Bead Manipulations on a Droplet Actuator

• US 20110086377A1
• Filed: 08/25/2008
• Published: 04/14/2011
• Est. Priority Date: 08/24/2007
• Status: Active Grant

• Current Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Thwar, Prasanna, Paik, Philip, Allen, Dwayne
• Granted Patent: US 8,591,830 B2
• Time in Patent Office: Days
• US Class Current: 435/29
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0424 Dielectrophoretic forces
• CPC Class Codes: B01L 2400/0427 Electrowetting
• 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: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B01L 3/52 Containers specially adapted for storing or dispensing a reagent B01L3/02 takes precedence; containers for medical or pharmaceutical purposes A61J1/00; containers in general B65D; storing or dispensing test elements G01N33/4875; automated reagent dispensing G01N35/1002

Abstract

A droplet actuator comprising: (a) a base substrate comprising electrodes configured for conducting droplet operations on a droplet operations surface thereof; (b) a droplet comprising one or more beads situated on the droplet operations surface; (c) a barrier arranged in relation to the droplet and the electrodes such that a droplet may be transported away from the beads using one or more droplet operations mediated by one or more of the electrodes while transport of the beads is restrained by a barrier. Related methods and kits are also provided.

Claims

1. A droplet actuator comprising:

(a) a base substrate comprising electrodes configured for conducting droplet operations on a droplet operations surface thereof;

(b) a droplet comprising one or more beads situated on the droplet operations surface;

(c) a barrier arranged in relation to the droplet and the electrodes such that a droplet may be transported away from the beads using one or more droplet operations mediated by one or more of the electrodes while transport of the beads is restrained by a barrier.

2. The droplet actuator of claim 1 further comprising a top substrate separated from the droplet operations surface to form a gap for conducting droplet operations.

3. The droplet actuator of claim 2 wherein the barrier is coupled to and extends downward from the top substrate.

4. The droplet actuator of claim 3 wherein the barrier is configured to leave a gap between a bottom edge of the barrier and the droplet operations surface.

5. The droplet actuator of claim 3 wherein the barrier comprises a vertical gap through which fluid may pass during a droplet operation mediated by one or more of the electrodes.

6. The droplet actuator of claim 5 wherein the vertical gap is situated over an electrode.

7. The droplet actuator of claim 5 wherein the vertical gap extends substantially from a surface of the top substrate facing the gap and the droplet operations surface.

8. The droplet actuator of claim 3 wherein the one or more beads are completely surrounded by the barrier.

9. The droplet actuator of claim 8 wherein the barrier comprises a rectangular barrier situated on a path of electrodes configured for transporting droplets.

10. The droplet actuator of claim 8 wherein the barrier comprises a rectangular barrier situated on a path of electrodes configured for transporting droplets, wherein one side of the rectangular barrier is situated about halfway across a first electrode and another side of the rectangular barrier situated about halfway across a second electrode.

11. The droplet actuator of claim 8 wherein the barrier comprises an angular barrier traversing an electrode path and pointing in a direction which is away from a bead retaining portion of the barrier.

12. The droplet actuator of claim 8 wherein the barrier comprises an angular barrier traversing an electrode path and pointing in a direction which is towards a bead retaining portion of the barrier.

13. The droplet actuator of claim 1 wherein the one or more beads are blocked by the barrier from being transported away from the barrier in any direction.

14. The droplet actuator of claim 1 wherein the one or more beads are blocked by the barrier from being transported away from the barrier in the first direction but not blocked by the barrier from being transported away from the barrier in the second direction.

15. The droplet actuator of claim 14 wherein the barrier comprises an opening which permits beads having a size which is below a predetermined size limit to traverse the barrier while retaining beads which are above the predetermined size limit.

16. The droplet actuator of claim 15 wherein the droplet actuator comprises two or more such barriers, wherein each barrier has a different predetermined size limit.

17. The droplet actuator of claim 1 wherein the barrier comprises an opening which permits beads having a size which is below a predetermined size limit to traverse the barrier while retaining beads which are above the predetermined size limit.

18. The droplet actuator of claim 17 wherein the droplet actuator comprises two or more such barriers, wherein each barrier has a different predetermined size limit.

19. The droplet actuator of claim 1 wherein the barrier is traversed by a first elongated, gradually narrowing droplet operations electrode, comprising a thick base at a first end thereof on a bead retaining side of the barrier and gradually narrowing to a narrow apex at a second end on an opposite side of the barrier.

20. The droplet actuator of claim 1 wherein the barrier is traversed by a first elongated, gradually narrowing droplet operations electrode, comprising a thick base at a first end thereof opposite a bead retaining side of the barrier and gradually narrowing to a narrow apex at a second end on a bead retaining side of the barrier.

21. The droplet actuator of claim 19 wherein the first droplet operations electrode has a generally triangular shape comprising two sides that are similar in length and substantially longer than a third side.

22. The droplet actuator of claim 20 wherein the first droplet operations electrode has a generally triangular shape comprising two sides that are similar in length and substantially longer than a third side.

23. The droplet actuator of claim 21 wherein triangular shape comprises an elongated right triangle, equilateral triangle, or scalene triangle.

24. The droplet actuator of claim 22 wherein triangular shape comprises an elongated right triangle, equilateral triangle, or scalene triangle.

25. The droplet actuator of claim 19 further comprising a second elongated, gradually narrowing droplet operations electrode oriented alongside the first gradually narrowing droplet operations electrode such that:

(a) the base of the first gradually narrowing droplet operations electrode is adjacent to the apex of the second gradually narrowing droplet operations electrode; and

(b) the apex of the first gradually narrowing droplet operations electrode is adjacent to the base of the second gradually narrowing droplet operations electrode.

26. The droplet actuator of claim 25 comprising two sets of the first and second elongated gradually narrowing droplet operations electrodes traversing the barrier.

27. The droplet actuator of claim 1 wherein the beads comprise biological cells bound thereto.

28. The droplet actuator of claim 1 wherein the beads comprise substantially pure populations biological cells bound thereto.

29. A droplet actuator comprising:

(a) a base substrate comprising electrodes configured for conducting droplet operations on a droplet operations surface thereof;

(b) a funnel-shaped reservoir comprising a narrow opening situated in proximity to the base substrate;

wherein the base substrate and the funnel-shaped reservoir are arranged such that a portion of a sample comprising beads loaded in the funnel will flow onto the droplet operations surface, and wherein the portion of the sample comprises a substantial amount of the beads.

30. The droplet actuator of claim 29 further comprising a magnetic field source situated in a manner which attracts magnetic beads from the funnel-shaped reservoir onto the substrate surface.

31. The droplet actuator of claim 29 further comprising a top substrate arranged in a manner which is parallel to the droplet operations surface, wherein the narrow opening of the funnel shaped reservoir passes through the top substrate.

32. A droplet actuator comprising:

(a) a base substrate comprising electrodes configured for conducting droplet operations on a droplet operations surface thereof;

(b) a top substrate arranged in a generally parallel fashion relative to the droplet operations surface; and

(c) beads trapped in a barrier on the droplet actuator, wherein the barrier permits droplets to be transported in to and out of the barrier using droplet operations mediated by one or more of the electrodes, while retaining one or more of the beads within the barrier.

33. The droplet actuator of claim 32 wherein the barrier retains substantially all of the beads within the barrier.

34. The droplet actuator of claim 32 wherein two or more of the electrodes are arranged for conducting droplet operations within the barrier.

35. The droplet actuator of claim 32 further comprising an array of barriers, each barrier retaining beads comprising a specific bead type, the array comprising a multiplicity of bead types.

36. The droplet actuator of claim 32 wherein the beads comprise biological cells bound thereto.

37. The droplet actuator of claim 32 wherein the beads comprise a substantially pure population of biological cells bound thereto.

38. A method of reducing a volume of fluid surrounding a bead, the method comprising transporting a portion of the volume of fluid past a barrier on a droplet actuator, where in the barrier restrains transport of the bead.

39. The method of claim 38 wherein the beads comprise biological cells bound thereto.

40. The method of claim 39 wherein the volume of fluid comprises culture medium selected for growing the biological cells.

41. The method of claim 38 further comprising using the portion of the volume of fluid for conducting one or more droplet operations in an assay protocol.

42. The method of claim 38 wherein the transporting is electrode mediated.

43. The method of claim 38 wherein the transporting is electrowetting mediated.

44. The method of claim 38 wherein the transporting is dielectrophoresis mediated.

45. A method of providing a nutrient to a biological cell, the method comprising:

(a) reducing a volume of fluid according to the method of claim 40; and

(b) conducting one or more droplet operations to bring into contact with the beads a fluid comprising the nutrient.

46. The method claim 38 wherein the beads comprise a substantially pure population of biological cells bound thereto.

47. The method claim 38 wherein the beads comprise interacting populations of cells.

48. A method of separating a volume of fluid from one or more beads, the method comprising transporting the volume of fluid past a barrier on a droplet actuator, wherein the barrier restrains transport of one or more of the one or more beads.

49. A method of transporting a droplet substantially free of beads away from a droplet containing beads, the method comprising:

(a) providing a droplet actuator comprising;

(i) a base substrate comprising electrodes configured for conducting droplet operations on a droplet operations surface thereof;

(ii) a droplet comprising one or more beads situated on the droplet operations surface;

(iii) a barrier arranged in relation to the droplet and the electrodes such that a droplet may be transported away from the beads using one or more droplet operations mediated by one or more of the electrodes while transport of the beads is restrained by a barrier; and

(b) transporting the droplet containing beads across the barrier, wherein the barrier retains the beads and a droplet substantially free of beads is formed on an opposite side of the barrier.

50. The method of claim 49 wherein the droplet actuator further comprises a top substrate separated from the droplet operations surface to form a gap for conducting droplet operations.

51. The method of claim 49 wherein the barrier is coupled to and extends downward from the top substrate.

52. The method of claim 49 wherein the barrier is traversed by a first elongated, gradually narrowing droplet operations electrode, comprising a thick base at a first end thereof opposite a bead retaining side of the barrier and gradually narrowing to a narrow apex at a second end on a bead retaining side of the barrier, and step 49(b) comprises activating the electrode to cause a droplet to traverse the barrier.

53. The method of claim 49 wherein the beads comprise a population of cells.

54. A method of washing beads on a droplet actuator, the method comprising:

(a) providing a droplet actuator comprising;

(i) a base substrate comprising electrodes configured for conducting droplet operations on a droplet operations surface thereof;

(ii) a droplet comprising one or more beads situated on the droplet operations surface;

(iii) a barrier arranged in relation to the droplet and the electrodes such that a droplet may be transported away from the beads using one or more droplet operations mediated by one or more of the electrodes while transport of the beads is restrained by a barrier; and

(b) transporting the droplet containing beads across the barrier, wherein the barrier retains the beads and a droplet substantially free of beads is formed on an opposite side of the barrier;

(c) transporting a wash droplet into contact with the beads;

(d) repeating steps (b) and (c) until washing of the beads is complete.

55. The method of claim 54 wherein step 54(c) comprises transporting the droplet across the barrier into contact with the beads.

56. A method of sorting beads on a droplet actuator, the method comprising:

(a) providing a droplet actuator comprising;

(i) a base substrate comprising electrodes configured for conducting droplet operations on a droplet operations surface thereof;

(ii) a first barrier arranged to permit beads having a size which is below a first predetermined size to traverse the barrier while retaining beads which are above the first predetermined size;

(b) transporting a droplet comprising beads having at least three sizes through the first barrier to provide a retained droplet comprising beads above the first predetermined size and a transmitted droplet comprising beads above the first predetermined size.

57. The method of claim 56 wherein:

(a) the droplet actuator further comprises a second barrier arranged to permit beads having a size which is below a second predetermined size to traverse the barrier while retaining beads which are above the second predetermined size;

(b) the method further comprises transporting a droplet comprising beads having at least three sizes through the first barrier to provide a retained droplet comprising beads above the first predetermined size and a transmitted droplet comprising beads above the first predetermined size;

(c) transporting the retained droplet of (b) through the second barrier to provide a retained droplet comprising beads above the first and second predetermined sizes and a transmitted droplet comprising beads above the first predetermined size and below the second predetermined size.

58. The method of claim 56 wherein the beads comprise biological cells adhered thereto.

59. A method of making a droplet actuator, the method comprising situating beads in a barrier on a droplet actuator between a top substrate and a droplet operations surface, wherein the barrier blocks transport of the beads outside of the barrier on all sides and permits fluid to be transported via droplet operations into and/or out of the barrier.

60. A kit comprising a droplet actuator comprising:

(a) beads situated within a barrier between the top substrate and a droplet operations surface of the droplet actuator; and

(b) a further component selected from the group consisting of;

(i) a filler fluid for use on the droplet actuator;

(ii) a reagent for use on the droplet actuator;

(iii) a device for use in loading a fluid onto the droplet actuator.