EP1407268A2

EP1407268A2 - Immobilized asymmetric double lipid layers

Info

Publication number: EP1407268A2
Application number: EP02758194A
Authority: EP
Inventors: Johannes Schmitt; Joachim NÖLLER
Original assignee: Nimbus Biotechnologie GmbH
Current assignee: Nimbus Biotechnologie GmbH
Priority date: 2001-05-21
Filing date: 2002-05-14
Publication date: 2004-04-14
Also published as: US20040171011A1; DE10125713A1; CA2448180A1; WO2002095406A2; WO2002095406A3

Abstract

The invention relates to a method suitable for the production of immobilized double lipid layers, comprising an inner and an outer lipid layer. The inner and outer lipid layer of said double lipid layers have a defined orientation on the surface of a solid body and are immobilized on the surface of said solid body in a substantially stable and dynamic manner. In order to carry out the inventive method, the surface of a solid body is initially modified in such a way that a surface is formed which preferably interacts with only one of the two lipid layers. In a second step, a double lipid layer is deposited on said modified surface. Preferably, other components, especially protein, are inserted into the double lipid layer.

Description

description

Immobilized asymmetric lipid bilayers

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.

Natural membranes, the so-called biomembranes, have long been the subject of intensive research as an important component of all organisms. In principle, 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. On the one hand, 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. Other membrane proteins are embedded in only part of the lipid bilayer, for example in the outer lipid layer. Furthermore, membrane proteins can also be attached to one or both sides of the membrane. In principle, membrane proteins can move laterally in the lipid matrix. However, 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.

An example of the asymmetry of biomembranes is the so-called Glykokalix on the outer side of membranes. This 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.

For example, 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.

Another very important example of the asymmetry of membranes are the transport proteins, which allow directed transport from outside to inside or from inside to outside. In summary, the asymmetry of a biomembrane is the crucial prerequisite for a membrane to be able to perform its complex tasks.

The properties of biomembranes are of great interest, for example, in the field of pharmaceutical screening and biosensor technology.

For example, long attempts have been made to understand the very complex transport processes on membranes. Furthermore, the receptors in the membrane are of course of great importance. Therefore there is an interest in suitable model systems for biomembranes.

Up to now, however, it was problematic that the asymmetry of the membrane or the lipid bilayer, which is crucial for many applications, is usually lost in the production of corresponding systems. Ultrasonic devices or polytrons are often used to isolate natural membranes from organisms. In such work-ups, vesicular structures are formed with a generally random orientation of the lipid bilayer. In this way, the lipid layer directed outwards in the natural system can also point outwards in the vesicles. In this case, one speaks of so-called "right-side-out" (RSO) vesicles. On the other hand, 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. As a rule, the vesicles resulting from such workup are a mixture of RSO and ISO vesicles. These preparations are not suitable for carrying out reliable studies on membrane properties because they are not defined vesicles.

Various methods are known for analyzing the asymmetry present in membranes and the orientation of the two lipid layers. For example, the activity of asymmetrically arranged enzymes (Ellman, GL; Courtney, A .; Valentino, Jr.; Featherstone, RM: Biochem. Pharmacol. 1961 (7), 88) or the release of membrane components oriented in a certain way by means of enzymes (Warren , LJ Biol. Chem. 1959 (234), 1971). Another possibility is the labeling of the Glykokalix with specifically labeled lectins or the labeling of membrane proteins from outside or inside protein domains with specific antibodies.

Very complex methods have been developed to produce vesicles of a certain orientation (e.g. Steck, TL; Kant, JA Methods in Enzymology 1974 (31), 172-180; DePierre, JW; Karnovsky, ML Journal of Cell Biology 1973 ( 56), 275-303). It was possible to achieve vesicular structures in which the asymmetry of the natural membrane was preserved. However, such vesicles are for investigations of the membrane properties or

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.

In order to take into account the problem of the low stability of vesicular structures and the poor handling, especially in automated processes, solid-state supported membrane systems have been developed. One possibility here is the immobilization of simple lipid layers, so-called monolayers, on a solid surface. For this purpose, the surface can first be made hydrophobic, for example with alkylsilanes or mercaptans. A lipid layer applied thereon orients with its hydrophobic part towards the carrier, and the hydrophilic side points outwards. However, this system is not suitable for simulating the natural properties of a membrane, since neither the dynamics nor the molecular order of a natural membrane is reflected. In addition, the installation of Transmembrane proteins in such a lipid monolayer are difficult or often not possible at all.

The immobilization of lipid bilayers on solid surfaces therefore offers a better approach. For the preparation of such solid-supported lipid bilayers, lipid vesicles 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)).

The details of the process of vesicle fusion and the formation of the lipid bilayer have not yet been fully understood. Two possible mechanisms are still discussed in the literature (Salafsky, J .; Groves, JT; Boxer, S. Biochemistry 1996 (35), 14773- 14781 and Puu, G .; Gustafson, I. Biochim. Biophys. Acta- Biomembranes 1997 (1327), 149-161). The main question here is whether the vesicle inverts or maintains its orientation when fused to a surface. However, everything indicates that the orientation of the vesicles occurs randomly during the fusion. The first randomness in orientation lies in the step of preparing the membrane vesicles, and the second randomness lies in the fusion with the solid surface. So far, the orientation of the immobilized lipid bilayers has not been controlled.

However, there are various indications that vesicles / cells are attached oriented to solid surfaces (Jacobson, Branton Science 1977 (195, 4275), 302-304; Wasserman, BP; Hultin, HO; Jacobson, BS Biotechnol. Bioeng. 1980 (22) 271-287 Through an electrostatic interaction of

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.

This object is achieved by an immobilized lipid bilayer that can be produced by a method as described in claim 1. Preferred embodiments of this method are set out in claims 2 to 8. Claims 9 to 13 relate to the use of a modified solid surface for the production an immobilized lipid bilayer. Claims 14 to 20 relate to an immobilized lipid bilayer or immobilized proteins and to a kit for producing immobilized lipid bilayers or immobilized proteins. Finally, claims 21 and 22 relate to a method for the analysis of proteins. The wording of all claims is hereby incorporated by reference into the content of the description.

The method according to the invention serves to produce a lipid bilayer which is immobilized on a solid. After completion of the process, 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. According to the method according to the invention, 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. In a further process step, 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.

Since 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.

In addition to the transmembrane membrane proteins which span the double layer in whole or in part in a preferred direction, 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. An example of such 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.

A large number of different lipid structures can be immobilized using the method according to the invention. According to the method, 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. For the fusion of the vesicles with the modified surface, the vesicles are advantageously provided in an aqueous dispersion.

The use of vesicles made of natural material is particularly preferred, so that the lipid bilayer immobilized according to the invention 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. Furthermore, 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.

In addition to the plasma membrane of cells, intracellular membranes that can originate, for example, from liver or kidney cells are of course also suitable as natural membranes. For example, cell organelles can be used for the preparation of intracellular membranes. These intracellular membranes are very interesting for various drug screening and / or test applications. In the intracellular membranes in particular, essentially all enzymes are located that play a decisive role in the metabolism of pharmaceuticals.

In addition to natural 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. Furthermore, 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.

In the process according to the invention, 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. Furthermore, 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.

In the molecular sense, 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. This results in denaturing effects of the solid surface on the lipid bilayer with the components contained therein avoided 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.

According to the invention, the final orientation of the lipid layers immobilized thereon is determined by the choice of a specific modification of the solid surface. For depositing the lipid bilayer on the modified 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. In a further particularly preferred embodiment of 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.

Various conventional methods can be used to check the final orientation of the lipid layers immobilized by the process according to the invention. For example, the functionality and / or activity of immobilized membrane proteins can be analyzed, 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. Furthermore, 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.

When the solid surface is modified according to the invention, an essentially hydrophilic surface is advantageously achieved. For this, 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. In a further step, 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. Various polymers, in particular polyelectrolytes, preferably anionic polyelectrolytes, can be used as interacting molecules. Polyampholytes, preferably proteins, and / or poly-zwitterions are also suitable according to the invention. Polystyrene sulfonate (PSS), in particular Na polystyrene sulfonate, and / or poly (styrene-co-maleic anhydride) (PSPMA) are particularly preferred. A particularly preferred substance that is chemisorbed is polyoxyethylene bis (glycidyl ether).

In a further preferred embodiment of the method according to the invention, further interacting molecules are used to modify the surface, which interact with the initially applied interacting molecules.

In a further preferred embodiment, 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.

According to the invention, 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. In a first embodiment of the invention, the modified surface preferably interacts with the originally inner lipid layer (inner leaflet) of the lipid bilayer. In this case, the modified surface preferably has a negative net charge. For this purpose, the modification of the solid surface is advantageously carried out by producing “ultra-soft” polymer surfaces with a negative net charge. 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.

In a second embodiment of the method according to the invention, the solid surface is modified such that it preferably interacts with the originally outer lipid layer of the lipid bilayer to be immobilized. In this case, the modified surface preferably has a positive net charge. For this purpose, 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. During the fusion, the vesicles align so that the originally outer lipid layer faces the solid. As a result, 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. These examples make it clear that, depending on the properties of the lipid bilayer to be immobilized, the orientation of the immobilized lipid bilayer on the solid can be controlled by selecting an appropriately suitable surface.

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.

Various conventional carrier materials can be used to carry out the method according to the invention. In a preferred embodiment of the invention, 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. In addition, 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.

It is particularly preferred to use 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. The use of silicate particles is very particularly preferred. These particles can be conventional silicate balls, for example.

In a particularly preferred embodiment of the invention, porous particles are used, which increases the surface area available for the immobilization.

In a further preferred embodiment, 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

Automation processes matter. Furthermore, the use of core shell polymer particles is preferred.

The solids used are preferably used in the form of a dispersion. This dispersion is advantageously produced in aqueous solutions. Furthermore, 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. These are 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. Furthermore, it may be preferred that 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. In a further preferred embodiment of the kit according to the invention, 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. This can be, for example, an aqueous dispersion which may contain appropriate additives which improve the stability and durability of the lipid vesicles. Of course, the 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. This kit 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. With regard to the various details with regard to the immobilized proteins, reference is made to the above description.

Finally, 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. With regard to the 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.

The described features and further features of the invention result from the figures and the following description of examples in conjunction with the subclaims. The various features can be implemented individually or in combination with one another.

The figures show:

Fig. 1 comparison immobilized according to the invention

Erythrocyte membranes in different orientations on the solid surface based on the activity of acetylcholinesterase,

2 Comparison of CACO-2 immobilized according to the invention

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),

Fig. 3 Comparison of immobilized plasma membranes according to the invention on HEK293 cells in different

Orientation on the solid surface based on N-acetylsialic acid released by neuromidase and

4 Comparison of erythrocyte membranes immobilized according to the invention in different orientations on the solid surface by means of the binding of

Peanuts Lectin-FITC to the Glykokalix

Neuramidasebehandlung.

Examples

1. MODIFYING THE SOLID SURFACE

1.1. Functionalization of the solid surface

1.1.1 Amine functionalization of powdery and porous silicate surfaces with N- (2-aminoethyl) -3- aminopropyltrimethoxysilane (EDA)

A freshly prepared silane solution consisting of 9.2 mL N- (2-aminoethyl) -3-aminopropyltrimethoxysilane (EDA) and 243 μL concentrated acetic acid in 450 mL deionized water. After 5 minutes, 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. In a similar way, further carrier materials with pore sizes between 5 and 400 nm were functionalized.

1.1.2. Amino functionalization of powdery and porous silicate surfaces with polyethyleneimine (PEI)

5 g of 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.

1.2. Setting of particularly preferred properties

1.2.1. Chemisorption of polyoxyethylene bis (glycidyl ether (PEG-DiGly) on EDA-functionalized silicate surfaces

To 10 mL of a 5% solution of polyoxyethylene bis (glycidyl ether PEG-DiGly) (Mw ~ 3350 SIGMA) in phosphate buffer (pH 7.4) was added 1 g of one according to Example 1.1.1. given amino-functionalized silicate material and shaken for three hours. The silicate material was then sedimented and washed three times with deionized water. The success of chemisorption was documented using DRIFT. 1.2.2. Adsorption of Na polystyrene sulfonate (PSS) on polyethyleneimine functionalized silicate surfaces

To a Na polystyrene sulfonate (PSS) solution consisting of 25 mg of PSS (Mw ~ 70,000, Aldrich, Steinheim) in 50 mL of a 3 molar NaCl solution was added 1 g of one according to Example 1.1.2. given amino-functionalized silicate material and shaken for three hours. The silicate material was then sedimented and washed three times with deionized water. The success of the adsorption was documented by means of DRIFT and the decrease in the PSS concentration in the solution.

2. DEPOSITION OF LIPID LAYERS ON THE MODIFIED SURFACES

2.1. Immobilization of native erythrocyte membranes on untreated silicate surfaces and measurement of the acetylcholine esterase function

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 μL 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. 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.

2.2. Immobilization of native CACO-2 membranes on different silicate surfaces and measurement of the ATPase function of the P-glycoprotein

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. To 900 μL of this solution (about 0.5 mg of total protein), 50 mg each of an example 1.2.1. or 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 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). In the case of the example 1.2.2. modified carrier, 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.

2.3. Immobilization of native HEK293 membranes on different silicate surfaces and measurement of the N-acetylsialic acid released by neuramidase

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. To 900 μL of this solution (about 0.5 mg of total protein), 50 mg each of an example 1.2.1. or 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 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 release of N-acetylsialic acid by neuramidase on the carrier materials was carried out after washing in the incubation buffer using a kit from CalBiochem (Bad Soden) in accordance with the attached protocol. In the case of the example 1.2.1. modified carrier, an ISO-oriented surface is obtained after immobilization, but because of the incorrect orientation of the glycocalyx to be examined, there is no appreciable release of N-acetylsilicic acid by neuramidase. First a comparable release can be achieved by adding detergent. Not so with the help of the carrier from example 1.2.2. manufactured RSO surface. It has a release (FIG. 3) comparable to that of the isolated vesicles on the carrier material.

2.4. Immobilization of native erythrocyte membranes on PEI / PSS-modified silicate surfaces and labeling of the glycocalyx with FITC lectins

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. To 900 μL of 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, Munich) 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).

3. PROPERTIES OF THE MEMBRANES ON OPTIMIZED SUPPORT MATERIAL 3.1. Stability in the flowing aqueous medium

The examples in 2.1. until 2.4. Systems described were exposed to a flowing medium (incubation buffer of the respective example) in a test bath for 24 hours. At intervals of 2 hours, equal amounts of support material were removed from the test bath, dried and then examined for their coating by means of DRIFT. No measurable decrease in membrane coating over time was observed.

3.2. Stability after freezing

The examples in 2.1. until 2.4. The systems described were frozen in the dispersed state at -80 ° C. and then brought to room temperature and dried. Comparative DRIFT measurements before and after freezing showed unchanged lipid and protein amounts on the carrier material. The systems described were additionally stored at -80 ° C. for 3 months after preparation. Samples were taken at intervals of one month and examined in accordance with the measured variables described in the examples. After 2 months, the enzyme activities had dropped to approx. 70% of the original value (measured immediately after the preparation and washing of the carrier material), while the labeling of the Glykokalix with FITC lectin brought unchanged results. No protein could be detected in the respective supernatants of the stored samples.

Claims

Expectations

1. A method for producing an immobilized lipid bilayer with an inner and an outer lipid layer, the lipid bilayer having a defined orientation with respect to the

Has lipid layers on a surface of a solid and is essentially stable and dynamically immobilized on the surface of the solid, comprising the following method steps: a) modifying the surface of the solid to form a surface which preferably interacts with only one of the two lipid layers and b) Deposit the lipid bilayer on the modified surface.

2. The method according to claim 1, characterized in that further components, in particular proteins, are inserted into the lipid bilayer.

3. The method according to claim 1 or claim 2, characterized in that the deposition of the lipid bilayer is carried out by fusion of lipid vesicles.

4. The method according to any one of the preceding claims, characterized in that the modification of the surface of the

Solid body comprises the following substeps: aa) functionalizing the surface and / or ab) adsorbing and / or chemisorbing interacting molecules.

5. The method according to any one of the preceding claims, characterized in that the surface modified according to process step a) interacts with the inner lipid layer.

6. The method according to claim 4 or claim 5, characterized in that the modification of the surface is carried out by applying polymer films with a negative net charge.

7. The method according to any one of claims 1 to 4, characterized in that the surface modified according to process step a) interacts with the outer lipid layer.

8. The method according to claim 7, characterized in that the modification of the surface is carried out by applying polymer films with a positive net charge.

9. Use of a modified surface of a solid to produce an immobilized lipid bilayer with an inner and an outer lipid layer, the

Lipid bilayer has a defined orientation with respect to the lipid layers on a surface of a solid and is essentially stable and dynamically immobilized on the surface of the solid.

10. Use according to claim 9, characterized in that the surface of the solid body is provided for interaction with the inner lipid layer.

11. Use according to claim 10, characterized in that the modified surface has a negative net charge, in particular a polymer film with a negative net charge.

12. Use according to claim 9, characterized in that the surface of the solid body is provided for interaction with the outer lipid layer.

13. Use according to claim 12, characterized in that the modified surface has a positive net charge, in particular a polymer film with a positive net charge.

14. Immobilized lipid bilayer, producible by a method according to one of claims 1 to 9.

15. Immobilized proteins, in particular membrane proteins, can be produced by a method according to one of claims 2 to 8.

16. Kit for producing immobilized lipid bilayers with inner and outer lipid layers, wherein the lipid bilayers have a defined orientation with regard to the lipid layers on the surface of solid bodies and are essentially stable and dynamically immobilized on the surface of the solid bodies, at least comprising a solid body with a modified one Surface that preferably interacts with only one of the two lipid layers.

17. Kit according to claim 16, further comprising lipid vesicles, which preferably have further components, in particular proteins.

18. Kit for the production of immobilized proteins in immobilized lipid bilayers with inner and outer

Lipid layers, the lipid bilayers being a defined one

Orientation with regard to the lipid layers on the surface of Have solids and on the surface of the solids are essentially stable and dynamically stabilized, at least comprising a solid with a modified surface that preferably interacts with only one of the two lipid layers, and preferably lipid vesicles that have proteins.

19. Kit according to one of claims 16 to 18, characterized in that the modified surface can be produced by a method according to claim 5 or claim 6.

20. Kit according to one of claims 16 to 18, characterized in that the modified surface can be produced by a method according to claim 7 or claim 8.

21. A method for the investigation of proteins, in particular membrane proteins, characterized in that these proteins are used as immobilized proteins in immobilized lipid bilayers with inner and outer lipid layers, the lipid bilayers having a defined orientation with respect to the lipid layers on the surface of solids and on the Surface of the solid are essentially stable and dynamically immobilized.

22. The method according to claim 21, characterized in that the immobilized lipid bilayers can be produced by a method according to one of claims 1 to 8.