We previously developed prototype oligodeoxyribonucleotide (ODN) duplex fluorescence energy transfer (FRET) reporters for optical sensing of NF-kappaB transcription factor. We report here a plate-binding assay designed for optimizing the above reporters. Nitrilotriacetate-bearing plates were prepared by using sequential (1) aminosilylation; (2) carboxylation; (3) coupling of Nalpha,Nalpha-bis(carboxymethyl)-L-lysine or, alternatively, by replacing steps 1 and 2 by treating the glass with 3-(triethoxysilyl)propylsuccinic anhydride. FRET reporters were obtained by covalent linking of Cy5.5 (fluorescence donor) and IRdye800CW (fluorescence acceptor) to complementary ODN strands encoding a high-affinity p50 binding site. Recombinant 6 x His tagged NF-kappaB p50 was used for immobilizing the protein on glass plates via linked NTA-Ni(II) groups. Imaging and quantification of the fluorescence intensity in the wells was performed in two channels (700 and 800 nm) using a near-infrared scanning device with microscopic resolution. The fluorescence intensity of the ODN duplex reporter was detectible in the plates at the concentration of 5 pM. NF-kappaB p50-ODN reporter interaction was studied by measuring the ratio of 700 nm (donor) to 800 nm (acceptor) fluorescence intensities. Using the plate assay, we were able to measure p50-mediated interference with FRET at low density of plate binding.