Biomembrane templates for nanoscale conduits and networks

Science

Volumn 273, Issue 5277, 1996, Pages 933-935

  Evans, Evan ^a   Bowman, Howard ^b   Leung, Andrew ^a   Needham, David ^c   Tirrell, David ^b  

^a UNIVERSITY OF BRITISH COLUMBIA   (Canada)

^b UNIVERSITY OF MASSACHUSETTS   (United States)

^c Duke University   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; LIPID BILAYER; POLYMERIZATION; PRIORITY JOURNAL; STRUCTURE ANALYSIS;

EID: 0029791320     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.273.5277.933     Document Type: Article

References (25)

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11. We prepared giant bilayer vesicles (20 to 40 μm) by a method similar to previously published methods [D. Needham and E. Evans, Biochemistry 27, 8261 (1988); D. Needham, Methods Enzymol. 220. 111 (1993)]. The lipid composition was a mixture of 66 mol% steatoyl-oleoyl phosphatidylcholine, 33 mol% cholesterol (Avanti Polar Lipids, Alabaster, AL), and 1 mol% N-([6-(biotinoyl)amino]hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3- phosphoethanolamine, triethylammonium salt (biotin-X-DHPE; Molecular Probes, Eugene, OR), in which biotin is conjugated to the DHPE lipid head group through a hydrocarbon spacer 10 to 15 Å long. The lipids were made up initially in chloroform-methanol (2:1) and spread on a Teflon disk. After evaporation of the volatile solvent for at least 6 hours in vacuum and constant darkness, the lipid paste was hydrated by a warm water-saturated argon jet for 15 min. Then the solution to be encapsulated (200 mM sucrose plus ingredients for polymerization) was gently added to the lipid multilayers. After the system was left to swell overnight, a sample of vesicles was harvested from the top of the beaker and resuspended in an equiosmolar glucose solution. In preparation for nanotube extrusion and photo-cross-linking, a small amount of the vesicle suspension was injected into a microchamber on the stage of an interference contrast videomicroscope. The glucose-sucrose differential ensured that the vesicles would sink to the bottom of the microscope chamber. For each test, a single vesicle was selected by micropipette and transferred into an adjacent test chamber, which contained avidin-coated microspheres as adhesive substrates for attachment to the vesicle surface. Details of the buffer solutions are described below.
12. We prepared giant bilayer vesicles (20 to 40 μm) by a method similar to previously published methods [D. Needham and E. Evans, Biochemistry 27, 8261 (1988); D. Needham, Methods Enzymol. 220. 111 (1993)]. The lipid composition was a mixture of 66 mol% steatoyl-oleoyl phosphatidylcholine, 33 mol% cholesterol (Avanti Polar Lipids, Alabaster, AL), and 1 mol% N-([6-(biotinoyl)amino]hexanoyl)-1,2-dihexadecanoyl-sn-glycero-3- phosphoethanolamine, triethylammonium salt (biotin-X-DHPE; Molecular Probes, Eugene, OR), in which biotin is conjugated to the DHPE lipid head group through a hydrocarbon spacer 10 to 15 Å long. The lipids were made up initially in chloroform-methanol (2:1) and spread on a Teflon disk. After evaporation of the volatile solvent for at least 6 hours in vacuum and constant darkness, the lipid paste was hydrated by a warm water-saturated argon jet for 15 min. Then the solution to be encapsulated (200 mM sucrose plus ingredients for polymerization) was gently added to the lipid multilayers. After the system was left to swell overnight, a sample of vesicles was harvested from the top of the beaker and resuspended in an equiosmolar glucose solution. In preparation for nanotube extrusion and photo-cross-linking, a small amount of the vesicle suspension was injected into a microchamber on the stage of an interference contrast videomicroscope. The glucose-sucrose differential ensured that the vesicles would sink to the bottom of the microscope chamber. For each test, a single vesicle was selected by micropipette and transferred into an adjacent test chamber, which contained avidin-coated microspheres as adhesive substrates for attachment to the vesicle surface. Details of the buffer solutions are described below.
13. 1) of Avidin NeutraliteT (Molecular Probes) in 150 mM NaCl. The avidin physiosorbed strongly to the bead surfaces. To prepare a pattern of sites for network formation, we coated the glass floor of the microchamber with a monolayer of biotinylated serum albumin; then the avidin-coated microspheres were positioned on the surface and immobilized by adhesive bonds. The avidin-biotin system was chosen only as a prototype to demonstrate attachments to solid surfaces. For microdevices, a more appropriate method would be to use thiol groups for bonding to gold or silver contacts (15). The thiol sulfur atoms form covalent bonds to these metal substrates. As such, thiol-conjugated amphiphiles could be synthesized and incorporated into the lipid bilayer of the vesicle to permit attachments to gold regions on a microchip surface.
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25. This development was motivated by the Canadian Institute for Advanced Research Program in Science of Soft Surfaces and Interfaces. Additional support came from Canadian Medical Research Council grant MT 7477 (E.E.), the University of Massachusetts Materials Research Science and Engineering Center (D.T.), and North Carolina Biotechnology Center grant 9413 ARG-0018 (D.N. and E.E.).