Hybrid glyco-nanolayers of two oligosaccharides with controlled surface sugar density were prepared on a gold surface by self-assembly chemisorption of chitohexaose and cellohexaose as bioactive carbohydrate and molecular spacer, respectively, whose reducing ends were site-selectively modified with thiosemicarbazide (TSC). Quartz crystal microbalance analysis and X-ray photoelectron spectroscopy suggested successful formation of self-assembled nanolayers consisting of chitohexaose–TSC and cellohexaose–TSC with various molar ratios. Human hepatocellular carcinoma cells which recognize an N-acetylglucosamine residue efficiently adhered to the hybrid nanolayers. The cells formed multicellular spheroids on nanolayers with particular molar ratios of the oligosaccharides. Relatively high enzymatic activity for the deethoxylation of ethoxyresorufinviacytochrome P450 1A1 was observed for the as-formed spheroids. The experimental data indicate that the surface density of bioactive chitohexaose, tailored by variation of the relative proportions of chitohexaose and cellohexaose, was a key factor in cell adhesion efficiency, morphological variation and some cellular responses, possibly due to carbohydrate–protein interactions. Architectural design of carbohydrate-based hybrid nanolayers via the vectorial chain immobilization method is expected to provide a new concept for the functional development of glyco-decorated biointerfaces.
