Towards the development of a sensitive electrochemical sensor for the determination of chloramphenicol residues in milk

Abstract

A simple and sensitive bioelectrochemical immunoassay method has been developed to detect chloramphenicol (CAP) residues in milk. Monoclonal anti-CAP antibodies and conjugates of ovalbumin–CAP (OVA–CAP) were used to establish the indirect competitive enzyme-linked immunosorbent reaction, in which the CAP in standard solutions or samples competed with the OVA–CAP immobilized on 96-well polystyrene reaction plates for the limited binding sites on the CAP monoclonal antibodies. After the competitive immunoreaction, the plate was rinsed and HRP-labeled goat anti-mouse IgG was added into the testing wells. o-Phenylenediamine (o-PD)-hydrogen peroxide was applied as the hydrogen donor. The reaction solutions were transferred into the testing cells of a voltammetric analyzer and the electrochemical signals (oxidation peak currents of o-PD) were recorded by Differential Pulse Voltammetry (DPV) after the HRP-catalyzed reaction on the plate was stopped by 1 M H₂SO₄. The results showed that the sensitivity of the electrochemical immunoassay was higher than that of c-ELISA (competitive ELISA) in detecting chloramphenicol residue in milk. This method also demonstrated a linear response of oxidation peak current to chloramphenicol concentration over the 0.1–300 ng mL⁻¹ range with a detection limit of 0.03 ng mL⁻¹ CAP. The average recovery rate reached 87.7% based on the milk samples. Furthermore, the immuno-voltammetric apparatus is portable and can be used on site for detecting chloramphenicol residue in milk.