| The use of glass substrates with bi-functional silanes for designing micropatterned cell-secreted cytokine immunoassays. | |
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MedLine Citation:
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PMID: 21550110 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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It is often desirable to sequester cells in specific locations on the surface and to integrate sensing elements next to the cells. In the present study, surfaces were fabricated so as to position cytokine sensing domains inside non-fouling poly(ethylene glycol) (PEG) hydrogel microwells. Our aim was to increase sensitivity of micropatterned cytokine immunoassays through covalent attachment of biorecognition molecules. To achieve this, glass substrates were functionalized with a binary mixture of acrylate- and thiol-terminated methoxysilanes. During subsequent hydrogel photopatterning steps, acrylate moieties served to anchor hydrogel microwells to glass substrates. Importantly, glass attachment sites within the microwells contained thiol groups that could be activated with a hetero-bifunctional cross-linker for covalent immobilization of proteins. After incubation with fluorescently-labeled avidin, microwells fabricated on a mixed acryl/thiol silane layer emitted ∼ 6 times more fluorescence compared to microwells fabricated on an acryl silane alone. This result highlighted the advantages of covalent attachment of avidin inside the microwells. To create cytokine immunoassays, micropatterned surfaces were incubated with biotinylated IFN-γ or TNF-α antibodies (Abs). Micropatterned immunoassays prepared in this manner were sensitive down to 1 ng/ml or 60 pM IFN-γ. To further prove utility of this biointerface design, macrophages were seeded into 30 μm diameter microwells fabricated on either bi-functional (acryl/thiol) or mono-functional silane layers. Both types of microwells were coated with avidin and biotin-anti-TNF-α prior to cell seeding. Short mitogenic activation followed by immunostaining for TNF-α revealed that microwells created on bi-functional silane layer had 3 times higher signal due to macrophage-secreted TNF-α compared to microwells fabricated on mono-functional silane. The rational design of cytokine-sensing surfaces described here, will be leveraged in the future for rapid detection of multiple cytokines secreted by individual immune cells. |
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Authors:
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Jeong Hyun Seo; Li-Jung Chen; Stanislav V Verkhoturov; Emile A Schweikert; Alexander Revzin |
Publication Detail:
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Type: Journal Article; Research Support, U.S. Gov't, Non-P.H.S. Date: 2011-05-07 |
Journal Detail:
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Title: Biomaterials Volume: 32 ISSN: 1878-5905 ISO Abbreviation: Biomaterials Publication Date: 2011 Aug |
Date Detail:
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Created Date: 2011-05-30 Completed Date: 2011-09-19 Revised Date: 2012-01-30 |
Medline Journal Info:
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Nlm Unique ID: 8100316 Medline TA: Biomaterials Country: England |
Other Details:
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Languages: eng Pagination: 5478-88 Citation Subset: IM |
Copyright Information:
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Copyright © 2011 Elsevier Ltd. All rights reserved. |
Affiliation:
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Department of Biomedical Engineering, University of California, Davis, CA 95616, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Acrylates
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chemistry Animals Antibodies / chemistry, immunology Avidin / chemistry Biotin / chemistry Cell Line, Tumor Cytokines / analysis*, secretion Eukaryotic Cells / drug effects, secretion* Fluorescent Dyes / chemistry Glass / chemistry* Humans Hydrogels / chemistry Immunoassay / methods Interferon-gamma / analysis, immunology Macrophages / drug effects, secretion Maleimides / chemistry Mice Microscopy, Atomic Force Microscopy, Fluorescence Microtechnology / methods Photochemical Processes Polyethylene Glycols / chemistry Silanes / chemistry* Spectrometry, Mass, Secondary Ion Streptavidin / chemistry Surface Properties Tetradecanoylphorbol Acetate / pharmacology Tumor Necrosis Factor-alpha / analysis, immunology |
| Grant Support | |
ID/Acronym/Agency:
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R01 DK079977-01A2/DK/NIDDK NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Acrylates; 0/Antibodies; 0/Cytokines; 0/Fluorescent Dyes; 0/Hydrogels; 0/Maleimides; 0/Polyethylene Glycols; 0/Silanes; 0/Tumor Necrosis Factor-alpha; 0/neutravidin; 1405-69-2/Avidin; 16561-29-8/Tetradecanoylphorbol Acetate; 4369-14-6/3-acryloxypropyltrimethoxysilane; 4420-74-0/(3-mercaptopropyl)trimethoxysilane; 58-85-5/Biotin; 82115-62-6/Interferon-gamma; 9013-20-1/Streptavidin |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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