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Sensitive Chemiluminescence Immunoassay for E. coli O157:H7 Detection with Signal Dual-Amplification Using Glucose Oxidase and Laccase
journal contribution
posted on 2014-01-21, 00:00 authored by Yun Zhang, Chen Tan, Ruihua Fei, Xiaoxiao Liu, Yuan Zhou, Jing Chen, Huanchun Chen, Rui Zhou, Yonggang HuA 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.