High throughput screen

• US 8,449,825 B2
• Filed: 11/05/2010
• Issued: 05/28/2013
• 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: Owen, David Geraint, Byrne, Nicholas Gerard
• Primary Examiner(s): BROWN, MELANIE YU
• Application Number:
  US12/940,564
• Publication Number:
  US 20110048939A1
• Time in Patent Office: 935 Days
• Field of Search: 422/82.01, 422/82.02, 422/101, 422/102, 422/103, 422/104, 435/7.2, 435/283.1, 435/288.4, 435/288.5
• 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 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

1. An apparatus for patch clamping measurements to detect and assay compounds with ion channel activity in a biological cellular membrane, 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 containing said compounds therethrough;

a first electrode disposed in said well; and

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

2. The apparatus of claim 1, 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.

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

4. The apparatus of claim 2, further comprising a biological cellular membrane adhered only to said upper side of the porous substrate with a high resistance seal.

5. The apparatus of claim 1, further comprising a voltage-clamp amplifier connected between said electrodes and configured for said patch clamping measurements.

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

m.

7. The apparatus of claim 6, wherein said at least one pore has a diameter of 1-2 μ

m.

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

9. The apparatus of claim 4, 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.

10. The apparatus of claim 1, further comprising a plurality of said wells.

11. The apparatus of claim 10, wherein said plurality of said wells are formed in a multi-well plate.

12. The apparatus of claim 11, wherein said first electrode is disposed on a moveable recording head that is moveable between the wells of the multi-well plate.

13. The apparatus of claim 12, 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.

14. The apparatus of claim 11, wherein said porous substrate in each said well includes plural pores.

15. The apparatus of claim 1, wherein said porous substrate includes plural pores.

21. The apparatus of claim 1, further comprising a solution containing said test compound disposed within said solution perfusion channel.

16. An apparatus, comprising:

a plate defining a plurality of wells;

a porous substrate disposed in a bottom of each said well, wherein the substrate in each well defines at least one pore with a diameter between 1 μ

m and 7 μ

m, and is configured and dimensioned to permit adherence of a biological cellular membrane on an upper side thereof with a high resistance seal between the membrane and said porous substrate having sufficient electrical resistance to facilitate patch clamping measurements across a biological membrane adhered to said substrate for detecting ion channel activity in the membrane;

a solution perfusion channel disposed below the porous substrate of each said well, said channel defining an inlet for flow of fluid therethrough across said pores and an outlet;

at least a first electrode disposable in each said well; and

at least a second electrode disposed in said solution perfusion channel in electrical communication with said first electrode across the porous substrate.

17. The apparatus of claim 16, wherein the porous substrate in each said well defines plural pores.

18. The apparatus of claim 16, wherein the porous substrate is glass, PTFE or PTEP and said pores are laser formed.

19. The apparatus of claim 16, wherein said at least one pore has a diameter between 1μ

and 2μ

.

20. The apparatus of claim 16, further comprising a voltage-clamp amplifier connected between said electrodes and configured for patch clamping measurements.