基于核酸外切酶-Ⅲ和金片基底捕获循环策略用于锰超氧化物岐化酶DNA的检测  

Exonuclease Ⅲ and Gold Substrate Capturing Cyclic Strategy for Detection of Manganese Superoxide Dismutase DNA

作　　者：王海飞[1] 唐英 闫晓梅 陈增萍[2] 陈瑶 WANG Hai-Fei;TANG Ying;YAN Xiao-Mei;CHEN Zeng-Ping;CHEN Yao(Hunan Key Lab of Biomedical Materials and Devices,College of Life Sciences and Chemistry,Hunan University of Technology,Zhuzhou 412007,China;State Key Laboratory of Chemo/Biosensing and Chemometrics,College of Chemistry and Chemical Engineering,Hunan University,Changsha 410082,China)

机构地区：[1]湖南工业大学生命科学和化学学院,湖南省生物医用纳米材料与器件重点实验室,株洲412007 [2]湖南大学化学化工学院,化学生物传感与计量学国家重点实验室,长沙410082

出　　处：《分析化学》2022年第5期720-727,共8页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(Nos.21705044,21804040);湖南省自然科学基金项目(Nos.2018JJ3114,2019JJ50134);湖南省教育厅科学研究项目(No.19C0554);化学生物传感与计量学国家重点实验室开放课题项目(No.2018015)资助。

摘　　要：利用核酸外切酶Ⅲ辅助信号放大技术和金片基底捕获循环策略,设计了一种灵敏且简单的锰超氧化物岐化酶基因序列(MnSOD DNA)检测方法。首先设计合成了标记荧光基团的发夹探针,在MnSOD DNA存在下,探针发夹结构被打开,然后在核酸外切酶Ⅲ的作用下,发夹探针部分被水解,释放出MnSOD DNA和标记荧光基团的残余发夹探针片段,MnSOD DNA可继续与剩余的完整发夹探针杂交,进入下一个循环。因此,反应一定时间后,反应液中含有大量的带有标记荧光基团的残余发夹探针片段,可与金片基底上修饰的捕获探针杂交而被捕获,通过检测其荧光,可实现对MnSOD DNA的定量分析,方法检出限为0.42 pmol/L。将本方法用于检测Hela、Jurkat和Ramos细胞裂解液中的MnSOD DNA,加标回收率在90.2%~111.2%之间。本方法可有效消除背景干扰和基质效应的影响,有望成为一种通用的短链DNA的检测方法。A sensitive and simple method was developed for detection of manganese superoxide dismutase(MnSOD)DNA using exonucleaseⅢassisted signal amplification technique and gold substrate capture cycle strategy.This strategy relied on three major steps in the experiment.First,the hairpin probe labeled with fluorophore was opened by MnSOD DNA.Second,with the help of exonucleaseⅢ,the hairpin probe was partially hydrolyzed to release MnSOD DNA and residual hairpin probe fragments labeled with fluorophore.The released MnSOD DNA could be recycled and continued to hybridize with the complete hairpin probe.And third,the reaction solution contained a large number of residual hairpin probe fragments labeled with fluorophore,which could be hybridized with capture probe modified on gold substrate,and then transferred to a new tube solution for fluorescence detection.Based on this principle,the method for MnSOD DNA detection was constructed,with detection limit of about 0.42 pmol/L.The method was also applied to the detection of MnSOD DNA in lysates of HeLa,Jurkat and Ramos cells,with recoveries ranging from 90.2%to 111.2%.This method could effectively eliminate the influence of background interference and matrix effect,and provide a new promising paradigm in developing universal methods for detection of short DNA.

关 键 词：核酸外切酶Ⅲ 金片基底 锰超氧化物岐化酶DNA 定量检测 

分 类 号：Q503[生物学—生物化学] O657.3[理学—分析化学]