Avidin molecules were immobilized on a COOH-modified SiO2/Si(100) surface with subnano-level flatness (root-mean-square (rms) roughness;approximate to 0.1 nm) forming covalent bonds between the COOH groups on the substrate surface and the NH2 groups of avidin molecules. The structures of avidin-immobilized surfaces were investigated by atomic force microscopy (AFM), ellipsometry, and infrared (IR) reflection absorption spectroscopy using buried metal layer substrate (BML-IRRAS), and transmission IR absorption spectroscopy (TIRAS). These data have evidenced that the avidin molecules are immobilized with the 2-fold symmetry axis of the tetramer almost perpendicularly to the substrate surface. (c) 2005 Elsevier B.V. All rights reserved.