Time-resolved surface enhanced resonance Raman spectro-electrochemistry of heme proteins

The membrane protein Cytochrome c Oxidase (CcO) is one of the most important functional bio-molecules. It appears in almost every eukaryotic cell and many bacteria. Although the different species differ in the number of subunits, the functional differences are merely marginal. CcO is the terminal link in the electron transfer pathway of the mitochondrial respiratory chain. Electrons transferred to the catalytic center of the enzyme conduce to the reduction of molecular oxygen to water. Oxygen reduction is coupled to the pumping of protons into the inter-membrane space and hence generates a difference in electrochemical potential of protons across the inner mitochondrial membrane. This potential difference drives the synthesis of adenosine triphosphate (ATP), which is the universal energy carrier within all biological cells. rnrnThe goal of the present work is to contribute to a better understanding of the functional mechanism of CcO by using time-resolved surface enhanced resonance Raman spectroscopy (TR-SERRS). Despite intensive research effort within the last decades, the functional mechanism of CcO is still subject to controversial discussions. It was the primary goal of this dissertation to initiate electron transfer to the redox centers CuA, heme a, heme a3 and CuB electrochemically and to observe the corresponding redox transitions in-situ with a focus on the two heme structures by using SERRS. A measuring cell was developed, which allowed combination of electrochemical excitation with Raman spectroscopy for the purpose of performing the accordant measurements. Cytochrome c was used as a benchmark system to test the new measuring cell and to prove the feasibility of appropriate Raman measurements. In contrast to CcO the heme protein cc contains only a single heme structure. Nevertheless, characteristic Raman bands of the hemes can be observed for both proteins.rnrnIn order to investigate CcO it was immobilized on top of a silver substrate and embedded into an artificial membrane. The catalytic activity of CcO and therefore the complete functional capability of the enzyme within the biomimetic membrane architecture was verified using cyclic voltammetry. Raman spectroscopy was performed using a special nano-structured silver surface, which was developed within the scope of the present work. This new substrate combined two fundamental properties. It facilitated the formation of a protein tethered bilayer lipid membrane (ptBLM) and it allowed obtaining Raman spectra with sufficient high signal-to-noise ratios.rnSpectro-electrochemical investigations showed that at open circuit potential the enzyme exists in a mixed-valence state, with heme a and and heme a3 in the reduced and oxidized state, respectively. This was considered as an intermediate state between the non-activated and the fully activated state of CcO. Time-resolved SERRS measurements revealed that a hampered electron transfer to the redox center heme a3 characterizes this intermediate state.rn

Raman-spektroskopische Untersuchungen an Alkali- und Erdalkalisilicatgläsern bei hohen Temperaturen

In der vorliegenden Arbeit wird die Struktur von Alkali- und Erdalkalisilicatglaesern bei hohen Temperaturen (bis 1800 K) mit Hilfe der Raman-Spektroskopie untersucht. Ein wesentlicher Teil der vorliegenden Arbeit besteht in dem Aufbau einer Hochtemperatureinrichtung, die es erlaubt, Raman-Spektren von Silicatglaesern bei sehr hohen Temperaturen zu messen. Mit der Hochtemperatur-Raman-Spektroskopie an Silicatglaesern sind erhebliche experimentelle Schwierigkeiten verbunden: Die thermische Strahlung der Probe überlagert sich mit dem Raman-Spektrum.Die Temperaturbestimmung der Glasprobe, die einen Durchmesser von nur 0,8 mm hat, erfolgt durch den Vergleich der Stokes- und Anti-Stokes-Raman-Intensitaeten einer intensiven Linie einer Referenzprobe. Die Natriumsilicatglaeser werden detailliert untersucht und die Verteilung der Struktureinheiten in den Natriumsilicatglaesern wird zwischen Zimmertemperatur und 900 K bestimmt. Aus der Verteilung der Strukturelemente wird eine Gleichgewichtskonstante K berechnet, welche die Disproportionierungsreaktion zwischen den Struktureinheiten in den Glaesern beschreibt. Der Wert für die Reaktionsenthalpie liegt im untersuchten Konzentrationsbereich zwischen 0 und 28 kJ/mol und haengt systematisch von der Zusammensetzung ab. Die Reaktionsenthalpie nimmt mit zunehmendem Natriumoxid-Gehalt zu.Die quantitative Auswertung der Raman-Spektren der Kaliumsilicatglaeser und der Bariumsilicatglaeser ist auf Grund deren Kristallisation bei hohen Temperaturen mit Problemen behaftet.

Structure and dynamics of supramolecular assemblies studied by advanced solid-state NMR spectroscopy

Ziel der vorliegenden Arbeit ist die Aufklärung von Struktur und Dynamik komplexer supramolekularer Systeme mittels Festkörper NMR Spektroskopie. Die Untersuchung von pi-pi Wechselwirkungen, welche einen entscheidenden Einfluss auf die strukturellen und dynamischen Eigenschaften supra- molekularer Systeme haben, hilft dabei, die Selbst- organisationsprozesse dieser komplexen Materialien besser zu verstehen. Mit dipolaren 1H-1H and 1H-13C Wiedereinkopplungs NMR Methoden unter schnellem MAS können sowohl 1H chemische Verschiebungen als auch dipolare 1H-1H und 1H-13C Kopplungen untersucht werden, ohne dass eine Isotopenmarkierung erforderlich ist. So erhält man detaillierte Informationen über die Struktur und die Beweglichkeit einzelner Molekül- segmente. In Verbindung mit sogenannten nucleus independent chemical shift (NICS) maps (berechnet mit ab-initio Methoden) lassen sich Abstände von Protonen relativ zu pi-Elektronensystemen bestimmen und so Strukturvorschläge ableiten. Mit Hilfe von homo- und heteronuklearen dipolaren Rotationsseitenbandenmustern könnenaußerdem Ordnungs- parameter für verschiedene Molekülsegmente bestimmt werden. Die auf diese Weise gewonnenen Informationen über die strukturellen und dynamischen Eigenschaften supramolekularer Systeme tragen dazu bei, strukturbestimmende Molekül- einheiten und Hauptordnungsphänomene zu identifizieren sowie lokale Wechselwirkungen zu quantifizieren, um so den Vorgang der Selbstorganisation besser zu verstehen.

Vibrational spectroscopic and electrochemical investigations of multi-centered heme proteins in biomimetic membrane architectures

Membrane proteins play a major role in every living cell. They are the key factors in the cell’s metabolism and in other functions, for example in cell-cell interaction, signal transduction, and transport of ions and nutrients. Cytochrome c oxidase (CcO), as one of the membrane proteins of the respiratory chain, plays a significant role in the energy transformation of higher organisms. CcO is a multi centered heme protein, utilizing redox energy to actively transport protons across the mitochondrial membrane. One aim of this dissertation is to investigate single steps in the mechanism of the ion transfer process coupled to electron transfer, which are not fully understood. The protein-tethered bilayer lipid membrane is a general approach to immobilize membrane proteins in an oriented fashion on a planar electrode embedded in a biomimetic membrane. This system enables the combination of electrochemical techniques with surface enhanced resonance Raman (SERRS), surface enhanced reflection absorption infrared (SEIRAS), and surface plasmon spectroscopy to study protein mediated electron and ion transport processes. The orientation of the enzymes within the surface confined architecture can be controlled by specific site-mutations, i.e. the insertion of a poly-histidine tag to different subunits of the enzyme. CcO can, thus, be oriented uniformly with its natural electron pathway entry pointing either towards or away from the electrode surface. The first orientation allows an ultra-fast direct electron transfer(ET) into the protein, not provided by conventional systems, which can be leveraged to study intrinsic charge transfer processes. The second orientation permits to study the interaction with its natural electron donor cytochrome c. Electrochemical and SERR measurements show conclusively that the redox site structure and the activity of the surface confined enzyme are preserved. Therefore, this biomimetic system offers a unique platform to study the kinetics of the ET processes in order to clarify mechanistic properties of the enzyme. Highly sensitive and ultra fast electrochemical techniques allow the separation of ET steps between all four redox centres including the determination of ET rates. Furthermore, proton transfer coupled to ET could be directly measured and discriminated from other ion transfer processes, revealing novel mechanistic information of the proton transfer mechanism of cytochrome c oxidase. In order to study the kinetics of the ET inside the protein, including the catalytic center, time resolved SEIRAS and SERRS measurements were performed to gain more insight into the structural and coordination changes of the heme environment. The electrical behaviour of tethered membrane systems and membrane intrinsic proteins as well as related charge transfer processes were simulated by solving the respective sets of differential equations, utilizing a software package called SPICE. This helps to understand charge transfer processes across membranes and to develop models that can help to elucidate mechanisms of complex enzymatic processes.

Structure formation as studied by EPR spectroscopy: From simple solutions to nanoparticles

In this thesis, three nitroxide based ionic systems were used to investigate structure and dynamics of their respective solutions in mixed solvents by means of electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopy at X- and W-band (9.5 and 94.5 GHz, respectively). rnFirst, the solvation of the inorganic radical Fremy’s salt (K2ON(SO3)2) in isotope substituted binary solvent mixtures (methanol/water) was investigated by means of high-field (W-band) pulse ENDOR spectroscopy and molecular dynamics (MD) simulations. From the analysis of orientation-selective 1H and 2H ENDOR spectra the principal components of the hyperfine coupling (hfc) tensor for chemically different protons (alcoholic methyl vs. exchangeable protons) were obtained. The methyl protons of the organic solvent approach with a mean distance of 3.5 Å perpendicular to the approximate plane spanned by ON(S)2 of the probe molecule. Exchangeable protons were found to be distributed isotropically, approaching closest to Fremy’s salt from the hydrogen-bonded network around the sulfonate groups. The distribution of exchangeable and methyl protons as found in MD simulations is in full agreement with the ENDOR results. The solvation was found to be similar for the studied solvent ratios between 1:2.3 and 2.3:1 and dominated by an interplay of H-bond (electrostatic) interactions and steric considerations with the NO group merely involved into H-bonds.rnFurther, the conformation of spin labeled poly(diallyldimethylammonium chloride) (PDADMAC) solutions in aqueous alcohol (methanol, ethanol, n-propanol, ethylene glycol, glycerol) mixtures in dependence of divalent sodium sulfate was investigated with double electron-electron resonance (DEER) spectroscopy. The DEER data was analyzed using the worm-like chain model which suggests that in organic-water solvent mixtures the polymer backbones are preferentially solvated by the organic solvent. We found a less serve impact on conformational changes due to salt than usually predicted in polyelectrolyte theory which stresses the importance of a delicate balance of hydrophobic and electrostatic interactions, in particular in the presence of organic solvents.rnFinally, the structure and dynamics of miniemulsions and polymerdispersions prepared with anionic surfactants, that were partially replaced by a spin labeled fatty acid in presence and absence of a lanthanide beta-diketonate complex was characterized by CW EPR spectroscopy. Such miniemulsions form multilayers with the surfactant head group bound to the lanthanide ion. Beta-diketonates were formerly used as NMR shift reagents and nowadays find application as luminescent materials in OLEDs and LCDs and as contrast agent in MRT. The embedding of the complex into a polymer matrix results in an easy processable material. It was found that the structure formation takes place in miniemulsion and is preserved during polymerization. For surfactants with carboxyl-head group a higher order of the alkyl chains and less lateral diffusion is found than for sulfat-head groups, suggesting a more uniform and stronger coordination to the metal ion. The stability of these bilayers depends on the temperature and the used surfactant which should be considered for the used polymerization temperature if a maximum output of the structured regions is wished.

An all-solid state laser system for the laser ion source RILIS and in-source laser spectroscopy of astatine at ISOLDE,CERN

This doctoral thesis describes the extension of the resonance ionization laser ion source RILIS at CERN/ISOLDE by the addition of an all-solid state tunable titanium:sapphire (Ti:Sa) laser system to complement the well-established system of dye lasers. Synchronous operation of the so called Dual RILIS system of Ti:Sa and dye lasers was investigated and the potential for increased ion beam intensity, reliability, and reduced setup time has been demonstrated. In-source resonance ionization spectroscopy was performed at ISOLDE/CERN and at ISAC/TRIUMF radioactive ion beam facilities to develop an efficient and selective three-colour ionization scheme for the purely radioactive element astatine. A LabVIEW based monitoring, control and measurement system was conceived which enabled, in conjunction with Dual RILIS operation, the spectroscopy of high lying Rydberg states, from which the ionization potential of the astatine atom was determined for the first time experimentally.

EPR spectroscopy as a tool for the elucidation of non-covalent structuring principles in complex soft matter

This thesis aims at connecting structural and functional changes of complex soft matter systems due to external stimuli with non-covalent molecular interaction profiles. It addresses the problem of elucidating non-covalent forces as structuring principle of mainly polymer-based systems in solution. The structuring principles of a wide variety of complex soft matter types are analyzed. In many cases this is done by exploring conformational changes upon the exertion of external stimuli. The central question throughout this thesis is how a certain non-covalent interaction profile leads to solution condition-dependent structuring of a polymeric system.rnTo answer this question, electron paramagnetic resonance (EPR) spectroscopy is chosen as the main experimental method for the investigation of the structure principles of polymers. With EPR one detects only the local surroundings or environments of molecules that carry an unpaired electron. Non-covalent forces are normally effective on length scales of a few nanometers and below. Thus, EPR is excellently suited for their investigations. It allows for detection of interactions on length scales ranging from approx. 0.1 nm up to 10 nm. However, restriction to only one experimental technique likely leads to only incomplete pictures of complex systems. Therefore, the presented studies are frequently augmented with further experimental and computational methods in order to yield more comprehensive descriptions of the systems chosen for investigation.rnElectrostatic correlation effects in non-covalent interaction profiles as structuring principles in colloid-like ionic clusters and DNA condensation are investigated first. Building on this it is shown how electrostatic structuring principles can be combined with hydrophobic ones, at the example of host-guest interactions in so-called dendronized polymers (denpols).rnSubsequently, the focus is shifted from electrostatics in dendronized polymers to thermoresponsive alkylene oxide-based materials, whose structuring principles are based on hydrogen bonds and counteracting hydrophobic interactions. The collapse mechanism in dependence of hydrophilic-hydrophobic balance and topology of these polymers is elucidated. Complementarily the temperature-dependent phase behavior of elastin-like polypeptides (ELPs) is investigated. ELPs are the first (and so far only) class of compounds that is shown to feature a first-order inverse phase transition on nanoscopic length scales.rnFinally, this thesis addresses complex biological systems, namely intrinsically disordered proteins (IDPs). It is shown that the conformational space of the IDPs Osteopontin (OPN), a cytokine involved in metastasis of several kinds of cancer, and BASP1 (brain acid soluble protein one), a protein associated with neurite outgrowth, is governed by a subtle interplay between electrostatic forces, hydrophobic interaction, system entropy and hydrogen bonds. Such, IDPs can even sample cooperatively folded structures, which have so far only been associated with globular proteins.

Structural analysis of the major lightharvesting complex II by electron paramagnetic resonance (EPR) : comparison between monomers and trimers

Der Haupt-Lichtsammenkomplex II (LHCII) höherer Pflanzen ist das häufigsternMembranprotein der Welt und in die chloroplastidäre Thylakoidmembran integriert. DerrnLHCII kann als Modellsystem genutzt werden, um die Funktionsweise vonrnMembranproteinen besser zu verstehen, da 96 % seiner Struktur kristallografisch aufgelöstrnist und er in rekombinanter Form in vitro rückgefaltet werden kann. Hierbei entsteht einrnvoll funktionaler Protein-Pigment.Komplex, der nahezu identisch mit der in vivo Varianternist.rnElektronenparamagnetischen Resonanz (EPR) Spektroskopie ist eine hoch sensitive undrnideal geeignete Methode, um die Strukturdynamik von Proteinen zu untersuchen. Hierzurnist eine ortsspezifische Markierung mit Spinsonden notwendig, die kovalent an Cysteinernbinden. Möglich wird dies, indem sorgfältig ausgewählte Aminosäuren gegen Cysteinerngetauscht werden, ohne dass die Funktionsweise des LHCII beeinträchtigt wird.rnIm Rahmen dieser Arbeit wurden die Stabilität des verwendeten Spinmarkers und diernProbenqualität verbessert, indem alle Schritte der Probenpräparation untersucht wurden.rnMithilfe dieser Erkenntnisse konnte sowohl die Gefahr einer Proteinaggregation als auchrnein Verlust des EPR Signals deutlich vermindert werden. In Kombination mit derrngleichzeitigen Etablierung des Q-Band EPR können nun deutlich geringer konzentrierternProben zuverlässig vermessen werden. Darüber hinaus wurde eine reproduzierbarernMethode entwickelt, um heterogene Trimere herzustellen. Diese bestehen aus einemrndoppelt markierten Monomer und zwei unmarkierten Monomeren und erlauben es, diernkristallografisch unvollständig aufgelöste N-terminale Domäne im monomeren undrntrimeren Assemblierungsgrad zu untersuchen. Die Ergebnisse konnten einerseits diernVermutung bestätigen, dass diese Domäne im Vergleich zum starren Proteinkern sehrrnflexibel ist und andererseits, dass sie in Monomeren noch mobiler ist als in Trimeren.rnZudem wurde die lumenale Schleifenregion bei unterschiedlichen pH Werten undrnvariierender Pigmentzusammensetzung untersucht, da dieser Bereich sehr kontroversrndiskutiert wird. Die Messergebnisse offenbarten, dass diese Region starre und flexiblerernSektionen aufweist. Während der pH Wert keinen Einfluss auf die Konformation hatte,rnzeigte sich, dass die Abwesenheit von Neoxanthin zu einer Änderung der Konformationrnführt. Weiterführende Analysen der strukturellen Dynamik des LHCII in einerrnLipidmembran konnten hingegen nicht durchgeführt werden, da dies eine gerichteternInsertion des rückgefalteten Proteins in Liposomen erfordert, was trotz intensiverrnVersuche nicht zum Erfolg führte.

Internal characteristics, chemical compounds and spectroscopy of sapphire as single crystals

In this study more than 450 natural sapphire samples (most of basaltic type) collected from 19 different areas were examined. They are from Dak Nong, Dak Lak, Quy Chau, two unknown sources from the north (Vietnam); Bo Ploi, Khao Ploi Waen (Thailand); Ban Huay Sai (Laos); Australia; Shandong (China); Andapa, Antsirabe, Nosibe (Madagascar); Ballapana (Sri Lanka); Brazil; Russia; Colombia; Tansania and Malawi. rnThe samples were studied on internal characteristics, chemical compositions, Raman-, luminescence-, Fourier transform infrared (FTIR)-, and ultraviolet-visible-near infrared (UV-Vis-NIR)- spectroscopy. The internal features of these sapphire samples were observed and identified by gemological microscope, con focal micro Raman and FTIR spectroscopy. The major and minor elements of the samples were determined by electron probe microanalysis (EPMA) and the trace elements by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). rnThe structural spectra of sapphire were investigated by con focal Raman spectroscopy. The FTIR spectroscopy was used to study the vibration modes of OH-groups and also to determine hydrous mineral inclusions in sapphire. The UV-Vis-NIR absorption spectroscopy was used to analyze the cause of sapphire color. rnNatural sapphires contain many types of mineral inclusions. Typically, they are iron-containing inclusions like goethite, ilmenite, hematite, magnetite or silicate minerals commonly feldspar, and often observed in sapphires from Asia countries, like Dak Nong, Dak Lak in the south of Vietnam, Ban Huay Sai (Laos), Khao Ploi Waen and Bo Ploi (Thailand) or Shandong (China). Meanwhile, CO2-diaspore inclusions are normally found in sapphires from Tansania, Colombia, or the north of Vietnam like Quy Chau. rnIron is the most dominant element in sapphire, up to 1.95 wt.% Fe2O3 measured by EPMA and it affects spectral characteristics of sapphire.rnThe Raman spectra of sapphire contain seven peaks (2A1g + 5Eg). Two peaks at about 418.3 cm-1 and 577.7 cm-1 are influenced by high iron content. These two peaks shift towards smaller wavenumbers corresponding to increasing iron content. This shift is showed by two equations y(418.3)=418.29-0.53x andy(577.7)=577.96-0.75x, in which y is peak position (cm-1) and x is Fe2O3 content (wt.%). By exploiting two these equations one can estimate the Fe2O3 contents of sapphire or corundum by identifying the respective Raman peak positions. Determining the Fe2O3 content in sapphire can help to distinguish sapphires from different origins, e.g. magmatic and metamorphic sapphire. rnThe luminescence of sapphire is characterized by two R-lines: R1 at about 694 nm and R2 at about 692 nm. This characteristic is also influenced by high iron content. The peak positions of two R-lines shift towards to smaller wavelengths corresponding to increasing of iron content. This correlation is showed by two equations y(R_2 )=692.86-0.049x and y(R_1 )=694.29-0.047x, in which y is peak position (nm) of respective R-lines and x is Fe2O3 content (wt.%). Two these equations can be applied to estimate the Fe2O3 content of sapphire and help to separate sapphires from different origins. The luminescence is also applied for determination of the remnant pressure or stress around inclusions in Cr3+-containing corundum by calibrating a 0-pressure position in experimental techniques.rnThe infrared spectra show the presence of vibrations originating from OH-groups and hydrous mineral inclusions in the range of 2500-4000 cm-1. Iron has also an effect upon the main and strongest peak at about 3310 cm-1. The 3310 cm-1 peak is shifted to higher wavenumber when iron content increases. This relationship is expressed by the equation y(3310)=0.92x+3309.17, in which y is peak position of the 3310 cm-1 and x is Fe2O3 content (wt.%). Similar to the obtained results in Raman and luminescence spectra, this expression can be used to estimate the Fe2O3 content and separate sapphires from different origins. rnThe UV-Vis-NIR absorption spectra point out the strong and sharp peaks at about 377, 387, and 450 nm related to dispersed Fe3+, a broad band around 557 and 600 nm related to intervalence charge transfer (IVCT) Fe2+/Ti4+, and a broader band around 863 nm related to IVCT of Fe2+/Fe3+. rnGenerally, sapphires from different localities were completely investigated on internal features, chemical compounds, and solid spectral characteristics. The results in each part contribute for identifying the iron content and separate sapphires from different localities order origins. rn

Festkörperunterstützte Lipid-Modellmembranen auf Gold zur Rekonstitution von Membranproteinen

Festkörperunterstützte Lipid-Modellmembranen auf Goldzur Rekonstitution von Membranproteinen Ziel der Arbeit war der Aufbau von Lipid-Modellmembranen auf Goldelektroden in welchen die funktionelle Aktivität von rekonstituierten Membranproteinen über elektrochemische Methoden nachgewiesen werden kann.Im Rahmen der Arbeit wurden Lipidbilayer mit und ohne hydrophile Ethylenglykol-Spacer durch Kombination von Selbstorganisation, Langmuir-Blodgett-Kuhn-Techniken und Vesikelfusion aufgebaut. Dabei dienten Thiolipide zur Verankerung der Membranen auf der Goldelektrode und es wurden diverse Wege verfolgt, deren Ankerdichte auf dem Substrat einzustellen.Eine Studie zum Aufbau von festkörperunterstützten Lipidbilayern durch Fusion von Vesikeln auf binäre Alkanthiol-/Hydroxythiol-Monolagen mit definierter Oberflächenenergie zeigte, daß eine minimale Grenzflächenenergie (Monolayer/Wasser) existiert, unterhalb welcher die Fusion nicht mehr zu einer zusätzlichen Monolage, sondern lediglich zur Ausbildung von adsorbierten oder teilgespreiteten Vesikeln führt.Zur Charakterisierung der Membranen wurden Oberflächenplasmonenresonanz, Impedanzspektroskopie, zyklische Voltammetrie, elektrochemische reduktive Desorption, Rasterkraftmikroskopie und Kontaktwinkelmessungen herangezogen.In die Modellmembranen wurden Membranproteine (Porin, Annexin V, H+-ATPase) sowie ganze Membranfragmente (Bande 3 aus roten Blutzellen) rekonstituiert und mittels elektrochemischer Methoden auf ihre funktionelle Aktivität überprüft.

Optische Spektroskopie an Fermium (Z=100)

Im Rahmen der vorliegenden Arbeit wurde erstmals Laser-Atomspektroskopie an einem Element durchgeführt, für das bisher keine atomaren Niveaus bekannt waren. Die Experimente wurden am Element Fermium mit der Ordnungszahl Z=100 mit der Resonanzionisationsspektroskopie (RIS) in einer Puffergaszelle durchgeführt. Verwendet wurde das Isotop 255Fm mit einer Halbwertszeit von 20.1 h, das im Hochflusskernreaktor des ORNL, Oak Ridge, USA, hergestellt wurde. Die von einem elektrochemischen Filament in das Argon-Puffergas bei einer Temperatur von 960(20)°C abgedampften Fm-Atome wurden mit Lasern in einem Zweistufenprozess resonant ionisiert. Dazu wurde das Licht eines Excimerlaser gepumpten Farbstofflasers für den ersten Anregungsschritt um die Wellenlänge 400 nm durchgestimmt. Ein Teil des Excimer (XeF) Laser Pumplichtes mit den Wellenlänge 351/353 nm wurde für die nicht-resonante Ionisation verwendet. Die Ionen wurden mit Hilfe elektrischer Felder aus der optischen Zelle extrahiert und nach einem Quadrupol Massenfilter mit einem Channeltron-Detektor massenselektiv nachgewiesen. Trotz der geringen Probenmenge von 2.7 x 10^10 eingesetzten Atomen wurden zwei atomare Resonanzen bei Energien von 25099.8(2) cm-1 und 25111.8(2) cm-1 gefunden und das Sättigungsverhalten dieser Linien gemessen. Es wurde ein theoretisches Modell entwickelt, dass sowohl das spektrale Profil der sättigungsverbreiterten Linien als auch die Sättigungskurven beschreibt. Durch Anpassung an die Messdaten konnten die partiellen Übergangsraten in den 3H6 Grundzustand Aki=3.6(7) x 10^6/s und Aki=3.6(6) x 10^6/s bestimmt werden. Der Vergleich der Niveauenergien und Übergangsraten mit Multikonfigurations Dirac-Fock Rechnungen legt die spektroskopische Klassifizierung der beobachteten Niveaus als 5f12 7s7p 5I6 und 5G6 Terme nahe. Weiterhin wurde ein Übergang bei 25740 cm-1 gefunden, der aufgrund der beobachteten Linienbreite von 1000 GHz als Rydbergzustand Zustand mit der Niveauenergie 51480 cm-1 interpretiert wurde und über einen Zweiphotonen Prozess angeregt werden kann. Basierend auf dieser Annahme wurde die Obergrenze für die Ionisationsenergie IP = 52140 cm-1 = 6.5 eV abgeschätzt. In den Messungen wurden Verschiebungen in den Zeitverteilungsspektren zwischen den mono-atomaren Ionen Fm+ und Cf+ und dem Molekül-Ion UO+ festgestellt und auf Driftzeitunterschiede im elektrischen Feld der gasgefüllten optischen Zelle zurückgeführt. Unter einfachen Modellannahmen wurde daraus auf die relativen Unterschiede Delta_r(Fm+,Cf+)/r(Cf+) ˜ -0.2 % und Delta_r(UO+,Cf+)/r(Cf+) ˜ 20 % in den Ionenradien geschlossen. Über die Bestimmung der Abnahme der Fm-a Aktivität des Filamentes auf der einen Seite und die Messung der Resonanzzählrate auf der anderen Seite, wurde die Nachweiseffizienz der Apparatur zu 4.5(3) x 10^-4 bestimmt. Die Nachweisapparatur wurde mit dem Ziel weiterentwickelt, Laserspektroskopie am Isotop 251Fm durchzuführen, das über die Reaktion 249Cf(a,2n)251Fm direkt in der optischen Zelle erzeugt werden soll. Das Verfahren wurde am chemischen Homolog Erbium getestet. Dabei wurde das Isotop 163Er über die Reaktion 161Dy(a,2n)163Er erzeugt und nach Resonanzionisation nachgewiesen. Die Nachweiseffizienz der Methode wurde zu 1 x 10^-4 bestimmt.

Determination of trace elemental species in periostracum of Cypraea shells

n this work, three Cypraea species (C. talpa, C. tigris and C. zebra) were exhaustively studied. The shells have been separated in the structural layers. The mineralogy, ultra- and micro-structure of each layer were analyzed by Confocal Laser Scanning Microscopy (CLSM), Scanning Electron Microscopy (SEM), X-Ray Diffractometry (XRD) and Raman Spectroscopy (RS). The presence of biologically relevant trace metals (Mn, Co, Fe, Zn, Cr, etc.) has been investigated using Instrumental Neutron Activation Analysis (INAA) and Inductively Coupled Plasma – Mass Spectrometry (ICP-MS) as detection tool. A new method has been developed and optimized to extract and analyze the soluble organic matrix (SOM) of the shell. Although the molecular nature of the SOM is not really known, it contains at least large protein fraction, if not only consists of proteins. The extracted matrices were compared between layers and species using Size Exclusion High Performance Liquid Chromatography coupled with Ultra Violet Spectrometry (SE-HPLC-UV), Gel electrophoresis (GE) and protein quantification tests. For the first time to our knowledge the association of trace elements to the protein in the SOM of the shell was studied using hyphenated on line as well as combined off line techniques and validated through inter-comparison tests between the different methods applied. Interesting correlations between the trace element concentration, the microstructure and the protein content were directly and indirectly detected. The metals Cu, Ni, Co and Zn have shown to bind to the SOM extracted from C. talpa, C. tigris and C. zebra shells. Within the conclusions of this work it was demonstrated that these protein-metal-complexes (or metal containing proteins) change from one layer to the other and are different between the three snails analyzed. In addition, the complexes are clearly related only to certain protein fractions of the SOM, and not to the whole SOM observed. These fractions and show not to be very metal-specific (i.e. some of these fractions bind two or three different metals).

Synthesis and characterization of Poly(vinylphosphonic acid) for proton exchange membranes in fuel cells

Vinylphosphonic acid (VPA) was polymerized at 80 ºC by free radical polymerization to give polymers (PVPA) of different molecular weight depending on the initiator concentration. The highest molecular weight, Mw, achieved was 6.2 x 104 g/mol as determined by static light scattering. High resolution nuclear magnetic resonance (NMR) spectroscopy was used to gain microstructure information about the polymer chain. Information based on tetrad probabilities was utilized to deduce an almost atactic configuration. In addition, 13C-NMR gave evidence for the presence of head-head and tail-tail links. Refined analysis of the 1H NMR spectra allowed for the quantitative determination of the fraction of these links (23.5 percent of all links). Experimental evidence suggested that the polymerization proceeded via cyclopolymerization of the vinylphosphonic acid anhydride as an intermediate. Titration curves indicated that high molecular weight poly(vinylphosphonic acid) PVPA behaved as a monoprotic acid. Proton conductors with phosphonic acid moieties as protogenic groups are promising due to their high charge carrier concentration, thermal stability, and oxidation resistivity. Blends and copolymers of PVPA have already been reported, but PVPA has not been characterized sufficiently with respect to its polymer properties. Therefore, we also studied the proton conductivity behaviour of a well-characterized PVPA. PVPA is a conductor; however, the conductivity depends strongly on the water content of the material. The phosphonic acid functionality in the resulting polymer, PVPA, undergoes condensation leading to the formation of phosphonic anhydride groups at elevated temperature. Anhydride formation was found to be temperature dependent by solid state NMR. Anhydride formation affects the proton conductivity to a large extent because not only the number of charge carriers but also the mobility of the charge carriers seems to change.

Synthesis and characterization of metal-chalcogenide MQ 2-Nanoparticles (M = Mo, W, Zr; Q = S, O)

Here, we present the adaptation and optimization of (i) the solvothermal and (ii) the metal-organic chemical vapor deposition (MOCVD) approach as simple methods for the high-yield synthesis of MQ2 (M=Mo, W, Zr; Q = O, S) nanoparticles. Extensive characterization was carried out using X-ray diffraction (XRD), scanning and transmission electron micros¬copy (SEM/TEM) combined with energy dispersive X-ray analysis (EDXA), Raman spectroscopy, thermal analyses (DTA/TG), small angle X-ray scattering (SAXS) and BET measurements. After a general introduction to the state of the art, a simple route to nanostructured MoS2 based on the decomposition of the cluster-based precursor (NH4)2Mo3S13∙xH2O under solvothermal conditions (toluene, 653 K) is presented. Solvothermal decomposition results in nanostructured material that is distinct from the material obtained by decomposition of the same precursor in sealed quartz tubes at the same temperature. When carried out in the presence of the surfactant cetyltrimethyl¬ammonium bromide (CTAB), the decomposition product exhibits highly disordered MoS2 lamellae with high surface areas. The synthesis of WS2 onion-like nanoparticles by means of a single-step MOCVD process is discussed. Furthermore, the results of the successful transfer of the two-step MO¬CVD based synthesis of MoQ2 nanoparticles (Q = S, Se), comprising the formation of amorphous precursor particles and followed by the formation of fullerene-like particles in a subsequent annealing step to the W-S system, are presented. Based on a study of the temperature dependence of the reactions a set of conditions for the formation of onion-like structures in a one-step reaction could be derived. The MOCVD approach allows a selective synthesis of open and filled fullerene-like chalcogenide nanoparticles. An in situ heating stage transmission electron microscopy (TEM) study was employed to comparatively investigate the growth mechanism of MoS2 and WS2 nanoparticles obtained from MOCVD upon annealing. Round, mainly amorphous particles in the pristine sample trans¬form to hollow onion-like particles upon annealing. A significant difference between both compounds could be demonstrated in their crystallization conduct. Finally, the results of the in situ hea¬ting experiments are compared to those obtained from an ex situ annealing process under Ar. Eventually, a low temperature synthesis of monodisperse ZrO2 nanoparticles with diameters of ~ 8 nm is introduced. Whereas the solvent could be omitted, the synthesis in an autoclave is crucial for gaining nano-sized (n) ZrO2 by thermal decomposition of Zr(C2O4)2. The n-ZrO2 particles exhibits high specific surface areas (up to 385 m2/g) which make them promising candidates as catalysts and catalyst supports. Co-existence of m- and t-ZrO2 nano-particles of 6-9 nm in diameter, i.e. above the critical particle size of 6 nm, demonstrates that the particle size is not the only factor for stabilization of the t-ZrO2 modification at room temperature. In conclusion, synthesis within an autoclave (with and without solvent) and the MOCVD process could be successfully adapted to the synthesis of MoS2, WS2 and ZrO2 nanoparticles. A comparative in situ heating stage TEM study elucidated the growth mechanism of MoS2 and WS2 fullerene-like particles. As the general processes are similar, a transfer of this synthesis approach to other layered transition metal chalcogenide systems is to be expected. Application of the obtained nanomaterials as lubricants (MoS2, WS2) or as dental filling materials (ZrO2) is currently under investigation.

New platforms for optical biosensing

Physicochemical experimental techniques combined with the specificity of a biological recognition system have resulted in a variety of new analytical devices known as biosensors. Biosensors are under intensive development worldwide because they have many potential applications, e.g. in the fields of clinical diagnostics, food analysis, and environmental monitoring. Much effort is spent on the development of highly sensitive sensor platforms to study interactions on the molecular scale. In the first part, this thesis focuses on exploiting the biosensing application of nanoporous gold (NPG) membranes. NPG with randomly distributed nanopores (pore sizes less than 50 nm) will be discussed here. The NPG membrane shows unique plasmonic features, i.e. it supports both propagating and localized surface plasmon resonance modes (p SPR and l-SPR, respectively), both offering sensitive probing of the local refractive index variation on/in NPG. Surface refractive index sensors have an inherent advantage over fluorescence optical biosensors that require a chromophoric group or other luminescence label to transduce the binding event. In the second part, gold/silica composite inverse opals with macroporous structures were investigated with bio- or chemical sensing applications in mind. These samples combined the advantages of a larger available gold surface area with a regular and highly ordered grating structure. The signal of the plasmon was less noisy in these ordered substrate structures compared to the random pore structures of the NPG samples. In the third part of the thesis, surface plasmon resonance (SPR) spectroscopy was applied to probe the protein-protein interaction of the calcium binding protein centrin with the heterotrimeric G-protein transducin on a newly designed sensor platform. SPR spectroscopy was intended to elucidate how the binding of centrin to transducin is regulated towards understanding centrin functions in photoreceptor cells.