,构建了基于8-17E脱氧核酶(8-17E DNAzyme)的pb2+荧光循环放大检测方法.pb2+可激活8-17E脱氧核酶水解RNA底物,产生并释放出的单链与分子信标探针( Molecular beacon,MB)杂交,导致其茎环结构被破坏,荧光信号恢复；同时形成含有核酸切割酶Nt.BbvCI识别位点的双链区域.在核酸切割酶Nt.BbvCI的作用下,分子信标探针被切割释放,游离出来的单链可与其它分子信标重新杂交,从而触发下一轮酶切,引起荧光检测信号的循环放大.本方法避免了8-17E脱氧核酶与底物链的修饰,最低可以检测出水溶液中1.0×10-10 mol/L Pb2+,并在2倍浓度的Zn2+,以及5倍浓度的其它干扰金属离子存在的情况下对pb2+显示出良好的选择性.本方法对环境水样中pb2+的标准加样回收率为96.1％～108.0％.

Amplified Fluorescence Detection of Pb2+ Using pb2+-dependent 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 8-17E DNAzyme and nicking endonuclease mediated fluorescence cyclic amplification strategy.In the presence of Pb2+,the 8-17E DNAzyme catalyzed the cleavage of RNA substrate.Subsequently,the partial substrate strand would dissociated from DNAzyme and hybridized with molecuar beacon(MB),resulting in the restoration of the fluorescence signal and the formation of the double-stranded recognition site for nicking endonuclease(Nt.BbvCI).After the Nt.BbvCI mediated cleavage of MB probes,the released partial substrate strand could then hybridize with another MB probe 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 cyclic amplified fluorescence detection of Pb2+.This new design both avoids the modification on 8-17E DNAzyme and substrate,and significantly improves the sensitivity with a detection limit down to 1.0×10-10 mol/L.Moreover,it also exhibited satisfactory selectivity for Pb2+ detection,even in the presence of 2 times of Zn2+ and 5 times of other interferential metal ions.Furthermore,the applicability of this proposed method for Pb2+ detection in environmental samples was demonstrated with a Pb2+ recovery from 96.1% to 108%.These advantages endow the sensing strategy with a great potential for the facile on-site and real-time Pb2+ sensing from a wide range of real samples.