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associated change in EV proteome varies with the EV type. Practically, it transforms a biological fluid as a
single source of biomarkers into multiple independent sources of biomarkers.

A third advantage is that as the different EV types are novel sources of biomarkers, the distribution of
known and well-characterized biomarkers and proteins in these EVs are not known. As we have shown,
examining their distribution in the different EV types as a function of the pathophysiological state is a
viable strategy to identify candidate biomarkers. Furthermore, it is easier to validate these known and
characterized biomarkers and proteins as the necessary assay reagents such as antibodies are available.

A fourth advantage is that the technique for EV isolation by lipid-binding ligands is akin to the commonly
used antibody capture techniques, and can be easily adapted for direct coupling to an ELISA for biomarker
assay. Hence isolating EV from biological fluids and assaying for biomarkers by ELISA can be readily
adapted for the standard automated 96 well ELISA platform used in routine clinical laboratory settings. It is
[38]
also amenable to the recent developments in nano-plasmonic technology .

Finally, the volume of plasma required for extracting EVs by their affinity for membrane lipid binding
ligands in sufficient quantity for a typical ELISA ranges from 10-30 µL biological fluids per assay [34,36] . As
each of the EV types identified so far are unique EVs [28,33] , the volume of biological fluid could be reduced
further by using multiplexed EV isolation system where the different membrane lipid-binding ligands are
added to the same sample and the bound EVs are then extracted according to the ligands bound to the
EVs.

Despite the advantages of lipid binding ligands for isolation of EVs, there are significant limitations with
this approach. The avidity of the ligands for the different lipids has turned out to be a double-edged sword.
The dissociation constants (K ) of CTB to GM1 is between 4.6 × 10 -12[39] and 7.3 × 10 -10[40] , K of AV to
D
D
[32]
phosphatidylserine is 5 × 10 M [41,42] and K of ST to GB3 is 5-30 × 10 M . These dissociation constants
-10
-10
D
[43]
-11
which are comparable to the median K of antibody to antigen is 5-30 × 10 M illustrate the difficulty
D
in dissociating the EVs from the ligands for subsequent characterisation. While this poses significant
challenge for biological studies of the different EV types, biomarker discovery and assay can be readily
performed on the ligand-bound EVs.

Another drawback of this approach is the limited number of suitable lipid binding ligands to isolate
EVs. Also, it is highly conceivable that many other EV types may not exhibit sufficiently distinctive lipid
molecules for EV stratification by this method. Also, the lipid binding characteristics of an EV is not likely
to be a characteristic of a specific cell type and is thus not likely to provide information on the cell source
of the EVs. Nevertheless, lipid binding ligands represent an additional tool to isolate and stratify EV types
that could increase the yield and specificity of EV types when complemented with present EV isolation
tools such as size exclusion fractionation or immunoaffinity isolation technology.

CONCLUSION
We propose that the use of membrane lipid binding ligands in EV isolation for liquid biopsy is a viable and
robust strategy to circumvent the present technical limitations in isolating bona fide EVs from biological
fluids for liquid biopsy. In addition, this isolation technique is versatile can be easily integrated into the
current clinical analysis platforms using 96 well assay plates and even newer analytical platforms such as
[44]
nano-plasmonics which uses the interaction of light and electronic oscillations in metallic substrates in a
nanometer-dimension to detect the presence of single EV.