Previous studies have shown that pullulan films are able to stabilize enzymes and other labile molecules from thermal and oxidative degradation. Solutions made with commercially available pullulan are extremely viscous and difficult to process limiting the ability to use low-cost printing systems, such as inkjet printers, to format pullulan-containing. In this work, we show that pullulan can be made printable by decreasing its chain length by acid hydrolysis. The acid hydrolysis reaction was modelled using statistical software; the molecular weight of pullulan decreased with increasing reaction time, temperature and acid concentration. Interactions between time and temperature, and temperature and acid concentration were determined to be significant to the reaction as well. The mechanical properties and oxygen permeability of films made from pullulan with different molecular weights were also measured. The films were found to have similar tensile properties and oxygen permeabilities to each other and to those obtained using native pullulan. Using a thermally unstable enzyme (acetylcholinesterase) and an easily oxidizable small molecule (indoxyl acetate) as test materials, it was found that these films have the same ability to stabilize the enzyme and to serve as an oxygen barrier, as the films made with native pullulan. It was also found that pullulan is inkjet printable as long as the molecular weight is 56 kDa. Poor jetting and clogging of the printhead was observed when pullulan with a molecular weight higher than this threshold was used. Microarray printing was also demonstrated by a printing acetylcholinesterase/pullulan in nano-sized volumes using a Dimatix inkjet printer and showing activity of the enzyme after printing and storage at ambient conditions. Proof of concept of microarray printing opens up the potential for future applications of pullulan in other high throughput applications. / Thesis / Master of Applied Science (MASc)

Microfluidic paper-based analytical devices (microPADs) are an emerging platform for point-of-care diagnostic tests for use by untrained users with potential applications in healthcare, environmental monitoring, and food safety. These devices can be developed for a multitude of different tests, many of which employ enzymes as catalysts. Without specialized treatment, some enzymes tend to lose their activity when stored on microPADs within 48 hours, which is a major hurdle for taking these types of devices out of the laboratory and into the real world. This work focused on the development of simple methods for stabilizing enzymes by applying polymers to chromatography paper. The longterm stabilization was exlored and SU-8 of various concentrations was found to stabilize horseradish peroxidase for times in excess of two weeks. A variety of microPAD fabrications, enzyme dispensing methods, and substrate delivery techniques were explored.

Lipases são enzimas que catalisam a hidrólise de triacilgliceróis em ácidos graxos, mono e diacilgliceróis e glicerol. Em contraste com as esterases, lipases são ativadas apenas quando estão adsorvidas a uma interface óleo-água. Lipases têm sido amplamente utilizadas em muitos processos industriais, tais como química orgânica, formulações de detergentes e de produtos como cosméticos e farmacêuticos. A principal preocupação na produção de enzimas comerciais é a proteção da sua estabilidade em solução aquosa. A água facilita ou medeia uma variedade de vias de degradação física e química, durante as etapas de purificação, transporte e armazenamento. Por conseguinte, formulações sólidas são desenvolvidas para alcançar uma vida útil aceitável para essas substâncias. Spray drying é comumente usado como uma técnica de desidratação na indústria farmacêutica para fabricação de produtos em pó diretamente do estado líquido. No presente trabalho, a purificação e caracterização bioquímica de lipases produzidas pelo fungo endofítico Cercospora kikuchii, bem como os efeitos de adjuvantes no processo de secagem destas enzimas foram estudados. A lipase bruta foi purificada à homogeneidade através de cromatografia de interação hidrofóbica e gel filtração. A lipase foi purificada 5,54 vezes, com rendimento de 9% e a atividade específica de 223,6 U/mg. O peso molecular da enzima foi estimado em 65,1 kDa por SDS-PAGE e 73,5 kDa utilizando cromatografia de gel filtração, indicando que provavelmente trata-se de um monômero. A lipase mostrou um pH ótimo em 4,6 e uma temperatura ótima de 35°C. Cerca de 80,2% de sua atividade foi mantida após incubação a 40°C durante 2 horas. A Vmax e Km foram 10,28 mmol/min/mg de proteína e 0,03240 mM, respectivamente, utilizando pNPP como substrato. As lipases presentes no extrato bruto e as lipases ligadas ao micélio foram caracterizadas para avaliar o potencial de utilização em biocatálise. A lipases no extrato bruto apresentaram atividade máxima a 60ºC e pH 6,2, enquanto que as lipases ligadas ao micélio apresentaram atividade máxima a 50ºC e pH 5,4. Nos estudos de efeito da temperatura sobre a atividade enzimática, as lipases no extrato bruto mantiveram-se estáveis a 50°C, com 85,3% de atividade residual após 2 horas de incubação. As lipases ligadas ao micélio mantiveram pelo menos 75,1% de atividade residual após 2 horas de incubação a 80°C. Estes resultados mostram que as lipases de C. kikuchii têm propriedades cinéticas e termoestabilidade desejáveis para aplicações em biocatálise. As lipases presentes no extrato bruto foram secas em spray dryer com diferentes adjuvantes, e sua estabilidade foi avaliada. A recuperação da atividade enzimática após a secagem, com a adição de 10% de lactose, -ciclodextrina, maltodextrina, manitol, goma arábica, e trealose variou de 63 a 100%. A atividade da enzima foi totalmente perdida durante a secagem do extrato bruto na ausência de adjuvantes. A maioria dos adjuvantes utilizados manteve pelo menos 50% da atividade enzimática a 5°C e 40% a 25°C, após 8 meses de armazenagem. As lipases secas com 10% de - ciclodextrina mantiveram 72% da atividade a 5°C no mesmo período. A partir destes resultados preliminares foi realizada a otimização do processo de secagem utilizando -ciclodextrina, maltodextrina e lactose como adjuvantes. A análise estatística dos resultados experimentais permitiu a determinação das condições ótimas para a retenção da atividade enzimática (RAE), a saber: concentração de adjuvantes de secagem de 12,05%, temperatura de entrada do gás de secagem em 153,6oC e vazão do extrato enzimático alimentado de 9,36 g/min, para - ciclodextrina e maltodextrina como adjuvantes. Para lactose, o estudo mostrou que o aumento da quantidade de adjuvante de secagem e/ou diminuindo a temperatura do gás de entrada tem um efeito positivo sobre a retenção da atividade enzimática do produto seco. Após o processo de purificação foi realizada a secagem da enzima parcialmente purificada e da lipase pura, com estes três adjuvantes. A manutenção da atividade enzimática variou 90,6-100% quando foram utilizadas as condições ótimas para cada adjuvante de secagem. Concluindo, as lipases produzidas por C. kikuchii podem ser eficientemente secas por spray dryer, uma vez que a atividade enzimática foi mantida no extrato bruto, na lipase pura e na lipase semi-purificada submetidas à secagem. / Lipases are enzymes that catalyze the hydrolysis of triacylglycerols to fatty acids, mono and diacylglycerols, and glycerol. In contrast to esterases, lipases are activated only when they are adsorbed to an oilwater interface. They have been widely used in many industrial processes such as organic chemical, detergent and cleaning formulations and in products like cosmetics and pharmaceutical products. The main concern in the production of commercial enzymes is to protect their stability in aqueous solution. Water facilitates or mediates a variety of physical and chemical degradation pathways, active during protein purification, shipping and storage. Consequently, dry solid formulations are developed to achieve an acceptable protein shelf life. Spray drying is commonly used as a dehydration technique in the pharmaceutical industry for making powdery products directly from the liquid. In the present work, the purification and biochemical characterization of lipases produced by endophytic fungus Cercospora kikuchii as well as the effects of adjuvants on the spray drying process of theses enzymes were studied. The crude lipase was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The lipase purified was 5.54-fold with 9% recovery and the specific activity was 223.6. The molecular mass of the lipase was estimated to be 65.1 kDa using SDS-PAGE and 73.5 using gel filtration chromatography, indicating that the lipase is a monomer. The lipase demonstrated an optimum pH at 4.6, an optimum temperature of 35°C. About 80.2% of its activity was retained after incubation at 40°C for 2 hours. The Vmax and Km were 10.28 mol/min/mg protein and 0.03240 mM, respectively, using pNPP as substrate. The lipases present in crude extract and the mycelium-bound lipases were characterized in order to evaluate the potential for use in biocatalysis. The crude extract showed maximum activity at 60ºC and pH 6.2 while the myceliumbound lipases showed maximum activity at 50ºC and pH 5.4. In tests of the temperature effect on the enzymatic activity, the lipases in the crude extract was stable at 50°C, with 85.3% residual activity after 2 hours of incubation. The mycelium-bound lipases maintained at least 75.1% of residual activity after 2 h incubation at 80°C. These results show that the lipases of C. kikuchii have kinetic properties and stability characteristics suitable to applications in biocatalysis. The lipases present in crude extract were spray dried with different adjuvants, and their stability was evaluated. The recovery of the enzyme after drying with 10% of lactose, -cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63% to 100%; but the enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5°C and 40% at 25°C after 8 months. The lipase dried with 10% of -cyclodextrin retained 72% of activity at 5°C. From these preliminary results the optimization of drying process using -cyclodextrin, maltodextrin and lactose as adjuvants was carried out. Statistical optimization of the experimental results allowed the determination of the processing conditions that maximized the retention of the enzymatic activity (RAE), namely: concentration of drying adjuvants of 12.05 %, inlet temperature of the drying gas of 153.6oC, and flow rate of the enzymatic extract fed to the dryer of 9.36 g/min, for the b-cyclodextrin and maltodextrin as adjuvants. For lactose as adjuvant the study showed that increasing the amount of drying adjuvant and/or decreasing the inlet gas temperature has positive effect on the retention of enzymatic activity of the dried product. After the purification process was carried out the drying of the partially purified enzyme and pure lipase, using these three adjuvants. The retention of enzymatic activity ranged from 90.6 to 100% when was used the optimal conditions for each drying adjuvant. Concluding, the lipases produced by C. kikuchii may be efficiently spray dried since its activity enzimatic was retained in crude extract, pure lipase and in semi-purified lipase after drying.

Lipases são enzimas que catalisam a hidrólise de triacilgliceróis em ácidos graxos, mono e diacilgliceróis e glicerol. Em contraste com as esterases, lipases são ativadas apenas quando estão adsorvidas a uma interface óleo-água. Lipases têm sido amplamente utilizadas em muitos processos industriais, tais como química orgânica, formulações de detergentes e de produtos como cosméticos e farmacêuticos. A principal preocupação na produção de enzimas comerciais é a proteção da sua estabilidade em solução aquosa. A água facilita ou medeia uma variedade de vias de degradação física e química, durante as etapas de purificação, transporte e armazenamento. Por conseguinte, formulações sólidas são desenvolvidas para alcançar uma vida útil aceitável para essas substâncias. Spray drying é comumente usado como uma técnica de desidratação na indústria farmacêutica para fabricação de produtos em pó diretamente do estado líquido. No presente trabalho, a purificação e caracterização bioquímica de lipases produzidas pelo fungo endofítico Cercospora kikuchii, bem como os efeitos de adjuvantes no processo de secagem destas enzimas foram estudados. A lipase bruta foi purificada à homogeneidade através de cromatografia de interação hidrofóbica e gel filtração. A lipase foi purificada 5,54 vezes, com rendimento de 9% e a atividade específica de 223,6 U/mg. O peso molecular da enzima foi estimado em 65,1 kDa por SDS-PAGE e 73,5 kDa utilizando cromatografia de gel filtração, indicando que provavelmente trata-se de um monômero. A lipase mostrou um pH ótimo em 4,6 e uma temperatura ótima de 35°C. Cerca de 80,2% de sua atividade foi mantida após incubação a 40°C durante 2 horas. A Vmax e Km foram 10,28 mmol/min/mg de proteína e 0,03240 mM, respectivamente, utilizando pNPP como substrato. As lipases presentes no extrato bruto e as lipases ligadas ao micélio foram caracterizadas para avaliar o potencial de utilização em biocatálise. A lipases no extrato bruto apresentaram atividade máxima a 60ºC e pH 6,2, enquanto que as lipases ligadas ao micélio apresentaram atividade máxima a 50ºC e pH 5,4. Nos estudos de efeito da temperatura sobre a atividade enzimática, as lipases no extrato bruto mantiveram-se estáveis a 50°C, com 85,3% de atividade residual após 2 horas de incubação. As lipases ligadas ao micélio mantiveram pelo menos 75,1% de atividade residual após 2 horas de incubação a 80°C. Estes resultados mostram que as lipases de C. kikuchii têm propriedades cinéticas e termoestabilidade desejáveis para aplicações em biocatálise. As lipases presentes no extrato bruto foram secas em spray dryer com diferentes adjuvantes, e sua estabilidade foi avaliada. A recuperação da atividade enzimática após a secagem, com a adição de 10% de lactose, -ciclodextrina, maltodextrina, manitol, goma arábica, e trealose variou de 63 a 100%. A atividade da enzima foi totalmente perdida durante a secagem do extrato bruto na ausência de adjuvantes. A maioria dos adjuvantes utilizados manteve pelo menos 50% da atividade enzimática a 5°C e 40% a 25°C, após 8 meses de armazenagem. As lipases secas com 10% de - ciclodextrina mantiveram 72% da atividade a 5°C no mesmo período. A partir destes resultados preliminares foi realizada a otimização do processo de secagem utilizando -ciclodextrina, maltodextrina e lactose como adjuvantes. A análise estatística dos resultados experimentais permitiu a determinação das condições ótimas para a retenção da atividade enzimática (RAE), a saber: concentração de adjuvantes de secagem de 12,05%, temperatura de entrada do gás de secagem em 153,6oC e vazão do extrato enzimático alimentado de 9,36 g/min, para - ciclodextrina e maltodextrina como adjuvantes. Para lactose, o estudo mostrou que o aumento da quantidade de adjuvante de secagem e/ou diminuindo a temperatura do gás de entrada tem um efeito positivo sobre a retenção da atividade enzimática do produto seco. Após o processo de purificação foi realizada a secagem da enzima parcialmente purificada e da lipase pura, com estes três adjuvantes. A manutenção da atividade enzimática variou 90,6-100% quando foram utilizadas as condições ótimas para cada adjuvante de secagem. Concluindo, as lipases produzidas por C. kikuchii podem ser eficientemente secas por spray dryer, uma vez que a atividade enzimática foi mantida no extrato bruto, na lipase pura e na lipase semi-purificada submetidas à secagem. / Lipases are enzymes that catalyze the hydrolysis of triacylglycerols to fatty acids, mono and diacylglycerols, and glycerol. In contrast to esterases, lipases are activated only when they are adsorbed to an oilwater interface. They have been widely used in many industrial processes such as organic chemical, detergent and cleaning formulations and in products like cosmetics and pharmaceutical products. The main concern in the production of commercial enzymes is to protect their stability in aqueous solution. Water facilitates or mediates a variety of physical and chemical degradation pathways, active during protein purification, shipping and storage. Consequently, dry solid formulations are developed to achieve an acceptable protein shelf life. Spray drying is commonly used as a dehydration technique in the pharmaceutical industry for making powdery products directly from the liquid. In the present work, the purification and biochemical characterization of lipases produced by endophytic fungus Cercospora kikuchii as well as the effects of adjuvants on the spray drying process of theses enzymes were studied. The crude lipase was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The lipase purified was 5.54-fold with 9% recovery and the specific activity was 223.6. The molecular mass of the lipase was estimated to be 65.1 kDa using SDS-PAGE and 73.5 using gel filtration chromatography, indicating that the lipase is a monomer. The lipase demonstrated an optimum pH at 4.6, an optimum temperature of 35°C. About 80.2% of its activity was retained after incubation at 40°C for 2 hours. The Vmax and Km were 10.28 mol/min/mg protein and 0.03240 mM, respectively, using pNPP as substrate. The lipases present in crude extract and the mycelium-bound lipases were characterized in order to evaluate the potential for use in biocatalysis. The crude extract showed maximum activity at 60ºC and pH 6.2 while the myceliumbound lipases showed maximum activity at 50ºC and pH 5.4. In tests of the temperature effect on the enzymatic activity, the lipases in the crude extract was stable at 50°C, with 85.3% residual activity after 2 hours of incubation. The mycelium-bound lipases maintained at least 75.1% of residual activity after 2 h incubation at 80°C. These results show that the lipases of C. kikuchii have kinetic properties and stability characteristics suitable to applications in biocatalysis. The lipases present in crude extract were spray dried with different adjuvants, and their stability was evaluated. The recovery of the enzyme after drying with 10% of lactose, -cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63% to 100%; but the enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5°C and 40% at 25°C after 8 months. The lipase dried with 10% of -cyclodextrin retained 72% of activity at 5°C. From these preliminary results the optimization of drying process using -cyclodextrin, maltodextrin and lactose as adjuvants was carried out. Statistical optimization of the experimental results allowed the determination of the processing conditions that maximized the retention of the enzymatic activity (RAE), namely: concentration of drying adjuvants of 12.05 %, inlet temperature of the drying gas of 153.6oC, and flow rate of the enzymatic extract fed to the dryer of 9.36 g/min, for the b-cyclodextrin and maltodextrin as adjuvants. For lactose as adjuvant the study showed that increasing the amount of drying adjuvant and/or decreasing the inlet gas temperature has positive effect on the retention of enzymatic activity of the dried product. After the purification process was carried out the drying of the partially purified enzyme and pure lipase, using these three adjuvants. The retention of enzymatic activity ranged from 90.6 to 100% when was used the optimal conditions for each drying adjuvant. Concluding, the lipases produced by C. kikuchii may be efficiently spray dried since its activity enzimatic was retained in crude extract, pure lipase and in semi-purified lipase after drying.

Ideally, every person in the world should have access to a safe and clean water supply; if not all sources of water are clean and safe, at the very least, an effective method to detect water contamination should be readily available. An effective detection method should not only be sensitive, rapid, robust, and affordable, but, ideally, it should also be equipment-free and easy to transport and deliver to the end-users. The main goal of this project is to develop a variety of bits and pieces of bioassay systems, with a particular focus on paper-based bioactive devices in order to provide portable and ready-to-use biosensors which can be useable by anyone anywhere around the world without requiring formal training. According to the World Health Organization (WHO), 76,000 people each year die in India alone because of pesticide poisoning. Long term exposure to organophosphate pesticides is known to have adverse effects on neurological function and can lead to Alzheimer's Disease, Attention Deficit Hyperactivity Disorder (ADHD), and reduced Intelligence Quotient (IQ). The likelihood of long term exposure to pesticides is heightened in developing countries, so a reliable and inexpensive pesticide sensor is a much-needed device in the developing world. To address this need, this project reports on the development of a fully-automated bioactive paper-based sensor for the detection of organophosphate pesticides. In the proposed biosensor, two innovations were implemented to achieve a full-automated format for the pesticide sensor: (I) First is a PUMP ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2012) that increases the flow rate of fluids within paper-based microfluidic analytical devices and sequentially brings two separate liquid streams to the enzyme test zone on the paper sensor, and (II) the second innovation is a PIPETTE ON A PAPER (Jahanshahi-Anbuhi et al., LOC, 2014) that involved the creation of a pullulan (a natural non-ionic polysaccharide) temporary bridge-system to transfer a known amount of solution to the sensing zone that, gives the enzyme zone a chance to dry and accept the substrate solution from the slow channel after a fixed period of time. This proposed format results in a simplified assay that detects the presence of pesticides automatically without any further manipulation from the user. However, the shelf life of this assay kit is challenging due to instability of both enzyme (AChE) and substrate (IDA) at room temperature. AChE loses its enzymatic activity when stored at room temperature and IDA becomes oxidized quickly. This problem is not unique to these two bio reagents, however; almost all bioassays which use bio-reagents (such as enzymes and small-molecular substrates) are unstable to varying degrees and require special shipping and storage. The instability of these molecules can arise from either thermal denaturation or chemical modification, such as oxidation or hydrolysis. Because of these issues, they often have to be shipped on dry ice with special packaging, which is costly. The cost of maintaining a cold chain for distributing bio-reagents accounts for up to 80% of the cost. Aside from the cost, these reagents also have to be stored in bulk in refrigerators or freezers to minimize the loss of activity, but they must be thawed and aliquoted for their intended tests. Repeated freezing and thawing can result in a significant loss of activity, which often leads to less reliable test results. These issues make running such assays in resource-limited settings a significant challenge. There is, therefore, an urgent need for an assay system with stable reagents that is easy to use, simple to read, inexpensive, and that includes a method for the long-term stabilization of enzymes and other unstable reagents in pre-measured quantities. To overcome to all these issues, pullulan is utilized for the development of pill-based-biosensors. Pullulan dissolves quickly in aqueous solutions and shows very high oxygen barrier properties in its film form. Considering the unique properties of pullulan, it is hypothesized that pullulan may be suitable for producing assay pills with encapsulated enzymes or other unstable molecules and may provide a simplified platform for carrying out bioassays in resource-limited settings. The application of these pill-based-biosensors is shown via the entrapment of AChE and IDA for the creation of an assay kit that can detect organophosphate pesticides (Jahanshahi-Anbuhi et al., Angew. Chem., 2014). Moreover, this thesis reports on the stabilization of highly unstable firefly luciferase for the detection of microorganisms and, more particularly, ATP. Through the use of pullulan, this thesis demonstrates that both the enzyme and the substrate can be protected, immobilized, and stabilized at room temperature, instead of the existing storage methods, which require temperatures <-20˚C. This innovation allows for a more convenient method of shipping the bioassay kits around the world without any extra care. Furthermore, pullulan-based films are utilized for the development of a method for controlled multidirectional flow within paper-based biosensors. This method provides the possibility of trapping labile and volatile reagents and stabilizing them by forming thin films with pullulan. The trapped reagents within pullulan films can be strategically stacked and assembled on a paper strip in different directions. Furthermore, should the need arise, these reagents can be released and delivered sequentially or simultaneously in both vertical and lateral directions through the paper. The application of this method is shown for: (I) creation of "ready-to-use" assay kit for the detection of Escherichia coli (E. Coli). This assay kit has the step of cell lysing and proceeds automatically to the step in which enzymes react. The second application (II) shows the trapping of Simon’s reagents, which is widely used for methamphetamine detection. Overall, these unique fabrication techniques can be widely used for the preparation of highly stable, ready-to-use, and user-friendly biosensors. We are currently working on the detection of other contaminants such as heavy metals, and we are starting on vaccine stabilization and delivery, which would have a tremendous impact for society. / Dissertation / Doctor of Engineering (DEng)

Living cells accumulate compensatory solutes for protection against the harmful effects of extreme environmental conditions such as high salinity, temperature and desiccation. Even at high concentrations these solutes do not disrupt the normal cellular functions and at times counteract by stabilizing the cellular components. These properties of compensatory solutes have been exploited for stabilizing proteins and cells in vitro. Betaines are widespread natural compensatory solutes that have also been used in other applications such as therapeutic agents and polymerase chain reaction (PCR) enhancers. Some biomedical applications of novel synthetic analogues of natural betaines were investigated. Natural compensatory solutes are either dipolar zwitterionic compounds or polyhydroxyl compounds, and the physical basis of compensation may differ between these, so one focus was on synthetic betaines with hydroxyl substituents. The majority of the synthetic solutes stabilized different model proteins against stress factors such as high and low temperatures. The presence of hydroxyl groups improved protection against desiccation. The observed stabilization effect is not just on the catalytic activity of the enzyme, but also on its structural conformation. Synthetic compensatory solutes have a potential application as protein stabilizers. Dimethylthetin was evaluated as a therapeutic agent and found to be harmful in a sheep model. However, from the study we were able to generate a large-animal continuous ambulatory peritoneal dialysis (CAPD) model and showed that glycine betaine could be added to the dialysis fluid in chronic renal failure. Some synthetic compensatory solutes reduce the melting temperatures of DNA better than most natural solutes. Synthetic solutes were identified that have potential to enhance PCR and could replace some reagents marketed by commercial suppliers. Density, viscosity and molecular model data on the solutes showed correlations with the biochemical effects of the solutes, but no physical measurements were found that reliably predicted their potential for biotechnological applications.