Development of an electrochemical immunoassay for detection of gatifloxacin in swine urine

Abstract

To detect gatifloxacin (GAT) residue in swine urine, an electrochemical immunoassay was established. An indirect competitive immunoassay was developed, in which the coating antigen is immobilized in an enzyme-linked immunosorbent assay (ELISA) plate and GAT residue from the sample competes with the limited binding sites in added anti-GAT antibody. Horseradish peroxidase (HRP) conjugated to goat anti-rabbit IgG was used as the enzymatic label. A carbon fiber working electrode was constructed and current signals were detected by using hydrogen peroxide as a substrate and hydroquinone as an electrochemical mediator. The electrochemical immunoassay was evaluated by analysis of GAT in buffer or swine urine and an average value of half inhibition concentration (IC(50)) of 8.9 ng/ml was obtained. Excellent specificity of the antibody was achieved with little cross-reaction with lomefloxacin (3.0%), ciprofloxacin (3.0%), and ofloxacin (1.9%) among commonly used (fluoro)quinolones. In conclusion, the immunoassay system developed in this research can be used as a rapid, powerful and on-site analytical tool to detect GAT residue in foods and food products.

Fig. 1

Structures of the gatifloxacin and related (fluoro)quinolones

Fig. 2

Fabrication of carbon fiber working electrode

Fig. 3

Electrochemical immunoassay procedure

Fig. 4

Optimization of analytical conditions The influences of (a) H₂O₂ (2.0 mmol/L HQ), (b) HQ (2.0 mmol/L H₂O₂), (c) incubation time (2.0 mmol/L HQ and 2.0 mmol/L H₂O₂), and (d) ultrasonic for the working electrode (2.0 mmol/L HQ, 2.0 mmol/L H₂O₂, and 10 min for incubation) on current response in the competitive assay are shown. The concentration of IgG-HRP conjugate was 1 μl/ml for protocols (a) to (c). The experiments were carried out in PBST (pH 7.4) containing NaCl (138 mmol/L), KH₂PO₄ (1.5 mmol/L), Na₂HPO₄·12H₂O (7 mmol/L), KCl (2.7 mmol/L), and Tween 20 (0.05%), with the applied potential of −0.15 V

Fig. 5

Competitive curve (a) and standard curve (b) of GAT determination with GAT as competitor in PBST c _GAT: concentration of GAT (ng/ml)

Fig. 6

Competitive curves of GAT in PBST and various dilutions of 1:2, 1:5, 1:10, and 1:20 of the extracted swine urine c _GAT: concentration of GAT (ng/ml)