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