Macromolécules et Microsystèmes en Biologie et en Médecine (MMBM)

Publications de l’équipe

Année de publication : 2012

Anaïs Ali-Cherif, Stefano Begolo, Stéphanie Descroix, Jean-Louis Viovy, Laurent Malaquin (2012 May 18)

Programmable magnetic tweezers and droplet microfluidic device for high-throughput nanoliter multi-step assays.

Angewandte Chemie (International ed. in English) : 10765-9 : DOI : 10.1002/anie.201203862 En savoir plus
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Mohamad Reza Mohamadi, Romain Verpillot, Myriam Taverna, Markus Otto, Jean-Louis Viovy (2012 May 16)

Microchip electrophoresis, with respect to « profiling of Aβ peptides in the cerebrospinal fluid of patients with Alzheimer’s disease ».

Methods in molecular biology (Clifton, N.J.) : 173-84 : DOI : 10.1007/978-1-61779-821-4_14 En savoir plus
Résumé

Aggregation of beta amyloid peptides especially Aβ1-42 in amyloid plaques is one of the major -neuropathological events in Alzheimer’s disease. This event is normally accompanied by a relative reduction of the concentration of Aβ1-42 in the cerebrospinal fluid (CSF) of patients developing the signs of Alzheimer’s disease. Here, we describe a microchip gel electrophoresis method in a polydimethylsiloxane (PDMS) chip that enables rapid profiling of major Aβ peptides. The method was applied to compare the relative concentration of Aβ1-42 with other Aβ peptides, for example, Aβ 1-40 in CSF. In order to increase the sensitivity of detection, Aβ peptides in the CSF samples were first captured and concentrated using magnetic beads coated with specific anti-Aβ antibodies.

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Sandrine Miserere, Guillaume Mottet, Velan Taniga, Stephanie Descroix, Jean-Louis Viovy, Laurent Malaquin (2012 Apr 11)

Fabrication of thermoplastics chips through lamination based techniques.

Lab on a chip : 1849-56 : DOI : 10.1039/c2lc21161k En savoir plus
Résumé

In this work, we propose a novel strategy for the fabrication of flexible thermoplastic microdevices entirely based on lamination processes. The same low-cost laminator apparatus can be used from master fabrication to microchannel sealing. This process is appropriate for rapid prototyping at laboratory scale, but it can also be easily upscaled to industrial manufacturing. For demonstration, we used here Cycloolefin Copolymer (COC), a thermoplastic polymer that is extensively used for microfluidic applications. COC is a thermoplastic polymer with good chemical resistance to common chemicals used in microfluidics such as acids, bases and most polar solvents. Its optical quality and mechanical resistance make this material suitable for a large range of applications in chemistry or biology. As an example, the electrokinetic separation of pollutants is proposed in the present study.

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Année de publication : 2011

Bruno Teste, Florent Malloggi, Jean-Michel Siaugue, Anne Varenne, Frederic Kanoufi, Stéphanie Descroix (2011 Oct 29)

Microchip integrating magnetic nanoparticles for allergy diagnosis.

Lab on a chip : 4207-13 : DOI : 10.1039/c1lc20809h En savoir plus
Résumé

We report on the development of a simple and easy to use microchip dedicated to allergy diagnosis. This microchip combines both the advantages of homogeneous immunoassays i.e. species diffusion and heterogeneous immunoassays i.e. easy separation and preconcentration steps. In vitro allergy diagnosis is based on specific Immunoglobulin E (IgE) quantitation, in that way we have developed and integrated magnetic core-shell nanoparticles (MCSNPs) as an IgE capture nanoplatform in a microdevice taking benefit from both their magnetic and colloidal properties. Integrating such immunosupport allows to perform the target analyte (IgE) capture in the colloidal phase thus increasing the analyte capture kinetics since both immunological partners are diffusing during the immune reaction. This colloidal approach improves 1000 times the analyte capture kinetics compared to conventional methods. Moreover, based on the MCSNPs’ magnetic properties and on the magnetic chamber we have previously developed the MCSNPs and therefore the target can be confined and preconcentrated within the microdevice prior to the detection step. The MCSNPs preconcentration factor achieved was about 35,000 and allows to reach high sensitivity thus avoiding catalytic amplification during the detection step. The developed microchip offers many advantages: the analytical procedure was fully integrated on-chip, analyses were performed in short assay time (20 min), the sample and reagents consumption was reduced to few microlitres (5 μL) while a low limit of detection can be achieved (about 1 ng mL(-1)).

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Jean-Michel Peyrin, Bérangère Deleglise, Laure Saias, Maéva Vignes, Paul Gougis, Sebastien Magnifico, Sandrine Betuing, Mathéa Pietri, Jocelyne Caboche, Peter Vanhoutte, Jean-Louis Viovy, Bernard Brugg (2011 Sep 15)

Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers.

Lab on a chip : 3663-73 : DOI : 10.1039/c1lc20014c En savoir plus
Résumé

Various experimental models are used to study brain development and degeneration. They range from whole animal models, which preserve anatomical structures but strongly limit investigations at the cellular level, to dissociated cell culture systems that allow detailed observation of cell phenotypes but lack the highly ordered physiological neuron connection architecture. We describe here a platform comprising independent cell culture chambers separated by an array of « axonal diodes ». This array involves asymmetric micro-channels, imposing unidirectional axon connectivity with 97% selectivity. It allows the construction of complex, oriented neuronal networks not feasible with earlier platforms. Different neuronal subtypes could be co-cultivated for weeks, and sequential seeding of different cell populations reproduced physiological network development. To illustrate possible applications, we created and characterized a cortico-striatal oriented network. Functional synaptic connections were established. The activation of striatal differentiation by cortical axons, and the synchronization of neural activity were demonstrated. Each neuronal population and subcompartment could be chemically addressed individually. The directionality of neural pathways being a key feature of the nervous system organization, the axon diode concept brings in a paradigmatic change in neuronal culture platforms, with potential applications for studying neuronal development, synaptic transmission and neurodegenerative disorder such as Alzheimer and Parkinson diseases at the sub-cellular, cellular and network levels.

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Naïs Coq, Antoine Bricard, Francois-Damien Delapierre, Laurent Malaquin, Olivia du Roure, Marc Fermigier, Denis Bartolo (2011 Jul 30)

Collective beating of artificial microcilia.

Physical review letters : 014501 En savoir plus
Résumé

We combine technical, experimental, and theoretical efforts to investigate the collective dynamics of artificial microcilia in a viscous fluid. We take advantage of soft lithography and colloidal self-assembly to devise microcarpets made of hundreds of slender magnetic rods. This novel experimental setup is used to investigate the dynamics of extended cilia arrays driven by a precessing magnetic field. Whereas the dynamics of an isolated cilium is a rigid body rotation, collective beating results in a symmetry breaking of the precession patterns. The trajectories of the cilia are anisotropic and experience a significant structural evolution as the actuation frequency increases. We present a minimal model to account for our experimental findings and demonstrate how the global geometry of the array imposes the shape of the trajectories via long-range hydrodynamic interactions.

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Paolo Pierobon, Giovanni Cappello (2011 Jul 7)

Quantum dots to tail single bio-molecules inside living cells.

Advanced drug delivery reviews : 167-78 : DOI : 10.1016/j.addr.2011.06.004 En savoir plus
Résumé

In the last two decades, the single particle and single molecule approach became more and more popular to investigate the activity and the mechano-chemical properties of biological molecules. The inherent limit of these assays was that the molecules of interest were observed in vitro, out of their natural environment, the cell. Several recent works have shown the possibility to overcome this limit, to extend this approach to living cells and to observe the details of many cellular processes at the molecular level. In this review we discuss the use of semiconductor quantum dots to perform single particle and single molecule tracking in the cell. We refer to other articles for the technical aspects of this method. Here, after an introduction on the advantages provided by these nanoparticles, we restrict ourselves to some examples, mainly related to intracellular transport and molecular motor activity. These will illustrate the important role played by semiconductor quantum dots as fluorescent nano-reporters in in cell single molecule approach in modern biology and biophysics.

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Pascaline Mary, Luce Dauphinot, Nadège Bois, Marie-Claude Potier, Vincent Studer, Patrick Tabeling (2011 Jul 1)

Analysis of gene expression at the single-cell level using microdroplet-based microfluidic technology.

Biomicrofluidics : 24109 : DOI : 10.1063/1.3596394 En savoir plus
Résumé

In the present work, we have measured the messenger RNA expression of specific genes both from total RNA and cells encapsulated in droplets. The microfluidic chip introduced includes the following functionalities: RNA∕cell encapsulation, lysis, reverse transcription and real-time polymerase chain reaction. We have shown that simplex and duplex gene expression measurements can be carried out over a population of 100 purified RNA samples encapsulated simultaneously in 2 nl droplets in less than 2 h. An analysis of 100 samples containing one to three cells has shown excellent consistency with standard techniques regarding average values. The cell-to-cell distributions of the E-cadherin expression suggest fluctuations on the order of 80% in the number of transcripts, which is highly consistent with the general findings from the literature. A mathematical model has also been introduced to strengthen the interpretation of our results. The present work paves the way for the systematic acquisition of such information in biological and biomedical studies.

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Pierre Recouvreux, Christophe Lavelle, Maria Barbi, Natalia Conde E Silva, Eric Le Cam, Jean-Marc Victor, Jean-Louis Viovy (2011 Jun 7)

Linker histones incorporation maintains chromatin fiber plasticity.

Biophysical journal : 2726-35 : DOI : 10.1016/j.bpj.2011.03.064 En savoir plus
Résumé

Genomic DNA in eukaryotic cells is organized in supercoiled chromatin fibers, which undergo dynamic changes during such DNA metabolic processes as transcription or replication. Indeed, DNA-translocating enzymes like polymerases produce physical constraints in vivo. We used single-molecule micromanipulation by magnetic tweezers to study the response of chromatin to mechanical constraints in the same range as those encountered in vivo. We had previously shown that under positive torsional constraints, nucleosomes can undergo a reversible chiral transition toward a state of positive topology. We demonstrate here that chromatin fibers comprising linker histones present a torsional plasticity similar to that of naked nucleosome arrays. Chromatosomes can undergo a reversible chiral transition toward a state of positive torsion (reverse chromatosome) without loss of linker histones.

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Marie Girardot, Hong-Yi Li, Stéphanie Descroix, Anne Varenne (2011 May 28)

Determination of binding parameters between lysozyme and its aptamer by frontal analysis continuous microchip electrophoresis (FACMCE).

Journal of chromatography. A : 4052-8 : DOI : 10.1016/j.chroma.2011.04.077 En savoir plus
Résumé

An original and simple methodology based on microchip electrophoresis (MCE) in a continuous frontal analysis mode (named frontal analysis continuous microchip electrophoresis, FACMCE) was developed for the simultaneous determination of the binding parameters, i.e. ligand-site dissociation constant (k(d)) and number of binding sites on the substrate (n). This simultaneous determination was exemplified with the interaction between an aptamer and its target. The selected target is a strongly basic protein, lysozyme, as its quantification is of great interest due to its antimicrobial and allergenic properties. A glass microdevice equipped with a fluorescence detection system was coated with hydroxypropylcellulose, reducing the electroosmotic flow and adsorption onto the channel walls. This microdevice allowed the continuous electrokinetic injection of a mixture of fluorescently labelled aptamer and non-labelled lysozyme. By determining the concentration of the free fluorescently labelled aptamer thanks to its corresponding plateau height, mathematical linearization methods allowed to determine a k(d) value of 48.4±8.0 nM, consistent with reported results (31 nM), while the average number of binding sites n on lysozyme, never determined before, was 0.16±0.03. These results seem to indicate that the buffer nature and the SELEX process should influence the number and affinity of the binding sites. In parallel it has been shown that the binding between lysozyme and its aptamer presents two sites of different binding affinities.

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Bruno Teste, Frédéric Kanoufi, Stéphanie Descroix, Pascal Poncet, Thomas Georgelin, Jean-Michel Siaugue, Jan Petr, Anne Varenne, Marie-Claire Hennion (2011 May 7)

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis.

Analytical and bioanalytical chemistry : 3395-407 : DOI : 10.1007/s00216-011-5021-4 En savoir plus
Résumé

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles’ colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core-shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient’s sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.

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Judith Miné-Hattab, Geneviève Fleury, Chantal Prevost, Marie Dutreix, Jean-Louis Viovy (2011 Apr 13)

Optimizing the design of oligonucleotides for homology directed gene targeting.

PloS one : e14795 : DOI : 10.1371/journal.pone.0014795 En savoir plus
Résumé

Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit.

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Fabien Montel, Morgan Delarue, Jens Elgeti, Laurent Malaquin, Markus Basan, Thomas Risler, Bernard Cabane, Danijela Vignjevic, Jacques Prost, Giovanni Cappello, Jean-François Joanny (2011 Mar 3)

Stress clamp experiments on multicellular tumor spheroids.

Physical review letters : 188102 En savoir plus
Résumé

The precise role of the microenvironment on tumor growth is poorly understood. Whereas the tumor is in constant competition with the surrounding tissue, little is known about the mechanics of this interaction. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spheroid cell aggregate. We observe that a stress of 10 kPa is sufficient to drastically reduce growth by inhibition of cell proliferation mainly in the core of the spheroid. We compare the results to a simple numerical model developed to describe the role of mechanics in cancer progression.

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Ayat Abbood, Christine Herrenknecht, Gaëlle Proczek, Stéphanie Descroix, Jordi Rodrigo, Myriam Taverna, Claire Smadja (2011 Feb 22)

Hexylacrylate-based mixed-mode monolith, a stationary phase for the nano-HPLC separation of structurally related enkephalins.

Analytical and bioanalytical chemistry : 459-68 : DOI : 10.1007/s00216-011-4762-4 En savoir plus
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The potential of an in situ photopolymerized hexylacrylate-based monolithic stationary phase-bearing sulfonic acid groups was investigated by studying the chromatographic retention of small structurally related peptides (enkephalins) by nano-LC. Several retention mechanisms were highlighted. First, a reverse-phase chromatographic behavior toward neutral solutes due to hexylacrylate-moieties was demonstrated. Second, an evaluation of the influences of buffer pH suggested the involvement of a cation-exchange mechanism due to the presence of 2-acrylamido-2-methyl-1-propanesulfonic acid. This cation-exchange phenomenon was confirmed by the clear influence of Na(+) concentration in the mobile phase on peptide retention.

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Jean-Louis Viovy (2011 Feb 11)

10th anniversary issue: France.

Lab on a chip : 775-6 : DOI : 10.1039/c1lc90006d En savoir plus
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