A reagentless bienzyme amperometric biosensor based on alcohol oxidase/peroxidase and an Os-complex modified electrodeposition paint

A highly stable and sensitive amperometric ethanol bi-enzyme biosensor was developed using alcohol oxidase isolated from thermotolerant methylotrophic yeast Hansenula polymorpha and horseradish peroxidase as biorecognition elements. Enzyme immobilization was performed by means of electrodeposition paints (EDP) with a first layer integrating horseradish peroxidase within an Os-complex modified EDP in order to assure fast electron transfer between the enzyme and the electrode surface. On top of this layer alcohol oxidase was entrapped within an EDP layer thus assuring fast substrate diffusion within the hydrogel layer concomitantly with a stabilization of the enzyme. A variety of sensor architectures were investigated aiming on the optimization of the electrochemical communication between the immobilized enzymes and the electrode surface. The immobilized enzymes activities were highly dependent on the applied potential during the electrochemically induced EDP precipitation and on the chemical composition of the used EDP. Bioanalytical properties of an optimized alcohol biosensor such as response time, dynamic range for different analytes (ethanol, methanol, n-propanol, n-butanol, formaldehyde), operational and storage stability were investigated. Moreover, the developed biosensor was applied for the determination of ethanol in wine samples.