Orientador: José Alberto Fracassi da Silva / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-20T05:22:01Z (GMT). No. of bitstreams: 1 Campos_RichardPifferSoaresde_M.pdf: 3349460 bytes, checksum: 5efdb4dd4409b9d1b6771593c8874edf (MD5) Previous issue date: 2012 / Resumo: Os micro sistemas de análise total consistem de dispositivos da ordem de centímetros que tem como objetivo a integração de várias etapas analíticas em um único chip, tais como etapas de tratamento de amostra, separação por eletroforese capilar, ou mesmo a integração de sensores em canais microfluídicos. O poli(dimetilsiloxano), PDMS, é um dos polímeros mais adotados para a fabricação destes microdispositivos, devido a suas propriedades elastoméricas, transparência óptica, permeabilidade gasosa, biocompatibilidade, fácil moldagem, relativa alta resistência química e baixo custo de fabricação, além de poder ser facilmente moldado e selado, resultando em microcanais com boa resolução. Além disso, é possível a fabricação de canais por ablação a laser sobre o polímero curado. Entretanto, a característica altamente hidrofóbica do PDMS faz com que sua aplicação para soluções aquosas seja problemática e analitos pouco polares possam sofrer forte adsorção nas paredes do canal, tornando pobre a reprodutibilidade do processo. Neste sentido, estratégias para modificar o material nativo ou mesmo a superfície dos canais vêm sendo estudadas. Neste trabalho, foi inicialmente estudada a modificação estrutural do PDMS, que consiste na utilização de um reticulante (contendo função orgânica polar metacrilato ou amina) na formação do substrato. Também foi realizada a modificação da superfície do substrato de PDMS por reação topológica, com a introdução de polietileno glicol, além da modificação do processo convencional de reticulação do PDMS Sylgard 184, pela adição do surfactante Silwet-L77 a este processo. O PDMS modificado foi avaliado quanto a sua hidrofobicidade, por medida do ângulo de contato com a água, em relação às propriedades do fluxo eletrosmótico gerado no microcanal e as modificações foram estudadas por métodos espectroscópicos. A reação de modificação de superfície do PDMS com divinil éter de polietileno glicol apresentou as melhores características hidrofílicas dentre as modificações estudadas e mobilidade do fluxo eletrosmótico com valor de 3,6x10 cm V s. Em adição, as modificações puderam ser caracterizadas por métodos de espectroscopia (IR e Raman), que se mostraram eficientes na avaliação tanto da rota de modificação quanto do produto final / Abstract: The micro total analysis systems consist of devices in the order of centimeters that aim to integrate several analytical steps on a single substrate, such as sample treatment, injection, or even integrated sensors on microfluidic channels. Poly(dimethylsiloxane), PDMS, is one of the most used polymers for microfabrication due to its elastomeric properties, optical transparency, gas permeability, biocompatility, relatively high chemical resistance and low fabrication costs. PDMS can also be easily cast and sealed, resulting in microchannels with good resolution. On top of that, it is possible to fabricate the microchannels using the lase ablation technique on the cured PDMS. However, the highly hydrophobic characteristic of PDMS makes its aqueous applications problematic. Moreover, non-polar analytes can adsorb on the channel walls, leading to poor reproducibility. In this sense, strategies to modify the raw material or channel surface have been proposed. In this work, the structural modification of PDMS, involving the use of a crosslinking agent (containing the methacrylate or amine polar functions) was studied. In addition, the surface modification of PDMS by topologic reaction with polyethylene glycol and the modification of the conventional PDMS Sylgard 184 crosslinking by the addition of Silwet-L77 surfactant were also performed. The hydrophobicity of modified PDMS was evaluated by water contact angle measurements and the modifications were studied by spectroscopic methods. The electroosmotic flow (EOF) generated in the microchannels was also evaluated. The best hydrophilic characteristic among the studied modifications were obtained with the polyethylene glycol divinyl ether PDMS modification. This device presented an EOF of 3,6x10 cm V s. In addition, the modifications could be characterized by spectroscopic methods (Raman and IR) and those techniques were efficient in the evaluation of the reaction routes as well as the final products / Mestrado / Quimica Analitica / Mestre em Química

Orientadores: Lucimara Gaziola de La Torre, Adriano Rodrigues Azzoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-20T20:39:39Z (GMT). No. of bitstreams: 1 Balbino_TiagoAlbertini_M.pdf: 2581790 bytes, checksum: 674a2d22b439ef811cbfb642774f6d4d (MD5) Previous issue date: 2012 / Resumo: Esta pesquisa teve como objetivo o desenvolvimento tecnológico de processo microfluídico para a obtenção de vetores não virais, baseados na complexação eletrostática entre lipossomas catiônicos (LC) e DNA plasmideal (pDNA) destinados à terapia e vacinação gênica. O desenvolvimento desse processo foi comparado ao processo convencional "bulk", que, a partir da simples mistura manual entre as soluções ou em sistema de vórtice, gera dificuldade no controle do tamanho destas estruturas e pode produzir variações nos resultados biológicos e na estabilidade coloidal. Já o processo microfluídico, que utiliza dispositivos que processam pequenas quantidades de fluidos (10-9 a 10-18 litros), permite a complexação eletrostática em regime contínuo, com o controle das condições difusionais, o que também permite melhor controle do tamanho destes complexos. Metodologicamente, o trabalho foi dividido em três principais etapas: na primeira parte, foi realizado o estudo físico-químico, estrutural e biológico dos complexos pDNA/LC obtidos por processo "bulk". Nessa etapa, verificou-se a correlação das propriedades físico-químicas e estruturais dos complexos com o processo de transfecção in vitro em células HeLa. A segunda parte do trabalho visou à otimização da produção de lipossomas catiônicos em dois dispositivos microfluídicos, com uma única e com dupla focalização hidrodinâmica, de modo a se obter lipossomas similares aos estudados na primeira parte do trabalho. Na utilização do segundo dispositivo, foi possível operar em vazões volumétricas mais altas quando comparadas ao primeiro. Por fim, na terceira parte, foi realizado o estudo da complexação entre LC e pDNA por processo microfluídico também em dois diferentes dispositivos, um similar ao utilizado na segunda parte do trabalho, com focalização hidrodinâmica única, e outro com blocos regulares nas paredes do microcanal, o que aumenta a área de contato entre os fluidos. Os complexos formados no primeiro dispositivo apresentaram melhores respostas biológicas in vitro, as quais foram similares às do processo "bulk". No segundo dispositivo, ensaios de acessibilidade de sonda de fluorescencência ao DNA indicaram alteração na associação entre LC e DNA. Dessa forma, a partir dos resultados, conclui-se que os dispositivos microfluídicos estudados são uma alternativa promissora para a formação de LC e também sua complexão com pDNA em modo contínuo, tanto pela potencialidade tecnológica quanto biológica, o que contribui para o desenvolvimento de produtos farmacêuticos que veiculam DNA e que são destinados à terapia e vacinação gênica / Abstract: This research aimed at the technological development of microfluidic process for nonviral carriers production based on the electrostatic complexation between cationic liposomes (CL) and plasmidal DNA (pDNA) for gene and vaccine therapy applications. The development of this process was compared to the conventional bulk process, in which the solutions are mixed followed by the simple hand shaking or brief vortexing, what generates difficulties on the particles sizes control and can affect the biological functionality and colloidal stability of the formulations. In contrast, microfluidic process, which uses devices that manipulate small amounts of fluids (10-9 to 10-18 liters), allows the electrostatic complexation in continuous mode, controlling diffusion conditions, which also allows the colloidal control of the obtained formulations. Furthermore, microfluidic devices have minimum dimensions and operate with low energy consumption. Methodologically, the present work was carried out in three mean steps: in the first step, the physicochemical, structural and biological characteristics of the pDNA/CL complexes obtained by the bulk process were studied. In this step, it was possible to verify the correlation of physicochemical and structural properties with the transfection phenomenon in vitro of HeLa cells. The second part of this work focused the optimization of the production of CL through two microfluidic devices, with single and double hydrodynamic focusing, to obtain similar CL to those of the first step of this work. By employing the second device, it was possible to operate at higher volumetric flow rates than the first one. Finally, in the third step, it was explored the complexation between CL and pDNA via microfluidic process also in two different microfluidic devices; the first was similar to that employed in the second part of this work, with a single hydrodynamic focusing, and a second one with patterned microchannel walls, which increase the surface contact area between the fluids. The complexes formed in the first device showed better biological results in vitro, which were similar to the complexes formed in the bulk complexation method. In the patterned device, the experiments of the DNA accessibility to fluorescent probe pointed out modifications between the pDNA and CL association in the complexes. In conclusion, we showed that the studied microfluidic devices are a promising alternative for the production of CL and the complexation with pDNA in continuous mode, because of the technological and biological potentialities, which contributes to the development of feasible processes, for the production of new pharmaceutical products for gene and vaccine therapies / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Cette thèse traite le développement de dispositifs, basés sur la technologie "laboratoire sur puce"(LOC) qui visent à contrôler l'environnement des systèmes biologiques pour des applications macro et microbiologiques. En effet, les caractéristiques de la microfluidique permettent de manipuler l'environnement cellulaire à un niveau supérieur à celui du degré de contrôle atteignable avec les techniques ordinaires. Dans ce travail de thèse sera explorée la possibilité de profiter de ces fonctions afin de développer des outils de diagnostic peu coûteux et pourtant efficaces. En particulier, on rapporte le développement de systèmes microfluidiques permettant une perfusion des médias fluide et rapide, ainsi qu'une plateforme LOC capable de réaliser des PCRq hautement multiplexes. Au sujet des systèmes de perfusion, le but était d'obtenir une substitution du médium entourant les particules afin d'augmenter les capacités de séparation des modules de tri microfluidiques couplés. L'efficacité de notre approche a été validée par les hauts taux de séparation obtenus (>90%) avec l'utilisation de notre système de perfusion microfluidique couplé à une puce d'acoustophorèse. De plus, nous avons conçu et développé un système de thermalisation microfluidique capable d'opérer des changements de température en moins de 1s. Plus spécifiquement, cette plateforme exploite l'échange de chaleur entre un liquide de thermalisation qui circule dans une puce microfluidique et l'échantillon. Ces performances de thermalisation, et le rapport surface/volume élevé typique des appareils microfluidiques, ont permis d'effectuer 50 cycles de PCRq et l'analyse de courbe de fusion en moins de dix minutes. / The topic of this manuscript is the development of microdevices, based on "lab on chip" (LOC) technology, aimed to the environmental control and regulation of biological systems for macro and microbiological applications. Indeed, microfluidics possesses some inherent features which allow the manipulation of the environment at the cell and sub-cell level which are superior than the degree of control achievable with standard techniques. In this thesis work the possibility to leverage these features to develop inexpensive yet effective diagnostic tools is explored. In particular, we report the development of microfluidic systems which allow seamless and fast media perfusion and a novel LOC platform capable of performing highly multiplexed real-time PCR assays. Concerning the microfluidic perfusion systems, the aim was to achieve in-flow substitution of the particles' surrounding media in order to enhance the separation capabilities of the coupled microfluidic sorting modules. The effectiveness of our approach was validated by obtaining high separation purities (>90%) using our microfluidic perfusion system coupled with an acoustophoresis chip to discern two population of micro-sized beads. Moreover, we conceived and developed a microfluidic thermalisation system capable of sub-second temperature switches. Specifically, this platform relies on conductive heat exchange between a thermalisation liquid flowing inside a microfluidic chip and the biological sample. These thermalisation performances, and the high surface to volume ratio typical of microfluidic devices, allowed to perform 50 qPCR cycles and subsequent melting curve analysis in less than ten minutes.

Les nanoparticules de polymère (NP) sont une technologie prometteuse pour la libération contrôlée d’actifs . Elles permettent de protéger les drogues et de les délivrer de façon continue. Toutefois pour que cette technologie devienne mature, il est nécessaire de mieux contrôler la synthèse de ces objets. Afin d'atteindre cet objectif, cette thèse propose deux nouvelles méthodes de synthèse basée sur les technologies microfluidiques. Ces méthodes permettent d’encapsuler des drogues hydrophiles et hydrophobes et permettent d’atteindre des taux d’encapsulation supérieurs à ceux mesurés dans la littérature (80% avec une masse de 20% de drogue). Ces approches ont été appliquées à l'encapsulation de l'oxyde de fer. / Polymeric nanoparticle (NP) drug carriers present a promising technology for controlled releasesince they are capable of improving the encapsulation efficiency and stability of the drugs inside theNPs and also able to provide effective drug levels over a longer period of time, compared totraditional therapy. However, before the NP drug delivery technology becomes a reality, importantparameters of NPs like size, drug loading ability and sustained release kinetics must be wellinvestigated and optimized in order to minimize the adverse effects of chemotherapeutic compoundsand prolong the drug releasing profile in a controlled manner.In order to accomplish this objective, this thesis proposed two novel methods for synthesis of NPs asdrug delivery carriers, with assistance from bulk and microfluidic technologies, for hydrophobic andhydrophilic drugs, individually.Encapsulation efficiency as high as 80% is reached with a mass loading of 20%. We extend ourapproaches to the encapsulation of iron oxide

La formation de bouchon est un problème récurrent et presque inévitable lors de l'écoulement de solutions diluées dans des milieux poreux. Actuellement, on ne sait pas comment, à partir du processus initial de déposition de particules à la paroi, ces dernières s'accumulent dans le pore et finissent par le boucher. L'idée générale de ce travail est d'étudier la dynamique de formation de bouchon lors l'écoulement de particules colloïdales au sein de matériaux poreux modèles (canaux microfluidiques). Nous décrivons dans un premier temps, les différents phénomènes physiques impliqués dans la capture de particules et dans l'agrégation colloïdale. Nous faisons également une brève présentation des différentes techniques d'imagerie utilisées dans ce travail et des méthodes de préparation des solutions colloïdales ainsi que des dispositifs microfluidiques. Le troisième chapitre est consacré à l'étude du processus de colmatage en situation de fort confinement (2d). Nous avons identifié deux régimes de colmatage (régime de ''ligne'' et ''d'invasion''). Nous avons ensuite déterminé les processus de capture de particules à l'origine de ces deux régimes, à l'échelle de la particule. Nous avons montré que le processus de colmatage correspond à un phénomène d'auto-filtration. Alors que les premières particules sont capturées de manière « directe » par les parois du pore, la déposition de toutes les suivantes résulte systématiquement d'une interaction avec ces dernières. Finalement, nous avons abordé le colmatage de pore 3d, dont la hauteur est égale à la largeur du pore. Nous avons fourni une description détaillée de l'ensemble du processus de colmatage, à l'échelle du pore et de la particule. Nous avons déterminé les conditions d'adhésion des premières particules à la paroi du pore, les propriétés de croissance des agrégats, ainsi que la manière dont ils se connectent pour obstruer le pore. Nous avons montré que cette dynamique de formation conduit à une structure finale de bouchon très ténue. / Clog formation is a recurring and almost inevitable issue when dilute solution of particles flows in porous media. Currently, we do not know how, from the initial process of particle deposition on the pore wall, particles accumulate in the pore leading to its blocking. The main idea of this work is to study the dynamics of the clog formation, when colloidal particles flow through a single pore (microfluidics channels). In a first part, we describe the various physical phenomenon involved in the particle capture and the colloidal aggregation. We also describe briefly the imaging techniques used in this work as well as the colloidal solution and micro-fluidics chips preparation. The third chapter is devoted to the study of the clogging process in high confinement (2d). We identified two clogging regimes (“line” and “invasion”). We then studied the underlying capture mechanisms, at the particle scale, related to both clogging regimes. We showed that the blockage process corresponds to a self-filtration process. The first particles are captured “directly” by the pore walls, while the deposition of all the following ones systematically results from hydrodynamic interactions with those first still particles. Finally, we addressed the clogging of a 3d pore, in which the height of the pore is equal to its width. We gave a detailed description of the whole clogging process at the pore and at the particle scale. We provided the conditions for the adhesion of the first particles on the pore walls, the properties of subsequent aggregates growth, and how the aggregates eventually merge in order to block the pore. We showed that this dynamics of formation leads to a very loose clog structure.

La microfluidique en gouttes permet l’analyse de systèmes biochimiques à grand débit, en utilisant de faibles volumes réactionnels, à faible coût. Dans l’état de l’art, le volume des gouttes varie entre 2 pL et 4 nL, un millier à million de fois inférieur au volume d’un puit de microplaque. Miniaturiser d’avantage les volumes réactionnels permettrait d’augmenter encore les débits d’analyse, de d’avantage réduire les coûts et ouvre également l’accès à de nouvelles études, telles que les études sur molécule unique ou la délivrance de médicaments. La première partie de ce travail de thèse concerne la miniaturisation des opérations classiques de la microfluidique en gouttes à l’échelle femtolitrique: production, stabilité, biocompatibilité, mélange en gouttes, coalescence, tri, division de gouttes, production à la demande ont été démontrés avec succès sur des gouttelettes femtolitriques. Le tout a été permis en restant dans les limites de la technologie PDMS et des standards classiques de la lithographie. La seconde partie s’intéresse à certaines applications biologiques issues du couplage de gouttelettes picolitriques et femtolitriques. Une plateforme permettant l’encodage in situ de gouttes à l’aide de codes barres d’ADN lisibles par séquençage a été construite. Deux autres applications ont été envisagées: un projet concernant l’émergence des chromosomes dans un monde prébiotique qui nécessitait des facteurs de dilution de l’ordre de 1:1000 et un projet de cartographie génotype-phénotype sur une enzyme qui nécessitait deux dilutions par 10 ont bénéficié de la miniaturisation à l’échelle femtolitrique. / Droplet-based microfluidics has demonstrated its multiple advantage over standard microtiter plates technologies by increasing analysis throughputs, decreasing costs and enabling the encapsulation of single cells into individual reservoirs. In the state-of-the-art, droplet volumes usually range from 2 pL to 4nL, one thousand to one million times smaller than microtiter plate wells. This PhD work focuses on the miniaturization of biological reservoirs down to the femtoliter scale which would enable an increase of throughputs of analysis and open up access to new studies, such as single-molecule studies or drug delivery. The first part of this manuscript concentrates on the miniaturization of elementary operations of droplet-based microfluidics down to the femtoliter scale. Production, mixing, electrocoalescence, DEP sorting, splitting, drop-on-demand, stability, biocompatibility were successfully demonstrated on droplets of a few micrometers diameter. The second part of this manuscript focuses on some biological applications that were developed with the LBC. A platform for the in situ encoding of droplets with DNA barcodes readable per sequencing was developped. Two other applications were envisioned: a project studying the conditions that prevailed the apparition of chromosomes in an early RNA world and a genotype-phenotype mapping project benefited from downscaling to the femtoliter scale.

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No description available.

Cette thèse est consacrée à l’étude en microfluidique de la fabrication de fibres. Les deux étapes critiques sont : - la mise en forme du matériau : nous avons étudié des instabilités qui peuvent se déclencher dans des coécoulements coeur/écorce faisant intervenir des fluides complexes (polymères, suspensions concentrées), celles-ci peuvent empêcher un contrôle de cette étape ; - le figeage de cette forme : nous avons étudié la gélification de l’alginate (un biopolymère formant un gel par l’ajout d’ions calcium) sous écoulement. Nous avons étudié des phénomènes de diffusion-réaction sous écoulement pour comprendre les points de fonctionnement de nos dispositifs. Une fois ces étapes contrôlées, nous nous sommes intéressés à la fabrication des fibres d’alginates fortement chargées en cellules pour l’ingénierie tissulaire. / Abstract

There are situations in which it is very important to quickly and positively identify an individual. Examples include suspects detained in the neighborhood of a bombing or terrorist incident, individuals detained attempting to enter or leave the country, and victims of mass disasters. Systems utilized for these purposes must be fast, portable, and easy to maintain. The goal of this project was to develop an ultra fast, direct PCR method for forensic genotyping of oral swabs. The procedure developed eliminates the need for cellular digestion and extraction of the sample by performing those steps in the PCR tube itself. Then, special high-speed polymerases are added which are capable of amplifying a newly developed 7 loci multiplex in under 16 minutes. Following the amplification, a postage stamp sized microfluidic device equipped with specially designed entangled polymer separation matrix, yields a complete genotype in 80 seconds. The entire process is rapid and reliable, reducing the time from sample to genotype from 1-2 days to under 20 minutes. Operation requires minimal equipment and can be easily performed with a small high-speed thermal-cycler, reagents, and a microfluidic device with a laptop. The system was optimized and validated using a number of test parameters and a small test population. The overall precision was better than 0.17 bp and provided a power of discrimination greater than 1 in 106. The small footprint, and ease of use will permit this system to be an effective tool to quickly screen and identify individuals detained at ports of entry, police stations and remote locations. The system is robust, portable and demonstrates to the forensic community a simple solution to the problem of rapid determination of genetic identity.