基于核酸外切酶Ⅲ辅助双循环等温信号放大的高灵敏Hg^(2+)传感方法研究  

Exonuclease Ⅲ-assisted Dual-Cycle Isothermal Signal Amplification for Highly Sensitive “Turn-on” Type Detection of Mercury Ion (Ⅱ)

作　　者：张何[1] 王青 杨梅 傅昕[1] ZHANG He;WANG Qing;YANG Mei;FU Xin(Hunan Province Key Laboratory of Environmental Catalysis & Waste Recycling, School of Chemistry andChemical Engineering, Hunan Institute of Engineering, Xiangtan 411104, China)

机构地区：[1]湖南省环境催化与废弃物再生化重点实验室湖南工程学院化学化工学院,湘潭411104

出　　处：《分析化学》2019年第6期899-906,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.21005067);湖南省自然科学基金项目(No.2019JJ40055);湖南省教育厅科研项目(No.17A044;16B060)资助~~

摘　　要：设计了一种Hg^(2+)触发的核酸外切酶Ⅲ(ExoⅢ)辅助的双发夹探针双循环等温信号放大策略,在此基础上发展了一种免标记、超灵敏的Hg^(2+)生物传感器。在Hg^(2+)存在情况下,发夹探针1的3'游离序列与汞离子识别探针通过T-Hg^(2+)-T配位结合形成双链DNA平末端,核酸外切酶ExoⅢ能识别双链DNA平末端并沿3'-5'方向催化水解茎部序列。释放的Hg^(2+)和识别探针进入新一轮反应循环(循环1);释放的发夹探针1未降解序列包含Ⅰ区序列和G-四链体形成序列,其中,Ⅰ区序列与发夹探针2的3'游离序列(Ⅰ~*区序列)完全互补,引发了ExoⅢ参与的双链DNA平末端水解,释放的Ⅰ区序列进入新一轮反应循环(循环2)。循环1和2为自发循环过程,经过多次循环可以产生大量单链的G-四链体序列。体系中加入氯化血红素,与G-四链体结合形成G-四链体-血红素-DNA酶,催化ABTS-H_2O_2反应体系显色。考察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Hg^(2+)的线性检测范围为0.3～50 pmol/L,检出限为0.25 pmol/L(S/N=3),回归方程为ΔA_(420 nm)=0.117+0.004C_(Hg^(2+))(pmol/L)。当水样中存在大量其它金属离子时,传感器对Hg^(2+)仍然具有较高的选择性。将此方法应用于环境水体中Hg^(2+)含量检测,加标回收率为96.0%～106.0%。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Hg^(2+)的高灵敏检测。An Hg2+-triggered exonuclease Ⅲ-assisted dual-cycle isothermal signal amplification was developed for label-free and ultrasensitive detection of Hg2+ using two designed hairpin probes. In the presence of Hg2+, the Hg2+discrimination probe could hybridize with the free 3’-terminus of hairpin probe 1 by T-Hg2+-T coordination and a blunt 3’-terminus is formed. In this case, Exo Ⅲ could preferentially bind to the blunt 3’-terminus and catalyze the stepwise hydrolysis of mononucleotides in the 3’-to-5’ direction. The released Hg2+ and Hg2+discrimination probe were recycled in a new round of reaction cycle(cycle 1). Undegraded sequence of hairpin probe 1 comprises G-quadruplex-forming sequence and I-region that was completely complementary to the free 3’-terminus of hairpin probe 2(Ⅰ* region) to form another blunt 3’-terminus. The new blunt 3’-terminus could trigger Exo Ⅲ-assisted cleavage again, and the I-region was released and recycled in a new round of reaction cycle(cycle 2). Cycles 1 and 2 were spontaneous cycle processes. After multiple cycles, a large number of single-stranded G-quadruplex-forming sequences could be generated. The released G-quadruplex-forming sequences could bind hemin to form stable G-quadruplex-hemin DNAzymes, which could efficiently catalyze ABTS-H2O2 system to produce the colored product. Under the optimal experimental conditions, the linear range for detection of Hg2+ was 0.3 pmol/L-50 pmol/L with a detection limit of 0.25 pmol/L, and the regression equation was ΔA420 nm=0.117+0.004CHg2+(pmol/L). This Hg2+ sensing system had excellent selectivity for Hg2+ against otherpossible competing ions. The fabricated sensor was applied to detection of Hg2+ in environmental water samples, and the recoveries were 96.0%-106.0%, respectively. This method was simple with good selectivity, high sensitivity and strong anti-interference ability, and was suitable for the sensitive detection of Hg2+ in environmental water samples.

关 键 词：等温信号放大 核酸外切酶Ⅲ G-四链体-血红素DNA酶 汞离子(Ⅱ) 比色分析 

分 类 号：O657.3[理学—分析化学] X832[理学—化学]