基于脱氧核酶-等温级联放大耦合的传感体系高灵敏检测水样中铅离子

A DNAzyme-isothermal cascade amplification sensing system for ultrasensitive detection of Pb2+ in water samples

摘要:

结合等温链替代扩增（ISDA）和指数扩增（EXPAR）设计一种非标记、高灵敏的Pb²⁺荧光生物传感体系。在Pb²⁺存在情况下，底物链被活化的GR-5 DNAzyme快速切割释放引物1。引物1与模板1杂交，并被DNA聚合酶（BSM）延伸形成带限制性内切酶（Nt.BbvCI）识别序列的双链核苷酸。BSM从Nt.BbvCI切割产生的切口再次延伸形成双链核苷酸，并释放信号G4-DNA片段。G4-DNA片段同时又可以作为模板2的引物，启动指数放大过程，从而释放更多的信号G4-DNA片段。扩增产物G4-DNA与原卟啉锌（ZnPPIX）相互结合从而产生强烈的荧光信号。详细优化了多种因素对检测体系的影响，在最优实验条件下，此方法对Pb²⁺的线性检测范围为0.1~50.0 nmol/L，检出限为0.03 nmol/L（S/N=3），回归方程为y=288.7x+744.7（y为荧光强度，x为Pb²⁺浓度）。干扰实验表明，该传感器对Pb²⁺具有良好的特异性和选择性。该传感体系成功应用于环境水体中Pb²⁺含量检测，加标回收率为94.0%~103.0%。本方法操作简单、选择性好、灵敏度高、有较强的抗干扰性能，可用于环境水样中Pb²⁺的高灵敏检测。

Abstract:

We exploited strand displacement amplification (ISDA) and exponential amplification (EXPAR) to design a label-free, ultrasensitive fluorescence sensing system for the detection of Pb²⁺. In the presence of Pb²⁺, the substrate strand is rapidly cleft by the activated GR-5 DNAzyme to release Primer 1. Primer 1 hybridizes to template 1 and is extended by DNA polymerase (BSM) to form a double-stranded nucleotide with a recognition sequence for restriction endonuclease (Nt. BbvCI). The nicks generated by BSM cleavage from Nt.BbvCI re-extended to form double-stranded nucleotides and release the signal G4-DNA fragment. The G4-DNA fragment can also be used as a primer for template 2 to initiate a series of amplification process, thereby releasing more signal G4-DNA fragments. G4-DNA binds to protoporphyrin zinc (ZnPPIX) to produce a strong fluorescent signal. The effects of various factors on the sensing system were investigated. Under the optimal experimental conditions, the linear detection range of Pb²⁺ was 0.1-50 nmol/L, and the LOD was 0.03 nmol/L (S/N=3). The regression equation is y=288.7x+744.7 (y is the fluorescence intensity and x is the Pb²⁺ concentration). Interference experiments show that the sensing system has good selectivity for Pb²⁺ against other metal ions. This method was applied to the detection of Pb²⁺ in environmental water, and the recoveries were obtained from 94.0% to 103.0%. The proposed sensing system has the advantages of simple operation, good selectivity, high sensitivity and strong anti-interference performance, and can be used for high-sensitivity detection of Pb²⁺ in environmental water samples.