1,721,035 research outputs found
A VERSTAILE POLYMERIC COATING FOR MICROARRAYS USING COPPER-MEDIATED CLICK CHEMISTRY
Full text linkIn this thesis we introduce a method to functionalize the surface of glassy materials with alkynes using a polymer that produces a coating by a facile 'dip and rinse' method. The alkyne groups on the tri-dimensional surface can be subsequently linked to azide-containing carbohydrates using Cu-catalyzed azide/alkyne cycloaddition (CuAAC, click chemistry)1,2. The research is aimed at developing a new strategy to generate a polymer coating enabling the attachment of complex sugars via click chemistry by a method that does not require skilled personnel and chemistry laboratories. The proposed approach combines the advantages of high sensitivity and superior signal-to-noise ratio of a Si-SiO2 substrate with the quality of a 3D coating. The Si/SiO2 surface was used as the substrate to take advantage from the superior optical properties of this material. A novel polymer named poly (DMA-PMA-MAPS), obtained from the polymerization of N,N-dimethylacrylamide (DMA), 3-trimethylsilanyl-prop-2-yn methacrylate (PMA) and 3(trimethoxysilyl)-propylmethacrylate (MAPS) was synthesized and characterized. It consists of: 1) a segment of polydimethylacrylamide that interacts with the surface by weak, non covalent interactions such as hydrogen bonding, Van der Waals or hydrophobic forces, 2) a pending silane hydrolysable monomers that promote condensation of the polymer with surface silanols or between contiguous chains and 3) chemically active monomers whose reactivity is selected on the basis of the reactivity of the molecules that have to be immobilized.
The polymer reported herein is similar to another polymer developed in 2004 by Pirri et al.3 to form a coating on glass slides by a combination of physi- and chemi-sorption. The novelty of this work consists in the presence of an alkyne functional monomer that replaces the succinimide active ester.
By exploiting the presence of a stable coating that allows regio-specific and bio-orthogonal immobilization, a glycan microarray was built and its performance was deeply investigated. First, a qualitative assay (fluorescence analysis) was carried out to obtain a fast screening of the affinity of the interaction of nine glycomimetics with Concanavlin A. Second, thanks to the high-sensitivity of the Si/SiO2 platform used, a study of the influence of the multivalency presentation of glycans during lectin interaction was made. The high-performing substrate used allows a dramatic decrease of glycan surface densities (from 1,96•1014 down to 5,07•1012 molecules/cm2), offering the possibility to calculate and compare the avidity in different conditions by providing density dependent surface dissociation constants (KD,surf).
In a second different application, the new poly(DMA-PMA-MAPS) copolymer coating was used to functionalize microarray slides with orientated antibodies taking advantage from the regio-specific reaction between alkyne on the surface and azido groups on the biomolecule.
Inspired by the work of Zeglis et al.4 an enzymatic procedure was devised to obtain site-specific modified antibodies using an unnatural UDP-6-azidogalactose and two commercially available enzymes: β-(1,4)-galactosidase and β-(1,4)-galactosyltransferase. As the 6-azidogalactose is sterically less hindered, it is expected to display a higher reactivity in the surface immobilization process. The strategy adopted was, in part, mutated from the procedure reported by Bosco et al.5.
To validate the methodology and highlight its advantages, a sandwich microray test for the detection of interleukin-6 (IL-6) was developed.
Cytokines, a set of proteins implicated in the onset and development of almost every major life-threatening disease are amongst the most intensively studied biomarkers. They play a prominent role in cancer, neurodegenerative diseases, cardiovascular diseases, sepsis and many other pathologies6. Although enzyme-linked immunosorbent assay (ELISA) is the gold standard for the measurement of a single cytokine concentration, the key to successful identification of biomarkers is the simultaneous detection of multiple cytokines with high sensitivity. IL-6 was chosen as a model of a typical inflammatory biomarker, to demonstrate the senistivity provided by an oriented immobilization of the capturing antibody in a microarray based immunoassay.
Our site-specifically modified antibody was compared to both a randomly azido-pegylated antibody and a site-specifically modified antibody derivatized by a commercial Kit (Site-Click Antibody Labelling purchase from Life Technology), which makes use of an unnatural UDP-2-azidogalactose instead of UDP-6-azidogalactose, and of a mutant GalT (Y289L) instead of a commercially available GalT. Furthermore, through the use of the label-free sensing platform IRIS, we have correlated the efficiency of the Ab-antigen interaction given by the fluorescence signal with the mass of antibody immobilized per surface unit (ng/mm2). The fluorescence per mass unit allows to assess the importance the antibody orientation on its capturing ability. In particular its was demonstrated that a higher amount of immobilized probe does not necessary lead to a higher antibody-antigen interaction.
(1) H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem. Int. Ed., 2001, 40, 2004.
(2) C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem., 2002, 67, 3057.
(3) G. Pirri, F. Damin, M. Chiari, E. Bontempi, L. E. Depero, Anal. Chem., 2004, 76, 1352.
(4) B. M. Zeglis, C. B. Davis, R. Aggeler, H. C. Kang, A. Chen, B. J. Anew and J. S. Lewis, Bioconjugate Chem., 2013, 24, 1057.
(5) M. Bosco, S. Le Gall, C. Rihouey, S. Couve-Bonnaire, M. Bardol, P. Lerouge, X. Pannecoucke, Tethraedron Lett., 2008, 49, 2294.
(6) R.P. Huang, B. Burkholder, V.S. Jones, W.D. Jiang, Y.Q. Mao, Q.L. Chen, Z. Shi, Current Proteomics, 2012, 9, 55
Composition shift in vapour compression-dry expansion refrigerating plants operating with refrigerant mixtures
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Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Universal hydrophilic coating of thermoplastic polymers currently used in microfluidics
Full text linkA number of materials used to fabricate disposable microfluidic devices are hydrophobic in nature with water contact angles on their surface ranging from 80 to over 100. This characteristic makes them unsuitable for a number of microfluidic applications. Both the wettability and analyte adsorption parameters are highly dependent on the surface hydrophobicity. In this article, we propose a general method to coat the surface of five materials: polydimethylsiloxane (PDMS), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), polycarbonate (PC), and polytetrafluoroethylene (PTFE). This fast and robust process, which is easily implementable in any laboratory including microfabrication clean room facilities, was devised by combining gas-phase and wet chemical modification processes. Two different coatings that improve the surface hydrophilicity were prepared via the "dip and rinse" approach by immersing the plasma oxidized materials into an aqueous solution of two different poly(dimethylacrylamide) copolymers incorporating a silane moiety and functionalized with either N-acryloyloxysuccinimide (NAS) (poly(DMA-NAS-MAPS) or glycidyl methacrylate (GMA) (poly(DMA-GMA-MAPS). The coating formation was confirmed by contact angle (CA) analysis comparing the variation of CAs of uncoated and coated surfaces subjected to different aging treatments. The antifouling character of the polymer was demonstrated by fluorescence and interferometric detection of proteins adsorbed on the surafce. This method is of great interest in microfluidics due to its broad applicability to a number of materials with varying chemical compositions
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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