Article Cite This: Langmuir XXXX, XXX, XXX−XXX
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Continuous Pore-Spanning Lipid Bilayers on Silicon Oxide-Coated Porous Substrates Nelli Teske,† Jeremias Sibold,† Johannes Schumacher, Nikolas K. Teiwes, Martin Gleisner, Ingo Mey, and Claudia Steinem* Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstraße 2, 37077 Göttingen, Germany S Supporting Information *
ABSTRACT: A number of techniques has been developed and analyzed in recent years to generate pore-spanning membranes (PSMs). While quite a number of methods rely on nanoporous substrates, only a few use micrometer-sized pores to be able to individually resolve suspending membranes by means of fluorescence microscopy. To be able to produce PSMs on pores that are micrometer in size, an orthogonal functionalization strategy resulting in a hydrophilic surface is highly desirable. Here, we report on a method to prepare PSMs based on the evaporation of a thin layer of silicon monoxide on top of the porous substrate. PM-IRRAS experiments demonstrate that the final surface is composed of SiOx with 1 < x < 2. The hydrophilic surface turned out to be well suited to spread giant unilamellar vesicles forming PSMs. As the method does not rely on a gold coating as frequently used for orthogonal functionalization, fluorescence micrographs provide information not only from the freestanding membrane areas but also from the supported ones. The observation of the entire PSM area enabled us to observe phase-separation in these membranes on the freestanding and supported parts as well as protein binding and possible lipid reorganization of the membranes induced by binding of the protein Shiga toxin.
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INTRODUCTION Planar lipid bilayers have been established as versatile tools to investigate structural properties of membranes as well as membrane-confined processes.1−4 A widely used system are supported lipid bilayers exhibiting high mechanical stability. Owing to the planar geometry and their attachment to a support, they allow to apply surface-sensitive methods such as surface plasmon resonance spectroscopy to investigate, e.g., membrane−protein interactions5−8 and microscopy methods such as fluorescence and atomic force microscopy to resolve, e.g., phase-separation phenomena.9−11 However, frictional coupling of the membrane to the support and the lack of a second aqueous compartment reduces the mobility of the membrane components and thus limits the suitability of this system. An alternative to supported lipid bilayers has been developed, termed pore-spanning membranes (PSMs). These membranes span nanometer-12−24 to micrometer-sized holes25−31 in silicon or aluminum substrates so that freestanding and supported membrane areas are generated. While nanometer-sized pores are highly advantageous as they provide high long-term stability of the membranes, allow preparation methods based on large unilamellar vesicle fusion,12−16,32 and enable one to apply particular readout methods such as optical waveguide spectroscopy,33 individual freestanding PSMs cannot be resolved by optical microscopy techniques. Here, substrates with highly ordered pores in the micrometer regime are most suited.27,28,34−37 © XXXX American Chemical Society
To be able to produce PSMs on pores that are micrometer in size, typically giant unilamellar vesicles (GUVs) are used that cannot enter the large pores as well as an orthogonal surface functionalization strategy or a particular pore geometry to prevent the lipid bilayers to align the inner pore walls.30,31 A widely used orthogonal functionalization strategy for micrometer-sized pores is the deposition of a thin gold layer on top of the porous substrate allowing the chemisorption of thiols to form a self-assembled monolayer (SAM).17,38,39 Hydrophobic SAMs such as 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol or octanethiol SAMs permit spreading of vesicles, which are larger in diameter than the pore diameters of the substrate,23 resulting in a bilayer structure on the pores and a hybrid layer composed of the SAM and a lipid monolayer on the pore rims.28 On hydrophilic SAMs composed of, e.g., mercaptoethanol or mercaptohexanol, PSMs are formed upon vesicle spreading leading to a continuous lipid bilayer on the pores and on the supported parts.34,35 Even mixed SAMs composed of a hydrophobic O-cholesteryl N-(8-mercapto-3,6-dioxaoctyl)carbamate and a hydrophilic mercaptohexanol are suited to form PSMs.35 A major disadvantage of strategies relying on gold coverage is the quenching of the fluorescence near the gold surface preventing the observation of the lipid bilayer on the pore rims by fluorescence microscopy, which renders the Received: August 4, 2017 Revised: November 5, 2017 Published: November 17, 2017 A
DOI: 10.1021/acs.langmuir.7b02727 Langmuir XXXX, XXX, XXX−XXX
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deposited on indium tin oxide (ITO) slides. After removal of the solvent under reduced pressure for 3 h, the ITO slides were assembled in a chamber sealed with a Teflon ring and filled with sucrose solution (1.7 mL, 298 mOsmol/L). A frequency generator was connected to the ITO slides to apply an ac field. GUVs composed of POPC/POPE/ cholesterol/Texas Red DHPE (79.5/10/10/0.5) were produced using the following parameters: U = 3 V, f = 5 Hz, t = 3 h. GUVs composed of POPC/POPE/POPG/Texas Red DHPE (59.8/30/10/0.2) or phase-separated GUVs composed of either DOPC/sphingomyelin/ cholesterol/Gb3/BODIPY-cholesterol/Texas Red DHPE (39.5/35/ 19.5/5/0.5/0.5) or DOPC/sphingomyelin/cholesterol/Gb3/Atto488DPPE (39.5/35/20/5/0.5) were prepared with the following parameters: U = 1.6 V, f = 12 Hz, t = 3 h. Phase-separated GUVs were prepared at T = 55 °C. Pore-Spanning Membranes (PSMs). For the preparation of PSMs on micrometer sized silicon nitride substrates, 60−80 μL of the GUV suspension were added to the SiO-coated substrates. To remove lipid debris, the GUVs were sedimented onto the surface via a 5 mL pipet tip filled with PBS. Because of the higher density of the sucrose filled GUVs, the vesicles move faster through the pipet tip than the lipid debris, which is thus separated. After 10 min of incubation, the substrates were rinsed with PBS to remove nonspread GUVs. To bind STxB to the Gb3 doped PSMs, bovine serum albumin (1 mg/mL) was first added for 45 min to the surface, rinsed with PBS, and then incubated with STxB (60 nM with respect to the molar mass of the STxB monomer) for 45 min. For the preparation of PSMs on AAO, the substrates were cleaned in oxygen plasma (Zepto LF PC, Diener electronic, Ebhausen, Germany, 0.25 mbar O2, 60 W, 30 s), mounted with double-sided adhesive tape in a Petri dish and rinsed with PBS containing 1 mM CaCl2. GUVs composed of POPC/POPE/POPG/Texas Red DHPE (59.8/30/10/0.2) or POPC/POPE/cholesterol/Texas Red DHPE (79.5/10/10/0.5) were added after 15 min and incubated for 10 min. After rinsing with buffer, 0.5 mM pyranine was added to the aqueous phase to indicate membrane-spanned pores. Scanning Electron Microscopy (SEM). The SiO-coated substrates were imaged using a LEO supra-35 microscope (Carl Zeiss, Jena, Germany). Samples were mounted in the chamber with a working distance of ∼3 mm and a pressure of
