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

通过静电引力，将阴离子或阳离子聚电解质和带相反电荷的酶逐层沉积在固体表面，可以制备含酶薄膜。膜的厚度主要取决于洗浴溶液的条件，如聚电解质和酶的浓度、离子强度和pH。亲和素和生物素标记酶的交替沉积也通过亲和素和生物素之间的强烈生物亲和力获得了酶薄膜。亲和素和酶在多层膜中形成单分子层，酶在膜中保持其催化活性，将膜包覆在电极表面可以构建电化学酶生物传感器。该生物传感器的优点是可以通过改变膜中酶的层数来任意调节输出电流的大小。以葡萄糖氧化酶、乳酸氧化酶、尿酸酶和过氧化物酶为原料，刀豆蛋白A和糖基酶通过刀豆蛋白A与糖的亲和力构建酶多层膜。非糖基化的酶在用活性糖试剂修饰后也可用于此目的。刀豆蛋白A为基础的酶薄膜也具有催化活性，本研究开发的逐层沉积技术适用于抗体薄膜的形成，为生物传感器的结构和功能设计提供了依据。

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.