Improvement of Electrochemical Biosensors Using Enzyme Immobilization from Water−Organic Mixtures with a High Content of Organic Solvent

We propose immobilizing enzymes into Nafion membranes by suspending the enzyme in a water−ethanol mixture with a high (>90%) ethanol content, followed by mixing with the dissolved polyelectrolyte, and finally allowing the enzyme−polyelectrolyte solution to dry at the target surface (electrode surface). Since Nafion solution was deposited from a solution where it is truly dissolved and without excessive dilution with water, the resulting membranes were more uniform and stable than those otherwise obtained. Enzyme suspensions in concentrated ethanol solutions were prepared without any prior modifications of the protein. The remaining activity after 30 min exposure to such solutions under optimal conditions was up to 100%. The stability of the enzymes in these suspensions was higher than that in aqueous solution. Electrochemical biosensors made accordingly showed a several times increased response compared with those of enzyme electrodes based on the traditional way of using excessive dilution of Nafion with water. The remaining activity, after the drying−washing cycle of the enzyme electrode made by enzyme immobilization from concentrated organic solvent, was at least 10 times higher than that of the traditional one.