Functional metal-phenolic cortical cytoskeleton for artificial cells.

Cortex-like cytoskeleton, a thin layer of cross-linked cytoplasmic proteins underlying the cell membrane, plays an essential role in modulating membrane behavior and cell surface properties. However, bottom-up construction of functional cortex-like cytoskeleton in artificial cells remains a challenge. Here, we present metal-phenolic networks as artificial cortical cytoskeletons in liposome-based artificial cells. The metal-phenolic cytoskeleton-reinforced artificial cells exhibit long-term stability, enhanced resistance to a variety of harsh environments, tunable permeability, and well-controlled morphologies. We anticipate that our stable artificial cell models will stride forward to practical applications of liposome-based microsystem.

Tunable Structural Coloration in Eccentric Water-in-Oil-in-Water Droplets.

Structural colors show diverse advantages such as fade resistance, eco-friendliness, iridescence, and high saturation in comparison with chemical pigments. In this paper, we show tunable structural coloration in colorless water-in-oil-in-water double emulsion droplets via total internal reflection and interference at the microscale concave interfaces. Through experimental work and simulations, we demonstrate that the shell thickness and the eccentricity of the core-shell structures are key to the successful formation of iridescent structural colors. Only eccentric thin-shell water-in-oil-in-water droplets show structural colors. Importantly, structural colors based on water-oil interfaces are readily responsive to a variety of environmental stimuli, such as osmotic pressure, temperature, magnetic fields, and light composition. This work highlights an alternative structural coloration that expands the applications of droplet-based structural colors to aqueous systems.

Division in synthetic cells.

The bottom-up construction of a living cell using non-living materials represents a grand challenge in science and technology. Reproduction of cells into similar offspring is key to life, and therefore, building a synthetic cell that can autonomously divide is one of the most fundamental tasks that need to be achieved in bottom-up synthetic biology. In this review, we summarize the strategies of inducing synthetic division by using physical, chemical, and biological stimuli, and highlight the future challenges to the construction of autonomous synthetic cell division.

Photoswitchable Molecular Communication between Programmable DNA-Based Artificial Membraneless Organelles.

Spatiotemporal organization of distinct biological processes in cytomimetic compartments is a crucial step towards engineering functional artificial cells. Mimicking controlled bi-directional molecular communication inside artificial cells remains a considerable challenge. Here we present photoswitchable molecular transport between programmable membraneless organelle-like DNA coacervates in a synthetic microcompartment. We use droplet microfluidics to fabricate membraneless non-fusing DNA coacervates by liquid-liquid phase separation in a water-in-oil droplet, and employ the interior DNA coacervates as artificial organelles to imitate intracellular communication via photo-regulated uni- and bi-directional transfer of biomolecules. Our results highlight a promising new route to assembly of multicompartment artificial cells with functional networks.

Elastic-instability-enabled locomotion.

Locomotion of an organism interacting with an environment is the consequence of a symmetry-breaking action in space-time. Here we show a minimal instantiation of this principle using a thin circular sheet, actuated symmetrically by a pneumatic source, using pressure to change shape nonlinearly via a spontaneous buckling instability. This leads to a polarized, bilaterally symmetric cone that can walk on land and swim in water. In either mode of locomotion, the emergence of shape asymmetry in the sheet leads to an asymmetric interaction with the environment that generates movement--via anisotropic friction on land, and via directed inertial forces in water. Scaling laws for the speed of the sheet of the actuator as a function of its size, shape, and the frequency of actuation are consistent with our observations. The presence of easily controllable reversible modes of buckling deformation further allows for a change in the direction of locomotion in open arenas and the ability to squeeze through confined environments--both of which we demonstrate using simple experiments. Our simple approach of harnessing elastic instabilities in soft structures to drive locomotion enables the design of novel shape-changing robots and other bioinspired machines at multiple scales.

Complex coacervates as artificial membraneless organelles and protocells.

Complex coacervates are water droplets dispersed in water, which are formed by spontaneous liquid-liquid phase separation of an aqueous solution of two oppositely charged polyelectrolytes. Similar to the membraneless organelles that exist in biological cells, complex coacervate droplets are membraneless and have a myriad of features including easy formation, high viscosity, selective encapsulation of biomolecules, and dynamic behaviors in response to environmental stimuli, which make coacervates an excellent option for constructing artificial membraneless organelles. In this article, I first summarize recent advances in artificial compartments that are built from coacervates and their response to changes in the surrounding environment and then show the advantages of microfluidic techniques in the preparation of monodisperse coacervates and encapsulation of coacervates in droplets and liposomes to construct complex cell-like compartments, and finally discuss the future challenges of such membraneless aqueous compartments in cell mimics and origin of life.

Trojan-Horse-Like Stimuli-Responsive Microcapsules.

Multicompartment microcapsules, with each compartment protected by a distinct stimuli-responsive shell for versatile controlled release, are highly desired for developing new-generation microcarriers. Although many multicompartmental microcapsules have been created, most cannot combine different release styles to achieve flexible programmed sequential release. Here, one-step template synthesis of controllable Trojan-horse-like stimuli-responsive microcapsules is reported with capsule-in-capsule structures from microfluidic quadruple emulsions for diverse programmed sequential release. The nested inner and outer capsule compartments can separately encapsulate different contents, while their two stimuli-responsive hydrogel shells can individually control the content release from each capsule compartment for versatile sequential release. This is demonstrated by using three types of Trojan-horse-like stimuli-responsive microcapsules, with different combinations of release styles for flexible programmed sequential release. The proposed microcapsules provide novel advanced candidates for developing new-generation microcarriers for diverse, efficient applications.

Macromolecularly Crowded Protocells from Reversibly Shrinking Monodisperse Liposomes.

The compartmentalization of cell-free gene expression systems in liposomes provides an attractive route to the formation of protocells, but these models do not capture the physical (crowded) environment found in living systems. Here, we present a microfluidics-based route to produce monodisperse liposomes that can shrink almost 3 orders of magnitude without compromising their stability. We demonstrate that our strategy is compatible with cell-free gene expression and show increased protein production rates in crowded liposome protocells.

Microfluidic Formation of Monodisperse Coacervate Organelles in Liposomes.

Coacervates have been widely studied as model compartments in protocell research. Complex coacervates composed of disordered proteins and RNA have also been shown to play an important role in cellular processes. Herein, we report on a microfluidic strategy for constructing monodisperse coacervate droplets encapsulated within uniform unilamellar liposomes. These structures represent a bottom-up approach to hierarchically structured protocells, as demonstrated by storage and release of DNA from the encapsulated coacervates as well as localized transcription.

Continuous fabrication of polymeric vesicles and nanotubes with fluidic channels.

Fluidic channels were employed to induce the self-assembly of poly(ethylene glycol)-b-polystyrene into polymeric vesicles and nanotubes. The laminar flow in the device enables controlled diffusion of two miscible liquids at the phase boundary, leading to the formation of homogeneous polymeric structures of different shapes. These structures could be easily loaded with small molecule cargoes and functionalized with nanometer sized catalytic platinum nanoparticles. This technique offers a facile methodology to rapidly and continuously produce well-defined polymeric structures for nanotechnology applications.

Microfluidic Assembly of Monodisperse Vesosomes as Artificial Cell Models.

Vesosomes are nested liposomal structures with high potential as advanced drug delivery vehicles, bioreactors and artificial cells. However, to date no method has been reported to prepare monodisperse vesosomes of controlled size. Here we report on a multistep microfluidic strategy for hierarchically assembling uniform vesosomes from dewetting of double emulsion templates. The control afforded by our method is illustrated by the formation of concentric, pericentric and multicompartment liposomes. The microfluidic route to vesosomes offers an exceptional platform to build artificial cells as exemplified by the in vitro transcription in "nucleus" liposomes and the mimicry of the architecture of eukaryotic cells. Finally, we show the transport of small molecules across the nucleic envelope via insertion of nanopores into the bilayers.

Spontaneous transfer of droplets across microfluidic laminar interfaces.

The precise manipulation of droplets in microfluidics has revolutionized a myriad of drop-based technologies, such as multiple emulsion preparation, drop fusion, drop fission, drop trapping and drop sorting, which offer promising new opportunities in chemical and biological fields. In this paper, we present an interfacial-tension-directed strategy for the migration of droplets across liquid-liquid laminar streams. By carefully controlling the interfacial energies, droplets of phase A are able to pass across the laminar interfaces of two immiscible fluids from phase B to phase C due to a positive spreading coefficient of phase C over phase B. To demonstrate this, we successfully perform the transfer of water droplets across an oil-oil laminar interface and the transfer of oil droplets across an oil-water laminar interface. The whole transfer process is spontaneous and only takes about 50 ms. We find that the fluid dynamics have an impact on the transfer processes. Only if the flowrate ratios are well matched will the droplets pass through the laminar interface successfully. This interfacial-tension-directed transfer of droplets provides a versatile procedure to make new structures and control microreactions as exemplified by the fabrication of giant unilamellar vesicles and cell-laden microgels.

Monodisperse Uni- and Multicompartment Liposomes.

Liposomes are self-assembled phospholipid vesicles with great potential in fields ranging from targeted drug delivery to artificial cells. The formation of liposomes using microfluidic techniques has seen considerable progress, but the liposomes formation process itself has not been studied in great detail. As a result, high throughput, high-yielding routes to monodisperse liposomes with multiple compartments have not been demonstrated. Here, we report on a surfactant-assisted microfluidic route to uniform, single bilayer liposomes, ranging from 25 to 190 µm, and with or without multiple inner compartments. The key of our method is the precise control over the developing interfacial energies of complex W/O/W emulsion systems during liposome formation, which is achieved via an additional surfactant in the outer water phase. The liposomes consist of single bilayers, as demonstrated by nanopore formation experiments and confocal fluorescence microscopy, and they can act as compartments for cell-free gene expression. The microfluidic technique can be expanded to create liposomes with a multitude of coupled compartments, opening routes to networks of multistep microreactors.

Plug-n-play microfluidic systems from flexible assembly of glass-based flow-control modules.

In this study, we report on a simple and versatile plug-n-play microfluidic system that is fabricated from flexible assembly of glass-based flow-control modules for flexibly manipulating flows for versatile emulsion generation. The microfluidic system consists of three basic functional units: a flow-control module, a positioning groove, and a connection fastener. The flow-control module that is based on simple assembly of low-cost glass slides, coverslips, and glass capillaries provides excellent chemical resistance and optical properties, and easy wettability modification for flow manipulation. The flexible combination of the flow-control modules with 3D-printed positioning grooves and connection fasteners enables creation of versatile microfluidic systems for generating various higher-order multiple emulsions. The simple and reversible connection of the flow-control modules also allows easy disassembly of the microfluidic systems for further scale-up and functionalization. We demonstrate the scalability and controllability of flow manipulation by creating microfluidic systems from flexible assembly of flow-control modules for controllable generation of multiple emulsions from double emulsions to quadruple emulsions. Meanwhile, the flexible flow manipulation in the flow-control module provides advanced functions for improved control of the drop size, and for controllable generation of drops containing distinct components within multiple emulsions to extend the emulsion structure. Such modular microfluidic systems provide flexibility and versatility to flexibly manipulate micro-flows for enhanced and extended applications.

In situ fabrication of a temperature- and ethanol-responsive smart membrane in a microchip.

Here we report a simple and versatile strategy for the in situ fabrication of nanogel-containing smart membranes in microchannels of microchips. The fabrication approach is demonstrated by the in situ formation of a chitosan membrane containing poly(N-isopropylacrylamide) (PNIPAM) nanogels in a microchannel of a microchip. The PNIPAM nanogels, that allow temperature- and ethanol-responsive swelling-shrinking volume transitions, serve as smart nanovalves for controlling the diffusional permeability of solutes across the membrane. Such self-regulation of the membrane permeability is investigated by using fluorescein isothiocyanate (FITC) as a tracer molecule. This approach provides a promising strategy for the in situ fabrication of versatile nanogel-containing smart membranes within microchips via simply changing the functional nanogels for developing micro-scale detectors, sensors, separators and controlled release systems.

Wetting-induced coalescence of nanoliter drops as microreactors in microfluidics.

Controllable one-to-one coalescence of surfactant-stabilized nanoliter water drops is successfully achieved from wetting-induced drop engulfing in microfluidics by surrounding one of the drops with a thin layer of immiscible wetting fluid. This wetting layer can spread over the other drop to drain away the liquid film between the two drops, thereby inducing coalescence. This innovative approach is totally spontaneous and highly potential in a myriad of fields, such as quantitative analysis, microreaction, and high-throughput injection. To demonstrate this potential, we successfully perform the drop-coalescence-triggered microreaction in microchannels for pH indicator and syntheses of functional materials including micro- and nanoparticles.

Monodisperse and fast-responsive poly(N-isopropylacrylamide) microgels with open-celled porous structure.

A simple and efficient method is developed to fabricate monodisperse and fast-responsive poly(N-isopropylacrylamide) (PNIPAM) microgels with open-celled porous structure. First, numerous fine oil droplets are fabricated by homogeneous emulsification method and are then evenly dispersed inside monodisperse PNIPAM microgels as porogens via the combination of microfluidic emulsification and UV-initiated polymerization methods. Subsequently, the embedded fine oil droplets inside the PNIPAM microgels are squeezed out upon stimuli-induced rapid volume shrinkage of the microgels; as a result, a spongelike open-celled porous structure is formed inside the PNIPAM microgels. The open-celled porous structure provides numerous interconnected free channels for the water transferring convectively inward or outward during the volume phase transition process of PNIPAM microgels; therefore, the response rates of the PNIPAM microgels with open-celled porous structure are much faster than that of the normal ones in both thermo-responsive shrinking and swelling processes. Because of the fast-responsive characteristics, the microgels with open-celled porous structure will provide ever better performances in their myriad applications, such as microsensors, microactuators, microvalves, and so on.

Wetting-induced formation of controllable monodisperse multiple emulsions in microfluidics.

Multiple emulsions, which are widely applied in a myriad of fields because of their unique ability to encapsulate and protect active ingredients, are typically produced by sequential drop-formations and drop-encapsulations using shear-induced emulsification. Here we report a qualitatively novel method of creating highly controlled multiple emulsions from lower-order emulsions. By carefully controlling the interfacial energies, we adjust the spreading coefficients between different phases to cause drops of one fluid to completely engulf other drops of immiscible fluids; as a result multiple emulsions are directly formed by simply putting preformed lower-order emulsion drops together. Our approach has highly controllable flexibility. We demonstrate this in preparation of both double and triple emulsions with a controlled number of inner drops and precisely adjusted shell thicknesses including ultra-thin shells. Moreover, this controllable drop-engulfing-drop approach has a high potential in further investigations and applications of microfluidics. Importantly, this innovative approach opens a window to exploit new phenomena occurring in fluids at the microscale level, which is of great significance for developing novel microfluidics.