Droplet-Based Nucleic Acid Amplification Method and Apparatus

Abstract

Aspects of embodiments of the invention relate to a simulator including a visual display capable of outputting to a user a display one or more effects of a command series selected and a system including a droplet microactuator electronically coupled to and controlled by a processor capable of executing instructions, the droplet microactuator including a substrate comprising electrodes for conducting droplet operations. Further aspects of embodiments of the invention relate to a droplet operations troubleshooting apparatus. Other aspects of embodiments of the invention relate to a computer implemented method of displaying simulated microactuator droplets.

1 - 17 . (canceled) 
     
     
         18 . A droplet actuation system comprising:
 (a) a droplet actuation device comprising:
 (i) a substrate comprising electrodes for conducting droplet operations; and 
 (ii) one or more temperature control means arranged in proximity with one or more of the electrodes for heating and/or cooling a region of the droplet actuation device and arranged such that a droplet can be transported on the substrate by the electrodes into the region for heating; and 
 (iii) a means for effecting a magnetic field in proximity to one or more of the electrodes, wherein the strength and proximity of the magnetic field is sufficient to substantially immobilize magnetically responsive beads in a droplet on the substrate during execution of a droplet operation; and 
   (b) a processor electronically coupled to and configured to control the electrodes and the temperature of one or more of the one or more temperature control means.   
     
     
         19 . The droplet actuation system of  claim 18  wherein the temperature control means comprises a heater. 
     
     
         20 . The droplet actuation system of  claim 19  wherein the temperature control means comprises a thin film heater. 
     
     
         21 . The droplet actuation system of  claim 18  wherein the temperature control means comprises one or more cooling elements. 
     
     
         22 . The droplet actuation system of  claim 21  wherein the one or more cooling elements comprise a thermoelectric cooler. 
     
     
         23 . The droplet actuation system of  claim 21  wherein the one or more cooling elements comprise a Peltier device. 
     
     
         24 . The droplet actuation system of  claim 18  wherein a region of the droplet actuation device is set to a temperature sufficient for annealing. 
     
     
         25 . The droplet actuation system of  claim 18  wherein a region of the droplet actuation device is set to a temperature sufficient for denaturation. 
     
     
         26 . The droplet actuation system of  claim 18  wherein a region of the droplet actuation device is set to a temperature sufficient for extension. 
     
     
         27 . The droplet actuation system of  claim 18  wherein one or more of the droplet operations is electrowetting-mediated. 
     
     
         28 . The droplet actuation system of  claim 18  wherein one or more of the droplet operations is dielectrophoresis-mediated. 
     
     
         29 . The droplet actuation system of  claim 19  wherein the temperature control means comprises at least two heaters:
 (a) at least one heater set to a temperature sufficient to effect denaturation of a nucleic acid in a nearby droplet on the droplet actuation device; and 
 (b) at least one heater set to a temperature sufficient to effect extension and/or annealing of a nucleic acid in a nearby droplet on the droplet actuation device. 
 
     
     
         30 . The droplet actuation system of  claim 18  further comprising a means for effecting a magnetic field in proximity to one or more of the electrodes, wherein the strength and proximity of the magnetic field is sufficient to immobilize magnetically responsive beads in a droplet during execution of a droplet operation. 
     
     
         31 . The droplet actuation system of  claim 30  wherein the means for effecting a magnetic field comprises an electromagnetic and a means for controlling power supply to the electromagnet. 
     
     
         32 . The droplet actuation system of  claim 30  wherein the means for effecting a magnetic field comprises a magnet and a means for moving the magnet into and out of proximity with the one or more of the electrodes. 
     
     
         33 . The droplet actuation system of  claim 18  comprising one or more droplet actuation device reservoirs in proximity to one or more of the electrodes arranged such that electrodes can dispense droplets from fluid in the droplet actuation device reservoirs, wherein the reservoirs comprise reagents therein each comprising one or more components selected from the group consisting of: buffers, primers, nucleotides, polymerases, reverse transcriptases and other nucleic acid amplification reagents. 
     
     
         34 . The droplet actuation system of claim  81  further comprising an amplification-ready droplet loaded thereon. 
     
     
         35 . The droplet actuation system of  claim 34  wherein the amplification-ready droplet has a volume which ranges from about 1 nL to about 10 μL. 
     
     
         36 . The droplet actuation system of  claim 34  wherein the amplification-ready droplet has a volume which ranges from about 10 nL to about 1 μL. 
     
     
         37 . The droplet actuation system of  claim 34  wherein the amplification-ready droplet has a volume which is less than about 100 nL. 
     
     
         38 . The droplet actuation system of  claim 18  programmed to execute steps in a nucleic acid amplification thermal cycling protocol on a droplet positioned in proximity to a temperature control means by controlling the temperature of the temperature control means. 
     
     
         39 . The droplet actuation system of  claim 38  wherein the nucleic acid amplification thermal cycling protocol comprises a PCR thermal cycling protocol. 
     
     
         40 . The droplet actuation system of  claim 38  wherein the nucleic acid amplification thermal cycling protocol comprises an RT-PCR thermal cycling protocol. 
     
     
         41 . The droplet actuation system of  claim 18  programmed to execute steps in a nucleic acid amplification thermal cycling protocol by transporting a droplet into and out of proximity with one or more temperature control means. 
     
     
         42 . The droplet actuation system of  claim 41  wherein the nucleic acid amplification thermal cycling protocol comprises a PCR thermal cycling protocol. 
     
     
         43 . The droplet actuation system of  claim 18  wherein the processor is programmed to execute droplet operations to combine one or more sample droplets with one or more amplification reagent droplets on the droplet actuation device in appropriate ratios to constitute one or more amplification-ready droplets. 
     
     
         44 . The droplet actuation system of  claim 18  wherein the processor is programmed to execute droplet operations controlled by the electrodes for a droplet-based washing protocol in which magnetically responsive beads are washed on the droplet actuation device. 
     
     
         45 . The droplet actuation system of  claim 18  wherein the processor is programmed to execute droplet operations for eluting one or more substances from the magnetically responsive beads. 
     
     
         46 . The droplet actuation system of  claim 33  wherein the processor is programmed to:
 (a) dispense reagent droplets from each of the one or more reservoirs; 
 (b) transport the dispensed reagent droplets; 
 (c) combine dispensed reagent droplets with one or more sample droplets potentially comprising a target nucleic acid to yield one or more amplification-ready droplets; and 
 (d) execute a thermal cycling protocol on the one or more amplification-ready droplets sufficient to amplify the target nucleic acid when present in the sample droplet. 
 
     
     
         47 . The droplet actuation system of  claim 46  wherein the thermal cycling protocol comprises a measurement step in which amplified target nucleic acid is quantified after a predetermined number of cycles. 
     
     
         48 . The droplet actuation system of  claim 47  wherein the processor is programmed to stop the thermal cycling when a predetermined quantity of target nucleic acid is detected. 
     
     
         49 . The droplet actuation system of  claim 47  wherein the processor is programmed to stop the thermal cycling when a predetermined quantity of target nucleic acid is not detected after a predetermined number of cycles. 
     
     
         50 . The droplet actuation system of  claim 46  wherein the processor is further programmed to transport the amplification-ready droplet for further processing during or after thermal cycling. 
     
     
         51 . The droplet actuation system of  claim 50  wherein the further processing comprises detection of one or more target nucleic acids. 
     
     
         52 . The droplet actuation system of  claim 51  wherein the processor is programmed to provide a user output indicating the presence or absence of one or more target nucleic acids. 
     
     
         53 . The droplet actuation system of  claim 51  wherein the target nucleic acid comprises a diagnostic nucleic acid. 
     
     
         54 . The droplet actuation system of  claim 51  wherein the target nucleic acid comprises a diagnostic nucleic acid the presence of which is indicative of the presence of a pathogenic organism. 
     
     
         55 . The droplet actuation system of  claim 33  comprising a cartridge comprising one or more cartridge reservoirs and a means for establishing a fluid path from the one or more cartridge reservoirs to one or more droplet microactuator reservoirs. 
     
     
         56 . The droplet actuation system of  claim 55  comprising one or more nucleic acid amplification reagents pre-loaded in the one or more cartridge reservoirs. 
     
     
         57 . The droplet actuation system of  claim 56  wherein the reagent(s) are selected from the group consisting of: buffers, primers, nucleotides, polymerases and other nucleic acid amplification reagents. 
     
     
         58 . A method of assembling a droplet actuation system for operation, the method comprising coupling the cartridge of  claim 55  to a droplet actuation device. 
     
     
         59 . A method of loading a droplet actuation system for operation, the method comprising flowing a fluid from the cartridge of  claim 55  to a droplet actuation device reservoir via the fluid path.