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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.

The genome sequencing programs have identified hundreds of thousands of genetic and proteomic targets for which there are presently no ascribed functions. The challenge for researchers now is to characterize them, as well as identify and characterize their natural variants. Historically, this has meant studying each individual target separately. However, due to the recent development of multi-analyte microarray devices, these characterizations can be performed in a combinatorial manner in which a single experiment provides information on thousands of targets at a time. In the past decade, microarray technology has settled in on two major designs. The first entails spotting individual receptor types onto a functionalized glass substrate. This is a simple and inexpensive process; however, due to the limited resolution of the mechanical devices used to do the spotting, the densities of these arrays are relatively low. Moreover, receptor preparation requires substantial time and effort. The second variety of microarray uses photolithographic techniques adapted from the semi-conductor industry to chemically synthesize the receptor elements in situ on the sensing surface. Because lithographic patterning is spatially very precise, these arrays achieve very high densities, with as many as one million features per square centimeter. Although these arrays obviate the necessity for laborious "off chip" probe preparation, they are expensive to produce and are limited to two types of receptors (oligonucleotides and peptides). This dissertation presents the development work performed on a hydrogel-based biosensor platform which provides a high density and low cost alternative to the two aforementioned designs. The array features are fabricated lithographically from a liquid pre-polymer doped with biologically active sensing elements at sizes as small as 50[micrometer]. Each of the feature types is uniquely shaped, which enables the features to be mass-produced in batches, pooled together and then assembled into randomly ordered arrays using highly-parallelized self-assembly techniques. The three-dimensional hydrogel features accommodate a wide variety of sensing elements, such as enzymes, antibodies and cells, which cannot be deployed using the traditional designs. This dissertation presents methods developed to integrate cellular and oligonucleotide sensing elements into the hydrogel features which preserve their biological activity and optimize the sensor's performance. / text

Water and methanol are common solvents used in liquid chromatographic (LC) separations. It is highly desirable to model .the interactions of these solvents in order to better understand the nature of analyte solvation and its effect on retention. Therefore, structure and frequencies of complexes of these solvent molecules have been studied from a theoretical perspective as a first step in this direction. Specifically, cluster structures have been optimized at the RHF and MP2 levels in various flexible basis sets and with the counterpoise correction for basis set superposition error, and trends in the structure and binding energies of several clusters are described. Good agreement wasobtained for the water dimer with the experimental value for the binding energy of D20 using MP2 energies from 6-3 11G**/6-3 l+G** basis sets in conjunction with counterpoise optimizations and full counterpoise corrections. In this investigation harmonic frequencies have been calculated and corrected for the effects of anharmonicity by several methods, two of which are original. The first new method fits a Morse potential function to the energy computed along each normal mode. A second new method is based on fitting a quartic polynomial to energies computed along each normal mode. In cases where the quartic potential function is not very different from the harmonic well, a second order perturbation formula provides a reasonable approximation to the anharmonic vibrational frequencies. When the quartic potential is very far from the harmonic potential, a variational treatment of the vibrations is required. We find that the Morse method delivers reasonable estimates of frequencies of anharmonic motions at lower cost than multi-point potential mapping/multiple geometry optimization/Taylor series methods, and is more successful at predicting intermolecular frequencies than the anharmonic VSCF methods found in GAMESS software. Variational calculations using the quartic polynomials produce estimates of frequencies comparable to the more costly VSCF method. Both the Morse method and polynomial method are very fast computationally relative to these and other methods found in the literature.

A solid-liquid two phase partitioning system (TPPS) is a new technology platform for destroying toxic organic compounds. TPPS have traditionally been operated by using an immiscible organic phase which partitions organic compounds into the aqueous phase. TPPS using an immiscible organic phase suffers from several limitations such as the organic phase could be biodegradable and hence only certain compatible microbial strains could be used. This therefore, eliminates the desired use of mixed microbial populations for efficient degradation. A solid-liquid two phase partitioning system, in which solid polymeric beads replace liquid organic phase, appears to have benefits over the traditional liquid-liquid partitioning systems. The choice of suitable polymeric material should have similar absorption properties as the liquid organic solvent but have the added benefit of being able to be used with mixed microbial population. In this study, poly (ethylene-co-vinyl acetate), brand name ELVAX 40W, was selected as the test polymer in an effort to lower the concentrations of selected analytes; phenol, 4- nitrophenol and o-cresol in aqueous solutions. Studies were performed to determine the degree of partitioning using HPLC and UV-VIS. Kinetic studies were also performed and illustrated a first order dependence on the absorption of the phenols tested. Activation energies were also determined for each analyte. Rate constants were on the order of 10-4 min-1. Activation energy ranged from 19-46 kJ/mol. Regeneration tests showed that a release of analyte from the polymer is possible when the beads are placed in water. Therefore the ability to reuse the polymer is possible and therefore cost efficient. The polymer was observed to lower high concentrations up to 2000 ppm suggesting its potential use to treat the high concentrations of toxic organic compounds.

An alternative molecular recognition approach was developed for sensing small molecule analytes using the differential binding of an allosteric transcription factor (TF, specifically TetR) to its cognate DNA as the molecular recognition element coupled with Förster resonance energy transfer (FRET) to yield an internally calibrated optical signal transduction mechanism. Sensors were evaluated comprising Cy5-modified DNA (FRET acceptor) with either a tdTomato-TetR fusion protein (FP-TF) or quantum dot-TetR conjugate (QD-TF) as the FRET donor by measuring the ratio of acceptor and donor fluorescence intensities (FA/FD) with titrations of a derivative of the antibiotic tetracycline, anhydrous tetracycline (aTc). A proof-of-concept FRET-based biosensor was successfully demonstrated through the modulation of FA/FD signal intensities based on varying analyte concentrations. Sensor design parameters affecting overall signal-to-noise ratio and sensitivity of the sensors are also identified. / 2020-06-03T00:00:00Z

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A Toxicologia tem desempenhado um importante papel na identificação de efeitos nocivos à população, gerando subsídios para a tomada de decisões na prática clínica, auxiliando desta forma no bom prognóstico de pacientes intoxicados. De acordo com a Pan American Health Organization, o sucesso em qualquer intervenção em saúde somente pode ser obtido com a criação e manutenção de um banco de dados confiável, que seja capaz de predizer as diversas particularidades das intoxicações, como a população-alvo e suas suscetibilidades. Assim, recomenda-se que médicos, especialistas, legisladores e administradores em saúde adotem em sua rotina uma coleta de dados sistêmica e integrada para o mapeamento e caracterização das intoxicações. Neste cenário, técnicas de análises multivariadas poderiam ser empregadas para evidenciar possíveis intercorrelações; entretanto, seu uso ainda não é comum na toxicologia clínica. Neste trabalho foram identificados e quantificados agentes exógenos em amostras biológicas (sangue e urina) provenientes do Centro de Controle de Intoxicações da cidade de São Paulo, correlacionando os dados obtidos dessas análises com o perfil clínico e prognóstico dos pacientes. Fármacos benzodiazepínicos, antidepressivos, anticonvulsivantes, paracetamol, drogas de abuso e praguicidas foram selecionados de acordo com a incidência reportada por esse centro no período de 2013 a 2014. Do total de amostras analisadas (n = 320), 192 foram positivas para alguma substância, sendo 101 positivas para etanol e 131 positivas para as demais substâncias. Os dados obtidos foram submetidos à análise de correspondência múltipla e análise hierárquica de cluster. A partir da análise multivariada foi possível agrupar os indivíduos em 3 clusters, o que correspondeu a 66,5% do total de informações. No primeiro eixo houve a separação dos pacientes do gênero feminino, que se intoxicaram ou foram expostos a medicamentos e drogas de abuso, por tentativa de suicídio, dos pacientes do gênero masculino, de idade entre 30 a 39 anos, que se intoxicaram com drogas de abuso. No segundo eixo fatorial foram agrupados os pacientes que se intoxicaram com etanol isoladamente, juntamente com pacientes que se intoxicaram com diazepam. Este trabalho contribuiu para o mapeamento dos casos de intoxicação atendidos pelo CCI-SP e foi um estudo inicial para a criação de um banco de dados que poderá ser alimentado constantemente e assim, oferecer ao sistema de toxicovigilância uma base para políticas educativas. / Toxicology has played an important role in the identification of harmful effects to the population, generating subsidies for decision making in clinical practice, helping in this way the good prognosis of acutely intoxicated patients. According to the Pan American Health Organization, success in any health intervention can only be achieved by creating and maintaining a reliable database that is capable of predicting the various characteristics of intoxications, such as the target population and their susceptibilities. Thus, it is recommended that doctors, specialists, legislators and health administrators adopt in their routine a systemic and integrated data collection for the mapping and characterization of intoxications. In this scenario, multivariate analysis techniques could be used to evidence possible intercorrelations; however, its use is not yet common in clinical toxicology. In this work, were identified and quantified exogenous agents in biological samples (blood and urine) from the Poison Control Center São Paulo city, correlating the data obtained from these analyzes with the clinical and prognostic profile of the patients. Benzodiazepines, antidepressants, anticonvulsants, acetaminophen, drugs of abuse and pesticides were selected according to the incidence reported by this center in the period from 2013 to 2014. Of the total number of samples analyzed (n = 320), 192 samples have shown to be positive for some of the analytes, from these 100 were positive for ethanol and 131 positive for other substances. The data were submitted to multiple correspondence analysis and hierarchical cluster analysis. From the multivariate analysis it was possible to group the individuals into 3 clusters, which corresponded to 66.5% of the total information. In the first axis, the patients were separated from the female gender, who were intoxicated or were exposed to drugs and suicide attempt of the male patients, aged between 30 and 39 years, who became intoxicated with drugs of abuse. In the second factorial axis were grouped the patients who were intoxicated with ethanol, together with patients who became intoxicated with diazepam. This work contributed to the mapping of intoxication cases attended by the CCI-SP and was a initial study for the creation of a database that could be fed constantly and thus provide the toxicovigilance system with a basis for educational policies.

A Toxicologia tem desempenhado um importante papel na identificação de efeitos nocivos à população, gerando subsídios para a tomada de decisões na prática clínica, auxiliando desta forma no bom prognóstico de pacientes intoxicados. De acordo com a Pan American Health Organization, o sucesso em qualquer intervenção em saúde somente pode ser obtido com a criação e manutenção de um banco de dados confiável, que seja capaz de predizer as diversas particularidades das intoxicações, como a população-alvo e suas suscetibilidades. Assim, recomenda-se que médicos, especialistas, legisladores e administradores em saúde adotem em sua rotina uma coleta de dados sistêmica e integrada para o mapeamento e caracterização das intoxicações. Neste cenário, técnicas de análises multivariadas poderiam ser empregadas para evidenciar possíveis intercorrelações; entretanto, seu uso ainda não é comum na toxicologia clínica. Neste trabalho foram identificados e quantificados agentes exógenos em amostras biológicas (sangue e urina) provenientes do Centro de Controle de Intoxicações da cidade de São Paulo, correlacionando os dados obtidos dessas análises com o perfil clínico e prognóstico dos pacientes. Fármacos benzodiazepínicos, antidepressivos, anticonvulsivantes, paracetamol, drogas de abuso e praguicidas foram selecionados de acordo com a incidência reportada por esse centro no período de 2013 a 2014. Do total de amostras analisadas (n = 320), 192 foram positivas para alguma substância, sendo 101 positivas para etanol e 131 positivas para as demais substâncias. Os dados obtidos foram submetidos à análise de correspondência múltipla e análise hierárquica de cluster. A partir da análise multivariada foi possível agrupar os indivíduos em 3 clusters, o que correspondeu a 66,5% do total de informações. No primeiro eixo houve a separação dos pacientes do gênero feminino, que se intoxicaram ou foram expostos a medicamentos e drogas de abuso, por tentativa de suicídio, dos pacientes do gênero masculino, de idade entre 30 a 39 anos, que se intoxicaram com drogas de abuso. No segundo eixo fatorial foram agrupados os pacientes que se intoxicaram com etanol isoladamente, juntamente com pacientes que se intoxicaram com diazepam. Este trabalho contribuiu para o mapeamento dos casos de intoxicação atendidos pelo CCI-SP e foi um estudo inicial para a criação de um banco de dados que poderá ser alimentado constantemente e assim, oferecer ao sistema de toxicovigilância uma base para políticas educativas. / Toxicology has played an important role in the identification of harmful effects to the population, generating subsidies for decision making in clinical practice, helping in this way the good prognosis of acutely intoxicated patients. According to the Pan American Health Organization, success in any health intervention can only be achieved by creating and maintaining a reliable database that is capable of predicting the various characteristics of intoxications, such as the target population and their susceptibilities. Thus, it is recommended that doctors, specialists, legislators and health administrators adopt in their routine a systemic and integrated data collection for the mapping and characterization of intoxications. In this scenario, multivariate analysis techniques could be used to evidence possible intercorrelations; however, its use is not yet common in clinical toxicology. In this work, were identified and quantified exogenous agents in biological samples (blood and urine) from the Poison Control Center São Paulo city, correlating the data obtained from these analyzes with the clinical and prognostic profile of the patients. Benzodiazepines, antidepressants, anticonvulsants, acetaminophen, drugs of abuse and pesticides were selected according to the incidence reported by this center in the period from 2013 to 2014. Of the total number of samples analyzed (n = 320), 192 samples have shown to be positive for some of the analytes, from these 100 were positive for ethanol and 131 positive for other substances. The data were submitted to multiple correspondence analysis and hierarchical cluster analysis. From the multivariate analysis it was possible to group the individuals into 3 clusters, which corresponded to 66.5% of the total information. In the first axis, the patients were separated from the female gender, who were intoxicated or were exposed to drugs and suicide attempt of the male patients, aged between 30 and 39 years, who became intoxicated with drugs of abuse. In the second factorial axis were grouped the patients who were intoxicated with ethanol, together with patients who became intoxicated with diazepam. This work contributed to the mapping of intoxication cases attended by the CCI-SP and was a initial study for the creation of a database that could be fed constantly and thus provide the toxicovigilance system with a basis for educational policies.

The development of a chip-based sensor array composed of individually addressable polystyrene-polyethylene glycol and agarose microspheres has been demonstrated. The microspheres are selectively arranged in micromachined cavities localized on silicon wafers. These cavities are created with an anisotropic etch and serve as miniaturized reaction vessels and analysis chambers. The cavities possess pyramidal pit shapes with trans-wafer openings that allow for both fluid flow through the microreactors/analysis chambers as well optical access to the chemically sensitive microspheres. Identification and quantification of analytes occurs via colorimetric and fluorescence changes to receptor and indicator molecules that are covalently attached to termination sites on the polymeric microspheres. Spectral data is extracted from the array efficiently using a charge-coupled device (CCD) allowing for the near-real-time digital analysis of complex fluids. The power and utility of this new microbead array detection methodology is demonstrated here for the analysis of complex fluids containing a variety of important classes of analytes including acids, bases, metal cations, sugars and antibody reagents. The application of artificial neural network analyses to the microbead array is demonstrated in the context of pH measurements. To assess the utility of the analysis and gain an understanding of the molecular level design of the sensor, parameters such as the choice of the indicator dyes, array size, data pre-processing techniques, as well as different network types and architectures were evaluated. Additionally, the development of miniaturized chromatographic systems localized within individual polymer microspheres and their incorporation into an array is reported. The integrated chromatographic and detection concept is based on the creation of distinct functional layers within the microspheres. Such beads have been incorporated into the array platform and used for speciation and concentration determination of aqueous metal cation solutions. / text