A simple and efficient technique for the concentration enrichment of charged species within a microfluidic device was developed. The functional component of the system is a hydrogel microplug photopolymerized inside the microfluidic channel. The fundamental properties of the nanoporous hydrogel microplug in modulating the electrokinetic transport during the concentration enrichment were investigated. The physicochemical properties of the hydrogel plug play a key role in determining the mode of concentration enrichment. A neutral hydrogel plug acts as a physical barrier to the electrophoretic transport of charged analytes resulting in size-based concentration enrichment. In contrast, an anionic hydrogel plug introduces concentration polarization effects, facilitating a size and charge-based concentration enrichment. The concentration polarization effects result in redistribution of the local electric field and subsequent lowering of the extent of concentration enrichment. In addition, an electroosmotic flow originating inside the pores of the anionic hydrogel manipulates the location of concentration enrichment. A theoretical model qualitatively consistent with the experimental observations is provided.

A simple and efficient technique for the concentration enrichment of charged species within a microfluidic device was developed. The functional component of the system is a hydrogel microplug photopolymerized inside the microfluidic channel. The fundamental properties of the nanoporous hydrogel microplug in modulating the electrokinetic transport during the concentration enrichment were investigated. The physicochemical properties of the hydrogel plug play a key role in determining the mode of concentration enrichment. A neutral hydrogel plug acts as a physical barrier to the electrophoretic transport of charged analytes resulting in size-based concentration enrichment. In contrast, an anionic hydrogel plug introduces concentration polarization effects, facilitating a size and charge-based concentration enrichment. The concentration polarization effects result in redistribution of the local electric field and subsequent lowering of the extent of concentration enrichment. In addition, an electroosmotic flow originating inside the pores of the anionic hydrogel manipulates the location of concentration enrichment. A theoretical model qualitatively consistent with the experimental observations is provided.

Continuous progress in the nanotechnology field has allowed for the emergence of powerful, nanopore-based detection technology. Solid-state nanopores were developed for next-generation sequencing and single-molecule detection. They are advantageous over their biological counterpart because they offer robustness, stability, tunable pore size and the ability to be integrated within a microfluidic device. With all of these attractive attributes, solid-state nanopores are a top contender for point-of-care diagnostic technologies. However, hindering their performance is an inability to distinguish between small molecules, pore-clogging, and the detection rate's dependence on sample concentration. The concentration-dependent detection rate becomes particularly evident at low sample concentrations (<1 nM), sometimes taking hours for the nanopore to sense a single molecule because of diffusion. The inability to distinguish between small molecules can be addressed using DNA nanostructures; however, pore-clogging and variable detection rates hinder its potential in a clinical setting. This thesis proposes a microfluidic device design and methodology that seeks to mitigate pore-clogging and improve the detection rate for dilute samples. DNA coated microbeads will create a bead column within the microfluidic device and confine the target molecules to an extremely small (20 nL) volume. The sample can be washed, ridding the contaminants, and eluted on-chip, so the sample is purified and concentrated, affording a more reliable sensing performance. First, a magnetic microbead DNA assay was optimized off-chip, and the capture and release efficiencies were monitored using a Biotek™ Epoch™ 2 spectrophotometer (Chapter 2). Next, a novel microfluidic device design was optimized and validated to ensure precise sample manipulation (Chapter 3). Finally, the microbead assay was incorporated into the microfluidic device for sample concentration (Chapter 4). Fluorescence microscopy results suggest successful DNA elution from the microbeads within the microfluidic device, allowing for a 28.5 X concentration increase. This platform shows promise for sample preconcentration by reducing the starting DNA sample volume of 25 µL to 20 nL, which could improve the speed of solid-state nanopore sensing.

No description available.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / Chemical separations are a necessary component in many scientific analyses. Microfluidics, the use of micron-sized fluidic channels defined in glass or polymer blends, is a powerful branch of separation science that is developing rapidly. Miniaturized analytical devices offer important advantages compared to traditional bench-top techniques, most notably capillary electrophoresis (CE). This dissertation was focused on developing several novel methods to improve microfluidic based separations and techniques. The electrophoretic separation of small similarly charged analytes can be very difficult. Chapter 2 discusses a new buffer that has been developed for fast, high efficiency separations of amino acids by micellar electrokinetic chromatography (MEKC). This buffer is more environmentally friendly than the most commonly used surfactant containing buffers for MEKC separations. It uses a commercially available dish washing soap by Seventh Generation™ Inc. that contains three micelle forming agents; sodium lauryl ether sulfate (anionic), cocamidopropyl betaine (zwitterionic), and cocamide monoethanolamine (MEA) (non-ionic), and is completely void of organic solvents. Many biological samples contain analytes below the limit of detection of traditional detection systems; therefore, chapter 3 reports the fabrication of nanoporous membranes on microfluidic devices that are capable of analyte concentration enrichment. Donnan exclusion is responsible for the preconcentration of fluorescent dyes near a charged, porous titania membrane. The level of analyte enrichment was monitored, and enrichment factors greater than 4000 in 400 s were obtained for 2,7-Dichlorofluorescein. Chapter 4 describes the fabrication and characterization of paper based microfluidic devices. Mixtures of acrylate modified photocurable polymers were used to photolithographically define channels on multiple paper substrates. Flow characteristics are described and their use for monitoring complications associated with type 1 diabetes is demonstrated. Finally in Chapter 5, Sol-gel modified gold surfaces for preventing protein adsorption during surface plasmon resonance (SPR) detection are also presented.

La nanofluidique est l’étude du transport de molécules au travers de nanostructures filtrantes dont la taille avoisine l'épaisseur de la double-couche diffuse à la surface du verre. A cette échelle de la centaine de nanomètres, la charge de surface qui induit une exclusion des ions négatifs à l'extérieur du "nanofiltre" produit un effet de rétention des biomolécules. Des études menées sur le transport électrocinétique au sein d’un nanocanal unique ont permis de montrer qu’il était théoriquement possible de concentrer des solutions, même fortement diluées, avec des taux élevés (jusqu’à 103) grâce à un effet de concentration de polarisation. Ce phénomène pourrait être exploité dans de nombreuses applications de diagnostic médical (analyses rapides et précoces d’échantillons bruts), de contrôle qualité (agroalimentaire) ou encore de défense (suivi continu de zones à risques pour la menace terroriste biologique).La modélisation de la dynamique des phénomènes d’électropréconcentration (sous champ électrique) et de rétention (sous gradient de pression) d’un nanocanal unique s’avère extrêmement ardue. Une multitude d’observations, souvent contradictoires quant au profil de préconcentration obtenu, ont été par ailleurs rapportées dans la littérature, avec des points focaux de préconcentration observés parfois du côté anodique ou du coté cathodique pour une même protéine. Certaines expériences observent ces points focaux soit très loin en amont dans le microcanal réservoir soit directement à proximité de l’entrée du nanocanal. C’est dans ce contexte qu’a été développé précédemment un modèle unidimensionnel permettant de prédire le profil de concentration de l’analyte en tout point de la structure MNM(Micro/Nano/Microcanal) proposée. Ce travail de modélisation a démontré l’existence de quatre régimes distincts: deux régimes coté anodique et deux régimes coté cathodique, plus ou moins éloignés du nanocanal. Ce modèle a mis en avant la sélectivité de ce processus vis-à-vis de la mobilité électrophorétique et de la valence des analytes préconcentrés, et il a permis d’appréhender un peu mieux la diversité des expériences rapportées. Cependant, le régime de préconcentration obtenu dépend du bioanalyte étudié. Il serait pourtant intéressant de ne plus être tributaire des caractéristiques intrinsèques de la solution analysée et de ne plus subir le régime obtenu mais, au contraire, d’effectuer ce que l’on pourrait appeler une électropréconcentration sélective du dit analyte. Ceci pourrait permettre d’effectuer deux étapes primordiales de tout diagnostic que sont la séparation et la préconcentration d’un mélange. Pour se faire, nous avons introduit un paramètre expérimental, une composante hydrodynamique (ou surpression), en sus du champ électrique, pour moduler la localisation de la préconcentration.A l’aide d’une technologie "tout verre" récemment brevetée, nous élaborons aujourd’hui des puces intégrant une nanofente dans un long microcanal. Ces puces sont parfaitement isolantes, biocompatibles et présentent une tenue exceptionnelle au cours du temps. Elles sont combinées à un banc expérimental "fait maison" complètement automatisé (interfacé avec Matlab), qui permet de contrôler les différents paramètres imposés. Les données recueillies sont ensuite prétraitées par d’autres codes Matlab que nous avons développés. Grâce à ces divers outils, de nombreuses expériences d’électropréconcentration "classique" (champ électrique seul) et assistée en pression ont été réalisées pour deux bioanalytes modèles : la fluorescéine et la BSA (Bovin Serum Albumin). Elles ont permis de déterminer les différents paramètres influant sur la préconcentration de ces deux analytes et de prouver la sélectivité et l’efficacité de la méthode proposée ici. Des régimes de préconcentration inattendus, stables et présentant des taux élevés ont en effet été obtenus au cours de cette thèse. / Nanofluidics is the study of the transport of molecules through filtering nanostructures whose size approximates the thickness of the diffuse double layer at the surface of the glass. At this scale of hundreds of nanometers, the surface charge induces an exclusion of negative species outside the "nanofilter" and a retention effect of biomolecule. Studies on the electrokinetic transport in a single nanochannel have shown that it was theoretically possible to concentrate solutions, even highly diluted, with high rates (up to 103) thanks to a concentration polarization effect. This phenomenon can be exploited in many medical diagnostic applications (early and fast sample analysis), quality control (food, water) or defense (continuous monitoring of risky areas for biological terrorist threat). Modeling the dynamics of electropreconcentration phenomena (under an electric field) and retention phenomena (under a pressure gradient) of a single nanochannel is extremely difficult. A multitude of observations, often contradictory regarding the obtained preconcentration profile, were also reported with focal points observed sometimes in the anodic side and other times in the cathodic side for the same protein . Some experiments observe these focal points either upstream in the microchannel reservoir or directly at the entrance of the nanochannel. In this context, a one-dimensional model was previously developed to predict the concentration profile of the analyte at any point of the proposed MNM (Micro/Nano/Microchannel) structure. This modeling work has demonstrated the existence of four distinct regimes: two regimes in the anodic side and two regimes in the cathodic side, more or less distant from the nanochannel. This model highlighted the selectivity of the process regarding the electrophoretic mobility and the valence of the preconcentrated analytes and allowed to understand a little better the diversity of reported experiments. However, the obtained regime depends on the bioanalyte. Though it would be interesting not to be dependent of the characteristics of the analyzed solution and, on the contrary, to realize a selective electropreconcentration of the analyte. This could allow to perform two important steps in any diagnosis: the separation and the preconcentration of a mixture. To do so, we introduced an experimental setting, a hydrodynamic component (or pressure) in addition to the electric field to modulate the localization of the preconcentration .Using an "all glass" technology patented in LPN, chips perfectly insulating, biocompatible and with an exceptional resistance over time are now manufactured. These chips presenting a nanoslit in a straight microchannel are combined with a "homemade" experimental set-up fully automated (interfaced with Matlab®) which allow a perfect control of the various experimental parameters. The data obtained during experiments are then preprocessed by other Matlab codes that we have developed. Thanks to these various tools, many electropreconcentration experiments were performed for two bioanalytes: fluorescein and BSA (Bovin Serum Albumin). They have identified different parameters affecting the preconcentration of these analytes and they have demonstrated the selectivity and the efficiency of the method proposed in this thesis. Unexpected and stable preconcentration regime have been obtained with high rates of preconcentration.

A pré-concentração consiste no descarte prévio de uma fração da ganga liberada, ou parcialmente liberada, com pouco ou nenhum conteúdo de interesse, antes da alimentação da usina. Tal prática pode levar a uma série de benefícios nas operações subsequentes, como redução do consumo de energia por tonelada de metal produzido, nos casos em que o material rejeitado apresentar WI (work Index) elevado; diminuição do desgaste abrasivo, quando a ganga rejeitada exibir AI (Abrasion Index) alto; além de aumento de recuperação na flotação e eliminação de flutuações no teor da alimentação. Neste sentido, a pré-concentração diminui custos operacionais e aumenta a rentabilidade da usina. Este trabalho teve por objetivo avaliar a aplicabilidade de pré-concentração para o minério de zinco silicatado de Vazante/MG. Em uma primeira etapa foram executados estudos de liberação da ganga no produto de britagem, explorando as propriedades luminescentes do minério. Para isto, imagens de contraste de cores luminescentes foram obtidas e tratadas no Image J. Obteve-se um valor de liberação da ganga da ordem de 85%. Em um segundo momento, foi investigado o desempenho de técnicas de separação densitária e seleção por sensores (ou ore sorting) como alternativas para a pré-concentração do minério de zinco e remoção de ganga carbonática, para isto, foram utilizados ensaios de afunda/flutua em líquidos densos, que simularam uma operação em meio denso, estratificação em jigue e ensaios em sorter com sensor de transmissão de Raios-X de energia dupla (DE- XRT). O melhor resultado foi obtido através do ensaio de afunda/flutua com densidade de separação de 2,95 g/cm³, no qual foi alcançado um descarte de 30% da massa de alimentação como produto flutuado, numa fração -25 +1,18 mm, removendo 60% dos carbonatos (CaO e MgO) e perdendo apenas 2% de zinco. Os ensaios do sorter também apresentaram resultados positivos, com aproximadamente 93% de recuperação metalúrgica de zinco e um descarte de aproximadamente 30% da massa, mas com uma perda de zinco considerável (aproximadamente 5% em teor). Para a jigagem, os resultados indicaram teor de zinco muito elevado no rejeito (aproximadamente 6%). Entretanto, uma jigagem adicional, realizada com uma segunda amostra numa granulometria menor (-12,7 +1,18 mm), proporcionou menores níveis de zinco perdido para o rejeito, apenas 3,6% em teor. Os impactos da pré-concentração na moagem e flotação também foram avaliados. Para isto foram executados ensaios de work index (WI), abrasion index (AI) e flotação com material tal qual (sem passar por pré-concentração) e com o material pré-concentrado de forma a se ter um comparativo. Como não houve variação de WI e nem de AI entre as amostras tal qual e pré-concentrada, a pré-concentração previamente à moagem, não irá impactar no consumo energético e tampouco no desgaste abrasivo, isto se for adotada a estratégia de manter a mesma vazão mássica de alimentação do circuito de moagem para ambas as alternativas. Em linhas gerais, a flotação após a pré-concentração aumentou a produção de concentrado de zinco com uma qualidade superior em termos de recuperação deste metal. Outro impacto positivo observado foi a redução da quantidade de rejeito fino de flotação e, consequentemente, o volume a ser estocado em barragem. / Preconcentration consists of the prior disposal of a fraction of the liberated or partially liberated gangue with low or no content of interest material before plant feed. Such practice can lead to a number of benefits in subsequent operations, such as reducing energy consumption per ton of metal produced, in cases where the rejected material presents a high WI (work index); decreased abrasive wear when the rejected presents high Abrasion Index (AI); increased recovery in flotation and elimination of fluctuations in feed content. Therefore, preconcentration reduces operating costs and increases the profitability of the plant. This work had the objective of evaluating the preconcentration applicability for a silicate zinc ore from Vazante/MG. In the first stage, grind release tests were performed on the crushing product, exploring the luminescent properties of the ore. For this, luminescent color contrast images were obtained and treated in Image J. A gangue release value of the order of 85% was obtained. Second, the performance of densification separation and sensor sorting (or ore sorting) techniques were investigated as alternatives for preconcentration of zinc ore and removal of carbonate gangue, for this, sink/float tests in dense liquids were used to simulate a dense medium operation, as well jig stratification and sorter tests with dual energy X-ray transmission sensor (DE-XRT). The best result was obtained by the sink/float test with separation density of 2.95 g / cm³, in which a 30% discard of the feed mass as a flotation product was achieved, in a fraction -25 +1.18 mm, removing 60% of the carbonates (CaO and MgO) and losing only 2% of zinc. The sorter tests also showed positive results, with approximately 93% metallurgical recovery and a discard of approximately 30% of the mass, but with a considerable loss of zinc. For jigging, the results indicated very high zinc content in the tailings (approximately 6%). However, additional jigging, performed with a second sample at a smaller particle size (-12.7 +1.18 mm), resulted in lower levels of zinc lost to the tailings, only 3.6% in content. The impacts of preconcentration on milling and flotation were also evaluated. For this, work index (WI), abrasion index (AI) and flotation tests were carried out with the material without preconcentration and with the preconcentrated material in order to compare. As there was no variation of WI and AI between the samples, the preconcentration prior to milling will not impact on energy consumption or on abrasive wear, if the strategy of maintaining the same mass flow rate of the milling circuit for both alternatives is adopted. In general terms, flotation followed by a preconcentration strategy can increase zinc production without requiring expansion of the plant\'s capacity. Another positive impact was the reduction of the amount of fine flotation tailings.

La electroforesis capilar presenta menos sensibilidad en términos de concentración, comparado con la cromatografía de líquidos debido principalmente al pequeño volumen de inyección de la muestra y a la pequeña longitud del camino óptico para la detección, el cual coincide con el diámetro interno del capilar. El objetivo de la presente Tesis es el estudio y desarrollo de diferentes estrategias para disminuir los límites de detección de la electroforesis capilar, concretamente técnicas de preconcentración electroforéticas y cromatográficas. Se presentan técnica basadas en isotacoforesis y SPE acoplado en línea a electroforesis capilar para la determinación de drogas de abuso y de anti-inflamatorios no esteroidales. El desarrollo fundamental de estas estrategias va, en muchos casos, relacionado con el desarrollo de las aplicaciones, por lo cual el trabajo con muestras reales es fundamental, concretamente se ha trabajado con muestras biológicas (plasma y orina) y con muestras de agua medioambientales (agua de rio y de grifo). / Capillary electrophoresis is a highly promising analytical separation technique with a wide range of applications in biological and environmental fields. However, Capillary electrophoresis suffers from a lack of sensitivity when compared with chromatographic techniques because, in general, it is not possible to inject high sample volumes and also due to the short optical path-length for on-capillary detection. The objective of this Doctoral Thesis is the development of analytical methods to increase sensitivity in capillary electrophoresis for the determination of pharmaceuticals and drugs of abuse in environmental and biological samples. To do that, several preconcentration techniques either stacking (based on isotachophoresis principles) or chromatographic (based on in-line SPE) have been studied. The compounds selected for the different studies in this Doctoral Thesis belong to the pharmaceutical compound group (in particularly in non-steroidal anti-inflammatory drugs) and to the drugs of abuse group.

This work begins with chemotaxis studies involving Salmonella typhimurium. Known chemical attractants (ribose, aspartic acid, etc.) and repellents (nickel chloride, sodium acetate, etc.) were tested to direct bacteria swimming patterns. It was found that high concentrations of both attractant and repellent, approximately 10% chemical in deionized (DI) water, yielded better separation results than lower concentrations, such as 1% and .1% chemical in DI water. Utilizing these attractants or repellents appropriately can allow live bacteria to be directed in a desired manner in a microfluidic device, while dead bacteria, which yield no response, can be separated into a waste reservoir. Another important aspect of bacteria separation is preconcentration, or the process of concentrating bacteria in a usable amount of liquid for further analysis in a microfluidic device. This study introduces a method of capturing Salmonella typhimurium through the use of magnetic nanoparticles (MNPs) without functionalizing them with antibody or amine coatings. Based on the work by Deng et al., MNPs were prepared in various ways to alter their diameter and surface characteristics to achieve optimal bacteria capture efficiency. A capture efficiency of approximately 94% has been achieved by altering chemical quantities in the MNP fabrication process. A macro-scale flow cell prototype was designed and characterized in order to ‘clean’ large volumes of buffer and separate the bacteria-MNP aggregates through the use of a magnetic field. Finally, intervention of bacteria is a significant topic in food safety applications. This study utilizes Fe (III) to inhibit bacteria growth. This chemical was used in the presence of Salmonella, E. coli, Staphylococcus, and Pseudomonas. Further experiments were conducted with raw chicken and lettuce contaminated with Salmonella typhimurium. Using as little as .005M Fe III in DI water, up to 5 orders of magnitude reduction in bacteria growth was seen on test plates as compared to control plates.

Submitted by Edileide Reis (leyde-landy@hotmail.com) on 2013-04-23T13:40:57Z No. of bitstreams: 1 Sergio Rocha.pdf: 458715 bytes, checksum: f65463df66bf4d66d87f86f79e12db98 (MD5) / Made available in DSpace on 2013-04-23T13:40:57Z (GMT). No. of bitstreams: 1 Sergio Rocha.pdf: 458715 bytes, checksum: f65463df66bf4d66d87f86f79e12db98 (MD5) Previous issue date: 2005 / No presente trabalho, é proposto o emprego do reagente 4-(2-tiazolilazo)-orcinol (TAO) como complexante no desenvolvimento de sistemas de separação e pré-concentração para determinação de metais em águas naturais. Inicialmente foi desenvolvido um método para separação, pré-concentração e determinação de traços de titânio(IV) em amostras de água de mar usando Espectrometria de Emissão Ótica com Plasma Indutivamente Acoplado (ICP OES). As amostras foram coletadas nos municípios de Lauro de Freitas e Salvador, localizados no estado da Bahia. Nesta região existe uma indústria de produção de dióxido de titânio, que libera efluentes para o mar. O método é baseado na reação de complexação de íons titânio(IV) com o reagente 4-(2-tiazolilazo)-orcinol (TAO), seguida por sorção em carvão ativo. Foram estudados os seguintes parâmetros: efeito do pH na complexação e sorção, quantidade de TAO, quantidade de cloridrato de hidroxilamina, tempo de agitação, massa de carvão ativo, dessorção do Ti(IV) do carvão ativo, volume de amostra, efeito de outros íons, sensibilidade analítica e precisão. Os resultados demonstraram que titânio(IV) pode ser sorvido quantitativamente (95-105%) dentro da faixa de pH que varia de 3,8 a 7,0. O tempo necessário para sorção foi de 5 minutos usando 100 mg de carvão ativo. A dessorção mostrou-se eficiente quando o carvão ativo foi aquecido, cerca de 10 minutos a 40oC, com uma solução de ácido nítrico 3 mol L-1. O procedimento apresentou boa precisão, RSD de 4,6 e 2,5 % para concentrações de titânio de 1,00 e 2,50 mg L-1, respectivamente. O limite de detecção foi de 0,01 mg L-1 para um volume de amostra de 400 mL. O método apresentou boa tolerância para outros íons normalmente presentes em amostras de água de mar. O procedimento foi aplicado para determinação deste elemento em amostras de águas coletadas nas praias de Arembepe (Lauro de Freitas), Stella Mares e Ondina (Salvador), Bahia. O teor de titânio(IV) nas amostras analisadas variou de 0,04 a 0,15 mg L-1. Em outro trabalho foi proposto um procedimento em linha para separação, pré-concentração e determinação de quantidades traços de cádmio(II) por espectrometria de absorção atômica com chama por injeção em fluxo. O método foi baseado na pré-concentração em linha de ions cádmio(II) em uma coluna empacotada com a resina Amberlite XAD-2 impregnada com o reagente 4-(2-tiazolilazo)-orcinol e conectada a uma válvula manual de injeção em fluxo. Durante a etapa de injeção quatro variáveis foram examinadas: pH, concentração do tampão, concentração do eluente e vazão de amostra. As interações entre os fatores analíticos e seus níveis ótimos foram investigados usando planejamento fatorial de dois níveis e matriz Doehlert. O método proposto apresenta faixa linear de calibração de 0,02 a 5 mg L-1 de cádmio(II), com limite de detecção de 0,02 mg L-1, e precisão, calculada como desvio padrão relativo (RSD) de 6,2 e 3,0% para concentração de cádmio de 2,0 e 4,0 mg L-1. O fator de enriquecimento obtido foi de 252 para tempo de pré-concentração de 3 minutos. A recuperação encontrada para determinação de cádmio(II) em presença de vários cátions demonstrou que este apresenta boa tolerância. O procedimento proposto foi aplicado para determinação de cádmio amostras de água mineral. O teor de cádmio(II) encontrado nas amostras analisadas variou de 0,11 a 0,15 mg L-1. / Salvador