(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
` CORRECTED VERSION
`
`(19) World Intellectual Property Organization
`International Bureau
`
`(43) International Publication Date
`12 September 2008 (12.09.2008)
`
` (10) International Publication Number
`
`WO 2008/109176 A2
`
`(51) International Patent Classification:
`CIZQ 1/68 (2006.01)
`301] 13/00 (2006.01)
`
`(21) International Application Number:
`PCT/US2008/003185
`
`(22) International Filing Date:
`
`7 March 2008 (07.03.2008)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/905,567
`
`English
`
`English
`
`7 March 2007 (07.03.2007)
`
`US
`
`(71) Applicant (for all designated States except US): PRES-
`IDENT AND FELLOWS 0F HARVARD COLLEGE
`
`[US/US]; 17 Quincy Street, Cambridge, MA 02138 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): AGRESTI, Jeremy
`[US/US]; 68 Dartmouth Street, Apt. #2, Belmont, MA
`02478 (US). CHU, Liang-yin [CN/CN]; School Of Chem—
`ical Engineering, Siehuan University, Chengdu 610065
`(CN). WEITZ, David, A.
`[US/US]; 213 Green Road,
`Bolton, MA 01740 (US). KIM, Jin-woong [KR/KR];
`303—806 Judong Apartment, Geumwha—village, Sang—
`galidong, Giheungrgu, Yonginrsi, Gyeonggiido 4467958
`(KR). ROWAT, Amy [CA/US]; 240 River Street, #8,
`Cambridge, MA 02139 (US). SOMMER, Morten
`[DK/USJ; 2 Brimmer Street #2, Boston, MA 02108 (US).
`DANTAS, Gautam [IN/US]; 14 Mead St Apt 2, Allston,
`MA 02134 (US). CHURCH, George [US/US]; 218 Kent
`Street, Brookline, MA 02446 (US).
`
`(74) Agent: OYER, Timothy, J.; Wolf, Greenfield & Sacks,
`P.C., Federal Reserve Plaza, 600 Atlantic Avenue, Boston,
`MA 02210—2206 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, CA,
`CH, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE,
`EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID,
`IL, IN, IS, JP, KE, KG, KM, KN, KP, KR, KZ, LA, LC,
`LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN,
`MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PG, PH,
`PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, SV,
`SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN,
`ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
`FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MT, NL,
`NO, PL, PT, RO, SE, SI, SK, TR), OAPI (BF, BJ, CF, CG,
`CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Published:
`
`— without international search report and to be republished
`upon receipt of that report
`
`(48) Date of publication of this corrected version:
`13 November 2008
`
`(15) Information about Correction:
`see Notice of 13 November 2008
`
`(54) Title: ASSAYS AND OTHER REACTIONS INVOLVING DROPLETS
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`Fig.1A
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`321
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`To Fig.1c
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`To Flg'. I D
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`(57) Abstract: The present invention generally relates to droplets and/or emulsions, such as multiple emulsions. In some cases, the
`droplets and/or emulsions may be used in assays, and in certain embodiments, the droplet or emulsion may be hardened to form a
`gel. In some aspects, a heterogeneous assay can be performed using a gel. For example, a droplet may be hardened to form a gel,
`where the droplet contains a cell, DNA, or other suitable species. The gel may be exposed to a reactant, and the reactant may interact
`with the gel and/or with the cell, DNA, etc., in some fashion. For example, the reactant may diffuse through the gel, or the hardened
`particle may liquefy to form a liquid state, allowing the reactant to interact with the cell. As a specific example, DNA contained
`within a gel particle may be subjected to PCR (polymerase chain reaction) amplification, e.g., by using PCR primers able to bind to
`the gel as it forms. As the DNA is amplified using PCR, some of the DNA will be bound to the gel via the PCR primer. After the
`PCR reaction, unbound DNA may be removed from the gel, e.g., via diffusion or washing. Thus, a gel particle having bound DNA
`may be formed in one embodiment of the invention.
`
`
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`WO 2008/109176
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`PCT/US2008/003185
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`-1-
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`ASSAYS AND OTHER REACTIONS INVOLVING DROPLETS
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`RELATED APPLICATIONS
`
`This application claims the benefit of U.S. Provisional Patent Application Serial
`
`No. 60/905,567, filed March 7, 2007, entitled “Assay and Other Reactions Involving
`
`Droplets,” by J.J. Agresti, et al., incorporated herein by reference.
`
`FIELD OF INVENTION
`
`The present invention generally relates to droplets and/or emulsions. In some
`
`cases, the droplets and/or emulsions may be used in assays.
`
`BACKGROUND
`
`An emulsion is a fluidic state which exists when a first fluid is dispersed in a
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`second fluid that is typically immiscible or substantially immiscible with the first fluid.
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`Examples of common emulsions are oil in water and water in oil emulsions. Multiple
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`emulsions are emulsions that are formed with more than two fluids, or two or more fluids
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`arranged in a more complex manner than a typical two-fluid emulsion. For example, a
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`multiple emulsion may be oil-in-water-in-oil, or water-in—oil-in-water. Multiple
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`emulsions are of particular interest because of current and potential applications in fields
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`such as pharmaceutical delivery, paints and coatings, food and beverage, and health and
`
`beauty aids.
`
`Typically, multiple emulsions consisting of a droplet inside another droplet are
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`made using a tw0-stage emulsification technique, such as by applying shear forces
`
`through mixing to reduce the size of droplets formed during the emulsification process.
`
`Other methods such as membrane emulsification techniques using, for example, a porous
`
`glass membrane, have also been used to produce water-in-oil-in-water emulsions.
`Microfluidic techniques have also been used to produce droplets inside ofdroplets using.
`a procedure including two or more steps. For example, see International Patent
`
`Application No. PCT/USZOO4/010903, filed April 9, 2004, entitled “Formation and
`
`Control of Fluidic Species,” by Link, et al. , published as WO 2004/091763 on October
`
`28, 2004; or International Patent Application No. PCT/USO3/20542, filed June 30, 2003,
`
`entitled “Method and Apparatus for Fluid Dispersion,” by Stone, et al. , published as WO
`
`2004/002627 on January 8, 2004, each of which is incorporated herein by reference. See
`
`also Anna, et al. , “Formation of Dispersions using ‘Flow Focusing’ in Microchannels,”
`
`Appl. Phys. Lett., 82:364 (2003) and Okushima, et al., “Controlled Production of
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`Monodispersed Emulsions by Two-Step Droplet Breakup in Microfluidic Devices,”
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`Langmuir 20:9905-9908 (2004). In some of these examples, a T-shaped junction in a
`
`microfluidic device is used to first form an aqueous droplet in an oil phase, which is then
`
`carried downstream to another T-junction where the aqueous droplet contained in the oil
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`phase is introduced into another aqueous phase. In another technique, co-axial jets can
`
`be used to produce coated droplets, but these coated droplets must be re-emulsified into
`
`the continuous phase in order to form a multiple emulsion. See Loscertales et al. ,
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`“Micro/Nano Encapsulation via Electrified Coaxial Liquid Jets,” Science 295:1695
`
`(2002).
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`Multiple emulsions and the products that can be made from them can be used to
`
`produce a variety of products useful in the food, coatings, cosmetic, or pharmaceutical
`
`industries, for example. Methods for producing multiple emulsions providing consistent
`
`droplet sizes, consistent droplet counts, consistent coating thicknesses, and/or improved
`
`control would make commercial implementation of these products more viable.
`
`SUMMARY OF THE INVENTION
`
`The present invention generally relates to droplets and/or emulsions. In some
`
`cases, the droplets and/or emulsions may be used in assays. The subject matter of the
`
`present invention involves, in some cases, interrelated products, alternative solutions to a
`
`particular problem, and/or a plurality of different uses of one or more systems and/or
`
`20
`
`articles.
`
`In one aspect, the invention is a method. In one set of embodiments, the method
`
`includes acts of providing a fluidic droplet containing a species, hardening the fluidic
`
`droplet containing the species, and exposing the species within the hardened fluidic
`
`droplet to a reactant. In another set of embodiments, the method includes acts of
`
`providing a gel droplet comprising a first nucleic acid and a second nucleic acid different
`
`from the first nucleic acid, the first nucleic acid being bound to the gel, and growing the
`
`first nucleic acid within the gel, using the second nucleic acid as a template. In still
`
`another set of embodiments, the method includes acts of providing a fluidic droplet
`
`containing a species, hardening the fluidic droplet containing the species, and liquefying
`
`the hardened fluidic droplet. In another set of embodiments, the method includes acts of
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`providing a first fluidic droplet and a second fluidic droplet, and causing the first fluidic
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`droplet and the second fluidic droplet to fuse, wherein the fused droplet hardens.
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`-3-
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`The method, in yet another set of embodiments, includes hardening a fluidic
`
`droplet containing cells, and causing the cells within the hardened fluidic droplet to
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`multiply.
`
`In another set of embodiments, the method includes an act of causing a PCR
`
`reaction to occur within a gel droplet. In still another set of embodiments, the method
`
`includes an act of forming a gel droplet containing a PCR primer bound to the gel.
`
`In one set of embodiments, the method includes acts of providing a fluidic
`
`droplet in a carrying fluid, the fluidic droplet substantially immiscible in water and the
`
`carrying fluid substantially immiscible in water, hardening the fluidic droplet, removing
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`the carrying fluid, and placing the hardened fluidic droplet in a third fluid.
`
`In another aspect, the invention is an article. In one embodiment, the article
`
`includes a gel droplet containing a clonal population of cells.
`
`In another embodiment, the article includes a gel droplet containing a PCR
`
`primer bound to the gel.
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`In another aspect, the present invention is directed to a method of making one or
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`more of the embodiments described herein. In another aspect, the present invention is
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`directed to a method of using one or more of the embodiments described herein.
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`Other advantages and novel features of the present invention will become
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`apparent from the following detailed description of various non-limiting embodiments of
`
`the invention when considered in conjunction with the accompanying figures. In cases
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`where the present specification and a document incorporated by reference include
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`conflicting and/or inconsistent disclosure, the present specification shall control. If two
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`or more documents incorporated by reference include conflicting and/or inconsistent
`
`disclosure with respect to each other, then the document having the later effective date
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`shall control.
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`BRIEF DESCRIPTION OF THE DRAWINGS
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`Non-limiting embodiments of the present invention will be described by way of
`
`example with reference to the accompanying figures, which are schematic and are not
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`intended to be drawn to scale. In the figures, each identical or nearly identical
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`component illustrated is typically represented by a single numeral. For purposes of
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`clarity, not every component is labeled in every figure, nor is every component of each
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`embodiment of the invention shown where illustration is not necessary to allow those of
`
`ordinary skill in the art to understand the invention. In the figures:
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`PCT/US2008/003185
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`-4-
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`Figs. lA-lD illustrates a microfluidic device of one embodiment of the invention;
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`Figs. 2A-2C illustrate the formation of droplets in accordance with another
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`embodiment of the invention;
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`Figs. 3A-3C illustrate temperature dependence of various microgels, in yet
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`another embodiment of the invention;
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`Figs. 4A-4B illustrate the probing of DNA in various hydro gel particles, in
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`another embodiment of the invention; and
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`Fig. 5 is a schematic illustration of a microfluidic device useful in making
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`multiple emulsions, according to one embodiment of the invention.
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`DETAILED DESCRIPTION
`
`The present invention generally relates to droplets and/or emulsions, such as
`
`multiple emulsions. In some cases, the droplets and/or emulsions may be used in assays,
`
`and in certain embodiments, the droplet or emulsion may be hardened to form a gel. In
`
`some aspects, a heterogeneous assay can be performed using the gel. For example, a
`
`droplet may be hardened to form a gel, where the droplet contains a cell, DNA, or other
`
`suitable species. The gel may be exposed to a reactant, and the reactant may interact
`
`with the gel and/or with the cell, DNA, etc., in some fashion. For example, the reactant
`
`may diffuse through the gel, or the hardened particle (or a portion thereof) may liquefy to
`
`form a liquid state, allowing the reactant to interact with the cell. As a specific example,
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`DNA contained within a gel particle may be subjected to PCR (polymerase chain
`
`reaction) amplification, e.g., by using PCR primers able to bind to the gel as it forms. As
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`the DNA is amplified using PCR, some of the DNA will be bound to the gel via the PCR
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`primer. After the PCR reaction, unbound DNA may be removed from the gel, e.g., via
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`diffusion or washing. Thus, a gel particle having bound DNA may be formed in one
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`embodiment of the invention.
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`Thus, in one aspect, the invention involves reactions involving liquid droplets, for
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`example, droplets contained within emulsions such as multiple emulsions. In certain
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`embodiments, systems and methods are providing for causing two or more droplets to
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`fuse or coalesce, e.g., in cases where the droplets ordinarily are unable to fuse or
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`coalesce, for example due to composition, surface tension, size, etc., e. g., to cause a
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`reaction to occur. For example, in a microfluidic system, the surface tension of the
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`fluidic droplets, relative to their size, may prevent fusion of the fluidic droplets. The
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`fluidic droplets may each independently contain gas or liquid.
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`PCT/US2008/003185
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`-5-
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`Fields in which droplets and emulsions may prove useful include, for example,
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`food, beverage, health and beauty aids, paints and coatings, and drugs and drug delivery.
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`For instance, a precise quantity of a drug, pharmaceutical, or other agent can be
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`encapsulated by a shell designed to release its contents under particular conditions, as
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`described in detail below. In some instances, cells can be contained within a droplet, and
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`the cells can be stored and/or delivered. Other species that can be stored and/or delivered
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`include, for example, biochemical species such as nucleic acids such as siRNA, RNAi
`
`and DNA, proteins, peptides, or enzymes. Additional species that can be incorporated
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`within a droplet or emulsion of the invention include, but are not limited to,
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`nanoparticles, quantum dots, fragrances, proteins, indicators, dyes, fluorescent species,
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`chemicals, or the like. A droplet or emulsion can also serve as a reaction vessel in
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`r
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`certain cases, such as for controlling chemical reactions, or for in vitro transcription and
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`translation, e. g., for directed evolution technology.
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`Using the methods and devices described herein, in some embodiments, a
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`consistent size and/or number of dr0plets can be produced, and/or a consistent ratio of
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`size and/or number of outer droplets to inner droplets, inner droplets to other inner
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`droplets, or other such ratios, can be produced. For example, in some cases, a single
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`droplet within an outer droplet of predictable size can be used to provide a specific
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`quantity of a drug. In addition, combinations of compounds or drugs may be stored,
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`transported, or delivered in a emulsion or droplet. For instance, hydrophobic and
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`hydrophilic species can be delivered in a single droplet, as the droplet can include both
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`hydrophilic and hydrophobic portions. The amount and concentration of each of these
`
`portions can be consistently controlled according to certain embodiments of the
`invention, which can provide for a predictable and consistent ratio of two or more
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`species in the multiple droplet.
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`Thus, in one aspect, the present invention is directed to systems, assays, methods,
`
`etc. involving gels produced as described herein, e.g., gel particles, gel capsules, and the
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`like. In some cases, a heterogeneous assay involving the gel can be performed. A gel
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`may allow easier use of a heterogeneous assay than by using a liquid emulsion drop
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`directly. That is, once a gel is formed, its composition can be changed by adding new
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`reagents or washing out old ones.
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`One non-limiting example of a heterogeneous assay in a microgel particle is
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`similar to a “polony,” as described in detail in U.S. Pat. Nos. 6,432,360, 6,485,944 and
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`6,511,803, and in PCT/USOS/06425, each incorporated by reference, as well as US. Pat.
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`Apl. Ser. No. 11/505,073, also incorporated herein by reference. Briefly, in one
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`embodiment, one or more DNA molecules (or other species, as described herein), either
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`naked, or contained within an intact cell, are encapsulated within a microgel at the time
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`of formation, e. g., by forming a droplet containing the cell, DNA molecule, other
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`species, etc., then hardening the droplet, e.g., to form a gel. In some cases, molecules of
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`one or more PCR primers having 5’ acrydite moieties may be included in the droplet,
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`prior to hardening. Upon polymerization of the gel, the primer may be covalently
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`coupled to the gel matrix, via the acrydite. The emulsion droplets may thus be collected
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`and caused to polymerize or gel. The surrounding fluid (e.g., an oil phase) can then be
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`removed, e. g., by washing away with a suitable solvent, leaving gel particles containing
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`the cell, DNA molecule, other species, etc., and the PCR primers. The gels can, in some '
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`cases, be resuspended in an aqueous solution containing reagents for PCR (e. g.,
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`buffering components, salts, dNTPs, DNA polymerase, and/or one or more PCR primers
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`with no acrydite modification). The suspension of gel particles can then be thermally
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`cycled in a test tube, e.g., using standard PCR cycling techniques known to those of
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`ordinary skill in the art, which may allow PCR amplification to occur, e.g., between pairs
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`of primers. The strand of DNA synthesized from the acrydite-modified primers may
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`thus be covalently coupled to the gel in some cases.
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`The gels can be washed to remove unreacted or unbound PCR components,
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`including DNA strands synthesized from non-acrydite primers. The remaining
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`covalently attached DNA strand can be probed using techniques known to those of
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`ordinary skill in the art, for example, by binding sequence-specific fluorescent
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`oligonucleotides and washing away unbound probes. As another example, a single-base
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`extension reaction can be used to probe the DNA sequence at a particular site. The
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`fluorescence of the gel from the bound oligonucleotides can be measured by measured
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`using techniques known to those of ordinary skill in the art, such as fluorescence
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`microscopy or by FACS. Either the presence or absence of a sequence can be
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`determined, and/or the sequence state at one or more positions can be determined, for
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`example, genotyping by SNPs. For instance, by using FACS, sub-populations of the gels
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`can be sorted and analyzed separately.
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`As discussed herein, in another set of embodiments, cells of any type
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`(prokaryotic or eukaryotic) can be encapsulated in the gel, and the DNA or RNA within
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`the cell can be used as a template for enzymatic amplification, in one embodiment of the
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`invention. This could be done, for example, by reverse transcription PCR (rtPCR) for
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`the study of RNA within the cell, or standard PCR for DNA. As another example, other
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`types of enzymatic amplification, such as whole-genome amplification by phi29
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`polymerase, can also be performed.
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`As yet another example, a collection of gels with covalently attached DNA may
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`be used as a library, e. g., that could be probed and washed many times in succession,
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`which would be especially useful when whole genomes are amplified in the gel, since
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`many regions of the DNA could be probed over time. Furthermore, in some cases, by
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`doing PCR from the library gels using unmodified primers, chosen sections of DNA can
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`be amplified away from the gel and analyzed further. For example, a gene can be
`
`amplified from a sub-population of library gels and then the supernatant from the PCR
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`could be sequenced by standard methods.
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`In another embodiment of the invention, particles, such as polymer beads, may be
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`encapsulated or incorporated into droplets which are then hardened into gels (e.g.,
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`forming gel particles, gel capsules, etc.). In one embodiment, the beads may be
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`magnetic, which could allow for the magnetic manipulation of the gels. The beads could
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`also be functionalized so that they could have other molecules attached, such as proteins,
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`nucleic acids or small molecules. One embodiment of the present invention is directed to
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`a set of beads encoding a library of, for example, nucleic acids, proteins, small
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`molecules, or other species as described herein, that would stay embedded in a gel
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`particle indefinitely.
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`As mentioned, in certain aspects, the invention generally relates to emulsions
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`and/or droplets. The emulsion may include droplets, such as those described above,
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`and/or colloid particles. As used herein, an “emulsion” is given its ordinary meaning as
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`used in the art, i.e., a liquid dispersion. In some cases, the emulsion may be a
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`“microemulsion” or a “nanoemulsion,” i.e., an emulsion having a dispersant on the order
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`of microns or nanometers, respectively. The dispersion or emulsion, in some cases, may
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`include droplets having a homogenous distribution of diameters, i.e., the droplets may
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`have a distribution of diameters such that no more than about 10%, about 5%, about 3%,
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`about 1%, about 0.03%, or about 0.01% of the droplets have an average diameter greater
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`than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the
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`average diameter of the droplets. As one example, such an emulsion may be created by
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`allowing fluidic droplets of the appropriate size or sizes (e. g., created as described
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`herein) to enter into a solution that is immiscible with the fluidic droplets.
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`Techniques for forming droplets have been disclosed in, for example, U.S. Patent
`
`Application Serial No. 11/360,845, filed February 23, 2006, entitled “Electronic Control
`
`of Fluidic Species,” by Link, et al. , published as U.S. Patent Application Publication No.
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`2007/000342 on January 4, 2007, incorporated herein by reference. For example,
`
`electric fields may be used to create droplets of fluid surrounded by a liquid. The fluid
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`and the liquid may be essentially immiscible in many cases, i.e., immiscible on a time
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`scale of interest (e. g., the time it takes a fluidic droplet to be transported through a-
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`IO
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`particular system or device). In certain cases, the droplets may each be substantially the
`
`same shape or size, as further described below. The fluid may also contain other species,
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`for example, certain molecular species (e.g., as further discussed below), cells, particles,
`
`etc.
`
`The electric field, in some embodiments, is generated from an electric field
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`generator, i.e., a device or system able to create an electric field that can be applied to the
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`fluid. The electric field generator may produce an AC field (i.e., one that varies
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`periodically with respect to time, for example, sinusoidally, sawtooth, square, etc.), a DC
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`field (i.e., one that is constant with respect to time), a pulsed field, etc. The electric field .
`
`generator may be constructed and arranged to create an electric field within a fluid
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`contained within a channel or a microfluidic channel. The electric field generator may
`
`be integral to or separate from the fluidic system containing the channel or microfluidic
`
`channel, according to some embodiments. As used herein, “integral” means that portions
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`of the components integral to each other are joined in such a way that the components
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`cannot be manually separated from each other without cutting or breaking at least one of
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`the components.
`
`Techniques for producing a suitable electric field (which may be AC, DC, etc.)
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`are known to those of ordinary skill in the art. For example, in one embodiment, an
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`electric field is produced by applying voltage across a pair of electrodes, which may be
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`positioned on or embedded within the fluidic system (for example, within a substrate
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`defining the channel or microfluidic channel), and/or positioned proximate the fluid such
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`that at least a portion of the electric field interacts with the fluid. The electrodes can be
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`fashioned from any suitable electrode material or materials known to those of ordinary
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`skill in the art, including, but not limited to, silver, gold, copper, carbon, platinum,
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`copper, tungsten, tin, cadmium, nickel, indium tin oxide (“ITO”), etc., as well as
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`combinations thereof. In some cases, transparent or substantially transparent electrodes
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`can be used.
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`In some embodiments of the invention, systems and methods are provided for at
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`least partially neutralizing an electric charge present on a fluidic droplet, for example, a
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`fluidic droplet having an electric charge, as described above. For example, to at least
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`partially neutralize the electric charge, the fluidic droplet may be passed through an
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`electric field and/or brought near an electrode, e. g., using techniques such as those
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`described herein. Upon exiting of the fluidic droplet from the electric field (i.e., such
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`that the electric field no longer has a strength able to substantially affect the fluidic
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`droplet), and/or other elimination of the electric field, the fluidic droplet may become
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`electrically neutralized, and/or have a reduced electric charge.
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`In another set of embodiments, droplets of fluid can be created from a fluid
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`surrounded by a liquid within a channel by altering the channel dimensions in a manner
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`that is able to induce the fluid to form individual droplets. The channel may, for
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`example, be a channel that expands relative to the direction of flow, e.g., such that the
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`fluid does not adhere to the channel walls and forms individual droplets instead, or a
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`channel that narrows relative to the direction of flow, e.g., such that the fluid is forced to
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`coalesce into individual droplets. In other embodiments, internal obstructions may also
`be used to cause droplet formation to occur. For instance, baffles, ridges, posts, or the
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`like may be used to disrupt liquid flow in a manner that causes the fluid to coalesce into
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`fluidic droplets.
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`In some cases, the channel dimensions may be altered with respect to time (for
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`example, mechanically or electromechanically, pneumatically, etc.) in such a manner as
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`to cause the formation of individual fluidic droplets to occur. For example, the channel
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`may be mechanically contracted (“squeezed”) to cause droplet formation, or a fluid
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`stream may be mechanically disrupted to cause droplet formation, for example, through
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`the use of moving baffles, rotating blades, or the like.
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`In some cases, an emulsion may include a larger fluidic droplet that contains one
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`or more smaller droplets therein which, in some cases, can contain even smaller droplets
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`therein, etc. In some cases, the droplet is surrounded by a liquid (e.g., suspended). Any
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`of these droplets may be of substantially the same shape and/or size (i.e.,
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`“monodisperse”), or of different shapes and/or sizes, depending on the particular
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`application. As used herein, the term “fluid” generally refers to a substance that tends to
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`flow and to conform to the outline of its container, i.e., a liquid, a gas, a viscoelastic
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`fluid, etc. Typically, fluids are materials that are unable to withstand a static shear stress,
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`and when a shear stress is applied, the fluid experiences a continuing and permanent
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`distortion.
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`The fluid may have any suitable viscosity that permits flow, for example, a
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`viscosity similar to water (e. g., as in an aqueous solution), oil, etc. In certain
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`embodiments of the invention, the liquid may include an oil or an organic solvent, such
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`as those known to ordinary skill in the art. If two or more fluids are present, each fluid
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`may be independently selected among essentially any fluids (liquids, gases, and the like)
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`by those of ordinary skill in the art, by considering the relationship between the fluids.
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`The fluids may each be miscible or immiscible. For example, two fluids can be selected
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`to be immiscible within the time frame of formation of a stream of fluids, or within the
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`time frame of reaction or interaction. As an example, where the portions remain liquid
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`for a significant period of time, the fluids may be immiscible. As another example,
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`where, after contact and/or formation, the dispersed portions are quickly hardened by
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`polymerization or the like, the fluids need not be as immiscible. Those of ordinary skill
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`in the art can select suitable miscible or immiscible fluids, using contact angle
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`measurements or the like, to carry out the techniques of the invention.
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`A “droplet,” as used herein, is an isolated portion of a first fluid that is
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`surrounded by a second fluid. It is to be noted that a droplet is not necessarily spherical,
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`but may assume other shapes as well, for example, depending on the external
`environment. In one embodiment, the droplet has a minimum cross-sectional dimension
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`that is substantially equal to the largest dimension of the channel perpendicular to fluid
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`flow in which the droplet is located.
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`As used herein, a first entity is “surrounded” by a second entity if a closed loop
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`can be drawn around the first entity through only the second entity. A first entity is
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`“completely surrounded” if closed loops going through only the second entity can be
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`drawn around the first entity regardless of direction. In one aspect, the first entity may
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`be a cell, for example, a cell suspended in media is surrounded by the media. In another
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`aspect, the first entity is a particle. In yet another aspect of the invention, the entities can
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`both be fluids. For example, a hydrophilic liquid may be suspended in a hydrophobic
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`liquid, a hydrophobic liquid may be suspended in a hydrophilic liquid, a gas bubble may
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`be suspended in a liquid, etc. Typically, a hydrophobic liquid and a hydrophilic liquid
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`are substantially immiscible with respect to each other, where the hydrophilic liquid has
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`a greater affinity to water than does the hydrophobic liquid. Examples of hydrOphilic
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`liquids include, but are not limited to, water and other aqueous solutions comprising
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`water, such as cell or biological media, ethanol, salt solutions, etc. Examples of
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`hydrophobic liquids include, but are not limited to, oils such as hydrocarbons, silicon
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`oils, fluorocarbon oils, organic solvents etc.
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`Many such oils are commercially available. As discussed above, the oil may be
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`chosen so as to be substantially immiscible in water, for instance, with solubilities of less
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`than about 50 ppb, less than about 25 ppb, or less than about 10 ppb (without
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`surfacntant). Examples of potentially suitable hydrocarbons include, but are not limited
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`to, light mineral oil (Sigma), kerosene (Fluka), hexadecane (Sigma), decane (Sigma),
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`undecane (Sigma), dodecane (Sigma), octane (Sigma), cyclohexane (Sigma), hexane
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`(Sigma), or the like. Non-limiting examples of potentially suitable silicone oils include 2
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`cst polydimethylsiloxane oil (Sigma). Non-limiting examples of fluorocarbon oils
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`include FC3283 (3M), FC4O (3M), Krytox GPL (Dupont), etc. In some cases, oils
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`potentially suitable for the invention include those that have viscosities of between about
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`0.8 cSt and about 1 cSt, or between about 0.7 cSt and about 0.9 cSt. In certain
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`embodiments