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Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces

[Image: see text] Protein chips are powerful tools as analytical and diagnostic devices for detection of biomolecular interactions, where the proteins are covalently or noncovalently attached to biosensing surfaces to capture and detect target molecules or biomarkers. Thus, fabrication of biosensing...

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Autores principales: Choi, Seoung-ryoung, Seo, Jin-soo, Bohaty, Rochelle F. H., Poulter, C. Dale
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2014
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983139/
https://www.ncbi.nlm.nih.gov/pubmed/24437976
http://dx.doi.org/10.1021/bc400413d
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author Choi, Seoung-ryoung
Seo, Jin-soo
Bohaty, Rochelle F. H.
Poulter, C. Dale
author_facet Choi, Seoung-ryoung
Seo, Jin-soo
Bohaty, Rochelle F. H.
Poulter, C. Dale
author_sort Choi, Seoung-ryoung
collection PubMed
description [Image: see text] Protein chips are powerful tools as analytical and diagnostic devices for detection of biomolecular interactions, where the proteins are covalently or noncovalently attached to biosensing surfaces to capture and detect target molecules or biomarkers. Thus, fabrication of biosensing surfaces for regio- and chemoselective immobilization of biomolecules is a crucial step for better biosensor performance. In our previous studies, a regio- and chemoselective immobilization strategy was demonstrated on glass surfaces. This strategy is now used to regioselectively attach proteins to self-assembled monolayers (SAMs) on gold surfaces. Recombinant green fluorescent protein (GFP), glutathione S-transferase (GST), and antibody-binding protein G, bearing a C-terminal CVIA motif, were prepared and a farnesyl analogue with an ω-alkyne moiety was attached to the sulfhydryl moiety in the cysteine side chain by protein farnesyltransferase. The proteins, modified with the bioorthogonal alkyne functional group, were covalently and regioselectively immobilized on thiol or dithiocarbamate (DTC) SAMs on a gold surface by a Huigsen [3 + 2] cycloaddition reaction with minimal nonspecific binding. A concentration-dependent increase of fluorescence intensity was observed in wells treated with GFP on both thiol- and DTC-SAMs. The highly ordered, densely packed layer allowed for a high loading of immobilized protein, with a concomitant increase in substrate binding capacity. The DTC-SAMs were substantially more resistant to displacement of the immobilized proteins from the gold surface by β-mercaptoethanol than alkane-thiol SAMs.
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spelling pubmed-39831392015-01-17 Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces Choi, Seoung-ryoung Seo, Jin-soo Bohaty, Rochelle F. H. Poulter, C. Dale Bioconjug Chem [Image: see text] Protein chips are powerful tools as analytical and diagnostic devices for detection of biomolecular interactions, where the proteins are covalently or noncovalently attached to biosensing surfaces to capture and detect target molecules or biomarkers. Thus, fabrication of biosensing surfaces for regio- and chemoselective immobilization of biomolecules is a crucial step for better biosensor performance. In our previous studies, a regio- and chemoselective immobilization strategy was demonstrated on glass surfaces. This strategy is now used to regioselectively attach proteins to self-assembled monolayers (SAMs) on gold surfaces. Recombinant green fluorescent protein (GFP), glutathione S-transferase (GST), and antibody-binding protein G, bearing a C-terminal CVIA motif, were prepared and a farnesyl analogue with an ω-alkyne moiety was attached to the sulfhydryl moiety in the cysteine side chain by protein farnesyltransferase. The proteins, modified with the bioorthogonal alkyne functional group, were covalently and regioselectively immobilized on thiol or dithiocarbamate (DTC) SAMs on a gold surface by a Huigsen [3 + 2] cycloaddition reaction with minimal nonspecific binding. A concentration-dependent increase of fluorescence intensity was observed in wells treated with GFP on both thiol- and DTC-SAMs. The highly ordered, densely packed layer allowed for a high loading of immobilized protein, with a concomitant increase in substrate binding capacity. The DTC-SAMs were substantially more resistant to displacement of the immobilized proteins from the gold surface by β-mercaptoethanol than alkane-thiol SAMs. American Chemical Society 2014-01-17 2014-02-19 /pmc/articles/PMC3983139/ /pubmed/24437976 http://dx.doi.org/10.1021/bc400413d Text en Copyright © 2014 American Chemical Society
spellingShingle Choi, Seoung-ryoung
Seo, Jin-soo
Bohaty, Rochelle F. H.
Poulter, C. Dale
Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces
title Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces
title_full Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces
title_fullStr Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces
title_full_unstemmed Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces
title_short Regio- and Chemoselective Immobilization of Proteins on Gold Surfaces
title_sort regio- and chemoselective immobilization of proteins on gold surfaces
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3983139/
https://www.ncbi.nlm.nih.gov/pubmed/24437976
http://dx.doi.org/10.1021/bc400413d
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