基于核酸外切酶Ⅰ辅助目标物循环放大策略的非标记适配体荧光传感器检测土霉素

引用本文: 孙春燕, 司金雨, 杜彩溢, 吕婷, 刘妮, 张晓光, 王作昭. 基于核酸外切酶Ⅰ辅助目标物循环放大策略的非标记适配体荧光传感器检测土霉素[J]. 分析化学, 2021, 49(9): 1488-1496. doi: 10.19756/j.issn.0253-3820.191720 shu

Citation: SUN Chun-Yan, SI Jin-Yu, DU Cai-Yi, LYU Ting, LIU Ni, ZHANG Xiao-Guang, WANG Zuo-Zhao. Label-free Fluorescent Aptasensor Based on Exonuclease-assisted Target Recycling Strategy for Sensitive Detection of Oxytetracycline[J]. Chinese Journal of Analytical Chemistry, 2021, 49(9): 1488-1496. doi: 10.19756/j.issn.0253-3820.191720 shu

基于核酸外切酶Ⅰ辅助目标物循环放大策略的非标记适配体荧光传感器检测土霉素

吉林大学食品科学与工程学院, 长春 130062

通讯作者: 张晓光,E-mail:xiaoguang61@jlu.edu.cn; 王作昭,E-mail:zuozhaowang@163.com

基金项目:
吉林省自然科学基金项目（No.20180101246JC）、吉林省教育厅"十三五"科学技术项目（No.JJKH20190170KJ）和中央高校基本科研业务费专项资金资助。

摘要: 建立了基于核酸适配体的特异性识别和核酸外切酶Ⅰ（Exo Ⅰ）辅助目标物循环放大的非标记荧光检测方法，用于土霉素的定量测定。体系中无土霉素时，SYBR Green I （SGI）分子可以插入土霉素适配体与其互补链形成的双链DNA （dsDNA）中，并在495 nm光激发下产生强烈的荧光发射。在土霉素存在时，由于核酸适配体与土霉素之间的强亲和力而特异性结合，dsDNA被打开并释放出SGI，游离的SGI荧光明显减弱。为了增强土霉素存在时引起的SGI荧光猝灭效应，采用Exo Ⅰ辅助目标物循环放大的策略。丙烯酰胺凝胶电泳实验结果证实，Exo Ⅰ可选择性降解单链DNA以及与土霉素结合的核酸适配体。被释放的土霉素参与下一个循环，不断破坏dsDNA，游离的SGI增多，荧光逐渐减弱。基于Exo Ⅰ辅助目标物循环放大的策略，检测灵敏度得到了有效提高。在最优实验条件下，本方法检测土霉素的线性范围为0.01~10 μg/mL，检出限（3σ）为6.77 ng/mL。采用本方法检测实际样品牛奶和蜂蜜中的土霉素，牛奶样品的加标回收率为93.0%~105.1%，相对标准偏差（RSD）为0.5%~6.2%，蜂蜜样品的加标回收率为94.0%~95.8%，RSD为0.3%~7.7%。此荧光传感器具有成本低、灵敏度高和特异性好等优点，在快速检测食品有害物残留方面具有良好的应用潜力。

关键词:

English

Label-free Fluorescent Aptasensor Based on Exonuclease-assisted Target Recycling Strategy for Sensitive Detection of Oxytetracycline

College of Food Science and Engineering, Jilin University, Changchun 130062, China

Corresponding author: ZHANG Xiao-Guang, ; WANG Zuo-Zhao,

Received Date: 05 December 2019
Revised Date: 05 April 2021
Fund Project:
Supported by the Natural Science Foundation of Jilin Provincial Technology Development Program (No.20180101246JC), the "Thirteenth Five-Year" Science and Technology Project of Jilin Provincial Department of Education (No.20190170KJ) and the Fundamental Research Funds for the Central Universities of China.

Abstract: A label-free fluorescence detection method was developed for quantitative determination of oxytetracycline (OTC) based on aptamer recognition capability and exonuclease Ⅰ (Exo Ⅰ)-assisted target recycling. In the absence of OTC, SYBR green I (SGI) molecules could insert the double-stranded DNA (dsDNA) of OTC aptamer and its complementary DNA, resulting in strong fluorescence emission of SGI with excitation wavelength of 495 nm. In the presence of OTC, due to the strong affinity of the aptamer to its target, aptamer preferentially bound with OTC and unwound the dsDNA. Thus, SGI molecules released from the hydrogen bonds of dsDNA, accompanied with the obvious decrease of SGI fluorescence. To amplify the fluorescence quenching effect of SGI caused by the addition of OTC, Exo Ⅰ-assisted target recycling was involved in. The results of acrylamide gel electrophoresis confirmed that Exo Ⅰ could digest single stranded DNA selectively and the aptamer bound with OTC. The released OTC participated in the next cycle, continuously destroyed the dsDNA structure, releasled the free SGI, and gradually weakened the fluorescence. Based on this Exo Ⅰ-catalyzed target recycling strategy, the sensitivity of this aptamer-based fluorescent strategy was significantly improved. The linear range of OTC was 0.01-10 μg/mL with a detection limit (3σ) of 6.77 ng/mL. The method realized the detection of OTC in milk and honey sample ssuccessfully. For milk samples, the recoveries were 93.0%-105.1% with relative standard deviations (RSD) of 0.5%-6.2%, and for honey samples, the recoveries were 94.0%-95.8% with RSD of 0.3%-7.7%, respectively. The fluorescence sensor had many advantages such as low cost, high sensitivity and good specificity, showing great potential in the rapid detection of residue of harmful substances in food.

Key words:

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沈阳化工大学材料科学与工程学院沈阳 110142

基于核酸外切酶Ⅰ辅助目标物循环放大策略的非标记适配体荧光传感器检测土霉素

通讯作者: 张晓光,E-mail:xiaoguang61@jlu.edu.cn; 王作昭,E-mail:zuozhaowang@163.com

English

Label-free Fluorescent Aptasensor Based on Exonuclease-assisted Target Recycling Strategy for Sensitive Detection of Oxytetracycline

Corresponding author: ZHANG Xiao-Guang, ; WANG Zuo-Zhao,

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

基于核酸外切酶Ⅰ辅助目标物循环放大策略的非标记适配体荧光传感器检测土霉素

通讯作者: 张晓光,E-mail:xiaoguang61@jlu.edu.cn; 王作昭,E-mail:zuozhaowang@163.com

English

Label-free Fluorescent Aptasensor Based on Exonuclease-assisted Target Recycling Strategy for Sensitive Detection of Oxytetracycline

Corresponding author: ZHANG Xiao-Guang, ; WANG Zuo-Zhao,

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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通讯作者: 陈斌, bchen63@163.com

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