基于核酸外切酶Ⅲ信号放大技术的荧光适配体传感器用于卡那霉素的检测  

A fluorescent aptasensor for kanamycin detection based on exonuclease Ⅲ assisted signal amplification

作　　者：冯婷婷 王峥 王晓华[3] 杨丽霞 FENG Tingting;WANG Zheng;WANG Xiaohua;YANG Lixia(Institute of Pharmaceutical and Food Engineering,Shanxi University of Chinese Medicine,Jinzhong 030619;College of Pharmaceutical Sciences,Zhejiang University,Hangzhou 310058;Pharmacy School of Guilin Medical University,Guilin Medical University,Guilin 541004)

机构地区：[1]山西中医药大学中药与食品工程学院,晋中030619 [2]浙江大学药学院,杭州310058 [3]桂林医学院药学院,桂林541004

出　　处：《分析试验室》2021年第10期1140-1146,共7页Chinese Journal of Analysis Laboratory

基　　金：中国博士后科学基金面上项目(2019M662089);国家自然科学基金(82003925);山西省卫健委课题(2019088);山西省教育厅科技创新计划项目(2020L0413)资助。

摘　　要：建立了一种基于核酸外切酶Ⅲ(Exo Ⅲ)和碳纳米颗粒(CNPs)的信号放大体系用于卡那霉素(KAN)检测的新方法。合成了水溶性的CNPs,并设计合成了不同序列的DNA,具体包括:卡那霉素适配体(Apt),羧基荧光素标记的信号DNA探针(FAM-DNA)和互补链cDNA。当体系中不存在KAN时,Apt与cDNA可以杂交形成双链DNA,体系中FAM-DNA处于单链状态,Exo Ⅲ不能水解单链DNA;此时,体系中加入CNPs,单链FAM-DNA被CNPs吸附,荧光发生淬灭;在KAN存在下,Apt与其靶标KAN特异性结合,此时FAM-DNA与cDNA杂交形成双链DNA,由于CNPs对双链DNA吸附较弱,DNA探针的荧光不发生淬灭。ExoⅢ可以特异性的从3’-端对FAM-DNA降解,释放FAM荧光团和cDNA,该体系通过"降解-杂交"循环,最终释放出大量的FAM荧光团。由于CNPs对FAM具有较低的亲和力,释放出的FAM不能吸附在CNPs表面,FAM荧光不会发生淬灭,实现荧光信号放大扩增作用。方法线性范围为50~100 nmol/L,检测限为2.5 nmol/L。该方法可用于实际样品牛奶中卡那霉素的检测。A carbon napoparticles( CNPs)-based fluorescent aptasensor to detect kanamycin by employing exonuclease Ⅲ( Exo Ⅲ) as a signal amplifying element was presented. In the absence of kanamycin,the kanamycin aptamers hybridized with the complementary DNA( cDNA),and the Exo Ⅲ could not cleave the single strand signal probes labeled with carboxylfluorescein( FAM) at its 5’ends. When the CNPs was finally added,it could strongly adsorb the single-strand signal probes and quenched the fluorophore effectively. In the presence of kanamycin,the aptamers associated with the targets,which led to the formation of duplex DNAs between the cDNAs and the signal probes. The Exo Ⅲ thereafter could digest the duplex DNAs from 3’blunt terminus of signal probes,liberating the fluorophore. Upon adding the CNPs,the fluorophore could not be adsorbed and quenched. By coupling cyclic enzymatic cleavage,a remarkable fluorescent increase was obtained.Due to the specific recognition ability of the aptamer for the target and the powerful quenching property of CNPs for signal probe,this proposed approach had a good selectivity and high sensitivity for kanamycin. In the optimum conditions,the linear range of the method was 50-100 nmol/L and the detection limit was 2. 5 nmol/L. The method can be used for the determination of kanamycin in actual milk samples.

关 键 词：核酸外切酶Ⅲ 碳纳米颗粒 信号放大 卡那霉素 荧光检测 

分 类 号：O657.39[理学—分析化学]