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Tuesday 25 April 2017

Electrochemistry of Biosystems

 

Stéphane Arbault, Neso Sojic, Alexander Kuhn, Laurent Bouffier, Patrick Garrigue


Our research activities are devoted to the development of sensors and methodologies for the detection of biological activities in order to elucidate with biologists and clinicians fundamental metabolic or genetic mechanisms.
We develop electrochemical and combined electrochemical/optical sensors, which dimensions and structuration range from micro- to nanoscale. These dimensions are adapted to the size of the biological entity under investigation (tissue, single cell, membrane, organelle, isolated DNA) and to the precision of localization required.

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Scheme describing our applications of micro- to nanoscale sensors for the bioactive species.


1. Electrochemical detection of mitochondria metabolites

A current project is focused on the monitoring of species from respiratory and energetic metabolism, including oxygen itself, ATP and Reactive Oxygen and Reactive Nitrogen Species (ROS & RNS). These latter ones are produced during oxidative and nitrosative stress processes. Their detection is of major interest to elucidate mechanisms involved in neurodegenerative or carcinogenetic situations. To do so, nanoparticle- or enzyme-modified microelectrodes and optoelectrodes are developed to measure selectively each species consumed or released by cells or by mitochondria. In particular, we quantify the ratio between O2 respiration and H2O2 release by mitochondria in physiological-signaling conditions and oxidative stress-induced situations. A further combination of the electrochemical and spectrophotometric approaches (see also the section “micro-nanoscale imaging” on our website) provides us relevant spectral signatures of species and give us the opportunity to follow-up their spatial distribution (typically from an intracellular source to an extracellular secretion).

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Left : A thermostated oxygraphy chamber (Clark electrode) integrating microsensor arrays for multi-parametric measurements of mitochondrial activities. Electrochemical sensor arrays were integrated on a silicon microchip (Collab. LAAS CNRS) and their surface individually modified by e.g. platinum deposits to increase their sensitivity or selectivity versus nitro-oxidant metabolites.

Right : example of a combined detection of oxygen consumption and hydrogen peroxide production by isolated mitochondria (yeast origin) under activation (ethanol: EtOH; ADP: adenosine-diphosphate) and inhibition (Antimycin A) of their respiratory chain.


2. Electrochemical arrays

Sensors are also integrated within microsystems to combine in situ the culture of cells or isolated organelles, their stimulation and the detection of biological responses. In particular, we have shown that a rational micro- and nanostructuration of the surface (highly organized meso-macroporous platinum deposits) of such devices allows reducing considerably the measurement noise and thus gives access to signals, like action potentials, that otherwise would not be detectable (see Figure below). Such optimized Bio-MEMS pave the way for clinical use and applications in diagnosis.

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Low-noise recording of neural activity using mesoporous MEAs. a) Whole embryonic (E13.5) hindbrain-spinal cord preparation on a 4x15 mesoporous MEA. b) Example of array-wide recording of rhythmic activity in such a preparation showing two wave-like episodes. c) Close-up view of the raw signal corresponding to the 2 channels highlighted in b). Each episode is composed of a local field potential on which a burst of spikes is superimposed. d) Same signals as in c) after high-pass filtering showing only the bursts of spikes. e) Same signals as in d) but superimposed on the background noise of the same microelectrodes before mesoporous modification. This last panel shows that bursting activity would not have been detected with these flat unmodified electrodes.


3. DNA biosensors

The development of DNA biosensors has been an expending field of research since decades motivated by constant needs for medical diagnosis and biochemical assays. Various analytical approaches including optics or spectroscopy are generally used to detect DNA hybridization events but electrochemistry is very often considered as a convenient alternative technique. In that context, an attractive route of DNA labelling, that does not require any chemical derivatization of the DNA complementary strand involves the use of small molecules interacting directly and selectively with the duplex, namely DNA intercalators. These natural or synthetic compounds were first studied as drugs for potential anticancer applications but are now also considered as interesting tools for DNA detection especially when they are redox-active. We are currently developing the synthetic access to pyridoacridine DNA intercalators. We are investigating the spectroelectrochemical properties in order to achieve multiple modes of detection based on DNA-mediated electron transfer but also in-situ fluorescence or Raman spectroscopy under electrochemical control.

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Chemical access to synthetic DNA intercalators from a commercially available acridine (left); Electrochemical and fluorescence detection of DNA hybridization on microelectrode arrays (right).

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