Determination of Cellular Oxygen Consumption Rate Based on Phosphorescence Intensity Measurements Using a...

Intracellular oxygen (O2) is closely associated with cellular metabolism, as represented by energy production, and the cellular O2 consumption rate (OCR) in particular serves as a valuable indicator of metabolic activity. Here, we developed a blue-emitting extracellular O2 probe based on Ir(III) complexes and estimated the OCR of cultured cells using a commercially available microplate reader. To achieve this goal, we first synthesized a series of heteroleptic tris-cyclometalated Ir(III) complexes bearing 2-phenylpyridine (ppy) ligands or ppy ligands with a difluoro or bistrifluoromethyl group in the phenyl ring. Comprehensive investigations of the spectral and photophysical properties of these complexes in acetonitrile and aqueous solutions identified 35CF3Irpic (35CF3 = 2-(3,5-bis(trifluoromethyl)phenyl)pyridine, pic = picolinate) as a blue-emitting Ir(III) complex with high phosphorescence quantum yield. We next developed a blue-emitting extracellular O2 probe (35CF3Irpic-PEG10000) by incorporating a polyethylene glycol (PEG) group (average molecular weight: 10 kDa) via a methylene chain containing an amide group into the 35CF3 ligands of 35CF3Irpic. 35CF3Irpic-PEG10000 exhibited bright blue emission with high quantum yield, suitable O2 sensitivity and selectivity, good biocompatibility, and high photostability in aqueous solutions. Phosphorescence intensity measurements of 35CF3Irpic-PEG10000 dissolved in culture medium using a microplate reader enabled tracking of real-time changes in extracellular O2 concentration after treatment with metabolic stimulants. The OCR of cultured cells could be determined from the calibration line based on the Stern-Volmer relationship. Furthermore, simultaneous measurements using intracellular O2 or mitochondrial membrane potential probes and 35CF3Irpic-PEG10000 allowed evaluation of the relationship between cellular activity and O2 status.