Publications by authors named "Stéphane Arbault"

68 Publications

Shadow Electrochemiluminescence Microscopy of Single Mitochondria.

Angew Chem Int Ed Engl 2021 Jun 11. Epub 2021 Jun 11.

University of Bordeaux, Bordeaux INP, ISM, UMR CNRS 5255, 33607, Pessac, France.

Mitochondria are the subcellular bioenergetic organelles. The analysis of their morphology and topology is essential to provide useful information on their activity and metabolism. Herein, we report a label-free shadow electrochemiluminescence (ECL) microscopy based on the spatial confinement of the ECL-emitting reactive layer to image single living mitochondria deposited on the electrode surface. The ECL mechanism of the freely-diffusing [Ru(bpy) ] dye with the sacrificial tri-n-propylamine coreactant restrains the light-emitting region to a micrometric thickness allowing to visualize individual mitochondria with a remarkable sharp negative optical contrast. The imaging approach named "shadow ECL" (SECL) reflects the negative imprint of the local diffusional hindrance of the ECL reagents by each mitochondrion. The statistical analysis of the colocalization of the shadow ECL spots with the functional mitochondria revealed by classical fluorescent biomarkers, MitoTracker Deep Red and the endogenous intramitochondrial NADH, validates the reported methodology. The versatility and extreme sensitivity of the approach are further demonstrated by visualizing single mitochondria, which remain hardly detectable with the usual biomarkers. Finally, by alleviating problems of photobleaching and phototoxicity associated with conventional microscopy methods, SECL microscopy should find promising applications in the imaging of subcellular structures.
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http://dx.doi.org/10.1002/anie.202105867DOI Listing
June 2021

Single-Particle Tracking Method in Fluorescence Microscopy to Monitor Bioenergetic Responses of Individual Mitochondria.

Methods Mol Biol 2021 ;2276:153-163

NSysA group, Univ. Bordeaux, CNRS, INP Bordeaux, ISM, UMR 5255, Talence, France.

The spectroscopic methods commonly used to study mitochondria bioenergetics do not show the diversity of responses within a population of mitochondria (isolated or in a cell), and/or cannot measure individual dynamics. New methodological developments are necessary in order to improve quantitative and kinetic resolutions and eventually gain further insights on individual mitochondrial responses, such as studying activities of the mitochondrial permeability transition pore (mPTP ). The work reported herein is devoted to study responses of single mitochondria within a large population after isolation from cardiomyocytes. Mitochondria were preloaded with a commonly used membrane potential sensitive dye (TMRM), they are then deposited on a plasma-treated glass coverslip and subsequently energized or inhibited by additions of usual bioenergetics effectors. Responses were analyzed by fluorescence microscopy over few thousands of mitochondria simultaneously with a single organelle resolution. We report an automatic method to analyze each image of time-lapse stacks based on the TrackMate-ImageJ plug-in and specially made Python scripts. Images are processed to eliminate defects of illumination inhomogeneity, improving by at least two orders of magnitude the signal/noise ratio. This method enables us to follow the track of each mitochondrion within the observed field and monitor its fluorescence changes, with a time resolution of 400 ms, uninterrupted over the course of the experiment. Such methodological improvement is a prerequisite to further study the role of mPTP in single mitochondria during calcium transient loading.
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http://dx.doi.org/10.1007/978-1-0716-1266-8_11DOI Listing
July 2021

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

Adv Biol (Weinh) 2021 Jul 9;5(7):e2100484. Epub 2021 May 9.

CNRS, LAAS, 7 avenue du colonel Roche, Toulouse, F-31400, France.

Microdevices composed of microwell arrays integrating nanoelectrodes (OptoElecWell) are developed to achieve dual high-resolution optical and electrochemical detections on single Saccharomyces cerevisiae yeast cells. Each array consists of 1.6 × 10 microwells measuring 8 µm in diameter and 5 µm height, with a platinum nanoring electrode for in situ electrochemistry, all integrated on a transparent thin wafer for further high-resolution live-cell imaging. After optimizing the filling rate, 32% of cells are effectively trapped within microwells. This allows to analyse S. cerevisiae metabolism associated with basal respiration while simultaneously measuring optically other cellular parameters. In this study, the impact of glucose concentration on respiration and intracellular rheology is focused. It is found that while the oxygen uptake rate decreases with increasing glucose concentration, diffusion of tracer nanoparticles increases. The OptoElecWell-based respiration methodology provides similar results compared to the commercial gold-standard Seahorse XF analyzer, while using 20 times fewer biological samples, paving the way to achieve single cell metabolomics. In addition, it facilitates an optical route to monitor the contents within single cells. The proposed device, in combination with the dual detection analysis, opens up new avenues for measuring cellular metabolism, and relating it to cellular physiological indicators at single cell level.
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http://dx.doi.org/10.1002/adbi.202100484DOI Listing
July 2021

Impacts of vesicular environment on Nox2 activity measurements in vitro.

Biochim Biophys Acta Gen Subj 2021 01 22;1865(1):129767. Epub 2020 Oct 22.

Université Paris-Saclay, CNRS, Institut de Chimie Physique, UMR8000, 91405, Orsay, France. Electronic address:

Background: The production of superoxide anions (O) by the phagocyte NADPH oxidase complex has a crucial role in the destruction of pathogens in innate immunity. Majority of in vitro studies on the functioning of NADPH oxidase indirectly follows the enzymatic reaction by the superoxide reduction of cytochrome c (cyt c). Only few reports mention the alternative approach consisting in measuring the NADPH consumption rate. When using membrane vesicles of human neutrophils, the enzyme specific activity is generally found twice higher by monitoring the NADPH oxidation than by measuring the cyt c reduction. Up to now, the literature provides only little explanations about such discrepancy despite the critical importance to quantify the exact enzyme activity.

Methods: We deciphered the reasons of this disparity in studying the role of key parameters, including. cyt c and arachidonic acid concentrations, in conjunction with an ionophore, a detergent and using Clark electrode to measure the O consumption rates.

Results: Our results show that the O low permeability of the vesicle membrane as well as secondary reactions (O and HO disproportionations) are strong clues to shed light on this inconsistency.

Conclusion And General Significance: These results altogether indicate that the cyt c reduction method underestimates the accurate Nox2 activity.
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http://dx.doi.org/10.1016/j.bbagen.2020.129767DOI Listing
January 2021

Dynamic monitoring of a bi-enzymatic reaction at a single biomimetic giant vesicle.

Analyst 2021 Jan;145(24):7922-7931

Univ. Bordeaux, CNRS, Bordeaux INP, ISM, UMR 5255, F-33400, Talence, France.

Giant unilamellar vesicles were used as individual biomimetic micro-reactors wherein a model bi-enzymatic reaction involving a glucose oxidase (GOx) and horseradish peroxidase (HRP) was monitored by confocal microscopy. These giant vesicles were formed from a natural mix of phospholipids in physiological conditions of pH and osmolarity (phosphate buffer, pH 7.4, 330 mOsm). The so-called Amplex Red assay, which generates the highly fluorescent resorufin species, was performed in individual vesicles and used to report on the progress of the whole reaction. We aimed at controlling kinetically and quantitatively the different steps of the bi-enzymatic reaction in vesicles. To do so, substrates (glucose and Amplex Red) were provided in individual reactors by two ways. Electro-microinjection allowed the control of volume variations owing to a reservoir of lipids connected to the vesicle membrane. Alternatively, substrates could passively diffuse from the outer solution to the vesicle compartment. The semi-permeability feature of the phospholipid membrane was characterized for all substrates and products while we demonstrated that enzymes remain sequestrated in the vesicles after their injection. The Amplex Red assay was thus achieved in individual vesicles under steady-state conditions, and could pursue over tens of minutes. Such giant vesicles are stable, fully compatible with media used for bioanalyses and allow out-of-equilibrium reactions at time scales compatible with living reaction dynamics, making them a good choice for the development of minimal cell-like systems.
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http://dx.doi.org/10.1039/d0an01273dDOI Listing
January 2021

Chemo- and Magnetotaxis of Self-Propelled Light-Emitting Chemo-electronic Swimmers.

Angew Chem Int Ed Engl 2020 May 11;59(19):7508-7513. Epub 2020 Mar 11.

Univ. Bordeaux, CNRS UMR 5255, Bordeaux INP, Site ENSCBP, 33607, Pessac, France.

Miniaturized autonomous chemo-electronic swimmers, based on the coupling of spontaneous oxidation and reduction reactions at the two poles of light-emitting diodes (LEDs), are presented as chemotactic and magnetotactic devices. In homogeneous aqueous media, random motion caused by a bubble-induced propulsion mechanism is observed. However, in an inhomogeneous environment, the self-propelled devices exhibit positive chemotactic behavior, propelling themselves along a pH or ionic strength gradient (∇pH and ∇I, respectively) in order to reach a thermodynamically higher active state. In addition, the intrinsic permanent magnetic moment of the LED allows self-orientation in the terrestrial magnetic field or following other external magnetic perturbations, which enables a directional motion control coupled with light emission. The interplay between chemotaxis and magnetotaxis allows fine-tuning of the dynamic behavior of these swimmers.
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http://dx.doi.org/10.1002/anie.201915705DOI Listing
May 2020

Remote Actuation of a Light-Emitting Device Based on Magnetic Stirring and Wireless Electrochemistry.

Chemphyschem 2020 Apr 4;21(7):600-604. Epub 2020 Mar 4.

Univ. Bordeaux, ISM, UMR 5255, 33400, Talence, France.

We propose a straightforward access to a rotating light-emitting device powered by wireless electrochemistry. A magnetic stirrer is used to rotate a light-emitting diode (LED) due to the intrinsic magnetic properties of the tips that contain iron. At the same time, the LED is submitted to an electric field and acts as a bipolar electrode. The electrochemical processes that are coupled on both extremities of the LED drive an electron flow across the device, resulting in light emission. The variation of the LED alignment in time enables an alternating light emission that is directly controlled by the rotation rate. The stirring also enables a continuous mixing of the electrolyte that improves the stability of the output signal. Finally, the LED brightness can readily reveal a change of chemical composition in the electrolyte solution.
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http://dx.doi.org/10.1002/cphc.202000019DOI Listing
April 2020

Reactive Oxygen Species Generated by Cold Atmospheric Plasmas in Aqueous Solution: Successful Electrochemical Monitoring in Situ under a High Voltage System.

Anal Chem 2019 07 20;91(13):8002-8007. Epub 2019 Jun 20.

Univ. Bordeaux , CNRS, Bordeaux INP, ISM, UMR 5255 , F-33400 Talence , France.

Many investigations are dedicated to the detection and quantification of reactive oxygen and nitrogen species (RONS), particularly when generated in liquids exposed to cold atmospheric plasmas (CAPs). CAPs are partially ionized gases that can be obtained by applying a high electric field to a gas. A challenge is to get better insights on the plasma-liquid interactions in order to understand the induced effects on different targets (liquid, cells, tissues, etc.). As RONS are biochemically reactive, the difficulty lies in finding efficient methods to get both dynamic and quantitative data. Herein, we developed an innovative setup aimed at performing an in situ electrochemical monitoring of redox species generated by CAPs in a physiological buffer (PBS, pH 7.4). The challenge was to apply millivolt-potential variations and measure nanoampere Faradaic currents in the presence of ionization waves generated by micropulsed electric fields of some 10 kV·cm amplitude and ampere-transient currents. This was fulfilled by using dedicated working ultramicroelectrodes (Pt-black UMEs) and protecting them, as well as the reference and counter electrodes, within insulated-earthed containers. In this condition, we succeeded in performing both cyclic voltammetry and chronoamperometry in situ, with a resolution equivalent to working in a static solution (subnanoampere currents). Thus, we monitored the accumulation over time of species (HO, NO) generated by CAPs in PBS and observed the mean dynamic of RONS chemistry during and after plasma exposition, particularly through the detection of a short-living species.
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http://dx.doi.org/10.1021/acs.analchem.9b01912DOI Listing
July 2019

Electrochemiluminescence Imaging for Bioanalysis.

Annu Rev Anal Chem (Palo Alto Calif) 2019 06 2;12(1):275-295. Epub 2019 Apr 2.

School of Chemistry and Chemical Engineering, State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing, Jiangsu 210093, China; email:

Electrochemiluminescence (ECL) is a widely used analytical technique with the advantages of high sensitivity and low background signal. The recent and rapid development of electrochemical materials, luminophores, and optical elements significantly increases the ECL signals and, thus, ECL imaging with enhanced spatial and temporal resolutions is realized. Currently, ECL imaging is successfully applied to high-throughput bioanalysis and to visualize the distribution of molecules at single cells. Compared with other optical bioassays, no optical excitation is involved in imaging, so the approach avoids a background signal from illumination and increases the detection sensitivity. This review highlights some of the most exciting developments in this field, including the mechanisms, electrode designs, and the applications of ECL imaging in bioanalysis and at single cells and particles.
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http://dx.doi.org/10.1146/annurev-anchem-061318-115226DOI Listing
June 2019

Highly parallel remote SPR detection of DNA hybridization by micropillar optical arrays.

Anal Bioanal Chem 2019 Apr 23;411(11):2249-2259. Epub 2019 Feb 23.

CEA, CNRS, INAC-SyMMES, Université Grenoble Alpes, 38000, Grenoble, France.

Remote detection by surface plasmon resonance (SPR) is demonstrated through microstructured optical arrays of conical nanotips or micropillars. Both geometries were fabricated by controlled wet chemical etching of bundles comprising several thousands of individual optical fibers. Their surface was coated by a thin gold layer in order to confer SPR properties. The sensitivity and resolution of both shapes were evaluated as a function of global optical index changes in remote detection mode performed by imaging through the etched optical fiber bundle itself. With optimized geometry of micropillar arrays, resolution was increased up to 10 refractive index units. The gold-coated micropillar arrays were functionalized with DNA and were able to monitor remotely the kinetics of DNA hybridization with complementary strands. We demonstrate for the first time highly parallel remote SPR detection of DNA via microstructured optical arrays. The obtained SPR sensitivity combined with the remote intrinsic properties of the optical fiber bundles should find promising applications in biosensing, remote SPR imaging, a lab-on-fiber platform dedicated to biomolecular analysis, and in vivo endoscopic diagnosis. Graphical abstract We present a single fabrication step to structure simultaneously all the individual cores of an optical fiber bundle composed of thousands of fibers. The resulting sensor is optimized for reflection mode (compatible with in vivo applications) and is used to perform for the first time highly parallel remote SPR detection of DNA via several thousands of individual optical fiber SPR sensors paving the way for multiplexed biological detection.
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http://dx.doi.org/10.1007/s00216-019-01689-2DOI Listing
April 2019

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

Biosens Bioelectron 2019 Feb 26;126:672-678. Epub 2018 Nov 26.

Univ. Bordeaux, ISM, CNRS UMR 5255, INP Bordeaux, Pessac, France. Electronic address:

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 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 half-height 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).
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http://dx.doi.org/10.1016/j.bios.2018.11.036DOI Listing
February 2019

Surface-Confined Electrochemiluminescence Microscopy of Cell Membranes.

J Am Chem Soc 2018 11 29;140(44):14753-14760. Epub 2018 Oct 29.

University of Bordeaux , Bordeaux INP, ISM, UMR CNRS 5255 , 33607 Pessac , France.

Herein is reported a surface-confined microscopy based on electrochemiluminescence (ECL) that allows to image the plasma membrane of single cells at the interface with an electrode. By analyzing photoluminescence (PL), ECL and AFM images of mammalian CHO cells, we demonstrate that, in contrast to the wide-field fluorescence, ECL emission is confined to the immediate vicinity of the electrode surface and only the basal membrane of the cell becomes luminescent. The resulting ECL microscopy reveals details that are not resolved by classic fluorescence microscopy, without any light irradiation and specific setup. The thickness of the ECL-emitting regions is ∼500 nm due to the unique ECL mechanism that involves short-lifetime electrogenerated radicals. In addition, the reported ECL microscopy is a dynamic technique that reflects the transport properties through the cell membranes and not only the specific labeling of the membranes. Finally, disposable transparent carbon nanotube (CNT)-based electrodes inkjet-printed on classic microscope glass coverslips were used to image cells in both reflection and transmission configurations. Therefore, our approach opens new avenues for ECL as a surface-confined microscopy to develop single cell assays and to image the dynamics of biological entities in cells or in membranes.
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http://dx.doi.org/10.1021/jacs.8b08080DOI Listing
November 2018

A snapshot of the electrochemical reaction layer by using 3 dimensionally resolved fluorescence mapping.

Chem Sci 2018 Aug 16;9(32):6622-6628. Epub 2018 Jul 16.

Univ. Bordeaux , CNRS , Bordeaux INP , ISM , UMR 5255 , F-33400 Talence , France . Email:

The coupling between electrochemistry and fluorescence confocal laser scanning microscopy (FCLSM) allows deciphering the electrochemical and/or redox reactivity of electroactive fluorophores. This is demonstrated with phenoxazine electrofluorogenic species frequently used in bioassays by mapping the variation of fluorescence intensity with respect to the distance from the electrode. The electrochemical conversion of resorufin dye (RF) to non-fluorescent dihydroresorufin (DH) leads to a sharp decrease of the fluorescence signal in the vicinity of the electrode. In contrast, the direct reduction of resazurin (RZ) to DH leads to an unexpected maximum fluorescence intensity localized further away from the surface. This observation indicates that the initial electron transfer (heterogeneous) is followed by a chemical comproportionation step (homogeneous), leading to the formation of RF within the diffusion layer with a characteristic concentration profile. Therefore, FCLSM affords a direct way to monitor such chemical reactivity in space and to decipher a new redox pathway that cannot be resolved solely by electrochemical means.
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http://dx.doi.org/10.1039/c8sc02011fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6115633PMC
August 2018

Electroformation of phospholipid giant unilamellar vesicles in physiological phosphate buffer.

Integr Biol (Camb) 2018 07;10(7):429-434

University of Bordeaux, ISM, CNRS UMR5255, NSysA group, ENSCBP, 16 avenue Pey Berland, 33607 Pessac, France.

Phospholipid Giant Unilamellar Vesicles (GUVs) are usually prepared by electroformation in water, that is in a low-conductivity solution. We developed a protocol allowing their electroformation in the most common physiological buffer, phosphate-buffered saline (PBS). This was achieved based on a specific sequence of increasing electrical fields and for the two usual electrode types for electroformation, namely Indium Tin oxide-coated glass slides and Pt electrodes. These GUVs are stable over time (hour time-scale) and they can be isolated or micro-injected. The membrane composition was modified by adding cholesterol in order to adjust its mechanical properties. The optimal proportion of cholesterol vs. total phospholipid concentration was a ratio of 20 mol%, which increases membrane rigidity and facilitates vesicle microinjection.
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http://dx.doi.org/10.1039/c8ib00074cDOI Listing
July 2018

Correlations between gaseous and liquid phase chemistries induced by cold atmospheric plasmas in a physiological buffer.

Phys Chem Chem Phys 2018 Apr;20(14):9198-9210

UPPA, IPREM, CNRS UMR 5254, 2 Avenue Président Angot, 64000 Pau, France.

The understanding of plasma-liquid interactions is of major importance, not only in physical chemistry, chemical engineering and polymer science, but in biomedicine as well as to better control the biological processes induced on/in biological samples by Cold Atmospheric Plasmas (CAPs). Moreover, plasma-air interactions have to be particularly considered since these CAPs propagate in the ambient air. Herein, we developed a helium-based CAP setup equipped with a shielding-gas device, which allows the control of plasma-air interactions. Thanks to this device, we obtained specific diffuse CAPs, with the ability to propagate along several centimetres in the ambient air at atmospheric pressure. Optical Emission Spectroscopy (OES) measurements were performed on these CAPs during their interaction with a liquid medium (phosphate-buffered saline PBS 10 mM, pH 7.4) giving valuable information about the induced chemistry as a function of the shielding gas composition (variable O2/(O2 + N2) ratio). Several excited species were detected including N2+(First Negative System, FNS), N2(Second Positive System, SPS) and HO˙ radical. The ratios between nitrogen/oxygen excited species strongly depend on the O2/(O2 + N2) ratio. The liquid chemistry developed after CAP treatment was investigated by combining electrochemical and UV-visible absorption spectroscopy methods. We detected and quantified stable oxygen and nitrogen species (H2O2, NO2-, NO3-) along with Reactive Nitrogen Species (RNS) such as the peroxynitrite anion ONOO-. It appears that the RNS/ROS (Reactive Oxygen Species) ratio in the treated liquid depends also on the shielding gas composition. Eventually, the composition of the surrounding environment of CAPs seems to be crucial for the induced plasma chemistry and consequently, for the liquid chemistry. All these results demonstrate clearly that for physical, chemical and biomedical applications, which are usually achieved in ambient air environments, it is necessary to realize an effective control of plasma-air interactions.
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http://dx.doi.org/10.1039/C8CP00264ADOI Listing
April 2018

Single Cell Electrochemiluminescence Imaging: From the Proof-of-Concept to Disposable Device-Based Analysis.

J Am Chem Soc 2017 11 8;139(46):16830-16837. Epub 2017 Nov 8.

University of Bordeaux, Bordeaux INP, ISM, UMR CNRS 5255, 33607 Pessac, France.

We report here the development of coreactant-based electrogenerated chemiluminescence (ECL) as a surface-confined microscopy to image single cells and their membrane proteins. Labeling the entire cell membrane allows one to demonstrate that, by contrast with fluorescence, ECL emission is only detected from fluorophores located in the immediate vicinity of the electrode surface (i.e., 1-2 μm). Then, to present the potential diagnostic applications of our approach, we selected carbon nanotubes (CNT)-based inkjet-printed disposable electrodes for the direct ECL imaging of a labeled plasma receptor overexpressed on tumor cells. The ECL fluorophore was linked to an antibody and enabled to localize the ECL generation on the cancer cell membrane in close proximity to the electrode surface. Such a result is intrinsically associated with the unique ECL mechanism and is rationalized by considering the limited lifetimes of the electrogenerated coreactant radicals. The electrochemical stimulus used for luminescence generation does not suffer from background signals, such as the typical autofluorescence of biological samples. The presented surface-confined ECL microscopy should find promising applications in ultrasensitive single cell imaging assays.
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http://dx.doi.org/10.1021/jacs.7b09260DOI Listing
November 2017

Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure.

Radiat Res 2018 01 23;189(1):95-103. Epub 2017 Oct 23.

a   Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France.

The existence of effects of radiofrequency field exposure at environmental levels on living tissues and organisms remains controversial, in particular regarding potential "nonthermal" effects produced in the absence of temperature elevation. Therefore, we investigated whether TRPV1, one of the most studied thermosensitive channels, can be activated by the heat produced by radiofrequency fields and by some specific nonthermal interaction with the fields. We have recently shown that TRPV1 activation can be assessed in real-time on live cells using the bioluminescence resonance energy transfer technique. Taking advantage of this innovative assay, we monitored TRPV1 thermal and chemical modes of activation under radiofrequency exposure at 1800 MHz using different signals (CW, GSM, UMTS, LTE, Wi-Fi and WiMAX) at specific absorption rates between 8 and 32 W/kg. We showed that, as expected, TRPV1 channels were activated by the heat produced by radiofrequency field exposure of transiently-transfected HEK293T cells, but found no evidence of TRPV1 activation in the absence of temperature elevation under radiofrequency field exposure. There was no evidence either that, at fixed temperature, radiofrequency exposure altered the maximal efficacy of the agonist Capsaicin to activate TRPV1.
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http://dx.doi.org/10.1667/RR14877.1DOI Listing
January 2018

Oxidative modification and electrochemical inactivation of Escherichia coli upon cold atmospheric pressure plasma exposure.

PLoS One 2017 30;12(3):e0173618. Epub 2017 Mar 30.

Faculté de Chirurgie Dentaire de Toulouse, centre Hospitalier Universitaire de Toulouse, Université Paul Sabatier, Toulouse, France.

Cold atmospheric pressure plasmas (CAPPs) are known to have bactericidal effects but the mechanism of their interaction with microorganisms remains poorly understood. In this study the bacteria Escherichia coli were used as a model and were exposed to CAPPs. Different gas compositions, helium with or without adjunctions of nitrogen or oxygen, were used. Our results indicated that CAPP induced bacterial death at decontamination levels depend on the duration, post-treatment storage and the gas mixture composition used for the treatment. The plasma containing O2 in the feeding gas was the most aggressive and showed faster bactericidal effects. Structural modifications of treated bacteria were observed, especially significant was membrane leakage and morphological changes. Oxidative stress caused by plasma treatment led to significant damage of E. coli. Biochemical analyses of bacterial macromolecules indicated massive intracellular protein oxidation. However, reactive oxygen and nitrogen species (RONS) are not the only actors involved in E. coli's death, electrical field and charged particles could play a significant role especially for He-O2 CAPP.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0173618PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373509PMC
August 2017

Mechanistic insights into the impact of Cold Atmospheric Pressure Plasma on human epithelial cell lines.

Sci Rep 2017 01 25;7:41163. Epub 2017 Jan 25.

IPREM, UMR 5254, Université de Pau et des Pays de l'Adour, 64000, Pau, France.

Compelling evidence suggests that Cold Atmospheric Pressure Plasma (CAPP) has potential as a new cancer therapy. However, knowledge about cellular signaling events and toxicity subsequent to plasma treatment is still poorly documented. The aim of this study was to focus on the interaction between 3 different types of plasma (He, He-O, He-N) and human epithelial cell lines to gain better insight into plasma-cell interaction. We provide evidence that reactive oxygen and nitrogen species (RONS) are inducing cell death by apoptosis and that the proteasome, a major intracellular proteolytic system which is important for tumor cell growth and survival, is a target of (He or He-N) CAPP. However, RONS are not the only actors involved in cell death; electric field and charged particles could play a significant role especially for He-O CAPP. By differential label-free quantitative proteomic analysis we found that CAPP triggers antioxidant and cellular defense but is also affecting extracellular matrix in keratinocytes. Moreover, we found that malignant cells are more resistant to CAPP treatment than normal cells. Taken together, our findings provide insight into potential mechanisms of CAPP-induced proteasome inactivation and the cellular consequences of these events.
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http://dx.doi.org/10.1038/srep41163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5264585PMC
January 2017

Full-Spectral Multiplexing of Bioluminescence Resonance Energy Transfer in Three TRPV Channels.

Biophys J 2017 Jan;112(1):87-98

Laboratoire de l'Intégration du Matériau au Système, Centre National de la Recherche Scientifique (CNRS) UMR 5218, Talence, France; Université de Bordeaux, Talence, France. Electronic address:

Multiplexed bioluminescence resonance energy transfer (BRET) assays were developed to monitor the activation of several functional transient receptor potential (TRP) channels in live cells and in real time. We probed both TRPV1 intramolecular rearrangements and its interaction with Calmodulin (CaM) under activation by chemical agonists and temperature. Our BRET study also confirmed that: (1) capsaicin and heat promoted distinct transitions, independently coupled to channel gating, and that (2) TRPV1 and Ca-bound CaM but not Ca-free CaM were preassociated in resting live cells, while capsaicin activation induced both the formation of more TRPV1/CaM complexes and conformational changes. The BRET assay, based on the interaction with Calmodulin, was successfully extended to TRPV3 and TRPV4 channels. We therefore developed a full-spectral three-color BRET assay for analyzing the specific activation of each of the three TRPV channels in a single sample. Such key improvement in BRET measurement paves the way for the simultaneous monitoring of independent biological pathways in live cells.
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http://dx.doi.org/10.1016/j.bpj.2016.11.3197DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5232350PMC
January 2017

Selective electrochemiluminescent sensing of saccharides using boronic acid-modified coreactant.

Chem Commun (Camb) 2016 Oct;52(87):12845-12848

University of Bordeaux, ISM, CNRS UMR 5255, Bordeaux INP, Pessac, France.

We report a strategy for modulating the electrogenerated chemiluminescence (ECL) response by integrating a boronic acid to the chemical structure of coreactants. Excellent selectivity for d-glucose was achieved by tuning the linker length of a bis-boronic acid amine coreactant.
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http://dx.doi.org/10.1039/c6cc07030bDOI Listing
October 2016

Direct oxidative pathway from amplex red to resorufin revealed by in situ confocal imaging.

Phys Chem Chem Phys 2016 Oct 29;18(37):25817-22. Epub 2016 Jul 29.

Univ. Bordeaux, ISM, UMR 5255, F-33400 Talence, France.

Amplex Red (AR) is a very useful chemical probe that is employed in biochemical assays. In these assays, the non-fluorescent AR is converted to resorufin (RS), which strongly absorbs in the visible region (λabs = 572 nm) and yields strong fluorescence (λfluo = 583 nm). Even if AR is commonly used to report on enzymatic oxidase activities, an increasing number of possible interferences have been reported, thus lowering the accuracy of the so-called AR assay. As a redox-based reaction, we propose here to directly promote the conversion of AR to RS by means of electrochemistry. The process was first assessed by classic electrochemical and spectroelectrochemical investigations. In addition, we imaged the electrochemical conversion of AR to RS at the electrode surface by in situ confocal microscopy. The coupling of methodologies allowed to demonstrate that RS is directly formed from AR by an oxidation step, unlike what was previously reported. This gives a new insight in the deciphering of AR assays' mechanism and about their observed discrepancy.
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http://dx.doi.org/10.1039/c6cp04438gDOI Listing
October 2016

PDMS microwells for multi-parametric monitoring of single mitochondria on a large scale: a study of their individual membrane potential and endogenous NADH.

Integr Biol (Camb) 2016 08 7;8(8):836-43. Epub 2016 Jul 7.

Univ. Bordeaux, CNRS, ISM UMR5255, NSYSA group, ENSCBP, 33607 Pessac, France.

Microwell arrays have been developed to monitor simultaneously, and on a large scale, multiple metabolic responses of single mitochondria. Wells of 50 to 1000 μm-diameter were prepared based on easy structuration of thin polydimethylsiloxane layers (PDMS; 100 μm thickness). Their surface treatment with oxygen plasma allowed the immobilization in situ and observation with time of populations of single isolated mitochondria. Their metabolic activities could be monitored individually by fluorescence microscopy under several activation/inhibition conditions. We measured the concomitant variations of two main metabolic parameters - the endogenous NADH level and the internal membrane potential difference Δψ owing to a cationic fluorescent probe (TMRM) - at energized, uncoupled and inhibited stages of the mitochondrial respiratory chain. Microwell arrays allowed analyses on large populations, and consequently statistical studies with a single organelle resolution. Thus, we observed rapid individual polarizations and depolarizations of mitochondria following their supply with the energetic substrate, while an averaged global polarization (increase of TMRM fluorescence within mitochondria) and NADH increase were detected for the whole population. In addition, statistical correlation studies show that the NADH content of all mitochondria tends toward a metabolic limit and that their polarization-depolarization ability is ubiquitous. These results demonstrate that PDMS microwell platforms provide an innovative approach to better characterize the individual metabolic status of isolated mitochondria, possibly as a function of their cell or organ origin or in different physio-pathological situations.
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http://dx.doi.org/10.1039/c6ib00064aDOI Listing
August 2016

Coupling Electrochemistry with Fluorescence Confocal Microscopy To Investigate Electrochemical Reactivity: A Case Study with the Resazurin-Resorufin Fluorogenic Couple.

Anal Chem 2016 06 1;88(12):6292-300. Epub 2016 Jun 1.

Univ. Bordeaux , ISM, UMR 5255, F-33400 Talence, France.

The redox couple resazurin-resorufin exhibits electrofluorochromic properties which are investigated herein by absorption and fluorescence spectroelectrochemistry and by electrochemically coupled-fluorescence confocal laser scanning microscopy (EC-CLSM). At pH 10, the highly fluorescent resorufin dye is generated at the electrode surface by the electrochemical reduction of the poorly fluorescent resazurin. Performing EC-CLSM at electrode surfaces allows to monitor spatially resolved electrochemical processes in situ and in real time. Using a small (315 μm diameter) cylindrical electrode, a steady-state diffusion layer builds up under potentiostatic conditions at -0.45 V vs Ag|AgCl. Mapping the fluorescence intensity in 3D by CLSM enables us to reconstruct the relative concentration profile of resorufin around the electrode. The comparison of the experimental diffusion-profile with theoretical predictions demonstrates that spontaneous convection has a direct influence on the actual thickness of the diffusion layer, which is smaller than the value predicted for a purely diffusional transport. This study shows that combining fluorescence CLSM with electrochemistry is a powerful tool to study electrochemical reactivity at a spatially resolved level.
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http://dx.doi.org/10.1021/acs.analchem.6b00477DOI Listing
June 2016

Dual Enzymatic Detection by Bulk Electrogenerated Chemiluminescence.

Anal Chem 2016 06 2;88(12):6585-92. Epub 2016 Jun 2.

University of Bordeaux , ISM, UMR 5255 CNRS, ENSCBP, 33607 Pessac, France.

The combination of enzymes, as recognition elements for specific analytes, and of electrogenerated chemiluminescence (ECL) as a readout method has proven to be a valuable strategy for sensitive and specific analytical detection. However, ECL is intrinsically a 2D process which could potentially limit the analysis of inhomogeneous samples. Here, we show how a bulk ECL signal, generated by thousands of carbon microbeads remotely addressed via bipolar electrochemistry, are implemented as a powerful tool for the concomitant ECL sensing and imaging of two enzymatic substrates. We selected two enzymes (glucose dehydrogenase and choline oxidase) that react with their respective model substrates and produce in situ chemical species (β-nicotinamide adenine dinucleotide (NADH) and H2O2) acting as coreactants for the ECL emission of different luminophores ([Ru(bpy)3](2+) at λ = 620 nm and luminol at λ = 425 nm, respectively). Both enzymes are spatially separated in the same capillary. We demonstrate thus the simultaneous quantitative determination of both glucose and choline over a wide concentration range. The originality of this remote approach is to provide a global chemical view through one single ECL image of inhomogeneous samples such as a biochemical concentration gradient in a capillary configuration. Finally, we report the first proof-of-concept of dual biosensing based on this bulk ECL method for the simultaneous imaging of both enzymatic analytes at distinct wavelengths.
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http://dx.doi.org/10.1021/acs.analchem.6b01434DOI Listing
June 2016

Generation of electrochemiluminescence at bipolar electrodes: concepts and applications.

Anal Bioanal Chem 2016 Oct 16;408(25):7003-11. Epub 2016 May 16.

Institut des Sciences Moléculaires, University of Bordeaux, Bordeaux INP, ENSCBP, 16 Avenue Pey Berland, 33607, Pessac, France.

Bipolar electrochemistry (BPE) is an unconventional technique where a conducting object is addressed electrochemically in an electrolyte without any wire connection with an external power supply. BPE has been known for decades but remained limited to only a couple of niche applications. However, it is now undergoing a true renewal of interest especially in the context of analytical chemistry. The bipolar electrode exhibits two distinct poles of opposite polarization with respect to the solution. This allows one to separate the localization of sensing elements versus reporting ones. Also, arrays of bipolar microelectrodes can be addressed simultaneously to perform parallel analyses. Among several reporting strategies, the combination of BPE with electro-chemiluminescence (ECL) is the most frequent choice owing to the very simple visual readout provided by ECL. This article reviews the field from the initial reports to the most recent ones, revealing numerous opportunities including novel analytical strategies for the detection of small molecular analytes and biorelevant molecules such as DNA, RNA, peptides, or other biomarkers. Graphical Abstract Principle of electrochemiluminescence generation at one extremity of a bipolar electrode.
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http://dx.doi.org/10.1007/s00216-016-9606-9DOI Listing
October 2016

Double remote electrochemical addressing and optical readout of electrochemiluminescence at the tip of an optical fiber.

Analyst 2016 Jul 16;141(14):4299-304. Epub 2016 May 16.

University of Bordeaux, ISM, CNRS UMR 5255, Bordeaux INP, ENSCBP, Pessac, France.

In this work, we report an original strategy for the wireless electrochemical generation of light at the tip of an optical fiber bundle, coupled with a simultaneous remote readout. An optical fiber bundle coated with a nanometer-thin gold film acts as a dual platform, on the one hand to locally generate electrochemiluminescence (ECL) in a wireless manner by bipolar electrochemistry, and on the other hand to guide the resulting ECL signal. The light emission is triggered and collected at one end, transmitted by the waveguide and remotely detected at the opposite end. Integration of both functionalities at the level of the same miniaturized object leads to an unprecedented bipolar opto-electrode, allowing one to quantify the ECL intensity as a function of different parameters in a double remote approach with interesting potential applications, ranging from high-throughput catalyst screening to massive parallel biochemical analysis.
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http://dx.doi.org/10.1039/c6an00652cDOI Listing
July 2016

Microscopic imaging and tuning of electrogenerated chemiluminescence with boron-doped diamond nanoelectrode arrays.

Anal Bioanal Chem 2016 Oct 2;408(25):7085-94. Epub 2016 Apr 2.

Department of Molecular Sciences and Nanosystems, University Ca' Foscari of Venice, Calle Larga S. Marta, 30123, Venice, Italy.

Nanoelectrode arrays (NEAs) are increasingly applied for a variety of electroanalytical applications; however, very few studies dealt with the use of NEAs as an electrochemical generator of electrogenerated chemiluminescence (ECL). In the present study, arrays of nanodisc and nanoband electrodes with different dimensions and inter-electrode distances were fabricated by e-beam lithography on a polycarbonate layer deposited on boron-doped diamond (BDD) substrates. In particular, NEAs with 16 different geometries were fabricated on the same BDD sample substrate obtaining a multiple nanoelectrode and ultramicroelectrode array platform (MNEAP). After electrochemical and morphological characterization, the MNEAP was used to capture simultaneously with a single image the characteristic behaviour of ECL emission from all the 16 arrays. Experiments were performed using Ru(bpy)3 (2+) as the ECL luminophore and tri-n-propylamine (TPrA) as the co-reactant. With a relatively limited number of experiments, such an imaging procedure allowed to study the role that geometrical and mechanistic parameters play on ECL generation at NEAs. In particular, at high concentrations of TPrA, well-separated individual ECL spots or bands revealed an ECL signal which forms a pattern matching the nanofabricated structure. The analysis of the imaging data indicated that the thickness of the ECL-emitting zone at each nanoelectrode scales inversely with the co-reactant concentration, while significantly stronger ECL signals were detected for NEAs operating under overlap conditions.
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http://dx.doi.org/10.1007/s00216-016-9504-1DOI Listing
October 2016

A Sensitive Electrochemiluminescence Immunosensor for Celiac Disease Diagnosis Based on Nanoelectrode Ensembles.

Anal Chem 2015 Dec 24;87(24):12080-7. Epub 2015 Nov 24.

Department of Molecular Sciences and Nanosystems, University Ca'Foscari of Venice , via Torino 155, 30172 Venezia Mestre, Italy.

We report here the design of a novel immunosensor and its application for celiac disease diagnosis, based on an electrogenerated chemiluminescence (ECL) readout, using membrane-templated gold nanoelectrode ensembles (NEEs) as a detection platform. An original sensing strategy is presented by segregating spatially the initial electrochemical reaction and the location of the immobilized biomolecules where ECL is finally emitted. The recognition scaffold is the following: tissue transglutaminase (tTG) is immobilized as a capturing agent on the polycarbonate (PC) surface of the track-etched templating membrane. It captures the target tissue transglutaminase antibody (anti-tTG), and finally allows the immobilization of a streptavidin-modified ruthenium-based ECL label via reaction with a suitable biotinylated secondary antibody. The application of an oxidizing potential in a tri-n-propylamine (TPrA) solution generates an intense and sharp ECL signal, suitable for analytical purposes. Voltammetric and ECL analyses evidenced that the ruthenium complex is not oxidized directly at the surface of the nanoelectrodes; instead ECL is generated following the TPrA oxidation, which produces the TPrA•+ and TPrA• radicals. With NEEs operating under total overlap diffusion conditions, high local fluxes of these reactive radicals are produced by the nanoelectrodes in the immediate vicinity of the ECL labels, so that they efficiently generate the ECL signal. The radicals can diffuse over short distances and react with the Ru(bpy)32+ label. In addition, the ECL emission is obtained by applying a potential of 0.88 V versus Ag/AgCl, which is about 0.3 V lower than when ECL is initiated by the electrochemical oxidation of Ru(bpy)3(2+). The immunosensor provides ECL signals which scale with anti-tTG concentration with a linearity range between 1.5 ng·mL–1 and 10 μg·mL–1 and a detection limit of 0.5 ng·mL–1. The sensor is finally applied to the analysis of anti-tTG in human serum samples, showing to be suitable to discriminate between healthy and celiac patients.
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http://dx.doi.org/10.1021/acs.analchem.5b02801DOI Listing
December 2015

Guiding pancreatic beta cells to target electrodes in a whole-cell biosensor for diabetes.

Lab Chip 2015 Oct;15(19):3880-90

CNRS UMR 5248, Chimie et Biologie des Membranes et Nano-objets, Allée Geoffroy Saint-Hilaire, Pessac, France.

We are developing a cell-based bioelectronic glucose sensor that exploits the multi-parametric sensing ability of pancreatic islet cells for the treatment of diabetes. These cells sense changes in the concentration of glucose and physiological hormones and immediately react by generating electrical signals. In our sensor, signals from multiple cells are recorded as field potentials by a micro-electrode array (MEA). Thus, cell response to various factors can be assessed rapidly and with high throughput. However, signal quality and consequently overall sensor performance rely critically on close cell-electrode proximity. Therefore, we present here a non-invasive method of further exploiting the electrical properties of these cells to guide them towards multiple micro-electrodes via electrophoresis. Parameters were optimized by measuring the cell's zeta potential and modeling the electric field distribution. Clonal and primary mouse or human β-cells migrated directly to target electrodes during the application of a 1 V potential between MEA electrodes for 3 minutes. The morphology, insulin secretion, and electrophysiological characteristics were not altered compared to controls. Thus, cell manipulation on standard MEAs was achieved without introducing any external components and while maintaining the performance of the biosensor. Since the analysis of the cells' electrical activity was performed in real time via on-chip recording and processing, this work demonstrates that our biosensor is operational from the first step of electrically guiding cells to the final step of automatic recognition. Our favorable results with pancreatic islets, which are highly sensitive and fragile cells, are encouraging for the extension of this technique to other cell types and microarray devices.
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http://dx.doi.org/10.1039/c5lc00616cDOI Listing
October 2015