Fabrication of Nanometer- and Micrometer-Scale ProteinStructures by Site-Specific Immobilization of Histidine-Tagged Proteinsto Aminosiloxane Films with Photoremovable Protein-Resistant ProtectingGroups

摘要

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 negligibleamounts of His-tagged proteins, indicating that the oligo(ethyleneglycol) adduct on the nitrophenyl protecting group confers excellentprotein resistance; in contrast, exposed regions bind His-GFP veryeffectively, yielding strong fluorescence that is almost completelyremoved on treatment of the surface with imidazole, confirming a degreeof site-specific binding in excess of 90%. This simple strategy offersa versatile generic route to the spatially selective site-specificimmobilization of proteins at surfaces.