Show simple item record

Phospholipidmembranen auf mikroporösen Substraten: in situ Bildung elektrochemischer Gradienten

dc.contributor.advisorSteinem, Claudia Prof.
dc.contributor.authorFrese, Danielde
dc.publisherNiedersächsische Staats- und Universitätsbibliothek Göttingende
dc.titlePhospholipidmembranen auf mikroporösen Substraten: in situ Bildung elektrochemischer Gradientende
dc.title.translatedPhospholipid membranes on microporous substrates: in situ generation of electrochemical gradientsde
dc.contributor.refereeSteinem, Claudia Prof.
dc.description.abstractengElectrochemical gradients are of utmost importance for cellular processes, such as the generation of action potentials or the production of ATP in mitochondria. They are established by membrane proteins, which consume energy to provide an active transport of e.g. ions across membranes. Membrane proteins are the main targets for pharmaceutical drugs and they are controlled very sensitively by external stimuli (e.g. light, potential differences, temperature), raising interest in their application as biosensors. A model system based on artificial membranes spanning well-defined picolitre cavities on microporous substrates was developed for the functional reconstitution of the membrane protein bacteriorhodopsin (bR) and the in situ generation of electrochemical gradients. The formation of pore-spanning membranes (PSMs), either by spreading of giant unilamellar vesicles (GUVs) onto hydrophilically functionalized substrates, or by application of lipids dissolved in n-decane onto hydrophobically functionalized substrates (painting-technique), was investigated by three-dimensional confocal laser scanning microscopy (CLSM), fluorescence recovery after photobleaching (FRAP) experiments and scanning ion conductance microscopy (SICM). The amount of residual solvent in painted PSMs could be reduced by solvent freeze-out. Spreading of GUVs on hydrophilically functionalized porous substrates yielded not only planar, but also hemispherical PSMs, protruding several micrometers into the bulk solution. The pH-sensitive dye pyranine was stably entrapped within membrane suspended pores derived from vesicle spreading as well as from the painting-technique. Changes in the intensity of entrapped pyranine were used to monitor the formation of nigericin-induced proton gradients across PSMs. Proton gradients of up to ΔpH = 2 units were generated and the initial rate for nigericin-mediated proton transport across PSMs was determined to be 10^14 H+∙s-1∙cm-2. Several strategies were used to reconstitute bR into liposomes and PSMs, conserving its function as a light-driven proton pump. Photocurrents were recorded, verifying the functionality of bR in large unilamellar vesicles (LUVs), as well as in GUVs. A net-transport of protons into the liposomes or from the liposomes into the bulk solution could neither be observed by changes in pyranine intensity, nor directly via pH-electrode. After its reconstitution into PSMs by spreading of proteo-GUVs, bR was shown to be laterally mobile with a diffusion coefficient of approximately D = 0,2 ± 0,1 μm2∙s-1 and a mobile fraction of M = 68 ± 16 %. The developed model system therefore provides highly ordered, well-defined picolitre cavities that can be addressed by optical microscopy and scanning probe techniques (e.g. SICM). Cavities can tightly be sealed with PSMs as indicated via the generation of electrochemical gradients. These features in combination with the ability to reconstitute membrane proteins (bR) make the developed setup a promising tool for pharmaceutical screening applications and biosensor
dc.contributor.coRefereeZweckstetter, Markus Prof.
dc.subject.engPore-spanning membranesde
dc.subject.engElectrochemical gradientsde
dc.subject.engMembrane transportde
dc.subject.engPorous silicon substratesde
dc.affiliation.instituteFakultät für Chemiede
dc.subject.gokfullChemie  (PPN62138352X)de

Files in this item


This item appears in the following Collection(s)

Show simple item record