Search

226 p., La aparición de nuevos conocimientos y tecnologías en microfabricación han permitido el desarrollo de nuevas herramientas miniaturizadas para ensayos de biología celular. El mayor progreso que estos dispositivos ofrecen es proveer a los investigadores con recursos para ensayos analíticos, útiles para un amplio rango de aplicaciones que pueden llegar a superar los métodos establecidos en términos de sensibilidad y rentabilidad. En la presente tesis, se han aprovechado algunas de estas microtecnologías para el desarrollo de plataformas biosensóricas simples pero versátiles y fiables, para su implementación en ensayos de alta relevancia biomédica. Concretamente, se han desarrollado dos tipos de biosensores. En primer lugar, se crearon biosensores para ensayos biomédicos de afinidad celular y citotoxicidad, basados en la adhesión de células a patrones de biomoléculas, sin y con integración en dispositivos microfluídicos. En segundo lugar, biosensores plasmónicos basados en fibras ópticas y nanopartículas de oro se desarrollaron y usaron para cuantificar agentes bioactivos como los iones de cobre y la proteína Citocromo c. Estos biosensores constituyen un paso adelante en un campo en auge que está llamado a cerrar la brecha entre la obtención de resultados cuantitativos y reproducibles y la democratización de los medios para obtener dichos resultados.

El capítulo 9 está sujeto a confidencialidad por la autora. 289 p., Esta tesis investiga diferentes aspectos de la manipulación de biofluidos complejos, como son el sudor, la sangre y el semen a microescala, para mejorar los enfoques de detección, separación y selección en dispositivos microfluídicos. Esto implica la fabricación de materiales sensores basados en alginato, así como su integración en diferentes plataformas microfluídicas para mejorar la detección de analitos. Además, también se investiga la generación de nuevas herramientas para la separación de plasma a partir de muestras de sangre humana completa mediante tecnología de impresión 3D. Por último, también se contempla el diseño y fabricación de novedosas configuraciones de dispositivos microfluídicos para la selección de espermatozoides viables con fines reproductivos.

224 p., Los dispositivos microfluídicos ideales deben ser pequeños, fáciles de operar, portátiles, autónomos y capaces de integrar todas las funciones analíticas requeridas dentro de un mismo chip. El control de flujo es un aspecto crítico a tener en cuenta al diseñar estos sistemas microfluídicos, sin embargo, a menudo se utilizan componentes convencionales y voluminosos que requieren fuentes de alimentación externas y reducen su portabilidad. En esta tesis se investigó la integración de microbombas poliméricas modulares en cartuchos microfluídicos plásticos para el desarrollo de dispositivos microfluídicos autónomos, dando como resultado el desarrollo de una arquitectura universal altamente versátil y adaptable para una amplia gama de volúmenes y tiempos de actuación. Además, los cartuchos microfluídicos se pueden fabricar fácilmente mediante técnicas de prototipado rápido y un proceso de fabricación capa-a-capa, lo que, en combinación con las bajas presiones generadas por las microbombas poliméricas, permite el desarrollo rápido de operaciones fluídicas complejas para el análisis de muestras. A modo de ejemplo, se demostró la aplicabilidad de estos sistemas autónomos para realizar la calibración de la reacción de Griess en el interior de ionogeles o la separación de plasma a partir de muestras de sangre completa. En conclusión, estas arquitecturas combinadas están destinadas a ser un referente en el diseño de operaciones microfluídicas autónomas y personalizables debido a su versatilidad y facilidad de fabricación.

Non-invasive collection of target cells is crucial for research in biology and medicine. In this work, we combine a thermo-responsive material, poly(N-isopropylacrylamide), with an electroactive material, poly(3,4-ethylene-dioxythiopene):poly(styrene sulfonate), to generate a smart and conductive copolymer for the label-free and non-invasive detection of the capture and release of cells on gold electrodes by electrochemical impedance spectroscopy. The copolymer is functionalized with fibronectin to capture tumor cells, and undergoes a conformational change in response to temperature, causing the release of cells. Simultaneously, the copolymer acts as a sensor, monitoring the capture and release of cancer cells by electrochemical impedance spectroscopy. It is expected that this platform has the potential to play a role in top-notch label-free electrical monitoring of human cells obtaining in clinic.

372 p., En las últimas décadas, se han realizado avances considerables en el desarrollo de dispositivos miniaturizados "lab-on-a-chip" integrados. Los sistemas miniaturizados pueden aportar información difícil de conseguir con otros medios, por lo que la utilización de protocolos de microfabricación de fácil implementación impulsaría el uso de estas plataformas como tecnologías de monitorización. En esta tesis, se han combinado la microfabricación y la ciencia de los materiales para desarrollar microsistemas para monitorización química y celular, dando lugar a estrategias novedosas como sensores, basadas en la integración de materiales funcionales en sistemas miniaturizados.

123 p.

In recent years, innovative cell-based biosensing systems have been developed, showing impact in healthcare and life science research. Now, there is a need to design mass-production processes to enable their commercialization and reach society. However, current protocols for their fabrication employ materials that are not optimal for industrial production, and their preparation requires several chemical coating steps, resulting in cumbersome protocols. We have developed a simplified two-step method for generating controlled cell patterns on PMMA, a durable and transparent material frequently employed in the mass manufacturing of microfluidic devices. It involves air plasma and microcontact printing. This approach allows the formation of well-defined cell arrays on PMMA without the need for blocking agents to define the patterns. Patterns of various adherent cell types in dozens of individual cell cultures, allowing the regulation of cell–material and cell–cell interactions, were developed. These cell patterns were integrated into a microfluidic device, and their viability for more than 20 h under controlled flow conditions was demonstrated. This work demonstrated the potential to adapt polymeric cytophobic materials to simple fabrication protocols of cell-based microsystems, leveraging the possibilities for commercialization.

Platelet-Rich Plasma (PRP) is an autologous biological product which, due to its regenerative capacity, is currently used in different fields of medicine. This biological treatment has proven to be effective in numerous research studies due to its high content of growth factors released by platelets. However, the current systems used to obtain PRP do not enrich the growth factors and cytokines outside platelets. Considering this, the present work aims to develop a new technique by which all the biomolecules present in plasma are enriched. Thus, a new method based on ultrafiltration has been developed for the obtaining of the novel PRP. By this method, ultrafiltration of the plasma water is carried out using a 3KDa filtering unit. The results showed that the technique was able to concentrate extraplatelet factors, such as IGF-1 and HGF, in contrast with conventional plasmas. Thus, the cultured cells responded with increased viability to this new PRP. These results could provide a new approach to the treatment of injuries requiring regenerative medicine, potentially improving the outcomes of the conventional PRPs.

Platelet-rich plasma (PRP) is an autologous biologic product used in several fields of medicine for tissue repair due to the regenerative capacity of the biomolecules of its formulation. PRP consists of a plasma with a platelet concentration higher than basal levels but with basal levels of any biomolecules present out of the platelets. Plasma contains extraplatelet biomolecules known to enhance its regenerative properties. Therefore, a PRP containing not only a higher concentration of platelets but also a higher concentration of extraplatelet biomolecules that could have a stronger regenerative performance than a standard PRP. Considering this, the aim of this work is to develop a new method to obtain PRP enriched in both platelet and extraplatelet molecules. The method is based on the absorption of the water of the plasma using hydroxyethyl acrylamide (HEAA)-based hydrogels. A plasma fraction obtained from blood, containing the basal levels of platelets and proteins, was placed in contact with the HEAA hydrogel powder to absorb half the volume of the water. The resulting plasma was characterized, and its bioactivity was analyzed in vitro. The novel PRP (nPRP) showed a platelet concentration and platelet derived growth factor (PDGF) levels similar to the standard PRP (sPRP), but the concentration of the extraplatelet growth factors IGF-1 (p < 0.0001) and HGF (p < 0.001) were significantly increased. Additionally, the cells exposed to the nPRP showed increased cell viability than those exposed to a sPRP in human dermal fibroblasts (p < 0.001) and primary chondrocytes (p < 0.01). In conclusion, this novel absorption-based method produces a PRP with novel characteristics compared to the standard PRPs, with promising in vitro results that could potentially trigger improved tissue regeneration capacity.

Glucose is an analyte of great importance, both in the clinical and sports fields. As an alternative to standard blood analysis, sweat arises as an easily accessible, non-invasive biofluid, as well as a great candidate to monitor human health and sport performance of athletes. However, there is a current need for the fabrication of low cost, easy to use technologies capable of sequential analysis of sweat biomarkers at different times. In this research, we developed a chrono-sampling microfluidic platform integrated with an alginate/TiO2 biosystem for sweat glucose determination. Glucose concentrations ranging from 10 to 1000 µM in artificial sweat were detected and quantified, both in a bead format and in the device, where the whole system was calibrated and verified. The PDMS microfluidic platform allowed chrono-sampling over more than 42 min. This research is oriented towards the development of a miniaturized device for fast analysis at the point and place of need. The integration of the chrono-sampling capacity opens the possibility for a continuous monitoring of sweat biomarkers during exercise, allowing a personalized-oriented training to improve the performance of athletes.

Biomachining has been investigated as a sustainable and effective alternative to conventional prototyping techniques for molding polymeric materials for their subsequent use as microfluidic devices. A novel and simple process based on the combination of a Pressure Sensitive Adhesive mask and a varnish has been proposed for preparing metal workpieces as an alternative to photolithography, with the latter being the most widely used technique for protecting workpieces. As far as the bioprocess is concerned, it has been applied in successive mold-etching and oxidant bio-regeneration stages. Metal solubilization has proven to be repeatable in several consecutive mold-etching stages when using the regenerated oxidant solution. As a result, the lifespan of the biomachining medium has been prolonged, contributing to process sustainability. An equation with two restrictions has been proposed to predict the time required to obtain a mold with a fixed height, as metal solubilization evolves differently between the first and subsequent hours. Finally, the bio-engraved copper pieces have acted as effective molds in the fabrication of self-powered polydimethylsiloxane microfluidic devices. This new biomachining application is therefore an effective and ecofriendly process for producing microfluidic devices.

This work presents a new architecture concept for microfluidic devices, which combines the conventional 3D printing fabrication process with the stable and precise integration of polymeric functional materials in small footprints within the microchannels in well-defined locations. The approach solves the assembly errors that normally occur during the integration of functional and/or sensing materials in hybrid microfluidic devices. The method was demonstrated by embedding four pH-sensitive ionogel microstructures along the main microfluidic channel of a complex 3D printed microfluidic device. The results showed that this microfluidic architecture, comprising the internal integration of sensing microstructures of diverse chemical compositions, highly enhanced the adhesion force between the microstructures and the 3D printed microfluidic device that contains them. In addition, the performance of this novel 3D printed pH sensor device was investigated using image analysis of the pH colour variations obtained from photos taken with a conventional camera. The device presented accurate and repetitive pH responses in the 2 to 12 pH range without showing any type of device deterioration or lack of performance over time.

Glucose is an analyte of great importance, both in the clinical and sports fields. Since blood is the gold standard biofluid used for the analytical determination of glucose, there is high interest in finding alternative non-invasive biofluids, such as sweat, for its determination. In this research, we present an alginate-based bead-like biosystem integrated with an enzymatic assay for the determination of glucose in sweat. The system was calibrated and verified in artificial sweat, and a linear calibration range was obtained for glucose of 10–1000 µM. The colorimetric determination was investigated, and the analysis was carried out both in the black and white and in the Red:Green:Blue color code. A limit of detection and quantification of 3.8 µM and 12.7 µM, respectively, were obtained for glucose determination. The biosystem was also applied with real sweat, using a prototype of a microfluidic device platform as a proof of concept. This research demonstrated the potential of alginate hydrogels as scaffolds for the fabrication of biosystems and their possible integration in microfluidic devices. These results are intended to bring awareness of sweat as a complementary tool for standard analytical diagnosis.

The effect of cell-cell contact on gene transfection is mainly unknown. Usually, transfection is carried out in batch cell cultures without control over cellular interactions, and efficiency analysis relies on complex and expensive protocols commonly involving flow cytometry as the final analytical step. Novel platforms and cell patterning are being studied in order to control cellular interactions and improve quantification methods. In this work, we report the use of surface patterning of fibronectin for the generation of two types of mesenchymal stromal cells patterns: single cell patterns without cell-to-cell contact, and small cell-colonypatterns. Both scenarios allowed the integration of the full transfection process and the continuous monitoring of thousands of individualized events by fluorescence microscopy. Our results showed that cell-to-cell contact clearly affected the transfection, as single cells presented a maximum transfection peak 6 hours earlier and had a 10 % higher transfection efficiency than cells with cell-to-cell contact.

Water quality monitoring of drinking, waste, continental and seawaters is of great importance to ensure safety and wellbeing for humans, fauna and flora. Researchers are developing robust water monitoring microfluidic devices but, the delivery of a cost-effective, commercially available platform has not been achieved yet. Conventional water monitoring is mainly based on laboratory instruments or on sophisticated and expensive handheld probes for on-site analysis, both requiring trained personnel and being time-consuming. As an alternative, microfluidics has emerged as a powerful tool with the capacity to displace conventional analytical systems. Nevertheless, microfluidic devices largely use conventional pumps and valves for operation, and electronics for sensing, that increment the dimensions and costs of the final platforms, diminishing their commercialization perspectives. In this review, we critically analyze the characteristics of conventional microfluidic devices for water monitoring, focusing on different water matrices (drinking, waste, continental and seawaters), towards their application in potential commercial products. Moreover, we introduce the revolutionary concept of using functional materials such as hydrogels, poly(ionic liquid) hydrogels and ionogels as alternatives to conventional fluidic handling and sensing tools for water monitoring in microfluidic devices.

The electrical and electrochemical properties of two ionogel materials based on the poly(N-isopropylacrylamide) gel with 1-ethyl-3-methylimidazolium ethyl sulfate and trihexyltetradecyl-phosphonium dicyanamide ionic liquids are investigated. The current-voltage curves show a current rectification for both ionogels and ionic liquids after inducing ion adsorption on gold electrode surface. The polymer matrix of the ionogels mitigates ion adsorption, resulting in lower rectification ratios when comparing with the corresponding ionic liquids. The stability and the electrochemical window (ECW) of the ionogels are calculated from cyclic voltammograms and compared with the ones obtained for the ionic liquids, achieving the widest electrochemical window the phosphonium based ionic liquid and its ionogel. Finally, the effect of the absorbed atmospheric water and the coefficient diffusion of the redox active species inside the IO is calculated. This work widens the knowledge of the behavior of ionogel materials characterized on gold electrode interfaces.

[EN] Nowadays, miniaturization and portability are crucial characteristics that need to be considered for the development of water monitoring systems. In particular, the use of handheld technology, including microfluidics, is exponentially expanding due to its versatility, reduction of reagents and minimization of waste, fast analysis times and portability. Here, a hybrid handheld miniaturized polymer platform with a paper-based microfluidic device was developed for the simultaneous detection of nitrite and nitrate in real samples from both, fresh and seawaters. The platform contains an ionogel-based colorimetric sensor for nitrite detection and a paper-based microfluidic device for the in situ conversion of nitrate to nitrite. The platform was fully characterized in terms of its viability as a portable, cheap and quick pollutant detector at the point of need. The calibration was carried out by multivariate analysis of the color of the sensing areas obtained from a taken picture of the device. The limits of detection and quantification, for nitrite were 0.47 and 0.68mgL-1, while for nitrate were 2.3 and 3.4mgL-1, found to be within the limits allowed by the environmental authorities, for these two pollutants. Finally, the platform was validated with real water samples, demonstrating its potential to monitor nitrite and nitrate concentrations on-site as a first surveillance step before performing extensive analysis.

[EN] Remote manipulation of superhydrophobic surfacesprovides fascinating features in water interface-related applications. Asuperhydrophobic magnetic nanoparticle colloid layer is able to float on the water-air interface and form a stable water-solid-air interface dueto its inherent water repulsion, buoyancy, and lateral capillarity properties. Moreover, it easily bends downward under an externally applied gradient magnetic field. Thanks to that, the layer creates a stabletwister-like structure with aflipped conical shape, under controlled waterlevels, behaving as a soft and elastic material that proportionally deformswith the applied magneticfield and then goes back to its initial state in the absence of an external force. When the tip of the twister structure touches the bottom of the water container, it provides a stable magnetomovable system, which has many applications in the microfluidicfield.We introduce, as a proof-of-principle, three possible implementations of this structure in real scenarios, the cargo and transport of water droplets in aqueous media, the generation of magneto controllableplugs in open surface channels, and the removal of floating microplastics from the air-water interface

[EN] Biocompatible magnetic hydrogels provide a great source of synthetic materials, which facilitate remote stimuli, enabling safer biological and environmental applications. Prominently, the ex situ and in situ magnetic phase integration is used to fabricate magneto-driven hydrogels, exhibiting varied behaviours in aqueous media. Therefore, it is essential to understand their physicochemical properties to target the best material for each application. In this investigation, three different types of magnetic alginate beads were synthesised. First, by direct, ex situ, calcium chloride gelation of a mixture of Fe3O4 nanoparticles with an alginate solution. Second, by in situ synthesis of Fe3O4 nanoparticles inside of the alginate beads and third, by adding an extra protection alginate layer on the in situ synthesised Fe3O4 nanoparticles alginate beads. The three types of magnetic beads were chemically and magnetically characterised. It was found that they exhibited particular stability to different pH and ionic strength conditions in aqueous solution. These are essential properties to be controlled when used for magneto-driven applications such as targeted drug delivery and water purification. Therefore, this fundamental study will direct the path to the selection of the best magnetic bead synthesis protocol according to the defined magneto-driven application.

[EN] Low cost and user-friendly paper microfluidic devices, combined with DNA-based biosensors with binding capacities for specific molecules, have been proposed for the developing of novel platforms that ease and speed-up the process of cell secretion monitoring. In this work, we present the first cellulose microfluidic paper-based analytical device for the single-step detection of cell secreted Vascular Endothelial Growth Factor through a self-reporting Structure Switching Signaling Aptamer. A three-part Structure Switching Signaling Aptamer was designed with an aptameric sequence specific for VEGF, which provides a quantifiable fluorescent signal through the displacement of a quencher upon VEGF recognition. The VEGF biosensor was integrated in cellulose paper, enabling the homogenous distribution of the sensor in the paper substrate and the detection of as low as 0.34ng of VEGF in 30min through fluorescence intensity analysis. As a proof-of-concept, the biosensor was incorporated in a microfluidic paper-based analytical device format containing a VEGF detection zone and a control zone, which was applied for the detection of cell secreted VEGF in the supernatant of mesenchymal stem cells culture plates, demonstrating its potential use in cell biology research.