High throughput screen

• US 7,846,389 B2
• Filed: 05/20/2005
• Issued: 12/07/2010
• Est. Priority Date: 06/12/1998
• Status: Expired due to Fees

• Current Assignee: Sophion Bioscience A/S
• Original Assignee: Xention Limited (Xention Pharma Ltd.)
• Inventors: Byrne, Nicholas Gerard, Owen, David Geraint
• Primary Examiner(s): Jung; Unsu
• Application Number:
  US11/133,808
• Publication Number:
  US 20050221282A1
• Time in Patent Office: 2,027 Days
• Field of Search: 435/7.2, 435/283.1, 435/288.4, 435/288.5, 422/82.01, 422/82.02, 422101-104
• US Class Current: 422/82.01
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0851 Bottom walls
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 2300/16 Surface properties and coatings
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: G01N 2500/04 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)
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 27/28 Electrolytic cell components
• CPC Class Codes: G01N 33/48728 Investigating individual cells, e.g. by patch clamp, voltage clamp investigating individual particles in general G01N15/10
• CPC Class Codes: G01N 33/502 for testing non-proliferative effects
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/6872 Intracellular protein regulatory factors and their receptors, e.g. including ion channels
• CPC Class Codes: Y10T 29/49826 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 inn 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

1. An apparatus for use with a biological cellular membrane in detecting and assaying compounds with ion channel activity, comprising:

a well with a porous substrate disposed therein, said substrate having at least one pore with a diameter between 0.5 μ

m and 10 μ

m;

a solution perfusion channel disposed below said porous substrate and defining an inlet and an outlet for flow of fluid therethrough;

a first electrode disposed in said well;

a second electrode disposed in said solution perfusion channel in electrical communication with the first electrode through said porous substrate;

wherein said substrate is configured to permit adherence of a biological cellular membrane on an upper side thereof opposite said perfusion channel with a seal between said cellular membrane and said porous substrate having sufficient electrical resistance to facilitate patch clamping measurements across said membrane; and

further comprising a biological cellular membrane adhered only to said upper side of the porous substrate with a high resistance seal.

4. The apparatus of claim 1 or 2, wherein said at least one pore has a diameter between 1 and 7 μ

m.

5. The apparatus of claim 4, wherein said at least one pore has a diameter of 1-2 μ

m.

6. The apparatus of claim 1 or 2, wherein said at least one pore is formed by a laser.

7. The apparatus of claim 1 or 2, wherein a pressure differential is applied across the substrate such that a lower pressure exists in the solution perfusion channel as compared to in the well above the substrate.

8. The apparatus of claim 1 or 2, further comprising a plurality of said wells.

9. The apparatus of claim 8, wherein said plurality of said wells are formed in a multi-well plate.

10. The apparatus of claim 9, wherein said first electrode is disposed on a moveable recording head that is moveable between the wells of the multi-well plate.

11. The apparatus of claim 10, wherein said movable recording head includes plural first electrodes such that more than one of said wells of the multi-well plate may be contacted with each movement of said recording head.

12. The apparatus of claim 9, wherein said porous substrate in each said well includes plural pores.

13. The apparatus of claim 1 or 2, wherein said porous substrate includes plural pores.

2. An apparatus for use with a biological cellular membrane in detecting and assaying compounds with ion channel activity, comprising:

a well with a porous substrate disposed therein, said substrate having at least one pore with a diameter between 0.5 μ

m and 10 μ

m;

a solution perfusion channel disposed below said porous substrate and defining an inlet and an outlet for flow of fluid therethrough;

a first electrode disposed in said well;

a second electrode disposed in said solution perfusion channel in electrical communication with the first electrode through said porous substrate;

wherein said substrate is configured to permit adherence of a biological cellular membrane on an upper side thereof opposite said perfusion channel with a seal between said cellular membrane and said porous substrate having sufficient electrical resistance to facilitate patch clamping measurements across said membrane; and

further comprising a voltage-clamp amplifier connected between said electrodes and configured for patch clamping measurements.

3. The apparatus of claim 2, wherein said seal is a high resistance seal.

14. An apparatus for use with a biological cellular membrane in detecting and assaying compounds with ion channel activity, comprising:

a multi-well plate defining a plurality of wells, each with a laser perforated substrate disposed therein, said substrate being perforated with plural pores in each said well having a pore diameter of 1 μ

m-2 μ

m, and said substrate being configured to permit adherence of a biological cellular membrane thereto with a seal having an electrical resistance sufficient to facilitate patch clamping measurements thereacross;

a solution perfusion channel disposed below said porous substrate with a differential pressure applied across said substrate to create lower pressure below the substrate to facilitate positioning of cells on said pores said perfusion channel having an inlet and an outlet;

at least a first electrode disposed on a moveable recording head for sequentially positioning in said plurality of wells;

a second electrode disposed in said solution perfusion channel in electrical communication with the first electrode through said porous substrate;

a voltage-clamp amplifier connected between said first and second electrodes for patch clamping measurements.

15. The apparatus of claim 14, wherein said movable recording head includes plural first electrodes such that more than one well of the multi-well plate may be contacted with each movement of said recording head.

16. The apparatus of claim 14, further comprising a biological cellular membrane adhered to said substrate with a high resistance seal.

17. The apparatus of claim 16, wherein said biological cellular membrane is adhered to the substrate with a high resistance seal.

18. The apparatus of claim 14, wherein said substrate is glass.

19. The apparatus of claim 14, wherein said substrate is plastic.

20. The apparatus of claim 19 wherein said substrate is polytetraflurotethylene (PTFE) or polyethylene terephthalate (PTEP).

21. The apparatus of claim 14, wherein said seal is a high resistance seal.

22. A high throughput apparatus for use with a biological cellular membrane in detecting and assaying compounds with ion channel activity, comprising:

a multi-well plate defining a plurality of wells;

a perforated plastic substrate disposed at a bottom of said wells, said substrate being perforated with plural pores in each said well, said pores having a diameter of 1 μ

m-2 μ

m;

a solution perfusion channel disposed below said porous substrate with a differential pressure applied across said substrate to create a lower pressure below the substrate to facilitate positioning of cells on said pores, said perfusion channel having an inlet and an outlet;

plural first electrodes disposed on a moveable recording head for sequentially positioning in said plurality of wells;

at least a second electrode disposed below said porous substrate in electrical communication with said first electrodes through said porous substrate; and

a voltage-clamp amplifier connected between said electrodes for patch clamping measurements.

23. The high throughput apparatus of claim 22, wherein said at least a second electrode is disposed in said solution perfusion channel.

24. The apparatus of claim 22 wherein said substrate is PTFE or PTEP.

25. The apparatus of claim 22, wherein the pores are arranged in a grid in each said well.