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

Abstract

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 Ni2+, 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.

Nanotechnology to the rescue: using nano-enabled approaches in microbiological food safety and quality

“…[53,54] More recently biofouling surfaces based on proteins have also been created. [55] Feng et al utilized aluminum oxide anodization surfaces to mitigate microbial attachment and biofilm formation, with promising results. Their findings open the way to the design and fabrication of nanoporous anodized surfaces as well as other engineered affordable antifouling surfaces for the food industry.…”

Fabrication of microstructured binary polymer brush “corrals” with integral pH sensing for studies of proton transport in model membrane systems

“…However, the large excess of this reagent combined with the poor solvent quality should ensure the formation of (near-)surface aldehyde groups. 60 …”

From Monochrome to Technicolor: Simple Generic Approaches to Multicomponent Protein Nanopatterning Using Siloxanes with Photoremovable Protein-Resistant Protecting Groups

“…Nevertheless, it was determined for OEG-NPEOC-APTES using XPS that ∼70% of adsorbates undergo complete deprotection to expose the amine group. 31 Xia et al demonstrated that this side reaction did not impede surface functionalization of deprotected films with nitrilotriaceticacid (NTA) or subsequent site-specific attachment of histidine-tagged proteins. Protein binding was reversed by treatment with imidazole, suggesting that the presence of nitroso byproducts did not have a significant impact on the utilization of the films form protein binding.…”

“…Photochemical methods are an attractive route to the patterning of biological interactions at surfaces, 46 − 55 offering the capacity to execute specific chemical transformations through strategies such as the use of nitrophenyl protecting groups, 56 − 64 and they have been found to facilitate the formation of multiple-component biological assemblies at micrometer length scales. 51 Photopatterning at the nanometer scale is feasible through the use of near-field optical methods 31 , 41 , 42 , 65 and through the use of interferometric lithography. 26 , 66 In the present study we demonstrate that both methods may be applied to enable selective deprotection of (methoxyheptaethylene glycol)nitrophenylethoxycarbonyl-protected aminopropyltriethoxysilane (henceforth OEG-NPEOC-APTES), an aminosilane bearing a photoremovable nitrophenyl group derivatized with an oligo(ethylene glycol) adduct.…”

“… 61 Intact OEG-NPEOC-APTES has been shown previously to be highly protein resistant. 31 , 61 However, on exposure to UV light, the nitrophenyl protecting group is removed, exposing an amine group ( Scheme 1 ). The deprotected surface is thus no longer protein-resistant.…”