This work reports for the first time on the fabrication of a bi-functional Slayer tandem fusion protein which is able to self-assemble on solid supports without losing its functionality. Two variants of the green fluorescent protein (GFP) were genetically combined with a self-assembly system having the remarkable opportunity to interact with each other and act as functional nanopatterning biocoating. The S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a was fused with the cyan ECFP donor protein at the SgsE N-terminus and with the yellow YFP acceptor protein at the C-terminus. The fluorescence energy transfer was studied with spectrofluorimetry, confocal microscopy and flow cytometry, whilst protein self-assembly (on silicon dioxide particles) and structural investigations were carried out with atomic force microscopy (AFM). The fluorescence resonance energy transfer efficiency of reassembled SgsE tandem protein was 20.0 +/- 6.1% which is almost the same transfer efficiency shown in solution (19.6 +/- 0.1%). This work shows that bi-fluorescent Slayer fusion proteins self-assemble on silica particles retaining their fluorescent properties. (C) 2010 Elsevier Inc. All rights reserved.