Visualization, manipulation and force spectroscopy of cylindrical polymer brushes

The Ph.D. thesis deals with the conformational study of individual cylindrical polymer brush molecules using atomic force microscopy (AFM). Imaging combined with single molecule manipulation has been used to unravel questions concerning conformational changes, desorption behavior and mechanical properties of individual macromolecules and supramolecular structures. In the first part of the thesis (chapter 5) molecular conformations of cylindrical polymer brushes with poly-(N-isopropylacrylamide) (PNIPAM) side chains were studied in various environmental conditions. Also micelle formation of cylindrical brush-coil blockcopolymers with polyacrylic acid side chains and polystyrene coil have been visualized. In chapter 6 the mechanical properties of single cylindrical polymer brushes with (PNIPAM) side chains were investigated. Assuming that the brushes adopt equilibrium conformation on the surface, an average persistence length of lp= (29 ± 3) nm was determined by the end-to-end distance vs. contour length analysis in terms of the wormlike chain (WLC) model. Stretching experiments suggest that an exact determination of the persistence length using force extension curves is impeded by the contribution of the side chains. Modeling the stretching of the bottle brush molecule as extension of a dual spring (side chain and main chain) explains the frequently observed very low persistence length arising from a dominant contribution of the side chain elasticity at small overall contour lengths. It has been shown that it is possible to estimate the “true” persistence length of the bottle brush molecule from the intercept of a linear extrapolation of the inverse square root of the apparent persistence length vs. the inverse contour length plot. By virtue of this procedure a “true” persistence length of 140 nm for the PNIPAM brush molecules is predicted. Chapter 7 and 8 deal with the force-extension behavior of PNIPAM cylindrical brushes studied in poor solvent conditions. The behavior is shown to be qualitatively different from that in a good solvent. Force induced globule-cylinder conformational changes are monitored using “molecule specific force spectroscopy” which is a combined AFM imaging and SMFS technique. An interesting behavior of the unfolding-folding transitions of single collapsed PNIPAM brush molecules has been observed by force spectroscopy using the so called “fly-fishing” mode. A plateau force is observed upon unfolding the collapsed molecule, which is attributed to a phase transition from a collapsed brush to a stretched conformation. Chapter 9 describes the desorption behavior of single cylindrical polyelectrolyte brushes with poly-L-lysine side chains deposited on a mica surface using the “molecule specific force spectroscopy” technique to resolve statistical discrepancies usually observed in SMFS experiments. Imaging of the brushes and inferring the persistence length from a end-to-end distance vs. contour length analysis results in an average persistence length of lp = (25 ± 5) nm assuming that the chains adopt their equilibrium conformation on the surface. Stretching experiments carried out on individual poly-L-lysine brush molecules by force spectroscopy using the “fly-fishing” mode provide a persistence length in the range of 7-23 nm in reasonable accordance with the imaging results. In chapter 10 the conformational behavior of cylindrical poly-L-lysine brush-sodium dodecyl sulfate complexes was studied using AFM imaging. Surfactant induced cylinder to helix like to globule conformational transitions were observed.

From lipid bilayers to synaptic vesicles: atomic force microscopy on lipid-based systems

The aim of this thesis was to apply the techniques of the atomic force microscope (AFM) to biological samples, namely lipid-based systems. To this end several systems with biological relevance based on self-assembly, such as a solid-supported membrane (SSM) based sensor for transport proteins, a bilayer of the natural lipid extract from an archaebacterium, and synaptic vesicles, were investigated by the AFM. For the characterization of transport proteins with SSM-sensors proteoliposomes are adsorbed that contain the analyte (transport protein). However the forces governing bilayer-bilayer interactions in solution should be repulsive under physiological conditions. I investigated the nature of the interaction forces with AFM force spectroscopy by mimicking the adsorbing proteoliposome with a cantilever tip, which was functionalized with charged alkane thiols. The nature of the interaction is indeed repulsive, but the lipid layers assemble in stacks on the SSM, which expose their unfavourable edges to the medium. I propose a model by which the proteoliposomes interact with these edges and fuse with the bilayer stacks, so forming a uniform layer on the SSM. Furthermore I characterized freestanding bilayers from a synthetic phospholipid with a phase transition at 41°C and from a natural lipid extract of the archaebacterium Methanococcus jannaschii. The synthetic lipid is in the gel-phase at room temperature and changes to the fluid phase when heated to 50°C. The bilayer of the lipid extract shows no phase transition when heated from room temperature to the growth temperature (~ 50°C) of the archeon. Synaptic vesicles are the containers of neurotransmitter in nerve cells and the synapsins are a family of extrinsic membrane proteins, that are associated with them, and believed to control the synaptic vesicle cycle. I used AFM imaging and force spectroscopy together with dynamic light scattering to investigate the influence of synapsin I on synaptic vesicles. To this end I used native, untreated synaptic vesicles and compared them to synapsin-depleted synaptic vesicles. Synapsin-depleted vesicles were larger in size and showed a higher tendency to aggregate compared to native vesicles, although their mechanical properties were alike. I also measured the aggregation kinetics of synaptic vesicles induced by synapsin I and found that the addition of synapsin I promotes a rapid aggregation of synaptic vesicles. The data indicate that synapsin I affects the stability and the aggregation state of synaptic vesicles, and confirm the physiological role of synapsins in the assembly and regulation of synaptic vesicle pools within nerve cells.

Atomic force microscopy of biomimetic systems

This thesis was driven by the ambition to create suitable model systems that mimic complex processes in nature, like intramolecular transitions, such as unfolding and refolding of proteins, or intermolecular interactions between different cell compo-nents. Novel biophysical approaches were adopted by employing atomic force mi-croscopy (AFM) as the main measurement technique due to its broad diversity. Thus, high-resolution imaging, adhesion measurements, and single-molecule force distance experiments were performed on the verge of the instrumental capabilities. As first objective, the interaction between plasma membrane and cytoskeleton, me-diated by the linker protein ezrin, was pursued. Therefore, the adsorption process and the lateral organization of ezrin on PIP2 containing solid-supported membranes were characterized and quantified as a fundament for the establishment of a biomimetic model system. As second component of the model system, actin filaments were coated on functionalized colloidal probes attached on cantilevers, serving as sensor elements. The zealous endeavor of creating this complex biomimetic system was rewarded by successful investigation of the activation process of ezrin. As a result, it can be stated that ezrin is activated by solely binding to PIP2 without any further stimulating agents. Additional cofactors may stabilize and prolong the active conformation but are not essentially required for triggering ezrin’s transformation into an active conformation. In the second project, single-molecule force distance experiments were performed on bis-loop tetra-urea calix[4]arene-catenanes with different loading rates (increase in force per second). These macromolecules were specifically designed to investigate the rupture and rejoining mechanism of hydrogen bonds under external load. The entangled loops of capsule-like molecules locked the unbound state of intramolecular hydrogen bonds mechanically, rendering a rebinding observable on the experimental time scale. In conjunction with Molecular Dynamics simulations, a three-well potential of the bond rupture process was established and all kinetically relevant parameters of the experiments were determined by means of Monte Carlo simulations and stochastic modeling. In summary, it can be stated that atomic force microscopy is an invaluable tool to scrutinize relevant processes in nature, such as investigating activation mechanisms in proteins, as shown by analysis of the interaction between F-actin and ezrin, as well as exploring fundamental properties of single hydrogen bonds that are of paramount interest for the complete understanding of complex supramolecular structures.

Mechanische Eigenschaften von freitragenden artifiziellen und nativen Membranen

This thesis presents a new method to explore the local mechanical properties such as bending modulus or surface tension of artificial and native pore-spanning membranes. Therefore the elastic response of a free-standing membrane to a local indentation by the means of atomic force microscopy is measured. Starting point are highly hexagonal ordered pores in alumina produced by electrochemical anodization of planar aluminium. The homogeneous pore radius can by tailored in the range of 10 nm up to 200 nm, but radius of 33 nm, 90 nm and 200 nm turned out to be best suited for investigation of the mechanical properties of pore-spanning native or artificial membranes. In this work artificial membrane systems consisting of DODAB as a bilayer in gel phase or DOTAP as a fluide membrane are spreaded by vesicle absorption on hexagonal structured pores after chemisorption of a 3-mercaptopropionic acid monolayer. Centrally indenting these nanodrums with an atomic force microscope tip yields force-indentation curves, which are quantitatively analyzed by solving the corresponding shape equations of continuum curvature elasticity. Since the measured response depends in a known way on the system geometry (pore size, tip radius) and on material parameters (bending modulus, lateral tension, adhesion), this opens the possibility to monitor local elastic properties of lipid membranes in a well-controlled setting. Additionally the locally distributed mechanical properties of pore-spanning artificial membranes are compared to those of native pore-spanning membranes. Therefore the basal membrane of MDCK II cells was prepared on porous alumina assays and their mechanical properties were analyzed by means of atomic force microscopy. Finally the elastic behavior such as the Young modulus of living MDCK II cells under various osmotic pressures is investigated. By changing the osmolarity in the extracellular region of MDCK II cells a volume change is induced according to hydration and dehydration of the cells, respectively. This volume change induces also a change in the elastic behavior of the cell, which is quantified by the means of force spectroscopy.

Eigenschaften von thermoresponsiven Bürstenpolymeren

In dieser Doktorarbeit werden die Eigenschaften von thermoresponsiven Bürstenpolymeren untersucht. Hierbei konnten erstmalig thermische Konformationsübergänge von zylindrischen Bürstenmolekülen auf Oberflächen beobachtet werden. Der Einfluss der Oberfläche auf die Umkehrbarkeit und die Kinetik der Übergänge wurde untersucht. Die dabei erhaltenen Erkenntnisse konnten verwendet werden, um das Verhalten der Polymere auf anderen Oberflächen vorherzusagen. Im zweiten Projekt wurde gezeigt, dass Einzelmolekül-Kraftspektroskopie eine gute Methode für die Untersuchung der mechanischen Eigenschaften von Bürstenpolymeren in guten Lösungsmitteln ist. Mit kleinen Substanzmengen kann die Persistenzlänge der Polymere bestimmt werden. Es ist möglich zu zeigen, dass die Persistenzlänge der Polymerbürsten von ihrer Seitenkettenlänge abhängt. Überraschenderweise funktioniert das Experiment nicht, wenn man die Bürsten aus einem Polymerfilm zieht anstatt ein Einzelmolekülexperiment durchzuführen. In diesem Fall zeigen die Kraft-Abstands-Kurven zu lange Kontur- und Persistenzlängen. Diese Beobachtung ist für lineare Polymere nicht gültig. Im dritten Teil der Doktorarbeit werden Kraft-Abstands-Experimente an einzelnen kollabierten Polymeren untersucht. In schlechten Lösungsmitteln zeigen die Bürsten ein moleküllängenabhängiges Kraft-Plateau, welches theoretisch vorausgesagt wurde und einen Phasenübergang von einem kollabierten zu einem entspannten Zustand der Polymerkette anzeigt. In Fly-Fishing-Experimenten kann man eine Hysterese zwischen den beiden Messkurven beobachten, welche bei mehrfachem Ziehen kleiner wird. Alle Experimente in schlechten Lösungsmitteln wurden mit linearen Polymeren reproduziert, um den Einfluss der Molekülarchitektur von den generellen Eigenschaften von Polymeren in schlechten Lösungsmitteln unterscheiden zu können. Zum Abschluss wird die Abhängigkeit der Polymerentfaltung von der Laderate des Experiments gemessen.

Rupture forces of split aptamers

Rupture forces of ligand-receptor interactions, such as proteins-proteins, proteins-cells, and cells-tissues, have been successfully measured by atomic force spectroscopy (AFS). For these measurements, the ligands and receptors were chemically modified so that they can be immobilized on the tip and on a substrate, respectively. The ligand interact the receptor when the tip approaches the substrate. This interaction can be studied by measuring rupture force upon retraction. However, this technique is not feasible for measurements involving small molecules, since they form only few H-bonds with their corresponding receptors. Modifying small molecules for immobilization on surfaces may block or change binding sites. Thus, recorded rupture forces might not reflect the full scope of the involved small ligand-receptor interactions.rnIn my thesis, a novel concept that allows measuring the rupture force of small involved ligand-receptor interactions and does not require molecular modification for immobilization was introduced. The rupture force of small ligand-receptor interaction is not directly measured but it can be determined from measurements in the presence and in the absence of the ligand. As a model system, the adenosine mono phosphate (AMP) and the aptamer that binds AMP were selected. The aptamer (receptor) is a single stranded DNA that can partially self-hybridize and form binding pockets for AMP molecules (ligands). The bonds between AMP and aptamer are provided by several H-bonds and pair stacking.rnIn the novel concept, the aptamer was split into two parts (oligo a and oligo b). One part was immobilized on the tip and the other one on the substrate. Approaching the tip to the substrate, oligo a and oligo b partially hybridized and the binding pockets were formed. After adding AMP into the buffer solution, the AMP bound in the pockets and additional H-bonds were formed. Upon retraction of the tip, the rupture force of the AMP-split aptamer complex was measured. In the presence of excess AMP, the rupture force increased by about 10 pN. rnThe dissociation constant of the AMP-split aptamer complex was measured on a single molecular level (~ 4 µM) by varying the AMP concentrations and measuring the rupture force at each concentration. Furthermore, the rupture force was amplified when more pockets were added to the split aptamer. rnIn the absence of AMP, the thermal off-rate was slightly reduced compared to that in the presence of AMP, indicating that the AMP stabilized the aptamer. The rupture forces at different loading rates did not follow the logarithmic fit which was usually used to describe the dependence of rupture forces at different loading rates of oligonucleotides. Two distinguished regimes at low and high loading rates were obtained. The two regimes were explained by a model in which the oligos located at the pockets were stretched at high loading rates. rnThe contribution of a single H-bond formed between the AMP molecule and the split aptamer was measured by reducing the binding groups of the AMP. The rupture forces reduce corresponding to the reduction of the binding groups. The phosphate group played the most important role in the formation of H-bond network between the AMP molecule and the split aptamer. rn

Vibrational spectroscopy on thermally and optically switchable spin crossover compounds

Oktaedrisch koordinierte Übergangsmetalle mit der Elektronenkonfiguration [Ar]3d4 - 3d7 können in zwei unterschiedlichen elektronischen Zuständen existieren: im High-Spin (HS) oder im Low-Spin (LS) Zustand. Zum Beispiel kann Fe(II) in 1A1g (LS) oder 5T2g (HS) Konfiguration auftreten.Besonderes Interesse besteht in der Aufklärung des Mechanismus der kooperativen Wechselwirkung, die den Spinübergang im Festkörper bestimmt. Hierzu müssen zunächst die internen Freiheitsgrade der molekularen Einheiten bekannt sein. Besonders der Beitrag der molekularen Schwingungen zur Entropiedifferenz, die die Triebkraft des Spinübergangs darstellt, ist von entscheidender Bedeutung. Bisher existieren nur wenige detaillierte Untersuchungen zu den Schwingungseigenschaften der Spincrossovermoleküle.In Rahmen der vorliegenden Arbeit wurden die Schwingungseigenschaften einiger Komplexverbindungen, die Spincrossover zeigen, im Detail untersucht. Dazu wurden temperaturabhängige Raman-, Fern- und Mittel-Infrarot-Spektroskopie, Isotopensubstitution und Normalkoordinatenanalysen (NKA) in Verbindung mit Dichtefunktional-Rechnungen (DFT) verwendet.Die gewonnenen Werte der zugeordneten Schwingungsfrequenzen und die bestimmten Kraftkonstantenänderungen können nun zur Verfeinerung von theoretischen Modellen zur Beschreibung des Spinübergangs verwendet werden.

Polyelectrolytes and their counterions studied by EPR spectroscopy

In this thesis methods of EPR spectroscopy were used to investigate polyion-counterion interactions in polyelectrolyte solutions. The fact that EPR techniques are local methods is exploited and by employing spin-carrying (i.e., EPR-active) probe ions it is possible to examine polyelectrolytes from the counterions’ point of view. It was possible to gain insight into i) the dynamics and local geometry of counterion attachment, ii) conformations and dynamics of local segments of the polyion in an indirect manner, and iii) the spatial distribution of spin probe ions that surround polyions in solution. Analysis of CW EPR spectra of dianion nitroxide spin probe Fremy’s salt (FS, potassium nitrosodisulfonate) in solutions of cationic PDADMAC polyelectrolyte revealed that FS ions and PDADMAC form transient ion pairs with a lifetime of less than 1 ns. This effect was termed as dynamic electrostatic attachment (DEA). By spectral simulation taking into account the rotational dynamics as a uniaxial Brownian reorientation, also the geometry of the attached state could be characterized. By variation of solvent, the effect of solvent viscosity and permittivity were investigated and indirect information of the polyelectrolyte chain motion was obtained. Furthermore, analysis of CW EPR data also indicates that in mixtures of organic solvent/water PDADMAC chains are preferentially solvated by the organic solvent molecules, while in purely aqueous mixtures the PDADMAC chain segments were found in different conformations depending on the concentration ratio R of FS counterions to PDADMAC repeat units.Broadenings in CW EPR spectra of FS ions were assigned to spin-exchange interaction and hence contain information on the local concentrations and distributions of the counterions. From analysis of these broadenings in terms of a modified cylindrical cell approach of polyelectrolyte theory, radial distribution functions for the FS ions in the different solvents were obtained. This approach breaks down in water above a threshold value of R, which again indicates that PDADMAC chain conformations are altered as a function of R. Double electron-electron resonance (DEER) measurements of FS ions were carried out to probe the distribution of attached counterions along polyelectrolyte chains. For a significant fraction of FS spin probes in solution with a rigid-rod model polyelectrolyte containing charged Ru2+-centers, a bimodal distance distribution was found that nicely reproduced the spacings of direct and next-neighbor Ru2+-centers along the polyelectrolyte: 2.35 and 4.7 nm. For the system of FS/PDADMAC, DEER data could be simulated by assuming a two-state distribution of spin probes, one state corresponding to a homogeneous (3-dimensional) distribution of spin probes in the polyelectrolyte bulk and the other to a linear (1-dimensional) distribution of spin probes that are electrostatically condensed along locally extended PDADMAC chain segments. From this analysis it is suggested that the PDADMAC chains form locally elongated structures of a size of at least ~5 nm.

Grating coupled surface plasmon enhanced fluorescence spectroscopy

Over the last three decades, sensors based on the phenomenon of surface plasmon resonance have proven particularly suitable for real time thin film characterization, gas detection, biomolecular interaction examination and to supplement electrochemical methods. Systems based on prism coupling have been combined with fluorescence detection under the name of surface plasmon fluorescence spectroscopy to increase sensitivity even further. Alternatively, metal gratings can be employed to match photons for plasmon resonance. The real time monitoring of binding reactions not yet been reported in the combination of fluorescence detection and grating coupling. Grating-based systems promise more competitive products, because of reduced operating costs, and offer benefits for device engineering. This thesis is comprised of a comprehensive study of the suitability of grating coupling for fluorescence based analyte detection. Fundamental properties of grating coupled surface plasmon fluorescence spectroscopy are described, as well as issues related to the commercial realization of the method. Several new experimental techniques are introduced and demonstrated in order to optimize performance in certain areas and improve upon capabilities in respect to prism-based systems. Holographically fabricated gratings are characterized by atomic force microscopy and optical methods, aided by simulations and profile parameters responsible for efficient coupling are analyzed. The directional emission of fluorophores immobilized on a grating surface is studied in detail, including the magnitude and geometry of the fluorescence emission pattern for different grating constants and polarizations. Additionally, the separation between the minimum of the reflected intensity and the maximum fluorescence excitation position is examined. One of the key requirements for the commercial feasibility of grating coupling is the cheap and faithful mass production of disposable samples from a given master grating. The replication of gratings is demonstrated by a simple hot embossing method with good reproducibility to address this matter. The in-situ fluorescence detection of analyte immobilization and affinity measurements using grating coupling are described for the first time. The physical factors related to the sensitivity of the technique are assessed and the lower limit of detection of the technique is determined for an exemplary assay. Particular attention is paid to the contribution of bulk fluorophores to the total signal in terms of magnitude and polarization of incident and emitted light. Emission from the bulk can be a limiting factor for experiments with certain assay formats. For that reason, a novel optical method, based on the modulation of both polarization and intensity of the incident beam, is introduced and demonstrated to be capable of eliminating this contribution.

Assessing the functional structure of molecular transporters by EPR spectroscopy

In this thesis, the self-assembled functional structure of a broad range of amphiphilic molecular transporters is studied. By employing paramagnetic probe molecules and ions, continuous-wave and pulse electron paramagnetic resonance spectroscopy reveal information about the local structure of these materials from the perspective of incorporated guest molecules. First, the transport function of human serum albumin for fatty acids is in the focus. As suggested by the crystal structure, the anchor points for the fatty acids are distributed asymmetrically in the protein. In contrast to the crystallographic findings, a remarkably symmetric entry point distribution of the fatty acid binding channels is found, which may facilitate the uptake and release of the guest molecules. Further, the metal binding of 1,2,3-triazole modified star-shaped cholic acid oligomers is studied. These biomimetic molecules are able to include and transport molecules in solvents of different polarity. A pre-arrangement of the triazole groups induces a strong chelate-like binding and close contact between guest molecule and metal ion. In absence of a preordering, each triazole moiety acts as a single entity and the binding affinity for metal ions is strongly decreased. Hydrogels based on N-isopropylacrylamide phase separate from water above a certain temperature. The macroscopic thermal collapse of these hydrogels is utilized as a tool for dynamic nuclear polarization. It is shown that a radical-free hyperpolarized solution can be achieved with a spin-labeled gel as separable matrix. On the nanoscale, these hydrogels form static heterogeneities in both structure and function. Collapsed regions protect the spin probes from a chemical decay while open, water-swollen regions act as catalytic centers. Similarly, thermoresponsive dendronized polymers form structural heterogeneities, which are, however, highly dynamic. At the critical temperature, they trigger the aggregation of the polymer into mesoglobules. The dehydration of these aggregates is a molecularly controlled non-equilibrium process that is facilitated by a hydrophobic dendritic core. Further, a slow heating rate results in a kinetically entrapped non-equilibrium state due to the formation of an impermeable dense polymeric layer at the periphery of the mesoglobule.

Investigation of the electric field enhanced PNA-DNA hybridization on Au surface by using surface plasmon field-enhanced fluorescence spectroscopy (SPFS)

Recently, the surface plasmon field-enhanced fluorescence spectroscopy (SPFS) was developed as a kinetic analysis and a detection method with dual- monitoring of the change of reflectivity and fluorescence signal for the interfacial phenomenon. A fundamental study of PNA and DNA interaction at the surface using surface plasmon fluorescence spectroscopy (SPFS) will be investigated in studies. Furthermore, several specific conditions to influence on PNA/DNA hybridization and affinity efficiency by monitoring reflective index changes and fluorescence variation at the same time will be considered. In order to identify the affinity degree of PNA/DNA hybridizaiton at the surface, the association constant (kon) and the dissociation constant (koff) will be obtained by titration experiment of various concentration of target DNA and kinetic investigation. In addition, for more enhancing the hybridization efficiency of PNA/DNA, a study of polarized electric field enhancement system will be introduced and performed in detail. DNA is well-known polyelectrolytes with naturally negative charged molecules in its structure. With polarized electrical treatment, applying DC field to the metal surface, which PNA probe would be immobilized at, negatively charged DNA molecules can be attracted by electromagnetic attraction force and manipulated to the close the surface area, and have more possibility to hybridize with probe PNA molecules by hydrogen bonding each corresponding base sequence. There are several major factors can be influenced on the hybridization efficiency.

Fluctuation spectroscopy of single plasmonic nanostructures

Plasmonic nanoparticles are great candidates for sensing applications with optical read-out. Plasmon sensing is based on the interaction of the nanoparticle with electromagnetic waves where the particle scatters light at its resonance wavelength. This wavelength depends on several intrinsic factors like material, shape and size of the nanoparticle as well as extrinsic factors like the refractive index of the surrounding medium. The latter allows the nanoparticle to be used as a sensor; changes in the proximate environment can be directly monitored by the wavelength of the emitted light. Due to their minuscule size and high sensitivity this allows individual nanoparticles to report on changes in particle coverage.rnrnTo use this single particle plasmon sensor for future sensing applications it has to meet the demand for detection of incidents on the single molecule level, such as single molecule sensing or even the detection of conformational changes of a single molecule. Therefore, time resolution and sensitivity have to be enhanced as today’s measurement methods for signal read-out are too slow and not sensitive enough to resolve these processes. This thesis presents a new experimental setup, the 'Plasmon Fluctuation Setup', that leads to tremendous improvements in time resolution and sensitivity. This is achieved by implementation of a stronger light source and a more sensitive detector. The new setup has a time resolution in the microsecond regime, an advancement of 4-6 orders of magnitude to previous setups. Its resonance wavelength stability of 0.03 nm, measured with an exposure time of 10 ms, is an improvement of a factor of 20 even though the exposure time is 3000 times shorter than in previous reports. Thus, previously unresolvable wavelength changes of the plasmon sensor induced by minor local environmental alteration can be monitored with extremely high temporal resolution.rnrnUsing the 'Plasmon Fluctuation Setup', I can resolve adsorption events of single unlabeled proteins on an individual nanorod. Additionally, I monitored the dynamic evolution of a single protein binding event on a millisecond time scale. This feasibility is of high interest as the role of certain domains in the protein can be probed by a study of modified analytes without the need for labels possibly introducing conformational or characteristic changes to the target. The technique also resolves equilibrium fluctuations in the coverage, opening a window into observing Brownian dynamics of unlabeled macromolecules. rnrnA further topic addressed in this thesis is the usability of the nanoruler, two nanospheres connected with a spacer molecule, as a stiffness sensor for the interparticle linker under strong illumination. Here, I discover a light induced collapse of the nanoruler. Furthermore, I exploit the sensing volume of a fixed nanorod to study unlabeled analytes diffusing around the nanorod at concentrations that are too high for fluorescence correlation spectroscopy but realistic for biological systems. Additionally, local pH sensing with nanoparticles is achieved.

Hundertneunundzwanzig Xe NMR for investigations of complex and dynamic systems

In this work, the remarkable versatility and usefulness of applications of Xe-129 NMR experiments is further extended. The application of Xe-129 NMR spectroscopy to very different system is studied, including dynamic and static, solid and liquid, porous and non-porous systems. Using the large non-equilibrium polarization created by hyperpolarization of Xe-129, time-resolved NMR measurements can be used for the online-monitoring of dynamic systems. In the first part of this work, several improvements for medical applications of hyperpolarized Xe-129 are achieved and their feasibility shown experimentally. A large gain in speed and reproducibility of the accumulation process of Xe-129 as ice and an enhancement of the usable polarization in any experiment requiring prior accumulation are achieved. An enhancement of the longitudinal relaxation time of Xe-129 is realized by admixture of a buffer gas during the storage of hyperpolarized Xe-129. Pursuing the efforts of simplifying the accumulation process and enhancing the storage time of hyperpolarized Xe-129 will allow for a wider use of the hyperpolarized gas in (medical) MRI experiments. Concerning the use of hyperpolarized Xe-129 in MRI, the influence of the diffusion coefficient of the gas on parameters of the image contrast is experimentally demonstrated here by admixture of a buffer gas and thus changing the diffusion coefficient. In the second part of this work, a polymer system with unique features is probed by Xe-129 NMR spectroscopy, proving the method to be a valuable tool for the characterization of the anisotropic properties of semicrystalline, syndiotactic polystyrene films. The polymer films contain hollow cavities or channels with sizes in the sub-nanometer range, allowing for adsorption of Xe-129 and subsequent NMR measurements. Despite the use of a ’real-world’ system, the transfer of the anisotropic properties from the material to adsorbed Xe-129 atoms is shown, which was previously only known for fully crystalline materials. The anisotropic behavior towards atomar guests inside the polymer films is proven here for the first time for one of the phases. For the polymer phase containing nanochannels, the dominance of interactions between Xe-129 atoms in the channels compared to interactions between Xe atoms and the channel walls are proven by measurements of a powder sample of the polymer material and experiments including the rotation of the films in the external magnetic field as well as temperature-dependent measurements. The characterization of ’real-world’ systems showing very high degrees of anisotropy by Xe-129 are deemed to be very valuable in future applications. In the last part of this work, a new method for the online monitoring of chemical reactions has been proposed and its feasibility and validity are experimentally proven. The chemical shift dependence of dissolved Xe-129 on the composition of a reaction mixture is used for the online monitoring of free-radical miniemulsion polymerization reactions. Xe-129 NMR spectroscopy provides an excellent method for the online monitoring of polymerization reactions, due to the simplicity of the Xe-129 NMR spectra and the simple relationship between the Xe-129 chemical shift and the reaction conversion. The results of the time-resolved Xe-129 NMR measurements are compared to those from calorimetric measurements, showing a good qualitative agreement. The applicability of the new method to reactions other than polymerization reactions is investigated by the online monitoring of an enzymatic reaction in a miniemulsion. The successful combination of the large sensitivity of Xe-129, the NMR signal enhancements due to hyperpolarization, and the solubility of Xe-129 gives access to the large new field of investigations of chemical reaction kinetics in dynamic and complex systems like miniemulsions.

Formation of DNA complexes in aqueous and organic solvents

From a physical-chemical point of view, it is challenging to form complexes with polyelectrolytes, consisting of only molecule of the largest component, i.e. the component with the highest number of charges. In this study, complexes are formed with DNA because of its potential applications as an artificial vector for gene delivery. The aim of this work is to prepare complexes in aqueous solutions as well as in organic solvents containing only one DNA molecule. For this purpose, the topology, equilibrium and conformation of complexes between a supercoiled DNA pUC19 (2686 base pairs) and spermine containing hydrophilic and/or hydrophobic moieties or a polylysine with a hydrophilic block are determined by means of dynamic (DLS) and static light scattering (SLS), atomic force microscopy (AFM), and circular dichroism (CD) spectroscopy. It is demonstrated that all of these complexes consisted of only one molecule of the polyanion. Only the polylysine-b-polyethylene glycol copolymer satisfied the conditions: 1) 100% neutralization of DNA charges and with a small excess of the cation (lower than 30%) and 2) form stable complexes at every charge ratio. rnDNA complex formation is also investigated in organic solvents. Precipitation is induced by neutralizing the charge of the supercoiled DNA pUC19 with the surfactants dodecyltrimethylammonium bromide (DTAB) and tetradecyltrimethylammonium bromide (TTAB). After isolation and drying of the solids, the complexes are dissolved in organic solvents. DNA-TTA complexes are only soluble in methanol and DNA-DTA in DMF. The complexes again consisted of only one DNA molecule. The final topology of the complexes is different in methanol than in DMF. In the former case, DNA seems to be compacted whereas in the latter case, the DNA-DTA complexes seem to have an expanded conformation. Upon complex formation with polycations in organic solvents (with polyvilylpyridine brush (b-PVP) in methanol and with a protected polylysine in DMF), DNA aggregates and precipitates. rnDNA is linearized with an enzyme (SmaI) to investigate the influence of the initial topology of the polyanion on the final conformation of the complexes in organic solvents. Two main differences are evidenced: 1. Complexes in organic solvents formed with linear DNA have in general a more expanded conformation and a higher tendency to aggregate. 2. If a polycation, i.e. the b-PVP, is added to the linear DNA-TTA complexes in methanol, complexes with the polycation are formed at a higher charge ratio. In DMF, the addition of the same b-PVP and of b-PLL did not lead to the formation of complexes.rn

Studies of degradation of organic solar cell materials using electrical scanning probe microscopy

Intense research is being done in the field of organic photovoltaics in order to synthesize low band-gap organic molecules. These molecules are electron donors which feature in combination with acceptor molecules, typically fullerene derivarntives, forming an active blend. This active blend has phase separated bicontinuous morphology on a nanometer scale. The highest recorded power conversionrnefficiencies for such cells have been 10.6%. Organic semiconductors differ from inorganic ones due to the presence of tightly bonded excitons (electron-hole pairs)resulting from their low dielectric constant (εr ≈2-4). An additional driving force is required to separate such Frenkel excitons since their binding energy (0.3-1 eV) is too large to be dissociated by an electric field alone. This additional driving force arises from the energy difference between the lowest unoccupied molecular orbital (LUMO) of the donor and the acceptor materials. Moreover, the efficiency of the cells also depends on the difference between the highest occupied molecular orbital (HOMO) of the donor and LUMO of the acceptor. Therefore, a precise control and estimation of these energy levels are required. Furthermore any external influences that change the energy levels will cause a degradation of the power conversion efficiency of organic solar cell materials. In particular, the role of photo-induced degradation on the morphology and electrical performance is a major contribution to degradation and needs to be understood on a nanometer scale. Scanning Probe Microscopy (SPM) offers the resolution to image the nanometer scale bicontinuous morphology. In addition SPM can be operated to measure the local contact potential difference (CPD) of materials from which energy levels in the materials can be derived. Thus SPM is an unique method for the characterization of surface morphology, potential changes and conductivity changes under operating conditions. In the present work, I describe investigations of organic photovoltaic materials upon photo-oxidation which is one of the major causes of degradation of these solar cell materials. SPM, Nuclear Magnetic Resonance (NMR) and UV-Vis spectroscopy studies allowed me to identify the chemical reactions occurring inside the active layer upon photo-oxidation. From the measured data, it was possible to deduce the energy levels and explain the various shifts which gave a better understanding of the physics of the device. In addition, I was able to quantify the degradation by correlating the local changes in the CPD and conductivity to the device characteristics, i.e., open circuit voltage and short circuit current. Furthermore, time-resolved electrostatic force microscopy (tr-EFM) allowed us to probe dynamic processes like the charging rate of the individual donor and acceptor domains within the active blend. Upon photo-oxidation, it was observed, that the acceptor molecules got oxidized first preventing the donor polymer from degrading. Work functions of electrodes can be tailored by modifying the interface with monomolecular thin layers of molecules which are made by a chemical reaction in liquids. These modifications in the work function are particularly attractive for opto-electronic devices whose performance depends on the band alignment between the electrodes and the active material. In order to measure the shift in work function on a nanometer scale, I used KPFM in situ, which means in liquids, to follow changes in the work function of Au upon hexadecanethiol adsorption from decane. All the above investigations give us a better understanding of the photo-degradation processes of the active material at the nanoscale. Also, a method to compare various new materials used for organic solar cells for stability is proposed which eliminates the requirement to make fully functional devices saving time and additional engineering efforts.