基于切刻内切酶信号放大的电致化学发光DNA生物传感器的研究

引用本文: 海洪, 黄文刚, 梁顺超, 李建平. 基于切刻内切酶信号放大的电致化学发光DNA生物传感器的研究[J]. 分析化学, 2016, 44(5): 779-786. doi: 10.11895/j.issn.0253-3820.150742 shu

Citation: HAI Hong, HUANG Wen-Gang, LIANG Shun-Chao, LI Jian-Ping. Ultrasensitive Electrochemiluminescence Biosensor for DNA Determination Based on Nicking Endonuclease Assisted Signal Amplification[J]. Chinese Journal of Analytical Chemistry, 2016, 44(5): 779-786. doi: 10.11895/j.issn.0253-3820.150742 shu

基于切刻内切酶信号放大的电致化学发光DNA生物传感器的研究

电磁化学功能物质广西重点实验室, 桂林理工大学化学与生物工程学院, 桂林 541004
基金项目:
本文系国家自然科学基金项目(Nos.21165007,21375031)资助

摘要: 利用切刻内切酶的酶切作用实现信号放大,结合量子点高效的电化学发光性能,构建了一种新型电化学发光DNA生物传感器。将捕获探针DNA(c-DNA)通过自组装的方式固定到金电极表面,后与目标DNA(t-DNA)互补杂交形成双链DNA,利用切刻内切酶Nt.BstNBI特异性识别双链上的酶切位点(5'-GAGTC-3'),然后在c-DNA相应的切割位点(识别序列3'端后的4个碱基处)对其进行剪切,释放出目标链,参与下一轮的杂交及酶切,通过目标物的循环利用,实现信号放大。利用N-羟基琥珀酰亚胺(NHS)和1-乙基-3-3-二甲基氨丙基碳化二亚胺(EDC)活化羧基化CdTe量子点表面的羧基,与电极表面残留的c-DNA末端的氨基共价交联,通过测定捕获的量子点的电化学发光信号对目标DNA进行检测。优化后的检测条件为:c-DNA浓度1μmol/L,杂交时间60 min,Nt.BstNBI浓度0.5 U/μL,酶切反应时间4 h。在优化条件下,目标DNA浓度在2.0×10^-13~2.0×10^-11 mol/L范围内,其对数与电化学发光强度呈线性关系,检出限为7.3×10^-14 mol/L。人体血样加标回收率为96.4%~108.0%。

关键词:

English

Ultrasensitive Electrochemiluminescence Biosensor for DNA Determination Based on Nicking Endonuclease Assisted Signal Amplification

Guangxi Key Laboratory of Electrochemical and Magnetochemical Function Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guilin 541004, China
Received Date: 19 September 2015
Revised Date: 29 December 2015
Fund Project:
This work was supported by the National Natural Science Foundation of China (Nos.21165007, 21375031)

Abstract: A novel electrochemiluminescence (ECL) DNA biosensor based on nicking endonuclease and the efficient ECL property of quantum dots(QDs) assisted signal amplification was developed. The capture probe DNA(c-DNA) was self-assembled on gold electrode via AuS bond, and then hybridized with target DNA (t-DNA) to form double-stranded DNA. Then nicking endonuclease Nt.BstNBI recognized a cutting site (5'-GAGTC-3') in the double chain and cleaved c-DNA strand at 4 bases away from the 3' end of its recognition site, thus releasing the t-DNA and achieving a recycle of t-DNA in the next hybridization and signal amplification. Finally, carboxyl groups on the surface of CdTe QDs were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS), and then reacted with amino groups at the terminal of residual c-DNA on the electrode surface. So the QDs were fabricated and the t-DNA concentration could be determined by measuring the ECL signal of the CdTe QDs. The experimental conditions were optimized and 1 μmol/L c-DNA, 60 min of hybridization time, 0.5 U/μL Nt.BstNBI and 4 h of endonuclease reaction time were chosen. The experimental results showed that under optimal conditions, t-DNA could be specifically assayed with a linear relationship between the ECL signal intensity and the logarithm of t-DNA concentration in the range of 2.0×10^-13-2.0×10^-11 mol/L, with a limit of detection of 7.3×10^-14 mol/L. The biosensor was successfully applied to determine t-DNA concentration in human blood sample with recoveries of 96.4%-108.0%.

Key words:

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基于切刻内切酶信号放大的电致化学发光DNA生物传感器的研究

English

Ultrasensitive Electrochemiluminescence Biosensor for DNA Determination Based on Nicking Endonuclease Assisted Signal Amplification

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中国化学会秘书处

基于切刻内切酶信号放大的电致化学发光DNA生物传感器的研究

English

Ultrasensitive Electrochemiluminescence Biosensor for DNA Determination Based on Nicking Endonuclease Assisted Signal Amplification

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

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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

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