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Fabricación y caracterización de nanobiohíbridos: Optimización de la interfaz entre nanoestructuras de oro y proteínas para posibles aplicaciones biosensoras
No full textTesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Pública de Navarra.--2017-09-08.--Sobresaliente cum laudem[ES] En las últimas décadas, la investigación en el campo de los nanomateriales se ha desarrollado considerablemente, debido a las potenciales aplicaciones de estos materiales en muy diversos campos de la ciencia. En muchas de estas aplicaciones los nanomateriales se combinan con biomoléculas para formar nuevas estructuras híbridas. Estos nanobiohíbridos (NBH), presenta nuevas propiedades y funciones, pues de la combinación de ambos, el nanomaterial y las biomoléculas, origina respuestas totalmente diferentes, lo cual incrementa el rango de aplicaciones. Sin embargo, conseguir interfaces funcionales entre proteínas y superficies presenta grandes desafíos. La estructura y función de las proteínas están fuertemente influenciadas por el material, el tamaño, la forma y por la química superficial, que puede ser altamente variable. Por lo tanto, la comprensión de los mecanismos implicados en la interacción de sistemas biológicos con nanomateriales es de interés tanto en las disciplinas fundamentales como en las aplicadas. El contenido de esta tesis está principalmente enfocado en llevar a cabo una eficiente inmovilización de proteínas en nanoestructuras de oro (nanopartículas de oro y superficies nanoestructuradas periódicas de oro), de manera que una vez unido al nanomaterial, éstas conserven su configuración funcional, y así poder entender los mecanismos relacionados con la interacción entre proteínas y nanoestructuras de oro. Para ello, la tesis se ha dividido en dos grandes bloques. En el primero de ellos se ha descrito el desarrollo de métodos de biofuncionalización de nanopartículas de oro con enzimas. Así, se ha estudiado la biofuncionalización de nanopartículas de oro de 9,6 nm de diámetro con la superóxido dismutasa de hierro, y la glucosa oxidasa, con el objetivo de optimizar la actividad y estructura de las enzimas en las nanopartículas. Se ha explorado el comportamiento de las proteínas en función de la química superficial de la nanopartícula, del microambiente, del uso de aditivos proteicos estabilizadores, y del método de unión (adsorción física y unión covalente). Se ha conjugado la proteína recombinante superóxido-dismutasa de hierro (rVuFeSOD) de caupí (Vigna unguiculata) a Au NPs covalentemente y electrostáticamente, utilizando ácido mercaptoundecanóico como nexo de unión. Se ha determinado el efecto de la concentración del ligando en la cobertura y la actividad de la proteína, produciendo un efecto contrario dependiendo del método de unión. Así, se ha comprobado que en la adsorción física, la concentración de ligando tiene un efecto más pronunciado en la cobertura que en la actividad mientras que en la unión covalente tiene un efecto más negativo en la actividad, llegando a producir la desnaturalización de la proteína debido a un exceso de puntos de unión con la superficie. Para este particular sistema, la adsorción física ha resultado un mejor método de unión. Con el objetivo de profundizar más en la adsorción física, la glucosa oxidasa se ha conjugado a nanopartículas de oro. Esta vez se ha caracterizado tanto la cobertura, la actividad enzimática, así como la estructura secundaria de la proteína una vez inmovilizada en la superficie de la NP. Se ha comprobado que las interacciones con las nanopartículas de oro pueden alterar significativamente la estructura y la función de la glucosa oxidasa. Las nanopartículas, pueden mejorar la unión del sustrato, al tiempo que reducen la actividad catalítica, indicando que los efectos estéricos no son tan importantes como la propia desnaturalización. La modificación del microambiente de la proteína mediante la adición de NaCl y glucosa mejora la actividad y el plegamiento. La glucosa es capaz de estabilizar el plegado de la proteína, pero no da lugar a grandes mejoras en la actividad, y el NaCl mejora la actividad, pero no la estructura secundaria. Esto significa que al planificar la inmovilización, es necesario investigar tanto la estructura como la actividad de la enzima, ya que no siempre se correlacionan. El segundo bloque del trabajo se ha centrado en la inmovilización covalente de anticuerpos monoclonales anti-caseína a superficies periódicas de nanodiscos de oro, para la construcción de un inmunosensor óptico basado en el fenómeno de la resonancia del plasmón superficial localizado (LSPR). La biofuncionalización de la superficie sensora es un paso clave en el desarrollo de un inmunosensor, ya que determina la especificidad, sensibilidad, reproducibilidad y estabilidad del mismo. Como paso previo a la biofuncionalización se ha realizado la caracterización de la respuesta óptica de obleas formadas por nanoestructuras de líneas y discos con un espectrofotómetro de laboratorio, siendo los nanodiscos 2,6 veces más sensibles al trabajar a una longitud de onda constante (6,7 Abs∙RIU-1). Para terminar, se ha desarrollado un protocolo de medida de caseína. El anticuerpo se ha unido covalentemente a la superficie a través de la química EDC/NHS. La optimización de proceso de inmovilización ha consistido en el estudio sistemático de parámetros tales como la composición de la SAM, diferentes soluciones tampón, la concentración de anticuerpo en la superficie, y el flujo de inmovilización. Asimismo se ha optimizado el proceso de unión antígeno-anticuerpo. Para ello se han estudiado el reconocimiento en función de la solución tampón, flujo de trabajo, solución de regeneración, y unión inespecífica. Se han reutilizado los chips durante más de 10 días llegando a realizar más de 40 ciclos de unión y regeneración con muy poca pérdida de señal, demostrando la robustez del método. Se ha obtenido un límite de detección de 0,5 μg∙mL-1 y un rango dinámico de 1-10 μg∙mL-1. La reproducibilidad del método queda probada por los bajos coeficientes de variación intra- e inter-ensayo.[EN] The research on nanomaterials has experienced, in the past years, a growing interest due to their potential applications in different industrial sectors and, in particular, in the field of biomedicine. The combination of nanomaterials and biomolecules to form new hybrid structures called nanobiohybirids (NBH) is considered a new strategy adopted to reach completely new properties and functions, extending the range of application of such structures. However, achieving functional interfaces between proteins and surfaces is not straightforward and requires extensive optimization and validation to control the structure and function of proteins linked to the nanomaterials which are strongly influenced by its size, shape and surface chemistry. Therefore, the understanding of the mechanisms involved in the interaction of biological systems with nanomaterials is of great interest in both the fundamental and applied disciplines. The content of this thesis is mainly focused on the development of an efficient immobilization of proteins on gold nanostructures (gold nanoparticles and periodic nanostructured surfaces). Once the protein is attached to the nanomaterial, retains its functional configuration and, hence, the mechanisms related to the interaction between proteins and gold nanostructures can be elucidated. The thesis has been divided into two main parts: In the first one, the development of biofunctionalisation methods of gold nanoparticles with enzymes is described. The second one relates to the optimization of covalent immobilization of anti-casein monoclonal antibodies to periodic surfaces of gold nanodiscs, for the development of an optical immunosensor based on localized superficial plasmon resonance (LSPR).Regarding the first part of the thesis, the biofunctionalisation of 9.6 nm diameter gold nanoparticles with iron superoxide dismutase and glucose oxidase has been studied in order to optimize the activity and structure of the enzymes on the nanoparticles. The behavior of proteins as a function of the nanoparticle surface chemistry, protein microenvironment, protein additives, and the binding method (physical adsorption and covalent bonding) have been explored. Recombinant iron superoxide dismutase (rVuFeSOD) protein from cowpea (Vigna unguiculata) has been conjugated to AuNPs covalently and electrostatically, using mercaptoundecanoic acid as a ligand. The effect of the concentration of the ligand on the coverage and activity of the protein has been determined, inducing an opposite effect depending on the binding method. Thus, it has been found that in physical adsorption, the concentration of the ligand has a more pronounced effect in the coverage than in the activity whereas in the covalent union has a more negative effect on the activity, leading to the denaturation of the protein due to an excess of attachment points to the surface. For this particular system, physical adsorption has resulted in a better binding method.
In order to gain more insight regarding the physical adsorption, glucose oxidase was conjugated to gold nanoparticles, following by the characterization of the coverage, enzymatic activity, and protein secondary structure onto AuNP. It has been shown that interactions with gold nanoparticles can significantly alter the structure and function of glucose oxidase. Nanoparticles can improve substrate binding while reducing catalytic turnover, indicating that steric effects are not as much of an issue as is denaturation. Modifying the protein microenvironment by addition of salt and the substrate glucose can help improve activity and folding. Incubation with its substrate glucose helps stabilize secondary structure, but does not result in major improvements in activity, and NaCl improves activity but not results in large improvements in the secondary structure. This means that when immobilization is planned, both structure and activity of the enzyme must be properly assessed, as they may not always be correlated.
The second part of work has focused on the covalent immobilization of anti-casein monoclonal antibodies to periodic surfaces of gold nanodiscs, for the construction of an optical immunosensor based on LSPR. The biofunctionalisation of the sensor is a key step in the development of an immunosensor, since it determines the specificity, sensitivity, reproducibility and stability of the system. As a preliminary step to the biofunctionalization, the characterization of the optical response of a wafer formed by nanostructures of lines and discs was performed by considering a laboratory spectrophotometer, being the nanodiscs configuration 2.6 times more sensitive when the system is working at a constant wavelength (6,7 Abs∙RIU-1).
Finally, a protocol for measuring casein was developed. The antibody has been covalently attached to the surface through the EDC / NHS chemistry. The optimization of the immobilization process was based on the systematic study of parameters such as the composition of the SAM, different buffer solutions, the concentration of antibody at the surface, and the immobilization flow. Additionally, the antigen-antibody binding process has also been optimized. For this purpose, recognition was monitored in terms of buffer solution, workflow, regeneration solution, and non-specific binding. The chips have been reused for more than 10 days, with more than 40 bonding and regeneration cycles with very little signal loss, demonstrating the robustness of the method. A detection limit of 0.5 g∙mL-1 and a dynamic range of 1-10 g∙mL-1 were obtained. The reproducibility of the method is proven by the low variation coefficients of intra- and inter-assay.Esta tesis ha sido financiada en gran parte por el Departamento de Innovación, Empresa y Empleo del Gobierno de Navarra a través de los siguientes proyectos de investigación, desarrollo e innovación y becas: SANBioNS: “Self assembly of nanoparticles and biomolecules on nanopatterned surfaces for catalytic and biochemical applications” (IIQ011902.RI1); SABioD: “Self Assembled Bio-Active Devices (IIQ14076.RI1); AGROSENSOR: “Biosensor LSPR para la industria alimentaria”; Beca Jeronimo de Ayanz 485/2010Peer reviewe
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Variations on the Author
Get PDF“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
Get PDFWe 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
Get PDFWe 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
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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