Patent Grant
9498761
The present invention relates generally to surfactants, and more specifically, to surfactants for stabilizing emulsions having a continuous fluorophilic phase.
Many emulsions comprise an aqueous phase and a hydrocarbon oil phase. Fluorocarbon oils are often immiscible with both water and hydrocarbon oils; thus, water or hydrocarbon oils may be dispersed as emulsion droplets in a fluorocarbon phase. The use of fluorocarbon oils as the continuous phase of an emulsion offers advantages over conventional hydrocarbon systems. For example, fluorocarbon oils may be well suited as the continuous phase for emulsions that require reduced diffusion and/or cross-contamination of hydrophilic or lipophilic material between droplets in the emulsion. Stabilizing such emulsions, however, sometimes requires the addition of appropriate surfactants, and these surfactants are often not available or do not have desirable physical characteristics. This is especially true because fluorocarbons are not commonly used as the continuous emulsion phase. Accordingly, new surfactants and surfactant systems for stabilizing droplets of water and hydrocarbon oils or organic solvents in a continuous fluorophilic phase are needed.
Surfactants for stabilizing emulsions having a continuous fluorophilic phase are presented. In one embodiment, a fluorosurfactant is provided. The fluorosurfactant has one of the general formulas: A-B-A or A-B or (A-B)n or B-(A)n, or (B)n-A or A-B-A′, where A (and A′, if present) comprises a fluorophilic component having a molecular weight greater than 1,000 or greater than 1,500 g/mol, the fluorophilic component comprising a (per)fluoropolyether or a different poly(perfluoroalkyl-methacrylates), etc., and B is one or both of: a) non-ionic and soluble in an aqueous phase; or b) a hydrocarbon soluble in a hydrocarbon phase, and n is an integer greater than 0. In other embodiments, the fluorosurfactant may have a formula such as those described herein.
In another embodiment, a macroemulsion is provided. The macroemulsion comprises an aqueous dispersed phase or a lipophilic dispersed phase having an average diameter greater than or equal to about 50 nanometers, a continuous phase comprising a fluorinated solvent or oil, and a fluorinated, non-ionic surfactant, where at least 95% of the dispersed phase does not coalesce for at least 30 minutes at 25 degrees C. and 1 atm.
In one aspect, the invention is directed to an article. In one set of embodiments, the article comprises a fluorosurfactant comprising the formula: A-B-A or A-B or (A-B)n or B-(A)n or (B)n-A or A-B-A′ or A-X—B or A-X1—B—X2-A or (A-X1—B—X2)n or B—X-(A)n or B—(X-A)n or B-(A-X)n or B—X1-(A-X2)n, wherein A and A′ comprise a fluorophilic component having a molecular weight greater than 1,000 g/mol, the fluorophilic component comprises a fluoropolyether; B is either: a) non-ionic and soluble in an aqueous phase or b) a hydrocarbon soluble in a hydrocarbon phase; X, when present, is either a covalent bond or a linking group, and X1 and X2, where present, may be the same or different; and n is an integer greater than 0.
In another set of embodiments, the fluorosurfactant comprises the formula: A-B-A or A-B or (A-B—)n or B-(A)n or (B)n-A or A-B-A′ or A-X—B or A-X1—B—X2-A or (A-X1—B—X2)n or B—X-(A)n or B—(X-A)n or B-(A-X)n or B—X1-(A-X2)n, wherein A and A′ comprise a component having a molecular weight greater than 1,000 g/mol, the component comprising a fluorophilic portion having fluorinated side chains; B is either: a) non-ionic and soluble in an aqueous phase or b) a hydrocarbon soluble in a hydrocarbon phase; X, when present, is either a covalent bond or a linking group, and X1 and X2, where present, may be the same or different; and n is an integer greater than 0.
In still another set of embodiments, the fluorosurfactant comprises the formula: A-B-A or A-B or (A-B—)n or B-(A)n or (B)n-A or A-B-A′ or A-X—B or A-X1—B—X2-A or (A-X1—B—X2)n or B—X-(A)n or B—(X-A)n or B-(A-X)n or B—X1-(A-X2)n, wherein A and A′ comprise a component having a molecular weight greater than 1,000 g/mol; B is either: a) non-ionic and soluble in an aqueous phase or b) a hydrocarbon soluble in a hydrocarbon phase; X, when present, is either a covalent bond or a linking group, and X1 and X2, where present, may be the same or different; and n is an integer greater than 0.
In yet another set of embodiments, the fluorosurfactant has a backbone comprising the formula: A-B-A or A-B or (A-B—)n or B-(A)n or (B)n-A or A-B-A′ or A-X—B or A-X1—B—X2-A or (A-X1—B—X2)n or B—X-(A)n or B—(X-A)n or B-(A-X)n or B—X1-(A-X2)n, wherein A and A′ comprise a fluorophilic component having a molecular weight greater than 1,000 g/mol, the fluorophilic component comprising a fluoropolyether; B is either: a) non-ionic and soluble in an aqueous phase or b) a hydrocarbon soluble in a hydrocarbon phase; X, when present, is either a covalent bond or a linking group, and X1 and X2, where present, may be the same or different; and n is an integer greater than 0.
In another set of embodiments, the fluorosurfactant having a backbone comprising the formula: A-B-A or A-B or (A-B—)n or B-(A)n or (B)n-A or A-B-A′ or A-X—B or A-X1—B—X2-A or (A-X1—B—X2)n or B—X-(A)n or B—(X-A)n or B-(A-X)n or B—X1-(A-X2)n, wherein A and A′ comprise a fluorophilic component comprising a (per)fluoropolyether; B is either: a) non-ionic and soluble in an aqueous phase or b) a hydrocarbon soluble in a hydrocarbon phase; X, when present, is either a covalent bond or a linking group, and X1 and X2, where present, may be the same or different; and n is an integer greater than 0.
In another aspect, the invention is directed to an emulsion. In one set of embodiments, the emulsion comprises an aqueous, polar, and/or hydrophilic dispersed phase or a lipophilic dispersed phase having an average diameter greater than or equal to about 50 nm; a continuous phase comprising a fluorinated solvent or oil; and a fluorinated, non-ionic surfactant. In some cases, at least 95% of the dispersed phase does not coalesce for at least 30 minutes at 25 degrees C. and 1 atm.
In another set of embodiments, the emulsion comprises an aqueous dispersed phase or a lipophilic dispersed phase having an average diameter greater than or equal to about 50 nm; a continuous phase comprising a fluorinated solvent or oil; and a fluorinated surfactant comprising a morpholino group. The emulsion, in still another set of embodiments, includes an aqueous dispersed phase or a lipophilic dispersed phase having an average diameter greater than or equal to about 50 nm; a continuous phase comprising a fluorinated solvent or oil; and a fluorinated surfactant comprising a phosphate group.
Yet another embodiment is directed to a method. The method, in one some cases, is generally directed to performing a chemical and/or biological reaction it he dispersed phase of any of the embodiments described above, or described herein.
In another aspect, the present invention is directed to a method of making one or more of the embodiments described herein. In another aspect, the present invention is directed to a method of using one or more of the embodiments described herein.
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.
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 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:
Surfactants (e.g., fluorosurfactants) for stabilizing aqueous or hydrocarbon droplets in a fluorophilic continuous phase are presented (or vice versa). In some embodiments, the fluorosurfactants include a fluorophilic tail soluble in a fluorophilic (e.g., fluorocarbon) continuous phase, and a headgroup soluble in either an aqueous phase or a lipophilic (e.g., hydrocarbon) phase. The headgroup and the tail may be directly linked, or linked via a linking moiety. The combination of a fluorophilic tail and a headgroup may be chosen so as to create a surfactant with a suitable geometry for forming stabilized reverse emulsion droplets having a disperse aqueous or lipophilic phase in a continuous, fluorophilic phase. In some embodiments, the headgroup is non-ionic and can prevent or limit the adsorption of molecules at the interface between the surfactant and the discontinuous phase. This configuration can allow the droplet to serve, for example, as a reaction site for certain chemical and/or biological reactions. In another embodiment, aqueous droplets are stabilized in a fluorocarbon phase at least in part by the electrostatic attraction of two oppositely charged or polar components, one of which is at least partially soluble in the dispersed phase, the other at least partially soluble in the continuous phase. One component may provide colloidal stability of the emulsion, and the other may prevent the adsorption of biomolecules at the interface between a component and the discontinuous phase. Advantageously, surfactants and surfactant combinations of the invention may provide sufficient stabilization against coalescence of droplets in certain embodiments of the invention, without interfering with processes that can be carried out inside the droplets.
An “emulsion,” as used herein, is a stable mixture of at least two immiscible liquids. In general, immiscible liquids tend to separate into two distinct phases. An emulsion is thus stabilized by the addition of a “surfactant” which functions to reduce surface tension between the at least two immiscible liquids and/or to stabilize the interface. In some embodiments, emulsion described herein include a discontinuous or disperse phase (i.e., the isolated phase stabilized by a surfactant) formed of an aqueous or lipophilic (e.g., hydrocarbon) substance. The continuous phase may be formed of a fluorophilic substance (e.g., a fluorocarbon). The present invention involves, in some embodiments, water-in-fluorocarbon emulsions and hydrocarbon-in-fluorocarbon emulsions having a disperse aqueous or hydrocarbon phase and a fluorocarbon continuous phase. The isolated disperse aqueous or lipophilic phase in a fluorophilic solvent can form a “reverse emulsion,” which is simply one example of an emulsion. In some particular embodiments, the emulsions described herein are macroemulsions. Macroemulsions are emulsions that are kinetically stable, as compared to microemulsions, which are thermodynamically stable and undergo spontaneous formation. In some cases, a microemulsion may include droplets having an average diameter of less than about 50 nm.
As used herein “droplet” means an isolated aqueous or lipophilic phase within a continuous phase having any shape, for example cylindrical, spherical, ellipsoidal, irregular shapes, etc. Generally, in emulsions of the invention, aqueous and/or lipophilic droplets are spherical or substantially spherical in a fluorocarbon, continuous phase.
As used herein, “surfactant” defines a molecule that, when combined with a first component defining a first phase, and a second component defining a second phase, will facilitate assembly of separate first and second phases. In some cases, a surfactant of the invention typically can have one or more main fluorophilic chain(s) where one end of the chain is soluble in the fluorophilic phase of the emulsion and one or more chains that are not soluble in the fluorophilic phase of the emulsion (e.g., those chains may be soluble in the aqueous or lipophilic phase). For instance, a surfactant may be a multi-block surfactant (e.g., ABABABA . . . ), where one component of the chain (e.g., “A”) is soluble in the fluorophilic phase and another component of the chain (e.g., “B”) is soluble in the other phase (e.g., the aqueous or lipophilic phase). As used herein, a multi-block surfactant is a surfactant having an alternating copolymeric structure or an (A-B—)n structure, i.e., ABA, ABAB, ABABA, ABABABA, etc.). In some cases, one block may be soluble in the fluorophilic phase of the emulsion and one block may be soluble in the other phase of the emulsion (e.g., the aqueous or lipophilic phase). In still other cases, additional components may be present within the surfactant. For example, a multi-block surfactant may have other groups present within its polymeric structure, for example, linking moieties connecting A and B, e.g., (A-X—B—)n, (A-B—X)n, (A-X1—B—X2)n, or the like, where “X” represents a covalent bond or a linking moiety, as described below, and X1 and X2, where present, may be the same or different.
As used herein, a “fluorophilic” component comprises any fluorinated compound such as a linear, branched, cyclic, saturated, or unsaturated fluorinated hydrocarbon. The fluorophilic, component can optionally include at least one heteroatom (e.g., in the backbone of the component). In some cases, the fluorophilic compound may be highly fluorinated, i.e., at least 30%, at least 50%, at least 70%, or at least 90% of the hydrogen atoms of the component are replaced by fluorine atoms. The fluorophilic component may comprise a fluorine to hydrogen ratio of, for example, at least 0.2:1, at least 0.5:1, at least 1:1, at least 2:1, at least 5:1, or at least 10:1. In some such embodiments, at least 30%, at least 50%, at least 70%, or at least 90% but less than 100% of the hydrogen atoms of the component are replaced by fluorine atoms. In other cases, the fluorophilic component is perfluorinated, i.e., the component contains fluorine atoms but contains no hydrogen atoms. Fluorophilic components compatible with the present invention may have low toxicity, low surface tension, and the ability to dissolve and transport gases. Examples of fluorophilic components are described below.
As mentioned, in some embodiments, the emulsions of the invention include discontinuous aqueous and/or lipophilic (e.g., hydrocarbon) droplets in a continuous, fluorophilic phase. This means that separate, isolated regions of droplets of an aqueous and/or lipophilic component are contained within a continuous fluorophilic phase, which may be defined by a fluorocarbon component. The discontinuous aqueous and/or lipophilic droplets in the nonaqueous phase typically have an average cross-sectional dimension of greater than 25 nm. In some embodiments, the average cross-sectional dimension of the droplets is greater than 50 nm, greater than 100 nm, greater than 250 nm, greater than 500 nm, greater than 1 micron, greater than 5 microns, greater than 10 microns, greater than 50 microns, greater than 100 microns, greater than 200 microns, or greater than 500 microns, etc. As used herein, the average cross-sectional dimension of a droplet is the diameter of a perfect sphere having the same volume as the droplet.
Compositions of the invention are, according to some embodiments, stable for at least about 1 minute, at least about 5 minutes, at least about 10 minutes, at least about 20 minutes, at least about 30 minutes, at least about 40 minutes, at least about 1 hour, at least about 2 hours, at least about 6 hours, at least about 12 hours, at least about 1 day, at least about 1 week, at least about 1 month, or at least about 2 months, at a temperature of about 25 degrees Celsius and a pressure of 1 atm. As used herein, a “stable emulsion” means that at least about 95% of the droplets of the emulsion do not coalesce, e.g., to form larger droplets over these periods of time.
As used herein, “nonaqueous” is meant to define material such as a fluid that is immiscible with water. That is, a liquid that when mixed with water will form a stable two-phase mixture. The non-aqueous phase need not be liquid, but can be a solid or semi-solid lipid or other nonpolar substance that is not soluble in water. In some instances, the nonaqueous phase can include a lipophilic component (e.g., a hydrocarbon) or a fluorinated component (e.g., a fluorocarbon). The aqueous phase can be any liquid miscible with water; that is, any liquid that, when admixed with water, can form a room-temperature, single-phase solution that is stable. In some cases, the aqueous phase can comprise one or more physiologically acceptable reagents and/or solvents, etc. Non-limiting examples of aqueous phase materials include (besides water itself) methanol, ethanol, DMF (dimethylformamide), or DMSO (dimethyl sulfoxide).
Referring now to
Also shown in the embodiment illustrated in
In another embodiment, a fluorosurfactant of the invention includes a linking moiety (which can be referred to as “X”), which may be hydrophilic or hydrophobic, etc. As shown in
One aspect of the invention involves the formation of stabilized emulsions using fluorosurfactants including those described herein. Surprisingly, in order to obtain long-term stabilized emulsions, certain geometries of the fluorosurfactants are needed in some cases. For instance, certain ratios of molecular weights of the fluorophilic component to the headgroup component may be required for steric stabilization of the droplets. In addition, fluorophilic components having large molecular weights can contribute to long term colloidal stabilization, according to certain embodiments. These and other considerations for choosing appropriate components of fluorosurfactants and suitable mixtures of fluorsurfactants may be suitable for forming certain emulsions, for instance, emulsions comprising droplets having an average diameter in the micron or micrometer range. These and other criteria are described in more detail below.
The fluorophilic component of surfactant molecules described herein typically comprise a fluorophilic chain at least C8 in length (i.e., contains at least 8 carbon atoms). In some embodiments, the fluorophilic chain is at least C10 in length, at least C15 in length, at least C20 in length, at least C25 in length, or at least C30 in length. In other embodiments, the fluorophilic chain is at least C50 in length, at least C75 in length, at least C100 length, or greater. As a non-limiting example, a fluorophilic component having the structure —(C3F6O)10— has 30 carbons equivalent to a C30 chain. The fluorophilic component may be linear, branched, cyclic, saturated, unsaturated, etc.
In some embodiments, the fluorophilic component of a fluorosurfactant includes a heteroatom (e.g., a non-carbon such as oxygen (e.g., divalent oxygen), sulfur (e.g., divalent or hexavalent sulfur), nitrogen (e.g., trivalent nitrogen), etc.) in the structure of the component. Such heteroatoms may be bonded, for example, to carbon atoms in the skeletal structure of the component. Additionally and/or alternatively, the fluorophilic component may include one or more branches extending from the main chain of the structure.
In some embodiments, the fluorophilic component of a surfactant is a fluorinated oligomer or polymer (i.e., a fluoropolymer). The fluoropolymer may include a (per)fluoropolyether chain, among other fluorinated polymers that are soluble in a fluorocarbon oil. The (per)fluoropolyether chain may comprise repeating units including, but not limited to, —(CnF2nO)x—, where n is an integer, for example, —(C3F6O)x—, —(C4F8O)x—, —(C5F10O)x—; —(CF(CF3)CF2O)n—; —(CF2CF2O)x—; —(CF(CF3)CF2O)x—CF(CF3)CONH—; —(CF2(CF2)z′CF2O)x—, where z′ is an integer; —(CFLO)x—, where L=—F or —CF3; and —(CH2CF2CF2O)x—. In some cases, (CnF2n+1O)x—, where n is an integer (for example, —(CF3O)x—, —(C2F5O)x—, —(C3F7O)x—, etc.), is used as a terminal group and may not be polymerizable. In some cases, the (per)fluoropolyether chain may have the structure (CnFmO)x—, where n and m are integers properly chosen to form a valid structure. In some embodiments, the fluoropolymer comprises poly((per)fluoromethylene oxide), poly((per)fluoroethylene oxide), poly((per)fluoropropylene oxide), and/or poly((per)fluorobutylene oxide). In one particular embodiment, the fluorophilic chain includes poly((per)fluoropropylene oxide). In another embodiment, the fluorophilic chain includes a poly((per)fluoroalkyl-methacrylate). Typically, x in the structures above is greater than or equal to 8. For example, x may be greater than or equal to 10, greater than or equal to 14, greater than or equal to 16, greater than or equal to 20, x greater than or equal to 30, greater than or equal to 40, or greater than or equal to 50.
Non-limiting examples of other types of fluorpolymers or oligomers that can be included in the fluorophilic chain, and/or as side chains, include vinylidene fluoride (VDF), (per)fluoroolefins (e.g., tetrafluoroethylene (TFE)), chlorotrifluoroethylene (CTFE), (per)fluoroalkylvinylethers (PAVE), e.g., CF2═CFORf, where Rf is a (per)fluoroether or a Cn (per)fluoroalkyl such as trifluoromethyl or pentafluoropropyl, where n is an integer; and perfluoro-oxyalkylvinylethers CF2═CFORx, where x is a C1-C12 perfluoro-oxyalkyl having one or more ether groups, for example, perfluoro-2-propoxy-propyl. Other examples of monomers present within the fluorophilic component include fluorinated acrylates and fluorinated methacrylates. In some cases, the fluorophilic component may be a component where at least about 10% or at least about 20% of the atoms defining the component are fluorine atoms.
A fluorophilic component of a surfactant may have any suitable mixture of hydrogen and fluorine atoms so long as the fluorophilic component is sufficiently soluble in a suitable fluorophilic continuous phase. The solubility of a component in a fluorophilic continuous phase can be readily determined by those of ordinary skill in the art using no more than routine experimentation. For instance, a fluorophilic component may comprise monomer units such as —(C3F5HO)x—(C3F4H6-mO)x—, —(C3F4H2O)x—, etc. In some embodiments, the fluorophilic component has a ratio of fluorine to hydrogen atoms of greater than 1:1, greater than or equal to 3:1, greater than or equal to 5:1, greater than or equal to 6:1, greater than or equal to 9:1, greater than or equal to 10:1, greater than or equal to 12:1, greater than or equal to 15:1, or greater than or equal to 20:1. In certain embodiments, the fluorophilic component is perfluorinated.
In some embodiments, the fluorophilic component of a surfactant includes one or more fluoropolymers, where the number of monomer units forming the fluoropolymer is greater than or equal to 8. For example, the polymer (or oligomer) may have greater than or equal to 5 monomers, greater than or equal to 10 monomers, greater than or equal to 14 monomers, greater than or equal to 16 monomers, greater than or equal to 20 monomers, greater than or equal to 30 monomers, greater than or equal to 40 monomers, or greater than or equal to 50 monomers, etc.
The fluorophilic component may have a molecular weight greater than or equal to 1,000 g/mol, greater than or equal to 1,200 g/mol, greater than or equal to 1,500 g/mol, greater than or equal to 1,700 g/mol, greater than or equal to 1,900 g/mol, greater than or equal to 2,000 g/mol, greater than or equal to 2,200 g/mol, greater than or equal to 2,500 g/mol, greater than or equal to 2,700 g/mol, greater than or equal to 3,000 g/mol, greater than or equal to 3,200 g/mol, greater than or equal to 3,500 g/mol, greater than or equal to 3,700 g/mol, greater than or equal to 4,000 g/mol, greater than or equal to 4,200 g/mol, greater than or equal to 4,500 g/mol, greater than or equal to 4,700 g/mol, greater than or equal to 5,000 g/mol, greater than or equal to 5,200 g/mol, greater than or equal to 5,500 g/mol, greater than or equal to 5,700 g/mol, greater than or equal to 6,000 g/mol, greater than or equal to 6,200 g/mol, greater than or equal to 6,500 g/mol, greater than or equal to 6,700 g/mol, greater than or equal to 7,000 g/mol, greater than or equal to 7,200 g/mol, greater than or equal to 7,500 g/mol, greater than or equal to 7,700 g/mol, greater than or equal to 8,000 g/mol, greater than or equal to 8,200 g/mol, greater than or equal to 8,500 g/mol, greater than or equal to 8,700 g/mol, greater than or equal to 9,000 g/mol, greater than or equal to 9,200 g/mol, greater than or equal to 9,500 g/mol, greater than or equal to 9,700 g/mol, or greater than or equal to 10,000 g/mol.
The surfactants described herein may have a hydrophilic headgroup in some cases. In some embodiments, the hydrophilic component of a surfactant is a polymer (or oligomer). The polymer may include, for example, a polyether. The polyether chain may comprise repeating units including, but not limited to, —(CnH2nO)x—, where n is an integer, for example, —(C3H6O)x—, —(C4H8O)x—, —(C5H10O)x—; —(C2H4O)x—(C3H6O)x—, —(C4H8O)x—, —(C5H10O)x—(CH(CH3)CH2O)x—; —(CH2CH2O)x—; —(CH(CH3)CH2O)x—CH(CH3)CONH—; —(CH2(CH2)z′CH2O)x—, where z′ is an integer; —(CHLO)x—, where L=—H or —CH3; or —(CH2CH2CH2O)x—. The polyether chain may include, in some cases, terminal groups such as (CnH2n+1O)x—, where n is an integer, for example, (CH3O)x—, (C2H5O)x—, (C3H7O)x—, etc. In some embodiments, the hydrophilic component comprises polymethylene oxide, polyethylene oxide, polybutylene oxide, and/or polyTHF, and/or various polymers thereof. A non-limiting example is a JEFFAMINE® amine. In one particular embodiment, the fluorophilic chain includes polyethylene glycol. Typically, x in the hydrophilic structures above is greater than or equal to 1. For example, x may be greater than or equal to 5, greater than or equal to 10, greater than or equal to 14, greater than or equal to 16, greater than or equal to 20, greater than or equal to 30, greater than or equal to 40, or greater than or equal to 50. In other embodiments, a headgroup can include a sugar (e.g., glucose, glucosamine, and sorbitol). Other polar headgroups known to those of ordinary skill in the art are also included within the scope of the present invention.
In one set of embodiments, a headgroup of a fluorosurfactant is connected to a linking moiety. In some cases, the linking moiety is a relatively small entity. The linking entity may comprise, for example, a morpholino group (e.g., dimorpholino and monomoropholino groups). The linking entity also may comprises a phosphate group in some instances. In certain embodiments, the linking entity comprises both a morpholino group and a phosphate group (e.g., a dimoporpholino phosphate).
In some embodiments, a linking moiety (e.g., positioned between A and B components of a fluorosurfactant) may be chosen to assist the self assembly and the packing of the fluorosurfactant at the interface. Additionally, a linking moiety may have a good impact on the CMC (critical micelle concentration), and therefore on the diffusion to a newly formed interface from the fluorophilic phase, which may be important for emulsification.
In one set of embodiments, the headgroups of the surfactants may be chosen to render the surfactant soluble in water and/or relatively biologically inert. As mentioned, headgroups such as dimorpholino phosphate (DMP) and polyethylene glycol (PEG) are examples of potentially suitable headgroups. A non-limiting example of a tail of a surfactant is Krytox FS(H) (manufactured by DuPont), a carboxylic acid-terminated perfluoropolyether. This tail may have stability in fluorocarbon oils, its length (which can provide steric repulsion between the surfactant molecules), and the fact that it possesses a terminal group that is suitable for the grafting of different headgroups.
Specific examples of potentially suitable headgroups include, but are not limited to:
Specific examples of potentially suitable tails include, but are not limited to:
where x is any positive integer, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.
In some cases, a surfactant of the instant invention may also include a linking moiety. Non-limiting examples of linking moieties include carbonyls (—C(O)—) and phosphates (PO4). In some cases, the linking moiety may connect more than one headgroup and/or more than one tail. For instance, a phosphate linking moiety may connect two headgroups (which each may the same or different), two tails (which each may the same or different) and a headgroup, etc.
Thus, in one set of embodiments, a surfactant of the present invention may include a headgroup (or more than one headgroup), a tail (or more than one tail), and optionally, a linking moiety (or more than one moiety), including the headgroups, tails, and linking moieties described above. Specific, non-limiting examples of some of the surfactants of the invention, comprising a head group, a tail, and optionally a linking moiety, follow:
The hydrophilic component of a surfactant of the present invention may have any molecular weight suitable for forming a water-in-fluorocarbon type emulsion. For instance, the molecular weight of the hydrophilic component may be greater than or equal to 100 g/mol, greater than or equal to 200 g/mol, greater than or equal to 300 g/mol, greater than or equal to 500 g/mol, greater than or equal to 800 g/mol, greater than or equal to 1,000 g/mol, greater than or equal to 1,500 g/mol, or greater than or equal to 2,000 g/mol, etc.
In some applications involving oil-in-fluorocarbon type emulsions and/or lipophilic solvent applications, a surfactant having a headgroup comprising a lipophilic component may be desired. The lipophilic component may include, for example, an alkyl block, —(CH2)x—. In some embodiments, x is less than or equal to 20, less than or equal to 18, less than or equal to 16, less than or equal to 14, less than or equal to 12, less than or equal to 10, less than or equal to 8, less than or equal to 6, less than or equal to 4, or less than or equal to 2. Other alkyl-containing and/or aromatic-containing components are known in the art and may be used in surfactants described herein.
Various fluorophilic components can be used as the fluorophilic continuous phase in emulsions described herein. In some cases, the fluorophilic component of the continuous phase and the fluorophilic components of the surfactant is the same. In other cases, however, they can be different. In addition to other structures described herein, the following are non-limiting examples of fluorophilic components that can be used in either a surfactant and/or a continuous phase: perfluorodecalin, perfluoromethyldecalin, perfluoroindane, perfluorotrimethyl bicyclo[3.3.1]nonane, perfluoromethyl adamantane, perfluoro-2,2,4,4-tetra-methylpentane; 9-12C perfluoro amines, e.g., perfluorotripropyl amine, perfluorotributyl amine, perfluoro-1-azatricyclic amines; bromofluorocarbon compounds, e.g., perfluorooctyl bromide and perfluorooctyl dibromide; F-4-methyl octahydroquinolidizine and perfluoro ethers, including chlorinated polyfluorocyclic ethers, perfluoro-4-methylmorpholine, perfluorotriethylamine, perfluoro-2-ethyltetrahydrofuran, perfluoro-2-butyltetrahydrofuran, perfluoropentane, perfluoro(2-methylpentane), perfluorohexane, perfluoro-4-isopropylmorpholine, perfluorodibutyl ether, perfluoroheptane, perfluorooctane, perfluorotripropylamine, perfluorononane, perfluorotributylamine, perfluorodihexyl ether, perfluoro[2-(diethylamino)ethyl-2-(N-morpholino)ethyl]ether, n-perfluorotetradecahydrophenanthrene, and mixtures thereof. In some instances, the fluorophilic component can be straight-chained, branched, cyclic, etc., and/or have a combination of such structures. Specific non-limiting examples include fluoroinert PFPEs (perfluoropolyethers), such as KRYTOX®, by DuPont or perfluoropolyethers and other fluoropolymers from Solvay Solexis.
In one particular embodiment, the choice of solvent and/or fluorophilic component may include approximately matching the average chain length of the fluorophilic portion of the surfactant with the average chain length of the continuous-phase component of the mixture.
In some embodiments, surfactants have a structure such as A-B, A-B-A, or (A-B—)n (i.e., A-B-A-B-A-B-A . . . ) or B-(A)n, or A-B-A′, or another multi-block configuration, where A (and A′, if present) comprises a fluorophilic component and B is the hydrophilic or lipophilic component. In some cases, the surfactant may include a linking moiety, such as A-X—B, A-X—B-A, A-X—B-A′, A-X1—B—X2-A, A-X1—B—X2-A′, B—X-(A)n-, B-(A-X)n—, B—(X-A)n, B—(X-A)n, B-(A-X)n, B—X1-(A-X2)n, (A-X—B)n, (A-B—X)n, (A-X1—B—X2)n, or the like, where “X” represents a covalent bond or a linking moiety, and X1 and X2, where present, may be the same or different. Some such structures may have a ratio of the molecular weights of the A:B portions of greater than or equal to 1:1, greater than or equal to 3:1, greater than or equal to 6:1, greater than or equal to 9:1, greater than or equal to 10:1, greater than or equal to 12:1, greater than or equal to 15:1, greater than or equal to 20:1; greater than or equal to 25:1, greater than or equal to 30:1, greater than or equal to 40:1, or greater than or equal to 50:1, etc. Additionally or alternatively, the structures may have a ratio of the molecular weights of the X:B portions of greater than or equal to 1:1, greater than or equal to 3:1, greater than or equal to 6:1, greater than or equal to 9:1, greater than or equal to 10:1, greater than or equal to 12:1, greater than or equal to 15:1, greater than or equal to 20:1; greater than or equal to 25:1, greater than or equal to 30:1, greater than or equal to 40:1, or greater than or equal to 50:1, etc.
As described herein, the performance of a surfactant may also depend on the absolute block length and on the geometry of the surfactant, which may be tuned by changing the block length ratio in some cases. For example, surfactants having a structure such as A-B, A-B-A, or (A-B—)n (i.e., A-B-A-B-A-B-A . . . ), B-(A)n, A-B-A′, A-X—B, A-X—B-A, A-X—B-A′, A-X1—B—X2-A, A-X1—B—X2-A′, B—X-(A)n-, B-(A-X)n—, (A-X—B—)n, (A-B—X)n, (A-X1—B—X2)n, etc., or another multi-block configuration, where A (and A′, if present) comprises a fluorophilic component and B is the hydrophilic or lipophilic component, may have a ratio of the lengths of the A:B portions of greater than or equal to 1:1, greater than or equal to 3:1, greater than or equal to 6:1, greater than or equal to 9:1, greater than or equal to 10:1, greater than or equal to 12:1, greater than or equal to 15:1, greater than or equal to 20:1; greater than or equal to 25:1, greater than or equal to 30:1, greater than or equal to 40:1, or greater than or equal to 50:1, etc. In these structures, “X” represents a covalent bond or a linking moiety, and X1 and X2, where present, may be the same or different. Additionally or alternatively, these structures may have a ratio of the lengths of the X:B portions of greater than or equal to 1:1, greater than or equal to 3:1, greater than or equal to 6:1, greater than or equal to 9:1, greater than or equal to 10:1, greater than or equal to 12:1, greater than or equal to 15:1, greater than or equal to 20:1; greater than or equal to 25:1, greater than or equal to 30:1, greater than or equal to 40:1, or greater than or equal to 50:1, etc.
In some embodiments, the fluorosurfactants described herein are characterized by a certain overall chain length of, for example, less than 100 monomers, less than 80 monomers, less than 60 monomers, less than 40 monomers, or less than 20 monomers. Additionally or alternatively, the fluorosurfactants may have an overall chain length of, for example, greater than 5 monomers, greater than 7 monomers, greater than 10 monomers, greater than 30 monomers, greater than 50 monomers, greater than 70 monomers, or greater than 90 monomers. In other embodiments, fluorosurfactants of the invention include only a few monomers, or one monomer such as a macromonomer.
The capability to tune the compositions, lengths, molecular weights, and/or the geometry of the surfactants as described herein (which may be synthesized by forming a covalent bond between the fluorophilic and hydrophilic/lipophilic components and/or by graft polymerization, for example) allows for the tailored stabilization of droplets according to certain embodiments of the present invention. In some embodiments, higher ratios of fluorophilic component to hydrophilic (or lipophilic) component may be appropriate for the stabilization of very small aqueous droplets. Lower ratios may be useful for stabilizing fluorocarbon-in-water emulsions, for example. Also, the stabilization of double- and multiple emulsions is possible, in certain cases.
Increasing the size and/or length of the fluorophilic component can, in some embodiments, increase steric repulsion and/or improve long term stability of the emulsions. In some cases, if surfactant molecules of relatively high molecular weight and/or with relatively large headgroups are used, the adsorption of the surfactant to the interfaces can be slowed down in some cases, which may be advantageous for droplet formation, for instance, if hydrodynamic flow focusing techniques are used. Larger surfactants of suitable geometry may include larger chemical moieties, such as poly(ethylene glycol), which can allow for an even more efficient shielding of the interfaces against the adsorption of biological material.
For instance, without wishing to be bound by theory, the inventors believe that stabilization of droplets in a fluorophilic continuous phase involves the factors and criteria described below and herein. As shown in the diagram illustrated in
Accordingly, in designing a suitable fluorosurfactant (e.g., a block-copolymer fluorosurfactant) for stabilizing droplets in a continuous fluorophilic phase, a suitable fluorophilic portion of a surfactant can be chosen such that sufficient steric stabilization of the emulsion is provided. Stabilization relies, at least in part, on the solubility of the outer-facing portion of the surfactant (i.e., the portion facing the continuous phase) in the fluorophilic continuous phase and on a sufficient thickness of the stabilizing layer. Long-term droplet stabilization may be achieved by droplets that include a thick steric stabilizing layer, e.g., a thick fluorophilic layer. The stabilizing layer may have a thickness of, for example, at least 10 Angstroms in some embodiments, or at least 20 Angstroms, at least 30 Angstroms, at least 40 Angstroms, or at least 50 Angstroms. In some cases, the thickness of the fluorophilic stabilization layer may be greater than about 7 to 10 Angstroms and less than about 5,000 Angstroms or less than about 1,000 Angstroms. In some embodiments involving emulsions comprising a fluorophilic continuous phase, long fluorophilic components are favorable. The fluorophilic component of a surfactant may be at least about 7 to 10 Angstroms in length in some embodiments, or at least about 50 Angstroms, at least about 100 Angstroms, at least about 300 Angstroms, at least about 500 Angstroms, at least about 1,000 Angstroms, or at least about 5,000 Angstroms in other embodiments.
In some cases, the outer-facing portion of the surfactant (e.g., the fluorophilic portion of a surfactant in an emulsion comprising a fluorophilic continuous phase) is larger, longer, and/or has a larger molecular weight than the counterpart of the headgroup portion. The hydrophilic or lipophilic headgroup portion may be long enough to keep the surfactant molecules anchored at the interface and to provide a sufficiently densely packed hydrophilic/lipophilic layer at the inner interface such that it is able to provide a barrier to interfacial adsorption, e.g., for biological applications. However, too long of a hydrophilic/lipophilic component may cause crowding on the inner interface, yielding an outer interface that is insufficiently covered with the fluorophilic component, and hence prone to coalescence. Sufficient coverage may be characterized by the absence of bare patches and by a brush of sufficiently extended fluorophilic molecules on the outer interface.
Moreover, the geometry of the surfactant may be important in preventing coalescence in some cases, which may start with the formation of a small neck connecting two droplets. Efficient surfactants that stabilize the droplet from coalescence may destabilize the neck. The surfactant geometry may fit the curvature of the undisturbed droplet and oppose the curvature of the neck. As an example, as shown in
A suitable headgroup portion of a fluorosurfactant described herein that is able to anchor the surfactant to an interface of the droplet may be immiscible with the continuous phase and, in some embodiments, may be soluble in the disperse phase. Solubility in the disperse phase, however, may allow for the potential of interaction of the headgroup portion with components or compounds contained in the droplet. Thus, in some embodiments, a fluorosurfactant described herein includes an headgroup portion that does not interact undesirably with a component contained in the disperse phase. For certain biological applications, for example, PEG has been found to efficiently suppress interactions of biomolecules and cells with interfaces. Being water soluble and non-toxic, PEG may be a suitable choice for an headgroup component of a surfactant in certain biological systems. Additionally, being chemically inert and soluble in certain organic solvents makes PEG attractive for performing certain organic chemistry processes inside droplets. Of course, other compositions such as those described herein can also be used as headgroup portions of fluorosurfactants of the invention. A non-limiting example of such a composition is a morpholinophosphate. In addition, in some cases, compounds may be adsorbed onto an inner surface or interface, which may prevent or reduce the adsorption of biological compounds, e.g., through coacervation processes. As an example, BSA may be used as an interfacially active compound for screening residual uncovered patches.
In some embodiments, fluorosurfactants of the invention comprise two oligomeric (or polymeric) components including a fluorophilic component (e.g., component “A”) and a hydrophilic component (e.g., component “B”). These components may form a diblock-copolymer (e.g., a “A-B” structure), or other structures including those described herein. In order to synthesize a fluorosurfactant including these components, an appropriate solvent/solvent mixture may be chosen so as to dissolve (or at least partially dissolve or stabilize) both fluorophilic and hydrophilic (or lipophilic) components to provide mobility, which may be required for the coupling reaction. In one embodiment, the fluorophilic component comprises a perfluorinated oligomer or polymer, such as poly(perfluoro propyleneoxide) (e.g., KRYTOX® by DuPont); the hydrophilic component may include a non-fluorinated oligomer or polymer, such as poly(ethylene glycol) (PEG). In some embodiments, 40:60 mixtures by volume of fluorophilic component, e.g., methyl nonafluoroisobutylether and/or methyl nonafluorobutylether (e.g., HFE 7100), and a solvent, e.g., THF, may be adequate to dissolve both components. In another embodiment, a 1:1 mixture of a fluorophilic component, e.g., methyl nonafluoroisobutylether and/or methyl nonafluorobutylether (e.g., HFE 7100), and a solvent, e.g., dichloromethane, may be adequate to dissolve both components.
Those of ordinary skill in the art can choose suitable solvents to dissolve or stabilize particular components used to synthesize fluorosurfactants described herein based on, for example, known solubility properties of the components or by simple experimentation in combination with description provided herein. For instance, solubility parameters (e.g., Hildebrand parameters), as described in Barton, Handbook of Solubility Parameters, CRC Press, 1983, may be used to determine the likelihood of solubility of one component in another. Typically, chemical components having similar values of solubility parameter are soluble in one another. Those of ordinary skill in the art can also choose an appropriate solvent by, e.g., knowing the fluorophilic and hydrophilic (or lipophilic) components and the likelihood of reactivity between the surfactant components and the solvent, and/or by a simple screening test. One simple screening test may include adding the surfactant components to the solvent and determining whether the surfactant components react with and/or are negatively effected by the solvent. A screening test for choosing appropriate surfactant components and solvent for the formation of an emulsion can include mixing the components to form the emulsion and varying either the material composition, quantities, and/or concentration of one component while keeping the others constant, and determining the stability of the emulsion. The surfactants may have different relative block lengths or geometries. Other simple tests can be identified and conducted by those of ordinary skill in the art with the benefit of the present disclosure.
In some embodiments, it is desirable to choose a particular solvent to be contained inside droplets such as those described herein. For example, in some embodiments, emulsion droplets may be used as separate compartments for particle synthesis or high throughput screening, or the particles can be employed as miniaturized reaction sites, as described in more detail below. Prerequisites of such use include, for example, control over the droplet size, compatibility of the components contained in the disperse phase with the disperse phase and with the fluorosurfactants, and compatibility between the disperse phase, fluorosurfactants, and the continuous phase. While droplets may be made using emulsification techniques in some conventional emulsion systems (e.g., hydrocarbon oil-in-water emulsions and water-in-hydrocarbon oil emulsions), chemical compatibility in these systems can be limited if certain solvents, such as solvents that are miscible with hydrocarbons and water, are used as the continuous phase. In some cases, for chemical compatibility, the dispersed phase and the continuous phase in various embodiments of the instant invention may be chosen to be immiscible, and thus, the solvent to be compartmentalized in droplets may be chosen to be immiscible in the continuous phase (e.g., oil or water). For example, it may be difficult to form emulsions comprising certain alcohols or other solvents (e.g., THF) as the disperse phase, as alcohols or other solvents may be miscible with either water or certain organic oils, e.g., mineral oils or silicon oils that are used as the continuous phase. However, using emulsions involving a fluorocarbon phase and fluorosurfactants as described herein can allow generation of emulsions containing a wide variety of solvents. In particular, the inventors have discovered that emulsions of solvent droplets in fluorocarbon oils such as those described herein can include a wide range of common solvents such as alcohols as a discontinuous phase, which may not be possible in conventional emulsion systems. This is because fluorocarbons are at the same time hydrophobic and lipophobic, and may thus be immiscible with alcohols.
The choice of size and geometry of a surfactant (including outer and headgroup components) as applied to the stabilization of emulsions including an alcohol as a discontinuous phase is one example of tailoring droplets using description contained herein. Without wishing to be bound by any theory, the inventors have discovered the following trends and observations. In order of decreasing polarity, methanol (MeOH), ethanol (EtOH), and i-propanol (i-PrOH) are similar in their chemical properties and each does not dissolve in certain fluorocarbon oils. In some cases, each of methanol, ethanol, and i-propanol dissolve in a substance that can be used as an headgroup component of a fluorosurfactant. In one embodiment, the substance is PEG or a derivative thereof, which suggests that PEG-fluorophilic (e.g., PFPE)-block copolymers can stabilize emulsions comprising the alcohols in a fluorophilic continuous phase. However, surprisingly, it was discovered that the fairly polar methanol group may be stabilized with any of the applied surfactants, Table 1 shows that surfactants of certain block lengths provided long-term stabilizing i-propanol emulsions. These observations may be applied to other emulsions including low polarity solvents as the discontinuous phase.
Surfactants with small PEG- and small PFPE-blocks may decrease the surface tension; however, they may not provide colloidal stabilization of the emulsion. Increasing the length of both the PEG- and the PFPE-blocks may improve the long-term stability of the emulsion. In some cases, the influence of the outer-facing portion of the surfactant (e.g., PFPE) may be more important for long term stabilization than that of the headgroup portion (e.g., PEG). This suggests that the failure of emulsion stabilization may be dominated by the formation of a bare patch on the interface of two adjacent droplets, giving rise to neck formation and subsequent coalescence. This may also suggest that an inappropriate surfactant geometry or too short of an headgroup portion of a surfactant (e.g., PEG) that will facilitate surfactant desorption may be counterbalanced by a thicker stabilizing (e.g., outer-facing portion) layer, such as longer fluorophilic components of a surfactant.
Table 1 shows a summary of the effects of solvent polarity of the solvents MeOH>EtOH>i-PrOH, listed from highest to lowest polarity, surfactant geometry and absolute chain lengths on emulsion forming and stabilization. The block-copolymers are labeled with a four-digit code, and the co-polymers can also be labeled with E2K (difunctional polyethylene glycol coupled to two PFPE blocks). The first two digits in the entries in Table 1 indicate the molecular weight of the PEG block divided by 100, the third and fourth digit indicate the molecular weight of the PFPE block divided by 100. For example: 0220 (or E2K 0220) is a surfactant including PEG having a molecular weight of 200 g/mol and a PFPE block having a molecular weight of 2000 g/mol. The notations are as follows: “E”—stable emulsion was formed; “T”—slightly less stable emulsion that “E”; “S”—least stable emulsion was formed. “Cyt” is Cytonix FluorN surfactant, commercially available from Cytonix.
As the difference in polarity of the disperse and continuous phases becomes smaller, larger PEG-blocks and/or larger PFPE-blocks may be required for stabilizing emulsions. Larger PEG-blocks may provide a better anchoring strength to the interface compared to smaller PEG-blocks. Larger PFPE-blocks may shield a greater interfacial area more efficiently against coalescence compared to smaller PFPE-blocks. In some cases, the effect of the PFPE-block variation is more pronounced than that of the PEG-block variation. This may be due to the capability of a greater stabilizing moiety to cover a nearby bare patch or prevent or inhibit the bare patch from forming. In some embodiments, an increase in the size of one or both of the blocks could decrease the surfactant mobility on the interface, making the formation of bare patches less likely.
In another embodiment, an emulsion of the present invention comprises THF as a discontinuous phase and a fluorophilic continuous phase stabilized by fluorosurfactants described herein. In one embodiment, the surfactant comprises PEG and PFPE. In some cases, both longer PEG and longer PFPE blocks may afford an improved stabilization; the effect of the PFPE blocks may be more pronounced. In other cases, however, there are exceptions to this trend. For instance, surfactants including a PEG portion that has a higher molecular weight than the PFPE portion may stabilize droplets better than surfactants including a PFPE portion that has a higher molecular weight that the PEG portion. This result may be associated with the pronounced geometry of the surfactant molecules that cause the formation of thermodynamically stable, swollen micelles that cannot coalesce.
In certain embodiments, fluorosurfactants of the invention include triblock-copolymers (e.g., A-B-A structures), whose mid-block is soluble in the discontinuous phase. This “double-tail” morphology is known to have advantages in the colloidal stabilization properties over certain “single-tail” (e.g., A-B) surfactants. In some embodiments, the mid-block can include a poly(ethylene glycol) moiety. Many poly(ethylene glycol)s are available with two reactive headgroups on either end of the polymer-chain, which can facilitate the synthesis of double-tail morphologies. However, the synthetic routes described herein may be used for the synthesis of other surface active morphologies, such as diblock-copolymers, multi-block-copolymers, polymer brushes, etc. In some cases, the triblock copolymer may also contain one or more linking moieties, for example, as in the structure (A-X1—B—X2)n, where each “X” independently represents a covalent bond or a linking moiety, and the each X may be the same or different.
The following example syntheses use poly(ethylene glycol) as the hydrophilic component of a headgroup; however, the synthesis can be applied to the formation of surfactants including other chemical moieties. In particular, the synthetic routes described herein can be easily applied to the coupling of a variety of other hydrophilic components.
In some embodiments, surfactants including poly(ethylene glycol) as the hydrophilic component of a headgroup are advantageous for performing chemical and/or biological reactions (e.g., enzymatic reactions and reactions involving cells and/or cellular components). Poly(ethylene glycol)s are well known and widely applied to “passivate” surfaces against non-specific adsorption of nucleic acids and proteins to solid surfaces. (Harris, J. M.; Zalipsky, S. on Poly(ethylene glycol): Chemistry and Biological Applications, American Chemical Society, Washington, D.C.; 1997). For the same reason, poly(ethylene glycol) surfactants such as Tween (Bernath, K.; Hai, M.; Mastrobattista, E.; Griffiths, A. D. Magdassi, S.; Tawfik D. S. in Analytical Biochemistry, 325: 151-157; 2004) and Triton (Mastrobattista, E.; Taly, V.; Chanudet, E.; Treacy, P.; Kelly, B. T.; Griffiths, A. D. in Chemistry & Biology, 12: 1291-1300; 2005) have been applied for in vitro compartmentalization in hydrocarbon oils.
In some instances, a fluorophilic component and a non-fluorophilic component (e.g., a hydrophilic or lipophilic component) of a surfactant are bonded covalently. However, in other embodiments, ionic bonds or other associations, such as linking moieties, can be used to combine the two components. Non-limiting examples of reactions for coupling two or more components together follows.
Three different, non-limiting coupling reactions are now described, which can be used to achieve coupling of the fluorophilic and hydrophilic components. These include the formation of an ester bond, an amide bond, or an ether bond between a hydrophilic and fluorophilic component. The chemicals used in the synthetic routes described herein are produced on the industrial scale and therefore readily available.
While the amide bond may be formed in a particularly simple reaction of, e.g., poly(ethylene glycol) diamine, the formation of ether bonds may involve the use of hydroxyl terminated poly(ethylene glycol) components, which are inexpensive and are available in a wide variety of chain lengths. In some instances, ether bonds are more stable to hydrolysis than esters or amides; thus they may allow for a wider range of applicable pH.
In one embodiment, an ester bond may be formed through the reaction of, for example, poly(ethylene glycol) dicarboxylic acid with thionylchloride to yield the poly(ethylene glycol) diacid chloride, with which a fluoropolymer with a terminal alcohol group may react in the presence of poly(vinyl pyridine). The inverse case of reacting the fluorophilic acid halide with the poly(ethylene glycol) dialcohol (PEG(OH)2) is possible as well.
In another embodiment, an amide bond may be formed through simple amidation reaction. For example, a poly(ethylene glycol) or other component comprising a mono- or diamine reacts on a fluoropoymer carrying a terminal methylester to yield the amide may be used. The formation of the inverse amide is possible as well.
In another embodiment, an ether bond may be synthesized using a Mitsunobu reaction, which links, for example, two components having hydroxyl functional groups. For instance a fluoropolymer and a poly(ethylene glycol) may both comprise hydroxyl functional groups and can be bonded using a Mitsunobu reaction. DEAD (diethyl azodicarboxylate) and triphenyl phosphine may be used for activating the less acidic poly(ethylene glycol) alcohol. Sorbitol may be applied as an optional hydrophilic group. Williamson ether synthesis is also possible with certain hydrophilic headgroups. A Williamson ether synthesis may also be used for linking a fluorophilic and lipophilic component. For instance, a tosylated fluoropolymer can be linked to an organic alcohol in the presence of an organic base.
Other synthetic routes may involve very reactive species, such as isocyanates (forming urethane bonds), or precursors that are amphiphiles by themselves, such as fluorocarbons with acid, nitrile or acid halogenide headgroups that may be coupled to the hydrophilic component yielding esters and amides. Various reagents may be used to activate carboxylic groups (e.g. BOP).
The details of some of these synthetic routes are described below, but are intended to be exemplary only and not limiting. Possible ways to purify the products are indicated in these procedures as well. A particularly interesting method of purification includes phase separation of the unreacted fluorophobic reactant from an appropriate low-polarity fluorosolvent mixture. The unreacted fluorophobic compound will cream, forming a supernatant that may be easily decanted. At the same time, block-copolymer surfactant may phase separate if the critical micelle concentration is exceeded, but it will sediment, rather than cream, due to the high density of the fluorocarbon components contained in the surfactant molecules.
In one particular embodiment, suitable geometries and acceptable stabilization of droplets in fluorocarbon oils have been achieved using the ether synthesized from the reaction of phytol and KRYTOX® alcohol with a molecular weight of 1,700 g/mol. The molecular weight ratio in this example was about 1:6. A molecular weight ratio of poly(ethylene glycol) to poly(perfluoropropylene oxide) of 1:4 to 1:5 was suitable for the stabilization of THF droplets in fluorocarbon oils. The applied poly(ethylene glycol) contained 75% diamine and 25% of monoamine, yielding 75% triblock and 25% diblock co-polymer.
In some embodiments, unreacted mixtures of the methyl ester of KRYTOX®-fluoropolymers and poly(ethylene glycol) diamines may reduce the surface tension between water and fluorocarbon oils and stabilize droplets. Therefore, interfacially active emulsifying systems that rely on the interaction of appropriately functionalized fluoropolymers with amines and polyethers across the interface are contemplated within the scope of the present invention, even if the fluorophilic and the hydrophilic (or lipophilic) components are not bound covalently. Similarly, the application of poly(ethylene glycol) and amines to screen the interface of droplets that are stabilized with conventional surfactant systems are contemplated within the scope of the present invention.
In one embodiment, a fluorosurfactant of the invention has the appropriate geometry for suitably stabilizing (e.g., sterically) droplets of hydrocarbon oils and organic solvents in fluorocarbon oils. In some cases, a fluorosurfactant includes lipophilic headgroups for stabilizing inverse emulsions that comprise hydrocarbon oils and organic solvents in addition to water. Also in these cases, the fluorosurfactants may comprise oligomeric or polymeric fluorocarbon tails, which can allow long term colloidal stability against coalescence. This may be achieved, for instance, through sufficient steric repulsion.
The surfactant syntheses described herein include, but are not limited to, coupling a perfluorinated component to a lipophilic component in a suitable solvent mixture. As a fluorophilic component, a perfluorinated oligo- or polymer, such as an poly(perfluoro propyleneoxide) (e.g. KRYTOX® by DuPont), was used. The lipophilic component can be another oligomeric component, such as poly(ethylene glycol), poly(propylene glycol) or poly(ethylene-co-butylene), poly-THF, or an alkyl component such as lauryl or phytol. Other examples include, but are not limited to, proteins, or aromatics. This may represent a convenient route to surfactants of low to intermediate chain lengths with a well defined structure and molecular weight distribution. Other suitable methods of synthesis are known to those of ordinary skill in the art. The solvent used for the coupling reaction may be chosen to dissolve both components to provide their mobility required for the coupling reaction. 40:60 mixtures by volume of methyl nonafluorobutyl ether (3M's HFE 7100) and THF showed good results in some experiments. Other non-limiting examples of suitable solvents include HFE 7100 (3M) and dichloromethane or fluorochlorocarbons.
Another aspect of the invention includes stabilizing emulsions involving electrostatic attraction of two oppositely charged components (e.g., a first surfactant including a headgroup and a tail, and a second surfactant including a headgroup and a tail), one of which is soluble in the dispersed phase, the other soluble in the continuous phase. The two components may combine to form at the interface between the continuous and discontinuous phases to stabilize an emulsion. The component soluble in the continuous phase may include an ionic surfactant suitable for stabilizing inverse emulsions. The component soluble in the disperse phase may assemble as a layer of counterions to the surfactant headgroups at the interface. Particularly suited are counterions carrying multiple charges as they cannot be replaced easily by other charged species. They may be chosen to fit the desired surface chemistry that allows for chemical/biochemical/biological reactions inside the droplets.
As a non-limiting example of such a system, poly(perfluoropropyleneoxide) carboxylic acid (KRYTOX® FS series) was dissolved in a continuous fluorous phase and a polycation was dissolved in the dispersed phase. Polycations may carry functionalities such as primary, secondary, tertiary, or quaternary amines. In some experiments, 0.01%-0.5% (w/w of the aqueous phase) low molecular weight chitosan (Aldrich) were dissolved in the aqueous phase. The precise weight fractions of each component will depend on, for example, the droplet size and on the cationic charge density. For chitosan, both the primary amine and the quaternary ammonium salt may be suitable for preventing the adsorption of biomolecules to the interfaces.
To produce stable droplets of organic solvents 0.01%-0.5% (w/w), polymeric ammonium salts with counterions which are highly soluble in the organic phase and inert to chemical reactions were added. Suitable polycations include, for example, poly-N-methyl-vinylpirydinium, quaternized poly-N-vinyl-imdazole, polyallylamine, or LUVIQUAT® (BASF). Other polycations are known in the art and can also be used. Suitable counter-anions include 4-(trifluoromethyl)-benzenesulfonate, or trifluoromethanesulfonate. Other counter-anions are known in the art and can also be used.
In another embodiment, two surfactants can be dissolved in a continuous fluorous phase and combined to assemble at the interface between the continuous and discontinuous phase of an emulsion. One surfactant can provides colloidal stability of the emulsion. For example, KRYTOX® 157 FSL can be used for steric stabilization of the droplets. The other surfactant can be chosen to prevent adsorption of components such as chemicals, reactants, and biomolecules to the interfaces. For example, small amounts (<0.5% by weight) of a commercial fluorous surfactant that comprises a poly(ethylene glycol) group, such as ZONYL® (DuPont) FSN, may be used to sterically block the charges of the KRYTOX® surfactant.
The emulsions of the present invention may be formed using any suitable emulsification procedure known to those of ordinary skill in the art. In this regard, it will be appreciated that the emulsions can be formed using microfluidic systems, ultrasound, high pressure homogenization, shaking, stirring, spray processes, membrane techniques, or any other appropriate method. In one particular embodiment, a micro-capillary or a microfluidic device is used to form an emulsion. The size and stability of the droplets produced by this method may vary depending on, for example, capillary tip diameter, fluid velocity, viscosity ratio of the continuous and discontinuous phases, and interfacial tension of the two phases.
Non-limiting examples of microfluidic systems potentially suitable for use with the instant invention include the following, each incorporated herein by reference: U.S. patent application Ser. No. 11/024,228, filed Dec. 28, 2004, entitled “Method and Apparatus for Fluid Dispersion,” by Stone, et al., published as U.S. Patent Application Publication No. 2005/0172476 on Aug. 11, 2005; U.S. patent application Ser. No. 11/246,911, filed Oct. 7, 2005, entitled “Formation and Control of Fluidic Species,” by Link, et al., published as U.S. Patent Application Publication No. 2006/0163385 on Jul. 27, 2006; U.S. patent application Ser. No. 11/360,845, filed Feb. 23, 2006, entitled “Electronic Control of Fluidic Species,” by Link, et al., published as U.S. Patent Application Publication No. 2007/0003442 on Jan. 4, 2007; and International Patent Application No. PCT/US2006/007772, filed Mar. 3, 2006, entitled “Method and Apparatus for Forming Multiple Emulsions,” by Weitz, et al., published as WO 2006/096571 on Sep. 14, 2006.
In some embodiments, an emulsion may be formed by mixing an aqueous or hydrocarbon component with the fluorocarbon component, the mixture having a water or hydrocarbon content of between about 1-40%, in some cases between about 5-25%, and in other cases between about 10-15%. In another embodiment, the emulsion has a water or hydrocarbon content of at least about 20%, at least about 30%, at least about 40%, or at least about 50%, or at least 80%, etc. However, certain embodiments of the invention provide an emulsion within reverse emulsion droplets having a disperse aqueous or lipophilic phase in a continuous, fluorocarbon phase. The criteria in accordance with certain embodiments of the invention that can be used to select suitable discontinuous phases, continuous phases, and surfactants suitable for use in the invention may include (but are not limited to) the description contained herein. A simple, non-limiting screening test to determine whether an aqueous (or hydrocarbon)-in-fluorocarbon emulsion has been created follows: If a water-soluble, fluorocarbon-insoluble dye is added to an emulsion, if the emulsion is an aqueous-in-fluorocarbon emulsion the dye may form a separate phase, since it is not miscible with the continuous, fluorocarbon phase. But in the case of a fluorocarbon-in-aqueous emulsion, the dye may dissolve in the continuous, aqueous phase giving the appearance of disillusion of the dye in the entire mixture. In a second non-limiting screening test, the aqueous phase can be made slightly electrically conductive and, if the emulsion is slightly electrically conductive, then the continuous phase is aqueous, i.e. a fluorocarbon-in-aqueous phase results. If the mixture is not electrically conductive, then an aqueous-in-fluorocarbon emulsion results. As a third non-limiting screening test, if the mixture is optically clear, then very small aqueous aggregates in a fluorocarbon, continuous phase may have been formed.
In one embodiment, emulsions of the invention are prepared using microfluidic systems. For instance, the formation of droplets at intersection 92 of device 90 is shown in
Droplets of varying sizes and volumes may be generated within the microfluidic system. These sizes and volumes can vary depending on factors such as fluid viscosities, infusion rates, and nozzle size/configuration. Droplets may be chosen to have different volumes depending on the particular application. For example, droplets can have volumes of less than 1 μL (microliter), less than 0.1 μL (microliter), less than 10 nL, less than 1 nL, less than 0.1 nL, or less than 10 pL.
Emulsions including a fluorophilic continuous phase may be useful in a variety of fields due to their unique properties, according to another aspect of the invention. For example, one or more of the following advantages may be present in certain emulsions of the invention. A fluorophilic continuous phase may be immiscible with water and hydrocarbons and may allow separation of both hydrophilic and lipophilic compounds that can be contained within the droplets. In this way, cross-contamination of materials, such as proteins or DNA, between droplets may be reduced or prevented. This efficient compartmentalization can be advantageous for drop-based biological and/or chemical assays and the like. In addition, fluorophilic solvents such as fluorocarbons may be chemically inert. Their immiscibility with common organic solvents allows for making droplets or organic solvents that may be used as reactors with volumes typically on the order of picoliters, in certain embodiments of the present invention. In some embodiments, these emulsions can provide the dry environment that is necessary for carrying out water-sensitive chemical reactions. Certain fluorophilic solvents may also have a solubility for gases and can allow transport of gaseous compounds to and from the droplets. For example, biological experiments carried out with cells inside the droplets may benefit from the enhanced transport of oxygen. Microfluidic experiments may also be performed in polydimethylsiloxane (PDMS) or other polymeric devices obtained through the widely applied methods of soft-lithography. For instance, in contrast to mineral and silicone oils, certain fluorophilic solvents such as perfluorinated oils do not swell the rubbers and avoid a number of problems associated therewith.
Accordingly, the droplets and emulsions produced in accordance with various embodiments of the present invention have a variety of uses. For example, in one embodiment, the droplets are used as reaction vessels for carrying out chemical and/or biological reactions within the droplet. Increasing effort is being put into investigating biological systems on very small scales. This involves the observation of cells and their interaction with the environment as well as the investigation of strands of DNA, even of single genes. There are certain advantages to the encapsulation of cells and DNA into aqueous droplets (e.g., a dispersed phase of an emulsion) that are separated from one another with oil (e.g., a continuous phase), as is discussed herein. This is called compartmentalization and it generally allows the screening of much larger numbers of cells or genes at greater rates using much less chemicals than in classical experimental setups, such as Petri-dishes or microtiter plates.
As mentioned above, fluorocarbon oils may be suited as the continuous phase in some of these experiments, as they may reduce cross-contamination, e.g., through the diffusion of hydrophilic or lipophilic biological material from one droplet to another. Furthermore, they may allow for an efficient transport of oxygen in some cases, which is vital to some types of encapsulated cells.
In some applications, a role of the fluorosurfactant may be to ensure colloidal stability of the emulsion upon collision of droplets and/or during incubation in a creamed state. The surfactant may also reduce or prevent biological molecules from adsorbing to the interface of the droplet, which adsorption could disturb their native three-dimensional structure or render them inactive. For example, PEG and other headgroups described herein may reduce or prevent adsorption of certain biological molecules to interfaces and hence may be used for these applications. In fact, a model reaction for the in-vitro translation and transcription of specific DNA sequences into fluorescent proteins inside emulsion droplets demonstrates the potential of various fluorosurfactants of the invention (e.g., non-ionic fluorosurfactants) for biological in-vitro experiments.
In some embodiments, such as certain bioapplications that involve incubation at elevated temperatures, fluorosurfactants of the invention may comprise a water soluble fluorosurfactant such as the Zonyl compounds sold by DuPont. Some such compounds have relatively long PEG-units (400 g/mol and greater) and a short fluorotelomer. The compounds may be added to the water phase, but in some cases, they may be added to the fluorophilic phase. Such compounds can, in some embodiments, help keep biomolecules, such as BSA, from adsorbing to the droplet interface. They may aid the emulsification process and may also help in stabilizing the droplets during emulsification in some cases.
In other embodiments, a high-molecular weight surfactant may be inserted into the adsorbed surfactant layer after emulsification. The high-molecular weight surfactant may increase long term stabilization of the emulsion. In some cases, such a surfactant can be mixed with a fluorosurfactant before dissolving it in the fluorophilic continuous phase. Alternatively, the surfactant can be added to the fluorophilic continuous phase separately or added after emulsification of an emulsion.
In some embodiments, in-vitro translation can be performed using emulsions described herein. A gene (e.g., a DNA sequence) can be contained in the droplets together with all the chemicals required for transcribing it into RNA and translating that into a protein. Such chemicals are known to those of ordinary skill in the art, and may be readily obtained commercially. This protein may be fluorescent itself (e.g., green fluorescent protein, GFP) or enzymatically active, catalyzing a reaction that yields a fluorescent product. A fluorescent product may not be formed if adsorption to the interfaces of one of the compounds takes place, disturbing the 3D-structure of the bioactive compound and rendering it inactive. Fluorescence indicates that the reactions took place and, therefore, that adsorption had been prevented successfully. This is demonstrated in the right-most vessel of an example as shown in
In certain embodiments, the discontinuous aqueous and/or lipophilic phase of a droplet/emulsion may include one or more physiologically acceptable reagents. The reagents may be dissolved or suspended in the discontinuous phase. In another set of embodiments, the discontinuous aqueous and/or lipophilic phase of a droplet/emulsion may include one or more reagents that can participate in a chemical and/or in a biological reaction of interest. Non-limiting examples of reagents that can be involved in a chemical and/or biological reaction, or other chemical and/or biological process, include: buffers, salts, nutrients, therapeutic agents, drugs, hormones, antibodies, analgesics, anticoagulants, anti-inflammatory compounds, antimicrobial compositions, cytokines, growth factors, interferons, lipids, oligonucleotides polymers, polysaccharides, polypeptides, protease inhibitors, cells, nucleic acids, RNA, DNA, vasoconstrictors or vasodilators, vitamins, minerals, stabilizers and the like. In other embodiments, the discontinuous aqueous and/or lipophilic phase can contain toxins and/or other substances to be tested, assayed, or reacted within the droplet. Accordingly, chemical and/or biological reactions may be performed within droplets of the invention. Because conditions of pH, temperature, reactant concentration, and the like will be adjusted for a particular reaction that is to take place within the disperse phase of the emulsion, in some cases, the surfactant system may be tailored so as to preserve the emulsion under these conditions.
As specific examples, in one embodiment, a therapeutic agent can be provided in the aqueous phase of a droplet and a reactant introduced into the aqueous phase, optionally via a carrier or by fusion of droplets, and allowed to react inside the droplet. In another embodiment, droplets of the invention can be used as drug-delivery carriers.
The invention also provides, according to another aspect, a technique for controlling the length of assembly of components within droplets. For instance, for polymerization reactions within a droplet, the size of the resulting particle can be controlled by confining the space within which the reaction can occur. The confined space may be defined by the size of the droplets, which can serve as microreactors in some cases. For example, when a polymeric precursor is provided in the discontinuous phase and a reactant is added to the discontinuous phase, the reactant may be allowed to interact with the precursor and cause polymerization, according to certain embodiments. Because of the size of the droplets and the limited amount of discontinuous phase, polymer particle size may be accordingly limited. Smaller particles than the precursor droplets may be prepared, for instance, by diluting the polymer precursor with appropriate solvents. In one particular embodiment, such a reaction can involve the polymerization of monomer units to form polyurethane.
Confining chemical reactions in small volumes is of interest for various reasons. For example, combinatorial approaches to chemical synthesis require that a large number of reactions be performed with slightly different compositions or under systematically changed conditions without using large amounts of chemicals. By screening the resulting products, as discussed herein, optimal reaction conditions can be found or scaled up to commercial scales. The droplets may also serve as suitable containers in some cases. 2) Certain products and intermediates are worth more if they are available as micron or nanometer sized objects (e.g., in lattices). These products span the range of, for example, micron-sized heterogeneous catalysts, crystalline nanoparticles with controlled electronic and photonic properties, polymeric dispersions, fillers used for nanocomposite materials, etc. One way to achieve such confined reactions is to carry them out in the droplets of an emulsion. Emulsions usually include the oil- or solvent phase and a continuous water phase. However, many organic reactions are sensitive to water and an aqueous continuous phase may not be used. Using a fluorophilic continuous phase (e.g., fluorocarbon oils) instead of water as the continuous phase for such systems, as described herein, may be used to circumvent this problem. In some cases, appropriate surfactants may also be added, e.g., to increase stability of the emulsion.
In one particular embodiment, the methods and components described herein can be used to stabilize droplets of organic solvents for combinatorial chemistry. Choosing a suitable combination of inner (e.g., lipophilic/hydrophilic) and outer (e.g., fluorophilic) components may allow for the stabilization of droplets of organic solvents, as required for performing many water-sensitive chemical reactions. For instance, this allows for the facilitated combinatorial screening of chemical reactions by using microfluidic devices. Note that the surfactants containing, e.g., poly(ethylene glycol) as the discontinuous phase as described herein, also can allow stabilization of droplets of a variety of organic solvents (e.g., THF and acetone).
In another embodiment, the methods and components described herein can be used for nanoparticle synthesis. Droplets of organic solvents may be stabilized (as the discontinuous phase) for the production of organic and inorganic micron or sub-micron particles. This makes the synthesis of particles formed from water-sensitive precursors possible, e.g., polyurethanes and polyesters. In another embodiment, crystalline silica and titania particles may be synthesized in water-free reactions. The absence of water, in certain embodiments, offers advantages over sol-gel methods, which may be used to yield entirely amorphous particles.
In one embodiment, particle synthesis can be achieved in a suspension polymerization process in which a liquid precursor is emulsified and then each of the emulsion droplets is converted, e.g., 1:1, yielding a polymer particle. Suspension polymerization is an industrially important method that can form particles with sizes ranging from less than a micron to tens of microns, for example. The suspension polymerization process can be used with a variety of chemical compositions. Suspension polymerization, in some cases, relies on phase separation (and/or chemical compatibility) between the precursor and the continuous emulsion phase.
Non-limiting examples of other materials that can be synthesized using methods described herein include silica, titania, vanadia, zirconia, etc., and combinations thereof, for instance, by sol-gel reactions, to form particles or the like. These and other reactions may be performed, for instance, acid or base-catalyzed. For example, base-catalyzed reactions may yield nanoparticles through nucleation and growth inside the droplets, according to some embodiments. In some cases, the nanoparticles may adhere to one another after solvent evaporation.
In one embodiment, a suitable continuous emulsion phase for polyurethane (PU) suspension polymerization may be perfluorinated oils, as these oils phase may separate from the hydrocarbon-based PU precursors, and may be water-free in some cases. Certain fluorosurfactants of the invention can achieve stabilization of PU droplets, as described herein. Examples of the synthesis of stabilized polyurethane particles and emulsions are described in more detail in the Examples section.
In some embodiments, polyurethanes or other polymer or polymer precursors within a dispersed phase may be modified by adding reagents, such as linking moieties, to the precursor that will be incorporated into the polymer backbone in the course of polymerization. In some cases, the composition of the precursor droplets is the same or similar to that of the precursor before emulsification and does not rely on the diffusion of precursors. This can make suspension polymerization more versatile than techniques such as emulsion or precipitation polymerization according to some embodiments. As another example, step growth polymerization reactions, including the polyaddition of polyurethanes, may be used. Such reactions can involve homogeneous distribution of added reagents along the polymer chains. In some embodiments, this approach is used to incorporate fluorescent dyes in the polyurethane, e.g., through urea links. Other compounds can also be incorporated using this approach.
An example is illustrated in
In another embodiment, cross-linked particles (e.g., cross-linked polyurethane particles) can be formed. Cross-linking may be possible, for example, by replacing a small amount of dialcohol or diisocyanate with a trifunctional reagent, while maintaining a generally stochiometric 1:1 ratio of alcohol and isocyanate groups. Suspension polymerization can produce cross-linked particles in a single polymerization step, in some embodiments. Additionally, if desired, fluorescent labeling may not require an additional processing step. In some embodiments involving the surfactant systems described herein, neither cross-linker molecules nor reactive dyes may significantly change the colloidal stability of these emulsions. Effective cross-linking can be shown by dissolution tests with an appropriate solvent, such as THF. The example as shown in
Certain cross-linked polymer particles synthesized in this way may not have gradients in the cross-linking density. Accordingly, a polymer network having homogeneously distributed cross-links may be produced in some embodiments. In some cases, homogeneously distributed cross-links can be formed using the step polymerization mechanism. Cross-linked, monodisperse, and fluorescently labeled organic microgel particles are shown in the example of
In some embodiments, various mechanical properties of the polymer particle can be tuned by reducing the effective volume fraction of the precursor within a particle. This may be achieved, for example, by diluting the precursor with an inert solvent that can dissolve the precursor as well as the polymer. In this way, suspension polymerization can allow control over the mesh size of a cross-linked polymer particle. Particles (e.g., microgel particles) that were polymerized with small volume fractions of precursor may swell upon addition of a suitable solvent. Depending on the degree of swelling, such particles can exhibit different stiffnesses or other physical properties. For instance,
In some embodiments, particles formed by methods such as those described herein may be used as scaffolds for adding additional coatings or features to the particles. For instance, particles may serve as scaffolds for composite particles; for example, the particle network may be swollen with a precursor of another polymer that is polymerized subsequently. The synthesis of such particles can benefit from the ability to stabilize organic droplets in a wide range of polarities, including DMSO, with PEG-based non-ionic fluorosurfactants, or other surfactants described herein.
In some cases, porous structures can be formed using methods described herein. For example, mesoporous structures may be formed inside a droplet through templated synthesis (e.g. through self assembly of block-copolymers). As described herein, fluorosurfactants including non-ionic fluorosurfactants can be used in a wide range of particle syntheses through conversion of precursor emulsion droplets. The immiscibility of fluorocarbon oils with hydrocarbons of many different polarities may allow the formation of droplets having certain hydrocarbon disperse phases that cannot be formed in certain other continuous phases. In addition, hydrophobic chemicals may be emulsified in a dry environment allowing water-sensitive reactions to be performed. While the benefits of such a system has been demonstrated in conjunction with the suspension polymerization of polyurethane particles, it should be understood that the invention is not limited in this respect and that articles and methods described herein can be used to form particles having other compositions in other embodiments. Furthermore, in a simple one-step process, solid particles can be made with the option of cross-linking, grafting fluorescent dyes or other moieties to the polymer network and controlling its mesh-size by applying an inert solvent during polymerization. This process may be used for producing a variety of other polymers that rely on water-sensitive syntheses, such as, for example, polyesters and polyureas, and for producing transition metal alkoxides. Organic droplets that allow for water sensitive reactions are useful in fields other than particle synthesis, such as heterogeneous catalysis or screening applications in drop-based microfluidics. In some instances, methods and components described herein can be used for reactions with gases. For example, reactions of liquids with gaseous reactants (such as hydrogenations and oxidations using hydrogen and oxygen, respectively) may be carried out very efficiently. Fluorocarbon oils can dissolve around an order of magnitude more gases than water and conventional solvents in some cases. The high gas solubility and the large interfacial area of emulsions comprising a fluorophilic continuous phase may enhance the transport of the gaseous reactant(s) to the reaction loci inside the droplets, which often is the rate limiting step in industrial processes. Transport across the interface can be facilitated by the enormous specific area of emulsion droplets. Thus, articles and methods described herein can be used for chemical heterophase reactions. Applications of water-sensitive catalysts are also possible.
In another embodiment, methods and components described herein can be used for studies of liquid crystalline behavior. Lyophilic mesophases of fluorocarbon surfactants are interesting because of their particular contrast in hydrophilicity. The synthetic routes described herein may be applied for the production of such materials.
In another embodiment, methods and components described herein can be used for modifying the wetting behavior of microchannels. Chemicals used as wetting agents are typically chemically inert and capable of changing the wetting properties of fluorous and silicone elastomers in a reversible manner. By adsorbing them onto a solid-liquid interface, they provide a surface that changes the wetting behavior in a reversible manner. For instance, silicone rubbers may be wet by fluorocarbon oils and fluorous rubbers may be wet by hydrocarbon oils and organic solvents.
In certain embodiments, colloidal stabilization of droplets can be achieved while preventing the adsorption of biological materials. For instance, using passivating agents such as PEGs as headgroups of surfactants, droplets containing solutions of biomolecules may be stabilized in fluorocarbon oils while preventing the adsorption of DNA, RNA, proteins or other materials to the interfaces. In some embodiments, cells may be encapsulated in aqueous droplets without adsorbing to the droplet interfaces. They may therefore be investigated as if they were floating in an aqueous bulk medium.
Some embodiments of the invention involve mixtures of more than one different surfactant. The combination of surfactants synthesized using methods described herein with similar or different geometry and different molecular weight allows for efficient emulsification. For instance, low molecular weight surfactants may lower the interfacial tension rapidly, while high molecular weight surfactants may provide long term stability. Such considerations are crucial to the velocity of an emulsification process, e.g., in industry.
The methods and components described herein can also be used for studies of liquid crystalline behavior. For example, lyophilic mesophases of fluorocarbon surfactants are interesting because of their large contrast in hydrophilicity. The synthetic routes described herein may be applied for the production of such materials, e.g., as previously discussed.
In some embodiments, surfactants having the following structures are contemplated:
1. PFPE (KRYTOX®)-PEG-PFPE (KRYTOX®), where PFPE is perfluoropolyether:
F—[CF(CF3)CF2O]x—CF(CF3)CONH—(CH2CH2O)yCH2CH2—NHCOCF(CF3)—[OCF2CF(CF3)]x—F
with various chain lengths, for example:
2. PFPE (KRYTOX®)-PolyTHF-PFPE (KRYTOX®), where PFPE is perfluoropolyether:
F—[CF(CF3)CF2O]x—CF(CF3)CONH—(CH2CH2CH2CH2O)yCH2CH2CH2CH2—NHCOCF(CF3)—[OCF2CF(CF3)]x-F
3. PFPE—alkyls:
F—[CF(CF3)CF2O]n—CF(CF3)CONH—(CH2)n—H
4. Phytol may be used in some cases. Phytol is a branched C18 hydrocarbon block that may be linked with an ether bond:
(CH3)2HC—(CH2)3CH(CH3)—(CH2)3CH(CH3)—(CH2)3C(CH3)═CH—O—CH2CF(CF3)—[OCF2CF(CF3)n]—F
5. PFPE—sugars:
For example, sorbitol can be linked to KRYTOX® with an ether bond:
H—[CH(OH)]5—CH2—O—CH2CF(CF3)—[OCF2CF(CF3)]n—F
6. Glucosamine is a glucose derivative that may be linked to KRYTOX® with an amide bond:
glucose-NHCOCF(CF3)—[OCF2CF(CF3)]n-F
Glucose is a 6 membered heterocycle of the sum formula C6O6H12, in which the first and the fifth carbon atom are linked to the oxygen atom of the ring. Glucosamine carries an amine group instead of the OH-group at the second carbon atom.
The following documents are incorporated herein by reference in their entirety: U.S. Provisional Patent Application Ser. No. 60/392,195, filed Jun. 28, 2002, entitled “Multiphase Microfluidic System and Method,” by Stone, et al.; U.S. Provisional Patent Application Ser. No. 60/424,042, filed Nov. 5, 2002, entitled “Method and Apparatus for Fluid Dispersion,” by Link, et al.; U.S. Provisional Patent Application Ser. No. 60/461,954, filed Apr. 10, 2003, entitled “Formation and Control of Fluidic Species,” by Link, et al.; U.S. Provisional Patent Application Ser. No. 60/498,091, filed Aug. 27, 2003, entitled “Electronic Control of Fluidic Species,” by Link, et al.; U.S. patent application Ser. No. 08/131,841, filed Oct. 4, 1993, entitled “Formation of Microstamped Patterns on Surfaces and Derivative Articles,” by Kumar, et al., now U.S. Pat. No. 5,512,131, issued Apr. 30, 1996; International Patent Application No. PCT/US96/03073, filed Mar. 1, 1996, entitled “Microcontact Printing on Surfaces and Derivative Articles,” by Whitesides, et al., published as WO 96/29629 on Jun. 26, 1996; U.S. patent application Ser. No. 09/004,583, filed Jan. 8, 1998, entitled “Method of Forming Articles Including Waveguides via Capillary Micromolding and Microtransfer Molding,” by Kim, et al., now U.S. Pat. No. 6,355,198, issued Mar. 12, 2002; International Patent Application No. PCT/US01/17246, filed May 25, 2001, entitled “Patterning of Surfaces Utilizing Microfluidic Stamps Including Three-Dimensionally Arrayed Channel Networks,” by Anderson, et al., published as Publication No. WO 01/89788 on Nov. 29, 2001; International Patent Application No. PCT/US01/46181, filed May 25, 2001, entitled “Methods and Compositions for Encapsulating Active Agents,” by Weitz, et al., published as Publication No. WO 02/47665 on Jun. 20, 2002; International Patent Application No. PCT/US02/23462, filed Jul. 24, 2002, entitled “Laminar Mixing Apparatus and Methods,” by Stroock, et al., published as WO 03/011443 on Feb. 13, 2003; and International Patent Application No. PCT/US03/20542, filed Jun. 30, 2003, entitled “Method and Apparatus for Fluid Dispersion,” by Stone, et al., published as Publication No. WO 2004/002627 on Jan. 8, 2004. Also incorporated herein by reference is U.S. patent application Ser. No. 11/246,911, filed Oct. 7, 2005, entitled “Formation and Control of Fluidic Species,” by Link, et al.; U.S. patent application Ser. No. 11/024,228, filed Dec. 28, 2004, entitled “Method and Apparatus for Fluid Dispersion,” by Stone, et al.; International Patent Application No. PCT/US2006/007772, filed Mar. 3, 2006, entitled “Method and Apparatus for Forming Multiple Emulsions,” by Weitz, et al.; and U.S. patent application Ser. No. 11/360,845, filed Feb. 23, 2006, entitled “Electronic Control of Fluidic Species,” by Link, et al.
The following applications are each incorporated herein by reference: U.S. Provisional Patent Application Ser. No. 60/659,045, filed Mar. 4, 2005, by Weitz, et al.; U.S. Provisional Patent Application Ser. No. 60/498,091, filed Aug. 27, 2003, by Link, et al.; U.S. Provisional Patent Application Ser. No. 60/392,195, filed Jun. 28, 2002, by Stone, et al.; U.S. Provisional Patent Application Ser. No. 60/424,042, filed Nov. 5, 2002, by Link, et al.; U.S. Pat. No. 5,512,131, issued Apr. 30, 1996 to Kumar, et al.; International Patent Publication WO 96/29629, published Jun. 26, 1996 by Whitesides, et al.; U.S. Pat. No. 6,355,198, issued Mar. 12, 2002 to Kim, et al.; International Patent Application Serial No.: PCT/US01/16973, filed May 25, 2001 by Anderson, et al., published as WO 01/89787 on Nov. 29, 2001; International Patent Application Serial No. PCT/US03/20542, filed Jun. 30, 2003 by Stone, et al., published as WO 2004/002627 on Jan. 8, 2004; International Patent Application Serial No. PCT/US2004/010903, filed Apr. 9, 2004 by Link, et al.; U.S. Provisional Patent Application Ser. No. 60/461,954, filed Apr. 10, 2003, by Link, et al.; International Patent Application Serial No. PCT/US2004/027912, filed Aug. 27, 2004, by Link, et al.; U.S. Provisional Patent Application Ser. No. 60/659,046, filed Mar. 4, 2005, entitled “Systems and Methods of Forming Particles,” by Garstecki, et al.; and a U.S. utility patent application, entitled “Systems and Methods of Forming Particles,” by Garstecki, et al., filed on even date herewith.
The following examples are intended to illustrate certain embodiments of the present invention, but are not to be construed as limiting and do not exemplify the full scope of the invention.
The following is an example of a procedure for forming an amide bond between a headgroup and a fluorophilic component of a surfactant:
Chemicals:
This example illustrates the following procedure for forming an ester bond between a headgroup and a fluorophilic component of a surfactant:
Chemicals:
For testing emulsion stabilities of the emulsions shown in Table 1, the emulsions were formulated containing Fluorinert Electronic Liquid FC 40 fluorocarbon oil (available from 3M) with 1.6 wt % of the respective fluorosurfactant. FC 40 is a perfluoro compound with primarily compounds with 12 carbons, with the hydrogen replace by F. 10% v/v with respect to the fluorocarbon phase and the alcohol was added. Emulsification was carried out by first turning over the vessel, then shaking it by hand for 15 s and finally shaking it vigorously by hitting the edge of a table for an additional 15 s and for an additional minute, if emulsification was insufficient.
This example describes the synthesis of polyurethane particles using articles and methods described herein, according to one embodiment of the invention. In this example, the emulsions were formed by shaking the precursor and a fluorocarbon oil in the presence of fluorosurfactant.
Polyurethane latexes were synthesized from a stochiometric ratio of polyethylene glycol (a dialcohol) of a molecular weight of about 200 g/mol with hexanediisocyanate to form urethane bonds. This was emulsified in a fluorocarbon phase comprising the oil FC 40 and 1.6 wt % of the surfactant E2K 0420.
Emulsification of the polyurethane precursor yielded separate droplets with a similar chemical composition as the precursor liquid. Crude emulsions were obtained by shaking up the precursor and a fluorocarbon oil in the presence of fluorosurfactant, yielding a polydisperse precursor emulsion.
The resulting rubbery polymer resembled soft blocks of Spandex. At high degrees of polymerization, the liquid precursor became solid. Therefore, the mechanical properties of the product was used as a measure of conversion.
One way to confirm that the use of a continuous fluorocarbon phase results in a high degrees of polymerization or solid particles is by removing the continuous fluorocarbon phase through evaporation. Only solid particles have the mechanical rigidity to resist coalescence and retain their spherical shape in spite of capillary forces. This was tested with a crude polydisperse precursor emulsion obtained by shaking.
An additional probe for conversion is the presence of fluorescein, a fluorescent dye added to the reaction mixture prior to emulsification. Fluorescein is quenched in the presence of isocyanates and does not fluoresce. Fluorescence reappears, however, as the isocyanate is used up in the course of the reaction. Cross-polarization microscopy (
This example describes the synthesis of polyurethane particles using articles and methods as described herein in various embodiments of the present invention. In this example, the emulsions were formed using flow focusing techniques.
Polyurethane latexes were synthesized from a generally stochiometric ratio of polyethylene glycol (a dialcohol) of a molecular weight of 200 g/mol with hexanediisocyanate to form urethane bonds. This was emulsified in a fluorocarbon phase comprising the oil FC 40 and 1.6 wt % of the surfactant E2K 0420.
Emulsions with control over the droplet size were obtained by hydrodynamic flow focusing in microfluidic devices. For the rapid prototyping of such devices, soft lithography was used to form devices in polydimethylsiloxane (PDMS). Monodisperse emulsion droplets were formed when co-flowing streams of the continuous and of the precursor phase were forced into one microfluidic output channel. Surface tension caused the disperse stream to break up into discrete droplets. A surfactant that is dissolved in the continuous phase adsorbed to the newly formed interfaces of the droplets, thereby stabilizing the droplets against coalescence.
These results demonstrate that control over the particle size is possible by combining microfluidic emulsification with suspension polymerization. Moreover, it shows that the applied surfactant allowed for emulsification through hydrodynamic flow focusing and that it stabilized the fluorocarbon based emulsions sufficiently against coalescence throughout the polymerization and even at elevated temperature.
This example illustrates the synthesis of certain surfactants of the present invention, specifically AEH12, AEH14, AEH19, AEH22, AEH23, AEH100, AEH101, AEH102, AEH103, AEH104, AEH105, AEH106, or AEH107.
The perfluoroether and Krytox FS(H) were purchased from Dupont. The PEG was PEG monomethoxy with a molar mass of 750 g/mol (purchased from Sigma-Aldrich). All other chemicals were purchased from Sigma-Aldrich.
Thin-layer chromatography (TLC) analyses were performed on RP-18 silica gel F-254 plates (Merck). Silica gel (70-230 mesh, Merck) was used for column chromatography.
Synthesis of AEH12 head group. 3.14 g of 2-phenylethanol (20.45 mmol) and 7.1 ml of triethylamine (2.5 eq) were dissolved in 50 ml of dry tetrahydrofuran (THF) and added dropwise, at 0° C. and under anhydrous nitrogen, to 3.14 g of phosphorus oxytrichloride (20.45 mmol) in 100 ml of THF. The mixture was stirred at 0° C. for 3 hours. A solution of 3.56 g morpholine (40.90 mmol) and 14.2 ml triethylamine (2.5 eq) in 50 ml of THF was then added dropwise to the stirred reaction mixture, which was maintained free of oxygen and cooled with an ice-bath. After stirring for 18 hours the mixture was allowed to warm to room temperature, triethylamine hydrochloride was filtered off, and the solvent and excess of amine were removed under vacuum. The clear oily residue was dried under reduced pressure and purified by chromatography (ethyl acetate/methanol 9:1). The yield of this reaction was 70% (P,P-dimorpholino P-(2-phenylethyloxyd) phosphate). Purity was determined by TLC (Rf value: 0.7, ethyl acetate/methanol 9:1).
Synthesis of other head groups. The same procedure was applied for the synthesis of the other head groups, substituting with the appropriate components where necessary. Following synthesis, the structures of some of the head groups were verified by 1H nuclear magnetic resonance (NMR). NMR spectra for AEH12, AEH14, and AEH19 were collected using a Bruker AC 400 spectrometer and are shown in
Preparation of the acyl chloride of Krytox FS(H). The acyl chloride of Krytox FS(H) was prepared by adding an excess of thionyl chloride (4 equivalents) under an anhydrous atmosphere and vigorously mixing for 1 hour. After this time, the excess of thionyl chloride was removed under vacuum.
Grafting the acyl chloride of Krytox FS(H) to the P,P-dimorpholino P-(2-phenylethyloxyd) phosphate. The acyl chloride of Krytox FS(H) tail was grafted to the P,P-dimorpholino P-(2-phenylethyloxyd) phosphate head group using a Friedel-Craft reaction. A solution of 0.78 g of the P,P-dimorpholino P-(2-phenylethyloxyd) phosphate head group (0.68 mmol) and 0.11 g (1.2 eq) of aluminium trichloride were mixed in 50 ml of THF and stirred under reflux for 1 hour. Then, under vigorous agitation, 2.27 g of the acyl chloride of Krytox FS(H) was then added and the reaction mixture was stirred under reflux overnight.
The THF was removed under vacuum by rotary evaporation. The residual white viscous mixture was resuspended in Fluorinert Electronic Liquid FC-3283 and filtered on Celite 545. The celite was washed with 200 ml of FC-3283 to recover all of the surfactant. The filtrate was then distilled under vacuum by rotary evaporation to remove the FC-3283 and recover the final product: AEH12.
Grafting the acyl chloride of Krytox FS(H) to the other head groups. The same procedure was applied for the synthesis of all the other surfactants, substituting with the appropriate components where necessary.
Characterization of the surfactants: Emulsion stability. Water-in-oil (w/o) emulsions were generated in a microfluidics device (a “droplet-former”) using a flow-focusing geometry to form the droplets. An aqueous stream was infused from one channel through a narrow constriction with oil streams on either side hydrodynamically-focusing the water stream. Different combinations of flow rates and/or device geometries generated steady streams of uniform droplets of water in oil with volumes in the range of 10-500 pl.
The oil phase in each experiment was formed from the fluorocarbon oil Fluorinert Electronic Liquid FC-40 (manufactured by 3M) containing the dissolved surfactant of interest. The aqueous phase was phosphate-buffered saline (PBS).
To determine the stability of each emulsion, the emulsion was collected in a Hamilton glass syringe and then reloaded into the droplet-forming device using the same oil phase to space out the droplets. An emulsion was considered stable in this example if more than 99% of the droplets were not coalesced on reloading.
Results. The following surfactants generated stable w/o emulsions using the methodology described above when the surfactant concentration was in the range 0.5-2.5% (w/w): AEH12, AE14, and AEH19. The other surfactants were not tested in this example.
Compatibility with mammalian cells. 100 microliters of FC-40 oil containing a surfactant of interest were pipetted into the well of a clear 96-well plate. 100 microliters of a cell suspension containing 1.5×104 cells in Dulbecco's Modified Eagle's Medium supplemented with 10% (v/v) fetal bovine serum were then layered on top of the oil in each well. The cells were detached human HEK 293T expressing green fluorescent protein (GFP). After 24 hours the cells were examined by bright-field and fluorescence microscopy.
After 24 hours, bright-field microscopy revealed that the cells seeded directly on the well bottom (a control), on FC-40 alone, and on FC-40 containing AEH12 were healthy (
Compatibility with in vitro transcription-translation. An in vitro transcription-translation reaction was prepared by adding 5 nM of “T7 promoter-evolved β-galactosidase (EBG) Class IV-T7 terminator” DNA fragments and 500 micromolar fluorescein di-β-D-galactopyranoside (FDG) to a Novagen EcoPro T7 transcription-translation reaction (EMD Biosciences). A control reaction containing no DNA fragments was prepared as well. Each reaction was emulsified in FC-40 containing 2.5% (w/w) AEH19 using a droplet-forming device. The droplets generated in each case were 10 pl in volume. Each emulsion was collected and incubated at 30° C. for 1.5 hours and then reloaded into the droplet-forming device. Several thousand droplets from each emulsion were stimulated with 488 nm-wavelength laser light and their emissions in the fluorescein channel were measured.
After 1.5 hours at 30° C. it was observed that the droplets containing the EBG Class IV genes exhibited much greater fluorescence than droplets containing no genes per droplet (
Compatibility with purified enzymes. An enzymatic reaction containing the following components was prepared: 50 mM Tris HCl pH 8, 10 mM calcium chloride, 10 mM ethylamine, 200 micromolar phenazine methosulphate (PMS), 100 micromolar resazurin, 10 micromolar pyrroloquinoline quinone (PQQ), and alcohol dehydrogenase (ADH) protein in 0.05% (v/v) ethanol. For each surfactant, 200 microliters of this aqueous solution were emulsified in 200 microliters of FC-40 containing the surfactant of interest by vigorously shaking for 1 minute. 50 microliters of each emulsion was transferred to the well of a black 96-well plate. Every 4 seconds, the wells of the plate were stimulated with 540 nm-wavelength light and the resulting emissions at 590 nm-wavelength were measured.
The enzymatic reaction described above should generate fluorescent resorufin by reducing the non-fluorescent resazurin when the ADH is active. The reaction profiles for the different surfactants revealed that emulsification of the reaction using the surfactants AEH12, AEH14, and AEH19 was not detrimental to its progress (
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.
This application is a continuation of U.S. application Ser. No. 12/310,048, filed Feb. 9, 2009, which is a 35 U.S.C. §371 National Phase Application of PCT/US2007/017617, filed Aug. 7, 2007, which claims priority to, and the benefit of, U.S. Provisional Patent Application No. 60/836,455, filed Aug. 7, 2006. Each of these applications is incorporated herein by reference in its entirety.
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| Number | Date | Country | |
|---|---|---|---|
| 20150217246 A1 | Aug 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60836455 | Aug 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12310048 | US | |
| Child | 14687388 | US |
