基于滚环合成的聚分子信标用于凝血酶的靶向检测

Rolling Circle Amplification-based Polyvalent Molecular Beacon Probe for Signal-amplifying and Sensitive-Detection of Thrombin

采用滚环扩增(RCA)合成得到的DNA长链打开带适配体的分子信标,由于RCA长链上带有多个与分子信标(MB)互补的重复序列,其打开分子信标的能力比单一互补短链提高了上百倍.所形成的聚多价分子信标组装体,在分子信标浓度相同的情况下,打开后的荧光强度也大幅上升;并且由于组装体上多价适配体的存在,聚分子信标对凝血酶的靶向能力显著增强.实验结果表明,聚分子信标结合凝血酶后,其荧光信号与凝血酶浓度呈线性关系,检测灵敏度达到0. 2 nmol/L,该体系的构建有利于实现对凝血酶的高灵敏、特异性检测.

A long chain structure of DNA synthesized by rolling circle amplification( RCA) was used to turn on aptamer-based molecular beacons( MBs). Owing to the multiple complementary repeat units,the RCA scaffold can hybridize tens or hundreds of MBs to form polyvalent aptamer probes. The unfold ability and the fluorescence intensity of MBs were both improved by RCA compared to short single chains. Experimental results demonstrated that the system is significantly more effective than monovalent molecular beacon in targeting thrombin and signal sensitivity because of multivalent effects. Once the poly valent molecular beacon binding to the thrombin,there was a linear relationship between fluorescence signal and thrombin concentration,and the detection limit of thrombin concentration was 0. 2 nmol/L. The establishment of this system is conducive to the realization of highly sensitive and specific detection of thrombin.