Micro-regional thermal control for digital microfluidics
A method of thermal cycling a droplet, including providing a droplet actuator with heaters establishing a first thermal zone and second thermal zone in a substantially oil-filled droplet operations gap; a thermal cycling path comprising droplet operations electrodes comprising a first droplet operations electrode in the first thermal zone and a second droplet operations electrode in the second thermal zone, wherein the first and second droplet operations electrodes are within 5 mm of each other; a first temperature at the first droplet operations electrode and a second temperature at the second droplet operations electrode, wherein the first and second temperatures differ by at least about 10° C.; and using the droplet operations electrodes, transporting the droplet in a cycling pattern for multiple cycles along the thermal cycling path between the first droplet operations electrode and the second droplet operations electrode. Cartridges and systems are also provided.
1 . A method of thermal cycling a droplet, the method comprising:
(a) providing a droplet actuator comprising:
(i) two or more integrated heaters establishing an integrated heater/sensor arrangement, and comprising a first integrated heater and sensor pair, and establishing at least a first thermal zone and a second integrated heater and respective sensor pair establishing a second thermal zone droplet operations gap, wherein the integrated heater sensor/arrangement provides closed-loop thermal control, and wherein the droplet operations gap is optionally substantially oil filled;
(ii) a thermal cycling path comprising a plurality of droplet operations electrodes comprising a first droplet operations electrode designated as a first detection electrode in the first thermal zone and a second droplet operations electrode in the second thermal zone, wherein the first and second droplet operations electrodes are within 5 mm of each other;
(iii) a first temperature at the first droplet operations electrode and a second temperature at the second droplet operations electrode, wherein the first and second temperatures differ by at least about 10° C.;
(iv) two or more integrated sensors, configured to, in part, measure the two or more heaters and/or the first thermal zone and the second thermal zone, and wherein the two or more integrated heaters and the two or more integrated sensors form at least a first heater/sensor arrangement pair and a second heater/sensor arrangement pair corresponding to at least the first thermal zone and the second thermal zone, wherein the two or more heater/sensor arrangement pairs are configured for micro-regional thermal control of at least the first thermal zone and the second thermal zone;
(b) using the droplet operations electrodes, transporting the droplet in a cycling pattern for multiple cycles along the thermal cycling path between the first droplet operations electrode and the second droplet operations electrode.
2 . The method of claim 1 wherein:
(a) the droplet comprises reagents for amplifying a nucleic acid;
(b) the first temperature is a denaturation temperature and the second temperature is an elongation temperature; and
(c) the transporting the droplet in a cycling pattern results in nucleic acid amplification.
3 . The method of claim 1 wherein the droplet actuator comprises 2 or more of the thermal cycling path.
4 . The method of claim 1 wherein the first droplet operations electrode is adjacent to the second droplet operations electrode, without any intervening droplet operations electrode.
5 . The method of claim 1 wherein the first and second droplet operations electrodes are separated by no more than one additional droplet operations electrode between them.
6 . The method of claim 1 wherein the first and second droplet operations electrodes are separated by no more than two additional droplet operations electrodes between them.
7 . The method of claim 2 wherein each cycle of the multiple cycles is completed in less than about 1 seconds and effects substantially complete amplification.
8 . The method of claim 2 wherein each cycle of the multiple cycles is completed in less than about 0.5 seconds and effects substantially complete amplification.
9 . The method of claim 1 wherein the thermal cycling path has a length of less than about 5,000 μm.
10 . The method of claim 1 wherein the thermal cycling path has a length of less than about 100 μm.
11 . The method of claim 1 wherein the thermal cycling path has a length of less than about 10 μm.
12 . The method of claim 1 wherein transporting the droplet between the first droplet operations electrode and the second droplet operations electrode is accomplished in a time of about 100 milliseconds or less.
13 . The method of claim 1 wherein transporting the droplet between the first droplet operations electrode and the second droplet operations electrode is accomplished in a time of about 50 milliseconds or less.
14 . The method of claim 1 wherein transporting the droplet between the first droplet operations electrode and the second droplet operations electrode is accomplished in a time of about 25 milliseconds or less.
15 . The method of claim 1 wherein:
(a) the first thermal zone is set at a nucleic acid annealing temperature; and
(b) the second thermal zone is set at a nucleic acid denaturation temperature.
16 . The method of claim 15 wherein the method comprises retaining the droplet at the first droplet operations electrode for a period of about 500 milliseconds or less.
17 . The method of claim 15 wherein the method comprises retaining the droplet at the first droplet operations electrode for a period of about 0 seconds.
18 . The method of claim 15 wherein the method comprises retaining the droplet at the second droplet operations electrode for a period of about 500 milliseconds or less.
19 . The method of claim 15 wherein the method comprises retaining the droplet at the second droplet operations electrode for a period of about 0 seconds.
20 . The method of claim 1 wherein each cycle takes less than about 1 seconds.
21 . The method of claim 1 wherein each cycle takes less than about 0.5 seconds.
22 . The method of claim 1 wherein the heaters are arranged such that:
(a) a first heater is associated with the first droplet operations electrode and establishes the first thermal zone;
(b) a second heater is associated with the second droplet operations electrode and establishes the second thermal zone; and
(c) a third heater is associated with a boundary region adjacent to the second heater and is set at a temperature selected to maintain the temperature of the second thermal zone.
23 . The method of claim 22 wherein the second heater and the third heater are set at the same temperature.
24 . The method of claim 22 wherein the second heater and the third heater are set at a higher temperature than the first heater.
25 . The method of claim 22 wherein the second heater and the third heater are set at a denaturation temperature.
26 . The method of claim 22 wherein the third heater stabilizes the second thermal zone.
27 . The method of claim 1 wherein the one or more integrated sensors are arranged for sensing temperature of the thermal zone, wherein each of the one or more sensors corresponds to each of the one or more integrated heaters.
28 . The method of claim 27 wherein the one or more sensors are calibrated to measure temperature in the range of room temperature to 100° C.
29 . The method of claim 27 wherein the one or more sensors are each situated to measure temperature within in close proximity to the droplet.
30 . The method of claim 27 wherein the one or more sensors are each situated to measure temperature at a distance of about 1 mm or less from the droplet.