Bioimpedance is the term given to the complex impedance value that is characteristic of the resistance that biological cells offer to the flow of electric current. The objective of this study is to analyze the differences in the bioimpedance of highly metastatic MDA-MB-231 and normal MCF 10A breast epithelial cells and use this information to detect a very small number of breast cancer cells present in a background of normal breast cells and other cells that are typically present in a human biopsy sample.To accomplish this, a bioimpedance sensor with flat gold microelectrodes on a silicon substrate was designed and fabricated. Suberoylanilide hydroxamic acid (SAHA), an FDA-approved anti-cancer agent was used to improve the sensitivity of the bioimpedance sensor towards cancer cells by selectively modifying their cytoarchitecture. / Master of Science

Orientador: Paulo Roberto Bueno / Coorientador: Flávio Cesar Bedatty Fernandes / Banca: Hideko Yamanaka / Banca: Marina Ribeiro Batistuti / Resumo: Desde a primeira descrição de biossensor reportada por Clark e Lyons em 1962, houve um extenso trabalho no desenvolvimento e aprimoramento de novas técnicas de biossensoriamento para detecção de biomarcadores com relevância médica. Destaca-se nesse processo o estudo de superfície de eletrodos, pois esse influencia diretamente em aspectos como; sensibilidade, estabilidade e qualidade do sinal. Portanto, este projeto consiste em avaliar comparativamente três superfícies de eletrodos baseadas em nanoestruturas contendo nanopartículas de azul da Prússia, funcionando com sonda redox do sistema, e materiais carbonáceos (como óxido de grafeno e nanotubos de carbono) para aplicação em biossensores. Foram avaliados aspectos como composição, características capacitivas redox e estabilidade de sinal. A técnica de análise utilizada é a espectroscopia de capacitância eletroquímica (ECE) que apresenta vantagens como não usar amplificadores de sinal (sondas redox) em solução, configuração esta, importante para métodos de diagnóstico point-of-care. Das superfícies analisadas, a composta por nanopartículas de azul da Prússia e óxido de grafeno (PBNP+GO) apresentou os melhores parâmetros de estabilidade e compatibilidade com os aspectos teóricos da técnica de ECE, sendo então selecionada para realização de testes de biossensoriamento que, através da funcionalização da superfície com anticorpos Anti-IL-6, detectaram seletivamente a presença do biomarcador IL-6. / Abstract: Since the first description of biosensor reported by Clark and Lyons in 1962, numerous works related to the development and enhancement of novel medical biosensing techniques have been published. In that context, it must be highlighted the study of electrode surfaces as it has direct influence in aspects like; sensitivity, stability and signal quality. Therefore, this project aims to evaluate three electrode surfaces based on nanostructures with Prussian blue nanoparticles, as redox probe, and carbonaceous materials (like graphene oxide and carbon nanotubes) and their application in biosensors. It was evaluated aspects like composition, redox capacitive characteristics and signal stability. The electrochemical capacitance spectroscopy technique (ECE) was used as it offers several advantages like no need of signal amplifiers (redox probes) in solution and, then, making this technique more adequate for point-of-care diagnosis. Among the analysed surfaces, the one composed by Prussian blue nanoparticles and graphene oxide (PBNP+GO) was identified as the best surface in terms of stability and compatibility to the theoretical aspects of ECE. Therefore, that structure was selected to further biosensing essays, by functionalizing the surface with Anti-IL-6 antibodies, that indicated the selective detection of the IL-6 biomarker. / Mestre

The term "Lab-on-a-Chip" (LoC) describes highly miniaturized systems in which the functionalities of entire laboratories are scaled down to the size of transportable microchips. Particularly in the field of chemical and bio-analysis, such platforms are desired for a fast and highly sensitive sample analysis at the point of care. This work focuses on silicon nanowire (SiNW) based sensors. Innovative device fabrication concepts are developed from various directions, for a facile and reliable assembly of LoC analysis systems. Firstly, a multifunctional microfluidic set-up is developed which allows for a facile reversible sealing of channel structures on virtually any kind of substrate while maintaining the possibility of a rapid prototyping of versatile channel designs and the applicability of high working pressures of up to 600 kPa. Secondly, a 3-(triethoxysilyl)propylsuccinic anhydride (TESPSA) based surface modification strategy for the attachment of specific receptor molecules without additional binding site passivation is explored. Thirdly, bottom-up grown SiNWs are utilized for producing parallel arrays of Schottky barrier field-effect transistors (FETs) via contact printing. Using the initially developed microfluidic set-up, the concept of the TESPSA-based receptor immobilization is proved via fluorescence microscopy and by applying the SiNW FETs as biosensors. Using a receptor-analyte system based on a set of antibodies and a peptide from human influenza hemagglutinin, it is shown that antibodies immobilized with the developed method maintain the specificity for their antigens. The fourth major research field in this work is the microfluidics-based alignment of one-dimensional nanostructures and their deposition at predetermined trapping sites for reliably fabricating single NW-based FETs. Such devices are expected to provide superior sensitivity over sensors based on parallel arrays of FETs. Consequently, within this work, innovative LoC devices fabrication approaches over a broad range of length scales, from micrometer scale down to the molecular level, are investigated. The presented methods are considered a highly versatile and beneficial tool set not only for SiNW-based biosensors, but also for any other LoC application. / Unter dem Begriff „Lab-on-a-Chip“ (LoC) fasst man stark miniaturisierte Systeme zusammen, die die Fähigkeiten eines ganzen Labors auf einen transportablem Mikrochip übertragen. Insbesondere im Bereich der Analyse chemischer und biologischer Proben werden solche Plattformen bevorzugt eingesetzt, da sie direkt am Ort der Probenentnahme schnelle, hoch sensible Messungen ermöglichen. Im Mittelpunkt dieser Doktorarbeit stehen Sensoren auf Basis von Siliziumnanodrähten (SiNWs). Auf verschiedenen Gebieten werden innovative Konzepte zur einfachen und zuverlässigen Herstellung von LoC Systemen entwickelt. Zu Beginn wird ein multifunktionaler Mikrofluidik-Aufbau vorgestellt, der ein einfaches reversibles Verschließen von Mikrofluidik-Kanälen auf nahezu allen möglichen Substraten erlaubt. Der Aufbau ermöglicht das schnelle Anfertigen und Testen verschiedener Kanalstrukturen sowie das Betreiben von Fluidik-Experimenten mit hohen Arbeitsdrücken von bis zu 600 kPa. Der zweite Schwerpunkt der Arbeit ist die Entwicklung einer Methode zur Funktionalisierung von Sensor-Oberflächen mittels 3-(Triethoxysilyl) Propyl Bernsteinsäure Anhydrid (TESPSA) für die Immobilisierung spezifischer Rezeptormoleküle. Bei dieser Methode entfällt die Notwendigkeit einer zusätzlichen Passivierung ungenutzter Anbindungsstellen. Des Weiteren erfolgt die Herstellung von Parallelschaltungen von Schottky-Barrieren-Feld-Effekt-Transistoren (SB-FETs) aus „bottom-up“ gewachsenen SiNWs durch mechanisches Abreiben der SiNWs vom Wachstumssubstrat auf ein Empfängersubstrat. Unter Verwendung des eingangs entwickelten Mikrofluidik-Aufbaus wird die prinzipielle Anwendbarkeit der TESPSA-basierten Rezeptor-Immobilisierung nachgewiesen, sowohl anhand von Fluoreszenzmikroskopie-Untersuchungen als auch mit Hilfe der SiNW FETs als Biosensoren. Mittels eines Rezeptor-Analyt-Systems, bestehend aus verschiedenen Antikörpern und einem Peptid des Influenzavirus A, wird gezeigt, dass Antikörper, die über TESPSA an Oberflächen gebunden werden, ihre Spezifizität für ihre Antigene beibehalten. Der vierte große Forschungsabschnitt dieser Arbeit widmet sich der mikrofluidischen Ausrichtung eindimensionaler Nanomaterialien und deren Ablage an vorgegebenen Fangstellen, wodurch eine zuverlässige Herstellung von FETs aus Einzelnanodrähten erreicht wird. Es wird davon ausgegangen, dass Einzelnanodraht-FETs gegenüber Parallelschaltungen von Nanodraht-FETs verbesserte Sensoreigenschaften aufweisen. Folglich beinhaltet diese Arbeit viele zukunftsweisende Ansätze für die Herstellung von LoC Systemen. Untersuchungen über eine Bandbreite von Längenskalen, von Mikrometer großen Strukturen bis hinab zur molekularen Ebene, werden präsentiert. Es wird davon ausgegangen, dass die vorgestellten Methoden als eine vielfältige Sammlung von Werkzeugen nicht nur bei der Herstellung von Biosensoren auf SiNW-Basis Einsatz finden, sondern ganz allgemein den Aufbau verschiedenster LoC Systeme vorantreiben.

Vários estudos que tentam compreender a gênese da aterosclerose têm demonstrado evidências que a oxLDL é peça importante para o desenvolvimento da doença, tornando-a um importante marcador. Os objetivos deste trabalho foram propor um novo imunossensor empregando anticorpos monoclonais anti-oxLDL e desenvolver um processo inovador de obtenção de anticorpos plásticos anti-oxLDL. Para construção do imunossensor, a região Fc do anticorpo foi ligada ao eletrodo de trabalho do dispositivo AuSPE (Screen-Printed Gold Electrodes), utilizando cisteamina. Posteriormente, foi adicionado BSA como bloqueador de possíveis regiões livres. Após o bloqueio, o imunossensor foi testado com oxLDL e outros antígenos, como forma de garantir a especificidade. Em relação aos anticorpos plásticos, chamados MAPS, estes foram desenvolvidos contruindo uma camada impressa de forma invertida ao anticorpo plástico SPAN, referência deste trabalho. Após obtenção dos MAPSs, estes passaram por diversos testes, similar ao imunossensor. Os resultados demonstraram excelente sensibilidade e especificidade às moléculas de oxLDL com detecção em tempo real em ambas as metodologias. / Increased levels of plasma oxLDL are associated with atherosclerosis, and the subsequent development of severe cardiovascular diseases that are today a major cause of death in modern countries. It is therefore important to find a reliable and fast assay to determine oxLDL. A new Immunosensor employing three monoclonal antibodies against oxLDL and a backside protein-surface imprinting process are proposed in this work. To generate the Immunosensor the mAbs were set-up by cysteamine on a gold layer of a disposable screen-printed electrode. BSA was immobilized further as bloker. All steps were followed by various characterization techniques such as electrochemical impedance spectroscopy and square wave voltammetry. To generate specific synthetic antibody materials, called MAPS, these were developed with a backside protein-surface imprinting process of the plastic antibody SPAN, the reference of the work. The devices were successfully applied to determine the oxLDL fraction in real serum, without prior dilution or necessary chemical treatment. Overall, these were promising results with the possibility to apply on the practical use of clinical.

Este trabalho descreve a preparação, a caracterização e o uso de um biossensor de pasta de carbono modificado com a enzima acetilcolinesterase para a quantificação de carbamatos em alimentos, bem como o estudo das constantes de velocidade para a reação enzima/substrato (iodeto de acetiltiocolina) em solução. Inicialmente foram realizados testes de estabilidade, tanto para o substrato quanto para a enzima. Nestes testes, a absorção na região do UV-vis do substrato não apresentou diminuição significativa em 11 dias de análises, já a enzima apresentou uma grande perda de sua atividade com apenas três dias de preparo da solução. Na preparação do eletrodo de trabalho alguns parâmetros foram otimizados, tais como: quantidade de enzima e de ftalocianina de cobalto (CoPC) no eletrodo, bem como a porcentagem de glutaraldeído utilizada. A melhor resposta ocorreu para adição de 2,40x10-3g de enzima, 0,90x10-3g de CoPC (referentes à massa de 0,017g de pasta de carbono) e solução de glutaraldeido 1%. A seguir, realizou-se um experimento baseado na inibição da atividade da enzima, causada pela imersão do eletrodo na solução contendo o pesticida carbaril nas concentrações 5,00x10-5 e 1,00x10-4 mol L-1. Notou-se que, com o aumento da concentração do carbaril, houve aumento na inibição da atividade enzimática. Desta forma o eletrodo apresentou-se apto para determinação analítica de pesticidas. Estas medidas foram realizadas em meio de tampão fosfato 0,1 mol L-1, pH 7,4 e com tempo de incubação para o carbaril, metomil e aldicarbe foram de 8, 12 e 15 minutos, respectivamente. Os limites de detecção (LD) e quantificação (LQ) obtidos utilizando-se o biossensor amperométrico para o carbaril foram de 2,00x10-6 mol L-1 (0,40 mg L-1) e 6,70 x 10-6 mol L-1 (1,30 mg L-1), para o metomil de 1,88 x 10-7mol L-1 (30,45 micro g L-1) e 6,26 x 10-7 mol L-1 (0,10 mg / L-1) e para o aldicarbe de 1,10x10-6 mol L-1 (0,20 mg L-1) e 3,60x10-6 mol L-1 (0,70 mg L-1). Para a formulação comercial Lannate (metomil) os LD e LQ foram 2,13x10-7 mol L-1 (34,50 micro g L-1) e 7,09x10-7 mol L-1(0,12 mg L-1), respectivamente. As medidas de HPLC apresentaram LD e LQ de 1,58 x 10-8 mol L-1 (3,18 micro g L-1) e 5,27x10-8 mol L-1 (10,60 micro g L-1) para o carbaril e de 9,02 x 10-10 mol L-1 (0,15 micro g L-1) e 3,00 x 10-9 mol L-1 (48,60 micro g L-1) para o metomil. Testes de recuperação foram realizados usando ambas as técnicas para o carbaril e Lannate. As recuperações utilizando-se o biossensor mostraram-se eficientes, variando de 76,83 a 106,67% para o carbaril e de 78,00 a 96,50% para a Lannate, enquanto que nas medidas de HPLC, as recuperações foram de 78,00 a 108,33% para o carbaril e de 57,00 a 99,50% para o Lannate. A recuperação para o aldicarbe no tomate foi de 62,40 %. As análises da enzima em solução mostraram que a metodologia empregada neste estudo é adequada para a determinação das constantes de velocidade para a etapa lenta da reação AchE/AchI. Observou-se que os valores destas constantes são dependentes da concentração dos pesticidas fenitrothion (organofosforado) e carbaril (carbamato), em baixa concentração ambos apresentaram constantes de velocidade similares, mas com o aumento dessa concentração, o fenitrothion apresentou menor constante de velocidade em relação ao carbaril, sugerindo que este apresenta maior inibição da enzima e por conseqüência maior toxicidade no organismo. Esses resultados mostraram uma possível metodologia analítica para a quantificação destes pesticidas, obtendo-se os valores das constantes de velocidade enzimática e suas dependências com as concentrações dos pesticidas em solução. / This work describes the development, characterization and utilization of a carbon paste biosensor based in the acetylcholinesterase enzyme for carbamates determinations in foodstuff, as well as the study of rate constants for enzyme/substrate reaction in solution. Stability tests were initially performed for both the substrate and the enzyme. In these tests, the signal for UV-vis adsorption for the substrate shows no inhibition during 11 days while for the enzyme it has been demonstrated that a considerable loss of activity occurs after three days from the solution preparation. In the electrode preparation, some experimental parameters were optimized, such as the amount of enzyme and the content of cobalt ftalocyanine (CoPC) in the electrode, as well as the employed percentage of glutaraldehide. The highest analytical signals were obtained for the addition of 2.40x10-3 g enzyme, 0.90x10-3 g CoPC (related to the massa of 0,017g of carbon paste) and a 1% glutaraldehide solution. The next step was to carry out an experiment based in the inhibition of enzyme activity by the pesticide. For this, the biosensor was immersed in 5.00x10-5 e 1.00x10-4 mol L-1 carbaryl solutions. It was observed that, by increasing the carbaryl concentration, the electrochemical signal of the sensor was inhibited proportionally. This was indicative that the sensor was adequate to be used in carbaryl monitoring and analytical determinations. The analytical determinations of carbamate pesticides were performed in 0.1 mol L-1 phosphate buffer, pH 7,4, with incubation time of 8, 12 and 15 minutes for carbaryl, metomil and aldicarb, respectively. The detection (LD) and quantification (LQ) limits obtained with the biosensor were 2.00x10-6 mol L-1 (0.40 mg L-1) and 6.70 x 10-6 mol L-1 (1.30 mg L-1) for carbaryl, 1.88x10-7mol L-1 (30.45 micro g L-1) and 6.26x10-7 mol L-1 (0.10 mg / L-1) for metomil and 1.10x10-6 mol L-1 (0.20 mg L-1) and 3.60x10-6 mol L-1 (0.70 mg L-1) for aldicarb. For the commercial formulation of metomil, Lannate, LD and LQ obtained were 2.13x10-7 mol L-1 (34.50 microg L-1) and 7.09x10-7 mol L-1(0.12 mg L-1), respectively. The HPLC measurements showed LD and LQ of 1.58x10-8 mol L-1 (3.18micro g L-1) and 5.27x10-8 mol L-1 (10.60 micro g L-1) for carbaryl and 9.02x10-10 mol L-1 (0.15 micro g L-1) and 3.00x10-9 mol L-1 (48.60 micro g L-1) for metomil. Recovering tests were also done with both analytical techniques for carbaryl and Lannate. The obtained recoveries using the biosensor were in the range of 76.83 to 106.67% for carbaryl and 78.00 to 96.50% for Lannate, while using the HPLC, the recoverings were 78.00 a 108.33% for carbaryl and 57.00 to 99.50% for Lannate. The recovering of aldicarb in tomatoes, with HPLC, were 62.40 %. The study of the enzymatic reaction in solution showed that the employed methodology allows to obtain the rate constant values for the rate determining step of the AchE/AchI reaction. It was observed that these rate constant values were strongly dependent in the pesticide concentrations for fenitrothion (organofosforous) and carbaryl (carbamate). At low concentration levels of the pesticide in the electrolyte, all the rate constants showed similar values but, when the pesticide concentration was raised, fenitrothion was found to exert a more powerful inhibition action for the enzyme activity than carbaryl, thus suggesting its higher toxic character. These results showed the development of a possible analytical methodology for quantification of these pesticides, by calculating the rate constant value and its dependence to the pesticide concentration in solution.

Vários estudos que tentam compreender a gênese da aterosclerose têm demonstrado evidências que a oxLDL é peça importante para o desenvolvimento da doença, tornando-a um importante marcador. Os objetivos deste trabalho foram propor um novo imunossensor empregando anticorpos monoclonais anti-oxLDL e desenvolver um processo inovador de obtenção de anticorpos plásticos anti-oxLDL. Para construção do imunossensor, a região Fc do anticorpo foi ligada ao eletrodo de trabalho do dispositivo AuSPE (Screen-Printed Gold Electrodes), utilizando cisteamina. Posteriormente, foi adicionado BSA como bloqueador de possíveis regiões livres. Após o bloqueio, o imunossensor foi testado com oxLDL e outros antígenos, como forma de garantir a especificidade. Em relação aos anticorpos plásticos, chamados MAPS, estes foram desenvolvidos contruindo uma camada impressa de forma invertida ao anticorpo plástico SPAN, referência deste trabalho. Após obtenção dos MAPSs, estes passaram por diversos testes, similar ao imunossensor. Os resultados demonstraram excelente sensibilidade e especificidade às moléculas de oxLDL com detecção em tempo real em ambas as metodologias. / Increased levels of plasma oxLDL are associated with atherosclerosis, and the subsequent development of severe cardiovascular diseases that are today a major cause of death in modern countries. It is therefore important to find a reliable and fast assay to determine oxLDL. A new Immunosensor employing three monoclonal antibodies against oxLDL and a backside protein-surface imprinting process are proposed in this work. To generate the Immunosensor the mAbs were set-up by cysteamine on a gold layer of a disposable screen-printed electrode. BSA was immobilized further as bloker. All steps were followed by various characterization techniques such as electrochemical impedance spectroscopy and square wave voltammetry. To generate specific synthetic antibody materials, called MAPS, these were developed with a backside protein-surface imprinting process of the plastic antibody SPAN, the reference of the work. The devices were successfully applied to determine the oxLDL fraction in real serum, without prior dilution or necessary chemical treatment. Overall, these were promising results with the possibility to apply on the practical use of clinical.

Das Superoxidradikal kann mit fast allen Bestandteilen von Zellen reagieren und diese schädigen. Die medizinische Forschung stellte eine Beteiligung des Radikals an Krebs, Herzinfarkten und neuraler Degeneration fest. Ein empfindlicher Superoxidnachweis ist daher zum besseren Verständnis von Krankheitsverläufen wichtig. Dabei stellen die geringen typischen Konzentrationen und seine kurze Lebensdauer große Anforderungen. Ziel dieser Arbeit war es zum einen, zwei neuartige Proteinarchitekturen auf Metallelektroden zu entwickeln und deren elektrochemisches Ansprechverhalten zu charakterisieren. Zum anderen waren diese Elektroden zur empfindlichen quantitativen Superoxiddetektion einzusetzen. Im ersten Teil der Arbeit wurde eine Protein-Multischichtelektrode aus Cytochrom c und dem Polyelektrolyten Poly(anilinsulfonsäure) nach dem Layer-by-layer-Verfahren aufgebaut. Für zwei bis 15 Schichten an Protein wurde eine deutliche Zunahme an elektrodenaktivem Cytochrom c mit jedem zusätzlichen Aufbringungsschritt nachgewiesen. Die Zunahme verlief linear und ergab bei 15 Schichten eine Zunahme der redoxaktiven Proteinmenge um deutlich mehr als eine Größenordnung. Während das formale Potential im Multischichtsystem sich im Vergleich zur Monoschichtelektrode nicht veränderte, wurde für die Kinetik eine Abhängigkeit der Geschwindigkeit des Elektronentransfers von der Zahl der Proteinschichten beobachtet. Mit zunehmender Scangeschwindigkeit trat ein reversibler Kontaktverlust zu den äußeren Schichten auf. Die lineare Zunahme an elektroaktivem Protein mit steigender Zahl an Depositionsschritten unterscheidet sich deutlich von in der Literatur beschriebenen Protein/Polyelektrolyt-Multischichtelektroden, bei denen ab etwa 6-8 Schichten keine Zunahme an elektroaktivem Protein mehr festgestelltwurde. Auch ist bei diesen die Zunahme an kontaktierbaren Proteinmolekülen auf das Zwei- bis Fünffache limitiert. <br><br> Diese Unterschiede des neu vorgestellten Systems zu bisherigen Multischichtassemblaten erklärt sich aus einem in dieser Arbeit für derartige Systeme erstmals beschriebenen Elektronentransfermechanismus. Der Transport von Elektronen zwischen der Elektrodenoberfläche und den Proteinmolekülen in den Schichten verläuft über einen Protein-Protein-Elektronenaustausch. Dieser Mechanismus beruht auf dem schnellen Selbstaustausch von Cytochrom c-Molekülen und einer verbleibenden Rotationsflexibilität des Proteins im Multischichtsystem. Die Reduzierung des Proteins durch das Superoxidradikal und eine anschließende Reoxidation durch die Elektrode konnten nachgewiesen werden. In einem amperometrischen Messansatz wurde das durch Superoxidradikale hervorgerufene elektrochemische Signal in Abhängigkeit von der Zahl an Proteinschichten gemessen. Ein maximales Ansprechverhalten auf das Radikal wurde mit 6-Schichtelektroden erzielt. Die Empfindlichkeit der 6-Schichtelektroden wurde im Vergleich zum Literaturwert der Monoschichtelektrode um Faktor 14, also mehr als eine Größenordnung, verbessert. <br><br> Somit konnte eine Elektrode mit 6 Schichten aus Cytochrom c und Poly(anilinsulfonsäure) als neuartiger Superoxidsensor mit einer 14-fachen Verbesserung der Empfindlichkeit im Vergleich zum bislang benutzten System entwickelt werden. <br><br> Der zweite Teil dieser Arbeit beschreibt die Auswahl, Gewinnung und Charakterisierung von Mutanten des Proteins Cu,Zn-Superoxiddismutase zur elektrochemischen Quantifizierung von Superoxidradikalen. Monomere Mutanten des humanen dimeren Enzyms wurden entworfen, die durch Austausch von Aminosäuren ein oder zwei zusätzliche Cysteinreste besaßen, mit welchem sie direkt auf der Goldelektrodenoberfläche chemisorbieren sollten. 6 derartige Mutanten konnten in ausreichender Menge und Reinheit in aktiver Form gewonnen werden. Die Bindung der Superoxiddismutase-Mutanten an Goldoberflächen konnte durch Oberflächen-plasmonresonanz und Impedanzspektroskopie nachgewiesen werden. Alle Mutanten wiesen einen quasi-reversiblen Elektronentransfer zwischen SOD und Elektrode auf. Durch Untersuchung von kupferfreien SOD-Mutanten sowie des Wildtyps konnte nachgewiesen werden, das die Mutanten über die eingefügten Cysteinreste auf der Elektrode chemisorptiv gebunden wurden und der Elektronentransfer zwischen der Elektrode und dem Kupfer im aktiven Zentrum der SOD erfolgte. <br><br> Die Superoxiddismutase katalysiert die Zersetzung von Superoxidmolekülen durch Oxidation und durch Reduktion der Radikale. Somit sind beide Teilreaktionen von analytischem Interesse. Zyklovoltammetrisch konnte sowohl die Oxidation als auch die Reduktion des Radikals durch die immobilisierten Superoxiddismutase-Mutanten nachgewiesen werden. In amperometrischen Messanordnungen konnten beide Teilreaktionen zur analytischen Quantifizierung von Superoxidradikalen genutzt werden. Im positiven Potentialfenster wurde die Empfindlichkeit um einen Faktor von etwa 10 gegenüber der Cytochrom c–Monoschichtelektrode verbessert. / The superoxide radical can react with almost all components of a cell and thus damage them. Enzymatic and non-enzymatic scavengers remove it from the body. An implication of the radical in cancer, heart disease, and neuronal degredation has been found in medical research. Therefore, a sensitive quantification of superoxide is necessary for a better understanding of diseases as well as for the study of biological degradation processes. <br><br> The aim of this work was to develop two new protein architectures on metal electrodes and to characterize their electrochemical behavior. Secondly, both electrodes were to be applied as superoxide biosensors. <br><br> In the first part of the work, a protein multilayer electrode consisting of cytochrome c and the polyelectrolyte poly(aniline sulfonated acid) was built up by the layer-by-layer procedure. SPR experiments proved the formation of multilayers. For 2 to 15 protein layers, a significant increase in electroactive protein was found with every deposition step in a linear fashion. For 15 layers, this increase was found to be more than one order of magnitude. While the formal potential did not change for the proteins in the layers, the rate of electron transfer was found to be dependent on the number of layers deposited. With increased scanning speed, a reversible loss of contact to the outer layers was noted. The linear increase in electroactive protein loading differed significantly from protein/polyelectrolyte electrodes described in the literature, where after 6-8 layers no further increase was found. Additionally, these systems increase the number of electroactive protein molecules only by a factor of 2 to 5. <br><br> These differences can be explained by an electron transfer mechanism which was demonstrated in this work for the first time. The transport of electrons between the electrode surface and the proteins in the layers takes place by a protein-protein electron transfer. This mechanism relies on the fast self-exchange of cytochrome c and a residual rotational flexibility of the protein molecules inside the structure. The reduction of the protein by the radical and its subsequent reoxidation by the electrode could be shown. In the amperometric mode, the sensor signal was determined for 2 to 15 layer electrodes. A maximum signal was found for 6 layers, where the sensitivity was improved by a factor of 14, compared to monolayer sensors. <br><br> The second part of this work describes the selection, production and characterization of mutants of the protein Cu,Zn-superoxide dismutase and their application as superoxide sensors. Monomeric mutants of the human dimeric enzyme were designed, which contained one ore two additional cysteines in order to chemisorb directly onto gold surfaces. 6 such mutants were gained in sufficient amount and purity. The binding to gold was characterized by surface plasmon resonance studies. All mutants showed quasi-reversible electrochemistry on gold electrodes. Experiments with copper-free mutants and the wildtype enzyme proved that the mutants bind to gold via the additional cysteines, while the electron transfer takes place between the electrode and the active site copper. Superoxide dismutases catalyze the removal of superoxide by both oxidation and reduction. Thus, both partial reactions are of analytical interest. In cyclic voltammetry, both oxidation and reduction of the radical could be proved. In amperometric experiments, both reactions were used for a quantification of superoxide concentrations. In the positive potential window, the sensitivity was found to be increased by about one order of magnitude, as compared to the cytochrome c monolayer electrode.<br> -----------<br><br> <b>Hinweis zum Copyright:</b><br>Einige Abbildungen dieser Arbeit sind in Artikeln des Verfassers in den Zeitschriften <i>Angewandte Chemie, Angewandte Chemie International Edition, Analytical Chemisty</i> und <i>Elektroanalysis</i> erschienen.<br> Ihre Darstellung im Rahmen dieser Arbeit erfolgt auch online mit ausdrücklicher Genehmigung der Verlage.

Biosensoren werden oft für die Messung einzelner Substanzen in komplexen Medien verwendet, wie z.B. bei der Blutzuckerbestimmung. Sie bestehen aus einem physikochemischen Sensor, dem Transduktionselement, und einer darauf immobilisierten biologischen Komponente, dem Erkennungselement. In dieser Arbeit wurde als Transduktionselement eine Elektrode und als Biokomponente das Enzym „Meerrettich Peroxidase“ (engl. horseradish peroxidase, HRP) verwendet. Solche HRP-Elektroden werden für die Messung von Wasserstoffperoxid (H2O2) eingesetzt. H2O2 wird im Körper von weißen Blutkörperchen produziert, um Bakterien abzutöten, wird teilweise ausgeatmet und kann in kondensierter Atemluft nachgewiesen werden. Da viele weiße Blutkörperchen bei einer Chemotherapie abgetötet und dadurch die Patienten anfälliger für Infektionen werden, muss ihre Anzahl regelmäßig überwacht werden. Dazu wird zurzeit Blut abgenommen. Im ersten Teil dieser Arbeit wurde untersucht, ob eine Überwachung der Anzahl an weißen Blutkörperchen ohne Blutabnahme durch eine H2O2-Messung erfolgen kann. Ein direkter Zusammenhang zwischen der ausgeatmeten H2O2-Menge und der Zahl der weißen Blutkörperchen konnte dabei nicht festgestellt werden. Für empfindliche H2O2-Messungen mit einer HRP-Elektrode ist ein schneller Austausch von Elektronen zwischen der Elektrode und dem Enzym notwendig. Eine Vorraussetzung dafür ist eine kurze Distanz zwischen dem aktiven Zentrum des Enzyms und der Elektrodenoberfläche. Um einen kurzen Abstand zu erreichen wurden im zweiten Teil dieser Arbeit verschiedene poröse graphitähnliche Materialien aus pyrolysierten Kobalt-Porphyrinen für die Elektrodenherstellung verwendet. Dabei stellte sich heraus, dass eines der untersuchten Materialien, welches Poren von etwa der Größe eines Enzyms hat, Elektronen etwa 200mal schneller mit dem Enzym austauscht als festes Graphit. Die HRP selbst enthält in seinem aktiven Zentrum ein Eisen-Protoporphyrin, also ein aus vier Ringen bestehendes flaches Molekül mit einem Eisenatom im Zentrum. Reagiert die HRP mit H2O2, so entzieht es dem Peroxid zwei Elektronen. Eines dieser Elektronen wird am Eisen, das andere im Ringsystem zwischengespeichert, bevor sie an ein anderes Molekül oder an die Elektrode weitergegeben werden. Im letzten Teil dieser Arbeit wurde das Eisen durch Osmium ausgetauscht. Das so veränderte Enzym entzieht Peroxiden nur noch ein Elektron. Dadurch reagiert es zwar langsamer mit Wasserstoffperoxid, dafür aber schneller mit tert-Butylhydroperoxid, einem organischen Vertreter der Peroxid-Familie. / Biosensors are often used for the measurement of specific substances in complex media, e.g. glucose in blood. They consist of a physicochemical sensor, the transducer, onto which a biological component, the recognition element, is immobilised. In this work, an electrode was used as transducer and the enzyme “horseradish peroxidase” (HRP) as biological component. Such HRP electrodes are used for the measurement of hydrogen peroxide (H2O2). H2O2 is produced in the body by white blood cells to destroy bacteria, is partially exhaled and can be measured in breath condensate. Since a lot of white blood cells are destroyed during chemotherapy and patients get more prone to infections, their amount must be checked regularly. Currently blood samples are taken for this purpose. In the first part of this work it was investigated, if the amount of white blood cells can be checked without taking blood by measuring H2O2. A correlation between the amount of exhaled H2O2 and the number of white blood cells could not be found. For a sensitive H2O2 measurement with an HRP electrode a quick exchange of electrons between electrode and enzyme is needed. One condition for this is a short distance between the active centre of the enzyme and the electrode surface. In order to achieve a short distance, several porous graphite-like materials made of pyrolysed cobalt porphyrins where used in the second part of this work for the electrode production. It turned out that one of the tested materials, which had pores about the same size as the enzyme, did exchange electrons with the enzyme about 200 times faster than solid graphite. HRP itself contains an iron protoporphyrin, i.e. a planar molecule consisting of four rings with an iron atom in the middle, its active centre. When HRP reacts with H2O2, it takes two electrons from the peroxide. One of these electrons is stored at the iron, the other in the ring system, until they are passed on to another molecule or the electrode. In the last part of this work, the iron was exchanged with osmium. The modified enzyme takes only one electron from peroxides. Thus it reacts slower with hydrogen peroxide, but faster with tert-butylhydroperoxide, an organic member of the peroxide family.

The aim of this thesis is the design, expression and purification of human cytochrome c mutants and their characterization with regard to electrochemical and structural properties as well as with respect to the reaction with the superoxide radical and the selected proteins sulfite oxidase from human and fungi bilirubin oxidase. All three interaction partners are studied here for the first time with human cyt c and with mutant forms of cyt c. A further aim is the incorporation of the different cyt c forms in two bioelectronic systems: an electrochemical superoxide biosensor with an enhanced sensitivity and a protein multilayer assembly with and without bilirubin oxidase on electrodes. The first part of the thesis is dedicated to the design, expression and characterization of the mutants. A focus is here the electrochemical characterization of the protein in solution and immobilized on electrodes. Further the reaction of these mutants with superoxide was investigated and the possible reaction mechanisms are discussed. In the second part of the work an amperometric superoxide biosensor with selected human cytochrome c mutants was constructed and the performance of the sensor electrodes was studied. The human wild-type and four of the five mutant electrodes could be applied successfully for the detection of the superoxide radical. In the third part of the thesis the reaction of horse heart cyt c, the human wild-type and seven human cyt c mutants with the two proteins sulfite oxidase and bilirubin oxidase was studied electrochemically and the influence of the mutations on the electron transfer reactions was discussed. Finally protein multilayer electrodes with different cyt form including the mutant forms G77K and N70K which exhibit different reaction rates towards BOD were investigated and BOD together with the wild-type and engineered cyt c was embedded in the multilayer assembly. The relevant electron transfer steps and the kinetic behavior of the multilayer electrodes are investigated since the functionality of electroactive multilayer assemblies with incorporated redox proteins is often limited by the electron transfer abilities of the proteins within the multilayer. The formation via the layer-by-layer technique and the kinetic behavior of the mono and bi-protein multilayer system are studied by SPR and cyclic voltammetry. In conclusion this thesis shows that protein engineering is a helpful instrument to study protein reactions as well as electron transfer mechanisms of complex bioelectronic systems (such as bi-protein multilayers). Furthermore, the possibility to design tailored recognition elements for the construction of biosensors with an improved performance is demonstrated. / Ziel dieser Arbeit ist es genetisch veränderte Formen von humanem Cytochrom c herzustellen und diese einerseits hinsichtlich der Reaktion mit dem Sauerstoff-Radikal Superoxid aber auch mit anderen Proteinen zu untersuchen. Zusätzlich sollen die verschiedenen Protein-Mutanten in neuartige bioelektronische Systeme eingebracht werden. Es wurden insgesamt 20 Cytochrome c Mutanten designt, rekombinant exprimiert und aufgereinigt. Es konnte in dieser Arbeit gezeigt werden, dass sich die Reaktion von Cytochrom c mit dem negativ geladenen Superoxid durch gezielte Mutationen, die zusätzliche positive Ladungen in das Molekül bringen, um bis zu 30 % erhöhen lässt. Es wurde aber auch deutlich, dass andere Eigenschaften des Proteins sowie dessen Struktur durch die Mutationen geändert werden können. Cytochrom c Mutanten mit einer erhöhten Reaktionsrate mit Superoxid konnten erfolgreich in einen Superoxid-Biosensor mit erhöhter Sensitivität eingebracht werden. Weiterhin wurde einige Mutanten hinsichtlich Ihrer Interaktion mit den zwei Enzymen Sulfitoxidase und Bilirubinoxidase untersucht. Hier konnten ebenfalls unterschiedliche Reaktivitäten festgestellt werden. Schließlich wurden ausgewählte Protein-Varianten mit und ohne den zuvor untersuchten Enzymen in ein Multischicht-Elektroden-System eingebettet und dessen kinetisches Verhalten untersucht. Es wurde gefunden, dass die Schnelligkeit mit der Cytochrom c mit sich selbst Elektronen austauschen kann, eine Limitierung der Größenordnung der katalytischen Ströme darstellt. Diese Selbstaustausschrate wurde durch die eingeführten Mutationen verändert. So verdeutlicht diese Arbeit, dass „Protein-Engineering“ ein gutes Hilfsmittel sein kann, um einerseits Proteinreaktionen und komplexe Elektronentransferreaktionen in Multischichten zu untersuchen, aber auch ein potentes Werkzeug darstellt mit dem zugeschnittene Biokomponenten für Sensoren mit erhöhter Leistungsfähigkeit generiert werden können.

<p>In this thesis, I present the electrochemical studies of three dendrimeric polypropylene imine (PPI) nanomaterials and their applications as a platform in the development of a novel label free DNA nanobiosensor based on electrochemical impedance spectroscopy. Cyclic voltammetry (CV), differentia pulse voltammetry (DPV), square wave voltammetry (SWV) and electrochemical impedance spectroscopy (EIS) techniques were used to study and model the electrochemical reactivities of the nanomaterials on glassy carbon electrode (GCE) as the working electrode.</p>