Droplet actuator devices, systems, and methods
The invention relates to certain novel approaches to reducing or eliminating the movement of contaminants from one droplet to another on a droplet actuator via liquid filler fluid. In one application, droplet actuators are used to conduct genetic analysis using polymerase chain reaction (PCR) techniques. The invention addresses the need for improved methods of performing PCR on a droplet actuator that provide for optimum amplification and detection of a sample target.
1. A method of detecting a target nucleic acid in a sample, the method comprising:
(a) providing a set of nucleic acid amplification reaction droplets, each droplet comprising a portion of the sample;
(b) treating two or more subsets of the amplification reaction droplets under conditions for amplifying the target nucleic acid to yield corresponding subsets of amplified droplets comprising amplified nucleic acid, wherein each subset of the amplification reaction droplets:
(i) comprises one or more of the amplification reaction droplets;
(ii) is treated under different conditions for amplifying the target nucleic acid;
(c) preparing the amplified droplets for detection;
(d) detecting a signal from the amplified droplets;
(e) based on the signal, determining the amount of the amplified nucleic acid present in the amplified droplets and/or the sample.
2. The method of claim 1 wherein providing a set of nucleic acid amplification reaction droplets comprises dispensing the set of nucleic acid amplification droplets from a sample droplet.
3. The method of claim 2 wherein the sample droplet is provided in a droplet operations gap of a droplet actuator, and the dispensing is electrode mediated.
4. The method of claim 2 wherein:
(a) the sample droplet is provided in a reservoir of a droplet actuator;
(b) the droplet actuator comprises a liquid path from the reservoir into the droplet operations gap; and
(c) dispensing the set of nucleic acid amplification droplets comprises:
(i) flowing the sample droplet through the liquid path into the droplet operations gap; and
(ii) using electrodes to dispense the amplification reaction droplets in the droplet operations gap.
5. The method of claim 1 wherein providing a set of nucleic acid amplification reaction droplets comprises:
(a) providing a sample droplet;
(b) dividing the sample droplet into multiple sample sub-droplets; and
(c) combining each of the sample sub-droplets with one or more droplets comprising amplification reagents to yield the amplification reaction droplets.
6. The method of claim 5 wherein steps 5(b) and/or 5(c) is/are effected in a droplet operations gap of a droplet actuator using droplet operations mediated by electrodes.
7. The method of claim 5 wherein the sample droplet, the multiple sub-droplets and the one or more droplets comprising amplification reagents, and the amplification reaction droplets are:
(a) arranged in the droplet operations gap; and
(b) at least partially surrounded by a liquid filler fluid.
8. The method of claim 1 wherein providing a set of nucleic acid amplification reaction droplets comprises:
(a) providing a sample droplet;
(b) combining the sample droplet with one or more droplets comprising amplification reagents to yield an amplification-ready droplet; and
(c) dividing the amplification-ready droplet to yield the amplification reaction droplets.
9. The method of claim 8 wherein the sample droplet, the one or more droplets comprising amplification reagents, the parent amplification reaction droplet, and the amplification reaction droplets are:
(a) arranged in a droplet operations gap of a droplet actuator; and
(b) at least partially surrounded by a liquid filler fluid.
10. The method claim 8 wherein step 8(b) and/or step 8(c) is effected using droplet operations mediated by electrodes.
11. The method claim 8 further comprising treating the amplification-ready droplet under conditions selected to yield enough amplified nucleic acid in the amplification-ready droplet to ensure that each amplification reaction droplet produced in step 8(c) will include target nucleic acid if the target nucleic acid is present in the sample.
12. The method of claim 11 , wherein the treating the amplification-ready droplet comprises thermal cycling the amplification-ready droplet for 1-20 cycles.
13. The method of claim 11 , wherein the treating the amplification-ready droplet comprises thermal cycling the amplification-ready droplet for 1-10 cycles.
14. The method of claim 11 , wherein the treating the amplification-ready droplet comprises thermal cycling the amplification-ready droplet for 1-5 cycles.
15. The method of claim 1 , wherein step 1(b) is effected in a droplet operations gap of a droplet actuator using droplet operations mediated by electrodes.
16. The method of claim 1 , wherein step 1(b) comprises cycling the amplification reaction droplets between two or more thermal zones by transporting the droplets along a plurality of electrode paths in a droplet operations gap of a droplet actuator.
17. The method of claim 1 , wherein step 1(b) comprises cycling subsets of two or more of the amplification reaction droplets between two or more thermal zones by transporting droplets in each subset along a common electrode path in a droplet operations gap of a droplet actuator.
18. The method of claim 17 wherein the one or more electrode paths establish one or more path loops between the two or more thermal zones.
19. The method of claim 18 wherein:
(a) step 1(b) comprises transporting multiple amplification reaction droplets about an electrode path loop; and
(b) the method further comprises removing each amplified droplet from the electrode path loop when its predetermined number of cycles has been completed.
20. The method of claim 19 wherein removing each amplified droplet from the electrode path loop comprises using electrode mediated droplet operations to transport the amplification reaction droplet to another region of the droplet operations gap.
21. The method of claim 20 wherein region of the droplet operations gap comprises a temperature controlled region having a temperature selected for storing the amplified droplet pending detection.
22. The method of claim 19 wherein removing each amplified droplet from the electrode path loop comprises removing each amplified droplet from the droplet operations gap of the droplet actuator.
23. The method of claim 17 wherein the electrode path meanders between two or more thermal zones.
24. The method of claim 17 wherein:
(a) transport time from one electrode on the electrode path to an adjacent electrode on the electrode path is substantially uniform for each pair of adjacent electrodes; and
(b) residence time in a thermal zone is established by the number of electrodes in each turn of the electrode path present in the thermal zone.
25. The method of claim 1 , wherein step 1(b) comprises transporting two or more subsets of the amplification reaction droplets between thermal zones in parallel.
26. The method of claim 1 , wherein step 1(b) comprises sequentially transporting two or more subsets of the amplification reaction droplets into a thermal zone.
27. The method of claim 1 , wherein step 1(b) comprises:
(a) transporting a first subset of the amplification reaction droplets into a first thermal zone while transporting a second subset of the amplification reaction droplets into a second thermal zone; and
(b) transporting the first subset of the amplification reaction droplets into the second thermal zone while transporting the second subset of the amplification reaction droplets into the first thermal zone.
28. The method of claim 26 wherein the subset of the amplification reaction droplets thermal cycled sequentially are thermal cycled along a common electrode path.
29. The method of claim 26 wherein step 1(b) further comprises amplifying two or more subsets in parallel.
30. The method of claim 1 , wherein step 1(b) comprises thermally synchronized thermal cycling for all amplification reaction droplets.
31. The method of claim 1 , wherein step 1(b) comprises thermal cycling that is not thermally synchronized for all amplification reaction droplets.
32. The method of claim 1 , wherein step 1(b) is effected by heating and cooling a thermal cycling region of a droplet actuator.
33. The method of claim 1 , wherein step 1(b) comprises varying amplification reaction droplet dwell times in thermal zones at one or more thermal cycling cycle numbers.
34. The method of claim 1 , comprising completing step 1(b) for all subsets of amplification reaction droplets prior to initiating step 1(d).
35. The method of claim 1 , comprising completing step 1(d) for a first subset of amplified droplets prior to completing step 1(b) for a second set of the amplification reaction droplets.
36. The method of claim 1 , wherein step 1(c) comprises transporting a set of two or more amplified droplets away from a thermal cycling region of the droplet actuator prior to initiating step 1(d) with respect to the set of two or more amplified droplets.
37. The method of claim 1 , wherein step 1(c) comprises transporting each amplified droplet away from a thermal cycling zone prior to initiating step 1(d) with respect to each such droplet.
38. The method of claim 1 , wherein step 1(c) comprises transporting each amplified droplet to a thermal zone having a temperature appropriate for conducting step 1(d).
39. The method of claim 1 , wherein step 1(c) comprises arraying at least a subset of the amplified droplets away from a thermal cycling zone prior to initiating step 1(d) with respect to each such subset of arrayed amplified droplets.
40. The method of claim 1 , wherein step 1(c) comprises separating unbound detection reagent from the amplified nucleic acid.
41. The method of claim 1 , wherein step 1(c) comprises substantially stopping amplification in the amplified droplet.
42. The method of claim 1 , wherein step 1(c) comprises transferring the amplified droplets from one droplet actuator to another droplet actuator.
43. The method of claim 41 wherein substantially stopping amplification in the amplified droplet comprises adjusting droplet temperature to substantially stop the amplification reaction.
44. The method of claim 43 wherein substantially stopping amplification in the amplified droplet comprises adding a reagent to the amplified droplet to substantially stop the amplification reaction.
45. The method of claim 44 wherein adding a reagent to the amplified droplet comprises combining the amplified droplet with a reagent droplet, the reagent droplet comprising reagent selected to substantially stop the amplification reaction.
46. The method of claim 45 wherein the reagent selected to substantially stop the amplification reaction comprises a reagent that substantially stops the amplification reaction by interfering with polymerase activity, interfering with polymerase cofactor activity, binding to nucleic acids, and/or releasing iron ions.
47. The method of claim 1 , wherein step 1(e) comprises determining the amount of the amplified nucleic present in the amplified droplets by determining an increase or decrease in signal at a thermal cycling end-point for each of the subsets of amplified droplets.
48. The method of claim 1 , wherein:
(a) the amplification reaction droplets lack a detection reagent; and
(b) step 1(c) comprises adding a detection reagent to the amplified droplets.
49. The method of claim 48 wherein adding a detection reagent to the amplified droplets comprises combining each amplified droplet with a droplet comprising a detection reagent.
50. The method of claim 1 , wherein:
(a) step 1(c) comprises combining each of the amplified droplets with a droplet comprising one or more detection reagents; and
(b) step 1(d) comprises detecting amplification based on a signal mediated by the detection reagent.
51. The method of claim 1 , wherein step 1(d) comprises detecting a signal from each amplified droplet following transport of such droplet into a detection window.
52. The method of claim 1 , wherein step 1(c) comprises transporting sets of one or more of the subsets of amplified droplets into a detection window for detection.
53. The method of claim 1 , wherein:
(a) step 1(b) is accomplished in a droplet operations gap of a droplet actuator; and
(b) step 1(c) comprises transporting one or more of the amplified droplets out of the droplet operations gap for detection.
54. The method of claim 53 wherein step 1(d) is effected in a reservoir exterior to the droplet operations gap of the droplet actuator.
55. The method of claim 1 , wherein step 1(c) comprises transporting subsets of amplified droplets into a detection window beginning with lower cycle number subsets and proceeding to higher cycle number subsets.
56. The method of claim 1 , wherein step 1(d) comprises scanning an array of amplified droplets.
57. The method of claim 1 , wherein step 1(d) comprises imaging an array of amplified droplets.
58. The method of claim 1 , wherein:
(a) the amplification reaction droplets enter step 1(b) in parallel;
(b) the amplified droplets enter step 1(c) in series; and
(c) the amplified droplets enter step 1(d) in series.
59. The method of claim 1 , wherein:
(a) the amplification reaction droplets enter step 1(b) in parallel;
(b) the amplified droplets enter step 1(c) in series; and
(c) the amplified droplets enter step 1(d) in parallel.
60. The method of claim 1 , wherein:
(a) the amplification reaction droplets enter step 1(b) in series; and
(b) the amplified droplets enter step 1(c) in parallel.
61. The method of claim 47 wherein steps 1(c) and 1(d) are effected for each of the subsets of the amplified droplets following completion of thermal cycling of the subset.
62. The method of claim 1 , wherein step 1(e) further comprises using the amount of amplified nucleic acid present in the amplified droplets to determine the amount of the target nucleic acid present in the sample droplet.
63. The method of claim 1 , wherein step 1(e) further comprises using the amount of amplified nucleic acid present in the amplified droplets after different numbers of thermal cycles to determine the amount of the target nucleic acid present in the sample droplet.
64. The method of claim 1 , wherein the different conditions for amplifying the target nucleic acid comprise different numbers of thermal cycles for each subset of the amplification reaction droplets.
65. The method of claim 1 , wherein the amplification reaction droplets comprise a detection reagent.
66. The method of claim 1 , wherein the amplification reaction droplets do not comprise a significant amount of a detection reagent.
67. The method of claim 1 , wherein the one or more droplets comprising amplification reagents further comprise a detection reagent.
68. The method of claim 1 , wherein the one or more droplets comprising amplification reagents do not comprise a significant amount of a detection reagent.
69. The method of claim 1 , wherein the detection reagent comprises a nucleic acid binding agent.
70. The method of claim 69 wherein the nucleic acid binding agent significantly inhibits the rate of nucleic acid amplification.
71. The method of claim 69 wherein the nucleic acid binding agent comprises an intercalating agent.
72. The method of claim 71 wherein the intercalating agent comprises a fluorescent dye.
73. The method of claim 1 , wherein the target nucleic acid is indicative of a genetic disorder or infectious disease.
74. The method of claim 1 , wherein the target nucleic acid is indicative of identification of an individual or subgroup of individuals from a biological population.
75. The method of claim 1 , wherein:
(a) one or more of the nucleic acid amplification reaction droplets is subjected to an initial detection step prior to step 1(b); and
(b) step 1(e) comprises a comparison between signal detected in the initial detection step and signal detected in step 1(d).
76. The method of claim 1 , further comprising stopping the method when sufficient data has been collected to quantify the target nucleic acid present in the starting sample within a predetermined range of statistical certainty.
77. The method of claim 1 , further comprising:
(a) selecting a first set of cycle numbers expected to provide sufficient data for determining the amount of the amplified nucleic present in the amplified droplets and/or the sample;
(b) for each of the selected cycle numbers, subjecting a subset of one or more amplification reaction droplet to steps 1(b), 1(c), 1(d), and 1(e);
(c) determining whether sufficient data has been collected to identify or quantify the target nucleic acid present in the sample within a predetermined range of statistical certainty;
(d) repeating steps (a), (b), and (c) with new sets of cycle numbers until:
(i) sufficient data has been collected to identify or quantify the target nucleic acid present in the starting sample within a predetermined range of statistical certainty; or
(ii) sufficient data has been collected to determine within a predetermined range of statistical certainty that the target nucleic acid is not present in the sample.
78. The method of claim 77 wherein step 77(c) comprises determining whether sufficient data has been collected to identify the target nucleic acid present in the sample within a predetermined range of statistical certainty.
79. The method of claim 77 wherein step 77(c) comprises determining whether sufficient data has been collected to quantify the target nucleic acid present in the sample within a predetermined range of statistical certainty.