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化学学报
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化学学报 ›› 2009, Vol. 67 ›› Issue (20): 2329-2334. 上一篇    下一篇

研究论文

Au纳米标记物增强电化学免疫分析大肠杆菌的研究

黄晶晶   程欲晓    刘亚军    李康    鲜跃仲    金利通   

  1. (华东师范大学化学系 上海 200062)
  • 投稿日期:2009-02-25 修回日期:2009-04-28 发布日期:2009-06-18
  • 通讯作者: 金利通 E-mail:ltjin@chem.ecnu.edu.cn,litongjin@263.net,51050606028@student.ecnu.edu.cn
  • 基金资助:

    上海世博会专项基金(No. 06dg05824)资助项目

Gold Nanolabels for Enhanced Electrochemical Immunoanalysis of Escherichia coli

Huang, Jingjing       Cheng, Yuxiao       Liu, Yajun         Li, Kang          Xian, Yuezhong           Jin, Litong   

  1. (Department of Chemistry, East China Normal University, Shanghai 200062)
  • Received:2009-02-25 Revised:2009-04-28 Published:2009-06-18

PDF

1775

被引次数

10

通过在Au纳米颗粒表面修饰辣根过氧化酶(HRP)标记的大肠杆菌抗体制备了一种新型的Au纳米标记物, 并将该纳米标记物应用于增强电化学免疫分析大肠杆菌. 经过酶联免疫反应后, Au纳米标记物、免疫磁性颗粒(IMB)和大肠杆菌形成了IMB/抗体-大肠杆菌-Au纳米标记物的三明治式免疫复合物. 以3,3,5,5-四甲基联苯二胺(TMB)溶液作为底物, 采用电化学与流动注射检测(FIA)相结合的技术测定HRP的活性. 检测到的电流大小与免疫复合物上HRP的量成正比, 从而与大肠杆菌的浓度成正比. Au纳米颗粒增加了HRP的负载量, 增强了电化学信号, 大大提高了大肠杆菌的检测灵敏度. 实验结果表明, 大肠杆菌浓度在 1.0×102～5.0×104 cfu•mL－1范围内与电流大小成线性相关, 最低检测限达50 cfu•mL－1, 若对大肠杆菌样品溶液进行预浓缩, 将得到更宽的检测范围和更低的检测限. 本方法总的分析时间比其他方法短, 在1 h内就能完成对大肠杆菌样品的快速检测.

关键词: Au纳米标记物, 大肠杆菌, TMB, 流动注射检测(FIA), 快速检测

A novel nanolabel based on gold nanoparticle modified with anti-Escherichia coli (E. coli) peroxidase (HRP)-conjugated antibody has been developed. It was used for enhanced electrochemical immunoanalysis of E. coli. After the immuno-reaction, gold nanolabel, immunoparamagnetic bead (IMB) and E. coli formed a sandwich-type immunocomplex. 3,3,5,5-Tetramethylbenzidine (TMB) was used as substrate for HRP in the presence of H2O2 and the enzyme activity of HRP was measured by electrochemical detection coupled with flow injection assay (FIA). The concentration of E. coli could be confirmed by amperometric response, which was in proportion to the quantity of HRP on the sandwich-type immunocomplex. Gold nanoparticles were used as carriers of antibodies resulting in the increase of antibodies attached to E. coli. Therefore, the amperometric signal was enhanced and the detection sensitivity of E. coli was improved greatly. It showed a good linear relationship between the amperometric responses and logarithmic value of E. coli concentration ranging from 1.0×102 to 5.0×104 cfu•mL－1, with a detection limit of 50 cfu•mL-1. By introducing a pre-enrichment step, a wider detection range and lower detection limit could be obtained. The rapid detection could be finished within 1 h, shorter than other methods.

Key words: gold nanolabel, Escherichia coli (E. coli), TMB, flow injection assay (FIA), rapid detection