U.S. patent application number 12/527687 was filed with the patent office on 2010-08-05 for formation of lipid bilayers.
Invention is credited to Terence Alan Reid, Gurdial Singh Sanghera, Steven Paul White.
Application Number | 20100196203 12/527687 |
Document ID | / |
Family ID | 39385567 |
Filed Date | 2010-08-05 |
United States Patent
Application |
20100196203 |
Kind Code |
A1 |
Sanghera; Gurdial Singh ; et
al. |
August 5, 2010 |
Formation of Lipid Bilayers
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.
Inventors: |
Sanghera; Gurdial Singh;
(Oxford, GB) ; White; Steven Paul; (Oxford,
GB) ; Reid; Terence Alan; (Bicester, GB) |
Correspondence
Address: |
BAKER BOTTS L.L.P.;PATENT DEPARTMENT
98 SAN JACINTO BLVD., SUITE 1500
AUSTIN
TX
78701-4039
US
|
Family ID: |
39385567 |
Appl. No.: |
12/527687 |
Filed: |
February 18, 2008 |
PCT Filed: |
February 18, 2008 |
PCT NO: |
PCT/GB2008/000563 |
371 Date: |
December 21, 2009 |
Current U.S.
Class: |
422/68.1 ;
427/2.11 |
Current CPC
Class: |
G01N 33/92 20130101;
G01N 33/48728 20130101 |
Class at
Publication: |
422/68.1 ;
427/2.11 |
International
Class: |
G01N 33/00 20060101
G01N033/00; B05D 3/00 20060101 B05D003/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 20, 2007 |
GB |
0703256.8 |
Feb 20, 2007 |
GB |
0703257.6 |
Claims
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 .mu.m 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 .alpha.-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 .mu.m 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 .alpha.-hemolysin or a
variant thereof.
37-41. (canceled)
Description
[0001] The invention relates to the formation of lipid bilayers. In
particular, the invention relates to the formation of a lipid
bilayer across an aperture.
[0002] 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.
[0003] 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.
[0004] 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.
[0005] Other common methods of bilayer formation include
tip-dipping, painting bilayers and patch-clamping of liposome
bilayers.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] In one aspect, the present invention provides a method for
forming a lipid bilayer across an aperture, comprising:
[0011] (a) providing a cell having a chamber adjacent to a septum
comprising a membrane having an aperture capable of supporting a
lipid bilayer;
[0012] (b) depositing one or more lipids on an internal surface of
the chamber;
[0013] (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
[0014] (d) moving the interface past the aperture at least once to
form a lipid bilayer across the aperture.
[0015] In another aspect, the invention provides a device for
forming a lipid bilayer comprising,
[0016] (a) a cell having a chamber adjacent to a septum comprising
a membrane having an aperture capable of supporting a lipid
bilayer; and
[0017] (b) one or more lipids deposited on an internal surface of
the chamber,
[0018] wherein the cell comprises an inlet for introducing an
aqueous solution into the chamber having lipid deposited
therein.
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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).
[0025] 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.
[0026] 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.
[0027] The cell comprises an internal chamber. The chamber may be
any size and shape. The volume of the chamber is typically 0.1
.mu.l 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.
[0028] 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.
[0029] 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 .mu.m to 25 .mu.m thick.
[0030] 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.
[0031] 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 .mu.l to
10 .mu.l. 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 .mu.l of 1% (v/v) hexadecane and 0.6
mg/ml lipid, such as DPhPC, in pentane.
[0032] Some specific pretreatments are set out in Table 1 by way of
example and without limitation.
TABLE-US-00001 TABLE 1 Volumes applied Pretreatment formulation by
capillary pipette 0.3% hexadecane in pentane 2x 1 .mu.l 1%
hexadecane in pentane 2x 0.5 .mu.l; 2x 0.5 .mu.l; 1 .mu.l; 2x 1
.mu.l; 2x 1 .mu.l; 2x .mu.l; 2x 2 .mu.l; 5 .mu.l 3% hexadecane in
pentane 2x 1 .mu.l; 2 .mu.l 10% hexadecane in pentane 2x 1 .mu.l; 2
.mu.l; 5 .mu.l 0.5% hexadecane + 5 mg/ml DPhPC 5 .mu.l lipid in
pentane 1.0% hexadecane + 0.6 mg/ml 2x 0.5 .mu.l DPhPC lipid in
pentane 1.5% hexadecane + 5 mg/ml DPhPC 2 .mu.l; 2x 1 .mu.l lipid
in pentane
[0033] 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.
[0034] 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.
[0035] Although the pretreatment provides a beneficial effect, it
is not essential.
[0036] 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.RTM. (polyoxymethylene or acetal homopolymer),
Mylar.RTM. (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).
[0037] 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.
[0038] 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
.mu.m 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.
[0039] The aperture may be created using any method. Suitable
methods include, but are not limited to, spark generation and laser
drilling.
[0040] 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.
TABLE-US-00002 TABLE 2 Septum Aperture 6 .mu.m thick biaxial 25
.mu.m diameter spark-generated hole polycarbonate 6 .mu.m thick
biaxial 20 .mu.m diameter laser-drilled tapered hole polycarbonate
6 .mu.m thick biaxial 10 .mu.m diameter laser-drilled tapered hole
polycarbonate 5 .mu.m thick PTFE 10 .mu.m diameter spark-generated
holes 5 .mu.m thick PTFE 10 .mu.m diameter laser-drilled tapered
hole 5 .mu.m thick PTFE 5 .mu.m diameter laser-drilled tapered hole
10 .mu.m thick HD polyethylene 15 .mu.m diameter spark-generated
hole 4 .mu.m thick Polypropylene 15 .mu.m diameter spark-generated
hole 25 .mu.m thick Nylon (6,6) 20 .mu.m diameter spark-generated
hole 1.3 .mu.m thick PEN 30 .mu.m diameter spark-generated hole 14
.mu.m thick conductive 30 .mu.m diameter spark-generated hole
polycarbonate 7 .mu.m thick PVC 20 .mu.m diameter laser-drilled
hole
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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).
[0053] 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.
[0054] 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.
[0055] 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.
[0056] 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.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] 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.
[0067] 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, .beta.-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.
[0068] The membrane proteins preferably comprise .alpha.-hemolysin
or a variant thereof. The .alpha.-hemolysin pore is formed of seven
identical subunits (heptameric). The polynucleotide sequence that
encodes one subunit of .alpha.-hemolysin is shown in SEQ ID NO: 1.
The full-length amino acid sequence of one subunit of
.alpha.-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 .alpha.-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.
[0069] 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 .alpha.-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.
[0070] 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.
[0071] 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-US-00003 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
[0072] Non-conservative substitutions may also be made at one or
more positions within SEQ ID NO: 2 or 3, wherein the substituted
residue is replaced with an amino acid of markedly different
chemical characteristics and/or physical size. One example of a
non-conservative substitution that may be made is the replacement
of the lysine at position 34 in SEQ ID NO: 2 and position 9 in SEQ
ID NO: 3 with cysteine (i.e. K34C or K9C). Another example of a
non-conservative substitution that may be made is the replacement
of the asparagine residue at position 43 of SEQ ID NO: 2 or
position 18 of SEQ ID NO: 3 with cysteine (i.e. N43C or 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.
[0073] 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.
[0074] 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 .beta.-barrel
domain of SEQ ID NO: 2 or 3.
[0075] 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 .beta.-barrel part of chimeric proteins are typically formed
by the fragments or portions of SEQ ID NO: 2 or 3.
[0076] 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.
[0077] 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/).
[0078] 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. .sup.125I,
.sup.35S, enzymes, antibodies, polynucleotides and linkers such as
biotin.
[0079] 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.
[0080] 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.
[0081] 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 NaBH.sub.4, amidination with methylacetimidate or
acylation with acetic anhydride.
[0082] 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.
[0083] 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.
[0084] 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:
[0085] FIG. 1 is a view of an example of a device of the
invention;
[0086] FIG. 2 is a schematic diagram of an electrical circuit that
can be used with the device of the invention;
[0087] FIG. 3 is a graph of the current response to a 20 mV 50 Hz
alternating current (a.c.) waveform in the absence of the high
resistance electrical sealing of the aperture by a bilayer;
[0088] 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;
[0089] FIG. 5 is a graph of the stepwise increase of 60 pA direct
current (d.c.) as .alpha.-hemolysin pores automatically insert into
the bilayer formed by the Montal and Mueller method;
[0090] FIG. 6 is a graph of the characteristic interruptions in the
current caused by single molecules of .gamma.-cyclodextrin
transiently binding to the .alpha.-hemolysin pores;
[0091] 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;
[0092] FIG. 8 shows an expanded view of the current (1 second full
scale) during the final minute of recording shown in FIG. 7;
[0093] FIG. 9 is a graph of the current recorded over the duration
of a single test using a specially constructed cell;
[0094] FIG. 10 is a graph of the characteristic square wave
indicative of bilayer formation in accordance with the invention in
a specially constructed cell;
[0095] FIG. 11 is a graph of the current response to a 20 mV 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;
[0096] FIG. 12 is a graph of step increases in the current
.about.100 pA as .alpha.-hemolysin pores automatically insert into
the bilayer in the specially constructed cell; and
[0097] FIG. 13 is a graph of the characteristic interruptions in
the current caused by single molecules of .gamma.-cyclodextrin
transiently binding to the .alpha.-hemolysin pores in the specially
constructed cell;
[0098] FIG. 14 is a perspective view of a sensor system;
[0099] FIG. 15 is a perspective view of a cell of the sensor
system;
[0100] FIG. 16 is a cross-sectional of the cell, taken along line
in FIG. 2;
[0101] FIG. 17 is a perspective view of a support sheet of the cell
in isolation;
[0102] FIG. 18 is a perspective view of a body of the cell in
isolation with a first arrangement for an inlet;
[0103] FIG. 19 is a perspective view of a cover sheet of the cell
in isolation with a second arrangement for an inlet;
[0104] FIG. 20 is a cross-sectional view of the cell similar to
that of FIG. 3 but showing introduction of a sample;
[0105] FIG. 21 is an expanded, partial cross-sectional view of a
cell containing gel with a gap between the gel and an aperture;
[0106] FIG. 22 is an expanded perspective view of the connector
portion of the reader unit;
[0107] FIG. 23 is a perspective view of a rigid metal body
connected to the reader unit;
[0108] FIG. 24 is a cross-sectional view of the rigid metal body,
taken along line XII-XII in FIG. 11;
[0109] FIG. 25 is a cross-sectional view of the cell contained in a
Faraday cage;
[0110] FIGS. 26 to 28 are diagrams of various forms of the
electrical circuit in the reader unit; and
[0111] FIG. 29 is a flow chart of the operation of the reader unit;
and
[0112] FIG. 30 is a graph of a bias voltage applied to the reader
unit; and
[0113] FIGS. 31 to 35 are graphs of the current signal generated in
the cell during operation.
[0114] 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.
[0115] A device 130 in accordance with the invention is illustrated
in FIG. 1. The device 130 includes an electrophysiology 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.
[0116] The electrophysiology cell 101 comprises two chambers body
portions 102 having constructions which are mirror images of each
other. The chamber body portions 102 may be made from Delrin.RTM.
(polyoxymethylene or acetal homopolymer). The chamber body portions
102 each define a chamber portion 103 having an opening in the
upper surface 104 of the respective chamber body portion 102. The
chamber portions 103 each have a volume of a few millilitres, for
example 1.5 ml. The chamber portions 103 have no wall on a side
surface 105 of the respective chamber body portion 102. To form a
chamber body, the two chamber body portions 102 are assembled
together with their side surfaces 105 facing one another so that
the respective chamber portions 103 are aligned and together form a
chamber. The chamber body portions 102 may be attached by any
suitable means, typically a clamp or an adhesive.
[0117] The electrophysiology cell 101 further comprises a membrane
106 made of polycarbonate or any other suitable polymer. Each face
of the membrane 106 may be pre-treated in a conventional manner,
for example with 10% (V/V) hexadecane in pentane. The membrane 106
is positioned between the facing side surfaces 105 of the two
chamber body portions 102, for example by adhering both chamber
body portions 102 to the membrane 106. Accordingly, the membrane
106 forms a wall which divides the chamber formed by the two
chamber portions 103.
[0118] The membrane 106 has an aperture 107 which is aligned with
the chamber portions 103 when the electrophysiology cell is
assembled. The membrane 106 is sufficiently thin to facilitate
formation of a lipid bilayer, for example being 25 .mu.m thick. The
aperture 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 .mu.m or less. The cell 101
comprises inlets for introducing an aqueous solution into each
chamber portion 103, namely the openings in the upper surface 104
of each chamber body portion 102.
[0119] The device 130 further comprises lipids 108 deposited in
each chamber portion 103 of each one of the chamber body portions
102. The shape of the patch of lipids 108 deposited in each chamber
portion 103 may vary.
[0120] 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 both chamber
portions 103 simultaneously via openings in the upper surface 104
of each chamber body portion 102. The aqueous solution will cover
the lipids 108 deposited in each chamber 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 both chamber portions 103 and move past
the aperture 107 on both sides of the membrane 106 thereby forming
a lipid bilayer across the aperture 107. In this example, the lipid
108 is covered by the aqueous solution before the aperture 107 is
covered.
[0121] The electrophysiology cell 101 can further includes
respective electrodes (not shown in FIG. 1) provided in each
chamber portion 103 of each one of the chamber body portions 102.
The electrodes may be Ag/AgCl electrodes. The electrodes may form
part of an electrical circuit 120 which is capable of measuring an
electrical signal across the lipid bilayer. A suitable electrical
circuit 120 is illustrated schematically in FIG. 2 and is of a
conventional type for performing stochastic sensing by detecting
the current flowing across the lipid bilayer.
[0122] The electrodes 109 are connected to an amplifier 121 such as
a patch-clamp amplifier (eg an Axopatch 200B supplied by Axon
Instruments) which amplifies the current signal output from the
electrodes 109.
[0123] The current signal output by the amplifier 121 is supplied
through a low-pass filter 122, such as a Bessel filter (eg with
characteristics 80 dB/decade with a corner frequency of 2 kHz).
[0124] 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 a computer 124 for analysis. The computer
124 may be a conventional personal computer running an appropriate
program to store the current signal and display it on a display
device.
[0125] As an alternative, the invention may be applied to a device
which is the cell of the sensor system described in detail
below.
[0126] 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.
[0127] 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 .mu.m. The pentane
was allowed to evaporate.
[0128] 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 .mu.l 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.
[0129] 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.
[0130] Formation of a bilayer across the aperture was confirmed by
creation of a high resistance sealing of the aperture
(>10.OMEGA.), by measurement of the capacitance of the high
resistance seal and by the subsequent successful insertion of
.alpha.-hemolysin (.alpha.-HL) pores into the bilayer which
resulted in a fixed current flow which is identical for each
pore.
[0131] 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
in FIG. 4.
[0132] Wild-type .alpha.-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.
[0133] Finally, confirmation that the stepwise increase in current
is specifically due to insertion of the wild-type .alpha.-HL pores
into the bilayer was provided by the addition of
.alpha.-cyclodextrin, a well-characterised analyte that transiently
binds to .alpha.-HL pores. A characteristic interruption in the
current through the pores of approximately 60% was recorded as
single molecules of .alpha.-cyclodextrin transiently bound to the
pores, with a spread in binding durations in the range .about.100
ms. This is presented in FIG. 6.
[0134] 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 .alpha.-HL dried on the membrane
around the aperture.
[0135] Two different polymer films were used to create the membrane
separating the two chambers of the test cells; a 6 .mu.m thick
biaxial polycarbonate film, and a 5 .mu.m thick
polytetrafluoroethylene (PTFE) film: both films were sourced from
Goodfellow Cambridge Ltd.
[0136] For each of these two polymer films, apertures were created
by one of two different methods: sparking and laser drilling.
[0137] 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 .mu.m in diameter with a tapered morphology in cross section.
Holes of this size allow a stable lipid bilayer to e formed more
easily.
[0138] Spark-generated holes also in the range of 5-30 .mu.m 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-US-00004 TABLE 4 Polymer film Aperture 6 .mu.m thick biaxial
polycarbonate 25 .mu.m diameter spark generated hole 6 .mu.m thick
biaxial polycarbonate 10 .mu.m diameter laser drilled tapered hole
5 .mu.m thick PTFE 10 .mu.m diameter spark generated hole 5 .mu.m
thick PTFE 10 .mu.m diameter laser drilled tapered hole
[0139] 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 .mu.l 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.
[0140] In addition to the chemical treatment during preparation of
the aperture, 1 .mu.l of 0.17 mg/ml wild-type .alpha.-HL was
subsequently applied to the aperture and allowed to dry at room
temperature.
[0141] 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.
[0142] The dry research cells, already loaded with lipid and
.alpha.-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.
[0143] 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 .mu.s is per point
using the equipment described previously.
[0144] 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 .mu.m 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.
[0145] 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.
[0146] The results for the second membrane in Table 2, with a
pre-treatment of 2 .mu.l 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.
[0147] 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 .alpha.-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 .alpha.-HL pore in the bilayer (as described above for FIG.
6). After .about.70 s a second pore inserts into the bilayer.
[0148] 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.
[0149] 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 .alpha.-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.
[0150] 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
.mu.l. Two alternative membrane materials were tested: a 6 um thick
biaxial polycarbonate film, and a 5 .mu.m 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 .mu.m in diameter with a tapered morphology in
cross section. Spark generated apertures in the 5 .mu.m PTFE film
membranes were approximately 10 .mu.m diameter circular holes,
whereas for the 6 .mu.m polycarbonate film the sparked apertures
were elliptical with dimensions approximately 20 .mu.m by 30 .mu.m.
Holes of this size allow a stable lipid bilayer to be easily
formed.
[0151] The apertures then received a chemical pre-treatment to
facilitate the bilayer formation process. This consisted of 2 .mu.l
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.
[0152] Once the pentane solvent had evaporated a 1 .mu.l drop of
aqueous protein solution (0.017 mg/ml w.t. .alpha.-HL) was applied
near to one side of the aperture and dried.
[0153] The interior of each chamber was then coated with 4 .mu.l of
10 mg/ml diphantytanoyl-sn-glycero-3-phosphocholine (DPhPC)
dissolved in pentane.
[0154] 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.
[0155] 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 .mu.m and the
membrane has been pre-treated to increase its affinity to
lipids.
[0156] FIG. 9 shows a typical current trace recorded over the
entire duration of one test. FIGS. 10, 11, 12 and 13 show expanded
areas of FIG. 9. Each Fig. contains two graphs: the upper plot
shows the current response to the applied potential, which is shown
in the lower plot.
[0157] Prior to arrow 1 in FIG. 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 .about.330 pA, as seen in FIG. 10, indicating bilayer
formation across the aperture. When the a.c. potential waveform is
then replaced by a d.c. potential of +100 mV (after arrow 1 in FIG.
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.
[0158] In the period immediately prior to arrow 2 in FIG. 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 in FIG. 11 (recorded between arrow 2 and
arrow 3 in FIG. 9).
[0159] Beyond arrow 3 in FIG. 9, a new bilayer is formed using the
Montal and Mueller method by lowering and then raising the
solution/air interface carrying the lipid monolayer past the
aperture. The square wave capacitive current is restored as the new
bilayer forms. The potential waveform is then turned off and +100
mV d.c. applied, which results in approximately 100 pA step
increases in the current as .alpha.-HL protein pores automatically
insert into the bilayer. An expanded view showing the current as
the pores insert into the bilayer is presented in FIG. 12 (after
arrow 4 in FIG. 9). Again, pores will spontaneously insert into the
lipid bilayer if they are deposited in dried form on an interface
of the cell. This avoids the need to actively insert the pores into
the lipid bilayer.
[0160] The .gamma.-cyclodextrin in the test solution binds
stochastically to the .alpha.-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.
[0161] 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.
[0162] A sensor system 1 is shown in FIG. 14 and comprises a cell 2
and an electrical reader unit 3 which may be connected together. In
use, sensing using a lipid bilayer is formed in the cell 2 and an
electrical current signal across the bilayer is monitored and
interpreted by the reader unit 3. The sensor 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 the sensor system 1 also make it
advantageous for laboratory use.
[0163] The cell 2 has a construction allowing it to be
mass-produced at a low cost, allowing it to be a disposable
product. The cell 2 is easily connected and replaced in the reader
unit 3. The reader unit 3 is sufficiently small to be hand-held and
portable.
[0164] The cell 2 is shown in FIGS. 15 and 16 and will now be
described in detail. The cell 2 has a layered construction formed
from a stack of layers fixed together.
[0165] The cell 2 comprises a membrane 10 having an aperture 11
across which a lipid bilayer is supported in use. Although only a
single aperture 11 is used in many applications, there may be
plural apertures 11. The membrane 10 may be made of any material
capable of supporting lipid bilayer across the aperture 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 the
membrane 10 is the affinity to the lipid which affects the ease of
bilayer formation. However the material of the membrane 10 has less
significance when a pretreatment is used as described below. The
choice of the material of the membrane 10 also affects the ease of
formation of the aperture 11.
[0166] 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 .mu.m, preferably
being at most 10 .mu.m thick, for example 5 .mu.m or 6 .mu.m. The
thickness of the membrane 10 is typically at least 0.1 .mu.m. The
aperture 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.
[0167] The thickness of the membrane 10 is also dependent on the
size of the aperture 11. As the aperture 11 decreases in size, the
membrane 10 also needs to decrease in thickness in order to assist
the formation of a lipid bilayer. Typically the thickness of the
membrane 10 is no more than the minimum diameter of the aperture
11. Another factor is the electrical resistance of the membrane 10
which changes with the thickness. It is desirable that the
resistance of the membrane 10 is sufficiently high relative to the
resistance of the ion channel in a membrane protein inserted in the
membrane 10 that the current flowing across the membrane 10 does
not mask the current through the ion channel.
[0168] The membrane 10 is supported by two support sheets 12,
provided on opposite sides of the membrane 10 and fixed thereto. As
described further below, the membrane 10 and the support sheets 12
together form a septum 17. The support sheets 12 each have a window
13 which is aligned with the aperture 11 in the membrane 10 but is
of larger size than the aperture 11 in order that the support
sheets 12 do not interfere with the formation of a lipid bilayer
across the aperture 11. The support sheets 12 have the function of
supporting and strengthening the membrane 10 and may be made of any
material suitable for achieving this purpose. Suitable materials
include, but are not limited to: Delrin.RTM. (polyoxymethylene or
acetal homopolymer), a polyester, eg Mylar.RTM. (biaxially-oriented
polyethylene terephthalate (boPET) polyester film), PC, PVC, PAN,
PES, polysulphone, polyimide, polystyrene, polyethylene, PVF, PET,
PTFE, PEEK, or FEP
[0169] The support sheets 12 are typically thicker than the
membrane 10, having a thickness typically at least 0.1 .mu.m,
preferably at least 10 .mu.m. The support sheets 12 are thinner
than the bodies 14 described below, having a thickness typically at
most 1 mm, preferably at most 0.5 mm.
[0170] The cell 1 further comprises two bodies 14 each fixed to one
of the support sheets 12. The bodies 14 are each formed from a
sheet of material having an aperture 15 extending therethrough. The
apertures 15 in the bodies 14 are of larger area, parallel to the
membrane 10, than the windows 13 in the support sheets 12 and are
aligned therewith. Thus, the apertures 15 in the bodies 14 each
define a respective chamber 16, the two chambers 16 being separated
by the septum 17 formed by the membrane 10 and the support sheets
12 together, and the aperture 11 in the membrane 10 opening into
each of the chambers 16.
[0171] The thickness of each body 14 is greater than the thickness
of the support sheets 12 and is chosen to provide a desired volume
for the chambers 16. In general, the bodies 14 may have any
thickness, but typically the thickness of each body 14 is in the
range from 1 .mu.m to 3 mm. Typically, for use in a disposable
portable sensing system, the chambers 16 have a volume of 0.1 .mu.l
to 250 .mu.l. However, a restricted thickness can be advantageous
as described further below. The bodies 14 may be formed of any
suitable material, for example silicone rubber.
[0172] The chambers 16 are closed by means of a respective closure
sheet 18 which is fixed to the outer surface of the respective body
14 covering the aperture 15 formed therein. The closure sheet 18
may be formed from any material, but may for convenience be the
same material as the support sheets 12.
[0173] The septum 17 including the membrane 10 is not electrically
conductive and is designed to have a high electrical resistance.
Consequently, in use, the only significant electrical connection
between the two chambers 17 is by ionic conduction of an
electrolyte solution in the chambers 17 through the aperture 11 in
the membrane 10. Formation of a lipid bilayer across the aperture
11 blocks the aperture 11 creating a high-resistance electrical
seal between the chambers 17. Insertion of a membrane protein which
is an ion channel, for example a pore, restores the electrical
connection between the two chambers 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.
[0174] In order to detect and monitor such electrical signals, each
of the chambers 16 is provided with an electrode 20 formed as part
of a layer 23 of conductive material deposited on the surface of
the respective support sheet 12 which is internal to the chamber
16. In particular, the electrodes 20 are illustrated in FIG. 17
which shows one of the support sheets 12 as viewed from the side
internal to the adjacent chamber 16. In FIG. 17, the positions of
the aperture 15 in the body 14 and the aperture 11 in the membrane
10 are shown in dotted outline. The conductive material of the
electrodes 20 may be for example Ag/AgCl.
[0175] As shown in FIG. 17, the support sheets 12 each include a
protruding portion 21 which extends beyond the periphery of the
body 14. The layer 23 of conductive material which is deposited on
the support sheet 12 to form the electrode 20 extends from the
chamber 16 across the support sheet 12 to the protruding portion
21. Accordingly each layer 23 of conductive material forms not only
an electrode 20 but also a contact 24 which is exposed on a
connector portion 22, and a track 25 which electrically connects
the contact 24 and the electrode 20. As described further below,
the two protruding portions 21 of the two support sheets 12
together form a connector portion 22 for connecting the cell 2 to
the reader unit 3, and the electrical signal received by the
electrodes 20 in each chamber 16 is supplied to the reader unit 3
via the contacts 24.
[0176] 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.
[0177] To allow introduction of the sample solution, the test
chamber 16-1 may be provided with an inlet 30 or 32 using either
one of the following two alternative arrangements.
[0178] In the first inlet arrangement, the inlet 30 is formed in
the body 14 as shown in FIG. 18. In particular, the inlet 30 is
formed in one of the surfaces of the body 14 which may in general
be either the inner or outer surface as a channel extending from
the periphery of the body 14 to the aperture 15. The sample may be
injected through the inlet 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 an
exhaust outlet 31 having an identical construction to the inlet
30.
[0179] In the second inlet arrangement, the inlet 32 is formed in
the closure sheet 18 as illustrated in FIG. 19. In particular, the
inlet 32 is formed as a hole extending through the closure sheet 18
and aligned with the aperture 15 in the body 14 which defines the
test chamber 16-1, as shown in dotted outline in FIG. 19. To allow
exhaust of air in the chambers 16 displaced by the sample, the test
chamber 16-1 is further provided with an exhaust outlet 33 having
an identical construction to the inlet 32.
[0180] Such an inlet 30 or 32 may be provided with a closure, or
may be omitted altogether by making a portion of the cell 2 of a
material which allows penetration by a syringe for filling the test
chamber 16-1.
[0181] As a result of the design of the electrode 20 as shown in
FIG. 17, the electrode 20 is arranged in the flow path between the
inlet 30 or 32 and the aperture 11. In other words, when an aqueous
solution is introduced into the test chamber 16-1 through the inlet
30 or 32 it contacts the electrode 20 before reaching the aperture
11. This means that the electrode 20 is wetted before the lipid
bilayer is formed, the formation of the bilayer being described in
more detail below. When the electrode 20 is wetted, there can occur
a pertubation in the potential across the electrodes 20 between the
two chambers 16, derived from the reader unit 3. If this occurs
before the lipid bilayer is formed, then this causes no difficulty.
However if the aqueous solution was to contact the electrode 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.
[0182] 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 an
inlet 30 or 32 as described above. Alternatively the 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 an inlet 30 or 32 as described above.
[0183] Thus the chambers 16 are closed except for an inlet 30 or 32
if provided. This contrasts with a conventional laboratory
apparatus in which chambers on either side of an aperture are
formed as recesses in a molded block which are open to the
atmosphere. Use of closed chambers 16 has the advantage of reducing
evaporation from the contents of the chambers 16. This in turn
reduces the cooling of the contents which is important to maintain
appropriate temperatures in the case of many membrane proteins
which may be inserted in the bilayer.
[0184] 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 the aperture 11.
[0185] 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 the septum 17 has been constructed by
fixing together the membrane 10 and the support sheets 12 but
before assembly of the septum 17 into the remainder of the cell 2.
Alternatively the lipid may be deposited on the internal walls of
the chamber 16 formed by the aperture 15 in the body 14 or the
closure sheet 18, either before or after the body 14 is fixed to
the closure sheet 18, but before assembly to the septum 17.
[0186] 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.
[0187] 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 the body
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 the septum 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.
[0188] This effect is illustrated in FIG. 20 which shows a cell 2
in which the liquid sample 40 has been introduced into one side of
the test chamber 16-1 through the inlet 30 or 32 (although for
simplicity the inlet 30 or 32 is not shown in FIG. 20). As can be
seen, instead of the liquid sample 40 falling under gravity to the
lowest possible level in the chamber 16, surface tension holds the
liquid interface 41 with the air in the chamber 16 extending across
the depth of the chamber 16 between the septum 17 and the closure
sheet 18. Thus, the interface 41 is generally perpendicular to the
septum 17 and the aperture 11 except for the formation of a
meniscus.
[0189] By applying pressure at the inlet 30 or 32 to introduce more
liquid or to withdraw the liquid, the interface 41 may be moved in
the direction of the arrow A along the chamber parallel to the
septum 17 and hence across the aperture 11. Once the liquid sample
40 has rehydrated the dried lipid inside the chamber 16 the liquid
interface 41 will support a layer of the lipid. Thus, such movement
of the liquid interface 41 across the aperture 11 in the membrane
10 may be used to form a lipid bilayer.
[0190] 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 the
cell 2 is illustrated in FIG. 14 with the aperture 11 extending
horizontally, the same effect occurs regardless of the orientation
of the cell 2. Thus the above-described process of forming a lipid
bilayer across the aperture 11 may be carried out with the cell 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.
[0191] The cell 2 is easy to manufacture simply by cutting and
affixing together the individual layers of the cell 2. For
convenience the layers of the cell 2 are affixed by adhesive,
although in principle some form of mechanical fixing could be used.
Conveniently due to the use of a layered construction plural cells
2 or parts thereof may conveniently be manufactured together from a
large sheet and subsequently cut out. As a result of these points,
the cell 2 is capable of mass production at relatively low
cost.
[0192] By way of example and without limitation, one particular
manufacturing method will now be described in detail.
[0193] 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 sixteen support sheets 12 are to be formed.
The cards were Mylar polyester sheet (DuPont) of thickness 250
.mu.m with a 467 MP self-adhesive coating of thickness 50 .mu.m on
one side. With the release-paper facing upwards, 4 mm diameter
holes are punched in the cards on the template to provide the
windows 13 of each support sheet 12 and any burring of the edges of
the punched holes removed using a scalpel blade.
[0194] 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 the layers 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.
[0195] With the release paper side facing upwards, the cards are
then cut using a guillotine lengthwise into the six rows of support
sheets 12.
[0196] In this example the membranes 10 are formed from either a 6
.mu.m thick biaxial polycarbonate film or a 5 .mu.m thick PTFE film
(Goodfellow Cambridge Ltd.). Prior to use the apertures 11 are
formed as discussed below. The membrane 10 around the apertures 11
then receives a chemical pretreatment to facilitate the bilayer
formation process. In this case, the pretreatment consists of 2
.mu.l of 1% hexadecane in pentane applied to either side of the
aperture by capillary pipette.
[0197] Once the pentane solvent had evaporated a 1 .mu.l drop of
aqueous protein solution (0.017 mg/ml w.t. .alpha.-HL) was applied
near to one side of the aperture and dried.
[0198] Next the films are cut into strips, cleaned on both sides by
rinsing with ethanol, and gently air-dried.
[0199] 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.
[0200] 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 the
apertures 11. Then the strips are cut using a guillotine into
septums 17 for individual cells 2.
[0201] 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 diameter circular apertures 15 for respective 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
.mu.l have been produced by punching 6 mm diameter holes through
the 2 mm thick spacer material.
[0202] The individual chambers 16 are then closed by sticking an A4
sized card of plain 250 .mu.m 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 the body 14.
Channels of width 1 mm, to form the inlet 30 and exhaust gas outlet
31 are then cut in the silicone rubber sheet material (but not
through the backing card).
[0203] The interior of each chamber 16 is then coated with a
solution of 4 .mu.l 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 form
cells 2.
[0204] The size and formation of the aperture 11 in the membrane 10
will now be considered further.
[0205] 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 .mu.m to 150 .mu.m and an aperture 11 of such a
size may used in the present cell 2.
[0206] 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 the sensor system 1 is used outside
a laboratory setting where it may be subject to external
forces.
[0207] The increased stability achieved by restricting the size of
the aperture 11 has been experimentally demonstrated as
follows.
[0208] A number of actual membranes 10 which have been tested are
listed in Table 2 above The apertures 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.
[0209] 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.
[0210] 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
the range 5 .mu.m-10% n diameter in PTFE film of 5 .mu.m thickness.
Further control of the aperture 11 diameter could easily be
introduced through limiting the sparking energy by gating the
discharge after detecting the onset of dielectric breakdown.
[0211] The laser-generated apertures 11 were produce by laser
drilling.
[0212] The morphology of the aperture 11 can been seen to vary with
the material of the membrane 10 and method used to form the
bilayer. For example, with biaxial polycarbonate film, the spark
generated apertures 11 were elliptical while the laser drilled
apertures 11 were mostly circular. Similarly the spark generated
apertures 11 generally had a uniform cross-section while the laser
drilled apertures 11 generally a cross section which tapered
through the thickness of the membrane 10.
[0213] The regularity of the inside edge of the aperture 11 is also
sensitive to the material of the membrane 10, the thickness of the
membrane 10, and the method of formation of the aperture 11. This
is expected to impact on the stability of bilayer formation at the
aperture.
[0214] However in all cases irrespective of the method of formation
of the aperture 11, it is apparent that restricting the diameter of
the aperture 11 results in increasing the stability of the
bilayers, in fact to a dramatic degree. For example with an
aperture 11 of diameter 10 .mu.m the cell 2 can firmly knocked
against the table or disconnected from the reader unit 3 and
carried by hand without breaking the bilayer. This is of
significant advantage in the context of use of the sensor system 1
outside the laboratory setting.
[0215] For these reasons it is preferred that the aperture 11 has a
restricted diameter, say of 20 .mu.m or less in at least one
dimension. The aperture 11 may have such a restricted diameter in
all dimensions, but the advantage of increased stability is
achieved provided the aperture 11 is relatively small in one
dimension, even if the aperture 11 is longer in another
dimension.
[0216] 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 the membrane 10 and methods capable of
generating the apertures 11 is considerably more extensive than
those considered above.
[0217] As mentioned above, in one type of cell 2, the secondary
chamber 16-2 may contain a gel 50 as shown for example in the cell
2 of FIG. 20. In particular, the gel 50 extends across the aperture
11 in the membrane 10. The presence of the gel acts to physically
support a lipid bilayer formed across the aperture 11. As a result,
the gel 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
the sensor system 1 in a non-laboratory setting, because it makes
the sensor system 1 easier to use and also more robust against
external forces of the type which may disturb the sensor system 1
in normal use. In addition, the gel 50 may act as a matrix for
controlling the supply of molecules to the lipid bilayer.
[0218] In order to support the lipid bilayer, the gel 50 may fill
the secondary chamber 16-2 such that the gel 50 contacts the
membrane 10. This case is illustrated in FIG. 20. In this case, the
gel 50 may directly support the lipid bilayer formed across the
aperture 11. This is preferred in order to improve bilayer
formation and stability.
[0219] However, in an alternative illustrated in FIG. 21, there may
remain a gap 51 between the gel 50 and the membrane 10. In this
case, the gel 50 may still support the lipid bilayer formed across
the aperture 11 by acting through a solution occupying the gap 51,
although this effect will reduce as the size of the gap 51
increases. The presence of the gap 51 means that a wider variety of
materials can be used to make the gel 50, including ionically
non-conductive materials.
[0220] The gel 50 may be ionically conductive and indeed this is
necessary if the gel 50 directly contacts the lipid bilayer. In
this case the gel 50 may be for example a hydrogel. Suitable
ionically conductive gels include, but are not limited to, agarose
polyacrylimide gel, Gellan.TM. gel or Carbomer gel. Particular gels
which have been used are 5% agarose doped with NaCl or Signa Gel
(Parker Laboratories Inc.). In one case agarose gel 50 was made
using 10 mM PBS to which 1M NaCl had been added. The gel 50 was
melted and then injected in the chamber 16 where it solidified upon
cooling.
[0221] It has been discovered that when one chamber 16 of the cell
2 is filled with a gel 50, formation of a lipid bilayer was
possible by moving the liquid interface 41 carrying a lipid
monolayer past the aperture 11 on only one side of the aperture 11,
as opposed to both sides of the aperture 11 as more commonly
performed in the Montal & Muller method. Further, bilayers
could be formed with or without pretreatment of the membrane 10 by
this method. However considerably more attempts were required
without the pretreatment. Pretreating only the top side of the
membrane 10 was found to be sufficient for reproducible bilayer
formation. Being able to apply the pretreatment to only one side of
the membrane 10 greatly simplifies the manufacturing process.
[0222] The cell 2 may be provided to the user with the secondary
chamber 16-2 already containing the gel 50. This improves the ease
of use of the cell 2 because no filling the secondary chamber 16-2
is necessary by the user.
[0223] Each of the features described above of (1) restricting the
size of the aperture 11, (2) use of a pretreatment and (3) use of a
gel 50 assist the formation of a lipid bilayer across the aperture
11 in the membrane 10. In particular, this reduces the number of
times in which the interface 41 carrying a lipid monolayer must be
moved past the aperture 11 in order to form the bilayer. This
improves the ease of use of the cell 2.
[0224] 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 the liquid interface
41 pass the aperture 11. This is of significant advantage because
it means that the lipid bilayer may be formed across the aperture
11 simply on insertion of the test solution 40 into the cell 2, for
example using a pipette or a syringe. This means that the user does
not need to repeatedly move the liquid interface 41 back and fourth
across the aperture 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 the liquid interface 41.
[0225] The reader unit 3 will now be described in detail.
[0226] The reader unit 3 has a connector portion 60 which is
arranged to make a physical connection with the connector portion
24 of the cell 2. The connector portion 60 of the reader unit 3 is
visible in FIG. 14 but is shown in expanded form in FIG. 22. In
particular, the connector portion 60 consists simply of a pair of
blocks 61 which are separated by a spacing designed to provide a
tight fit for the connector portion 24 of the cell 2. Thus, the
connector portion 24 of the cell 2 may be plugged into the
connector portion 60 in between the blocks 61 by insertion of the
cell 2 in the direction of arrow B, thereby providing mating
between the connector portions 24 and 60.
[0227] In addition, respective contacts 62 and 63 are provided on
each of the facing surfaces of the block 61 or the connector
portions 60. The contacts 62 and 63 are simply pieces of metal,
typically gold-plated to assist formation of good electrical
contact. The contacts 62 and 63 may be sprung. When the connector
portion 24 of the cell 2 is plugged into the connector portion 60
of the reader unit 3, the contacts 24 of the cell 2 make an
electrical connection with the contacts 62 and 63 of the reader
unit 3. The reader unit 3 includes an electrical circuit 90
described further below which is connected to the contacts 62 and
63. In this manner, the connection together of the cell 2 in the
reader unit 3 allows the electrical signal generated between the
chambers 16 to be supplied from the electrodes 20 to the reader
unit 3.
[0228] 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 the cell 2 when it is connected to the reader
unit 3. Two alternative approaches are as follows.
[0229] The first approach uses a rigid metal body 70 as the Faraday
cage. The rigid metal body has an internal cavity 71 sufficient to
accommodate the cell 2. At one end 72, the rigid metal body 70 is
open and connected to the body 73 of the reader unit 3 so that the
cavity 71 is aligned with the connection portions 60. In this way,
the cell 2 is accommodated inside the cavity 71 when it is
connected to the reader unit 3, as shown in FIG. 24.
[0230] However, rather than entirely enclosing the cell 2, the
rigid metal body 70 has an aperture 74 facing the connector portion
60. The aperture 74 is of sufficient size to allow passage of the
cell 2 when the cell 2 is connected to the reader unit 3.
Therefore, an individual cell 2 may be connected to the reader unit
3 and replaced by another cell 3 by insertion through the aperture
74 without removal of the rigid metal body 70. It has been
appreciated that surprisingly the presence of the aperture 74 does
not prevent the operation of the rigid metal body 70 as a Faraday
cage. In particular, this is because the aperture 74 may be of
sufficiently small size that any electrical interference caused by
electro magnetic radiation penetrating the aperture 74 is at a
sufficient high frequency that it does not significantly degrade
the quality of the electrical signal of interest. In particular,
the aperture 74 of the rigid metal body 70 may have a maximum
dimension (horizontally in FIG. 23) of 50mm or less, preferably 20
mm or less.
[0231] The rigid metal body 70 also has a sample introduction hole
76 which is aligned with the inlet 30 or 32 when the cell 2 is
connected to the reader unit 3. The sample introduction hole 76
allows the sample to be introduced into the cell 2 after the cell 2
has been connected to the reader unit 3. The sample introduction
hole 76 is smaller than the aperture 74, typically having a maximum
dimension of 5 mm or less. Thus the sample introduction hole 76 is
also of sufficiently small size that any electrical interference
caused by electro magnetic radiation penetrating the sample
introduction hole 76 is at a sufficient high frequency that it does
not significantly degrade the quality of the electrical signal of
interest.
[0232] The second alternative approach is to provide a Faraday cage
75 fixed around the periphery of the cell 2, for example as shown
in FIG. 25. In this case, the Faraday cage 75 entirely encloses the
cell 2, except for the connector portion 24 which protrudes out of
the Faraday cage 75. In this case, the Faraday cage 75 may be
formed by a solid metal body. Alternatively, the Faraday 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 the cell 2.
[0233] 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.
[0234] The reader unit 3 houses an electrical circuit 90 which will
now be described in detail. The primary function of the electrical
circuit 90 is to measure the electrical current signal developed
across the electrodes 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.
[0235] The electrical circuit 90 may take various different forms
and some possible circuit designs are shown in FIGS. 14 to 16. In
each design there are some common elements as follows.
[0236] The two contacts 62 and 63 of the connector portion 60 will
be referred to as a reference contact 62 and a working contact 63.
Although the electrodes 62 and 63 are physically the same, in
operation the reference contact 62 provides a bias voltage
potential relative to the working contact 63, whilst the working
contact 63 is at virtual ground potential and supplies the current
signal to electrical circuit 90.
[0237] A possible alternative which is not illustrated would be for
the reference contact 62 to be held at ground and working contact
63 to be offset by the bias voltage.
[0238] The reader circuit 90 has a bias circuit 91 connected to the
reference contact 62 and arranged to apply a bias voltage which
effectively appears across the two contacts 62 and 63 and hence
across the electrodes 20 of a cell 2 connected to the reader unit
3. The bias circuit 91 may take different forms as described
below.
[0239] The reader circuit 90 also has an amplifier circuit 92
connected to the working contact 63 for amplifying the electrical
current signal the electrodes 20 of the cell 2 and appearing across
the two contacts 62 and 63. In each design of the electrical
circuit 90, the amplifier circuit 92 consists of a first amplifier
stage 93 and a second amplifier stage 94.
[0240] The first amplifier stage 93 is connected to the working
electrode 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 M.OMEGA., to provides the gain necessary to amplify the current
signal which typically has a magnitude of the order of tens to
hundreds of picoamps.
[0241] The second amplifier stage 94 is connected to the output of
the first amplifier stage 93 and arranged to amplify and filter the
voltage signal voltage. The second amplifier stage 94 provides
sufficient gain to raise the signal to a sufficient level for
processing in the microcontroller 95 described below. For example
with a 500 M.OMEGA. feedback resistance in the first amplifier
stage 93, the input voltage to the second 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 the second 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 the second
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.
[0242] To save power, the analogue circuitry in the bias circuit 91
and the amplifier 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.
[0243] 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
the second amplifier stage 94 equal to half reference voltage value
of the microcontroller 95.
[0244] The first design of the electrical circuit 90 shown in FIG.
26 and will now be described. This design is intended for a
stand-alone battery-operated reader unit 3 with PC connectivity. In
this case, the bias circuit 91 and the amplifier circuit 92 are
connected to a microcontroller 95. The microcontroller 95 has a
power control circuit 96 which supplies power from a battery. The
microcontroller 95 incorporates an analog-to-digital converter 97
which receives the output of the amplifier 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 the second amplifier stage 94 to prevent
aliasing.
[0245] In this case the analog-to-digital converter 97 is embedded
on the same silicon die as the microcontroller 95, but it could
alternatively be a separate circuit element.
[0246] The microcontroller 95 incorporates a microprocessor 98
which runs code to process and analyse the digital signal. The
microcontroller 95 has a display 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.
[0247] The microcontroller 95 receives commands from a keypad 100.
Of course other input and output devices could be used in addition
to, or instead of, the display 99 and keypad 100, for example LEDs
used as indicators or an audio generator 105.
[0248] The microcontroller 95 also has an interface 101 to provide
data communication with another digital device, for example a
computer. The interface 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.
[0249] The microcontroller 95 is connected to the bias circuit 91
as follows. The microcontroller 95 has a PWM generator 102 which
generates a PWM (pulse width modulation) voltage waveform, that is
a digital signal with fixed frequency but varying duty cycle. The
PWM 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.
[0250] The bias circuit 91 includes a low-pass filter 103 connected
to low-pass filter the PWM signal output by the PWM 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. The PWM
generator 102 built in this manner has a resolution equivalent to
the smallest duty cycle change possible with the microcontroller
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 in FIG. 26.
[0251] The bias circuit 91 further includes an output amplifier 104
for amplifying the output of the low-pass filter 103. In the case
described above that a bipolar output is required, the output
amplifier 104 is a summing amplifier arranged to subtract the
output of one of the pair of low pass filters 103 from the
other.
[0252] For systems requiring multiple or arrayed cells 2, the
microcontroller 95 can be chosen with an embedded analogue
multiplexer. In this case multiple analogue input circuits are
required and the output of each second amplifier stage 94 is
sampled by the analog-to-digital converter 97 through the
multiplexer.
[0253] The second design of the electrical circuit 90 is shown in
FIG. 27 and will now be described. This design is intended for a
reader 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 the microcontroller 95 interfaces with a
PDA device 106 which is a conventional PDA. This allows the reader
unit 3 to take advantage of the existing functionality of PDAs. The
PDA 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 the reader unit 2 to provide the functions of a
large interactive display with key or touch entry and a
rechargeable power source.
[0254] In this case, one option is for the connector portion 60,
the amplifier circuit 92, the bias circuit 91 and the
microcontroller 95 to be mounted within an electrical assembly
shaped to fit in an SD card slot or other card format slot. This
allows the reader unit 2 to be formed by an existing PDA device
with the assembly fitted in a card slot.
[0255] The third design of the electrical circuit 90 is shown in
FIG. 28 and will now be described. This design is intended for a
reader unit 3 which is based on a data acquisition card 107 to be
plugged into a computer 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 the amplifier circuit 92 is arranged as described above, but
the bias circuit 91 is simply formed by an inverting amplifier 109
supplied with a signal from a digital-to-analog converter 110 which
may be either a dedicated device or a part of the data acquisition
card 107 and which provides a voltage output dependent on the code
loaded into the data acquisition card 107 from software.
[0256] The third design of the electrical circuit 90 shown in FIG.
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 the data acquisition card 107 is modified to provide the
transport interface allowing multiple data streams into the
computer.
[0257] 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 the microprocessor 98 in the microcontroller 95 or the
PDA 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 the cell 2. Accordingly,
the state of the cell 2 can be detected from the electrical signal
by the electrical circuit 90.
[0258] 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:
[0259] 1) the chambers 16 in the cell 2 being dry;
[0260] 2) the chambers 16 in the cell 2 containing an aqueous
solution without a lipid bilayer being formed across the aperture
11 in the membrane 10;
[0261] 3) a lipid bilayer being formed across the aperture 11 in
the membrane 10 without a membrane protein being inserted
therein;
[0262] 4) a lipid bilayer being formed across the aperture 11 in
the membrane 10 with a membrane protein being inserted therein
without an analyte binding to the membrane protein; and
[0263] 5) a lipid bilayer being formed across the aperture 11 in
the membrane 10 with a membrane protein being inserted therein with
an analyte binding to the membrane protein.
[0264] 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, the electrical
circuit 90 then produces an output indicative of the detected
state, for example by displaying the detected state on the display
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.
[0265] 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 the cell 2 is
operating correctly from the moment it is connected to the reader
unit 3 until the end of the measurement assay. The reader 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), the reader unit 3
may output a signal reporting an error mode, or alternatively may
perform an automated remediation.
[0266] 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.
[0267] 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.
[0268] 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 .alpha.-HL to sense the presence of the
analyte .gamma.-cyclodextrin. The electrical circuit 90 performs
the process as shown in FIG. 29.
[0269] In an initialisation step S1 performed before connection of
the cell 2 to the reader unit 3, the electrical circuit 17 applies
a bias voltage as shown in FIG. 30 having a waveform which is a 50
Hz triangular AC signal with 20 mV amplitude, superimposed on +100
mV DC potential.
[0270] 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 the cell 2. In the
absence of the cell 2, the contacts 62 and 63 of the reader unit 3
behaves as a capacitor and produce a square wave current response
to the applied triangular AC potential, as shown in FIG. 31. In
particular the square wave has a 20 pA amplitude centred on 0 pA.
This waveform is characteristic of normal operation of the
electrical 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, the electrical circuit 90 outputs a signal indicate
indicative of a circuit error. Otherwise in step S4, the user
connects a cell 2 to the reader unit 3. The electrical circuit 90
may for example await a user input to indicate this.
[0271] 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 the cell 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 the cell 2.
[0272] 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 in FIG. 30. In step S9, the user
introduces the test solution into the cell 2.
[0273] 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, the aperture 11 provides a conductive
path between the electrodes 20 and so the cell 2 provides a current
response. Typically the current saturates the amplifier, for
example as shown in the typical response shown in FIG. 32.
[0274] In contrast, formation of the lipid bilayer prevents flow of
ionic current through the aperture 11 and so the cell 2 provides a
capacitive response. As a result, the resultant current signal is a
squarewave as shown in FIG. 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 G.OMEGA..
[0275] 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.
[0276] 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.
[0277] 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 in FIG. 34 which shows the current
increasing by a step of the order of 95 pA on insertion of single
.alpha.-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.
[0278] 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.
[0279] 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 in FIG. 35. The analyte
.gamma.-cyclodextrin causes a decrease in the current of the order
of 60 pA. Four such binding events are evident in FIG. 35. The
electrical 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.
[0280] Finally in step S21 the concentration of the analyte
.gamma.-cyclodextrin is calculated based on the kinetics of the
measured analyte binding.
Sequence CWU 1
1
31960DNAStaphylococcus aureusCDS(1)..(960) 1atg aaa aca cgt ata gtc
agc tca gta aca aca aca cta ttg cta ggt 48Met Lys Thr Arg Ile Val
Ser Ser Val Thr Thr Thr Leu Leu Leu Gly1 5 10 15tcc ata tta atg aat
cct gtc gct aat gcc gca gat tct gat att aat 96Ser Ile Leu Met Asn
Pro Val Ala Asn Ala Ala Asp Ser Asp Ile Asn 20 25 30att aaa acc ggt
act aca gat att gga agc aat act aca gta aaa aca 144Ile Lys Thr Gly
Thr Thr Asp Ile Gly Ser Asn Thr Thr Val Lys Thr 35 40 45ggt gat tta
gtc act tat gat aaa gaa aat ggc atg cac aaa aaa gta 192Gly Asp Leu
Val Thr Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val 50 55 60ttt tat
agt ttt atc gat gat aaa aat cac aat aaa aaa ctg cta gtt 240Phe Tyr
Ser Phe Ile Asp Asp Lys Asn His Asn Lys Lys Leu Leu Val65 70 75
80att aga aca aaa ggt acc att gct ggt caa tat aga gtt tat agc gaa
288Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser Glu
85 90 95gaa ggt gct aac aaa agt ggt tta gcc tgg cct tca gcc ttt aag
gta 336Glu Gly Ala Asn Lys Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys
Val 100 105 110cag ttg caa cta cct gat aat gaa gta gct caa ata tct
gat tac tat 384Gln Leu Gln Leu Pro Asp Asn Glu Val Ala Gln Ile Ser
Asp Tyr Tyr 115 120 125cca aga aat tcg att gat aca aaa gag tat atg
agt act tta act tat 432Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr Met
Ser Thr Leu Thr Tyr 130 135 140gga ttc aac ggt aat gtt act ggt gat
gat aca gga aaa att ggc ggc 480Gly Phe Asn Gly Asn Val Thr Gly Asp
Asp Thr Gly Lys Ile Gly Gly145 150 155 160ctt att ggt gca aat gtt
tcg att ggt cat aca ctg aaa tat gtt caa 528Leu Ile Gly Ala Asn Val
Ser Ile Gly His Thr Leu Lys Tyr Val Gln 165 170 175cct gat ttc aaa
aca att tta gag agc cca act gat aaa aaa gta ggc 576Pro Asp Phe Lys
Thr Ile Leu Glu Ser Pro Thr Asp Lys Lys Val Gly 180 185 190tgg aaa
gtg ata ttt aac aat atg gtg aat caa aat tgg gga cca tac 624Trp Lys
Val Ile Phe Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr 195 200
205gat cga gat tct tgg aac ccg gta tat ggc aat caa ctt ttc atg aaa
672Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly Asn Gln Leu Phe Met Lys
210 215 220act aga aat ggt tct atg aaa gca gca gat aac ttc ctt gat
cct aac 720Thr Arg Asn Gly Ser Met Lys Ala Ala Asp Asn Phe Leu Asp
Pro Asn225 230 235 240aaa gca agt tct cta tta tct tca ggg ttt tca
cca gac ttc gct aca 768Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe Ser
Pro Asp Phe Ala Thr 245 250 255gtt att act atg gat aga aaa gca tcc
aaa caa caa aca aat ata gat 816Val Ile Thr Met Asp Arg Lys Ala Ser
Lys Gln Gln Thr Asn Ile Asp 260 265 270gta ata tac gaa cga gtt cgt
gat gat tac caa ttg cat tgg act tca 864Val Ile Tyr Glu Arg Val Arg
Asp Asp Tyr Gln Leu His Trp Thr Ser 275 280 285aca aat tgg aaa ggt
acc aat act aaa gat aaa tgg aca gat cgt tct 912Thr Asn Trp Lys Gly
Thr Asn Thr Lys Asp Lys Trp Thr Asp Arg Ser 290 295 300tca gaa aga
tat aaa atc gat tgg gaa aaa gaa gaa atg aca aat taa 960Ser Glu Arg
Tyr Lys Ile Asp Trp Glu Lys Glu Glu Met Thr Asn305 310
3152319PRTStaphylococcus aureus 2Met Lys Thr Arg Ile Val Ser Ser
Val Thr Thr Thr Leu Leu Leu Gly1 5 10 15Ser Ile Leu Met Asn Pro Val
Ala Asn Ala Ala Asp Ser Asp Ile Asn 20 25 30Ile Lys Thr Gly Thr Thr
Asp Ile Gly Ser Asn Thr Thr Val Lys Thr 35 40 45Gly Asp Leu Val Thr
Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val 50 55 60Phe Tyr Ser Phe
Ile Asp Asp Lys Asn His Asn Lys Lys Leu Leu Val65 70 75 80Ile Arg
Thr Lys Gly Thr Ile Ala Gly Gln Tyr Arg Val Tyr Ser Glu 85 90 95Glu
Gly Ala Asn Lys Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val 100 105
110Gln Leu Gln Leu Pro Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr Tyr
115 120 125Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr Met Ser Thr Leu
Thr Tyr 130 135 140Gly Phe Asn Gly Asn Val Thr Gly Asp Asp Thr Gly
Lys Ile Gly Gly145 150 155 160Leu Ile Gly Ala Asn Val Ser Ile Gly
His Thr Leu Lys Tyr Val Gln 165 170 175Pro Asp Phe Lys Thr Ile Leu
Glu Ser Pro Thr Asp Lys Lys Val Gly 180 185 190Trp Lys Val Ile Phe
Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr 195 200 205Asp Arg Asp
Ser Trp Asn Pro Val Tyr Gly Asn Gln Leu Phe Met Lys 210 215 220Thr
Arg Asn Gly Ser Met Lys Ala Ala Asp Asn Phe Leu Asp Pro Asn225 230
235 240Lys Ala Ser Ser Leu Leu Ser Ser Gly Phe Ser Pro Asp Phe Ala
Thr 245 250 255Val Ile Thr Met Asp Arg Lys Ala Ser Lys Gln Gln Thr
Asn Ile Asp 260 265 270Val Ile Tyr Glu Arg Val Arg Asp Asp Tyr Gln
Leu His Trp Thr Ser 275 280 285Thr Asn Trp Lys Gly Thr Asn Thr Lys
Asp Lys Trp Thr Asp Arg Ser 290 295 300Ser Glu Arg Tyr Lys Ile Asp
Trp Glu Lys Glu Glu Met Thr Asn305 310 3153294PRTStaphylococcus
aureus 3Met Ala Asp Ser Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile
Gly1 5 10 15Ser Asn Thr Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp
Lys Glu 20 25 30Asn Gly Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp
Asp Lys Asn 35 40 45His Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly
Thr Ile Ala Gly 50 55 60Gln Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn
Lys Ser Gly Leu Ala65 70 75 80Trp Pro Ser Ala Phe Lys Val Gln Leu
Gln Leu Pro Asp Asn Glu Val 85 90 95Ala Gln Ile Ser Asp Tyr Tyr Pro
Arg Asn Ser Ile Asp Thr Lys Glu 100 105 110Tyr Met Ser Thr Leu Thr
Tyr Gly Phe Asn Gly Asn Val Thr Gly Asp 115 120 125Asp Thr Gly Lys
Ile Gly Gly Leu Ile Gly Ala Asn Val Ser Ile Gly 130 135 140His Thr
Leu Lys Tyr Val Gln Pro Asp Phe Lys Thr Ile Leu Glu Ser145 150 155
160Pro Thr Asp Lys Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val
165 170 175Asn Gln Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro
Val Tyr 180 185 190Gly Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser
Met Lys Ala Ala 195 200 205Asp Asn Phe Leu Asp Pro Asn Lys Ala Ser
Ser Leu Leu Ser Ser Gly 210 215 220Phe Ser Pro Asp Phe Ala Thr Val
Ile Thr Met Asp Arg Lys Ala Ser225 230 235 240Lys Gln Gln Thr Asn
Ile Asp Val Ile Tyr Glu Arg Val Arg Asp Asp 245 250 255Tyr Gln Leu
His Trp Thr Ser Thr Asn Trp Lys Gly Thr Asn Thr Lys 260 265 270Asp
Lys Trp Thr Asp Arg Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu 275 280
285Lys Glu Glu Met Thr Asn 290
* * * * *
References