Protein coverage on silicon surfaces modified with amino-organic films: A study by AFM and angle-resolved XPS

An approach to determine structural features, such as surface fractional coverage F and thickness d of protein layers immobilized on silicon substrates coated with amino-organic films is presented. To demonstrate the proposed approach rabbit gamma globulins (RgG) are adsorbed from a 0.66 μM solution onto SiO2 and Si3N4 modified with (3-aminopropyl)triethoxysilane (APTES). Atomic force microscopy data are analyzed by applying an integral geometry approach to yield average coverage values for silanized Si3N4 and SiO2 coated with RgG, F = 0.99 ± 0.01 and 0.76 ± 0.08, respectively. To determine the RgG thickness d from angle-resolved X-ray photoelectron spectroscopy (ARXPS), a model of amino-organic bilayer with non-homogeneous top lamellae is introduced. For an APTES layer thickness of 1.0 ± 0.1 nm, calculated from independent ARXPS measurements, and for fractional surface RgG coverage determined from AFM analysis, this model yields d = 1.0 ± 0.2 nm for the proteins on both silanized substrates. This value, confirmed by an evaluation (1.0 ± 0.2 nm) from integral geometry analysis of AFM images, is lower than the RgG thickness expected for monomolecular film (∼4 nm). Structures visible in phase contrast AFM micrographs support the suggested sparse molecular packing in the studied RgG layers. XPS data, compared for bulk and adsorbed RgG, suggest preferential localization of oxygen- and nitrogen-containing carbon groups at silanized silicon substrates. These results demonstrate the potential of the developed AFM/ARXPS approach as a method for the evaluation of surface-protein coverage homogeneity and estimation of adsorbed proteins conformation on silane-modified silicon substrates used in bioanalytical applications.