Université PSL



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Quantitative and sensitive detection of rare mutations using droplet microfluidics
Laboratoire Biochimie - D. Pekin, Y. Skhiri, J.-C. Baret, D. Le Corre, L. Mazutis, C. Ben Salem, F. Millot, A. El Harrak, J.B. Hutchison, J.W. Larson, D.R. Link, P. Laurent-Puig, A.D. Griffiths and V. Taly
Lab. Chip - 11(13) :2156-66 - DOI: 10.1039/c1lc20128j - 2011
Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(®) assays and pyrosequencing, while quantitative, cannot detect less than ∼1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(®) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(®) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.
External forces control mitotic spindle positioning
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Fink J, Carpi N, Betz T, Bétard A, Chebah M, Azioune A, Bornens M, Sykes C,Fetler L, Cuvelier D, Piel M*. (*corresponding authors)
Nat. Cell Biol. - 13(7) :771-8 - DOI:10.1038/ncb2269 - 2011
The response of cells to forces is essential for tissue morphogenesis and homeostasis. This response has been extensively investigated in interphase cells, but it remains unclear how forces affect dividing cells. We used a combination of micro-manipulation tools on human dividing cells to address the role of physical parameters of the micro-environment in controlling the cell division axis, a key element of tissue morphogenesis. We found that forces applied on the cell body direct spindle orientation during mitosis. We further show that external constraints induce a polarization of dynamic subcortical actin structures that correlate with spindle movements. We propose that cells divide according to cues provided by their mechanical micro-environment, aligning daughter cells with the external force field.
Cell migration in confinement: a micro-channel-based assay
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Heuzé ML, Collin O, Terriac E, Lennon-Duménil AM, Piel M
Methods Mol Biol. - 769 :415-434 - DOI:10.1007/978-1-61779-207-6_28. - 2011
This chapter describes a method to study cells migrating in micro-channels, a confining environment of well-defined geometry. This assay is a complement to more complex 3D migration systems and provides several advantages even if it does not recapitulate the full complexity of 3D migration. Important parameters such as degree of adhesion, degree of confinement, mechanical properties, and geometry can be varied independently of each other. The device is fully compatible with almost any type of light microscopy and the simple geometry makes automated analysis very easy to perform, which allows screening strategy. The chapters is divided into five parts describing the design of different types of migration chambers, the fabrication of a mold by photolithography, the assembly of the chamber, the loading of cells, and finally the imaging on live or fixed cells.
Millifluidic droplet analyser for microbiology
Laboratoire Colloïdes et Matériaux Divisés - L. Baraban, F. Bertholle, M. Salverda, N. Bremond, P. Panizza, J. Baudry, A.G.M. de Visser, J. Bibette
Lab. Chip - 11(23) :4057-62 - DOI:10.1039/C1LC20545E - 2011
We present a novel millifluidic droplet analyser (MDA) for precisely monitoring the dynamics of microbial populations over multiple generations in numerous (>103) aqueous emulsion droplets (~100 nL). As a first application, we measure the growth rate of a bacterial strain and determine the minimal inhibitory concentration (MIC) for the antibiotic cefotaxime by incubating bacteria in a fine gradient of antibiotic concentrations. The detection of cell activity is based on the automated detection of an epifluorescent signal that allows the monitoring of microbial populations up to a size of ~106 cells. We believe that this device is helpful for the study of population dynamic consequences of microbe-environment interactions and of individual cell differences. Moreover, the fluidic machine may improve clinical tests, as it simplifies, automates and miniaturizes the screening of numerous microbial populations that grow and evolve in compartments with a finely tuned composition.
Force-Velocity Measurements of a Few Growing Actin Filaments
Laboratoire Colloïdes et Matériaux Divisés - C. Brangbour, O. du Roure, E. Helfer, D. Demoulin, A. Mazurier, M. Fermigier, M.F. Carlier, J. Bibette, J. Baudry
Plos Biology - 9(4) :e10000613 - DOI:10.1371/journal.pbio.1000613 - 2011
The polymerization of actin in filaments generates forces that play a pivotal role in many cellular processes. We introduce a novel technique to determine the force-velocity relation when a few independent anchored filaments grow between magnetic colloidal particles. When a magnetic field is applied, the colloidal particles assemble into chains under controlled loading or spacing. As the filaments elongate, the beads separate, allowing the force-velocity curve to be precisely measured. In the widely accepted Brownian ratchet model, the transduced force is associated with the slowing down of the on-rate polymerization. Unexpectedly, in our experiments, filaments are shown to grow at the same rate as when they are free in solution. However, as they elongate, filaments are more confined in the interspace between beads. Higher repulsive forces result from this higher confinement, which is associated with a lower entropy. In this mechanism, the production of force is not controlled by the polymerization rate, but is a consequence of the restriction of filaments' orientational fluctuations at their attachment point.
Method and device for Reading an emulsion
Laboratoire Colloïdes et Matériaux Divisés - J. Baudry, J. Sylvestre, J. Bibette, L. Boitard, N. Bremond
Brevets - PCT/FR2009/051396 - - 2011
A method for reading an emulsion (3) including droplets and a continuous phase surrounding the droplets, the method includes: two-dimensional scanning of the emulsion (3), and construction of a two-dimensional image of the emulsion (3) based on the scanning. Preferably, the droplets do not move during scanning, for example by solidifying the continuous phase or by using a two-dimensional compact or semi-compact network of droplets. The method can further include time-based monitoring of a chemical or biological reaction taking place in at least one of the droplets. A device implementing this method is also described. The method is applicable for the detection and/or sorting of microdroplets performing the role of microreactors or containing specific cells or molecules, in fields such as gene expression or diagnosis.
Method for manufacturing capsules having controlled head
Laboratoire Colloïdes et Matériaux Divisés - N. Bremond, E. Santanach Carreras, J. Bibette
Brevets - PCT/FR2011/050064 - - 2011
The invention relates to a method which includes separately conveying a first liquid solution (36) and a second liquid solution (40) in a double shell (32). The invention includes forming a series of drops (78) at the outlet of the double shell (32), each drop (78) including a central core (80) made of a first solution (36) and a peripheral film (82) made of a second solution (40). The method comprises dropping each drop (78) into a gaseous volume at the outlet of the double shell (32) and submerging each drop (78) in a gelling solution (70) in order to form a gelled shell. The ratio of the kinetic energy of the drop (78) to the square of the radius thereof, when said drop comes into contact with the gelling solution (70), is greater than 1 J/m2.
(FR)Ce procédé comprend le convoyage séparé dans une double enveloppe (32) d'une première solution liquide (36) et d'une deuxième solution liquide (40). Il comprend la formation à la sortie de la double enveloppe (32) d'une série de gouttes (78), chaque goutte (78) comprenant un noyau central (80) formé de première solution (36) et une pellicule périphérique (82) formée de deuxième solution (40). Le procédé comporte la chute de chaque goutte (78) dans un volume gazeux à la sortie de la double enveloppe (32) et l'immersion de chaque goutte (78) dans une solution gélifiante (70) pour former une enveloppe gélifiée. Le rapport de l'énergie cinétique de la goutte (78), au carré de son rayon, lorsqu'elle entre en contact avec la solution gélifiante (70) est supérieur à 1 J/m2.
New family of fluorinated polymer chips for droplet and organic solvent microfluidics
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Begolo S, Colas G, Viovy JL, Malaquin L.
Lab. Chip - 11(3) :508-12 - DOI:10.1039/c0lc00356e - 2011
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.
Microchip integrating magnetic nanoparticles for allergy diagnosis
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Teste B, Malloggi F, Siaugue JM, Varenne A, Kanoufi F, Descroix S.
Lab. Chip - 11(24) :4207-13 - DOI:10.1039/C1LC20809H - 2011
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)).
Stress Clamp Experiments on Multicellular Tumor Spheroids
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Montel F, Delarue M, Elgeti J, Malaquin L, Basan M, Risler T, Cabane B, Vignjevic D, Prost J, Cappello G, Joanny JF.
Phys. Rev. Lett. - 107(18) :188102 - PMID:22107677 - 2011
Pour croître une tumeur doit « faire sa place » au sein d’un tissu : c’est tout un jeu de forces mécaniques qui s’exercent alors entre la tumeur et le tissu sain et dont le perdant pourrait être la tumeur. L’application d’une pression même faible sur un tissu tumoral bloque son expansion et lorsque l’on cesse d’exercer cette pression, la croissance tumorale repart. La pression stoppe la prolifération des cellules, mais pas de façon homogène : les cellules au centre de leur amas cellulaire ne se divisent plus, alors que celles en périphérie continuent. Dans l’organisme, seules les tumeurs capables de soutenir cette pression pourraient continuer à proliférer. L’environnement mécanique pourrait un jour devenir un outil à prendre en compte dans le diagnostic.

410 publications.