Method of mixing fluids by coalescence of multiple emulsions

• US 9,194,861 B2
• Filed: 08/24/2010
• Issued: 11/24/2015
• Est. Priority Date: 09/02/2009
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Hindson, Benjamin J., Colston, Billy W. Jr., Ness, Kevin D., Masquelier, Donald A.
• Primary Examiner(s): MARSCHEL, ARDIN H
• Application Number:
  US12/862,542
• Publication Number:
  US 20110053798A1
• Time in Patent Office: 1,918 Days
• Field of Search: 422/417
• US Class Current: 1/1
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/3033 using heat to mix or move the fluids
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00418 using pressure
• CPC Class Codes: B01J 2219/00495 Means for heating or cooling the reaction vessels
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00599 Solution-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00873 Heat exchange
• CPC Class Codes: B01J 2219/00894 More than two inlets
• CPC Class Codes: B01J 2219/00903 Segmented flow
• CPC Class Codes: B01J 2219/00957 Compositions or concentrations
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/0883 Serpentine channels
• CPC Class Codes: B01L 2300/18 Means for temperature control
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0677 : phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• 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 7/525 : with physical movement of samples between temperature zones
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6876 : Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2547/107 : Use of permeable barriers, e.g. waxes
• CPC Class Codes: C40B 50/08 : Liquid phase synthesis, i.e. wherein all library building blocks are in liquid phase or in solution during library creation; Particular methods of cleavage from the liquid support
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 1/30 : Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 1/44 : Sample treatment involving radiation, e.g. heat
• CPC Class Codes: G01N 21/75 : Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated systems in which material is burnt in a flame or plasma G01N21/72, G01N21/73
• CPC Class Codes: G01N 33/50 : Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing measuring or testing processes involving enzymes or microorganisms, compositions or test papers therefor; processes for forming such compositions, condition responsive control in microbiological or enzymological processes C12Q
• CPC Class Codes: G01N 35/08 : using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis

Abstract

System, including methods, apparatus, compositions, and kits, for the mixing of small volumes of fluid by coalescence of multiple emulsions.

Claims

1. A method of sample analysis, comprising:

obtaining a plurality of compound droplets each including at least one sample-containing droplet, at least one reagent-containing droplet, and a barrier fluid that separates the at least one sample-containing droplet from the at least one reagent-containing droplet;

heating compound droplets to induce fusion of at least one of the sample-containing droplet(s) with at least one of the reagent-containing droplet(s) within individual compound droplets to form fused droplets that combine sample and reagent to create assay mixtures for performing an assay;

collecting data from a plurality of the fused droplets; and

determining a result of the assay based on the collected data.

2. The method of claim 1, wherein the step of heating compound droplets creates an amplification mixture in fused droplets, and wherein the amplification mixture is capable of amplifying a nucleic acid target, if present, in the fused droplets.

3. The method of claim 2, wherein the at least one reagent-containing droplet of each of the plurality of compound droplets includes at least one primer for amplifying the nucleic acid target.

4. The method of claim 1, further comprising a step of thermally cycling the fused droplets after the step of heating compound droplets.

5. The method of claim 1, wherein the step of obtaining includes a step of forming a multiple emulsion including a hydrophilic surfactant and a hydrophobic surfactant.

6. The method of claim 1, wherein the at least one sample-containing droplet and the at least one reagent-containing droplet of each of the plurality of compound droplets are both encapsulated by the barrier fluid.

7. The method of claim 1, wherein the barrier fluid is encapsulated by the at least one sample-containing droplet or the at least one reagent-containing droplet of each of the plurality of compound droplets.

8. The method of claim 1, wherein the step of collecting data including a step of detecting fluorescence intensity signals from a plurality of the fused droplets.

9. The method of claim 1, wherein the step of determining a result includes a step of determining a presence and/or an activity of an analyte in the sample.

10. The method of claim 1, wherein the plurality of compound droplets includes a first type of compound droplets and a second type of compound droplets, and wherein each of the first and second types of compound droplets includes a respective distinct code that enables differentiation of the first and second types of droplets, wherein collecting data includes detection of test signals, further comprising (a) a step of detecting code signals from the fused droplets and (b) a step of utilizing the code signals to correlate test signals with the first type and the second type of compound droplets.

11. The method of claim 10, wherein the first and second types of compound droplets contain distinct reagents or distinct samples, and wherein the step of utilizing the code signals includes a step of correlating test signals with the distinct reagents or samples.

12. The method of claim 10, wherein the first and second types of compound droplets contain distinct primers for amplification of respective distinct nucleic acid targets, and wherein the step of utilizing the code signals includes a step of correlating test signals with a presence of the distinct nucleic acid targets in the fused droplets.

18. The method of claim 1, wherein the step of collecting data includes a step of detecting light from a plurality of the fused droplets, and wherein the step of determining a result of the assay is based on the light detected.

13. A method of sample analysis, comprising:

selecting an emulsion from a set of pre-formed emulsions each containing a different type of reagent-containing droplet;

forming compound droplets each including at least one sample-containing droplet, at least one reagent-containing droplet from the selected emulsion, and a barrier fluid that separates the at least one sample-containing droplet from the at least one reagent-containing droplet; and

heating compound droplets to induce fusion of at least one of the sample-containing droplet(s) and at least one of the reagent-containing droplet(s) within individual compound droplets to form fused droplets that combine reagent from the selected emulsion with sample to create assay mixtures for performing an assay.

14. The method of claim 13, wherein the step of selecting an emulsion includes a step of selecting an emulsion from an array of pre-formed emulsions.

15. The method of claim 13, wherein the step of selecting an emulsion includes a step of selecting one or more primers from a set of different primers contained by the pre-formed emulsions.

16. The method of claim 13, wherein the step of forming compound droplets includes a step of forming compound droplets each having the at least one sample-containing droplet and the at least one reagent-containing droplet encapsulated by the barrier fluid.

17. The method of claim 13, the emulsion being a first emulsion, further comprising a step of selecting a second emulsion from a set of pre-formed emulsions each containing a different type of sample-containing droplet, wherein the step of forming disposes at least one droplet of each of the first and second emulsions in each compound droplet.

19. The method of claim 13, further comprising:

detecting light from a plurality of the fused droplets; and

determining a result of the assay based on the light detected.