催化活性可视化的葡萄糖氧化酶纳米胶囊的制备与研究

Preparation and study of glucose oxidase nanocapsules with catalytic activity visualization

葡萄糖氧化酶(GOx)是一种具有重要工业应用价值的催化酶,能够将葡萄糖高效且特异性地催化氧化为葡萄糖酸.但GOx的环境稳定性差易于失活,并且其催化活性的检测步骤繁琐,这些缺陷都限制了GOx的实际应用.该文工作首先合成了一种与GOx辅酶FAD结构类似的可交联化合物DAA-Flavin,之后将其与主要单体丙烯酰胺(AAm)在GOx表面共聚合而形成交联聚合物外壳,制备得到了具有均匀尺寸分布的酶纳米胶囊GOx@Fla-c-PAAm.其中,交联聚合物外壳限制了内部GOx的取向,使酶的环境稳定性得到了提升.同时,分布在聚合物外壳中的类辅酶荧光化合物DAA-Flavin可作为GOx的电子传递媒介,赋予体系氧化还原荧光特性.在有底物葡萄糖存在条件下,DAA-Flavin能够持续接受由GOx活性中心给出的电子而被还原,纳米胶囊的荧光强度也随之逐渐降低,且荧光衰减速率(反映为荧光强度-时间曲线的斜率)与酶的催化活性之间呈现高度的线性相关(R^(2)>0.990),具有催化活性可视化的特性.

Glucose oxidase(GOx)is a type of catalytic enzyme which has important value in industrial applications.GOx is capable of catalyzing glucose into gluconic acid with high efficiency and selectivity.However,its actual applications are limited by the poor environmental stability and tedious operating steps of activity determination.In this work,a cross-linkable molecule DAA-Flavin was synthesized,which had similar structure with the cofactor of glucose oxidase(GOx).DAA-Flavin was then copolymerized with main monomer acrylamide(AAm)around GOx surface to fabricate core-shell GOx nanocapsules.The enzyme serving as core was encapsulated in the functional polymer shell,which protected the enzyme from denaturation and improved its environmental stability.Moreover,cofactor-like fluorescent molecules DAA-Flavin distributed in the polymer shell could act as mediators to accept electron given by the enzymatic active center,thus endowing the nanocapsules redox fluorescent property.In the presence of substrate,fluorescence intensity of the nanocapsules was attenuated gradually due to the continuous reduction of DAA-Flavin,and there was a highly linear correlation between the fluorescence decay rate and enzyme activity(R^(2)>0.990).Furthermore,attenuated fluorescence intensity at reducing state could restore under oxidation condition,making the redox fluorescence reversible.The fabrication of this novel kind of core-shell enzyme nanocapsules enabled the conversion from biochemical signals to photoelectric signals,and endowed the enzymatic activity visual properties.