Silicone-based bolaform 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 bolaform silicone surfactants comprising a polysiloxane backbone and two terminal hydrophilic groups.

Description

SILICONE-BASED BOLAFORM SURFACTANTSFIELD 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] 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

[0007] 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.

[0008] In some embodiments, the silicone surfactants of the disclosure are bolaform surfactants comprising a polysiloxane backbone and hydrophilic terminal groups. Stated differently, in some embodiments, the silicone surfactants of the disclosure comprise a poly siloxane backbone and two hydrophilic groups, one on each end of the backbone.

[0009] In some embodiments, the silicone surfactants comprise a backbone of repeating units (-Si-O-), a polysiloxane. In some embodiments, the silicone surfactant may contain in a range of about 1 to about 1000 backbone units. In some embodiments, a backbone unit may be grafted with an alkyl group comprising 1-18 carbon atoms or an aryl group.

[0010] In some embodiments, each backbone unit is grafted with the same group. In some embodiments, each backbone unit is substituted with two methyl groups, e.g., a polydimethylsiloxane (PDMS). In some embodiments, different backbone groups are present in the silicone surfactant. In these embodiments, the groups may be presented in a pattern (e.g., alternating groups) or be positioned randomly.

[0011] In some embodiments, the terminal groups are independent hydrophilic groups. In some embodiments, the hydrophilic groups comprise oxygen atoms. Exemplary groups include hydrophilic polymers including 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. In some embodiments, the hydrophilic groups are terminated with hydroxyl (-OH), methoxy (-OCH3), or ethoxy (-OC2H5) groups.

[0012] In some embodiments, the silicone surfactant is:HCk I P V ! \7 si-o-Usi-O+Si— \VI V / 73I0HorOH , where n is 74 or 108 and p is 12 or 16.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1 shows data from a formulation.

[0014] FIG. 2 shows a 74 backbone unit surfactant.

[0015] FIG. 3 shows data from formulations containing the 74 backbone unit surfactant.

[0016] FIG. 4 shows a 108 backbone unit surfactant.

[0017] FIG. 5 gives data from formulations containing the 108 backbone unit surfactant.DETAILED DESCRIPTION OF THE INVENTION

[0018] 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 asilicone 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 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.

[0019] Some embodiments of the disclosure relate to silicone surfactants. The silicone surfactants of the disclosure are bolaform surfactants comprising a poly siloxane backbone and terminal hydrophilic groups.

[0020] The silicone surfactants of the disclosure may correspond to Formula I^2 / R2 \R2 R]— Si— o4-Si-O-]-Si-R-|

[0021] In some embodiments, the silicone surfactant may contain in a range of 1-1000 backbone units a, or in a range of 1-200 backbone units, or in a range of 20-150 backbone units, or in a range of 50-125 backbone units, or in a range of 70-110 backbone units. In some embodiments, the silicone surfactant may contain in a range of 50-100 backbone units, or in a range of 60-90 backbone units, or in a range of 70-80 backbone units, or in a range of 70-75 backbone units. In some embodiments, the silicone surfactant may contain in a range of 80-140 backbone units, or in a range of 90-130 backbone units, or in a range of 100-120 backbone units, or in a range of 105-115 backbone units.

[0022] In the context of Formula I, the number of backbone units is designated as "a." In some embodiments, the silicone surfactant contains 73 backbone units. In some embodiments, the silicone surfactant contains 74 backbone units. In some embodiments, the silicone surfactant contains 108 backbone units.

[0023] Each of the backbone units of the silicone surfactant is grafted with two groups (R2) independently selected from alkyl groups comprising 1-18 carbon atoms or aryl group. In some embodiments, each backbone unit is grafted with the same group. In some embodiments, each backbone unit of is grafted with two methyl groups. In some embodiments, the backbone units are grafted with two or more different groups. In some embodiments, the distribution of groupsis patterned (e.g., alternating). In some embodiments, the different backbone groups are distributed randomly.

[0024] In some embodiments, the hydrophilic terminal groups (Ri) comprise oxygen atoms. In some embodiments, the hydrophilic side chains comprise 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.

[0025] Exemplary hydrophilic side chains (Ri) are shown below. As illustrated, attachment to the backbone occurs at the wavy line with the terminal groups, if present, designated by Y.

[0026] The length of the hydrophilic polymer segments 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 5-20 repeating units, or in a range of 8-12 repeating units, or in a range of 10-20 repeating units, or in a range of 11-19 repeating units. Accordingly, in the context of the structures above, j-u are independently selected from the ranges above.

[0027] In some embodiments, the silicon surfactant is:H0\ / ^ S li O ) [S li 0 \ - S li \ / \VI \ l / 3I ^9 °H

[0028] 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.

[0029] 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 of the disclosure which aids in droplet formation and stability. Data related to an analyte disposed in the dispersed droplets may be collected.

[0030] 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 include pus, sputum, semen, urine, blood, saliva, and cerebrospinal fluid.

[0031] 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 dropletcontaining the single nucleic acid template and a heterogeneous mixture of primer pairs and probes. The second fluid can also contain reagents for conducting aPCR reaction, such as a polymerase and dNTPs.

[0032] 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.

[0033] 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 intersects two 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 place.EXAMPLE 1

[0034] Surfactants of the disclosure may be synthesized by the following exemplary scheme. A polymer backbone containing two terminal unsubstituted silicon atoms (hydrosilanes) is exposed to an allylated hydrophilic group in the presence of a hydrosilylation metal catalyst and toluene.EXAMPLE 2

[0035] 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.

[0036] 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 poly dispersity or droplet instability.

[0037] In contrast, silicone surfactants that include a polysiloxane backbone and two hydrophilic groups on each end of the backbone maintain a good separation of droplets during a fluorescence analysis as shown as follows.

[0038] FIG. 2 shows a 74 backbone unit (a = 74) surfactant 201 according to certain embodiments.

[0039] FIG. 3 shows data from formulations containing the surfactant 201, showing that formulations maintain a good separation of droplets during a fluorescence analysis.

[0040] FIG. 4 shows a 108 backbone unit surfactant 401 of certain embodiments.

[0041] FIG. 5 gives data from formulations containing the surfactant 401, showing that formulations maintain a good separation of droplets during a fluorescence analysis. The results show that silicone surfactants that include a polysiloxane backbone and two hydrophilic groups on each end of the backbone maintain a good separation of droplets during a fluorescence analysis. In both cases, 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

[0042] 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

[0043] 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

ClaimsWhat is claimed is:

1. A silicone surfactant comprising a polysiloxane backbone and two hydrophilic groups on each end of the backbone.

2. The surfactant of claim 1, wherein the surfactant corresponds to Formula I:R2 / R2 \R2R-|— Si— O-HSi-O-j-Si-R-iR2 \R2 / aR2(i)wherein each Ri is an independent hydrophilic terminal group, each R2 is an independent linear aliphatic group or an aryl group, and a is 1-1000.

3. The surfactant of claim 2, wherein a is 70-110.

4. The surfactant of claim 2, wherein each Ri is independently selected from the group consisting of:wherein j, k, 1, m, n, o, p, q, r, s, t, and u are between about 1 and about 1000, and Y is selected from the group consisting of hydroxy or alkoxy groups.

5. The surfactant of claim 4, wherein Ri isand j is 11-19.

6. The surfactant of claim 5, wherein Y is -OH.

7. The surfactant of claim 4, wherein Y is selected from the group consisting of hydroxy, methoxy, and ethoxy.

8. The surfactant of claim 2, wherein Ri is independently selected from the group consisting of hydroxy, polyethylene glycol (PEG), polypropylene glycol (PPG), polyglycerin, linear polyethylenimine, branched polyethylenimine, and their respective block copolymers.

9. The surfactant of claim 2, wherein each R2 is independently selected from the group consisting of a linear aliphatic group, and a phenyl group.

10. The surfactant of claim 9, wherein the selected groups are spaced randomly along the polysiloxane backbone.

11. The surfactant of claim 9, wherein the aliphatic group is -CnH2n+2 and n=l-18.

12. The surfactant of claim 1, having a formula:

13. The surfactant of claim 1, having a formula:

14. The surfactant of claim 1, having a formula:

15. A composition comprising the surfactant of claim 1, a silicone oil, and aqueous droplets disposed in the silicone oil.

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