WO2018115978A2

WO2018115978A2 - Water-in-oil-in water emulsions for analysis of biological and chemical samples

Info

Publication number: WO2018115978A2
Application number: PCT/IB2017/001740
Authority: WO
Inventors: Mark Davies; Daniel Murphy
Original assignee: University of Limerick
Current assignee: University of Limerick
Priority date: 2016-12-23
Filing date: 2017-12-19
Publication date: 2018-06-28
Anticipated expiration: 2019-06-23
Also published as: WO2018115978A3

Abstract

The invention provides water-in-oil-in-water emulsions having large water-in-oil droplets in which chemical or biological reactions can be conducted and analyzed. The invention also provides fluidic systems comprising water-in-oil-in-water emulsions contained within the channel of a substrate. Also provided are methods of making water-in-oil-in-water emulsions having large water-in-oil droplets.

Description

WATER-IN-OIL-IN-WATER EMULSIONS FOR ANALYSIS OF BIOLOGICAL AND

CHEMICAL SAMPLES

Cross -Reference to Related Applications

This application claims the benefit of priority of U.S. Provisional Application No.

62/438,525, filed December 23, 2016, the contents of which are incorporated by reference.

Technical Field

The invention generally relates to water-in-oil-in-water emulsions having large water-in- oil droplets.

Background

Deadly diseases often fail to respond to a single therapeutic agent. For example, tuberculosis is caused by infection with the bacterium Mycobacterium tuberculosis. Derivatives of penicillin have historically been ineffective against M. tuberculosis because the bacterium has an active β-lactamase enzyme, and an increasing number of M. tuberculosis isolates from patients are resistant to other antibiotics as well. However, the combination of meropenem, an antibiotic that is metabolized slowly by M. tuberculosis β-lactamase, and clavulanate, an antibiotic that irreversibly inhibits the enzyme, is effective at treating tuberculosis.

Identifying new combinations of compounds that act synergistically to combat a disease poses both logistical and scientific problems. Vast numbers of experimental samples must be processed and analyzed. Existing robotic machines for liquid handling can be used to create fluidic droplets, but they do not have the mechanical means to combine drugs as part of droplet creation. Moreover, the droplets formed are not enclosed, so they require physical barriers to prevent their contents from mixing. Consequently, current methods for high-volume screening are performed in multi-well sample plates, and the cost of consumable products, such as sample plates and pipet tips, is significant. In addition, the user input required for preparation of robotic assays and analysis of the results makes the screening process laborious and time-consuming.

Many other endeavors to improve human health encounter similar problems related to consumable products, complex robotic machinery, and the human oversight required to perform large-scale processing. For example, some types of cancer have poor prognoses because existing assays are not sensitive enough to detect the disease in early stages. Early disease detection would be possible if large populations of biological components (e.g., cells, tissues, DNA molecules, proteins, antibodies, etc.) could be separated into individual members that could be analyzed, but large-scale screening for individual diagnostics is not feasible for the

aforementioned reasons. As another example, much biomedical research, particularly identification of new therapeutic compounds, relies on libraries that contain a large number of members of a class, such as chemical compounds, strains of a microorganism, etc. The individual members can be costly to produce in significant quantities, and size of the library (i.e., the number of members) is often limited by the logistics of preparing and maintaining it.

Consequently, people continue to suffer from diseases such as cancer and infectious diseases due to a dearth of new methods for identifying and treating these diseases.

Summary

The invention provides water-in-oil-in-water emulsions that include large, stable, and uniformly- sized water-in-oil droplets that have a single, continuous aqueous droplet surrounded by an oil layer. In preferred embodiments, the water-in-oil droplets are formed by mixing defined volumes of two or more aqueous solutions in an oil medium. The water-in-oil droplets are then extruded into an aqueous medium flowing through a channel of defined dimensions. By controlling the flow rate, channel size, and chemical properties of the channel wall, water-in-oil droplets having an inner aqueous volume of 200 nL or greater can be stably maintained in the aqueous medium. Because the oil layer forms a diffusion barrier to large molecules (e.g., proteins, antibodies, nucleic acids, carbohydrates, other macromolecules, etc.), cells, and viruses, the water-in-oil droplets serve as discrete chambers for separate, individual biological or chemical reactions. In addition, by using an oil that is permeable to small molecules, e.g., oxygen, carbon dioxide, metabolites, etc., chemical reactions or living cells can be sustained for hours or days within the water-in-oil droplet. Moreover, the dimensions and properties of the water-in-oil droplets permit the detection of a light-based signal (e.g., a fluorescent, luminescent, spectrophotometric, colorimetric, or other signal) while the sample remains in the water-in-oil droplet suspended in the aqueous medium. These features make the water-in-oil-in-water emulsions provided herein useful for rapid, high-volume analysis of biological or chemical reactions, such as the ability of various combinations of drug candidates to affect a particular target.

In certain aspects, the invention provides water-in-oil-in-water emulsions that include water-in-oil droplets, each having a single, continuous aqueous droplet surrounded by an oil layer, dispersed in an aqueous medium. The water-in-oil droplets may have any shape that droplets in an emulsion form naturally. For example and without limitation, the droplets may be spherical, ellipsoidal, or spheroidal. The water-in-oil droplets may have a maximum diameter of at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 μιη.

The aqueous droplet within each water-in-oil droplet may have a volume of at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 nL. The aqueous droplets in an emulsion may have uniform or nearly uniform volumes. For example, the aqueous droplets in an emulsion may have volumes that vary by less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5%, 4%, 3%, 2%, or 1%. The aqueous droplets may assume a shape similar that of the water-in-oil droplet, such as spherical, ellipsoidal, or spheroidal. The aqueous droplets may have a maximum diameter of at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 μιη.

The aqueous droplets may contain a member of population from a biological sample. For example and without limitation, the population from a biological sample may include nucleic acids, proteins, antibodies, carbohydrates, cells, cell clusters (such as islets), tissues, virus particles, macromolecular complexes, or organelles. To allow analysis of individual members of a population, a fraction of the aqueous droplets in the emulsion may contain a single member of the population, for example, one molecule, one cell, or one macromolecular complex. For example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the aqueous droplets in the emulsion may contain a single member of the population. The aqueous droplets may contain therapeutic agents or candidates for therapeutic agents, e.g., drugs, antibiotics, antiviral agents, enzyme inhibitors, etc. The aqueous droplets may contain multiple therapeutic agents or candidates for therapeutic agents. An emulsion may have droplets that all contain multiple therapeutic agents or candidates for therapeutic agents, but each aqueous droplet in the emulsion may contain a different combination of therapeutic agents or candidates for therapeutic agents The oil layer has a thickness sufficient to maintain the stability of the water-in-oil droplet. For example, the oil layer may be at least 5, 10, 20, 30, 40, 60, 80, 100, 200, or 400 nm thick. The oil layer may be less than 50, 30, 40, 10, 5, or 1 μηι thick. The oil layer may be between 10 nm and 50 μηι, between 40 nm and 20 μηι, between 100 nm and 10 μηι, or between 200 nm and 1 μηι thick. The oil layer may contain an oil with high permeability to small, water-soluble molecules, e.g., oxygen, carbon dioxide, metabolites, etc. For example, the oil layer may contain corn oil, mineral oil, or fluorinated oil. The oil layer may be substantially free of glycerides.

In certain aspects, the invention provides fluidic systems that include a channel in a substrate that holds a water-in-oil-in-water emulsion in which each water-in-oil droplet has a single, continuous aqueous droplet surrounded by an oil layer. The channel may have any shape that allows the structural integrity of individual water-in-oil droplets to be maintained and does not cause them to fuse or break apart. For example and without limitation, the cross-section of the channel may be circular, elliptical, oval, square, or rectangular. In a preferred embodiment, the channel is cylindrical with a circular cross-section. Preferably, the cross-section of the channel has a maximum diameter that is uniform along the length of the channel. The channel may have a maximum diameter of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1500 μηι. The cross-sectional dimensions of the channel influence the shape and size of the water-in-oil droplets. Preferably, the maximum diameter of the water-in-oil droplets is at least half the maximum diameter of the channel. Consequently, only one water-in-oil droplet can pass through a region of the channel at a time, i.e., the water-in-oil droplets move in single file as the emulsion flows through the channel.

The substrate may be made of any material suitable for formation of a channel. The substrate may be coated with a substance that stabilizes the emulsion, e.g., stabilizes the water- in-oil droplets within the aqueous medium. Thus, the substrate may be made of, or coated with, a material that prevents the oil layer of the water-in-oil droplets from contacting or interacting with the substrate. For example, the substrate may have a hydrophilic coating.

Because the oil layer of the water-in-oil droplets may be permeable to small, water- soluble molecules, the fluidic system may contain barriers that prevent diffusion of these small molecules between different water-in-oil droplets. For example, adjacent water-in-oil droplets in the fluidic system may be separated by one or more oil droplets. The oil droplets may contain an oil that is impermeable to small, water-soluble molecules. For example, the oil droplets may contain a silicone oil, such as pentamethyl cyclopentasiloxane.

In another aspect, the invention provides methods of making water-in-oil-in-water emulsions in which each water-in-oil droplet has a single, continuous aqueous droplet surrounded by an oil layer. The methods include adding a volume of an aqueous solution to the opening of a channel containing oil; optionally, adding a second volume of a second aqueous solution to the opening of the channel, thereby allowing the two aqueous solutions to mix;

adding oil to the opening of the channel, thereby creating an aqueous droplet surrounded by oil in a channel; and forcing the aqueous droplet surrounded by oil into a channel that contains a flowing aqueous medium, thereby creating a water-in-oil droplet in the aqueous medium to produce a water-in-oil-in-water emulsion.

Brief Description of the Drawings

FIG. 1 shows a water-in-oil-in-water emulsion according to embodiments of the invention.

FIG. 2 shows a fluidic system according to embodiments of the invention

FIG. 3 shows a fluidic system according to embodiments of the invention.

FIG. 4 shows a fluidic device for forming water-in-oil-in-water emulsions in which the aqueous droplets are a mixture of two different aqueous solutions.

FIG. 5 shows a microfluidic device for making water-in-oil-in-water emulsions according to certain embodiments.

FIG. 6 gives a top view of the microfluidic device of FIG. 5.

FIG. 7 gives a cutaway view of the microfluidic device of FIG. 5.

FIG. 8 shows a diagram of fluidic channel of a device that can be used to make emulsions according to some embodiments.

FIG. 9 shows slidable channels in a device that can be used to make emulsions according to some embodiments.

FIG. 10 diagrams steps of a method for combining aqueous solutions to form a water-in- oil-in-water emulsion in which the water-in-oil droplets contain a mixture of the aqueous solutions. Detailed Description

The invention provides water-in-oil-in-water emulsions that include large water-in-oil droplets dispersed in an aqueous medium. By using mechanical devices described previously (see WO 2015/173658 and PCT/IB2016/000980) and controlling the parameters of droplet formation, water-in-oil droplets that are uniform in size and contain a single, continuous aqueous droplet surrounded by an oil layer can be formed. The dimensions of the channel in which the water-in-oil droplets are formed and the flow rate through the channel are selected to prevent water-in-oil droplets from fusing or breaking into smaller droplets. In addition, aqueous droplets can be made by mixing sub-microliter volumes of two or more aqueous solutions. Consequently, each water-in-oil droplet serves as a discrete chamber in which the contents of the aqueous droplet can mix freely but are separated from the bulk aqueous medium and from other water-in- oil droplets. The sub-microliter volumes and physical properties of the water-in-oil droplets permit rapid processing of large numbers of samples. Thus, the water-in-oil-in-water emulsions provided herein are useful as a platform for conducting high-throughput analysis of biological and chemical reactions that can occur in a liquid aqueous environment.

FIG. 1 shows a water-in-oil-in-water emulsion 101 according to embodiments of the invention. The emulsion 101 is composed of water-in-oil droplets 109 dispersed in an aqueous medium 107. Each water-in-oil droplet 109 includes a single, continuous aqueous droplet 103 surrounded by an oil layer 105. The water-in-oil droplets 109 may have any shape that droplets in an emulsion form naturally, such as spherical, ellipsoidal, or spheroidal. The shape of the droplets may change as they flow through channels of fluidic device. The water-in-oil droplets 109 may have a maximum diameter of at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 μιη.

The aqueous droplet 103 within each water-in-oil droplet 109 is a single, continuous compartment, i.e., it is not sub-divided by internal oil layers. Consequently, the contents of the aqueous droplet 103 can diffuse throughout the droplet and mix freely with each other. The oil layer 105 forms a boundary that partitions the aqueous droplet 103 from the aqueous medium 107 and prevents mixing between these two aqueous phases. However, the oil layer 105 may be permeable to small molecules, such as oxygen and carbon dioxide, and thus allow the exchange of such molecules between the aqueous droplet 103 and the aqueous medium 107. The aqueous droplet 103 assumes a shape similar that of the water-in-oil droplet 109, such as spherical, ellipsoidal, or spheroidal, and the shape is dynamic. The aqueous droplets 103 may have a maximum diameter of at least 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 μιη. However, the volume of the aqueous droplet 103 remains constant even as its shape changes. The aqueous droplet 103 within each water-in-oil droplet 109 may have a volume of at least 50, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, or 1000 nL. Within an emulsion 101, the aqueous droplets 103 may have uniform or nearly uniform volumes. For example, the aqueous droplets 103 in the emulsion may have volumes that vary by less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.

The aqueous droplets 103 may contain components from a biological sample. For example and without limitation, the components from a biological sample may be nucleic acids, nucleotides, proteins, antibodies, amino acids, carbohydrates, cells, cell clusters (such as islets), tissues, virus particles, macromolecular complexes, or organelles. Biological samples include, without limitation, bodily fluids, such as blood, plasma, serum, urine, semen, sputum, saliva, tears, etc., cells and any components thereof, tissues, and cultures. It is understood that because each aqueous droplet is surrounded by an oil layer, the contents of an aqueous droplet 103 and the contents of the water-in-oil droplet 109 may be equivalent for water-soluble or water-residing components, such as nucleic acids, nucleotides, proteins, antibodies, amino acids, carbohydrates, cells, cell clusters (such as islets), tissues, virus particles, macromolecular complexes, or organelles. Therefore, in such instances, the contents of an aqueous droplet 103 and the contents of the water-in-oil droplet 109 are used interchangeably herein.

To allow analysis of individual members of a population of a component, i.e., individual molecules, cells, particles, etc., it is desirable that each aqueous droplet 103 within an emulsion 101 contain a single member of a population of a component. The parameters which govern this relationship are the volume of the droplets and the concentration of the components in the sample solution. The probability that a droplet will contain two or more molecules, cells, particles, etc. (P>₂) can be expressed as >₂=l-{ l+[cell]x } xe^_l where "[cell]" is the concentration of molecules, cells or particles in units of number of molecules, cells or particles per cubic micron (μιη ), and V is the volume of the droplet in units of μιη³.

Therefore, in certain embodiments of the water- in-oil-in- water emulsion 101, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the aqueous droplets 103 in the emulsion contain a single member of the population. Alternatively, many or most aqueous droplets 103 may contain 2, 3, 4, 5, 10, 20, 30, 50, or 100 members of the population. For example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the aqueous droplets 103 may contain 2, 3, 4, 5, 10, 20, 30, 50, or 100 members of the population. In other embodiments, the aqueous droplets 103 may contain a number of members above or below a certain threshold value. For example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the aqueous droplets 103 may contain fewer than 3, fewer than 4, fewer than 5, fewer than 10, fewer than 20, fewer than 30, fewer than 50, or fewer than 100 members of the population, or at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the aqueous droplets 103 may contain more than 1, more than 2, more than 3, more than 4, more than 5, more than 10, more than 20, more than 30, more than 50, or more than 100 members of the population.

In emulsions having a mixed population of aqueous droplets 103 in which some contain a species of interest and some do not contain the species of interest, the droplets may be screened or sorted for those droplets containing the species using light-based methods, as described below. Alternatively, droplets may be screened or sorted for those droplets that contain a particular number or range of the species of interest. Thus, in some cases, a plurality or series of aqueous droplets 103, some of which contain the species and some of which do not, may be enriched (or depleted) in the ratio of droplets that do contain the species. For example, a sub-population of droplets may be enriched by a factor of at least about 2, at least about 3, at least about 5, at least about 10, at least about 15, at least about 20, at least about 50, at least about 100, at least about 125, at least about 150, at least about 200, at least about 250, at least about 500, at least about 750, at least about 1000, at least about 2000, or at least about 5000 or more in some cases. In other cases, the enrichment may be in a ratio of at least about 10⁴, at least about 10⁵, at least about 10⁶, at least about 10⁷, at least about 10^s, at least about 10⁹, at least about 10¹⁰, at least about 10¹¹, at least about 10¹², at least about 10¹³, at least about 10¹⁴, at least about 10¹⁵, or more. For example, a droplet containing a particular species may be selected from a library of fluidic droplets containing various species, where the library may have about 100, about 10³, about 10⁴, about 10⁵, about 10⁶, about 10⁷, about 10⁸, about 10⁹, about 10¹⁰, about 10¹¹, about 10¹², about 10¹³, about 10¹⁴, about 10¹⁵ items. For example and without limitation, the library may be a DNA library, an RNA library, a protein library, or a combinatorial chemistry library.

P>2 can be minimized by decreasing the concentration of molecules, cells or particles in the sample solution. For sorting applications, however, decreasing the concentration of molecules, cells or particles in the sample solution also results in an increased volume of solution processed through the device and can result in longer run times. Accordingly, it may be desirable to minimize to presence of multiple molecules, cells or particles in the droplets (thereby increasing the accuracy of the sorting) and to reduce the volume of sample, thereby permitting a sorted sample in a reasonable time in a reasonable volume containing an acceptable

concentration of molecules, cells or particles.

The maximum tolerable P>₂ depends on the desired purity of the sorted sample. The purity in this case refers to the fraction of sorted molecules, cells or particles that possess a desired characteristic (e.g., display a particular antigen, are in a specified size range or are a particular type of molecule, cell, or particle). The purity of the sorted sample is inversely proportional to P>₂. For example, in applications where high purity is not needed or desired a relatively high P>₂ (e.g., P>₂=0.2) may be acceptable. For most applications, maintaining P>₂ at or below about 0.1, preferably at or below about 0.01, provides satisfactory results.

The aqueous droplets 103 may contain therapeutic agents or candidates for therapeutic agents, e.g., drugs, antibiotics, antiviral agents, enzyme inhibitors, etc. The aqueous droplets 103 may contain multiple therapeutic agents or candidates for therapeutic agents. An emulsion 101 may have aqueous droplets 103 that all contain multiple therapeutic agents or candidates for therapeutic agents, but each aqueous droplet 103 in the emulsion may contain a different combination of therapeutic agents or candidates for therapeutic agents.

The oil layer 105 of the water- in-oil droplet 109 has a thickness sufficient to maintain the stability of the water-in-oil droplet. For example, the oil layer 105 may be at least 5, 10, 20, 30, 40, 60, 80, 100, 200, or 400 nm thick. The oil layer 105 may be less than 50, 30, 40, 10, 5, or 1 μηι thick. The oil layer 105 may be between 10 nm and 50 μιη, between 40 nm and 20 μιη, between 100 nm and 10 μιη, or between 200 nm and 1 μιη thick.

In preferred embodiments, the oil layer 105 of the water-in-oil droplet 109 is a three- dimension fluid phase, i.e., it is not a bilayer. Biological membranes are bilayers composed substantially of glycerides, such as monoglycerides, diglycerides, and triglycerides. Therefore, the oil layer 105 may be substantially free of glycerides. Preferably, the oil layer 105 contains an oil with high permeability to small, water-soluble molecules, e.g., oxygen, carbon dioxide, metabolites, etc. For example, the oil layer may contain corn oil, mineral oil, or fluorinated oil. Alternatively, the oil layer may contain an oil that is impermeable or poorly permeable to small, water-soluble molecules. For example, the oil layer may contain a silicone oil, such as pentamethyl cyclopentasiloxane.

Preferably, the water-in-oil droplets 109 are transparent or translucent. The ability of light to pass through the water-in-oil droplets 109 depends on the thickness and type of oil in the oil layer 105, so these parameters may be adjusted to optimize light transmission. In certain embodiments, the aqueous droplet 103 contains a compound that produces or modifies a light- detectable signal when a chemical or biological reaction has occurred. Thus, the contents of the aqueous droplet 103 may be assayed by detection of a light-based signal in intact water-in-oil droplets 109. For example and without limitation, fluorescent, luminescent, spectrophotometric, or colorimetric signals may be detected in the aqueous droplet 103.

The water-in-oil droplets 109 can be used as chambers to conduct any type of biological or chemical reaction that can occur in a liquid aqueous environment. For example and without limitations, the water-in-oil droplets 109 can be used to assay chemical reactions, enzymatic reactions, binding of molecules (e.g., proteins, antibodies, ligands, etc.) or cells to other molecules or cells, cell growth, cell division, cell survival, cell death, cell differentiation, cell development, synthesis of macromolecules (e.g., nucleic acids, proteins, carbohydrates, polymers, etc.), or degradation of macromolecules (e.g., nucleic acids, proteins, carbohydrates, polymers, etc.).

To facilitate detection, one or more of the biological or chemical components may be labeled with a tag, dye, or quantum dot. Examples of fluorescent labels include, but are not limited to, Atto dyes, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid; acridine and derivatives: acridine, acridine isothiocyanate; 5-(2'-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino- l-naphthyl)maleimide; anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives; coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumaran 151); cyanine dyes; cyanosine; 4',6-diaminidino-2- phenylindole (DAPI); 5'5"-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red); 7- diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulfonic acid; 5-[dimethylamino]naphthalene-l-sulfonyl chloride (DNS, dansylchloride); 4- dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and derivatives; eosin, eosin isothiocyanate, erythrosin and derivatives; erythrosin B, erythrosin, isothiocyanate; ethidium; fluorescein and derivatives; 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2- yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein, fluorescein isothiocyanate, QFITC, (XRITC); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4-methylumbelliferoneortho cresolphthalein; nitrotyrosine; pararosaniline;

Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives: pyrene, pyrene butyrate, succinimidyl 1-pyrene; butyrate quantum dots; Reactive Red 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives: 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid; terbium chelate derivatives; Cy3; Cy5; Cy5.5; Cy7; IRD 700; IRD 800; La Jolta Blue; phthalo cyanine; and naphthalo cyanine. Preferred fluorescent labels are FAM and VIC (fluorescent label, commercially available from Applied Biosystems, Inc.). Labels other than fluorescent labels are contemplated by the invention, including other optically-detectable labels. See for example, United States Patent Application 20110244455.

The aqueous medium 107 may be any aqueous solution that supports formation of water- in-oil droplets 109. As indicated above, the oil layer 105 of the water- in-oil droplets 109 may be permeable to small molecules, and thus the contents of the aqueous medium may affect the chemical or biological reactions that occur in the aqueous droplets 103. Therefore, the contents aqueous medium may be selected to support the chemical or biological reactions that occur in the aqueous droplets 103. For example, the aqueous medium may contain buffers, salts, nutrients, drugs, growth factors, hormones, or other solutes. Preferably, the aqueous medium 107 is similar to the solution in the aqueous droplet 103 but for the specific analytes contained in the aqueous droplet 103. The aqueous medium may be replenished periodically or continuously to remove metabolites or waste products generated by the biological or chemical reactions that occur within the aqueous droplets and/or to provide fresh nutrients, substrates, etc.

FIG. 2 shows a fluidic system 201 according to embodiments of the invention. The fluidic system 201 includes a water-in-oil-in-water emulsion 101 contained within a channel 203 in a substrate 205. The water-in-oil-in-water emulsion 101 is composed of water-in-oil droplets 109 dispersed in an aqueous medium 107. Each water-in-oil droplet 109 includes a single, continuous aqueous droplet 103 surrounded by an oil layer 105.

The stability of water-in-oil droplets in a fluidic system is reflected in the Bond-Capillary (BC) number, which is represented by the following equation:

L²APgμu

D L —

r² where L is the diameter of the droplet, ΔΡ is the pressure differential, g is the

gravitational acceleration, μ is the dynamic viscosity, u is the flow velocity, and γ is the surface tension. A water-in-oil droplet is more stable in a system that has a lower BC number. As evident from the equation, increasing the droplet size leads to a higher BC number and thus lower droplet stability. However, by adjusting other elements in the system, water-in-oil droplets with diameters up to approximately 1 mm can be stably maintained.

A primary factor that affects the BC number is the surface tension. This parameter can be altered by the addition of selected surfactants to the aqueous medium 107 and to the oil that makes up the oil layer 105. Surfactants are agents that reduce the surface tension between two liquids, such as an aqueous solution and an oil. For example and without limitation, useful surfactants include sorbitan-based carboxylic acid esters (e.g., the "Span" surfactants, Fluka Chemika), including sorbitan monolaurate (Span 20), sorbitan monopalmitate (Span 40), sorbitan monostearate (Span 60) and sorbitan monooleate (Span 80), and perfluorinated polyethers (e.g., DuPont Krytox 157 FSL, FSM, and/or FSH), polyoxyethylenated alkylphenols (for example, nonyl-, p-dodecyl-, and dinonylphenols), polyoxyethylenated straight chain alcohols, polyoxyethylenated polyoxypropylene glycols, polyoxyethylenated mercaptans, long chain carboxylic acid esters (for example, glyceryl and polyglycerl esters of natural fatty acids, propylene glycol, sorbitol, polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters, etc.) and alkanolamines (e.g., diethanolamine-fatty acid condensates and isopropanolamine-fatty acid condensates).

Another factor that affects droplet stability is the surface modification of the substrate 205. If the oil layer 105 contacts the substrate 205, the water-in-oil droplet 109 can shear.

However, if the substrate 205 is highly hydrophilic, the oil layer 105 of the droplet will avoid contacting the substrate 205. Therefore, use of a hydrophilic substrate or substrate with a hydrophilic coating results in greater droplet stability. The substrate 205 may be made from polycyclic olefin polyethylene co-polymers, poly methyl methacrylate (PMMA), polycarbonate, polyalkanes and polystyrenes. The substrate may have one or more walls with multiple layers. For example, layers may include borosilicate glasses, pyrex, borofloat glass, Corning 1737, Corning Eagle 2000, silicon acrylic, polycarbonate, liquid crystal polymer,

polymethylmethoxyacrylate (PMMA), Zeonor, polyolefin, polystyrene, polypropylene, and polythiols.

The channel 203 may have any shape that provides a boundary for a fluid. The channel may be open, i.e., allow the fluid to be exposed to the external environment surrounding the channel, or closed. The channel may be a hybrid that includes one or more open portions and one or more closed portions. Preferably, the channel is closed and has a shape that promotes droplet stability, i.e., a shape that tends not to increase the pressure differential, gravitational acceleration, dynamic viscosity, or flow velocity of the system. For example and without limitation, the cross-section of the channel may be circular, elliptical, oval, square, or rectangular. In a preferred embodiment, the channel is cylindrical with a circular cross-section.

Preferably, the cross-section of the channel has a maximum diameter that is uniform along the length of the channel. The channel may have a maximum diameter of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1500 μιη. The cross-sectional dimensions of the channel influence the shape and size of the water-in-oil droplets. Preferably, the maximum diameter of the water-in-oil droplets 109 is at least half the maximum diameter of the channel 203. In this configuration, only one water-in-oil droplet 109 passes through a region of the channel at a time, i.e., the droplets move in single file as the emulsion flows through the channel 203, and fusion of droplets is avoided.

FIG. 3 shows a fluidic system according to embodiments of the invention. Because the oil layer 105 of the water-in-oil droplets 109 may be permeable to small, water-soluble molecules, fluidic systems in certain embodiments include barriers that prevent diffusion of these small molecules between different water-in-oil droplets 109. In the illustrated system, an oil droplet 303 suspended in the aqueous medium 107 is positioned in the channel 203 between two adjacent water-in-oil droplets 109. Adjacent water-in-oil droplets 109 may be separated by multiple oil droplets 303. Preferably, the oil droplets contain an oil that is impermeable to small, water-soluble molecules. For example, the oil droplets may contain a silicone oil, such as pentamethyl cyclopentasiloxane.

FIG. 4 illustrates a device for forming water-in-oil-in-water emulsions 101 in which the aqueous droplets 103 are a mixture of two different aqueous solutions. The device includes a first well 403 holding a first aqueous solution 411, a second aqueous well 405 holding a second aqueous solution 413, and a third well 407 holding oil 415. Each well has a port (not indicated) at the bottom. The device also includes a gap switch 419 with a vertical channel. The gap switch 419 can be moved so that the channel is alternately positioned below the first well 403, second well 405, or third well 407. Preferably, the channel of the gap switch has a surface material that is hydrophobic, such as polytetrafluoroethylene (PTFE). The device further includes a three-way crosspiece 427 that has a primary channel that extends horizontally in the illustration and a secondary channel that extends vertically and is positioned below the port of the third well. Preferably, the primary channel of the three-way crosspiece 427 has a surface material that is hydrophilic. The three-way crosspiece 427 may be made of a material, such as poly(methyl methacrylate) (PMMA), that can be readily coated with a hydrophilic coating

When the device illustrated in FIG. 4 is used for making a water-in-oil-in-water emulsion 101, the gap switch 419 partially loaded with oil 415 is placed under the port of the first well 403, and a defined volume of the first aqueous solution 411 is deposited from the first well 403 into the upper opening of the gap switch 419. The gap switch 419 is then positioned under the port of the second well 405, and a defined volume of the second aqueous solution 413 is deposited into the upper opening of the gap switch 419. The gap switch 419 is then positioned under the port of the third well 407, and oil 415 is allowed to flow into the upper opening of the gap switch 419 while oil simultaneously drains from a lower opening in the gap switch 419 and into the vertical channel of the three-way crosspiece 427. Flow rates of the first aqueous solution 411, second aqueous solution 413, and oil 415 may be determined by gravitational forces or by application of other forces, for example and without limitation, pressure, capillary action, electrophoresis, dielectrophoresis, or optical tweezers. Because the channel in the gap switch 419 is hydrophobic, the mixture of aqueous solutions in the gap switch 419 becomes surrounded by oil 415 to form a water-in-oil droplet. The oil containing the droplet exits the gap switch 419 and enters the secondary channel of the three-way crosspiece 427. Aqueous medium 107 flows through the horizontal channel of the three-way crosspiece 427. As the water-in-oil droplet 109 encounters the flowing aqueous medium 107, the oil 415 forms a layer that surrounds the aqueous droplet and is itself surrounded by the aqueous medium, thus forming the water-in-oil- in-water emulsion.

The aqueous droplet may be made by adding equal volumes of the first aqueous solution 411 and second aqueous solution 413. For example, 50, 100, 150, 200, 250, 300, 400, or 500 nL of each solution may added. Alternatively, different volumes of the two aqueous solutions may be mixed. For example, 50, 100, 150, 200, 250, 300, 400, or 500 nL of either solution can be added. The total volume of the aqueous droplet is the sum of volumes of each solution added. Thus, the total volume of the aqueous droplet may be 50, 100, 150, 200, 250, 300, 400, or 500, 600, 700, 800, 900, or 1000 nL. Mixture of the two aqueous solutions may cause a chemical or biological reaction to start or stop.

Because the water-in-oil droplets 109 are useful for performing chemical and biological assays as described above, the methods of making water-in-oil-in-water emulsions are broadly applicable. For example, the methods described herein may be used for DNA sequencing, microarray sample preparation, genotyping, gene expression, biodefense, food monitoring, forensics, disease modeling, drug investigations, proteomics, and cell biology.

Microfluidic devices that can be used to make the water-in-oil-in-water emulsions described herein have been described, for example, in WO 2015/173658 and

PCT/IB2016/000980, which are incorporated by reference.

FIG. 5 shows a microfluidic device 501 for making water-in-oil-in-water emulsions according to certain embodiments. The microfluidic device 501 includes a platform 545 with an axle member 521 extending upwards therefrom and a slot wheel 507 engaged to the axle member 521. A slot wheel 507 is capable of rotation relative to the platform 545 and may be rotated by the axle member 521. The ring assembly 505 includes the first ring 513 and the second ring 537, both of which are supported by the axle member 121 and disposed about the slot wheel 507. The first ring 513 includes at least one well 525 therein, open at the top. At least one shuttle 519 is engaged with the slot wheel 507 such that rotation of the slot wheel 507 converts to linear motion of the shuttle 519. The device comprises a shuttle carrier ring 531 to support shuttle 519. By inclusion of one or more motors connected to the rings and slot wheel, or by manual control, the microfluidic device 501 may be used form liquid volumes containing combinatorial libraries of numerous agents.

The first ring 513 may be configured to rotate relative to the platform 545, the slot wheel 507, or both. In order to rotate rings independently of each other, the devices may use a plurality of axle members 521. Additionally, the devices may comprise a plurality of gears coupled to the drive shafts or axles to turn the rings at various speeds.

FIG. 6 gives a top view of the microfluidic device 501, showing details of the ring assembly 505, as well as four shuttles 519. An end of the axle member 521 can be seen supporting first ring 513. Also visible is a portion of the second ring 537. Preferably the second ring 537 is concentric to the first ring 513. The first and second rings may be rotated at the same speed or at different speeds. The device may comprise several drive shafts and gears to rotate the rotor and rings at various speeds. One or more motors may be used to turn the drive shafts, which are coupled to gears, which are coupled to rotors and rings of the device to accomplish rotation. The slot wheel 507 comprises curved slots for receiving a pin of a shuttle. Each ring may comprise at least one liquid well 525.

FIG. 7 gives a cutaway view of the microfluidic device 501. At the top of the device 501 sits the ring assembly 505. The first ring 513 includes at least one well 525 open at the top and having an open dispensing port 529 at the bottom. The shuttle 519 is engaged with the slot wheel 507 such that rotation of the slot wheel 507 converts to linear motion of the shuttle 519. The shuttle 519 has a collection port 549 and the linear motion of the shuttle 519 aligns the collection port 549 with the dispensing port 529, causing liquid to flow from the well 525 into the collection port 549 of the shuttle 519.

The base 545 of the microfluidic device 501 includes a shuttle carrier ring 531 to aid in the movement of the shuttles 519 through the positioning of shuttle guides 593. When the shuttle 519 is pushed or pulled towards the center of the slot wheel 507 by the curved grooves, the shuttle guides 593 constrain the total movement of the shuttle such that the shuttle 519 slides back and forth under the rings.

The shuttle 519 has an elongated body having a distal portion 707 and proximal portion 709. The proximal portion 709 of the shuttle 519 has a pin 515. A flat, circular upper surface of the slot wheel 507 includes at least one curved groove to receive the pin 515. The pin 515 is configured to slide within the curved groove of the slot wheel 507. The microfluidic device 501 may include at least one motor to drive rotation of the slot wheel 507.

Preferably, the curved grooves of the slot wheel 507 are configured to convert the rotation of the slot wheel 507 to the linear motion of the shuttle 519. In some embodiments, the dispensing port 529 at the bottom of the well 525 has an opening that is less than 1.0 mm in any direction. In preferred embodiments, the dispensing port 529 at the bottom of the well 525 has an opening that is less than 0.5 mm in any direction. Preferably, the first ring 513 includes a plurality of wells. In the embodiment shown, the first ring 513 has 4 wells.

The proximal portion 707 of the shuttle 519 may include a dispensing port. The collection port 549 and the dispensing port of the shuttle 519 are in liquid communication through a channel that extends through the shuttle 519 (e.g., with the dispensing port located on surface opposite to the collection port 549). The dispensing port may configured to align with a collection channel located outside of the platform, or on the platform outside of the ring assembly 545, such that when aligned, liquid flows from the dispensing port of the shuttle into the collection channel.

Each ring and the slot wheel may be driven by its own motor, drive shaft, and

engagement surface. As shown in FIG. 7. motor 795 is built into the base 545 and the motor turns drive shaft 791. The drive shaft 791 turns an inner base member 733 via an engagement surface 799. The engagement surface 799 may be provided by meshing gears on the drive shaft 791 and the inner base member 733, or the engagement surface 799 may be provided by friction. The first ring 513 and the slot wheel may be driven by other instances of such mechanisms arrayed around the base 545.

FIG. 8 shows a diagram of microfluidic channel of a device that can be used to make emulsions according to some embodiments. The microfluidic channels of the devices are configured such that liquid is retained within the microfluidic channel when it is completely out of alignment with another microfluidic channel (e.g., no overlap between open ends of channels). Liquid may be retained within the microfluidic channel due to surface tension. The flow in a microfluidic channel system, as shown in FIG. 8, with a height of h, an internal diameter of d, a length of L, a fluid velocity of u, a fluid density of p, gravitation force of g, fluid viscosity of μ, and surface tension of y, can be represented by the equation:

or, rearranged as:

pghd

u

2γμ(2Η + L)

When fluid does not flow in the system, at maximum height, the equation becomes h = 4y/dpg.

FIG. 9 shows slidable channels in a device that can be used to make emulsions according to some embodiments. Importantly, channels and ports of the devices are configured so that when not aligned with another channel or port, there is no liquid flow there between. When aligned with another channel or port, liquid flows from the channel or port into the other channel or port. Alignment of channels can include complete or partial alignment. In complete alignment, the center axes of two microfluidic channels are aligned. In partial alignment, the center axes are not aligned, however, there is partial overlap of the first and second channels such that the distance between the center axes is sufficiently small so that flow between the two microfluidic channels occurs. In complete misalignment, there is no overlap between the channels and the distance between the center axes is sufficiently great so that flow between the two microfluidic channels does not occur. Alignment is meant to encompass both complete and partial alignment. The devices flow liquid between two microfluidic channels or ports even in the cases of partial alignment.

In some devices, when a channel or port is aligned with another channel or port, a gap or an air gap may exist there between. For example an air gap may exist between one port and another port of the device. The air gap may comprise any known gas, at any temperature and pressure. The air gap may be at atmospheric pressure and be comprised of air. However, the air gap is not limited to atmospheric pressure or air. The devices may be completely or partially enclosed within a chamber, and the chamber may be filled with a gas other than air. The pressure can be above or below atmospheric pressure and the temperature can be at, above, or below room temperature. In some devices, gravitational force is used to produce and control flow within the system. Thus, gravity drives the flow between the liquid compartment within a ring and the channel with the shuttle.

The volume of fluid that flows from one channel to another channel depends on the amount of time that the channels are aligned. As shown in FIG. 9, two channels 2800 and 2801 are aligned. Q is the flow rate in each channel, v is the velocity of the sliding channel, and r is the radius of the channel. Time when flowing is equal to nr/v, where n is the fraction of the lateral distance. As channel 2801 moves at a velocity relative to channel 2800, a volume of fluid flows from channel 2800 into channel 2801. G is the gap between the channels, and g is the force of gravity. The following equations denote the time required to dispense a volume, V from one channel to another channel. R is the resistance, P is the pressure, and u is the velocity. 8μ/Λ 8μΜ

"^'■ "^{h = Q} \nr*) ⁼ \nr*)

.·. pgh = u (^j

Qt = V where V=volume dispensed.

V 8iL\

For a given volume displaced we look to minimise time t.

t₌

Q LV

pghnr⁴

h = L for vertical channels

·'· t ⁼ pgnr* ^"^is equation denotes the time required to dispense a volume, V.

The liquid compartments or channels form the boundary for a liquid. A channel generally refers to a feature on or in the system (sometimes on or in a substrate) that at least partially directs the flow of a liquid. In some cases, the channel may be formed, at least in part, by a single component, e.g., an etched substrate or molded unit. The channel can have any cross-sectional shape, for example, circular, oval, triangular, irregular, square or rectangular (having any aspect ratio), or the like, and can be covered or uncovered (i.e., open to the external environment surrounding the channel).

The liquid compartments or channels may be partially or completely filled with liquid. In some cases the liquid may be held or confined within the liquid compartment or channel or port, for example, using surface tension. The channels or ports may be of a particular size or less, for example, having a largest dimension perpendicular to liquid flow of less than or equal to about 5 mm, less than or equal to about 2 mm, less than or equal to about 1 mm, less than or equal to about 500 microns, less than or equal to about 200 microns, less than or equal to about 100 microns, less than or equal to about 60 microns, less than or equal to about 50 microns, less than or equal to about 40 microns, less than or equal to about 30 microns, less than or equal to about 25 microns, less than or equal to about 10 microns, less than or equal to about 3 microns, less than or equal to about 1 micron, less than or equal to about 300 nm, less than or equal to about 100 nm, less than or equal to about 30 nm, or less than or equal to about 10 nm or less in some cases. Of course, in some cases, larger channels, tubes, etc. can be used to store liquids in bulk and/or deliver a liquid to the channel. The channels of the device can be of any geometry as described. However, the channels of the device can comprise a specific geometry such that the contents of the channel are manipulated, e.g., sorted, mixed, prevent clogging, etc. For example, for channels that are configured to carry droplets, the channels of the device may preferably be square, with a diameter between about 2 microns and 1 mm. This geometry facilitates an orderly flow of droplets in the channels. In some embodiments, the channel is a capillary.

The dimensions of the channels and ports, along with the time, control the amount or volume of liquid that is able to flow from a liquid compartment into the shuttle. The dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flow rate of liquid in the channel. The number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. The amount of liquid that flows from the liquid compartment into the shuttle can also be controlled by the speed of the shuttle. The longer time the shuttle is partially and completely aligned with the open bottom port of a liquid compartment, the greater the volume of liquid flows there between. Thus, one of skill in the art will appreciate that the speed of the shuttle can be controlled in order to control the amount of liquid from a liquid compartment flows into a port or channel of the shuttle. To prevent material (e.g., cells and other particles or molecules) from adhering to the sides of the channels, ports, or liquid compartments, they may have a coating which minimizes adhesion. Such a coating may be intrinsic to the material from which the device is manufactured, or it may be applied after the structural aspects of the channels have been fabricated. TEFLON (polymer, commercially available from DuPont, Inc.), or polytetrafluoroethylene (PTFE), is an example of a coating that has suitable surface properties. Channels, ports, or liquid compartments may be constructed from PTFE, or PTFE-containing materials. Additionally, or in the alternative, channels, ports, or liquid compartments may be coated with PTFE. Preferably, the walls of the interior portion of the microfluidic channels are composed of PTFE, or a material containing PTFE, to render the interiors of the microfluidic channels hydrophobic.

The surface of the channels, ports, or liquid compartments of the microfluidic system can be coated with any anti-wetting or blocking agent for the dispersed phase. The channels, ports, or liquid compartments can be coated with any protein to prevent adhesion of the biological or chemical sample. For example, in one embodiment the channels are coated with BSA, PEG- silane and/or fluorosilane. For example, 5 mg/ml BSA is sufficient to prevent attachment and prevent clogging. In another embodiment, the channels, ports, or liquid compartments can be coated with a cyclized transparent optical polymer obtained by copolymerization of perfluoro (alkenyl vinyl ethers), such as the type sold by Asahi Glass Co. under the trademark Cytop. In such an embodiment, the coating is applied from a 0.1-0.5 wt % solution of Cytop CTL-809M in CT-Solv 180. This solution can be injected into the channels, ports, or liquid compartments of a microfluidic device via a plastic syringe. The device can then be heated to about 90°C for 2 hours, followed by heating at 200°C for an additional 2 hours. In another embodiment, the channels, ports, or liquid compartments can be coated with a hydrophobic coating of the type sold by PPG Industries, Inc. under the trademark Aquapel (e.g., perfluoroalkylalkylsilane surface treatment of plastic and coated plastic substrate surfaces in conjunction with the use of a silica primer layer) and disclosed in U.S. Pat. No. 5,523,162, which patent is hereby incorporated by reference. By fluorinating the surfaces of the channels, ports, or liquid compartments, the continuous phase preferentially wets the channels and allows for the stable generation and movement of droplets through the device. The low surface tension of the channel walls thereby minimizes the accumulation of channel clogging particulates. The surface of the channels, ports, or liquid compartments in the microfluidic device can be also fluorinated to prevent undesired wetting behaviors. For example, a microfluidic device can be placed in a polycarbonate desiccator with an open bottle of (tridecafluoro-1,1,2,2- tetrahydrooctyl) trichlorosilane. The desiccator is evacuated for 5 minutes, and then sealed for 20-40 minutes. The desiccator is then backfilled with air and removed. This approach uses a simple diffusion mechanism to enable facile infiltration of channels of the microfluidic device with the fluorosilane and can be readily scaled up for simultaneous device fluorination.

The liquid compartments of the devices may contain any type of liquid. As discussed above, the direction and flow of liquids and entities within the device can be controlled. The term "flow" generally refers to any movement of liquid or solid through a device and encompasses without limitation any liquid stream, and any material moving with, within or against the stream, whether or not the material is carried by the stream. The application of any force may be used to provide a flow, including without limitation, pressure, capillary action, electro-osmosis, electrophoresis, dielectrophoresis, optical tweezers, gravity, and combinations thereof, without regard for any particular theory or mechanism of action, so long as molecules, cells or virus particle are directed for detection, measurement or sorting.

FIG. 10 diagrams steps of a method 801 for combining aqueous solutions to form a water-in-oil-in-water emulsion in which the water-in-oil droplets contain a mixture of the aqueous solutions. The method 801 includes providing 813 a first aqueous solution, for example, a solution with a first drug. The first aqueous solution is deposited 825 over oil in a channel, as illustrated in FIG. 4. A second aqueous solution, for example, a solution containing one or more of a second drug, a cell, a nucleic acid, a molecule, a protein, a virus, and a pathogen, is provided 829. The second aqueous solution is deposited 835 over oil in the channel with the first aqueous solution, allowing the two aqueous solutions to mix. Oil is then deposited 839 over the mixture of aqueous solutions to create a water-in-oil droplet. The water-in-oil suspension is then flowed 845 into the aqueous medium. As the water-in-oil droplet enters the aqueous medium, it forms 851 a water-in-oil-in-water emulsion. Incorporation by Reference

References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

Equivalents

Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Claims What is claimed is:

1. A water-in-oil-in-water emulsion comprising:

an aqueous medium; and

water-in-oil droplets dispersed in the aqueous medium, each water-in-oil droplet comprising:

a single, continuous aqueous droplet having a volume of at least 200 nL; and an oil layer surrounding the aqueous droplet.

2. The water-in-oil-in-water emulsion of claim 1, wherein each water-in-oil droplet has a maximum diameter of at least 250 μιη.

3. The water-in-oil-in-water emulsion of claim 1, wherein the volume of the aqueous droplets varies by less than 20%.

4. The water-in-oil-in-water emulsion of claim 1, wherein the oil layer has a thickness of at least 10 nm.

5. The water-in-oil-in-water emulsion of claim 1, wherein the oil layer is substantially free of glycerides.

6. The water-in-oil-in-water emulsion of claim 1, wherein the oil layer comprises a permeable oil.

7. The water-in-oil-in-water emulsion of claim 6, wherein the permeable oil is selected from the group consisting of corn oil, mineral oil, and fluorinated oil.

8. The water-in-oil-in-water emulsion of claim 1, wherein at least 20% of the water-in-oil droplets comprise aqueous droplets that comprise exactly one member of a population from a biological sample.

9. The water-in-oil-in-water emulsion of claim 8, wherein the population comprises nucleic acids, proteins, antibodies, carbohydrates, cells, cell clusters, virus particles, macromolecular complexes, or organelles.

10. A fluidic system comprising:

a channel in a substrate, the channel having a maximum diameter of at least 500 μιη; and a water-in-oil-in-water emulsion disposed within the channel, the emulsion comprising: an aqueous medium; and

water-in-oil droplets dispersed in the aqueous medium, each water-in-oil droplet having a maximum diameter of at least 250 μιη and comprising:

a single, continuous aqueous droplet; and

an oil layer surrounding the aqueous droplet.

11. The fluidic system of claim 10, wherein the channel is cylindrical.

12. The fluidic system of claim 10, wherein the substrate has a hydrophilic coating.

13. The fluidic system of claim 10, wherein each aqueous droplet has a volume of at least 200 nL.

14. The fluidic system of claim 13, wherein the volume of the aqueous droplets varies by less than 20%.

15. The fluidic system of claim 10, wherein the oil layer has a thickness of at least 10 nm.

16. The fluidic system of claim 10, wherein the oil layer is substantially free of glycerides.

17. The fluidic system of claim 10, wherein the oil layer comprises a permeable oil.

18. The fluidic system of claim 17, wherein the permeable oil is selected from the group consisting of corn oil, mineral oil, and fluorinated oil.

19. The fluidic system of claim 10, wherein at least 20% of the water-in-oil droplets comprise aqueous droplets that comprise exactly one member of a population from a biological sample.

20. The fluidic system of claim 19, wherein the population comprises nucleic acids, proteins, antibodies, carbohydrates, cells, cell clusters, tissues, virus particles, macromolecular complexes, or organelles.

21. The fluidic system of claim 10, wherein adjacent water-in-oil droplets within the channel are separated by at least one oil droplet.

22. The fluidic system of claim 21, wherein the oil droplet comprises a silicone oil.

23. The fluidic system of claim 22, wherein the silicone oil is pentamethyl

cyclopentasiloxane.