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

Abstract

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).

Microwell Array Based Opto‐Electrochemical Detections Revealing Co‐Adaptation of Rheological Properties and Oxygen Metabolism in Budding Yeast

“…As a sampling method to sense local oxygen changes around the entrapped cells, within microwells, we chose continuous cyclic voltammetry over chronoamperometry. We noticed that under optimized conditions–higher scan rates and gaps between each scan (4 V s −1 and 20 s gap), CV allows to monitor the local chemical changes happening within the microwells, while allowing the oxygen concentration to get to the equilibrium before next measurement, [ 38 ] i.e., no competition between working electrode and yeast cells in terms of oxygen usage. We first investigated the oxygen sensing capacity of OptoElecWells by performing the electrochemical reduction of dissolved oxygen under different oxygen concentrations in yeast buffer using cyclic voltammetry at a scan rate of 4 V s −1 .…”

“…The design and fabrication of the microwell array reported here is inspired from the previous works where the first generation of OptoElecWell is reported and its design by multiphysics simulation, manufacturing process, and characterization is discussed. [ 30,38 ]…”

Advances in Microtechnology for Improved Cytotoxicity Assessment

“…At the microscale, any small perturbation on the flow or a high density of adhered cells can cause huge stress to them, resulting on exhaustion of nutrients or even migration, which can be misunderstood as a cellular response to the toxic (Wang et al, 2007). These phenomena can be avoided through microfabrication; on one hand, inducing physical cell adhesion control by patterning small microwells on polymeric materials (Vajrala et al, 2019;Zhang et al, 2019) or on C- (Lee et al, 2006) and on U-shaped (Wang et al, 2007) microsieves in the culturing chambers. On the other hand, cell adhesion can be chemically controlled with micropatterns of proteins (Liu et al, 2013;Hamon et al, 2016) or hydrophilic-hydrophobic sites (Nath et al, 2004;Zhang et al, 2007), modulating cell adhesion by enhancing cell affinity for specific areas, keeping them comfortably located.…”

Microwell array based opto-electrochemical detections revealing co-adaptation of rheological properties and oxygen metabolism in budding yeast

“…However, this approach is still been faced with challenges in dealing with the low throughput owing to the manual positioning of the electrode near the cell and UME spatial coverage over the cell, i.e., partial detection of targeted cellular events due to lateral diffusion phenomenon. To overcome these limitations, we have recently developed a microwell array integrated with recessed ring-type micro/nanoelectrodes (RME/RNE) (Sékli Belaïdi et al, 2016;Vajrala et al, 2019). A functional and intricate integration of these RNE-based devices within microwells allowed us to entrap individual mitochondria in a high-throughput manner, and followed their respiration under several metabolic activation and inhibition conditions.…”

“…As a sampling method to sense local oxygen changes around the entrapped cells, within microwells, we chose continuous cyclic voltammetry over chronoamperometry. We noticed that under optimized conditions -higher scan rates and gaps between each scan (4 V/s and 20 s gap), CV allows to monitor the local chemical changes happening within the microwells, while allowing the oxygen concentration to get to the equilibrium before next measurement(Vajrala et al, 2019) i.e., no competition between working electrode and yeast cells in terms of oxygen usage. We first investigated the oxygen sensing capacity of OptoElecWells by performing the electrochemical reduction of dissolved oxygen under different oxygen concentrations in yeast buffer using cyclic voltammetry at a scan rate of 4 V/sec.…”