Designade ankyrinupprepningsproteiner (DARPiner) är små, mycket stabila antikroppsmimetiska proteiner. I det här projektet användes anti-EpCAM-DARPiner tillsammans med mikrofluidik för att avgära om de kunde fånga upp HCT116-celler mer effektivt än anti-EpCAM-antikroppar. Ytorna på insidan av mikroffluidikkanaler förändrades genom bindning av N-γ-maleimidobutyryl-oxysuccinimidester (GMBS) och merkaptopropyltrietoxysilan (MPTES) för anti-EpCAM-antikroppar och GMBS och (3-aminopropyl)trietoxysilan (APTES) för DARPiner. Båda kanaltyperna testades genom inflöde av cancerceller och helblod blandat med cancerceller. Ingen effektiv och konsekvent celluppfångst åstadkoms trots att det visades att antikropparna och DARPinerna kunde binda till cellerna direkt och att test med fluorescenta DARPiner och antikroppar visade att ytförändringskemin var fungerande. Slutsatsen blev att de mest troliga orsakerna till misslyckandena var att ytförändringskemin påverkade proteinernas bindningsförmåga negativt eller att proteinerna bands till kanalernas yta i fel riktning. DARPiner är fortfarande intressanta för tillämpningar inom mikrofluidik, men vidare förbättring av det experimentella protokollet behövs. / Designed ankyrin repeat proteins (DARPins) are small and highly stable antibody mimetics. In this project, anti-EpCAM DARPins were used in conjunction with microfluidics to determine if they could capture HCT116 cells more effectively than anti-EpCAM antibodies. The inside surfaces of microfluidic chips were modified using N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) and mercaptopropyltriethoxysilane (MPTES) for anti-EpCAM antibodies, and surface modifications for anti-EpCAM DARPins were made using GMBS and (3-aminopropyl)triethoxysilane (APTES). Both chip types were tested using cancer cells and whole blood mixed with cancer cells. No effective and consistent cell capture was achieved, despite the antibodies and DARPins being shown to be able to bind to the cells directly and tests with fluorescently labelled DARPins and antibodies showing that the surface modification chemistry used was functional. It was concluded that the most likely causes of the failures were surface modifications interfering with the binding ability of the proteins, or improper orientation of the bound proteins. The DARPin remains a protein of interest for microfluidic applications, but further changes and optimisation of the experimental protocol is necessary.

Switchable hydrogel-layered composite membranes can be used for the analysis of particle size distributions. This functionality is provided by pores with controllable diameter. In order to obtain a device that can be used to measure the cell size distribution in native biological samples, lots of switchable pores are required. In the current work, we model and simulate the mechanical behavior of active composite membranes with switchable pores. This is done in order to find the maximum number of pores that can be integrated into a membrane without cross-influencing effects on the actuation of the pores. Therefore, we investigate (1) the interaction of active pores inside the multifunctional composite and (2) the membrane bending under microfluidic pressure load. We show that through miniaturization, sufficient pores can be added to a permeation control membrane for processing native blood samples. The envisioned device allows a parallelized measurement of cell sizes in a simple lab-on-a-chip setup.

Membranes separate uid compartments and can comprise transport structures for selective permeation. In biology, channel proteins are specialized in their atomic structure to allow transport of specific compounds (selectivity). Conformational changes in protein structure allow the control of the permeation abilities by outer stimuli (gating). In polymeric membranes, the selectivity is due to electrostatic or size-exclusion. It can thus be controlled by size variation or electric charges. Controllable permeation can be useful to determine particle-size distributions in continuous ow, e.g. in micro uidics and biomedicine to gain cell diameter profiles in blood. The present approach uses patterned polyethylene terephthalate (PET) membranes with hydrogel surface coating for permeation control by size-exclusion. The thermosensitive hydrogel poly(N-isopropylacrylamide) (PNIPAAm) is structured with a cross-shaped pore geometry. A change in the temperature of the water ow through the membrane leads to a pore shape variation. The temperature dependent behavior of PNIPAAm can be numerically modeled with a temperature expansion model, where the swelling and deswelling is depicted by temperature dependent expansion coefficients. In the present study, the free swelling behavior was implemented to the Finite Element tool ABAQUS for the complex composite structure of the permeation control membrane. Experimental values of the geometry characteristics were derived from microscopy images with the tool ImageJ and compared to simulation results. Numerical simulations using the derived thermomechanical model for different pore geometries (circular, rectangle, cross and triangle) were performed. With this study, we show that the temperature expansion model with values from the free swelling behavior can be used to adequately predict the deformation behavior of the complex membrane system. The predictions can be used to optimize the behavior of the membrane pores and the overall performance of the smart membrane.

The fast development of DNA-encoded chemical libraries (DECL) in the past 10 years has received great attention from pharmaceutical industries. It applies the selection approach for small molecular drug discovery. Because of the limited choices of DNA-compatible chemical reactions, most DNA-encoded chemical libraries have a narrow structural diversity and low synthetic yield. There is also a poor correlation between the ranking of compounds resulted from analyzing the sequencing data and the affinity measured through biochemical assays. By combining DECL with dynamical chemical library, the resulting DNA-encoded dynamic library (EDCCL) explores the thermodynamic equilibrium of reversible reactions as well as the advantages of DNA encoded compounds for manipulation/detection, thus leads to enhanced signal-to-noise ratio of the selection process and higher library quality. However, the library dynamics are caused by the weak interactions between the DNA strands, which also result in relatively low affinity of the bidentate interaction, as compared to a stable DNA duplex. To take advantage of both stably assembled dual-pharmacophore libraries and EDCCLs, we extended the concept of EDCCLs to heat-induced EDCCLs (hi-EDCCLs), in which the heat-induced recombination process of stable DNA duplexes and affinity capture are carried out separately. To replace the extremely laborious and repetitive manual process, a fully automated device will facilitate the use of DECL in drug discovery. Herein we describe a novel lab-on-a-chip platform for high throughput drug discovery with hi-EDCCL. A microfluidic system with integrated actuation was designed which is able to provide a continuous sample circulation by reducing the volume to a minimum. It consists of a cooled and a heated chamber for constant circulation. The system is capable to generate stable temperatures above 75 °C in the heated chamber to melt the double strands of the DNA and less than 15 °C in the cooled chamber, to reanneal the shuffled library. In the binding chamber (the cooled chamber) specific retaining structures are integrated. These hold back beads functionalized with the target protein, while the chamber is continuously flushed with library molecules. Afterwards the whole system can be flushed with buffer to wash out unspecific bound molecules. Finally the protein-loaded beads with attached molecules can be eluted for further investigation

No description available.

This thesis, written by Ingrid Öberg Månsson at KTH Royal Institute of Technology and entitled “Electroanalytical devices with fluidic control using textile materials and methods”, presents experimental studies on the development of textile based electronic devices and biosensors. One of the reasons why this is of interest is the growing demand for integrated smart products for wearable health monitoring or energy harvesting. To enable such products, new interdisciplinary fields arise combining traditional textile technology and electronics. Textile based devices have garnered much interest in recent years due to their innate ability to incorporate function directly into, for example, clothing or bandages by textile processes such as weaving, knitting or stitching. However, many modifications of yarns required for such applications are not available on an industrial scale. The major objective of this work has been to study how to achieve the performance necessary to create electronic textile devices by either coating yarns with conductive material or using commercially available conductive yarns that are functionalized to create sensing elements. Further, liquid transport within textile materials has been studied to be able to control the contact area between electrolyte and electrodes in electrochemical devices such as sensors and transistors. Yarns with specially designed cross-sections, traditionally used in sportswear to wick sweat away from the body and enhance evaporation, was used to transport electrolyte liquids to come in contact with yarn electrodes. The defined area of the junction where the fluidic yarn meets the conductive yarn was shown to increase stability of the measurements and the reproducibility between devices. The results presented in the two publications of this thesis as well as additional results presented in the thesis itself show the promising potential of using textile materials to integrate electronic and electrochemical functionality in our everyday life. This is shown by using basic textile materials and processing techniques to fabricate complex devices for various application areas such as sensors and diagnostics as well as electrical and energy harvesting components. / Denna avhandling, skriven av Ingrid Öberg Månsson vid Kungliga Tekniska Högskolan och titulerad ”Elektroanalytiska sensorer med vätskekontroll integrerad genom användande av textila material och metoder”, presenterar experimentella studier inom utvecklingen av textilbaserade elektroniska komponenter och biosensorer. Detta är av intresse på grund av den ökade efterfrågan på integrerade smarta produkter som till exempel bärbara sensorer för hälsoövervakning eller för att samla upp och konvertera energi till elektricitet. För att möjliggöra denna typ av produkter föds nya interdisciplinära fält där traditionell textilteknologi och elektronik möts. Textilbaserade enheter har väckt stort intresse under de senaste åren på grund av den naturliga förmågan att integrera funktion i till exempel kläder eller förband genom textila tillverkningsprocesser som väveri, stickning eller sömnad. Många modifikationer hos garner som krävs för att möjliggöra sådana tillämpningar är dock inte tillgängliga i större skala. Därför har det huvudsakliga syftet med denna studie varit att undersöka hur man kan uppnå den prestanda som krävs för att tillverka elektroniska textila komponenter, antingen genom att belägga garner med elektroniskt ledande material eller genom att använda kommersiellt tillgängliga ledande garner som sedan modifieras kemiskt för att skapa sensorer. Utöver detta har vätsketransport inom textila material studerats för att kunna styra och kontrollera kontaktytan mellan elektrolyt och elektroder i elektrokemiska enheter så som sensorer och transistorer. Garner med speciella tvärsnitt, som traditionellt använts i sportkläder för att transportera svett bort från kroppen och underlätta avdunstning, har använts för att transportera elektrolytvätska till elektroder av garn. Den definierade kontaktytan där det vätsketransporterade garnet korsar elektrodgarnet har visats öka stabiliteten av mätningen och reproducerbarheten mellan mätenheter. Resultaten som presenteras i de två artiklar som denna avhandling bygger på samt i avhandlingen själv visar på lovande potential för användandet av textila material för att integrera elektronisk och elektrokemisk funktionalitet i våra vardagsliv. Detta har uppnåtts genom att använda grundläggande textila material och tillverkningsprocesser för att tillverka komplexa enheter för olika tillämpningsområden så som sensorer för diagnostik samt elektroniska komponenter. / <p>QC 2020-08-21</p>

[pt] Estima-se que 50 por cento das reservas mundiais de petróleo e gás sejam mantidas em reservatórios carbonáticos naturalmente fraturados. Um dos maiores desafios neste tipo de formações é a sua heterogeneidade. Além da presença de fraturas que conectam longitudinalmente o meio poroso, vugs em diferentes escalas e distribuições estão espalhados por toda a matriz porosa. Essas cavidades tornam as características do escoamento de fluidos significativamente diferentes daquelas dos reservatórios convencionais de estrutura porosa homogênea, e trazem a necessidade de avaliar propriedades petrofísicas equivalentes para o meio heterogêneo. Neste estudo, uma abordagem microfluídica é usada para determinar as curvas de permeabilidade relativa de água e óleo e os perfis de distribuição das fases em micromodelos 2D de meios porosos vugulares. Experimentos de injeção simultânea de água-óleo em estado estacionário foram realizados a diferentes fluxos fracionários de água, monitorando a dinâmica da queda de pressão e visualizando o deslocamento de fluidos na escala de poros. A aquisição de imagens em tempo real por microscopia de fluorescência permitiu examinar a evolução da saturação das fases. A comparação direta entre as curvas de permeabilidade relativa dos meios porosos vugulares com aquela da matriz porosa mostrou que a incorporação de vugs leva a (i) maior permeabilidade absoluta equivalente, especialmente com cavidades mais longas e em maior número, (ii) aumento da ocupação de óleo na matriz porosa, devido à invasão de água menos eficiente, e (iii) maior permeabilidade relativa à água, que flui preferencialmente pelo espaço vugular. Esses resultados são consistentes com a natureza molhável ao óleo dos micromodelos, uma vez que os vugs oferecem menor resistência capilar ao fluxo da fase não molhante. Nossa abordagem microfluídica de baixo custo provavelmente nos permitirá estudar sistematicamente configurações mais complexas de meios porosos heterogêneos / [en] It is estimated that 50 percent of world s oil and gas reserves are held in naturally fractured carbonate reservoirs. One of the biggest challenges in this type of formation is its heterogeneous nature. Besides the presence of fractures that longitudinally connect the porous medium, vugs at different scales and distributions are scattered throughout the porous matrix. These cavities cause fluid flow characteristics to significantly differ from those of conventional homogeneous pore structure reservoirs and bring the need to evaluate equivalent petrophysical properties of the heterogeneous medium. In this study, a microfluidic approach is used to determine the water and oil relative permeability curves and phase distribution profiles in 2D micromodels of vugular porous media. Steady-state water-oil injection experiments were performed in these devices at different fractional flows, while monitoring the dynamics of the pressure drop and visualizing the fluid displacement at the pore scale. Live-image acquisition through fluorescence microscopy made it possible to examine the evolution of the saturation of water and oil phases. The direct comparison between the relative permeability curves of well-characterized vugular porous media and their porous matrix showed that the incorporation of vugs leads to (i) higher equivalent absolute permeability, especially with longer cavities and higher vug density, (ii) increased oil occupancy in the porous matrix, due to less efficient water invasion into the porous matrix, and (iii) higher relative permeability to water, which flows preferentially through the vugular space. These results are consistent with the oil-wet nature of micromodels, since the vugs are offering less capillary resistance to the flow of the non-wetting phase. Our low-cost microfluidic approach will likely allow us to systematically study more complex vugular-fractured systems.

No description available.

[pt] Devido ao seu comportamento físico-químico, os polímeros solúveis em água são utilizados em várias fases de perfuração, completação, e produção de poços de petróleo. Portanto, é fundamental prever e controlar o comportamento em meio poroso para entender o desempenho do polímero. Experimentos foram conduzidos para estudar a degradação de uma solução aquosa semi-diluída de PEO, usando dois capilares com diâmetros de entrada diferentes (100 micrômetros e 200 micrômetros) ambos com constrição de 50 micrômetros, criando fluxos transientes rápidos em seu centro. Diferentes vazões foram impostas a fim de observar diferentes taxas de cisalhamento e de alongamento no sistema. O efluente do fluxo foi coletado e reinjetado, e suas propriedades reológicas foram utilizadas como proxies para a degradação. Observamos que, para a contração mais abrupta, a vazão mínima necessária para degradar a solução é menor. Este resultado, analisado apenas sob a perspectiva da taxa de cisalhamento, não é razoável, já que a taxa de cisalhamento na constrição a que o polímero é submetido é igual em ambos os capilares. Portanto, inferimos que a brusquidão da contração desempenha um papel na degradação, o que significa que a taxa de alongamento pode ser responsável pela menor taxa de fluxo crítico. Também foi observado um padrão de como ocorre a degradação com as injeções subsequentes. Podemos inferir que injeções subsequentes causam degradação incremental antes de se aproximar de um patamar de estabilização e que vazões mais altas geram patamares de degradação mais baixos. / [en] Due to their physical-chemical behavior, water-soluble polymers are used extensively in various phases of drilling, completion, workover, and production of oil and gas wells. Therefore, it is fundamental to predict and to control in-situ porous medium behavior in order to understand polymer performance. Experiments were conducted to study the degradation of a semi diluted (2000 ppm) aqueous solution of PEO, using two capillaries with different entrance diameter (100 micrometers and 200 micrometers) both with 50 micrometers radius constriction, creating Fast-Transient Flows in their center. Different injection rates were imposed in order to observe different shear and extensional rates in the system. The effluent of the flow was collected, and reinjected, and rheological properties of the fluids were used as proxies for the degradation of the solution. We observed that for the more abrupt contraction, the minimum flow rate needed for degrading the polymer solution is lower. This result, when analyzed purely under shear rate perspective, is not reasonable, since the constriction shear rates to which the polymer is subjected are equal at both capillaries. Therefore, we inferred that the abruptness of the contraction plays a role in the degradation, which means elongational rate may be responsible for the lower critical flow rate. It was also observed a pattern for how the degradation occurs with subsequent injections. We could infer that subsequent injections cause incremental degradation before approaching a stabilization plateau and that higher flow rates generated lower degradation plateaus.

The rise of superbugs, including antibiotic-resistant bacteria, and virus outbreaks, such as the recent coronavirus scare, illustrate the need for rapid detection of disease pathogens. Widespread availability of rapid disease identification would facilitate outbreak prevention and specific treatment. The ARROW biosensor microchip can directly detect single molecules through fluorescence-based optofluidic interrogation. The nature of the microfluidic channels found on optofluidic sensor platforms sets some of the ultimate sensitivity and accuracy limits and can result in false negative test results. Yet higher sensitivity and specificity is desired through hydrodynamic focusing. Novel 3D hydrodynamic focusing designs were developed and implemented on the ARROW platform, an optofluidic lab-on-a-chip single-molecule detector device. Microchannels with cross-section dimensions smaller than 10 μm were formed using sacrificial etching of photoresist layers covered with plasma-enhanced chemical-vapor-deposited silicon dioxide on a silicon wafer. Buffer fluid carried to the focusing junction enveloped an intersecting sample fluid, resulting in 3D focusing of the sample stream. The designs which operate across a wide range of fluid velocities through pressure-driven flow were integrated with optical waveguides in order to interrogate fluorescing particles and confirm 3D focusing, characterize diffusion, and quantify optofluidic detection enhancement of single viruses on chip.