US20100196203A1 - Formation of Lipid Bilayers - Google Patents
Formation of Lipid Bilayers Download PDFInfo
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- US20100196203A1 US20100196203A1 US12/527,687 US52768708A US2010196203A1 US 20100196203 A1 US20100196203 A1 US 20100196203A1 US 52768708 A US52768708 A US 52768708A US 2010196203 A1 US2010196203 A1 US 2010196203A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
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.
Description
- 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-channel 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: 3561-3566), 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 Langmuir-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 electrochemical 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,
- 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.
- Advantageously, 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, lamellar 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 be 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 0.1 μl to 10 ml. 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), polyvinylchloride (PVC), polyacrylonitrile (PAN), polyether sulphone (PES), polyimide, polystyrene, polyvinylfluoride (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 10 nm to 1 mm. The membrane is preferably 0.1 μm to 25 μm 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 be 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, iso-eicosane, octadecane, squalene, fluorinated 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 0.1 μl to 10 μl. 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 diphantytanoyl-sn-glycero-3-phosphocoline (DPhPC). Preferably, the aperture is treated with 2 μl of 1% (v/v) hexadecane and 0.6 mg/ml lipid, such as DPhPC, in pentane.
- Some specific pretreatments are set out in Table 1 by way of example and without limitation.
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TABLE 1 Volumes applied Pretreatment formulation by capillary pipette 0.3% hexadecane in pentane 2x 1 μl 1% hexadecane in pentane 2x 0.5 μl; 2x 0.5 μl; 1 μl; 2x 1 μl;2x 1 μl; 2x μl;2x 2 μl; 5μl 3% hexadecane in pentane 2x 1 μl; 2 μl 10% hexadecane in pentane 2x 1 μl; 2 μl; 5 μl 0.5% hexadecane + 5 mg/ ml DPhPC 5 μl lipid in pentane 1.0% hexadecane + 0.6 mg/ml 2x 0.5 μl DPhPC lipid in pentane 1.5% hexadecane + 5 mg/ ml DPhPC 2 μl; 2x 1 μllipid in pentane - The precise volume of pretreatment substance required depends on the pretreatment both the size of the aperture, the formulation of the pretreatment, and the amount and distribution of the pretreatment when it dries around the aperture. In general increasing the amount of pretreatment (i.e. by volume and/or by concentration) improves the effectiveness, but too much pretreatment can block the aperture. As the diameter of the aperture is decreased, the amount of pretreatment required also decreases. The distribution of the pretreatment can also affect effectiveness, this being dependent on the method of deposition, and the compatibility of the membrane surface chemistry.
- The relationship between the pretreatment and the ease and stability of bilayer formation is therefore complex, depending on a complex cyclic interaction between the aperture dimensions, the membrane surface chemistry, the pretreatment formulation and volume, and the method of deposition. The temperature dependent stability of the pretreated aperture further complicates this relationship. However, the pretreatment may be optimised by routine trial and error to enable bilayer formation immediately upon first exposure of the dry aperture to the lipid monolayer at the liquid interface.
- Although the pretreatment provides a beneficial effect, it is not essential.
- 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, Delrin® (polyoxymethylene or acetal homopolymer), Mylar® (biaxially-oriented polyethylene terephthalate (boPET) polyester film), polycarbonate (PC), polyvinylchloride (PVC), polyacrylonitrile (PAN), polyether sulphone (PES), polysulphone, polyimide, polystyrene, polyethylene, polyvinylfluoride (PVF), polyethylene telephthalate (PET), polytetrafluoroethylene (PTFE), polyetheretherketone (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 a lipid bilayer. The aperture preferably has a diameter in at least one dimension which is 20 μm 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 2 which sets out in the first column the thickness and material of the membrane and in the second column the diameter and method of forming the aperture.
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TABLE 2 Septum Aperture 6 μm thick biaxial 25 μm diameter spark-generated hole polycarbonate 6 μm thick biaxial 20 μm diameter laser-drilled tapered hole polycarbonate 6 μm thick biaxial 10 μm diameter laser-drilled tapered hole polycarbonate 5 μm thick PTFE 10 μm diameter spark-generated holes 5 μm thick PTFE 10 μm diameter laser-drilled tapered hole 5 μm thick PTFE 5 μm diameter laser-drilled tapered hole 10 μm thick HD polyethylene 15 μm diameter spark-generated hole 4 μm thick Polypropylene 15 μm diameter spark-generated hole 25 μm thick Nylon (6,6) 20 μm diameter spark-generated hole 1.3 μm thick PEN 30 μm diameter spark-generated hole 14 μm thick conductive 30 μm diameter spark-generated hole polycarbonate 7 μm thick PVC 20 μm diameter laser-drilled hole - 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 50 wt % solvent, such as less than 40 wt %, less than 30 wt %, less than 20 wt %, less than 15 wt %, less than 10 wt % or less than 5 wt % 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 1 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 diacylglycerides (DG) and ceramides (CM); zwitterionic head groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM); negatively charged head groups, such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid (PA) and cardiolipin (CA); and positively charged headgroups, such as trimethylammonium-Propane (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-Hexadecanoic 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 1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000]; functionionalised PEG Lipids, such as 1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N-[Biotinyl(Polyethylene Glycol)2000]; and lipids modified for conjugation, such as 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl). Suitable lipids whose tail groups have been chemically-modified include, but are not limited to, polymerisable lipids, such as 1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinated lipids, such as 1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine; deuterated lipids, such as 1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linked lipids, such as 1,2-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 stigmasterol; 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-Hydroxyethylpiperazine-N′-2-Ethanesulfonic Acid (HEPES) buffered saline, piperazine-1,4-Bis-2-Ethanesulfonic Acid (PIPES) buffered saline, 3-(n-Morpholino)Propanesulfonic Acid (MOPS) buffered saline and Tris(Hydroxymethyl)aminomethane (TRIS) buffered saline. By way of example, in one implementation, the aqueous solution may be 10 mM PBS containing 1.0M sodium chloride (NaCl) 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 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 lipid bilayer by physically 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 20 wt % solvent, such as less than 15 wt %, less than 10 wt % or less than 5 wt % 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 and/or artificial proteins. Suitable membrane proteins include, but are not limited to, β-barrel membrane proteins, such as non-constitutive toxins, porins and relatives and autotransporters; membrane channels, such as ion channels and aquaporins; bacterial rhodopsins; G-protein coupled receptors; and antibodies. Examples of non-constitutive toxins include hemolysin and leukocidin, such as Staphylococcal leukocidin. Examples of porins include maltoporin, OmpG, OmpA and OmpF. Examples of autotransporters include the NalP 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 mechanosensitive 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 α-hemolysin or a variant thereof. The α-hemolysin pore is formed of seven identical subunits (heptameric). The polynucleotide sequence that encodes one subunit of α-hemolysin is shown in SEQ ID NO: 1. The full-length amino acid sequence of one subunit of α-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 α-hemolysin without the signal peptide is shown in SEQ ID NO: 3. SEQ ID NO: 3 has a methionine residue at
position 1 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 α-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 Table 3. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:
-
TABLE 3 NON-AROMATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E H K R 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 orposition 18 of SEQ ID NO: 3 with cysteine (i.e. N43C or N17C). 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 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 β-barrel domain of SEQ ID NO: 2 or 3.
- Variants include chimeric 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 β-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 and/or 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 al (1984)
Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be 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 et al (1990) Mol Biol 215:403-10. 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. 125I, 35S, enzymes, antibodies, polynucleotides and linkers such as biotin.
- 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 in 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 as studying the properties of molecules known to be present (e.g. DNA sequencing or 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. 1 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 20mV 50 Hz 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 20 mV amplitude triangular waveform at 50 Hz indicative of bilayer formation across the aperture; -
FIG. 5 is a graph of the stepwise increase of 60 pA direct current (d.c.) as α-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 γ-cyclodextrin transiently binding to the α-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 inFIG. 7 ; -
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. 11 is a graph of the current response to a 20mV 50 Hz a.c. waveform in the absence of the high resistance electrical sealing of the aperture by the bilayer in the specially constructed cell; -
FIG. 12 is a graph of step increases in the current ˜100 pA as α-hemolysin pores automatically insert into the bilayer in the specially constructed cell; and -
FIG. 13 is a graph of the characteristic interruptions in the current caused by single molecules of γ-cyclodextrin transiently binding to the α-hemolysin pores in the specially constructed cell; -
FIG. 14 is a perspective view of a sensor system; -
FIG. 15 is a perspective view of a cell of the sensor system; -
FIG. 16 is a cross-sectional of the cell, taken along line inFIG. 2 ; -
FIG. 17 is a perspective view of a support sheet of the cell in isolation; -
FIG. 18 is a perspective view of a body of the cell in isolation with a first arrangement for an inlet; -
FIG. 19 is a perspective view of a cover sheet of the cell in isolation with a second arrangement for an inlet; -
FIG. 20 is a cross-sectional view of the cell similar to that ofFIG. 3 but showing introduction of a sample; -
FIG. 21 is an expanded, partial cross-sectional view of a cell containing gel with a gap between the gel and an aperture; -
FIG. 22 is an expanded perspective view of the connector portion of the reader unit; -
FIG. 23 is a perspective view of a rigid metal body connected to the reader unit; -
FIG. 24 is a cross-sectional view of the rigid metal body, taken along line XII-XII inFIG. 11 ; -
FIG. 25 is a cross-sectional view of the cell contained in a Faraday cage; -
FIGS. 26 to 28 are diagrams of various forms of the electrical circuit in the reader unit; and -
FIG. 29 is a flow chart of the operation of the reader unit; and -
FIG. 30 is a graph of a bias voltage applied to the reader unit; and -
FIGS. 31 to 35 are graphs of the current signal generated in the cell during operation. - In all of the graphs, the x-axis shows time in ms, the top portion of the y-axis shows the current in pA, and the bottom portion of the y-axis shows potential in mV.
- A
device 130 in accordance with the invention is illustrated inFIG. 1 . Thedevice 130 includes anelectrophysiology cell 101 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 101 comprises twochambers body portions 102 having constructions which are mirror images of each other. Thechamber body portions 102 may be made from Delrin® (polyoxymethylene or acetal homopolymer). Thechamber body portions 102 each define achamber portion 103 having an opening in theupper surface 104 of the respectivechamber body portion 102. Thechamber portions 103 each have a volume of a few millilitres, for example 1.5 ml. Thechamber portions 103 have no wall on aside surface 105 of the respectivechamber body portion 102. To form a chamber body, the twochamber body portions 102 are assembled together with theirside surfaces 105 facing one another so that therespective chamber portions 103 are aligned and together form a chamber. Thechamber body portions 102 may be attached by any suitable means, typically a clamp or an adhesive. - The
electrophysiology cell 101 further comprises amembrane 106 made of polycarbonate or any other suitable polymer. Each face of themembrane 106 may be pre-treated in a conventional manner, for example with 10% (V/V) hexadecane in pentane. Themembrane 106 is positioned between the facing side surfaces 105 of the twochamber body portions 102, for example by adhering bothchamber body portions 102 to themembrane 106. Accordingly, themembrane 106 forms a wall which divides the chamber formed by the twochamber portions 103. - The
membrane 106 has anaperture 107 which is aligned with thechamber portions 103 when the electrophysiology cell is assembled. Themembrane 106 is sufficiently thin to facilitate formation of a lipid bilayer, for example being 25 μm thick. Theaperture 107 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 20 μm or less. Thecell 101 comprises inlets for introducing an aqueous solution into eachchamber portion 103, namely the openings in theupper surface 104 of eachchamber body portion 102. - The
device 130 further compriseslipids 108 deposited in eachchamber portion 103 of each one of thechamber body portions 102. The shape of the patch oflipids 108 deposited in eachchamber portion 103 may vary. - The
electrophysiology cell 101 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 bothchamber portions 103 simultaneously via openings in theupper surface 104 of eachchamber body portion 102. The aqueous solution will cover thelipids 108 deposited in eachchamber portion 103 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 bothchamber portions 103 and move past theaperture 107 on both sides of themembrane 106 thereby forming a lipid bilayer across theaperture 107. In this example, thelipid 108 is covered by the aqueous solution before theaperture 107 is covered. - The
electrophysiology cell 101 can further includes respective electrodes (not shown inFIG. 1 ) provided in eachchamber portion 103 of each one of thechamber body portions 102. The electrodes may be Ag/AgCl electrodes. The electrodes may form part of anelectrical circuit 120 which is capable of measuring an electrical signal across the lipid bilayer. A suitableelectrical circuit 120 is illustrated schematically inFIG. 2 and is of a conventional type for performing stochastic sensing by detecting the current flowing across the lipid bilayer. - The
electrodes 109 are connected to anamplifier 121 such as a patch-clamp amplifier (eg an Axopatch 200B supplied by Axon Instruments) which amplifies the current signal output from theelectrodes 109. - The current signal output by the
amplifier 121 is supplied through a low-pass filter 122, such as a Bessel filter (eg withcharacteristics 80 dB/decade with a corner frequency of 2 kHz). - The current signal output by the low-
pass filter 122 is supplied to an A/D convertor 123, such as a Digitata 1320 A/D converter supplied by Axon Instruments. The A/D convertor 123 might typically operate with a sampling frequency of 5 kHz. The A/D convertor 123 converts the current signal into a digital signal which is then supplied to acomputer 124 for analysis. Thecomputer 124 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 detail below.
- 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 Delrin (acetal homopolymer) blocks, each machined to create an open-sided 700 ul 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. 2-5 ul of 10% hexadecane dissolved in pentane was applied to either side of a dry aperture having a diameter of approximately 50 μm. 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 10 mM Phosphate Buffered Saline (PBS) solution at pH 7.2, spiked with 1M NaCl. A 10 μl drop of 1,2-diphytanoyl-sn-glycero-3-phosphocholine lipid dissolved in pentane (10 mg/ml) 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. Natl. 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/AgCl 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 1322A 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 1322A is interfaced to a computer using pClamp 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 (>10Ω), by measurement of the capacitance of the high resistance seal and by the subsequent successful insertion of α-hemolysin (α-HL) pores into the bilayer which resulted in a fixed current flow which is identical for each pore.
- An electrical potential difference of +100 mV was applied between the two chambers once the electrolyte solution has been added, and a recorded current <10 pA 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 20 mV amplitude triangular waveform at 50 Hz in the presence of a lipid bilayer across the aperture is presented inFIG. 4 . - Wild-type α-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 α-HL pores into the bilayer was provided by the addition of α-cyclodextrin, a well-characterised analyte that transiently binds to α-HL pores. A characteristic interruption in the current through the pores of approximately 60% was recorded as single molecules of α-cyclodextrin transiently bound to the pores, with a spread in binding durations in the range ˜100 ms. 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 α-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 6 μm thick biaxial polycarbonate film, and a 5 μm 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 5-30 μm 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 μm in diameter were produced using a spark-generating device. The four polymer film/aperture combinations that were used are summarised below in Table 4.
-
TABLE 4 Polymer film Aperture 6 μm thick biaxial polycarbonate 25 μm diameter spark generated hole 6 μm thick biaxial polycarbonate 10 μm diameter laser drilled tapered hole 5 μm thick PTFE 10 μm diameter spark generated hole 5 μm thick PTFE 10 μm 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 μl 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, 1 μl of 0.17 mg/ml wild-type α-HL was subsequently applied to the aperture and allowed to dry at room temperature.
- The test cells were then loaded with 20 ul of the lipid solution (10 mg/ml of 1,2-diphytanoyl-sn-glycero-3-phosphocholine 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 α-HL, were re-hydrated by injecting a test solution (10 mM Phosphate Buffered Saline solution, 1.0M NaCl, and 0.25 mM g-cyclodextrin, at pH 6.9) into the base of each chamber of the cell, raising the lipid/solution 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 250 μs is 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 lipid/solution interface past aperture if the solution covers the dried lipid before it covers the aperture, the aperture has a diameter of less than 20 μm 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 the second membrane in Table 2, with a pre-treatment of 2 μl of 1% hexadecane in pentane, are shown in
FIG. 7 .FIG. 7 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 +100 mV. - The current remains at <10 pA, consistent with the GO seal of a bilayer, and then rises in a single step to approximately 90 pA, consistent with insertion of an α-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 α-HL pore in the bilayer (as described above forFIG. 6 ). After ˜70 s 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 the characteristic 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 α-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 2 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 12 mm and a length of 2 mm. The volume of each chamber was approximately 56 μl. Two alternative membrane materials were tested: a 6 um thick biaxial polycarbonate film, and a 5 μm 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 10 μm in diameter with a tapered morphology in cross section. Spark generated apertures in the 5 μm PTFE film membranes were approximately 10 μm diameter circular holes, whereas for the 6 μm polycarbonate film the sparked apertures were elliptical with dimensions approximately 20 μm by 30 μm. 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 2 μl 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 1 μl drop of aqueous protein solution (0.017 mg/ml w.t. α-HL) was applied near to one side of the aperture and dried.
- The interior of each chamber was then coated with 4 μl of 10 mg/ml diphantytanoyl-sn-glycero-3-phosphocholine (DPhPC) dissolved in pentane.
- The lipid re-hydrated by injecting test solution (10 mM Phosphate Buffered Saline solution, 1.0M NaCl, and 0.25 mM g-cyclodextrin, at pH 6.9) into each chamber.
- Control of the applied potential between the cell Ag/AgCl electrodes and recording of the subsequent current was with the same equipment described above. An electrical potential difference of +100 mV was applied between the two chambers after the test solution had been added, and a measured current <10 pA 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 20 μm 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 ofFIG. 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 1 inFIG. 9 , a 50 Hz triangular a.c. potential waveform of 20 mV 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 ˜330 pA, as seen inFIG. 10 , indicating bilayer formation across the aperture. When the a.c. potential waveform is then replaced by a d.c. potential of +100 mV (afterarrow 1 inFIG. 9 ), a constant current of <10 pA is recorded, confirming that the aperture is sealed with >10 GO resistance as would be expected with a bilayer. - In the period immediately prior to
arrow 2 inFIG. 9 , the bilayer is deliberately broken by ‘zapping’ with a 50 ms potential pulse of 1V d.c. applied on top of 50 Hz 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 inFIG. 11 (recorded betweenarrow 2 andarrow 3 inFIG. 9 ). - Beyond
arrow 3 inFIG. 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 +100 mV d.c. applied, which results in approximately 100 pA step increases in the current as α-HL protein pores automatically insert into the bilayer. An expanded view showing the current as the pores insert into the bilayer is presented inFIG. 12 (afterarrow 4 inFIG. 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 γ-cyclodextrin in the test solution binds stochastically to the α-HL pores causing characteristic interruptions in the pore current, seen as approximately 60 pA step drops in the current which last 50-500 ms. This is presented in
FIG. 13 . - The alternative device mentioned above which is the cell of a sensor system and to which the present invention may alternatively be applied will now be described. The sensor system is also described in a co-pending application being filed simultaneously with this application [J A Kemp & Co Ref: N.99663A; Oxford Nanolabs Ref: ONL IP 002] which is incorporated herein by reference. All the teachings of that application may be applied equally to the present invention.
- A
sensor system 1 is shown inFIG. 14 and comprises acell 2 and anelectrical reader unit 3 which may be connected together. In use, sensing using a lipid bilayer is formed in thecell 2 and an electrical current signal across the bilayer is monitored and interpreted by thereader unit 3. Thesensor system 1 has been designed for use outside of a laboratory setting. Some examples include use in medicine for point of care testing (POCT), use in environmental protection for a field-based test for pollutants, use for counter bioterrorism for the detection of explosives and chemical and biological agents at the “point of terror”. Nonetheless, some of features of thesensor system 1 also make it advantageous for laboratory use. - The
cell 2 has a construction allowing it to be mass-produced at a low cost, allowing it to be a disposable product. Thecell 2 is easily connected and replaced in thereader unit 3. Thereader unit 3 is sufficiently small to be hand-held and portable. - The
cell 2 is shown inFIGS. 15 and 16 and will now be described in detail. Thecell 2 has a layered construction formed from a stack of layers fixed together. - The
cell 2 comprises amembrane 10 having anaperture 11 across which a lipid bilayer is supported in use. Although only asingle aperture 11 is used in many applications, there may beplural apertures 11. Themembrane 10 may be made of any material capable of supporting lipid bilayer across theaperture 11. Some examples include but are not limited to: a biaxial polycarbonate, PTFE, polyethylene, polypropylene, nylon, PEN, PVC, PAN, PES, polyimide, polystyrene, PVF, PET, aluminized PET, nitrocellulose, PEEK, or FEP. One factor in the choice of the material of themembrane 10 is the affinity to the lipid which affects the ease of bilayer formation. However the material of themembrane 10 has less significance when a pretreatment is used as described below. The choice of the material of themembrane 10 also affects the ease of formation of theaperture 11. - Similarly, the thickness of the
membrane 10 is made sufficiently small to facilitate formation of the lipid bilayer across the aperture, typically being at most 25 μm, preferably being at most 10 μm thick, for example 5 μm or 6 μm. The thickness of themembrane 10 is typically at least 0.1 μm. Theaperture 11 may in general be of any shape or size which it is capable of supporting a lipid bilayer, although it preferably has a restricted size as discussed further below. - The thickness of the
membrane 10 is also dependent on the size of theaperture 11. As theaperture 11 decreases in size, themembrane 10 also needs to decrease in thickness in order to assist the formation of a lipid bilayer. Typically the thickness of themembrane 10 is no more than the minimum diameter of theaperture 11. Another factor is the electrical resistance of themembrane 10 which changes with the thickness. It is desirable that the resistance of themembrane 10 is sufficiently high relative to the resistance of the ion channel in a membrane protein inserted in themembrane 10 that the current flowing across themembrane 10 does not mask the current through the ion channel. - The
membrane 10 is supported by twosupport sheets 12, provided on opposite sides of themembrane 10 and fixed thereto. As described further below, themembrane 10 and thesupport sheets 12 together form aseptum 17. Thesupport sheets 12 each have awindow 13 which is aligned with theaperture 11 in themembrane 10 but is of larger size than theaperture 11 in order that thesupport sheets 12 do not interfere with the formation of a lipid bilayer across theaperture 11. Thesupport sheets 12 have the function of supporting and strengthening themembrane 10 and may be made of any material suitable for achieving this purpose. Suitable materials include, but are not limited to: Delrin® (polyoxymethylene or acetal homopolymer), a polyester, eg Mylar® (biaxially-oriented polyethylene terephthalate (boPET) polyester film), PC, PVC, PAN, PES, polysulphone, polyimide, polystyrene, polyethylene, PVF, PET, PTFE, PEEK, or FEP - The
support sheets 12 are typically thicker than themembrane 10, having a thickness typically at least 0.1 μm, preferably at least 10 μm. Thesupport sheets 12 are thinner than thebodies 14 described below, having a thickness typically at most 1 mm, preferably at most 0.5 mm. - The
cell 1 further comprises twobodies 14 each fixed to one of thesupport sheets 12. Thebodies 14 are each formed from a sheet of material having anaperture 15 extending therethrough. Theapertures 15 in thebodies 14 are of larger area, parallel to themembrane 10, than thewindows 13 in thesupport sheets 12 and are aligned therewith. Thus, theapertures 15 in thebodies 14 each define a respective chamber 16, the two chambers 16 being separated by theseptum 17 formed by themembrane 10 and thesupport sheets 12 together, and theaperture 11 in themembrane 10 opening into each of the chambers 16. - The thickness of each
body 14 is greater than the thickness of thesupport sheets 12 and is chosen to provide a desired volume for the chambers 16. In general, thebodies 14 may have any thickness, but typically the thickness of eachbody 14 is in the range from 1 μm to 3 mm. Typically, for use in a disposable portable sensing system, the chambers 16 have a volume of 0.1 μl to 250 μl. However, a restricted thickness can be advantageous as described further below. Thebodies 14 may be formed of any suitable material, for example silicone rubber. - The chambers 16 are closed by means of a
respective closure sheet 18 which is fixed to the outer surface of therespective body 14 covering theaperture 15 formed therein. Theclosure sheet 18 may be formed from any material, but may for convenience be the same material as thesupport sheets 12. - The
septum 17 including themembrane 10 is not electrically conductive and is designed to have a high electrical resistance. Consequently, in use, the only significant electrical connection between the twochambers 17 is by ionic conduction of an electrolyte solution in thechambers 17 through theaperture 11 in themembrane 10. Formation of a lipid bilayer across theaperture 11 blocks theaperture 11 creating a high-resistance electrical seal between thechambers 17. Insertion of a membrane protein which is an ion channel, for example a pore, restores the electrical connection between the twochambers 17 but only by ionic conduction through the membrane protein. Subsequently, binding events between an analyte and a membrane protein cause a characteristic interruption of the current flowing between the chambers under an applied electrical potential difference. - In order to detect and monitor such electrical signals, each of the chambers 16 is provided with an
electrode 20 formed as part of alayer 23 of conductive material deposited on the surface of therespective support sheet 12 which is internal to the chamber 16. In particular, theelectrodes 20 are illustrated inFIG. 17 which shows one of thesupport sheets 12 as viewed from the side internal to the adjacent chamber 16. InFIG. 17 , the positions of theaperture 15 in thebody 14 and theaperture 11 in themembrane 10 are shown in dotted outline. The conductive material of theelectrodes 20 may be for example Ag/AgCl. - As shown in
FIG. 17 , thesupport sheets 12 each include a protrudingportion 21 which extends beyond the periphery of thebody 14. Thelayer 23 of conductive material which is deposited on thesupport sheet 12 to form theelectrode 20 extends from the chamber 16 across thesupport sheet 12 to the protrudingportion 21. Accordingly eachlayer 23 of conductive material forms not only anelectrode 20 but also acontact 24 which is exposed on aconnector portion 22, and atrack 25 which electrically connects thecontact 24 and theelectrode 20. As described further below, the two protrudingportions 21 of the twosupport sheets 12 together form aconnector portion 22 for connecting thecell 2 to thereader unit 3, and the electrical signal received by theelectrodes 20 in each chamber 16 is supplied to thereader unit 3 via thecontacts 24. - In use, a sample solution is introduced into the chamber 16 on one side of the
membrane 10. The chamber 16 which receives the sample solution will now be referred to as the test chamber 16-1 and the other chamber will now be referred to as the secondary chamber 16-2, although in many embodiments both chambers 16 will be identical in size and construction. - To allow introduction of the sample solution, the test chamber 16-1 may be provided with an
inlet - In the first inlet arrangement, the
inlet 30 is formed in thebody 14 as shown inFIG. 18 . In particular, theinlet 30 is formed in one of the surfaces of thebody 14 which may in general be either the inner or outer surface as a channel extending from the periphery of thebody 14 to theaperture 15. The sample may be injected through theinlet 13, for example using a pipette or syringe. To allow exhaust of air in the chambers 16 displaced by the sample, the test chamber 16-1 is further provided with anexhaust outlet 31 having an identical construction to theinlet 30. - In the second inlet arrangement, the
inlet 32 is formed in theclosure sheet 18 as illustrated inFIG. 19 . In particular, theinlet 32 is formed as a hole extending through theclosure sheet 18 and aligned with theaperture 15 in thebody 14 which defines the test chamber 16-1, as shown in dotted outline inFIG. 19 . To allow exhaust of air in the chambers 16 displaced by the sample, the test chamber 16-1 is further provided with anexhaust outlet 33 having an identical construction to theinlet 32. - Such an
inlet cell 2 of a material which allows penetration by a syringe for filling the test chamber 16-1. - As a result of the design of the
electrode 20 as shown inFIG. 17 , theelectrode 20 is arranged in the flow path between theinlet aperture 11. In other words, when an aqueous solution is introduced into the test chamber 16-1 through theinlet electrode 20 before reaching theaperture 11. This means that theelectrode 20 is wetted before the lipid bilayer is formed, the formation of the bilayer being described in more detail below. When theelectrode 20 is wetted, there can occur a pertubation in the potential across theelectrodes 20 between the two chambers 16, derived from thereader unit 3. If this occurs before the lipid bilayer is formed, then this causes no difficulty. However if the aqueous solution was to contact theelectrode 20 after reaching the bilayer, such a pertubation in the potential across the electrodes could occur after the lipid bilayer is formed and risk rupturing the lipid bilayer. - The secondary chamber 16-2 may, in use contains a buffer solution or a gel. The
cell 2 may be supplied to users with the secondary chamber 16-2 already containing the buffer solution or gel. In this case, the secondary chamber 16-2 does not need aninlet cell 2 may be supplied with the secondary chamber 16-2 empty. In this case, the user must introduce a buffer solution or gel into the secondary chamber 16-2. To facilitate this the secondary chamber 16-2 may also be provided with aninlet - Thus the chambers 16 are closed except for an
inlet - An internal surface of the test chamber 16-1 has a lipid deposited thereon. When the sample is inserted into the test chamber 16-1, the sample rehydrates the lipids and forms a lipid/solution interface between the sample and the air in the test chamber 16-1. This interface is subsequently moved across the
aperture 11, either once or repeatedly, in order to form the lipid bilayer across theaperture 11. - In general, the lipid may be applied to any internal surface of the test chamber 16-1. The lipid may be deposited on the
septum 17 during manufacture after theseptum 17 has been constructed by fixing together themembrane 10 and thesupport sheets 12 but before assembly of theseptum 17 into the remainder of thecell 2. Alternatively the lipid may be deposited on the internal walls of the chamber 16 formed by theaperture 15 in thebody 14 or theclosure sheet 18, either before or after thebody 14 is fixed to theclosure sheet 18, but before assembly to theseptum 17. - The deposition may be achieved by coating the
septum 17 with a solution of the lipid dissolved in an organic solvent such as pentane and then subsequently allowing evaporation of the solvent, although other techniques could equally be applied. - In general the chambers 16 may be of any size. However, particular advantage is achieved by restricting the depth of the test chamber 16-1 in the direction perpendicular to the
septum 17. This depth is controlled by selection of the thickness of thebody 14. In particular, the depth is restricted to a level at which the surface tension of a sample solution introduced into the test chamber 16-1 prevents the liquid from flowing across the test chamber 16-1 and instead contains the liquid in part of the test chamber 16-1 across its area parallel to theseptum 17. In this state, the liquid interface with the air in the chamber 16 extends across the depth of the chamber 16, perhaps with some meniscus forming depending on the relative pressures of the liquid and the air. - This effect is illustrated in
FIG. 20 which shows acell 2 in which theliquid sample 40 has been introduced into one side of the test chamber 16-1 through theinlet 30 or 32 (although for simplicity theinlet FIG. 20 ). As can be seen, instead of theliquid sample 40 falling under gravity to the lowest possible level in the chamber 16, surface tension holds theliquid interface 41 with the air in the chamber 16 extending across the depth of the chamber 16 between theseptum 17 and theclosure sheet 18. Thus, theinterface 41 is generally perpendicular to theseptum 17 and theaperture 11 except for the formation of a meniscus. - By applying pressure at the
inlet interface 41 may be moved in the direction of the arrow A along the chamber parallel to theseptum 17 and hence across theaperture 11. Once theliquid sample 40 has rehydrated the dried lipid inside the chamber 16 theliquid interface 41 will support a layer of the lipid. Thus, such movement of theliquid interface 41 across theaperture 11 in themembrane 10 may be used to form a lipid bilayer. - A particular advantage of such a restricted depth for the chamber 16 is that the above-described effect of surface tension occurs irrespective of the orientation of the
cell 2. Although thecell 2 is illustrated inFIG. 14 with theaperture 11 extending horizontally, the same effect occurs regardless of the orientation of thecell 2. Thus the above-described process of forming a lipid bilayer across theaperture 11 may be carried out with thecell 2 in any orientation. This reduces the degree of care needed by the user and enhances the ability to use the sensor system outside of a laboratory setting. - The
cell 2 is easy to manufacture simply by cutting and affixing together the individual layers of thecell 2. For convenience the layers of thecell 2 are affixed by adhesive, although in principle some form of mechanical fixing could be used. Conveniently due to the use of a layered constructionplural cells 2 or parts thereof may conveniently be manufactured together from a large sheet and subsequently cut out. As a result of these points, thecell 2 is capable of mass production at relatively low cost. - By way of example and without limitation, one particular manufacturing method will now be described in detail.
- Firstly, a template for
plural cells 2 is inkjet printed onto the release paper of adhesive-coated polyester A4 sized cards from which six rows of sixteensupport sheets 12 are to be formed. The cards were Mylar polyester sheet (DuPont) ofthickness 250 μm with a 467 MP self-adhesive coating ofthickness 50 μm on one side. With the release-paper facing upwards, 4 mm diameter holes are punched in the cards on the template to provide thewindows 13 of eachsupport sheet 12 and any burring of the edges of the punched holes removed using a scalpel blade. - The
layers 23 of conductive material are then stencil screen-printed onto the cards using a 60/40 composition silver/silver chloride paste (Gwent Electronic Materials Ltd.), and left overnight to dry at room temperature. The registration and electrical resistance of thelayers 23 of conductive material is checked and the surface of the cards covered with a sheet of A4 paper, to keep the surface clean in subsequent stages of sensor production. - With the release paper side facing upwards, the cards are then cut using a guillotine lengthwise into the six rows of
support sheets 12. - In this example the
membranes 10 are formed from either a 6 μm thick biaxial polycarbonate film or a 5 μm thick PTFE film (Goodfellow Cambridge Ltd.). Prior to use theapertures 11 are formed as discussed below. Themembrane 10 around theapertures 11 then receives a chemical pretreatment to facilitate the bilayer formation process. In this case, the pretreatment consists of 2 μl of 1% hexadecane in pentane applied to either side of the aperture by capillary pipette. - Once the pentane solvent had evaporated a 1 μl drop of aqueous protein solution (0.017 mg/ml w.t. α-HL) was applied near to one side of the aperture and dried.
- Next the films are cut into strips, cleaned on both sides by rinsing with ethanol, and gently air-dried.
- A tape-laying jig with a rubber coated veneer roller is used to roll the membrane film strips evenly over the self-adhesive of one half of the card rows. Care is taken to ensure that the film above the punched holes in the card remained flat and free from creases.
- To complete the
septums 17, the other half of the card rows are stuck back to back to sandwich the membrane film strips, with the punched holes carefully aligned on either side with theapertures 11. Then the strips are cut using a guillotine intoseptums 17 forindividual cells 2. - In this example the
body 14 is formed from a 2 mm thick solid silicone rubber sheet with self-adhesive coating on both sides. A large such sheet is cut into A4 sized sheets. An array of 12 mm diametercircular apertures 15 forrespective cells 2 are formed by removal of the material of the sheet, in particular by hollow punching the spacer sheets. Chamber volumes as low as 56 μl have been produced by punching 6 mm diameter holes through the 2 mm thick spacer material. - The individual chambers 16 are then closed by sticking an A4 sized card of plain 250 μm thick Mylar polyester sheet (DuPont), which ultimately forms the
closure sheets 18, to one side of the silicone rubber sheet. This sheet is then cut using a guillotine lengthwise into rows having the desired width of thebody 14. Channels ofwidth 1 mm, to form theinlet 30 andexhaust gas outlet 31 are then cut in the silicone rubber sheet material (but not through the backing card). - The interior of each chamber 16 is then coated with a solution of 4 μl of 10 mg/ml DPhPC lipid dissolved in pentane. The rows of lipid-loaded chambers are cut using a guillotine into individual chambers 16 according to the template and then bonded symmetrically to each side of the
individual septums 17 to formcells 2. - The size and formation of the
aperture 11 in themembrane 10 will now be considered further. - In general, the
aperture 11 may be of any size capable of supporting a lipid bilayer. By way of comparison, the diameter of an aperture in a conventional laboratory apparatus is typically in the order of 30 μm to 150 μm and anaperture 11 of such a size may used in thepresent cell 2. - However, it has been appreciated that particular advantage may be achieved by restricting the size of the
aperture 11. In particular, this has been found to increase the mechanical stability of the bilayers formed. The increased stability reduces the number of passes of the liquid interface supporting the lipid past the aperture necessary to allow formation of the bilayer. Furthermore, the increased stability increases the robustness of the bilayer and reducing the chances of the bilayer rupturing. This is of particular advantage when thesensor system 1 is used outside a laboratory setting where it may be subject to external forces. - The increased stability achieved by restricting the size of the
aperture 11 has been experimentally demonstrated as follows. - A number of
actual membranes 10 which have been tested are listed in Table 2 above Theapertures 11 which are sparked-generated were produced by a spark generating device which comprises an adjustable high voltage generator that charges a storage capacitor, with feedback control. The storage capacitor is then switched to discharge into a high voltage transformer coil to rapidly produce a large potential difference between the points of two electrodes attached to the transformer output. Dielectric breakdown between the electrode points results in a spark. The energy of the spark is controlled by switching the value of the storage capacitor (33 nF-300 nF), by adjusting the capacitor charging voltage (200 nV-500V), and by changing the distance between the output electrode points. - The polymer film from which a
membrane 10 is subsequently cut is mounted flat on the sparking platform and the two output electrodes of the sparking device are positioned opposite each other, above and below the film. - To form
apertures 11 of small diameter the spark energy is minimised by choosing the lowest storage capacitor and lowest charging potential that can create a spark that penetrates through the film, and by controlling the dielectric resistance between the two electrodes. For example, decreasing the thickness of the membrane film enabled the use of lower energy sparks and produced smaller apertures, such that it was possible to create apertures in therange 5 μm-10% n diameter in PTFE film of 5 μm thickness. Further control of theaperture 11 diameter could easily be introduced through limiting the sparking energy by gating the discharge after detecting the onset of dielectric breakdown. - The laser-generated
apertures 11 were produce by laser drilling. - The morphology of the
aperture 11 can been seen to vary with the material of themembrane 10 and method used to form the bilayer. For example, with biaxial polycarbonate film, the spark generatedapertures 11 were elliptical while the laser drilledapertures 11 were mostly circular. Similarly the spark generatedapertures 11 generally had a uniform cross-section while the laser drilledapertures 11 generally a cross section which tapered through the thickness of themembrane 10. - The regularity of the inside edge of the
aperture 11 is also sensitive to the material of themembrane 10, the thickness of themembrane 10, and the method of formation of theaperture 11. This is expected to impact on the stability of bilayer formation at the aperture. - However in all cases irrespective of the method of formation of the
aperture 11, it is apparent that restricting the diameter of theaperture 11 results in increasing the stability of the bilayers, in fact to a dramatic degree. For example with anaperture 11 ofdiameter 10 μm thecell 2 can firmly knocked against the table or disconnected from thereader unit 3 and carried by hand without breaking the bilayer. This is of significant advantage in the context of use of thesensor system 1 outside the laboratory setting. - For these reasons it is preferred that the
aperture 11 has a restricted diameter, say of 20 μm or less in at least one dimension. Theaperture 11 may have such a restricted diameter in all dimensions, but the advantage of increased stability is achieved provided theaperture 11 is relatively small in one dimension, even if theaperture 11 is longer in another dimension. - The work described above demonstrates that
apertures 11 of small diameter may be formed using cheap off-the-shelf materials and processes adaptable for mass production. Nonetheless, the choice of materials for themembrane 10 and methods capable of generating theapertures 11 is considerably more extensive than those considered above. - As mentioned above, in one type of
cell 2, the secondary chamber 16-2 may contain agel 50 as shown for example in thecell 2 ofFIG. 20 . In particular, thegel 50 extends across theaperture 11 in themembrane 10. The presence of the gel acts to physically support a lipid bilayer formed across theaperture 11. As a result, thegel 50 assists the formation of the lipid bilayer and furthermore provides the lipid bilayer with increased stability. Both of these advantages are significant in the context of using thesensor system 1 in a non-laboratory setting, because it makes thesensor system 1 easier to use and also more robust against external forces of the type which may disturb thesensor system 1 in normal use. In addition, thegel 50 may act as a matrix for controlling the supply of molecules to the lipid bilayer. - In order to support the lipid bilayer, the
gel 50 may fill the secondary chamber 16-2 such that thegel 50 contacts themembrane 10. This case is illustrated inFIG. 20 . In this case, thegel 50 may directly support the lipid bilayer formed across theaperture 11. This is preferred in order to improve bilayer formation and stability. - However, in an alternative illustrated in
FIG. 21 , there may remain agap 51 between thegel 50 and themembrane 10. In this case, thegel 50 may still support the lipid bilayer formed across theaperture 11 by acting through a solution occupying thegap 51, although this effect will reduce as the size of thegap 51 increases. The presence of thegap 51 means that a wider variety of materials can be used to make thegel 50, including ionically non-conductive materials. - The
gel 50 may be ionically conductive and indeed this is necessary if thegel 50 directly contacts the lipid bilayer. In this case thegel 50 may be for example a hydrogel. Suitable ionically conductive gels include, but are not limited to, agarose polyacrylimide gel, Gellan™ gel or Carbomer gel. Particular gels which have been used are 5% agarose doped with NaCl or Signa Gel (Parker Laboratories Inc.). In one caseagarose gel 50 was made using 10 mM PBS to which 1M NaCl had been added. Thegel 50 was melted and then injected in the chamber 16 where it solidified upon cooling. - It has been discovered that when one chamber 16 of the
cell 2 is filled with agel 50, formation of a lipid bilayer was possible by moving theliquid interface 41 carrying a lipid monolayer past theaperture 11 on only one side of theaperture 11, as opposed to both sides of theaperture 11 as more commonly performed in the Montal & Muller method. Further, bilayers could be formed with or without pretreatment of themembrane 10 by this method. However considerably more attempts were required without the pretreatment. Pretreating only the top side of themembrane 10 was found to be sufficient for reproducible bilayer formation. Being able to apply the pretreatment to only one side of themembrane 10 greatly simplifies the manufacturing process. - The
cell 2 may be provided to the user with the secondary chamber 16-2 already containing thegel 50. This improves the ease of use of thecell 2 because no filling the secondary chamber 16-2 is necessary by the user. - Each of the features described above of (1) restricting the size of the
aperture 11, (2) use of a pretreatment and (3) use of agel 50 assist the formation of a lipid bilayer across theaperture 11 in themembrane 10. In particular, this reduces the number of times in which theinterface 41 carrying a lipid monolayer must be moved past theaperture 11 in order to form the bilayer. This improves the ease of use of thecell 2. - In fact, in actual embodiments of the
cell 2 employing each of features (1) to (3) there has been demonstrated reliable formation of lipid bilayer on a single pass of theliquid interface 41 pass theaperture 11. This is of significant advantage because it means that the lipid bilayer may be formed across theaperture 11 simply on insertion of thetest solution 40 into thecell 2, for example using a pipette or a syringe. This means that the user does not need to repeatedly move theliquid interface 41 back and fourth across theaperture 11 whilst monitoring the formation of the lipid bilayer, and so the required user skill level is greatly reduced. Furthermore, it is not necessary to employ any complicated fluidics control to so move theliquid interface 41. - The
reader unit 3 will now be described in detail. - The
reader unit 3 has aconnector portion 60 which is arranged to make a physical connection with theconnector portion 24 of thecell 2. Theconnector portion 60 of thereader unit 3 is visible inFIG. 14 but is shown in expanded form inFIG. 22 . In particular, theconnector portion 60 consists simply of a pair ofblocks 61 which are separated by a spacing designed to provide a tight fit for theconnector portion 24 of thecell 2. Thus, theconnector portion 24 of thecell 2 may be plugged into theconnector portion 60 in between theblocks 61 by insertion of thecell 2 in the direction of arrow B, thereby providing mating between theconnector portions - In addition,
respective contacts block 61 or theconnector portions 60. Thecontacts contacts connector portion 24 of thecell 2 is plugged into theconnector portion 60 of thereader unit 3, thecontacts 24 of thecell 2 make an electrical connection with thecontacts reader unit 3. Thereader unit 3 includes anelectrical circuit 90 described further below which is connected to thecontacts cell 2 in thereader unit 3 allows the electrical signal generated between the chambers 16 to be supplied from theelectrodes 20 to thereader unit 3. - There will now be described some alternatives for providing the
cell 2 with a Faraday cage to produce electrical interference from ambient electrical magnetic radiation with the electrical signals generated in thecell 2 when it is connected to thereader unit 3. Two alternative approaches are as follows. - The first approach uses a
rigid metal body 70 as the Faraday cage. The rigid metal body has aninternal cavity 71 sufficient to accommodate thecell 2. At oneend 72, therigid metal body 70 is open and connected to the body 73 of thereader unit 3 so that thecavity 71 is aligned with theconnection portions 60. In this way, thecell 2 is accommodated inside thecavity 71 when it is connected to thereader unit 3, as shown inFIG. 24 . - However, rather than entirely enclosing the
cell 2, therigid metal body 70 has anaperture 74 facing theconnector portion 60. Theaperture 74 is of sufficient size to allow passage of thecell 2 when thecell 2 is connected to thereader unit 3. Therefore, anindividual cell 2 may be connected to thereader unit 3 and replaced by anothercell 3 by insertion through theaperture 74 without removal of therigid metal body 70. It has been appreciated that surprisingly the presence of theaperture 74 does not prevent the operation of therigid metal body 70 as a Faraday cage. In particular, this is because theaperture 74 may be of sufficiently small size that any electrical interference caused by electro magnetic radiation penetrating theaperture 74 is at a sufficient high frequency that it does not significantly degrade the quality of the electrical signal of interest. In particular, theaperture 74 of therigid metal body 70 may have a maximum dimension (horizontally inFIG. 23 ) of 50mm or less, preferably 20 mm or less. - The
rigid metal body 70 also has asample introduction hole 76 which is aligned with theinlet cell 2 is connected to thereader unit 3. Thesample introduction hole 76 allows the sample to be introduced into thecell 2 after thecell 2 has been connected to thereader unit 3. Thesample introduction hole 76 is smaller than theaperture 74, typically having a maximum dimension of 5 mm or less. Thus thesample introduction hole 76 is also of sufficiently small size that any electrical interference caused by electro magnetic radiation penetrating thesample introduction hole 76 is at a sufficient high frequency that it does not significantly degrade the quality of the electrical signal of interest. - The second alternative approach is to provide a
Faraday cage 75 fixed around the periphery of thecell 2, for example as shown inFIG. 25 . In this case, theFaraday cage 75 entirely encloses thecell 2, except for theconnector portion 24 which protrudes out of theFaraday cage 75. In this case, theFaraday cage 75 may be formed by a solid metal body. Alternatively, theFaraday cage 75 may be formed by a metal foil which has the advantage of being easy to manufacture, for example simply by adhering the metal foil to the exterior of thecell 2. - It is noted that the provision of a Faraday cage attached around the exterior of the
cell 2 is equally applicable to other types of electrical sensor cell which are operative to detect an analyte by measurement of an electrical signal developed in the cell. - The
reader unit 3 houses anelectrical circuit 90 which will now be described in detail. The primary function of theelectrical circuit 90 is to measure the electrical current signal developed across theelectrodes 20 to provide a meaningful output to the user. This may be simply an output of the measured signal or may involve further analysis of the signal. - The
electrical circuit 90 may take various different forms and some possible circuit designs are shown inFIGS. 14 to 16 . In each design there are some common elements as follows. - The two
contacts connector portion 60 will be referred to as areference contact 62 and a workingcontact 63. Although theelectrodes reference contact 62 provides a bias voltage potential relative to the workingcontact 63, whilst the workingcontact 63 is at virtual ground potential and supplies the current signal toelectrical circuit 90. - A possible alternative which is not illustrated would be for the
reference contact 62 to be held at ground and workingcontact 63 to be offset by the bias voltage. - The
reader circuit 90 has abias circuit 91 connected to thereference contact 62 and arranged to apply a bias voltage which effectively appears across the twocontacts electrodes 20 of acell 2 connected to thereader unit 3. Thebias circuit 91 may take different forms as described below. - The
reader circuit 90 also has anamplifier circuit 92 connected to the workingcontact 63 for amplifying the electrical current signal theelectrodes 20 of thecell 2 and appearing across the twocontacts electrical circuit 90, theamplifier circuit 92 consists of afirst amplifier stage 93 and asecond amplifier stage 94. - The
first amplifier stage 93 is connected to the workingelectrode 63 and arranged to convert the current signal into a voltage signal in a first stage amplifier. It may comprise an electrometer operational amplifier configured as an inverting amplifier with a high impedance feedback resistor, of for example 500 MΩ, to provides the gain necessary to amplify the current signal which typically has a magnitude of the order of tens to hundreds of picoamps. - The
second amplifier stage 94 is connected to the output of thefirst amplifier stage 93 and arranged to amplify and filter the voltage signal voltage. Thesecond amplifier stage 94 provides sufficient gain to raise the signal to a sufficient level for processing in themicrocontroller 95 described below. For example with a 500 MΩ feedback resistance in thefirst amplifier stage 93, the input voltage to thesecond amplifier stage 94, given a typical current signal of the order of 100 pA, will be of the order of 50 mV, and in this case thesecond amplifier stage 94 must provide a gain of 50 to raise the 50 mV signal range to 2.5V. If the signal contains frequencies beyond the bandwidth limit of the first stage then analogue filtering is provided in thesecond amplifier stage 94 to increase gain at frequencies beyond the first stage bandwidth limitation. The filtering results in a combined first and second stage frequency response with constant gain beyond the first stage limitation. - To save power, the analogue circuitry in the
bias circuit 91 and theamplifier circuit 92 is shutdown when not being used. Each power rail is connected to bipolar PNP switching transistors for low leakage switching of the analogue circuitry. - Typically the signal will be unipolar, but if bipolar current signals are required the gain of the
second amplifier stage 94 can be halved and a DC offset applied to the inverting input of thesecond amplifier stage 94 equal to half reference voltage value of themicrocontroller 95. - The first design of the
electrical circuit 90 shown inFIG. 26 and will now be described. This design is intended for a stand-alone battery-operatedreader unit 3 with PC connectivity. In this case, thebias circuit 91 and theamplifier circuit 92 are connected to amicrocontroller 95. Themicrocontroller 95 has apower control circuit 96 which supplies power from a battery. Themicrocontroller 95 incorporates an analog-to-digital converter 97 which receives the output of theamplifier circuit 92 and converts it into a digital signal. The analog-to-digital converter 97 may be of a successive approximation type or of a voltage-to-frequency type, both resulting in a digital word for each conversion. A sampling rate is chosen that is at least twice the bandwidth of the signal at the output of thesecond amplifier stage 94 to prevent aliasing. - In this case the analog-to-
digital converter 97 is embedded on the same silicon die as themicrocontroller 95, but it could alternatively be a separate circuit element. - The
microcontroller 95 incorporates amicroprocessor 98 which runs code to process and analyse the digital signal. Themicrocontroller 95 has adisplay 99 which is conveniently an LCD display, and on which the microcontroller causes display of the signal itself or other analysis results such as temporal results of the signal analysis. - The
microcontroller 95 receives commands from akeypad 100. Of course other input and output devices could be used in addition to, or instead of, thedisplay 99 andkeypad 100, for example LEDs used as indicators or anaudio generator 105. - The
microcontroller 95 also has aninterface 101 to provide data communication with another digital device, for example a computer. Theinterface 101 may be of any type, for example a UART interface. This allows the received signal to be supplied to another device for display, storage and/or further analysis. - The
microcontroller 95 is connected to thebias circuit 91 as follows. Themicrocontroller 95 has aPWM generator 102 which generates a PWM (pulse width modulation) voltage waveform, that is a digital signal with fixed frequency but varying duty cycle. ThePWM generator 102 is of conventional construction. Generally, an internal timer is set running to generate the PWM signal frequency and a register is loaded with the count at which the PWM output is switched and a comparator detects when the count is reached. - The
bias circuit 91 includes a low-pass filter 103 connected to low-pass filter the PWM signal output by thePWM generator 102. The duty cycle of the PWM signal varies with time so that the output of the low-pass filter is the desired analog signal, which is the average voltage over one period of the PWM cycle. ThePWM generator 102 built in this manner has a resolution equivalent to the smallest duty cycle change possible with themicrocontroller 95. Bipolar outputs can be achieved by using a pair of PWM signals each connected to one of a pair of low pass filters 103 and one fed to the positive input and the other the negative input of a summing amplifier, this being shown inFIG. 26 . - The
bias circuit 91 further includes anoutput amplifier 104 for amplifying the output of the low-pass filter 103. In the case described above that a bipolar output is required, theoutput amplifier 104 is a summing amplifier arranged to subtract the output of one of the pair of low pass filters 103 from the other. - For systems requiring multiple or arrayed
cells 2, themicrocontroller 95 can be chosen with an embedded analogue multiplexer. In this case multiple analogue input circuits are required and the output of eachsecond amplifier stage 94 is sampled by the analog-to-digital converter 97 through the multiplexer. - The second design of the
electrical circuit 90 is shown inFIG. 27 and will now be described. This design is intended for areader unit 3 which is a derivative of a standard Personal Digital Assistant (PDA) architecture. The second design is identical to the first design except that themicrocontroller 95 interfaces with aPDA device 106 which is a conventional PDA. This allows thereader unit 3 to take advantage of the existing functionality of PDAs. ThePDA device 106 may have input/output facilities based on a variety of protocols, such as universal connectors, Secure Digital cards (SD), Compact Flash cards (CF, CF2), MultiMedia cards (MMC), memory stick cards or SIM card. Such functionality may be used to provide a framework for thereader unit 2 to provide the functions of a large interactive display with key or touch entry and a rechargeable power source. - In this case, one option is for the
connector portion 60, theamplifier circuit 92, thebias circuit 91 and themicrocontroller 95 to be mounted within an electrical assembly shaped to fit in an SD card slot or other card format slot. This allows thereader unit 2 to be formed by an existing PDA device with the assembly fitted in a card slot. - The third design of the
electrical circuit 90 is shown inFIG. 28 and will now be described. This design is intended for areader unit 3 which is based on adata acquisition card 107 to be plugged into acomputer 108 such as a desktop or laptop. This design is the simplest in terms of hardware development requiring only three amplifier stages and the data acquisition card. In this case theamplifier circuit 92 is arranged as described above, but thebias circuit 91 is simply formed by an invertingamplifier 109 supplied with a signal from a digital-to-analog converter 110 which may be either a dedicated device or a part of thedata acquisition card 107 and which provides a voltage output dependent on the code loaded into thedata acquisition card 107 from software. - The third design of the
electrical circuit 90 shown inFIG. 28 may be modified to provide a multi-port reader system connected through a fast transport interface such as the Universal Serial Bus or Ethernet for the purpose of analysing many cells at once. In work involving drug-screening or an industrial manufacturing environment there is a need for multiple readers connected to a central computer for research, analysis and quality control. In this case thedata acquisition card 107 is modified to provide the transport interface allowing multiple data streams into the computer. - The
electrical circuit 90 may provide analysis of the received signal. Such analysis may be performed, for example, by programming one of the microprocessors in the electrical circuit, for example themicroprocessor 98 in themicrocontroller 95 or thePDA device 106 in the above described designs of the electrical circuit. In particular the analysis may involve interpretation of the electrical signal. As already described, the electrical signal is characteristic of the physical state of thecell 2. Accordingly, the state of thecell 2 can be detected from the electrical signal by theelectrical circuit 90. - For example, when the
cell 2 is used as described above, the following states each have a characteristic electrical signal which may be detected by the electrical circuit 90: - 1) the chambers 16 in the
cell 2 being dry; - 2) the chambers 16 in the
cell 2 containing an aqueous solution without a lipid bilayer being formed across theaperture 11 in themembrane 10; - 3) a lipid bilayer being formed across the
aperture 11 in themembrane 10 without a membrane protein being inserted therein; - 4) a lipid bilayer being formed across the
aperture 11 in themembrane 10 with a membrane protein being inserted therein without an analyte binding to the membrane protein; and - 5) a lipid bilayer being formed across the
aperture 11 in themembrane 10 with a membrane protein being inserted therein with an analyte binding to the membrane protein. - Such states may be detected based on predetermined thresholds or adaptive thresholds, which may be derived from scientific study of the membrane protein and physical system being used in the
cell 2. On detection of such a state, theelectrical circuit 90 then produces an output indicative of the detected state, for example by displaying the detected state on thedisplay 99 or some other audio and/or visual output, or by outputting a signal indicative of the detected state, for example to a computer device connected thereto. - By detecting the continuous sequence of states (1) to (5) in order, the
reader unit 2 may also monitor the correct performance of the sensing process to check and ensure that thecell 2 is operating correctly from the moment it is connected to thereader unit 3 until the end of the measurement assay. Thereader unit 3 may apply a bias potential and continuously monitor the resultant signal. If the signal falls outside the expected levels showing a proper progress through the states (1) to (5), thereader unit 3 may output a signal reporting an error mode, or alternatively may perform an automated remediation. - As each state is detected the time duration of the state will be stored for subsequent or continuous statistical analysis. This may provide further information. For example, signals derived from single molecule binding events in or near multiple membrane protein channels will result in a time-varying current based on the number of binding events.
- Another example is where the membrane protein includes a tether. Signals derived from either single or multiple binding events to either single or multiple tethers attached to single or multiple membrane protein channels will appear as noisy signals which become less noisy when the tether or tethers are bound to a target analyte. Each tether will have a binding site for the target analyte. These signals will be analysed with an algorithm to detect the reduction in noise and as each event is detected the time duration of the event or the time course of noise reduction will be stored for subsequent or continuous statistical analysis.
- There will now be described an actual example of the algorithm used to monitor of the state of the
cell 2 in the case using the membrane protein α-HL to sense the presence of the analyte γ-cyclodextrin. Theelectrical circuit 90 performs the process as shown inFIG. 29 . - In an initialisation step S1 performed before connection of the
cell 2 to thereader unit 3, theelectrical circuit 17 applies a bias voltage as shown inFIG. 30 having a waveform which is a 50 Hz triangular AC signal with 20 mV amplitude, superimposed on +100 mV DC potential. - In step S2 it is detected whether the received signal is representative of a current and impedance within the respective limits for the
reader unit 3 in the absence of thecell 2. In the absence of thecell 2, thecontacts reader unit 3 behaves as a capacitor and produce a square wave current response to the applied triangular AC potential, as shown inFIG. 31 . In particular the square wave has a 20 pA amplitude centred on 0 pA. This waveform is characteristic of normal operation of theelectrical circuit 90 and so in step S2 it is detected whether this waveform is produced, within a reasonable margin. If not, then in step S3, theelectrical circuit 90 outputs a signal indicate indicative of a circuit error. Otherwise in step S4, the user connects acell 2 to thereader unit 3. Theelectrical circuit 90 may for example await a user input to indicate this. - Subsequently in step S5, there is detected state (1) that the chambers 16 in the
cell 2 are dry. In this case, the expected signal is the same as that detected in step S2 except that the insertion of thecell 2 causes an increase, for example the order of 25%, in the amplitude of the resultant squarewave, for example to provide an amplitude of 27 pA. If state (1) is not detected, then in step S6 and there is output an error signal indicating malfunctioning of thecell 2. - Otherwise, in step S7 there is output a signal indicating state (1) and in step S8 the
electrical circuit 90 changes the bias potential by removing the DC component, but maintaining the AC voltage of the waveform shown inFIG. 30 . In step S9, the user introduces the test solution into thecell 2. - In this particular implementation, state (2) is not detected, but in step S10 there is detected state (3) of the lipid bilayer being formed across the
aperture 11, as follows. In the absence of a lipid bilayer, theaperture 11 provides a conductive path between theelectrodes 20 and so thecell 2 provides a current response. Typically the current saturates the amplifier, for example as shown in the typical response shown inFIG. 32 . - In contrast, formation of the lipid bilayer prevents flow of ionic current through the
aperture 11 and so thecell 2 provides a capacitive response. As a result, the resultant current signal is a squarewave as shown inFIG. 33 typically having an amplitude of around 250 pA centred on 0 pA. State (3) is detected in step S10 by detecting a current signal showing this capacitive response. Typically the DC resistance is greater than 10 GΩ. - If state (3) is not detected, then in step S11 the detected current is compared to a threshold and then depending on whether the threshold is exceed or not there is output one of two possible error signals in steps S12 and S13 which indicate the absence of bilayer formation.
- However, if state (3) is detected in step S10, then in step S14 there is output a signal indicating that state (3) has been detected and in step S15 the bias voltage is changed by removing the AC waveform and instead applying a DC waveform.
- In step S16 there is detected state (4) of a membrane protein being inserted into the lipid bilayer formed across the
aperture 11. This is detected by detection of the predictable step increases in the DC current response which occurs on insertion of the membrane protein due to the ionic current flowing through the ion channel. This is shown inFIG. 34 which shows the current increasing by a step of the order of 95 pA on insertion of single α-HL membrane protein. In this example, one such insertion occurs at around 0.1 minutes and a second insertion occurs at around 1.7 minutes. Since the electrical composition of the solution and the bias potential are known, the total current reflects the total number of membrane proteins inserted and this information may be determined and subsequently used to calibrate the assay calculations. - If state (4) is not detected within a reasonable period then there is output in step S17 an error signal indicating failure of insertion. Otherwise, in step S18 there is output a signal indicating that state (4) has been detected.
- Thereafter, in step S19 there is detected state (5) of an analyte binding to the membrane protein. This may be detected as follows. When the analyte binds to the membrane protein this temporarily interrupts the ironic current passing through the ion channel causing a characteristic step decrease in the current. Prior knowledge of the analyte binding characteristics (eg current deflection and distribution in event duration) allows the
electrical circuit 90 to identify the relevant binding events. An example of the current is shown inFIG. 35 . The analyte γ-cyclodextrin causes a decrease in the current of the order of 60 pA. Four such binding events are evident inFIG. 35 . Theelectrical circuit 90 detects these characteristic changes as binding events. A signal indicative of this is output in step S20. To detect successive binding events, steps S19 and S20 are repeated. - Finally in step S21 the concentration of the analyte γ-cyclodextrin is calculated based on the kinetics of the measured analyte binding.
Claims (22)
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:
(a) the lipids are dried;
(b) the lipids are dried and comprise less than 50 wt % solvent;
(c) the aqueous solution covers the internal surface before it covers the aperture;
(d) the internal surface on which the lipids are deposited is on the septum;
(e) the aperture has a diameter in at least one dimension which is 20 μm or less;
(f) the method further comprises pre-treating the membrane to increase its affinity to lipids;
(g) the method further comprises pre-treating the membrane with hexadecane;
(h) 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;
(i) the septum further comprises a support sheet on at least one side of the membrane;
(j) the membrane is made from polycarbonate (PC), polytetrafluoroethylene (PTFE), polyethylene, polypropylene, nylon and polyethylene naphthalate (PEN), polyvinylchloride (PVC), polyacrylonitrile (PAN), polyether sulphone (PES), polyimide, polystyrene, polyvinylfluoride (PVF), polyethylene telephthalate (PET), aluminized PET, nitrocellulose, polyetheretherketone (PEEK) or fluoroethylkene polymer (FEP); and/or
(k) the lipids comprise diphantytanoyl-sn-glycero-3-phosphocholine.
3-8. (canceled)
9. A method according to claim 1 , 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 , wherein the aqueous solution is introduced into one chamber in step (c) and the other chamber comprises a gel, or a hydrogel.
12. (canceled)
13. A method according to claim 11 , wherein the gel or hydrogel comprises one or more membrane proteins.
14. (canceled)
15. A method according to claim 2 , wherein the internal surface on which the one or more membrane proteins are deposited is on the septum.
16. A method according to claim 2 , wherein the membrane proteins are dried.
17. A method according to claim 16 , wherein the membrane proteins comprise less than 20 wt % solvent.
18. A method according to any one of claims 2 , wherein the one or more membrane proteins comprise α-hemolysin or a variant thereof.
19-21. (canceled)
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:
(a) the lipids are dried;
(b) the lipids are dried and comprise less than 50 wt % solvent;
(c) 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;
(d) the internal surface on which the lipids are deposited is on the septum;
(e) the aperture has a diameter in at least one dimension which is 20 μm or less;
(f) the membrane has a pre-treatment to increase its affinity to lipids;
(g) the membrane has a hexadecane pre-treatment;
(h) the cell has two chambers;
(i) the cell has two chambers and the inlet opens into one chamber and the other chamber comprises a gel or a hydrogel;
(j) the device further comprises one or more membrane proteins deposited on the same or different internal surface;
(k) the septum further comprises a support sheet material on at least one side of the membrane;
(l) the membrane is made from polycarbonate (PC), polytetrafluoroethylene (PTFE), polyethylene, polypropylene, nylon and polyethylene naphthalate (PEN), polyvinylchloride (PVC), polyacrylonitrile (PAN), polyether sulphone (PES), polyimide, polystyrene, polyvinylfluoride (PVF), polyethylene telephthalate (PET), aluminized PET, nitrocellulose, polyetheretherketone (PEEK) or fluoroethylkene polymer (FEP); and/or
(m) the lipids comprise diphantytanoyl-sn-glycero-3-phosphocholine.
24-32. (canceled)
33. A device according to claim 23 , wherein the gel or the hydrogel comprises one or more membrane proteins.
34. (canceled)
35. A device according to claim 23 , wherein the internal surface on which the one or more membrane proteins are deposited is on the septum.
36. A device according to claim 23 , wherein the one or more membrane proteins:
(a) are dried;
(b) are dried and comprise less than 20 wt % solvent; or
(c) comprise α-hemolysin or a variant thereof.
37-41. (canceled)
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GB0703256.8 | 2007-02-20 | ||
GB0703257A GB2446823A (en) | 2007-02-20 | 2007-02-20 | Formulation of lipid bilayers |
PCT/GB2008/000563 WO2008102121A1 (en) | 2007-02-20 | 2008-02-18 | Formation of lipid bilayers |
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EP2122344B1 (en) | 2019-05-08 |
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US20150268256A1 (en) | 2015-09-24 |
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IL200384A0 (en) | 2010-04-29 |
IL200476A0 (en) | 2010-04-29 |
US20190242913A1 (en) | 2019-08-08 |
US10215768B2 (en) | 2019-02-26 |
EP2122344B8 (en) | 2019-08-21 |
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