Sensitive Chemiluminescence Immunoassay for E. coli O157:H7 Detection with Signal Dual-Amplification Using Glucose Oxidase and Laccase

化学 检出限 葡萄糖氧化酶 辣根过氧化物酶 色谱法 鲁米诺 化学发光 漆酶 免疫分析 大肠杆菌 生物传感器 比色法 一级和二级抗体 生物化学 抗体 免疫学 基因 生物
作者
Yun Zhang,Chen Tan,Ruihua Fei,Xiaoxiao Liu,Yuan Zhou,Jing Chen,Huanchun Chen,Rui Zhou,Yonggang Hu
出处
期刊:Analytical Chemistry [American Chemical Society]
卷期号:86 (2): 1115-1122 被引量:105
标识
DOI:10.1021/ac4028774
摘要

A novel, sensitive chemiluminescence (CL) immunoassay for Escherichia coli O157:H7 detection with signal dual-amplification using glucose oxidase (GOx) and laccase was investigated. The method was based on the characterization of a luminol–H2O2–laccase reaction. Compared with the horseradish peroxidase-based biosensor, laccase exhibited high catalytic activity in strong alkaline medium, which was compatible with the luminol system. The capture antibody was immobilized onto the magnetic bead (MB) surfaces. The detection antibody was linked with GOx through biotin–avidin recognition. Accordingly, the bioconjugation of MB–caputure antibody–E. coli O157:H7–detection antibody–GOx catalyzed the substrate glucose, thereby generating H2O2. E. coli O157:H7 was then detected by measuring the CL intensity after H2O2 formation. Under optimal conditions, the calibration plot obtained for E. coli O157:H7 was approximately linear from 4.3 × 103 colony-forming unit (CFU) mL–1 to 4.3 × 105 CFU mL–1, and the total assay time was <2.0 h without any enrichment. The limit of detection for the assay was 1.2 × 103 CFU mL–1 (3σ), which was considerably lower than that of enzyme-linked immunosorbent assay method (1.0 × 105 CFU mL–1) (3σ). A series of repeatability measurements of using 1.7 × 104 CFU mL–1 E. coli O157:H7 exhibited reproducible results with a relative standard deviation (RSD) of 3.5% (n = 11). Moreover, the proposed method was successfully used to detect E. coli O157:H7 in synthetic samples (spring water, apple juice, and skim milk), which indicated its potential practical application. This protocol can be applied in various fields of study.
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