EP1373550A1 - Procede de preparation de membranes a film lipidique sur support et utilisation de celles-ci - Google Patents

Procede de preparation de membranes a film lipidique sur support et utilisation de celles-ci

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Publication number
EP1373550A1
EP1373550A1 EP02708868A EP02708868A EP1373550A1 EP 1373550 A1 EP1373550 A1 EP 1373550A1 EP 02708868 A EP02708868 A EP 02708868A EP 02708868 A EP02708868 A EP 02708868A EP 1373550 A1 EP1373550 A1 EP 1373550A1
Authority
EP
European Patent Office
Prior art keywords
substrate surface
lipid
detergent
phosphatidyl
biomolecule
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.)
Withdrawn
Application number
EP02708868A
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German (de)
English (en)
Inventor
Olof Karlsson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytiva Sweden AB
Original Assignee
Biacore AB
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Filing date
Publication date
Priority claimed from SE0100875A external-priority patent/SE0100875D0/xx
Application filed by Biacore AB filed Critical Biacore AB
Publication of EP1373550A1 publication Critical patent/EP1373550A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • 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
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • the present invention relates generally to a preparation method for lipid film membrane type structures on substrate surfaces, particularly sensor surfaces, a supported lipid film membrane prepared by the method, the use of such supported lipid film membranes in lipid membrane interaction studies, and a substrate surface for use in the method.
  • Membranes play a central role in the structure and function of all living cells. Defining the boundaries of the cells as well as compartments within the cells, the membranes not only have a barrier function but also control the transport of substances across the membrane, mediate information between the compartments and are the site of enzymatic reactions.
  • the membranes consist of a lipid molecule bilayer with proteins and other components, such as e.g. lipopolysaccharides.
  • the membrane lipids have a polar (hydrophilic) part and a non-polar (hydrophobic) part.
  • the non-polar parts of the lipids are turned towards each other in the middle of the bilayer with the polar lipid parts forming the external surfaces of the membrane.
  • Proteins in the membranes which may extend through the membrane (transmembrane proteins) or be anchored to one of the membrane surfaces, are generally bound to the membrane through noncovalent forces, such as the hydrophobic force or electrostatic interactions, but there are also examples of proteins which are covalently bound to lipids.
  • BLM's Artificial supported bilayer lipid membranes
  • Such BLM's may be used for studying, for example, ligand-receptor interactions at the membrane- water interface.
  • EP-A-441120 proposes to join the lower lipid layer to the support by hydrophilic spacer arms.
  • the assembly of BLM's on a solid support may be obtained in various ways. Basically, however, three different approaches have generally been used.
  • a solid support is immersed into an aqueous solution and a lipid monolayer is then formed at the air-water interface. When the support is retracted from the solution, a lipid monoloayer is adhered to the support. The support is then re- immersed into the solution which results in the formation of a lipid bilayer on the support (Suarez-Isla, B. A., et al., Biochemistry 22, 2319-2323 (1983)).
  • a solid support surface is contacted with an aqueous solution of lipid vesicles whereby lipid bilayers under certain conditions may be formed by spontaneous fusion of the lipid vesicles, or liposomes, to the surface (a liposome is a lipid bilayer enclosing a volume).
  • lipid bilayers floating on a thin water film may be formed on a hydrophilic support, such as Si ⁇ 2-
  • lipid bilayer membranes may be produced which rest on an ultrathin hydrated polymer film, or hydrogel (see e.g. Sackmann, E., Science 271, 43-48 (1996)).
  • liposomes contacted with a hydrophobic substrate will build up a monolayer on the hydrophobic surface (Cooper, M. A., et al., Biochim. Biophys. Ada 1373. 101-111 (1998)).
  • the membrane may be linked more stably to the support, for instance, through hydrophilic spacers as mentioned above, or as disclosed in, for example, US-A-5,922,594 by covalently binding the lipid bilayer to a self-assembled monolayer of straight long chain molecules coated on the substrate surface.
  • an aqueous solution of micellar or vesicle liposomes is contacted with the monolayer- supporting surface so that a majority of the liposomes bind covalently to the monolayer to form an anchored bilayer lipid membrane. Due to the covalent anchoring of the membrane to the support, the process may be performed in a flow cell using a controlled laminar flow.
  • a streptavidin- coated surface is contacted with biotinylated liposomes.
  • a solid substrate surface is contacted with an aqueous solution of mixed micelles formed by codispersion of detergent with lipid in a receptacle, and the formation of a lipid bilayer on the surface is then effected by selective removal of the detergent by either diluting the micellar solution with buffer, "the micellar dilution technique", or by dialysis.
  • Micellar dilution may be exemplified by Lang, H., et al., Langmuir JO, 197-210 (1994), where a supported mixed bilayer was formed by contacting a gold electrode supporting a thiolipid monolayer with an aqueous solution of mixed micelles of lipid and detergent, and then stepwise diluting the solutions with electrolyte below the critical micelle concentration (CMC) of the detergent.
  • CMC critical micelle concentration
  • Supported lipid bilayer membranes containing proteins or peptides have been prepared by including the protein or peptide in the lipid layer and lipid vesicles used in the first and second approaches, respectively, mentioned above, or in the mixed micelles used in the above third approach.
  • WO 96/38726 discloses a solid device having a covalently attached coating of a lipid bilayer containing a protein.
  • a proximal phospholipid layer which may contain e.g. a transmembrane protein, was first covalently attached to a linker layer on the solid device.
  • a distal lipid layer was then deposited by vesicle or mixed micelle fusion to give a lipid bilayer structure.
  • Mixed micelle fusion was effected by depositing a mixed micelle dispersion onto the proximal phospholipid layer, and then diluting with an aqueous buffer.
  • US-A-5,765,355 discloses preparation of a bilayer lipid membrane sensor by contacting a gold surface with a thiolipid/detergent solution to covalently bind a thiolipid layer to the surface. After washing with detergent solution, a transmembrane protein-containing phospholipid/detergent solution was added and stepwise diluted with potassium chloride solution. The resulting lipid bilayer consisted of a mixed monolayer of thiolipid and phospholipid, and a second phospholipid monolayer containing the transmembrane protein.
  • a detergent/lipid mixed micelle preparation is deposited from an aqueous dispersion thereof onto a substrate surface, and the substrate surface is then contacted with an aqueous liquid substantially free from detergent to elute detergent from the micelles, the remaining lipid molecules thereby forming a lipid film on the substrate surface.
  • the present invention provides a method of preparing a substrate surface supporting a lipid film membrane structure, which method comprises the steps of: a) contacting a substrate surface with an aqueous liquid containing detergent/lipid mixed micelles to adhere detergent/lipid mixed micelles to the substrate surface, and then b) contacting the substrate surface having detergent/lipid mixed micelles adhered thereto with an aqueous liquid substantially free from detergent to elute the detergent molecules from the adhered mixed micelles and make the remaining lipid molecules assemble into a lipid film membrane structure on the substrate surface.
  • the invention provides a substrate supporting a lipid film membrane structure as prepared according to the first aspect above.
  • the invention provides the use of the method for reconstituting membrane protein function.
  • the invention provides the use of a substrate supporting a lipid film membrane structure as prepared according to the first aspect above for interaction studies with membrane associated proteins or peptides, e.g. in screening of drug candidate molecules.
  • the invention provides a substrate surface for use in the method.
  • Figs. 1 A to 1C are a schematic illustrations of the reconstitution of membrane proteins by deposition of detergent/lipid mixed micelles on an amphiphilic biosensor sensing surface with immobilized proteins, and subsequent elution of the detergent to form a lipid bilayer.
  • Fig. 2 is a sensorgram showing the response (RU) versus time (s) for the injection of three different mixtures of lipid and detergent into the flow cell of a SPR- biosensor instrument.
  • Fig. 3 is a diagram wherein the ratio of the difference between the concentration of detergent (OG) and its CMC to the concentration of lipid (POPC) is plotted against the deposition level of lipid on a sensing surface when passed by aqueous samples of detergent/lipid mixed micelles.
  • Fig. 4 is a diagram representing the signaling of reconstituted rhodopsin receptor on a SPR biosensor sensing surface.
  • the relative response (RU) is plotted against time (s) for the illumination of a POPC-reconstituted rhodopsin surface (1) relative to the signal in a POPC-only reference surface (2).
  • the present invention relates to the preparation of lipid film membranes, especially lipid bilayer membranes, supported on a solid substrate surface.
  • Membrane lipids are amphiphilic molecules, or amphiphiles, comprising a hydrophilic (water soluble) part and a hydrophobic (water insoluble) part.
  • the lipids form together a characteristic bilayer where the hydrophobic parts are directed towards the middle and the hydrophilic parts form the two surfaces of the membrane.
  • this bilayer there may also be biomolecules, such as proteins or peptides, partly or fully inserted therein.
  • the invention is based on the idea of reconstituting lipid membranes, with or without proteins or peptides, by adhering mixed micelles of detergent and lipid to a substrate surface with subsequent depletion of the detergent by aqueous liquid substantially free from detergent.
  • detergent/lipid mixed micelles in an aqueous solution are contacted with the substrate surface, they attach thereto. With a proper detergent/lipid ratio, this attachment is strong enough to allow selective elution of the detergent from the adhered detergent/lipid mixed micelles even when the liquid containing mixed micelles (and detergent) is rapidly replaced by aqueous liquid substantially free from detergent, such as by a liquid flow.
  • the replacement of the mixed micelle-containing liquid by detergent-free liquid may be performed in a static system by removal of the micelle-containing liquid from the substrate surface, e.g. by a pipette, with subsequent addition of the detergent-free liquid to the substrate surface.
  • the substrate having detergent/lipid mixed micelles adhered thereto is moved to another compartment or receptacle with detergent- free liquid.
  • the depletion of the detergent from the substrate surface is performed by a liquid flow over the substrate surface. If proteins or peptides are attached to the substrate surface prior to forming the lipid film thereon, the proteins or peptides are reconstituted with lipids when the detergent is selectively eluted from the adhered detergent/lipid mixed micelles.
  • lipid film membrane that is formed on the substrate surface, i.e. a monolayer or bilayer, depends mainly on the character of the surface. While the method of the invention performed on a hydrophobic substrate surface usually will produce a lipid monolayer, the formation of a lipid bilayer, which is (at least currently) preferred, requires an amphiphilic substrate surface as will be explained in more detail below.
  • the procedure of the invention applied to the preparation of a supported lipid bilayer membrane is schematically illustrated in Figs. 1A to lC.
  • substrate refers to any material body or layer onto which it is desired to apply a lipid film membrane.
  • Exemplary substrate materials are gels, beads, polymers, stationary hydrophobic or amphiphilic phases, etc.
  • Reference to the "surface" of the substrate or support includes, for porous substrates, the interior surfaces as well. Currently preferred substrates are sensor surfaces and chromatographic particles.
  • the substrate surface should be amphiphilic for a lipid bilayer to be formed.
  • amphiphilic is, however, to be construed broadly herein. Basically, the term means that the surface should exhibit hydrophilic and hydrophobic chemical structures (i.e. chemical groups or residues, including whole molecules, e.g. biomolecules) in ratios which may vary within a wide range, including surfaces ranging from partially hydrophobic to partially hydrophilic.
  • the hydrophobic structures of an amphiphilic surface constitute chemical projections capable of interacting with hydrophobic parts of the lipid bilayer.
  • the surface may support a self-assembled layer of hydrophilic residues mixed with hydrophobic residues (e.g. alkyl groups), the latter preferably extending out from the hydrophilic residues.
  • hydrophobic residues serve to adhere the mixed micelles whereas the hydrophilic residues aid in the formation of the lipid bilayer upon the depletion of the detergent.
  • the supported membrane bilayer includes a biomolecule, such as a membrane protein or peptide (the term peptide including oligopeptides and polypeptides).
  • Such proteins or peptides may be the species that provide the amphiphilic character to an otherwise hydrophilic surface.
  • a hydrophobic protein or peptide may be attached to a self-assembled layer of hydrophilic residues supported on the substrate surface.
  • the amphiphilic character of the surface may be provided by both hydrophobic chemical groups and membrane proteins or peptides.
  • hydrophobic may be defined as water-repelling whereas hydrophilic may be defined as water-attracting. It is also customary to define hydrophilicity and hydrophobicity with regard to the contact angle for a droplet of a liquid on a planar solid surface, the contact angle being measured from the plane of the surface, tangent to the water surface at the three phase boundary line.
  • a hydrophilic liquid will thus have a low contact angle on a hydrophilic surface, whereas a hydrophobic liquid will have a high contact angle.
  • hydrophobic surfaces typically have contact angles in the range of 40 to 110°, while the contact angles with water for hydrophilic surfaces typically are in the range of 1 to 25°.
  • hydrophilic moieties to hydrophobic moieties on the amphiphilic substrate surface will depend on the particular moieties as well as on the components of the mixed micelles used and may readily be determined by the skilled person for each particular situation.
  • a presently preferred substrate surface comprises a biocompatible porous matrix, preferably a hydrogel, modified to contain a certain amount of hydrophobic groups.
  • a “hydrogel” may be defined as presenting a surface layer of bound molecules which by reason of their chemical nature hold a large fraction of water, in which the molecules are predominantly in an amorphous, water-solvated state, and in which the thickness of the layer is of the order of 30 A minimum up to any indefinitely higher limit (Merrill, E.W., et al., (1986), Hydrogels in Medicine and Pharmacy, Vol. HI, Ed. Peppas, N. A., Chapter I, CRC Press, Inc., Boca Raton, Florida).
  • An exemplary such modified hydrogel is a carboxymethyl-modified dextran polymer hydrogel on which a substantial fraction of the glucose moieties have been modified by alkyl groups.
  • micelle mixed micelle
  • lipid lipid
  • detergent lipid
  • Micelles are aggregates of amphiphilic molecules (amphiphiles), such as detergents, which aggregates do not enclose an aqueous volume. They are formed when the concentration of the amphiphilic molecule in a liquid exceeds a critical value called the “critical micelle concentration", or CMC.
  • CMC critical micelle concentration
  • the micelles are usually globular but also other shapes, such as e.g. rod-shaped micelles, may form at high amphiphile concentrations .
  • detergents are in a broad sense defined as substances capable of lowering the surface tension of liquids, but the term relates in a more narrow sense to surfactants as means for purification purposes.
  • the detergents are amphiphiles and may form micelles. They may be anionic, cationic, zwitterionic or uncharged. In the context of the present invention, detergents may be said to be micelle-forming amphiphiles.
  • lipids are generally esters of long-chain carboxylic esters and include fats, waxes and cell lipids.
  • the lipids are usually membrane lipids (cell and organelle membrane lipids), which exhibit an enormous diversity, are more or less amphiphilic and include inter alia the following classes: phospholipids, lysophospholipids, glycosyl diacylglycerols, plasmalogens, sphingomyelins, gangliosides, and sterols.
  • phospholipids cell and organelle membrane lipids
  • lysophospholipids glycosyl diacylglycerols
  • plasmalogens sphingomyelins
  • gangliosides gangliosides
  • sterols sterols.
  • lysophospholipids form micelles, whereas some phospholipids under certain conditions form bilayers.
  • a detergent or detergent mixture
  • a membrane lipid or lipid membrane mixture
  • mixed micelles may be formed consisting of alternating bilayer-prone lipid molecules and detergent molecules.
  • a lipid bilayer will form on an amphiphilic support if the detergent is eluted from the micelles, such as by a continuous flow of substantially detergent-free liquid.
  • Lipids suitable for reconstitution purposes according to the present invention may readily be selected by the skilled person and are basically those able to (i) form lamellar aggregation structures, at least somewhere in the temperature interval 15 - 40 °C; and (ii) be solubilised by any suitable detergent to constitute a nonturbid micellar suspension.
  • These lipids, or lipid mixtures should preferably have a relatively low CMC and may be selected from natural or synthetic lipids or mixtures thereof.
  • such lipids may be selected from natural or synthetic lipid molecules such as glycerophospholipids, glyceroglycolipids, sphingophospholipids and sphingoglycolipids, and from the classes phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl acid, phosphatidyl inositol, galactopyranoside, glucopyranoside, digalactopyranoside, diglucopyranoside, ceramide-phosphatidyl choline, ceramide-phosphatidyl ethanolamine, ceramide-phosphatidyl serine, ceramide-phosphatidyl glycerol, ceramide- phosphatidyl acid, ceramide-phosphatidyl inositol, sphingomyelin molecules, glucosylceramides, gluco
  • Phosphatidylcholines with acyl chains ranging in length from 14 to 18 carbons such as di-l,2-myristoyl-SN- phosphatidylcholine, di- 1 ,2-oleoyl-SN-phosphatidylcholine, 1 -palmitoyl-2-oleoyl-SN- phosphatidylcholine (POPC) and l-stearoyl-2-oleoyl-SN-phosphatidylcholine; glyceroglycolipids like di-l,2-myristoyl-3-diglucopyranosyl-SN-glycerol, di-l,2-oleoyl- 3-diglucopyranosyl-SN-glycerol, l-palmitoyl-2-oleoyl-3-diglucopyranosyl-SN-glycerol, l-stearoyl-2-oleoyl-3-diglucopyranosyl-SN
  • detergents, or detergent mixtures, suitable for use in the present invention may readily be selected by the skilled person.
  • the CMC of the detergent is relatively high.
  • the detergent CMC usually should be at least about 1 mM, it is often preferable that the CMC is higher, such as at least about 10 mM.
  • Exemplary detergents are 3-[(3-cholamidopropyl)dimethylammonio]-l-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy- 1 -propane sulfonate (CHAPSO), ⁇ , ⁇ -bis-(3-D-gluconeamidopropyl)-deoxycholamide (deoxy- BIGCHAP), sodium taurocholate, cholic acid, deoxycholic acid, n-octylglucoside (OG), n-octylthioglucoside, N-decyl-N,N-dimethyl-3-ammonio- 1 -propane sulfonate (Zwittergent 3-10), N-dodecyl-N,N-dimethyl-3-ammonio-l -propane sulfonate (Zwittergent 3-12), octanoyl-N
  • the selective elution of detergent from the mixed micelles in accordance with step c) in the method of the invention as defined above is basically driven by the difference in critical micelle concentration, or CMC, of the detergent and lipid, respectively.
  • CMC critical micelle concentration
  • the ratio of detergent to lipid in the mixed micelles may be a critical factor in the method of the invention. Too detergent rich preparations may result in poor or no attachment at all of mixed micelles to the surface (note that liquids of a high detergent concentration, well above the CMC, are usually used to completely wash away deposited lipids from a sensor surface in a flow cell). On the other hand, too lipid rich preparations tend to be turbid and adhere slowly to the solid support, building aberrant aggregate structures. Therefore, it is crucial that the lipid/detergent ratio in the mixed micelles be balanced to obtain a maximal level of attachment to the solid support while still having a clear, non-turbid solution.
  • the approach of the present invention is to use mixed micelles of a predetermined detergent/lipid ratio known to give a clear micellar solution which attaches the micelles to the surface sufficiently strongly not to be released when contacted with detergent-free solution.
  • the desired ratio may be determined by relating the ratio of the excess concentration of detergent over CMC, i.e. ([detergent] - CMC)/[lipid], to the amount of lipid deposited on the solid support.
  • the optimum ratio varies depending on the particular lipid and detergent selected but can readily be determined by the skilled person, as will be further described below.
  • the above ratio is in the range of from about 0.1 to about 100, preferably from about 0.5 to about 100, and more preferably from about 0.5 to about 10.
  • the optimum ratio may be in the range from about 0.5 to about 5, an exemplary range being from about 0.5 to about 3, which is applicable to inter alia octylglucoside and POPC.
  • the concentration of lipid (or lipids) is usually in the range of from about 0.1 to about 50 mM, preferably from about 0.1 to about 10 mM.
  • the concentration of detergent is usually in the range of from about 0.5xCMC to about lOxCMC, preferably from about 0.5xCMC to about 5xCMC for the detergent.
  • the method of the invention should generally be performed at conditions, such as e.g. temperature, where the lipid (or lipid mixture) may form a liquid lamellar phase, i.e. above the main transition temperature of the lipid (or lipid mixture). In many cases it may be satisfactory to carry out the method at room temperature.
  • conditions such as e.g. temperature, where the lipid (or lipid mixture) may form a liquid lamellar phase, i.e. above the main transition temperature of the lipid (or lipid mixture). In many cases it may be satisfactory to carry out the method at room temperature.
  • elute is used herein basically in the commonly acknowledged sense, i.e. to denote the removal of an adsorbed substance from an adsorbent by means of a solvent, the adsorbent in the present case being the substrate surface.
  • the liquid used for eluting the detergent molecules from the mixed micelles should be substantially free from detergent, which means that the eluent liquid should contain no detergent at all or contain only trace amounts thereof. Otherwise the composition of the eluent liquid may vary depending on the particular application of the method of the invention. For example, in the case of chromatographic applications, the eluent liquid may be a conventional type eluent, and when the method is used in flow- cell-based biosensor applications, the eluent liquid may be the conventionally used running buffer.
  • the eluent liquid should provide a mobile phase for detergent removal, a salient feature of the invention being that micelle-containing liquid above the substrate surface is completely replaced (e.g. displaced) rather than diluted by the detergent-free liquid.
  • the flow of eluent liquid should be substantially continuous, i.e. it should preferably not be arrested, and if so, at least not for any long time periods, and it should also not be interrupted by air bubbles.
  • the liquid flow rate may vary during the elution, it is preferred that the flow rate is substantially constant. The optimum flow rate, which may vary within wide limits, depends on ter alia the flow system used and may readily be chosen by the skilled person for each particular situation.
  • step a) of the method While it is possible to carry out the deposition of the mixed micelles on the substrate surface according to step a) of the method in a stationary liquid, and only perform the elution step b) under flow conditions, it is preferred that the whole procedure is performed using a continuous liquid flow.
  • the method of the invention may be used for chromatographic applications.
  • the lipid bilayer membranes may be applied to hydrophobic or amphiphilic chromatographic particles, preferably in place in a column or in a channel of a micro- or nanofluidic device. In the latter types of devices, the lipid bilayer membranes may alternatively be applied to a channel wall.
  • a biosensor is an analytical device for analyzing minute quantities of sample solution having an analyte of interest, wherein the interaction of the analyte with a sensing surface is detected by a detection device.
  • the sensing surface or surfaces of the biosensor are preferably located in a flow cell or flow cells, i.e. broadly a channel part(s) or compartment(s) through which a liquid flow may be maintained.
  • the lipid bilayer contains a biomolecule(s), usually a protein or peptide, preferably a so-called membrane protein.
  • a protein or peptide may be applied to the bilayer lipid membrane by including the protein or peptide in the mixed micelle preparation to be deposited on the substrate surface, or by attaching the protein or peptide to the bilayer lipid membrane after the formation thereof, e.g. by adsorption or covalent binding.
  • the lipid bilayer will then reconstitute the protein or peptide by being formed around the protein or peptide molecules.
  • the protein or peptide may then constitute the sole hydrophobic elements of an amphiphilic surface.
  • Immobilization of the biomolecule to the substrate surface may be performed by methods well known in the art. For instance, groups on a protein or peptide may be coupled directly to active functional groups on the substrate, for example by amine coupling to surface carboxyl groups as described in the Examples below. It may, however, at least in some cases be advantageous to attach the biomolecule, such as a protein or peptide, to the substrate surface via a coupling member, such as, for instance, a spacer, a linker, or another protein or peptide.
  • a coupling member such as, for instance, a spacer, a linker, or another protein or peptide.
  • a specific binding pair may be used for attaching the biomolecule to the substrate surface.
  • exemplary specific binding pairs include antibody-antigen, antibody-hapten, biotin-avidin (or streptavidin), carbohydrate-protein, carbohydrate- lectin, nucleic acid duplexes, oligonucleotide pairs, oligonucleotide-polynucleotide pairs, polynucleotide pairs, such as DNA-DNA and DNA-RNA, protein nucleic acid (PNA) pairs, protein-RNA, interacting peptide pairs, and protein-metal chelate.
  • GPCRs G-protein coupled receptors
  • GPCRs G-protein coupled receptors
  • a histidine-tagged protein or peptide may be captured on the substrate surface by an immobilized metal chelate, such as, e.g., a nitrilotriacetic acid (NT A) nickel complex.
  • an immobilized metal chelate such as, e.g., a nitrilotriacetic acid (NT A) nickel complex.
  • Antibody as used herein means an immunoglobulin which may be natural or partly or wholly synthetically produced and also includes active fragments, including Fab antigen-binding fragments, univalent fragments and bivalent fragments. The term also covers any protein having a binding domain which is homologous to an immunoglobulin binding domain. Such proteins can be derived from natural sources, or partly or wholly synthetically produced. Exemplary antibodies are the immunoglobulin isotypes and the Fab, Fab', F(ab')2, scFv, Fv, dAb, and Fd fragments.
  • Hapten as used herein means a low molecular species that may give rise to an immune response only when coupled to a larger molecule or cell or by aggregation. After immunisation, however, free haptens may react with antibodies.
  • a substrate surface having an array of different (or the same) proteins or peptides in a lipid bilayer membrane may be prepared by attaching these proteins or peptides to the substrate surface, and then depositing a mixed micelle preparation to the surface to reconstitute the proteins or peptides.
  • "On surface reconstitution" according to the present invention permits the same handling of membrane proteins as for soluble proteins by standard procedures. No reconstitutions, into more or less stable proteoliposomes, have to be prepared in advance and when the protein is ready for reconstitution on a substrate surface, no elaborate detergent dilution steps are needed. Should it, for some reason, be desired that the lipid bilayer membrane contain other biomolecules than proteins or peptides, such molecules may be provided in the same ways as those outlined for proteins and peptides.
  • lipid bilayer membranes use a lipid bilayer without any protein or peptide.
  • the international patent application WO 00/79268 discloses a method of assaying drug candidates (usually small molecules) with regard to inter alia absorption, i.e. the uptake of a drug compound from the site of administration into the systemic circulation, by estimating the absorption from biosensor data associated with a sensor chip having lipids immobilized thereon.
  • absorption i.e. the uptake of a drug compound from the site of administration into the systemic circulation
  • biosensor data associated with a sensor chip having lipids immobilized thereon e.g. the analysis of interactions of a drug candidate with immobilized liposomes can be used to predict whether or not the drug candicate is absorbed by the small intestine.
  • the method of the invention may advantageously be used for such absorption assaying of drug candidates.
  • a great advantage of the present method of invention is the promptness by which the lipid reconstitution can be accomplished.
  • the present process of forming a lipid bilayer membrane on a support surface may be performed in a very short time. This means among other things that a fresh lipid bilayer may be formed for each assay, which may be desired for several reasons. For example, an accidentally introduced air bubble may tear away a part of the bilayer, it may not be possible to completely wash away absorbed small molecules (candidate molecules often tend to remain bound to the lipid bilayer in ligand binding assays), etc. Many approaches that before were either very laborious or impossible have now become both possible and easy to carry out. This is important for applications demanding standardized and high throughput processing, like screening and array assays.
  • Biosensors may be based on a variety of detection methods. Typically such methods include, but are not limited to, mass detection methods, such as piezoelectric, optical, thermo-optical and surface acoustic wave (SAW) device methods, and electrochemical methods, such as potentiometric, conductometric, amperometric and capacitance methods.
  • mass detection methods such as piezoelectric, optical, thermo-optical and surface acoustic wave (SAW) device methods
  • electrochemical methods such as potentiometric, conductometric, amperometric and capacitance methods.
  • representative methods include those that detect mass surface concentration, such as reflection-optical methods, including both internal and external reflection methods, angle, wavelength or phase resolved, for example ellipsometry and evanescent wave spectroscopy (EWS), the latter including surface plasmon resonance (SPR) spectroscopy, Brewster angle refractometry, critical angle refractometry, frustrated total reflection (FTR), evanescent wave ellipsometry, scattered total internal reflection (STIR), optical wave guide sensors, evanescent wave-based imaging such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, and the like.
  • SPR surface plasmon resonance
  • FTR frustrated total reflection
  • evanescent wave ellipsometry evanescent wave ellipsometry
  • scattered total internal reflection (STIR) scattered total internal reflection
  • optical wave guide sensors evanescent wave-based imaging such as critical angle resolved imaging, Brewster angle resolved imaging, SPR angle resolved imaging, and the like.
  • photometric methods based on
  • SPR surface plasmon resonance
  • the BIACORE instrument includes a light emitting diode (LED), a sensor chip including a glass plate covered with a thin gold film, an integrated fluid cartridge providing a liquid flow over the sensor chip, and a photo detector. Incoming light from the LED is totally internally reflected at the glass/gold interface and detected by the photo detector. At a certain angle of incidence ("the SPR angle"), a surface plasmon wave is set up in the gold layer which is detected as an intensity loss "or dip" in the reflected light.
  • the SPR angle depends on the refractive index of the medium close to the gold layer.
  • dextran is typically coupled to the gold surface, with the analyte-binding ligand being bound to the surface of the dextran layer.
  • the analyte of interest is injected in solution form onto the sensor surface through the fluid cartridge. Because the refractive index in the proximity of the gold films depends on (i) the refractive index of the solution (which is constant), and (ii) the amount of material bound to the surface, the binding interaction between the bound ligand and analyte can be monitored as a function of the change in SPR angle.
  • a lipid bilayer membrane is bound to a modified such dextran layer.
  • a typical output from the BIACORE instrument is a "sensorgram", which is a plot of response (measured in “resonance units” or “RU”) as a function of time.
  • An increase of 1,000 RU corresponds to an increase of mass on the sensor surface of about 1 ng/mm ⁇ .
  • This example describes the preparation of mixed micelles of detergent and lipid, deposition of the mixed micelles on a sensor chip surface, and elution of detergent to form a lipid bilayer on the surface.
  • a BIACORE 3000 instrument (Biacore AB, Uppsala, Sweden) was used. BIACORE instruments are based on surface plasmon resonance (SPR) detection at gold surfaces, and a micro-fluidic system is used for passing samples and running buffer through four individually detected flow cells (one by one or in series), with very high precision and with small sample volumes needed.
  • SPR surface plasmon resonance
  • HBS-OG was added to the dry lipid film and the mixtures were shaken every 10 min for at least 45 min at room temperature. In this way a number of combinations with 0.12 - 10 mM POPC and 5 - 50 mM octylglucoside were prepared. The preparations were checked for turbidity by the eye.
  • BIACORE 3000 for 8 min (during optimization, otherwise 1 min) at 5 ⁇ l/min.
  • the washing of the flow system was delayed for 2 min after injection.
  • Lipid deposition quantity data were collected 100 s after the end of injection.
  • the sensor surface was regenerated by two 1 min injections of 20 mM CHAPS or optimally 50 mM octylglucoside. The results are shown in Figs. 2 and 3.
  • the ratio of detergent to lipid in the mixed micelles seems to be a major factor in determining to which of the three groups above the sample belongs.
  • the ([octylglucoside]-CMC)/[POPC] ratio is related to the amount of POPC deposited after injection, it appears that the optimum is between 0.5 and 3 octylglucoside molecules per POPC in the mixed micelles, as illustrated in Fig 3 (only clear preparations are included in the diagram). Since this equation is dependent on the CMC of the detergent and the solubility of the current lipid mixture, which can vary considerably (Sch ⁇ rholz, T., Biophys. Chem. 58, 87-96 (1996)), the fine-tuning of composition has to be done in each instance.
  • Rhodopsin a G-protein coupled receptor; GPCR
  • GPCR G-protein coupled receptor
  • sensor chip Pioneer Chip LI (Biacore AB, Uppsala, Sweden) was first washed by two 1 min injections of 20 mM CHAPS (Sigma, U.S.A.).
  • the carboxymethyl- modified dextran polymer which is partially substituted with alkyl groups on Pioneer Chip LI, was activated with an injection of 0.2 M N-ethyl-N-dimethylamino- propylcarbodiimide (EDC) and 50 mM N-hydroxysuccinimide ( ⁇ HS) for 7 min.
  • EDC N-ethyl-N-dimethylamino- propylcarbodiimide
  • ⁇ HS N-hydroxysuccinimide
  • the amine coupling of rhodopsin above resulted in immobilization levels close to 4000 RU on the LI chip, which corresponds to 4 ng/mm 2 (Stenberg, E., et al., J. Colloid Interface Sci. 143, 513-526) or 0.1 pmol/mm 2 of rhodopsin. Since the amine coupling is not site specific but can involve any free amine group on the protein (mostly lysine residues), the rhodopsin is not uniformly oriented on the chip surface. However, most of the lysines in rhodopsin are positioned on the C-terminal side (cytosolic side) according to sequence based models of the structure (Hargrave, P. A., et al., Biophys. Struct. Mech. 9, 235-244 (1983)). Hence, the C-terminal side is probably dominating as the side of attachment, keeping the outside out orientation in favour.
  • step B Reconstitution of rhodopsin by lipid bilayer formation
  • the immobilized rhodopsin obtained in step A above was immediately reconstituted by a 2 min injection of mixed micelles, 3.3 mM POPC and 25 mM octylglucoside in HBS-N, prepared as in Example 1 above. Around 4500 RU of POPC were deposited. In a reference flow cell with unmodified Pioneer Chip LI -surface, about 5000 RU of lipid was simultaneously deposited. The always lower amount of lipids that bound in the rhodopsin flow cell is probably due to the space occupied by the immobilized protein, which indicates that lipid and protein coexist in each other's proximity.
  • the lipids deposited this way could be completely removed by two consecutive 1 min injections of 50 mM octylglucoside, and then resupplemented with a new 1 min injection of 3.3 mM POPC in 25 mM octylglucoside. This was indicated by the very stable responses achieved during 10 cycles of removal and supplementation.
  • rhodopsin In order to judge if the rhodopsin has a native and functional conformation after immobilization and supplementation with lipids as described in steps A and B above, its signaling capacity was assayed. When rhodopsin is activated by light, it transmits its signal by activating transducin. Activated transducin dissociates from the membrane under consumption of GTP (Heyse, S., et al., Biochemistry 37, 507-522 (1998)).
  • Fig. 4 shows the response signal for the POPC- rhodopsin reconstituted surface (curve 1) relative to the signal for the POPC reference surface. The flow was stopped at 459 s (Fig. 4). A few minutes after the flow-stop, at 902 s (Fig. 4), the level of bound transducin was stabilized in both the reference and rhodopsin flow cells.
  • the flow cells were then illuminated with an Ocean Optics 5 W halogen lamp via an optic fibre.
  • the activation of the receptor was recorded as a surface mass decrease caused by dissociation of the activated transducin from the membrane. Since this decrease was not observed in the reference flow cell or in absence of GTP it was concluded that it displays the signalling capacity of the receptor. After the signal decrease had levelled out significantly, the flow was resumed and the injection terminated (Fig. 4).
  • 9-cis-retinal Sigma, St. Louis, MO, U.S.A.
  • 10 ⁇ M in HBS-MDE and 0.7 % DMSO were injected over both flow cells for 13 min.
  • Membrane bound retinal was allowed to dissociate for about 40 min before next round of the assay. Following extended illumination of the rhodopsin and over night incubation, the signaling capacity of the receptor was exhausted. However, after injection of 10 ⁇ M 9-cw-retinal a recovery of the signalling capacity was detected. This showed that also the ligand binding capacity of the receptor was preserved when reconstituted by the method presented here. From these results it was concluded that rhodopsin in a micellar environment can be covalently attached to the Pioneer Chip LI chip surface by a commonly employed protocol for protein immobilization without irreversible loss of function. Since rhodopsin is sensitive to its lipid environment (Brown, M.F., Chem. Phys.

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Abstract

L'invention concerne un procédé de préparation de surface de substrat pour structure de membrane à film lipidique, qui comporte les étapes consistant à : a) mettre en contact une surface de substrat avec un liquide aqueux contenant des micelles mélangées à un détergent/lipide afin que des micelles adhèrent à la surface du substrat ; et b) mettre en contact la surface de substrat, sur laquelle ont adhéré des micelles mélangées à un détergent/lipide, avec un liquide aqueux sensiblement exempt de détergent pour éluer les molécules de détergent des micelles mélangées, et faire en sorte que les molécules lipidiques restantes s'assemblent en une structure de membrane à film lipidique sur la surface du substrat. L'invention concerne aussi le substrat pour structure de membrane à film lipidique préparé selon le procédé, l'utilisation d'un tel substrat pour étudier des interactions moléculaires, et une surface de substrat destinée à reconstituer la surface d'une protéine ou d'un polypeptide.
EP02708868A 2001-03-14 2002-03-14 Procede de preparation de membranes a film lipidique sur support et utilisation de celles-ci Withdrawn EP1373550A1 (fr)

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SE0100875A SE0100875D0 (sv) 2001-03-14 2001-03-14 Method of preparing supported lipid film membranes and use thereof
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US276729P 2001-03-16
PCT/SE2002/000481 WO2002072873A1 (fr) 2001-03-14 2002-03-14 Procede de preparation de membranes a film lipidique sur support et utilisation de celles-ci

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WO2003052420A2 (fr) * 2001-10-03 2003-06-26 Purdue Research Foundatio Dispositif et procedes utilisant une membrane biofonctionnalisee asymetrique
DE602004011198T2 (de) * 2003-03-25 2008-12-24 Biacore Ab Immobilisierungsverfahren und kit dafür
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JP4484553B2 (ja) * 2004-03-22 2010-06-16 幸宏 杉山 膜たんぱく質固定化基板および固定化方法
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JP2007003439A (ja) * 2005-06-27 2007-01-11 Hitachi Software Eng Co Ltd バイオセンサの製造方法
AT502713B1 (de) * 2005-10-19 2008-08-15 Univ Wien Bodenkultur Verfahren zur herstellung von lipid-membranen
GB0716264D0 (en) * 2007-08-21 2007-09-26 Isis Innovation Bilayers
EP2746772B1 (fr) 2012-12-20 2016-03-23 AIT Austrian Institute of Technology GmbH Particules enveloppées de membrane lipidique avec des protéines de membrane
EP2803372A1 (fr) 2013-05-16 2014-11-19 Universiteit Twente Procédé pour la préparation d'un objet portant une bicouche lipidique

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SE9403245D0 (sv) * 1994-09-26 1994-09-26 Pharmacia Biosensor Ab Improvements relating to bilayer lipid membranes
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