The long-term objective of our research is to design, synthesize, and characterize selective and catalytic molecular systems for bioanalytical applications. The specific aim of this competitive continuation proposal is the development of enzyme electrodes (biosensors) for the determination of clinically important species such as lactate, glucose, glutamate, and ethanol. The experimental design is based on the integration of dehydrogenase enzymes and electrodes using thin surface films of a biopolymer chitosan. While most of the current biosensors are based on oxidases, the use of dehydrogenases has a potential to expand the scope of biosensing because dehydrogenases are more abundant, less prone to interferences from oxygen, and have higher substrate specificity. In addition, such biosensors are expected to have improved operational stability, as the chitosan matrix will provide a biocompatible microenvironment for enzymes. Chitosan is a promising structural material for designing functional layers on electrode surfaces because it displays excellent membrane forming ability, good adhesion, and susceptibility to chemical modifications due to the presence of amino and hydroxyl groups. In a series of synthetic steps, the components necessary for the operation of a biosensor will be covalently linked to chitosan scaffold using bifunctional tethering molecules. The tethering chemistry will involve the Schiff base, urethane, and urea bonds. The biosensing films will be characterized using electrochemical and spectroscopic techniques in order to determine the composition-structure-activity patterns. The optimized biosensors will be used in a biological fluid (serum) to evaluate the effects of a sample matrix on their performance. The proposed enzyme electrodes will provide analytical access to a large group of dehydrogenases for applications in bioelectronics, and specifically, in reagentless biosensors, i.e. devices that do not require dissolved enzymes and cofactors to operate.