Simultaneous pH and Temperature Measurements Using Pyranine as a Molecular Probe

Steep variations in concentration and temperature frequently occur in small fluid compartments such as those found in cells or microfluidic devices. A quantitative characterization of concentration and temperature gradients is therefore required before these systems can be fully understood. Although different spatially resolved fluorescence methods have been developed to measure either the temperature or the concentration of ions such as proton or calcium, often concentration measurements depend on temperature and vice versa. Here, we describe a method allowing simultaneous measurement of pH and temperature. This method is based on the detection of the blinking of the fluorescent pH indicator pyranine, a process due to its alternating between a basic form and an acidic form. Fluorescence correlation spectroscopy allows measuring both the protonation and deprotonation rates of pyranine, and each pair of rates can be uniquely related to a pair of pH and temperature values. We show, however, that the relationship between rates, pH and temperature, is very sensitive to the presence of other acid-base molecules in solution. We also show that it is influenced by the overall ionic strength of the solution, in a manner that depends on buffer composition.