Exosomes are nanoscale vesicles found in many bodily fluids which play a significant role in cell-to-cell signaling and contain biomolecules indicative of their cells of origin. Recently, microfluidic devices have provided the ability to efficiently capture exosomes based on specific membrane biomarkers, but releasing the captured exosomes intact and label-free for downstream characterization and experimentation remains a challenge. We present a herringbone-grooved microfluidic device which is covalently functionalized with antibodies against general and cancer exosome membrane biomarkers (CD9 and EpCAM) to isolate exosomes from small volumes of high-grade serous ovarian cancer (HGSOC) serum. Following capture, intact exosomes are released label-free using a low pH buffer and immediately neutralized downstream to ensure their stability. Characterization of captured and released exosomes was performed using fluorescence microscopy, nanoparticle tracking analysis, flow-cytometry, and SEM. Our results demonstrate the successful isolation of intact and label-free exosomes, indicate that the amount of both total and EpCAM+ exosomes increases with HGSOC disease progression, and demonstrate the downstream internalization of isolated exosomes by OVCAR8 cells. This device and approach can be utilized for a nearly limitless range of downstream exosome analytical and experimental techniques, both on and off-chip.
Because of limits on specificity and purity to allow for indepth protein profiling, a standardized method for exosome isolation has yet to be established. In this study, we describe a novel, in-house microfluidic-based device to isolate exosomes from culture media and patient samples. This technology overcomes contamination issues because sample separation is based on the expression of highly specific surface markers CD63 and EpCAM. Mass spectrometry revealed over 25 exosome proteins that are differentially expressed in high-grade serous ovarian cancer (HGSOC) cell lines compared with normal cells-ovarian surface epithelia cells and fallopian tube secretory epithelial cells (FTSEC). Top exosome proteins were identified on the basis of their fold change and statistical significance between groups. Ingenuity pathway analysis identified STAT3 and HGF as top regulator proteins. We further validated exosome proteins of interest (pSTAT3, HGF, and IL6) in HGSOC samples of origin-based cell lines (OVCAR-8, FTSEC) and in early-stage HGSOC patient serum exosome samples using LC/MS-MS and proximity extension assay. Our microfluidic device will allow us to make new discoveries for exosome-based biomarkers for the early detection of HGSOC and will contribute to the development of new targeted therapies based on signaling pathways that are unique to HGSOC, both of which could improve the outcome for women with HGSOC. Significance: A unique platform utilizing a microfluidic device enables the discovery of new exosome-based biomarkers in ovarian cancer.
There
is a significant and growing research interest in the isolation of
extracellular vesicles (EVs) from large volumes of biological samples
and their subsequent concentration into clean and small volumes of
buffers, especially for applications in medical diagnostics. Materials
that are easily incorporated into simple sampling devices and which
allow the release of EVs without the need for auxiliary and hence
contaminating reagents are particularly in demand. Herein, we report
on the design and fabrication of a flexible, microporous, electrochemically
switchable cloth that addresses the key challenges in diagnostic applications
of EVs. We demonstrate the utility of our electrochemically switchable
substrate for the fast, selective, nondestructive, and efficient capture
and subsequent release of EVs. The substrate consists of an electrospun
cloth, infused with a conducting polymer and decorated with gold particles.
Utilizing gold–sulfur covalent bonding, the electrospun substrates
may be functionalized with SH-terminated aptamer probes selective
to EV surface proteins. We demonstrate that EVs derived from primary
human dermal fibroblast (HDFa) and breast cancer (MCF-7) cell lines
are selectively captured with low nonspecific adsorption using an
aptamer specific to the CD63 protein expressed on the EV membranes.
The specific aptamer–EV interactions enable easy removal of
the nonspecifically bound material through washing steps. The conducting
polymer component of the cloth provides a means for efficient (>92%)
and fast (<5 min) electrochemical release of clean and intact captured
EVs by cathodic cleavage of the Au–S bond. We demonstrate successful
capture of diluted EVs from a large volume sample and their release
into a small volume of clean phosphate-buffered saline buffer. The
developed cloth can easily be incorporated into different designs
for separation systems and would be adaptable to other biological
entities including cells and other EVs. Furthermore, the capture/release
capability holds great promise for liquid biopsies if used to targeted
disease-specific markers.
Electrochemical techniques offer great opportunities
for the capture
of chemical and biological entities from complex mixtures and their
subsequent release into clean buffers for analysis. Such methods are
clean, robust, rapid, and compatible with a wide range of biological
fluids. Here, we designed an electrochemically addressable system,
based on a conducting terpolymer [P(EDOT-co-EDOTSAc-co-EDOTEG)] coated onto a carbon cloth substrate, to selectively
capture and release biological entities using a simple electrochemical
redox process. The conducting terpolymer composition was optimized
and the terpolymer-coated carbon cloth was extensively characterized
using electrochemical analysis, Raman and Fourier transform-infrared
spectroscopy, water contact angle analysis, and scanning electron
microscopy. The conductive terpolymer possesses a derivative of EDOT
with an acetylthiomethyl moiety (EDOTSAc), which is converted into
a “free” thiol that then undergoes reversible oxidation/reduction
cycles at +1.0 V and −0.8 V (vs Ag/AgCl), respectively. That
redox process enables electrochemical capture and on-demand release.
We first demonstrated the successful electrochemical capture/release
of a fluorescently labeled IgG antibody. The same capture/release
procedure was then applied to release extracellular vesicles (EVs),
originating from both MCF7 and SKBR3 breast cancer cell line bioreactors.
EVs were captured using the substrate-conjugated HER2 antibody which
was purified from commercially available trastuzumab. Capture and
release of breast cancer EVs using a trastuzumab-derived HER2 antibody
has not been reported before (to the best of our knowledge). A rapid
(2 min) release at a low potential (−0.8 V) achieved a high
release efficiency (>70%) of the captured, HER2+ve,
SKBR3
EVs. The developed system and the electrochemical method are efficient
and straightforward and have vast potential for the isolation and
concentration of various biological targets from large volumes of
biological and other (e.g., environmental) samples.
Extracellular vesicles (EVs) are micro and nanoscale lipid-enclosed packages that have shown potential as liquid biopsy targets for cancer because their structure and contents reflect their cell of origin. However, progress towards the clinical applications of EVs has been hindered due to the low abundance of disease-specific EVs compared to EVs from healthy cells; such applications thus require highly sensitive and adaptable characterization tools. To address this obstacle, we designed and fabricated a novel space curvature-inspired surfaced-enhanced Raman spectroscopy (SERS) substrate and tested its capabilities using bioreactor-produced and size exclusion chromatography-purified breast cancer EVs of three different subtypes. Our findings demonstrate the platform’s ability to effectively fingerprint and efficiently classify, for the first time, three distinct subtypes of breast cancer EVs following the application of machine learning algorithms on the acquired spectra. This platform and characterization approach will enhance the viability of EVs and nanoplasmonic sensors towards clinical utility for breast cancer and many other applications to improve human health.
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