Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 95-99). Also available on the World Wide Web.

The use of traditional tools for the discovery and characterization of biological systems has resulted in a wealth of biological knowledge. Unfortunately, only a small portion of the biological world is well-understood to date, and the study of the rest remains a daunting task. This work involves using time-varying stimuli in order to more rapidly interrogate and characterize signaling pathways. The time-dependent stimulation of a signaling pathway can be used in conjunction with a model of the pathway to efficiently evaluate and test hypotheses. We are developing this technology using the yeast pheromone signal transduction pathway as a model system. The time-varying stimuli will be applied to the yeast cells via a novel microfluidic device, and the pathway output will be measured via various fluorescent reporters. The output of the pathway can then be compared to the output from a computational model of the pathway in order to test hypotheses and constrain our knowledge of the pathway. Initial work shows that a computational model can be used to identify stimuli time-courses that increase the parameter sensitivity, meaning that corresponding experiments could potentially be much more informative. / Poster presented at the 2005 ICSB meeting, held at Harvard Medical School in Boston, MA.

Thesis (M.S.)--University of Akron, Dept. of Mechanical Engineering, 2009. / "August, 2009." Title from electronic thesis title page (viewed 11/11/2009) Advisor, Alper Buldum; Co-Advisor, S. Graham Kelly; Faculty Reader, Fred Choy; Department Chair, Celal Batur; Dean of the College, George K. Haritos; Dean of the Graduate School, George R. Newkome. Includes bibliographical references.

Thesis (M.S.)--Oregon State University, 2009. / Printout. Includes bibliographical references (leaves 62-63). Also available on the World Wide Web.

La nucléation est l’étape est à l’origine de la cristallisation et c’est généralement elle qui va dicter les propriétés finales d’un cristal. Il est alors intéressant de comprendre les mécanismes qui gouvernent cette étape. Deux théories se confrontent aujourd’hui avec d’une part la théorie classique de la nucléation et d’autre part un modèle de nucléation en deux étapes. La théorie classique considère l’addition des monomères un par un pour former des clusters directement cristallins. Le modèle en deux étapes considère une première étape de concentration, au cours de laquelle les monomères vont former des agrégats denses, mais désordonnés, et une seconde étape de structuration qui permettra de réarranger les molécules au sein d’un cluster, pour obtenir une structure cristalline. Il semblerait que, dans le cas des protéines, le passage du mécanisme classique à celui en deux étapes se fasse par une augmentation des fluctuations de concentration en solution, ce qui se fait en se rapprochant de la spinodale de décomposition présente dans le diagramme de phase de certaines protéines. Ainsi, dans cette étude, nous développons des systèmes microfluidiques qui permettent d’étudier ces différents mécanismes. Dans un premier temps, nous avons conçus un dispositif utilisé pour déterminer des cinétiques de nucléation à l’aide de modèles probabilistes. Ensuite, nous avons adapté des systèmes microfluidiques à des grands équipements synchrotron, ce qui donne la possibilité de réaliser de la diffusion de rayons X aux petits angles. Ces deux approches complémentaires permettent alors d’obtenir des informations à l’échelle macroscopique et à l’échelle microscopique, et ainsi de participer à l’éclaircissement des zones d’ombre qui demeurent autour des mécanismes de nucléation. / Nucleation is the first step of crystallization and is the step that will give the final properties of crystals. It is therefore interesting to understand the mechanisms of this step. Nowadays, two theories exist : the classical nucleation theory and the two step theory. In the classical nucleation theory, monomers are added one by one to directly form crystalline clusters. In the two step theory, there is a first step of densification, in which monomers form dense amorphous aggregates and a second step of structuration in which aggregates arrange themselves to form crystalline clusters. It seems that for proteins, the two step mechanism is obtained by an increase of concentration fluctuations in solution, that happens when experimental conditions get closer to spinodal decomposition. In this study, experimental microfluidic devices are developped to study these mechanisms. Firstly, we developped microfluidic devices to determine nucleation kinetics with probabilistic models. Then, we coupled microfluidic devices with small angle X rays scattering from synchrotron sources. These two complementary studies give information on nucleation mechanisms on the macroscopic and microscopic scales.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / In research and industry, almost all chemical analysis methods involve the separation and detection of compounds. Typically, these separations are performed using traditional methods that require volumes in the 10 μL to 10 mL range of sample and in the 200 mL to 2 L range for solvents. These methods are not suitable for low-concentration, volume-limited samples frequently associated with biochemical studies. One way to overcome these limitations is to move the separation and detection to the microscale. The use of the microscale separation technologies enables the study of biological systems that have, until now, been out of reach due to their small volumes or low concentrations. The research presented in this dissertation will discuss two examples of this shift to microscale separation technologies which can solve some small volume sample challenges. These include the detection of protease activity in blood samples for use in cancer detection and the identification of immune system cascade proteins in the mosquito Anopheles gambiae. In Chapter 2 a microfluidic method and device is proposed to monitor protease activities for cancer detection. In this method nanobiosensors are used to measure enzyme activity in biological fluids. These nanobiosensors consist of iron-iron oxide magnetic nanoparticles that are attached to peptide substrates specific for proteases through a disulfide bond. The nanobiosensors are controlled using a neodymium magnet which is attached through a 3D printed adaptor to a rotating motor for mixing and a linear stage to move the nanoparticles between different sections of the device. The separation and detection sections of the device are explained in Chapter 3. Chapter 3 describes the fabrication and optimization of a simple device for microfluidic isoelectric focusing(IEF). IEF is a separation method in which analytes are separated based upon their isoelectric, i.e. neutral charge, points. A reducing agent can be added to the IEF buffer to detach the nanoparticle from the peptide substrate, releasing it for focusing. IEF is also a concentration as well as separation method that will allow the peptide substrates to be focused up to 10⁶ fold. It has a high peak capacity and produces reliable, reproducible separation patterns based on the isoelectric point of the peptide. To meet the detection limits required for cancer detection with proteases, scanning laser induced fluorescence is selected as the method of detection. This scanning system can monitor the separation over time to observe the parameters affecting the separation which cannot be done with typical point or imaging detection systems and allows better separation. This custom automatic detection system can distinguish focused samples of 500 fM from the background with minimal noise from the scanning system. In Chapter 4 the identification of serine protease and inhibitor binding complexes in A. gambiae hemolymph using magnetic bead immunoaffinity chromatography was attempted. These proteases play a key role in the insect innate immunity system and form irreversible complexes. These complexes can be purified from a complex hemolymph sample using an antibody to one of the complex members. To separate the complexes from the hemolymph, Serpin 2 antibodies were attached to protein A coated magnetic beads and then incubated with the hemolymph. Once the purified complexes and Serpin 2 were eluted, the purified proteases were identified on Orbitrap MS. In an attempt to simplify the isolation of the complexes, a magnetic bead mixing rotor column was developed to help reduce the volume of the elution to increase the concentration. This method, however, was not robust and did not improve the concentration.

Orientador: Fabiano Fruett / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-21T04:02:00Z (GMT). No. of bitstreams: 1 Ribeiro_LuizEduardoBento_M.pdf: 4022818 bytes, checksum: d2a40b9cee4f59bc80ec0b09a97c31a8 (MD5) Previous issue date: 2012 / Resumo: A integração de sistemas microeletrônicos em lab-on-a-chip está sendo cada vez mais necessária para concretizar novas aplicações dentro do emergente campo da microfluídica. Tanto na química quanto na bioquímica e até mesmo na medicina e bioengenharia, a microfluídica evolui conquistando um espaço crescente. Entretanto, desafios tecnológicos residem na sua complexa fabricação e integração com sistemas eletrônicos. Neste trabalho, foi desenvolvido um sistema sensor que emprega métodos de fabricação compatíveis tanto com a microeletrônica quanto com a microfluídica. Este sistema sensor é baseado em uma microponte de impedância composta por quatro capacitores interdigitados. Neste sistema, o fluido, guiado por um canal ou armazenado em um reservatório fabricado em polidimetilsiloxano (PDMS), passa sobre a microponte enquanto um termistor, fabricado no mesmo substrato, permite monitorar a temperatura do sistema durante a medida. A microponte é formada de eletrodos interdigitados arranjados de forma a permitir a utilização de um circuito eletrônico de condicionamento que pode ser construído bem próximo do elemento sensor. O trabalho foi validado comparando-se a função de transferência experimental do sensor, usando como analito a mistura etanol-água, com a função de transferência teórica obtida através de simulação baseada em elementos finitos. Identificamos a importância da deposição de um filme fino de boa qualidade para a proteção dos eletrodos de referência e sua influência na função de transferência experimental. Ainda, devido à utilização de materiais inertes como ouro, vidro e PDMS, o sistema sensor, com alguns ajustes, pode ser empregado para outras aplicações: desde o monitoramento da pureza e concentração de líquidos até a caracterização de filmes finos sensíveis a patógenos e fármacos / Abstract: The integration of microelectronic systems in lab-on-a-chip is being increasingly required to implement new applications on the emerging field of microfluidics. Both in chemistry and biochemistry, and even in medicine and bioengineering, microfluidics evolves gaining a growing space. However, technological challenges lie in its complex manufacturing and integration with electronic systems. In this work, we developed a sensor system that employs both fabrication methods compatible with microelectronics and with microfluidics. This sensor system is based on an impedance microbridge composed of four interdigitated capacitors. In this system, the fluid which is guided by a channel or is stored in a reservoir made of polydimethylsiloxane (PDMS), passes over the microbridge while a thermistor fabricated on the same substrate allows monitoring of the system temperature during the measurement. The microbridge is made of interdigitated electrodes arranged so as to allow the use of an electronic conditioning circuit that can be built very close to the sensor element. The study was validated by comparing experimental transfer function of the sensor, using the ethanol-water mixture as analyte, with the theoretical transfer function obtained by simulation based on finite element method. We identified the importance of depositing a good quality thin film for the protection of reference electrodes and its influence on experimental transfer function. Also, due to the use of inert materials such as gold, glass and PDMS, the sensor system, with some adjustments, can be used for other applications: from monitoring of the concentration and purity of liquid to the characterization of thin films sensitive to drugs and pathogenic agents / Mestrado / Eletrônica, Microeletrônica e Optoeletrônica / Mestre em Engenharia Elétrica

Cette thèse porte sur le développement d’une plateforme microfluidique polyvalente pour la cristallisation des protéines alliant études statistiques, économie de matière, rapidité d’exécution et facilité d’utilisation. L’objectif est de développer un outil unique pour répondre aux différentes problématiques de la cristallisation des protéines : identification d’une condition de cristallisation robuste via le criblage et l’optimisation et co-cristallisation d’une protéine et de ligands pour le structure-based drug design. Une méthode microfluidique versatile à base de gouttes sans tensioactif est utilisée. Elle permet la génération de centaines voire milliers de gouttes de quelques nL dans lesquelles la cristallisation peut avoir lieu indépendamment. La composition des gouttes est contrôlée par les débits des différentes solutions et vérifiée en ligne par spectrométrie UV-vis. De nombreuses conditions de cristallisation différentes peuvent ainsi être testées rapidement : nature et concentration de(s) agent(s) de cristallisation, ajout d’un ligand... Les cristaux obtenus à l’aide de cette plateforme sont caractérisés in situ et ex situ par DRX.Cette plateforme est appliquée à la cristallisation de deux protéines, une protéine modèle, le lysozyme, et une protéine d’intérêt pharmaceutique, la quinone réductase 2. Ainsi, nous avons développé un outil adapté aux contraintes de l’industrie pharmaceutique pouvant être transféré dans un laboratoire de recherche pour une utilisation de routine par des non-spécialistes de la microfluidique. La plateforme permet une approche de criblage à haut débit (HTS) et tend vers l’automatisation à la fois du criblage et de la DRX. / The aim of this work is the development of a versatile microfluidique platform for protein crystallization, combining statistical studies, material saving, speed of execution and ease of use. The aim is to develop a unique tool to address the different issues of proteins crystallization: identification of a robust crystallization condition via screening and optimization, and co-crystallization of a protein with ligands for structure-based drug design. For this purpose, a versatile droplet-based microfluidic method without adding any surfactant is used. It allows the generation of hundreds or even thousands of droplets of a few nanoliters in which the crystallization takes place independently. Droplets composition is controlled by the flow rates of the different solutions using programmable syringe pumps and checked on-line by UV-visible spectroscopy. Many different crystallization conditions can thus be tested quickly: nature and concentration of crystallization agent(s), addition of a ligand, etc. In addition, the crystals obtained using this microfluidic platform are characterized in situ and ex situ by X-ray diffraction.The platform is applied to the crystallization of two proteins, first a model protein, lysozyme, and then a protein of pharmaceutical interest, quinone reductase 2. Thus we have developed a tool suitable to constraints of pharmaceutical industry, in order to be transferred to research laboratories for routine use by non-specialists of microfluidics. This platform permits a high throughput screening approach, or HTS, and tends to the automation of both screening and X-ray diffraction.

EN: There is an industrial need for new technologies that can manipulate objects in the micrometric scale (1-1000 μm). In this thesis, an original non-contact actuation technique for the manipulation of microscale objects is proposed. The proposal is to use laser-induced thermocapillary convective flows to manipulate particles at the fluid/gas interface. These flows are generated when a surface tension stress is generated at the fluid/gas interface due to a thermal gradient. Laser heating is used because the generated thermal gradients produce fast, localized flows that improve the actuation performance. The particles are manipulated at the interface because the flow generated there is the fastest in the entire fluid. To ensure the precise positioning of a particle, closed-loop controllers are implemented in the system which are designed based on models proposed for the system. Experimental tests are performed that show that positioning precision can be ensured. In addition, the interaction forces between particles have been studied which is a preliminary step towards parallel manipulation. To counteract those forces during the manipulation, a different control strategy has been proposed, implemented and tested using simulations. Overall, the results obtained are comparable to the ones obtained with the other techniques. Therefore, the proposed technique can be considered as an attractive alternative that offers different advantages and disadvantages. FR: Il existe un besoin industriel croissant de nouvelles technologies capables de manipuler des objets à l’échelle micrométrique (1-1000 μm). Dans cette thèse, une technique originale d’actionnement sans contact pour la manipulation d’objets à l’échelle micrométrique est proposée. Elle est basée sur les écoulements thermocapillaires convectifs induits par un laser pour manipuler des particules à l’interface fluide/gaz. Le laser chauffe localement la surface de l’eau, ce qui induit un gradient de tension de surface. Ce gradient génère un écoulement fluidique. Ces écoulements sont rapides et localisés, ce qui confère des performances intéressantes à cette technique d’actionnement. Les particules sont manipulées à l’interface fluide/gaz, où l’écoulement généré est le plus rapide.Pour assurer le positionnement précis d’une particule, des contrôleurs en boucle fermée sont implémentés dans le système. Ils sont basés sur les modèles développés dans cette thèse. Des essais expérimentaux montrent que le positionnement précis de particules peut être assuré. De plus, les forces d’interaction entre des particules placées à l’interface ont été étudiées, et une stratégie de contrôle a été proposée, en vue de la manipulation en parallèle de plusieurs particules. Tant les études analytiques et les simulations numériques que les tests expérimentaux soulignent l’intérêt des écoulements thermocapillaires convectifs pour la manipulation contrôlée d’objets micrométriques. Cette technique est donc une alternative prometteuse aux approches classiques d’actionnement sans contact. / Doctorat en Sciences de l'ingénieur et technologie / info:eu-repo/semantics/nonPublished