Grafted siloxane surfactants

Abstract

System, including methods and compositions, for making and using emulsions that include a silicone oil and a silicone surfactant. The emulsions may include aqueous droplets disposed in a continuous phase that includes a silicone oil and a silicone surfactant. The aqueous droplets may contain an analyte, optionally at partial occupancy, and / or a luminescent (e.g., photoluminescent) reporter. An assay of the analyte may be performed with the droplets. In some cases, signals may be detected from the droplets, and a characteristic of the analyte, such as an analyte level or activity, may be determined based on the signals. The silicone surfactants of the disclosure are heterografted surfactant block copolymers comprising a polysiloxane backbone, and a plurality of hydrophilic and hydrophobic side chains grafted to said backbone.

Description

GRAFTED SILOXANE SURFACTANTS FIELD OF THE INVENTION

[0001] Embodiments of the invention relate to siloxane surfactants and their use in methods of analysis for biological assays. BACKGROUND OF THE INVENTION

[0002] An emulsion is a mixture of two or more liquids that are normally immiscible. Typically, one liquid, referred to as the dispersed phase, is dispersed into the other liquid, referred to as the continuous phase. Emulsions have had substantial impact in revolutionizing high-throughput assays, as emulsification techniques can create thousands, millions, or even billions of discrete aqueous droplets from a single sample for single cell or isolated nucleic acid analysis.

[0003] The resulting aqueous droplets, due to their isolation from each other within an immiscible continuous phase, can function as independent reaction chambers for biochemical reactions. Small aqueous samples can be partitioned into a vast number of droplets. In this way, individual biological components (e.g., cells, nucleic acids, proteins, etc.) can be manipulated, processed, and studied discretely and individually in a massively high-throughput manner.

[0004] Emulsions for assays are often formulated to have a continuous phase that includes a perfluorinated oil and a perfluorinated surfactant. The use of such a fluorophilic continuous phase around droplets can provide a permissive surrounding environment for certain biochemical reactions, such as PCR amplification, to occur in the droplets.

[0005] However, emulsions containing perfluorinated oil can suffer from various problems. For example, aqueous droplets are typically buoyant in perfluorinated oil, which can create problems during droplet manipulation. The buoyant droplets may be more likely to be damaged by exposure to air above the emulsion, particularly when heated. Also, such emulsions may require removal of excess oil below the droplets to position the droplets closer to a heat source. Furthermore, the droplets may be difficult to stabilize for heat treatment, such as thermocycling to promote amplification, and may be difficult to preserve for later detection.

[0006] Additionally, perfluorinated oils and surfactants have recently come under scrutiny as a source of halogens which have detrimental effects on the environment. Some locales have even recently sought to limit or ban the use of perfluorinated materials in laboratories.

[0007] Accordingly, there is a need in the art for novel surfactants for biological assays which do not rely on perfluoro- chemistries. SUMMARY OF THE INVENTION

[0008] The present disclosure provides systems, including methods and compositions, for making and using emulsions that comprise a silicone oil and a silicone surfactant. In a preferred embodiment, emulsions of the invention may include aqueous droplets disposed in a continuous phase that includes a silicone oil and a silicone surfactant. In some embodiments, the aqueous droplets contain an analyte, optionally at partial occupancy, and / or a luminescent (e.g., photoluminescent) reporter. An assay to detect the analyte may be performed with the droplets. In some cases, signals may be detected from the droplets, and a characteristic of the analyte, such as an analyte level or activity, may be determined based on the signals. In other embodiments, the emulsion is broken and contents of droplets are pooled for analyte detection.

[0009] In some embodiments, the silicone surfactants of the disclosure are heterografted block copolymer surfactants comprising a polysiloxane backbone, and a plurality of hydrophilic and hydrophobic side chains grafted to the backbone.

[0010] In some embodiments, the silicone surfactants are characterized as a di-block copolymer comprising a siloxane backbone with at least a first segment (A) and a second segment (B). Each segment is comprised of repeating backbone units (-Si-O-).

[0011] In some embodiments, the first segment may contain in a range of 2-100 backbone units. Each of the backbone units of the first segment is grafted with either a hydrophobic side chain or a hydrophilic side chain. In some embodiments, a backbone unit may also be grafted with an alkyl group comprising 1-18 carbon atoms or an aryl group. In some embodiments, the distribution of the hydrophobic and hydrophilic side chains is random within the first segment.

[0012] In some embodiments, the second segment may contain in a range of 1-1000 backbone units. Each of the backbone units of the second segment are grafted with an alkyl group comprising 1-18 carbon atoms, preferably 1-6 carbon atoms, or aryl group. In some embodiments, each backbone unit of second segment is grafted with the same group.

[0013] In some embodiments, the hydrophobic side chains are selected from alkyl capped polysiloxanes. In some embodiments, the polysiloxane of the hydrophobic side chain is apolydimethylsiloxane (PDMS). The alkyl capped polysiloxane may contain 1, 2, or 3 terminal groups (-X) each bound to a silicon atom.

[0014] In some embodiments, the hydrophobic side chain is symmetrical. Stated differently, in those embodiments where the alkyl capped polysiloxane contains 2 or 3 terminal groups, the silicon atom grafted to the polysiloxane backbone may be linked to the terminal group by polysiloxane segments of approximately equal length. In contrast, in some embodiments, the length of the polysiloxane segments attaching the grafted silicon atom to the terminal groups are different (i.e., an asymmetrical hydrophobic side chain). The length of the polysiloxane segments between the grafted silicon atom and the terminal groups is in a range of 1-1000 repeating units.

[0015] In some embodiments, the terminal groups are independently selected from alkyl groups comprising 1-18 carbon atoms, optionally substituted with one or more phenyl, and phenyl. In some embodiments, the alkyl groups are linear or branched. In specific embodiments, the terminal groups are independently selected from butyl (-C4H9), octyl (-C8H17), octadecyl (-C18H37), phenethyl ((C6H5)C2H4-), and phenhexyl ((C6H5)C6H12-).

[0016] In some embodiments, the hydrophilic side chains comprise oxygen atoms. Exemplary groups include hydrophilic polymers comprising one or more of polyethylene glycol (PEG), polypropylene glycols (PPG), polyglycerins, polyethyleneimines (PEI). In some embodiments, the hydrophilic polymers comprise block copolymers of more than one of the species identified above. The hydrophilic polymers are terminated with hydroxyl (-OH), methoxy (-OCH3), or ethoxy (-OC2H5) groups.

[0017] In some embodiments, the ratio of hydrophobic to hydrophilic side chains is controlled. In some embodiments, the ratio is in a range of 0.01 to 100, or in a range of 0.1-10. In some embodiments, the ratio is selected from the group consisting of 0.6, 1.0, 1.67, 2 and 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG.1 depicts fluorescence results having poor quality due to unreliable droplet formation.

[0019] FIG.2 depicts fluorescence results having improved quality and reliable droplet formation.DETAILED DESCRIPTION OF THE INVENTION

[0020] The present disclosure provides systems, including methods and compositions, for making and using emulsions that include a silicone oil and a silicone surfactant. The emulsions may include aqueous droplets disposed in a continuous phase that includes a silicone oil and a silicone surfactant. In some embodiments, the aqueous droplets contain an analyte, optionally at partial occupancy, and / or a luminescent (e.g., photoluminescent) reporter. An assay of the analyte may be performed within the droplets. In some cases, signals may be detected from the droplets, and a characteristic of the analyte, such as an analyte level or activity, may be determined based on the signals.

[0021] Some embodiments of the disclosure relate to silicone surfactants. As used in this regard, a “silicone” comprises repeating units of (Si-O). A silicone oil comprises a plurality of these units, with each silicon atom similarly bonded to hydrogen or a small alkyl group (e.g., PDMS). Given the relatively easy control over the length of these molecules, they can be used in a variety of applications and formulated to various viscosities.

[0022] This disclosure more particularly relates to silicone surfactants. As used in this regard, silicone surfactants are silicone oils which possess both hydrophilic and hydrophobic regions. Accordingly, they contain one or more hydrophobic moiety and one or more hydrophilic moiety. As the backbone of a silicone oil is known to possess some hydrophobic nature, a silicon surfactant likely contains at least one hydrophilic moiety apart from the backbone chain.

[0023] The silicone surfactants of the disclosure are heterografted block copolymer surfactants. As used in this regard, a block copolymer comprises at least two distinct regions (e.g., di-block, tri-block, etc.) of the polymer. These regions are separate and do not intermix. The term “heterografted” is used to indicate that more than one sidechain (or “graft”) is present within a given region.

[0024] Specifically, embodiments of the disclosure comprise a polysiloxane backbone and a plurality of hydrophilic and hydrophobic side chains grafted to the backbone.

[0025] Accordingly, some embodiments of the disclosure provide silicone surfactants that are characterized as a di-block copolymer. These copolymers comprise a siloxane backbone with at least a first segment (A) and a second segment (B). Each segment is comprised of repeating backbone units (-Si-O-) which are grafted.

[0026] The silicone surfactants of the disclosure may correspond to Formula I (I)

[0027] As shown above, R1,2

[0028] In some embodiments, units, or in a range of 2-50 backbone units, or in a range of 2-20 backbone units, or in a range of 5-10 backbone units, or in a range of 6-9 backbone units. In the context of Formula I, the number of backbone units in the first segment corresponds to a.

[0029] As illustrated above, each of the backbone units of the first segment is grafted with either a hydrophobic side chain (R1) or a hydrophilic side chain (R2). In some embodiments, the distribution of the hydrophobic and hydrophilic side chains is random within the first segment.

[0030] In some embodiments, the ratio of hydrophobic to hydrophilic side chains is controlled. In some embodiments, the ratio is in a range of 0.01 to 100, or in a range of 0.1-10. In some embodiments, the ratio is selected from the group consisting of 0.6, 1.0, 1.67, 2 and 5.

[0031] The backbone units of the first segment are also grafted (R3) with an alkyl group comprising 1-18 carbon atoms or an aryl group. In some embodiments, the backbone units of the first segment are grafted with a methyl group (R3is methyl). In some embodiments, R3is selected from aliphatic chains from about 1 to about 18 carbons, about 1 to about 6 carbons, about 1 to about 4 carbons, about a to about 2 carbons, phenyls or a mixture thereof.

[0032] In some embodiments, the second segment may contain in a range of 1-1000 backbone units, or in a range of 1-100 backbone units, or in a range of 1-50 backbone units, or in a range of 5-100 backbone units, or in a range of 5-50 backbone units, or in a range of 10-50 backbone units, or in a range of 10-30 backbone units, or in a range of 11-28 backbone units, or in a range of 12-27 backbone units. Each of the backbone units of the second segment are grafted with two groups (R3) independently selected from alkyl groups comprising 1-18 carbon atoms, 1-8 carbon atoms, 1-4 carbon atoms, 1-2 carbon atoms, or an aryl group. In some embodiments, each backbone unit of second segment is grafted with the same group. In some embodiments, each backbone unit of second segment is grafted with two methyl groups.

[0033] In some embodiments, the hydrophobic side chains are selected from alkyl capped polysiloxanes. Exemplary alkyl capped siloxanes are shown below. As illustrated, the terminal alkyl groups are designated by X and the bond interrupted by the wavy line connects to a silicon atom of the silicone backbone.

[0034] In some embodiments,side chain is a polydimethylsiloxane (PDMS, as shown above). The alkyl capped polysiloxane may contain 1, 2, or 3 terminal alkyl groups (-X).

[0035] In some embodiments, the hydrophobic side chain is symmetrical. Stated differently, in those embodiments where the alkyl capped polysiloxane contains 2 or 3 terminal groups, the silicon atom grafted to the polysiloxane backbone may be linked to the terminal group by polysiloxane segments of approximately equal length. With reference to the structures above, for symmetrical hydrophobic side chains e and f would be approximately equal, or g, h, and i would be approximately equal.

[0036] In contrast, in some embodiments, the length of the polysiloxane segments attaching the grafted silicon atom to the terminal groups are different (i.e., an asymmetrical hydrophobic side chain). The length of the polysiloxane segments between the grafted silicon atom and the terminal groups is in a range of 1-1000 repeating units, or in a range of 2-100 repeating units, or in a range of 2-50 repeating units, or in a range of 2-20 repeating units, or in a range of 5-10 repeating units, or in a range of 5-8 repeating units, or in a range of 6-7 repeating units. Accordingly, in the context of the structures above, d-i are independently selected from the ranges above.

[0037] In some embodiments, the terminal groups (X) are independently selected from alkyl groups comprising 1-18 carbon atoms, optionally substituted with one or more phenyl, and phenyl. In some embodiments, the alkyl groups are linear or branched. In specific embodiments, the terminal groups (X) are independently selected from butyl (-C4H9), octyl (- C8H17), octadecyl (-C18H37), phenethyl ((C6H5)C2H4-), and phenhexyl ((C6H5)C6H12-).

[0038] In some embodiments, the comprise oxygen atoms. In someembodiments, the hydrophilic side nitrogen atoms. Exemplary groups include hydrophilic polymers comprising one or more of polyethylene glycol (PEG), polypropylene glycols (PPG), polyglycerins, polyethyleneimines (PEI). In some embodiments, the hydrophilic polymers comprise block copolymers of more than one of the species identified above. The hydrophilic polymers are terminated with hydroxyl (-OH), methoxy (-OCH3), or ethoxy (-OC2H5) groups.

[0039] Exemplary hydrophilic side chains are shown below. As illustrated, the terminal groups are designated by Y.

[0040] The length of theof 1-1000 repeating units, or in a range of 2-100 repeating units, or in a range of 2-50 repeating units, or in a range of 2-20 repeating units, or in a range of 5-20 repeating units, or in a range of 8-12 repeating units, or in a range of 10-15 repeating units. Accordingly, in the context of the structures above, j-u are independently selected from the ranges above.

[0041] A composition is also provided. The composition may comprise a continuous phase that includes a silicone oil and a silicone surfactant. In some embodiments, the composition further comprises aqueous droplets disposed in the continuous phase. The droplets may include an analyte at partial occupancy.

[0042] Additional embodiments of the invention relate to methods of performing an assay. In the methods, an emulsion may be formed that includes droplets disposed in a continuous phase. The continuous phase may include a silicone oil and at least one silicone surfactant which aids in droplet formation and stability. Data related to an analyte disposed in the dispersed droplets may be collected.

[0043] The present invention additionally relates to a method for detecting a plurality of targets in a biological sample using digital PCR in microfluidic droplets. The sample may be a human tissue or body fluid. Exemplary body fluids pus, sputum, semen, urine, blood, saliva, and cerebrospinal fluid.

[0044] One or more droplets are formed, each containing a single nucleic acid template and a heterogeneous mixture of primer pairs and probes, each specific for multiple target sites on the template. For example, a first fluid (either continuous, or discontinuous as in droplets) containing a single nucleic acid template (DNA or RNA) is merged with a second fluid (also either continuous, or discontinuous as in droplets) containing a plurality of primer pairs and a plurality of probes, each specific for multiple targets sites on the nucleic acid template to form a droplet containing the single nucleic acid template and a heterogeneous mixture of primer pairs and probes. The second fluid can also contain reagents for conducting a PCR reaction, such as a polymerase and dNTPs.

[0045] Certain members of the plurality of probes include a detectable label. Members of the plurality of probes can each include the same detectable label, or a different detectable label. The detectable label is preferably a fluorescent label. The plurality of probes can include one or more groups of probes at varying concentrations. The one or more groups of probes can include the same detectable label which varies in intensity upon detection, due to the varying probe concentrations.

[0046] The first and second fluids can each be in droplet form. Any technique known in the art for forming droplets may be used with methods of the invention. An exemplary method involves flowing a stream of the sample fluid containing the nucleic acid template such that it intersectstwo opposing streams of flowing carrier fluid. The carrier fluid is immiscible with the sample fluid. Intersection of the sample fluid with the two opposing streams of flowing carrier fluid results in partitioning of the sample fluid into individual sample droplets containing the first fluid. The carrier fluid may be any fluid that is immiscible with the sample fluid. An exemplary carrier fluid is a silicone oil. In certain embodiments, the carrier fluid includes a surfactant, such as a silicone surfactant, disclosed herein. The same method may be applied to create individual droplets from the second fluid containing the primer pairs (and, in some implementations, the amplification reagents). Either the droplets containing the first fluid, the droplets containing the second fluid, or both, may be formed and then stored in a library for later merging, aspects of certain implementations of which are described in U.S. patent application Ser. No.12 / 504,764, hereby incorporated herein in its entirety for all purposes. Once formed, droplets containing the first and second fluids can be merged to form single droplets containing the single nucleic acid template and heterogeneous mixture of primer pairs and probes. Merging can be accomplished, for example, in the presence of an electric field. Moreover, it is not required that both fluids be in the form of droplets when merging takes places. EXAMPLES EXAMPLE 1

[0047] Surfactants of the disclosure may be synthesized by the following exemplary scheme (Scheme 1). A polymer backbone containing unsubstituted silicon atoms (designated (-H)) is exposed to an allylated hydrophobic side chain and an allylated hydrophilic side chain in the presence of a hydrosilylation metal catalyst and toluene. In a specific embodiment, shown in Scheme 2, the polymer backbone is a methylhydrosiloxane-dimethylsiloxane block copolymer, the hydrophobic side chain is an allylated bifurcated branched polydimethylsiloxane with butyl terminal groups, and the hydrophilic side chain is an allylated PEG monomethoxy. The resulting grafting of the side chain groups occurs randomly. The ratio of the side chains may be controlled by limiting the quantities of individual reactants.SCHEME 1 H Si O Si O Si O Si+ YSCHEME 2 H Si O Si O Si O Si+ +EXAMPLE 2

[0048] Embodiments of the disclosure are able to create suitable formulations for biological assays as described herein. These formulations are able to maintain the separation of droplets during analysis.

[0049] FIG.1 shows data from a formulation which maintained a poor separation of droplets during a fluorescence analysis. Note that the amplitude of the positive and negative droplets varies widely and shows poor separation. This results in difficulty in setting a threshold between positive and negative droplet partitions due to polydispersity or droplet instability.

[0050] In contrast, FIG.2 shows data from a formulation which maintains a good separation of droplets during a fluorescence analysis. Note the tight and consistent amplitudes for both positive and negative droplet partitions. Additionally, it is easier to determine a clear threshold between positive and negative partitions. INCORPORATION BY REFERENCE

[0051] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. EQUIVALENTS

[0052] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification, and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

CLAIMS What is claimed is:

1. A heterografted surfactant block copolymer comprising a polysiloxane backbone, and a plurality of hydrophilic and hydrophobic side chains grafted to said backbone.

2. The surfactant of claim 1, wherein the surfactant comprises a segment A and a segment B having a structure wherein a=2-100 and b=1-1000,chain, R2is a hydrophilic side chain, and R3 is a linear aliphatic group or a phenyl group.

3. The surfactant of claim 1, wherein R1and R2are randomly distributed in the segment A.

4. The surfactant of claim 1, wherein each R1is independently selected from the group consisting of. ;wherein d, e, f, g, h and i are between about 1 and about 1000 units in length and X is selected from the group consisting of linear alkanes, branched alkanes, phenyl, and phenyl substituted alkanes.

5. The surfactant of claim 1, wherein each R2 is independently selected from the group consisting of. ;between about 1 and about 1000, and Y is selected from the group consisting of hydroxy, methoxy, and ethoxy.

6. The surfactant of claim 1, wherein each R3 is independently selected from the group consisting of a linear alkyl group or a phenyl group.

7. The surfactant of claim 6, wherein the selected R3groups are spaced randomly along the polysiloxane backbone.

8. The surfactant of claim 6, wherein the linear aliphatic group is -CnH2n+2, and n=1-18.

9. The surfactant of claim 2, selected from:and .

10. The surfactant of claim 1, having the following structure:

11. The surfactant of claim 1, wherein a ratio of hydrophilic to hydrophobic side chains is between about 0.01 and about 100.

12. The surfactant of claim 4, wherein X is selected from the group consisting of linear alkanes and phenyl alkanes.

13. The surfactant of claim 5, wherein Y is selected from the group consisting of hydroxy and methoxy.

14. The surfactant of claim 1, wherein the polysiloxane backbone comprises between about 5 and about 12 side chains.

15. The surfactant of claim 1, wherein the copolymer comprises polyether and alkyl capped polysiloxane side chains grafted to the polysiloxane backbone.

16. The surfactant of claim 1, wherein the hydrophobic sidechain is a butyl-terminated polydimethylsiloxane grafted to the polysiloxane backbone at a midpoint.

17. The surfactant of claim 1, wherein the segment B comprises a PDMS chain.

18. The surfactant of claim 1, the hydrophilic sidechain is a polyethylene glycol.

19. The surfactant of claim 2, wherein each R3 is methyl.

20. The surfactant of claim 19, wherein a ratio of hydrophilic to hydrophobic side chains is about 5:

3.

21. The surfactant of claim 1, wherein the hydrophilic side chain is selected from the group consisting of a hydroxyl group, a methoxy group, PEG, PPG, and polyglycerins.

22. The surfactant of claim 1, wherein said hydrophilic side chains are selected from the group consisting of hydroxy, polyethylene glycol (PEG), polypropylene glycol (PPG), polyglycerin, linear polyethylenimine, and their respective block copolymers.

23. The surfactant of claim 1, having the structure: wherein a=6-9, b=11-28, R1isa hydrophilic side chain, and R3is a linear aliphatic group or a phenyl group.

24. The surfactant of claim 4, wherein R1 has the structure:wherein e=5-8 and f=5-8.

25. The surfactant of claim 5, wherein said side chains (R2) has the structurewherein j=8-12.

26. The surfactant of claim 2, wherein R3 is methyl.

27. The surfactant of claim 12, wherein X is butyl.

28. A composition comprising the surfactant of claim 1.

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