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Along with an increasing understanding of the harmful effects on the environment of a wide range of pollutants has come the need for more sensitive, faster and less expensive detection methods of identification and quantitation. Many environmental pollutants occur in low levels and often in complex matrices thus analysis can be difficult, time consuming and costly. Because of the availability and easy cultivation of the microorganisms with potentially high specificity, there is considerable interest in the use of living microorganisms as the analytical component (the biocomponent) of sensors for pollutants. While a number of biosensors using bacteria have been developed, yeast has been comparatively rarely used as the biocomponent. Yeast are attractive because they are easy to culture and they are eukaryotes which means their biochemistry is in many respects closer to that of higher organisms. This thesis describes the development of whole cell bioassays that use yeast cells as a sensing element and redox mediators to probe the intracellular redox reactions to monitor the catabolic activity of the yeast resulting from the external substrate, steady-state voltammetry is utilised as the electrochemical detection technique. The isogenic differential enzyme analysis (IDEA) concept of Lincoln Ventures Limited, lead NERF funded research consortium uses bacteria that have been cultured using specific organic pollutants as the carbon source which are the biocomponent in sensors. The use of wild type yeast Arxula adeninivorans that has the ability to use a very wide variety of substrates as sources of carbon and nitrogen was used as an alternative to bacteria to validate the “IDEA” concept. Naphthalene and di-butyl phthalate were chosen as model target contaminant molecules. The performance, detection limits and the usefulness of yeast based biosensor applications for environmental analysis are discussed. This thesis also describes the development and optimisation of a simple, cost effective in vivo estrogens bioassay for the detection of estrogens using either genetically modified or a wild type yeast Saccharomyces cerevisiae. In this study, catabolic repression by glucose was exploited to achieve specificity to estrogens in complex environmental samples that eliminates the requirement for conventional sample preparation. This is the first time that the use of wild type yeast to quantify estrogens has been reported. The attractive features of the bioassay are its use of a non-GMO organism, its speed, its high specificity and sensitivity with a detection limit of 10-15 M. The similarity of binding affinities for major estrogens to those of human estrogens receptors makes this in vivo estrogen bioassay very useful for analytical/screening procedures. The electrochemical detection method also makes it easy to interface with a variety of electronic devices.

Along with an increasing understanding of the harmful effects on the environment of a wide range of pollutants has come the need for more sensitive, faster and less expensive detection methods of identification and quantitation. Many environmental pollutants occur in low levels and often in complex matrices thus analysis can be difficult, time consuming and costly. Because of the availability and easy cultivation of the microorganisms with potentially high specificity, there is considerable interest in the use of living microorganisms as the analytical component (the biocomponent) of sensors for pollutants. While a number of biosensors using bacteria have been developed, yeast has been comparatively rarely used as the biocomponent. Yeast are attractive because they are easy to culture and they are eukaryotes which means their biochemistry is in many respects closer to that of higher organisms. This thesis describes the development of whole cell bioassays that use yeast cells as a sensing element and redox mediators to probe the intracellular redox reactions to monitor the catabolic activity of the yeast resulting from the external substrate, steady-state voltammetry is utilised as the electrochemical detection technique. The isogenic differential enzyme analysis (IDEA) concept of Lincoln Ventures Limited, lead NERF funded research consortium uses bacteria that have been cultured using specific organic pollutants as the carbon source which are the biocomponent in sensors. The use of wild type yeast Arxula adeninivorans that has the ability to use a very wide variety of substrates as sources of carbon and nitrogen was used as an alternative to bacteria to validate the “IDEA” concept. Naphthalene and di-butyl phthalate were chosen as model target contaminant molecules. The performance, detection limits and the usefulness of yeast based biosensor applications for environmental analysis are discussed. This thesis also describes the development and optimisation of a simple, cost effective in vivo estrogens bioassay for the detection of estrogens using either genetically modified or a wild type yeast Saccharomyces cerevisiae. In this study, catabolic repression by glucose was exploited to achieve specificity to estrogens in complex environmental samples that eliminates the requirement for conventional sample preparation. This is the first time that the use of wild type yeast to quantify estrogens has been reported. The attractive features of the bioassay are its use of a non-GMO organism, its speed, its high specificity and sensitivity with a detection limit of 10-15 M. The similarity of binding affinities for major estrogens to those of human estrogens receptors makes this in vivo estrogen bioassay very useful for analytical/screening procedures. The electrochemical detection method also makes it easy to interface with a variety of electronic devices.

Ce travail concerne l'étude de la dégradation enzymatique de micropolluants pharmaceutiques récalcitrants présents dans l'eau. Tout d’abord, les efficacités de trois laccases différentes issues respectivement de : Pycnoporus sanguineus CS43, Trametes versicolor (Tv) et Myceliophtora thermophila ont été comparés lors d’essais de dépollution de solutions modèles renfermant trois antibiotiques (amoxicilline, ciprofloxacine et sulfaméthoxazole) et un antiépileptique (carbamazépine). Les essais ont été réalisés avec les laccases libres en présence ou non de médiateurs redox. L'impact de plusieurs paramètres opératoires sur les performances des enzymes a également été étudié. Puis, une nouvelle méthode d’immobilisation des laccases impliquant l’activation du support (microparticules à base de silice commerciales) par du glutaraldéhyde en phase vapeur a été mise au point et optimisée en utilisant la méthodologie de plans d’expériences. Après immobilisation, la laccase Tv s’est avérée être la plus active. Des essais de dégradation en présence de médiateurs redox ont confirmé l’efficacité de l’enzyme immobilisée et sa possible réutilisation lors de cycles successifs. La toxicité des solutions après traitement a été évaluée par des tests Microtox®. La laccase Tv a également été immobilisée sur des nanoparticules non commerciales à base de silice ou d’argile ainsi que sur des composites à base de silice et d’argile. La laccase Tv immobilisée sur les supports composites riches en silice a montré une plus grande réactivité et de meilleures performances pour l'élimination des composés cibles. / This work is focused on the study of the enzymatic depletion of recalcitrant pharmaceutical micropollutants in water. The potential degradation of three antibiotics (amoxicillin, ciprofloxacin and sulfamethoxazole) and one anti-epileptic (carbamazepine) was studied with three laccases: Pycnoporus sanguineus CS43, Trametes versicolor (Tv) and Myceliophtora thermophila. Free laccase systems were evaluated for pharmaceuticals depletion on model solutions in the presence or absence of redox mediators and the impact of several parameters on the performance of laccases for degradation were studied. The enzymes were then immobilized on different solid supports: commercial silica, laboratory synthetized nano-silica and clay based composite nanomaterials and used for degradation tests. A novel methodology for the covalent binding of laccases onto carriers was developed by using glutaraldehyde in vapour phase and the best immobilization conditions were determined through a 23 full factorial design. The immobilized Tv shown the highest activity and was tested in presence of redox mediators. Moreover, the reusability was evaluated in several degradation cycles and the toxicity of the solutions after treatment was assessed with the Microtox® test. In comparison to laccase immobilized on commercial silica, the Tv supported on laboratory synthetized materials showed higher activity and a better performance for the removal of target compounds.

Ce mémoire est consacré à l'optimisation de la connexion enzymatique d'enzymes pour l'oxydation du glucose et la réduction de O2 sur matrices de nanotube de carbone (CNT) dans les biopiles à glucose.Premièrement, le transfert électronique indirect de la glucose oxydase (GOx) est optimisé dans une matrice nanostructurée de CNT contenant la 1,4-naphtoquinone comme médiateur rédox. Cette bioanode a ensuite été combinée avec des biocathodes similaires à bases d'enzymes à cuivre (laccase et tyrosinase). La biopile GOx-NQ/Lac a permis d'obtenir des puissances maximales de l'ordre de 1,5 mW.cm-2. Les utilisations de cette pile en décharge courte, longue et sa stabilité dans le temps ont également été étudiées. La seconde partie présente la préparation d'une autre anode basée sur la connexion indirecte d'une glucose déshydrogènase NAD+-dépendante (GDH-NAD+) comme alternative pour l'oxydation du glucose. La GDH-NAD+ a été combinée avec un catalyseur d'oxydation de NADH par différentes méthodes. Tout d'abord, elle a été encapsulée au sein du métallopolymère rédox, puis, la modification supramoléculaire a dans un second temps permis d'immobiliser le catalyseur moléculaire et l'enzyme à la surface des CNTs. Ces deux bioanodes ont permis respectivement l'obtention de courants catalytiques d'oxydation du glucose de 1,04 et 6 mA.cm-2. La seconde bioanode a été combinée avec une biocathode à base de BOD et a permis l'obtention de densités de courants maximales de l'ordre de 140 µW.cm-2 La dernière partie concerne l'élaboration d'une biocathode bienzymatique pour la réduction de O2. Le DET de la HRP sur CNTs a dans un premier temps été optimisé par modification de la surface par différents dérivés pyrène. Ensuite, la combinaison de la GOx et de la HRP sur la même électrode a permis de réduire efficacement O2 en 2 étapes. La biocathode est capable de délivrer une densité de courant maximale de l'ordre de 200 µA.cm-2. Cette dernière, combinée avec la bioanode GDH présentée précédemment a permis d'obtenir une biopile opérationnelle en conditions physiologiques et 10 mM de NAD+, en étant capable de débiter une densité de puissance maximale de l'ordre de 57 µW.cm-2. / This work focuses on the optimization of the electrical wiring of glucose oxidizing and dioxygen reducing enzymes on carbon nanotube (CNT) matrixes for glucose biofuel cells.In the first part, glucose oxidase (GOx) mediated electron transfer (MET) is optimized in nanostructured CNTs matrixes by mechanical compression of a CNTs/GOx composite containing 1,4-naphtoquinone as redox mediator. This bioanode was then combined with MCOs (laccase and tyrosinase) based biocathodes. The GOx-NQ/Lac biofuel cell was able to deliver a maximum power density of 1.5 mW.cm-2. The use of this biofuel cell in short/long time discharge and in storage has also been studied. The second part presents the preparation of another bioanode based on the indirect wiring of a NAD+-dependant glucose dehydrogenase (GDH-NAD+) as an alternative for glucose oxidation. The GDH-NAD+ has been combined with an NADH oxidation catalyst by two different techniques. The first one involves the encapsulation of the protein in the metallopolymer redox film, whereas the second one relies on the supramolecular modification of the CNTs by the molecular catalyst and the enzyme. Both bioanodes showed good catalytic properties toward glucose oxidation in presence of NAD+ with respectively 1.04 mA cm-2 and 6 mA cm-2. The latter has been combined with a BOD based biocathode to form a biofuel cell exhibiting maximum power densities of 140 µW cm-2. The last part of this work focuses on the design of a bienzymatic biocathode for O2 reduction. The DET of horseradish peroxidase (HRP) was first investigated and optimized by modification of the CNTs with pyrenes derivatives. The combination of the HRP with the GOx on the same electrode enables an efficient reduction of O2 in a 2-step process. The biocathode could exhibit maximum currents densities of 200 µA cm-2. This cathode along with the previous GDH bioanode formed a biofuel cell functional in physiological conditions and 10 mM NAD+ showing maximum power densities of 57 µW cm-2.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Nesta pesquisa, foram realizados experimentos em batelada e em fluxo contÃnuo com o objetivo de esclarecer diversos aspectos quÃmicos e biolÃgicos envolvidos no processo de remoÃÃo de cor de corantes tÃxteis sob condiÃÃes anaerÃbias na presenÃa de mediadores redox. Os testes em batelada possibilitaram a avaliaÃÃo do efeito de diferentes substratos, dos mediadores redox AQDS e riboflavina e do AQDS imobilizado em esferas de alginato no processo de remoÃÃo de cor de corantes. Foram realizadas tÃcnicas eletroquÃmicas para melhorar o entendimento da relaÃÃo estrutura/atividade. Adicionalmente, foram utilizados quatro reatores UASB para tratamento de efluente contendo corante, avaliando-se principalmente o efeito do substrato etanol, da presenÃa de sulfato e do mediador redox AQDS. Verificou-se que a glicose foi um excelente substrato doador de elÃtrons para reduÃÃo do corante azo Reactive Red 2 ou RR2 (0,3 mM), sendo obtida constante cinÃtica (k1) igual a 1,30 dia-1 e eficiÃncia de remoÃÃo de cor de 97,8%, o que foi atribuÃdo ao fato da reaÃÃo de oxidaÃÃo da glicose ser altamente favorÃvel em meio anaerÃbio. Na presenÃa de formiato, a eficiÃncia de remoÃÃo de cor de RR2 que havia sido de apenas 75,6% na ausÃncia de AQDS, foi de 92,7% quando se adicionou AQDS (50 ÂM), mostrando que este mediador redox canalizou os elÃtrons, oriundos da oxidaÃÃo do substrato, para a molÃcula de corante RR2, corrigindo o problema da competiÃÃo por elÃtrons. O poder catalÃtico dos mediadores redox AQDS e riboflavina foi comprovado para todos os corantes da classe azo, sendo mais marcante para os corantes recalcitrantes aos processos redutivos, baseado nas diferenÃas estruturais. Na presenÃa de 50 ÂM de AQDS, a constante cinÃtica do corante RR2 aumentou 3,5 vezes, em relaÃÃo ao controle (sem mediador redox). Para o corante azo Reactive Red 120 (RR120) obteve-se um aumento da constante cinÃtica de 10,1 e 7,6 vezes na presenÃa de riboflavina e AQDS, respectivamente, em relaÃÃo ao controle. O corante antraquinÃnico Reactive Blue 4 (RB4) foi o corante tÃxtil mais recalcitrante a processos redutivos (k1 = 0,09 dia-1) e mesmo na presenÃa de mediadores redox foram obtidas reduzidas taxas de descoloraÃÃo deste. Os resultados das tÃcnicas eletroquÃmicas esclareceram a resistÃncia deste corante à biodegradaÃÃo. Verificou-se que este corante apresenta uma regiÃo de passivaÃÃo, o que garante maior estabilidade e recalcitrÃncia. O processo redutivo à mais favorÃvel termodinamicamente no corante azo Reactive Orange 16 (RO16) do que no corante antraquinÃnico RB4, como foi comprovado pelas curvas de polarizaÃÃo destes corantes. Os reatores apresentaram eficiÃncias de remoÃÃo de cor do corante azo Congo Red (CR) superiores a 90%, mesmo para elevadas concentraÃÃes de corante, sendo o impacto do AQDS significativo apenas quando havia disponibilidade de elÃtrons no meio para reduÃÃo do corante. Nos reatores em que foi adicionada uma fonte extra de sulfato, interessantemente, mesmo na ausÃncia de substrato doador de elÃtrons, a eficiÃncia de remoÃÃo de cor do corante CR foi de 47,8% (reator livre de AQDS) e 96,5% (reator suplementado com AQDS). A presenÃa de sulfato pode garantir elevadas eficiÃncias de descoloraÃÃo, pois o sulfeto biogÃnico pode funcionar como doador de elÃtrons, sobretudo na presenÃa de mediador redox AQDS. O efeito catalÃtico do AQDS imobilizado em esferas de alginato foi comprovado na descoloraÃÃo dos corantes azo RR2 e RB5 por lodo anaerÃbio, o que traz boas perspectivas para a imobilizaÃÃo de mediadores redox em bioreatores. / In this research, experiments were performed in batch and continuous flow in order to clarify various aspects of chemical and biological processes involved in color removal of textile dyes under anaerobic conditions in the presence of the redox mediators. Batch tests allowed the evaluation of the effect of different substrates, redox mediator AQDS and riboflavin and AQDS immobilized in alginate beads in the process of color removal of dyes. Electrochemical techniques were conducted to improve understanding of the structure/activity. Additionally, we used four UASB reactors for treatment of wastewater containing dye, evaluating mainly the effect of ethanol substrate, the presence of sulfate and redox mediator AQDS. It was found that glucose was indeed an excellent substrate electron donor for azo Reactive Red 2 ou RR2 reduction (0.3 mM), the rate constant (k1) found was equal to 1.30 day-1 and the color removal efficiency was 97.8%, which was mainly because the reaction of glucose oxidation is highly favorable in anaerobic environment. For formate-supplied bottles, efficiency of RR2 color removal was only 75.6% in absence of AQDS, and 92.7% when AQDS was added (50 ÂM). This result indicated that this redox mediator channeled electrons from oxidation substrate towards RR2 molecule, correcting the electrons competition problem. Catalytic power of the redox mediators AQDS and riboflavin was demonstrated for all azo dyes tested, being more pronounced for dyes recalcitrant to reductive processes, based on structural differences. For 50 M AQDS, RR2 kinetic constant increased 3.5 times compared to control (without AQDS). For azo dye Reactive Red 120 (RR120), a rate constant increase of 10.1 and 7.6 fold was found for riboflavin and AQDS bottles, respectively, compared to controls. The anthraquinone dye reactive dye Reactive Blue 4 (RB4) was the most recalcitrant to reductive processes (k1 = 0.09 day-1) providing reduced rates of decolorization even by redox mediators supplementation. The results obtained with the electrochemical techniques clarified the resistance of RB4 to biodegradation. It was found that this dye has a passive region, which ensures greater stability and recalcitrance. The reductive process is thermodynamically more favorable for the azo dye Reactive Orange 16 (RO16) than for the anthraquinone dye RB4, as evidenced by the polarization curves. The reactors showed that Congo Red (CR) color removal efficiencies were above 90% even for high dye concentrations, while AQDS impact was significant only when electrons donor were availability to reduce the dye. In reactors that was added an extra sulfate dosage, interestingly, CR color removal was 47.8% (AQDS-free reactor) and 96.5% (reactor supplemented with AQDS), even during electron donor absence. The presence of sulfate can ensure high efficiency of decolorization, as the biogenic sulfide can act as electron donor, especially channeled by redox mediators. The catalytic effect of AQDS immobilized in alginate beads on color removal could be verified with the azo dyes RR2 and RB5, in bottles incubated with anaerobic sludge, which brings good prospect for redox mediators immobilization in bioreactors.

Le travail présenté dans ce manuscrit est une contribution à la recherche sur la mise en forme d'une nouvelle classe de bioélectrodes nanostructurées, principalement à base de nanotubes de carbone (NTCs). L'oxyde de graphène (GO) a été également évalué pour des applications bioélectrochimiques. Les procédés de fabrication développés autorisent l'ajout d'additifs tels que des médiateurs et des polymères. L'optimisation de la connexion enzymatique de la laccase pour la réduction de l'O2 sur des matrices de nanotubes de carbone ainsi que de la polyphénol oxydase (PPO) pour la détection électrochimique de l'ortho-quinone généré enzymatiquement a été étudiée. Dans un premier temps, le transfert d'électrons direct avec la laccase a été optimisé dans une matrice nanostructurée de NTCs. Dans ce contexte, nous avons examiné plusieurs approches pour immobiliser la laccase tout en l'orientant grâce à l'utilisation de dérivés de l'anthraquinone afin d'améliorer les performances catalytiques de la biocathode. L'immobilisation et l'orientation de l'enzyme ont été réalisées par fonctionnalisation des électrodes par le pyrène-mono-anthraquinone et le pyrène-bis-anthraquinone. La seconde partie présente la préparation d'une autre cathode basée sur la connexion indirecte de la laccase à une matrice nanostructurée de NTCs (buckypaper) contenant du bis-pyrène-ABTS comme médiateur rédox et comme réticulant pour la stabilité mécanique améliorée de ce buckypaper. La dernière partie de ce travail a été consacrée à la production de fibres par filage électrostatique à partir de deux mélanges différents: NTCs/ PAN(polyacrylonitrile) et GO/PAN. De telles fibres ont été utilisées comme électrodes pour des applications bioanalytiques et la bioconversion d'énergie. / This thesis is devoted to the development of a new class of freestanding nanostructured bioelectrodes mainly based on carbon nanotubes (CNTs) Graphene oxide (GO) was also evaluated for its appropriateness for the treated bioelectrochemical approaches. The developed manufacturing processes forming CNTs slides (Buckypapers) or electrospun tissues also allow the confinement with additives like mediators or polymers. The optimization of the enzymatic connection of laccase, for O2 reduction on carbon nanotube arrays, and the polyphenol oxidase (PPO) for the electrochemical detection of enzymatically generated electroactive ortho-quinone was studied. Initially, direct electron transfer of laccase is optimized in a nanostructured CNTs matrix. We examined several approaches to immobilize and orient the laccase using anthraquinone derivatives while improving the catalytic performance of the biocathode. These immobilisation and orientation strategies on electrodes are performed by functionalization using pyrene-mono-Anthraquinone and pyrene-bis-anthraquinone. The second part of this thesis shows the preparation of another biocathode based on the indirect connection of laccase in nanostructured CNT buckypapers containing bis-pyrene-ABTS as a redox mediator and cross-linker, enhancing the mechanic stability of the buckypaper. The last part of this work was devoted to the production of nanofibers by electrospinning from two different blends: CNT / PAN and GO / PAN. Such fiber electrodes were used as bioelectrodes for bioanalytical applications and biological energy conversion.

A presente investigaÃÃo teve como objetivo estudar o efeito do nitrato na remoÃÃo de cor de efluente tÃxtil em reatores anaerÃbios suplementados ou nÃo com mediadores redox. Dois reatores anaerÃbios em paralelo foram operados com um tempo de detenÃÃo hidrÃulica (TDH) de 10 horas, com etanol como co-substrato. O experimento em fluxo contÃnuo foi dividido em trÃs etapas apÃs a obtenÃÃo de condiÃÃes estÃveis de operaÃÃo. Na primeira etapa, os reatores receberam o afluente contendo o corante azo Reactive Red 2 (RR2) em concentraÃÃes variadas. Na segunda fase, um dos reatores foi inoculado com o mediador redox AQDS para testar seu efeito na aceleraÃÃo dos processos de remoÃÃo de cor. Finalmente na terceira fase do experimento, ambos os reatores (suplementado e livre de AQDS) foram inoculados com nitrato em concentraÃÃes variadas. Os resultados provaram que os reatores eram eficientes na remoÃÃo de cor, em que o composto etanol mostrou-se um eficiente doador de elÃtrons para sustentar a reduÃÃo do corante azo sob condiÃÃes anaerÃbias. O mediador redox AQDS aumentou as taxas de reduÃÃo do corante azo, mas o seu efeito nÃo foi tÃo marcante comparado com os experimentos conduzidos anteriormente. Contrariamente Ãs hipÃteses levantadas de que a adiÃÃo de nitrato poderia interferir nas taxas de remoÃÃo de cor e propriedades catalÃticas do mediador redox, nÃo foi verificado nenhum efeito deste composto. Tal observaÃÃo sugeriu que a presenÃa do nitrato em esgotos tÃxteis nÃo diminuirà a capacidade dos reatores anaerÃbios, suplementados ou nÃo com mediador redox, em descolorir corantes azo. Finalmente, os experimentos em batelada avaliaram o efeito do gradiente de concentraÃÃo do doador de elÃtrons (etanol) nas taxas de remoÃÃo de cor, os quais indicaram que as taxas eram proporcionais Ãs concentraÃÃes de etanol testadas e que o mediador redox AQDS teve um efeito mais marcante na cinÃtica de descoloraÃÃo. / The present investigation aimed to study the effect of nitrate on colour removal of dyes in anaerobic bioreactors supplemented or not with redox mediators. Two anaerobic bioreactors in parallel were operated at a hydraulic retention time (HRT) of 10 h, with ethanol as co-substrate. The contÃnuous flow experiment was divided in three steps after steady-state conditions were reached. In the first step, the reactors were fed with an affluent containing the azo dye Reactive Red 2 (RR2) tested in different concentrations. In the second step, one of the reactors was supplemented with the redox mediator AQDS, in order to evaluate its effect on the decolourisation process. Finally, in the third step of the experiment, both reactors (AQDS-supplemented and AQDS-free) received different concentrations of nitrate. The results showed that the reactors were efficient on colour removal, in which the compound ethanol was a good electron donor to sustain colour removal under anaerobic conditions. The redox mediator AQDS increased the decolourisation rates, but its effect was not so evident compared to the previous investigations. Contrary to the raised hypothesis that the nitrate addition could decrease decolourisation rates and catalytic properties of the redox mediators, no effect of nitrate was observed in the bioreactors, suggesting that the presence of nitrate in textile wastewaters will not decrease the capacity of anaerobic reactors supplemented or not with redox mediators to decolourize azo dyes. Finally, batch experiments evaluated the effect of an electron donor concentration gradient on colour removal, which indicated that the decolourisation rates were proportional to the concentration of ethanol tested and the redox mediator AQDS had a more evident effect on the colour removal kinetics.

By flavin's unique structure, nature predestined riboflavin and its derivatives to the participation in redox processes within the bodies of all the living organisms. These biomolecules draw attention with intriguing optical properties and photosensitising abilities. Nature-inspired flavin derivatives share these qualities, and there is also a possibility of fine-tuning for the particular application from the chemical point of view. The thesis deals with two main aims. The first aim handles the synthesis of the trimer heteroaromatic precursor and 1,2-diketone. These key intermediates are essential for the future synthesis of the central aromatic core of the novel NH-free non-fused flavin derivative. The thesis introduces and verifies three approaches, including oxidation of diarylalkynes, nucleophilic addition of a corresponding organolithium compound to a Weinreb amide and benzoin condensation. The second aim covers the properties customization of NH-free fused systems by implementation of linear and bulky alkyl side-chains on the nitrogen atoms N1 and N3 of the alloxazine dilactam. N,N-alkylation introduced an increase in solubility in common organic solvents dichloromethane and chloroform. For the derivatives with 2-(adamantan-1-yl)ethyl substituents, high thermal stability was observed via TGA.