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International audience; A regenerable bulk acoustic wave (BAW) biosensor is developed for the rapid, label-free and selective detection of

International audience; In this work, we present a pre-analytical module based on the combination of microfluidic and electroacoustic technologies to intercept and sort submicron biological particles. It is composed by assembling a lithium niobate (LN) substrate for acoustic function and a micromachined glass substrate for microfluidic channel. The interference between the two surface acoustic waves (SAWs) generated by inter digital transducers (IDTs) create a distribution of an acoustic radiation force (ARF). This force affects differently particles depending on their physical proprieties. The device is powered by an electronic circuit with a phase shifter to move the node of the standing surface acoustic wave (SSAW) along the channel width. When the device is powered at resonance frequency, experience has shown a submicron particles alignement at a fixed position along the channel. By shifting the SSAW phase, the particles are driven to one of the three channel outlets.

International audience; Introduction: The NanoBioAnalytical (NBA) platform is an established, calibrated and label-free system to characterize Extracellular Vesicles (EVs), without limitation in size, in different biological samples [1, 2]. NBA benefits were recently highlighted in latest MISEV guidelines [3]. The NBA platform combines biodetection and phenotyping of EVs subsets by immunocapture monitored by Surface Plasmon Resonance (SPR) on biochip, followed by EVs quantitation and sizing thanks to metrological evaluation by Atomic Force Microscopy (AFM). Our aim is to push the limit of the NBA to address clinical studies involving EVs.Methods: We emphasise here the performance of the NBA platform for establishing its dynamic range and limit of detection (LOD) for blood derived EVs. Concentration of EVs was first determined in solution by Tunable Resistive Pulse Sensing; NBA sensitivity and reliability was then studied by SPR on biochips presenting a-CD41 antibody arrays. Finally, even on 1000-fold diluted samples, reliable and complementary information to SPR measurements on size distribution, counting and shape deciphering could be obtained by AFM.Results: Optimizing different factors (flow rate, density of receptors on the surface, etc.) enabled detection of blood derived EVs at dynamic range from 106 to 109 particles /mL on a-CD41 surface. The determination of the LOD of EVs and their subsets size distribution at different capture levels are currently in progress.Summary/Conclusion: The NBA platform is modular and capable of detecting EVs reliably even in highly diluted samples. Such characterization and correlation studies are crucial for accurate and comprehensive characterization of EVs in biological samples with good reproducibility.

International audience; Manipulating micro and nano-biological particles like extracellular vehicles (EVs), without extracting them from their biological media, presents a big challenge for diagnosis purposes. Here we present the design and fabrication of a sorting device based on the combination of microfluidic and electroacoustic modules that is capable of aligning and sorting submicron biological particles according to their size, compressibility or mass density, all in a tunable way. The device relies on a lithium niobate (LN) substrate to generate acoustic waves assembled with a micromachined glass layer for microfluidic circuits. The interference between the two surface acoustic waves (SAWs) generated by interdigitated transducers (IDTs) create a distribution of an acoustic radiation force (ARF). This force affects differently particles depending on their physical proprieties. The device is powered by an electronic circuit with a phase shifter to move the node of the standing surface acoustic wave (SSAW) along the channel width. When the device is powered at resonance frequency of the IDTs, experiment shows submicron particles alignment along the channel. By shifting the electrical signal between the two IDTs we can translate the pressure node at any targeted position in the channel width. The particles are then driven to one selected outlet.

International audience; Extracellular vesicles (EVs) are vesicles shed by almost all cells with nanometric size and heterogeneous characteristics (biogenesis, shape…). EVs allow cell-to-cell communication and have a key role in lot of (patho)physiological reactions1. Since EVs cover a large size range (from 30 to 1000 nm) and originate from every cells, their characterization in their entirety represents a real challenge. Indeed, there is a lack of method enabling their isolation and analysis, in complex media, that impairs EVs to be qualified and used as biomarkers. Succeeding in EVs detection from their physiological environment and analyzing them in different subsets represent ambitious objective of many research groups. To reach this goal, one solution is to combine several technics and/or improve technical and preanalytical steps. We propose here to develop a microfluidic device, based on hydrodynamic filtration, also called deterministic lateral displacement : the aim of this system is to separate EVs to desired size if EVs are between sidewall and the cutoff radius (Rc), which are the maximum radius value allowing particle to pass in the lateral channel. A fluid flow, containing particles, is introduced into the main channel. In the cross section, only small portion of fluid pass in lateral channel: particles with radius smaller than Rc. This system will be coupled to a home-made gold biochip presenting microarrays enabling specific EVs subsets capture. Then, this lab-on-chip would make possible the recovery and analysis of EVs subsets at once.

International audience; Workshop « À la conquête des nanovésicules biologiques »

During the skin ageing which is accelerated by environmental factors, the generated reactive oxygen species make unstable the "proliferation/differentiation/apoptosis" coupling of epidermal cells. A biochemical study, previously realized on human keratinocyte cultures treated by a pesticide, glyphosate, allowed the validation of an in vitro model of skin ageing. In this work, we propose to study the loss of HaCaT cell integrity appearing after this chemical induced-oxidative stress. An original approach, combining a micro- to nanoscale cell characterization and analysis of fluctuating protein expression, is proposed: a) the exploration of cell growth and morphology through fluorescence and AFM imaging, b) a functional investigation using force spectroscopy for detection of oxidative stress biomarkers in membranes and c) the establishment of proteomic comparative profiles between safe and glyphosate-treated cells. Our results revealed up to now that a glyphosate treatment induced cell impairment in an acute dose-dependency. Indeed, we observed a breakage of plasma membrane and a release of cytosol, microtubules disorganization and fragmentation, and finally aggregation of chromatin. These observations characterized apoptotic key events. Our first force spectroscopy results revealed that a glyphosate coated tip interacted specifically with keratinocyte membrane structures. This nanobiotechnological tool will allow cell mapping establishment and screening original anti-oxidant molecules. The proteomic profiling would highlight cell oxidative stress cascade and identify corresponding major biomarkers in our skin ageing model. Original molecules with cytoprotective potentials will be tested and compared to well-known antioxidants, in order to correct pesticide induced cutaneous disorders. These data all together will contribute to better encircle the mechanisms of chemical and environmental hazards acting on the human skin.

We present herein an original approach, consisting to lead in parallel a global analysis (SPR) and nanoscale characterizations (AFM, mass spectrometry, TOF-SIMS) of bio-molecules on the biochip surface. The questions we want to answer concern especially the organization, orientation and identification of immobilized and captured biomolecules. In order to characterize the sensing layer in terms of molecular structure and orientation, we engaged a SPR-TOF SIMS first set of experiments (collaboration: Pr Aoyagi, Japan). Our results demonstrated that analysis of orientation of the immobilized cytochrome b5 is possible (ref2&3) and help to optimize and develop our sensing layer. We also recently developed an innovative concept to identify directly « on the chip » (without any elution) the biomolecules retained by mass spectrometry. We first validated the concept on a simple model (monolayer of albumin) with sensitivity for MS identification less than 1 fmol/mm2 (ref1). We further continued by developing immunosensors to catch specifically, in complex biological fluids like sera, low concentrated biomarkers (lag3 protein). Controlling the surface coverage and repartition of molecules by AFM, mass spectrometry “on the chip” allowed to identify without ambiguity both antibody and biomarker. Our objective is now to increase the throughput of analysis. For that, we are currently working on micro-arrays. Our expertise in molecule grafting and nanocharacterization will make our clinical proteomic research relevant, thanks to deeply controlled steps of biosensor development and use. References (1) Lucchi G., Rouleau A., Boireau W., Ducoroy P. SMAP 2007 (Congress on Mass Spectrometry and Proteomics Analysis), Pau (France). (2) S. Aoyagi, W. Boireau, C. Elie-Caille, A. Rouleau, M. Dohi, N. Kato, M. Kudo. 45th Annual Meeting of the Biophysical Society of Japan (Dec 2007), Pacifico Yokohama. (3) S. Aoyagi, A. Rouleau, W. Boireau. Applied Surface Science, (in press).

International audience; Single-strand annealing proteins, such as Redbeta from lambda phage or eukaryotic Rad52, play roles in homologous recombination. Here, we use atomic force microscopy to examine Redbeta quaternary structure and Redbeta-DNA complexes. In the absence of DNA, Redbeta forms a shallow right-handed helix. The presence of single-stranded DNA (ssDNA) disrupts this structure. Upon addition of a second complementary ssDNA, annealing generates a left-handed helix that incorporates 14 Redbeta monomers per helical turn, with each Redbeta monomer annealing approximately 11 bp of DNA. The smallest stable annealing intermediate requires 20 bp DNA and two Redbeta monomers. Hence, we propose that Redbeta promotes base pairing by first increasing the number of transient interactions between ssDNAs. Then, annealing is promoted by the binding of a second Redbeta monomer, which nucleates the formation of a stable annealing intermediate. Using threading, we identify sequence similarities between the RecT/Redbeta and the Rad52 families, which strengthens previous suggestions, based on similarities of their quaternary structures, that they share a common mode of action. Hence, our findings have implications for a common mechanism of DNA annealing mediated by single-strand annealing proteins including Rad52.

National audience; De la nanotechnologie ... Les nanotechnologies recouvrent l'ensemble des techniques, outils et procédés qui permettent de manipuler la matière à une échelle en dessous de la centaine de nanomètres (1 nanomètre = 1 milliardième de mètre), d'élaborer de nouveaux matériaux et composants toujours plus petits, de construire atome par atome de nouvelles molécules et d'exploiter leurs propriétés en vue de nouvelles applications. Les nanotechnologies devraient constituer dans les prochaines années un marché considérable. À l'horizon 2015, 15% de l'activité manufacturière mondiale serait concernée par des dispositifs ou des matériaux utilisant des avancées issues des nanotechnologies. En 2008, le montant du marché mondial est estimé à 500 milliards de dollars et pourrait doubler en 2015, selon la National science foundation. Toutefois, les nanotechnologies suscitent également des inquiétudes. Manipuler la matière à l'échelle moléculaire et interférer avec le monde du vivant soulève des questions éthiques et sanitaires qui doivent être prises en considération par les pouvoirs publics et les acteurs concernés. La nanotechnologie peut être considérée comme une évolution logique de la microélectronique dont l'objectif est la fabrication de circuits intégrés, familièrement appelés « puces électroniques » dont la référence en matériau de base est le silicium. A côté de la nanoélectronique industrielle a émergé la nanotechnologie moléculaire. Le but de cette dernière discipline est de pouvoir assurer un contrôle parfait sur la structure de la matière et de pouvoir construire des objets complexes avec une précision à l'échelle de l'atome ou de la molécule. ... aux nanoBIOtechnologies... Un important challenge dans les dispositifs de BIOcapteurs et de diagnostic est de savoir comment reconstituer/étudier des mécanismes biologiques pertinents à la surface d'une biopuce et quels outils analytiques sont convenables pour fournir rapidement des informations justes sur la structure des molécules attachées à la surface. Un meilleur contrôle dans la réalisation de biopuces peut être obtenu en combinant différentes approches interdisciplinaires, avec pour préoccupation de répondre à une question clé biologique. Les recherches menées au sein de la plateforme CLIPP (Clinical & Innovation Proteomic Platform), rassemblant des compétences multidisciplinaires, sont focalisées sur cet objectif. L'ensemble « conception, réalisation et caractérisation » de puces à protéines représente un des axes de recherche de la plateforme CLIPP. Nous développons différentes stratégies d'ingénierie/structuration de matériaux, de fonctionnalisation chimique et d'immobilisation de biomolécules qui conduit à l'optimisation d'évènements de reconnaissance et leur caractérisation à la surface de la puce. Dernièrement, nous avons développé une méthode simple pour la préparation de substrat d'or présentant des terrasses atomiquement planes, et permettant une caractérisation biomoléculaire à l'échelle nanométrique1. Le mode d'action du taxol sur les microtubules et les conséquences sur leur conformation a pu également être élucidé par observation par microscopie à force atomique (AFM) en conditions physiologiques2. Nous avons également développé des approches originales, consistant à combiner une analyse « globale » par Surface Plasmon Resonance (SPR) et un moyen de caractérisation à l'échelle nanométrique (AFM, Spectroscopie de Photo-électrons X, spectrométrie de masse (MS)). Nous avons en particulier démontré que l'orientation d'une protéine immobilisée en monocouche sur une surface, peut être déterminée par la détection de fragments secondaires spécifiques de la protéine par TOF-SIMS3. Ces développements ont contribué à l'établissement d'une nouvelle combinaison « Analyse des interactions Biomoléculaires / spectrométrie de masse (BIA-MS) basée sur une procédure totalement « on the chip »4. Nous proposons une approche bas coût combinant l'analyse sur un BIAcore 2000 sur puces synthétisées par nos soins (réalisation du substrat et greffage d'anticorps) avec l'identification par MS, directement sur la puce et sans étape d'élution. Utilisant cette technique, l'identification de complexes protéiques a pu être obtenue, et permet ainsi de compléter l'approche BIA-MS dans la découverte et l'analyse de complexes protéiques. Dernièrement, ces développements ont été transposés à de l'analyse multiplexée sur micro-arrays. Les premiers résultats de détection de biomarqueurs dans du plasma s'avèrent être très prometteurs pour une approche diagnostic moyen/haut débit.

During the skin ageing, the produced radical species make unstable the "proliferation / differentiation / apoptosis" coupling of epidermal cells; we propose to study this loss of cell integrity, by a double original and complementary approach: - the establishment of proteomic comparative profiles, by spectrofluorimetric, 2D chromatographic/mass spectrometry techniques, - the exploration of cell growth and morphology, by a spectroscopic and topographic study by atomic force microscopy. This work will allow identifying the better biomarker(s) of skin ageing modeled in vitro by a chosen chemical treatment using glyphosate. Original molecules with cell protection effect, able to correct the induced morphological and/or functional disorders could also been identified. Our first results revealed cell morphology of a line of keratinocytes grown on different substrates. A glyphosate dose-effect was noted through an important cell destructuration. Preliminary force spectroscopy measurements on cells revealed also specific interactions with glyphosate. These experimental results based on AFM investigation should highlight the first steps of the oxidative stress mechanism, by visualization and quantification of the phenomenon after its induction at the individual cell scale.

International audience; Redβ anneals DNA to initiate homologous recombination ined recent prominence through the development of the DNA engineering technology known as ‘recombineering' or ‘Red/ET'. It originates from the red-operon of λ phage where it is co-expressed in the early life cycle stage with Redα a processive 5'-3' exonuclease and Redγ, a DNA mimetic and RecBCD inhibitor. Unlike RecA/RAD51, Redβ is not an ATPase and it's mechanism for initiating homologous recombination is poorly understood. To examine the structure and dynamics of Redβ complexes at sub-molecular resolution we performed tapping mode atomic force microscopy (AFM) of Redβ protein alone and in complex with DNA. Without DNA, Redβ forms a ‘split lock washer' structure with a shallow right-handed helicity. Sequentially adding complementary ssDNA generates a stable left-handed helical filament. Importantly, the contour length of the helical filament equated linearly to the lengths of complementary ssDNA, giving the number of nucleotides per Redβ monomer. Additionally, the monomer width along the filament was quantified. These new quantities as well as the observed helical transition reveals new insights into the mechanism of DNA annealing mediated by Red and led us to suggest new mechanistic models.

International audience; A great challenge in biosensors and diagnosis devices relies on the way to reconstitute relevant biological mechanisms on surface of the biochips and which analytical tools are convenient to provide accurate and rapid information on the structures and function of molecules attached to this surface. A better control in the realization of biochips can be obtained in combining different complementary approaches while always keeping in mind the biological key point. Researches in CLIPP are focused towards this objective. Conception, realization and characterization of protein and cell chips are presented. We detail different strategies of materials engineering1,2,3, chemical functionalizations and biomolecular graftings4, molecular and cellular characterization in physiological conditions5,6, which lead to the optimization of “biorecognitions events” at the surface of the chip. We present herein an original interdisciplinary approach, consisting to carry out in parallel a micro-scale analysis (SPR, fluorescence microscopy) and nano-scale characterizations (AFM, XPS, TOF-SIMS). Concerning protein interfaces, we demonstrated in particular that the molecular orientation in a protein monolayer can be determined based on the specific fragment ions from the protein in TOF-SIMS spectra7. These developments have also contributed to the establishment of a new biomolecular interaction analysis/mass spectrometry (BIA-MS) combination based on an entire “on-a-chip” procedure8. We report a low-cost approach combining Biacore 2000 analysis with homemade chips and MS and MS/MS identification directly onto the chips without elution step. Using this technique, identification of protein complexes were routinely obtained giving the opportunity to the “on-a-chip” processing to complete the BIA-MS approach in the discovery and analysis of protein complexes in biological fluids. Our interest is also focused on cell/surface interaction. The cell biochips we are developing consist either of circulating or adherent cells, that we characterized in terms of cell capture on biofunctionalized surface or growth with substrate dependency respectively. Parameters such as cell spreading, growth, morphology, and topography are particularly investigated and controlled by atomic force microscopy in physiological conditions6. With the aim to increase the throughput of analysis, we are currently working on cell and protein micro-arrays. Our expertise in cell and protein biochip preparation, and competences in micro- to nanoscale characterization in liquid conditions, represents precious assets enabling a relevant clinical proteomic research, thanks to deeply controlled steps of biosensor development and use.

The approach lies on the development of an original DNA biosensor. This self-assembled DNA biochip is constituted of a tethered lipid membrane presenting « protein/DNA » complexes. This unique assembly, using a highly flexible bifunctional molecular spacer and a fluidic matrix, allows the control of oligonucleotides surface density and their mobility thus favouring hybridization of the DNA probes in liquid environment and under weak constraints. Recent Surface Plasmon Resonance experiments lead to building of protein/DNA dimers presenting two special DNA sequences recognized by regulation proteins. We demonstrated the capacity and relevance of this biosensor in the case of an estrogen receptor/DNA specific recognition, opening the possibility of specific drugs screening. We present here preliminary characterizations of these thin biomolecular films through Atomic Force Microscopy in liquid. Homogeneity of the lipid film was controlled, the surface coverage in protein was evaluated and specificity of binding is under examination at the present time. Indeed, the visualization of the self-assembled « edifice » at a molecular scale and in a bio-mimetic environment is a precious and complementary way to improve the understanding of the protein/DNA interaction mechanisms.