Synthesis, characterization, and application of fluorescence sensing lipobeads for intracellular pH measurements

This paper describes the synthesis and characterization of micrometric phos pholipid-coated polystyrene particles, named lipobeads, with pH-sensing cap ability and their application for intracellular pH measurements in murine m acrophages, The phospholipids used to coat the particles are labeled with f luorescein (a pll-sensitive dye) and tetramethylrhodamine (a pH-insensitive dye), which serves as a referencing fluorophore for increased accuracy of the pH measurements. The synthesis of the pH-sensing lipobeads is realized by the covalent attachment of the fluorescent phospholipids to the surface of carboxylated polystyrene particles. The pH dynamic range of the sensing particles is between 5.5 and 7.0 with a sensitivity of 0.1 pH unit. The exc itation light intensity is reduced to minimize photobleaching of the fluore scein-phospholipid conjugates. The fluorescent lipobeads are used to measur e the pH in single macrophages. The lipobeads are ingested by the macrophag es and directed to lysosomes, which are the cellular organelles involved in the phagocytosis process. Despite the high lysosomal levels of digestive e nzymes and acidity, the absorbed particles remain stable for over 6 h in th e cells when they are stored in a phosphate-buffered saline solution at pH 7.4.