surfactant

Abstract

The present invention provides a surfactant of formula (I): (A) m -X-(B) n (I), wherein X is a linking group; each A is independently a fluorocarbon or perfluoropolyether; each B is independently (II), wherein a is an integer from 3 to 50, and each R is independently C 1‑6 alkyl, CH2CH2OC 1‑6 alkyl, or CH2CH(CH3)OC 1‑6 alkyl; m is an integer from 1 to 10; and n is an integer from 1 to 10.

Description

[0001] INTRODUCTION

[0002] The present invention relates to surfactants of formula (I) which are particularly useful for the stabilization of water-in-oil emulsions, for example in microfluidic devices. The surfactants of formula (I) generally have a star-like structure. The present invention also relates to processes for the preparation of surfactants of formula (I) and compositions, such as emulsions, comprising surfactants of formula (I). In addition, the present invention relates to the use of compounds of formula (I) as surfactants and various methods in which said surfactants and / or emulsions comprising said surfactants are employed, for example in droplet generation, droplet sorting, coalescing droplets, splitting droplets, dispensing of droplets, etc. BACKGROUND

[0003] Surfactants have been used for many years to produce stable emulsions for various applications. General background prior art relating to emulsions can be found in US 5,587,153; US 6,017,546; WO 2005 / 099661; US 2004 / 081633; US 6,379,682; US 2002 / 172703; WO 2004 / 038363; US 2005 / 087122; US 2007 / 275415 and US 2008 / 053205. Conventional surfactants generally comprise a hydrophilic head group that is soluble in the aqueous phase of the emulsion and one or more lipophilic tails that are soluble in the oil phase of the emulsion.

[0004] Recently, surfactant stabilized emulsions comprising droplets of water in a continuous oil phase have been applied to microfluidic technology, enabling, for example, high-throughput screening, enzyme studies, nucleic acid amplification and other biological processes. For example, biological assays can be performed in microfluidic devices using very small amounts of biological material. Further information on microfluidic technology can be found in the prior applications WO 2009 / 050512 and WO 2015 / 015199. Further general background prior art on droplets can be found in patents / applications of RainDance Technologies Inc, for example WO 2008 / 063227.

[0005] In microfluidic applications, it is advantageous to use oils, and especially fluorine-containing oils, as the continuous phase in emulsion formation and preparation, because they have useful microfluidic properties, such as low friction, non-volatility (unlike alcohols), temperature resistance, and oil-water emulsions can be readily generated.

[0006] However, conventional surfactants are generally unsuitable for stabilising emulsions comprising a fluorochemical oil phase due to solubility issues. Furthermore, many conventional surfactants are toxic to biological molecules and cells and can hinder gas transfer from the external environment to the interior region of the emulsion.

[0007] There is therefore a need for new surfactants suitable for stabilising water-in-oil emulsions, for example of fluorochemical oils, and in particular such emulsions comprising hydrophobic small organic molecules in the aqueous phase. SUMMARY

[0008] Viewed from a first aspect, the present application provides a surfactant of formula (I):

[0009] (A) m -X-(B) n (I)

[0010] wherein

[0011] X is a linking group;

[0012] each A is independently a fluorocarbon or perfluoropolyether;

[0013] each B is independently

[0014]

[0015] wherein a is an integer from 3 to 50 and each R is independently C 1-6 alkyl, CH2CH2OC 1-6 alkyl or CH2CH(CH3)OC 1-6 alkyl;

[0016] m is an integer from 1 to 10; and

[0017] n is an integer from 1 to 10.

[0018] Viewed from a further aspect, the present application provides a method for preparing a surfactant as described hereinabove, the method comprising:

[0019] reacting a compound of formula (II)

[0020] (A) m -X-(OH) n (II)

[0021] wherein

[0022] X is a linking group;

[0023] each A is independently a fluorocarbon or perfluoropolyether;

[0024] m is an integer from 1 to 10;

[0025] n is an integer from 1 to 10;

[0026] in reaction with a compound of formula (III)

[0027]

[0028] wherein each R is independently C 1-6 alkyl, CH2CH2OC 1-6 alkyl, CH2CH(CH3)OC 1-6 alkyl.

[0029] Viewed from a further aspect, the present application provides a method for preparing a surfactant as hereinbefore described, the method comprising reacting a compound comprising (A)m with a compound comprising (B)n, wherein the reaction is a coupling reaction and a linker X is formed between the (A)m and the (B)n.

[0030] Viewed from a further aspect, the present application provides a composition comprising a surfactant of formula (I) as hereinbefore described, preferably an emulsion.

[0031] Viewed from a further aspect, the present application provides an emulsion comprising a surfactant of formula (I) as hereinbefore described.

[0032] Viewed from a further aspect, the present application provides the use of a compound as hereinbefore defined as a surfactant of formula (I).

[0033] Viewed from a further aspect, the present application provides the use of a surfactant of formula (I) as hereinbefore described in the preparation of an emulsion.

[0034] Viewed from a further aspect, the present application provides a method of preparing an emulsion as hereinbefore defined, the method comprising:

[0035] (i) providing an aqueous phase;

[0036] (ii) providing an oil phase, preferably a fluorine-containing oil phase; and

[0037] (iii) mixing the aqueous phase, the oil phase and a surfactant of formula (I) as hereinbefore defined to form the emulsion.

[0038] Viewed from a further aspect, the present application provides a method comprising carrying out one or more chemical and / or biological reactions, and / or biological processes in a discontinuous aqueous phase of an emulsion as hereinbefore defined.

[0039] Viewed from a further aspect, the present application provides a method for classifying droplets in a microfluidic device, the method comprising:

[0040] (i) providing a stream of aqueous droplets in an emulsion as defined above in a channel of the microfluidic device;

[0041] (ii) illuminating the stream from a first direction;

[0042] (iii) detecting light from an analyte within the droplet in a second direction; and

[0043] (iv) classifying the droplet into one of a plurality of differential streams in response to the detected light or measurable signal.

[0044] Viewed from another aspect the present application provides a method of coalescing a droplet in a microfluidic device, the method comprising:

[0045] (i) providing at least two aqueous droplets in an emulsion as defined above in a channel of the microfluidic device; and

[0046] (ii) exposing the aqueous droplets to an electric field, thereby coalescing the at least two aqueous droplets into a single droplet.

[0047] Viewed from another aspect the present application provides a method of introducing a fluid into a droplet in a microfluidic device, the method comprising:

[0048] (i) providing an aqueous droplet in an emulsion as defined above in a channel of the microfluidic device; and

[0049] (ii) contacting the aqueous droplet with a stream of fluid, thereby introducing the fluid into the aqueous droplet.

[0050] Viewed from another aspect the present application provides a method of splitting a droplet in a microfluidic device, the method comprising:

[0051] (i) providing a microfluidic device comprising a microfluidic junction, the microfluidic junction comprising a first microfluidic channel, a second microfluidic channel and a third microfluidic channel;

[0052] (ii) providing an aqueous droplet in an emulsion as defined above in the first microfluidic channel; and

[0053] (iii) passing the aqueous droplet through the microfluidic junction, thereby splitting the aqueous droplet into at least a first sub-droplet and a second sub-droplet, the first sub-droplet being in the second microfluidic channel and the second sub-droplet being in the third microfluidic channel.

[0054] Viewed from another aspect the present application provides a method of dispensing a droplet of an emulsion as described above in a microfluidic device, the method comprising:

[0055] receiving a droplet of the emulsion as defined above from a droplet feed line into a droplet outlet line;

[0056] ejecting the droplet from the droplet outlet line through a droplet outlet by providing pressurised dispensing fluid into the droplet outlet line;

[0057] receiving the droplet from the droplet outlet line into a waste line when pressurised dispensing fluid is not provided into the droplet outlet line; and

[0058] protecting the droplet upstream of the droplet outlet line by providing pressurised dispensing fluid upstream of the droplet outlet line.

[0059] Viewed from another aspect, the present application provides a method of dispensing a droplet of an emulsion as described above in a microfluidic device, the method comprising:

[0060] receiving a droplet of the emulsion as defined above from a droplet feed line into a droplet outlet line;

[0061] ejecting the droplet from the droplet outlet line through a droplet outlet by providing pressurised dispensing fluid into the droplet outlet line;

[0062] receiving the droplet into a waste line when pressurised dispensing fluid is provided into the droplet outlet line; and

[0063] injecting fluid into the waste line when pressurised dispensing fluid is provided into the droplet outlet line.

[0064] Viewed from another aspect, the present application provides a method of classifying a droplet in a microfluidic device, the method comprising:

[0065] (i) providing a microfluidic device comprising a microfluidic junction, the microfluidic junction comprising a first microfluidic channel, a second microfluidic channel and a third microfluidic channel;

[0066] (ii) providing an aqueous droplet in an emulsion as defined above in the first microfluidic channel;

[0067] (iii) passing the aqueous droplet through the microfluidic junction, whereby the aqueous droplet is split into at least a first daughter droplet and a second daughter droplet, the first daughter droplet being in the second microfluidic channel and the second daughter droplet being in the third microfluidic channel;

[0068] (iv) detecting the first daughter droplet by mass spectrometry; and

[0069] (v) classifying the second daughter droplet into one of a plurality of differentiated streams in response to the mass spectrometry performed on the first daughter droplet.

[0070] Viewed from a further aspect, the present application provides a method of extracting a molecule from a fluid, the method comprising:

[0071] (i) dissolving a surfactant of formula (I) as hereinbefore defined in carbon dioxide to form a mixture of carbon dioxide / surfactant of formula (I);

[0072] (ii) adding a fluid comprising said molecule to the mixture of carbon dioxide / surfactant of formula (I), thereby extracting said molecule from the fluid into the carbon dioxide.

[0073] Viewed from a further aspect, the present application provides the use of a surfactant of formula (I) as hereinbefore defined in a carrier fluid (e.g. a fluorous oil) for droplet partitioning during droplet processing.

[0074] Viewed from a further aspect, the present application provides the use of a surfactant of formula (I) as hereinbefore defined in a microfluidic channel or device, in molecular isolation in a larger fluidic device, vessel or tank, or in an automated device with associated software controlling the microfluidic channel or device.

[0075] Viewed from a further aspect, the present application provides the use of an emulsion as hereinbefore defined in a microfluidic channel or device, or in an automated device with associated software controlling the microfluidic channel or device.

[0076] Definitions

[0077] As used herein, the term "fluorocarbon" refers to a hydrocarbon group in which one or more hydrogen atoms have been replaced by a fluorine atom. In a "perfluorocarbon", all hydrogen atoms have been replaced by fluorine atoms.

[0078] As used herein, the term "perfluoropolyether" refers to a polyether compound in which all hydrogen atoms have been replaced by fluorine atoms.

[0079] As used herein, the term "polyether" refers to an organic compound comprising two or more -O- linkages.

[0080] As used herein, the term "star surfactant" refers to a surfactant having a star-like shape. A star surfactant comprises a core as a linking group and at least two arms extending from the core, and more preferably at least 3 arms, wherein each arm is linear. The number of arms is determined by the core and in particular depends on the number of branching points in the core.

[0081] As used herein, the term "star polymer surfactant" refers to a surfactant having a core as a linking group and at least two arms and more preferably at least 3 arms extending from the core, wherein each arm is a linear oligomer or polymer. Each arm is polymerized in a linear fashion by successive polymerization at a single functional group in the growing arm. Thus, linear arms cannot give rise to branched arms.

[0082] As used herein, the term "charged group" refers to a group comprising at least one atom or group of atoms that carries a positive or negative charge. The term encompasses groups in which both positive and negative charges are present, i.e. zwitterionic groups.

[0083] As used herein, a wavy bond denotes the point of attachment of a group of a compound to another moiety that is a constituent of the compound. Thus, a group having one wavy bond is a terminal group, while a group having two wavy bonds is typically a linking group.

[0084] As used herein, the term "oligomer" when used in relation to group A of formula (I) refers to a linear chain of 3 to 10 glycerol units.

[0085] As used herein, the term "polymer" when used in relation to group A of formula (I) refers to a linear chain of 11 to 50 glycerol units.

[0086] As used herein, the term "linear" refers to a linear oligomer or polymer. A linear polymer does not contain side chains that are themselves oligomers or polymers.

[0087] As used herein, the term "alkyl" refers to a saturated linear, branched, or cyclic group. The alkyl group can be substituted or unsubstituted.

[0088] As used herein, the term "alkylene" refers to a divalent alkyl group.

[0089] As used herein, the term "substituted" refers to a group in which one or more, for example up to 6, more particularly 1, 2, 3, 4, 5, or 6, of the hydrogen atoms in the group are independently of each other replaced by a corresponding number of said substituents. The term "optionally substituted" as used herein means substituted or unsubstituted.

[0090] As used herein, the term "fluorine-containing" refers to any group or substance containing one or more fluorine atoms. Typically, the group or substance contains multiple fluorine atoms. For example, a fluorine-containing oil refers to any oil containing fluorine atoms, including partially fluorinated hydrocarbons, perfluorocarbons, hydrofluoroethers, and mixtures thereof.

[0091] As used herein, the term "leaving group" refers to any atom or group that can dissociate from a molecule upon heterolysis of a covalent bond that joins the leaving group to the remainder of the molecule, while the atom or group carries the bonding electrons from the covalent bond. DETAILED DESCRIPTION

[0092] The present invention relates to surfactants that are particularly useful for the stabilization of water-in-oil emulsions. The surfactants include one or more lipophilic perfluoropolyether or fluorocarbon tail that extends into the oil phase or "faces" the oil phase and one or more hydrophilic tail that extends into the water phase or "faces" the water phase, and a linking group that links the one or more lipophilic tail and the one or more hydrophilic tail. In preferred surfactants of the present invention, the surfactants include at least one lipophilic tail and at least one hydrophilic tail, and a linking group that links the one or more lipophilic tail and the one or more hydrophilic tail, and the total amount of lipophilic tails and hydrophilic tails is at least two, and more preferably at least three. Such surfactants are referred to herein as star surfactants due to their resulting structure's resemblance to a star.

[0093] The surfactants of the present invention have the formula (I):

[0094] (A) m -X-(B) n (I)

[0095] wherein

[0096] X is a linking group;

[0097] each A is independently a fluorocarbon or perfluoropolyether;

[0098] each B is independently

[0099]

[0100] wherein a is an integer from 3 to 50, and each R is independently a C 1-6 alkyl, CH2CH2OC 1-6 alkyl, or CH2CH(CH3)OC 1-6 alkyl;

[0101] m is an integer from 1 to 10; and

[0102] n is an integer from 1 to 10.

[0103] In formula (I), the A group is a lipophilic tail, and the B group is a hydrophilic tail. In formula (I), B is a linear glycerol oligomer or polymer comprising at least 3 glycerol repeat units. As shown in the formula above, the oligomer or polymer is linear rather than branched. This is because there is only one reactive group (i.e. -OH) in the B group. The linear form of the oligomer or polymer is important for achieving the star structure of the surfactant of the present application.

[0104] A preferred surfactant of the present application is a star surfactant. A particularly preferred surfactant of the present application is a star polymer surfactant. The latter term reflects the fact that at least some of the arms in the surfactant of the present application are oligomeric or polymeric.

[0105] In a preferred surfactant of formula (I), a is an integer from 3 to 30, more preferably from 3 to 20, and still more preferably from 4 to 20. Thus, the surfactant of formula (I) comprises a linear oligomer and / or a linear polymer of glycerol.

[0106] In a preferred surfactant of formula (I), each R is a C 1-6 alkyl group. Preferably, each R is the same. Representative examples of suitable C 1-6 alkyl groups include methyl, ethyl, propyl, butyl, pentyl and hexyl. More preferably, each R is selected from methyl and ethyl. Particularly preferably, each R is methyl.

[0107] In a further preferred surfactant of formula (I), a is an integer from 3 to 30, more preferably from 3 to 24, and still more preferably from 4 to 20, and each R is a C 1-6 alkyl group, preferably methyl. In a still further preferred surfactant of formula (I), a is an integer from 4 to 20, and each R is methyl.

[0108] In a preferred surfactant of formula (I), the sum of n and m is greater than 2. In a more preferred surfactant, the sum of n and m is an integer from 3 to 10. Preferably, the sum of n and m is 3, 4, 5, 6 or 7. Particularly preferably, the sum of n and m is 3 or 4.

[0109] In a preferred surfactant of formula (I), n is an integer from 1 to 6. In other words, the number of hydrophilic arms formed from linear glycerol oligomers or polymers is from 1 to 6.

[0110] In a preferred surfactant of formula (I), m is 1, 2 or 3. In other words, the number of lipophilic arms formed from perfluoropolyethers and / or fluorocarbons is 1, 2 or 3. More preferably, m is 1 or 2.

[0111] In some particularly preferred surfactants of formula (I), m is 1 and n is 2 or 3, or m is 2 and n is 1 or 2.

[0112] In further preferred surfactants of formula (I), a is an integer from 4 to 20, each R is methyl, and m is 1 and n is 2 or 3, or m is 2 and n is 1 or 2.

[0113] In some preferred surfactants of formula (I), at least one A is a fluorocarbon compound. In some preferred surfactants of formula (I), each A is a fluorocarbon compound. Preferred fluorocarbon compounds are C 1-18 fluorocarbon compounds, and in particular C 1-18 perfluorocarbon compounds. More preferred fluorocarbon compounds are C 2-16 fluorocarbon compounds, more preferably C 4-14 fluorocarbon compounds, and still more preferably C 4-12 fluorocarbon compounds. Yet more preferably, the fluorocarbon compound is C 2-16 perfluorocarbon compounds, especially C 4-14 fluorocarbon compounds, and yet more preferably C 4-12 perfluorocarbon compounds.

[0114] In other preferred surfactants of formula (I), at least one A is a perfluoropolyether. In further preferred surfactants of formula (I), each A is a perfluoropolyether.

[0115] Preferred perfluoropolyethers present in surfactants of formula (I) comprise repeat units of formula -[CF(CF3)CF2O] b where b is a positive integer. More preferably, the perfluoropolyether present in surfactants of formula (I) comprises units of formula -[CF2CF2O] c -[CF(CF3)CF2O] b where b and c are each 0 or a positive integer, provided that b and c are not simultaneously 0. c is preferably 0 or an integer from 1 to 100, for example an integer from 5 to 50. In preferred surfactants, c is 0. Particularly preferred perfluoropolyethers present in surfactants of formula (I) consist of the formula CF3CF2CF2O-[CF(CF3)CF2O] m -CF(CF3)- where b is a positive integer. In preferred perfluoropolyethers present in surfactants of formula (I), b is preferably an integer from 1 to 100 (for example from 1 to 50), more preferably an integer from 5 to 50, and particularly preferably an integer from 10 to 25. Preferred perfluoropolyethers present in surfactants of formula (I) have a weight average molecular weight of from 166 to 16,600 Da, more preferably from 800 to 9,000 Da, and yet more preferably from 1,500 to 6,000 Da.

[0116] In further preferred surfactants of formula (I), a is an integer from 4 to 20, each R is methyl, m is 1 and n is 2 or 3, or m is 2 and n is 1 or 2, and each A is a perfluoropolyether, preferably a perfluoropolyether comprising repeating units of formula -[CF(CF3)CF2O] b - wherein b is a positive integer.

[0117] In the surfactants of the present application, and as described above, the linking group 'X' links one or more lipophilic perfluoropolyether and / or fluorocarbon tail to one or more hydrophilic tail comprising a linear glycerol oligomer or polymer. In preferred surfactants of formula (I) of the present application, the main chain structure of the linking group (i.e. excluding hydrogen and halogen atoms and any atoms on pendant or branched groups) contains 1-50 atoms, more preferably 2-30 atoms, and still more preferably 3-15 atoms. In preferred surfactants of the present application, the ratio of the number of atoms in the main chain structure of the linking group (i.e. excluding hydrogen and halogen atoms and any atoms on pendant or branched groups) to the total number of atoms in the main chain structure of groups A and B (i.e. excluding hydrogen and halogen atoms and any atoms on pendant or branched groups) is 1 :2 to 1 :500, more preferably 1 :3 to 1 :100, and still more preferably 1 :5 to 1 :50. This ratio reflects the polymeric nature of the hydrophilic arm of the surfactants of the present application.

[0118] In preferred surfactants of the present application, the linking group 'X' comprises a group selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -C(O)S-, -SC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)NH-, -OC(O)NMe-, -O-, -S-, -NHC(O)NH-, -NMeC(O)NH-, -NHC(O)NMe-, -NHC(O)O-, -NMeC(O)O-, -SO2NH-, -NHSO2-, -NHSO2-C6H4-O-, and -O-C6H4-SO2NH-. In further preferred surfactants of the present application, the linking group 'X' comprises a group selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -OC(O)NH-, -C(O)O-, -NHC(O)O-, -NMeC(O)O-, and -O-.

[0119] In some preferred surfactants of formula (I) of the present application, the linking group 'X' comprises a charged group. The charged group can be a positively charged group, a zwitterionic group or a negatively charged group, but is preferably a positively charged group. One preferred positively charged group is a quaternary ammonium group. Some preferred surfactants of formula (I) of the present application comprise a linking group 'X' which comprises a group of formula (II):

[0120]

[0121] wherein

[0122] R 1 and R 2 are independently selected from H and C 1-6 alkyl; and

[0123] W - is a counterion.

[0124] Preferably, R 1 and R 2 are independently selected from C 1-6 alkyl. More preferably, R 1 and R 2 are selected from methyl, ethyl, propyl and butyl. Still more preferably, R 1 and R 2 are methyl.

[0125] W - may be any counterion. Representative examples of W - include halide (e.g. Br, I, CI ), tosylate, mesylate and acetate. In addition, W - may be a polycharged compound (e.g. di- or tri-carboxylate) or a polymer such as a polycarboxylate.

[0126] The wavy line in formula (II) indicates the position at which the charged group is attached to the remainder of the linking group 'X' or to A or B as defined in formula (I). In preferred surfactants of formula (I), the wavy line indicates the position at which the charged group is attached to the remainder of the linking group 'X'.

[0127] In other preferred surfactants of the present application, the linking group 'X' comprises a heterocycle. Examples of heterocycles which can be present in the linking group 'X' include 1,2,3-triazole, 1,2,3-triazole fused to a 5-10 membered ring and 1,2,3-triazole fused to a bicyclic structure. Representative examples of suitable heterocycles are shown below:

[0128]

[0129] In other preferred surfactants of the present application, the linking group 'X' comprises a C1-6 alkylene. In some preferred surfactants of the application, the linking group 'X' consists of C 1-6 alkylene. More preferably still, the linking group 'X' comprises or consists of C1, C2, C3or C4alkylene.

[0130] The following shows preferred surfactants of the application of formula (I) wherein the linking group 'X' consists of C 1-6 alkylene. In some preferred surfactants of the application, the linking group 'X' consists of C

[0131]

[0132] wherein each of a, b and R is as defined above in relation to formula I.

[0133] Further preferred surfactants of the application of formula (I) comprise a linking group 'X' comprising a group of formula (III):

[0134]

[0135]

[0136] wherein

[0137] each p is independently 0 or an integer from 1 to 6;

[0138] each q is independently 0 or an integer from 1 to 6;

[0139] each r is independently an integer from 1 to 6;

[0140] s is an integer from 1 to 6, preferably 1 or 2, still more preferably 1 ;

[0141] each Z is independently selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O- and O;

[0142] each U is independently a heterocycle; and

[0143] each Y is independently selected from

[0144]

[0145] wherein W, R 1 and R 2 and the wavy line are as defined above in relation to formula (II).

[0146] Further preferred surfactants of the application of formula (I) comprise a linking group 'X' comprising a group of formula (IIIo-q):

[0147]

[0148] wherein

[0149] each p is independently 0 or an integer from 1 to 6;

[0150] each q is independently 0 or an integer from 1 to 6; and

[0151] each Z is independently selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O-, and O.

[0152] Still further preferred surfactants of formula (I) of the present application comprise a linking group 'X' comprising a group of formula (IIIa)-(IIIq) as shown above.

[0153] Some preferred surfactants of the present application comprise a linking group 'X' of formula (IIIa), (IIIb), (IIIc), or (IIId) (preferably (IIIb), (IIIc), or (IIId)):

[0154]

[0155] wherein

[0156] p is 0 or 1 ;

[0157] q is 0, 1, or 2, preferably 0 or 1 ; and

[0158] Z is selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O-, and O.

[0159] The following shows preferred surfactants of formula (I) of the present application comprising a linking group 'X' of formula (IIIa), (IIIb), (IIIc), or (IIId):

[0160]

[0161]

[0162] wherein each of a, b, and R is as defined above with respect to formula (I).

[0163] Some preferred surfactants of the present application comprise a linking group 'X' of (IIIe), (IIIf), or (IIIg) (preferably (IIIf) or (IIIg)):

[0164]

[0165] wherein

[0166] p is 0 or 1 ;

[0167] q is 0, 1 or 2, preferably 0 or 1 ;

[0168] r is an integer from 1 to 6, preferably 1, 2 or 3;

[0169] Z is selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O- and O;

[0170] Y is selected from

[0171]

[0172] wherein W, R 1 and R 2 are as defined above in relation to formula (II).

[0173] The following shows preferred surfactants of formula (I) of the application comprising linking group 'X' of formula (IIIe), (IIIf) or (IIIg):

[0174]

[0175] wherein each of a, b and R is as defined above in relation to formula (I).

[0176] Some preferred surfactants of the application comprise linking group 'X' of formula (IIIh), (IIIi) or (IIIj) (preferably (IIIi) or (IIIj)):

[0177]

[0178] wherein

[0179] p is 0 or 1 ;

[0180] q is 0, 1 or 2, preferably 0 or 1 ;

[0181] r is an integer from 1 to 6, preferably 1, 2 or 3;

[0182] Z is selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O- and O; and

[0183] U is a heterocycle.

[0184] The following shows a preferred surfactant of formula (I) of the application comprising a linking group 'X' of formula (IIIh), (IIIi) or (IIIj):

[0185]

[0186] wherein each of a, b and R is as defined above in relation to formula (I).

[0187] Some preferred surfactants of the application comprise a linking group 'X' of formula (IIIk):

[0188]

[0189] wherein

[0190] each p is 0 or 1 ;

[0191] each q is 0, 1 or 2, preferably 0 or 1 ; and

[0192] each Z is independently selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O- and O.

[0193] The following shows a preferred surfactant of formula (I) of the application comprising a linking group 'X' of formula (IIIk):

[0194]

[0195] wherein each of a, b and R is as defined above in relation to formula (I).

[0196] Some preferred surfactants of the application comprise a linking group 'X' of formula (IIIk):

[0197]

[0198] wherein

[0199] each p is 0 or 1 ;

[0200] each q is 0, 1 or 2, preferably 0 or 1 ;

[0201] each r is an integer from 1 to 6, preferably 1, 2 or 3;

[0202] each Z is independently selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O- and O.

[0203] each U is a heterocycle; and

[0204] each Y is independently selected from:

[0205]

[0206] wherein W, R 1 and R 2 as defined above in relation to formula (II).

[0207] The following shows preferred surfactants of formula (I) of the application comprising a linking group 'X' of formula (III) or (III):

[0208]

[0209]

[0210]

[0211] wherein each of a, b and R is as defined above in relation to formula (I). Some preferred surfactants of the application comprise a linking group 'X' of formula (III):

[0212]

[0213] wherein

[0214] each p is 0 or 1 ;

[0215] each q is 0, 1 or 2, preferably 0 or 1 ;

[0216] each r is an integer from 1 to 6;

[0217] s is an integer from 1 to 6; preferably 1, 2 or 3;

[0218] each Z is independently selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O- and O; and

[0219] each Y is independently selected from:

[0220]

[0221] wherein W, R 1 and R 2 as defined above in relation to formula (II).

[0222] The following shows preferred surfactants of formula (I) of the application comprising a linking group 'X' of formula (III):

[0223]

[0224]

[0225] wherein each of a, b and R is as defined above in relation to formula (I).

[0226] Some preferred surfactants of the application comprise a linking group 'X' of formula (IIIo):

[0227]

[0228] wherein

[0229] each p is independently 0 or an integer from 1 to 6;

[0230] each q is independently 0 or an integer from 1 to 6; and

[0231] each Z is independently selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O- and O.

[0232] A preferred surfactant of formula (I) of the application comprising a linking group 'X' of formula (IIIo) is shown below:

[0233]

[0234] Some preferred surfactants of the application comprise a linking group 'X' of formula (IIIp):

[0235]

[0236] wherein

[0237] each p is independently 0 or an integer from 1 to 6;

[0238] each q is independently 0 or an integer from 1 to 6; and

[0239] each Z is independently selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O- and O.

[0240] A preferred surfactant of formula (I) of the application comprising a linking group 'X' of formula (IIIp) is shown below:

[0241]

[0242] Some preferred surfactants of the application comprise a linking group 'X' of formula (IIIq):

[0243]

[0244] wherein

[0245] each p is independently 0 or an integer from 1 to 6;

[0246] each q is independently 0 or an integer from 1 to 6; and

[0247] each Z is independently selected from C(O)NH, C(O)NMe, NHC(O), NMeC(O), OC(O)NH, C(O)O, -NHC(O)O-, -NMeC(O)O-, and O.

[0248] The following shows preferred surfactants of formula (I) of the present application comprising a linking group 'X' of formula (IIIq):

[0249]

[0250] Preferred surfactants of formula (I) of the present application are selected from the group consisting of:

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] The preferred surfactants of the present application have a weight average molecular weight of from 1,000 to 100,000 Dalton, preferably from 2,000 to 30,000 Dalton, and further preferably from 5,000 to 15,000 Dalton.

[0263] The present application also relates to a process for preparing a surfactant of formula (I) as defined above. The surfactants can be prepared from commercially available starting materials using conventional reactions.

[0264] A preferred method of making the surfactant as described above comprises:

[0265] reacting a compound of formula (II)

[0266] (A) m -X-(OH) n (II)

[0267] wherein

[0268] X is a linker;

[0269] each A is independently a fluorocarbon or a perfluoropolyether;

[0270] m is an integer from 1 to 10;

[0271] n is an integer from 1 to 10;

[0272] with a compound of formula (III)

[0273]

[0274] wherein each R is independently C 1-6 alkyl, CH2CH2OC 1-6 alkyl or CH2CH(CH3)OC 1-6 alkyl.

[0275] Another further preferred method for making the surfactant as described above comprises reacting a compound comprising (A)m with a compound comprising (B)n, wherein the reaction is a coupling reaction and a linker X is formed between the (A)m and the (B)n.

[0276] The compound of formula (I) as defined above is used as a surfactant. Thus, in another aspect, the present invention relates to the use of a compound of formula (I) as defined above as a surfactant. The surfactant of the present invention can be used to stabilize emulsions, more particularly to stabilize a discontinuous aqueous phase (e.g. one or more aqueous droplets) in a continuous oil phase (e.g. a continuous oil phase comprising a fluorine-containing oil). The perfluoropolyether and / or fluorocarbon compound component of the surfactant of the present invention acts as a lyophobic tail and is soluble in the oil phase (e.g. the continuous oil phase) of the emulsion, particularly wherein the oil phase comprises a fluorine-containing oil, e.g. a fluorine-containing oil phase. The one or more hydrophilic tails of the surfactant of the present invention act as ionic groups and are soluble in the aqueous phase (e.g. the discontinuous aqueous phase) of the emulsion.

[0277] The surfactant of the present invention can be used in emulsion preparation. The present invention thus also relates to the use of a surfactant as described above in emulsion preparation.

[0278] The present application also relates to an emulsion comprising a surfactant as described above. Preferred emulsions of the present application comprise a discontinuous aqueous phase, a continuous oil phase, and a surfactant as described above. The emulsion can comprise any amount of aqueous phase, oil phase, and surfactant suitable for forming an emulsion. The skilled person will be able to readily determine such amounts.

[0279] Preferably, the continuous oil phase of the emulsions of the present application comprises a fluorine-containing oil. The fluorine-containing oil is preferably a partially fluorinated hydrocarbon, a perfluorocarbon, a hydrofluoroether, or a mixture thereof. Particularly preferably, the fluorine-containing oil is a hydrofluoroether. Preferred fluorine-containing oils present in the continuous oil phase of the emulsions of the present application are Novec TM 7500 (3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane), Novec TM 7300 (1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl)-pentane), Novec TM 7200 (C4F9OC2H5), Novec TM 7100 (C4F9OCH3), Fluorinert TM FC-72, Fluorinert TM FC-84, Fluorinert TM FC-77, Fluorinert TM FC-40, Fluorinert TM FC3283, Fluorinert TM FC-43, Fluorinert TM FC-70, perfluoro-decalin, and mixtures thereof. More preferred fluorine-containing oils are Novec TM 7500 (3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane), Fluorinert TM FC-40, Fluorinert TM FC3283 and perfluoro-decalin, and still more preferred is Novec TM 7500 (3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane).

[0280] In preferred emulsions of the application comprising a plurality of droplets, at least some of the droplets further comprise an aqueous phase and a non-aqueous phase, a chemical buffer, a biochemical buffer, or a culture medium (media) or other medium. 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.

[0281] In preferred emulsions of the application comprising a plurality of droplets, at least some of the droplets further comprise an aqueous phase and a non-aqueous phase, a chemical buffer, a biochemical buffer, or a culture medium (media) or other medium. 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.

[0282] In emulsions of the application comprising a plurality of droplets, at least some of which comprise one or more analytes, the analyte can be any entity of interest. In one set of emulsions of the application comprising a plurality of droplets, at least some of which comprise one or more analytes, the analyte is preferably a biological molecule selected from the group consisting of small molecules, amino acids, peptides, proteins, antibodies, enzymes, monosaccharides, disaccharides, oligosaccharides, polysaccharides, nucleic acids, oligonucleotides, nucleotides, metabolites, cofactors, and artificially engineered molecules. More preferably, the biological molecule is selected from the group consisting of antibodies, enzymes, oligonucleotides, and metabolites, and still more preferably from the group consisting of antibodies and metabolites. Optionally, the biological molecule can be contained within a cell (e.g., a mammalian cell, a plant cell, an algal cell, a yeast cell, a hybridoma, a microorganism), an organelle (e.g., a nucleus, a mitochondrion), a virus, or a prion.

[0283] In another set of emulsions of the application comprising a plurality of droplets, at least some of which contain one or more analytes, the analyte is a biological analyte, such as a cell, a subcellular complex of cellular building blocks or components. The biological analyte is preferably selected from a cell (such as a mammalian cell, a plant cell, an algal cell, a microbial cell, a yeast cell), a pro-B cell, a T cell, a hybridoma, a microorganism, a virus, a bacterium, or a prion, an organelle (such as a nucleus, a mitochondrion) or an exosome, more preferably from a B cell, a T cell, a hybridoma and a microorganism, and still more preferably from a hybridoma and a microorganism. When the biological analyte is a cell, the cell is preferably selected from a mammalian cell, a plant cell, an algal cell, a microbial cell, more preferably from a mammalian cell and a microbial cell, and still more preferably from a mammalian cell. Preferably, the molecule is produced in the cell, excreted or secreted from the cell, such as the molecule is excreted or secreted from the cell. When the biological analyte is an organelle, the organelle is preferably selected from a nucleus and a mitochondrion.

[0284] In another set of emulsions of the application comprising a plurality of droplets, at least some of which contain one or more analytes, the analyte is an assay component, preferably selected from a bead, a nanoparticle, a crystal, a micelle, a quantum dot, a detection reagent, an antibody, an enzyme cofactor, a nucleic acid amplification reagent, an oligonucleotide sequencing reagent, a cell transformation reagent, a cell transduction mixture and a genome editing reagent. More preferably, the assay component is selected from a bead, a detection reagent, a nucleic acid amplification reagent and a genome editing reagent, still more preferably a detection reagent.

[0285] When at least some of the droplets contain a living entity (such as a cell or a bacterium), the aqueous phase preferably comprises a culture or growth medium. Any conventional medium can be used. The medium can for example comprise glucose, vitamins, amino acids, proteins, salts, pH indicators and density matching agents such as Ficoll. Sufficient medium must be provided to keep the entity alive for the duration of the analysis, reaction or other process of interest (such as sorting in a microfluidic device).

[0286] The application also relates to a method of preparing an emulsion as described above, the method comprising:

[0287] (i) preparing an aqueous phase;

[0288] (ii) preparing an oil phase; and

[0289] (iii) mixing the aqueous phase, the oil phase and a surfactant as described above to form the emulsion.

[0290] In one preferred method of preparing the emulsion, the surfactant is mixed with (e.g. dissolved in) the oil phase prior to mixing with the water phase. Preferably, the surfactant is dissolved in the oil phase at a concentration of 0.001% (w / w) to 20% (w / w), more preferably 0.1% (w / w) to 10% (w / w) and still more preferably 0.5% (w / w) to 5% (w / w). Preferably, the water phase comprises at least one analyte. In some preferred methods, the oil phase can be a solution of surfactant in a fluorosolvent. In other words, the surfactant can be dissolved in a fluorosolvent to give the oil phase.

[0291] In an alternative preferred method of preparing the emulsion, the surfactant is mixed with (e.g. dissolved in) the water phase prior to mixing with the oil phase.

[0292] In a further preferred method of preparing the emulsion, the surfactant is mixed with (e.g. dissolved in) the water phase and separately mixed with (e.g. dissolved in) the oil phase prior to mixing of the water phase with the oil phase. Any conventional mixing method can be used, for example a T-junction method, a step emulsification method, a flow focusing junction method, etc.

[0293] In a preferred method of preparing the emulsion as described above, the mixing is carried out by a flow focusing junction of a microfluidic device, for example a microfluidic device disclosed in WO2012 / 022976 and WO2015 / 015199. This is advantageous because it enables very small water phases, for example droplets, to be produced, typically on the picolitre or nanolitre scale.

[0294] Further preferred features of the method of preparing the emulsion are the same as the preferred features of the emulsion described above. Thus, preferably, the emulsion, the water phase and the oil phase are as defined above in relation to the emulsion.

[0295] Experiments, assays, reactions and processes can be carried out in the emulsion of the application. The discontinuous water phase (e.g. aqueous droplets) of the emulsion can be used as a locus for experiments, assays, reactions and processes. The surfactant of the application stabilises the emulsion (e.g. the discontinuous water phase in the oil phase) so that experiments, assays, reactions or processes can be carried out in the emulsion. The experiments, assays, reactions or processes can thus be carried out without coalescence of the discontinuous water phase (e.g. aqueous droplets). The experiments, assays, reactions or processes can involve one or more analytes present in the water phase of the emulsion. Thus, a method of carrying out one or more experiments, assays, reactions and processes within the emulsion (e.g. within the discontinuous water phase (preferably aqueous droplets) of the emulsion as described above) forms a further aspect of the application. The experiments, assays, reactions and processes carried out in the emulsion of the application can be carried out in a microfluidic channel or in a microfluidic device, for example, the experiments, assays, reactions and processes can be carried out in one or more channels of a microfluidic device.

[0296] The present application therefore also relates to a method of performing one or more chemical and / or biological reactions, and / or biological processes in the discontinuous aqueous phase of an emulsion as described above.

[0297] In one aspect, the method of performing one or more chemical and / or biological reactions, and / or biological processes in the discontinuous aqueous phase of an emulsion as described above is preferably a method of performing one or more chemical and / or biological reactions. The chemical and / or biological reaction can be an enzymatic reaction. Alternatively, the chemical and / or biological reaction is a molecular binding, molecular interaction, cellular interaction or conformational change that produces a measurable signal. Preferably, the chemical and / or biological reaction is an enzymatic reaction, molecular binding or molecular / cellular interaction.

[0298] In another aspect, the method of performing one or more chemical and / or biological reactions, and / or biological processes in the discontinuous aqueous phase of an emulsion as described above is preferably a method of performing one or more biological processes. The biological process can be antibody secretion or enzyme secretion by a cell, or enzyme production inside a cell. Alternatively, the biological process is antibody binding. In alternative methods, the biological process can be a nucleic acid amplification process, a partial or complete nucleic acid replication process or a nucleic acid transcription process. Alternatively, the biological process can be cell proliferation, cell metabolism, cell transfection, cell signalling, cell apoptosis or cell death. Preferably, the biological process is PCR. The process used can be for digital PCR.

[0299] The present application therefore also relates to a method of performing one or more drug screening assays for cells, molecules or cellular components in the discontinuous aqueous phase of an emulsion as described above.

[0300] In another aspect of the method of performing one or more biological processes, the biological process can be a genome editing process. The biological process can be sample preparation, for example oligonucleotide sample preparation for sequencing. The biological process can be nucleic acid sequencing. The molecule sequenced can be RNA or DNA, and the sequencing can be at the genomic, epigenomic or transcriptomic level.

[0301] The method of performing one or more chemical and / or biological reactions, and / or biological processes in the discontinuous aqueous phase of an emulsion as described above can 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.

[0302] Preferably, the method of performing one or more chemical and / or biological reactions, and / or biological processes in the discontinuous aqueous phase of an emulsion as described above is performed 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 droplets), thus requiring very little material, e.g. biological material. The microfluidic channel or device is preferably controlled by an automated device and software.

[0303] Preferably, the method of performing one or more chemical and / or biological reactions, and / or biological processes in the discontinuous aqueous phase of an emulsion as described above is performed under thermal, pH or environmental cycling conditions.

[0304] The surfactants and emulsions of the application have a variety of useful applications. They have particular potential use in microfluidic applications. For example, the surfactants and / or emulsions as defined above can be used in a method of classifying droplets, coalescing droplets or introducing a fluid into a droplet. The surfactants and / or emulsions can also be used in a method of extracting a protein from a fluid. These methods are preferably performed in a microfluidic device.

[0305] The methods of the application described herein (e.g. the method of making an emulsion, the method of performing one or more chemical and / or biological reactions, and / or biological processes in the discontinuous phase of an emulsion, the method of classifying droplets in a microfluidic device, the method of coalescing droplets in a microfluidic device, the method of introducing a fluid into a droplet in a microfluidic device, the method of partitioning a droplet in a microfluidic device, the method of extracting a molecule from a fluid) can be performed simultaneously or sequentially (e.g. in sequence) in any combination and order. Performing two or more methods of the application can be referred to as a functional workflow.

[0306] A preferred functional workflow comprises the following steps:

[0307] (i) making an emulsion as defined above, comprising: a) making an aqueous phase, b) making an oil phase, and c) mixing the aqueous phase, the oil phase and a surfactant as defined above in a microfluidic device to form the emulsion, wherein the aqueous phase contains a cell (e.g. mammalian cell, plant cell, algal cell, yeast cell, hybridoma, microorganism), organelle (e.g. nucleus, mitochondrion), virus or prion in a biological medium; the oil phase consists of a fluorosurfactant as defined above and a surfactant as defined above; the resulting emulsion comprises a plurality of droplets, and each droplet contains one cell or a small group of cells such as up to 50 (e.g. mammalian cell, plant cell, algal cell, yeast cell, hybridoma, microorganism), organelle (e.g. nucleus, mitochondrion), virus or prion;

[0308] (ii) performing a first biological process as defined above inside the droplets from step (i), wherein the biological process is cell proliferation, antibody production by cells, antibody secretion by cells, genome editing by cells, enzyme secretion by cells, enzyme production in cells, and enzyme reactions;

[0309] (iii) classifying droplets as defined above in a microfluidic device, comprising: a) providing a stream of the aqueous droplets from step (ii) in an emulsion as defined above in a channel of the microfluidic device; illuminating the stream from a first direction; detecting light from an analyte inside the droplets in a second direction, wherein the detected light is scattered light or fluorescence from the analyte; classifying the droplets into one of a plurality of differentiated streams in response to the detected light or measurable signal;

[0310] (iv) optionally, introducing a fluid into the classified droplets from step (iii) as defined above in a microfluidic device, wherein the fluid comprises at least one biological molecule, wherein the biological molecule is selected from the group consisting of small molecules, proteins, enzymes, peptides, amino acids, polysaccharides, oligosaccharides, disaccharides, monosaccharides, nucleic acids, oligonucleotides, nucleotides, cofactors, and cell lysis reagents;

[0311] (v) optionally, performing a second biological process as defined above inside the droplets from step (iv), wherein the biological process is cell lysis and enzyme reactions, wherein the enzyme is secreted by the cells in step (ii) or produced inside the cells, and the enzyme reaction serves to convert the biological molecule in step (iv) into its corresponding product;

[0312] (vi) optionally, quenching the enzyme reaction in step (v) by: a) treating the droplets from step (v) at an elevated temperature for a period of time, wherein the temperature is from 50 °C to 98 °C, and the period of time is from 10 seconds to 1 hour; b) introducing a fluid into the droplets from step (v) as defined above in a microfluidic device, wherein the fluid comprises an acid, a base, or an enzyme inhibitor; c) classifying the droplets from step (v) at a temperature from 4 °C to 10 °C;

[0313] (vii) partitioning droplets as defined above from step (iii) or (vi) in a microfluidic device, comprising: a) providing a droplet from step (iii) or (vi) in a first microfluidic channel of a microfluidic junction comprising three microfluidic channels on the microfluidic device; and passing the aqueous droplet through the microfluidic junction, thereby partitioning the droplet into two daughter droplets, a first daughter droplet in a second microfluidic channel and a second daughter droplet in a third microfluidic channel;

[0314] (viii) analysing the product molecules produced by the enzyme reaction in step (iii) or (v) inside the first daughter droplet using mass spectrometry (MS) after evaporation and ionisation of the contents of the first daughter droplet by microfluidic electrospray ionisation (i.e. ESI) emitter;

[0315] (ix) classifying a corresponding second daughter droplet in response to the MS analysis in step (viii) in a microfluidic device.

[0316] The application will now be described by way of the following non-limiting examples and figures, in which:

[0317] Figure 1 showing surfactant stabilised picodroplets at the flow focusing junction of a microfluidic device;

[0318] Figure 2 showing the same surfactant stabilised picodroplets at the outlet of a microfluidic device;

[0319] Figure 3 showing sample picodroplets stabilised by the surfactant removed from a collection vial;

[0320] Figure 4 showing CHO media droplet generation using surfactant 37 at the flow focusing junction (a), droplets at the outlet (b), and sample picodroplets removed from a collection vial (c);

[0321] Figure 5 showing hybridoma media droplet generation using surfactant 37 at the flow focusing junction (a), droplets at the outlet (b), and sample picodroplets removed from a collection vial (c);

[0322] Figure 6 shows (a) images of droplets in a flow focusing device, (b) surfactant 22 at the outlet of a generation chip, and (c) images of the droplets shown in images (a) and (b) stored in a 100 pm tall measurement chip;

[0323] Figure 7 shows images of droplets made from a 5 wt% solution of Ingredient B (surfactant 22) in Novec 7500. Image 7a: droplet generation of 294 pL droplets using a 60 pm x 60 pm flow focusing device, 5% surfactant 22 in Novec 7500, flow rate 700 pL / h and hybridoma media 500 pL / h. Image 7b generated at the outlet of a generation chip under the same conditions as image a. Image 7c shows 294 pL stored picodroplets generated in images a and b stored in a 100 pm tall measurement chip; and

[0324] Figure 8 shows microdroplets made from Krytox-Double-Click Surfactant 41 (1% surfactant (w:w) in Novec 7500) under conditions of oil flow 2600 pL / h, DPBS 333 pL / h, volume 243.3 pL (n=4), 77.4 pm droplet generation. Figure 8b Figure 8 shows microdroplets made from Krytox-Double-Click Surfactant 41 (1% surfactant (w:w) in Novec 7500) under conditions of oil flow 2600 pL / h, DPBS 333 pL / h, volume 243.3 pL (n=4), 77.4 pm droplet generation. Figure 8c Figure 8 shows microdroplets made from Krytox-Double-Click Surfactant 41 (1% surfactant (w:w) in Novec 7500) under conditions of oil flow 2600 pL / h, DPBS 333 pL / h, volume 243.3 pL (n=4), 82.4 pm droplet generation. Figure 8d Figure 8 shows microdroplets made from Krytox-Double-Click Surfactant 41 (1% surfactant (w:w) in Novec 7500) under conditions of oil flow 2600 pL / h, DPBS 333 pL / h, volume 243.3 pL (n=4), 77.4 pm droplet generation.

[0325] Example

[0326] Example 1: Synthesis of Surfactant 3 by polymerization, where R is Me

[0327]

[0328] To synthesize Surfactant 3, Krytox alcohol (3a) was first reacted with allyl chloride. The resulting intermediate 3b was then oxidized to a 1,2-diol, followed by polymer chain extension from both hydroxyl groups of the Krytox diol intermediate 3c.

[0329] Example 2: Synthesis of Surfactant 3 by ether coupling, where R is Me

[0330]

[0331] Surfactant 3 can also be synthesized starting from the polymer chain extension of both hydroxyl groups of 3-chloro-1,2-propanediol followed by a coupling reaction between the resulting intermediate 3d and Krytox alcohol in the presence of a base.

[0332] Example 3: Synthesis of surfactant 4 by amide coupling, wherein R is Me

[0333]

[0334] For the synthesis of surfactant 4, 3-chloro-1,2-propanediol is first converted into 3-azido-1,2-propanediol followed by a polymer chain extension from both hydroxyl groups. The azido group of the resulting intermediate 4a is reduced to an amino group. Finally, intermediate 4b is then coupled with Krytox acid chloride to give surfactant 4.

[0335] Example 4: Synthesis of surfactant 11 by amide coupling, wherein R is Me

[0336]

[0337] The synthesis of surfactant 11 starts from the Boc protection of the amino group of a base followed by a polymer chain extension from three hydroxyl groups. After deprotection of the Boc group of intermediate 11a, a coupling reaction between intermediate 11b and Krytox acid chloride generates the target surfactant 11.

[0338] Example 5: Synthesis of surfactant 5 by carbamate coupling, wherein R is Me

[0339]

[0340] Surfactant 5 is synthesized by coupling intermediate 4b with 4-nitrophenyl chloroformate activated Krytox alcohol.

[0341] Example 6: Synthesis of surfactant 8 by coupling, wherein R is Me

[0342]

[0343] Surfactant 8 is synthesized by coupling intermediate 3d and intermediate 8a, which is prepared by reacting Krytox acid chloride with 1-(2-aminoethyl)piperidine.

[0344] Example 7: Synthesis of surfactant 23 by coupling, wherein R is Me

[0345]

[0346] ​The synthesis of surfactant 23 starts from the polymer chain extension of two hydroxyl groups from 2,2-bis(bromomethyl)-1,3-propanediol to give intermediate 23a, followed by coupling with intermediate 23b prepared according to the following procedure.

[0347] Synthesis of N-[3-(dimethylamino)propyl]Krytox amide (23b)

[0348] To a stirred solution of 3-(dimethylamino)-1 -propylamine (50.61 g, 62.3 mL, 495 mmol) in anhydrous THF (60 mL) at 50 °C under nitrogen, a solution of krytox acid chloride (2, 192.39 g, 82.55 mmol) in Novec 7500 (203 mL) was added dropwise via cannula over 1 h. After stirring the mixture at 50 °C under nitrogen for 48 h, the mixture was cooled to room temperature and the yellow solid was removed by filtration and washed with Novec 7500 (30 ml). The filtrate was stirred with methanol (4 x 100 mL) each time the fluorine-containing phase was separated at the bottom in a separatory funnel. The fluorine-containing layer was then evaporated to dryness in vacuo to give (6) as a light yellow oil (189.7 g, 95.9%). IR (cm -1 ) 2955.5 (bw), 2832.0 (bw), 1729.6 (sm). 1 H NMR (400 MHz, 5% C6D 12 in FC72; volume: volume): 9.506 (1 H, bs, NH), 3.493 (2H, m, CONHCH2), 2.488 (2H, t, CH2-NMe2), 2.246 (6H, s, NMe2), 1.692 (2H, m, CH2-CH2NMe2).

[0349] Example 8: Synthesis of surfactant 27 by coupling, wherein R is Me

[0350]

[0351] The synthesis of surfactant 27 starts from the polymer chain extension of two hydroxyl groups from 1,4-dibromo-2,3-butanediol to give intermediate 27a, followed by coupling with intermediate 23b prepared according to the procedure above.

[0352] Example 9: Synthesis of surfactant 34 by click chemistry, wherein R is Me

[0353]

[0354] First, Krytox amide is reduced to Krytox amine, followed by coupling with 4-nitrophenyl chloroformate-activated (1R)-(-)-nopol. The resulting intermediate 34a is then subjected to a click reaction (linking reaction) with intermediate 4a to give the target surfactant 34.

[0355] Example 10: Synthesis of surfactant 36 by click chemistry where R is Me

[0356]

[0357] First, 2,3-dibromo-1-propanol is reacted with sodium azide to give 2,3-diazido-1- propanol, followed by polymer chain extension. The resulting intermediate 36a is then subjected to a click reaction with Krytox nopol (36b) prepared from Krytox alcohol to give the target surfactant 36.

[0358] Example 11: Synthesis of surfactant 35 by click chemistry where R is Me

[0359]

[0360] The synthesis of surfactant 35 starts from the reaction between 4-nitrophenyl chloroformate-activated (1R)-(-)-nopol and intermediate 11b. The resulting intermediate 35a is then subjected to a click reaction with Krytox azide (35b) prepared from Krytox alcohol to give the target surfactant 35.

[0361] Example 12:

[0362] Synthesis of Krytox-FSL-PEG4-11-azido-1-amide

[0363]

[0364] To a stirred solution of Krytox FSL 2,2,2-trifluoroethyl ester (57.26 g, 23.97 mmol) in Novec 7100 (58.0 mL) at 38 °C under nitrogen, a solution of 11-azido-3,6,9-trioxaundecan-1-amine (5.393 g, 24.71 mmol) in dry THF (25.5 mL) including 1.5 mL for washing was added via syringe. This was then stirred at 40 °C overnight, where TLC indicated the presence of significant unreacted amine. Triethylamine (3.512 mL, 25.2 mmol) was added via syringe and the block temperature was raised to 60 °C. After 48 h the reaction was cooled to room temperature and evaporated to dryness in vacuo. The resulting oil was dissolved in Novec 7500 (200 mL) and extracted in a separatory funnel with methanol (4 x 50 mL). The fluorous layer was then evaporated in vacuo to give a thick yellow oil (69.0 g). IR (cm -1 ) 2115.0 (N3), 1790.5 (C=0, TFE-ester), 1730.62 (C=0, product). The two carbonyl stretches were approximately the same in intensity. A portion of the starting material (20.95 g) was dissolved in Novec 7100 (15.0 mL) and loaded onto an 80 g Interchim 50 pm HC cartridge pre-washed with Novec 7100. The column was then washed as follows: a. Novec 7100 (250 mL), b. 0.5% MeOH / Novec 7100 (250 mL), c. 1.0% MeOH / Novec 7100 (250 mL), d. 1.5% MeOH / Novec 7100 (250 mL), e. 2.0% MeOH / Novec 7100 (250 mL), f. 3.0% MeOH / Novec 7100 (250 mL), g. 5.0% MeOH / Novec 7100 (250 mL), h. 10.0% MeOH / Novec 7100 (500 mL). The starting material eluted in Novec 7100 was evaporated in vacuo to give a colourless oil (12.224 g). The product fractions from the later runs were combined and evaporated in vacuo to give the product as a clear oil (7.317 g, 12.15%). IR (cm -1 ): 2112.3 (N3, mBr), (C=0, 1717.15). NMR (400 MHz, 5% C6D 12 (w / w) containing 0.04% TMS as internal standard, Delta (ppm) 7.80 (1H, s, NH), 3.72 (14H, s), 3.60 (2H, s), 3.35 (2H, s).

[0365] Polymerization of 1-(2-(6,6-dimethyldicyclo[3.1.1]hept-2-en-2-yl)ethoxy)- poly(2-methoxymethyl oxirane) Click reaction between Krytox-FSL-PEG4-11-azido-1-amide and 1-(2-(6,6- dimethyldicyclo[3.1.1]hept-2-en-2-yl)ethoxy)-poly(2-methoxymethyl oxirane)

[0366]

[0367] t-Bu-P4 phosphazene base (10.48 ml of 0.8 M in hexanes, 8.4 mmol) was added to a solution of (1R)-(-)-nopol (1.40 g, 8.4 mmol) in toluene (40 ml). Methyl glycidyl ether (19.6 g, 222.4 mmol) was added dropwise and the resulting mixture was stirred at 40 °C under N2for 2 days. The reaction was quenched with benzoic acid (2.0 g), concentrated under reduced pressure, and redissolved in THF. The product was purified via passage through neutral alumina, filtration, and concentration under reduced pressure to give a light brown oil (11.33 g).

[0368] 2,2-bis(azidomethyl)-1,3-propanediol 2,2-bis(azidomethyl)-1,3-propanediol-di-O-polymer

[0369]

[0370] To a solution of crude norbornanol polymer (0.93 g) in Novec 7500 (5.4 mL) was added a solution of Krytox-FSL-PEG4-11-azido-1-amide (1.303 g, 5.18 mmol) in anhydrous DMF (2.0 mL) via syringe and under a nitrogen atmosphere. The heating zone was set to 105 °C. After 3 hours, TLC (4% MeOH in Novec 7100) indicated that all azide had reacted, but heating at 105 °C was continued overnight. After cooling to room temperature, Novec 7500 (20 mL) was added and the solution was extracted with methanol (3 x 50 mL) in a separatory funnel. The fluorous phase was evaporated to dryness in vacuo to give the product (1.156 g). A portion of the starting material (1.00 g) was dissolved in Novec 7100 (3.0 mL) and loaded onto a 25 g Interchim 50 μm HC cartridge pre-washed with Novec 7100. The cartridge was then washed as follows: a. Novec 7100 (100 mL), b. 1.0% MeOH / Novec 7100 (100 mL), c. 2.0% MeOH / Novec 7100 (100 mL), d. 3.0% MeOH / Novec 7100 (100 mL), e. 4.0% MeOH / Novec 7100 (100 mL), f. 5.0% MeOH / Novec 7100 (100 mL), g. 6.0% MeOH / Novec 7100 (100 mL), h. 7.0% MeOH / Novec 7100 (100 mL), and i. 8.0% MeOH / Novec 7100 (100 mL). The polar fractions were combined and evaporated in vacuo to give the product as a light yellow oil (365 mg). A 5.0% stock solution was prepared by dissolving this oil in Novec 7500 (6.935 g) and used for microdroplet generation.

[0371] Example 13

[0372] 2,2-bis(azidomethyl)-1,3-propanediol-di-O-polymer

[0373]

[0374] To a stirred solution of 2,2-bis(hydroxymethyl)-1,3-propanediol (24.06 g, 91.86 mmol) in water (50 mL) at room temperature under nitrogen was added a solution of sodium azide (18.19 g, 275.6 mmol) in water (50 mL) and the remaining azide in the beaker was washed out with water (40.0 mL) and added to the reaction. The stirred solution was then heated to a block temperature of 100 °C under nitrogen and when the solution became slightly cloudy, additional water (30.0 mL) was added and the temperature raised to 105 °C. After 46 h the reaction was cooled to room temperature and extracted with DCM (3 x 100 mL). The combined organic extracts were dried over sodium sulfate, filtered and evaporated to give the product as a pale yellow oil (16.24 g, 95.0%). NMR (400 MHz, CDC13, contains 0.04% TMS as internal standard, delta (ppm) 3.65 (4H, t, CH2-O), 3.43 (4H, t, CH2-N3), 2.60 (2H, bs, OH).

[0375] Synthesis of Surfactant 22 where R is Me

[0376]

[0377] To a stirred solution of 2,2-bis(hydroxymethyl)-1,3-propanediol (24.06 g, 91.86 mmol) in water (50 mL) at room temperature under nitrogen was added a solution of sodium azide (18.19 g, 275.6 mmol) in water (50 mL) and the remaining azide in the beaker was washed out with water (40.0 mL) and added to the reaction. The stirred solution was then heated to a block temperature of 100 °C under nitrogen and when the solution became slightly cloudy, additional water (30.0 mL) was added and the temperature raised to 105 °C. After 46 h the reaction was cooled to room temperature and extracted with DCM (3 x 100 mL). The combined organic extracts were dried over sodium sulfate, filtered and evaporated to give the product as a pale yellow oil (16.24 g, 95.0%). NMR (400 MHz, CDC13, contains 0.04% TMS as internal standard, delta (ppm) 3.65 (4H, t, CH2-O), 3.43 (4H, t, CH2-N3), 2.60 (2H, bs, OH). -1)2101.0 (moderate, s, N3).

[0378] Example 14: Water Pico-Droplet Generation

[0379]

[0380] To a solution of crude 2,2-bis(aminomethyl)-1,3-propane-di-O-polymer (0.86 g, ca. 0.27 mmol) in methanol (3.0 mL) was added solid ammonium formate (85 mg, 1.35 mmol) followed by zinc dust (118 mg, 1.80 mmol) and the reaction was started at room temperature under nitrogen. After 20 h the solids were filtered off through celite and the filtrate was evaporated in vacuo to give a light yellow oil (0.731 g). This was dissolved onto a 1 g SCX cartridge and eluted first with methanol (15 mL) then with ammonia in methanol (7.0 M, 15.0 mL). The methanol ammonia fractions were evaporated to give a green oil (90 mg).

[0381] Figure 1

[0382]

[0383] To a stirred solution of 2,2-bis(aminomethyl)-1,3-propane-di-O-polymer (90 mg, 28.8 mmol) in THF (2.5 mL) at room temperature under nitrogen was added N-methylmorpholine (50 μL, 0.54 mmol). A solution of Krytox Carbonic acid-4-nitrophenyl ester (133 mg, 71.94 μmol, 2.5 mol equiv) in Novec 7100 (3.0 mL) was added and during the addition the solution changed from green to yellow. The batch temperature was then raised to 40 °C and stirred for ca. 112 h then cooled to room temperature. The solution was evaporated to dryness in vacuo then dissolved in Novec 7500 (12.0 mL) and washed with methanol (3 x 10.0 mL). The fluorous fraction was evaporated in vacuo to give the crude product as a colourless oil (219 mg). IR (cm -1 )1779.8 (weak, s, unreacted Krytox carbonate) and 1744.6 (moderate, s, carbamate).

[0384] Figure 2

[0385] To generate droplets with a volume of 300-400 pL, a fluorosurfactant containing 5% (w / w) of the surfactant synthesized in Example 12 (e.g. Novec 7500) was used as the continuous carrier oil phase, while a biochemical buffer solution was used as the dispersed aqueous phase. Both phases were injected using Cetoni GmbH syringe pumps connected via polyethylene tubing (ID: 0.38 mm) to a PDMS microfluidic chip with a single flow-focusing nozzle (nozzle dimensions: 60 x 60 pm). The typical flow rate of the fluorosurfactant phase was in the range of 1000-1500 pL / hr, and the typical flow rate of the aqueous phase was kept constant at 500 pL / hr.

[0386] The figures show the stabilization of the picodroplets by the surfactant. Figure 3 The droplet generation at the flow-focusing junction is shown, and Figure 4 The droplets at the outlet are shown. It is clear that the size of the droplets is the same. Figure 4 The sample droplets taken out of the collection bottle are shown, which further confirm the stability of the droplets.

[0387] Example 15: Synthesis of surfactant 37, wherein R is Me

[0388] Synthesis of 2,3-diazidopropan-1-ol

[0389]

[0390] 2,3-dibromopropan-1-ol (4.36 g, 20 mmol) was added to a stirred solution of NaN3in DMSO (120 mL, 60 mmol) and stirred at 75 °C for 2 days. TLC showed only one product spot. The reaction was quenched with water (250 mL), extracted with diethyl ether (3 x, 150 mL), washed with water (2 x, 150 mL) and saturated brine (150 mL), and dried over sodium sulfate (50 g). Filtration gave the product as a yellow oil (2.231 g, 15.7 mmol, 79%). IR (cm -1 ) 3374.8 (broad peak, m, OH), 2932.8 (b, m, CH), 2085.0 (sharp peak, s, N3). NMR (400 MHz, CDC13, with 0.04% TMS as internal standard, Delta (ppm) 3.82 (3H, m, CH2OH & CHN3), 3.45 (1H, m, CH2N3), 2.00 (1H, s, OH).

[0391] Synthesis of 1-(2,3-diazidopropoxy)-poly(3-methyloxetane)

[0392]

[0393] To a solution of 2,3-diamino-propan-l-ol (0.51 g, 3.59 mmol) in toluene (20 mL) was added 2-(methoxymethyl)oxirane (8.38 g, 95.1 mmol). Tert-Bu-P4 base (4.49 mL, 0.8 M in hexanes, 3.59 mmol) was added dropwise and the mixture was stirred at room temperature for 6 days. The reaction was quenched with benzoic acid (1.00 g) and evaporated to give a crude oil (8.183 g). This was purified by column chromatography eluting with THF (100%) to give the product (2.989 g). NMR (400 MHz, CDC13, contains 0.04% TMS as internal standard, delta (ppm) 3.94 (2H, broad s, 2H, CH2OH), 3.64 (broad m, 4H), 3.56-3.40 (m, 7.5H), 3.39-3.36 (m, 5H), 1.89 (2H, s, OH).

[0394] Synthesis of 1-(2,3-diaminopropoxy)-poly(3-methyloxetane)

[0395]

[0396] To a stirred solution of 1-(2,3-diazido-propoxy)-poly(3-methyloxetane) (2.0 g) in methanol (50 mL) was added triphenylphosphine (1.20 g, 4.56 mmol) and heated to 50 °C. The solution was stirred under N2at 50 °C for two weeks. The mixture was evaporated and redissolved in dichloromethane (30 mL). This was acidified with HC1 (1.0 M, 15 mL) and brine (5 mL), washed with dichloromethane (2 x 15 mL), basified with NaOH (10 M) and extracted with dichloromethane (3 x 20 mL). The combined organic fractions were dried over Na2S04and evaporated to give the product as a yellow / brown oil (0.31 g). NMR (400 MHz, CDC13, contains 0.04% TMS as internal standard, delta (ppm) 3.94 (2H, s, CH20), 3.68 (26H, broad s), 3.55-3.28 (60H, broad m), 2.80 (4H, bm, (NH2)2), 0.8 (2H, broad s, OH).

[0397] Synthesis of Surfactant 37

[0398]

[0399] A solution of 1-(2,3-diaminopropoxy)-poly(3-methyloxetane) (0.294 g, 0.639 mmol) and 4-methylmorpholine (0.21 mL, 1.1917 mmol) in THF (25 mL) was added to a solution of PFPE-CH2OC(O)O-Ph-4-NO2(2.36 g, 1.278 mmol) in Novec 7100 (15 mL) and stirred at 40 °C under N2for 1 week. The reaction mixture was filtered, evaporated, and re-dissolved in Novec 7100. The solution was then washed followed by filtration with aminopropyl functionalized silica (20 g, 10 g and 10 g) until no yellow residue remained. The resulting solution was evaporated to give a light yellow oil (0.765 g) which was purified by column chromatography on 20 g silica (30 um) eluted with 2% MeOH / Novec 7100 (75 mL), 5% MeOH / Novec 7100 (50 mL), 7% MeOH / Novec 7100 (50 mL) and 10% MeOH / novec 7100 (100 mL) to give the product (0.667 g). NMR (400 MHz, 5% C6D 12 (Weight: Weight) containing 0.04% TMS as internal standard, Delta (ppm) 4.78 (4H, broad m, (Krytox-CH2)2), 4.10-3.70 (broad m, 16H), 3.68-3.40 (broad m, 9.2H), 3.39-3.25 (broad m, 16H).

[0400] Microdroplet generation

[0401] To generate droplets with a volume of 300-400 pL, a fluorosurfactant 37 containing 5% (w / w) of the surfactant synthesized in Example 15 was used in a fluorosurfactant containing oil (e.g. Novec 7500) as the continuous carrier oil phase, while cell culture medium was used as the dispersed aqueous phase. Both phases were injected using Cetoni GmbH syringe pumps connected via polyethylene tubing (ID: 0.38 mm) to a PDMS microfluidic chip with a single stream focusing nozzle (nozzle dimensions: 60 x 60 pm). Typical flow rates for the fluorosurfactant phase were in the range of 800 - 1000 pL / hr and for the aqueous phase were kept constant at 500 pL / hr. Figure 5 and 5 Microdroplets are shown to be stabilized by the surfactant. Alternative Procedure Microdroplet generation of CHO culture medium at the stream focusing junction (a), microdroplets at the outlet (b), and sample microdroplets taken from the collection bottle are shown, which demonstrate the stability of the microdroplets. Figure 6aHybridoma culture media microdroplets at the flow-focussing junction are shown to be generated (a), microdroplets at the exit (b), and sample microdroplets removed from the collection vial, which demonstrates the stability of the microdroplets.

[0402] Example 16: Synthesis of Surfactant 38 where R is Me

[0403] Synthesis of 2,3-diazidobutane-1,4-diol

[0404]

[0405] A solution of 2,3-dibromo-1,4-butanediol (4.96 g, 20 mmol) in water (25 mL) was added to a solution of NaN3(3.90 g, 60 mmol) in water (25 mL) and heated at 80 °C under N2for 5 days. The reaction mixture was cooled to room temperature and washed with chloroform / isopropanol (3:1, 4 x 50 mL). The combined organic extracts were dried over Na2S04and evaporated to give a yellow oil (2.95 g, 17.2 mmol, 86%).

[0406] Synthesis of 1,4-(2,3-diazidobutoxy)-dipoly(3-methyloxetane)

[0407]

[0408] 2-(Methoxymethyl)oxirane (13.57 g, 154.0 mmol) was added to a solution of 2,3-diazidobutane-1,4-diol (1.00 g, 5.81 mmol) in toluene (50 mL). Tert-Bu-P4base (7.26 mL, 5.81 mmol) was added dropwise, resulting in a change from colourless to clear yellow. The reaction was stirred at room temperature under N2for 1 week. The reaction was quenched with benzoic acid (1.50 g) and evaporated. The crude oil was purified by column chromatography on neutral alumina eluted with THF (100%) to give the product (8.389 g).

[0409] Synthesis of 1,4-(2,3-diaminobutoxy)-dipoly(3-methyloxetane)

[0410]

[0411] To a stirred solution of 1,4-(2,3-diaminobutoxy)-dimeric(3-methyloxetane) (0.8 g, 0.735 mmol) and 4-methylmorpholine (0.22 g, 2.206 mmol) in THF (25 mL) was added to a solution of PFPE-CH2OC(O)O-Ph-4-NO2(2.71 g, 1.471 mmol) in Novec 7100 (15 mL) and stirred at room temperature under N2for 1 week. The reaction mixture was evaporated and re-dissolved in Novec 7100 (50 mL) then washed with 2- aminopropyl functionalised silica (2 x 20 g) until no yellow residue. The resulting solution was evaporated to give a light yellow oil (0.43 g) which was purified by column chromatography on 20 g silica (30 um) eluted with 2% MeOH / Novec 7100 (75 mL), 5% MeOH / Novec 7100 (50 mL), 7% MeOH / Novec 7100 (50 mL) and 10% MeOH / novec 7100 (100 mL) to give the product.

[0412] Synthesis of surfactant 38

[0413]

[0414] To a stirred solution of 1,4-(2,3-diaminobutoxy)-dimeric(3-methyloxetane) (0.8 g, 0.735 mmol) and 4-methylmorpholine (0.22 g, 2.206 mmol) in THF (25 mL) was added to a solution of PFPE-CH2OC(O)O-Ph-4-NO2(2.71 g, 1.471 mmol) in Novec 7100 (15 mL) and stirred at room temperature under N2for 1 week. The reaction mixture was evaporated and re-dissolved in Novec 7100 (50 mL) then washed with 2- aminopropyl functionalised silica (2 x 20 g) until no yellow residue. The resulting solution was evaporated to give a light yellow oil (0.43 g) which was purified by column chromatography on 20 g silica (30 um) eluted with 2% MeOH / Novec 7100 (75 mL), 5% MeOH / Novec 7100 (50 mL), 7% MeOH / Novec 7100 (50 mL) and 10% MeOH / novec 7100 (100 mL) to give the product.

[0415] Example 17: Synthesis of surfactant 39 where R is Me

[0416] Polymerisation of 1-(2-(6,6-dimethyldicyclo[3.1.1]hept-2-en-2-yl)ethoxy)- poly(2-methoxymethyloxetane)

[0417]

[0418] t-Bu-P4 phosphazene base (10.48 ml of 0.8 M in hexanes, 8.4 mmol) was added to a solution of (1R)-(-)-nopol (1.40 g, 8.4 mmol) in toluene (40 ml). Methyl glycidyl ether (19.6 g, 222.4 mmol) was added dropwise and the resulting mixture was stirred at 40 °C under N2for 2 days. The reaction was quenched with benzoic acid (2.0 g), concentrated under reduced pressure and re-dissolved in THF. The product was purified by passing through neutral alumina, filtration and concentration under reduced pressure to give a light brown oil (11.33 g).

[0419] Alternative method

[0420] To a solution of (1R)-(-)-nopol (0.181 g, 1.09 mmol) in toluene (2.6 ml) was added 2-(methoxymethyl)glycidol (0.959 g, 10.89 mmol) followed by a solution of aluminium trifluoromethanesulfonate (0.0181 g, 3.817 x 10 -5 mol) in di chloromethane (0.31 mL) and the solution was warmed on a heating block at 75 °C. After 18 hours the reaction mixture was cooled to room temperature to give a light brown oil (1.011 g) which was passed over neutral alumina (12 g) and eluted with THF. Fractions 1-3 were combined and evaporated to dryness to give a light yellow oil (0.198 g). NMR (400 MHz, CDC13, contains 0.04% TMS as internal standard, delta (ppm) 5.107 (0.39H, m, nopol = C-H, minor isomer), 5.022 (0.61H, m, nopol = C-H, major isomer), 4.35-4.22 (m, 1H), 4.12 (m, 1H), 3.956-3.71 (m, 5H), 3.67-3.40 (m, 8H), 3.395-3.383 (m, OMe, 14.6H, n = 4.86), 2.10-1.80 (m, 3.0H), 1.776 (1H, s, OH), 1.73-1.51 (m, 3H), 1.292-1.09 (m, 4H), 1.07-0.95 (m, 4H), 0.85-0.80 (m, 2H).

[0421] Synthesis of Krytox-FSL-PEG4-11-azido-1-amide

[0422]

[0423] To a stirred solution of Krytox FSL 2,2,2-trifluoroethyl ester (57.26 g, 23.97 mmol) in Novec 7100 (58.0 mL) at 38 °C under nitrogen, a solution of 11-azido-3,6,9-trioxaundecan-1-amine (5.393 g, 24.71 mmol) in dry THF (25.5 mL) was added by syringe, including 1.5 mL for washing. This was then stirred at 40 °C overnight, with TLC indicating that some unreacted amine still remained. Triethylamine (3.512 mL, 25.2 mmol) was added by syringe and the block temperature raised to 60 °C. After 48 h the reaction was cooled to room temperature and evaporated to dryness in vacuo. The resulting oil was dissolved in Novec 7500 (200 mL) and extracted in a separatory funnel with methanol (4 x 50 mL). The fluorous layer was then evaporated in vacuo to give a thick yellow oil (69.0 g). IR (cm -1 ) 2115.0 (N3), 1790.5 (C=0, TFE-ester), 1730.62 (C=0, product). The two carbonyl stretches were approximately the same in intensity. A portion of the starting material (20.95 g) was dissolved in Novec 7100 (15.0 mL) and loaded onto an 80 g Interchim 50 pm HC cartridge pre-washed with Novec 7100. The cartridge was then washed as follows: a. Novec 7100 (250 mL), b. 0.5% MeOH / Novec 7100 (250 mL), c. 1.0% MeOH / Novec 7100 (250 mL), d. 1.5% MeOH / Novec 7100 (250 mL), e. 2.0% MeOH / Novec 7100 (250 mL), f. 3.0% MeOH / Novec 7100 (250 mL), g. 5.0% MeOH / Novec 7100 (250 mL), h. 10.0% MeOH / Novec 7100 (500 mL). The starting material eluted in Novec 7100 was evaporated in vacuo to give a colourless oil (12.224 g). The product fractions from the later runs were combined and evaporated in vacuo to give the product as a clear oil (7.317 g, 12.15%). IR (cm -1 ): 2112.3 (N3, mBr), (C=0, 1717.15). NMR (400 MHz, 5% C6D 12 (w / w) containing 0.04% TMS as internal standard, Delta (ppm) 7.80 (1H, s, NH), 3.72 (14H, s), 3.60 (2H, s), 3.35 (2H, s).

[0424] Figure 7a

[0425] Under nitrogen atmosphere and at room temperature, Krytox FSL acyl chloride (24.46 g, 12.25 mmol, IR 1806.7 cm⁻¹) was injected via syringe. -1 11-Azide-3,6,9-trioxaundecan-1-amine (2.674 g, 12.25 mmol) was added to a solution in Novec 7500 (50.0 mL) in anhydrous 1,4-dioxane-1-amine. The solution was prepared in alkane (20.0 mL, with 5.0 mL of washing buffer). The mixture was then stirred overnight at 40 °C, where TLC showed the presence of some unreacted amines. PS-piperidine (3.0–4.0 mmol / g, 5.249 g) was added to the resulting solution, and the reaction temperature was raised to 75 °C and stirred for 72 hours. The reaction was cooled to room temperature, and the polystyrene beads were removed by filtration. The reaction flask and beads were washed with Novec 7500 (2 × 40 mL). The filtrate was shaken with methanol (30 mL), and the bottom fluorinated layer was separated and evaporated under vacuum to obtain a mixture of Krytox methyl ester (30.5%) and the desired Krytox-FSL-PEG4-11-azido-1-amide (26.36 g), as determined by proton NMR. IR (cm) -1 ): 2109.9(N3),1785.7(CO2Me),1723.0(CONH).

[0426] Synthesis of surfactant 39:

[0427] Click reaction between Krytox-FSL-PEG4-11-azido-1-amide and 1-(2-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)ethoxy)-poly(2-methoxymethylethylene oxide)

[0428]

[0429] To a solution of crude nootkatol polymer (0.93 g) in Novec 7500 (5.4 mL) was added a solution of Krytox-FSL-PEG4-11-azido-1 -amide (1.303 g, 5.18 mmol) in anhydrous DMF (2.0 mL) via syringe and under a nitrogen atmosphere. The heating block was set to 105 °C. After 3 hours, TLC (4% MeOH in Novec 7100) indicated that all azide had reacted, but heating at 105 °C was continued overnight. After cooling to room temperature, Novec 7500 (20 mL) was added and the solution was extracted with methanol (3 x 50 mL) in a separatory funnel. The fluorous phase was evaporated to dryness in vacuo to give the product (1.156 g). A portion of the starting material (1.00 g) was dissolved in Novec 7100 (3.0 mL) and loaded onto a 25 g Interchim 50 μιη HC cartridge pre-washed with Novec 7100. The column was then washed as follows: a. Novec 7100 (100 mL), b. 1.0% MeOH / Novec 7100 (100 mL), c. 2.0% MeOH / Novec 7100 (100 mL), d. 3.0% MeOH / Novec 7100 (100 mL), e. 4.0% MeOH / Novec 7100 (100 mL), f. 5.0% MeOH / Novec 7100 (100 mL), g. 6.0% MeOH / Novec 7100 (100 mL), h. 7.0% MeOH / Novec 7100 (100 mL), and i. 8.0% MeOH / Novec 7100 (100 mL). The polar fractions were combined and evaporated in vacuo to give the product as a light yellow oil (365 mg). A 5.0% stock solution was prepared by dissolving this oil in Novec 7500 (6.935 g) and used for microdroplet generation.

[0430] Example 18: Synthesis of Surfactant 40 where R is Me

[0431] 2,2-Bis(azidomethyl)-1,3-propanediol

[0432]

[0433] To a solid 2,2-bis(azidomethyl)-l,3-propanediol (24.06 g, 91.86 mmol) at room temperature under nitrogen was added a solution of sodium azide (18.19 g, 275.6 mmol) in water (50 mL) and the remaining azide in the beaker was washed out with water (40.0 mL) and added to the reaction. The stirred solution was then heated to a block temperature of 100 °C under nitrogen and when the solution became slightly cloudy, additional water (30.0 mL) was added and the temperature raised to 105 °C. After 46 hours the reaction was cooled to room temperature and extracted with DCM (3 x 100 mL). The combined organic extracts were dried over sodium sulfate, filtered and evaporated to give the product as a pale yellow oil (16.24 g, 95.0%). NMR (400 MHz, CDC13, contains 0.04% TMS as internal standard, delta (ppm) 3.65 (4H, s, CH2-0), 3.43 (4H, s, CH2-N3), 2.60 (2H, bs, OH).

[0434] 2,2-bis(azidomethyl)-l,3-propanediol

[0435]

[0436] To a stirred solution of 2,2-bis(azidomethyl)-l,3-propanediol (200 mg, 1.075 mmol) in anhydrous toluene (10 mL) at room temperature under nitrogen, was added a solution of P4-t-Bu in hexanes (0.8 M, 2.68 mL, 2.15 mmol, phosphorane base) via syringe. Then, neat 2-(methoxymethyl)oxirane (2.215 mL, 36.49 mmol, 34.0 mol equiv) was added to the stirred solution via syringe at room temperature. After 48 h, the reaction was quenched by the addition of solid benzoic acid (0.934 g, 7.65 mmol) and, after dissolution, the reaction was evaporated in vacuo. The crude product was dissolved in DCM (50 mL) and washed with water (50 mL) and the aqueous phase back-extracted with DCM (2 x 15 mL). The combined organic phases were washed with saturated sodium bicarbonate. The organic fractions were then stirred with aminopropyl silica (1.64 g, 1.78 mmol) for ten minutes, then filtered and evaporated in vacuo to give a pale yellow oil (2.985 g). This was dissolved in THF (3.0 mL) and loaded onto a gravity column of alumina (34 g) pre-washed with THF. The column was first washed with THF (300 mL) and the THF evaporated in vacuo to give a yellow oil (0.722 g, IR (cm"1) 2101.0 (weak, s, N3). Washing the column with 10% methanol in THF (250 mL) gave the crude product (1.876 g, IR (cm"1) 2101.0 (moderate, s, N3).

[0437] 2,2-bis(amino methyl)-l,3-propane-di-O-poly(2-methoxymethyloxirane)

[0438]

[0439] To a solution of crude 2,2-bis(azidomethyl)-l,3-propane-di-O-poly(2- methoxymethyloxirane) (2.564 g, ca. 1.74 mmol) in methanol (35.0 mL) was added triphenylphosphine (0.913 g, 3.48 mmol). Water (0.626 mL) was then added and the heating block was warmed to 77 °C and it was stirred for 19 h. The solution was evaporated to dryness to give an oily solid (3.325 g) which was dissolved in DCM and washed with 2.0 M HC1 (1 x 10 mL) and (1 x 20 mL). The combined acid layers were neutralized with sodium hydroxide (10 M, 10.62 mL) and extracted with DCM (3 x 20 mL). The combined organic fractions were dried over sodium sulfate, filtered, and evaporated to give a light yellow oil (1.132 g). NMR (400 MHz, CDC13, contains 0.04% TMS as internal standard, delta (ppm) 3.95 (broad singlet), 3.8-3.40 (broad multiplet), 3.378 (-OMe, broad singlet).

[0440] Alternative Method

[0441] To a stirred solution of crude 2,2-bis(azidomethyl)-l,3-propane-di-O-poly(2- methoxymethyloxirane) (0.86 g, ca. 0.27 mmol) (columned over neutral alumina) in methanol (3.0 mL) under nitrogen was first added ammonium formate (solid, 85.2 mg, 1.35 mmol) followed by zinc dust (<10 μm, 110 mg, 1.68 mmol) at room temperature. The crude reaction mixture was filtered through celite and washed with methanol and the filtrate was evaporated in vacuo to give a light yellow oil (0.731 g). The crude oil was purified on a 1 g SCX cartridge eluted with methanol followed by ammonia in methanol (7 M, 15 mL) and concentrated in vacuo to give a green oil (90 mg).

[0442] Surfactant 40 - 2,2-bis(krytox carboxamido-methyl)-l,3-propane-di-O-poly(2- methoxymethyloxirane)

[0443]

[0444] To a stirred solution of crude 2,2-bis(azidomethyl)-l,3-propane-di-O-poly(2- methoxymethyloxirane) (0.86 g, ca. 0.27 mmol) (columned over neutral alumina) in methanol (3.0 mL) under nitrogen was first added ammonium formate (solid, 85.2 mg, 1.35 mmol) followed by zinc dust (<10 μm, 110 mg, 1.68 mmol) at room temperature. The crude reaction mixture was filtered through celite and washed with methanol and the filtrate was evaporated in vacuo to give a light yellow oil (0.731 g). The crude oil was purified on a 1 g SCX cartridge eluted with methanol followed by ammonia in methanol (7 M, 15 mL) and concentrated in vacuo to give a green oil (90 mg). In a stirred solution of anhydrous alkylene (17.5 mL), PS-piperidine (0.652 g, approximately 3.5 mmol / g) was added. Then, a solution of Krytox acyl chloride (3.416 g) in Novec 7500 (15.0 mL, with 2.5 mL of washing solution) was added via syringe. The aluminum block was heated to 80 °C and stirred for approximately 60 hours. The reaction mixture was then cooled to room temperature, filtered, and the filtrate was transferred to a separatory funnel. The lower fluorinated layer was then compared with... The alkyl layer was separated and extracted once with methanol (25 mL). The fluorinated layer was separated and evaporated to dryness to give a pale yellow oil (3.456 g). IR (cm⁻¹) 1790.7 (weak, s, CO, ester) and 1738.9 (weak, sharp, CO, amide). The crude material was passed through a silica column (25 g) and eluted stepwise with 1% methanol in Novec 7100 to 15% methanol in Novec 7100. The eluent fraction was evaporated to give a brown oil (0.54 g), IR (cm⁻¹) 3369.8 (weak, b, -OH), 2891.3 (strong, br, CH), 1699.6 (strong, s, CO, amide). NMR (400MHz, CDCl3, containing 0.04% TMS as internal standard), Δ(ppm) 5.35 (broad multiplet, NH), 3.82 (s, CH2O), 3.77 (s, CH2O), 3.72 (s, CH2O), 3.72 (s, CH2O), 3.7-3.5 (broad multiplet, CHO), 3.46 (s, OMe), 3.45-3.10 (broad multiplet, CHO). The sample contained P4-t-Bu impurity.

[0445] Example 19: Alternative synthesis of surfactant 22 of Me, where R is R.

[0446]

[0447] To a stirred solution of chloroformic acid 4-nitrophenyl ester (25.24 g, 125.22 mmol) in THF (54.5 mL) at room temperature under nitrogen, Novec 7100 (32.0 mL) was added via syringe. A solution of Krytox alcohol (52.63 g, 31.3 mmol) was then added via syringe to a mixture of Novec 7100 (15.0 mL) and THF (24.5 mL). Finally, a solution of pyridine (5.1 mL, 62.6 mmol) in a mixture of Novec 7100 (4.0 mL) and THF (6.5 mL) was added via syringe, whereupon a white solid began to precipitate from the solution. The reaction was then warmed to 40 °C and stirred for 48 h. The reaction was cooled to room temperature and filtered through celite and the bed washed with Novec 7100 (50 mL). The filtrate was evaporated in vacuo and dissolved in Novec 7100: FC 72 (1 : 1, vol:vol, 200 mL), whereupon a precipitate began to form. After 1 h, the solution was filtered through celite and the bed washed with a small amount of FC 72 (30 mL). The filtrate was concentrated in vacuo and redissolved in FC 72 (180 mL) and allowed to stand overnight. Finally, the solution was filtered through celite and evaporated to dryness to give a colourless oil (54.88 g, 95.0 %). NMR (400 MHz, 5% C6D 12 (Weight: Weight) containing 0.04% TMS as internal standard, delta (ppm) 8.21 (2H, d, aromatic), 7.30 (2H, d, aromatic), 4.80 (2H, m, CH2).

[0448]

[0449] To a stirred solution of crude 2,2-bis(amino methyl)-1,3-propane-di-O- polymer (1.072 g, ca. 0.87 mmol) in THF (anhydrous, 12.5 mL) under nitrogen at room temperature, N-methylmorpholine (0.48 mL, 4.35 mmol) was added by syringe. Then a solution of Krytox carbonic acid 4-nitrophenyl ester (3.53 g, 19.15 mmol) was added to Novec 7100 (14.0 mL plus 1.0 ml wash) by syringe and the heating block set to 42°C and stirred for 48 hours. The reaction was cooled to room temperature and evaporated to dryness in vacuo to give an oil which was washed with THF (2 x 20 mL) and the residue decanted. The residue was dissolved in Novec 7500 (50 mL) and extracted with methanol (1 x 50 mL and 2 x 30 mL). The fluorous phase was concentrated in vacuo to give a dark oil (4.54 g). The oil was dissolved in Novec 7100 (50 mL) with stirring and amino propyl silica (2.52 g, 1.78 mml / g) was added and stirred for 30 minutes. The mixture was filtered and the silica washed with a small amount of Novec 7100 (10 mL). The filtrate was re-treated with amino propyl silica (2.52 g, 1.78 mml / g) and after 30 minutes the solution was filtered and the silica washed with a small amount of Novec 7100 (10 mL). After the third filtration the filtrate was concentrated under low vacuum. The resulting oil was dissolved in Novec 7100 (4.0 mL) and loaded onto a silica cartridge (25 g, SIHC, Interchim) packed in Novec 7100. A gradient from Novec 7100 to 15% methanol in 7100 was run collecting 50 mL fractions. Fractions 12 and 13 were combined and concentrated in vacuo to give a light brown oil, fraction A (0.84 g). IR (cm -1 ) 1737.9 (Krytox-CH2C=0, carbamate). NMR (400 MHz, 5% C6D 12 (w / w) with 0.04% TMS as internal standard, delta (ppm) 5.47 (4H, s, Krytox-CH2), 5.05 (6H, s), 4.78 (12H, m), 4.00-3.48 (68H, m), 3.47-3.25 (47.5H, bs, OMe). Fractions 14-18 were concentrated in vacuo to give a light brown oil, fraction B (0.685 g). IR (cm -1 ) 1768.3 (Krytox-CH2O, carbonate). NMR 400 MHz, 5% C6D 12(Weight: Weight), containing 0.04% TMS as internal standard, delta (ppm) 5.45 (4H, s, Krytox-CH20), 5.05 (10H, s) 4.75 (25H, s), 4.00-3.48 (96H), 3.47-3.28 (100H, bs, OMe). Figure 8a and 6b Microdroplets made from a 5 wt% solution of Fraction B in Novec 7500 are shown. Image 6a: Droplet generation to produce 308 pL microdroplets using a 60 pm x 60 pm flow focusing device, 5% surfactant 22 in Novec 7500, flow rate 700 pL / h and CHO media 500 pL / h. Image 6b produced under the same conditions as image a but at the generation chip outlet. Image 6c shows the 308 pL stored microdroplets produced in images A and B stored in a 100 pm high measurement chip.

[0450] Figure 8b and 7b Microdroplets made from a 5 wt% solution of Fraction B in Novec 7500 are shown. Image 7a: Droplet generation to produce 294 pL microdroplets using a 60 pm x 60 pm flow focusing device, 5% surfactant 22 in Novec 7500, flow rate 700 pL / h and hybridoma media 500 pL / h. Image 7b produced under the same conditions as image a but at the generation chip outlet. Image 7c shows the 294 pL stored microdroplets produced in images a and b stored in a 100 pm high measurement chip.

[0451] Example 20

[0452]

[0453] Under nitrogen atmosphere and at room temperature, a solution of diacetylacetamide (0.60 mL, 5.81 mmol) in anhydrous THF (4.0 mL, plus 1.00 mL washing buffer) was added to a stirred solution of Krytox acyl chloride (12.893 g, 5.54 mmol) in Novec 7100, where a fine white precipitate initially formed. Finally, a solution of triethylamine (1.16 mL, 8.30 mmol) in anhydrous THF (4.0 mL) was added, and the solution was heated to 55 °C. After 20 hours, the reaction was cooled to room temperature, filtered through diatomaceous earth, washed with Novec 7100 (20 mL), and the filtrate was evaporated under vacuum to dryness, giving a pale brown oil (13.009 g, 98.5%). IR (cm⁻¹): 3320.2 (weak, CH), 1697.3 (moderate, s, CO). NMR (400MHz, CDCl3, containing 0.04% TMS as internal standard, Δ(ppm)(4H,dt,N(CH2)2), 2.08(2H,d,CH).

[0454] 1-Azide-3-methoxy-prop-2-ol & 2-Azide-3-methoxy-prop-1-ol

[0455]

[0456] At room temperature and under nitrogen atmosphere, a solution of 2-(methoxymethyl)ethylene oxide (1.547 g, 17.56 mmol) in 1,2-DME (74.0 mL) (anhydrous, 9.0 mL with 1.0 mL washing buffer) was added via syringe to a stirred solution of tetra-n-butylammonium azide (5.245 g, 18.44 mmol) in 1,2-DME (74.0 mL). Finally, a solution of aluminum trifluoromethanesulfonate (10 mM, 1.756 mmol) in 1,2-DME was added, and the temperature of the heating block was raised to 65 °C. The reaction was stirred for 48 hours and then cooled to room temperature. The crude reaction mixture was cooled to room temperature and concentrated under vacuum to give a yellow oil (7.60 g). Finally, the desired diastereomeric mixture (0.798 g, 34.9%) free of tetra-n-butylammonium salt was obtained by multiplex chromatography using an ethyl acetate gradient in hexane. IR (cm) -1 3421.3 (OH, bm), 2927.7 (CH, bm), 2094.0 (N3, ss). NMR (400MHz, CDCl3, containing 0.04% TMS as internal standard, Δ (ppm) 3.94 (1H, m, CH-OH), 3.55-3.42 (2H, m, CH2OMe), 3.40 (3H, s, OMe), 3.38-3.32 (2H, m, CH2-N3), 2.46 (1H, s, OH).

[0457] 1 -azido-3-methoxy-propane-2-0-poly(2-methoxymethyloxirane)

[0458]

[0459] To a stirred solution of tetra-n-butylammonium azide (1.493 mmol, 0.25 M) in 1,2-DME (5.97 mL) at room temperature under nitrogen, a solution of 2- (methoxymethyl)oxirane (1.973 g, 22.40 mmol) in 1,2-DME (anhydrous, 4.0 mL plus 1.0 mL wash) was added via syringe. Finally, a solution of aluminium trifluoromethanesulfonate (10 mM, 44.8 μmol, 4.48 mL) in 1,2-DME was added and the heating block temperature was raised to 75 °C. The reaction was stirred for 48 h before cooling to room temperature. The crude reaction mixture was cooled to room temperature and concentrated in vacuo to give a yellow oil (2.1 g). Purification on a 40 g silica body (SIHC, Interchim) using ethyl acetate in hexane gave the desired azido-polymer as a light yellow oil (0.473 g, 33.7%, n = 10.2, mw 941, using internal standard). IR (cm -1 ) 3460.0 (OH, bm), 2877.7 (CH, bm), 2097.2 (N3, ss). NMR (400 MHz, CDC13, contains 0.04% TMS as internal standard, Delta (ppm) 4.03-3.93 (1H, m, CH-OH), 3.77-3.54 (6H, m, CH2OMe), 3.53-3.42 (12.5H, s, OMe), 3.38-3.32 (2H, m, CH2-N3), 1.96 (1H, s, OH).

[0460] Krytox-Bis-Click Surfactant 41

[0461]

[0462] To a stirred solution of 1-azido-3-methoxy-propane-2-0-poly(2-methoxymethyl oxirane) (0.257 g, Mn 836, 0.307 mmol) in t-butanol (2.70 mL) at 36 °C under nitrogen was added a solution of Krytox dipropargylamide (1.10 g, 0.4611 mmol) in Novec 7100 (5.0 mL). Then a solution of copper (II) acetate:THPTA (1 : 1, 9.357 pmol each) in t-butanol:water (5:1, vol:vol, 0.783 mL) was added by pipette. The next morning the reaction was cooled to room temperature and evaporated to dryness in vacuo to give a blue / green oil (1.294 g) which was dissolved in Novec 7100 and purified on a 25 g silica column (SiHC, Interchim) eluting with a gradient of 100% Novec 7100 to 12% MeOH in Novec 7100. The later running fractions were combined to give the bis-1,4-triazole surfactant (0.884 g). NMR 400 MHz, 5% C6D 12 (Weight:Weight) containing 0.04% TMS as internal standard, delta (ppm) 8.08 (2H, broad m, (triazole-H5)2), 5.40-4.26 (5H, broad m), 4.25-3.90 (3H, broad m), 3.90-3.15 (25H, broad m, OMe), 1.37 (1H, broad m, OH), 0.85 (1H, broad m, OH).

[0463] Figure 8c Microdroplets of Krytox-Bis-Click surfactant (1% surfactant (w:w) in Novec 7500) are shown to be produced under conditions of oil flow 2600 pL / h, DPBS 333 pL / h, volume 243.3 pL (n=4), 77.4 pm. Figure 8d Microdroplets of Krytox-Bis-Click surfactant (1% surfactant (w:w) in Novec 7500) are shown to be produced under conditions of oil flow 2600 pL / h, DPBS 333 pL / h, volume 243.3 pL (n=4), 77.4 pm. Figure 8d Microdroplets of Krytox-Bis-Click surfactant (1% surfactant (w:w) in Novec 7500) are shown to be produced under conditions of oil flow 2000 pL / h, DPBS 333 pL / h, volume 293.5 pL (n=4), 82.4 pm.​ Microdroplets made from Krytox-Double-Click surfactant (1% surfactant (w:w) in Novec 7500) in a high reservoir of 100 μιη are shown. The microdroplets were made under the conditions of oil flow 2000 μί / η, DPBS 333 μί / η, volume 293.5 pL (n = 4), Microdroplets made from Krytox-Double-Click surfactant (1% surfactant (w:w) in Novec 7500) in a high reservoir of 100 μιη are shown. The microdroplets were made under the conditions of oil flow 2000 μί / η, DPBS 333 μί / η, volume 293.5 pL (n = 4),

Claims

1. A surfactant of formula (I): wherein (A) m -X-(B) n (I) X is a linking group; each A is independently a fluorocarbon or a perfluoropolyether; each B is independently m is an integer from 1 to 10; and wherein a is an integer from 3 to 50, and each R is independently C 1-6 alkyl, CH2CH2OC 1-6 alkyl or CH2CH(CH3)OC 1-6 alkyl; n is an integer from 1 to 10.

2. The surfactant of claim 1, which is a star surfactant.

3. The surfactant of claim 2, which is a star polymer surfactant.

4. The surfactant of any one of claims 1-3, wherein the sum of n and m is greater than 2.

5. The surfactant of any one of claims 1-3, wherein the sum of n and m is an integer from 3 to 10.

6. The surfactant of any one of claims 1-3, wherein the sum of n and m is 3, 4, 5, 6, or 7.

7. The surfactant of any one of claims 1-3, wherein a is an integer from 3 to 24.

9. The surfactant of any one of claims 1-3, wherein n is an integer from 1 to 6.

8. The surfactant of any one of claims 1-3, wherein each R is C 1-6 alkyl.

10. The surfactant of any one of claims 1-3, wherein m is 1, 2, or 3.

11. The surfactant of any one of claims 1-3, wherein m is 1 and n is 2 or 3, or m is 2 and n is 1 or 2.

12. The surfactant of any one of claims 1-3, wherein at least one A is a perfluoropolyether.

13. The surfactant of claim 12, wherein each A is a perfluoropolyether.

14. The surfactant of any one of claims 1-3, wherein the perfluoropolyether comprises a repeating unit of the formula: wherein b is a positive integer.

15. The surfactant of any one of claims 1-3, wherein the linking group is one selected from the group consisting of -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -C(O)S-, -SC(O)-, -C(O)O-, -OC(O)-, -OC(O)O-, -OC(O)NH-, -OC(O)NMe-, -O-, -S-, -NHC(O)NH-, -NMeC(O)NH-, -NHC(O)NMe-, -NHC(O)O-, -NMeC(O)O-, -SO2NH-, -NHSO2-, -NHSO2-C6H4-O-, or -O-C6H4-SO2NH-. -[CF(CF3)CF2O] b -, 16. The surfactant of any one of claims 1-3, wherein the linking group comprises a charged group.

17. The surfactant of claim 16, wherein the linking group comprises a positively charged group.

18. The surfactant of any one of claims 1-3, wherein the linking group is one selected from the group consisting of: wherein 19. The surfactant of any one of claims 1-3, wherein the linking group comprises a heterocycle.

21. The surfactant of any one of claims 1-3, wherein the linking group is one selected from the group consisting of: R 1 and R 2 are independently selected from H or C 1-6 alkyl; and W - are counterions. wherein 20. The surfactant of any one of claims 1-3, wherein the linking group comprises C 1-6 alkylene, or consists of C 1-6 alkylene. ​ ​ each p is independently 0 or an integer from 1 to 6; each q is independently 0 or an integer from 1 to 6; each r is independently an integer from 1 to 6; s is an integer from 1 to 6; each Z is independently selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -OC(O)NH-, -C(O)O-, -NHC(O)O-, -NMeC(O)O-, or -O-; each U is independently a heterocycle; and each Y is a group independently selected from: wherein W - , R 1 and R 2 are as defined in claim 18.

22. The surfactant of any one of claims 1-3, wherein the linking group is a group of formula (Ilia), (Illb), (IIIc), or (IIId): wherein p is 0 or 1 ; q is 0, 1, or 2; and Z is selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -OC(O)NH-, -C(O)O-, -NHC(O)O-, -NMeC(O)O-, or -O-; or the linking group is a group of formula (IIIe), (IIIf), or (IIIg): (IIIe) (IIIf) (IIIg) wherein p is 0 or 1 ; q is 0, 1, or 2; r is an integer from 1 to 6; Z is selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -OC(O)NH-, -C(O)O-, -NHC(O)O-, -NMeC(O)O-, or -O-; Y is a group selected from: wherein W - , R 1 and R 2 are as defined in claim 18; or the linking group is a group of formula (IIIh), (IIIi), or (IIIj): (IIIh) (IIIi) (IIIj) wherein p is 0 or 1 ; q is 0, 1, or 2; r is an integer from 1 to 6; Z is selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -OC(O)NH-, -C(O)O-, -NHC(O)O-, -NMeC(O)O-, or -O-; and U is a heterocycle; or the linking group is a group of formula (IIIk): (IIIk) wherein each p is 0 or 1 ; each q is 0, 1, or 2; and each Z is independently selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -OC(O)NH-, -C(O)O-, -NHC(O)O-, -NMeC(O)O-, or -O-; or the linking group is a group of formula (III) or (IIIm): wherein each p is 0 or 1 ; each q is 0, 1, or 2; each r is an integer from 1 to 6; each Z is independently selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -OC(O)NH-, -C(O)O-, -NHC(O)O-, -NMeC(O)O-, or -O-; each U is a heterocycle; and each Y is a group independently selected from: wherein W - , R 1 and R 2 are as defined in claim 18; or the linking group is a group of formula (IIIn): (III) wherein each p is 0 or 1 ; each q is 0, 1, or 2; each r is an integer from 1 to 6; s is an integer from 1 to 6; each Z is independently selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -OC(O)NH-, -C(O)O-, -NHC(O)O-, -NMeC(O)O-, or -O-; and each Y is a group independently selected from: wherein W - , R 1 and R 2 are as defined in claim 18.

23. The surfactant of any one of claims 1 to 3, wherein the linking group is a group of formula (IIIo), (IIIp) or (IIIq): wherein each p is independently 0 or an integer from 1 to 6; each q is independently 0 or an integer from 1 to 6; and each Z is independently selected from -C(O)NH-, -C(O)NMe-, -NHC(O)-, -NMeC(O)-, -OC(O)NH-, -C(O)O-, -NHC(O)O-, -NMeC(O)O-, or -O-.

24. The surfactant of any one of claims 1-3, wherein the ratio of the number of atoms in the backbone structure of the linking group, excluding hydrogen and halogen atoms and any atoms on pendant or branched groups, to the total number of atoms in the backbone structure of the groups A and B, excluding hydrogen and halogen atoms and any atoms on pendant or branched groups, is from 1 :2 to 1 :

500.

25. The surfactant of any one of claims 1-3, selected from the group consisting of: 39 41 。 26. A method for preparing a surfactant according to any one of claims 1-25, the method comprising: reacting a compound of formula (II) (A) m -X-(OH) n (II) wherein X is a linking group; each A is independently a fluorocarbon or perfluoropolyether; m is an integer from 1 to 10; n is an integer from 1 to 10; with a compound of formula (III) (III) wherein each R is independently C 1-6 alkyl, CH2CH2OC 1-6 alkyl or CH2CH(CH3)OC 1-6 alkyl.

27. A method for making the surfactant of any one of claims 1-25, the method comprising: reacting a compound comprising (A)m with a compound comprising (B)n, wherein the reaction is a coupling reaction and a linker X is formed between the (A)m and the (B)n.

28. A composition comprising a surfactant of formula (I) according to any one of claims 1 to 25.

29. The composition of claim 28, wherein the composition is an emulsion.

30. Use of a surfactant as defined in any one of claims 1 to 25 as a surfactant.

31. Use of a surfactant of formula (I) as defined in any one of claims 1 to 25 in the preparation of an emulsion.

32. A method of preparing an emulsion according to claim 29, the method comprising: (i) providing an aqueous phase; (ii) providing an oil phase; and (iii) mixing the aqueous phase, the oil phase and a surfactant of formula (I) as defined in any one of claims 1 to 25 to form the emulsion.

33. A method of performing one or more chemical and / or biological reactions, and / or biological processes, the method comprising: The one or more chemical and / or biological reactions, and / or biological processes are carried out in the discontinuous aqueous phase of the emulsion of claim 29.

34. A method for classifying droplets in a microfluidic device, the method comprising: (i) providing a stream of aqueous droplets in an emulsion according to claim 29 in a channel of the microfluidic device; (ii) illuminating the stream from a first direction; (iii) detecting light from an analyte within the droplets in a second direction; and (iv) classifying the droplets into one of a plurality of differentiated streams in response to the detected light or measurable signal.

35. A method of coalescing droplets in a microfluidic device, the method comprising: (i) providing at least two aqueous droplets in an emulsion according to claim 29 in a channel of the microfluidic device; and (ii) exposing the aqueous droplets to an electric field, thereby coalescing the at least two aqueous droplets into a single droplet.

36. A method of introducing a fluid into a droplet in a microfluidic device, the method comprising: (i) providing an aqueous droplet in an emulsion according to claim 29 in a channel of the microfluidic device; and (ii) contacting the aqueous droplet with a stream of fluid, thereby introducing the fluid into the aqueous droplet.

37. A method of splitting a droplet in a microfluidic device, the method comprising: (i) providing a microfluidic device comprising a microfluidic junction, the microfluidic junction comprising a first microfluidic channel, a second microfluidic channel, and a third microfluidic channel; (ii) providing an aqueous droplet in an emulsion according to claim 29 in the first microfluidic channel; and (iii) passing the aqueous droplet through the microfluidic junction, thereby splitting the aqueous droplet into at least a first sub-droplet and a second sub-droplet, the first sub-droplet being in the second microfluidic channel and the second sub-droplet being in the third microfluidic channel.

38. A method of dispensing a droplet of an emulsion according to claim 29 in a microfluidic device, the method comprising: receiving a droplet of an emulsion as defined in claim 29 from a droplet feed line into a droplet outlet line; expelling a droplet from the droplet outlet line through a droplet outlet by providing pressurized dispensing fluid into the droplet outlet line; receiving a droplet from the droplet outlet line into a waste line when pressurized dispensing fluid is not provided into the droplet outlet line; and protecting a droplet upstream of the droplet outlet line by providing pressurized dispensing fluid upstream of the droplet outlet line.

39. A method of dispensing a droplet of an emulsion according to claim 29 in a microfluidic device, the method comprising: receiving a droplet of an emulsion as defined in claim 29 from a droplet feed line into a droplet outlet line; expelling a droplet from the droplet outlet line through a droplet outlet by providing pressurized dispensing fluid into the droplet outlet line; receiving a droplet into a waste line when pressurized dispensing fluid is not provided into the droplet outlet line; and injecting a fluid into the waste line when pressurized dispensing fluid is provided into the droplet outlet line.

40. A method of classifying droplets in a microfluidic device, the method comprising: (i) providing a microfluidic device comprising a microfluidic junction, the microfluidic junction comprising a first microfluidic channel, a second microfluidic channel and a third microfluidic channel; (ii) providing an aqueous droplet in an emulsion according to claim 29 in the first microfluidic channel; (iii) passing the aqueous droplet through the microfluidic junction, thereby splitting the aqueous droplet into at least a first daughter droplet and a second daughter droplet, the first daughter droplet being in the second microfluidic channel and the second daughter droplet being in the third microfluidic channel; (iv) detecting the first daughter droplet by mass spectrometry; and (v) classifying the second daughter droplet into one of a plurality of differential streams in response to the mass spectrometry performed on the first daughter droplet.

41. Use of a surfactant of formula (I) according to any one of claims 1 to 25 in a carrier fluid for droplet partitioning during droplet processing.

42. Use of an emulsion according to claim 29 in a microfluidic channel or device or in an automated device with associated software to control the microfluidic channel or device.

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