Blanc SVSE 5 - Blanc - SVSE 5 - Physique, chimie du vivant et innovations biotechnologiques

Nano-sensors for metabolic studies at the single mitochondrion level – NANOMITO

Nanosensors of Oxidative Stress: Deciphering Mechanisms of Mitochondrial Pathologies

Micro and nanotechnologies are used to design and build systems dedicated to the analysis of oxidative stress. These systems provide the monitoring of activities of individual mitochondria, in order to go further into the understanding of mitochondrial pathologies.

How can we analyze Oxidative Stress status of each mitochondrion ?

Our fundamental research project positions upstream from biomedical research on mitochondrial pathologies. These pathologies include myopathies, neuropathies and cancers due to deficiencies of a major cell sub-unit: mitochondria. Mitochondria are named the cell powerhouses because they transform breather oxygen and substrates (sugars, fatty acids) into ATP, this molecule being the chemical energy substrate for many biological processes. <br />However, mitochondrial respiration can also lead to the production of « free radicals » derived from oxygen or nitrogen. These molecules might be harmful for mitochondria themselves or for the host cell, when produced in high quantities. Such dysfunction leads eventually to pathological processes in muscle cells, neurons and many other cell types. Consequently, it is essential to understand these mechanisms in order to propose pharmacological or medical actions. Understanding means first detecting and analyzing. Our project is precisely focused on the development of novel analytical devices to monitor with quantitative and temporal accuracy several mitochondrial metabolic parameters (respiration, energetic production of ATP and “free radicals” levels). We will develop sensors, using micro and nanotechnologies, for these metabolites and assemble them for multi-parametric analyses. In particular, we will focus on optical and electrochemical analyses at single mitochondria (few hundred nanometer size), so as to unravel unexpected properties of this organelle.

Our project is funded on the development of sensing electrodes and optical fibers, which dimension is in the range between few micrometers to a few tens nanometers. Individual sensors will be used to proceed single mitochondria analysis, or assembled in an array to get analytical microchips. These chips will afford large scale measurements of several mitochondrial activities, possibly from pathological origin.

-We developed microarrays for large scale-single mitochondria analyses. These were based on arrays of microwells, builded-up upon soft lithography (molding of a biocompatible polymer) or optical glass fiber structuration, for the isolation and observation of small populations of mitochondria in vitro. We succeeded in detecting the activity of single mitochondria disposed in each microwell of a large array. This permitted the monitoring of metabolic state variations (NADH, FAD, membrane potential, free radicals) of each mitochondrion under respiratory modulation. The goal of such analytical platforms is to compare metabolic states of mitochondria originating from different sources, tissues or cells.

- In parallel, we developed electrochemical microsystems, based on silicon chips integrating microelectrode arrays, for the simultaneous monitoring of oxygen and oxidative metabolites in vitro. We worked on the surface structuration and modification (platinum nanodeposits, enzymes) of platinum microelectrode arrays so as to detect hydrogen peroxide at nanomolar level in biological conditions in vitro. Our electrochemical chips allowed a direct quantification and kinetic study of the ratio between O2 consumption and H2O2 release by mitochondria, under modulation by respiratory activators and inhibitors.

Major perspectives of our micro and nanosensor systems include further studies and understanding of the correlations between metabolic parameters controlling the balance between redox signaling and oxidative stress in mitochondria.
Our studies are focused on single mitochondria detection since it should allow characterizing metabolic heteroplasmy between these organelles in a single cell or a tissue in diverse physio-pathological situations.

-Development of microwell arrays for single mitochondria studies with fluorescence detection:
Suraniti E. et al., Analytical Chemistry, 2013, 85, 5146-5152. Suraniti E. et al., Analytical & Bioanalytical Chemistry, 2013, in press, “paper in Forefront”
- Development of integrated electrochemical microsystems for the detection of oxidative stress markers:
Ben-Amor S. et al, Electroanalysis, 2013, 3, 656-663. Ben-Amor S. et al, Electrochimica Acta, 2013, accepted.

The NANOMITO project aims at developing novel analytical tools and biophysical approaches to study the mitochondrial metabolism at micro to nanoscale.
A literature survey shows that most of the current studies on mitochondrial activities are based on the use of classical spectrophotometric or electrochemical assays. As a consequence, seminal questions concerning the metabolic pathways of mitochondria are still unsolved, or even wrongly defined. In particular, it has been demonstrated that mitochondria are a major source of chemical reactive oxygen and nitrogen species (ROS & RNS), these species being involved in oxidative and nitrosative stress processes, but also in cell signaling and homeostasis. However, the exact nature of the ROS or RNS initiating these pathways, as well as their kinetic and quantitative features, are fairly unknown, which is critical for mechanistic studies or therapeutic approaches specifically targeting the mitochondria.
The NANOMITO project aims at developing novel analytical tools and methodological approaches to ascertain these issues. To do so, we have defined several challenging goals : (1) the development of selective sensors for metabolites of nitro-oxidative (ROS & RNS), respiratory and energetic pathways; (2) a simultaneous detection of these species on the same population of mitochondria to achieve a multi-parametric analysis of the mitochondrial metabolism; (2) measuring exact concentrations or fluxes of these species (quantitative information); (3) achieving in situ measurements (real-time detection) of the species released by mitochondria, that is at the single mitochondrion level, in order to ascertain the dynamics of production/release/diffusion of each in the framework of reactive- chemical signaling waves in cells.
To achieve these goals, The NANOMITO project will be based on the development of electrochemical sensors of micrometric to nanometric size. Their surface will be chemically modified and structured to detect selectively the major ROS or RNS- O2•-, H2O2, NO, ONOO- , oxygen or ATP, under physiological conditions. They will be used at three different levels of the NANOMITO project, funded on a top-down approach:
- First, the different sensors will be assembled on a microsystem platform and integrated within oxygraphy chambers of millimetric dimensions, in order to achieve multi-parametric analyses on the same population of mitochondria.
- Then, chambers will be miniaturized to perform analyses in situ on a small population composed of a few hundreds to a few mitochondria. Because of the decrease of space and time dimensions, species with a short life-time will become detectable and the variations due to statistical effect observed. These electrochemical microcells will be assembled as an array to perform studies in parallel on multiple samples of mitochondria.
- In addition, arrays of optical microcells will be developed based on the surface structuration of optical fibers. Mitochondrial activities will be analyzed in situ with these spectro-photometric microsystems following a staining of mitochondria by permeant or non-permeant probes. The comparison of data obtained by each method will afford a comprehensive view of the intra or extra-mitochondrial localization of the generation of species.
- Finally, our efforts will be concentrated on the development of sub-micrometric electrochemical sensors, i.e. nanoelectrodes, in order to analyze the production of some active metabolites at the single mitochondrion level. This goal corresponds to the name of our project: NANOMITO. We will perform detection either on single isolated mitochondria or on mitochondria directly within a cardiac muscle fiber (in cellulo). Though it represents a high analytical challenge, because minute amounts of metabolites will have to be detected, we expect analyzing some nitro-oxidative activities at a sub-cellular structure in space and time conditions that are the ones of biological events.

Project coordinator


The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 549,920 euros
Beginning and duration of the scientific project: January 2012 - 36 Months

Useful links

Explorez notre base de projets financés



ANR makes available its datasets on funded projects, click here to find more.

Sign up for the latest news:
Subscribe to our newsletter