High throughput screen

• US 8,759,017 B2
• Filed: 05/02/2013
• Issued: 06/24/2014
• 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:
  US13/875,860
• Publication Number:
  US 20130288289A1
• Time in Patent Office: 418 Days
• Field of Search: 422/82.01, 422/82.02, 435/7.2, 435/283.1, 435/288.4, 435/288.5
• US Class Current: 435/7.2
• 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. A method of screening for the detection or assay of compounds with activity on ion channels or transporters, comprising:

placing a biological cellular membrane in contact with a pore in a porous substrate, the pore having a diameter between 0.5 μ

m and 10 μ

m;

flowing a solution containing at least one said compound from an inlet to an outlet through a perfusion channel disposed below the porous substrate, said channel communicating with said pore and said biological cellular membrane; and

performing patch clamping measurement across the biological cellular membrane between an electrode positioned below the substrate in contact with said flowing solution and an electrode positioned above the substrate electrically communicating with the biological cellular membrane.

2. The method of screening of claim 1, wherein the substrate is disposed in a well of a multi-well plate.

3. The method of screening of claim 1, further comprising adhering said biological cellular membrane to said porous substrate over said pore with a seal between said membrane and said porous substrate having sufficient electrical resistance to facilitate said patch clamping measurements.

4. The method of screening of claim 3, wherein said adhering forms a high resistance seal.

5. The method of screening of claim 3, further comprising:

applying a pressure differential across said porous substrate; and

forming a lower pressure in the solution perfusion channel as compared to above the substrate.

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

m.

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

m.

8. The method of screening of claim 1, further comprising said pore is with a laser.

9. The method of screening of claim 8, further comprising selecting the porous substrate from a material comprising one of glass, PTFE or PTEP.

10. The method of screening of claim 1, further comprising:

connecting a voltage-clamp amplifier connected between said electrodes; and

configuring said voltage-clamp amplifier for patch clamping measurements.