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Currently, three major circumstances threaten the management of bacterial infections: increasing antimicrobial resistance, expansion of chronic biofilm-associated infections, and lack of an appropriate approach to treat them. To date, the development of accelerated drug susceptibility testing of biofilms and of new antibiofouling systems has not been achieved despite the availability of different methodologies. There is a need for easy-to-use methods of testing the antibiotic susceptibility of bacteria that form biofilms and for screening new possible antibiofilm strategies. Herein, we present a microfluidic platform with an integrated interdigitated sensor (BiofilmChip). This new device allows an irreversible and homogeneous attachment of bacterial cells of clinical origin, even directly from clinical specimens, and the biofilms grown can be monitored by confocal microscopy or electrical impedance spectroscopy. The device proved to be suitable to study polymicrobial communities, as well as to measure the effect of antimicrobials on biofilms without introducing disturbances due to manipulation, thus better mimicking real-life clinical situations. Our results demonstrate that BiofilmChip is a straightforward tool for antimicrobial biofilm susceptibility testing that could be easily implemented in routine clinical laboratories.

Suspended planar-array (SPA) chips embody millions of individual miniaturized arrays to work in extremely small volumes. Here, the basis of a robust methodology for the fabrication of SPA silicon chips with on-demand physical and chemical anisotropies is demonstrated. Specifically, physical traits are defined during the fabrication process with special focus on the aspect ratio, branching, faceting, and size gradient of the final chips. Additionally, the chemical attributes augment the functionality of the chips with the inclusion of complete coverage or patterns of selected biomolecules on the surface of the chips with contact printing techniques, offering an extremely high versatility, not only with the choice of the pattern shape and distribution but also in the choice of biomolecular inks to pattern. This approach increases the miniaturization of printed arrays in 3D structures by two orders of magnitude compared to those previously demonstrated. Finally, functional micrometric and sub-micrometric patterned features are demonstrated with an antibody binding assay with the recognition of the printed spots with labeled antibodies from solution. The selective addition of physical and chemical attributes on the suspended chips represents the basis for future biomedical assays performed within extremely small volumes.

Here we devise a multiprobe electrical measurement system based on quartz tuning forks (QTFs) and metallic tips capable of having full 3D control over the position of the probes. The system is based on the use of bent tungsten tips that are placed in mechanical contact (glue-free solution) with a QTF sensor. Shear forces acting in the probe are measured to control the tip-sample distance in the Z direction. Moreover, the tilting of the tip allows the visualization of the experiment under the optical microscope, allowing the coordination of the probes in X and Y directions. Meanwhile, the metallic tips are connected to a current-voltage amplifier circuit to measure the currents and thus the impedance of the studied samples. We discuss here the different aspects that must be addressedwhenconductingthesemultiprobeexperiments,suchastheamplitudeofoscillation,shear force distance control, and wire tilting. Different results obtained in the measurement of calibration samples and microparticles are presented. They demonstrate the feasibility of the system to measure the impedance of the samples with a full 3D control on the position of the nanotips.

The present paper reports a bacteria autonomous controlled concentrator prototype with a user-friendly interface for bench-top applications. It is based on a micro-fluidic lab-on-a-chip and its associated custom instrumentation, which consists in a dielectrophoretic actuator, to pre-concentrate the sample, and an impedance analyser, to measure concentrated bacteria levels. The system is composed by a single micro-fluidic chamber with interdigitated electrodes and a instrumentation with custom electronics. The prototype is supported by a real-time platform connected to a remote computer, which automatically controls the system and displays impedance data used to monitor the status of bacteria accumulation on-chip. The system automates the whole concentrating operation. Performance has been studied for controlled volumes of Escherichia coli (E. coli) samples injected into the micro-fluidic chip at constant flow rate of 10 μL/min. A media conductivity correcting protocol has been developed, as the preliminary results showed distortion of the impedance analyser measurement produced by bacterial media conductivity variations through time. With the correcting protocol, the measured impedance values were related to the quantity of bacteria concentrated with a correlation of 0.988 and a coefficient of variation of 3.1%. Feasibility of E. coli on-chip automated concentration, using the miniaturized system, has been demonstrated. Furthermore, the impedance monitoring protocol had been adjusted and optimized, to handle changes in the electrical properties of the bacteria media over time.

El creciente desarrollo y la mejora en cuanto a innovación de dispositivos basados en la convergencia de tecnologías emergentes ha dado lugar a un uso cada vez mayor de los nanosensores en la comunidad biomédica. Sin embargo, los nanosensores implantables aún tienen que afrontar ciertos retos como la biocompatibilidad y la seguridad de datos. En este artículo se abordan el progreso y los principales desafíos para esta clase de dispositivos nanomédicos y se analizan además las principales aplicaciones médicas con especial énfasis en la teragnosis, término que integra el concepto de diagnosis y terapia en un mismo dispositivo. De este modo, se traza el proceso desde la investigación aplicada hasta la comercialización del producto, que es cuando el retorno social puede ser estimado. Finalmente, se contempla la gestión de la tecnología dentro de un ecosistema de innovación, cuya cadena de valor incluye una integración multidisciplinaria y el flujo del conocimiento.

La nanobiotecnologia és la confluència entre la nanotecnologia i la biotecnologia. Inclou tots els mètodes i tècniques a nanoescala (10–9 m) que poden servir per controlar i caracteritzar la matèria biològica. Aquestes tècniques són emprades actualment per estudiar i modificar biomolècules i cèŀlules. A més de l'aplicació per estudiar biosistemes, els investigadors també aprenen de la biologia per crear nous dispositius a micronanoescala i per comprendre els processos relacionats amb la vida a l'escala nanomètrica. La nanotecnologia aplicada a la salut —usualment denominada nanomedicina— és una de les tecnologies clau per aconseguir sistemes de diagnosi precoç més precisos (quan més aviat es diagnostica, les opcions i eficiència dels tractaments augmenten), tractaments més orientats a les dianes terapèutiques (que disminueixen efectes secundaris) i millor seguiment de la teràpia. És evident que la nanomedicina serà un instrument fonamental per al desenvolupament d'una medicina personalitzada i sostenible., Nanobiotechnology represents the convergence between nano- and biotechnology and includes all methods and techniques at the nanoscale (10–9 m) that can serve to control and characterize biological matter. These techniques are therefore used to study and modify biomolecules and cells. Besides the application to study biosystems, researchers also learn defrom biology to create new micro-nanoscale devices and better understand life processes at the nanoscale. Nanotechnology applied to healthcare —usually called nanomedicine— is one of the key enabling technologies to achieve and enable earlier and more precise, individual diagnosis (the sooner, the better the treatment), better targeted therapies (fewer side effects) and better therapy monitoring. Thus, nanomedicine is understood to be a fundamental instrument for personalized and sustainable medicine.

La bioenginyeria o enginyeria biomèdica és una disciplina que cerca la millora de la salut, mitjançant la integració de coneixements de les enginyeries, de les ciències biomèdiques i la pràctica clínica. La bioenginyeria combina les capacitats de l'enginyeria amb els requeriments mèdics per millorar la salut. És una branca de l'enginyeria en què el coneixement i les habilitats es desenvolupen i s'apliquen a definir i solucionar qüestions de la biologia i la medicina. El segle xxi ha estat denominat el segle biològic, atès que s'esperen avenços profunds en els àmbits mèdics i dels sectors industrials relacionats, que marquin canvis rellevants. La bioenginyeria esdevé essencial per comprendre la ingent quantitat d'informació que està sent generada per la recerca bàsica, per entendre la complexitat dels éssers vius i per proveir solucions innovadores que es puguin convertir en productes comercials. En aquest sentit l'aplicació de la nanotecnologia a la millora de la salut, àmbit que es coneix com a nanomedicina, ofereix opcions nombroses i prometedores per millorar de manera significativa el diagnòstic i la terapèutica, i permeten assolir una millor qualitat de vida per a tothom. Nous sistemes de diagnòstic que fan ús de la nanotecnologia per quantificar biomarcadors indicatius de malalties permetran una millor valoració dels riscs abans que els símptomes apareguin completament., Bioengineering or biomedical engineering is a discipline that improves human health through cross-disciplinary activities that integrate the engineering sciences with the biomedical sciences and clinical practice. Bioengineering combines engineering expertise with medical needs for the enhancement of health care. It is a branch of engineering in which knowledge and skills are developed and applied to define and solve problems in biology and medicine. The 21st century has been labeled as the “Biological Century” with the expectation of profound implications to future technological breakthroughs both in the medical and other industrial sectors. The bioengineering is becoming essential to understanding the enormous amount of information that is being generated by basic research, to using quantitative approaches, to integrating disparate components in order to understand complex living systems, to providing truly innovative solutions and to translating these to commercial products. In this sense, the application of nanotechnology in healthcare, also know as nanomedicine, offers numerous very promising possibilities to significantly improve medical diagnosis and therapy, leading to an affordable higher quality of life for everyone. New diagnostic tests making use of nanotechnology to quantify disease-related biomarkers could offer an earlier and more personalised risk assessment before symptoms show up.

Al principio de los años 60, aparecieron los primeros transistores FET en Arseniuro de Galio. En el instante en que el silicio se imponía de forma definitiva al germanio, la aparición de este nuevo material venía justificada por múltiples razones: a) El material GaAs de tipo N presenta unas propiedades de transporte muy buenas; b) Se pueden conseguir alturas de barrera Schottky elevadas, con diversidad de metales (Al,Pt,Ti,...), obteniéndose diodos con un factor de calidad excelente y un valor de la corriente en inverso baja; c) La estructura de bandas del GaAs que presenta un gap directo, favorece las transiciones ópticas entre las bandas. Este hecho, ha determinado que el GaAs sea el material base, sobre el que se ha desarrollado la Optoelectrónica; d) La posibilidad de obtener un substrato semi-aislante, es una importante ventaja para simplificar la tecnología de circuitos integrados en GaAs, al permitir utilizar el substrato para realizar el aislamiento entre dispositivos. Las diferentes propiedades que hemos enunciado, hacen del GaAs, además de su aplicación generalizada en optoelectrónica, el candidato idóneo para aplicaciones de alta velocidad como amplificadores hiperfrecuencia (Gigahertz) y circuitos integrados lógicos de gran velocidad (Gigabits). En esta memoria, analizaremos el aislamiento entre dispositivos MESFET obtenido mediante la implantación iónica de boro en comparación con otros métodos de aislamiento. La evaluación de los resultados obtenidos, se realizará en función de la corriente de fugas existente entre dispositivos - capítulo III- y el mayor o menor grado de backgating que presenten los mismos-capítulo IV-. No es posible lograr una comprensión de los efectos tecnológicos de un proceso como la implantación iónica, sin estudiar en primer lugar aspectos propios de la física de semiconductores como es los defectos producidos por la irradiación, en particular los eléctricamente activos. Así, en el capítulo I, se estudia las características de los defectos producidos por la implantación iónica de boro, mediante espectroscopía de emisión térmica (DLTS). Este análisis no sería completo sin el estudio de los parámetros ópticos asociados a los mismos. Por ello, en el capítulo II, se analizan los espectros de fotocapacidad, mediante la aplicación de una nueva técnica (Optical Isothermal Transient Spectroscopy), cuyos resultados nos han permitido confirmar la existencia de interacción entre los defectos producidos.

[cat] Els polímers són uns excel·lents candidats per a la producció de dispositius biomèdics que incorporin estructures nanomètriques. Característiques com ara bones propietats òptiques i de segellament, cost de fabricació baix, disseny ràpid i, sobretot, biocompatibilitat són avantatges que poden fer decantar els científics cap a la producció d’aquests dispositius. Aquest article recopilatori vol mostrar alguns dels mètodes i les tècniques que es fan servir per a la fabricació de nanoestructures amb polímers mitjançant tècniques de replicació que poden ser rellevants per a la producció de dispositius biomèdics. Es posa l’èmfasi en la producció de rèpliques polimèriques, per mètodes d’estampació i amb l’ús per a la fabricació de motius de la tecnologia del «focused ion beam» com a mètode senzill per a l’obtenció de manera reproduïble de gran quantitat de nanoestructures. Es descriu l’ús d’aquestes estructures en les tècniques d’estampació, juntament amb consideracions de fabricació específiques. La maduresa assolida per la nanotecnologia basada en els polímers, conjuntament amb les primeres aplicacions d’aquests en l’anàlisi de cèl·lules aïllades i del comptatge de molècules d’ADN, ens indica que aquests materials constitueixen una alternativa viable a les nanotecnologies basades en el silici per a aplicacions biomèdiques., [eng] Polymers are excellent candidates for the production of biomedical devices incorporating nanometric structures. Good optical transparency and sealing properties, low fabrication costs, fast design realization times, and, crucially, biocompatibility are all advantages that can be exploited by scientists for the production of such devices. Here, we review some of the methods and techniques used in the fabrication of polymeric nanostructures by pattern replication techniques that may be of relevance in the production of biomedical devices. Emphasis is placed on imprint production of polymeric replicas, with master fabrication using focussed ion-beam technology, as a relatively simple method for reproducibly obtaining large numbers of nanostructures. The use of these structures in polymercasting techniques is also described, together with some specific fabrication considerations. The maturity reached by polymer-based nanotechnologies, together with the first polymer-based applications for single-cell analysis and for counting single DNA molecules, demonstrates that polymers constitute a viable alternative to silicon-based nanotechnologies for biomedical applications.

Low-cost tin oxide gas sensors are inherently nonspecific. In addition, they have several undesirable characteristics such as slow response, nonlinearities, and long-term drifts. This paper shows that the combination of a gas-sensor array together with self-organizing maps (SOM's) permit success in gas classification problems. The system is able to determine the gas present in an atmosphere with error rates lower than 3%. Correction of the sensor's drift with an adaptive SOM has also been investigated

[eng] The composition and architecture of the extracellular matrix (ECM), and their dynamic alterations, play an essential regulatory role in numerous cellular processes. Furthermore, structural, and biochemical properties of the ECM are central in regulating cell response via mechanical, chemical, and topological cues detected by receptors on the cell membrane, which induce cytoskeleton and/or cell nucleus rearrangements affecting gene expression. Indeed, distinct ECM architectures in the native stroma depend on tissue type, function, and composition. For instance, ECM anisotropy and stiffness are associated with altered ECM degradation and remodeling in cancer. In turn, abnormal ECM architecture favors tumor progression and invasion. Moreover, numerous diseases are associated with mutations in genes encoding ECM components, leading to deficient mechanical properties and altered ECM structure. Thus, there is an increasing interest to exploit and consolidate this knowledge to improve patients’ diagnosis, treatment, and care. In this work, we examined the use of cutting-edge open-source software to characterize the properties of ECM fibers and fibril bundles in patients’ samples and in vitro model tissues. We focused on Collagen I-III in lung cancer and on Collagen VI-related muscular dystrophies (COL6-RMD) because these pathologies present a clear link between altered ECM architecture and patients’ outcomes, which requires further investigation and may be exploited at the clinical level. For this purpose, we initially generated phantom images of fibrils networks (created artificially in silico or obtained from bioengineered models in vitro mimicking isotropic/anisotropic ECM) to test and validate the analysis of ECM fibrils with different bioengineering tools. Then, we focused on ECM images obtained from patient cell cultures or tissue biopsies and evaluated ECM descriptors as novel potential biomarkers. We collaborated with the Institut de Recerca Sant Joan De Déu (IRSJD) to analyze ECM models obtained from fibroblasts of patients affected by COL6-RMD. This is a rare subset of neuromuscular diseases related to deficiency in collagen type VI (COL6) expression, which affects young patients causing a broad range of severe disabilities, which worsen with time and shorten their lifespan. Currently, analysis of in vitro cultures of patients’ fibroblasts is employed as the first screening to select cases of COL6-RMD, and image-based classification has been suggested to improve this assay. Employing similar protocols, fibroblasts of different sources and malignant origin have been used to obtain decellularized cell-derived matrices (CDM), which retain topography and biochemical features, preserving their native microenvironment. Therefore, we produced and analyzed patients’ CDM to provide new in vitro personalized ECM models and improve early recognition of COL6-RMD subtypes with novel fiber-based ECM descriptors. In parallel, in collaboration with the CIBERES (Centro de Investigación Biomédica en Red de Enfermedades Respiratorias), we characterized the fibrillar properties of Non-Small Cell Lung Cancer (NSCLC) patients’ tissues. NSCLC is among the tumors with the highest death rate and recurrence; therefore, novel biomarkers may provide helpful complementary data with clinical impact. Fiber descriptors extracted by CT-FIRE have been demonstrated to capture relevant prognostic changes in the collagen structure of several tumors. In NSCLC, collagen architecture influences immune system activation, and increased fibrosis is already reported to correlate with patients’ prognosis. Therefore, we studied alterations of collagen fiber networks in the two most common NSCLC subtypes, Adenocarcinoma and Squamous Cell Carcinoma, to provide novel collagen structure-based biomarkers. Ultimately this work, along with the tools presented, paves the way for addressing the need for novel ECM-based descriptors, to stratify patients and evaluate their response to experimental treatments., [spa] La composición y arquitectura de la matriz extracelular (MEC), y sus alteraciones dinámicas, juegan un papel regulador importante en numerosos procesos patológicos. Por ejemplo, la anisotropía y la rigidez de la MEC están asociadas con la remodelación alterada de la MEC en el cáncer. A su vez, esta arquitectura favorece la progresión e invasión tumoral. Además, numerosas enfermedades están asociadas con mutaciones en los genes que codifican los componentes de la MEC, lo que conduce a propiedades mecánicas deficientes y una estructura alterada de la MEC. Por lo tanto, existe un interés creciente por explotar y consolidar este conocimiento para mejorar la atención de los pacientes. En este trabajo, nos centramos en las distrofias musculares relacionadas con el colágeno VI (COL6-RMD) y el cáncer de pulmón de células no pequeñas (NSCLC), para investigar biomarcadores relacionados con la MEC in vitro y en los tejidos de los pacientes que se implementarán en el entorno clínico. Empleamos la segmentación automática de imágenes para cuantificar las características fibrilares e investigamos la asociación con la información clinicopatológica de los pacientes. Por lo tanto, producimos y analizamos matrices derivadas de células de pacientes para proporcionar nuevos modelos de ECM personalizados in vitro y nuevos descriptores de ECM basados en fibra de los subtipos COL6-RMD. Se ha demostrado que los descriptores de fibra basados en imágenes capturan cambios pronósticos relevantes en la estructura del colágeno de varios tumores. En el NSCLC, la arquitectura del colágeno influye en la activación del sistema inmunitario y ya se reconoce que el aumento de la fibrosis se correlaciona con el pronóstico de los pacientes. Por lo tanto, estudiamos las alteraciones de las redes de fibras de colágeno en los dos subtipos de NSCLC más comunes, el adenocarcinoma y el carcinoma de células escamosas, y demostramos que los descriptores de la estructura del colágeno son posibles biomarcadores basados en imágenes con aplicaciones de diagnóstico y pronóstico. En última instancia, este análisis, junto con las herramientas presentadas, es prometedor para abordar la necesidad de nuevos descriptores, para estratificar a los pacientes y evaluar su respuesta a los tratamientos experimentales.

[eng] In vitro engineered three-dimensional tissue models are attracting an increasing interest due to their potential applications in preclinical assays. On the one hand, they are an alternative to the high costs, ethical issues and time-consuming experiments associated with animal models. On the other hand, unlike traditional monolayer cultures, 3D models are fabricated with polymer matrices that can mimic the spatial organization and physiological environment of native tissue. The scalability of these models to the market is currently limited by the fabrication methods. Additive manufacturing techniques, as extrusion bioprinting, provide the automated and controlled deposition of biomaterials with encapsulated cells to fabricate 3D models with unlimited shapes. However, few biomaterials fulfill the rheological, mechanical and biological needs for tissue engineering approaches. Printable biomaterials are commonly highly concentrated viscous fluids that could resemble the mechanical properties of stiff tissues, as skeletal muscle. However, they result in restrictive matrices with closed pores that can hamper the migration and proliferation of cells. As a solution, biomaterials have been chemically modified to obtain photocrosslinkable hydrogels, which provide 3D cultures with flexible physical properties. Nevertheless, current bioprinted muscle tissue models show poorly differentiated fibers and lack of functionality. Based on these precedents, this thesis is focused on the development of photocrosslinkable bioinks with tunable physical properties to fabricate customized in vitro 3D models of skeletal muscle tissue and neuroblastoma. To that end, gelatin, alginate and cellulose natural polymers are chemically modified to obtain UV- crosslinkable hydrogels with disparate physical properties. It is found that composite biomaterials of gelatin methacryloyl and alginate methacrylate present the best mechanical properties for stiff tissues as skeletal muscle and tumors. On this basis, the physical properties and composition of GelMA-AlgMA are modified to obtain matrices that resemble the physiological conditions of each tissue. It is found that neuroblastoma models require dense polymer networks to mimic the restrictive matrices found in solid tumors of high-risk patients. Neuroblastoma cell clusters in bioprinted cultures with a high concentration of AlgMA display the characteristic phenotype of aggressive solid tumors. Therefore, this bioink is proposed for the fabrication of stiff neuroblastoma tumor models. In contrast, this formulation was found unsuitable for skeletal muscle models, which present low proliferation and differentiation. Instead, the physical properties of GelMA-AlgMA bioink are tuned by changing the fabrication parameters, and fibrin is added to the composition to obtain a highly porous bioprinted model that resembles the mechanical properties of muscle tissue. As a result, muscle precursor cells are spontaneously differentiated into highly aligned mature fibers that, in combination with an electric pulse stimulation system, develop into mature muscle fibers with contraction capability and pronounced sarcomere units. The functionality of the bioprinted tissue agrees with the metabolic activity analysis, which corresponds to the behavior of native tissue. Hence, this model could be used to monitor the effects of drugs in the metabolic respiration of muscle mitochondria, simplifying the traditional protocols based on the isolation of single fibers. As an approach to obtain faithful platforms for the study of muscle pathologies as cancer cachexia, bioprinted muscle rings are treated with medium conditioned with colorectal cancer cells. This study shows that soluble factors secreted by cancer cells induce the upregulation of protein degradation pathways and degeneration of muscle fibers. In particular, high levels of soluble TNFRI are associated with severe cachexia, which correlates with the plasma level of tumor-bearing mice. The results indicate a close resemblance between the gene expression pattern of the bioprinted model and muscle tissue of cachectic mice, whereas monolayer cultures present several disparities. Together, GelMA-AlgMA-Fibrin is presented as a promising biomaterial for the fabrication of bioprinted models of healthy skeletal muscle tissue and muscle wasting on cancer cachexia disease., [spa] La bioimpresión por extrusión es una técnica prometedora para el escalado y la fabricación automatizada de modelos tisulares in vitro. Sin embargo, las propiedades fisicoquímicas de las biotintas actuales pocas veces cumplen los requisitos de los tejidos naturales. Esta tesis describe la modificación química de polímeros naturales como gelatina y alginato para obtener biotintas fotopolimerizables con propiedades físicas maleables. La composición de las biotintas y los parámetros de fabricación de los diseños bioimpresos son modulados para conseguir dos tipos de formulaciones adecuadas para el desarrollo de modelos de músculo esquelético y neuroblastoma. En el estudio, ambos modelos muestran mayor semblanza a los tejidos originales que los tradicionales cultivos en monocapa. Por un lado, los modelos de neuroblastoma recapitulan el comportamiento de los tumores sólidos en pacientes de alto riesgo. Por otro lado, la composición de la biotinta para modelos musculares induce la diferenciación de células precursoras a fibras musculares maduras y alineadas que, en combinación con un sistema de estimulación por pulsos eléctricos, dan como resultado fibras musculares funcionales con capacidad contráctil. Este modelo imita el comportamiento del músculo esquelético y se puede utilizar para monitorizar los efectos de compuestos químicos sobre el tejido. Al final del estudio, se trabaja en la generación de un modelo de músculo atrofiado por caquexia derivada del cáncer. El modelo bioimpreso reproduce la característica sobreexpresión de genes relacionados con la degradación proteica hallada en ratones. En conjunto, se adaptan la composición y propiedades físicas de biotintas basadas en gelatina y alginato para la fabricación de modelos de tejido muscular y neuroblastoma por medio de fabricación aditiva. Además, el modelo muscular se presenta como un posible candidato para realizar estudios in vitro del tejido sano y atrofiado por caquexia derivada de cáncer.

[eng] Injuries and conditions of musculoskeletal tissues are among the main causes of disability worldwide and there is a wide need for improved regenerative therapies. In vitro platforms and biomaterials that aim to guide stem cell differentiation should be designed considering that cells interact with their surroundings through nanoscale receptors at the membrane, attaching to ligands in the extracellular matrix. In this thesis we employ uneven dendrimer-nanopatterned substrates to modulate local cell-surface adherence of cells undergoing differentiation towards cartilage, tendon and bone. We aim to elucidate nanoscale adherence cues driving stem cell behaviour during the formation of these musculoskeletal tissues, and the corresponding mechanisms of sensing and transduction. Single and collective cell migration are an essential part of biological processes such as tissue development, wound healing and disease. In the first stages of cartilage formation, mesenchymal stem cells gather into clusters that set the structural bases for subsequent steps in morphogenesis. This process of mesenchymal condensation is limited by the ability of cells to migrate across the extracellular environment and establish cell-cell contacts. We live-imaged the onset of chondrogenesis on nanopatterns of cell-adhesive matrix ligand and found that local ligand density modulates both single and collective stem cell migration through directionality and velocity, impacting the rate of cell-cell collisions. The progressive transition from single cells to multicellular condensates is also guided by cell-cell contacts mediated by N-cadherin and gap junctions. We pose that, once two cells collide on nanopatterned substrates, mesenchymal condensation is regulated by the balance between contact inhibition of locomotion (cells resuming single migration) and contact following of locomotion (cells establishing a new condensation unit). Gap junction intercellular communication (GJIC) provides a continuous and efficient flow of biological information during tissue formation and is essential to sustain homeostasis and function in living organisms. This mode of intercellular communication is particularly important in avascular tissues such as cartilage. We use nanopatterned substrates to study how local ligand density modulates the structure, mechanical stability and protein network architecture within multicellular mesenchymal condensates in early chondrogenesis. We show that nanopatterns of high ligand density facilitate condensate growth and generate condensates that are more stable in culture, as well as increasing cell compaction in them. We demonstrate that high local ligand density nanopatterns promote gap junction protein expression, improve the architecture of the intercellular protein network, and promote GJIC in mesenchymal condensates. We then design a condensate transplantation assay and show that cell sensing of ligand density is a continuous process, with cells responding to changing substrate conditions even if they are not in direct contact with it. Finally, we confirm that substrate information is sensed by integrin adhesions and propagated into the forming tissue through cytoskeletal tensions. As an application of nanopatterned substrates in regenerative therapies of musculoskeletal tissues, we induce cell differentiation towards cartilage, tendon and bone to unveil the optimal local ligand density that promotes it. Nanopatterns of high local ligand density promote chondrogenic differentiation through nuclear translocation of a mechanosensitive transcriptional activator. The same nanopattern configuration also promotes osteogenesis, although in this case cells form smaller adhesions than on low- and medium-density substrates. Tenogenesis is seemingly not affected by substrate ligand density. Given that tension exerted from cell membrane receptors is transmitted by the cytoskeleton to the cell nucleus, we analyse nuclear morphology on each condition according to cell fate. We find that cell response to nanoscale ligand density depends on both size and distribution of integrin adhesions around the cell, in a different manner for each of the three analysed lineages, and that nuclear stretching modulates the observed effects on differentiation., [cat] Les lesions i les condicions dels teixits musculoesquelètics es troben entre les principals causes de discapacitat a tot el món i hi ha una gran necessitat de millorar les teràpies regeneratives. Les plataformes in vitro que tenen com a objectiu guiar la diferenciació de cèl·lules mare s'han de dissenyar tenint en compte que les cèl·lules interaccionen amb el seu entorn mitjançant receptors a nanoescala de la membrana, unint-se als lligands extracel·lulars. En aquesta tesi, utilitzem substrats amb nanopatrons de dendrímers per modular l'adhesió local en cèl·lules que s'estan diferenciant cap a cartílag, tendó i os. Dilucidem les condicions d'adhesió a nanoescala que modulen el comportament de les cèl·lules mare durant la formació d'aquests teixits musculoesquelètics, i els mecanismes corresponents de detecció i transducció. La migració cel·lular és una part essencial de processos biològics com el desenvolupament dels teixits. En les primeres etapes de formació del cartílag, les cèl·lules mare mesenquimals migren per formar condensats pluricel·lulars. Vam fer filmacions de l'inici de la condrogènesi i vam trobar que la densitat de lligands afecta la migració de cèl·lules mare individuals i col·lectives, i la formació de condensats. La comunicació intercel·lular d'unió bretxa (GJIC) proporciona un flux continu i eficient d'informació biològica durant la formació de teixit. Mostrem que els nanopatrons d'alta densitat de lligands generen condensats mesenquimàtics que són més compactes i estables en cultiu. També promouen l'expressió de proteïnes d'unió bretxa, milloren l'arquitectura de la xarxa de proteïnes intercel·lulars i promouen GJIC en condensats. Confirmem que la informació del substrat és detectada per les adhesions d'integrines i es propaga al teixit en formació a través de tensions citoesquelètics. Finalment, induïm la diferenciació cel·lular cap a cartílag, tendó i os per revelar la densitat de lligand local òptima que l'afavoreix. L'alta densitat de lligands promou la condrogènesi i l'osteogènesi, però no la tenogènesi. Trobem que la resposta cel·lular a la densitat de lligands a nanoescala depèn tant de la mida com de la distribució de les adhesions d'integrines al voltant de la cèl·lula, d'una manera diferent per a cadascun dels tres llinatges analitzats, i que l'estirament nuclear modula la diferenciació.

[eng] The skeletal muscle is the largest tissue of the human body. Its main function is to generate contractile forces, essential for locomotion, thermogenesis, and metabolism. Fundamental research on skeletal muscle in health and disease, and preclinical research for new therapies, are currently based on 2D in vitro cell cultures and in vivo animal models. However, these strategies have important shortcomings. For instance, conventional cell culture models cannot emulate the complex 3D architecture of native skeletal muscle, and the species-specific differences in animal models limit their relevance to humans. In contrast, engineered skeletal muscle tissues are emerging as in vitro 3D cell culture models that complement existing 2D strategies. These engineered tissues can offer an improved microenvironment resembling native muscle tissue, comprised of bundles of aligned, multinucleated fibers. Therefore, the main objective of this thesis was to develop 3D skeletal muscle tissues for in vitro studies of muscle metabolism and disease modeling. Skeletal muscle precursor cells were encapsulated in microfabricated hydrogel scaffolds, introducing the appropriate topographical and microenvironmental cues to guide muscle fiber formation. First, photocrosslinkable gelatin methacryloyl (GelMA)-based composite hydrogels were synthesized and evaluated as cell-laden bioinks for 3D bioprinting of murine skeletal muscle tissue. The fabrication conditions were optimized to ensure the biocompatibility of the process and promote in vitro myogenesis. Our results demonstrated that the composite hydrogels have a higher resistance to degradation than GelMA hydrogels. Thus, the bioprinted scaffolds maintained their 3D structure over a prolonged culture period. Furthermore, the shear stress during extrusion bioprinting combined with the appropriate scaffold geometry resulted in highly aligned myoblasts that correctly differentiated into multinucleated myotubes. Considering these results, GelMA-carboxymethylcellulose methacrylate (CMCMA) hydrogels were then used to generate skeletal muscle microtissues in long-lasting cell cultures. Photomold patterning of cell-laden GelMA-CMCMA filaments led to the formation of highly aligned 3D myotubes expressing sarcomeric proteins. Moreover, the presented protocols were highly biocompatible and reproducible. Murine skeletal muscle microtissues were fabricated in a microfluidic platform integrated with an electrical stimulation system and biosensors for monitoring muscle metabolism in situ. Here, we measured the contraction-induced release of muscle-secreted cytokines upon electrical or biological stimulation. The obtained results confirmed the endocrine function of the bioengineered tissues, obtaining in vivo-like responses upon exercise or endotoxin-induced inflammation. Then, the photomold patterning protocol was optimized for human cells to develop the first in vitro 3D model of myotonic dystrophy type 1 (DM1) human skeletal muscle. DM1 is the most prevalent hereditary myopathy in adults, and there is no effective treatment to date. We proved that 3D micropatterning enhances DM1 myotube formation compared to 2D cultures. Furthermore, we detected the reduced thickness of 3D DM1 myotubes compared to healthy controls, which was proposed as a new in vitro structural phenotype. Thus, as a proof-of-concept, we demonstrated that treatment with an antisense oligonucleotide, antagomiR-23b, could rescue both molecular and structural phenotypes in these bioengineered DM1 muscle tissues. Finally, animal-derived components were eliminated to develop in vitro functional tissues in xeno-free cell culture as a next step towards improving bioengineered human skeletal muscle tissues. Cell-laden nanocomposite hydrogels consisting of human platelet lysate and functionalized cellulose nanocrystals (HUgel) were fabricated in hydrogel casting platforms that implemented uniaxial tension during matrix remodeling. We modulated the content of cellulose nanocrystals to tune the mechanical and biological properties of HUgel and favor the formation of long, highly aligned myotube bundles. Additionally, we performed in situ force measurements of electrical stimulation-induced contractions. Altogether, the results presented in this thesis provide promising approaches to advanced cell culture models of skeletal muscle tissue that could be valuable tools for fundamental studies, disease modeling, and future personalized medicine., [spa] El músculo esquelético tiene funciones esenciales para la salud que pueden verse afectadas por enfermedades neuromusculares o metabólicas. Actualmente, la investigación fundamental y preclínica se basa en cultivos celulares en 2D y modelos animales. Sin embargo, estos ensayos tienen relevancia limitada para la salud humana. En cambio, modelos in vitro de tejidos 3D que mimeticen la arquitectura y funcionalidad del músculo esquelético, podrían complementar las estrategias 2D tradicionales. Por lo tanto, el objetivo principal de esta tesis fue desarrollar tejidos de músculo esquelético en 3D para estudios sobre el metabolismo muscular y modelos de enfermedades in vitro. Los tejidos fueron desarrollados mediante diferentes técnicas de microfabricación de hidrogeles, en los que se encapsularon células precursoras del músculo esquelético introduciendo las señales topográficas adecuadas para guiar la formación de fibras musculares. Las propiedades de estos biomateriales fueron optimizadas para garantizar su biocompatibilidad y promover la miogénesis. Estos biomateriales mantienen su estructura durante periodos de cultivo prolongados, permitiendo la formación y diferenciación de miotubos 3D altamente alineados. La función endócrina de los tejidos fue evaluada utilizando un dispositivo músculo-en-un-chip, con el que fue posible medir la liberación de citoquinas secretadas tras estimulación eléctrica o biológica. Posteriormente, se desarrolló el primer modelo 3D de músculo esquelético humano para la distrofia miotónica tipo 1. Como prueba de concepto, demostramos que el tratamiento con un oligonucleótido antisentido, antagomiR-23b, podría rescatar fenotipos moleculares y estructurales en los tejidos fabricados a partir de células de pacientes. Finalmente, se desarrollaron tejidos funcionales en cultivos celulares xeno-free, con el objetivo de incrementar la relevancia de modelos humanos en los que fue posible medir las fuerzas generada por tejidos contráctiles. En conjunto, los resultados de esta tesis proporcionan enfoques prometedores para modelos avanzados de músculo esquelético que podrían ser herramientas valiosas para estudios fundamentales, modelos de enfermedades y medicina personalizada.

[eng] The advancements in nanotechnology enabled the development of new diagnostic tools and drug delivery systems based on nanosystems, which offer unique features such as large surface area to volume ratio, cargo loading capabilities, increased circulation times, as well as versatility and multifunctionality. Despite this, the majority of nanomedicines do not translate into clinics, in part due to the biological barriers present in the body. Synthetic nano- and micromotors could be an alternative tool in nanomedicine, as the continuous propulsion force and potential to modulate the medium may aid tissue penetration and drug diffusion across biological barriers. Enzyme-powered motors are especially interesting for biomedical applications, owing to their biocompatibility and use of bioavailable substrates as fuel for propulsion. This thesis aims at exploring the potential applications of urease-powered nanomotors in nanomedicine. In the first work, we evaluated these motors as drug delivery systems. We found that active urease- powered nanomotors showed active motion in phosphate buffer solutions, and enhanced in vitro drug release profiles in comparison to passive nanoparticles. In addition, we observed that the motors were more efficient in delivering drug to cancer cells and caused higher toxicity levels, due to the combination of boosted drug release and local increase of pH produced by urea breakdown into ammonia and carbon dioxide. One of the major goals in nanomedicine is to achieve localized drug action, thus reducing side-effects. A commonly strategy to attain this is the use moieties to target specific diseases. In our second work, we assessed the ability of urease-powered nanomotors to improve the targeting and penetration of spheroids, using an antibody with therapeutic potential. We showed that the combination of active propulsion with targeting led to a significant increase in spheroid penetration, and that this effect caused a decrease in cell proliferation due to the antibody’s therapeutic action. Considering that high concentrations of nanomedicines are required to achieve therapeutic efficiency; in the third work we investigated the collective behavior of urease-powered nanomotors. Apart from optical microscopy, we evaluated the tracked the swarming behavior of the nanomotors using positron emission tomography, which is a technique widely used in clinics, due to its noninvasiveness and ability to provide quantitative information. We showed that the nanomotors were able to overcome hurdles while swimming in confined geometries. We observed that the nanomotors swarming behavior led to enhanced fluid convection and mixing both in vitro, and in vivo within mice’s bladders. Aiming at conferring protecting abilities to the enzyme-powered nanomotors, in the fourth work, we investigated the use of liposomes as chassis for nanomotors, encapsulating urease within their inner compartment. We demonstrated that the lipidic bilayer provides the enzymatic engines with protection from harsh acidic environments, and that the motility of liposome-based motors can be activated with bile salts. Altogether, these results demonstrate the potential of enzyme-powered nanomotors as nanomedicine tools, with versatile chassis, as well as capability to enhance drug delivery and tumor penetration. Moreover, their collective dynamics in vivo, tracked using medical imaging techniques, represent a step-forward in the journey towards clinical translation., [spa] Recientes avances en nanotecnología han permitido el desarrollo de nuevas herramientas para el diagnóstico de enfermedades y el transporte dirigido de fármacos, ofreciendo propiedades únicas como encapsulación de fármacos, el control sobre la biodistribución de estos, versatilidad y multifuncionalidad. A pesar de estos avances, la mayoría de nanomedicinas no consiguen llegar a aplicaciones médicas reales, lo cual es en parte debido a la presencia de barreras biológicas en el organismo que limitan su transporte hacia los tejidos de interés. En este sentido, el desarrollo de nuevos micro- y nanomotores sintéticos, capaces de autopropulsarse y causar cambios locales en el ambiente, podrían ofrecer una alternativa para la nanomedicina, promoviendo una mayor penetración en tejidos de interés y un mejor transporte de fármacos a través de las barreras biológicas. En concreto, los nanomotores enzimáticos poseen un alto potencial para aplicaciones biomédicas gracias a su biocompatibilidad y a la posibilidad de usar sustancias presentes en el organismo como combustible. Los trabajos presentados en esta tesis exploran el potenical de nanomotores, autopropulsados mediante la enzima ureasa, para aplicaciones biomédicas, y investigan su uso como vehículos para transporte de fármacos, su capacidad para mejorar penetración de tejidos diana, su versatilidad y movimiento colectivo. En conjunto, los resultados presentados en esta tesis doctoral demuestran el potencial del uso de nanomotores autopropulsados mediante enzimas como herramientas biomédicas, ofreciendo versatilidad en su diseño y una alta capacidad para promover el transporte de fármacos y la penetración en tumores. Por último, su movimiento colectivo observado in vivo mediante técnicas de imagen médicas representan un significativo avance en el viaje hacia su aplicación en medicina.

Influenza A virus is one of the most outstanding human viruses. The major treatments against influenza are small analogues, monoclonal antibodies and vaccines, however, due to the fast mutation of the seasonal influenza strain, these methods are easily outdated, so vaccine production and antiviral development need to be in continuous growth and study to improve immunity and fight against influenza disease. The characterization of the viral structure and the identification of the mechanisms of action of newly synthesized antivirals are crucial to develop fast and powerful treatments, nonetheless, due to the small size of viruses, conventional fluorescence techniques are lacking the resolution power to resolve individual viral structures. In this context, super-resolution microscopy has positioned in the last decade as a powerful technique to characterize viral constructs by achieving resolutions up to 10 nm. Here, we have optimized and established a type of super-resolution microscopy technique entitled single-molecule localization microscopy (SMLM) to study viral structures at single-particle level by characterizing several viral structures, antivirals and vaccines. Firstly, we could characterize the filament formation of influenza virus and described how monoclonal antibodies disturbed the development of those filaments, deforming them. Further, we could relate this malformation with an inhibition of the infectivity, suggesting the crucial role of filament formation in the infectivity of influenza. Moreover, we optimized and implemented a novel SMLM called DNA-PAINT in the study of the target distribution and quantification in the nanoscale, validating this method using commercial nanoparticles for its further implementation in the study of the expression of recombinant proteins of influenza and the corresponding virus-like particle produced. In addition, we studied and identified several other viral structures and antivirals interactions using SMLM such as the distribution of Hepatitis B and Hepatitis Delta on paraffin tissues, the interaction of analogues of sialic acid on four strains of influenza and the uptake of nanovaccines from antigen-presenting cells, obtaining features on how these viruses and antivirals interact in order to produce a smart design of antivirals and vaccines, corroborating how SMLM could increase the knowledge of the mechanism of action of viruses.

[eng] Two decades ago, microfluidic technology begun to make its appearance in the fields of drug delivery and biomedical engineering to irrevocably revolutionize them. It was quickly realized how microchannels can aid formulation of microdroplets, microparticles and nanoparticles (NPs). They offer very small and controlled environment for reaction, that is unreproduced in bulk methods. As a result, the formulation is not limited only to the modification of compounds, but the flowing microvolumes open gates to the unexplored world of controllable mixing time and diffusion region impacting the formation of nanoparticles. Beyond the drug delivery systems formulation, the microfluidic technology is emerging as a gap-bridging element of the in vitro and in vivo tests in preclinical trials. Biocompatible and microscopy- friendly microfluidic chips are used to reconstruct physiological elements of human tissues (organ-on-a- chip). They recapitulate 3D, dynamic in vivo environment, that is lacking in 2D cell culture, revealing their relevance in understanding the development of a disease and screening of drug delivery candidates. This work presents the use of microfluidic technology in the formulation of tunable size amphiphilic block co-polymer nanoparticles for drug delivery. The particle diameter is modified in the response to studied phase flow rates. The impact of fluidic parameters on drug/dyes encapsulation efficiency and NP size are analyzed using traditional bulk methods, as well as techniques with single particle resolution, such as Transmission Electron Microscopy (TEM) and Total Internal Reflection Fluorescence (TIRF). Furthermore, the NPs are bioevaluated with in vitro tests performed on MCF-7 cell line. Following the NPs formulation, a chip for combinatorial mixing of NP precursors is presented. A passive micromixer is designed, prototyped and evaluated with fluorescent dyes, to visualize the mixing efficiency. Finally, the model is microfabricated in glass and re-assessed in terms of mixing and cleaning efficiency, which previously was poor due to the absorption of small molecules by PDMS. The micromixer is built into a platform for NPs formulation and first proof-of-concept experiments are performed, yielding monodisperse nanoparticles with encapsulated fluorescent dyes. The encapsulation of dyes is visualized in single particles with TIRF microscopy. The last part of the thesis takes the microfluidic technology into organ-on-a-chip, where a reconstruction of tumor blood vessel model is presented. It recapitulates elements of tumor 3D microenvironment such as blood vessel, endothelial barrier, extracellular matrix and cancer cell spheroid. Observed in vivo leakiness of endothelial barrier is reproduced here in the presence of cancer cells. In this work the microscopy- friendly chip is used as a platform for time- and space-resolved monitoring of micelles stability followed during their interaction with the reconstructed barriers mentioned above. The special optical properties of perfused micelles allow to distinguish assembled from disassembled form. The results are consulted with previously reported observations in 2D cell culture, revealing significant difference in cellular uptake between the two studies. Overall, this work demonstrates how multidisciplinary approach of incorporation of microfluidic technology into formulation and screening of potential drug nanocarriers can accelerate development of nanomedicine. The proposed solutions deliver tunability of nanoparticle properties, combinatorial formulation to create library of NPs and a complementary method in in vitro screening., [spa] Hace dos décadas, la tecnología microfluídica hizo su aparición en los campos de la industria farmacéutica y la ingeniería biomédica de manera revolucionaria. Rápidamente se descubrió cómo los microcanales pueden ayudar a la formulación de microgotas, micropartículas y nanopartículas. Ofrecen entornos de reacción muy pequeños y controlados comparados con la formulación de los métodos tradicionales. En consecuencia, la formulación no sólo se limita a la modificación de compuestos, sino que los flujos de microvolúmenes posibles con la tecnología abren puertas a un mundo inexplorado para la formulación de nanopartículas a través del control del tiempo de mezcla y el área de difusión. Más allá de la formulación de los sistemas de fármacos, la tecnología microfluídica está emergiendo como un elemento puente de las pruebas in vitro e in vivo en los ensayos preclínicos. Los chips de microfluidica biocompatibles y aptos para microscopía se utilizan para reconstruir elementos fisiológicos de tejidos humanos (órgano en un chip). Recapitulan el entorno dinámico in vivo en 3D, carente en el cultivo celular en 2D, desvelando su relevancia para comprender el desarrollo de una enfermedad y la detección de fármacos candidatos para la administración. Este trabajo presenta el uso de la tecnología microfluídica en la formulación de nanopartículas de copolímeros de bloques anfifílicos de tamaño ajustable en respuesta a los caudales de las fases estudiadas. Se estudia el impacto de los parámetros de flujo sobre la eficiencia de encapsulación de fármacos/colorantes y el tamaño de NP. Además, se presenta un chip para la formulación combinatoria de nanopartículas fluorescentes, con potenciales aplicaciones en medicina personalizada. La última parte de la tesis traslada la tecnología de microfluidos a órgano en un chip, donde se presenta la reconstrucción del modelo de vaso sanguíneo tumoral. Recapitula las fugas observadas in vivo de la barrera endotelial en presencia de células tumorales. En este trabajo, se utiliza como una plataforma para el monitorización en el tiempo y en el espacio de la estabilidad de las micelas, mientras interactúan con las barreras reconstruidas que se encuentran en el cuerpo humano: vasos sanguíneos, barrera endotelial, matriz extracelular y esferoide multicelular de células cancerosas.

[eng] Movement of skeletal-muscle fibres is generated by the locomotion circuit, in which many cells play a different role. Failures in any part of the circuit can cause or define the severity of neuromuscular diseases (NMD), such as amyotrophic lateral sclerosis (ALS). Conventional in vitro study models are based on cocultures of motoneurons and skeletal muscle cells from animal origin in 2D. These models have proved to be quite limited for the understanding of neuromuscular connection and NMD. They do not consider that: i) neural somas and muscles or peripheral glia are physically separated in vivo and have different microenvironment requirements; ii) both sensory and motor neurons can be altered in particular NMD; iii) glial cells are also affected and involved in several neuromuscular pathologies; iv) 2D cultures do not mimic physiological conditions; v) rodent models offer limited benefit translated into clinic research, as they do not carry human genetic background. Later progresses in neuromuscular-mimicking in vitro systems, have been achieved incorporating increasingly evolving technologies, such as 3D cell-culture techniques, human induced pluripotent stem cells (hiPSC) and compartmentalised microfluidic culture systems (cµFCS). The later ones are microfluidic devices for 2D or 3D cell- cultures, with several interconnected compartments, each mimicking different microenvironments or functional units in organ or tissue level. 3D cell culture techniques make cells acquire more in vivo like phenotype and genotype patterns. And finally, hiPSC serve to create study models that mimic the human physiology in both healthy and pathological conditions. This thesis, entitled “Compartmentalised microfluidic culture systems for in vitro modelling of neurological and neuromuscular microenvironments”, aims to study the neuromuscular context in vitro through cµFCS and to create physiologically relevant models. It offers an evolving prospective of in vitro models, moving from mice to human cells, from 2D to 3D cell cultures, from primary cells to hiPSC, and analysing both healthy and diseased cells. Chapter 1 reviews the state of the art in the neuromuscular circuit, amyotrophic lateral sclerosis, and the evolution of in vitro techniques available for their study. Chapter 2 presents the first approach of the neuromuscular in vitro connection model on a chip, showing the relevance of myelin in the peripheral nervous system and in the neuromuscular circuit. Chapter 3 moves to study the proprioception, the differentiation of human neural stem cells to proprioceptive sensory neurons, and their role in ALS. These concepts, together with the ones introduced in Chapter 2, are integrated in Chapter 4, presenting the development of a physiological human neuromuscular circuit on a microfluidic device, that integrates neuromuscular motor and sensory pathways in a 3D cell culture system. Lastly, in a context of neuromuscular vascularisation, Chapter 5 studies the blood-brain barrier and the techniques to monitor its permeability, known to be affected in some NMD such as ALS. This thesis presents the use of several cµFCS for different purposes, incorporating the study of several neuromuscular key role players and obtaining the following results. Myelination induction was successfully incorporated in a designed and fabricated compartmentalised microfluidic culture system (a PDMS device with two compartments connected through microchannels). This system was capable of a simplified mimicking of both peripheral nervous system and neuromuscular afferent or efferent pathways. To move onto human models, first proprioceptive sensory neuron (pSN) differentiation protocol was established. Genetic comparative analysis between healthy and ALS diseased samples revealed differences among pSN related genetic patterns and those involved in the communication between pSN and motoneurons (MN). Human pSN differentiation was combined with skeletal muscle cells to create sensorimotor units in a two-compartment commercial microfluidic device, showing for the first time the formation of synaptic bouton like structures in the contact points of an annulospiral wrapping. Then, a human neuromuscular circuit model was created, integrating for the first time human motor and sensory pathways in 3D cultures in tailored microfluidic devices. Finally, the blood-brain barrier was studied as an example of neural vascularisation, within the framework of potentially affected components in NMD. To that end, a new technology for the in vitro monitorisation of blood-brain barrier permeability was created and implemented in a device previously developed in the lab. This system could easily be translated for blood-spinal cord barrier studies. This thesis gathers many technological innovations from a Bioengineering point of view, paving the way for future studies in the neuromuscular field. It shows that the integration of the entire neuromuscular circuit components in the developed in vitro systems provides a wider view of the neuromuscular physiology and the pathological processes. These results show first steps towards future 3D physiological neuromuscular circuit models on a chip for NMD studies., [spa] El movimiento de las fibras musculoesqueléticas está generado por el circuito locomotor. Los fallos en cualquier parte del circuito pueden originar la aparición de enfermedades neuromusculares (ENM) — como la esclerosis lateral amiotrófica (ELA) — o determinar su gravedad. Los modelos de estudio in vitro convencionales, basados en cocultivos de motoneuronas con células de músculo esquelético, no son representativos de la fisiología humana. Además, el estudio de fragmentos del circuito no puede aportar una visión global y exhaustiva del proceso patológico. Esta tesis tiene como objetivo estudiar el contexto neuromuscular in vitro a través de sistemas microfluídicos de cultivo compartimentado (cµFCS) y crear modelos de estudio fisiológicamente relevantes. Ofrece una perspectiva evolutiva de modelos in vitro, pasando de células murinas a humanas, de cultivos celulares 2D a 3D, de células primarias a células madre humanas pluripotentes inducidas (hiPSC), y analizando tanto células sanas como enfermas. El Capítulo 1 hace una revisión bibliográfica del estado del arte en el circuito neuromuscular, la ELA, y la evolución de las técnicas in vitro disponibles para su estudio. El Capítulo 2 presenta la optimización inicial del modelo de conexión neuromuscular in vitro en un chip, junto con el estudio de la relevancia de la mielina en el sistema nervioso periférico y en el circuito neuromuscular. El Capítulo 3 pasa a estudiar la propiocepción, la diferenciación de células madre neurales humanas a neuronas sensoriales propioceptivas, y su papel en el desarrollo de la ELA. Estos conceptos, junto con los introducidos en el Capítulo 2, se integran en el Capítulo 4, presentando el desarrollo de un circuito neuromuscular humano fisiológico en un dispositivo microfluídico, que integra las vías sensoriales y motoras neuromusculares en un sistema de cultivo celular en 3D. Finalmente, en el contexto de la vascularización neuromuscular, el Capítulo 5 estudia la barrera hematoencefálica y las técnicas para monitorizar su permeabilidad, afectada en ciertas ENM como la ELA. Esta tesis recoge muchas innovaciones tecnológicas desde el punto de vista de la Bioingeniería, abriendo nuevas puertas para futuros estudios en el campo neuromuscular. Muestra que la integración de todos los componentes del circuito neuromuscular en los sistemas in vitro aquí desarrollados aporta una visión más amplia de la fisiología neuromuscular y los procesos patológicos. Estos resultados representan un primer paso hacia futuros modelos de circuitos neuromusculares 3D fisiológicos en un chip para estudios de ENM.

[eng] Intestinal cells self-organize into 3-dimensional (3D) organoids that recapitulate the in vivo structural and functional characteristics when embedded in a 3D cell-derived protein mixture (Matrigel). However, these very same cells self-organize into 2-dimensional (2D) intestinal epithelial monolayers that recapitulate the in vivo-like cell organization when seeded on thin layers of the same cell-derived protein mixture. Moreover, in vivo, the intestine integrates regulation from paracrine signals to establish its characteristic crypt-villus axis self- organization. However, direct experimental manipulation of these paracrine signals, as well as their functional concentrations and effects at the cellular level, has been hampered by limitations of the in vivo and in vitro currently available systems. In general, changes in epithelial cell organization are characterized by a cross-talk between cell-substrate and cell-cell interactions, but the role of the ECM dimensionality, protein composition and spatial distribution in the intestinal epithelial cells’ organization is not fully understood. In this thesis, we show that intestinal epithelial cells self-organize in 2D-monolayers or 3D-tubular networks depending on the Matrigel protein concentration when the dimensionality is fixed. This self-assembles tubular networks have inner apical polarization and are similar to soap foams or de-wetted collagen networks. They have well defined topological and metrical properties and become spontaneously ordered at large length scales. Interestingly, stem cells have a particular dynamic during the formation of each self-organized patterns. On low Matrigel concentration, stem cells present a confined random movement to form a 2D-monolayer. In contrast, on higher Matrigel concentration, stem cells perform a direct motion towards a specific target to form the 3D- tubular networks. By reducing the proportion of stem cells in the culture, the formation of 3D-tubular networks is impaired. Instead, primary cells form aggregates when seeded above the transition protein concentration, similar to two other epithelial cell types (Caco-2 and MDCK cells). On the other hand, the 2D-monolayers formed on low Matrigel concentration contain crypt- and villus-like domains resembling those found in vivo. These compartments are randomly distributed and their shape is not uniform. However, by producing localized micropatterns of immobilized Wnt and ephrin factors on freeze-dried Matrigel-coated substrates by microcontact printing, we can drive the compartmentalization of the intestinal epithelial monolayers by spatially positioning the crypt- and villus-like domains. Finally, by changing the shape and dimension of the patterns we can control the distance between the crypt-like domains as well as their dimensions and shape. Overall, our experiments illustrate how Matrigel concentration regulates intestinal epithelial cell organization as a function of cell-substrate adhesion, and show that primary intestinal epithelial cells self-organize in structures with well-defined sizes and shapes independently of dimensionality or external signaling gradients. Also, we show that the amount of stem cells in the culture regulates the geometry of those self-organized structures. On the other hand, micropatterns of immobilized proteins to the ECM provides accurate control of the crypt-villus domain positioning in our epithelial monolayers. Thus, our work could yield insights about the roles of stem cells and protein concentration in tissue morphogenesis and their influence in the in vivo tissue morphological features such as the dimension of the crypts. In addition, we believe our platform will allow an easy and reliable manner to analyze the effect of relevant proteins on the epithelial cell compartmentalization, as well as the study of important intestinal epithelial processes such as stem cells proliferation, cell migration and differentiation both in homeostasis and pathological processes., [spa] Las células intestinales cultivadas dentro de Matrigel se auto-organizan en organoides tridimensionales (3D) que recapitulan la organización del tejido in vivo. Sin embargo, estas mismas células cultivadas sobre láminas del mismo sustrato se auto-organizan en monocapas bidimensionales (2D) que también recapitulan la organización del tejido in vivo. Además, in vivo, el intestino integra señales paracrinas para establecer su característica auto- organización en criptas y vellosidades. Sin embargo, la manipulación de estás señalizaciones se ha visto obstaculizada por limitaciones en los sistemas in vivo e in vitro actuales. En general, la organización epitelial se caracteriza por interacciones célula-célula y célula-sustrato. Sin embargo, no se acaba de entender el rol de la dimensión, la composición y la distribución de la matriz extracelular (ECM) sobre la organización de las células epiteliales del intestino. En esta tesis, se analiza la organización de células epiteliales en monocapas 2D o redes tubulares 3D en función de la adhesión célula-sustrato. De esta manera, se ilustra la organización en estructuras con tamaños y formas bien definidas independientemente de la dimensionalidad o señalizaciones externas. Además, la proporción de células madre regula la geometría de dichas estructuras. Por otro lado, en contraste con lo que se observa in vivo, los dominios de cripta de las monocapas están desordenados y su forma no es uniforme. Mediante una plataforma que localiza micropatrones de proteínas sobre la ECM, controlamos el posicionamiento de los dominios de cripta-vellosidad. Para concluir, nuestro trabajo proporciona información sobre como influencia la composición y la distribución de la ECM y las células madre en la morfología del tejido in vivo, como la dimensión de las criptas. Además, la plataforma permite analizar el efecto de diferentes proteínas en la compartimentación de las células y en otros procesos epiteliales como proliferación, migración o diferenciación celular, tanto en homeostasis como en proceso patológicos.

[eng] Conventional in vitro cell culture models do not possess the complexity that the native tissues offer. Because of this, the functional properties of the tissues are not properly mimicked, which causes poorly predictive capabilities. Engineered tissues, which combine biofabrication and tissue engineering techniques, try to overcome this gap by providing the cells with an environment similar to the native tissue, recapitulating (I) the physicochemical and mechanical properties of the cellular matrix, (II) the multicellular complexity of the different tissue compartments, and (III) the 3D structures of the tissues. These new engineered models are key factors to improve the platforms for basic research studies, testing new drugs or modelling diseases. Among all the engineered tissues, the intestinal mucosa is not well represented. The intestinal mucosa is formed by the epithelium, which is a multicellular monolayer laying on top of the lamina propria, a connective tissue containing several cell types (mesenchymal cells, immune cells). The gold standard intestinal models are based on epithelial cell lines derived from colon cancer cells grown on the hard porous membranes of the Transwell® inserts. The lack of the intestinal stromal compartment and the growth on a hard surface give high transepithelial electrical resistance and low apparent permeability. Therefore, the development of better in vitro platforms, which integrates both compartments and provides epithelium-lamina propria cell interactions, is highly desirable. In this work, we describe an easy and cost-effective method to engineer a 3D intestinal mucosa model that combines both the epithelium and the lamina propria compartments. To build the 3D scaffolds we chose hydrogels as materials to mimic the physicochemical and mechanical properties of intestinal tissue. Thus, hydrogel co- networks of gelatin methacryolyl (GelMA), a natural polymer, and poly(ethylene glycol) diacrylate (PEGDA), a synthetic polymer, are photopolymerized. On one hand, GelMA provides biodegradation and cell adhesion sequences but it lacks long-term mechanical stability. On the other hand, PEGDA, is non-biodegradable and does not present cell adhesion motifs. Nevertheless, it has good mechanical properties. By this technique, the lamina propria compartment of the intestinal mucosa can be reproduced in vitro. To do that, GelMA and PEGDA polymers are laden with mesenchymal cells (fibroblasts or myofibroblasts) and/or immune cells (macrophages). We demonstrated that GelMA – PEGDA hydrogel co-networks support the growth of these cells and epithelial monolayers on top of the scaffolds. Embedding fibroblasts or myofibroblasts on the hydrogel co-networks enhance the formation and the maturity of the Caco-2 epithelial monolayers, providing barrier properties similar to in vivo. The presence of the stromal cells, also enhances the recovery of the epithelial integrity when the epithelium is temporally damaged. Finally, an immunocompetent model is obtained by the encapsulation of macrophages in the constructs. The presence of macrophages does not influence the formation of the epithelium. However, when the epithelial monolayer is disrupted, the presence of mesenchymal and immune cells in the stromal compartment increases cytokine secretion in a synergistic manner. Our model can successfully mimic the interactions between stromal and epithelial compartments found in vivo intestinal tissue, offering a potential platform to be used to study absorption and toxicity of drugs, as well as cell behaviour under physiological and pathological conditions., [cat] En l’intestí prim trobem la mucosa, la capa més externa de la paret intestinal, formada per dos compartiments, la lamina propia i l’epiteli, en els quals resideixen diferents tipus cel·lulars. La interacció entre els dos compartiments és essencial pel funcionament correcte del intestí. Actualment, els models intestinals in vitro es basen en el cultiu de línies cel·lulars epitelials sobre membranes dures i de plàstic, els quals no representen correctament ni la complexitat ni la organització cel·lular trobada en el intestí prim, ja que manquen de la lamina propria. Conseqüentment, els resultats obtinguts utilitzant aquests models són significativament poc fisiològics i no comparables amb els trobats en condicions in vivo (alts valors de resistència elèctrica transepitelial, subestimació dels valors d’absorció de molècules a través de la ruta paracel·lular i alteració en la expressió enzims digestius). Per reduir les distàncies entre els models intestinals in vitro i l’intestí, s’han de desenvolupar plataformes que modelitzin: (I) les propietats fisicoquímiques i mecàniques de la matriu extracel·lular, (II) els dos compartiments de la mucosa intestinal, i si pogués ser (III) l’arquitectura tridimensional. Per aconseguir aquests requeriments, en aquesta tesi s’utilitzen hidrogels formats per polímers naturals (gelatina metacrilada (GelMA)) co-polimeritzats amb polímers sintètics (poly(ethylene glycol) diacrylate (PEGDA)) com a substrat per imitar els dos compartiments intestinals. Per reproduir la lamina propria, els polímers de GelMA i PEGDA es dissolen juntament amb el fotoiniciador. Tot seguit, les cèl·lules residents de la lamina propria (fibroblasts, miofibroblasts o macròfags) es barregen amb la solució de pre-polimer, s’aboca a les piscines de PDMS, s’exposa a llum ultraviolada, i s’obté l’hidrogel amb les cèl·lules en el seu interior. Finalment, les cèl·lules epitelials es sembren a la superfície del hidrogel. En aquesta tesi, s’ha obtingut un hidrogel amb propietats fisicoquímiques i mecàniques similars a la matriu extracel·lular del intestí humà, i que permeten el cultiu cel·lular fins a 21 dies, imitant els dos compartiments. A més, la el co-cultiu de cèl·lules residents de la lamina propria juntament amb les cèl·lules epitelials, ens ha permès obtenir un model 3D de la mucosa intestinal in vitro amb propietats fisiològiques més semblants a la del intestí prim.

[eng] The precise delivery of therapeutic agents to their specific site of action is a big challenge in cancer treatment, which would enhance the efficacy and reduce the side effects of drugs. In this framework, nanotechnology can greatly contribute to the development of novel drug delivery systems. Nowadays, a large number of nanoparticles, differing in chemical nature, have been synthesized and evaluated for their therapeutic performances. However, most of the newly developed delivery systems are ineffective in the clinic because they don’t reach the specific cancer cells. One of the main reasons of this failure is the lack of knowledge about the interactions between the designed nanosystems and the biological media before getting to the targeted cells, the so-called nanobiointeractions. In particular, undesired interactions with blood proteins and other molecules in vasculature are often responsible for the poor performance of nanocarriers. Further research about the biological interactions occurring in the blood vessels is needed in order to design novel and improved therapeutic nanoparticles. We believe that the understanding of these critical steps together with an in-depth study of the structural composition of nanoparticles will guide a rational design of systems, increasing their applicability and performance in the clinic. To accomplish a comprehensive study, in this thesis, we propose the use of advanced optical microscopy techniques to investigate the chemical and biological identity of nanomaterials and to understand their role with a nanometric precision. One of the first biological barriers nanoparticles encounter when introduced intravenously to the body are proteins which travel through the blood stream. These molecules form the so-called protein corona: a shell of proteins attached to the surface of the nanoparticle. One of the main drawbacks caused by protein corona formation is the hindering of the nanoparticle’s surface, reducing the specific interactions of nanosystems with the cancer cells they are targeting. Protein corona formation is mainly studied using ensemble techniques which give only an approximate idea of the molecules interacting with the surface of the nanoparticles. In chapter two, STORM imaging of corona is presented as a new methodology to obtain an in situ characterization of protein corona on individual nanoparticles. This study reveals a high interparticle heterogeneity regarding the number of proteins per nanoparticle, which may be one of the causes of their poor clinical performance. Protein interactions are not only responsible of reducing specific interactions but can dramatically affect the stability of nanosized delivery systems. Therefore, it is important to study their stability in the blood complex environment. Polyplexes are nanocarriers characterized by the electrostatic interactions between the carrier and the nucleic acid. These systems need to be fully complexed during their circulation in the blood vessels in order to protect their cargo from degradation. Up to now, the challenges in characterizing the molecular distribution of the individual components have limited the rational design of nanosystems. In the third chapter, dSTORM imaging is used to visualize the exact molecular composition of polyplexes. dSTORM imaging unveiled the differences in the stoichiometry of individual systems, revealing a heterogeneity inside the same population. Once the system is fully characterized, his complexation can be followed under different blood-like conditions thanks to the molecular resolution of the technique. This new method allows to determine the real molecular stability of the system in contact with serum proteins and provides mechanistic insights into the disassembly process. The stability in complex biological media is a determining factor of the good performance of drug delivery systems, especially in the use of supramolecular structures, due to their dynamic nature. Therefore, it is necessary to understand the behavior of self-assembled nanoparticles in conditions close to the ones they would confront in vivo. Serum proteins can prematurely disassemble the system, as seen in the previous chapter, and lead to non-selective release of the cargo in healthy tissues. Another critical issue of self-assembling systems is the strong dilution they undergo when injected in blood, which may severely affect the supramolecular stability. Hence, it is of crucial importance to investigate the effect of dilution in biologically relevant media. These issues are often overlooked in the literature, most likely due to the difficulties of studying supramolecular assemblies in complex biological media. In chapter four, micelles that change their fluorescent properties upon disassembly are characterized under different blood- like conditions using a combination of fluorescence spectroscopy and microscopy techniques, allowing to predict the system with the best properties. A last critical step nanoparticles face when injected into the blood vessels is the flow, which may also affect the stability of the system. Moreover, their efficiency is directly proportional to the ability of extravasation from the blood vessel across the tight endothelial layer before reaching the cancer cells. In each of these barriers, the stability of supramolecular systems may be compromised. In chapter five a microfluidic chip mimicking the vascular tumor microenvironment is optimized to study the ability and stability of supramolecular structures during extravasation. A monolayer of human umbilical vein endothelial cells are grown in the microfluidic device forming a blood-vessel-like channel. Moreover, the chip contains a second channel of tumorigenic cells to test the stability of the nanocarrier in the extracellular matrix close to cancer cells after extravasation. This device allows to screen the behavior of the different delivery systems and predicts the most stable and promising system thus optimizing and reducing the pre-clinical and clinical testing., [cat] La nanomedicina pot contribuir en la millora dels tractaments de càncer gràcies al ús de nanopartícules per el lliurament controlat de fàrmacs. En els últims anys s’ha dut a terme un gran nombre d’investigacions però fins ara la majoria de nanopartícules investigades són ineficients en pacients ja que no arriben a les cèl·lules específiques del càncer. Una de les raons principals d’aquest fracàs és la manca de coneixement sobre les interaccions entre les nanopartícules i els medis biològics amb els quals es troben dins el cos. En particular, les interaccions no desitjades amb les proteïnes de la sang i altres molècules en els vasos sanguínies són sovint responsables del seu mal funcionament. La comprensió d’aquests passos crítics juntament amb un estudi en profunditat de la composició estructural de les nanopartícules guiarà un disseny racional dels sistemes, augmentant la seva traducció a les clíniques. Actualment, una de les limitacions principals d’aquestes investigacions és la manca de tècniques experimentals capaces de proporcionar informació del comportament de les nanopartícules a escala nano, així com, que es puguin realitzar en medis biològics complexes. L'objectiu principal d'aquesta tesi és l'ús de noves tècniques de microscòpia òptica avançades per donar a conèixer les interaccions biològiques de les nanopartícules en medis biològics complexos. Per a aquest propòsit, el capítol 2, descriu l'ús de la imatge dSTORM com a nova metodologia per obtenir una caracterització in situ de la corona proteica. En conjunt, revela una alta heterogeneïtat pel que fa al nombre de proteïnes per nanopartícula, que pot ser una de les causes del seu mal rendiment clínic. En el capítol 3, la tècnica dSTORM s'utilitza per visualitzar la composició molecular exacta de poliplexes. Una vegada caracteritzats aquests sistemes es pot seguir la seva complexació en sèrum per estudiar la seva estabilitat. En el capítol 4, micel·les, que canvien la seva fluorescència en funció de si estan intactes o no, es caracteritzen en diferents condicions similars a la sang mitjançant una combinació d’espectroscòpia de fluorescència i tècniques de microscòpia espectral. En aquest capítol es mostra una relació entre l'estabilitat de les micel·les, i la seva internalització cel·lular. Finalment, el capítol 5 té com a objectiu desenvolupar un xip microfluídic imitant els vasos sanguinis tumorals per estudiar la capacitat i l'estabilitat de les nanopartícules durant l'extravasació mitjançant tècniques de microscòpia espectral.

[eng] Despite the last 60 years have seen major advances in many scientific and technological inputs of drug Research and Development, the number of new molecules hitting the market per billion US dollars of R&D spending has been declined steadily during the same period. The current scenario highlights the need for new research tools to enable reduce costly animal and clinical trials while providing a better prediction about drug efficacy and security in humans A recent emerging approach to improve the current models is emerging from the field of microfluidics, which studies systems that process or manipulate tiny amounts of fluids using channels with dimensions of tens to hundreds of micrometers. Combining microfluidics with cell culture, scientists gave rise to a new field named “Organ-on-chip” (OOC). Microfluidic OOCs are advanced platforms designed to mimic physiological structures and continuous flow conditions, thus allowing the culture of cells in a friendlier microenvironment. This thesis, titled “Cell culture interfaces for different organ-on-chip applications: from photolithography to rapid-prototyping techniques with sensor embedding”, aims to design, simulate and test new OOC devices to reproduce cell culture interface under flow conditions. The work has a focus on the exploration of novel fabrication techniques which enable rapid prototyping of OOC devices, reducing costs, time and human labor associated to the fabrication process. The final objective is to demonstrate the viability of the devices as research tools for biological problems, applying them to the tubular kidney and the blood brain barrier (BBB). To achieve the objective, at least three device version have been developed: 1) OOCv1, fabricated by multilayer PDMS soft lithography; 2) OOCv2, fabricated in thermoplastic by layered object manufacturing using both a vinyl cutter and a laser cutter, integrating standard fluidic connectors alone (OOCv2.1) or together with embedded electrodes (OOCv2.2); 3) OOCv3 using a mixed technique of laser cut and 3D printing by stereolithography. All devices are fabricated using biocompatible materials with high optical quality and an embedded commercial membrane. The biological experiments with renal tubular epithelial cells, realized on OOCv1 and OOCv2.1 devices, demonstrated the viability of the device for culturing cells under flow conditions. The study realized on fatty acid oxidation and accumulation in cells exposed to physiological and diabetogenic oscillating levels of glucose suggest a possible positive role of shear stress in activation of fatty acid metabolism. The studies were performed using a compact experimental unit with embedded flow control which reduce significatively the complexity and cost of the fluidic experimental setup. The biological experiments on the BBB confirmed viability of OOCv2.1 and OOCv2.2 for compartmentalized co-culturing of endothelial cells and pericytes. The formation and recovery of the barrier after disruptive treatment has been assessed using different techniques, including immunostaining, fluorescence and live phase contrast imaging, and electrical impedance spectroscopy. The repeatability of measurements using electrodes was verified. A model to classify measurements from different timepoints has been developed, resulting in accuracy of 100% in learning and 90% in testing case. Results are confirmed by imaging data, which also suggest a critical role of pericytes in the development, maintenance, and regulation of BBB, in accordance with the literature., [spa] En los últimos años está emergiendo una nueva propuesta para mejorar los modelos actuales en el estudio de nuevos fármacos. Mediante la fusión de cultivos celulares y microfluídica ha nacido un nuevo campo de aplicación denominado “Órgano-en-un-chip” (OOC), donde se recrea un entorno fisiológico capaz de reproducir unidades funcionales mínimas de diversos órganos del cuerpo humano. Un elemento importante para el desarrollo de dispositivos OOC es la reproducción de zonas de interacción entre varios tejidos formados por diferentes tipos celulares. Esta tesis, titulada “Interfaces de cultivo celular para diferentes aplicaciones de OOC: desde fotolitografía a técnicas de prototipado rápido con inclusión de sensores”, tiene como objetivo el diseño, simulación y evaluación de dispositivos OOC capaces de reproducir superficies de contacto de tejidos contiguos expuestos a flujo. El trabajo está enfocado a la exploración de nuevas técnicas de fabricación que permitan el prototipado rápido de dispositivos OOC, reduciendo costes, tiempo y mano de obra asociada a dicha fabricación. El objetivo final es demostrar la utilidad de los dispositivos como herramientas de investigación para problemas biológicos, aplicándolos en esta tesis al estudio del túbulo renal y de la barrera hematoencefálica. Para ello se han fabricado tres versiones de dispositivos: 1) OOCv1 fabricado por litografía suave en múltiples capas de PDMS; 2) OOCv2 fabricado con cortadora de vinilo y cortadora láser en múltiples capas de materiales termoplásticos y con electrodos integrados en la versión OOCv2.2; 3) OOCv3 fabricado mediante impresión 3D por esterolitografía. Todos los dispositivos están hechos de materiales biocompatibles de alta calidad óptica, con conectores fluídicos y una membrana comercial integrada. Los experimentos biológicos sobre túbulo renal, realizados en los dispositivos OOCv1 y OOCv2, han demostrado la viabilidad de los dispositivos, integrados con un sistema de flujo, para estudios de la metabolización de ácidos grasos en el riñón relacionados con condiciones diabetogénicas. Los experimentos biológicos sobre la barrera hematoencefálica han confirmado la viabilidad de OOCv2 para el cocultivo compartimentado de células endoteliales de cerebro y pericitos. La integración de electrodos en el OOCv2.2 ha demostrado ser una técnica fiable para la medición de la integridad de barreras biológicas de modo no-invasivo, libre de etiqueta (“label-free”), y a tiempo real gracias a la espectroscopía de impedancia.

[eng] Intestinal epithelium is highly specialized tissue organized into crypt-villus units relevant for their effective barrier function and nutrient absorption. In the crypt units reside the proliferative intestinal stem cells (ISCs) that divide and differentiate while migrating along the villi to generate the epithelium. The proliferation, migration and differentiation of ISCs is governed by the tightly controlled spatio-chemical gradients of ISC niche factors; bone morphogenic protein (BMP), wingless/Int (Wnt) and epidermal growth factor (EGF) pathway modulators. In vitro models of the intestinal epithelium, for the most part, based on culturing of intestinal stem cells/crypts in 3D cultures forming structures called organoids. These structures faithfully recapture diverse cell populations and their multicellular organization of native intestinal epithelium. However, 3D closed geometry of intestinal organoids prevents access to the apical region of the epithelium, making them unsuitable for conventional functionality assays. Experimental modeling of intestinal epithelial biology and physiology are limited due to the lack in vitro platforms that recapitulate these key aspects of the small intestinal epithelium: its distinct cell populations, 3D architecture and the gradients of ISC niche biochemical factors along the crypt-villus axis. Here, we describe development of in vitro models of intestinal epithelium obtained from intestinal organoid-derived crypts. First, we present a method that takes the advantage of substrate stiffness to dictate the formation of monolayers with accessible lumen rather than 3D organoids with a closed geometry. The 2D intestinal epithelium model has in vivo-like crypt-villus cellular organization with all major epithelial cell types and show physiologically relevant tissue barrier function. Then, we describe the development of a more complex model of intestinal epithelium by incorporating a 3D villus-like basement membrane substitute fabricated on hydrogels. For that, poly(ethylene glycol) diacrylate (PEGDA) hydrogels are chosen due to their highly tunable chemical, and mechanical properties, porosity and photocrosslinkable nature allowing easy microstructuring. The formation of 3D bullet-like complex shapes was achieved by photolithography-based crosslinking of PEGDA, a simple, cost-effective approach. The bioactive functionalization of otherwise inert PEGDA for cell adhesion, was achieved by copolymerizing it with acrylic acid and a variety of cell adhesion proteins can be covalently anchored to the 3D villus-like hydrogels. We establish the optimal conditions for the growth of intestinal organoid-derived epithelial monolayers and demonstrated that organoid-derived intestinal epithelial cells successfully formed epithelial monolayers on collagen type I functionalized 3D villus-like PEGDA-acrylic acid hydrogels. Finally, we describe methods to create spatiotemporal gradients of biochemical ISC niche factors on 3D villus-like hydrogels and demonstrate that these gradients can be used to compartmentalize the differentiated epithelial cells. The spatio-chemical gradients of ISC niche biochemical factors on PEGDA hydrogels with proper porosity were successfully generated based on the free diffusion of the factors from a source to a sink chamber in a custom-made microfluidic device allocating the hydrogel and visualized with light-sheet fluorescence microscopy. In silico models were developed to simulate the spatio-chemical gradients formed within the hydrogels. The 3D villus-like PEGDA hydrogels were fabricated on porous membranes and successfully adapted to Transwell® inserts that permitted access to both sides of the hydrogel and the generation of spatio-chemical gradients. The gradients generated in this fashion can be used to compartmentalize the differentiated epithelial cells more towards the tips of the villus-like microstructures. The 3D villus-like platform improves the current models in providing cells with physiologically representative topographical and mechanical cues and biochemical gradients. Due to its utility, this platform might find uncountable applications. It can be used for the understanding of the basic biology of the intestinal epithelium. In addition, it can be used to culture human intestinal stem cells allowing for the screening of novel therapies and disease modeling., [spa] El epitelio intestinal es un tejido altamente especializado, organizado en unidades de criptas y vellosidades que son relevantes para sus eficaces funciones de barrera y absorción de nutrientes. En las unidades de criptas residen las células madre intestinales (ISC) proliferativas que se dividen y diferencian mientras migran a lo largo de las vellosidades, las cuales generan el epitelio maduro. En el epitelio maduro, las ISC y las células proliferativas se localizan en las criptas y las células absorbentes y secretoras diferenciadas en las vellosidades. La proliferación, migración y diferenciación de las ISC se rigen por los gradientes químicos espaciales altamente controlados de los factores de nicho de la ISC; Moduladores de la vía de bone morphogenic protein (BMP), wingless/Int (Wnt) y epidermal growth factor (EGF). El modelado experimental de la biología y la fisiología del epitelio intestinal está limitado debido a la falta de plataformas in vitro que recapitulan estos aspectos clave del epitelio del intestino delgado: sus distintas poblaciones celulares, la arquitectura 3D y los gradientes de factores bioquímicos de nicho ISC a lo largo del eje cripta-vellosidad. Aquí, describimos el desarrollo de modelos in vitro de epitelio intestinal obtenidos de criptas derivadas de organoides intestinales. En primer lugar, presentamos un método para obtener monocapas epiteliales intestinales 2D con lumen accesible y función de barrera fisiológica. A continuación, describimos el desarrollo de andamios biomiméticos 3D similares a vellosidades en hidrogeles de diacrilato de polietilenglicol (PEGDA) utilizando un enfoque fotolitográfico simple y rentable. Demostramos que nuestra plataforma de vellosidades sintéticas apoya la formación de monocapas epiteliales de células epiteliales intestinales derivadas de organoides. Finalmente, describimos métodos para crear gradientes espaciotemporales de factores nicho bioquímicos ISC en hidrogeles 3D similares a vellosidades y demostramos que estos gradientes se pueden usar para compartimentar las células epiteliales diferenciadas. La plataforma 3D que recrea las vellosidades intestinalesmejora los modelos actuales al proporcionar a las células las señales topográficas y mecánicas y los gradientes bioquímicos fisiológicamente representativos. Debido a su utilidad, esta plataforma puede encontrar innumerables aplicaciones. Puede ser utilizada para la comprensión de la biología básica del epitelio intestinal. Además, se puede utilizar para cultivar células madre intestinales humanas que permitan la detección de nuevas terapias y el modelado de enfermedades.

[eng] Scarce supply of clean water and rising water pollution are key global challenges for water sustainability. Much of the wastewater generated by human agricultural and industrial activity is left untreated. Nanotechnological materials and systems have emerged as new tools for improving the efficiency of water treatment. Among those, self-propelled micromotors have shown several advantageous characteristics. Micromotors are autonomously propelled systems which either use chemical energy present in their environment or are propelled via external force fields. Diverse designs, materials composition and mechanisms of propulsion are reported for micromotors found in the literature. Among them, bubble-propelled micromotors, which move due to the generation and release of gas bubbles from their surface, are the main type of motors used for water remediation applications. In addition to the motion in fluids, the bubbles generated by the motors, also contribute with additional mixing of the fluid and enhance the mass transfer between active material and pollutant at the microscale. Additionally, the structure of micromotors can be modified to target a wide variety of pollutants, almost on demand. The micromotors that we synthesized during the research work for this thesis can remove organic and heavy metal pollutants, as well as exhibit bactericidal activity. We studied Iron/Platinum (Fe/Pt) micromotors for their reusability, effect of sizes, swimming behaviors and catalytic properties. These micromotors were fabricated by spontaneous roll-up of iron and platinum nanomembranes, deposited on the pre-fabricated patterns of a photoresist substrate. The iron layer present as the outer surface of these micromotors can degrade organic pollutants via Fenton-like reaction and the inner platinum layer acts as the engine decomposing hydrogen peroxide to oxygen for bubble propulsion. We observed that Fe/Pt micromotors can swim continuously for hours, and can be stored for weeks before reuse, without sacrificing much of their activity. The results suggested that Fe/Pt micromotors act as a heterogeneous catalyst due to in situ generated iron oxide species on the surface, without leaching high concentration of iron in the media. We developed graphene oxide-based micromotors (GOx-micromotors) for heavy metal removal, consisting of nanosized multilayers of graphene oxide, nickel, and platinum. These micromotors can capture, transfer, and remove heavy metals (i.e. lead) from contaminated water. GOx-micromotors are synthesized by electrodepositions of electro-reducible graphene oxide, nickel and platinum layers in the polycarbonate porous templates. The outer layer of graphene oxide captures lead on their surface, and the inner layer of platinum provides self-propulsion in hydrogen peroxide, while the middle layer of nickel enables external magnetic control of the micromotors. We observed that the mobile GOx-micromotors can remove lead 10 times more efficiently than non-motile GOx-micromotors, cleaning water from 1000 ppb down to below 50 ppb. We have demonstrated control of their motion and directionality in a proof of concept microfluidic system. Silver nanoparticles (AgNPs) decorated Magnesium Janus micromotors were designed for disinfection and remove of Escherichia coli (E. coli) bacteria from contaminated water. Magnesium present in the micromotors functions as both, the template for the spherical shape and propulsion source by producing hydrogen bubbles while in contact with water. The inner layer of iron provides functionality for the magnetic remote guidance, and an outer AgNP coated gold layer facilitates adhesion of bacteria and gives bactericidal properties to the micromotors. We observed that the AgNPs-coated Au cap of the micromotors shows dual capabilities, capturing bacteria and killing them. In our efforts to develop multifunctional micromotors and scalable synthesis methods, we developed two types of micromotors. (i) Mesoporous silica-based micromotors with manganese dioxide (MnO2) layer on the inner surface and coated with γ-Fe2O3 nanoparticles (FeSiMnOx micromotors). These micromotors can remove both organic and heavy metal pollutants, and they are synthesized using only template-assisted chemical methods. (ii) Cobalt ferrite micromotors (CFO micromotors) synthesized by template-free chemical synthesis approach. They are made up of aggregated cobalt ferrite nanoparticles, which act as the catalyst for propulsion and for Fenton-like reactions. We qualitatively measured the generation of hydroxyl radicals by CFO micromotors and studied the effect of surfactants on the degradation efficiency of CFO micromotors. We hope that such approach of synthesizing micromotors via relatively facile methods will push the use of micromotors towards commercially practical solutions for water treatment. Overall, our results show that the multifunctional self-propelled micromotors have potential to become an effective tool for water remediation in the near future., [spa] El escaso suministro de agua limpia y el aumento de la contaminación del agua son desafíos globales clave para la sostenibilidad del agua, sobre todo teniendo en cuenta que gran parte del agua residual generada por la actividad agrícola e industrial humana no se trata. Los materiales y sistemas nanotecnológicos han surgido como nuevas herramientas para mejorar la eficiencia del tratamiento de aguas. Entre ellos, los micromotores autopropulsados han mostrado varias características ventajosas. Los micromotores son sistemas de propulsión autónoma que utilizan energía química presente en su entorno o pueden también ser propulsadas a través de campos de fuerzas aplicadas externamente. Varios diseños, composición de materiales y mecanismos de propulsión se han sido reportados en el campo de los micromotores. Entre ellos, principalmente los micromotores propulsados por burbujas, los cuales se mueven debido a la generación y liberación de burbujas de gas de su superficie, se utilizan como una herramienta para aplicaciones de remediación de aguas. Esto se debe a la eficacia añadida de la transferencia de masa a la microescala, que se origina a partir de su movimiento y el movimiento de las burbujas liberadas. Además, la estructura de los micromotores se puede modificar para dirigirse a una amplia variedad de contaminantes, según los requerimientos. Los micromotores que sintetizamos durante el trabajo de investigación para esta tesis pueden eliminar contaminantes orgánicos y metales pesados, así como exhibir actividad anti bactericida. Estudiamos micromotores de hierro / platino (Fe / Pt) por su reutilización, efecto de tamaños, su comportamiento durante su movimiento y propiedades catalíticas. Estos micromotores se fabricaron mediante enrollamiento espontáneo de nanomembranas de hierro y platino, depositadas en los patrones prefabricados definidos en una capa sacrificial fotorresistente. La capa de hierro presente como superficie externa de estos micromotores puede degradar los contaminantes orgánicos a través de la reacción tipo Fenton y la capa interna de platino actúa como el motor, siendo el catalizador que descompone el peróxido de hidrógeno en oxígeno para generar una propulsión por burbujas. Observamos que los micromotores Fe / Pt pueden nadar continuamente durante horas y pueden almacenarse durante semanas antes de volver a ser usados, sin que esto repercuta de manera significativa en su actividad. Los resultados de nuestros experimentos sobre el análisis de superficie de micromotores, estudio de nanoindentación y liberación de hierro sugirieron que los micromotores Fe / Pt actúan como un catalizador heterogéneo debido a las especies de óxido de hierro generadas in situ en la superficie, sin lixiviación de alta concentración de hierro en los medios. Desarrollamos micromotores basados en óxido de grafeno (micromotores GOx) para la eliminación de metales pesados que consisten en multicapas nanométricas de óxido de grafeno, níquel y platino. Estos micromotores pueden capturar, transferir y eliminar metales pesados (es decir, plomo) del agua contaminada. Los micromotores GOx se sintetizan mediante electrodeposiciones de capas de óxido de grafeno, níquel y platino, los cuales son electroreducidos en la parte interior de membranas de policarbonato porosas. La capa externa de óxido de grafeno captura el plomo en su superficie, y la capa interna de platino proporciona autopropulsión en presencia de peróxido de hidrógeno, mientras que la capa intermedia de níquel permite el control magnético externo de los micromotores. Observamos que los micromotores móviles GOx pueden eliminar el plomo hasta 10 veces más que los micromotores GOx no móviles (Figura 1B), limpiando el plomo en agua de 1000 ppb a menos de 50 ppb en menos de 60 min. Hemos demostrado el control de su movimiento y direccionalidad en un sistema microfluídico como prueba de concepto. Diseñamos también micromotores tipo Janus decorados con nanopartículas de plata (AgNP) para la desinfección y eliminación de la bacteria Escherichia coli (E. coli) en agua contaminada. Los micromotores Janus se sintetizaron recubriendo un lado de una micro-partícula de magnesio con capas de hierro y oro, las cuales posteriormente se funcionalizaron con AgNP. El magnesio presente en los micromotores funciona no sólo como estructura principal para conseguir una forma esférica, sino también como fuente de propulsión mediante la producción de burbujas de hidrógeno al entrar en contacto con el agua. La capa interna de hierro proporciona la funcionalidad requerida para el posterior control magnético externo, mientras que la capa de oro externa decorada con AgNPs promueve la adhesión de bacterias y dota de propiedades bactericidas a los micromotores. En nuestro esfuerzo por desarrollar micromotores multifuncionales y métodos de síntesis escalables, desarrollamos dos tipos de micromotores. (i) Micromotores mesoporosos basados en sílice con una capa de dióxido de manganeso (MnO2) en la superficie interna y recubiertos con nanopartículas γ-Fe2O3 (micromotores FeSiMnOx). Estos micromotores pueden eliminar contaminantes orgánicos y metales pesados, y se sintetizan utilizando solo métodos químicos asistidos por un molde (por ejemplo, una membrana porosa). (ii) Los micromotores de ferrita de cobalto (micromotores CFO) fueron sintetizados sin necesidad de utilizar ningún molde. Están formados por nanopartículas de ferrita de cobalto agregadas, que actúan como catalizadores para la propulsión y para reacciones tipo Fenton. Medimos cualitativamente la generación de radicales hidroxilos por micromotores CFO y estudiamos el efecto de los tensioactivos sobre la eficiencia de degradación de los micromotores CFO. Esperamos que la síntesis de micromotores a través de métodos relativamente fáciles empuje la implementación de micromotores en soluciones comercialmente prácticas para el tratamiento del agua. En general, nuestros resultados muestran que los micromotores autopropulsados multifuncionales tienen el potencial de convertirse en una herramienta efectiva para la limpieza de aguas en el futuro.

[spa] El objetivo general de esta tesis doctoral fue el desarrollo de biosensores basados en aptámeros para la detección de bacterias enteropatógenas. Para ello se estudiaron diferentes estrategias de detección gracias a las propiedades únicas de los aptámeros, tales como su capacidad de adoptar diferentes estructuras conformacionales tras el enlace con la molécula diana, su estabilidad y la facilidad con que se pueden modificar químicamente, introduciendo marcajes o ciertos grupos funcionales. Las bacterias utilizadas en todas las estrategias de detección fueron: Salmonella typhimurium (diana del aptámero seleccionado, específicamente las proteinas transportador ABC, OmpA y precursor OmpD), Eschericia coli O157 Shiga, Shigella sonnei, Escherichia coli k5, Proteus mirabilis, Bacillus cereus y Kocuria lutea. En la primera fase de la tesis se realizó la caracterización de la bacteria Salmonella typhimurium y de la afinidad del aptámero por este microorganismo. La afinidad del aptámero por la bacteria se comprobó utilizando las técnicas de impresión por microcontacto (μCP), la microscopía de fuerza atómica (AFM) y la microscopía de fluorescencia. Posteriormente en la fase siguiente se desarrolló un sistema de detección basado en partículas magnéticas como plataforma de captura, preconcentración y detección basada en un ensayo competitivo indirecto. Se llevó a cabo la detección colorimétrica, por absorbancia, y electroquímica, por voltametría de pulso diferencial (DPV). En ambas alternativas de detección se observó reactividad cruzada de la bacteria Salmonella typhimurium con E. coli O157 Shiga y con Shigella sonnei, no presentándose dicha reactividad con las otras bacterias. El porcentaje de similitud de las proteinas diana análogas fue mayor en relación a las proteinas correspondientes a E. coli O157 Shiga y Shigella sonnei, y menor respecto a las proteinas análogas de las otras bacterias. En la tercera fase de la tesis se desarrollaron dos biosensores electroquímicos. Uno basado en la detección directa del enlace con la bacteria por espectroscopía de impedancia electroquímica (EIS). El otro biosensor se basó en la inhibición enzimática causada por el cambio conformacional del aptámero tras su enlace con la bacteria. En este caso se utilizó la técnica electroquímica DPV para medir la actividad de la enzima fosfatasa alcalina. Con el primero se lograron los mejores resultados, por cuanto los parámetros de comportamientos fueron los mejores (menor LOD, mayor sensibilidad, menor tiempo de análisis), pero además es una estrategia en la cual la construcción del aptasensor es simple puesto que consta de un solo paso. Finalmente los ensayos de selectividad llevados a cabo en todas las estrategias de detección muestran que todos los sistemas de detección fueron capaces de discriminar el grupo de bacterias clasificadas como enteropatógenos (Salmonella typhimurium, Escherichia coli O157 Shiga y Shigella sonnei) del resto de bacterias (Escherichia coli k5, Proteus mirabilis, Bacillus cereus y Kocuria lutea), demostrando que la afinidad del aptámero por Salmonella typhimurium no es específica para esta bacteria, sino que también existe una afinidad equivalente por E. coli O157 Shiga y Shigella sonnei., [eng] The overall objective of this Thesis was to develop different biosensors based on aptamers to detect enteropathogen bacteria. This objective was accomplished through the study of different detection strategies which were proposed based on the unique properties of aptamers such as the capacity to present different conformational structures after linking to the target molecule, stability and ease of chemical functionalization. The affinity of the aptamer against the Salmonella typhimurium was confirmed by Microcontact Printing (μCP), Atomic Force Microscopy (AFM) and Fluorescence Microscopy. Then, a magnetic particle detection system was developed as a capture, pre-concentration and detection platform based on an indirect competitive test. Colorimetric and electrochemical detection, using differential pulse voltammetry (DPV) were performed. In both detection alternatives, cross-reactivity of Salmonella typhimurium with Escherichia coli O157 Shiga and Shigella sonnei was observed, with no cross- reactivity with Proteus mirabilis, Bacillus cereus, Kocuria lutea and Escherichia coli k5. In the next phase of the thesis, two electrochemical biosensors were developed, one based on the direct detection of the binding to the bacterium by electrochemical impedance spectroscopy (EIS) and the other based on the enzymatic inhibition caused by the conformational change of the aptamer after binding with the bacterium. In the second case the DPV technique was used to measure the activity of alkaline phosphatase. The best results were obtained with the impedance biosensor because as it showed improved behavior parameters (lower LOD, higher sensitivity and shorter analysis time). In addition, the construction of the aptasensor is simple as it consists of a single step. Selectivity tests carried out on all detection strategies showed that the developed detection systems were able to discriminate the bacterial group classified as enteropathogens (Salmonella typhimurium, Escherichia coli O157 Shiga and Shigella sonnei) from other bacteria (Escherichia coli K5, Proteus mirabilis, Bacillus cereus and Kocuria lutea). Hence, the affinity of the aptamer for Salmonella typhimurium is not specific for this bacterium and there is also an equivalent affinity for E. coli O157 Shiga and Shigella sonnei.

[eng] Most of the current in vitro cell culture models do not reproduce the anatomy of tissues and physiological behavior of cells in vivo and provide misleading results when compared to the real tissue. Microfabrication technologies can be used to go one step beyond the conventional 2D in vitro tissue culture models and reliably reproduce in vivo tissue microenvironments.1 For the small intestinal epithelium, this approach has been explored through the fabrication of collagen and poly (lactic-co-glycolic acid) villi-like scaffolds, using sequential micromolding.2–4 Although that has been an innovative and interesting approach, the nature of the employed materials does not allow fine-tuning of the system properties and the molding method is laborious, requiring several intermediate molding steps. Here we describe a simple and cost-effective method to fabricate soft 3D villi-like microstructures with the anatomical architecture. We use poly (ethylene glycol) diacrylated (PEGDA) and acrylic acid that copolymerize and form soft hydrogels upon crosslinking through UV photolithography. And we demonstrate that our technology allows producing functional monolayers of polarized Caco-2 epithelial cells. Hydrogels are networked materials with high water content, which allow easy diffusion of soluble factors and oxygen.5,6 PEG-based hydrogels possess highly tunable chemical and mechanical properties and have become trendy materials to mimic and are the extracellular matrix and tissue basement membranes.7,8 To get 3D villi-like microstructures, 2D photomasks were used in a UV light-dependent polymerization process. During this process, several variable factors, such as UV exposure time, photoinitiator concentration, and PEGDA molecular weight and concentration, were studied to understand the mechanisms that allow the formation of high aspect ratio soft microstructures. Then, these variables were adjusted to control the microstructure dimensions and to obtain villi-like microstructures resembling the in vivo villi dimensions, physical and mechanical properties. To allow cell adhesion and growth, we copolymerize PEGDA (non-bioactive) with acrylic acid and we took advantage of the exposed carboxylic groups of the acrylic acid to covalently incorporate laminin through the EDC/NHS coupling reaction. We have stablished the ratio between PEGDA and acrylic acid to obtain on the one hand, a successful copolymerization and villi-like structures formation and, on the other a proper protein functionalization of the hydrogel. Our results show that the amount of covalent bound protein to the hydrogel depends on the amount of acrylic acid employed which offers a way to control ligand density on the hydrogel, independently on the mechanical properties Functionalized villi-like microstructured hydrogels have proven suitable for cell growth. First, cell adhesion on the hydrogels was tested by culturing fibroblasts. Then, MDCK cells were used to determine the suitability of the microstructured hydrogels of growing epithelial monolayers. Finally, we used Caco-2 cells (widely used in intestinal barrier models)9 to validate the functionality of our construct. Caco-2 cells were seeded on the villi-like hydrogel and maintained for 21 days. Our results show that Caco-2 cells were able to adhere and divide, lining the villi-like microstructures and forming a monolayer of polarized epithelial cells, as shown by the expression of actin and villin in the apical part of the monolayer and the shape, position and orientation of cell nuclei. We have also been able to successfully transfer the hydrogel platform to polycarbonate membranes and Transwell® inserts. With this technology, we are currently working on performing paracellular and transcellular permeability tests to evaluate the performance of our system in comparison with standard Caco-2 monolayers. Furthermore, we have evaluated the transepithelial electrical resistance (TEER) of the resulting monolayers, and our TEER measurements results confirmed the formation of functional epithelial cell barriers., [cat] La major part dels actuals models in vitro de cultiu cel·lular no reprodueixen l'anatomia dels teixits i el comportament fisiològic de les cèl·lules in vivo. Les tècniques de microfabricació es poden utilitzar per anar un pas més enllà dels models in vitro convencionals, basats en cultius 2D, i reproduir de forma fiable microambients del teixit viu. Per l'epiteli de l'intestí prim, aquest enfocament s'ha explorat a través de la fabricació de scaffolds de col·lagen i poly (làctic-co àcid glicòlic) que imiten les vellositats, utilitzant micromolding seqüencial. Encara que ha estat un enfocament innovador i interessant, la naturalesa dels materials emprats no permet l'ajust de les propietats del sistema i el mètode d'emmotllament és laboriós, requerint diverses etapes de intermedies. Aquí es descriu un mètode senzill i ràpid per fabricar microestructures 3D similars a les vellositats. Utilitzem poli (etilenglicol) diacrylat (PEGDA) i àcid acrílic per formar hidrogels tous mitjançant fotolitografia. Hem demostrat que el nostre mètode permet produir monocapes funcionals de celules epitelials polaritzades(Caco-2). Els hidrogels són materials polimèrics que formen xarxes 3D amb un alt contingut d'aigua, que permeten una fàcil difusió de factors solubles i oxygen. Els hidrogels fets de PEG posseeixen propietats químiques i mecàniques que es poden ajustar i s'han convertit en materials molt itilitzats com a substrat per cultivar cèl·lules. Per obtenir microestructures 3D similars a les vellositats, s’han utilitzat fotomáscares en un procés de polimerització depenent de llum UV. Per permetre l'adhesió cel·lular i el seu creixement, el PEGDA es va copolimeritzar amb l’àcid acrílic per treure profit dels grups carboxílics de l'àcid acrílic i incorporar-hi covalentment proteines de la matrix extracel·lular. Després de la fabricació i caracterització dels hidrogels microestructurats, es va optimitzar la fabricació per obtenir microestructures similars a les vellositats. Per validar la funcionalitat del nostre sistema, es van utilitzar cèl·lules epitelials (Caco-2). Els nostres resultats mostren que les Caco-2 van ser capaces d'adherir-se i dividir-se, i formar monocapes de cèl·lules epitelials polaritzades. També hem estat capaços de transferir amb èxit la plataforma dels hidrogels microestructurats als sistemes convencionals (Transwell®) per fer mesures quantitatives de la integritat de la barrera epitelial.

[eng] Cells in tissues are exposed to extracellular signals that integrate and appropriately translate into specific responses. Receptors at the cell membrane recognize a variety of soluble ligands, extracellular matrix proteins and molecules presented by the neighboring cells. Ligand-receptor recognition event triggers intracellular signal transduction pathways modulating the resulting cell function. Some receptors do not function individually as signaling units but require interactions and associations with other receptors in multimolecular complexes. This process is known as receptor clustering and is an evolutionarily preserved mechanism responsible for the integration of highly complex signals. Increasing evidences suggest that this exceptional integration is subjected to spatially controlled ligand distribution at the nanoscale. Recent developments in highly sophisticated nanofabrication approaches have allowed to experimentally address this detailed spatial regulation on cell signaling. However, it is still unclear how the nanoscale distribution of ligands can impact on the dynamics of receptor activation and signaling processes. Herein we present a nanostructured platform to create patterns of ligands in regular nanosized (< 30 nm) clusters. We based our platform in self-assembled diblock copolymers composed of poly(styrene) (PS) and poly(methyl methacrylate) (PMMA) that tend to segregate into nanodomains. The hexagonal arrangement of the PMMA domains acts as template to be replicated by the ligand distribution. Thanks to the versatile functionalization strategy developed, any amine-bearing molecule can be covalently immobilized. The spatial distribution of ligand was analyzed by Atomic force microscopy (AFM) and stochastic reconstruction microscopy (STORM), unveiling the high level of fidelity between the nanopatterned ligands and the underlying polymeric template. To validate these substrates as platforms for systematic study of receptor clustering processes, an adhesive peptide which promotes focal adhesion formation, was immobilized on the nanopatterned surfaces. While the overall ligand surface density was maintained constant, the spatial distribution of ligands showed a remarkable impact on focal adhesion formation. Cells on nanopatterns showed an increased number of focal contacts, which were, in turn, more matured than those found in cells cultured on randomly presenting ligands. These findings suggest that ligand presentation in a clustered format might promote multivalent ligand-receptor interactions which can help to shed light on receptor oligomerization processes. In addition, the nanopatterned substrates developed were used to investigate the dynamics of the process of Eph receptor assembly into oligomeric clusters upon stimulation with ephrin ligands. It is known that Eph receptor oligomer composition is crucial in the fine-tuning of receptor signaling, as it will trigger intracellular signals feedback which will modulate cell response. Oligomerization processes, which imply resolving the temporal evolution of nanometric size objects in diffusive environments such as cell membranes are beyond the reach of live-imaging tools. We in here resolve the oligomerization kinetics of the Eph receptor in live cells with the required spatial and temporal resolution by using an enhanced version of the Number and Brightness (eN&B) technique, which can discriminate with molecular sensitivity the oligomeric species. The results demonstrated that stimulation through surface-bound ligands with a random distribution was not sufficient to activate the receptor signaling. Conversely, when nanopatterned on our substrates, ligands effectively induced receptor oligomerization. In addition, surface-induced ligand clustering by our nanopatterning approach accelerated the dynamics of receptor oligomerization process when compared to antibody-induced ligand clustering. Such an efficiency was induced even when ligand surface coverage was 9-fold lower in the nanopatterned presentation. Therefore, our ligand presenting platform is thought to induce multivalent ligand-receptor interactions, and might be a useful strategy to precisely tune and potentiate receptor responses. It has promising applications in biotechnology and biomedicine, such as cell culture systems to provide insight into relevant receptor clustering processes and design of new bioactive materials and drug-delivery systems., [spa] En los tejidos, las células reciben múltiples señales tanto de naturaleza física como química del entorno que las rodea. Inmersas en un entorno tridimensional, las células interactúan entre sí y con la matriz proteica que las envuelve. Además, hasta ellas difunden diversos factores solubles que transmiten señales químicas revelantes implicadas en el correcto funcionamiento celular. Ante tan complejo entorno, las células son capaces de reconocer de manera diferencial los estímulos que reciben y responder a todos ellos a través de complejos mecanismos intracelulares de señalización. Recientemente, se han desarrollado herramientas altamente sofisticadas que permiten estudiar el comportamiento celular ante una presentación definida de ligandos. Se ha demostrado que fenómenos tan relevantes como la adhesión, la proliferación o la diferenciación celular son sensibles a la distribución espacial nanométrica de ligandos en superficie. Múltiples receptores celulares, cuando son estimulados por sus correspondientes ligandos, necesitan agruparse y formar clústers que modulan la transmisión de la señal. Desafortunadamente, todavía se desconocen los pormenores de la activación y la dinámica de agregación de los mismos ante las múltiples combinaciones espaciales de ligandos. Por este motivo, este trabajo tiene como objetivo el desarrollo de superficies que permitan la presentación controlada de ligandos en grupos nanométricos para analizar el efecto de los mismos en los procesos de señalización intracelular. Para abordar este ambicioso objetivo, se desarrolló una plataforma a partir de copolímeros en bloque cuya principal particularidad es que se autoensamblan, generando estructuras nanométricas. El copolímero en bloque más utilizado en este ámbito es el compuesto por poliestireno y poli(metil metacrilato) (PS-b-PMMA). En este estudio se utilizaron dos copolímeros en bloque con distinta fracción volumétrica de cada uno de los componentes, de manera que se autoensamblan generando cilindros nanométricos de PMMA inmersos en una matriz de PS. Cuando se depositan en una capa fina sobre un sustrato de silicio o de vidrio, y se controla tanto el grosor de la capa como la energía superficial del sustrato, se puede conseguir que los cilindros se posicionen de forma perpendicular y ordenada sobre la superficie. Para ello, en primer lugar se modificó la energía superficial del sustrato mediante el anclaje de polímeros con una disposición de monómeros aleatoria. Por otro lado, el grosor de la capa fina se controló mediante la concentración de la solución empleada y esta capa fina se sometió a un tratamiento térmico a 220°C en vacío que permite equilibrar las tensiones superficiales del PS y del PMMA. De este modo, se fabricaron dos plataformas nanoestructuradas con patrones circulares compuestos de cilindros de PMMA (21 y 28 nm de diámetro) separados por una matriz de PS. Una vez obtenidas las plataformas nanostructuradas, se diseñó un proceso de funcionalización que permitiera la localización de pequeños grupos de ligandos sobre los dominios nanométricos de PMMA. Para ello, se realizó una hidrólisis superficial de los grupos metilos del PMMA, generando así grupos ácidos más reactivos que posibilitan la unión covalente de cualquier molécula con un grupo amino terminal. En este tipo de moléculas se incluyen todas las proteínas y pequeños péptidos, lo cual pone de manifiesto la gran versatilidad de la estrategia de funcionalización. La caracterización de la disposición espacial de los ligandos se realizó mediante microscopía de fuerzas atómicas, y se corroboró utilizando una novedosa técnica de alta resolución denominada microscopía de reconstrucción óptica estocástica, que permite confirmar el estado de agregación de los ligandos biológicamente activos. Para validar la utilidad de estas superficies nanoestructuradas, primeramente se inmovilizó un conocido ligando de adhesión celular y se monitorizó la respuesta celular, en concreto evaluando la formación de contactos focales. Los resultados demostraron que sobre estas superficies, los fibroblastos se expandían de tal manera que el área ocupada por las células era equivalente en todos los sustratos. En cambio, cuando se analizó en detalle las estructuras macromoleculares que forman los receptores en la membrana celular tras la activación por parte del ligando, se observaron diferencias significativas. El número de contactos focales formados en la superficie donde los grupos de ligandos estaban más separados, era menor que en aquellos cuya distancia entre ligandos era menor. Por otro lado, aquellas superficies donde los ligandos se presentaban en grupos fomentaban la maduración de los contactos focales, revelando de este modo que este proceso puede manipularse utilizando estrategias de presentación de ligandos como la desarrollada en esta tesis. Tras verificar el potencial de nuestras plataformas, se indagó en el proceso de agregación del receptor EphB2 ante ligandos (efrinas) con una distribución nanométrica variada. Para alcanzar la resolución espacio-temporal necesaria y ser capaces de distinguir entre los diferentes oligómeros formados por el receptor, se empleó una innovadora técnica que analiza las fluctuaciones en intensidad de cada uno de los pixeles de imágenes de fluorescencia. En combinación con un modelo matemático, se demostró que la agregación de receptores para formar hexámeros y octámeros impulsa la activación máxima del receptor EphB2. Anteriormente, se había descrito que los ligandos solubles individuales eran incapaces de activar el receptor y de promover su oligomerización. En cambio, la presentación controlada de ligandos en grupos nanométricos, no sólo fomenta la activación del receptor, sino que además acelera la formación de clústers, demostrando nuevamente la efectividad de los ligandos nanoagrupados como moduladores y potenciadores del dinámico proceso de oligomerización. A la vista de los resultados obtenidos, se puede concluir que hemos sido capaces de desarrollar una plataforma nanoestrucuturada mediante copolímeros en bloque para su posterior modificación covalente con ligandos celulares cuya distribución en nanoagregados favorece las interacciones multivalentes con los receptores. De este modo, estas plataformas tienen potenciales aplicaciones a la hora de promover una respuesta concreta de los receptores, en función del tamaño del grupo de ligandos y del espaciado entre ellos. Este tipo de ligandos multivalentes se presentan como una atractiva estrategia para activar los complejos receptor-ligando de manera más potente, y por lo tanto, menos costosa. Por lo tanto, las posibles aplicaciones de estos sistemas de presentación de ligandos comprenden desde aplicaciones biotecnológicas a aplicaciones biomédicas, incluyendo sistemas de cultivo celular, materiales bioactivos y administración de fármacos

[eng] Ischemic heart disease is a major cause of human death worldwide owing to the heart minimal ability to repair following injury. Other than heart transplantation, there are currently no effective or long-lasting therapies for end-stage heart failure. Therefore, it is crucial to develop not only alternative therapies that potentiate heart regeneration or repair, but also new tools to study human cardiac physiology and pathophysiology in vitro. In this context, cardiac tissue engineering arises a promising strategy, as it is aimed at generating cardiac tissue analogues that would act as in vitro models of human cardiac tissue or as surrogates for heart repair. Thus, having 3D human cardiac tissue constructs resembling human myocardium could revolutionize drug discovery and toxicity testing, cardiac disease modelling and regenerative medicine. An strategy to obtain reliable cardiac tissue constructs is to mimic the native cardiac environment. The classical approach is based on seeding cardiomyocytes in biocompatible 3D scaffolds, and then culturing the construct in a biomimetic signaling system, usually a bioreactor. Although major advances have been made, the generation of thick and mature tissue constructs from human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CM) is still a challenge. Therefore, the hypothesis of our study is that the combination of hiPSC-CM with 3D scaffolds and appropriate regulatory signals may lead to the generation of mature human cardiac tissue constructs resembling human myocardium, both functionally and structurally. To address this, we have characterized a collagen-based 3D scaffold and established an efficient method for cell seeding into the scaffold. We have also developed a parallelized perfusion bioreactor system, which ensures an effective mass transport between cells and culture medium and allows culturing multiple replicas of tissue constructs. In addition, we have designed and fabricated a perfusion chamber including electrodes to electrically stimulate cells during culture, as well as to monitor tissue function. With this advanced 3D culture system, we have been able to generate thick 3D human cardiac constructs with tissue-like functionality. Our results indicate that perfusion of culture medium combined with electrical stimulation and collagen-based scaffold improve the structural and functional maturation of hiPSC-CM. In general terms, electrical stimulation has improved the structural organization, alignment and coupling of cardiomyocytes in our cardiac tissue constructs. Moreover, electrical stimulation has promoted the formation of synchronous contractile constructs at the macroscale with improved electrophysiological functions. Through the development of a new electrophysiological recording system, we report for the first time to our knowledge a technique that provides information about the electrical activity of intact cardiac tissue constructs in real time. Specifically, the combination of action potentials generated by hiPSC-CM composing cardiac constructs produces ECG-like signals, which could be monitored online. Finally, we have demonstrated the ability of stimulated human cardiac tissue constructs to detect drug-induced cardiotoxicity, as typical features of arrhythmias (e.g. prolongation of RR intervals and regular blockades) could be observed upon treatment with sotalol. Taken together, results indicate that macroscopic human cardiac tissue constructs with tissue-like functionality can be obtained through the use of our advanced 3D culture system. We have studied the effects of electrical stimulation on cardiomyocytes at multiple levels: molecular (presence, distribution and expression of cardiac proteins), ultrastructural (sarcomere width and presence of specialized cellular junctions), cellular (morphology and alignment), and functional (amplitude, directionality and strain of contractions, and electrophysiological recordings). Findings validate our in vitro approach as a valuable system to obtain 3D cardiac patches with an improved maturity and functionality. Importantly, the online monitoring system developed in this study can provide essential electrophysiological information of intact cardiac tissue constructs, which opens up myriad possibilities in the field of cardiovascular research., [cat] La cardiopatia isquèmica és una de les principals causes de mort a nivell mundial. Exceptuant el trasplantament de cor, les teràpies actuals són insuficients per restablir la funció cardíaca. Per tant, cal desenvolupar teràpies alternatives que fomentin la regeneració i/o reparació del cor, així com també noves eines per estudiar la fisiologia i fisiopatologia cardíaca in vitro. Una de les estratègies més prometedores és l’enginyeria tissular cardíaca, ja que té com a finalitat generar constructes de teixit cardíac que mimetitzin el teixit real. Aquests constructes podrien utilitzar-se com a models in vitro del miocardi humà i també com a empelts per reparar el cor malmès. Per obtenir constructes de teixit cardíac humà cal reproduir l’entorn cardíac real. Una de les estratègies més habituals consisteix en sembrar cardiomiòcits en una estructura 3D (bastida), i després cultivar el constructe en un sistema de senyalització biomimètic, normalment un bioreactor. Tanmateix, generar constructes grans i semblants al miocardi humà adult a partir de cardiomiòcits humans derivats de cèl·lules mare de pluripotència induïda (hiPSC-CM) segueix sent un repte. Així doncs, la hipòtesi d’estudi és que combinant hiPSC-CM amb una bastida 3D i estímuls biofísics adequats, es podrien generar constructes de teixit cardíac semblants al miocardi humà tant a nivell estructural com funcional. Per abordar la hipòtesi, en aquest treball s’ha caracteritzat una bastida 3D constituïda principalment per col·lagen i s’ha definit un mètode eficient per sembrar cardiomiòcits dins l’estructura. A més a més, s’ha desenvolupat un bioreactor de perfusió de sistema en paral·lel que assegura un transport de massa efectiu entre les cèl·lules i el medi de cultiu. També s’ha dissenyat una càmera de perfusió que inclou elèctrodes per estimular elèctricament les cèl·lules durant el cultiu, així com també per monitorar la funció del teixit artificial. Amb aquest avançat sistema de cultiu, s’han generat constructes de teixit cardíac humà 3D amb una funcionalitat semblant a la del teixit real. A més a més, el sistema ha permès monitorar l’electrofisiologia del teixit artificial en temps real, així com també demostrar el paper crucial de l’estimulació elèctrica per obtenir constructes amb una funcionalitat òptima.

[eng] Transferring nanotechnology into marketable products and services is still considered a major challenge. In Europe, this issue has been identified as a weakness, not only for nanotechnology, but also for the other five Key Enabling Technologies (KETs), strategic for the economic growth of the region. In this regard, the current European Funding Programme Horizon 2020 is making great efforts with their action lines in order to prioritize the industrial implementation of KETs, and in this manner, address major economic and societal needs. This initiative is also fostering the cross-fertilization of KETs, since it has been determined that the sum of individual technologies increases the potential for innovation, optimizes technological development, and allows the creation of new markets. This thesis has been developed on the basis of this scenario. The aim is to analyse innovation and technology transfer challenges for the successful commercialization of nanotechnologies by emphasizing the process of cross-fertilization of KETs. The research is focused on healthcare due to the great impact that nano-scale is having on this field. For this reason, the present work has considered two approaches: from a technological perspective and from a management perspective. The analysis is comprised of a state-of-the-art and theoretical framework review, followed by a multiple case-study approach where several nano-enabled sensor-based devices are analysed at diverse levels of technological maturity. In addition, an empirical study of European nano-related innovation projects was undertaken in order to determine which projects’ characteristics are influencing the creation of technological diversity; a critical element for the long-term success of an emergent technology. Finally, project leaders were interviewed in order to gain insights about the managerial strategies that are boosting the process of cross-fertilization of KETs. Findings have shown that a multi-disciplinary, collaborative and integrated community of innovators is necessary for the effective transference and commercialization of multi-KET nanotechnologies. Additionally, the degree of multi-disciplinary projects was identified as significantly contributing to the creation of technological diversity. Furthermore, higher levels of cross-fertilization were found in market and customer-oriented projects, with actors strongly motivated to search for ideas from broad informal networks, and where technological knowledge is moderately heterogeneous. Lastly, it has been found that the cross-fertilization of KETs is boosted by actors with a high involvement of nanotechnologies in their industries. With these outcomes, this thesis has sought to contribute to the analysis of the successful transference and commercialization of multi-KET nanotechnologies in the field of healthcare by understanding the processes and ecosystems of innovation. The outcomes of this thesis have sought to contribute to the analysis of the successful transference and commercialization of multi-KET in the field of nanotechnologies applied to healthcare by understanding the processes and ecosystems of innovation. Accordingly, it is aimed to contribute to the reduction of the gap between research and the marketplace and to expand the knowledge of current interest regarding innovation ecosystems of emergent technologies, regional systems of innovation and strategic innovation management. [, [cat] La transferència de productes i serveis basats en la nanotecnologia representa un gran repte. A Europa, aquest fet ha estat identificat com a punt dèbil, no només per a les nanotecnologies si no també per a les altres cinc tecnologies facilitadores transversales (KETs per les seves sigles en anglès), considerades estratègiques pel creixement econòmic de la regió. En aquest sentit, l’actual programa marc Europeu Horitzó 2020 està redirigint les seves línies d’acció per a prioritzar la implementació de les KETs i, d’aquesta manera, poder fer front a les necessitats econòmiques i socials més imperatives d’Europa. Aquesta iniciativa també pretén fomentar la fertilització creuada de les KETs, ja que s’ha establert que la suma de tecnologies individuals incrementa el potencial d’innovació, optimitza el desenvolupament de tecnologies i permet la creació de nous mercats. Sobre aquesta base es desenvolupa aquest treball d’investigació, el qual té la finalitat d’analitzar els reptes relacionats amb la innovació i la transferència tecnològica per a assolir amb èxit la comercialització de les nanotecnologies, posant de relleu el procés de fertilització creuada de les KETs en el camp de la salut. Amb aquesta finalitat, s’han considerat dues aproximacions: d’una banda una perspectiva tecnològica i, de l’altra, una perspectiva de gestió de la innovació. Els resultats obtinguts fan aportacions per l’anàlisi i identificació dels reptes que cal afrontar per a una favorable transferència i comercialització de les nanotecnologies multi-KET en el camp de la salut mitjançant la comprensió dels processos i ecosistemes d’innovació i, d’aquesta manera, contribuir a la reducció de la separació entre el laboratori i el mercat. Finalment també es pretén ampliar el coneixement sobre temàtiques d’interès actual respecte els ecosistemes d’innovació de les tecnologies emergents, els sistemes regionals d’innovació i la gestió estratègica de la innovació tecnològica.

[eng] The present doctoral thesis is framed in the research and development (R & D) project between a private biotechnology company of molecular diagnostics Genomica SAU, the Institute for Bioengineering of Catalonia (IBEC), the University of Barcelona, and the Microfluidics ChipShop Company. The main objective of the project is making, implementation and marketing of a diagnostic device for early detection of DNA sequences involved with cancer. The multi device, or lab-on-chip (LOC), consists of a central automation unit (CAU), a system in miniature of DNA amplification or chain reaction polymerase (mini-PCR), and a biosensing platform (DNA chip) that consisting of a matrix or electrochemical array. The three elements are integrated by a microfluidic system in sandwich format cartridge. For this purpose, the aim of this thesis was the creation, characterization and optimization of the biochemical recognition platform between two single strands of DNA of dissimilar lengths but with some complementary sequences for the subsequent electrochemical detection of a hybridization event between them. Then, the integration into the cartridge of above platform was done. For the creation of this platform, we chose to use a self-assembled monolayer (SAM) of thiols as biorecognition interface of the 14 DNA sequences that are part of the project. During optimization of the interface chips individual gold and various molecules were used being chosen the molecule with two arms disulfide of polyethylene glycol (PEG) and a malaimida group at the end of one of them. This linker (or MalPEG linker) reacts with the gold surface due to the dative interaction between the sulfur atoms of the disulfide and the gold atoms from the surface of the chips. At the same time, the malaimida group reacts with the thiol group of the capture probes, joining. The PEG groups function as anti-adhesion molecules. Surface plasmon resonance (SPR) and cyclic voltammetry (CV) were techniques used to characterize the substrate and the hybridization event. For the manufacture of the cartridge, this was divided into two main blocks, the biosensing or electrochemical block and PCR block. The electrochemical block is composed of 4 layers, one of 64 working electrodes and gold paths for contact with the potentiostat, another layer that defines the area of the sensors must be functionalized gold and isolating the gold surface of the tracks. The third layer is a double-sided adhesive that has a hexagonal hole working as hybridization chamber, and the last layer is a screen printing layer with the reference electrode (RE) and counter electrodes. The above layers form an electrochemical cell wherein the hybridization will occurs. Regarding the PCR block, this is a system of two layers with a type microfluidic channel kind loop and its function is to contain the solutions during the process of DNA amplification by the mini-PCR. During the integration of the optimized SAM into an electrochemical cartridge a manual and automated ways were used to immobilize the capture probes. Several tests were performed in order to obtain the best conditions and ratios between the molecules to maximize the hybridization signal during the electrochemical detection., [spa] El presente trabajo de tesis está enmarcado en un proyecto de investigación y desarrollo (I+D) entre la empresa privada Genomica S.A.U., el Instituto de Bioingeniería de Cataluña (IBEC), la Universidad de Barcelona y la empresa alemana ChipShop Microfluidics. El objetivo principal es el desarrollo, puesta a punto y comercialización de un dispositivo electroquímico de diagnóstico médico para etapas tempranas de cáncer. El objetivo de la tesis es la creación, optimización y posterior integración de una interfaz de biosensado de ADN en el dispositivo de diagnóstico, siendo pieza fundamental en el desarrollo de éste. La interfaz escogida fue una monocapa autoensamblada (SAM) que hace las veces de biosensor y que es capaz de anclar secuencias de ADN como sondas de captura y así poder detectar, selectivamente, las secuencias objetivo complementarias. El dispositivo también cuenta con un sistema microfluídico y un sistema de amplificación de ADN de reacción en cadena de la polimerasa en miniatura. La SAM esta inmovilizada en un array electroquímico que consta de 64 electrodos de trabajo que funcionan como elemento transductor de la señal electroquímica redox de los eventos de hibridación que ocurren sobre ellos. La funcionalización y puesta a punto del dispositivo se llevó a cabo inmovilizando múltiples sondas de captura después de una optimización de las concentraciones entre las diferentes partes constituyentes de la monocapa. Técnicas ópticas y electroquímicas fueron utilizadas para la caracterización de cada etapa y técnicas de fotolitografiado y de impresión por pantalla fueron utilizadas para la fabricación de los componentes del dispositivo. Finalmente, y después de algunos cambios surgidos durante el desarrollo del dispositivo, se llega a un diseño final y a las pruebas con muestras reales, proceso que aún está en etapa experimental.

The search for analytical tools suitable for wide-scale application of DNA analysis is an hot research topic, although thanks to well-established microarray based technology, analysis of DNA sequences and SNP detection can be worked out through a fairly laboratory routine. DNA analysis has nowadays become of increasing interest for several different purposes, mainly thanks to the successful employment of microarray technology, characterized by high sensitiveness and high-throughput analysis, which rapidly advanced genetics leading to devel- opment of many fields of application of DNA analysis, which keeps high the trend in alternative technologies, which could overcome inherent limitations of microarrays technology. In this regard, many efforts have been spent to study electrochemical/electrical based detection strategies by means of which it could be possible to accomplish sensitive analysis by using portable equipments, cheaper and more practical than optical ones, and with scalable-devices compatibles with standard microelectronic processing. Emerging nano-probes with increased chemical-physical properties are considered with growing interest in DNA biosensors as ideal candidates to enhance electrochemical/electrical based detection systems. Among these, nano-gaps adjusted to fit DNA, or in general analyte molecules sizes, are very promising because they can enable direct electrical detec- tion schemes, thus providing a straightforward electronic analogue of the successful DNA microarray standard. Electrical properties of DNA have been the principal focus of many experimental and theoretical research, since early experimental founding confirmed an old hypothesis, for its relevance in the biological function of DNA, being related both with damage and base repair, but also for the appealing potential for biosensor and, in general, in bioelectronic applications. Thanks to its peculiar interactions, it allows versatile manipulations of the structure, compared to other organic and synthetic polymers which have been considered for such purposes. Even though many questions still are open on electrical properties of DNA, it is generally accepted that DNA's conductivity is intimately linked with details of the sequences involved, its length and the overall environment in which molecule is found. The sensitivity to structure's alteration, as that induced by the presence of a mutation, confirmed by experimental and theoretical works allows to exploit DNA electrical properties for biosensor applications. Relying on this agreed description of DNA electrical properties features, the general aim of this thesis was to explore the possibility of developing a platform for the direct transduction of DNA hybridization event based on a nano-gap device and electrical signaling enabled by long range electron transport through DNA molecules., La detección de hibridación de cadenas de ADN es un reto relevante científicamente y tecnológicamente, que puede aprovechar de las posibilidades proporcionadas por los alcances en los procesos de nano fabricación y caracterización, inspiradores de la idea de una medicina en el punto de atención. El propósito de este trabajo es de establecer un sistema de detección de hibridación de ADN, y polimorfismo de un solo nucleótido (SNP), basado en la medida eléctrica de la reasistencia de un nano-gap funcional izado con el ADN diana. El desarrollo y test del sistema se ha llevado a cabo fijando diferentes objetivos. Un estudio preliminar de la literatura relacionada con las propiedades eléctricas del ADN se ha conducido con la finalidad de establecer el marco de factibilidad del proyecto. De acuerdo con los resultados de este estudio ha sido posible idear el sistema y optimizar su eficacia respeto a las experiencias reportadas. Fijar una estrategia de fabricación de los dispositivos capaz de proveer nano-gaps aptos a la medida de conductividad muy baja, según una rutina de fácil implementación y con alta reproducibilidad de los resultados. Estos se han caracterizados mediante el utilizo de diferentes técnicas basadas primariamente en métodos de detección Óptica y Eléctrica/Electro-química. Obtener la bio-funcionalización selectiva de los electrodos en el nano-gap testando y caracterizando métodos diferentes. Probar el principio de funcionamiento del sistema a través de la medida de la conductividad en los nano-gap durante las diferentes etapas de funcionalización con los bio-receptores y el DNA target. Optimizar el sensor testando su selectividad respeto a la presencia de mutaciones, la sensibilidad a medir diferentes concentraciones del target, y finalmente la posibilidad de regeneración del dispositivo después desnaturalización del ADN hibridado

[spa] El objetivo de esta tesis es el desarrollo de nuevas metodologías en el procesado de señal multivariante en espectros IMS. En este trabajo se ha realizado una comparación entre tres espectrómetros IMS. Esta labor comparativa, mediante procesado multivariante, es prácticamente inédita en este ámbito. En este caso se realizó un estudio con 3 aminas y se determinó el límite de detección. Los resultados mostraron que los 3 espectrómetros tuvieron un rendimiento similar, a pesar de que sus condiciones de operación son distintas. Se propuso una técnica específica para eliminar ruido de baja frecuencia acoplado al espectro de IMS. Se observó que utilizar PCA o ICA (métodos multivariantes) mejora notablemente la relación señal ruido si se compara con las técnicas convencionales. Se ha estudiado el alineamiento de los espectros y se han propuesto soluciones basadas en los diferentes métodos del estado del arte. Se ha evidenciado que incluir compuestos de referencia para garantizar que el proceso de alineamiento es el adecuado es ventajoso. En el caso de que esto no fuese posible se aconseja realizar el alineamiento por etapas, primero un alineamiento en una misma muestra, y luego entre muestras. Se realizaron modelos cualitativos para diferenciar o discriminar clases a partir de medidas de IMS. Se propusieron dos modelos multivariantes con técnicas de validación cruzada. Los resultados obtenidos muestran el gran potencial de IMS en este sentido. Se evaluó el rendimiento cuantitativo de los IMS al utilizar métodos multivariantes y fueron comparados con métodos univariantes habituales en el ámbito de IMS. De los resultados obtenidos se observó que los modelos univariantes no son capaces de resolver comportamientos típicos de IMS como son el comportamiento no lineal y el efecto en mezclas. En este sentido las técnicas multivariantes mostraron mejores prestaciones. Se comparó la utilización de técnicas multivariantes que proyectan los datos en un nuevo subespacio como lo es PLS con técnicas de deconvolución como lo es MCR en sus dos versiones ALS y Lasso. Los resultados obtenidos fueron bastante similares, sin embargo MCR ofrece una ventaja importante ya que permite interpretar de mejor manera los resultados, [eng] There are several applications where the measurement of VOC results to be useful, such as: toxic leaks, air quality measurements, explosive detection, monitoring of food and beverages quality, diagnosis of diseases, etc. Some of this applications claim for fast responses or even real time responses. In this context, there are few analytical techniques for performing gas phase analysis, among of them Ion Mobility Spectrometry (IMS). IMS is a fast analytical device based on the time of flight of ions in a drift tube. The response of IMS lasts typically few seconds, but it can be even less than a second. This fast response has drifted its use towards novel applications, such as biomedical and food applications (bio-related applications). Nonetheless, it has also brought the need to analyze complex spectra with hundreds of compounds. In fact, tackling this disadvantage is the main focus of this thesis, where new algorithms for enhancing the IMS performance are investigated when are applied to bio-related applications. Nonlinear behavior and charge competitions of IMS responses are important issues that need to be addressed. Both effects have a direct impact in the IMS spectra interpretation —especially when real dataset are studied. Additionally, the use of univariate spectra analysis, where peaks information is extracted manually, becomes unfeasible in bio-related applications. In this context, this work introduces multivariate methodologies focused on quantitative and qualitative analysis. In the case of quantitative analysis, calibration models were built using univariate methodology, Partial Leas Squares (PLS) and Multivariate Curve Resolution techniques (MCR). The quantitative analysis aims tackling the main issues of IMS such as non linearities and mixture effect. Definitely, univariate techniques provides poor or overoptimistic results that minimize the impact of the IMS use. The results show a really improvement on the performance when multivariate techniques were used. Regarding the results between MCR and PLS, the main difference is the interpretability that offers MCR. In the case of qualitative analysis, two different approaches were planned for building models for classes' discrimination. The first approach consisted on building a model through principal component analysis and linear discriminant analysis, besides of using robust cross validation methodology for obtaining reliable results. This methodology were implemented in samples of wine, where main motivation was found discrimination regarding to their origin. The results were fully satisfactory because the model was able to separate four groups with a high accuracy rate. The second approach involves the use of Multivarite Curve Resolution — Lasso algorithm for extracting pure components of samples from rats' breath and then use a feature selection technique for obtaining the most representative features subset. In this case, the objective of the application was to find a model that discriminate rats with sepsis from control rats. The results shows there were few pure components of IMS that generate a discriminatory model that means there are specific compounds in the breath linked with the disease. Summarizing, the following proposal has as main objective resolving open issues in stand-alone IMS that are applied to the analysis of bio-related applications. Two major investigation lines were proposed in this thesis: (i) qualitative analysis and (ii) quantitative analysis. The qualitative analysis covers pre-processing algorithms and the developing of new methodologies for building models in bio-related applications. The quantitative analysis are focused on highlighting the importance of the use of multivariate techniques instead of univariate techniques. In order to reach the objectives of this thesis, a set of datasets were created, which are detailed on the content of this thesis. The results and main conclusions are deeply explained in the extended proposal.

Patterns are a collection of forming units predictably repeated over a defined magnitude. Researchers have used patterns to guarantee the functionality and repeatability of their study. For that, the obtained data is purposely compared over and over in hope that the results are comparable. Two main research approaches are based on patterns: The initial requires a single substrate with localized and repeated units to create multiple testing sites, obtaining a repeated, multi-analysis system. The second approach uses fixed localization with different testing motifs, creating a diverse multi-analysis platform. The miniaturization of these assays provides an alternative to reduce cost, maximize efficiency, and increase repeatability. Micropatterns consist on immobilized (bio)molecular motifs constrained in small areas over a solid substrate. These fixed spots provide up to thousands of reaction sites for parallel detection. Micropatterns were first developed to study the interaction between Deoxyribonucleic acid (DNA) strands and the study of the genome. Afterwards, this technology was used to create miniaturized protein patterns. Today, this technology is essential for large-scale and high-throughput biological and biochemical studies. Single-feature microarrays are routinely reproduced at many laboratories using various contact, non-contact, or alternatively methods. The foundation is to transfer a (bio)molecule in a solution onto a solid substrate obtaining a defined feature shape. This Thesis aims to expand the current contact replication techniques for microarray fabrication. Initially, an automatized microcontact printing tool was characterized to create complex patterns on a wide range of substrates. Thiols, silanes, and various biomolecules were printed on glass, silicon oxide or gold. The printing properties were explored to create a definitive protocol for further applications. The effect of the printing force and dwell time were thoroughly studied to form a mathematical expression to understand all the variables involved during contact printing. The miniscule resolution provided by the automatized tool allowed the creation of complex micropatterns with single or multiple printings steps. This tool was later upgraded and fitted with new controllers to create smaller patterns. An alternatively contact printing technique called polymer pen lithography was used to pattern the surface of specialized substrates to create micropatterns on constricted areas. The miniaturized microarrays were later liberated to create functionalized microparticles. These microparticles can be tuned for many biochemical applications, such as protein interaction studies, drug discovery or life science. Lastly, a new contact replication method was established to fabricate DNA arrays. An initial DNA master arrays was fabricated with known contact printing techniques. Then, either hybridized or in situ synthesized strands were transported to an intermediate substrate. A second hybridization or synthesis was used to transport a replica of the master array to a new substrate, maintaining the chemical and spatial information present on the original array., Un patrón es una colección de unidades formadoras que se repiten predeciblemente en una magnitud definida. Los investigadores han utilizado patrones para garantizar la funcionalidad y repetitividad de sus estudios. Para conseguir eso, los datos obtenidos de los estudios se comparan entre varios resultados, esperando así una correlación. Dos métodos de investigación están basados en patrones: uno requiere un sustrato con unidades repetidas localizadas en un plano cartesiano definido, obteniendo una plataforma de análisis múltiple. El segundo método utiliza localizaciones definidas con diferentes áreas de prueba, creando así una plataforma de multianálisis. La miniaturización de estas pruebas permiten reducir el costo, maximizar la eficiencia e incrementar la repetitividad de los ensayos. Los micropatrones consisten en puntos de (bio)moléculas limitados en pequeñas áreas para crear zonas de reacción múltiples. Esta tecnología fue inicialmente utilizada para crear las interacciones del ADN para estudios genómicos. La técnica evolucionó para crear patrones de proteínas y actualmente se utiliza para estudios bioquímicos a gran escala y de muy alto rendimiento. Patrones de una (bio)molécula repetida a través del sustrato son fabricados rutinariamente en muchos laboratorios utilizando técnicas de impresión por contacto, por inyección u otro métodos. El cimiento de estas técnicas es transferir una (bio)molécula de una solución a un sustrato. Esta Tesis pretende expandir los métodos de creación de micropatrones por técnicas de impresión por contacto. Inicialmente se caracterizó una máquina automatizada de impresión por microcontacto para crear patrones y estudiar las variables que afectan al momento de la impresión. Se correlacionaron la presión y el tiempo de impresión para entender la morfología del patrón resultante. Igualmente se caracterizó el posicionamiento micrométrico de los patrones para crear estructuras complejas. Posteriormente, la máquina se modificó para incluir la técnica de impresión con plumas poliméricas. Esta técnica permitió crear micropatrones en superficies minúsculas. Estos micropatrones fueron luego liberados para crear micropartículas que pueden ser personalizadas para aplicaciones diversas. Finalmente, se formuló una nueva técnica de replicación de patrones de ADN desde un patrón inicial, manteniendo la información química y espacial presente en éste.

[cat]Vesícules naturals produïes a partir de cèl·lules modificades genèticament són prometedors components de sensat per utilitzar com a detectors en biodispositius. Això és particularment cert en el cas de receptors adjuntats a proteïna G (GPCRs) presents en molts processos cel·lulars, on la seva funcionalitat depèn estrictament del seu entorn lipídic. Els receptors de membrana estan involucrats en una gran varietat de vies bioquímiques i per tant són objectiu d’estudi per teràpia i desenvolupament de nous fàrmacs. Per tant, plataformes bioanalítiques i assajos d’unió receptor-lligand, utilitzant receptors transmembrana, requereixen la construcció de matrius de membranes lipídiques ben caracteritzades, actuant com a suport per evitar la desnaturalització de proteïnes durant el processament del bioxip. En aquesta tesi es presenta la producció i caracterització de nanovesícules de membrana (NV) provinents de cèl·lules de llevat Saccharomyces cerevisiae que contenen receptors olfactius (un membre de la família de GPCRs) heteròlogament expressats a la membrana. Hem demostrat que les fraccions de membrana, a partir de cèl·lules de llevat, en solució formen espontàniament nanovesícules esfèriques tancades. També s’ha demostrat, que després d’un procés de enginyeria genètica els receptors olfactius van ser expressats correctament a la membrana del llevat. També s’ha presentat un mètode simple per homogeneïtzar la mida de les nanovesícules. A més a més, es presenta per primer cop un nou mètode immunoquímic per la quantificació directa de les proteïnes transmembrana (GPCR) en el seu ambient lipídic natural. El mètode utilitza anticossos monoclonals en un assaig basat en ELISA amb alta detectabilitat. L’aplicació del mètode es demostra a través de la quantificació del receptors olfactius OR1740 i OR7D4 expressats en nanovesícules de membrana plasmàtica. També es presenta, mitjançant observació directa amb AFM, com les nanovesícules es depositen i s’aplanen sense trencar-se sobre substrats de vidre i or seguint la llei de difusió. Es demostra com en el cas del vidre els màxims recobriments superficials obtinguts són del 20-25% i en el cas del or funcionalitzat del 10-15%, controlant la concentració de nanovesícules, el temps de depòsit, la presència de residus procedents del procés de producció de les nanovesícules, la química de la superfície, la força iònica del medi, etc. Finalment, s’ha demostrat per SPR que els receptors expressats eren funcionals i que aquesta tècnica òptica permet la detecció de petites molècules, com són els odorants, a les concentracions en el rang micromolar. Els resultats presentats en aquesta tesis contribueixen donant un pas important a la realització de dispositius biosensors basats en nanovesícules naturals que integren receptors de membrana adjuntats a proteïna G., [eng] Natural vesicles produced from genetically engineered cells with tailored membrane receptor composition are promising building blocks for sensing biodevices. This is particularly true for the case of G-protein coupled receptors (GPCRs) present in many sensing processes in cells, whose functionality crucially depends on their lipid environment. Membrane receptors are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays, using these transmembrane receptors, for drug screening or diagnostic require building well-characterized lipid membrane arrays, acting as supports to prevent protein denaturation during biochip processing. The controlled production of natural vesicles containing GPCRs, their characterization and their reproducible deposition on surfaces are among the outstanding challenges in the road map to realize practical biomolecular devices based on GPCRs. In addition, quantification of the protein receptors in such lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. In this thesis we present the production and characterization of membrane nanovesicles (NV) from Saccaromyces Cerevisiae containing heterologously expressed olfactory receptors - a member of the family of GPCRs. We have demonstrated that membrane fractions from yeast cells spontaneously form closed spherical nanovesicles in solution. A simple method to homogenize the size of the nanovesicles to a diameter of around 100 nm at a concentration of more than 1010 nanovesicles mL-1 is also presented. It is also showed that after a genetic engineering process the olfactory receptors of interest were well expressed in the yeast membrane. Furthermore, we report for the first time a novel immunochemical analytical approach for the quantification of transmembrane proteins (i.e. GPCR) in their natural lipid environment. The procedure allows direct determination of tagged receptors (i.e. c-myc tag) without any previous protein purification or extraction steps. The proposed approach uses monoclonal antibodies addressed against the c-myc tag, frequently used in protein expression, on a microplate-based ELISA format with high detectability. The immunochemical method quantifies this tag on proteins or bioreceptors embedded in nanovesicles with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptors (ORs, c-myc-OR1740 and c-myc-OR7D4) in plasma membrane nanovesicles (NVs). We also show by direct observation with Atomic Force Microscopy that nanovesicles deposit and flatten without rupturing on glass and gold substrates following approximately a diffusive law. We show that on glass surface coverages larger than 20-25% of the substrate can be reproducibly achieved under practical nanovesicle concentrations and reasonable time scales, while keeping to the minimum the presence of background residuals coming from the nanovesicles production process. On the other hand, on functionalized gold substrates surface coverages around 10-15% were achieved. Then, the role of surface chemistry was studied showing that modification of gold substrates indicates a higher affinity of natural nanovesicles for acid modified surfaces as compared to amino or alcohol modified surfaces. Nanovesicles deposition in acid modified gold surfaces and glass have been exploited for the generation of an array of multiple nanovesicles. Present results constitute an important step in the practical realization of biosensor devices based on natural nanovesicles integrating G-protein coupled membrane receptors. When olfactory receptors are genetically expressed in closed vesicles from natural yeast membrane fractions the verification of their capability for capturing specific odorant molecules are critical for the design of artificial noses. Thus, we demonstrated by Surface Plasmon Resonance (SPR) measurements on L1 Biacore chips that the receptors were functional. Despite the fact that the expression of olfactory receptors in nanovesicles is low, a fact that is coherent with the general expression level of GPCRs proteins in cells, the integration in nanovesicles together with a careful choice of the SPR experimental conditions and data analysis allowed us to obtain a concentration-dependent SPR response vs. odorant concentration with a sensitivity of 0.5-1.8RU/micromolar. The selectivity of OR carrying NV towards its specific odorant was proved in cross-check experiments with unspecific odorant molecules and control receptors. These results constitute a proof of concept that ORs embedded in nanovesicles properly respond to odorants and definitely open the perspective to use the surface plasmon resonance technique for the detection of small odorants at concentration in the micromolar range.

This thesis aims at providing a better understanding of the micro- and nanofabrication of conducting polymers for biomedical devices and presents novel processes that widen the application range of conducting polymers in this field. The thesis is divided in four chapters, namely “Materials and Methods”, “Biocatalytically-produced polypyrrole thin films and microelectrodes on insulating surfaces”, “Azide-PEDOT electrodes. Application to DNA sensors” and “Fabrication of polypyrrole single nanowire devices”. Chapter 1, entitled “Materials and Methods”, describes the materials used in this work and the fabrication and characterization methods required for the development of the thesis. Here, theoretical and experimental details about the techniques employed, are provided. Chapter 2, entitled “Biocatalytically-produced polypyrrole thin films and microelectrodes on insulating surfaces”, presents a new on-surface biocatalytical procedure for the fabrication of polypyrrole microelectrodes on insulating surfaces, with resolutions comparable to the ones of conventional photolitography. This is an environmentally respectful microfabrication method that allows the entrapment of biomolecules during the polymer synthesis in a single step. As a proof of concept, biotin was trapped in the polypyrrole matrix and then released in a controlled way through electrical stimulation. It was proven that the polymer keeps its electroactivity after the fabrication and functionalization processes. This biocatalytical-based technique represents a straightforward method for the microfabrication of biological-active conducting polymers, which could be implemented in implantable devices for remotely controlled tissue interactions. Chapter 3, entitled “Azide-PEDOT electrodes. Application to DNA sensors”, describes the fabrication and testing of an electrochemical label-free DNA hybridization sensor, based on novel azidomethyl-modified poly(3,4-ethylenedioxythiophene) electrodes (azide-PEDOT electrodes). These azide-PEDOT electrodes were used as platforms for the immobilization of acetylene-DNA probes, complementary to the “Hepatitis C” virus. The acetylene-DNA probes were covalently grafted to the polymer backbone via the robust “Click” reaction, which a part from being a very selective functionalization method, preserves DNA from denaturation during the synthesis of the polymer. DNA hybridization was detected by Differential Pulse Voltammetry (DPV), where the electrochemical change of the polymer behaviour, produced by the recognition event, was directly evaluated. This fabrication procedure is a powerful tool for the preparation of label-free DNA sensors able to selectively recognize a specific DNA sequence, down to the nanomolar range. Finally, Chapter 4, entitled “Fabrication of polypyrrole single nanowire devices”, discusses the fabrication of polypyrrole at the nanoscale. Two fabrication techniques were investigated here, namely dip pen nanolithography and electrochemical polymerization on template-assisted surfaces. On one hand, the dip pen nanolithography proved to be a simple deposition technique with good control over size and location of the polypyrrole nanowires. On the other hand, the electrochemical polymerization on template-assisted surfaces provided as well nanoscaled polypyrrole, but added the possibility to chemically manipulate the polymer. This chemical manipulation was translated into polymer devices with different electrical properties. By the use of these techniques, the capability of fabricating single nanowire devices (ready to use in different applications) and arrays of ordered nanowires based on conducting polymers is demonstrated. Additionally, two appendixes can be found at the end of the thesis: Appendix A: “Fabrication of azide-PEDOT microwire-based devices” and Appendix B: “Fabrication of nanopatterns by electron-sensitive silanes”. They provide short experimental results obtained during the course of this work, which are first steps for future investigations. A general conclusions section can be found at the end of the thesis, where a summary of the main achievements and contributions of this thesis are listed., Aunque los polímeros conductores se presentan como una alternativa viable a los materiales convencionalmente usados en aplicaciones biomédicas, las técnicas de fabricación adaptadas a ellos y el aprovechamiento de sus propiedades están lejos de ser completos. Existen importantes limitaciones en la fabricación de micro y nano estructuras basadas en polímeros conductores. Debido a la agresividad de las técnicas tradicionalmente usadas en microelectrónica, se hace necesaria la búsqueda de nuevas estrategias de fabricación adaptadas a polímeros conductores, así como de nuevos procesos que puedan mejorar el rendimiento de los dispositivos diseñados. En esta tesis titulada “Conducting polymers micro and nano electrodes. Application to biomolecule sensing and release”, se han investigado nuevas técnicas de fabricación y de funcionalización de polímeros conductores, poniendo un especial interés en su aplicación biomédica. Una nueva técnica de fabricación de microestructuras de polipirrol por método biocatalítico sobre superficies aislantes ha sido desarrollada con resoluciones comparables a las de la litografía óptica. Dicha técnica es compatible con la incorporación de biomoléculas durante el proceso de síntesis, lo que garantiza su utilización en entornos biológicos. Esto fue demostrado mediante la incorporación de biotina durante el proceso de polimerización y su posterior liberación, mediante estimulo eléctrico. También se ha desarrollado un nuevo sensor de ADN sin marcaje basado en electrodos de azida-PEDOT, para la detección de secuencias basadas en la “Hepatitis C”. Estos electrodos, permiten la directa y covalente funcionalización con secuencias de ADN, modificadas con grupos acetileno, por medio de la química “Click”. La hibridación fue detectada mediante la evaluación de la electroactividad del polímero tras el suceso de reconocimiento. Esta novedosa modalidad de sensores demostró ser selectiva y sensible, siendo capaz de detectar secuencias complementarias en el rango nM, sin necesidad de marcajes, ni complejas técnicas de microfabricación. Finalmente, se estudiaron dos técnicas de fabricación de nanohilos de polímero conductor: nanolitografía de dip-pen y electropolimerización sobre superficies con plantillas. Estos estudios proveen al incompleto campo de la fabricación de nanoestructuras de polímeros conductores de resultados adicionales, que amplían el campo de aplicación de dichos materiales.

[spa] La detección de bacterias patógenas y micotoxinas es la clave para la prevención y la identificación de los problemas relacionados con la salud pública y seguridad alimentaria. En esta tesis hemos desarrollado una nueva plataforma de múltiples electrodos (multi-electrodo) para detección impedimétrica de bacterias patógenas y micotoxinas. Nos hemos centrado en la detección de bacterias E. coli O157: H7, ya que son responsable por brotes de origen alimentario graves. Se han caracterizado y estudiado la influencia de la (bio)interfaz del sensor para el desarrollo de un biosensor altamente sensible. Por esta razón, se probaron diferentes estrategias y materiales (óxido de indio - estaño y oro). Todos los pasos fueron completamente caracterizados por medio de múltiples técnicas y los resultados obtenidos mostraron que los biosensores desarrollados tenían una excelente respuesta en términos de sensibilidad y selectividad. Además, se obtuvieron resultados prometedores usando multi-electrodos. Estos fueron fabricados en oro y consistían en múltiples electrodos iguales e independientes que permiten el alto rendimiento y experimentos en paralelo en las mismas condiciones experimentales. Los multi-electrodos fueron caracterizados por varias técnicas de análisis de la superficie y técnicas electroquímicas, confirmando la calidad del proceso de fabricación. Hemos demostrado las capacidades de biosensores del multi-electrodo para la detección de bacterias patógenas utilizando diferentes bio-receptores, incluyendo anticuerpos y péptidos antimicrobianos. También se aplicaron los multi-electrodos para el desarrollo de sensores basados en aptámeros para la detección de micotoxinas. Nos centramos en concreto en el caso de la ocratoxina A (OTA), una de las más abundantes que contaminan los alimentos. Se presentaron dos estrategias para la inmovilización de aptámeros, ambas basadas en la hibridación de la superficie del biosensor a través de oligonucleótidos parcialmente complementarios. Se utilizaron técnicas electroquímicas para caracterizar todas las etapas de funcionalización. El multi-sensor desarrollado es capaz de detectar concentraciones de OTA y los prometedores resultados obtenidos demuestran la aplicación para la detección de micotoxinas y las ventajas de utilizar multi-electrodos., [eng] This thesis aims the development of a multi-electrode platform with applications on different biosensing fields: (i) detection of pathogenic bacteria E.coli O157:H7 and (ii) detection of the mycotoxin Ochratoxin A. For most label-free biosensors, including impedimetric biosensors, the principal limitation on multiplexing arises from the affinity step. Therefore, a great part of the research work described here deals with the characterization, optimization and evaluation of different functionalization strategies for biosensing. These surface functionalization strategies developed here are applied for the final development and fabrication of the multi­electrode biosensor. In Chapter 2, we take advantage of the outstanding properties of indium tin oxide (ITO) material for the development of an ITO-based immunosensor for detecting pathogenic E. coli O157:H7 bacteria. The sensor build-up consisted on a simple, efficient and direct covalent binding of anti-E. coli O157 antibodies onto the ITO substrates. The functionalization methodology was fully characterized by multiple techniques, showing the specific binding of E. coli O157:H7 to the antibody­functionalized surface. The detection capacity of the ITO-based immunosensor was finally tested by EIS and a novel highly sensitive and selective sensor was obtained. In Chapter 3, we develop a gold-based electrochemical immunosensor for the detection of pathogenic E. coli O157:H7 bacteria. Gold is bio-compatible, can be easy obtained and it is easy to pattern using photolithography. In order to enhance the sensor performance, the functionalization protocol was optimized and antibodies were immobilized onto gold electrodes following two different strategies. Both functionalization strategies were evaluated and characterized by several techniques and the strategy showing better antibody immobilization was selected for the development of a highly sensitive label-free immunosensor. The immunosensor showed a very low limit of detection and low interference with other pathogenic bacteria. In Chapter 4, we take advantage of the functionalization strategies developed in the previous chapter 3 to develop a miniaturized multi-electrodes array for the detection of pathogenic bacteria. The multi-electrodes were fabricated in gold and consisted of multiple equally independent electrodes. This allowed high-throughput and independent experiments, in parallel and under the same experimental conditions. Multi-electrodes were fabricated by standard photolithography techniques and characterized by several surface analysis and electrochemical techniques, confirming the quality of the fabrication process. We demonstrated the biosensing capabilities of the multi­electrode platform for the detection of pathogenic bacteria using different bioreceptors, including antibodies and antimicrobial peptides. In Chapter 5, we applied the multi-electrodes platform for the development of an aptamer­based sensor for the detection of mycotoxins. We focused on the specific case of ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins. Two strategies for aptamer immobilization were presented, both based on the hybridization onto the biosensor surface through partially complementary oligonucleotides. Electrochemical techniques were used to characterize all the functionalization steps. The developed multi-sensor was capable to detect OTA concentrations and the promising results obtained prove the successful application of the multi-electrodes strategy for the detection of mycotoxins and the advantages of using multi-electrode platform.

En esta tesis doctoral se ha estudiado de forma sistemática la relación entre los campos eléctricos y las células, y los efectos que éstos tienen a diferentes niveles (supervivencia, metabolismo, expresión proteica, morfología), en dispositivos de tipo Lab-on-a-Chip y aplicándolo a un caso concreto de un concentrador basado en dielectroforesis. Se ha realizado de manera que se ha podido diferenciar la aportación de cada uno de los parámetros eléctricos a los cambios. Se ha diseñado, fabricado y comprobado un chip que permite la aplicación de campos eléctricos controlados a células así como la evaluación de parámetros objetivos. Se han realizado dos versiones del mismo chip, con electrodos en oro y en ITO. El sistema se ha controlado en sus aspectos fluídicos y eléctricos, así como en el comportamiento térmico y la variación de la conductividad con el tiempo. Se ha realizado una simulación en COMSOL para determinar cuál es el rango en el que es más interesante centrar el estudio. Para ello se han simulado las condiciones de campo eléctrico sobre un modelo de la bacteria Escherichia coli como una elipsoide alargada con 3 capas. El análisis de los resultados en frecuencia de la intensidad de campo, densidad de corriente y disipación de potencia muestra que la corriente comienza a atravesar la célula a una frecuencia entre 100 KHz y 1 MHz, y otorga un rol importante a la pared bacteriana en cuanto a diana de la corriente eléctrica. Se ha realizado una simulación complementaria de la variación de la impedancia del sistema formado por el chip y la solución de las bacterias. La combinación de ambas informaciones nos permite escoger como frecuencias más relevantes para el estudio las comprendidas entre 100KHz y 1 MHz. El primer bloque de investigación se ha realizado sobre el modelo biológico bacteriano: Escherichia coli. Las magnitudes evaluadas se han dividido en dos grupos: eléctricas (Intensidad de campo, Corriente y Frecuencia) y no eléctricas (tiempo, temperatura y concentración). Los resultados principales son los siguientes: Se puede establecer como umbral de letalidad de la intensidad de campo eléctrico un valor inferior al considerado habitualmente de 1000 V/cm, ya que se consigue una mortalidad del 80% de la población con una intensidad de campo de 660 V/cm, y de un 50 % con campos de 400 V/cm. Se han evidenciado daños subletales en el aumento de los tiempos de generación, así como en la dinámica de crecimiento. A frecuencias altas la mortalidad se hace más elevada. La corriente eléctrica se evidencia a través de los resultados como la magnitud principal en relación a la supervivencia celular. La aplicación de la temperatura amplifica el efecto del campo eléctrico, mostrando una sinergia entre ambas magnitudes. En general, el tiempo no afecta de forma lineal. Parece haber una disminución de la viabilidad en el primer instante de aplicación del campo eléctrico. Posteriormente las células se adaptan y mayor tiempo de aplicación no repercute en una mayor letalidad. Analizando los valores de la supervivencia a diferentes concentraciones, encontramos que concentraciones muy altas producen un efecto de “protección” sobre las células. Posteriormente se ha profundizado en las implicaciones biológicas subletales que tienen los dispositivos LOC, aplicándolo a un dispositivo concentrador de bacterias, y se han hecho estudios proteómicos y de alteraciones morfológicas. Para valorar el efecto del campo eléctrico se ha realizado un análisis de expresión proteica mediante un SDS-PAGE. A las intensidades utilizadas en el dispositivo concentrador no se observan cambios en el proteograma. Si aumentamos la corriente eléctrica, observamos variaciones en la expresión de las proteínas, correspondientes mayoritariamente a dos grupos: Outer Membrane Proteins A (ompA) y Proteínas 2 ribosomales implicadas en el proceso de translación. También se ha observado que las muestras irradiadas tienen una longitud promedio mayor que las de la muestra control, siendo esto un indicio de posibles mecanismos SOS de reparación. El segundo modelo biológico sobre el que se ha trabajado es la línea celular C2C12 de premioblastos inmortalizados de ratón. En este caso, el estudio ha consistido en el diseño y puesta a punto del sistema capaz de albergar las células, permitir su crecimiento normal y aplicar de forma controlada los campos eléctricos. Se han determinado las pruebas a realizar para estudiar un posible cambio de linaje de destino inducido por campos eléctricos, obteniéndose un patrón de referencia con el cual comparar los resultados obtenidos. El sistema ha demostrado ser adecuado para estos estudios y queda preparado para posteriores investigaciones.

[spa] En años recientes, la comunidad de LOC ha enfocado todos sus esfuerzos en la investigación de nuevas aplicaciones para la biomedicina y biotecnología. Algunos países en vías de desarrollados no tienen tecnologías de diagnóstico adecuadas, además el suministro y almacenamiento de los reactivos es en muchos casos limitado, y en ocasiones cuentan con un acceso limitado al consumo de energía. Por otra parte, los países desarrollados se han encontrado con una población envejecida, y por lo tanto se ha generado la necesidad de contar con nuevas tecnologías para el diagnóstico de enfermedades las cuales sean accesibles y orientadas a una terapia más personalizada. Tanto la microfluídica como los LOC han permitido la integración de funciones de análisis complejas capaces de desarrollar herramientas de diagnostico más precisas, de bajo coste y confiables. Actualmente toda la atención se ha centrado en el diseño de aplicaciones para administración de fármacos 1, análisis celular 2 y diagnostico de enfermedades 3. La introducción de la microfluídica ha servido para mejorar el desarrollo de nuevos dispositivos point-of-care, pero todavía existen algunos problemas que han evitado la producción masiva de estos LOC. Las áreas en las que se pretende conseguir una mejora son la recolección de la muestra, mejora de la interfaz entre el chip y el usuario, tratamiento previo de la muestra, mejorar la estabilidad de los reactivos, trabajo con muestras complejas, detección múltiple de biomarcadores y simplificación del sistema de medida 4. Nuestros esfuerzos se han dedicado en desarrollar un sistema LOC con capacidad de detección electroquímica ajustable a cualquier biomarcador, dependiendo únicamente en la cantidad de muestra y los tiempos de análisis. 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Chin, V. Linder and S. K. Sia, Lab on a Chip, 2012, 12, 2118-2134. 5. R. Rodriguez-Trujillo, C. A. Mills, J. Samitier and G. Gomila, Microfluidics and Nanofluidics, 2007, 3, 171-176. 6. R. Rodriguez-Trujillo, O. Castillo-Fernandez, M. Garrido, M. Arundell, A. Valencia and G. Gomila, Biosensors and Bioelectronics, 2008, 24, 290-296. 7. O. Castillo-Fernandez, R. Rodriguez-Trujillo, G. Gomila and J. Samitier, Microfluidics and Nanofluidics, 2014, 16, 91-99. 8. J. Comelles, V. Hortigüela, J. Samitier and E. Martínez, Langmuir, 2012, 28, 13688-13697. 9. E. Prats-Alfonso, F. García-Martín, N. Bayo, L. J. Cruz, M. Pla-Roca, J. Samitier, A. Errachid and F. Albericio, Tetrahedron, 2006, 62, 6876-6881. 10. J. Vidic, M. Pla-Roca, J. Grosclaude, M.-A. Persuy, R. Monnerie, D. Caballero, A. Errachid, Y. Hou, N. Jaffrezic-Renault, R. Salesse, E. Pajot-Augy and J. Samitier, Analytical Chemistry, 2007, 79, 3280-3290. 11. Y. Hou, S. Helali, A. Zhang, N. Jaffrezic-Renault, C. Martelet, J. Minic, T. Gorojankina, M.-A. Persuy, E. Pajot-Augy, R. Salesse, F. Bessueille, J. Samitier, A. Errachid, V. Akimov, L. Reggiani, C. Pennetta and E. Alfinito, Biosensors and Bioelectronics, 2006, 21, 1393-1402. 12. S. Rodríguez Seguí, M. Pla, J. Minic, E. Pajot‐Augy, R. Salesse, Y. Hou, N. Jaffrezic‐Renault, C. A. Mills, J. Samitier and A. Errachid, Analytical Letters, 2006, 39, 1735-1745. 13. A. Lagunas, J. Comelles, E. Martínez and J. Samitier, Langmuir, 2010, 26, 14154-14161. 14. A. Lagunas, J. Comelles, S. Oberhansl, V. Hortigüela, E. Martínez and J. Samitier, Nanomedicine: Nanotechnology, Biology and Medicine, 2013, 9, 694-701. 15. M. Castellarnau, N. Zine, J. Bausells, C. Madrid, A. Juárez, J. Samitier and A. Errachid, Materials Science and Engineering: C, 2008, 28, 680-685. 16. M. Castellarnau, N. Zine, J. Bausells, C. Madrid, A. Juárez, J. Samitier and A. Errachid, Sensors and Actuators B: Chemical, 2007, 120, 615-620. 17. M. Kuphal, C. A. Mills, H. Korri-Youssoufi and J. Samitier, Sensors and Actuators B: Chemical, 2012, 161, 279-284. 18. D. Caballero, E. Martinez, J. Bausells, A. Errachid and J. Samitier, Analytica Chimica Acta, 2012, 720, 43-48. 19. M. Barreiros dos Santos, J. P. Agusil, B. Prieto-Simón, C. Sporer, V. Teixeira and J. Samitier, Biosensors and Bioelectronics, 2013, 45, 174-180., [eng] In recent years, the LOC community has focused most of its research in the biomedical and biotechnology fields, due to the need of portable, low power consumption and low cost theranostics microdevices. Some developing countries do not have suitable medical diagnostics technologies and the supply and storage of the reagents is in many cases limited as well as the access to energy. Furthermore, developed countries are experimenting population aging needing novel low cost efficient disease-screening technologies. The introduction of LOC and microfluidics allow the integration of complex functions that could lead to the developing of more accurate, cheap and reliable theranostic tools. Current focus of application is focused mostly in drug delivery 1, cellular analysis 2, and disease diagnosis 3. Microfluidics is improving the developing of novel point-of-care devices, but there are some challenges that are slowing down the massive production of these LOC. These areas include new methods for sample collection, world-to-chip interfaces, sample pre-treatment, improvement of long-term stability of reagents, working with complex sample specimens, multiple detection of biomarkers and simplify the read-out 4. The main aim of this thesis work was to create novel, cheap and with a high degree of automatization miniaturized biosensing devices with the objective to facilitate Point-of-Care diagnostics in the near future. Our efforts have been focused into developing a LOC system with electrochemical sensing capabilities adjustable to any biomarker, depending only on sample volumes and required analysis times. The devices integrate low-cost label-free biosensors exploiting microfluidics-based self-functionalization, or specialization. The biosensor functionalization takes place in situ and selectively, just before the sensing, and their area keeps dry and inactive until the test starts. The reagents and the sensing parts are kept separated and brought into contact just before the test, avoiding the need of complex fabrication and storage methods to guarantee functionalization integrity. The novel design reduces the cost of the final instrumentation, by simplifying the measurements, while keeping sensitivities and LODs relevant for the application. Furthermore, since the interaction of antibody and protein is time and concentration dependent, our device has the capability to adjust its sensitivity. We have tuned and characterized our system sensitivity using different biomarkers. The development of our novel devices was possible by exploiting synergies in disciplines previously studied in our group. Particularly, in fields such as microfluidics 5-8, surface functionalization 9-14 and electrochemical biosensors 15-19. Summarizing, we are proposing novel microfluidic devices with integrated biosensors. The systems are based on the principle of laminar co-flow in order to perform an on-chip selective surface bio-functionalization of LOC integrated biosensors. This method has the advantage of performing the surface modification protocols “in situ” before the detection. The system can be easily scaled to incorporate several sensors with different biosensing targets in a single chip. We are proposing a novel voltage and impedance differential measurements; that allow us to simplify the read-out. As biomedical application we focus our attention on the detection of prostate cancer biomarkers. Bibliography 1. I. U. Khan, C. A. Serra, N. Anton and T. Vandamme, Journal of Controlled Release, 2013, 172, 1065-1074. 2. H. Andersson and A. Van den Berg, Sensors and Actuators B: Chemical, 2003, 92, 315-325. 3. M. J. Cima, Annual Review of Chemical and Biomolecular Engineering, 2011, 2, 355-378. 4. C. D. Chin, V. Linder and S. K. Sia, Lab on a Chip, 2012, 12, 2118-2134. 5. R. Rodriguez-Trujillo, C. A. Mills, J. Samitier and G. Gomila, Microfluidics and Nanofluidics, 2007, 3, 171-176. 6. R. Rodriguez-Trujillo, O. Castillo-Fernandez, M. Garrido, M. Arundell, A. Valencia and G. Gomila, Biosensors and Bioelectronics, 2008, 24, 290-296. 7. O. Castillo-Fernandez, R. Rodriguez-Trujillo, G. Gomila and J. Samitier, Microfluidics and Nanofluidics, 2014, 16, 91-99. 8. J. Comelles, V. Hortigüela, J. Samitier and E. Martínez, Langmuir, 2012, 28, 13688-13697. 9. E. Prats-Alfonso, F. García-Martín, N. Bayo, L. J. Cruz, M. Pla-Roca, J. Samitier, A. Errachid and F. Albericio, Tetrahedron, 2006, 62, 6876-6881. 10. J. Vidic, M. Pla-Roca, J. Grosclaude, M.-A. Persuy, R. Monnerie, D. Caballero, A. Errachid, Y. Hou, N. Jaffrezic-Renault, R. Salesse, E. Pajot-Augy and J. Samitier, Analytical Chemistry, 2007, 79, 3280-3290. 11. Y. Hou, S. Helali, A. Zhang, N. Jaffrezic-Renault, C. Martelet, J. Minic, T. Gorojankina, M.-A. Persuy, E. Pajot-Augy, R. Salesse, F. Bessueille, J. Samitier, A. Errachid, V. Akimov, L. Reggiani, C. Pennetta and E. Alfinito, Biosensors and Bioelectronics, 2006, 21, 1393-1402. 12. S. Rodríguez Seguí, M. Pla, J. Minic, E. Pajot‐Augy, R. Salesse, Y. Hou, N. Jaffrezic‐Renault, C. A. Mills, J. Samitier and A. Errachid, Analytical Letters, 2006, 39, 1735-1745. 13. A. Lagunas, J. Comelles, E. Martínez and J. Samitier, Langmuir, 2010, 26, 14154-14161. 14. A. Lagunas, J. Comelles, S. Oberhansl, V. Hortigüela, E. Martínez and J. Samitier, Nanomedicine: Nanotechnology, Biology and Medicine, 2013, 9, 694-701. 15. M. Castellarnau, N. Zine, J. Bausells, C. Madrid, A. Juárez, J. Samitier and A. Errachid, Materials Science and Engineering: C, 2008, 28, 680-685. 16. M. Castellarnau, N. Zine, J. Bausells, C. Madrid, A. Juárez, J. Samitier and A. Errachid, Sensors and Actuators B: Chemical, 2007, 120, 615-620. 17. M. Kuphal, C. A. Mills, H. Korri-Youssoufi and J. Samitier, Sensors and Actuators B: Chemical, 2012, 161, 279-284. 18. D. Caballero, E. Martinez, J. Bausells, A. Errachid and J. Samitier, Analytica Chimica Acta, 2012, 720, 43-48. 19. M. Barreiros dos Santos, J. P. Agusil, B. Prieto-Simón, C. Sporer, V. Teixeira and J. Samitier, Biosensors and Bioelectronics, 2013, 45, 174-180.

In this thesis, pH and potassium all-solid-state ISE based on potentiometry and bioimpedance sensors were designed, fabricated and integrated in a miniaturized array for its application in endoscopic surgery for in vivo ischemia detection inside the stomach. To achieve this goal, the developed array withstood the low pH and corrosive condition in the gastric juice of the stomach, by modifying the surface with a conductive Ag/AgCl ink containing hydrophilic and hydrophobic groups. That creates a stable and robust candidate for low pH applications. However, these sensors have to demonstrate besides stability, high sensitivity, and selectivity. For this purpose, different ionophores specific to a single ion were tested. Octadecyl isonicotinate was the one that shown better results as pH ionophore and valinomycin, bis [(benzo-15-crown-4)-4-ylmethyl] pimelate for potassium detection. All these ionophores were embedded in PVC polymer membrane containing also plasticizers such as 2-nitrophenyl octyl ether, bis (1-butylpentyl) adipate (BBPA) and liphophilic anionic additives such as potassium tetrakis (4-chlorophenyl) borate (KTpClPB). The specific compositions of membranes to detect potassium or pH were optimized for the better performance of the sensors. pH ISE sensor shows a nernstian behavior (-54,38 mV/pH) at low pH and a nearly nernstian behavior at physiological pH (-34,899 mV/pH). Bioimpedance sensor was tested and optimized in vitro with different solutions of ions concentration to mimic ischemia detection and with different kinds of tissues from different nature. For this purpose, chicken fat and breast tissues were taken as a model for mimicking non-ischemic and ischemic states respectively. The effect of electrodes insulation as well as the pressure applied on the tissue was studied. The dependence of the impedance response with different pressure applied to the sensor was overcome by applying magnetic field attachment. The sensor array was modified with ring magnets which were attracted by an external magnet, giving stable and reliable signal discarding mechanical motion. The shape and size of the sensor array were designed for being adapted to the commercially available gastroendoscopes. Round shaped cylinder of 7 mm diameter was fabricated with 12 electrodes pin of 600 µm diameter, containing 3 RE, 3 pH and 2 potassium all-solid-state sensors and 4 electrodes in a row for impedance measurements. The sensor array was successfully integrated in commercial endoscope and inserted inside the pig stomach. The blood flow of certain area of the stomach was interrupted by ligating or crossclamping vessels and organ wall. Ischemia and reperfusion steps were sensed successfully with potassium and pH sensors. These results also indicate that information about hypoxic tissue damage can be collected with this array. Ischemia was also sensed on small intestine tissue by opening the abdominal part of the body and getting the sensor array in contact with the intestine. By crossclamping of mesenteric artery by tourniquets and scissors, ischemic and reperfusion states were controlled. Results proved that ischemia and reperfusion can be monitored by our integrated sensor array. As a conclusion, a novel all-solid-state potentiometric, miniaturized, low cost and mass producible pH, potassium all-solid-state ISE and impedance sensors integrated in an array was successfully fabricated for detecting ischemia inside the stomach by means of endoscopic techniques and also on small intestine. This array was tested in vitro and vivo giving reproducible and reliable results. The developed all-solid-state pH sensors permit low pH sensing from 0.7-2.5, which is the only example in the literature that allows so low pH detection, and so make this sensor a unique device for stomach detection., El diagnóstico médico es uno de los campos que han obtenido más ventajas de la capacidad de los electrodos selectivos de iones (ESI) para la detección de iones, ya que los cambios en la concentración de estos elementos están directamente relacionados con diferentes enfermedades. La detección de isquemia es una de las favorecidas por estos sensores. La isquemia es una disminución del suministro de sangre a un órgano y se requiere una detección rápida y precisa. Los métodos de detección in situ en el tejido de los órganos conllevan una detección temprana de la isquemia y el estómago es uno de los órganos más importantes en la detección de Ischemia. Sin embargo, el bajo pH del jugo gástrico del estómago hace difícil la fabricación de sensores de estado sólido con ESI estables y funcionales, principalmente debido a la interferencia de aniones y al problema de la adhesión entre la membrana ESI y la superficie del electrodo. En esta tesis, se han diseñado y fabricado electrodos selección de iones de pH y potasio ESI de estado sólido basados en la potenciometría y sensores de bioimpedancia y se han integrado en una matriz en miniatura para su aplicación en la cirugía endoscópica para la detección de isquemia in vivo en el interior del estómago. El conjunto de sensores se integró con éxito en endoscopio comercial y se inserto en el interior del estómago de un cerdo. El flujo de sangre de cierta área del estómago se interrumpió mediante la ligación o pinzamiento de los vasos sanguineos y la pared del órgano. Los pasos de isquemia y reperfusión fueron detectados con éxito con los sensores de potasio y de pH. Estos resultados también indican que se puede obtener información sobre el daño en el tejido hipóxico recogido con esta matriz. Los sensores de pH de sólido desarrollados permiten la detección pH bajos de 0,7 a 2,5, que es el único ejemplo en la literatura de detección de pH tan bajos con este tipo de sensores y por lo tanto hacen que sea este sensor de un dispositivo único para la detección de isquemia en el estómago.

[spa] La presente tesis titulada "Construcción de nanoplataformas biomoleculares versátiles vía Nanolitografía Dip-pen y su aplicación en bio-sensing y diferenciación celular" pretende contribuir al campo de la nanobiotecnología, definida como la biotecnología en miniatura donde se utiliza la tecnología de nanofabricación con fines biológicos. Con este objectivo, este trabajo de investigación emplea la relativamente novedosa técnica de nanofabricación llamada Dip-Pen Nanolitografía (DPN) para obtener un patrón de moléculas biológicamente relevantes a micro y nanoescala, tanto para aplicaciones de biosensores como para los estudios de diferenciación celular. - El primer capítulo trata de los desafíos encontrados al trabajar con Dip-Pen Nanolitografía y los llamados multi-pens. Con el fin de facilitar la nivelación de la punta con respecto al sustrato y obtener un control constante de la fuerza ejercida desde las puntas al sustrato, se desarrolló y fabricó un dispositivo. El dispositivo piezo-resistivo resultante se pudo aplicar con éxito y se utilizó para la fabricación de patrones de dos moléculas diferentes en sustratos de oro. Se obtuvo una mejora de la sensiblidad en la detección del punto de contacto en comparación con puntas convencionales - El segundo capítulo trata de la miniaturización de una plataforma biosensora que ya se había desarollado previamente en el laboratorio. Se evaluaron varios métodos diferentes para la fabricación de un patrón de oligonucleótidos mediante DPN sobre oro o sobre vidrio modificado químicamente con el fin de asegurar una unión covalente. La estrategia más exitosa resultó ser la ruta de química click, donde un oligonucleótido con un grupo azida se acopla a un sustrato de vidrio que presenta un grupo alquino, utilizando un catalizador de cobre (cicloadición 1,3-dipolar) y produciendo un triazol. De esta manera, se pudo establecer una plataforma sensora y se pudo evaluar su sensibilidad - El tercer capítulo trata del desarrollo y la fabricación sustratos con patrón mediante DPN para experimentos de diferenciación celular. Una molécula de biotina-tiol fue elegida como patrón, ya que permite la derivatización con estreptavidina y, en un segundo paso, cualquier tipo de molécula de biotina. El patrón se llevó a cabo usando un lípido como molécula para facilitar el transporte de la tinta. De esta manera, el tamaño de la característica del patrón fue de alrededor de 5 micras y el área de patrón general más grande que 1 mm ². Los sustratos se aplicaron con éxito en experimentos de diferenciación celular, usando la proteína BMP-2 biotinilada inmovilizada sobre los sustratos., [eng] The present thesis entitled “Construction of versatile biomolecule nano-platforms via Dip-pen Nanolithography and their application in bio-sensing and cell differentiation” aims at contributing to the field of Nanobiotechnology, best defined as miniaturized biotechnology where nanofabrication technology is used for biological purposes. To this end, this research work employs the relatively novel nanofabrication technique called Dip-pen Nanolithography (DPN) for direct patterning of biologically relevant molecules at the micro- and nanoscale, both for biosensor applications and cell differentiation studies. - The first chapter deals with the challenges encountered when working with Dip-pen Nanolithography and so called multi-pens. In order to facilitate the levelling of the tips with respect to the substrate and also to obtain a constant feedback of the force exerted from the tips to the substrate, a device was developed and fabricated together with a collaboration. The resulting piezoresistive device could be implemented successfully and was used to pattern two different molecules on gold substrates. An improved sensitivity at detecting the touching point could be shown when compared to conventional tips. - The second chapter deals with the miniaturization of an already in-house developed biosensor platform. Therefore, several different approaches were evaluated for the DPN patterning of oligonucleotides on gold or chemically modified glass in order to ensure a covalent attachment. The most successful strategy was the click chemistry route, where an azide bearing oligonucleotide was coupled to an alkyne bearing glass substrate, using a copper catalyst (1,3-dipolar cycloaddition) yielding an triazole. This way, a sensor platform could be established and the sensitivity could be assessed. - The third chapter deals with the development and fabrication of DPN patterned substrates for cell differentiation experiments. A biotin-thiol was chosen for patterning because it allows derivatization with streptavidin and any type of biotinylated molecule. The patterning was accomplished, using a lipid as a carrier molecule. This way, the feature size of the pattern was around 5 µm and the overall pattern area was bigger than 1 mm². The substrates were applied successfully in cell differentiation experiments, having a biotinylated BMP-2 immobilized on the substrates.

[eng] The present thesis work titled “Analysis and characterisation of biological samples in nano and microfluidic devices using AC and DC electric fields” has as main objective study the effects that electric fields have biologic samples to develop microfluidic tools (lab-on-a-chip) that allow to manipulate and sensing these samples. This work is divided in three main issues oriented to develop different modules for a diagnostic device. In the first block, we studied the movement of DNA molecules confined in a nanochannel of 20 nm height under the effect of DC and AC electric fields. The objective is to determine the mechanism behind the size separation of DNA molecules, in gel-free environments. The second block presents the development of a new dielectrophoretic system for size sorting of cells. It is based in the competence between dielectrophoretic forces and the dragging forces generated by the fluid motion. The system is able to separate red blood cells form monocytes in physiological conditions (high conductivity) in continuously flow. And finally, in the third block it is developed a new instrumentation for microcytometry applications based on impedance detection. The electronic device is validated by using a synchronised optical detection system that allow us relate the obtained signal with the position of the cell on the sensing area. The system demonstrated good sensing and sizing capabilities. We also used hydrodynamic focusing, to increase the sensing capabilities of a sensing geometry base don coplanar electrodes., [spa] Esta tesis titulada “Analysis and characterisation of biological samples in nano and microfluidic devices using AC and DC electric fields” tiene como objetivo el estudiar los efectos que tienen los campos eléctricos sobre muestras biológicas con el fin de desarrollar herramientas microfluídicas (lab-on-a-chip) para la manipulación y detección de las muestras biológicas. El trabajo está dividido en tres ámbitos orientados a desarrollar dispositivos o módulos en un dispositivo para diagnóstico. En el primer bloque estudiamos el movimiento de moléculas de ADN (-DNA) en el interior de nanocanales de 20 nm de alto bajo la influencia de campos DC y AC. El objetivo es determinar el mecanismo que hay detrás de la separación por tamaño de esta molécula en ausencia de geles y matrices. En el segundo bloque desarrollamos un nuevo sistema dielectroforético de separación celular por tamaño, basado en la competencia entre la fuerza dielectroforética y las fuerzas fluídicas de arrastre. El sistema se utiliza para separar glóbulos rojos de monocitos en condiciones fisiológicas (alta conductividad) y en flujo continuo. En el tercer bloque desarrollamos una instrumentación para un microcitómetro de flujo basado en medidas de impedancia. El sistema electrónico se valida mediante la utilización de un sistema de óptico sincronizado que nos permite relacionar la señal obtenida con el paso de las células sobre el área de detección. Mediante este sistema se ponen aprueba las capacidades de detección, así como la capacidad de distinguir células por tamaño. Finalmente utilizamos el efecto de la focalización hidrodinámica para mejorar las prestaciones de sensibilidad de un sistema de electrodos coplanares.

[spa] El desarrollo de microambientales artificiales ordenados en un formato de microarray (microarrays celulares, también conocidos en inglés como "cellular microarrays") se ha propuesto como una herramienta que permite el análisis a gran escala de combinaciones de factores que afectan la diferenciación de células madre. Este novedoso acercamiento puede implementarse para imitar superficies planas y de esta forma estudiar su diferenciación, este fin tiene directas implicaciones en el desarrollo de nuevos biomateriales y en ingeniería de tejidos. El objetivo general del presente trabajo de tesis consistió en el diseño y evaluación de microarrays celulares construidos con células madre con el objetivo de estudiar su diferenciación hacia osteoblastos (células óseas) en función de los factores impresos. A estos fines, se utilizó una trazadora de microarrays (microarray plotter) para realizar los microarrays donde diversos factores inmovilizados en micro islas impresas sobre substratos activados químicamente (vidrio y polimetilmetacrilato (PMMA), entre otros). Sobre estos substratos impresos se cultivaron células por períodos de 1 a 8 días y se evaluó su diferenciación al cabo de este tiempo de cultivo. En la presente tesis se hizo una comparación de substratos en función de la cantidad de proteína impresa que retienen inmovilizada en su superficie. Otro objetivo consistió en optimizar los parámetros para la formación del microarray celular (tales como tiempo y concentración de sembrado de células, medio de cultivo, etc..). Finalmente se evaluó la diferenciación de una línea celular hacia osteoblastos en función de la presencia o no de un factor de crecimiento inmovilizado en las islas de microarray.

Projecte Final de Carrera d'Enginyeria Electrònica, Universitat de Barcelona, Facultat de Física. Director: Samitier i Martí, Josep. Any: 2001

La tecnología microelectrónica esta entrando a formar parte de la vida cotidiana. Así, además del ordenador PC, tanto en los vehículos, como en las viviendas es difícil encontrar equipos o subsistemas eléctricos que no incluyan algún tipo de circuito integrado para su control.Aspectos como la automatización de viviendas y edificios se empieza a considerar una cuestión clave para la industria informática. Los cuales se basan en buses de campo. Estos precisan de sensores que le informen del estado de la vivienda, industria, vehículo... que se desea controlar, y actuadores que permitan modificar dicho estado.Sin embargo, los sensores y actuadores son, en general, sistemas analógicos, con salidas difícilmente estandarizables si no se les incluye una electrónica que acondicione la señal, la convierta en digital, y la adapte a dicho estándar.La respuesta a esta necesidad la han dado los microsistemas, que permiten incluir en una oblea de silicio sensores, actuadores y la electrónica de control necesaria dando lugar a los llamados sensores y actuadores inteligentes.Sin embargo, la fabricación de estos microsistemas suele precisar de procesos tecnológicos complejos y costosos, que requieren de etapas que normalmente no se realizan en los procesos CMOS estándar. Esto puede implicar la necesidad de realizar el sensor/actuador en una oblea distinta de la del circuito, por problemas de compatibilidad entre procesos.Esta tesis también se enmarca dentro de este campo, y busca el diseño de un circuito de interfaz para sensores capacitivos micromecanizados en silicio.Teniendo en cuenta la posible utilización posterior, se ha impuesto que el diseño debe ser compatible con una tecnología CMOS de bajo coste. Esto lleva a realizar el diseño en tecnologías concebidas inicialmente para la implementación de circuitos digitales.La salida del interfaz debe ser digital, para poder ser conectado fácilmente a un bus digital. Por este motivo, se ha realizado un estudio de las posibles metodologías de conversión.Por último, se ha buscado un diseño siguiendo una metodología Top-Down, acorde con las utilizadas en las tecnologías digitales. Esto simplifica el proceso de diseño cuando se hacen sistemas mixtos, ya que se pueden realizar los diferentes pasos en paralelo.A la hora de escribir esta tesis también se ha seguido este esquema, organizando los capítulos en el mismo orden.Medida de Microsensores Capacitivos: Describe el problema de la medida, haciendo hincapié en los problemas que se pueden encontrar con un microsensor capacitivo. También realiza el estudio de un microsensor concreto, un acelerómetro xyz para el automovil.Diseño Funcional: En este capítulo se estudian las posibles opciones para el diseño de la interfaz. Y posteriormente se analiza la estabilidad del sistema sensor-interfaz.Diseño Estructural: A partir de las decisiones tomadas en el diseño funcional, se baja al nivel de bloques y se estudia que componentes y dispositivos son más adecuados para el diseño a partir de la modelización y simulaciones realizadas.Diseño Físico: Se explica todo el proceso seguido para el diseño de las máscaras de la interfaz. Para ello se presenta una explicación más detallada de aquellos dispositivos que por sus características son más dependientes de las condiciones de las máscaras.Test: Este último capítulo presenta la metodología seguida para la caracterización de los circuitos, así como los resultados obtenidos