Real time electronic cell sensing system and applications for cell-based assays

• US 20070172939A1
• Filed: 03/15/2007
• Published: 07/26/2007
• Est. Priority Date: 12/20/2002
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: ACEA Biosciences, Inc (Agilent Technologies, Inc.)
• Inventors: Wang, Xiaobo, Xu, Xiao, Gan, Jiangbo, Abassi, Yama
• Granted Patent: US 8,026,080 B2
• Time in Patent Office: Days
• US Class Current: 435/287.100
• CPC Class Codes: C12M 25/08 electrically charged
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/4836 using multielectrode arrays
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/6854 Immunoglobulins

Abstract

The present invention includes devices, systems, and methods for assaying cells using cell-substrate impedance monitoring. In one aspect, the invention provides cell-substrate impedance monitoring devices that comprise electrode arrays on a nonconducting substrate, in which each of the arrays has an approximately uniform electrode resistance across the entire array. In another aspect, the invention provides cell-substrate monitoring systems comprising one or more cell-substrate monitoring devices comprising multiple wells each having an electrode array, an impedance analyzer, a device station that connects arrays of individual wells to the impedance analyzer, and software for controlling the device station and impedance analyzer. In another aspect, the invention provides cellular assays that use impedance monitoring to detect changes in cell behavior or state. In some preferred aspects, the assays are designed to investigate the affects of compounds on cells, such as cytotoxicity assays. In other preferred aspects, the assays are designed to investigate the compounds that effect IgE-mediated responses of cells to antigens.

Claims

1. A device for monitoring cell-substrate impedance, comprising:

a) a nonconducting substrate;

b) two or more electrode arrays fabricated on the substrate, wherein each of said two or more electrode arrays comprises two electrode structures; and

c) at least two connection pads;

wherein for each of said two or more electrode arrays, each of said two electrode structures comprises multiple electrode elements and the first of said two electrode structures of each of said at least two electrode arrays is connected to one of said at least two connection pads, and the second of said two electrode structures of each of said at least two electrode arrays is connected to another of said at least two connection pads;

further wherein the electrode resistance at a first location on any one array of said two or more electrode arrays and the electrode resistance at a second location on said any one array do not differ by more than 30%; and

further wherein said substrate has a surface suitable for cell attachment or growth;

wherein said cell attachment or growth on said substrate can result in a detectable change in impedance between or among said electrode structures within each electrode array.

2. The device of claim 1, wherein the electrode structures of each of said two or more electrode arrays have substantially the same surface area.

3. The device of claim 1, wherein at least two of said two or more electrode arrays are individually addressed.

5. The device of claim 1, wherein the electrode resistance at a first location on any one of said two or more arrays and the electrode resistance at a second location on said any one of said two or more arrays do not differ is by more than 15%.

6. The device of claim 5, wherein the electrode resistance at a first location on any one of said two or more arrays and the electrode resistance at a second location on said any one of said two or more arrays do not differ is by more than 5%.

7. The device of claim 6, wherein the electrode resistance at a first location on any one of said two or more arrays and the electrode resistance at a second location on said any one of said two or more arrays is do not differ by more than 2%.

8. The device of claim 1, wherein for each of said two or more electrode arrays, said first electrode structure is connected to a first electrode bus that is connected to one of said at least two connection pads via at least one electrical trace, and said second electrode structure is connected to a second electrode bus that is connected to another of said at least two connection pads via at least one electrical trace.

9. The device of claim 8, wherein one electrode structure of each of at least two of said two or more electrode arrays is connected to a common connection pad.

10. The device of claim 9, wherein for each of said two or more electrode arrays, one of said two electrode structures is connected to a connection pad that also connects to an electrode structure of at least one other of said two or more electrode arrays, and the other of said two electrode structures is connected to a connection pad that is not connected to any other electrode structures.

11. The device according to claim 1, wherein the substrate comprises glass, sapphire, silicon dioxide on silicon, or a polymer.

12. The device according to claim 11, wherein the substrate is configured as a flat surface.

13. The device according to claim 12, wherein the electrode elements of each of said two or more electrode arrays are of approximately equal widths.

14. The device according to claim 13, wherein said electrode elements have a width of greater than 30 microns.

15. The device according to claim 14, wherein said electrode elements have a width of between about 50 microns and about 300 microns.

16. The device according to claim 15, wherein said electrode elements have a width of about 90 microns.

17. The device according to claim 14, wherein the electrode elements of each of said two or more electrode arrays are approximately evenly spaced.

18. The device according to claim 17, wherein the gap between adjacent electrode elements is less than 50 microns.

19. The device according to claim 18, wherein the gap between adjacent electrode elements is between about 5 microns and about 30 microns.

20. The device according to claim 18, wherein the gap between adjacent electrode elements is about 20 microns.

21. The device according to claim 17, wherein said two or more electrode arrays comprise circle-on-line electrode elements.

22. The device according to claim 21, wherein for each array of said two or more electrode arrays, said first electrode bus comprises an electrode that extends around at least a portion of the perimeter of said each of said each array and said second electrode bus comprises an electrode that extends around at least a portion of the perimeter of said each array, wherein said first electrode bus and said second electrode bus do not overlap.

23. The device according to claim 22, wherein electrode elements of the same electrode structure of said two or more electrode arrays are connected to the same electrode bus.

24. The device according to claim 13, wherein each array of said two or more electrode arrays is organized in a concentric, sinusoidal, interdigitated, or castellated fashion.

25. The device according to claim 8, wherein said at least one electrical trace is covered with an insulating layer.

26. The device according to claim 22, wherein said at least one electrical trace is disposed in a second plane of the substrate.

27. The device according to claim 1, further comprising a plurality of receptacles, wherein each receptacle is disposed on the nonconductive substrate in a perpendicular orientation thereto, further wherein each receptacle forms a fluid-impermeable container and each of said two or more electrode arrays on the substrate is associated with a fluid-impermeable container.

28. The device according to claim 27, wherein each container is shaped as a tube with opposing open ends, one end of which being in fluid-tight contact with the substrate.

29. The device according to claim 28, further wherein the diameter of the container at the end in contact with the substrate is smaller than the diameter of the opposing end.

30. The device according to claim 28, wherein the diameter of one or more containers is, at the container end disposed on the substrate, between about 1 millimeter and about 20 millimeters.

31. The device according to claim 28, wherein the containers together form a multi-well bottomless microtiter plate.

32. The device according to claim 27, wherein the number of wells present is between 2 and 1,536.

33. The device according to claim 32, wherein the number of wells present is between 4 and 384.

34. The device according to claim 27, wherein the number of wells present is between 16 and 96.

35. The device according to claim 31, wherein less than all of the wells are associated with an electrode array.

36. The device of claim 27, wherein said two or more electrode arrays comprise circle-on-line elements.

37. The device of claim 36, further wherein two electrode buses are associated with each of said electrode arrays, wherein for each electrode array, multiple electrode elements of a first electrode structure connect to the first of said two electrode buses and multiple electrode elements of a second electrode structure connect to the second of said two electrode buses.

38. The device of claim 37, wherein said two electrode buses are on the perimeter of said each of said electrode arrays.

39. The device of claim 38, wherein each of said two electrode buses associated with each of said electrode arrays is connected to one of said at least two connection pads via at least one electrical trace.

40. The device of claim 39, wherein each of said electrode arrays comprises a first electrode structure comprising multiple electrode elements connected to a first electrode bus and a second electrode structure comprising multiple electrode elements connected to a second electrode bus, and said first electrode bus and said second electrode bus are connected to separate connection pads.

41. The device of claim 40, wherein a first electrode structure of each of at least two of said electrode arrays is connected to a common connection pad.

42. The device of claim 41, wherein for each of said at least two electrode arrays, a first electrode structure is connected to a connection pad that also connects to an electrode structure of at least one other of said electrode arrays, and a second of said electrode structures is connected to a connection pad that is not connected to an electrode structure of any other electrode array.

43. The device of claim 42, further comprising at least one printed circuit board (PCB) attached to the bottom surface of said substrate.

44. The device of claim 43, further comprising metal clips that contact said at least two connection pads and said at least one PCB.

45. The device of claim 44, further comprising metal connector pins that contact said at least one PCB, wherein said connector pins can be connected to an impedance analyzer.

46. The device of claim 45, comprising 16 wells.

47. The device of claim 45, comprising 96 wells.

48. The device of claim 47, comprising 95 electrode arrays, each of which is localized to the bottom of one of said 96 wells.

49. The device according to claim 1, further comprising an impedance analyzer electrically connected to said at least two connection pads.

50. The device according to claim 36, wherein said impedance analyzer can measure impedance at a frequency ranging from about 1 Hz to about 1 MHz.

4. (canceled)

51-159. -159. (canceled)