The specific binding of receptor to ligand covalently attached to surface with different surface densities was studied using streptavidin-biotin model pair. Biotinylated substrates with different spacer thicknesses as formed through a simple reaction between amine immobilized surfaces and N-hydroxysucciimide groups at the end of biotin modifi ed PEG in anhydrous organic solutions(“grafting to” technique). The amount of the specifi cally adsorbed protein was measured as a function of spacer thickness between hard surface and biotin moieties. It has been shown that the amount of specifically adsorbed streptavidin decreases with the increase spacer thickness and the protein adsorbs onto the functionalized surfaces in a single molecular manner. It provides an interesting model system for studying single molecular interactions. The specific binding of receptor to ligand covalently attached to surface with different surface densities was studied using streptavidin-biotin model pair. Biotinylated substrates with different spacer thicknesses as formed through a simple reaction between amine immobilized surfaces and N-hydroxysucciimide groups at the end of biotin The amount of the specifi cally adsorbed protein was measured as a function of spacer thickness between hard surfaces and biotin moieties. It has been shown that the amount of specifically adsorbed streptavidin decreases with the increase spacer thickness and the protein adsorbs onto the functionalized surfaces in a single molecular manner. It provides an interesting model system for studying single molecular interactions.