Carboxy SNARF-4F as a fluorescent pH probe for ensemble and fluorescence correlation spectroscopies

The optical spectroscopic properties of 1,4(and 5)-benzenedicarboxylic acid, 2-[10-(dimethylamino)-4-fluoro-3-oxo-3H-benzo[c]xanthen-7-yl] (carboxy SNARF-4F), a commercial promising fluorescent pH probe, are investigated in buffered aqueous solutions in the 5.6-8.2 pH range by steady-state and time-resolved spectroscopy. A multiexponential global analysis of the picosecond time-resolved data is performed. The nonprotonated A species decays monoexponentially (0.73 ns), while the protonated species AH decays following a biexponential law with time constants of 0.40 and 1.87 ns. A kinetic scheme is proposed to explain the observations, which involves AH in equilibrium with a species denoted Y. The nature of Y is discussed in terms of a possible structural change in the molecule producing the lactone form, although the formation of a hydrogen-bonded complex to the solvent cannot be ruled out. Finally, the ability of the dye to probe pH at the single-molecule level is explored using fluorescence correlation spectroscopy.