Mass transport and reaction kinetic parameters determined electrochemically for immobilized glucose oxidase

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Abstract
Mass-transfer resistances often have pronounced effects on the overall reaction rates of enzymes immobilized at interfaces or in polymeric matrices. In the present work glucose oxidase was immobilized on the surface of a Pt disk electrode by 1 of 3 attachment techniques: silane-glutaraldehyde, allylamine-glutaraldehyde and albumin-glutaraldehyde. In 1 group of studies the electordes were rotated, and methods were employed to determine the diffusion and shielding coefficients for transport of a model electroactive compound, i.e., potassium ferrocyanide, through the enzyme matrix. A model electrochemically active compound was used because glucose exhibits a very slow rate of electron transfer at a Pt-surface. The diffusion coefficient for ferrocyanide was reduced 7% by the silane-enzyme and 25% by the allylamine-enzyme matrices. In a 2nd group of studies the electrodes were held stationary. Marked internal diffusional resistance was noted for the alubmin-glutaraldehyde-enzyme matrix. The calculated flux of ferrocyanide was decreased by a factor of 2000-8500 for transport through alubmin-enzyme matrices 0.21-0.063 cm thick, as compared to transport through free solution. In a 3rd group of studies the rotating enzyme-matrix electrode was utilized in determining apparent values of the Michaelis constant for glucose. The velocity of the reaction was determined by amperometric measurement of the concentration of hydrogen peroxide reaching the ring electrode. The results, determined from Eadie-Hofstee type plots of reaction current and substrate concentration, gave values between 12 and 36 mM for the 3 methods of immobilization. The results show that the mass-transfer resistance for diffusion of small molecules through immobilized glucose oxidase varied with the method of enzyme coupling. This study also shows that electrochemical techniques are useful in defining the relative rates of mass transfer and enzymatic reaction.