**3.1 Conventional FCM combined with amplified single EV signals**

In the first approach, several methods are feasible for signal amplification to produce detectable structures via FCM. The proteins on single EVs could be detected

#### **Figure 6.**

*Colocalization of two proteins on the same individual EV were detected via proximity ligation assay (PLA) and the signal was amplified via rolling circle amplification (RCA) to form a detectable structure under FCM.*

#### **Figure 7.**

*The principle of flow cytometer (FCM) and an example of detected EV samples with scattered signals and fluorescently labeled Her-2 antigen.*

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

with aptamers with a trigger sequence at the end, which then trigger hybridization chain reaction via DNA hairpins with biotin labels. Addition of avidin-fluorescent labels lead to the formation of an 500 nm fluorescently labeled EV-centered complex to be detect in FCM. Signal amplification via hybridization chain reaction occurred after single EV biomarker recognition by a conformation switchable aptamer. Two biomarkers could be targeted and analyzed simultaneously [12]. In another study, investigators utilized DNA oligonucleotides labeled antibodies to detect two proteins on the same EV. Proximity ligation assay occurs and rolling circle amplification (RCA) reaction produce thousands of copies of DNA sequences for fluorescent labels to bind and consequently forms a structure detectable in FCM (**Figure 6**) [13].

### **3.2 High resolution FCM and nano FCM**

In the second approach, FCM were configured to overcome the limitations of detectable particle size and adapt to the analysis of nanoscale EVs. The improved sensitivity for EV detection includes higher laser power, slower flow and longer signal integration times and so on [14, 15]. Fluorescence based triggering could be superior over light scatter based triggering [16, 17]. Short wavelength laser can detect smaller particle size, for example 405 nm violet side scatter (VSSC) in Beckman Coulter CytoFLEX S [18]. As particle size decreases, the scattered light decreases rapidly. Data acquisition always needs to be optimized for better SSC resolution and efficacy of the detection of dim fluorescent single EVs. Therefore, novel noise reduction algorithm is applied for the high sensitivity detection of small particles. Calibrating light scatter detection for EV analysis have been proposed [19]. Nowadays, Apogee flow cytometry with micro-PLUS mode announced a detectable size of as low as 80 nm [20]. Yan's lab developed nano flow cytometer (nFCM) employing single molecule fluorescence detection in a sheathed flow. Two single-photon counting avalanche photodiodes (APDs) were used to detect side scatter and fluorescence of individual EVs, respectively, which enables phenotyping of single EVs as small as 40 nm (**Figure 7**) [21].
