Highly fluorescent and water-soluble diketopyrrolopyrrole dyes for bioconjugation

The preparation of highly water-soluble and strongly fluorescent diketopyrrolopyrrole (DPP) dyes using an unusual taurine-like sulfonated linker has been achieved. Exchanging a phenyl for a thienyl substituent shifts the emission wavelength to near λ=600 nm. The free carboxylic acid group present in these new derivatives was readily activated and the dyes were subsequently covalently linked to a model protein (bovine serum albumin; BSA). The bioconjugates were characterized by electronic absorption, fluorescence spectroscopy and MALDI-TOF mass spectrometry, thus enabling precise determination of the labeling density (ratio DPP/BSA about 3 to 8). Outstanding values of fluorescence quantum yield (30% to 59%) for these bioconjugates are obtained. The photostability of these DPP dyes is considerably greater than that of fluorescein under the same irradiation conditions. Remarkably low detection limits between 80 and 300 molecules/μm(2) were found for the BSA bioconjugates by fluorescence imaging with a epifluorescence microscope.