U.S. patent application number 12/339956 was filed with the patent office on 2009-07-02 for formation of layers of amphiphilic molecules.
Invention is credited to James Anthony Clarke, Stuart William Reid, Terence Alan Reid, Gurdial Singh Sanghera, Steven Paul White.
Application Number | 20090167288 12/339956 |
Document ID | / |
Family ID | 39048345 |
Filed Date | 2009-07-02 |
United States Patent
Application |
20090167288 |
Kind Code |
A1 |
Reid; Stuart William ; et
al. |
July 2, 2009 |
Formation of Layers of Amphiphilic Molecules
Abstract
To form a layer separating two volumes of aqueous solution,
there is used an apparatus comprising elements defining a chamber,
the elements including a body of non-conductive material having
formed therein at least one recess opening into the chamber, the
recess containing an electrode. A pre-treatment coating of a
hydrophobic fluid is applied to the body across the recess. Aqueous
solution, having amphiphilic molecules added thereto, is flowed
across the body to cover the recess so that aqueous solution is
introduced into the recess from the chamber and a layer of the
amphiphilic molecules forms across the recess separating a volume
of aqueous solution introduced into the recess from the remaining
volume of aqueous solution.
Inventors: |
Reid; Stuart William;
(Chester, GB) ; Reid; Terence Alan; (Bicester,
GB) ; Clarke; James Anthony; (Colchester, GB)
; White; Steven Paul; (Oxford, GB) ; Sanghera;
Gurdial Singh; (Oxford, GB) |
Correspondence
Address: |
BAKER BOTTS L.L.P.;PATENT DEPARTMENT
98 SAN JACINTO BLVD., SUITE 1500
AUSTIN
TX
78701-4039
US
|
Family ID: |
39048345 |
Appl. No.: |
12/339956 |
Filed: |
December 19, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61080492 |
Jul 14, 2008 |
|
|
|
Current U.S.
Class: |
324/72 ; 427/331;
427/532; 427/58 |
Current CPC
Class: |
B01L 2300/161 20130101;
B01L 2400/0421 20130101; B01L 2300/0645 20130101; B01L 2400/0427
20130101; B01L 3/502707 20130101; G01N 33/48721 20130101; B01L
3/50273 20130101; G01N 27/44791 20130101; G01N 27/3278 20130101;
C12Q 1/6869 20130101; G01N 27/453 20130101; G01N 27/333
20130101 |
Class at
Publication: |
324/72 ; 427/331;
427/532; 427/58 |
International
Class: |
G01R 29/00 20060101
G01R029/00; B05D 1/40 20060101 B05D001/40; B05D 3/00 20060101
B05D003/00; B05D 5/12 20060101 B05D005/12 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2007 |
GB |
0724736.4 |
Claims
1. A method of forming a layer separating two volumes of aqueous
solution, the method comprising: (a) providing an apparatus
comprising elements defining a chamber, the elements including a
body of non-conductive material having formed therein at least one
recess opening into the chamber, the recess containing an
electrode; (b) applying a pre-treatment coating of a hydrophobic
fluid to the body across the recess; (c) flowing aqueous solution,
having amphiphilic molecules added thereto, across the body to
cover the recess so that aqueous solution is introduced into the
recess from the chamber and a layer of the amphiphilic molecules
forms across the recess separating a volume of aqueous solution
introduced into the recess from the remaining volume of aqueous
solution.
2. A method according to claim 1, wherein step (c) comprises: (c1)
flowing aqueous solution across the body to cover the recess so
that aqueous solution flows into the recess; (c2) flowing the
aqueous solution to uncover the recess, leaving some aqueous
solution in the recess; and (c3) flowing aqueous solution, having
amphiphilic molecules added thereto, across the body and to
re-cover the recess so that a layer of the amphiphilic molecules
forms across the recess separating a volume of aqueous solution
inside the recess from the remaining volume of aqueous
solution.
3. A method according to claim 2, wherein the apparatus is provided
with a further electrode in the chamber outside said recess, in
step (c1), the aqueous solution is flowed also to contact the
further electrode, and step (c) further comprises, between steps
(c1) and (c2): (c4) applying a voltage across said electrode
contained in the recess and said further electrode sufficient to
reduce the amount of excess hydrophobic fluid covering said
electrode contained in the recess.
4. A method according to claim 2, wherein the aqueous solution
caused to flow in steps (c1) and (c2) is the same aqueous
solution.
5. A method according to claim 1, wherein surfaces including one or
both of (a) the outermost surface of the body around the recess,
and (b) at least an outer part of the internal surface of the
recess extending from the rim of the recess, are hydrophobic.
6. A method according to claim 5, wherein the body comprises an
outermost layer formed of a hydrophobic material, the recess
extending through the outermost layer and said outer part of the
internal surface of the recess being a surface of the outermost
layer.
7. A method according to claim 5, wherein an inner part of the
internal surface of the recess inside the outer part is
hydrophilic.
8. A method according to claim 7, wherein the body comprises an
outermost layer formed of a hydrophobic material and an inner layer
formed of a hydrophilic material, the recess extending through the
outermost layer and inner layer, said outer part of the internal
surface of the recess being a surface of the outermost layer, and
said inner part of the internal surface of the recess being a
surface of the inner layer.
9. A method according to claim 5, wherein said surfaces are
modified by a fluorine species.
10. A method according to claim 9, wherein said surfaces are
modified by a fluorine species by treatment with a fluorine
plasma.
11. A method according to claim 1, wherein the electrode contained
in the recess is provided on the base of the recess.
12. A method according to claim 1, wherein the body comprises a
substrate and at least one further layer attached to the substrate,
the recess extending through the at least one further layer.
13. A method according to claim 1, wherein the electrode has
provided thereon a hydrophillic surface which repels the
hydrophobic fluid applied in step (c) whilst allowing ionic
conduction from the aqueous solution to the electrode.
14. A method according to claim 13, wherein the hydrophillic
surface is the surface of protective material provided on the
electrode.
15. A method according to claim 14, wherein the protective material
is a covalently-attached hydrophillic species or a conductive
polymer.
16. A method according to claim 1, wherein the electrode has a
conductive polymer provided thereon.
17. A method according to claim 1, wherein the elements defining
the chamber further include a cover extending over the body so that
the chamber is a closed chamber.
18. A method according to claim 17, wherein the cover comprises at
least one inlet and at least one outlet, the aqueous solution being
introduced into the chamber through the inlet in step (c) and the
outlet venting fluid displaced by the aqueous solution thus
introduced.
19. A method according to claim 1, wherein the internal surface of
the recess has no openings capable of fluid communication.
20. A method according to claim 1, wherein the at least one recess
comprises plural recesses.
21. A method according to claim 1, wherein the layer of the
amphiphilic molecules is a bilayer of the amphiphilic
molecules.
22. A method according to claim 21, wherein the amphiphilic
molecules are lipids.
23. A method according to claim 1, wherein the layer of the
amphiphilic molecules has an electrical resistance of at least 1
G.OMEGA..
24. A method according to claim 1, further comprising, before step
(c), depositing the amphiphilic molecules on an internal surface of
the chamber or on an internal surface in the flow path of the
aqueous solution into the chamber, the aqueous solution covering
the internal surface during step (c) whereby the amphiphilic
molecules are added to the aqueous solution.
25. A method according to claim 1, further comprising inserting a
membrane protein into the layer of amphiphilic molecules.
26. A method according to claim 25, wherein the aqueous solution
has a membrane protein added thereto, whereby the membrane protein
is inserted spontaneously into the layer of amphiphilic
molecules.
27. A method according to claim 25, further comprising, before step
(c), depositing the membrane protein on an internal surface of the
chamber, the aqueous solution covering the internal surface during
step (c) whereby the membrane protein is added to the aqueous
solution.
28. A method according to claim 1, wherein the at least one recess
comprises plural recesses and the method comprises inserting
different membrane protein into the layers of amphiphilic molecules
formed in different recesses.
29. A method according to claim 25, wherein the apparatus is
provided with a further electrode in the chamber outside the
recess, and the method further comprises applying a potential
across the electrode in the recess and the further electrode and
monitoring an electrical signal developed between the electrode in
the recess and the further electrode.
30. An apparatus for supporting a layer separating two volumes of
aqueous solution, the apparatus comprising: elements defining a
chamber, the elements including a body of non-conductive material
having formed therein at least one recess opening into the chamber;
and an electrode contained in the recess.
31. An apparatus according to claim 30, wherein surfaces including
either or both of (a) the outermost surface of the body around the
recess, and (b) at least an outer part of the internal surface of
the recess extending from the rim of the recess, are
hydrophobic.
32. An apparatus according to claim 31, wherein the body comprises
an outermost layer formed of a hydrophobic material, the recess
extending through the outermost layer and said outer part of the
internal surface of the recess being a surface of the outermost
layer.
33. An apparatus according to claim 31, wherein an inner part of
the internal surface of the recess inside the outer part is
hydrophilic.
34. An apparatus according to claim 33, wherein the body comprises
an outermost layer formed of a hydrophobic material and an inner
layer formed of a hydrophilic material, the recess extending
through the outermost layer and inner layer, said outer part of the
internal surface of the recess being a surface of the outermost
layer, and said inner part of the internal surface of the recess
being a surface of the inner layer.
35. An apparatus according to claim 31, wherein said surfaces are
modified by a fluorine species.
36. An apparatus according to claim 35, wherein said surfaces are
modified by a fluorine species by treatment with a fluorine
plasma.
37. An apparatus according to claim 30, wherein the electrode
contained in the recess is provided on the base of the recess.
38. An apparatus according to claim 30, wherein the body comprises
a substrate and at least one further layer attached to the
substrate, the recess extending through the at least one further
layer.
39. An apparatus according to claim 38, wherein the at least one
further layer is: polycarbonate; poly-vinyl chloride; polyester; a
thermal laminating film; a photoresist; or an ink.
40. An apparatus according to claim 38, wherein the substrate
comprises at least one of silicon, silicon oxide, silicon nitride
or a polymer.
41. An apparatus according to claim 30, wherein the body has a
conductive path extending from the electrode in the chamber to a
contact allowing connection to an electrical circuit.
42. An apparatus according to claim 41, wherein the conductive path
extends through the body to a contact disposed on the opposite side
of the body from the recess.
43. An apparatus according to claim 41, wherein the conductive path
extends across a surface of the substrate under the at least one
further layer.
44. An apparatus according to claim 30, wherein the electrode has
provided thereon a hydrophillic surface which repels the
hydrophobic fluid applied in step (c) whilst allowing ionic
conduction from the aqueous solution to the electrode.
45. An apparatus according to claim 44, wherein the hydrophillic
surface is the surface of protective material provided on the
electrode.
46. An apparatus according to claim 45, wherein the protective
material is a covalently-attached hydrophillic species or a
conductive polymer.
47. An apparatus according to claim 30, wherein the electrode has a
conductive polymer provided thereon.
48. An apparatus according to claim 30, further comprising a
further electrode in the chamber outside said recess.
49. An apparatus according to claim 30, wherein the elements
defining the chamber further include a cover extending over the
body so that the chamber is a closed chamber.
50. An apparatus according to claim 49, wherein the cover comprises
at least one inlet and at least one outlet, the aqueous solution
being introduced into the chamber through the inlet in step (c) and
the outlet venting fluid displaced by the aqueous solution thus
introduced.
51. An apparatus according to claim 30, wherein the internal
surface of the recess has no openings capable of fluid
communication.
52. An apparatus according to claim 30, wherein the recess has a
width of at most 500 .mu.m.
53. An apparatus according to claim 30, wherein the at least one
recess is plural recesses.
54. An apparatus according to claim 30, further comprising
amphiphilic molecules deposited on an internal surface of the
chamber.
55. An apparatus according to claim 54, wherein the amphiphilic
molecules are lipids.
56. An apparatus according to claim 30, further comprising a
membrane protein deposited on the on an internal surface of the
chamber.
57. An apparatus according to claim 30, further comprising a
pre-treatment coating of a hydrophobic fluid applied to the body
across the recess.
58. An apparatus according to claim 57, wherein the recess and the
chamber contain aqueous solution.
59. An apparatus according to claim 58, further comprising a layer
of amphiphilic molecules extending across the opening of the
recess.
60. An apparatus according to claim 59, wherein the layer of the
amphiphilic molecules has an electrical resistance of at least 1
G.OMEGA..
61. An apparatus according to claim 59, wherein the amphiphilic
molecules are lipids.
62. An apparatus according to claim 59, the layer of amphiphilic
molecules having a membrane protein inserted therein.
63. A method of using an apparatus according to claim 59, wherein
the apparatus is provided with a further electrode in the chamber
outside the recess, and the method comprises applying a potential
across the electrode in the recess and the further electrode and
monitoring an electrical signal developed between the electrode in
the recess and the further electrode.
64. A method of improving the performance of an electrode in a
recess in conducting electro-physiological measurements, the method
comprising depositing a conductive polymer on the electrode.
65. A method according to claim 64, wherein the electrode is made
of metal.
66. A method according to claim 65, wherein the electrode is made
of silver, gold or platinum.
67. A method according to claim 64, wherein the conductive polymer
is a polypyrrole.
68. An apparatus for conducting electro-physiological measurements,
the apparatus comprising, a body having a recess in which an
electrode is located, wherein a conductive polymer is deposited on
the electrode.
69. A method according to claim 68, wherein the electrode is made
of metal.
70. A method according to claim 69, wherein the electrode is made
of silver, gold or platinum.
71. A method according to claim 68, wherein the conductive polymer
is a polypyrrole.
Description
RELATED APPLICATIONS
[0001] The present application claims priority to United Kingdom
Patent Application No. 0724736.4 filed on Dec. 19, 2007. The
present application also claims priority to U.S. Provisional Patent
Application Ser. No. 61/080,492 filed Jul. 14, 2008. The entire
contents of the above referenced applications are incorporated
herein by reference.
FIELD OF THE DISCLOSURE
[0002] In one aspect, the present disclosure relates to the
formation of layers of amphiphilic molecules such as lipid
bilayers. It is particularly concerned with the formation of high
quality layers suitable for applications requiring measurement of
electrical signals with a high degree of sensitivity, for example
single channel recordings and stochastic sensing for biosensor or
drug screening applications. In some aspects, it is concerned with
applications employing arrays of layers of amphiphilic molecules,
for example lipid bilayers. In another aspect, the present
disclosure relates to the performance of an electrode provided in a
recess, for example for conducting electro-physiological
measurements.
BACKGROUND OF THE DISCLOSURE
[0003] The potential for using cellular proteins for biosensing and
drug discovery applications has long been appreciated. However
there are many technical challenges to overcome in developing this
technology to fully realise the potential. There is a wealth of
literature on using fluorescent and optical approaches, but the
focus of this document is on the measurement of electrical signals
to recognise analytes in biosensing.
[0004] In one type of technique, a layer of amphiphilic molecules
may be used as the layer separating two volumes of aqueous
solution. The layer resists the flow of current between the
volumes. A membrane protein is inserted into the layer to
selectively allow the passage of ions across the layer, which is
recorded as an electrical signal detected by electrodes in the two
volumes of aqueous solution. The presence of a target analyte
modulates the flow of ions and is detected by observing the
resultant variations in the electrical signal. Such techniques
therefore allow the layer to be used as a biosensor to detect the
analyte. The layer is an essential component of the single molecule
biosensor presented and its purpose is two-fold. Firstly the layer
provides a platform for the protein which acts as a sensing
element. Secondly the layer isolates the flow of ions between the
volumes, the electrical resistance of the layer ensuring that the
dominant contribution of ionic flow in the system is through the
membrane protein of interest, with negligible flow through the
bilayer, thus allowing detection of single protein channels.
[0005] A specific application is stochastic sensing, where the
number of membrane proteins is kept small, typically between 1 and
100, so that the behaviour of a single protein molecule can be
monitored. This method gives information on each specific molecular
interaction and hence gives richer information than a bulk
measurement. However, due to the small currents involved, typically
a few pA, requirements of this approach are a very high resistance
seal, typically at least 1 G.OMEGA. and for some applications one
or two orders of magnitude higher, and sufficient electrical
sensitivity to measure the currents. While the requirements for
stochastic sensing have been met in the laboratory, the conditions
and expertise required limit its use. In addition, the laboratory
methods are laborious and time-consuming and are not easily
scalable to high-density arrays, which are desirable for any
commercial biosensor. Furthermore, the fragility of single bilayer
membranes means that anti-vibration tables are often employed in
the laboratory.
[0006] By way of background, existing techniques for forming layers
of amphiphilic molecules such as lipid bilayers will be
reviewed.
[0007] Several methods for forming planar artificial lipid bilayers
are known in the art, most notably including folded bilayer
formation (e.g. Montal & Mueller method), tip-dipping,
painting, patch clamping, and water-in-oil droplet interfaces.
[0008] At present, the bulk of routine single ion channel
characterisation in research labs is performed using folded
bilayers, painted bilayers or tip-dip methods. These methods are
used either for the ease of bilayer formation, or for the high
resistive seals that can be formed (e.g. 10-100 G.OMEGA.). Tip-dip
bilayers and bilayers from patch-clamping of giant unilamellar
liposomes are also studied as they can be formed in a solvent free
manner, which is thought to be important for the activity of some
protein channels. The method of Montal & Mueller (Proc. Natl.
Acad. Sci. USA. (1972), 69, 3561-3566) is popular as a
cost-effective and relatively straightforward method of forming
good quality folded lipid bilayers suitable for protein pore
insertion, in which a lipid monolayer is carried on the water/air
interface past either side of an aperture in a membrane which is
perpendicular to that interface. Typically, the lipid is added to
the surface of the aqueous electrolyte solution by first dissolving
it in an organic solvent, a drop of which is then allowed to
evaporate on the surface of the aqueous solution on either side of
the aperture. Once the organic solvent has been evaporated, the
solution/air interfaces are physically moved up and down past
either side of the aperture until a bilayer is formed. The
technique requires the presence of a hydrophobic oil applied as a
pre-treatment coating to the aperture surface. The primary function
of the hydrophobic oil is to form an annulus region between the
bilayer and the aperture film where the lipid monolayers must come
together over a distance typically between 1 and 25 .mu.m.
[0009] Tip-dipping bilayer formation entails touching the aperture
surface (e.g. 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 evaporating a drop
of lipid dissolved in organic solvent applied to the solution
surface. The bilayer is then formed by mechanical actuation to move
the aperture into/out of the solution surface.
[0010] For painted bilayers, the drop of lipid dissolved in organic
solvent is applied directly to the aperture, which is submerged in
the aqueous test solution. The lipid solution is spread thinly over
the aperture using a paint brush or 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 is less stable and more noise prone
during measurement.
[0011] Patch-clamping is commonly used in the study of biological
cell membranes, whereby 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 artificial
bilayer studies by clamping liposomes which then burst to leave a
lipid bilayer sealing over the aperture of the pipette. This
requires stable giant unilamellar liposomes and the fabrication of
small apertures in glass surfaced materials.
[0012] Water-in-oil droplet interfaces are a more recent disclosure
in which two aqueous samples are submerged in a reservoir of
hydrocarbon oil containing lipid. The lipid accumulates in a
monolayer at the oil/water interface such that when the two samples
are brought into contact a bilayer is formed at the interface
between them.
[0013] In any of these techniques, once the bilayer has been
formed, the protein is then introduced to the bilayer either by
random collision from the aqueous solution, by fusion of vehicles
containing the protein, or by mechanically transporting it to the
bilayer, for example on the end of a probe device such as an agar
tipped rod.
[0014] There have been great efforts recently to increase the ease
of bilayer formation using micro fabrication. Some techniques have
attempted essentially to miniaturise standard systems for folded
lipid bilayers. Other techniques include bilayer formation on solid
substrates or directly on electrode surfaces, through either
covalent attachment or physical adsorption.
[0015] A large proportion of the devices that are capable of
performing stochastic sensing form a bilayer by using a variant of
the folded lipid bilayers technique or the painted bilayer
technique. To date most have concentrated either on novel methods
of aperture formation or on utilising the emerging technologies in
micro fabrication to miniaturise the device or to create a
plurality of addressable sensors.
[0016] An example is Suzuki et al., "Planar lipid bilayer
reconstitution with a micro-fluidic system", Lab Chip, (4),
502-505, 2004. Herein, an aperture array is created by etching a
silicon substrate, followed by a surface treatment to encourage the
bilayer formation process, although the disclosed rate of
successful bilayer formation is very low (two out of ten).
[0017] A more recent example is disclosed in Sandison, et al., "Air
exposure technique for the formation of artificial lipid bilayers
in Microsystems", Langmuir, (23), 8277-8284, 2007. Herein the
device fabricated from poly(methylmethacrylate) contains two
distinct aqueous chambers. Problems with the reproducibility of
bilayer formation are attributed to the difficulty in removing the
excess hydrophobic material from the aperture, and tackled by using
a period of air exposure to aid the bilayer formation process to
thin the pre-treatment.
[0018] The devices of both Sandison et al. and Suzuki et al. are
both miniaturised versions of a standard painted bilayer technique
with two distinct fluidic chambers separated by a septum containing
an aperture across which the bilayer is formed, one chamber being
filled before the other. This presents a number of difficulties for
scaling up the system to a large number of individually addressable
bilayers, as at least one of the aqueous chambers must be a
distinct chamber with no electrical or ionic connectivity to any
other chamber. Sandison et al. created a device with three fluid
chambers, each with separate fluidics, an approach which would be
difficult to scale to large numbers of bilayers. Suzuki et al.
tried to address this problem by using a hydrophobic photoresist
layer to create small aqueous chambers on top of the aperture
containing substrate. In this case, it is difficult to control the
flow of solution across the aperture containing interface and the
use of small volumes exposed to air makes the apparatus susceptible
to evaporation effects. In both cited examples, the need for the
individual aqueous chambers for each bilayer means that a large
sample volume must be used to fill all the chambers.
[0019] An example of biosensor device using a supported lipid
bilayer is disclosed in U.S. Pat. No. 5,234,566. The device is
capacitive. A gated ion channel responds to an analyte, the binding
of this analyte causes a change in the gating behavior of the ion
channel, and this is measured via the electrical response of the
membrane capacitance. To support the lipid bilayer, there is used a
monolayer of alkane-thiol molecules on a gold electrode, which
provides a scaffold for a lipid monolayer to self-assemble onto.
This monolayer can incorporate ion channels such as gramicidin
which are used as the sensing element of the device. Variations on
this method have been used to create a tethered lipid bilayer onto
an electrode surface to incorporate other membrane proteins.
However, the approach has a number of drawbacks, the first is that
the small aqueous volume present under the lipid bilayer, typically
of the order of 1 nm to 10 nm thick, does not contain enough ions
to perform a direct current measurement for any useful period of
time. This is an effect common to nearly all tethered bilayer
systems on solid supports. For recordings of any meaningful
duration, an alternating current measurement must be used to
overcome the ionic depletion at the electrode, but that limits the
sensitivity of the device.
[0020] An example of a biosensor device using a supported lipid
bilayer is disclosed in Urisu et al., "Formation of high-resistance
supported lipid bilayer on the surface of a silicon substrate with
micro electrodes", Nanomedicine, 2005, (1), 317-322. This device
exploits the strong surface adhesion between phospholipid molecules
and a SiO.sub.2 surface to form a supported bilayer. A silicon
oxide surface is modified, using etching techniques common in
silicon chip production, to expose small channels to an electrode
surface. A bilayer is then formed on the silicon oxide surface,
resulting in an electrical resistance of a few M.OMEGA.. In this
system, the wells created by this process could not be individually
addressed.
[0021] In both of the cited examples using a supported lipid
bilayer, it is very difficult to form a high resistive seal using
these methods. Although the resistance may be sufficient to observe
a change arising from a large number of ion channels, single
channel or stochastic measurements, which are inherently more
sensitive, are incredibly challenging using this methodology.
[0022] There are a number of problems with the supported bilayer
approach in these documents and in general, which makes this system
unsuitable. The first problem lies with the resistance of the
bilayer membrane which is typically about 100 M.OMEGA.. While this
may be suitable for examining protein behaviour at large protein
concentrations, it is not sufficient for a high-fidelity assay
based on single molecule sensing, typically requiring a resistance
of at least 1 G.OMEGA. and for some applications one or two orders
of magnitude higher. The second problem is the small volume of
solution trapped in the short distance between the bilayer and the
solid support, typically of the order of 1 nm. This small volume
does not contain many ions, affecting the stability of the
potential across the bilayer and limiting the duration of the
recording.
[0023] A number of methods have been proposed to overcome the
problems with solid supported bilayers. One option is to
incorporate a chemical linkage between the bilayer and the surface,
either a small polyethylene glycol layer is introduced (polymer
cushioned bilayers), or the lipid is chemically modified to contain
a small hydrophilic linkage and reacted with the surface providing
a scaffold for vehicle deposition (tethered bilayers). While these
methods have increased the ionic reservoir beneath the lipid
bilayer, they are inconvenient to implement and have done little to
decrease the current leakage across the bilayer.
[0024] The techniques used in the silicon chip industry provide an
attractive technology for creating a large number of electrodes
that could be used in biosensor applications. This approach is
disclosed in the related applications U.S. Pat. No. 7,144,486 and
U.S. Pat. No. 7,169,272. U.S. Pat. No. 7,144,486 discloses a method
of fabricating a microelectrode device containing microcavities
etched into layers of insulator material. The devices are said to
have a wide range of electrochemical applications in which
electrodes in the cavities measure electrical signals. It is stated
that thin films may be suspended across the cavities. Several types
of film are mentioned, including being a lipid bilayer. However
this is merely a proposal and there is no disclosure of any
technique for forming the lipid bilayer, nor any experimental
report of this. Indeed the related application U.S. Pat. No.
7,169,272, which does report experimental formation of lipid
bilayers in the same type of device, discloses the supported lipid
bilayers being chemically attached directly on the electrodes. This
uses similar techniques to those presented in Osman et al. cited
above and suffers from the same drawbacks relating to the lack of a
sufficiently high resistive seal for stochastic measurements and
the lack of an ionic reservoir for recording ionic flow across the
bilayer system.
[0025] To summarize, the technologies described above either
present methods of bilayer formation which can not reproducibly
achieve high resistance, or suffer from low ionic reservoirs and
are not capable of high duration direct current measurements, or
require a separate fluidic chamber for each array element, limiting
the scale up of that device to a high-density array. It would be
desirable to reduce these problems.
BRIEF SUMMARY OF THE DISCLOSURE
[0026] According to a first aspect of the present disclosure, there
is provided a method of forming a layer separating two volumes of
aqueous solution, the method comprising:
[0027] (a) providing an apparatus comprising elements defining a
chamber, the elements including a body of non-conductive material
having formed therein at least one recess opening into the chamber,
the recess containing an electrode;
[0028] (b) applying a pre-treatment coating of a hydrophobic fluid
to the body across the recess;
[0029] (c) flowing aqueous solution, having amphiphilic molecules
added thereto, across the body to cover the recess so that aqueous
solution is introduced into the recess from the chamber and so that
a layer of the amphiphilic molecules forms across the recess
separating a volume of aqueous solution introduced into the recess
from the remaining volume of aqueous solution.
[0030] Such a method allows the formation of layers of amphiphilic
molecules which are of sufficiently high quality for sensitive
techniques such as stochastic sensing whilst using apparatus and
techniques which are straightforward to implement.
[0031] The apparatus used is relatively simple, and comprises a
body of ionically non-conductive material having formed therein at
least one recess. It has been demonstrated, surprisingly, that it
is possible to form a layer of the amphiphilic molecules across
such a recess simply by flowing the aqueous solution across the
body to cover the recess. To achieve this, a pre-treatment coating
of a hydrophobic fluid is applied to the body across the recess.
The pre-treatment coating assists formation of the layer. The layer
is formed without any need for a complicated apparatus involving
two chambers separated by a septum and requiring a complicated
fluidics arrangement to achieve separate filling. This is because
the method does not require the recess to be pre-filled prior to
introducing aqueous solution into the chamber above. Instead, the
aqueous solution is introduced into the recess from the chamber.
Despite this, it is still possible to form the layer by mere
control of the aqueous solution flowing into the chamber. Such flow
control is a straightforward practical technique.
[0032] In some embodiments, the method may allow the formation of
layers of amphiphilic molecules which are suitable for high
sensitivity biosensor applications such as stochastic sensing and
single channel recording. It has been demonstrated possible to form
layers of high resistance providing highly resistive electrical
seals, having an electrical resistance of 1 G.OMEGA. or more,
typically at least 100 G.OMEGA., which, for example, enable
high-fidelity stochastic recordings from single protein pores. In
some embodiments, this maybe achieved whilst trapping a volume of
aqueous solution in the recess between the layer and the electrode.
This maintains a significant supply of electrolyte. For example,
the volume of aqueous solution is sufficient to allow stable
continuous do current measurement through membrane proteins
inserted in the layer. This contrasts significantly with the known
techniques described above using supported lipid bilayers.
[0033] Furthermore, the simple construction of the apparatus allows
the formation of a miniaturized apparatus having an array of plural
recesses and allowing the layer across each recess to be
electrically isolated and individually addressed using its own
electrode, such that the miniaturized array is equivalent to many
individual sensors measuring in parallel from a test sample. The
recesses may be relatively densely packed, allowing a large number
of layers to be used for a given volume of test sample. Individual
addressing may be achieved by providing separate contacts to each
electrode which is simple using modern microfabrication techniques,
for example lithography.
[0034] Furthermore, in some embodiments, the method may allow the
formation of multiple layers of one or more amphiphilic molecules
within a single apparatus across the plural recesses in an array
using a very straightforward technique.
[0035] In most applications, one or more membrane proteins may be
subsequently inserted into the layer. Certain membrane proteins
that may be used in accordance with the disclosure are discussed in
more detail below.
[0036] According to further aspects of the disclosure, there is
provided an apparatus suitable for implementing such methods of
formation of a layer of amphiphilic molecules.
[0037] Further details and features of the disclosure will now be
described.
[0038] The amphiphilic molecules are typically a lipid. In some
embodiments, the layer is a bilayer formed from two opposing
monolayers of lipid. The lipids may comprise one or more lipids.
The lipid bilayer may also contain additives that affect the
properties of the bilayer. Certain lipids and other amphiphilic
molecules, and additives that can be used in accordance with the
disclosure are discussed in more detail below.
[0039] Various techniques may be applied to add the amphiphilic
molecules to the aqueous solution.
[0040] A first technique is simply to add the amphiphilic molecules
to the aqueous solution outside the apparatus before introducing
the aqueous solution into the chamber.
[0041] A second technique comprises before introducing the aqueous
solution into the chamber, to deposit the amphiphilic molecules on
an internal surface of the chamber, or elsewhere in the flow path
of the aqueous solution, for example in a fluidic inlet pipe
connected to the inlet. In this case, the aqueous solution covers
the internal surface during step (c) whereby amphiphilic molecules
are added to the aqueous solution. In this manner the aqueous
solution is used to collect the amphiphilic molecules from the
internal surface. Such deposition of the amphiphilic molecules has
several advantages. It allows the formation of layer of amphiphilic
molecules in the absence of large amounts of organic solvent, as
would typically be present if the amphiphilic molecules were added
directly to the aqueous solution. This means that it is not
necessary to wait for evaporation of the organic solvent before the
layer can be formed. In addition, this means that the apparatus is
not required to be made from materials that are insensitive to
organic solvents. For instance, organic-based adhesives can be used
and screen-printed conductive silver/silver chloride paste can be
used to construct electrodes.
[0042] In some embodiments, the deposited amphiphilic molecules may
be dried. In such embodiments, an aqueous solution may be used to
rehydrate the amphiphilic molecules. This allows amphiphilic
molecules to be stably stored in the apparatus before use. In some
embodiments, it also avoids the need for wet storage of amphiphilic
molecules. Such dry storage of amphiphilic molecules increases
shelf life of the apparatus.
[0043] Several techniques may be used to insert a membrane protein
into the layer of amphiphilic molecules.
[0044] A first technique is simply for the aqueous solution to have
a membrane protein added thereto, whereby the membrane protein is
inserted spontaneously into the layer of amphiphilic molecules. One
or more membrane protein(s) may be added to the aqueous solution
outside the apparatus before introducing the aqueous solution into
the chamber. Alternatively a membrane protein may be deposited on
an internal surface of the chamber before introducing the aqueous
solution into the chamber. In this case, the aqueous solution
covers the internal surface during step (c), whereby one or more
membrane protein(s) is added to the aqueous solution.
[0045] A second technique is for the aqueous solution to have
vesicles containing a membrane protein added thereto, whereby the
membrane protein is inserted on fusion of the vesicles with the
layer of amphiphilic molecules.
[0046] A third technique is to insert one or more membrane protein
by carrying the membrane protein to the layer on a probe, for
example an agar-tipped rod.
[0047] To form a layer of amphiphilic molecules, the aqueous
solution is flowed across the body to cover the recess. Formation
is improved if a multi-pass technique is applied in which aqueous
solution covers and uncovers the recess at least once before
covering the recess for a final time. This is thought to be because
at least some aqueous solution is left in the recess which assists
formation of the layer in a subsequent pass.
[0048] The pre-treatment coating is a hydrophobic fluid which
assists formation of the layer by increasing the affinity of the
amphiphilic molecules to the surface of the body around the recess.
In general any pre-treatment that modifies the surface of the
surfaces surrounding the aperture to increase its affinity to
lipids may be used. Certain exemplary materials for the
pre-treatment coating that may be used in accordance with the
disclosure are discussed in more detail below.
[0049] To assist in the spreading of the pre-treatment coating,
surfaces including either or preferably both of (a) the outermost
surface of the body around the recess and (b) at least an outer
part of the internal surface of the recess extending from the rim
of the recess may be hydrophobic. This may be achieved by making
the body with an outermost layer formed of a hydrophobic
material.
[0050] Another way to achieve this is for the surfaces to be
treated by a fluorine species, such as a fluorine radical, for
example by treatment with a fluorine plasma during manufacture of
an apparatus of the disclosure.
[0051] The application of the pre-treatment coating may leave
excess hydrophobic fluid covering said electrode contained in the
recess. This potentially insulates the electrode by reducing ionic
flow, thereby reducing the sensitivity of the apparatus in
measuring electrical signals. However various different techniques
may be applied to minimize this problem.
[0052] A first technique may comprise applying a voltage across an
electrode in a recess and a further electrode in the chamber
sufficient to reduce the amount of excess hydrophobic fluid
covering said electrode contained in the recess. This produces a
similar effect to electro-wetting. The voltage is applied after
flowing aqueous solution across the body to cover the recess so
that aqueous solution flows into the recess. As the voltage will
rupture any layer formed across the recess, subsequently the
aqueous solution is flowed to uncover the recess, and then aqueous
solution, having amphiphilic molecules added thereto, is flowed
across the body to re-cover the recess so that a layer of the
amphiphilic molecules forms across the recess.
[0053] A second technique may comprise making an inner part of the
internal surface of the recess hydrophilic. Typically this may be
applied in combination with making the outer part of the internal
surface of the recess hydrophobic. This may be achieved by making
the body with an inner layer formed of a hydrophilic material and
an outermost layer formed of a hydrophobic material.
[0054] A third technique may comprise providing on the electrode a
hydrophilic surface, for example a protective material, which
repels the hydrophobic fluid applied in step (c) whilst allowing
ionic conduction from the aqueous solution to the electrode. The
protective material may be a conductive polymer, for example
polypyrrole/polystyrene sulfonate. Alternatively, the protective
material may be a covalently attached hydrophilic species, such as
thiol-PEG.
[0055] In general, a wide range of constructional features may be
employed in the apparatus to form the body of non-conductive
material, the at least one recess formed therein and the other
elements defining the chamber. Examples are described in further
detail below.
[0056] According to a second aspect of the present disclosure,
there is provided a method of improving the performance of an
electrode in a recess in conducting electro-physiological
measurements, the method comprising depositing a conductive polymer
on the electrode.
[0057] Further according to a second aspect of the present
disclosure, there is provided an apparatus for conducting
electro-physiological measurements, the apparatus comprising, a
body having a recess in which an electrode is located, wherein a
conductive polymer is provided on the electrode.
[0058] It has been discovered that the providing a conductive
polymer on an electrode in a recess can improve the performance of
the electrode in conducting electro-physiological measurements. One
advantage is to improve the electrode's performance as a stable
electrode for conducting electro-physiological measurements. A
further advantage is to increase the charge reservoir available to
the electrode within the recess without increasing the volume of
aqueous solution contained in the recess.
[0059] To allow better understanding, embodiments of the present
disclosure will now be described by way of non-limitative example
with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] Some embodiments of the disclosure may be understood by
referring, in part, to the present disclosure and the accompanying
drawings, wherein:
[0061] FIG. 1 is a perspective view of an apparatus;
[0062] FIG. 2 is a cross-sectional view of the apparatus of FIG. 1,
taken along line II-II in FIG. 1, and showing the introduction of
an aqueous solution;
[0063] FIG. 3 is a cross-sectional view of the apparatus, similar
to that of FIG. 2 but showing the apparatus full of aqueous
solution;
[0064] FIG. 4 is sequence of a cross-sectional, partial views of
the recess in the apparatus over an electrochemical electrode
modification process;
[0065] FIG. 5 is an SEM image of a recess formed by CO.sub.2 laser
drilling;
[0066] FIG. 6 is an OM image of a recess formed using
photolithography;
[0067] FIGS. 7A and 7B are 3D and 2D LP profiles, respectively, of
a recess formed using photolithography;
[0068] FIGS. 8A and 8B are 3D and 2D LP profiles, respectively, of
a recess formed using photolithography, after electroplating;
[0069] FIG. 9 is a cross-sectional, partial view of the recess in
the apparatus with a pre-treatment coating applied;
[0070] FIGS. 10A, 10B, 10C, 10D and 10E are a sequence of
cross-sectional, partial view of the recess in the apparatus during
a method of removing excess pre-treatment coating;
[0071] FIG. 11 is a cross-sectional, partial view of the recess in
the apparatus having plural further layers in the body;
[0072] FIG. 12 is a diagram of an electrical circuit;
[0073] FIG. 13 is a perspective view of the apparatus and
electrical circuit mounted on a printed circuit board;
[0074] FIG. 14 is a diagram of an electrical circuit for acquiring
plural signals in parallel;
[0075] FIG. 15 is a graph of the applied potential and current
response for a dry apparatus;
[0076] FIG. 16 is a graph of the applied potential and current
response for a wet apparatus;
[0077] FIG. 17 is a graph of the applied potential and current
response on electro-wetting of the apparatus;
[0078] FIG. 18 is a graph of the applied potential and current
response on formation of a layer of amphiphilic molecules;
[0079] FIGS. 19, 20, 21 and 22 are graphs of the applied potential
and current response for various different apparatuses;
[0080] FIGS. 23, 24 and 25 are plan views of a further layer in a
modified apparatus having plural recesses;
[0081] FIGS. 26, 27 and 28 are plan views of the substrate in the
modified apparatuses having plural recesses;
[0082] FIGS. 29 and 30 are graphs of the current response for two
different apparatuses having plural recesses;
[0083] FIG. 31 is a cross-sectional view of a portion of a modified
apparatus;
[0084] FIG. 32 is a cross-sectional view of another modified
apparatus;
[0085] FIG. 33 is a flow chart of a method of manufacture of the
apparatus;
[0086] FIGS. 34A and 34B are 3D- and 2D surface profiles of a
recess having an electrode modified by electropolymerisation of
polypyrrole, measured by profilometry;
[0087] FIG. 35 is a graph of current recorded on an array of
recesses having an electrode modified by electropolymerisation of
polypyrrole.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0088] An apparatus 1 which may be used to form a layer of
amphiphilic molecules is shown in FIG. 1. The apparatus 1 includes
a body 2 having layered construction as shown in FIGS. 2 and 3
comprising a substrate 3 of non-conductive material supporting a
further layer 4 also of nonconductive material. In the general
case, there may be plural further layers 4, as described further
below.
[0089] A recess 5 is formed in the further layer 4, in particular
as an aperture which extends through the further layer 4 to the
substrate 3. In the general case, there may be plural recesses 5,
as described further below.
[0090] The apparatus 1 further includes a cover 6 which extends
over the body 2. The cover 6 is hollow and defines a chamber 7
which is closed except for an inlet 8 and an outlet 9 each formed
by openings through the cover 6. The lowermost wall of the chamber
7 is formed by the further layer 4 in FIG. 2, but as an alternative
the further layer 4 could be shaped to provide side walls.
[0091] As described further below, in use aqueous solution 10 is
introduced into the chamber 7 and a layer 11 of amphiphilic
molecules is formed across the recess 5 separating aqueous solution
10 in the recess 5 from the remaining volume of aqueous solution in
the chamber 7. The apparatus includes the following electrode
arrangement to allow measurement of electrical signals across the
layer 11 of amphiphilic molecules.
[0092] Use of a chamber 7 which is closed makes it very easy to
flow aqueous solution 10 into and out of the chamber 7. This is
done simply by flowing the aqueous solution 10 through the inlet 8
as shown in FIG. 2 until the chamber 7 is full as shown in FIG. 3.
During this process, gas (typically air) in the chamber 7 is
displaced by the aqueous solution 10 and vented through the outlet
9. For example, a simple fluidics system attached to the inlet 8
may be used. This may be as simple as a plunger, although more
complicated systems may be used to improve the control. However,
the chamber 7 is not necessarily closed and may be open, for
example by forming the body 2 as a cup.
[0093] The substrate 3 has a first conductive layer 20 deposited on
the upper surface of the substrate 3 and extending under the
further layer 4 to the recess 5. The portion of the first
conductive layer 20 underneath the recess 5 constitutes an
electrode 21 which also forms the lowermost surface of the recess
5. The first conductive layer 20 extends outside the further layer
4 so that a portion of the first conductive layer 20 is exposed and
constitutes a contact 22.
[0094] The further layer 4 has a second conductive layer 23
deposited thereon and extending under the cover 6 into the chamber
7, the portion of the second conductive layer 23 inside the chamber
7 constituting an electrode 24. The second conductive layer 23
extends outside the cover 6 so that a portion of the second
conductive layer 23 is exposed and constitutes a contact 25.
[0095] The electrodes 21 and 24 make electrical contact with
aqueous solution in the recess 5 and chamber 7. This allows
measurement of electrical signals across the layer 11 of
amphiphilic molecules by connection of an electrical circuit 26 to
the contacts 22 and 25. The electrical circuit 26 may have
basically the same construction as a conventional circuit for
performing stochastic sensing across a lipid bilayer formed in a
conventional cell by the Montal & Mueller method.
[0096] An example design of the electrical circuit 26 is shown in
FIG. 12. The primary function of the electrical circuit 26 is to
measure the electrical current signal developed between the
electrodes 21 and 24 to provide a meaningful output to the user.
This may be simply an output of the measured signal, but in
principle could also involve further analysis of the signal. The
electrical circuit 26 needs to be sufficiently sensitive to detect
and analyze currents which are typically very low. By way of
example, an open membrane protein might typically pass current of
100 pA to 200 pA with a 1M salt solution.
[0097] In this implementation, the electrode 24 in the chamber 7 is
used as a reference electrode and the electrode 21 in the recess 5
is used as a working electrode. Thus the electrical circuit 26
provides the electrode 24 with a bias voltage potential relative to
the electrode 21 which is itself at virtual ground potential and
supplies the current signal to the electrical circuit 26.
[0098] The electrical circuit 26 has a bias circuit 40 connected to
the electrode 24 in the chamber 7 and arranged to apply a bias
voltage which effectively appears across the two electrodes 21 and
24.
[0099] The electrical circuit 26 also has an amplifier circuit 41
connected to the electrode 21 in the recess 5 for amplifying the
electrical current signal appearing across the two electrodes 21
and 24. Typically, the amplifier circuit 41 consists of a two
amplifier stages 42 and 43.
[0100] The input amplifier stage 42 connected to the electrode 21
converts the current signal into a voltage signal.
[0101] The input amplifier stage 42 may comprise transimpedance
amplifier, such as an electrometer operational amplifier configured
as an inverting amplifier with a high impedance feedback resistor,
of for example 500 M.OMEGA., to provide the gain necessary to
amplify the current signal which typically has a magnitude of the
order of tens to hundreds of picoamps.
[0102] Alternatively, the input amplifier stage 42 may comprise a
switched integrator amplifier. This is preferred for very small
signals as the feedback element is a capacitor and virtually
noiseless. In addition, a switched integrator amplifier has wider
bandwidth capability. However, the integrator does have a dead time
due to the necessity to reset the integrator before output
saturation occurs. This dead time may be reduced to around a
microsecond so is not of much consequence if the sampling rate
required is much higher. A transimpedance amplifier is simpler if
the bandwidth required is smaller. Generally, the switched
integrator amplifier output is sampled at the end of each sampling
period followed by a reset pulse. Additional techniques can be used
to sample the start of integration eliminating small errors in the
system.
[0103] The second amplifier stage 43 amplifies and filters the
voltage signal output by the first amplifier stage 42. The second
amplifier stage 43 provides sufficient gain to raise the signal to
a sufficient level for processing in a data acquisition unit 44.
For example with a 500 M.OMEGA. feedback resistance in the first
amplifier stage 42, the input voltage to the second amplifier stage
43, 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
43 must provide a gain of 50 to raise the 50 mV signal range to
2.5V.
[0104] The electrical circuit 26 includes a data acquisition unit
44 which may be a microprocessor running an appropriate program or
may include dedicated hardware. The data acquisition unit 44 may be
a card to be plugged into a computer 45 such as a desktop or
laptop. In this case, the bias circuit 40 is simply formed by an
inverting amplifier supplied with a signal from a digital-to-analog
converter 46 which may be either a dedicated device or a part of
the data acquisition unit 44 and which provides a voltage output
dependent on the code loaded into the data acquisition unit 44 from
software. Similarly, the signals from the amplifier circuit 41 are
supplied to the data acquisition card 40 through an
analog-to-digital converter 47.
[0105] The various components of the electrical circuit 26 may be
formed by separate components or any of the components may be
integrated into a common semiconductor chip. The components of the
electrical circuit 26 may be formed by components arranged on a
printed circuit board. An example of this is shown in FIG. 13
wherein the apparatus 1 is bonded to a printed circuit board 50
with aluminum wires 51 connecting from the contacts 22 and 25 to
tracks 52 on the printed circuit board. A chip 53 incorporating the
electrical circuit 26 is also bonded to the printed circuit board
50. Alternatively the apparatus 1 and the electrical circuit 26 may
be mounted on separate printed circuit boards.
[0106] In the case that the apparatus 1 contains plural recesses 5,
each having a respective electrode 21, then the electrical circuit
26 is modified essentially by replicating the amplifier circuit 41
and A/D converter 47 for each electrode 21 to allow acquisition of
signals from each recess 5 in parallel. In the case that the input
amplifier stage 42 comprises switched integrators then those would
require a digital control system to handle the sample-and-hold
signal and reset integrator signals. The digital control system is
most conveniently configured on a field-programmable-gate-array
device (FPGA). In addition the FPGA can incorporate processor-like
functions and logic required to interface with standard
communication protocols i.e. USB and Ethernet.
[0107] FIG. 14 shows a possible architecture of the electrical
circuit 26 and is arranged as follows. The respective electrodes 21
of the apparatus 1 are connected to the electrical circuit 26 by an
interconnection 55, for example the aluminum wires 51 and the
printed circuit board in the arrangement of FIG. 13. In the
electrical circuit 26, the amplifier circuits 41 may be formed in
one or more amplifier chips 56 having plural channels. The signals
from different electrodes 21 may be on separate channels or
multiplexed together on the same channel. The outputs of the one or
more amplifier chips 56 are supplied via the A/D converter 47 to a
programmable logic device 57 for receiving the signal on each
channel. For example to handle signals from an apparatus having
1024 recesses, the programmable logic device 57 might operate at a
speed of the order of 10 Mbits/s. The programmable logic device 57
is connected via an interface 58, for example a USB interface, to a
computer 59 to supply the signals to the computer 59 for storage,
display and further analysis.
[0108] During use the apparatus 1 may be enclosed in a Faraday cage
to reduce interference.
[0109] Various materials for the components of the apparatus 1 will
now be discussed. The materials for each component of apparatus 1
are determined by the properties required to enable the component
to function correctly during operation, but the cost and
manufacturing throughput are also considered. All materials may be
chosen to provide sufficient mechanical strength to allow robust
handling, and surfaces compatible with bonding to the subsequent
layers.
[0110] The material of the substrate 3 is chosen to provide a rigid
support for the remainder of the apparatus 1. The material is also
chosen to provide a high resistance and low capacitance electrical
insulation between adjacent electrodes 21 when there are plural
recesses 5. Possible materials include without limitation:
polyester (e.g. Mylar), or another polymer; or silicon, silicon
nitride, or silicon oxide. For example, the substrate may comprise
a silicon wafer with a thermally grown oxide surface layer.
[0111] The material of the further layer 4 (or in the general case
layers) are chosen to provide a high resistance and low capacitance
electrical insulation between the electrodes 21 and 24 and also,
when there are plural recesses 5, between the electrodes 21 and 24
of adjacent recesses 5. Also the surface of the further layer 4
should be chemically stable both to the pre-treatment coating
applied before operation (as discussed below) and to the aqueous
solution 10. Lastly, the further layer 4 should be mechanically
robust in order to maintain its structural integrity and coverage
of the first conductive layer 20, and should be suitable for
subsequent attachment of the cover 6.
[0112] The following is a list of possible materials for the
further layer 4, together with thicknesses which have been
successfully employed experimentally, although these thicknesses
are not limitative: photoresist (e.g. SU8 photoresist or Cyclotene)
with a variety of thicknesses; polycarbonate, 6 .mu.m thick film;
PVC, 7 .mu.m thick film; polyester, 50 .mu.m thick film; adhesive
backed polyester, 25 .mu.m and 50 .mu.m thick film; thermal
laminating films, e.g. Magicard 15 .mu.m thick and Murodigital 35
.mu.m; or a screen-printed dielectric ink.
[0113] Advantageously, surfaces including (a) the outermost surface
of the body 2 around the recess and (b) the outer part of the
internal surface of the recess 5 extending from the rim of the
recess 5 are hydrophobic. This assists in the spreading of the
pre-treatment coating and therefore also formation of a lipid
bilayer. One particular way to achieve this is to modify these
surfaces by a fluorine species. Such a fluorine species is any
substance capable of modifying the surfaces to provide a
fluorine-containing layer. The fluorine species is preferably one
containing fluorine radicals. For example the modification may be
achieved by treating the body 2 with a fluorine plasma, for example
a CF.sub.4 during manufacture.
[0114] The conductive layers 20 and 23 will now be discussed
further.
[0115] The material of the electrodes 21 and 24 should provide an
electrochemical electrode in contact with the aqueous solution 10,
enabling measurement of low currents, and should be stable to the
pre-treatment coating and aqueous solution 10. The material of the
remainder of the conductive layers 20 and 23 (usually but not
necessarily the same as the electrodes 21 and 24) also provides
electrical conductance from the electrodes to the contacts 22 and
25. The first conductive layers 20 will also accept bonding of the
further layers 4. The conductive layers 20 and 23 can be
constructed with plural overlapping layers and/or an appropriate
surface treatment. One possible material is platinum, coated with
silver at the area exposed to the test solution and then silver
chloride formed on top of the silver. Possible materials for the
first conductive layer 20 include without limitation: Silver/silver
chloride electrode ink; silver with or without a surface layer, for
example of silver chloride formed by chloridisation or of silver
fluoride formed by fluoridisation; gold with or without redox
couple in solution; platinum with or without redox couple in
solution; ITO with and without redox couple in solution; gold
electrochemically coated with conductive polymer electrolyte; or
platinum electrochemically coated with conductive polymer
electrolyte. Possible materials for the second conductive layer 23
include without limitation: silver/silver chloride electrode ink;
silver wire; or chloridised silver wire.
[0116] Some specific examples of include: the substrate 3 being
silicon and the conductive layer 20 being a metal conductor
(diffusion or polysilicon wires are poor methods) buried in a
silicon oxide insulating layer (e.g. using typical semiconductor
fabrication technology); the substrate 3 being glass and the
conductive layer 20 being metal conductors (e.g. using typical LCD
display technology); or the substrate 3 being a polymeric
substrates and the conductive layer 20 being an ablated metal or
printed conductor (e.g. using typical glucose biosensor
technology).
[0117] The requirements for the material of the cover 6 are to be
easily attached to create a seal for the chamber 7, to be
compatible with both the pre-treatment coating and the aqueous
solution 10. The following are possible materials, together with
thicknesses which have been successfully employed experimentally,
although these thicknesses are not limitative: silicone rubber,
0.5, 1.0, 2.0 mm thick; polyester, 0.5 mm thick; or PMMA (acrylic)
0.5 mm to 2 mm thick.
[0118] Various methods of manufacturing the apparatus 1 will now be
discussed. In general terms, the layered construction of the
apparatus 1 is simple and easy to form by a variety of methods.
Three different fabrication technologies which have actually been
applied are: lamination of polymer films; printed circuit board
manufacture with high resolution solder mask formation and
photolithography using silicon wafers or glass.
[0119] An example of a lamination process is as follows.
[0120] The substrate 3 is a 250 .mu.m thick polyester sheet
(Mylar), and the first conductive layer 20 is deposited by either:
screen printing silver/silver chloride electrode ink; adhesion of
metal foil; or vapour deposition (sputtering or evaporation). The
further layer 4 is then laminated onto the substrate 3 by either: a
pressure-sensitive adhesive; a thermally activated adhesive; or
using the wet silver/silver chloride ink as the adhesive painted
directly onto the dielectric before lamination (referred to as
"painted electrodes"). The aperture in the further layer 4 that
forms the recess 5 is created with 5-100 .mu.m diameter either
before or after lamination to the substrate 3 by either: electrical
discharge (sparking); or laser drilling, for example by an excimer,
solid state or CO.sub.2 laser. An apparatus created by lamination
of polymer films sometimes requires an additional sparking step to
activate the electrodes prior to use. The second conductive layer
23 is formed on top of the further layer 4 by screen printing. The
cover 6 is laminated on top using pressure sensitive adhesive.
[0121] An example of a process employing photolithography using
silicon wafers is as follows.
[0122] The substrate 3 is a silicon wafer with an oxide surface
layer. The first conductive layer 20 is formed by gold, silver,
chloridised silver, platinum or ITO deposited onto the substrate 3.
Photoresist (e.g. SU8) is then spin-coated over the substrate 3 to
form the further layer 4. The recess 5 is formed with 5-100 .mu.m
diameter by removal of the photoresist following UV exposure using
a mask to define the shape of the recess 5. The second conductive
layer 23 is formed on top of the further layer 4, for example by
screen printing. The cover 6 is laminated on top using pressure
sensitive adhesive.
[0123] The ability to use this type of process is significant
because it allows the apparatus to be formed on silicon chips using
standard silicon wafer processing technology and materials.
[0124] The electrodes 21 and 24 will now be discussed further.
[0125] For stable and reliable operation, the electrodes 21 and 24
should operate at the required low current levels with a low
over-potential and maintain their electrode potential value over
the course of the measurement. Further, the electrodes 21 and 24
should introduce a minimum amount of noise into the current signal.
Possible materials for the electrodes 21 and 24 include without
limitation: Silver/silver chloride electrode ink; silver with or
without a surface layer, for example of silver chloride formed by
chloridisation or of silver fluoride formed by fluoridisation; gold
with or without redox couple in solution; platinum with or without
redox couple in-solution; ITO with and without redox couple in
solution; palladium hydride, gold electrochemically coated with
conductive polymer electrolyte; or platinum electrochemically
coated with conductive polymer electrolyte.
[0126] Silver is a good choice for the material of electrodes 21
and 24 but is difficult to incorporate in a silicon wafer
manufacturing process due to its tendency to undergo oxidation on
exposure to light, air and high temperatures. To avoid this problem
it is possible to manufacture the apparatus with an inert
conductive material (e.g. Pt or Au) in the recess, and then change
the surface type or properties of the inert conductive material
using methods including but not limited to electroplating,
electropolymerisation, electroless plating, plasma modification,
chemical reaction, and other coating methods known in the art.
[0127] Electroplating of silver may be achieved, for example, using
a modified version of the method of Polk et al., "Ag/AgCl
microelectrodes with improved stability for microfluidics", Sensors
and Actuators B 114 (2006) 239-247. A plating solution is prepared
by addition of 0.41 g of silver nitrate to 20 ml of 1M ammonium
hydroxide solution. This is rapidly shaken to avoid precipitation
of the insoluble silver oxide, and to facilitate the formation of
the diammine silver complex. The solution is always fresh to avoid
fall in plating efficiency. The plating is performed using
conventional equipment, connecting the electrode 21 as the cathode
and using a platinum electrode is used as the anode. For example in
the case of plating on Pt electrodes, a potential of -0.58V is
applied to the cathode, with the anode being held at ground
potential, whereas in the case of plating on Au electrodes, the
potential is held at -0.48V with respect to ground. A target charge
of 5.1.times.10.sup.3 C/m.sup.2 has been found empirically to
result in a silver deposition of between 1 .mu.m and 2 .mu.m for a
100 .mu.m diameter electrode, typically taking of the order of 60
s.
[0128] In performing such plating it is desirable to achieve
uniform penetration of the aqueous plating solution to the bottom
of the recess 5. In the case that the layer 4 is formed from a
naturally hydrophobic material (e.g. SU8 photoresist) and in order
to ensure uniform wetting of the recess, desirably the degree of
hydrophilicity can be increased. Three methods to achieve this are
as follows. A first method is application of a lipid to the surface
of the layer 4, so that the lipid acts as a surfactant,
facilitating the entry of the plating solution. A second method is
exposure of the layer 4 to oxygen plasma which activates the
material of the layer and produces hydrophilic functional groups.
This produces a well defined hydrophilic and clean surface. A third
method is to add ethanol to the plating solution.
[0129] Where the electrode 21 is made of silver (or indeed other
metals), the outer surface of the electrode is desirably converted
to a halide, in order for the electrode 21 to function efficiently
as a provider of a stable reference potential. In common usage, the
halide used is chloride, since the conversion of silver to silver
chloride is relatively straightforward to achieve, for example by
electrolysis in a solution of hydrochloric acid. Alternative
chemical methods avoiding the use of a potentially corrosive acid
which may affect the surface condition of the layer 4 include a)
sweeping voltammetry in 3M sodium chloride solution, and b) a
chemical etching by immersion of the electrode 21 in 50 mM ferric
chloride solution.
[0130] An alternative halogen for the halidisation is fluorine. The
choice of fluorine has the significant advantage that the silver
fluoride layer can be formed in the same step as modification of
surfaces (a) and (b) of the body 2 to make them hydrophobic, as
discussed above. For example this may be achieved during
manufacture of the apparatus 1 by treatment of the body 2 by a
fluorine plasma for example a CF.sub.4 plasma. This is effective to
modify the surfaces of the body 2, particularly in the case that
the layer 4 is a photoresist such as SU8 to achieve a sufficient
degree of hydrophobicity to support the formation of a stable lipid
bilayer. At the same time exposure to the fluorine plasma converts
the metal of the electrode 21 into an outer layer of metal
fluoride.
[0131] There will now be discussed some possible adaptations of the
electrode 21 in the recess 5 as alternatives to the use of a
fluorine plasma as discussed above.
[0132] The electrode 21 may be electrochemically modified to change
the surface-type. This allows use of additional materials with good
bulk properties but poor surface properties, such as gold. Possible
electrochemical surface modifications include without limitation:
silver electroplating; electrochemical chloridisation of silver;
electropolymerisation of a polymer/polyelectrolyte.
[0133] By way of further example, one possible sequence of
modification is shown in FIG. 4 in which a coating 37 of silver is
formed on the electrode 21 formed of gold or platinum by
electrochemical deposition. Electroplating may typically be
performed in 0.2M AgNO.sub.2, 2M KI, 0.5 mM Na.sub.2S.sub.2O.sub.3
at -0.48V using a standard single liquid junction Ag/AgCl reference
electrode and a platinum counter electrode. A typical thickness of
the coating 37 is estimated to be 750 nm with a deposition time of
about 50 s and about 50 .mu.C charge passed. Subsequently a
chloridised layer 38 is formed by chloridisation, typically at +150
mV in 0.1M HCl for 30 s.
[0134] Another possible surface modification is to apply a
conductive polymer. The conductive polymer may be any polymer which
is conductive. A suitable conductive polymer will have mobile
charge carriers. Typically such a conductive polymer will have a
backbone having delocalised electrons which are capable of acting
as charge carriers, allowing the polymer to conduct. The conductive
polymer may be doped to increase its conductivity, for example by a
redox process or by electrochemical doping. Suitable conductive
polymers include, without limitation: polypyrroles, polyacetylenes,
polythiophenes, polyphenylenes, polyanilines, polyfluorenes,
poly(3-alkylthiophene)s, polytetrathiafulvalenes, polynaphthalenes,
poly(p-phenylene sulfide)s, polyindoles, polythionines,
polyethylenedioxythiophenes, and poly(para-phenylene
vinylene)s.
[0135] One possible conductive polymer is a polypyrrole, which may
be doped, for example with polystyrene sulfonate. This may be
deposited, for example, on an electrode 21 of gold by
electrooxidizing an aqueous solution of 0.1M pyrrole+90 mM
polystyrene sulfonate in 0.1M KCl at +0.80V vs. Ag/AgCl reference
electrode. The estimated thickness of polymer deposited is 1 .mu.m
at 30 .mu.C, based on an assumption that 40 mC/cm.sup.2 of charge
produces a film of thickness around 0.1 .mu.m. The polymerization
process can be represented as follows, where PE stands for
polystyrene sulfonate:
##STR00001##
[0136] One advantage of using a conductive polymer deposited on an
inert electrode, such as polypyrrole doped with polystyrene,
electropolymerised onto gold or platinum, is to improve the
electrode's performance as a stable electrode for conducting
electrophysiological measurements. A further advantage is to
increase the charge reservoir available to the electrode within the
recess without increasing the volume of aqueous solution contained
in the recess. These advantages are generally applicable when
conducting electrophysiological measurements using an electrode in
a recess, such as the electrode 21 in the apparatus 1.
[0137] Other advantages of using a conductive polymer on the
electrode 21 in the recess 5 of the apparatus 1 include but are not
limited to control of the hydrophilic nature of the electrode
surface to aid wetting of the electrode surface by the aqueous
buffer solution and similarly prevention of blocking of the
electrode by the chemical pre-treatment prior to bilayer
formation.
[0138] FIGS. 34A and 34B are 3D and 2D surface profiles of an
example electrode modified by electropolymerisation of polypyrrole,
measured by profilometry. The thickness of electrochemically
deposited polymer film in this example is about 2 .mu.m. FIG. 35
shows the current recorded on an array of recesses modified by
electropolymerisation of polypyrrole, showing stable lipid bilayers
and single molecule detection of cyclodextrin from inserted protein
pores.
[0139] In all embodiments, an alternative to the second conductive
layer 23 is to form an electrode in the chamber 7 simply by
insertion through the cover 6 of a conductive member, such as a
chloridised silver wire.
[0140] In order to characterise the electrodes 21, visualisation of
recesses 5 formed in a body 2 has been conducted using optical
microscopy (OM), scanning electron microscopy (SEM), and laser
profilometry (LP).
[0141] FIG. 5 shows an SEM image of a recess 5 formed by drilling
with a CO.sub.2 laser in an apparatus 1 formed by lamination of
polymer layers, with subsequent application of electrical discharge
to activate the electrode 21. The image illustrates that the
geometry of the recess 5 is poorly defined using this method of
formation, with considerable surface damage therearound and
variability in diameter, although it is hoped this maybe improved
through optimisation of the laser characteristics.
[0142] FIG. 6 shows an OM image of a recess 5 formed using
photolithography of a further layer 4 of SU8 photoresist over an
electrode 21 of vapour deposited gold on a substrate 3 of silicon.
Similarly, FIGS. 7A and 7B are 3D and 2D LP profiles of a similarly
manufactured recess 5. FIGS. 8A and 8B are 3D and 2D LP profiles of
the same recess 5 after electroplating to form a coating 38 of
silver. These images show that photolithography provides a high
degree of control of the geometry and dimensions of the recess.
[0143] Excimer laser methods also produce a controlled geometry
similar to photolithography.
[0144] There will now be described an example of a method of
manufacture of the apparatus 1, as shown in FIG. 33. The rationale
of this method is to provide high throughput manufacture. This is
achieved by processing a wafer of silicon which forms the substrate
3 of plural apparatuses 1 and which is subsequently diced. The
wafer is prepared with an insulating layer, for example a thermally
grown silicon-oxide.
[0145] First the wafer is prepared. In step S1, the wafer is
cleaned. In step S2, the wafer is subjected to a BF dif to improve
adhesion of metals and resist. Typical conditions are a 3 minute
dip in 10:1 buffered oxide etch. In S3, the wafer is subjected to a
bake as a dehydration step. Typical conditions are baking for 1
hour at 200.degree. C. in an oven.
[0146] Next, the wafer is metallised to provide the first
conductive layer 20 of each apparatus 1. In step S4, photoresist is
spun onto the wafer which is then subjected to UV light to form the
desired pattern. In step S5, the conductive layers 20 are
deposited, for example consisting of successive layers of Cr and
Au. Typically of respective thicknesses 50 nm and 300 nm. In step
S6 the resist is removed for example by soaking in acetone.
[0147] Next, the layers 4 and recesses 5 are formed. In step S7,
photoresist adhesion is improved by the application of an O.sub.2
plasma and dehydration bake for example in an oven. In step S8, the
wafer has applied thereto photoresist which is then subjected to UV
exposure to form the layers 4 and recesses, for example SU8-10 with
a thickness of 20 m. In step S9 an inspection and measurement of
the recesses is performed.
[0148] Next, the electrodes 21 are plated. In step S10, the surface
is prepared for plating by performing an O.sub.2 plasma descum. In
step S11, silver plating of the electrode is performed, as
described above, for example to form a plating thickness of 1.5
.mu.m.
[0149] In step S12, the wafer is diced to form the bodies 2 of
separate apparatuses 1.
[0150] Lastly, the bodies 2 are treated by a CF.sub.4 plasma which
modifies the surfaces of the body 2 and the electrode 21 as
discussed above. A typical exposure is for 12 minutes at 70 W and
160 mTorr.
[0151] In practice with an apparatus 1 manufactured using this
method, the results of bilayer formation and pore current stability
have been comparable to those achieved with bodies plated and
chloridised by wet chemical means.
[0152] The method of using the apparatus 1 to form a layer 11 of
amphiphilic molecules will now be described. First the nature of
the amphiphilic molecules that may be used will be considered.
[0153] The amphiphilic molecules are typically a lipid. In this
case, the layer is a bilayer formed from two opposing monolayers of
lipid. The two monolayers 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).
[0154] Any lipids that may form a lipid bilayer may be used. The
lipids are chosen such that a lipid bilayer having the required
properties, such as 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.
[0155] 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.
[0156] 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.
[0157] 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.
[0158] However, although lipids are commonly used to form bilayers,
it is expected that in general the method is applicable to any
amphiphilic molecules which may form a layer.
[0159] As to the aqueous solution 10, in general a wide range of
aqueous solutions 10 that are compatible with the formation of a
layer 11 of amphiphilic molecules may be used. The aqueous solution
10 is typically a physiologically acceptable solution. The
physiologically acceptable solution is typically buffered to a pH
of 3 to 11. The pH of the aqueous solution 10 will be dependent on
the amphiphilic molecules used and the final application of the
layer 11. Suitable buffers include without limitation: 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 10 may be 10 mM PBS containing 1.0M sodium
chloride (NaCl) and having a pH of 6.9.
[0160] The method of using the apparatus 1 is as follows.
[0161] First, a pre-treatment coating 30 is applied to the body 2
across the recess 5, as shown in FIG. 9. The pre-treatment coating
30 is a hydrophobic fluid which modifies the surface of the body 2
surrounding the recess 5 to increase its affinity to the
amphiphilic molecules.
[0162] The pre-treatment coating 30 is typically an organic
substance, usually having long chain molecules, in an organic
solvent. Suitable organic substances include without limitation:
n-decane, hexadecane, isoecoisane, squalene, fluoroinated oils
(suitable for use with fluorinated lipids), alkyl-silane (suitable
for use with a glass membrane) and alkyl-thiols (suitable for use
with a metallic membrane). Suitable solvents include but are not
limited to: pentane, hexane, heptane, octane, decane, and toluene.
The material might typically be 0.1 .mu.l to 10 .mu.l of 0.1% to
50% (v/v) hexadecane in pentane or another solvent, for example 2
.mu.l of 1% (v/v) hexadecane in pentane or another solvent, in
which case lipid, such as
diphantytanoyl-sn-glycero-3-phosphocholine (DPhPC), might be
included at a concentration of 0.6 mg/ml.
[0163] Some specific materials for the pre-treatment coating 30 are
set out in Table 1 by way of example and without limitation.
TABLE-US-00001 TABLE 1 Pre-treatment formulation Volumes applied
0.3% hexadecane in pentane 2 .times. 1 .mu.l 1% hexadecane in
pentane 2 .times. 2 .times. 0.5 .mu.l; 2 .times. 0.5 .mu.l; 1
.mu.l; 2 .times. 1 .mu.l; 2 .times. 1 .mu.l; 2 .mu.l; 2 .times. 2
.mu.l; 5 .mu.l 3% hexadecane in peatane 2 .times. 1 .mu.l; 2 .mu.l
10% hexadecane in pentane 2 .times. 1 .mu.l; 2 .mu.l; 5 .mu.l 0.5%
hexadecane + 0.6 mg/ml 5 .mu.l DPhPC lipid in pentane 1.0%
hexadecane + 0.6 mg/ml 2 .times. 2 .times. 0.5 .mu.l DPhPC lipid in
pentane 1.5% hexadecane + 0.6 mg/ml 2 .mu.l; 2 .times. 1 .mu.l
DPhPC lipid in pentane
[0164] The pre-treatment coating 30 may be applied in any suitable
manner, for example simply by capillary pipette. The pre-treatment
coating 30 may be applied before or after the cover 6 is attached
to the apparatus 1.
[0165] The precise volume of material of the pre-treatment coating
30 required depends on the size of the recess 5, the formulation of
the material, and the amount and distribution of the when it dries
around the aperture. In general increasing the amount (by volume
and/or by concentration) improves the effectiveness, although
excessive material can cover the electrode 21 as discussed below.
As the diameter of the recess 5 is decreased, the amount of
material of the pre-treatment coating 30 required also varies. The
distribution of the pre-treatment coating 30 can also affect
effectiveness, this being dependent on the method of deposition,
and the compatibility of the membrane surface chemistry. Although
the relationship between the pre-treatment coating 30 and the ease
and stability of layer formation is complex, it is straightforward
to optimize the amount by routine trial and error. In another
method the chamber 7 can be completely filled by pre-treatment in
solvent followed by removal of the excess solvent and drying with a
gas flow.
[0166] The pre-treatment coating 30 is applied across the recess 5.
As a result and as shown in FIG. 9, the pre-treatment coating 30
covers the surface of the body 2 around the recess 5. The
pre-treatment coating 30 also extends over the rim of the recess 5
and desirably covers at least the outermost portion of the side
walls of the recess 5. This assists with formation of the layer 11
of amphiphilic molecules across the recess 5.
[0167] However, the pre-treatment coating 30 also has a natural
tendency during application to cover the electrode 21. This is
undesirable as the pre-treatment coating 30 reduces the flow of
current to the electrode 21 and therefore reduces the sensitivity
of measurement of electrical signals, in the worst case preventing
any measurement at all. A number of different techniques may be
employed to reduce or avoid this problem, and will be discussed
after the description of forming the layer 11 of amphiphilic
molecules.
[0168] After application of the pre-treatment coating 30, the
aqueous solution 10 is flowed across the body 2 to cover the recess
5 as shown in FIG. 3. This step is performed with the amphiphilic
molecules added to the aqueous solution 10. It has been
demonstrated that, with an appropriate pre-treatment coating 30
this allows the formation of the layer 11 of amphiphilic molecules
across the recess 5. Formation is improved if a multi-pass
technique is applied in which aqueous solution 10 covers and
uncovers the recess 5 at least once before covering the recess 5
for a final time. This is thought to be because at least some
aqueous solution is left in the recess 5 which assists formation of
the layer 11 in a subsequent pass. Notwithstanding this, it should
be noted that the formation of the layer 11 is reliable and
repeatable. This is despite the fact that the practical technique
of flowing aqueous solution 10 across the body 2 through the
chamber 7 is very easy to perform. Formation of the layer 11 may be
observed by monitoring of the resultant electrical signals across
the electrodes 21 and 24, as described below. Even if a layer 11
fails to form it is a simple matter to perform another pass of the
aqueous solution 10. Such reliable formation of a layer 11 of
amphiphilic molecules using a simple method and a relatively simple
apparatus 1 is a particular advantage of the present
disclosure.
[0169] Furthermore, it has been demonstrated that the layers 11 of
amphiphilic molecules are of high quality, in particular being
suitable for high sensitivity biosensor applications such as
stochastic sensing and single channel recording. The layers 11 have
high resistance providing highly resistive electrical seals, having
an electrical resistance of 1 G.OMEGA. or more, typically at least
100 G.OMEGA. which, for example, enables high-fidelity stochastic
recordings from single protein pores.
[0170] This is achieved whilst trapping a volume of aqueous
solution 10 in the recess 5 between the layer 11 and the electrode
21. This maintains a significant supply of electrolyte. For
example, the volume of aqueous solution 10 is sufficient to allow
stable continuous do current measurement through membrane proteins
inserted in the layer. Experimental results demonstrating these
advantages are set out later.
[0171] There are various techniques for adding the amphiphilic
molecules to the aqueous solution 10, as follows.
[0172] A first technique is simply to add the amphiphilic molecules
to the aqueous solution 10 outside the apparatus 1 before
introducing the aqueous solution 10 into the chamber 7.
[0173] A second technique which has particular advantage is, before
introducing the aqueous solution 10 into the chamber 7, to deposit
the amphiphilic molecules on an internal surface of the chamber 7,
or on an internal surface elsewhere in the flow path of the aqueous
solution 10 into the chamber 7, for example in a fluidic inlet pipe
connected to the inlet. The amphiphilic molecules can be deposited
on any one or more of the internal surfaces of the chamber 7,
including a surface of the further layer 4 or of the cover 6. The
aqueous solution 10 covers the internal surface during its
introduction, whereby the amphiphilic molecules are added to the
aqueous solution 10. In this manner, the aqueous solution 10 is
used to collect the amphiphilic molecules from the internal
surface. The aqueous solution 10 may cover the amphiphilic
molecules and the recess 5 in any order but preferably covers the
amphiphilic molecules first. If the amphiphilic molecules are to be
covered first, the amphiphilic molecules are deposited along the
flow path between the inlet 8 and the recess 5.
[0174] Any method may be used to deposit the lipids on an internal
surface of the chamber 7. 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. An apparatus
1 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.
[0175] The deposited amphiphilic molecules are preferably dried. In
this case, the aqueous solution 10 is used to rehydrate the
amphiphilic molecules. This allows the amphiphilic molecules to be
stably stored in the apparatus 1 before use. It also avoids the
need for wet storage of amphiphilic molecules. Such dry storage of
amphiphilic molecules increases shelf life of the apparatus. Even
when dried to a solid state, the amphiphilic molecules 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.
[0176] In most practical uses, a membrane protein is inserted into
the layer 11 of amphiphilic molecules. There are several techniques
for achieving this.
[0177] A first technique is simply for the aqueous solution 10 to
have a membrane protein added thereto, whereby the membrane protein
is inserted spontaneously into the layer 11 of amphiphilic
molecules after a period of time. The membrane protein may be added
to the aqueous solution 10 outside the apparatus 1 before
introducing the aqueous solution 10 into the chamber 7.
Alternatively the membrane protein may be added after formation of
the layer 11.
[0178] Another way of adding the membrane protein to the aqueous
solution 10 is to deposit it on an internal surface of the chamber
7 before introducing the aqueous solution 10 into the chamber 7. In
this case, the aqueous solution 10 covers the internal surface
during its introduction, whereby the membrane protein is added to
the aqueous solution 10 and subsequently will spontaneously insert
into layer 11. The membrane proteins may be deposited on any one or
more of the internal surfaces of the chamber 7, including a surface
of the further layer 4 or of the cover 6. The membrane proteins can
be deposited on the same or different internal surface as the
amphiphilic molecules (if also deposited). The amphiphilic
molecules and the membrane proteins may be mixed together.
[0179] Any method may be used to deposit the membrane proteins on
an internal surface of the chamber 7. Suitable methods include, but
are not limited to, drop coating, various printing techniques,
spin-coating, painting, dip coating and aerosol application.
[0180] The membrane proteins are preferably dried. In this case,
the aqueous solution 10 is used to rehydrate the membrane proteins.
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 100 wt % or
less than 5 wt % solvent.
[0181] A second technique is for the aqueous solution 10 to have
vesicles containing the membrane protein added thereto, whereby the
membrane protein is inserted on fusion of the vesicles with the
layer 11 of amphiphilic molecules.
[0182] A third technique is to insert the membrane protein by
carrying the membrane protein to the layer 11 on a probe, for
example an agar-tipped rod, using the techniques disclosed in
WO-2006/100484. Use of a probe may assist in selectively inserting
different membrane proteins in different layers 11, in the case
that the apparatus has an array of recesses. However, this requires
modification to the apparatus 1 to accommodate the probe.
[0183] 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, P-barrel membrane proteins, such
as 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 anthrax
protective antigen, 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.
[0184] 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.
[0185] 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.
[0186] The variant may be a naturally-occurring variant which is
expressed by an organism, for instance by a Staphylococcus
bacterium. Variants also include non-naturally occurring variants
produced by recombinant technology. Over the entire length of the
amino acid sequence of SEQ ID NO: 2 or 3, a variant will preferably
be at least 50% homologous to that sequence based on amino acid
identity. More preferably, the subunit polypeptide is at least 80%,
at least 90%, at least 95%, at least 98%, at least 99% homologous
based on amino acid identity to the amino acid sequence of SEQ ID
NO: 2 or 3 over the entire sequence. Amino acid substitutions may
be made to the amino acid sequence of SEQ ID NO: 2 or 3, for
example a single amino acid substitution may be made or two or more
substitutions may be made. Conservative substitutions may be made,
for example, according to the following table. Amino acids in the
same block in the second column and preferably in the same line in
the third column may be substituted for each other:
TABLE-US-00002 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
[0187] 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.
[0188] 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.
[0189] 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 may 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.
[0190] 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.
[0191] 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 may 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
may be added between residues 127 and 130 of SEQ ID NO: 3,
following removal of residues 128 and 129. Additions may be made at
the equivalent positions in SEQ ED NO: 2. A carrier protein may be
fused to an amino acid sequence according to the disclosure.
[0192] 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, p 387-395). 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) J Mol Biol 215:403-10. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (http://www.ncbi.nhn.nih.gov/).
[0193] The membrane proteins can be labelled with a revealing or
detection/detectable label. The detectable 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.
[0194] 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
transcription translation. 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.
[0195] 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.
[0196] 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.
[0197] Recombinant membrane proteins may be produced using standard
methods known in the art. Nucleic acid sequences encoding a protein
may be isolated and replicated using standard methods in the art.
Nucleic acid sequences encoding a protein may be expressed in a
bacterial host cell using standard techniques in the art. The
protein may 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.
[0198] Thus an apparatus of the disclosure, such as apparatus 1 can
be used for a wide range of applications. Typically a membrane
protein is inserted in the layer 11. An electrical signal,
typically a current signal, developed between the electrode 21 in
the recess 5 and the further electrode 24 in the chamber 7 is
monitored, using the electrical circuit 26. Often a voltage is also
applied between the electrodes 21 and 24, whilst monitoring the
electrical signal. The form of the electrical signal, and in
particular changes therein, provide information about the layer 11
and any membrane protein inserted therein.
[0199] Some non-limitative examples of uses will now described. One
use is in vitro investigation of membrane proteins by
single-channel recording. An important commercial use is as a
biosensor to detect the presence of a range of substances. The
apparatus 1 may be used to detect an analyte molecule that binds
with an inserted membrane protein, or another stimulus, using
stochastic sensing by detecting a change in the current flow
indicating the presence of the analyte molecule or other stimulus.
Similarly, the apparatus 1 may be used to detect the presence or
absence of membrane pores or channels in a sample, by detecting a
change in the current flow as the pore or channel inserts. 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.
[0200] Some techniques to reduce or avoid the problem of the
pre-treatment coating 30 covering the electrode 21 will now be
discussed.
[0201] A first technique may comprise the following: after
application of the pre-treatment coating 30 to apply a voltage
across the electrode 21 in the recess 5 and the further electrode
24 in the chamber 7 sufficient to reduce the amount of excess
hydrophobic fluid covering the electrode 21 in the recess 5. This
is produces a similar effect to electro-wetting.
[0202] This technique is illustrated in FIGS. 10A, 10B, 10C, 10D
and 10E. First, as shown in FIG. 10A, the pre-treatment coating 30
is applied as shown in FIG. 10A where the pre-treatment coating 30
covers the electrode 21. Next, as shown in FIG. 10B, aqueous
solution 10 is flowed across the body 2 to cover the recess 5 so
that aqueous solution 10 flows into the recess 5. Next, a voltage
is applied which removes the pre-treatment coating 30 covering the
electrode 21, as shown in FIG. 10C. This voltage will rupture any
layer of amphiphilic molecules formed across the recess 5.
Therefore, next, as shown in FIG. 10D, the aqueous solution 10 is
flowed out of the chamber 7 to uncover the recess 5. Typically an
amount of aqueous solution 10 will remain in the recess 5. Lastly,
as shown in FIG. 10E, aqueous solution 10, having amphiphilic
molecules added thereto, is flowed across the body 2 to re-cover
the recess 5 so that the layer 11 of the amphiphilic molecules
forms.
[0203] This is most simply performed by flowing the same aqueous
solution 10 in and out of the chamber 7. However, in principle, the
aqueous solution 10 flowed into the chamber 7 to re-covering the
recess 5 (in FIG. 10E) could be different from the aqueous solution
10 flowed into the chamber 7 to first cover the recess 5 (in FIG.
10B) before applying the voltage. Similarly, there could be no
amphiphilic molecules added to the aqueous solution 10 flowed into
the chamber 7 to first cover the recess 5 (in FIG. 10B) before
applying the voltage.
[0204] A second technique comprises making an inner part of the
internal surface of recess 5 hydrophilic. This may be achieved by
making body 2 with two (or in general more) further layers 4a and
4b as shown in FIG. 11, of which the innermost further layer 4a (or
layers) formed of a hydrophilic material, for example SiO.sub.2.
Typically but without limitation, the innermost further layer 4a
might have a thickness of 2 .mu.m.
[0205] The outermost further layer 4b (or layers) is formed of a
hydrophobic material and as a result both of (a) the outermost
surface of the body 2 around the recess and (b) the outer part of
the internal surface of the recess 5 extending from the rim of the
recess 5 is hydrophobic. This assists in the spreading of the
pre-treatment coating. In some embodiments, indeed this property of
these surfaces of the body 2 is desirable even if there is not an
inner further layer 4a formed of a hydrophilic material. Typically
but without limitation, outermost further layer 4b might have a
thickness of 1 .mu.m, 3 .mu.m, 5 .mu.m, 10 .mu.m, 20 .mu.m or 30
.mu.m.
[0206] A third technique comprises providing a hydrophillic surface
on electrode 21 which repels an applied pre-treatment coating 30,
whilst allowing ionic conduction from aqueous solution 10 to
electrode 2. This may be achieved by depositing a protective
material on electrode 21. A range of protective materials may be
used. One possibility is a conductive polymer, for example
polypyrrole/polystyrene sulfonate as discussed above. Another
possibility is a covalently attached hydrophilic species, such as
thiol-PEG.
[0207] As will be understood by those skilled in the art who have
the benefit of the instant disclosure, other equivalent or
alternative devices, methods, and systems for formation of layers
of amphiphilic molecules can be envisioned without departing from
the description contained herein. Accordingly, the manner of
carrying out the disclosure as shown and described is to be
construed as illustrative only. Persons skilled in the art may make
various changes in the shape, size, number, and/or arrangement of
parts of one or more of the apparatus of the disclosure without
departing from the scope of the instant disclosure. Similarly one
or more methods of the disclosure may be changed by varying the
steps without departing from the scope of the disclosure. Also,
where ranges have been provided, the disclosed endpoints may be
treated as exact and/or approximations as desired or demanded by
the particular embodiment. In addition, it may be desirable in some
embodiments to mix and match range endpoints. A device or parts
thereof may be configured and arranged to be disposable,
serviceable, interchangeable, and/or replaceable. These equivalents
and alternatives along with obvious changes and modifications are
intended to be included within the scope of the present disclosure.
Accordingly, the foregoing disclosure is intended to be
illustrative, but not limiting, of the scope of the disclosure as
illustrated by the following examples and claims.
Example 1
[0208] The apparatus 1 described above has been made and used
experimentally to demonstrate formation of a layer 11, in
particular being a lipid bilayer, and insertion of a membrane
protein, for example .alpha.-hemolysin. The following procedure was
followed after manufacture of the apparatus 1:
[0209] 1) apply pre-treatment coating 30 to body 2;
[0210] 2) introduce aqueous solution 10 into chamber 7 to cover
recess 5;
[0211] 3) electro-wet the electrode 21;
[0212] 4) remove aqueous solution 10 to un-cover recess 5 and re
introduce aqueous solution 10 into chamber 7 to cover recess 5 and
form the layer 11;
[0213] 5) add a-hemolysin free into aqueous solution 10 and monitor
insertion into layer 11.
[0214] In step 1), the pre-treatment coating 30 was hexadecane
dissolved in pentane. The quantity and volume of the pre-treatment
coating 30 was varied for each test to obtain the optimum
conditions for formation of the layer 11. Insufficient
pre-treatment coating 30 prevented formation of the layer 11 while
excess pre-treatment coating 30 caused blocking of the recesses.
However routine variation of the amount allowed optimisation.
[0215] The amphiphilic molecules were a lipid, in particular
1,2-diphytanoyl-sn-glycero-3-phosphocholine. The lipid was
dissolved in pentane and then dried onto the surface of the cover 6
defining an internal surface of the chamber 7 before attaching the
cover 6 on top of the body 2. In step 2), the aqueous solution 10
collected the lipid.
[0216] Step 3) was performed by application of a large potential to
across the electrodes 21 and 24. This removed excess pre-treatment
coating 30 from the electrode 21. Although not required in every
case, when performed this stage helped to condition the recess 5
for formation of the layer 11 and assisted subsequent measurement
of electrical signals.
[0217] By monitoring of the electrical signals developed across the
electrodes 21 and 24, in steps 4) and 5), formation of the layer 11
and insertion of the membrane protein was observed.
[0218] The procedure was successfully performed for an apparatus 1
of the type described above formed by lamination onto a polymer
substrate 3. Formation of the layer 11 and insertion of the
membrane protein was observed using all the fabrication variables
described above, albeit with varying degrees of repeatability and
signal quality.
Example 2
[0219] An example will now be described for a typical apparatus 1,
in which the first conductive layer 20 was formed by a silver foil
strips (25 .mu.m thick, from Goodfellow) thermally laminated onto
the substrate 3 using a 15 .mu.m thick laminating film (Magicard)
to form the further layer 4. A circular recess 5 of diameter 100
.mu.m was created further layer 4 using an excimer laser, exposing
a circular silver electrode 21 of diameter 100 .mu.m. The exposed
silver was chloridised electrochemically as described previously.
The second conductive layer 23 was a screen printing silver/silver
chloride ink printed on the top side of the body 2.
[0220] The pre-treatment coating 30, comprising 0.5 .mu.l of 1%
heaxadecane+0.6 mg/ml DPhPC in pentane, was then applied to the
body 2 and dried at room temperature.
[0221] The cover 6 comprised a 1 mm thick silicon rubber body with
a 250 .mu.m thick Mylar lid. Lipid (4 .mu.l of 10 mg/ml DPhPC in
pentane) was applied to the inside of the cover 6 and allowed to
dry at room temperature before attachment to the body 2 with
self-adhesive.
[0222] A typical successful test proceeded as follows.
[0223] The dry contacts 22 and 25 were attached to the electrical
circuit 26 enclosed in a Faraday cage and a 20 mV 50 Hz triangular
potential waveform applied. FIG. 15 shows the applied waveform and
the resultant current signal which is indicative of the expected
capacitive response.
[0224] Addition of the aqueous solution 10 creates an "open
circuit" connection between the electrodes, such that the current
response to the applied potential waveform is large, typically
saturating the current amplifier. A typical trace is shown in FIG.
16, involving a current response greater than 20,000 pA to the 20
mV potential. This corresponds to a resistance of less than 1
M.OMEGA., which is sufficiently small for use in conjunction with
bilayer formation and pore current measurement.
[0225] In the event that the electrode 21 does not initially form a
proper electrical connection with the aqueous solution 10,
application of a -1V DC potential can be used to increase in the
available active electrode area. This is illustrated in FIG. 17, in
which the electrode begins partially active and is then fully
activated after around 4 s of the applied potential.
[0226] Following open-circuit connection between the aqueous
solution 10 and the electrode 21, the aqueous solution 10 is
removed from the chamber 7 and reintroduced. On re-introduction, a
layer 11 of the lipid collected from the internal surface of
chamber 7 is formed across recess 5. The formation is observed by
an increase in the capacitive squarewave current response to just
under 500 pA, for example as shown in FIG. 18. This value is
consistent with the capacitance expected for a circular lipid
bilayer of diameter of order 100 .mu.m and varies predictably for
different geometries.
[0227] Subsequent addition of .alpha.-hemolysin to the aqueous
solution 10 creates a current response typical of pore insertion
under an applied potential of 100 mV. For example FIG. 19 is a
typical example with cyclodextrin present in the aqueous solution
10 and shows an expected current response with binding events
confirming that the current is through the pores.
[0228] Although the example above shows data for the thermally
laminated apparatus 1, the other systems investigated also produced
successful formation of the layer 11 and pore insertion. For
example, this was also successfully demonstrated for an apparatus 1
formed by lamination using pressure-sensitive adhesive bonding of
the further layer 4. However, the adhesive layer was found to
complicate formation of the recess 5 both in terms of the resulting
aspect ratio and spreading of the adhesive across the electrode 21.
This problem was overcome by electrical sparking to "activate" the
electrode 21.
[0229] The impact of the quality of the recess 5 is evident by
comparing results from recesses formed by a CO.sub.2 laser and an
excimer laser, as shown in FIGS. 20 and 21, respectively. In both
cases formation of the layer 11 and pore insertion is successful
and evident in the response, but more reproducible apertures were
produced using the excimer laser. Recesses 5 formed by the CO.sub.2
laser tended to form relatively leaky layers 11 with more noisy
pore signals and were also susceptible to blocking. Recesses 5
formed by the excimer laser produced well sealed layers 11 with
good pore signals.
[0230] Formation of the layer 11 and pore insertion was similarly
observed with an apparatus 1 formed as described above using high
definition printed circuit board manufacture. In this case, to form
apparatus 1, the first conductive layer 20 was formed by etching
the copper foil on an FR4 substrate typically used in printed
circuit board manufacture. The board was then screen printed with a
Ronascreen SPSR.TM. photoimageable solder mask to a depth of 25
.mu.m and exposed to UV light on an Orbotech Paragon 9000 laser
direct imaging machine and developed with KaCO.sub.3 solution to
create 100 .mu.,m circular apertures over the electrodes 21.
[0231] Formation of the layer 11 and pore insertion was similarly
observed with an apparatus 1 formed as described above using
photolithography. In this case, to form the apparatus 1, the first
conductive layer 20 was formed by gold vapour deposited using
clean-room facilities onto the substrate 3 and a further layer 4 of
SU8 photoresist of thickness 12.5 .mu.m was spin-coated on top.
Recesses 5 were formed by curing of the photoresist by UV exposure
with a mask and subsequent removal of the uncured photoresist.
Recesses 5 had a diameter of 100 .mu.m, exposing an electrode 21 of
diameter 100 .mu.m. After baking to set the photoresist, the wafer
was diced to form separate substrates each with a single recess 5.
The electrodes 21 were electroplated with silver and then
chloridised electrochemically as described previously. The second
electrode 24 was screen printed silver/silver chloride ink printed
on the top side of the body 2.
[0232] The pre-treatment coating 30, comprising 0.5 .mu.l of 0.75%
hexadecane in pentane, was then applied to the body 2 and dried at
room temperature.
[0233] The cover 6 comprised a 1 mm thick silicon rubber body with
a 250 .mu.m thick Mylar lid. Lipid (4 .mu.l of 10 mg/ml DPhPC in
pentane) was applied to the inside of the cover 6 and allowed to
dry at room temperature before attachment to the body 2 with
self-adhesive.
[0234] Testing was performed as described above and successful
formation of the layer 11 and pore insertion was observed. For
example, FIG. 22 shows a typical current trace showing cyclodextrin
binding events with wild-type .alpha.-hemolysin pores.
[0235] These results generally show the ease with which the method
of formation of the layer 11 may be performed. In particular
formation of the layer 11 is achieved with a wide range of
materials of the apparatus 1, dimensions (width and depth) of the
recess 5, and methods of manufacture. Some variation in success
rate is evident but in general this can be optimised by routine
testing of different apparatuses 1. In particular the formation of
the layer 11 is not overly dependent on the width of the recess 5.
Formation has been demonstrated over widths from 5 .mu.m to 100
.mu.m and in view of the ease of formation it is expected that
formation is possible at higher widths up to 200 .mu.m, 500 .mu.m
or higher. Also in view of this ease of formation of the layer 11,
it is expected that variations of the shape of the recess 5 could
also be accommodated.
Example 3
[0236] There will now be discussed modifications to the apparatus 1
to include plural recesses 5, commonly referred to as an array of
recesses 5. The ability to easily form an array of layers 11 across
an array of recesses 5 in a single apparatus 1 is a particular
advantage of the present disclosure. By contrast to traditional
methods of formation of lipid bilayers, the apparatus 1 has a
single chamber 7, but creates the layer 11 in situ during the test
and captures a reservoir of electrolyte in the recess 5 under the
layer 11 which allows continuous stable measurement of current
passing through protein pores inserted in the layer 11. Further the
layer 11 formed is of high quality and is localised to the area of
the recess 5, ideal for high-fidelity current measurements using
membrane protein pores. These advantages are magnified in an
apparatus 1 which forms an array of layers 11 because this allows
measurements to be taken across all the layers 11 in parallel,
either combining the current signals to increase sensitivity or
monitoring the current signals separately to perform independent
measurements across each layer 11.
[0237] Apparatuses having an array of recesses 5 have been tested
and demonstrated successful formation of an array of layers 11,
showing the possibility of creating a miniaturized array of close
packed individually addressable layers recording current signals in
parallel from a test sample.
[0238] Essentially an apparatus 1 having an array of recesses 5 can
be formed simply using the manufacturing techniques described above
but instead forming plural recesses 5. In this case, the first
conductive layer 20 is divided to form a separate electrode 21,
contact 22 and intermediate conductive track 27 in respect of each
recess 5. The apparatus 1 has a single chamber 7 with a single
electrode 24 common to all the recesses 5.
[0239] FIGS. 23, 24 and 25 show first to third designs in which the
apparatus 1 is modified by providing, respectively, four, nine and
128 recesses 5 in the further layer 4. In each of the first to
third designs, the first conductive layer 20 is divided, as shown,
respectively, in FIGS. 26, 27 and 28 being plan views of the
substrate 3. The first conductive layer 20 provides, in respect of
each recess 5: an electrode 21 underneath the recess 5; a contact
22 exposed for connection of the external circuit 26 and a track 27
between the electrode 21 and the contact 22. Thus each electrode
21, and its associated track 27 and contact 22, is electrically
insulated from each other allowing separate measurement of current
signals from each recess 5.
[0240] Manufacture of the apparatus 1 may be performed using the
techniques described above using lamination of polymer films or
photolithography using silicon wafers.
[0241] Apparatuses 1 having plural recesses 5 have been made and
used experimentally to demonstrate formation of a layer 11, in
particular being a lipid bilayer, and insertion of a membrane
protein, in particular .alpha.-hemolysin. The experimental
procedure was as described above for an apparatus 1 having a single
recess 5, except that formation of the layer 5 and membrane protein
insertion was observed at plural recesses 5. Some examples are as
follows.
[0242] An apparatus of the first design having four recesses 5 was
manufactured by the technique described above of lamination onto a
polymer substrate 3. The first conductive layer 20 was silver
vapour deposited on a polyester sheet substrate 3. The further
layer 4 was a 15 .mu.m thick laminating film thermally laminated on
top. The four recesses 5 of 100 .mu.m diameter were formed at a
pitch of 300 .mu.m by an excimer laser.
[0243] For recording of from each recess 5 simultaneously in
parallel, multiple Axon current amplifier devices were operated in
parallel with a single silver/silver chloride electrode 24 in the
chamber 7 as the ground electrode common to all channels. Formation
of layers 11 and insertion of membrane proteins at plural recesses
5 was successfully recorded in parallel. Often this occurred at
each recess 5 although sometimes a layer 11 failed to form at one
or more recesses 5. For example typical current traces are shown in
FIG. 29 demonstrating simultaneous formation of four layers 11,
each having one or two .alpha.-hemolysin pores inserted, with
cyclodextrin binding events. Notably there is no cross-talk between
the signals. This confirms that the layers 11 are operating
independently and can produce meaningful measurements in parallel
while being individually addressed and using a common second
electrode 24.
[0244] An apparatus of the second design having nine recesses 5 was
manufactured by the technique described above of photolithography
using silicon wafer substrates 2. The further layer 4 was 5 .mu.m
thick SU8 photoresist. The nine circular recesses 5 were formed at
a pitch of 300 .mu.m by photolithography. In this case, the
recesses 9 had different diameters, in particular of 5 .mu.m, 10
.mu.m, 15 .mu.m, 20 .mu.m, 20 .mu.m, 30 .mu.m, 40 .mu.m, 50 .mu.m,
and 100 .mu.m. The substrate 3 was bonded to a printed circuit
board with separate tracks connected to each contact 22 and 25.
Epoxy was added across the contacts 22 and 25 for protection.
[0245] In order to control the applied potential and record the
current response in parallel, a multichannel electrical circuit 26
was created with corresponding software. Testing was computer
automated using a syringe pump to provide fluidics control of the
repeated application and removal of the aqueous solution 10.
[0246] Formation of layers 11 and insertion of membrane proteins at
plural recesses 5 was successfully recorded in parallel. Often this
occurred at each recess 5 although sometimes a layer 11 failed to
form at one or more recesses 5. For example, typical current traces
for recesses 5 constructed with gold electrodes and operating
without a redox couple in solution are shown in FIG. 30
demonstrating simultaneous formation of eight layers 11, each
having one or two a-hemolysin pores inserted, with cyclodextrin
binding events. Again there is no cross-talk and this confirms that
the layers 11 are operating independently and can produce
meaningful measurements in parallel.
[0247] Furthermore the apparatus 1 demonstrates successful
formation of a layer 11 across the recess 5 of each diameter in the
range of 5 .mu.m to 100 .mu.m. Accordingly the apparatus 1 was used
to investigate the role of the diameter of the recess 5 and the
quantity of pre-treatment coating applied, by experimentally
testing the percentage success rate of forming a layer 5 with three
different concentrations of pre-treatment coating 30, namely 0.5%,
1.0%, and 2.0% hexadecane in pentane. The results showed that in
the case of too little pretreatment coating 30, it was not possible
to form the layer 11 across the range of diameters of recess 5.
Furthermore in the case of too much pretreatment coating 30, it was
not possible to wet the electrode 21 and formation of the layer 11
could not be observed. In this particular configuration, the yield
of formation of layers 11 was greater than 60% for the range of
diameters 15 .mu.m to 100 .mu.m. Factors affecting layer formation,
some of which were investigated in this experiment include, but are
not limited to, pretreatment coating 30, diameter of recess 5,
depth of recess 5, aspect ratio of recess 5, surface properties of
the recess 5, surface properties of the surfaces around the recess,
fluid flow within the chamber 7, the amphiphilic molecules used in
the layer formation and the physical and electrical properties of
the electrode 21 within the recess 5. Subsequent experiments have
demonstrated yield of formation of layers 11, verified by
stochastic binding signals of inserted membrane channels, greater
than 70% using the 128 recesses, each 100 .mu.m in diameter, of the
device of FIG. 28.
[0248] In the apparatus 1 described above, the conductive tracks 27
from the electrode 21 to the contact 22 is formed on a surface of
the substrate 3 under the further layer. This may be referred to as
a planar escape route for the conductive track 27. As previously
described the separate conductive tracks 27 allow each electrode 21
to be connected individually to a dedicated low-noise high-input
impedance picoammeter in the circuit 26 whilst minimizing the
signal deterioration due to noise and bandwidth reduction. Such
planar conductive tracks 27 are ideal for an apparatus 1 having a
small number of recesses 5 and a thick layer between the tracks 27
and the aqueous solution 10.
[0249] However, for uses where high sensitivity is required, the
electrical connection between the electrodes 21 and the amplifier
circuit desirably has low parasitic capacitance and low leakage to
the surroundings. Parasitic capacitance causes noise and hence
signal deterioration and bandwidth reduction. Leakage also
increases noise, as well as introducing an offset current. In the
apparatus 1, the conductive tracks 27 experience some degree of
parasitic capacitance and leakage, both between tracks 27 and
between track and aqueous solution 10. As the number of recesses in
the array increases, the number of electrical connections to escape
increases and with a planar escape route, a practical limit is
reached where the density of the conductive tracks 27 creates too
much parasitic capacitance and/or leakage between tracks.
Furthermore as the thickness of the layer 4 decreases the
capacitance and/or leakage between the tracks 27 and the aqueous
solution 10 increases.
[0250] By way of example, typical figures may be obtained by
modelling the lipid bilayer as a capacitive element with a typical
value for the capacitance per unit area of 0.8 .mu.F/cm.sup.2. The
parasitic capacitance between track 27 and aqueous solution 10 can
be crudely modelled as a capacitative element with the area of
track 27 exposed, through the layer, to the aqueous solution.
Typical values for the track 27 may be 50 .mu.m wide with 2 mm
exposed and a relative permittivity (dielectric constant) of the
layer around 3. For a 100 .mu.m diameter bilayer and 20 .mu.m deep
recess the capacitance is 63 pF with a track-solution parasitic
capacitance of 0.13 pF. However scaling to smaller bilayers of 5
.mu.m diameter and 1 .mu.m deep the capacitance is 0.16 pF with
parasitic capacitance 0.53 pF. For smaller bilayers and thinner
layers the parasitic capacitance dominates.
[0251] To reduce this problem, a modification shown in FIG. 31
comprises, replacing the conductive track 27 by a conductive path
28 which extends through the body 2 to a contact 29 on the opposite
side of the body 2 from the electrode 21. In particular, the
conductive path 28 extends through the substrate 3. As this
substrate 3 provides a thicker dielectric between the conductive
paths 28 than is possible between the planar conductive paths 27, a
much lower parasitic capacitance is achieved. Also, the leakage is
low due to the thickness and dielectric properties of the substrate
3. Consequently, the use of the conductive paths 28 effectively
increases the number of recesses 5 which may be accommodated in the
body 2 before the practical limits imposed by parasitic capacitance
and/or leakage are met. This form of interconnect can be attached
to a low-capacitance multi-layer substrate 61, which allows a far
greater number of electrical escape routes by virtue of the number
of layers and the low dielectric constant of the material. In
addition the use of solder bump technology (also known as "flip
chip" technology) and a suitable connector allows the apparatus 1
shown in FIG. 31, excluding the substrate 61, to be made as low
cost disposable part.
[0252] The conductive path 28 may be formed using known
through-wafer interconnection technology. Types of through-wafer
interconnects which may be applied to form the conductive path
include without limitation:
[0253] on substrates 3 of silicon, through-wafer interconnects
formed by producing a via through the silicon wafer, isolating the
internal surface of via and filling the via with a conducting
material, or alternatively the conductive path 28 is formed by
producing a semiconductor PN junction in the form of a cylindrical
via through the silicon substrate; on substrates 3 of glass,
through-wafer interconnects formed by methods including laser
drilling, wet etching and filling vias with metal or doped
semiconductor material; and on substrates 3 made of polymers,
through-wafer interconnects formed by methods including laser
drilling, laser ablation, screen printed conductors and known
printed circuit board techniques.
[0254] As the opposite side of the body 2 from the electrode 21 is
dry, an electrical point contact array can be used to make
connections to the electrical circuit 26. By way of example, FIG.
31 illustrates the use of solder bump connections. In particular,
deposited on each contact 29 are respective solder bumps 60 on
which a circuit element 61 is mounted so that the solder bumps 60
make electrical contact with a track 62 on the circuit element
61.
[0255] The circuit element 61 may be a printed circuit board for
example as shown in FIG. 13.
[0256] Alternatively, the circuit element 61 could be an integrated
circuit chip or a laminate, for example a low temperature cured
ceramic package. Such an integrated circuit chip or laminate may be
used as a method of spreading out connections, connecting to a
further solder bump array on the opposite side of the integrated
circuit chip or laminate with a greater pitch. An example of this
is shown in FIG. 32 in which the circuit element 61 is an
integrated circuit chip or a laminate providing connections from
the solder bumps 60 deposited on the body 2 to further solder bumps
63 arrayed at a greater pitch and used to connect to a further
circuit element 64, for example a printed circuit board. The
circuit element 61 being an integrated circuit chip or laminate may
also be used to escape connections sideways in a multi-layer
format.
[0257] In the case of a substrate 3 of semiconductor material such
as silicon, two types of through-wafer interconnect which may be
applied to make the conductive path 28 are
Metal-Insulator-Semiconductor (MIS), and a PN junction type. In
MIS, a hole is drilled through the silicon chip by Deep Reactive
Ion Etching (DRIE) process and this hole is coated with insulator
and then filled with metal to form a the conductive path 28. The PN
junction type of through-wafer interconnect is a semiconductor
junction formed into a cylindrical via through a silicon chip. Each
type of through-wafer interconnection is formed on silicon wafers
that have been thinned down to less than 0.3 mm to save DRIE
processing time in making the holes. The important feature of PN
junction type through-wafer interconnects is the low capacitance
provided by having a large depletion region compared to the MIS
type of interconnect. This is partially helped by increasing the
reverse-bias of the junction.
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 Asn20 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 Thr35 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 Val50 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
Glu85 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 Val100 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 Tyr115 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 Tyr130 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 Gln165 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 Gly180 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 Tyr195 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
Lys210 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 Thr245 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 Asp260 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 Ser275 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 Ser290 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 Asn20 25 30Ile Lys Thr Gly Thr Thr
Asp Ile Gly Ser Asn Thr Thr Val Lys Thr35 40 45Gly Asp Leu Val Thr
Tyr Asp Lys Glu Asn Gly Met His Lys Lys Val50 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 Glu85 90 95Glu
Gly Ala Asn Lys Ser Gly Leu Ala Trp Pro Ser Ala Phe Lys Val100 105
110Gln Leu Gln Leu Pro Asp Asn Glu Val Ala Gln Ile Ser Asp Tyr
Tyr115 120 125Pro Arg Asn Ser Ile Asp Thr Lys Glu Tyr Met Ser Thr
Leu Thr Tyr130 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 Gln165 170 175Pro Asp Phe Lys Thr Ile
Leu Glu Ser Pro Thr Asp Lys Lys Val Gly180 185 190Trp Lys Val Ile
Phe Asn Asn Met Val Asn Gln Asn Trp Gly Pro Tyr195 200 205Asp Arg
Asp Ser Trp Asn Pro Val Tyr Gly Asn Gln Leu Phe Met Lys210 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 Thr245 250 255Val Ile Thr Met Asp Arg Lys Ala Ser Lys
Gln Gln Thr Asn Ile Asp260 265 270Val Ile Tyr Glu Arg Val Arg Asp
Asp Tyr Gln Leu His Trp Thr Ser275 280 285Thr Asn Trp Lys Gly Thr
Asn Thr Lys Asp Lys Trp Thr Asp Arg Ser290 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 Glu20 25 30Asn Gly Met His Lys Lys
Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn35 40 45His Asn Lys Lys Leu
Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly50 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 Val85 90 95Ala
Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys Glu100 105
110Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val Thr Gly
Asp115 120 125Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala Asn Val
Ser Ile Gly130 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 Val165 170 175Asn Gln Asn Trp Gly Pro
Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr180 185 190Gly Asn Gln Leu
Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala195 200 205Asp Asn
Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly210 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 Asp245 250 255Tyr Gln Leu His Trp Thr Ser Thr Asn Trp
Lys Gly Thr Asn Thr Lys260 265 270Asp Lys Trp Thr Asp Arg Ser Ser
Glu Arg Tyr Lys Ile Asp Trp Glu275 280 285Lys Glu Glu Met Thr
Asn290
* * * * *
References