An amperometric biosensor has been developed, incorporating the electroactive polymer, polyaniline (PANI), which undergoes redox cycling, and can couple electrons directly from the enzyme active site, to the electrode surface. Construction of this sensor was achieved by electropolymerisation of polyvinylsulphonate-doped aniline onto the surface of a screen-printed carbon-paste electrode. Biomolecules could then be doped onto the surface of the polymer by electrostatic interactions with the polymer backbone. A key component in a biosensor is the recognition molecule and its immobilisation. This study investigates this process of protein immobilisation using amperometric and colorimetric techniques. Immobilisation of protein (enzyme or antibody) onto the transducer is achieved by electrostatic interactions. By applying bovine serum albumin (BSA) electrostatically at the electrode, efficient blocking of the electrode surface from the bulk solution was achieved above approximately 0.75 mg/ml. When horseradish peroxidase was immobilised on the electrode surface at various concentrations, optimal amperometric responses were achieved at approximately the same protein concentration. Determination of the number of molecules of protein immobilised on the surface of the electrode at this concentration was done using a colorimetric enzyme assay. It was found that under optimal immobilisation conditions, a protein monolayer was formed at the electrode surface. In the case of enzymes such as horseradish peroxidase (HRP), this provides simultaneous blocking of the electrode surface from bulk solution interactions as well as yielding optimal electron transfer properties
