**5. Barcoding based single EV analysis**

In the methods described above, the number of simultaneously analyzed biomarkers is limited to types of distinguishable readout signals like fluorescent dyes etc.

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

To achieve multiplexed high-throughput single EV analysis, researchers unitized molecular tools named Proximity Barcoding Assay (PBA) to detect colocalized proteins on the same EV [33]. The principle is that only proteins on the same individual EV are in proximity and therefore labeled with the same EV tags in PBA.

PBA probes were prepared by labelling of selected antibodies with DNA oligonucleotides containing unique 8-nt protein tag (**Figure 14a**). Rolling circle amplification

**Figure 14.** *Schematic illustration of proximity barcoding assay (PBA) and 38-plex analysis of 18 EV samples.*




*Comparison of single EV analysis methods.*
