Here's the brief review of the background of exosome and related isolation, quantification and detection methodology.

1. A brief review of
exosome & its
detection
Presenter: Peng-Wen Liu
Advisor: Hsien-Chang Chang

2. Introduction
• Extracellular vesicles are lipid bilayer-closed
structures derived from endocytosis and
secreted by almost all types of cells,
including ectosomes and exosomes.
• The heterogeneity of exosomes is likely
reflective of their size, content, functional
impact on recipient cells, and cellular origin.
• Research in this field is stimulated by the
potential of exosomes as diagnostic and
therapeutic tools for the treatment of
various diseases, including
neurodegeneration, cardiovascular
dysfunction, and cancer.
Membrane budding
MVB fusion and
exosome release
Exosome vs. Ectosome:
Some studies proposed that ectosomes bear
CD9 or CD81 but not CD63 while exosomes
bear CD63 and other tetranspanins.

3. Hallmarks of exosomes
• Exosomes can contain
membrane proteins,
cytosolic and nuclear
proteins metabolites,
and nucleic acids, which
renders exosome
different regulatory
functions.
• CD9, CD63 and CD81 are
the tetraspanins
enriched in the
membrane of exosomes,
which are regarded as
exosome biomarkers.
ALIX: apoptosis-linked gene 2-interacting protein X
HSP: heat shock protein

4. Zhu, L. et al. J Hematol Oncol 13, 152 (2020).
• Exosomes can be detected in
invasive liquid biopsies for
disease diagnosis and prognosis:
• Elevated urine-derived
exosomal miR-21 has been
associated with bladder and
prostate cancer.
• Exosomes for the delivery of
drug payload(s) are being
actively explored as therapeutic
agents:
• Clinical-grade MSC derived
exosomes with KrasG12D siRNA
payload (iExosomes) for
pancreatic cancer treatment in
multiple animal models
without any obvious toxicity.
Clinical applications
of exosomes

5. Common exosomal
separation techniques
Fig. Schematic representation of common
exosomal separation techniques.
(A) Ultracentrifugation (Gold standard method)
(B) Density gradient centrifugation
(C) Dead-end filtration (DEF)
(D)Tangential flow filtration (TFF)
(E) Size-exclusion chromatography
(F) Immunoaffinity
Limitations:
1. Time-consuming
2. Requires large sample volume
3. Need specialized equipment
4. Low yield and purity
Chen J, et al. Front Bioeng Biotechnol. 2022;9:811971
> 4 hr
> 16 hr
Prepare discontinuous
gradient of sucrose or
iodixanol

6. Common exosomal
separation techniques- ExoQuik kit
Manufacture: System Biosciences (SBI); 台灣代理商：騰達行
Peterson MF, Otoc N, Sethi JK, Gupta A, Antes TJ. Integrated systems for exosome investigation. Methods. 2015;87:31-45.
• Captures and collects exosomes of a certain size
range (60 – 150 nm) in ‘‘polymer nets’’ that can
be recovered by a simple, low speed
centrifugation on the bench top at 1500g.
• Once the exosome pellet is obtained, the
supernatant containing excess polymer is removed
and the exosomes can then be resuspended in a
suitable solution, such as PBS.
• This resuspension process dilutes the residual
polymer in the exosome pellet enough to dissolve
the polymer net and liberate intact exosomes.
The entire process can take as little at 30 min.

7. Methods for exosome quantification-
Nanoparticle tracking analysis (NTA)
Principle:
Provides particle size and concentration
in liquid suspension using the properties
of Brownian motion and light scattering.
Advantages:
1. Does not rely on detection of a
specific marker
2. Direct quantification
Disadvantages:
1. Expensive instrument
2. Photobleaching and potential
background from dye aggregates
Microscope
(20x)
Particles suspended in liquid
Laser beam (Approx 50 μm)
Metalized surface
Glass
Particles scattering from laser beam
This instrument is available in NCKU center for Micro/Nano science technology.

8. Principle:
Detects particles suspended in a fluid
by their interaction with a laser beam
as they flow through a detection cell.
Advantages:
1. Direct quantification
Disadvantages:
1. Insensitivity to smaller exosomes.
2. Requires binding to fuorophore-
conjugated antibody-coated beads
3. Swarm effect that means multiple
smaller vesicles are counted as
single particle. This may provide
false positive result
Methods for exosome quantification-
Flow cytometry
Flow cytometry typically has a lower practical size limit of
around 300 nm at which point the signal is indistinguishable
from the baseline noise level.

9. Scheme 1. The electrochemical aptasensor for exosome detection based on click chemistry and HCR for signal amplification.
An Y, Jin T, Zhu Y, Zhang F, He P. An ultrasensitive electrochemical aptasensor for the determination
of tumor exosomes based on click chemistry. Biosens Bioelectron. 2019;142:111503.
Signal amplification
CuAAC click
chemistry
HRP catalysis
3 hr for hybridization
chain reaction
0.5 hr for streptavidin-
HRP conjugation
OPD: o-phenylenediamine

10. Fig. 2. CV (A) and EIS (B) of different modified GCEs in 0.1M KCl containing 5.0mM [Fe(CN)6]3-/4- solution: bare
GCE (a), DenAu/rGO/GCE (b), CD63 aptamer/DenAu/rGO/GCE (c), MCH/CD63 aptamer/DenAu/rGO/GCE (d),
exosomes/MCH/CD63 aptamer/DenAu/rGO/GCE (e), and HCR-exosomes/MCH/CD63 aptamer/DenAu/rGO/GCE (f).
An Y, Jin T, Zhu Y, Zhang F, He P. An ultrasensitive electrochemical aptasensor for the determination
of tumor exosomes based on click chemistry. Biosens Bioelectron. 2019;142:111503.
a. Bare GCE
b. Reduction of graphene oxide and deposition of dendritic gold nanostructure
c. CD63 aptamer immobilation
d. MCH blocking
e. Exosome binding
f. HCR reaction

11. Fig. 3.
(C) DPV responses with (a) and
without (b) signal amplification by HCR;
(D) DPV responses with (a) and without
(b) the recognition of exosomes
Fig. 4.
(A) DPV responses of the
electrochemical aptasensor for the
exosomes at different
concentrations;
(B) linear relationship between the
electrochemical signal and the
logarithm of the exosome
concentration.
Error bars represent the relative
standard deviation of
measurements (%RSD ≤7.6%,
n=3).
An Y, Jin T, Zhu Y, Zhang F, He P. An ultrasensitive electrochemical aptasensor for the determination
of tumor exosomes based on click chemistry. Biosens Bioelectron. 2019;142:111503.
1.12×10
2
1.12×10
8
The DPV was performed with
HAc/NaAc buffer solution containing
2mM OPD and 4 mM H2O2.

12. Pang Y, Shi J, Yang X, Wang C, Sun Z, Xiao R. Personalized detection of circling exosomal PD-L1 based on Fe3O4@TiO2 isolation and
SERS immunoassay. Biosens Bioelectron. 2020;148:111800.
Scheme 1. A schematic view of the nanoparticles synthesis (a) and SERS tag-based exosomal PD-L1 detection (b).
Exosome
enrichment (5 min)
Detection time: 40 min
LoD: 1 PD-L1+ exosome/μl
4 μl
TBOT: Tetrabutyl titanate (Titanium tetrabutoxide)
The incubation time:
1. MBA immobilization: 2 hr
2. EDC/NHS activation: 15 min
3. Incubation with anti-PD-L1: overnight
(785 nm)
(300 nm)
(20 nm)

13. Fig. 3. (c) Capture efficiency comparison of the Fe3O4@TiO2 nanospheres and UC in serum and PBS respectively.
Fig. 4. SEM images and SERS signal of (a)Fe3O4@TiO2 (b) Fe3O4@TiO2/A549 exosome (c)Fe3O4@TiO2/A549 exosome/SERS tags.
(Ultracentrifugation)
Optimization for exosome isolation:
1. The quantity of Fe3O4@TiO2: 0.8 mg
2. Incubation time: 5 min

14. Fig. 5. (a) The SERS spectra with different A549 exosome concentrations (b) The correlation analysis of SERS intensity (at 1074 cm-1)
and the logarithm of exosome concentrations. Blank represents SERS intensity without any exosome added.
Fig. 6. (b) Scatter plots of the log [intensity] in the serum samples from the controls and the early-stage (stage I/II) and advanced
(stage III/IV) patients measured by the SERS system. Error bars are the mean and one standard deviation of the mean (SD).
Based on the personalized SERS (Surface-Enhanced Raman Scattering) signal analysis,
NSCLC patients can be distinguished from the healthy controls easily.

15. Conclusion
1. Exosomes are extracellular vesicles generated by all cells and they carry nucleic
acids, proteins, lipids, and metabolites. They are mediators of near and long-
distance intercellular communication in health and disease and affect various
aspects of cell biology.
2. The ExoQuik kit can greatly simply the isolation process and the operation time
only requires 30 min. This method may be the suitable exosome separation
method for our future study.
3. The nanoparticle tracking analysis is widely used as the comparison methods for
exosome quantification. And the instrument is also available in NCKU.
4. The detection of exosomes depends on recognition of selected biomarkers, such as
tetraspanin CD63 or PD-L1 on the exosome plasma membrane. Hence, the
selection of target biomarkers should be taken seriously for future studies.