文章摘要
陆云飞,贾敏,吴继魁.基于脱氧核酶-等温级联放大耦合的传感体系高灵敏检测水样中铅离子[J].上海海洋大学学报,2020,29(6):840-846
基于脱氧核酶-等温级联放大耦合的传感体系高灵敏检测水样中铅离子
A DNAzyme-isothermal cascade amplification sensing system for ultrasensitive detection of Pb2+ in water samples
投稿时间:2019-05-20  修订日期:2020-03-11
DOI:10.12024/jsou.20190502664
中文关键词: Pb2+  荧光传感器  等温信号放大  G-四联体  原卟啉锌  GR-5 DNAzyme
英文关键词: Pb2+  fluorescence sensor  isothermal signal amplification  G-quadruplex  ZnppIX  GR-5 DNAzyme
基金项目:国家自然科学基金(31972772);上海自然科学基金(11ZR415400)
作者单位E-mail
陆云飞 上海海洋大学 食品学院, 上海 201306  
贾敏 上海海洋大学 食品学院, 上海 201306  
吴继魁 上海海洋大学 食品学院, 上海 201306
上海水产品加工及贮藏工程技术中心, 上海 201306
农业农村部水产品贮藏保鲜质量安全风险评估实验室, 上海 201306 
jkwu@shou.edu.cn 
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中文摘要:
      结合等温链替代扩增(ISDA)和指数扩增(EXPAR)设计一种非标记、高灵敏的Pb2+荧光生物传感体系。在Pb2+存在情况下,底物链被活化的GR-5 DNAzyme快速切割释放引物1。引物1与模板1杂交,并被DNA聚合酶(BSM)延伸形成带限制性内切酶(Nt.BbvCI)识别序列的双链核苷酸。BSM从Nt.BbvCI切割产生的切口再次延伸形成双链核苷酸,并释放信号G4-DNA片段。G4-DNA片段同时又可以作为模板2的引物,启动指数放大过程,从而释放更多的信号G4-DNA片段。扩增产物G4-DNA与原卟啉锌(ZnPPIX)相互结合从而产生强烈的荧光信号。详细优化了多种因素对检测体系的影响,在最优实验条件下,此方法对Pb2+的线性检测范围为0.1~50.0 nmol/L,检出限为0.03 nmol/L(S/N=3),回归方程为y=288.7x+744.7(y为荧光强度,x为Pb2+浓度)。干扰实验表明,该传感器对Pb2+具有良好的特异性和选择性。该传感体系成功应用于环境水体中Pb2+含量检测,加标回收率为94.0%~103.0%。本方法操作简单、选择性好、灵敏度高、有较强的抗干扰性能,可用于环境水样中Pb2+的高灵敏检测。
英文摘要:
      We exploited strand displacement amplification (ISDA) and exponential amplification (EXPAR) to design a label-free, ultrasensitive fluorescence sensing system for the detection of Pb2+. In the presence of Pb2+, the substrate strand is rapidly cleft by the activated GR-5 DNAzyme to release Primer 1. Primer 1 hybridizes to template 1 and is extended by DNA polymerase (BSM) to form a double-stranded nucleotide with a recognition sequence for restriction endonuclease (Nt. BbvCI). The nicks generated by BSM cleavage from Nt.BbvCI re-extended to form double-stranded nucleotides and release the signal G4-DNA fragment. The G4-DNA fragment can also be used as a primer for template 2 to initiate a series of amplification process, thereby releasing more signal G4-DNA fragments. G4-DNA binds to protoporphyrin zinc (ZnPPIX) to produce a strong fluorescent signal. The effects of various factors on the sensing system were investigated. Under the optimal experimental conditions, the linear detection range of Pb2+ was 0.1-50 nmol/L, and the LOD was 0.03 nmol/L (S/N=3). The regression equation is y=288.7x+744.7 (y is the fluorescence intensity and x is the Pb2+ concentration). Interference experiments show that the sensing system has good selectivity for Pb2+ against other metal ions. This method was applied to the detection of Pb2+ in environmental water, and the recoveries were obtained from 94.0% to 103.0%. The proposed sensing system has the advantages of simple operation, good selectivity, high sensitivity and strong anti-interference performance, and can be used for high-sensitivity detection of Pb2+ in environmental water samples.
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