Advanced multimodal solid-state optochemical pH and dual pH/O₂ sensors for cell analysis

Optochemical sensors are actively used in cell analysis, however existing systems have limitations with respect to their robustness and analytical performance. We have developed advanced multimodal and multi-parametric solid-state pH sensors for cell analysis based on hydrophobic protonable metal-free porphyrins, such as octaethylporphine (OEP), and octaethylporphine-ketone (OEPK), as fluorescent pH indicators. The internally referenced ratiometric intensity and nanosecond lifetime-based versions of the pH sensors were developed and also multiplexed with the O₂ sensors based on phosphorescent PtOEP dye. We optimised the key parameters of the pH sensor, including: dye encapsulation matrix, the type and concentration of proton transfer reagent, measurement pH range and pKa, dye concentrations and cross-talk with the O₂ sensor. Subsequently, sensor coatings were deposited on common substrates used in cell analysis (96-well plates), fine-tuned for their operational performance, dual O₂/pH sensing functionally and the ability to measure Extracellular Acidification (ECAR) and Oxygen Consumption (OCR) rates in biological samples containing cells. The optimised sensors with stable internally referenced calibrations, convenient spectral characteristics and low cytotoxicity, were demonstrated with cultured cells and 3D spheroid structures, measuring their ECAR, OCR and responses to stimulation. These pH and dual pH/O₂ sensors are well-suited for detailed metabolic studies of biological samples on widely available laboratory equipment.