ABSTRACT
The mechanical behavior of lipid bilayers spanning the pores of highly ordered porous silicon substrates was scrutinized by local indentation experiments as a function of surface functionalization, lipid composition, solvent content, indentation velocity, and pore radius. Solvent-containing nano black lipid membranes (nano-BLMs) as well as solvent-free pore-spanning bilayers were imaged by fluorescence and atomic force microscopy prior to force curve acquisition, which allows distinguishing between membrane-covered and uncovered pores. Force indentation curves on pore-spanning bilayers attached to functionalized hydrophobic porous silicon substrates reveal a predominately linear response that is mainly attributed to prestress in the membranes. This is in agreement with the observation that indentation leads to membrane lysis well below 5% area dilatation. However, membrane bending and lateral tension dominate over prestress and stretching if solvent-free supported membranes obtained from spreading giant liposomes on hydrophilic porous silicon are indented. An elastic regime diagram is presented that readily allows determining the dominant contribution to the mechanical response upon indentation as a function of load and pore radius.
Subject(s)
Lipid Bilayers/chemistry , Gold/chemistry , Hydrophobic and Hydrophilic Interactions , Membranes/chemistry , Microscopy, Atomic Force , Microscopy, Fluorescence , Models, Chemical , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/chemistry , Silicon/chemistry , Stress, MechanicalABSTRACT
Nano-BLMs (black lipid membranes) suspending the pores of highly ordered porous silicon substrates have been proven useful for functional investigations of ion channel proteins by electrical readouts. With the aim to monitor the resistive behavior of nano-BLMs spatially resolved in a contact-free manner, we report here on the visualization of nano-BLMs by means of scanning ion conductance microscopy (SICM). Silicon surfaces with highly ordered pore arrays were coated with a gold layer and functionalized with octadecanethiol before a droplet of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) (2% w/v) dissolved in n-decane was applied. The topography of DPhPC membranes suspending the pores was stably imaged for hours without mechanical contact using SICM. This suggests that SICM provides a significant advantage over atomic force microscopy, where mechanical interactions occur that easily damage the suspended membranes. Dynamic processes such as spreading and rupturing of membranes were spatially and temporally resolved. Furthermore, SICM was used to individually manipulate membranes suspending single pores, thereby writing lithographic patterns into the lipid. The process of local membrane manipulation was correlated to a characteristic signature in the simultaneously recorded ion current. The results show that SICM is well-suited both for contact-free imaging of soft suspended membranes and for local membrane manipulation.
Subject(s)
Chemistry/methods , Lipid Bilayers , Phosphatidylcholines/chemistry , Silicon/chemistry , Alkanes/chemistry , Cations , DNA/chemistry , Electrodes , Equipment Design , Ions , Lipids/chemistry , Membranes/chemistry , Microscopy, Atomic Force , Microscopy, Electron, Scanning/methods , SynchrotronsABSTRACT
Pore-suspending membranes on porous alumina substrates with pore diameters of 60 nm were prepared by fusion of vesicles on a (cholesterylpolyethylenoxy)thiol-functionalized surface, and their functionality demonstrated by the activity of the proton pump bacteriorhodopsin.
ABSTRACT
In a recent paper, we hypothesized that the continuous increase in membrane conductance observed for nano-BLMs is the result of an independent rupturing of single membranes or membrane patches covering the pores of the porous material. To prove this hypothesis, we prepared micro-BLMs on porous silicon substrates with a pore size of 7 mum. The upper surface of the silicon substrate was coated with a gold layer, followed by the chemisorption of 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) and subsequent addition of a droplet of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) dissolved in n-decane. The lipid membranes were fluorescently labeled and investigated by means of fluorescence microscopy and impedance spectroscopy. Impedance spectroscopy revealed the formation of pore-suspending bilayers with high membrane resistance. Increases in membrane capacitance and membrane conductance were observed. This increase in membrane conductance could be unambiguously related to the individual rupturing of membranes suspending the pores of the porous material as visualized by means of fluorescence microscopy. Moreover, by fluorescence recovery after photobleaching experiments, we investigated the lateral mobility of the lipids within the micro-BLMs leading to a mean effective diffusion coefficient of Deff = (14 +/- 1) microm2/s.
Subject(s)
Nanostructures/chemistry , Silicon/chemistry , Electric Impedance , Microscopy, Fluorescence , Photobleaching , Porosity , Spectrophotometry , Stress, MechanicalABSTRACT
Free-standing lipid bilayer membranes can be formed on small apertures (60 nm diameter) on highly ordered porous alumina substrates. The formation process of the membranes on a 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol submonolayer was followed by impedance spectroscopy. After lipid bilayers had thinned, the reconstitution and ionic conducting properties of the outer membrane protein OmpF of E. coli were monitored using single-channel recordings. The characteristic conductance states of the three monomers, fast kinetics, and subconductance states were observed. Blockade of the ion flow as a result of interaction of the antibiotic ampicillin with the protein was verified, indicating the full functionality of the protein channel in nanometer-scale bilayer membranes.
