A.; and the support from the Environmental Toxicology Program fund for Q. human serum and cell culture medium without tedious sample preparation was demonstrated, consuming much less sample compared to ELISA and Western Blot. We envision that our method will be valuable for quick quantification Rimonabant hydrochloride of EVs Rimonabant hydrochloride in biological samples, benefiting disease monitoring and functional study. Graphical Abstract INTRODUCTION Extracellular vesicles (EVs) are membrane-enclosed vesicles with sizes ranging from 50 to 200 nm; can be secreted by most cell types;1,2 and are found in various body fluids like blood, bile and serum.3 They carry miscellaneous molecular cargos including proteins, metabolites, and nucleic acids inherited from the parent cells;2 and can transfer them to recipient cells, serving as a new route for cell-to-cell communication.3 It is also believed that, EVs could mediate the tumor-related functions like formation, progression, and metastasis of malignant cells.4 Besides, cells under pathological conditions could produce a large number of EVs loaded with unique cargoes reflecting disease development.5,6 Thus, EVs are considered as promising markers in liquid biopsy for clinical diagnosis and prognosis. However, the abundance of the tumor-specific EVs in bio-fluids is very low, and their signals are Rimonabant hydrochloride buried within a large number of heterogeneous EVs from diverse sources, imposing great challenges to EV detection and quantification. Enzyme-linked immunosorbent assays (ELISAs) and Western blots (WB) are the gold standards for detection of EVs through specific protein recognition.7,8 Nanoparticle-tracking analysis (NTA) is another common method for EV analysis which measures the size distribution and particle concentration of clean EV samples.9 But these methods all require purification and enrichment of EVs prior to detection, adding complexity to the assay.10,11 In addition, without prior EV isolation, ELISA and WB could not differentiate the free and EV-bound proteins, making protein concentration detected not truly representative to that of the EVs.6 Conventional isolation methods like ultracentrifugation consume large amounts of samples and yield low recovery. Recently, some advanced approaches and devices have been developed for EVs detection, such as DNA nanodevices,9 electrochemistry,12 microfluidics,13 and surface enhanced Raman scattering.14 These advancements have greatly enhanced researchers capability to analyze EVs and identify the ones specifically related to pathological development. Still, methods that can avoid the labor-intensive EV purification steps and still provide specific and sensitive detection of the EV-bound proteins, are in high demand, for practical applications of EVs as the markers in liquid biopsy. Herein, we report that the CuS-enclosed microgels can enable rapid EV isolation and sensitive quantification Rimonabant hydrochloride in complex biological samples. The microgels are designed to carry out dual functions in EV analysis: they can facilitate EV isolation through membrane filtration, while they contain a large number of Cu2+ ions per particle to produce strong chemiluminescence (CL) for EV detection. Only proteins on the EVs but not those freely suspended in samples are detected at very low Rimonabant hydrochloride concentrations with our method, which represents a beneficial tool for discovery of EV-based biomarkers and study of EV functions in disease development. EXPERIMENTAL Materials. N-(4-Aminobutyl)-N-ethylisoluminal (ABEI), sodium sulfide nonahydrate (SDS), potassium persulfate (KPS), N, N-Methylenebisacrylamide (bis), acrylic acid (AA), allylamine (ALA) and N-isopropylacrylamide (NIPAM) were purchased from Sigma Aldrich. The biotinylated mouse anti-human ErbB2/HER2 (Recombinant Monoclonal Human IgG1 Clone Hu5), mouse anti-human CD63 (Clone mem-259), and biotinylated mouse anti-human CD63 (Clone NVG-2) was obtained from R&D systems, Sino Biological, and BioLegend, respectively. Horseradish peroxidase (HRP)-conjugated secondary antibody was from Cell Signaling Technology; and the streptavidin-HRP-conjugate was attained from Invitrogen. All other chemicals, including 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), and N-hydroxysulfosuccinimide (Sulfo-NHS), were purchased from ThermoFisher Scientific. Cell culture. Human breast cell lines MCF-10A, MDA-MB-231 and SK-BR-3 were obtained from ATCC and cultured in the recommended media containing 1% penicillin streptomycin. MCF-10A cells were cultured in the Dulbecco’s Modified Eagle Medium (DMEM)/Nutrient Mixture F-12 supplied Rabbit Polyclonal to EPHB1/2/3/4 with 5% horse serum, 0.1 g/ml cholera toxin, 10 g/ml insulin, 0.5 g/ml hydrocortisone, and 20 ng/ml.