System for mixing fluids by coalescence of multiple emulsions

• US 10,166,522 B2
• Filed: 11/11/2015
• Issued: 01/01/2019
• Est. Priority Date: 09/02/2009
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated, Cadence Design Systems Incorporated (Cadence Group)
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Hindson, Benjamin J., Colston, Jr., Billy W., Ness, Kevin D., Masquelier, Donald A.
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US14/938,470
• Publication Number:
  US 20160059204A1
• Time in Patent Office: 1,147 Days
• 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:

forming a first type and a second type of reagent droplets, wherein the first type has a first code and the second type has a second code to identify each type;

encapsulating the first and second types of reagent droplets in sample droplets; and

inducing fusion of the sample droplets with the encapsulated first and second types of reagent droplets to produce fused droplets each having the first code or the second code.

2. The method of claim 1, wherein the first type and the second type of reagent droplets respectively contain a first pair of primers for amplification of a first nucleic acid target and a second pair of primers for amplification of a second nucleic acid target, and wherein the sample droplets contain nucleic acid.

3. The method of claim 1, wherein each code is provided by at least one particle in each type of reagent droplet.

4. The method of claim 1, wherein the step of inducing fusion includes a step of heating the first and second types of reagent droplets and the sample droplets after the step of encapsulating.

5. The method of claim 1, wherein the step of inducing fusion includes a step of applying an electric field to the first and second types of reagent droplets and the sample droplets after the step of encapsulating.

6. The method of claim 2, wherein the fused droplets contain a respective probe for each nucleic acid target and a thermostable polymerase, further comprising a step of thermally cycling the fused droplets after the step of inducing fusion.

7. The method of claim 6, further comprising a step of collecting data from a label of each respective probe after the step of thermally cycling.

8. The method of claim 7, wherein the step of collecting data includes a step of detecting fluorescence from the label of each respective probe present in a plurality of the fused droplets.

9. The method of claim 7, further comprising a step of determining a concentration of each nucleic acid target using the collected data.

10. The method of claim 7, wherein the step of collecting data includes a step of detecting 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 and second types of reagent droplets.

11. The method of claim 10, wherein the step of utilizing the code signals includes a step of correlating test signals with a presence of the first and second nucleic acid targets in the fused droplets.

15. The method of claim 8, wherein the step of detecting fluorescence includes a step of detecting fluorescence 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 and second types of reagent droplets.

12. A method of sample analysis, comprising:

obtaining an emulsion including a plurality of outer droplets disposed in a liquid carrier phase and each encapsulating at least one inner droplet, wherein the outer droplets are sample-containing droplets and the at least one inner droplet is at least one reagent-containing droplet or wherein the outer droplets are reagent-containing droplets and the at least one inner droplet is at least one sample-containing droplet; and

heating the emulsion to induce fusion of the outer droplets with the at least one inner droplet to form fused droplets that combine sample and reagent to create assay mixtures for performing an assay.

13. The method of claim 12, wherein the step of heating creates an amplification mixture in fused droplets, wherein the amplification mixture includes primers for amplifying a nucleic acid target, a probe for the nucleic acid target, and a polymerase, and wherein the amplification mixture is capable of amplifying the nucleic acid target, if present, in the fused droplets.

14. The method of claim 13, further comprising a step of collecting data from a label of the probe, and a step of determining a concentration of the nucleic acid target based on the collected data.