基于适配体/金纳米粒子的“开-关”型荧光探针快速测定水胺硫磷  

A“Turn on-off”Fluorescent Probe Based on Aptamer/Gold Nanoparticles for Rapid Detection of Isocarbophos

作　　者：邹小波[1] 蒋彩萍 李志华[1] 孙悦 石吉勇[1] 李艳肖[2] 黄晓玮[1] 张迪[1] 胡雪桃 翟晓东[1] 魏晓鸥 ZOU Xiao-Bo;JIANG Cai-Ping;LI Zhi-Hua;SUN Yue;SHI Ji-Yong;LI Yan-Xiao;HUANG Xiao-Wei;ZHANG Di;HU Xue-Tao;ZHAI Xiao-Dong;WEI Xiao-Ou(School of Food and Biological Engineering,Jiangsu University,Zhenjiang 212013,China;Analytical Instrumentation Center,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]江苏大学食品与生物工程学院,镇江212013 [2]江苏大学分析测试中心,镇江212013

出　　处：《分析化学》2020年第4期507-515,共9页Chinese Journal of Analytical Chemistry

基　　金：国家重点研发计划项目(Nos.2017YFC1600806,2016YFD0401104);国家自然科学基金项目(Nos.31801631,31671844,31601543);江苏省自然科学基金(Nos.BK20160506,BE2016306,BK20180865);江苏省国际科技合作项目(No.BZ2016013)资助。

摘　　要：基于适配体和金纳米粒子(Gold nanoparticles,AuNPs)合成“开-关”型荧光探针,用于快速、灵敏、高选择性测定水胺硫磷。5'端连接荧光素(6-Carboxyfluorescein,FAM)的发卡型水胺硫磷适配体互补链(Complementary strand,cDNA)通过修饰于3'端的巯基连接到AuNPs表面,FAM(荧光信号分子)与AuNPs(荧光猝灭剂)间发生荧光共振能量转移效应(Fluorescence resonance energy transfer,FRET),导致荧光猝灭。加入水胺硫磷适配体(Isocarbophos-binding aptamer,ICP-Aptamer)与cDNA杂交,cDNA发卡构型被打开,使荧光信号恢复。当体系中存在水胺硫磷时,ICP-Aptamer与其特异性结合,导致与cDNA解离,cDNA发卡型结构重新恢复,使荧光信号猝灭。采用透射电镜、紫外-可见光谱、Zeta电位表征纳米粒子特性,优化了实验参数,包括pH值、ICP-Aptamer浓度、温育时间和温育温度。在优化的实验条件下,在0.02~10μmol/L线性范围内,水胺硫磷浓度与荧光抑制率呈良好的线性关系,检出限为17.8 nmol/L(3σ)。将此荧光探针应于大米、菠菜样品中水胺硫磷的测定,回收率均为92.9%~107.0%,相对标准偏差小于4.2%,表明此荧光探针在农药的检测和食品安全的监测中具有潜在的应用价值。A“turn on-off”fluorescent probe based on aptamer/gold nanoparticles(AuNPs)was developed for rapid,highly sensitive and selective detection of isocarbophos(ICP).Hairpin complementary strand(cDNA)with a fluorescein label(FAM)at 5'end was attached to the surface of AuNPs through thiol group.Subsequently,ICP-binding aptamer(ICP-Aptamer)was added and hybridized with cDNA,leading to open of hairpin assembly and"turn on"of fluorescent signal quenched by AuNPs.ICP-Aptamer specifically bound to the target ICP with restoration of configuration and“turn off”of the fluorescent signal.The properties of nanoparticles were characterized by transmission electron microscopy,ultraviolet-visible absorption spectroscopy and zeta potential.Experimental parameters influencing fluorescence inhibition rate,including pH value,concentration of ICP-Aptamer,incubation time and incubation temperature,were investigated and optimized.The corresponding fluorescence inhibition rate had a good linear relationship with concentration of ICP in the range of 0.02-10μmol/L,and the limit of detection was 17.8 nmol/L.Furthermore,the fluorescent probe was applied to determination of ICP in rice and spinach samples.The recoveries were from 92.9%to 107.0%and the relative standard deviations were lower than 4.2%.It could be concluded that the fluorescence probe had good application potential for detection of pesticides and supervision of food safety.

关 键 词：荧光探针 “开-关”型 适配体 金纳米粒子 水胺硫磷 

分 类 号：O657.3[理学—分析化学] TS207.53[理学—化学]