Microwell array integrating nanoelectrodes for coupled opto-electrochemical monitorings of single mitochondria

Chips composed of microwell arrays integrating nanoelectrodes (OptoElecWell) were developed to achieve dual optical and electrochemical detections on isolated biological entities. Each array consists in 10(6) microwells of 6 pm diameter x 5.2 mu m height each, with a transparent bottom surface for optical observations, a platinum nano-ring electrode at its half-height for in situ electrochemistry, and a top open surface to inject solutions. Then, populations of individual mitochondria isolated from yeasts (Saccharomyces cerevisiae) were let to sediment on the array and be trapped within microwells. The trapping efficiency reached 20% but owing to the large number of microwells on the platform, hundreds of them could be filled simultaneously by single mitochondria. This allowed to follow up their individual energetic status based on fluorescence microscopy of their endogenous NADH. Simultaneously, the array of interconnected Pt nanoelectrodes in the microwells was used to monitor in situ variations of dioxygen consumed by all mitochondria captured in the device. Mitochondrial bioenergetics were modulated sequentially using respiratory chain-ATP synthase substrates (ethanol and ADP) and inhibitor (antimycin A). Overall, we show how two complementary analytical approaches, fluorescence and electrochemical detections, can be coupled for a multi-parametric monitoring of mitochondria] activities, with a resolution ranging from a small population (whole device) to the single mitochondrion level (unique well).