In HPLC grade DMSO, a Stock Solution of Mito-pH2 dye (1 mM) was made. Analyte stock solutions were made with Milli-Q water. The Mito-pH2 dye was diluted to 2 μM in a 40 mM BR buffer solution to perform spectral measurements. Every time, a quartz cuvette with a 10 mm optical path length was filled with 600 μL of the dye solution. Every spectroscopic experiment was carried out at ambient temperature.