Desmulsification of water-in-silicon-oil emulsion

Abstract

Fluorocarbon free emulsions, and compositions and methods for breaking fluorocarbon free emulsions are provided. Many compositions comprise silicone or siloxane based emulsions, surfactants and emulsion breakers.

Description

EMULSIFIERS, SURFACTANTS AND EMULSION BREAKINGCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This document claims the benefit of priority to US Provisional Application Serial No.63 / 778,646, filed March 27, 2025, the contents of which are hereby incorporated by reference in their entirety.BACKGROUND

[0002] Emulsions facilitate a broad range of high throughput biomolecular analyses by allowing one to segregate biomaterials such as nucleic acids or cells, among others, and to perform analyses in parallel in individual emulsion droplets.

[0003] However, disruption of emulsions may be challenging, in some cases requiring heat or addition of reagents harmful to the biomolecules being studied. Furthermore, many emulsions are formed using fluorocarbon oil carriers, which complicates waste disposal.SUMMARY

[0004] Disclosed herein are methods of breaking a water-in-oil emulsion comprising a silicone carrier and in some cases a surfactant. Some such methods comprise contacting the water-in-oil emulsion to a detergent such as a dimethyl siloxane polymer having at least one hydrophilic side branch, or a non-silicone surfactant.

[0005] Some such methods comprise adding a excess of an aqueous composition so as to dissolve the oil carrier into the aqueous composition. Some such methods comprise adding a detergent or surfactant such as an ionic surfactant, such as SDS, or a nonionic surfactant or detergent, such as Triton X-100, IGEPAL, or Tween-20.

[0006] Also disclosed herein are emulsions, such as emulsions comprising a silicone oil carrier and in some cases a siloxane surfactant.

[0007] Similarly, disclosed herein are single phase liquid compositions. Some such compositions comprise a silicone oil carrier and an aqueous biomaterial, and in some cases further comprising a siloxane surfactant.

[0008] Similarly, disclosed herein are methods of emulsion breaking. Some such methods comprise adding to an emulsion an emulsion breaking oil and an emulsion breaker, and adding an aqueous phase. Some such methods comprise adding to an emulsion an emulsion breaking aqueous phase.

[0009] Further, disclosed herein are methods of breaking a water-in-oil emulsion comprising a silicone carrier. Some such methods of breaking a water-in-oil emulsion comprise contacting the water-in-oil emulsion comprising a silicone carrier to an emulsion breaker.

[0010] Also, disclosed herein are emulsions, such as emulsions comprising a silicone oil carrier and a dispersed aqueous droplet population.

[0011] Also, disclosed herein are single phase liquid compositions. Some such single phase liquid compositions comprise a silicone oil, a surfactant, and an aqueous biomaterial, wherein the silicone oil and the surfactant are dissolved in a single aqueous phase.

[0012] Also, disclosed herein are methods of emulsion breaking. Some such methods of emulsion breaking comprise adding to an emulsion an emulsion breaking oil and an emulsion breaker, and adding an aqueous phase.

[0013] Similarly, disclosed herein are methods of emulsion breaking. Some such methods of emulsion breaking comprise adding to a silicone oil emulsion an emulsion breaking aqueous phase.INCORPORATION BY REFERENCE

[0014] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] At Fig. 1 A, one sees a silicone monomer dimethyl silane (DMS) which is iterated “y” times in a carrier oil of the disclosure herein.

[0016] At Fig. IB, one sees a silicone monomer phenyl methyl silane (FMS) which is iterated “x” times in a carrier oil of the disclosure herein.

[0017] At Fig. 2A, one sees a surfactant compatible with the carrier oils disclosed herein.

[0018] At Fig. 2B, one sees a surfactant compatible with the carrier oils disclosed herein.

[0019] At Fig. 3, one sees an emulsion breaking compound or demulsifier compatible with the carrier oils disclosed herein.

[0020] At Fig. 4, one sees a cartoon depiction of two-phase emulsion breaking.

[0021] At Fig. 5, one sees emulsions contacted to demulsifiers heptanol, octanol, and nonanol pursuant to two phase emulsion breaking.

[0022] At Fig. 6 one sees a schematic single phase demulsification.

[0023] At Fig. 7, one sees a compound or group of compounds consistent with the disclosure herein.

[0024] At Figure 8, one sees the results of emulsion breaking.

[0025] At Figure 9, one sees structures of representative detergents.

[0026] At Figure 10, one sees emulsion breaking using high concentration detergents.

[0027] At Figure 11, one sees emulsion breaking using low concentration detergents.

[0028] At Figure 12, one sees a silicone oil emulsion comprising microcapsules.DETAILED DESCRIPTION

[0029] Introduction. Disclosed herein are compositions and methods related to silicone carrier emulsions, including use in generating and utilizing microdroplets as segregated reaction chambers, and recovery of contents for downstream analysis such as bulk analysis. Through the disclosure herein, one may used silicone based carriers and surfactants for emulsion generation, bioparticle manipulation, and release for downstream analysis such as bulk downstream analysis, in some cases without reliance upon fluorocarbons or chlorofluorocarbons and the associated fluoro-organic waste.

[0030] Compositions. A range of compositions are disclosed or consistent with the disclosure herein. Some such compositions are molecules or individual constituents, while other compositions relate to mixtures of molecules or products of reactions, or phase changes or reaction products resulting from contact of compositions herein to one another.

[0031] Emulsions. Disclosed herein are emulsions, such as water-in-oil emulsions, and oil carrier constituents for such emulsions. Notably, the carriers oils used for these emulsions often comprise silicone oils rather than fluorinated oils or fluorocarbons.

[0032] Carrier oils are often silicone oils, comprising an iteratively repeated [-Si-O-] backbone, the Si of which further comprising two carbon moieties bound thereto. Exemplary silicone oils include poly-dimethyl silane (DMS), as shown in Fig. 1 A. Variants where one or both methyl groups are replaced, such as using a hydrophobic alternate moiety, are also contemplated.Various alternatives include, in place of one or both methyl groups, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or higher order linear or branched alkanes, or cyclic moieties such as phenyl moieties. For example, one sees at Fig. IB a monomer unit of a silicone oil for which onemethyl group of the monomer unit is substituted with a phenyl group, so as to constitute phenyl methyl silicone (FMS) monomer units.

[0033] Carrier oils further comprise capping or bookend moieties such as methyl (CH3) capping moieties or silicone trimethyl Si (CH3)3 moieties at the ends of the poly-monomer chain.

[0034] Carrier oils are in some cases homogeneous, such that they comprise individual molecules comprising repetitions of a single monomer unit such as DMS polymers or FMS polymers. Polymers variously comprise about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 monomer units.

[0035] Carriers are in some cases heterogeneous, in that they comprise a nonuniform population of individual polymer molecules. Carriers comprise, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, for example by weight, by volume or by molar concentration, of a first uniform polymer molecule such as poly DMS or another silicone oil and a remaining portion of one or more second uniform polymers, or second and third uniform polymers. One representative heterogeneous oil comprises 66% poly DMS and 33% FMS.

[0036] Individual molecules are in some cases heterogeneous in that they comprise more than one type of individual monomer constituent. An individual molecule comprises, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 33%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 66%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of afirst monomer constituent such as DMS or another silicone oil monomer and a remaining portion of one or more second monomer constituent, or second and third monomer constituent. One representative heterogeneous molecule comprises 66% DMS monomer constituents and 33% FMS monomer constituents. Heterogeneous individual molecules in some cases are segregated as to their monomer constituents, such that a first monomer subpopulation forms a first block while a second monomer subpopulation forms a second block adjacent or separate rather than intermingled with the first subpopulation. A first subpopulation block variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 monomer units. Similarly, a second subpopulation block variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 monomer units.

[0037] A number of alternate carriers are consistent with the disclosure herein. Generally, carriers share the properties that they lack or are clear of fluorocarbons, which are popular or common carriers in biological emulsions, but which are problematic because their disposal is complicated by their environmental impact.

[0038] Alternate carriers often share the additional trat of comprising silicone or a silicone component. Silicone oils are well known but not employed in the emulsion space because of a perceived difficulty in breaking emulsions or recovering emulsion droplet contents.

[0039] Exemplary silicone oils consistent with the disclosure herein include linear siloxane polymers, such as tri-siloxane, tetra-siloxane, or other length siloxanes such as di-siloxane, penta-siloxane, hexa-siloxane, hepta-siloxane or higher order siloxanes.

[0040] Alternatively or in addition to linear siloxanes, some oils comprise circular siloxanes, such as cyclical siloxanes (D3 - tri, D4 - tetra, D5 - penta, or larger cyclical siloxanes).

[0041] Siloxanes having ethyl side groups are also contemplated, as are siloxanes comprising any of a broad range of alkyl or phenyl side chains. Some additional siloxanes suitable with some embodiments of the technology include, among others, Methyltris(trimethylsiloxy)silane, Octyltris(trimethylsiloxy)silane, Phenyltris(trimethylsiloxy)silane, and Phenethyltris(trimethylsiloxy)silane, among others.

[0042] Exemplary siloxane carriers exhibit a viscosity suitable for droplet and emulsion formation, such as a viscosity in the range of 0.5-10cSt, such as 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, lOcSt, or a number spanned by or outside of the given range. In some cases, the silicone oil carrier has a viscosity of 0.5-1 OcSt, including 0.5-9.5cSt, 0.5-9cSt, 0.5-8.5cSt, 0.5-8cSt, 0.5-7.5cSt, 0.5-7cSt, 0.5-6.5cSt, 0.5-6cSt, 0.5-5.5cSt, 0.5-5cSt, 0.5-4.5cSt, 0.5-4cSt, 0.5-3.5cSt, 0.5-3cSt, 0.5-3cSt, 0.5-2.5cSt, 0.5-2cSt, 0.5-1.5cSt, and 0.5-lcSt, 2-10cSt, 2-9cSt, 2-8cSt, 2-7cSt, 2-6cSt, 2-5cSt, 2-4cSt, 2-3cSt, l-2cSt, 3-10cSt, 4-10cSt, and 5-10cSt. Similarly, exemplary linear silicone oils in some cases exhibit an ability to dissolve a surfactant or surfactants added to the silicone oil carrier.

[0043] Silicone oils are a homogeneous liquid or are alternatively are a mixture of two or more siloxane oils, at least one of which is a linear siloxane. Siloxane oil mixtures are often selected from monomer lengths and proportions so as to achieve a target, selected or desired property or properties, such as viscosity of, for example, no greater than lOcSt, no greater than 9, no greater than 8, no greater than 7, no greater than 6, no greater then 5, no greater than 4, no greater than 3, no greater than 2, or no greater than 1, among others. Some blended or homogeneous siloxane oils are selected to achieve a specific viscosity number of 1 , 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9 or 2.0cSt, or a number spanned by or adjacent to that range. Blending is achieved in some cases through adding relative proportions of tri-siloxane, and tetra-siloxane, though other siloxanes are also contemplated and suitable in some embodiments.

[0044] Some compositions comprise two linear siloxanes, such as tri-siloxane and tetra-siloxane, at a molar percent or a percent mass of l%-99%, 2%-98%, 5%-95%, 10%-90%, 20%-80%, 30%-70%, 40%-60%, 50%-50%, 60%-40%, 70%-30%, 80%-20%, 90%-10%, 95%-5%, 98%-2%, 99%-l%, or a percent ratio spanned by the rage provided herewith. Some compositions comprise two siloxanes at a proportion as indicated above relative to one another, and a third siloxane at a proportion indicated above relative to the combination of the first and second siloxane. Some compositions comprise four or more types of siloxanes.

[0045] A carrier in some cases further comprises a surfactant or other moiety to reduce tension between the aqueous and oil phases of an emulsion. Some surfactants, like some of the silicone oil constituents above, are not fluorinated or do not comprise a fluorocarbon moiety or moieties.

[0046] Representative surfactants as disclosed herein often comprise multiple functional or structural units or blocks, such as a lipophilic block, hydrophilic block, and a core or backbone such as a silane block. Schematics of representative surfactants re shown in Fig. 2A and Fig. 2B.

[0047] An exemplary lipophilic bloc comprises, for example, a cetyl lipid moiety tethered to one or more monomer units of a silane backbone. A more full range of alternatives include, for example, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or higher order linear or branched alkanes, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more than 30 member linear or beached alkanes, or a ring motif such as a phenyl ring.

[0048] Lipophilic moieties are attached to one or more monomer units of a backbone such as a silane or siloxane backbone. A hydrophobic bloc variously comprises about, at most, at least orexactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 monomer units. Similarly, a hydrophobic bloc variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 polymeric moieties attached to a silane, siloxane or other backbone.

[0049] An exemplary hydrophilic bloc comprises, for example, a methyl capped poly-PPG moiety tethered to one or more monomer units of a silane or siloxane backbone. Suitable block constituents include PPG or PPE, as shown in Fig. 2A, and an alternative in Fig. 2B, as well as a number of alcohols, ethers, esters, amines, or ketones, among others, such as polyvinyl alcohol, polyethylenimine, polyacrylic acid, PEG, or other hydrophilic monomer unit. A hydrophilic bloc variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 monomer units. Similarly, a hydrophilic bloc variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 polymeric moieties attached to a silane, siloxane or other backbone.

[0050] An exemplary core bloc comprises ‘copies’ of the core backbone attached to the core backbone as polymers branching from the core. See again Fig. 2 A and Fig. 2B. In that figure, the core backbone and third block comprise siloxane DMS monomer blocks, with the side chains tethered to the core backbone through C-C linkages. A siloxane bloc variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150monomer units. Similarly, a siloxane bloc variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 polymeric moieties attached to a silane, siloxane or other backbone.

[0051] As alternatives to siloxane, variant blocks may comprise monomeric units where one or both methyl group are replaced, such as using a hydrophobic alternate moiety, are also contemplated. Various alternatives include, in place of one or both methyl groups, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl or higher order linear or branched alkanes, or cyclic moieties such as phenyl moieties as in phenyl methyl silicone (FMS) monomer units.

[0052] Although substantial variation exists in the identity of block side chains, size of blocks as measured in number of side chains per block, and length of block side chains, many surfactants share the common structure exemplified in Fig. 2A or Fig. 2B.

[0053] Some such molecules, such as molecules of Fig. 2B have a molecular weight ranging from, for example, 3000-6000, such as about 3000, 3500, 4000, 4500, 5000, 5500, 6000, or a value spanned by or adjacent to the given range. In some cases, the molecular weight can range from 3000 to 6000, including from 3000 to 5500, from 3000 to 5000, from 3000 to 4500, from 3000 to 4000, from 3000 to 3500, from 3500 to 6000, from 3500 to 5500, from 3500 to 5000, from 3500 to 4500, from 3500 to 4000, from 4000 to 6000, from 4000 to 5500, from 4000 to 5000, from 4000 to 4500, from 4500 to 6000, from 4500 to 5500, from 4500 to 5000, from 5000 to 6000, and from 5500 to 6000.

[0054] Some such molecules, such as molecules of Fig. 2B have an m polymer value ranging from 1-100, 1-50, 1-20, 2-20, 5-20, 8-18, or 10-16, for example, as well as an n polymer valueranging from 1-100, 1-50, 1-20, 1-10, 1-8, or 2-6, for example, as well as a p polymer value ranging from 1-100, 1-50, 5-50, 10-40, or 15-35, for example.

[0055] Some such molecules exhibit a molecular weight of 3000-6000, m in the range of 10-16, n is in the range 2-9, 2-6 or 6-9, and p in the range of 15-35.

[0056] A broad range of variant surfactants are also consistent with the disclosure herein.Surfactants are generally free of fluorocarbons or fluorination, and are soluble in the oil carrier with which they are used. Exemplary surfactants comprise di-block or tri-block siloxane polymer. Exemplary surfactants comprise tri-block siloxane polymer.

[0057] The carrier and surfactants serve in some cases to facilitate formation of emulsions, such as water-in-oil emulsions. The water or aqueous droplets of the emulsion may comprise biological material and may further comprise at least one reagent for processing or analysis of the biological material, such as a protease, cell lysis reagent, polymerase and nucleotides, reverse transcriptase, or barcodes, such as droplet identifying barcodes as may be used to barcode or otherwise tag the contents of the droplet so as to distinguish them from contents of at least one other droplet of the emulsion.

[0058] That is, droplet contents are in some cases barcoded such that contents of a first droplet share a common first barcode that allows one to distinguish the contents of the first droplet from contents of a second droplet, that is either unbarcoded or barcoded using a second barcode that identifies barcoded biomaterial as not being from the first droplet or as being from the second droplet to the exclusion of the first droplet. Barcoded contents often comprise nucleic acids, such as nucleic acids, for example at least one category selected from the list including genomic fragments, exposed nucleic acid segments in chromatin, organellar genomic fragments, reverse-transcribed nucleic acids such as cDNA, organellar reverse transcribed nucleic acids, among others.

[0059] Barcoding in some cases is effected by co-segregation of a biological material and a barcode-carrying particle to a common droplet of an emulsion, such that the barcode information of the bead may be transferred to nucleic acids of the droplet. Often, specifically or uniquely tagging nucleic acids of a particular biological material, for example a cell, requires that the material and the barcoded bead be co-segregated to a bead to the exclusion of a second, separately segregated or unsegregated biological material such as a second droplet.

[0060] The water or aqueous droplets of the emulsion may comprise biological material and may further comprise at least one reagent for formation of a microcapsule such as a porous hydrogel microcapsule having an aqueous core, for example as described in PCT Publication No W02023 / 099610, published June 8, 2023, which is hereby incorporated by reference in its entirety.

[0061] The emulsions are formed, again, in some cases without reliance upon fluorocarbons or chlorofluorocarbons and the associated fluoro-organic waste.

[0062] Emulsions are in some cases ‘broken’ so as to allow one to bulk the emulsion contents for downstream analysis, such as packaging with appropriate library ends for bulk sequencing.

[0063] Emulsions are broken through any of a number of approaches. Some such approaches comprise adding aqueous phase in excess so that the carrier oil dissolves thereinto, rendering the carrier and the aqueous droplets mutually miscible, such that the composition becomes a single phase comprising carrier, surfactant, droplet contents and an emulsion breaking reagent.

[0064] Emulsion breaking is effected in some cases by adding to a composition an emulsion breaking reagent or demulsifier, such as that shown in Fig. 3 or any of those shown in Fig. 9.

[0065] A broad range of demulsifiers or emulsion breaking molecules are consistent with the disclosure herein. Generally, demulsifiers exhibit an ability to dissolve or to draw silicone oils into the aqueous phase. Some well known examples include Triton, IGEPAL, Tween and SDS, Brij-35 / Brij-58, n-Dodecyl-P-D-maltoside (DDM), Digitonin, Sodium Deoxy cholate (DOC), Laurosylsarcosine (Sarkosyl), CHAPS / CHAPSO, and Sulfobetaines (SB3-10, SB3-12) among others. Milder demulsifiers include, for example, Pluronic F-68, Pluronic F-127, Tetronics (Poloxamines), Polyvinyl Alcohol (PVA) polymers, Methylcellulose & Hydroxypropyl Methylcellulose (HPMC), Polyethylene Glycol (PEG) / PEG-Stearates and biosurfactants like Lecithin, Sophorolipids, Rhamnolipids, Surfactin, Aescin, or mixtures comprising one or more of phospholipids, triglycerides, fatty acids, carbohydrates, and phosphatidylcholine.

[0066] In some cases an emulsion breaker is ionic, while alternate emulsion breakers are nonionic.

[0067] Some exemplary demulsifiers or emulsion breaking reagents comprise at least two blocks and Si(Me3) end blocking moieties.

[0068] The first block comprises a polymer of DMS monomer or siloxane units. The siloxane bloc variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 monomer units. Conceptually, one may understand the first block in some cases as an extended unmodified backbone of monomeric units such as DMS monomeric units.

[0069] As alternatives to silane or siloxane, variant blocks may comprise monomeric units where one or both methyl group are replaced, such as using a hydrophobic alternate moiety, are also contemplated. Various alternatives include, in place of one or both methyl groups, ethyl, propyl,butyl, pentyl, hexyl, heptyl, octyl, nonyl or higher order linear or branched alkanes, or cyclic moieties such as phenyl moieties as in phenyl methyl silicone (FMS) monomer units.

[0070] The second block comprises a polymer of modified monomer units such as modified DMS monomer or siloxane units to which a hydrophilic moiety is attached. An exemplary moiety is a PEG polymer having a methyl terminal cap.

[0071] Suitable block constituents include PEG, as shown in Fig. 3, as well as a number of alcohols, ethers, esters, amines, or ketones, among others, such as polyvinyl alcohol, polyethylenimine, polyacrylic acid, PPG, or other hydrophilic monomer unit. A hydrophilic bloc variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 monomer units. Similarly, a hydrophilic bloc variously comprises about, at most, at least or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, or greater than 150 polymeric moieties attached to a silane, siloxane or other backbone.

[0072] Block constituents are tethered directly to the DMS core, or alternatively are tethered via an alkyl moiety, such as a propyl moiety as seen in Fig. 3.

[0073] Without being bound by theory, an effect of the first and second block of the emulsion breaking reagent is to create a molecule having a first region that is hydrophobic so as to be miscible with the carrier, while the second block is hydrophilic so as to be miscible with the aqueous droplet, such that the molecule facilitates miscibility between emulsion droplets and carrier, so as to bring the aqueous droplets and hydrophilic carrier into a single phase emulsion breaking composition.

[0074] The emulsion breaking reagent is in some cases at least, at most or about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% hydrophobic, such as 15%-45% hydrophobic, 20%-40% hydrophobic, 25% - 35% hydrophobic, or 30% hydrophobic. Exemplary molecules have a molecular weight ranging from, for example, 400-800, 500-700550-650 or 600, or a number spanned by or adjacent to the previous range.

[0075] Emulsion breaking reagent m, n, and p values vary across broad ranges in various embodiments, such as up to 75, up to 50, up to 35, up to 20, up to 10, or for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Exemplary n values are variously 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or greater, such as 1-5, 1-4, 1-3, 2 or 1. Exemplary m values are variously 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or greater, such as 1-5, 1-4, 1-3, 2 or 1. Exemplary p values are variously 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or greater, such as 1-5, 1-4, 1-3, 2 or 1.

[0076] An exemplary emulsion breaking reagent is 30% hydrophobic, has a molecular weight of 600 and has n, m and p values of 1, 2, and 2.

[0077] A broad range of emulsion breaking molecule concentrations are consistent with embodiments of the disclosure herein. Concentrations range variously from 0.01%, 0.02%, 0.03%, 0.033%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, 15%, or a value spanned by this range or falling outside of this rage. In some instances, the emulsion breaking molecule concentration ranges from 0.01% to 15%, including 0.01% to 10%, 0.01% to 5%, 0.01% to 1%, 0.01% to 0.8%, 0.01% to 0.5%, 0.01% to 0.4%, 0.1% to 1%, 0.1% to 0.8%, 0.1% to 0.5%, 0.5% to 1%, 0.7% to 1%, 0.8% to 1%, 1% to 15%, 5% to 15%, 5% to 10%, 10% to 15%, 0.05% to 1%, 0.05% to 5%, 1% to 5%, and 1% to 10%. In some instances, the emulsion breaking molecule concentration isno greater than 0.01%, 0.02%, 0.03%, 0.033%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, or 15%. In some instances, the emulsion breaking molecule concentration is at least 0.01%, 0.02%, 0.03%, 0.033%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10%, 11%, 12%, 13%, 14%, or 15%.

[0078] The single phase composition variously comprises carrier oil, surfactant, former droplet contents such as water and in some cases biomaterial such as nucleic acids, cell lysates, enzymes, intact cells, or other biomaterials either obtained from a sample or introduced pursuant to manipulation of the sample. In some cases the single composition comprises a first nucleic acid population that shares a common first tag and a second nucleic acid population that shares a second tag in the single phase population. The first tag may indicate that the first nucleic acid population arises from a common first source or was contained in a first emulsion droplet prior to forming the single phase composition. Similarly, the second tag may indicate that the second nucleic acid population arises from a common second source or was contained in a second emulsion droplet prior to forming the single phase composition. The first nucleic acid population variously comprises genomic segments, cDNA, or amplicons, for example, that may arise from a single cell or a single emulsion droplet.

[0079] In many cases the first population and the second population cannot be tagged in the single phase composition such that the first tag indicates a common origin for the first population and the second tag indicates a common origin for the second population. Rather, addition of the first tag and the second tag must occur prior to intermingling of the first nucleic acid populationand the second nucleic acid population in the single phase composition. In these cases, differential tagging indicates that the first nucleic acid population and the second nucleic acid population were previously segregated, such as into first and second emulsion droplets.

[0080] The single phase composition is in some cases subjected to further processing, such as nucleic acid purification, library end addition or other processing that does not rely upon segregation of nucleic acid or other biomaterial contents.

[0081] Single phase compositions in some cases comprise beads or microcapsules, such as hydrogel microcapsules, that comprise a hydrogel shell and a liquid core, such as those that may be formed in emulsion droplets prior to forming a single phase from the emulsions. In these cases, as in cases involving biomaterials, the ‘single phase’ refers to the liquid of the composition and does not exclude solid biomaterial or hydrogel material in the composition.

[0082] Single phase compositions resulting from broken water in oil emulsions are generated in various cases by vortexing, heating, or incubating at room temperature.

[0083] Single phase compositions generally are characterized by no longer comprising an oil carrier. The single phase composition may in some cases be nonuniform and may comprise, for example, hydrogel microcapsules or oil droplets that may be residual components of the formerly continuous oil carrier. In some cases the single phase composition is an aqueous carrier harboring one or more than one oil droplet. In many cases, a single phase composition is characterized by an aqueous carrier which is largely or fully in continuous liquid communication throughout most or all of its volume. Thus, for example, the compositions of Fig. 10-11 are in some cases considered single phase compositions even if residual or other oil droplets remain. Alternately, in some cases single phase compositions do not comprise or do not comprise substantial amounts of oil droplets.

[0084] The single phase composition are formed in some cases without reliance upon fluorocarbons or chlorofluorocarbons and the associated fluoro-organic waste. Similarly, bioparticles are recovered from the single phase composition without requiring special recovery or disposal of any fluorocarbons or chlorofluorocarbons and the associated fluoro-organic waste.Methods related to emulsion breaking

[0085] Disclosed herein are methods for performing reactions in segregated regions such as emulsion droplets, and recovery of the reaction products. Some such methods are consistent with the compositions above, or come to outcomes similar to outcomes consistent with the compositions above. Methods are practiced in many cases without reliance upon fluorocarbons or chlorofluorocarbons and without generation of the associated fluoro-organic waste. Methods are practiced in some cases without a distinct emulsion breaking step separate from downstream sample processing.

[0086] Bioparticles are suspended in an aqueous solution and introduced into a hydrophobic carrier such as the carriers disclosed above to form an emulsion.

[0087] A broad range of bioparticles are consistent with the disclosure herein. Some bioparticles comprise whole cells, cell lysates, proteins, chromosomes, nucleic acids or other material suitable for segregated analysis.

[0088] Some aqueous carriers comprise reactants for analysis of the bioparticles, such as lysozyme, polymerase, reverse transcriptase or other enzymes, nucleotides, oligos such as bead bound uniform populations of oligos, such as those that may allow later correlation of individual molecules to a particular droplet of origin, oligo randomers for specific target counting or other reagents.

[0089] Some methods comprise merging droplets, so as to successively deliver reagents, for example reagents for incompatible reactions to be performed successively on a single biomaterial.

[0090] Accordingly, some methods comprise encapsulating cells or other biomaterials, lysing the biomaterials using a lysozyme, for example, and then delivering reagents for nucleic acid labeling, such as barcoding the contents, for example using a barcoded bead as may be delivered in the second droplet via merger.

[0091] Some aqueous carriers comprise reagents for partial or total solidification of droplet contents, so as to form permeable or impermeable hydrogel beads or microcapsules having permeable or impermeable hydrogel shells enclosing aqueous or liquid interiors.

[0092] Accordingly, some methods comprise segregation of a biomaterial into a droplet followed by encasing the biomaterial in a hydrogel bead or enclosing it in the liquid or aqueous interior of a microcapsule such as a hydrogel microcapsule.

[0093] A central step in many of the methods herein is the breaking of any water-in-oil emulsion so as to facilitate collection or bulking of droplet contents. Some emulsion breaking approaches disrupt or dissolve water-oil boundaries, such that the water-in-oil two phase emulsion forms a single phase into which the aqueous droplet carrier and oil emulsion carrier become mutually miscible.

[0094] Some methods for breaking an emulsion comprise introducing an emulsion breaking molecule. Such a molecule comprises a hydrophobic moiety or moiety block and a hydrophilic moiety or block. Without being bound by theory, the molecule may render the aqueous and oil phases miscible by having its hydrophobic bloc introduced into the oil carrier while concurrently introducing its hydrophilic block into the aqueous drop, such that the molecule spans the formermiscibility border between the two liquid phases, thereby breaching this border and rendering the aqueous and oil phases miscible.

[0095] An example of a molecule or group of molecules consistent with this step of the method is seen at Fig. 3. It is further observed that surfactants such as the molecule or group of molecules seen in Fig. 2A or Fig. 2B similarly comprise a hydrophobic moiety or moiety block and a hydrophilic moiety or block.

[0096] A key feature of some embodiments of the emulsion breaking molecule or the emulsion breaking step of some methods herein is that the blocks introduce themselves into the aqueous and oil components of the emulsion, rather than localizing themselves to the interface. It is observed that molecules having more strongly hydrophobic and hydrophilic blocks exhibit a more disruptive effect on the aqueous / oil border so as to render the emulsion into a single phase liquid.

[0097] Emulsion breaking is in some cases effected without introducing a change of temperature, or without vortexing or shaking, or without physical disruption, or without introduction of a fluorocarbon or chlorofluorocarbon. Alternatively, some emulsion breaking comprises vortexing, shaking or other physical disruption.

[0098] Emulsion breaking in some cases comprises incubation at a temperature of at least 15 °C, at least 20 °C, at least 25 °C, at least 30 °C, at least 35 °C, at least 40 °C, at least 45 °C, at least 50 °C, at least 55 °C, at least 60 °C, at least 65 °C, at least 70 °C, at least 80 °C, at least 90 °C, at least 100 °C or higher than 100 °C, or at a temperature spanned by or outside of the range presented herein. Alternately, some methods comprise incubation at room temperature or below room temperature.

[0099] Emulsion breaking in some cases comprises fractionation, for example so as to allow residual oil droplets to be more conveniently collected and removed.

[0100] Fractionation in some cases comprises centrifugation. Duration and strength of centrifugation may vary according to one or more factors such as oil characteristics such as viscosity, demulsifier strength or concentration, or contents of the emulsion contents to be recovered. That is, for example, emulsions comprising live cells are centrifuged in some cases under conditions that maintain the viability of some or all of the cells. Alternatively, some emulsion compositions are centrifuged under harsh conditions, so as to increase the stringency of separation of the carrier from aqueous phase, or to facilitate collection of residual oil droplets in an otherwise uniform single phase product of emulsion breaking. Exemplary centrifuge conditions include centrifugation for 15, 30, or 45 seconds, or for 1, 2, or 5 minutes, or longer, or a duration spanned by our outside of the listed range, at a centrifugation intensity of 2000g, or for example 200g, 500g, 1000g, 2000g, 5000g, or a g-force spanned by or outside of the listed range.

[0101] Samples are in some cases vortexed prior to centrifugation, such as for 30 seconds at 1000 rpm, or alternately 5, 10, 15, 30, 60 seconds, or 2, 3,4 ,5, or more than 5 minutes, or a value spanned by or outside of this range, at 100, 200, 500, 1000, 2000 or greater than 2000 rpm, or a value spanned by or outside of this range.

[0102] Fractionation in some cases comprises allowing the phases to separate or the carrier to be dissolved without external separation. Separation may proceed for at most, at least, about, or for a period of 30 seconds, 1, 2, 5, 10, 20, or 30 minutes, or 1, 2, or more than 2 hours.

[0103] Through practice of some methods herein, no chlorofluorocarbon or fluorocarbon waste is generated.

[0104] Emulsion breaking or microparticle or microcapsule recovery as may occur pursuant to emulsion breaking in some cases comprises centrifuging.

[0105] Upon generation of a single phase liquid, such as a single phase liquid comprising a first population of nucleic acids having a first tag indicative of a first droplet localization and a second population of nucleic acids having a second tag indicative of a second droplet localization, the nucleic acids or other biomaterials may be further processed for sequencing. Alternatively, hydrogel beads or hydrogel shell microcapsules may be isolated from the single phase liquid, and subjected to further processing such as by introduction of iterative reaction environments, buffers or reagents so as to iteratively process the hydrogel or hydrogel shell microcapsule biomaterial contents.

[0106] Silicone oil emulsions may be broken, such that the emulsion droplet contents are released, though an number of approaches consistent with the disclosure herein.

[0107] Some approaches comprise ‘two-phase’ emulsion breaking. In these approaches, two phases are generated, with water in oil emulsion droplets being released into the aqueous phase of the emulsion two phase system. Emulsion contents may then be analyzed in bulk or recovered for downstream analysis.

[0108] In these approaches, a water-in-silicone-oil emulsion composition serves as the breakage target. To the composition is added an emulsion breaker or demulsifier, such as an emulsion breaker comprising an intermediate length alkyl alcohol. A broad range of linear and branched alkyl alcohols are consistent with the disclosure herein, with some exemplary alcohols comprising nonanol, octanol, or heptanol. Alternatively, longer alcohols are contemplated, such as decyl-alcohol, lauryl-alcohol, cetyl-alcohol, or an alcohol having an alkyl group of comparable length to those listed herein. Alcohols consistent with the disclosure comprise, invarious cases, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25 or greater than 25 carbon atoms. The emulsion breaker is added alone or in combination with or mixed into a oil carrier, such as a silicone oil carrier. An aqueous phase is added concurrently, previously or subsequently, so as to form a two phase liquid composition, with emulsion droplets accumulating at the water-oil junction.

[0109] In some cases a second emulsion breaker is added, concurrently or subsequently, such that ‘combination breaking’ is effected. Exemplary second breakers include the alcohols mentioned above, silane terminated siloxanes, or carbon - silicon compounds such as a compound of Fig. 2A, Fig. 2B, or Fig. 3, or the compounds of Fig. 9, or emulsion breakers or demulsifiers as listed elsewhere herein.

[0110] The composition is subjected to settling condition such as centrifugation, resulting in emulsion droplet contents being released into the aqueous phase.

[0111] These approaches convey one or more of at least two benefits in some implementations. Firstly, the emulsions breakers are in some cases not fluorinated or perfluorinated alcohols, substantially reducing challenges relating to waste creation or disposal.

[0112] Secondly, some emulsion breakers exhibit low cell toxicity, such that emulsions comprising live cells may be broken and the cells collected without substantial cell mortality. In various embodiments, cell viability is observed to be at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% up to 100% of the cell viability of cells in emulsions prior to emulsion breaking or prior to cell encapsulation in emulsions.

[0113] Alternate emulsion breaking approaches comprise ‘single phase’ emulsion breaking. In single phase emulsion breaking, a water in oil emulsion and its silicone oil carrier are rendered soluble in an excess of an aqueous phase added thereto. That is, the carrier oil, such as a siliconeoil of a water in oil silicone emulsion, is dispersed into an aqueous phase comprising a demulsifier, pursuant to which the emulsion droplets break and release their contents into the aqueous phase.

[0114] Demulsifiers suitable for single phase emulsion breaking include water soluble relatives of emulsifiers such as silicone emulsifiers. Representative molecules include molecules of Fig. 3 comprising at least 60% hydrophilic block, such as polyethylene glycol. Similarly, molecules resembling those of Fig. 3 for which the polyethylene glycol block is replaced with, bound to or intermingled with polypropylene glycol, as seen in Fig. 7, are also representative demulsifiers compatible with single phase emulsion breaking.

[0115] A broad range of demulsifiers are effective in single phase emulsion breaking. Many such demulsifiers exhibit the structural core of the molecules of Fig. 3. For example, some such molecules exhibit 60%-70% PEG, or 70%-80% PEG, or 80-85% PEG. Molecules of Fig. 3 having PEG percentages outside of these ranges are also contemplated.

[0116] Similarly, many such demulsifiers exhibit the structural core of the molecules of Fig. 7. For example, some such molecules exhibit 60%-85%, or 65%-85%, or 65%-80%, or 65%-75%, or 65%-70%, or 60%-65%, or 60%-70%, or 60%-75%, or 60%-80%, or 70%-75%, or 70%-80%, or 70%-85%, or 75%-80%, or 80-85% of the poly-glycol branches, such as PPG and / or PEG, such as 60% PPG / 40% PEG, or similarly 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater PPG, or similarly 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater PEG. In some cases, some such molecules exhibit 60%-85%, or 65%-85%, or 65%-80%, or 65%-75%, or 65%-70%, or 60%-65%, or 60%-70%, or 60%-75%, or 60%-80%, or 70%-75%, or 70%-80%, or 70%-85%, or 75%-80%, or 80-85% of PEG. some such molecules exhibit 60%-85%, or 65%-85%, or 65%-80%, or 65%-75%, or 65%-70%, or 60%-65%, or 60%-70%, or 60%-75%, or 60%-80%, or 70%-75%, or 70%-80%, or 70%-85%, or 75%-80%, or 80-85% of PPG. In some cases, some such molecules exhibit no more than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of PEG. In some cases, some such molecules exhibit no more than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of PPG. In some cases, a proportion of 65%-80% PEG / PPG is preferred.

[0117] Some emulsion breaking compositions comprise a detergent, such as Triton X-100, IGEPAL CA-630, or other detergent comprising a PEG tail, a tween such as Tween-20, or an ionic detergent such as Sodium dodecyl sulfate (SDS). Other detergents are compatible with the methods or as constituents of the compositions disclosed herein. The detergent may be added to a concentration of from less than 0.1%, to 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10%, or greater than 10%, or a percentage spanned by or outside of this range.

[0118] Often, the detergent or demulsifier or emulsion breaker is added at a volume in excess of the volume of the silicone oil emulsion, such that the final concentration of the detergent in the composition at breaking is about or only slightly less than 0.1%, to 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10%, or greater than 10%, or a percentage spanned by or outside of this range.

[0119] Microcapsule formation and recovery. Emulsions are sometimes used to form droplets in which solid beads or hydrogel-shell microcapsules such as semi-porous microcapsules having a hydrogel shell and an aqueous or non-hydrogel interior may form. Such solid beads or hydrogel shell microcapsules are useful in that, being porous to buffers and in some cases to reagents, one may perform successive reactions on their contents in series by washing reagents in and out of the beads or microcapsules rather than through iterative droplet merger and reagent dilution, as is the case for droplets in an emulsion.

[0120] However, to make use of this benefit of the permeability of beads or hydrogel shell microcapsules, one must recover the beads or microcapsules from the hydrophobic carrier of the emulsion in which they are formed. Particle recovery, such as of bead or hydrogel shell microcapsule, is effected in some cases through the emulsion breaking approaches as disclosed herein.Turning to the Figures, one sees the following.

[0121] At Fig. 1 A, one sees a dimethyl silane (DMS) monomer unit of a silicone oil consistent with the disclosure herein. The oil comprises “Y” units of the DMS, and is in some cases capped at each end with CH3 methyl cap.

[0122] At Fig. IB, one sees a variant on the silicone oil of Fig. 1A, a phenyl modified silicone oil monomer unit of a silicone oil consistent with the disclosure herein. The oil comprises “X” units of the DMS, and is in some cases capped at each end with CH3 methyl cap.

[0123] Not shown but consistent with the disclosure herein are other silicone oils, such as those comprising monomer units differing from that of Fig. 1 A in that one or more methyl groups of the DMS monomer is modified to be an ethyl, propyl, butyl, pentyl or other linear, branched or right hydrophobic moiety.

[0124] Also consistent with the disclosure herein are heterologous silicone oils, such as those wherein the oil comprises more than one species of monomer unit, such as a ratio of monomer units shown in Fig. 1 A and Fig. IB, or otherwise disclosed herein or contemplated. The heterologous silicone oils may comprise distinct runs of uniform monomers tethered to one another or may comprise ordered or random interleaving of two or more monomer types. An exemplary silicone oil comprises 66% DMS and 33% phenyl modified silicone oil.

[0125] At Fig. 2A, one sees a surfactant consistent with the disclosure herein. The surfactant comprises “a” multimers of a silicone methyl silone monomer to which an alkane is tethered, such as a cetyl group or other alkane, “b” multimers of a silicone methyl siloxane monomer to which a hydrophilic PPG is tethered, and “c” multimers of a silicone methyl siloxane monomer to which is tethered a siloxane branch of “d” DMS monomers is tethered. Methyl caps on the silicone side branches are shown, while in some cases the ends of the molecule are similarly methyl capped. Alternates varying in organization or identity of side chains are also consistent with the disclosure herein.

[0126] At Fig. 2B, one sees a surfactant consistent with the disclosure herein. The surfactant comprises “m” multimers of a silicone methyl silone monomer to which an alkane is tethered, such as a CIO alkene a shown or alkenes of length, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or greater, for example a range of 1-20, 5-20, or 10-16 for example, “n” multimers of a silicone methyl siloxane monomer to which a hydrophilic PPE is tethered, such as 6-9 as shown, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, or a range such as 1-10, 2-9, 2-6, 3-5, 6-9 or other range; “p” DMS monomers in a range of, for example 1-50, 5-50, 10-40 or 15-35, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or greater.

[0127] At Fig. 3, one sees a compound or group of compounds consistent with the disclosure herein. The scaffold for these molecules is shown, including “n” units of a DMS monomer, adjacent to “n” units of a silicone methyl siloxane monomer to each of which a methyl-capped polyethylene glycol PEG multimer of “p” monomer units is tethered. The scaffold is capped bySi(CH3)3 silicon trimethyl end caps as shown in the example in this figure, though variants are also contemplated.

[0128] At Fig. 4, one sees a cartoon depiction of two-phase emulsion breaking. An oil and oil emulsion breaker are added to a silicone oil carrier emulsion, followed by addition of an aqueous phase. In alternative embodiments the aqueous phase is added prior to addition of the oil and emulsion breaker. Similarly, the emulsion breaker may be added separately from the oil.

[0129] The mixture resolves into two phases, with emulsion droplets aligned at the water-oil interface. The mixture is subjected to centrifugation or otherwise allowed to separate, such that emulsion droplets are released into the aqueous phase. The oil phase is then removed.

[0130] At Fig. 5, one sees emulsions contacted to demulsifiers heptanol, octanol, and nonanol pursuant to two phase emulsion breaking. The hydrogel particles (capsules), which were originally in the oil phase droplets have been released to the aqueous phase. Additionally, some of the small oil droplets attached to hydrogel particles are visible, which indicates that the emulsion breaking has not been complete.

[0131] At Fig. 6 one sees a schematic single phase demulsification. A silicone oil water-in-oil emulsion is contacted to an aqueous buffer and a demulsifier. The oil carrier and emulsion droplets briefly from a second phase. Upon centrifugation or other settling, the oil carrier disperses into the aqueous phase, which releases the emulsion droplet contents.

[0132] At Fig. 7, one sees a compound or group of compounds consistent with the disclosure herein. The scaffold for these molecules is shown, including “n” units of a DMS monomer, adjacent to “n” units of a silicone methyl siloxane monomer to each of which a methyl-capped polyethylene glycol PEG multimer of “p” monomer units and polypropylene glycol PPGmultimer of “q” units is tethered. The scaffold is capped by Si(CH3)3 silicon trimethyl end caps as shown in the example in this figure, though variants are also contemplated.

[0133] At Figure 8, one sees the results of emulsion breaking. From left to right, one sees the effects of A, a molecule of Fig. 3 showing 60%-70% PEG; B, a molecule of Fig. 3 showing 80% PEG; C, a molecule of Fig. 3 showing 80-85% PEG; and D, a molecule of Fig. 7 showing 65%-70% (60% PPG / 40% PEG). The hydrogel particles (capsules), which were originally in the oil phase droplets have been released to the aqueous phase. In this case there are no small attached oil droplets to the hydrogels, which indicates a successful and complete transfer of the SPCs from droplets into the aqueous phase.

[0134] At Fig. 9, one sees structures of a partial list of exemplary demulsifiers, including Triton X-100 as a representative or reference PEG detergent, IGEPAL CA-630, exhibiting a similar structure but a slightly different “n” PEG polymer length, poloxamer as an alternative PEP chain compound and Tween-20 as nonionic detergent representatives, and sodium dodecyl sulfate as a representative demulsifier or detergent. Triton X-100 and IGEPAL are commercial names for molecules of identical head group but which are manufactured to have slightly differing hydrophobicity due to variation in average or exact “n” values for their PEG polymers - Triton X-100 is slightly more hydrophilic and has a greater “n” value. IGEPAL is believed to be synonymous in structure and properties with Nonidet P-40 (NP-40).

[0135] At Fig. 10, one sees the result of silicone oil treatment with high concentrations of the detergents or demulsifiers listed. For each detergent, microcapsule recovery is assessed in ‘emulsion broken’ compositions. The top row represents an upper stratum upon centrifugation, while the bottom row represents the particulate pellet in the bottom of the tube uponcentrifugation. Results are shown for, from left to right, 10% Tween-20 in IxPBS, 5% SDS in lx PBS, 10% IGEPAL CA-630 m lx PBS, and 9% Poloxamer 188 m IxPBS.

[0136] For all emulsion breakers tested, the silicone oil emulsion is substantially broken such that the composition no longer comprises microcapsules in aqueous droplets of a silicone oil carrier. For Tween-20, SDS and IGEPAL, first three from left, microcapsules accumulated in the bottom pellet of upon centrifugation, while various degrees of incompletely dissolved oil droplets, representing a substantially smaller proportion of a field of view than would be seen in an unbroken emulsion, are seen in the upper portion of the centrifugate. For Poloxamer, one again sees that the silicone oil emulsion is substantially broken such that the composition no longer comprises microcapsules in aqueous droplets of a silicone oil carrier. However, microcapsules were seen in both the lower and, in higher numbers, in the upper portion of the centrifugate, and that some microcapsules remain associated with vestigial oil droplets.

[0137] These results indicate the efficacy of all detergents tested as emulsion breakers and the variation in their efficacy in microcapsule recovery.

[0138] At Fig. 11, one sees results of silicone oil treatment with low concentrations of some of the detergents or demulsifiers of Fig. 8. . The top row represents an upper stratum upon centrifugation, while the bottom row represents the particulate pellet in the bottom of the tube upon centrifugation. Results are shown for, from left to right, 0.1% IGEPAL CA-630 in IxPBS, 0.1% Triton X-100 in lx PBS, and 01.% SDS in IxPBS.

[0139] For all emulsion breakers tested, the silicone oil emulsion is substantially broken such that the composition no longer comprises microcapsules in aqueous droplets of a silicone oil carrier. For all detergents shown, microcapsules accumulated in the bottom pellet of upon centrifugation.

[0140] At Figure 12, one sees a silicone oil emulsion of microcapsules formed within aqueous droplets. This is provided as a reference for comparison to the compositions of broken emulsions seen in the previous figures such as Fig. s 10 and 11, while also providing evidence of microcapsule formation in a silicone oil emulsion.

[0141] As used herein, the term “about” in the context of a number refers to a range spanning from 10% below that number to 10% above that number, while in the context of a range refers to an extended range spanning from 10% below the lower listed value to 10% above the higher listed number.

[0142] A percentage “%” in the context of a detergent in a solution refers to a weight per unit volume measurement as is standard in the field.Numbered embodiments

[0143] The technology is further understood in light of the following partial list of numbered embodiments. 1. A method of breaking a water-in-oil emulsion comprising a silicone carrier, the method comprising contacting the water-in-oil emulsion comprising a silicone carrier to an emulsion breaker. 2. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the water-in-oil emulsion comprises a surfactant. 3. The method of numbered embodiment 1 or of any embodiment recited herein, comprising vortexing the water-in-oil emulsion. 4. The method of numbered embodiment 1 or of any embodiment recited herein, comprising centrifuging the water-in-oil emulsion. 5. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises silicone. 6. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsionbreaker does not comprise silicone. 7. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises a detergent. 8. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises an ionic detergent. 9. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises a nonionic detergent. 10. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises a PEG chain. 11. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises IGEPAL CA-630. 12. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises Triton X-100. 13. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises poloxamer. 14. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises SDS. 15. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker is present at a concentration of no greater than 1%. 16. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker is present at a concentration of no greater than 0.1%. 17. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker is present at a concentration of at least 0.03%. 18. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the emulsion breaker comprises a dimethyl siloxane polymer having at least one hydrophilic side branch. 19. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the dimethyl siloxane polymer having at least one hydrophilic sideO(CH2CH2O)p— CH3branch comprises wherein m is no greater than 50; wherein n is no greater than 50; and wherein p is no greater than 50. 20. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the silicone carrier comprises a silicone oil. 21. The method of numbered embodiment 20 or of any embodimentrecited herein, wherein the silicone oil is, wherein x is within a range of 5-20. 22. The method of numbered embodiment 20 or of any embodiment recited herein, wherein thesilicone oil is , wherein y is within a range of 3-25. 23. The method of numberedembodiment 20 or of any embodiment recited herein, wherein the silicone oil iswherein y is within a range of 3-10. 24. The method of numbered embodiment 20 or of anyembodiment recited herein, wherein the silicone oil is , wherein y is 5. 25. Themethod of numbered embodiment 1 or of any embodiment recited herein, wherein the surfactantcomprises . 26. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the surfactant comprisesOHO. 27. The method of numbered embodiment 26 or of any embodiment recited herein, wherein the surfactant has a molecular weight of 3000-6000. 28. The method of numbered embodiment 26 or of any embodiment recited herein, wherein the surfactant comprises m in the range of 10-16, n in the range 2-6, and p in the range of 15-35. 29. The method of numbered embodiment 1 or of any embodiment recited herein, comprising performing a biological manipulation on the water-in-oil emulsion prior to contacting the water-in-oil emulsion to the emulsion breaker. 30. The method of numbered embodiment 1 or of any embodiment recited herein, wherein the water-in-oil emulsion comprises a first nucleic acid population sharing a first nucleic acid tag and a second nucleic acid population sharing a second tag. 31. The method of numbered embodiment 30 or of any embodiment recited herein, wherein the water-in-oil emulsion comprises hydrogel particles. 32. The method of numbered embodiment 30 or of any embodiment recited herein, wherein the water-in-oil emulsion comprises hydrogel microcapsules. 33. The method of numbered embodiment 30 or of any embodiment recited herein, wherein the first nucleic acid population is contained within a first hydrogel particle. 34. The method of numbered embodiment 30 or of any embodiment recited herein, wherein the first nucleic acid population is contained within a first hydrogel microcapsule. 35. The method of numbered embodiment 26 or of any embodiment recited herein, wherein the first nucleic acid population arises from a first cell and the second nucleic acid population arises from a second cell. 36. The method of numbered embodiment 26 or of any embodiment recited herein, wherein the first nucleic acid population is a cDNA population. 37. The method of numbered embodiment 26 or of any embodiment recited herein, wherein the first nucleic acid population is a genomic nucleic acid population. 38. The method of numbered embodiment 26 or of any embodiment recited herein, wherein the water-in-oil emulsion comprises a first clonal cell population and a second clonal cell population. 39. The method of numbered embodiment 26 or of any embodiment recited herein, wherein the water-in-oil emulsion does not comprise a chlorinated oil. 40. The method of numbered embodiment 26 or of any embodiment recited herein, wherein the water-in-oil emulsion does not comprise a fluorinated oil. 41. The method of numbered embodiment 26 or of any embodiment recited herein, wherein the water-in-oil emulsion does not comprise a fluorocarbon.

[0144] The technology is further understood in light of the following partial list of numbered embodiments. 42. An emulsion comprising a silicone oil carrier and a dispersed aqueous droplet population. 43. The emulsion of numbered embodiment 42 or of any embodiment recited herein, further comprising a siloxane surfactant. 44. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier has a viscosity of from 0.5 to 10 cSt. 45. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier has a viscosity of from 0.5 to 2.0 cSt. 46. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises a linear siloxane. 47. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises di-siloxane. 48. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises tri-siloxane. 49. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises a tri-siloxane and a disiloxane. 50. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises a cyclical siloxane. 51. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises a cyclical tri-siloxane. 52. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises a cyclical tetra-siloxane. 53. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises a cyclical penta-siloxane. 54. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises a siloxane having an ethyl side group. 55. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier comprises a phenyl modified silicone oil. 56. Theemulsion of numbered embodiment 55 or of any embodiment recited herein, wherein the phenyl modified silicone oil comprises multimers of a repeating phenyl modified silicone unit. 57. The emulsion of numbered embodiment 56 or of any embodiment recited herein, wherein the multimers comprise 5-20 monomer units. 58. The emulsion of numbered embodiment 57 or ofany embodiment recited herein, wherein a monomer unit of the silicone oil isThe emulsion of numbered embodiment 58 or of any embodiment recited herein, wherein the phenyl modified silicone oil comprises multimers of a repeating phenyl modified silicone unit.60. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil comprises a dimethyl siloxane oil. 61. The emulsion of numbered embodiment 60 or of any embodiment recited herein, wherein the multimers comprise 3-25 monomer units. 62. The emulsion of numbered embodiment 60 or of any embodiment recited herein, wherein a monomerunit of the silicone oil is 63. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the siloxane surfactant comprises a lipophilic block, a hydrophilic block and a siloxane block. 64. The emulsion of numbered embodiment 63 or of any embodiment recited herein, wherein the lipophilic bloc comprises an alkane. 65. The emulsion of numbered embodiment 64 or of any embodiment recited herein, wherein the alkane comprises a cetyl block. 66. The emulsion of numbered embodiment 64 or of any embodiment recited herein, wherein the alkane comprises a C-16 carbon constituent. 67. The emulsion of numbered embodiment 64 or of any embodiment recited herein, wherein the alkane comprises a carbonconstituent of 10-15 carbons. 68. The emulsion of numbered embodiment 64 or of any embodiment recited herein, wherein the alkane comprises a carbon constituent of 17-25 carbons.69. The emulsion of numbered embodiment 63 or of any embodiment recited herein, wherein the hydrophilic block comprises PPG. 70. The emulsion of numbered embodiment 69 or of any embodiment recited herein, wherein the siloxane surfactant comprises. 71. The emulsion of numbered embodiment 69 or of any embodiment recited herein, wherein the siloxane surfactant comprisesOH. 72. The emulsion of numbered embodiment 70 or of any embodiment recited herein, wherein the surfactant has a molecularweight of 3000-6000. 73. The emulsion of numbered embodiment 70 or of any embodiment recited herein, wherein the surfactant comprises m in the range of 10-16, n in the range 2-6, and p in the range of 15-35. 74. The emulsion of numbered embodiment 42 or of any embodiment recited herein, comprising at least one aqueous droplet. 75. The emulsion of numbered embodiment 74 or of any embodiment recited herein, wherein the aqueous droplet comprises a biomaterial. 76. The emulsion of numbered embodiment 74 or of any embodiment recited herein, wherein the aqueous droplet comprises a hydrogel shell and a fluid core. 77. The emulsion of numbered embodiment 74 or of any embodiment recited herein, wherein the aqueous droplet harbors a cell. 78. The emulsion of numbered embodiment 74 or of any embodiment recited herein, wherein the aqueous droplet harbors a bioreaction. 79. The emulsion of numbered embodiment 42 or of any embodiment recited herein, comprising an emulsion breaking component. 80. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises silicone. 81. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component does not comprise silicone. 82. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises a detergent. 83. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises an ionic detergent. 84. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises a nonionic detergent. 85. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises a PEG chain.86. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises IGEPAL CA-630. 87. The emulsion of numberedembodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises Triton X-100. 88. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises poloxamer. 89. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises SDS. 90. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component is present at a concentration of no greater than 1%. 91. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component is present at a concentration of no greater than 0.1%. 92. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component comprises a dimethyl siloxane polymer backbone and at least one PEG modified side branch. 93. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the at least one PEG modified side branch comprises a methyl cap. 94. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the dimethyl siloxane polymer backbone comprises n adjacent dimethyl siloxane oil monomers. 95. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the dimethyl siloxane polymer backbone comprises m adjacent dimethyl siloxane oil monomers having at least one PEG modified side branch. 96. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein theO(CH2CH2O)p— CH3CH3 / (CH2)3\ / (j)H3\ CH3CH3— SSi— O-f— ^i— O++-^i-O-W— CH3CH3\ CH3 / m\CH3 / „ CH3emulsion breaking component comprises . 97. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein theUnoemulsion breaking component comprises .98. The emulsion of numbered embodiment 97 or of any embodiment recited herein, wherein the surfactant has a molecular weight of 3000-6000. 99. The emulsion of numbered embodiment 97 or of any embodiment recited herein, wherein the surfactant comprises m in the range of 10-16, n in the range 2-6, and p in the range of 15-35. 100. The emulsion of numbered embodiment 79 or of any embodiment recited herein, wherein the emulsion breaking component destabilizes the emulsion. 101. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier does not comprise chlorine. 102. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the silicone oil carrier does not comprise fluorine. 103. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the siloxane surfactant does not comprise chlorine. 104. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the siloxane surfactant does not comprise fluorine. 105. The emulsion of numbered embodiment 42 or of any embodiment recited herein, wherein the composition does not comprise a fluorocarbon.

[0145] The technology is further understood in light of the following partial list of numbered embodiments. 106. A single phase liquid composition comprising a silicone oil, a surfactant, and an aqueous biomaterial, wherein the silicone oil and the surfactant are dissolved in a single aqueous phase. 107. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, comprising a dimethyl siloxane polymer having at least one hydrophilic side branch. 108. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises a linear siloxane. 109. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises di-siloxane. 110. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises tri-siloxane. 111. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises a tri-siloxane and a disiloxane. 112. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises a cyclical siloxane. 113. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises a cyclical tri-siloxane. 114. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises a cyclical tetra-siloxane. 115. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises a cyclical penta-siloxane. 116. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises a siloxane having an ethyl side group. 117. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises aphenyl modified silicone oil. 118. The single phase liquid composition of numbered embodiment 117 or of any embodiment recited herein, wherein the phenyl modified silicone oil comprises multimers of a repeating phenyl modified silicone unit. 119. The single phase liquid composition of numbered embodiment 118 or of any embodiment recited herein, wherein the multimers comprise 5-20 monomer units. 120. The single phase liquid composition of numbered embodiment 119 or of any embodiment recited herein, wherein a monomer unit of the siliconeoil is121. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the silicone oil comprises a dimethyl siloxane oil.122. The single phase liquid composition of numbered embodiment 121 or of any embodiment recited herein, wherein the phenyl modified silicone oil comprises multimers of a repeating phenyl modified silicone unit. 123. The single phase liquid composition of numbered embodiment 122 or of any embodiment recited herein, wherein the multimers comprise at least 3-25 twenty monomer units. 124. The single phase liquid composition of numbered embodiment 123 or of any embodiment recited herein, wherein a monomer unit of the silicone oil is. 125. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the siloxane surfactant comprises a lipophilic block, a hydrophilic block and a siloxane block. 126. The single phase liquid composition of numbered embodiment 125 or of any embodiment recited herein, wherein the lipophilic bloc comprises a fatty alcohol. 127. The single phase liquid composition of numbered embodiment 126 or of anyembodiment recited herein, wherein the fatty alcohol comprises a cetyl block. 128. The single phase liquid composition of numbered embodiment 126 or of any embodiment recited herein, wherein the fatty alcohol comprises a C-16 carbon constituent. 129. The single phase liquid composition of numbered embodiment 126 or of any embodiment recited herein, wherein the fatty alcohol comprises a carbon constituent of 10-15 carbons. 130. The single phase liquid composition of numbered embodiment 126 or of any embodiment recited herein, wherein the fatty alcohol comprises a carbon constituent of 17-25 carbons. 131. The single phase liquid composition of numbered embodiment 125 or of any embodiment recited herein, wherein the hydrophilic block comprises PPG. 132. The single phase liquid composition of numbered embodiment 125 or of any embodiment recited herein, wherein the siloxane surfactant comprises. 133. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the at least one hydrophilic side branch comprises PEG. 134. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the dimethyl siloxane polymercomprises . 135. The single phase liquid composition of numbered embodiment 134 or of any embodiment recited herein, wherein m is no greater than 50, wherein n is no greater than 50, and wherein p is no greater than 50. 136. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the aqueous biomaterial comprises a first nucleic acid population sharing a first nucleic acid tag and a second nucleic acid population sharing a second tag. 137. The single phase liquid composition of numbered embodiment 136 or of any embodiment recited herein, wherein the first nucleic acid population arises from a first cell and the second nucleic acid population arises from a second cell. 138. The single phase liquid composition of numbered embodiment 136 or of any embodiment recited herein, wherein the first nucleic acid population is a cDNA population.139. The single phase liquid composition of numbered embodiment 136 or of any embodiment recited herein, wherein the first nucleic acid population is a genomic nucleic acid population. 140. The single phase liquid composition of numbered embodiment 106 or of any embodiment recited herein, wherein the aqueous biomaterial comprises a first clonal cell population and a second clonal cell population. 141. The single phase liquid composition of numbered embodiment 136 or of any embodiment recited herein, wherein the first nucleic acid population and the second nucleic acid population are intermingled. 142. The single phase liquid composition of numbered embodiment 108 or of any embodiment recited herein, wherein the composition does not comprise chlorine. 143. The single phase liquid composition of numbered embodiment 108 or of any embodiment recited herein, wherein the composition does notcomprise fluorine. 144. The single phase liquid composition of numbered embodiment 108 or of any embodiment recited herein, wherein the composition does not comprise a fluorocarbon.

[0146] The technology is further understood in light of the following partial list of numbered embodiments. 145. A method of emulsion breaking, comprising adding to an emulsion an emulsion breaking oil and an emulsion breaker, and adding an aqueous phase. 146. The method of numbered embodiment 145 or of any embodiment recited herein, comprising centrifuging. 147. The method of numbered embodiment 145 or of any embodiment recited herein, wherein the emulsion is a water in oil emulsion for which the oil is a silicone oil. 148. The method of numbered embodiment 145 or of any embodiment recited herein, wherein the emulsion breaking oil is a silicone oil. 149. The method of numbered embodiment 145 or of any embodiment recited herein, wherein the emulsion breaker is a fatty alcohol. 150. The method of numbered embodiment 149 or of any embodiment recited herein, wherein the fatty alcohol is heptanol. 151. The method of numbered embodiment 149 or of any embodiment recited herein, wherein the fatty alcohol is octanol. 152. The method of numbered embodiment 149 or of any embodiment recited herein, wherein the fatty alcohol is nonanol. 153. The method of numbered embodiment 149 or of any embodiment recited herein, wherein the fatty alcohol does not comprise fluorine.154. The method of numbered embodiment 145 or of any embodiment recited herein, comprising recovering emulsion contents from the aqueous phase. 155. The method of numbered embodiment 145 or of any embodiment recited herein, comprising assaying for the emulsion contents in the aqueous phase. 156. The method of numbered embodiment 145 or of any embodiment recited herein, wherein the emulsion comprises microcapsules.

[0147] The technology is further understood in light of the following partial list of numbered embodiments. 157. A method of emulsion breaking, comprising adding to a silicone oil emulsionan emulsion breaking aqueous phase. 158. The method of numbered embodiment 157 or of any embodiment recited herein, wherein the emulsion breaking aqueous phase comprises a demulsifier. 159. The method of numbered embodiment 158 or of any embodiment recited herein, wherein the demulsifier comprises a molecule of Fig. 3. 160. The method of numbered embodiment 159 or of any embodiment recited herein, wherein the molecule of Fig. 3 comprises 60%-70% PEG. 161. The method of numbered embodiment 159 or of any embodiment recited herein, wherein the molecule of Fig. 3 comprises 70%-80% PEG. 162. The method of numbered embodiment 159 or of any embodiment recited herein, wherein the molecule of Fig. 3 comprises 80%-85% PEG. 163. The method of numbered embodiment 159 or of any embodiment recited herein, wherein the molecule of Fig. 3 comprises 60%-70% PEG. 164. The method of numbered embodiment 158 or of any embodiment recited herein, wherein the demulsifier comprises 65%-70% of a side chain comprising PEG and PPG. 165. The method of numbered embodiment 164 or of any embodiment recited herein, wherein the side chain comprises at least 50% PPG. 166. The method of numbered embodiment 164 or of any embodiment recited herein, wherein the side chain comprises at least 60% PPG. 167. The method of numbered embodiment 164 or of any embodiment recited herein, wherein the side chain comprises no more than 50% PPG. 168. The method of numbered embodiment 164 or of any embodiment recited herein, wherein the side chain comprises no more than 40% PPG.EXAMPLES

[0148] Example 1. Two phase emulsion breaking. Emulsions were subjected to two phase emulsion breaking as depicted in Fig. 4. Water in oil emulsions in a silicone oil carrier were contacted to an oil carrier in which heptanol, octanol or nonanol was added as a demulsifier, andto an aqueous phase. The emulsion droplets gathered at the oil-aqueous interface, and upon centrifugation the emulsion droplets largely released their contents into the aqueous phase in bulk.

[0149] Results of the treatment are shown in Fig. 5. One observes that in each case microcapsule SPCs exhibit nonuniform shell exteriors. Additionally, smaller oil droplets are present in all three treatments (most abundant with octanol and also abundant with heptanol, but also present, at right, with nonanol treatment).

[0150] Example 2. Single phase emulsion breaking. Emulsions were subjected to single phase emulsion breaking as depicted in Fig. 6. Water in oil emulsions in a silicone oil carrier were contacted to an aqueous phase comprising a demulsifier. The mixture was centrifuged and the silicone oil was observed to dissolve into the aqueous phase, while the emulsion droplets largely released their contents into the aqueous phase in bulk.

[0151] Results of the treatment are shown in Fig. 8. From left to right, one sees the effects of A, a molecule of Fig. 3 showing 60%-70% PEG; B, a molecule of Fig. 3 showing 80% PEG; C, a molecule of Fig. 3 showing 80-85% PEG; and D, a molecule of Fig. 7 showing 65%-70% (60% PPG / 40% PEG). One observes that in each case microcapsule SPCs exhibit nonuniform oil exteriors, indicative of droplet instability. Additionally, smaller oil droplets are present in all three treatments.

[0152] Example 3. Example 3. Emulsion breaking and microcapsule recovery with high concentration demulsifiers. Water in silicone oil emulsions were subjected to emulsion breaking using the compounds presented in Fig. 9 at high concentration. To a 10 uL microcapsule emulsion was added 100 uL of, from left to right, 10% Tween-20 in IxPBS, 5% SDS in lx PBS, 10% IGEPAL CA-630 m lx PBS, and 9% Poloxamer 188 m IxPBS.

[0153] The resulting composition was centrifuged to sediment microcapsule. Compositions were assayed at their top and bottom portions, seen as the top and bottom images of each image column of Fig. 10, and assessed for emulsion breaking and microcapsule recovery. Results are shown for, from left to right, 10% Tween-20 in IxPBS, 5% SDS in lx PBS, 10% IGEPAL CA-630 in lx PBS, and 9% Poloxamer 188 in IxPBS. This set of detergents included both nonionic detergents and the representative ionic detergent SDS. Final concentrations for the compositions were 9.1% Tween-20, 4.55% SDS, 9.1% IGEPAL, and 8.2% Poloxamer.

[0154] One sees that, for all emulsion breakers tested, the silicone oil emulsion is substantially broken such that the composition no longer comprises microcapsules in aqueous droplets of a silicone oil carrier. For Tween-20, SDS and IGEPAL, first three from left, microcapsules accumulated in the bottom pellet of upon centrifugation, while various degrees of incompletely dissolved oil droplets, representing a substantially smaller proportion of a field of view than would be seen in an unbroken emulsion, are seen in the upper portion of the centrifugate. The top and bottom portions of the compositions are separated by a middle portion lacking both oil droplets and microcapsules.

[0155] For Poloxamer, one again sees that the silicone oil emulsion is substantially broken such that the composition no longer comprises microcapsules in aqueous droplets of a silicone oil carrier. However, microcapsules were seen in both the lower and, in higher numbers, in the upper portion of the centrifugate, and that some microcapsules remain associated with vestigial oil droplets.

[0156] These results indicate the efficacy of all detergents tested as emulsion breakers. That is, at each case, treatment resulted in a composition that no longer comprised a silicone oil carrier inwhich were dispersed aqueous droplets in which were formed hydrogel microcapsules, as seen in reference Fig. 12.

[0157] The results further indicate that, at high concentrations, both ionic and nonionic detergents facilitated microcapsule recovery from the silicone oil emulsions, as indicated by accumulation of microcapsules in aqueous solution at the bottom portion of tubes, free of oil.

[0158] Example 4. Emulsion breaking and microcapsule recovery with low concentration demulsifiers. High-performing emulsion breakers from Example 3 were assayed at low concentrations to test their efficacy in emulsion breaking and microcapsule recovery. To a 10 uL microcapsule emulsion was added 100 uL of, from left to right, 0.1% IGEPAL CA-630 in IxPBS, 0.1% Triton X-100 in lx PBS, and 0.1% SDS in IxPBS. Samples were vortexed for 30 seconds at 1000 rpm and then centrifuged for 1 minute at 2000g.

[0159] Compositions were assayed at their top and bottom portions, and assessed for emulsion breaking and microcapsule recovery. Results are shown for, from left to right, 0.1% IGEPAL CA-630 in IxPBS, 0.1% Triton X-100 in IxPBS, and 0.1% SDS in IxPBS. This set of detergents included both nonionic detergents and the representative ionic detergent SDS. Final concentrations for the compositions were 0.091% Tween-20, 0.0455% SDS, and 0.091% IGEPAL.

[0160] Much like the high concentration assay of Example 3 discussed above, one sees that for all emulsion breakers tested the silicone oil emulsion is substantially broken such that the composition no longer comprises microcapsules in aqueous droplets of a silicone oil carrier. For all detergents shown, microcapsules accumulated in the bottom pellet of upon centrifugation, in aqueous solution and free of oil, indicating efficient microcapsule recovery in addition to efficient emulsion breaking.

[0161] Results were replicated at different emulsion scales. To a 27.5 uL microcapsule emulsion in silicone oil was added 500 uL of emulsion breaker. Samples were vortexed for 30 seconds at 1000 rpm and then centrifuged for 1 minute at 2000g.

[0162] Results are shown for, from left to right, 0.1% IGEPAL CA-630 in IxPBS, 0.1% Triton X-100 in lx PBS, and 0.1% SDS in IxPBS. Final concentrations were 0.095% IGEPAL, Triton-X-100 or SDS. Again, compositions subsequent to centrifugation are shown, with views of the top and bottom images of each image column presented in Fig. 11. For all detergents shown, microcapsules accumulated in the bottom pellet of upon centrifugation, in aqueous solution and free of oil, indicating efficient microcapsule recovery in addition to efficient emulsion breaking.

[0163] Compare again with the water in oil emulsion depiction of Fig. 12.

[0164] Example 5. Emulsion breaking at lower concentrations.

[0165] Lower concentrations were also tested and showed efficacy for emulsion breaking.

[0166] High-performing emulsion breakers from Example 3 were assayed at lower concentrations to test their efficacy in emulsion breaking and microcapsule recovery. To a 27.5 uL microcapsule emulsion was added 500 uL of 0.1% Tween 20 in IxPBS, 0.033% IGEPAL CA-630 in lx PBS, 0.033% Triton X-100 in lx PBS, and 0.033% SDS in IxPBS. Samples were vortexed for 30 seconds at 1000 rpm and then centrifuged for 1 minute at 2000g.

[0167] Compositions were assayed at their top and bottom portions, and assessed for emulsion breaking and microcapsule recovery. This set of detergents included both nonionic detergents and the representative ionic detergent SDS. Final concentrations for the compositions were 0.091% Tween-20, 0.0455% SDS, 0.091% Triton X-100, and 0.091% IGEPAL.

[0168] Much like the high concentration assay of Example 3 discussed above, for all emulsion breakers tested the silicone oil emulsion is substantially broken such that the composition nolonger comprises microcapsules in aqueous droplets of a silicone oil carrier. For all detergents shown, microcapsules accumulated in the bottom pellet of upon centrifugation, in aqueous solution and free of oil, indicating efficient microcapsule recovery in addition to efficient emulsion breaking. However, for Tween-20 microcapsules were also seen intermingled with oil droplets in the upper phase, and in many cases microcapsule sedimentation was less efficient.

Claims

CLAIMS1. A method of breaking a water-in-oil emulsion comprising a silicone carrier, the method comprising contacting the water-in-oil emulsion comprising a silicone carrier to an emulsion breaker.

2. The method of claim 1, wherein the water-in-oil emulsion comprises a surfactant.

3. The method of claim 1, comprising vortexing the water-in-oil emulsion.

4. The method of claim 1 , comprising centrifuging the water-in-oil emulsion.

5. The method of claim 1, wherein the emulsion breaker comprises silicone.

6. The method of claim 1 , wherein the emulsion breaker does not comprise silicone.

7. The method of claim 1, wherein the emulsion breaker comprises a detergent.

8. The method of claim 1, wherein the emulsion breaker comprises an ionic detergent.

9. The method of claim 1 , wherein the emulsion breaker comprises a nonionic detergent.

10. The method of claim 1 , wherein the emulsion breaker comprises a PEG chain.

11. The method of claim 1, wherein the emulsion breaker comprises IGEPAL CA-630.

12. The method of claim 1, wherein the emulsion breaker comprises Triton X-100.

13. The method of claim 1, wherein the emulsion breaker comprises poloxamer.

14. The method of claim 1, wherein the emulsion breaker comprises SDS.

15. The method of claim 1, wherein the emulsion breaker is present at a concentration of no greater than 1%.

16. The method of claim 1, wherein the emulsion breaker is present at a concentration of no greater than 0.1%.

17. The method of claim 1, wherein the emulsion breaker is present at a concentration of at least 0.03%.

18. The method of claim 1, wherein the emulsion breaker comprises a dimethyl siloxane polymer having at least one hydrophilic side branch.

19. The method of claim 1, wherein the dimethyl siloxane polymer having at least one hydrophilic side branch comprisesO(CH2CH2O)p-CH3wherein m is no greater than 50;wherein n is no greater than 50; andwherein p is no greater than 50.

20. The method of claim 1, wherein the silicone carrier comprises a silicone oil.

21. The method of claim 20, wherein the silicone oil is• SrO •X X, wherein x is within a range of 5-20.

22. The method of claim 20, wherein the silicone oil iswherein y is within a range of 3-25.

23. The method of claim 20, wherein the silicone oil iswherein y is within a range of 3-10.

24. The method of claim 20, wherein the silicone oil iswherein y is 5.

25. The method of claim 1, wherein the surfactant comprises26. The method of claim 1, wherein the surfactant comprisesOHO27. The method of claim 26, wherein the surfactant has a molecular weight of 3000-6000.

28. The method of claim 26, wherein the surfactant comprises m in the range of 10-16, n in the range 2-6, and p in the range of 15-35.

29. The method of claim 1, comprising performing a biological manipulation on the water-in-oil emulsion prior to contacting the water-in-oil emulsion to the emulsion breaker.

30. The method of claim 1, wherein the water-in-oil emulsion comprises a first nucleic acid population sharing a first nucleic acid tag and a second nucleic acid population sharing a second tag.

31. The method of claim 30, wherein the water-in-oil emulsion comprises hydrogel particles.

32. The method of claim 30, wherein the water-in-oil emulsion comprises hydrogel microcapsules.

33. The method of claim 30, wherein the first nucleic acid population is contained within a first hydrogel particle.

34. The method of claim 30, wherein the first nucleic acid population is contained within a first hydrogel microcapsule.

35. The method of claim 26, wherein the first nucleic acid population arises from a first cell and the second nucleic acid population arises from a second cell.

36. The method of claim 26, wherein the first nucleic acid population is a cDNA population.

37. The method of claim 26, wherein the first nucleic acid population is a genomic nucleic acid population.

38. The method of claim 26, wherein the water-in-oil emulsion comprises a first clonal cell population and a second clonal cell population.

39. The method of claim 26, wherein the water-in-oil emulsion does not comprise a chlorinated oil.

40. The method of claim 26, wherein the water-in-oil emulsion does not comprise a fluorinated oil.

41. The method of claim 26, wherein the water-in-oil emulsion does not comprise a fluorocarbon.

42. An emulsion comprising a silicone oil carrier and a dispersed aqueous droplet population.

43. The emulsion of claim 42, further comprising a siloxane surfactant.

44. The emulsion of claim 42, wherein the silicone oil carrier has a viscosity of from 0.5 to 10 cSt.

45. The emulsion of claim 42, wherein the silicone oil carrier has a viscosity of from 0.5 to 2.0 cSt.

46. The emulsion of claim 42, wherein the silicone oil carrier comprises a linear siloxane.

47. The emulsion of claim 42, wherein the silicone oil carrier comprises di-siloxane.

48. The emulsion of claim 42, wherein the silicone oil carrier comprises tri-siloxane.

49. The emulsion of claim 42, wherein the silicone oil carrier comprises a tri-siloxane and a di-siloxane.

50. The emulsion of claim 42, wherein the silicone oil carrier comprises a cyclical siloxane.

51. The emulsion of claim 42, wherein the silicone oil carrier comprises a cyclical trisiloxane.

52. The emulsion of claim 42, wherein the silicone oil carrier comprises a cyclical tetrasiloxane.

53. The emulsion of claim 42, wherein the silicone oil carrier comprises a cyclical pentasiloxane.

54. The emulsion of claim 42, wherein the silicone oil carrier comprises a siloxane having an ethyl side group.

55. The emulsion of claim 42, wherein the silicone oil carrier comprises a phenyl modified silicone oil.

56. The emulsion of claim 55, wherein the phenyl modified silicone oil comprises multimers of a repeating phenyl modified silicone unit.

57. The emulsion of claim 56, wherein the multimers comprise 5-20 monomer units.

58. The emulsion of claim 57, wherein a monomer unit of the silicone oil is59. The emulsion of claim 58, wherein the phenyl modified silicone oil comprises multimers of a repeating phenyl modified silicone unit.

60. The emulsion of claim 42, wherein the silicone oil comprises a dimethyl siloxane oil.

61. The emulsion of claim 60, wherein the multimers comprise 3-25 monomer units.

62. The emulsion of claim 60, wherein a monomer unit of the silicone oil is63. The emulsion of claim 42, wherein the siloxane surfactant comprises a lipophilic block, a hydrophilic block and a siloxane block.

64. The emulsion of claim 63, wherein the lipophilic bloc comprises an alkane.

65. The emulsion of claim 64, wherein the alkane comprises a cetyl block.

66. The emulsion of claim 64, wherein the alkane comprises a C-16 carbon constituent.

67. The emulsion of claim 64, wherein the alkane comprises a carbon constituent of 10-15 carbons.

68. The emulsion of claim 64, wherein the alkane comprises a carbon constituent of 17-25 carbons.

69. The emulsion of claim 63, wherein the hydrophilic block comprises PPG.

70. The emulsion of claim 69, wherein the siloxane surfactant comprises71. The emulsion of claim 69, wherein the siloxane surfactant compriseso72. The emulsion of claim 71, wherein the surfactant has a molecular weight of 3000-6000.

73. The emulsion of claim 71, wherein the surfactant comprises m in the range of 10-16, n in the range 2-6, and p in the range of 15-35.

74. The emulsion of claim 42, comprising at least one aqueous droplet.

75. The emulsion of claim 74, wherein the aqueous droplet comprises a biomaterial.

76. The emulsion of claim 74, wherein the aqueous droplet comprises a hydrogel shell and a fluid core.

77. The emulsion of claim 74, wherein the aqueous droplet harbors a cell.

78. The emulsion of claim 74, wherein the aqueous droplet harbors a bioreaction.

79. The emulsion of claim 42, comprising an emulsion breaking component.

80. The emulsion of claim 79, wherein the emulsion breaking component comprises silicone.

81. The emulsion of claim 79, wherein the emulsion breaking component does not comprise silicone.

82. The emulsion of claim 79, wherein the emulsion breaking component comprises a detergent.

83. The emulsion of claim 79, wherein the emulsion breaking component comprises an ionic detergent.

84. The emulsion of claim 79, wherein the emulsion breaking component comprises a nonionic detergent.

85. The emulsion of claim 79, wherein the emulsion breaking component comprises a PEG chain.

86. The emulsion of claim 79, wherein the emulsion breaking component comprises IGEPAL CA-630.

87. The emulsion of claim 79, wherein the emulsion breaking component comprises Triton X-100.

88. The emulsion of claim 79, wherein the emulsion breaking component comprises poloxamer.

89. The emulsion of claim 79, wherein the emulsion breaking component comprises SDS.

90. The emulsion of claim 79, wherein the emulsion breaking component is present at a concentration of no greater than 1%.

91. The emulsion of claim 79, wherein the emulsion breaking component is present at a concentration of no greater than 0.1%.

92. The emulsion of claim 79, wherein the emulsion breaking component comprises a dimethyl siloxane polymer backbone and at least one PEG modified side branch.

93. The emulsion of claim 79, wherein the at least one PEG modified side branch comprises a methyl cap.

94. The emulsion of claim 79, wherein the dimethyl siloxane polymer backbone comprises n adjacent dimethyl siloxane oil monomers.

95. The emulsion of claim 79, wherein the dimethyl siloxane polymer backbone comprises m adjacent dimethyl siloxane oil monomers having at least one PEG modified side branch.

96. The emulsion of claim 79, wherein the emulsion breaking component comprises O(CH2CH2O)p-CH397. The emulsion of claim 79, wherein the emulsion breaking component comprisesOHO98. The emulsion of claim 97, wherein the surfactant has a molecular weight of 3000-6000.

99. The emulsion of claim 97, wherein the surfactant comprises m in the range of 10-16, n in the range 2-6, and p is the range of 15-35.

100. The emulsion of claim 79, wherein the emulsion breaking component destabilizes the emulsion.

101. The emulsion of claim 42, wherein the silicone oil carrier does not comprise chlorine.

102. The emulsion of claim 42, wherein the silicone oil carrier does not comprise fluorine.

103. The emulsion of claim 42, wherein the siloxane surfactant does not comprise chlorine.

104. The emulsion of claim 42, wherein the siloxane surfactant does not comprise fluorine.

105. The emulsion of claim 42, wherein the composition does not comprise a fluorocarbon.

106. A single phase liquid composition comprising a silicone oil, a surfactant, and an aqueous biomaterial, wherein the silicone oil and the surfactant are dissolved in a single aqueous phase.

107. The single phase liquid composition of claim 106, comprising a dimethyl siloxane polymer having at least one hydrophilic side branch.

108. The single phase liquid composition of claim 106, wherein the silicone oil comprises a linear siloxane.

109. The single phase liquid composition of claim 106, wherein the silicone oil comprises disiloxane.

110. The single phase liquid composition of claim 106, wherein the silicone oil comprises trisiloxane.

111. The single phase liquid composition of claim 106, wherein the silicone oil comprises a tri-siloxane and a di-siloxane.

112. The single phase liquid composition of claim 106, wherein the silicone oil comprises a cyclical siloxane.

113. The single phase liquid composition of claim 106, wherein the silicone oil comprises a cyclical tri-siloxane.

114. The single phase liquid composition of claim 106, wherein the silicone oil comprises a cyclical tetra-siloxane.

115. The single phase liquid composition of claim 106, wherein the silicone oil comprises a cyclical penta-siloxane.

116. The single phase liquid composition of claim 106, wherein the silicone oil comprises a siloxane having an ethyl side group.

117. The single phase liquid composition of claim 106, wherein the silicone oil comprises a phenyl modified silicone oil.

118. The single phase liquid composition of claim 108, wherein the phenyl modified silicone oil comprises multimers of a repeating phenyl modified silicone unit.

119. The single phase liquid composition of claim 118, wherein the multimers comprise 5-20 monomer units.

120. The single phase liquid composition of claim 119, wherein a monomer unit of the silicone oil is121. The single phase liquid composition of claim 106, wherein the silicone oil comprises a dimethyl siloxane oil.

122. The single phase liquid composition of claim 121, wherein the phenyl modified silicone oil comprises multimers of a repeating phenyl modified silicone unit.

123. The single phase liquid composition of claim 122, wherein the multimers comprise at least 3-25 twenty monomer units.

124. The single phase liquid composition of claim 123, wherein a monomer unit of thesilicone oil is125. The single phase liquid composition of claim 106, wherein the siloxane surfactant comprises a lipophilic block, a hydrophilic block and a siloxane block.

126. The single phase liquid composition of claim 125, wherein the lipophilic bloc comprises a fatty alcohol.

127. The single phase liquid composition of claim 126, wherein the fatty alcohol comprises a cetyl block.

128. The single phase liquid composition of claim 126, wherein the fatty alcohol comprises a C-16 carbon constituent.

129. The single phase liquid composition of claim 126, wherein the fatty alcohol comprises a carbon constituent of 10-15 carbons.

130. The single phase liquid composition of claim 126, wherein the fatty alcohol comprises a carbon constituent of 17-25 carbons.

131. The single phase liquid composition of claim 125, wherein the hydrophilic block comprises PPG.

132. The single phase liquid composition of claim 125, wherein the siloxane surfactant+• o-fi O-SKCM, cwtICM,Mjf O*| |comprises133. The single phase liquid composition of claim 106, wherein the at least one hydrophilic side branch comprises PEG.

134. The single phase liquid composition of claim 106, wherein the dimethyl siloxane polymer comprisesO(CH2CH2O)p-CH3135. The single phase liquid composition of claim 134,wherein m is no greater than 50,wherein n is no greater than 50, andwherein p is no greater than 50.

136. The single phase liquid composition of claim 106, wherein the aqueous biomaterial comprises a first nucleic acid population sharing a first nucleic acid tag and a second nucleic acid population sharing a second tag.

137. The single phase liquid composition of claim 136, wherein the first nucleic acid population arises from a first cell and the second nucleic acid population arises from a second cell.

138. The single phase liquid composition of claim 136, wherein the first nucleic acid population is a cDNA population.

139. The single phase liquid composition of claim 136, wherein the first nucleic acid population is a genomic nucleic acid population.

140. The single phase liquid composition of claim 106, wherein the aqueous biomaterial comprises a first clonal cell population and a second clonal cell population.

141. The single phase liquid composition of claim 136, wherein the first nucleic acid population and the second nucleic acid population are intermingled.

142. The single phase liquid composition of claim 108, wherein the composition does not comprise chlorine.

143. The single phase liquid composition of claim 108, wherein the composition does not comprise fluorine.

144. The single phase liquid composition of claim 108, wherein the composition does not comprise a fluorocarbon.

145. A method of emulsion breaking, the method comprising adding to an emulsion an emulsion breaking oil and an emulsion breaker, and adding an aqueous phase.

146. The method of claim 145 comprising centrifuging.

147. The method of claim 145, wherein the emulsion is a water in oil emulsion for which the oil is a silicone oil.

148. The method of claim 145, wherein the emulsion breaking oil is a silicone oil.

149. The method of claim 145, wherein the emulsion breaker is a fatty alcohol.

150. The method of claim 149, wherein the fatty alcohol is heptanol.

151. The method of claim 149, wherein the fatty alcohol is octanol.

152. The method of claim 149, wherein the fatty alcohol is nonanol.

153. The method of claim 149, wherein the fatty alcohol does not comprise fluorine.

154. The method of claim 145, comprising recovering emulsion contents from the aqueous phase.

155. The method of claim 145, comprising assaying for the emulsion contents in the aqueous phase.

156. The method of claim 145, wherein the emulsion comprises microcapsules.

157. A method of emulsion breaking, the method comprising adding to a silicone oil emulsion an emulsion breaking aqueous phase.

158. The method of claim 157, wherein the emulsion breaking aqueous phase comprises a demulsifier.

159. The method of claim 158, wherein the demulsifier comprises a molecule of Fig.

3.

160. The method of claim 159, wherein the molecule of Fig. 3 comprises 60%-70% PEG.

161. The method of claim 159, wherein the molecule of Fig. 3 comprises 70%-80% PEG.

162. The method of claim 159, wherein the molecule of Fig. 3 comprises 80%-85% PEG.

163. The method of claim 159, wherein the molecule of Fig. 3 comprises 60%-70% PEG.

164. The method of claim 158, wherein the demulsifier comprises 65%-70% of a side chain comprising PEG and PPG.

165. The method of claim 164, wherein the side chain comprises at least 50% PPG.

166. The method of claim 164, wherein the side chain comprises at least 60% PPG.

167. The method of claim 164, wherein the side chain comprises no more than 50% PPG.

168. The method of claim 164, wherein the side chain comprises no more than 40% PPG.

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