skip to main content

DOE PAGESDOE PAGES

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

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 excessmore » of 90%. As a result, this simple strategy offers a versatile generic route to the spatially selective site-specific immobilization of proteins at surfaces.« less
Authors:
 [1] ;  [1] ;  [1] ;  [2] ;  [1] ;  [1]
  1. Univ. of Sheffield, Sheffield (United Kingdom)
  2. The Pennsylvania State Univ., University Park, PA (United States); Montana State Univ., Bozeman, MT (United States)
Publication Date:
Grant/Contract Number:
SC0001035; SC 0001035
Type:
Published Article
Journal Name:
Langmuir
Additional Journal Information:
Journal Volume: 32; Journal Issue: 7; Journal ID: ISSN 0743-7463
Publisher:
American Chemical Society
Research Org:
Pennsylvania State Univ., University Park, PA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
OSTI Identifier:
1249749
Alternate Identifier(s):
OSTI ID: 1344925