Design and Preparation of Lifetime‐Based Dual Fluorescent/Phosphorescent Sensor of pH and Oxygen and its Exploration in Model Physiological Solutions and Cells

Abstract In the present report, a novel dual pH‐O₂ sensor based on covalent conjugate of rhodamine 6G and cyclometalated iridium complex with poly(vinylpyrrolidone‐block‐vinyltetrazole) copolymer is reported. In model physiological solutions the sensor chromophores display independent phosphorescent and fluorescent lifetime responses onto variations in oxygen concentration and pH, respectively. Colocalization studies on Chinese hamster ovary cells demonstrate the preferential localization in endosomes and lysosomes. The fluorescent lifetime imaging microscopy‐phosphorescent lifetime imaging microscopy (FLIM‐PLIM) experiments show that the phosphorescent O₂ sensor provides unambiguous information onto hypoxia versus normoxia cell status as well as semi‐quantitative data on the oxygen concentration in cells in between these two states. However, the results of FLIM measurements indicate that dynamic lifetime interval of the sensor (≈0.5 ns between pH values 5.0 and 8.0) is insufficient even for qualitative estimation of pH in living cells because half‐width of lifetime distribution in the studied samples is higher than the sensor dynamic interval. Nevertheless, the variations in rhodamine emission intensity are much higher and allow rough discrimination of acidic and neutral cell conditions. Thus, the results of this study indicate that the suggested approach to the design of dual pH‐O₂ sensors makes possible to prepare the biocompatible and water‐soluble conjugate with fast cellular uptake.