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

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.