APPLICATION NOTE

Step up your EV characterization with Leprechaun
Introduction
Analyzing all the EVs in your sample without missing
critical particles is a challenge due to the small sizeof EVs and the abundance of similar-sized, non-EV
nanoparticles present in complex samples. This
problem is made even trickier by contaminants that
co-purify with EVs – including lipoproteins, protein
aggregates and bovine EVs 1. Most common particle
characterization tools, such as NTA and TRPS, can’ttell EVs apart from other particles meaning theycount the garbage as well as the gold.
Leprechaun utilizes an immuno-capture step toseparate EVs from other particles to ensure you
only count the vesicles you care about. It is the only
platform that provides vesicle concentration, sizeand phenotypic analysis (including cargo) on a single
vesicle basis, without the need for sample purification(Figure 1). Building on the technology underpinning its
predecessor, the ExoView R200, Leprechaun boasts
an improved lower sizing limit of 35 nm, opening thedoor to comprehensive, purification-free analysis ofEVs from exosomes to exomeres.

Figure 1: Leprechaun: The next step in EV analysis.

imaging while fluorescence microscopy provides
information on EV phenotype and cargo content.
For this experiment an EV standard with endogenous
GFP was analyzed (Sigma-Aldrich) and fluorescentanti-CD81-CF555 added to confirm tetraspanin
expression. The EV standard was diluted 1 in 25 in kit
provided Incubation Solution (Unchained Labs) andincubated on the Luni for 1 hour prior to washing andstaining. Data was analyzed in Leprechaun Exosome
Analysis (Unchained Labs) where colocalization
analysis was performed to determine the number oftetraspanin particles which are GFP tagged.

This app note describes how Leprechaun sizes up
even the smallest EVs to shed light on previously
undetected particles, and compares Leprechaun
performance relative to the ExoView R200.

EV analysis on Leprechaun

Leprechaun vs ExoView R200

Leprechaun’s Human Tetraspanin Luni consumablecaptures EVs on anti-CD81, -CD9 and -CD63 antibody spots. This immuno-capture step on the Luni
surface means it is not necessary to purify samples
prior to analysis, allowing Leprechaun to characterize
EVs from crude or pure samples. Once EVs have been
captured on the Luni surface the Luni Washer performs automated washing to remove nonspecificallybound particles, EVs are then counterstained with fluorescent antibodies, before being dried by the washer
ready for analysis on Leprechaun. Particles are sizedusing single particle interferometric reflectanceThe biggest step forward with Leprechaun is theability to size EVs all the way down to 35 nm – soparticles invisble to the ExoView R200 can now
be seen, sized, and added up. Both instruments
utilize single particle interferometry to measure
the diameter of individual vesicles captured on theLuni surface. The signal enhancing silicon dioxidesurface of the Luni maximizes the interference oflight reflected from the Luni surface and scatteredby the captured EV 2. Updated optics and refinedbackground subtraction allow Leprechaun to reduce

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STEP UP YOUR EV CHARACTERIZATION WITH LEPRECHAUN

A

B

Figure 2: (A) Total EV concentration as determined by Leprechaun and the ExoView R200. (B) Comparison of the concentration of GFP
positive and GFP negative EVs calculated by each platform. For both plots n=3 and error bars are SD, green bars = Leprechaun, bluebars = ExoView R200.

there are significantly fewer GFP negative EVs
detected by the ExoView (27% of total CD81+ EVs)
compared to Leprechaun (45%) (Figure 2B).

the lower limit of particle sizing from 50 nm, as
found on the R200, to the improved 35 nm.
The total concentration of EVs in the sample, asdetermined by each platform, is shown in Figure 2A.
Here, an EV is defined as a tetraspanin positiveparticle, which has a diameter within the sizing
range of the respective platform and may or may
not express GFP. There is no difference between theconfiguration, power, or sensitivity in the fluorescencechannels of the Leprechaun and ExoView, meaning fluorescence detection is not a driver of any
differences in measured EV concentration.

Particle size analysis performed on both instruments
reveals that the GFP positive and negative populations have distinct size profiles (Figure 3A & 3B).
On both platforms the mean diameter of GFP
positive EVs is measured to be 82 nm, compared
to under 60 nm for GFP negative EVs. While thesizing peak for the GFP positive EVs sits comfortably in the 50 – 200 nm range of the ExoView, asignificant proportion of the smaller, GFP negative
EVs appear to be below the 50 nm cut off.

Subpopulation analysis shows that while theconcentration of GFP positive EVs is comparable
between both platforms (6.5 x 109 particles/ml),
A

Leprechaun’s ability to detect and size sub 50 nm
particles means the true mean and mode of theB

Leprechaun

ExoView R200Figure 3: Size distributions of GFP positive and GFP negative EVs, acquired on Leprechaun (A) and ExoView R200 (B). For both plots
green line = GFP positive EVs, blue line = GFP negative EVs.

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STEP UP YOUR EV CHARACTERIZATION WITH LEPRECHAUN

Leprechaun

ExoView R200

Mean (nm)Mode (nm)Mean (nm)Mode (nm)

GFP+ EVs

82

72

82

75

GFP- EVs

48

42

58

50

Table 1: Mean and mode comparison for both GFP subpopulations on Leprechaun and ExoView R200.

GFP negative population can be obtained (Table 1).
The inclusion of these small EVs accounts for thedifference in total EV concentration between Leprechaun and ExoView (Figure 2A). Based on particle counts from Leprechaun, 61% of GFP negative
EVs are smaller than 50 nm, equivalent to 3.35 x
109 particles/ml. This value matches the differencein the concentration of GFP negative EVs between
ExoView (2.33 x 109) and Leprechaun (5.50 x 109).

Conclusion
There is an increasing appreciation of the heterogeneity of EVs, not only in terms of surface markers
expressed, but also vesicle size. The discovery ofexomeres in 2018 highlighted the abundance ofvesicles at the lower end of the size range 3. Most
analytical tools however, struggle to detect and size
biological particles below 50 nm in diameter, and so
provide no information on this particle population,
leading to inaccurate characterization of EV samples.

Titration of the EV standard highlights the linear
nature of the Leprechaun readout (Figure 4). EV
concentration can be determined from crude orpure samples down to a lower limit of detection of
5 x 106 particles/ml.

Leprechaun provides the full picture for your EV
sample, by sizing and phenotyping even the smallest
vesicles on a single particle basis. Building on thetechnology of its predecessor, the ExoView R200,
Leprechaun serves up EV concentration, size,
surface marker phenotyping and cargo detection
without the need for sample purification. No matter
how rare or small your EV population, Leprechaun
can give you the low down.

References
1 Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during
isolation and detection. Sodar, B et al. ScientificReports, 2016. 6:24316.
2 Digital detection of exosomes by interferometric
imaging. Daaboul, G et al. Scientific Reports, 2016.
6:37426.

Figure 4: Linearity of EV concentration on Leprechaun. N = 3,
mean values displayed, error bars = SD.

3 Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow
field-flow fractionation. Zhang, H et al. Nature Cell
Biology, 2018. 332-343.
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