Development of new method for the preparation of enzyme thin films

研究了亲和素-生物素技术制备酶薄膜的实验参数。在酶液浓度、温度、pH、处理时间等条件下制备葡萄糖氧化酶（GOx）酶薄膜，可以使用数月。为了将大量的酶固定在耐用的薄膜中，采用了几种合成聚合物作为薄膜的材料。生物素修饰的多胺（聚乙烯亚胺（PEI）和聚烯丙胺（PAA））可以通过亲和素与生物素的相互作用来固定化大量亲和素。另一方面，聚阴离子如聚乙烯醇硫酸盐（PVS）和聚苯乙烯磺酸盐（PSS）可通过静电引力使聚阴离子和亲和素层层沉积，从而固定化亲和素。PVS和PSS的柔性骨架是薄膜中亲和素高负载的主要原因。在这些聚阴离子/亲和素薄膜中，亲和素的生物活性（或与生物素的结合能力）被保留，并且有可能在聚阴离子/亲和素薄膜中容纳一些生物素标记的功能分子。在利用亲和素-生物素相互作用制备酶薄膜的过程中，必须保持薄膜的湿润，才能得到组织良好、具有生物活性的薄膜。如果将膜干燥，酶的催化活性会迅速下降，这可能是由于膜结构的降解。在本研究过程中，我们获得了通过亲和素-生物素相互作用开发生物活性酶薄膜的有用信息。

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.