Reconfigurable complex emulsions: Design, properties, and applications

Emulsification is a widespread technique for mixing immiscible fluids and is central to many industrial products and applications¹ including cosmetics,² food,^3,4 and pharmaceuticals.^2,5 The simplest emulsions consist of two immiscible liquids, such as oil and water, where one liquid is the dispersed droplet phase and the other is the continuous phase. Complex emulsions, in which the droplets contain two or more phase-separated liquids, are also common;⁶ complex droplets can have different morphologies, such as multiple emulsions [e.g., oil-in-water-in-oil (O/W/O)] or Janus droplets (e.g., a biphasic oil droplet in water, where each oil has an interface with the water).^7,8 Reconfigurable complex emulsions, which are composed of multiphase droplets that exhibit dynamic changes in morphology, were first described⁹ in 2015 and have recently been of significant interest due to their tunable physical and chemical properties resultant from the ability to alter the ordering and curvature of the droplets' fluid interface. The droplets' morphological changes, which are triggered by alterations in the balance of interfacial tensions,⁹ can be easily manipulated in response to diverse stimuli such as light, pH, enzymes, chemical analytes, temperature, and magnetic fields. As such, reconfigurable droplets present numerous new opportunities for emulsions ranging from tunable optics to sensors to controlled release.

Here, we review the chemistry, properties, and applications of emulsions that exhibit reconfigurable droplet morphology. We focus on emulsions in which all phases in a droplet are immiscible with each other and with the continuous phase, such as multiphase oil droplets dispersed in water or multiphase aqueous droplets dispersed in oil. Thus, although there are many other types of complex droplets, including precisely structured multiple emulsions containing alternating water and oil interfaces (e.g., W/O/W/O), we do not cover these emulsions here and point the reader to other reviews.^6–8,10 We summarize the chemistries of reconfigurable complex emulsions discussed in the literature thus far, the considerations for methods of emulsion fabrication, as well as the mechanisms for droplet reconfiguration. We describe the surfactants and emulsion formulation strategies used for sensitizing the droplets to various stimuli and highlight research which has exploited reconfigurable droplets for more specialized applications, including for optics and sensing.

Reconfigurable complex emulsions should contain at least three immiscible fluids: two or more liquids within each droplet, and another immiscible fluid as the continuous phase. Surfactants or stabilizers (i.e., polymers, particles)¹¹ are usually necessary to kinetically stabilize droplets of the dispersed phase within the continuous phase, but often, surfactants are not added inside the droplets because the interfaces between the dispersed phases tend to have inherently low interfacial tension (a few mN/m or less).⁹ Dispersed phase oils that have garnered significant interest for use in reconfigurable complex emulsions are combinations of fluorinated oils (e.g., perfluorohexane, perfluorooctane, and methoxyperfluorobutane) and hydrocarbon or other organic oils. Many such oil combinations are immiscible at room temperature but have a low upper critical solution temperature (UCST), meaning that with gentle heating, these oils can be readily mixed.⁹ Hexane and perfluorohexane, for example, have a UCST just above room temperature (∼23 °C).¹² Combinations of other classes of fluids, such as silicone oils, vegetable oils, mineral oils, liquid crystals, and ionic liquids, have also been employed in the formation of multiphase oil-in-water complex emulsions.^13–17 Complex emulsions are not limited to oils dispersed in water and can also be formed via the aqueous phase separation of polymer mixtures dispersed in an immiscible water or oil phase.^18–20 An example aqueous two-phase system is poly(ethylene glycol) and dextran.^21–23 In Table I, we highlight several examples of reconfigurable complex emulsion formulations that have been studied so far.

An emulsion is a thermodynamically unstable mixture comprised of multiple immiscible liquid phases. With the exception of microemulsions, emulsions do not form spontaneously and require an input of energy to induce droplet breakup. Emulsifying agents are usually added to increase the kinetic stability of the droplets formed. Here, we describe typical methods that have been utilized for the formation of complex reconfigurable emulsions.

The simplest emulsification techniques for complex emulsion fabrication are bulk homogenization methods using a mechanical device capable of applying high-shear forces to induce droplet breakup (e.g., vortex mixer, homogenizer, shaker, and sonicator). These homogenization methods typically form droplets with high size dispersity and compositional heterogeneity [Fig. 1(a)]. Researchers have demonstrated the formation of both hydrocarbon-fluorocarbon^9,24,25 biphasic droplets and silicone oil-vegetable oil^15,26 biphasic droplets utilizing bulk homogenization techniques. For example, silicone oil-vegetable oil Janus droplets of various sizes and volume ratios can be formed upon the addition of both oils to water in the presence of a stabilizing surfactant, such as Tween 80, and subsequent application of shear force.¹⁵ To improve the droplet compositional uniformity, researchers have exploited a temperature-induced phase separation method in conjunction with bulk emulsification techniques, which we will elaborate on in Sec. II B 2 to yield polydisperse biphasic hydrocarbon-fluorocarbon complex emulsions.

Another conventional bulk emulsification technique for emulsion formation is membrane emulsification,^27,28 which works by using an applied pressure to force the dispersed phase (either a single fluid or a pre-made emulsion) through a porous membrane into the continuous phase [Fig. 1(b)]. In comparison to most bulk homogenization methods, this technique offers better control over individual droplet size and size distribution. Aqueous two-phase droplets, for example, have been shown to form via this method by generating first single emulsions of premixed poly(ethylene glycol) and dextran in water and observing subsequent phase separation upon the concentration of droplet components by extracting water.²⁹ 

A more precise, small-volume method of emulsion generation is microfluidics, which relies on the focusing of flow streams of both the dispersed and continuous phases through a constricted junction.³⁰ At this junction point, the symmetric shear force from the continuous phase is generated on the dispersed phase, inducing steady breakup of droplets with a narrow size distribution. Common microfluidic platforms include plastic or glass chips with embedded channels, polydimethylsiloxane channels on glass substrates, and co-axially assembled glass capillaries. A number of publications^7,8,30–32 have discussed microfluidic approaches to droplet fabrication in great detail, so here we only focus on some key considerations about the microfluidic formation of reconfigurable complex droplets. In general, when using microfluidics to make complex droplets with several encapsulated phases (e.g., O/W/O) the channel designs and flow requirements may get somewhat complex with multiple junctions required to sequester the miscible phases from one another during droplet formation. However, for most of the reconfigurable complex droplet systems discussed in this review, all liquids in the complex emulsion are immiscible with each other, an advantage that allows the microfluidic droplet fabrication to be relatively simple with only one junction needed. For example, a simple four-inlet microfluidic chip can be used to generate biphasic complex droplets,^24,25,33 where the two immiscible dispersed phases are introduced together at a single junction before the droplet is formed, as depicted in Fig. 1(c). Variation of the flow rates of the multiple phases allows for tunability of both the droplet size and the ratio of the liquids inside each droplet.

Exploitation of phase transitions or phase separations within single emulsion droplets has often been utilized for the facile formation of complex droplets.^9,18,34–36 The general principle involves the temporary mixing of the dispersed phases prior to emulsification, such as by the addition of a co-solvent or application of heat, with the phase separation triggered subsequent to droplet formation. In this way, all droplets in the emulsion are formed from the same single phase, have the same composition, and will phase separate into the same volume ratios leading to consistent droplet morphology. Phase separation of single emulsions can be used in combination with bulk homogenization methods or microfluidics to generate either monodisperse or polydisperse droplets with controlled morphology and volume ratios of droplet-internal components. Phase separation methods of complex droplet formation have thus attracted interest as a simplified route to the generation of multiphase droplets.

To produce complex droplets using solvent-assisted phase separation, immiscible liquids that are to become the dispersed phase (e.g., immiscible polymers, oils) are made miscible by the addition of a co-solvent; the co-solvent should be chosen to be mutually miscible with all dispersed phase liquids and should also be sufficiently soluble in the continuous phase to enable extraction subsequent to droplet formation. This fully-miscible mixture is emulsified into the continuous phase containing a surfactant to form droplets. As the co-solvent leaves the droplets over time, the dispersed phase liquids within each droplet become concentrated and eventually phase separate to create a complex droplet. Researchers who have employed this method to generate complex oil-in-water emulsions have frequently used co-solvents of chloroform,³⁴ dichloromethane,^37–39 toluene,^40,41 or ethanol^42,43 [Fig. 1(d)]. Other researchers have used similar approaches for microcapsule formation,^37,38 such as the polymerization of a polystyrene shell following the phase separation of a ternary mixture of polystyrene, hexadecane, and dichloromethane,³⁷ or the formation of a poly(methyl methacrylate) (PMMA) capsule upon phase separation of PMMA from a mixture of dichloromethane, PMMA, and a silicone oil, methylhydrosiloxane dimethylsiloxane.³⁹ Solvent-assisted phase separation is not limited to oil-in-water systems and can be used to form two-phase aqueous droplets in a continuous oil phase as well.²⁹ Phase separation of aqueous systems containing two water-soluble polymers or a water-soluble polymer and a salt-rich phase has been studied for decades: a classic system of this type includes poly(ethylene glycol) (PEG) and a polyglucose, usually dextran (DEX).^21,22 Above critical concentrations, solutions of these two polymers phase separate into PEG-rich and DEX-rich phases. Researchers have demonstrated complex droplet formation containing these two polymers by extracting water from aqueous single emulsions containing both of these polymers, thereby concentrating the droplet and inducing phase separation.²⁹ 

In temperature-induced phase separation methods, droplets are formed at a temperature at which the dispersed phase fluids are miscible (i.e., single emulsion droplets are produced), and then the temperature is subsequently changed to induce phase separation within each droplet, thereby forming complex emulsions. With a temperature-induced phase separation approach, it is straightforward to create complex droplets of controllable morphology and droplet-internal volume ratios, since each single emulsion droplet formed would initially have the sample composition and hence would phase separate into the same complex droplet morphology when triggered by temperature.⁹ In order to utilize this fabrication approach, the dispersed phase liquids should have a critical temperature, such as an upper critical solution temperature (UCST) or a lower critical solution temperature (LCST) that is accessible. Researchers have exploited, for example, the low UCST of alkanes and fluorinated oils to generate complex oil-in-water emulsion droplets.^{9,24,25,33,44–48} As mentioned previously, many hydrocarbon-fluorocarbon combinations have a UCST above room temperature, such that gentle heating induces mixing. Example oil pairings and their UCSTs include: hexane and perfluorohexane (23 °C),^9,12 diethylbenzene and 2-trifluoromethyl-3-ethoxyperfluorohexane (HFE-7500) (37 °C),⁴⁹ hexane and perfluorooctane (34–37 °C).^24,50 Single emulsion drops are thus produced when emulsification takes place above the UCST, and when the droplets are cooled, the oils phase separate to form complex droplets. Figure 1(e) illustrates the formation of complex biphasic droplets using this temperature-induced complex droplet forming technique in conjunction with a bulk homogenization emulsification method. In addition to two-phase systems, researchers have similarly demonstrated the temperature-induced formation of droplets containing three immiscible phases, such as hydrocarbon oils, fluorocarbon oil, and silicone oil.⁹ 

It has also been shown that the application of heat can be used to, instead, induce the phase separation. Such an approach has been used in droplets composed of liquids crystals and fluorocarbon oils.¹⁶ Researchers found that mixtures of nematic liquid crystal E7 with isotropic hexafluorobenzene form a single phase at room temperature, but heating induced the droplets to phase separate into Janus morphologies.¹⁶ Temperature-induced phase separation methods have also been used to form complex droplets with aqueous two-phase systems.^23,51,52 For example, at particular PEG/DEX concentrations and molecular weights, the polymer mixture is temperature-sensitive and miscibility can be induced upon cooling.^20,23 This temperature-sensitivity has been exploited using PEG (35 kDa) and DEX (40 kDa) to form complex biphasic droplets whose morphology is dependent on the volume ratio of constituent phases. To form these droplets, researchers cooled mixtures of PEG (35 kDa) and DEX (40 kDa) until miscible, and then subsequently emulsified the temporarily-miscible polymer solutions in tetradecane containing either Span 80 or Span 85 as the oil-soluble surfactant.²⁰ Upon return to room temperature, phase separation was induced, allowing the formation of complex aqueous two-phase Janus emulsions.

It can be experimentally challenging to generate high-order multiple emulsions of controlled size and composition with bulk or microfluidic approaches alone, because dispersity would be a prevalent problem or because necessary microfluidic geometries would be complicated. It is, thus, important to elucidate strategies that may be employed for the generation of complex biphasic droplets with controlled structure and composition.⁵³ As illustrated in Figs. 1(d) and 1(e), it is possible to combine emulsification methods for the facile and scalable production of complex droplets. It is feasible, for example, to combine a phase-separation approach in conjunction with a bulk emulsification technique, as has been shown in several works^{9,25,46,54,55} which form biphasic hydrocarbon-fluorocarbon droplets; desired oils are heated above their UCST, emulsified in a warm surfactant solution, then allowed to cool to room temperature to induce phase separation. Droplets formed via this method are homogenous in composition, but polydisperse. If, however, both compositional and size homogeneity are desired, it is possible to combine microfluidic techniques with phase-separation methods as well. Researchers have utilized solvent-extraction-induced phase separation techniques in conjunction with microfluidics to form uniform single emulsions of two oils, made temporarily miscible with the introduction of an extractable co-solvent (as described in more detail in Sec. II B 1). Depending on the desired droplet size, composition, and dispersity, there are many possible routes to the fabrication of complex reconfigurable emulsions.

Equilibrium complex droplet morphology is determined by the balance of interfacial tensions at the droplet interfaces. A number of publications have examined the equilibrium morphologies of biphasic droplets in detail^9,56–58 and here we summarize some of the key considerations governing droplet structure and shape reconfiguration. In our discussion, we consider a droplet composed of two immiscible liquids (L1 and L2), and in a third immiscible liquid (L3). Biphasic droplets containing an interface between L1 and L2 exist in three general configurations: (1) a fully-engulfing double emulsion configuration where L1 encapsulates L2, (2) partial engulfment (i.e., Janus) where both L1 and L2 have an interface with L3, and (3) a fully-engulfing double emulsion configuration where L2 encapsulates L1. Biphasic droplet morphology is dependent on the balance of interfacial tensions at these interfaces present within the droplet. Thus, we consider the interfacial tensions of the following interfaces: the L1-L3 interface, γ_L1, the L2-L3 interface, γ_L2, and the L1-L2 interface, γ_L1L2, such that L1 and L2 remain in contact and do not separate into two single emulsion droplets. In the case that γ_L1 > γ_L2 + γ_L1L2, then the encapsulation of L1 inside of L2 is favored. In the case of γ_L2 > γ_L1 + γ_L1L2, then the opposite encapsulating geometry is favored, with L2 inside L1. At values of γ_L1, γ_L2, and γ_L1L2 wherein a Janus morphology is expected, we can configure all three respective interfacial tensions into a Neumann's triangle, solvable for θ_L1 and θ_L2, where θ_L1 is the contact angle between L1-L3 and L1-L2 interfaces and θ_L2 as the contact angle between the L2-L3 and L1-L2 interfaces [Fig. 2(a)]. The morphology of a complex droplet can be expressed using the following equations when the droplet is in equilibrium:^9,58

Understanding how the balance of interfacial tensions affects droplet morphology has made it possible for researchers to induce morphological transitions in complex droplets. By dynamically altering the interfacial tensions in situ, the droplet will respond by changing shape and adapting to the new balance of forces. Notably, if γ_L1 and γ_L2 are significantly larger than γ_L1L2, then small shifts in the balance of γ_L1 and γ_L2 while γ_L1L2 is held constant would induce dramatic changes in droplet morphology, allowing biphasic droplets to be reconfigured between fully engulfed and Janus shapes [Fig. 2(b)]. If instead, γ_L1 and γ_L2 are held constant but γ_L1L2 is altered, biphasic droplets can be switched between a fully engulfed and Janus shape, but cannot be switched between the two fully engulfed states [Fig. 2(c)]. In theory, it would be ideal to be able to manipulate the interfacial tension at each interface independently when tuning droplet shape. In practice, it is challenging or impossible to achieve in most situations. Fortunately, however, surfactants do tend to affect the interfacial tensions of different liquid interfaces to varying degrees depending on their chemistry, allowing for tuning of the balance of the interfacial tensions. Strategic surfactant choice is thus necessary to direct the balance of interfacial tensions and bias complex droplet geometry toward desired morphologies.

A straightforward way to modify the balance of interfacial tensions and tune droplet morphology between double emulsion and Janus states is by changing the concentration of surfactants present in the continuous phase to modify the balance of γ_L1 and γ_L2. Typically, the surfactants are chosen to preferentially lower the interfacial tension of either the L1 or L2 interface. Likely, one would choose combinations of surfactants to be able to address γ_L1 and γ_L2 as independently as possible and maximize the change of the droplet shape with the smallest amount of surfactant. For example, when stabilizing a complex droplet containing hydrocarbon and fluorocarbon oils, the use of an aqueous surfactant with a hydrocarbon tail [e.g., sodium dodecyl sulfate (SDS)] would favor the reduction of the hydrocarbon-water interfacial tension over that of the fluorocarbon-water interface; this would in turn favor formation of a droplet with more hydrocarbon-water interfacial area.⁹ Increasing the concentration of a surfactant with a fluorinated tail (e.g., Zonyl FS-300 or Capstone FS-30, both nonionic fluorosurfactants) would preferentially lower the fluorocarbon-water interfacial tension favoring greater fluorocarbon-water interfacial area.⁹ Considering as an example a droplet composed of hexane and perfluorohexane (with interfacial tension of 0.4 mN/m),⁹ using 0.1 wt. % SDS generates a double emulsion, perfluorohexane-in-hexane-in-water. When a fluorinated surfactant is used, such as 0.1 wt. % Zonyl FS-300, then hexane-in-perfluorohexane-in-water droplets are formed.⁹ Use of a single surfactant alone at varying concentration may have little impact on drop shape (e.g., droplets in 0.1 and 1 wt. % SDS are both perfluorohexane-in-hexane-in-water), but mixing of SDS and Zonyl in varying concentration ratios allows significant shape tuning of the droplet morphology to various Janus configurations.⁹ Thus, to induce dynamic droplet reconfiguration, it is often necessary to employ mixed surfactant systems. Surfactants should be strategically chosen to address the different dispersed-phase interfaces such that changes in the concentration ratio of those two surfactants affect the balance of interfacial tensions and drop morphology.

Instead of changing the surfactant concentrations as just discussed in Sec. IV A, use of responsive surfactants that, at a fixed concentration, change effectiveness in response to an applied stimulus (e.g., light, pH, presence of a specific reagent or analyte) can also be employed to affect the interfacial tension balance and droplet shape. Other reviews^59–63 have summarized the interfacial properties of stimuli-responsive surfactants in detail, so here we focus on how such surfactants have been used thus far to trigger shape changes in complex droplets.

Photoresponsive surfactants^59,60 exhibit altered effectiveness when illuminated with specific wavelengths of light, and hence their chemical structures usually have chromophores in either their tails or headgroups. In particular, there are a number of variations of photo-responsive surfactants based on spiropyan^59,64 and azobenzene.^{9,33,60,65,66} Azobenzene-based surfactants undergo a reversible cis-trans isomerization dependent on the wavelength of incident light; one of the most common azobenzene-based surfactants is the cationic surfactant (4-butylphenyl)-2-(4-trimethylammoniumpropoxyphenyl)diazene, often abbreviated “AzoTAB”, which adopts a cis conformation upon irradiation with UV light (∼360 nm), and a trans conformation upon introduction of blue light (∼440 nm)⁶⁶ [Fig. 3(a)]. The trans form of the surfactant is more effective than the cis form at reducing the oil-water interfacial tension. Surfactants containing azobenzene moieties have been demonstrated to be effective at tuning droplet morphology in biphasic fluorocarbon-hydrocarbon emulsion systems.^9,33,65 Spiropyrans are another common class of photosensitive molecules used to generate light-responsive surfactants, though, to our knowledge, they have not yet been applied toward the reconfiguration of complex droplets. Irradiation of spiropyans in solution with UV light within an approximate range of 250 to 380 nm breaks a C-O bond, allowing the formation of an open-structured merocyanine form which is charged and more hydrophilic. Diblock copolymers formed from poly(ethylene oxide) and a spiropyran-containing methacrylate monomer⁶⁴ have been used to create photoresponsive micelle-forming macromolecules. Irradiation with UV light (365 nm) and visible light (620 nm) allows for photoswitching between hydrophobic spiropyran and hydrophilic merocyanine that reversibly affects the polymer amphiphilicity and micelle assembly. Other work using a spiropyran-based surfactant, 1′(6-trimethylammoniododecyl)-3′,3′-dimethyl-6-nitrospiro-(2H-1-benzopyran-2,2′-indoline) bromide, demonstrated a reversible change in the surface tension of the surfactant solution upon photo-induced isomerization. Visible light irradiation (>420 nm) was shown to result in a significant decrease in the surface tension of the surfactant solution.⁶⁷ 

pH-responsive surfactants can be reversible (e.g., triggered by protonation or deprotonation) or irreversible (e.g., acid or base-catalyzed reactions, which cleave parts of the surfactant).^59,62,63 Researchers have used the pH-responsive surfactant N-dodecylpropane-1,3-diamine⁵⁹ in conjunction with fluorosurfactant Zonyl FS-300, for example, to reversibly tune the morphology of hexane-perfluorohexane droplets between opposite core-shell geometries upon addition of acid and base.⁹ Decreasing the pH of the continuous phase below the surfactant's pKa of 4.7 significantly reduces the effectiveness of the N-dodecylpropane-1,3-diamine but not the Zonyl FS-300, thereby affecting the balance of interfacial energies and droplet shape [Fig. 3(b)]. An acid-cleavable surfactant, sodium 2,2-bis(hexyloxy)propyl sulfate, in combination with Zonyl FS-300, has been used to induce an irreversible droplet morphological change over time at a pH of 3 for hexane-perfluorohexane droplets.⁹ There are many other pH-responsive surfactants reported, which have not, to our knowledge, been utilized in complex droplet systems but could be considered; examples include pH-responsive gemini surfactants N,N′-dialkyl-N,N′-di(ethyl-2-pyrrolidone)ethylenediamine (Di-CnP, where n = 6, 8, 10, 12), or pH-responsive copolymer surfactants, such as one made of methacrylic acid and poly(ethylene glycol) methacrylate.⁵⁹ 

The need for more analyte-specific molecular detection has led to the study of how substrate-specific surfactant binding or complexation can be used to generate changes in droplet configuration by affecting the balance of relevant interfacial tensions. For example, host-guest interactions between cyclodextrins and surfactants, where the hydrophobic tail of the surfactant is inserted into the hydrophobic core of the cyclodextrin, can be used to modify surfactant effectiveness. For instance, the hydrophobic tail of Triton X-100 becomes complexed into the hydrophobic core of γ-cyclodextrin when both molecules are present in the continuous phase, and this complexation greatly diminishes the Triton X-100's effectiveness as a surfactant⁶⁸ [Fig. 3(c)]. Disruption of the host-guest interaction, either through competitive interactions or host molecule degradation, can then be used to generate responsive changes in interfacial tensions and droplet shapes. For the case of the cyclodextrin and Triton X-100, researchers were able to break down the cyclodextrin with α-amylase and use the resultant shape change of heptane-FC770 droplets over time to measure the enzyme activity.⁶⁹ 

Other examples utilizing non-covalent interactions for morphological change in biphasic droplets are bacterial or protein detection schemes (which we discuss at length in Sec. V B 2). Researchers, in one example, used a boronic acid surfactant and carbohydrate binding to induce a droplet morphology change for Salmonella enterica detection.⁴⁷ Hydrocarbon-fluorocarbon double emulsions were stabilized in Zonyl FS-300 while two boronic acid cosurfactants (3,4-dibutoxyphenylboronic acid polystyrene-block-poly(acrylic acid-co-acrylamidophenylboronic acid) were dissolved in the hydrocarbon phase to localize at the hydrocarbon-water interface. Upon addition of carbohydrates (mannan, mannose, glucose) to the continuous phase, droplets would adopt a Janus morphology, however, when Salmonella was introduced, carbohydrates would preferentially bind to Salmonella and leave the droplet interface, inducing a corresponding morphology change in the droplet.⁴⁷ 

Synthesizing surfactants via chemical reactions in situ within the emulsion can be used to trigger droplet morphological changes. Since a surfactant molecule is typically composed of a hydrophilic portion and a hydrophobic portion, one strategy for in situ formation of surfactants is to bring together two reagents (one hydrophilic and one hydrophobic) that upon reaction, create a molecule with enhanced amphiphilicity. However, it can be difficult to have those two reagents together within a single phase due to solubility limitations. Thus, a strategy for the formation of a surfactant in an emulsion is to have the reaction localized at an oil-water interface, where one reagent is present in the dispersed droplet phase in contact with the second reagent in the continuous phase. In this strategy, droplets of the reagents are first stabilized by one surfactant and as the second surfactant is formed in situ, the droplet morphology will change over time.

In one study, a water-soluble amine and an oil-soluble aldehyde underwent an interfacial reaction to produce imine surfactants [Fig. 3(d)].⁴⁶ Researchers demonstrated that aldehyde precursors could not successfully stabilize droplets on their own and did not display any surfactant-like behavior pre-reaction. In the chosen system comprised of hydrocarbon oil (diethylbenzene) and fluorocarbon oil (HFE-7500), a monofunctional amine polymer monomethoxypolyethylene glycol amine (MW 1000) was reacted with either a hydrocarbon-soluble carbonyl-containing compound or a fluorocarbon-soluble aldehyde or dialcohol. The dynamic nature of formed imine surfactants was probed through the addition of excess propylamine (post-formation of the imine surfactant) to the continuous phase, leading to imine exchange, thereby also affecting droplet morphology by affecting the balance of interfacial tensions.⁴⁶ Another work examining the in situ formation of surfactants showed the creation of surfactants which are programed to respond to nucleophilic triggers. Researchers utilized SN₂ Michael addition chemistry to affect surfactant stabilizing ability.⁷⁰ The designed surfactant structure was based on a quaternary ammonium moiety linked to the α-methyl carbon of an alkyl-methacrylate molecule, where the quaternary ammonium moiety served as the hydrophilic head group and the long alkyl chain behaved as the hydrophobic tail. More specifically, a triggerable Michael acceptor (TMAc) functionality was used as a head group component in the surfactant molecules that assembled at the emulsion interface. Further nucleophile-induced modification of the TMAc surfactant was shown to affect the ability to stabilize the complex droplet interface, leading to macroscopic changes in emulsion geometry. The structure of the surfactant molecules was found to impart tunable kinetic control on the emulsions' response through changes in leaving group ability.⁷⁰ 

Cleavable surfactants,^62,63 which typically include a weak bond deliberately built into the surfactant's structure that can break down in a controllable and predictable way, are also employed as a way to tune droplet morphology. Surfactants of this type can either become better or worse surfactants upon cleavage, thus, triggering a morphology change in the complex emulsion droplet as the balance of interfacial tensions is changed dependent on surfactant effectiveness. In one example, researchers use an acid-cleavable surfactant, sodium 2,2-bis(hexyloxy)propyl sulfate, to induce a morphological change over time at a pH of 3 in hexane-perfluorohexane droplets co-stabilized by Zonyl FS-300 [Fig. 3(e)].⁹ Researchers have also shown surfactants to be cleaved upon exposure to certain enzymes. In one example, researchers utilized an ester-linked hydrocarbon surfactant [tetra(ethylene glycol) mono-n-octanoate] to measure the activity of lipase from Candida sp. Upon addition of the lipase, the ester-linkage in the surfactant was cleaved and the droplet changed shape.⁶⁹ 

Interfacial tensions are dependent on temperature, especially the interfacial tensions of near-critical fluids,⁷¹ such as the combinations of hydrocarbons and fluorocarbons often used in reconfigurable droplets. Changes in temperature can also be used to induce phase transitions within droplets, which would similarly change the interfacial tensions and droplet shapes as the chemical compositions of the interfaces are altered. Thus, variations in temperature can be used to induce morphological changes in complex droplets. A simple switch between a single emulsion and complex droplet shape can be induced by heating and cooling above the critical solution temperature of the droplet-internal liquids.⁹ Researchers have shown that in a double emulsion droplet formed from a fluorocarbon oil (perfluorobenzene) and hydrocarbon liquid crystal mesogens (E7) in an aqueous SDS solution, droplets are found to reconfigure into different Janus morphologies over a range of approximately 10 °C.⁷² This is because E7, a mixture of hydrogenated cyanobiphenyl and terphenyl mesogens, forms a nematic liquid crystal at room temperature, however, in combination with perfluorobenzene, demonstrates a much wider nematic-isotropic coexistence range than is typical for hydrocarbon oils with E7. This broad coexistence allowed researchers to precisely manipulate droplet shape with minute changes in temperature.

While reversible changes between different emulsion core-shell and Janus morphologies are usually induced chemically, it has been demonstrated that morphological changes can be generated by mechanical confinement as well. Researchers created droplets of silicone oil and paraffin oil in aqueous solutions of SDS, dioctyl sodium sulfosuccinate, and cetyltrimethylammonium bromide, and confined the droplets between two parallel hydrophilic plates.⁷³ In an unconfined state, droplets existed in a core-shell geometry with a paraffin oil core and a silicone oil shell. Upon sandwiching droplets between two rigid plates, the gap between which was varied to different heights using spacers, researchers found that the meniscus of the continuous phase liquid, which forms between the parallel plates, influences the total energy of the droplet, triggering a morphological change to a Janus state.⁷³ 

Unlike molecular surfactants that stabilize droplets through the reduction in interfacial tensions, solid particles stabilize droplet interfaces by replacing the liquid-liquid interfacial area with solid-liquid interfaces of favorable wettability.^74–76 This different stabilization mechanism affords opportunities for using particles to manipulate complex droplet morphology in situations where molecular surfactants are not very suitable. For example, most complex droplets have low interfacial tension at the droplet-internal L1-L2 interface, on the order of a few mN/m or less; while traditional molecular surfactants are generally ineffective under those conditions, particles have been successfully used to stabilize many such interfaces and provide a route to manipulate the droplet-internal interface's surface energy.^24,76,77 For example, researchers used functionalized silica nanoparticles to selectively stabilize the interface between hexadecane and perfluorooctane oils in order to form double emulsions of hexadecane-in-perfluorooctane-in-water (aqueous interface stabilized by Capstone FS-30) or perfluorooctane-in-hexadecane-in-water (aqueous interface stabilized by SDS);²⁴ hexadecane and perfluorooctane would naturally form “snowman” type Janus droplets in the absence of the particles. Not only were the particles used at the oil−oil interface shown to affect the interfacial energy (thus, influencing the overall droplet shape), but the particles were also demonstrated to trap complex droplets into metastable geometries, wherein changes in temperature could trigger their reconfiguration under varying aqueous surfactant conditions.²⁴ 

The properties of complex emulsions are inherently tied to their chemical composition and morphology such that they exhibit strong structure-function relationships. In particular, the optical properties of complex droplets have received significant attention—the optical properties depend largely on the curvature, ordering, and orientation of droplet interfaces as well as the physical properties of the liquids themselves, such as the refractive indices. Tailoring droplet properties through tuning of the interfacial tensions and droplet shapes facilitates the use of complex emulsions for applications in which responsive or dynamic behavior is beneficial, such as for tunable lenses or chemical sensors.

Liquids, in particular droplets, have long been explored for dynamic optical applications.^78,79 The notion of dynamically tunable optics, in particular dynamic lenses,^80,81 finds significance in several ways, including in imaging devices^82,83 and biomedical diagnostics.^82,84 Droplets smaller than the capillary length of fluids, wherein surface tension is the dominant force, form curved interfaces⁸⁵ between fluid volumes which can be harnessed for light refraction and focusing. Liquid droplets have minimal surface roughness⁸⁶ even if the interfacial tension is low, they have varied and tunable refractive indices, can be easily mixed with dyes or pigments to alter their absorption or fluorescence characteristics, and can be produced with precise radius of curvature and size using microfluidic methods.^7,8,32 The ease by which the shapes and interfacial curvatures of complex emulsion droplets can be precisely manipulated has made such droplets of significant interest for use as responsive optical materials. Two significant tunable optical properties relevant to multiphase droplets are the ability of the droplets to act as dynamic lenses and the ability to generate tunable interference colors. Both properties are influenced by the droplet interfacial curvature, index contrast at the droplet interfaces, and the droplet orientation. Importantly, in nearly all of the complex droplet optical systems to be discussed, there is a significant density difference between the droplet-internal phases, such as the orientation of droplets is biased by gravity and all droplets when dispersed in a monolayer will have the same orientation. This natural alignment is quite useful, and often necessary, to control and tune the optical properties of the emulsions.

Designing dynamic micro-lenses which controllably bend, scatter, or focus light can be a challenge because of the necessity to alter either the lens structure or composition.^87,88 Optofluidic approaches provide an opportunity to create reconfigurable micro-optical components that rely on the intrinsic deformability of liquids to reshape and reconfigure optical interfaces in response to various stimuli.⁸⁹ Likewise, reconfigurable complex droplets have allowed researchers to controllably manipulate the curvature and ordering of liquid interfaces in biphasic emulsion droplets and demonstrate the use of droplets as tunable fluidic compound micro-lenses [Fig. 4(a)].⁸¹ It has been shown that complex emulsions containing fluorocarbon (FC-770, refractive index = 1.27) and hydrocarbon (hexane, refractive index = 1.375) oils can be used to create variable focusing lenses that switch between converging and diverging upon changes in droplet shape.⁸¹ Adjustment of the droplet's oil-water interfacial tensions with aqueous phase surfactants (SDS and Zonyl FS-300) allowed controllable variation between double emulsion and Janus droplet morphologies to precisely tune the curvature of the oil-oil interface. Double emulsions with the optically-denser fluid (hexane) as the core phase acted as a converging lens and focused light, while inverted double emulsions with an optically-denser shell acted as a diverging lens. Tuning the shape of the oil-oil interface through various Janus morphologies allowed variation in the lens focal length and numerical aperture which was consistent with ray-tracing results [Figs. 4(b)–4(d)]. Controlled modification of complex emulsion morphology, thus, resulted in predictable variation of the droplet light focusing and scattering behavior.⁸¹ The optical changes caused by variation in droplet morphology are visible to the naked eye when collections of monodisperse or polydisperse droplets are used, as long as all the droplets are oriented in the same direction; layers of hexane-perfluorohexane double emulsion droplets are, for example, more scattering and opaque, whereas Janus droplets are more transmissive.⁶⁹ Researchers have since extended the usefulness of complex droplets as responsive lenses to sensing platforms for the detection of diagnostic analytes in point-of-care sensors⁶⁹ by utilizing this change in transmission as a readout mechanism. Further description of the approaches for these optical sensing platforms will be discussed in Sec. V B 1.

It was recently discovered that microscale concave interfaces, such as those present in a Janus droplet, can generate iridescent structural color through optical interference occurring between light rays traveling by different paths of total internal reflection (TIR) [Figs. 5(a)–5(c)].^33,90 Although it is well known that microscale spherical droplets can generate color through refraction and dispersion (such as an atmospheric rainbow), the broken symmetry of the Janus droplets allows them to behave very differently than a perfectly spherical single emulsion droplet.^33,91 Researchers first observed this structural color in monodisperse, biphasic Janus droplets containing heptane (refractive index = 1.37) and perfluorohexane (refractive index = 1.27) when the droplets were illuminated by collimated white light from above.³³ Droplets which had an upward-facing concave interfacial geometry between the constituent oils exhibited size and angle-dependent iridescent color in reflection with the light emanating from near the droplets' three-phase contact line. Researchers determined that the curvature of the droplet-internal hydrocarbon-fluorocarbon interface with respect to the light was essential to generating this optical effect [Figs. 5(d) and 5(e)]. Although this coloration phenomenon is not exclusive to biphasic droplets and can be reproduced at many different microscale concave interfaces where light undergoes multiple total internal reflections,⁹⁰ a unique aspect of using the complex emulsions is that the colors can be easily changed by modulating the droplet shape. By introducing a light-responsive azobenzene surfactant, (4-butylphenyl)-2-(4-trimethylammoniumpropoxyphenyl) diazene, researchers could use ultraviolet and blue light to photo-pattern regions of different droplet morphologies and create colored images [Fig. 5(e)].³³ This unique mechanism for generating optical interference in liquids enables dynamic manipulation of color and has promise for further exploration in color-based droplet sensors or liquid displays.

Liquid crystals (LCs) are condensed fluid phases that exist as an intermediary between crystalline solids and isotropic liquids. Due to the molecular organization, LCs can have quite different optical properties compared to isotropic fluids, such as birefringence and thermochromism.^92,93 LCs are attractive for use in responsive soft material systems because of the ability for constituent molecules (mesogens) to flow and be reconfigured while still maintaining long-range order.^94–96 Confinement or anchoring of LCs at liquid-liquid interfaces, especially those decorated with surfactants, imposes interfacial constraints on mesogen organization that changes LC alignment.^95,97–99 LCs are hence of interest for incorporation within droplets where dynamic changes to the liquid-liquid interfacial curvature and chemistry affect the LC properties and have potential use in photonic elements¹⁰⁰ and sensors.^101,102 Multi-compartment droplets which contain both an LC and isotropic immiscible oil potentially offer the responsiveness and optical properties not found in spherically symmetric single-phase LC droplets or biphasic droplets containing two isotropic oils.^103–107 

Initial work exploring LCs in reconfigurable biphasic droplet systems used the LC 4′-pentyl-4-biphenylcarbonitrile (5CB) combined with several different hydrofluoroethers to produce complex emulsions using a solvent-induced phase separation method with dichloromethane as the co-solvent.¹⁰⁸ LC-containing droplets were stabilized with 0.1 wt. % non-ionic, hydrocarbon-favoring surfactant Tween 20 in the continuous phase and a surfactant containing both LC and fluorinated components was synthesized and dissolved into the dispersed oil phases to reduce the droplet-internal interfacial tension and enhance droplet reconfigurability. Researchers were able to utilize fluorosurfactant Zonyl FS-300 to reconfigure LC-fluorocarbon droplets from a fluorocarbon-core and LC-shell double emulsion morphology to the completely inverted LC-core and fluorocarbon-shell morphology. Polarized light optical microscopy images were used to visualize differences in LC ordering resultant from droplet morphology changes.¹⁰⁸ Other work has also looked at reconfigurable biphasic droplets containing LCs as one of the dispersed phases, including a study that used 5CB in conjunction with perfluorononane.¹⁶ By employing a combination of fluorocarbon (perfluorooctanoic acid) and hydrocarbon (sodium dodecyl sulfate) surfactants in a continuous aqueous or glycerol phase, researchers observed a range of emulsion droplet morphologies to form, including core−shell and Janus structures. It was observed that while both surfactants induced Janus droplet morphologies, the internal organization of 5CB mesogen constituents depended on the surfactant type and concentration. Furthermore, it was noted that 5CB anchors homeotropically and weakly at liquid perfluorononane interfaces, which is consistent with the smectic layering of 5CB molecules. The anchoring of 5CB with the fluorocarbon oil interface is noted by the authors to be unusually weak, giving rise to shells of 5CB with either quadrupolar (r/R > 0.4) or dipolar symmetry (r/R < 0.4). Most significantly, it was observed that while SDS and perfluorooctanoic acid gave the same internal configurations for single-phase LC droplets, LC domains in multiphase droplets give rise to distinguishable internal organizations. This result has important implications and suggests that confinement of LCs within multiphase droplets provides new opportunities to differentiate interfacial phenomena that would not be otherwise distinguishable in single-phase LC droplets.¹⁶ 

Chemical sensors play an important role in scientific fields with applications such as healthcare, food safety, and environmental monitoring. Especially for point-of-care chemical sensors, the sensitivity, selectivity, and ease of use are important.¹⁰⁹ Complex droplets have proven of interest as a sensing platform because of the ability to tune droplet morphology in response to a wide range of stimuli and the ability to characterize droplet shapes through changes in optical properties visible to the naked eye. In principle, any stimulus that can be correlated with a change in the balance of interfacial tensions or droplet orientations can be sensed, while the readout mechanisms can be as simple as changes in transparency, fluorescence intensity, or color (Fig. 6).

The ability to reconfigure droplet interfaces, by modulating their curvature and modifying their ordering within a droplet, provides unprecedented opportunities to tune the light manipulation characteristics of liquid droplets. Previously, in Sec. V A, we discussed the lensing behavior of complex emulsion droplets and how the droplets direct light depending on their configuration. The microscopic structure of the droplet lenses affects the emulsion's macroscale optical properties, providing a route to coupling changes in measurable optical properties to droplet morphology. For example, layers of double emulsion droplets appear scattering and largely opaque in transmission when viewed macroscopically⁸¹ [Fig. 6(a)]. In contrast, Janus droplets' optical properties are dependent on the orientation and curvature of the droplet-internal oil-oil interface with respect to the light; in some shapes and orientations Janus droplets are mostly transparent,⁶⁹ while the same droplet tipped on its side will look opaque⁴⁵ [Fig. 6(b)]. Modification of complex emulsion morphology results in a predictable variation of light focusing and scattering behavior that can be harnessed as a macroscale optical indicator for droplet shape and orientation, even when the droplets are polydisperse.

Optical changes caused by variations in droplet morphology are visible to the naked eye and provide an optical readout mechanism for droplet shape that can be as simple as measuring the degree of light transmission [Figs. 6(a) and 6(b)]. This correlation between droplet shape and optical transmission has allowed researchers to utilize complex emulsions as sensors and couple analyte concentrations in the continuous phase to droplet morphology via the optical readout. Researchers demonstrated this transmission measurement method to quantify the activity of α-amylase, lipase, and sulfatase enzymes in the emulsion continuous phase via the use of enzyme-degradable surfactants and enzyme-uncaging of host-guest complexes containing surfactant.⁶⁹ For instance, to measure α-amylase activity, polydisperse droplets of heptane and FC-770 were used in a solution of Capstone FS-30, Triton X-100, and γ-cyclodextrin (an amylase substrate), where the Triton X-100 forms an inclusion complex⁶⁸ with the cyclodextrin [Fig. 7(a)]. As the amylase degraded the cyclodextrin, the droplet shape and optical transmission changed over time, which could be measured either with a spectrometer or with a personal electronic device such as a smartphone or tablet [Figs. 7(b) and 7(c)]. The droplet shape transitioned from double emulsion (opaque) to the translucent, flat-interface Janus, and finally ended in the inverted opaque double emulsion morphology [Fig. 7(d)]. To quantitatively utilize the transmission over time data to describe the rate of droplet shape change (and hence, the enzyme activity), researchers defined a parameter t^* as the time difference between when maximum and half-maximum transmission was reached [Fig. 7(e)]. The parameter t^* was measured for a range of α-amylase activities, and 1/t^* vs enzyme activity was plotted to create a linear standard curve. This approach highlights how the rates of change in transmission, and, correspondingly, how fast a complex biphasic droplet changes shape, can be used to give information about reaction rates or binding or reaction events.

Typically, biphasic hydrocarbon-fluorocarbon Janus droplets align naturally with their symmetry axis perpendicular to the substrate due to gravity, because the hydrocarbon oil's density is often less than that of the continuous phase, and the fluorocarbon oil's density is often greater than that of the continuous phase. However, one way to disrupt this natural droplet orientation is through droplet agglutination, where the biphasic droplets are induced to aggregate and form droplet clumps that include droplets, which are tipped on their side. Optically, hydrocarbon-fluorocarbon Janus droplets are most optically transmissive when their oil-oil interface is perpendicular to the direction of illumination; when the droplets are tipped on their side, they macroscopically appear more opaque and scattering, providing a means to optically distinguish between agglutinated and non-agglutinated droplets. In order to trigger the agglutination, droplets are functionalized with a surface-active molecule designed to bind with analytes present in the continuous phase via multi-valent interactions.

Agglutination of Janus droplets has been used for the detection of a variety of different bacteria, viruses, enzymes, and small molecules (see Sec. V B 2 for further details).^45,48 For example, researchers demonstrated that a mannose-binding lectin, concanavalin A (ConA, used as a functional substitute for E. coli bacteria) could induce agglutination of hexane-FC770 Janus droplets stabilized by a surfactant containing a mannose head group and C-14 alkyl tail.⁴⁵ Upon agglutination, there was an observable difference in transparency between aligned and agglutinated Janus droplets which could be detected by placing the droplets in a transparent analysis chamber positioned over a quick response (QR) code; upon the addition of ConA, the Janus droplets agglutinated in 5 s and the chamber became opaque, rendering the QR code unreadable by a smartphone [Figs. 8(a) and 8(b)]. Authors found that the degree of optical scattering was dependent on the ConA concentration, as well as the distance between the QR code and smartphone [Fig. 8(c)].⁴⁵ Other work has used a Janus droplet agglutination assay for the detection of the Zika virus via protein-protein interactions.⁴⁸ Droplets were functionalized with a binding protein rcSso7d variant (rcSso7d-ZNS1) for the detection of Zika NS1 protein. The addition of tetravalent streptavidin to the rcSso7d-SA functionalized droplets triggered agglutination by linking rcSso7d from different droplets together. In this system, agglutination was observed within 30 min of the addition of the streptavidin. The sensor had a detection limit of 100 nM Zika NS1 protein.⁴⁸ 

As noted previously in Sec. V A 2, reflected structural coloration is created in Janus droplets from light interference due to different paths of TIR along the droplet-internal interface between a high and low index liquid.³³ The coloration in biphasic Janus droplets is affected by the refractive index contrast between the liquids, the shape and size of the droplet, and the orientation of the droplets with respect to the illumination and viewing direction. The color is very sensitive to droplet morphology; while Janus droplets may be brightly iridescent, double emulsions do not reflect color. Even small alterations in interfacial tensions can induce morphological changes in the droplets that affect the path lengths of light and hence alter the colors [Fig. 6(c)]. Proof-of-concept work for a colorimetric droplet assay has been demonstrated for the detection of α-amylase activity.¹¹⁰ Researchers employed the same inclusion-complex mechanism detailed in Secs. IV B 3 and V B 1 a to observe the uncaging of Triton X-100 from γ-cyclodextrin in the presence of α-amylase. When emulsion droplets were placed in the enzyme-responsive surfactant solution (containing Triton X-100, γ-cyclodextrin, and Capstone FS-30) and exposed to α-amylase, droplets went from colorless to colored and changed color over time. Researchers could record changes in the color of the droplets by using a cell phone camera and flash for both viewing and illumination. Given that the specific reflected colors vary with droplet size and volume ratio of constituent oils, if specific color changes are desired for sensing, then each droplet morphology would need to be calibrated prior to use.¹¹⁰ 

In addition to changes in optical transmission and color, changes in fluorescence intensity of fluorophores located within the droplets can also be correlated to changes in droplet morphology or orientation and used as a sensor readout mechanism [Figs. 6(d) and 9]. Fluorescence characteristics of the multicompartment droplets are dependent on the droplets' interfacial curvatures, droplet orientation, refractive index contrast, and exposure to chemicals that lead to fluorescence quenching. Several works have measured changes in emission intensity from fluorescent dyes dissolved in one of the dispersed droplet phases as a function of droplet shape.^47–49 In one example, researchers employed the use of fluorescent perylene dye, which partitioned into the higher-index hydrocarbon phase of a hydrocarbon-fluorocarbon biphasic droplet, as well as a fluorescent fluorinated perylene diimide dye, which partitioned into the fluorinated phase.⁴⁷ When excited by 400 nm light, the intensity of emitted light (475 nm) in the direction normal to the sample surface varied as a function of droplet morphology. Researchers observed brighter emission in a ring near the three-phase contact line of the Janus droplets, which they speculated was due to light undergoing TIR in this region and confining the perylene emission. As a result, researchers were able to detect the binding of poly- and monosaccharaides to boronic acid surfactant by generating a curve of normalized emission intensity vs contact angle, analyte concentration, and surfactant concentration. However, the measurement of droplets containing only a dye in the fluorinated phase revealed inconsistencies in the emission intensity as a function of the morphology. To remedy this, using a ratiometric readout between the emission intensities of a fluorescent dye in the hydrocarbon phase (λ = 475 nm) and a second fluorescent dye in the fluorocarbon phase (λ = 580 nm) rather than the absolute emissions, negated size effects arising from polydisperse droplets.⁴⁷ In a similar study, researchers also employed a two-dye system for the detection of Listeria monocytogenes using biphasic complex droplets. A subphthalocyanine (sub-PC) dye was dissolved in the hydrocarbon phase, and a fluorous soluble perylene PBI dye (F-PBI) was placed in the fluorocarbon droplet phase. In the absence of Listeria, droplets naturally aligned with gravity such that the fluorocarbon phase containing F-PBI was on the bottom and the hydrocarbon phase containing sub-PC was on the top due to density differences between dispersed phase liquids. When aligned, excitation and emission collection from the top gave only a small signal from the F-PBI as a result of the absorptive blocking of both the excitation and emission of the F-PBI by the sub-PC dye in the top hydrocarbon phase. When Listeria was added into the solution, and droplet agglutination was triggered, the resulting tilted droplet orientation allowed for unobstructed excitation of the F-PBI and the detection of its emission.⁴⁴ 

To develop a sensing approach which takes advantage of morphological reconfiguration of biphasic droplets, but is not dependent on droplet orientation with gravity, researchers employed the use of quenchable fluorescent surfactants. Specifically, researchers used meta-amino substituted green fluorescence protein chromophore (GFPc) surfactants, because they exhibit hydrogen-bonding mediated fluorescence quenching.¹¹¹ The meta-amino substituted amphiphilic GFPc used contained octyl chains and triethylene glycol and was organic oil-soluble but localized at the hydrocarbon-water interface. The fluorescence intensity was dependent on the hydrocarbon-water interfacial surface area where hydrogen bonding-induced quenching occurred. Thus, when a biphasic hydrocarbon-fluorocarbon droplet existed in a double morphology with a hydrocarbon shell, fluorescence was quenched due to the hydrogen bonding of surfactants at the aqueous interface. Fluorescence intensity returned as the droplet changed shape to Janus and was most fluorescent when in a double emulsion geometry with a fluorocarbon oil shell to entirely remove the fluorophore contact with water.¹¹¹ 

Recently, approaches exploiting evanescent wave sensing schemes combined with biphasic complex droplet reconfiguration for the detection of chemical and biological analytes have become of interest. In general, whenever light undergoes TIR at an interface, such as within a waveguide or an optical resonator, an evanescent wave is created at the interface that penetrates a finite distance into the external medium. The evanescent wave is highly sensitive to local perturbations, such as the presence of absorbing molecules or refractive index changes, providing a basis for sensing. This method of detection is best used for sensing environments immediately surrounding the waveguide because the amplitude of the evanescent field decreases exponentially with distance from the waveguide surface with typical penetration depths into the external medium falling in the nanometer scale.¹¹² In one example, researchers explored how reconfiguration of droplets containing dibutyl phthalate and methoxyperfluorobutane sitting on the surface of a waveguiding substrate could be used to attenuate light through evanescent wave coupling.¹¹³ Laser radiation was directed into a glass prism at an incidence angle above the critical angle such that the light bounced by TIR at the glass-emulsion interface; if the droplets contacting the glass prism had a high-index oil shell with absorbing dye (e.g., dibutyl phthalate containing a perylene, n = 1.49), then the laser radiation would be out-coupled into the droplets, exciting the dye. The fluorophore emission was tracked as a function of droplet geometry, and indeed the emission was highest for double emulsions with the high index dibutyl phthalate shell and lowest for droplets with the low index methoxyperfluorobutane shell. This detection mechanism was then used to sense the presence of caffeine via a caffeine-sensitive surfactant designed using an amphiphilic Pd-pincer complex.¹¹³ Another example of evanescent wave sensing applied to biphasic complex droplets relied on the use of microcavity resonators.¹¹⁴ Authors used a silicon nitride (Si₃N₄)-based racetrack optical resonator cavity in combination with hydrocarbon-fluorocarbon biphasic complex droplets.¹¹⁴ Changes in the effective refractive index caused by reconfiguration of biphasic droplets in response to an external stimulus led to shifts in the cavity's resonance wavelength. Researchers speculated that combining these “hard” and “soft” modes of chemical sensing in the future will allow for the detection of a wide variety of analytes.

Complex droplets are promising for the development of low-cost, easy to use sensors that are compatible with a wide range of analytes and optical readout mechanisms. There is a plethora of ways to sensitize the balance of interfacial tensions in a reconfigurable droplet system to the presence of a desired analyte. Here, we discuss some of the different analytes and transduction mechanisms developed for reconfigurable complex droplet-based sensors. In general, strategies employ the use of two surfactants: an inert surfactant that is necessary to maintain the stability of the complex droplets during stimulation and a responsive surfactant that is designed to be sensitive to the analyte. Usually, these two surfactants are chosen to selectively reduce interfacial tension at different interfaces of the complex droplet (Fig. 10).

Bioassays are frequently employed in healthcare for the identification and quantification of biochemical markers and molecules, which are diagnostic indicators for monitoring diseases and health conditions. Enzymes are particularly important targets for sensor development, not only because enzymes themselves are diagnostic indicators but enzymes can be used in enzyme-linked assays to detect other molecules as well. Reconfigurable complex emulsions have been used for the quantification of enzyme activity by correlating changes in optical properties of droplet lenses to changes in the balance of interfacial tensions in a complex droplet system triggered by enzymatic reactions.⁶⁹ To link enzyme activity to interfacial tension changes, the enzymes participate in either formation or degradation of a surface-active molecule, thereby changing the surfactant's efficacy. Researchers have used that general strategy to quantify the activity of α-amylase, lipase, and sulfatase with transmission data collected from a spectrometer or with a smartphone or tablet.⁶⁹ To measure the activity of lipase from Candida sp., researchers utilized an ester-linked hydrocarbon surfactant [tetra(ethylene glycol) mono-n-octanoate] paired with a fluorosurfactant to initially stabilize biphasic hydrocarbon-fluorocarbon droplets into a Janus morphology. Upon addition of the lipase, the ester-linkage in the surfactant was cleaved and the droplet changed shape to a double emulsion morphology with a hydrocarbon oil core and fluorocarbon oil shell⁶⁹ [Fig. 10(a)]. Sensing of sulfatase from Helix pomatia was achieved by exploiting a similar approach in which the sulfatase degraded a surfactant, 4-decyl-2,6-difluorophenyl sulfate, resulting in optically detectable droplet morphological reconfiguration.⁶⁹ In another example, researchers were able to sense for the enzyme trypsin using a peptide-linked GFPc surfactant (D8AP).¹¹¹ Trypsin is a pancreatic digestive enzyme and cleaves specific peptide bonds in proteins. The employed D8AP surfactant dissolved into the hydrocarbon phase of the biphasic heptane-FC-770 emulsion droplet. The GFPc exhibited hydrogen-bonding-mediated fluorescence quenching, thus the droplet was most fluorescent when adopting an heptane/FC-770/water morphology and almost all fluorescence was quenched when the GFPc surfactant localized at the hydrocarbon-water interface in an FC-770/heptane/water morphology. When trypsin was added to biphasic droplets containing D8AP stabilized with a fluorosurfactant, the hydrophilic peptide in D8AP was cleaved and the fluorocarbon surfactant in the aqueous phase promoted a transition to a comparatively much more emissive droplet shape.¹¹¹ 

Rapid detection of certain bacteria and viruses, such as foodborne pathogens like E. coli or the highly infectious Zika virus, has emerged as a global health priority in recent years.¹¹⁵ Agglutination of Janus droplets has been used as a sensing platform for the detection of both of these aforementioned analytes.^45,47,48 Disruption of the density-driven orientation of biphasic Janus droplets through aggregation is referred to as agglutination, which is discussed more extensively in Sec. V B 1 b. Agglutination results in changes in the optical scattering properties of the emulsion which makes layers of the agglutinated Janus droplets appear more opaque compared to the non-agglutinated state.^45,48 With respect to sensor development, agglutination is achieved when Janus droplets are stabilized with a surface-active molecule that is functionalized to bind to a specific multivalent analyte in the continuous phase. If the multivalent analyte is present, it will simultaneously bind with several surfactants and cause droplets to stick together upon agitation and tip into large clumps. For example, to develop an agglutination assay for E. coli, researchers used mannose-based surfactants that bind to the lectin concanavalin A (ConA), a functional substitute for E. coli bacteria⁴⁵ [Fig. 10(b)]. Janus emulsions in a transparent analysis chamber were positioned over a quick response (QR) code printed on paper, and when the droplets were highly agglutinated due to the presence of ConA and optically scattering, the QR code became unreadable. Concentrations as low as 10⁴ CFU/mL were shown to induce agglutination and affect QR code visibility. A different study used two different fluorescent dyes dissolved into each of a hydrocarbon-fluorocarbon droplet's constituent dispersed phases for the rapid detection of Listeria monocytogenes via an emissive signal produced in response to Listeria binding. Listeria antibodies were attached to a functional surfactant polymer with a tetrazine/transcyclooctene click reaction. The strong binding between Listeria and the Listeria antibody located at the hydrocarbon surface of the biphasic emulsions resulted in the tilting of the Janus droplets from their density-aligned position to produce emission that would ordinarily be obscured by a blocking dye. Researchers observed a detection limit of less than 100 CFU/mL Listeria in 2 h.⁴⁴ In a final example, researchers used a Janus drop agglutination assay for the detection of interfacial protein-protein interactions in the sensing of the Zika virus. Janus droplets were functionalized with a binding protein rcSso7d variant (rcSso7d-ZNS1) for the detection of Zika NS1 protein. The addition of tetravalent streptavidin to the rcSso7d-SA functionalized droplets triggered agglutination by linking rcSso7d from different droplets together [Fig. 10(c)]. In this system, agglutination was observed within 30 min of the addition of the streptavidin. Researchers were able to detect up to 100 nM Zika NS1 protein.⁴⁸ 

In principle, it is possible to detect any desired analyte using a complex droplet platform if a surface-active component in the system is made sensitive to the desired analyte. Caffeine, for example, is a previously studied target in molecular recognition which researchers have detected using complex biphasic droplets.¹¹³ A caffeine-sensitive surfactant was designed around an amphiphilic Pd-pincer complex C₁₀P containing two hydrophilic ethylene glycol chains and one hydrophobic aliphatic moiety. The synthesized surfactant was found to bind caffeine in its hydrophobic binding pocket at the droplet interface, resulting in both an increase in hydrophobicity as well as a decrease in its surfactant strength. Thus, the addition of small amounts of caffeine to the continuous phase induced changes in the balance of interfacial tensions and biphasic droplet morphology. In another study, researchers utilized EDC [N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride]/NHS (N-hydroxysuccinimide) coupling chemistry to functionalize the interfaces of complex biphasic droplets for the biosensing of several different small molecules and proteins.⁴⁹ The authors identified trialkylgallic acid (GA) groups as being ideal for the synthesis of oil-soluble surfactants, which preferentially localize at the biphasic droplet's oil-water interface, because of the ability to functionalize the carboxylic acid in the surfactant's headgroup. This method was used for the detection of anti-mouse immunoglobulin (IgG) using Protein A functionalized emulsion assays. Protein A reacted with GA12-NHS, a trialkylgallic acid surfactant with an amine group which localizes at the surface of Janus emulsion droplets. Subsequent addition of fluorescein isothiocyanate-labelled anti-mouse IgG to the continuous phase resulted in the binding to Protein A on the surface of droplets. Droplet morphology was reconfigured from transmissive Janus to a fluorocarbon-in-hydrocarbon-in-water double emulsion, and protein binding was confirmed using fluorescence microscopy. Macroscopically, after IgG was bound to protein A, the double emulsion droplets became less transmissive and blurred the image of a QR code beneath a monolayer of droplets.⁴⁹ 

Complex emulsions have often been used as templates¹¹⁶ for capsules^25,55 and particles,^117–120 which are used in a range of applications such as interfacial stabilization¹²¹ and controlled release.^122,123 To form particles from droplets, typically at least one of the dispersed droplet phases contains a monomer that can be polymerized into solid particles. The ability to reconfigure the biphasic droplets into a specific double emulsion or Janus morphologies before solidification has introduced unique templating opportunities. Small and precise variations in droplet morphology can be controllably induced, facilitating the formation of particles with a customizable structure.

Anisotropic particles are of interest for a broad range of applications including the design of colloidal stabilizers^{121,124–126} for uses in active matter.^127–129 Typically, droplets can serve as precursors to particles when one or more polymerizable liquid monomers are used as the droplet dispersed phases. The droplet shape is tuned by varying the balance of interfacial tensions with surfactants, and upon polymerization (often initiated by UV light when a photoinitiator is present) the geometric shape of the particle is templated by the droplet.⁹ Not all droplet phases have to be polymerizable, presenting a facile route to the fabrication of anisotropic, non-spherical particles with tunable shapes and precise morphologies. To generate solid hemispherical particles, a biphasic emulsion droplet consisting of a liquid monomer, 1,6-hexanediol diacrylate with a photoinitiator, and immiscible, inert methoxyperfluorobutane was photopolymerizated.⁹ By replacing methoxyperfluorobutane with a polymerizable fluorinated acrylate oligomer and cross-linker, researchers also showed that spherical solid Janus particles with fluorinated and non-fluorinated sides could be easily formed [Figs. 11(a) and 11(b)].⁹ Another work utilized two immiscible polymerizable monomers [tripropylene glycol diacrylate (TPGDA) and methacryl oxypropyl dimethylsiloxane (DMS)] as part of a Janus emulsion for the production of anisotropic particles. The geometry of the resultant Janus particles was controlled from a described “snowman” shape to a “dumbbell” morphology by adjusting the mass ratio of two oils in the initial emulsion.¹²¹ Further refining of particle shape was achieved by tuning surface coverage of one lobe to the other by adjusting the combination of employed mixed surfactants (Pluronic F127 and Tween 80).¹²¹ 

Droplets are thermodynamically unstable, and a limitation of emulsions for long-term use applications is their eventual breakdown over time by pathways such as coalescence and Ostwald ripening that lead to variations in droplet size and composition.^11,130–132 Many of the potential uses of complex droplets, such as for optics or sensors, require precision in the droplet composition or size. Strategies to enhance droplet stability and longevity while not compromising the fluidity and reconfigurability or responsive character that makes the droplets unique, are therefore important. A general strategy for droplet stabilization that has been explored is the use of thin polymeric shells^25,55 formed at the oil-water interface of biphasic oil droplets. Thin, clear membranes are preferred as they do not significantly alter the optical properties of the droplets. In one example, researchers stabilized biphasic Janus droplets by forming a hemispherical shell of the cross-linked polymer at the aqueous interface of one of the dispersed phases.⁵⁵ Specifically, droplets containing hexadecane with 20 wt. % dibutyl maleate as the hydrocarbon phase and ethyl nonafluorobutylether as the fluorocarbon phase were made in a continuous aqueous phase containing polyethylene glycol (PEG)-divinylether as the water-soluble monomeric component. Interfacial polymerization at the hydrocarbon-water interface was triggered when a colocalized cationic interfacial initiator was used [Fig. 12(a)]. Triton X-100 and Zonyl FS-300 were utilized as cosurfactants to help stabilize biphasic droplets into the desired Janus morphology before polymerization was induced. Researchers noted Janus droplets with the hemispherical polymer shell displayed resistance to morphological reconfiguration upon changes in surfactant concentrations.⁵⁵ Another work demonstrated that the encapsulation of hydrocarbon-fluorocarbon biphasic droplets with hydrogel capsules provided enhanced droplet stability while still allowing reconfiguration of droplet morphology upon addition of surfactants.²⁵ Researchers encapsulated the droplets in a thin calcium alginate polyelectrolyte hydrogel capsule formed phase through the use of a Pickering emulsion intermediate. Droplets were first stabilized with calcium carbonate nanoparticles and dispersed in a sodium alginate aqueous phase; upon addition of hydrochloric acid, the calcium carbonate dissolved, releasing calcium ions into the water and ionically cross-linking the alginate to form a hydrogel capsule [Fig. 12(b)]. Covalent cross-linking of the hydrogel's carboxylic acids with the amine groups of polyethyleneimine enhanced capsule stability. These droplets showed better stability over time and better stability on various treated glass surfaces in comparison to droplets stabilized by the SDS alone [Fig. 12(c)]. In combination with the introduction of oil-soluble surfactant Capstone FS-66 dissolved into the fluorocarbon oil, the complex droplets could be reconfigured across the full spectrum of droplet shapes from a double emulsion to Janus.²⁵ 

Particles can adsorb to fluid interfaces and stabilize emulsions (i.e., Pickering emulsions) by replacing the droplet fluid-fluid interface with a fluid-solid interfacial area. Much work has been done on the particle stabilization of single emulsion droplets,^133–136 and more recently, researchers have utilized particles to stabilize reconfigurable complex droplets as well.^24,54 Platinum on carbon (Pt/C) particles have been demonstrated to stabilize fluorocarbon-in-hydrocarbon-in-water droplets via a temperature-induced phase separation fabrication method with the particles stabilizing the hydrocarbon-water interface.⁵⁴ Droplets reconfigured to a Janus morphology upon addition of Zonyl FS-300 with the particles still preferentially located on the hydrocarbon-water interface⁵⁴ [Fig. 13(a)]. The Pt/C particles served as a nucleation site for the templated growth of gold hemispherical shells. Concentrations ranging from 10 to 200 × 10⁻³ M of gold were used, with much denser and thicker capsules forming at higher gold concentrations.⁵⁴ Furthermore, researchers also demonstrated that through an interfacial reaction at the biphasic complex droplet's interface between oil-soluble aldehydes and amine-functionalized magnetic nanoparticles in the aqueous continuous phase, the directed motion of complex droplets using magnets was possible.⁹⁸ Specifically, Fe₃O₄ particles were confined to the interface of either diethylbenzene or 2-trifluoromethyl-3-ethoxyperfluorohexane (HFE-7500), with water in both Janus and double emulsion morphologies, and magnetization curves of free nanoparticles in solution, as well as confined particles in double emulsion and Janus morphologies, obtained and compared. Authors were also able to demonstrate more precise nanoparticle assembly in biphasic droplets containing liquid crystals as one of the dispersed phases.⁹⁸ Inclusion of hydrophobic magnetite nanoparticles into the hydrocarbon phase of Janus hydrocarbon-fluorocarbon biphasic droplets in water also allowed the facile orientation and movement of the droplets with an external magnet.⁹ 

Researchers have also explored the use of particles inside biphasic droplets to affect the droplet-internal interface and influence reconfigurability while traditional molecular surfactants are still used in the continuous phase.²⁴ Researchers used silica nanoparticles functionalized with both lipophilic and fluorophilic moieties, hexadecyltrimethoxysilane and 1H,1H,2H,2H-perfluorooctyltriethoxysilane, respectively, to stabilize a wide range of hydrocarbon-fluorocarbon interfaces. Hexadecane and perfluorooctane, which have an interfacial tension of 6.2 mN/m,¹³⁷ could be formed into double emulsions when particles were used at the oil-oil interface. Not only do particles at the oil−oil interfaces affect the interfacial energy to influence the overall droplet shape, but the particles were also used to “trap” the complex droplets in geometries that were unexpected considering the specific surfactants present in the continuous phase [Fig. 13(b)]; heating above the oils' UCST then cooling triggered reconfiguration of such particle-containing droplets, as shown in Fig. 13(c).²⁴ In general, the use of particles to address the interfacial energy and stability of complex droplet interfaces expands the scope of droplet chemical compositions accessible for reconfigurable biphasic droplets.

Complex droplets of controllable composition and reconfigurable morphology provide a new dynamic element to tuning the properties of emulsions and enable numerous directions for fundamental exploration and application. Reconfigurable droplets have so far been used to sense a variety of analytes and stimuli, ranging from environmental (pH and light) to biological (enzymes, proteins, bacteria, and viruses); changes in droplet color, transparency, or fluorescence as a function of droplet shape and orientation have been used for tunable lenses and sensors; and shape-tunable droplets have served as templates in the formation of anisotropic particles. The emulsion fabrication strategies and droplet sensitization mechanisms are generalizable to many chemicals, materials, liquids, surfactants, stimuli, and analytes beyond what has already been demonstrated in the literature. Emulsions with the characteristic ability to selectively “present” and “hide” specific liquid interfaces and controllably alter droplet morphology may also find novel applications in fields such as active matter, microreactors, or in other devices which may necessitate reconfigurable liquids.¹³⁸ Living organisms, which harness multicomponent fluids to generate dynamic hierarchical structures and chemical networks, serve as future inspiration for the construction and study of architectured fluids. Potential pathways coupling synthetic biology with soft fluids-based materials science may be fruitful, with the use of reconfigurable complex droplets as exemplar non-equilibrium compartmentalized liquid domains. Systems, for example, built around unilamellar vesicles^139,140 share fundamental physical properties with droplets stabilized by complex interfaces in which compartmentalization and communication between compartments give rise to nature-inspired advanced materials. There are numerous ways in which dynamic, complex emulsions can be designed, functionalized, and applied toward important new scientific discoveries and technologies.

The authors gratefully acknowledge funding support from the Army Research Office, Grant No. W911NF-18-1-0414.

Data sharing is not applicable to this article as no new data were created or analyzed.