Towards homogenous multiwell plate based pH sensors using a responsive triangulenium dye and an ATTO-647 reference dye.

pH remains the most important chemical parameter and must be monitored for positive outcomes in areas as different as cheese making and fertilisation (IVF). Where blood gas analysers enable patient monitoring, starter cultures in cheese manufacturing are still monitored using conventional pH electrodes. Here, we present a homogeneous multiwell plate sensor for monitoring pH, with the same sensitivity as a pH electrode. The homogenous sensor operates in small liquid samples and uses two components: a pH responsive triangulenium dye on a polystyrene nanoparticle, and a freely diffusing commercial reference dye. Sensor measurements were made in triplicate to investigate and document the performance and robustness of the individual components, before we moved on to investigate the multiwell plate sensor design. The pH sensor was first tested in cuvettes, before moving to microwells and smaller volumes. The target is to monitor pH in IVF cultures, and the sensor proved to be operational in the pH range found in fertilisation buffers, but the references dye was shown not to be suitable for sensors. We conclude that the homogeneous sensor design is sound, but reaching the required precision of ΔpH = 0.01 can only be done with a different reference dye.