化学学报 ›› 2012, Vol. 70 ›› Issue (21): 2251-2256.DOI: 10.6023/A12080567 上一篇    下一篇

研究论文

基于场放大进样及Au纳米粒子双重富集用于大肠杆菌检测的毛细管电泳电化学免疫分析法

张召香, 张飞, 刘营   

  1. 青岛科技大学化学与分子工程学院 青岛 266042
  • 收稿日期:2012-08-19 出版日期:2012-11-14 发布日期:2012-10-08
  • 通讯作者: 张召香 E-mail:zhangzhx@qust.edu.cn
  • 基金资助:
    项目受国家自然科学基金(No. 21105051)和山东省优秀中青年科学家科研奖励基金(No. BS2009HZ009)资助.

Electrochemical Immunoassay by Capillary Electrophoresis for E. coli Enhanced by Field-ampli?ed Sample Injection and Gold Nanoparticles

Zhang Zhaoxiang, Zhang Fei, Liu Ying   

  1. College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042
  • Received:2012-08-19 Online:2012-11-14 Published:2012-10-08
  • Supported by:
    Project supported by the National Natural Science Foundation of China (No. 21105051) and the Foundation for Outstanding Young Scientist in Shandong Province (No. BS2009HZ009).

利用Au纳米粒子作为辣根过氧化物酶(HRP)标记抗体的载体, 结合电堆积预富集技术, 发展了一种基于场放大进样及Au纳米粒子双重富集的毛细管电泳电化学免疫分析技术用于大肠杆菌的检测. 大肠杆菌与酶标抗体免疫反应后直接进行场放大进样预富集, 免疫样品快速迁移并堆积在毛细管入口端, 同时带负电荷的金纳米粒子向阳极端迁移, 在样品与缓冲溶液的界面处吸附样品离子. 金纳米粒子作为多酶载体使检测信号进一步放大. 以标记在抗体上的HRP催化H2O2氧化邻苯二胺产生的电流信号来检测大肠杆菌. 同常规电动进样毛细管电泳相比, 该双重富集技术可使灵敏度提高1400倍. 该方法对大肠杆菌检测的线性范围为2.0~2000.0 cfu·mL-1, 检出限为1.0 cfu·mL-1, 实现了对扇贝样品中大肠杆菌的快速、灵敏检测.

关键词: 大肠杆菌, 毛细管电泳, 场放大进样, 金纳米粒子, 双重富集, 电化学检测

A dual concentration technique combining ?eld-ampli?ed sample injection and gold nanoparticles as multi-enzyme carriers was developed in this paper. A novel E. coli detection platform based on immunoreaction, dual amplification focusing, capillary electrophoresis separation, and electrochemical detection was proposed for sensitive detection of E. coli in scallop samples. After noncompetitive immunoreaction between free E. coli antigen and excessive amount of horseradish peroxidase (HRP)-labeled anti-E. coli antibody tracer (Ab*) in liquid phase, the immune sample was directly introduced into the separation capillary. The ?eld-ampli?ed sample injection process allows introducing large amount of analytes into capillary to accumulate at the capillary inlet. Meanwhile, the negative charged gold nanoparticles migrated to the anode and attracted the immune sample ions onto its surface at the boundary of sample and buffer solution. Gold nanoparticles were used as multienzyme carriers of the signaling Ab* and the bound enzyme-labeled complex (Ag-Ab*) in order to achieve a further amplification of the electrochemical detection signal. Then the bound Ag-Ab* and unbound Ab* were separated by capillary electrophoresis, and the E. coli could be detected according to the H2O2/o-phenylenediamine reaction currents catalyzed by HRP labeled on anti-E. coli antibody. The assay adopting ?eld-ampli?ed sample injection preconcentration and gold nanoparticles as enhancer resulted in the improved sensitivity of 1400 fold when compared with traditional 10 kV electrokinetic injection for 10 s. The method allowed quantitative determination of E. coli concentration from 2.0 to 2000 cfu·mL-1, with a detection limit of 1.0 cfu·mL-1. The formed complex (Ag-Ab*) and the excessive Ab* were baseline separated with the separation efficiencies (theoretical plate number, N) greater than 104 plates/m. The relative standard deviation (RSD) values of the peak height, peak area and migration time were 2.6%, 3.5% and 3.1%, respectively. The proposed ?eld-ampli?ed sample injection and gold nanoparticles enhanced capillary electrophoresis based immunoassay with electrochemical detection method was successfully applied for the determination of E. coli in scallop samples.

Key words: E. coli, capillary electrophoresis, ?eld-ampli?ed sample injection, Au nanoparticle, dual concentration, electrochemical detection