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The pattern for the geometrical Brownian ratchet was defined by electron beam lithography. First a 200-nm-thick poly(methyl methacrylate) resist layer was spun onto a glass microscope coverslip, which was subsequently baked and coated with an 80-nm-thick layer of a conducting polymer (11) to prevent charging of the insulating glass coverslip during exposure. After electron beam exposure, the conducting polymer was dissolved in water and the resist was developed. Finally a 30-nm film of Ti was evaporated onto the coverslip. After liftoff, the Ti was oxidized for several hours at 400°C. Before deposition of the lipid bilayer, the patterned coverslips were rinsed for 15 min in hot 7X detergent (ICN Biochemicals, Aurora, OH) and then rinsed with purified water. The coverslips were dried under a nitrogen stream and then heated to 400°C for 4 hours.
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0344528098
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The supported lipid bilayer was formed by fusion of small unilamellar vesicles (SUVs) with the patterned coverslips. The SUVs were prepared according to the Barenholtz procedure; for a detailed description of the procedure see (13-15). The SUVs consist of 99 mol% of the neutral (zwitterionic) phospholipid L-α-phosphatidylcholine (egg-PC; Avanti Polar Lipids, Alabaster, AL) and 1 mol% of the negatively charged, labeled lipids. The bilayer was produced by putting the patterned coverslip on a 70-μl drop of small unilamellar vesicles in a petri dish, which was then filled with purified water and shaken gently to remove nonfused vesicles. Under water, a clean nonpatterned coverslip was placed at the side of the bilayer and this sandwich was transferred to an electrophoresis cell. The electrophoresis cell consists of two reservoirs filled with purified water and each contains a thin platinum electrode (15). The sandwich was placed between the two reservoirs; the water sandwiched between the coverslips provides electrical contact between the two Pt electrodes. All experiments were performed at room temperature. The diffusion and drift of the charged lipids were monitored with a Nikon E800 epifluorescence microscope with a X 4 or X 10 objective and a charge-coupled device camera. The negatively charged fluorescent probes were covalently attached to the phospholipid headgroup (Texas Red-DHPE from Molecular Probes, Eugene, OR; NBD-PE and NBD-PS from Avanti Polar Lipids, Alabaster, AL). The labeled lipids NBD-PE and Texas Red-DHPE carry a net single negative charge, whereas NBD-PS has a double negative charge.
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2, the membranes are conformal and will follow the contour of corrugated surfaces without disruption.
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The typical drift velocities of GPI-linked proteins are significantly larger than was observed for charged lipids, which facilitates separation and relaxes the need for submicrometer lithography
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Supported in part by a grant from the NSF Biophysics Program, by the Materials Research Science and Engineering Center Program of the NSF under award DMR-9808677, and by a grant from SmithKline Beecham. A.v.O. acknowledges the Netherlands Organization for Scientific Research (NWO) for financial support. We acknowledge very useful discussions and insights from J. T. Groves in the development of this project and valuable comments and suggestions on the manuscript by R. D. Astumian. The Stanford Nanofabrication Facility is acknowledged for support in fabrication.
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