TABLE 2.

Summary of biosensing techniques for single particle analysis of extracellular vesicles

Biosensing technique	EV characteristic	Advantages	Limitations	References
Atomic force microscopy	Size distribution, morphology, surface topography, and mechanical properties	• Minimal sample prep • High resolution • Measures EV stiffness and elasticity • Can be analyzed in solution	• Expensive equipment • Destructive • Low throughput • EVs must be immobilized to a surface • Nonquantitative	Beekman et al. (2019) and Sharma et al. (2020)
Cryogenic electron microscopy	Individual EV size and native morphology	• Minimal sample prep • High resolution • Accurate sizing/morpohology • Can use stains or labels to observe specific proteins	• Expensive equipment • Destructive • Low throughput • Nonquantitative	Emelyanov et al. (2020) and Cizmar and Yuana (2017)
Digital ELISA	Surface proteins	• Fluorescent labels can identify singular proteins of interest • Semi‐quantitative (estimate concentration)	• EVs must be isolated into single droplets for single EV analysis (can be done through microfluidics) • Expensive materials (i.e. antibodies) • No multiplexed detection for single EV	Liu et al. (2018) and Yang et al. (2022)
Digital PCR	Nucleic acid and surface proteins	• Quantitative • Multiplexed detection • Simultaneous tracking of multiple surface protein and RNA cargo	• EVs must be isolated into single droplets for single EV analysis (can be done through microfluidics) • Expensive equipment	Liu et al. (2021) and Ko et al. (2020)
Flow cytometry	Size distribution, surface/soluble markers	• High throughput • Low sample volume • Multiplexed detection	• Diffraction‐limited: >100 nm • Usually ideal for micron‐scale particles, rather than nanoparticles. Specialized flow cytometer is likely needed. • Cannot distinguish between EVs and aggregates • Typically requires fluorescent labeling	Campos‐Silva et al. (2019) and Görgens et al. (2019) and Tian et al. (2018)
Interferometric reflectance imaging sensing	Surface proteins and receptors	• Minimal sample prep • Nondestructive • High sensitivity • Low sample volume • Quantitative and qualitative • Multiplexed detection • High throughput	• Requires expensive analytical chips with conjugated antibodies • Size limitation: > 50 nm	Deng et al. (2022); Mizenko et al. (2021) and Yang et al. (2018)
Laser trapping Raman spectroscopy	Chemical fingerprint	• Minimal sample prep • Label‐free • Nondestructive • Low sample volume • Quantitative and qualitative • Direct imaging	• Long acquisition times • Low throughput • Expensive equipment	Carney et al. (2017), Enciso‐Martinez et al. (2020), and Penders et al. (2018, 2021)
Nanoparticle tracking analysis	Size distribution and concentration	• Minimal sample prep • Low sample concentration • Nondestructive • High throughput	• Performance fluenced by aggregates and larger nanoparticles • Cannot distinguish between EVs and protein aggregates • Size‐limited: >70 nm	Bachurski et al. (2019), Comfort et al. (2021), Maas et al. (2015) and Serrano‐Pertierra et al. (2020)
Resistive Pulse Sensing	Size distribution, concentration	• Quantitative • Does not rely on diffraction limited measurement • Analyzed in solution	• Size‐limited: >50 nm • Expensive equipment	Maas et al. (2017) and Vogel et al. (2017)
Scanning electron microscopy	Individual EV size, morphology, and surface topography	• High resolution • Direct imaging	• Expensive equipment • Destructive • Low throughput • Nonquantitative	Han et al. (2021) and Hartjes et al. (2019)
Surface enhanced Raman spectroscopy	Chemical fingerprint	• Minimal sample prep • Label‐free • Nondestructive • High sensitivity • Low sample volume • Rapid acquisition • High throughput	• Expensive lithographic substrates required • Issues with reproducibility	Jones et al. (2015) and Zhang et al. (2021)
Total internal reflection fluorescence microscopy	Surface/soluble markers, surface binding kinetics	• High throughput • Multiplexed detection • Quantitative • Direct imaging	• EVs must be immobilized at a surface • Expensive materials (i.e. antibodies)	He et al. (2019) and Zhou et al. (2020)
Transmission electron microscopy	Individual EV size, morphology, and inner structure	• High resolution • Direct imaging	• Expensive equipment • Extensive sample prep (staining, fixation) • Destructive • Low throughput • Expensive nanoparticle labels required for chemical specificity	Lennon et al. (2019) and Malenica et al. (2021)