10-(2-Biotinyloxyethyl)-9-acridone: A novel fluorescent label for (strept)avidin–biotin based assays

A novel biotinylated fluorophore, 10-(2-biotinyloxyethyl)-9-acridone 3 has been synthesized and its fluorescent properties were examined in the presence and absence of avidin or streptavidin. In aqueous solutions the novel fluorophore 3, as well as its precursor 10-(2-hydroxyethyl)-9-acridone 2 exhibit intense fluorescence and can be detected down to 8.62 × 10−10 and 1.90 × 10−10 M, respectively. A short spacer was chosen in order to minimize steric repulsion between the adjacently bound to avidin or streptavidin residues of acridone. The novel biotinylated fluorophore 3 exhibits rapid, specific and stoichiometric binding to the four biotin binding sites in avidin or streptavidin tetramers, even at low concentrations (20 nM). Preliminary measurements showed that the new conjugate can be applied for the fluorimetric determination of solid-phase immobilized mouse IgG with detection limits down to 1.3 ng per assay.