Table 4.

Methods of exosome quantification

Method	Principle	Advantages	Disadvantages	References
Nanoparticle tracking analysis (NTA)	Based on the detection of light scatter by particles in suspension and their Brownian motion to estimate the number and volume distribution of EVs	Does not rely on detection of a specific marker Direct quantification	Expensive instrument Photobleaching and potential background from dye aggregates Measures non-exosomal contaminants also	[73, 74]
Flow cytometry	Flow cytometry detects particles suspended in a fluid by their interaction with a laser beam as they flow through a detection cell	Direct quantification	Insensitivity to smaller exosomes. Requires binding to fluorophore-conjugated antibody-coated beads Swarm effect that means multiple smaller vesicles are counted as single particle. This may provide false positive result	[75–77]
Tunable resistive pulse sensing	Detects the passage of individual particles through a pore in a membrane	Direct quantification	Pore clogging. Insensitivity to smaller exosomes. Measures non-exosomal contaminants also	[78, 79]
Electron microscopy	Imaging of individual exosomes under scanning electron microscope	Exosomes are manually counted	Labor intensive, slow process	[80, 81]
Dynamic light scattering	Evaluates fluctuations in the light scattering intensity of particles	High sensitivity, simple sample preparation, rapid	Heterogeneous exosome populations cannot be analyzed, difficulty with polydisperse samples	[82]
Microfluidics-based detection	Transport of fluids controlled by capillary forces	Product purity, high throughput analysis	Not ready for industrialization yet, increase in the signal/noise ratio is encountered	[83, 84]
Surface plasmon resonance (SPR)	A light is focused to a metal film through a prism and the reflected light is detected which is collective oscillation of free electrons. It is sensitive to change in refractive index of the media	Label-free and real-time quantitative analysis technique High sensitivity of up to 1 nM for a 20 kDa protein Specific to the binding event	Difficult to discriminate between specific and non-specific interactions Mass sensitive limitation Limited sensor area Expensive instrument and sensor cost	[85–87]
Single particle interferometric reflectance imaging sensor (SP-IRIS)	A monochromatic light illuminates on sensor surface and scattering signal from individual nanovesicles is detected by CMOS camera The signal is enhanced due to the interferometric phenomena	Quantitative, label-free and dynamic detection method Multiplexed phenotyping and digital counting of individual EVs with diameters of 50–200 nm	Detection limit of 3.94E+09 particles/mL	[88–90]