Polydimethylsiloxane (PDMS) is an inexpensive robust polymer that is commonly used as the fundamental fabrication material for soft‐lithography‐based microfluidic devices. Owing to its versatile material properties, there are some attempts to use PDMS as a porous 3D structure for sensing. However, reliable and easy fabrication has been challenging along with the inherent hydrophobic nature of PDMS hindering its use in biomedical sensing applications. Herein, a cleanroom‐free inexpensive method to create 3D porous PDMS structures, “ExoSponge” and the effective surface modification to functionalize its 3D porous structure is reported. The ability of ExoSponge to recover cancer‐associated extracellular vesicles (EVs) from complex biological samples of up to 10 mL in volume is demonstrated. When compared to ultracentrifugation, the ExoSponge showes a significant increase in cancer EV isolation of more than 210%. Targeted ultra‐high pressure liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) is further employed to profile 70 metabolites in the EVs recovered from the lung cancer patient's plasma. The resulting profiles reveal the potential intraexosomal metabolite biomarker, phenylacetylglutamine (PAG), in non‐small cell lung cancer. The high sensitivity, simple usage, and cost‐effectiveness of the ExoSponge platform creates huge potential for rapid, economical and yet specific isolation of exosomes enabling future diagnostic applications of EVs in cancers.
Extracellular vesicles (EVs) are emerging as a potential diagnostic test for cancer. Owing to the recent advances in microfluidics, on‐chip EV isolation is showing promise with respect to improved recovery rates, smaller necessary sample volumes, and shorter processing times than ultracentrifugation. Immunoaffinity‐based microfluidic EV isolation using anti‐CD63 is widely used; however, anti‐CD63 is not specific to cancer‐EVs, and some cancers secrete EVs with low expression of CD63. Alternatively, phosphatidylserine (PS), usually expressed in the inner leaflet of the lipid bilayer of the cells, is shown to be expressed on the outer surface of cancer‐associated EVs. A new exosome isolation microfluidic device (newExoChip), conjugated with a PS‐specific protein, to isolate cancer‐associated exosomes from plasma, is presented. The device achieves 90% capture efficiency for cancer cell exosomes compared to 38% for healthy exosomes and isolates 35% more A549‐derived exosomes than an anti‐CD63‐conjugated device. Immobilized exosomes are then easily released using Ca2+chelation. The recovered exosomes from clinical samples are characterized by electron microscopy and western‐blot analysis, revealing exosomal shapes and exosomal protein expressions. ThenewExoChip facilitates the isolation of a specific subset of exosomes, allowing the exploration of the undiscovered roles of exosomes in cancer progression and metastasis.
more » « less- NSF-PAR ID:
- 10372436
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Small
- Volume:
- 15
- Issue:
- 47
- ISSN:
- 1613-6810
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
more » « less
Abstract -
The analysis of membranous extracellular vesicles, such as exosomes vesicles (EV) opens a new direction for the rapid disease diagnosis because EVs can carry molecular constituents of their originating cells. Secreted by mammalian cells, the size of most membrane-bound phospholipid EVs ranges from 50 to 150 nm in diameter. Recent studies have demonstrated the potential of using EVs for cancer diagnosis and treatment monitoring. To diagnose infectious diseases using EVs, the ability to discriminate EVs from host cells and parasites is key. Here, we report a rapid EV analysis assay that can discriminate EVs based on a host-specific transmembrane protein (CD63 antigen) using a label-free optical biosensor.more » « less
-
There is great interest in advancing methodologies for the isolation and characterization of exosomes (30–150 nm, extracellular vesicles (EVs)) for fundamental biochemical research and liquid biopsy applications. This is due to the accessibility of exosomal surface biomarkers, providing relevant biochemical information from their cells of origin. Exosome-based techniques hold potential for diagnostic applications through less invasive sampling ( versus the physical extraction methods of pathology). This study demonstrates a simple spin-down tip methodology for generic exosome capture, followed by immunoaffinity-based fluorescent labeling to classify EVs captured on a polyester capillary-channeled polymer (C-CP) fiber stationary phase. An antibody to the generic EV tetraspanin protein (CD81) is employed to confirm the presence of biologically active EVs on the fiber surface. An antibody to the CA125 protein, upregulated in the case of ovarian cell stress, is included as a cancer marker protein. Scanning electron microscopy and confocal fluorescence microscopy were performed directly on the capture fibers to visualize the morphology and assess the bioactivity/identity of captured vesicles. This report provides a proof-of-concept for an efficient means of isolating, purifying, immunolabeling, and fluorescent imaging for the biomarker assessment of extracellular vesicles on a single platform . Herein lies the novelty of the overall approach. The ability to affect the entire isolation, immunolabeling, and imaging process in <5 hours is demonstrated. The C-CP fiber spin-down tip is an efficient exosome isolation methodology for microliter samples from diverse media (human urine and cell culture media here) towards diverse means of characterization and identification.more » « less
-
more » « less
Abstract This work presents chemically stable and biodegradable hydrogel beads for the isolation of circulating tumor cells (CTCs) and circulating exosomes in liquid biopsy. The liquid biopsy hydrogel beads (LBbeads) consisting of alginate and poly(vinyl alcohol) hydrogels show both chemical stability and stimuli‐degradable characteristics. Unlike single‐component hydrogels, this hybrid form is not easily degraded by buffers or cell culture media while its degradable characteristic remains; thus, it is useful in bio‐applications requiring multi‐step processes with various reagents and lengthy incubation periods. We applied our platform to clinical samples for isolating two promising circulating biomarkers for a liquid biopsy, CTCs and exosomes, by conjugating the hydrogel surface with anti‐EpCAM and anti‐CD63 antibodies, respectively, thus achieving 37.4 CTCs and comparable amount of exosome recovery per 1 milliliter of blood. The results show easy device‐free isolation and retrieval of CTCs and exosomes, with recovered circulating biomarkers successfully analyzed by western blot analysis and fluorescence microscopy. We believe that this simple and versatile platform enables us to isolate prominent circulating biomarkers for clinical use in cancer diagnosis.
-
more » « less
Abstract Exosomes, a subset of extracellular vesicles (EVs, 30–200‐nm diameter), serve as biomolecular snapshots of their cell of origin and vehicles for intercellular communication, playing roles in biological processes, including homeostasis maintenance and immune modulation. The large‐scale processing of exosomes for use as therapeutic vectors has been proposed, but these applications are limited by impure, low‐yield recoveries from cell culture milieu (CCM). Current isolation methods are also limited by tedious and laborious workflows, especially toward an isolation of EVs from CCM for therapeutic applications. Employed is a rapid (<10 min) EV isolation method on a capillary‐channeled polymer fiber spin‐down tip format. EVs are isolated from the CCM of suspension‐adapted human embryonic kidney cells (HEK293), one of the candidate cell lines for commercial EV production. This batch solid‐phase extraction technique allows 1012EVs to be obtained from only 100‐µl aliquots of milieu, processed using a benchtop centrifuge. The tip‐isolated EVs were characterized using transmission electron microscopy, multi‐angle light scattering, absorbance quantification, an enzyme‐linked immunosorbent assay to tetraspanin marker proteins, and a protein purity assay. It is believed that the demonstrated approach has immediate relevance in research and analytical laboratories, with opportunities for production‐level scale‐up projected.
