Apparatus, system, and method using immiscible-fluid-discrete-volumes

• US 10,041,113 B2
• Filed: 10/28/2015
• Issued: 08/07/2018
• Est. Priority Date: 08/22/2005
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Linda G., Woo, Sam L., Ma, Congcong, Reel, Richard T., Oldham, Mark F., Cox, David M., Schroeder, Benjamin G., Sorenson, Jon M., Wiyatno, Willy
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/925,656
• Publication Number:
  US 20160194697A1
• Time in Patent Office: 1,014 Days
• Field of Search: 435 912, 435 61, 435 61
• CPC Class Codes: B01J 2219/00353 Pumps
• CPC Class Codes: B01J 2219/0036 Nozzles
• CPC Class Codes: B01J 2219/00364 Pipettes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00657 One-dimensional arrays
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2400/0421 electrophoretic flow
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/0293 for liquids
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502784 : specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6806 : Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 : Methods for sequencing
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2535/101 : Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: F15C 5/00 : Manufacture of fluid circuit elements; Manufacture of assemblages of such elements integrated circuits
• CPC Class Codes: F16K 2099/0084 : Chemistry or biology, e.g. "lab-on-a-chip" technology
• CPC Class Codes: F16K 99/0001 : Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0011 : Gate valves or sliding valves
• CPC Class Codes: F16K 99/0013 : Rotary valves
• CPC Class Codes: G01N 1/14 : Suction devices, e.g. pumps; Ejector devices
• CPC Class Codes: G01N 27/44743 : Introducing samples
• CPC Class Codes: G01N 27/44769 : Continuous electrophoresis, i.e. the sample being continuously introduced, e.g. free flow electrophoresis [FFE]
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
• CPC Class Codes: Y10T 137/0318 : Processes
• CPC Class Codes: Y10T 137/4259 : With separate material addition
• CPC Class Codes: Y10T 137/85978 : With pump
• CPC Class Codes: Y10T 137/85986 : Pumped fluid control
• CPC Class Codes: Y10T 137/86863 : Rotary valve unit
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

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.

Claims

1. A method comprising:

within a first conduit having a circular cross section having a maximum dimension, flowing a sample including a first plurality of aqueous droplets comprising a single molecule of a target nucleic acid sequence and a second plurality of aqueous droplets containing no molecules of the target nucleic acid sequence, the aqueous droplets being separated within the conduit by an immiscible fluid that is immiscible with the aqueous droplets, the aqueous droplets having a maximum dimension that is less than the maximum dimension of the first conduit;

amplifying the target nucleic acid sequence in each of the first plurality of aqueous droplets to form an amplicon;

while flowing the aqueous droplets within a second conduit, using a fluorescent signal detector to detect and identify the first plurality of aqueous droplets by detecting fluorescence from the first plurality of aqueous droplets.

8. The method of claim 1, wherein less than 37% of aqueous droplets comprise a single molecule of the target nucleic acid sequence.

9. The method of claim 8, wherein 1% or more of aqueous droplets comprise a single molecule of the target nucleic acid sequence.

10. The method of claim 8, wherein 10% or more of aqueous droplets comprise a single molecule of the target nucleic acid sequence.

11. The method of claim 1, wherein the aqueous droplets and the immiscible fluid form a plurality of emulsified droplets, the method further comprising:

dispensing emulsified droplets from an end of the conduit and into one or more containers; and

thermal cycling the container and the emulsified droplets in a thermal cycler.

12. The method of claim 11, wherein the containers comprises wells of a multiwell plate.

13. The method of claim 11, wherein at least some of the emulsified droplets have a volume that is less than about 50 nanoliters.

14. The method of claim 11, wherein at least some of the emulsified droplets have a volume of between about 1 femtoliter and 1 nanoliter.

15. The method of claim 11, further comprising, for at least some of the first plurality of aqueous droplets, detecting with a detector the amplicon or a derivative thereof in a second conduit.

16. The method of claim 11, wherein the detector is a fluorescent signal detector.

17. The method of claim 11, further comprising, after thermal cycling in the thermal cycler, running the sample through a capillary.

18. The method of claim 11, further comprising, after thermal cycling in the thermal cycler, running the sample through on a capillary electrophoretic sequencer.

2. A method comprising:

sequentially contacting an aqueous sample fluid in a conduit with a non-aqueous spacing fluid that is immiscible with the aqueous sample fluid, to form discrete volumes of the aqueous sample fluid separated from one another by the non-aqueous spacing fluid, the aqueous sample fluid comprising a target nucleic acid sequence, wherein a first plurality of the discrete volumes contains at least one molecule of the target nucleic acid sequence and a second plurality of the discrete volumes contains no molecules of the target nucleic acid sequence;

amplifying the target nucleic acid sequence in one or more of the first plurality of discrete volumes to form an amplicon; and

in a second conduit, detecting a fluorescence signal from the amplicon in each of the one or more first plurality of discrete volumes; and

based on the detecting, discriminating between the first plurality of discrete volumes and the second plurality of discrete volumes.

3. The method of claim 2, wherein the contacting comprises continuously flowing at least one of the aqueous sample fluid and the non-aqueous spacing fluid into the conduit.

4. The method of claim 2, further comprising sorting the second plurality of discrete volumes from the first plurality of discrete volumes.

5. The method of claim 2, wherein less than 37% of the discrete volumes comprise a single molecule of the target nucleic acid sequence.

6. The method of claim 5, wherein 1% or more of the discrete volumes comprise a single molecule of the target nucleic acid sequence.

7. The method of claim 5, wherein 10% or more of the discrete volumes comprise a single molecule of the target nucleic acid sequence.