A novel electrochemical immunosensor for ochratoxin A with hapten immobilization on thionine/gold nanoparticle modified glassy carbon electrode

Abstract

A novel electrochemical sensor was developed based on the combination of polythionine (PTH)/gold nanoparticle (nanoAu) composite film and enzyme catalysis amplification for the indirect competitive immunoassay of ochratoxin A (OTA). The sensing substrate was fabricated using a glassy carbon electrode (GCE) modified with an electropolymerized film of polythionine, which then mediated the self-assembly of nanoAu monolayer. After OTA–ovalbumin conjugates (OTA–OVA) were adsorbed onto the surface-confined gold nanoparticles, the analyte OTA in sample solution competed for the limited anti-OTA monoclonal antibodies with the immobilized hapten. Alkaline phosphatase (ALP)-labeled horse anti-mouse IgG antibodies were selectively bound onto the surface of the electrode, which afforded an indicator for OTA concentration in the sample. Under the optimal conditions, electrochemical response arising from the oxidation of enzymatic product of 1-naphthyl phosphate was observed to be inversely proportional to OTA concentration in the range from 1 ng mL⁻¹ to 1000 ng mL⁻¹ with a detection limit as low as 0.2 ng mL⁻¹. In addition, good reproducibility, high selectivity, sensitivity and stability were achieved. The recovery test demonstrated the feasibility of the proposed method for accurate determination of OTA contaminated samples.