The purpose of this thesis project is to demonstrate and evaluate an enzyme-linked immunosorbent assay (ELISA) on a paper microfluidic device platform. The integration of ELISA technology onto paper microfluidic chips allows for a quantitative detection of stroke biomarkers, such as glial fibrillary acidic protein (GFAP). Dye experiments were performed to confirm fluid connectivity throughout the 3D chips. Several chip and housing designs were fabricated to determine an optimal design for the microfluidic device. Once this design was finalized, development time testing was performed. The results confirmed that the paper microfluidic device could successfully route fluid throughout its channels at a reasonable rate. For the biochemistry portion of this thesis project, antibodies were selected to target the intended stroke biomarker: GFAP. However, due to antibody pairing complications, the protein chosen for this project was natural human cardiac troponin T, which is elevated in the bloodstream of patients who have suffered a stroke. Several antibody experiments were performed to help finalize the procedure for performing an ELISA on the paper chip. The final antibody experiment was able to demonstrate that a paper microfluidic device utilizing ELISA techniques can successfully detect a stroke biomarker at physiologically relevant concentrations. Overall, this project supported the ability to accurately and effectively diagnose stroke in a timely manner through the use of a paper microfluidic device.

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<p>Research in paper-based analytical devices has been increasing rapidly in recent years. Manyof these devices are used as low-cost alternatives for diagnostics and biosensing. In this work,two novel paper-based technologies were developed.</p> <p>The first paper-based technology achieved was measuring streaming potential on paper-based microfluidic devices. The streaming potential measurements were able to detect the presence of adsorbed polyvinylamine or potassium polyvinylsulfate in paper-based microfluidic channels.</p> <p>The measured streaming potential ranged from -80 mV to 80 mV and the polarity was sensitive to the adsorbed polymer. Furthermore, the measured streaming potential on paper treated with BSA showed a polarity switch when the pH was changed from below the pKa to above the pKa of BSA. Lastly, streaming potential measurements may provide an electronic interface for paperbased sensors.</p> <p>The second technology developed was a paper-based chromatographic pre-concentration device for biological and chemical applications. The device successfully concentrated a protein, streptavidin, via biotinylated microgels immobilized onto a selected area of the filter paper. The device was able to process a large volume of fluid with the incorporation of a passive pump made of superabsorbent polymer. The concentration factor achieved by the device was over 3000-fold. The flow dynamics through the paper was modeled using Darcy’s law. This technology could be an excellent low-cost alternative for biochemical analysis for samples thatrequire preconcentration, especially for the analysis of trace compounds in wastewater and drinking water.</p> / Master of Applied Science (MASc)

Microbial biosensor systems (MBS) are useful for analyte detection owing to their low cost, sensitivity, and selectivity for bioavailable analytes. Due to typically poor shelf-life and sensitivity to external conditions, there are few reports of MBS technology applied to simple analytical devices. The effectiveness of air-drying MBS in natural polymers was investigated as a novel preservation technique. Two colorimetric Escherichia coli MBS, a tetracycline-inducible reporter and an arsenate-inducible reporter, were dried on various substrates yielding novel MBS platforms. In proof-of-concept experiments performed in 96-well microplates, both systems demonstrated responsivity after air-drying in low concentrations of pullulan. However, the MBS were unresponsive following brief storage of 1 week. To improve the preservation of MBS, sensing strips were created by air-drying concentrated acacia gum-based MBS suspensions onto paper. Cells dried on these strips demonstrated responsivity upon solubilization in various tube-based assays. MBS sensing strip responsivity was demonstrated following storage for 6 weeks at 4 °C. Tetracycline-responsive sensing strips also performed well in assays using spiked lake water samples. Air-drying in natural polymers was an effective MBS preservation technique, and allowed for the creation of “mix and read” style assays which were simple, equipment-free and ready-to-use. / Thesis / Master of Science (MSc)

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.

O objetivo principal desta tese foi o de se desenvolver uma biocélula a combustível enzimática em microfluídica, utilizando a glicose como combustível e o oxigênio como oxidante. Foram utilizadas as enzimas Glicose Oxidase ou Glicose Desidrogenase em um bioânodo, de forma a promover reações bioeletrocatalíticas de oxidação da glicose e as enzimas Lacase ou Bilirrubina Oxidase, de forma a promover reações bioeletrocatalíticas de redução do oxigênio molecular. O trabalho se procedeu por tentativas de imobilizar estas enzimas, de forma a promover o mecanismo de transferência eletrônica direta com um eletrodo. Nas situações as quais isso não foi possível, foram utilizados mediadores eletrônicos, de forma a promover o mecanismo de transferência eletrônica mediada. O melhor par de sistemas de bioeletrodos e mediadores foi escolhido para serem aplicados em uma biocélula a combustível. O trabalho se procedeu em adaptar este par de bioeletrodos desenvolvidos para um sistema de microfluídica em papel, sendo ambos biocátodo e bioânodo em papel. Como as condições de concentração de combustível e de cofatores foram otimizadas para o bioânodo, foi necessário trabalhar com os biocátodos, de forma a apresentar as características de um biocátodo respirador, para melhor utilizar o oxigênio presente no ar e a apresentar um desempenho tão bom quanto o dos bioânodos. A biocélula a combustível em papel possibilitou a geração de energia elétrica por até 18 dias, utilizando uma resistência de 1.7 k&Omega;, nas condições experimentais ideais. De forma a provar o conceito da tecnologia para aplicações reais, a biocélula a combustível em papel foi demonstrada a ter a capacidade de geração de energia elétrica suficiente para fazer um relógio funcionar por pelo menos 36 horas, utilizando a bebida isotônica Gatorade&reg;, como combustível. / The main objective of this thesis is to develop a microfluidic biofuel cell using glucose as the fuel and oxygen as the oxidant. The enzymes Glucose Oxidase or Glucose Dehydrogenase were used in a bioanode to promove the bioelectrocatalytic oxidation of glucose and the enzymes Laccase or Bilirubin Oxidase to promove the bioelectrocatalytic reduction of the molecular oxygen. The work was conducted by attempts to immobilize these enzymes in order to promote the mechanism of direct electron transfer with the electrode. For the situations where this was not observed, mediators were used in a way to promote the mechanism of mediated electron transfer. The best pair of bioelectrodes and mediatores was chosen to be applied in a biofuel cell. The work was carried out to adapt this par of developed bioelectrodes to a paper based microfluidic system, using both biocathode and bioanode in a paper-like design. As the conditions for concentration of fuel and cofactors were optimized for the bioanode, it was necessary to work on these biocathodes so as to have the characteristics of an air-breathing biocathode for a better use of the oxygen present in the air and to work with a performance as good as the bioanode. The paper based biofuel cell enabled the generation of electricity for up to 18 days using a resistance of 1.7 k&Omega; within the optimum experimental conditions. In order to prove the concept of this technology for real applications, the paper based biofuel cell was demonstrated to have the capacity for generation of enough electrical energy to power up a clock for at least 36 hours using the isotonic drink Gatored&reg; as fuel.

Conductive paper materials consisting of conductive polymers and cellulose are promising for high-tech applications (energy storage and biosciences) due to outstanding aspects of environmental friendliness, mechanical flexibility, electrical conductivity and efficient electroactive behavior. Recently, a conductive composite paper material was developed by covering the individual nanofibers of cellulose from the green algae Cladophora with a polypyrrole (PPy) layer. The PPy-Cladophora cellulose composite paper is featured with high surface area (80 m2 g-1), electronic conductivity (~2 S cm-1), thin conductive layer (~50 nm) and easily up-scalable manufacturing process. This doctoral thesis reports the development of the PPy-Cladophora composite as an electrode material in electrochemically controlled solid phase ion-exchange of biomolecules and all-polymer based energy storage devices. First, electrochemical ion-exchange properties of the PPy-Cladophora cellulose composite were investigated in electrolytes containing three different types of anions, and it was found that smaller anions (nitrate and chloride) are more readily extracted by the composite than lager anions (p-toluene sulfonate). The influence of differently sized oxidants used during polymerization on the anion extraction capacity of the composite was also studied. The composites synthesized with two different oxidizing agents, i.e. iron (III) chloride and phosphomolybdic acid (PMo), were investigated for their ability to extract anions of different sizes. It was established that the number of absorbed ions was larger for the iron (III) chloride-synthesized sample than for the PMo-synthesized sample for all four electrolytes studied. Further, PPy-Cladophora cellulose composites have shown remarkable electrochemically controlled ion extraction capacities when investigated as a solid phase extraction material for batch-wise extraction and release of DNA oligomers. In addition, composite paper was also investigated as an electrode material in the symmetric non-metal based energy storage devices. The salt and paper based energy storage devices exhibited charge capacities (38−50 mAh g−1) with reasonable cycling stability, thereby opening new possibilities for the production of environmentally friendly, cost efficient, up-scalable and lightweight energy storage systems. Finally, micron-sized chopped carbon fibers (CCFs) were incorporated as additives to improve the charge-discharge rates of paper-based energy storage devices and to enhance the DNA release efficiency. The results showed the independent cell capacitances of ~60-70 F g-1 (upto current densities of 99 mA cm2) and also improved the efficiency of DNA release from 25 to 45%.

Paper is renewable, recyclable, sustainable and biodegradable material and, as a result, paper-based materials are widely used in the world packaging market. However, paper-based materials cannot compete with plastics in terms of processability into various 3D shapes. This is due to poor formability of paper, which is closely associated with its toughness. To improve paper formability, we report on a facile and green method that combines fiber and paper mechanical modifications at different structural levels as well as biopolymer treatment via spraying. As a result, a remarkable elongation of ∼30% was achieved after proposed combined approach on the laboratory scale. At the same time, a significant increase in tensile strength and stiffness (by ∼306% and ∼690%, respectively) was observed. Overall, an inexpensive, green, and scalable approach is introduced to improve formability of fiber networks that in turn allows preparation of 3D shapes in the processes with fixed paper blanks such as vacuum forming, hydroforming, hot pressing, etc.