Droplet Actuator Devices and Methods Employing Magnetic Beads

• US 20100068764A1
• Filed: 02/11/2008
• Published: 03/18/2010
• Est. Priority Date: 02/09/2007
• Status: Active Grant

• Current Assignee: Duke University, Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Original Assignee: Advanced Liquid Logic, Inc. (Illumina Incorporated)
• Inventors: Sista, Ramakrishna, Pamula, Vamsee K., Eckhardt, Allen E., Srinivasan, Vijay, Pollack, Michael G.
• Granted Patent: US 9,046,514 B2
• Time in Patent Office: Days
• US Class Current: 435/79
• CPC Class Codes: B01L 2200/025 Align devices or objects to ensure defined positions relative to each other
• CPC Class Codes: B01L 2200/028 Modular arrangements
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/141 Preventing contamination, tampering
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/043 magnetic forces
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• 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 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: C12M 25/01 Drops
• CPC Class Codes: C12M 25/16 Particles; Beads; Granular material; Encapsulation chemical or physical processes conducted in the presence of fluids and solid particles B01J8/00
• CPC Class Codes: G01N 2035/1046 Levitated, suspended drops
• CPC Class Codes: G01N 27/44717 Arrangements for investigating the separated zones, e.g. localising zones
• CPC Class Codes: G01N 27/44786 of the magneto-electrophoresis type
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/54386 : Analytical elements
• CPC Class Codes: G01N 35/0098 : involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C

Abstract

A method of providing a droplet in contact with a magnetically responsive bead and having a reduced quantity of a substance. The method generally includes the steps of (a) providing a droplet actuator (200) comprising: (i) a substrate (210) comprising electrodes (214) arranged for conducting droplet operations on a surface; (ii) a starting droplet (218/222) comprising: (1) one or more magnetically responsive beads (220); (2) a starting quantity of the substance; and (3) a starting volume; (b) magnetically immobilizing the one or more magnetically responsive beads at a location which is at a distance from a target droplet splitting zone, (224); (c) conducting one or more droplet operations comprising droplet dividing operations selected to divide the combined droplet to yield a set of droplets comprising: (i) a droplet (218) comprising substantially all of the one or more magnetically responsive beads and having a decreased quantity of the substance relative to the starting concentration; and (ii) a droplet (222) substantially lacking the magnetically responsive beads.

Claims

1. A method of providing a droplet in contact with a magnetically responsive bead and having a reduced quantity of a substance, the method comprising:

(a) providing a droplet actuator comprising;

(i) a substrate comprising electrodes arranged for conducting droplet operations on a surface; and

(ii) a starting droplet comprising;

(1) one or more magnetically responsive beads;

(2) a starting quantity of the substance; and

(3) a starting volume;

(b) magnetically immobilizing the one or more magnetically responsive beads at a location which is at a distance from a target droplet splitting zone; and

(c) conducting one or more droplet operations comprising droplet dividing operations selected to divide the combined droplet to yield a set of droplets comprising;

(i) a droplet comprising substantially all of the one or more magnetically responsive beads and having a decreased quantity of the substance relative to the starting concentration; and

(ii) a droplet substantially lacking the magnetically responsive beads.

2. The method of claim 1 wherein the starting droplet comprises a starting concentration of the substance, and the one or more droplet operations yield a droplet comprising a reduced concentration and reduced quantity of the substance relative to the starting concentration and starting quantity.

3. The method of claim 1 further comprising conducting one or more droplet operations to merge a wash droplet with the droplet provided in step 1(a) to yield a combined droplet.

4. The method of claim 1 wherein the one or more magnetically responsive beads comprises one or more beads having a diameter ranging from about 0.1 nm to about 25 mm.

5. The method of claim 1 further comprising releasing or resuspending one or more of the beads following step 1(c).

6. The method of claim 5 further comprising providing a hydrophilic polymer in the droplet that improves resuspension relative to a corresponding droplet lacking the hydrophilic polymer.

7. The method of claim 5 further comprising providing a surfactant in the droplet that improves resuspension relative to a corresponding droplet lacking the hydrophilic polymer.

8. The method of claim 5 further comprising providing a surfactant in the droplet that:

(a) reduces bead clumping in the droplet in a non-gaseous filler fluid; and

(b) does not serve to reduce molecular adsorption of droplet components to a surface of the droplet actuator.

9. The method of claim 5 further comprising agitating one or more of the beads following step 1(c) using a sonicator.

10. The method of claim 1 wherein:

(a) step 1(b) comprises situating a droplet comprising the beads in proximity with a means for effecting a magnetic field in proximity to one or more of the electrodes; and

(b) the strength and location of the magnetic field are selected to immobilize magnetically responsive beads in the droplet at a distance from a target splitting zone which is sufficient to retain substantially all of the magnetic beads in one droplet.

11. The method of claim 1 wherein:

(a) step 1(b) comprises situating a droplet comprising the beads in proximity with a means for effecting a magnetic field in proximity to one or more of the electrodes; and

(b) the location of the source of the magnetic field is selected to centrally locate the magnetic beads along a lateral dimension of the droplet.

12. The method of claim 1 wherein:

(a) step 1(b) comprises situating a droplet comprising the beads in proximity with a means for effecting a magnetic field in proximity to one or more of the electrodes; and

(b) the location of the source of the magnetic field is selected to centrally locate the magnetic beads along a vertical dimension of the droplet.

13. The method of claim 1 wherein:

(a) step 1(b) comprises situating a droplet comprising the beads in proximity with a means for effecting a magnetic field in proximity to one or more of the electrodes; and

(b) the location of the source of the magnetic field is selected to centrally locate the magnetic beads along;

(i) a vertical dimension of the droplet; and

(ii) a lateral dimension of the droplet.

14. The method of claim 1 wherein step 1(b) comprises magnetically immobilizing the one or more magnetically responsive beads using a means comprising:

(a) an electromagnet; and

(b) a means for controlling power supply to the electromagnet.

15. The method of claim 1 wherein step 1(b) comprises magnetically immobilizing the one or more magnetically responsive beads using a means comprising:

(a) means for effecting a magnetic field comprises a magnet; and

(b) a means for moving the magnet into and out of proximity with the one or more of the electrodes.

16. The method of claim 1 wherein the droplet of 1(c)(i) comprises more than 99% of beads present in the combined droplet.

17. The method of claim 1 wherein the droplet of 1(c)(i) comprises more than 99.9% of beads present in the combined droplet.

18. The method of claim 1 wherein the droplet of 1(c)(i) comprises more than 99.999% of beads present in the combined droplet.

19. The method of claim 1 wherein step 1(c) is electrode-mediated.

20. The method of claim 1 wherein step 1(c) is electrowetting-mediated.

21. The method of claim 1 wherein step 1(c) is dielectrophoresis-mediated.

22. The method of claim 1 wherein step 1(c) is electric field-mediated.

23. The method of claim 1 wherein the substance comprises one or more contaminants.

24. The method of claim 1 wherein the substance comprises one or more analytes.

25. The method of claim 1 wherein the magnetically responsive bead comprises a first component having specific or non-specific affinity for a second component provided in the droplet of 1(a).

26. The method of claim 1 wherein the beads comprise one or more populations of pre-selected biological cells.

27. The method of claim 25 wherein the affinity comprises an affinity for non-specific adsorption.

28. The method of claim 25 wherein the affinity comprises an affinity for specific adsorption.

29. The method of claim 25 wherein the first component comprises an antigen binding region.

30. The method of claim 25 wherein the first component comprises an antibody binding region.

31. The method of claim 25 wherein the first component comprises an antibody.

32. The method of claim 25 wherein the first component comprises two or more antibodies, each having affinity for a different second component.

33. The method of claim 25 wherein the first component comprises an analyte.

34. The method of claim 25 wherein the first component comprises two or more analytes, each having affinity for a different second component.

35. The method of claim 25 wherein the first component comprises an antigen.

36. The method of claim 25 wherein the first component comprises a nucleic acid.

37. The method of claim 25 wherein the first component comprises a biotin compound or streptavidin compound.

38. The method of claim 25 wherein the second component comprises an antigen binding region.

39. The method of claim 25 wherein the second component comprises an antibody.

40. The method of claim 25 wherein the second component comprises two or more antibodies, each having affinity for a different type of second component.

41. The method of claim 25 wherein the second component comprises an analyte.

42. The method of claim 25 wherein the second component comprises two or more analytes, each having affinity for a different type of second component.

43. The method of claim 25 wherein the second component comprises an antigen.

44. The method of claim 25 wherein the second component comprises a nucleic acid.

45. The method of claim 25 wherein the second component comprises amplified nucleic acid.

46. The method of claim 25 wherein the second component comprises a biotin compound or streptavidin compound.

47. The method of claim 25 wherein the second component comprises a contaminant

48. The method of claim 25 wherein:

(a) the first component comprises a compound which binds to the second component; and

(b) the second component comprises a compound that binds to the first component.

49. The method of claim 1 wherein the beads comprise biological cells bound thereto.

50. The method of claim 1 wherein the beads comprise one or more populations of pre-selected biological cells.

51. The method of claim 49 wherein the droplet comprises culture medium selected for growing the biological cells.

52. The method of claim 51 further comprising using the droplet substantially lacking the magnetically responsive beads for conducting one or more droplet operations in an assay protocol.

53. The method of claim 51 wherein the beads comprise a substantially pure population of biological cells bound thereto.

54. The method of claim 51 wherein the beads comprise interacting populations of cells.

55. The method of claim 1 wherein the droplet substantially lacking the magnetically responsive beads comprises one or more beads that are not substantially magnetically responsive.

56. The method of claim 55 wherein the one or more beads that are not substantially magnetically responsive are bound to one or more second target substances.

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

(a) providing a droplet in contact with a magnetically responsive bead according to the method of claim 1, where the beads comprise one or more populations of biological cells; and

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

58. A method of splitting a first droplet comprising beads, the method comprising:

(a) positioning one or more magnets in proximity to the first droplet sufficient to result in a centralized localization of the beads within the droplet; and

(b) splitting the droplet to yield one or more droplets comprising beads and one or more droplets substantially lacking beads.

59. A method of splitting a first droplet comprising beads, the method comprising:

(a) providing a droplet comprising;

(i) beads; and

(ii) an additive selected and provided in an amount sufficient to enhance bead immobilization in the presence of a magnetic field and/or resuspension following removal of a magnetic field relative to a corresponding control droplet lacking such additive; and

(b) splitting the droplet to yield one or more droplets comprising beads and one or more droplets substantially lacking beads.

60. A method of splitting a first droplet comprising beads, the method comprising:

(a) providing a magnetic field in proximity to the first droplet sufficient to substantially immobilize the beads within the droplet; and

(b) splitting the droplet at a position sufficiently distant from the immobilized beads to yield one or more droplets comprising beads and one or more droplets substantially lacking beads.

61. A droplet actuator device comprising:

(a) an array of electrodes associated with a substrate and configured for conducting one or more droplet operations;

(b) a magnet arranged to exert a magnetic field on a droplet positioned on a first set of one or more of the electrodes; and

(c) a magnetic shield arranged to shield a droplet positioned on a second set of one or more of the electrodes from the magnetic field.

62. The droplet actuator device of claim 61 further comprising:

(a) a second magnet arranged to exert a magnetic field on a droplet positioned on a second set of one or more of the electrodes; and

(b) a magnetic shield, which may be the same or different from the magnetic shield of 61(c), arranged to shield a droplet positioned on the first set of one or more of the electrodes from the magnetic field.

63. The droplet actuator device of claim 62 comprising a series of magnet sets and magnetic shield sets, wherein:

(a) each magnet set comprises one or more magnets;

(b) each magnetic shield set comprises one or more magnetic shields; and

(c) the magnet sets and magnetic shield sets are arranged to permit independent droplet operations on a series of droplet sets, wherein;

(i) each droplet set comprises one or more droplets; and

(ii) each droplet set is influenced by a single magnet set and is blocked by one or more magnetic shield sets from influence by other magnet sets.

64. The droplet actuator device of claim 61 comprising a droplet:

(a) comprising one or more magnetically responsive beads thereon; and

(b) positioned on one or more electrodes from the first set, so that the magnetically responsive beads are attracted by the magnetic field.

65. The droplet actuator device of claim 61 comprising a droplet:

(a) comprising one or more magnetically responsive beads thereon; and

(b) positioned on one or more electrodes from the second set, so that the magnetically responsive beads are not attracted by the magnetic field.

66. The droplet actuator device of claim 61 wherein the magnetic shield comprises a material having high magnetic permeability.

67. The droplet actuator device of claim 61 wherein the magnetic shield comprises a material selected from the group consisting of Mu metals, permalloys, iron, steel, nickel, and alloys substantially comprising any of the foregoing.

68. A method of conducting a droplet operation on a droplet actuator device using a droplet comprising magnetic beads, the method comprising:

(a) providing droplet actuator device comprising;

(i) an array of electrodes associated with a substrate and configured for conducting one or more droplet operations;

(ii) a magnet arranged to exert a magnetic field on a droplet positioned on a first set of one or more of the electrodes; and

(iii) a magnetic shield arranged to shield a droplet positioned on a second set of one or more of the electrodes from the magnetic field;

(b) positioning a first droplet comprising one or more magnetically responsive beads on one or more electrodes from the first set, so that the magnetically responsive beads are attracted by the magnetic field;

(c) positioning a second droplet comprising one or more magnetically responsive beads on one or more electrodes from the second set, so that the magnetically responsive beads are not attracted by the magnetic field; and

(d) conducting a droplet operation on the first droplet and/or conducting a droplet operation on the second droplet.

69. A droplet actuator device comprising magnet sets and electrode sets, wherein:

(a) each electrode set comprises one or more electrodes associated with a substrate;

(b) two or more electrode sets are;

(i) arranged to form an array of electrodes; and

(ii) configured for conducting one or more droplet operations;

(c) each magnet set comprises one or more magnets;

(d) each magnet set is arranged in a relationship with an electrode set such that magnetic field(s) exerted by the magnet set restricts mobility of magnetically responsive particles to a locus which is proximate to the electrode set; and

(e) each magnet set is arranged in relationship to its adjacent magnet set(s), which relationship a polar opposite to the polar orientation of the adjacent magnet set.

70. The droplet actuator device of claim 69 wherein each magnet set is oriented with a pole facing an electrode set, and each adjacent magnet set is oriented with an opposite pole facing the electrode set.

71. The droplet actuator device of claim 69 wherein each magnet has a sufficiently strong field to immobilize magnetically responsive particles during a splitting operation, but not sufficiently strong to immobilize magnetically responsive particles during a droplet transport operation.

72. A droplet actuator device comprising:

(a) electrodes arranged in association with a substrate to conduct droplet operations on the substrate, the droplet operations comprising droplet splitting operations and droplet transport operations;

(b) a magnet associated with a subset or electrodes comprising one or more of the electrodes; and

(c) a droplet on one or more electrodes of the subset of electrodes, the droplet comprising one or more magnetically responsive particles or substances;

wherein the magnet is oriented to exert a magnetic field in proximity to the droplet, the magnetic field exerting sufficient force on the magnetically responsive particles or substances to immobilize the magnetically responsive particles or substances during a droplet splitting operation, but not sufficiently strong to immobilize magnetically responsive particles during a droplet transport operation.

73. A method of reducing carryover in a droplet actuator, the method comprising:

(a) transporting a droplet comprising a target substance past a locus;

(b) transporting a wash droplet not comprising the target substance past the locus; and

(c) repeating steps (a) and (b), wherein the target substance may be the same or different.

74. The method of claim 73 wherein the locus comprises a locus in proximity to a sensor arranged to sense a property of the droplet.

75. The method of claim 73 wherein the wash droplet comprises a buffer droplet.

76. A method of eliminating a satellite droplet, the method comprising:

(a) conducting a droplet splitting operation at a locus on a droplet in a filler fluid on a droplet actuator, the splitting operation yielding two or more sub-droplets and one or more satellite droplets;

(b) conducting a droplet transport operation on one or more of the sub-droplets yielded in step (a) to transport the one or more sub-droplets away from the locus; and

(c) transporting a wash droplet into proximity with the locus to merge the one or more of the satellite droplets with the wash droplet.

77. The method of claim 76 wherein the wash droplet comprises a buffer droplet.

78. A method of exposing a droplet to a sensor, the method comprising:

(a) providing droplet on a substrate in sensing proximity to a sensor, the droplet having a starting surface area exposed to the sensor; and

(b) effecting a change in the droplet'"'"'s shape and producing an expanded surface area of the droplet in sensing proximity to the sensor, wherein the expanded surface area exposed to the sensor is greater than the starting surface area exposed to the sensor.

79. The method of claim 78 wherein step 78(b) is effected by steps comprising activating one or more electrodes associated with the substrate.

80. The method of claim 78 wherein step 78(b) is effected by an effect comprising an electrowetting mediated effect.

81. The method of claim 78 where the sensor comprises a photon sensor, and the droplet comprises a substance that emits photons.

82. The method of claim 78 where the change in the droplet'"'"'s shape produces a slug.

83. The method of claim 78 where the change in the droplet'"'"'s shape produces a series of droplets.

84. A method of exposing a surface area of a droplet to a sensor, the method comprising exposing to a sensor a droplet on a substrate, the droplet having an expanded surface area effected at least in part by activation of one or more electrodes associated with the substrate to effect a change in the droplet'"'"'s shape and producing the expanded surface area.

85-114. -114. (canceled)