Université PSL

Publications

RECHERCHER

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Frontal analysis capillary electrophoresis hyphenated to electrospray ionization mass spectrometry for the characterization of the antithrombin/heparin pentasaccharide complex
Laboratoire Synthèse, Electrochimie, Imagerie et Systèmes Analytiques... - Fermas S, Gonnet F, Varenne A, Gareil P, Daniel R.
Anal. Chem. - 79(13) :4987-93 - PMID:17536781 - 2007
The interaction of proteins with polysaccharides represents a major and challenging topic in glycobiology, since such complexes mediate fundamental biological mechanisms. A new strategy based on the hyphenation of frontal analysis capillary electrophoresis (FACE) with electrospray ionization mass spectrometry (ESIMS) is reported for the characterization of protein/carbohydrate complexes. While most of the previously reported CE-MS experiments were performed using capillary electrophoresis in zone format, we report for the first time CE-MS experiments in which CE was performed in frontal analysis (FACE-MS). We showed that the frontal mode offered a better sensitivity than zone mode and was well suited for the CE-MS coupling. This FACE-MS coupling was applied to the analysis of the complex between antithrombin and the sulfated pentasaccharide reproducing the antithrombin-binding sequence in heparin. The mixture of coincubated antithrombin and heparin pentasaccharide was continuously injected into the capillary, and the electrophoretic separation of the free and bound forms of the protein was achieved. The intact noncovalent complex antithrombin/heparin pentasaccharide was detected on-line by ESIMS in positive ionization mode and in nondenaturing sheath liquid conditions. The complex stoichiometry was determined from the mass measurement of the complex. In addition, the characterization of the sulfated pentasaccharide ligand dissociated from the complex was performed in negative ionization mode using a denaturing sheath liquid, allowing the determination of its molecular mass and sulfation features. This FACE-ESIMS strategy opens the way to ligand fishing experiments performed on heterogeneous carbohydrate mixtures and subsequent characterization of specifically bound carbohydrates.
Stable Modification of PDMS surface properties by plasma polymerization: Application to the formation of double emulsions in microfluidic systems
Laboratoire Procédés, Plasmas, Microsystèmes - Vanessa Barbier, Michaël Tatoulian, Hong Li, Farzaneh Arefi-Khonsari ,Armand Ajdari and Patrick Tabeling
Langmuir - 22(12) :5230-2 - DOI:10.1021/la053289c - 2006
We describe a method based on plasma polymerization for the modification and control of the surface properties of poly(dimethylsiloxane) (PDMS) surfaces. By depositing plasma polymerized acrylic acid coatings on PDMS, we succeeded to fabricate stable (several days) hydrophilic and patterned hydrophobic/hydrophilic surfaces. We used this approach to generate direct and (for the first time in this material) double emulsions in PDMS microchannels.
A conductive hydrogel based on alginate and carbon nanotubes for probing microbial electroactivity
- Léopold Mottet, Domitille Le Cornec, Jean-Marc Noël, Frédéric Kanoufi, Brigitte Delord, Philippe Poulin, Jérôme Bibettea and Nicolas Bremond
- 14 1434-1441 - 10.1039/C7SM01929G -
Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential of generating electrical current. Despite their high value for sustainable energy, organic compound production and bioremediation, a tool to probe the natural biodiversity and to select most efficient microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes. Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the bacterial culture (Geobacter sulfurreducens). Bacteria can indeed colonize the outer wall of plain beads or the inner wall of the conductive capsules' shell that function as an anode from which electrons produced by the cells are collected.
Micropipette-powered droplet based microfluidics
Laboratoire Colloïdes et Matériaux Divisés - Krzysztof Langer, Nicolas Bremond, Laurent Boitard, Jean Baudry, Jerome Bibette
Biomicrofluidics - 12, 044106 - https://doi.org/10.1063/1.5037795 -
Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is
becoming a widespread method for performing assays and especially in the cell biol-
ogy field. Making a simple and highly portable system for creating emulsion droplets
would help to continue the popularization of such a technique. Also, the ability to
emulsify all the samples would strengthen this compartimenlization technique to han-
dle samples with limited volume. Here, we propose a strategy of droplet formation
that combines a classical flow-focusing microfluidic chip, which could be commer-
cially available, with a standard laboratory adjustable micropipette. The micropipette
is used as a negative pressure generator for controlling liquid flows. In that way, emul-
sification does neither require any electrical power supply nor a cumbersome device
and functions with small liquid volumes. Droplet formation can be easily and safely
performed in places with limited space, opening a wide range of applications espe-
cially in biological laboratory environments with higher level of safety regulations,
i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid vol-
umes, and thus possible time dependent flow conditions, allows to minimize dead
volume while keeping drops’ size homogeneous. A physical characterization
of droplet production and a model that describes the emulsion features, in terms of
drop size and size distribution, are proposed for rationalizing the performances of
the micropipette-powered emulsification process.
Published by AIP Publishing.
A conductive hydrogel based on alginate and carbon nanotubes for probing microbial electroactivity
Laboratoire Colloïdes et Matériaux Divisés - Léopold Mottet, Domitille Le Cornec, Jean-Marc Noël, Frédéric Kanoufi, Brigitte Delord, Philippe Poulin, Jérôme Bibette and Nicolas Bremond
- 14 1434 - DOI: 10.1039/c7sm01929g -
Some bacteria can act as catalysts to oxidize (or reduce) organic or inorganic matter with the potential
of generating electrical current. Despite their high value for sustainable energy, organic compound
production and bioremediation, a tool to probe the natural biodiversity and to select most efficient
microbes is still lacking. Compartmentalized cell culture is an ideal strategy for achieving such a goal but
the appropriate compartment allowing cell growth and electron exchange must be tailored. Here, we
develop a conductive composite hydrogel made of a double network of alginate and carbon nanotubes.
Homogeneous mixing of carbon nanotubes within the polyelectrolyte is obtained by a surfactant
assisted dispersion followed by a desorption step for triggering electrical conductivity. Dripping the
mixture in a gelling bath through simple extrusion or a double one allows the formation of either plain
hydrogel beads or liquid core hydrogel capsules. The process is shown to be compatible with the
bacterial culture (
Geobacter sulfurreducens
). Bacteria can indeed colonize the outer wall of plain beads
or the inner wall of the conductive capsules’ shell that function as an anode from which electrons
produced by the cells are collected
Micropipette-powered droplet based microfluidics
Laboratoire Colloïdes et Matériaux Divisés - Krzysztof Langer, Nicolas Bremonda), Laurent Boitard, Jean Baudry, and Jérôme Bibette
Biomicrofluidics - 044106 - 10.1063/1.5037795 -
Droplet-based microfluidics, using water-in-oil emulsion droplets as micro-reactors, is becoming a widespread method for performing assays and especially in the cell biology field. Making a simple and highly portable system for creating emulsion droplets would help to continue the popularization of such a technique. Also, the ability to emulsify all the samples would strengthen this compartimenlization technique to handle samples with limited volume. Here, we propose a strategy of droplet formation that combines a classical flow-focusing microfluidic chip, which could be commercially available, with a standard laboratory adjustable micropipette. The micropipette is used as a negative pressure generator for controlling liquid flows. In that way, emulsification does neither require any electrical power supply nor a cumbersome device and functions with small liquid volumes. Droplet formation can be easily and safely performed in places with limited space, opening a wide range of applications especially in biological laboratory environments with higher level of safety regulations, i.e., BSL-3/4. Fortunately, the present methodology that involves small fluid volumes, and thus possible time dependent flow conditions, allows to minimize dead volume while keeping drops' size homogeneous. A physical characterization of droplet production and a model that describes the emulsion features, in terms of drop size and size distribution, are proposed for rationalizing the performances of the micropipette-powered emulsification process.
Laboratoire Colloïdes et Matériaux Divisés - Rouzeau C, Dagkesamanskaya A, Langer K, Bibette J, Baudry J, Pompon D, Anton-Leberre V.
- 169(6) 335-342. - doi: 10.1016/j.resmic.2018.06.002. -
Adjustment of plasmid copy number resulting from the balance between positive and negative impacts of borne synthetic genes, plays a critical role in the global efficiency of multistep metabolic engineering. Differential expression of co-expressed engineered genes is frequently observed depending on growth phases, metabolic status and triggered adjustments of plasmid copy numbers, constituting a dynamic process contributing to minimize global engineering burden. A yeast model involving plasmid based expression of phosphoribulokinase (PRKp), a key enzyme for the reconstruction of synthetic Calvin cycle, was designed to gain further insights into such a mechanism. A conditional PRK expression cassette was cloned either onto a low (ARS-CEN based) or a high (2-micron origin based) copy number plasmid using complementation of a trp1 genomic mutation as constant positive selection. Evolution of plasmid copy numbers, PRKp expressions, and cell growth rates were dynamically monitored following gene de-repression through external doxycycline concentration shifts. In the absence of RubisCO encoding gene permitting metabolic recycling, PRKp expression that led to depletion of ribulose phosphate, a critical metabolite for aromatic amino-acids biosynthesis, and accumulation of the dead-end diphosphate product contribute to toxicity. Triggered copy number adjustment was found to be a dynamic process depending both on plasmid types and levels of PRK induction. With the ARS-CEN plasmid, cell growth was abruptly affected only when level PRKp expression exceeded a threshold value. In contrast, a proportional relationship was observed with the 2-micron plasmid consistent with large copy number adjustments. Micro-compartment partitioning of bulk cultures by embedding individual cells into inverse culture medium/oil droplets, revealed the presence of slow and fast growing subpopulations that differ in relative proportions for low and high copy number plasmids.
Laboratoire Colloïdes et Matériaux Divisés - Dagkesamanskaya A, Langer K, Tauzin AS, Rouzeau C, Lestrade D, Potocki-Veronese G, Boitard L, Bibette J, Baudry J, Pompon D, Anton-Leberre V.
J Microbiol Methods. - 147 59-65 - doi: 10.1016/j.mimet.2018.03.001. -
Application of droplet-based microfluidics for the screening of microbial libraries is one of the important ongoing developments in functional genomics/metagenomics. In this article, we propose a new method that can be employed for high-throughput profiling of cell growth. It consists of light-driven labelling droplets that contain growing cells directly in a microfluidics observation chamber, followed by recovery of the labelled cells. This method is based on intracellular expression of green-to-red switchable fluorescent proteins. The proof of concept is established here for two commonly used biological models, E. coli and S. cerevisiae. Growth of cells in droplets was monitored under a microscope and, depending on the targeted phenotype, the fluorescence of selected droplets was switched from a "green" to a "red" state. Red fluorescent cells from labelled droplets were then successfully detected, sorted with the Fluorescence Activated Cell Sorting machine and recovered. Finally, the application of this method for different kind of screenings, in particular of metagenomic libraries, is discussed and this idea is validated by the analysis of a model mini-library.

Copyright © 2018 Elsevier B.V. All rights reserved.
Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.
Laboratoire Colloïdes et Matériaux Divisés - Eyer K, Doineau RCL,Castrillon C, Briseño-Roa L, Menrath V, Mottet G, England P, Godina A, Brient-Litzler E, Nizak C, Jensen A, Griffiths AD, Bibette J, Bruhns P, Baudry J.
Nat Biotechnol. - 35(10) 977-982 - doi: 10.1038/nbt.3964. -
Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening.
Hydrophobization of Silica Nanoparticles in Water: Nanostructure and Response to Drying Stress
Laboratoire Colloïdes et Matériaux Divisés - Solenn Moro, Caroline Parneix, Bernard Cabane, Nicolas Sanson , and Jean-Baptiste d’Espinose de Lacaillerie
Langmuir - 33 (19) 4709–4719 - DOI: 10.1021/acs.langmuir.6b04505 -
We report on the impact of surface hydrophobization on the structure of aqueous silica dispersions and how this structure resists drying stress. Hydrophilic silica particles were hydrophobized directly in water using a range of organosilane precursors, with a precise control of the grafting density. The resulting nanostructure was precisely analyzed by a combination of small-angle X-ray scattering (SAXS) and cryo-microscopy (cryo-TEM). Then, the dispersion was progressively concentrated by drying, and the evolution of the nanostructures as a function of the grafting density was followed by SAXS. At the fundamental level, because the hydrophobic character of the silica surfaces could be varied continuously through a precise control of the grafting density, we were able to observe how the hydrophobic interactions change particles interactions and aggregates structures. Practically, this opened a new route to tailor the final structure, the residual porosity, and the damp-proof properties of the fully dried silica. For example, regardless of the nature of the hydrophobic precursor, a grafting density of 1 grafter per nm2 optimized the interparticle interactions in solution in view to maximize the residual porosity in the dried material (0.9 cm3/g) and reduced the water uptake to less than 4% in weight compared to the typical value of 13% for hydrophilic particles (at T = 25 °C and relative humidity = 80%).

410 publications.