**4. Nano-sensing technology**

#### **4.1 Nanoparticle tracking analysis (NTA)**

Nanoparticle tracking analysis (NTA) is well acknowledged and commercially available technique for EV characterization. By tracking the Brownian motion of each particle, the hydrodynamic diameter of each particle is calculated with Stockes-Einstein equation. Thereafter, size distribution and concentration of EVs in a fluid sample could be obtained. Combined with fluorescent labeled antibodies or molecular beacons, EV subpopulation with expression of certain protein or miRNAs, respectively, could be quantified (**Figure 8**).

As a modified tracking analysis of EVs, an on-chip microcapillary electrophoresis system was built with a laser dark-field microscope. The tracking analysis of the electrophoretic migration of individual exosomes were performed and the zeta potential distribution of exosomes were able to be analyzed. The system consists of a chip, a pair of platinum electrodes, a DC power supply, a laser source, an inverted microscope, and an EMCCD camera (**Figure 9**) [22].

**Figure 8.**

*The principle of nanoparticle tracking analysis (NTA) and its obtained EV size distribution.*

**Figure 9.** *Tracking analysis combined with zeta potential measurement of single EVs.*

### **4.2 Fluorescence correlation spectroscopy (FCS)**

Fluorescence Correlation Spectroscopy (FCS) were set up for characterize the GFP labeled vesicles at the single molecule – single vesicle level. FCS detects temporal fluorescence fluctuations in a defined confocal volume at a single photon sensitivity [23].

#### **4.3 Surface plasmon resonance (SPR)**

Surface plasmon resonance (SPR) detects real-time interactions between the receptors and the targeted biomolecules. SPR sensors show high sensitivity for labelfree exosome quantification, which could be attributed to a nanoscale sensing range closing to exosome size. The receptors were immobilized on the surface of the biosensor. When a solution of biomolecules flows across, the targeted biomolecules interact with the receptors to change the refractive index of the sensor surface. The response of the sensor is measured in resonance units (RU) and is proportional to the mass of molecules interacting with the immobilized receptor. (**Figure 10**) Nano-plasmonic exosome (nPLEX) system was developed based on SPR principles and utilized 200 nm plasmonic nanoholes for EV capture and detection. nPLEX enables quantitative detection and proteomic profiling of EVs at the throughput of 12 biomarkers

*High-Throughput Single Extracellular Vesicle Profiling DOI: http://dx.doi.org/10.5772/intechopen.97544*

#### **Figure 10.**

*The priciple of surface plasmon resonance (SPR).*

detection with antibodies immobilized at 12 channels of a microfluidic chip [24]. The nPLEX-FL assay enables multiplexed single EV analysis of targeted markers with improved sensitivities. In nPLEX-FL assay, EVs are biotinylated and then captured on the nanohole surface with avidin coating. EVs are immunostained by fluorescent labeled antibodies. Plasmon-enhanced fluorescence detection can amplify fluorescence signals using plasmonic metallic nanostructures. The labeled EVs are imaged, and their fluorescent intensities are analyzed. Therefore, biomarker distribution analysis could be performed on a single-EV level [25]. A localized surface plasmon resonance imaging (LSPRi) platform improves the limit of detection down to the single exosome limit. With a 400 nanopillar array sensor chip, single EVs are captured by nanopillar for both imaging and spectrometer measurement (**Figure 11**) [26].

### **4.4 Raman spectroscopy**

Raman spectroscopy is a spectral analysis method that determines the chemical properties of samples by measuring the vibration mode of covalent bonds caused by a radiation laser. Raman spectroscopy is non-destructive label-free analysis and therefore is suitable for analysis of biological samples. Laser tweezers Raman spectroscopy (LTRS)explore the chemical content of individual EVs, in which a tightly focused laser beam traps small particles at the laser's focal point. A confocal detection of Raman scattering from the precise focal volume allows EVs to be studied individually. Based on the exosomal chemical differences, EVs could be classified into subpopulations (**Figure 12**) [27–29].

#### **Figure 11.**

*A localized surface plasmon resonance imaging (LSPRi) nanopillar platform enables both spectrum and image readout.*

**Figure 12.**

*The principle of laser tweezers Raman spectroscopy (LTRS).*

**Figure 13.** *Multi modal characterization of antibody captured individual EVs via SEM, AFM and Raman spectroscopy.*

Surface-enhanced Raman spectroscopy (SERS) is a type of molecular vibration spectrum with enhanced Raman scattering signals from molecules adsorbed on noble metal (Ag, Au) nanostructures. SERS has been applied for the detection of cancer exosomes due to its high sensitivity, specificity, and multiplexing capability. Single EV analysis was performed in several studies. An graphene-coated periodic goldpyramid were used as SERS substrate to detect single EVs and EV samples from different biological sources were distinguished in their Raman signature [30]. In another study, gold nanoparticles (AuNP) coated single EVs were formed due to electrostatic interaction between cationic AuNP and anionic EV membrane. SERS spectra of individual ELVs with a high density of AuNP were then acquired and variations in the SERS spectra of individual ELVs could be used for EV heterogeneity analysis [31]. Multi modal characterization of individual EVs could be achieved after EV capture on antibody functionalized stainless steel substrate followed by characterization via SEM, AFM and Raman spectroscopy (**Figure 13**) [32].
