Controllable Production Of Hydrogel Microspheres Based On Microfluidics And Their Application In Enzyme Immobilization

As a natural and degradable biocatalyst,enzymes have gained more and more attention in recent years.However,due to their water solubility and environmental sensitivity,recycling and usage under non-mild conditions have become two major obstacles in their applications.In order to improve both of them,enzyme immobilization is currently the most widely selected method.At the same time,the physical and chemical properties of immobilization carriers are mainly affected by their sizes.In this thesis,a large quantity of monodisperse chitosan/β-sodium glycerophosphate composite hydrogel microspheres were prepared using droplet microfluidic technology,which were used as embedding and immobilization carriers.Glucose oxidase was successfully immobilized and its enzymatic properties were characterized.Our work has resulted in significantly improved catalytic activity,pH stability,temperature stability and recyclability.This thesis is mainly divided into the following three parts:In the first part,monodisperse chitosan/β-sodium glycerophosphate composite hydrogel microspheres were prepared using a self-made co-flow microfluidic device,in which the continuous phase fluid was the dichloromethane solution of glutaraldehyde obtained by extraction,while the dispersed phase fluid is chitosan/β-sodium glycerophosphate solution.The obtained hydrogel was characterized by polarized light microscope,scanning electron microscope,and Fourier transform infrared spectroscopy.According to the followed data analysis,the microspheres were almost of the same size,while the standard deviation was less than 5%.This indicated a good monodispersity.Under the scanning electron microscope,there observed gaps and microporous structures,which respectively ensure the loading of the enzymes and the exchange of substrate and product.The weakening of the primary amino peak and the appearance of the imine bond peak are observed in the infrared spectrum of after-gelation sample,indicating that glutaraldehyde cross-links are formed between the chitosan chains.In the next part,the previously prepared composite hydrogel microspheres were used as the immobilization carriers of glucose oxidase under different immobilization conditions.To study the immobilization under different glutaraldehyde concentrations,different enzyme addition amounts and different immobilizing times,as well as the catalytic performance of immobilized enzyme,several sets of samples were prepared and characterized by ultraviolet-visible spectrophotometry.The results show that glutaraldehyde concentration and immobilization time can be regarded as a set of equivalent parameters on the immobilization effect on enzymes.When the concentration is low or the time is short,the cross-linking is of low degree,leading to an unideal result of the enzyme immobilization.When the concentration is high or the time is long,the excessively high degree of cross-linking occupies the active sites on the enzyme surface and weakens its catalytic activity.The enzyme addition amount determines the ratio of intra-molecular cross-linking to inmolecular cross-linking.Low addition drives glutaraldehyde to operate inside few enzyme molecules and reduce enzyme activity.The third part explores the performance of immobilized enzymes under different conditions and was compared with free enzymes.The result provides a reference for industrial production.The characterization results of UV-Vis spectrophotometry show that the immobilized enzyme is more adaptable to alkaline condition than free enzymes.What’s more,the immobilized enzymes maintained high catalytic performance in a wider pH range,whose maximum hits 3.55 times of free enzyme.Under different temperatures,the immobilized enzyme and the free enzyme have an almost same applicable temperature range,but obviously,the immobilized enzyme has higher catalytic activity which is up to 2.4 times of free enzyme.After 10 times of washing and recycling,the immobilized enzyme maintained 43.28%of the initial catalytic activity.Between every two cycles,the catalytic properties decreased only a tiny bit that can almost be ignored.In short,we have successfully prepared immobilized enzyme on chitosan/β-sodium glycerophosphate composite hydrogel microspheres.The immobilized enzymes have higher and catalytic properties than free enzymes.Meanwhile,they possess recyclable and controllable activity and better tolerance to extreme conditions.Therefore,they would have broad application prospects in the field of industrial catalysis.