Orientador: Eneida de Paula / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-27T06:03:07Z (GMT). No. of bitstreams: 1 Couto_VeronicaMuniz_M.pdf: 2320216 bytes, checksum: fdeaa0b57898e701ff2245fa5c39eded (MD5) Previous issue date: 2015 / Resumo: Os anestésicos locais (AL) são utilizados para se bloquear a sensação de dor em procedimentos clínicos e odontológicos. Porém, os AL são moléculas pequenas que facilmente se redistribuem no sitio de ação, limitando a duração da anestesia. A dibucaína (DBC) é um anestésico local da família das amino-amidas e tem uso predominante como anestésico tópico. Este trabalho teve por finalidade desenvolver um novo sistema de liberação sustentada para o anestésico local dibucaína baseado em lipossomas de fosfatidilcolina de soja hidrogenada e colesterol com gradiente iônico transmembranar, para uso parenteral. Primeiramente, avaliamos uma metodologia analítica para quantificação de DBC por cromatografia líquida de alta eficiência. Entao, foram então desenvolvidas formulações de lipossomas unilamelares (LUV) e multivesiculares (LMVV) com gradiente iônico (citrato e sulfato), para encapsulação da DBC a 0,012%. As formulações foram caracterizadas quanto à eficiência de encapsulação (%EE) potencial zeta, diâmetro e polidispersão. O potencial zeta de todas as formulações foi sempre negativo (ca. -30 mV). Formulações de LUV contendo sulfato (LUVDBC5.5+sulfin/7.4ex) apresentaram %EE (62,6% ± 4,3) significativamente maior que as demais formulações. As LUV apresentaram tamanho médio de 500 nm, sendo mais estáveis e menos polidispersas que as LMVV. As formulações LUV foram selecionadas para a continuidade do estudo, sendo caracterizadas morfologicamente por microscopia eletrônica de transmissão e testadas quanto a sua estabilidade química, liberação in vitro, viabilidade celular e atividade antinociceptiva em camundongos. As micrografias das LUV confirmaram a formação de vesículas esféricas e unilamelares. Nenhuma formulação apresentou níveis de peroxidação lipídica significativa (> 0,5% de lipídios totais) durante 150 dias de armazenamento a 4oC. A formulação LUVDBC5.5+sulfin/7.4ex apresentou o melhor perfil de liberação sustentada in vitro e aumento significativo no tempo de bloqueio sensorial (27 h) in vivo, em comparação com solução de DBC (11 h) de igual concentração. Nos testes de viabilidade celular não houve mudança significativa no perfil da citotoxicidade induzida por DBC, encapsulada ou não. Portanto, nesse trabalho reportamos o preparo e caracterização de uma formulação lipossomal de liberação sustentada para DBC, com alta eficiência de encapsulação e capaz de promover um significativo aumento (ca. 2,5 vezes) no tempo de analgesia in vivo / Abstract: Local anesthetics (LA) are used in medical and dental procedures to decrease pain sensation. However, LA are small molecules that easily diffuse in the site of action, restricting the duration of anesthesia. Dibucaine (DBC) is an amino-amide local anesthetic, which major use as a topical agent. This study aimed the development of a new drug delivery system for dibucaine based on liposomes with transmembrane ion-gradient, for parenteral drug administration. First, an analytical methodology for DBC quantification through high performance liquid chromatography was determined. Then, large unilamellar (LUV) and multivesicular (MLVV) liposome formulations were prepared with internal ionic-gradients for the encapsulation of DBC (0.012 %). The formulations were characterized regarding their encapsulation efficiency (%EE), zeta potential, average size and polydispersity. The zeta potential of all formulations was always negative (ca. -30 mV). Sulfate-containing LUV formulations (LUVDBC5.5 sulfin/7.4out) showed 62.6% ± 4.3 EE, which was significantly higher than all other formulations. LUV presented an average size of 500 nm and proved to be less polidisperse and more stable than LMVV, being selected for further morphological (by transmission electron microscopy), storage chemical stability, in vitro release, cell viability and in vivo analgesic tests. Micrographs proved that LUVs were spherical and unilamellar shape. No formulation showed lipid peroxidation level higher than 0.5 % of total lipids during 150 days of storage at 4oC. LUVDBC5.5 sulfin/7.4out presented increased in vitro sustained release profile and significant increase in sensory block duration (27 h) in vivo, than a equivalent DBC solution (11 h). No changes in the cytotoxicity effect of the anesthetic were observed for DBC free or encapsulated. Therefore, this study reports the successful preparation and characterization of a liposomal formulation for the sustained release of DBC with high encapsulation efficiency and that is able to significantly prolong (ca. 2.5 times) the duration of analgesia in vivo / Mestrado / Fármacos, Medicamentos e Insumos para Saúde / Mestra em Ciências

Reações de oxidação lipídica são responsáveis pelo desenvolvimento de sabores e odores desagradáveis tornando os alimentos impróprios para consumo, sendo necessário o uso de antioxidantes. O ácido ascórbico (AA) é um antioxidante muito eficaz, que exibe função vitamínica, no entanto é relativamente instável. Com o objetivo de aumentar a estabilidade do AA e, consequentemente, facilitar sua aplicação em produtos alimentícios, os métodos de encapsulação por coacervação complexa e por dispositivos microfluídicos foram testados. Primeiramente foi apresentada a Revisão Bibliográfica no Capítulo 1, e em seguida, a encapsulação por coacervação complexa, como será visto no Capítulo 2. Neste caso, nove tratamentos foram obtidos utilizando-se gelatina e goma arábica como materiais de parede e analisados com relação à morfologia, por microscopia ótica e eletrônica de varredura, umidade, atividade de água, higroscopicidade, solubilidade, potencial zeta, espectroscopia no infravermelho por transformada de Fourier (Ftir), tamanho e distribuição de tamanho de partículas, cor instrumental, eficiência de encapsulação e estabilidade do material encapsulado. No capítulo 3 serão apresentados resultados obtidos na encapsulação do AA pelo método de dispositivos microfluídicos. Cinco tratamentos foram obtidos, sendo analisados com relação à morfologia, por microscopia ótica, eletrônica de varredura e confocal, eficiência de encapsulação, tamanho e distribuição de tamanho de partícula e estabilidade do material encapsulado. A obtenção das microcápsulas de AA pelos dois métodos citados foi viável uma vez que apresentaram altos valores de eficiência de encapsulação e ótima atuação em relação à proteção do AA durante estocagem. Comparando-se os dois métodos, as cápsulas obtidas por dispositivos microfluídicos conferiram melhor estabilidade ao ácido ascórbico, no entanto amostras obtidas por coacervação complexa foram aplicadas em salsicha devido a maior quantidade de AA presente em sua constituição. O efeito da aplicação das microcápsulas nas salsichas foi avaliado durante 40 dias de armazenamento refrigerado como será visto no Capítulo 4. Cinco tratamentos foram elaborados e analisados de acordo com a estabilidade da emulsão cárnea durante o processamento, umidade, atividade de água, alteração do pH, determinação da cor instrumental, perfil de textura instrumental, estabilidade oxidativa pelo método de substâncias reativas ao ácido tiobarbitúrico (TBARS) e aceitação sensorial. A aplicação das microcápsulas de AA em salsicha foi possível sem comprometer a qualidade do produto final. Todos os dados obtidos foram analisados estatisticamente por análise de variância ANOVA e teste de Tukey, ao nível de 5% de significância com auxílio do programa SAS. Os experimentos relacionados à encapsulação por coacervação complexa e aplicação das microcápsulas em salsicha foram realizados no Laboratório de Produtos Funcionais, nas dependências do Departamento de Engenharia de Alimentos da FZEA/USP. Os experimentos relacionados à utilização de dispositivos microfluídicos foram realizados nos laboratórios do professor David A. Weitz, da Escola de Engenharia e Ciências Aplicadas de Harvard, da Universidade de Harvard, Cambridge, Estados Unidos. / Lipid oxidation reactions are responsible for the development of unpleasant tastes and odors making food unfit for consumption. For this reason, the use of antioxidant is necessary. Ascorbic acid (AA) is a very effective antioxidant with vitamin function, however it is relatively unstable. With the aim of increasing the stability of AA and thus improve its application in food products, the methods of encapsulation by complex coacervation and microfluidic devices were tested. First of all the Literature Review is presented in Chapter 1. The encapsulation by complex coacervation can be seen in Chapter 2. For this methodology, nine treatments were obtained using gelatin and gum Arabic as encapsulant agent and analyzed regarding to morphology by optical and scanning electron microscopy, moisture, water activity, hygroscopicity, solubility, Zeta Potential, Fourier transform infrared Spectroscopy (FTIR), particle size and particle size distribution, instrumental color, encapsulation efficiency and stability of the encapsulated material. The results obtained for AA encapsulation by microfluidic device will be presented in Chapter 3. Five treatments were obtained and analyzed regarding to morphology by optical, scanning electron and confocal microscopy, encapsulation efficiency, particle size and particle size distribution and stability of the encapsulated material. The production of AA microcapsules by the two methods mentioned was feasible once that showed high levels of encapsulation efficiency and optimal performance regarding to the protection of AA during storage. Comparing the two methods, the microcapsules obtained by microfluidic device conferred better stability to AA, however samples obtained by complex coacervation were applied in sausage due to the greater amount of AA in its constitution. The effect of the application of microcapsules in sausages was evaluated during 40 days at refrigerated storage as it will be seen in Chapter 4. Five treatments were prepared and analyzed according to the stability of the meat emulsion during processing, moisture, water activity, pH changes, determination of instrumental color, instrumental texture profile, oxidative stability by the method of thiobarbituric acid reactive substances (TBARS) and sensory acceptance. The application of AA microcapsules in sausage was possible without compromising the quality of the final product. All data were statistically analyzed by ANOVA and Tukey test, at 5% of significance with the use of SAS software. The experiments related to encapsulation by complex coacervation and application of microcapsules in sausage were carried out at Laboratory of Functional Products, at Department of Food Engineering, FZEA / USP. The experiments related to the use of microfluidic devices were performed in the laboratories of Professor David A. Weitz, at School of Engineering and Applied Sciences of Harvard, at Harvard University, Cambridge, USA.

This diploma thesis deals with the determination of nicotine in different products. The theoretical part summarizes review on nicotine, smoking and opportunities how to quit. In the practical part a method for the determination of nicotine by HPLC / PDA was optimized. As the most suitable stationary phase was selected a Kinetex 5u C18 100A 150 x 4.6 mm column, as the optimal mobile phase was chosen a pure methanol with a flow rate of 1 ml min-1 and a temperature of 25 °C. For the analysis of nicotine were chosen: 18 kinds of cartridges for electronic cigarettes, two kinds of nicotine gum, nicotine spray, nicotine pastilles, nicotine orodispersible film and ten species of classic cigarettes. For each type of product the most appropriate method for extracting nicotine and its subsequent analysis by HPLC / PDA was found. For tobacco 24 hour extraction in methanol and 10s ultrasound was selected. The nicotine spray and electronic cigarette refills without flavours were only diluted with methanol. Flavoured refills were first diluted by sodium hydroxide and then with methanol. For chewing gums, pastilles and nicotine film extraction with 5% sodium hydroxide was chosen. In this study also new experimental nicotine product was designed. Nicotine has been encapsulated in alginate-starch material to form small gel particles. As the most suitable medium for storage the water medium was determined.

This diploma thesis deals with application of microencapsulation techniques in development of hydrogel controlled-release systems in which the main role is played by humic acids, biopolymer chitosan, compound fertilizer NPK and 3-indoleacetic acid. This paper continues my bachelor thesis topic about utilization of polyelectrolyte complexes. The aim of this work was to develop a literature review focusing on the microencapsulation techniques and according to its results optimize the method. Microencapsulation was performed with a commercial encapsulator BUSCHI B-395 Pro and a release of individual components into a water was measured. An amout of released substances was measured by UV-VIS method and HPLC analysis. Practical part also included testing of repeated swelling and drying. It was found that suitable composition and combination of ingredients form hydrogels for further use in agriculture.

Plusieurs catégories de MEMS nécessitent un environnement sous vide pour fonctionner de manière optimale, tel le micro-bolomètre. Le fonctionnement optimal de ce détecteur, à la base des imageurs infrarouge non refroidis, nécessite qu’il soit thermiquement isolé, et donc qu’il évolue dans une atmosphère raréfiée (< 10-2 mbar). Le maintien sous vide d’une matrice bolométrique durant la durée de vie d'une dizaine d’années du composant est réalisé par une encapsulation dans un boîtier de très faible volume (de 0,5 à 30 µL).Cette encapsulation sous vide fait appel à deux techniques complémentaires : le scellement hermétique sous vide et l’intégration d’un dispositif d’absorption du gaz dans la cavité, appelé getter. La technique de scellement donnant un joint de scellement suffisamment hermétique (<10-14 atm.cm3.s-1) est la soudure métallique. Le getter est un film mince métallique à base de métaux de transition. Il acquiert une activité de sorption lorsqu’il est chauffé.Les procédés d’encapsulation sous vide de l’état de l’art permettent l’encapsulation de micro-bolomètres à des températures de 300°C. Mais il est fort probable que les futurs matériaux micro-bolométriques en cours de développement ne supporteront pas des températures de recuit supérieures à 280°C. Leur encapsulation demande donc la mise à disposition d’un nouveau procédé de scellement sous vide à plus basse température et d’un nouveau film getter s’activant aussi à basse température.Ces deux techniques ont par conséquent été développées, au moyen de caractérisations en laboratoire et de tests sur composants industriels. / Some kinds of MEMS like micro-bolometers require vacuum to operate optimally. This IR sensor is the cornerstone for uncooled infrared detection. Its best sensing capacity is achieved by thermal insulation, which is realized by placing it under vacuum (< 10-2 mbar). The vacuum is maintained throughout the camera lifetime thanks to a microvolume packaging (0.5 to 30 µL).The MEMS vacuum packaging implies the combination of two complementary technical solutions: first hermetic sealing, then getter device integration absorbing internal gas. The sealing technique retained (which enables leak rate <10-14 atm.cm3.s-1) is the metallic bonding. The getter is a thin transition metal film. When activated by an annealing, its surface traps gaseous molecules. The sorption process of the getter is ideally activated during the sealing process of the bonding.The typical temperature packaging process for micro-bolometers is 300°C. It is expected that sensibility of new types of micro-bolometers materials will be degraded if they are exposed to temperatures higher than 280°C. Consequently, their encapsulation require the elaboration of a new low temperature packaging technology.Such a technology has been developed based on experimental studies in laboratory and tests under industrial conditions.

Microspheres are spherically shaped particles within the size range of 1-1000 μm in diameter. Due to the their small size and round shape, microspheres show many advantages in various applications such as pharmaceuticals, composites and coatings. The microspheres can be customized to fit a specific application and are manufactured in various forms such as solid, hollow and encapsulating. Encapsulating cellulose microspheres have been produced in this project by the emulsionsolvent evaporation technique. The purpose of this study was to further investigate the possibility of producing encapsulating microspheres with a size range of 10-50 μm that will have a high encapsulation. A second purpose of this study was optimizing the emulsifier system for the preparation of these spheres. This has been accomplished by varying several process parameters such as type of emulsifiers and solvents to study the effect on morphology and encapsulation efficiency. The analyses of the spheres were performed with optical microscopy, thermal gravimetric analyzer (TGA) and scanning electron microscopy (SEM). The emulsifier type and concentration affected the encapsulation and size distribution but had no direct effect on the internal and external structure, which was multi-cellular and porous, respectively. The highest encapsulation in relation to average size was obtained with 0.1 v/v- % of the emulsifier mixture Emulsifier 1 (E1)/Emulsifier 2 (E2) (70/30 %). The solvent used to dissolve the polymer had a direct effect on encapsulation, a combination of Solvent 2 (S2) and Solvent 1 (S1) proved best for the three tested cellulose derivatives with low, medium and high number average molecular weight. The solvent also had an effect on the internal structure of the microspheres, becoming more core-shell when using the S1/S2 combination.

För att använda sig av tekniska produkter krävs en nätverksuppkoppling.Nätverksinfrastrukturen blir idag allt mer beroende av datacenter, en fysiskt eller virtuell platsdär datorer, servrar, lagringsenheter och nätverkskomponenter är samlade. I takt med attkraven på framtidens nätverk ökar blir virtuella nätverk och molnbaserade tjänster alltvanligare. Virtualisering av datacenter har medfört hinder för traditionella tekniker inomnätverkssegmentering och tunnling. Med hjälp av virtuella maskiner och containrar, fårdatacenter möjlighet att övergå till ett mer flexibelt användande av hårdvara och resursersom dessutom vid behov kan frångå den traditionella IP-strukturen. Lösningen äranvändandet av ett overlay-nätverk, ett virtuellt nätverk byggt ovanpå den fysiskainfrastrukturen. Overlay-protokollen Virtual Extensible Local Area Network (VXLAN), NetworkVirtualization using Generic Routing Encapsulation (NVGRE) och Generic NetworkVirtualization Encapsulation (GENEVE) kan tunnla trafiken över det underliggande fysiskanätverket. Det tillåter en större flexibilitet i form av segmentering i kommunikationen mellannätverksenheter. De olika overlay-protokollen kan framstå som väldigt lika och valet av ettspecifikt protokoll kan kompliceras. Rapporten använder befintlig litteratur för att analyseraoch jämföra tre aktuella overlay-protokoll i syfte att ge stöd till företag inför valet avoverlay-protokoll. För att möjliggöra detta krävs en underliggande kunskap om varproblemen med traditionella tekniker uppstår, varför overlay-protokoll är en fördel i dagensnätverk, vilka likheter och skillnader som finns samt hur dessa kan användas för attunderlätta valet av overlay-protokoll. Slutsatsen är att den tekniska specifikationen för varjeprotokoll stödjer ett generiskt användande. För att få en tillräcklig uppfattning om vilketoverlay-protokoll som är att föredra i en specifik situation måste både den tekniska designenav protokollen och syftet med användningen vara fastställd. Arbetet bidrar med endjupgående analys av hur varje protokoll är designat, hur dess metoder skiljer sig och vilkeninnebörd det har, både för dagens nätverk men också för framtidens. / To be able to use the technical products there has to be an internet connection. The networkinfrastructure is relying more and more on data centers, a physical or virtual place wherecomputers, servers, storage is gathered. In tune with the requirements of the future networksincreasing, virtual networks and cloud-based services are getting more common.Virtualization of data centers has brought complications for traditional technologies innetwork segmentation and tunneling. With the help of virtual machines and containers, thedata centers get an opportunity to pass over to a more flexible usage of hardware andresources that also, if needed, can leave the traditional IP-framework. The solution resides inthe usage of an overlay network, a virtual network built on top of the physical infrastructure.The overlay protocols Virtual Extensible Local Area Network (VXLAN), Network Virtualizationusing Generic Routing Encapsulation (NVGRE) and Generic Network VirtualizationEncapsulation (GENEVE) can tunnel the traffic over the underlying physical network. Thisallows for a bigger flexibility in the shape of segmentation in the communication betweennetworking units. The different overlay protocols can at first seem very similar and the choiceof a specific protocol can get complicated. This report uses available literature to analyzeand compare three active overlay protocols in the purpose of giving support to enterpriseswhen choosing an overlay protocol. To make this possible, a basic knowledge is needed ofwhere the problems with traditional technologies occur, why an overlay protocol is anadvantage in today's networking, what similarities and differences there are and also howthey can be used to support in the choice of an overlay protocol. The conclusion is that thetechnical specification of each protocol supports a generic usage. To get a reasonableperception of which protocol is preferred in a specific situation, both the technical design ofthe protocols and the purpose of the usage must be established. The work contributes with adeep analysis of how each protocol is designed, how its methods differ and what meaning ithas, both for today's network but also for the future.

DEVELOPMENT AND CHARACTERIZATION OF PHOSPHOLIPID ENCAPSULATED QUANTUM DOT CONSTRUCTS FOR BIOLOGIC APPLICATIONS The American Cancer Society predicts that 577,190 cancer-related deaths and 1,638,910 newly diagnosed cases of cancer will occur in 2012. As these statistics show, cancer is a prevalent and devastating health issue; determined by the Mayo Clinic to be the second leading cause of death in the United States. Skin cancer is the most common form of cancer in the United States. In 2012 more than 68,000 Americans will be diagnosed with melanoma, 48,000 will be diagnosed with an early form of the disease that has not yet reached the lower levels of the epidermis, and more than 2 million people will be treated for basal cell or squamous cell skin cancer. Early and accurate detection is the most reliable way to ensure a positive outcome and the ultimate survival of the patient. As the most aggressive form of skin cancer, survival of melanoma is especially connected to early detection. Current methods for the initial detection of potential cancerous masses and lesions rely on visual examination, palpitation, and biopsy. Accurate determination of the presence of cancerous cells in a biopsy is especially difficult at the early stages when only a small percentage of cells in the biopsied mass show the morphological traits associated with being cancerous. This circumstance often results in a false negative (FN), delaying the necessary treatment until the cancer has reached a more developed stage. Developing more accurate methods for the detection of cancerous cells within a biopsy would aid in alleviating this problem. An improvement to the conventional method of visually examining biopsied tissues for the presence of cells with abnormal morphologies can be offered by utilizing the model of functionalized quantum dot (QD) constructs. Quantum dots are nano-particles composed of semi-conducting materials that fluoresce at discrete wavelengths when irradiated by a high energy UV source. QD constructs are cadmium-selenium/zinc-sulfide (CdSe/ZnS) quantum dots encapsulated within a bovine derived milk phospholipid micelle. QD constructs provide a potential mechanism for the identification of cancerous cells within a biopsy. Appreciating the scope of the clinical problem and understanding the potential of QDs, the objective of this thesis is to develop a primary model for the solubilization, encapsulation, and primary phospholipid functionalization of two distinct sizes of CdSe/ZnS QDs. The first stage of this thesis optimized the currently utilized protocol for synthesizing cadmium-selenium (CdSe) quantum dots to develop a set of parameters for consistently producing white fluorescing CdSe cores (WFCs) and CdSe/ZnS QDs of 505nm and 555nm (+/- 10nm). The application of synthesis times, temperatures, and quenching methods were employed to achieve this. The second stage developed a phospholipid encapsulation method for the initial functionalization and suspension of the hydrophobic QDs in aqueous media via encapsulation within phospholipid micelles. The final stage of this thesis focused on the successful introduction of the QD constructs into keratinocyte cells. Calcein and Ethidium homodimer-1 stains were applied to determine cell viability, Histochoice was applied as a fixative, and Hoechst staining was employed for cell nuclei identification. Analysis using confocal microscopy suggests successful attachment of QD constructs, in 0.1% w/v keratinocyte media, to the exterior of keratinocyte cell membranes with a 30% average cell survival rate at 24 hours after sample introduction. Future research investigating the interaction of QD constructs with biologic mediums of greater physiological complexity, as well as application of a secondary functionalization, are the next steps on the path toward achieving a viable mechanism for targeting and identifying cancerous cells within a biopsy.

Polyelectrolyte complex coacervates (PECCs) result from liquid-liquid phase separation (LLPS) in solutions containing oppositely charged polymers 1. Multiphase polyelectrolyte complex coacervates (MPECCs) result from the combination of multiple, specific PECCs 2. The encapsulation of proteins in PECCs can serve as promising vehicles for the effective delivery of protein-based therapeutics, which are notoriously difficult to deliver. The encapsulation of model proteins, such as Bovine Serum Albumin (BSA) 3 or Human Hemoglobin (Hb) 4 have illustrated the protein-encapsulating capabilities of these PECC systems. The encapsulation of proteins in MPECCs is a topic that has yet to be explored; however, it can serve to mimic the structure and function of multiphase membraneless organelles, which are abundantly available in cells. This project sought to understand and quantify the encapsulation of enzymes in both PECC and MPECC models; as well as evaluate their efficiency upon encapsulation, as enzymes are simply proteins with catalytic functions 5. A synthesized library of charged, heterochiral polypeptides were used to form both PECC and MPECC systems. Glucose oxidase (GOx) and horseradish peroxidase (HRP) were the enzymes chosen to be assessed in both PECC and MPECC systems. Turbidity measurements, in terms of percent mole of polycation, were used to determine the optimal stoichiometric ratio between the polyanion and polyanion, in the presence of a given concentration of both or either enzyme, in which maximum complex formation occurred. Here we report that a 1:1 stoichiometric ratio of polycation to polyanion in either a solution with 25ug/mL HRP and 25ug/mL GOx, a solution with 50ug/mL GOx, or a solution with 50ug/mL HRP leads to the highest level of complexation. Enzyme encapsulation efficiency of individual PECCs for both enzymes was assessed using the Bradford assay, in which the supernatant was used to determine the concentration of enzyme left in the PECC post-centrifugation. Here we report that all PECC systems were able to encapsulate both GOx and HRP. Higher encapsulation efficiencies were seen with GOx samples compared to HRP samples. Enzymatic activity and efficiency were assessed using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay in the presence of ß-D-glucose. The chromogenic change in intensity over time of each sample was assessed using optical microscopy. Michaelis-Menten graphs were made from the data collected. The resulting data was used to evaluate the Km and Vmax of the enzyme cascade in PECC and MPECC systems compared to a control. Here we report that enzyme cascade efficiency varied among PECC and MPECC samples, with some being more efficient than others. We find that both PECC and MPECC systems generally have lower enzyme-substrate affinity (higher Km) compared to performing the reactions in water. However, this may be related to the need for the substrate to diffuse into a different phase or phases. Interestingly, many of the PECC and MPECC systems have lower Vmax values compared to the water control, indicating a faster enzyme saturation. The enzyme kinetics and efficiency could also be controlled by varying the location of the enzymes in each phase within the MPECC systems. Overall, we show that using MPECC systems allows one to select advantageous properties of individual PECCs and combine them together.

No description available.