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.

Herstellung und physikochemische Charakterisierung von planaren gestützten Lipid-Modellmembran-Systemen

Es wurden funktionalisierte polymerunterstützte planare Phospholipid-Modellmembran-Systeme hergestellt und auf jeder Präparationsstufe eingehend charakterisiert. Dünne Polysaccharidfilme wurden in der Form von quellbaren Gelen auf oxidische Oberflächen aufgebracht und bezüglich ihres Quellungsverhaltens und der Oberflächeneigenschaften in Abhängigkeit vom Wassergehalt untersucht. Lipidmonoschichten unterschiedlicher Zusammensetzung wurden mittels Langmuir-Blodgett-Tranfer auf Polymersubstrate übertragen und bezüglich der Stärke der Lipid/Polymer Wechselwirkung, der lateralen Selbstdiffusion in Abhängigkeit von der Wasseraktivität, dem Spreitverhalten der monomolekularen Membran auf dem Substrat in Abhängigkeit von der Wasseraktivität und dem Lateraldruck der Monoschicht, sowie des Ausmaßes der Hydratation im Kopfgruppenbereich der Lipidmembran in Abhängigkeit von der Wasseraktivität mittels Fluoreszensondenmethoden (Fluoreszenzerholung nach Photobleichung (FRAP), Fluoreszenzmikroskopie und Fluoreszenzspektroskopie) untersucht. Diffusions- und Spreitverhalten von amphiphilen Monoschichten auf Polymersubstraten wurden auf der Basis von in dieser Arbeit entwickelten physikalischen Modellen diskutiert. Mittels Langmuir-Schäfer Transfer wurde auf polymerunterstützte Lipidmonoschichten eine zweite Monoschicht übertragen. Die somit erhaltenen Lipid-Doppelschichtmembranen wurden bezüglich ihrer Stabilität, der lateralen Struktur, der lateralen Selbstdiffusion, des Spreitverhaltens auf unbedeckte Bereiche sowie der Stärke der Membran/Substrat Wechselwirkung vermittels Fluoreszenzmikroskopie, FRAP und Interferenz-Kontrast-Mikroskopie (RICM) untersucht. Schließlich wurden substratgestützte Doppelschicht-Lipidmembranen mit als Protonenpumpen fungierenden integralen Membranproteinen versehen. Die laterale Selbstdiffusion der rekonstituierten Proteinmoleküle wurde mittels FRAP, die funktionale Aktivität der Protonenpumpen mit einem Ionen-sensitiven Feldeffekttransistor-Array analysiert.

Thin functional conducting polymer films

In the present study, thin functional conducting polyaniline (PANI) films, either doped or undoped, patterned or unpatterned, were prepared by different approaches. The properties of the obtained PANI films were investigated in detail by a combination of electrochemistry with several other techniques, such as SPR, QCM, SPFS, diffraction, etc. The sensing applications (especially biosensing applications) of the prepared PANI films were explored. Firstly, the pure PANI films were prepared by the electropolymerisation method and their doping/dedoping properties in acidic conditions were investigated in detail by a combination of electrochemistry with SPR and QCM. Dielectric constants of PANI at different oxidation states were obtained quantitatively. The results obtained here laid a good foundation for the following investigations of PANI films in neutral pH conditions. Next, PANI multilayer films doped by a variety of materials were prepared by the layer-by-layer method in order to explore their biosensing applications, because of the loss of redox activity of pure PANI in neutral pH conditions. The dopants used include not only the traditionally used linear polyelectrolytes, but also, for the first tim, some other novel materials, like modified gold nanoparticles or modified carbon nanotubes. Our results showed that all the used dopants could form stable multilayer films with PANI. All the obtained PANI multilayer films showed good redox activity in a neutral pH environment, which makes them feasible for bioassays. We found that all the prepared PANI multilayer films can electrocatalyze the oxidation of NADH in neutral conditions at a low potential, although their catalytic efficiencies are different. Among them, PANI/carbon nanotube system showed the highest catalytic efficiency toward the oxidation of NADH, which makes it a good candidate as a NADH sensor. Besides, because some of the prepared PANI multilayer systems were end-terminated with –COOH groups (like PANI/Au nanoparticles system), which can be utilized to easily link biomolecules for biosensing applications. Here, we demonstrated, for the first time, to use the prepared PANI multilayer films for the DNA hybridisation detection. The detection event was monitored either by direct electrochemical method, or by enzyme-amplified electrochemical method, or by surface plasmon enhanced fluorescence spectroscopic method. All the methods can effectively differentiate non-complementary DNA from the complementary ones, even at the single-base mismatch level. It should also be noted that, our success in fabricating PANI multilayer films with modified Au nanoparticles or carbon nanotubes also offered another novel method for incorporating such novel materials into (conducting) polymers. Because of the unique electrochemical and optical properties of each component of the obtained PANI multilayer films, they should also find potential applications in many other fields such as microelectronics, or for electrochromic and photovoltaic devices. Finally, patterned PANI films were fabricated by the combination of several patterning techniques, such as the combination of electrocopolymerization with micromolding in capillaries (EP-MIMIC), the combination of microcontact printing with the layer-by-layer technique (µCP-LBL), and the polystyrene (PS) template induced electropolymerisation method. Using the obtained stripe-shaped PANI/PSS film, a redox-switchable polymer grating based on the surface-plasmon-enhanced mode was constructed and its application in the field of biosensing was explored. It was found that the diffraction efficiency (DE) of the grating was very sensitive to the applied potential (i.e. redox state of the film) as well as the pH environment of the dielectric medium. Moreover, the DE could also be effectively tuned by an electrocatalytic event, such as the electrocatalytic oxidation of NADH by the grating film. By using PS colloidal crystal assemblies as templates, well-ordered 3D interconnected macroporous PANI arrays (PANI inverse opals) were fabricated via electropolymerisation method. The quality of the obtained inverse opals was much higher than those reported by chemical synthesis method. By electrochemical method, the structures of the prepared inverse opals can be easily controlled. To explore the possible biosensing applications of PANI inverse opals, efforts were also done toward the fabrication of PANI composite inverse opals. By selecting proper dopants, high quality inverse opals of PANI composites were fabricated for the first time. And the obtained opaline films remained redox-active in neutral pH conditions, pointing to their possible applications for electrobioassays.

Neural network activity in the neonatal rat barrel cortex in vivo

Coordinated patterns of electrical activity are important for the early development of sensory systems. The spatiotemporal dynamics of these early activity patterns and the role of the peripheral sensory input for their generation are essentially unknown. There are two projects in this thesis. In project1, we performed extracellular multielectrode recordings in the somatosensory cortex of postnatal day 0 to 7 rats in vivo and observed three distinct patterns of synchronized oscillatory activity. (1) Spontaneous and periphery-driven spindle bursts of 1–2 s in duration and ~10 Hz in frequency occurred approximately every 10 s. (2) Spontaneous and sensory-driven gamma oscillations of 150–300 ms duration and 30–40 Hz in frequency occurred every 10–30 s. (3) Long oscillations appeared only every ~20 min and revealed the largest amplitude (250–750 µV) and longest duration (>40 s). These three distinct patterns of early oscillatory activity differently synchronized the neonatal cortical network. Whereas spindle bursts and gamma oscillations did not propagate and synchronized a local neuronal network of 200–400 µm in diameter, long oscillations propagated with 25–30 µm/s and synchronized 600-800 µm large ensembles. All three activity patterns were triggered by sensory activation. Single electrical stimulation of the whisker pad or tactile whisker activation elicited neocortical spindle bursts and gamma activity. Long oscillations could be only evoked by repetitive sensory stimulation. The neonatal oscillatory patterns in vivo depended on NMDAreceptor-mediated synaptic transmission and gap junctional coupling. Whereas spindle bursts and gamma oscillations may represent an early functional columnar-like pattern, long oscillations may serve as a propagating activation signal consolidating these immature neuronal networks. In project2, Using voltage-sensitive dye imaging and simultaneous multi-channel extracellular recordings in the barrel cortex and somatosensory thalamus of newborn rats in vivo, we found that spontaneous and whisker stimulation induced activity patterns were restricted to functional cortical columns already at the day of birth. Spontaneous and stimulus evoked cortical activity consisted of gamma oscillations followed by spindle bursts. Spontaneous events were mainly generated in the thalamus or by spontaneous whisker movements. Our findings indicate that during early developmental stages cortical networks self-organize in ontogenetic columns via spontaneous gamma oscillations triggered by the thalamus or sensory periphery.

Functional characterisation of in vitro synthesised G-protein coupled receptors in polymersomes

Membrane proteins play an indispensable role in physiological processes. It is, therefore, not surprising that many diseases are based on the malfunction of membrane proteins. Hence membrane proteins and especially G-protein coupled receptors(GPCRs)- the largest subfamily- have become an important drug target. Due to their high selectivity and sensitivity membrane proteins are also feasible for the detection of small quantities of substances with biosensors. Despite this widespread interest in GPCRs due to their importance as drug targets and biosensors there is still a lack of knowledge of structure, function and endogenous ligands for quiet a few of the previously identified receptors.rnBottlenecks in over-expression, purification, reconstitution and handling of membrane proteins arise due to their hydrophobic nature. Therefore the production of reasonable amounts of functional membrane proteins for structural and functional studies is still challenging. Also the limited stability of lipid based membrane systems hampers their application as platforms forrnscreening applications and biosensors.rnIn recent years the in vitro protein synthesis became a promising alternative to gain better yields for expression of membrane proteins in bio-mimetic membrane systems. These expression systems are based on cell extracts. Therefore cellular effects on protein expression are reduced. The open nature of the cell-free expression systems easily allows for the adjustment of reactionrnconditions for the protein of interest. The cell-free expression in the presence of bio-mimetic membrane systems allows the direct incorporation of the membrane proteins and therefore skips the time-consuming purification and reconstitution processes. Amphiphilic block-copolymers emerged as promising alternative for the less stable lipid-based membrane systems. They, likernlipids, form membraneous structures in aqueous solutions but exhibit increased mechanical and chemical stability.rnThe aim of this work was the generation of a GPCR-functionalised membrane system by combining both promising alternatives: in vitro synthesis and polymeric membrane systems. This novel platform should be feasible for the characterisation of the incorporated GPCR. Immunodetection of Dopamine receptor 1 and 2 expressed in diblock- and triblock-polymersomes demonstrated the successful in vitro expression of GPCRs in polymeric membranes. Antibodyrnbinding studies suggested a favoured orientation of dopamine receptors in triblockpolymersomes.rnA dopamine-replacement assay on DRD2-functionalised immobilised triblockpolymersomes confirmed functionality of the receptor in the polymersomes. The altered binding curve suggests an effect of the altered hydrophobic environment presented by the polymer membrane on protein activity.