Electrochemical indirect competitive immunoassay for ultrasensitive detection of zearalenone based on a glassy carbon electrode modified with carboxylated multi-walled carbon nanotubes and chitosan

The authors describe an electrochemical immunoassay for ultrasensitive detection of the mycotoxin zearalenone (ZEA). A nanocomposite was prepared from carboxy-functionalized multi-walled carbon nanotubes and chitosan (cMWCNTs/Chit). The morphology and electrochemical performance of the materials was characterized by field-emission scanning electron microscopy, atomic force microscopy, differential pulse voltammetry, cyclic voltammetry, and electrochemical impedance spectroscopy. In this assay, ZEA–BSA conjugated covalently to activated cMWCNTs/Chit film, then the indirect competition between ZEA–BSA and free ZEA when immobilization of excess anti-ZEA. The secondary antibody is labeled with the enzyme alkaline phosphatase which can hydrolyze the substrate 1-naphthylphosphate to produce 1-naphthol which gives a stable and strong anodic electrochemical signal at a low working voltage of 0.3 V (vs. Ag/AgCl). The use of the modified GCE results in a strongly enhanced electrochemical current response. Compared with conventional methods, the established immunosensor exhibited a high level of sensitivity. Under optimal conditions, this immunoassay can quantify ZEA in the 10 pg·mL−1 to 1000 ng·mL−1 concentration range with a detection limit of 4.7 pg·mL−1 and the sensitivity is 0.51 μA·μM−1·cm−2. The method was applied to the determination of ZEA in cereal and feedstuff samples. Results showed satisfactory recovery and good consistency with high-performance liquid chromatography. Therefore, the indirect competitive electrochemical immunosensor provide a viable tool based on bioanalysis.