Specific Adsorption of Histidine-Tagged Proteins on Silica Surfaces Modified with Ni²⁺/NTA-Derivatized Poly(ethylene glycol)

Silica surfaces modified with nitrilotriacetic acid (NTA)-polyethylene glycol (PEG) derivatives were used to immobilize hexahistidine-tagged green fluorescent protein (His₆-GFP), biotin/streptavidin-AlexaFluor555 (His₆-biotin/SA-AF), and gramicidin A-containing vesicles (His₆-gA). Three types of surface-reactive PEG derivatives−NTA-PEG3400-Si(OMe)₃, NTA-PEG3400-vinylsulfone, and mPEG5000-Si(OMe)₃ (control)−were grafted onto silica and tested for their ability to capture His₆-tag species via His₆/Ni²⁺/NTA chelation. The composition and thicknesses of the PEG-modified surfaces were characterized using X-ray photoelectron spectroscopy, contact angle, and ellipsometry. Protein capture efficiencies of the NTA-PEG-grafted surfaces were evaluated by measuring fluorescence intensities of these surfaces after exposure to His₆-tag species. XPS and ellipsometry data indicate that surface adsorption occurs via specific interactions between the His₆-tag and the Ni²⁺/NTA-PEG-grafted surface. Protein immobilization was most effective for NTA-PEG3400-Si(OMe)₃-modified surfaces, with maximal areal densities achieved at 45 pmol/cm² for His₆-GFP and 95 fmol/cm² for His₆-biotin/SA-AF. Lipid vesicles containing His₆-gA in a 1:375 gA/lipid ratio could also be immobilized on Ni²⁺/NTA-PEG3400-Si(OMe)₃-modified surfaces at 0.5 mM total lipid. Our results suggest that NTA-PEG-Si(OMe)₃ conjugates may be useful tools for immobilizing His₆-tag proteins on solid surfaces to produce protein-functionalized surfaces.