In this work, we report the fabrication and the electrochemical characterization of recessed disk microelectrodes (DME) and ring nanoelectrodes (RNE) integrated in microwell arrays. Such configuration has all advantages of microelectrodes arrays but is more suitable for electrochemical measurement in sub-picolitre volumes (~ 0.3 pL). The technological process based on the reactive ion etching of a SiO2/Ti/Pt/Ti/SiO2 stack is optimized in order to integrate RNE arrays on transparent glass substrate. Multiphysic simulations and electrochemical characterizations are conducted in order to study and improve the amperometric behaviour of recessed ring nanoelectrodes according to their geometry. A good fit is shown between experimental, theoretical and simulation results, allowing full understanding of the electrochemical detection properties of RNE-based microwell arrays. Then, a "generationcollection mode" chronoamperometric approach is proposed to evaluate experimentally the collection ratio of RNE arrays and compare it with simulation results. Finally, first electrochemical characterizations in sub-picolitre volumes are conducted with anti-oxidant species. All these results demonstrate that recessed ring nanoelectrode arrays are fitted to the detection of bio-electrochemical species at the microscale and, consequently to single mitochondrion or single sub-cellular organelle analysis.

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 µm diameter × 5.2 µm height each, with a transparent bottom surface for optical observations, a platinum nano-ring electrode at its halfheight 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 mitochondrial activities, with a resolution ranging from a small population (whole device) to the single mitochondrion level (unique well).

This work deals with the development of electrochemical transducers for the analysis of the metabolic status of mitochondria isolated from leukemic cells. It proposes the use of ring nanoelectrodes (RNE) integrated into microwell arrays for the simultaneous monitoring of the oxygen (O2) consumption and the hydrogen peroxide (H2O2) production. The sensor enabled the real-time recording of the oxygen consumption of approximately 10,000 isolated mitochondria. Solutions are now proposed to detect H2O2 production and to reduce the number of mitochondria under test, targeting the single mitochondrion analysis.