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Oukacine F.,Montpellier University | Romestand B.,Montpellier University | Goodall D.M.,Paraytec | Massiera G.,CNRS Charles Coulomb Laboratory | And 2 more authors.
Analytical Chemistry | Year: 2012

A new methodology for an antibacterial assay based on capillary electrophoresis with multiple UV detection points has been proposed. The possible antibacterial activity of cationic molecules on bacteria (Gram-positive and Gram-negative) is studied by detecting the bacteria before, during, and after their meeting with the cationic antibacterial compound. For that, a UV area imaging detector having two loops and three detection windows was used with a 95 cm × 100 μm i.d. capillary. In the antibacterial assay, the bacteria (negatively charged) and the cationic molecules were injected separately from each end of the capillary. The bacteria were mobilized by anionic ITP mode while cationic molecules migrate in the opposite direction under conditions close to CZE. The cationic molecules were injected into the capillary as a broad band (injected volume about 16% of the volume of the capillary) to prevent dilution of the sample during the electrophoretic process. Bacteriolytic activity, as well as strong interactions between the small antibacterial molecules and the bacteria, can be investigated within a few minutes. The assay was used to study the antibacterial activity of dendrigraft poly-l-lysines on Micrococcus luteus and Erwinia carotovora. Because dendrigraft poly-l-lysines are nonimmunogenic and have low toxicity, this new class of dendritic biomacromolecules is very promising for antibacterial applications. © 2012 American Chemical Society.

Ibrahim A.,Montpellier University | Koval D.,Czech Institute of Organic Chemistry And Biochemistry | Kasicka V.,Czech Institute of Organic Chemistry And Biochemistry | Faye C.,COLCOM | Cottet H.,Montpellier University
Macromolecules | Year: 2013

In this work, capillary isotachophoresis (ITP) was used to determine the effective charge of the first five generations of dendrigraft poly-l-lysines. This approach, which is based on the linear dependence of ITP zone length of the solute on its concentration and effective charge, offers a simple and straightforward method for effective charge determination. The cationic ITP system employed in this work yields good linearity, repeatability and sharp zones. The value of effective charge number per one lysine residue obtained for long linear poly-l-lysine is in a good agreement with the Manning theoretical value (0.5). Results obtained for dendrigraft poly-l-lysines show a dramatic decrease in the effective charge number per lysine residue with increasing generation number, from 0.84 for short oligolysines (generation 1) down to 0.08 for the fifth generation. This decrease in effective charge is due to the proximity of charged groups in the dendrigraft structure of higher generation number. © 2012 American Chemical Society.

Bondia A.M.,French National Center for Scientific Research | Larcher N.,French National Center for Scientific Research | Garrelly L.,Colcom | Rossi J.C.,French National Center for Scientific Research | Pascal R.,French National Center for Scientific Research
Tetrahedron Letters | Year: 2010

A bifunctional tetraethylene glycol (TEG) linker was prepared and used as an initiator for the synthesis of the first two generations of dendrigraft poly-l-lysines (DGL). The key steps involved the desymmetrization of TEG by introduction of an amine group after activation of a terminal hydroxyl group and of a conveniently protected aminooxy functionality at the other end. Initiation of l-lysine N-carboxyanhydride polymerization by the terminal amine yielded generations 1 and 2 of DGL in which a subsequent functionalization of the aminooxy group by ligation with entities bearing an aldehyde group turned out to be feasible. © 2010 Elsevier Ltd. All rights reserved.

Coussot G.,Montpellier University | Ladner Y.,Montpellier University | Bayart C.,Montpellier University | Faye C.,Colcom | And 2 more authors.
Journal of Chromatography A | Year: 2015

This work aims at studying the potentialities of an on-line capillary electrophoresis (CE)-based digestion methodology for evaluating polymer-drug conjugates degradability in the presence of free trypsin (in-solution digestion). A sandwich plugs injection scheme with transverse diffusion of laminar profile (TDLFP) mode was used to achieve on-line digestions. Electrophoretic separation conditions were established using poly-. l-Lysine (PLL) as reference substrate. Comparison with off-line digestion was carried out to demonstrate the feasibility of the proposed methodology. The applicability of the on-line CE-based digestion methodology was evaluated for two PLL-drug conjugates and for the four first generations of dendrigraft of lysine (DGL). Different electrophoretic profiles presenting the formation of di, tri, and tetralysine were observed for PLL-drug and DGL. These findings are in good agreement with the nature of the linker used to link the drug to PLL structure and the predicted degradability of DGL. The present on-line methodology applicability was also successfully proven for protein conjugates hydrolysis. In summary, the described methodology provides a powerful tool for the rapid study of biodegradable polymers. © 2014 Elsevier B.V.

Coussot G.,Max Mousseron Institute of Biomolecules | Faye C.,Max Mousseron Institute of Biomolecules | Ibrahim A.,Max Mousseron Institute of Biomolecules | Ramonda M.,Montpellier University | And 6 more authors.
Analytical and Bioanalytical Chemistry | Year: 2011

The functionalization of surfaces with amino groups is used in many application areas such as in industrial biocatalytic processes for the development of medical biomaterials and in the environment for removing pollutants from water. Amino group density and grafting stability are often related to functionalized material performances; thus, their characterizations are of prime importance. The determination of amino density and grafting stability on polymeric material (e.g. polypropylene, polystyrene and cylco olefin copolymer) is often time consuming and sometimes presents technical constraints, more particularly with non-flat materials. In this paper, we report a novel colorimetric assay using the Coomassie Brilliant Blue dye for both amino density determination and grafting stability measurement. The assay named ADECA for "Amino Density Estimation by Colorimetric Assay" is sensitive, rapid, robust and versatile. We demonstrate that ADECA makes the evaluation of aminated materials performances possible for numerous material compositions, formats and chemistries used for grafting. Our study focuses on dendrigraft of poly-l-lysine and poly(amidoamine) dendrimers dendritic materials. © Springer-Verlag 2011.

Oukacine F.,Montpellier University | Garrelly L.,COLCOM | Romestand B.,Montpellier University | Goodall D.M.,Paraytec | And 2 more authors.
Analytical Chemistry | Year: 2011

An isotachophoretic method has been developed for mobilizing and focusing bacteria. This allows quantification and detection of bacteria in a narrow zone. Very good linearity was obtained for Micrococcus lysodeikticus (also called Micrococcus luteus, studied as a model of Gram+ bacteria) in the range of 0.4 × 108 cells/mL to 2.9 × 108 cells/mL, with correlation coefficients for peak height and peak area as a function of cell concentration of 0.999 and 0.998, respectively. This method is usable on both bare and hydroxypropyl cellulose-coated fused silica capillaries. The best results were obtained using 13.6 mM Tris, 150 mM boric acid as terminating electrolyte, and 4.5 mM Tris, 50 mM boric acid, and 3.31 mM HCl as leading electrolyte. With a 33.5 cm ×100 μm i.d. capillary, short migration times were obtained while maintaining very low electrical current in order to minimize any Joule heating and lysis of the bacteria. A UV area imaging detector (ActiPix D100, Paraytec) was used with a 109 cm × 100 μm i.d. capillary having three loops and four detection windows to monitor the migration behavior of M. luteus and to show the stability of the zone of the focused bacteria along the capillary. Similar results were obtained for Erwinia carotovora (a model of Gram- bacteria), and for Enterobacter cloacae and Vibrio splendidus. © 2011 American Chemical Society.

Couturaud B.,Charles Gerhardt Institute | Bondia A.M.,Montpellier University | Faye C.,COLCOM | Garrelly L.,COLCOM | And 2 more authors.
Journal of Colloid and Interface Science | Year: 2013

The present work describes a new environmental friendly strategy for the development of surfaces with high amine density via the grafting of native or modified poly-L-lysine dendrigraft (DGL G3) onto plasma activated polypropylene (PP), polystyrene (PS), polyimide, and polytetrafluoroethylene (PTFE) surface. Modified DGL G3 was prepared by replacement of few peripheral amines by various functionalities. Grafting efficiency was determined by wettability measurements, IRTF, XPS, AFM, and by colorimetry using optimized Coomassie Brilliant Blue method tailored for surface analysis. It was shown that a 4-7 nm DGL G3 monolayer with 4 × 1014 amine cm-2 was covalently grafted onto various surfaces. Immobilization of adenosine triphosphate on the DGL-g-PP material from dilute solution was studied by bioluminescence and proved the ability of the material to interact with polyanionic biological compounds: 1 ATP complex with 5 amine groups. So, this material has a potential use in diagnostic and more widely for biotechnology due to its high capacity for biomolecule immobilization. © 2013 Elsevier Inc.

Jebors S.,Max Mousseron Institute of Biomolecules | Cecillon S.,École Centrale Lyon | Faye C.,Colcom | Enjalbal C.,Max Mousseron Institute of Biomolecules | And 4 more authors.
Journal of Materials Chemistry B | Year: 2013

A straightforward method for the preparation of hybrid bioorganic-inorganic materials is reported. Common strategies to synthesize such promising materials require special surface modifications of silica followed by grafting of the organic moiety via chemoselective ligation. In this context, we set up a general and bottom-up strategy relying on modified peptides functionalized with a trialkoxysilane group. Used in mixtures with TEOS and a surfactant as the structure directing agent, these hybrid building blocks allow one step direct synthesis of bioorganic-inorganic hybrid materials. Two examples were chosen to demonstrate our general approach. (1) An antifouling surface was prepared by dip coating of a sol containing an antibacterial silylated peptide. (2) Organized mesoporous silica displaying a peptide catalyst in the pores was prepared in one step and tested. © 2013 The Royal Society of Chemistry.

Romestand B.,Montpellier University | Rolland J.-L.,Montpellier University | Commeyras A.,COLCOM | Coussot G.,Max Mousseron Institute of Biomolecules | And 3 more authors.
Biomacromolecules | Year: 2010

An easily synthesized DendriGraft poly-lysine DGL-G3 (third generation) was shown to act as an efficient carrier for raising antibodies directed against small molecules. The immunological properties of three different forms of DGL-G3 were investigated: the native form (molecular weight 22 kDa bearing a mean number of 123 surface amino groups as TFA salts), a form modified at the C-terminus by fluorescein (fluorescein-DGL-G3), and last a surface-modified form bearing histamine (DGL-G3-Histamine). Our studies demonstrate the native DGL-G3 to be inefficient in eliciting antibody production in rabbits. Immunizations of rabbits using the core-modified fluorescein-DGL-G3 or the surface-modified DGL-G3-histamine conjugate failed in eliciting antibody production. Conversely, following a primary immunization using a BSA-histamine conjugate, a second immunization with DGL-G3-histamine conjugate improved the production of specific hapten-directed antibodies, which demonstrates the utility of DGL-G3 as a carrier for the production of highly specific antibody against haptens. © 2010 American Chemical Society.

Faye C.,Max Mousseron Institute of Biomolecules | Chamieh J.,CNRS Analytical Sciences Lab | Moreau T.,Max Mousseron Institute of Biomolecules | Granier F.,Colcom | And 4 more authors.
Analytical Biochemistry | Year: 2012

The efficient immobilization of antibodies on monolithic support is one of the most critical steps when preparing immunoaffinity supports. In this work, the ADECA (amino density estimation by colorimetric assay) method was adapted to tridimensional supports (in a dynamic mode) and proved to be efficient to characterize the antibodies grafting efficiency on 15.3 ± 0.9 mg porous glycidyl methacrylate (GMA)-co-ethylene dimethacrylate (EDMA) monolithic columns. The amount of grafted antibodies measured in situ on the monolith by ADECA (8.2 ± 0.2 μg of antibodies per milligram of monolith) was consistent with values obtained by bicinchoninic acid assay (BCA) after crushing the monolith. ADECA was shown to be less time-consuming and more versatile than BCA. The ADECA method was further implemented to thoroughly study and optimize the antibody grafting conditions (influence of pH and kinetics of the grafting step) on GMA-based monoliths and to check the covalent nature of the antibody/surface linking and its stability. Using the total amount of grafted antibodies and the amount of recognized antigen, we found that 65 ± 6% of antibodies were able to capture their antigen. Finally, the grafting of Fab and F(ab′)2 fragments demonstrated that no significant improvement of the global binding capacity of the monolith was obtained. © 2011 Elsevier Inc. All rights reserved.

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