Protein-polymer hybrids can act as giant monolayer-forming amphiphiles at the air-water interface. Using biotinylated polystyrene (PSb) as the hydrophobic part and streptavidin (SAv) as a hydrophilic end of the giant amphiphile, monolayer formation and subsequent deposition leads to a well defined solid-supported monolayer, in which the remaining free sites of the polymer-bound SAv are accessible to biotin binding. The deposition conditions are evaluated by investigating the supported films by atomic force microscopy (AFM). Imaging of the PSb without protein reveals uniform monolayer formation. In situ binding of SAv indicates that the biotin is accessible. Deposition of the SAv-polymer hybrid film results primarily in a very similar morphology, indicative of the dominant role of the polymer in the monolayer formation. High resolution AFM revealed a homogeneously dense packing. This is confirmed by combined AFM and scanning confocal fluorescence microscopy (SCFM) on hybrid monolayers containing rhodamine-red-X labeled SAv. The biotin binding capability of the monolayer was assessed by binding of fluorescein labeled biotin and performing dual color excitation and detection. Biotin binding was uniform and homogeneous. Local spectra revealed that the fluorescein was largely quenched by the rhodamine, indicating an efficient energy transfer. (C) 2003 Elsevier B.V. All rights reserved.