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A cysteine mutant of a monomeric human Cu, Zn-SOD (Glycine 61, Serine 142) has been immobilized directly on gold electrodes using the thiol groups introduced. The electrochemical behavior of the surface confined protein was studied in mixtures of aqueous buffer and DMSO up to an organic solvent content of 60%. The formal potential was found to be rather independent of the DMSO content. However, half peak width increased and the redoxactive amount clearly decreased with raising DMSO content. In addition, the kinetics of the heterogeneous electron transfer became slower; but still a quasireversible electrochemical conversion of the mutant SOD was feasible. Thus, the electrodes were applied for sensorial superoxide detection. At a potential of +220 mV vs. Ag/AgCl advantage was taken of the partial oxidation reaction of the enzyme. A defined superoxide signal was obtained in solutions up to 40% DMSO. The sensitivity of the mutant electrodes decreased linearly with the organic solvent content in solution but was still higher compared to conventional cyt.c based sensors. At DMSO concentrations higher than 40% no sensor response was detected.
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.
Surface-Tuned Electron Transfer and Electrocatalysis of Hexameric Tyrosine-Coordinated Heme Protein
(2015)
Molecular modeling, electrochemical methods, and quartz crystal microbalance were used to characterize immobilized hexameric tyrosine-coordinated heme protein (HTHP) on bare carbon or on gold electrodes modified with positively and negatively charged self-assembled monolayers (SAMs), respectively. HTHP binds to the positively charged surface but no direct electron transfer (DET) is found due to the long distance of the active sites from the electrode surfaces. At carboxyl-terminated surfaces, the neutrally charged bottom of HTHP can bind to the SAM. For this "disc" orientation all six hemes are close to the electrode and their direct electron transfer should be efficient. HTHP on all negatively charged SAMs showed a quasi-reversible redox behavior with rate constant k(s) values between 0.93 and 2.86 s(-1) and apparent formal potentials E-app(0)' between -131.1 and -249.1 mV. On the MUA/MU-modified electrode, the maximum surface concentration corresponds to a complete monolayer of the hexameric HTHP in the disc orientation. HTHP electrostatically immobilized on negatively charged SAMs shows electrocatalysis of peroxide reduction and enzymatic oxidation of NADH.
Diese Arbeit befasst sich mit der Herstellung und Charakterisierung von thermoresponsiven Filmen auf Goldelektroden durch Fixierung eines bereits synthetisierten thermoresponsiven Polymers. Als Basis für die Entwicklung der responsiven Grenzfläche dienten drei unterschiedliche Copolymere (Polymere I, II und III) aus der Gruppe der thermisch schaltbaren Poly(oligo(ethylenglykol)methacrylate).
Die turbidimetrischen Messungen der Copolymere in Lösungen haben gezeigt, dass der Trübungspunkt vom pH-Wert, der Gegenwart von Salzen sowie von der Ionenstärke der Lösung abhängig ist. Nach der Charakterisierung der Polymere in Lösung wurden Experimente der kovalenten Kopplung der Polymere I bis III an die Oberfläche der Gold-Elektroden durchgeführt. Während bei Polymeren I und II die Ankopplung auf einer Amidverbrückung basierte, wurde bei Polymer III als alternative Methode zur Immobilisierung eine photoinduzierte Anbindung unter gleichzeitiger Vernetzung gewählt. Der Nachweis der erfolgreichen Ankopplung erfolgte bei allen Polymeren elektrochemisch mittels Cyclovoltammetrie und Impedanzspektroskopie in K3/4[Fe(CN)6]-Lösungen. Wie die Ellipsometrie-Messungen zeigten, waren die erhaltenen Polymer-Filme unterschiedlich dick. Die Ankopplung über Amidverbrückung lieferte dünne Filme (10 – 15 nm), während der photovernetzte Film deutlich dicker war (70-80 nm) und die darunter liegende Oberfläche relativ gut isolierte.
Elektrochemische Temperaturexperimente an Polymer-modifizierten Oberflächen in Lösungen in Gegenwart von K3/4[Fe(CN)6] zeigten, dass auch die immobilisierten Polymere I bis III responsives Temperaturverhalten zeigen. Bei Elektroden mit den immobilisierten Polymeren I und II ist der Temperaturverlauf der Parameterwerte diskontinuierlich – ab einem kritischen Punkt (37 °C für Polymer I und 45 °C für Polymer II) wird zunächst langsame Zunahme der Peakströme wird deutlich schneller. Das Temperaturverhalten von Polymer III ist dagegen bis 50 °C kontinuierlich, der Peakstrom sinkt hier durchgehend.
Weiterhin wurde mit den auf Polymeren II und III basierten Elektroden deren Anwendung als responsive Matrix für Bioerkennungsreaktionen untersucht. Es wurde die Ankopplung von kleinen Biorezeptoren, TAG-Peptiden, an Polymer II- und Polymer III-modifizierten Elektroden durchgeführt. Das hydrophile FLAG-TAG-Peptid verändert das Temperaturverhalten des Polymer II-Films unwesentlich, da es die Hydrophilie des Netzwerkes nicht beeinflusst. Weiterhin wurde der Effekt der Ankopplung der ANTI-FLAG-TAG-Antikörper an FLAG-TAG-modifizierte Polymer II-Filme untersucht. Es konnte gezeigt werden, dass die Antikörper spezifisch an FLAG-TAG-modifiziertes Polymer II binden. Es wurde keine unspezifische Anbindung von ANTI-FLAG-TAG an Polymer II beobachtet. Die Temperaturexperimente haben gezeigt, dass die thermische Restrukturierung des Polymer II-FLAG-TAG-Filmes auch nach der Antikörper-Ankopplung noch stattfindet. Der Einfluss der ANTI-FLAG-TAG-Ankopplung ist gering, da der Unterschied in der Hydrophilie zwischen Polymer II und FLAG-TAG bzw. ANTI-FLAG-TAG zu gering ist.
Für die Untersuchungen mit Polymer III-Elektroden wurde neben dem hydrophilen FLAG-TAG-Peptid das deutlich hydrophobere HA-TAG-Peptid ausgewählt. Wie im Falle der Polymer II Elektrode beeinflusst das gekoppelte FLAG-TAG-Peptid das Temperaturverhalten des Polymer III-Netzwerkes nur geringfügig. Die gemessenen Stromwerte sind geringer als bei der Polymer III-Elektrode. Das Temperaturverhalten der FLAG-TAG-Elektrode ähnelt dem der reinen Polymer III-Elektrode – die Stromwerte sinken kontinuierlich bis die Temperatur von ca. 40 °C erreicht ist, bei der ein Plateau beobachtet wird. Offensichtlich verändert FLAG-TAG auch in diesem Fall nicht wesentlich die Hydrophilie des Polymer III-Netzwerkes. Das an Polymer III-Elektroden gekoppelte hydrophobe HA-TAG-Peptid beeinflusst dagegen im starken Maße den Quellzustand des Netzwerkes. Die Ströme für die HA-TAG-Elektroden sind deutlich geringer als die für die FLAG-TAG-Polymer III-Elektroden, was auf geringeren Wassergehalt und dickeren Film zurückzuführen ist. Bereits ab 30 °C erfolgt der Anstieg von Stromwerten, der bei Polymer III- bzw. bei Polymer III-FLAG-TAG-Elektroden nicht beobachtet werden kann. Das gekoppelte hydrophobe HA-TAG-Peptid verdrängt Wasser aus dem Polymer III-Netzwerk, was in der Stauchung des Films bereits bei Raumtemperatur resultiert. Dies führt dazu, dass der Film im Laufe des Temperaturanstieges kaum noch komprimiert. Die Stromwerte steigen in diesem Fall entsprechend des Anstiegs der temperaturabhängigen Diffusion des Redoxpaares. Diese Untersuchungen zeigen, dass das HA-TAG-Peptid als Ankermolekül deutlich besser für eine potentielle Verwendung der Polymer III-Filme für sensorische Zwecke geeignet ist, da es sich deutlich in der Hydrophilie von Polymer III unterscheidet.
The heme-undecapeptide microperoxidase-11 (MP-11) was immobilized on mesoporous antimony-doped tin oxide (ATO) thin-film electrodes modified with the positively charged binding promotor polydiallyldimethylammonium chloride. Surface concentrations of MP-11 of 1.5 nmol cm(-2) were sufficiently high to enable spectroelectrochemical analyses. UV/Vis spectroscopy and resonance Raman spectroscopy revealed that immobilized MP-11 adopts a six-coordinated low-spin conformation, as in solution in the presence of a polycation. Cathodic reduction of hydrogen peroxide at potentials close to +500mV versus Ag/AgCl indicates that the reaction proceeds via a Compound I-type like intermediate, analogous to natural peroxidases, and confirms mesoporous ATO as a suitable host material for adsorbing the heme-peptide in its native state. A hydrogen peroxide sensor is proposed by using the bioelectrocatalytic properties of the MP-11-modified ATO.
The homodinuclear ruthenium(II) complex [{Ru(l-N4Me2)}(2)(-tape)](PF6)(4) {[1](PF6)(4)} (l-N4Me2=N,N-dimethyl-2,11-diaza[3.3](2,6)-pyridinophane, tape=1,6,7,12-tetraazaperylene) can store one or two electrons in the energetically low-lying * orbital of the bridging ligand tape. The corresponding singly and doubly reduced complexes [{Ru(l-N4Me2)}(2)(-tape(.-))](PF6)(3) {[2](PF6)(3)} and [{Ru(l-N4Me2)}(2)(-tape(2-))](PF6)(2) {[3](PF6)(2)}, respectively, were electrochemically generated, successfully isolated and fully characterized by single-crystal X-ray crystallography, spectroscopic methods and magnetic susceptibility measurements. The singly reduced complex [2](PF6)(3) contains the -radical tape(.-) and the doubly reduced [3](PF6)(2) the diamagnetic dianion tape(2-) as bridging ligand, respectively. Nucleophilic aromatic substitution at the bridging tape in [1](4+) by two sulfite units gave the complex [{Ru(l-N4Me2)}(2){-tape-(SO3)(2)}](2+) ([4](2+)). Complex dication [4](2+) was exploited as a redox mediator between an anaerobic homogenous reaction solution of an enzyme system (sulfite/sulfite oxidase) and the electrode via participation of the low-energy *-orbital of the disulfonato-substituted bridging ligand tape-(SO3)(2)(2-) (E-red1=-0.1V versus Ag/AgCl/1m KCl in water).
MoS2, a typical layered transition-metal dichalcogenide, is promising as an electrode material in supercapacitors. However, its low electrical conductivity could lead to limited capacitance if applied in electrochemical devices. Herein, a new nanostructure composed of hollow carbon-MoS2-carbon was successfully synthesized through an L-cysteine-assisted hydrothermal method by using gibbsite as a template and polydopamine as a carbon precursor. After calcination and etching of the gibbsite template, uniform hollow platelets, which were made of a sandwich-like assembly of partial graphitic carbon and two-dimensional layered MoS2 flakes, were obtained. The platelets showed excellent dispersibility and stability in water, and good electrical conductivity due to carbon provided by the calcination of polydopamine coatings. The hollow nanoplate morphology of the material provided a high specific surface area of 543 m(2) g(-1), a total pore volume of 0.677 cm(3) g(-1), and fairly small mesopores (approximate to 5.3 nm). The material was applied in a symmetric supercapacitor and exhibited a specific capacitance of 248 F g(-1) (0.12 F cm(-2)) at a constant current density of 0.1 Ag-1; thus suggesting that hollow carbon-MoS2 carbon nanoplates are promising candidate materials for supercapacitors.
The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for animal health, food, and environmental matters. The active agent Monensin (MON) belongs to the ionophore antibiotics and is widely used as a veterinary drug against coccidiosis in broiler farming. However, no electrochemically (EC) generated TPs of MON have been described so far. In this study, the online coupling of EC and mass spectrometry (MS) was used for the generation of oxidative TPs. EC-conditions were optimized with respect to working electrode material, solvent, modifier, and potential polarity. Subsequent LC/HRMS (liquid+ chromatography/high resolution mass spectrometry) and MS/MS experiments were performed to identify the structures of derived TPs by a suspected target analysis. The obtained EC-results were compared to TPs observed in metabolism tests with microsomes and hydrolysis experiments of MON. Five previously undescribed TPs of MON were identified in our EC/MS based study and one TP, which was already known from literature and found by a microsomal assay, could be confirmed. Two and three further TPs were found as products in microsomal tests and following hydrolysis, respectively. We found decarboxylation, O-demethylation and acid-catalyzed ring-opening reactions to be the major mechanisms of MON transformation
The knowledge of transformation pathways and identification of transformation products (TPs) of veterinary drugs is important for animal health, food, and environmental matters. The active agent Monensin (MON) belongs to the ionophore antibiotics and is widely used as a veterinary drug against coccidiosis in broiler farming. However, no electrochemically (EC) generated TPs of MON have been described so far. In this study, the online coupling of EC and mass spectrometry (MS) was used for the generation of oxidative TPs. EC-conditions were optimized with respect to working electrode material, solvent, modifier, and potential polarity. Subsequent LC/HRMS (liquid chromatography/high resolution mass spectrometry) and MS/MS experiments were performed to identify the structures of derived TPs by a suspected target analysis. The obtained EC-results were compared to TPs observed in metabolism tests with microsomes and hydrolysis experiments of MON. Five previously undescribed TPs of MON were identified in our EC/MS based study and one TP, which was already known from literature and found by a microsomal assay, could be confirmed. Two and three further TPs were found as products in microsomal tests and following hydrolysis, respectively. We found decarboxylation, O-demethylation and acid-catalyzed ring-opening reactions to be the major mechanisms of MON transformation.
Moxidectin (MOX) is a widely used anthelmintic drug for the treatment of internal and external parasites in food-producing and companion animals. Transformation products (TPs) of MOX, formed through metabolic degradation or acid hydrolysis, may pose a potential environmental risk, but only few were identified so far. In this study, we therefore systematically characterized electro- and photochemically generated MOX TPs using high-resolution mass spectrometry (HRMS). Oxidative electrochemical (EC) TPs were generated in an electrochemical reactor and photochemical (PC) TPs by irradiation with UV-C light. Subsequent HRMS measurements were performed to identify accurate masses and deduce occurring modification reactions of derived TPs in a suspected target analysis. In total, 26 EC TPs and 59 PC TPs were found. The main modification reactions were hydroxylation, (de-)hydration, and derivative formation with methanol for EC experiments and isomeric changes, (de-)hydration, and changes at the methoxime moiety for PC experiments. In addition, several combinations of different modification reactions were identified. For 17 TPs, we could predict chemical structures through interpretation of acquired MS/MS data. Most modifications could be linked to two specific regions of MOX. Some previously described metabolic reactions like hydroxylation or O-demethylation were confirmed in our EC and PC experiments as reaction type, but the corresponding TPs were not identical to known metabolites or degradation products. The obtained knowledge regarding novel TPs and reactions will aid to elucidate the degradation pathway of MOX which is currently unknown.