Emulsion chemistry for encapsulated droplets

• US 9,598,725 B2
• Filed: 12/22/2010
• Issued: 03/21/2017
• 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:
  US12/976,827
• Publication Number:
  US 20110217712A1
• Time in Patent Office: 2,281 Days
• Field of Search: None
• US Class Current: 1/1
• 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 amplifying target nucleic acids in skin-encapsulated capsules comprising:

a) providing an aqueous phase comprising at least about 0.01% by weight of a skin-forming protein, an amplification reagent mix and target nucleic acids;

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

c) heating said emulsion to at least about 50°

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

d) amplifying said target nucleic acids in said capsules; and

e) detecting amplification of said target nucleic acids.

2. A method of amplifying according to claim 1 wherein said oil is a fluorinated oil.

3. A method of amplifying according to claim 1 wherein said step (d) includes a step of thermally cycling said capsules through multiple rounds of heating and cooling after the step of heating.

4. A method of amplifying according to claim 1 wherein said skin forming protein is selected from the group consisting of albumin, casein, gelatin, and globulin.

5. A method of amplifying according to claim 4 wherein said protein is bovine serum albumin (BSA).

6. A method of amplifying according to claim 1 wherein said amplification reagent mix comprises dNTPs, a polymerase and nucleic acid primers.

7. A method of amplifying according to claim 1 wherein said aqueous phase comprises said skin-forming proteins at a concentration of above about 0.03% by weight.

8. A method of amplifying according to claim 1 wherein said aqueous phase comprises said skin-forming proteins at a concentration of above about 0.1% by weight.

9. A method of amplifying according to claim 1 wherein said aqueous phase further comprises an aqueous phase surfactant comprising a block copolymer of polypropylene oxide and polyethylene oxide.

10. A method of amplifying according to claim 9 wherein said aqueous phase surfactant is at a concentration of about 0.01% to 10% by weight.

11. A method of amplifying according to claim 1 wherein said negatively charged ionic surfactant is a fluorinated surfactant.

12. A method of amplifying according to claim 1 wherein said negatively charged ionic surfactant is a carboxylate.

13. A method of amplifying according to claim 1 wherein said negatively charged ionic surfactant is present in said continuous phase at a concentration of about 0.02% to 0.5% by weight.

14. A method of amplifying according to claim 1 further comprising a step of selectively removing a portion of the continuous phase after the step of forming an emulsion and before the step of heating the emulsion.

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

a) providing an aqueous phase comprising at least about 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; and

c) heating said emulsion to at least about 50°

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

16. A method according to claim 15 wherein said oil is a fluorinated oil.

17. A method according to claim 15 wherein said ionic surfactant is a negatively charged surfactant.

18. A method according to claim 17 wherein said surfactant is a fluorinated surfactant.

19. A method according to claim 15 wherein said oil is a fluorinated oil and said ionic surfactant is a negatively charged fluorinated surfactant.