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

引用本文: 傅昕, 邹婷, 张何, 张培柔, 蒋序春. 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[J]. 分析化学, 2022, 50(7): 1022-1031. doi: 10.19756/j.issn.0253-3820.210793 shu

Citation: FU Xin, ZOU Ting, ZHANG He, ZHANG Pei-Rou, JIANG Xu-Chun. Ultrasensitive Colorimetric Sensor for Detection of Thrombin Based on Loop Mediated Isothermal Signal Amplification Triggered by Aptamer Sandwich-Mediated Enzymatic Digestion[J]. Chinese Journal of Analytical Chemistry, 2022, 50(7): 1022-1031. doi: 10.19756/j.issn.0253-3820.210793 shu

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

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

通讯作者: 张何,E-mail:mzhang_he@126.com

基金项目:
国家自然科学基金项目(No.21005067)和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-H₂O₂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(R²=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。

关键词:

English

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

Hunan Provincial Key Laboratory of Environmental Catalysis and Waste Recycling, College of Materials and Chemical Engineering, Hunan Institute of Engineering, Xiangtan 411104, China

Corresponding author: ZHANG He, mzhang_he@126.com

Received Date: 14 October 2021
Revised Date: 20 December 2021
Fund Project:
Supported by the National Natural Science Foundation of China (No.21005067) and the Natural Science Foundation of Hunan Province, China (No.2019JJ40055).

Abstract: 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 H₂O₂, 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 was ΔA_{420 nm}=0.187C_thrombin+0.171 (R²=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.

Key words:

Thrombin
/ Loop mediated isothermal amplification
/ G-quadruplex-hemin DNAzyme
/ Colorimetric analysis

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沈阳化工大学材料科学与工程学院沈阳 110142

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

通讯作者: 张何,E-mail:mzhang_he@126.com

English

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

Corresponding author: ZHANG He, mzhang_he@126.com

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

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

通讯作者: 张何,E-mail:mzhang_he@126.com

English

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

Corresponding author: ZHANG He, mzhang_he@126.com

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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