Annually, about 80,000 people die in the United Kingdom due to myocardial infarction, congestive heart failure, stroke, or from other diseases related to blood vessels. The current gold standard treatment for replacing the damaged blood vessel is by autograft procedure, during which the internal mammary artery (IMA) graft or saphenous vein graft (SVG) are usually employed. However, some limitations are associated with this type of treatment, such as lack of donor site and post-surgery problems that could negatively affect the patient’s health. Therefore, this present work aims to fabricate a synthetic blood vessel that mimics the natural arteries and to be used as an alternative method for blood vessel replacement. Polymeric materials intended to be used for this purpose must possess several characteristics including: (1) Polymers must be biocompatible; (2) Biodegradable with adequate degradation rate; (3) Must maintain its structural integrity throughout intended use; (4) Must have ideal mechanical properties; and (5) Must encourage and enhance the proliferation of the cells. The feasibility of using synthetic biodegradable polymers such as poly (ε- caprolactone) (PCL) and poly (lactide-co-glycolic acid) (PLGA) for fabricating tubular vascular grafts was extensively investigated in this work. Many fundamental experiments were performed to develop porous tissue- engineered polymeric membranes for vascular graft purposes through electrospinning technique to achieve the main aim. Electrospinning was selected since the scaffolds produced by this method usually resemble structural morphology similar to the extracellular matrix (ECM). Hence, four 6mm in diameter tubular shape vascular grafts PCL only, PLGA only, coaxial (core-PCL and shell-PLGA), and bilayer (inner layer-PCL and outer layer-PLGA) was designed and fabricated successfully. The structure and properties of each scaffold membrane were observed by scanning electron microscopy (SEM), and these scaffolds were fully characterized by Fourier-transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), water contact angle measurements, mechanical tensile test, as well as cell culture studies were carried out by seeding human umbilical vein cells (HUVEC) and human vascular Fibroblast cells (HVF). Moreover, all polymeric grafts underwent degradation process, and the change in their morphological structure properties was studied over 12 weeks at room temperature. All scaffolds were also exposed to a controlled temperature of 37°C for four weeks, in phosphate-buffered saline solution (pH, 7.3). It was found that all scaffolds displayed exceptional fibre structure and excellent degradability with adequate steady weight-loss confirming the suitability of the fabricated scaffolds for tissue engineering applications. The coaxial and bilayer scaffolds degraded at a much slower (and steadier) rate than the singular PCL and PLGA tubular scaffolds. Coaxial grafts fabricated via coaxial needle showed an increase in their fibre diameter and pore size volume than other membranes, but also showed to have significant tensile strength, elongation at fracture, and Young’s modulus. To conclude, all scaffolds have demonstrated to be reliable to adhere and proliferate HUVEC, and HVF cells, but these cells were attracted to the PLGA membrane more than other fabricated membranes.

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[ES] Los biosensores son dispositivos analíticos con aplicabilidad en diferentes campos y con numerosas ventajas frente a otros métodos analíticos convencionales, como son el uso de pequeños volúmenes de muestra y reactivos, su sensibilidad y su rápida respuesta, sin necesidad de pretratamiento de la muestra, equipos caros o personal especializado. Sin embargo, se trata de un campo de investigación relativamente nuevo en el que todavía queda mucho camino por andar. Esta Tesis doctoral pretende aportar un granito de arena a este campo de conocimiento mediante el estudio del potencial de diferentes materiales porosos como transductores para el desarrollo de biosensores ópticos con respuesta en tiempo real y sin marcajes. Los materiales propuestos van desde aquellos artificialmente sintetizados, como silicio poroso (SiP), nanofibras (NFs) poliméricas o membranas poliméricas comerciales, hasta materiales naturales con propiedades fotónicas que todavía no habían sido explotadas para el sensado, como son los exoesqueletos de biosílice de diatomeas. Todos ellos tienen en común la simplicidad en su obtención, evitando costosos y laboriosos procesos de nanofabricación. Para su estudio, se analizará su respuesta óptica y, en aquellos casos en los que ésta permita llevar a cabo experimentos de detección, se desarrollarán estrategias para su biofuncionalización y su implementación en experimentos de biosensado. En el caso del SiP y las NFs se han optimizado los parámetros de fabricación para obtener una respuesta óptica adecuada que permita su interrogación. A continuación, se ha llevado a cabo su biofuncionalización empleando métodos covalentes y no covalentes, así como diferentes bioreceptores (aptámeros de ADN y anticuerpos) para estudiar su potencial y sus limitaciones como biosensores. En el caso de las membranas comerciales y el exoesqueleto de sílice de diatomeas, se ha caracterizado su respuesta óptica y se han llevado a cabo experimentos de sensado de índice de refracción para estudiar su sensibilidad. Así mismo, se ha desarrollado un método de funcionalización de la superficie del exoesqueleto de diatomeas basado en el uso de polielectrolitos catiónicos. Como resultado, se ha demostrado el potencial tanto de NFs para el desarrollo de biosensores, como el de membranas comerciales para sensores cuya aplicación no requiera una elevada sensibilidad pero sí un bajo coste. Además, se ha puesto de manifiesto el gran potencial del exoesqueleto de diatomeas para el desarrollo de sensores basados en su respuesta óptica. Por el contrario, las limitaciones encontradas en el desarrollo de biosensores basados en SiP han evidenciado la necesidad de un estudio riguroso y la optimización de la estructura de materiales porosos previamente a ser usados en (bio)sensado. / [CA] Els biosensors són dispositius analítics amb aplicabilitat en diferents camps i amb nombrosos avantatges enfront d'altres mètodes analítics convencionals, com són l'ús de xicotets volums de mostra i reactius, la seua sensibilitat i la seua ràpida resposta, sense necessitat de pretractament de la mostra, equips cars o personal especialitzat. No obstant això, es tracta d'un camp d'investigació relativament nou en el qual encara queda molt camí per fer. Aquesta Tesi doctoral pretén aportar el seu òbol a aquest camp de coneixement mitjançant l'estudi del potencial de diferents materials porosos com a transductors per al desenvolupament de biosensors òptics amb resposta en temps real i sense marcatges. Els materials proposats van des d'aquells artificialment sintetitzats, com a silici porós (SiP), nanofibras (NFs) polimèriques o membranes polimèriques comercials, fins a materials naturals amb propietats fotòniques que encara no havien sigut explotades per al sensat, com són els exoesquelets de biosílice de diatomees. Tots ells tenen en comú la simplicitat en la seua obtenció, evitant costosos i laboriosos processos de nanofabricació. Per al seu estudi, s'analitzarà la seua resposta òptica i, en aquells casos en els quals aquesta permeta dur a terme experiments de detecció, es desenvoluparan estratègies per a la seua biofuncionalizació i la seua implementació en experiments de biosensat. En el cas del SiP i les NFs s'han optimitzat els paràmetres de fabricació per a obtenir una resposta òptica adequada que permeta la seua interrogació. A continuació, s'ha dut a terme la seua biofuncionalizació emprant mètodes covalents i no covalents, així com diferents bioreceptors (aptàmers d'ADN i anticossos) per a estudiar el seu potencial i les seues limitacions com a biosensors. En el cas de les membranes comercials i l'exoesquelet de sílice de diatomees, s'ha caracteritzat la seua resposta òptica i s'han dut a terme experiments de sensat d'índex de refracció per a estudiar la seua sensibilitat. Així mateix, s'ha desenvolupat un mètode de funcionalizació de la superfície de l'exoesquelet de diatomees basat en l'ús de polielectròlits catiònics. Com a resultat, s'ha demostrat el potencial tant de NFs per al desenvolupament de biosensors, com el de membranes comercials per a sensors amb una aplicació que no requerisca una elevada sensibilitat però sí un baix cost. A més, s'ha posat de manifest el gran potencial de l'exoesquelet de diatomees per al desenvolupament de sensors basats en la seua resposta òptica. Per contra, les limitacions trobades en el desenvolupament de biosensors basats en SiP han evidenciat la necessitat d'un estudi rigorós i l'optimització de l'estructura dels materials porosos prèviament a ser usats en (bio)sensat. / [EN] Biosensors are analytical devices with application in diverse fields and with several advantages relative to other conventional methods, such as the use of small volumes of sample and reagents, their sensitivity and their fast response, without the need of the sample pretreatment, expensive equipments or specialised technicians. Nevertheless, this is a relatively new research field in which there is a long way to go yet. This doctoral Thesis aims at doing its bit to this field of knowledge by studying the potential of different porous materials as transducers for the development of real-time and label-free optical biosensors. The proposed materials range from those artificially synthesised, such as porous silicon (pSi), polymeric nanofibres (NFs) or commercial polymeric membranes, to natural materials with photonic properties that had not been exploited for sensing yet, such as biosilica exoskeletons of diatoms. All of them have in common its simple production, avoiding expensive and laborious nanofabrication processes. For their study, their optical response will be analysed and, in those cases in which such optical response allows performing detection experiments, strategies for their biofunctionalisation and their implementation in biosensing experiments will be developed as well. Regarding pSi and NFs, the fabrication parameters were optimised to get a suitable optical response for their interrogation. Afterwards, their surface functionalisation was carried out by covalent and non-covalent methods, as well as different bioreceptors (DNA aptamers and antibodies), to study their potential and their constraints as biosensors. Concerning commercial membranes and the biosilica exoskeleton of diatoms, their optical response was characterised and refractive index sensing experiments were carried out to study their sensitivity. Additionally, a biofunctionalisation method for the surface of the diatoms exoskeleton was developed based on the use of cationic polyelectrolytes. As a result, it was demonstrated the potential of NFs for the development of biosensors, as well as the potential of commercial membranes for developing sensors for an application that does not require a high sensitivity but a low cost. Furthermore, the great potential of biosilica exoskeleton of diatoms for the development of sensors based on their optical response has been revealed. By contrast, the constraints found in the development of pSi illustrate the importance of an accurate study and optimisation of porous materials structure before using them for (bio)sensing. / Martínez Pérez, P. (2021). Development and Optimization of Experimental Biosensing Protocols Using Porous Optical Transducers [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/172541 / TESIS