Preparation Of Multi-compartment Microcapsules Via Microfluid-generated W/W/O Emulsions For Enzyme Cascade Reaction

Water-in-water-in-oil（W/W/O）emulsions are a kind of dual emulsions,which have been widely applied in some fields of cell extraction,functional material fabrication,delivering of drug and artificial cell.In this thesis,we design microfluidic device to fabricate monodisperse W/W/O emulsion and explore the general rules for regulating emulsion size,alginate microcapsules are prepared to encapsulate bioactive molecules using W/W/O emulsion as template by virtue of ATPS partitioning effect and is applied to enhance enzyme cascade reaction.In this experiment,the mechanism of protein partition encapsulation and enzyme catalyzed tandem reactions are studied.In the part of preparation and regulation of microfluidic W/W/O emulsion,the monodisperse emulsion is prepared using microfluidics assembled with glass capillary tubes and needle.The influences of the flow rates on size of W/W/O emulsion with the increase of flow rate of oil phase,the inner and outer diameter of droplets with a slight decrease when ratio of both remains unchanged.The sum of flow rate of middle and inner phase has little effect on the size of W/W/O emulsion.The ratio of inner and outer diameter of emulsion can be precisely controlled from 0.44 to 0.76 with the flow rates range of middle phase(1～3μL·min^-1)and inner phase(0.5～1.5μL·min^-1).Therefore the morphology of microcapsules can be regulated to meet a variety of requirements by controlling the flow rate of each phase.In addition,due to the selective distribution of ATPS,W/W/O emulsion can enrich bioactive molecules in the inner phase droplets.This double emulsion is expected to be widely used in the fields of analysis and detection,biomedicine and food chemical industry.In the part of the preparation of compartmentalization microcapsule and mechanism of partition encapsulation,alginate is dissolved to the middle phase of W/W/O emulsion,which dripped into a PEG-rich solution containing Ca Cl₂ to form microcapsules.It has been proved that this method enriches bioactive molecules in the core of microcapsule,which is not able to realize for traditional transient W/W/O emulsion（inner and middle phases are aqueous solutions）.The prepared microcapsules of about 430μm have a good monodispersion.Moreover,another bioactive molecules can be encapsulated in alginate microcapsule shell.Fluorescence images show that bovine serum albumin（BSA）and catalase（CAT）are spatially confined in core and shell of microcapsules with the encapsulation efficiency of 77.5%and71.0%respectively at 30 minutes of cross-linking curing.This study provides a new platform for artificial cells,multi-compartment reactors,drug delivery systems,multi-compartmental reactors,etc.In the intensification of enzyme cascade reaction with double-compartment microcapsule,we chose enzyme cascade reaction system of glucose oxidase and horseradish peroxidase（GOX-HRP）as the model reactions.Fluorescence images show that HRP and GOX are spatially confined in core and shell of microcapsules and proceed enzyme cascade reaction with glucose as the substrate and Amplex Red as the indicator.In C_s=200 mmol·L^-1,C_GOX=C_HRP=50μg·m L^-1,t=5 min,this showing～51 times higher overall activity than that of microcapsules co-immobilized with GOX and HRP.Compared with other compartmentalized hydrogel microcapsules,we prepared microcapsules with the liquid core,which reduced diffusion resistance,helping intermediate product rapidly transfers among compartments,increasing the rate of enzyme cascade reaction.It is expected that the method can be widely used in the fields of biocatalysis,analysis,detection and functional materials.