Дисертації з теми "Point-of-care devices"

Developing non-invasive and accurate diagnostics that are easily manufactured, robust and reusable will provide monitoring of high-risk individuals in any clinical or point-of-care environment, particularly in the developing world. There is currently no rapid, low-cost and generic sensor fabrication technique capable of producing narrow-band, uniform, reversible colorimetric readouts with a high-tuneability range. This thesis aims to present a theoretical and experimental basis for the rapid fabrication, optimisation and testing of holographic sensors for the quantification of pH, organic solvents, metal cations, and glucose in solutions. The sensing mechanism was computationally modelled to optimise its optical characteristics and predict the readouts. A single pulse of a laser (6 ns, 532 nm, 350 mJ) in holographic “Denisyuk” reflection mode allowed rapid production of sensors through silver-halide chemistry, in situ particle size reduction and photopolymerisation. The fabricated sensors consisted of off-axis Bragg diffraction gratings of ordered silver nanoparticles and localised refractive index changes in poly(2-hydroxyethyl methacrylate) and polyacrylamide films. The sensors exhibited reversible Bragg peak shifts, and diffracted the spectrum of narrow-band light over the wavelength range λpeak ≈ 500-1100 nm. The application of the holographic sensors was demonstrated by sensing pH in artificial urine over the physiological range (4.5-9.0), with a sensitivity of 48 nm/pH unit between pH 5.0 and 6.0. For sensing metal cations, a porphyrin derivative was synthesised to act as the crosslinker, the light absorbing material, the component of a diffraction grating, as well as the cation chelating agent. The sensor allowed reversible quantification of Cu2+ and Fe2+ ions (50 mM - 1 M) with a response time within 50 s. Clinical trials of a glucose sensor in the urine samples of diabetic patients demonstrated that the glucose sensor has an improved performance compared to a commercial high-throughput urinalysis device. The experimental sensitivity of the glucose sensor exhibited a limit of detection of 90 µM, and permitted diagnosis of glucosuria up to 350 mM. The sensor response was achieved within 5 min and the sensor could be reused about 400 times without compromising its accuracy. Holographic sensors were also tested in flake form, and integrated with paper-iron oxide composites, dyed filter and chromatography papers, and nitrocellulose-based test strips. Finally, a generic smartphone application was developed and tested to quantify colorimetric tests for both Android and iOS operating systems. The developed sensing platform and the smartphone application have implications for the development of low-cost, reusable and equipment-free point-of-care diagnostic devices.

Els test de tipus 'point-of-care' (POC) presenten un gran potencial per al maneig i el diagnòstic de malalties. Els dispositius POC permeten la realització de proves clíniques prop del pacient, permetent així un diagnòstic ràpid, una ràpida iniciació de tractaments, i en cas necessari, una derivació ràpida a altres centres mèdics. Aquests dispositius tenen a més el potencial de ser més econòmics, més robustos, i més fàcils d'usar que els dispositius mèdics tradicionals. Per aquests motius, els dispositius mèdics de tipus POC es consideren prometedors per als països en vies de desenvolupament, els quals són també els que necessiten de forma més urgent noves tecnologies mèdiques. En aquest context, aquesta tesi se centra en el desenvolupament de dispositius mèdics de diagnòstic in-vitro de tipus POC per salut global. Tenint en compte que els recursos per al desenvolupament i explotació de dispositius POC per a països amb baixos recursos són limitats, el Capítol 2 s'enfoca en el desenvolupament de prioritats d'investigació en salut. Mitjançant l'establiment d'aquestes prioritats es pretén facilitar la selecció d'objectius a fabricants d'instruments mèdics, així com incrementar l'impacte de les noves tecnologies desenvolupades. Els criteris de priorització considerats són molt amplis i inclouen l'impacte d'un nou test en la incidència d'una malaltia, la disponibilitat i preu dels tractaments de les malalties, la inversió tecnològica per al desenvolupament d'un nou dispositiu, i els principis bioètics. El tercer capítol descriu el desenvolupament d'un dispositiu mèdic senzill que pot ser fabricat fàcilment en laboratoris amb escassos recursos: tires reactives de diagnòstic de paper per a la detecció de biomarcadors presents en fluids biològics fabricats amb impressores de raig de tinta domèstiques i amb receptes senzilles per la preparació de les tintes. Aquesta tècnica de fabricació de tires reactives de diagnòstic va ser provada per a la detecció de deficiència de iode, un problema sever de salut global al món. En aquest capítol es presenten experiments de preparació de tintes químiques, impressió en paper, detecció de iode en les concentracions presents en l'orina, i consells per al desenvolupament de noves tintes per a la detecció d'altres biomarcadors de malalties. Aquest simple i versàtil procés de fabricació de tests de diagnòstic permetria a hospitals i laboratoris amb pocs recursos dissenyar els seus propis diagnòstics per a malalties rellevants, i en la forma i quantitat adaptada a les necessitats de cada comunitat. Desafortunadament, no totes les malalties es poden diagnosticar usant senzilles tires reactives de diagnòstic, i freqüentment es necessiten dispositius més complexos. El capítol 4 està enfocat en el desenvolupament de dispositius de PCR i RT-PCR de baix cost, a temps real, i de tipus POC que permeten detectar quantitativament patògens basats en DNA i RNA respectivament. El nostre sistema es basa en PCR de flux continu, el qual manté zones de temperatura fixes i empeny la solució de PCR entre les àrees calefactades, permetent així una transferència de calor més ràpida i conseqüentment, una PCR més veloç. Tots dos sistemes de PCR i RT-PCR van ser fabricats a partir d'un xip microfluídic sol ús dissenyat per a ser produït a baix cost industrialment mitjançant mètodes de 'roll-to-roll'. El sistema òptic permet la detecció de patògens en temps real mitjançant mesures de fluorescència. Per demostrar la funció del xip, dos bacteris infeccioses i un virus van ser seleccionats: Chlamydia trachomatis, Escherichia coli O157: H7, i Ebola virus. Per als tres patògens, es van provar diferents velocitats de flux, es va determinar el límit de detecció del sistema, i es van calcular les eficiències de les PCRs. L'èxit dels resultats obtinguts i la versatilitat del sistema, fa que aquests dispositius es considerin prometedors per al diagnòstic d'altres patògens com Zika o chikungunya, que constitueixen amenaces mundials a la salut pública. Tots dos dispositius de diagnòstic in vitro presentats en aquesta tesi són bons exemples de dispositius de diagnòstic apropiats per a salut global.<br>Los test de tipo ‘point-of-care’ (POC) presentan un gran potencial para el manejo y el diagnóstico de enfermedades. Los dispositivos POC permiten la realización de pruebas clínicas cerca del paciente, permitiendo así un diagnóstico rápido, una pronta iniciación de tratamientos, y en caso necesario, una derivación rápida a otros centros médicos. Estos dispositivos tienen además el potencial de ser más económicos, más robustos, y más fáciles de usar que los dispositivos médicos tradicionales. Por estos motivos, los dispositivos médicos de tipo POC se consideran prometedores para los países en vías de desarrollo, los cuales son también los que necesitan de forma más urgente nuevas tecnologías médicas. En este contexto, esta tesis se centra en el desarrollo de dispositivos médicos de diagnóstico in vitro de tipo POC para salud global. Teniendo en cuenta que los recursos para el desarrollo de dispositivos POC para países con bajos recursos son limitados, el Capítulo 2 se enfoca en el desarrollo de prioridades de investigación en salud. Mediante el establecimiento de estas prioridades se pretende facilitar la selección de objetivos a fabricantes de dispositivos médicos, así como incrementar el impacto de las nuevas tecnologías desarrolladas. Los criterios de priorización considerados son muy amplios e incluyen el impacto de un nuevo test en la incidencia de una enfermedad, la disponibilidad y precio de los tratamientos de las enfermedades, la inversión tecnológica para el desarrollo de un nuevo dispositivo, y los principios bioéticos. El segundo Capítulo 3 describe el desarrollo de un dispositivo médico sencillo que puede ser fabricado fácilmente en laboratorios con escasos recursos: tiras reactivas de diagnóstico de papel para la detección de biomarcadores presentes en fluidos biológicos fabricados con impresoras de chorro de tinta domésticas y con recetas sencillas para la preparación de las tintas. Esta técnica de fabricación de tiras reactivas de diagnóstico fue probada para la detección de deficiencia de yodo, un problema severo de salud global en el mundo. En este capítulo se presentan experimentos de preparación de tintas químicas, impresión en papel, detección de yodo en las concentraciones presentes en la orina, y directrices para el desarrollo de nuevas tintas para la detección de otros biomarcadores de enfermedades. Este simple y versátil proceso de fabricación de tests de diagnóstico permitiría a hospitales y laboratorios con pocos recursos diseñar sus propios diagnósticos para enfermedades relevantes, en una forma y cantidad adaptada a las necesidades de cada comunidad. Desafortunadamente, no todas las enfermedades pueden diagnosticarse usando sencillas tiras reactivas de diagnóstico, y frecuentemente se necesitan dispositivos más complejos. El Capítulo 4 está enfocado en el desarrollo de dispositivos de PCR y RT-PCR de bajo coste, de tiempo-real, y de tipo POC que permiten detectar cuantitativamente patógenos basados en DNA y RNA respectivamente. Nuestro sistema se basa en PCR de flujo continuo, el cual mantiene zonas de temperatura fijas y empuja la solución de PCR entre las áreas calefactadas, permitiendo así una transferencia de calor más rápida y consecuentemente, PCR más veloces. Ambos sistemas de PCR y RT-PCR fueron fabricados en base a un chip microfluídico desechable diseñado para ser producido a bajo coste industrialmente mediante métodos de ‘roll-to-roll’. El sistema óptico permite la detección de patógenos en tiempo real mediante medidas de fluorescencia. Para demostrar la función del chip, dos bacterias infecciosas y un virus fueron seleccionados: Chlamydia trachomatis, Escherichia coli O157:H7, y Ebola virus. Para los tres patógenos, se probaron diferentes velocidades de flujo, se determinó el límite de detección del sistema, y se calcularon las eficiencias de las PCRs. El éxito de los resultados obtenidos y la versatilidad del sistema, hace que estos dispositivos se consideren prometedores para el diagnóstico de otros patógenos como Zika o chikungunya, que constituyen amenazas mundiales a la salud pública. Ambos dispositivos de diagnóstico in vitro presentados en esta tesis son buenos ejemplos de dispositivos de diagnóstico apropiados para salud global.<br>Point-of-care (POC) testing has great potential for the management and diagnosis of disease. POC devices allow for testing close to the patient permitting rapid diagnosis, prompt treatment initiation, and when needed, quick referral to other health-care units. They have the potential to be lower-cost, more robust, and more user-friendly than traditional medical devices. For these reasons, POC diagnostic tests are a promising approach for the developing world, where there is also the most urgent need for new health technologies. In this context, this thesis is focused in the development of POC in vitro diagnostic tests for global health. Considering that the resources for developing POC devices for low-resource settings are limited, during Chapter 2 we focused on setting health research priorities to aid test developers setting their targets to increase the impact of the technology. The criteria for prioritization considered were very broad and took into account the impact of a new test on the burden of disease, the availability and expense of disease treatments, the technological investment to develop a new device, and the bioethical principles. Chapter 3 describes the development of a medical device that can be easily manufactured in limited resources laboratories: paper diagnostic chemical dipsticks to detect biomarkers present in biological fluids produced with domestic inkjet printers and simple ink preparation recipes. This fabrication technique for diagnostic strips was tested for the detection of iodine deficiency, a severe global health problem worldwide. In this chapter we present successful experiments for chemical inks preparation, printing on paper, detection of iodine in the concentrations present in the urine, and guidelines for new ink development to target other disease biomarkers. This simple and versatile manufacturing process for diagnostic tests would allow hospitals and laboratories with limited infrastructure to design diagnostics for relevant diseases in a format and quantity adapted to each community needs. Unfortunately, not all diseases can be diagnosed using simple chemical dipstick assays and more complex diagnostic devices are required. Chapter 4 is focused on the development of a low-cost, real-time, point-of-care PCR and RT-PCR systems for quantitative detection of DNA and RNA-based pathogens. Our systems are based on continuous-flow PCR which maintains fixed temperatures zones and pushes the PCR solution between heated areas allowing for faster heat transfer and as a result, faster PCRs. Both PCR and RT-PCR systems were built around disposable microfluidic chips designed to be economically produced industrially by roll-to-roll embossing methods. The optical system allows for pathogen detection via real-time fluorescence measurements. To demonstrate the function of the chips, two infectious bacteria and one viral target were selected: Chlamydia trachomatis, Escherichia coli O157:H7, and Ebola virus. For the three pathogens, different flow velocities were tested, the limit of detection of the system was determined, and PCR efficiencies were calculated. Our successful results, and the versatility of our system, make it promising for the detection of other DNA and RNA-based pathogens such as Zika or chikungunya, which constitute global health threats worldwide. The two in vitro diagnostic tests presented in this thesis are good examples of promising POC diagnostic devices appropriate for global health.

De nos jours, efficacité et précision des diagnostics médicaux sont des éléments essentiels pour la prévention en termes de santé et permettre une prise en charge rapide des maladies des patients. Les récentes innovations technologiques, particulièrement dans les domaines de la microélectronique et des sciences des matériaux ont permis le développement de nouvelles plateformes personnalisées de diagnostics portatifs. Les matériaux électroniques organiques qui ont déjà par le passé démontré leur potentiel en étant intégrés dans des produits de grande consommation tels que les écrans de smartphones ou encore les cellules solaires montrent un fort potentiel pour une intégration dans des dispositifs biomédicaux. En effet, de par leurs natures et leurs propriétés physiques et chimiques, ils peuvent être à la fois en contact avec les milieux biologiques et constituer l’interface entre les éléments biologiques à l’étude, et les dispositifs électroniques. L’objectif de mes travaux de thèse et d’étudier et évaluer les performances des matériaux organiques électroniques intégrés dans des dispositifs biomédicaux en étudiant leurs interactions avec des milieux biologiques et par l’utilisation et l’optimisation de ces dispositifs permettre la détection de métabolites tel que le glucose ou lactate par exemple. Pendant ma thèse, j’ai notamment créé une plateforme de diagnostics combinant à la fois microfluidique et électronique organique permettant la multi détection de métabolites présents dans des fluides corporels humains, j’ai également conçu des capteurs intégrant des transistors organiques au sein des circuits électroniques classiques afin de détecter la présence des cellules tumorales. D’autres applications biologiques ont également été envisagées telles que la détection d’acides nucléiques par l’utilisation d’une approche simple de biofonctionnalisation. Bien que l’objectif ma thèse était de de créer des capteurs biomédicaux en utilisant une approche in vitro, il pourrait être également possible d’intégrer ces dispositifs « in vivo » ou encore dans des e-textiles<br>Rapid and early diagnosis of disease plays a major role in preventative healthcare. Undoubtedly, technological evolutions, particularly in microelectronics and materials science, have made the hitherto utopian scenario of portable, point-of-care personalized diagnostics a reality. Organic electronic materials, having already demonstrated a significant technological maturity with the development of high tech products such as displays for smartphones or portable solar cells, have emerged as especially promising candidates for biomedical applications. Their soft and fuzzy nature allows for an almost seamless interface with the biological milieu rendering these materials ideally capable of bridging the gap between electronics and biology. The aim of this thesis is to explore and validate the capabilities of organic electronic materials and devices in real-world biological sensing applications focusing on metabolite sensing, by combining both the right materials and device engineering. We show proof-of-concept studies including microfluidic integrated organic electronic platforms for multiple metabolite detection in bodily fluids, as well as more complex organic transistor circuits for detection in tumor cell cultures. We finally show the versatility of organic electronic materials and devices by demonstrating other sensing strategies such as nucleic acid detection using a simple biofunctionalization approach. Although the focus is on in vitro metabolite monitoring, the findings generated throughout this work can be extended to a variety of other sensing strategies as well as to applications including on body (wearable) or even in vivo sensing

Although advanced medical technology has been progressively developed, access to quality healthcare services is still a major problem, especially in developing countries. Advanced medical technology requires enormous and expensive resources; therefore, affordable, easy to use and accessible technology could bridge the gap, improving people’s lives, and attracting venture capital investment. Moreover, it is expected that the diagnostic device market would grow exponentially, particularly for paper-based technology. As a result, technology development could promote the local economy by creating jobs. Paper-based analytical devices have been introduced and developed over the last decades. This technology has been widely used as a tool for diagnosis in fields such as environment, food quality and healthcare. Point-of-care (POC) diagnosis has attracted a great deal of attention from the research community, eventually aiming for the development of a platform that can evaluate biological markers in body fluids such as saliva and urine. Paper-based diagnostic devices make an impact because of their low cost, environmentally friendliness and biodegradability. The significant advantage of paperbased devices is the capillarity driven fluid transport through the paper network without the need for additional equipment. This thesis starts with a detailed literature review on paper-based devices. The literature review includes material selection, fundamentals, applications and design criteria, and mainly discusses the technical challenges in engineering and biochemical aspects. Additionally, this section will discuss the current research trends and perspectives of advanced technologies for enhancing assay performance. The study of wicking in paper strips predicts the flow behaviour to control fluid flow and perform programmable fluid handling tasks. An accurate model to predict flow in paper is required for designing paper-based devices. In this thesis, a novel model explains wicking in paper strips altogether with liquid absorption capacity. Based on observation, paper can store liquid in a matrix because after removing the reservoir, the fluid continues flowing. We employ the electrical circuit analogy to formulate the model. The capacitance should be included in the model because the capability to store and release charges is analogous to liquid absorption capacity in the matrix which contain and discharge the fluid. The theoretical data from the model agrees well with the experimental data obtained for wicking in paper strips in a vertical configuration. Additionally, fitting the model with experimental data confirms the critical parameters of liquid absorption capacity and capillary pressure. Considering liquid absorption capacity as a capacitance in electrical circuit analogy could elucidate the relationship between materials and wicking mechanism. Next, the thesis focuses on developing of paper-based analytical device fabrication technology that allows the defined hydrophobic pattern on paper to guide fluid along a hydrophilic path in a controlled manner. There have been many fabrication processes reported in recent decades. However, some methods using harsh chemicals result in contamination of the subsequent reagents for analytical assays. Some other techniques have complicated processes requiring expensive equipment, impractical for mass production. The subsequent study focused on a parametric fabrication of parafilm hot pressing, which is inexpensive, rapid, and straightforward. The basic concept is providing heat and pressure to melt and squeeze parafilm into the paper matrix resulting in a hydrophobic pattern defined by a laminate mask. The smallest hydrophobic barrier made by this technique is 821 um, resulting from the resolution of the laminate mask. Likewise, this study also demonstrated the suitability of paper for both physical and biochemical functions. In terms of physical function, the wicking speed on fabricated paper is slower than on non-fabricated paper because the pore could be reduced due to pressure. Diffusive mixing in 2D and 3D paperfluidics are also reported. We employed a sandwich immunological assay for biochemical functions to evaluate protein binding capacity on the paper. Demonstrating the paper device from this fabrication process is potentially applicable to analytical instrument for wicking studies and biomolecule detection. Besides investigating wicking in paper strips and the fabrication process to handle engineering challenges, biomarker detection has been studied to demonstrate diagnostic applications for the developed devices. Biomarkers used in this study include SARSCoV-2 humanised antibody and cell-derived exosomes. The readout methods implemented in this study are colourimetric, fluorescent, and electrochemical. First, we employed a paper-based colorimetric assay using the horseradish peroxidase and 3,3’,5,5’-tetramethylbenzidine (TMB)/hydrogen peroxide system. The colourimetric readout was obtained from a self-made image acquisition system and is quantified using the MATLAB program. The detection limit of SARS-CoV-2 humanised antibody assay was 9.00 ng/uL, which is lower than commercially available kits (0.112 IU/mL vs 5 IU/mL). However, the result for exosome detection encountered many challenges. Firstly, the exosome concentration may be inadequate to reach a detectable range. Secondly, high background signal resulting from non-specific binding on the platform leads to a lack of sensitivity and specificity for exosome detection. A paper-based colourimetric assay has the potential to be further developed into a point-of-care diagnostic device. Further modification of the paper may be required to promote protein binding for specific targets and prevent non-specific binding to reduce the background signal. Next, the thesis reports a paper-based immunofluorescent assay for biomarker detection using fluorophore conjugation with detecting antibodies. The fluorescent-based assay requires a specific excitation wavelength and retrieves a wavelength of emission. Fluorescent microscopy was used to observe the readout. Before, the images were processed and quantified using a MATLAB program. The assay selectively detects SARS-CoV-2 humanised antibodies spiked in PBS and healthy human serum samples. The limit of detection of the assay was 2 ng/uL (0.025 IU/mL) and 10 ng/uL (0.125 IU/mL) in PBS and human serums, respectively. This assay can detect 1010 exosome/mL obtained from cell culture media, but also faced many obstacles. First, exosome concentration prepared from cell culture media may be insufficient to reach the detectable range. Second, minimising chemical contamination could enhance assay specificity and sensitivity to prevent non-specific absorption. Therefore, a paper-based fluorescent assay could be further developed into a portable device. The light source to excite the fluorophore and to emit the signal, including an optical system, could be scaled down from fluorescent microscopy into a handheld-size device. Lastly, this thesis presents a proof-of-concept electrochemical paper-based device for biomarker detection. The paper-based device is fabricated using parafilm hot pressing, as reported previously. The electrochemical chacracterisation on paper-based carbon electrodes was thoroughly investigated using cyclic voltammetry. The detection employed a sandwich immunological assay using carbon electrodes on paper. Differential pulse voltammetry was used to observe the current response in the subsequent steps. The stepwise addition of biomolecules on paper-based carbon electrodes results in the attenuation of the current response caused by stepwise biomolecules binding on the electrode surface. The current reaction after target binding corresponds to the target concentrations. In addition, electrochemical impedance spectroscopy is utilised to affirm the validity of the assay by observing the electron transfer resistance coming from the interfacial electron transfer at the electrode surface. The assay for SARS-CoV-2 antibody detection in PBS samples detected the target concentration in the range of 10 to 100 ng/uL. The detection limit is estimated to be 9.37 ng/uL. For exosome detection prepared from cell culture media, this assay quantified the total exosome and ovarian cell-derived exosome concentration with a limit of detection of 9.3 X 10 exosomes/mL and 7.1 x 10 exosomes/mL with < 10% relative standard deviation for samples of n =3. However, the limit of detection can be enhanced by strengthening antibody immobilisation on the paper-based device and stabilising the carbon electrodes on paper to be conductive enough to sense the change of the subsequent loading of biomolecules. Our electrochemical paper-based assay could be an alternative tool for detecting diseasespecific exosomes in biological samples for point-of-care diagnosis. In conclusion, this study aims to overcome engineering and biochemical challenges posed by paper-based analytical devices. To tackle the engineering issues, a novel wicking model with consideration of liquid absorption capacity offers an alternative way to predict flow behaviour in capillary rise experiments and explain the material characteristics. Additionally, the thesis studies the fabrication parameters to control the paper-based device fabrication better using parafilm hot pressing, demonstrating the functionality of paper for both physical and biochemical applications. Regarding biochemical challenges, the thesis reports colourimetric, fluorescent, and electrochemical techniques implemented on paper-based platforms, employing a sandwich immunological assay to detect biomarkers, which include SARS-CoV-2 humanised antibody and cell-derived exosome samples. These established protocols have the potential to be further improved for automation and portability, which could be compatible with other advanced technologies such as wearable sensing devices, artificial intelligence and machine learning.<br>Thesis (PhD Doctorate)<br>Doctor of Philosophy (PhD)<br>School of Eng & Built Env<br>Science, Environment, Engineering and Technology<br>Full Text

La tecnologia "a inkjet-Printing" està cridada a liderar la propera generació d'electrònica flexible capaç de realitzar funcions a què només es podia accedir amb tecnologies de microfabricació d'última generació. El inkjet-Printing és un mètode de fabricació additiva que es basa en imprimir sense contacte microgotes d'un material funcional amb precisió micromètrica en l'àrea de l'substrat desitjat, a través d'un disseny digital. A més, és capaç de modificar el disseny d'impressió en temps real. En conseqüència, es permet crear dissenys personalitzats amb característiques úniques. Avui en dia, el inkjet-Printing a nivell industrial ha assolit alts estàndards de rendiment, flexibilitat, robustesa i confiança. Es preveu és que el inkjet-Printing facilitarà la producció de productes electrònics flexibles de forma rendible, basant-se conceptes d'economia circular i reduint el malbaratament, el que permetrà el desenvolupament de dispositius portàtils i d'un sol ús no disponibles actualment. Aquest és el punt en el qual els dispositius de Point-of-Care (PoC) entren en l'equació causa de la seva importància en les proves mèdiques. Aquests dispositius es defineixen com a proves de diagnòstic mèdic en el pacient o prop d'ell mateix. Els dispositius PoC es basen en una mesura ràpida i precisa basada en sensors que ofereixen a el metge dades importants per realitzar un diagnòstic. No obstant això, les principals limitacions d'aquests dispositius, fins i tot avui dia, són el cost, la disponibilitat, la biodegradabilitat i la fiabilitat. El inkjet-Printing ofereix solucions per abordar aquests problemes on els seus grans promeses són la fabricació ràpida de prototips, de baix cost, utilitzant una gran varietat de materials i una àmplia gamma de substrats. No obstant això, la nostra comprensió de la tecnologia i les seves capacitats roman en una etapa prometedora, i cal adquirir més experiència per facilitar el desenvolupament de dispositius PoC complets i totalment impresos. Identificant aquests problemes i possibles solucions, aquesta tesi se centra en mostrar el potencial de l'inkjet Printing per desenvolupar sensors sobre substrats de plàstic flexible i paper porós, desafiant la tecnologia fins al seu límit actual. En una primera part s'aborda la formulació, impressió i caracterització de noves tintes funcionals que permetin obtenir noves tintes conductores per a ser usades en l'àrea de l'sensat d'analits d'interès. Sobre plàstic flexible, s'han desenvolupat dos sensors de pH potenciomètrics. El primer mostra la importància de la propietat de rugositat intrínseca d'una nova tinta de platí en base a nanopartícules per dotar d'estabilitat mecànica a l'òxid d'iridi, material sensible a l'pH, crescut electroquímicament sobre ell. Per a aquest propòsit, es va desenvolupar un sensor de pH en PEN utilitzant la nova tinta de Pt i es va estudiar l'estabilitat al llarg d'un any d'aquesta capa d'òxid d'iridi, que va mostrar una clara millora del seu rendiment. El segon sensor de pH va un pas més i és totalment imprès per a inkjet-Printing. Per complir aquest objectiu, es va formular una nova tinta polimèrica sensible a el pH. En una segona part es va abordar el repte de la impressió d'un sensor sobre un substrat més ecosostenible com és el paper, factor important per als PoC sol ús. En un primer treball es va abordar el desafiament d'imprimir tintes funcionals conductores com l'or o la plata, i dielèctriques com el SU8 sobre el substrat de manera eficient i fàcil de reproduir per a l'obtenció d'un sensor electroquímic. Finalment, en un segon treball es va implementar un immunosensor de cortisol sobre aquests sensors impresos sobre substrat de paper i es va caracteritzar i comparar la seva resposta respecte d'altres sensors reportats, demostrant el bon rendiment d'aquesta tecnologia en la detecció de molècules biològiques.<br>La tecnología "Inkjet-Printing" está llamada a liderar la próxima generación de electrónica flexible capaz de realizar funciones a las que sólo se podía acceder con tecnologías de microfabricación de última generación. El Inkjet-Printing es un método de fabricación aditiva que se basa en imprimir sin contacto microgotas de un material funcional con precisión micrométrica en el área del sustrato deseado, a través de un diseño digital. Además, es capaz de modificar el diseño de impresión en tiempo real. En consecuencia, se permite crear diseños personalizados con características únicas. Hoy en día, el Inkjet-Printing a nivel industrial ha alcanzado altos estándares de rendimiento, flexibilidad, robustez y confianza. Se prevé es que el Inkjet-Printing facilitará la producción de productos electrónicos flexibles de forma rentable, en base a conceptos de economía circular y reduciendo el desperdicio, lo que permitirá el desarrollo de dispositivos portátiles y desechables no disponibles actualmente. Este es el punto en el que los dispositivos de Point-of-Care (PoC) entran en la ecuación debido a su importancia en las pruebas médicas. Estos dispositivos se definen como pruebas de diagnóstico médico en el paciente o cerca del mismo. Los dispositivos PoC se basan en una medición rápida y precisa basada en sensores que ofrecen al médico datos importantes para realizar un diagnóstico. Sin embargo, las principales limitaciones de estos dispositivos, incluso hoy en día, son el coste, la disponibilidad, la biodegradabilidad y la fiabilidad. El Inkjet-Printing ofrece soluciones para abordar estos problemas donde sus grandes promesas son la fabricación rápida de prototipos, de bajo coste, usando una gran variedad de materiales y una amplia gama de sustratos. Sin embargo, nuestra comprensión de la tecnología y sus capacidades permanece en una etapa prometedora, y es necesario adquirir más experiencia para facilitar el desarrollo de dispositivos PoC completos y totalmente impresos. Identificando estos problemas y posibles soluciones, esta tesis se centra en mostrar el potencial del Inkjet Printing para desarrollar sensores sobre sustratos de plástico flexible y papel poroso, desafiando la tecnología hasta su límite actual. En una primera parte se aborda la formulación, impresión y caracterización de nuevas tintas funcionales que permitan obtener nuevas tintas conductoras para ser usadas en el área del sensado de analitos de interés. Sobre plástico flexible, se han desarrollado dos sensores de pH potenciométricos. El primero muestra la importancia de la propiedad de rugosidad intrínseca de una nueva tinta de platino en base a nanopartículas para dotar de estabilidad mecánica al óxido de iridio, material sensible al pH, crecido electroquímicamente sobre él. Para este propósito, se desarrolló un sensor de pH en PEN utilizando la nueva tinta de Pt y se estudió la estabilidad a lo largo de un año de esta capa de óxido de iridio, que mostró una clara mejora de su rendimiento. El segundo sensor de pH va un paso más y es totalmente impreso por Inkjet-Printing. Para cumplir este objetivo, se formuló una nueva tinta polimérica sensible al pH. En una segunda parte se abordó el reto de la impresión de un sensor sobre un sustrato más ecosostenible como es el papel, factor importante para los PoC desechables. En un primer trabajo se abordó el desafío de imprimir tintas funcionales conductoras como el oro o la plata, y dieléctricas como el SU8 sobre el sustrato de manera eficiente y fácil de reproducir para la obtención de un sensor electroquímico. Finalmente, en un segundo trabajo se implementó un immunosensor de cortisol sobre estos sensores impresos sobre sustrato de papel y se caracterizó y comparó su respuesta respecto a otros sensores reportados, demostrando el buen rendimiento de esta tecnología en la detección de moléculas biológicas en matrices fisiológicas.<br>Universitat Autònoma de Barcelona. Programa de Doctorat en Enginyeria Electrònica i de Telecomunicació

La thrombine est l'enzyme principale dans le processus d'hémostase. Les dérèglements de la concentration de thrombine clinique prédisposent les patients à des complications hémorragiques ou thromboemboliques. Le suivi en temps réel de la thrombine dans le sang est donc nécessaire pour améliorer le traitement de patients en état critique. Les aptamères, qui sont de courts nucléotides monobrins semblent constituer des candidats prometteurs pour la reconnaissance moléculaire dans les biocapteurs. L'objectif de ces travaux est l'étude de différentes solutions d'intégration des aptamères dans des dispositifs de diagnostic de type "point of care" pour le suivi en continu de la thrombine dans le plasma. La cinétique d'interaction des aptamères avec la thrombine et leur spécificité vis-à-vis de la prothrombine et des inhibiteurs de la thrombine ont été étudiés par résonance par plasmons de surface. Ces travaux ont démontré la faible spécificité de l'aptamère HD1 vis-à-vis de la thrombine, et la présence d'interactions non-spécifiques avec la prothrombine, les inhibiteurs naturels de la thrombine et l'albumine. Inversement, nous avons observé une bonne affinité de l'aptamère HD22 avec la même liste de cible. Parallèlement, nous avons évalué des stratégies d'intégration d'aptamères dans des dispositifs d'analyse. Le principe de reconnaissance a ensuite été validé et la possibilité de détecter la thrombine dans des gammes de concentration de 5 à 500nM a été démontrée. Enfin, afin d'augmenter la spécificité de la détection de la thrombine, nous avons proposé une nouvelle approche basée sur l'ingénierie de structures dimères interconnectant HD1 et HD22<br>Thrombin is the central enzyme in the process of hemostasis. Normally, in vivo concentration of thrombin is rigorously regulated; however, clinically impaired or unregulated thrombin generation predisposes patients either to hemorrhagic or thromboembolic complications. Monitoring thrombin in real-time is therefore needed to enable rapid and accurate determination of drug administration strategy for patients under vital threat. Aptamers, short single-stranded oligonucleotide ligands represent promising candidates as biorecognition elements for new-generation biosensors. The aim of this PhD work therefore is to investigate different solutions for the integration of thrombin-binding aptamers in point-of-care devices for continuous monitoring of thrombin in plasma. The kinetics of aptamer interaction with thrombin and specificity towards prothrombin and thrombin - inhibitor complexes was rigorously investigated using Surface Plasmon Resonance. These experiments unveiled the complex character of interaction of the HD1 with thrombin, confirming nonspecific interactions with prothrombin, natural inhibitors of thrombin, serum albumin whereas another 29-bp aptamer HD22 proved to be highly affine and specific towards thrombin. On the other hand we explored aptamer integration options. We validated the principle and at the same managed to detect different concentrations of thrombin (5-500 nM). We finally proposed a novel approach to increase sensitivity and specificity for thrombin detection based on the engineering of aptadimer structures bearing aptamers HD1and HD22 interconnected with a nucleic acid spacer

Un dels majors reptes per a monitoritzar i millorar la salut de la població a nivell mundial és la manca de proves de diagnòstic apropiades per a la detecció primerenca de malalties, la selecció de tractaments apropiats i el seguiment de pacients al llarg del temps. La disponibilitat d’eines de diagnòstic prou ràpides, sensibles i robustes és crucial per aconseguir el benestar dels pacients a tot el món. En aquest context, la nanotecnologia i el desenvolupament de biosensors són camps en ràpida evolució que han generat grans expectatives, produint proves més ràpides i més fàcils de realitzar que la majoria dels mètodes clàssics. Els biosensors s’han descrit en base a l’ús d’una àmplia varietat d’elements de biotecnologia i tipus de transducció de senyals. Entre ells, els biosensors electroquímics són el tipus més comú en ús avui en dia gràcies a la portabilitat i el baix cost de l’equip de mesura, les mesures ràpides, robustes i quantitatives proporcionades, i la facilitat de miniaturització de tot el sistema de detecció. La recent incorporació de paper per a la producció d’elèctrodes impresos en paper i assajos electroquímics de flux lateral està fomentant el desenvolupament de dispositius extremadament econòmics que, gràcies a les propietats fluídicas del paper, permeten reduir la complexitat de l’assaig i el nivell de manipulació per al usuari final. Això afavoreix el desenvolupament de dispositius de diagnòstic de \”Point-of-care\” (POC), que poden ser utilitzats directament pel pacient o als centres d’atenció primària de salut. D’altra banda, les partícules magnètiques (PM) s’han utilitzat amb gran èxit en l’optimització dels magneto-biosensors. Les PM són atractives per a aquest propòsit perquè, un cop modificades amb un bioreceptor apropiat, atorguen una preconcentració simple, ràpida i específica de l’analit objectiu. Les PM també ofereixen superfícies actives en 3D relativament grans, que es barregen amb agitació constant amb les mostres i permeten una ràpida unió amb els analits. No obstant això, les PM també presenta limitacions, com el seu maneig tediós i lent que només està a l’abast d’usuaris altament capacitats. L’objectiu principal d’aquest projecte de tesi doctoral va ser la producció de magneto-biosensors electroquímics ràpids, fàcils de realitzar, robustos i sensibles per a la detecció de biomarcadors de diagnòstic en mostres de sèrum, plasma i sang. Com es mostrarà, això s’ha aconseguit en dos nivells. Primer, desenvolupant un format de magneto-immunoassaig extremadament ràpid i simple. En segon lloc, fabricant elèctrodes de paper microfluids simples i econòmics, que van ser explotats per dur a terme en el xip la majoria dels passos del magneto-immunoassaig simplificat amb la mínima intervenció de l’usuari.<br>Uno de los mayores desafíos para monitorear y mejorar la salud de la población a nivel mundial es la falta de pruebas de diagnóstico apropiadas para la detección temprana de enfermedades, la selección de tratamientos apropiados y el seguimiento de pacientes a lo largo del tiempo. La disponibilidad de herramientas de diagnóstico suficientemente rápidas, sensibles y robustas es crucial para lograr el bienestar de los pacientes en todo el mundo. En este contexto, la nanotecnología y el desarrollo de biosensores son campos en rápida evolución que han generado grandes expectativas, produciendo pruebas más rápidas y más fáciles de realizar que la mayoría de los métodos clásicos. Los biosensores se han descrito en base al uso de una amplia variedad de elementos de biotecnología y tipos de transducción de señales. Entre ellos, los biosensores electroquímicos son el tipo más común en uso hoy en día gracias a la portabilidad y el bajo costo del equipo de medición, las medidas rápidas, robustas y cuantitativas proporcionadas, y la facilidad de miniaturización de todo el sistema de detección. La reciente incorporación de papel para la producción de electrodos impresos en papel y ensayos electroquímicos de flujo lateral está fomentando el desarrollo de dispositivos extremadamente económicos que, gracias a las propiedades fluídicas del papel, permiten reducir la complejidad del ensayo y el nivel de manipulación para el usuario final. Esto favorece el desarrollo de dispositivos de diagnóstico de \”Point-of-care\” (POC), que pueden ser utilizados directamente por el paciente o en los centros de atención primaria de salud. Por otro lado, las partículas magnéticas (PM) se han utilizado con gran éxito en la optimización de los magneto-biosensores. Las PM son atractivos para este propósito porque, una vez modificados con un bioreceptor apropiado, otorgan una preconcentración simple, rápida y específica del analito objetivo. Las PM también ofrecen superficies activas en 3D relativamente grandes, que se mezclan bajo agitación constante con las muestras y permiten una rápida unión con los analitos. Sin embargo, las PM también presenta limitaciones, como su manejo tedioso y lento que solo está al alcance de usuarios altamente capacitados. El objetivo principal de este proyecto de tesis doctoral fue la producción de magneto-biosensores electroquímicos rápidos, fáciles de realizar, robustos y sensibles para la detección de biomarcadores de diagnóstico en muestras de suero, plasma y sangre. Como se mostrará, esto se ha logrado en dos niveles. Primero, desarrollando un formato de magneto-inmunoensayo extremadamente rápido y simple. En segundo lugar, fabricando electrodos de papel microfluidos simples y económicos, que fueron explotados para llevar a cabo en el chip la mayoría de los pasos del magneto-inmunoensayo simplificado con la mínima intervención del usuario.<br>One of the greatest challenges for monitoring and improving the health of the population at a global level is the lack of appropriate diagnostic tests for early detection of diseases, selection of appropriate treatments and patient follow-up over time. The availability of sufficiently fast, sensitive and robust diagnostic tools will be crucial to achieve patients’ well-being worldwide. In this context, nanotechnology and biosensor development are rapidly evolving fields that have generated great expectations, producing tests faster and easier to carry out than most classical methods. Biosensors have been described based on the use of a wide variety of biotechnology elements and types of signal transduction. Among them, electrochemical biosensors are the most common type in use today thanks to the portability and low cost of the measuring equipment, fast, robust and quantitative measures provided, and easiness of miniaturization of the whole detection system. The recent incorporation of paper and paper-like materials for the production of paper printed electrodes and lateral flow electrochemical assays is fostering the development of extremely inexpensive devices that, thanks to the fluidic properties of paper, allow reducing assay complexity and level of manipulation for the end user. This favours the development of "Point-of-Care" diagnostic devices (POC), which can be used directly by the patient or at primary health care centres. On the other hand, magnetic beads (MB) have been used with great success in the optimization of magneto-biosensors. MB are attractive for this purpose because, once modified with an appropriate bioreceptor, they grant simple, rapid and specific preconcentration of the targeted analyte. MB offer also relatively large 3D active surfaces, which mixed under constant agitation with the sample supply efficient and fast analyte binding as well. Nevertheless, MB display limitations too, requiring tedious and time-consuming handling that is only at reach of highly trained users. The main objective of this PhD Thesis project was the production of rapid, easy to perform, robust and sensitive electrochemical magneto-biosensors for the detection of diagnostic biomarkers in serum, plasma and blood samples. As it will be shown, this has been achieved at two levels. First, by developing an extremely fast and simple magnetoimmunoassay format. Second, by fabricating simple and inexpensive microfluidic paper electrodes, which were exploited to carry out on-chip most of the steps of the simplified magneto-immunoassay with minimal user intervention.

The recent growing interest and development of microfluidic paper-based analytical devices (μPADs) for point-of-care (POC) testing in human health in low-resource settings has great potential for the exploitation of these technologies in animal disease diagnosis. Sheep scab is a highly infectious, widespread and notifiable disease of sheep, which poses major economic and welfare concerns for the UK farming industry. The possibility of diagnosing sheep scab at the POC is, consequently, very important to controlling this disease. The overall aim of this project was, therefore, to develop μPADs based on a novel method of fabrication, in order to translate the existing lab-based sheep scab ELISA (Pso o 2) and a biomarker test for haptoglobin (Hp) into paper-based ELISA (P-ELISA), to enable POC diagnosis of this animal disease. In Chapter 3, the novel fabrication method is described, in Chapters 4 and 5, the translation of the lab-based ELISAs (Hp and Pso o 2 respectively) are explained and in Chapter 6 the development of a μPAD for incorporation of the POC tests into a multiplexed, rapid assay is covered. Experiments showed that both ELISAs were successfully transferred onto paper and that the devices developed were suitable for POC testing. This study has resulted in a novel fabrication method for μPADs, in successfully translated existing ELISAs to P-ELISA and in novel solutions for the POC diagnosis of an important veterinary disease.

This dissertation explores the different ways to create thin film-based biosensors that are capable of rapid and label-free detection of cortisol, a non-specific biomarker closely linked to stress, within the physiological range of 10pM to 10 uM. Increased cortisol levels have been linked to stress-related diseases, such as chronic fatigue syndrome, irritable bowel syndrome, and post-traumatic stress disorder. It also plays a role in the suppression of the immune system as well. Therefore, accurate measurement of cortisol in saliva, serum, plasma, urine, sweat, and hair, is clinically significance to predict physical and mental diseases. In this dissertation, thin film-based electrochemical immunosensors were fabricated using a self-assembled monolayer (SAM) functionalized by cortisol specific antibodies to detect cortisol at 10 pM level sensitivities in the presence of a redox probe. The fabricated electrochemical cortisol immunosensors were able to detect cortisol in human saliva samples and the outcomes were validated using the standard Enzyme Linked Immuno Sorbent Assay (ELISA) technique. With the aim of improving signal amplification and label-free cortisol detection, copper nanoparticles were incorporated on screen-printed carbon electrodes (SPCE) for the fabrication of electrochemical cortisol immunosensor. This SPCE-based sensor showed a sensitivity of 4.21µA/M and the limit of detection 6.6nM. Both the SAM and SPCE-based immunosensors were not thermally stable due to the instability of antibodies at room temperature. To address this issue, an antibody-free immunosensor was fabricated. Molecular Imprinted Polymer (MIP) was used to template the target cortisol molecule. The MIP-based sensing platform was prepared using polypyrrole, a thermally stable conducting polymer. The conductivity of the polymer ensured good electrical performance. The polypyrrole-based MIP was synthesized by means of electrochemical polymerization and was used to detect cortisol within the physiological range at room temperature. MIP-based sensors exhibited the detection limit of 1 pM, and were cost-effective, easy to fabricate, temperature stable, and reusable. The sensing performance of the resulting sensors was comparable to those of commercially available technologies, such as ELISA. Aiming to perform cortisol sensing at point-of-care (POC), an Extended Gate Field Effect Transistor (EGFET) was integrated with a developed MIP cortisol sensor. The as developed MIP-EGFET sensor was used to detect the cortisol concentration in the range of 1 pM to 100 nM. A few of the major advantages of the developed sensor are its ability to provide a direct readout and simpler electronic systems, which are necessary for miniaturized Point of Care devices.

This dissertation describes the development of a label-free, electrochemical immunosensing platform integrated into a low-cost microfluidic system for the sensitive, selective and accurate detection of cortisol, a steroid hormone co-related with many physiological disorders. Abnormal levels of cortisol is indicative of conditions such as Cushing’s syndrome, Addison’s disease, adrenal insufficiencies and more recently post-traumatic stress disorder (PTSD). Electrochemical detection of immuno-complex formation is utilized for the sensitive detection of Cortisol using Anti-Cortisol antibodies immobilized on sensing electrodes. Electrochemical detection techniques such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) have been utilized for the characterization and sensing of the label-free detection of Cortisol. The utilization of nanomaterial’s as the immobilizing matrix for Anti-cortisol antibodies that leads to improved sensor response has been explored. A hybrid nano-composite of Polyanaline-Ag/AgO film has been fabricated onto Au substrate using electrophoretic deposition for the preparation of electrochemical immunosening of cortisol. Using a conventional 3-electrode electrochemical cell, a linear sensing range of 1pM to 1µM at a sensitivity of 66µA/M and detection limit of 0.64pg/mL has been demonstrated for detection of cortisol. Alternately, a self-assembled monolayer (SAM) of dithiobis(succinimidylpropionte) (DTSP) has been fabricated for the modification of sensing electrode to immobilize with Anti-Cortisol antibodies. To increase the sensitivity at lower detection limit and to develop a point-of-care sensing platform, the DTSP-SAM has been fabricated on micromachined interdigitated microelectrodes (µIDE). Detection of cortisol is demonstrated at a sensitivity of 20.7µA/M and detection limit of 10pg/mL for a linear sensing range of 10pM to 200nM using the µIDE’s. A simple, low-cost microfluidic system is designed using low-temperature co-fired ceramics (LTCC) technology for the integration of the electrochemical cortisol immunosensor and automation of the immunoassay. For the first time, the non-specific adsorption of analyte on LTCC has been characterized for microfluidic applications. The design, fabrication technique and fluidic characterization of the immunoassay are presented. The DTSP-SAM based electrochemical immunosensor on µIDE is integrated into the LTCC microfluidic system and cortisol detection is achieved in the microfluidic system in a fully automated assay. The fully automated microfluidic immunosensor hold great promise for accurate, sensitive detection of cortisol in point-of-care applications.

Background: At the end of 2012, worldwide, approximately 35.5 million people were living with HIV. 1 HIV mortality rates, in resource limited settings are now starting to improve as antiretroviral therapy (ART) is becoming universally available. Now, these settings need to improve the care provided to patients. Good quality care requires timely detection, staging and initiation of therapy. CD4+ cell counting, point-of-care (POC) devices could improve the quality of care in resource limited settings, by allowing for the decentralization of HIV care. As a result, these POC CD4+ cells assays could circumvent patient barriers to care, and relieve building pressure on regional laboratories. Several POC CD4+ devices are available, but an independent comparison of performance has not yet been done. The aim of this thesis is to evaluate the current evidence for POC CD4+ cell counting technologies and determine whether their performance would allow them to be used interchangeably with the current gold standard.Methods: To attain our objective we completed a systematic review and meta-analysis of the evidence relating to POC CD4+ cell assays. Our populations of interest were global populations of adults with a HIV+ status. Our outcome of interest was to the absolute Bland Altman mean bias, which represents the agreement between the POC device and the gold standard. We systematically searched 19 databases, relevant conferences and grey literature for the period 2000 to 2013. Of 4154 citations found, 16 articles were selected. A Bayesian hierarchical normal-normal model was used to meta-analyze data.Findings: POC devices appear to perform best in capillary samples. Only sufficient data was available to allow for a meta-analysis of the PIMA device; a smaller BA mean bias in capillary blood vs. venous specimens was found (-3.0 cells/μL; 95% CrI: -28•2 to 22•8 vs. -26•5 cells/μL; 95% CrI: -46•7 to -6•8). Insufficient data was available for other POC devices (the MiniPOC and the MBio) to allow for a meta-analysis; however they also appear to perform well when considered graphically in a forest plot.Conclusion: The PIMA CD4 device was comparable to flow cytometry as the estimated difference between the CD4+ cell count of the device and the reference fell within a range of acceptable accuracy (+/- 30 cells/μL). The miniPOC and the MBio devices also appear to perform well. Devices appear to better estimate capillary specimens compared to venous specimens. POC CD4+ cell count devices are a rapidly developing field and so providers need reliable evidence for technology selection decisions. The synthesis of evidence relating to the accuracy of POC CD4+ cell counting devices may be of interest for initiatives that are scaling up the use of these devices globally.<br>Contexte : À la fin de 2012, il y avait 35,5 millions de personnes à travers le monde vivant avec le VIH.1 Le taux de mortalité du VIH dans des milieux à faibles ressources commence maintenant à s'améliorer avec la disponibilité de la thérapie antirétrovirale (TAR) qui est en train de devenir universelle. Maintenant ces milieux doivent améliorer les soins donnés aux patients. Les soins de bonne qualité nécessitent un dépistage, une détermination du stade et un début de traitement rapides. Les appareils de diagnostic qui comptent les cellules CD4+ au point de service (POC) pourraient améliorer la qualité des soins dans les milieux à faibles ressources en permettant la décentralization des soins VIH. Par conséquent, ces tests effectués par ces dispositifs POC de diagnostic et de comptage de cellules CD4+ pourraient contourner les obstacles aux soins des patients et atténuer la pression de devoir construire des laboratoires régionaux. Plusieurs dispositifs POC de diagnostic de CD4+ sont disponibles mais une comparaison indépendante de leur performance n'a pas encore été faite. Le but de cette thèse est d'évaluer les éléments dont on dispose déjà concernant les technologies POC comptage de cellules CD+4 et de déterminer si leur performance leur permettrait d'être utilisées de façon interchangeable avec la norme de référence actuelle.Méthodes : Afin d'atteindre notre objectif nous avons procédé à un examen méthodique et à une méta-analyse afin d'évaluer les tests POC comptage de cellules CD4+. Notre résultat d'intérêt était selon le biais moyen absolu de Bland Altman, qui représente l'accord entre le dispositif POC et la norme de référence. Nous avons examiné systématiquement 19 bases de données, ainsi que des conférences et de la littérature grise pertinentes datant de 2000 à 2013. 16 articles ont été sélectionnés sur 4154 citations. Nous nous sommes servis d'un modèle bayésien hiérarchique de distribution normale-normale afin de faire la méta-analyse des données.Résultats : les dispositifs POC semblent fonctionner mieux avec des prélèvements de sang capillaire. Il y avait seulement des données disponibles pour effectuer une méta-analyse du dispositif PIMA ; on a trouvé un plus petit biais moyen Bland Altman dans les prélèvements de sang capillaire par rapport aux échantillons de sang veineux (-3,0 cellules/ μL; 95% CrI : de -28,2 à 22,8 contre -26,5 cellules/ μL; 95% CrI : de -46,7 à -6,8). Les données disponibles pour les autres dispositifs POC (le MiniPOC et le MBio) n'étaient pas suffisantes pour permettre une méta-analyse, pourtant ces dispositifs semblaient bien fonctionner selon la représentation graphique en forêt.Conclusion : Le dispositif PIMA CD4 était comparable à la cytométrie en flux parce que la différence estimée entre le comptage de cellules CD4 du dispositif et de la référence a donné une précision suffisante (+/- 30 cellules/ μL). Les dispositifs miniPOC et MBio semblent bien fonctionner aussi. Il semble que les dispositifs donnent une meilleure estimation de prélèvements de sang capillaire que pour les prélèvements de sang veineux. Les dispositifs POC de comptage de cellules CD4+ font partie d'un secteur en pleine croissance, donc les fournisseurs de soins de santé ont besoin de preuves fiables pour la prise de décision en matière de technologie. La synthèse des données probantes portant sur l'exactitude des dispositifs POC de comptage de cellules CD4+ pourrait intéresser les initiatives qui intensifient l'utilisation de ces dispositifs à l'échelle mondiale.

Point-of-care testing (POCT) is defined as medical testing at or near the site of patient care and has become a critical component of the diagnostic industry. POCT has many advantages over tests in centralized laboratories including small reagent volumes, small size, rapid turnaround time, cost-effectiveness, low power consumption and functional integration of multiple devices. Paper-based POCT sensors are a new alternative technology for fabricating simple, low-cost, portable and disposable analytical devices for clinical diagnosis. The focus of this dissertation was to develop simple, rapid and low cost paper-based POCT sensors with high sensitivity and portability for disease biomarker detection. Lateral flow strips (LFS) were used as the basic platform as it provides several key advantages such as simplicity, fast response time, on site and cost-effectiveness, and it can be used to detect specific substances including small molecules, large proteins and even whole pathogens, in a sample by immunological reactions. Earlier designs of paper strips lacked the quantitative information of the analyte concentration and could only provide single analyte detection at a time. In this study, a series of modifications were made to upgrade the platform to compensate for these limitations. First, we developed a gold nanoparticle based LFS for qualitative colorimetrical detection of bladder cancer related biomarkers in standard solutions and in urine samples. Second, by incorporating an image processing program “ImageJ”, a semi-quantitative LFS platform was established. The capability of the strip was evaluated by testing a small DNA oxidative damage biomarker in urine and cell culture models. Third, we combined the electrochemical method and colorimetrical method for quantitative biomarker detection. Finally, we integrated a commercialized blood glucose meter to quantitatively detection of two non-glucose biomarkers by converting their signals to that of glucose. The upgraded sensor could provide a noninvasive, rapid, visual, quantitative and convenient detection platform for various disease biomarkers. In addition, this platform does not require expensive equipments or trained personnel, deeming it suitable for use as a simple, economical and portable field kit for on-site biomarker monitoring in a variety of clinical settings.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-05-04T14:00:33Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE VERSÃO DEFINITIVA COM ARTIGO 2 (1).pdf: 3543569 bytes, checksum: 401f073227f2753f70b28b7a15be070e (MD5)<br>Made available in DSpace on 2017-05-04T14:00:33Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE VERSÃO DEFINITIVA COM ARTIGO 2 (1).pdf: 3543569 bytes, checksum: 401f073227f2753f70b28b7a15be070e (MD5) Previous issue date: 2016-03-02<br>FACEPE<br>A infecção pelo vírus da Hepatite B (HBV) é considerada uma enfermidade de alta morbimortalidade, apresentando diagnóstico complexo e quadro de persistência, fatores que dificultam a detecção, terapêutica e cura. Relatos variados têm apontado os imunossensores como importantes ferramentas de auxílio no diagnóstico de doenças, definido como um dispositivo que converte respostas de eventos biológicos a partir da interação antígenoanticorpo em sinal elétrico. No presente estudo foram desenvolvidos biossensores para detecção de anticorpos contra o nucleocapsídeo do HBV (Anti-HBc) mais perene apresentado no diagnóstico da doença. Recentemente, o emprego de nanomateriais no desenvolvimento de tais dispositivos tem despertado interesse devido às propriedades destes materiais. Particularmente, os nanotubos de carbono (NTCs) têm oferecido aos imunossensores melhoria na condutividade, aumento na velocidade de transferência de carga, aumento da área eletródica com maior possibilidade de imobilização de biomoléculas. Nesta tese, foram empregados o ácido hialurônico e o náfion como suporte para forte interação com os NTC funcionalizados em eletrodos de carbono vítreo e de ouro fabricado sobre folha de acetato. Os dispositivos foram caracterizados por técnicas de imagem (microscopia de força atômica) e eletroquímicas (voltametrias de onda quadrada e cíclica), as quais demonstraram a estabilidade da plataforma, imobilização eficaz e sensibilidade. O primeiro protótipo em eletrodos de carbono vítreo modificado com filme de ácido hialurônico associado a nanotubos funcionalizados apresentou resposta linear de 1 a 6ng/ml com limite de detcção de 0,03ng/ml. No segundo protótipo com eletrodos impressos de ouro modificado com filme etanólico de náfion associado a nanotubos funcionalizados, o imunossensor apresentou resposta linear de 0,5 até 2ng/ml, com limite de detecção de 0,15 ng/ml de anti-HBc. Os protótipos desenvolvidos apresentam-se como potenciais para diagóstico da HBV.<br>Infection with hepatitis B virus (HBV) is considered a high mortality disease, with complex diagnosis and persistence framework, factors that hinder detection, therapy and cure. various reports have pointed out the immunosensors as important aid tools in the diagnosis of disease, defined as a device that converts biological events of answers from the electrical signal in antigen-antibody interaction. In the present study biosensors have been developed for the detection of antibodies to the HBV nucleocapsid (anti-HBc) Perennial presented in the diagnosis of disease. Recently, the use of nanomaterials in the development of such devices has aroused interest because of the properties of these materials. Particularly, carbon nanotubes (CNTs) have offered immunosensors improvement in conductivity, increased charge transfer speed, increased electrodic area with the highest possibility of immobilization of biomolecules. In this thesis, we employed hyaluronic acid and nafion as support for strong interaction with the NTC functionalized glassy carbon electrodes and manufactured gold on acetate sheet. The devices were characterized by imaging techniques (atomic force microscopy) and electrochemical (cyclic and square wave voltammetry), which demonstrated the platform stability, effective restraint and sensitivity. The first prototype on glassy carbon electrode modified with hyaluronic acid film associated with functionalized nanotubes showed a linear response of 1 to 6ng/ ml with detction limit 0,03ng / ml. In the second prototype printed gold electrodes modified with ethanolic nafion film associated with functionalized nanotubes, the immunosensor showed a linear response of 0.5 to 2 ng / ml, the detection limit of 0.15 ng / ml of anti-HBc. The developed prototype is present as diagnostic potential for HBV.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2018-02-21T17:01:13Z No. of bitstreams: 2 Tese - Paulo de Tarso Garcia - 2017.pdf: 4177950 bytes, checksum: b76155aa4a091b54d3ad6a2ff6f1e6c6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)<br>Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2018-02-21T17:02:33Z (GMT) No. of bitstreams: 2 Tese - Paulo de Tarso Garcia - 2017.pdf: 4177950 bytes, checksum: b76155aa4a091b54d3ad6a2ff6f1e6c6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5)<br>Made available in DSpace on 2018-02-21T17:02:33Z (GMT). No. of bitstreams: 2 Tese - Paulo de Tarso Garcia - 2017.pdf: 4177950 bytes, checksum: b76155aa4a091b54d3ad6a2ff6f1e6c6 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-12-15<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES<br>This study describes the development of low cost colorimetric and electrochemical sensors aiming clinical and forensic applications. Firstly, a microfluidic paper-based analytical device (μPAD) was developed as colorimetric sensor for rapid estimation of post-mortem interval (PMI) on the crime scene using human vitreous-humour (VH) samples. Experimental parameters were optimized and the best conditions were: paper 1 CHR, 5 mm microzone diameter, 4 μL sample volume and 0.05 mol/L chromogen concentration. μPADs were coupled to colorimetric detection and the feasibility was demonstrated by Fe2+ determination in VH samples, in which the data were not statistically different from conventional technique (ICP-MS). It is important to highlight that Fe2+ levels were proportional to PMI. A color scale was also developed to help the forensic teams in order to estimate the PMI with a simple, quick and visual way. For electrochemical sensors, two different sensors were proposed to determine salivary α-amylase (sAA) levels in human saliva samples, aiming help in the diagnostic of pancreatitis and periodontitis. The first sensor based in a carbon screen-printed electrode (SPE) associated to amperometric detection. Experimental parameters were optimized and the best conditions were: 5 mmol L-1 NaOH (pH= 12), 20 min reaction time, 15 μL sAA volume and 0.5% (w/v) starch concentration. The feasibility of the sensor was demonstrated by sAA determination in five saliva samples (two from male donators and three from female individuals). The sAA concentrations ranged between 182.1 e 1117.1 U mL-1; once two female samples presented high sAA levels because the use of oral contraceptive. The other proposed electrochemical sensor was based in a Batch Injection Analysis with Amperometric Detection (BIA-AD) system using copper oxide (CuO) as working electrode (WE). Through experimental optimization was selected the potential that generate the best current signal. The WE obtained by a chemical/thermal treatment present good stability, once the relative standard deviation (RSD) value was 0.3%, which is ca. 75 fold lower than the RSD obtained with the electrochemical procedure to generate CuO in the electrode surface. The feasibility of the sensor was demonstrated by sAA determination in four human saliva samples. Was possible distinguish patients with and without periodontitis, obtaining thus a quick information about periodontal state of the patients. In general, the three proposed sensors in this study offered good precision, accuracy and specificity. Furthermore, the sensors are simple, portables, low cost and not requires none sophisticate instrumentation. Therefore, they present as promising alternatives to be used in point-of-care clinical and forensic analysis.<br>O trabalho descrito nesta tese demonstra o desenvolvimento de sensores colorimétricos e eletroquímicos de baixo custo, para aplicações nas áreas clínica e forense. Inicialmente, foi desenvolvido um dispositivo microfluídico de papel (μPAD, do inglês microfluidic paper-based analytical device) como sensor colorimétrico, visando a estimativa rápida do intervalo post-mortem (IPM) na cena do crime usando amostras de humor vítreo (HV) humano. Parâmetros experimentais foram otimizados e as melhores condições foram: papel tipo 1 CHR, microzona com 5 mm de diâmetro, volume de amostra de 4 μL e concentração de cromógeno de 0,05 mol/L. Os μPADs foram acoplados à detecção colorimétrica e a viabilidade foi demonstrada através da determinação dos níveis de Fe2+ em amostras de HV, onde os dados não diferiram estatisticamente da técnica convencional (ICP-MS). Vale ressaltar que os níveis de Fe2+ foram proporcionais ao IPM. Também foi desenvolvida uma escala de cor, para auxiliar as equipes forenses a estimar o IPM de maneira simples, rápida e visual. Em relação aos sensores eletroquímicos, foram propostos dois diferentes sensores para realizar a dosagem de α-amilase salivar (sAA, do inglês salivary α-amylase) em amostras de fluido oral humano, visando auxiliar no diagnóstico de doenças como pancreatite e periodontite. O primeiro sensor baseou-se em um eletrodo impresso (SPE, do inglês screen-printed electrode) de carbono associado a detecção amperométrica. Otimizou-se parâmetros experimentais e as melhores condições foram: concentração de NaOH igual a 5 mmol L-1 (pH= 12), tempo reacional de 20 min, volume de sAA de 15 μL e concentração de amido igual a 0,5% (m/v). A viabilidade do sensor foi demonstrada através da determinação de sAA em cinco amostras de fluido oral (duas de indivíduos do gênero masculino e três do gênero feminino). Os valores de concentração de sAA variaram entre 182,8 e 1117,1 U mL-1; sendo que duas amostras do gênero feminino exibiram elevados níveis de sAA devido ao uso de contraceptivo oral. O outro sensor eletroquímico proposto baseou-se em um sistema de análise por injeção em batelada com detecção amperométrica (BIA-AD, do inglês Batch Injection Analysis with Amperometric Detection) usando eletrodo de trabalho (ET) de óxido de cobre (CuO). Através de uma otimização experimental foi possível selecionar o potencial que fornece o melhor sinal de corrente. O ET obtido por um tratamento químico/térmico apresentou boa estabilidade, onde o valor de desvio padrão relativo (DPR) foi de 0,3%, que é cerca de 75 vezes menor do que o DPR obtido com o procedimento eletroquímico para gerar o CuO na superfície do eletrodo. A viabilidade do sensor foi demonstrada através da dosagem de sAA em quatro amostras de fluido oral humano. Foi possível diferenciar pacientes com e sem periodontite, obtendo assim uma informação rápida sobre a situação periodontal dos pacientes. De maneira geral, os três sensores propostos neste trabalho ofereceram boa precisão, exatidão e especificidade. Além disso, são simples, portáteis, de baixo custo e não requerem nenhuma instrumentação sofisticada. Sendo assim, se apresentam como alternativas promissoras para serem utilizados em análises clínicas e forenses no point-of-care.

Rapid determination of the concentrations of molecules related to diseases can provide timely information for treatment options. However, most biomarker quantitation methods require costly and complex equipment. On the other hand, point-of-care systems have less complex instrumentation needs than laboratory-based equipment, but often provide less information; for example, biomarker presence or absence instead of concentration. A complete analysis setup addressing key limitations of both laboratory-based and portable systems is highly desirable. I developed microfluidic devices with visual inspection readout of a target’s concentration from microliter volumes of solution flowed into a microchannel. Microchannels are formed within polydimethylsiloxane (PDMS), and the surfaces are coated with receptors. Capillary flow of target solution in the channel crosslinks the top and bottom surfaces, which constricts the channel and stops flow. The flow distance of the target solution in the channel before flow stops indicates the target’s concentration, enabling simple visual inspection readout without complex detection instrumentation. Because of its easy readout and portability, my system has great potential for use in point-of-care diagnostics. I initially demonstrated a proof-of-concept assay using biotin-streptavidin. Solution capillary flow distances scaled linearly with the negative logarithm of streptavidin concentration over a 100,000-fold range. I measured streptavidin concentrations as low as 1 ng/mL using these microsystems, demonstrating low detection limits. I also characterized the mechanism wherein time-dependent channel constriction in the first few millimeters leads to concentration-dependent flow distances. I demonstrated the visual detection and quantification capability of my system to determine an antigen target, thymidine kinase 1 (TK1). I developed surface modification methods for carrying out flow assays and verified receptor attachment on channel surfaces using fluorescence imaging. I obtained a 1 ng/mL TK1 detection limit in flow assays. I also demonstrated nucleic acid quantitation in my flow devices. I detected specific DNA targets in buffer and synthetic urine at 10 pg/mL levels. A dynamic range of 106 was obtained with single-base mismatch specificity. DNA analogues of two miRNA biomarkers were measured near clinically significant levels, showing great promise for future medical application. The promising results demonstrate that this diagnostic tool offers a simple route to analyte quantitation in microliter volumes, with excellent potential for point-of-care application.

Preliminary investigation at the beginning of this research showed that informatics on point-of-care (POC) devices was limited to basic data generation and processing. This thesis is based on publications of several studies during the course of the research. The aim of the research is to model and analyse information generation and exchange in telehealth systems and to identify and analyse the capabilities of these systems in managing chronic diseases which utilise point-of-care devices. The objectives to meet the aim are as follows: (i) to review the state-of-the-art in informatics and decision support on point-of-care devices. (ii) to assess the current level of servitization of POC devices used within the home environment. (iii) to identify current models of information generation and exchange for POC devices using a telehealth perspective. (iv) to identify the capabilities of telehealth systems. (v) to evaluate key components of telehealth systems (i.e. POC devices and intermediate devices). (vi) to analyse the capabilities of telehealth systems as enablers to a healthcare policy. The literature review showed that data transfer from devices is an important part of generating information. The implication of this is that future designs of devices should have efficient ways of transferring data to minimise the errors that may be introduced through manual data entry/transfer. The full impact of a servitized model for point-of-care devices is possible within a telehealth system, since capabilities of interpreting data for the patient will be offered as a service (c.f. NHS Direct). This research helped to deduce components of telehealth systems which are important in supporting informatics and decision making for actors of the system. These included actors and devices. Telehealth systems also help facilitate the exchange of data to help decision making to be faster for all actors concerned. This research has shown that a large number of capability categories existed for the patients and health professionals. There were no capabilities related to the caregiver that had a direct impact on the patient and health professional. This was not surprising since the numbers of caregivers in current telehealth systems was low. Two types of intermediate devices were identified in telehealth systems: generic and proprietary. Patients and caregivers used both types, while health professionals only used generic devices. However, there was a higher incidence of proprietary devices used by patients. Proprietary devices possess features to support patients better thus promoting their independence in managing their chronic condition. This research developed a six-step methodology for working from government objectives to appropriate telehealth capability categories. This helped to determine objectives for which a telehealth system is suitable.

Mobile computing devices are capable of changing how healthcare is delivered in the future, since they aim to merge and integrate all services into one device that is versatile, customisable, and portable. The aim of this study was to explore and describe the experiences of professional nurses with regard to accessing information at the point-of-care of the patient, in order to develop guidelines that could assist other professional nurses with implementing the mobile computing device for accessing information at the point-of-care of patients. To achieve the purpose of the study, a qualitative, explorative, descriptive, and contextual design was used to conduct this research – to gain an understanding of how the professional nurses experienced accessing information at the point-of-care via mobile computing devices. The study was conducted among the professional nurses employed at the public hospital, who were trained and provided with the mobile computing device for accessing information at the point-of-care for more than two years. In-depth interviewing was conducted to obtain the data. Data analysis was done using Tesch‘s method to make sense out of text and data. Four themes were identified, namely, the professional nurses‘ expression of various experiences regarding the training received; the need for support in implementing the mobile computing device; the accessing of information at the point-of-care as beneficial for educational purposes; and the accessing of information at the point-of-care as beneficial to patient care. Two main guidelines were developed. The study concludes with recommendations made with regard to the areas of nursing practice, education and research. Throughout the study, the researcher abided by the ethical considerations. The aspects of trustworthiness implemented in this study, included dependability, credibility, transferability and confirmability (Holloway & Wheeler, 2010:298).

Thesis (MBA)--Stellenbosch University, 2014.<br>There is a growing trend towards the use of point of care testing in resource poor settings, in particular in the diagnosis and treatment of infectious diseases such as Human Immunodeficiency Virus (HIV), Tuberculosis (TB) and Malaria. The Alere PIMA CD4 counter is widely used as a point of care device in the staging and management of HIV. While the instrument has been extensively validated and shown to be comparable to central laboratory testing, little is known about the error rates of these devices, as well as the factors that contribute to error rates. This research was a retrospective analysis of error rates from 61 PIMA point of care devices deployed in nine African countries belonging to Medisciens Sans Frontiers. The data was collected between January 2011 and June 2013. The objectives of the study were to determine the overall error rate and, where possible, determine the root cause. Thereafter the study aimed to determine the variables that contribute to the root causes and make recommendations to reduce the error rate. The overall error was determined to be 13.2 percent. The errors were further divided into four root causes and error rates assigned to each root cause based on the error codes generated by the instrument. These error rates were found to be operator error (48.4%), instrument error (2.0%), reagent/cartridge error (1%) and sample error (4.3%). It was found that a high percentage of the errors were ambiguous (44.3%), meaning that they had more than one possible root cause. A systems-based knowledge management approach was used to create a qualitative politicised influence diagram, which described the variables that affect each of the root causes. The influence diagram was subjected to loop analysis where individual loops were described in terms of the knowledge type (tacit or explicit), the knowing type (know-how, know-who, know-what and know-why), and the actors involved with each variable. Where possible, the variable was described as contributing to pre-analytical, analytical or post-analytical error. Recommendations to reduce the error rates for each of the variables were then made based on the findings.

Microfluidics miniaturizes many benchtop processes and provides advantages of low cost, reduced reagent usage, process integration, and faster analyses. Microfluidic devices have been fabricated from a wide variety of materials and methods for many applications. This dissertation describes four such examples, each employing different features and fabrication methods or materials in order to achieve their respective goals. In the first example of microfluidic applications in this dissertation, thermoplastics are hot embossed to form t-shaped channels for microchip electrophoresis. These devices are used to separate six preterm birth (PTB) biomarkers and establish a limit of detection for each. The next chapter describes 3D printed devices with reversed-phase monoliths for solid-phase extraction and on-chip fluorescent labeling of PTB biomarkers. I demonstrate the optimization of the monolith and selective retention of nine PTB biomarkers, the first microchip study to perform an analysis on this entire panel. The third project describes the iterative design and fabrication of glass/polydimethylsiloxane (PDMS) devices with gold and nickel electrodes for the self-assembly of DNA nanotubes for site-selective placement of nanowires. Simple flow channels and “patch electrode” devices were successfully used, and DNA seeding was achieved on gold electrodes. Finally, a 3D printed device for cancer drug screening was developed as a replacement for one previously fabricated in PDMS. Devices of increasing complexity were fabricated, and those tested found to give good control over fluid flow for multiple inlets and valves. Although the applications and methods of these projects are varied, the work in this dissertation demonstrates the potential of microfluidics in several fields, particularly for diagnostics, therapeutics, and nanoelectronics. Furthermore, it demonstrates the importance of applying appropriate tools to each problem to gain specific advantages. Each of the described devices has the potential for increased complexity and integration, which further emphasizes the advantages of miniaturized analyses and the potential for microfluidics for analytical testing in years to come.

År 2020 listade Världshälsoorganisationen (WHO) cancer som den globalt ledande dödsorsaken med över 10 miljoner dödsfall årligen. Av dessa 10 miljoner fall förekommer nästan 70% i låg- till medelinkomstländer - en siffra som på grund av den låga prioriteringen av cancerbehandling- och diagnostik förväntas öka till 85% redan år 2030. Att utveckla enkla, specifika och prisvärda verktyg för diagnostik kommer därför att bli avgörande för förebyggandet av cancer på en global nivå. För att komma ett steg närmare denna utveckling optimerades och testades i denna studie ett mikrofluidiskt system, utvecklat genom layer-bylayer- metoden, baserat på cellulosa nanofibriller med förmågan att isolera och fånga cirkulerande tumörceller. För att uppnå en termodynamisk jämvikt optimerades systemets hydrodynamiska parametrar optimerades för att uppnå en homogen fördelning med hög densitet av det cellulosa-baserade systemet i det mikrofluidiska chippet. Då jämvikt är grundläggande för att maximera det efterföljande beläggningen av antikroppar, och därmed hur effektivt celler isoleras, modifierades parametrar såsom koncentration, flödeshastighet, inkubationstid med fler tills att önskad effekt uppnåtts. Således koncepttestades systemet genom att fånga celler spetsade i blod och därmed demonstrera att systemet kan användas i syfte att isolera cancerceller från blodprov. Detta öppnar upp för utveckling av liknande diagnostiska verktyg som kan användas för att isolera lågfrekventa celler direkt från blod.<br>In 2020, the World Health Organization (WHO) listed cancer as the leading cause of death worldwide, reaching a staggering number of 10 million cancer-related deaths annually. Of these 10 million deaths, nearly 70% occurred in low- and middle-income countries; a number that is expected to increase to 85% by 2030 due to the lack of resources as well as low priority of the development of cancer treatment and diagnosis. Hence, the development of a sophisticated, specific and affordable diagnostic tool will be crucial for global cancer prevention and control. In this study, a cellulose nanofibril-based Layer-by-Layer system for immuno-capture of tumour cells in a microfluidic device was optimized and tested for the development of a simple and cost-effective diagnostic tool for use in resource-limited areas. In the pursuit of a thermodynamic equilibrium, the hydrodynamic parameters of the system were optimized to achieve a homogeneous distribution with a high surface density of the cellulose-based system across the microfluidic channels. Since an equilibrated system is essential to maximize the antibody coating, and thereby cell capture efficiency, parameters including but not limited to concentration, flow rate and incubation time were altered until a desired effect had been achieved. Thus, as proof-of-concept, the system was tested by capturing cancer cells spiked into whole blood, thereby demonstrating that the system can be utilized for the purpose of isolating cancer cells from blood samples. This paves the way for the development of similar clinical diagnostic tools for the isolation of rare cells directly from whole blood.

Introduction/Aims: Patients undergoing interventional neuroradiology (IVN) procedures to treat intracranial aneurysms experience a minor, but potentially life threatening risk of thromboembolic or haemorrhagic complications. Antiplatelet medications, such as clopidogrel and aspirin, are routinely administered pre-operatively for elective IVN procedures to reduce the risk of thromboembolic events. There are known hyper- or hypo-effective responses to these pre-operative antiplatelet medications, notably clopidogrel, which may insufficiently inhibit platelet function in some patients and increase the risk of complication. The point-of-care (POC) Multiplate® analyser was used to assess platelet function (aggregation) at numerous time points. Patient outcome (ie. those who suffered surgical complications, and those who did not) was assessed to establish whether platelet functionality was related to IVN complication. Additionally, analysing platelet response data in patients with and without complications aimed to establish the potential diagnostic value of Multiplate® response for negative clinical outcomes. In addition, platelet aggregation as recorded by Multiplate® analysis was with platelet activation as assessed using flow cytometry in an attempt to compare functional Multiplate® data to sensitive molecular activation markers. Methods: This project received ethical approval from Griffith University and the Gold Coast University Hospital (GCUH), including amendments for the inclusion of flow cytometric analysis. Demographic and surgical data, as well as blood samples were collected from adult patients admitted to the GCUH for elective or emergent treatment of intracranial aneurysms between March 2015 and April 2018. Blood samples were collected at five separate time points: admission (pre-antiplatelet administration), pre-operatively (post-antiplatelet administration), post-surgically, and at days 1 and 4 post-operatively. The samples of 120 patients and 131 procedures were analysed on Multiplate®, while 12 matched patient samples were also analysed using a BD LSRFortessa™ Cell Analyser for flow cytometric analysis. Samples were collected from elective and emergent (ruptured aneurysm) patients. The main variable of interest were the extent of platelet aggregation/activation at each time point, as well as any peri- or post—surgical complications the patient experienced. Results: Analysis of the 120 patients showed that pre-operative aspirin and clopidogrel administration significantly reduced platelet aggregation (Multiplate®) and platelet activation (flow cytometry). A total of 26 complications were recorded including ischaemic, haemorrhagic events, vasospasm and evidence of temporary neurological deficit. Elective patients who suffered complications (composite score of all adverse events) had significantly higher pre-operative, ADP-induced Multiplate® scores compared to their non-complication counterparts (P<0.05). There were no significant differences in Multiplate® scores in the elective cohort when comparing the non-complication group to those who suffered ischaemic or haemorrhagic complications specifically (P>0.05). Furthermore, Multiplate® scores did not differ in patients in non-complication versus complications within the emergent cohort. ROC analysis of pre-operative Multiplate® data demonstrated that a ADP-induced platelet aggregation score of >31.5AUC was a significant predictor of elective complications (i.e. all complications excluding haemorrhagic). This allowed successful identification of 88.89% (i.e. sensitivity) of surgical complications pre-surgically by assessing their platelet aggregation with a 34.62% false positive rate. There were no significant predictors of haemorrhagic complication, or for any type of emergent complication. Platelet activation recorded by flow cytometry followed a similar trend over time to platelet aggregation as recorded by Multiplate® analyser. “A significant but weak correlation between flow cytometric and Multiplate® analysis was established for agonists including ADP (p=>0.01, r2=0.32) and TRAP-6 (p=0.05, r 2= 0.12). Conclusion: Elective patients experience significant inhibition of platelet function following antiplatelet administration for IVN procedures as recorded by Multiplate®. Furthermore, this study demonstrated, for the first time, that pre-operative ADP induced platelet aggregation can sensitively predict patients’ risk of experiencing thromboembolic events and/or vasospasm. However, the predictive values established are only reliable for elective patients and do not predict haemorrhagic complications. This is likely due to the small number of patients suffering complication throughout the study. This thesis also presents the first data to compare and potentially validate Multiplate® recorded platelet aggregation against flow cytometry analysis for platelet activation.<br>Thesis (Masters)<br>Master of Medical Research (MMedRes)<br>School of Medical Science<br>Griffith Health<br>Full Text

Thesis (MEng)--Stellenbosch University, 2015.<br>ENGLISH ABSTRACT: South Africa has a severe HIV (human immunodeficiency virus) burden and the management of the disease is a priority, especially in the public healthcare sector. One element of managing the disease, is determining when to initiate an HIV positive individual onto anti-retroviral therapy (ART), a treatment that the patient will remain on for the remainder of their lifetime. For the majority of HIV positive individuals in the country, this decision is governed by the results of a CD4 (cluster of differentiation 4) test that is performed at set time intervals from the time that the patient is diagnosed with HIV until the patient is initiated onto ART. A device for CD4 measurement at the point of care (POC), the Alere PIMA™, has recently become commercially available. This has prompted a need to evaluate whether CD4 testing at the POC (i.e. at the patient serving healthcare facility) should be incorporated into the South African public healthcare sector's HIV diagnostic service provision model. One challenge associated with the management of HIV in the country is the relatively large percentage of patients that are lost to follow-up at various points in the HIV treatment process. There is extensive evidence that testing CD4 levels at the POC (rather than in a laboratory, as is the current practice) reduces the percentage of patients that are lost to follow-up before being initiated onto ART. Therefore, though POC CD4 testing is more expensive than laboratory-based CD4 testing, the use of this technology in South Africa should be investigated for its potential to positively influence health outcomes. In this research, a multi-objective location science model is used to generate scenarios for the provision of CD4 testing capability. For each scenario, CD4 testing provision at 3 279 ART initiation facilities is considered. For each facility, either (i) a POC device is placed at the site; or (ii) the site's testing workload is referred to one of the 61 CD4 laboratories in the country. To develop this model, the characteristics of eight basic facility location models are compared to the attributes of the real-world problem in order to select the most suitable one for application. The selected model's objective, assumptions and inputs are adjusted in order to adequately model the realworld problem. The model is solved using the cross-entropy method for multi-objective optimisation and the results are verified using a commercial algorithm. Nine scenarios are selected from the acquired Pareto set for detailed presentation. In addition, details on the status quo as well as a scenario where POC testing is used as widely as possible are also presented. These scenarios are selected to provide decision-makers with information on the range of options that should be considered, from no or very limited use to widespread use of POC testing. Arguably the most valuable contribution of this research is to provide an indication of the optimal trade-off points between an improved healthcare outcome due to POC CD4 testing and increased healthcare spending on POC CD4 testing in the South African public healthcare context. This research also contributes to the location science literature and the metaheuristic literature.<br>AFRIKAANSE OPSOMMING: Suid-Afrika gaan gebuk onder `n swaar MIV- (menslike-immuniteitsgebreksvirus-) las en die bestuur van die siekte is `n prioriteit, veral in die openbare gesondheidsorgsektor. Een element in die bestuur van die siekte is om te bepaal wanneer `n MIV-positiewe individu met antiretrovirale- (ARV-)behandeling behoort te begin, waarop pasiënte dan vir die res van hul lewens bly. Vir die meeste MIV-positiewe individue in die land word hierdie besluit bepaal deur die uitslae van `n CD4- (cluster of differentiation 4-)toets wat met vasgestelde tussenposes uitgevoer word vandat die pasiënt met MIV gediagnoseer word totdat hy of sy met ARV-behandeling begin. `n Toestel vir CD4-meting by die punt van sorg (\POC"), die Alere PIMA™, is onlangs kommersieel beskikbaar gestel. Dit het `n behoefte laat ontstaan om te bepaal of CD4-toetsing by die POC (met ander woorde, by die gesondheidsorgfasiliteit waar die pasiënt bedien word) by die MIV-diagnostiese diensleweringsmodel van die Suid-Afrikaanse openbare gesondheidsorgsektor ingesluit behoort te word. Een uitdaging met betrekking tot MIV-bestuur in die land is die betreklik groot persentasie pasiënte wat verlore gaan vir nasorg in die verskillende stadiums van die MIV-behandelingsproses. Heelwat bewyse dui daarop dat die toetsing van CD4-vlakke by die POC (eerder as in `n laboratorium, soos wat tans die praktyk is) die persentasie pasiënte wat verlore gaan vir nasorg voordat hulle met ARV-behandeling kan begin, verminder. Daarom, hoewel CD4-toetsing by die POC duurder is as toetsing in `n laboratorium, behoort die gebruik van hierdie tegnologie in Suid-Afrika ondersoek te word. In hierdie studie is `n meerdoelige liggingswetenskapmodel gebruik om scenario's vir die voorsiening van CD4-toetsvermoë te skep. Vir elke scenario word CD4-toetsvermoë by 3 279 ARV-inisiasie fasiliteite oorweeg. Vir elke fasiliteit word toetsvermoë verskaf deur (i) die plasing van POC-toestelle by die fasiliteit, of (ii) verwysing vir laboratoriumgebaseerde toetsing by een van die 61 CD4-laboratoriums in die land. Die kenmerke van agt basiese fasiliteitsliggingsmodelle is met die kenmerke van die werklike probleem vergelyk om die mees geskikte model vir toepassing op die werklike probleem te bepaal. Die doelwitte, aannames en insette van die gekose model is daarna aangepas om die werklike probleem voldoende te modelleer. Die model is opgelos met behulp van die kruis-entropie-metode vir meerdoelige optimering, waarna die resultate deur middel van `n kommersiële algoritme bevestig is. Nege scenario's uit die verworwe Pareto-stel word uitvoerig aangebied. Daarbenewens beskryf die studieresultate die besonderhede van die status quo sowel as `n scenario waar POC-toetsing so wyd moontlik gebruik word. Hierdie scenario's word aangebied om besluitnemers van inligting te voorsien oor die verskeidenheid moontlikhede wat oorweeg kan word, wat wissel van geen of baie beperkte tot wydverspreide gebruik van POC-toetsing. Die mees beduidende bydrae van hierdie navorsing is stellig dat dit `n aanduiding bied van die optimale kompromie tussen `n verbeterde gesondheidsorguitkoms weens CD4-toetsing by die POC, en verhoogde gesondheidsorgbesteding aan CD4-toetsing by die POC, in die konteks van Suid-Afrikaanse openbare gesondheidsorg. Die navorsing dra ook by tot die ligingswetenskapliteratuur sowel as tot die metaheuristiekliteratuur.

N,N′-methylenebisacrylamide-crosslinked poly(N-isopropylacrylamide), also known as P(NIPAM), was developed as a fluid delivery system for use with microfluidic paper-based analytical devices (microPADs). MicroPADs are postage-stamp-sized devices made out of paper that can be used as platforms for low-cost, simple-to-use point-of-care diagnostic assays. P(NIPAM) is a thermally responsive polymer that absorbs aqueous solutions at room temperature and will expel the solutions to microPADs when heated. The fluid delivery characteristics of P(NIPAM) were assessed, and P(NIPAM) was able to deliver multiple solutions to microPADs in specific sequences or simultaneously in a laminar-flow configuration. P(NIPAM) was then shown to be suitable for delivering four classes of reagents to microPADs: small molecules, enzymes, antibodies and DNA. P(NIPAM) successfully delivered a series of standard concentrations of glucose (0 – 5 mM) to microPADs equipped to perform a colorimetric glucose assay. The results of these tests were used to produce an external calibration curve, which in turn was used to determine accurately the concentrations of glucose in sample solutions. P(NIPAM) successfully delivered fluorescein-labeled IgG and fluorescein-labeled oligonucleotides (20 base pairs) to microPADs in a variety of concentrations. P(NIPAM) also successfully delivered horseradish peroxidase (HRP) to microPADs, and it was determined that HRP could be stored in P(NIPAM) for 35 days with minimal loss in activity. The combination of P(NIPAM) with microPADs will allow for more complex assays to be performed with minimal user input, will facilitate the preparation of external calibration curves in the field, and may be useful in extending the shelf life of microPADs by stabilizing reagents.

Master of Science<br>Department of Biological & Agricultural Engineering<br>Mei He<br>Anemia affects about 25% of the world’s population and causes roughly 8% of all disability cases. The development of an affordable point-of-care (POC) device for detecting anemia could be a significant for individuals in underdeveloped countries trying to manage their anemia. The objective of this study was to design and fabricate a 3D printed, low cost microfluidic mixing chip that could be used for the diagnosis of anemia. Microfluidic mixing chips use capillary flow to move fluids without the aid of external power. With new developments in 3D printing technology, microfluidic devices can be fabricated quickly and inexpensively. This study designed and demonstrated a passive microfluidic mixing chip that used capillary force to mix blood and a hemoglobin detecting assay. A 3D computational fluid dynamic simulation model of the chip design showed 96% efficiency when mixing two fluids. The mixing chip was fabricated using a desktop 3D printer in one hour for less than $0.50. Blood samples used for the clinical validation were provided by The University of Kansas Medical Center Biospecimen Repository. During clinical validation, RGB (red, green, blue) values of the hemoglobin detection assay color change within the chip showed consistent and repeatable results, indicating the chip design works efficiently as a passive mixing device. The anemia detection assay tended to overestimate hemoglobin levels at lower values while underestimating them in higher values, showing the assay needs to go through more troubleshooting.

<p> This dissertation is composed of two projects dedicated to the development of techniques and technologies for improving the quality of life for patients in both clinical and resource-limited settings. </p><p> The purpose of the first project was to design a rapid diagnostic device to screen whole blood samples for the presence of infectious agents. Point-of-care (PoC) technologies are becoming increasingly important for the detection of infectious agents in resource-limited settings (RSLs) where state-of-the-art blood screening practices are not feasible for implementation. For this project, a rapid diagnostic device was developed to directly detect pathogen content within freshly drawn whole blood samples using a ligand-binding assay format. The assay is completely self-contained within a hermetically sealed device to minimize operational complexity and ensure operator safety. The diagnostic device is capable of processing complex sample matrices by selectively capturing, concentrating, and labeling infectious agents upon functionalized surfaces. Following sample processing, the assay is optically interrogated with a fluorescence-based reader to provide rapid feedback regarding sample purity. Designs of the rapid diagnostic platform evolved over several prototype generations corresponding to project milestones emphasizing ergonomic performance, military specification testing for environmental resilience, and manufacture to yield production-grade devices for future diagnostic performance data collection. </p><p> The goal of the regenerative therapy-based portion of this research was to develop a novel technique for the selective enrichment of cells demonstrating enhanced regenerative capacity in tissue-extracted cell samples. Adherent cell cultures of stromal vascular fractions (SVFs) extracted from adipose tissues were exposed to nutrient deficient conditions &ndash; eliciting a bimodal cellular response between two dissimilar cell culture subpopulations. The regenerative capacity of these two distinct subpopulations was evaluated by assessing their characteristic morphology, metabolic activity, and ability to undergo multilineage differentiation. The SVF subpopulation which demonstrated sensitivity to the nutrient deficient conditions expressed typical morphological expression of adherent cell cultures, elevated metabolic activity, and the ability to differentiate along adipogenic, chondrogenic, and osteogenic lineages. The SVF subpopulation which demonstrated resistance to the nutrient deficient conditions, however, expressed atypical morphologies, impaired metabolic activity, and did not survive culture with differentiation growth media. Based on the data, the &lsquo;treatment-sensitive&rsquo; SVF subpopulation demonstrated a greater regenerative capacity than the &lsquo;treatment-resistant&rsquo; subpopulation. Furthermore, the treatment-resistant subpopulation of the SVF may be representative of the damaged, senescent, and otherwise less-functional cells that comprise a significant portion of tissue-extracted cell samples and pose a significant risk to therapeutic efficacy and reproducibility. Ultimately, this expedient and inexpensive bioprocessing technique may serve to improve cell-based regenerative therapies by eliminating undesirable cells from culture.</p>

This dissertation is composed of two projects dedicated to the development of techniques and technologies for improving the quality of life for patients in both clinical and resource-limited settings. The purpose of the first project was to design a rapid diagnostic device to screen whole blood samples for the presence of infectious agents. Point-of-care (PoC) technologies are becoming increasingly important for the detection of infectious agents in resource-limited settings (RSLs) where state-of-the-art blood screening practices are not feasible for implementation. For this project, a rapid diagnostic device was developed to directly detect pathogen content within freshly drawn whole blood samples using a ligand-binding assay format. The assay is completely self-contained within a hermetically sealed device to minimize operational complexity and ensure operator safety. The diagnostic device is capable of processing complex sample matrices by selectively capturing, concentrating, and labeling infectious agents upon functionalized surfaces. Following sample processing, the assay is optically interrogated with a fluorescence-based reader to provide rapid feedback regarding sample purity. Designs of the rapid diagnostic platform evolved over several prototype generations corresponding to project milestones emphasizing ergonomic performance, military specification testing for environmental resilience, and manufacture to yield production-grade devices for future diagnostic performance data collection. The goal of the regenerative therapy-based portion of this research was to develop a novel technique for the selective enrichment of cells demonstrating enhanced regenerative capacity in tissue-extracted cell samples. Adherent cell cultures of stromal vascular fractions (SVFs) extracted from adipose tissues were exposed to nutrient deficient conditions' eliciting a bimodal cellular response between two dissimilar cell culture subpopulations. The regenerative capacity of these two distinct subpopulations was evaluated by assessing their characteristic morphology, metabolic activity, and ability to undergo multilineage differentiation. The SVF subpopulation which demonstrated sensitivity to the nutrient deficient conditions expressed typical morphological expression of adherent cell cultures, elevated metabolic activity, and the ability to differentiate along adipogenic, chondrogenic, and osteogenic lineages. The SVF subpopulation which demonstrated resistance to the nutrient deficient conditions, however, expressed atypical morphologies, impaired metabolic activity, and did not survive culture with differentiation growth media. Based on the data, the 'treatment-sensitive' SVF subpopulation demonstrated a greater regenerative capacity than the‘treatment-resistant' subpopulation. Furthermore, the treatment-resistant subpopulation of the SVF may be representative of the damaged, senescent, and otherwise less-functional cells that comprise a significant portion of tissue-extracted cell samples and pose a significant risk to therapeutic efficacy and reproducibility. Ultimately, this expedient and inexpensive bioprocessing technique may serve to improve cell-based regenerative therapies by eliminating undesirable cells from culture.

Ammonia is produced in the body during the metabolism of amino acids. In the liver, it is converted to urea via the urea cycle and excreted by the kidneys as urine. Normal levels are between 11 to 50 µM, whereas a blood ammonia level of approximately 100 µM indicates pathology. Elevated blood ammonia is associated with a number of pathological conditions including liver and kidney dysfunction. Conditions such as these can affect brain function and can be fatal. Current blood ammonia analysis requires a laboratory blood test. Few, if any of the techniques used are suitable for point of care (POC) testing. The development of a reliable and simple method for blood ammonia determination is essential for clinical diagnosis and management of patient progress in order to prevent further debilitating illnesses developing, and extending life. This is particularly critical in many disorders such as hyperammonaemia of the newborn, inborn errors of metabolism including urea cycle defects, organic acidaemias, hyperinsulinism/hyperammonaemia, liver disease and other cause of hyperammonaemic encephalopathy. This thesis investigates the development of an electrochemical sensor for the measurement of ammonia in blood. Polyaniline has a known affinity for ammonia which operates on the deprotonation of the polyaniline backbone forming an ammonium ion. In this work, polyaniline nanoparticles were fabricated and inkjet-printed onto silver screen printed electrodes. The sensors were then incorporated into devices containing a gas-permeable membrane, which facilitated the measurement of gaseous ammonia from a liquid sample (blood) using electrochemical impedance spectroscopy. The combination of impedance spectroscopy with a gas-permeable membrane allowed the measurement of gaseous ammonia from solution. The ammonia device developed possessed refinements to enhance its sensitivity and included careful optimisation of other aspects of the measurement. For example, an air purge through the device gas chamber was employed to remove matrix interferences from the sensor and improve the specificity to ammonia. The pH of the sample to be analysed was modified in order to increase the mass of ammonia in solution, thus lowering the limit of detection (LOD) of the device. Finally, assay timings were optimised in order to increase the impedimetric response of ammonia. These optimisations resulted in the effective detection of ammonia in a liquid sample down to the lowest clinically relevant levels found in blood. The devices displayed an impedimetric baseline intra- and inter-variability of 25 and 6.9%, respectively for n = 15 over a period of 160 s. A calculated limit of LOD of 12 µM was achieved for human serum measurements. A coefficient of determination of 0.9984, slope of 0.0046 and an intercept of 1.1534 was obtained in human serum across the linear range of 25 to 200 μM ammonia (n = 3). The device was validated against a commercial spectrophotometric assay which resulted in excellent correlation (0.9699, p < 0.0001) with a slope of 1.4472 and an intercept of 0.5631 between both methods (n = 3). The devices could be stored in desiccant for up to five months and displayed minimal variation (0.64%) over time (n = 12).

Actualment el diagnòstic de Tuberculosi (TB) es realitza en laboratoris centralitzats, emprant equips voluminosos, reactius complexos i personal capacitat, augmentant els costos i el temps per obtenir els resultats. Per aquesta raó, l’objectiu d’aquesta tesi doctoral és el desenvolupament d’una plataforma point-of-care (POC) capaç d’oferir una resposta ràpida i fiable en el diagnòstic de TB. Per dur a terme aquest objectiu, la plataforma POC integra un nou sensor fotònic incorporat en un cartutx de micofluídica d’un sol ús. El sensor fotònic consisteix en un conjunt de interferòmetres Mach-Zehnder que ofereixen una alta sensibilitat. En primer lloc, es va dur a terme una caracterització òptica per estudiar el rendiment de la plataforma POC i la seva capacitat per a ser emprada en aplicacions biosensoras. Un cop caracteritzada òpticament, es van avaluar diferents estratègies de biofuncionalització per incorporar anticossos específics com a bioreceptors a la superfície del sensor. Després d’un estudi en profunditat, es va seleccionar i es va emprar l’estratègia de biofuncionalització òptima per l’anàlisi dels biomarcadors de TB. Els biomarcadors de TB es van avaluar tant en solució tampó com en mostres biològiques, particularment en orina humana. El biomarcador més prometedor i conegut de TB és el lipoarabinomanan (LAM), un component de la paret cel·lular bacteriana. En concret, la detecció d’aquest biomarcador va ser validada amb mostres clíniques de pacients amb TB i donants sans, mostrant la capacitat de la nostra plataforma POC per discriminar aquells pacients amb tuberculosi activa. A més, el disseny del sensor fotònic permet la detecció simultània de sis biomarcadors diferents. Tenint en compte això, hem dut a terme una prova de concepte de l’ús de la plataforma biosensora POC per a la detecció d’un panell de biomarcadors de TB utilitzant nanolitografía Dip-Pen per a la deposició de cada bioreceptor en cada sensor. Els nostres resultats, validats en estudis clínics més amplis, podrien tenir importants implicacions diagnòstiques. A més, el nostre biosensor POC ofereix una sèrie d’avantatges en comparació amb els mètodes recomanats per l’Organització Mundial de la Salut.<br>Actualmente el diagnóstico de Tuberculosis (TB) se realiza en laboratorios centralizados, empleando equipos voluminosos, reactivos complejos y personal capacitado, aumentando los costes y el tiempo para obtener los resultados. Por esta razón, el objetivo de esta Tesis Doctoral es el desarrollo de una plataforma point-of-care (POC) capaz de ofrecer una respuesta rápida y fiable en el diagnóstico de TB. Para llevar a cabo este objetivo, la plataforma POC integra un novedoso sensor fotónico incorporado en un cartucho de micofluídica desechable. El sensor fotónico consiste en un conjunto de interferómetros Mach-Zehnder que ofrecen una alta sensibilidad. En primer lugar, se llevó a cabo una caracterización óptica para estudiar el rendimiento de la plataforma POC y su capacidad para ser empleada en aplicaciones biosensoras. Una vez caracterizada ópticamente, se evaluaron distintas estrategias de biofuncionalización para incorporar anticuerpos específicos como bioreceptores a la superficie del sensor. Después de un estudio en profundidad, se seleccionó y empleó la estrategia de biofuncionalización óptima para el análisis de los biomarcadores de TB. Los biomarcadores de TB se evaluaron tanto en solución tampón como en muestras biológicas, particularmente en orina humana. El biomarcador más prometedor y conocido de TB es el lipoarabinomanano (LAM), un componente de la pared celular bacteriana. En concreto, la detección de este biomarcador fue validada con muestras clínicas de pacientes con TB y donantes sanos, mostrando la capacidad de nuestra plataforma POC para discriminar a aquellos pacientes con Tuberculosis activa. Además, el diseño del sensor fotónico permite la detección simultánea de seis biomarcadores distintos. Teniendo esto en cuenta, hemos llevado a cabo una prueba de concepto del empleo de la plataforma biosensora POC para la detección de un panel de biomarcadores de TB utilizando nanolitografía Dip-Pen para la deposición de cada bioreceptor en cada sensor. Nuestros resultados, validados en estudios clínicos más amplios, podrían tener importantes implicaciones diagnósticas. Además, nuestro biosensor POC ofrece una serie de ventajas en comparación con los métodos recomendados por la Organización Mundial de la Salud.<br>Nowadays, Tuberculosis (TB) diagnosis is carried out at centralised laboratories, employing bulky equipment, complex reagents, and trained staff, increasing costs and the time to obtain the results. For that reason, the aim of this Doctoral Thesis is to develop a point-of-care (POC) platform able to deliver a prompt and reliable response to TB diagnosis, taking advantage of a highly sensitive evanescent wave optical sensor. The POC platform integrates a novel photonic sensor consisting of a Mach-Zehnder Interferometer transducer array incorporated in a disposable microfluidic cartridge. Firstly, an optical characterisation was carried out to study the new POC performance and its ability to be employed for biosensing applications. Once the POC platform was optically characterised, diverse biofunctionalisation strategies were tested in order to incorporate specific antibodies as bioreceptors to the sensor surface. After an in-depth study, the optimal biofunctionalisation strategy was selected and employed for the analysis of the TB biomarkers. The TB biomarkers were evaluated in both buffer and biological samples, particularly human urine. The most promising and well-known TB biomarker was lipoarabinomannan (LAM), a bacterial cell wall component. In particular, this biomarker detection was validated with clinical samples from TB patients and healthy donors, showing the ability of our POC platform to discriminate those patients with active TB. Moreover, taking advantage of the photonic sensor design, which allows the simultaneous detection of six different biomarkers, we initiated the proof-of-concept of the POC platform for a TB biomarker panel detection using Dip-Pen Nanolithography for each corresponding bioreceptor deposition. Our results, if validated with larger clinical studies, could have important diagnostic implications taking into account the advantages added by our POC biosensor in comparison with the methods recommended by the World Health Organisation.<br>Universitat Autònoma de Barcelona. Programa de Doctorat en Biotecnologia

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2011.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references (p. 77).<br>As part of the development of a point of care complete blood count device, I designed a single use lancet integrated with a blood collection mechanism and interface and successfully tested a prototype. High speed video was taken of lancet motion at 50,000 frames per second, and lancet velocities were found to be -11 m/s as compared to ~3m/s for a commonly used lancet. As faster lancet velocity reduces pain, this device will be less painful than comparable devices on the market. Work with an industrial design consultant has begun, and the lancet prototype will be modified for production. I built a prototype of the blood collection mechanism and integrated it with the single-use lancet into a single device. Additionally, I designed the loading mechanism to interface collected blood with the machine. Several concepts were considered, and the best concept was selected and prototyped. The loading mechanism was tested to ensure proper mixing between collected blood and reagents required for the complete blood count.<br>by Julia C. Zimmerman.<br>S.M.

Diseases are often diagnosed by detection of disease-specific biomarkers in fluid samples. However, many state-of-the-art detection methods require a lab with complex machinery, trained operators, and/or lengthy analysis time. In contrast, point-of-care (POC) devices are brought to the patient's location, they are easy to use, and results are obtained almost immediately. Many current POC devices are too difficult to be used without a skilled assistant, and although many are able to detect analytes above a threshold value, they give little or no quantitative information. This work presents the development of polymer-based microfluidic devices capable of sensing and quantifying biomarkers in fluid samples in a straightforward manner using a novel biomarker assay termed "flow valve diagnostics". In this assay, an antibody-modified polydimethylsiloxane (PDMS) microchannel constricts due to the binding force between antibodies and antigens, stopping fluid flow. The flow distance is measured and correlated to antigen concentration. This detection method is an improvement over other methods because it is an innovative, non-instrumented, label-free, easy-to-use approach. These devices are small, portable, disposable, inexpensive, and thus ideal for use in POC testing. I have successfully fabricated flow valve devices with standard micromachining techniques, including photolithography, replica molding with PDMS, and plasma oxidation. Following fabrication, I compared two methods for attaching receptor biomolecules (e.g., antibodies) to the microchannel surfaces: non-specific adsorption and silanization with 3-glycidoxytrimethoxypropylsilane (GOPS). I used laser-induced fluorescence to determine that silanization with GOPS was the better method for biomolecule attachment. Finally, I tested antibody-modified flow valve devices with target antigens to determine if the antibody/antigen binding force was strong enough to cause channel pinching and flow stoppage. By modifying the device design and using higher antigen concentrations, I was able to show that flow valve devices can detect antigens in a concentration-dependent manner. Future work to improve the device design and to modify and test these devices with different receptor/target pairs will bring flow valve diagnostics closer to becoming a valuable asset in biomarker detection and POC testing.

Individual genetic variations are suspected to be the most influential factor with respect to drug efficacy, specifically, single nucleotide polymorphisms (SNP) in genes responsible for the transportation and metabolism of a particular drug. Correlations have been discovered between the genotype of the various SNPs located within the drug-metabolising enzymes and the overall efficacy of a particular drug. This information could then potentially be used to prescribe personalised medication based on an individual's genetic profile.

Background: Globally, 422 million adults have type 2 diabetes mellitus (T2DM), causing 1.5 million deaths per year. Kuwait has one of the highest T2DM prevalence in the world and determining the proportion of patients and relatives who have pre-diabetes (PDM), insulin resistance (IR) and T2DM is crucial to inform preventive activities and curative services. Study objectives: The study describes the prevalence and risk factors of PDM, IR and T2DM, in adult patients attending a primary health care facility in Kuwait and the prevalence and risk factors of the same conditions among the patients’ first-degree relatives. The study also describes the degree of glycaemic control achieved by patients with T2DM and risk factors for poor glycaemic control. Finally, we assessed the agreement of a point of care (POC) device to measure glycated haemoglobin (HbA1c) to monitor T2DM control. Methods We conducted cross-sectional surveys of patients and first-degree relatives attending Nuzha health care facility in Kuwait and case-control studies of participants attending Nuzha’s diabetic clinic. Diabetic participants were consecutively tested by the Quo-test (POC) device to compare its agreement with a reference test. Results The prevalence of T2DM, IR and PDM among patients attending the clinics were 29.6% (95% CI: 25.1%-34.1%), 34.6% (95% CI: 29.1%-40.2%) and 26.0% (95% CI: 21.6%-30.4%), respectively. The proportion of patients with T2DM increased with age (AOR=5.4), with the highest prevalence occurring at 60-69 years of age. T2DM was associated with hypertension (AOR=1.95) and being a widow (AOR=6.11). IR was associated with low HDL (AOR=1.96), overweight (OR=8.25), obesity (OR=18.33) and increased waist circumference (OR= 5.5). Sugar-sweetened beverages were associated with IR. The prevalence of T2DM, IR and PDM among first-degree relatives of T2DM patients were 29.1% (95%CI: 23.7%-34.5%), 32.8% (95%CI: 26.2%-39.4%), 20.4% (95%CI: 15.6%-25.2%). The risk factors for T2DM were similar among patients and first-degree relatives, but IR was associated with manual labour occupations (AOR=3.6). Only 30% of T2DM patients achieved good glycaemic. Poor control was associated with high triglycerides (AOR=2.2), smoking (AOR=4.1) and the number of years since diagnosis (AOR=4). The Quo-Test had comparable performance to the reference test, with a Coefficient of Variation of 2.1% (r2 = 935, Kappa 90% and 87% at HbA1c cut-offs of 7.0 and 9.0% respectively). The POC and the reference tests performed poorly in patients with haemoglobinopathies. Conclusion This study demonstrates that a high proportion of patients and first-degree relatives attending one of the main primary health care centres in Kuwait have T2DM. Many patients and relatives were unaware of their condition. There was also a very high prevalence of IR and PDM suggesting the burden of T2DM will increase further in the future. Major efforts are needed to upscale detection, and preventive programmes for IR, PDM and T2DM and the quality of T2DM management needs to improve. The POC device tested could provide timely information for the management of T2DM.

A dynamic model is developed and presented that predicts the voltage response for a Severinghaus electrode-based point-of-care pCO2 sensor. Eight partial differential equations are derived to describe the diffusion and reaction phenomena in the sensor. The model is able to predict the potential response versus time behaviour from different CO2 concentrations in the calibration fluid and control fluids. The two most influential and uncertain parameters in the model are determined to be the forward rate constant for benzoquinone consumption at the gold surface ( k_(f_Au ) ), and the partition coefficient for CO2 between the membrane and the electrolyte (κ_(〖CO〗_(2_m ) )). These parameters were adjusted heuristically to obtain a good fit (within 2 mV) between the dynamic voltage response data and the model predictions during a critical 4 second period. The model predictions are sufficient for design sensitivity studies, however an improved fit might be possible using a formal least-squares parameter estimation approach, or if additional parameters were estimated. Several design parameters are varied to study the influence of the electrolyte concentration and the sensor geometry on the voltage response. The most influential design parameter studied is the amount of water present in the electrolyte during sensor operation. This can be affected by the amount of water evaporated during manufacturing and storage, and by the amount of water present when the sensor “wets up” again during operation. The amount of water picked up by the sensor in turn is affected by design parameters such as component/membrane dimensions and thicknesses. The initial buffer concentration in the electrolyte is the second most influential parameter. The resulting model can be used to perform “what if” analyses in order to understand the impact of design decisions on the sensor performance, and to potentially improve the sensor from performance and manufacturing cost perspectives.<br>Thesis (Master, Chemical Engineering) -- Queen's University, 2014-02-06 15:00:47.555

In the developing world, there are large populations suffering from infectious diseases, many of whom are located in remote regions. With the rapid growth in microfluidic systems in recent years, complex functions of conventional diagnostic equipment have been miniaturized and integrated into small devices at the size of a credit card (so-called portable Point-of-care (POC) devices). In this thesis a novel approach to overcoming the challenge of in-field biological sample processing and preparation to produce high quality fluids that can be readily used for downstream testings is described and proof of concept experiments presented. This approach uses hydrodynamic effects and combines nanoporous membrane with microfluidic systems and to filter the cellular component of blood. Experiments presented here demonstrate successful cells filtration from whole blood. Employing hydrodynamic effects is also shown to be an effective and potentially useful technique to isolate cells and plasma within appropriate micro-architectures.