Coating processes – Applying superposed diverse coating or coating a coated base – Synthetic resin coating
Reexamination Certificate
2000-08-28
2003-04-01
Cameron, Erma (Department: 1762)
Coating processes
Applying superposed diverse coating or coating a coated base
Synthetic resin coating
C427S409000, C427S412100
Reexamination Certificate
active
06541071
ABSTRACT:
BACKGROUND OF THE INVENTION
As a large fraction of drug targets are membrane bound proteins (e.g, G-protein coupled receptors, ion-channels, etc.), there is a need for the development of surfaces that bind lipids incorporating membrane bound targets. For example, bilayer-lipid membranes adsorbed onto solid supports, referred to as supported bilayer-lipid membranes, can mimic the structural and functional role of biological membranes. See Bieri, C. et al.,
Nature Biotech
, 1999, 17, 1105-1108; Groves, J. T. et al.,
Science
1997, 275, 651-653; Lang, H. et al.,
Langmuir
1994, 10, 197-2 10; Plant, A. L. et al.,
Langmuir
1999, 15, 5128-5135; and
Raguse, B. et al.,
Langmuir
1998, 14, 648-659. These hybrid surfaces were developed to overcome the fragility of black lipid membranes while preserving aspects of lateral fluidity observed in native biological membranes. The properties of supported membranes are determined by the. nature of the adsorbing surface. Self-assembled monolayers may be used to coat a substrate and are capable of further derivatization to attach membranes.
Surfaces binding lipid membranes can be broadly classified into three categories: (i) hydrophobic surfaces (e.g., self-assembled monolayers presenting terminal methyl groups) which support the adsorption of lipid monolayers are of limited utility as they cannot be used to incorporate membrane-spanning proteins (Plant, A. L.,
Langmuir
1999, 15, 5128-5135); (ii) hydrophilic surfaces (e.g., glass surfaces) which bind bilayer-lipid membranes are also of limited utility as they can only be used to incorporate membrane-spanning proteins with extra-membrane domains that are less thicker than the layer of adsorbed water (~10° A) (Groves, J. T. et al.,
Science
1997, 275, 651-653; and Groves, J. T. et al.,
Langmuir
1998, 14, 3347-3350); and (iii) hybrid surfaces presenting amphiphilic anchor lipids that bind bilayer-lipid membranes offer the potential for incorporating a wide variety of membrane-spanning proteins (Lang, H. et al.,
Langmuir
1994, 10, 197-210; Raguse, B. et al.,
Langmuir
1998, 14, 648-659; and Vanderah, D. J. et al.,
Materials Research Society Fall Meeting Abstracts
, Boston, 1999).
Self-assembled monolayers (“SAMs”) of alkanethiolates on gold are well suited for studying biomolecular recognition at surfaces because the well-defined structures are amenable to detailed characterization at a molecular level (e.g., Scanning Tunneling Microscopy “STM,” Atomic Force Microscopy “AFM,” etc.). See Widrig, C. A. et al.,
J. Am. Chem. Soc.
1991, 113, 2805-2810; and Alves, C. A. et al.,
J. Am. Chem. Soc.
1992, 114, 1222-1227. They may also be addressed by a variety of bioanalytical techniques (e.g., optical, electrochemical, etc.). See Lahiri, J. et al.,
Anal. Chem.
1999, 71, 777-790; Plant, A. L.,
Langmuir
1998, 14, 3347-3350; Rueda, M. et al.,
Langmuir
1999, 15, 3672-3678; and Steinem, C. et al.,
Bioelectrochem. and Bioenerg.
1997, 42, 213-220.
The importance of a hydrophilic spacer between the substrate and the adsorbed lipid has been studied. The use of thiolated anchor lipids consisting of dipalmitoylphosphatidic acid extended at the hydrocarbon end by a hydrophilic ethyleneoxy group linked at the other end to a terminal disulfide has been shown (Lang, H. et al.,
Langmuir
1994, 10, 197-210; Plant et al.,
Materials Research Society Fall Meeting Abstracts
, Boston, 1999; and Raguse et al.,
Langmuir
1998, 14, 648-659). Similar anchor lipids containing thiaoligoethyleneoxide (HS(CH
2
CH
2
O)
n
—) moieties have also been used. However, these approaches have two disadvantages: first, they require the laborious synthesis of the oligo(ethylene oxide) containing thiols, and second, the structures of the SAMs formed from these thiols may not be well-defined. Biotinylated anchor lipids have been used to immobilize streptavidin on self-assembled monolayers presenting biotin groups (Bieri, C. et al.,
Nature Biotech.
1999, 17, 1105-1108). Although this strategy circumvents issues regarding the structure and stability of putative self-assembled monolayers containing thiaoligoethyleneoxide moieties, the approach itself is cumbersome and requires the synthesis of biotinylated thiols. A simpler method for fabricating a supported membrane is desired.
Methods to create arrays of membranes would enable high-throughput screening of multiple targets against multiple drug-candidates. Arrays of membranes may be obtained by fabricating grids of titanium oxide on a glass substrate as titanium oxide resists the adsorption of lipids (Boxer, S. G. et al.
Science
1997, 275, 651-653; and Boxer, S. G. et al.
Langmuir
1998, 14, 3347-3350). Micropipeting techniques have been used to spatially address each corralled lipid-binding region (Cremer, P. S. et al.,
J. Am. Chem. Soc.
1999, 121, 8130-8131). However, these methods are cumbersome and require the fabrication of patterned surfaces. To make membrane arrays by printing membranes on unpatterned surfaces, it would be necessary to confine the membrane to the printed areas without lateral diffusion of the membrane molecules to the unprinted areas. Boxer et al. demonstrated that it was possible to pattern lipids on glass surfaces by microcontact printing using poly-dimethylsiloxane (PDMS) stamps “inked” with phosphatidylcholine (“PC”). They attributed the lateral confinement of the lipids to the stamped regions, to the self-limiting expansion of PC membranes to ~106% of the original printed areas (Hovis, J. et al.,
Langmuir
2000, 16, 894-897).
SUMMARY OF THE INVENTION
The present invention relates to a method and composition for producing a supported membrane. In one embodiment, the present invention relates to a method for producing a supported membrane comprising the steps: (i) providing a substrate coated with a monolayer having reactive functional groups; (ii) contacting the reactive groups with a linker compound to form a derivatized monolayer having covalently bonded linker moieties; and (iii) contacting the derivatized monolayer with a membrane solution to produce a supported membrane.
In another embodiment, the present invention relates to a method for producing a supported membrane comprising the steps: (i) providing a substrate coated with a monolayer having reactive functional groups; (ii) activating the reactive functional groups on the monolayer coated substrate to form an activated monolayer; (iii) contacting the activated monolayer with a linker compound to form a derivatized monolayer having covalently bonded linker moieties; and (iv) contacting the derivatized monolayer with a membrane solution to produce a supported membrane.
In yet another embodiment, the present invention relates to a method of producing a supported membrane comprising the steps: (i) providing a gold substrate coated with a monolayer having carboxylic acid groups; (ii) activating the carboxylic acid groups to form an activated monolayer having anhydride groups; (iii) contacting the activated monolayer with a linker compound to form a derivatized monolayer having covalently bonded linker moieties; and (iv) contacting a membrane solution with the derivatized monolayer to produce a supported membrane.
In a further embodiment, the present invention relates to a supported membrane produced by any of the methods of the present invention.
The method of the present invention offers several advantages over the previous approaches: (i) the method requires minimal organic synthesis as most of the reagents are commercially available; (ii) the method uses a reactive intermediate onto which any potential linker moiety with a terminal nucleophilic functional group can be attached; the use of a common reactive intermediate obviates the need for the individual synthesis of thiolated linker moieties; (iii) the method separates the covalent attachment of the linker moiety from the initial formation of a well-defined and stable monolayer; and (iv) the method allows control of the surface density of the adsorbed membrane, where the adsorbed membranes retain natural form and function.
REF
Bookbinder Dana C.
Lahiri Joydeep
Beall Thomas R.
Cameron Erma
Nixon & Peabody LLP
Turner Patricia W.
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