The carboxylic acid of NBD-X (6-(N-(7-nitrobenz-2-oxa- 1,3-diazol-4-yl)amino) hexanoic acid) can be activated in an aqueous solution with a water-soluble carbodiimide such as EDAC (E2247) in order to couple this environment sensitive fluorophore to amines, hydrazines or hydroxylamines on biomolecules of interest. NBD is a functional analog of the dinitrophenyl hapten, and its fluorescence is quenched upon binding to anti-dinitrophenyl (anti-DNP) antibodies.