High-performance biosensors based on molecular orientation in enzyme thin films

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Enzyme-containing thin films can be prepared by a layer-by-layer deposition of anionic or cationic polyelectrolyte and oppositely charged enzymes on a solid surface,through a electrostatic force of attraction. The thickness of the film depended significantly on the conditions of bathing solutions,such as polyelectrolyte and enzyme concentration,ionic strength,and pH. An alternate deposition of avidin and biotin-labeled enzymes also gave enzyme thin films through strong biological affinity between avidin and biotin. Avidin and enzyme formed a monomolecular layer in the multilayer film.The enzymes retained their catalytic activity in the film and thus it was possible to construct electrochemical enzyme biosensors by coating the film on the surface of electrodes.The biosensors thus prepared had an advantage that the size of output current can be regulated arbitrarily by changing the number of enzyme layers in the film. Glucose oxidase, lactate oxidase,uricase, and peroxidase were used as materials.Similarly, concanavalin A and glyco-enzymes can be used for constructing enzyme multilayer films,through affinity between concanavalin A and sugar. Non-glycosylated enzymes can also be used for this purpose after being modified with reactive sugar reagent. The concanavalin A-based enzyme thin films also exhibited catalytic activity.The layer-by-layer deposition technique developed in this study is applicable to the formation of thin film of antibody.The present technique would be useful for designing structure and function of biosensors.