Development of new method for the preparation of enzyme thin films

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Experimental variables for the preparation of enzyme thin films using avidin-biotin technology were investigated in detail. The enzyme thin films containing glucose oxidase (GOx) were found to be used for several months if the GOx films were prepared under the optimum conditions, including the concentration of enzyme solution, temperature, pH, and treatment time. In order to immobilize much amount of enzymes in durable thin films, some kinds of synthetic polymer were employed as material for the film. Biotin-modified polyamines (polyethyleneimine (PEI) and polyallylamine (PAA)) can be used for immobilizing much amount of avidin through avidin-biotin interactions. On the other hand, polyanions such as polyvinylsulfate (PVS) and polystyrenesulfonate (PSS) were useful for immobilize avidin through electrostatic force of attraction, by a layer-by-layer deposition of the polyanion and avidin. The flexible backbone of PVS and PSS is responsible for high loading of avidin in the thin films. In these polyanion/avidin thin films, the biological activity of avidin (or binding ability to biotin) was retained and it was possible to accommodate some biotin-tagged functional molecules in the polyanion/avidin thin films. During the preparation of enzyme thin films by avidin-biotin interactions, it necessary to keep the film wet for the development of well-organized and biologically-active films. If the film was dried, the catalytic activity of enzyme was degraded rapidly, probably due to the degradation of the structure of the film. During the course of this study, we obtained useful information for the development of biologically active enzyme thin films through avidin-biotin interactions.