基于核酸适配体夹心结构介导酶切引发环介导等温信号放大的超灵敏凝血酶比色传感方法研究  

Ultrasensitive Colorimetric Sensor for Detection of Thrombin Based on Loop Mediated Isothermal Signal Amplification Triggered by Aptamer Sandwich-Mediated Enzymatic Digestion

作　　者：傅昕[1] 邹婷 张何[1] 张培柔 蒋序春 FU Xin;ZOU Ting;ZHANG He;ZHANG Pei-Rou;JIANG Xu-Chun(Hunan Provincial Key Laboratory of Environmental Catalysis and Waste Recycling,College of Materials and Chemical Engineering,Hunan Institute of Engineering,Xiangtan 411104,China)

机构地区：[1]湖南工程学院材料与化工学院,湖南省环境催化与废弃物再生化重点实验室,湘潭411104

出　　处：《分析化学》2022年第7期1022-1031,共10页Chinese Journal of Analytical Chemistry

基　　金：国家自然科学基金项目(No.21005067);湖南省自然科学基金项目(No.2019JJ40055)资助。

摘　　要：设计了一种双核酸适配体夹心识别凝血酶并通过酶切开启环介导等温扩增实现信号放大的高灵敏凝血酶比色传感方法。在凝血酶存在下,利用双核酸适配体构建三明治夹心结构识别并捕获凝血酶,通过Nb.BsrDI切刻内切酶的酶切作用引发环介导等温扩增实现信号放大,使G-四链体-血红素DNA酶催化ABTS-H_(2)O_(2)体系显示特征绿色。在最优实验条件下,本方法检测凝血酶的线性范围为0.01~1.0 ag/mL,检出限为0.008 ag/mL,回归方程为ΔA_(420 nm)=0.187C_(thrombin)+0.171(R^(2)=0.991),具有超高的灵敏度和良好的线性度。在血清样品中存在大量干扰蛋白时,传感器对凝血酶仍然具有较高的选择性。将本方法应用于人血清样品中凝血酶含量检测,加标回收率为96.1%~103.2%。本方法将核酸适配体夹心识别技术、环介导等温扩增技术和G-四链体-血红素DNA酶酶促信号放大技术相结合,实现了凝血酶的超灵敏检测;同时,本方法具有操作简便、稳定性好且成本低的优点,可望应用于临床人血清中凝血酶的高灵敏检测。An ultrasensitive colorimetric sensor for detection of thrombin was designed based on loop mediated isothermal signal amplification(LAMP)triggered by aptamer sandwich-mediated enzymatic digestion.In the presence of thrombin,aptamer capable of recognizing thrombin heparin site and fibrinogen site was used to recognize thrombin and construct a sandwich structure.The Nb.BsrDI nicking endonuclease could recognize and cleave this complementary sequence to start-up loop mediated isothermal amplification.G-quadruplex-hemin DNAzyme could catalyze the oxidation of ABTS to ABTS^(·+) in the presence of H_(2)O_(2),and the color changed to green.Under the optimal experimental conditions,the linear detection range of this method for thrombin was 0.01-1.0 ag/mL,the detection limit(3σ)was 0.008 ag/mL,and the regression equation wasΔA_(420 nm)=0.187 C_(thrombin)+0.171(R^(2)=0.991).When there were a large number of other interfering proteins in the serum sample,the sensor still had a high selectivity to thrombin.When this method was applied to the detection of thrombin content in serum samples,the recoveries were 96.1%-103.2%.This method innovatively integrated aptamer-based sandwich assay,loop-mediated isothermal amplification technology,and G-quadruplex-hemin DNAzyme enzymatic signal amplification technology that caused the ultrasensitive detection of thrombin.The method showed many advantages such as lower cost,simple operation and good stability,and could be used for highly sensitive clinical detection of thrombin in human serum.

关 键 词：凝血酶 环介导等温扩增 G-四链体-血红素DNA酶 比色分析 

分 类 号：R313[医药卫生—基础医学] O657.3[理学—分析化学]