Macromolécules et Microsystèmes en Biologie et en Médecine (MMBM)

Publications de l’équipe

Année de publication : 2014

Charles-Edouard Leroux, Sylvain Monnier, Irène Wang, Giovanni Cappello, Antoine Delon (2014 Nov 1)

Fluorescent correlation spectroscopy measurements with adaptive optics in the intercellular space of spheroids.

Biomedical optics express : 3730-8 : DOI : 10.1364/BOE.5.003730 En savoir plus
Résumé

In this study we demonstrate the use of adaptive optics to correct the biasing effects of optical aberrations when measuring the dynamics of molecules diffusing between cells in multicellular spheroids. Our results indicate that, on average, adaptive optics leads to a reduction of the 3D size of the point spread function that is statistically significant in terms of measured number of molecules and diffusion time. The sensorless approach, which uses the molecular brightness as optimization metric, is validated in a complex, highly heterogeneous, biological environment. This work paves the way towards the design of accurate diffusion measurements of molecules in thick biological specimens.

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Jana Kucerova, Zuzana Svobodova, Petr Knotek, Jiri Palarcik, Milan Vlcek, Miloslav Kincl, Daniel Horak, Julien Autebert, Jean-Louis Viovy, Zuzana Bilkova (2014 May 27)

PEGylation of magnetic poly(glycidyl methacrylate) microparticles for microfluidic bioassays.

Materials science & engineering. C, Materials for biological applications : 308-15 : DOI : 10.1016/j.msec.2014.04.011 En savoir plus
Résumé

In this study, magnetic poly(glycidyl methacrylate) microparticles containing carboxyl groups (PGMA-COOH) were coated using highly hydrophilic polymer poly(ethylene glycol) (PEG). PEG was used to reduce nonspecific interactions with proteins and cells while decreasing adhesion of particles to the walls of a microfluidic devices from poly(dimethylsiloxane) (PDMS) and cyclic olefin copolymer (COC). Zeta potential measurement, infrared spectroscopy, scanning electron microscopy, anti-PEG ELISA assay, and bioaffinity interactions between biotin and streptavidin-HRP successfully proved the presence of PEG on the surface of microspheres. Both neat and PEGylated microspheres were then incubated with the inert protein bovine serum albumin or cells to evaluate the rate of nonspecific adsorption (NSA). PEG with Mr of 30,000 Da was responsible for 45% reduction in NSA of proteins and 74% for cells compared to neat particles. The microspheres’ behavior in PDMS and COC microchannels was then evaluated. Aggregation and adhesion of PEGylated microspheres significantly decreased compared to neat particles. Finally, the model enzyme horseradish peroxidase was immobilized on the microspheres through the heterobifunctional PEG chain. The possibility for subsequent covalent coupling of the ligand of interest was confirmed. Such PEGylated microparticles can be efficiently used in PDMS microchips as a carrier for bioaffinity separation or of enzyme for catalysis.

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Morgan Delarue, Fabien Montel, Danijela Vignjevic, Jacques Prost, Jean-François Joanny, Giovanni Cappello (2014 Apr 2)

Compressive stress inhibits proliferation in tumor spheroids through a volume limitation.

Biophysical journal : 1821-8 : DOI : 10.1016/j.bpj.2014.08.031 En savoir plus
Résumé

In most instances, the growth of solid tumors occurs in constrained environments and requires a competition for space. A mechanical crosstalk can arise from this competition. In this article, we dissect the biomechanical sequence caused by a controlled compressive stress on multicellular spheroids (MCSs) used as a tumor model system. On timescales of minutes, we show that a compressive stress causes a reduction of the MCS volume, linked to a reduction of the cell volume in the core of the MCS. On timescales of hours, we observe a reversible induction of the proliferation inhibitor, p27Kip1, from the center to the periphery of the spheroid. On timescales of days, we observe that cells are blocked in the cell cycle at the late G1 checkpoint, the restriction point. We show that the effect of pressure on the proliferation can be antagonized by silencing p27Kip1. Finally, we quantify a clear correlation between the pressure-induced volume change and the growth rate of the spheroid. The compression-induced proliferation arrest that we studied is conserved for five cell lines, and is completely reversible. It demonstrates a generic crosstalk between mechanical stresses and the key players of cell cycle regulation. Our results suggest a role of volume change in the sensitivity to pressure, and that p27Kip1 is strongly influenced by this change.

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Année de publication : 2013

Anthony Bruchet, Vélan Taniga, Stéphanie Descroix, Laurent Malaquin, Florence Goutelard, Clarisse Mariet (2013 Oct 24)

Centrifugal microfluidic platform for radiochemistry: potentialities for the chemical analysis of nuclear spent fuels.

Talanta : 488-94 : DOI : 10.1016/j.talanta.2013.06.064 En savoir plus
Résumé

The use of a centrifugal microfluidic platform is for the first time reported as an alternative to classical chromatographic procedures for radiochemistry. The original design of the microfluidic platform has been thought to fasten and simplify the prototyping process with the use of a circular platform integrating four rectangular microchips made of thermoplastic. The microchips, dedicated to anion-exchange chromatographic separations, integrate a localized monolithic stationary phase as well as injection and collection reservoirs. The results presented here were obtained with a simplified simulated nuclear spent fuel sample composed of non-radioactive isotopes of Europium and Uranium, in proportion usually found for uranium oxide nuclear spent fuel. While keeping the analytical results consistent with the conventional procedure (extraction yield for Europium of ≈97%), the use of the centrifugal microfluidic platform allowed to reduce the volume of liquid needed by a factor of ≈250. Thanks to their unique « easy-to-use » features, centrifugal microfluidic platforms are potential successful candidates for the downscaling of chromatographic separation of radioactive samples (automation, multiplexing, easy integration in glove-boxes environment and low cost of maintenance).

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Karla Perez-Toralla, Jérôme Champ, Mohamad Reza Mohamadi, Olivier Braun, Laurent Malaquin, Jean-Louis Viovy, Stéphanie Descroix (2013 Sep 25)

New non-covalent strategies for stable surface treatment of thermoplastic chips.

Lab on a chip : 4409-18 : DOI : 10.1039/c3lc50888a En savoir plus
Résumé

In order to be more extensively used outside of research laboratories, lab-on-chip technologies must be mass-produced using low-cost materials such as thermoplastics. Thermoplastics, however, are generally hydrophobic in their native state, which makes them unsuitable for direct use with biological samples in aqueous solution, and thus require surface coating. This coating should be robust, inexpensive and simple to implement, in order not to hinder the industrial advantage of thermoplastic chips. Cyclic Olefin Copolymer (COC) is a particularly appealing polymer, but it is also difficult to functionalize due to its chemical inertness. Here we introduce and compare the performance of two new approaches for COC coating. One relies on the use of a commercial triblock copolymer, Pluronic® F127. The second approach uses new copolymers synthesized by radical polymerization, and consisting of a dimethylacrylamide (DMA) backbone carrying aliphatic side chains (C22). Two DMA-C22 copolymers were synthesized with various C22/DMA ratios: DMA-S at 0.175% and DMA-M at 0.35%. Different physicochemical properties of the polymers such as critical micellar concentration (CMC), water contact angle, electroosmosis were investigated. Coated COC chips were then tested for their ability to reduce the adsorption of proteins, microparticles, and for protein electrophoresis. For each application we found an optimal treatment protocol to considerably improve the performance of the thermoplastic chip. These treatments use physisorption in situ which requires no photografting or chemical reaction and can be performed by a simple incubation either after chip production, or just prior to use.

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Sébastien Magnifico, Laure Saias, Bérangère Deleglise, Eric Duplus, Devrim Kilinc, Marie-Christine Miquel, Jean-Louis Viovy, Bernard Brugg, Jean-Michel Peyrin (2013 Aug 27)

NAD+ acts on mitochondrial SirT3 to prevent axonal caspase activation and axonal degeneration.

FASEB journal : official publication of the Federation of American Societies for Experimental Biology : 4712-22 : DOI : 10.1096/fj.13-229781 En savoir plus
Résumé

In chronic degenerative syndromes, neuronal death occurs over long periods, during which cells progressively lose their axons and, ultimately, their cell bodies. Although apoptosis is recognized as a key event in neuronal death, the molecular mechanisms involved in CNS axons degeneration are poorly understood. Due to the highly polarized phenotypes of CNS neurons, the different neuronal subcompartments are likely to be targeted by light repetitive and localized aggression. Such locally initiated deleterious signal transduction pathways could theoretically spread through the cytoplasm. However, where axon-degenerative signals initiate, what these early signals are, and how they lead to axon degeneration are unanswered questions that limit our understanding of neurodegenerative diseases and our ability to identify novel therapeutic targets. Using a microfluidic culture device adapted to CNS primary neurons, allowing specific access to the axonal and somatodendritic compartments, we analyzed the molecular pathways involved in axonal degeneration of differentiated neurons. We show here that local application of proapoptotic stimuli on the somatodentritic compartment triggers a dying-back pattern involving caspase-dependent axonal degeneration. Using complementary pharmacological and genetic approaches, we further demonstrate that NAD(+) and grape wine polyphenols prevent axonal apoptosis and act via mitochondrial SirT3 activation in axons.

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Bérangère Deleglise, Benjamin Lassus, Vaneyssa Soubeyre, Aurélie Alleaume-Butaux, Johannes J Hjorth, Maéva Vignes, Benoit Schneider, Bernard Brugg, Jean-Louis Viovy, Jean-Michel Peyrin (2013 Aug 27)

Synapto-protective drugs evaluation in reconstructed neuronal network.

PloS one : e71103 : DOI : 10.1371/journal.pone.0071103 En savoir plus
Résumé

Chronic neurodegenerative syndromes such as Alzheimer’s and Parkinson’s diseases, or acute syndromes such as ischemic stroke or traumatic brain injuries are characterized by early synaptic collapse which precedes axonal and neuronal cell body degeneration and promotes early cognitive impairment in patients. Until now, neuroprotective strategies have failed to impede the progression of neurodegenerative syndromes. Drugs preventing the loss of cell body do not prevent the cognitive decline, probably because they lack synapto-protective effects. The absence of physiologically realistic neuronal network models which can be easily handled has hindered the development of synapto-protective drugs suitable for therapies. Here we describe a new microfluidic platform which makes it possible to study the consequences of axonal trauma of reconstructed oriented mouse neuronal networks. Each neuronal population and sub-compartment can be chemically addressed individually. The somatic, mid axon, presynaptic and postsynaptic effects of local pathological stresses or putative protective molecules can thus be evaluated with the help of this versatile « brain on chip » platform. We show that presynaptic loss is the earliest event observed following axotomy of cortical fibers, before any sign of axonal fragmentation or post-synaptic spine alteration. This platform can be used to screen and evaluate the synapto-protective potential of several drugs. For instance, NAD⁺ and the Rho-kinase inhibitor Y27632 can efficiently prevent synaptic disconnection, whereas the broad-spectrum caspase inhibitor zVAD-fmk and the stilbenoid resveratrol do not prevent presynaptic degeneration. Hence, this platform is a promising tool for fundamental research in the field of developmental and neurodegenerative neurosciences, and also offers the opportunity to set up pharmacological screening of axon-protective and synapto-protective drugs.

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Bruno Teste, Anaïs Ali-Cherif, Jean Louis Viovy, Laurent Malaquin (2013 May 4)

A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets.

Lab on a chip : 2344-9 : DOI : 10.1039/c3lc50353d En savoir plus
Résumé

Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.

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Marie Girardot, Fanny d'Orlyé, Stéphanie Descroix, Anne Varenne (2013 Jan 22)

Aptamer-conjugated nanoparticles: preservation of targeting functionality demonstrated by microchip electrophoresis in frontal mode.

Analytical biochemistry : 150-2 : DOI : 10.1016/j.ab.2012.12.022 En savoir plus
Résumé

Aptamer-conjugated nanoparticles (Apt-NPs) are increasingly being developed for biomedical purposes and especially for diagnosis and therapy. However, there is no quantitative study of the targeting functionality of such grafted aptamers compared with free aptamers. Thus, we report the first determination of binding parameters for Apt-NP/target complexes, thanks to a continuous frontal analysis in a microchip electrophoresis format (named FACMCE), based on a methodology previously developed by our group. As a model system, the targeting ability of a lysozyme-binding aptamer conjugated to fluorescent maghemite nanoparticles was evaluated and showed evidence that the conjugation does not alter the affinity of this aptamer.

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Année de publication : 2012

Scott Atwell, Ludovic Disseau, Alicja Z Stasiak, Andrzej Stasiak, Axelle Renodon-Cornière, Masayuki Takahashi, Jean-Louis Viovy, Giovanni Cappello (2012 Nov 28)

Probing Rad51-DNA interactions by changing DNA twist.

Nucleic acids research : 11769-76 : DOI : 10.1093/nar/gks1131 En savoir plus
Résumé

In eukaryotes, Rad51 protein is responsible for the recombinational repair of double-strand DNA breaks. Rad51 monomers cooperatively assemble on exonuclease-processed broken ends forming helical nucleo-protein filaments that can pair with homologous regions of sister chromatids. Homologous pairing allows the broken ends to be reunited in a complex but error-free repair process. Rad51 protein has ATPase activity but its role is poorly understood, as homologous pairing is independent of adenosine triphosphate (ATP) hydrolysis. Here we use magnetic tweezers and electron microscopy to investigate how changes of DNA twist affect the structure of Rad51-DNA complexes and how ATP hydrolysis participates in this process. We show that Rad51 protein can bind to double-stranded DNA in two different modes depending on the enforced DNA twist. The stretching mode is observed when DNA is unwound towards a helical repeat of 18.6 bp/turn, whereas a non-stretching mode is observed when DNA molecules are not permitted to change their native helical repeat. We also show that the two forms of complexes are interconvertible and that by enforcing changes of DNA twist one can induce transitions between the two forms. Our observations permit a better understanding of the role of ATP hydrolysis in Rad51-mediated homologous pairing and strand exchange.

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Sandrine Morlot, Valentina Galli, Marius Klein, Nicolas Chiaruttini, John Manzi, Frédéric Humbert, Luis Dinis, Martin Lenz, Giovanni Cappello, Aurélien Roux (2012 Oct 30)

Membrane shape at the edge of the dynamin helix sets location and duration of the fission reaction.

Cell : 619-29 : DOI : 10.1016/j.cell.2012.09.017 En savoir plus
Résumé

The GTPase dynamin polymerizes into a helical coat that constricts membrane necks of endocytic pits to promote their fission. However, the dynamin mechanism is still debated because constriction is necessary but not sufficient for fission. Here, we show that fission occurs at the interface between the dynamin coat and the uncoated membrane. At this location, the considerable change in membrane curvature increases the local membrane elastic energy, reducing the energy barrier for fission. Fission kinetics depends on tension, bending rigidity, and the dynamin constriction torque. Indeed, we experimentally find that the fission rate depends on membrane tension in vitro and during endocytosis in vivo. By estimating the energy barrier from the increased elastic energy at the edge of dynamin and measuring the dynamin torque, we show that the mechanical energy spent on dynamin constriction can reduce the energy barrier for fission sufficiently to promote spontaneous fission. :

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Mohamed Lemine Youba Diakité, Jerôme Champ, Stephanie Descroix, Laurent Malaquin, François Amblard, Jean-Louis Viovy (2012 Sep 13)

A low-cost, label-free DNA detection method in lab-on-chip format based on electrohydrodynamic instabilities, with application to long-range PCR.

Lab on a chip : 4738-47 : DOI : 10.1039/c2lc40372b En savoir plus
Résumé

In order to evolve from a « chip in the lab » to a « lab on a chip » paradigm, there is still a strong demand for low-cost, portable detection technologies, notably for analytes at low concentrations. Here we report a new label-free DNA detection method with direct electronic read, and apply it to long-range PCR. This method uses a nonlinear electrohydrodynamic phenomenon: when subjected to high electric fields (typically above 100 V cm(-1)), suspensions of large polyelectrolytes, such as long DNA molecules, create « giant » dynamic concentration fluctuations. These fluctuations are associated with large conductivity inhomogeneities, and we use here a contact-mode local conductivity detector to detect these fluctuations. In order to decouple the detection electronics from the high voltage excitation one, an original « doubly symmetric » floating mode battery-operated detection scheme was developed. A wavelet analysis was then applied, to unravel from the chaotic character of the electohydrodynamic instabilities a scalar signal robustly reflecting the amplification of DNA. As a first proof of concept, we measured the products of the off-chip amplification of 10 kbp DNA from lambda phage DNA, achieving a sensitivity better than 100 fg DNA in the original 50 μl sample. This corresponds to the amplification products of less than 100 initial copies of target DNA. The companion enabling technologies developed to implement this new concept, i.e. the doubly symmetric contact conductivity detection and wavelet analysis, may also find various other applications in lab-on-chips.

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Julien Autebert, Benoit Coudert, François-Clément Bidard, Jean-Yves Pierga, Stéphanie Descroix, Laurent Malaquin, Jean-Louis Viovy (2012 Jul 17)

Microfluidic: an innovative tool for efficient cell sorting.

Methods (San Diego, Calif.) : 297-307 : DOI : 10.1016/j.ymeth.2012.07.002 En savoir plus
Résumé

At first mostly dedicated to molecular analysis, microfluidic systems are rapidly expanding their range of applications towards cell biology, thanks to their ability to control the mechanical, biological and fluidic environment at the scale of the cells. A number of new concepts based on microfluidics were indeed proposed in the last ten years for cell sorting. For many of these concepts, progress remains to be done regarding automation, standardization, or throughput, but it is now clear that microfluidics will have a major contribution to the field, from fundamental research to point-of-care diagnosis. We present here an overview of cells sorting in microfluidics, with an emphasis on circulating tumor cells. Sorting principles are classified in two main categories, methods based on physical properties of the cells, such as size, deformability, electric or optical properties, and methods based on biomolecular properties, notably specific surface antigens. We document potential applications, discuss the main advantages and limitations of different approaches, and tentatively outline the main remaining challenges in this fast evolving field.

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Daniel Horák, Zuzana Svobodová, Julien Autebert, Benoit Coudert, Zdeněk Plichta, Karel Královec, Zuzana Bílková, Jean-Louis Viovy (2012 Jul 7)

Albumin-coated monodisperse magnetic poly(glycidyl methacrylate) microspheres with immobilized antibodies: application to the capture of epithelial cancer cells.

Journal of biomedical materials research. Part A : 23-32 : DOI : 10.1002/jbm.a.34297 En savoir plus
Résumé

Monodisperse (4 μm) macroporous crosslinked poly(glycidyl methacrylate) (PGMA) microspheres for use in microfluidic immunomagnetic cell sorting, with a specific application to the capture of circulating tumor cells (CTCs), were prepared by multistep swelling polymerization in the presence of cyclohexyl acetate porogen and hydrolyzed and ammonolyzed. Iron oxide was then precipitated in the microspheres to render them magnetic. Repeated precipitation made possible to raise the iron oxide content to more than 30 wt %. To minimize nonspecific adsorption of the microspheres in a microchannel and of cells on the microspheres, they were coated with albumin crosslinked with glutaraldehyde. Antibodies of epithelial cell adhesion molecule (anti-EpCAM) were then immobilized on the albumin-coated magnetic microspheres using the carbodiimide method. Capture of breast cancer MCF7 cells as a model of CTCs by the microspheres with immobilized anti-EpCAM IgG was performed in a batch experiment. Finally, MCF7 cells were captured by the anti-EpCAM-immobilized albumin-coated magnetic microspheres in an Ephesia chip. A very good rejection of lymphocytes was achieved. Thus, albumin-coated monodisperse magnetic PGMA microspheres with immobilized anti-EpCAM seem to be promising for capture of CTCs in a microfluidic device.

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Zuzana Svobodova, Mohamad Reza Mohamadi, Barbora Jankovicova, Hermann Esselmann, Romain Verpillot, Markus Otto, Myriam Taverna, Jens Wiltfang, Jean-Louis Viovy, Zuzana Bilkova (2012 Jun 20)

Development of a magnetic immunosorbent for on-chip preconcentration of amyloid β isoforms: Representatives of Alzheimer’s disease biomarkers.

Biomicrofluidics : 24126-2412612 : DOI : 10.1063/1.4722588 En savoir plus
Résumé

Determination of amyloid β (Aβ) isoforms and in particular the proportion of the Aβ 1-42 isoform in cerebrospinal fluid (CSF) of patients suspected of Alzheimer’s disease might help in early diagnosis and treatment of that illness. Due to the low concentration of Aβ peptides in biological fluids, a preconcentration step prior to the detection step is often necessary. This study utilized on-chip immunoprecipitation, known as micro-immunoprecipitation (μIP). The technique uses an immunosorbent (IS) consisting of magnetic beads coated with specific anti-Aβ antibodies organized into an affinity microcolumn by a magnetic field. Our goal was to thoroughly describe the critical steps in developing the IS, such as selecting the proper beads and anti-Aβ antibodies, as well as optimizing the immobilization technique and μIP protocol. The latter includes selecting optimal elution conditions. Furthermore, we demonstrate the efficiency of anti-Aβ IS for μIP and specific capture of 5 Aβ peptides under optimized conditions using various subsequent analytical methods, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), capillary electrophoresis, microchip electrophoresis, and immunoblotting. Synthetic Aβ peptides samples prepared in buffer and spiked in human CSF were analyzed. Finally, on-chip immunoprecipitation of Aβ peptides in human CSF sample was performed.

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