WO2009061372A1

WO2009061372A1 - Systems and methods for creating multi-phase entities, including particles and/or fluids

Info

Publication number: WO2009061372A1
Application number: PCT/US2008/012384
Authority: WO
Inventors: Rhutesh Kishorkant Shah; Jin-Woong Kim; David A. Weitz
Original assignee: President And Fellows Of Harvard College
Priority date: 2007-11-02
Filing date: 2008-10-31
Publication date: 2009-05-14
Also published as: WO2009061372A9

Abstract

The present invention generally relates to multi-phase entities, which may include one or more phases containing particles. The particles may be agglomerated in some cases. In one embodiments, the multi-phase entity contains one or more phases and/or regions, which each may independently be a solid or a liquid. For example, a multi-phase entity may contain a solid phase and a liquid phase, a first solid phase and a second solid phase, a first liquid phase and a second liquid phase, etc., and the phases may be present within one or more phases within the entity. In some aspects of the invention, the hydrophobicities/hydrophilicities of one or more phases of the multi- phase entity are sensitive to temperature, pH, and/or an analyte, etc. Still other aspects of the invention generally relate to systems and methods of making and using such multi-phase entities, kits involving such entities, or the like.

Description

SYSTEMS AND METHODS FOR CREATING MULTI-PHASE ENTITIES, INCLUDING PARTICLES AND/OR FLUIDS

RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application Serial

No. 61/001,601, filed November 2, 2007, entitled "Systems and Methods for Creating Multi-Phase Entities, Including Particles and/or Fluids," by Shah, et al, the entirety of which is incorporated herein by reference.

FIELD OF INVENTION

The present invention generally relates to multi-phase entities, which may include one or more phases containing particles. In some cases, the particles may be agglomerated.

BACKGROUND The manipulation of fluids to form fluid streams of desired configuration, discontinuous fluid streams, droplets, particles, dispersions, etc., for purposes of fluid delivery, product manufacture, analysis, and the like, is a relatively well-studied art. For example, highly monodisperse gas bubbles, less than 100 microns in diameter, have been produced using a technique referred to as capillary flow focusing. In this technique, gas is forced out of a capillary tube into a bath of liquid, the tube is positioned above a small orifice, and the contraction flow of the external liquid through this orifice focuses the gas into a thin jet which subsequently breaks into equal-sized bubbles via a capillary instability. In a related technique, a similar arrangement was used to produce liquid droplets in air. SUMMARY OF THE INVENTION

The present invention generally relates to multi-phase entities, which may include one or more phases containing particles. The particles may be agglomerated in some cases. 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 articles.

In one aspect, the invention relates to an article. In one embodiment, the article comprises a multi-phase entity comprising at least a first phase and a second phase, wherein at least one of the phases comprises colloidal particles, and at least some of the colloidal particles have a first hydrophobicity/hydrophilicity at a first temperature and a second hydrophobicity/hydrophilicity different from the first hydrophobicity/hydrophilicity at a second temperature. In another embodiment, the article comprises a multi-phase entity comprising at least a first phase and a second phase, wherein at least one of the phases comprises colloidal particles, and at least some of the colloidal particles have a first hydrophobicity/hydrophilicity at a first pH and a second hydrophobicity/hydrophilicity different from the first hydrophobicity/hydrophilicity at a second pH. The article, in yet another embodiment, comprises a multi-phase entity comprising at least a first phase and a second phase, wherein at least one of the phases comprises colloidal particles, and at least some of the colloidal particles are able to bind to an analyte.

In another embodiment, the article comprises a multi-phase entity having a maximum dimension of no more than about 1 mm, wherein the multi -phase entity comprises at least three phases, and at least one phase is defined by an agglomeration of sub-particles.

The article, according to one embodiment comprises an outer fluid droplet containing one or more first fluid droplets, at least one of which contains one or more second fluid droplets, wherein at least one of the second fluid droplets contains agglomerated particles.

In accordance with another embodiment, the article comprises a fluid droplet containing more than one nesting level of inner fluid droplets therein, wherein a nesting level is defined by one or more fluid droplets each contained within a surrounding fluid droplet, and wherein at least one nesting level is defined by an agglomeration of particles.

The article comprises a microparticle containing a first phase surrounding a second phase, wherein the second phase is defined by an agglomeration of sub-particles, according to another embodiment. In another embodiment, the article comprises a microparticle containing at least three phases with one of the phases surrounding the other phases, and at least one of the phases is defined by an agglomeration of sub-particles. The article, in yet another embodiment, comprises a particle having a first, generally hemispherical phase and a second, generally hemispherical phase, wherein the second generally hemispherical phase is porous.

Another aspect of the invention relates to a method. In one embodiment, the method comprises providing a multi-phase entity comprising at least a first phase and a second phase, and altering the hydrophobicity/hydrophilicity of the first phase by altering the temperature of the multi-phase entity.

The method comprises providing a multi-phase entity comprising at least a first phase and a second phase, and altering the hydrophobicity/hydrophilicity of the first phase by altering pH, according to another embodiment of the invention.

In yet another embodiment, the method comprises providing a fluid containing an emulsion defined by a continuous and a discontinuous phase defined by a plurality of fluid droplets, and solidifying at least a portion of the discontinuous phase without solidifying the continuous phase. The method, in another embodiment, comprises providing emulsified fluid droplets, each droplet defined by a continuous and a discontinuous phase, condensing the discontinuous phase in each fluid droplet in one portion of the fluid droplet, and polymerizing the continuous phase in each fluid droplet.

Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where the present specification and a document incorporated by reference include conflicting and/or inconsistent disclosure, the present specification shall control. If two 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 shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

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 intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each - A - 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:

FIGS. 1 A-IC illustrate the agglomeration of microgels according to one set of embodiments of the invention; FIGS. 2A-2E illustrate the method of producing articles according to one set of embodiments of the invention;

FIG. 3 is a photograph of a microreactor used to produce articles according to one set of embodiments of the invention;

FIGS. 4A-4E are a series of micrographs showing articles according to one set of embodiments of the invention;

FIG. 5 is a schematic illustrating a method of producing articles according to one set of embodiments of the invention;

FIGS. 6A-6B are plots illustrating the dependence of a property of an article on system parameters according to one set of embodiments of the invention; FIGS. 7A-7E are schematics of methods of producing articles and resultant articles according to one set of embodiments of the invention;

FIGS. 8A-8B are micrographs of articles according to one set of embodiments of the invention;

FIGS. 9A-9C are confocal images illustrating the effect of a system parameter on an article property according to one set of embodiments of the invention;

FIGS. 10A- 1OB are (A) a micrograph and (B) a confocal image of an article according to one set of embodiments of the invention;

FIGS. 1 IA-I IE are micrographs of an article according to one set of embodiments of the invention; FIG. 12 is a schematic representation of a set of embodiments used to make particles;

FIGS. 13A-13C illustrate particles formed by photopolymerizing monomers in phase separated droplets according to one set of embodiments;

FIGS. 14A-14B illustrate closely packed microgels forming a raspberry-like structure, according to one set of embodiments;

FIG. 15A-15B illustrate the swelling kinetics of a PNIPAm phase within a droplet upon cooling to room temperature, according to one set of embodiments; FIG. 16 is a photograph illustrating the behavior of magnetic particles, according to one set of embodiments;

FIG. 17 is a fluorescent microscope image displaying an array of microgels synthesized by precipitation polymerization, according to one set of embodiments; FIG. 18A-18B illustrate capillary based microfluidic device that uses flow focusing geometry, according to one set of embodiments; and

FIG. 19 includes a micrograph of a microfluidic device used to generate droplets, according to one set of embodiments.

DETAILED DESCRIPTION

The present invention generally relates to multi-phase entities, which may include one or more phases containing particles. The particles may be agglomerated in some cases. In one embodiment, the multi-phase entity contains one or more phases and/or regions, which each may independently be a solid or a liquid. For example, a multi- phase entity may contain a solid phase and a liquid phase, a first solid phase and a second solid phase, a first liquid phase and a second liquid phase, etc., and the phases may be present within one or more regions within the entity. In some aspects of the invention, the hydrophobicities/hydrophilicities of one or more phases of the multi-phase entity are sensitive to temperature, pH, and/or an analyte, etc. Still other aspects of the invention generally relate to systems and methods of making and using such multi -phase entities, kits involving such entities, or the like.

Fields in which various multi-phase entities of the present invention may be useful include (but are not limited to), for example, food, beverage, health and beauty aids, paints and coatings, chemical separations, and drugs and drug delivery. For instance, a precise quantity of a drug, pharmaceutical, or other agent can be contained within an entity by a phase designed to release its contents under particular conditions, as described in detail below. Other species that can be stored and/or delivered in an entity include, for example, biochemical species such as nucleic acids such as RNA or DNA, proteins, peptides, or enzymes. Additional species that can be incorporated within a multi-phase entity include, but are not limited to, nanoparticles, quantum dots, charged particles, magnetic particles, fragrances, proteins, indicators, dyes, fluorescent species, chemicals, or the like. Various aspects of the present invention are generally directed to a multi-phase entity. As used herein, a "multi-phase entity" is an entity containing a least a first phase and a second phase, and in some cases, a third phase, a fourth phase, etc. The "phases" will have distinct chemical and/or physical properties. For instance, each of the phases may independently be a solid or a fluid such as a liquid. For example, the multi-phase entity may contain a solid phase and a liquid phase, a first solid phase and a second solid phase, a first liquid phase and a second liquid phase, etc. As non-limiting examples, FIG. IB shows a multi-phase entity having a liquid phase and a solid phase. As another example, FIGS. 4A-4B show a collection of multi-phase entities having a first solid phase and a second solid phase. The phases may be positioned in any location within the multi-phase entity, for example, one phase may be surrounded by a second phase (e.g. as shown in FIGS. lOA-1 IE), two phases may be positioned side-by-side (for example, as in a Janus particle, where each phase has a generally hemispherical shape, e.g., as is shown in FIG. IB, or the like). In some cases, multiple phases may be nested, as discussed below. In certain instances, a combination of these and/or other phases may be present within the multi-phase entity. In some cases, the multi-phase entity has a maximum dimension of no more than about 1 mm. In other cases, the multi-phase entity may have a maximum dimension of less than about 500 micrometers, less than about 300 micrometers, less than about 100 micrometers, less than about 10 micrometers, or less than about 1 micrometer or smaller, in some cases.

In some cases, one or more phases of a multi -phase entity may be present as a single continuous region, or as multiple, discrete regions within the multi-phase entity. As a specific, non-limiting example, a multi-phase entity may be droplet containing an emulsion therein (e.g., such that the continuous phase of the emulsion is the first phase, and the discontinuous phase of the emulsion is the second phase, present as a plurality of discrete regions contained within the first phase). As another non-limiting example, a multi-phase entity may be a droplet or a particle containing (sub-)particles, as discussed below, where the (sub-)particles define a phase within the entity. The (sub-)particles may be present as discrete particles (thereby defining discrete regions within the droplet or particle), and/or as one or more agglomerations of particles. In some cases, a plurality of sub-particles may be agglomerated or immobilized relative to each other in some fashion, thereby defining a solid phase within the multi-phase entity. In one set of embodiments, the multi-phase entity may be created using a microfluidic system. Non-limiting examples of such a system are shown in FIGS. 3, 4A- 4B, and 8A-8B. FIG. 2 A schematically illustrates the creation of multi -phase entities in a microfluidic system, according to one embodiment of the invention. In this non- limiting example, an aqueous emulsion phase containing PNIPAM (poly(N-isopropyl acrylamide)), PAA (poly(acrylic acid)), acrylamide, a crosslinker, and a photoinitiator are fed into a capillary channel. The aqueous phase, in this example, is surrounded by a continuous phase of silicon oil (e.g., DC550 from Dow Corning). Downstream of the inlet, phase separation of the emulsion is induced by, for example, heating the system. Upon phase separation, the PNIPAM may form an agglomerated microgel which can condense to one side of the multi-phase entity. Next, the residual polymer in the aqueous phase may be polymerized, forming a multi-phase entity where the two phases are positioned side-by-side. This technique can produce multi-phase entities such as the examples shown in FIGS. 2C-2E. In other embodiments, however, a microfluidic system is not necessary to create the multi-phase entities.

One aspect of the present invention is generally directed to multi -phase entities having at least three phases. The phases may be positioned in any orientation within the entity relative to each other. For instance, one of the phases may surround the other phases. The multi-phase entity may be created from multiple emulsions such as those described in International Patent Application No. PCT/US2006/007772, filed March 3, 2006, entitled "Method and Apparatus for Forming Multiple Emulsions," published as WO 2006/096571 on September 14, 2006 or U.S. Provisional Patent Application Serial No. 60/920,574, filed March 28, 2007, entitled "Multiple Emulsions and Techniques for Formation," each incorporated herein by reference, and may include any number of phases, which may be side-by-side or nested in some cases. For example, an entity may contain one, two, three, four, or more phases therein, some or all of which can contain additional phases therein. There can be any number of nestings present within a multiphase entity of the invention. For example, a first phase may contain a second phase, which may contain a third phase, etc. A nesting level can be defined by one or more phases each contained within a surrounding phase.

In some cases, as mentioned, one phase may be present within a multi-phase entity as a plurality of discrete regions, e.g., as a plurality of droplets, particles, or the like. Any of these may be of substantially the same shape and/or size (i.e., "monodisperse"), or of different shapes and/or sizes, depending on the particular application. For instance, the droplets, particles, etc. may have a homogenous distribution of diameters, i.e., having a distribution of diameters such that no more than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the droplets have an average diameter greater than about 10%, about 5%, about 3%, about 1%, about 0.03%, or about 0.01% of the average diameter.

The "average diameter" of a plurality or series of droplets or particles is the arithmetic average of the average diameters of each of the droplets or particles. Those of ordinary skill in the art will be able to determine the average diameter (or other characteristic dimension), for example, using laser light scattering, microscopic examination, or other known techniques. The average diameter of a single entity, in a non-spherical entity, is the diameter of a perfect sphere having the same volume as the non-spherical entity. The average diameter may be, for example, less than about 1 mm, less than about 500 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 25 micrometers, less than about 10 micrometers, or less than about 5 micrometers in some cases. The average diameter may also be at least about 1 micrometer, at least about 2 micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 15 micrometers, or at least about 20 micrometers in certain cases.

As used herein, the term "fluid" generally refers to a substance that tends to flow and to conform to the outline of its container, i.e., a liquid, a gas, a viscoelastic fluid, etc. Typically, fluids are materials that are unable to withstand a static shear stress, and when a shear stress is applied, the fluid experiences a continuing and permanent distortion. The fluid may have any suitable viscosity that permits flow. If two or more fluids are present, each fluid may be independently selected among essentially any fluids (liquids, gases, and the like) by those of ordinary skill in the art, by considering the relationship between the fluids. In some cases, the droplets (or other multi-phase entities) may be contained within a carrier fluid, e.g., a liquid. A "droplet," as used herein, is an isolated portion of a first fluid that is surrounded, e.g., by a second fluid or a solid, etc. It is to be noted that a droplet is not necessarily spherical, but may assume other shapes as well, for example, depending on the external environment.

In one set of embodiments, two (or more) of the phases within the multi-phase entity may be present as an emulsion or a suspension, e.g., where one phase (the continuous phase) contains a second phase (the discontinuous phase) that is present as discrete regions within the continuous phase. For instance, in one embodiment, a fluid may contain therein fluidic droplets that form an emulsion with the fluid. Emulsions or multiple emulsions may be formed using techniques such as such as those described in International Patent Application No. PCT/US2006/007772, filed March 3, 2006, entitled "Method and Apparatus for Forming Multiple Emulsions," published as WO

2006/096571 on September 14, 2006 or U.S. Provisional Patent Application Serial No. 60/920,574, filed March 28, 2007, entitled "Multiple Emulsions and Techniques for Formation," each incorporated herein by reference.

In some cases, a phase defined by particles may be created by solidifying a liquid phase or a discontinuous phase of an emulsion, e.g., to form solid particles. Thus, in one set of embodiments, at least a portion of the discontinuous phase is solidified. Examples of solidification techniques are described herein. In another set of embodiments, a phase may be defined by a gel, which can be created, for example, by causing gelling of a discontinuous phase of an emulsion. The gel, in some cases, may be condensed to form a single phase within the multi-phase entity.

For instance, in one embodiment, a phase may be solidified by reducing the temperature of the phase to a temperature that causes the phase to reach a solid state. Any technique able to solidify a fluid can be used. For example, the phase may be solidified by cooling the phase to a temperature that is below the melting point or glass transition temperature of the phase, thereby causing the phase to become solid. As non- limiting examples, an emulsion may be formed at an elevated temperature (e.g., above room temperature, about 25 ⁰C), then cooled, e.g., to room temperature or to a temperature below room temperature; an emulsion may be formed at room temperature, then cooled to a temperature below room temperature, or the like. In some embodiments, a plurality of regions and/or phases may be solidified to form a plurality of discrete particles, which can be agglomerated in some cases to produce an agglomeration of particles. As another example, in one embodiment, a phase is solidified using a chemical and/or a polymerization reaction that causes solidification of the phase to occur. For example, two or more fluids may react to produce a solid product, thereby causing formation of a solid phase. As another example, a first reactant may be reacted with a second reactant surrounding the fluidic droplet to produce a solid, which may cause a solid "shell" to form in some cases, thereby forming a core/shell structure having a solid shell or exterior, and a fluidic core or interior, e.g., containing liquid or gas. As yet another example, a polymerization reaction may be initiated within a phase, thereby causing the formation of a polymeric particle. For instance, the phase may contain one or more monomer or oligomer precursors (e.g., dissolved and/or suspended within the fluidic droplet), which may polymerize to form a polymer that is solid. The polymerization reaction may occur spontaneously, or be initiated in some fashion, e.g., during formation of the phase, or after the phase has been formed. For instance, the polymerization reaction may be initiated by adding an initiator to the phase, by applying light or other electromagnetic energy to the phase (e.g., to initiate a photopolymerization reaction), or the like.

In some cases, the phase may comprise a material having a sol state and a gel state (e.g., a hydrogel), such that the conversion of the material from the sol state into a gel state causes the phase to solidify. The conversion of the sol state of the material within the fluidic droplet into a gel state may be accomplished through any technique known to those of ordinary skill in the art, for instance, by cooling the phase, by initiating a polymeric reaction within the phase, etc. For example, if the material includes agarose, the phase containing the agarose may be produced at a temperature above the gelling temperature of agarose, then subsequently cooled, causing the agarose to enter a gel state. As another example, if the phase contains acrylamide (e.g., dissolved within the fluidic droplet), the acrylamide may be polymerized (e.g., using tetramethylethylenediamine) to produce a polymeric particle comprising polyacrylamide, for example, as a hollow particle containing a fluid therein.

Accordingly, in one set of embodiments, one or more phases of the multi-phase entity may be defined by particles, such as an agglomeration of particles. Any of the phases present within a multi-phase entity may be defined by particles. For instance, in one embodiment, a multi-phase entity may contain a first phase and a second phase, where the second phase is defined by an agglomeration of sub-particles. In some cases, the first phase is also solid, i.e., the multi-phase entity is a particle, such as a microparticle. In this embodiment, the first phase may surround the second phase (or vice versa), or both phases may be generally hemispherical, e.g., as in a Janus particle, as shown in FIGS. 1B-2E or FIGS. 4A-4E. As another example, three or more phases may be present within the multi-phase entity, and the phases may be present in any configuration, where at least one of the phases is defined by particles, such as an agglomeration of particles. In some cases, the agglomeration is porous, e.g., the particles are not connected together as an impenetrable mass, and channels or "pores" may be present within the agglomeration of particles.

In one set of embodiments, multi-phase entities comprise one or more phases that comprise colloidal particles. The term "colloid" is given its ordinary meaning in the art, and is generally used to refer to a type of mechanical mixture where one substance (e.g., colloidal particles) is dispersed evenly throughout another (e.g., a fluid medium). For example, colloidal particles may refer to particles that form a colloid when dispersed in a medium, such as water.

In some embodiments, at least some of the phases of and/or particles in the multiphase entity have a first hydrophobicity/hydrophilicity at a first temperature and a second hydrophobicity/hydrophilicity at a second temperature. Accordingly, by altering the temperature, the hydrophobicity/hydrophilicity of the multi-phase entity may be altered. In some embodiments, the difference between the first temperature and the second temperature is at least about 1 ⁰C, at least about 5 ⁰C, at least about 10 ⁰C, at least about 25 ⁰C, at least about 50 °C, at least about 100 ⁰C, or at least about 250 ⁰C. The hydrophobicity of the phases and/or particles can be measured using techniques known to those of ordinary skill in the art, for example, by the contact angle of a water droplet on a surface coated with the material. Non-limiting examples of materials that exhibit a temperature-dependent hydrophibicity/hydrophilicity include poly(N-isopropyl acrylamide) (PNIPAM) and poly(n-vinylcaprolactam) (PVCL). It should be understood that the "hydrophobicity/hydrophilicity" is defined relatively as a material property, i.e., a particle or a phase within the multi-phase entity may have a first hydrophobicity/hydrophilicity at a first temperature and a second hydrophobicity/hydrophilicity at a second temperature. The particle or phase may be hydrophobic relative to water, or hydrophilic relative to water, etc. In other cases, the hydrophobicity/hydrophilicity at the first temperature may be hydrophobic (relative to water) and the hydrophobicity/hydrophilicity at the second temperature may be hydrophilic, etc. In yet another set of these embodiments, at least some of the particles and/or phases have a first hydrophobicity/hydrophilicity at a first pH and a second hydrophobicity/hydrophilicity at a second pH. Thus, in some cases, by changing the pH, the hydrophobicity/hydrophilicity of the multi-phase entity may be altered. In some embodiments, the difference between the first pH and the second pH is at least about 0.1, at least about 0.5, at least about 1 , at least about 1.5, at least about 2, at least about 3, at least about 4, at least about 5, or at least about 7, etc. Non-limiting examples of materials that exhibit a pH-dependent hydrophobicity/hydrophilicity are poly(vinyl pyridine), poly(acrylic acid), poly(methacrylic acid), poly(alylamine hydrochloride), and poly(ethyleneimine) . In still another set of these embodiments, at least some of the particles and/or phases are able to bind to an analyte. The analyte may be one that is able to bind, typically specifically, or otherwise interact with the colloidal particle, or at least a portion thereof. Non-limiting examples include dyes sensitive to pH, temperature, ions, or the like. Examples of such compounds include, but are not limited to, pH-sensitive dyes such as phenol red, bromothymol blue, chlorophenol red, fluorescein, HPTS, 5(6)- carboxy-2',7'-dimethoxyfluorescein SNARP, and phenothalein; dyes sensitive to calcium such as Fura-2 and Indo-1; dyes sensitive to chloride such as 6-methoxy-N-(3- sulfopropyl)-quinolinim and lucigenin; dyes sensitive to nitric oxide such as 4-amino-5- methylamino-2',7'-difluorofluorescein; dyes sensitive to oxygen such as tris(4,4'- diphenyl-2,2'-bipyridine) ruthenium (II) chloride pentahydrate; or the like.

In some cases, at least one of the particles and/or phases within a multi -phase entity comprises magnetic and/or charged particles. For example, in one set of embodiments, a multi-phase entity may comprise at least one phase comprising charged particles and, at the same time, at least one phase substantially free of charged particles. In some instances, the multi-phase entity comprises one or more phase comprising magnetic particles and, at the same time, one or more phase substantially free of magnetic particles. In one aspect, the discontinuous phase of an emulsion used to form a multi-phase entity may be condensed, i.e., to form a separate, continuous phase. The discontinuous phase may be liquid (e.g., forming a new continuous, liquid phase, which may be contained within the continuous phase of the emulsion), or a solid (e.g., forming an agglomeration of particles). Examples of forming a solid discontinuous phase are discussed in detail herein. In one embodiment, as a non-limiting example, poly(N- isopropyl acrylamide) (PNIPAM) can be used to form microgels in droplets. The PNIPAM microgels may, for example, be synthesized with allyl amine and have NH₂ functionality on the surface. Addition of small amounts of polyacrylic acid to the PNIPAM suspension may cause the PNIPAM microgels to aggregate, e.g., due to electrostatic interactions. The aggregation process can also be accelerated in some cases by heating. For instance, the microgels may be heated to about 65 ⁰C. A schematic of this process is shown in FIG. IA.

In some embodiments, the condensation of a discontinuous phase of an emulsion to form a separate, continuous phase is induced mechanically. In other embodiments, the condensation of a discontinuous phase of an emulsion to form a separate, continuous phase is induced by heating. Condensation can be induced, for example, by heating to temperatures of at least about 20 ⁰C, at least about 25 ⁰C, at least about 50 ⁰C, at least about 100 ⁰C, or at least about 250 ⁰C. In some cases, a continuous phase may be polymerized, e.g., to produce a multi-phase particle. In some embodiments the relative volumes of the continuous and discontinuous phases are controlled by varying the temperature at which the condensation step occurs. In other embodiments, the relative volumes of the two phases are controlled by varying the time between the condensation and polymerization steps. In still other embodiments, the relative volumes of the two phases are controlled by varying the chemical composition of the emulsified fluid droplet. In yet other embodiments, combinations of these and/or other techniques can be used. Example 2 includes examples of detailed descriptions of the control of relative volumes using process parameters.

As mentioned above, in one aspect of the present invention, multi-phase entities may be formed by flowing two, three, or more fluids through a system of conduits, and optionally solidifying and/or condensing one or more of the fluids. The system may be a microfluidic system. "Microfluidic," as used herein, refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than about 1 millimeter (mm), and in some cases, a ratio of length to largest cross- sectional dimension of at least 3:1. One or more conduits of the system may be a capillary tube. In some cases, multiple conduits are provided, and in some embodiments, at least some are nested, as described herein. The conduits may be in the microfluidic size range and may have, for example, average inner diameters, or portions having an inner diameter, of less than about 1 millimeter, less than about 300 micrometers, less than about 100 micrometers, less than about 30 micrometers, less than about 10 micrometers, less than about 3 micrometers, or less than about 1 micrometer, thereby providing droplets having comparable average diameters. One or more of the conduits may (but not necessarily), in cross section, have a height that is substantially the same as a width at the same point. Conduits may include an orifice that may be smaller, larger, or the same size as the average diameter of the conduit. For example, conduit orifices may have diameters of less than about 1 mm, less than about 500 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 20 micrometers, less than about 10 micrometers, less than about 3 micrometers, etc. In cross-section, the conduits may be rectangular or substantially non-rectangular, such as circular or elliptical. The conduits of the present invention can also be disposed in or nested in another conduit, and multiple nestings are possible in some cases. In some embodiments, one conduit can be concentrically retained in another conduit and the two conduits are considered to be concentric. In other embodiments, however, one conduit may be off-center with respect to another, surrounding conduit. By using a concentric or nesting geometry, the inner and outer fluids, which are typically miscible, may avoid contact, which can facilitate great flexibility in making multi-phase entities such as those described herein.

FIG. 19 is a non-limiting example of a micrograph of an apparatus used to make multiple emulsions. In this figure, a coaxial flow geometry and hydrodynamic focusing are used to produce multiple droplets within a single droplet. Many parameters of the multi-phase entities, including both inner droplets and middle layer droplets (outer droplets), can be controlled using hydrodynamic focusing. For instance, droplet diameter, outer droplet volume and the total number of inner droplets per outer droplet can be controlled.

In some embodiments, the multi-phase entities comprise phases that are miscible. Some embodiments may also comprise multi-phase entities in two or more phases are substantially immiscible. As used herein, two fluids are "immiscible," or not miscible, with each other when one is not soluble in the other to a level of at least 10% by weight at the temperature and under the conditions at which the multiple emulsion is produced. For instance, two fluids may be selected to be immiscible within the time frame of a particular technique carried out in accordance with the invention. A "channel," as used herein, means a feature on or in an article (substrate) that at least partially directs flow of a fluid. The channel can have any cross-sectional shape (circular, oval, triangular, irregular, square or rectangular, or the like) and can be covered or uncovered. In embodiments where it is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlet(s) and/or outlet(s). A channel may also have an aspect ratio (length to average cross sectional dimension) of at least 2: 1 , more typically at least 3: 1, 5: 1, 10: 1, 15: 1, 20:1, or more. An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics that can exert a force (e.g., a containing force) on a fluid. The fluid within the channel may partially or completely fill the channel. In some cases where an open channel is used, the fluid may be held within the channel, for example, using surface tension (i.e., a concave or convex meniscus). The channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm or 2 mm, or less than about 1 mm, or less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns, less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about 100 nm, less than about 30 nm, or less than about 10 nm. In some cases the dimensions of the channel may be chosen such that fluid is able to freely flow through the article or substrate. The dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flowrate of fluid in the channel. Of course, 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. In some cases, more than one channel or capillary may be used. For example, two or more channels may be used, where they are positioned inside each other, positioned adjacent to each other, positioned to intersect with each other, etc.

A variety of materials and methods, according to certain aspects of the invention, can be used to form systems (such as those described above) able to produce the multiphase entities. For example, various components can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et al). In one embodiment, at least a portion of the fluidic system is formed of silicon by etching features in a silicon chip. Technologies for precise and efficient fabrication of various fluidic systems and devices of the invention from silicon are known. In another embodiment, various components of the systems and devices of the invention can be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane ("PDMS"), polytetrafluoroethylene ("PTFE" or Teflon®), or the like. Different components can be fabricated of different materials. For example, a base portion including a bottom wall and side walls can be fabricated from an opaque material such as silicon or PDMS, and a top portion can be fabricated from a transparent or at least partially transparent material, such as glass or a transparent polymer, for observation and/or control of the fluidic process. Components can be coated so as to expose a desired chemical functionality to fluids that contact interior channel walls, where the base supporting material does not have a precise, desired functionality. For example, components can be fabricated as illustrated, with interior channel walls coated with another material. Material used to fabricate various components of the systems and devices of the invention, e.g., materials used to coat interior walls of fluid channels, may desirably be selected from among those materials that will not adversely affect or be affected by fluid flowing through the fluidic system, e.g., material(s) that is chemically inert in the presence of fluids to be used within the device. In one embodiment, various components of the invention are fabricated from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, etc.). The hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the fluidic network. In one embodiment, the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a "prepolymer"). Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, or mixture of such polymers heated above their melting point. As another example, a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation. Such polymeric materials, which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art. A variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material. A non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers. Epoxy polymers are characterized by the presence of a three- membered cyclic ether group commonly referred to as an epoxy group, 1 ,2-epoxide, or oxirane. For example, diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones. Another example includes the well-known Novolac polymers. Non-limiting examples of silicone elastomers suitable for use according to the invention include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes, phenylchlorosilanes, etc.

Silicone polymers are preferred in one set of embodiments, for example, the silicone elastomer polydimethylsiloxane. Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, MI, and particularly Sylgard 182, Sylgard 184, and Sylgard 186. Silicone polymers including PDMS have several beneficial properties simplifying fabrication of the microfluidic structures of the invention. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat. For example, PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65 °C to about 75 °C for exposure times of, for example, about an hour. Also, silicone polymers, such as PDMS, can be elastomeric, and thus may be useful for forming very small features with relatively high aspect ratios, necessary in certain embodiments of the invention. Flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.

One advantage of forming structures such as microfluidic structures of the invention from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non-polymeric materials. Thus, components can be fabricated and then oxidized and essentially irreversibly sealed to other silicone polymer surfaces, or to the surfaces of other substrates reactive with the oxidized silicone polymer surfaces, without the need for separate adhesives or other sealing means. In most cases, sealing can be completed simply by contacting an oxidized silicone surface to another surface without the need to apply auxiliary pressure to form the seal. That is, the pre- oxidized silicone surface acts as a contact adhesive against suitable mating surfaces. Specifically, in addition to being irreversibly sealable to itself, oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma). Oxidation and sealing methods useful in the context of the present invention, as well as overall molding techniques, are described in the art, for example, in an article entitled "Rapid Prototyping of Microfluidic Systems and Polydimethylsiloxane," Anal. Chem., 70:474-480, 1998 (Duffy, et al.), incorporated herein by reference. In some embodiments, certain microfluidic structures of the invention (or interior, fluid-contacting surfaces) may be formed from certain oxidized silicone polymers. Such surfaces may be more hydrophilic than the surface of an elastomeric polymer. Such hydrophilic channel surfaces can thus be more easily filled and wetted with aqueous solutions.

In one embodiment, a bottom wall of a microfluidic device of the invention is formed of a material different from one or more side walls or a top wall, or other components. For example, the interior surface of a bottom wall can comprise the surface of a silicon wafer or microchip, or other substrate. Other components can, as described above, be sealed to such alternative substrates. Where it is desired to seal a component comprising a silicone polymer (e.g. PDMS) to a substrate (bottom wall) of different material, the substrate may be selected from the group of materials to which oxidized silicone polymer is able to irreversibly seal (e.g., glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, epoxy polymers, and glassy carbon surfaces which have been oxidized). Alternatively, other sealing techniques can be used, as would be apparent to those of ordinary skill in the art, including, but not limited to, the use of separate adhesives, bonding, solvent bonding, ultrasonic welding, etc. The following are each incorporated herein by reference: U.S. Patent Application

Serial No. 11/246,911, filed October 7, 2005, entitled "Formation and Control of Fluidic Species," published as U.S. Patent Application Publication No. 2006/0163385 on July 27, 2006; U.S. Patent Application Serial No. 11/024,228, filed December 28, 2004, entitled "Method and Apparatus for Fluid Dispersion," published as U.S. Patent Application Publication No. 2005/0172476 on August 11, 2005; U.S. Patent Application Serial No. 11/360,845, filed February 23, 2006, entitled "Electronic Control of Fluidic Species," published as U.S. Patent Application Publication No. 2007/000342 on January 4, 2007; International Patent Application No. PCT/US2006/007772, filed March 3, 2006, entitled "Method and Apparatus for Forming Multiple Emulsions," published as WO 2006/096571 on September 14, 2006; U.S. Patent Application Serial No. 11/368,263, filed March 3, 2006, entitled "Systems and Methods of Forming Particles," published as U.S. Patent Application Publication No. 2007/0054119 on March 8, 2007; U.S. Provisional Patent Application Serial No. 60/920,574, filed March 28, 2007, entitled "Multiple Emulsions and Techniques for Formation"; International Patent Application No. PCT/US2006/001938, filed January 20, 2006, entitled "Systems and Methods for Forming Fluidic Droplets Encapsulated in Particles Such as Colloidal Particles," published as WO 2006/078841 on July 27, 2006; and U.S. Provisional Patent Application Serial No. 61/001,601, filed November 2, 2007, entitled "Systems and Methods for Creating Multi-Phase Entities, Including Particles and/or Fluids," by Shah, et al.

The following examples are intended to illustrate certain embodiments of the present invention, but do not exemplify the full scope of the invention.

EXAMPLE 1

This example describes a multi-phase entity that can serve as a temperature sensitive surfactant. FIGS. 2B-2E illustrate a multi-phase entity comprising polymerized polyacrylamide in the first phase and cross-linked poly(N-isopropyl acrylamide) (PNIPAM) aggregate in the second phase. The polyacrylamide was hydrophilic at all temperatures. The cross-linked PNIPAM, on the other hand, was hydrophilic at low temperatures (i.e. less than about 32 ⁰C) and hydrophobic at high temperatures (e.g. greater than about 32 ⁰C). FIG. 2C shows a PNIPAM suspension in water with polyacrylamide, acrylamide, BIS (a cross-linking agent), and Darocur 1173 (a photoinitiator). Upon application of heat, the PNIPAM polymerized, aggregated and separated from the polyacrylic acid. The PAA was then crosslinked together, e.g., using ultraviolet light (e.g., as shown schematically in FIG. 2B). Alternatively, the PNIPAM was chemically cross-linked with an aldehyde such as, for example, glutaric aldehyde. Consequently, at temperatures above about 32 ⁰C, the multi-phase article was amphiphilic and could be used in a surfactant. At temperatures below about 32 ⁰C, the entire article was hydrophilic.

EXAMPLE 2

This example illustrates a technique for producing various multi-phase entities. FIG. 2A illustrates the production of multi-phase entities. In this illustrative process, an aqueous phase containing poly(N-isopropyl acrylamide) (PNIPAM), poly acrylic acid (PAA), acrylamide, a cross-linking agent, and Darocur (a photoinitiator), was surrounded by a continuous silicon oil phase. The droplets were formed at the inlet of a capillary. The presence of photopolymerizable monomer, cross-linker, and photoinitiator did not affect the phase separation of the microgels. This is illustrated in FIGS. 2C-2E. Downstream of the capillary inlet, PNIPAM microgels, which had NH₂ functionality on the surface, were phase-separated from the aqueous PAA phase. The addition of PAA caused the PNIPAM microgels to aggregate due, e.g., to electrostatic interactions. The aggregation process was accelerated by heating the microgels from 20 ⁰C to about 65 ⁰C. A schematic of this process is shown in FIGS. 1A-1C. When the PNIPAM microgels aggregated, the aqueous components - including water, PAA, acrylamide, the cross- linking agent, and the photoinitiator - were expelled from the microgels. The aqueous component formed a distinct phase that appeared to be substantially free of PNIPAM microgels. Downstream of the phase-separation step, the emulsion was exposed to UV radiation, polymerizing the bubbles to form solid articles. The solid particles were then washed with isopropanol to remove oil and were then redispersed in water.

The chemical composition of these microspheres was such that one phase was relatively hydrophilic whereas the other one was relatively hydrophobic at high tempereatures (> 35 ⁰C) and relatively hydrophilic at low temperatures. Thus, the particles can be used, for example, as thermosensitive surfactants.

The particles can be functionalized in different ways for different applications. For example, in this example cationic PNIPAM microgels (determined by zeta potential measurements) were made. When anionic magnetic particles are added to these microgels, the particles were trapped in the PNIPAM phase, as shown schematically in FIG. 5. Thus, magnetically anisotropic particles can be made for various applications, such as for use in magnetically controlled displays.

EXAMPLE 3 In this example, microfiuidics were used for the manufacture of monodisperse

Janus particles. In the process discussed herein, the phase separation process was performed downstream of microfiuidics. Hence, there were no limitations on flow conditions, which could lead to higher production rates.

The relative volumes of the separated PNIPAM phase and the PAA phase were controlled by modifying a variety of process parameters. As shown in FIG. 2A, phase separation occurred at high temperatures. If the droplets ware allowed to cool in water, the PNIPAM phase expanded, incorporating water. Thus, by simply controlling the time interval between the phase separation stage and the UV irradiation, the relative volumes of the two phases was controlled. As illustrated in FIG. 5, the fraction of the final article comprising PNIPAM decreased as the time between the phase separation step and the polymerization step was increased. Additionally, the fraction of the final article comprising PNIPAM increased as the temperature of the emulsion during polymerization was increased. Also, as illustrated in FIG. 6B, increasing the concentration of acrylamide in the aqueous phase led to a decrease in the fraction of the final article comprising PNIPAM.

EXAMPLE 4 In this example, a versatile and robust technique to fabricate Janus particles with a novel, highly anisotropic, and finely tunable internal architecture is described. Microparticles with one side composed of a hydrogel and the other side composed predominantly of aggregated colloidal nanoparticles have been generated. The creation of Janus particles with such a unique internal morphology was facilitated by the induced phase separation of colloidal nanoparticles in droplets. By using microfluidic devices, this technique was used to make extremely monodisperse particles; moreover, this technique could also be combined with bulk emulsification methods, such as membrane emulsification, to produce Janus particles in large quantities for more commercially viable applications. The technique was demonstrated by forming Janus particles with polyacrylamide (PAAm) as the hydrogel and poly(N-isopropylacrylamide), PNIPAm, microgels as the nanoparticles. The thermosensitive nature of the PNIPAm microgels offered a means of control for precisely tuning the relative volumes of the two phases. The functional dichotomy of the Janus particles was further enhanced by embedding different functional materials selectively into the two sides of the particles as illuistrated by the incorporation of magnetic nanoparticles in the microgel-rich phase of the particles.

The process began with an aqueous suspension of ~500-nm-diameter PNIPAm microgels prepared by precipitation polymerization of N-isopropylacrylamide, NIP Am, monomer. 5 mol % allylamine along with the NIPAm monomer were copolymerized to incorporate reactive amine groups into and onto the microgel particles. The cationic nature of the microgels was confirmed by measuring the electrophoretic mobility of the particles which was found to be 0.9 x 10^'8 m² v^'1 s^"1. A small amount of an oppositely charged water soluble polymer, a high molecular weight polyacrylic acid (PAAc), was added to the microgel suspension to induce clustering of the microgels by electrostatic interactions between the ammonium ions of the microgels and the carboxyl groups of PAAc. 10 wt% acrylamide was also dissolved in the microgel suspension along with a crosslinker (methylene-bis-acrylamide) and a photoinitiator. This aqueous mixture was emulsified in an oil and heated at 65 ⁰C in an oven. PNIPAm is a thermosensitive polymer that exhibits a phase transition at -32 °C; the microgels were hydrophobic above this transition temperature and hydrophilic below this temperature. When the emulsion was heated to 65 °C, the weakly associated PNIPAm microgel aggregate shrank and became compacted on one side of the droplets by pushing the acrylamide containing water to the other side, thus forming phase-separated Janus droplets. The acrylamide monomer was then polymerized and cross-linked by exposure to ultraviolet (UV) radiation, forming Janus supraparticles with a PNIPAm microgel-rich side and an PAAm-rich side. The overall scheme is presented in FIG. 12. Since the particles were generated by inducing phase separation in preformed droplets, the technique could be combined with any emulsification method to produce Janus particles. Thus, bulk emulsification methods could be employed to produce Janus particles on a large scale.

However, since the particles use the droplets as templates, the polydispersity of the Janus particles was set by that of the droplets. For applications that require extremely monodisperse particles, monodisperse droplets made using microfiuidic techniques should be used as templates. Drop formation in microfiuidic devices results from a balance of the interfacial tension between the two phases and the shear exerted by the continuous phase on the dispersed phase. Since, the interfacial tension between the two fluids was constant and the shear rate could be precisely adjusted in these devices, droplets with less than 1% polydispersity could be efficiently made. Here, the fabrication of monodisperse Janus particles using a capillary based microfiuidic device was demonstrated.

The microfiuidic device was composed of coaxially aligned glass microcapillaries. The outer capillary had a square cross-section and the inner capillary had a round cross-section. The coaxial alignment was achieved by matching the outer dimensions of the round capillary with the inner dimensions of the square capillary. Prior to its placement within the square capillary, the round capillary was heated and pulled using a pipette puller to create a gradual taper that culminated in a much finer circular orifice. The square capillary served as a flow channel for the two individual fluid streams while the circular capillary served as a collection tube for the emulsion. A schematic representation of the device and a camera image of the actual device are included in FIGS. 18A-18B. The surfactant-containing silicon oil flowed from one end of the square capillary and focused the monomer-containing aqueous phase flowing from the opposite end into the orifice of the collection tube. The aqueous phase broke into monodisperse droplets upon entering the collection tube to form an emulsion of monodisperse droplets as shown in FIG. 13 A. Once collected, the emulsion was heated at 65 °C in an oven, which caused the microgels to shrink and compact on one side of the droplets, thus forming monodisperse Janus droplets (FIG. 13B). Monodisperse Janus gel microparticles were formed when the acrylamide was polymerized by exposing the phase separated droplets to UV radiation. At this point, the dimensions and the positions of the microgels became fixed and they no longer changed volume in response to changes in the external temperature. A fluorescent microscope image of the Janus particles is presented in FIG. 13C. The PNIPAm microgels were tagged with rhodamine B to enhance visual contrast between the two phases. Since, the phase separation process occurred downstream of the microfluidics process, the problems associated with the convective mixing of the monomers, often encountered during the co-flow microfluidic techniques for making Janus particles, were eliminated. This technique was not just limited to the fabrication of spherical Janus particles but could also be extended to other shapes. The versatile applicability of this technique was demonstrated by forming a Janus structure in bulk by separating the two hydrogel phases in a glass vial (FIGS. 2C- 2E).

The microstructure and phase boundaries of these particles were further probed using a scanning electron microscope (SEM). A larger sized Janus particle (~2 mm diameter) was used for this purpose to visually allow suitable orientation of the particle on the SEM specimen stub. The images revealed a rougher surface for the PNIPAm phase in contrast to a relatively smooth surface for the PAAm phase (FIG. 14A). A high magnification micrograph of the PNIPAm phase revealed a raspberry-like structure formed by the aggregation of the PNIPAm microgels (FIG. 14B). The surfaces of both sides revealed wrinkles and stretch marks caused by the dehydration of the hydrogels during sample preparation for electron microscopy. The micrographs also revealed a somewhat continuous phase surrounding the PNIPAm microgels, suggesting that not all of the acrylamide was pushed to the opposite side of the drop but some of it was trapped and subsequently polymerized between the microgels. Not wishing to be bound by any theory, this may explain why the volume of the microgel-aggregate did not change with temperature once the phase-separated droplets were UV-irradiated.

The thermosensitive nature of the PNIPAm microgels could be effectively exploited to adjust the relative volumes of the two phases of these Janus particles. When the phase-separated Janus droplets were cooled from 65 ⁰C to below the phase transition temperature of PNIPAm, the microgels became hydrophilic and began to absorb water from the other side of the drop. As a result, the internal morphology of the Janus droplets evolved dynamically during the cooling process. The PNIPAm phase compacted on one side of the drops swelled and occupied an increasingly larger volume of the droplet with time, as shown in FIGS. 15A-15B. Thus, by controlling the elapsed time between heating the emulsion and exposing it to UV radiation, the relative volumes of the two phases could be effectively tuned. This range of phase ratios could be further expanded by varying the concentration of the microgels in the aqueous mixture. Alternatively, the size ratios of the two phases could also be adjusted by varying the crosslinker concentration of the PNIPAm microgels. Microgels with a lower crosslinker concentration exhibited a greater equilibrium size change compared to those with a higher crosslinker concentration. Hence, the use of PNIPAm microgels with a lower crosslinker concentration resulted in Janus particles with a wider range of phase ratios as compared to those made using microgels with a higher crosslinker concentration. Different materials were selectively incorporated into the two phases to enhance the functional dichotomy of the particles and to tailor them to suit specific applications. As an example, magnetically anisotropic particles were made by embedding magnetic nanoparticles only in the PNIPAm-rich side of the Janus particles. Anionic magnetic beads were added to the aqueous mixture of the PNIPAm microgels and other monomers. Since the microgel particles were cationic, the magnetic beads covalently bound to the surface of the microgels, and were thus trapped only in the PNIPAm phase of the Janus particles, as shown in FIG. 16. Such magnetically anisotropic particles could be used to make magnetically actuated displays or other applications that require directional orientation or transportation of particles. Cationic materials repelled by the cationic PNIPAm microgels could also be successfully embedded into the PAAm gel of the Janus particles. Although this example describes the fabrication of spherical Janus microparticles, the concept could be extended to generate Janus structures of various shapes. Experimental

Microgel Synthesis: The PNIPAm microgels were synthesized by precipitation polymerization. NIPAm monomer (4g, 99% purity, Acros) was dissolved along with N, N'- methylene bisacrylamide (0.16g, 99.5% purity, Fluka) and allyl amine (0.14 ml, Sigma- Aldrich) in deionized water (150 ml). The solution was filtered with a 1 -micron filter to remove any particulate impurities and then transferred to a round bottom flask (250 ml) fitted with a stirrer, glass thermometer, condenser, and a nitrogen purge line. The solution was heated to 70 ⁰C and allowed to equilibrate for 5 minutes under nitrogen. A fluorescent dye, methacryloxy thiocarbonyl rhodamine B (0.0002 g) and a reaction initiator, K₂S₂O₈ (0.12 g, 99% purity, Sigma- Aldrich) dissolved in water (5 ml), were then added to the heated solution. The monomers were allowed to polymerize for ~90 minutes. After polymerization, the microgel suspension was filtered using a filter cloth (100 micron mesh size) and quickly cooled down to room temperature using a water bath. The suspension was dialyzed for 72 hours to remove the unreacted monomers. The microgel particle size was determined using dynamic light scattering (ALV 5000, 532 nm laser, 90° scattering angle) and fluorescence optical microscopy (Leica TCS-SP5 scanning confocal microscope). A fluorescent micrograph of the synthesized microgels is shown in FIG. 17.

Microfluidics: A detailed description of the fabrication technique of glass capillary based microfluidic devices has been presented in previous publications. A schematic and an image of an actual capillary based microfluidic device with a flow focusing geometry used for making single emulsions are included in FIGS. 18A-18B. The aqueous phase for microfluidic emulsification was prepared by dissolving PAAc (2xlO^"3 wt%, 1.25 xlO⁶ g/mol, Carbopol 941, Noveon), acrylamide (10 wt%, 99 wt% purity, Sigma-aldrich), N, N'- methylene bisacrylamide (1 wt%, 99.5% purity, Fluka), and a photoinitiator (2xlO^'3 wt%, Darocur 1173) in the microgel suspension. The continuous phase was poly(dimethylsiloxane) fluid (500 cSt, Sigma-Aldrich) containing 0.3 wt% DC547 for emulsion stabilization and the photoinitiator (2x10^'3 wt%, Darocur 1173). The addition of the photoinitiator to the oil phase helped prevent the diffusion of the photoinitiator, which is soluble in both water and oil, out of the drops into the continuous phase. The fluids were pumped into the microfluidic device using syringe pumps (Harvard PHD 2000 series). The flow rates for the continuous and the dispersed fluids were set at 3000 and 500 microliter/hr, respectively. The collected emulsion was heated in an oven at 65 ⁰C for 45 minutes and was then placed under a UV lamp (Rad-Free, Schleicher & Schuell, 365 nm wavelength). The solidified Janus particles were removed from the silicon oil and repeatedly flushed with isopropanol to remove any adsorbed oil. The particles were then washed and stored under water. The microfluidics process was monitored using an inverted optical microscope (DM-IRB, Leica) fitted with a fast camera (Phantom V5, Vision Research). Characterization: The images for the phase-separated droplets and the Janus particles were taken using an inverted optical microscope fitted with an EMCCD camera (Rolera MGi, Qlmaging). SEM images were taken using a Zeiss Ultra55 field emission microscope (FESEM). To enhance image contrast, the samples were coated with -5-10 nm of gold after dehydration prior to SEM analysis. To effectively capture the temporal evolution of the internal morphology of a Janus drop, a large drop was made by pipetting 25 microliter of the aqueous mixture in Ig of silicon oil (DC550, 125 cSt, 1.07 g/cc, Dow corning) in a glass vial. After thermally inducing phase separation, the vial was immediately placed in front of a Pulnix TM-200 camera mounted with a Pextax-FA 1 :3.5 macro lens and images were taken at regular intervals of 10s each. An image-processing software package, Image J, was used to calculate the ratio of the area occupied by the PNIPAm phase to that occupied by the entire drop for each image. Magnetically anisotropic particles were fabricated by dissolving a ferro-fluid (EMG 708, Ferrotec) in the aqueous mixture. For ease of visualization, large Janus particles made using 25- microliter droplets as templates were used for this purpose.

While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law. As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as

"comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

What is claimed is:

Claims

1. An article comprising: a multi-phase entity comprising at least a first phase and a second phase wherein: at least one of the phases comprises colloidal particles; and at least some of the colloidal particles have a first hydrophobicity/hydrophilicity at a first temperature and a second hydrophobicity/hydrophilicity different from the first hydrophobicity/hydrophilicity at a second temperature.

2. A collection of articles as in claim 1, wherein the multi-phase entities have a distribution of diameters such that no more than about 10% of the multi-phase entities has an average diameter greater than about 10% of the average diameter.

3. The article of claim 1, wherein at least one phase of the multi -phase entity is liquid.

4. The article of claim 1, wherein at least one phase of the multi -phase entity is solid.

5. The article of claim 1, wherein each phase of the multi-phase entity is solid.

6. The article of claim 1, wherein the multi-phase entity further comprises a third phase.

7. The article of claim 6, wherein the third phase surrounds the first phase and the second phase.

8. The article of claim 1, wherein the colloidal particles have an average diameter of no more than about 100 micrometers.

9. The article of claim 1, wherein the colloidal particles have an average diameter of no more than about 10 micrometers.

10. The article of claim 1, wherein the colloidal particles have an average diameter of no more than about 1 micrometer.

11. The article of claim 1 , wherein the first temperature and the second temperature are separated by at least about 10 ⁰C.

12. The article of claim 1, wherein the first temperature and the second temperature are separated by at least about 25 ⁰C.

13. The article of claim 1, wherein the first temperature and the second temperature are separated by at least about 50 ⁰C.

14. The article of claim 1, wherein at least some of the colloidal particles comprise poly(N-isopropyl acrylamide).

15. The article of claim 1, wherein at least some of the colloidal particles are agglomerated.

16. The article of claim 1, wherein substantially all of the colloidal particles are agglomerated.

17. The article of claim 1, wherein at least one phase comprises a gel.

18. The article of claim 1 , wherein the first phase at least partially surrounds the second phase.

19. The article of claim 1 , wherein the first phase and the second phase are each generally hemispherical.

20. The article of claim 1, wherein the multi-phase entity is contained in a liquid.

21. The article of claim 1, wherein the average diameter of the multi -phase entity is less than about 1 mm.

22. The article of claim 1, wherein the average diameter of the multi-phase entity is less than about 500 micrometers.

23. The article of claim 1, wherein the average diameter of the multi-phase entity is less than about 100 micrometers.

24. The article of claim 1, wherein the average diameter of the multi-phase entity is less than about 75 micrometers.

25. The article of claim 1, wherein the average diameter of the multi-phase entity is less than about 50 micrometers.

26. The article of claim 1 , wherein the average diameter of the multi-phase entity is less than about 25 micrometers.

27. The article of claim 1, wherein the average diameter of the multi-phase entity is less than about 10 micrometers.

28. The article of claim 1, wherein the average diameter of the multi-phase entity is at least about 3 micrometers.

29. The article of claim 1, wherein the average diameter of the multi-phase entity is at least about 1 micrometer.

30. A method, comprising: providing a multi-phase entity comprising at least a first phase and a second phase; and altering the hydrophobicity/hydrophilicity of the first phase by altering the temperature of the multi -phase entity.

31. The method of claim 30, wherein at least one phase is defined by a plurality of particles.

32. The method of claim 30, wherein the temperature is altered by at least about 1 ⁰C.

33. The method of claim 30, wherein the temperature is altered by at least about 1O ⁰C.

34. The method of claim 30, wherein the temperature is altered by at least about 100 ⁰C.

35. The method of claim 30, wherein the temperature is altered by at least about 250 ⁰C.

36. An article comprising: a multi-phase entity comprising at least a first phase and a second phase wherein: at least one of the phases comprises colloidal particles; and at least some of the colloidal particles have a first hydrophobicity/hydrophilicity at a first pH and a second hydrophobicity/hydrophilicity different from the first hydrophobicity/hydrophilicity at a second pH.

37. A collection of articles as in claim 36, wherein the multi-phase entities have a distribution of diameters such that no more than about 10% of the sub-particles has an average diameter greater than about 10% of the average diameter.

38. The article of claim 36, wherein the first pH and the second pH are separated by at least about 1.

39. The article of claim 36, wherein the first pH and the second pH are separated by at least about 3.

40. The article of claim 36, wherein the first pH and the second pH are separated by at least about 7.

41. A method, comprising: providing a multi-phase entity comprising at least a first phase and a second phase; and altering the hydrophobicity/hydrophilicity of the first phase by altering pH.

42. The method of claim 41, wherein the pH is altered by at least about 0.1.

43. The method of claim 41, wherein the pH is altered by at least about 1.

44. The method of claim 41, wherein the pH is altered by at least about 3.

45. The method of claim 41 , wherein the pH is altered by at least about 7

46. An article comprising: a multi-phase entity comprising at least a first phase and a second phase wherein: at least one of the phases comprises colloidal particles; and at least some of the colloidal particles are able to bind to an analyte.

47. The article of claim 46, wherein the analyte is a nucleic acid.

48. The article of claim 46, wherein the analyte is a protein or a peptide.

49. An article, comprising: a multi-phase entity having a maximum dimension of no more than about 1 mm, the multi-phase entity comprises at least three phases, wherein at least one phase is defined by an agglomeration of sub-particles.

50. The article of claim 49, wherein at least some of the sub-particles comprise a polymer.

51. The article of claim 49, wherein the multi-phase entity has an average diameter of no more than about 100 micrometers.

52. The article of claim 49, wherein at least one phase of the multi-phase entity has an average diameter of no more than about 100 micrometers.

53. An article, comprising: an outer fluid droplet containing one or more first fluid droplets, at least one of which contains one or more second fluid droplets, wherein at least one of the second fluid droplets contains agglomerated particles.

54. An article, comprising: a fluid droplet containing more than one nesting level of inner fluid droplets therein, wherein a nesting level is defined by one or more fluid droplets each contained within a surrounding fluid droplet, and wherein at least one nesting level is defined by an agglomeration of particles.

55. An article, comprising: a microparticle containing a first phase surrounding a second phase, wherein the second phase is defined by an agglomeration of sub-particles.

56. An article, comprising: a microparticle containing at least three phases, one of the phases surrounding the other phases, wherein at least one of the phases is defined by an agglomeration of sub-particles.

57. A collection of articles as in claim 56, wherein the outer fluid droplets have a distribution of diameters such that no more than about 10% of the sub-particles has an average diameter greater than about 10% of the average diameter.

58. The article of claim 56, wherein the first phase is solid.

59. The article of claim 56, wherein the sub-particles have a distribution of diameters such that no more than about 10% of the sub-particles has an average diameter greater than about 10% of the average diameter.

60. A method comprising: providing a fluid containing an emulsion defined by a continuous and a discontinuous phase defined by a plurality of fluid droplets; and solidifying at least a portion of the discontinuous phase without solidifying the continuous phase.

61. The method of claim 60, further comprising polymerizing the continuous phase.

62. The method of claim 61, comprising polymerizing the continuous phase using ultraviolet light.

63. The method of claim 60, wherein the fluid droplets have an average dimension of no more than about 100 run.

64. The method of claim 60, further comprising agglomerating the solidified discontinuous phase.

65. A method, comprising: providing emulsified fluid droplets, each droplet defined by a continuous and a discontinuous phase; condensing the discontinuous phase in each fluid droplet in one portion of the fluid droplet; and polymerizing the continuous phase in each fluid droplet.

66. The method of claim 65, wherein the continuous phase and the discontinuous phase are substantially immiscible.

67. The method of claim 65, wherein the discontinuous phase is liquid.

68. The method of claim 65, wherein the discontinuous phase is solid.

69. The method of claim 65, comprising polymerizing the continuous phase using ultraviolet light.

70. The method of claim 65, wherein the fluid droplets have an average dimension of no more than about 100 nm.

71. An article, comprising: a particle having a first, generally hemispherical phase and a second, generally hemispherical phase, wherein the second generally hemispherical phase is porous.

72. The article of claim 71 , wherein the second generally hemispherical phase has a porosity of at least about 10% by volume.

73. The article of claim 71, wherein the second generally hemispherical phase has a porosity of at least about 30% by volume.

74. The article of claim 71 , wherein the second generally hemispherical phase has a porosity of at least about 50% by volume.

75 The article of claim 71, wherein the particle is a microparticle.

76. The article of claim 71, wherein the particle has a maximum dimension of no more than about 100 nm.

77. The article of claim 71, wherein the particle comprises a polymer.