Fluorescence Spectra of Prototropic Forms of Fluorescein and Some Derivatives and Their Potential Use for Calibration-Free pH Sensing.

Fluorescence pH sensing has proven to be efficient but with the drawback that molecules photobleach, requiring frequent calibrations. Double-emission peak molecules allow ratiometric measurements and theoretically avoid calibration. However, they are often expensive and fragile and usually have very low quantum yields. Single emission peaks such as fluorescein and derivatives are inexpensive and have very high quantum yields. Because they are single emission peaks, the pH is assumed to be derived from the ratio of emitted intensities at measured pH and at high pH values, i.e., they require frequent calibration. However, the shape of their single emitted peak evolves slightly with pH. In this paper, we first demonstrate a simple method to calculate the emission spectrum shape of each prototropic form of fluorescein (and derivatives) as well as the values of the pKa. A complete model of the evolution of the emission spectrum shape with pH is then constructed. Second, we evaluate the potential of these molecules for pH sensing by fitting the experimental spectra with the complete emission model. The method is applied to fluorescein, FITC and FAM. Depending on the molecule, pH can be measured from pH 1.9 to pH 7.3 with standard deviations between 0.06 and 0.08 pH units. Estimating pH and pKas from shape instead of intensity allows calibration-free measurements even with single-emission peak molecules.