Surface Response Methodology for the Study of Supported Membrane Formation
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- 作者：Claire Rossi ; Elisabeth Briand ; Pierre Parot ; Michael Odorico ; Joë ; l Chopineau
- 刊名：Journal of Physical Chemistry B
- 出版年：2007
- 出版时间：July 5, 2007
- 年：2007
- 卷：111
- 期：26
- 页码：7567 - 7576
- 全文大小：568K
- 年卷期：v.111,no.26(July 5, 2007)
- ISSN：1520-5207
文摘
We report on the investigations of the formation of the tethered lipid bilayer by vesicle deposition on amine-functionalized surfaces. The tethered bilayer was created by the deposition of egg-PC vesicles containing1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly-(ethyleneglycol)-N-hydroxysuccinimide as anchoringmolecules on an amine-coated surface. This approach is an easy route for the formation of a biomimetic-supported membrane. A Doelhert experimental design was applied to determine the conditions leading to theformation of a continuous and defect-free tethered bilayer on different surfaces (gold and glass). Doehlertdesigns allow modeling of the experimental responses by second-order polynomial equations as a function ofexperimental factors. Four factors expected to influence bilayer formation were studied: the lipid concentrationin the vesicle suspension, the mass percentage of anchoring molecules in the vesicles, the contact time betweenthe vesicles and the surface, and the resting time of the membrane after buffer rinse. The optimization of themembrane preparation parameters was achieved by monitoring lipid assembly formation using surface plasmonresonance spectroscopy on gold and by fluorescence recovery after photobleaching on glass. Three characteristicresponses were systematically measured: the bilayer thickness, the lipid diffusion coefficient, and the lipidmobile fraction. The simultaneous inspection of the three characteristics revealed that a restricted experimentaldomain leads to properties that are in accordance with a bilayer presence. The factors of this domain are alipid concentration from 0.1 to 1 mg/mL, 4-8% of anchoring molecules in the vesicles, 1-4 h of contacttime between vesicles and surface, and 21-24 h of resting time after buffer rinse. Under these conditions, amembrane having a lipid mass per surface between 545 ± 5 and 590 ± 10 ng/cm2, a diffusion coefficient ofbetween 2.5 ± 0.3 × 10-8 and 3.60 ± 0.5 × 10-8 cm2/s, and a mobile fraction between 94 ± 2 and 99 ± 1%was formed. These findings were confirmed by atomic force microscopy observations, which showed thepresence of a continuous and homogeneous bilayer in the determined experimental domain. This formationprocedure presents many advantages; it provides an easily obtainable biomimetic membrane model for proteinsstudies and offers a versatile tethered bilayer because it can be adapted easily to various types of supports.