A tris(2,2′-bipyridyl)cobalt(III)-bovine serum albumin composite membrane for biosensors

A concept based on a novel redox-biocompatible composite protein membrane fabrication, double enzyme membrane modification technique and antibody immobilization, was exploited to develop a highly sensitive amperometric enzyme immunosensor for detection of carcinoembryonic antigen (CEA). In this concept, a solution of bovine serum albumin (BSA) containing horseradish peroxidase (HRP) is coated on the gold electrode in such a way that the first enzyme membrane is achieved. Then tris(2,2′-bipyridyl) cobalt(III) (Co(bpy)33+), as a mediator, was embedded in BSA–HRP composite membrane vis the electrostatic force and hydrophobe functions. Later a self-assembled conductive nano-Au monolayer was constructed onto the resultant electrode surface by electrostatic interaction between the negatively charged nano-Au and positively charged Co(bpy)33+. Protein A is used as a binding material to achieve an adjusted (but not random) orientation of the antibodies surface for efficient combination of antigens. Finally, the HRP, was employed to block the possible remaining active sites and avoid the non-specific adsorption, which acts not only as a blocking reagent instead of the commonly used BSA but also as the conventional enzyme-labeling to amplify the response of the antigen–antibody reaction. The immunosensor constructed with the double layer biocatalytic HRP membranes and the desirable Co(bpy)33+/BSA redox-biocompatible composite membrane performed high sensitivity and a wide linear response to CEA in the range of 0.50–80.00 ng/mL with a limit of detection of 0.14 ng/mL, as well as good stability and long-term life.