Université PSL

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Feasibility of ultra high performance supercritical neat carbon dioxide. chromatography at conventional pressures
Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation - C. Sarazin, D. Thiebaut, P. Sassiat,J. Vial
J. Sep. Sci. - 34(19) :2773-8 - DOI:10.1002/jssc.201100332 - 2011
The implementation of columns packed with sub-2 µm particles in supercritical fluid chromatography (SFC) is described using neat carbon dioxide as the mobile phase. A conventional supercritical fluid chromatograph was slightly modified to reduce extra column band broadening. Performances of a column packed with 1.8 µm C18-bonded silica particles in SFC using neat carbon dioxide as the mobile phase were compared with results obtained in ultra high performance liquid chromatography (UHPLC) using a dedicated chromatograph. As expected and usual in SFC, higher linear velocities than in UHPLC must be applied in order to reach optimal efficiency owing to higher diffusion coefficient of solutes in the mobile phase; similar numbers of theoretical plates were obtained with both techniques. Very fast separations of hydrocarbons are presented using two different alkyl-bonded silica columns.
Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte
Laboratoire Spectrométrie de masse biologique et protéomique - Kanhoush, R; Praseuth, D; Perrin, C; Chardard, D; Vinh, J; Penrad-Mobayed, M.
Nucleic Acids Res. - 39(10) :4109-21 - DOI:10.1093/nar/gkq1315 - 2011
A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.
T Cells Recognizing a Peptide Contaminant Undetectable by Mass Spectrometry
Laboratoire Spectrométrie de masse biologique et protéomique - Brezar V, Culina S, Osterbye T, Guillonneau F, Chiappetta G, Verdier Y, Vinh J, Wong FS, Buus S, Mallone R.
PLoS One - 6(12) :e28866 - DOI:10.1371/journal.pone.0028866 - 2011
Synthetic peptides are widely used in immunological research as epitopes to stimulate their cognate T cells. These preparations are never completely pure, but trace contaminants are commonly revealed by mass spectrometry quality controls. In an effort to characterize novel major histocompatibility complex (MHC) Class I-restricted ß-cell epitopes in non-obese diabetic (NOD) mice, we identified islet-infiltrating CD8+ T cells recognizing a contaminating peptide. The amount of this contaminant was so small to be undetectable by direct mass spectrometry. Only after concentration by liquid chromatography, we observed a mass peak corresponding to an immunodominant islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214 epitope described in the literature. Generation of CD8+ T-cell clones recognizing IGRP206-214 using a novel method confirmed the identity of the contaminant, further underlining the immunodominance of IGRP206-214. If left undetected, minute impurities in synthetic peptide preparations may thus give spurious results.
Reversed-phase HPLC and hyphenated analytical strategies for peptidomics
Laboratoire Spectrométrie de masse biologique et protéomique - Hesse AM, Ndiaye S, Vinh J.
Methods Mol Biol. - 789 :203-21 - DOI:10.1007/978-1-61779-310-3_13. - 2011
Peptide study and analysis widely involve liquid chromatography. Among the different strategies available, reversed-phase liquid chromatography (RP-HPLC) is one of the methods of choice to separate species in a nontargeted approach. The compounds are sorted according to their hydrophobicity, even though the experimental order of elution could change according to the nature of the mobile phase and the stationary phase. In our work, we have developed protocols to resolve hundred of peptidic species. To overcome the limitations of peak capacity of RP-HPLC alone, it has been coupled downstream to tandem mass spectrometry using two different ionization modes. To overcome the limitations of peak capacity of RP-HPLC MS/MS, it has been coupled upstream to strong cation exchange liquid chromatography. Multidimensional analysis allows for a deeper description of a sample because the limit of detection is often due to a lack of dynamic range of the detection itself rather than due to a lack of sensitivity. In this chapter, different protocols are presented. They should be considered as examples that could be used as starting point for new protocols optimization. Even if RP-HPLC is a universal peptide separation method, it should be optimized according to the specific characteristics of the peptide(s) of interest.
Microchip integrating magnetic nanoparticles for allergy diagnosis
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - 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)).
On-chip multi-electrochemical sensor array platform for simultaneous screening of nitric oxide and peroxynitrite
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Quinton D., Girard A., Kim LTT., Raimbault V., Griscom L., Razan F., Griveau S., Bedioui F.
Lab. Chip - 11 :1342-50 - DOI:10.1039/c0lc00585a - 2011
In this work we report on the design, microfabrication and analytical performances of a new electrochemical sensor array (ESA) which allows for the first time the simultaneous amperometric detection of nitric oxide (NO) and peroxynitrite (ONOO(-)), two biologically relevant molecules. The on-chip device includes individually addressable sets of gold ultramicroelectrodes (UMEs) of 50 µm diameter, Ag/AgCl reference electrode and gold counter electrode. The electrodes are separated into two groups; each has one reference electrode, one counter electrode and 110 UMEs specifically tailored to detect a specific analyte. The ESA is incorporated on a custom interface with a cell culture well and spring contact pins that can be easily interconnected to an external multichannel potentiostat. Each UME of the network dedicated to the detection of NO is electrochemically modified by electrodepositing thin layers of poly(eugenol) and poly(phenol). The detection of NO is performed amperometrically at 0.8 V vs. Ag/AgCl in phosphate buffer solution (PBS, pH = 7.4) and other buffers adapted to biological cell culture, using a NO-donor. The network of UMEs dedicated to the detection of ONOO(-) is used without further chemical modification of the surface and the uncoated gold electrodes operate at -0.1 V vs. Ag/AgCl to detect the reduction of ONOOH in PBS. The selectivity issue of both sensors against major biologically relevant interfering analytes is examined. Simultaneous detection of NO and ONOO(-) in PBS is also achieved.
Magnetic core shell nanoparticles trapping in a microdevice generating high magnetic gradient
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Teste B, Malloggi F, Gassner AL, Georgelin T, Siaugue JM, Varenne A, Girault H, Descroix S.
Lab. Chip - 11(5) :833-40 - DOI:10.1039/c0lc00510j - 2011
Magnetic core shell nanoparticles (MCSNPs) 30 nm diameter with a magnetic weight of 10% are usually much too small to be trapped in microfluidic systems using classical external magnets. Here, a simple microchip for efficient MCSNPs trapping and release is presented. It comprises a bed of micrometric iron beads (6-8 µm diameter) packed in a microchannel against a physical restriction and presenting a low dead volume of 0.8 nL. These beads of high magnetic permeability are used to focus magnetic field lines from an external permanent magnet and generate local high magnetic gradients. The nanoparticles magnetic trap has been characterised both by numerical simulations and fluorescent MCSNPs imaging. Numerical simulations have been performed to map both the magnetic flux density and the magnetic force, and showed that MCSNPs are preferentially trapped at the iron bead magnetic poles where the magnetic force is increased by 3 orders of magnitude. The trapping efficiency was experimentally determined using fluorescent MCSNPs for different flow rates, different iron beads and permanent magnet positions. At a flow rate of 100 µL h(-1), the nanoparticles trapping/release can be achieved within 20 s with a preconcentration factor of 4000.
Determination of binding parameters between lysozyme and its aptamer by frontal analysis continuous microchip electrophoresis (FACMCE)
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Girardot M, Li HY, Descroix S, Varenne A.
J. Chrom. A - 1218 :4052-8 - DOI:10.1016/j.chroma.2011.04.077 - 2011
An original and simple methodology based on microchip electrophoresis (MCE) in a continuous frontal analysis mode (named frontal analysis continuous microchip electrophoresis, FACMCE) was developed for the simultaneous determination of the binding parameters, i.e. ligand-site dissociation constant (k(d)) and number of binding sites on the substrate (n). This simultaneous determination was exemplified with the interaction between an aptamer and its target. The selected target is a strongly basic protein, lysozyme, as its quantification is of great interest due to its antimicrobial and allergenic properties. A glass microdevice equipped with a fluorescence detection system was coated with hydroxypropylcellulose, reducing the electroosmotic flow and adsorption onto the channel walls. This microdevice allowed the continuous electrokinetic injection of a mixture of fluorescently labelled aptamer and non-labelled lysozyme. By determining the concentration of the free fluorescently labelled aptamer thanks to its corresponding plateau height, mathematical linearization methods allowed to determine a k(d) value of 48.4±8.0 nM, consistent with reported results (31 nM), while the average number of binding sites n on lysozyme, never determined before, was 0.16±0.03. These results seem to indicate that the buffer nature and the SELEX process should influence the number and affinity of the binding sites. In parallel it has been shown that the binding between lysozyme and its aptamer presents two sites of different binding affinities.
Microelectrochemical patterning of gold surfaces using 4-azidobenzenediazonium and scanning electrochemical microscopy
Laboratoire Synthèse Electrochimie Imagerie et Systèmes Analytiques - Coates M., Cabet E., Griveau S., Nyokong T., Bedioui F.
Electrochemistry Communications - 13 :150-3 - DOI:10.1016/j.elecom.2010.11.037 - 2011
This work describes for the first time the possibility of performing local micro electrochemical grafting of a gold substrate by 4-azidobenzenediazonium by SECM in a single and simple one step without complications from adsorption. The electrografted spots of diazonium were performed by positioning a Pt tip at a given distance above the gold substrate and the SECM was used in a three-electrode configuration (the Pt tip serving as the microanode) in acetonitrile containing 5 mM 4-azidobenzenediazonium and 0.1 M Bu4NBF4 during 10 ms. The dimensions of the derivatized areas of the substrates were finely tuned by using different experimental conditions (tip distance above the substrate, tip diameter, presence or absence of supporting electrolyte). The use of the azido-derivated diazonium molecule and these preliminary results open the gate to important applications and developments devoted to the local micro functionalization of electrodes by thin layers that allow the implementation of the emerging and attractive interfacial click reaction.
Ultra-high-throughput screening in drop-based microfluidics for directed evolution of peroxidases
Laboratoire Biochimie - J.J. Agresti, E. Antipov, A.R. Abate, K. Ahn, A.C. Rowat, J.-C. Baret, M. Marquez, A.M. Klibanov, A.D. Griffiths, David A. Weitz
Proc. Nat. Acad. Sci. USA - 107(9) :4004-9 - DOI:10.1073/pnas.0910781107 - 2010
The explosive growth in our knowledge of genomes, proteomes, and metabolomes is driving ever-increasing fundamental understanding of the biochemistry of life, enabling qualitatively new studies of complex biological systems and their evolution. This knowledge also drives modern biotechnologies, such as molecular engineering and synthetic biology, which have enormous potential to address urgent problems, including developing potent new drugs and providing environmentally friendly energy. Many of these studies, however, are ultimately limited by their need for even-higher-throughput measurements of biochemical reactions. We present a general ultrahigh-throughput screening platform using drop-based microfluidics that overcomes these limitations and revolutionizes both the scale and speed of screening. We use aqueous drops dispersed in oil as picoliter-volume reaction vessels and screen them at rates of thousands per second. To demonstrate its power, we apply the system to directed evolution, identifying new mutants of the enzyme horseradish peroxidase exhibiting catalytic rates more than 10 times faster than their parent, which is already a very efficient enzyme. We exploit the ultrahigh throughput to use an initial purifying selection that removes inactive mutants; we identify ∼100 variants comparable in activity to the parent from an initial population of ∼107. After a second generation of mutagenesis and high-stringency screening, we identify several significantly improved mutants, some approaching diffusion-limited efficiency. In total, we screen ∼108 individual enzyme reactions in only 10 h, using < 150 μL of total reagent volume; compared to state-of-the-art robotic screening systems, we perform the entire assay with a 1,000-fold increase in speed and a 1-million-fold reduction in cost.
Quantitative cell-based reporter gene assays using droplet-based microfluidics
Laboratoire Biochimie - J.-C. Baret, Y. Beck, I. Billas-Massobrio, D. Moras and A.D. Griffiths
Chem. Biol. - 17(5) :528–36 - DOI: 10.1016/j.chembiol.2010.04.010 - 2010
We used a droplet-based microfluidic system to perform a quantitative cell-based reporter gene assay for a nuclear receptor ligand. Single Bombyx mori cells are compartmentalized in nanoliter droplets which function as microreactors with a >1000-fold smaller volume than a microtiter-plate well, together with eight or ten discrete concentrations of 20-hydroxyecdysone, generated by on-chip dilution over 3 decades and encoded by a fluorescent label. The simultaneous measurement of the expression of green fluorescent protein by the reporter gene and of the fluorescent label allows construction of the dose-response profile of the hormone at the single-cell level. Screening approximately 7500 cells per concentration provides statistically relevant data that allow precise measurement of the EC(50) (70 nM +/- 12%, alpha = 0.05), in agreement with standard methods as well as with literature data.
Microfluidic Sorting and High Content Multimodal Typing of Cancer Cells in Self-Assembled Magnetic Arrays
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Saliba AE, Saias L, Psichari E, Minc N, Simon, D, Mathiot C, Bidard FC, Pierga JY,Fraisier V, Salamero J, Saada V, Farace F, Vielh P, Malaquin L, Viovy JL.
Proc. Nat. Acad. Sci. USA - 107(33) :14524–9 - DOI:10.1073/pnas.1001515107 - 2010
We propose a unique method for cell sorting, “Ephesia,” using columns of biofunctionalized superparamagnetic beads self-assembled in a microfluidic channel onto an array of magnetic traps prepared by microcontact printing. It combines the advantages of microfluidic cell sorting, notably the application of a well controlled, flow-activated interaction between cells and beads, and those of immunomagnetic sorting, notably the use of batch-prepared, well characterized antibody-bearing beads. On cell lines mixtures, we demonstrated a capture yield better than 94%, and the possibility to cultivate in situ the captured cells. A second series of experiments involved clinical samples—blood, pleural effusion, and fine needle aspirates— issued from healthy donors and patients with B-cell hematological malignant tumors (leukemia and lymphoma). The immunophenotype and morphology of B-lymphocytes were analyzed directly in the microfluidic chamber, and compared with conventional flow cytometry and visual cytology data, in a blind test. Immunophenotyping results using Ephesia were fully consistent with those obtained by flow cytometry. We obtained in situ high resolution confocal three-dimensional images of the cell nuclei, showing intranuclear details consistent with conventional cytological staining. Ephesia thus provides a powerful approach to cell capture and typing allowing fully automated high resolution and quantitative immunophenotyping and morphological analysis. It requires at least 10 times smaller sample volume and cell numbers than cytometry, potentially increasing the range of indications and the success rate of microbiopsy-based diagnosis, and reducing analysis time and cost.
Microchip electrophoresis profiling of AB peptides in the cerebrospinal fluid of patients with Alzheimer’s disease
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Mohamadi MR, Svobodova Z, Verpillot R, Esselmann H, Wiltfang J, Otto M, Taverna M, Bilkova Z, Viovy JL.
Anal. Chem. - 82(18) :7611-7 - DOI:10.1063/1.4722588 - 2010
The preferential aggregation of Aß1-42 in amyloid plaques is one of the major neuropathological events in Alzheimer's disease. This is accompanied by a relative reduction of the concentration of Aß1-42 in the cerebrospinal fluid (CSF) of patients developing the signs of Alzheimer's disease. Here, we describe a microchip gel electrophoresis method in polydimethylsiloxane (PDMS) chip that enables rapid profiling of major Aß peptides in cerebrospinal fluid. To control the electroosmotic flow (EOF) in the PDMS channel and also to reduce the adsorption of the peptides to the surface of the channel, a new double coating using poly(dimethylacrylamide-co-allyl glycidyl ether) (PDMA-AGE) and methylcellulose-Tween-20 was developed. With this method, separation of five synthetic Aß peptides (Aß1-37, Aß1-38, Aß1-39, Aß1-40, and Aß1-42) was achieved, and relative abundance of Aß1-42 to Aß1-37 could be calculated in different standard mixtures. We applied our method for profiling of Aß peptides in CSF samples from nonAlzheimer patients and patients with Alzheimer's disease. Aß peptides in the CSF samples were captured and concentrated using a microfluidic system in which magnetic beads coated with anti-Aß were self-organized into an affinity microcolumn under the a permanent magnetic field. Finally, we could detect two Aß peptides (Aß1-40 and Aß1-42) in the CSF samples.
Monodisperse Colloids Synthesized with Nanofluidic Technology
Laboratoire Microfluidique MEMS et nanostructures - F. Malloggi, N. Pannacci, R. Attia, F. Monti, P. Mary, H. Villaime, P. Tabeling, B. Cabane, P. Poncet
Langmuir - 26(4) :2369-73 - DOI:10.1021/la9028047 - 2010
Limitations in the methods employed to generate micrometric colloidal droplets hinder the emergence of key applications in the fields of material science and drug delivery. Through the use of dedicated nanofluidic devices and by taking advantage of an original physical effect called capillary focusing, we could circumvent some of these limitations. The nanofluidic (i.e., submicrometric) devices introduced herein are made of soft materials, and their fabrication relies upon rapid technologies. The objects that we have generated are simple droplets, multiple droplets, particles, and Janus particles whose sizes lie between 900 nm and 3 µm (i.e., within the colloidal range). Colloidal droplets have been assembled on-chip into clusters and crystals, yielding discrete diffraction patterns. We illustrate potential applications in the field of drug delivery by demonstrating the ability of multiple droplets to be phagocytosed by murine macrophage-type cells.
Interfacially Driven Instability in the Microchannel Flow of a Shear-Banding Fluid
Laboratoire Microfluidique MEMS et nanostructures - P. Ngher, S. Fielding, A. Ajdari, P. Tabeling
Phys. Rev. Lett. - 104(24) :248303 - DOI:10.1103/PhysRevLett.104.248303 - 2010
Using microparticle image velocimetry, we resolve the spatial structure of the shear-banding flow of a wormlike micellar surfactant solution in a straight microchannel. We reveal an instability of the interface between the shear bands, associated with velocity modulations along the vorticity direction. We compare our results with a detailed theoretical study of the diffusive Johnson-Segalman model. The quantitative agreement obtained favors an instability scenario previously predicted theoretically but hitherto unobserved experimentally, driven by a normal stress jump across the interface between the bands.
Wettability Patterning by UV-initiated graft polymerization of PAA in microfluidic systems of complex geometries
Laboratoire Microfluidique MEMS et nanostructures - M. Schneider, H. Willaime, Y. Tran, F. Rezgui, P. Tabeling
Anal. Chem. - 82(21) :8848-55 - DOI:10.1021/ac101345m - 2010
Many microfluidic applications require modified surface wettability of the microchannels. Patterning of wettability within enclosed microfluidic structures at high spatial resolution has been challenging in the past. In this paper, we report an improved method for altering the surface wettability in poly(dimethylsiloxane) (PDMS) microchannels by UV-induced graft polymerization of poly(acrylic acid). Our method presents significant improvements in terms of wettability contrast and spatial resolution of the patterned structures as compared to recent literature and is in particular applicable to complex microfluidic structures with a broad range of channel sizes and aspect ratios. A key part of our work is the clear description of the surface treatment process with the identification of key parameters, some of which have been overlooked, neglected, or misinterpreted in previous works. We have studied these key parameters in detail and provide recommended values for each parameter supported by experimental results. This detailed understanding of the treatment process and the effects of the critical parameters on it allowed us to significantly improve quality and reliability of the treatment process.
Thermocapillary actuation by optimized resistor pattern: bubbles and droplets displacing, switching and trapping
Laboratoire Microfluidique MEMS et nanostructures - B. Selva, V. Miralles, I. Cantat, M. C Jullien
Lab. Chip - 10(14) :1835-40 - DOI:10.1039/c001900c - 2010
We report a novel method for bubble or droplet displacement, capture and switching within a bifurcation channel for applications in digital microfluidics based on the Marangoni effect, i.e. the appearance of thermocapillary tangential interface stresses stemming from local surface tension variations. The specificity of the reported actuation is that heating is provided by an optimized resistor pattern (B. Selva, J. Marchalot and M.-C. Jullien, An optimized resistor pattern for temperature gradient control in microfluidics, J. Micromech. Microeng., 2009, 19, 065002) leading to a constant temperature gradient along a microfluidic cavity. In this context, bubbles or droplets to be actuated entail a surface force originating from the thermal Marangoni effect. This actuator has been characterized (B. Selva, I. Cantat, and M.-C. Jullien, Migration of a bubble towards a higher surface tension under the effect of thermocapillary stress, preprint, 2009) and it was found that the bubble/droplet (called further element) is driven toward a high surface tension region, i.e. toward cold region, and the element velocity increases while decreasing the cavity thickness. Taking advantage of these properties three applications are presented: (1) element displacement, (2) element switching, detailed in a given range of working, in which elements are redirected towards a specific evacuation, (3) a system able to trap, and consequently stop on demand, the elements on an alveolus structure while the continuous phase is still flowing. The strength of this method lies in its simplicity: single layer system, in situ heating leading to a high level of integration, low power consumption (P < 0.4 W), low applied voltage (about 10 V), and finally this system is able to manipulate elements within a flow velocity up to 1 cm s(-1).
Force fluctuations assist nanopore unzipping of DNA
Laboratoire Nanobiophysiques - V Viasnoff, N Chiaruttini, J Muzard, and U Bockelmann
Journal of Physics : Condensed Matter - 22(45) :454122 - DOI:10.1088/0953-8984/22/45/454122 - 2010
We experimentally study the statistical distributions and the voltage dependence of the unzipping time of 45 base-pair-long double-stranded DNA through a nanopore. We then propose a quantitative theoretical description considering the nanopore unzipping process as a random walk of the opening fork through the DNA sequence energy landscape biased by a time-fluctuating force. To achieve quantitative agreement fluctuations need to be correlated over the millisecond range and have an amplitude of order kBT/bp. Significantly slower or faster fluctuations are not appropriate, suggesting that the unzipping process is efficiently enhanced by noise in the kHz range. We further show that the unzipping time of short 15 base-pair hairpins does not always increase with the global stability of the double helix and we theoretically study the role of DNA elasticity on the conversion of the electrical bias into a mechanical unzipping force.
DNA translocation and unzipping through a nanopore : some geometrical effects
Laboratoire Nanobiophysiques - J. Muzard, M. Martinho, J. Mathe, U. Bockelmann, and V. Viasnoff
Biophys. J. - 98(10) :2170–8 - DOI:10.1016/j.bpj.2010.01.041. - 2010
This article explores the role of some geometrical factors on the electrophoretically driven translocations of macromolecules through nanopores. In the case of asymmetric pores, we show how the entry requirements and the direction of translocation can modify the information content of the blocked ionic current as well as the transduction of the electrophoretic drive into a mechanical force. To address these effects we studied the translocation of single-stranded DNA through an asymmetric alpha-hemolysin pore. Depending on the direction of the translocation, we measure the capacity of the pore to discriminate between both DNA orientations. By unzipping DNA hairpins from both sides of the pores we show that the presence of single strand or double strand in the pore can be discriminated based on ionic current levels. We also show that the transduction of the electrophoretic drive into a denaturing mechanical force depends on the local geometry of the pore entrance. Eventually we discuss the application of this work to the measurement of energy barriers for DNA unzipping as well as for protein binding and unfolding.
Pre-Dispositions and Epigenetic Inheritance in the Escherichia Coli Lactose Operon Bistable Switch
Laboratoire Nanobioscience et Microsystèmes group - L. Robert, G. Paul, Y. Chen, F. Taddei, D. Baigl and A. B. Lindner
Molecular Systems Biology - 6 :357 - DOI:10.1038/msb.2010.12 - 2010
The lactose operon regulation in Escherichia coli is a primary model of phenotypic switching, reminiscent of cell fate determination in higher organisms. Under conditions of bistability, an isogenic cell population partitions into two subpopulations, with the operon's genes turned on or remaining off. It is generally hypothesized that the final state of a cell depends solely on stochastic fluctuations of the network's protein concentrations, particularly on bursts of lactose permease expression. Nevertheless, the mechanisms underlying the cell switching decision are not fully understood. We designed a microfluidic system to follow the formation of a transiently bimodal population within growing microcolonies. The analysis of genealogy and cell history revealed the existence of pre-disposing factors for switching that are epigenetically inherited. Both the pre-induction expression stochasticity of the lactose operon repressor LacI and the cellular growth rate are predictive factors of the cell's response upon induction, with low LacI concentration and slow growth correlating with higher switching probability. Thus, stochasticity at the local level of the network and global physiology are synergistically involved in cell response determination.

638 publications.