Study On Enzymatic Reaction For Aqueous Two-Phase Systems In Microchannel And Preparation Of Gel Composite Microspheres

The aqueous two-phase system?ATPS?is composed of two aqueous solutions of hydrophilic compounds mixed at a certain concentration,due to the steric hindrance effect,ATPS is naturally divided into extraction systems of two aqueous phase which are insoluble and have a clear phase interface.It can be widely used in the extraction of biologically active substances,metal ions,proteins and cells,as well as combine the biochemical reaction and separation process.However,ATPS is difficult to develop and apply on the large scale due to the fact that the phase forming material and delamination resulting in high energy and time consumed.ATPS combines the microchannel devices not only reduce the cost,but also strengthen the interface reaction and separation process.In this dissertation,we base on the mechanism of the selective distribution of macromolecule proteins in PEG-Dex ATPS,the enzymatic reaction in parallel laminar flow and droplet flow microchannels are strengthened,and the composite calcium alginate microspheres are prepared by combining an ATPS with gel materials.In the research of the aqueous two-phase system enhance enzymatic reaction in a parallel laminar flow microchannel.A double Y-type parallel laminar flow microchannel device is fabricated,which makes the aqueous two-phase form a stable and clear interface in the microchannel.We add the substrate urea and catalyst urease to the PEG phase and Dex phase respectively,and coupling reaction and separation is realized at the two-aqueous phase interface of the Y-type bifurcation microchannel.The enzymatic reaction rate and conversion rate in the parallel laminar microchannel are 1.37?mol·m L^-1·s^-1 and 7.69%.While the enzymatic reaction rate in beaker is only reach 0.0016?mol·mL^-1·s^-1.The beaker is nearly800 times lower than microchannel.It main reason is fluid in the microchannel has a large specific surface area and the short mass transfer distance.In addition,the flowing state of ATPS interface has the high surface renewal rate,so the material diffusion rate is high,and then the enzymatic reaction process is strengthened.Moreover,due to the urease macromolecule is tend assigned to Dex phase,the enzyme and product can be separated at the end of the Y-shaped microchannel.Based on above issue,the microchannel enhancement platform provide the foundation for the subsequent application of the microchannel aqueous two-phase system.In the research of the aqueous two-phase system enhance enzymatic reactions in droplet flow microchannel.A coaxial micro-channel droplet flow microchannel device is fabricated,and the stable and uniform size PEG droplets are generated in the microchannel.Firstly,urease is added to the outer phase which contains the Dex phase and urea is added to the PEG phase,the neutral red indicator is added in the droplets and used to verify the progress of the urease-catalyzed reaction.Then,the effects of droplet size,contact time,flow ratio of two-phase flow and other conditions on the reaction rate and conversion rate of enzyme are investigated.Finally,the enzymatic reaction rate of 1.12?mol·mL^-1·s^-1 and the conversion rate of 56.03%are obtained in the microchannel.Compared with parallel laminar flow,the reaction rate do not have much difference,in addition to,the conversion rate is increased by nearly 8 times.The main reason is that the droplet flow has a larger specific surface area than the parallel laminar flow,and there is an internal field circulation during the flow of the droplets,which will effectively accelerate the surface update rate so that more substrates are reacted adequately.Therefore,the enzyme reaction can get a higher conversion rate.As a result,this study provides a more efficient and convenient microchannel platform for the study of enzymatic reactions at the microscale.In the research of the aqueous two-phase system prepare the composite gel microspheres.We use self-made micrometric needles and principle of instillation method to prepare micro-scale and composite calcium carbonate alginate microspheres.The composite microspheres can be systematically observed the surface morphology and calcium carbonate particle adhesion by scanning electron microscope and optical microscope.In addition,the encapsulation of macromolecule protein BSA in composite calcium alginate microspheres and calcium alginate microspheres is investigated.Through the measurement of fluorescence photos and UV absorbance,the BSA is found in the composite calcium alginate microspheres and it has a high encapsulation.Moreover,by adjusting the concentration of urease in the preparation system,the degree of calcium carbonate adhesion can be changed,and the release rate of the BSA in composite microspheres can be adjusted accordingly.This is mainly due to the fact that the Dex phase in the ATPS of PEG-Dex has a very high distribution ratio for macromolecular proteins.Combination of ATPS and calcium alginate can improve the encapsulation efficiency of BSA to more than 90%,and the calcium carbonate particles are attached on the microspheres and increased the mechanical strength.So that the release rate of the composite microspheres can be directionally controlled.In order to recycle the composite microspheres,the nano-magnetic Fe₃O₄ particles are added into the composite microspheres.The composite microspheres are found to have the good magnetic response and facilitated recovery and transfer.The research results successfully demonstrate that the prepared composite calcium carbonate-alginate microspheres have high encapsulation efficiency,adjustable release rate and recyclability.This method can further apply for the field of drug encapsulation carriers and enzyme immobilization.