Methods and apparatus for manipulating droplets by electrowetting-based techniques

• US 20040055891A1
• Filed: 09/24/2002
• Published: 03/25/2004
• Est. Priority Date: 09/24/2002
• Status: Active Grant

• Current Assignee: Duke University
• Original Assignee: Duke University
• Inventors: Pamula, Vamsee K., Ren, Hong, Paik, Philip Y., Pollack, Michael G., Fair, Richard B.
• Granted Patent: US 6,911,132 B2
• Time in Patent Office: Days
• US Class Current: 205/98
• CPC Class Codes: B01F 31/65 the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3031 using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
• CPC Class Codes: B01F 33/30351 using hydrophilic/hydrophobic surfaces
• CPC Class Codes: B01L 2200/06 Fluid handling related problems
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2300/161 Control and use of surface tension forces, e.g. hydrophobic, hydrophilic
• CPC Class Codes: B01L 2300/165 Specific details about hydrophobic, oleophobic surfaces
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 3/00 Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• 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 3/502784 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: B01L 3/502792 for moving individual droplets on a plate, e.g. by locally altering surface tension
• CPC Class Codes: B41J 2002/14395 Electrowetting
• CPC Class Codes: C25B 9/00 Cells or assemblies of cells; Constructional parts of cells; Assemblies of constructional parts, e.g. electrode-diaphragm assemblies; Process-related cell features
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• CPC Class Codes: G02B 26/005 : based on electrowetting

Abstract

An apparatus is provided for manipulating droplets. The apparatus is a single-sided electrode design in which all conductive elements are contained on one surface on which droplets are manipulated. An additional surface can be provided parallel with the first surface for the purpose of containing the droplets to be manipulated. Droplets are manipulated by performing electrowetting-based techniques in which electrodes contained on or embedded in the first surface are sequentially energized and de-energized in a controlled manner. The apparatus enables a number of droplet manipulation processes, including merging and mixing two droplets together, splitting a droplet into two or more droplets, sampling a continuous liquid flow by forming from the flow individually controllable droplets, and iterative binary or digital mixing of droplets to obtain a desired mixing ratio.

Claims

1. An apparatus for manipulating droplets, comprising:

(a) a substrate comprising a substrate surface;

(b) an array of electrodes disposed on the substrate surface;

(c) an array of reference elements settable to a reference potential disposed in substantially co-planar relation to the electrode array, each reference element adjacent to at least one of the electrodes;

(d) a dielectric layer disposed on the substrate surface and patterned to cover the electrodes; and

(e) an electrode selector for sequentially activating and de-activating one or more selected electrodes of the array to sequentially bias the selected electrodes to an actuation voltage, whereby a droplet disposed on the substrate surface moves along a desired path defined by the selected electrodes.

2. The apparatus according to claim 1 comprising a plate spaced from the substrate surface by a distance to define a space between the plate and the substrate surface, wherein the distance is sufficient to contain a droplet disposed in the space.

3. The apparatus according to claim 2 wherein the plate comprises a plate surface facing the substrate surface, and the plate surface is hydrophobic.

4. The apparatus according to claim 2 comprising a filler fluid disposed in the space.

5. The apparatus according to claim 1 wherein at least outer portions of the electrodes and the reference elements are respectively hydrophobized.

6. The apparatus according to claim 1 comprising a hydrophobic film disposed on the electrodes and the reference elements.

7. The apparatus according to claim 1 wherein the array of reference elements comprises a grid of elongate structures.

8. The apparatus according to claim 1 wherein the reference elements are set to a reference voltage less than the actuation voltage.

9. The apparatus according to claim 1 wherein the reference elements are set to ground potential.

10. The apparatus according to claim 1 wherein at least a portion of the dielectric layer is hydrophobic.

11. The apparatus according to claim 1 wherein the electrode selector comprises an electronic processor.

12. The apparatus according to claim 1 comprising a droplet inlet communicating with the surface.

13. The apparatus according to claim 12 comprising a droplet outlet communicating with the surface.

14. A method for actuating a droplet comprising the steps of:

(a) providing the droplet on a surface comprising an array of electrodes and a substantially co-planar array of reference elements, wherein the droplet is disposed on a first one of the electrodes, and the droplet at least partially overlaps a second one of the electrodes and an intervening one of the reference elements disposed between the first and second electrodes;

(b) activating the first and second electrodes to spread a least a portion of the droplet across the second electrode; and

(c) de-activating the first electrode to move the droplet from the first electrode to the second electrode.

15. The method according to claim 14 wherein the second electrode is adjacent to the first electrode along a first direction, the array comprises one or more additional electrodes adjacent to the first electrode along one or more additional directions, the droplet at least partially overlaps the one or more additional electrodes, and the method comprises the steps of:

(a) selecting the first direction as a desired direction along which the droplet is to move; and

(b) selecting the second electrode for activation based on the selection of the first direction.

16. The method according to claim 14 wherein the activating step comprises selectively biasing the first and second electrodes to a drive voltage, and the de-activating step comprises de-coupling the first electrode from the drive voltage.

17. A method for splitting a droplet into two or more droplets, comprising the steps of:

(a) providing a starting droplet on a surface comprising an array of electrodes and a substantially co-planar array of reference elements, wherein the electrode array comprises at least three electrodes comprising a first outer electrode, a medial electrode adjacent to the first outer electrode, and a second outer electrode adjacent to medial electrode, and the starting droplet is initially disposed on at least one of the three electrodes and at least partially overlaps at least one other of the three electrodes;

(b) activating each of the three electrodes to spread the starting droplet across the three electrodes; and

(c) de-activating the medial electrode to split the starting droplet into first and second split droplets, whereby the first split droplet is disposed on the first outer electrode and the second split droplet is disposed on the second outer electrode.

18. The method according to claim 17 wherein the activating step comprises selectively biasing the three electrodes to a drive voltage, and the de-activating step comprises de-coupling the medial electrode from the drive voltage.

19. The method according to claim 17 comprising the step of using an electrode selector to control the activating and de-activating steps.

20. The method according to claim 19 wherein the electrode selector comprises an electronic processor.

21. A method for merging two or more droplets into one droplet, comprising the steps of:

(a) providing first and second droplets on a surface comprising an array of electrodes and a substantially co-planar array of reference elements, wherein the electrode array comprises at least three electrodes comprising a first outer electrode, a medial electrode adjacent to the first outer electrode, and a second outer electrode adjacent to the medial electrode, the first droplet is disposed on the first outer electrode and at least partially overlaps the medial electrode, and the second droplet is disposed on the second outer electrode and at least partially overlaps the medial electrode;

(b) selecting one of the three electrodes as a destination electrode;

(c) selecting two or more of the three electrodes for sequential activation and de-activation based on the selection of the destination electrode; and

(d) sequentially activating and de-activating the electrodes selected for sequencing to move one of the first and second droplets toward the other droplet or both of the first and second droplets toward each other, whereby the first and second droplets merge together to form a combined droplet on the destination electrode.

22. The method according to claim 21 wherein the first outer electrode is selected as the destination electrode, and the sequencing step comprises activating the second outer electrode and the medial electrode to spread the second droplet across the medial electrode, de-activating the second outer electrode to move the second droplet away from the second outer electrode, activating the first outer electrode to spread the first and second droplets into each other, and de-activating the medial electrode to form the combined droplet on the first outer electrode.

23. The method according to claim 21 wherein the second outer electrode is selected as the destination electrode, and the sequencing step comprises activating the first outer electrode and the medial electrode to spread the first droplet across the medial electrode, de-activating the first outer electrode to move the first droplet away from the first outer electrode, activating the second outer electrode to spread the first and second droplets into each other, and de-activating the medial electrode to form the combined droplet on the second outer electrode.

24. The method according to claim 21 wherein the medial electrode is selected as the destination electrode, and the sequencing step comprises activating the first outer electrode, the medial electrode, and the second outer electrode to spread the first and second droplets across the medial electrode and into each other, and de-activating the first and second outer electrodes to move the first and second droplets away from the first and second outer electrodes, respectively, and form the combined droplet on the medial electrode.

25. The method according to claim 21 comprising the step of sequentially activating and de-activating other electrodes of the electrode array to move the first droplet into electrical communication with the first outer electrode prior to forming the combined droplet.

26. The method according to claim 21 wherein the step of sequentially activating and de-activating the electrodes selected for sequencing comprises sequentially biasing one or more of the selected electrodes to a drive voltage and de-coupling one of more of the selected electrodes from the drive voltage.

27. The method according to claim 21 wherein the first droplet comprises a first composition, the second droplet comprises a second composition, and the combined droplet comprises the first and second compositions, the method further comprising the step of mixing the first and second compositions together.

28. The method according to claim 27 wherein the step of forming the combined droplet mixes the first and second compositions together.

29. The method according to claim 27 wherein the mixing step comprises passively mixing the first and second compositions together by allowing diffusion to occur within the combined droplet.

30. The method according to claim 27 wherein the mixing step comprises moving the combined droplet on a two-by-two sub-array of four electrodes by sequentially activating and de-activating the four electrodes to rotate the combined droplet.

31. The method according to claim 30 wherein at least a portion of the combined droplet remains substantially stationary at or near an intersecting region of the four electrodes while the combined droplet rotates.

32. The method according to claim 27 wherein the mixing step comprises sequentially activating and de-activating a linearly arranged set of electrodes of the electrode array to oscillate the combined droplet back and forth along the linearly arranged electrode set a desired number of times and at a desired frequency.

33. The method according to claim 27 wherein the mixing step comprises selecting a set of electrodes of the electrode array as mixing electrodes, and sequentially activating and de-activating one or more of the mixing electrodes to split the combined droplet into two or more split droplets and oscillate the split droplets along one or more linear paths a desired number of times and at a desired frequency.

34. The method according to claim 33 comprising the step of merging the split electrodes to form a new combined droplet.

35. The method according to claim 34 comprising the steps of splitting the new combined droplet into two or more new split droplets and oscillating the new split droplets.

36. The method according to claim 27 wherein the mixing step comprises selecting a set of electrodes of the electrode array as transport electrodes, and sequentially activating and de-activating one or more of the transport electrodes to actuate the combined droplet along a transport path defined by the transport electrodes, whereby the first and second compositions of the combined droplet become mixed together as the combined droplet moves along the transport path.

37. The method according to claim 36 wherein the transport path comprises a repeatable loop on the electrode array, and the combined droplet is actuated along the loop a desired number of times.

38. The method according to claim 27 wherein the mixing step comprises selecting a set of electrodes of the electrode array as mixing electrodes, and sequentially activating and de-activating one or more of the mixing electrodes to split the combined droplet into two or more split droplets and move the split droplets along two or more paths.

39. An apparatus for manipulating droplets, comprising:

(a) a substrate comprising a substrate surface;

(b) an array of electrodes disposed on the substrate surface;

(c) a dielectric layer disposed on the substrate surface and covering the electrodes; and

(d) an electrode selector for dynamically creating a sequence of electrode pairs, each electrode pair comprising a selected first one of the electrodes biased to a first voltage and a selected second one of the electrodes disposed adjacent to the selected first electrode and biased to a second voltage less than the first voltage, whereby a droplet disposed on the substrate surface moves along a desired path running between the electrode pairs created by the electrode selector.

40. The apparatus according to claim 39 comprising a plate spaced from the substrate surface by a distance to define a space between the plate and the substrate surface, wherein the distance is sufficient to contain a droplet disposed in the space.

41. The apparatus according to claim 39 wherein the plate comprises a plate surface facing the substrate surface, and the plate surface is hydrophobic.

42. The apparatus according to claim 39 comprising a filler fluid disposed in the space.

43. The apparatus according to claim 39 wherein the array comprises a plurality of linearly arranged groups of electrodes and each group is offset in relation to adjacent groups.

44. The apparatus according to claim 39 wherein at least outer portions of the electrodes are hydrophobized.

45. The apparatus according to claim 39 comprising a hydrophobic film disposed on the electrodes.

46. The apparatus according to claim 39 wherein at least a portion of the dielectric layer is hydrophobic.

47. The apparatus according to claim 39 wherein the electrode selector comprises an electronic processor.

48. The apparatus according to claim 39 wherein the second voltage is a reference voltage.

49. The apparatus according to claim 39 wherein the second voltage is a ground state.

50. A method for actuating a droplet comprising the steps of:

(a) providing the droplet on a surface comprising an array of electrodes, wherein the droplet is initially disposed on a first one of the electrodes and at least partially overlaps a second one of the electrodes separated from the first electrode by a first gap;

(b) biasing the first electrode to a first voltage and the second electrode to a second voltage lower than the first voltage, whereby the droplet becomes centered on the first gap;

(c) biasing a third one of the electrodes proximate to the first and second electrodes to a third voltage higher than the second voltage to spread the droplet onto the third electrode; and

(d) removing the bias on the first electrode to move the droplet away from the first electrode, whereby the droplet becomes centered on a second gap between the second and third electrodes.

51. The method according to claim 50 wherein the second voltage is a ground state.

52. The method according to claim 50 wherein the first and third voltages are substantially equal.

53. A method for splitting a droplet into two or more droplets, comprising the steps of:

(a) providing a starting droplet on a surface comprising an array of electrodes, wherein the electrode array comprises at least three electrodes comprising a first outer electrode, a medial electrode adjacent to the first outer electrode, and a second outer electrode adjacent to medial electrode, and the starting droplet is initially disposed on at least one of the three electrodes and at least partially overlaps at least one other of the three electrodes;

(b) biasing each of the three electrodes to a first voltage to spread the initial droplet across the three electrodes; and

(c) biasing the medial electrode to a second voltage lower than the first voltage to split the initial droplet into first and second split droplets, whereby the first split droplet is formed on the first outer electrode and the second split droplet is formed on the second outer electrode.

54. The method according to claim 53 wherein the step of biasing the three electrodes to the first voltage comprises selectively coupling the three electrodes with a voltage source.

55. The method according to claim 53 wherein the second voltage is approximately zero.

56. A method for merging two or more droplets into one droplet, comprising the steps of:

(a) providing first and second droplets on a surface comprising an array of electrodes, wherein the electrode array comprises at least three electrodes comprising a first outer electrode, a medial electrode adjacent to the first outer electrode, and a second outer electrode adjacent to the medial electrode, the first droplet is disposed on the first outer electrode and at least partially overlaps the medial electrode, and the second droplet is disposed on the second outer electrode and at least partially overlaps the medial electrode;

(b) selecting one of the three electrodes as a destination electrode;

(c) selecting two or more of the three electrodes for sequential biasing based on the selection of the destination electrode; and

(d) sequentially biasing the electrodes selected for sequencing between a first voltage and a second voltage to move one of the first and second droplets toward the other droplet or both of the first and second droplets toward each other, whereby the first and second droplets merge together to form a combined droplet on the destination electrode.

57. The method according to claim 56 wherein the first outer electrode is selected as the destination electrode, and the sequential biasing step comprises biasing the second outer electrode and the medial electrode to the first voltage to spread the second droplet across the medial electrode, biasing the second outer electrode to the second voltage to move the second droplet away from the second outer electrode, biasing the first outer electrode to the first voltage to spread the first and second droplets into each other, and biasing the medial electrode to the second voltage to form the combined droplet on the first outer electrode.

58. The method according to claim 56 wherein the second outer electrode is selected as the destination electrode, and the sequential biasing step comprises biasing the first outer electrode and the medial electrode to the first voltage to spread the first droplet across the medial electrode, biasing the first outer electrode to the second voltage to move the first droplet away from the first outer electrode, biasing the second outer electrode to the first voltage to spread the first and second droplets into each other, and biasing the medial electrode to the second voltage to form the combined droplet on the second outer electrode.

59. The method according to claim 56 wherein the medial electrode is selected as the destination electrode, and the sequential biasing step comprises biasing the first outer electrode, the medial electrode, and the second outer electrode to the first voltage to spread the first and second droplets across the medial electrode and into each other, and biasing the first and second outer electrodes to the second voltage to move the first and second droplets away from the first and second outer electrodes, respectively, and form the combined droplet on the medial electrode.

60. The method according to claim 56 comprising the step of sequentially biasing other electrodes of the electrode array to move the first droplet into electrical communication with the first outer electrode prior to forming the combined droplet.

61. The method according to claim 56 wherein the second voltage is approximately zero.

62. The method according to claim 56 wherein the first droplet comprises a first composition, the second droplet comprises a second composition, and the combined droplet comprises the first and second compositions, the method further comprising the step of mixing the first and second compositions together.

63. The method according to claim 62 wherein the step of forming the combined droplet mixes the first and second compositions together.

64. The method according to claim 62 wherein the mixing step comprises passively mixing the first and second compositions together by allowing diffusion to occur within the combined droplet.

65. The method according to claim 62 wherein the mixing step comprises moving the combined droplet on a two-by-two sub-array of four electrodes by sequentially biasing each of the four electrodes to rotate the combined droplet.

66. The method according to claim 65 wherein at least a portion of the combined droplet remains substantially stationary at or near an intersecting region of the four electrodes while the combined droplet rotates.

67. The method according to claim 62 wherein the mixing step comprises sequentially activating and de-activating a linearly arranged set of electrodes of the electrode array to oscillate the combined droplet back and forth along the linearly arranged electrode set a desired number of times and at a desired frequency.

68. The method according to claim 62 wherein the mixing step comprises selecting a set of electrodes of the electrode array as mixing electrodes, and sequentially biasing one or more of the mixing electrodes to split the combined droplet into two or more split droplets and oscillate the split droplets along one or more linear paths a desired number of times and at a desired frequency.

69. The method according to claim 68 comprising the step of merging the split electrodes to form a new combined droplet.

70. The method according to claim 69 comprising the steps of splitting the new combined droplet into two or more new split droplets and oscillating the new split droplets.

71. The method according to claim 62 wherein the mixing step comprises selecting a set of electrodes of the electrode array as transport electrodes, and sequentially biasing one or more of the transport electrodes to actuate the combined droplet along a transport path defined by the transport electrodes, whereby the first and second compositions of the combined droplet become mixed together as the combined droplet moves along the transport path.

72. The method according to claim 71 wherein the transport path comprises a repeatable loop on the electrode array, and the combined droplet is actuated along the loop a desired number of times.

73. The method according to claim 62 wherein the mixing step comprises selecting a set of electrodes of the electrode array as mixing electrodes, and sequentially biasing one or more of the mixing electrodes to split the combined droplet into two or more split droplets and move the split droplets along two or more paths.

74. A method for sampling a continuous liquid flow, comprising the steps of:

(a) supplying a liquid flow to a surface along a first flow path, the surface comprising an array of electrodes and a substantially co-planar array of reference elements, wherein at least a portion of the liquid flow is disposed on a first one of the electrodes and at least partially overlaps a second one of the electrodes and a reference element between the first and second electrodes;

(b) activating the first electrode, the second electrode, and a third one of the electrodes adjacent to the second electrode to spread the liquid flow portion across the second and third electrodes;

(c) de-activating the second electrode to form a droplet from the liquid flow on the third electrode, whereby the droplet is distinct from and controllable independently of the liquid flow.

75. The method according to claim 74 comprising the step of moving the droplet on the surface along a second flow path.

76. The method according to claim 75 wherein the step of moving the droplet comprises sequentially activating and de-activating a set of electrodes of the electrode array.

77. The method according to claim 75 comprising the step of activating a set of electrodes of the electrode array to create a processing area, and the droplet is moved along the second flow path to the processing area.

78. The method according to claim 74 wherein the first flow path flows along the surface along an input direction, and the second and third electrodes are disposed along the input direction.

79. The method according to claim 74 wherein the first flow path flows along the surface along an input direction, and the second and third electrodes are disposed along a transport direction different from the input direction.

80. The method according to claim 74 comprising the step of combining the droplet with one or more additional droplets on the surface to form a liquid output flow stream.

81. A method for sampling a continuous liquid flow, comprising the steps of:

(a) supplying a liquid flow to a surface along a first flow path, the surface comprising an array of electrodes, wherein at least a portion of the liquid flow is disposed on a first one of the electrodes and at least partially overlaps a second one of the electrodes;

(b) biasing the first electrode, the second electrode, and a third one of the electrodes adjacent to the second electrode to a first voltage to spread the liquid flow portion across the second and third electrodes; and

(c) biasing the second electrode to a second voltage less than the first voltage to form a droplet from the liquid flow on the third electrode, whereby the droplet is distinct from and controllable independently of the liquid flow.

82. A binary mixing apparatus comprising:

(a) first mixing unit comprising a first surface area, an array of first electrodes disposed on the first surface area, and an array of first reference elements disposed in substantially co-planar relation to the first electrodes;

(b) a second mixing unit comprising a second surface area, an array of second electrodes disposed on the second surface area, an array of second reference elements disposed in substantially co-planar relation to the second electrodes, and a droplet outlet area communicating with the second surface area and with the first mixing unit; and

(c) an electrode selector for sequentially activating and de-activating one or more selected first electrodes to mix together two droplets supplied to the first surface area, and for sequentially activating and de-activating one or more selected second electrodes to mix together two other droplets supplied to the second surface area.

83. The apparatus according to claim 82 comprising a buffer unit communicating with the first mixing unit and the droplet outlet area and controlled by the electrode selector.

84. A binary mixing apparatus comprising:

(a) first mixing unit comprising a first surface area and an array of first electrodes disposed on the first surface area;

(b) a second mixing unit comprising a second surface area, an array of second electrodes disposed on the second surface area, and a droplet outlet area communicating with the second surface area and with the first mixing unit; and

(c) an electrode selector for dynamically creating a sequence of first electrode pairs on the first surface area and a sequence of second electrode pairs on the second surface area, each first electrode pair comprising a selected first electrode biased to a first voltage and a selected first electrode biased to a second voltage less than the first voltage, each second electrode pair comprising a selected second electrode biased to a third voltage and a selected second electrode biased to a fourth voltage less than the third voltage, whereby two droplets supplied to the first surface area are actuated by the first electrode pairs to mix together and two other droplets supplied to the second surface area are actuated by the second electrode pairs to mix together.

85. A method for producing a droplet having a desired mixing ratio, comprising the steps of:

(a) providing a surface, an array of electrodes disposed on the surface, and an array of conducting elements disposed in substantially co-planar relation to the electrode array;

(b) providing a sample droplet having an initial concentration and a diluent droplet on the surface;

(c) merging the sample droplet with the diluent droplet to form a combined droplet by sequentially energizing and de-energizing a selected set of the electrodes; and

(d) mixing the combined droplet to reduce its concentration below the initial concentration of the sample droplet, whereby the reduced concentration of the combined droplet corresponds to an approximate mixing ratio.

86. The method according to claim 85 comprising the step of repeating the merging and mixing steps for a determined number of times using one or more additional diluent droplets to form one or more new combined droplets until the reduced concentration of the last combined droplet approaches the desired mixing ratio within a desired range of accuracy.

87. The method according to claim 85 comprising the steps of splitting the mixed combined droplet into two mixed droplets, merging at least one of the two mixed droplets with an additional diluent droplet to form a new combined droplet, and mixing the new combined droplet.

88. The method according to claim 85 comprising the step of, after mixing the combined droplet, determining whether the approximate mixing ratio of the combined droplet approaches the desired mixing ratio within the desired range of accuracy.

89. The method according to claim 88 wherein the step of determining comprises measuring a value representative of the reduced concentration of the combined droplet and comparing the measured value to a determined set point value representative of the desired mixing ratio.

90. The method according to claim 88 wherein, if it is determined that the approximate mixing ratio of the combined droplet has not approached the desired mixing ratio within a desired range of accuracy, merging the combined droplet with a new diluent droplet to form a new combined droplet having a concentration more closely approaching the desired mixing ratio.

91. A method for producing a droplet having a desired mixing ratio, comprising the steps of:

(a) providing an array of electrodes disposed on a surface;

(b) providing a sample droplet having an initial concentration and a diluent droplet on the surface;

(c) merging the sample droplet with the diluent droplet to form a combined droplet by dynamically creating a sequence of electrode pairs from the array, each electrode pair comprising a selected first one of the electrodes biased to a first voltage and a selected second one of the electrodes biased to a second voltage less than the first voltage, whereby one of or both the sample droplet and the diluent droplet are actuated along a path defined by the sequence of electrode pairs; and

(d) mixing the combined droplet to reduce its concentration below the initial concentration of the sample droplet, whereby the reduced concentration of the combined droplet corresponds to an approximate mixing ratio.

92. The method according to claim 91 wherein the mixing step comprises dynamically creating an additional sequence of electrode pairs from the array to actuate the combined droplet.

93. A method for producing a droplet having a desired final mixing ratio, comprising the steps of:

(a) in a first mixing unit comprising a first surface area, an array of first electrodes disposed on the first surface area, and an array of first conducting elements disposed in substantially co-planar relation to the first electrodes, mixing a first sample droplet with a first diluent droplet to form a first combined droplet having a desired first intermediate mixing ratio;

(b) in a second mixing unit comprising a second surface area, an array of second electrodes disposed on the second surface area, and an array of second conducting elements disposed in substantially co-planar relation to the second electrodes, mixing a second sample droplet with a second diluent droplet to form a second combined droplet having a desired second intermediate mixing ratio;

(c) transporting the second combined droplet to the first mixing unit; and

(d) in the first mixing unit, combining the first combined droplet with the second combined droplet to form a third combined droplet having the desired final mixing ratio.

94. A method for producing a droplet having a desired final mixing ratio, comprising the steps of:

(a) in a first mixing unit comprising an array of first electrodes disposed on a first surface area, mixing a first sample droplet with a first diluent droplet by dynamically creating a first sequence of first pairs of first electrodes, each first pair comprising a first drive electrode biased to a first voltage and a first reference electrode biased to a second voltage less than the first voltage, whereby the first sample droplet and the first diluent droplet are actuated to form a first combined droplet having a desired first intermediate mixing ratio;

(b) in a second mixing unit comprising an array of second electrodes disposed on a second surface area, mixing a second sample droplet with a second diluent droplet by dynamically creating a second sequence of second pairs of second electrodes, each second pair comprising a second drive electrode biased to a third voltage and a second reference electrode biased to a fourth voltage less than the third voltage, whereby the second sample droplet and the second diluent droplet are actuated to form a second combined droplet having a desired second intermediate mixing ratio;

(c) transporting the second combined droplet to the first mixing unit; and

(d) in the first mixing unit, combining the first combined droplet with the second combined droplet to form a third combined droplet having the desired final mixing ratio.