Study On Enzymatic Catalysis Enhanced By Microfluidic Droplets Flow With Aqueous Two-phase Systems And Application In Urea Detection

The enzyme reaction is the core and key of the biochemical process.However,the enzymatic catalysis process is limited by mass transfer and product inhibition usually.The purpose of this study is to design a“selective permeability”microfluidic ATPS（Aqueous two phase system）droplet flow interface to eliminate product inhibition,break through the limit of reaction equilibrium,and explore the motion state of ATPS droplets flow at the microscale.The mechanism of the enhanced enzymatic catalysis by ATPS droplets flow is revealed.On this basis,a microfluidic ATPS droplets flow enzymatic reaction method based on rapid detection of urea is developed.The research results provide theoretical support and application guidance for the enhancement of enzymatic catalysis process in the fields of pharmaceuticals,food,sensors,analysis and detection.In the part of the construction of the microfluidic ATPS droplets flow,ATPS based on Dex and PEG,two high-molecular polymers.The microfluidic device which is self-made with loop constructed with Dex/PEG droplets of air pump/syringe pump and PEG/Dex droplets of two syringe pumps.The effect of different pressures on the diameter of Dex/PEG droplets is explored.The experimental results show that under certain conditions of PEG phase flow,the diameter of Dex/PEG droplets increases as the pressure of Dex phase increases.At the same time,the effect of different flow rates on the diameter of PEG/Dex droplets is also explored.That is,under certain conditions of Dex phase flow,the diameter of PEG/Dex droplets increases with the increase of PEG phase flow;under certain conditions of PEG phase flow,the diameter of PEG/Dex droplets decreases with the increase of Dex phase flow.By controlling the pressure and the two-phase flow rates,droplets of different diameters can be realized to meet various needs.Due to the distribution characteristics of ATPS,enzyme is enriched in Dex phase,and the distribution behavior of small molecular substances in the two phases is relatively close.In addition,the diffusion phenomenon of the molecules at the interface of Dex/PEG droplets and PEG/Dex droplets is also explored.This selective distribution provides the possibility of achieving process intensification in the enzymatic catalysis of microfluidic ATPS droplets flow.In the part of microfluidic ATPS droplets flow intensification enzymatic catalysis reaction,the author builds Dex/PEG droplets flow,using the urease catalytic reaction as a model,adding urease to Dex,and adding urea to PEG.When the reaction is carried out for 3 min,the reaction rate could still be maintained at 1μmol·m L^-1·min^-1,and the conversion reaches up to 40%.Compared with traditional beaker ATPS（conversion<1%）,the conversion has significantly been improved by the microfluidic ATPS droplets flow.Moreover,its reaction rate and conversion are about 3 times that of the water/oil droplets flow.This is because the droplet flow has a great specific surface area,internal circulation and a faster surface renewal rate.Moreover the selective distribution performance of ATPS droplets eliminates the interface product inhibition in time,breaking the limit of the reaction equilibrium.Therefore,it has advantages in reaction rate and conversion.We believe that the study can provide a new way to intensify the process of enzymatic catalysis at a micro-scale.In the part of microfluidic ATPS droplets detect urea,on the basis of constructing PEG/Dex droplets flow,author adds urea and neutral red to PEG,and adds urease to Dex.Utilizing urease to hydrolyze urea to produce ammonium carbonate to discolor the neutral red indicator in droplets,and analyze the color intensity of droplets to determine the concentration of urea in the sample.The detection range can reach 0～50 mg·m L^-1.ATPS overcomes the shortcomings of low biocompatibility of traditional oil-water analysis and detection platforms.The droplets flow reduces the consumption of reagents,the large specific surface area,and the mass transfer distance in the micrometer range,which greatly improves the reaction rate and shortens the detection time to about 20 s.In this study,a simple and fast microfluidic enzymatic catalysis reaction method is used to realize the visual detection of urea,in order to provide a reference for the rapid analysis and detection of urea in the field of applied chemistry.