基于生物酶介导的DNA循环及滚环复制双重放大检测DNA

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牛淑妍， 张梦华， 房亚杰, 娄晓飞

（青岛科技大学 化学与分子工程学院， 山东 青岛 266042） 

摘要： 利用Nb.BbvC I限制性内切酶和Klenow聚合酶的特性设计了一个DNA的聚合剪切循环反应，再利用环DNA的滚环放大效应在金电极表面生成一条长的ssDNA。然后以氯化六氨合亚钌为电信号指示剂，设计了一个灵敏的DNA电化学生物传感器。通过循环伏安法（CV）和交流阻抗法（EIS）对电极进行表征以确保DNA的连接及杂交循环反应正常进行。在优化条件下考察了对目标DNA的响应范围及工作曲线。其检测线性范围为1.0～15 nmol·L-1，检测限为6.62×10-11 mol·L-1（S/N=3）。

关键词：  DNA电化学生物传感器； 限制性内切酶； 聚合酶； 氯化六氨合亚钌

中图分类号： O 657.32文献标志码： A

Detection of DNA Based on the Enzyme Mediated Circulation and Hoop Replication Double Amplification

NIU Shuyan, ZHANG Menghua, FANG Yajie, LOU Xiaofei

(CollegeofChemistryand Molecular Engineering,QingdaoUniversityof Science and Technology,Qingdao266042,China)

Abstract: A novel and sensitive electrochemical DNA biosensor was developed for the detection of DNA based on the enzyme mediated circulation and hoop replication double amplification. The biosensor was proposed by using Hexaamimineruthenium(Ⅱ) chloride (Ru(NH₃)²⁺₆) as an electroactive indicator and based on the fellow two reactions: First, a cycle of DNA hybridization reaction happened by using Nb.BbvC I restriction endonuclease. Then a long ssDNA generated on gold electrode surface by using Klenow polymerase based on hoop replication. Electrodes were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) to ensure that DNA was modified on the electrode surface and the hybridization reaction was carried out. The electrode response signal was detected under optimized conditions. The target DNA was quan -tified in a linear range from 1.0 to 15 nmol·L^-1, with a detection limit of 6.62× 10^-11mol·L^-1(S/N = 3).

Key words: DNA electrochemical biosensor; restriction endonuclease; Klenow polymerase; hexaamimineruthenium(Ⅱ) chloride

收稿日期：    20150317

基金项目： 国家自然科学基金项目(21075073)； 山东省自然科学基金重点项目（ZR2010BZ006）.

作者简介： 牛淑妍(1963—)，女，教授.

文章编号： 16726987（2016）05047905； DOI： 10.16351/j.16726987.2016.05.002