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Micro-BLMs on Highly Ordered Porous Silicon Substrates: Rupture Process and Lateral Mobility Daniela Weiskopf,† Eva K. Schmitt,† Marco H. Klu¨hr,‡,§ Stephan K. Dertinger,‡,| and Claudia Steinem*,† Institut fu¨r Organische und Biomolekulare Chemie, Georg-August UniVersita¨t, Tammannstrasse 2, 37077 Go¨ttingen, Germany, and Qimonda AG, Otto-Hahn-Ring 6, 81739 Mu¨nchen, Germany ReceiVed April 13, 2007. In Final Form: June 11, 2007 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 µm. The upper surface of the silicon substrate was coated with a gold layer, followed by the chemisorption of 1,2dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) and subsequent addition of a droplet of 1,2-diphytanoyl-snglycero-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) µm2/s.
I. Introduction Lipid bilayers attached to a support are invaluable tools for investigating the physical and mechanical properties of membranes, their molecular dynamics, and the interaction of proteins.1,2 In general, lipid bilayers are immobilized on a planar support, which might, however, alter their physical properties such as the lateral diffusion of lipids and hampers the insertion of transmembrane proteins such as ion channels and transporters.3 Several groups have addressed these major drawbacks by separating the lower leaflet of the bilayer from the substrate using long hydrophilic spacers and tethering soft polymer cushions.4,5 A different approach makes use of membranes that suspend the pores of a porous material.6-13 In this case, the porous material * To whom correspondence should be addressed. E-mail: claudia.
[email protected]. Phone: + 49 551 393294. Fax: + 49 551 393228. † Georg-August Universita ¨ t. ‡ Qimonda AG. § Present address: Agrobiogen GmbH, Thalmannsdorf 25, 86567 Hilgertshausen, Germany. | Present address: Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany. (1) Brian, A. A.; McConnell, H. M. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 6159-6163. (2) Sackmann, E. Science 1996, 271, 43-48. (3) Janshoff, A.; Steinem, C. Anal. Bioanal. Chem. 2006, 385, 433-451. (4) Sackmann, E.; Tanaka, M. Trends Biotechnol. 2000, 18, 58-64. (5) Knoll, W.; Morigaki, K.; Naumann, R.; Sacca, B.; Schiller, S.; Sinner, E.-K., Functional Tethered Bilayer Lipid Membranes. In Ultrathin Electrochemical Chemo- and Biosensors; Mirsky, V. M., Ed.; Springer: Berlin, 2004; pp 239253. (6) Favero, G.; Capanella, L.; Cavallo, S.; D’Annibale, A.; Perrella, M.; Mattei, E.; Ferri, T. J. Am. Chem. Soc. 2005, 127, 8103-8111. (7) Favero, G.; Capanella, L.; D’Annibale, A.; Santucci, R.; Ferri, T. Microchem. J. 2003, 74, 141-148. (8) Hemmler, R.; Bo¨se, G.; Wagner, R.; Peters, R. Biophys. J. 2005, 88, 40004007. (9) Hennesthal, C.; Drexler, J.; Steinem, C. ChemPhysChem. 2002, 3, 885889. (10) Hennesthal, C.; Steinem, C. J. Am. Chem. Soc. 2000, 122, 8085-8086. (11) Quist, A. P.; Chand, A.; Ramachandran, S.; Daraio, C.; Jin, S.; Lal, R. Langmuir 2007, 23, 1375-1380. (12) Worsfold, O.; Voelcker, N. H.; Nishiya, T. Langmuir 2006, 22, 70787083.
is intended to provide a support to increase the stability of the membranes, whereas they are in part still freestanding and thus their properties are not influenced by a support. In particular, we have demonstrated that lipid membranes can be suspended on highly ordered porous alumina substrates with pore sizes of 60 and 280 nm, respectively, and porous silicon with pores of 1 µm diameter.14-17 Such membranes, which we term nano- and microBLMs, respectively, are achieved by first functionalizing the upper porous surface with gold and then chemisorbing 1,2dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) from ethanol, followed by spreading 1,2-diphytanoyl-sn-glycero-3phosphocholine (DPhPC) dissolved in n-decane. An impedance spectroscopy investigation revealed a mean specific capacitance of the membranes that is characteristic of the formation of single lipid bilayers. Membrane resistances were on the order of gigaohms,whichenabledustoperformsingle-channelrecordings.15-17 An interesting observation is that the membrane resistance does not decrease in an all-or-none process as is found for membranes suspending a single aperture but decreases continuously over time.15 We hypothesized that the continuous decrease is a result of the individual rupturing of suspending lipid membranes on the porous material. The aim of this study was to visualize the proposed rupturing process by fluorescence microscopy. A prerequisite for a fully functional lipid bilayer is that the lipids diffuse laterally within the plane of the membrane. Model membranes provide an excellent opportunity to study the lateral diffusion at the molecular level. Over the past 30 years, a number of techniques have been developed and applied to determine the diffusion constants of lipids in membranes, such as nuclear magnetic and electron spin resonance, fluorescence correlation spectroscopy, and fluorescence recovery after photobleaching (13) Steltenkamp, S.; Mu¨ller, M.; Deserno, M.; Hennesthal, C.; Steinem, C.; Janshoff, A. Biophys. J. 2006, 91, 217-226. (14) Horn, C.; Steinem, C. Biophys. J. 2005, 89, 1046-1054. (15) Ro¨mer, W.; Lam, Y. H.; Fischer, D.; Watts, A.; Fischer, W. B.; Go¨ring, P.; Wehrspohn, R. B.; Go¨sele, U.; Steinem, C. J. Am. Chem. Soc. 2004, 126, 16267-16274. (16) Ro¨mer, W.; Steinem, C. Biophys. J. 2004, 86, 955-965. (17) Schmitt, E. K.; Vrouenraets, M.; Steinem, C. Biophys. J. 2006, 91, 21632171.
10.1021/la701080u CCC: $37.00 © 2007 American Chemical Society Published on Web 07/27/2007
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(FRAP). In this study, we applied FRAP to determine the lateral diffusion coefficient of lipids in our micro-BLMs. II. Experimental Section Materials. 1,2-Dipalmitoyl-sn-glycero-3-phosphothioethanol (DPPTE) and 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) were obtained from Avanti Polar Lipids (Alabaster, AL). Texas red DHPE (1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) and β-BODIPY 500/510C12-HPC were from Sigma-Aldrich, Germany. The water used was ion exchanged and filtered with a Millipore filter (Millipore, Milli-Q system, Molsheim, France, specific resistance R > 18 MΩ cm-1, pH 5.5). For all measurements, a 0.5 M KCl solution was used. Formation of Micro-BLMs. Highly ordered macroporous silicon substrates were used for the preparation of micro-BLMs. Fabrication of the macroporous silicon membrane starts with a photolithographic definition of the pore pattern in n-type FZ silicon (100) with a resistivity of 90 Ω cm. The start pits for the growth of the pores were etched into the silicon surface using anisotropic KOH etching (cKOH ) 10 wt %). The pores were arranged in an orthogonal lattice with a pitch of 12 µm. Electrochemical etching was carried out in HF under backside illumination (cHF ) 6 wt %, T ) 10 °C, U ) 2.5 V),18,19 resulting in 430-µm-deep pores. To obtain a membrane with through pores, the bulk silicon on the back side of the wafer was removed by KOH etching (cKOH ) 10 wt %). The silicon membrane was oxidized in a dry oxygen atmosphere (Tempress Systems TS 6304) at 900 °C for 10 min to grow a thermal oxide layer of 10 nm. The final thickness of the membrane was 420 µm. The diameter of the square pores was 7 µm. Single chips were obtained by dicing or breaking. The top side of the porous material was then coated with a thin titanium layer (2.5 nm) as an adhesive to deposit a gold layer of 180 nm thickness. The gold layer allowed for the chemisorption of DPPTE from a 0.5 mM ethanol solution (t > 12 h), rendering the surface hydrophobic. Micro-BLMs were prepared by adding a small droplet of DPhPC (2% w/v in n-decane) to the hydrophobic submonolayer. Impedance Spectroscopy. Rupturing of the micro-BLMs was monitored by impedance spectroscopy. The experimental setup is shown in Figure 1d. The porous substrate was clamped between two O-rings with a diameter of 3 mm and bathed in 0.5 M KCl solution. Two platinized platinum wires served as working and counter electrodes. An SI 1260 impedance gain/phase analyzer and an SI 1296 dielectric interface (Solartron Instruments, Farnborough, U.K.) controlled by a personal computer were used for ac impedance analysis. The absolute value of impedance |Z|(f) and the phase angle Φ(f) between voltage and current were recorded within a frequency range of 10-3-106 Hz, with equally scaled data points per decade, which took approximately 30 min. All data was obtained at zero offset potential by applying a small ac voltage of 30 mV to avoid nonlinear responses. Data recording was performed using the Solartron impedance measurement software (version 3.5.0). For data analysis, the Zview 2.9c software package with calc-modulus data weighting was used. Fluorescence Microscopy. By means of fluorescence microscopy, the process of membrane rupturing on the porous substrate was visualized. An AxioTech Vario (Zeiss, Jena, Germany) was used and was equipped with a 40× water immersion objective (ACHROPLAN, na ) 0.8 water, Zeiss, Hamburg, Germany) and a filter set allowing the excitation of fluorescence at around 560 nm and emission at around 630 nm (filter no. 45, Zeiss, Jena, Germany). Membranes were fluorescently labeled by adding 0.1 mol % Texas red DHPE. For fluorescence recovery after photobleaching (FRAP) experiments, an argon ion laser (JDS Uniphase, Milpitas, CA) with a wavelength of 488 nm was coupled into the optical path of the fluorescence microscope. A filter set was used for fluorescence excitation at around 475 nm and emission at around 530 nm (18) Klu¨hr, M. H.; Sauermann, A.; Elsner, C. A.; Thein, K. H.; Dertinger, S. K. AdV. Mater 2006, 23, 3135-3139. (19) Lehmann, V. J. Electrochem. Soc. 1993, 140, 2836-2843.
Figure 1. Impedance spectroscopy analysis of a pore-suspending membrane on porous silicon immersed in a 0.5 M KCl solution over a time period of 8.3 h. (a) Absolute value of the impedance |Z|(f) and (b) phase angle Φ(f) 0.6 h after the micro-BLM has been formed (9), after 2.2 h (2), and after 8.3 h ((). The solid lines are the results of a fitting procedure using the equivalent circuit (1) depicted in part c with the following fit parameters: (9) Cm ) 0.008 µF, Rel ) 220 Ω; (2) Cm ) 0.012 µF, Rm ) 2.7 GΩ, and Rel ) 190 Ω; and (() Cm ) 0.022 µF, Rm ) 51 MΩ, and Rel ) 172 Ω. The dashed line is the result of fitting equivalent circuit (2) to the data with the following fit parameters: A ) 0.034 µF, R ) 0.92, Rm ) 57 MΩ, and Rel ) 160 Ω. (c) Schematic representations of the equivalent circuits used to model the impedance data. (d) Experimental setup. Two Teflon half-cells with a porous silicon substrate clamped between two O-rings with a diameter of 3 mm that were used for impedance measurements. Electrical contact was obtained by platinized platinum electrodes on both sides. (filter no. 44, Zeiss, Jena, Germany). Membranes were fluorescently labeled with 0.2 mol % β-BODIPY 500/510C12-HPC. Uniform circular photobleaching (spot size ∼20 µm in diameter) was obtained with a laser intensity of ∼35 mW at the surface with a bleaching time of 3 s. Fluorescence images were recorded using a 40× water
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immersion objective (ACHROPLAN, na ) 0.8 water, Zeiss, Hamburg, Germany) every second for about 100 s. From the recovery curves, diffusion coefficients were obtained using the theory of Axelrod et al.20 for a uniform circular laser profile.
III. Results and Discussion Rupturing of Micro-BLMs Monitored by Impedance Analysis. The electrical properties of the micro-BLMs on porous silicon with an etch length of 7 µm were determined using impedance spectroscopy. After the addition of the lipid-solvent droplet and the thinning process of the membranes, impedance spectra were obtained from which the membrane capacitance Cm as well as the membrane resistance Rm could be extracted using equivalent circuit (1) shown in Figure 1c.15 It is composed of a parallel connection with an ohmic resistance Rm and a capacitance Cm representing the electrical properties of the lipid membrane in series to an ohmic resistance Rel representing the electrolyte solution. If resistance Rm was larger than 3 GΩ and could not to be detected within the observed frequency range of 10-3-106 Hz, it was omitted in equivalent circuit (1) (Figure 1c). Characteristic impedance spectra of micro-BLMs are depicted in Figure 1. Figure 1a,b show the absolute value of the impedance |Z|(f) and the phase angle Φ(f) as a function of time, respectively. Directly after the formation of the micro-BLM, the impedance spectra are characterized by the electrolyte resistance in the highfrequency regime and a capacitance in the frequency range below 105 Hz. Membrane resistance is not defined in the observed frequency range. Fitting a simple serial connection of a capacitance Cm and an electrolyte resistance Rel to the data results in a membrane capacitance of 8 nF. This translates into a specific membrane capacitance of Cm ) 0.7 µF/cm2 when taking the total porous area of 17% into account, which was obtained from scanning electron microscopy images.15 The average specific membrane capacitance after the formation of a micro-BLM was determined to be (0.7 ( 0.3) µF/cm2 as obtained from 67 independent measurements. Over time, the membrane resistance decreases and becomes discernible in the impedance spectra. Fitting equivalent circuit (1) (Figure 1c) to the data results in good agreement between fit and data and allows the extraction of the membrane capacitance as well as the membrane resistance, which are both plotted as a function of time (Figure 2). We attribute the increase in membrane conductance and capacitance to a gradual rupturing of the bilayers covering the pores. As a result, the surface becomes more inhomogeneous as more membranes rupture, hence the impedance behavior becomes less ideal. In this case, fitting equivalent circuit (1) to the data does not lead to good agreement (Figure 1a,b). Hence, it might be more adequate to apply an alternative model for data analysis. By replacing the ideal capacitance by a constant-phase element (CPE), which accounts for the inhomogeneity of the surface,21 good agreement between data and fit can be achieved (Figure 1a,b, dashed line). The deviation from the ideal capacitance is given by the parameter R, which turned out to be R ) 0.92 for the impedance spectra obtained 8.3 h after micro-BLM formation and is still close to 1, justifying that the parameter A is given in Farad. As expected, the membrane resistance decreases over time (Figure 2a). Directly after micro-BLM formation, Rm was not detectable within the observed frequency range, indicating that it is well above 3 GΩ. After 2.2 h, Rm was determined to be 2.7 GΩ and decreases to 51 MΩ at t ) 8.3 h. The principal time (20) Axelrod, D.; Koppel, D. E.; Schlessinger, J.; Elston, E.; Webb, W. W. Biophys. J. 1976, 16, 1055-1069. (21) Kerner, Z.; Pajkossy, T. J. Electroanal. Chem. 1998, 448, 139-142.
Figure 2. (a) Change in membrane resistance Rm as a function of time. Rm was extracted from time-resolved impedance spectra by fitting equivalent circuit (1) (Figure 1c) to the data. (b) Change in the specific membrane capacitance Cm within a time period of 18 h extracted from impedance spectra.
course of membrane resistance change is the same as we have observed for nano-BLMs on porous alumina substrates with pore sizes of 280 nm.16 However, by taking the time period in which the membrane resistance decreases by 1 order of magnitude after its preparation as a measure of its stability, it can be stated that smaller pores increase the stability of the membrane. Although the membrane resistance on 7 µm pores decreases within (5 ( 3) hours by 1 order of magnitude, the time period is about 10 times larger on 280 nm pores.16 This result demonstrates that smaller pores are advantageous for obtaining long-term stable membranes with high resistance. The capacitance Cm also increases with time (Figure 2b). Although directly after micro-BLM formation the specific membrane capacitance is 0.7 µF/cm2, it increases up to 1.9 µF/cm2 after 8.3 h. The increase is presumably due to the occurrence of defects in the bilayer over the pores. To investigate the influence of the functionalization of the porous silicon substrate on the rupturing process, similar experiments were performed in which the submonolayer DPPTE was replaced by an octadecanethiol submonolayer. Within the error of the experiments, no significant difference was observed. The average capacitance of the micro-BLMs was determined to be (1.0 ( 0.4) µF/cm2 for seven independent measurements, whereas the membrane resistance was initially in the gigaohm regime and decreased over time (Supporting Information). Rupturing of Micro-BLMs as Monitored by Fluorescence Microscopy. By means of fluorescence microscopy, we were able to visualize micro-BLMs on DPPTE-functionalized porous silicon substrates and the rupturing process as a function of time. To obtain fluorescence images, the membrane was labeled with the Texas red DHPE fluorophore. Painting the fluorescently labeled lipid solution in n-decane onto the functionalized porous silicon substrate results in an immediate spreading of the solution across the porous material as observed by fluorescence microscopy. After the thinning process, fluorescently labeled membranes
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Figure 4. Pixel analysis of fluorescence images obtained from microBLMs on porous silicon substrates as shown in Figure 3. The observed fluorescence at time t ) 0 h was set to 100%.
Figure 3. Fluorescence micrographs of micro-BLMs composed of DPhPC doped with 0.1 mol % Texas red DHPE on functionalized porous silicon immersed in 0.5 M KCl over a time period of 4 h. Membrane-covered pores are bright, and non-covered pores are black. t(h) ) (a) 0, (b) 0.5, (c) 1, (d) 1.5, (e) 2.5, (f) 3, (g) 3.5, (h) 4. The scale bar represents 20 µm.
are observed, which are separated by the gold-covered pore rims on which the fluorescence is quenched (Figure 3). This quenching on the pore rims enables us to monitor every membrane covering an individual pore. In Figure 3, characteristic fluorescence micrographs are displayed, as monitored over a time period of 4 h. Dependent on the exact focal plane, the pores appear either rather quadratic or circular. Directly after the micro-BLMs have been formed, almost all pores are covered with lipid membranes (Figure 3a). Thirty minutes after membrane formation, dark spots occur as observed in the lower left corner of Figure 3b, indicative of the rupturing of fluorescently labeled individual micro-BLMs. During the next 3.5 h, more and more black spots appear as a result of membranes that have been ruptured (Figure 3c-h). Very similar results were obtained for micro-BLMs prepared on porous silicon substrates functionalized with an octadecanethiol monolayer (Supporting Information) instead of DPPTE, stressing the point that the functionalization of the porous silicon substrate does not significantly influence the rupturing process of micro-BLMs on 7 µm pores. The fluorescence images were analyzed in more detail by means of pixel analysis (Figure 4). At time t ) 0, the observed fluorescence was set to 100%. Over time, the number of pores that are not covered by a membrane increases, as plotted in Figure 4. Within the first 3 h, already 70% of the micro-BLMs covering the pores have ruptured. The fraction of non-covered
pores appears to be rather large compared to the results obtained from impedance analysis. However, one should be aware that by fluorescence microscopy only part of the porous material is analyzed whereas impedance analysis is an integral method and hence the changes in capacitance and resistance are monitored over the entire porous area. On one substrate, there exist areas where no membranes have ruptured, whereas other areas are already almost free of pore-suspending membranes. To illustrate the difference in coverage on one substrate, three fluorescence micrographs of a micro-BLM preparation monitored 17 h after the formation process are shown in Figure 5. Figure 5a displays a region where most of the micro-BLMs have ruptured, and Figure 5b shows an area where the membranes have partially ruptured. Figure 5c is an example of an area where even after 17 h the micro-BLMs suspending the pores of the porous silicon substrate are mostly intact. This result demonstrates that it will be very useful to apply a local electrical method to micro- and nano-BLMs22 to exploit the fact that even if membranes have ruptured in certain areas there are other areas where the membranes are still fully intact and thus exhibit a large membrane resistance, which might even be sufficient for single-channel recordings. Fluorescence Recovery after Photobleaching on MicroBLMs. The membrane’s fluidity is a prerequisite for and an indicator of a functional membrane system. To elucidate the lateral mobility of our micro-BLMs, we performed fluorescence recovery after photobleaching (FRAP) experiments. After preparation of the micro-BLMs and the thinning process, photobleaching was accomplished with a 3 s laser pulse focused on a small spot on the sample. In Figure 6, a typical FRAP experiment is displayed. Before bleaching, the micro-BLMs appear as described above with a rather homogeneous fluorescence of the membranes covering the pores (Figure 6a, A). Directly after the bleaching pulse, the fluorescence obtained from membranes covering the pores is bleached (Figure 6a, B) and recovers almost completely after 100 s (Figure 6a, D). From the recovery curve, the diffusion coefficient can be extracted using the theory developed by Axelrod et al.20 for a circular bleaching spot. An effective diffusion coefficient of Deff ) (10.9 ( 0.7) µm2/s is obtained with an immobile fraction of about 6% (Figure 6b). From 10 independent measurements, an average effective diffusion coefficient of Deff ) (14 ( 1) µm2/s and an immobile fraction of (15 ( 5)% were obtained. This diffusion coefficient is very similar to what has been obtained for classical (22) Bo¨cker, M.; Anczykowski, B.; Wegener, J.; Scha¨ffer, T. E. Nanotechnology 2007, 18, 1-6. (23) Ladha, S.; Mackie, A. R.; Harvey, L. J.; Clark, D. C.; Lea, E. J. A.; Brullemans, M.; Duclohier, H. Biophys. J. 1996, 71, 1364-1373. (24) Lalchev, Z. I.; Mackie, A. R. Colloids Surf., B 1999, 15, 147-160.
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Figure 5. Fluorescence micrographs of micro-BLMs composed of DPhPC on a functionalized porous silicon substrate doped with 0.1 mol % Texas red DHPE immersed in 0.5 M KCl 17 h after preparation. Depending on the observed area, micro-BLMs are (a) mostly ruptured, (b) in part ruptured, and (c) mostly intact and suspend the pores. The scale bars represent 20 µm.
Figure 6. (a) Fluorescence micrographs of micro-BLMs composed of DPhPC on a functionalized porous silicon substrate doped with 0.2 mol % β-BODIPY 500/510C12-HPC during a FRAP experiment. (A) t < 0 s, (B) t ) 0 s, (C) t ) 20 s, and (D) t ) 100 s after the bleaching pulse. The region of interest is marked with a white circle. The scale bars represent 20 µm. (b) (b) Fluorescence recovery curve extracted from the fluorescence images after a bleaching pulse, normalized to the initial flourescence before bleaching. The solid line is the result of the fitting routine according to Axelrod et al.20 resulting in an effective diffusion coefficient of Deff ) (10.9 ( 0.7) µm2/s and an immobile fraction of about 6%.
BLMs23,24 and is larger than those obtained for membranes immobilized on glass supports.25 However, one has to be aware
of the fact that we expect to have two different diffusion coefficients on the surface, namely, one of the freely moving
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lipids in the pore-suspending region and the second one on the pore rims, which experiences enhanced friction as a result of the interaction with the functionalized solid support. Thus, we refer to the determined diffusion coefficient as an effective diffusion coefficient. Experiments and simulations are currently underway to address this problem in more detail. In conclusion, we were able to demonstrate by means of fluorescence microscopy that the observed continuous change in membrane resistance is a result of the individual rupturing of membranes suspending the pores of a porous material. Moreover, by using fluorescence recovery after photobleaching, we were able to demonstrate that the lipids within the micro-BLMs are (25) Groves, J. T.; Boxer, S. G. Biophys. J. 1995, 69, 1972-1975.
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laterally mobile with a diffusion coefficient similar to those obtained in classical BLMs. Acknowledgment. The DFG (STE 884/5-1) is gratefully acknowledged for financial support. E.K.S. thanks the Fonds der Chemischen Industrie for a fellowship. We also thank H. Adam from the University of Mainz for providing us software written in IGOR for the FRAP data workup. Supporting Information Available: Impedance analysis and fluorescence micrographs of micro-BLMs prepared on octadecanethiol submonolayers. This material is available free of charge via the Internet at http://pubs.acs.org. LA701080U
