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

孙春燕 司金雨 杜彩溢 吕婷 刘妮 张晓光 王作昭

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

    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。

English


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  • 收稿日期:  2019-12-05
  • 修回日期:  2021-04-05
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