基于磁珠和双链特异性核酸酶辅助目标循环技术构建荧光传感器用于MicroRNA的检测  

A fluorescence sensor based on magnetic beads and duplex-specific nuclease assisted target recycling for MicroRNA detection

作　　者：陈秋香[1,2] 赵燕苹 吴冬枝 蔡淑贤[1] 夏垚坤 陈梅[1] 陈敬华[1] 

机构地区：[1]福建医科大学药学院,福州350108 [2]晋江市疗养院,泉州362200

出　　处：《分析试验室》2015年第8期869-873,共5页Chinese Journal of Analysis Laboratory

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

摘　　要：基于磁珠（MBs）的分离富集和双链特异性核酸酶（DSN）选择性切割DNA单链的特性,建立了信号增强型荧光生物传感器用于microRNA-21（miR-21）的检测。荧光素（FAM）修饰的捕获探针（Cps）,通过亲和素-生物素的特异识别作用固定在磁珠表面。当miR-21存在时,Cps与其杂交形成DNA/RNA双螺旋结构,DSN能特异性水解杂合双链中的DNA,同时释放出荧光标记片段和完整的miR-21。被释放出来的miR-21与另一Cp再次杂交并被DSN酶切,如此循环,从而实现恒温条件下一个miR-21与多个Cp杂交、酶切,释放出大量的荧光标记片段的循环过程,最终使体系的荧光强度明显变大。相反,当miRNA-21不存在时,Cps无法形成双螺旋结构,DSN对单链DNA无酶切作用,不能水解Cps,经磁分离,上清液没有荧光标记片段,所以检测不到荧光信号。最佳条件下,miR-21浓度在100~5×104fmol/L范围内,荧光强度与其浓度呈良好的线性关系,检测限达80 fmol/L。该传感器可以识别单碱基错配序列,有望为肿瘤早期诊断提供新思路。In this paper,a fluorescent biosensor was developed for microRNA-21（ miR-21） detection based on magnetic beads（ MBs） and duplex-specific nuclease（ DSN） assisted target recycling. Through the specific binding of the avidin-biotin,fluorescein functionalized capture probes（ Cps） were immobilized on MBs. In the presence of target miR-21,it hybridized with the target-binding part of a Cp to form DNA： RNA heteroduplex.Due to the considerable cleavage preference for DNA in DNA： RNA hybrids,DSN hydrolyzed the target-binding fragment of Cp while liberated fluorescein labeled fragment and the intact target miR-21 to hybridize with a new Cp and initiate the second cycle of hydrolysis. Eventually,through magnetic separation,only fluorescein labeled fragment of Cp could remain in solution and function as a signaling flare to increase the fluorescence intensity of the sensor dramatically. However,in the absence of the target miR-21,Cps were unable to form a double helix structure and could not be hydrolyzed due to the low activity of DSN against single-stranded DNA. Therefore,after magnetic separation,and the supernatant liquid contained no fluorescein labeled fragment,the fluorescence signal was not detected. Under the optimized conditions,a good linear relationship was obtained between the fluorescence intensity and the concentration of the miR-21 in the range of 100 ~ 5 × 104 fmol/L,and the limit of detection was calculated to be 80 fmol/L. In addition,the sensor has good selectivity and even identify singlebase mismatched sequences. It may provide a new method for the early diagnosis of cancer.

关 键 词：磁珠 荧光生物传感器 MIR-21 双链特异性核酸酶 目标循环 

分 类 号：TQ460.7[化学工程—制药化工] O657.3[理学—分析化学]