An electrochemical immunosensor for ochratoxin A based on immobilization of antibodies on diazonium-functionalized gold electrode

A novel electrochemical immunosensor for sensitive detection of ochratoxin A (OTA) was reported. An electrochemical and chemical reaction protocol was elaborated to modify the gold electrode. A screen-printed gold electrode (SPGE) was modified with a layer of 4-nitrophenyl, assembled from 4-nitrophenyl diazonium salt synthesized in situ in acidic aqueous solution. Next, the nitro groups were electrochemically reduced to amines followed by activation with glutaraldehyde to give a stable intermediate derivative that covalently binds antibodies against OTA during the second step, thereby tailoring an immunosensor for ochratoxin A. The utility of the electrochemical immunosensor for a competitive immunoassay was demonstrated. A competition between OTA and fixed concentration of a horseradish peroxidase-labeled OTA (OTA-HRP) for the immobilized antibodies was realized. The activity of the bound OTA-HRP was electrochemically measured by chronoamperometry (CA) using 3,3′,5,5′-tetramethylbenzidine (TMB) as substrate. The immunosensor obtained using this novel approach enabled a detection limit of 12 ng mL−1 and a dynamic range up to 60 ng mL−1 of OTA. Precision, accuracy and stability studies were satisfactory for the developed immunosensor.