外泌體生物分析及其臨床應(yīng)用研究進展

張怡萌，張慧欣，2，劉 洋

（1.清華大學(xué)化學(xué)系,微量分析測試方法與儀器研制北京市重點實驗室,生物有機磷化學(xué)及化學(xué)生物學(xué)教育部重點實驗室,北京100084；2.青島大學(xué)化學(xué)化工學(xué)院,山東省中日碳納米材料合作研究中心,青島266071）

1 Introduction

Exosomes belong to a category of extracellular vesicles（EVs）with a size range ofca.30 to 200 nm in diameter［1—4］［Fig.1（A）］.They are membrane-bound phospholipid vesicles with cup-shaped structure derived by all eukaryotic cells including tumors，neutrophils，and dendritic cells［5，6］.Exosomes are generated through invagination of the plasma membrane，leading to the formation of intracellular multivesicular bodies（MVBs）.MVBs would fuse with cellar plasma membrane and proceeded exocytosis to release exosomes to the extracellular microenvironment［7—9］.Consistent with the biogenesis，abundant molecular information of exosomes reflects closely the parent cells［10—12］.They are enriched in proteins and genetic materials including lipids，membrane proteins，nuclear proteins，nucleic acids namely miRNAs，mRNAs，DNA and other noncoding RNAs，which offers the important information related to various physiological and pathological processes［2］［Fig.1（B）］.It was that exosomes participate in the intercellular communication，mammalian reproduction and development，immune responses and infection［13—16］.Meanwhile，they play the key role in the development of the diseases including the metabolic and cardiovascular diseases，neurodegeneration and cancer［17—19］.Exosomes exist in all biological fluids，such as blood，saliva，sweat，urine and breast milk，which render them as ideal biomarkers to follow disease diagnosis，treatment and prevention［20—23］.

Fig.1 TEM image of exosomes derived by HeLa cells[4](A)and exosomal structure[2](B)

At present，there is increasing evidence that exosomes may play a positive role in diagnosing or solving specific diseases［24，25］.Especially，they have been shown to have a special and important role in cellular signal transduction，intercellular communication and related biological processes.Therefore，the analysis of exosomes has become an attractive research field.At the same time，recent reviews involve multiple aspects，including the exosomes isolation techniques，analysis of exosomal contents and the application as therapeutics.The biology，function and biomedical applications of exosomes suggesting that they have a role in regulating intercellular communication［26，27］.Unfortunately，the small size and low buoyant density of exosomes pose significant challenges of detection in the complex biofluids.To improve the sensitivity，speed and efficiency of quantitative detection of exosomes，many researchers attempted to use fluorescence，colorimetric and electrochemical analysis methods to construct the sensitive biosensor with novel signal amplification strategies for detecting exosomes［28］.These exosome analysis platforms based on novel signal transduction pathways and signal amplification strategies hold promise for advanced diagnostic tools.This review summarizes the recent advances in quantitative detection methods of exosomes including fluorescence，electrochemistry，electrogenerated chemiluminescence，surface enhanced Raman spectra，colorimetry and microfluidic analysis and so on.In addition，the clinic diagnostic and diseases therapies based on exosomes were also described.Finally，the perspective on exosome analysis as well as the clinic diagnostics and therapies were also proposed.

2 Quantitative Analysis of Exosomes

In these years，numerous researches have shown that exosomes play an important role in immune response，cell migration，cell differentiation，cardiovascular diseases，central nervous system-related diseases，and cancer progression.Exosome-mediated responses are related with disease promoting or restraining and they are distributed in all biological fluids.Exosome-based liquid biopsy highlights their potential aid in diagnosis and therapy.As a result，the high sensitivity detection technology of exosomes is crucial for their applications in clinical diagnosis，biomedical and biochemical research.Numerous biosensors have been established for the detection of exosomes.The optical，electrochemical and mechanical technologies have been applied in the signal transduction and biosensors construction，including nanoparticle tracking analysis（NTA），electrochemistry，fluorescence，colorimetric assays，surface-enhanced Raman scattering（SERS），surface plasmon resonance（SPR）and microfluidics.For the design of the biosensors，newly identified biomarkers，signal transduction pathway and signal amplification strategies have been developed rapidly to improve the sufficient selectivity，diversity of analysis，high sensitivity and rapid response time.

2.1 Nanoparticle Tracking Analysis

NTA is a method for measuring the concentration and size distribution of exosomes based on Brownian motion［29，30］.The principle of NTA is that a light beam is used to illuminate the exosomes particles in the sample and then perform Brownian motion.The Brownian motion of the exosomes particles is tracked by using Nanosight software with the Stokes-Einstein equation to calculate the hydrodynamic diameter of exosomes particles based on their average velocity and diffusivity.The NTA method shows great potential to become a standard for measuring the size and concentration of exosomes in clinical samples［31，32］.Unlike dynamic light scattering（DLS）technique which measures the bulk scattered light from exosomes when they are illuminated by a monochromatic light source，NTA can enable sample visualization and provides the information about exosomes particles concentrations and size distribution（Fig.2）［33］.These also put higher demands on operators to obtain the accurate result with high reproducibility.

Fig.2 Nanoparticle tracking analysis[33]

2.2 Electrochemical Analysis

Electrochemical biosensors can convert the identification of biomolecules into electrical signals，including current，potential，and impedance with rapid response，simplicity and low cost［34，35］.Electrochemical biosensors have been used in the detection of exosomes in recent years for the high sensitivity and selectivity，low cost and facility［36，37］.In order to promote the performances，nanomaterials-based biosensor with superior performances have been continuously reported due to the excellent properties of nanomateirals，such as excellent conductivity，large surface area，good biocompatibility，and unique optical-electrochemical features and so on［38，39］.

Exosomes contain a variety of proteins，which not only reflect the origin of exosomes，but also affect homeostasis and disease development.As the research progresses，more and more cancer-related proteins are found in exosomes，including transmembrane protein，epithelial cell adhesion molecule（EpCAM），prostate specific membrane antigen（PSMA）and so on［40—44］.Therefore，exosomes proteins analysis and quantification are essential for the diagnosis of cancer.Numerous of strategies were designed to be applied in electrochemical biosensors for exosomes determination based on the antibody and aptamer recognition toward the proteins expressed on exosomes surface.

The electrochemical immunosensors are the most traditional tools for a wide range of biomarkers detection based on the high affinity antibody-antigen reaction.CD63，a member of the transmembrane family protein，is considered a classic marker for exosomes.The Revzin’s group［45］has developed an aptamer-based electrochemical biosensor with specificity to CD63 for quantitative detection of exosomes.In the presence of exosomes，the CD63 aptamer can specifically recognize the CD63 protein on the surface of exosomes，the DNA probe with methylene blue（MB）were released，leading to a reduced electrochemical signal［Fig.3（A）］.This method was much simpler than standard immunoassay and was much more sensitive with 100 times lower detection limit compared to commercial CD63 antibody-based immunoassay.CD9 belonging to the transmembrane proteins is also a well-known exosomal marker.Doldanet al.［46］described a sandwich type electrochemical biosensor for detection of exosomes based on theα-CD9 antibodies and a horseradish peroxidase（HRP）-conjugatedα-mouse IgG antibody，and the electrochemical signal of HRP-oxidized 3，3，5，5-tetramethylbenzidine（TMB） was monitored.This sandwich type electrochemical biosensor can discriminate between exosomes and other EVs，and show a limit of detection of 200 particles/mL with a dynamic range spanning almost 4 orders of magnitude［Fig.3（B）］.Additionally，Shiddiky’s group［47］reported an electrochemical detection method to directly quantify the exosomes in cell culture media with a two-step design［Fig.3（C）］.Where exosomes were capturedviaCD9 antibody，and then using human epidermal growth factor receptor 2（HER-2）antibody to identify the exosomes，achieving quantification of HER-2-postive breast-cancer-derived exosomes.The detection limit was 4.7×105exosomes/μL.In addition，by using the electrochemical probes with different antibody labelings，the multichannel electrochemical detection can be realized to exosomes derived from various cell lines as well as their multiple surface proteins.For example，Kelley’s group［48］used bare gold electrodes to fabricate a biosensor chip containing 11 individual circular gold electrodes as substrates and reported a multiplexed electrochemical sensor for the detection and characterization of exosomes microsomes by direct electro-oxidation of metal nanoparticles（MNPs）labeled with antibodies which can specifically recognize the proteins expressed on exosomes and microsomes［Fig.3（D）］.The electrochemical assay exhibited a limit of detection（LOD）of 50 exosomes.In addition，the expression of epithelial cell adhesion molecule（EpCAM，a ubiquitously expressed epithelial cancer marker）and PSMA can be analyzed simultaneously by labeling the metallic naonoparticles such as silver nanoparticles（AgNPs）and copper nanoparticles（CuNPs）with EpCAM and PSMA recognized antibodies，respectively.Based on the electrochemical biosensor，serumderived exosomes and microsomes from prostatic carcinoma（PCa）clinical samples can be analyzed.In addition，it was observed that the expression levels of both EpCAM and PSMA on the vesicles from PCa patients were higher than those healthy controls，which shows a great potential in the accurate analysis of exosomes from clinic sample with high sensitivity.

Fig.3 Scheme of an Au electrode array patterned on a glass surface for detection of exosomes[45](A),scheme of the electrochemical sensor using CD9 antibody for detection of exosomes[46](B),scheme of the electrochemical sensor using CD9 antibody for detection of exosomes in cell culture media[47](C),scheme of the multiplexed electrochemical sensor for detection of exosomes[48](D),scheme of the elec?trochemical sensor for detection of exosomes based on the gold electrodes[36](E),scheme of the iMEX for detection of exosomes[41](F),ECL biosensor for detection of HeLa cell derived exosomes[4](G),ECL biosensor for detection of OVCAR cell derived exosomes[53](H)

Besides the immunosensor for exosomes detection，aptamers-based biosensor attracted considerable attention recently owing to their ability to bind target proteins with high affinity and specificity.In addition，compared with the antibodies，aptamer also has some advantages including easier storage，simpler synthesis，excellent reproducibility and so on.As a result，various strategies based on the aptamer configuration changes and the aptamer modification nanomaterials were designed to fabricate the electrochemical biosensor with high sensitivity and selectivity.Tan’s group［36］designed a nano tetrahedron（NTH）-assisted aptasensor for the direct capture and detection of hepatocellular exosomes［Fig.3（E）］.Compared with single-chain aptamerfunctionalized aptamer biosensor，NTH-assisted aptamer biosensor can detect exosomes with high sensitivity（100 times），resulting in the electrochemical determination of exosomes derived from HepG2 with a LOD of 2.09×104particles/mL and a wide linear range of 105—1012particles/mL.In addition，Jeong and colleagues［41］reported a novel magnetic-electrochemical exosome（iMEX）approach for exosomes detection by the magnetic enrichment，antibody recognition，and enzymatic signal amplification［Fig.3（F）］.This method can enable highly sensitive and high-throughput measurements of exosome detection by combining the magnetic enrichment and enzymatic amplification.

Electrogenerated chemiluminescence（ECL）is a technique that combines the advantages of the electrochemistry and luminescence［49，50］.Compared to other electrochemical methods，the ECL assay has advantages in a wide range of concentration response and high sensitivity［51—53］.What is more，it is a potential and spatial controlled technique.These features make it great potential in the design of biosensor with high sensitivity and selectivity.Liuet al.［4］presented an ECL biosensor for detection of HeLa cell derived exosomes by combining the graphitic carbon nitride nanosheets conjugated polydopamine coated Galinstan liquid metal shell-core nanohybrids（g-C3N4@Galinstan-PDA）nanoprobes and multivalent PAMAM-AuNPs electrode interface.The antibody modified multivalent PAMAM-AuNP interface can improve the capture efficiency of exosomes on the electrode，and the antibody modified g-C3N4@Galinstan-PDA nanoprobe was adsorbed on the electrode based on the antibody on the nanoprobes and the proteins expressed on exosomes surface.In this way，stable and strong ECL signals were obtained resulted from the excellent features of the Galinstan NPs in facilitating electron transfer and suppressing the g-C3N4passivation［Fig.3（G）］.The ECL biosensor exhibited high specificity and sensitivity toward HeLa cell derived exosomes with the detection limit of 31 particles/μL.In addition，an ultrasensitive ECL biosensor for the detection of exosomes and their surface proteins were also designed by Zhanget al.［53］by thein situformation of gold nanoparticles（AuNPs）decorated Ti3C2MXenes hybrid with aptamer modification（AuNPs-MXenes-Apt）［Fig.3（H）］.The in situ formed AuNPs-MXenes-Apt hybrid not only presented highly efficient recognition of exosomes specifically，but also provide naked catalytic surface with high electrocatalytic activity of gold nanoparticles with predominated（111）facets that significantly improved the ECL signal of luminol.The LOD was calculated to be 30 particles/μL.

2.3 Fluorescence Analysis

Fluorescence biosensor is usually a method of detecting the target using the fluorescence change emitted by the interaction between the fluorescent molecule and the target［54—56］.Because of its low cost，no damage，multiplex detection capability，ease of automation and other advantages，it has become a very competitive analysis method［57，58］.

The fluorescence biosensor developed for the detection of exosomes shows great advantages due to the above characteristics.The performances of the fluorescence biosensors are generally depended on the features of the fluorophores such as dyes，fluorescent proteins or fluorescent nanoparticles［59，60］.Among them，fluorescence resonance energy transfer（FRET）is a very popular strategy in the fluorescence biosensor for biomolecular analysis based on the distance-dependent fluorescence quenching coupling with the recognition of biomolecules labeled with fluorophore-quencher pair respectively.The energy of an excited donor molecule is transferred to the acceptor by resonance coupling of the donor dipole and the acceptor dipole.Two dimensional（2D）nanomaterials have unique nanobiointerface units for the biosensor construction and exhibit intrinsic fluorescence quenching ability，which makes them become the attractive candidate applied in the exosomal biosensor［61，62］.For instance，Liuet al.［63］reported a fluorescent biosensor using unique Cy3 labeled CD63 aptamer（Cy3-CD63 aptamer）/Ti3C2MXenes nanocomplex as a nanoprobe to detect exosomes.The Cy3-CD63 aptamer can be selectively adsorbed onto the Ti3C2MXenes nanosheets and then the fluorescence was quenched quickly.The addition of exosomes turned on the recovery of Cy3 fluorescence due to the strong specific recognition between proteins on exosomes and CD63 aptamers，which resulted in the release of CD63 aptamers from the Ti3C2MXenes the surface.This fluorescence biosensor showed a high sensitivity and a LOD of 1.4×103particles/mL with a wide dynamic ranged from 104to 109particles/mL［Fig.4（A）］.Similar strategy was also used using the graphene oxide（GO）as the quencher，in which fluorophore-labeled aptamers were adsorbed on GO and the fluorescence signal were quenched，then released in the present of exosomes.Moreover，a deoxyribonuclease I（DNase I）enzyme-aided fluorescence amplification method was applied for the colorectal cancer exosome detection based on GO-DNA aptamer（CD63 and EpCAM aptamers）interactions［Fig.4（B）］.Meanwhile，without GO protection，DNase I enzyme digested the aptamers and released the fluorophores from exosome surface，enabling exosomal marker recycling to amplify the marker-aptamer recognition events.Then，the fluorescence quenched by GO was restored，the released exosomes entered a new detection cycle to generate an enhanced fluorescent signal［64］.Additionally，Jinet al.［65］also used the singlestranded fluorescent aptamer，GO and DNase I to construct the exosome-oriented，aptamer nanoprobe-based profiling（ExoAPP）assay to phenotype surface proteins and quantify cancerous exosomes［Fig.4（C）］.The ExoAPP interfaces GO with target-responsive aptamers can profile exosomal markers across five cell types.This approach was versatile，since the aptamer probes can be changed based on the specific exosome markers，which allowed identification of types of exosomes.

Fig.4 Scheme of the fluorescent biosensor based on the Cy3?CD63 aptamer/Ti3C2MXenes nanocomplex as nanoprobe to detect exosomes[63](A),scheme of the fluorescent biosensor based on the fluorophore?labeled aptamers[64](B),scheme of the fluorescent ExoAPP[65](C),scheme of the fluorescent biosensor based on the aptamer/DNA nanoassemblies on the surfaces of exosomes[66](D),scheme of fluorescent biosensor based on a copper?mediated signal amplification strategy[67](E),scheme of fluorescent bio?sensor based on zirconium?phosphate coordination[68](F)

A mild，precise，but versatile method for exosomal surface modification as a signal amplification strategy is expected to have broad biomedical and bioanalytical applications.Tan’s group［66］also developed a fluorescence biosensor on account of an aptamer-based DNA nano assemblies on exosome surface.In this method，they introduced DNA hybridization chain reaction initiated by an aptamer-chimeric trigger and realized thein situselective DNA nanoassembly on membrane surfaces of nanosized exosomes［Fig.4（D）］.Thisin situassembly method based on molecular recognition between DNA aptamers and the exosome surface markers as well as DNA hybridization chain reaction initiated by an aptamer-chimeric trigger as signal amplification strategy is useful for exosome modification and functionalization，extending applications of exosome-based disease theragnostic.

Recently，according to the structure of exosomes，such as the membrane lipid bilayer，some low-cost assay for direct capture and rapid detection of exosomes based on the signal amplification strategy were put forward.Ye’s group［67］presented a low-cost amplification strategy for direct capture and rapid detection of exosomes based on fluorescent CuNPs［Fig.4（E）］.In the design，magnetic beads（MBs）and copper（II）oxide nanoparticles（CuO NPs）modified with a cholesterol anchor and CD63 aptamer were built a sandwich structure.When exosomes were present，the MB probe and CuO NPs probe can form sandwich complexes（MB-exosome-CuO NPs），and then the CuO NPs were digested into copper（II）ions（Cu2+）by acidolysis.The reduction of Cu2+into fluorescent CuNPs generated fluorescent signal by sodium ascorbate in the presence of poly（thymine）.The fluorescence signal of CuNPs increased with the increase of Cu2+concentration，which was directly proportional to the concentration of exosomes with a detection of limit of 4.8×104particles/μL in biological sample.In addition，Wang’s group［68］has proposed a exosomes-zirconium-liposomes sandwich structure to detect exosomes by zirconium-phosphate coordination chemistry.The combined use of the strong electrostatic interaction between the Zr4+ion and phospholipid membranes of exosomes，and calcein-packaged liposomes can endow the fluorescence biosensor without modification probe［Fig.4（F）］.

2.4 Colorimetric Analysis

Colorimetric analysis is a method to determine the content of the component to be measured by comparing or measuring the color changes of the colored substance solution［69，70］.Generally，colorimetric biosensors can easily observe color changes in real time with the naked eye that yields a“yes/no”answer or semiquantitative result without any additional analytical instrumentation.Colorimetric biosensors used to detect exosomes are great important owing to their easy operation and convenient readout［71］.A colorimetric method based on aptamer modified AuNPs was developed for the profiling of exosomal proteins.

In this strategy，the mixture solution of 13-nm gold nanoparticles（AuNPs）and several aptamers that can recognized specifically with the proteins were prepared firstly［Fig.5（A）］［72］.The complexation of aptamers with AuNPs protected the nanoparticles from aggregating in a high-salt solution.In the presence of exosomes，the aptamer released from the surface of gold nanoparticles owing to the strong binding affinity between the aptamer and exosomes.The non-specific and weaker binding between aptamers and the AuNPs was broken，inducing the aggregation of gold nanoparticles.This aggregation led to the color variations，which can be used to identify multiple proteins on the surface of exosomes.Moreover，the colorimetric biosensor can also be applied for the analysis of expression levels of proteins such as CD63，PTK7 and EpCAM，etc.on exosomes，which offered the possibility for early detection of cancer and help in designing potential curative options.Chen’s group［71］has developed a visible and simple method for the detection of exosomes by integrating single-walled carbon nanotubes that being excellent water solubility（s-SWCNTs）and aptamer［Fig.5（B）］.Aptamers were absorbed onto the surface of s-SWCNTs，and improved the peroxidase activity of s-SWCNTs，which efficiently catalyzed H2O2-mediated oxidation of 3，3′，5，5′-tetramethylbenzidine（TMB）and led to a change from colorless to blue in solution.As a while，after the addition of exosomes，the color of solution turned light.In a similar design，the g-C3N4NSs were also used for the solution-based colorimetric biosensor.When the concentration of exosomes increased，the solution showed a clearly color change from dark blue to light blue［73］［Fig.5（C）］.The nanoenzyme colorimetric methods show a potential for the detection of exosomes due to their simplicity，low cost，and ease of preparation.

Fig.5 Scheme of the colorimetric biosensor based on the aptamer/AuNPs complex for molecular profi?ling of exosomal proteins[72](A),(B)scheme of the colorimetric biosensor based on DNA?capped single?walled carbon nanotubes[71],scheme of the colorimetric biosensor based on g?C3N4NSs[73](C)

2.5 Surface-enhanced Raman Scattering(SERS)Analysis

SERS is a popular optical technique used in bio-analysis which can enhance Raman scattering by molecules adsorbed on rough metal surfaces or by nanostructures.SERS can provide unique spectral signals in complex biological environments［74，75］.In addition，it also has excellent multiplexing capability and high sensitivity［76，77］.SERS platforms include two system formats，the solid-based SERS system and the solution-based system.The solid substrate and the SERS nanoprobe can specifically adsorbed with exosomes to form sandwich structures.The glass slide substrate as the substrate of the SERS biosensor has been improved by being coated with a rough nanoscale metal layer to enhance the sensitivity.The SERS nanoprobes are formed by a metal core with various shapes and sizes，a Raman reporter，and a recognition element.The SERS nanoprobes employed in the biosensors are critical to the performance of the biosensors.Kwizeraet al.reported a SERS biosensor for exosomes detection based on the standard gold-coated glass microscopy slide and the gold nanorods coated with QSY21 Raman reporters［Fig.6（A）］［78］.The gold nanorods were used as the label agent to quantitatively detect the expressions of several surface protein markers on exosomes based on surface enhanced Raman scattering spectroscopy.This assay can specifically detect exosomes with an LOD of 2×106exosomes/mL.To improve the performance of the biosensors，many researchers have devoted to changing the shape of the gold metallic core and fabricated a nanoprobe for the better SERS property.For instance，Tianet al.［79］constructed a SERS biosensor based on the surfaces of gold nanostar@4-mercaptobenzoic acid@nanoshell structures（AuNS@4-MBA@Au）with a bivalent cholesterol（B-Chol）-labeled DNA anchor as the SERS nanoprobes［Fig.6（B）］.Exosomes were specifically captured by immunomagnetic beads，and then SERS nanoprobes were fixed on the surface of exosomes to form the sandwich structure.The range of the biosensor correlated with the exosome concentration was over a wide linear range of 40—4 ×107particles/μL and the LOD is as low as 27 particles/μL.In the solution-based SERS biosensors，nanoparticles with magnetic properties were used as substrates by an external magnetic field to separate the exosome.What is more，the magnetic nanoparticle-substrate has a larger surface area than the glass slide-substrate to improve efficiency and sensitivity.In solution-based system，the nanoparticles with magnetic properties are the critical element.Zonget al.［80］have used SERS nanoprobes and magnetic nanobeads to detect exosomes derived from human breast cancer cells［Fig.6（D）］.In this work，anti-HER2 antibody was modified on the surfaces of the SERS nanobeads to recognize the proteins of tumor derived exosomes.They immobilized anti-CD63 antibody on the surface of magnetic nanobeads which could recognize and capture all types of exosomes.According to the specific recognition of proteins on exosomes and antibodies，exosomes could combine magnetic nanobeads and SERS nanoprobes to form a sandwich structure，separated out by the external magnetic field.Therefore，a strong SERS signal may be detected in the separated fraction.Instead of antibodies，aptamers as recognition elements are also effective for the SERS platform.For example，Wang’s group［81］proposed the screening and simultaneous multiple detection of exosomes using magnetic substrates and SERS probes［Fig.6（D）］.The gold nanoparticles decorated with a Raman reporter and a specific aptamer as the SERS probes were used for targeting exosomes.The capturing substrates using gold shell magnetic nanobeads modified by aptamers could capture most kinds of exosomes by recognizing the generic surface protein CD63.When the target exosome was present，the target exosomes，the substrate and the corresponding kind of SERS probes can form an apt-immunocomplex. This detection method has also been used to detect single kind of exosome，and the LODs are 32，73，and 203 exosomes/mL for the SKBR3，T84，and LNCaP exosomes，respectively.

Fig.6 Scheme of the solid?based SERS biosensors based on the gold nanorods coated with QSY21 Raman reporters[78](A);scheme of the solid?based SERS biosensors based on AuNS@4?MBA@Au[79](B);scheme of the solution?based SERS biosensors based on the nanobeads with anti?HER2 antibody[80](C);scheme of the solution?based SERS biosensors based on the gold shell magnetic nanobeads with aptamers[81](D)

2.6 Surface Plasmon Resonance Analysis

Surface plasmon resonance（SPR）is a label-free real-time analysis technique that detects local refractive index changes upon the binding of target substances to a sensing surface.SPR can reflect the interaction information between positive and negative permittivity materials at the interface through the resonant oscillation of stimulated electrons［82—85］.SPR is extremely sensitive to the biological binding phenomenon of the gold layer with other substances of 200 nm（wave depth）.The size of exosomes just meets this distance.SPR biosensing is not only a label-free and real-time molecular sensing technique，but also a non-destructive method with no need of tedious sample preparation steps.Therefore，SPR-based biosensors are very suitable for the study of exosomes.

Gold substrate is the most common used for reported SPR biosensors for exosome detection.For example，Hu’s group［86］reported that the surface plasmon resonance imaging（SPRi）with antibody microarrays could detect exosomes in tumor cell culture medium characteristically by the proteins on the exosome membrane［Fig.7（A）］.The antibodies specific to exosome transmembrane proteins were printed on the gilded gold chip.Upon the injection of sample，exosomes can be captured by antibodies on the chip，resulting in changes in the refractive index and the reflection intensities monitored by the charge coupled device（CCD）camera.To enhance the signal，the two-step SPR biosensors where additional components typically injected onto a gold chip are developed.For example，Piccioliniet al.［87］have designed the surface plasmon resonance imaging（SPRi）analysis biosensor based on the gold chip to take advantage of the fact that the size of exosomes was completely suitable for the surface plasmon wave depth.The biosensor isolated and fixed exosomes derived from neurons and oligodendrocytes by using a series of antibodies and quantified the amount of CD81 and GM1 which were the membrane components of exosomes.In this way，secondary antibodies were injected into the system to generate an additional SPR signal，which demonstrated that exosomal proteins were not expressed homogeneously but exhibited variable abundance［Fig.7（B）］.This work paved the way for more precise clinical research using exosomes as potential biomarkers for neurodegenerative diseases.In addition to a regular gold chip，random arrays of self-assembly gold nanoislands（SAM-AuNIs）chip with sensitive and low-cost properties were introduced for exosome detection.Thakuret al.［88］reported that the localized surface plasmon resonance（LSPR）biosensor with SAM-AuNIs can be used to detect and distinguish exosomes from A-549 cells，SH-SY5Y cells，blood serum，and urine from a lung cancer mouse model［Fig.7（C）］.This method can distinguish the two different tumor-derived EVs such as exosomes and multivesicular bodies by bared SAM-AuNIs LSPR without functionalization with an antibody.The zeta potential values of exosomes were more negative than that of MVs，indicating that exosomal membranes have a more aggregative property，tending towards being more attracted to bare gold nanoislands.Recently，besides the gold materials，other chemical composition materials have also been exploited for SPR biosensor to improve the performance.For instance，Qiuet al.［89］have constructed a novel biotinylated antibody functionalized titanium nitride（TiN）biosensor as an advanced plasmonic material for quantifying malignant U251 glioma cells（GMs）-derived exosomes［Fig.7（D）］.The TiN film was functionalized with biotinylated-anti-CD63 and biotinylated anti-EGFRvIII antibody to capture the GMs-derived exosomes.And the total internal reflected light from the TiN-exosomes interface was monitored to correlate with the quantity of the exosomes.

Fig.7 Scheme of the SPRi biosensor on the gilded gold chip[86](A),scheme of the SPRi biosensor based on the CD81 and GM1 antibodies[87](B),scheme of the LSPR based on the SAM?AuNIs[88](C),scheme of the SPR biosensor based on the TiN[89](D)

2.7 Microfluidics Analysis

Microfluidic techniques can manipulate precisely fluids and own the high-level of integration，which provide the opportunities for the separation and detection of exosomes based on the size of the exosomes and the affinity between the specific binding of antibodies or aptamers and surface biomarkers on exosomes［90—92］.Microfluidics analysis relies on a variety of fundamental techniques，such as electrochemistry，fluorescence，colorimetric assays，SERS and SPR.It utilizes the plastic including poly（dimethylsiloxane）（PDMS），poly（N，N′-dimethylacrylamide）（PDMA）and silicon with multiple channels as the substrate for isolating，washing，processing，and analyzing samples.In addition，they require minimal amounts of sample.Microfluidics offers a potentially rapid and cost-effective analysis of exosomes.

Exosomes are commonly present in different complex biological fluids which contain amounts of biomacromolecules interfering with the detection and analysis.To decrease the interference from other biomacromolecules and improve the sensitivity of the exosomes，it is necessary to isolate exosomes for biomedical investigation and clinical applications.For example，Liuet al.［92］designed a viscoelastic microfluidic system for sizedependent and field-free separation of exosomes from other large EVs.The viscoelastic microfluidic device was composed of a high-aspect-ratio straight microchannel，two inlets，and three outlets for viscoelastic separation［Fig.8（A）］.The microfluidic device was assembled by bonding a PDMS layer containing the embedded microchannel with a glass substrate.Inlet I and inlet II were used for the introduction of sample and sheath fluids containing a diluted poly-（oxyethylene）（PEO）solution.The sample solution was introduced into the microchannel from inlet I，and a low concentration of PEO solution was introduced into the microchannel from inlet II.Exosomes were pushed forward in the microchannel by the sample fluid.Under the optimal conditions with a size of 200 nm，the exosomes were continuously separated from large EVs of the cell culture medium or serum.The microfluidic system is the versatile and label-free separation tool for exosome analyses.In addition to the isolation，the microfluidic can be applied for the further detection.Xuet al.［93］have proposed a twostage microfluidic platform（ExoPCD chip）with an integrated on-chip isolation andin situelectrochemical analysis of exosomes in serum［Fig.8（B）］.The strategy promoted exosomes capture efficiency and improved without any surface modification.In this method，the ExoPCD chip combined magnetic enrichment based on specific phosphatidylserine-Tim4 protein recognition and the electrochemical signal transition resulted in the high sensitivity detection of exosomes with the detection limit of 4.39×103particles/mL and 5 orders of linear range.Moreover，the method can efficiently capture tumor-derived exosomes and response rapidly within 3.5 h in a small-volume sample（30 μL）.Most importantly，clinical sample analysis showed satisfactory results to distinguish between liver cancer patients and healthy serum.On the basis of quantitative analysis，the analysis of the nucleic acid and proteins has attracted more and more attention，which reveals the biological significance of exosomes further.Talleret al.［94］have designed a microfluidics-based approach to the analysis of the exosomal RNA for pancreatic cancer study and diagnosies［Fig.8（C）］.In this work，surface acoustic wave（SAW）exosome lysis and ion-exchange nanomembrane were on two separate chips and microRNA hsa-miR-550，raw cell media from pancreatic cancer cell lines were chosen as a model target and a biological sample，respectively.The biosensor was shown to have an LOD of 2 pmol/L.And the total analysis time of the platform wasca.1.5 h which was improved significantly over other methods.For point-of-care（POC）analysis，Shaoet al.［95］developed a comprehensive microfluidic platform realizing three functions including targeted enrichment of exosomes，on-chip RNA isolation，and real-time RNA analysis［Fig.8（D）］.The antibody-functionalized magnetic beads were used to separate cancer-specific exosomes.After that，the exosomes passed through a glass-bead filter，RNA was selectively adsorbed and eluted from the filter for reverse transcription and qPCR analysis.

Fig.8 Scheme of the microfluidic biosensors for isolation of the exosomes[92](A);scheme of the ExoPCD?chip for the isolation and in situ detection of the exosomes[93](B);scheme of the microfluidic chip with SAW for exosomal RNA[94](C);scheme of the microfluidic chip for POC[95](D)

Exosomes have been shown to provide good identify early diseases and real-time information.However，the extremely small size will increase the difficulty of exosome detection.Therefore，sensitive detection and quantification techniques are needed to accurately quantify exosomes extracted from patient samples.Various types of sensors based on novel signal transduction and amplification strategies are the future development trend.

3 Clinical Applications

The biological significance of exosomes has been investigated substantially，their clinical applications in the diagnosis and treatment have grown tremendously.Disease-derived exosomes，an emerging liquid biopsy tool，have been used for detection and monitoring［96—98］.Furthermore，they are now being researched as delivery vehicles for therapeutics［99—101］.

3.1 Clinic Diagnostics

Exosomes are secreted by all cells and harvested from all biological fluids，favoring them to follow disease progression.Exosomes with molecular cargo are beneficial to realize the multiparameter and precise diagnostic testing，while surface proteins on exosomes as biomarkers could help their immune capture.Diagnostic applications of diseases including cancer，neurodegenerative diseases and cerebrovascular disease have been exploited through exosomes［102—104］.

Tumor-derived exosomes facilitate tumor growth and metastasis through intercellular communication transferring of biomolecules，which could modulate disease initiation and progression.Moreover，exosomes of biofluids from the patient carry information in the vesicular contents that evaluates the origins of the cancer cells.Comparing to traditional tissue biopsy，liquid biopsy is superior in the early diagnosis of tumors，the tracking of disease prognosis and treatment responses.The detection of exosomes has already been explored in the diagnosis of many types of cancers，such as breast cancer，lung cancer，ovarian cancer，prostate cancer and melanoma cancer［19，105，106］.For instance，Meloet al.［107］reported GPC1+circulating exosomes were detected in the serum from patients of pancreas cancer with specificity and sensitivity.GPC1 is a cell surface proteoglycan overexpressed on the cancer cells and can regulate cancer progression.Thus，GPC1+circulating exosomes may serve as a potential non-invasive diagnostic.It is demonstrated that the circulating exosomes GPC1+level of breast cancer patients is higher than that of healthy individuals.The method based on the GPC1+circulating exosomes can distinguish healthy subjects from patients with pancreas cancer and monitor the stages of diseases.Exosomal microRNAs（miRNAs）are reliable and non-invasive biomarkers for the early diagnosis of cancer.Besides the analysis of the protein expression，exosomal microRNAs are reliable and non-invasive biomarkers for the early diagnosis of cancer.Sunet al.［108］designed a strategy of detection of the exosomal miRNAsin situby a thermophoretic sensor implemented with NanoFlares.The LOD was down to 0.36 fmol/L in 0.5 μL serum samples without RNA extraction or target amplification.To further demonstrate the clinical application，the estrogen receptor（ER）-positive breast cancer（BC）patients and healthy donors（HD）as control were chosen to provide the serum samples where four breast cancer-associated miRNAs（miR-375，miR-221，miR-210，and miR-10b）in exosomes were isolated.Compared to the HD cohort，the levels of exosomal miR-375，miR-221，and miR-10b in the ER+BC cohort detected were statistically higher.Particularly，exosomal miR-375 showed a sensitivity of 88%，a specificity of 83%，an accuracy of 85%，and the area under the curve value of 0.94 for early detection of ER+BC（stage I—II）［Fig.9（A）］.

The analysis of proteins and nucleic acid reflects the genetic information which is significant for understanding the occurrence and progress.Therefore，exosomes are the attractive candidate for the early diagnosis，allowing the earlier access to treatment decrease the mortality.

3.2 Diseases Therapeutic

Exosomes are endogenous carrier for various biomolecules，such as proteins，lipids and nucleic acid，which avail them to participate in information interchange to regulate pathophysiological course［109］.They contain the cell-surface compounds and thus have the ability to cross biological barriers［110］.Moreover，exosome can avoid the mononuclear phagocytic system by the surface protein and own intrinsic stability in circulation due to their negatively charged.In addition，they are less cytotoxicity than foreign substances［111］.Exosomes’features qualify them a potential avenue for therapy.They have been used as therapeutic agents in regenerative medicine，cancer therapy and immune modulation［112］.

The lack of ideal delivery systems impedes the efficient therapeutic delivery to tumor tissue.The safety，biodegradation，stability and targeting are necessary for the ideal delivery systems.Coincidently，exosomes offer several advantages to meet the above requirements for therapeutic delivery.Tianet al.［113］reported the engineered exosomes deliver chemotherapeutic drug doxorubicin（Dox）to tumor tissue in BALB/c nude mice［Fig.9（B）］.Exosomes were separated from immature dendritic cells（imDCs）which were engineered to express a well-characterized exosomal membrane protein（Lamp2b）fused to αv integrin-specific iRGD peptide（CRGDKGPDC）to realize the tumor targeting.Then，the engineered exosomes were loaded with Doxviaelectroporation.Compared to the untransfected cellular exosomes，the targeted exosomes bound toαvintegrinpositive breast cancer cells with high affinity.The effectiveness of therapy was improved with exosomeencapsulated Dox relative to free Dox at the same dose.After injecting exosome-encapsulated Dox to the tumor-bearing mice，the tumor volumes were inhibited availably，while causing no obvious toxicity.Recently，nucleic acid-based therapeutics has become a promising approach for cancer therapy.Exosomes also has been paid attention to deliver RNA to tumor for treatment.Huanget al.［114］reported exosomes loaded and delivered RNA to the leukemia cells and induce significant cell apoptosis［Fig.9（C）］.According to the biogenesis of the exosomes，endogenous microRNA 21（miR-21）sponges，inhibitors of miR-21，were enriched on the plasma membrane and were allotted into exosomes.Simultaneously，AS1411 aptamers were modified on the surface of the engineered exosomes to realize the targeted delivery of RNA into leukemia cells.The miR-21 in leukemia cells was blocked by the miR-21 sponges on the exosomes，which stimulated cell apoptosis to cure tumors.Besides the above delivery therapy system，immunotherapy based on the exosomes has attracted a lot of attention.Exosomes are shown to mediate immune responses for killing and eliminating diseased cells.Poggioet al.［115］reported the exosomal PD-L1 was suppressed which induced systemic anti-tumor immunity.PD-L1 on the surface of tumor cells would bind to the receptor PD-1 on T cells，which inhibited to activate immune system，similar to the effect of anti-PD-L1 antibodies.Removal of exosomal PD-L1 could inhibit the tumor growth and prolong the survival.Therefore，previous findings show that exosomes have the potential to become the immunological tool for the disease treatment.

Fig.9 Clinical use of thermophoretic sensor for profiling exosomal miRNAs in breast cancer patients[108](A);antitumor activity of Exos?Dox[113](B);design of exosomes loading system[114](C)

4 Conclusions and Perspective

Exosomes are types cell derived vesicles with diverse cellular constituents which not only reflect the information of the parent cells，but also influence the intrinsic microenvironment and disease progression.Exosomes as biomarkers have shown the ability of offering the information in diagnostics and therapeutic response non-invasively.Moreover，they have been proved to serve as a promising therapeutic tool through delivery the drugs and mediating immune responses.The number of studies around the exosomes has increased substantially at the basic and applied levels.Hence，we reviewed the most recent advances in exosomes research including analysis and clinical applications.The biosensors with different techniques are being developed and optimized for exosomes，which provides their prospects into the diagnostics and therapeutic potential.To overcome the extant challenges of the exosomes research，several pivotal aspects for further improvements are proposed.Firstly，the standard of all processing steps should be established.The isolation，count and storage are fundamental and critical for the subsequent studies and applications.Secondly，single exosomes profiling should be paid attention.Individual exosome analysis could help us to obtain the comprehensive biological map and develop valuable clinical applications.Finally，biosensor with better performance should be fabricated with specificity，stability，sensitivity and economics.It is essential to select the specific exosome markers according to different requirements for enhancing the specificity.The poor reproducibility could be improved by controlling the synthesized nanoparticles with stability.The sensitivity of real clinical samples，the cost of equipment and labor are vital for the point-of-care testing.Exosomes will have made the contribution to the clinical diagnosis and therapy in the future.

This paper is supported by the National Key Research and Development Program of China（No.2016YFA0203101）and the National Natural Science Foundation of China（Nos.21874080，21622506，21621003）.

（Ed.：N，K）