Extraction And Determination Of Exosomes Based On Gel Electrophoresis Microfluidic Chip And Biosensor

Background:The morbidity and mortality of cancer in the worldwide are increasing year by year,which has threatened human life and health seriously.Early diagnosis and treatment is one of the most effective ways to reduce the mortality of cancer.In recent years,the early molecular diagnosis technology based on"liquid biopsy"has gradually attracted increasing attention.Liquid biopsy is different from traditional surgical tissue biopsy and puncture biopsy,it mainly uses blood,urine,saliva,milk and other biological fluids to detect the biomarkers that fall off from primary tumor cells and enter the peripheral blood circulation.It focuses on the detection of biomarkers that including circulating tumor cells（CTCs）,free DNA,RNA,exosomes and so on.In recent years,extracellular vesicles,especially exosomes,have attracted more and more attention due to the important roles in tumor cell transport and intercellular communication.As one of the most prospective objects in liquid biopsy,exosomes are extracellular vesicles with phospholipid bilayer membrane-bound structure（30-200 nm）.Exosomes are secreted by most types of cells into the biological fluids,they are rich in content,with high specificity,uniform in size and contain abundant gene information associated with tumor.The contents of exosomes include many specific biomarkers,such as RNA,DNA,lipids and proteins.They regulate the function and phenotype of receptor cells,and play an extremely important role in regulating the occurrence and development of tumors.At present,a series of clinical researches have used exosomes as biomarkers to analyze the types and contents of exosomes for early diagnosis of cancer accurately and quantitatively.High-purity extraction and accurate quantitation of exosomes are the key steps of these studies.However,up to now,there is still lacks the standardized technical methods to isolate and quantitatively determinate exosomes.How to extract and determinate exosomes efficiently is a great challenge for the current liquid biopsy of exosomes and tumor.Objective:In order to solve the two technical problems of high-purity extraction and accurate quantitative analysis of exosomes,gel electrophoresis microfluidic chip and immunoassay-type biosensor were established respectively in this study.An gel electrophoresis microfluidic chip with excellent performance and portable size is used for the extraction and separation of exosomes in human plasma.Extraction of exosomes from human plasma samples efficiently and rapidly,it can make a high production of exosomes and low cost.It aims to solve the problems of exosome extraction,such as time-consuming,laborious and high cost,it also provide the possibility for the real-time diagnosis technology based on exosome.An immunoassay biosensor with high sensitivity and specificity is used for the quantitative determination of exosomes in various biological samples,especially the tumor-derived exosomes.The immunoassay biosensor is expected used for the ultrasensitive determination of tumor-related exosomes and other biomarkers in the early diagnosis of cancer,and provide a new prospect for the early diagnosis and clinical research of cancer.Methods:1.Collected peripheral blood from normal human,designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes.The extracted exosomes were characterized by transmission electron microscopy,nanosight and western blot,the morphology,concentration and particle size of exosomes were identified and analyzed.At the same time,use ultracentrifugation and microfluidic chip to isolate exosomes separately.The particle size and concentration of the exosomes extracted by two methods were compared and analyzed,and their respective extraction efficiency was discussed.Finally,the expression level of mi RNA-21 in exosomes was analyzed by RT-PCR,and discuss the feasibility of downstream analysis.2.Based on the capture of magnetic nanoparticles（MNPs）and the catalysis of horseradish peroxidase（HRP）,an immunoassay fluorescent biosensor was designed for the determination of tumor-related exosomes.MNPs were used as substrates to capture exosomes by modifying CD63 antibody on the surface of MNPs.Then the biotinylated Ep CAM antibody was used to capture the tumor associated exosomes that expressing Ep CAM specifically,and forming the sandwich structure of“antibody-exosome-antibody”.Then adding SA-HRP,SA could bind with biotin and thus connected HRP to the sandwich structure of"antibody-exosome-antibody".Finally,adding tyramine as substrate and H₂O₂ as catalyzer,in the presence of H₂O₂,SA-HRP can catalyze tyramine to form a dimer and generate fluorescent signals.The content of tumor-associated exosomes can be quantified specifically by the measurement of the final fluorescence intensity.Results:1.The microfluidic chip can isolate（in 1 hour）high-purity exosomes with size ranging from 30-200 nm directly from human plasma,which allows downstream exosomal mi RNA analysis.2.By comparing with ultracentrifugation,the isolation yield of microfluidic chip is3.80 times higher than ultracentrifugation when the volume of plasma sample less than100μL.The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip:voltage:100 V;concentration of agarose gel:1.0%;flow rate of injection pump:0.1 m L/h.3.Immunoassay-type biosensor can be used for the determination of tumor-related exosomes.There is a linear relationship of this sensor between the fluorescence intensity and the content of tumor-related exosomes,with a detection limit as low as200（±9）particles m L^-1 and the analytical range of this method is from 576（±15） particles m L^-1 to 5.76×10⁷（±5.1×10⁵）particles m L^-1.4.For the fluorescence measurement,the excitation wavelength was set to 320 nm.Fluorescent spectra were collected at emission wavelength in the range 370 to 550 nm.Immunoassay-type biosensor is stable within 24 h,this sensor can also be used to determinate exosomes in plasma of patients with HCC.Conclusions:1.The gel electrophoresis microfluidic chips can isolate the exosomes in human plasma rapidly and efficiently.2.Immunoassay-type biosensor can be used to determinate tumor-related exosomes with high sensitivity and specificity.