Apparatus, system, and method using immiscible-fluid-discrete-volumes
Various embodiments of the teachings relate to a system or method for sample preparation or analysis in biochemical or molecular biology procedures. The sample preparation can involve small volume processed in discrete portions or segments or slugs, herein referred to as discrete volumes. A molecular biology procedure can be nucleic acid analysis. Nucleic acid analysis can be an integrated DNA amplification/DNA sequencing procedure.
1. A system comprising:
a first conduit having a circular cross section having a maximum dimension and configured to flow an aqueous sample fluid containing at least one molecule of a target nucleic acid;
an intersecting conduit intersecting the first conduit and configured to flow a spacing fluid that is immiscible with the aqueous sample fluid;
a spacing fluid introduction unit configured to inject the spacing fluid by contacting the aqueous sample fluid in the first conduit to provide a first plurality of aqueous droplets comprising a single molecule of the target nucleic acid and a second plurality of aqueous droplets containing no molecules of the target nucleic acid, the aqueous droplets being separated within the first conduit by the spacing fluid, the conduits configured so that the aqueous droplets have a maximum dimension that is less than the maximum dimension of the first conduit;
a thermal cycler configured to amplify the target nucleic acid in each of the first plurality of aqueous droplets to form an amplicon; and
a fluorescent signal detector and a second conduit, the second conduit configured to receive and flow the aqueous droplets from the thermal cycler, the fluorescent signal detector configured to detect the first plurality of aqueous droplets by detecting fluorescence from the first plurality of aqueous droplets while flowing in the second conduit.
2. The system of claim 1 , wherein the fluorescent signal detector is further configured to identify the first plurality of aqueous droplets by detecting fluorescence from the first plurality of aqueous droplets.
3. A method of using the system of claim 1 , the method comprising:
in the spacing fluid introduction unit, sequentially contacting the aqueous sample fluid in the first conduit with the spacing fluid from the intersecting conduit to form a plurality of discrete volumes of the aqueous sample fluid separated from one another by the spacing fluid, the aqueous sample fluid comprising the target nucleic acid, wherein at least one of the discrete volumes contains at least one molecule of the target nucleic acid and one or more of the discrete volumes contains no molecules of the target nucleic acid;
amplifying the target nucleic acid in the conduit thermal cycler to form an amplicon in the at least one discrete volume;
moving the at least one discrete volume to the second conduit; and
using the fluorescent signal detector to detect the amplicon as the plurality of discrete volumes flow in the second conduit.
4. A system comprising:
a first conduit configured to flow an aqueous sample fluid containing at least one molecule of a target nucleic acid;
an intersecting conduit intersecting the first conduit and configured to flow a spacing fluid that is immiscible with an aqueous sample fluid;
a spacing fluid introduction unit configured to inject the spacing fluid by contacting the aqueous sample fluid in the first conduit to provide a first plurality of aqueous droplets comprising at least one molecule of the target nucleic acid and a second plurality of aqueous droplets containing no molecules of the target nucleic acid;
a thermal cycler configured to amplify the target nucleic acid in each of the first plurality of aqueous droplets to form an amplicon; and
a fluorescent signal detector and a second conduit, the second conduit configured to receive and flow the droplets from the thermal cycler, the fluorescent signal detector configured to detect the first plurality of aqueous droplets by detecting fluorescence from the first plurality of aqueous droplets while flowing in the second conduit.
5. The system of claim 4 , wherein the system is configured to use the detected fluorescence to discriminate between the first plurality of aqueous droplets and the second plurality of aqueous droplets.
6. The system of claim 4 , wherein the first conduit has a circular cross section having a maximum dimension and the conduits are configured so that the aqueous droplets have a maximum dimension that is less than the maximum dimension of the first conduit.
7. The system of claim 4 , wherein the first conduit has a circular cross section having a maximum dimension and the aqueous droplets have a maximum dimension that is less than the maximum dimension of the first conduit.
8. The system of claim 4 , wherein the conduits are configured to sequentially contact the aqueous sample fluid with the spacing fluid to form the aqueous droplets.
9. The system of claim 4 , wherein less than 37% of the first and second pluralities of aqueous droplets comprise a single molecule of the target nucleic acid.
10. The system of claim 9 , wherein 1% or more of the first and second pluralities of aqueous droplets comprise a single molecule of the target nucleic acid.
11. The system of claim 9 , wherein 10% or more of the first and second pluralities of aqueous droplets comprise a single molecule of the target nucleic acid.
12. The system of claim 4 , wherein the conduits are configured to provide a plurality of emulsified droplets comprising the aqueous droplets and the spacing fluid, the system configured to:
dispense the emulsified droplets from an end of an output conduit and into one or more containers; and
thermal cycle the container and the emulsified droplets in a thermal cycler.
13. The system of claim 12 , wherein at least some of the emulsified droplets have a volume that is less than about 50 nanoliters.
14. The system of claim 12 , wherein at least some of the emulsified droplets have a volume of between about 1 femtoliter and 1 nanoliter.
15. The system of claim 4 , wherein:
the intersecting conduit contains the spacing fluid;
the first conduit contains one or more of the first plurality of aqueous droplets, or one or more of the second plurality of aqueous droplets, or both;
the second conduit contains one or more of the first plurality of aqueous droplets, or one or more of the second plurality of aqueous droplets the aqueous droplets, or both; and
during operation, the spacing fluid introduction unit injects the spacing fluid by contacting the aqueous sample fluid in the first conduit to provide the aqueous droplets.
16. The system of claim 15 , further comprising, during operation, an additional spacing fluid added to the spacing fluid between adjacent aqueous droplets of one or both of the first plurality of aqueous droplets and the second plurality of aqueous droplets, so as to increase the spacing between the adjacent aqueous droplets produced by the spacing fluid introduction unit.
17. The system of claim 15 , wherein:
each of the aqueous droplets has an outer dimension;
the first conduit has a circular cross section having a maximum dimension; and
the outer dimension is equal to about 95% or more of the maximum inner cross-sectional dimension.
18. The system of claim 1 , wherein:
the intersecting conduit contains the spacing fluid;
the first conduit contains one or more of the first plurality of aqueous droplets, or one or more of the second plurality of aqueous droplets, or both;
the second conduit contains one or more of the first plurality of aqueous droplets, or one or more of the second plurality of aqueous droplets the aqueous droplets, or both; and
during operation, the spacing fluid introduction unit injects the spacing fluid by contacting the solution aqueous sample fluid in the first conduit to provide the aqueous droplets.
19. The system of claim 18 , further comprising, during operation, an additional spacing fluid added to the spacing fluid between adjacent aqueous droplets of one or both of the first plurality of aqueous droplets and the second plurality of aqueous droplets, so as to increase the spacing between the adjacent aqueous droplets produced by the spacing fluid introduction unit.