Establishment And Application Analysis Of A Multi-channel Automated Isolationtion Method For Extracellular Vesicles

Extracellular vesicles are membranous structures released by a cells and exist in a variety of body fluids.It can help to transfer of proteins,nucleic acids and other substances between cels,regulate physiological or pathological related cell behavior,and plays an important role in the occurrence,development,diagnosis and treatment of cancer.Therefore,it is highly desirable to quickly isolate extracel ular vesicles for downstream molecular analysis.However,most of the existing extracel ular vesicle isolation methods,such as ultracentrifugation,immunoseparation and polymer precipitation are usually time-consuming and irreproducible.To address those,we have developped an automatically muti-channel isolation method for extracel ular vesicles.Based on EVRTAP(Extracellular Vesicles Total Recovery and Purification),a new material for Extracel ular Vesicles enrichment and prepared by our research group,the automatic mechanical processing platform was used in this paper.A multi-channel,automated and efficient isolation method for extracellular vesicles from urine samples was developed.and then optimized and analyzed.Optimization conditions include:enrichment material amount,reaction solution amount,reaction time,washing speed and elution speed;The analytical methods included Western blot,Nanoparticle Tracking Analysis(NTA),transmission electron microscopy(TEM),and LC-MS/MS characterization.The characterization results showed that compared with the over-speed centrifugal method and manual extraction of extracellular vesicles,the extracellular vesicles extracted by automatic extraction method were complete in morphology,and more stable and high-throughput extracellular vesicles extraction could be achieved.Further,the optimized automated isolation method was used to isolate urine samples from6 groups,including the disease group and the healthy control group.The results of mass spectrometry showed that the total protein and phosphorylated protein extracted from extracel ular vesicles of the disease group and the control group reached a good level,indicating the high efficiency of automatic isolation.Proteomic analysis of extracel ular vesicles subsequently revealed that 155 differential total proteins and 81 differential phosphorylated proteins were upregulated in the disease group,and several potential biomarkers related to prostate cancer were found,demonstrating the significance of automated isolation for clinical research.In order to have a more comprehensive analysis of extracellular vesicles,we explored methods for extracting nucleic acid from extracel ular vesicles.At present,the extraction method of extracellular vesicle nucleic acid mostly uses the Trizol method.This method is easy to use toxic solvents and cannot be automated.Therefore,we make full use of the advantages of magnetic nanoparticles that are easy to separate,high surface area,and easy to automate.Magnetically separated extracel ular vesicle nucleic acid extraction technology.First,we performed the synthesis and characterization of Fe₃O₄ and Fe₃O₄@Si O₂ magnetic nanoparticles.Furthermore,we applied the synthesized Fe₃O₄@Si O₂ magnetic nanoparticles to extracting nucleic acid from cels and extracellular vesicles,and the results of PCR and RT-PCR proved the reliability of this method.The method of extracting nucleic acid from extracel ular vesicle based on magnetic separation is expected to realize the automatic extraction of nucleic acid in the future.In summary,in this paper,an automatic isolation method of extracel ular vesicles was established by mechanized treatment platform,which was further applied in clinical practice,at the same time,it explored the extraction of extracellular vesicle nucleic acid.Through a series of characterization,the automatic isolation has the advantages of high enrichment efficiency,good stability,time saving,and so on.And the clinical prostate cancer urine sample extraction experiment proved its practicability.Finally,Fe₃O₄@Si O₂ magnetic nanoparticles were synthesized for the preliminary extraction of nucleic acid from extracel ular vesicles,and a method of nucleic acid extraction based on magnetic particles was explored.In the future,it is expected that the automatic isoaltion of extracellular vesicles will be combined with nucleic acid extraction,multi-parameter extraction and multi-omics analysis of the same sample,and comprehensive and accurate analysis of the molecular characteristics of disease patients'samples.