WO2025114425A1 - Dendronized surfactants - Google Patents

Abstract

The present invention provides a surfactant comprising a poly(amidoamine) dendrimer and a perfluoropolyether, a polydimethylsiloxane or both, wherein said dendrimer comprises the unit (I).

Description

Dendronized surfactants

INTRODUCTION

The present invention relates to surfactants that are particularly useful for the stabilisation of water in oil emulsions, e.g. in microfluidic devices. The surfactants generally have a dendrimer structure, in particular a poly(amidoamine) (PAMAM) based structure. The present invention also relates to a method for making the surfactant as well as to compositions, such as emulsions, comprising the surfactant. Additionally, the present invention relates to use of the compound described as a surfactant and to various methods, wherein the surfactant and/or emulsions comprising the surfactant, are employed, e.g. in droplet generation, droplet sorting, coalescing droplets, splitting droplets, dispensing of droplets, etc.

BACKGROUND

Surfactants have been used for many years in the production of stable emulsions for various applications. General background prior art relating to emulsions can be found in the following: US5,587,153; US6,017,546; W02005/099661 ; US2004/081633; US6,379,682; US2002/172703; W02004/038363; US2005/087122; US2007/275415 and US2008/053205. Conventional surfactants generally comprise a hydrophilic head group soluble in an aqueous phase of an emulsion and one or more lipophilic tails soluble in an oil phase of an emulsion.

More recently, surfactant-stabilised emulsions comprising droplets of water in a continuous oil phase have found applications in microfluidic technologies, enabling, for example, high-throughput screening, enzyme studies, nucleic acid amplification and other biological processes to be conducted. Biological assays may, for example, be performed in microfluidic devices using a very small quantity of biological material. Further information relating to microfluidic technology can be found in our previous applications W02009/050512 and W02015/015199. Other general background prior art on droplets can be found in patents/applications in the name of RainDance Technologies Inc., for example W02008/063227.

In microfluidic applications the use of oils and especially fluorous oils as the continuous phase in emulsion formation and production is beneficial because they have useful microfluidic properties, such as low friction, non-volatility (unlike alcohols), temperature-resistance and can easily create oil-water emulsions.

However, conventional surfactants are generally not suitable for stabilising emulsions comprising a fluorous oil phase due to solubilty issues. Furthermore, many conventional surfactants are toxic to biological molecules and to cells and can hinder gas transfer from the external environment to the inner regions of the emulsion.

A particular challenge that surfactants for use in water in oil emulsions must address is preventing leakage of molecules present in the water phase into the continuous oil phase and/or between different droplets. Such leakage is a huge problem for the various analysis methods employing these emulsions, which are underpinned by the separation of the molecules into different droplets. However, the molecules (e.g. DNA, small organic molecule drugs, etc) would often prefer to be in the oil phase, and have a tendency to leak through the membranes formed by the surfactants.

New surfactants suitable for stabilising water in oil emulsions, and in particular such emulsions comprising organic molecules in the aqueous phase, are therefore required. Ideally, such surfactants should prevent or minimise leakage of the organic molecules into the continuous oil phase and/or between droplets.

SUMMARY OF INVENTION

Viewed from a first aspect the present invention provides a surfactant comprising a poly(amidoamine) dendrimer and a perfluoropolyether, a polydimethylsiloxane or both, wherein said dendrimer comprises the unit (I):

Preferably the surfactant is of formula (II):

X(R)_n (II) wherein

X is a poly(amidoamine) dendrimer comprising the unit (I):

each R is independently selected from a perfluoropolyether and polydimethylsiloxane; and n is an integer between 1 and 8, preferably 2 and 6.

Viewed from a further aspect, the present invention provides a method of making a surfactant as hereinbefore defined, comprising:

(i) forming a poly(amidoamine) dendrimer comprising unit (I); and

(ii) connecting said dendrimer comprising unit (I) to a perfluoropolyether, a polydimethylsiloxane or both.

Viewed from another aspect, the present invention provides a method of making a surfactant of formula (VII) comprising:

(i) providing H2N-(CH2)r-[(Si(CH3)2O)_pSi(CH2)_q]s-(CH2)r-NH2 wherein wherein p is 1 to 100, q is 1 to 100, r is 1 to 12 and s is 1 to 100_; and

(ii) converting it into a compound of formula (VII), preferably by reacting it with methyl methacrylate in a 1 ,4-Michael addition to form an ester-terminated intermediate, followed by an amination with an amine, e.g. optionally substituted alkyl amine.

Viewed from a further aspect the present invention provides a composition, preferably an emulsion, comprising a surfactant as hereinbefore described.

Viewed from a further aspect the present invention provides an emulsion comprising a surfactant as hereinbefore described.

Viewed from a further aspect the present invention provides the use of a compound as hereinbefore defined as a surfactant.

Viewed from a further aspect the present invention provides the use of a surfactant as hereinbefore described in the preparation of an emulsion. Viewed from a further aspect the present invention provides a method of preparing an emulsion as hereinbefore defined comprising:

(i) providing an aqueous phase;

(ii) providing an oil phase; and

(iii) mixing said aqueous phase, said oil phase and a surfactant as hereinbefore defined to form said emulsion.

Viewed from a further aspect the present invention provides a method comprising performing one or more chemical and/or biological reactions, and/or biological processes in the discontinuous aqueous phase of an emulsion as hereinbefore defined.

Viewed from a further aspect the present invention provides a method for sorting droplets in a microfluidic device, the method comprising:

(i) providing a stream of aqueous droplets in an emulsion as hereinbefore defined in a channel of the microfluidic device;

(ii) illuminating the stream from a first direction;

(iii) detecting light from analytes within the droplets in a second direction; and

(iv) sorting the droplets into one of a plurality of differentiated streams responsive to the detected light or a measurable signal.

Viewed from a further aspect the present invention provides a method of coalescing droplets in a microfluidic device, the method comprising:

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

(ii) exposing the aqueous droplets to an electric field, thereby causing coalescence of the at least two aqueous droplets into a single droplet.

Viewed from a further aspect the present invention provides a method of introducing a fluid into a droplet in a microfluidic device, the method comprising:

(i) providing an aqueous droplet in an emulsion as hereinbefore defined in a channel of the microfluidic device; and

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

Viewed from a further aspect the present invention provides a method of splitting droplets in a microfluidic device, the method comprising:

(i) providing a microfluidic device comprising a microfluidic junction, said microfluidic junction comprising a first microfluidic channel, a second microfluidic channel and a third microfluidic channel; (ii) providing an aqueous droplet in an emulsion as hereinbefore defined in said first microfluidic channel; and

(iii) passing the aqueous droplet through the microfluidic junction, thereby splitting said aqueous droplet into at least a first daughter droplet and a second daughter droplet, the first daughter droplet in the second microfluidic channel and the second daughter droplet in the third microfluidic channel.

Viewed from a further aspect the present invention provides a method of dispensing droplets of an emulsion as hereinbefore defined in a microfluidic device, the method comprising: receiving droplets of an emulsion as hereinbefore defined in a droplet outlet line from a droplet feed line; ejecting a droplet from said droplet outlet line through droplet outlet by providing a pressurised dispensing fluid into said droplet outlet line; receiving droplets in a waste line from droplet outlet line when pressurised dispensing fluid is not provided into droplet outlet line; and protecting droplets upstream of said droplet outlet line by providing pressurised dispensing fluid upstream of droplet outlet line.

Viewed from a further aspect the present invention provides a method of dispensing droplets of an emulsion as hereinbefore defined in a microfluidic device, the method comprising: receiving droplets of an emulsion as hereinbefore defined in a droplet outlet line from a droplet feed line; ejecting a droplet from said droplet outlet line through droplet outlet by providing a pressurised dispensing fluid into said droplet outlet line; receiving droplets in a waste line when pressurised dispensing fluid is not provided into droplet outlet line; and injecting fluid into the waste line when pressurised dispensing fluid is provided into droplet outlet line.

Viewed from a further aspect the present invention provides a method of sorting droplets in a microfluidic device, the method comprising:

(i) providing a microfluidic device comprising a microfluidic junction, said microfluidic junction comprising a first microfluidic channel, a second microfluidic channel and a third microfluidic channel;

(ii) providing an aqueous droplet in an emulsion as hereinbefore defined in said first microfluidic channel; (iii) passing the aqueous droplet through the microfluidic junction, thereby splitting said aqueous droplet into at least a first daughter droplet and a second daughter droplet, the first daughter droplet in the second microfluidic channel and the second daughter droplet in the third microfluidic channel;

(iv) detecting said first daughter droplet by mass spectroscopy; and

(v) sorting said second daughter droplets into one of a plurality of differentiated streams responsive to the mass spectroscopy on said first daughter droplet.

Viewed from a further aspect the present invention provides a method of extracting a molecule from a fluid, the method comprising:

(i) dissolving a surfactant as hereinbefore defined in carbon dioxide to form a carbon dioxide/surfactant mixture;

(ii) adding a fluid comprising the molecule to the carbon dioxide/surfactant mixture, thereby extracting the molecule from the fluid into the carbon dioxide.

Viewed from a further aspect the present invention provides the use of a surfactant as hereinbefore defined in a carrier fluid (e.g. oil) for droplet spacing during droplet processing.

Viewed from a further aspect the present invention provides the use of a surfactant as hereinbefore defined in a microfluidic channel or device, in a molecular isolation in larger fluidic devices, containers or vats, or in an automated device with associated software that controls a microfluidic channel or device.

Viewed from a further aspect the present invention provides the use of an emulsion as hereinbefore defined in a microfluidic channel or device or in an automated device with associated software that controls a microfluidic channel or device.

DEFINITIONS

As used herein the term “fluorocarbon” refers to a hydrocarbyl group wherein one or more hydrogen atoms are replaced by fluorine atoms. In a “perfluorocarbon”, all of the hydrogen atoms are replaced by fluorine atoms.

As used herein the term “perfluoropolyether” refers to a polyether compound wherein all of the hydrogen atoms have been replaced by fluorine atoms.

As used herein the term “polyether” refers to an organic compound comprising two or more -O- linkages.

As used herein the term “dendrimer” refers to an oligomer or polymer with a repeat unit that forms at least two branches. The term “dendrimer” encompasses dendrimers and dendrons. As used herein the term “poly(amidoamine) dendrimer” refers to a dendrimer which comprises, e.g. consisting of, repeat units that are branched and which comprise amido and amino functional groups. Poly(amidoamine) dendrimers are often referred to as PAMAM dendrimers.

As used herein, a wavy bond indicates the point of attachment to another part of the compound (e.g. atom or group) of which it is a constituent part. Thus, an entity with one wavy bond is a terminal group whereas an entity with two or more wavy bonds is generally a non-terminal group.

As used herein the term “fluorous” refers to any group or substance which contains one or more fluorine atoms. Generally, the group or substance contains multiple fluorine atoms. For example, a fluorous oil refers to any oil containing fluorine atoms, including partially fluorinated hydrocarbons, perfluorocarbons, hydrofluoroethers and mixtures thereof.

As used herein the term “silicone oil” refers to an oil comprising silicon atoms.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to surfactants, which are particularly useful for the stabilisation of water in oil emulsions. The surfactants comprise a poly(amidoamino) dendrimer and a perfluoropolyether and/or a polydimethylsiloxane, preferably a perfluoropolyether.

The perfluoropolyether and/or polydimethylsiloxane are lipophilic and extend out into and/or “face” the oil phase and the poly(amidoamino) dendrimer is hydrophilic and extends into or “faces” the aqueous phase. The presence of the poly(amidoamino) dendrimer units in the surfactants of the present invention, which have multiple amido and amino functional groups, means that they are able to interact with organic molecules present via a plurality of “arms”. This is particularly beneficial in water-in-oil emulsions, wherein organic molecules present in the water phase, have a tendency to leak into the oil phase. The surfactants of the present invention have been found to form very stable water in oil emulsions, wherein leakage of organic molecules from the discontinuous aqueous phase is minimised or avoided, even at high temperatures (e.g. PCR temperatures) and during temperature cycling.

The surfactants of the present invention comprise a poly(amidoamine) dendrimer and a perfluoropolyether, a polydimethylsiloxane or both, wherein said dendrimer comprises the unit (I):

The perfluoropolyether and/or polydimethylsiloxane may be present as one or more terminal groups and/or as one or more linking groups. Some preferred surfactants of the invention comprise perfluoropolyether and polydimethylsiloxane is absent. Other preferred surfactants of the invention comprise polydimethylsiloxane and perfluoropolyether is absent. In less preferred surfactants, both perfluoropolyether and polydimethylsiloxane are present. Generally, surfactants comprising perfluoropolyether are preferred.

In formula (I) each wavy bond independently denotes a connection to another atom (e.g. H), group (e.g. R or a group comprising R or other functional group), or dendrimer unit. In some preferred surfactants, each amino group with two wavy bonds is connected to H and a R group (i.e. the functionality NHR is present), wherein each R is independently selected from perfluoropolyether and polydimethylsiloxane. In other preferred surfactants, each amino group with two wavy bonds is connected to H and dendrimer (i.e. the functionality NH-dendrimer is present), wherein dendrimer is a poly(amidoamino) dendrimer.

In other preferred surfactants comprising a dendrimer unit of formula (I), the amino group with a single wavy bond is connected to C1-6 alkyl (preferably CH3), COOH, OH, COOR¹⁰, CONH, CONHR¹⁰, wherein R¹⁰ is C1-6 alkyl or C1-6 alkylene-dendrimer, wherein dendrimer is a poly(amidoamino) dendrimer.

Preferred surfactants of the present invention are of formula (II):

X(R)_n (II) wherein X is a poly(amidoamine) dendrimer comprising the unit (I):

each R is independently selected from a perfluoropolyether and polydimethylsiloxane, preferably perfluoropolyether; and n is an integer between 2 and 8, preferably 2 to 6.

In formula (I) each amino groups with two wavy bonds is preferably connected to H and a R group (i.e. the functionality NHR is present), wherein each R is independently selected from perfluoropolyether and polydimethylsiloxane. In other preferred surfactants, each amino groups with two wavy bonds is preferably connected to H and dendrimer (i.e. the functionality NH-dendrimer is present), wherein dendrimer is a poly(amidoamino) dendrimer.

In preferred surfactants of formula (II), n is 1 , 2 or 4. “n” denotes the number of perfluoropolyether and/or polydimethylsiloxane groups present.

In a preferred surfactant of formula (II) X is a poly(amidoamine) dendrimer comprising the unit (III):

wherein R¹ is C_1-6 alkyl (preferably CH₃), OH, COOH, COOR¹⁰, CONH, CONHR¹⁰, wherein R¹⁰ is _{C1-6 alkyl; or}

wherein a is an integer between 1 and 6, preferably 2, 3 or 4. When R¹ is the latter structure, the connection to the unit of formula (III) is via the (CH2)a group. I_{n formula (III) each amino group with two wavy bonds is preferably connected to} H and a R group (i.e. the functionality NHR is present), wherein each R is independently selected from perfluoropolyether and polydimethylsiloxane, preferably _{perfluoropolyether. In other preferred surfactants, each amino groups with two wavy bonds is preferably connected to H and dendrimer (i.e. the functionality NH-dendrimer is} present), wherein dendrimer is a poly(amidoamino) dendrimer. 14358744-1 In some preferred surfactants of formula (II) comprising a unit of formula (III), R¹ is CH3, OH, or COOH. In such surfactants, when R¹ is CH3, OH, or COOH, n is preferably 1 , 2 or 4, and more preferably 2 or 4.

In some preferred surfactants of formula (II) comprising a unit of formula (III), R¹ is

wherein a is an integer between 1 and 6, preferably 2, 3 or 4. In such surfactants, n is preferably 4. When R¹ is this structure, the connection to the unit of formula (III) is via the (CH2)_a group.

A further preferred surfactant of formula (II) is a poly(amidoamine) dendrimer of formula (IV):

wherein R¹ is C1-6 alkyl (preferably CH3), OH, COOH, COOR¹⁰, CONH, CONHR¹⁰, wherein R¹⁰ is C1-6 alkyl; or

wherein a is an integer between 1 and 6, preferably 2, 3 or 4, each R is independently perfluoropolyether or polydimethylsiloxane, preferably perfluoropolyether, and at least one R group is present. When R¹ is the latter structure, the connection to the unit of formula (IV) is via the (CH₂)_a group. In some preferred surfactants of formula (IV), R¹ is CH₃, OH, or COOH. In such surfactants, when R¹ is CH₃, OH, or COOH, n is preferably 1 or 2. In some preferred surfactants of formula (IV), R¹ is:

_{wherein a is an integer between 1 and 6, preferably 2, 3 or 4 and each R is independently perfluoropolyether or polydimethylsiloxane. In such surfactants, n is preferably 4. When} R¹ is this structure, the connection to the unit of formula (IV) is via the (CH2)a group. The surfactants of the present invention may also comprise a second generation 5 pol(amidoamine) dendrimer. In some preferred surfactants of formula (II), X is a poly(amidoamine) dendrimer comprising the unit (V):

wherein each Z is independently selected from OC_1-6 alkyl (e.g. OMe), NH(CH₂)_kNH-R and NH(CH₂)_k-R; each R is independently perfluoropolyether or polydimethylsiloxane; k is an integer from 1 to 6; with the proviso that at least one Z is NH(CH₂)_kNH-R or NH(CH₂)_k-R. In formula (V) the wavy bond is preferably connected to C_1-6 alkyl, COOH, OH, 15 COOR¹⁰, CONH, CONHR¹⁰, wherein R¹⁰ is C_1-6 alkyl, or C_1-6 alkylene-dendrimer, wherein dendrimer is a poly(amidoamino) dendrimer. A _{further preferred surfactant of formula (II) is a poly(amidoamine) dendrimer of formula (VI):}

wherein R¹ is C1-6 alkyl (preferably CH3), OH, COOH, COOR¹⁰, CONH, CONHR¹⁰, wherein R¹⁰ is C1-6 alkyl; or

wherein a is an integer between 1 and 6, preferably 2, 3 or 4; and Z is OC_1-6 alkyl (e.g. OMe), NH(CH₂)_kNH-R and NH(CH₂)_k-R wherein each R is i_{ndependently perfluoropolyether or polydimethylsiloxane; and k is an integer from 1 to 6. When R1 is the latter structure, the connection to the unit of formula (VI) is via the} (CH2)a group. In some preferred surfactants of formula (II) comprising a unit of formula (VI), R¹ i_{s CH3, OH, or COOH. In such surfactants, when R1 is CH3, OH, or COOH, n is preferably}10 1, 2 or 4, and more preferably 1, 2 or 4. In some preferred surfactants of formula (VI), R¹ is:

w_{herein a is an integer between 1 and 6, preferably 2, 3 or 4; and} Z is OC_1-6 alkyl (e.g. OMe), NH(CH₂)_kNH-R and NH(CH₂)_k-R, wherein each R is i_{ndependently perfluoropolyether or polydimethylsiloxane; and k is an integer from 1 to 6. When R1 is this structure, the connection to the unit of formula (VI) is via the (CH2)a} group. I_{n such surfactants, n is preferably 4 or 8.} In some preferred surfactants of formula (II) comprising a unit of formula (VI), one 10 Z group is NH(CH2)kNH-R or NH(CH2)k-R, wherein each R is independently perfluoropolyether or polydimethylsiloxane and k is an integer from 1 to 6. In such surfactants, the other Z groups are preferably OMe. In some preferred surfactants of formula (II) comprising a unit of formula (VI), at l_{east two (e.g. two) Z groups are NH(CH2)kNH-R or NH(CH2)k-R, wherein each R is}15 independently perfluoropolyether or polydimethylsiloxane and k is an integer from 1 to 6. In such surfactants, other Z groups present are preferably OMe. I_{n some preferred surfactants of formula (II) comprising a unit of formula (VI), at} independently perfluoropolyether or polydimethylsiloxane and k is an integer from 1 to 6. In such surfactants, other Z groups present are preferably OMe. In some preferred surfactants of formula (II) comprising a unit of formula (VI), at _{least four (e.g. four) Z groups are NH(CH2)kNH-R or NH(CH2)k-R, wherein each R is} independently perfluoropolyether or polydimethylsiloxane and k is an integer from 1 to 6. In such surfactants, any other Z groups are preferably OMe. In preferred surfactants of formula (II), R is a perfluoropolyether. In further preferred surfactants of formula (II), each R is a perfluoropolyether. Preferred perfluoropolyether present in surfactants of the invention (e.g. formula (II)) comprise a repeat unit of the formula -[CF(CF3)CF2O]b-, wherein b is a positive _{integer. More preferably the perfluoropolyether present in surfactants of the invention} (e.g. formula (II)) comprise a unit of the formula -[CF₂CF₂O]_c-[CF(CF₃)CF₂O]_b-, wherein _{b and c are each 0 or a positive integer, with the proviso that b and c are not both 0. c is} preferably 0 or an integer from 1 to 100, e.g. an integer from 5 to 50. In preferred surfactants c is 0. Particularly preferred perfluoropolyether present in surfactants of the invention (e.g. formula (II)) consists of the formula CF3CF2CF2O-[CF(CF3)CF2O]b- _{CF(CF3)- or CF3CF2CF2O-[CF(CF3)CF2O]b-CF(CF3)-CO- or CF3CF2CF2O-} [CF(CF₃)CF₂O]_b-CF₂-CO-, wherein b is a positive integer. In preferred perfluoropolyether _{present in the surfactants of the invention b is preferably an integer from 1 to 100 (e.g.1 to 50), more preferably an integer from 5 to 50 and particularly preferably an integer from} 10 to 25. Preferred perfluoropolyether present in surfactants of the present invention _{(e.g. formula (II)) has a weight average molecular weight of 166 to 16,600 Da, more preferably 800 to 9,000 Da and yet more preferably 1,500 to 6,000 Da.} In some preferred surfactants of formula (II), R is a polydimethylsiloxane. In further preferred surfactants of formula (II), each R is a polydimethylsiloxane. Preferred polydimethylsiloxane present in surfactants of the invention (e.g. _{formula (II)), comprise a repeat unit of the formulae -Si(CH3)2O)p(CH2)q- or - (Si(CH3)2O)pSi(CH3)2(CH2)q- wherein p is a positive integer and q is 0 or a positive} integer. More preferably the polydimethylsiloxane present in surfactants of the invention _{(e.g. formula (II)) comprise, preferably consists of, a unit of the formulae Y-(CH2)r- (Si(CH3)2O)p(CH2)q- or Y-(CH2)r-(Si(CH3)2O)pSi(CH3)2(CH2)q- or Y-(CH2)r- (Si(CH3)2O)p(CH2)q-CO- or Y-(CH2)r-(Si(CH3)2O)pSi(CH3)2(CH2)q-CO- wherein p is a positive integer; q and r are 0 or a positive integer; and Y is H, NH2, OH, or COOH.} When the surfactant of the invention comprises polydimethylsiloxane (e.g. and perfluoropolyether is absent), one preferred group of compounds are those of formula (IV) wherein each R is independently selected from: Y-(CH2)r-(Si(CH3)2O)p(CH2)q-; Y-(CH2)r-(Si(CH3)2O)pSi(CH3)2(CH2)q-; Y-(CH2)r-(Si(CH3)2O)p(CH2)q-CO-; or Y-(CH2)r-(Si(CH3)2O)pSi(CH3)2(CH2)q-CO- wherein p is a positive integer; q and r are 0 or a positive integer; and Y is H, NH2, OH, or COOH. I_{n the above formulae, preferably p is 1 to 100, more preferably 5 to 50 and still more preferably 10 to 25. Preferably q is 1 to 100, more preferably 5 to 50 and still more preferably 10 to 25. Preferably s is 1 to 100, more preferably 5 to 50 and still more preferably 10 to 25. Preferably r is 1 to 12, more preferably 2 to 10 and still more} preferably 2 to 6. Preferred surfactants of formula (II) include the following compounds (1)-(12):

_{Wherein Rf is F(CFCF3CF2O)bCF2CO- and R is CH3, COOH or OH. Preferably b is 1 to} 50.

Rsi = Wherein Rsi is

and R is CH₃, COOH or OH. Preferably p is 1 to 100.

(_{3) (4) Wherein Rf is F(CFCF3CF2O)bCF2CO- and R is CH3, COOH or OH. Preferably b is 1 to} 50.

W_{herein Rf is F(CFCF3CF2O)bCF2CO- and R is CH3, COOH or OH. Preferably b is 1 to} 50. 15 14358744-1

Preferably p is 1 to 100. C_{ompounds 1, 2, 3, 5, 8, 10, and 11 are particularly preferred.} Preferably the surfactant is not of the following formulae:

100. Other preferred surfactants of the present invention are of formula (VII), wherein X is a poly(amidoamine) dendrimer comprising the unit:

wherein each of e and g are independently an integer from 1 to 6 and f is a positive integer, preferably 3-100. In formula (VII) the amino groups with two wavy bonds are preferably connected to H and a R group (i.e. the functionality NHR is present), wherein each R is independently selected from perfluoropolyether and polydimethylsiloxane. In other preferred surfactants of formula (VII), the amino groups with two wavy bonds are preferably connected to H and dendrimer (i.e. the functionality NH-dendrimer is present), wherein dendrimer is a poly(amidoamino) dendrimer. Preferably each of e and g are 2, 3, or 4. Preferred surfactants of formula (VII) include the following compounds:

wherein each of e and g are independently an integer from 1 to 6, preferably 2, 3, or 4 _{and f a positive integer, preferably 3-100.}

wherein f is a positive integer, preferably 3-100. The present invention also relates to a method of making a surfactant as hereinbefore described, comprising: _{(i) forming a poly(amidoamine) dendrimer comprising unit (I); and (ii) connecting said dendrimer comprising unit (I) to a perfluoropolyether, a} polydimethylsiloxane or both. In preferred methods of the invention, the step of forming a poly(amidoamine) dendrimer comprising unit (I) comprises reacting 1,2-diethylamine and methyl _{methacrylate in a 1,4-Michael addition to form an ester-terminated intermediate, followed} by an amination with an amine, e.g. optionally substituted alkyl amine. This process is _{shown in Figure 1 and produces a generation 1 (G1) dendrimer.} Optionally, the resulting dendrimer undergoes one or more successive 1,4- Michael additions followed by amination with an amine, e.g. optionally substituted alkyl amine. This produces 2^nd (G2) dendrimers and so on. In preferred methods of the invention step (ii) comprises reacting the dendrimer comprising unit (I) with an activated perfluoropolyether or polydimethylsiloxane, e.g. the acid chloride or ester. The present invention also relates to a method of making a surfactant as hereinbefore described, comprising: _{(i) providing H2N-(CH2)r-[(Si(CH3)2O)pSi(CH2)q]s-(CH2)r-NH2 wherein p is 1 to 100, q} is 1 to 100, r is 1 to 12 and s is 1 to 100; and _{(ii) converting it into a compound of formula (VII), preferably by reacting it with} methyl methacrylate in a 1,4-Michael addition to form an ester-terminated intermediate, followed by an amination with an amine, e.g. optionally substituted alkyl amine. When providing H2N-(CH2)r-[(Si(CH3)2O)pSi(CH2)q]s-(CH2)r-NH2, preferably p is 5 to 50 and more preferably 10 to 25. Preferably q is 5 to 50 and more preferably 10 to 25. Preferably s is 5 to 50 and more preferably 10 to 25. Preferably r is 2 to 10 and more preferably 2 to 6. T_{he compounds (e.g. of formula (II), (IV), (VI) and (VII)) hereinbefore defined are} for use as surfactants. Thus, in another aspect the present invention relates to the use _{of a compound as hereinbefore defined (e.g. of formula (II), (IV), (VI) and (VII)) as a} surfactant. The surfactants of the invention may be used to stabilise an emulsion, more particularly to stabilise a discontinuous aqueous phase, e.g. one or more aqueous _{droplets, in a continuous oil phase, e.g. a continuous oil phase comprising a fluorous oil, silicone oil or hydrocarbon oil. The perfluoropolyether and/or polydimethylsiloxane} component of the surfactants of the present invention acts as a lipophilic component, and is soluble in an oil phase, e.g. the continuous oil phase of an emulsion, particularly _{wherein the oil phase comprises a fluorous oil, , silicone oil or hydrocarbon oil e.g. a fluorous oil phase, silicone oil phase or hydrocarbon oil phase. The hydrophilic component (i.e. the dendrimer unit(s)) of the surfactants of the invention acts as an ionic} group, and is soluble in an aqueous phase, e.g. the discontinuous aqueous phase of an emulsion. T_{he surfactants of the present invention (e.g. of formula (II), (IV), (VI) and (VII))} may be used in the preparation of an emulsion. The present invention thus also relates _{to the use of a surfactant as hereinbefore described (e.g. of formula (II), (IV), (VI) and} (VII)) in the preparation of an emulsion. The present invention also relates to an emulsion comprising a surfactant as _{hereinbefore described (e.g. of formula (II), (IV), (VI) and (VII)). Preferred emulsions of} the present invention comprise a discontinuous aqueous phase, a continuous oil phase _{and a surfactant (e.g. of formula (II), (IV), (VI) and (VII)) as hereinbefore described. The emulsions may comprise aqueous phase, oil phase and surfactants (e.g. of formula (II), (IV), (VI) and (VII)) in any amounts suitable to form an emulsion. The skilled person will} be readily able to determine such amounts. 14358744-1 Preferably, the continuous oil phase of the emulsions of the invention comprises a fluorous oil, silicone oil or hydrocarbon oil. The fluorous oil is preferably a partially fluorinated hydrocarbon, a perfluorocarbon, a hydrofluoroether, or a mixture thereof. Particularly preferably the _{fluorous oil is a hydrofluoroether. Preferred fluorous oils present in the continuous oil phase of the emulsions of the present invention are Novec™ 7500 (3-ethoxy- 1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-hexane), Novec™ 7300 (1,1,1,2,2,3,4,5,5,5-decafluoro-3-methoxy-4-(trifluoromethyl)-pentane), Novec™ 7200 (C4F9OC2H5), Novec™ 7100 (C4F9OCH3), Fluorinert™ FC-72, Fluorinert™ FC-84,} Fluorinert™ FC-77, Fluorinert™ FC-40, Fluorinert™ FC3283, Fluorinert™ FC-43, _{Fluorinert™ FC-70, perfluorodecalin and mixtures thereof. More preferred fluorous oils are Novec™ 7500 (3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-(trifluoromethyl)-} hexane), Fluorinert™ FC-40, Fluorinert™ FC3283 and perfluorodecalin, and still more _{preferred is Novec™ 7500 (3-ethoxy-1,1,1,2,3,4,4,5,5,6,6,6-dodecafluoro-2-} (trifluoromethyl)-hexane). In preferred emulsions of the present invention, the discontinuous aqueous phase comprises a plurality of droplets. The droplets preferably have an average diameter of 1 µm to 500 µm, more preferably 10 to 150 µm and still more preferably 30 to 120 µm. This is advantageous because the volume of a droplet is therefore small, and thus the amount of material, e.g. biological material, needed is small. It is preferred that at least some of the droplets comprise one or more analytes. Preferably each droplet comprises an average number of 0 to 100 analytes, more preferably 1 to 20 and still more preferably 1 to 5, e.g.1 analyte. In preferred emulsions of the present invention comprising a plurality of droplets, at least some of the droplets further comprise an aqueous and non-aqueous phase, a _{chemical buffer, a biochemical buffer or a culture or other media. 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. In emulsions of the invention comprising a plurality of droplets wherein at least some of the droplets comprise one or more analytes, the analyte may be any entity of interest. In one group of emulsions of the invention comprising a plurality of droplets _{wherein at least some of the droplets comprise one or more analytes, the analytes are preferably biological molecules selected from small molecules, amino acids, peptides,} proteins, antibodies, enzymes, monosaccharides, disaccharides, oligosaccharides, 14358744-1 polysaccharides, nucleic acids, oligonucleotides, nucleotides, metabolites, cofactors and _{artificially engineered molecules. More preferably the biological molecules are selected from antibodies, enzymes, oligonucleotides and metabolites and still more preferably from antibodies and metabolites. Optionally the biological molecules may be contained in cells (e.g. mammalian cells, plant cells, algal cells, yeast cells, hybridomas, microorganisms), cell organelles (e.g. cell nuclei, mitochondria), viruses or prions.} In another group of emulsions of the invention comprising a plurality of droplets _{wherein at least some of the droplets comprise one or more analytes, the analytes are} biological analytes, e.g. cells, sub-cellular complexes of cellular building blocks or _{components. The biological analytes are preferably selected from cells (e.g. mammalian} cells, plant cells, algal cells, microbial cells, yeast cells), primary B-cells, T-cells, _{hybridomas, microorganisms, viruses, bacteria, or prions, cell organelles (e.g. cell nuclei, mitochondria) or exosomes, more preferably from B-cells, T-cells, hybridomas and microorganisms, and still more preferably from hybridomas and microorganisms. When} the biological analyte is a cell, the cell is preferably selected from mammalian cells, plant _{cells, algal cells, microbial cells, more preferably from mammalian cells and microbial} cells and still more preferably from mammalian cells. Preferably molecules are produced in, excreted or secreted from the cells, e.g. molecules are excreted or secreted from the cells. When the biological analyte is a cell organelle, the cell organelle is preferably selected from cell nuclei and mitochondria. In a further group of emulsions of the invention comprising a plurality of droplets _{wherein at least some of the droplets comprise one or more analytes, the analytes are assay components which are preferably selected from beads, nanoparticles, crystals,} micelles, quantum dots, detection reagents, antibodies, enzyme co-factors, nucleic acid amplification reagents, oligonucleotide sequencing reagents, cell transformation _{reagents, cell transduction mixtures and genome editing reagents. More preferably the} assay components are selected from beads, detection reagents, nucleic acid _{amplification reagents and genome editing reagents, still more preferably detection} reagents. W_{hen at least some of the droplets contain a living entity, e.g. cell or bacterium,} the aqueous phase preferably comprises a culture or growth medium. Any conventional medium may be used. The medium may, for example, comprise glucose, vitamins, amino _{acids, proteins, salts, pH indicators and density matching reagents, e.g. Ficoll. Sufficient} medium must be provided to keep the entity alive for the duration of the analysis, reaction _{or other process of interest, e.g. sorting in a microfluidic device.} 14358744-1 The present invention also relates to a method of preparing an emulsion as hereinbefore described, comprising: (i) preparing an aqueous phase; (_{ii) preparing an oil phase; and (iii) mixing the aqueous phase, the oil phase and a surfactant as hereinbefore described (e.g. of formula (II), (IV), (VI) and (VII)) to form the emulsion. In one group of preferred methods of preparing an emulsion the surfactant (e.g. of formula (II), (IV), (VI) and (VII)) is mixed with (e.g. dissolved in) the oil phase prior to mixing with said aqueous 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 aqueous phase _{comprises at least one analyte. In some preferred methods the oil phase may be a solution of the surfactant in a fluorous, silicone or hydrocarbon solvent. In other words, the surfactant may be dissolved in a, e.g. fluorous, solvent to give the oil phase. In alternative preferred methods of preparing an emulsion the surfactant (e.g. of formula (II), (IV), (VI) and (VII)) is mixed with (e.g. dissolved in) the aqueous phase prior} to mixing with the oil phase. I_{n further preferred methods of preparing an emulsion the surfactant (e.g. of formula (II), (IV), (VI) and (VII)) is mixed with (e.g. dissolved in) the aqueous phase and} is separately mixed with (e.g. dissolved in) the oil phase prior to mixing of the aqueous phase with the oil phase. Any conventional mixing method may be used, e.g. T-junction, step emulsification, flow focus junction etc. In preferred methods of preparing an emulsion as hereinbefore described the mixing is by a flow focus junction of a microfluidic device, e.g. a microfluidic device as _{disclosed in WO2012/022976 and WO2015/015199. This is advantageous because it} enables very small aqueous phases, e.g. droplets, to be produced, with volumes typically in the order of picolitres or nanoliters. Further preferred features of the method of preparing an emulsion are the same as the preferred features of the emulsion described above. Thus preferably the emulsion, the aqueous phase and the oil phase are as defined above in relation to the emulsion. Experiments, assays, reactions and processes may be carried out in the emulsions of the present invention. The discontinuous aqueous phase of the emulsion, e.g. aqueous droplets, may serve as the site for the experiments, assays, reactions and _{processes. The surfactants of the present invention (e.g. of formula (II), (IV), (VI) and} (VII)) stabilise the emulsion, e.g. a discontinuous aqueous phase in an oil phase, allowing 14358744-1 the experiment, assay, reaction or process to be carried out in the emulsion. The experiment, assay, reaction or process may therefore be carried out without the discontinuous aqueous phase, e.g. aqueous droplets, coalescing. The experiment, assay, reaction or process may involve one or more analytes present in the aqueous phase of the emulsion. Thus a method of performing one or more experiments, assays, _{reactions and processes within an emulsion, e.g. within the discontinuous aqueous} phase (preferably aqueous droplets) of an emulsion as hereinbefore described forms _{another aspect of the present invention. The surfactants of the present invention} advantageously prevent or minimise the leakage of analytes from the aqueous phase to the oil phase. The experiments, assays, reactions and processes carried out in the emulsions of the present invention may be carried out in a microfluidic channel or in a microfluidic device, e.g. the experiments, assays, reactions and processes may be carried out in one or more channels of a microfluidic device. The present invention thus 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 hereinbefore described. I_{n 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 hereinbefore described is preferably a method of performing one or more chemical and/or biological reactions. The chemical and/or biological reaction may be an enzymatic reaction. Alternatively, the chemical and/or biological reaction is a molecular} binding, molecular interaction, cellular interaction or conformational change resulting in _{a measurable signal. Preferably the chemical and/or biological reaction is an enzyme} reaction, a molecular binding or a molecular/cellular interaction. I_{n 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 hereinbefore described is preferably a method of performing one or more biological processes. The biological process may be antibody secretion or enzyme} secretion by cells, or enzyme production inside cells. Alternatively, the biological process _{is antibody binding. In alternative methods the biological process may be a nucleic acid} amplification process, partial or full nucleic acid replication process or nucleic acid _{transcription process. Alternatively, the biological process may be cell proliferation, cell} metabolism, cell transfection, cell transduction, cell signalling, cell apoptosis or cell _{death. Preferably the biological process is PCR. The process used could be for digital} PCR. A significant advantage of the surfactants of the invention (e.g. of formula (II), (IV), 14358744-1 _{(VI) and (VII)) is that they are stable to temperature cycling and the temperature} conditions of PCR. The present invention thus also relates to a method of performing one or more _{drug screening tests against cells, molecules or cell constituents in the discontinuous} aqueous phase of an emulsion as hereinbefore described. I_{n another aspect of the method of performing one or more biological processes the biological process may be a genome editing process. The biological process may be sample preparation, e.g. oligonucleotide sample preparation process for sequencing.} The biological process may be nucleic acid sequencing. The molecules being sequenced could be RNA or DNA and the sequencing could be at the genomic, epigenomic or transcriptomic level. T_{he method of performing one or more chemical and/or biological reactions,} and/or biological processes in the discontinuous aqueous phase of an emulsion as hereinbefore described may comprise one or more chemical reactions, one or more biological reactions, one or more biological processes or a mixture thereof. Preferred _{chemical and/or biological reactions, and/or biological processes are as described} above. P_{referably, 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 hereinbefore described is carried out in a microfluidic channel or microfluidic device. This enables chemical and/or biological reactions and/or biological processes to _{be performed on a very small scale, e.g. in droplets, and so very little material, e.g.} biological material, is required. The microfluidic channel or device is preferably controlled by an automated device and software. P_{referably, 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 hereinbefore described is carried out under thermal, pH or environmental cycling conditions. T_{he surfactants (e.g. of formula (II), (IV), (VI) and (VII)) and emulsions of the} present invention have many useful applications. They particularly have many potential uses in microfluidics applications. For example, the surfactants (e.g. of formula (II), (IV), _{(VI) and (VII)) and/or emulsions hereinbefore defined may be used in methods of sorting} droplets, coalescing droplets or introducing fluid into a droplet. The surfactants (e.g. of _{formula (II), (IV), (VI) and (VII)) and/or emulsions may also be used in methods of} 14358744-1 extracting a protein from a fluid. These methods are preferably carried in a microfluidic device. The methods of the invention described herein (e.g. method of preparing an emulsion, method comprising performing one or more chemical and/or biological reactions, and/or biological processes in the discontinuous phase of an emulsion, method for sorting droplets in a microfluidic device, method of coalescing droplets in a microfluidic device, method of introducing a fluid into a droplet in a microfluidic device, method of splitting droplets in a microfluidic device, method of extracting a molecule from a fluid) may be carried out simultaneously or sequentially (e.g. sequentially) in any combination and order. The carrying out of two or more methods of the invention may be known as a workflow of functions. A preferred workflow of functions comprises the steps of: (i) preparing an emulsion as hereinbefore defined, comprising a) preparing an _{aqueous phase, b) preparing an oil phase, and c) mixing said aqueous phase, said oil phase and a surfactant as hereinbefore defined (e.g. of formula (II), (IV) and (VII)) to form said emulsion in a microfluidic device, wherein the aqueous phase contains cells (e.g. mammalian cells, plant cells, algal cells, yeast cells, hybridomas, microorganisms), cell organelles (e.g. cell nuclei, mitochondria), viruses, or prions in a biological media;} the oil phase consists of a fluorous solvents as hereinbefore defined and a surfactant as hereinbefore defined; the resultant emulsion comprises a plurality of droplets, and each _{droplet contains one cell or a small pool or cells, such as up to 50 (e.g. mammalian cells, plant cells, algal cells, yeast cells, hybridomas, microorganisms), cell organelle (e.g. cell} nuclei, mitochondria), virus, or prion; (ii) performing a first biological process as hereinbefore defined inside the said droplets from step (i), wherein the biological processes are cell proliferation, antibody production by cells, antibody secretion by cells, genome editing of cells, enzyme secretion by cells, enzyme production in cells and enzyme reaction; (iii) sorting droplets as hereinbefore defined in a microfluidic device, comprising a) providing a stream of said aqueous droplets from step (ii) in an emulsion as _{hereinbefore defined in a channel of the microfluidic device; illuminating the stream from} a first direction; detecting light from analytes within the droplets in a second direction, wherein detecting light is a scattered light or a fluorescence from analytes; sorting the droplets into one of a plurality of differentiated streams responsive to the detected light or a measurable signal; 14358744-1 _{(iv) optionally introducing a fluid into the said sorted droplets from step (iii) as hereinbefore defined in a microfluidic device, wherein the fluid comprises at least one} biological molecule, wherein the biological molecule is selected from small molecules, proteins, enzymes, peptides, amino acids, polysaccharides, oligosaccharides, _{disaccharides, monosaccharides, nucleic acids, oligonucleotides, nucleotides, cofactors,} and cell lysing reagents; (v) optionally performing a second biological process as hereinbefore defined inside the said droplets from step (iv), wherein the said biological processes are cell lysis and an enzyme reaction, wherein the said enzyme is secreted by the said cell or produced inside the said cell in step (ii), and the said enzyme reaction is to convert a _{said biological molecule in step (iv) into its corresponding products;} (vi) optionally quenching the said enzyme reaction in step (v) by a) treating the said droplets from step (v) at an elevated temperature for a certain period of time, wherein the temperature is from 50^oC to 98^oC, and the period of time is from 10 seconds _{to 1 hour; b) introducing a fluid into the said droplets from step (v) as hereinbefore defined} in a microfluidic device, wherein the fluid comprises an acid, an alkaline, or an enzyme inhibitor; c) storing the said droplets from step (v) at a temperature from 4^oC to 10^oC; (vii) splitting droplets from step (iii) or (vi) as hereinbefore defined in a microfluidic device comprising a) providing droplets from step (iii) or (vi) in a first microfluidic channel of a microfluidic junctions comprising three microfluidic channels on the microfluidic device; and passing the aqueous droplet through the microfluidic junction, thereby splitting the said droplet into two daughter droplets, the first daughter droplet in the second microfluidic channel and the second daughter droplet in the third microfluidic channel; (viii) analysing the product molecule produced from the said enzyme reaction in step (iii) or (v) inside the first daughter droplet using mass spectrometry (MS) method _{after evaporating and ionizing the contents of the first daughter droplet via a microfluidic electrospray ionization (i.e. ESI) emitter;} (ix) sorting the corresponding second daughter droplet in a microfluidic device responsive to MS analysis results in step (viii). The invention will now be described with reference to the following non-limiting _{examples and Figures, wherein: Figure 1 shows general methodology for the synthesis of PAMAM dendrimers;} Figure 2 shows the synthesis of surfactants 1 and 2 of the present invention; 14358744-1 Figure 3 shows general methodology for the synthesis of further surfactants 3-6 of the invention; Figure 4 shows general methodology for the synthesis of further surfactants 7-10 of the invention; Figure 5 shows alternative methodology for the synthesis of surfactants 11 and 12 of the invention; Figure 6 shows alternative methodology for the synthesis of yet further surfactants 13 and 14 of the invention; Figure 7 shows alternative methodology for the synthesis of surfactant 15 of the invention; Figure 8 shows examples of surfactants of the present invention; F_{igure 9 shows a bright field image of picodroplets stabilised with a surfactant of} the invention after 10 days at room temperature; F_{igure 10a shows a bright field image of picodroplets stabilised with a surfactant of the invention after PCR thermal cycling; and} Figure 10b shows a southern blot wherein lanes 1 and 12 are DNA ladders, lanes _{2-4 are controls; and lane 10 is 5% surfactant (4).} EXAMPLES Synthesis of surfactants T_{he surfactants are synthesised using a divergent approach. This method requires the smallest generation dendrimer or dendron to be formed, starting from (G0.5), and successive reactions to create higher generations, e.g. extending to large generation dendrimers (G4.5) or dendron. The preparation of the dendrimer or dendron surfactants involves the repetitive} sequence of two simple reactions; 1,4 Michael addition to form methyl ester-terminated PAMAM dendrimers or dendrons (Half generation, G0.5) followed by an amination reaction for amine terminated PAMAM dendrimers or dendrons (Whole generation, G1.0). This is shown in Figure 1. D_{ifferent dendrimers or dendrons can be coupled to, e.g. PFPE or PDMS chains.} As shown in Figure 2, the amine-functionalized PAMAM dendrimer (whole generation) reacts with the activated carboxy terminus (e.g., acid chloride, active esters, etc) of the PFPE or PDMS chains. Example 1: Synthesis of compounds (1) and (2)

(₁₎ wherein Rf is F(CFCF3CF2O)bCF2CO-, Rsi i

90 g of Krytox™ 157 FSL (Mw = 2103 Daltons) were placed in a 250 mL round bottom flask, equipped with a magnetic stirrer bar and sealed with a rubber seal. The flask was evacuated and refilled with nitrogen three times to de-gas the Krytox™ polymer. 75 mL of anhydrous Novec™ 7100 was added by syringe to dissolve the Krytox™. Then 105 mL of oxalyl chloride was added by syringe at room temperature followed by catalytic amounts of anhydrous DMF (one drop from a syringe needle). The reaction was stirred at room temperature overnight, decanted into a clean 250 mL round bottom flask and evaporated to dryness. Yield of acyl chloride (off-white opaque oil): quantitative. IR carbonyl stretch at 1807 cm-1. P_{AMAM dendrimer G1.0 intermediate (holding 4 terminal NH2 groups) was added to a 100 mL 2-necked round bottom flask. The reaction flask connected to the nitrogen and dry THF was added to dissolve PAMAM dendrimer G1.0 intermediate. Krytox acid} chloride solution (dissolved in Novec 7100) was added in the reaction flask. The reaction _{was allowed to react for 2 days. Purification yielded thick transparent/white viscos oil as} Compound (1). T_{he same methodology was used for synthesis of compound (2). PAMAM} dendrimer G1.0 intermediate (holding 4 terminal NH2 groups) was added to a 100 mL 2- _{necked round bottom flask. The reaction flask connected to the nitrogen and dry THF was added to dissolve PAMAM dendrimer G1.0 intermediate. PDMS acid chloride} solution (prepared from carboxylate-functional silicones, and dissolved in THF) was added in the reaction flask. The reaction was allowed to react for 2 days. Purification _{yielded thick transparent/white viscos oil as Compound (2).} Example 2: Synthesis of compounds (3), (4), (5) and (6)

or OH The general methodology for the preparation of compounds (3)-(6) is shown in _{Figure 3. R-NH2, wherein R is CH3, COOH or OH, was used in the initial 1,4 Michael} addition reaction. PAMAM dendrone G1.0 (holding 2 terminal NH2 groups) was dissolved in dry _{THF in a 2-necked round bottom flask. Krytox acid chloride (2.0 mol equivalent) was} dissolved in dry Novec 7100 and added to the reaction flask. The reaction was allowed _{to react for 2 days. Purification yielded thick transparent/white viscos oil as Compound} (3). The same methodology may be used for synthesis of compounds (4), (5) and (6): PAMAM dendrone G1.0 (holding 2 terminal NH2 groups) was dissolved in dry _{THF in a 2-necked round bottom flask. Krytox acid chloride (1.0 mol equivalent) was} dissolved in dry Novec 7100 and added to the reaction flask. The reaction was allowed _{to react for 2 days. Purification yielded thick transparent/white viscos oil as Compound} (4). PAMAM dendrone G1.0 (holding 2 terminal NH2 groups) was dissolved in dry _{THF in a 2-necked round bottom flask. PDMS acid chloride (2.0 mol equivalent, prepared from carboxylate-functional silicones) was dissolved in dry THF and added to the} reaction flask. The reaction was allowed to react for 2 days. Purification yielded thick transparent/white viscos oil as Compound (5). PAMAM dendrone G1.0 (holding 2 terminal NH2 groups) was dissolved in dry _{THF in a 2-necked round bottom flask. PDMS acid chloride (1.0 mol equivalent, prepared} from carboxylate-functional silicones) was dissolved in dry THF and added to the reaction flask. The reaction was allowed to react for 2 days. Purification yielded thick transparent/white viscos oil as Compound (6). _{Example 3: Synthesis of compounds (7), (8), (9) and (10)}

The general methodology for the preparation of compounds (7)-(10) is shown in Figure 4. They undergo a second 1,4-Michael addition, followed by reaction of the _{methyl ester-functionalised dendrimer intermediate (1.5 generation) with the amino} termini of PFPE or PDMS chains. K_{rytox acid methyl ester was reacted with excess ethylene diamine and the reaction left stirring for 2 days. Purification yielded viscos oil as amine terminal Krytox. PAMAM dendrone G1.5 (holding 4 terminal methyl ester groups, prepared via} 1,4-Michael addition from PAMAM dendrone G1.0 with methyl methacrylate) was _{dissolved in dry THF in a 2-necked round bottom flask. Amine terminal Krytox} intermediate (1.0 mol equivalent) was dissolved in dry Novec 7100 and added to the reaction flask. The reaction was allowed to react for 2 days. Purification yielded thick _{transparent/white viscos oil as Compound (7). The same methodology may be used for synthesis of compounds (8), (9) and} (10): PAMAM dendrone G1.5 was dissolved in dry THF in a 2-necked round bottom flask. Amine terminal Krytox intermediate (4.0 mol equivalent) was dissolved in dry Novec 7100 and added to the reaction flask. The reaction was allowed to react for 2 days. Purification yielded thick transparent/white viscos oil as Compound (8). PAMAM dendrone G1.5 was dissolved in dry THF in a 2-necked round bottom _{flask. Amine terminal silicone (1.0 mol equivalent) was dissolved in dry THF and added} to the reaction flask. The reaction was allowed to react for 2 days. Purification yielded thick transparent/white viscos oil as Compound (9). PAMAM dendrone G1.5 was dissolved in dry THF in a 2-necked round bottom flask. Amine terminal silicone (4.0 mol equivalent) was dissolved in dry THF and added to the reaction flask. The reaction was allowed to react for 2 days. Purification yielded thick transparent/white viscos oil as Compound (10). Example 4: Synthesis of compounds (11) an

The general methodology used for the preparation of compounds (11) and (12) _{was is shown in Figure 5. Amine terminal silicone was reacted with the methyl ester-} functionalised intermediates. PAMAM dendrone G1.5 was dissolved in methanol in a 2-necked round bottom flask. Amine terminal silicone (1x terminal NH2 group, 4.0 mol equivalent) was dissolved in DCM and added to the reaction flask. The reaction was allowed to react for 2 days. _{Purification yielded clear viscos oil as Compound (11).} PAMAM dendrone G1.5 was dissolved in methanol in a 2-necked round bottom flask. Amine terminal silicone (2x terminal NH2 group, 4.0 mol equivalent) was dissolved in DCM and added to the reaction flask. The reaction was allowed to react for 2 days. Purification yielded clear viscos oil as Compound (12). Example 5: Synthesis of compounds (13) and (14) A_{nother approach is to grow dendrimer moieties from the either amino terminuses or methyl ester terminuses as shown in Figure 6.}

M_{ethyl methacrylate was added drop wise to a solution of amine terminal silicone (2x terminal NH2 groups) dissolved in DCM in a 2-necked round bottom flask. The} reaction was then stirred at room temperature for 2 days. The excess methyl _{methacrylate was removed by rotary evaporation and then placed under a high vacuum to give the desired half generation PAMAM-PDMS dendrimer G0.5. The ester terminated PAMAM-PDMS dendrimer G0.5 intermediate was dissolved in DCM in a 2-necked round bottom flask. To the reaction flask, ethylene diamine (EDA)} was added dropwise. The reaction was subsequently stirred at room temperature for 4 days. The solvent was removed via rotary evaporation and excess EDA was removed by washing the product with an azeotropic mixture of toluene:methanol in the ratio 9:1. The purification, by way of an azeotropic wash, was repeated until no further trace of EDA could be detected analytically, generating the desired whole generation PAMAM- _{PDMS dendrimer G1.0 as Compound (13).} Synthesis of compound 14: C_{ompound (13) was dissolved in DCM in a 2-necked round bottom flask. Methyl methacrylate was added dropwise and reaction stirred at room temperature for 5 days.} The excess methyl methacrylate was removed by rotary evaporation and then placed under a high vacuum to give the desired one and half generation PAMAM-PDMS _{dendrimer G1.5 as Compound (14).} Example 6: Synthesis of compound (15) The synthesis of this compound is shown in Figure 7.

The ester terminated PAMAM-PDMS dendrimer G0.5 intermediate was dissolved in DCM in a 2-necked round bottom flask. To the reaction flask, amine terminal silicone _{(2x terminal NH2 groups, 4 equivalents) was added dropwise. The reaction was} subsequently stirred at room temperature for 4 days. The solvent was removed via rotary _{evaporation and the residue was purified yielding the desired whole generation PAMAM-} PDMS dendrimer G1.0. PAMAM-PDMS dendrimer G1.0 intermediate was dissolved in DCM in a 2- necked round bottom flask. Methyl methacrylate was added dropwise and reaction stirred at room temperature for 5 days. The excess methyl methacrylate was removed by rotary evaporation and then placed under a high vacuum to give the desired one and half generation PAMAM-PDMS dendrimer G1.5 as Compound (15). Testing of Surfactants Stability in emulsion _{Emulsion droplets (54 µm diameter) were generated on a glass focused flow droplet generator chip (Cat. No. 00877-F, Micronit) with a nozzle of 50 µm. Compound} (13) was dissolved in octamethylcyclotetrasiloxane (D4) at 4% (w/w) as the oil phase. _{PBS buffer was used as the aqueous phase.} Generated droplets were incubated at room temperature for 10 days. Inspected _{droplet integrity under a Zeiss microscope, and took images using a Mikrotron Hi-Speed camera. Figure 9 shows images of the stabilised picodroplets after 10 days at room} temperature. The image shows that the droplets are stable. Stability in PCR thermal cycling Emulsion droplets were generated on a polydimethylsiloxane (PDMS) Pico- Gen™ biochip (Sphere Fluidics Limited) with a flow focusing cross junction nozzle of 40 _{µm x 40 µm. Novec™ 7500 was used as the continuous oil phase and polymerase chain reaction (PCR) mix solution (see Table 1) was used as the aqueous phase. 5% (w/w) of} purified Compound (4) was dissolved in the continuous oil phase prior to mixing of the oil and aqueous phases in the microfluidic device. Table 1 shows the composition of the _{PCR mix solution.} PCR mix solution Platinum® Taq DNA Polymerase kit (Life Technologies, #10966) Jurkat genomic DNA sample (Thermo Fisher Scientific, #SD1111) ACTB primer set (Jena Bioscience GmbH, # PCR-253) dNTP Mix, 10 mM each (Thermo Fisher Scientific, #R0191) Nuclease-free Water, 50 mL (Life Technologies, #AM9937 Table 1

The oil flow rate was 300 mL/hr and the aqueous flow rate was 300 mL/hr. Droplet _{generation frequency was about 1,000 Hz, and droplet volume was around 80 - 87 pL (53.5 - 55 µm in diameter).} The droplet emulsion samples generated were each placed in a G-Strom Thermal Cycler System (Labtech.com), and the thermal cycle program shown in Table 2 was run. Table 2

Droplet images were taken under a Zeiss microscope with a Mikrotron Hi-Speed _{camera before and after the PCR thermal cycles. Figure 10a shows a microscope image} of the droplet emulsion sample comprising Compound (4) after the PCR thermal cycles _{were run. The image shows that Compound (4) was functionally active by stabilising the} droplets and stopping coalescence even during thermal cycles. The PCR product was then analysed with standard agarose gel DNA _{electrophoresis. Figure 9b shows the electrophoresis result for the emulsion PCR product resulting from the droplet emulsion sample comprising Compound (4). In Figure} 10b, Lanes 1 and 12 are DNA ladders, Lanes 2-4 are the controls, Lane 10 is PCR _{product in emulsion stabilized with Compound (4). This shows that the PCR product in} Compound (4) stabilized droplet emulsion gives a clear product band as bright as that of _{the PCR product from control, i.e., Pico-Surf™ 1 stabilized droplet emulsions.} Resorufin leak test T_{o investigate the ability of the present invention to circumvent the issue of inter- droplet molecular exchange, Resorufin (10 µM) in phosphate buffer saline (PBS) as the aqueous phase, Compound (1) in Novec 7500 at 5% (w/w) as the oil phase, and droplets} were generated as described above. The positive emulsions (with Resorufin) were carefully pipetted into an Eppendorf tube containing the corresponding negative emulsions (without Resorufin). The tube was rotated slowly in order to fully mix the emulsions, and left to stand _{at room temperature over 24 hours. The droplets were then inspected under fluorescence microscope showing resorufin was still retained in original positive droplets.} Control emulsion samples were also made using 2 commercial surfactants. After 24 hours incubation at room temperature, most of resorufin had leaked out original positive _{droplets and into neighbour negative droplets.}

Claims

CLAIMS: _{1. A surfactant comprising a poly(amidoamine) dendrimer and a perfluoropolyether, a polydimethylsiloxane or both, preferably perfluoropolyether, wherein said dendrimer}

_{2. A surfactant as claimed in claim 1 of formula (II):} X(R)_n (II) wherein _{X is a poly(amidoamine) dendrimer comprising the unit (I):}

each R is independently selected from a perfluoropolyether and polydimethylsiloxane; and n is an integer between 1 and 8, preferably 2 to 6. _{3. A surfactant as claimed in claim 2, wherein X is a poly(amidoamine) dendrimer comprising the unit (III):}

wherein R¹ is CH3, OH, COOH or

wherein a is an integer between 1 and 6. _{4. A surfactant as claimed in claim 2 or 3, wherein R1 is CH3, OH, or COOH.}

_{5. A surfactant as claimed in claim 2 or 3, wherein R1 is}

wherein a is an integer between 1 and 6. _{6. A surfactant as claimed in any one of claims 3 to 5, of formula (IV):}

wherein at least one R group is present. _{7. A surfactant as claimed in claim 6, wherein R1 is CH3, OH, or COOH. 8. A surfactant as claimed in claim 6, wherein R1 is:}

wherein a is an integer between 1 and 6. _{9. A surfactant as claimed in claim 2, wherein X is a poly(amidoamine) dendrimer comprising the unit (V):}

wherein each Z is independently selected from OC1-6 alkyl (e.g. OMe), NH(CH2)kNH-R and NH(CH2)k-R; _{each R is independently perfluoropolyether or polydimethylsiloxane; k is an integer from 1 to 6;} with the proviso that at least one Z is NH(CH2)kNH-R or NH(CH2)k-R.

wherein

wherein a is an integer between 1 and 6; and _{Z is OC1-6alkyl (e.g. OMe), NH(CH2)kNH-R and NH(CH2)k-R wherein each R is independently perfluoropolyether or polydimethylsiloxane and k is an integer from 1 to 6. 11. A surfactant as claimed in claim 9 or 10, wherein R1 is CH3, OH, or COOH. 12. A surfactant as claimed in claim 9 or 10, wherein R1 is:}

_{wherein a is an integer between 1 and 6; and} Z is OC_1-6alkyl (e.g. OMe), NH(CH₂)_kNH-R and NH(CH₂)_k-R, wherein each R is _{independently perfluoropolyether or polydimethylsiloxane and k is an integer from 1 to 6. 13. A surfactant as claimed in any one of claims 9 to 12, wherein one Z group is} NH(CH2)kNH-R or NH(CH2)k-R, wherein each R is independently perfluoropolyether or _{polydimethylsiloxane and k is an integer from 1 to 6. 14. A surfactant as claimed in any one of claims 9 to 12, wherein at least two Z groups} are NH(CH2)kNH-R or NH(CH2)k-R, wherein each R is independently perfluoropolyether _{or polydimethylsiloxane and k is an integer from 1 to 6. 15. A surfactant as claimed in any one of claims 9 to 12, wherein at least three Z} groups are NH(CH2)kNH-R or NH(CH2)k-R, wherein each R is independently _{perfluoropolyether or polydimethylsiloxane and k is an integer from 1 to 6.}

_{16. A surfactant as claimed in any one of claims 9 to 12, wherein at least four Z} groups are NH(CH2)kNH-R or NH(CH2)k-R, wherein each R is independently _{perfluoropolyether or polydimethylsiloxane and k is an integer from 1 to 6. 17. A surfactant as claimed in any one of claims 1 to 16, wherein said perfluoropolyether comprises a repeat unit of the formula:} -[CF(CF3)CF2O]b-, wherein b is a positive integer, preferably from 1 to 100. _{18. A surfactant as claimed in any one of claims 1 to 17, wherein said} polydimethylsiloxane comprises a repeat unit of the formulae: -(Si(CH₃)₂O)_p(CH₂)_q- or -(Si(CH₃)₂O)_pSi(CH₂)_q- _{wherein p is a positive integer and q is 0 or a positive integer. 19. A surfactant as claimed in any one of claims 6-8, wherein each R is independently} selected from: Y-(CH₂)_r-(Si(CH₃)₂O)_p(CH₂)_q-; Y-(CH₂)_r-(Si(CH₃)₂O)_pSi(CH₃)₂(CH₂)_q-; Y-(CH₂)_r-(Si(CH₃)₂O)_p(CH₂)_q-CO-; or Y-(CH₂)_r-(Si(CH₃)₂O)_pSi(CH₃)₂(CH₂)_q-CO- wherein p is a positive integer; q and r are 0 or a positive integer; and Y is H, NH₂, OH, or COOH. _{20. A surfactant as claimed in claim 1 of formula (VII), wherein X is a} poly(amidoamine) dendrimer comprising the unit:

wherein each of e and g are an integer from 1 to 6 and f is a positive integer. _{21. A method of making a surfactant as claimed in any one of claims 1 to 19,} comprising: _{(i) forming a poly(amidoamine) dendrimer comprising unit (I); and (ii) connecting said dendrimer comprising unit (I) to a perfluoropolyether, a} polydimethylsiloxane or both. _{22. A method of making a surfactant as claimed in claim 20 comprising: (i) providing H2N-(CH2)r-[(Si(CH3)2O)pSi(CH2)q]s-(CH2)r-NH2 wherein wherein p is 1} to 100, q is 1 to 100, r is 1 to 12 and s is 1 to 100; and _{(ii) converting it into a compound of formula (VII), preferably by reacting it with} methyl methacrylate in a 1,4-Michael addition to form an ester-terminated intermediate, followed by an amination with an amine, e.g. optionally substituted alkyl amine. _{23. A composition, preferably an emulsion, comprising a surfactant as claimed in any} one of claims 1 to 20. _{24. Use of a compound as defined in any one of claims 1 to 20 as a surfactant.}

_{25. Use of a surfactant as defined in any one of claims 1 to 20 in the preparation of} an emulsion. _{26. A method of preparing an emulsion as claimed in claim 23 comprising: (i) providing an aqueous phase; (ii) providing an oil phase; and (iii) mixing said aqueous phase, said oil phase and a surfactant as claimed in any one of claims 1 to 20 to form said emulsion.}