Jean-Christophe Baret

Surfactants in droplet-based microfluidics

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Introduction

Exactly 10 years ago, in 2001, Todd Thorsen, Richard W. Roberts, Frances H. Arnold and Stephen R. Quake reported in Physical Review Letters the first use of microfluidic chips for the production of monodisperse droplets 1 (see Figure 1). At the end of the Letter, the authors stated: 'This system may also find application as a component in a microfluidic screening chip, since it has been shown that subnanoliter vesicles have significant potential as tools for screening of biological and synthetic compounds'. Interestingly, the parallelisation of reactions in small droplets is also thought to have a role in the prebiotic evolution of life 2 : among the billions of small reservoirs generated at the surface of oceans and stabilized by small amphiphilic molecules, a couple of favorable conditions might have arisen for the early synthesis of short polymers while the dynamics of coalescence and splitting of droplets would represent basic reproduction mechanisms. Droplet-based microfluidics is now indeed used as a component for many applications in biotechnology. Recent reviews summarize the basics of droplet-based microfluidics and of droplet manipulation techniques [3][4][5][6] , as well as the range of applications of the technology [7][8][9] . To summarize, the interest of the technology lies in the fact that (i) droplets are microreactors of a size perfectly suited for the manipulation of single genes, cells or organisms (typically 1 to 100 microns) and that (ii) these droplets can be manipulated in an automated manner in microfluidic channels at a very high throughput (typically 1 000 to 10 000 droplet per second).

If it becomes clear that droplet-based microfluidics has a great potential for many applications in biotechnology -some of them will be discussed in the following -questions might arise on the limits of the technology: can this be a universal system for the miniaturization and automation of biochemical assays ? In order to answer this question, the essential characteristics of the system -namely the manipulation of emulsion -has to be considered: understanding how general the approach can be, lies in the understanding and the control of the properties of emulsions.

In the present manuscript, we want to focus on one central ingredient of the droplet-based microfluidic technology: the surfactant. These amphiphilic molecules are commonly used to stabilize the droplet interface and prevent coalescence of droplets. However, their use has some impacts on the feasibility of biochemical assays. In the first section of the manuscript, we will summarize the use of various emulsification systems through the applications they are used for, and the limitations encountered. In the second section of the manuscript, we will review how microfluidic devices are now taylored to study surfactants and emulsions, especially their dynamic behaviors, for a better understanding of the physics of interfaces.

Surfactants and emulsions

The term 'surfactant' is the contraction of 'surface active agent' [START_REF] Rosen | Surfactants and Interfacial phenomena[END_REF] . It describes an amphiphilic molecule, i.e. with different groups having affinities for different immiscible phases (water/air, water/oil, oil/air. . . ). This property drives the molecule to the interface: the surface tension between the two phases is decreased. The decrease of the surface tension is directly given by the amount of molecules adsorbed at the interface as given by the Gibbs adsorption isotherm for dilute solutions [START_REF] Cabane | Liquides: Solutions, dispersions, emulsions, gels[END_REF] :

Γ = - c RT dγ dc
where Γ is the surface concentration, c the surfactant bulk concentration and γ the surface tension (T is temperature and R the gas constant). Other properties of the interface (flow, interactions. . . )

are also modified by surfactant as seen in the simple experiment of rising bubble [START_REF] Ybert | [END_REF] . In the presence of surfactant (for example a protein), after a transient decay the speed of the bubble rising in the protein solution stabilises to a value smaller than in the pure water case. As surfactant adsorbs to the interface, the interface rigidifies: the loss of mobility imposes a change in the boundary condition at the bubble interface which slows it down [START_REF] Ybert | [END_REF] . In the viscous case, the drag force on the droplet is intermediate between those of a solid or a gas body [START_REF] Guyon | Physical Hydrodynamics[END_REF] . The origin of the rigidification is the so-called Marangoni effect: as the bubble moves, the surfactant distribution is non-uniform, with an excess at the rear of the bubble. The non-uniform surface concentration leads to a gradient in surface tension (the surface tension is decreased at the rear of the bubble) which generate a stress opposed to the flow (Figure 2 (a,b)). Interestingly, this Marangoni force already rigidifies the interface when the surfactant concentration at the interfaces is less than half the equilibrium concentration [START_REF] Ybert | [END_REF] . The motion of surfactant at the interface, its exchange properties with the bulk phase, and the response of surfactant layers to deformations of the interface are dynamic processes controlling the rheological properties of the interface: surfactants modify not only the behavior of interfaces but also the behavior of the whole liquid structure through the coupling with the flow around the interface [START_REF] Erni | [END_REF] .

In emulsions 15 , surfactants have a basic role: they guarantee that droplets do not coalesce, which is the minimal requirement for the use of droplets as microreactors. The dispersion of a fluid into another is a system out of thermodynamic equilibrium: the total energetical cost for the formation of the interfacial area is defavorable and the minimum of energy of the system is a configuration where the two liquids are separated in two phases. The driving force acting towards the homogeneisation of the system is balanced by the action of surfactants. Adding surfactants provides an energy barrier to stabilize the dispersion in a metastable state 15,16 . Dynamic processes are then controlling the stability of the emulsion. One aging mechanism of emulsions is induced by coalescence of droplets. Surfactants hinder such process through two (not mutually exclusive) mechanisms: (i) droplet are stabilized by steric repulsion of the surfactant molecules as in colloidal dispersions and (ii) surfactant gradients at the droplet interface induced by the drainage of the continuous film between two droplets will result in a force acting against the drainage, according to the previously discussed Marangoni effect 17 (Figure 2 (c,d)).

Besides the coalescence mechanism, Ostwald ripening 15,18 is another aging mechanisms: small droplets of the emulsion having a higher Laplace pressure than larger droplets, they will then tend to dissolve in the larger ones leading to an increase of droplet size in time and coarsening of the emulsion. This is possible when the dispersed phase is even slightly soluble in the continuous phase. This effect can be reduced by adding to the droplet species that are not soluble in the continuous phase. A loss of the dispersed phase in the small droplets leads to an increase of osmotic pressure that hinders the exchange of the dispersed phase. These osmotic effects can lead to a polydisperse emulsion with well defined droplet sizes 19 . Finally, in other cases, the solute of the dispersed phase can be exchanged by phase partitioning, either directly in the continuous phase or in the surfactant micelles of the continuous phase [19][20][21] . In all these cases, the surfactant plays a key role in these mechanisms as depicted in the case of oil-in-water emulsion in Figure 3 22 .

Figure 2 Marangoni effects in the presence of surfactant. (a) The flow pattern in the absence of surfactant for a droplet moving in a surrounding fluid is given by the black arrows (in the reference frame of the droplet). (b) in the presence of surfactant, the flow induces a heterogeneous surfactant distribution causing a Marangoni stress opposing to the flow (red arrow): the viscous drag is then modified by the presence of surfactant which rigidifies the interface. (c) Upon collision of droplets, the continuous phase has to be drained before droplets coalesce. (d) in the presence of surfactants, the gradient of surface density of the surfactant generates a Marangoni stress (red arrows) which counteracts the film drainage, increasing this drainage time 17 and therefore stabilising the emulsion against coalescence.

Microreactors for biochemical reactions

The parallelisation and automation of liquid handling in droplet-based microfluidics makes it appealing for many biotechnology applications. The droplets can be produced on chip using various mechanisms 1,23,24 , splitted to increase production rates 25,26 , reinjected 27 , incubated 28,29 , fused 24,30,31 , analyzed and sorted 27,[32][33][34] . The parallelisation of the reactions in the droplets has the advantage that different reactions can proceed in different droplets: different chemicals or, alternatively, one single compound at different concentrations, can be screened [35][36][37] providing automated tools for optimizing chemical synthesis. For example, droplet-based microfluidics provides tools to obtain informations at the single cell level and on large populations which is extremely powerful as an analytical tool to find extraordinary variants or obtain statistically relevant data 36,37 . Millions of different gene variants can be encapsulated in droplet and individually amplified for quantitative emulsion PCR [38][39][40] . At the single gene level, such systems are especially interesting for diagnostics, in order to find mutants of genes among a large population of normal genes [41][42][43] . Finally, by enabling the selection of specific variants in a population, successive rounds of mutagenesis and selection can be performed in an automated manner and on small volume for directed evolution of enzymes or micro-organisms 27,34 .

For these biochemical applications, the surfactant -and, in general, the emulsification system (i.e. the combination of oil phase, aqueous phase and surfactant) -must be biocompatible. The oil-water interface must appear as inert as possible to the droplet content. Indeed with the miniatur-ization of the assay in micrometer-sized droplets, interfacial effects become predominant compared to bulk and the downscaling of the assay can only be achieved with a minimisation of the surface effects. Biocompatibility can basically be summarized as the fact that the bio-chemical reaction performed in droplets must be as efficient as in bulk. The best examples of non-biocompatible systems are given by the adsorption or the denaturation of proteins at the water / oil interface 44 (Figure 4) or the interactions of surfactants with cell membranes or proteins 45 . In order to use droplets as microreactors, these three major points -stability (Figure 2), control of exchange (Fig-

ure 3) and biocompatibility (Figure 4)-have to be addressed at the same time which gives a very stringent constraint on the surfactant and the emulsification system to be used.

Although many emulsification systems have been described and are commercially available, only a few have been successfully used for biochemical applications in microfluidics. One of the original application for emulsion is amplification of DNA in droplets (emulsion PCR) used as a sample preparation for DNA sequencing. In this case, single genes are encapsulated and amplified in parallel within the emulsion. One advantage that microfluidics brings to PCR from single genes is the monodispersity of the droplet microreactors: it ensures that each single DNA fragment is encapsulated in a reservoir identical to the other. The monodispersity of the emulsion is then a key for quantitative amplification from single genes. PCR is a very good example of the constraints on the emulsification system since the multiple cycles of temperature up to 95 degrees are a very strong constraint on the system. The interfacial activity and the physical-chemical parameters of the surfactant such as the critical micellar concentration are a function of the temperature and obtaining a stable system over a wide range of temperature is difficult. A recent review summarizes the efforts and progress in the field 46 . Emulsion PCR has been originally reported in silicone oils (Table 1) in non-microfluidic systems 47 and later in microfluidics 48,49 where the poor compatibility of silicone oils with microfluidic chips produced in PDMS 50 requires the use of glass devices 48,49,51 . Alternative systems are based on hydrocarbon oils which have been successfully used for PCR applications 52,53 (Table 2) and for directed evolution of enzymes 54 . However in hydrocarbon systems, hydrophobic compounds can phase partition into the oil which limits the range of applications 55 . With a leaking microreactor, bio-chemical assays can only be performed provided that the exchange of molecules is not a limiting step. They are therefore limited to the applications such as PCR where the objects of interest (the DNA or RNA fragments) do not exchange between the droplets. Finally, fluorinated oils and fluorosurfactants are also suitable for single gene amplification [41][42][43]56 . The droplets can be thermocycled on chip 56 or off-chip [41][42][43] and further reinjected in other microfluidic chips for analysis. Figure 5 shows the example of an emulsion plated after a PCR amplification with Taqman probes for the detection of mutant genes in a pool of normal genes 42 . Table 1 Reported surfactants (all commercially available) and corresponding applications for water-in-silicon oil emulsions in microfluidics.

Surfactant

Silicon Oil DC200 PDMS AR20 Triton X-100 PCR 48,49,51 (complex oil mix, see 47 ) SDS (in water)

Oil-in-water emulsification 57

ABIL EM90

Directed evolution 54

No surfactant

Chemical compound storage 58 Raman measurement 59 Emulsification 60 64,65 Droplet patterns 66 Droplet patterns 67 Droplet pairing 68 Droplet splitting 25,69 Chemical coupling 70 Molecular exchange 20 Electrocoalescence 63 Interfacial instabilities 71 Cell encapsulation 72,73 Laser manipulation 74 Chemical reactions 75 Electrocoalescence 31,76 Electricaly-assisted emulsification 77 Droplet sorting 32 Electrowetting in emulsification 78 89,90 microPIV 91 Compound screening 92 Protein cristallisation 93 Protein cristallisation 93 Cell sorting 33

PF-decanol

Commercial PCR in droplets 94

PF-TD acid

Commercial Protein adsorption 44 PF-TD OEG Home made Protein adsorption 44 (from above)

Chemistrode 95

PFPE-COOH Commercial

Splitting droplets 26 Electrocoalescence 96 (Krytox, Dupont) Droplet detection 97

PFPE-COONH4

Home-made Cells in droplets 36 Multiple emulsions 98 Cells in droplets 99 (from Krytox, Dupont) Droplet hydrodynamics 100 Chemical reaction 101

PFPE-PEG

Home-made Cells in droplets 36 Directed evolution 34 Cells in droplets 99 (from Krytox, Dupont)

Picoinjector 102 Dropspot 103 EA, Proprietary PCR 56 PCR and diagnostics 42 Coalescence 104 Molecular exchange 20 (Raindance Tech.)

Diagnostics 43 DNA amplification 105 Chemical gradients 106 Chemical gradients 106 Sorting microorganisms 27,107 Droplet separation 108 Yeasts in droplets 19 Electrocoalescence 96 PFPE-DMP Home-made Cells in droplets 99,109 Bacteria in droplets 110 (from Krytox, Dupont)

Coalescence 111

Short chains

Home-made synthesis Surface tension and emulsification 112,113 No surfactant Bacteria / antibiotics 114 Many other applications of droplet-based microfluidics rely on the use of fluorinated oils and fluorosurfactants (Table 3). The choice of fluorinated oils is mainly driven by two points: first, they are appealing since most of organic compounds are insoluble in these oils. The compounds encapsulated in the droplet should not phase partition and therefore remain in the droplet which solves the exchange limitations of organic and silicone oils. The second advantage of fluorinated oils is biocompatibily. The first experiments showing the growth of cells on fluorocarbon liquid interfaces dates back to the 80's 115 . These oils are compatible with cell culture but the importance of surfactants on the cell growth has been reported 116,117 . The cell spreading on the surface is linked to the ability of the cells to grow on a protein film at the oil interface (Figure 6). At the same

Biocompatible surfactants

Although fluorinated oils represent a very promising component in the system, the range of surfactants available for the stabilization of water in oil interfaces is still limited. Fluorosurfactants (Perfluoropolyethers -PFPE -with hydrophilic head groups) are used as emulsifiers for supercritical CO 2 124 and are perfectly suited to stabilize water in fluorinated oil emulsions. Although the carboxylic terminated PFPE has questionnable biocompatibility properties 125 , many other fluorinated surfactants have been reported to show very interesting biocompatibility properties 126 . For example some fluorinated surfactants with short fluorinated tails are used as therapeutic agents in lung deficiencies cases or for carrier of drugs through the pulmonary route 127,128 . Surfactants with short fluorinated tails have been used in microfluidic systems: perfluorooctanol (PFO) was, for example, used for the study of chemical reactions and protein cristallisation 89,93 . However, the system showed some limitations in terms of biocompatibility, for example, non-specific interaction of the surfactant with fibrinogen 44 . Using various head groups on the short fluorinated chain of PFO, it has also been shown that the biocompatibility (measured through protein adsorption at interfaces) is directly linked to the chemistry of the hydrophilic head group 44 . Chemical modifications of the head of the surfactant lead to an increase of protein adsorption for a carboxylic head-group or to a decrease of adsorption (enhanced biocompatibility) with a short ethylenoxide chain 44 (Figure 4).

Over the past years, other molecules have been developed specifically for their use in microfluidic applications. Amphiphilic molecules with perfluoropolyether chains or perfluoroalkyl chains and hydrophilic head groups have been reported 99,112,113,129,[START_REF]A1, Fluorocarbon emulsion stabilizing surfactants[END_REF] . Currently, the most promising molecules consist in a block copolymer of perfluoropolyether and polyethylenoxide. These block copolymers reduce protein adsorption or the interactions with cell membranes. Such molecules have been used to screen mammalian cells, yeasts, bacteria, viruses or for the encapsulation of hybridoma cells 27,34,36,37,99,103,107,[START_REF] Koester | [END_REF] (Figure 8). Mineral oils have been used with cells, for example focusing on encapsulation mechanisms 72,73 , and also as double-emulsification system to screen bacteria 132 with incubation times of a few minutes and limited to ∼2 hours 132 . Fluorinated oils are -to date -the examples where cell survival and proliferation has been demonstrated with long-term incubation of organisms where gas exchange becomes crucial 99,103 .

Molecular exchange between droplets

Adding surfactant to the oil-water system has another consequence. Although the exchange of molecules through phase partitioning is reduced by the use of fluorinated oils, surfactant have an effect on this process. Surfactant bilayers can form between droplet and those can be porous to certain molecules, especially small molecules, as observed during thermocycling 55 . More recently, it has also been shown that the formation of a lipid bilayer between droplets is required to obtain synchronization of droplet assemblies in which an oscillating reaction is taking place 70,79 : transport through bilayers is therefore an important point to consider. It is however not the only mechanism -besides simple phase partitioning -since it has been shown that micelles solubilised in the continuous phase can act as carrier for small molecules 133,134 . This transport mechanism is function of the lipophobicity of the molecules to be transported, as demonstrated in the case of a mineral oil and non-ionic surfactant (polyoxyethylen oleyl ester) 135 .

In water in perfluorinated oil, it has been demonstrated recently that the exchange rate is a function of the hydrophobicity of the molecules: small fluorescent molecules (coumarine) were used as a model for the exchange: by grafting sulfonate groups on the coumarine, the exchange rate has been drastically reduced 136 . The size of the molecule is also a key point to consider, as demonstrated in numerical models: larger molecules have to overcome a higher energetic barrier to be transported through the surfactant layer 21 . Other mechanisms of exchange have been reported

in microemulsions with the possible coalescence and subsequent division of droplets 137 . However, in the case of microfluidic emulsion, this process can be ruled out at the droplet level (such process would lead quickly to a polydisperse emulsion), but might still happen at the micelles level.

Solvent is also transported between droplets containing different concentrations of non diffusive compounds. This phenomenon has been observed with yeast cells metabolising sugar in droplets during their growth phase. In order to maintain the chemical potential of the sugar constant, the droplet containing yeast shrinks in order to increase the concentration of remaining sugar 19 . Since water is in this experiment insoluble in the fluorinated oil, the exchange is here again mainly mediated by the micelles and driven by the osmotic pressure difference between the droplets. It is therefore important to understand and control such processes in order to guarantee that the microreactors remain intact. Alternatively, one can make use of such transport properties for the control of reactions 20,138 .

Studying emulsions and surfactants in microfluidics

Surfactants play a ubiquitous role in droplet-based microfluidic technology. It is therefore important to understand the behavior of these molecules at the time-scale and at the length-scale at which they are used. New characterizations approaches for surfactant and emulsion sciences are required to complement the existing methods and microfluidic systems provide an interesting platform for such studies.

Emulsion stability and surfactant dynamics

The water in oil emulsions should of course be stable for times sufficiently long to perform the bio-chemical operations. If surfactant free systems might even be usable 40,88,[139][140][141] surfactants are required for long-term stability, for example, for storage applications. The stability of the reactors is also a very stringent conditions for PCR applications where cycles of temperature have to reach 95 degrees. Long term stability of fluorinated emulsions 142 have already been reported which, again, makes them an appealing system. It is important to understand the stability of droplet, for example when they flow in microchannels and experience high shear rates. Microfluidic systems are perfectly suited for this type of study because they work exactly at the length and time-scale at which these effects should be studied. The interest of microfluidics for the study of interfaces can be highlighted by the simple problem of the kinetics of surfactant adsorption to interfaces 143 .

In standard emulsification systems or in flow focusing geometries, the time scale at which new interfaces are produced is typically in the range of the millisecond or below. However, there is a very limited number of experimental methods that can address the dynamics of surfactant at timescales close to the millisecond 144 . Therefore microfluidic systems themselves are now developed towards a better understanding of the basic principles of the physics of interfaces at these short time-scale.

In parallel, the question of the length-scale is relevant. It has indeed been shown that the kinetics of surfactant adsorption is controlled by different processes at large or small scale. The kinetics of adsorption involves two processes, (i) diffusion to the interface and (ii) binding to the interface, each of them with a typical time-and length-scale. Above a critical droplet size, surfactant adsorption is controlled by diffusive processes, while below, the process is controlled by the adsorption/desorption kinetics 145 . For classical surfactants, a crossover is found around 20 to 70 microns droplet size. Hence, understanding surfactant kinetics at the right time and length-scale is important. Microfluidics provides a very powerful platform to create and control interfaces, to visualize them, as well as to place these interfaces in external fields (thermal gradients, electric fields, external flow fields...). New experimental approaches are now developed for a better understanding of the physical-chemistry of interfaces.

Coalescence and electrocoalescence

Understanding coalescence of droplets -with or without surfactants -has been extensively studied. Two approaching droplets merge when the thin oil film drained between them becomes unstable 17,146 : the coalescence is controlled by the capillary number 17,147 and adding surfactant delays the coalescence process 17 . Recent studies demonstrated that the instability of the film can be triggered by the forcing of droplet separation 64,148,149 . This process leads to cascades of coalescence in a line of droplets and is the source of massive destabilization of emulsions, even in the presence of surfactant 64 (Figure 9). Interestingly, these microfluidic experiments bring additional quantitative insights into the physics of emulsions. The cascade of coalescence observed in the microfluidic systems could be a mechanism leading to phase inversion in emulsification which is still an open problem 65 .

The kinetics of surfactant adsorption has also an influence on coalescence. Indeed at the timescale of droplet production (typically ∼ ms), the surfactant does not fully cover the interface.

Therefore droplets colliding right after production have a higher chance to coalescence than if the surfactant has been given time to adsorb at the interface 111 : microfluidic systems provide means to study such processes by decoupling droplet production and droplet collisions showing the importance of surfactant adsorption kinetics on the coalescence in emulsions. For applications, where the fusion of droplets has to be controlled, it is possible to use this dynamic process for a passive control of the coalescence of droplet pairs 104 . This system complements several other passive systems developed for the control droplets coalescence, for example based on wetting patches 95,150,151 . Applications, however, often require an active control of the coalescence.

A stable interface covered by surfactants can be destabilized in controlled manner in electric fields. Electrocoalescence consists in the destabilization of the thin oil film between two aqueous droplets 30 . By the control of single oil lamelae between two droplets, it has been shown that the destabilization of the oil layer is linked to an electro-hydrodynamic instability of the oil film and is not induced by a dielectric breakdown of the insulating oil 63 . Collective behavior can also be observed with propagation of coalescence events 76,96 leading to destabilization of large parts of an emulsion. The principle of electrocoalescence has been used to trigger the fusion of droplet for the study and control of very fast reactions 31,101,152,153 or to perform several bio-chemical reactions in series 104 . Electric field can also be used to tune the adhesion properties of surfactant bilayers 86 and direct measurement of the electrical properties of lipid bilayers have also been recently performed 79 . Reciprocally, the fine control of the adhesion properties of droplets and a microfluidic control of those droplets in microfluidics allows quantitative measurement of the behavior of the formed lipid bilayers in electric fields, providing insights to a better understanding of electroporation processes 154 . The electric fields generated on chip are therefore versatile tools to control interfacial layers and can be used to modulate the energetic barrier linked to the surfactant at interfaces. In general, microfluidic devices provide new methods to quantitatively analyse dynamic interfacial phenomena.

Surface tension measurement and interfacial rheology

Understanding coalescence also requires a better understanding of the dynamics of surfactant-laden interfaces. As discussed, the coalescence mechanism is linked to the dynamics of the surfactant layers 17 . It becomes then crucial to determine the rheological properties of the interface: how does the surfactant flow at the interface as a function of the applied external shear stress ? What is the boundary condition at the interface ? These questions can be answered by interfacial rheology methods [START_REF] Erni | [END_REF]144,155,156 . However, once again, it is extremely difficult to obtain informations on the interfacial properties at the time and length scales reached in microfluidic applications 144 . The study in microfluidic of these quantities is therefore extremely interesting. The possibility to perform micro Particle Image Velocimetry (microPIV) measurements of the droplet flow 91 provides informations on the boundary conditions at the interface which is linked to the way surfactant are flowing. Coupling these microPIV informations with in situ measurements of droplet deformations provides means to extract more information on the rheological properties of interfaces 84,[157][158][159] .

Surface tension measurement are feasible in microfluidics: using electrowetting surface tension in oil have been measured with droplet sizes down to 20 nL 160 . In microfluidic channels, the effect of surfactant on surface tension is also measurable. As already discussed, surfactant is not fully covering the droplet interface right after droplet production 111 . The surface tension of the interface is then comprised between the surface tension without surfactant and the equilibrium surface tension in the presence of surfactant. The surface tension being one of the parameter controlling droplet size in a T-junction or in a flow focusing junction, a measurement of droplet size is an indirect way to access the surface tension at the time-scale of droplet production 81,82 . More complex dynamic processes such as tip-streaming 83,161 which is an instability of the surfactant layer in an external flow field or other interfacial instabilities at low surface tensions 71 can also be addressed in microfluidic environments. Microfluidic systems are therefore now also used to access informations on surface tension at small scales and with dynamics much below 1 s 162 .

They offer a versatile platform for the study of dynamic processes at short length-scale in a well controlled environment and should provide new insights into the dynamics of interfaces [START_REF] Erni | [END_REF] .

Active and reactive surfactants: Towards complex systems

Finally, recent systems have been described to actively control the interfacial properties of surfactants. The aggregation of droplets -and therefore their adhesion properties -can be controlled by external stimuli such as pH variations in the continuous phase 163 . This aggregation is reversible and could be used to control self-organization in emulsion. In microfluidic channels the addition of chemicals such as EDTA or salt has been shown to influence droplet stability against coalescence or the adhesion properties of the droplets in a reversible manner 164 . Such systems could represent interesting opportunities as environmental sensors in liquid systems. Droplet interfaces can be used to display catalytic parts for chemical reactions and organic synthesis 165 . In such cases, the increase of surface area per unit of volume coming from the reaction in emulsion is beneficial.

The possibility for surfactants to react with other components of the systems also provides new actuation mechanisms. Indeed it is well known that surface tension gradients can induce a liquid flow through Marangoni force 166 . When two surfactants are mixed at the surface of a droplet with an heterogeneous distribution of the molecule, the droplet can also move. In the presence of a dynamic exchange of the surfactant at the interface and a chemical reaction which switches the interfacial properties of the surfactant, the motion can be sustained for a few minutes and the speed of the droplet can be controlled by the concentration of the surfactant 167 . In a population of monodisperse droplets, collective dynamics can then be observed, similar to the collective motion that can happen in ensemble of micro-organisms. A combination of reactive surfactants 79 , environ-mental sensing surfactants 163 , surfactant-induced locomotion 166,167 , and communication between agents (here the droplets) through chemical signaling 20,167 should provide means to produce, in the future, powerful biomimetic systems for applications as biosensors, autonomous microsystems or programmable microcontrollers for soft-matter.

Conclusions and outlook

Surfactants are an essential part of the droplet-based microfluidic technology. They are involved in the stabilization of droplet interfaces, in the biocompatibility of the system and in the exchange rate of molecules between droplets. The recent progress in the applications of droplet-based microfluidics have been possible by the development of new molecules and their charaterisations either off-chip or on-chip. Fluorosurfactants for water in fluorinated oil emulsions are very interesting systems for biotechnology applications. They can be used as systems for thermocycling, cell encapsulation and proliferation and are therefore the most promising system for the miniaturization of biochemical assays in micron-sized emulsion droplets. The use of such micron sized droplets and their actuation at time-scale of the order of a millisecond in microchannels requires the characterisation of surfactants at these time and length-scales. In this respect, microfluidic systems appear to be very powerful tools to study surfactant dynamics at the time-and length-scale relevant for the corresponding microfluidic applications. Finally, the control of interfaces in droplet-based microfluidics offers means to produce new materials in a very controlled maner. Reactions can be performed in droplets but also at interfaces for the synthesis of core-shell particles 168 or polymerosomes 169 . Double and multiple emulsions can also be produced 98,170 . In such systems, surfactants are essential since the interfacial properties of the different phases will determine whether or not multiple emulsions can be stably generated 171 . Highly complex systems -including for example oscillating chemical reactions -can be studied and controlled in microfluidics. The capability of microfluidics to generate controlled liquid structures with complex functionalities at interfaces, in the continuous phase, or in the dispersed phase, should bring this technology towards the creation of autonomous, biomimetic microsystems that could be used as model systems of micro-organisms or to programmable microcontroller for soft-matter.

Figure 1

 1 Figure 1 Droplet production in a microfluidic chip. The monodispersity of the droplets makes it appealing for biochemical applications but also as a model tool to understand the physics and physical-chemistry of interfaces. Figure reprinted with permission from Thorsen et al., PRL, 86, 4163, 2001. Copyright (2001) by the American Physical Society 1

Figure 3

 3 Figure 3 Role of surfactant micelles in the transport between droplets in the case of Ostwald ripening 22 . Oil molecules solubilised in micelles (1a,1b) diffuse through the oil (2) and are uptaken by another droplet (3a, 3b). Reprinted from J. Colloid Interface Sci, Ariyaprakai and Dungan, 343, 102 (2010), Copyright (2010), with permission from Elsevier 22 .

Figure 4

 4 Figure 4 Protein adsorption in droplets as a function of the polar head group of the surfactant. (a) adsorption of fibrinogen at the interface with an alcool head group. (b) Polyethylenoxide head groups reduce protein adsorption as revealed by the distribution of fluorescence at the droplet-oil interface. Reprinted with permission from Roach et al., Anal. Chem., 77, 785 (2005). Copyright (2005) American Chemical Society 44 .

Figure 5

 5 Figure 5 Water in fluorinated oil emulsion after thermocycling for PCR. The droplet color indicates the presence of a normal gene in the droplet (a -red) or mutant (b -green) using Taqman probe. The ratio of green droplets to red droplets (c) gives a measurement of the ratio of Mutant to Wild-type gene in a population. This system used for diagnostic relies on the possibility to perform billions of reaction in parallel in droplets for analysis at the single gene level. Pekin et al., Lab Chip, 11, 2156 (2011) -Reproduced by permission of The Royal Society of Chemistry 42

Figure 6

 6 Figure6Example of cell growth on polylysine coated droplet of fluorinated oil (FC-70) . The cells adhere well at the droplet surface and show the same division rate as in conventional cell culture116 . Figure fromKeese and Giaver, Science, 219, 1448 (1983). Reprinted with permission from AAAS.116

Figure 7

 7 Figure 7 Water in fluorinated oil emulsion for the control of the oxygenation of red blood cells. The cells appear as black dots in their native state and as white dots when hemoglobin fiber polymerize (as in the case of sickle cell anemia). By controlling the amount of oxygen in the oil as a function of time, several cycles of oxygenation -deoxygenation of the cells are performed, mimicking the cycles the cells would undergo in the human body 109,123 . Abbyad et al., Lab Chip, 11, 813 (2011)-Reproduced by permission of The Royal Society of Chemistry 123

Figure 8

 8 Figure 8 Example of cell growth on surfactant layers using fluorinated oil and fluorosurfactant generated from the same PFPE chain (red) after deposition (0d), after 1 day of incubation (1d) and after 2 days (2d). Polyethylenoxide head groups and dimorpholinophosphate groups are shown to be biocompatible: (c) and (d) show the same behavior as the control without surfactant (a). Polylysine head group (not shown here) or carboxylic head groups (b) was shown to kill cells 99 . Adapted from Chemistry and Biology, Clausell-Tormos et al. 15, 427 (2008), Copyright (2008), with permission from Elsevier.99

Figure 9

 9 Figure 9 Quantitative analysis of droplet coalescence in microfluidic channels. The separation of droplets can induce the coalescence (a). In a stream of droplets, a first coalescence will force the separation of the next interface. This separation leads to a second coalescence and then to collective coalescence by the cascade of successive separations/coalescence events (b). Figure reprinted with permission from Bremond et al., PRL, 100, 024501, 2008. Copyright (2008) by the American Physical Society 64

Table 2

 2 Reported surfactants (all commercially available) and corresponding applications for water-in-hydrocarbon oil emulsions in microfluidics.

	Surfactant			Hydrocarbon Oil
		Hexadecane	Tetra/Octa/Dodecane Mineral oil	Isopar M	Vegetable / organic
	Span80	Emulsification 1,61	Emulsification 1	Protein expression 62	Emulsification 63
		Coalescence

Table 3

 3 Reported surfactants and corresponding applications for water-in-fluorinated oil emulsions in microfluidics. ( * PF=Perfluoro-, PFPE=Perfluoropolyether, TD=tetradecanoic- * * Perfluoro-hexane perfluoro-cyclohexane perfluoro-decaline perfluoro-perhydrophenantren)

	Surfactant *	Source					Fluorinated Oil
			PFH / PFC / PFD /	Carrier oil (Rain-	HFE / Novec	FC40	FC70 / FC77	FC3283
			PFPH * *		dance Technolo-
					gies)
	PF-octanol	Commercial	Reaction	kinet-
			ics

Acknowledgments

The author thanks Dr. J. Brian Hutchison (Raindance Technologies), Dr. Valérie Taly (ISIS-UdS-CNRS) and Dr. S. Vessigaud for helpful comments as well as Prof. Dr. Stephan Herminghaus (MPI-ds Goettingen) for his support and Dr. E. Rio (Universit Paris Sud) for helpful discussions.

Financial support from the Max-Planck Gesellschaft and from the Deutsche Forschunggemeinschaft through the Sonderforschungbereich 755 (Nanoscale Photonic Imaging) are also acknowledged.