New fluorescent pH sensors based on covalently linkable PET rhodamines

A new class of rhodamines for the application as indicator dyes in fluorescent pH sensors is presented. Their pH-sensitivity derives from photoinduced electron transfer between non-protonated amino groups and the excited chromophore which results in effective fluorescence quenching at increasing pH. The new indicator class carries a pentafluorophenyl group at the 9-position of the xanthene core where other rhodamines bear 2-carboxyphenyl substituents instead. The pentafluorophenyl group is used for covalent coupling to sensor matrices by "click" reaction with mercapto groups. Photophysical properties are similar to "classical" rhodamines carrying 2'-carboxy groups. pH sensors have been prepared with two different matrix materials, silica gel and poly(2-hydroxyethylmethacrylate). Both sensors show high luminescence brightness (absolute fluorescence quantum yield Φ(F)≈0.6) and high pH-sensitivity at pH 5-7 which makes them suitable for monitoring biotechnological samples. To underline practical applicability, a dually lifetime referenced sensor containing Cr(III)-doped Al(2)O(3) as reference material is presented.