The past few years have seen a rapid expansion in interest in the characterization of the entire complement of proteins, or proteome. Micro total analysis systems (μTAS) are an emerging promising method, offering rapid, sensitive and low sample consumption separations. I have demonstrated microchip capillary electrophoresis (CE) devices made of CaF2. New methods have been developed for micromachining enclosed capillaries in CaF2. CE analysis of fluorescently labeled amino acids was used to illustrate bioanalytical applications of these microdevices. Initial on-chip infrared spectroscopy results for qualitative analyte identification were achieved in microfluidic CaF2 channels. I have also shown the evaluation of poly(methylmethacrylate) (PMMA) and thermoset polyester (TPE) microchips for use in protein profiling. To improve separation efficiency and reduce protein adsorption, dynamic coating and poly(ethylene glycol) (PEG) grafting using atom transfer radical polymerization (ATRP) have been used in PMMA microdevices. Proteins, peptides and protein digests have been separated electrophoretically in these PMMA microchips. My results demonstrate that PMMA microdevices should be well suited as microfluidic systems for high performance separations of complex biological mixtures. In-channel ATRP has been developed for the surface modification of TPE microdevices. Characterization indicates that PEG-modified microchannels have much lower and more pH-stable electroosmotic flow, more hydrophilic surfaces and reduced nonspecific protein adsorption. CE of amino acid and peptide mixtures in these PEG-modified TPE microchips had good reproducibility. Phosducin-like protein and phosphorylated phosducin-like protein were also separated to measure the phosphorylation efficiency. My results show that PEG-grafted TPE microchips have broad potential application in biomolecular analysis. Cancer marker analysis is important for medical research and applications. I report a method that can covalently attach appropriately oriented antibodies of interest on monolith surfaces. To reduce nonspecific adsorption, protein solutions were used to effectively block the monolith surface. Selective preconcentration and elution of human chorionic gonadotropin have been performed in my affinity columns, demonstrating that this type of system should have promising applications in cancer marker detection.

Developing microdevices on flexible material attracts scientific community to explore applications in different aspects of health and point of care diagnostics. Flexible substrates offer unique characteristics such as flexibility, stretchability, portability, low-cost, and simple fabrication. Fabrication of cost-effective paper-based analytical devices by wax printing has recently become popular using cellulose filter papers. Paper-based devices need higher temperature to form hydrophobic barrier across paper substrate, rely on large working channels (≥ 500 μm) for liquid handling, and exhibit lower efficiency (~50%) of sample mobility. Such limitations confine applications of wax-based fabrication. In this dissertation, we report printability, fidelity, and applications of wax micropatterns on polyethylene terephthalate-based substrate (PET), which is a a non-cellulosic, non-fibrous, and non-porous material. Resolution, sustainability against heat and biocompatibility was tested on wax micro-features. The patterned devices were explored for variety of applications.First, wax microwells on PET showed mouse embryonic stem cell (mESC) self-renewal or direct differentiation. Second, microfluidic flow was demonstrated on wax printed microchannels on PET which was used to develop distance-based assay. Third, fluidic properties of trinucleotide repeat sequences were investigated on wax microchannels. Fourth, multilayer wax-on-plastic device was fabricated using wax printing with hand painting of conductive materials for electrochemical immunosensing.

No description available.

Microfluidics is a continually growing field covering a wide range of applications, such as cellular analysis, biomarker quantification, and drug discovery; but in spite of this, the field of microfluidics remains predominately academic. New materials are pivotal in providing tailored properties to improve device integration and decrease prototype turnaround times. In biosensing, nonspecific adsorption in microfluidic systems can deplete target molecules in solution and prevent analytes, especially those at low concentrations, from reaching the detector. Polyethylene glycol diacrylate (PEGDA) mixed with photoinitiator forms, on exposure to ultraviolet (UV) radiation, a polymer with inherent resistance to nonspecific adsorption. Optimization of the polymerized PEGDA (poly-PEGDA) formula imbues this material with some of the same properties, including optical clarity, water stability, and low background fluorescence, that makes polydimethylsiloxane (PDMS) a widely used material for microfluidics. Poly-PEGDA demonstrates less nonspecific adsorption than PDMS over a range of concentrations of flowing fluorescently tagged bovine serum albumin solutions, and poly-PEGDA has greater resistance to permeation by small hydrophobic molecules than PDMS. Poly-PEGDA also exhibits long-term (hour scale) resistance to nonspecific adsorption compared to PDMS when exposed to a low (1 μg/mL) concentration of a model adsorptive protein. Electrophoretic separations of amino acids and proteins resulted in symmetrical peaks and theoretical plate counts as high as 4 × 105/m. Pneumatically actuated, non-elastomeric membrane valves fabricated from poly-PEGDA have been characterized for temporal response, valve closure, and long-term durability. A ∼100 ms valve opening time and a ∼20 ms closure time offer valve operation as fast as 8 Hz with potential for further improvement. Comparison of circular and rectangular valve geometries indicates that the surface area for membrane interaction in the valve region is important for valve performance. After initial fabrication, the fluid pressure required to open a closed circular valve is ∼50 kPa higher than the control pressure holding the valve closed. However, after ∼1000 actuations to reconfigure polymer chains and increase elasticity in the membrane, the fluid pressure required to open a valve becomes the same as the control pressure holding the valve closed. After these initial conditioning actuations, poly-PEGDA valves show considerable robustness with no change in effective operation after 115,000 actuations.Often, localized areas of surface functionalization are desired in biosensing, necessitating site-specific derivatization. Integration of poly-PEGDA with different substrates, such as glass, silicon, or electrode-patterned materials, allows for broad application in biosensing and microfluidic devices. Deposition of 3-(trimethoxysilyl) propyl methacrylate or (3-acryloxypropyl) dimethylmethoxysilane onto these substrates makes bonding to poly-PEGDA possible under UV exposure. Primary deposition of (3-acryloxypropyl) dimethylmethoxysilane, followed by photolithographic patterning, allows for silane removal through HF surface etching in the exposed areas and subsequent deposition of 3 aminopropyldiisopropylethoxysilane on the etched regions. Fluorescent probes are used to evaluate surface attachment methods. Primary attachment via reaction of Alexa Fluor 488 TFP ester to the patterned aminosilane demonstrates excellent fluorescent signal. Initial results with glutaraldehyde were demonstrated but require more optimization before this method for secondary attachment is viable. Fabrication of 3D printed microfluidic devices with integrated membrane-based valves is performed with a low-cost, commercially available stereolithographic 3D printer and a custom PEGDA resin formulation tailored for low non-specific protein adsorption. Horizontal microfluidic channels with designed rectangular cross sectional dimensions as small as 350 µm wide and 250 µm tall are printed with 100% yield, as are cylindrical vertical microfluidic channels with 350 µm designed (210 µm actual) diameters. Valves are fabricated with a membrane consisting of a single build layer. The fluid pressure required to open a closed valve is the same as the control pressure holding the valve closed. 3D printed valves are successfully demonstrated for up to 800 actuations. Poly-PEGDA is a versatile material for microfluidic applications ranging from electrophoretic separations, valve implementation, and heterogeneous material integration. Further improvements in PEGDA resin formulation, in combination with a UV source 3D printer, will provide poly-PEGDA devices that are not only rapidly fabricated (<40 min per device), but that also include pumps and valves and are usable with a variety of detection methods, such as laser-induced fluorescence and immunoassays, for broad application in biosensing.

Enzymes are implicated in many diseases including neurodegenerative, cancer, immune deficiency, and inflammatory disorders. There is a constant need to develop novel drug compounds that target enzymes in order to modulate their function, thus treating the disease state. These compounds are typically small molecules with affinity to the enzyme active site or an allosteric site. In order to discover novel compounds for treating disease, the interaction between an enzyme and a small molecule must first be identified and then characterized. With the target enzyme known, it is beneficial to screen libraries of compounds against the target. Immobilizing the enzyme allows for pre-concentration of ligands on the surface and therefore increased signal enhancement, as well as permitting multiple wash steps and enzyme reuse. Immobilized enzyme columns are optimal for coupling to a variety of detection devices by way of liquid chromatography, including absorbance or mass spectrometric detection. Immobilized enzyme reactors (IMERs) were generated and optimized for two target molecules, acetylcholinesterase (AChE) and adenosine deaminase (ADA), for rapid function-based screening of enzyme inhibitors in mixtures. The IMER mode is useful for increasing throughput and facilitating the identification of hit mixtures, but it is slow and tedious to manually deconvolute hit compounds from mixtures and the IMER method is not amenable to natural product extracts, which are good sources of structurally diverse compounds that are more likely to result in a hit compound. Bio-selective solid-phase extraction (BioSPE) is an orthogonal method of isolating and identifying enzyme inhibitors in a single step, and was used to easily deconvolute complex mixtures, rapidly identifying to key compounds EHNA and MAC-0038732 out of mixtures using ADA columns. A data dependent acquisition MS method was developed and used to screen a set of fungal endophyte extracts, identifying two potentially novel inhibitors that were confirmed by IMER-MS/MS. / Thesis / Doctor of Philosophy (PhD) / The discovery of new drug compounds is crucial for the treatment of diseases. Enzymes are proteins that turn a substrate into a product; and in diseases they can often malfunction, overproducing the product. Small molecule compounds can sometimes inhibit enzyme function and can be further developed into therapeutic drugs. This thesis describes a method for detecting small molecule inhibitors that bind to an enzyme that is immobilized in a small column. Once the small molecule is bound to the immobilized enzyme, it can be detected by either showing that enzyme function is inhibited or by removing the compound from the enzyme and identifying the compound by mass spectrometry. These methods can quickly identify compounds at extremely low levels from complex mixtures, such as natural product extracts.

No description available.

No description available.

A reação peroxioxalato é o sistema quimiluminescente não enzimático de maior eficiência, alcançando rendimentos quânticos de até 50%. A quimiluminescência deste sistema vem sendo amplamente utilizada em aplicações analíticas e bioanalíticas visando a detecção de analitos de interesses biológicos e medicinais. O presente trabalho consistiu em se estudar a reação peroxioxalato com diferentes catalisadores e determinação de seu mecanismo em condições experimentais distintas das que vinham sendo estudadas até o momento, visando sua adaptação a meios aquosos para futuras aplicações. Foi estudada a utilização do salicilato de sódio como catalisador deste sistema em meio puramente orgânico, em substituição ao imidazol, e verificou-se a atuação do salicilato como catalisador básico geral e/ou específico. O rendimento quântico máximo desta transformação foi da ordem de 10-3 E mol-1. Ensaios cinéticos de emissão também foram realizados com ésteres oxálicos de diferentes reatividades em um sistema binário composto por 1,2-dimetoxietano/água contendo tampão fosfato como catalisador, em diferentes valores de pH. Nestas condições se observou a ocorrência de catálise ácida geral e catálise básica geral, uma vez que as constantes de velocidade se mostraram dependentes da concentração de tampão no meio reacional. Além disso, também se utilizou salicilato de sódio como catalisador no sistema binário 1,2-dimetoxietano/água, o qual também apresentou um papel catalítico importante no percurso desta transformação com diferentes ésteres oxálicos. Verificou-se nestes estudos a boa reprodutibilidade da reação peroxioxalato com salicilato de sódio como catalisador em meios parcialmente aquosos, que pode ampliar a utilização deste sistema quimiluminescente em aplicações analíticas e bioanalíticas. Quando se realizou os ensaios em um meio majoritariamente aquoso, o salicilato não se mostrou um catalisador eficiente, porém, mesmo sem catalisador, a reação peroxioxalato se mostrou reprodutível nestas condições / The peroxyoxalate reaction is the non-enzymatic chemiluminescence system with the highest efficiency, achieving quantum yields of up to 50%. The chemiluminescence of this system has been widely used in analytical and bionanalytical applications in order to detect analytes of biological and medicinal interests. The present work consisted in a study of the peroxyoxalate reaction with different catalysts and the determination of its mechanism in experimental conditions different from those studied before, aiming its adaptation to aqueous media for future applications. The use of sodium salicylate as base catalyst for this system in pure organic medium, in substitution to imidazole, was studied and shown that salicylate acts as a general and/or specific base catalyst. The maximum quantum yield obtained for the transformation in these conditions was in the order of 10-3 E mol-1. Emission experiments were also performed in a binary solvent system composed of 1,2- dimethoxyethane/aqueous phosphate buffer at different pH values as catalyst, using oxalic esters with different reactivities. In these conditions the occurrence of general acid catalysis and general base catalysis was observed, since the rate constants proved to be dependent on the buffer concentration. In addition, sodium salicylate was also used as catalyst in the binary 1,2-dimethoxyethane/water system with different oxalic esters, indicating its important catalytic role in the transformation. These studies allowed it to establish a reproducible peroxyoxalate system in partially aqueous media using sodium salicylate as base catalyst, which may increase the use of the chemiluminescence of this system in analytical and bioanalytical applications. However, when the experiments were performed in a medium containing mostly water, salicylate did not act as an efficient catalyst in these conditions. Even in the absence of catalyst, the reaction proved to be reproducible in the medium containing mostly water as solvent

Os imunoensaios e os ensaios bioquímicos são amplamente utilizados em clinica médica. Os dispositivos fabricados em papel devido ao seu baixo custo, portabilidade, todas as etapas serem realizadas em temperatura ambiente, e possibilidade da produção local dos dispositivos, tornam-se ideais para serem aplicados em regiões carentes. Assim, desenvolvemos um ensaio imunocromatográfico que permitiu a detecção de IgG de coelho em um dispositivo com uma única camada de papel impressa por cera, mostrando que esse protótipo tem potencial de ser aplicado em diferentes ensaios imunológicos. Pela primeira vez foi utilizado um teste enzimático colorimétrico (sarcosina oxidase, peroxidase e o indicador redox (ABTS) em plataforma de papel, impressa por cera, para detecção de sarcosina, o qual detectou um potencial marcador de tumor de câncer de próstata, a sarcosina, com limite de detecção (LD) = 0,21 mmol L-1 e limite de quantificação (LQ) = 0,61 mmol L-1, constatando que a intensidade da cor formada foi proporcional a concentração de sarcosina presente na amostra. Os imunoensaios em papel se mostraram extremamente versáteis, capazes de detectar diferentes analitos. O primeiro dispositivo foi capaz de detectar toxoplasmose (IgG contra T. gondii presente nas amostras). A avaliação da performance do teste nos forneceu um cut-off =21,73 U.A, sensibilidade = 0,96, especificidade = 0,87, AUC = 0,97, além de uma criação de uma zona cinza utilizando uma tolerância em porcentagem sobre a o cut-off de 15%. Desenvolvemos também uma macro no excel qye calcula a acurácia, m-Acuraccy, a qual nos forneceu um valor de 0,88 U.A. O segundo dispositivo permitiu a detecção do marcador tumoral CEA, através de um ensaio do tipo sanduíche, com um cut-off =68,28 U.A, sensibilidade = 0,86, especificidade = 1, AUC = 0,97. A tolerância em porcentagem sobre a o cut-off para a criação da zona cinza foi de 12%, e a m-Acuraccy calculou uma acurácia de 0,90 U.A. Pela primeira vez, foi aplicada essa completa avaliação estatística em testes em papel. Mais do que isso, trazemos com a m-Acuraccy uma nova forma de calcular a acurácia, com grande inovação na clínica médica. Portanto, torna-se evidente o grande potencial que os dispositivos fabricados em papel possuem para ser aplicados como ferramentas diagnósticas. / Immunoassays and bioassays are broadly used in clinical medicine. Paper-based devices are ideal to be used in remote regions due to their low-cost, portability and the possibility of in loco manufacture. Paper-based immunoassays are extremely versatile, capable of detecting distinct analytes: initially we have developed an immunochromatographic assay to detect rabbit IgG in a paper-based device fabricated using wax printing technology, and we have shown that this prototype has potential to be applied in distinct immunoassays. The second developed paper-based device was an enzymatic colorimetric assay for the detection of a potential prostate cancer biomarker - sarcosine (sarcosine oxidase, peroxidase and redox indicator (ABTS)), obtaining good figures or merit (LOD = 0.21 mmol L-1; LOQ = 0.61 mmol L-1, r² = 0.890). The third developed paper-based device was capable of detecting toxoplasmosis (IgG against Toxoplasma gondii in human serum samples). The performance evaluation showed a cut-off = 21.73 A.U., sensitivity = 0.96, specificity = 0.87, AUC = 0.97, besides defining the gray zone as the zone comprehended in-between 15% over the cut-off value. We also have developed a Microsoft Excel® macro to calculate diagnostic test\'s accuracy - m-Accuracy - that is a new way to calculate accuracy with great innovation for clinical medicine, which resulted in an accuracy of 0.88. for toxoplasmosis assay. The fourth developed paper-based device was used to detect CEA tumor biomarker using a sandwich ELISA assay, with a cut-off = 68.28 A.U., sensitivity = 0.86, specificity = 1.0, AUC = 0.97. The defined gray zone to this test was the zone comprehended in-between 12% over the cut-off value, with an accuracy of 0.90. To the best of our knowledge, this is the first complete statistical evaluation of paper-based diagnostic devices, which showed the great potential of this technology to be used as a new point-of care diagnostic tool.

A reação peroxioxalato é o sistema quimiluminescente não enzimático de maior eficiência, alcançando rendimentos quânticos de até 50%. A quimiluminescência deste sistema vem sendo amplamente utilizada em aplicações analíticas e bioanalíticas visando a detecção de analitos de interesses biológicos e medicinais. O presente trabalho consistiu em se estudar a reação peroxioxalato com diferentes catalisadores e determinação de seu mecanismo em condições experimentais distintas das que vinham sendo estudadas até o momento, visando sua adaptação a meios aquosos para futuras aplicações. Foi estudada a utilização do salicilato de sódio como catalisador deste sistema em meio puramente orgânico, em substituição ao imidazol, e verificou-se a atuação do salicilato como catalisador básico geral e/ou específico. O rendimento quântico máximo desta transformação foi da ordem de 10-3 E mol-1. Ensaios cinéticos de emissão também foram realizados com ésteres oxálicos de diferentes reatividades em um sistema binário composto por 1,2-dimetoxietano/água contendo tampão fosfato como catalisador, em diferentes valores de pH. Nestas condições se observou a ocorrência de catálise ácida geral e catálise básica geral, uma vez que as constantes de velocidade se mostraram dependentes da concentração de tampão no meio reacional. Além disso, também se utilizou salicilato de sódio como catalisador no sistema binário 1,2-dimetoxietano/água, o qual também apresentou um papel catalítico importante no percurso desta transformação com diferentes ésteres oxálicos. Verificou-se nestes estudos a boa reprodutibilidade da reação peroxioxalato com salicilato de sódio como catalisador em meios parcialmente aquosos, que pode ampliar a utilização deste sistema quimiluminescente em aplicações analíticas e bioanalíticas. Quando se realizou os ensaios em um meio majoritariamente aquoso, o salicilato não se mostrou um catalisador eficiente, porém, mesmo sem catalisador, a reação peroxioxalato se mostrou reprodutível nestas condições / The peroxyoxalate reaction is the non-enzymatic chemiluminescence system with the highest efficiency, achieving quantum yields of up to 50%. The chemiluminescence of this system has been widely used in analytical and bionanalytical applications in order to detect analytes of biological and medicinal interests. The present work consisted in a study of the peroxyoxalate reaction with different catalysts and the determination of its mechanism in experimental conditions different from those studied before, aiming its adaptation to aqueous media for future applications. The use of sodium salicylate as base catalyst for this system in pure organic medium, in substitution to imidazole, was studied and shown that salicylate acts as a general and/or specific base catalyst. The maximum quantum yield obtained for the transformation in these conditions was in the order of 10-3 E mol-1. Emission experiments were also performed in a binary solvent system composed of 1,2- dimethoxyethane/aqueous phosphate buffer at different pH values as catalyst, using oxalic esters with different reactivities. In these conditions the occurrence of general acid catalysis and general base catalysis was observed, since the rate constants proved to be dependent on the buffer concentration. In addition, sodium salicylate was also used as catalyst in the binary 1,2-dimethoxyethane/water system with different oxalic esters, indicating its important catalytic role in the transformation. These studies allowed it to establish a reproducible peroxyoxalate system in partially aqueous media using sodium salicylate as base catalyst, which may increase the use of the chemiluminescence of this system in analytical and bioanalytical applications. However, when the experiments were performed in a medium containing mostly water, salicylate did not act as an efficient catalyst in these conditions. Even in the absence of catalyst, the reaction proved to be reproducible in the medium containing mostly water as solvent