Vibrational information recorded by infrared-visible sum frequency generation spectroscopy was used to study the adsorption of a derivated vitamin (biocytin) on different substrates and its subsequent reaction with a protein (avidin). No reaction is observed on metallic substrates. When the experiments are carried out with a CaF2 substrate in the total internal reflection configuration, significant changes of the CH and NH vibrations can be related to the specific bonding of avidin to biocytin. (C) 2004 Elsevier B.V. All rights reserved.