EP1407268A2 - Doubles couches lipidiques asymetriques immobilisees - Google Patents

Doubles couches lipidiques asymetriques immobilisees

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Publication number
EP1407268A2
EP1407268A2 EP02758194A EP02758194A EP1407268A2 EP 1407268 A2 EP1407268 A2 EP 1407268A2 EP 02758194 A EP02758194 A EP 02758194A EP 02758194 A EP02758194 A EP 02758194A EP 1407268 A2 EP1407268 A2 EP 1407268A2
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EP
European Patent Office
Prior art keywords
lipid
immobilized
solid
proteins
modified
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP02758194A
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German (de)
English (en)
Inventor
Johannes Schmitt
Joachim NÖLLER
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Nimbus Biotechnologie GmbH
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Nimbus Biotechnologie GmbH
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Publication date
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Publication of EP1407268A2 publication Critical patent/EP1407268A2/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/553—Metal or metal coated
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
    • G01N33/552—Glass or silica
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00—Magnetic particle immunoreagent carriers
    • G01N2446/80—Magnetic particle immunoreagent carriers characterised by the agent used to coat the magnetic particles, e.g. lipids

Definitions

  • the invention relates to a method for producing immobilized lipid bilayers, the lipid bilayers having a defined orientation with regard to the lipid layers on the surface of a solid.
  • the invention further relates to the use of modified solid surfaces for the production of such lipid bilayers and to a kit for the production of immobilized lipid bilayers.
  • membranes are made up of two layers of lipid molecules that form a lipid bilayer.
  • This lipid bilayer is hydrophobic on the inside and hydrophilic on both outer sides.
  • the structural and functional asymmetry of this lipid bilayer is decisive for the different functions of a membrane.
  • this consists in a different distribution of different lipid molecules in the two lipid layers (“inner leaflet” and “outer leaflet”), which form the lipid bilayer.
  • Other components are various membrane proteins that are located in the biomembrane. These can be transmembrane membrane proteins that span the entire lipid bilayer.
  • membrane proteins are embedded in only part of the lipid bilayer, for example in the outer lipid layer.
  • membrane proteins can also be attached to one or both sides of the membrane.
  • membrane proteins can move laterally in the lipid matrix.
  • the proteins can also be more or less fixed at a certain point through specific interactions. In general, however, it is not possible for membrane proteins to migrate from one lipid layer to the other, so that the asymmetry formed by the various lipids and membrane components of the membrane is a long-term structural and thus of course also functional property of the membrane.
  • glycocalix which performs certain recognition and identification functions, is formed by glycoproteins on the outer side of the lipid bilayer and glycolipids in the outer lipid bilayer.
  • phosphate idylserine lipids are typical of the inner lipid layer of an animal cell. During the programmed cell death (apoptosis), these specific lipids are also built into the outer lipid layer and serve there as a recognition pattern for further processes.
  • asymmetry of membranes are the transport proteins, which allow directed transport from outside to inside or from inside to outside.
  • transport proteins which allow directed transport from outside to inside or from inside to outside.
  • the asymmetry of a biomembrane is the crucial prerequisite for a membrane to be able to perform its complex tasks.
  • biomembranes are of great interest, for example, in the field of pharmaceutical screening and biosensor technology.
  • the inner side of the original membrane can also be directed outwards when the vesicles are formed, whereby one then calls "in-side-out” ISO) vesicles speaks.
  • ISO in-side-out
  • the vesicles resulting from such workup are a mixture of RSO and ISO vesicles.
  • Membrane protein properties are only suitable to a limited extent, since the size of the vesicles and morphology can hardly be controlled and the system therefore has no defined and reproducible properties. Furthermore, such vesicular structures are not accessible to automation because of their low stability and poor manageability. A high-throughput screening, as is necessary in the field of functional tests and pharmaceutical screening, is therefore not possible with vesicles.
  • lipid bilayers are fused on a solid surface, whereby a lipid bilayer is spontaneously formed on the surface (Tamm, LK; McConnell, HM Biophysical Journal 47, 105-113 (1985); Salafsky, J .; Groves, JT ; Boxer, S. Biochemistry 35, 14773-14781 (1996)).
  • Solid surface with transmembrane proteins could Orientation of the lipid bilayer can be achieved in which the originally inner side of the lipid bilayer in the immobilized form points outwards (Kalish, DI; Cohen, CM; Jacobson, BS; Branton, D. Biochim. Biophys. Acta 1978 (506), 97- 1 10). Although the asymmetry of the membrane could be preserved here, it was only possible to orient the inner side of the membrane outwards. Orientation of the membrane with the originally outer side to the outside was not possible. In addition, the enzymatic activities decreased due to the electrostatic interactions of the membrane with the solid surface, so that this system is not suitable for functional studies of the membrane.
  • the object of the invention is therefore to provide a lipid bilayer system which has a defined one
  • Orientation of the lipid layers on a surface of a solid body ensured.
  • This system should be supported by solid bodies, so that it is stable and easy to handle and is also suitable for automation.
  • the orientation, ie the asymmetry of the lipid bilayer, should be freely selectable, so that either the originally outer side of the lipid bilayer or the membrane points outwards or that this side faces the surface of the solid.
  • the functional and structural properties of the lipid bilayer should also be retained in the immobilized form in order to be able to investigate the natural properties of the lipid bilayer or the proteins contained therein or the like in vitro.
  • the method according to the invention serves to produce a lipid bilayer which is immobilized on a solid.
  • the lipid bilayer covers the solid as an essentially continuous membrane film.
  • the lipid bilayer consists of an inner and outer lipid layer (“inner leaflet” or “outer leaflet”), these two lipid layers being oriented in a certain way, so that the immobilized lipid bilayer has a defined orientation or asymmetry with respect to the solid surface.
  • the lipid bilayer is essentially stable and dynamically immobilized on the surface of the solid, so that the structural and functional properties of a natural lipid bilayer, in particular a membrane, are reflected.
  • the surface of the solid is first modified in such a way that a surface is formed which can preferably interact with one of the two lipid layers, that is to say with the inner or the outer lipid layer.
  • the lipid bilayer is deposited on this modified solid surface.
  • the modification of the solid surface according to the invention enables the lipid bilayer to align itself on the solid surface in such a way that a certain orientation of the lipid bilayer is achieved. According to the invention, this is mainly due to electrostatic interactions between the modified solid surface and the two layers of the Lipid bilayer, whereby the interactions can be both attractive and repulsive in nature.
  • the electrostatic interactions in particular in combination with the “ultra-soft” surface explained in more detail below, lead to the desired orientation of the lipid bilayer on the solid. Further details can be found in the description below and in particular in the examples.
  • the asymmetry of the lipid bilayer can be caused on the one hand by different lipids or lipid-analogous molecules in the two lipid layers of the lipid bilayer. Furthermore, the asymmetry can also be based on further components in the lipid layers, which are distributed differently in the two lipid layers. These further components are advantageously proteins, in particular membrane proteins. These can be so-called transmembrane membrane proteins that span the entire lipid bilayer in a certain orientation. Examples of this are various transport proteins that allow directed transport through a membrane or a lipid bilayer. An example of such transmembrane proteins are G-protein coupled receptors (GPCR), which are very interesting targets for potential drugs. The binding sites of these receptors are on the outside of membranes. The method according to the invention is therefore particularly suitable for investigating the interaction of these receptors with potential active substances, since the orientation of the lipid bilayers containing such receptors can be controlled in such a way that these binding sites in the immobilized membrane system are accessible from the outside.
  • GPCR G-protein coupled
  • a solid-supported lipid bilayer can be produced with the aid of the method according to the invention has a certain orientation, this can of course also be used to control the orientation of the components integrated in the lipid bilayer, for example the transport proteins.
  • the method according to the invention therefore provides a membrane model system which is outstandingly suitable for the investigation and characterization of the membrane properties and / or the properties of individual membrane components.
  • the lipid double layer can also have so-called peripheral components, in particular proteins, which are only in one of the two lipid layers or on one of the two surfaces of the lipid double layer.
  • peripheral proteins are G-protein coupled receptors (GPR), which are also very interesting targets for potential drugs. They are located on the inside of the cell membrane and are therefore only accessible after inversion of the original orientation.
  • the lipid structures to be immobilized are first converted into the form of vesicles which, by fusion with the modified solid surface, form the lipid bilayer according to the invention supported by the solid.
  • the vesicles are advantageously provided in an aqueous dispersion.
  • vesicles made of natural material represents a model of the natural membrane in which either the originally inner or the originally outer membrane surface ultimately points outwards.
  • Membrane vesicles for example, which are obtained from erythrocytes or culturable cells, for example CACO-2 cells, are particularly suitable for this.
  • the lipid bilayer can be at least partially constructed from membrane fragments of natural cells.
  • the vesicles are prepared using conventional methods. According to the invention, the vesicles are brought into contact with the modified solid surface, as a result of which the vesicles fuse spontaneously with one another and form the lipid bilayer or the membrane layer. By choosing an appropriately modified surface, the final orientation of the lipid bilayer or membrane layer can be determined in advance.
  • intracellular membranes that can originate, for example, from liver or kidney cells are of course also suitable as natural membranes.
  • cell organelles can be used for the preparation of intracellular membranes.
  • artificial lipid layers or membranes are also suitable for the process according to the invention.
  • Such artificial structures can be assembled (reconstituted) from various components in vitro, so that this provides a corresponding membrane vesicle which can be used in the method according to the invention for producing an immobilized lipid bilayer.
  • Artificial lipid layers as well as natural lipid layers can be made from various lipids, lipid derivatives, lipid-like and / or lipid-analogous Be composed of substances.
  • the lipid layers can have peptides, proteins, nucleic acids, ionic or nonionic surfactants and / or polymers.
  • These additional components of the lipid layers can advantageously be enzymes, receptors and / or ligands which more or less span the lipid layer or the lipid bilayer, are embedded therein and / or are superficially attached to the lipid bilayer.
  • the surface of the solid is first modified in such a way that the orientation of the subsequent formation of a lipid bilayer as a result of the fusion of vesicles is controlled.
  • the modification of the solid surface according to the invention offers optimal conditions for the gentle immobilization of a lipid bilayer or a membrane. These optimal conditions relate to a combination of different intermolecular interactions, the superposition of which results in an attractive force between the lipid bilayer to be applied and the solid. This simultaneously prevents direct contact of the lipid bilayer with the solid. In this way, the characteristic dynamic movement properties of the lipid bilayer, in particular the lateral diffusion of the various lipid components, are largely left unchanged.
  • the modification forms an “ultra-soft” surface which, on the one hand, largely leaves the immobilized lipid bilayer unaffected and, on the other hand, ensures a sufficient distance between the solid surface and the lipid bilayer.
  • Modification of the solid surface according to the invention has the advantage that interactions of components of the lipid bilayer, in particular proteins, with the solid surface are largely minimized or eliminated, so that the membrane proteins, for example, retain their activity, for example their enzyme activity, even in the new environment.
  • the immobilization according to the invention also maintains the lipid properties of the lipid bilayer, as a result of which the natural properties of a membrane are perfectly imitated. Overall, the surface modification thus inter alia decouples the lipid bilayer from the solid surface.
  • This is a very decisive advantage, for example, for an application in the field of biosensor technology, since here immobilized lipid bilayers, in particular membranes, which retain their native properties, for example enzymatic activities and / or channel activities, are required.
  • This advantage of the invention can be used particularly advantageously, for example, in an application with biochips.
  • the final orientation of the lipid layers immobilized thereon is determined by the choice of a specific modification of the solid surface.
  • membrane vesicles of random orientation that is to say so-called ISO and RSO vesicles, can advantageously be produced and brought into contact with the modified solid. Due to the surface properties of the modified solid, essentially only the fusion of the vesicles of a certain orientation takes place. This results in a solid-supported lipid bilayer which has a defined orientation is obtained by the method according to the invention.
  • the modified solid is offered a vesicle preparation with only one orientation. In this way, greater yields of immobilized lipid bilayer of a certain orientation can be obtained if necessary.
  • lipid layers immobilized by the process according to the invention can be checked, which are known to be on the originally inner side, that is to say in particular on the cytoplasmic side, or on the outer side of the membrane or the lipid bilayer.
  • the N-acetylsialic acid released by neuramidase can be examined.
  • the glycocalyx on the outside of the original membrane layer can also be marked using fluorescence-labeled lectins. Details of these analysis methods can be found in the examples.
  • the solid surface is modified according to the invention, an essentially hydrophilic surface is advantageously achieved.
  • the surface is first functionalized. For example, amino functions, epoxy functions,
  • Haloalkyl functions and / or thio functions are applied to the surface.
  • Silanes can advantageously be used for the functionalization.
  • Mercaptans and / or disulfides, in particular alkyl disulfides are also preferred.
  • N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (EDA), polyethyleneimine (PEI) and / or cysteamine, in particular cysteamine hydrochloride are very particularly preferred for the functionalization.
  • interacting molecules with these functions are adsorbed and / or chemisorbed.
  • a previous functionalization of the surface can also be omitted if an interaction of other molecules is readily possible due to the material properties of the solid.
  • polystyrene sulfonate PSS
  • PSPMA poly (styrene-co-maleic anhydride)
  • a particularly preferred substance that is chemisorbed is polyoxyethylene bis (glycidyl ether).
  • further interacting molecules are used to modify the surface, which interact with the initially applied interacting molecules.
  • functional substances are used as interacting molecules which can be used for an analysis of the properties of the lipid bilayer or of the components contained in the lipid bilayer.
  • These are advantageously dyes, in particular fluorescent dyes, and / or enzymatically, chemically and / or photochemically reactive molecules.
  • the modification of the surface is chosen such that the modified surface essentially only interacts with one of the two lipid layers of the lipid bilayer, the interaction being essentially electrostatic in nature, which can be both repulsive and attractive. This ensures that the lipid bilayer aligns itself in a certain orientation during the fusion of the vesicles as the lipid bilayer is deposited on the solid.
  • the modified surface preferably interacts with the originally inner lipid layer (inner leaflet) of the lipid bilayer.
  • the modified surface preferably has a negative net charge.
  • the modification of the solid surface is advantageously carried out by producing “ultra-soft” polymer surfaces with a negative net charge.
  • oriented vesicle systems For depositing the lipid double layer on this modified solid surface, for example, oriented vesicle systems are used that carry a negative net charge on the outside provided essentially hydrophilic surface which reacts so repulsively with the outer lipid layer that the lipid bilayer aligns itself in a fusion of a vesicle preparation essentially so that the originally outer side of the lipid bilayer points outwards, i.e. faces away from the solid, and so is accessible for various examinations.
  • the solid surface is modified such that it preferably interacts with the originally outer lipid layer of the lipid bilayer to be immobilized.
  • the modified surface preferably has a positive net charge.
  • the modification of the surface is preferably carried out by producing soft polymer surfaces with a positive net charge.
  • Oriented vesicle systems which carry a negative net charge on the outside are advantageously used for depositing the lipid bilayers on this modified solid surface.
  • the vesicles align so that the originally outer lipid layer faces the solid.
  • the components that were originally in or on the inner side of the lipid bilayer, for example on the cytoplasmic side are accessible for examination.
  • the solid surfaces modified in this way are preferably brought into contact with the vesicular structures of the lipid bilayer to be immobilized within a medium, preferably an aqueous medium, as a result of which the vesicles spontaneously fuse with one another and with the modified surface and thus form an immobilized lipid bilayer due to the properties of the modified surface , which has a defined asymmetry.
  • the method according to the invention is an extremely easy-to-use system, which requires only little preparative effort.
  • an essentially planar carrier is used as the solid.
  • This can consist of different materials.
  • the surface can advantageously also be made of a different material than the rest of the carrier.
  • the solid surface preferably consists of silicate, a semiconductor, in particular silicon, a noble metal, for example gold, and / or a polymer, for example polystyrene. Other surfaces are of course also possible.
  • films made of powdery metals, semiconductors, noble metals and / or polymers can be used as surface materials, which are applied to largely planar carrier materials or can be applied thereon.
  • These carrier materials can be, for example, documents made of paper, glass, plastic or the like, with which the solid bodies are connected in a suitable manner, in particular by gluing or fusing.
  • dispersible solids which advantageously consist of particles of, for example, microscopic dimensions.
  • Preferred materials for such solids are aluminates, borates and / or zeolites. Colloidal solutions of precious metals, metals etc. are also suitable.
  • silicate particles is very particularly preferred. These particles can be conventional silicate balls, for example.
  • porous particles are used, which increases the surface area available for the immobilization.
  • the particles used are magnetic particles, as a result of which the handling in various applications can be made considerably easier. This plays in particular when using the lipid bilayers immobilized according to the invention
  • the solids used are preferably used in the form of a dispersion.
  • This dispersion is advantageously produced in aqueous solutions.
  • the invention comprises the use of a modified surface of a solid to produce an immobilized lipid bilayer with a defined orientation with regard to the lipid layers on the surface of the solid.
  • the modified surface is suitable for essentially stable and dynamic immobilization of the lipid bilayer. With regard to the further properties of the use of the modified surface, reference is made to the above description.
  • the invention further comprises an immobilized lipid bilayer which can be produced by a method as described above.
  • This immobilized lipid bilayer has a defined orientation and is essentially stable and dynamically immobilized on a solid.
  • the invention further comprises immobilized proteins which can be produced by a method described above.
  • proteins that are associated with a lipid bilayer, which in turn is immobilized on a solid.
  • the proteins are in particular membrane proteins that span the lipid bilayer in whole or in part, that span only one of the lipid layers forming the lipid bilayer or are embedded here, or that are embedded or attached to one or both sides of the lipid bilayer.
  • the invention also includes a kit for producing immobilized lipid bilayers.
  • This kit is intended to immobilize lipid bilayers, so that these lipid bilayers with their inner and outer lipid layers have a defined orientation on the surface of a solid. The orientation can be determined in advance.
  • the immobilized in this way Lipid bilayers are essentially stable and dynamically fixed on the solid, so that they are particularly suitable for functional and structural investigations of the lipid bilayer, for example a membrane.
  • This kit comprises at least one solid with a modified surface which is suitable for the immobilization of the lipid bilayer in the sense of the invention. With regard to the properties of this modified solid surface, reference is made to the above description.
  • the kit does not contain the finished modified surface, but rather the various components that are necessary for producing such a modified surface. This can be advantageous for storage and / or stability reasons, for example.
  • lipid vesicles are also present in this kit, the lipid vesicles preferably having additional components, in particular proteins. These lipid vesicles are intended for fusion on the modified solid surface and thus form the material for the immobilized lipid bilayer. In this case, it may be preferred that further components for the lipid bilayer are provided by the user himself and introduced into the lipid vesicles.
  • the lipid vesicles are advantageously in the form of a dispersion.
  • lipids can also be provided in a form other than vesicles, for example in a dry form.
  • the invention further comprises a kit for the production of immobilized proteins which are immobilized in or on their part
  • Lipid bilayers with a defined orientation comprises at least one solid with a modified surface and preferably lipids, in particular in the form of vesicles, which have the proteins to be immobilized.
  • These proteins are preferably membrane proteins which find their natural environment through immobilization in lipid bilayers and can thus be analyzed under almost native conditions.
  • the invention comprises a method for the investigation of proteins, in particular membrane proteins, these proteins being used as immobilized proteins in their immobilized lipid bilayers.
  • proteins in particular membrane proteins, these proteins being used as immobilized proteins in their immobilized lipid bilayers.
  • immobilized proteins used in lipid bilayers reference is also made to the above description.
  • the further implementation of the method is carried out using customary methods known to a person skilled in the art.
  • Plasma membranes in different orientations on the Solid surface based on the hydrolysis activity of the P-glycoprotein in the presence of a modulator (Verapamil),
  • a freshly prepared silane solution consisting of 9.2 mL N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (EDA) and 243 ⁇ L concentrated acetic acid in 450 mL deionized water.
  • EDA N- (2-aminoethyl) -3-aminopropyltrimethoxysilane
  • 2 g of a porous silicate material (Nucleosil 50-10 from Macherey-Nagel, Duren) was added to the silane solution and slurried with shaking. This dispersion was slowly rotated for three hours, after which the silicate material is sedimented and three times washed with deionized water.
  • the success of the silanization is documented by means of infrared spectroscopy in diffuse reflection (DRIFT) on the dried silicate material.
  • DRIFT diffuse reflection
  • a porous silicate material (Nucleosil 4000-10) from Macherey-Nagel, Duren) were added to a polyethyleneimine (PEI) solution consisting of 250 mg PEI (50% solution in water, Aldrich, Steinheim) in 50 ml deionized water and rotated slowly for three hours. The silicate material was then sedimented and washed three times with deionized water. The success of the implementation was documented using infrared spectroscopy in diffuse reflection (DRIFT) on the dried silicate material. In a similar way, further carrier materials with pore sizes between 5 and 400 nm were functionalized.
  • PEI polyethyleneimine
  • PSS polystyrene sulfonate
  • Oriented RSO and ISO plasma membrane vesicles were produced by a method by Steck and Kant (Methods in Enzym. 1974, 31, 172-180). This dispersion was then converted into small, single-shell vesicles with a diameter of 20-90 nm by extrusion. 50 mg of a porous silicate carrier (Mache-Rey-Nagel, Düren) were added to each 900 ⁇ L of these solutions (approx. 0.5 mg total protein) and incubated for 18 hours at 4 ° C., 100 mM triethanolamine (pH 7, 4) and 100 mM NaCl (incubation buffer) was used. After that it was The carrier material sedimented and washed three times with incubation buffer.
  • a porous silicate carrier Mache-Rey-Nagel, Düren
  • the success of the coating was documented using DRIFT on the dried material.
  • the activity of the acetylcholinesterase occurring in the outer layer was determined by a method by Ellman et. al. (Biochem. Pharmacol. 1961, 7, 88) on the support material after washing in the incubation buffer. It is important to make holes in the lipid bilayer in a control by means of detergent and thus to measure enzyme molecules possibly facing away from the surface (FIG. 1). These functional tests demonstrated an inversion in the fusion with the solid surface offered, as well as the uniformity (> 90%) of the surface obtained.
  • ISO-oriented plasma membrane vesicles from CACO-2 cells were produced by a method by Schlemmer and Sirotnak (Anal. Biochem. 1995, 228, 226-231). This dispersion was then converted into small, single-shell vesicles with a diameter of 20-90 nm by extrusion.
  • 900 ⁇ L of this solution about 0.5 mg of total protein
  • Modified porous silicate carrier added and incubated for 18 hours at 4 ° C, using 100 mM triethanolamine (pH 7.4) and 100 mM NaCl (incubation buffer) as the buffer solution. The carrier materials were then sedimented and washed three times with incubation buffer.
  • the success of the coating was documented using DRIFT on the dried material.
  • the activity of the P-glycoprotein on the carrier materials was determined after washing in the incubation buffer by determining the ATP hydrolysis activity as a function of known modulator (Verapamil).
  • Verapamil known modulator
  • an RSO-oriented surface is obtained after immobilization, which, however, has no appreciable enzyme activity due to the incorrect orientation of the protein to be examined. Not so with the help of the carrier from example 1.2.1. manufactured ISO surface. It has a hydrolysis activity (FIG. 2) on the carrier material that is comparable to that of the isolated vesicles.
  • RSO-oriented plasma membrane vesicles from HEK293 cells were produced by a method by DePierre and Karnovsky (Journal of Cell Biology 1973, 56, 275-303). This dispersion was then converted into small, single-shell vesicles with a diameter of 20-90 nm by extrusion.
  • 900 ⁇ L of this solution about 0.5 mg of total protein
  • Modified porous silicate carrier added and incubated for 18 hours at 4 ° C, using 100 mM triethanolamine (pH 7.4) and 100 mM NaCl (incubation buffer) as the buffer solution. The carrier materials were then sedimented and washed three times with incubation buffer.
  • Oriented RSO and ISO plasma membrane vesicles were produced by a method by Steck and Kant (Methods in Enzym. 1974, 31, 172-180). These dispersions were then converted into small, single-shell vesicles with a diameter of 20-90 nm by extrusion.
  • a 1: 1 (v / v) mixture of these solutions (a total of about 0.5 mg of total protein) was 50 mg of one according to Example 1.2.2.
  • Modified porous silicate carrier added and incubated for 18 hours at 4 ° C, using 100 mM triethanolamine (pH 7.4) and 100 mM NaCl (incubation buffer) as the buffer solution.
  • the carrier material was then sedimented and washed three times with incubation buffer. The success of the coating was documented using DRIFT on the dried material.
  • the glycocalix (outside of the RSO vesicles) was labeled with the help of peanut lectin (FITC-coupled, Sigma-Aldrich GmbH, Kunststoff) on the support material after washing in the incubation buffer and documented in the fluorescence microscope. This marking shows that only an RSO surface with a uniformity of at least 90% was produced (FIG. 4).

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  • Inorganic Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)

Abstract

L'invention concerne un procédé pour réaliser des doubles couches lipidiques immobilisées au moyen d'une couche lipidique intérieure et d'une couche lipidique extérieure. Au niveau de la couche lipidique intérieure et de la couche lipidique extérieure, ces doubles couches lipidiques ont une orientation définie sur la surface d'un solide, sur laquelle elles sont pratiquement immobilisées de manière stable et dynamique. Ce procédé consiste d'abord à modifier la surface d'un solide, de façon à former une surface qui est en interaction, de préférence, avec une seule des deux couches lipidiques. Dans une deuxième étape, une double couche lipidique est déposée sur cette surface modifiée. D'autres composants, notamment des protéines, sont de préférence insérés dans la double couche lipidique.
EP02758194A 2001-05-21 2002-05-14 Doubles couches lipidiques asymetriques immobilisees Withdrawn EP1407268A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10125713A DE10125713A1 (de) 2001-05-21 2001-05-21 Immobilisierte asymmetrische Lipiddoppelschichten
DE10125713 2001-05-21
PCT/EP2002/005268 WO2002095406A2 (fr) 2001-05-21 2002-05-14 Doubles couches lipidiques asymétriques immobilisées

Publications (1)

Publication Number Publication Date
EP1407268A2 true EP1407268A2 (fr) 2004-04-14

Family

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Family Applications (1)

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EP02758194A Withdrawn EP1407268A2 (fr) 2001-05-21 2002-05-14 Doubles couches lipidiques asymetriques immobilisees

Country Status (5)

Country Link
US (1) US20040171011A1 (fr)
EP (1) EP1407268A2 (fr)
CA (1) CA2448180A1 (fr)
DE (1) DE10125713A1 (fr)
WO (1) WO2002095406A2 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102005002469B3 (de) * 2005-01-18 2006-05-11 Abnoba Heilmittel Gmbh Verfahren und Vorrichtungen zur Einkapselung von Stoffen in Liposomen mit frei einstellbarem Membranaufbau
AT502713B1 (de) * 2005-10-19 2008-08-15 Univ Wien Bodenkultur Verfahren zur herstellung von lipid-membranen
WO2010052337A2 (fr) * 2008-11-10 2010-05-14 Ecole Polytechnique Federale De Lausanne (Epfl) Procédure bioanalytique pour détecter une activation des récepteurs membranaires
WO2011123525A1 (fr) * 2010-04-01 2011-10-06 The Regents Of The University Of California Formation d'une cellule artificielle ayant une composition de membrane, une asymétrie et un contenu contrôlés
US20210341401A1 (en) * 2016-10-28 2021-11-04 Arizona Board Of Regents On Behalf Of The University Of Arizona Scintillant nanoparticles for detection of radioisotope activity

Family Cites Families (5)

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Publication number Priority date Publication date Assignee Title
DD297170A5 (de) * 1990-08-13 1992-01-02 Martin-Luther-Universitaet Halle Wittenberg,De Verfahren zur fixierung von lipidschichten an polymeren traegeroberflaechen
EP0723549B1 (fr) * 1993-08-30 2003-12-17 Promega Corporation Compositions et procede de purification d'acides nucleiques
DE10027705A1 (de) * 2000-06-03 2001-12-13 Erich Sackmann Verfahren zur elektrophoretischen Auftrennung von Membranproteinen
DE10048822A1 (de) * 2000-09-29 2002-04-18 Nimbus Biotechnologie Gmbh Verfahren zur Immobilisierung von Lipidschichten
DE10121903A1 (de) * 2001-04-27 2002-10-31 Nimbus Biotechnologie Gmbh System für die Membranpermeationsmessung

Non-Patent Citations (1)

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Title
See references of WO02095406A3 *

Also Published As

Publication number Publication date
WO2002095406A3 (fr) 2004-01-29
US20040171011A1 (en) 2004-09-02
DE10125713A1 (de) 2002-11-28
CA2448180A1 (fr) 2002-11-28
WO2002095406A2 (fr) 2002-11-28

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