High-throughput electrophysiological measurement system

• US 20030070923A1
• Filed: 09/05/2002
• Published: 04/17/2003
• Est. Priority Date: 08/04/2000
• Status: Active Grant

• Current Assignee: Essen Instruments, Inc. (Sartorius AG)
• Original Assignee: Essen Instruments, Inc. (Sartorius AG)
• Inventors: Neagle, Bradley D., Schroeder, Kirk S.
• Granted Patent: US 7,270,730 B2
• Time in Patent Office: Days
• US Class Current: 204/400
• CPC Class Codes: G01N 2035/00237 Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/1034 Transferring microquantities of liquid
• 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 35/10 Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices G01N35/0099 takes precedence

Abstract

Systems, including apparatus and methods, for performing electrophysiological measurements on membranous samples, including living cells, isolated cell fragments (such as organelles), and/or artificial membranes (such as vesicles). The apparatus may include a high-throughput electrophysiological measurement system, and components thereof. This measurement system may include, among others, (1) a fluidics head for transferring samples and/or other compounds to a perforated measurement substrate, (2) a pressure-regulated plenum system for positioning samples on the substrate and subsequently forming a high-resistance electrical seal, (3) an activation system (such as a computer-controlled pulsed UV illumination module) for activating caged compounds, (4) an electronics head for applying and/or measuring voltage and/or current, and/or (5) a computer-controlled analysis system for collecting and/or analyzing data. The methods may include methods for performing high-throughput electrophysiological measurements on transporters and/or voltage or ligand-gated ion channels, sequentially and/or simultaneously.

Claims

1. Electrophysiological measurement apparatus, comprising:

a first fluid chamber configured to support a cell or membrane to be measured;

a second fluid chamber;

a thin substrate separating the two chambers, the substrate having an aperture formed therethrough, wherein the diameter of the aperture is less than the diameter of the cell or membrane, thereby enabling a high-resistance seal to be formed between the cell or membrane and the substrate;

an electrode disposed in each of the fluid chambers; and

electrophysiological measurement circuitry in electrical communication with the electrodes.

2. The electrophysiological measurement apparatus of claim 1, further comprising a single aperture formed in the substrate separating the first and second chambers.

3. The electrophysiological measurement apparatus of claim 1, wherein the substrate is a plastic substrate having a glass coating at least in the region where the high-resistance seal is formed between the cell or membrane and the substrate.

4. The electrophysiological measurement apparatus of claim 3, wherein the substrate is selected from the group consisting of polyethylene terephthalate (PET) and polyimide.

5. The electrophysiological measurement apparatus of claim 1, wherein the diameter of the aperture is in the range of about 1 to 10 micrometers.

6. The electrophysiological measurement apparatus of claim 1, wherein the aperture is tapered.

7. The electrophysiological measurement apparatus of claim 1, wherein a differential pressure is applied between the first and second chambers, causing the cell or membrane to migrate to the aperture.

8. The electrophysiological measurement apparatus of claim 1, wherein a differential pressure is maintained between the first and second chambers until the high-resistance seal is formed between the cell or membrane and the substrate.

9. The electrophysiological measurement apparatus of claim 1, wherein the second fluid chamber includes a chemical reagent that electrically permeabilizes the biological membrane in the vicinity of the aperture.

10. The electrophysiological measurement apparatus of claim 1, wherein a high voltage is temporarily applied across the electrodes to permeabilize the biological membrane in the vicinity of the aperture.

11. The electrophysiological measurement apparatus of claim 1, further comprising:

a plurality of first chambers forming a multi-well plate; and

a substrate having a plurality of apertures, wherein the substrate is bonded to the multi-well plate such that the apertures are in alignment with the chambers thereof.

12. The electrophysiological measurement apparatus of claim 11, further comprising a mechanism for moving the electrode into the chambers of the multi-well plate so as to automate the measurement of the cells or membranes contained therein.

13. The electrophysiological measurement apparatus of claim 11, further comprising:

a plurality of electrodes in alignment with a plurality of the chambers of the multi-well plate; and

a mechanism for moving the electrodes into the chambers of the multi-well plate to perform simultaneous measurements on the cells or membranes contained therein.

14. The electrophysiological measurement apparatus of claim 11, further comprising a system for transferring fluids from one or more sources to the chambers of the multi-well plate.

15. Electrophysiological measurement apparatus, comprising:

a multi-well plate having a plurality of fluid-carrying chambers;

a thin substrate having an array of apertures in alignment with the chambers of the multi-well plate;

wherein the substrate is bonded to the multi-well plate such that the chambers are open at the top and sealed at the bottom except for the apertures; and

wherein the diameter of the apertures is in the range of about 1 to 10 micrometers.

16. The electrophysiological measurement apparatus of claim 15, wherein the substrate is plastic.

17. The electrophysiological measurement apparatus of claim 15, wherein the plastic is selected from the group consisting of polyethylene terephthalate (PET) and polyimide.

18. The electrophysiological measurement apparatus of claim 15, wherein a laser is used to photomachine the apertures.

19. The electrophysiological measurement apparatus of claim 15, wherein the plastic is glass coated on the side that is bonded to the multi-well plate.

20. The electrophysiological measurement apparatus of claim 15, wherein the diameter of the apertures is in the range of about 1 to 3 micrometers.

21. The electrophysiological measurement apparatus of claim 15, wherein the diameter of the apertures is less than the diameter of the cells or membranes used in performing an electrophysiological measurement with the apparatus.

22. The electrophysiological measurement apparatus of claim 15, wherein the apertures are tapered.

23. The electrophysiological measurement apparatus of claim 15, wherein the substrate is bonded to the multi-well plate using a non-toxic adhesive.

24. The electrophysiological measurement apparatus of claim 15, wherein the substrate is silicon.

25. The electrophysiological measurement apparatus of claim 24, wherein the apertures are formed using photolithographic techniques.