State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University , Wuhan 430070, China.
Anal Chem. 2014 Jan 21;86(2):1115-22. doi: 10.1021/ac4028774. Epub 2014 Jan 9.
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.
一种基于葡萄糖氧化酶（GOx）和漆酶信号双重放大的新型、灵敏的化学发光（CL）免疫分析方法用于检测大肠杆菌 O157:H7。该方法基于鲁米诺-H2O2-漆酶反应的特性。与基于辣根过氧化物酶的生物传感器相比，漆酶在强碱性介质中表现出高催化活性，与鲁米诺体系兼容。捕获抗体固定在磁珠（MB）表面。检测抗体通过生物素-亲和素识别与 GOx 连接。因此，MB-捕获抗体-大肠杆菌 O157:H7-检测抗体-GOx 的生物缀合物催化底物葡萄糖，从而产生 H2O2。然后通过测量 H2O2 形成后 CL 强度来检测大肠杆菌 O157:H7。在最佳条件下，大肠杆菌 O157:H7 的校准曲线从 4.3 × 10(3)菌落形成单位（CFU）mL(-1)到 4.3 × 10(5) CFU mL(-1)大致呈线性，总测定时间<2.0 小时，无需任何富集。该测定的检测限为 1.2 × 10(3) CFU mL(-1)（3σ），明显低于酶联免疫吸附测定方法（1.0 × 10(5) CFU mL(-1)）（3σ）。使用 1.7 × 10(4) CFU mL(-1)大肠杆菌 O157:H7 进行的一系列重复性测量结果具有可重复性，相对标准偏差（RSD）为 3.5%（n = 11）。此外，该方法成功用于检测合成样品（泉水、苹果汁和脱脂牛奶）中的大肠杆菌 O157:H7，表明其具有潜在的实际应用。该方案可应用于各个研究领域。
