In vitro studies which focus on cellular metabolism can benefit from time-resolved readouts from the living cells. pH and O₂ concentration are fundamental parameters upon which cellular metabolism is often inferred. This work demonstrates a novel use of a ruthenium oxide (RuO_x) electrode for in vitro studies. The RuO_x electrode was characterized to measure both pH and O₂ using two different modes. When operated potentiometrically, continuous pH reading can be obtained, and O₂ concentration can be measured chronoamperometrically. In this work, we demonstrate the use of the RuO_x electrodes in inferring two different types of metabolism of human pluripotent stem cell-derived cardiomyocytes. We also show and discuss the interpretation of the measurements into meaningful extracellular acidification rates and oxygen consumption rates of the cells. Overall, we present the RuO_x electrode as a versatile and powerful tool in in vitro cell metabolism studies, especially in comparative settings.

A ruthenium oxide (RuOx) sensor for potentiometric pH sensing is currently being developed for organs-on-chip purposes. The sensor was fabricated from a Ru(OH)₃ precursor, resulting in RuOx nanorods after heating. An open-circuit potential of the RuOx electrode showed a near-Nernstian response of −58.05 mV/pH, with good selectivity against potentially interfering ions (lithium, sulfate, chloride, and calcium ions). The preconditioned electrode (stored in liquid) had a long-term drift of −0.8 mV/h, and its response rate was less than 2 s. Sensitivity to oxygen was observed at an order of magnitude lower than other reported metal-oxide pH sensors. Together with miniaturizability, the RuOx pH sensor proves to be a suitable pH sensor for organs-on-chip studies.

A ruthenium oxide (RuOx) electrode was used to monitor contractile events of human pluripotent stem cells-derived cardiomyocytes (hPSC-CMs) through electrical impedance spectroscopy (EIS). Using RuOx electrodes presents an advantage over standard thin film Pt electrodes because the RuOx electrodes can also be used as electrochemical sensor for pH, O₂, and nitric oxide, providing multisensory functionality with the same electrode. First, the EIS signal was validated in an optically transparent well-plate setup using Pt wire electrodes. This way, visual data could be recorded simultaneously. Frequency analyses of both EIS and the visual data revealed almost identical frequency components. This suggests both the EIS and visual data captured the similar events of the beating of (an area of) hPSC-CMs. Similar EIS measurement was then performed using the RuOx electrode, which yielded comparable signal and periodicity. This mode of operation adds to the versatility of the RuOx electrode’s use in in vitro studies.

A ruthenium oxide (RuOx) electrode is being developed as potentiometric pH sensor for organs-on-chip applications. Open-circuit potential (OCP) of the RuOx electrode showed a response of −58.05 mV/pH, with no cross-sensitivity to potentially interfering/complexing ions (tested were lithium, sulfate, chloride, and calcium ions). Similar response was observed in complex biological medium. The electrode stored in liquid had a long-term drift of −0.8 mV/hour (corresponding to ΔpH of 0.013/hour) and response time in complex biological medium was 3.7 s. Minimum cross-sensitivity to oxygen was observed as the OCP shifted ~3 mV going from deoxygenated to oxygenated solution. This response is one magnitude lower than previously reported for metal- oxide pH sensors. Overall, the RuOx pH sensor has proven to be a suitable pH sensor for organs- on-chip applications.