New synthetic strategies to tethered bilayer lipid membranes

Tethered bilayer lipid membranes provide an efficient, stable and versatile platform for the investigation of integrated membrane proteins. However, the incorporation of large proteins, as well as of proteins with a large submembrane part is still a very critical issue and therefore, further optimisation of the system is necessary. The central element of a tBLM is a lipid bilayer. Its proximal leaflet is, at least to some extend, covalently attached to a solid support via a spacer group. The anchor lipid consists of three distinct parts, a lipid headgroup, a spacer group and an anchor. All parts together influence the final bilayer properties. In the frame of this work, the synthesis of new thiolipids for tBLMs on gold has been investigated. The aim was to obtain molecules with longer spacers in order to increase the submembrane space. The systems obtained have been characterized using SPR and EIS. The results obtained during this study are multiple. First, the synthesis of a previously synthesized architecture was successfully scaled up in an industrial lab using a new synthetic approach. The synthesis of large amounts is now feasible. Then, the synthesis of the new thiolipids was carried out taking into account the following requirements: the increase of the submembrane space by having longer ethyleneglycol spacers, the attachment of the molecules to a gold substrate via a thiol bond, and the tunability of the lateral mobility by changing the lipid headgroup. Three different synthetic strategies have been investigated. The polymeric approach did not prove to be successful, merely because of the broad molecular weight distribution. The synthesis of heterofunctionally protected oligoethyleneglycols allowed to obtain ethyleneglycol moieties with 6 and 8 units, but the tedious purification steps gave very low yields. Finally, the block by block synthesis using ethyleneglycol precursors proved to be an efficient and fast method to synthesize the target molecules. Indeed, these were obtained with very high yields, and the separation was very efficient. A whole family of new compounds was obtained, having 6, 8 and 14 ethyleneglycol units and with mono- or diphytanyl lipid headgroups. This new pathway is a very promising synthetic strategy that can be used further in the development of new compounds of the tether system. The formation of bilayers was investigated for the different thiolipids mainly by using EIS. The electrical properties of a bilayer define the quality of the membrane and allow the study of the functionality of proteins embedded in such a system. Despite multiple trials to improve the system using self assembly, Langmuir Blodgett transfer, and detergent mixed vesicles, the new polymer thiolipids did not show as high electrical properties as tBLMs reported in the literature. Nevertheless, it was possible to show that a bilayer could be obtained for the different spacer lengths. These bilayers could be formed using self assembly for the first monolayer, and two different methods for bilayer formation, namely vesicle fusion and solvent exchange. We could furthermore show functional incorporation of the ion carrier valinomycin: the selective transport of K+ ions could be demonstrated. For DPHL, it was even possible to show the functional incorporation of the ion channel gramicidin. The influence of the spacer length is translated into an increase of the spacer capacitance, which could correspond to an increase in the capacity of charge accumulation in the submembrane space. The different systems need to be further optimised to improve the electrical properties of the bilayer. Moreover, the incorporation of larger proteins, and proteins bearing submembrane parts needs to be investigated.

Surface-plasmon optical and electrochemical characterization of biofunctional surface architectures

The development and characterization of biomolecule sensor formats based on the optical technique Surface Plasmon Resonance (SPR) Spectroscopy and electrochemical methods were investigated. The study can be divided into two parts of different scope. In the first part new novel detection schemes for labeled targets were developed on the basis of the investigations in Surface-plamon Field Enhanced Spectroscopy (SPFS). The first one is SPR fluorescence imaging formats, Surface-plamon Field Enhanced Fluorescence Microscopy (SPFM). Patterned self assembled monolayers (SAMs) were prepared and used to direct the spatial distribution of biomolecules immobilized on surfaces. Here the patterned monolayers would serve as molecular templates to secure different biomolecules to known locations on a surface. The binding processed of labeled target biomolecules from solution to sensor surface were visually and kinetically recorded by the fluorescence microscope, in which fluorescence was excited by the evanescent field of propagating plasmon surface polaritons. The second format which also originates from SPFS technique, Surface-plamon Field Enhanced Fluorescence Spectrometry (SPFSm), concerns the coupling of a fluorometry to normal SPR setup. A spectrograph mounted in place of photomultiplier or microscope can provide the information of fluorescence spectrum as well as fluorescence intensity. This study also firstly demonstrated the analytical combination of surface plasmon enhanced fluorescence detection with analyte tagged by semiconducting nano- crystals (QDs). Electrochemically addressable fabrication of DNA biosensor arrays in aqueous environment was also developed. An electrochemical method was introduced for the directed in-situ assembly of various specific oligonucleotide catcher probes onto different sensing elements of a multi-electrode array in the aqueous environment of a flow cell. Surface plasmon microscopy (SPM) is utilized for the on-line recording of the various functionalization steps. Hybridization reactions between targets from solution to the different surface-bound complementary probes are monitored by surface-plasmon field-enhanced fluorescence microscopy (SPFM) using targets that are either labeled with organic dyes or with semiconducting quantum dots for color-multiplexing. This study provides a new approach for the fabrication of (small) DNA arrays and the recording and quantitative evaluation of parallel hybridization reactions. In the second part of this work, the ideas of combining the SP optical and electrochemical characterization were extended to tethered bilayer lipid membrane (tBLM) format. Tethered bilayer lipid membranes provide a versatile model platform for the study of many membrane related processes. The thiolipids were firstly self-assembled on ultraflat gold substrates. Fusion of the monolayers with small unilamellar vesicles (SUVs) formed the distal layer and the membranes thus obtained have the sealing properties comparable to those of natural membranes. The fusion could be monitored optically by SPR as an increase in reflectivity (thickness) upon formation of the outer leaflet of the bilayer. With EIS, a drop in capacitance and a steady increase in resistance could be observed leading to a tightly sealing membrane with low leakage currents. The assembly of tBLMs and the subsequent incorporation of membrane proteins were investigated with respect to their potential use as a biosensing system. In the case of valinomycin the potassium transport mediated by the ion carrier could be shown by a decrease in resistance upon increasing potassium concentration. Potential mediation of membrane pores could be shown for the ion channel forming peptide alamethicin (Alm). It was shown that at high positive dc bias (cis negative) Alm channels stay at relatively low conductance levels and show higher permeability to potassium than to tetramethylammonium. The addition of inhibitor amiloride can partially block the Alm channels and results in increase of membrane resistance. tBLMs are robust and versatile model membrane architectures that can mimic certain properties of biological membranes. tBLMs with incorporated lipopolysaccharide (LPS) and lipid A mimicking bacteria membranes were used to probe the interactions of antibodies against LPS and to investigate the binding and incorporation of the small antimicrobial peptide V4. The influence of membrane composition and charge on the behavior of V4 was also probed. This study displays the possibility of using tBLM platform to record and valuate the efficiency or potency of numerous synthesized antimicrobial peptides as potential drug candidates.

Structure and dynamics of ionic liquid,electrode interfaces

In the early 20th century, Gouy, Chapman, and Stern developed a theory to describe the capacitance and the spatial ion distribution of diluted electrolytes near an electrode. After a century of research, considerable progress has been made in the understanding of the electrolyte/electrode interface. However, its molecular-scale structure and its variation with an applied potential is still under debate. In particular for room-temperature ionic liquids, a new class of solventless electrolytes, the classical theories for the electrical double layer are not applicable. Recently, molecular dynamics simulations and phenomenological theories have attempted to explain the capacitance of the ionic liquid/electrode interface with the molecular-scale structure and dynamics of the ionic liquid near the electrode. rnHowever, experimental evidence is very limited. rnrnIn the presented study, the ion distribution of an ionic liquid near an electrode and its response to applied potentials was examined with sub-molecular resolution. For this purpose, a new sample chamber was constructed, allowing in situ high energy X-ray reflectivity experiments under potential control, as well as impedance spectroscopy measurements. The combination of structural information and electrochmical data provided a comprehensive picture of the electric double layer in ionic liquids. Oscillatory charge density profiles were found, consisting of alternating anion- and cation-enriched layers at both, cathodic and anodic, potentials. This structure was shown to arise from the same ion-ion correlations dominating the liquid bulk structure that were observed as a distinct X-ray diffraction peak. Therefore, existing physically motivated models were refined and verified by comparison with independent measurements. rnrnThe relaxation dynamics of the interfacial structure upon potential variation were studied by time resolved X-ray reflectivity experiments with sub-millisecond resolution. The observed relaxation times during charging/discharging are consistent with the impedance spectroscopy data revealing three processes of vastly different characteristic time-scales. Initially, the ion transport normal to the interface happens on a millisecond-scale. Another 100-millisecond-scale process is associated with molecular reorientation of electrode-adsorbed cations. Further, a minute-scale relaxation was observed, which is tentatively assigned to lateral ordering within the first layer.

Entwicklung eines schnellen, piezobasierten Frequenztuners für supraleitende CH-Kavitäten

Im Rahmen der vorliegenden Arbeit wurde ein schnelles, piezobasiertes Frequenztuningsystem für aktuelle sowie zukünftige supraleitende (sl) CH-Kavitäten entwickelt. Die Grundlage des hierbei verwendeten Tuningkonzepts unterscheidet sich von bisherigen, konventionellen Tuningmethoden supraleitender Kavitäten grundlegend. Zum Ausgleichen von unerwünschten Frequenzverstimmungen während des Beschleunigerbetriebes werden sogenannte bewegliche Balgtuner in das Innere der Resonatorgeometrie geschweißt. Aufgrund ihrer variablen Länge können diese die Kapazität der Kavität und somit die Resonanzfrequenz gezielt beeinflussen. Die Antriebsmechanik, die für die Auslenkung bzw. Stauchung der Balgtuner sorgt, besteht aus einer langsamen, schrittmotorbetriebenen und einer schnellen, piezobasierten Tuningeinheit, welche auf der Außenseite des Heliummantels der jeweiligen CH-Kavität installiert wird. Zur Überprüfung dieses neuartigen Tuningkonzepts wurde in der Werkstatt des Instituts für Angewandte Physik (IAP) der Goethe Universität Frankfurt ein Prototyp der gesamten Tuningeinheit aus Edelstahl gefertigt. Die Funktionsweise der langsamen sowie schnellen Tuningeinheit konnten hierbei in ersten Messungen bei Raumtemperatur erfolgreich getestet werden. Somit stellt die in dieser Arbeit entwickelte Tuningeinheit eine vielversprechende Möglichkeit des dynamischen Frequenztunings supraleitender CH-Strukturen dar. rnDes Weiteren wurden im Rahmen der Arbeit mit Hilfe der Simulationsprogramme ANSYS Workbench sowie CST MicroWave Studio gekoppelte strukturmechanische und elektromagnetische Simulationen der sl 217 MHz CH sowie der sl 325 MHz CH-Kavität durchgeführt. Hierbei konnte zum einen der Frequenzbereich und somit der notwendige mechanische Hub der jeweiligen Tuningeinheit durch Bestimmung der Frequenzverstimmungen signifikant reduziert werden. Zum anderen war es möglich, die mechanische Stabilität der beiden Kavitäten zu untersuchen und somit plastische Deformationen von vornherein auszuschließen. Zur Überprüfung der Genauigkeit sämtlicher getätigter Simulationsrechnungen wurde das strukturmechanische Verhalten in Abhängigkeit äußerer Einflüsse und die daraus resultierenden Frequenzverstimmungen der CH-Kavitäten sowohl bei Raumtemperatur als auch bei kryogenen Temperaturen von 4.2 K gemessen. Hierbei zeigten sich zum Teil hervorragende Übereinstimmungen zwischen den simulierten und gemessenen Werten mit Diskrepanzen von unter 10%. Mit Hilfe dieser Ergebnisse konnte gezeigt werden, dass die gekoppelte Simulation ein essentielles Werkzeug während der Entwicklungsphase einer supraleitenden Beschleunigungsstruktur darstellt, so dass die für den Betrieb erforderliche mechanische Stabilität einer supraleitenden Kavität erreicht werden kann. rn