Table 1.

Advantage and disadvantages of methods for characterization of EVs.

Method	Characteristic of EVs	Advantages	Disadvantages	Ref.
NTA	Particle size distribution; concentration (number of particles)	Detect particle in the size range of 10–1000 nm diameter	Requires sample volumes around 500 µL Requires optimization for collection of data and parameters of analysis	(Soo et al., 2012) (Palmieri et al., 2014) (Filipe et al., 2010)
DLS	Size distribution; zeta potential	Requires very small sample volume (70 µL) Easy to use (requires optimization for a few parameters)	Poor analysis of heterogeneous populations of particles	(Palmieri et al., 2014) (Filipe et al., 2010)
Tunable Resistive Pulse Sensing (tRPS)	Size distribution, concentration of particles	Length of the resistive pulse is correlated with the particle size Rate of resistive pulses reveals concentration of particles	An indirect method that requires a series of standard sample	(Maas et al., 2014)
TEM	Size, morphology	Produces high resolution images Electrons that pass through the sample are detected	EVs typically have a divot in their center due to the drying process associated with the sample preparation	(Wu et al., 2015)
SEM	Size, morphology	Produces high resolution images Scattered electrons are detected	Requires extensive sample preparation EVs typically have a divot in their center due to the drying process associated with the sample preparation	(Wu et al., 2015)
Cryo-EM	Size, morphology	Samples can be conserved in their native hydrated state Produces better quality and preserved morphology Artifacts can be avoided In combination with TEM, cryo-EM can detect proteins in EVs, and uptake by cells	Requires extensive sample preparation	(Chernyshev et al., 2015; Choi and Mun 2017; György et al., 2017)
Immunogold-EM	Specific protein detection qualitatively	Requires small volume of EVs Can detect the proteins in EVs Can detect multiple proteins in EVs by using different size secondary gold particles Quantify disease specific markers in EVs Better for molecular characterization of EVs	Requires extensive sample preparation	(Cappello et al., 2016)
Western blot	Specific protein detection quantitatively	Allows molecular characterization of EVs. Allows quantification of proteins in EVs.	Does not allow observation of intact vesicles Not well multiplexed The specificity and reproducibility are limited by the quality of the antibody used Requires large sample volume Extensive sample processing is required Specialized instruments are needed	(Gallagher et al., 2008)
ELISA	Specific protein detection quantitatively	Allows quantification of protein in EVs, crucial for molecular characterization of EVs	Requires a large sample volume Extensive sample processing is required Specialized instruments are needed	(Witwer et al., 2013)
Flow cytometry	Specific protein detection quantitatively	Detection limit is 100–200 nm Allows for high throughput analysis of exosomes Allows for quantification or classification of exosomes based on the antigen expression	Requires a single particle suspension Aggregation of vesicles results in the observation of multiple particles at a single time Requires the immobilization of exosomes on the surface of beads	(Szatanek et al., 2017) (Ko et al., 2016)
Thermophoretic aptasensor (TAS)	Profile EVs as a function of surface protein expression	Inexpensive, fast, and requires small serum volume (less than1 µL)	Currently, TAS profiles one marker per run. Therefore, further development is necessary for high throughput Accuracy needs to be further improved.	(Liu et al., 2019)
Mass spectroscopy	Proteomic analysis of EVs	Allows the identification and quantification of thousands of EV proteins Can identify missing proteins in the human protein map	Protein interference issue due to the identification of peptides as protein surrogate sequence coverage Requires isolation of homogeneous EV population Characterization of the proteome of EVs isolated from primary cell lines and tissues is challenging	(Rosa-Fernandes et al., 2017)
SPR	Membrane protein analysis, biophysical properties, protein-protein interaction	Real-time measurement Able to detect low affinity antibodies or antigens, a calibration-free concentration analysis Elimination of labels Requires low sample volume	Requires high sensitivity and specificity for the detection of biomarker at the early stage of disease progression The sensor chip requires functionalization of ligands	(Thakur et al., 2017; Hosseinkhani et al., 2017; Im et al., 2014)
AFM	Membrane protein analysis, biophysical properties, topology, surface characteristics	Can detect EVs in liquid as well as air mode Produces topographical pictures of EVs Allows quantification and imaging of EVs Specific EVs can be detected via immobilization of antibodies Extensive sample preparation is not required Resolution limit is around 1 nm	Requires specific stages such as mica for immobilization. Requires probe for the detection of EVs, which can damage the EVs	(Klinov and Magonov 2004; Sharma et al., 2010, 2011; Yuana et al., 2010; Hardij et al., 2013)
Raman spectroscopy	Detects membrane protein, functionality	Simple, inexpensive, highly efficient, and portable method	Analysis of a single vesicle is time-consuming because of the weak Raman signals that often need enhancement via the nanostructured substrates or nanoparticles for a more effective analysis	(Kwizera et al., 2018; Gualerzi et al., 2019)
Quantum dots	Detection of disease specific exosomes	Sensitive detection of 100 exosomes per μL Facilitates better tracking of EVs and more specific targeting QDs have strong resistance to photobleaching	In the context of QD-EV conjugation chemistry, the NHS-ester used for QDs and EV modification can react with primary amines.	(Boriachek et al., 2017; Goreham et al., 2020; Zhang et al., 2020) (Takov et al., 2017)
Integrated magneto-electrochemical sensor (i-MEX)	Fast and streamlined analysis of EVs Cell-specific exosomes can be isolated High detection sensitivity through magnetic enrichment and enzymatic amplification Sensors can be miniaturized	Fast, high-throughput, and on-the-spot analysis Cell-specific exosomes can be isolated directly from complex media High detection sensitivity via magnetic enrichment and enzymatic amplification Can be miniaturized and expanded for simultaneous measurements	The iMEX system has lower sensitivity and throughput than nPLEX	(Jeong et al., 2016)
Aptamer based biosensor	Quantitative detection of exosomes	Requires small sample volume Application of aptamer instead of antibody, improves the stability of the system, resulting in better sensitivity Due to label-free approach, the cost is reduced. The aptasensor detected exosomes in a homogeneous system	Lack of a reliable process to obtain aptamers to be specifically used in electrochemical sensors	(Zhou et al., 2016; Rozenblum et al., 2019; Xia et al., 2017)
Aptasensor	Detects exosomes by integrating single-walled carbon nanotubes	Visible and simple method Can be applied to detect other targets by changing the aptamer	Requires development of a “signal-on” strategy to replace “signal-off” strategy, susceptible to interference	(Xia et al., 2017)