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Total on-line analysis of a target protein from plasma by immunoextraction, digestion and liquid chromatography-mass spectrometry
A. Cingöz, F. Hugon-Chapuis, V. Pichon
J. Chrom. B - 878(2) :213-21 - DOI:10.1016/j.jchromb.2009.07.032 - 2010
A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion followed by LC-MS/MS analysis. For the development of this device, cytochrome c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome c from 8.5pmol to 1.7nmol in buffer medium. Finally, this device was applied to the analysis of only 85pmol of cytochrome c from plasma with a RSD value lower than 10% (n=3).
Supercritical fluid chromatography hyphenated with twin comprehensive two-dimensional gas chromatography for ultimate analysis of middle distillates
F. Adam, D. Thiébaut, F. Bertoncini, M. Courtiade, M.C. Hennion
J. Chrom. A - 1217(8) :1386-94 - DOI:10.1016/j.chroma.2009.11.092 - 2010
This paper reports the conditions of online hyphenation of supercritical fluid chromatography (SFC) with twin comprehensive two-dimensional gas chromatography (twin-GCxGC) for detailed characterization of middle distillates; this is essential for a better understanding of reactions involved in refining processes. In this configuration, saturated and unsaturated compounds that have been fractionated by SFC are transferred on two different GC x GC columns sets (twin-GCxGC) placed in the same GC oven. Cryogenic focusing is used for transfer of fractions into the first dimension columns before simultaneous GCxGC analysis of both saturated and unsaturated fractions. The benefits of SFC-twin-GC x GC are demonstrated for the extended alkane, iso-alkane, alkene, naphthenes and aromatics analysis (so-called PIONA analysis) of diesel samples which can be achieved in one single injection. For that purpose, saturated and unsaturated compounds have been separated by SFC using a silver loaded silica column prior to GC x GC analysis. Alkenes and naphthenes are quantitatively recovered in the unsaturated and saturated fractions, respectively, allowing their identification in various diesel samples. Thus, resolution between each class of compounds is significantly improved compared to a single GCxGC run, and for the first time, an extended PIONA analysis of diesel samples is presented.
Indirect Grafting of Acetonitrile-Derived Films on Metallic Substrates
A. Berisha, C. Combellas, F. Kanoufi, J. Pinson, S. Ustaze, F.I. Podvorica
Chem. Mater - 22(9) :2962-9 - DOI:10.1021/cm100295n - 2010
Strongly bonded organic films with amino groups are obtained on gold, copper, and silicon surfaces by reduction of 2,6-dimethyl benzenediazonium in acetonitrile (ACN). The sterically hindered 2,6-dimethylphenyl radical is unable to attach to the surface, but it abstracts an hydrogen atom from ACN to give the cyanomethyl radical (·CH2CN) that reacts with the surface. A spontaneous reaction is also possible on copper. The film is characterized by IR spectroscopy, scanning electron microscopy, ellipsometry, water contact angles, and cyclic voltammetry. A mechanism is elaborated that accounts for the formation, grafting of the cyanomethyl radical, and finally formation of amino multilayers.
Ultra-high-throughput screening in drop-based microfluidics for directed evolution of peroxidases
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
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
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
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
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
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
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.

410 publications.