Parameters which influence the optimal immobilisation of oxidase type enzymes on methacrylate copolymers as demonstrated for amperometric biosensors

Optimisation of the covalent attachment of enzymes to copolymers of methyl and glycidyl methacrylates is explored. The importance of ionic strength and pH were shown to influence the immobilisation efficiency, as well as enzyme concentration and immobilisation time. It was seen that for the case of glucose oxidase, the protein immobilisation efficiency correlated with the isoelectric point, whereas the immobilised enzyme activity, measured in terms of an amperometric response to glucose correlated with an immobilisation pH of maximum enzyme activity. Conditions for the maximum reactivity of the glycidyl group were also considered but found not to be the major determinant of an optimum immobilised enzyme. The conditions were evaluated for five oxidase type enzymes: glucose oxidase, lactate tyrosinase (polyphenol oxidase), cholesterol oxidase and alcohol oxidase in terms of their immobilisation efficiency. The immobilisation process has also been shown to stabilise the enzyme with respect to activity loss with time of storage, especially in the case of lactate oxidase.