Control of the biosensor surface is required for developing high performance sensing systems. The optic immunosensor based on a solid-liquid and antigen-antibody reaction, which requires homogeneous immobilization of a probe protein, is used in this study. Distribution of fluorochrome-labeled protein A-immobilized on the biosensor surface and immunoglobulin G (IgG) reacted with protein A were estimated by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS) spectra. Specific chemical distribution including amino acid fragments related to proteins will provide useful information on immobilization of proteins and their reactions. Peak selection of proteins for chemical imaging is difficult because proteins generate similar fragment ions. In this study, specific peaks of fragment ions from different proteins were determined by means of mutual information, one of useful applications of information theory. Aammosilanized glass plates immobilized with protein A and immunoglobulin G were measured with TOF-SIMS, respectively. TOF-SIMS spectra of the protein immobilized glass plates were analyzed with information theory and principal component analysis (PCA). Both methods suggest differences between proteins and mutual information clearly indicates specific peaks from each protein. Thus the analysis of protein immobilization on the biosensor surface with TOF-SIMS suggests that this technique will contribute to the improvement and the development of a sophisticated biosensor. (C) 2004 Elsevier B.V. All rights reserved.