Microfabricated cell sorter

• US 7,214,298 B2
• Filed: 08/13/2001
• Issued: 05/08/2007
• Est. Priority Date: 09/23/1997
• Status: Expired due to Fees

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Quake, Stephen R., Arnold, Frances H., Fu, Anne Y., Spence, Charles F.
• Primary Examiner(s): Nguyen; Nam
• Assistant Examiner(s): BARTON, JEFFREY THOMAS
• Application Number:
  US09/928,590
• Publication Number:
  US 20020005354A1
• Time in Patent Office: 2,094 Days
• Field of Search: 435/5, 435/6, 435/283.1, 435/286.1, 435/287.3, 204451-455, 204601-605, 204/547, 204/643
• US Class Current: 204/450
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0418 electro-osmotic flow [EOF]
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/149 specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 27/26 by investigating electrochemical variables; by using electrolysis or electrophoresis

Abstract

The invention provides a microfabricated device for sorting cells based on a desired characteristic, for example, reporter-labeled cells can be sorted by the presence or level of reporter on the cells. The device includes a chip having a substrate into which is microfabricated at least one analysis unit. Each analysis unit includes a main channel, having a sample inlet channel, typically at one end, and a detection region along a portion of its length. Adjacent and downstream from the detection region, the main channel has a discrimination region or branch point leading to at least two branch channels. The analysis unit may further include additional inlet channels, detection points, branch points, and branch channels as desired. A stream containing cells is passed through the detection region, such that on average one cell occupies the detection region at a given time. The cells can be sorted into an appropriate branch channel based on the presence or amount of a detectable signal such as an optical signal, with or without stimulation, such as exposure to light in order to promote fluorescence.

Claims

1. A microfluidic system for processing a flow of biological material in a fluid, wherein said system comprises:

(i) a substrate having an analysis unit microfabricated thereon, wherein said analysis unit comprises

(a) a main channel in communication with a sample inlet,

(b) a detection region downstream of the sample inlet,

(c) a branch point discrimination region adjacent to and downstream of the detection region; and

(d) at least two branch channels originating at the branch point discrimination region and in communication with the main channel;

(ii) a detection apparatus for evaluating the biological material according to at least one characteristic as the material passes through the detection region;

(iii) a processor configured to receive a signal from the detection apparatus and in response to the signal, actuate the flow control system to reverse the flow of fluid in a branch channel; and

(iv) a flow control system, wherein said flow control system is responsive to the processor and is adapted to direct biological material into a selected branch channel and to reverse the direction of movement of biological material that has been directed into a selected branch channel.

2. A device of claim 1, wherein at least one of the main and branch channels communicates with a reservoir.

3. A device of claim 1, wherein the substrate comprises of silicon.

4. A device of claim 1, wherein the substrate comprises a silicone elastomer.

5. A device of claim 4 wherein the silicone elastomer substrate is made from an impression of an etched silicon wafer.

6. A device according to claim 4 wherein the silicon elastomer comprises polydimethylsiloxan (PDMS).

7. A device of claim 1 wherein the biological material comprises cells.

8. A device of claim 1 wherein the flow control system is electro-osmotic.

9. A device of claim 1 wherein the flow control system is electrophoretic.

10. A device of claim 1 wherein the flow control system is dielectrophoretic.

11. A device of claim 1 wherein the flow control system is pressure driven.

12. A device of claim 1 wherein the flow control system is microvalve.

13. A device of claim 1 wherein the flow control system is optical trapping.

14. A device of claim 1 wherein the flow control system is flow stoppage-based control.

15. A device according to claim 1 wherein the flow control system provides a voltage gradient between the branch channels and the junction.

16. A device according to claim 15 wherein the voltage gradient is generated by electrodes in the branch channels.

17. A device of claim 1 wherein the flow control system provides a pressure gradient between one or more channels and the junction.

18. A device of claim 17 wherein the pressure gradient is provided by capillary action at one or more channels of the substrate.

19. A device of claim 1 wherein the flow control system comprises one or more valves.

20. A device of claim 1 wherein the characteristic is optically detectable.

21. A device of claim 1 wherein the characteristic is determined by a fluorescent reporter.

22. A device of claim 1 wherein the characteristic is determined by a chemiluminescent reporter.

23. A device of claim 1 wherein the characteristic is determined by a radioactive reporter.

24. A device of claim 1 wherein the characteristic is determined by a spectroscopically detectable reporter.

25. A device according to claim 1 wherein the characteristic is size.

26. A device of claim 1 wherein the detection apparatus comprises a light scattering apparatus.

27. A device of claim 1 wherein the detection apparatus comprises an apparatus for recognizing electromagnetic radiation.

28. A device of claim 27 wherein the detection apparatus further comprises a source of electromagnetic excitation.

29. A device of claim 28 wherein the excitation source is a light source and the recognizing apparatus is a charge coupled device.

30. A device of claim 1 wherein the detection apparatus comprises at least one of photomultiplier tubes and photodiodes.

31. A device of claim 1 wherein the detection apparatus is positioned to target the biological material within the detection region.

32. A device of claim 1, wherein the width and height of at least one of the channels of the device is at least about two times as large as the diameter of the largest material to be sorted.

33. A device of claim 1, wherein at least one of the channels about 20 μ

m to 200 μ

m wide and about 20 μ

m to 200 μ

m deep.

34. A device of claim 1, wherein the biological material is a cell having a predetermined characteristic that is identified according to a reporter signal selected from a dye, fluorescent agent, chemiluminescent agent, chromophore, radio-isotope, and optically detectable protein.

35. A device of claim 34, wherein the control of flow is selected from electro-osmotic, electrophoretic, dielectrophoretic, pressure driven, microvalve, laser trapping and flow stoppage-based control.

36. A method for sorting cells according to a predetermined characteristic, which method comprises:

(a) flowing a sample of cells through the main channel of a device according to claim 1 so that on average one cell at a time is placed within the detection region;

(b) interrogating each cell for the predetermined characteristic as it passes through the detection region;

(c) directing the flow of each cell into a selected branch channel according to the results of the interrogation;

(d) directing at least one cell out of the selected branch channel so that it passes through the detection region a second time; and

(e) interrogating the cell a second time.

37. A method of claim 36 wherein the width and height of each channel is at least about two times as large as the diameter of the largest cell in the sample of cells.

38. A method of claim 36 wherein the predetermined characteristic is an optically detectable reporter in or on the cells.

39. A method of claim 38 wherein the reporter is selected from a dye, fluorescent agent, chemiluminescent agent, chromophore, radio-isotope, and optically detectable protein.

40. A method of claim 36 wherein the cells are interrogated by at least one device selected from the group of microscopes, diodes, light stimulating devices, lasers, light scattering apparatuses, electromagnetic excitation sources, electromagnetic radiation detector apparatuses, photomultiplier tubes, and processors.

41. A method of claim 36 wherein the flow is controlled by electro-osmosis, electrophoresis, dielectrophoresis, pressure gradient, microvalve, optical trapping or flow stoppage.

42. A method of claim 41 wherein the flow control is provided by a voltage gradient between the branch channels and the junction.

43. A method of claim 42 wherein the voltage gradient is generated by electrodes in the branch channels.

44. A method of claim 42 wherein the main channel comprises an electrode.

45. A method of claim 41 wherein the flow control is by a pressure gradient between one or more channels and the junction.

46. A method of claim 41 wherein the pressure gradient is provided by capillary action at one or more channels of the substrate.

47. A method of claim 36 wherein the flow control comprises one or more valves.