1、1903916(1 of 18)2019 WILEY-VCH Verlag GmbH&Co.KGaA,Weinheimwww.small-REVIEWProgress in Microfluidics-Based Exosome Separation and Detection Technologies for Diagnostic ApplicationsShujing Lin,Zixian Yu,Di Chen,*Zhigang Wang,Jianmin Miao,Qichao Li,Daoyuan Zhang,Jie Song,and Daxiang CuiDr.S.J.Lin,Z.X.
2、Yu,Prof.D.Chen,Prof.J.M.Miao,Q.C.Li,D.Y.Zhang,Prof.J.Song,Prof.D.X.CuiSchool of Electronic Information and Electrical EngineeringShanghai Engineering Research Center for Intelligent Diagnosis and Treatment InstrumentKey Laboratory for Thin Film and Microfabrication Technology of Ministry of Educatio
3、nShanghai Jiao Tong UniversityShanghai 200240,ChinaE-mail:Prof.Z.G.WangDepartment of General SurgeryShanghai Jiao Tong University Affiliated Sixth Peoples HospitalShanghai 200233,ChinaThe ORCID identification number(s)for the author(s)of this article can be found under https:/doi.org/10.1002/smll.20
4、1903916.DOI:10.1002/smll.201903916various contents,such as lipids,mem-brane proteins,cytosolic proteins,and RNA,between cells.25 Exosomes contain signaling proteins,RNA and lipids within a type of vesicle.6,7 Although exosomes were initially thought of as cellular debris,they are now increasingly re
5、cognized as important mediators that regulate biolog-ical behaviors of cells.8 In recent years,exosomes have been considered prom-ising biomarkers for diagnosis of various diseases and treatment monitoring in diabetic cardiomyopathy,rheumatoid arthritis,skin diseases,cancer,urinary tract infection,a
6、sthma pathology,and even orthopedic conditions.918 Moreover,study of the bioactivity of exosomes in cell cultures or in vivo has become popular in an effort to understand their physiological functions.1923 Recent studies have shown that exosomes can be found in various body fluids such as blood,seme
7、n,plasma,saliva,and urine.2428 Thus,separation of exosomes from body fluids is indispensable to the exosome research field.Although much progress has been achieved in the field of exosome separation,there are still significant chal-lenges due to the complex nature of exosome.29To data,several conven
8、tional techniques have been employed to separate exosomes,including ultracentrifugation-based sepa-ration,immunological separation,ultrafiltration separation,size-exclusion chromatography,and polymer-based precipita-tion separation.3035 Among these techniques,the ultracen-trifugation-based separatio
9、n technique is the most commonly used technique for separating exosomes.36 Currently,several commercial kits for exosome separation have been developed to avoid time-consuming steps.37 Simultaneously,detection techniques for analyzing and quantifying exosomes have been developed for both research an
10、d clinical applications.These widely used detection techniques include scanning electron microscopy(SEM),transmission electron microscopy(TEM),atomic force microscopy(AFM),enzyme-linked immuno-sorbent assay(ELISA),dynamic light scattering(DLS),and nanoparticle tracking analysis(NTA).3842 Despite the
11、 great advances in exosome detection techniques,further develop-ment is needed to meet the requirements of rapid,sensitive,and low-cost detections.Microfluidics has been rapidly developed over the past dec-ades and has enormous potential for application in optics,Exosomes are secreted by most cell t
12、ypes and circulate in body fluids.Recent studies have revealed that exosomes play a significant role in intercellular communication and are closely associated with the pathogenesis of dis-ease.Therefore,exosomes are considered promising biomarkers for disease diagnosis.However,exosomes are always mi
13、xed with other components of body fluids.Consequently,separation methods for exosomes that allow high-purity and high-throughput separation with a high recovery rate and detec-tion techniques for exosomes that are rapid,highly sensitive,highly specific,and have a low detection limit are indispensabl
14、e for diagnostic applications.For decades,many exosome separation and detection techniques have been developed to achieve the aforementioned goals.However,in most cases,these two techniques are performed separately,which increases operation complexity,time consumption,and cost.The emergence of micro
15、fluidics offers a promising way to integrate exosome separation and detection func-tions into a single chip.Herein,an overview of conventional and microflu-idics-based techniques for exosome separation and detection is presented.Moreover,the advantages and drawbacks of these techniques are compared.
16、1.IntroductionIntercellular communication is a fundamental behavior in mul-ticellular organisms.The modalities of intercellular communi-cations are realized through direct interaction with neighboring cells or transfer of chemical substances secreted by specific cells.1 Multiple types of vesicles se
17、creted by cells play a key role in intercellular communication by serving as carriers to transfer Small 2019,19039161903916(2 of 18) 2019 WILEY-VCH Verlag GmbH&Co.KGaA,Weinheimwww.small-information technology,chemical synthesis and biological analysis.43 As a technology,microfluidics offers many adv
18、antages,such as very small consumption of samples and reagents,high resolution and sensitivity,low cost,and significant reduction in assay time.44,45 Because of these advantages,microfluidics has been successfully applied in the biomedical field,for example,in particle separation,cell separation,DNA
19、/RNA manipulation,polymerase chain reac-tion(PCR),detection of circulating tumor cells(CTCs)and droplet generation.4655 In recent years,increasing attention has been paid to the development of new exosome separation and detection techniques based on microfluidics.5662 These new techniques have exhib
20、ited high potential application prospects because of their simple,fast,and time-saving pro-cedures,as well as the minimal consumption of samples and reagents.Currently,although several reviews have reported and discussed the advances in exosomes,most have focused on specific aspects of the exosomes
21、field,such as conventional exosome separation or enrichment techniques,the biological role of exosomes,and exosome detection techniques.6366To better understand the significance of exosomes in diag-nostic application fields,we will discuss the biogenesis and contents of exosomes.Progresses in exosom
22、e separation and detection techniques will also be reviewed based on certain rep-resentative exosome studies.In particular,microfluidics-based exosome separation and detection technologies will be inten-sively discussed alongside their limitations and challenges.It is highly expected that by taking
23、advantage of microfluidics a deeper understanding of exosomes for diagnostics can be achieved.In the coming decades,studies on microfluidics-based exosome separation and detection techniques will likely become common.2.MicrofluidicsMicrofluidics allows manipulation of small fluid volumes using micro
24、channels with dimensions of tens to hundreds of microns.43 In the early 1990s,Manz et al.first verified that elec-trophoretic separations in a micro total analysis system(-TAS)is more efficient than conventional electrophoresis.67 Since then,microfluidics has been considered as a promising tech-niqu
25、e to miniaturize the bulk of conventional equipment.68,69 For instance,Quake and co-workers reported a large-scale inte-grated microfluidic chip in 2002.70 This large-scale integrated microfluidic chip enabled analysis of the expression of a par-ticular enzyme.Microfluidic chips now are employed in
26、the fields of chemistry,physics,biology,medicine,agriculture,food,and environmental monitoring.7175 Among them,applica-tions in biomedical research and related fields are the most widely and thoroughly studied.Because of the merits of microfluidics,for example,high-throughput capacity and low-time r
27、equirements,microfluidics-based devices are able to handle and analyze more/multiple samples in less time than conventional methods.76,77 Currently,microfluidic chips are widely applied in the separation and detection of micro/nanoparticles,biomarkers,cells,and proteins.7884 A study conducted by Lin
28、 et al.utilized a flyover microfluidic chip to separate white blood cells(WBCs)from mouse peripheral blood through a two-stage magnetic separation methods(Figure 1A).85 A local magnetically enhanced nickel microarray in the first separation stage and vertical cell separation in the second stage ensu
29、re high-purity WBC separation.The separation purity of WBCs was 93%at a flow rate of 20 L min1.Exosomes are now considered an important biological sub-stance with sizes in the range of 30150 nm.Thus,even in microfluidic chips,it is much more difficult to separate and detect exosomes than cells.Some
30、microfluidics-based exosome separation and detection techniques have been reported.How-ever,the separation efficiency and detection ability of these microfluidics-based techniques needs to be improved.For instance,Wang et al.reported a 3D nanostructured microfluidic Shujing Lin received his Ph.D.fro
31、m Shanghai Jiao Tong University,China in 2019.He is currently a post-doctoral researcher in Shanghai Jiao Tong University.His research fields are bio-microfluidic,soft/stretchable electronics,and radio-frequency micro-electromechanical systems(RF MEMS).Zixian Yu is currently a master student in Shan
32、ghai Jiao Tong University,China.He received his Bachelors degree in Shanghai University,China in 2016.His research fields are microelectrome-chanical systems(MEMS)and microfluidics.Di Chen received his diploma and doctoral degree from the University of Hamburg,Germany in 1986 and 1990 respectively.F
33、rom 1990 to 1992 he was post-doctoral researcher in Micro Parts Company,Germany.Since 1993 he has been associate professor,professor,and vice director of Research Institute of Micro/Nano Science and Technology at Shanghai Jiao Tong University.His research fields are implantable microsystems,micro-fl
34、uidics,and RF MEMS.Small 2019,19039161903916(3 of 18) 2019 WILEY-VCH Verlag GmbH&Co.KGaA,Weinheimwww.small-chip to purify chemically edited exosomes for drug delivery(Figure 1B).86 In the exosome separation microfluidic chip,functionalized,multiwalled carbon nanotubes(MWCNTs)with anti-CD63 antibody
35、were immobilized onto PDMS pillars to capture exosomes.The exosome capture efficiency was 90%at a flow rate of 1.0 mL h1.3.ExosomesExtracellular vesicles(EVs)were first found in 1946 by Chargaff and West87 EVs are secreted into the extracellular microenvi-ronment by most cell types88 and can be furt
36、her sorted into three subclasses based on their biogenesis:i)apoptotic bodies(15 m),ii)microvesicles(150 nm to 1 m),and iii)exosomes(30150 nm in diameter).893.1.Biogenesis of ExosomesThe term“exosome”was coined to describe a cluster of nano-vesicles that are derived from the endolysosome and release
37、d into the extracellular microenvironment.90,91 The forma-tion of exosomes involves three representative stages:i)early endosomes,ii)late endosomes,and iii)late multivesicular bodies(MVBs)(Figure 2A).63,92,93 The biogenesis of exosomes begins with early endosomes.When early endosomes mature into lat
38、e endosomes,also known as MVBs,the location(from the outer cytoplasm to the nuclear periphery)and shape(from tube-like to spherical)are changed.There are two potential growth directions for MVBs:fuse with lysosomes or with the plasma membrane.If an MVB fuses with a lysosome,it will be hydrolyzed by
39、the lysosome.In following the other pathway,the MVB fuses with the plasma membrane and then releases Small 2019,1903916Figure 1.A)Optical micrograph of the flyover microfluidic chip.This microfluidic chip includes a lateral separation zone and a vertical separation zone.By introducing gravity,the ce
40、ll separation purity can reach up to 93%at a flow rate of 20 L min1.Reproduced with permission.85 Copyright 2019,Elsevier.B)Schematic describing the process of plating MWCNTs onto 3-amino-propyl-triethoxysilane(APTES)functionalized polydimethylsi-loxane(PDMS)pillars and of functionalizing the multiw
41、alled carbon nanotubes(MWCNTs)with anti-CD63 antibody for immune-capture of exosomes.Reproduced with permission.86 Copyright 2017,American Chemical Society.1903916(4 of 18) 2019 WILEY-VCH Verlag GmbH&Co.KGaA,Weinheimwww.small-intraluminal vesicles(ILVs)into the extracellular space.The released vesic
42、les are considered exosomes.Some reports have shown that the small Rab guanosine triphosphatases(GTPases)play a key role in the exosome release process.These GTPases include RAB27A,RAB27B,RAB11,and RAB31.3.2.Contents and Functions of ExosomesExosomes are considered to be somewhat unique in their pro
43、tein and other biomolecule composition,which pro-vides additional characteristics for their identification.Cur-rently,it is mostly agreed that exosomes contain proteins,microRNA(miRNA),noncoding RNA,mRNA,DNA,and lipids(Figure 2B).94 In particular,the proteins they contain are derived from MVBs,the p
44、lasma membrane,the cytosol,and the nucleus,as well as some from mitochondria and the Golgi apparatus.Currently,two databases(ExoCarta and Vesiclepedia database)for recording the contents of exosomes are available.However,the data in the databases are submitted by users and lack verification.Some rev
45、iews have elaborated on the proteins that exosome contain.9597 In short,the proteins in exosomes can be divided into common proteins(e.g.,CD9,CD63,CD81,and CD82)and specific proteins(e.g.,EpCAM).Consequently,exosomes can be traced to their source cells by detecting their specific proteins.Since miRN
46、A and mRNA were identified in exosomes,98 other nucleic acids,including noncoding RNAs,transfer RNAs(tRNAs),and DNA have gradually been found in exosomes.98100 The detection of nucleic acids showed that exosomes play a role in intracellular transfer of genetic information.64 Recently,many studies ha
47、ve demonstrated that in addition to proteins and nucleic acids,exosomes also carry lipids.101,102 The lipids in exosomes include cholesterol,sphingomyelin,cholesterol,phosphatidylserine,and saturated fatty acids and have also been listed in the abovementioned databases.Currently,exosomes have been f
48、ound in various body fluids,such as blood,semen,saliva,and urine.The role of exosomes in intercellular communication has been revealed.4,88,103,104 Tumor-secreted exosomes can transfer oncogenetic cargoes from their parent cells and modulate gene expression in recip-ient cells.105,106 Therefore,thes
49、e tumor-secreted exosomes play a significant role in the progression and metastasis of tumors.107 Recently,exosomes have emerged as potential bio-markers for early detection,diagnosis,and treatment moni-toring of cancer and other diseases.107109 In addition,urinary exosomes can be used as biomarkers
50、 for diagnosis of urinary tract infection.16 Moreover,Zakeri et al.reported that exosomes are significant messengers in rheumatoid arthritis.10 The diag-nostic functions of exosomes in the fields of pregnancy,infec-tions,tumor,stem cells,heart,and brain have already been summarized in previous revie