Study On Design Of Integrated Microfluidic Chip For Separation And Purification Of Exosomes And Concentration Calibration Method

As a kind of nano-sized vesicles,exosomes are widely present in various body fluids of organisms.Studies have shown that the content and composition of exosomes are closely related to the development of various diseases.The premise of studying exosomes is the efficient extraction of high purity exosomes.Conventional exosomes separation methods include ultracentrifugation,sucrose density gradient centrifugation,ultrafiltration,polymer precipitation,and immunoaffinity.However,traditional exosomes separation methods have some drawbacks,such as expensive equipment,the need for professional operators,time-consuming separation and extraction,and low purity of exosomes.Based on the mechanism of separation of exosomes by immunomagnetic beads,a microfluidic chip capable of separating and purifying exosomes was designed.The microfluidic chip combines the advantages of high-purity extraction of exosomes from immunomagnetic beads,and utilizes the characteristics of automated continuous reaction of microfluidic chips to improve the extraction efficiency and purity of exosomes In view of the difficulty in accurately measuring the concentration of exosomes,this paper established a method for measuring the concentration of nanoparticles based on AFM.This article has conducted in-depth research on the following four aspects:Firstly,in the research on mechanism analysis and modeling simulation of microfluidic chip mixing reaction,the mechanism of capture exosomes by microfludic chip was analyzed.According to the advantages and disadvantages of exosomes separation by immunomagnetic beads,a combination of imununomagnetic beads and microfluidic chips was proposed to achieve integrated high-efficiency extraction of exosomes.The mixed reaction of exosomes and immunomagnetic beads in microfluidic chips is very important,so three microchannel structures(Y-shaped,S-shaped,embedded obstacle S-shaped)were established,and fluid simulation software Fluent was used to simulate the mixing efficiency of the three structural microchannels.The experimental results show that the embedded barrier s-shaped microchannels have the best mixing effect under the same conditions compared with the Y-shaped and S-shaped microchannel structures.Secondly,in the research on design and processing of integrated chip for separation and purification of exosomes,the microfluidic chip is designed according to the mixed reaction mechanism of the microfluidic chip and the embedded obstacle S-shaped microchannel structure is selected.Next,the processing technology of the chip was studied,including the selection of processing materials,the fabrication of photolithographic masks,the processing of microchannel molds,the processing of PDMS channels,and the bonding of PDMS channels to glass substrates.In addition,the magnetic beads capture experiment and the two-phase liquid mixing experiment were carried out on the fabricated chip.The experimental results were consistent with the simulation results,which proved the rationality of the design of the exosome separation and purification integrated microfluidic chip.Thirdly,in the research on establishment and experimental verification of nanoparticle concentration measurement method based on AFM,the concentration and morphology of exosomes need to be quantitatively calibrated after separation and purification.Therefore,the method of measuring the concentration of nanoparticles based on AFM is studied.First,standard commercial nanospheres were selected as research objects,and absolute ethanol was used as a dilute solution to prepare samples.Due to the rapid volatility of anhydrous ethanol,the sample preparation process is simple and the nanoparticles are less prone to agglomeration.The number of nanoparticles increases gradually along the radius of the circular pattern of the sample.According to this distribution law,the calculation model of nanoparticle concentration measurement is established.Based on this calculation model,the number of nanoparticles with different sizes and different dilution concentrations was calculated using Matlab.The consistency between the concentration measurement and the mass fraction estimation was higher than 75%.This study not only laid the foundation for exosome concentration measurement,but also provided a new way for quantitative calibration of nanoparticles.At last,in the research on exosomes isolation,purification and concentration calibration experiments,the operation platform of exosome separation and purification system based on microfluidic chip and the specific operation process of extracting exosomes using microfluidic chip are introduced.Next,the solution extracted from the microfluidic chip was identified by TEM and SEM,and it was confirmed that the vesicles in the extraction solution were exosomes which have a diameto of 30 nm to 150 nm.Compared with the traditional exosooe extraction method,the microfluidic chip designed in this paper is used to extract exosomes faster and with higher purity.The process of extracting 150 ?l of serum samples using a microfluidic chip to extract exosomes takes only 50 minutes.Finally,the concentration and morphology of the exosomes were successfully characterized according to the established nanoparticle concentration measurement method.