CA2334770A1 - High throughput screen - Google Patents
High throughput screen Download PDFInfo
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- CA2334770A1 CA2334770A1 CA002334770A CA2334770A CA2334770A1 CA 2334770 A1 CA2334770 A1 CA 2334770A1 CA 002334770 A CA002334770 A CA 002334770A CA 2334770 A CA2334770 A CA 2334770A CA 2334770 A1 CA2334770 A1 CA 2334770A1
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- Prior art keywords
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- structure according
- biological membrane
- membrane
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- 239000000758 substrate Substances 0.000 claims abstract 25
- 102000004310 Ion Channels Human genes 0.000 claims abstract 23
- 239000012528 membrane Substances 0.000 claims abstract 22
- 238000000034 method Methods 0.000 claims abstract 19
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract 15
- 150000001875 compounds Chemical class 0.000 claims abstract 10
- 230000000694 effects Effects 0.000 claims abstract 6
- 238000004519 manufacturing process Methods 0.000 claims abstract 6
- 239000011521 glass Substances 0.000 claims abstract 5
- 238000012216 screening Methods 0.000 claims abstract 4
- 210000004027 cell Anatomy 0.000 claims 25
- 108090000862 Ion Channels Proteins 0.000 claims 20
- 239000011148 porous material Substances 0.000 claims 9
- 210000000170 cell membrane Anatomy 0.000 claims 4
- 229920000139 polyethylene terephthalate Polymers 0.000 claims 4
- 239000005020 polyethylene terephthalate Substances 0.000 claims 4
- 229930183010 Amphotericin Natural products 0.000 claims 3
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 claims 3
- QRLVDLBMBULFAL-UHFFFAOYSA-N Digitonin Natural products CC1CCC2(OC1)OC3C(O)C4C5CCC6CC(OC7OC(CO)C(OC8OC(CO)C(O)C(OC9OCC(O)C(O)C9OC%10OC(CO)C(O)C(OC%11OC(CO)C(O)C(O)C%11O)C%10O)C8O)C(O)C7O)C(O)CC6(C)C5CCC4(C)C3C2C QRLVDLBMBULFAL-UHFFFAOYSA-N 0.000 claims 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims 3
- 108090000790 Enzymes Proteins 0.000 claims 3
- 102000004190 Enzymes Human genes 0.000 claims 3
- 229940009444 amphotericin Drugs 0.000 claims 3
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims 3
- 239000003599 detergent Substances 0.000 claims 3
- UVYVLBIGDKGWPX-KUAJCENISA-N digitonin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)C[C@@H](O)[C@H](O[C@H]5[C@@H]([C@@H](O)[C@@H](O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)CO7)O)[C@H](O)[C@@H](CO)O6)O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O7)O)[C@@H](O)[C@@H](CO)O6)O)[C@@H](CO)O5)O)C[C@@H]4CC[C@H]3[C@@H]2[C@@H]1O)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 UVYVLBIGDKGWPX-KUAJCENISA-N 0.000 claims 3
- UVYVLBIGDKGWPX-UHFFFAOYSA-N digitonine Natural products CC1C(C2(CCC3C4(C)CC(O)C(OC5C(C(O)C(OC6C(C(OC7C(C(O)C(O)CO7)O)C(O)C(CO)O6)OC6C(C(OC7C(C(O)C(O)C(CO)O7)O)C(O)C(CO)O6)O)C(CO)O5)O)CC4CCC3C2C2O)C)C2OC11CCC(C)CO1 UVYVLBIGDKGWPX-UHFFFAOYSA-N 0.000 claims 3
- 229960001760 dimethyl sulfoxide Drugs 0.000 claims 3
- 230000006862 enzymatic digestion Effects 0.000 claims 3
- 150000002500 ions Chemical class 0.000 claims 3
- 238000005259 measurement Methods 0.000 claims 3
- 229960000988 nystatin Drugs 0.000 claims 3
- VQOXZBDYSJBXMA-NQTDYLQESA-N nystatin A1 Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/CC/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 VQOXZBDYSJBXMA-NQTDYLQESA-N 0.000 claims 3
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims 3
- 229930182490 saponin Natural products 0.000 claims 3
- 150000007949 saponins Chemical class 0.000 claims 3
- 108091006146 Channels Proteins 0.000 claims 2
- 230000003115 biocidal effect Effects 0.000 claims 2
- 239000002299 complementary DNA Substances 0.000 claims 2
- 239000004744 fabric Substances 0.000 claims 2
- 230000003834 intracellular effect Effects 0.000 claims 2
- 230000002093 peripheral effect Effects 0.000 claims 2
- 239000004033 plastic Substances 0.000 claims 2
- 229920003023 plastic Polymers 0.000 claims 2
- -1 polyethylene terephthalate Polymers 0.000 claims 2
- 239000005060 rubber Substances 0.000 claims 2
- 238000007789 sealing Methods 0.000 claims 2
- 210000001578 tight junction Anatomy 0.000 claims 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims 1
- 241000699802 Cricetulus griseus Species 0.000 claims 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims 1
- 102000000591 Tight Junction Proteins Human genes 0.000 claims 1
- 108010002321 Tight Junction Proteins Proteins 0.000 claims 1
- 230000004913 activation Effects 0.000 claims 1
- 239000003242 anti bacterial agent Substances 0.000 claims 1
- 238000003556 assay Methods 0.000 claims 1
- 230000015572 biosynthetic process Effects 0.000 claims 1
- 239000011575 calcium Substances 0.000 claims 1
- 229910052791 calcium Inorganic materials 0.000 claims 1
- 238000005266 casting Methods 0.000 claims 1
- 238000010367 cloning Methods 0.000 claims 1
- 238000012258 culturing Methods 0.000 claims 1
- 238000001514 detection method Methods 0.000 claims 1
- 210000001320 hippocampus Anatomy 0.000 claims 1
- 210000002865 immune cell Anatomy 0.000 claims 1
- 230000002779 inactivation Effects 0.000 claims 1
- 239000000463 material Substances 0.000 claims 1
- 239000011159 matrix material Substances 0.000 claims 1
- 210000004165 myocardium Anatomy 0.000 claims 1
- 230000001537 neural effect Effects 0.000 claims 1
- 210000001672 ovary Anatomy 0.000 claims 1
- 238000001259 photo etching Methods 0.000 claims 1
- 239000011591 potassium Substances 0.000 claims 1
- 229910052700 potassium Inorganic materials 0.000 claims 1
- 210000002027 skeletal muscle Anatomy 0.000 claims 1
- 210000002460 smooth muscle Anatomy 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 210000003594 spinal ganglia Anatomy 0.000 claims 1
- 210000002222 superior cervical ganglion Anatomy 0.000 claims 1
- 210000001519 tissue Anatomy 0.000 claims 1
- 238000001890 transfection Methods 0.000 claims 1
- 102000040811 transporter activity Human genes 0.000 abstract 2
- 108091092194 transporter activity Proteins 0.000 abstract 2
- 238000012544 monitoring process Methods 0.000 abstract 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
- G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0829—Multi-well plates; Microtitration plates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0848—Specific forms of parts of containers
- B01L2300/0851—Bottom walls
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0893—Geometry, shape and general structure having a very large number of wells, microfabricated wells
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T29/00—Metal working
- Y10T29/49—Method of mechanical manufacture
- Y10T29/49826—Assembling or joining
Abstract
The present invention relates to a structure comprising a biological membrane and a porous or perforated substrate, a biological membrane, a substrate, a high throughput screen, methods for production of the structure membrane and substrate, and a method for screening a large number of test compounds in a short period. More particularly it relates to a structure comprising a biological membrane adhered to a porous or perforated substrate, a biological membrane capable of adhering with high resistance seals to a substrate such as perforated glass and the ability to form sheets having predominantly an ion channel or transporter of interest, a high throughput screen for determining the effect of test compounds on ion channel or transporter activity, methods for manufacture of the structure, membrane and substrate, and a method for monitoring ion channel or transporter activity in a membrane.
Claims (46)
1. A structure which comprises a biological membrane adhered with a high resistance seal to a porous or perforated substrate for use in a high throughput screen wherein the biological membrane comprises an ion channel or transporter.
2. A structure according to claim 1 wherein the biological membrane comprises a contiguous layer of cells which is capable of adhering to a substrate with a high resistance seal wherein each cell forms a tight junction with adjacent cells and expresses an ion channel or transporter which is localised in the cell membrane.
3. A structure according to claim 1 or 2 which comprises cells having an ion channel or transporter which naturally resides in the cell membrane thereof or, it can be inserted by transfection with cDNA and/or cRNA
encoding the ion channel or transporter.
encoding the ion channel or transporter.
4. A structure according to any preceding claim which comprises a plurality or ions channels or transporters which are predominantly preselected ion channels or transporters of interact.
5. A structure according to any preceding claim which comprises genetically engineered cells which have been engineered to predominantly express an ion channel or transporter.
6. A structure according to any preceding claim which comprises voltage gated ion channels.
7. A structure according to any one of claims 2 to 5 wherein the cells are selected from the group which comprises HEK-293 cells, genetically modified Chinese hamster ovary (CHO) cells, primary neuronal tissue such as hippocampus, dorsal root ganglia, superior cervical ganglia etc.; skeletal muscle; smooth muscle; cardiac muscle; immune cells; epithelia; endothelia.
8. A structure according to any preceding claim which comprises an ion channel having rapid activation and inactivation kinetics.
9. A structure according to any preceding claim having an ion channel which shows specificity for an ion selected from the group which comprises sodium, potassium, calcium, chloride.
10. A structure according to any one of claims 2 to 9 wherein the contiguous layer of cells is capable of adhering with a high resistance seal to a substrate selected from the group which comprises glass, plastics, rubber, polytetraflurotethylene (PTFE), PTFE/glass fabric and polyethylene terephthalate (PETP).
11. A structure according to any preceding claim which comprises a pseudo-epithelium wherein one face of a contiguous layer of cells is permeabilized thereby providing access to the interior of the cells.
12. A structure according to claim 11 which comprises a pseudo-epithelium wherein one face of the contiguous layer of cells is permeabilized by an antibiotic selected from the group which comprises amphotericin and nystatin; or detergent selected from the group which comprises digitonin and saponin; or physical disruption using a high voltage field; or by enzymatic digestion of a part of the membrane using an appropriate enzyme.
13. A structure according to any preceding claim wherein the substrate is perforated.
14. A structure according to any preceding claim which comprises a perforated coverslip.
15. A structure according to any preceding claim wherein the substrate has pores of diameters between 0.5µm and 10µm.
16. A structure according to claim 15 wherein the pores are of diameter between 1µm and 7µm.
17. A structure according to claim 15 or 16 wherein the pores are of diameter 1-2µm.
18. A structure according to any preceding claim which comprises a coverslip having a grid of pores.
19. A structure according to any preceding claim which comprises a perforated substrate which is manufactured of a material selected from the group which comprises glass, plastics, rubber, polytetraflurotethylene (PTFE), PTFE/glass fabric and polyethylene terephthalate (PETP).
20. A biological membrane for use in the structure according to any preceding claim.
21 A substrate for use in the structure according to any one of claim 1 to 19.
22. A high throughput screen for detecting and assaying compounds with activity on voltage gated ions channels which comprises a structure according to any one of claims 1 to 19, a biological membrane according to any claim 20 or a substrate according to claim 21.
23. A high throughput screen according to claim 22 which comprises :
a plurality of chambers, each having a permeable peripheral surface providing a substrate for the biological membrane;
a plurality of wells each capable of receiving a chamber and a test compound in a physiological solution or non-physiological solution comprising dimethyl sulphoxide;
a plurality of reference electrodes, having electrical contact with each well;
a movable recording head carrying at least one recording electrode;
means for measuring electrical resistance or impedance between the recording and reference electrodes; wherein electrical current may pass between the recording and reference electrodes through the permeable peripheral surface of each chamber only via ion channels or transporters in the biological membrane.
a plurality of chambers, each having a permeable peripheral surface providing a substrate for the biological membrane;
a plurality of wells each capable of receiving a chamber and a test compound in a physiological solution or non-physiological solution comprising dimethyl sulphoxide;
a plurality of reference electrodes, having electrical contact with each well;
a movable recording head carrying at least one recording electrode;
means for measuring electrical resistance or impedance between the recording and reference electrodes; wherein electrical current may pass between the recording and reference electrodes through the permeable peripheral surface of each chamber only via ion channels or transporters in the biological membrane.
24. A high throughput screen according to claim 23 wherein the wells are provided by a multiwell plate.
25. A high throughput screen according to claim 22 which comprises an array of droplets on a porous substrate.
26. A high throughput screen according to any one of claims 22 to 25 which comprises a recording head having a single recording electrode capable of being moved to visit each chamber sequentially.
27. A high throughput screen according to any one of claims 22 to 25 which comprises a recording head having a plurality of recording electrodes arranged in a line.
28. A high throughput screen according to any one of claims 22 to 25 which comprises a recording head having a plurality of recording electrodes arranged in a matrix.
29. A method of manufacturing the structure of any one of claims 1 to 19 which comprises he steps of selecting a substrate, perforating it, introducing a biological -46- ~
membrane to the substrate and sealing each pore with biological membrane.
membrane to the substrate and sealing each pore with biological membrane.
30. A method according to claim 29 which comprises the steps of simultaneously perforating d substrate and sealing the pores with biological membrane.
31. A method of manufacturing a biological membrane according to claim 20 which comprises the steps of selecting a cell type, evaluating it for ability to form contiguous layers of cells with tight junctions and for low to negligible numbers of voltage gated ion channels, culturing the cells on a substrate and ensuring that a contiguous layer of cells is grown.
32. A method according to claim 31 which includes the step of permeabilizing one surface of the contiguous layer of cells thereby providing access to the interior of the cells.
33. A method according to claim 32 wherein the step of permeabilizing one surface of the contiguous layer of cells is carried out by the step of contacting the surface with do antibictic selected from the group which comprises amphotericin and nystatin; or detergent selected from the group which comprises digitonin and saponin; or physical disruption using a high voltage field; or by enzymatic digestion of a part of the cell membrane using an appropriate enzyme.
34. A method according to any one of claims 31 to 33 which comprises the steps of transfecting cells with cDNA or cRNA encoding an ion channel of interest and cloning cells expressing the ion channel or interest.
35. A method of manufacturing a substrate according to claim 21 which comprises the steps of shining a laser of preselected focal area, power or time of exposure at a coverslip to perforate it.
36. A method according to claim 35 which comprises the steps of adjusting the profile, taper or diameter of the pore with a laser.
37. A method according to claim 35 or 36 wherein the laser source is controlled by an automated stage under control of a computer and inverted optics microscope.
38. A method of manufacturing a substrate according to claim 21 which comprises a non-laser method selected from the group which comprises photo-etching, casting and physical piercing of the substrate.
39. A method of screening for the detection or assay of compounds with activity on ion channels or transporters which comprises the steps of placing a biological membrane which expresses ion channels or transporters of interest in contact with test compound in physiological solution or non-physiological solution comprising dimethyl sulphoxide and measuring the resistance or impedance of the biological membrane under the influence of test compound.
40. A method according to claim 39 wherein ion channel activity is monitored by trans-epithelial resistance measurements (TERM) across an intact cell layer.
41. A method according to claim 39 which comprises the step of permeabilizing a contiguous cell layer to provide access to the interior of the cells permitting intracellular voltage and current measurements to be made.
42. A method according to claim 41 wherein a contiguous cell layer is permeabilized by antibiotic selected from the group which comprises amphotericin and nystatin;
or detergent selected from the group which comprises digitonin and saponin; or physical disruption using a high voltage field; or by enzymatic digestion of a part of the cell membrane using an appropriate enzyme -49- ~
thereby permitting intracellular voltage or current measurements to made.
or detergent selected from the group which comprises digitonin and saponin; or physical disruption using a high voltage field; or by enzymatic digestion of a part of the cell membrane using an appropriate enzyme -49- ~
thereby permitting intracellular voltage or current measurements to made.
43. A method according to any one of claims 39 to 42 which includes the step of multiplexing up to 384 recording elements to a data acquisition system utilizing multiple voltage-clamp amplifiers.
44. A method according to any one of claims 39 to 43 which comprises the step of placing an array of droplets having ion channels or transporters of interest therein on a porous substrate and screening test compounds for activity on the ion channels or transporters.
45. A method according to any one of claims 39 to 43 which comprises the step of placing biological membrane having ion channels or transporters of interest and test compounds in physiological solution, or non-physiological solution comprising dimethyl sulphoxide, in a plurality of chambers and screening the test compounds for activity on the ion channels or transporters.
46. A method according to any of claims 21, 30 and 35 to 38 wherein, subsequent to pore formation, the substrate is exposed to localised heat and/or to electrical plasma in order to impart an appropriate raised level of smoothness to the pore(s).
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB9812783.0A GB9812783D0 (en) | 1998-06-12 | 1998-06-12 | High throuoghput screen |
GB9812783.0 | 1998-06-12 | ||
PCT/GB1999/001871 WO1999066329A1 (en) | 1998-06-12 | 1999-06-14 | High throughput screen |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2334770A1 true CA2334770A1 (en) | 1999-12-23 |
CA2334770C CA2334770C (en) | 2011-04-12 |
Family
ID=10833717
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2334770A Expired - Fee Related CA2334770C (en) | 1998-06-12 | 1999-06-14 | High throughput screen |
Country Status (17)
Country | Link |
---|---|
US (5) | US6936462B1 (en) |
EP (2) | EP1084410B3 (en) |
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1999
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