New solid-state optochemical pH sensors for the analysis of cell bioenergetics

Monitoring pH and extracellular acidification rate (ECA) in biological samples containing live mammalian cells can provide valuable information on the glycolytic activity and bioenergetic status of cells. Compared to pH electrodes, optochemical pH sensors look more advantageous, since they allow rapid, non-invasive parallel analysis of multiple samples with stable readout of pH. We have developed new fluorescent pH sensors based on hydrophobic protonable metal-free porphyrins,OEP and OEPK, embedded in a plasticized PVC matrix containing a proton transfer agent. These pH sensors provide internally-referenced calibration-free operation, both in ratiometric intensity and lifetime-based detection modes. Sensor development included optimization of the indicator dye and its photophysical characteristics, screening of different proton transfer agents to minimize sensor toxicity, tuning of the protonation range and pKa, long-term storage stability and response time studies. Optimised pH sensor coatings were then deposited on plastic substrates (96-well microplates) and used for real-time monitoring of Extracellular Acidification Rate (ECAR) for cultured cancer cells and 3D spheroid structures on standard laboratory equipment (multi-label plate reader and confocal FLIM microscope). The advanced pH sensors tailored for use with biological samples have high potential for cell analysis and related applications.