基于竞争触发滚环扩增的荧光适配体传感器高灵敏检测凝血酶  

Highly Sensitive Fluorescent Aptasensor for Thrombin Detection Based on Competition Triggered Rolling Circle Amplification

作　　者：张松柏[1] 郑丽英[1] 胡霞[1] 沈广宇[1] 刘学文[1] 沈国励[2] 俞汝勤[2] 

机构地区：[1]湖南文理学院化学化工学院,常德415000 [2]湖南大学化学化工学院,长沙410082

出　　处：《分析化学》2015年第11期1688-1694,共7页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金(No.21205039);湖南省自然科学基金(Nos.13JJ6071;14JJ7073);湖南大学化学生物传感与计量学国家重点实验室开放基金(No.2013004);湖南省教育厅项目(Nos.15C035;13C622;13C620)资助~~

摘　　要：利用目标蛋白、适体探针、挂锁探针和与适体探针匹配的互补序列之间的竞争反应,发展了一种基于滚环扩增的高灵敏荧光适配体传感器。在不含目标蛋白时,互补序列由于与适体探针杂交形成双链,因此不能与挂锁探针杂交以触发连接-滚环放大反应。相反,有目标蛋白存在时,由于目标蛋白与适体探针结合,使得互补序列被置换下来,从而可以与挂锁探针杂交。在DNA连接酶的作用下,挂锁探针被进一步环化并在Phi 29 DNA聚合酶的作用下发生滚环扩增反应。扩增产物含有许多能与分子信标检测探针环状部分杂交的重复序列,从而分子信标被打开产生荧光信号。对互补序列的长度及挂锁探针的浓度进行了考察。在优化的实验条件下,本传感系统可以实现对的凝血酶高灵敏检测,线性范围为0.067~32 nmol/L,检出限为0.03 nmol/L（约90 amol目标分子）。通过设计不同的适体探针和相关核酸序列,本传感系统可以作为一种通用型方法用于其它目标物的分析。Based on the competition reaction of target protein, aptamer probe, padlock probe and complementary sequence,a highly sensitive fluorescent aptasensor was developed in this study in combination with rolling circle amplification. In the absence of target protein,the ligation-rolling circle amplification reaction was repressed because the complementary sequence hybridized with aptamer probe to form doublestranded duplex. While in the presence of target protein,the target molecules bound specifically with aptamer probe,inducing displacement of the complementary sequence and hybridization with padlock probe. The padlock probe was circularized with the assistance of E. coli DNA ligase,and the rolling circle amplification process could be accomplished by Phi 29 DNA polymerase. The amplification product contained thousands of repeated sequences which could hybridize with the loop of molecular beacon（ the detection probes）,resulting in a significant fluorescence signal. The effects of length of complementary DNA（ CDNA） sequence and concentration of padlock probe were investigated. Under the optimized experimental conditions,the model target protein thrombin could be highly sensitively detected by the proposed aptasensing system in a linear range of 0. 067- 32. 4 nmol / L with a detection limit of 0. 03 nmol / L（ approximately 90 amol target molecules）. Moreover,the presented sensing method was universal for other target analysis by skillfully design of the sequence of aptamer probe and related oligonucleotides.

关 键 词：核酸适配体 滚环扩增 分子信标 荧光 凝血酶 

分 类 号：R730.43[医药卫生—肿瘤] O657.3[医药卫生—临床医学]