How liquid-liquid phase separation induces active spreading.

The interplay between phase separation and wetting of multicomponent mixtures is ubiquitous in nature and technology and recently gained significant attention across scientific disciplines, due to the discovery of biomolecular condensates. It is well understood that sessile droplets, undergoing phase separation in a static wetting configuration, exhibit microdroplet nucleation at their contact lines, forming an oil ring during later stages. However, very little is known about the dynamic counterpart, when phase separation occurs in a nonequilibrium wetting configuration, i.e., spreading droplets. Here we show that liquid-liquid phase separation strongly couples to the spreading motion of three-phase contact lines. Thus, the classical Cox-Voinov law is not applicable anymore, because phase separation adds an active spreading force beyond the capillary driving. Intriguingly, we observe that spreading starts well before any visible nucleation of microdroplets in the main droplet. Using high-speed ellipsometry, we further demonstrate that the evaporation-induced enrichment, together with surface forces, causes an even earlier nucleation in the wetting precursor film around the droplet, initiating the observed wetting transition. We expect our findings to improve the fundamental understanding of phase separation processes that involve dynamical contact lines and/or surface forces, with implications in a wide range of applications, from oil recovery or inkjet printing to material synthesis and biomolecular condensates.

Spontaneous oscillation of an active filament under viscosity gradients.

We investigate the effects of uniform viscosity gradients on the spontaneous oscillations of an elastic, active filament in viscous fluids. Combining numerical simulations and linear stability analysis, we demonstrate that a viscosity gradient increasing from the filament's base to tip destabilises the system, facilitating its self-oscillation. This effect is elucidated through a reduced-order model, highlighting the delicate balance between destabilising active forces and stabilising viscous forces. Additionally, we reveal that while a perpendicular viscosity gradient to the filament's orientation minimally affects instability, it induces asymmetric ciliary beating, thus generating a net flow along the gradient. Our findings offer new insights into the complex behaviours of biological and artificial filaments in complex fluid environments, contributing to the broader understanding of filament dynamics in heterogeneous viscous media.

Emerging aqueous two-phase systems: from fundamentals of interfaces to biomedical applications.

Aqueous two-phase systems (ATPSs) have been recognized for their applications in extraction, separation, purification, and enrichment of (bio)molecules and cells. Recently, their unique ability to create aqueous-aqueous interfaces through phase separation and the characteristics of these interfaces have created new opportunities in biomedical applications. In this review, we summarize recent progress in understanding the dynamics at aqueous-aqueous interfaces, and in developing interface-assisted design of artificial cells and cyto-mimetic materials, fabrication of cyto- and bio-compatible microparticles, cell micropatterning, 3D bioprinting, and microfluidic separation of cells and biomolecules. We also discuss the challenges and perspectives to leverage the unique characteristics of ATPSs and their interfaces in broader applications.

Droplet and Microchamber-Based Digital Loop-Mediated Isothermal Amplification (dLAMP).

Digital loop-mediated isothermal amplification (dLAMP) refers to compartmentalizing nucleic acids and LAMP reagents into a large number of individual partitions, such as microchambers and droplets. This compartmentalization enables dLAMP to be an excellent platform to quantify the absolute number of the target nucleic acids. Owing to its low requirement for instrumentation complexity, high specificity, and strong tolerance to inhibitors in the nucleic acid samples, dLAMP has been recognized as a simple and accurate technique to quantify pathogenic nucleic acid. Herein, the general process of dLAMP techniques is summarized, the current dLAMP techniques are categorized, and a comprehensive discussion on different types of dLAMP techniques is presented. Also, the challenges of the current dLAMP are illustrated together with the possible strategies to address these challenges. In the end, the future directions of the dLAMP developments, including multitarget detection, multisample detection, and processing nucleic acid extraction are outlined. With recently significant advances in dLAMP, this technology has the potential to see more widespread use beyond the laboratory in the future.

Flower-like droplets obtained by self-emulsification of a phase-separating (SEPS) aqueous film.

Self-emulsification, referring to the spontaneous formation of droplets of one phase in another immiscible phase, is attracting growing interest because of its simplicity in creating droplets. Existing self-emulsification methods usually rely on phase inversion, temperature cycling, and solvent evaporation. However, achieving spatiotemporal control over the morphology of self-emulsified droplets remains challenging. In this work, a conceptually new approach of creating both simple and complex droplets by self-emulsification of a phase-separating (SEPS) aqueous film, is reported. The aqueous film is formed by depositing a surfactant-laden aqueous droplet onto an aqueous surface, and the fragmentation of the film into droplets is triggered by a wetting transition. Smaller and more uniform droplets can be achieved by introducing liquid-liquid phase separation (LLPS). Moreover, properly modulating quadruple LLPS and film fragmentation enables the creation of highly multicellular droplets such as flower-like droplets stabilized by the interfacial self-assembly of nanoparticles. This work provides a novel strategy to design aqueous droplets by LLPS, and it will inspire a wide range of applications such as membraneless organelle synthesis, cell mimics and delivery.

Diffusion-Dominated Pinch-Off of Ultralow Surface Tension Fluids.

We study the breakup of a liquid thread inside another liquid at different surface tensions. In general, the pinch-off of a liquid thread is governed by the dynamics of fluid flow. However, when the interfacial tension is ultralow (2-3 orders lower than normal liquids), we find that the pinch-off dynamics can be governed by bulk diffusion. By studying the velocity and the profile of the pinch-off, we explain why the diffusion-dominated pinch-off takes over the conventional breakup at ultralow surface tensions.

Picoinjection-Enabled Multitarget Loop-Mediated Isothermal Amplification for Detection of Foodborne Pathogens.

In this study, we develop a method to detect multiple DNAs of foodborne pathogens by encapsulating emulsion droplets for loop-mediated isothermal amplification (LAMP). In contrast to the traditional bulk-phase LAMP, which involves a labor-intensive mixing process, with our method, different primers are automatically mixed with DNA samples and LAMP buffers after picoinjection. By directly observing and analyzing the fluorescence intensity of the resultant droplets, one can detect DNA from different pathogens, with a detection limit 500 times lower than that obtained by bulk-phase LAMP. We further demonstrate the ability to quantify bacteria concentration by detecting bacterial DNA in practical samples, showing great potential in monitoring water resources and their contamination by pathogenic bacteria.

Generation of High-Order All-Aqueous Emulsion Drops by Osmosis-Driven Phase Separation.

Droplets containing ternary mixtures can spontaneously phase-separate into high-order structures upon a change in composition, which provides an alternative strategy to form multiphase droplets. However, existing strategies always involve nonaqueous solvents that limit the potential applications of the resulting multiple droplets, such as encapsulation of biomolecules. Here, a robust approach to achieve high-order emulsion drops with an all-aqueous nature from two aqueous phases by osmosis-induced phase separation on a microfluidic platform is presented. This technique is enabled by the existence of an interface of the two aqueous phases and phase separation caused by an osmolality difference between the two phases. The complexity of emulsion drops induced by phase separation could be controlled by varying the initial concentration of solutes and is systematically illustrated in a state diagram. In particular, this technique is utilized to successfully achieve high-order all-aqueous droplets in a different aqueous two-phase system. The proposed method is simple since it only requires two initial aqueous solutions for generating multilayered, organic-solvent-free all-aqueous emulsion drops, and thus these multiphase emulsion drops can be further tailored to serve as highly biocompatible material templates.

Control of Particle Adsorption for Stability of Pickering Emulsions in Microfluidics.

Studying the stability of Pickering emulsion is of great interest for applications including catalysis, oil recovery, and cosmetics. Conventional methods emphasize the overall behavior of bulk emulsions and neglect the influence of particle adsorbing dynamics, leading to discrepancies in predicting the shelf-life of Pickering emulsion-based products. By employing a microfluidic method, the particle adsorption is controlled and the stability of the Pickering emulsions is consequently examined. This approach enables us to elucidate the relationship between the particle adsorption dynamics and the stability of Pickering emulsions on droplet-level quantitatively. Using oil/water emulsions stabilized by polystyrene nanoparticles as an example, the diffusion-limited particle adsorption is demonstrated and investigated the stability criteria with respect to particle size, particle concentration, surface chemistry, and ionic strength. This approach offers important insights for application involving Pickering emulsions and provides guidelines to formulate and quantify the Pickering emulsion-based products.

Droplet Formation by Rupture of Vibration-Induced Interfacial Fingers.

By imposing vibration to a core-annular flow of an aqueous two-phase system (ATPS) with ultralow interfacial tension, we observe a liquid finger protruding from the interface of an expanding jet. We find that the protruded finger breaks up only when its length-to-width ratio exceeds a threshold value. The breakup follows a constant wavelength-to-width ratio that is consistent with that of breakup under Rayleigh-Plateau instability. The mechanism is applicable to aqueous two-phase systems with a large range of viscosity ratios. The protruded finger can break up into small droplets that are monodisperse in size, controllable in generation frequency under a wide range of flow rates. This work suggests a way to generate small water-water droplets with high monodispersity and production rate from a single nozzle.

Emergence of Droplets at the Nonequilibrium All-Aqueous Interface in a Vertical Hele-Shaw Cell.

The interfacial phenomena at liquid-liquid interfaces remain the subject of constant fascination in science and technology. Here, we show that fingers forming at the interface of nonequilibrium all-aqueous systems can spontaneously break into an array of droplets. The dynamic formation of droplets at the water-water (w/w) interface is observed when a less dense aqueous phase, for instance, the dextran solution, is placed on a denser aqueous phase, the polyethylene glycol solution, in a vertical Hele-Shaw cell. Because of the gradual diffusion of water from the upper phase into the lower phase, a dense layer appears at the nonequilibrium w/w interface. As a result, a periodic array of fingers emerge and sink. Remarkably, these fingers break up and an array of droplets are emitted from the interface. We characterize the wavelength of fingering by measuring the average distance between the dominant fingers. By varying the initial concentrations of the two nonequilibrium aqueous phases, we identify experimentally a phase diagram with a wide parameter space in which finger breaking occurs. Finally, plenty of droplets, spontaneously formed when one phase is continuously deposited onto another aqueous phase, further confirm the robustness of our experimental results. Our work suggests a simple yet efficient approach with a potential upscalability to generate all-aqueous droplets.

Partitioning-dependent conversion of polyelectrolyte assemblies in an aqueous two-phase system.

Partitioning refers to the distribution of solute molecules in the two immiscible phases of a mixture of two solutions, such as an aqueous two-phase system (ATPS). The partitioning of RNA and peptide has been adjusted in situ to facilitate their assembly into intracellular membraneless organelles. Despite the immense potential of this approach in artificial systems, a partitioning-dependent assembly of macromolecules has been limited, due to the sophisticated processing associated with their in situ modification. Here we demonstrate an approach to direct the assembly of polyelectrolytes in an ATPS through varying their partitioning via pH changes. Microcapsules can be converted to microgel particles as the polyelectrolytes selectively partition to different emulsion phases when changing pH. Such partitioning-dependence can also be equally applied for complexing hydrophilic nanoparticles with polyelectrolytes in an ATPS. By enabling access of hydrophilic materials across the aqueous interface freely, the ATPS allows modification of their intrinsic properties in situ; this advantage will inspire more versatile control over the partitioning of hydrophilic materials and will create new multi-functional biomaterials.

Moving wetting ridges on ultrasoft gels.

The surface mechanics of soft solids are important in many natural and technological applications. In this context, static and dynamic wetting of soft polymer gels has emerged as a versatile model system. Recent experimental observations have sparked controversial discussions of the underlying theoretical description, ranging from concentrated elastic forces over strain-dependent solid surface tensions to poroelastic deformations or the capillary extraction of liquid components in the gel. Here we present measurements of the shapes of moving wetting ridges with high spatiotemporal resolution, combining distinct wetting phases (water, FC-70, air) on different ultrasoft PDMS gels (∼100Pa). Comparing our experimental results to the asymptotic behavior of linear viscoelastocapillary theory in the vicinity of the ridge, we separate reliable measurements from potential resolution artifacts. Remarkably, we find that the commonly used elastocapillary scaling fails to collapse the ridge shapes, but, for small normal forces, yields a viable prediction of the dynamic ridge angles. We demonstrate that neither of the debated theoretical models delivers a quantitative description, while the capillary extraction of an oil skirt appears to be the most promising.

Effects of particle size on the electrocoalescence dynamics and arrested morphology of liquid marbles.

HYPOTHESIS: The coalescence of bare droplets when surface tension dominates always results in one larger spherical droplet. In contrast, droplets coated with particles may be stabilized into non-spherical structures after arrested coalescence, which can be achieved by different approaches, such as changing the particle surface coverage. The size of particles coating the initial liquid marbles can be used to control the coalescence dynamics and the resulting morphology of arrested droplets. EXPERIMENT: We characterized the electrocoalescence of liquid marbles coated with particles ranging from hundred nanometers to hundred micrometers. The electrocoalescence was recorded using high-speed imaging. FINDINGS: When the electrocoalescence initiates, particles jam and halt the relaxation of the marbles at different stages, resulting in four possible final morphologies that are characterized using the Gaussian curvature at the neck region. The four regimes are total coalescence, arrested puddle coalescence, arrested saddle coalescence, and non-coalescence. The coalescence is initiated at the center of the contact zone, independent of the particle size. Small particles show little resistance to the coalescence, while marbles coated by large particles demonstrate a viscous-like behavior, indicated by the growth of the liquid bridge and the damping. The present study provides guidelines for applications that involve the formulation of liquid marbles with complex morphologies.

Aquabots.

Soft robots, made from elastomers, easily bend and flex, but deformability constraints severely limit navigation through and within narrow, confined spaces. Using aqueous two-phase systems we print water-in-water constructs that, by aqueous phase-separation-induced self-assembly, produce ultrasoft liquid robots, termed aquabots, comprised of hierarchical structures that span in length scale from the nanoscopic to microsciopic, that are beyond the resolution limits of printing and overcome the deformability barrier. The exterior of the compartmentalized membranes is easily functionalized, for example, by binding enzymes, catalytic nanoparticles, and magnetic nanoparticles that impart sensitive magnetic responsiveness. These ultrasoft aquabots can adapt their shape for gripping and transporting objects and can be used for targeted photocatalysis, delivery, and release in confined and tortuous spaces. These biocompatible, multicompartmental, and multifunctional aquabots can be readily applied to medical micromanipulation, targeted cargo delivery, tissue engineering, and biomimetics.

Controlled Formation of All-Aqueous Janus Droplets by Liquid-Liquid Phase Separation of an Aqueous Three-Phase System.

Janus droplets have been demonstrated in a wide range of applications, ranging from drug delivery, to biomedical imaging, to bacterial detection. However, existing fabrication strategies often involve nonaqueous solvents, such as organic solvent or oil, which largely limits their use in fields that require a high degree of biocompatibility. Here, we present a method to achieve all-aqueous Janus droplets by liquid-liquid phase separation of an aqueous three-phase system (A3PS). An aqueous droplet containing two initially miscible polymers is first injected into an aqueous solution of another concentrated polymer, and then it spontaneously phase-separates into a Janus droplet due to the diffusive mass exchange between the drop and bulk phases during equilibration. To achieve continuous generation of the Janus droplets, the A3PS is further integrated with microfluidics and electrospray. The size and shape of the phase-separated Janus droplets can be easily controlled by tuning the operation parameters, such as the flow rate and/or the initial composition of the drop phases. Dumbbell-shaped and snowman-shaped Janus droplets with average sizes between 100 and 400 µm can be generated by both coflow microfluidics and electrospray. In particular, the phase-separated Janus droplets can simultaneously load two different liposomes into each compartment, which are promising carriers for combination drugs. The obtained Janus droplets are superior templates for biocompatible materials, which can serve as building blocks such as high-order droplet patterns for constructing advanced biomaterials.

Hand-Powered Microfluidics for Parallel Droplet Digital Loop-Mediated Isothermal Amplification Assays.

Droplet digital loop-mediated isothermal amplification (ddLAMP) is an important assay for pathogen detection due to its high accuracy, specificity, and ability to quantify nucleic acids. However, performing ddLAMP requires expensive instrumentation and the need for highly trained personnel with expertise in microfluidics. To make ddLAMP more accessible, a ddLAMP assay is developed, featuring significantly decreased operational difficulty and instrumentation requirements. The proposed assay consists of three simplified steps: (1) droplet generation step, in which a LAMP mixture can be emulsified just by manually pulling a syringe connected to a microfluidic device. In this step, for the first time, we verify that highly monodispersed droplets can be generated with unstable flow rates or pressures, allowing untrained personnel to operate the microfluidic device and perform ddLAMP assay; (2) heating step, in which the droplets are isothermally heated in a water bath, which can be found in most laboratories; and (3) result analysis step, in which the ddLAMP result can be determined using only a fluorescence microscopy and an open-source analyzing software. Throughout the process, no droplet microfluidic expertise or equipment is required. More importantly, the proposed system enables multiple samples to be processed simultaneously with a detection limit of 10 copies/µL. The test is simple and intuitive to operate in most laboratories for multi-sample detection, significantly enhancing the accessibility and detection throughput of the ddLAMP technique.

Rapid Multilevel Compartmentalization of Stable All-Aqueous Blastosomes by Interfacial Aqueous-Phase Separation.

Producing artificial multicellular structures to process multistep cascade reactions and mimic the fundamental aspects of living systems is an outstanding challenge. Highly biocompatible, artificial systems consisting of all-aqueous, compartmentalized multicellular systems have yet to be realized. Here, a rapid multilevel compartmentalization of an all-aqueous system where a 3D sheet of subcolloidosomes encloses a mother colloidosome by interfacial phase separation is demonstrated. These spatially organized multicellular structures are termed "blastosomes" since they are similar to blastula in appearance. The barrier to nanoparticle assembly at the water-water interface is overcome using oppositely charged polyelectrolytes that form a coacervate-nanoparticle-composite network. The conditions required to trigger interfacial phase separation and form blastosomes are quantified in a mapped state diagram. We show a versatile model for constructing artificial multicellular spheroids in all-aqueous systems. The rapid interfacial assembly of charged particles and polyelectrolytes can lock in nonequilibrium shapes of water, which also enables top-down technologies, such as 3D printing and microfluidics, to program flexible compartmentalized structures.

Thermocapillary thin-film flows on a compliant substrate.

We study the dynamics of a thin liquid film on a compliant substrate in the presence of thermocapillary effect. A set of long-wave equations are derived to investigate the effects of fluid gravity (G), fluid inertia (Re), and Marangoni stresses (Ma) on the dynamics of the liquid film and the compliant substrate. By performing linear stability analysis and time-dependent computations of the long-wave equations, we examine two different cases: thin-film flows on a horizontally compliant substrate (ß=0, where ß is the inclined angle) and down a vertically compliant substrate (ß=π/2), respectively. For ß=0, we neglect fluid inertia and identify two different modes: (1) sinuous mode, where the deformations of liquid-air and liquid-substrate interfaces are in phase, which is induced by the fluid gravity, and (2) varicose mode, where the deformations of two interfaces are in phase opposition, which is induced by the Marangoni stresses. For ß=π/2, we consider a weak fluid inertia and only observe the varicose mode driven by fluid inertia and Marangoni stresses. However, because the gravity direction is parallel to the substrate, the fluid gravity modifies the varicose mode, making the deformations of two interfaces out of phase. In particular, we also seek the nonlinear traveling-wave solutions in the case of ß=π/2, revealing that fluid inertia and/or heating effect enhance the height and speed of the traveling waves. In both cases, the introduction of a strong wall heating gives rise to large deformations of both the thin liquid film and the compliant substrate.

All-Aqueous Thin-Film-Flow-Induced Cell-Based Monolayers.

Cells in vitro usually require a solid scaffold to attach and form two-dimensional monolayer structures. To obtain a substrate-free cell monolayer, long culture time and specific detaching procedures are required. In this study, a thin-film-flow-induced strategy is reported to overcome the challenges of assembling in vitro scaffold-free monolayered cell aggregates. The assembly is driven by a dewetting-like thin-film withdrawal along all-aqueous interfaces characterized by a low interfacial tension. The withdrawal process drives the cells adsorbed on the liquid film to aggregate and assemble into an organized and compact monolayer. This strategy is not limited to biological cells but also colloidal particles, as demonstrated by the assembly of hybrid cell-particle monolayers. The versatility offered by this approach suggests new opportunities in understanding early tissue formation and functionalizing cell monolayer aggregates by colloidal particles with customized functions.