Macromolécules et Microsystèmes en Biologie et en Médecine (MMBM)

Publications de l’équipe

Année de publication : 2011

Bruno Teste, Florent Malloggi, Anne-Laure Gassner, Thomas Georgelin, Jean-Michel Siaugue, Anne Varenne, Hubert Girault, Stéphanie Descroix (2011 Jan 22)

Magnetic core shell nanoparticles trapping in a microdevice generating high magnetic gradient.

Lab on a chip : 833-40 : DOI : 10.1039/c0lc00510j En savoir plus
Résumé

Magnetic core shell nanoparticles (MCSNPs) 30 nm diameter with a magnetic weight of 10% are usually much too small to be trapped in microfluidic systems using classical external magnets. Here, a simple microchip for efficient MCSNPs trapping and release is presented. It comprises a bed of micrometric iron beads (6-8 μm diameter) packed in a microchannel against a physical restriction and presenting a low dead volume of 0.8 nL. These beads of high magnetic permeability are used to focus magnetic field lines from an external permanent magnet and generate local high magnetic gradients. The nanoparticles magnetic trap has been characterised both by numerical simulations and fluorescent MCSNPs imaging. Numerical simulations have been performed to map both the magnetic flux density and the magnetic force, and showed that MCSNPs are preferentially trapped at the iron bead magnetic poles where the magnetic force is increased by 3 orders of magnitude. The trapping efficiency was experimentally determined using fluorescent MCSNPs for different flow rates, different iron beads and permanent magnet positions. At a flow rate of 100 μL h(-1), the nanoparticles trapping/release can be achieved within 20 s with a preconcentration factor of 4000.

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Laure Saias, Julien Autebert, Laurent Malaquin, Jean-Louis Viovy (2011 Jan 18)

Design, modeling and characterization of microfluidic architectures for high flow rate, small footprint microfluidic systems.

Lab on a chip : 822-32 : DOI : 10.1039/c0lc00304b En savoir plus
Résumé

We propose a strategy for optimizing distribution of flow in a microfluidic chamber for microreactor, lateral flow assay and immunocapture applications. It is aimed at maximizing flow throughput, while keeping footprint, cell thickness, and shear stress in the distribution channels at a minimum, and offering a uniform flow field along the whole analysis chamber. In order to minimize footprint, the traditional tree-like or « rhombus » design, in which distribution microchannels undergo a series of splittings into two subchannels with equal lengths and widths, was replaced by a design in which subchannel lengths are unequal, and widths are analytically adapted within the Hele-Shaw approximation, in order to keep the flow resistance uniform along all flow paths. The design was validated by hydrodynamic flow simulation using COMSOL finite element software. Simulations show that, if the channel is too narrow, the Hele-Shaw approximation loses accuracy, and the flow velocity in the chamber can fluctuate by up to 20%. We thus used COMSOL simulation to fine-tune the channel parameters, and obtained a fluctuation of flow velocity across the whole chamber below 10%. The design was then implemented into a PDMS device, and flow profiles were measured experimentally using particle tracking. Finally, we show that this system can be applied to cell sorting in self-assembling magnetic arrays, increasing flow throughput by a factor 100 as compared to earlier reported designs.

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Paolo Pierobon, Judith Miné-Hattab, Giovanni Cappello, Jean-Louis Viovy, Marco Cosentino Lagomarsino (2011 Jan 15)

Separation of time scales in one-dimensional directed nucleation-growth processes.

Physical review. E, Statistical, nonlinear, and soft matter physics : 061904 En savoir plus
Résumé

Proteins involved in homologous recombination such as RecA and hRad51 polymerize on single- and double-stranded DNA according to a nucleation-growth kinetics, which can be monitored by single-molecule in vitro assays. The basic models currently used to extract biochemical rates rely on ensemble averages and are typically based on an underlying process of bidirectional polymerization, in contrast with the often observed anisotropic polymerization of similar proteins. For these reasons, if one considers single-molecule experiments, the available models are useful to understand observations only in some regimes. In particular, recent experiments have highlighted a steplike polymerization kinetics. The classical model of one-dimensional nucleation growth, the Kolmogorov-Avrami-Mehl-Johnson (KAMJ) model, predicts the correct polymerization kinetics only in some regimes and fails to predict the steplike behavior. This work illustrates by simulations and analytical arguments the limitation of applicability of the KAMJ description and proposes a minimal model for the statistics of the steps based on the so-called stick-breaking stochastic process. We argue that this insight might be useful to extract information on the time and length scales involved in the polymerization kinetics.

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Année de publication : 2010

Virginie Caux-Moncoutier, Laurent Castéra, Carole Tirapo, Dorothée Michaux, Marie-Alice Rémon, Anthony Laugé, Etienne Rouleau, Antoine De Pauw, Bruno Buecher, Marion Gauthier-Villars, Jean-Louis Viovy, Dominique Stoppa-Lyonnet, Claude Houdayer (2010 Dec 2)

EMMA, a cost- and time-effective diagnostic method for simultaneous detection of point mutations and large-scale genomic rearrangements: application to BRCA1 and BRCA2 in 1,525 patients.

Human mutation : 325-34 : DOI : 10.1002/humu.21414 En savoir plus
Résumé

The detection of unknown mutations remains a serious challenge and, despite the expected benefits for the patient’s health, a large number of genes are not screened on a routine basis. We present the diagnostic application of EMMA (Enhanced Mismatch Mutation Analysis(®) , Fluigent, Paris, France), a novel method based on heteroduplex analysis by capillary electrophoresis using innovative matrices. BRCA1 and BRCA2 were screened for point mutations and large rearrangements in 1,525 unrelated patients (372 for the validation step and 1,153 in routine diagnosis) using a single analytical condition. Seven working days were needed for complete BRCA1/2 screening in 30 patients by one technician (excluding DNA extraction and sequencing). A total of 137 mutations were found, including a BRCA2 duplication of exons 19 and 20, previously missed by Comprehensive BRACAnalysis(®) . The mutation detection rate was 11.9%, which is consistent with patient inclusions. This study therefore suggests that EMMA represents a valuable short-term and midterm option for many diagnostic laboratories looking for an easy, reliable, and affordable strategy, enabling fast and sensitive analysis for a large number of genes.

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Stefano Begolo, Guillaume Colas, Jean-Louis Viovy, Laurent Malaquin (2010 Nov 30)

New family of fluorinated polymer chips for droplet and organic solvent microfluidics.

Lab on a chip : 508-12 : DOI : 10.1039/c0lc00356e En savoir plus
Résumé

We present a new family of microfluidic chips hot embossed from a commercial fluorinated thermoplastic polymer (Dyneon THV). This material shares most of the properties of fluoro polymers (very low surface energy and resistance to chemicals), but is easier to process due to its relatively low melting point. Finally, as an elastic material it also allows easy world to chip connections. Fluoropolymer films can be imprinted by hot embossing from PDMS molds prepared by soft lithography. Chips are then sealed by an original technique (termed Monolithic-Adhesive-Bonding), using two different grades of fluoropolymer to obtain uniform mechanical, chemical and surface properties. This fabrication process is well adapted to rapid prototyping, but it also has potential for low cost industrial production, since it does not require any curing or etching step. We prepared microfluidic devices with micrometre resolution features, that are optically transparent, and that provide good resistance to pressure (up to 50 kPa). We demonstrated the transport of water droplets in fluorinated oil, and fluorescence detection of DNA within the droplets. No measurable interaction of the droplets with the channels wall was observed, alleviating the need for surface treatment previously necessary for droplet applications in microfluidic chips. These chips can also handle harsh organic solvents. For instance, we demonstrated the formation of chloroform droplets in fluorinated oil, expanding the potential for on chip microchemistry.

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N Ktari, P Poncet, H Sénéchal, L Malaquin, F Kanoufi, C Combellas (2010 Oct 16)

Patterning of polystyrene by scanning electrochemical microscopy. Biological applications to cell adhesion.

Langmuir : the ACS journal of surfaces and colloids : 17348-56 : DOI : 10.1021/la1028564 En savoir plus
Résumé

Polystyrene surfaces may be patterned by Ag(II), NO(3)(•), and OH(•) electrogenerated at the tip of a scanning electrochemical microscope. These electrogenerated reagents lead to local surface oxidation of the polymer. The most efficient surface treatment is obtained with Ag(II). The patterns are evidenced by XPS and IR and also by the surface wettability contrast between the hydrophobic virgin surface and the hydrophilic pattern. Such Ag(II) treatment of a polystyrene Petri dish generates discriminative surfaces able to promote or disfavor the adhesion of proteins and also the adhesion and growth of adherent cells. The process is also successfully applied to a cyclo-olefin copolymer and should be suitable to pattern any hydrogenated polymer.

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Mohamad Reza Mohamadi, Zuzana Svobodova, Romain Verpillot, Hermann Esselmann, Jens Wiltfang, Markus Otto, Myriam Taverna, Zuzana Bilkova, Jean-Louis Viovy (2010 Aug 21)

Microchip electrophoresis profiling of Aβ peptides in the cerebrospinal fluid of patients with Alzheimer’s disease.

Analytical chemistry : 7611-7 : DOI : 10.1021/ac101337n En savoir plus
Résumé

The preferential aggregation of Aβ1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer’s disease. This is 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 polydimethylsiloxane (PDMS) chip that enables rapid profiling of major Aβ peptides in cerebrospinal fluid. To control the electroosmotic flow (EOF) in the PDMS channel and also to reduce the adsorption of the peptides to the surface of the channel, a new double coating using poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE) and methylcellulose-Tween-20 was developed. With this method, separation of five synthetic Aβ peptides (Aβ1-37, Aβ1-38, Aβ1-39, Aβ1-40, and Aβ1-42) was achieved, and relative abundance of Aβ1-42 to Aβ1-37 could be calculated in different standard mixtures. We applied our method for profiling of Aβ peptides in CSF samples from nonAlzheimer patients and patients with Alzheimer’s disease. Aβ peptides in the CSF samples were captured and concentrated using a microfluidic system in which magnetic beads coated with anti-Aβ were self-organized into an affinity microcolumn under the a permanent magnetic field. Finally, we could detect two Aβ peptides (Aβ1-40 and Aβ1-42) in the CSF samples.

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Claude Houdayer, Virginie Moncoutier, Jérôme Champ, Jérémie Weber, Jean-Louis Viovy, Dominique Stoppa-Lyonnet (2010 Aug 20)

Enhanced mismatch mutation analysis: simultaneous detection of point mutations and large scale rearrangements by capillary electrophoresis, application to BRCA1 and BRCA2.

Methods in molecular biology (Clifton, N.J.) : 147-80 : DOI : 10.1007/978-1-60761-759-4_9 En savoir plus
Résumé

We present the routine diagnostic application of EMMA (Enhanced Mismatch Mutation Analysis, Fluigent), a new, fast, reliable, and cost-effective method for mutation screening. This method is based on heteroduplex analysis by capillary electrophoresis and relies on the use of innovative matrices increasing the electrophoretic mobility differences between homoduplex and heteroduplex DNA, which is further enhanced by the addition of nucleosides in the separation matrix. Nucleosides interact with heteroduplex mismatched bases, hence increasing mobility difference with homoduplex. As separations are performed by multi-capillary electrophoresis, it allows for high automation, low cost, and high throughput. Moreover, EMMA, in combination with limiting PCR conditions, can be used to achieve the simultaneous detection of point mutation and large scale rearrangement in a single run.We now report on the routine diagnostic use of this method for BRCA1 and BRCA2 screening. The coding sequence and exon-intron junctions of BRCA1 and BRCA2 were amplified in 24 multiplex PCRs using a single condition. PCRs were electrophoresed with a single analytical condition on an ABI3100, and data were analyzed using dedicated software (Emmalys).The strength of this new method relies on the following assets: (1) a single condition of analysis: modeling related to melting domain is not required (2) simultaneous detection of point mutations and large scale rearrangements, (3) optimized and ready-to-use polymer that can be used on various ABI sequencers, (4) easy to use, (5) low reagent costs, and (6) throughput.

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Elisabetta Fanizza, Laurent Malaquin, Tobias Kraus, Heiko Wolf, Marinella Striccoli, Norberto Micali, Antonietta Taurino, Angela Agostiano, M Lucia Curri (2010 Aug 7)

Precision patterning with luminescent nanocrystal-functionalized beads.

Langmuir : the ACS journal of surfaces and colloids : 14294-300 : DOI : 10.1021/la1023339 En savoir plus
Résumé

A reliable strategy is presented to combine the preparation of functional building blocks based on polymer beads decorated with luminescent nanocrystals (NCs) and their precise positioning onto suitable patterns by capillary assembly technique. In particular, a layer-by-layer (LbL) polyelectrolyte (PE) deposition procedure has been implemented to provide uniform NC coverage on PS beads, thus conveying the optical properties of luminescent nanocrystals to highly processable PS beads. The latter have then been integrated into patterned stamps by means of template-driven capillary assembly. Their selective positioning has been directed by means of pattern geometry. The use of luminescent (CdSe)ZnS NCs offers a direct optical probe to evaluate the efficiency of the positioning procedure on the substrate, enabling the extension of the method to a wide range of materials, i.e., NCs with different compositions and specific geometry-dependent properties. Moreover, the precise control over the pattern geometry and the micrometer accuracy in positioning achieved by capillary assembly make such functional patterned structures excellent candidates for integration into devices exploiting specific size-dependent NC properties.

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Antoine-Emmanuel Saliba, Laure Saias, Eleni Psychari, Nicolas Minc, Damien Simon, François-Clément Bidard, Claire Mathiot, Jean-Yves Pierga, Vincent Fraisier, Jean Salamero, Véronique Saada, Françoise Farace, Philippe Vielh, Laurent Malaquin, Jean-Louis Viovy (2010 Aug 2)

Microfluidic sorting and multimodal typing of cancer cells in self-assembled magnetic arrays.

Proceedings of the National Academy of Sciences of the United States of America : 14524-9 : DOI : 10.1073/pnas.1001515107 En savoir plus
Résumé

We propose a unique method for cell sorting, « Ephesia, » using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples–blood, pleural effusion, and fine needle aspirates–issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.

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L Malaquin, T Charitat, J Daillant (2010 Mar 23)

Supported bilayers: combined specular and diffuse X-ray scattering.

The European physical journal. E, Soft matter : 285-301 : DOI : 10.1140/epje/i2010-10578-2 En savoir plus
Résumé

A method is proposed for the analysis of specular and off-specular reflectivity from supported lipid bilayers. Both thermal fluctuations and the « static » roughness induced by the substrate are carefully taken into account. Examples from supported bilayers and more complex systems comprising a bilayer adsorbed or grafted on the substrate and another « floating » bilayer are given. The combined analysis of specular and off-specular reflectivity allows the precise determination of the structure of adsorbed and floating bilayers, their tension, bending rigidity and interaction potentials. We show that this new method gives a unique opportunity to investigate phenomena like protrusion modes of adsorbed bilayers and opens the way to the investigation of more complex systems including different kinds of lipids, cholesterol or peptides.

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Devrim Kilinc, Jean-Michel Peyrin, Vanessa Soubeyre, Sébastien Magnifico, Laure Saias, Jean-Louis Viovy, Bernard Brugg (2010 Feb 17)

Wallerian-like degeneration of central neurons after synchronized and geometrically registered mass axotomy in a three-compartmental microfluidic chip.

Neurotoxicity research : 149-61 : DOI : 10.1007/s12640-010-9152-8 En savoir plus
Résumé

Degeneration of central axons may occur following injury or due to various diseases and it involves complex molecular mechanisms that need to be elucidated. Existing in vitro axotomy models are difficult to perform, and they provide limited information on the localization of events along the axon. We present here a novel experimental model system, based on microfluidic isolation, which consists of three distinct compartments, interconnected by parallel microchannels allowing axon outgrowth. Neurons cultured in one compartment successfully elongated their axons to cross a short central compartment and invade the outermost compartment. This design provides an interesting model system for studying axonal degeneration and death mechanisms, with a previously impossible spatial and temporal control on specific molecular pathways. We provide a proof-of-concept of the system by reporting its application to a well-characterized experimental paradigm, axotomy-induced Wallerian degeneration in primary central neurons. Using this model, we applied localized central axotomy by a brief, isolated flux of detergent. We report that mouse embryonic cortical neurons exhibit rapid Wallerian-like distal degeneration but no somatic death following central axotomy. Distal axons show progressive degeneration leading to axonal beading and cytoskeletal fragmentation within a few hours after axotomy. Degeneration is asynchronous, reminiscent of in vivo Wallerian degeneration. Axonal cytoskeletal fragmentation is significantly delayed with nicotinamide adenine dinucleotide pretreatment, but it does not change when distal calpain or caspase activity is inhibited. These findings, consistent with previous experiments in vivo, confirm the power and biological relevance of this microfluidic architecture.

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Irene Dujovne, Jacob Kerssemakers, G Cappello, Cees Dekker (2010 Feb 2)

Note: Interference technique for minimally invasive, subnanometer, microsecond measurements of displacements.

The Review of scientific instruments : 016103 : DOI : 10.1063/1.3280167 En savoir plus
Résumé

We present a novel high-resolution technique for single-molecule experiments, viz., differential traveling wave tracking. This is an interference-based scattering technique where we use gold nanoparticles for high scattering intensities and incorporate differential measurements along one in-plane direction to subtract mechanical noise and drift of the system. In addition, out-of-plane distances are measured via scattered light intensity in a total internal reflectance illumination field. In plane, we demonstrate a rms noise level of only 0.10 nm at 10 kHz and less than 0.5 nm at 600 kHz.

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Christophe Lavelle, Pierre Recouvreux, Hua Wong, Aurélien Bancaud, Jean-Louis Viovy, Ariel Prunell, Jean-Marc Victor (2010 Jan 13)

Right-handed nucleosome: myth or reality?

Cell : 1216-7; author reply 1217-8 : DOI : 10.1016/j.cell.2009.12.014 En savoir plus
Résumé

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Année de publication : 2009

Martine Poitevin, Yuliya Shakalisava, Sandrine Miserere, Gabriel Peltre, Jean-Louis Viovy, Stephanie Descroix (2009 Dec 17)

Evaluation of microchip material and surface treatment options for IEF of allergenic milk proteins on microchips.

Electrophoresis : 4256-63 : DOI : 10.1002/elps.200900254 En savoir plus
Résumé

The use of glass and PDMS microchips has been investigated to perform rapid and efficient separation of allergenic whey proteins by IEF. To decrease EOF and to limit protein adsorption, two coating procedures have been compared. The first one consists in immobilizing hydroxypropyl cellulose (HPC) and the second one poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE). EOF limitation has been evaluated using frontal electrophoresis of a fluorescent marker of known effective mobility. EOF velocity was decreased by a factor about 100 and 30, respectively. pH gradient formation has been evaluated for each microchip using fluorescent pI markers. It was demonstrated that as expected a coating was essential to avoid pH gradient drift. Both coatings were efficient on glass microchips, but only PDMA-AGE allowed satisfying focusing of pI markers on PDMS microchips. Fluorescent covalent and noncovalent labelings of milk proteins have been compared by IEF on slab-gels. IEF separation of three major allergenic whey proteins [beta-lactoglobulin A (pI 5.25) and B (pI 5.35) and alpha-lactalbumin (pI 4.2-4.5)] was performed in both microchips. Milk proteins were separated with better resolution and shorter analysis time than by classical CIEF. Finally, better resolutions for milk allergens separation were obtained on glass microchips.

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