Emulsion chemistry for encapsulated droplets

• US 10,378,048 B2
• Filed: 03/07/2017
• Issued: 08/13/2019
• Est. Priority Date: 03/02/2010
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Hiddessen, Amy L., Hindson, Benjamin J.
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/452,582
• Publication Number:
  US 20170175179A1
• Time in Patent Office: 889 Days
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components

Abstract

System, including methods, apparatus, compositions, and kits, for making and using a stabilized emulsion. A method of generating a stabilized emulsion is provided. In the method, an aqueous phase may be provided. The aqueous phase may include an effective concentration of one or more skin-forming proteins. An emulsion may be formed. The emulsion may include droplets of a dispersed phase disposed in a continuous phase, with the aqueous phase being the continuous phase or the dispersed phase. The emulsion may be heated to create an interfacial skin between each droplet and the continuous phase, to transform the droplets into capsules.

Claims

1. A method of generating skin-encapsulated capsules comprising:

a) providing an aqueous phase comprising;

i) at least one reagent;

ii) at least 0.01% by weight of a skin-forming protein;

b) forming an emulsion comprising droplets of said aqueous phase disposed in a continuous phase comprising an oil and an ionic surfactant;

c) heating said emulsion to at least 50°

C. to create an interfacial skin between each droplet and the continuous phase, such that said droplets form skin-encapsulated capsules.

2. A method according to claim 1 wherein said reagent comprises nucleic acid.

3. A method according to claim 2 wherein said nucleic acid is selected from the group comprising a nucleic acid target, a nucleic acid primer and a nucleic acid probe.

4. A method according to claim 2 wherein said reagent comprises a nucleic acid primer.

5. A method according to claim 2 wherein said reagent comprises a pair of nucleic acid primers.

6. A method according to claim 2 wherein said reagent comprises an amplification mixture comprising at least one primer, at least one probe, a polymerase enzyme and deoxynucleotides.

7. A method according to claim 6 wherein said method further comprises:

a) thermocycling said capsules through multiple rounds of heating and cooling, such that amplicons are formed; and

b) detecting said amplicons.

8. A method according to claim 7 wherein said detecting is done by flowing said capsules through a detection region of a detector.

9. A method according to claim 1 wherein said oil is a fluorinated oil.

10. A method according to claim 1 wherein said ionic surfactant is a negatively charged ionic surfactant.

11. A method according to claim 1 wherein said skin-forming protein is selected from the group comprising albumin, casein, gelatin and globulin.

12. A method according to claim 11 wherein said albumin is bovine serum albumin (BSA).

13. A stabilized emulsion comprising:

a) a continuous phase comprising an oil and an ionic surfactant;

b) a plurality of capsules disposed in said continuous phase, each capsule including a proteinaceous, interfacial skin encapsulating an aqueous phase.

14. A stabilized emulsion according to claim 13 wherein a plurality of said capsules have an indicator dye.

15. A method of calibrating a detector comprising adding a stabilized emulsion according to claim 14 to a detector and collecting data.