A dual emission ratiometric nanothermometer for evaluating cell temperature during energy metabolism

ATP synthesis and thermogenesis are two key outputs of intracellular oxidative phosphorylation (OXPHOS), and simultaneous monitoring of changes in ATP and temperature will facilitate understanding of the energy metabolism mechanisms. Herein, a ratiometric probe was developed to measure temperature variations during ATP synthesis/hydrolysis. Firstly, poly(methyl methacrylate) (PMMA) copolymers with different types of charged groups (carboxylate, sulfonate and trimethylammonium) were prepared to obtain size-controllable nanoparticles by nanoprecipitation. The back energy transfer between excited state of Eu³⁺ and the ligand triplet state was sensitive to heat, so Eu³⁺ exhibited temperature-dependent emission behavior. Eu-complex-loaded polymer PMMA-trimethylammonium (PMMA-NMe₃) nanoparticles (Eu@PMMA-NMe₃ NPs) showed the strongest fluorescence and the smallest particle size, making them suitable as labels for cell imaging. Eu@PMMA-NMe₃ NPs were subsequently doped with temperature-insensitive rhodamine dye to construct ratiometric temperature probes. The maximum relative thermal sensitivity of the temperature probe was 2.7 % °C⁻¹ with a temperature resolution of 0.1–0.3 °C in the range of cell temperatures from 30.9 to 40.1 °C. Combining the temperature probe with an ATP indicator successfully monitored changes in temperature and ATP during OXPHOS and Ca²⁺ burst. When OXPHOS was inhibited, cell temperature increased by about 2.5 °C and ATP levels decreased by about 13 % within 360 s. The Ca²⁺ burst also induced a decrease in ATP levels and an increase in temperature attributed to the process of Ca²⁺ transport from the cytoplasm to the endoplasmic reticulum, which promoted ATP hydrolysis. These results have important implications for understanding energy conversion and metabolic pathways.