The Detection Method of Cell-Derived Exosomes- Juniper Publishers
Juniper Publishers- Journal of Cell Science
Abstract
Exosomes are small extracellular vesicles (30-100nm)
from various cells, and have emerged as a promising biomarker of some
diseases. Quantitative detection of cell-derived exosome is appealing
and challenging. In recent years, many kinds of methods have emerged for
the detection and analysis of exosomes, including electrochemical
method, microfluidic technology, microarray chip and colorimetric
method. We summarize the current detection methods of exosomes and
highlight the development direction of detection method for exosomes.
Keywords: Exosomes; Electrochemical biosensors; Microarray chip; Microfluidic platform; Flow immunoassay; Colorimetric methodIntroduction
Exosomes were initially reported in 1983 by Johnstone and colleagues while culturing reticulocytes [1]. It is already very clear that exosomes are cell-derived lipid bilayer vesicles that range from 30 to 100nm in diameter [2], are released by many types of cells, such as immune cells and tumor cells [3].
And Exosomes can be found in most bodily fluid, including blood, urine,
saliva, amniotic fluid, breast milk, hydrothoracic fluid, and ascitic
fluid, as well as in culture medium of most cell types [4].
Exosomes are also an extracellular subset containing microRNAs,
messenger RNAs (mRNA), DNA fragments, lipids and proteins for cell
communication [5], are transported through the bloodstream and other body fluids [6].
A series of emerging evidence have suggested that exosomes may play an
important role in the interaction between tumor cells and their
surrounding cells in the tumor microenvironment. However, the detection
of exosomes remains challenging and restricts its clinical application.
According to the existing literatures, the review summarizes and
analyzes the latest detection methods of exosomes, and prospects the
detection technology of the exosomes.
Electrochemical Biosensor
Electrochemical biosensors combine the sensitivity of
electroanalytical methods with the inherent bioselectivity of the
biological component [7]. Wang et al. [8]
developed a nanotetrahedron (NTH)-assisted electrochemical aptasensor
for direct capture and detection of hepatocellular exosomes, and
provided a proof-of-concept for sensitive and efficient quantification
of tumor-derived exosomes [8].
The NTH-assisted electrochemical aptasensor combined the strengths of
advanced aptamer technology, DNA-based nanostructure and portable
electrochemical devices. Aptamer LZH8 with superior binding selectivity
on target hepatocarcinoma cells (HepG2) was used for the proposed
electrochemical aptasensor. Yadav et al. [9] reported an electrochemical method to directly quantify the breast cancer cell-derived exosomes in cell culture media [9].
The method exhibited an excellent specificity for human epidermal
growth factor receptor 2 (+) BT474 cell-derived exosomes (detection
limit of 4.7*105 exosomes/μL) with an RSD of < 4.9% (n=3). Zhou et al. [10]
developed an electrochemical biosensor based on aptamers specific to
exosome transmembrane protein CD63 for the quantitative detection of
exosomes [10].
The biosensors functioned well both in “clean” (HEPES Buffer) and
“dirty” (DMEM cell culture media containing 10% fetal bovine serum)
solutions with a detection limit of 1x106 particles/mL and a linear
range extending two orders of magnitude to 1x108 particles/mL. These
three studies only detected exosomes in cell culture media, and didn't
involve the detection of exosomes in body fluid samples.
Microarray Chip
Microarrays with biomolecules, cells and tissues
immobilized on solid substrates are important tools for biological
research, including genomics, proteomics, and cell [11]. Ibsen et al. [12]
developed an alternating current electrokinetic (ACE) microarray chip
device to rapidly isolate and recover glioblastoma exosomes from
undiluted human plasma samples [12].
The detection of the exosome specific pattern of external CD63 membrane
protein and internal TSG101 protein17 confirms the presence of
glioblastoma exosomes among the collected extracellular vesicles by the
ACE microarray chip device. The ACE device achieved the direct detection
of exosomes in human plasma. Daaboul et al. [15]
presented a method based on Single Particle Interferometric Reflectance
Imaging Sensor (SP-IRIS) that allowed multiplexed phenotyping and
digital counting of various populations of individual exosomes
(>50nm) captured on a microarray-based solid phase chip [13].
The SP-IRIS for the detection of exosomes purified from HEK cell line
showed a good correlation for both capture antibodies CD63 and CD81, and
detection limits of 5.07x109 particles/mL for CD63 antibody
and 3.94x109 particles/mL for CD81 antibody. The imaging method could
capture exosomes from a very small volume (20uL) of human cerebrospinal
fluid by using antibodies directed against tetraspanins.
Microfluidic Platform
Microfluidic systems can be designed to obtain and
process measurements from small volumes of complex fluids with
efficiency and speed [14]. Zhao et al. [15]
employed a microfluidic ExoSearch chip for diagnosis of ovarian cancer
by multiplexed measurement of three exosomal tumor markers (CA-125,
EpCAM, CD24) using a training set of plasmas from ovarian cancer
patients [15].
The ExoSearch chip combined on-chip continuous-flow mixing and
immunomagnetic isolation with an in situ, multiplexed exosome
immunoassay, and was applied for ovarian cancer diagnosis via
quantifying a panel of tumor markers from exosomes in 20μL of blood
plasma within 40min. Zhang et al. [16]
developed a microfluidic platform based on a new graphene
oxide/polydopamine (GO/PDA) nano-interface for the analysis of
circulating exosomes [16].
CD81 mAb served for the capture antibody, and a cocktail of
biotinylated mAbs of CD9 and CD81 and EpCAM acted as the detection
antibody in the study. The platform was applied to discriminate ovarian
cancer patients from healthy controls by the quantitative detection of
circulating exosomes directly from 2μL of plasma without sample
processing. These two studies provided a feasible microfluidic platform
to directly detect exosomes in blood plasma from patients and to
facilitate basic investigation of exosomes.
Flow Immunoassay
Lateral flow immunoassay represents a well
established and appropriate technology among rapid assays because of its
low cost and user-friendliness [17].
Oliveira-Rodriguez et al. developed a novel lateral flow immunoassay
(LFIA) for the detection of exosomes based on the use of tetraspanins as
targets [18].
The platform was used to detect exosomes purified from different
sources, including cell culture supernatants, human plasma and urine,
and completed an one-step assay of exosomes in 15 min with detection
limit of 8.54x105 exosomes/μL. And the platform selected a
blend of anti-CD9 and anti-CD81 as capture antibodies and anti-CD63
labeled with gold nanoparticles as detection antibody. The work achieved
a rapid quantification of exosome from different biological samples and
provided promising techniques for clinical diagnosis.
Colorimetric Method
Colorimetry is that a well-designed chemical
interaction between the analyte and NPs surroundings leads to a change
of color allowing the visual detection of the target analyte [19]. Yao et al. [20]
reported a visible and colorimetric aptasensor based on DNA-capped
single-walled carbon nanotubes for the detection of exosomes, and
estimated the linear range from 1.84x106 to 2.21x107 particles/μL with
detection limit of 5.2 x10s particles/ μL [20].
An aptamer specific to exosomes transmembrane protein CD63 was used for
the proposed colorimetric aptasensor. The aptasensor was also used to
quantify exosomes in serum from breast cancer patients and healthy
individuals.
Conclusion and Outlook
The importance of exosomes is self-evident for cancer
research. At present, many methods, including electrochemical
biosensor, microarray chip, microfluidic platform, flow immunoassay and
colorimetric method, provide the detection technology of exosomes.
Electrochemical methods afford a sensitive and rapid analysis strategy,
but cannot directly detect exosomes in the biological sample.
Colorimetric method possesses a simple visual advantage, but is helpless
for the direct detection of exosomes in serum. Lateral flow immunoassay
is homogeneous and heterogeneous analytical methods which enables the
separation of reacted products from unreacted products without any
additional precipitation or washing procedure, and provide a promising
platform for detecting exosomes. Microarray chip can afford many
molecular signatures for cells, tissues and disease states that can be
used for disease diagnosis, prediction, prevention and drug discovery,
and is more suitable for the analysis and detection of exosomes in
complex samples. Microfluidic chip have had a considerable impact on the
fields of biomedical diagnostics, and is better to analyze the exosomes
in different body fluid. According to the above analysis and summary,
the studies of exosomes need to combine various advanced techniques to
obtain more comprehensive information of exosomes and to achieve the
diagnosis of related diseases in clinical.
Acknowledgement
We gratefully acknowledge the financial support of
the Natural Science Foundation of Fujian Province (2017J01547 and
2017J01346), the Key Projects of Science and Technology Plan of Fujian
Province (2015Y0030), the Scientific Research Project of Fujian
Provincial Colleges and Universities (JK2014045), and the Science and
Technology Plan Projects of Putian City (2013S01(2)).
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