U.S. patent application number 10/690618 was filed with the patent office on 2004-03-25 for substrate and a method for determining and/or monitoring electrophysiological properties of ion channels.
This patent application is currently assigned to Sophion Bioscience A/S. Invention is credited to Bech, Morten, Christophersen, Palle, Due, Jorgen, Hansen, Ole, Olesen, Soren Peter, Petersen, Jon Wulff, Telleman, Pieter, Thomsen, Lars.
Application Number | 20040055901 10/690618 |
Document ID | / |
Family ID | 30118738 |
Filed Date | 2004-03-25 |
United States Patent
Application |
20040055901 |
Kind Code |
A1 |
Petersen, Jon Wulff ; et
al. |
March 25, 2004 |
Substrate and a method for determining and/or monitoring
electrophysiological properties of ion channels
Abstract
The present invention relates to a substrate and a method for
obtaining an electrophysiological measuring configuration in which
a cell forms a high resistive seal (giga-seal) around a measuring
electrode making it suitable for determining and monitoring a
current flow through the cell membrane. The substrate is typically
part of an apparatus for studying electrical events in cell
membranes, such as an apparatus for carrying out patch clamp
techniques utilised to study ion transfer channels in biological
membranes. The substrate has a plurality or an array of measuring
sites with integrated measuring and reference electrodes formed by
wafer processing technology. The electrodes are adapted to conduct
a current between them by delivery of ions by one electrode and
receipt of ions by the other electrode and are typically
silver/silver halide electrodes. This allows for effective and fast
measuring of cells in configurations where the there is a direct
electrical connection between the measuring electrode and the cell
interior, a whole-cell measuring configuration.
Inventors: |
Petersen, Jon Wulff;
(Lyngby, DK) ; Telleman, Pieter; (Lyngby, DK)
; Hansen, Ole; (Lyngby, DK) ; Christophersen,
Palle; (Ballerup, DK) ; Bech, Morten;
(Ballerup, DK) ; Olesen, Soren Peter; (Ballerup,
DK) ; Due, Jorgen; (Ballerup, DK) ; Thomsen,
Lars; (Ballerup, DK) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Sophion Bioscience A/S
|
Family ID: |
30118738 |
Appl. No.: |
10/690618 |
Filed: |
October 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10690618 |
Oct 23, 2003 |
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09676814 |
Oct 2, 2000 |
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6682649 |
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60157847 |
Oct 6, 1999 |
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Current U.S.
Class: |
205/789 ;
204/416; 205/792.5 |
Current CPC
Class: |
G01N 33/48728
20130101 |
Class at
Publication: |
205/789 ;
205/792.5; 204/416 |
International
Class: |
G01N 027/26 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 1, 1999 |
DK |
1999 01407 |
Claims
1. An assembly comprising: a plane substrate having a plurality of
sites in the first surface part of the substrate each of which is
adapted to hold an ion channel-containing structure contained in a
liquid, and each of which has a passage therein through the
substrate connecting the first surface part and the second surface
part, which passage has walls and is dimensioned to hold an ion
channel-containing structure and to form a high resistance seal
between said ion channel-containing structure and said substrate
around, or along the walls of said passage, a plurality of
measuring electrodes, each of which is associated with a respective
site, one or more reference electrodes, the measuring electrodes
and the respective reference electrode or reference electrodes
being electrodes capable of passing, when in electrolytic contact
with each other and when a potential difference is applied between
them, a current between them by delivery of ions by one electrode
and receipt of ions by the other electrode, each of the sites being
adapted to provide a high electrical resistance seal established
between an area of contact of the outer surface of an ion
channel-containing structure held at the site, and a first surface
part of the substrate around, or along the walls of said passage,
the seal, when established, separating a domain defined on one side
of the seal established by the ion channel-containing structure and
in electrolytic contact with the measuring electrode, from a domain
defined on the opposite side of the seal established by the ion
channel-containing structure and in electrolytic contact with the
respective reference electrode, whereby a current flowing between
the reference and respective measuring electrodes and through the
ion channel-containing structure can be determined and/or
monitored, the electrodes being integrated with the assembly,
wherein the assembly further comprises a plurality of flow channel
structures created in the substrate for delivering liquid to said
plurality of sites.
2. The assembly according to claim 1, wherein the plane substrate
has a first side and a second side, the first and second sides
being spaced apart from one another and defining a first substrate
thickness, the first surface part and the opposite second surface
part being spaced apart from one another and defining a second
substrate thickness, the second substrate thickness being less than
the first substrate thickness.
3. The assembly according to claim 2, wherein the plane substrate
comprises a first substrate component, and a second substrate
component, the first substrate component having a first face
forming the first side of the plane substrate, and an opposite
face, and the second substrate having a first face defining the
second side of the plane substrate, and an opposite, second face,
the first and second substrate components being attached to one
another at their respective second faces.
4. The assembly according to claim 2 or claim 3, wherein the
plurality of flow channel structures is formed in the first side of
the plane substrate.
5. The assembly according to claim 1, wherein the substrate is a
silicon substrate, and the surface part of the site with which the
high electrical resistance seal is to be established is a silica
surface part.
6. The assembly according to claim 1 or 2, wherein the plurality of
sites is arranged in an array on the first surface part of the
substrate.
7. The assembly according to claim 6, wherein the array of sites
comprises at least 9 sites.
8. The assembly according to claim 1, wherein the measuring and
reference electrodes are silver/silver halide electrodes.
9. The assembly according to claim 8, wherein the measuring and
reference electrodes are silver/silver chloride electrodes.
10. The assembly according to claim 1 or claim 2, comprising a
first layer of hydrophobic material positioned on or above the
surface of the substrate, said first layer covering only parts of
the surface of the substrate.
11. The assembly according to claim 10, where one or more sites are
located within parts of the surface of the substrate not covered by
said first layer.
12. The assembly according to claim 1 or claim 2, comprising one or
more wells extending into the substrate and having well openings
defined in the first surface part, each having a bottom part and a
side part, at least some of the sites of the first surface part
being positioned within the bottom parts of the wells such that a
well and a passage together define a funnel.
13. The assembly according to claim 12, wherein the plurality of
flow channel structures enable liquid to be added to the one or
more funnels.
14. The assembly according to claim 13, wherein the plurality of
flow channel structures comprises an inflow and an outflow port
to/from the one or more funnels.
15. The assembly according to claim 12, wherein the wells have been
formed by a process comprising a photolithography/etching
process.
16. The assembly according to claim 15, wherein the substrate is a
silicon substrate, and wherein the wells are shaped as truncated
pyramids the bottoms of which are constituted by the well openings,
and the side parts of which have a slope of 54.7.degree..
17. The assembly according to claim 12, wherein a reference
electrode is positioned on the side part of each well.
18. The assembly according to claim 1 or claim 2, wherein the
measuring electrode associated with each site is positioned at each
respective site.
19. The assembly according to claim 1, wherein the transverse
dimension of the passage is 1-5 .mu.m.
20. The assembly according to claim 2, wherein the measuring
electrode associated with each site is positioned on the opposite
second surface part of the substrate.
21. The assembly according to claim 20, wherein the measuring
electrode associated with each site is positioned adjacent to an
opening of the passage defined at the respective site.
22. The assembly according to claim 1 or claim 2, further
comprising, for each of the sites, an electronic circuit that is
connected with the respective measuring electrode and with the
reference electrode or one of the reference electrodes for
generation of an amplified signal that is a unique function of a
current flowing through ion channels between said electrodes.
23. An assembly according to claim 1, further comprising a
connection means for connecting the substrate to a suction means
for creating a suction on said ion channel-containing structure and
through said passage so as to enable the ion channel-containing
structure to be positioned, sealed and ruptured by the suction,
wherein, in use the first surface part is in contact with the
liquid containing the ion channel containing structure, the
connection means forming an integral part of the assembly and
extending from the second surface part of the substrate to the
first side of the substrate.
24. A method of establishing a whole cell measuring configuration
for determining and/or monitoring an electrophysiological property
of one or more ion channels of one or more ion channel-containing
structures, said method comprising the steps of: providing an
assembly comprising: a first surface part and an opposite second
surface, said substrate having a plurality of sites in the first
surface part of the substrate, each of which is adapted to hold an
ion channel-containing structure and each of which has a passage
therein through the substrate connecting the first surface part and
the second surface part, which passage has walls and is dimensioned
to hold an ion channel-containing structure and to form a high
resistance seal between said ion channel-containing structure and
said substrate, around or along the walls of said passage, a
plurality of measuring electrodes, each of which is associated with
a respective site, and one or more reference electrodes; supplying
a carrier liquid at one or more sites using a plurality of flow
channel structures created in the substrate, said carrier liquid
containing one or more ion channel-containing structures the
carrier liquid contacting the first surface part of the substrate;
positioning at least one of the ion channel-containing structures
at a corresponding number of sites; forming a high electrical
resistance seal between an area of contact of the outer surface of
an ion channel-containing structure held at the site and a first
surface part of the substrate around or along the walls of said
passage, the seal, when established, separating a domain defined on
one side of the seal established by the ion channel-containing
structure and in electrolytic contact with the measuring electrode
from a domain defined on the opposite side of the seal established
by the ion channel-containing structure and in electrolytic contact
with the respective reference electrode; checking for a high
electrical resistance seal between an ion channel-containing
structure held at a site and the first surface part of the
substrate or along the walls of said passage with which the high
electrical resistance seal is to be established by successively
applying a first electric potential difference between the
measuring electrode associated with the site and a reference
electrode, monitoring a first current flowing between said
measuring electrode and said reference electrode, and comparing
said first current to a predetermined threshold current and, if the
first current is at most the predetermined threshold current, then
approving the site as having an acceptable seal between the ion
channel-containing structure and the first surface part of the
site; and establishing a whole-cell configuration at approved
sites, whereby a third current flowing through ion channels of the
ion channel-containing structure between the measuring electrode
and the reference electrodes can be determined and/or
monitored.
25. The method according to claim 24, wherein the substrate
comprises a first side and second side spaced apart from the first
side, the first and second sides defining a first substrate
thickness, and the first and second surface parts of the substrate
defined in the second substrate thickness, the second substrate
thickness being less than the first substrate thickness.
26. The method according to claim 25, wherein the substrate is
formed from a first substrate component and a second substrate
component, the first substrate component having a first face formed
from the first side of the plane substrate, and an opposite face,
the second substrate having a first face defining the second side
of the plane substrate, and an opposite, second face, the first and
second substrate components being attached to one another at their
respective second faces.
27. The method according to claim 25 or 26, wherein the flow
channel structures are formed on the first side of the plane
substrate.
28. The method according to claim 24, wherein the step of
establishing a whole-cell configuration at approved sites comprises
applying, between the measuring electrode associated with each
approved site and a reference electrode, a series of second
electric potential difference pulses, monitoring a second current
flowing between the measuring electrode and the reference
electrode, and interrupting the series of second electric potential
difference pulses whenever said second current exceeds a
predetermined threshold value, thereby rupturing a part of the ion
channel-containing structure.
29. The method according to claim 24, wherein the assembly further
comprises connection means for connecting the substrate to a
suction means, the connecting means extending from the second
surface part to the first side of the substrate, and the step of
establishing a whole cell configuration at approved sites comprises
forming the high electrical resistance seal between an area of
contact of the outer surface of an ion channel-containing structure
held at the site and a first part of the substrate around or along
the walls of said passage being formed by applying a suction to
said passage via said connection means.
30. The method according to claim 24, wherein the step of
establishing a whole-cell configuration at approved sites comprises
subjecting a part of the ion channel-containing structure with a
pore forming substance.
Description
[0001] This application claims priority on U.S. application Ser.
No. 09/676,814, filed on Oct. 2, 2000, which claims priority on
Danish Application No. 1999 01407 filed on Oct. 1, 1999, and U.S.
Provisional Application No. 60/157,847, filed on Oct. 6, 1999. The
entire contents of each of these applications is hereby
incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to a substrate and a method
for determining and/or monitoring electrophysiological properties
of ion channels of ion channel-containing structures, typically
lipid membrane-containing structures such as cells, by establishing
an electrophysiological measuring configuration in which a cell
membrane forms a high resistive seal around a measuring electrode,
making it possible to determine and monitor a current flow through
the cell membrane. The substrate is typically part of an apparatus
for studying electrical events in cell membranes, such as an
apparatus for carrying out patch clamp techniques utilised to study
ion transfer channels in biological membranes. More particularly,
the invention relates to a substrate for such patch clamp apparatus
having high through-put and utilising only small amounts of test
compounds, only small amounts of liquid carrier, and being capable
of carrying out many tests in a short period of time by performing
parallel tests on a number of cells simultaneously and
independently.
BACKGROUND ART
[0003] The general idea of electrically insulating a patch of
membrane and studying the ion channels in that patch under
voltage-clamp conditions was outlined by Neher, Sakmann, and
Steinback in "The Extracellular Patch Clamp, A Method For Resolving
Currents Through Individual Open Channels In Biological Membranes",
Pflueger Arch. 375; 219-278, 1978. They found that, by pressing a
pipette containing acetylcholine (ACh) against the surface of a
muscle cell membrane, they could see discrete jumps in electrical
current that were attributable to the opening and closing of
ACh-activated ion channels. However, they were limited in their
work by the fact that the resistance of the seal between the glass
of the pipette and the membrane (10-50 M.OMEGA.) was very small
relative to the resistance of the channel (10 G.OMEGA.). The
electrical noise resulting from such a seal is inversely related to
the resistance and was large enough to obscure the currents flowing
through ion channels, the conductance of which are smaller than
that of the ACh channel. It also prohibited the clamping of the
voltage in the pipette to values different from that of the bath
due to the large currents through the seal that would result.
[0004] It was then discovered that by fire polishing the glass
pipettes and by applying suction to the interior of the pipette a
seal of very high resistance (1-100 G.OMEGA.) could be obtained
with the surface of the cell. This Giga-seal reduced the noise by
an order of magnitude to levels at which most channels of
biological interest can be studied and greatly extended the voltage
range over which these studies could be made. This improved seal
has been termed a "giga-seal", and the pipette has been termed a
"patch pipette". A more detailed description of the giga-seal may
be found in O. P. Hamill, A. Marty, E. Neher, B. Sakmann & F.
J. Sigworth: Improved patch-clamp techniques for high resolution
current recordings from cells and cell-free membrane patches.
Pflugers Arch. 391, 85-100, 1981. For their work in developing the
patch clamp technique, Neher and Sakmann were awarded the 1991
Nobel Prize in Physiology and Medicine.
[0005] Ion channels are transmembrane proteins which catalyse
transport of inorganic ions across cell membranes. The ion channels
participate in processes as diverse as generating and timing action
potentials, synaptic transmission, secretion of hormones,
contraction of muscles, etc. Many drugs exert their specific
effects via modulation of ion channels. Examples are antiepileptic
compounds like phenytoin and lamotrigine which block
voltage-dependent Na.sup.+-channels in the brain, antihypertensive
drugs like nifedipine and diltiazem which block voltage dependent
Ca.sup.2+-channels in smooth muscle cells, and stimulators of
insulin release like glibenclamide and tolbutamide which block an
ATP-regulated K.sup.+-channel in the pancreas. In addition to
chemically induced modulation of ion-channel activity, the patch
clamp technique has enabled scientists to perform manipulations
with voltage dependent channels. These techniques include adjusting
the polarity of the electrode in the patch pipette and altering the
saline composition to moderate the free ion levels in the bath
solution.
[0006] The patch clamp technique represents a major development in
biology and medicine, since this technique allows measurement of
ion flow through single ion channel proteins, and also allows the
study of the single ion channel responses to drugs. Briefly, in
standard patch clamp technique, a thin (app. 0.5-2 .mu.m in
diameter) glass pipette is used. The tip of this patch pipette is
pressed against the surface of the cell membrane. The pipette tip
seals tightly to the cell and isolates a few ion channel proteins
in a tiny patch of membrane.
[0007] The activity of these channels can be measured individually
(single channel recording) or, alternatively, the patch can be
ruptured allowing measurements of the channel activity of the
entire cell membrane (whole cell recording). High-conductance
access to the cell interior for performing measurements can be
obtained, e.g., by rupturing the membrane by applying
subatmospheric pressure in the pipette.
[0008] During both single channel recording and whole-cell
recording, the activity of individual channel subtypes can be
characterised by imposing a "voltage clamp" across the membrane. In
the voltage clamp technique the membrane current is recorded at a
constant membrane potential. Or--to be more precise--the amplifier
supplies exactly the current, which is necessary to keep the
membrane potential at a level determined by the experimenter.
Hence, currents resulting from opening and closing of ion channels
are not allowed to recharge the membrane.
[0009] FIG. 1 shows a simplified diagram of the basic operation of
a standard prior art voltage clamp amplifier such as the EPC-9
amplifier from HEKA Elektronik. An electrode 6 inside a pipette 4
is connected to the negative terminal of a feedback amplifier,
while the clamping voltage (referred to a grounded bath electrode
(8)) is connected to a positive terminal (from Stim. In.) and made
available at a voltage monitor output. Since the measured pipette
voltage and the clamp voltage are supposed to be identical, a
correction potential is constantly supplied at the pipette
electrode as a current forced through the large feedback resistor.
After inversion, the current is made available as an analogue
voltage at the Current Monitor output.
[0010] The time resolution and voltage control in such experiments
are impressive, often in the msec or even .mu.sec range. However, a
major obstacle of the patch clamp technique as a general method in
pharmacological screening has been the limited number of compounds
that could be tested per day (typically no more than 1 or 2). Also,
the very slow rate of solution change that can be accomplished
around cells and patches may constitute a major obstacle.
[0011] A major limitation determining the throughput of the patch
clamp technique is localisation and clamping of cells and pipette,
and the nature of the feeding system, which leads the dissolved
compound to cells and patches.
[0012] In usual patch clamp setups, cells are placed in
experimental chambers which are continuously perfused with a
physiological salt solution. The establishment of the cell-pipette
connection in these chambers is time-consuming and troublesome.
Compounds are applied by changing the inlet to a valve connected to
a small number of feeding bottles. The required volumes of the
supporting liquid and the sample to be tested are high.
[0013] High throughput systems for performing patch clamp
measurements have been proposed, which typically consist of a
substrate with a plurality of sites adapted to hold cells in a
measuring configuration where the electrical properties of the cell
membrane can be determined.
[0014] U.S. Pat. No. 5,187,096, Rensselaer, discloses an apparatus
for monitoring cell-substrate impedance of cells. Cells are
cultured directly on the electrodes which are then covered with a
plurality of cells, thus, measurements on individual cells can not
be performed.
[0015] WO 98/54294, Leland Stanford, discloses a substrate with
wells containing electrode arrays. The substrate with wells and
electrodes (metal electrodes) is made of silicon using CVD
(Chemical Vapor Deposition) and etching techniques and comprises
Silicon Nitride "passivation" layers surrounding the electrodes.
The cells are cultivated directly on the electrode array. The
substrate is adapted to measure electrophysiological properties and
discloses a variety of proposed measuring schemes.
[0016] WO 99/66329, Cenes, discloses a substrate with perforations
arranged in wells and electrodes provided on each side of the
substrate. The substrate is made by perforating a silicon substrate
with a laser and may be coated with anti-adhesive material on the
surface. The substrate is adapted to establish giga seals with
cells by positioning the cells on the perforations using suction
creating a liquid flow through the perforations, providing the
anti-adhesion layer surrounding the perforations, or by guiding the
cells electrically. The cells can be permeabilised by EM fields or
chemical methods in order to provide a whole-cell measuring
configuration. All perforations, and hence all measurable cells, in
a well share one working electrode and one reference electrode, see
FIG. 1, hence measurements on individual cells can not be
performed.
[0017] WO 99/31503, Vogel et al., discloses a measuring device with
an aperture arranged in a well on a substrate (carrier) and
separating two compartments. The measuring device comprises two
electrodes positioned on either side of the aperture and adapted to
position a cell at the aperture opening. The substrate may have
hydrophobic and hydrophilic regions in order to guide the
positioning of the cells at the aperture opening.
SUMMARY OF THE INVENTION
[0018] The present invention provides a substrate and a method
optimised for determining or monitoring current flow through ion
channel-containing structures such as cell membranes, with a high
throughput and reliability and under conditions that are realistic
with respect to the influences to which the cells or cell membranes
are subjected. Thus, the results determined using the substrate and
the method of the invention, e.g., variations in ion channel
activity as a result of influencing the cell membrane with, e.g.,
various test compounds, can be relied upon as true manifestations
of the influences proper and not of artefacts introduced by the
measuring system, and can be used as a valid basis for studying
electrophysiological phenomena related to the conductivity or
capacitance of cell membranes under given conditions.
[0019] This is because the current through one or more ion channels
is directly measured using reversible electrodes as characterized
below, typically silver/silver halide electrodes such as silver
chloride electrodes, as both measuring electrodes and reference
electrodes.
[0020] The substrate and method of the invention may be used not
only for measurements on cell membranes, but also on other ion
channel-containing structures, such as artificial membranes. The
invention permits performing several tests, such as
electrophysiological measurements on ion transfer channels and
membranes, simultaneously and independently. The substrate of the
invention constitutes a complete and easily handled microsystem
which uses only small amounts of supporting liquid (a physiological
salt solution, isotonic with the cells, that is, normally having an
osmolarity of 150 millimolar NaCl or another suitable salt) and
small amounts of test samples.
[0021] In one aspect, the invention relates to a plane substrate
having an first surface part and an opposite second surface part,
the first surface part having a plurality of sites each of which is
adapted to hold an ion channel-containing structure, each site
having a measuring electrode associated therewith, the substrate
carrying one or more reference electrodes, the measuring electrodes
and the respective reference electrode or reference electrodes
being electrodes capable of generating, when in electrolytic
contact with each other and when a potential difference is applied
between them, a current between them by delivery of ions by one
electrode and receipt of ions by the other electrode, each of the
sites being adapted to provide a high electrical resistance seal
between an ion channel-containing structure held at the site and a
surface part of the site, the seal, when provided, separating a
domain defined on one side of the ion channel-containing structure
and in electrolytic contact with the measuring electrode from a
domain defined on the other side of the ion channel-containing
structure and in electrolytic contact with the respective reference
electrode so that a current flowing through ion channels of the ion
channel-containing structure between the electrodes can be
determined and/or monitored, the electrodes being integrated with
the substrate and having been formed by a wafer processing
technology.
[0022] In another aspect, the invention relates to a method of
establishing a whole cell measuring configuration for determining
and/or monitoring an electrophysiological property of one or more
ion channels of one or more ion channel-containing structures, said
method comprising the steps of
[0023] providing a substrate as defined above,
[0024] supplying a carrier liquid at one or more sites, said
carrier liquid containing one or more ion channel-containing
structures,
[0025] positioning at least one of the ion channel-containing
structures at a corresponding number of sites,
[0026] checking for a high electrical resistance seal between an
ion channel-containing structure held at a site and the surface
part of the site with which the high electrical resistance seal is
to be provided by successively applying a first electric potential
difference between the measuring electrode associated with the site
and a reference electrode, monitoring a first current flowing
between said measuring electrode and said reference electrode, and
comparing said first current to a predetermined threshold current
and, if the first current is at most the predetermined threshold
current, then approving the site as having an acceptable seal
between the ion cannel-containing structure and the surface part of
the site, and
[0027] establishing a whole-cell configuration at approved
sites,
[0028] whereby a third current flowing through ion channels of the
ion channel-containing structure between the measuring electrode
and the reference electrodes can be determined and/or
monitored.
[0029] An ion channel-containing structure in a solution may be
guided towards a site on a substrate either by active or passive
means. When the ion channel-containing structure makes contact with
the site, e.g., substrate around an electrode, the contact surfaces
form a high electrical resistance seal (a giga-seal) at the site,
e.g., surrounding the electrode, so that an electrophysiological
property of the ion channels can be measured using the respective
electrode. Such electrophysiological property may be current
conducted through the part of membrane of the ion
channel-containing structure that is encircled by the
giga-seal.
[0030] In the present context, the term "giga-seal" normally
indicates a seal of a least 1 G ohm, and this is the size of seal
normally aimed at as a minimum, but for certain types of
measurements where the currents are large, lower values may be
sufficient as threshold values.
[0031] The whole-cell configuration may be obtained by applying,
between the measuring electrode associated with each approved site
and a reference electrode, a series of second electric potential
difference pulses, monitoring a second current flowing between the
measuring electrode and the reference electrode, and interrupting
the series of second electric potential difference pulses whenever
said second current exceeds a predetermined threshold value,
thereby rupturing the part of the ion channel-containing structure
which is closest to the measuring electrode.
[0032] Alternatively, the whole-cell configuration may be obtained
by subjecting the part of the ion channel-containing structure
which is closest to the measuring electrode to interaction with a
pore forming substance.
[0033] It should be noted that in the present context, the term
"whole-cell configuration" denotes not only configurations in which
a whole cell has been brought in contact with the substrate at a
measuring site and has been punctured or, by means of a
pore-forming substance, has been opened to electrical contact with
the cell interior, but also configurations in which an excised cell
membrane patch has been arranged so that the outer face of the
membrane faces "upwardly", towards a test sample to be applied.
[0034] As the measuring electrode associated with a site is one of
a plurality of electrodes on the substrate, and the ion
channel-containing structure is one of many in a solution, it is
possible to obtain many such prepared measuring set-ups on a
substrate. A typical measurement comprises adding a specific test
sample to the set-up, for which reason each measuring set-up is
separated from other measuring set-ups to avoid mixing of test
samples and electrical conduction in between set-ups.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The present invention will now be described in greater
detail with reference to the accompanying drawings, in which:
[0036] FIG. 1, as mentioned above, shows a diagram of a typical
known electronic circuit for voltage clamp measurements;
[0037] FIG. 2 shows a schematic view of examples of substrates
having sites with electrodes for holding cell membranes or
artificial membranes;
[0038] FIGS. 3A-3D shows cross-sectional side views of various
embodiments of substrates of the invention, showing the different
layers produced in wafer processing technology
(deposition/photolithography/etching technology);
[0039] FIG. 4A shows a cross-sectional side view of another design
for a substrate having sites with electrodes for holding cell
membranes or artificial membranes; FIG. 4B shows a top view of the
structure of FIG. 4A;
[0040] FIG. 5 shows a close-up of sites enclosed by a region of
hydrophobic material;
[0041] FIG. 6 shows a test confinement with an array of electrodes
connected to a line of contacts; and
[0042] FIG. 7 shows a flow diagram of a procedure for detecting
when a cell forms a giga-seal with an substrate, e.g. around an
electrode.
[0043] The reference numbers in the drawings refer to the
following:
1 No. Description 2 cell 4 pipette 6 pipette measuring electrode 8
reference electrode 10 voitage clamp amplifier 11 edge of
hydrophobic region 12 substrate 13 substructure 14 site 15 test
confinement 16 electrode 17 second structure part 18 lines of
conducting material 20 contacts 22 insulating film 24 Silver 26
hydrophobic region 28 AgCl layer 30 aperture 31 SiO.sub.2 layer 32
piping
DESCRIPTION OF PREFERRED EMBODIMENTS
[0044] The present invention relates to a substrate with a
plurality of electrodes at sites adapted to hold cells (or other
ion channel-containing structures), such that the cell membrane and
the substrate interface creates a giga-seal around an electrode,
making it possible to determine or monitor electrophysiological
properties of the cell membrane. It will be understood that when
the term "cell" or "cell membrane" is used in the present
specification, it will normally, depending on the context, be
possible to use any other ion channel-containing structure, such as
another ion channel-containing lipid membrane or an ion
channel-containing artificial membrane. Electrophysiological
properties can be, e.g., current flow through an ion channel or
capacitance of an ion channel-containing membrane. It is possible
to add individual test samples (typically pharmacological drugs) at
each cell-holding location so that individual experiments can be
carried out on each cell. An experiment can be to measure the
response of the ion transfer channel to the addition of test
sample. In order to carry out individual experiments, different
test samples could be added to different cell-holding sites. One or
more cell holding sites where a specific test sample is (going to
be) added is hereafter called a test confinement.
[0045] The substrate of the invention will typically be a component
used in an apparatus for carrying out measurements of the
electrophysiological properties of ion transfer channels in lipid
membranes such as cells.
[0046] The apparatus will be designed to provide means for carrying
out a large number of individual experiments in a short period of
time. This is accomplished by providing a microsystem having a
plurality of test confinements each of which having sites
comprising integrated measuring electrodes, and providing and
suitable test sample supply. Each test confinement may comprise
means for positioning cells, for establishment of giga-seal, for
selection of sites at which giga-seal has been established,
measuring electrodes and one or more reference electrodes. Thereby
it is possible to perform independent experiments in each test
confinement, and to control the preparation and measurements of all
experiments from a central control unit such as a computer. Due to
the small size of the test confinements, the invention permits
carrying out measurements utilising only small amounts of
supporting liquid and test sample. The present invention also
provides several different procedures for carrying out
measurements; these include measurements on fragments of cells and
artificial membranes.
[0047] The substrate having sites with measuring electrodes
(electrodes hereafter) can be designed in a number of ways, of
which three are illustrated in FIGS. 2A-2C, and further ones are
illustrated in FIGS. 3A-3D and 4A-4B. The distinction between the
embodiments is the design of the sites on the substrate. Sites are
adapted to hold an ion channel-containing structure, such as a
cell, in that the surface material at the site is well suited for
creating a seal with the cell (or structure) membrane as described
in the prior art. Such materials include silicon, plastics, pure
silica and other glasses such as quarts and pyrex or silica doped
with one or more dopants selected from the group of Be, Mg, Ca, B,
Al, Ga, Ge, N, P, As and oxides from any of these. The substrate
proper can be made of any material suitable for a wafer processing
technology, such as silicon, plastics, pure silica and other
glasses such as quarts and pyrex or silica doped with one or more
dopants selected from the group of Be, Mg, Ca, B, Al, Ga, Ge, N, P,
As. Silicon is the presently preferred substrate material.
[0048] In the designs of FIGS. 2A-2C, the sites 14 are arranged on
a locally flat surface of the substrate 12. Locally flat indicates
that the surface of the substrate may have some substructure 13 on
a scale larger than one or more sites, as seen in FIG. 2B. Sites,
and thereby electrodes 16, can be arranged alone or in groups
within this substructure.
[0049] The methods for production of the three designs of FIG. 2
are analogous to each other. FIGS. 2A and 2B simply includes some
subdivision of the basic design of FIG. 2C. The manufacture of the
designs is now described with reference to FIGS. 3A and 6:
[0050] Lines 18 of conducting material are formed on the surface of
the substrate by first depositing a layer of conducting material on
the substrate. Deposition of materials on the substrate, and on
other surfaces throughout the description, can be made using one of
several deposition techniques, such as Physical Vapour Deposition
which includes 1) application of material from a vapour phase, 2)
spottering and 3) laser ablation; Chemical Vapour Deposition
techniques which include 1) atmospheric pressure chemical vapour
deposition (APCVD), 2) low pressure chemical vapour deposition
(LPCVD), 3) plasma enhanced chemical vapour deposition (PECVD) and
4) photo enhanced chemical vapour deposition; as well as spin
coating and growth techniques. Second, the individual wires are
defined in a photolithography step, and third, conducting material
not being a part of the wires is removed by etching. The wires are
preferably defined so that one part of the wires forms a line of
contact pads 20 whereas another part forms an array of measuring
electrode parts 16 and one or more reference electrodes 8. The
array of electrode parts is not necessarily an ordered pattern. The
contact pad and electrode part are preferably the two end parts of
the wire, but may be any parts of e.g. a pattern of conducting
strips. Preferably, the conducting material consists of metals or
doped silicon.
[0051] In order to establish the electrodes and contacts, the
conducting material not forming part of the electrode or of the
contact part of each wire is covered with an insulating
(hydrophilic) film 22, e.g. silicon dioxide, or multiple layers of
silicon nitride and silicon dioxide. This is carried out by
covering the whole surface with a layer of the insulating film
using either thermal oxidation of silicon, physical or chemical
vapour deposition, or spin coating. Using photolithography and an
etching step, parts of the insulating film are removed to expose
the wire and thereby form electrodes 16 and 8 and contacts 20. For
a better electrical contact, electrodes (and contacts) can be
covered with silver 24. Alternatively, lift-off techniques might be
used in these cases where several layers of material are to be
deposited in several thin layers. Here a photoresist is deposited
over the substrate and the pattern to be formed is defined in the
resist by illumination through a mask followed by etching. A layer
of material, typically a metal, is vapour deposited onto the
structure, and the photoresist is dissolved, thereby leaving metal
in the defined pattern. At this stage, the substrate will appear as
shown in FIG. 7, the thin lines 18 connecting electrodes and
contacts being covered by insulating film.
[0052] Optionally, but shown in FIG. 3A, hydrophobic regions 26
completely surrounding electrode sites or groups of sites are
formed using a combination of deposition of a hydrophobic material
like Teflon and photolithography. The hydrophobic material is
deposited using either spin coating, chemical vapour deposition or
plasma enhanced chemical vapour deposition. FIG. 2A shows a
possible use of such regions.
[0053] Finally, before use, a silver chloride layer 28 is formed on
the electrode 16 using electrolytic treatment. The same procedure
is normally followed for all measuring and reference electrodes in
the substrates of the invention to establish them as silver/silver
halide electrodes, such as silver/silver chloride electrodes.
[0054] Using the same production scheme as described above, a
number of different electrode designs shown in FIGS. 3B-3D can be
applied. The designs shown imply some differences in the wafer
processing described above, however, given the design, the
adaptation of the wafer processing steps is obvious to the person
skilled in wafer processing technology.
[0055] FIG. 3B shows a close-up of a site holding a cell 2 where
the seal 25 is formed at the site surrounding the AgCl layer 28. In
the production of the electrode, a large volume of AgCl layer 28 is
formed on top of the silver 24 prior to deposition of the silica
layer 22, thereby ensuring a large supply of AgCl.
[0056] FIG. 3C shows another embodiment wherein the measuring
electrode is positioned in a small well 27 whereby the seal is
formed between the membrane and the rim of the well 27. Depending
on the size of the well 27, this embodiment allows for a greater
separation of the membrane and the working electrode as well as for
a larger volume of the carrier liquid surrounding the
electrode.
[0057] FIG. 3D shows yet another embodiment wherein the working
electrode is positioned in a small well 27 as in FIG. 3C. Here, a
pore-forming substance 40 has been deposited at the site in order
to establish, by the action of dissolved pore-forming substance on
the cell, a whole-cell measuring configuration when a cell is
positioned.
[0058] In the design of FIG. 4A, a site is positioned at the bottom
of a well, a geometrically shaped structure on the substrate. The
function of the well is both to position the cell 2 at the site and
to separate test confinements, which in this case consist of single
sites.
[0059] A substrate with a well shaped as a truncated pyramid is
shown in FIG. 4A, an aperture or passage 30 from the narrow end of
the truncated pyramid to the bottom surface part of the substrate
is also defined in the substrate, the well and the passage thereby
creating a funnel. A measuring electrode 16 is provided on the
bottom surface part of the substrate close to the aperture or
passage, and a reference electrode 8 is provided at a side surfaces
of the well, as shown in FIG. 4A. Preferably there is provided
piping 32 for applying suction to the passage on the bottom side of
the substrate. In a preferred embodiment, this piping leads to the
upper side of the substrate, and may include the electrical wiring
to the measuring electrode.
[0060] When the term "bottom" is applied above, this merely refers
to the orientation of the drawing. In the use of the substrate
according to the invention, it is not a condition that the first
surface part of the substrate is the upper surface part and the
second surface part the lower surface part. In other words, gravity
is not utilized to any substantial extent in connection with these
very small structures, and, as an example, the design of FIG. 4A
could also be used in an orientation corresponding to the figure
having been rotated an angle of 180 degrees (or any other angle,
for that matter).
[0061] The well shown in FIG. 4A is basically a truncated pyramidal
cavity with a hole 30 at the apex. The base of the pyramid is a
square. The top angle of the pyramid is 2.times.54.7.degree., the
wafer thickness d=350-650 .mu.m, the side-length at the apex of the
pyramid is w.apprxeq.30 .mu.m in order to allow room for a cell.
The apex of the pyramid is covered with a Silicon-dioxide membrane
31 of thickness h.apprxeq.3 .mu.m. In this membrane, a hole of
diameter a.apprxeq.0.1-10 .mu.m, such as 1-51 .mu.m, is formed.
[0062] The structure comprising a well or wells can be made in
several quite different ways. Below, two different production
processes for the basic structure are summarised, the oxide first
process and the oxide last process, respectively
[0063] Oxide First Process
[0064] Grow 31 .mu.m wet thermal SiO.sub.2 covering whole
substrate.
[0065] Define the hole on the bottom side of the substrate by
photomasking and Reactive Ion Etching to make the hole through the
oxide to the silicon substrate.
[0066] Deposit LPCVD Silicon-nitride for an etch mask on both sides
of the substrate.
[0067] Define nitride windows to form pyramid base plane on the
upper side of the substrate by photomasking and Reactive Ion
Etching and wet oxide etching (buffered Fluoric Acid)
[0068] Etch pyramidal cavities through the windows by anisotropic
etching in the silicon. This creates pyramid sides with a slope of
54.7.degree..
[0069] Strip nitride etch stop using hot H.sub.3PO.sub.4.
[0070] Grow 1 .mu.m wet thermal SiO.sub.2 to electrically insulate
the bulk silicon wafer in order to cover the sides of the pyramid.
Other SiO.sub.2 regions will not grow considerably.
[0071] Oxide Last Process
[0072] Form an etch-stop layer in silicon (boron doping) on the
bottom side of the substrate, using either doping by implantation
or epitaxial growth. The etch stop layer will typically be around 1
.mu.m thick.
[0073] Deposit LPCVD silicon nitride for an etch mask on both sides
of the substrate.
[0074] Define nitride windows to form pyramid base plane on the
upper side of the substrate by photomasking and Reactive Ion
Etching and wet oxide etching (buffered Fluoric Acid)
[0075] Etch pyramidal cavities through the windows by anisotropic
etching in the silicon. This creates pyramid sides with a slope of
54.7.degree.. The etching stops on the boron-doped etch stop to
form an .about.1 .mu.m thick silicon membrane.
[0076] Strip nitride etch stop using Hot H.sub.3PO.sub.4.
[0077] Define the hole on the bottom side by photomasking and
Reactive Ion Etching of Silicon
[0078] Grow wet thermal SiO.sub.2 to convert the Silicon membrane
into an oxide everywhere on the substrate. This process shrink the
hole since SiO.sub.2 is also formed inside the hole, which thereby
can be made smaller compared to what is possible using
photolithography.
[0079] For both production processes the main concern during
processing is the mechanical stability of the SiO.sub.2 membrane
with the hole during the final high temperature oxidation step. The
surface material (here SiO.sub.2) can optionally be coated with
silicon nitride, in order to prevent a contribution to the
electrical conductivity.
[0080] Measuring and reference electrodes can now be formed. The
measuring electrode on the bottom side can be formed using standard
deposition and photolithography techniques. The reference electrode
is preferably formed using evaporation of conducting material
through a shadow mask, or through use of an electrophoretic resist
technique.
[0081] Further, flow channel structures for adding liquid to the
funnel may possibly be created in the substrate, giving an in-flow
and an out-flow port to/from the funnel and elsewhere on the
substrate. Alternatively, the flow channels are made on another
substrate to be applied on top of the substrate, using normal
etching techniques.
[0082] The features described are preferably arranged such that
there is an easy access to all connection in- and outlets from
above the assembly, as illustrated in FIG. 4B (suction outlet 32,
contacts to measuring electrode 16 and reference electrode 8). This
preferred configuration is adapted for applying a unit, having
similar but reverse in- and outlets, on top of the assembly.
[0083] It is an important aspect that the substrate can provide
some means for separating test confinements 15 as in FIG. 2. Test
confinements preferably hold volumes as small as nanolitres. This
is convenient considering the necessary amounts of the often
expensive test samples; moreover, the time needed for mixing the
solution by diffusion decreases with decreasing volume.
[0084] In FIG. 2A, the test confinements are defined using surface
materials to define hydrophobic regions 26 and hydrophilic sites 14
on the substrate, as described previously. If the surface is wetted
(but not flooded) by an aqueous solution such as saline, the liquid
will confine itself to the hydrophilic areas, thereby defining the
test confinements. Each hydrophilic area includes some sites 14
with electrodes 16 and may also include smaller scale hydrophobic
areas.
[0085] On the substrate shown in FIG. 2B, the test confinements are
separated by subdivisions 13 formed on the surface of the
substrate. These subdivisions can be produced on the raw substrate
by covering the substrate surface with a resist, and define the
well openings using photolithography. An etch step followed by
removal of the remaining resist leaves the substrate ready for
formation of sites and electrodes.
[0086] FIG. 2C shows a substrate covered with electrodes, without
any substantial subdivision. In this case the test confinements are
defined using a structure part 17 with hollow
subdivisions/chambers, to be applied on top of the substrate. By
making a tight mechanical contact with the substrate, the structure
part forms closed chambers each holding one or more sites with
electrodes. If convenient, a similar structure part can be applied
on top of any of the substrates shown in FIGS. 2A and B.
[0087] In all of the embodiments shown in FIG. 2, a reference
electrode has to be located within each test confinement. This can
be realised either by having an electrode at a site where no cell
can cover it, an electrode so large that no cell can cover it, or,
by dosing the number of cells in such a way that cells can not
cover all electrodes. This last option allows for any of the
measuring electrodes to function as reference electrode.
[0088] Depending on the specific shape of the substrate with
electrodes, the addition of cell-supporting liquid and cells is
carried out in one of the following ways. In a preferred
embodiment, the test confinements are accessible from above, and
droplets of supporting liquid and cells can be supplied at each
test confinement by means of a dispensing or pipetting system.
Systems such as an ink jet printer head or a bubble jet printer
head can be used. Another possibility is an nQUAD aspirate
dispenser or any other dispensing/pipetting device adapted to dose
small amounts of liquid. Alternatively, supporting liquid and cells
are applied on the substrate as a whole (e.g. by pouring supporting
liquid containing cells over the substrate or immersing the
substrate in such), thereby providing supporting liquid and cells
to each test confinement. Since the volumes of supporting liquid
and later test samples are as small as nanolitres, water
vaporisation could represent a problem. Therefore, depending of the
specific volumes, handling of liquids on the substrate should
preferably be carried out in high humidity atmospheres.
[0089] In the case of the test confinements being closed chambers,
they might only be accessible through a system of channels, i.e. a
microliquid handling system. This is the case when a second
structure part 17 (FIG. 2C) is applied on top of any of the
substrates with or without test confinements. In this case
supporting liquid and cells must be provided through inlet channels
typically defined in the second structure part 17. Such a second
structure part can be made of, e.g., silicon in which case flow
channels can be formed using standard photolithography and etching
techniques. Such a second structure part can be applied on top of
any of the embodiments.
[0090] In another aspect, the cells are cultivated directly on the
substrate, while immersed in growth medium. In the optimal case,
the cells will form a homogeneous monolayer (depending on the type
of cells to be grown) on the entire surface, except at regions
where the surface intentionally is made unsuitable for cell growth.
The success of cultivation of cells on the substrate depends
strongly on the substrate material.
[0091] In still another aspect, an artificial membrane with
incorporated ion channels may be used instead of a cell. Such
artificial membrane can be made from a saturated solution of
lipids, by positioning a small lump of lipid over an aperture. This
technique is thoroughly described in e.g., "Ion Channel
Reconstitution" by Christopher Miller, Plenum 1986, p. 577. If the
aperture size is appropriate, and a polar liquid such as water is
present on both sides of the aperture, a lipid bilayer can form
over the aperture. The next step is to incorporate a protein ion
channel into the bilayer. This can be achieved by supplying lipid
vesicles with incorporated ion channels on one side of the bilayer.
The vesicles can be drawn to fusion with the bilayer by e.g.
osmotic gradients, whereby the ion channels are incorporated into
the bilayer.
[0092] Obtaining good contact between the cell and a glass pipette,
and thereby creating a giga-seal between a cell and the tip the
pipette, is well described in the prior art. In order to draw the
cell to the tip of the pipette, as well as to make the necessary
contact for obtaining the giga-seal, it is normal to apply suction
to the pipette.
[0093] In the case of the substrates described in FIGS. 2A-C, no
suction is provided, and the positioning of the cells is carried
out by other means. Moreover, it has been shown that the mere
contact between the cell membrane and the substrate, typically
ultra-pure silica, is sufficient for the cell to make some bonding
to the surface and create a giga-seal.
[0094] The positioning can be carried out by electrophoresis, where
an electric field from an electrode draws the charged cell towards
it. Negatively charged cells will be drawn towards positive
electrodes and vice versa. The electrostatic pull can also act as
guiding means for a group of electrodes. Alternatively, within a
test confinement, a hydrophobic material 26 may cover the surface
of the substrate except at areas just around electrodes. This is
shown in FIG. 5. Thereby, cells can only bind themselves on
electrode sites 14. It is possible to apply both of these methods
simultaneously or optionally in combination with a suitable
geometrical shape of the substrate surface around electrodes, to
guide the sinking cells towards the electrode.
[0095] In another embodiment, the density and pattern of sites and
measuring electrodes is close to or higher than the density of
cells when these are packed to make closest packing on the surface
of the substrate. This ensures that when a sufficient number of
cells is supplied, at least one electrode is covered by a cell
without further guiding means.
[0096] In the embodiment shown in FIG. 4A, one or more cells 2 in a
supporting liquid are applied and sink to the bottom end of the
funnel, this being an example of positioning by geometrical
shaping. If suction is applied, it draws the cell to the aperture
30 and establishes a connection between the cell and the aperture,
creating a giga-seal separating the aperture inside and the
solution. The giga-seal may take any form, e.g., circular, oval or
rectangular. The supporting liquid makes electrical contact between
the cell membrane and the reference electrode. The cell may be
deformed by the suction, and a case where the cell extends into the
aperture may be desired if controlled.
[0097] Each test confinement preferably holds several electrode
sites. In order to detect whether an electrode is covered by a cell
and insulated by a giga-seal, leak currents are measured between
electrodes or between electrodes and the reference electrode. Even
though a test confinement may include numerous electrodes, it is a
simple task to search for electrodes insulated by giga-seals, a
task well suited for a computer.
[0098] FIGS. 6 and 7 proposes a scheme for doing so, where the
electrodes 16 in a test confinement form an n.times.m matrix (here
3.times.3). The electrode connections 18 lead to a line of contacts
20 (No. 1 to 9) on the substrate that can be individually addressed
by a computer with means for measuring currents. A list of
giga-sealed electrodes can be made using a simple method sketched
in the flow diagram of FIG. 7. First (1), two loops are established
for going through all entries in the matrix of electrodes. In (2),
the n.times.m array of the matrix is unfolded to provide an
individual addressing (3) of electrode contacts with an electrode
contact number N (No. 1 to 9). The current, at an applied voltage
between contact N and the reference electrode 8, contact No. 0, is
measured (4), and its value is compared to some threshold current
I.sub.threshold (5) for determining whether the electrode is
giga-sealed. If a giga-seal is detected, the contact number is
added to a list of suitable electrodes (6) from which a measuring
electrode is selected (7). This scheme carries some information on
the relative positions n,m of suitable electrodes. This information
can be used for selecting the optimal measuring electrode in (7),
but can be omitted so that each electrode is simply known by its
contact number N. Typically, only one electrode per test
confinement is chosen.
[0099] The activity of these channels can be measured electrically
(single channel recording) or, alternatively, the patch can be
ruptured allowing measurements of the channel activity of the
entire cell membrane (whole cell recording). High-conductance
access to the cell interior for performing whole cell measurements
can be obtained in at least 3 different ways (all methods are
feasible, but various cells may work better with different
approaches):
[0100] a) In the embodiment shown in FIG. 4A, the membrane can be
ruptured by suction from the aperture side. Subatmospheric
pressures are applied either as short pulses of increasing strength
or as ramps or steps of increasing strength. Membrane rupture is
detected by highly increased capacitative current spikes
(reflecting the total cell membrane capacitance) in response to a
given voltage test pulse.
[0101] b) Membrane rupture by applied voltage pulses. Voltage
pulses are applied either as short pulses of increasing strength
(mV to V) and duration (u- to msec), or as ramps or steps of
increasing strength, between the electrodes. The lipids forming the
membrane of a typical cell will be influenced by the large
electrical field strength from the voltage pulses, whereby the
membrane to disintegrates in the vicinity of the electrode.
Membrane rupture is detected by highly increased capacitative
current spikes in response to a given voltage test pulse.
[0102] c) Permeabilization of membrane. Application of pore-forming
substances (for example antibiotics such as nystatin or
amphotericin B), by e.g. prior deposition of these at the site.
Rather than by rupturing the membrane, the membrane resistance is
selectively lowered by incorporation of permeabilizing molecules,
resulting in effective cell voltage control via the electrode pair.
The incorporation is followed by a gradually decreasing total
resistance and an increasing capacitance.
[0103] At this stage, a substrate with some electrodes each holding
a cell is provided, the selected cells form a giga-seal around
their respective electrodes, allowing for the electrode to measure
electrophysiological properties of the ion transfer channels in the
cell membrane. This represents the main aspect of the invention,
the making available of a plurality of prepared sample cells for
performing electro-physiological experiments. Moreover, each cell
is confined in order to permit individual testing of the cells.
[0104] The remaining of this description will focus on the
application of the substrate made ready in this way.
[0105] The test samples must be added to each test confinement
individually, with different test samples for each test
confinement. This can be carried out using the methods for applying
supporting liquid, with the exception of the methods where
supporting liquid are applied on the substrate as a whole.
[0106] Upon positioning the cell in a measuring configuration,
several electrophysiological properties can be measured, such as
current through ion channels (voltage clamp), or capacitance of ion
channels containing membranes. In any case, a suitable electronic
measuring circuit should be provided. The person skilled in the art
will be able to select such suitable measuring circuit. One such
possible circuit for voltage clamp measurements is described above
with reference to FIG. 1.
[0107] In the case of voltage clamp measurements, the electrical
current carried by the ion transfer channels in the cell membrane
results in a charge transfer from the solution (reference
electrode) to the measuring electrode, typically of the order of pA
to .mu.A (picoampere--10.sup.-12A- ). A low noise amplifier is
provided for measuring these currents. The electronic circuits can
be integrated in a separate standard unit having contact to the two
electrodes and possibly flow channels for drug application.
* * * * *