Die vorliegende Arbeit befasst sich mit der Entwicklung und Anwendung elektrochemischer Biosensoren zur Erweiterung oder zum Ersatz herkömmlicher Diagnostikverfahren. Als Basis für die Biosensoren wurden Elektrodenarraychips entworfen und im Reinraum gefertigt. Die als 9WPtE bezeichneten Elektrodenarrays waren aus 3 x 3 Elektrodenpaaren im 96-well-Maßstab (ANSI-Standard) aufgebaut. Jedes Elektrodenpaar bestand aus einer kreisrunden Arbeitselektrode mit einem Durchmesser von 1,9 mm und einer Gegenelektrode als offenem Kreisring um die Arbeitselektrode mit einem Durchmesser von 7 mm. Außerhalb des Reinraums wurden separate Messkammern und Ag/AgCl-Referenzelektroden integriert. Sowohl das Referenzsystem als auch die Signalqualität der 9WPtE-Elektrodenarraychips wurden mittels Zyklovoltammetrie, Impedanzspektroskopie und Rasterkraftmikroskopie analysiert und anhand dieser Untersuchungen optimiert. Das Augenmerk lag hierbei auf den Produktionsprozessen zur Herstellung der Elektrodenarraychips, auf den Elektrolytbedingungen für die elektrochemischen Messungen und auf der Recyclebarkeit der Chips. Die Funktionalisierung der Arbeitselektroden der 9WPtE-Chips erfolgte mit sich selbst-organisierenden Schichten aus Thiolen. An die Thiole wurden mittels Chemoligation die biologischen Erkennungskomponenten kovalent gekoppelt. Mit dem 9WPtE-Elektrodenarray wurde auf diese Weise ein funktionsfähiger kompetitiver Immunosensoren gegen den Tumormarker Tenascin C entwickelt. Außerdem wurden der 9WPtE-Chip und ein zusätzlich entwickelter Durchflusssensor, basierend auf dem Prinzip des 9WPtE, genutzt, um die Möglichkeit der Detektion ganzer eukaryotischer Zellen zu untersuchen.

Glutamine (Gln) is a critical intermediate in nitrogen metabolism in all organisms. Here, a whole cell biosensor (GlnLux) for Gln was constructed by transforming a bacterial Gln auxotroph with a constitutive lux reporter. The biosensor was optimized for sensitivity, linearity, efficiency, specificity and robustness to permit detection of Gln in vitro and in vivo. The optimized GlnLux biosensor achieved nanomolar sensitivity with Gln standards. Extracts from only 1 mg of maize (Zea mays L.) leaf tissue were sufficient for Gln detection by GlnLux. Measurements of Gln in leaf extracts by GlnLux correlated with quantification by high performance liquid chromatography (Spearman r = 0.95). GlnLux permitted indirect in planta imaging of Gln using a CCD camera, enabling identification of plants that had been fertilized with nitrogen. Imaging using GlnLux also resolved predicted spatial differences in leaf Gln concentration. In a second application, it was demonstrated that GlnLux embedded into agar permits non-destructive screening of co-inoculated bacterial colonies for biological nitrogen fixation (BNF). GlnLux agar was able to distinguish a Bradyrhizobium japonicum wild type strain (nif+) from a mutant strain defective in nitrogenase (nif-) following ≥8 h of co-incubation. The technology was used to screen a bacterial endophyte diversity library cultured from Zea mays (L.) seeds for biological nitrogen fixation. / OMAFRA

<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>

Das Influenzavirus infiziert Säugetiere und Vögel. Der erste Schritt im Infektionszyklus ist die Anbindung des Viruses über sein Oberflächenprotein Hämagglutinin (HA) an Zuckerstrukturen auf Epithelzellen des respiratorischen Traktes im Wirtsorganismus. Aus den drei komplementaritätsbestimmenden Regionen (complementarity determining regions, CDRs) der schweren Kette eines monoklonalen Hämagglutinin-bindenden Antikörpers wurden drei lineare Peptide abgeleitet. Die Bindungseigenschaften der drei Peptide wurden experimentell mittels Oberflächenplasmonenresonanzspektroskopie untersucht. Es zeigte sich, dass in Übereinstimmung mit begleitenden Molekulardynamik-Simulationen zwei der drei Peptide (PeB und PeC) analog zur Bindefähigkeit des Antikörpers in der Lage sind, Influenzaviren vom Stamm X31 (H3N2 A/Aichi/2/1968) zu binden. Die Interaktion des Peptids PeB, welches potentiell mit der konservierten Rezeptorbindestelle im HA interagiert, wurde anschließend näher charakterisiert. Die Detektion der Influenzaviren war unter geeigneten Immobilisationsbedingungen im diagnostisch relevanten Bereich möglich. Die Spezifität der PeB-Virus-Bindung wurde mittels geeigneter Kontrollen auf der Seite des Analyten und des Liganden nachgewiesen. Des Weiteren war das Peptid PeB in der Lage die Bindung von X31-Viren an Mimetika seines natürlichen Rezeptors zu inhibieren, was die spezifische Interaktion mit der Rezeptorbindungsstelle im Hämagglutinin belegt. Anschließend wurde die Primärsequenz von PeB durch eine vollständige Substitutionsanalyse im Microarray-Format hinsichtlich der Struktur-Aktivitäts-Beziehungen charakterisiert. Dies führte außerdem zu verbesserten Peptidvarianten mit erhöhter Affinität und breiterer Spezifität gegen aktuelle Influenzastämme verschiedener Serotypen (z.B. H1N1/2009, H5N1/2004, H7N1/2013). Schließlich konnte durch Verwendung einer in der Primärsequenz angepassten höher affinen Peptidvariante die Influenzainfektion in vitro inhibiert werden. Damit stellen die vom ursprünglichen Peptid PeB abgeleiteten Varianten Rezeptormoleküle in biosensorischen Testsystemen sowie potentielle Wirkstoffe dar. / The influenza virus infects mammals and birds. The first step of the infection cycle comprises the attachment of the viral surface protein hemagglutinin (HA) on glycan structures on epithelial cells within the respiratory tract of the host organism. Starting from the complementarity determining regions (CDRs) of the heavy chain of a monoclonal hemagglutinin-binding antibody three linear peptides were derived. The binding properties of these peptides was characterized experimentally using surface plasmon resonance spectroscopy. In accordance with accompanying molecular dynamics simulation it was shown, that two of the three peptides (PeB and PeC) were able to bind the influenza virus of the strain X31 (H3N2 A/Aichi/2/1968) comparably to the antibody itself. The interaction of peptide PeB, which was supposed to bind to the conserved receptor binding site at the HA, was then characterized more in detail. The detection of influenza viruses was achieved within the diagnostically relevant concentration range using defined immobilization conditions. The specificity of the peptide-virus-binding was proven by appropriate control experiments. Additionally, peptide PeB was able to inhibit the binding of X31 viruses to mimics of its natural receptor. Furthermore the structure-activity-relationship within all the amino acids of peptide PeB was characterized using a full substitutional analysis in a microarray format. This led to improved peptidic variants, which were able to bind different influenza serotypes and inhibit the influenza infection in vitro. The found peptides and their variants can now be used as receptor molecules in biosensors and also represent potential drug candidates.

Philosophiae Doctor - PhD / 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. / South Africa

Biosensoren stellen technische Geräte dar, die im Zentrum ihres Wirkmechanismus ein Biomolekül, also ein Molekül mit einer biologischen Funktion, aufweisen. Eine Vielzahl von Biosensoren findet in der Wissenschaft bereits Verwendung. Viele dieser Sensoren werden an planen Grenzflächen mit hoher Steifigkeit angewandt, welche weit von der in vivo Situation entfernt sind. Hierdurch ist unklar, ob diese Vereinfachung von natürlichen Prozessen inkorrekte oder in der biologischen Funktionalität veränderte Ergebnisse liefert. Aus diesem Grund soll mit dieser Arbeit die Vielseitigkeit eines neuen Biosensorsystems aufgezeigt werden, mit dem physikalische Phänomene von biologischen Systemen mimetisch nachgestellt und untersucht werden können. Der auf Hydrogelmikropartikeln basierende Biosensor bewies hierbei seine Verwendbarkeit für die Quantifizierung von unspezifischen Adhäsionsvorgängen an Materialgrenzflächen, wie auch für spezifische Rezeptor-Ligand-Wechselwirkungen von lebenden Zellen. Weiterhin konnte mit zwei verschiedenen Nachweismethoden, einer auf Reflektionskontrastmikroskopie basierenden und einer kraftspektroskopischen Methode, die breitgefächerte Anwendung der Hydrogelmikropartikel für biophysikalische Fragestellungen aufgezeigt werden. Die multivalenten und biomimetischen Eigenschaften der Mikropartikel erlauben hierbei ein quantitatives Verständnis von Wechselwirkungsvorgängen von Biomolekülen mit Materialgrenzflächen und spezifischen Rezeptorstrukturen, wobei das Biosensorsystem selbst einfach und leicht zu kontrollieren bleibt.

No description available.

Conducting polymers have emerged as a promising material for optoelectronics and chemical sensing application. Polyaniline (PANI) is a conductive polymer which can be easily functionalized to be specific for various biomolecules and has ideal sensor characteristics. The protonation and deprotonation of the polyaniline’s backbone by derivatization can result in color and conductive change responses. This makes it ideal for the construction of a real time, naked eye sensor. Derivatized polyaniline has previously been reported as a colorimetric sensor in solution. We plan to create a more practical sensor by synthesizing hydroxyl functionalized polyaniline thin films. In this study, we designed a process to functionalize polyaniline and deposit it as a thin film on quartz or silicon substrate via a dip coating process. To demonstrate the use of derivatized PANI in biosensing applications, derivatized and underivatized PANI thin films were treated with solutions of L-aspartic (Asp) acid at concentrations ranging from 10-8 mM to 103 mM and monitored utilizing UV-Vis spectroscopy. We found that the derivatized thin films change from deep blue to green color upon addition of Asp solution and showed a decrease in the characteristic quinoid ring peak at 600nm and the appearance of a new polaron peak at 425nm. The underivatized PANI films showed no colorimetric response indicating the hydroxyl functionalized PANI films are a more ideal material for a biosensing and naked eye detection. The polyaniline derivative was characterized using FT-IR spectroscopy, 1H NMR spectroscopy, UV-VIS spectroscopy, and Scanning Electron Microscopy. Additionally, conductivity studies were utilized to explore the material’s effectiveness as an electronic sensor using a 4-point probe to measure resistance.

A urease (Canavalia ensiformis DC.) foi fisicamente imobilizada em matrizes de polipirrol (PPI) com o objetivo de se detectar uréia em amostras padrão. A eletropolimerização do pirrol foi realizada por voltametria cíclica em uma faixa de potencial de -1,0 a 1,0 V vs. ECS em um meio aquoso contendo 0,2 mol/L de \'LI\'CL\'O IND.4\' e 0,1 mol/L de pirrol. Este procedimento permitiu também a imobilização da enzima na matriz polimérica em suas formas, urease purificada (comercial) e como extrato bruto obtido a partir do feijão de porco (Jack Bean), após a adição de 300 \'mü\'g/mL de urease purificada ou 100 \'mü\'L de extrato bruto de feijão de porco. A urease purificada possui 34.375 U/g de sólido e o extrato bruto, 13.000 UA/mL, valores obtidos por titrimetria. A presença da enzima imobilizada nos filmes de PPI foi verificada por voltametria cíclica, FTIR, microscopia eletrônica de varredura (MEV), microscopia de força atômica (AFM) e por uma microbalança de cristal de quartzo (MCQE). A atividade da enzima após a imobilização nos filmes de PPI foi confirmada pela presença de íons amônio em solução, que são formados como produtos da reação de hidrólise da uréia catalisada pela enzima. Como o transdutor influencia a eficiência e a sensibilidade do biossensor, dois métodos de transdução foram estudados: cronoamperometria, aplicando-se um potencial de -0,28 V durante 120 s em tampão fosfato pH 7,0 e a cronopotenciometria, aplicando-se uma corrente de 1,0 mA durante 120 s em tampão fosfato pH 7,0 variando-se a concentração de uréia. O principal objetivo deste trabalho foi avaliar a eficiência do biossensores para a detecção de uréia por meio de transdutores potenciométricos e amperométricos e depois comparar as eficiências dos filmes de PPI/urease purificada e PPI/extrato bruto como biosensores. / Urease (Canavalia ensiformis DC.) was physically immobilized on polypyrrole (PPy) films aiming at detecting urea in standard samples. The electropolymerization of pyrrole was performed by cyclic voltammetry at a potential range from -1.0 to 1.0 V vs SCE in an aqueous medium containing \'LI\'CL\'O IND.4\' 0.2 mol/L and 0.1 mol/L pyrrole. This procedure also allowed us to immobilize the enzyme into the PPy matrix in forms, commercially purified and crude extract of urease obtained from Jack Bean (Canavalia ensiformis) after adding into the electropolymerization media 300 \'mü\'g/mL of purified urease or 100 \'mü\'L of crude extract. The urease solutions had units of active enzyme of 34.375 U/g (purified) and 13.000 UA/mL (crude extract), and the crude extract was obtained from Jack beans by titrimétric methods. The presence of urease immobilized into the PPy film was verified by cyclic voltammetry, FTIR, scanning electron microscopy (SEM), atomic force microscopy (AFM), and by electrochemical quartz crystal microbalance (EQCM) The activity of the enzyme after immobilizing into the PPy films was confirmed by the presence of ammonium ions in solution, since they are formed as catalytic products by urea hydrolysis reaction catalyzed enzyme. The transducer element influences the efficiency and sensitivity of the biosensor, and two transducer methods were studied: chronoamperometry, by applying a potential of -0.28 V during 120 s in buffer phosphate at pH 7.0 and chronopotentiometry, by applying a current of 1.0 mA during 120 s in buffer phosphate at pH 7.0 both after varying the urea concentration. Our main purpose was to evaluate the efficiency of the biosensors for detecting urea by means of potentiometric and amperometric transducers and then compare the efficiencies of PPY/purified urease e PPY/crude extract as biosensors.

Esta tese descreve a esfoliação e modificação do grafeno oxidado (GO) na obtenção de grafeno em sua forma reduzida (RGO) para aplicações biomédicas, que envolve sensoriamento e biossensoriamento, além de aplicação em fototerapia. Nas aplicações em sensores, inicialmente o RGO juntamente com o surfactante aniônico Dihexadecilfosfato (DHP), foi utilizado na fabricação de filmes por drop casting em eletrodo de carbono vítreo (CGE), na detecção do hormônio Estradiol. O eletrodo modificado (RGO-DHP/CGE) foi caracterizado por voltametria cíclica e impedância de espectroscopia eletroquímica. Os resultados mostraram uma corrente de pico de oxidação irreversível em 0,6 V. Sob as condições experimentais ideais, usando a voltametria linear, o limite de detecção para este hormônio foi de 7,7 × 10-8 mol L-1. Foram fabricados também dispositivos de efeito de campo (FET) de RGO via porta líquida em eletrodos interdigitados, para a detecção de Cistatina-C, um marcador de doença renal crônica. Os filmes foram fabricados utilizando a técnica de automontagem de interação eletrostática, nos quais, como polieletrólito de carga positiva foi utilizado o RGO modificado via ligação covalente de APTES, e como polieletrólito de carga negativa, o RGO dopado com nitrogênio, através da redução via micro-ondas. Estes dispositivos apresentaram uma sensibilidade de (1,94 &plusmn; 0,29) &Delta;IDS(%)ngmL-1. O LD foi de 0,39 ngmL-1 e a região linear entre 5 ngmL-1 100 ngmL-1, quando utilizados em urina sintética. Avaliamos também o uso de RGO em sistemas de fototerapia, utilizando GO reduzido com NH4OH na presença de L-Glutamina (RGO-Glu), onde observamos um aumento de temperatura localizado quando o material é irradiado por um laser (808 nm). Este sistema apresentou uma boa estabilidade e baixa agregação em dispersão aquosa e em meio de cultura, devido à formação de uma corona proteica. O RGO-Glu mostrou-se mais eficiente para o aquecimento que o RGO sem a modificação, na absorção do laser em 808 nm, com valores de eficiência de conversão de energia de 63% e 50% respectivamente. Estudos utilizando célula HeLa mostram que a internalização do RGO-Glu foi mais eficiente do que o RGO sem a modificação. Estes estudos mostram a versatilidade do grafeno quimicamente esfoliado em aplicações biomédicas quando convenientemente modificado, que pode ser utilizado em diagnóstico e em terapia. / This thesis describes the exfoliation and modification of graphene oxide (GO) to obtain reduced graphene oxide (RGO), for biomedical applications, namely: (bio)sensing for diagnostics and as active material in phototherapy. For (bio)sensing applications, RGO was used in combination with the anionic surfactant Dihexadecylphosphate (DHP) in the fabrication of drop-cast thin films onto carbon glass electrode (CGE), to be used in the detection of the hormone Estradiol. The modified electrode (RGO-DHP/CGE) was characterized by cyclic voltammetry and electrochemical spectroscopy impedance (EIS). The results showed an irreversible oxidation peak current at 0.6 V. Under ideal experimental conditions, and using linear voltammetry, the detection limit obtained for this sensor was 7.7 × 10-8 mol L-1. In the second part of the study, RGO was used in the fabrication of field effect transistors (FETs) via liquid gate, and the devices were applied in the detection of Cystatin-C, a biomarker for chronic renal disease. The films were made using the electrostatic layer-by-layer technique, in which APTES-modified RGO was used as positive polyelectrolyte, whereas nitrogen-doped RGO was used as the negative species. These devices exhibited a sensitivity of (1,94 &plusmn; 0,29) &Delta;IDS(%)ngmL-1, whereas LD was 0,39 ng.mL-1 and the linear region of detection was between 5 ng.mL-1 100 ngmL-1 when used in synthetic urine. The studies on the use of RGO in phototherapy were carried out using NH4OH -reduced GO in the presence of L-Glutamine (RGO-Glu) for subsequent cell internalization and irradiation under an 808 nm lase line to promote hiperthermia. This system showed good stability and low aggregation in aqueous dispersions and culture medium, due to the formation of a protein corona. RGO-Glu was more efficient than the RGO without the modification in the absorption of the laser at 808 nm, resulting in an efficiency of heat generation (energy conversion efficiency) of 63% and 50% respectively. Cytotoxicity studies using HeLa cell lines revealed that the internalization of RGO-Glu was more efficient than RGO without modification. These studies show the versatility of chemically exfoliated graphene oxides for biomedical applications, including diagnosis and therapy.