Emulsions with improved stability

Abstract

The present invention relates to an emulsion, preferably a water-in-oil emulsion. comprising: a continuous phase comprising a conductivity improving compound, a dispersed phase suspended in the continuous phase, and a surfactant. The present invention also relates to a population of droplets comprising an aqueous phase, dispersed in a continuous oily phase comprising an oil and a conductivity-improving compound. The present invention also relates to a microfluidic chip comprising a hydrophobic composition comprising an oil, a surfactant and a conductivity improving compound in an injection chamber configured so that injecting a hydroplilic composition through said injection means will generate an emulsion in the injection chamber; and to a kit comprising a microfluidic chip and a container comprising an aqueous composition. The present invention also relates to a process for manufacturing an emulsion according to the invention. The present invention also provides methods for analyzing biological material, for example for analyzing biological material within the emulsion of the invention.

Claims

1. An emulsion comprising: a continuous oily phase which is a solution comprising a fluorinated oil and a conductivity-improving compound, wherein said conductivity-improving compound is an ionic liquid, a dispersed aqueous phase dispersed in said continuous oily phase, and a fluorinated surfactant.

2. The emulsion according to claim 1, wherein said fluorinated surfactant is selected from the group consisting of perfluoro-octanol; 1H,1H,2H,2H-perfluoro-1-octanol; perfluoro-decanol; 1H,1H,2H,2H-perfluoro-1-decanol; perfluoro-tetradecanoic acid; perfluoro-tetradecanoic oligo ethylene glycol; perfluoropolyether; perfluoropolyether-polyethylene glycol; perfluoropolyether-polyethylene glycol-perfluoropolyether; perfluoropolyether-dimorpholinophosphate; polyhexafluoropropylene oxide carboxylate; polyhexafluoropropylene oxide-polyethylene glycol-polyhexafluoropropylene oxide; polyhexafluoropropylene oxide-polyether-polyhexafluoropropylene oxide; polyhexafluoropropylene oxide-polypropylene glycol-polyethylene glycol-polypropylene glycol-polyhexafluoropropylene oxide and mixtures thereof.

3. The emulsion according to claim 1, wherein said fluorinated oil is selected from the group consisting of perfluoro-hexane; perfluoro-cyclohexane; perfluoro-decaline; perfluoro-perhydrophenantrene; poly-hexafluoropropylene oxide; perfluoro polytrimethylene ether; poly perfluoroalkylene oxide; fluorinated amines, tri(perfluoropentyl)amine, mixture of perfluorooctane amine and perfluoro-1-oxacyclooctane amine, perfluorotripropylamine; fluorinated ethers, 3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane, 2,3,3,4,4-pentafluorotetrahydro-5-methoxy-2,5-bis[1,2,2,2-tetrafluoro-1-trifluoromethyl) ethyl]-furan; and mixtures thereof.

4. The emulsion according to claim 1, wherein said ionic liquid comprises a fluorinated anion, a fluorinated cation, or a mixture thereof.

5. The emulsion according to claim 1, wherein said ionic liquid is selected from the group consisting of N-trimethyl-N-butylammonium bis(trifluoromethanesulfonyl)imide, 1-butyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, 1-hexyl-3-methylimidazolium tris(pentafluoroethyl)trifluorophosphate, 2,3-dimethylimidazolium tetracyanoborate, N-tributyl-N-methylammonium bis(trifluoromethanesulfonyl)imide, and mixtures thereof.

6. The emulsion according to claim 1, wherein said ionic liquid is N-tributyl-N-methylammonium bis(trifluoromethanesulfonyl)imide.

7. The emulsion according to claim 1, wherein said aqueous phase comprises biological material, reagents, or a mixture thereof for performing a chemical reaction, a biological reaction, or a chemical and biological reaction.

8. A microfluidic device comprising: a microchannel, a droplet generator means, and the emulsion according to claim 1 in the microchannel.

9. A method for analyzing a biological material, comprising: forming the emulsion according to claim 1, the emulsion comprising a biological material; processing said biological material in said emulsion, and detecting said biological material in said emulsion.

10. The method according to claim 9, wherein said biological material is cells or nucleic acids.

11. The method according to claim 9, wherein the step of processing of the biological material comprises amplifying the biological material.

12. The method according to claim 11, wherein the step of processing of the biological material comprises amplifying the biological material by PCR.

13. The method according to claim 11, wherein the step of processing of the biological material comprises amplifying the biological material by droplet digital PCR (ddPCR).

14. The emulsion according to claim 1, wherein said ionic liquid comprises bis(trifluoromethanesulfonyl)imide.

15. The emulsion according to claim 7, wherein said biological material is a nucleic acid template.

16. The emulsion according to claim 3, wherein said poly-hexafluoropropylene oxide is a poly-hexafluoropropylene oxide with carboxylic end group; or said fluorinated amine is N-bis(perfluorobutyl)N-trifluoromethyl amine; or said fluorinated ethers is a mixture of methyl nonafluorobutyl ether and methyl nonafluoroisobutyl ether.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a photograph showing an epifluorescence imaging of an emulsion with Phase 1 as a continuous phase immediately after emulsification and thermic treatment.

(2) FIG. 2 is a photograph showing an epifluorescence imaging of an emulsion with Phase 1 as a continuous phase after rubbing with a lab glove.

(3) FIG. 3 is a photograph showing an epifluorescence imaging of an emulsion with Phase 2 as a continuous phase immediately after emulsification and thermic treatment.

(4) FIG. 4 is a photograph showing an epifluorescence imaging of an emulsion with Phase 2 as a continuous phase after rubbing with a lab glove.

(5) FIG. 5 is a photograph showing an epifluorescence imaging of an emulsion with Phase 3 as a continuous phase immediately after emulsification and thermic treatment.

(6) FIG. 6 is a photograph showing an epifluorescence imaging of an emulsion with Phase 3 as a continuous phase after rubbing with a lab glove.

EXAMPLES

(7) The present invention is further illustrated by the following examples.

Example 1: Emulsions without Conductivity-Improving Compound

(8) The Applicant has studied the occurrence and intensity of electro-coalescence in emulsions of a given aqueous phase (a PCR mix, hereafter named Aqueous Phase A) and two different continuous phases (hereafter named Oily Phase 1 and Oily Phase 2).

(9) As showed in Table 2 below, FC-40 and Novec-7500 are two fluorinated oil presenting very similar physical properties. The only physical property for which the difference between the oils is higher than the order of magnitude is the electrical conductivity: the electrical conductivity of Novec-7500 is more than a million times higher than that of FC-40.

(10) TABLE-US-00002 TABLE 2 Novec-7500 FC-40 Boiling point (° C.) 128 155 Melting point (° C.) −100 −57 Molecular weight (g/mol) 414 650 Critical Temperature (° C.) 261 270 Critical Pressure (MPa) 1.55 1.18 Vapor Pressure (kPa) 2.1 0.43 Heat of Vaporization (kj/kg) 89 68 Liquid Density (kg/m3) 1614 1850 Coefficient of Expansion (/K) 0.0013 0.0012 Kinematic Viscosity (cSt) 0.77 1.8 Absolute Viscosity (cP) 1.24 3.4 Specific Heat (J/kg-K) 1128 1100 Thermal Conductivity (W/m-K) 0.0065 0.065 Surface Tension (mN/m) 16.2 16 Solubility in Water (ppm by weight) <3 <5 Dielectric Strength, 0.1″ gap (kV) ~40 >40 Dielectric Constant @ 1 kHz 5.8 1.9 Electrical conductivity (S/m) 45 × 10.sup.−8 25 × 10.sup.−15

(11) Behavior of two water-in-oil emulsions having two different continuous phases was studied.

(12) Materials and Methods

(13) Aqueous Phase A was manufactured by diluting in Nuclease-Free water: 20% v/v Perfecta PCR Toughmix Mastermix 5x (from Quantaboisciences, USA), fluorescein sodium salt (100 nM final concentration), PCR primers (final concentration 500 nM), and FAM-labelled probes to amplify and detect in Blue epifluorescence the gene that encodes for the protein BRAF in human genomic DNA (final concentration 250 nM).

(14) Various quantity of human genomic DNA (200 to 400 copies per microliter) was then added depending on the experiment.

(15) Oily Phase 1 was manufactured by diluting 5% w/w of fluorinated surfactant polyhexafluoropropylene oxide-polyethylene glycol-polyhexafluoropropylene oxide (PHFPO-PEG-PHFPO) in fluorinated oil 3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane (3M™ Novec™ 7500). PHFPO-PEG-PHFPO is described in European patent application EP2315629 (in particular in Example 1, reaction 2), which is hereby entirely incorporated by reference.

(16) Oily Phase 2 was manufactured by diluting 5% w/w of fluorinated surfactant polyhexafluoropropylene oxide-polyethylene glycol-polyhexafluoropropylene oxide (PHFPO-PEG-PHFPO) in fluorinated oil N-bis(perfluorobutyl)N-trifluoromethyl amine (3M™ Fluorinert™ FC 40).

(17) Two Sapphire chip (from Stilla Technologies SAS, France) were each loaded with 123 μL of oily Phase 1 or of oily Phase 2.

(18) 25 μL of aqueous Phase A was pipetted in the inlet port of each chip. A difference of pressure between the inlet and the outlet port was applied on each chip in order to produce two-dimensional water-in-oil emulsions of droplets within the chip. The aqueous Phase A constituted the discontinuous phase and the oily Phase 1 or 2 constituted the continuous phase of these emulsion.

(19) A thermal treatment (1—heating up to 95° C. for 5 minutes; 2—45 cycles of 95° C. for 30 seconds and 58° C. for 15 seconds; 3—cooling down to 25° C.) was then applied to the emulsions to activate the PCR reaction in the dispersed phases. Each of the chip was then loaded into an inverted epifluorescence microscope (Prism3—Stilla Technologies, France) and microscope images were recorded.

(20) After imaging, each of the chip was taken out of the microscope and rubbed with a nitrile laboratory glove, in order to simulate experimental manipulation of the Sapphire chips which is known to cause electrocoalescence. Each of the chip was then loaded back into the microscope and microscope images were recorded.

(21) Results

(22) The microscope images of the emulsions before rubbing for the experiments using the oily Phases 1 and 2 are displayed respectively in FIG. 1 and FIG. 3.

(23) As shown in FIG. 1, the emulsion comprising oily Phase 1 withstood thermal treatment for PCR amplification and was suitable for subsequently imaging. By contrast, as shown in FIG. 3, the emulsion comprising the oily Phase 2 displayed large regions of coalesced droplets, indicating that previous electro-coalescence events took place within the emulsion.

(24) The microscope images of the emulsions after rubbing for the experiments using the emulsions comprising oily Phase 1 and 2 are displayed respectively in FIG. 2 and FIG. 4.

(25) As shown in FIG. 2, the emulsion comprising oily Phase 1 displayed many large droplets due to electrocoalescence. However, it was still suitable for quantify DNA by digital PCR. By contrast, as shown in FIG. 4, the emulsion comprising oily Phase 2 had almost totally coalesced into a few large droplets, and could no more be used for quantifying DNA by digital PCR.

(26) This experiment thus evidences the technical problem of the sensitivity of emulsions to electrocoalescence, to be solved within the present invention.

Conclusion

(27) The experiments thus showed that: 1. The occurrence frequency of electro-coalescence events under a given electrical forcing is higher in the emulsion wherein the continuous phase is FC-40. 2. The threshold of the intensity of the electrical forcing required to induce an electro-coalescence event is lower in the emulsion wherein the continuous phase is FC-40. 3. The scale of an electro-coalescence event, i.e. the number of droplets that coalescence during an electro-coalescence event, is higher in the emulsion wherein the continuous phase is FC-40.

(28) In conclusion, the emulsion wherein the continuous phase (Novec-7500) has a relatively higher electrical conductivity is less sensitive to electro-coalescence than an emulsion wherein the continuous phase (FC-40) has a relatively lower electrical conductivity.

(29) Therefore, the reduction of sensitivity for emulsions with highly conductive continuous phases is apparent through three parameters: (1) the occurrence frequency of electro-coalescence events under a given stimulation decreases as the electrical conductivity of the continuous phase increases; (2) the threshold of the intensity of the stimulation required to induce electro-coalescence events can occur increases as the electrical conductivity of the continuous phase increases; (3) the scale of an electro-coalescence event decreases as the electrical conductivity of the continuous phase increases.

Example 2: Emulsion with Conductivity-Improving Compound

(30) Materials and Methods

(31) Oily Phase 3 was manufactured by diluting 5% w/w of fluorinated surfactant polyhexafluoropropylene oxide-polyethylene glycol-polyhexafluoropropylene oxide (PHFPO-PEG-PHFPO) and 5% w/w of ionic liquid N-tributyl-N-methylammonium bis(trifluoromethanesulfonyl)imide (99.5% purity from Solvionics, France), in fluorinated oil 3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane (3M™ Novec™ 7500). Oily Phase 3 was thus equivalent to oily Phase 1 wherein a conductivity-improving compound had been added.

(32) A two-dimensional emulsion of aqueous Phase A in oily Phase 3 is manufactured within a Sapphire Chip in the conditions described in Example 1. A thermal treatment, imaging, rubbing and imaging were applied to this emulsion as described in Example 1.

(33) Results

(34) The microscope images of the emulsions before rubbing for the experiments using the oily Phase 1 (of Example 1) and oily Phase 3 are displayed respectively in FIG. 1 and FIG. 5.

(35) FIG. 1 and FIG. 5 show than in both cases, the emulsions were sufficient stable to withstand thermal treatment for PCR amplification and to be subsequently imaged. The presence of the ionic liquid as conductivity-improving compound at 6% v/v in the oily Phase 3 did not destabilize the emulsion compared to the emulsion comprising oily Phase 1 which did not comprise the conductivity-improving compound.

(36) The microscope images of the emulsions after rubbing for the experiments using the emulsions comprising oily Phase 1 and 2 are displayed respectively in FIG. 2 and FIG. 6.

(37) As shown in FIG. 2 and FIG. 6, the emulsion comprising oily Phase 3 was almost not affected by rubbing and electro-coalescence as it displayed only a few small coalesced droplets, whereas the emulsion comprising oily Phase 1 displayed many large coalesced droplets. Emulsion comprising oily Phase 3

(38) This experiment evidences that the presence in the continuous phase of an emulsion of a conductivity-improving compound, such as an ionic liquid, significantly reduce the sensitivity of the emulsion to electrocoalescence.

Example 3: ddPRC Assay

(39) Materials and Methods

(40) The ability to quantify DNA by digital PCR using emulsions comprising the oily Phases 1, 2 and 3 (of Examples 1 and 2) was measured using Sapphire Chips primed each with one of the oily Phases 1, 2 and 3 and by running a digital PCR assay on the Naica System, a digital PCR system provided by Stilla Technologies.

(41) To quantify DNA by digital PCR, an emulsion was formed inside the Sapphire Chip using the aqueous phase of Example 1 as dispersed phase and one of the oily Phase 1, 2 or 3 as the continuous phase.

(42) The Sapphire Chips underwent a thermal treatment using the Naica Geode instrument of the Naica System to induce PCR amplification of the DNA targets in the emulsion and were subsequently imaged using the Naica Prism3 instrument.

(43) In the fluorescence images, droplets having a strong fluorescent signal after thermocycling are droplets that initially contained at least one amplifiable target DNA in each of them. These droplets are called positive droplets. Droplets having a low fluorescent signal are droplets that initially did not contain an amplifiable target DNA. These droplets are called negative droplets.

(44) Consequently, the initial amount of DNA target in the aqueous solution can be measured by counting the number of positive and negative partitions in the images and applying a statistical correction.

(45) Image analysis and DNA quantification is automatically performed using the Crystal Miner software from Stilla Technologies for the experiments using emulsions comprising the oily Phases 1, 2 and 3.

(46) Results

(47) Following Table 3 shows the results of the analysis.

(48) TABLE-US-00003 TABLE 3 Contin- DNA Incer- uous Ana- quanti- titude oily lyzable Lost droplets fication coef- phase droplets after rubbing (copies/μL) ficient Phase 1 Before 26557 — 336.0 3.2% rubbing After 5061 21496 (81%) 353.6 7.3% rubbing Phase 2 Before 18808 — 235.1 4.8% rubbing After 1065 17743 (94%) 251.5 19% rubbing Phase 3 Before 18336 — 328.7 3.8% rubbing After 17831    505 (2.8%) 331.7 3.8% rubbing

(49) Using emulsions comprising oily Phases 1 and 2, rubbing leads to a loss of 80% to nearly 95% of analyzable droplets, leading to a significant increase (from twice to 4-times) in the uncertainty coefficient of the DNA quantification.

(50) For the emulsion comprising the oily Phase 3, rubbing only leads to a loss of 3% of the total analyzable droplets, with insignificant effects on the uncertainty of the measurement.

(51) This experiment evidences that the presence of conductivity-improving compound, such as an ionic liquid, does not inhibit PCR amplification within the droplets while successfully preventing electro-coalescence of the emulsion during handling of the Sapphire Chip.