Optical determination of pH on surfaces using immobilized Fluorescent dyes

The attachment of pH-sensitive Fluorescent dyes on to hydroxylapatite for measuring the pH of small volumes is described. Fluorescein and acridine were used, both activated with reactive functions, such as isothiocyanate or succinimidyl ester groups, to enhance the possibility of a covalent linkage to the surface. First investigations were carried out on synthetic hydroxylapatite as the surface material. After the preparation of a particular surface with the fluorescent dye, steady-state and time-resolved fluorescence spectroscopies were employed for estimating the pH value of a solution applied to the surface. In this paper we present the results of our investigations done with both methods. Fluorescein shows significant variation in excitation spectra with pH, whereas in the case of acridine, the fluorescence lifetimes are very sensitive to pH.