基于核酸外切酶Ⅲ及DNAzyme的铅离子荧光传感器的研究  

A Fluorescence Biosensor for Lead Ion Detection Based on DNAzyme and Exonuclease Ⅲ

作　　者：刘涛 李丹 梁杰 汪秀妹 LIU Tao;LI Dan;LIANG Jie;WANG Xiu-Mei(Fujian Provincial Key Laboratory of Ecology-Toxicological Effects&Control for Emerging Contaminants College of Environmental and Biological Engineering,Putian University,Putian 351100,China)

机构地区：[1]福建省新型污染物生态毒理效应与控制重点实验室莆田学院环境与生物工程学院

出　　处：《分析化学》2020年第2期248-254,共7页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.31801462);福建省教育厅项目(No.JT180466);莆田学院校内项目(No.2017015)资助~~

摘　　要：利用DNAzyme及核酸外切酶Ⅲ(ExoⅢ)构建了荧光生物传感器用于检测铅离子(Pb^2+)。DNAzyme与底物探针结合并在Pb^2+辅助作用下切割底物,使发夹结构的底物探针在环上修饰有RNA碱基处断裂,切割断裂底物探针后,DNAzyme被释放,继续与下一个底物探针结合并切割,利用DNAzyme可循环反复催化裂解底物的特性实现循环反应。底物探针被切割断裂后,形成的Y字形探针可与信标探针结合并打开其发夹结构,产生荧光信号,同时在ExoⅢ的作用下降解,从3′端开始切割水解被打开的信标探针,释放出底物探针,继续与下一个信标探针结合切割,形成第二步的循环信号放大。经过两步的循环反应,荧光信号得到不断增强,从而达到高灵敏检测的目的。200μL反应体系在37℃反应60 min后,其荧光信号与Pb^2+浓度在0.05~200 nmol/L范围内呈良好的线性关系,检出限为0.01 nmol/L。用于实际样品中Pb 2+的检测,加标回收率为96.3%~108.3%。本方法具有简单、快速、高选择性、高灵敏的特点,在Pb^2+检测方面有良好的应用潜力。A fluorescence sensor for Pb^2+ detection was developed on the basis of a DNAzyme cleavage and exonuclease Ⅲ assistant amplification strategy.In the presence of Pb^2+,the DNAzyme hybridized with substrate strand and catalyzed the hydrolytic cleavage of the substrate strand,and then the DNAzyme released from the substrate strand and bound another substrate strand to trigger another cycle of hydrolytic cleavage.The DNAzymes were used as catalysts for amplified sensing through multiple turnover reactions.The substrate probe was cleavaged and broken to form a Y-shaped probe which could hybridize and open the molecular beacon,resulting in the increase of the fluorescence signal.At the same time,exonuclease Ⅲ catalytically digested the molecular beacon from 3′-end and released the Y-shaped substrate strand.The released Y-shaped substrate strand could directly hybridize with another molecular beacon to generate fluorescence signal,and thus was further recognized and cleaved by exonuclease Ⅲ from the second step of cyclic signal amplification.Accompanying with each cleavage toward molecular probe by exonuclease Ⅲ,the fluorescence signal was accumulated,which resulted in a cyclic amplification format for the fluorescence response toward Pb 2+detection.The fluorescence response was detected in a 200μL reaction system that was incubated at 37℃ for 60 min.The linear range for detection of Pb^2+was 0.05-200 nmol/L with a detection limit of 0.01 nmol/L,and the recoveries of environmental water samples were 96.3%-108.3%.This method had many advantages such as simple operation,rapid detection,high selectivity and high sensitivity,and showed great application potential in Pb 2+detection.

关 键 词：铅离子 脱氧核酸酶 核酸外切酶Ⅲ 荧光生物传感器 

分 类 号：O657.3[理学—分析化学] O614.433[理学—化学]