Optimization of a polypyrrole glucose oxidase biosensor.

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of +0.65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentiostating the enzyme electrode at a potential of +0.7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determine the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0.3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0.01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0.17 microns (75 mC/cm2) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1.0 mM to 7.5 mM glucose and kinetic parameters determined for the optimized biosensors were 33.4 mM for the Km and 7.2 microA for the Imax. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.