Université PSL



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Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome
Laboratoire Physique des biomolécules - M. Manosas, M. M. Spiering, Z. Zhuang, S. J. Benkovic and V. Croquette
Nature Chemical Biology - 5(12) :904–12 - DOI:10.1038/nchembio.236 - 2009
The unwinding and priming activities of the bacteriophage T4 primosome, which consists of a hexameric helicase (gp41) translocating 5' to 3' and an oligomeric primase (gp61) synthesizing primers 5' to 3', have been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a primosome disassembly mechanism or a new DNA looping mechanism. A fused gp61-gp41 primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms by disfavoring primosome disassembly, thereby increasing primase processivity. In contrast to T4, priming in bacteriophage T7 and Escherichia coli involves discrete pausing of the primosome and dissociation of the primase from the helicase, respectively. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.
Photomanipulation of a Droplet by the Chromocapillary Effect
Laboratoire Pôle Microfluidique - A. Diguet, R. M. Guillermic, N. Magome, A. Saint-Jalmes, Y. Chen, K. Yoshikawa and D. Baigl
Angew Chem Int Ed Engl. - 48(49) :9281-4 - DOI:10.1002/anie.200904868 - 2009
Ace of hearts: Liquid droplets can be manipulated in a controlled fashion along trajectories of any desired shape (such as a heart, see picture) by using light to create a wavelength-dependent interfacial tension gradient at a liquid/liquid interface. In this new phenomenon, the “chromocapillary effect”, an interfacial flow generates droplet motion in the direction opposite to the gradient.
Sequence-Independent and Reversible Photocontrol of Transcription/Expression Systems Using a Photosensitive Nucleic Acid Binder
Laboratoire Pôle Microfluidique - A. Estevez-Torres, C. Crozatier, A. Diguet, T. Hara, H. Saito, K. Yoshikawa and D. Baigl
Proc. Nat. Acad. Sci. USA - 106(30) :12219-23 - DOI:10.1073/pnas.0904382106 - 2009
To understand non-trivial biological functions, it is crucial to develop minimal synthetic models that capture their basic features. Here, we demonstrate a sequence-independent, reversible control of transcription and gene expression using a photosensitive nucleic acid binder (pNAB). By introducing a pNAB whose affinity for nucleic acids is tuned by light, in vitro RNA production, EGFP translation, and GFP expression (a set of reactions including both transcription and translation) were successfully inhibited in the dark and recovered after a short illumination at 365 nm. Our results indicate that the accessibility of the protein machinery to one or several nucleic acid binding sites can be efficiently regulated by changing the conformational/condensation state of the nucleic acid (DNA conformation or mRNA aggregation), thus regulating gene activity in an efficient, reversible, and sequence-independent manner. The possibility offered by our approach to use light to trigger various gene expression systems in a system-independent way opens interesting perspectives to study gene expression dynamics as well as to develop photocontrolled biotechnological procedures.
Wetting and spreading
Laboratoire Procédés - Plasmas - Microsystèmes - Bonn D., Eggers J., Iindekeu J., Meunier J., Rolley E.
REVIEWS OF MODERN PHYSICS - 81(2) :739-805 - DOI:10.1103/RevModPhys.81.739 - 2009
Wetting phenomena are ubiquitous in nature and technology. A solid substrate exposed to the environment is almost invariably covered by a layer of fluid material. In this review, the surface forces that lead to wetting are considered, and the equilibrium surface coverage of a substrate in contact with a drop of liquid. Depending on the nature of the surface forces involved, different scenarios for wetting phase transitions are possible; recent progress allows us to relate the critical exponents directly to the nature of the surface forces which lead to the different wetting scenarios. Thermal fluctuation effects, which can be greatly enhanced for wetting of geometrically or chemically structured substrates, and are much stronger in colloidal suspensions, modify the adsorption singularities. Macroscopic descriptions and microscopic theories have been developed to understand and predict wetting behavior relevant to microfluidics and nanofluidics applications. Then the dynamics of wetting is examined. A drop, placed on a substrate which it wets, spreads out to form a film. Conversely, a nonwetted substrate previously covered by a film dewets upon an appropriate change of system parameters. The hydrodynamics of both wetting and dewetting is influenced by the presence of the three-phase contact line separating ``wet'' regions from those that are either dry or covered by a microscopic film only. Recent theoretical, experimental, and numerical progress in the description of moving contact line dynamics are reviewed, and its relation to the thermodynamics of wetting is explored. In addition, recent progress on rough surfaces is surveyed. The anchoring of contact lines and contact angle hysteresis are explored resulting from surface inhomogeneities. Further, new ways to mold wetting characteristics according to technological constraints are discussed, for example, the use of patterned surfaces, surfactants, or complex fluids.
Development of oligonucleotide microarray involving plasma polymerized acrylic acid” Thin Solid Films
Laboratoire Procédés - Plasmas - Microsystèmes - R. Jafari , M. Tatoulian, D. Le Clerre, L. Talini, F. Richard, F. Arefi-Khonsari
Thin Solid Films - 517(19) :5763-8 - DOI:10.1016/j.tsf.2009.03.217 - 2009
This paper presents the manufacturing of biochips by using the COOH– derived polymer coating deposited by plasma polymerization of acrylic acid. This technology is based on depositing a thin layer obtained by plasma polymerization of acrylic acid which allows a further covalent immobilization of biomolecules on glass substrates. The plasma power value was optimized to maximize the stability of plasma polymerized acrylic acid (PPAA) coatings in water, which has a very important role for such applications. In order to obtain a covalent immobilization of DNA probes on the PPAA coated surface, the activation protocol of carboxylic function was carried out with the help of N-Hydroxy Succinimide and 1-Ethyl-3-(3-DimethylAminopropyl) Carbodiimide. The efficiency of PPAA coated in microarray applications was compared with two types of commercial slides. Such surfaces have shown very interesting results in terms of relative density of attached DNA probe molecules and signal-to-background ratio measured for target DNA hybridization. Nonspecific DNA bonding measurements showed only a small amount of nonspecific physisorption between the DNA probe and the PPAA-activated surfaces. This work shows that the plasma polymerization technique can be successfully applied to produce a high-quality glass surface for the manufacturing of DNA arrays.
Determination of Cocaine in Human Plasma by Selective Solid-Phase Extraction Using an Aptamer-Based Sorbent
Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation - B. Madru, F. Chapuis-Hugon, E. Peyrin, V. Pichon
Anal. Chem. - 81(16) :7081-6 - DOI:10.1021/ac9006667 - 2009
A complete characterization is presented of a highly selective solid-phase extraction (SPE) sorbent which exploits the properties of aptamers. An oligosorbent based on aptamers immobilized on a solid support was synthesized and tested for the selective extraction of cocaine from human plasma. Anticocaine aptamers were immobilized to CNBr-activated Sepharose, and an extraction procedure was developed in pure media. Specific retention of cocaine on the oligosorbent was demonstrated, and the capacity of the support was determined. This oligosorbent was then applied to the selective extraction of cocaine from plasma at a concentration of 0.4 mg L(-1), i.e., corresponding to the plasma concentration reached after an intake of a single dose of cocaine. Extraction recovery close to 90% was obtained. Moreover, interfering compounds that perturbed cocaine quantification when using a standard SPE sorbent were not retained on the oligosorbent, thus allowing fast and reliable analyses of plasma samples with an estimated limit of detection of 0.1 microg mL(-1).
Charge-based characterization of nanometric cationic bifunctional maghemite/silica core/shell particles by capillary zone electrophoresis
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - d'Orlye F, Varenne A, Georgelin T, Siaugue JM, Teste B, Descroix S, Gareil P
Electrophoresis - 30(14) :2572-82 - DOI:10.1002/elps.200800835. - 2009
In view of employing functionalized nanoparticles (NPs) in the context of an immunodiagnostic, aminated maghemite/silica core/shell particles were synthesized so as to be further coated with an antibody or an antigen via the amino groups at their surface. Different functionalization rates were obtained by coating these maghemite/silica core/shell particles with 3-(aminopropyl)triethoxysilane and 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane at different molar ratios. Adequate analytical performances with CE coupled with UV-visible detection were obtained through semi-permanent capillary coating with didodecyldimethyl-ammonium bromide, thus preventing particle adsorption. First, the influence of experimental conditions such as electric field strength, injected particle amount as well as electrolyte ionic strength and pH, was evaluated. A charge-dependent electrophoretic mobility was evidenced and the separation selectivity was tuned according to electrolyte ionic strength and pH. The best resolutions were obtained at pH 8.0, high ionic strength (ca. 100 mM), and low total particle volume fraction (ca. 0.055%), thus eliminating interference effects between different particle populations in mixtures. A protocol derived from Kaiser's original description was performed for quantitation of the primary amino groups attached onto the NP surface. Thereafter a correlation between particle electrophoretic mobility and the density of amino groups at their surface was established. Eventually, CE proved to be an easy, fast, and reliable method for the determination of NP effective surface charge density.
Droplet-based microfluidic platforms for the encapsulation and screening of mammalian cells and multicellular organisms
Laboratoire Biochimie - J. Clausell-Tormos, D. Lieber, J.-C. Baret, A. El-Harrak, O.J. Miller, L. Frenz, J. Blouwolff, K.J. Humphry, S. Köster, H. Duan, C. Holtze, D.A. Weitz, A.D. Griffiths and C.A. Merten
Chem. Biol. - 15(5) :427-37 - DOI: 10.1016/j.chembiol.2008.04.004 - 2008
High-throughput, cell-based assays require small sample volumes to reduce assay costs and to allow for rapid sample manipulation. However, further miniaturization of conventional microtiter plate technology is problematic due to evaporation and capillary action. To overcome these limitations, we describe droplet-based microfluidic platforms in which cells are grown in aqueous microcompartments separated by an inert perfluorocarbon carrier oil. Synthesis of biocompatible surfactants and identification of gas-permeable storage systems allowed human cells, and even a multicellular organism (C. elegans), to survive and proliferate within the microcompartments for several days. Microcompartments containing single cells could be reinjected into a microfluidic device after incubation to measure expression of a reporter gene. This should open the way for high-throughput, cell-based screening that can use >1000-fold smaller assay volumes and has approximately 500x higher throughput than conventional microtiter plate assays.
Droplet-based microreactors for the synthesis of magnetic iron oxide nanoparticles. Angewandte
Laboratoire Biochimie - L. Frenz, A. El Harrak, M. Pauly, S. Bégin-Colin, A.D. Griffiths and J.-C. Baret
Chimie Int. Ed. - 47(36) :6817-20 - DOI: 10.1002/anie.200801360 - 2008
Microdroplets for nanoparticles: An extremely reliable method to create droplet pairs, based on hydrodynamic coupling of two spatially separated nozzles, has been developed. Droplets containing the reagents for the precipitation of iron oxide are electrocoalesced to synthesize iron oxide nanoparticles in a very fast (millisecond-scale) and reproducible reaction (see picture).
Flourescence-activated droplet sorting (FADS): efficient microfluidic cell sorting based on enzymatic activity
Laboratoire Biochimie - J.-C. Baret, O.J. Miller, V. Taly, M. Ryckelynck, A. El-Harrak, L. Frenz, C. Rick, M.L. Samuels, J. B. Hutchison, J.J. Agresti, D.R. Link, D.A. Weitz and A.D. Griffiths
Lab. Chip - 9 :1850-8 - DOI: 10.1039/B902504A - 2008
We describe a highly efficient microfluidic fluorescence-activated droplet sorter (FADS) combining many of the advantages of microtitre-plate screening and traditional fluorescence-activated cell sorting (FACS). Single cells are compartmentalized in emulsion droplets, which can be sorted using dielectrophoresis in a fluorescence-activated manner (as in FACS) at rates up to 2000 droplets s−1. To validate the system, mixtures of E. coli cells, expressing either the reporter enzyme β-galactosidase or an inactive variant, were compartmentalized with a fluorogenic substrate and sorted at rates of 300 droplets s−1. The false positive error rate of the sorter at this throughput was <1 in 104 droplets. Analysis of the sorted cells revealed that the primary limit to enrichment was the co-encapsulation of E. coli cells, not sorting errors: a theoretical model based on the Poisson distribution accurately predicted the observed enrichment values using the starting cell density (cells per droplet) and the ratio of active to inactive cells. When the cells were encapsulated at low density ( 1 cell for every 50 droplets), sorting was very efficient and all of the recovered cells were the active strain. In addition, single active droplets were sorted and cells were successfully recovered.

391 publications.