Fluorinated surfactants and uses thereof

Abstract

The present disclosure relates to fluorosurfactants useful for the preparation of emulsions, which may be used in droplet-based microfluidics. The present disclosure also relates to emulsions comprising such surfactants. and methods of preparing such emulsions. The present disclosure further relates to methods and uses of such surfactants and emulsions.

Description

FLUORINATED SURFACTANTS AND USES THEREOF FIELD

[0001] The present disclosure relates to fluorosurfactants useful for the preparation of emulsions, which may be used in droplet-based microfluidics. The present disclosure also relates to emulsions comprising such surfactants, and methods of preparing such emulsions. The present disclosure further relates to methods and uses of such surfactants and emulsions.BACKGROUND

[0002] Microfluidics is a process that can generate thousands of droplets per second via a chip in which two immiscible fluids circulate in a network of micro-channels, forming an emulsion. The microfluidic droplets can essentially act as individual microreactors and can be manipulated in an automated manner in microfluidic channels at a very high throughput. Thus, droplet-based microfluidic technology has allowed for significant improvements in the throughput of chemical and biological reactions and processes, including digital PCR, directed evolution of enzymes, screening of pharmaceuticals including antibiotics. Other useful applications of droplet-based microfluidics include high-throughput single-cell cultivation, microbial co-cultivation, targeted sequencing and single-cell sequencing.

[0003] To stabilise the microfluidic droplets, a surfactant is usually required. Surfactants (surface-active agents) are amphiphilic molecules that are typically used to stabilise droplet interfaces and prevent coalescence of droplets. Because the microfluidic droplets may be used as microreactors, ideally the surfactants should be capable of stabilising the droplets under the reaction conditions. The surfactants should also preferably be biocompatible (e.g., they should not interfere with the chemical / biological processes taking place in the droplet), prevent or suppress molecular exchange between droplets, and provide uniform droplet size.

[0004] In microfluidic applications, the use of oils as the continuous phase in emulsion formation / production is beneficial because they have useful microfluidic properties, including low friction, non-volatility (unlike alcohols), temperature-resistance and the ability to easily create oil-water emulsions. The use of fluorinated oils is of particular interest in certain applications, in part due to most organic compounds being insoluble in these oils and their biocompatibility. Conventional surfactants are generally not suitable for stabilising emulsions comprising a fluorous oil phase due to factors such as solubility issues, poor stability and poor biocompatibility. However, fluorinated surfactants (fluorosurfactants), which typically comprise a fluorous tail group and a hydrophilic head group, have been shown to be effective in stabilising water-in-fluorous oil emulsions.

[0005] Over the past few years, various fluorosurfactants have been developed for use in microfluidics applications. Currently, the most successful fluorosurfactant is a tri -block copolymer PFPE2-PEG, which consists of highly fluorinated perfluoropolyethers (PFPE) and polyethylene glycol (PEG) and is commercially available (e.g. FluoSurf™ (Emulseo)). Another commercially available fluorosurfactants is PicoSurf™ (Sphere Fluidics), which is a PFPE2- Jeffamine tri-block copolymer. However, these commercially available fluorosurfactants suffer from various drawbacks, such as poor stability (including at high temperatures, for example in PCR thermal cycling), inter-droplet transfer and producing uneven droplet size. They are also expensive.

[0006] Accordingly, there remains a need for new fluorosurfactants and emulsions suitable for use in microfluidics applications. It would be advantageous to provide surfactants which are capable of stabilising emulsions, particularly water-in-fluorous oil emulsions, which prevent droplets from coalescing, which are biocompatible, which prevent or reduce inter-droplet transfer, and / or which provide uniform droplet size; or which may provide a useful alternative to existing fluorosurfactants.SUMMARY

[0007] The present inventors have undertaken extensive research into the development of fluorosurfactants and have identified that the surfactants as described herein are capable of stabilising emulsions and can be useful in microfluidics applications. Beneficially, surfactants described herein exhibited comparable or improved effects compared to the commercially available fluorosurfactant PicoSurf ™. The present disclosure relates to applications of the surfactants described herein in emulsions and in various methods and uses.

[0008] In one aspect of the present disclosure, there is provided an emulsion comprising:(a) a dispersed phase which is an aqueous phase or a lipophilic phase;(b) a continuous phase comprising a solvent or an oil; and(c) a surfactant which is a block copolymer having a backbone comprising:(i) a first block which is soluble in an aqueous phase or soluble in a hydrocarbon phase; and(ii) a second block which is a fluoropolyalkyl block or a fluoropolyether block; wherein one or both of the following apply: the first block presents its functionality pendant to the block copolymer backbone; the second block presents its functionality pendant to the block copolymer backbone.

[0009] The block copolymer may be prepared using a living polymerisation method.

[0010] In some embodiments, the first block presents its functionality pendant to the block copolymer backbone.

[0011] In some embodiments, the first block is a hydrophilic block.

[0012] In some embodiments, the second block is a (per)fluoropolyether block.

[0013] In some embodiments, the dispersed phase is an aqueous phase, and the continuous phase is an oil phase. The oil phase may be a fluorinated oil.

[0014] The emulsion may be a single emulsion or a multiple emulsion (e.g. a double emulsion).

[0015] The emulsion may comprise a plurality of stabilised droplets which coalesce in response to a stimulus (e.g. temperature and / or pH).

[0016] In another aspect of the present disclosure, there is provided a method of preparing an as emulsion described herein, the method comprising: providing an aqueous phase or a lipophilic phase; providing a solvent or an oil phase; and mixing the aqueous or lipophilic phase, the solvent or oil phase, and a surfactant as described herein to form the emulsion.

[0017] In some embodiments, the method of preparing the emulsion comprises: providing an aqueous phase; providing an oil phase, which is preferably a fluorinated oil; and mixing the aqueous phase, the oil phase, and the surfactant as described herein to form the emulsion.

[0018] In some embodiments, the mixing is by a flow focus junction of a microfluidic device.

[0019] In another aspect of the present disclosure, there is provided a method of demulsifying an emulsion, the method comprising: providing an emulsion described herein, or prepared according to a method described herein, wherein the emulsion comprises a plurality of stabilised droplets which coalesce in response to a stimulus (e.g. temperature and / or pH); and exposing the emulsion to said stimulus.

[0020] In another aspect of the present disclosure, there is provided the use of a surfactant as described herein for the preparation of an emulsion.

[0021] In another aspect of the present disclosure, there is provided a surfactant as described herein for use in the preparation of an emulsion.

[0022] In another aspect of the present disclosure, there is provided the use of a surfactant as described herein in a microfluidic channel or device, in a molecular isolation in larger fluidic devices, containers or vats, or in an automated device with associated software that controls a microfluidic channel or device.

[0023] In another aspect of the present disclosure, there is provided a surfactant as described herein for use in a microfluidic channel or device, in a molecular isolation in larger fluidic devices, containers or vats, or in an automated device with associated software that controls a microfluidic channel or device.

[0024] In another aspect of the present disclosure, there is provided the use of an emulsion as described herein in a microfluidic channel or device, or in an automated device with associated software that controls a microfluidic channel or device.

[0025] In another aspect of the present disclosure, there is provided an emulsion as described herein for use in a microfluidic channel or device, or in an automated device with associated software that controls a microfluidic channel or device.

[0026] In another aspect of the present disclosure, there is provided a method comprising performing a chemical and / or biological reaction in a dispersed phase as described herein. The dispersed phase may be the dispersed phase of an emulsion as described herein.

[0027] In some embodiments, the chemical and / or biological reaction is a polymerisation reaction. In some embodiments, the chemical and / or biological reaction is an enzymatic reaction.

[0028] In some embodiments, the chemical and / or biological reaction involves a cell. In some embodiments, the chemical and / or biological reaction involves a cellular component.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1. (a) 'H and (b)19F NMR spectra in CDCh and assignments to the spectra of poly(OEGMA-co-PFPEMA). (c) The chemical structure of poly(OEGMA-co-PFPEMA) showing labels for assignment of the NMR spectra.

[0030] Figure 2. (a) 'H and (b)19F NMR spectra in CDCh and assignments to the spectra of poly(2-HEA)s-PFPE. (c) The chemical structure of poly(2-HEA)s-PFPE showing labels for assignment of the NMR spectra.

[0031] Figure 3. (a) 'H and (b)19F NMR spectra in CDCh and assignments to the spectra of poly(OEGA)s-PFPE. (c) The chemical structure of poly(OEGA)s-PFPE showing labels for assignment of the NMR spectra.

[0032] Figure 4. (a) 'H and (b)19F NMR spectra in CDCh and assignments to the spectra of poly(MSEA)s-PFPE. (c) The chemical structure of poly(MSEA)s-PFPE showing labels for assignment of the NMR spectra.

[0033] Figure 5. (a) Micrographs displaying the size distribution of the Pico-Surf™ surfactant stabilized droplets, showing steady conditions during 24 h incubation at 4 °C under water, PBS and DMEM+10% FBS dispersed phase, (b) Box and scatterplot of droplet size distribution during 24 h incubation at 4 °C. The Image J line profding tool was applied to measure 25 droplets to determine the mean average droplet diameter value. Scale bar, 100 pm.

[0034] Figure 6. (a) Micrographs displaying the size distribution of the PFPE-(2-HEA)4 surfactant stabilized droplets during 24 h incubation at 4 °C under water, PBS and DMEM+10% FBS dispersed phase. The Image J line profding tool was applied to measure 100 droplets to determine the mean average droplet diameter value. Scale bar, 100 pm.

[0035] Figure 7. (a) Micrographs displaying the size distribution of the PFPE-(OEGA)e surfactant stabilized droplets during 24 h incubation at 4 °C under water, PBS and DMEM+10% FBS dispersed phase. The Image J line profding tool was applied to measure 100 droplets to determine the mean average droplet diameter value. Scale bar, 100 pm.

[0036] Figure 8. (a) Micrographs displaying the size distribution of the PFPE-(MSEA)2 surfactant stabilized droplets during 24 h incubation at 4 °C under water, PBS and DMEM+10% FBS dispersed phase. The Image J line profding tool was applied to measure 100 droplets to determine the mean average droplet diameter value. Scale bar, 100 pm.

[0037] Figure 9. (a) Micrographs displaying the size distribution of PFPE-(2-HEA)4, PFPE- (MSEA)s, PFPE-(OEGA)e, Pico-Surf™ surfactant stabilized droplets during 24 h incubation at 4 °C. (a) Micrographs displaying the size distribution of PFPE-(2-HEA)4, PFPE-(MSEA)3, PFPE- (OEGA)e, Pico-Surf™ surfactant stabilized droplets during 24 h incubation at 4 °C after 35 cycles of PCT. (c) Box and scatterplot of droplet size distribution during PCR. The Image J line profding tool was applied to measure 25 droplets to determine the mean average droplet diameter value. Scale bar, 100 pm.

[0038] Figure 10. (a) Micrographs of inter-droplet diffusion of fluorescein salt after 3 days of stabilisation with PFPE-(2-HEA)4 or Pico-Surf™ of groups of empty and dye-containing droplets. Scale bar, 100 pm. (b) Box-plot of PBS -only-droplets fluorescence intensity during 72 h incubation at 37 °C. The Image J line profiling tool was applied to measure 5 PBS-only- droplets droplets to quantify the fluorescence intensity, (c) The dynamic surface tension of PFPE-(2-HEA)4-based surfactant solution compared to Pico-Surf™.

[0039] Figure 11. (a) Micrographs displaying the size distribution of the PFPE-(2-HEA)4 surfactant stabilized w / o / w double emulsion, showing steady conditions during 24 h incubation at 4 °C under yeast cell medium dispersed phase. Scale bar, 50 pm. (b) Micrographs displaying the bright field and fluorescence of yeast cells within double emulsion at 4 time points. Scalebar, 30 un. (c) Micrographs displaying the fluorescence of yeast cells per droplet at 6 time points. The Image J line profiling tool was applied to quantify the fluorescence intensity. Scale bar, 50 pm. (d) Box-plot showing fluorescence intensity of PBS-only-droplets during 24 h incubation at 30 °C. (e) Box-plot showing number of yeast cells per droplet during 24 h incubation at 30 °C.

[0040] Figure 12. (a)JH spectrum in CDCh and assignments to the spectra of p(DMAEA)2- PFPE. (b) The chemical structure of p(DMAEA)2-PFPE showing labels for assignment of the NMR spectra.

[0041] Figure 13. (a)JH spectrum in CDCh and assignments to the spectra of p(NIPAM)s- PFPE. (b) The chemical structure of p(NIPAM)3-PFPE showing labels for assignment of the NMR spectra.

[0042] Figure 14. (a) Micrographs displaying p(DMAEA)2-PFPE surfactant stabilized w / o droplets at pH 7.0 and coalescence at pH 5.0 under PBS dispersed phase, (b) Micrographs displaying Pico-Surf™ surfactant stabilized w / o droplets at pH 7.0 and pH 5.0. Scale bar, 100 pm.

[0043] Figure 15. (a) Micrographs displaying p(NIPAM)3-PFPE surfactant stabilized w / o droplets at room temperature and coalescence at 40 °C under PBS dispersed phase, (a) Micrographs displaying Pico-Surf™ surfactant stabilized w / o droplets at room temperature and at 40 °C under PBS dispersed phase. Scale bar, 100 pm.DETAILED DESCRIPTION

[0044] Reference will now be made in detail to certain embodiments of the present disclosure. While the present disclosure will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the present disclosure to those embodiments. On the contrary, the present disclosure is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present disclosure as defined by the claims.

[0045] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. The present disclosure is in no way limited to the methods and materials described. It will be understood that the present disclosure extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the present disclosure.

[0046] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0047] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

[0048] For the purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0049] The present inventors have identified that the fluorosurfactants described herein are capable of stabilising emulsions, including water-in-fluorous oil emulsions, and may be useful in various applications, including as droplet stabilisers in microfluidics applications. The surfactants of the present disclosure are block copolymers having a general A-B structure, where A constitutes the first block (which is soluble in an aqueous phase or a hydrocarbon phase) and B constitutes the second block (which is a fluorinated block), and one or both of A and B present their functionalities pendant to the polymer backbone. This is in contrast to commercially available fluorosurfactants used in microfluidics applications, such as FluoSurf™ (PFPE2-PEG) and PicoSurf™ (PFPE2-Jeffamine) which are tri-block copolymers having a general B-A-B structure where the functionalities of both A and B are in the backbone.

[0050] The surfactants of the present disclosure may advantageously be prepared by living radical polymerisation methods. For example, as described herein and shown in the Examples, the surfactants of the present disclosure may be prepared by reversible addition fragmentation chain transfer (RAFT) polymerisation and atom transfer radical polymerisation (ATRP). The present inventors believe this is the first disclosure of the use of fluorosurfactants prepared by living polymerisation methods in droplet microfluidics applications. Using living polymerisation methods can provide surfactants that present functionality pendant to the polymer backbone. Without wishing to be bound by theory, the present inventors hypothesise that this structural characteristic of the surfactants of the present disclosure contribute to their ability to act as effective droplet stabilisers. Further, using living polymerisation methods can allow the surfactants of the present disclosure to be easily tuned for a desired application, for example by altering the monomer(s) used to prepare the first and / or second blocks (especially the first block), the degrees of polymerisation of the first and / or second blocks, the ratio of molecular weight of the first block to the second block, and the extent of fluorination of the second block. This can advantageously allow for the generation of surfactants having different hydrophilicity / lipophilicity, distribution of functionalities and fluorine content, which can influence their ability to act as droplet stabilisers in under different conditions.

[0051] As described herein and as shown in the Examples, the surfactants of the present disclosure are capable of stabilising emulsion droplets and preventing coalescence of droplets over at least a 24 hour period, demonstrating steady conditions under different dispersed aqueous phases at 4 °C and under PCR cycling conditions. In addition, the surfactants of the present disclosure were capable of producing monodisperse droplets under various dispersed aqueous phases. The surfactants were also found to minimise inter-droplet molecular transfer or a fluorescent dye. Further, the surfactants also exhibited low or reduced interfacial tension, which can help prevent subsequent coalescence of formed droplets. Moreover, the surfactants were found to be biocompatible and capable of growing yeast cells in double emulsions droplets. Advantageously, surfactants of the present disclosure were found to have comparable or improved effects over the commercially available fluorosurfactant PicoSurf™.

[0052] Additional advantages of the surfactants of the present disclosure include one or more of the following:The surfactants can be less expensive than current commercially available fluorosurfactants such as FluoSurf™ and PicoSurf™. Because the surfactants can be prepared by living polymerisation methods, they can be manufactured more cost effectively.Using living polymerisation methods can allow for controlled polymerisation and the generation of various block copolymer structures with predictable molecular weight, low molar mass dispersity, high end-group fidelity, and capacity for continued chain growth.The surfactants can be used with current microfluidics chips without physical alterations, as shown in the Examples. The surfactants can be suitably tailored to be used with currently available microchips.General definitions

[0053] Unless otherwise herein defined, the following terms will be understood to have the general meanings which follow.

[0054] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., chemistry, polymer chemistry, and the like).

[0055] As used herein, the term “and / or”, e.g., “X and / or Y” shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

[0056] As used herein, singular forms “a”, “an” and “the” include plural aspects, unless the context clearly indicates otherwise. Thus, for example, a reference to “a surfactant” may includea plurality of surfactants and a reference to “at least one monomer” may include one or more monomers, and so forth.

[0057] The term “(s)” following a noun contemplates the singular or plural form, or both.

[0058] Various features of the present disclosure are described with reference to a certain value, or range of values. These values are intended to relate to the results of the various appropriate measurement techniques, and therefore should be interpreted as including a margin of error inherent in any particular measurement technique. Some of the values referred to herein are denoted by the term “about” to at least in part account for this variability. The term “about”, when used to describe a value, may mean an amount within ±10%, ±5%, ±1% or ±0.1% of that value.

[0059] As used herein, except where the context requires otherwise, the term “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0060] As used herein, the term “alkyl”, used either alone or in compound words, denotes straight chain, branched or cyclic alkyl, preferably C1-20 alkyl, e.g. C1-10 or C1-6. Examples of straight chain and branched alkyl include methyl, ethyl, w-propyl. isopropyl, w-butyl. scc-butyl. t- butyl, M-pentyl, 1,2-dimethylpropyl, 1, 1 -dimethyl -propyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2-methylpentyl, 3 -methylpentyl, 1,1 -dimethylbutyl, 2,2-dimethylbutyl, 3, 3 -dimethylbutyl, 1,2- dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, heptyl, 5- methylhexyl, 1 -methylhexyl, 2,2-dimethylpentyl, 3, 3 -dimethylpentyl, 4,4-dimethylpentyl, 1,2- dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethyl -pentyl, 1,2,3-trimethylbutyl, 1,1,2- trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1 -methylheptyl, 1, 1,3,3- tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-methyloctyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or 3 -propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8 -methylnonyl, 1-, 2-, 3-, 4-, 5- or 6- ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-ethylnonyl, 1-, 2-, 3-, 4- or 5 -propyloctyl, 1-, 2- or 3 -butylheptyl, 1- pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methyhmdecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl, 1-2- pentylheptyl and the like. Examples of cyclic alkyl include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like. Where an alkyl group is referred to generally as "propyl", butyl" etc, it will be understood that this can refer to any of straight, branched and cyclic isomers whereappropriate. An alkyl group may be optionally substituted by one or more optional substituents as herein defined. The term “alkylene” is intended to denote the divalent form of alkyl.

[0061] The term “alkenyl” as used herein denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon to carbon double bond including ethylenically mono-, di- or polyunsaturated alkyl or cycloalkyl groups as defined herein, preferably C2-20 alkenyl (e.g. C2-10 or C2-6). Examples of alkenyl include vinyl, allyl, 1- methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1 -pentenyl, cyclopentenyl, 1 -methyl - cyclopentenyl, 1 -hexenyl, 3 -hexenyl, cyclohexenyl, 1 -heptenyl, 3 -heptenyl, 1 -octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl, 1,4- pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,3-cyclohexadienyl, 1,4- cyclohexadienyl, 1,3 -cycloheptadienyl, 1,3,5-cycloheptatrienyl and 1,3,5,7-cyclooctatetraenyl. An alkenyl group may be optionally substituted by one or more optional substituents as herein defined.

[0062] As used herein the term “alkynyl” denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon-carbon triple bond including ethylenically mono-, di- or polyunsaturated alkyl or cycloalkyl groups as defined herein. Unless the number of carbon atoms is specified the term preferably refers to C2-20 alkynyl (e.g. C2-10 or C2-6). Examples include ethynyl, 1-propynyl, 2-propynyl, and butynyl isomers, and pentynyl isomers. An alkynyl group may be optionally substituted by one or more optional substituents as herein defined.

[0063] The term “halogen” (“halo”) denotes fluorine, chlorine, bromine or iodine (fluoro, chloro, bromo or iodo). Preferred halogens are chlorine and bromine.

[0064] The term “aryl” (or “carboaryl”) denotes any of single, polynuclear, conjugated and fused residues of aromatic hydrocarbon ring systems (e.g. Ce-is aryl). Examples of aryl include phenyl, biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, idenyl, azulenyl, chrysenyl. Preferred aryl include phenyl and naphthyl. An aryl group may be optionally substituted by one or more optional substituents as herein defined. The term “arylene” is intended to denote the divalent form of aryl.

[0065] The term “carbocyclyl” includes any of non-aromatic monocyclic, polycyclic, fused or conjugated hydrocarbon residues, preferably C3-20 (e.g. C3-10 or C3-8). The rings may be saturated, e.g. cycloalkyl, or may possess one or more double bonds (cycloalkenyl) and / or one or more triple bonds (cycloalkynyl). Particularly preferred carbocyclyl moieties are 5-6- membered or 9- 10 membered ring systems. Suitable examples include cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, cyclopentadienyl, cyclohexadienyl, cyclooctatetraenyl, indanyl, decalinyl and indenyl. A carbocyclyl group may be optionally substituted by one or more optional substituents as herein defined. The term “carbocyclylene” is intended to denote the divalent form of carbocyclyl.

[0066] The term “heterocyclyl” when used alone or in compound words includes any of monocyclic, polycyclic, fused or conjugated hydrocarbon residues, preferably C3-20 (e.g. C3-10 or C3-8) wherein one or more carbon atoms are replaced by a heteroatom so as to provide a non aromatic residue. Suitable heteroatoms include O, N, S, P and Se, particularly O, N and S. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms. The heterocyclyl group may be saturated or partially unsaturated, i.e. possess one or more double bonds. Particularly preferred heterocyclyl are 5-6 and 9-10 membered heterocyclyl. Suitable examples of heterocyclyl groups may include azridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 2H-pyrrolyl, pyrrolidinyl, pyrrolinyl, piperidyl, piperazinyl, morpholinyl, indolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, thiomorpholinyl, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrolyl, tetrahydrothiophenyl, pyrazolinyl, dioxalanyl, thiazolidinyl, isoxazolidinyl, dihydropyranyl, oxazinyl, thiazinyl, thiomorpholinyl, oxathianyl, dithianyl, trioxanyl, thiadiazinyl, dithiazinyl, trithianyl, azepinyl, oxepinyl, thiepinyl, indenyl, indanyl, 3H-indolyl, isoindolinyl, 4H- quinolazinyl, chromenyl, chromanyl, isochromanyl, pyranyl and dihydropyranyl. A heterocyclyl group may be optionally substituted by one or more optional substituents as herein defined. The term “heterocyclylene” is intended to denote the divalent form of heterocyclyl.

[0067] The term “heteroaryl” includes any of monocyclic, polycyclic, fused or conjugated hydrocarbon residues, wherein one or more carbon atoms are replaced by a heteroatom so as to provide an aromatic residue. Preferred heteroaryl have 3-20 ring atoms, e.g. 3-10. Particularly preferred heteroaryl are 5-6 and 9-10 membered bicyclic ring systems. Suitable heteroatoms include, O, N, S, P and Se, particularly O, N and S. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms. Suitable examples of heteroaryl groups may include pyridyl, pyrrolyl, thienyl, imidazolyl, furanyl, benzothienyl, isobenzothienyl, benzofuranyl, isobenzofuranyl, indolyl, isoindolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, quinolyl, isoquinolyl, phthalazinyl, 1,5-naphthyridinyl, quinozalinyl, quinazolinyl, quinolinyl, oxazolyl, thiazolyl, isothiazolyl, isoxazolyl, triazolyl, oxadialzolyl, oxatriazolyl, triazinyl, and furazanyl. A heteroaryl group maybe optionally substituted by one or more optional substituents as herein defined. The term “heteroarylene” is intended to denote the divalent form of heteroaryl.

[0068] The term “acyl” either alone or in compound words denotes a group containing the moiety C=O (and not being a carboxylic acid, ester or amide). Preferred acyl includes C(O)-Re, wherein Reis hydrogen or an alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, or heterocyclyl residue. Examples of acyl include formyl, straight chain or branched alkanoyl (e.g. C1-20) such as acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2- dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl and icosanoyl; cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl and cyclohexylcarbonyl; aroyl such as benzoyl, toluoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e.g. phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutylyl, phenylpentanoyl and phenylhexanoyl) and naphthylalkanoyl (e.g. naphthylacetyl, naphthylpropanoyl and naphthylbutanoyl] ; aralkenoyl such as phenylalkenoyl (e.g. phenylpropenoyl, phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e.g. naphthylpropenoyl, naphthylbutenoyl and naphthylpentenoyl); aryloxyalkanoyl such as phenoxyacetyl and phenoxypropionyl; arylthiocarbamoyl such as phenylthiocarbamoyl; arylglyoxyloyl such as phenylglyoxyloyl and naphthylglyoxyloyl; arylsulfonyl such as phenylsulfonyl and napthylsulfonyl; heterocycliccarbonyl; heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl; heterocyclicalkenoyl such as heterocyclicpropenoyl, heterocyclicbutenoyl, heterocyclicpentenoyl and heterocyclichexenoyl; and heterocyclicglyoxyloyl such as thiazolyglyoxyloyl and thienylglyoxyloyl. The Reresidue may be optionally substituted as described herein.

[0069] The term “sulfoxide”, either alone or in a compound word, refers to a group - S(O)Rfwherein Rfis selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl. Examples of preferred Rfinclude C1-20 alkyl, phenyl and benzyl.

[0070] The term “sulfonyl”, either alone or in a compound word, refers to a group S(O)2-Rf, wherein Rfis selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl and aralkyl. Examples of preferred Rfinclude C1-20 alkyl, phenyl and benzyl.

[0071] The term “sulfonamide”, either alone or in a compound word, refers to a group S(O)NRfRfwherein each Rfis independently selected from hydrogen, alkyl, alkenyl, alkynyl,aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl. Examples of preferred Rfinclude hydrogen, C 1-20 alkyl, phenyl and benzyl.

[0072] The term “amino” is used here in its broadest sense as understood in the art and includes groups of the formula NRaRb, wherein Raand Rbmay be independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, arylalkyl, and acyl. Raand Rb, together with the nitrogen to which they are attached, may also form a monocyclic, or polycyclic ring system e.g. a 3-10 membered ring, particularly, 5-6 and 9-10 membered systems. Examples of “amino” include NH2, NHalkyl (e.g. Ci-2oalkyl), NHaryl (e.g. NHphenyl), NHaralkyl (e.g. NHbenzyl), NHacyl (e.g. NHC(0)Ci-2oalkyl, NHC(O)phenyl), Nalkylalkyl (wherein each alkyl, for example C1-20, may be the same or different) and 5 or 6 membered rings, optionally containing one or more same or different heteroatoms (e.g. O, N and S).

[0073] The term “amido” is used here in its broadest sense as understood in the art and includes groups having the formula C(O)NRaRb, wherein Raand Rbare as defined as above. Examples of amido include C(O)NH2, C(O)NHalkyl (e.g. Ci-2oalkyl), C(O)NHaryl (e.g. C(O)NHphenyl), C(O)NHaralkyl (e.g. C(O)NHbenzyl), C(O)NHacyl (e.g. C(0)NHC(0)Ci-2oalkyl, C(O)NHC(O)phenyl), C(O)Nalkylalkyl (wherein each alkyl, for example C1-20, may be the same or different) and 5 or 6 membered rings, optionally containing one or more same or different heteroatoms (e.g. O, N and S).

[0074] The term “carboxy ester” is used here in its broadest sense as understood in the art and includes groups having the formula CChRg, wherein Rgmay be selected from groups including alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, aralkyl, and acyl. Examples of carboxy ester include COrCi-roalkyl. CCharyl (e.g. CChphenyl), COraralkyl (e.g. CChbenzyl).

[0075] As used herein, the term “optionally substituted” is taken to mean that a group may or may not be substituted or fused (so as to form a condensed polycyclic group) with one, two, three or more of organic and inorganic groups, including those selected from: alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, heteroaryl, acyl, aralkyl, alkaryl, alkheterocyclyl, alkheteroaryl, alkcarbocyclyl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, halocarbocyclyl, haloheterocyclyl, haloheteroaryl, haloacyl, haloaryalkyl, hydroxy, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxycarbocyclyl, hydroxyaryl, hydroxyheterocyclyl, hydroxyheteroaryl, hydroxyacyl, hydroxyaralkyl, alkoxyalkyl, alkoxyalkenyl, alkoxyalkynyl, alkoxycarbocyclyl, alkoxyaryl, alkoxyheterocyclyl, alkoxyheteroaryl, alkoxyacyl, alkoxyaralkyl, alkoxy, alkenyloxy, alkynyloxy, aryloxy, carbocyclyloxy, aralkyloxy, heteroaryloxy, heterocyclyloxy, acyloxy, haloalkoxy, haloalkenyloxy, haloalkynyloxy, haloaryloxy, halocarbocyclyloxy, haloaralkyloxy,haloheteroaryloxy, haloheterocyclyloxy, haloacyloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, nitroheteroayl, nitrocarbocyclyl, nitroacyl, nitroaralkyl, amino (Nth), alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, aralkylamino, diaralkylamino, acylamino, diacylamino, heterocyclamino, heteroarylamino, carboxy, carboxyester, amido, alkylsulphonyloxy, arylsulphenyloxy, alkylsulphenyl, arylsulphenyl, thio, alkylthio, alkenylthio, alkynylthio, arylthio, aralkylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, acylthio, sulfoxide, sulfonyl, sulfonamide, aminoalkyl, aminoalkenyl, aminoalkynyl, aminocarbocyclyl, aminoaryl, aminoheterocyclyl, aminoheteroaryl, aminoacyl, aminoaralkyl, thioalkyl, thioalkenyl, thioalkynyl, thiocarbocyclyl, thioaryl, thioheterocyclyl, thioheteroaryl, thioacyl, thioaralkyl, carboxyalkyl, carboxyalkenyl, carboxyalkynyl, carboxycarbocyclyl, carboxyaryl, carboxyheterocyclyl, carboxyheteroaryl, carboxyacyl, carboxyaralkyl, carboxyesteralkyl, carboxyesteralkenyl, carboxyesteralkynyl, carboxyestercarbocyclyl, carboxyesteraryl, carboxyesterheterocyclyl, carboxyesterheteroaryl, carboxyesteracyl, carboxyesteraralkyl, amidoalkyl, amidoalkenyl, amidoalkynyl, amidocarbocyclyl, amidoaryl, amidoheterocyclyl, amidoheteroaryl, amidoacyl, amidoaralkyl, formylalkyl, formylalkenyl, formylalkynyl, formylcarbocyclyl, formylaryl, formylheterocyclyl, formylheteroaryl, formylacyl, formylaralkyl, acylalkyl, acylalkenyl, acylalkynyl, acylcarbocyclyl, acylaryl, acylheterocyclyl, acylheteroaryl, acylacyl, acylaralkyl, sulfoxidealkyl, sulfoxidealkenyl, sulfoxidealkynyl, sulfoxidecarbocyclyl, sulfoxidearyl, sulfoxideheterocyclyl, sulfoxideheteroaryl, sulfoxideacyl, sulfoxidearalkyl, sulfonylalkyl, sulfonylalkenyl, sulfonylalkynyl, sulfonylcarbocyclyl, sulfonylaryl, sulfonylheterocyclyl, sulfonylheteroaryl, sulfonylacyl, sulfonylaralkyl, sulfonamidoalkyl, sulfonamidoalkenyl, sulfonamidoalkynyl, sulfonamidocarbocyclyl, sulfonamidoaryl, sulfonamidoheterocyclyl, sulfonamidoheteroaryl, sulfonamidoacyl, sulfonamidoaralkyl, nitroalkyl, nitroalkenyl, nitroalkynyl, nitrocarbocyclyl, nitroaryl, nitroheterocyclyl, nitroheteroaryl, nitroacyl, nitroaralkyl, cyano, sulfate and phosphate groups. Optional substitution may also be taken to refer to where a -CH2- group in a chain or ring is replaced by a group selected from -O-, -S-, - NRa-, -C(O)- (i.e. carbonyl), -C(O)O- (i.e. ester), and -C(O)NRa- (i.e. amide), where Rais as defined herein.

[0076] Preferred optional substituents include alkyl, (e.g. C1-6 alkyl such as methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), hydroxyalkyl (e.g. hydroxymethyl, hydroxyethyl, hydroxypropyl), alkoxyalkyl (e.g. methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl etc) alkoxy (e.g. C1-6 alkoxy such as methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy), halo, trifluoromethyl, trichloromethyl, tribromomethyl, hydroxy, phenyl (which itself may be further substituted e.g.,by Ci-6 alkyl, halo, hydroxy, hydroxyCi-6 alkyl, Ci-6 alkoxy, haloCi-6 alkyl, cyano, nitro OC(O)Ci-6 alkyl, and amino), benzyl (wherein benzyl itself may be further substituted e.g., by Ci-6 alkyl, halo, hydroxy, hydroxyCi-6 alkyl, Ci-6 alkoxy, haloCi-6 alkyl, cyano, nitro OC(O)Ci- 6 alkyl, and amino), phenoxy (wherein phenyl itself may be further substituted e.g., by Ci-6 alkyl, halo, hydroxy, hydroxyCi-6 alkyl, Ci-ealkoxy, haloCi-6 alkyl, cyano, nitro OC(O)Ci-6 alkyl, and amino), benzyloxy (wherein benzyl itself may be further substituted e.g., by Ci-6 alkyl, halo, hydroxy, hydroxyCi-6 alkyl, Ci-6 alkoxy, haloCi-6 alkyl, cyano, nitro OC(O)Ci-6 alkyl, and amino), amino, alkylamino (e.g. Ci-6 alkyl, such as methylamino, ethylamino, propylamino etc), dialkylamino (e.g. Ci-6 alkyl, such as dimethylamino, diethylamino, dipropylamino), acylamino (e.g. NHC(0)CH3), phenylamino (wherein phenyl itself may be further substituted e.g., by Ci- 6 alkyl, halo, hydroxy, hydroxyCi-6 alkyl, Ci-6 alkoxy, haloCi-6 alkyl, cyano, nitro OC(O)Ci- 6 alkyl, and amino), nitro, formyl, -C(O)-alkyl (e.g. Ci-6 alkyl, such as acetyl), O-C(O)-alkyl (e.g. Ci-6 alkyl, such as acetyloxy), benzoyl (wherein the phenyl group itself may be further substituted e.g., by Ci-6 alkyl, halo, hydroxy, hydroxyl Ci-6 alkyl, Ci-6 alkoxy, haloCi-6 alkyl, cyano, nitro OC(O)Ci-6 alkyl, and amino), replacement of CH2 with C=O, CO2H, CO2 alkyl (e.g. C1-6 alkyl such as methyl ester, ethyl ester, propyl ester, butyl ester), CChphenyl (wherein phenyl itself may be further substituted e.g., by C1-6 alkyl, halo, hydroxy, hydroxyl C1-6 alkyl, Ci- 6 alkoxy, halo Ci-6 alkyl, cyano, nitro OC(0)Ci-6 alkyl, and amino), C0NH2, CONHphenyl (wherein phenyl itself may be further substituted e.g., by Ci-6 alkyl, halo, hydroxy, hydroxyl Ci- 6 alkyl, C1-6 alkoxy, halo C1-6 alkyl, cyano, nitro OC(O)Ci-6 alkyl, and amino), CONHbenzyl (wherein benzyl itself may be further substituted e.g., by C1-6 alkyl, halo, hydroxy, hydroxyl C1-6 alkyl, C1-6 alkoxy, halo C1-6 alkyl, cyano, nitro OC(O)Ci-6 alkyl, and amino), CONHalkyl (e.g. C1-6 alkyl such as methyl ester, ethyl ester, propyl ester, butyl amide) CONHdialkyl (e.g. C1-6 alkyl), aminoalkyl (e.g., HN C1-6 alkyl-, Ci-ealkylHN-Ci-6 alkyl- and (C1-6 alkyl)2N-Ci-6 alkyl-), thioalkyl (e.g., HS C1-6 alkyl-), carboxyalkyl (e.g., HO2CC1-6 alkyl-), carboxyesteralkyl (e.g., Ci- 6 alkylChCCi-6 alkyl-), amidoalkyl (e.g., H2N(O)CCI-6 alkyl-, H(CI-6 alkyl)N(O)CCi-6 alkyl-), formylalkyl (e.g., OHCC1-6 alkyl-), acylalkyl (e.g., C1-6 alkyl(O)CCi-6 alkyl-), nitroalkyl (e.g., O2NC1-6 alkyl-), sulfoxidealkyl (e.g., Rf(O)SCi-6 alkyl, such as Ci-6alkyl(O)SCi-6 alkyl-), sulfonylalkyl (e.g., Rf(O)2SCi-6 alkyl- such as C1-6 alkyl(O)2SCi-6 alkyl-), sulfonamidoalkyl (e.g., HRfN(O)SCi-6 alkyl such as H(CI-6 alkyl)N(O)SCi-6 alkyl-).

[0077] The term “heteroatom” or “hetero” as used herein in its broadest sense refers to any atom other than a carbon atom which may be a member of a cyclic organic group. Particular examples of heteroatoms include nitrogen, oxygen, sulfur, phosphorous, boron, silicon, selenium and tellurium, more particularly nitrogen, oxygen and sulfur.

[0078] For monovalent substituents, terms written as “[group A] [group B]” refer to group A when linked by a divalent form of group B. For example, “[group A] [alkyl]” refers to a particular group A (such as hydroxy, amino, etc.) when linked by divalent alkyl, i.e. alkylene (e.g. hydroxyethyl is intended to denote HO-CH2-CH-). Thus, terms written as “[group]oxy” refer to a particular group when linked by oxygen, for example, the terms “alkoxy” or “alkyloxy”, “alkenoxy” or “alkenyloxy”, “alkynoxy” or “alkynyloxy”, “aryloxy” and “acyloxy”, respectively, denote alkyl, alkenyl, alkynyl, aryl and acyl groups as hereinbefore defined when linked by oxygen. Similarly, terms written as “[group]thio” refer to a particular group when linked by sulfur, for example, the terms “alkylthio”, “alkenylthio”, “alkynylthio” and “arylthio”, respectively, denote alkyl, alkenyl, alkynyl and aryl groups as hereinbefore defined when linked by sulfur.Surfactants

[0079] The present disclosure relates to a surfactant, which is a block copolymer. The block copolymer has a backbone comprising:(i) a first block which is either soluble in an aqueous phase (and / or polar phase and / or hydrophilic phase) or ii) soluble in a hydrocarbon phase (and / or lipophilic phase); and(ii) a second block which is a fluoropolyalkyl block or a fluoropolyether block; wherein one or both of the following apply:(a) the first block presents its functionality pendant to the block copolymer backbone;(b) the second block presents its functionality pendant to the block copolymer backbone.

[0080] As used herein, the term “block copolymer” is intended to mean a copolymer having two or more polymer chains (or blocks) that are chemically different and covalently attached to each other, either directly or indirectly via a linking moiety. In the context of the present disclosure, the block copolymer has a first block which is soluble in either an aqueous (and / or polar and / or hydrophilic) phase or a hydrocarbon (and / or lipophilic) phase covalently attached to a second block which is fluorinated.

[0081] The “backbone” of the block copolymer represents the main chain of its molecular structure.

[0082] The first block may itself be polymeric or oligomeric. The first block may be a homopolymer or a copolymer (including a random or block copolymer).

[0083] The first block may be soluble in an aqueous phase. Accordingly, in some embodiments, the first block is a hydrophilic block. A “block which is soluble in an aqueous phase”, also referred to herein as a “hydrophilic block”, is intended to refer to a section or region of the copolymer (i.e. a block) that exhibits overall hydrophilic character. By having“hydrophilic” character is meant the block, if isolated, would be soluble in an aqueous, polar and / or hydrophilic liquid. For the purpose of determining such hydrophilic character, the term “aqueous liquid” is intended to mean at least 50 vol%, at least 60 vol%, at least 70 vol%, at least 80 vol%, at least 90 vol%, at least 95 vol%, or at least 98 vol% of water. The aqueous liquid may comprise one or more other components, such as aqueous soluble liquids, for example methanol, ethanol and / or tetrahydrofuran (THF). The block will itself be polymeric or oligomeric.

[0084] The hydrophilic block of the present disclosure will comprise hydrophilic (and / or polar and / or hydrophilic) moiety or functionality that imparts overall hydrophilic character to the first block. The hydrophilic block may comprise other moieties or functionality that in their own right do not impart hydrophilic character per se. The hydrophilic block will be defined by a polymeric or oligomeric section or region that overall presents the required hydrophilic character.

[0085] The first block may be soluble in a hydrocarbon phase. Accordingly, in some embodiments, the first block is a lipophilic block. A “block which is soluble in a hydrocarbon phase”, also referred to herein as a “lipophilic block”, is intended to refer to a section or region of the copolymer (i.e. a block) that exhibits overall lipophilic character. By having “lipophilic” character is meant the block, if isolated, would be soluble in a hydrocarbon, non-polar and / or lipophilic liquid. The lipophilic block may be a hydrocarbon block.

[0086] The lipophilic block of the present disclosure will comprise lipophilic (and / or non-polar and / or lipophilic) moiety or functionality that imparts overall lipophilic character to the lipophilic block. The lipophilic block may comprise other moieties or functionality that in their own right do not impart lipophilic character per se. The lipophilic block will be defined by a polymeric or oligomeric section or region that overall presents the required lipophilic character.

[0087] In preferred embodiments, the first block is non-ionic.

[0088] The second block is a fluorinated block. A “fluorinated block” is intended to refer to a section or region of the copolymer (i.e. a block) that is replete with fluorine functionality. The fluorinated block may be a fluoropolyalkyl block or a fluoropolyether block. A fluoropolyalkyl block will comprise a repeat hydrocarbon group in which one or more hydrogen atoms have been replaced with a fluorine atom. A fluoropolyether block will comprise a repeat group having two or more hydrocarbon moieties linked with an oxygen atom defining the "ether" of the fluoropolyether. At least one and typically two or more of the hydrocarbon groups has at least one hydrogen atom replaced with a fluorine atom or a fluoroalkyl group.

[0089] In the block copolymer, either one or both of the first block and the second block present(s) their respective functionalities pendant to the block copolymer backbone.

[0090] In one embodiment, the first block presents its (hydrophilic or lipophilic) functionality pendant to the block copolymer backbone. In another embodiment, the first block presents its functionality in the block copolymer backbone.

[0091] In one embodiment, the second block presents its (fluorinated) functionality pendant to the block copolymer backbone. In another embodiment, the second block presents its functionality in the block copolymer backbone.

[0092] In one embodiment, the first block presents its functionality pendant to the block copolymer backbone and the second block presents its functionality pendant to the block copolymer backbone. In another embodiment, the first block presents its functionality in the block copolymer backbone and the second block presents its functionality in the block copolymer backbone. In another embodiment, both the first block and the second block present their respective functionalities pendant to the block copolymer backbone.

[0093] Molecular structural variations of the blocks are explained in further detail with reference to Scheme 1.Scheme 1. Pendant functionality (1) and in chain functionality (2). Backbone structure (1)- B - B - B - B - B - B - B - Backbone structure (2)

[0094] With reference to Scheme 1, structure (1) is a simplistic illustration of a block that presents its functionality (B) pendant to the block copolymer backbone (only part of which is shown). Structure (2) is a simplistic illustration of a block that presents its functionality (B) in the block copolymer backbone (only part of which is shown).

[0095] In preferred embodiments, the block copolymer has a backbone comprising:(i) a hydrophilic block; and(ii) a fluorinated block which is a fluoropolyalkyl block or fluoropolyether block, preferably a fluoropolyether block, wherein one or both of the following apply:(a) the hydrophilic block presents hydrophilic functionality pendant to the block copolymer backbone;(b) the fluorinated block presents fluorinated functionality pendant to the block copolymer backbone.

[0096] In these embodiments, the hydrophilic block preferably presents hydrophilic functionality pendant to the block copolymer backbone. In some embodiments, the hydrophilic block presents hydrophilic functionality pendant to the block copolymer backbone and the fluorinated block presents fluorinated functionality in the block copolymer backbone. In some embodiments, both the hydrophilic block and the fluorinated block present their respective functionalities pendant to the block copolymer backbone.

[0097] The first block that forms part of the block copolymer may be prepared by polymerising a monomer composition that comprises one or more monomers. Any monomer(s) suitable for preparing the first block may be used. The monomer(s) may be suitably selected depending on the desired properties of the block copolymer. In some embodiments, the first block is prepared by polymerising one monomer (i.e., one species of monomer). In some embodiments, the monomer is a hydrophilic monomer.

[0098] Examples of suitable monomers which may be used in preparing the first block include, but are not limited to, alkylene oxides, alkylene glycols, and ethylenically unsaturated monomers. Any suitable ethylenically unsaturated monomers may be used, including those known in the art and those described herein. The ethylenically unsaturated monomers may be selected from any acrylate, methacrylate, acrylamide, methacrylamide, vinyl ester, vinyl amide and styrene monomers. Examples of suitable ethylenically unsaturated monomers include acrylic acid, methacrylic acid, isopropyl acrylate, isopropyl methacrylate, N-isopropylacrylamide, N- isopropyhnethacrylamide, hydroxyethyl methacrylate, hydroxypropyl acrylate, hydroxypropyl methacrylate, oligo(alkylene glycol) methylether acrylate, methacrylate, acrylamide and methacrylamide, oligo(2-alkyl-2-oxazoline) acrylate, methacrylate, acrylamide and methacrylamide, oligo(2-aryl-2-oxazoline) acrylate, methacrylate, acrylamide and methacrylamide, hydroxyethyl acrylate, hydroxyethyl acrylamide, hydroxyethyl methacrylamide, methyl acrylate, methyl methacrylate, N-methylacrylamide, N-methyl methacrylamide, N,N-dimethylacrylamide, N,N-dimethyl methacrylamide, 2- (methylamino)ethyl acrylate, 2-(methylamino)ethyl methacrylate, (N-2-(N- methylamino)ethyl)acrylamide, (N-2-(N-methylamino)ethyl)methacrylamide, N,N- dimethylaminoethyl acrylate, N,N-dimethylaminoethyl methacrylate, N,N-dimethylaminoethyl acrylamide, N,N-dimethylaminoethyl methacrylamide, N,N-dimethylaminopropyl acrylate, N,N- dimethylaminopropyl methacrylate, N,N -dimethylaminopropyl acrylamide, N,N- dimethylaminopropyl methacrylamide, N-hydroxypropyl acrylamide, N-hydroxypropyl methacrylamide, 4-acryloylmorpholine, 2-acrylamido-2-methyl-l-propanesulfonic acid, N- vinylpyrolidone, oligo(2-methyl-2-oxazoline) (meth)acrylate, oligo(2-ethyl-2-oxazoline)(meth)acrylate, oligo(2-(n-propyl)-2-oxazoline) (meth)acrylate, 2-(methylsulfmyl)ethyl acrylate, (N-2-(methylsulfinyl)ethyl) acrylamide, (N-2-(methylsulfinyl)ethyl) methacrylamide, N-(2- acryloyloxyethyl)-N,N -dimethyl-N -(sulfopropyl)betain, 3 - [N,N -dimethyl-N - (methacryloyloxyethyl)ammonium]propanesulfonate, carboxybetaine acrylate, carboxybetaine methacrylate, 2-acryloyloxyethylphosphorylcholine and 2-methacryloyloxyethyl phosphatidylcholine. For example, the ethylenically unsaturated monomers may be selected from acrylic acid, methacrylic acid, isopropyl acrylate, isopropyl methacrylate, N- isopropylacrylamide, N-isopropylmethacrylamide, hydroxyethyl methacrylate, hydroxypropyl methacrylate, oligo(alkylene glycol) methylether (meth)acrylate, acrylamide and methacrylamide, hydroxyethyl acrylate, N-methylacrylamide, N,N-dimethylacrylamide, N,N- dimethylaminoethyl acrylate, N,N-dimethylaminoethyl methacrylate, N,N-dimethylaminopropyl methacrylamide, N-hydroxypropyl methacrylamide, 4-acryloylmorpholine, 2-acrylamido-2- methyl-l-propanesulfonic acid, N-vinylpyrolidone, oligo(2-methyl-2-oxazoline) (meth)acrylate, oligo(2-ethyl-2-oxazoline) (meth)acrylate, oligo(2-(n-propyl)-2-oxazoline) (meth)acrylate, 2- (methylsulfinyl)ethyl acrylate, 3-[N,N-dimethyl-N- (methacryloyloxyethyl)ammonium]propanesulfonate, carboxybetaine methacrylate, and 2- methacryloyloxyethyl phosphatidylcholine .

[0099] Examples of suitable monomers which may be used in preparing the first block also include monomers having the following structure of Formula (A):whereinRlais H or CHs; andRHis selected frompreferablywherein each Y is independently NH or O; andeach X is independently selected from H, Ci-ealkyl (for example isopropyl) and any one of the following:wherein each y is independently an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10; each W is independently Ci-3 alkylene, preferably C1-2 alkylene;R2is H or C1-6 alkyl, preferably H or C1-3 alkyl, more preferably H;R3aand R3bare independently H or C1-3 alkyl, preferably H or CH3; andR5is Ci-ealkyl, preferably C2-ealkyl.

[0100] In some embodiments, each X is independently selected from H, Ci-ealkyl (preferably isopropyl) and any one of the following:wherein is an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10; and R5is Ci-ealkyl, preferably C2-ealkyl.

[0101] In some embodiments, the first block is prepared by polymerising one or more monomers selected from N-isopropylacrylamide, N-isopropyhnethacrylamide, polyethylene glycol) methyl ether acrylate, polyethylene glycol) methyl ether methacrylate, poly[oligo(2- ethyl-2-oxazoline) acrylate], poly[oligo(2-ethyl-2-oxazoline) methacrylate], hydroxyethyl acrylate, hydroxyethyl methacrylate, 2-(methylsulfmyl)ethyl acrylate, 2-(methylsulfmyl)ethyl methacrylate, N,N-dimethylaminoethyl acrylate, N,N-dimethylaminoethyl methacrylate, N-(2- acryloyloxyethyl)-N,N -dimethyl-N -(sulfopropyl)betain, 3 - [N,N -dimethyl-N - (methacryloyloxyethyl)ammonium]propanesulfonate, carboxybetaine acrylate, carboxybetaine methacrylate, 2-acryloyloxyethylphosphorylcholine and 2-methacryloyloxyethyl phosphatidylcholine .

[0102] In some embodiments, the one or more monomers are selected from N- isopropylacrylamide, polyethylene glycol) methyl ether acrylate, poly[oligo(2-ethyl-2- oxazoline) acrylate], hydroxyethyl acrylate, 2-(methylsulfinyl)ethyl acrylate, N,N- dimethylaminoethyl acrylate, 3-[N,N-dimethyl-N- (methacryloyloxyethyl)ammonium]propanesulfonate, carboxybetaine methacrylate and 2- methacryloyloxyethyl phosphatidylcholine .

[0103] In some embodiments, the first block comprises the following structure:wherein* represents a covalent connection point to the remainder of the block co-polymer structure; g is an integer ranging from 1 to about 1000, for example from 1 to about 500, from 1 to about 400, from 1 to about 200, from 1 to about 100, from 1 to about 50, from 1 to about 40, from 1 to about 20, or from 1 to about 10;Rlais H or CHs; andRHis selected frompreferablywherein each Y is independently NH or O; and each X is independently H, Ci-ealkyl (for example isopropyl) or is selected from any one of the following:wherein each y is independently an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10; each W is independently C1-3 alkylene, preferably C1-2 alkylene;R2is H or C1-6 alkyl, preferably H or C1-3 alkyl, more preferably H;R3aand R3bare independently H or C1-3 alkyl, preferably H or CHs; andR5is Ci-ealkyl, preferably C2-ealkyl.

[0104] In some embodiments, each X is independently selected from H, Ci-ealkyl (preferably isopropyl) and any one of the following:wherein is an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10; and R5is Ci-ealkyl, preferably C2-ealkyl.

[0105] In some embodiments, the first block comprises a structure selected from any one of the following:wherein * represents a covalent connection point to the remainder of the block co-polymer structure; each g is independently an integer ranging from 1 to about 1000, for example from 1 to about 400, from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10; each y is independently an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10; and R5is Ci-ealkyl, preferably C2-ealkyl.

[0106] In some embodiments, the first block comprises a structure selected from any one of the following:wherein * represents a covalent connection point to the remainder of the block co-polymer structure; each g is independently an integer ranging from 1 to about 1000, for example from 1 to about 400, from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10; and v is independently an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10.

[0107] In some embodiments, the first block comprises a polyoxyalkylene moiety.

[0108] Examples of suitable polyoxyalkylene moieties include those comprising an oxyalkylene group of formula: -O(CRxRY)i-, where Rxand RYare each independently selected from hydrogen and optionally substituted alkyl, and i is an integer ranging from 1 to 10, for example from 1 to 8, or from 1 to 6, or from 1 to 4. Generally, Rxand RYare each independently selected from hydrogen and optionally substituted Ci-6 alkyl, and i is an integer selected from 2, 3, and 4. When i > 1, each (CRXRY) may be the same or different. For example, when the oxyalkylene unit is an oxyethylene unit, Rxand RYare both hydrogen and i=2 (i.e. -O(CH2)2-), and where the oxyalkylene unit is an oxypropylene unit, i=2 and Rxand RYof the first “i” areboth hydrogen and Rxand RYof the second “i” can respectively be hydrogen and methyl (i.e. - OCH2CH(CH3)-).

[0109] In one embodiment, the first block comprises an oxyethylene group, an oxypropylene group or a combination thereof.

[0110] Each oxyalkylene group or unit within the polyoxyalkylene may be the same or different. In other words, the polyoxyalkylene may be a homopolymer or a copolymer (including a random or block copolymer). The oxyalkylene units may be derived, for example, from an alkylene oxide such as ethylene oxide, propylene oxide, or butylene oxide.[oni] In one embodiment, the first block comprises an oxyethylene group, an oxypropylene group or a combination thereof.

[0112] The first block of the present disclosure (in isolation), which may be a hydrophilic block or a lipophilic block, will generally have a number average molecular weight ranging from about 100 to about 100,000 g / mol, and all combinations and sub-combinations of ranges therein. The number average molecular weight of the first block may be at least about 100, 150, 200, 300, 400, 500, 600, 700, 800, 900 or 1,000 g / mol. The number average molecular weight of the first block may be up to about 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 20,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000 or 2,000 g / mol. Any minimum and maximum amount may be combined to form a range, provided the range is between 100 to about 100,000 g / mol, for example from about 100 to about 5,000 g / mol and from about 100 to about 3,000 g / mol.

[0113] In the context of the present disclosure, the number-average molecular weight (An) and molecular weight distribution (molar mass dispersity, £> = v / A / n) of a polymer (or polymer block in isolation) are those determined by SEC using a Waters Alliance 2690 separation module equipped with a Waters 2414 differential refractive index (RI) detector, a Waters 2489 UV / vis detector, a Waters 717 Plus autosampler, and a Waters 1515 isocratic HPLC pump. THF was used as the mobile phase with a flow rate of 1 mL / min. The system is calibrated using polystyrene standards with molecular weights ranging from 6.82 x 102to 1.67 x 106g / mol. The polymers are dissolved in THF, filtered through a PTFE membrane (0.45 pm pore size), and then subjected to injection.

[0114] As described herein, the second block is a fluorinated block, which may be a fluoropolyalkyl block or a fluoropolyether block. In some embodiments, at least about 20% of the atoms of the second block are fluorine atoms.

[0115] The second block may be partially hydrogenated. In these embodiments, the second block preferably has a fluorine to hydrogen ratio of at least 1: 1.

[0116] The second block may be perfluorinated. In this context, the term “perfluorinated” is intended to mean all hydrogen atoms on the hydrocarbon groups of the second block are replaced with fluorine atoms. Accordingly, in some embodiments, the second block is a perfluoroalkyl block or a perfluoropolyether block. In preferred embodiments, the second block is a perfluoropolyether block.

[0117] The second block that forms part of the block copolymer may be prepared by polymerising ethylenically unsaturated monomer that comprises fluoropolyether functionality, for example fluoroether substituted vinyl ethers, perfluoroether substituted vinyl ethers, fluoroether substituted styrenes, perfluoroether substituted styrenes, fluoroether substituted norbomyl, perfluoroether substituted norbomyl, fluoropolyether (meth) acrylates and perfluoropolyether (meth) acrylates.

[0118] In preferred embodiments, the second block is a fluoropolyether block, which may be a perfluoropolyether block. In some embodiments, the (per)fluoropolyether block comprises a moiety selected from — (CPF2 O) — , — (CF(J)O) — , — (CF(J)CPF2 O) — , — (CPF2 CF(J)O) — , — CF2CF(J)O) — , or combinations thereof, where p is an integer ranging from 1 to 10, and where J is selected from a fluoroalkyl group, a fluoroether group, a fluoropolyether group, and a fluoroalkoxy group. In these embodiments, J may be selected from a perfluoroalkyl group, a perfluoroether group, a perfluoropolyether group, and a perfluoroalkoxy group. In some embodiments, the (per)fluoropolyether block comprises a moiety selected from C3F7O(CF(CF3)CF2O)qCF(CF3)— , C3F7O(CF2CF2CF2O)qCF2CF2— , and CF3O(C2F4O)qCF2— , where q has an average value of 1 to 50, for example 3 to 30, 3 to 15, or 3 to 10.

[0119] In some embodiments, the second block comprises the following structure:wherein* represents a covalent connection point to the remainder of the block co-polymer structure; and / is an integer ranging from 1 to about 100, for example from 1 to about 50, or from 1 to about 20.

[0120] In some embodiments, the second block comprises the following structure:wherein * represents a covalent connection point to the remainder of the block co-polymer structure; n is an integer ranging from 1 to about 30, for example from 1 to about 15; x is an integer ranging from 1 to about 100, for example from 1 to about 50, or from 1 to about 20; and Rlbis H or CHs.

[0121] The second block of the present disclosure (in isolation), which may be a fluoropolyalkyl block or a fluoropolyether block, will generally have a number average molecular weight ranging from about 1,000 to about 100,000 g / mol, and all combinations and sub-combinations of ranges therein. The number average molecular weight of the second block may be at least about 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 5,000 or 6,000 g / mol. The number average molecular weight of the second block may be up to about 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 20,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000 or 3,000 g / mol. Any minimum and maximum amount may be combined to form a range, provided the range is between 1,000 to about 100,000 g / mol, for example from about 1,000 to about 4,000 g / mol, from about 2,000 to about 3,000, from about 5,000 to about 10,000 g / mol, from about 2,500 to about 7,000 g / mol, and from about 6,000 to about 8,000.

[0122] Reference herein to a given block of the block copolymer having a number average molecular weight “in isolation” is intended to mean the molecular weight of that block alone as if it is not associated with any other block of the block copolymer.

[0123] The block copolymer of the present disclosure (overall) will generally have a molecular weight ranging from about 1,500 g / mol to about 110,000 g / mol.

[0124] Reference to the number average molecular weight of the block copolymer “overall” is intended to mean the molecular weight of the block copolymer inclusive of both the first and second blocks.

[0125] The first and / or second blocks of the block copolymer may form part of a linear, branched, hyperbranched, star or dendritic polymer structure. In preferred embodiments, the block copolymer has a linear structure.

[0126] More specific examples of the block copolymer used in accordance with the present disclosure include, but are not limited to, those comprising Formula (I), (II) or (III):wherein in Formula (I) m is an integer ranging from 1 to about 1000; n is an integer ranging from 1 to about 30; x is an integer ranging from 1 to about 100; and Rlbis H or CHs;in Formula (II) Z is NH or O, R4is H or CHs; a is an integer ranging from 1 to about 40; and b is an integer ranging from 1 to about 100; in Formula (III) is an integer ranging from 1 to about 40; and e is an integer ranging from 1 to about 100; and in Formulas (I), (II) and (III) * represents a covalent connection point to the remainder of the block co-polymer structure; each Rlais independently H or CHs; and each RHis independentlywherein each Y is independently NH or O; and each X is independently selected from H, Ci- ealkyl and any one of the following:wherein each y is independently an integer ranging from 1 to about 500; each W is independently Ci-3 alkylene; R2is H or Ci-6 alkyl; R3aand R3bare independently H or C1-3 alkyl; and R5is C1-6 alkyl.

[0127] In some embodiments, in Formula (I) one or more of the following apply: m is an integer ranging from 1 to about 400; n is an integer ranging from 1 to about 15; and x is an integer ranging from 1 to about 50. In some embodiments, Rlbis H. In some embodiments, Rlbis CH3.

[0128] In some embodiments, in Formula (II) one or both of the following apply: a is an integer ranging from 1 to about 20; and b is an integer ranging from 1 to about 50. In some embodiments, R4is H. In some embodiments, R4is CH3.

[0129] In some embodiments, in Formula (III) one or both of the following apply: is an integer ranging from 1 to about 20; and e is an integer ranging from 1 to about 50.

[0130] In some embodiments, in any one of Formulas (I), (II) and (III) Rlais H. In some embodiments, Rlais CH3.

[0131] In some embodiments, in any one of Formulas (I), (II) and (III) RHis

[0132] In some embodimentssome embodiments, Y is NH.

[0133] In some embodiments of RH, X is H. In some embodiments, X is Ci-ealkyl, for example isopropyl. In some embodiments, X is selected from any one of the following:wherein each y is independently an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10; and R5is Ci-ealkyl, preferably C2-ealkyl.

[0134] In some embodiments, in any one of Formulas (I), (II) and (III) Rlais H; RHisselected from any one of the following:wherein is independently an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10.

[0135] The block copolymers of the present disclosure may be prepared using polymerisation techniques well known in the art.

[0136] The block copolymers may be prepared in full or in part by the polymerisation of ethylenically unsaturated monomers, including those described herein. Polymerisation of such ethylenically unsaturated monomers is preferably conducted using a living or a so called "quasi" living polymerisation technique. Accordingly, in some embodiments, the block copolymer of the present disclosure is prepared using a living polymerisation method.

[0137] Living or quasi living polymerisation is generally considered in the art to be a form of chain polymerisation in which irreversible chain termination is substantially absent. An important feature of living polymerisation is that polymer chains will continue to grow while monomer and reaction conditions to support polymerisation are provided. Polymer chains prepared by living polymerisation can advantageously exhibit a well-defined molecular architecture, a predetermined molecular weight and narrow molecular weight distribution or low molar mass dispersity. Examples of living polymerisation include ionic polymerisation and controlled radical polymerisation (CRP). Examples of CRP include, but are not limited to, iniferter polymerisation, stable free radical mediated polymerisation (SFRP), atom transfer radical polymerisation (ATRP), and reversible addition fragmentation chain transfer (RAFT) polymerisation.

[0138] Equipment, conditions, and reagents for performing living polymerisation are well known to those skilled in the art.

[0139] Where ethylenically unsaturated monomers are polymerised by a living polymerisation technique, it will generally be necessary to make use of a so-called living polymerisation agent. The term “living polymerisation agent” is intended to refer to a compound that can participate in and control or mediate the living polymerisation of one or more ethylenically unsaturated monomers so as to form a living polymer chain (i.e. a polymer chain that has been formed according to a living polymerisation technique).

[0140] Living polymerisation agents include, but are not limited to, those which promote a living polymerisation technique selected from ionic polymerisation and CRP.

[0141] In one embodiment, the block copolymer is prepared using ionic polymerisation. In another embodiment, the block copolymer is prepared using CRP. In a further embodiment, the block copolymer is prepared using iniferter polymerisation. In another embodiment, the block copolymer is prepared using SFRP. In a further embodiment, the block copolymer is prepared using ATRP. In yet a further embodiment, the block copolymer is prepared using RAFT polymerisation.

[0142] A polymer prepared by RAFT polymerisation may conveniently be referred to as a RAFT polymer. By virtue of the mechanism of polymerisation, such polymers will comprise residue of the RAFT agent that facilitated polymerisation of the monomer.

[0143] RAFT agents suitable for use in accordance with the present disclosure comprise a thiocarbonylthio group (which is a divalent moiety represented by: -C(S)S-). RAFT polymerisation and RAFT agents are described in numerous publications such as WO98 / 01478, Moad G.; Rizzardo, E; Thang S, H. Polymer 2008, 49, 1079-1131 and Aust. J. Chem., 2005, 58, 379-410; Aust. J. Chem., 2006, 59, 669-692; and Aust. J. Chem., 2009, 62, 1402-1472. Suitable RAFT agents for use in preparing the polymers described herein include xanthate, dithioester, dithiocarbamate and trithiocarbonate compounds.

[0144] In some embodiments, the block copolymer described herein is prepared using a RAFT agent having the following structure:whereinRzis Ph or CnFhnS — wherein n is an integer ranging from 1 to 20, preferably from 4 to 12; and Rwis selected from:

[0145] The component Rwmay be functionalised by methods known in the art. For example, Rwmay be directly or indirectly linked to one of the first or the second block of the blockcopolymer of the present disclosure, as described herein (e.g. as used to prepare block copolymers having the structure of Formula (II)).

[0146] Accordingly, in some embodiments, the block copolymer described herein comprises within its structure -C(S)S-RZ, as residue of the RAFT agent that facilitated polymerisation to prepare the block copolymer. The component -C(S)S-RZmay also be referred to as the “end group”. The end group can be removed and / or functionalised by methods known in the art.

[0147] Further, in some embodiments, the block copolymer described herein comprises within its structure Rw, as residue of the RAFT agent that facilitated polymerisation to prepare the block copolymer. The component Rwmay be functionalised by methods known in the art. For example, Rwmay be directly or indirectly linked to one of the first or the second block of the block copolymer of the present disclosure, as described herein (e.g. as per block copolymers having the structure of Formula (II)).

[0148] Where a free radical polymerisation technique is to be used in polymerising one or more ethylenically unsaturated monomers so as to form all or part of the block copolymer, the polymerisation will usually require initiation from a source of free radicals.

[0149] A source of initiating radicals can be provided by any suitable means of generating free radicals, such as the thermally induced homolytic scission of suitable compound(s) (thermal initiators such as peroxides, peroxyesters, or azo compounds), the spontaneous generation from monomers (e.g. styrene), redox initiating systems, photochemical initiating systems or high energy radiation such as electron beam, X-ray or gamma-radiation.

[0150] Suitable initiating systems are described in commonly available texts. See, for example, Moad and Solomon "The Chemistry of Free Radical Polymerisation", Pergamon, London, 1995, pp 53-95.

[0151] A polymer prepared by ATRP polymerisation may conveniently be referred to as a ATRP polymer. By virtue of the mechanism of polymerisation, such polymers will comprise residue of the ATRP agent that facilitated polymerisation of the monomer.

[0152] ATRP initiators suitable for use in accordance with the present disclosure comprise an alkyl halide, which allows for introduction of a-fimctionality into a linear copolymer chain as known in the art. The halide is preferably a bromide or a chloride, more preferably a bromide. The ATRP initiator may be functionalised by methods known in the art. For example, the ATRP initiator may be directly or indirectly linked to one of the first or the second block of the block copolymer of the present disclosure, as described herein (e.g. as used to prepare block copolymers having the structure of Formula (III)).

[0153] Any suitable ATRP catalyst and ligand known in the art may be used. Examples of suitable catalysts include transition metal catalysts, including copper catalysts such as copper halides. Examples of suitable ligands include those described in Macromolecules 2000, 33, 1628-1635 and Macromolecular Chemistry and Physics 2004, 205, 551-566.

[0154] Methods for preparing block copolymers suitable for use in accordance with the present disclosure are also described in Macromolecules 2017, 50, 5953-5963, Nanoscale 10(17): 8226- 8239, ACS Nano 2018, 12, 9162-9176, Journal of the American Chemical Society 2021, 143, 35, 14106-14114 and Australian Journal of Chemistry 2012, 65, 1186-1190.

[0155] In some embodiments, the block copolymer of the present disclosure has a structure of Formula (la), (Ila) or (Illa):whereinin Formula (la) m is an integer ranging from 1 to about 1000; n is an integer ranging from 1 to about 30; x is an integer ranging from 1 to about 100; Rlbis H or CHs; Rzis Ph or CnFbnS — wherein n is an integer ranging from 1 to 20, preferably from 4 to 12; and Rwis selected fromin Formula (Ila) Z is NH or O; R4is H or CHs; Rzis Ph or CnFbnS — wherein n is an integer ranging from 1 to 20, preferably from 4 to 12; a is an integer ranging from 1 to about 40; and b is an integer ranging from 1 to about 100; in Formula (Illa) is an integer ranging from 1 to about 40; and e is an integer ranging from 1 to about 100; and in Formulas (la), (Ila) and (lib), Rlais H or CHs, and RHis selected fromwherein each Y is independently NH or O; and each X is independently selected from H, Ci-6 alkyl and any one of the following:wherein each y is independently an integer ranging from 1 to about 500; each W is independently C1-3 alkylene; R2is H or C1-6 alkyl; and R3aand R3bare independently H or C1-3 alkyl.

[0156] In some embodiments, in Formula (la) one or more of the following apply: m is an integer ranging from 1 to about 400, n is an integer ranging from 1 to about 15, and x is an integer ranging from 1 to about 50. In some embodiments, Rlbis H. In some embodiments, Rlbis CHs. In some embodiments, Rzis CnFbnS — wherein n is an integer ranging from 1 to 20, preferably from 4 to 12. In some embodiments, Rzis C12H25S — . In some embodiments, Rzis C4H9S — . In some embodiments, Rzis Ph. In some embodiments, Rwis. , . In someembodiments, Rwis. In some embodiments, Rwis. In some embodiments,

[0157] In some embodiments, in Formula (Ila) one or both of the following apply: a is an integer ranging from 1 to about 20, for example from 1 to about 10; and b is an integer ranging from 1 to about 50, for example from 1 to about 20. In some embodiments, R4is H. In some embodiments, R4is CHs . In some embodiments, Rzis CnFbnS — wherein n is an integer ranging from 1 to 20, preferably from 4 to 12. In some embodiments, Rzis C12H25S — . In some embodiments, Rzis C4H9S — . In some embodiments, Rzis Ph.

[0158] In some embodiments, in Formula (Illa) one or both of the following apply: is an integer ranging from 1 to about 20, for example from 1 to about 10; and e is an integer ranging from 1 to about 50, for example from 1 to about 20.

[0159] In some embodiments, in any one of Formulas (la), (Ila) and (Illa) Rlais H. In some embodiments, Rlais CHs.

[0160] In some embodiments, in any one of Formulas (la), (Ila) and (Illa) RHis

[0161] In some embodimentssome embodiments, Y is NH.

[0162] In some embodiments of RH, X is H. In some embodiments, X is Ci-ealkyl, for example isopropyl. In some embodiments, X is selected from any one of the following:wherein each y is independently an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10; and R5is Ci-ealkyl, preferably C2-ealkyl.

[0163] In some embodiments of RH, X is selected from any one of the following:wherein is independently an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10.

[0164] In some embodiments, the block copolymer of the present disclosure has the following structure:wherein a is an integer ranging from 1 to about 40, preferably 1 to about 20, more preferably 1 to about 10; and b is an integer ranging from 1 to about 100, preferably from 1 to about 50, more preferably from 1 to about 20.

[0165] In some embodiments, the block copolymer of the present disclosure has the following structure:wherein a is an integer ranging from 1 to about 40, preferably 1 to about 20, more preferably 1 to about 10; and b is an integer ranging from 1 to about 100, preferably from 1 to about 50, more preferably from 1 to about 20.

[0166] In some embodiments, the block copolymer of the present disclosure has the following structure:wherein a is an integer ranging from 1 to about 40, preferably 1 to about 20, more preferably 1 to about 10; b is an integer ranging from 1 to about 100, preferably from 1 to about 50, more preferably from 1 to about 20; and y is an integer ranging from 1 to about 500, for example from 1 to about 200, from 1 to about 100, from 1 to about 40, from 1 to about 20, or from 1 to about 10.

[0167] In some embodiments, the block copolymer of the present disclosure has the following structure:wherein a is an integer ranging from 1 to about 40, preferably 1 to about 20, more preferably 1 to about 10, most preferably 2 to 8; and b is an integer ranging from 1 to about 100, preferably from 1 to about 50, more preferably from 1 to about 20, most preferably from 2 to about 8.

[0168] In some embodiments, the block copolymer of the present disclosure has the following structure:wherein a is an integer ranging from 1 to about 40, preferably 1 to about 20, more preferably 1 to about 10, most preferably 2 to 3; and b is an integer ranging from 1 to about 100, preferably from 1 to about 50, more preferably from 1 to about 20, most preferably from 8 to about 20.Emulsions

[0169] As described herein and shown in the Examples, the block copolymer described herein is useful as a surfactant for stabilising an emulsion. The term “emulsion” as used herein, is intended to refer a stable mixture of at least two immiscible liquids. In general, immiscible liquids tend to separate into two distinct phases. An emulsion may thus be stabilised by the addition of a surfactant which can reduce the surface tension between the at least two immiscible liquids and / or to stabilise the interface.

[0170] Accordingly, the present disclosure provides the use of the surfactant described herein for the preparation of an emulsion. The emulsion may include any emulsion as described herein.

[0171] The present disclosure also provides an emulsion comprising:(a) a dispersed phase;(b) a continuous phase; and(c) a surfactant which is a block copolymer as described herein.

[0172] The emulsions of the present disclosure may comprise dispersed phase, continuous phase and surfactants in any amounts suitable to form an emulsion. A person skilled in the art will be readily able to determine such amounts.

[0173] The emulsion may be a single emulsion or a multiple emulsion (e.g. a double emulsion or a triple emulsion).

[0174] In embodiments where the emulsion is a single emulsion, the emulsion may be a water- in-oil (w / o) emulsion, which comprises an aqueous dispersed phase and an oil continuous phase. Alternatively, the single emulsion may be an oil-in-water (o / w) emulsion, which comprises an oil dispersed phase and an aqueous continuous phase. In some embodiments, the emulsion of the present disclosure is a single emulsion, which is preferably a water-in-oil emulsion.Advantageously, as shown in the Examples, block copolymers of the present disclosure are capable of stabilising aqueous droplets in water-in-oil emulsions under various dispersed aqueous phases and conditions. The block copolymers also exhibited comparable or improved activity compared to a commercially available fluorosurfactant.

[0175] Advantageously, as described herein, the surfactant of the present disclosure may be suitably tailored for an intended application in an emulsion. Methods are known in the art for predicting the suitability of a surfactant for a desired emulsion (e.g. a w / o emulsion or a o / w emulsion). For example, as described in the Examples herein, the hydrophilic -lipophilic balance(HLB) of a surfactant, which is a measure of its degree of hydrophilicity or lipophilicity, can be used to predict whether a surfactant may be suitable for a w / o emulsion or a o / w emulsion.

[0176] In embodiments where the emulsion is a double emulsion, the emulsion may be a water- in-oil-in-water (w / o / w) emulsion, which comprises aqueous droplets dispersed in oil droplets, which in turn are dispersed in a continuous aqueous phase; or an oil-in-water-in-oil (o / w / o) emulsion, which comprises oil droplets dispersed within water droplets, which in turn are dispersed in a continuous oil phase. In some embodiments, the emulsion of the present disclosure is a double emulsion, which is preferably a water-in-oil-in-water emulsion. Advantageously, as shown in the Examples, block copolymers of the present disclosure were capable of stabilising a water-in-oil-in-water emulsion and also allowed for cell growth in the droplets.

[0177] The emulsion may be a nanoemulsion, microemulsion or macroemulsion.

[0178] The emulsion may comprise one or more surfactants (i.e. one or more species of surfactant) as described herein. The surfactant(s) may be present in any concentration suitable for stabilising the emulsion. For example, the surfactant(s) may be present in an amount ranging from about 1 to about 10% w / w, based on the total weight of the continuous phase, and all combinations and sub-combinations of ranges therein. The surfactant(s) may be present in an amount of at least about 1, 2, or 3 % w / w, based on the total weight of the continuous phase. The surfactant(s) may be present in an amount up to about 10, 9, 8, 7, 6, 5, 4 or 3 % w / w, based on the total weight of the continuous phase. Any minimum and maximum amount may be combined to form a range, provided the range is between 1 to 10% w / w, for example from about 1 to about 5% w / w, or from about 3 to about 10% w / w, based on the based on the total weight of the continuous phase. The concentration of surfactant(s) may be suitably selected depending on the type of emulsion (e.g. single or double emulsion). For example, for a single emulsion, the surfactant(s) may be present in an amount ranging from about 1 to about 5% w / w, preferably from about 2 to about 4% w / w, based on the based on the total weight of the continuous phase. For a double emulsion, the surfactant(s) may be present in an amount ranging from about 2 to about 10% w / w, preferably from about 3 to about 10% w / w, based on the based on the total weight of the continuous phase.

[0179] Advantageously, as described herein and as shown in the Examples, the ability to tune the compositions, lengths, molecular weights, and / or the geometry of the surfactants as described herein can allow for the tailored stabilisation of emulsion droplets prepared with the block copolymers of the present disclosure. For example, higher ratios of the second (fluorinated) block to first (hydrophilic or lipophilic) block may be appropriate for stabilising very small aqueous droplets, whereas lower ratios may be useful for stabilising oil-in-water emulsions.

[0180] The continuous phase of the emulsion may comprise a solvent or an oil. In preferred embodiments, the continuous phase is an oil phase. The oil phase preferably comprises a fluorinated oil. The fluorinated oil is preferably a partially fluorinated hydrocarbon, a perfluorocarbon, a hydrofluoroether, or a mixture thereof. Particularly preferably the fluorinated oil is a hydrofluoroether. Preferred fluorinated oils include Novec™ 7500 (3-ethoxy- l,l,l,2,3,4,4,5,5,6,6,6-dodecafhroro-2-(trifluoromethyl)-hexane), Novec™ 7300 (l,l,l,2,2,3,4,5,5,5-decafhroro-3-methoxy-4-(trifluoromethyl)-pentane), Novec™ 7200 (C4F9OC2H5), Novec™ 7100 (C4F9OCH3), Fluorinert™ FC-72, Fluorinert™ FC-84, Fluorinert™ FC-77, Fluorinert™ FC-40, Fluorinert™ FC3283, Fluorinert™ FC-43, Fluorinert™ FC-70, perfluorodecalin and mixtures thereof. More preferred fluorous oils are Novec™ 7500, Fluorinert™ FC-40, Fluorinert™ FC3283 and perfluorodecalin, and still more preferred is Novec™ 7500. Further examples of suitable fluorinated oils include those and those described herein and those known in the art, such as those described in Lab on a Chip, Royal Society of Chemistry, 2012, 12 (3), pp.422-433.

[0181] The dispersed phase of the emulsion may be an aqueous phase, which may also be referred to as a polar phase or a hydrophilic phase; or a lipophilic phase, which may also be referred to as a non-polar phase or lipophilic phase. In preferred embodiments, the dispersed phase is an aqueous phase.

[0182] The dispersed phase may have any suitable diameter. The diameter of the dispersed phase may be suitably selected depending on the desired application of the emulsion. In some embodiments, the dispersed phase has an average diameter of from about 50 nm to about 1000 pm, and all combinations and sub-combinations of ranges therein. The dispersed phase may have an average diameter of from about 50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 800 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm or 100 pm. The dispersed phase may have an average diameter of up to about 1000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 550 pm, 400 pm, 450 pm, 400 pm, 350 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm or 150 pm. Any minimum and maximum amount may be combined to form a range, provided the range is between 50 nm to 1000 pm, for example about 50 nm to about 500 pm, or from about 50 nm to about 200 pm.

[0183] In preferred emulsions of the present disclosure, the dispersed phase is an aqueous phase, which comprises a plurality of droplets. In some embodiments, the droplets have an average diameter of from about 50 nm to about 1000 pm, and all combinations and subcombinations of ranges therein. The droplets may have an average diameter of from about 50nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 800 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 20 pm, 30 pm, 40 pm, 50 pm, 60 pm, 70 pm, 80 pm, 90 pm or 100 pm. The droplets may have an average diameter of up to about 1000 pm, 900 pm, 800 pm, 700 pm, 600 pm, 500 pm, 550 pm, 400 pm, 450 pm, 400 pm, 350 pm, 300 pm, 250 pm, 200 pm, 190 pm, 180 pm, 170 pm, 160 pm or 150 pm. Any minimum and maximum amount may be combined to form a range, provided the range is between 50 nm to 1000 pm, for example about 50 nm to about 500 pm, or from about 50 nm to about 200 pm.

[0184] The droplets may comprise one or more analytes. The analyte may be any entity of interest. Preferably each droplet comprises an average number of 0 to 100 analytes, more preferably 1 to 20 and still more preferably 1 to 5, e.g. 1 analyte.

[0185] In one embodiment, the analytes may be biological molecules, for example small molecules, amino acids, peptides, proteins, antibodies, enzymes, monosaccharides, disaccharides, oligosaccharides, polysaccharides, nucleic acids, oligonucleotides, nucleotides, metabolites, cofactors and artificially engineered molecules. Optionally the biological molecules may be contained in cells (e.g. mammalian cells, plant cells, algal cells, yeast cells, hybridomas, microorganisms), cell organelles (e.g. cell nuclei, mitochondria), viruses or prions.

[0186] In another embodiment, the analytes are biological analytes, for example cells, sub- cellular complexes of cellular building blocks or components. In some embodiments, the biological analytes are selected from cells, primary B-cells, T- cells, hybridomas, microorganisms, viruses, bacteria, or prions, cell organelles (e.g. cell nuclei, mitochondria) and exosomes. When the biological analyte is a cell, the cell may be selected from mammalian cells, plant cells, algal cells, microbial cells and yeast cells. Preferably molecules are produced in, excreted or secreted from the cells, e.g. molecules are excreted or secreted from the cells. When the biological analyte is a cell organelle, the cell organelle may be selected from cell nuclei and mitochondria.

[0187] In another embodiment, the analytes are assay components, for example beads, nanoparticles, crystals, micelles, quantum dots, detection reagents, antibodies, enzyme cofactors, nucleic acid amplification reagents, oligonucleotide sequencing reagents, cell transformation reagents, cell transduction mixtures and genome editing reagents. In some embodiments, the assay components are selected from beads, detection reagents, nucleic acid amplification reagents and genome editing reagents.

[0188] When at least some of the droplets contain a living entity, e.g. cell or bacterium, the aqueous phase preferably comprises a culture or growth medium. Any conventional medium may be used. The medium may, for example, comprise glucose, vitamins, amino acids, proteins,salts, pH indicators and density matching reagents, e.g. Ficoll. Sufficient medium may be provided to keep the entity alive for the duration of the analysis, reaction or other process of interest, e.g. sorting in a microfluidic device.

[0189] The droplets may further comprise one or more of an aqueous and non-aqueous phase, a chemical buffer, a biochemical buffer or a culture or other media, for example as required to carry out a reaction conducted therein. Examples of suitable chemical buffers include ammonium bicarbonate, ammonium acetate and phosphate- buffered saline (PBS). Examples of suitable biochemical buffers include HEPES, PBS and Trizma.

[0190] The present disclosure also provides an emulsion comprising the surfactant as described herein. The emulsion may further comprise a dispersed phase, as described herein. The emulsion may further comprise a continuous phase, as described herein.

[0191] Emulsions of the present disclosure may be 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, for example at a temperature of about 25 °C and a pressure of 1 atm. As used herein, a “stable emulsion” is intended to mean 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.Advantageously, as shown in the Examples, block copolymers of the present disclosure were capable of stabilising emulsion droplets for at least 24 hours under various dispersed phases and conditions.

[0192] Emulsions of the present disclosure may also be capable of on-demand demulsification, that is, demulsification in response to a stimulus. Such emulsions may comprise a plurality of stabilised droplets which coalesce upon exposure to a stimulus. Thus, the demulsification occurs in a controlled manner. The stimulus may be, for example, temperature (e.g., an increase in temperature, which may be an increase from room temperature to above about 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C or 60 °C) or pH (e.g., a decrease in pH, which may be a decrease from neutral pH to an acidic pH, for example a decrease to about pH 6.0, 5.5, 5.0, 4.5, 4.0, 3.5 or 3.0). Advantageously, as described herein and as shown in the Examples, surfactants of the present disclosure were capable of stabilising emulsion droplets at room temperature and neutral pH, with the droplets shown to coalesce at higher temperature and acidic pH.

[0193] The emulsion of the present disclosure may be prepared by methods known in the art, including methods as described herein.

[0194] The present disclosure provides a method of preparing an emulsion as described herein, the method comprising: providing a first phase which is an aqueous phase or a lipophilic phase; providing a second phase which is a solvent or an oil phase; and mixing the first phase, the second phase and the surfactant as defined herein to form the emulsion.

[0195] The aqueous or lipophilic phase, the solvent or oil phase and the surfactant may be any of those as described herein.

[0196] In preferred embodiments, the method comprises: providing an aqueous phase; providing an oil phase, which is preferably a fluorinated oil; and mixing the aqueous phase, the oil phase and the surfactant as defined herein to form the emulsion.

[0197] In one embodiment, the surfactant is mixed with (e.g. dissolved in) the oil phase, prior to mixing with the aqueous phase. In another embodiment, the surfactant is mixed with (e.g. dissolved in) the aqueous phase, prior to mixing with the oil phase. In another embodiment, the surfactant is mixed with (e.g. dissolved in) the aqueous phase and is separately mixed with (e.g. dissolved in) the oil phase, prior to mixing of the aqueous phase with the oil phase. Any conventional mixing method may be used, e.g. T-junction, step emulsification, flow focus junction etc.

[0198] Emulsions of the present disclosure may be formed using any suitable emulsification procedure known to those of ordinary skill in the art. In this regard, it is known that the emulsions can be formed using microfluidic systems, ultrasound, high pressure homogenisation, shaking, stirring, spray processes, membrane techniques, or any other appropriate method. For example, a micro-capillary or a microfluidic device may be 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.

[0199] In some embodiments, the mixing is by a flow focus junction of a microfluidic device, for example a microfluidic device as disclosed in US patent publication No. 2013 / 0139477 (corresponding to international publication WO 2012 / 022976) and US patent publication No. 2016 / 0252446 (corresponding to international publication WO 2015 / 015199). In particular, US 2013 / 0139477 paragraphs

[0051] and

[0066] and US 2016 / 0252446 paragraphs

[0176] to

[0180] ,This advantageously allows very small aqueous phases (e.g. microdroplets) to be produced, with volumes typically in the order of picolitres or nanoliters.

[0200] As described herein, emulsions of the present disclosure are also capable of on-demand demulsification. Accordingly, the present disclosure also provides a method of demulsifying an emulsion, the method comprising: providing an emulsion as described herein, or prepared according to a method as described herein, wherein the emulsion comprises a plurality of stabilised droplets which coalesce in response to a stimulus; and exposing the emulsion to said stimulus.Applications

[0201] As described herein and as shown in the Examples, various experiments, assays, reactions and processes may be carried out in the emulsions of the present disclosure. The dispersed phase of the emulsion (e.g. aqueous droplets) can serve as the site for the experiments, assays, reactions and processes. The surfactants of the present disclosure can stabilise the emulsion (e.g. a dispersed aqueous phase in an oil phase), allowing the experiment, assay, reaction or process to be carried out in the emulsion. The experiment, assay, reaction or process may therefore be carried out without the dispersed phase coalescing. The experiment, assay, reaction or process may involve one or more analytes present in the aqueous phase of the emulsion.

[0202] Accordingly, the present disclosure provides a method of performing one or more experiments, assays, reactions and processes within an emulsion as described herein (e.g. within the dispersed phase, which is preferably an aqueous phase, which may be aqueous droplets). The experiments, assays, reactions and processes carried out in the emulsions of the present disclosure may be carried out in a microfluidic channel or in a microfluidic device, e.g. the experiments, assays, reactions and processes may be carried out in one or more channels of a microfluidic device.

[0203] The present disclosure also provides a method of performing a chemical and / or biological reaction in the dispersed phase of an emulsion as described herein. In these embodiments, the dispersed phase is preferably an aqueous phase.

[0204] Examples of suitable chemical and / or biological reactions include chemical and / or biological reactions capable of being carried out in droplet based microfluidics applications. In some embodiments, the chemical and / or biological reaction is an enzymatic reaction. In some embodiments, the chemical and / or biological reaction is a polymerisation reaction. In anotherembodiment, the chemical and / or biological reaction is a molecular binding, molecular interaction, cellular interaction or conformational change resulting in a measurable signal.

[0205] The present disclosure also provides a method of preforming a biological processes in the dispersed phase of an emulsion as described herein. In these embodiments, the dispersed phase is preferably an aqueous phase.

[0206] Examples of suitable biological processes include biological processes capable of being carried out in droplet based microfluidics applications. In some embodiments, the biological process is antibody secretion or enzyme secretion by cells, or enzyme production inside cells. In some embodiments, the biological process is antibody binding. In some embodiments, the biological process may be a nucleic acid amplification process, partial or full nucleic acid replication process or nucleic acid transcription process. In some embodiments, the biological process may be cell proliferation, cell metabolism, cell transfection, cell transduction, cell signalling, cell apoptosis or cell death. In preferred embodiments, the biological process is PCR. The process used could be for digital PCR, for example droplet digital PCR. In some embodiments, the biological process is a genome editing process. The biological process may be sample preparation, e.g. oligonucleotide sample preparation process for sequencing. The biological process may be nucleic acid sequencing. The molecules being sequenced could be RNA or DNA and the sequencing could be at the genomic, epigenomic or transcriptomic level.

[0207] In some embodiments, the biological process is a bioassay, such as biosensing, cell assay, energy transfer-based assay, probing, protein / immunological assay, and microarray / biochip assay. Among the arrays used in microarray / biochip assay, fluorescencebased microarrays / biochips, such as antibody / protein microarrays, bead / suspension arrays, capillary / sensor arrays, DNA microarrays / polymerase chain reaction (PCR)-based arrays, glycan / lectin arrays, immunoassay / enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy / poisoning / toxicity, contamination and efficacy / mechanism, and quality control / safety.

[0208] The methods of performing described herein may comprise one or more chemical reactions, one or more biological reactions, one or more biological processes or a mixture thereof. Preferred chemical and / or biological reactions, and / or biological processes are as described above. Preferably, the method of performing one or more chemical and / or biological reactions, and / or biological processes in the dispersed phase of an emulsion as described herein (which is preferably an aqueous phase) is carried out in a microfluidic channel or microfluidic device. This enables chemical and / or biological reactions and / or biological processes to be performed on a very small scale, e.g. in microdroplets, and so very little material, e.g. biologicalmaterial, is required. The microfluidic channel or device is preferably controlled by an automated device and software.

[0209] Preferably, the method of performing one or more chemical and / or biological reactions, and / or biological processes in the dispersed phase of an emulsion as described herein is carried out under thermal, pH or environmental cycling conditions.

[0210] As described herein, the surfactants and emulsions of the present disclosure may be particularly useful in microfluidics applications. For example, the surfactants and / or emulsions described herein may be used in methods of sorting droplets, coalescing droplets or introducing fluid into a droplet. The surfactants and / or emulsions may also be used in methods of extracting a protein from a fluid. These methods are preferably carried in a microfluidic device. Examples of such methods are described in US patent publication No. 2020 / 0017635. In particular, US 2020 / 0017635 paragraphs

[0174] to

[0231] ,

[0211] Accordingly, the present disclosure also relates to various uses of the surfactants and emulsions of the present disclosure as described herein.

[0212] The present disclosure provides the use of a surfactant as described herein in a microfluidic channel or device. The present disclosure also provides the use of a surfactant as described herein in a molecular isolation in larger fluidic devices, containers or vats. Earger fluidic devices, containers or vats refers to devices, containers or vats which are larger than microfluidic devices. The skilled person will readily be able to distinguish between a microfluidic device and a larger device, container or vat. Preferably the larger fluidic devices, containers or vats are multi-litre sized, i.e. they have a multi-litre capacity. The present disclosure also provides the use of a surfactant as described herein in an automated device with associated software that controls a microfluidic channel or device.

[0213] The present disclosure also provides a surfactant as described herein for use in a microfluidic channel or device. The present disclosure also provides a surfactant as described herein for use in a molecular isolation in larger fluidic devices, containers or vats. The present disclosure also provides a surfactant as described herein for use in an automated device with associated software that controls a microfluidic channel or device.

[0214] Accordingly, the present disclosure also provides the use of a surfactant of the present disclosure in any one or more of the following: in a microfluidic channel or device, in a molecular isolation in larger fluidic devices, containers or vats, or in an automated device with associated software that controls a microfluidic channel or device.

[0215] The present disclosure also provides the use of an emulsion as described herein in a microfluidic channel or device. The present disclosure also provides the use of an emulsion asdescribed herein in an automated device with associated software that controls a microfluidic channel or device.

[0216] The present disclosure also provides an emulsion as described herein for use in a microfluidic channel or device. The present disclosure also provides an emulsion as described herein for use in an automated device with associated software that controls a microfluidic channel or device.

[0217] The present disclosure still further provides a method of using a surfactant as described herein in microfluidics, as well as the use of an emulsion as described herein in droplet-based microfluidic applications.

[0218] The term “microfluidics” as used herein refers to volumes of sample, and / or reagent, and / or amplified polynucleotide which are from about 0.1 pL to about 999 pL, such as from 1- 100 pL, or from 2 pL. Similarly, as applied to a cartridge, the term microfluidic means that various components and channels of the cartridge, as further described herein, are configured to accept, and / or retain, and / or facilitate passage of microfluidic volumes of sample, reagent, or amplified polynucleotide.

[0219] Non-limiting examples of microfluidic systems which may be suitable for use with the present disclosure include those described in the following: U.S. patent publication No. 2005 / 0172476; US patent publication No. 2006 / 0163385; US patent publication No.2007 / 0003442; US patent publication No. 2009 / 0131543 (corresponding to international publication WO 2006 / 096571); and US patent publication No. 2023 / 0279295 (corresponding to international publication WO 2022 / 029088). In particular, US 2005 / 0172476 paragraphs

[0059] ,

[0060] ,

[0065] to

[0068] ,

[0085] ,

[0086] , and

[0088] to

[0095] ; US 2006 / 0163385 paragraphs

[0076] to

[0095] ; US 2007 / 0003442 paragraphs

[0037] to

[0039] ,

[0045] to

[0048] ,

[0051] ,

[0056] to

[0059] ,

[0061] to

[0064] ,

[0066] to

[0068] ,

[0070] to

[0089] ,

[0105] and

[0106] ; US 2009 / 0131543 paragraphs

[0046] to

[0062] ; and US 2023 / 0279295 paragraph

[0018] ,

[0220] 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 ranging from about 100 pU to about 1 aU, and all combinations and sub-combinations of ranges therein. The volume may be at least about 1 aU, 5 aU, 10 aU, 50 aU, 100 aU, 500 aU, 1 fL, 5 fL, 10 fL, 50 fL, 100 fL, 500 fL, 1 pL or 5 pL. The volume may be up to about 100 pL, 50 pL, 10 pL, 5 pL, 1 pL, 500 nL, 100 nL, 50 nL, 10 nL, 5 nL 1 nL, 500 pL, 100 pL, 50 pL or 10 pL. Any minimum andmaximum amount may be combined to form a range, provided the range is between 100 pL to 1 aL, for example from about 1 nL to about 1 aL, or from about 10 pL to about 1 aL.

[0221] The microfluidic device for use according to the present disclosure may include one or more analysis units. An “analysis unit” is a microelement, e.g. a microchip. The analysis unit includes at least one inlet channel, at least one main channel, at least one inlet module, at least one coalescence module, and at least one detection module. The analysis unit may further include one or more sorting modules. The sorting module may be in fluid communication with branch channels which are in fluid communication with one or more outlet modules (collection module or waste module). For sorting applications, at least one detection module cooperates with at least one sorting module to divert flow via a detector-originated signal. It will be appreciated that the “modules” and “channels” are in fluid communication with each other and therefore may overlap, i.e., there may be no clear boundary where a module or channel begins or ends. A plurality of analysis units of the disclosure may be combined in one device. The microfluidic device for use according to the present disclosure include channels that form the boundary for a fluid. A “channel” as used herein is intended to mean a feature on or in a substrate that at least partially directs the flow of a fluid. In some cases, the channel may be formed, at least in part, by a single component, e.g. an etched substrate or molded unit. The channel can have any cross- sectional shape, for example, circular, oval, triangular, irregular, square or rectangular (having any aspect ratio), or the like, and can be covered or uncovered (i.e., open to the external environment surrounding the channel). In embodiments where the channel is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, and / or the entire channel may be completely enclosed along its entire length with the exception of its inlet and outlet. An open channel generally will include characteristics that facilitate control over fluid transport, e.g. structural characteristics (an elongated indentation) and / or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) and / or other characteristics that can exert a force (e.g., a containing force) on a fluid. The fluid within the channel may partially or completely fill the channel. In some cases, the fluid may be held or confined within the channel or a portion of the channel in some fashion, for example, using surface tension (e.g., such that the fluid is held within the channel within a meniscus, such as a concave or convex meniscus). In an article or substrate, some (or all) of the channels may be of a particular size or less, for example, having a largest dimension perpendicular to fluid flow of less than about 5 mm, less than about 2 mm, less than about 1 mm, less than about 500 microns, less than about 200 microns, less than about 100 microns, less than about 60 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 25 microns,less than about 10 microns, less than about 3 microns, less than about 1 micron, less than about 300 nm, less than about lOOnm, less than about 30 nm, or less than about 10 nm or less in some cases. Of course, in some cases, larger channels, tubes, etc. can be used to store fluids in bulk and / or deliver a fluid to the channel. In one embodiment, the channel is a capillary. The dimensions of the channel may be chosen such that fluid is able to freely flow through the channel, for example, if the fluid contains cells. The dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flow rate of fluid in the channel. Of course, the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel or capillary may be used. For example, two or more channels may be used, where they are positioned inside each other, positioned adjacent to each other, etc.

[0222] A preferred configuration of microfluidic device according to the present disclosure is a droplet microfluidizer. A “droplet” as used herein, is an isolated portion of a first fluid that completely surrounded by a second fluid. In some cases, the droplets may be spherical or substantially spherical; however, in other cases, the droplets may be non-spherical, for example, the droplets may have the appearance of "blobs" or other irregular shapes, for instance, depending on the external environment. As used herein, a first entity is “surrounded” by a second entity if a closed loop can be drawn or idealized around the first entity through only the second entity. The dispersed phase fluid can include a biological / chemical material, as described herein. The biological / chemical material can include tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, and pharmaceuticals. The biological / chemical material can include one or more labels known in the art. The label can be a DNA tag, dyes or quantum dot, or combinations thereof. The fluidic droplets may each be substantially the same shape and / or size. The shape and / or size can be determined, for example, by measuring the average diameter or other characteristic dimension of the droplets. The "average diameter" of a plurality or series of droplets is the arithmetic average of the average diameters of each of the droplets. Those of ordinary skill in the art will be able to determine the average diameter (or other characteristic dimension) of a plurality or series of droplets, for example, using laser light scattering, microscopic examination, or other known techniques. The diameter of a droplet, in a non- spherical droplet, is the mathematically defined average diameter of the droplet, integrated across the entire surface. The average diameter of a droplet (and / or of a plurality or series of droplets) may be, for example, less than about 1 mm, less than about 500 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 25 micrometers, less than about 10 micrometers, orless than about 5 micrometers in some cases. The average diameter may also be at least about 1 micrometer, at least about 2 micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 15 micrometers, or at least about 20 micrometers in certain cases. The droplet forming liquid is typically an aqueous buffer solution, such as ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example by column chromatography), 10 mM Tris HC1 and 1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetate buffer. Any liquid or buffer that is physiologically compatible with the population of molecules, cells or particles to be analyzed and / or sorted can be used. The fluid passing through the main channel and in which the droplets are formed is one that is immiscible with the droplet forming fluid. The fluid passing through the main channel can be a non-polar solvent, decane (e g., tetradecane or hexadecane), fluorocarbon oil, silicone oil or another oil (for example, mineral oil). The dispersed phase fluid may also contain biological / chemical material (e.g., molecules, cells, or other particles) for combination, analysis and / or sorting in the device. The droplets of the dispersed phase fluid can contain more than one particle or can contain no more than one particle. For example, where the biological material comprises cells, each droplet preferably contains, on average, no more than one cell. However, in some embodiments, each droplet may contain, on average, at least 1000 cells. The droplets can be detected and / or sorted according to their contents. The concentration (i.e., number) of molecules, cells or particles in a droplet can influence sorting efficiently and therefore is preferably optimized, in particular, the sample concentration should be dilute enough that most of the droplets contain no more than a single molecule, cell or particle, with only a small statistical chance that a droplet will contain two or more molecules, cells or particles. This is to ensure that for the large majority of measurements, the level of reporter measured in each droplet as it passes through the detection module corresponds to a single molecule, cell or particle and not to two or more molecules, cells or particles. The fluids used to generate droplets in microfluidic devices are typically immiscible liquids such as oil and water. These two materials generally have very different dielectric constants associated with them. These differences can be exploited to determine droplet rate and size for every drop passing through a small section of a microfluidic device. One method to directly monitor this variation in the dielectric constant measures the change in capacitance over time between a pair of closely spaced electrodes.

[0223] The present disclosure also provides solid phase particles and methods for the forming solid phase particles on a microfluidic device for downstream analysis. The solid phase particles can be used for various biological or chemical analysis (e.g., DNA or protein analyses). For DNA analysis, post amplification encapsulation of amplicons occurs within a gel or polymermatrix prior to breaking of the droplet emulsion. Amplification reactions within droplets using one of several amplification type methods including, but not limited to; PCR, Rolling Circle Amplification (RCA), Nucleic Acid Sequence Based Amplification (NASBA), ligase chain reaction, etc. followed by encapsulation / solidification of the amplified reaction within the droplets by either polymerizing the droplets using chemical or physical means. A physical means might be termed “gelling” whereby one incorporates low temperature agarose within the droplet during formulation and keeping the droplet above the solidification temperature until one desires the droplet to solidify.

[0224] The microfluidic device of the present disclosure can also include one or more detection modules. A “detection module” is a location within the device, typically within the main channel where molecules, cells, small molecules, or particles are to be detected, identified, measured or interrogated on the basis of at least one predetermined characteristic. The molecules, cells, small molecules, or particles can be examined one at a time, and the characteristic is detected or measured optically, for example, by testing for the presence or amount of a reporter. For example, the detection module is in communication with one or more detection apparatuses. The detection apparatuses can be optical or electrical detectors or combinations thereof. Examples of suitable detection apparatuses include optical waveguides, microscopes, diodes, light stimulating devices, (e.g., lasers), photo multiplier tubes, and processors (e.g., computers and software), and combinations thereof, which cooperate to detect a signal representative of a characteristic, marker, or reporter, and to determine and direct the measurement or the sorting action at the sorting module. However, other detection techniques can also be employed. The term “determining” as used herein, generally refers to the analysis or measurement of a species, for example, quantitatively or qualitatively, and / or the detection of the presence or absence of the species. “Determining” may also refer to the analysis or measurement of an interaction between two or more species, for example, quantitatively or qualitatively, or by detecting the presence or absence of the interaction. Examples of suitable techniques include, but are not limited to, spectroscopy such as infrared, absorption, fluorescence, UV / visible, FTIR ("Fourier Transform Infrared Spectroscopy"), or Raman; gravimetric techniques; ellipsometry; piezoelectric measurements; immunoassays; electrochemical measurements; optical measurements such as optical density measurements; circular dichroism; light scattering measurements such as quasielectric light scattering; polarimetry; refractometry; or turbidity measurements as described further herein. A detection module is within, communicating or coincident with a portion of the main channel at or downstream of the inlet module and, in sorting embodiments, at, proximate to, or upstream of, the sorting module or branch point. Thesorting module may be located immediately downstream of the detection module, or it may be separated by a suitable distance consistent with the size of the molecules, the channel dimensions and the detection system. Precise boundaries for the detection module are not required but are preferred.

[0225] As a matter of convenience, predetermined amounts of the surfactants and / or emulsions described herein and employed in the present disclosure can be optionally provided in a kit in packaged combination to facilitate the application of the various assays and methods described herein.

[0226] Accordingly, the present disclosure also provides a kit comprising a surfactant as described herein. The present disclosure also provides a kit comprising an emulsion as described herein.

[0227] Such kits also typically include instructions for carrying out the subject assay, reaction or process, and may optionally include the fluid receptacle, e.g., the cuvette, multiwell plate, microfluidic device, etc. in which the assay, reaction or process is to be carried out. The kit may also comprise reagents including tissues, cells, particles, proteins, antibodies, amino acids, nucleotides, small molecules, substrates, and / or pharmaceuticals. The surfactants and / or reagents may be provided in the emulsion. The surfactants, reagents and / or emulsions may be provided in pre-measured container (e.g., vials or ampoules) which are co-packaged in a single box, pouch or the like that is ready for use. The container holding may be configured to readily attach to the fluid receptacle of the device in which the reaction is to be carried out (e.g., the inlet module of the microfluidic device as described herein).

[0228] It will be understood that the present disclosure extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the present disclosure.

[0229] It will be appreciated by persons skilled in the art that numerous variations and / or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

[0230] The surfactants, emulsions, methods, uses and kits described herein are described by the following illustrative and non-limiting examples.EXAMPLESEXAMPLE 1MATERIALS AND METHODSMaterials

[0231] Hydroxy-terminated perfluorinated poly (propylene ether) (PFPE-OH, MW approx. 2000 g / mol) was purchased from The Chemours Company. 2-Hydroxyethyl acrylate (96%), acryloyl chloride (97%), poly (ethylene glycol) methyl ether acrylate (OEGA, MW 480 g / mol), and 2,2'-azobis(2-methylpropionitrile) (AIBN) were purchased from Sigma Aldrich and used as received. 2-(Methylsulfmyl)ethyl acrylate was prepared according to Fu et al., Macromolecules, 2018, 51, 15, 5875-5882. RAFT agent 2-(n-butyltrithiocarbonate)propionic acid (BTPA) was prepared according to Ferguson et al., Macromolecules , 2005, 38, 6, 2191— 2204.Nuclear Magnetic Resonance (NMR)

[0232] 1H NMR spectra of polymer solutions in CDCh were acquired on a Bruker AVANCE 400 MHz spectrometer at 25 °C. The solvent was deuterated chloroform (CDCh). A 90° pulse width 14 ps, relaxation delay 1 s, acquisition time 4.1 s, and 64 scans were used in all measurements.

[0233] 19F NMR spectra were acquired on a Bruker AVANCE 400 MHz spectrometer at 25 °C. The solvent was deuterated chloroform (CDCh). A 90° pulse width 14 ps, relaxation delay 1 s, acquisition time 4. 1 s, and 128 scans were used in all measurements.Size Exclusion Chromatography (SEC)

[0234] SEC was used to determine molecular weights and molecular weight distributions using a Waters Alliance 2690 separations module outfitted with a Waters 2414 refractive index (RI) detector, a Waters 2489 UV / vis detector, a Waters 717 Plus autosampler, and a Waters 1515 isocratic HPLC pump. The mobile phase was THF at a flow rate of 1 mL / min. Before testing, the samples were dissolved in THF at a predetermined concentration (1 mg / mL) and passed through 0.45 m PTFE filters. The molecular weight was determined in comparison to polystyrene standards.Flash Chromatography

[0235] Automatic flash chromatography was carried out using a compact Pure Chromatography Systems (BUCHI) equipped with an ELSD and UV detection, utilising FlashPure Silica Cartridge (80g, 40UM irregular particle shape; BUCHI) eluted with appropriate solvent pairings. All of the chromatographic solvents were ACS grade or above and were utilised without additional purification.Cell Preparation

[0236] GFP -tagged .S', cerevisiae strain (CEN.PK2-1C-GFP) was used in the cell growth assays. CEN.PK2-1C-GFP were pre-cultured overnight at 30 °C, 200 rpm in synthetic drop-out medium (Xu X et al., Biotechnology for Biofuels, 2019, 12, 1), washed cell twice and re-inoculated into 5 ml uracil drop-out medium containing defined concentrations of potassium (l x translucent K+-free YNB, 1% glucose, and 0, 1, 10, and 50 mM potassium chloride).Chip fabrication

[0237] The flow-focusing water-oil (single) or water-oil-water (double) emulsion droplet generator used in this study was fabricated by standard photolithography techniques. A designed channel was patterned to a silicon wafer with SU-8 2100 or 2035 (MicroChem) to obtain a mold. Then, polydimethylsiloxane (PDMS) in liquid form was prepared by mixing the based and curing agent (10: 1 in weight, respectively) to duplicate the pattern on the mold. After a plasma cleaning process on the surface of glass slide and PDMS, the surface energy was increased and finally pressured to each other for an irreversible bonding.Single and double emulsion generation

[0238] Water-oil single droplets were generated using one of water, PBS and DMEM+10% FBS as the dispersed phase, and 2% fluorosurfactant as the continuous phase. Two syringe pumps (Harvard Apparatus, USA) were used to inject the dispersed phase and continuous phase respectively. The flow rates of the two phases were set as 1:3 (dispersed phase: 200 pl / h; continuous phase: 600 pl / h).

[0239] Double emulsion (DE) droplets were generated using yeast cell medium added with 30% OptiPrep™ (D1556; Sigma-Aldrich) as the dispersed (inner) phase, 3% PFPE-HEA4-added Novec 7500 oil (3M, St. Paul, MN, USA) as the middle phase, and 1% w / v Pluronic F-127 and 1% v / v Tween 20-added PBS as the outer phase. Three syringe pumps (Harvard Apparatus, USA) were used to inject the inner phase, oil, and outer phase, respectively. The flow rates of the three phases were set as 1:4: 10 (inner phase: 60 pl / h; oil: 180 pl / h; outer phase: 600 pl / h). A PTFE tubing (Cole-Parmer, Illinois, U.S.A.) with inner diameter 0.06-inch, outer diameter 0.02 inch was used to transfer DE microdroplets into a 2 ml Eppendorf TM safe-lock tube for storage.Image acquisition and data processing

[0240] A Nikon optical microscope (Eclipse TS100 Inverted Microscope, Nikon Inc., Japan) equipped with a digital camera (Canon, Tokyo, Japan) was used to capture the droplets and images. The number of yeast cell clusters in the droplets were counted by comparing the fluorescence images to bright filed images. The size distribution and average fluorescence intensity of the micro droplets was analysed by Image J (National Institute of Health, USA).HLB value

[0241] The hydrophilic-lipophilic balance (HLB) value for surfactants was determined by Griffin’s method using the following equation:HLB = 20 * Mh / Mwhere Mh is the molecular mass of the hydrophilic portion of the molecule, and M is the molecular mass of the whole molecule, giving a result on a scale of 0 to 20.

[0242] The HLB value can be used to predict the surfactant properties of a molecule. For example, a value from 3 to 6 may indicate a W / O emulsifier, and value from 8 to 18 may indicate an O / W emulsifier.Interfacial tension

[0243] The interfacial tension for surfactant enriched O / W interfaces were measured by a pendant drop method using a KRUSS DSA-10 droplet-shape analysis (DSA) instrument.PCR experiments

[0244] Microfluidic drop making devices were used to prepare -120 pm diameter monodisperse droplets stabilised by the indicated surfactants (2% w / w) in HFE7500 carrier oil. Each emulsion of droplets was prepared from 40 pl PCR mix comprising 32 pl water, 8 pl 5x Phusion HF detergent-free Buffer (F-520L, Thermo Fisher). The PCR program used was 98 °C for 30 s; then 35 cycles of 98 °C for 7 s, 60 °C for 30 s, and 72 °C for 20 s; then a final step of 72 °C for 10 min.Dye diffusion experiment

[0245] Syringe pumps (Harvard Apparatus, United States) were used to regulate the flow of various liquid streams. For imaging, emulsion droplets were pulled by capillary force into a hollow rectangular capillary tube (dimensions: ID 0.2 x 2.0 x 50 mm; source ProSciTech Pty Ltd) and then the open ends were sealed with Vaseline grease before being affixed to microscope cover glass (thickness: 0.13-0.16 mm). At days 0, 1, 2, and 3, Zeiss LSM 710 (Germany) was used to produce bright field and fluorescence images of fluorescein sodium salt. For the recording of green fluorescence signals, a 488 nm excitation wavelength and a 520 / 55 nm band pass filter were used. ImageJ was used to determine the fluorescence intensity of PBS-only droplets (National Institute of Health, USA).GFP-tagged S. cerevisiae cell growth experiment

[0246] The growth of a GFP-tagged .S', cerevisiae strain (GEN.PK2-1C) cultured in water-in- oil-in-water (w / o / w) double emulsions (DEs) was investigated. After cell encapsulation, the collected DEs were stored in a centrifugal tube and incubated for 24 h at 30 °C and 50 rpm. The total fluorescence intensity of yeast cells per droplet was then measured at 2 h, 4 h, 6 h, 10 h, 18 h and 24 h. The number of yeast cells per droplet was also measured by comparing the bright filed and fluorescence images. A Nikon optical microscope (Eclipse TS100 Inverted Microscope, Nikon Inc., Japan) equipped with a digital camera (Canon, Tokyo, Japan) was used to capture the droplets and images. The number of yeast cell clusters in the droplets was counted by comparingthe fluorescence images to bright filed images. The size distribution and average fluorescence intensity of the micro droplets was analyzed by Image J (National Institute of Health, USA). RESULTSSynthesis of perfluoropolyether methacrylate and oligo(ethylene glycol) methyl ether acrylate copolymers; where perfluoropolyether is pendant to the backbone (Formula I).Synthesis of perfluoropolyether methacrylate (PFPEMA)

[0247] Into a two-neck flask fitted with a dropping funnel, stirrer bar and purged with N2, monohydroxy-terminated perfluoropolyether (PFPE-ol) (1.65 kDa, 2.0 g, 1.21 mmol), triethylamine (211 pL, 0.153 g, 1.52 mmol), 2,6-di-t-butyl-4-methylphenol (20 mg) in a,a,a- trifluorotoluene (2 mL) (TFT) were added. Methacryloyl chloride (142 pL, 0.152 g, 1.45 mmol) was added dropwise at 0 °C, stirred for 1 h at this temperature, and then allowed to warm to room temperature overnight. The mixture was then diluted with TFT (20 mL), poured into water (50 mL) and the organic phase was washed with HC1 (5%, 2 x 50 mL), NaOH (2 x 50 mL) and water to neutrality followed by drying over MgSOi. The product was collected by filtration through a sintered glass funnel, before being reduced to dryness under vacuum (0. 1 mmHg). The perfluoropolyether methacrylate (PFPEMA), a colorless oil, was treated with another portion of 2,6-di-t-butyl-4-methylphenol (20 mg), sealed under N2 and stored below 5 °C. The resultant polymer was characterised by solution-state 'H and19F NMR (Figure 1).Production of perfluoropolyether methacrylate and oligo(ethylene glycol) methyl ether acrylate copolymers through RAFT polymerization

[0248] The copolymerization of OEGMA and PFPEMA was conducted as follows. In a typical experiment, OEGMA (1.2 g, 5 mmol), PFPEMA (1.73 g, 1 mmol), V40 (4.89 mg, 0.02 mmol), and CPADB (27.9 mg, 0.1 mmol) were dissolved in 5 mL TFT and sealed in a 25 mL flask fitted with a magnetic stirrer bar. The solution was then deoxygenated by purging thoroughly with nitrogen for 15 min, heated to 90 °C in an oil bath, and allowed to react for 24 h. The resulting solution was centrifuged for 5 min at 4000 rpm and the supernatant was precipitated into hexane and dissolved in THF three times. The precipitate was then dissolved in water and purified by dialysis, yielding a pink viscous solid after freeze drying.Synthesis of chain-end functionalized perfluoropolyether polymers with perfluoropolyether in the backbone (Formula II).Synthesis of PABTC-PFPE macro-RAFT agent 2-(butylthiocarbonothioylthio)propionic acid- perfluoropolyeth er

[0249] The PABTC-PFPE macro-RAFT agent was synthesised via a dicyclohexylcarbodiimide / 4-(dimethylamino)pyridine (EDC1 / DMAP) esterification couplingreaction of carboxylic acid from PABTC RAFT agent with PFPE-OH as shown in Scheme 1. Briefly, a solution of A-(3-dimcthylaminopropyl)-A' - ethylcarbodiimide hydrochloride (EDCI) (0.288 g, 1.50 mmol) in trifluorotoluene (TFT, 5 mL) was added dropwise to a solution of PFPEs (1.7 g, 1.0 mmol), 2- (butylthiocarbonothioylthio)propionic acid (PABTC, 0.381 g, 1.60 mmol) and 4-dimethyl aminopyridine (DMAP, 0.019 g, 0.16 mmol) in TFT (10 mL) at 0 °C. After complete addition, the reaction mixture was allowed to stir for 20 hours at room temperature. The reaction mixture was washed twice with a 1 M sodium hydroxide solution then twice with distilled water. The organic layer was dried over anhydrous magnesium sulfate, filtered, concentrated under vacuum and subjected to precipitation against methanol to remove the unreacted PABTC RAFT agent. The desired fraction was concentrated under vacuum to afford the product as a yellow oil. Synthesis of the macro-RAFT agent was validated byJH NMR and19F NMR.Scheme 1. Synthesis of macro-RAFT agentPFPE-OH

[0250] The RAFT agent can be varied so as to provide macro-RAFT agents with different end groups, for example 2-[[(dodecylthio)thioxomethyl]thio]-2-methylpropanoic acid or 4-cyano-4- (phenylcarbonothioylthio)pentanoic acid could be used instead of PABTC used in this example. Synthesis of chain-end functionalized perfluoropolyether polymers through RAFT polymerization

[0251] In this study, three distinct hydrophilic and molecular size segments were chosen for the preparation of fluorosurfactants: 2-hydroxyethyl acrylate (2-HEA), polyethylene glycol) methyl ether acrylate (OEGA) (Mn = 480), and 2-(methylsulfmyl)ethyl acrylate (MSEA). RAFT polymerisation was used to produce PFPE-(2-HEA), PFPE-(OEGA) and PFPE-(MSEA)-based surfactants, with the aim of preparing surfactants each having 5 degrees of polymerisation as shown in Scheme 2. In a typical experiment, the PABTC-PFPE macro-RAFT agent (400 mg, 0.18 mmol), AIBN (6.0 mg, 0.036 mmol) and a monomer (one of 2-HEA (104.6 mg, 0.90 mmol), OEGA (1.5 g, 3.13 mmol) and MSEA (146.1 mg, 0.90 mmol)) were dissolved in trifluorotoluene (TFT, 2 ml) and sealed in a glass flask fitted with a magnetic stirrer bar. Thesolution was then deoxygenated by purging thoroughly with argon for 15 minutes, heated to 70 °C in an oil bath, and allowed to react for 4 hours. Finally, the products PFPE-(2-HEA)s, PFPE- (OEGA)s and PFPE-(MSEA)s were respectively dried at 60 °C under vacuum to provide a viscous yellow liquid. Synthesis of PFPE-(2-HEA)s, PFPE-(OEGA)s and PFPE-(MSEA)s was confirmed byJH NMR and19F NMR (Figures 2-4).Scheme 2. Synthesis of PFPE-(2-HEA)s, PFPE-(OEGA)s and PFPE-(MSEA)s-based surfactants via RAFT polymerisation

[0252] The ratio of PABTC-PFPE macro-RAFT agent to hydrophilic monomer (e.g., 2-HEA, OEGA, MSEA) can be varied so as to produce block copolymer of varying weight ratios of PFPE to hydrophilic block, for example on average 5, 10, 20 or 40 monomer units. In this example, the ratio of hydrophilic monomer to macro-RAFT agent provided block copolymers having OEGA blocks with on average 1-10 monomer units.

[0253] The obtained copolymers were separated based on degree of polymerization using automated flash chromatography. After identifying fractionated elution settings by TLC analysis, these parent copolymers were dissolved in a THF solvent. Using a DCM / THF / MeOH solvent gradient, a commercially available silica chromatography column, and an evaporative lightscattering detector, an automated fractionation of parent copolymers was finished within 7 column volumes. Data fromJH NMR showed great separation with a different DPs of each copolymer. Besides, each parent copolymer exhibited a low molar mass dispersity (D < 1.1) and molecular weights that matched predictions.

[0254] The molecular characterisation data for PFPE-(2-HEA), PFPE-(OEGA) and PFPE- (MSEA)-based surfactants are summarised in Table 1.Table 1. Molecular characterization data for PFPE-(2-HEA), PFPE-(2-HEA), PFPE-(OEGA) and PFPE-(MSEA)-based surfactantsSynthesis of chain-end functionalized perfluoropolyether polymers with perfluoropolyether in the backbone (Formula III).Synthesis ofPFPE-Br

[0255] A suspension of monohydroxy PFPE (PFPE-OH; Ma= 2000 g / mol, 15.0 g, 7.50 mmol) in a,a,a-trifluorotoluene (5 mL) was cooled to 0 °C under argon and a solution of 2- bromoisobutyryl bromide (1.55 mL, 13.63 mmol) in trifluorotoluene (5 mL) was added dropwise over 0.5 min. The reaction mixture was stirred at 0 °C for 3 h followed by 18 h at room temperature. The mixture was allowed to phase separate and the bottom layer was collected and washed with saturated NaHCOs solution (2 x 100 mL) and water (2 x 100 mL). The organicphase was concentrated in vacuo to afford PFPE-Br as a clear oil, which was stored at < 5 °C until required.Synthesis of chain-end functionalized perfluoropolyether polymers through ATRP polymerization

[0256] In this study, a PFPE-(2-HEA)-based surfactant was prepared via ATRP polymerisation as shown in Scheme 3. PFPE-Br (An= 2.2 kg / mol, 300 mg, 0.136 mmol, 1 equiv.), N,N,N',N'',N"-pentamethyldiethylenetriamine (PMDETA, 31 pL, 0.136 mmol, 1 equiv.) and HEA (78.9 mg, 0.68 mmol, 5 equiv.) were dissolved in a trifluorotoluene solution. The solution was then added to a dried Schlenk tube complete with stirrer bar. The mixture was subjected to three freeze pump-thaw cycles and backfdled with argon. The Schlenk tube was then immersed in liquid nitrogen and once the solution had frozen CuBr (19.5 mg, 0.136 mmol) was added under a flow of argon. Another three freeze-pump-thaw cycles the Schlenk tube was backfdled with argon and the mixture stirred at room temperature for 10 mins to ensure homogeneity. The reaction mixture was then heated at 80 °C for 8 h. The reaction mixture was diluted with CHCh (1 mb) and passed through a column of basic AI2O3 to remove the copper catalyst. The fdtrate was precipitated into hexane (50 mb), collected via centrifugation and dried in vacuo (0. 1 mbar, 50 °C) to afford PFPE-HEA polymer.Scheme 3. Synthesis of PFPE-(2-HEA)-based surfactant via ATRP polymerisationEmulsion droplet stability test

[0257] The influence of the PFPE-(2-HEA), PFPE-(OEGA) and PFPR-(MSEA)-based surfactants on emulsion droplet stability was investigated. The surfactants were prepared in HFE 7500 solvent at 2% w / w concentration. PDMS microfluidic chips (channel width 100 pm, height 100 pm) were used to generate encapsulated 100 pm droplets (PBS, water, and DMEM+10% FBS dispersed phases). Pico-Surf™ (2% w / w in Novec™ 7500) was used as a reference standard.

[0258] The emulsion droplet stability after 24 h cultivation at 4 °C was tested. The PFPE-(2-HEA)4-based surfactant stabilized droplets demonstrated stable conditions among other fractioned PFPE-(2-HEA), similar to the commercial Pico-Surf™ surfactant. The PFPE-(OEGA)e-based surfactant stabilized droplets showed stable conditions among other fractioned PFPE-(OEGA). The PFPE-(MSEA)2-based surfactant stabilized droplets showed steady conditions among other fractioned PFPE-(MSEA). In contrast to 2-HEA and OEGA-based surfactants, the droplet size of fractioned PFPE-(MSEA) decreased from 150 pm to 100 pm, while the DP increased from 2 to 4 under three dispersion conditions. This may indicate that when the hydrophilicity of the PFPE-(MSEA) surfactant increases, the droplet size may decrease. The results for Pico-Surf™, PFPE-(2-HEA)4, PFPE-(OEGA)e, PFPE-(MSEA)2 are shown in Figures 5-8 respectively.

[0259] The emulsion droplet stability during PCR was also tested. In general, systems with high interfacial tensions are known to tend to separate out or coalesce more easily since higher interfacial energy drives a reduction in the interfacial area. In this example, both PFPE-(2-HEA)4 and PFPE-(MSEA)3-based surfactants showed the lowest interfacial tension among other fractioned surfactants, with droplets stabilised with these surfactants shown to exhibit practically negligible merging after 35 cycles of PCR. The data indicated that PFPE-(2-HEA)4 and PFPE- (MSEA)s showed the most stable during PCR reaction for 35 cycles at 60 °C to 98 °C. Surprisingly, these two surfactants outperformed the Pico-Surf™ surfactant, with droplets stabilised with Pico-Surf™ shown to be slightly merged. In contrast, the droplets made by fractioned PFPE-(OEGA)-based surfactants were shown to completely merge after PCR. The HLB value of the PFPE-(OEGA)-based surfactants having 2-6 DPs ranged from 7-10 (see Table 1 below), indicating these surfactants may be more suitable for oil-in-water emulsions. The results for PFPE-(2-HEA)4, PFPE-(MSEA)3, PFPE-(OEGA)e and Pico-Surf™ are shown in Figure 9.

[0260] The HLB and IFT values for PFPE-(2-HEA), PFPE-(OEGA) and PFPE-(MSEA)-based surfactants are summarised in Table 2.Table 2. HLB and IFT values for PFPE-(2-HEA), PFPE-(OEGA) and PFPE-(MSEA)-based surfactants.

[0261] In these tests, the PFPE-(2-HEA)4-based surfactant was shown to have the lowest IFT among other fractioned surfactant and the best droplet stability both under three dispersed conditions during 24 h at 37 °C and PCRtest. This surfactant was used in subsequent studies. Dye diffusion (inter droplet transfer) test

[0262] The PFPE-(2-HEA)4-based surfactant was used in dye diffusion experiments to investigate its influence on inter-droplet molecular transport. For the tests, 3 pM sodium fluorescein salt was used as a water-soluble and less-leaky dye. Equal amounts of PBS-only and PBS + fluorescein dye droplets were collected. Generated droplets were collected with Eppendorf tubes and incubated at 37 °C. Pico-Surf™ (2% w / w in Novec™ 7500) was used as a reference standard.

[0263] The results are illustrated in Figure 10. The PFPE-(2-HEA)4 showed a higher capacity to inhibit inter-droplet dye diffusion compared to Pico-Surf™. Quantitatively, on day 1, the PicoSurf™ demonstrated 3 times the fluorescence intensity of the PFPE-(2-HEA)4-based surfactant, indicating a significant quantity of leakage. On day 3, the mean green fluorescence signal of Pico-Surf™ stabilized PBS-only drops was around 2 times the signal detected in PBS-only dropsstabilized by PFPE-(2-HEA)4. In addition, the analysis of inter-droplet transport kinetics using the 3 pM sodium fluorescein salt revealed that for these two surfactants, a fewer amount of leakage occurs on day 1 in comparison to the leakage that takes place on day 3. Furthermore, testing of the interfacial tension of both fluorosurfactants showed there was no significant difference in their measured surface tension values.Double emulsion yeast stability test

[0264] The growth of a fluorescence GFP-tagged S. cerevisiae strain (GEN.PK2-1C) was investigated to test the biocompatibility of the PFPE-(2-HEA)4-based surfactant in a double emulsion. Water-in-oil-in-water (w / o / w) double emulsions can improve emulsion stability compared to single emulsions (e.g., droplet shrinkage) and can be useful for quick on-chip analytical procedures. Although they require an additional emulsification process, w / o / w double emulsions on- and off-chip can provide advantages, including compatibility with commercial fluorescence-activated cell sorting (FACS) sorting systems.

[0265] The results are illustrated in Figure 11. The results show that the number of fluorescent yeast cells per droplets increased overtime. In addition, the number of yeast cells per droplet increased from ~2 to ~12 after 24 hours incubation (50 rpm, 30 °C). Further, the average intensity of cells in droplets increased to 3 times more than original state, indicating the variation of fluorescence intensity can be also used to quantitatively describe cell growth in the droplets.EXAMPLE 2MATERIALS AND METHODSNuclear Magnetic Resonance (NMR)

[0266] 1H NMR spectra of polymer solutions in deuterated chloroform (CDCh) were acquired on a Bruker AVANCE 400 MHz spectrometer at 25 °C. A 90° pulse width 14 ps, relaxation delay 1 s, acquisition time 4.1 s, and 64 scans were used in all measurements.19F NMR spectra were acquired on a Bruker AVANCE 400 MHz spectrometer at 25 °C. The solvent was CDCh. A 90° pulse width 14 ps, relaxation delay 1 s, acquisition time 4.1 s, and 128 scans were used in all measurements.Image acquisition and analysis of droplets size

[0267] Images of droplets were taken by using a Nikon optical microscope (Eclipse TS 100 Inverted Microscope, Nikon Inc., Japan) equipped with a digital camera (Canon, Tokyo, Japan). Image J was used to analyze droplets size.RESULTSSynthesis of chain-end functionalized perfluoropolyether polymers with perfluoropolyether in the backbone (Formula II).Synthesis ofBTPA-PFPE macro-RAFT agent 2-(butylthiocarbonothioylthio)propionic acid- perfluoropolyeth er

[0268] The BTPA-PFPE macro-RAFT agent (also called PABTC-PFPE macro-RAFT agent) was synthesized by an EDCI / DMAP esterification reaction as generally described in Example 1. Typically, BTPA / PABTC (435 mg, 1.83 mmol), PFPE-OH (2 g, 1.52 mmol), and DMAP (56 mg, 0.46 mmol) were dissolved in 20 mL of TFT, followed by dropwise addition of another solution of EDCI (584 mg, 3.05 mmol) dissolved in DCM (5 mL) to the mixed reaction solution at 0 °C with stirring. Upon complete addition, the reaction mixture was reacted at room temperature for 48 h. The crude solution mixture was then precipitated into a large quantity of methanol five times for purification. BTPA-PFPE macro-RAFT agent, a yellow oil, was dried under high vacuum at 25 °C for 24 h using an oil pump to remove any residual solvent.Synthesis of p(DMAEA)2-PFPE Block Copolymers

[0269] P(DMAEA)2-PFPE was synthesized by RAFT polymerization as generally described in Example 1. The BTPA-PFPE macro-RAFT agent (700 mg, 0.315 mmol), DMAEA (N,N- dimethylaminoethyl acrylate, 150.3 mg, 1.050 mmol) and AIBN (10.34mg, 0.0631 mmol) were dissolved in 1.5 mL of TFT solution in a glass vial equipped with a suitable magnetic stirrer bar and sealed well using a specific septum. Then the solution blend underwent deoxygenation through argon venting for approximately 15 min while placed on ice. Subsequently, it underwent a reaction within an oil bath at 70°C for 4 h and 20 min with continuous stirring. Then the reaction was terminated by placing it in an ice bath and exposing it to the air. Subsequently, the solution was concentrated and precipitated into a large amount of hexane. After completing the centrifugation process, the precipitated polymer was redissolved in THF, and it was further subjected to centrifugation at 1500 rpm at room temperature for 10 min followed by undergoing another precipitation into the excessive hexane. This whole cycle was repeated three times for full purification. Finally, p(DMAEA)2-PFPE was acquired by evaporating remaining solvent under house vacuum at 30°C overnight. The assignedJH NMR spectrum of purified p(DMAEA)2-PFPE is shown in Figure 12.

[0270] The polymerization degrees of p(DMAEA)n-PFPE surfactants can preferably range from 2 to 3. In this example, the ratio of hydrophilic monomer to macro-RAFT agent provided block copolymers having DMAEA blocks with on average 2 monomer units.Synthesis of p(NIPAM) s-PFPE Block Copolymers

[0271] P(NIPAM)3-PFPE was synthesized by RAFT polymerization as generally described in Example 1. The BTPA-PFPE macro-RAFT agent (700 mg, 0.315 mmol), NIP AM (N- isopropylacrylamide, 118.8 mg, 1.328 mmol) and AIBN (10.34 mg, 0.0631 mmol) weredissolved in 1.5 mL of TFT solution in a glass vial equipped with a suitable magnetic stirrer bar and sealed well using a specific septum. Then the solution blend underwent deoxygenation through argon venting for approximately 15 min while placed on ice. Subsequently, it underwent a reaction within an oil bath at 70°C for 4 h and 20 min with continuous stirring. Then the reaction was terminated by placing it in an ice bath and exposing it to the air. Subsequently, the solution was concentrated and precipitated into a large amount of hexane. After completing the centrifugation process, the precipitated polymer was redissolved in THF, and it was further subjected to centrifugation at 1500 rpm at room temperature for 10 min followed by undergoing another precipitation into the excessive hexane. This whole cycle was repeated three times for full purification. Finally, p(NIPAM)3-PFPE was acquired by evaporating remaining solvent under house vacuum at 30°C overnight. The assignedJH NMR spectrum of purified p(NIPAM)3- PFPE is shown in Figure 13.

[0272] The polymerization degrees of p(NIPAM)n-PFPE surfactants can preferably range from 3 to 8. In this example, the ratio of hydrophilic monomer to macro-RAFT agent provided block copolymers having NIP AM blocks with on average 3 monomer units.Emulsion droplet studies

[0273] The development of droplet systems with on-demand demulsification (coalescence) can enable applications in, e.g., precise manipulation and encapsulation of cells or bioactive cargos.

[0274] In this study, DMAEA and NIP AM monomers were used to produce functional copolymers as surfactants for generating w / o droplets with different functions, in particular pH- and temperature-responsive droplets. The height and width of chips used were 50 pm. pH-responsive droplet formation

[0275] PBS buffer was used as the aqueous phase. pH 5.0 and 7.0 PBS buffer were prepared using 1 mg / mL HC1 and 1 mg / mL NaOH solution. P(DMAEA)2-PFPE dissolved in Novec™ HFE7500 at 2% w / w concentration was used as the oil phase. Commercial surfactant PicoSurf™ (2 % (w / w) in Novec™ HFE7500) was used as a reference standard. The specific speed of the oil phase was 600 pL per hour and the aqueous phase was 200 pL per hour. After the droplets came out, continued to run the pump for 5 minutes, a 1.5 mL Eppendorf tube was then used to collect the droplets for 15 minutes. Finally, pictures of droplets were taken. The magnification of the microscope was 4X.

[0276] As shown in Figure 14(a), p(DMAEA)2-PFPE stabilized droplets at neutral pH and lead to coalescence at an acidic pH of 5.0. In comparison, as shown in Figure 14(b), Pico-Surf™ stabilised droplets at neutral and acidic pH. This indicates that pDMAEA-PFPE is capable ofacting as a pH-responsive fluorosurfactant for controlled, on-demand demulsification of droplets with controlled droplet stability.Temperature-responsive droplet stability

[0277] PBS buffer was used as the aqueous phase. P(NIPAM)3-PFPE dissolved in Novec™ HFE7500 at 2% concentration was used as the oil phase. Commercial surfactant Pico-Surf™ (2 % (w / w) in Novec™ HFE7500) was used as a reference standard. The speed of the water and oil phases was the same as above. After the droplets came out, continued to run the pump for 5 minutes, a 1.5 mb Eppendorf tube was then used to collect the droplets for 15 minutes. Then droplets were placed at 40 °C for 1 hour. Finally, pictures of droplets at 25 °C and 40 °C were taken. The magnification of the microscope was 4X.

[0278] As shown in Figure 15(a), p(NIPAM)3-PFPE stabilized droplets at room temperature and coalesced at 40 °C. In comparison, as shown in Figure 15(b), Pico-Surf™ stabilised droplets at room temperature and 40 °C. This indicates that pNIPAM-PFPE is capable of acting as a thermo-responsive fluorosurfactant for on-demand demulsification of droplets with controlled droplet stability.

Claims

CLAIMS:

1. An emulsion comprising:(a) a dispersed phase which is an aqueous phase or a lipophilic phase;(b) a continuous phase comprising a solvent or an oil; and(c) a surfactant which is a block copolymer having a backbone comprising:(i) a first block which is soluble in an aqueous phase or soluble in a hydrocarbon phase; and(ii) a second block which is a fluoropolyalkyl block or a fluoropolyether block; wherein one or both of the following apply:- the first block presents its functionality pendant to the block copolymer backbone;- the second block presents its functionality pendant to the block copolymer backbone.

2. The emulsion of claim 1, wherein the first block presents its functionality pendant to the block copolymer backbone.

3. The emulsion of claim 1 or claim 2, wherein the first block is a hydrophilic block.

4. The emulsion of any one of claims 1 to 3, wherein the first block is prepared by polymerising one or more monomers selected from alkylene oxides, alkylene glycols, and ethylenically unsaturated monomers.

5. The emulsion of any one of claims 1 to 4, wherein the first block comprises the following structure:wherein * represents a covalent connection point to the remainder of the block co-polymer structure; g is an integer ranging from 1 to about 1000; Rlais H or CHs; andRHis selected fromwherein each Y is independently NH or O, and each X is independently selected from H, Ci- ealkyl and any one of the following:wherein each y is independently an integer ranging from 1 to about 500; each W is independently Ci-3 alkylene; R2is H or Ci-6 alkyl; R3aand R3bare independently H or C1-3 alkyl; and R5is Ci- ealkyl.

6. The emulsion of any one of claims 1 to 5, wherein the first block has a number average molecular weight ranging from about 100 to about 100,000 g / mol.

7. The emulsion of any one of claims 1 to 6, wherein at least about 20% of the atoms of the second block are fluorine atoms.

8. The emulsion of any one of claims 1 to 7, wherein the second block is a perfluoroalkyl block or a perfluoropolyether block.

9. The emulsion of any one of claims 1 to 8, wherein the second block is a fluoropolyether block.

10. The emulsion of claim 9, wherein the fluoropolyether block comprises a moiety selected from — (CPF2pO)— , — (CF(J)O)— , — (CF(J)CPF2pO)— , — (CPF2pCF(J)O)— , — CF2CF(J)O)— ,or combinations thereof, where p is an integer ranging from 1 to 10, and where J is selected from a fluoroalkyl group, a fluoroether group, a fluoropolyether group, and a fluoroalkoxy group.

11. The emulsion of any one of claims 1 to 10, wherein the second block has a number average molecular weight ranging from about 1,000 to about 100,000 g / mol.

12. The emulsion of any one of claims 1 to 11, wherein the block copolymer has a molecular weight ranging from about 1,500 g / mol to about 110,000 g / mol.

13. The emulsion of any one of claims 1 to 12, wherein the block copolymer comprises a structure of Formula (I), (II) or (III):wherein in Formula (I) m is an integer ranging from 1 to about 1000; n is an integer ranging from 1 to about 30; x is an integer ranging from 1 to about 100; and Rlbis H or CHs; in Formula (II) Z is NH or O, R4is H or CHs; a is an integer ranging from 1 to about 40; and b is an integer ranging from 1 to about 100; in Formula (III) d is an integer ranging from 1 to about 40; and e isan integer ranging from 1 to about 100; and in Formulas (I), (II) and (III) * represents a covalent connection point to the remainder of the block co-polymer structure; each Rlais independently H or CHs; and each RHis independently selected fromwherein each Y is independently NH or O; Rlais H or CHs; and each X is independently selected from H, Ci -ealkyl and any one of the following:wherein each y is independently an integer ranging from 1 to about 500; each W is independently Ci-3 alkylene; R2is H or Ci-6 alkyl; R3aand R3bare independently H or C1-3 alkyl; and R5is C1-6 alkyl.

14. The emulsion of any one of claims 1 to 13, wherein the block copolymer is prepared using a living polymerisation method.

15. The emulsion of any one of claims 1 to 14, wherein: the dispersed phase is an aqueous phase; and the continuous phase is an oil phase.

16. The emulsion of any one of claims 1 to 15, wherein the continuous phase is an oil phase which is a fluorinated oil.

17. The emulsion of any one of claims 1 to 16, wherein the emulsion is a single emulsion or a multiple emulsion.

18. The emulsion of any one of claims 1 to 17, wherein the emulsion comprises a plurality of stabilised droplets which coalesce in response to a stimulus, optionally wherein the stimulus is an increase in temperature and / or a decrease in pH of the emulsion.

19. A method of preparing an emulsion according to any one of claims 1 to 18, the method comprising: providing an aqueous phase or a lipophilic phase; providing a solvent or an oil phase; and mixing the aqueous or lipophilic phase, the solvent or oil phase, and a surfactant as defined in any one of claims 1 to 18 to form the emulsion.

20. The method of claim 19, wherein the mixing is by a flow focus junction of a microfluidic device.

21. A method of demulsifying an emulsion, the method comprising: providing an emulsion according to any one of claims 1 to 17, or prepared according to claim 19 or claim 20, wherein the emulsion comprises a plurality of stabilised droplets which coalesce in response to a stimulus, optionally wherein the stimulus is an increase in temperature and / or a decrease in pH of the emulsion; and exposing the emulsion to said stimulus.

22. Use of a surfactant as defined in any one of claims 1 to 14 for the preparation of an emulsion.

23. Use of a surfactant as defined in any one of claims 1 to 14 in a microfluidic channel or device, in a molecular isolation in larger fluidic devices, containers or vats, or in an automated device with associated software that controls a microfluidic channel or device.

24. A method comprising performing a chemical and / or biological reaction in the dispersed phase of any one of claims 1 to 18.

25. The method of claim 24, wherein any one or more of the following apply: the chemical and / or biological reaction is a polymerisation reaction; the chemical and / or biological reaction is an enzymatic reaction; the chemical and / or biological reaction involves a cell or a cellular component.

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