The assemblies of optically active gold nanoparticles on a functionalized silicon planar substrate were considered in terms of the interface depth density distribution obtained by atomic-force microscopy, X-ray diffraction/reflectometry and neutron reflectometry. Two layers of nanoparticles were sequentially deposited using an additional cross-linking agent. First layer was made of nanoparticles being synthesized in light water while the second one in light/heavy water mixture. The isotope difference in layers observed in the neutron reflectometry experiment was related to water molecules incorporation into the organic shell around nanoparticles. The results are discussed with respect to the possibilities in resolving the structure of complex multilayer assemblies. (C) 2018 Elsevier B.V. All rights reserved.