Bioelectrochemical Applications of Reactions Catalyzed by Immobilized Enzymes

Abstract: The sensitivity of biosensors can be increased substantially by incorporation in a substrate recycling scheme. Based on the amplification of NAD+/NADH by the recycling reaction catalyzed by glycerol dehydrogenase (GDH) and diaphorase, NAD+/NADH can be determined amperometrically with around 1000 times higher sensitivity than for the substrate sensing mode. The sensitivity of enzyme electrodes were significantly enhanced compared to the enzyme reactor for the same recycling reaction scheme. An enzyme electrode was made from a glassy carbon electrode covered with a gelatin membrane containing entrapped GDH and diaphorase and put into a flow injection analysis system. Acetate is an important carbon source in fermentations. Methods that can be used for automatic process monitoring are particularly attractive. Flow injection determinations of acetate were carried out using immobilized acetate kinase, pyruvate kinase and lactate dehydrogenase. The acetate was determined by the depletion of NADH which was monitored amperometrically with a graphite electrode modified with a mediator. The application of this method is exemplified by the off-line determination of acetic acids in Escherichia coli cultivation. The results indicate that the enzyme-based flow injection analysis (FIA) is in principle suitable for the determination of acetic in fermentations. Simultaneous detection of multiple signals for common biochemical/electrochemical reaction is a challenging area of research for comparative studies. A biosensor has been developed for the simultaneous monitoring of the electrochemically generated current and the thermal signal produced in association with enzymatic catalysis. The method was based on the amplification of catechol/ benzoquinone by the tyrosinase immobilized on a reticulated vitreous carbon. The thermal signal could benefit in sensitivity from the electrochemical reaction which resulted in catechol/ benzoquinone recycling. The hybrid biosensor provides a useful tool for comparative studies. Polyethyleneimine (PEI) is a weak, polybasic aliphatic amine. PEI with a high concentration of amino groups has been widely used in a number of industrial immobilized biosystems. The immobilized diaphorase on reticulated vitreous carbon (RVC) was stabilized by the use of PEI coating. The PEI- coated RVC showed good properties compared to an uncoated one, such as long life-time and constant response to a series of injections in a flow injection system.