Design and Application of Probes of Novel Bioactivities

Whilst methods for the isolation and optimisation of bacterial enzymes for preparative biotransformations are well established the use of plant biocatalysts is relatively unexplored. Since the plant kingdom contains a greater diversity of potential biocatalysts this represents an untapped resource. Two simple plant enzyme systems were investigated: AtSFGH and AtCXE12. A series of profluorescent esters based on 4 methylumbelliferone and fluorescein were synthesised and used in screening experiments against these enzymes. To produce libraries of recombinant proteins molecular biology techniques such as site-directed mutagenesis and error-prone PCR were utilised and Agrobacterium tumefaciens infiltration methods developed to increase the levels of transformation within plant protoplasts. In order to isolate specific enzymes probe molecules were developed in which a desired bioactivity resulted in the ‘switching on’ of a fluorophore during enzymatic processes. Several reactivity probes were synthesised which contained a fluorophore, a fluorescent quencher moiety and a functional group which was able to react with a specific protein target. An ester probe was developed which reacted with porcine liver esterase, with the enzymatic response causing the molecule to fragment in such a way as to release the fluorescent quencher and result in a concomitant increase in fluorescence. The modular synthesis and structure of the probe allowed for a variety of reactive groups to be introduced. Several BODIPY analogues were synthesised that were able to exclusively target the peroxisome organelle, as demonstrated by co-localisation studies. Their proliferation in the presence of external signals, such as the drug clofibrate and biotic stress, were studied. Confocal microscopy enabled the dynamics of the peroxisome to be visualised in a variety of cell types and species, including both plant and mammalian cells. In addition to this computational studies were carried out to enable the rational design of probes based on their fluorescent properties. Systematic alteration of the probe molecule has highlighted areas of the compound which are amenable to adaptation. This includes the ability to extend the conjugation of the pyrrole functionality which enables the emission wavelength, and hence colour, to be changed

Photochemical and Enzymatic Method for DNA Methylation Profiling and Walking Approach for Increasing Read Length of DNA Sequencing by Synthesis

The first half of this dissertation demonstrates development of a novel method for DNA methylation profiling based on site specific conversion of cytosine in CpG sites catalyzed by DNA methyltransferases. DNA methylation, a chemical process by which DNA bases are modified by methyl groups, is one of the key epigenetic mechanisms used by cells to regulate gene expression. It predominantly occurs at the 5-position of cytosines in CpG sites and is essential in normal development. Aberrant methylation is associated with many diseases including cancer. Bisulfite Genomic Sequencing (BGS), the gold standard in DNA methylation profiling, works on the principle of converting unmethylated cytosines to uracils using sodium bisulfite under strong basic conditions that cause extensive DNA damage limiting its applications. This dissertation focuses on the research and development of a new method for single cell whole-genome DNA methylation profiling that will convert the unmethylated cytosines in CpG sites to thymine analogs with the aid of DNA methyltransferase and photo-irradiation. Previously we synthesized a model deoxycytidine containing an optimized allyl chemical group at the 5-position and demonstrated that this molecule undergoes photo-conversion to its deoxythymidine analog (C to T conversion) with irradiation at 300 nm. The C to T conversion also proved feasible using synthetic DNA molecules. In this thesis, we demonstrate the conversion of a novel modified deoxycytidine molecule (PhAll-dC) using 350 nm photo-irradiation and a triplet photosensitizer (thioxanthone, TX) to avoid potential DNA damage. The new photoproduct was identified as the deoxythymidine analog of the starting molecule as assessed by IR, MS and NMR. An AdoMet analog containing the optimized chemical group was also synthesized and tested for enzymatic transfer to the C5-position of CpG cytosines using DNA methyltransferases. DNA methyltansferase M.SssI was engineered for more efficient enzymatic transfer. In the future, we will incorporate a triplet photosensitizer into the photoreactive moiety on AdoMet to increase energy transfer efficiency for photo-conversion of C to the T analog. Incorporating this into an overall method followed by amplification and sequencing should allow us to assess the methylation status of all CpGs in the genome in an efficient manner. The second half of this dissertation demonstrates development of a DNA sequencing by synthesis (SBS) method, The Sequence Walking Approach, using novel nucleotide reversible terminators (NRTs) together with natural nucleotides. Following the completion of The Human Genome Project, next generation DNA sequencing technologies emerged to overcome the limitations of Sanger Sequencing, the prominent DNA sequencing technology of the time. These technologies led to significant improvements in throughput, accuracy and economics of DNA sequencing. Today, fluorescence-based sequencing by synthesis methods dominate the high-throughput sequencing market. One of the major challenges facing fluorescence-based SBS methods is their read length limitation which constitutes a big barrier for applications such as de novo genome assembly and resolving structurally complex regions of the genome. In this regard, we have developed a novel SBS method called ‘The Sequence Walking Approach’ to overcome current challenges in increasing the single pass read length of DNA sequencing. Our method utilizes three dNTPs together with one nucleotide reversible terminator in reactions called ‘walks’ that terminate at predetermined bases instead of after each incorporation. In this method, the primer extended via 4-color SBS is stripped off and replaced by the original primer for walking reactions. By reducing the accumulation of cleavage artifacts of incorporated NRTs in a single run, our method aims to reach longer read lengths. In this thesis, we have demonstrated a variation of The Sequence Walking Approach in which 4-color sequencing steps are interspersed with walking steps over a continuous length of DNA without stripping off extended primers and reannealing the original primer. The improvements introduced with this method will enable the use of fluorescence-based SBS in many applications such as detection of genomic variants and de novo genome assemblies while preserving low costs and high accuracy

Molecular Probes, Chemosensors, and Nanosensors for Optical Detection of Biorelevant Molecules and Ions in Aqueous Media and Biofluids

Synthetic molecular probes, chemosensors, and nanosensors used in combination with innovative assay protocols hold great potential for the development of robust, low-cost, and fast-responding sensors that are applicable in biofluids (urine, blood, and saliva). Particularly, the development of sensors for metabolites, neurotransmitters, drugs, and inorganic ions is highly desirable due to a lack of suitable biosensors. In addition, the monitoring and analysis of metabolic and signaling networks in cells and organisms by optical probes and chemosensors is becoming increasingly important in molecular biology and medicine. Thus, new perspectives for personalized diagnostics, theranostics, and biochemical/medical research will be unlocked when standing limitations of artificial binders and receptors are overcome. In this review, we survey synthetic sensing systems that have promising (future) application potential for the detection of small molecules, cations, and anions in aqueous media and biofluids. Special attention was given to sensing systems that provide a readily measurable optical signal through dynamic covalent chemistry, supramolecular host–guest interactions, or nanoparticles featuring plasmonic effects. This review shall also enable the reader to evaluate the current performance of molecular probes, chemosensors, and nanosensors in terms of sensitivity and selectivity with respect to practical requirement, and thereby inspiring new ideas for the development of further advanced systems

Applications of Graphene Quantum Dots in Biomedical Sensors

Due to the proliferative cancer rates, cardiovascular diseases, neurodegenerative disorders, autoimmune diseases and a plethora of infections across the globe, it is essential to introduce strategies that can rapidly and specifically detect the ultralow concentrations of relevant biomarkers, pathogens, toxins and pharmaceuticals in biological matrices. Considering these pathophysiologies, various research works have become necessary to fabricate biosensors for their early diagnosis and treatment, using nanomaterials like quantum dots (QDs). These nanomaterials effectively ameliorate the sensor performance with respect to their reproducibility, selectivity as well as sensitivity. In particular, graphene quantum dots (GQDs), which are ideally graphene fragments of nanometer size, constitute discrete features such as acting as attractive fluorophores and excellent electro-catalysts owing to their photo-stability, water-solubility, biocompatibility, non-toxicity and lucrativeness that make them favorable candidates for a wide range of novel biomedical applications. Herein, we reviewed about 300 biomedical studies reported over the last five years which entail the state of art as well as some pioneering ideas with respect to the prominent role of GQDs, especially in the development of optical, electrochemical and photoelectrochemical biosensors. Additionally, we outline the ideal properties of GQDs, their eclectic methods of synthesis, and the general principle behind several biosensing techniques.DFG, 428780268, Biomimetische Rezeptoren auf NanoMIP-Basis zur Virenerkennung und -entfernung mittels integrierter Ansätz

ENVIRONMENTAL IMPLICATIONS AND APPLICATIONS OF NANOMATERIALS

Recent advances in material science and nanotechnology have given rise to a myriad of developments, while in the meantime call for research into the impacts of nanomaterials on the environment and human health. Although considerable progress has been made in the past decade concerning the behavior of nanomaterials in biological systems, such understanding is critically lacking with respect to the fate of nanomaterials in ecosystems. Accordingly, this dissertation addresses the interactions between nanomaterials and algae--the major constituent of the aquatic food chain (Part I, Chapter two), and exploits the physicochemistry of nanoscaled synthetic dendritic polymers for environmental applications, especially for water purification that is a focused theme of the entire dossier (Part II, Chapters two-five). This dissertation is organized as follows. Chapter one presents a general review of the physical/physicochemical properties, characterizations, implications--especially ecological implication, and applications of a host of most produced and studied nanomaterials. In addition, advances in environmental applications of nanomaterials are discussed. Chapter two examines algal responses to two major types of engineered nanomaterials - quantum dots and polystyrene. Inhibited photosynthetic activities of green algae are observed as a result of the physical adsorption of the nanomaterials. Chapter three elucidates the physicochemical properties of poly(amidoamine)-tris(hydroxymethyl)amidomethane- and amine-terminated dendrimers towards their applications in water remediation. Here, the capacities and mechanisms of the dendrimers in hosting cationic copper, anionic nitrate, polyaromatic phenanthrene, and the more heterogeneous humic acids are discussed. Based on the results of Chapter three, Chapter four presents a dendrimer-based novel optical scheme for improving the detection sensitivity and selectivity of environmental pollutants. Specifically, the surface plasmon resonance of a gold nanowire and the high hosting capacity of dendrimers are utilized for enhancing the detection limit of copper down to the nanomolar level. Chapter five exploits a promising use of dendrimers for the removal of potentially harmful discharged nanoparticles. Here fullerenols are used as a model nanomaterial, and their interactions with dendrimers of two different generations are studied using spectrophotometry and thermodynamics methods. Chapter six summarizes the key findings in this dissertation and presents future work that is stimulated by this Doctor of Philosophy (PhD) research

Sensores ópticos de gases tóxicos basados en reactivos selectivos soportados en películas porosas

Programa de Doctorado en Biotecnología, Ingeniería y Tecnología QuímicaLínea de Investigación: Tecnología Química y de MaterialesClave Programa: DBICódigo Línea: 112En esta memoria se han estudiado distintos tipos de materiales con potencial para el desarrollo de sensores ópticos de gases tóxicos que han sido soportados sobre películas porosas para su posterior verificación como sensores de gases. Además, se han establecido distintos procedimientos de calibración para la cuantificación de los analitos estudiados. El Capítulo 1 o Introducción se ha dividido en tres grandes bloques. En primer lugar, se ha elaborado una introducción general sobre la importancia y repercusión que tienen los distintos gases contaminantes. Seguidamente se han expuesto algunos de los métodos analíticos desarrollados para la detección de gases tóxicos. En el apartado tres, se describen las distintas moléculas sensoras que se han usado en el desarrollo de los sensores descritos en esta tesis, así como la razón que ha motivado su selección como candidatas para su uso como parte del sensor en soporte sólido. En el Capítulo 2 o Experimental, se detallan y especifican las características de los materiales utilizados, así como el montaje experimental y la metodología empleados para la caracterización del proceso sensor. Se exponen los detalles técnicos de los equipos y de los reactivos usados. Además de la información que se encuentra en este capítulo, estos datos se complementan en cada uno de los apartados correspondientes de los capítulos de resultados. Los capítulos que se resumen a continuación contienen los resultados obtenidos durante la consecución de esta tesis. En el Capítulo 3, se han usado películas finas nanocristalinas de TiO2 creadas mediante screen printing como matriz para el reactivo de Harrison. Esta película ha sido empleada como sensor óptico de fosgeno gaseoso. Se ha demostrado la capacidad sensora de esta película frente a diferentes concentraciones del analito estudiado, estableciendo, además, un calibrado que permite la cuantificación de fosgeno, tanto en muestras comerciales de este compuesto como en muestras generadas a partir de la descomposición de cloroformo. La capacidad sensora del sistema no se ve mermada por ninguno de los potenciales interferentes que pudieran afectar a la detección de fosgeno, por lo que se puede concluir en que el sensor tiene una excelente selectividad frente al analito estudiado. Además, se ha estudiado la capacidad de incorporar este sensor a un dispositivo portátil que se pueda ser usado de sonda para la detección de compuestos de interés ambiental o industrial. En el Capítulo 4, se ha estudiado la capacidad sensora del (bis(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)sulfano, (NBD)2S, depositado sobre una tira de papel de cromatografía Whatman, frente a H2S gaseoso. El (NBD)2S ha sido sintetizado a partir de su precursor NBD-Cl mejorando el método propuesto por Montoya y col. La exposición al H2S gaseoso se realizó usando la técnica passive sampling o exposición pasiva en la que el sensor se encuentra dentro de una cubeta de volumen conocido y dentro de la misma cubeta se genera el gas a la vez que se monitoriza el cambio que este gas provoca en la alguna propiedad de la muestra. Se estudiaron numerosos interferentes observando que el sensor es selectivo al H2S, sin que ninguno de los posibles interferentes que se puedan encontrar en la detección de H2S le afecte de manera significativa. Se ha establecido un calibrado para un rango de concentraciones que va desde las ppb a las ppm mediante el que, para una muestra problema, se puede determinar la cantidad de H2S presente y/o el tiempo que se tardaría en detectar una muestra con una concentración determinada. Estos límites de cuantificación y detección se encuentran muy por debajo de los límites establecidos como potencialmente peligrosos y también muy por debajo que los límites de detección de algunos dispositivos ópticos comerciales. Además, se ha establecido un calibrado que permite usar las coordenadas del espacio de color Lab extraídas de imágenes de smartphone para la cuantificación del H2S, lo que amplía la versatilidad del sistema diseñado. En el Capítulo 5, se ha desarrollado un sensor de triperóxido de triacetona (TATP) a partir de la incorporación de una clorina, sintetizada por el grupo de Ana M. G. Silva en la Universidad de Oporto en películas de TiO2 creadas mediante screen printing. El anclaje de esta clorina ha sido verificado mediante espectroscopía infrarroja (IR) y la pureza del TATP mediante ESI-MS. Esta clorina tiene un rendimiento cuántico de fluorescencia muy elevado, lo que la hace muy buena candidata para su uso como sonda fluorescente. Se ha probado la capacidad sensora de esta molécula al TATP de manera directa, estableciendo tiempos de respuesta muy cortos a concentraciones muy bajas de este compuesto en estado gaseoso, lo que supone un avance notable a la hora de su detección, puesto que en la mayoría de los casos se reportan detecciones indirectas del TATP a través de los productos de su descomposición. Además, se han realizado pruebas con sus principales interferentes viendo que, incluso a concentraciones relativas altas, estos no causan interferencia en su detección. De esta forma podemos afirmar que el sensor desarrollado es un método muy fiable, selectivo y rápido para la detección de este potente explosivo. En el Capítulo 6, se describe la síntesis y caracterización de dos nuevos ligandos fluorescentes, preparados mediante la combinación de la estructura base de una rosamina a la que se le une un receptor de isoftalato utilizando enlaces de amina y amida (Rosamina-aip1 y aip2). Posteriormente se observó la diferencia espectral, tanto en el espectro UV-Vis como en el espectro de fluorescencia, que existe entre estas dos moléculas. Esta caracterización se llevó a cabo tanto en disolución como en películas sólidas nanocristalinas de TiO2, preparadas mediante la técnica de screen printing. Estas películas fueron estudiadas para maximizar su capacidad sensora, evitando, entre otros, los efectos de agregación. Se determinó la capacidad de la Rosamina-aip2 para ser usada como sonda de fluorescencia, mostrando una menor agregación y una mayor capacidad de emisión que la Rosamina-aip1. Posteriormente se realizarán estudios de sensing con posibles analitos que puedan causar en la molécula algún tipo de variación espectral, bien sea en el espectro UV-Vis o en el espectro de fluorescencia. De esta forma se podrá desarrollar un sensor óptico a partir de la Rosamina-aip2.Universidad Pablo de Olavide de Sevilla. Departamento de Sistemas Físicos, Químicos y Naturale

Advancements in the Synthesis and Application of Near-Infrared Imaging Reagents: A Dissertation

Fluorescence-based imaging techniques provide a simple, highly sensitive method of studying live cells and whole organisms in real time. Without question, fluorophores such as GFP, fluorescein, and rhodamines have contributed vastly to our understanding of both cell biology and biochemistry. However, most of the fluorescent molecules currently utilized suffer from one major drawback, the use of visible light. Due to cellular autofluorescence and the absorbance of incident light by cellular components, fluorescence imaging with visible wavelength fluorophores often results in high background noise and thus a low signal-to-noise ratio. Fortunately, this situation can be ameliorated by altering the wavelength of light used during imaging. Near-infrared (NIR) light (650-900 nm) is poorly absorbed by cells; therefore, fluorophores excited by this light provide a high signal-to-noise ratio and low background in cellular systems. While these properties make NIR fluorophores ideal for cellular imaging, most currently available NIR molecules cannot be used in live cells. The first half of this thesis addresses the synthetic difficulties associated with preparing NIR fluorophores that can be used within living systems. Small molecule NIR fluorophores are inherently hydrophobic which makes them unsuitable for use in the aqueous environment of the cell. Water-solubility is imparted to these dyes through highly polar sulfonates, which subsequently prevents the dyes from entering the cell. The novel work presented here details vii synthetic routes to aid in the development of sulfonated NIR fluorophores, which can be delivered into live cells through the inclusion of an esterase-labile sulfonate protecting group. Application of these synthetic techniques should allow for the development of novel NIR fluorophores with intracellular applications. The second half of this thesis addresses the need for novel NIR imaging reagents. Although several classes of NIR scaffolds do exist, most NIR probes are derivatives of a single class, heptamethine indocyanines. The work described here increases this palette by displaying the ability of NIR oxazines to function as an imaging reagent in live cells and in vivo and as a molecular sensor of biologically-relevant environmental conditions. Combined, the work contained herein has the capacity to not only advance the current NIR toolkit, but to expand it so that fluorescence imaging can move out of the dark and into the NIR light

Interfacing fluorescent DNA oligonucleotides with graphene oxide and metal oxides: from adsorption to sensing

DNA, apart from being the mode of genomic information storage, has found several uses in catalysis (DNAzymes) and target detection (aptamers). Developing novel biosensors utilizing these properties has therefore been a significant avenue for research in recent decades. Of these avenues, interfacing fluorescent dye-labelled DNA with various nanomaterials has birthed many sensors which have been implemented in several environments such as lake water, food, and even within the cell. In this thesis, we provide an improved understanding of DNA adsorption on such nanomaterials and interpretation of sensor results. In Chapter 1, background information related to DNA, fluorescence and nanomaterials are introduced, with associated examples of different biosensor design. The fundamental questions arising from these sensor designs are also stated, along with thesis objectives. In Chapter 2, a comparison is made between graphene oxide and inorganic metal oxides for aptamer-based fluorescence sensing. It was found that, for graphene oxide, target/aptamer interactions dominate the sensor response. This is in contrast to the metal oxide nanoparticles, where sensing is achieved through the target simply displacing DNA from the nanomaterial surface. In Chapter 3, the properties of carboxyfluorescein-labelled poly-C DNA are explored in detail. Through fluorescence and circular dichroism experiments, it was seen that carboxyfluorescein stabilizes i-motif formation in poly-C DNA, even at neutral pH. This folding was irreversible upon heating. Unfolding of the structure led to improved adsorption on GO demonstrated through fluorescence desorption experiments. In Chapter 4, the anomalously high affinity of poly-C adsorption was investigated using both fluorescence experiments and simulations. It was found that the arrangement of cytosines within the chain did not affect affinity, merely their total number. Through simulations, it was determined that poly-C DNA spreads out on the GO surface due to its lack of intrastrand interactions. This results in more phosphate-backbone hydrogen bond sites and a more favourable bond. At lower pH, i-motif formation drastically reduces poly-C affinity to GO; intrastrand interactions dominate over GO/DNA binding. In Chapter 5, fluorescence polarization was used to characterize labelled DNA interactions with various nanomaterials. First, it was determined that, at low labelled-DNA concentrations, polarization is artificially increased by scattering of incident polarized light. Polarization is also increased with the addition of GO to this DNA. Through a simple mathematical derivation, it was shown that the increase in polarization with this kind of surface was due to low concentration of free DNA, rather than adsorption to the GO surface. This was compared to a low-quenching surface (Yttrium oxide), in which the total polarization observed was dominated by the binding DNA rather than free DNA. Overall, the work presented in this thesis improves the current understanding of both fundamental DNA/nanomaterial interactions, as well as its implementation in fluorescence-based sensor designs. Future biosensor construction can incorporate these concepts for better sensitivity, specificity and signal interpretation

The synthesis and characterisation of inorganic and organic luminophores suitable for biomolecule conjugation

Inorganic transition metal complexes have been under extensive investigation for many years in supramolecular assemblies due to their favourable photophysical and redox properties including; absorbance and emission in the visible region of the spectrum, large stokes shifts, long lifetimes, intense luminescence, good photostability and useful photosensitising properties for photodynamic therapy. Their properties make them potentially very valuable biological probes but to date relatively little application of transition metals in this area have been made. This thesis focuses on a range of novel ruthenium and iridium luminophores, their bioconjugates and nanoparticle conjugates which were prepared for applications in cell imaging. A key aim of this thesis was the synthesis, characterisation and identification of novel bioconjugates suitable for applications in cellular imaging. Some preliminary studies of their application in cell imaging are also presented. Chapter 1 outlines how metal complexes have been used previously in cellular imaging and how conjugation of these transition metal complexes to biomolecules has lead to more targeted and improved applications in medical diagnostics, photodynamic therapy, cellular imaging and pharmaceutical drug delivery. Chapters 3 & 4 detail the synthesis and photophysical characterisation of a series of Raman and oxygen sensitive, water soluble and water insoluble ruthenium (II) and novel iridium (III) polypyridyl complexes suitable for biomolecule coupling. Following conjugation of these luminophores to gold nanoparticles in Chapter 3 and cell penetrating peptides in Chapter 5, the dye-conjugates were shown to transport efficiently across the cellular membrane of mammalian SP2 and CHO cells and locate throughout the cell’s organelles. Whereas, using confocal fluorescence microscopy, the parent complexes were shown not to internalise within the cellular structures. The inherent properties of the dyes, such as Raman and lifetime sensitivity, may then be used to determine pH and oxygen levels inside the cell. This could provide critical information for the early detection of certain diseases, as abnormal pH and oxygen levels are indicative of cancerous tumours. Furthermore, the generation of singlet oxygen following light absorption by the luminophores is known to cause additional cell apoptosis. Finally, Chapter 6 describes attempts to functionalise the nucleobase guanine with a fluorescent fluorescein molecule through a short and rigid linker. A range of synthetic techniques such as Suzuki coupling, Sonogashira coupling, click chemistry and Buchwald-Hartwig coupling were used in an effort to achieve this. Using DNA as a scaffold for the first time, the modified nucleoside may be incorporated into the sequence of DNA which may be surface immobilised. Thus, providing an efficient light harvesting supramolecular assembly for the conversion of solar energy into electrical potential

Flavin-binding fluorescent proteins as genetically encoded singlet oxygen photosensitizers

Aquesta tesi profunditza en l'estudi de les proteïnes fluorescents que uneixen flavina com a fotosensibilitzadors d'oxigen singlet codificables genèticament per a la teràpia fotodinàmica. Els fotosensibilitzadors biològics són una alternativa poderosa als fàrmacs convencionals sensibles a la llum degut a la major especificitat front a cèl·lules sanes i a la capacitat d’acumular-se preferentment en orgànuls crítics, gràcies al control genètic de l'expressió cel·lular. Una nova família de proteïnes fluorescents que contenen mononucleòtid de flavina com a cromòfor ha despertat molt d’interès ja que produeixen majors quantitats d'oxigen singlet que la proteïna verda fluorescent i els seus derivats. En aquest treball s'han avaluat les propietats fotofísiques, fotosensibilitzants i antimicrobianes d'onze flavoproteïnes provinents de diferents organismes. Totes les proteïnes estudiades són capaces de produir oxigen singlet i la majoria són altament fototòxiques quan s'expressen en cèl·lules E. coli. Malgrat que comparteixen el mateix cromòfor, les propietats fotofísiques de les proteïnes difereixen notablement d'una a l'altra. Per exemple, algunes són fotosensibilitzadors eficients i són capaces de provocar la mort de cèl·lules bacterianes però fotoblanquegen ràpidament. D’altres produeixen petites quantitats d'oxigen singlet però exhibeixen una elevada fotostabilitat. Els resultats presentats en aquest treball amplien el ventall de flavoproteïnes fotoactives i proporcionen una guia útil per triar la millor opció per a cada aplicació. MiniSOG va ser la primera flavoproteïna desenvolupada racionalment amb l’objectiu de produir d'oxigen singlet. Des de llavors ha estat emprada en nombrosos estudis, però les seves propietats fotoquímiques són complexes i encara resten qüestions pendents de resoldre. Mitjançant estudis estructurals i espectroscòpics s’ha aconseguit racionalitzar la seva modesta producció d’oxigen singlet, dilucidar les transformacions que experimenta quan s’irradia amb llum i establir una base científica sòlida pel desenvolupament racional de nous fotosensibilitzadors d'oxigen singlet codificats genèticament. Així mateix, s'han caracteritzat nous mutants de miniSOG més eficients, així com heterodímers de flavoproteïnes que combinen propietats fotofísiques complementàries. També s’ha demostrat que miniSOG i certs mutants indueixen selectivament la mort de cèl·lules de mamífer quan s’il·luminen amb llum blava, i que és possible combinar proteïnes fotoactives amb reporters fluorescents de processos cel·lulars. L'aplicació de la teràpia fotodinàmica pel tractament del melanoma continua sent un dels principals reptes biomèdics. En aquest estudi, s’ha proposat utilitzar miniSOG per tractar cèl·lules de melanoma. Lamentablement, no ha estat possible comprovar l’expressió correcta de la proteïna fotoactiva, pel que no s’ha pogut avaluar la idoneïtat de la proposta ni extreure’n conclusions.Esta tesis profundiza en el estudio de proteínas fluorescentes que unen flavina como compuestos fotosensibilizadores de oxígeno singlete codificables genéticamente para su uso en terapia fotodinámica. Los fotosensibilizadores biológicos son una poderosa alternativa a los fármacos convencionales sensibles a la luz debido a su mayor especificidad frente a células sanas y a su capacidad para acumularse en orgánulos críticos, gracias al control genético de la expresión celular. Una nueva familia de proteínas fluorescentes que encapsulan el mononucleótido de flavina ha despertado un gran interés por su mayor capacidad de generar oxígeno singlete en comparación con la proteína verde fluorescente y sus derivados. En este trabajo se han evaluado las propiedades fotofísicas, fotosensibilizantes y antimicrobianas de once flavoproteínas provenientes de diferentes organismos. Todas ellas producen oxígeno singlete y la mayoría son altamente fototóxicas una vez expresadas en células E. coli. Aunque comparten el mismo cromóforo, las propiedades fotofísicas difieren notablemente de una a otra proteína. Por ejemplo, algunas son fotosensibilizadores eficientes y destruyen las células bacterianas, pero fotoblanquean rápidamente. Otras producen pequeñas cantidades de oxígeno singlete, pero son más fotoestables. Los resultados obtenidos amplían el abanico de flavoproteínas fotoactivas disponibles y proporcionan una guía útil para elegir la mejor opción para cada aplicación. MiniSOG fue la primera flavoproteína racionalmente desarrollada para generar oxígeno singlete. A pesar de haber sido analizada en numerosos estudios, su fotoquímica es compleja y hay observaciones controvertidas e incógnitas que restan por resolver. Su caracterización estructural y espectroscópica ha permitido comprender los factores que limitan su modesta producción de oxígeno singlete, dilucidar las transformaciones que sufre bajo irradiación y establecer una base científica sólida para el desarrollo de nuevos fotosensibilizadores codificados genéticamente. Además, se han caracterizado nuevos mutantes de miniSOG más eficientes así como heterodímeros de flavoproteínas que combinan propiedades fotofísicas complementarias. También se ha demostrado que miniSOG y ciertos mutantes inducen selectivamente la muerte de células de mamífero cuando se iluminan con luz azul y que es posible combinar proteínas fotoactivas con reporteros fluorescentes de procesos celulares. La aplicación de la terapia fotodinámica para el tratamiento del melanoma sigue siendo uno de los principales desafíos en la biomedicina. En este estudio se ha propuesto la expresión de miniSOG para tratar celulas de melanoma. Sin embargo, no ha sido posible comprobar la expresión correcta de la proteína fotoactiva, por lo que no se ha podido evaluar la idoneidad del enfoque ni sacar conclusiones adicionales.This thesis reports the study of flavin-binding fluorescent proteins as genetically encodable singlet oxygen photosensitizers in photodynamic therapy. Biological photosensitizers are a powerful alternative to conventional light-sensitive drugs owing to their superior targeting potential and localized accumulation in critical organelles, conferred by the genetic control of cell expression. A novel family of fluorescent proteins encasing flavin mononucleotide as the chromophore is gaining much attention since flavoproteins produce higher amounts of singlet oxygen than the proteins derived from the green fluorescent protein family. In this work, the photophysical, photosensitizing and antimicrobial properties of eleven flavoproteins derived from different organisms have been evaluated. All the proteins studied are capable of producing singlet oxygen and most of them are highly phototoxic when expressed in E. coli cells. Although they encase the same chromophore, their photophysical properties differ remarkably from one protein to another. For example, some of them are efficient photosensitizers and kill bacterial cells but show rapid photobleaching. Others produce little amounts of singlet oxygen but exhibit high photostability. The results presented herein expand the toolbox of photoactive flavoproteins and provide valuable guides for choosing the best candidate for a given application. MiniSOG was the first flavoprotein rationally developed for singlet oxygen applications. Since then, it has been a matter of intense research; however, its photochemistry is complex and a number of controversial observations and fundamental questions remain unexplained. The combination of structural and spectroscopic studies has allowed to rationalize its modest singlet oxygen production, elucidate the transformations that it undergoes upon photolysis and establish a sound scientific basis for the rational design and development of new genetically encoded singlet oxygen photosensitizers. Novel miniSOG mutants with improved photosensitizing properties have been characterized as well as flavoprotein heterodimers that combine complementary photophysical properties. It has also been demonstrated that miniSOG and selected mutants induce mammalian cell killing selectively upon light exposure and that it is possible to combine photoactive proteins with fluorescent cell reporters. The application of photodynamic therapy for the treatment of melanoma remains one of the main challenges in the biomedical sciences. In this study, miniSOG has been selected to test the suitability of the genetically encoded approach in melanoma cell lines. Unfortunately, it has not been possible to ascertain the correct expression of the photoactive protein, which has prevented further studies and the possibility of drawing conclusions