Non-aqueous enzymology approach for the development of electrochemical biosensors was proposed earlier by our group [1]. Water insoluble polymers, which can be dissolved in organic media, seem to accomplish the requirements of the optimal matrix for enzyme immobilisation. Nafion is commercially available as a solution in water-alcohol mixtures with a high (85-90%) content of organic solvent. We proposed the method of enzymes immobilisation into Nafion membranes by suspending the enzyme in a water-ethanol mixture with a high (>90%) ethanol content, mixing with the poly-electrolyte solution, and drying at the target surface. Electrochemical biosensors made according to the proposed method of enzyme immobilisation showed both increased response and improved stability as compared with the enzyme electrodes prepared in the traditional way using excessive dilution of Nafion with water[1].

The aim of the present investigation was the detailed study of the following basic factors, which affect both the stability and the activity of enzyme immobilised in Nafion membrane: the sequence of preparing of enzyme-polyelectrolyte complex in water-ethanol mixture, the alkaline used for Nafion neutralisation, the concentrations of all components in the enzyme-polyelectrolyte complex.

The activity of GOD was measured with a Clark-type oxygen electrode. The GOD-Nafion membrane was formed on the bottom of 1 ml polymethylmetacrylate chamber. The enzyme activity was determined due to oxygen consumption after injection of glucose. The stability of GOD immobilised in the Nafion membrane was studied by repeated measurements of its activity. Prior to each measurement chamber was thoroughly washed with water. The stability of GOD-Nafion films have decreased in raw of NH₄OH, LiOH, NaOH, KOH used as neutralising agents. Water solution of NH4OH had promoted the most stable GOD-Nafion films: 100% of initial GOD activity were preserved during 3-5 hours of repeated activity measurements (more then 10 measurements).

In contrast to our previous report [1], the formation of the enzyme-polyelectrolyte complex was carried out suspending the enzyme with Nafion solution in ethanol instead of using of pure organic solvent. The existence of polyelectrolyte in suspending solvent caused enzyme stabilisation in organic medium. As a result the homogeneous solutions of GOD-Nafion complex in water-organic mixtures were stable over weeks. The GOD-Nafion membranes obtained from such complexes exhibited the improved operational stability.

The content of water, enzyme and polyelectrolyte in ethanol were found to be crucial factors affecting the stability and the activity of the resulting membranes. The multidimensional patterns taking into account all mentioned factors were made. The content of the enzyme-polyelectrolyte complex concerning the optimal surrounding of GOD in Nafion membrane was found.

[1] Karyakin, A. A., Karyakina, E. E., Gorton, L., Bobrova, O. A., Lukachova, L. V.,. Gladilin, A. K., Levashov, A. V. Anal. Chem., 68, 4335-4341, 1996.