Dispersing an aqueous medium into an immiscible oil phase allows producing droplets whose interface is subjected to a high surface tension. As a consequence, droplets tend to coalesce to minimize oil/water surface area, a phenomenon that disrupts genotype/phenotype linkage. Nevertheless, droplet stability can be preserved by supplementing the oil phase with a “surface active agent” (or surfactant). Surfactants are amphiphilic molecules composed of different groups having affinity for each phase (oil and aqueous) and that are able to reduce the surface tension by partitioning at the oil/aqueous interface (
Figure 5a). Once at the interface, the surfactant was proposed to prevent droplet coalescence by steric hindrance as well as by the establishment of a Marangoni flow, counteracting the oil drainage between droplets contacting each other [
109]. A variety of oils and surfactants can be used as reviewed elsewhere [
109,
110] and, whereas IVC protocols usually employ non-fluorinated oils [
111], nowadays droplet-based microfluidic approaches mainly use fluorinated carrier oils (e.g., FC40 and Novec 7500) since they both limit droplet–droplet molecular exchanges, as organic molecules are poorly soluble in these oils [
112], and they are well compatible with PDMS (avoiding PDMS swelling [
113] as mineral oil would [
52]). In addition, fluorinated oils efficiently dissolve respiratory gases [
114], a particularly interesting feature when cells need to be grown in the droplets. Most of the fluorinated surfactants are based on the perfluorinated polyether (PFPE) Krytox™ (
Figure 5b) that possesses a carboxylic head group. However, the ammonium salt of the molecule has poor biocompatibility [
115], a key parameter that can be efficiently assessed using cell-based [
115] or in vitro gene expression [
116] assays. The toxicity of Krytox™ was likely due to the nature of the counter cation used, since a recent study showed that the addition of free polyetherdiamine in the aqueous phase (
Figure 5c) restores the biocompatibility of droplets stabilized by Krytox™ [
117]. However, most of the surfactants used nowadays consist of PFPE tails covalently conjugated with polar heads like dimorpholinophosphate [
115], polyethylene glycols [
118], or polyetherdiamine [
71]. Several of these block co-polymers are now commercially available (e.g., from RAN biotechnologies (Beverly, MA, USA) or Sphere Fluidics (Cambridge, UK)) which eases the access to the technology. Furthermore, since surfactant toxicity may be due to non-specific adhesion to surface, additives such as Pluronic®, can be added to the aqueous phase to limit adsorption of cells or molecules [
119].
Besides its function in droplet stabilization, the surfactant may also fulfill additional roles. For instance, using a surfactant affording a polar head functionalized with a nitrilotriactetate group was used to both stabilize the droplets and capture his-tagged green fluorescent protein at their surface [
120]. Similarly, gold-conjugated surfactant was used to immobilize peptides for capturing cells at the droplet surface [
121]. Finally, inner droplet surface could also be functionalized using surfactant affording hydroxyl groups such as the fluorinated polyglycerols [
122] or
via bio-orthogonal click chemistry using azide surfactant [
123], both approaches opening up exciting perspectives in future applications exploiting the large inner droplet surface.