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

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.