GB2446823A - Formulation of lipid bilayers - Google Patents

Abstract

A method for forming a lipid bilayer across an aperture, comprises: (a) providing a cell having a chamber adjacent to a septum comprising a membrane having an aperture capable of supporting a lipid bilayer; (b) depositing one or more lipids on an internal surface of the chamber; (c) introducing an aqueous solution into the chamber to cover the aperture and the internal surface and to form an interface between the solution and lipids; and (d) moving the interface past the aperture at least once to form a lipid bilayer across the aperture. Also claimed is the device for forming a lipid bilayer.

Description

FORMATION OF LIPID BILAYERS

The invention relates to the formation of lipid bilayers. In particular, the invention relates to the formation of a lipid bilayer across an aperture.

Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-chaimel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. In particular, lipid bilayers can used to detect the presence of membrane pores or channels or can be used in stochastic sensing in which the response of a membrane protein to a molecule or physical stimulus is used to perform sensing of that molecule or stimulus.

Lipid bilayers are commonly formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 356 1-3566), in which a lipid monolayer is carried on aqueous solution/air interface past either side of an aperture which is perpendicular to that interface. The lipid is normally added to the surface of an aqueous electrolyte solution by first dissolving it in an organic solvent and then allowing a drop of the solvent to evaporate on the surface of the aqueous solution on either side of the aperture. Once the organic solvent has evaporated, the solution/air interfaces on either side of the aperture are physically moved up and down past the aperture until a bilayer is formed.

The method of Montal & Mueller is popular because it is a cost-effective and relatively straightforward method of forming good quality lipid bilayers that are suitable for protein pore insertion.

Other common methods of bilayer formation include tip-dipping, painting bilayers and patch-clamping of liposome bilayers.

Tip-dipping bilayer formation entails touching the aperture surface (for example, a pipette tip) onto the surface of a test solution that is carrying a monolayer of lipid. Again, the lipid monolayer is first generated at the solution/air interface by allowing a drop of lipid dissolved in organic solvent to evaporate at the solution surface. The bilayer is then formed by the Langrnuir-Schaefer process and requires mechanical automation to move the aperture relative to the solution surface.

For painted bilayers, a drop of lipid dissolved in organic solvent is applied directly to the aperture, which is submerged in an aqueous test solution. The lipid solution is spread thinly over the aperture using a paintbrush or an equivalent.

Thinning of the solvent results in formation of a lipid bilayer. However, complete removal of the solvent from the bilayer is difficult and consequently the bilayer formed by this method is less stable and more prone to noise during electrochemjcal measurement.

Patch-clamping is commonly used in the study of biological cell membranes.

The cell membrane is clamped to the end of a pipette by Suction and a patch of the membrane becomes attached over the aperture. The method has been adapted for producing lipid bilayers by clamping liposomes which then burst to leave a lipid bilayer sealing over the aperture of the pipette. The method requires stable, giant and unilamellar liposomes and the fabrication of small apertures in materials having a glass surface.

These common methods of forming lipid bilayers are complicated and time consuming. For instance, it is normally necessary to wait for the evaporation of an organic solvent in which the lipids are dissolved before a bilayer can be formed. There is therefore a need for simple and rapid methods of forming a lipid bilayer that do not involve the use of organic solvents.

In one aspect, the present invention provides a method for forming a lipid bilayer across an aperture, comprising: (a) providing a cell having a chamber adjacent to a septum comprising a membrane having an aperture capable of supporting a lipid bilayer; (b) depositing one or more lipids on an internal surface of the chamber; (c) introducing an aqueous solution into the chamber to cover the aperture and the internal surface and to form an interface between the solution and lipids; and (d) moving the interface past the aperture at least once to form a lipid bilayer across the aperture.

In another aspect, the invention provides a device for forming a lipid bilayer comprising, (a) a cell having a chamber adjacent to a septum comprising a membrane having an aperture capable of supporting a lipid bilayer; and (b) one or more lipids deposited on an internal surface of the chamber,

F

wherein the cell comprises an inlet for introducing an aqueous solution into the chamber having lipid deposited therein.

The inventors have shown that a lipid bilayer will form across an aperture following the deposition of lipids on a surface adjacent to the aperture. They have shown that an aqueous solution can be used to collect the lipids from the surface and form a lipid/solution interface. The lipid bilayer forms across an aperture as the interface passes the aperture.

Advatageously, the lipids can be dried. The inventors have also shown that a lipid bilayer will form across an aperture following the rehydration of dried lipids.

They have shown that an aqueous solution can be used to rehydrate the lipids and form a lipid/solution interface. The lipid bilayer forms across an aperture as the interface passes the aperture.

The invention has several advantages. The invention allows the formation of a lipid bilayer in the absence of large amounts of organic solvent. This means that a lipid bilayer can be formed rapidly because it is not necessary to wait for evaporation of the organic solvent before the lipid bilayer can be formed.

In addition, this means that the cell in the device of the invention can be made from materials that may be sensitive to organic solvents. For instance, organic-based adhesives can be used to construct the cell and screen-printed conductive silver/silver chloride paste can be used to construct electrodes within the cell. This means that the device can be cheaply manufactured in a straightforward manner. In addition, the use of organic solvent-sensitive polymers to construct the membrane comprising the aperture facilitates manufacture of the device.

As lipid bilayers are preferably formed from dried lipid, this allows the lipid to be stably stored in the cell until it is needed to form a lipid bilayer. This also avoids the need for wet storage of lipid in the device prior to use. Dry storage of lipids means that the device has a long shelf life.

The invention generally concerns the formation of a lipid bilayer across an aperture. A lipid bilayer is formed from two opposing layers of lipids. The two layers of lipids are arranged such that their hydrophobic tail groups face towards each other to form a hydrophobic interior. The hydrophilic head groups of the lipids face outwards towards the aqueous environment on each side of the bilayer. The bilayer may be present in a number of lipid phases including, but not limited to, the liquid disordered phase (fluid lamellar), liquid ordered phase, solid ordered phase (lamellar gel phase, interdigitated gel phase) and planar bilayer crystals (lamellar sub-gel phase, laniellar crystalline phase).

The lipid bilayer can be formed from one or more lipids. The lipid bilayer can also contain additives that affect the properties of the bilayer. In many applications, the lipid bilayer has one or more membrane proteins inserted therein.

Certain lipids, additives and proteins that can be used in accordance with the invention are discussed in more detail below.

The lipid bilayer is formed inside a cell. In general, any cell can be used.

The cell may be any shape or size. The cell may he a conventional electrophysiology cell or a specially-constructed cell, such as a biosensor chip.

The cell comprises an internal chamber. The chamber may be any size and shape. The volume of the chamber is typically 0. lizl to lOmi. The chamber is adjacent to a septum. In a preferred embodiment, the cell comprises a septum which divides the cavity into two chambers. The two chambers may have equal volumes or different volumes.

The septum comprises a membrane. The membrane can be made from any material including, but not limited to, a polymer, glass and a metal. The membrane is preferably made from a material that forms a barrier to the flow of ions from the chamber. Suitable materials include, but are not limited to, polycarbonate (PC), polytetrafluoroethylene (PTFE), polyethylene, polypropylene, nylon and polyethylene naphthalate (PEN), polyvinylchlorjcje (PVC), polyacrylonitri le (PAN), polyether sulphone (PES), polyimide, polystyrene, polyvinyl fluoride (PVF), polyethylene telephthalate (PET), aluminized PET, nitrocellulose, polyetheretherketone (PEEK) and fluoroethylkene polymer (FEP). The membrane is preferably made from polycarbonate or PTFE.

The membrane is sufficiently thin to facilitate formation of the lipid bilayer across an aperture as described below. Typically the thickness will be in the range of I Onm to 1mm. The membrane is preferably 0.1 zm to 25m thick.

The membrane is preferably pre-treated to make the lipids and the aperture more compatible such that the lipid bilayer forms more easily that it would in the absence of pre-treatment. The membrane is preferably pre-treated to increase its affinity to lipids. The inventors have shown that pre-treatment of the membrane to increase its affinity to lipids allows the lipid bilayer to form more easily. In particular, it allows the formation of a lipid bilayer across the aperture following a single pass of the lipid/solution interface. The removal of the need to move the lipid/solution interface back and forth past the aperture means that the method of the invention is simplified. It also means that there is no need for fluidics control in the device of the invention. Hence, the cost and size of the device of the invention are reduced. The inventors have also shown that pre-treatment of the membrane to increase its affinity to lipids results in the formation of a lipid bilayer with increased stability. This means that the method of the invention can be used to form stable lipid bilayers. It also means that the device of the invention can he used in situations where the lipid bilayer is likely to encounter mechanical or other forces. For instance, the device of the invention can be used as a hand-held device.

Any treatment that modifies the surface of the membrane surrounding the aperture to increase its affinity to lipids may be used. The membrane is typically pre-treated with long chain organic molecules in an organic solvent. Suitable long chain organic molecules include, but are not limited to, n-decane, hexadecane, hexadecance mixed with one or more of the lipids discussed below, squalene, fluoroinated oils (suitable for use with fluorinated lipids), alkyl-silane (suitable for use with a glass membrane) and alkyl-thiols (suitable for use with a metallic membrane). Suitable solvents include, but are not limited to, pentane, hexane, heptane, octane, decane, iso-ecoisane and toluene. The membrane is typically pre-treated with from 0.1 % (v/v) to 50% (v/v), such as 0.3%, 1 % or 3% (v/v), hexadecane in pentane. The volume of hexadecane in pentane used is typically from 0.ljl to lOl. The hexadecane can be mixed with one or more lipids. For instance, the hexadecane can be mixed with any of the lipids discussed below. The hexadecane is preferably mixed with diphantytanoylsnglycero3 -phosphocoline (DPhPC). Preferably, the aperture is treated with 2d of 1% (vlv) hexadecane and I 0mg/mi lipid, such as DPhPC, in pentane.

The septum preferably further comprises a support sheet on at least one side of the membrane. The septum preferably comprises a support sheet on both sides of the membrane. The support sheet may be of any material. Suitable materials include, but are not limited to, Deirin (polyoxymethylene or acetal homopolymer), Mylar (biaxially-oriented polyethylene terephthalate (boP ET) polyester film), polycarbonate (PC), polyvinylchlorjde (PVC), polyacrylonitrile (PAN), polyether sulphone (PES), polysuiphone, polyimide, polystyrene, polyethylene, polyvinyifluoride (PVF), polyethylene telephthalate (PET), polytetrafluoroethylene (PTFE), pol yetheretherketone (PEEK) and fluoroethylkene polymer (FEP).

The membrane has an aperture which is capable of supporting a lipid bilayer.

The septum typically has one aperture but can have more than one aperture. A lipid bilayer will form across each of the apertures in the membrane.

If the membrane is made from a material that forms a barrier to the flow of ions, the aperture allows the movement of ions between from the chamber. The aperture may be any size and shape which is capable of supporting lipid bilayer.

The aperture preferably has a diameter in at least one dimension which is 2Otm or less. The inventors have shown that this preferred size of aperture results in the formation of a lipid bilayer with increased stability. This means that the method of the invention can form stable lipid bilayers and that the device of the invention can be used in situations where the lipid bilayer is likely to encounter mechanical or other forces. For instance, it can be used as a hand-held device. The preferred size of aperture also allows the lipid bilayer to form more easily. In particular, it allows the formation of a lipid bilayer across the aperture following a single pass of the lipid/solution interface and removes the need to move the lipid/solution interface back and forth past the aperture.

The aperture may be created using any method. Suitable methods include, but are not limited to, spark generation and laser drilling.

Preferred combinations of membrane and aperture for use in accordance with the invention are shown in the Table below.

Septum Aperture 6jtm thick biaxial polycarbonate 25gm diameter spark-generated hole 6m thick biaxial polycarbonate 2Ojm diameter laser-drilled tapered hole 6m thick biaxial polycarbonate I Ozm diameter laser-drilled tapered hole 5m thick PTFE I Ojm diameter spark-generated holes 5m thick PTFE 1 O4um diameter laser-drilled tapered hole 5m thick PTFE 5zm diameter laser-drilled tapered hole I Ozm thick HD polyethylene 15zm diameter spark-generated hole b 4j.tm thick Polypropylene i 5zm diameter spark-generated hole 7 25iim thick Nylon (6,6) 2Ozm diameter spark-generated hole I.3tm thick PEN 3Om diameter spark-generated hole I 4zm thick conductive 3Om diameter spark-generated hole polycarbonate One or more lipids are deposited on an internal surface of the chamber. The lipids can be deposited on one or more of any of the internal surfaces of the chamber. If the cell has two chambers, one or more lipids are deposited on an internal surface of one or both chambers. The lipids can be deposited on one or more of any of the internal surfaces of one or both chambers. The lipids can be deposited on one or both sides of the septum and on the membrane and/or the support sheet. The lipids are deposited in such a manner that the aqueous solution covers the lipids and the apertures as discussed in more detail below. The lipid can be deposited on the septum and/or one or more internal walls of the chamber but are preferably deposited on the septum. The lipids can be deposited on one or both sides of the septum and on the membrane and/or the support sheet. The lipids are deposited in such a manner that the aqueous solution covers the lipids and the apertures as discussed in more detail below.

Any method may be used to deposit the lipids on an internal surface of the chamber. Suitable methods include, but are not limited to, evaporation or sublimation of a carrier solvent, spontaneous deposition of liposomes or vesicles from a solution and direct transfer of the dry lipid from another surface. Cells having lipids deposited on an internal surface may be fabricated using methods including, but not limited to, drop coating, various printing techniques, spin-coating, painting, dip coating and aerosol application.

The lipids are preferably dried. Even when dried to a solid state, the lipids will typically contain trace amounts of residual solvent. Dried lipids are preferably lipids that comprise less than 5Owt% solvent, such as less than 4Owt%, less than 3Owt%, less than 2Owt%, less than l5wt%, less than lOwt% or less than 5wt% solvent.

Any lipids that form a lipid bilayer may be deposited. The lipids deposited in the cell are chosen such that a lipid bilayer having the required properties, such surface charge, ability to support membrane proteins, packing density or mechanical properties, is formed. The lipids can comprise one or more different lipids. For instance, the lipids can contain up to 100 lipids. The lipids preferably contain I to 10 lipids. The lipids may comprise naturally-occurring lipids and/or artificial lipids.

The lipids typically comprise a head group, an interfacial moiety and two hydrophobic tail groups which may be the same or different. Suitable head groups include, but are not limited to, neutral head groups, such as diacyiglycerides (DG) and ceramides (CM); zwitterionjc head groups, such as phosphatidylcholine (PC), phosphatidylethariolamine (PE) and sphingomyelin (SM); negatively charged head groups, such as phosphatidyiglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (P1), phosphatic acid (PA) and cardiolipin (CA); and positively charged headgroups, such as trimethylammoniumpropane (TAP). Suitable interfacial moieties include, but are not limited to, naturally-occurring interfacial moieties, such as glycerol-based or ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains, such as lauric acid (n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmitic acid (n-Hexadecanojc acid), stearic acid (n-Octadecanoic) and arachidic (n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid (cis-9-Octadecanoic); and branched hydrocarbon chains, such as phytanoyl. The length of the chain and the position and number of the double bonds in the unsaturated hydrocarbon chains can vary. The length of the chains and the position and number of the branches, such as methyl groups, in the branched hydrocarbon chains can vary. The hydrophobic tail groups can be linked to the interfacial moiety as an ether or an ester.

The lipids can also be chemically-modified. The head group or the tail group of the lipids may be chemically-modified. Suitable lipids whose head groups have been chemically-modified include, but are not limited to, PEG-modified lipids, such as I,2-Diacyl-sn-Glycero-3 -PhosphoethanolamineN -[Methoxy(Polyethylene glycol)-2000]; functionionalised PEG Lipids, such as I,2-Distearoyl-sn-Glycero..3 PhosphoethanolamineN[Biotinyl(polyethylene Glycol)2000]; and lipids modified for conjugation, such as l,2-DioleoylsnGlycero3.phosphoethafloIamjfleN..

(succinyl) and I,2-Dipalmitoyl-sn-Gl ycero-3 -Phosphoethanolamine-N.(Biotinyl).

Suitable lipids whose tail groups have been chemically-modified include, but are not limited to, polymerisable lipids, such as I,2-bis( 10,1 2-tricosadiynoyl).sn-Qlycero3 -Phosphocholine; fluorinated lipids, such as l-Palmitoyl-2-(16-Fluoropalmitoyl)sn Glycero-3-Phosphocholine; deuterated lipids, such as l,2-Dipalmitoyl-D62-sn.

Glycero-3-Phosphocholjne and ether linked lipids, such as l,2-Di-O-phytanyl-sn.

Glycero-3 -Phosphocholine.

The lipids typically comprise one or more additives that will affect the properties of the lipid bilayer. Suitable additives include, but are not limited to, fatty acids, such as palmitic acid, myristic acid and oleic acid; fatty alcohols, such as palmitic alcohol, myristic alcohol and oleic alcohol; sterols, such as cholesterol, ergosterol, lanosterol, sitosterol and stigrnasterol; lysophospholipids, such as 1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine* and ceramides. The lipid preferably comprises cholesterol and/or ergosterol when membrane proteins are to be inserted into the lipid bilayer.

The lipid bilayer is formed by introducing an aqueous solution into the chamber. The aqueous solution covers both the internal surface on which the lipids are deposited and the aperture. The chamber may be completely filled with the aqueous solution or may be partially filled with the aqueous solution, as long as the both the lipids and the aperture are covered with the aqueous solution. If the cell has two chambers, one chamber may be completely filled, while the other is only partially filled.

The aqueous solution may cover the lipids and the aperture in any order but preferably covers the lipids before the aperture. The inventors have shown that covering the lipids before the aperture allows the lipid bilayer to form more easily.

In particular, it allows the formation of a lipid bilayer across the aperture following a single pass of the lipid/solution interface. The removal of the need to move the lipid/solution interface back and forth past the aperture means that the method of the invention is simplified. It also means that there is no need for fluidics control in the device of the invention, thereby reducing its cost and size.

The design of the chamber and the position of the lipids may be chosen to determine the order in which the aqueous solution covers the lipids and aperture. For instance, if the lipids are to be covered first, a chamber is provided in which the lipids are positioned along the flow path between the point at which the aqueous solution is introduced to the chamber and the aperture.

Any aqueous solution that collects the lipids from the internal surface and allows the formation of a lipid bilayer may be used. The aqueous solution is typically a physiologically acceptable solution. The physiologically acceptable solution is typically buffered to a pH of 3 to 9. The pH of the solution will be dependent on the lipids used and the final application of the lipid bilayer. Suitable buffers include, but are not limited, to phosphate buffered saline (PBS), N-2-HydroxyethyIpjperazineN2Ethanesulfofljc Acid (FIEPES) buffered saline, Piperazine-I,4-Bis-2-Ethanesujfonic Acid (PIPES) buffered saline, 3-(n-Morpholino)Propanesulfonic Acid (MOPS) buffered saline and (TRIS) buffered saline. By way of example, in one implementation, the aqueous solution may be 10mM PBS containing I.OM sodium chloride (NaCI) and having a pH of 6.9.

The introduction of the aqueous solution collects the lipids from the internal surface. The immiscibility of the rehydrated lipids and the aqueous solution allows the formation of an interface between the lipids and the solution. The interface can be any shape and size. The interface typically separates a layer of lipids from the aqueous solution. The layer of lipids preferably forms on the top of the solution.

The layer of lipid typically separates the solution from any air in the chamber(s).

The lipid bilayer is formed as the interface moves past the aperture. The interface moves past the aperture in such a way that the layer of lipids contacts the membrane material surrounding the aperture and a lipid bilayer is formed. The interface can be at any angle relative to the membrane as it moves past the aperture.

The interface is preferably perpendicular to the membrane as it moves past the aperture.

The interface may move past the aperture as many times as is necessary to form the lipid bilayer. The interface moves past the aperture at least once. The interface can move past the aperture more than once, such as twice, three times or more. The interface can move past the aperture on one side or on both sides of the membrane.

If the aqueous solution covers the internal surface on which the lipids are deposited before the aperture, the lipid bilayer may form as the interface moves past the aperture as the chamber fills. Hence, if the lipid bilayer can be formed by a I0 single pass of the interface past the aperture, the step of moving the interface past the aperture may be performed by the filling of the chamber.

In other embodiments, it will be necessary to move the interface back and forth past the aperture. For instance, if the aqueous solution covers the aperture before the lipids or covers the aperture and lipids simultaneously, it may be necessary to move the interface back and forth past the aperture.

In a preferred embodiment, the cell has two chambers and one of the chambers contains a gel. The chamber is typically filled with the gel such that the gel contacts the membrane. The presence of the gel contacting the membrane facilitates the formation of the!ipid hilayer by physical!y supporting the bilayer. The presence of the gel allows the lipid bilayer to form more easily. In particular, it allows the formation of a lipid bilayer across the aperture following a single pass of the lipid/solution interface and removes the need to move the lipid/solution interface back and forth past the aperture. It also means that there is no need for fluidics control in the device of the invention, thereby reducing its cost and size. The presence of the gel also results in the formation of a lipid bilayer with increased stability. This means that the method of the invention can be used to form stable lipid bilayers. It also means that the device of the invention can be used in situations where the lipid bilayer is likely to encounter mechanical or other forces. For instance, it can be used as a hand-held device.

In another embodiment, there can remain a gap between the gel and the membrane. The presence of the gap means that a wider variety of materials can be used to make the gel, including ionically non-conductive materials.

The gel is preferably a hydrogel. The gel is typically ionically conductive.

Suitable ionically conductive gels include, but are not limited to, agarose, polyacrylamide gel, Gellan gel and carbomer gel. However, if there is a gap present between the gel and the aperture, the gel can be ionically non-conductive.

The invention preferably also involves inserting membrane proteins into the lipid bilayer once it has been formed. The membrane proteins are deposited within the chamber and spontaneously insert into the lipid bilayer following the introduction of the aqueous solution. The inventors have shown that membrane proteins will spontaneously insert into the lipid bilayer following their removal from an internal surface of the chamber by the aqueous solution. This avoids the need to actively insert the membrane proteins into the lipid bilayer by introducing the proteins into the solution surrounding the bilayer or physically carrying the protein through the solution to the bilayer. Again, this simplifies the method of the invention as well as removes the need for wet storage of the proteins and the need for automation within a device of the invention.

In one embodiment, the gel described above comprises one or more membrane proteins. The membrane proteins can be deposited on the surface of the gel and/or can be present within the body of gel. Once the lipid bilayer has formed, the membrane proteins move from the gel and spontaneously insert themselves into the lipid bilayer. The gel can comprise one or more different membrane proteins.

In another embodiment, one or more membrane proteins are deposited on an internal surface of the chamber. The aqueous solution collects the membrane proteins from the surface and allows them to insert into the lipid bilayer. The membrane proteins may be deposited anywhere within the cell such that, once they have been collected from the surface, they can diffuse to and spontaneously insert into the lipid bilayer. The membrane proteins can be deposited on the same or different internal surface as the lipids. The lipids and the membrane proteins may be mixed together. The membrane proteins can be deposited on the septum and/or one or more internal walls of the chamber, but are preferably deposited on the septum.

They may be deposited on one or both sides of the septum and on the membrane or the support sheet. The lipids, the aperture and the membrane proteins may be covered by

the aqueous solution in any order, although as already discussed the aqueous solution preferably covers the lipids first. The design of the cell and the position of the membrane proteins may be chosen to determine the order in which the aqueous solution covers the lipids, the aperture and the membrane proteins.

Any method may be used to deposit the membrane proteins on an internal surface of the cell. Suitable methods include, but are not limited to, drop coating, various printing techniques, spin-coating, painting, dip coating and aerosol application.

The membrane proteins are preferably dried. Even when dried to a solid state, the membrane proteins will typically contain trace amounts of residual solvent.

Dried membrane proteins are preferably membrane proteins that comprise less than b 2Owt% solvent, such as less than l5wt% , less than lOwt% or less than 5wt% solvent.

In a further embodiment, the gel comprises one or more membrane proteins and one or more membrane proteins are deposited on an internal surface of one or both chambers.

Any membrane proteins that insert into a lipid bilayer may be deposited. The membrane proteins may be naturally-occurring proteins andlor artificial proteins.

Suitable membrane proteins include, but are not limited to, (3-barrel membrane proteins, such as non-constitutive toxins, porins and relatives and autotransporters; membrane channels, such as ion channels and aquaporins; bacterial rhodopsins; 0-protein coupled receptors; and antibodies. Examples of non-constitutive toxins include hemolysin and leukocidin, such as Staphylococcal leukocidin. Examples of porins include maltoporin, 0mpG, OmpA and OmpF. Examples of autotransporters include the NaIP and Hia transporters. Examples of ion channels include the NMDA receptor, the potassium channel from Streptomyces lividans (KcsA), the bacterial mechanosensitive membrane channel of large conductance (MscL), the bacterial mechanosensjtive membrane channel of small conductance (MscS) and gramicidin.

Examples of G-protein coupled receptors include the metabotropic glutamate receptor. The membrane protein can also be the anthrax protective antigen.

The membrane proteins preferably comprise a-hemolysin or a variant thereof. The a-hemolysin pore is formed of seven identical subunits (heptameric).

The polynucleotide sequence that encodes one subunit of a-hemolysin is shown in SEQ ID NO: 1. The full-length amino acid sequence of one subunit of a-hemolysin is shown in SEQ ID NO: 2. The first 26 amino acids of SEQ ID NO: 2 correspond to the signal peptide. The amino acid sequence of one mature subunit of a-hemolysin without the signal peptide is shown in SEQ ID NO: 3. SEQ ID NO: 3 has a methionine residue at position I instead of the 26 amino acid signal peptide that is present in SEQ ID NO: 2.

A variant is a heptameric pore in which one or more of the seven subunits has an amino acid sequence which varies from that of SEQ ID NO: 2 or 3 and which retains pore activity. 1,2, 3, 4, 5,6 or 7 of the subunits in a variant a-hemolysin may have an amino acid sequence that varies from that of SEQ ID NO: 2 or 3. The seven subunits within a variant pore are typically identical but may be different.

The variant may be a naturally-occurring variant which is expressed by an organism, for instance by a Staphylococcus bacterium. Variants also include non-naturally occurring variants produced by recombinant technology. Over the entire length of the amino acid sequence of SEQ ID NO: 2 or 3, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the subunit polypeptide is at least 80%, at least 90%, at least 95%, at least 98%, at least 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 2 or 3 over the entire sequence.

Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 2 or 3, for example a single amino acid substitution may be made or two or more substitutions may be made. Conservative substitutions may be made, for example, according to the following table. Amino acids in the same block in the second column and preferably in the same line in the third column may be NON-AROMATIC Non-polar G A P

ILV

Polar -uncharged C S T M

NQ

Polar -charged D E

HKR

AROMATIC H F W Y

Non-conservative substitutions may also be made at one or more positions within SEQ ID NO: 2 or 3, wherein the substituted residue is replaced with an amino acid of markedly different chemical characteristics and/or physical size. One example of a non-conservative substitution that may be made is the replacement of the lysine at position 34 in SEQ ID NO: 2 and position 9 in SEQ ID NO: 3 with cysteine (i.e. K34C or K9C). Another example of a non-conservative substitution that may be made is the replacement of the asparagine residue at position 43 of SEQ ID NO: 2 or position 18 of SEQ ID NO: 3 with cysteine (i.e. N43C or NI 7C). The inclusion of these cysteine residues in SEQ ID NO: 2 or 3 provides thiol attachment points at the relevant positions. Similar changes could be made at all other positions, and at multiple positions on the same subunit.

One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 or 3 may alternatively or additionally be deleted. Up to 50% of the residues residues may be deleted, either as a contiguous region or multiple smaller regions distributed throughout the length of the amino acid chain.

Variants can include subunits made of fragments of SEQ ID NO: 2 or 3.

Such fragments retain their ability to insert into the lipid bilayer. Fragments can be at least 100, such as 150, 200 or 250, amino acids in length. Such fragments may be used to produce chimeric pores. A fragment preferably comprises the i3-barrel domain of SEQ ID NO: 2 or 3.

Variants include chimenc proteins comprising fragments or portions of SEQ ID NO: 2 or 3. Chimeric proteins are formed from subunits each comprising fragments or portions of SEQ ID NO: 2 or 3. The j3-barrel part of chimeric proteins are typically formed by the fragments or portions of SEQ ID NO: 2 or 3.

One or more amino acid residues may alternatively or additionally be inserted into, or at one or other or both ends of, the amino acid sequence SEQ ID NO: 2 or 3.

Insertion of one, two or more additional amino acids to the C terminal end of the peptide sequence is less likely to perturb the structure andlor function of the protein, and these additions could be substantial, but preferably peptide sequences of up to 10, 20, 50, 100 or 500 amino acids or more can be used. Additions at the N terminal end of the monomer could also be substantial, with one, two or more additional residues added, but more preferably 10, 20, 50, 500 or more residues being added.

Additional sequences can also be added to the protein in the trans-membrane region, between amino acid residues 119 and 139 of SEQ ID NO: 3. More precisely, additional sequences can be added between residues 127 and 130 of SEQ ID NO: 3, following removal of residues 128 and 129. Additions can be made at the equivalent positions in SEQ ID NO: 2. A carrier protein may be fused to an amino acid sequence according to the invention.

Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et a! (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be

F

used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F eta! (1990) J Mol Biol 2 15:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

The membrane proteins can be labelled with a revealing label. The revealing label can be any suitable label which allows the proteins to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. 125j, 35S, enzvmes antibodies. polynucleotides and linkers such as hiotin The membrane proteins may be isolated from an organism, such as Staphylococcus aureus, or made synthetically or by recombinant means. For example, the protein may be synthesized by in vitro translation transcription. The amino acid sequence of the proteins may be modified to include non-naturally occurring amino acids or to increase the stability of the proteins. When the proteins are produced by synthetic means, such amino acids may be introduced during production. The proteins may also be modified following either synthetic or recombinant production.

The proteins may also be produced using D-amino acids. In such cases the amino acids will be linked in reverse sequence in the C to N orientation. This is conventional in the art for producing such proteins.

A number of side chain modifications are known in the art and may be made to the side chains of the membrane proteins. Such modifications include, for example, modifications of amino acids by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4, amidination with methylacetimidate or acylation with acetic anhydride.

Recombinant membrane proteins can be produced using standard methods known in the art. Nucleic acid sequences encoding a protein can be isolated and replicated using standard methods in the art. Nucleic acid sequences encoding a protein can be expressed in a bacterial host cell using standard techniques in the art.

The protein can be introduced into a cell by in Situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide.

The lipid bilayer may be used for a variety of purposes. The lipid bilayer may be used for fl Vitro investigation of membrane proteins by single-channel recording. The lipid bilayer may be used as a biosensor to detect the presence of a range of substances. The lipid bilayer may be used to detect the presence or absence of membrane pores or channels in a sample. The presence of the pore or channel may be detected as a change in the current flow across the lipid bilayer as the pore or channel inserts into the lipid bilayer. The lipid bilayer preferably contains membrane protein and is used to detect the presence or absence of a molecule or stimulus using stochastic sensing. The lipid bilayer may be used for a range of other purposes, such In c thijivino th nrnnprfjp of mnlpi-ii1pc known to hp nrpQpnf (p flNA nh1Pnrn or r -- -----r--------* -drug screening), or separating components for a reaction.

To allow further understanding, embodiments of the invention will now be described by way of a non-limiting example with reference to the drawings, in which: Fig. I is a view of an example of a device of the invention.

Fig. 2 is a schematic diagram of an electrical circuit that can be used with the device of the invention.

Fig. 3 is a graph of the current response to a 2OmV 50Hz alternating current (a.c.) waveform in the absence of the high resistance electrical sealing of the aperture by a bilayer.

Fig. 4 is a graph of the characteristic square wave capacitive current in response to a 2OmV amplitude triangular waveform at 50Hz indicative of bilayer formation across the aperture.

Fig. 5 is a graph of the stepwise increase of 6OpA direct current (d.c.) as a-hemolysin pores automatically insert into the bilayer formed by the Montal and Mueller method.

Fig. 6 is a graph of the characteristic interruptions in the current caused by single molecules of'y-cyclodextnn transiently binding to the a-hemolysin pores.

Fig. 7 is a graph of the current response to an applied potential before, during and following spontaneous bilayer formation and pore insertion in accordance with the invention in a standard two-chamber research cell.

Fig. 8 shows an expanded view of the current (1 second full scale) during the final minute of recording shown in Fig. 5.

Fig. 9 is a graph of the current recorded over the duration of a single test using a specially constructed cell.

Fig. 10 is a graph of the characteristic square wave indicative of bilayer formation in accordance with the invention in a specially constructed cell. Fig. 8 shows an expanded version of the portion of Fig. 7 immediately before arrow I. Fig. 11 is a graph of the current response to a 2OmV 50Hz a.c. waveform in the absence of the high resistance electrical sealing of the aperture by the bilayer in the specially constructed cell. Fig. 9 shows an expanded version of the portion of Fig. 7 between arrows 2 and 3.

r; 1) ,c, . ,-ç ,, _...1 flfl,. A e I. S . AU 5.4 S FtS J U I I S/s S/t4_IS/..5 SSIs 5' bAA S/ASS S 5/ ta5J *.A, S 5/5455/5) UI 5 J5JI automatically insert into the bilayer in the specially constructed cell. Fig. 10 shows an expanded version of the portion of Fig. 7 after arrow 4.

Fig. 13 is a graph of the characteristic interruptions in the current caused by single molecules of"y-cyclodextrin transiently binding to the a-hemolysin pores in the specially constructed cell.

In all of the graphs, the x-axis shows time in ms. The top portion of the y-axis shows the current in pA. The bottom portion of the y-axis shows potential in mV.

A device 30 in accordance with the invention is illustrated in Fig. 1. The device 30 includes an electrophysiology cell I which is of a conventional type and construction for the performance of stochastic sensing using a membrane protein inserted in a lipid bilayer.

The electrophysiology cell I comprises two chambers body portions 2 having constructions which are mirror images of each other. The chamber body portions 2 may be made from Delrin (polyoxymethylene or acetal homopolymer). The chamber body portions 2 each define a chamber portion 3 having an opening in the upper surface 4 of the respective chamber body portion 2. The chamber portions 3 each have a volume of a few millilitres, for example 1.5 ml. The chamber portions 3 have no wall on a side surface 5 of the respective chamber body portion 2. To form a chamber body, the two chamber body portions 2 are assembled together with their side surfaces 5 facing one another so that the respective chamber portions 3 are aligned and together form a chamber. The chamber body portions 2 may be attached by any suitable means, typically a clamp or an adhesive.

The electrophysiology cell 1 further comprises a membrane 6 made of polycarbonate or any other suitable polymer. Each face of the membrane 6 may be pre-treated in a conventional manner, for example with 10% (VIV) hexadecane in pentane. The membrane 6 is positioned between the facing side surfaces 5 of the two chamber body portions 2, for example by adhering both chamber body portions 2 to the membrane 6. Accordingly, the membrane 6 forms a wall which divides the chamber formed by the two chamber portions 3.

The membrane 6 has an aperture 7 which is aligned with the chamber portions 3 when the electrophysiology cell is assembled. The membrane 6 is sufficiently thin to facilitate formation of a lipid hilayer; for example being 25pm thick. The aperture 7 may in general be of any shape or size which is capable of supporting the lipid bilayer, but preferably has a diameter in one dimension of 20j.tm or less. The cell comprises inlets for introducing an aqueous solution into each chamber portion 3, namely the openings in the upper surface 4 of each chamber body portion 2.

The device 30 further comprises lipids 8 deposited in each chamber portion 3 of each one of the chamber body portions 2. The shape of the patch of lipids 8 deposited in each chamber portion 3 may vary.

The electrophysiology cell I may be used to form a lipid bilayer in accordance with the method of the invention. For example, an aqueous solution may be introduced into both chamber portions 3 simultaneously via openings in the upper surface 4 of each chamber body portion 2. The aqueous solution will cover the lipids 8 deposited in each chamber portion 3 and a lipid/solution interface will form with a layer of lipid resting on top of the solution. As more aqueous solution is introduced, the interface will rise within both chamber portions 3 and move past the aperture 7 on both sides of the membrane 6 thereby forming a lipid bilayer across the aperture 7. In this example, the lipid 8 is covered by the aqueous solution before the aperture 7 is covered.

The electrophysiology cell 1 can further includes respective electrodes (not shown in Fig. 1) provided in each chamber portion 3 of each one of the chamber body portions 2. The electrodes may be Ag/AgCl electrodes. The electrodes may form part of an electrical circuit 20 which is capable of measuring an electrical signal across the lipid bilayer. A suitable electrical circuit 20 is illustrated schematically in b Fig. 2 and is of a conventional type for performing stochastic sensing by detecting the current flowing across the lipid bilayer.

The electrodes 9 are connected to an amplifier 21 such as a patch-clamp amplifier (eg an Axopatch 200B supplied by Axon Instruments) which amplifies the current signal output from the electrodes 9.

The current signal output by the amplifier 21 is supplied through a low-pass filter 22, such as a Bessel filter (eg with characteristics 80dB/decade with a corner frequency of 2kHz).

The current signal output by the low-pass filter 22 is supplied to an AID convertor 23, such as a Digitata 1320 A/F) converter supplied by Axon Instruments.

The A/D convertor 23 might typically operate with a sampling frequency of 5kHz.

The A/D convertor 23 converts the current signal into a digital signal which is then supplied to a computer 24 for analysis. The computer 24 may be a conventional personal computer running an appropriate program to store the current signal and display it on a display device.

As an alternative, the invention may be applied to a device which is the cell of the sensor system described in a co-pending application being filed simultaneously with this application [J A Kemp & Co Ref: N.99663; Oxford Nanolabs Ref: ONL IP 001] which is incorporated herein by reference. All the teachings of that application may be applied equally to the present invention.

For comparative purposes, a bilayer was first formed using the common Montal and Mueller method. Bilayer formation was performed using a standard two-chamber research cell. The research cell is typical of those used in laboratory bilayer tests and comprises two Deirin (acetal homopolymer) blocks, each machined to create an open-sided 700ul chamber with appropriate access portals. The blocks are clamped together on either side of a polymer film which thereby separates the two chambers. The only electrical connection between the two chambers is by ionic conduction of the electrolyte solution through a small aperture created in the polymer film.

In order to facilitate bilayer formation, it is first necessary to apply a chemical surface treatment (commonly called the "pre-treatment") to either side of the aperture.

2-5ul of 10% hexadecane dissolved in pentane was applied to either side of a dry aperture having a diameter of approximately 50/Lm. The pentane was allowed to evaporate.

Once the pre-treatment on the apertures had dried, both chambers of the research cell were filled with electrolyte solution comprising 10mM Phosphate Buffered Saline (PBS) solution at pH 7.2, spiked with IM NaCI. A lOjd drop of 1,2-diphytanoyl.snglycero3phosphochoIjn lipid dissolved in pentane (10mg/mI) was then carefully applied to the surface of the solution in both chambers of the cell, and left to stand at room temperature for 15 minutes to allow the pentane to evaporate.

Bilayers were subsequently formed by sequentially lowering and raising the air/solution interface past either side of the aperture, as described in Montal and Mueller, Proc. Nati. Acad. Sci. USA., 1972; 69: 3561-3566.

An electrical potential difference was applied across the membrane between the chambers of the test cell using Ag/AgCI electrodes, one immersed in each chamber.

Control of the applied potential and recording of the subsequent current was carried out using a current amplifier (MultiClamp700B from Axon Instruments with a CV 7B/BL headstage), coupled to a data acquisition system (DigiData 1 322A also from Axon Instruments). The headstage and the test cell were housed in a Faraday cage to prevent interference from external electromagnetic noise. The DigiData I 322A is interfaced to a computer using plamp version 9.2 software, and data acquired at 4 kHz, with a 2 kHz Bessel filter.

Formation of a bilayer across the aperture was confirmed by creation of a high resistance sealing of the aperture (>l0Gç), by measurement of the capacitance of the high resistance seal and by the subsequent successful insertion of cx-hemolysin (a-HL) pores into the bilayer which resulted in a fixed current flow which is identical for each pore.

An electrical potential difference of+lOOmV was applied between the two chambers once the electrolyte solution has been added, and a recorded current <lOpA is consistent with bilayer formation. Evidence that the bilayer has sealed the aperture was provided by measurement of a predictable capacitive current in response to applying an alternating current (a.c.) potential perturbation. The current response to the applied a.c. waveform when the aperture was not sealed is given in Fig. 3. The squareware current response to a 2OmV amplitude triangular waveform at 50Hz in the presence of a lipid bilayer across the aperture is presented in Fig. 4.

Wild-type a-HL pores were injected into the bulk of the test solution.

Confirmation that the high resistance seal across the aperture was caused by a lipid bilayer was provided by the successful insertion of pores in the bilayer. This insertion was seen as a stepwise increase in the direct current (d.c.) across the bilayer and is presented in Fig. 5.

Finally, confirmation that the stepwise increase in current is specifically due to insertion of the wild-type a-HL pores into the bilayer was provided by the addition of a-cyclodextrin, a well-charactei-ised analyte that transiently binds to a-I-IL pores. A characteristic interruption in the current through the pores of approximately 60% was recorded as single molecules of a-cyclodextnn transiently bound to the pores, with a spread in binding durations in the range -lOOms. This is presented in Fig. 6.

In the following Examples, a lipid bilayer was formed in accordance with the invention. The research test cell and apparatus described above was used to investigate the use of lipid dried to the base of the cell chambers and a-HL dried on the membrane around the aperture.

Two different polymer films were used to create the membrane separating the two chambers of the test cells; a 6jm thick biaxial polycarbonate film, and a 5m thick polytetrafluoroethylene (PTFE) film: both films were sourced from Goodfellow Cambridge Ltd. For each of these two polymer films, apertures were created by one of two different methods: sparking and laser drilling.

Laser-drilled membranes were produced using an Excimer laser at the UK Laser Micromachining Centre, Bangor, Wales. The laser-drilled holes used in these experiments were in the range of 5-30gm in diameter with a tapered morphology in cross section. Holes of this size allow a stable lipid bilayer to e formed more easily.

Spark-generated holes also in the range of 5-30,um in diameter were produced using a spark-generating device. The four polymer film/aperture combinations that were used are summarised below in Table I. Table 1 -Different substrate/aperture combinations used.

Polymer film Aperture A 6jm thick biaxial polycarbonate 25gm diameter spark generated hole B 6jcm thick biaxial polycarbonate l0zm diameter laser drilled tapered hole C 5izm thick PTFE I Ojzm diameter spark generated hole D 5zm thick PTFE l0/Lm diameter laser drilled tapered hole Holes of this size allow a stable lipid bilayer to be formed more easily.

Part of the aperture construction involved a chemical surface treatment to facilitate the bilayer formation process. This involved application of 2/LI of 1% hexadecane in pentane to either side of the aperture. This was then allowed to evaporate. Pre-treatment also allows the easy formation of a stable lipid bilayer.

In addition to the chemical treatment during preparation of the aperture, ld of 0.1 7mg/mI wild-type a-HL was subsequently applied to the aperture and allowed to dry at room temperature.

The test cells were then loaded with 20u1 of the lipid SOiUtjOfl (10mg/mi of 1,2-diphytanoyl-sn-glycero-3phosphocholjne in pentane) applied to the base of each chamber and stored at room temperature to allow the pentane to evaporate, leaving dry lipid coated on the base of each chamber.

The dry research cells, already loaded with lipid and a-HL, were re-hydrated by injecting a test solution (10mM Phosphate Buffered Saline solution, l.OM NaCI, and 0.25mM g-cyclodextrin, at pH 6.9) into the base of each chamber of the cell, raising the Iipidlsolution interface past the aperture only once on either side sequentially.

The electrical potential difference was applied across the membrane using Ag/AgCl electrodes, as in the traditional set up, and data recorded at a sampling rate of 2SOjis per point using the equipment described previously.

The first recorded evidence of spontaneous bilayer formation and pore insertion upon re-hydration of a test cell, which had been pre- loaded with dried lipid and protein pores, is presented in Fig. 7. Hence, a lipid bilayer can be formed by one pass of the lipidlsolution interface past aperture if the solution covers the dried lipid before it covers the aperture, the aperture has a diameter of less than 20/Lm and the membrane has been pre-treated to increase its affinity to lipids. This removes the need to move the interface back and forth past the aperture.

Further testing was performed to confirm bilayer formation and pore insertion.

Hence, pores will spontaneously insert into the lipid bilayer if they are deposited in dried form on an internal surface of the cell. This avoids the need to actively insert the pores into the lipid bilayer. The bilayer formation and pore insertion were consistent with the results for the traditional Montal and Mueller method that are described above and shown in Figs 4, 5 and 6.

The results for Membrane B in Table 1, with a pre-treatment of 2itl of 1% hexadecane in pentane, are shown in Fig. 7. This Fig. shows the current response (pA, upper portion) and the applied potential (mV, lower portion) recorded over a period of 180 seconds. Over the first 40 seconds of recording the cell is dry and the Faraday cage is open. Solution is injected on either side of the membrane just prior to the first marker on the plot (approximately 45 seconds). After a period of fluctuation as the electrodes are wetted and the Faraday cage is closed, the applied potential is then increased to +lOOmV.

The current remains at <lOpA, consistent with the Gf = seal of a bilayer, and then rises in a single step to approximately 9OpA, consistent with insertion of an cx-HL pore and confirming that the aperture was blocked with a lipid bilayer (as described above for Fig. 5). The stepwise fluctuation in the current is from binding events with cyclodextrin causing transient partial blockage of the pore, and confirms that the current is due to an a-HL pore in the bilayer (as described above for Fig. 6). After -70s a second pore inserts into the bilayer.

Fig. 8 shows an expanded view of the current (1 second full scale) during the final minute of recording, again illustrating thecharacteristic step-like profile of the analyte binding events.

The results presented therefore illustrate that bilayer formation and subsequent pore insertion is possible directly upon re-hydration of the dry test cell with test solution using lipid and a-HL dried in the test cell. By this method the bilayers can be formed on the first exposure of the aperture to the solution/air interface carrying the lipid and a variety of apertures can be used including different membrane materials and aperture formation methods. Although the results are not presented here, bilayers have been formed on all the membrane/aperture combinations presented in Table I above.

A cell having a much smaller scale that the two-chamber research cell used above was constructed. The cell contained two cylindrical chambers having a cross-sectional diameter of 12mm and a length of 2mm. The volume of each chamber was approximately 56il. Two alternative membrane materials were tested: a 6um thick biaxial polycarbonate film, and a 5/Lm thick PTFE film (Goodfellow Cambridge Ltd.).

Apertures were formed in the centre of the membrane by one of two different methods described above, sparking and laser drilling. The laser-drilled holes used in these experiments were I Ojtm in diameter with a tapered morphology in cross section.

Spark generated apertures in the 5m PTFE film membranes were approximately I Ozm diameter circular holes, whereas for the 6jm polycarbonate film the sparked apertures were elliptical with dimensions approximately 2Ojm by 3Ozm. Holes of this size allow a stable lipid bilayer to be easily formed.

The apertures then received a chemical pre-treatment to facilitate the bilayer formation process. This consisted of 2z1 of 1% hexadecane in pentane applied to either side of the aperture by capillary pipette. Pre-treatment also allows the easy formation of a stable lipid bilayer.

Once the pentane solvent had evaporated a I l drop of aqueous protein solution (0.017 mg/mI w.t. a-HL) was applied near to one side of the aperture and dried.

The interior of each chamber was then coated with 4d of 10mg/mI diphantytanoyl.snglycero..3..phosphocholine (DPhPC) dissolved in pentane.

The lipid re-hydrated by injecting test solution (10mM Phosphate Buffered Saline solution, I.OM NaCI, and 0.25mM g-cyclodextrin, at pH 6.9) into each chamber.

Control of the applied potential between the cell Ag/AgCI electrodes and recording of the subsequent current was with the same equipment described above. An electrical potential difference of +1 OOmV was applied between the two chambers after the test solution had been added, and a measured current <lOpA was consistent with bilayer formation. Formation of a bilayer across the aperture was confirmed as discussed above. Hence, a lipid bilayer can be formed by one pass of the lipid/solution interface past the aperture if the solution covers the dried lipid before the aperture, the aperture has a diameter of less than 20tm and the membrane has been pre-treated to increase its affinity to lipids.

Fig. 9 shows a typical current trace recorded over the entire duration of one test. Figs 10, 11, 12 and 13 show expanded areas of Fig. 9. Each Fig. contains two graphs: the upper plot shows the current response to the applied potential, which is shown in the lower plot.

Prior to arrow I in Fig. 9, a 50Hz triangular a.c. potential waveform of 2OmV amplitude is applied between the electrodes, which are initially dry. When the test solution is then injected into each chamber of the cell, and the Faraday cage is closed, a square-wave capacitive current response is recorded with amplitude -33OpA, as seen in Fig. 10, indicating bilayer formation across the aperture. When the a.c. potential waveform is then replaced by a d.c. potential of+lOOmV (after arrow I in Fig. 9), a constant current of <lOpA is recorded, confirming that the aperture is sealed with >10 Gf resistance as would be expected with a bilayer.

In the period immediately prior to arrow 2 in Fig. 9, the bilayer is deliberately broken by zapping' with a 5Oms potential pulse of I V d.c. applied on top of 50Hz triangular a.c. waveform. The potential pulse is sufficient to permanently disrupt the high resistance electrical seal of the aperture, causing the current to go off scale, as seen in Fig. 11 (recorded between arrow 2 and arrow 3 in Fig. 9).

Beyond arrow 3 in Fig. 9, a new bilayer is formed using the Montal and Mueller method by lowering and then raising the solution/air interface carrying the lipid monolayer past the aperture. The square wave capacitive current is restored as the new bilayer forms. The potential waveform is then turned off and + I OOmV d.c.

applied, which results in approximately lOOpA step increases in the current as ct-HL protein pores automatically insert into the bilayer. An expanded view showing the current as the pores insert into the bilayer is presented in Fig. 12 (after arrow 4 in Fig. 9). Again, pores will spontaneously insert into the lipid bilayer if they are deposited in dried form on an interface of the cell. This avoids the need to actively insert the pores into the lipid bilayer.

The y-cyclodextrin in the test solution binds stochastically to the ct-HL pores causing characteristic interruptions in the pore current, seen as approximately 6OpA step drops in the current which last 50-500ms. This is presented in Fig. 13.

SEQUENCE LISTING

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(960) <400> 1 atg aaa aca cgt ata gtc agc tca gta aca aca aca cta ttg cta ggt 48 Met Lys Thr Arg lie Val Ser Ser Val Thr Thr Thr Leu Leu Leu Gly 1 5 10 15 tcc ata tta atg aat cct gtc gct aat gcc gca gat tct gat aCt aat 96 Ser lie Leu Met Asn Pro Val Ala Asn Ala Ala Asp Ser Asp lie Asn 25 30 att aaa acc ggt act aca gat aCt gga agc aat act aca gta aaa aca 144 lie Lys Thr Gly Thr Thr Asp lie Gly Ser Asn Thr Thr Val Lys Thr 40 45 ggt gat tta gtc act tat gat aaa gaa aat ggc atg cac aaa aaa gta 192 Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val 55 60 ttt tat agt ttt atc gat gat aaa aat cac aat aaa aaa ctg cta gtt 240 Phe Tyr Ser Phe lie Asp Asp Lys Asn His Asn Lys Lys Leu Leu Vai 70 75 80 att aga aca aaa ggt acc att gct ggt caa tat aga gtt tat agc gaa 288 lie Arg Thr Lys Gly Thr lie Ala Gly Gin Tyr Arg Val Tyr Ser Glu 90 95 gaa ggt gct aac aaa agt ggt tta gcc tgg cct tca gcc ttt aag gta 336 Glu Giy Ala Asn Lys Ser Giy Leu Ala Trp Pro Ser Ala Phe Lys Val 105 110 cag ttg caa cta cct gat aat gaa gta gct caa ata tct gat tac tat 384 Gin Leu Gin Leu Pro Asp Asn Glu Val Ala Gin lie Ser Asp Tyr Tyr 120 125 cca aga aat tcg att gat aca aaa gag tat atg agt act tta act tat 432 Pro Arg Asn Ser lie Asp Thr Lys Glu Tyr Met Ser Thr Leu Thr Tyr 135 140 gga ttc aac ggt aat get act ggt gat gat aca gga aaa att ggc ggc 480 Gly Phe Asn Gly Asn Val Thr Gly Asp Asp Thr Gly Lys lie Gly Gly 150 155 160 ctt att ggt gca aat gtt tcg att ggt cat aca ctg aaa tat gtt caa 528 Leu lie Giy Ala Asn Val Ser lie Gly His Thr Leu Lys Tyr Val Gin 170 175 cct gat ttc aaa aca att tta gag agc cca act gat aaa aaa gta ggc 576 Pro Asp Phe Lys Thr lie Leu Glu Ser Pro Thr Asp Lys Lys Val Gly 185 190 tgg aaa gtg ata ttt aac aat atg gtg aat caa aat tgg gga cca tac 624 Trp Lys Val lie Phe Asn Asn Met Val Asn Gin Asn Trp Giy Pro Tyr 200 205 gat cga gat tct tgg aac ccg gta tat ggc aat caa ctt ttc atg aaa 672 Asp Arg Asp Ser Trp Asn Pro Val Tyr Giy Asn Gin Leu Phe Met Lys 210 215 220 act aga aat ggt tct atg aaa gca gca gat aac ttc ctt gat cct aac 720 Thr Arg Asn Giy Ser Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn 225 230 235 240 aaa gca agt tct cta tta tct tca ggg ttt tca cca gac ttc gct aca 768 Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr 245 250 255 gtt att act atg gat aga aaa gca tcc aaa caa caa aca aat ata gat 816 Val lie Thr Met Asp Arg Lys Ala Ser Lys Gin Gin Thr Asn lie Asp 260 265 270 gta ata tac gaa cga gtt cgt gaL gat tac caa ttg cat tgg act tca 864 Val lie Tyr Glu Arg Val Arg Asp Asp Tyr Gin Leu His Trp Thr Ser 275 280 285 aca aat tgg aaa ggt acc aat act aaa gat aaa tgg aca gat cgt tct 912 Thr Asn Trp Lys Gly Thr Asn Thr Lys Asp Lys Trp Thr Asp Arg Ser 290 295 300 tca gaa aga tat aaa atc gat tgg gaa aaa gaa gaa atg aca aat taa 960 Ser Glu Arg Tyr Lys lie Asp Trp Glu Lys Giu Glu Met Thr Asn 305 310 315 <210> 2 <211> 319 <212> PRT <213> Staphylococcus aureus <400> 2 Met Lys Thr Arg lie Vai Ser Ser Val Thr Thr Thr Leu Leu Leu Giy 1 5 10 15 Ser lie Leu Met Asn Pro Val Ala Asn Ala Ala Asp Ser Asp Ile Asn 25 30 lie Lys Thr Gly Thr Thr Asp lie Gly Ser Asn Thr Thr Vai Lys Thr 40 45 Giy Asp Leu Vai Thr Tyr Asp Lys Giu Asn Giy Met His Lys Lys Vai 55 60 Phe Tyr Ser Phe lie Asp Asp Lys Asn His Asn Lys Lays Leu Leu Val 70 75 80 lie Arg Thr Lys Gly Thr lie Ala Gly Gin Tyr Arg Val Tyr Ser Glu 90 95 Giu Gly Ala Asn Lys Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val 105 110 Gin Leu Gin Leu Pro Asp Asn Glu Val Ala Gin lie Ser Asp Tyr Tyr 120 125 Pro Arg Asn Ser lie Asp Thr Lys Giu Tyr Met Ser Thr Leu Thr Tyr 135 140 Gly Ph Asn Gly Asn Val ThL Gly Ap Asp Thr Gly Lys lie Gly Gly 150 155 160 Leu lie Gly Ala Asn Val Ser lie Gly His Thr Leu Lys Tyr Vai Gin 170 175 Pro Asp Phe Lys Thr lie Leu Giu Ser Pro Thr Asp Lys Lys Val Gly 185 190 Trp Lys Val lie Phe Asn Asn Met Val Asn Gin Asn Trp Giy Pro Tyr 200 205 Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly Asn Gin Leu Phe Met Lys 210 2i5 220 Thr Arg Asn Giy Ser Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn 225 230 235 240 Lys Aia Ser Ser Leu Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala Thr 245 250 255 Val lie Thr Met Asp Arg Lys Ala Ser Lys Gin Gin Thr Asn lie Asp 260 265 270 Vai lie Tyr Giu Arg Val Ar Asp Asp Tyr Gin Leu His Trp Thr Ser 275 280 285 Thr Asn Trp Lys Giy Thr Asn Thr Lys Asp Lys Trp Thr Asp Arg Ser 290 295 300 Ser Giu Arg Tyr Lys lie Asp Trp Glu Lys Giu Giu Met Thr Asn 305 3i0 315 <210> 3 <211> 294 <212> PRT <2i3> Staphylococcus aureus <400> 3 Met Ala Asp Ser Asp lie Asn lie Lys Thr Gly Thr Thr Asp lie Gly 1 5 10 15 Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu 25 30 Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe lie Asp Asp Lys Asn 40 45 His Asn Lys Lys Leu Leu Val lie Arg Thr Lys Gly Thr lie Ala Gly 55 60 Gin Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala 70 75 80 Trp Pro Ser Ala Phe Lys Val Gin Leu Gin Leu Pro Asp Asn Glu Vai 90 95 Ala Gin lie Ser Asp Tyr Tyr Pro Arg Asn Ser lie Asp Thr Lys Glu 105 110 Tyr Met Ser Thr Leu Thr Tyr Giy Phe Asn Giy Asn Val Thr Gly Asp 120 125 Asp Thr Giy Lys lie Gly Giy Leu lie Giy Ala Asn Vai Set Ile Gly 135 140 His Thr Leu Lys Tyr Vai Gin Pro Asp Phe Lys Thr lie L.eu Glu Set i45 150 155 160 Pro Thr Asp Lys Lys Vai Gly Trp Lys Val lie Phe Asn Asn Met Val i65 170 175 Asn Gin Asn Trp Gly Pro Tyr Asp Arg Asp Set Trp Asn Pro Val Tyr 185 190 Gly Asn Gin Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala 200 205 Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Set Gly 210 215 220 Phe Ser Pro Asp Phe Ala Thr Val lie Thr Met Asp Arg Lys Ala Ser 225 230 235 240 Lys Gin Gin Thr Asn lie Asp Val Ile Tyr Glu Arg Vai Arg Asp Asp 245 250 255 Tyr Gin Leu His Trp Thr Ser Thr Asn Trp Lys Giy Thr Asn Thr Lys 260 265 270 Asp Lys Trp Thr Asp Arg Set Set Giu Arg Tyr Lys lie Asp Trp Giu 275 280 285 Lys Giu Giu Met Thr Asn

Claims (41)

1. A method for forming a lipid bilayer across an aperture, comprising: (a) providing a cell having a chamber adjacent to a septum comprising a membrane having an aperture capable of supporting a lipid bilayer; (b) depositing one or more lipids on an internal surface of the chamber; (c) introducing an aqueous solution into the chamber to cover the aperture and the internal surface and to form an interface between the solution and lipids; and (d) moving the interface past the aperture at least once to form a lipid bilayer across the aperture.

2. A method according to claim 1, wherein the lipids are dried.

3. A method according to claim 2, wherein the dried lipids comprises less than 5Owt% solvent.

4. A method according to any one of the preceding claims, wherein the aqueous solution covers the internal surface before it covers the aperture.

5. A method according to any one of the preceding claims, wherein the internal surface on which the lipids are deposited is on the septum.

6. A method according to any one of the preceding claims, wherein the aperture has a diameter in at least one dimension which is 20/hm or less.

7. A method according to any one of the preceding claims, further comprising pre-treating the membrane to increase its affinity to lipids.

8. A method according to claim 7, comprising pre-treating the membrane with hexadecane.

9. A method according to any one of the preceding claims, wherein the cell has two chambers.

10. A method according to claim 9, wherein the method comprises depositing the lipids on an internal surface of both chambers.

11. A method according to claim 9 or 10, wherein the aqueous solution is introduced into one chamber in step (c) and the other chamber comprises a gel.

12. A method according to claim 11, wherein the gel is a hydrogel.

13. A method according to claim 11 or 12, wherein the gel comprises one or more membrane proteins.

14. A method according to any one of the preceding claims, wherein step (b) further comprises depositing one or more membrane proteins on the same or different internal surface, step (c) further comprises introducing the aqueous solution into the chamber to cover the membrane proteins and the method further comprises allowing the membrane proteins to insert into the lipid bilayer.

15. A method according to claim 14, wherein the internal surface on which the one or more membrane proteins are deposited is on the septum.

16. A method according to claim 14 or 15, wherein the membrane proteins are dried.

17. A method according to claim 16, wherein the membrane proteins comprise less than 2Owt% solvent.

18. A method according to any one of claims 13 to 17, wherein the one or more membrane proteins comprise a-hemolysin or a variant thereof.

19. A method according to any one of the preceding claims, wherein the septum further comprises a support sheet on at least one side of the membrane.

20. A method according to any one of the preceding claims, wherein the membrane is made from polycarbonate (PC), polytetrafluoroethylene (PTFE), polyethylene, polypropylene, nylon and polyethylene naphthalate (PEN), polyvinyichloride (PVC), polyacrylonitrile (PAN), polyether suiphone (PES), polyimide, polystyrene, polyvinyifluoride (PVF), polyethylene telephthalate (PET), aluminized PET, nitrocellulose, polyetheretherketone (PEEK) or fluoroethyl kene _i. ---rr,n\ puiyiizi I1Ir).

21. A method according to any one of the preceding claims, wherein the lipids comprise diphantytanoyl-sn-glycero-3 -phosphocholine.

22. A device for forming a lipid bilayer comprising: (a) a cell having a chamber adjacent to a septum comprising a membrane having an aperture capable of supporting a lipid bilayer; and (b) one or more lipids deposited on an internal surface of the chamber, wherein the cell comprises an inlet for introducing an aqueous solution into the chamber having lipid deposited therein.

23. A method according to claim 22, wherein the lipids are dried.

24. A method according to claim 23, wherein the dried lipids comprises less than 5Owt% solvent.

25. A device according to any one of claims 22 to 24, wherein the inlet, internal surface and aperture are arranged in such a manner that the aqueous solution covers the internal surface before it covers the aperture.

26. A device according to any one of claims 22 to 25, wherein the internal surface on which the lipids are deposited is on the septum.

27. A device according to any one claims 22 to 26, wherein the aperture has a diameter in at least one dimension which is 20jm or less.

28. A device according to any one of claims 22 to 27, wherein the membrane has a pre-treatment to increase its affinity to lipids.

29. A device according to claim 28, wherein the membrane has a hexadecane.

30. A device according to any one of claims 22 to 29, wherein the cell has two chambers.

31. A device according to claim 30, wherein the inlet opens into one chamber and the other chamber comprises a gel.

32. A device according to claim 31, wherein the gel is a hydrogel.

33. A device according to claim 31 or 32, wherein the gel comprises one or more membrane proteins.

34. A device according to any one of claims 22 to 33, further comprising one or more membrane proteins deposited on the same or different internal surface.

35. A device according to claim 34, wherein the internal surface on which the one or more membrane proteins are deposited is on the septum.

36. A device according to claim 34 or 35, wherein the membrane proteins are dried.

37. A device according to claim 36, wherein the dried membrane proteins comprise less than 2Owt% solvent.

38. A device according to any one of claims 33 to 37, wherein the one or more membrane proteins comprise a-hemolysin or a variant thereof.

39. A device according to any one of claims 22 to 38, wherein the septum further comprises a support sheet material on at least one side of the membrane.

40. A device according to any one of claims 22 to 39, wherein the membrane is made from pol ycarbonate (PC), pol ytetrafluoroethylene (PTFE), polyethylene, polypropylene, nylon and polyethylene naphthal ate (PEN), polyvinyl chloride (PVC), polyacrylonitrile (PAN), polyether sulphone (PES), polyimide, pOIyStTCnC, p01 yvinylfluoride (PVF), polyethylene telephthalate (PET), aluminized PET, nitrocellulose, polyetheretherketone (PEEK) or fluoroethylkene polymer (FEP).

41. A device according to any one of claims 22 to 40, wherein the lipids comprise diphantytanoyl-sn-gl ycero-3-phosphocholine.