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1.
ACS Sens ; 5(9): 2820-2833, 2020 09 25.
Article in English | MEDLINE | ID: mdl-32935542

ABSTRACT

For more effective early-stage cancer diagnostics, there is a need to develop sensitive and specific, non- or minimally invasive, and cost-effective methods for identifying circulating nanoscale extracellular vesicles (EVs). Here, we report the utilization of a simple plasmonic scaffold composed of a microscale biosilicate substrate embedded with silver nanoparticles for surface-enhanced Raman scattering (SERS) analysis of ovarian and endometrial cancer EVs. These substrates are rapidly and inexpensively produced without any complex equipment or lithography. We extensively characterize the substrates with electron microscopy and outline a reproducible methodology for their use in analyzing EVs from in vitro and in vivo biofluids. We report effective chemical treatments for (i) decoration of metal surfaces with cysteamine to nonspecifically pull down EVs to SERS hotspots and (ii) enzymatic cleavage of extraluminal moieties at the surface of EVs that prevent localization of complementary chemical features (lipids/proteins) to the vicinity of the metal-enhanced fields. We observe a major loss of sensitivity for ovarian and endometrial cancer following enzymatic cleavage of EVs' extraluminal domain, suggesting its critical significance for diagnostic platforms. We demonstrate that the SERS technique represents an ideal tool to assess and measure the high heterogeneity of EVs isolated from clinical samples in an inexpensive, rapid, and label-free assay.


Subject(s)
Extracellular Vesicles , Metal Nanoparticles , Biocompatible Materials , Liquid Biopsy , Porosity , Silver
2.
Anal Chem ; 89(10): 5357-5363, 2017 05 16.
Article in English | MEDLINE | ID: mdl-28345878

ABSTRACT

Extracellular vesicles (EVs), including exosomes, are circulating nanoscale particles heavily implicated in cell signaling and can be isolated in vast numbers from human biofluids. Study of their molecular profiling and materials properties is currently underway for purposes of describing a variety of biological functions and diseases. However, the large, and as yet largely unquantified, variety of EV subpopulations differing in composition, size, and likely function necessitates characterization schemes capable of measuring single vesicles. Here we describe the first application of multispectral optical tweezers (MS-OTs) to single vesicles for molecular fingerprinting of EV subpopulations. This versatile imaging platform allows for sensitive measurement of Raman chemical composition (e.g., variation in protein, lipid, cholesterol, nucleic acids), coupled with discrimination by fluorescence markers. For exosomes isolated by ultracentrifugation, we use MS-OTs to interrogate the CD9-positive subpopulations via antibody fluorescence labeling and Raman spectra measurement. We report that the CD9-positive exosome subset exhibits reduced component concentration per vesicle and reduced chemical heterogeneity compared to the total purified EV population. We observed that specific vesicle subpopulations are present across exosomes isolated from cell culture supernatant of several clonal varieties of mesenchymal stromal cells and also from plasma and ascites isolated from human ovarian cancer patients.


Subject(s)
Exosomes/metabolism , Optical Tweezers , Tetraspanin 29/analysis , Animals , Antibodies/immunology , Female , Fluorescent Dyes/chemistry , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Nanoparticles/chemistry , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Principal Component Analysis , Rats , Spectrum Analysis, Raman , Tetraspanin 29/immunology
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