Photophysics of a xanthenic derivative dye useful as an "on/off" fluorescence probe

The photophysical behavior of a new fluorescein derivative has been explored by using absorption and steady-state and time-resolved fluorescence measurements. The influence of ionic strength, as well as total buffer concentration, on both the absorbance and fluorescence has been investigated. The apparent acidity constant of the dye determined by absorbance is almost independent of the added buffer and salt concentrations. A semiempirical model is proposed to rationalize the variations in the apparent pKa values. The excited-state proton-exchange reaction around the physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the steady-state fluorescence and decay times. Fluorescence decay traces, collected as a function of total buffer concentration and pH, were analyzed by global compartmental analysis, yielding the following values of the rate constants describing excited-state dynamics: k01 = 1.29 x 10(10) s(-1), k02 = 4.21 x 10(8) s(-1), k21 approximately 3 x 10(6) M(-1) s(-1), k12B= 6.40 x 10(8) M(-1) s(-1), and k21B = 2.61 x 10(7) M(-1) s(-1). The decay rate constant values of k01, k21, k21B, along with the low molar absorption coefficient of the neutral form, mean that coupled decays are practically monoexponential at buffer concentrations higher than 0.02 M and any pH. Thus, the pH and buffer concentration can modulate the main lifetime of the dye

Fluorescein Excited-State Proton Exchange Reactions: Nanosecond Emission Kinetics and Correlation with Steady-State Fluorescence Intensity

Fluorescein is a complex fluorophore that can exist in one or more of four different prototropic forms (cation, neutral, dianion, and monoanion) depending on pH. In the pH range 6-10, only the dianion and monanion forms are important. In a previous article, we showed by steady-state fluorescein measurements that an excited fluorescein molecule displays excited-state proton transfer reactions which interconvert the monoanion and dianion forms. However, we found that these reactions can occur only in the presence of a suitable proton donor-acceptor buffer such as phosphate buffer. Assuming that, at 1 M phosphate buffer concentration, the excited-state proton exchange reaction of fluorescein rapidly equilibrates during the lifetime of fluorescein, we were able to fit quantitatively steady-state fluorescence intensity vs pH titration graphs to a relatively simple reaction model. In this article, we use nanosecond emission (decay time) methods to study the excitedstate proton reactions of fluorescein in the pH range 6-10 and in the presence of a phosphate buffer concentration. Fluorescein is a challenging fluorophore for the study of excited-state proton reactions because of the strong overlap of the absorption and emission spectra of the monoanion and dianion forms of fluorescein. However by recording nanosecond emission graphs and using methods of analysis of high precision, we have been able to test kinetic mechanisms and evaluate the specific rate constants for the excited-state proton reactions as well as the lifetimes of the monoanion and dianion. Using these values for lifetimes and rate constants, we discuss the process of equilibration in the excited-state and derive expressions which allow us to predict how quickly the excited-state reactions can reach equilibrium. Moreover, we use the above kinetic and spectral parameters to calculate steady-state fluorescence intensity FS vs pH at 1 M phosphate buffer concentration and compare this theoretically calculated graph with the experimental graph

Tuned lifetime, at the ensemble and single molecule level, of a xanthenic fluorescent dye by means of a buffer-mediated excited-state proton exchange reaction

The photophysical behaviour of the new fluorescein derivative 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one has been explored by using absorption, and steady-state, time-resolved and single-molecule fluorescence measurements. The apparent ground-state acidity constant of the dye determined by both the absorbance and steady-state fluorescence is almost independent of the added buffer and salt concentrations. The excited-state proton exchange reaction around the physiological pH becomes reversible upon addition of phosphate buffer, inducing a pH-dependent change of the steady-state fluorescence and decay times. Fluorescence decay traces, collected as a function of total buffer concentration and pH, were analyzed by global compartmental analysis (GCA) to elucidate the values of the excited-state rate constants. The features of this system make the fluorescence decays monoexponential at pH values and phosphate buffer concentrations higher than 6.10 and 0.2 M respectively, with the possibility of tuning the fluorescence lifetime value by changing pH or buffer concentrations. The tuned lifetimes obtained by means of phosphate concentration at constant pH have also been recovered at the single-molecule level