基于酶辅助循环放大和银纳米簇技术构建荧光传感器用于MicroRNA的检测  

A Fluorescence Sensor for MicroRNA Detection Based on Nuclease Assisted Target Recycling and Silver Nanoclusters

作　　者：刘景荣[1] 潘云苓[1] 赵燕苹 陈梅[2] 夏垚坤 兰建明[2] 韩志钟[2] 吴芳[2] 陈敬华[2] 李春艳[2] 

机构地区：[1]福州市第一医院中西医结合肿瘤科,福建福州350009 [2]福建医科大学药学院,福建福州350108

出　　处：《化学试剂》2016年第4期345-348,352,共5页Chemical Reagents

基　　金：国家自然科学基金资助项目(21375017;21105012);福州市科技计划项目(2013-S-122-4);福建省自然科学基金杰出青年人才项目(2013J06003);福建省自然科学基金(2015J01596;2015J05020);福建省科技厅重点项目(2013Y0045);福建省卫生系统中青年骨干人才培养项目(2014-ZQN-ZD-26);福建省高等学校新世纪优秀人才支持计划资助(JAl3130)

摘　　要：基于双链特异性核酸酶(Duplex-specific nuclease,DSN)选择性切割DNA单链和DNA保护的银纳米簇的特性,建立了一种新型的荧光传感器用于MicroRNA(MiR-21)的高灵敏检测。当MiR-21存在时,与Cp杂交形成DNA/RNA双链结构,此时在DSN的水解下,释放出探针DNA部分片段和完整的MiR-21。释放出来的MiR-21可再次发生与Cp杂交、DSN酶切等反应,如此反复,可实现一条MiR-21分子与多条探针杂交、酶切的循环过程。经磁分离,上清液中的探针DNA部分片段与Ag+结合并在硼氢化钠(Na BH4)的还原下产生银簇,检测到较强的荧光信号。相反,当MiR-21不存在时,DSN对单链探针无酶切作用,不能水解Cp,经磁分离,上清液中检测不到荧光信号。在最佳条件下,该传感器检测MiR-21的线性范围为50 fmol/L^10 pmol/L,检测限达6 fmol/L,而且具有较好的选择性,有望用于临床实际样本中微量MiR-21的检测。A novel fluorescence sensor was developed for microRNA（ MiR-21） detection based on duplex-specific nuclease（ DSN） which can selectively cut the DNA single strand and DNA protected silver nanoclusters. In the presence of target MiR-21,Cp hybridized with MiR-21 to form a double-stranded DNA,which was recognized and digested by DSN,then the no complementary part of the Cp and the complete MiR-21 were released. The intact target MiR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. Eventually,through magnetic separation,the released DNA sequences can be combined with the Ag＋and produced silver nanoclusters under the reduction of sodium borate,and a strong fluorescent signal was detected. However,in the absence of the target MiR-21,Cps cannot be hydrolyzed by DSN,after separation,no fluorescence signal was detected from the supernatant solution. Under the optimized conditions,the sensor showed a good linear relationship for MiR-21 detection in the range of 50 fmol / L ~ 10 pmol / L with a detection limit as low as 6 fmol / L. Furthermore,the sensor exhibited a good selectivity,it＇ s expected to be used for the ultra-low detection of MiR-21 in clinical real samples.

关 键 词：磁珠 双链特异性核酸酶 银纳米簇 MIR-21 荧光生物传感器 

分 类 号：O657.3[理学—分析化学]