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Fabrication of Nanometer- and Micrometer-Scale Protein Structures by Site-Specific Immobilization of Histidine-Tagged Proteins to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting Groups
[Image: see text] The site-specific immobilization of histidine-tagged proteins to patterns formed by far-field and near-field exposure of films of aminosilanes with protein-resistant photolabile protecting groups is demonstrated. After deprotection of the aminosilane, either through a mask or using...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2016
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848731/ https://www.ncbi.nlm.nih.gov/pubmed/26820378 http://dx.doi.org/10.1021/acs.langmuir.5b04368 |
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author | Xia, Sijing Cartron, Michaël Morby, James Bryant, Donald A. Hunter, C. Neil Leggett, Graham J. |
author_facet | Xia, Sijing Cartron, Michaël Morby, James Bryant, Donald A. Hunter, C. Neil Leggett, Graham J. |
author_sort | Xia, Sijing |
collection | PubMed |
description | [Image: see text] The site-specific immobilization of histidine-tagged proteins to patterns formed by far-field and near-field exposure of films of aminosilanes with protein-resistant photolabile protecting groups is demonstrated. After deprotection of the aminosilane, either through a mask or using a scanning near-field optical microscope, the amine terminal groups are derivatized first with glutaraldehyde and then with N-(5-amino-1-carboxypentyl)iminodiacetic acid to yield a nitrilo-triacetic-acid-terminated surface. After complexation with Ni(2+), this surface binds histidine-tagged GFP and CpcA-PEB in a site-specific fashion. The chemistry is simple and reliable and leads to extensive surface functionalization. Bright fluorescence is observed in fluorescence microscopy images of micrometer- and nanometer-scale patterns. X-ray photoelectron spectroscopy is used to study quantitatively the efficiency of photodeprotection and the reactivity of the modified surfaces. The efficiency of the protein binding process is investigated quantitatively by ellipsometry and by fluorescence microscopy. We find that regions of the surface not exposed to UV light bind negligible amounts of His-tagged proteins, indicating that the oligo(ethylene glycol) adduct on the nitrophenyl protecting group confers excellent protein resistance; in contrast, exposed regions bind His-GFP very effectively, yielding strong fluorescence that is almost completely removed on treatment of the surface with imidazole, confirming a degree of site-specific binding in excess of 90%. This simple strategy offers a versatile generic route to the spatially selective site-specific immobilization of proteins at surfaces. |
format | Online Article Text |
id | pubmed-4848731 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | American
Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-48487312016-04-29 Fabrication of Nanometer- and Micrometer-Scale Protein Structures by Site-Specific Immobilization of Histidine-Tagged Proteins to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting Groups Xia, Sijing Cartron, Michaël Morby, James Bryant, Donald A. Hunter, C. Neil Leggett, Graham J. Langmuir [Image: see text] The site-specific immobilization of histidine-tagged proteins to patterns formed by far-field and near-field exposure of films of aminosilanes with protein-resistant photolabile protecting groups is demonstrated. After deprotection of the aminosilane, either through a mask or using a scanning near-field optical microscope, the amine terminal groups are derivatized first with glutaraldehyde and then with N-(5-amino-1-carboxypentyl)iminodiacetic acid to yield a nitrilo-triacetic-acid-terminated surface. After complexation with Ni(2+), this surface binds histidine-tagged GFP and CpcA-PEB in a site-specific fashion. The chemistry is simple and reliable and leads to extensive surface functionalization. Bright fluorescence is observed in fluorescence microscopy images of micrometer- and nanometer-scale patterns. X-ray photoelectron spectroscopy is used to study quantitatively the efficiency of photodeprotection and the reactivity of the modified surfaces. The efficiency of the protein binding process is investigated quantitatively by ellipsometry and by fluorescence microscopy. We find that regions of the surface not exposed to UV light bind negligible amounts of His-tagged proteins, indicating that the oligo(ethylene glycol) adduct on the nitrophenyl protecting group confers excellent protein resistance; in contrast, exposed regions bind His-GFP very effectively, yielding strong fluorescence that is almost completely removed on treatment of the surface with imidazole, confirming a degree of site-specific binding in excess of 90%. This simple strategy offers a versatile generic route to the spatially selective site-specific immobilization of proteins at surfaces. American Chemical Society 2016-01-28 2016-02-23 /pmc/articles/PMC4848731/ /pubmed/26820378 http://dx.doi.org/10.1021/acs.langmuir.5b04368 Text en Copyright © 2016 American Chemical Society This is an open access article published under an ACS AuthorChoice License (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) , which permits copying and redistribution of the article or any adaptations for non-commercial purposes. |
spellingShingle | Xia, Sijing Cartron, Michaël Morby, James Bryant, Donald A. Hunter, C. Neil Leggett, Graham J. Fabrication of Nanometer- and Micrometer-Scale Protein Structures by Site-Specific Immobilization of Histidine-Tagged Proteins to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting Groups |
title | Fabrication of Nanometer- and Micrometer-Scale Protein
Structures by Site-Specific Immobilization of Histidine-Tagged Proteins
to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting
Groups |
title_full | Fabrication of Nanometer- and Micrometer-Scale Protein
Structures by Site-Specific Immobilization of Histidine-Tagged Proteins
to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting
Groups |
title_fullStr | Fabrication of Nanometer- and Micrometer-Scale Protein
Structures by Site-Specific Immobilization of Histidine-Tagged Proteins
to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting
Groups |
title_full_unstemmed | Fabrication of Nanometer- and Micrometer-Scale Protein
Structures by Site-Specific Immobilization of Histidine-Tagged Proteins
to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting
Groups |
title_short | Fabrication of Nanometer- and Micrometer-Scale Protein
Structures by Site-Specific Immobilization of Histidine-Tagged Proteins
to Aminosiloxane Films with Photoremovable Protein-Resistant Protecting
Groups |
title_sort | fabrication of nanometer- and micrometer-scale protein
structures by site-specific immobilization of histidine-tagged proteins
to aminosiloxane films with photoremovable protein-resistant protecting
groups |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4848731/ https://www.ncbi.nlm.nih.gov/pubmed/26820378 http://dx.doi.org/10.1021/acs.langmuir.5b04368 |
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