Lifetime-based pH sensors: Indicators for acidic environments

We characterized the pal-dependent intensity decays of three fluorophores, Oregon green 514 carboxylic acid, Cl-NERF, and DM-NERF, using frequency-dom ain fluorometry, with the objective of identifying lifetime-based sensors f or low pH values. These three probes were originally designed as dual excit ation wavelength-ratiometric probes, with high photostability and high quan tum yields in aqueous solutions. We found that their fluorescence intensity decays were strongly dependent on pH. Moreover, global intensity decays an alysis reveals that these probes have double exponential intensity decays a t intermediate pH values and that the decay time amplitudes are greatly dep endent on pH. The longer lifetime components originated from the unprotonat ed forms and the shorter components from the protonated forms. Both forms c an emit fluorescence at intermediate pH values. The apparent pK(a) values w ere also determined from the titration curves of phase angles and modulatio ns versus pH for the purpose of pH sensing. The apparent pK(a) values range from pH 3 to 5, a range where lifetime-based sensors are not presently rep orted. Since these probes show low pK(a) values and display substantial pha se and modulation changes with pH, they are suitable as lifetime-based pH s ensors to monitor the pH changes in acidic environments. One potential appl ication of these probes is to trace the pH in different cellular compartmen ts, (C) 1999 Academic Press.