title: Sensitive chemiluminescence immunoassay for E. coli O157:H7 detection with signal dual-amplification using glucose oxidase and laccase.
abstract: 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 × 10(3) colony-forming unit (CFU) mL(-1) to 4.3 × 10(5) 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 × 10(3) CFU mL(-1) (3σ), which was considerably lower than that of enzyme-linked immunosorbent assay method (1.0 × 10(5) CFU mL(-1)) (3σ). A series of repeatability measurements of using 1.7 × 10(4) 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.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University , Wuhan 430070, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Chen
authors.initials: C
authors.lastname: Tan
authors.forename: Ruihua
authors.initials: R
authors.lastname: Fei
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Liu
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhou
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Huanchun
authors.initials: H
authors.lastname: Chen
authors.forename: Rui
authors.initials: R
authors.lastname: Zhou
authors.forename: Yonggang
authors.initials: Y
authors.lastname: Hu
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000737:chemistry; D055910:Antibodies, Immobilized / Q000737:chemistry; D001628:Beverages / Q000382:microbiology*; D015374:Biosensing Techniques; D002138:Calibration; D015169:Colony Count, Microbial; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D005618:Fresh Water / Q000382:microbiology*; D005949:Glucose Oxidase / Q000737:chemistry*; D006861:Hydrogen Peroxide / Q000737:chemistry; D007118:Immunoassay; D042845:Laccase / Q000737:chemistry*; D057230:Limit of Detection; D008163:Luminescent Measurements; D008165:Luminol / Q000737:chemistry; D059346:Magnets; D027845:Malus; D008892:Milk / Q000382:microbiology*; D015203:Reproducibility of Results
chemical_list: D000907:Antibodies, Bacterial; D055910:Antibodies, Immobilized; D008165:Luminol; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D042845:Laccase