Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Patent
1997-03-25
1999-07-13
Tsang, Cecilia J.
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
436 13, 422 57, 264 41, 424420, 424450, 427 214, C12M 134
Patent
active
059225947
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to the preparation of lipid membranes, and more particularly to the preparation of lipid membrane supporting surfaces suitable for use in biosensors.
BACKGROUND OF THE INVENTION
There is currently a general demand for sensors based on the integration of lipid membrane components, such as membrane bound receptor proteins, into planar bilayer lipid membranes, so-called BLM's. Such lipid bilayers may form spontaneously, and are self-assembling under suitable conditions and with suitable surfaces. The BLM's formed may then be used for studying ligand-receptor interactions at the lipid-water interface.
Brian and McConnell, Proc. Natl. Acad. Sci. USA (1984) 81, 6159-6163 describes the spontaneous fusion of phospholipid vesicles to hydrophilic glass surfaces for studies with fluorescence techniques.
Poglitsch and Thompson (1990) Biochemistry 29, 248-254 describes the spontaneous fusion of phospholipid vesicles to hydrophilic glass surfaces by passing the vesicle solution through an assembly of a fused silica substrate and a microscope slide mounted together with a spacer of about 100 .mu.m thickness.
Zot et al. (1992) J. Cell Biol. 116, 367-376 discloses the preparation of planar lipid surfaces in a flow cell and the study of actin filament gliding on the lipid layer by fluorescence microscopy.
Terrettaz et al. (1993) Langmuir 9, 1361-1369 describes the formation of lipid monolayers by the adsorption of alkanethiols with hydrophobic terminal groups in a discontinuous dilution procedure. Interactions with membrane components were studied by surface plasmon resonance and impedance measurements.
Gitler et al., Bridging Research and Applications, 43-61, Eds. D. Kamely et al., 1991 Kluwer Ac Publ., and Vogel et al. (1994) 10, 197-210 disclose approaches to provide for a water layer between the support and the BLM which is desirable in order to obtain conditions suitable for transmembrane proteins. To this end, lipids are modified with an oligoethylene spacer and a thio group (thiol or disulphide). These thiolipids are then anchored to a gold surface together with an unmodified lipid, thereby spontaneously forming a BLM anchored via the thio groups to the metal surface. The oligoethylene spacer was introduced in order to create the desired water layer spacing.
Stelzle, M., et al. (1993) J. Phys. Chem. 97, 2974-2981 discloses the preparation of a bilayer lipid membrane on a biosensor device by first depositing a negatively charged monolayer of a carboxy mercaptan onto gold and then adding vesicles of positively charged dioctadecyldimethylammonium bromide which fuse spontaneously to the negative layer, or alternatively, fusing negatively charged dimyristoylphosphatidylglycerol to the negative layer by the of addition of calcium ions.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a sensorgram showing the response vs. time for three consecutive contactings of liposome solution with a hydrophilic sensor surface to form a lipid bilayer thereon containing ganglioside G.sub.M1.
FIG. 2 is a corresponding sensorgram to that in FIG. 1 and shows the response when sequentially contacting the lipid surface with negative control (BSA), cholera toxin, and hydrochloric acid.
FIG. 3 is a corresponding sensorgram to those in FIGS. 1 and 2 and shows the response for the formation of a highly hydrophobic lipid layer and the subsequent contacting of the lipid layer with cholera toxin.
DETAILED DESCRIPTION OF THE INVENTION
The object of the present invention is to provide improvements in the preparation and use of planar BLM's, and more particularly to improve the stability of the formed BLM's. According to the present invention, this and other objects and advantages are achieved by covalently binding the BLM to a self-assembled monolayer on a substrate surface.
The present invention therefore provides a method of producing a substrate surface supporting a continuous planar bilayer lipid membrane by fusing a micellar or vesicle preparation, preferably containing a membrane protein or othe
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Biacore AB
Lukton David
Tsang Cecilia J.
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