Fabrication of dense, uniform aminosilane monolayers: A platform for protein or ligand immobilization

Designing biointerfaces with enhanced biomolecule binding abilities, along with their distribution and presentation on the surface, largely requires control over underlying surface chemistries. In an effort to correlate experimental parameters to surface properties, ligand distribution and biomolecule binding, the influence of (3-aminopropyl)triethoxysilane (APTES) concentration (1, 2 and 4vol%) and duration of the silanization reaction (5-60min) on the number of available NH₂ groups were determined and correlated with the amount of surface bound human immunoglobulin G (HIgG). Surfaces silanized with 2vol% APTES for 30min yielded a densely populated silane monolayer (1.0-1.2nm) where the average molecular orientation was 38±2° with respect to the surface normal. A surface density of NH₂ moieties of ~10¹⁵/cm² was obtained and was significantly higher compared to other conditions evaluated. The combined data from ellipsometry and atomic force microscopy (AFM) analyses supports our findings. Coupling of HIgG to surfaces silanized with 2vol% APTES for 30min yielded ~10¹³IgG/cm², which was significantly higher than values obtained at 1vol% APTES for 30min. The distribution of immobilized HIgG was noted to be dense and uniform on surfaces silanized with 2vol% APTES when visualized using Nanogold^® antibody conjugates. This study highlights the critical role of the experimental parameters that impact the biomolecule immobilization process on functionalized surfaces.