Analysis of mitochondrial oxygen consumption and hydrogen peroxide release from cardiac mitochondria using electrochemical multi-sensors

Mitochondria are the primary sites of oxygen (O2) consumption and energy metabolism in most cell types, but also produce reactive oxygen species (ROS) that contribute to a wide array of pathological and physiological processes. Accordingly, simultaneous monitoring of mitochondrial ROS release and oxygen consumption rate (OCR) from cells and mitochondrial preparations is an attractive investigative approach in biological research, particularly when sample quantity is scarce. This paper presents the development of a sensitive multi-sensor device capable of measuring ROS production and OCR from biological samples in a single micro-chamber assay. Sensor sensitivities for O2 and hydrogen peroxide (H2O2; the major ROS species released by mitochondria and cells) are 4.32 nA/μM and 54.89 nA/μM, respectively, with limits of detection of 2.9 μM and 58.36 nM, respectively. Proof-of-concept studies in isolated mitochondria from rat cardiac tissue (5 µg protein) demonstrate an expected 3 – 4 fold increase in H2O2 release over the basal rate following addition of respiratory substrates, with a comparatively small change in OCR. The subsequent addition of adenosine diphosphate (ADP) decreased H2O2 release by 73% (p < 0.01) and increased OCR by 168% (p < 0.01), consistent with established shifts in mitochondrial membrane potential and electron flow from an ADP-limited (State 4) to ADP-stimulated (State 3) respiratory state. These studies validate the results from the use of a novel multi-sensor device capable of monitoring OCR and H2O2 simultaneously in scarce biological samples, with potential utility in the non-destructive integrative study of cellular metabolism and mitochondrial function.