Method of performing droplet-based assays
Method of analysis. In the method, a first emulsion and a second emulsion substantially separated from one another by a spacer fluid may be formed. The first emulsion, the spacer fluid, and the second emulsion may be flowed in a channel from a fluid inlet to a fluid outlet of a heating and cooling station having two or more temperature-controlled zones, such that each emulsion is thermally cycled to promote amplification of a nucleic acid target in droplets of the emulsion. Amplification data may be collected from individual droplets of each emulsion downstream of the heating and cooling station. A level of the nucleic acid target present in each emulsion may be determined based on the amplification data collected from the individual droplets of the emulsion.
1. A method of analysis, the method comprising:
forming a first emulsion and a second emulsion substantially separated from one another by a spacer fluid;
flowing the first emulsion, the spacer fluid, and the second emulsion in a channel from a fluid inlet to a fluid outlet of a heating and cooling station having two or more temperature-controlled zones, the channel forming a single-pass continuous fluid route from the fluid inlet to the fluid outlet, the fluid route traversing the temperature-controlled zones serially and repeatedly, such that each emulsion is thermally cycled to promote amplification of a nucleic acid target in droplets of the emulsion;
collecting amplification data from individual droplets of each emulsion downstream of the heating and cooling station; and
determining a level of the nucleic acid target present in each emulsion based on the amplification data collected from the individual droplets of the emulsion.
2. The method of claim 1 , wherein the spacer fluid is miscible with a continuous phase of each emulsion.
3. The method of claim 1 , wherein the spacer fluid is gaseous.
4. The method of claim 1 , wherein the spacer fluid includes more than one spacer fluid that creates at least two spacer segments between the emulsions.
5. The method of claim 1 , wherein the spacer fluid is labeled.
6. The method of claim 5 , wherein the spacer fluid includes a dye configured to permit the spacer fluid to be distinguished from a carrier fluid of each emulsion.
7. The method of claim 1 , wherein flowing includes flowing the first emulsion and the second emulsion through at least three temperature-controlled zones of the heating and cooling station.
8. The method of claim 1 , wherein flowing drives a polymerase chain reaction in droplets of each emulsion.
9. The method of claim 1 , wherein the fluid route is helical.
10. The method of claim 1 , wherein a different nucleic acid target is amplified in each of the emulsions.
11. The method of claim 1 , wherein collecting amplification data includes detecting fluorescence from droplets of each emulsion.
12. The method of claim 1 , wherein determining a level of the nucleic acid target includes assigning individual droplets of each emulsion as amplification-positive or amplification-negative for the nucleic acid target based on the amplification data collected.