Systems and methods for continuous flow digital droplet polymerase chain reaction bioanalysis
Systems and methods for continuous flow polymerase chain reaction (PCR) are provided. The system comprises an injector, a mixer, a coalescer, a droplet generator, a detector, a digital PCR system, and a controller. The injector takes in a sample, partitions the sample into sample aliquots with the help of an immiscible oil phase, dispenses waste, and sends the sample aliquot to the mixer. The mixer mixes the sample aliquot with a PCR master mix and diluting water, dispenses waste, and sends the sample mixture (separated by an immiscible oil) to the coalescer. The coalescer coalesces the sample mixture with primers dispensed from a cassette, dispenses waste, and sends the reaction mixture (separated by an immiscible oil) to the droplet generator. The droplet generator converts the sample mixture into an emulsion where aqueous droplets of the reaction mixture are maintained inside of an immiscible oil phase and dispenses droplets to the digital PCR system. The digital PCR system amplifies target DNAs in the droplets. The detector detects target DNAs in the droplets. The controller controls the system to run automatically and continuously.
1. A method for analyzing a sample, the method comprising the steps of:
receiving the sample in an injector;
partitioning the sample with a first immiscible oil to obtain a sample aliquot;
delivering the sample aliquot to a mixer;
mixing the sample aliquot with a plurality of PCR reagents to obtain a sample mixture;
mixing the sample mixture with a plurality of PCR primers for performing PCR to obtain a reaction mixture;
generating a plurality of droplets of the reaction mixture;
amplifying at least one molecular target contained in the plurality of droplets of the reaction mixture by directing the plurality of droplets through a channel wrapped in an interwoven configuration around at least two separate heater cores; and
quantifying the at least one molecular target contained in the plurality of droplets of the reaction mixture.
2. The method of claim 1 , wherein receiving the sample further comprises lysing the sample and isolating a plurality of nucleic acids.
3. The method of claim 2 , wherein isolating the plurality of nucleic acids comprises isolating deoxyribonucleic acid (DNA) of the sample.
4. The method of claim 2 , wherein isolating the plurality of nucleic acids comprises isolating ribonucleic acid (RNA) of the sample.
5. The method of claim 1 , wherein partitioning the sample with the first immiscible oil to obtain the sample aliquot further comprises dispensing a first waste.
6. The method of claim 1 , wherein mixing the sample aliquot with the plurality of PCR reagents to obtain the sample mixture further comprises dispensing a second waste.
7. The method of claim 1 , wherein mixing the sample mixture with the plurality of PCR primers to obtain the reaction mixture further comprises dispensing a third waste.
8. The method of claim 1 , wherein generating the plurality of droplets of the reaction mixture comprises using voltage electrocoalescence to generate the plurality of droplets of the reaction mixture.
9. The method of claim 1 , wherein mixing the sample mixture with the plurality of PCR primers comprises mixing the sample mixture with a plurality of fluorescently labeled PCR primers.
10. The method of claim 1 , wherein quantifying the at least one molecular target contained in the plurality of droplets of the reaction mixture comprises measuring fluorescent strength of a fluorescent labeled for at least one molecular target.
11. The method of claim 1 , wherein generating the plurality of droplets of the reaction mixture comprises using a droplet generator.
12. The method of claim 11 , wherein the droplet generator comprises a fluorophilic surface.
13. The method of claim 1 , wherein a first heater of the at least two separate heater cores comprises a temperature of 95° C. and a second heater of the at least two separate heater cores comprises a temperature of 60° C.