Synthesis, Characterization, and Application of Fluorescence Sensing Lipobeads for Intracellular pH Measurements

This paper describes the synthesis and characterization of micrometric phospholipid-coated polystyrene particles, named lipobeads, with pH-sensing capability and their application for intracellular pH measurements in murine macrophages. The phospholipids used to coat the particles are labeled with fluorescein (a pH-sensitive dye) and tetramethylrhodamine (a pHinsensitive 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 excitation light intensity is reduced to minimize photobleaching of the fluorescein-phospholipid conjugates. The fluorescent lipobeads are used to measure the pH in single macrophages. The lipobeads are ingested by the macrophages and directed to lysosomes, which are the cellular organelles involved in the phagocytosis process. Despite the high lysosomal levels of digestive enzymes and acidity, the absorbed particles remain stable for over 6 h in the cells when they are stored in a phosphate-buffered saline solution at pH 7.4.