As biocélulas a combustível proporcionam meios de se obter energia de maneira mais sustentável, limpa e renovável e apresentam grande potencial para serem usadas como fontes de energia alternativas para dispositivos eletrônicos de baixa demanda energética. Esta dissertação investiga a bioeletro-oxidação de etanol pela enzima Álcool Desidrogenase (ADH) empregando-se bioânodos com polimerização simultânea do mediador (poli - verde de metileno) e polímero condutor (polipirrol). Uma vez preparado o bioânodo foram realizados estudos para verificar a oxidação do cofator (NADH), formação do produto da bio-oxidação de etanol, estabilidade e também estudos visando aumentar a eficiência energética gerada empregando nanotubos de carbono para aumento de área eletroquímicamente ativa e melhor comunicação com os sítios ativos das enzimas. Foi também preparada uma biocélula completa sem membrana polimérica visando diminuir resistência de transferência de prótons do sistema, com a configuração final constituída de ADH/NAD+, etanol, Lacase/O2 com e sem MWCNT. O bioânodo se mostrou relativamente estável, apresentando um decaimento médio de 36% do valor inicial da densidade de potência após 20 semanas de estocagem. Quando operado em condição contínua (tempo = 4h, E (V)= ½ PCA), o decaimento foi de 39% e de 66% em 12 horas de operação. Os valores de densidade de potência foram melhorados com a adição de MWCNTs sobre o suporte de C antes da eletropolimerização do filme simultâneo, em pH 8,9, obteve-se 275 + 12 ?W cm-2. A eletrólise para este sistema mostrou a formação de acetaldeído com conversão de 18% de etanol. Para a biocélula completa, os melhores resultados foram com a presença de MWCNTs no bioânodo, obtendo-se uma potência de 12,5 +0,9 ?W cm-2. Os resultados obtidos são bastante promissores comparado com a literatura atual e mostram que esse sistema pode ser empregado para construção de BFC. / Biofuel cells provide the means to obtain energy in a more sustainable, clean, and renewable way and have great potential as alternative energy sources for low-energy electronic devices. This dissertation investigates ethanol bioelectrooxidation by the enzyme Alcohol Dehydrogenase (ADH) at bioanodes with simultaneous polymerization of the mediator (polymethylene green) and conductive polymer (polypyrrole). After preparing the bioanode, we investigated cofactor (NADH) oxidation, the ethanol biooxidation product,and bioanode stability. We also conducted studies to increase the generated energy efficiency by using carbon nanotubes to augment the electrochemically active area and to improve communication with the enzyme active sites. We also prepared a complete biofuel cell without polymer membrane to decrease the system proton transfer resistance: the final configuration consisted of ADH / NAD +, ethanol, Laccase , and O2 with or without MWCNTs. The bioanode was relatively stable. The mean decay was 36% of the initial power density after 20 weeks of storage. When the bioanode was operated in continuous condition (time = 4h, E (V) = ½ PCA), the decay was 39% and 66% in 12 hours of operation. The power density values increased upon addition of MWCNTs to the C-support before simultaneous film electropolymerization at pH 8.9, to give 275 + 12 ?W cm-2. Under electrolysis conditions, this system produced acetaldehyde and converted 18% of ethanol. For the complete biofuel cell, the best results were with the presence of MWCNTs in the bioanode, which provided a power of 12.5 +0.9 ?W cm-2. The results obtained here are quite promising if compared to the current literature and show that this system can be used to construct a BFC.

The objectives of the doctoral thesis are following: (i) to design carbohydrate/oxygen enzymatic biofuel cells (EBFCs); (ii) to determine the factors limiting the performance of EBFCs; (iii) to characterise the bioelectrochemical properties of the enzymes adsorbed at conductive nanostructures and evaluate the viscoelasticity of these nanostructures. In this work 5-amino-1,10-phenanthroline (5AP) has been found to be the best redox mediator for glucose oxidase (GOx) enzyme among five studied phenanthroline derivatives with different functional groups. Later the 5AP cross-linked with GOx enzyme on a graphite rod electrode (GRE) was employed as an anode while GRE with co-immobilised horseradish peroxidase (HRP) and GOx was exploited as a cathode in order to design a glucose powered EBFC. A positively charged bi-functional thiol, N-(6-mercapto)hexylpyridinium (MHP), was exploited to electrostatically attach the cellobiose dehydrogenase (CDH) enzymes from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) to the gold nanoparticle (AuNP) surface. This coupling enabled a sufficient direct electron transfer between the enzymes and the AuNP-modified gold surface. Therefore, the HiCDH enzyme, showing better performance characteristics, was employed as an anodic biocatalyst in the designing of a mediatorless carbohydrate (glucose or lactose)/oxygen EBFC. The biocathode of the EBFC was based on bilirubin oxidase from Myrothecium verrucaria directly immobilised on the surface... [to full text] / Disertacinio darbo tikslai: (i) sukonstruoti fermentinius angliavandenių/deguonies biokuro elementus (FBKE); (ii) nustatyti FBKE veikimą ribojančius faktorius; (iii) apibūdinti fermentų, adsorbuotų laidžiose nanostruktūrose, bioelektrokatalizines charakteristikas ir įvertinti šių nanostruktūrų viskoelastines savybes. 5-amino-1,10-fenantrolino (5AF) junginys, iš penkių šiame darbe tirtų fenantrolinų junginių besiskiriančių funkcinėmis grupėmis, įvertintas kaip geriausias elektronų pernašos (EP) tarpininkas gliukozės oksidazės (GO) katalizuojamoje heterogeninėje reakcijoje. 5AF junginys kartu su GO fermentu (5AF/GO) buvo panaudotas anodinio elektrodo konstrukcijoje, o atitinkamai bifermentinė krienų peroksidazės (KP) ir GO sistema (KP/GO) – katodinio elektrodo konstrukcijoje. Šie elektrodai panaudoti gliukozės FBKE kūrimui. Teigiamą krūvį turintis bifunkcinis tiolinis N-(6-merkapto)heksilopiridinio (MHP) junginys panaudotas fermentų imobilizacijai aukso nanodalelių (AuND) paviršiuje elektrostatinės sąveikos būdu. AuND paviršiuje imobilizuoti celiobiozės dehidrogenazės (CDH) fermentai, išskirti iš Corynascus thermophilus (CtCDH) ir Humicola insolens (HiCDH) kamienų, sudarė fermentas-AuND sąsają įgalinančią tiesioginę EP. HiCDH fermentas kaip biokatalizatorius pritaikytas anodinio elektrodo konstrukcijoje AuND/MHP/HiCDH kuriant tiesiogine EP paremtus angliavandenių (gliukozės, laktozės)/deguonies FBKE. Bilirubino oksidazė (BO), tiesiogiai imobilizuota AuND paviršiuje (AuND/BO)... [toliau žr. visą tekstą]

Disertacinio darbo tikslai: (i) sukonstruoti fermentinius angliavandenių/deguonies biokuro elementus (FBKE); (ii) nustatyti FBKE veikimą ribojančius veiksnius; (iii) apibūdinti fermentų, adsorbuotų laidžiose nanostruktūrose, bioelektrokatalizines charakteristikas ir įvertinti šių nanostruktūrų viskoelastines savybes. 5-amino-1,10-fenantrolino (5AF) junginys, iš penkių šiame darbe tirtų fenantrolinų junginių besiskiriančių funkcinėmis grupėmis, įvertintas kaip geriausias elektronų pernašos (EP) tarpininkas gliukozės oksidazės (GO) katalizuojamoje heterogeninėje reakcijoje. 5AF junginys kartu su GO fermentu (5AF/GO) buvo panaudotas anodinio elektrodo konstrukcijoje, o atitinkamai bifermentinė krienų peroksidazės (KP) ir GO sistema (KP/GO) – katodinio elektrodo konstrukcijoje. Šie elektrodai panaudoti gliukozės FBKE kūrimui. Teigiamą krūvį turintis bifunkcinis tiolinis N-(6-merkapto)heksilopiridinio (MHP) junginys panaudotas fermentų imobilizacijai aukso nanodalelių (AuND) paviršiuje elektrostatinės sąveikos būdu. AuND paviršiuje imobilizuoti celiobiozės dehidrogenazės (CDH) fermentai, išskirti iš Corynascus thermophilus (CtCDH) ir Humicola insolens (HiCDH) kamienų, sudarė fermentas-AuND sąsają įgalinančią tiesioginę EP. HiCDH fermentas kaip biokatalizatorius pritaikytas anodinio elektrodo konstrukcijoje AuND/MHP/HiCDH kuriant tiesiogine EP paremtus angliavandenių (gliukozės, laktozės)/deguonies FBKE. Bilirubino oksidazė (BO, tiesiogiai imobilizuota AuND paviršiuje (AuND/BO)... [toliau žr. visą tekstą] / The objectives of the doctoral thesis are following: (i) to design carbohydrate/oxygen enzymatic biofuel cells (EBFCs); (ii) to determine the factors limiting the performance of EBFCs; (iii) to characterise the bioelectrochemical properties of the enzymes adsorbed at conductive nanostructures and evaluate the viscoelasticity of these nanostructures. In this work 5-amino-1,10-phenanthroline (5AP) has been found to be the best redox mediator for glucose oxidase (GOx) enzyme among five studied phenanthroline derivatives with different functional groups. Later the 5AP cross-linked with GOx enzyme on a graphite rod electrode (GRE) was employed as an anode while GRE with co-immobilised horseradish peroxidase (HRP) and GOx was exploited as a cathode in order to design a glucose powered EBFC. A positively charged bi-functional thiol, N-(6-mercapto)hexylpyridinium (MHP), was exploited to electrostatically attach the cellobiose dehydrogenase (CDH) enzymes from Corynascus thermophilus (CtCDH) and Humicola insolens (HiCDH) to the gold nanoparticle (AuNP) surface. This coupling enabled a sufficient direct electron transfer between the enzymes and the AuNP-modified gold surface. Therefore, the HiCDH enzyme, showing better performance characteristics, was employed as an anodic biocatalyst in the designing of a mediatorless carbohydrate (glucose or lactose)/oxygen EBFC. The biocathode of the EBFC was based on bilirubin oxidase from Myrothecium verrucaria directly immobilised on the surface... [to full text]

Les avancées dans le domaine de l'analyse médicale et environnementale exigent des méthodes sensibles et précises pour la détection de molécules organiques nocives. Les travaux de recherche de cette thèse présentent un nouveau système d'affinité pour l'immobilisation de biorécepteurs, de nouvelles stratégies d'amplification du signal et de nouveaux nanomatériaux à base de modèles bio-inspirés afin d'améliorer les performances des biocapteurs ou des biopiles.Dans une première partie, un nouveau système d'affinité supramoléculaire entre la biotine et la beta-cyclodextrine a été étudié et a permis de déterminer une constante d'association de 3 x 10² L.mol-1. Ce système permet l'immobilisation d'une grande variété de biorécepteurs commerciaux marqués avec une biotine sur des surfaces éléctrogénérées pour l'élaboration de biocapteurs. Au-NPs modifiés par des beta-CD a été utilisé avec succès pour "optical antenna" pour l'amplification additionnelle du signal SPRi utilisant des marqueurs QD. La combinaison de ses nano-objets permettent la construction d'immunocapteurs ou de capteurs à ADN très sensible.Dans une deuxième partie, différentes variétés de nanomatériaux tels que les nanodiamants, les nanoparticules magnétiques, les nanotubes de carbone (CNT) et graphène ont été utilisé pour modifier la surface des transducteurs suivies par leur fonctionnalisation non-covalentes par des dérivés pyrène. Le nouveau dépôt de nanotubes « layer-by-layer » et les différentes tailles de nanoparticules avec des porosité variable présentent une approche flexible pour la construction de capteurs enzymatiques et d'immunocapteurs. Le graphène est un matériel d'épaisseur atomique qui doublent la sensibilité SPR pour la détection d'anticorps et d'antigène. Enfin, une réduction bioélectrocatalytique efficace de l'oxygène est reporté en utilisant des CNT fonctionnalisés par les pyrènes pour une application comme biocathode dans les biopiles. / The advancing field of medicine and environmental analysis demands sensitive and accurate methods for sensing harmful organic molecules The research work in this thesis presents a novel affinity system for immobilization of bioreceptors, a novel signal amplification strategy, and novel nanomaterials based bio-designs (architectures) with the improved biosensor or bio-fuel cell (bio-cathode) performances.Firstly, a new affinity system based on supramolecular host-guest interactions between biotin and & beta-CD with an association constant of 3 x 10² M-1 is studied. This allows immobilization of a variety of commercially available biotin labelled bioreceptors for biosensing application. beta-CD modified Au-NPs were successfully applied as optical antenna for additional SPRi signal amplification using QD labels. The beneficial effect of the combination of these nano-objects enables the construction of highly sensitive DNA or immunosensors.Secondly, various kinds of nanomaterials such as nanodiamonds, carbon nanotubes, magnetic nanoparticles, graphene and are employed to modify transducer surface followed by non-covalent functionalization with pyrene derivatives. The novel 3D layer-by-layer deposition of nanotubes and different sized nanoparticles with varying porosity presents a flexible approach towards construction of enzymatic or immuno-sensors. Graphene, a material with atomic thickness doubles the SPR sensitivity towards detection of antibody, anti-CT. Finally, an efficient bioelectrocatalytic reduction of oxygen is reported using pyrene functionalized CNT forest as a bio-cathode for bio-fuel cell applications.

Les biopiles microbiennes sont des sources d’énergies renouvelables utilisant des bactéries qui convertissent une énergie chimique en électricité. Pour cela, l’anode doit collecter les électrons issus des microorganismes. La sélection d’un matériau d’anode possédant de grandes performances est d’une importance cruciale dans la construction d’une biopile microbienne. Le graphène est considéré comme un matériau prometteur avec de grandes possibilités d’application dans de nombreux domaines tels que les batteries Li-ion, les cellules photovoltaïques et les super condensateurs électrochimiques en raison de sa structure nanométrique. Ainsi, la modification de surface par de l’oxyde de graphène réduit a été appliquée à la construction d’anodes pour biopiles microbiennes. La cathode abiotique a aussi été étudiée puisqu’elle présente souvent une limitation cinétique vis-à-vis de la réduction de l’oxygène. Les potentialités des complexes organométalliques, et en particulier les phthalocyanines de nickel, ont été étudiés et appliquées à la construction d’une cathode pour biopile. Ainsi, une biopile hybride à deux compartiments a été construite en combinant une bioanode en mousse d’acier inoxydable modifiée par de l’oxyde de graphène réduite et une cathode en feutre de carbone modifiée avec de la phthalocyanine de nickel. La biopile microbienne ainsi construite utilise du lixiviat de terreau, comme source de microorganismes, pour le développement d’un biofilm électroactif à l’anode et présente une bonne stabilité dans le temps. Le graphène a permis d’obtenir une densité de puissance stable pendant une période 40 jours (24.8 mW/m² en présence d’oxygène pur). La cathode présentée dans ce travail a permis d’obtenir une densité de puissance supérieure à une cathode de platine (7.5 fois supérieur). Par ailleurs, un nouveau design de biopile à deux compartiments a été construit, afin de produire de l’électricité à partir d’une souche pure : Pseudomonas aeruginosa qui est connu pour la formation de biofilm électroactive. Un nouveau design a été proposé, permettant de travailler à alimentation constante et non plus en batch comme cela se fait de manière classique. Cette configuration permet de ne plus avoir de diminution de courant liée à un manque d’apport en carburant. Différents paramètres ont ainsi été explorés tel que le débit d’alimentation, la consommation en glucose dans le compartiment anodique, la variation de pH au cours du temps ainsi que l’évolution de la biomasse. Une première approche d’étude de corrélation de ces différents paramètres est proposée. / Microbial fuel cell (MFC) has been considered as a renewable energy source which uses bacteria to convert chemical energy into electricity. Since the anode, as the electron acceptor for the electroactive bacteria, directly interacts with microorganisms, the selection of high performance anode materials is of crucial importance in the design of a MFC. Recently, graphene has been considered as the intriguing material, attracting strong scientific and technological interest with great application potentials in various fields, such as lithium ion batteries, solar cells and electrochemical super-capacitors, for its unique nanostructure and extraordinary properties. Therefore, surface modification using reduced graphene oxide has been investigated for the construction of anodes. An abiotic cathode has also been investigated since it often has a kinetic limitation regarding the oxygen reduction reaction. The potential of the use of organometallics complexes, and more particularly nickel phthalocyanines (poly-NiTSPc), has been studied and applied to the fabrication of cathodes for biofuel cells applications. Thereby, a dual chamber hybrid MFC has been constructed combining a reduced graphene oxide modified bioanode with a chemical poly-NiTSPc modified carbon felt. This MFC uses compost garden leachate, as source of microorganisms, for the growth of an electroactive biofilm onto the anode and presents an excellent lifetime. Indeed, graphene allowed to obtain a power density stable for 40 fays (24.8 mW/m² with pure oxygen). When the modified carbon felt was used as cathode, the power densities obtained were 7.5 higher than the use of platinum cathode. Furthermore, a new design of a dual chamber MFC has been built up in order to work with a constant flow, to supply continuously substrates to the biofilm formed onto the anode from a pure strain, Pseudomonas aeruginosa, and to avoid to work in a batch as it is usually done. The interest of this configuration is to prevent any current loss due to a lack in supply of substrates. Using this bioreactor as a MFC, different parameters have been explored such as the feed rate and the glucose consumption in the anodic compartment, the evolution of the pH as well as the biomass between the entrance and the exit of the chamber. A first study of the correlation between all these parameters has been proposed.

O objetivo principal desta tese foi o de se desenvolver uma biocélula a combustível enzimática em microfluídica, utilizando a glicose como combustível e o oxigênio como oxidante. Foram utilizadas as enzimas Glicose Oxidase ou Glicose Desidrogenase em um bioânodo, de forma a promover reações bioeletrocatalíticas de oxidação da glicose e as enzimas Lacase ou Bilirrubina Oxidase, de forma a promover reações bioeletrocatalíticas de redução do oxigênio molecular. O trabalho se procedeu por tentativas de imobilizar estas enzimas, de forma a promover o mecanismo de transferência eletrônica direta com um eletrodo. Nas situações as quais isso não foi possível, foram utilizados mediadores eletrônicos, de forma a promover o mecanismo de transferência eletrônica mediada. O melhor par de sistemas de bioeletrodos e mediadores foi escolhido para serem aplicados em uma biocélula a combustível. O trabalho se procedeu em adaptar este par de bioeletrodos desenvolvidos para um sistema de microfluídica em papel, sendo ambos biocátodo e bioânodo em papel. Como as condições de concentração de combustível e de cofatores foram otimizadas para o bioânodo, foi necessário trabalhar com os biocátodos, de forma a apresentar as características de um biocátodo respirador, para melhor utilizar o oxigênio presente no ar e a apresentar um desempenho tão bom quanto o dos bioânodos. A biocélula a combustível em papel possibilitou a geração de energia elétrica por até 18 dias, utilizando uma resistência de 1.7 kΩ, nas condições experimentais ideais. De forma a provar o conceito da tecnologia para aplicações reais, a biocélula a combustível em papel foi demonstrada a ter a capacidade de geração de energia elétrica suficiente para fazer um relógio funcionar por pelo menos 36 horas, utilizando a bebida isotônica Gatorade®, como combustível. / The main objective of this thesis is to develop a microfluidic biofuel cell using glucose as the fuel and oxygen as the oxidant. The enzymes Glucose Oxidase or Glucose Dehydrogenase were used in a bioanode to promove the bioelectrocatalytic oxidation of glucose and the enzymes Laccase or Bilirubin Oxidase to promove the bioelectrocatalytic reduction of the molecular oxygen. The work was conducted by attempts to immobilize these enzymes in order to promote the mechanism of direct electron transfer with the electrode. For the situations where this was not observed, mediators were used in a way to promote the mechanism of mediated electron transfer. The best pair of bioelectrodes and mediatores was chosen to be applied in a biofuel cell. The work was carried out to adapt this par of developed bioelectrodes to a paper based microfluidic system, using both biocathode and bioanode in a paper-like design. As the conditions for concentration of fuel and cofactors were optimized for the bioanode, it was necessary to work on these biocathodes so as to have the characteristics of an air-breathing biocathode for a better use of the oxygen present in the air and to work with a performance as good as the bioanode. The paper based biofuel cell enabled the generation of electricity for up to 18 days using a resistance of 1.7 kΩ within the optimum experimental conditions. In order to prove the concept of this technology for real applications, the paper based biofuel cell was demonstrated to have the capacity for generation of enough electrical energy to power up a clock for at least 36 hours using the isotonic drink Gatored® as fuel.

Biofuel cells (BFCs) that utilize enzymes as catalysts represent a new sustainable and renewable energy technology. Numerous efforts have been directed to improve the performance of the enzymatic BFCs (EBFCs) with respect to power output and operational stability for further applications in portable power sources, self-powered electrochemical sensing, implantable medical devices, etc. This concept article details the latest advances about the EBFCs based on porous nanoarchitectures over the past 5 years. Porous matrices from carbon, noble metal, and polymer promote the development of EBFCs through the electron transfer and mass transport benefits. We will also discuss some key issues on how these nanostructured porous media improve the performance of EBFCs in the end.

A miniaturização de uma biocélula a combustível (BC) enzimática de glicose/O2 para aplicação em dispositivos bioeletrônicos implantáveis representa um grande desafio em eletroquímica moderna. Isso porque é preciso desenvolver bioeletrodos com alta atividade bioeletrocatalítica, com enzimas fortemente ligadas a superfície eletródica. Além disso, o próprio processo de micromanipulação é desafiador, uma vez que é desejável obter biocélulas miniaturizadas e com alta densidade de potência. Assim, propõe-se aqui o desenvolvimento de uma BC que atenda os requisitos supracitados. Para isso, desenvolveram-se bioânodos e biocátodos compostos por folhas de grafeno individuais modificadas com as enzimas glicose desidrogenase (GDh) e bilirrubina oxidase (BOx), respectivamente. Eletrodos de grafeno com área de 10-3 cm2 e espessura de 0,9 ± 0,2 nm foram utilizados em um microchip de Si/SiO2. Observou-se que o grafeno transferido para o microchip permanecia com contaminações de cobre, mesmo após a utilização dos métodos químicos tradicionais de remoção desse metal. A presença de cobre é decorrente do processo de fabricação do grafeno, neste caso, a deposição química em fase vapor (CVD). Para remover qualquer resíduo deste metal, submeteu-se o grafeno a um procedimento de remoção eletroquímica de cobre, denominada aqui como e-etching. Uma vez não observada qualquer corrente faradaica residual associada às impurezas, obtiveram-se os bioeletrodos com a GDh e a BOx. Para a imobilização enzimática, utilizou-se a ligação covalente via funcionalização com o ácido 4-aminobenzóico. As curvas de polarização de estado quase-estacionário obtidas com os bioeletrodos em tampão fosfato pH 7,0 revelaram correntes de onset para oxidação de glicose em -0,13 V e redução de oxigênio em 0,45 V. Por fim, os eletrodos foram utilizados em uma BC sem membrana, operando no microchip de Si/SiO2, em eletrólito tampão fosfato saturado com O2 e glicose 8,0 mmol L-1. A BC apresentou um potencial de circuito aberto em 0,55 V, com densidade de potência volumétrica igual a 1,7 W cm-3, o maior valor reportado até os dias de hoje para uma BC. / The miniaturization of a glucose/O2 enzymatic biofuel cell (BFC) for application in implantable bioelectronic devices is a challenge in electrochemistry. For this purpose, the necessity of bioelectrodes development with high biocatalytic activity such as enzymes strongly attached to electrode surfaces is a current trend. Moreover, the micromanipulation procedure itself is a challenge since the obtention of BFCs with high power density is desirable. Then, the present study shows the partial results obtained in the development of a glucose/O2 BFC with the characteristics exemplified. For the later, bioanodes and biocathodes were obtained with single graphene flakes modified with the enzymes glucose dehydrogenase (GDh) and bilirubin oxidase (BOx), respectively. Graphene flakes electrodes with area of about 10-3 cm2 and thickness of 0,9 ± 0,2 nm were used in a Si/SiO2 microchip. It was observed that transferred graphene to the microchip remained with copper/copper oxide contamination even after the use of conventional methodologies for the remotion of the metal from single graphene foils. The presence of the remaining copper is due to the fabrication process of graphene by chemical vapor deposition (CVD). For the remotion of remaining impurities from graphene, the electrochemical remotion of copper from graphene was carried out in acidic media by the so called e-etching procedure. Since no residual faradaic current was observed due to metal/metal oxide impurities in graphene electrodes, the bioelectrodes were obtained with the enzymes GDh and BOx. The covalent functionalisation of graphene with 4-aminobenzoic acid via diazonium coupling reaction was used for the enzymatic immobilization. The quasi-stationary polarization curves obtained with the bioelectrodes in phosphate buffer pH = 7,0 showed onset oxidation current for glucose at -0.13V and reduction of molecular oxygen starting at +0.45V. Finally, the bioelectrodes were used in a membraneless BFC operating in a Si/SiO2 microchip under saturated oxygen and glucose 8 mmol L-1 in the electrolyte media. The BFC showed an open circuit potential at 0.55V and volumetric power density of 1.7 W cm-3, the highest value reported for an enzymatic BFC so far.

La puissance de la biopile enzymatique à glucose/oxygène est limitée par sa partiecathodique. Afin de contourner cette limitation, nous avons étudié les enzymescathodiques : les Bilirubine oxydases (BODs). Dans le but de mieux appréhender ces BODs, lemécanisme réactionnel, la nature de l’étape limitante et l’effet des sels ont alors été étudiés.Deux mécanismes différents sont retrouvés en fonction du mode de transfert des protons etdes électrons (4 fois 1H+/1e- ou 2 fois 2H+/2e-). De plus, nous avons démontré que l’étapelimitante est l’oxydation du substrat pour les trois substrats testés et que les sels agissent auniveau du cuivre T1. Les principales limitations des BODs sont leur stabilité à 37 °C ainsi queleur inhibition par le NaCl. Deux techniques ont alors été utilisées pour identifier des BODsplus résistantes. La première méthode est l’extraction de nouvelles enzymes à partird’organismes extremophiles. Elle a permis d’isoler la BOD d’Anaerophaga thermohalophilaqui possède une bonne résistance au NaCl mais une densité de courant faible. Dans unsecond temps, afin de reconstruire des séquences ancestrales, la phylogénie de la familledes Bacillus Bacterium a été effectuée. Cette technique a permis l’identification de troisBODs possédant des caractéristiques très intéressantes : la BOD de Bacillus nakamurai etdeux BODs ancestrales (Noeud 10 et Noeud 13). Par exemple, après une heure à 37°C et 140mM de NaCl, le Noeud 10 possède une meilleure densité de courant que la BOD de Bacilluspumilus, qui est l’enzyme utilisée comme base de la phylogénie. La seconde technique estdonc une méthode de choix permettant la découverte de nouvelles enzymes à la fois plusstables et plus résistantes que les enzymes actuelles. Elle ouvre de grandes perspectivespour l’utilisation des BODs comme enzymes cathodiques ou pour d’autres applicationsbiotechnologiques. Enfin, nous avons montré que l’immobilisation de la BOD de B. pumilusdans le matériau Si-(HIPE) permet la décoloration cyclique de colorants chimiques surplusieurs mois. / Power of glucose/oxygen enzymatic biofuel cell is limited by the cathodic part. In order to prevent this limitation, we studied cathodic enzymes: Bilirubin oxidases (BODs). For this purpose, the kinetic mechanism, rate-limiting step and salts effect were determined. Two different mechanisms are observed depending on the electron/proton transfer (4 times1H+/1e- or 2 times 2H+/2e-). We also demonstrated that the rate-limiting step is the substrate oxidation for the three substrates tested and salts act around the T1 copper. Main BODs limitations are their stability at 37°C and their inhibition by NaCl. Two methods were used toidentify the most resistant BODs. The first one was the identification of new enzymes from extremophile organisms. It allows to isolate BOD from Anaerophaga thermohalophila whichhas good NaCl resistance but low current density. In addition, in order to reconstructancestral sequences, phylogeny of Bacillus Bacterium family was performed. This methodidentified three BODs with interesting features: BOD from Bacillus nakamurai and twoancestral BODs (Noeud 10 and Noeud 13). For example, after one hour at 37°C and 140 mMNaCl, Noeud 10 has a better current density than the BOD from Bacillus pumilus, which is theenzyme used as basis for the phylogeny. This second method allowed the discovery of newenzymes that were both more stable and more resistant than actual enzymes. Thistechnique opens up valuable prospects for the use of BODs as cathodic enzymes or for otherbiotechnological applications. In the end, we demonstrated that BOD from B. pumilusimmobilization in Si-(HIPE) materials allows cyclic discoloration of chemical dyes duringseveral months.

Dans la nature, la réduction du dioxygène est catalysée par des enzymes de la famille des oxydoréductases. A l’heure actuelle, ces protéines spécifiques et efficaces sont envisagés comme biocatalyseurs au sein de biopile enzymatique. Dans ce contexte, l’optimisation de l’orientation et de la connexion d’oxydases multi-cuivre (MCOs) pour la réduction d’O2 sur des matrices de nanotubes carbone (CNTs) fonctionnalisées a été étudiée. Dans un premier temps, le transfert électronique direct de la laccase est optimisé par la fonctionnalisation non covalente de CNTs par divers dérivés hydrophobes. La dynamique moléculaire ainsi que la modélisation électrochimique ont permis la rationalisation des performances des différentes biocathodes développées. Dans une seconde approche, la modification spécifique par des groupements pyrène de la surface de laccases modifiées par mutagénèse a également été envisagée. La fonctionnalisation supramoléculaire de CNTs par des feuillets de graphène fonctionnalisés d’une part, et par des nanoparticules d’or d’autre part, a également permis de favoriser la connexion de laccases. La seconde partie présente l’élaboration d’autres types de biocathodes basées sur la connexion directe de bilirubines oxydases. Plusieurs stratégies de fonctionnalisation covalente et non covalente de CNTs ont été envisagées. Les différentes biocathodes élaborées par l’assemblage supramoléculaire de MCOs et de matériaux nanostructurés délivrent des densités de courant de réduction du dioxygène de plusieurs mA cm-2. Ces nouvelles bioélectrodes combinées à une bioanode qui catalyse l’oxydation du glucose ont permis le développement de biopiles enzymatiques glucose/O2 délivrant des densités maximales de puissances allant de 250 µW cm-2 à 750 µW cm-2 selon les conditions expérimentales. Enfin une bioanode à base d’une hydrogénase hyperthermophile a été développée et associée à une biocathode à base de bilirubine oxydase pour former un nouveau design de biopile H2/O2. Au sein de ce dispositif, la biocathode à diffusion de gaz réduit directement l’oxygène provenant de l’air, ce qui permet de s’affranchir de l’utilisation d’une membrane séparatrice tout en protégeant l’hydrogénase de sa désactivation en présence d’oxygène. Cette nouvelle biopile délivre une densité maximale de puissance de 750 µW cm-2. / The reduction of oxygen is realized in nature by oxidoreductase enzymes. Currently, these highly specific and efficient proteins are considered as biocatalysts for the development of biofuel cells. In this context, optimizing the orientation and the connection of multicopper oxidase (MCOs) for the reduction of O2 on functionalized carbon nanotubes was studied. In the first part of this manuscript, direct electron transfer of laccase is assessed and optimized by the non-covalent functionalization of CNTs by various hydrophobic derivatives. Electrochemical modeling and molecular dynamics enabled the rationalization of the developed biocathodes efficiency. In a second approach, the specific modification by pyrene moieties of laccases surface modified by protein engineered has also been considered. Additionally, supramolecular functionalization of CNTs by modified graphene sheets and gold nanoparticles also helped to promote laccase connection. The second part presents the development of other types of biocathodes based on the direct connection of bilirubin oxidase. Several strategies of covalent and non-covalent CNTs functionalization have been considered. The different biocathodes developed by the supramolecular assembly of nanostructured materials and MCOs delivered current density of several mA cm-2 for oxygen reduction. These new bioelectrodes combined with a bioanode which catalyzes the glucose oxidation have enabled the development of glucose/O2 enzymatic biofuel cells; delivering maximum power densities from 250 µW cm-2 to 750 µW cm-2 depending on the experimental conditions. Finally a hyperthermophilic hydrogenase based bioanode was developed and associated with a bilirubin oxidase-based biocathode to form a new design of H2/O2 biofuel cell. Within this device, the gas diffusion biocathode directly reduces oxygen from the air, which eliminates the use of a separation membrane while protecting the hydrogenase from its deactivation in the presence oxygen. This new biofuel cell delivers a maximum power density of 750 µW cm-2.