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Analysis of single extracellular vesicles for bioapplication

已有 1830 次阅读 2022-4-15 17:28 |系统分类:论文交流

Analysis of Single Extracellular Vesicles for Biomedical Applications with Especial Emphasis on Cancer Investigations

Ting Wang【王婷】, Yanlong Xing【邢艳珑】*, Ziyi Cheng【程子译】, Fabiao Yu【于法标】*

Laboratory of Neurology, The First Affiliated Hospital of Hainan Medical University, Key Laboratory of Emergency and Trauma, Ministry of Education, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China

TrAC Trends in Analytical Chemistry Volume 152, July 2022, 116604



•A comprehensive review covering the recent progresses in single extracellular vesicle-based analysis.

  • •Discussion on the significance of single extracellular vesicle analysis.

  • •Focusing on the advanced analysis of single extracellular vesicles, especially in combination with microfluidic platform.

  • •Review on the single extracellular vesicle analysis-based biomedical application with emphasis on cancer investigations.

  • •Discussion on the existing obstacles and future perspectives on single extracellular vesicle research.


Extracellular vesicles (EVs) are lipid membrane enclosed nano-sized vesicles that are secreted by all known organisms. These vesicles are increasingly recognized as important circulating biomarkers for the diagnosis and prognosis of different diseases including various types of cancer, owing to their essential role in intercellular communication. EVs preserve heterogeneity in both physical properties and cargos, which makes it extraordinarily tough to fully exploit their clinical potential. Therefore, comprehensive characterization of single EVs and their sensitive detection are urgently demanded. In this article, we survey the latest progress in single EVs analysis with innovative discoveries in heterogeneity and highlight the various label-free and labelling approaches of single EVs detection. Furthermore, the state-of-the-art advances in single EV-detection based biomedical applications with especial emphasis on cancer investigations are summarized. To the end, the challenges and prospects for exploiting new system in the field of single EVs study are discussed.

1.     Introduction

EVs are heterogenous, lipid-bilayer-phospholipid membranous vesicles generated by various living cells through active secretion [1, 2]. Although initially thought to be cell debris, EVs have been discovered as vital biological species, owing to their physiological and pathological function in organisms. These vesicles carry bioactive molecules such as proteins and nucleic acids that are inherited from parental cells, and thus, affect microenvironment locally and at a distance by transferring cargos to recipient cells [3]. EVs can mediate intercellular communication and have been regarded as potential biomarkers for the diagnosis and treatment of diseases [4, 5]. 

EVs can be released by cells to the extracellular space via different ways. Based on the currently known origin mechanism of EVs, these vesicles can be divided into three categories: exosomes (30-200 nm in diameter), microvesicles (100-1000 nm in diameter) and apoptotic bodies (500-2000 nm in diameter) [2]. In this review, we concern on exosomes and microvesicles and collectively define them as EVs. Exosomes and microvesicles have different modes of biogenesis. In one aspect, exosomes are originated from endocytic pathway. Initially, inward budding of cellular plasma membrane results in the formation of early endosome. Further inward invagination and budding of membrane inside early endosome leads to the formation of multivesicular body (MVB) bearing intraluminal vesicles that carry transmembrane, cytosolic contents, and peripheral proteins. MVBs may then partially fuse with lysosomes and degrades inside cell. Alternatively, MVBs can fuse with plasma membrane and release vesicles to the extracellular environment, which are defined as exosomes. In another aspect, the direct outward budding of the plasma membrane induces the formation of microvesicles [6, 7]. Therefore, EVs preserve high heterogeneity in physical characteristics (size, density, morphology) and cargos (protein, lipid content, nucleic acids), mainly owing to their intricate biogenesis processes [8].

Fig. 1. Various characterization and detection methods of single EVs

Growing evidence has demonstrated the role of EVs in the development of various diseases such as neurodegenerative diseases, acute organ injury and cancer, owing to the bioactive cargos carried and transferred by EVs [5]. In particular, tumour secreted EVs effect critical functions in facilitating intercellular communication in tumour microenvironment and modulate tumour initiation and progression [9]. Additionally, EVs are widely present in various bodily fluids and have advantages in high concentration (up to 1011/mL) and stability in the blood circulation. As a result, tumour derived EVs can be used as promising biomarkers for liquid biopsy in cancer patients [4, 10]. Notably, many tumour-associated protein biomarkers have been identified in EVs from clinical blood samples, and their types and expression levels are strongly correlated with the presence and progression of certain cancer, which makes the investigation on EVs’ heterogeneity important [11]. Since EVs are heterogeneous in sizes and contents, it is of utmost importance to investigate the molecular composition of EVs at single vesicle level in order to completely understand the biological function of various EV subtypes in disease development and exploit their clinical value [11]. Conventional techniques such as western blotting (WB), enzyme linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (RT-PCR) have been utilized to detect EVs contents (protein and mRNA), however, mainly for bulk vesicles, which are unsuitable for single EV analysis [2, 5]. Recent years, research progress has been made in analysis of single EV, using various advanced detection technologies [12, 13]. In general, detection approaches of single EVs fall into two main categories, label-free and label-based techniques. Label-free methods such as Raman spectroscopy and plasmon resonance are based on the physical properties of EVs, offering non-destructive approaches for single EV detection [14]. Leveraging fluorescently labelled antibody or aptamer, label-based methods can achieve the detection of individual EVs, in conjugation with flow cytometry or fluorescence imaging techniques etc. [15]. Herein, we intend to summarize the recent progress in single EVs analysis and the latest advances in biomedical applications, with especial emphasis on cancer diagnostics based on EV-derived biomarker discovery. In detail, the influence of EVs’ heterogeneity on their function and the significance of single EVs analysis are discussed. Additionally, various characterization and detection methods are listed and compared. And the state-of-the-art clinical application in cancer diagnosis based on single EVs analysis are exemplified. Finally, we propose the current challenges and future perspectives in single EV-related research.

2. Significance of single EVs analysis

Cells actively release a large number of EV populations with distinct biomechanical properties and biological functions into the extracellular environment, which exerts diverse biological effects on recipient cells [16]. Exosomes and microvesicles are most concerned EV populations, which are discriminated based primarily on their cellular origin [3]. Nonetheless, increasing evidence has indicated that these EV populations contain various subpopulations with unique function in bioprocesses. For instance, non-membranous nanovesicles and exosome subsets including small and large exosome vesicles from various cancer cell lines have been identified [17]. Additionally, in a recent study, it has been observed that tetraspanins are unevenly distributed across single EVs [18]. The EV subpopulations can reflect the associated biological processes, which enables them to be promising biomarkers for clinical diagnosis. However, these intrinsic diversity and heterogeneity of EVs make it more complex and difficult for investigating their biology and function. Typically, in cancer biology, the various EV subtypes may have unique biological roles in the development of cancer, and the varied distribution of membrane proteins on single EVs may bias sensitivity to multiplexed cancer biomarkers [18, 19].

Therefore, one of the major concerns in EV research is to address the heterogeneity within EV populations and investigate the molecular composition of single EVs in detail [12]. Clarifying EVs’ diversity will help to better understand the exact function of EV in the physiological and pathophysiological processes, and ultimately accelerate the use of EVs in diagnostics and therapeutics. The significant progress has been made in the field of EVs, owing to the improved characterization and detection techniques, which enable deciphering of the heterogeneity of single EVs for biomedical applications [20].

3. Characterization techniques of single EVs

The characterization of EVs include two parts, one is the physical characterization including morphology, size and distribution, the other is the characterization of the molecular composition [21]. The physical characterization can only be employed to examine the physical characteristics of EVs, while complementary techniques are needed to examine the molecular composition e.g., protein, nucleic acids etc. to ensure the successful isolation of the desired vesicles [22]. There are multiple methods for the physical characterization of EVs including scanning electron microscopy (SEM), transmission electron microscopy (TEM) and dynamic light scattering (DLS) [5]. Different from the above techniques that are commonly employed for bulk EVs examination, as illustrated in Fig. 1, nanoparticle tracking analysis, atomic force microscopy and cryogenic transmission electron microscopy have been proved to be valuable methods for characterizing single EVs [23, 24]. Characterization of the molecular composition of single EVs requires optical techniques such as Raman or fluorescence spectroscopy to obtain the molecular features of individual vesicles, either with or without exogenous labels. Commonly, Raman tweezers microspectroscopy and nanoscale flow cytometry have been employed for the molecular characterization of single EVs [25, 26]. Mass spectrometry has also been employed to characterize EV proteins specifically expressed by single EV subpopulations [27]. The description and comparison of different characterization techniques are summarized in Table 1. 

Table 1. Summary of characterization techniques for single EVs


Working principle

Information obtained




Physical characterization (morphologies, size and distribution)

Nanoparticle tracking analysis (NTA)

Tracking and recording the Brownian motion of nanoparticles in   suspension

Size distribution and concentration of particles

Minimum sample preparation, easy to operate, less time-consuming

Low specificity, possible false signal from protein aggregates or other   nanoparticles, interference of scattered light from adjacent particles

[5, 32]

Atomic   force microscopy (AFM)

Measuring the interaction between the probing tip and sample surface

Morphological and mechanical characteristics of single EVs

No special sample preparation, high resolution, high throughput,   integration with other techniques

Limited information obtained; perfectly flat substrate required, time   consuming, labour intensive

[28, 29]

Cryogenic transmission electron microscopy (cryo-TEM)

Observing frozen   samples under transmission electron microscopic

Morphology and   structure

No complicated   sample preparation, retain the native structure of EVs, high resolution

Limited information obtained, low throughput, low contrast image

[31, 33]

Molecular composition characterization

Raman tweezers   microspectroscopy (RTM)

Measuring the Raman   spectra of vesicles using laser mediated tweezing of the object

Raman fingerprints   of the sample EV’s chemical constituents

No exogenous label,   non-destructive, timesaving, no sample treatment

Weak signal, low throughput

[25, 34]

Mass spectrometry

Measuring mass-to-charge ratio after ionization and fragmentation of   sample molecule in the gas phase

Molecular mass and   ion   fragmentation pattern of   given molecule showing the structural information

No exogenous label,   rapid, sensitive, specific, high throughput

Limited information obtained, strict   operation condition, coupling with other techniques required

[27, 35]

Nanoscale flow cytometry   (nFC)

Detecting the   multiparametric scattered light and fluorescence signal emitted by labeled   vesicles on a nanoscale flow cytometer

The size   distribution and diversity of EV populations, and the protein or nucleic acid   content of single EVs

High resolution,   high throughput

Interference from unbound dye, highly purified labelled EVs and properly   diluted samples required

[26, 36]

Multiparametric characterization



Different techniques are coupled on one setup to measure the same   sample simultaneously and measure by two techniques

membrane protein composition and abundance, the size and mechanical   properties etc.

Multiparametric analysis of single   EVs, detailed information on morphology and structure

Special setup required, different software for processing and analysis


Table 2. A summary of the various detection methods for single EVs

Detection methods

Working principle

Information detected




Label-free approaches


Refer to Table 1


Surface-enhanced Raman spectroscopy   (SERS)

Exposing EVs to signal-enhancing   nanoparticles to obtain amplified Raman signal

SERS spectra of   individual EVs

Enhanced Raman signal, high   sensitivity, high throughput

Limited   information of EV surface proteins, low reproducibility, complicated data   processing

[43, 44]

Surface plasmon resonance imaging   (SPRi)

A technique which detects the   changes in refractive index induced by molecular binding to noble metal   surface

SPR spectra and   SPR imaging upon binding of EVs on substrate

High   sensitivity, multiplexed data collection, compatibility with microfluidic systems

Low throughput, uniform substrate and   modified sensor surface required

[45, 46]

Interferometric plasmonic   microscopy (iPM)

Combined surface plasmon stimulation   and interferometric scattering effect to detect single EVs

iPM images reflecting the   adsorption and binding events of single exosomes

High   sensitivity, high spatial resolution, in situ visualization

Low   specificity, low throughput


Single-particle interferometric   reflectance imaging sensor (SP-IRIS)

Detecting individual enhanced   scattering signals generated by bound vesicles on layered substrate

Size and   multiplexed profiling of membrane biomarkers of EV groups in a single   measurement

High   sensitivity, high specificity, high throughput, compatibility with   microfluidics

Limited lateral   resolution, difficult to detect small nanovesicles

[18, 48]

Frequency locking optical   whispering evanescent resonator (FLOWER)

Recognizing EVs by tracking changes   in resonant frequency of the microtoroid optical resonators

Resonance   frequency changes over time indicating the binding of EVs to resonator

High signal-to-noise ratio, high   sensitivity

Difficult to identify particle   size, limited information obtained


Reflection enhanced dark field   scattering microscopy (REDFSM)

Recording scattering

signal of single EVs after   illumination on a reflective surface

The size and scattering intensity of single exosomes

High sensitivity, simplicity, high spatial resolution

Low   throughput, low specificity


Labelling approaches

Imaging flow cytometry (IFCM)

Detection by   combined flow cytometry and imaging techniques

Fluorescence   images for qualitative and quantitative evaluation of single EVs

Multi-parametric   detection, high throughput, stable signal

Dependence on   biological reference materials, time consuming

[51, 52]

Micro/ Nano-Flow cytometry (MFC/nFC)

Refer to Table 1


Detection methods (continued)

Working principle

Information detected




Fluorescence microscopic imaging

Fluorescence   from external labels for in vitro and in vivo tracking

Fluorescence   images of single EVs both in vitro and in vivo

Rapid response,   multi-colour labelling, high sensitivity, compatibility with microfluidics

Low   signal-to-noise ratio, fluctuation of fluorescence induced by low   photostability of dyes


Super-resolution microscopy (SRM)

Fluorescence   from external labels detected below the optical diffraction limit

Fluorescence   images of single EVs in biological samples

High   resolution, in situ visualization, less invasive

Fixed   samples required, interference from fixing regent, low throughput


Total internal reflection   fluorescence (TIRF) microscopy

Imaging of   fluorescence signal generated by the evanescent wave induced excitation of   external labels at glass-water interface

Fluorescence   images of single EVs in aqueous environment

High axial   resolution, high signal-to-noise ratio

Limited specimen detected, fluorophore   instability and photobleaching

[63, 64]

DNA points accumulation for imaging   in nanoscale topography (DNA-PAINT)

Combination of   DNA-PAINT and TIRF to measure exosomal biomarkers

Quantitative   analysis of exosomal surface biomarkers on individual vesicles

High accuracy, high resolution, multiplexed   profiling

Complicated   data analysis


Droplet digital exosome   enzyme-linked immunosorbent assay (ExoELISA)

Detection of the enzymatic   fluorescent reporter tagged on sandwich ELISA complexes of exosomes in single   droplets

Fluorescence   signals from droplets indicating the expression levels of biomarker on single   exosomes

High   specificity, high sensitivity, high accuracy, high throughput

Magnetic beads dependence, diluted   sample required


Immune droplet digital polymerase   chain reaction (iddPCR)

PCR   amplification of the genetic barcode of labelled EVs in single droplets

Absolute   quantification of specific targets in individual EVs

Multiplexed   analysis, high sensitivity, high throughput

Difficulty in   obtaining absolute expression level of biomarker, diluted sample required


Proximity   barcoding assay (PBA)

Profiling surface proteins of individual   EVs using antibody-DNA conjugates and next-generation sequencing

Surface   protein patterns of individual EVs

Multiple-recognition   assay, high throughput, high specificity

Complicated   processing, time consuming


Nanoplasmon-enhanced scattering   (nPES)

Local plasmon-coupling effect   induced by the binding of Au nanostructures to immunocaptured EVs

Dark field   images and scattering spectra indicating the concentration of EV and   biomarker

High   specificity, high sensitivity, no sample pre-treatment, little sample   required

Low throughput


Nano-plasmonic EV analysis with   enhanced fluorescence detection (nPLEX-FL)

Fluorescence   signals of the immunostained EVs are amplified by SPR excited by the Au   nanohole structures

Enhanced   fluorescence intensity indicating the biomarker profiling of single EVs

Multiplexed analysis, high   sensitivity

Complex   substrate modification, limited enhancement of fluorescence