Two-component membrane lithography via lipid backfilling

Herein, we describe the development of a novel lithographic technique to pattern artificial lipid bilayer microdomains by exploiting the limited mobility of gel-phase phospholipids in two-component mixtures. Reconstituted supported phospholipid bilayers are formed by the self-assembly of lipids into two opposing leaflets on a hydrophilic surface. They are useful model systems for studying the physics and chemistry of biological membranes because of similarities with natural analogues, as well as accessibility to fluorescence microscopy and atomic force microscopy. Spatially addressable supported lipid bilayers hold great promise for the development of integrated biological solid-state devices, such as rapid screening assays for proteins or other biomolecules that associate with membranes, enabling new high-throughput studies for many important cellular functions. They are also highly ordered structures that effectively resist nonspecific binding of many molecules. Patterning of supported lipid bilayer microarrays has been demonstrated with various lateral diffusion barriers, such as metals or metal oxides, proteins, photoresist, mechanical scratches, and voids following either resist lift-off or photo-oxidative degradation of lipids. Patterning has also been demonstrated by direct deposition or removal of lipid material with polymeric stamps. Recently, metastable membrane microdomains with lipid mixtures that resemble the composition of putative natural lipid rafts were patterned photolithographically into supported bilayers. Our approach is to use kinetically “trapped” separation of components in a binary mixture to form stable lipid patterns. Previous investigators have shown that microdomains can spontaneously form in supported bilayer membranes, which contain two or more lipid components. Lipid phases segregate due to different head groups, chain lengths, and/or varying degrees of saturation of their hydrocarbon chains. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), which has a longer chain length and a higher transition temperature (Tm 41 8C), was used to form ordered gel-like regions, while 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) (Tm 1 8C) was used to form liquid crystalline regions. Differential scanning calorimetry of multi-bilayers, as well as confocal microscopy and fluorescence correlation spectroscopy of giant unilamellar vesicles, have shown that DLPC and DPPC lipids have limited miscibility and can mix over time to form a two-phase region of coexisting ordered (rich in DPPC) and fluid (rich in DLPC) phases at equilibrium. However, the lateral mobility of lipid molecules in the ordered phase is 250 times below that of the fluid phase. Lipid mixing in patterned areas in this case will be an activated process with a large energy barrier, which suggests that, at room temperature, segregated regions of pure DLPC and pure DPPC will remain kinetically stable. Poly(dimethylsiloxane) (PDMS) stamps were used to create both positive and negative pattern transfers of bilayer microdomains, as depicted in Figure 1. Fluid microdomains could be patterned into a solid gel-like matrix (Figure 1A). Alternatively, gel-like lipids could be patterned in a sea of fluid lipids (Figure 1B). For the first scenario shown in Figure 1A, a PDMS stamp with square features in bas relief was held in contact with a clean planar glass coverslip in aqueous solution. This served as a mold around which the gel-phase DPPC bilayer was cast, using the vesicle fusion method. A solution containing small unilamellar vesicles (SUVs) of DPPC at 45 8C, which is above the transition temperature for this lipid, formed a fluid bilayer around the PDMS posts on the coverslip. Upon cooling to room temperature, the DPPC bilayer solidified into a gel. After rinsing away any residual debris and removal of the PDMS, a solution containing DLPC vesicles was introduced, which resulted in the formation of fluid bilayers in the areas previously protected by the PDMS posts. Generally, the fluid-to-gel phase transition in supported phospholipid bilayers results in the formation of submicron [a] Dr. S.-Y. Jung, Prof. C. P. Collier Division of Chemistry and Chemical Engineering California Institute of Technology, M/C 127–72 Pasadena, CA 91125–7200 (USA) Fax: (+1)626-568-8824 E-mail : collier@caltech.edu [b] Dr. M. A. Holden University of Oxford, Chemistry Research Laboratory Mansfield Road, Oxford, OX1 3TA (UK) [c] Prof. P. S. Cremer Department of Chemistry, Texas A&M University, P.O. Box 30012 College Station, Texas 77843–3012 (USA) Supporting information for this article is available on the WWW under http://www.chemphyschem.org or from the author.