WO2003051506A1

WO2003051506A1 - Membrane

Info

Publication number: WO2003051506A1
Application number: PCT/GB2002/005795
Authority: WO
Inventors: James Richard Bushby; John Colyer; Stephen J. Evans
Original assignee: University Of Leeds
Priority date: 2001-12-19
Filing date: 2002-12-19
Publication date: 2003-06-26
Also published as: GB0130318D0; AU2002358214A1

Abstract

There is described a membrane adapted to be supported on a patterned substrate characterised in that the pattern comprises bilayer regions the size of which is less than 20µm in at least one dimension.

Description

Membrane

The present invention relates to a novel form of membrane, methods of preparing such membranes, uses of such membranes and devices comprising such membranes.

Biological membranes play a crucial role in regulating cellular activity. They consist of a flexible phospholipid bilayer that is given shape/support by the cytoskeleton and which is impervious to the flow of ions and large molecules. Such bilayers contain numerous proteins, peptides, sugars, carbohydrates, etc. These proteins are involved in a wide range of cellular activities, such as, signal transduction and immunoresponse, for example, the opening of an acetylcholine receptor channel permits Na⁺ to diffuse into a cell.

One way to mimic events happening in natural cells is to incorporate membrane proteins and receptors into artificial lipid bilayers, attached onto solid supports (sBLM) or suspended over an aperture (BLM). The activities of proteins and receptors can then be detected using electrical techniques.

There are numerous known methods of creating bilayers on surfaces, and of isolating portions of these from their neighbours. One of these is to exploit mixed SAMs.

Self-assembled monolayers (SAMs), are capable of modifying the chemical and physical properties of surfaces in a reproducible, well-defined, and robust fashion.

Generally, a mixed SAM will comprise mixtures of anchor molecules and packing molecules, the packing molecules will often be hydrophilic in character.

The anchor molecule will generally contain two parts, a hydrophobic moiety (e.g. cholesterol, fatty acid or phospholipid moiety), whose function is to insert into the hydrophobic region of the inner leaflet of the bilayer and to anchor or tether the bilayer to the surface, and a hydrophilic, "spacer" segment, which is used to create a hydrophilic region between the bilayer leaflet and the substrate surface. The "packing" molecules, serve to control the density of anchor groups present on the surface of the substrate as well as to help maintain the hydrophilic nature of the substrate surface.

More recently, patterned SAMs have been prepared, for example using microcontact printing and used to support biomembranes. Such patterned SAMs generally comprise regions of free standing lipid bilayers separated by lipid monolayers on top of anchor molecules (Figure 2).

Recently, Cheng, et al. in "Discrete Membrane Arrays" Reviews in Molecular Biotechnology, 74, (2000) 159 - 174 described a typical structure formed in which the square hydrophilic wells were dimensional to cover a, 20 x 20 μm, area and were generally spaced 200 μm apart.

We have now surprisingly found that significantly improved membranes can be prepared by limiting the bilayer region to wells with a diameter of less than 20μm.

Thus, according to a first aspect of the invention we provide a membrane adapted to be supported on a patterned substrate characterised in that the pattern comprises bilayer regions the size of which is less than 20μm in at least one dimension.

It should be understood that the bilayer regions need not be circular in section. Furthermore, the diameter of the bilayer regions should be understood to mean that a substantial proportion of the bilayer regions have the diameter quoted and/or are seeded. In addition, the diameter is generally a mean diameter, that is, for example, at least 50% of the bilayer regions may have a mean diameter of 20μm or less, preferably 60%, more preferably 70%, most preferably 80% and especially at least 90%. The membrane of the invention may, preferentially comprise a size of less than 20μm wherein, this represents the largest (in plane) dimension, i.e. in the plane of the substrate.

The, size of the smallest dimensions (in plane) of the bilayer regions may vary, but they are preferentially less than 15μm, more preferably less lOμm, most preferably less than 5μm and especially less than lμm.

Thus the smallest (in plane) dimension of the bilayer region may be from lnm to lOOOnm and especially from 500nm to 750nm.

In a further aspect of this invention, the dimensions above represent the largest (in plane) dimension of the bilayer region. Thus, in each of the respective embodiments hereinbefore described, the dimension referred to may comprise the largest respective dimension.

Any conventionally known anchoring and/or packing molecules may be used. Such molecules include, by way of example only, anchoring molecules, such as thiol derivatives of cholesterol and lipid disulphides; packing molecules, such as mercapto alcohols, e.g. mercapto ethanol, or thiol derivatives of polyethylene oxide. Thus, preferentially, the anchoring and/or packing molecules may include thiol, disulphide, sulphide groups (for anchoring to Au, Ag and other noble metals), organosilanes (for anchoring to SiO₂, or other hydroxylated surfaces) and alkenes (for anchoring to Si[H]).

Thus, the anchor molecules will generally comprise a lipophilic "head" region and a hydrophilic tail region, functionalised to bond the substrate.

The lipophilic head group region may comprise one or more of the groups normally associated with naturally occurring or synthetic lipids. Naturally occurring lipid molecules include cholesterol or other sterols, phosphatidyl choline, phosphatidyl ethanolamine, mono-, di- or tri-methylated phosphatidyl ethanolamine, phosphatidic acid, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol, distributed head groups as found in cardiolipins, ganglioside head groups, sphingomyelin head groups, plasmalogen head groups, glycosyl, galactosyl, digalactosyl, sulfosugar, phosphosugar, N-acetyl neuramic acid, sialic acid, aminosugar head groups carbohydrate head groups, gal(betal-3)galNAc(betal-4) [NAcNeu(alpha2-3] gal (betal-4) glc-ceramide. The hydrophilic tail region may comprise saccharides, polysaccharides, oligomers of ethylene glycol, ethylene glycol, oligomers of propylene glycol, propylene glycol, amino acids, oligomers of amino acids, combinations of oligomers of ethylene glycol or propylene glyco functionalised with amino acids or other ionic species or any combination or derivative of the above.

The hydrophilic region of the packing molecules may comprise one or more hydrophilic compounds. The hydrophilic region of the packing molecule may be composed of ethers, peptides, amides, amines, esters, saccharides, polyols, charged groups (positive and/or negative), electroactive species or combinations thereof. The main requirement of the hydrophilic region of the packing molecule is that it provides a water-filled space between the bilayer (or the anchor:lipid hybrid layer) and the substrate.

The membranes of the invention are advantageous in that, inter alia, the stability, function and quality of the membrane bilayer is improved over prior art membranes.

Prior art mixed SAMs have generally comprised random array bilayer regions. Thus, the use of a pre-defined patterned SAM in a controlled, i.e. non-random, fashion is especially advantageous.

Thus, the controlled patterned SAMs in the membranes of the invention will generally comprise bilayer regions and monolayer regions. In a further aspect of the invention, the monolayer or hydrophilic regions may be "seeded" with a proportion of a ligand. This is a novel and especially advantageous aspect of the present invention which improves bilayer formation on the substrate and improves the stability of such bilayers.

Thus, according to this aspect of the invention we provide a membrane adapted to be supported on a patterned substrate, characterised in that the membrane supporting regions of the substrate are provided with a proportion of ligands.

The membrane may therefore be adapted to be anchored to a substrate surface and may, optionally, comprise a self-assembled monolayer (SAM).

The nature of the ligands may vary. Thus, for example, the ligands may comprise lipophilic anchor molecules as hereinbefore described. Alternatively, the ligands may comprise fusogens. In a yet further alternative, the ligands may comprise a mixture of lipophilic anchors and fusogens. Furthermore, the fusogens may be non- fusogenic compounds that become fusogenic by triggering, for example, chemical triggering by exposure to low pH or an oxidative environment.

In this aspect of the invention the patterned bilayer regions may comprise a random pattern or an ordered e.g. controlled pattern. Furthermore, the size of bilayer regions may vary and may be up to lOOμm in their smallest dimension. In particular, the "seeded" bilayer regions need not be limited to a minimum dimension of less than 20μm.

Nevertheless, in a preferred embodiment of this aspect of the invention, the "seeded" bilayer regions may still be less than 20μm in their smallest dimension.

When the bilayer regions are provided with a proportion of ligands, the ligands themselves may be randomly scattered or may be positioned in a controlled pattern. The proportion of ligands "seeded" in the bilayer region may vary, but may be, for example, from up to 50% of the bilayer region of the substrate.

The lipids used in the membranes of the present invention may comprise any conventionally known lipids, for example, those described in International Patent Application No. WO 94/07593.

When the ligand is a fusogen it may comprise any conventionally known fusogen. In a preferred embodiment, the fusogen may be a non-metallic fusogen, e.g. polyethylene glycol. Alternatively, the fusogen may be a cation, plus a cation binding ligand, such as a carboxylic acid. When a cation binding ligand is used, the cation may vary, but may be, for example, Ca²⁺, Mg²⁺, Al³⁺, etc.

Alternatively, the fusogen may comprise a cation and a phosphate moiety. It is within the scope of the present invention to use a mixture of fusogens, for example, a mixture of one or more carboxylic acids and one or more phosphate moieties.

The membranes of the invention may be manufactured using conventional processes known per se, for example by vesicle fusion.

The membranes of the invention are advantageous in that they may be useful, inter alia, in the manufacture of biosensors or in the field of drug discovery. Other uses not mentioned here can also be contemplated.

A variety of substrates may be used in the manufacture of the membranes of the invention. Such substrates include, but shall not be limited to noble metals, such as gold, silver, platinum, or palladium; or other metals/metal derivatives, e.g. silicon, silicon oxide or silicon nitride. Alternatively, non-metal substrates may be used, for example, a glass or a polymer substrate, such as a plastics substrate. In addition, other substrates may be contemplated. In a further aspect of the invention, the substrate may be a coated substrate, e.g., a noble metal substrate coated with a photoresist, such as SU8.

The membrane material itself may comprise any conventionally known lipid, such lipids may be synthetic lipids or naturally occurring lipids. Examples as in claim (1) where the biomembrane is a bilayer, monolayer or hybrid monolayer/bilayer structure.

The membrane may, for example, comprise a fragment of a natural cell, h this case the cell may contain a channel or channel modulator, in some cases these components may be engineered to possess non-natural sequences or structures.

The membranes of the invention are advantageous in that, inter alia, the fabrication, stability, and performance of the membrane bilayer is improved over prior art membranes.

The membranes may be useful, inter alia, in the manufacture of electrodes and/or biosensors or in the field of drug discovery. Other uses not mentioned here can also be contemplated.

Thus, according to a yet further aspect of the invention we provide an electrode which comprises a membrane as hereinbefore described.

The device could be used to identify compounds which alter the function of the membrane (e.g. membrane potential, pore-forming agents, ion channel activity, systems engineered to report through ion channels, transport enzymes). An array of pixels containing the membrane, with for example, an ion channel incorporated in the membrane could be fabricated to permit the high throughput exposure of the ion channel to many diverse compounds in a search to identify compounds which affect ion channel function. Patterned membranes can be formed by creating wells, or corrals, out of resist or other materials. Membranes can then be formed at the bottom of the wells via vesicle rupture at the substrate surface. The patterning thus arises due to the barriers separating the neighbouring wells.

Such electrodes are especially advantageous in that they may be used in the manufacture of sensors, such as biosensors. Therefore, in a yet further aspect of the invention we provide a sensor, e.g. a biosensor, which comprises an electrode as hereinbefore described.

The sensor of the invention may comprise a means of detecting the amount of a species of interest in a sample and a membrane in accordance with the invention, the membrane providing both a barrier function and a biocompatible interface function between the detecting means and the sample.

According to this aspect of the invention the biosensor may comprise a reference electrode, e.g. a silver/silver chloride electrode. When used as a sensor, the membrane may have incorporated in it one or more proteins, peptides, ionophores and/or other bioactive molecules.

In this aspect of the invention, we also provide a method of determining the amount of a selected component in a sample, the method comprising using a sensor device as hereinbefore described.

The electrodes and/or sensors of the invention offer a reduced capacitance and/or increased resistance giving improved signal/noise ration in the detection of electronic events. The electrodes and/or sensors may also provide enhanced -detection of ion- selectivity of ion channel proteins (or peptides/ionophores) wherein the activity of individual ion channel molecules can be recorded and/or other electronic events, such as pore formation, transport processes, changes in membrane potential etc. may be monitored. According to a further aspect of the invention, we provide a method of manufacturing a biosensor comprising a membrane of the invention.

According to a further aspect of the invention, we provide a method of manufacturing a membrane of the invention which comprises generating a pattern by a method selected from the group, photolithography, e-beam lithography, microcontact printing, fluid flow, ink-jet printing and deposition of Langmuir Blodget films.

The invention will now be described by way of example only and with reference to the accompanying drawings, in which;

Figure 1 is a schematic representation of a solid supported bilayer;

Figure 2 is a schematic representation of a bilayer supported on a patterned SAM;

Figure 3 is an illustration of a lipid vesicle unrolling on a micropatterned SAM; and Figure 4 is a graph illustrating the variance of capacitance with bilayer diameter.

Referring to Figure 1, the substrate (1) is provided with anchor molecules (2) and the packing molecules (3), with hydrophilic portions (4 and 5). The bilayer (6) comprises an inner leaflet (7) and an outer leaflet (8). The hydrophilic portions (4 and 5) serve to provide an aqueous environment between the bilayer(6) and the solid support substrate (1).

Referring to Figure 2, the SAM was produced using microcontact printing. A cholesterol was stamped on to a bare gold surface, the sample was then placed into a solution containing a short chain ethyleneoxy derivative. Upon incubation with vesicles a lipid monolayer was adsorbed on the hydrophobic surfaces (region 1) and a bilayer was formed on the hydrophilic areas (region 2).

Experiment 1 Formation of supported bilayer of improved quality 1. Manufacture of mask/stamp capable of forming pixels of lOOnm to 20μm size

Chromium-glass photo-lithography masks were constructed using electron beam lithography at the Rutherford-Appleton Laboratory, Didcot, UK. The mask consists of seven separate patterns each being an array of circles spaced on a regular square lattice. The circle diameters range from 0.1 μm to 16 μm. The ratio of the circle area to surrounding area is kept constant.

Patterned stamp

The mask was used to create arrays of raised columns in SU18 photoresist (Chestech, Rugby, UK) using standard lithographic techniques. Poly-dimethylsiloxane (PDMS) was applied to the patterned photoresist and baked at 60° C for 1 hour. The baked PDMS was peeled off and examined under an optical microscope to ensure the pattern had been effectively reproduced in the stamp. Each individual patterned stamp has gross dimensions of 7 x 7 mm.

2. Creation of a patterned gold surface (pixels of a range of sizes)

Gold coating

120nm of gold was thermally evaporated (Balzers thermal evaporator at pressures < 4 x 10^"6mbar) onto a 5nm chromium adhesion on cleaned (2% Helmanex, sonication, ethanol) glass microscope slides.

Microcontact printing

The gold surfaces were cleaned in piranha solution (30% H₂O₂ / 70% H₂SO ) for 1 minute followed by washing in pure water. The PDMS stamps were 'inked' with

5mM cholesteroyl thiol in ethanol for 1 minute, dried under a stream of nitrogen for ~ 30s and applied to the cleaned gold surfaces. The stamp was left on the surface for 1 minute to allow time for transfer. The stamp was carefully peeled off, the substrate rinsed with ethanol and then immersed for 2 min in 5mM mercaptoethanol solution in ethanol followed by rinsing in copious quantities of ethanol.

3. Preparation of lipid vesicles (SUV, state lipid mixture used)

Lipid vesicles were prepared by hydrating egg-phosphatidylcholine in 0.1M KC1 for 1 hour to give a lmg / ml dispersion, then extruding through 50 nm diameter polycarbonate membranes for 18 cycles. The resultant vesicle diameters were 60-70 nm. The vesicles were diluted to a working concentration of 0.2mg / ml with 0.1M KC1.

4. Depositing vesicles on patterned surface

The vesicle solution was applied to the patterned surface and left for 90min followed by rinsing in 0.1M KC1.

5. Measurement of capacitance of bilayer over pixels of different sizes

Electrochemical impedance measurements were made on a Solartron 1260 frequency response analyser coupled to an EG&G 273A potentiostat. The cell was operated in two electrode mode with a coiled platinum wire counter electrode. A 12 mV r.m.s. AC potential was applied at the open circuit potential of the cell. The applied AC frequency was swept between 50kHz and 300 MHz. Measurements were first made on the bare SAM in 0.1 M KC1, then measurements made at regular intervals during the lipid deposition. Subsequently, the cell was rinsed with 0.1M KC1 and a final impedance measurement taken.

The high frequency part of the impedance spectrum pertains to the organic film whilst the low frequency impedance can be ascribed to the gold double layer capacitance. Fitting a simple RC series circuit to the data over the 50kHz - lMHz range gives a reasonable measure of the capacitance of the organic film (figure 4).

Experiment 2 Measuring ion channel activity in bilayer pixels of differing size

1. Formation of patterned surface

As in Experiment 1.1 and 1.2 above. This would give patterned SAMs with the 'tethering' regions ranging in diameter from 0.1 μm to 16 μm.

2. Incorporation of the ion channel gramicidin

This was done at a gramicidin peptide:lipid ratio of O.lmole % into egg phosphatidylcholine lipid vesicles by sonication.

3. Depositing ion channel containing vesicles onto patterned surface

Vesicles incorporating gramicidin allowed to react with the patterned SAMs for 90min then washed with 0.1M KC1 to remove excess vesicles.

4. Measurement of ion channel activity; measurement of ion selectivity of ion channel

Ion channel activity measured by impedance methods. Measurements were carried out in different chloride salt solutions (KC1, NaCl, CsCl, BaCl₂) at 0.1M depending on the desired cation. The cell was thoroughly flushed between different electrolytes with Millipore water and four cell volumes of the desired electrolyte.

Re 1. Microcontact Printing of Lipophilic Self-assembled Monolayers for attachment of Biomimetic lipid Bilayers to surfaces A.T.A. Jenkins, N. Boden, RJ. Bushby, S.D. Evans*, P.F. Knowles, R.E.Miles, S.D. Ogier, H. Schδnherr, G.J. Vancso J. Am. Chem. Soc. 1999 , 121, 521

2. Ion-Selective lipid Bilayers Tethered to Microcontact Printed Self- Assembled Monolayers Containing Cholesterol Derivatives A.T.A. Jenkins, RJ. Bushby, N. Boden, S.D. Evans*, P.F. Knowles, Q. Liu, R.E.Miles, S.D. Ogier Langmuir Letters 1998, 14

Experiment 3

Detection of single channel activity in supported bilayers

1. Formation of patterned surfaces defining a range of pixel sizes (lOOnm to 20 μm)

As in experiment 1.1 & 1.2.

2. Incorporation of ion channel into egg phosphatidylcholine sonicated vesicles at 0.01 to 0.1 molar %

As in experiment 2.2

3. Bilayer formation on patterned surface

As in Experiment 2.3.

4. Detection and analysis of single ion channel activity

Ion conductance and impedence spectroscopy measurements determine bilayer characteristics and ion channel activity. Single ion channel sensitivity results in A scale changes in current. Experiment 4

Improved vesicle fusion onto pixels using mixtures of anchor/packing molecule or fusogen molecules within the pixel zone

1. Manufacture of patterned stamps

As in Experiment 1.1 to give pixel sizes in range 0.1 μm to 16 μm.

2. Preparation of 5 mM concentration mixtures of cholesteroyl thiol/mercaptoethanol and fusogen/mercaptoethanol

This was done in ethanol in proportions ranging from 20% to 50% of the cholesteroyl thiol component; or with fusogen/mercaptoethanol (in ImM ethanol solution). Fusogen consists of (a) the metal salt of a carboxylic acid functionalised SAM (20- 50mole%) or (b) polyethylene glycol functionalised thio derivatives (20-50mole%).

3. Patterning of the gold coated surface

As in Experiment 1.2 but using the cholesterol-based anchor molecules and mercaptoethanol from Experiment 4.2 deposited on the pixels.

4. Preparation of vesicles and depositing of vesicles on patterned surface

As in Experiment 1.3 and 1.4.

5. Measurement of the kinetics of bilayer formation on pixels

Impedance spectroscopy, as in Experiment 1.5, and comparison with similar data where the pixels are composed of homogeneous cholesterol- based anchor or mecaptoethanol. 6. Measurement of capacitance and resistance of bilayers as a function of pixel size

Comparison with data for same pixel size with homogeneous cholesterol- based anchor or mecaptoethanol

7. Measurement of stability of bilayer once formed

Comparison with data for same pixel size with homogeneous cholesterol- based anchor or mecaptoethanol pixels by monitoring changes in impedance with time.

8. Test ion selectivity behaviour of ion channelling peptide gramicidin in these mixed composition pixel zones

As in Experiment 2.2 to 2.4.

9. Test single ion channel behaviour of gramicidin in these mixed composition pixel zones

As in Experiment 3.2 to 3.4.

Claims

1. A membrane adapted to be supported on a patterned substrate characterised in that the pattern comprises bilayer regions the size of which is less than 20μm in at least one dimension.

2. A membrane according to claim 1 characterised in that the size of less than 20μm represents the largest in plane dimension.

3. A membrane according to claims 1 or 2 characterised in that the size of each of the bilayer regions in at least one dimension is less than 15μm.

4. A membrane according to claims 1 or 2 characterised in that the size of each of the bilayer regions in at least one dimension is less lOμm .

5. A membrane according to claim 4 characterised in that the size of each of the bilayer regions in at least one dimension is less than lμm.

6. A membrane according to claim 5 characterised in that the size of each of the bilayer regions in at least one dimension is from 500nm to 750nm.

7. A membrane according to claim 1 characterised in that the membrane utilises a controlled pattern.

8. A membrane according to claim 1 characterised in that the membrane comprises a self-assembled monolayer (SAM).

9. A membrane according to claim 1 wherein the substrate is provided with anchor and/or packing molecules characterised in that the anchor and/or packing molecules contain a functional group to facilitate surface attachment.

10. A membrane according to claim 9 characterised in that the functional group is selected from the group thiols, organosilanes, phosphoric and alkanoic acids and others known to those skilled in the art.

11. A membrane according to claim 1 characterised in that the head region of the anchor molecule is a lipophilic head group, selected from the group of synthetic lipids or natural lipids including cholesterol or other sterol, phosphatidyl choline, phosphatidyl ethanolamine, mono-, di- or tri-methylated phosphatidyl ethanolamine, phosphatidic acid, phosphatidyl serine, phosphatidyl glycerol, phosphatidyl inositol, distributed head groups as found in cardiolipins, ganghoside head groups, sphingomyelin head groups, plasmalogen head groups, glycosyl, galactosyl, digalactosyl, sulfosugar, phosphosugar, N-acetyl neuramic acid, sialic acid, aminosugar head groups carbohydrate head groups, gal(betal-3)galNAc(betal-4) [NAcNeu(alpha2-3] gal (betal-4) glc-ceramide.

12. A membrane according to claim 1 characterised in that the tail region of the anchor molecule is a saccharide, polysaccharide, oligomer of ethylene glycol, ethylene glycol, oligomers of propylene glycol, propylene glycol, amino acids, oligomers of amino acids, combinations of oligomers of ethylene glycol or propylene glycol functionalised with amino acids or other ionic species or any combination or derivative.

13. A membrane according to claim 1 characterised in that the anchor molecule selected is a thiol cholesterol derivative or a lipid sulphide.

14. A membrane according to claim 1 characterised in that the hydrophilic region of the packing molecule comprises one or more hydrophilic compounds.

15. A membrane according to claim 14 characterised in that the hydrophilic region of the hydrophilic compounds is composed of ethers, peptides, amides, amines, esters, saccharides, polyols, charged groups (positive and/or negative), electroactive species or combinations thereof.

16. A membrane according to claim 1 characterised in that the packing molecule is an alcohol.

17. A membrane according to claim 16 characterised in that the packing molecule is a functionalised polyethylene oxide derivative.

18. A membrane according to claim 16 characterised in that the alcohol is mercapto ethanol.

19. A membrane adapted to be supported on a patterned substrate, characterised in that the membrane supporting regions of the substrate are provided with a proportion of ligands.

20. A membrane according to claim 19 characterised in that the membrane is a SAM.

21. A membrane according to claim 19 characterised in that the ligands comprise lipophilic anchors.

22. A membrane according to claim 19 characterised in that the ligands comprise fusogens.

23. A membrane according to claim 21 characterised in that the ligands comprise a mixture of lipophilic anchors and fusogens.

24. A membrane according to claim 22 characterised in that the fusogens comprise non-fusogenic compounds that become fusogenic by triggering,

25. A membrane according to claim 19 characterised in that the bilayer regions comprise a controlled pattern..

26. A membrane according to claim 19 characterised in that the bilayer regions are up to lOOμm in at least one dimension.

27. A membrane according to claim 26 characterised in that a substantial portion the bilayer regions are less than 20μm in at least one dimension.

28. A membrane according to claim 19 characterised in that the proportion of ligands "seeded" in the bilayer region is from up to 50 mole% of the bilayer region.

29. A membrane according to claim 22 characterised in that the fusogen is a non- metallic fusogen.

30. A membrane according to claim 22 characterised in that the fusogen is a cation.

31. A membrane according to claim 30 characterised in that the cation is selected from Ca²⁺, Mg²⁺, Al³⁺.

32. A membrane according to claim 22 characterised in that the fusogen comprises a phosphate moiety.

33. A membrane according to claim 22 characterised in that the fusogen is a mixture of fusogens.

34. A membrane according to any one of claims 1 and 19 characterised in that the substrate surface is: a noble metal, such as gold, silver, platinum, or palladium; an oxide surface such as silicon oxide, Al_xO_y; a nitride surface, such as silicon nitride; a glass or an organic polymer.

35. A membrane according to any one of claims 1 and 19 characterised in that the substrate is a polymer.

36. A membrane according to any one of claims 1 and 19 characterised in that the substrate is coated with a photoresist.

37. A membrane according to any one of claims 1 and 19 characterised in that the photoresist such as SU8.

38. A membrane according to any one of claims 1 and 19 characterised in that the membrane material comprises a synthetic lipid or a naturally occurring lipid, or a modified lipid or a mixture thereof.

39. A membrane according to claim 1 characterised in that the membrane has incorporated in it one or more proteins, peptides, ionophores and/or other bioactive molecules.

40. A membrane according to any one of claims 1 and 19 characterised in that the membrane comprises a fragment of a natural cell, or contains a natural or engineered channel or a natural or engineered channel modulator.

41. An electrode which comprises a membrane according to claim 1 or claim 19.

42. A device, which comprises an electrode according to claim 1 or claim 19.

43. A device according to claim 42 characterised in that the device comprises a means for detecting the amount of a species of interest in a sample.

44. A device according to claim 42 characterised in that the sensor comprises a reference electrode.

45. A device according to claim 41 which is adapted for use in the screening of drugs or the identification of potential pharmaceutical products.

46. A method of manufacturing a device according to claim 42, which uses microcontact printing to create a SAM support onto which a lipid bilayer is fonned by exposure to a suspension of phospholipid vesicles.

47. A method of manufacturing a membrane according to claim 1 or claim 19 which comprises generating a membrane supporting pattern by a method selected from the group, photolithography, e-beam lithography, microcontact printing, fluid flow, and ink-jet printing.

48. A device according to claim 42 which permits the measurement of electrical events with improved signahnoise ratio.

49. A device according to claim 42 winch permits the measurement of the activity of a single ion channel.

50. A device according to claim 42 characterised in that the device has increased lifetime.

51. A device comprising an areay of membranes according to either of claim 1 or claim 19.

52. A membrane or a sensor device substantially as described with reference to the accompanying examples and drawings.

P36722 O