Droplet-based microfluidics provides an excellent platform for high-throughput biological assays. Each droplet serves as a reaction vessel with a volume as small as a few picolitres. This is an important technology for a high variety of applications. However this technology is restricted to homogeneous assays as it is very difficult to wash reagents from the reaction vessel. To help overcome this limitation, we introduce a method to effectively dilute the content of a droplet while retaining the high throughput. We use electrocoalescence to merge the parent drop with a much larger drop containing only solvent, thereby increasing the volume of the drop by as much as a factor of 14. Three T-junctions then break the larger drop into eight smaller droplets. This dilution and break-up process can be repeated, thus leading to many drops comparable in size to the original one but with much lower concentration of reagents. The system is fully integrated in a PDMS device. To demonstrate its power, we perform a labelling reaction at the surface of the cells by coencapsulating yeast cells expressing S6 peptide tags with the enzyme SFP synthase and the fluorescent substrate CoA 488. After reaction, the droplets are diluted twice using the system and the intensity of their fluorescence is measured. This noise reduction method enables us to more easily distinguish the fluorescence at the surface of a single cell from the fluorescent background inside the droplet.
We measure the elastic properties of composite cytoskeletal networks consisting of cross-linked actin filaments and microtubules. We show that even a small concentration of microtubules leads to dramatic and qualitative changes in the non-linear elastic properties of the actin filament networks. Specifically, we find that microtubules promote non-linear stiffening of F-actin networks because they are much stiffer than actin filaments and therefore suppress non-uniform strain. This finding may be relevant for interpretation of the mechanical behavior of cells, while also suggesting a new way to reinforce the non-linear elasticity of semiflexible polymer materials.
Digital microfluidic devices allow the manipulation of droplets between two parallel electrodes. These electrodes can act as mirrors generating a micro-cavity, which can be exploited for a droplet dye-laser. Three representative laser-dyes with emission wavelengths spanning the whole visible spectrum are chosen to show the applicability of this concept. Sub-microlitre droplets of laser-dye solution are moved in and out of a lasing site on-chip to down-convert the UV-excitation light into blue, green and red laser-pulses.
A facile method for preparing highly monodisperse, sub-micrometre conjugated polymer particles is reported. The particles are prepared through emulsification of a conjugated polymer solution on a microfluidic chip followed by solvent evaporation. The particle size is tuned between 150 nm to 2 mu m, by controlling the polymer concentration.
Long-term storage and controlled release of multiple components while avoiding cross-contamination have potentially important applications for pharmaceuticals and cosmetics. Polymersomes are very promising delivery vehicles but cannot be used to encapsulate multiple independent components and release them in a controlled manner. Here, we report a microfluidic approach to produce multiple polymersomes, or polymersomes-in-polymersome by design, enabling encapsulation and programmed release of multiple components. Monodisperse polymersomes are prepared from templates of double-emulsion drops, which in turn are injected as the innermost phase to form the second level of double-emulsion drops, producing double polymersomes. Using the same strategy, higher-order polymersomes are also prepared. In addition, incorporation of hydrophobic homopolymer into the different bilayers of the multiple polymersomes enables controlled and sequential dissociation of the different bilayer membranes in a programmed fashion. The high encapsulation efficiency of this microfluidic approach, as well as its programmability and the biocompatibility of the materials used to form the polymersomes, will provide new opportunities for practical delivery systems of multiple components.
The crucial early stages of amyloid growth, in which normally soluble proteins are converted into fibrillar nanostructures, are challenging to study using conventional techniques yet are critical to the protein aggregation phenomena implicated in many common pathologies. As with all nucleation and growth phenomena, it is difficult to track individual nuclei in traditional macroscopic experiments, which probe the overall temporal evolution of the sample, but do not yield detailed information on the primary nucleation step as they mix independent stochastic events into an ensemble measurement. To overcome this limitation, we have developed microdroplet assays enabling us to detect single primary nucleation events and to monitor their subsequent spatial as well as temporal evolution, both of which we find to be determined by secondary nucleation phenomena. By deforming the droplets to high aspect ratio, we visualize in real-time propagating waves of protein assembly emanating from discrete primary nucleation sites. We show that, in contrast to classical gelation phenomena, the primary nucleation step is characterized by a striking dependence on system size, and the filamentous protein self-assembly process involves a highly nonuniform spatial distribution of aggregates. These findings deviate markedly from the current picture of amyloid growth and uncover a general driving force, originating from confinement, which, together with biological quality control mechanisms, helps proteins remain soluble and therefore functional in nature.
We use a microfluidic device to prepare monodisperse amphiphilic particles in the shape of a crescent-moon and use these particles to stabilize oil droplets in water. The microfluidic device is comprised of a tapered capillary in a theta (theta) shape that injects two oil phases into water in a single receiving capillary. One oil is a fluorocarbon, while the second is a photocurable monomer, which partially wets the first oil drop; silica colloids in the monomer migrate and adsorb to the interface with water but do not protrude into the oil interface. Upon UV-induced polymerization, solid particles with the shape of a crescent moon are formed; removal of fluorocarbon oil yields amphiphilic particles due to the selective adsorption of silica colloids. The resultant amphiphilic microparticles can be used to stabilize oil drops in a mixture of water and ethanol; if they are packed to sufficient surface density on the interface of the oil drop, they become immobilized, preventing direct contact between neighboring drops, thereby providing the stability.
We introduce an emulsification technique that creates monodisperse double-emulsion drops with a core-shell geometry having an ultra-thin wall as a middle layer. We create a biphasic flow in a microfluidic capillary device by forming a sheath flow consisting of a thin layer of a fluid with high affinity to the capillary wall flowing along the inner wall of the capillary, surrounding the innermost fluid. This creates double-emulsion drops, using a single-step emulsification, having a very thin fluid shell. If the shell is solidified, its thickness can be small as a hundred nanometres or even less. Despite the small thickness of this shell, these structures are nevertheless very stable, giving them great potential for encapsulation. We demonstrate this by creating biodegradable microcapsules of poly(lactic acid) with a shell thickness of a few tens of nanometres, which are potentially useful for encapsulation and delivery of drugs, cosmetics, and nutrients.
Mechanical stresses elicit cellular reactions mediated by chemical signals. Defective responses to forces underlie human medical disorders(1-4) such as cardiac failure(5) and pulmonary injury(6). The actin cytoskeleton's connectivity enables it to transmit forces rapidly over large distances(7), implicating it in these physiological and pathological responses. Despite detailed knowledge of the cytoskeletal structure, the specific molecular switches that convert mechanical stimuli into chemical signals have remained elusive. Here we identify the actin-binding protein filamin A (FLNA)(8,9) as a central mechanotransduction element of the cytoskeleton. We reconstituted a minimal system consisting of actin filaments, FLNA and two FLNA-binding partners: the cytoplasmic tail of beta-integrin, and FilGAP. Integrins form an essential mechanical linkage between extracellular and intracellular environments, with beta-integrin tails connecting to the actin cytoskeleton by binding directly to filamin(4). FilGAP is an FLNA-binding GTPase-activating protein specific for RAC, which in vivo regulates cell spreading and bleb formation(10). Using fluorescence loss after photoconversion, a novel, high-speed alternative to fluorescence recovery after photobleaching(11), we demonstrate that both externally imposed bulk shear and myosin-II-driven forces differentially regulate the binding of these partners to FLNA. Consistent with structural predictions, strain increases beta-integrin binding to FLNA, whereas it causes FilGAP to dissociate from FLNA, providing a direct and specific molecular basis for cellular mechanotransduction. These results identify a molecular mechanotransduction element within the actin cytoskeleton, revealing that mechanical strain of key proteins regulates the binding of signalling molecules.
We show how attractive interactions dramatically influence emulsion rheology. Unlike the repulsive case, attractive emulsions below random close packing, phi(RCP), can form soft gel-like elastic solids. However, above phi(RCP), attractive and repulsive emulsions have similar elasticities. Such compressed attractive emulsions undergo an additional shear-driven relaxation process during yielding. Our results suggest that attractive emulsions begin to yield at weak points through the breakage of bonds, and, above phi(RCP), also undergo droplet configurational rearrangements.
This study presents a simple microfluidic approach to the rapid fabrication of complex-shaped microfibers (e.g., single hollow, double hollow, and microbelt), with highly uniform structures, based on a combination of the spontaneous formation of polymeric jet streams and in situ photopolymerization. Two laminar flows of a photocurable fluid and a liquid template (nonpolymerizing fluid) spontaneously form jet streams in equilibrium states in microfluidic channels because of the minimization of the interfacial energy between the two fluids. The formation of the jet streams strongly depends on the spreading coefficients and the evolution time along the downstream of the microfluidic system. Thus, the simple control of the spreading coefficients can guide microfibers into various shapes. The sizes of the core and shell of the hollow fibers can also be readily manipulated by the flow rates of the polymerizing fluid and the liquid template phase. Asymmetric hollow fibers can also be produced in different evolutionary states in the microfluidic system. The microfluidic approach shown here represents a significant step toward the easy fabrication of microfibers with readily controllable structures and geometries. We anticipate that this novel fabrication approach and the prediction method based on spreading coefficients presented in this work can be applied to produce a wide variety of functional microfibrous materials.
We report the controlled breakup of double emulsion droplets as they flow through an orifice of a tapered nozzle. The results are summarized in a phase diagram in terms of the droplet-to-orifice diameter ratio and the capillary number. We identify a flow regime where the inner aqueous phase is released.
We demonstrate a microfluidic method to first generate double emulsion droplets containing two different inner drops, and to then control the internal coalescence of the encapsulated drops. The advantages of the core-coalescence method are illustrated by fabricating high viscosity particles and by demonstrating the dissolution of cell membranes.
Collective cell migration in tissues occurs throughout embryonic development, during wound healing, and in cancerous tumor invasion, yet most detailed knowledge of cell migration comes from single-cell studies. As single cells migrate, the shape of the cell body fluctuates dramatically through cyclic processes of extension, adhesion, and retraction, accompanied by erratic changes in migration direction. Within confluent cell layers, such subcellular motions must be coupled between neighbors, yet the influence of these subcellular motions on collective migration is not known. Here we study motion within a confluent epithelial cell sheet, simultaneously measuring collective migration and subcellular motions, covering a broad range of length scales, time scales, and cell densities. At large length scales and time scales collective migration slows as cell density rises, yet the fastest cells move in large, multicell groups whose scale grows with increasing cell density. This behavior has an intriguing analogy to dynamic heterogeneities found in particulate systems as they become more crowded and approach a glass transition. In addition we find a diminishing self-diffusivity of short-wavelength motions within the cell layer, and growing peaks in the vibrational density of states associated with cooperative cell-shape fluctuations. Both of these observations are also intriguingly reminiscent of a glass transition. Thus, these results provide a broad and suggestive analogy between cell motion within a confluent layer and the dynamics of supercooled colloidal and molecular fluids approaching a glass transition.
Microfluidic devices can form emulsions in which the drops have an intricate, controlled structure; however, a challenge is that the droplets are produced slowly, typically only a few millilitres per hour. Here, we present a simple technique to increase the production rate. Using a large drop maker, we produce large drops at a fast volumetric rate; by splitting these drops several times in a splitting array, we create drops of the desired small size. The advantage of this over forming the small drops directly using a small drop maker is that the drops can be formed at much faster rates. This can be applied to the production of single and multiple emulsions.
In microfluidic devices, droplets are normally formed using T-junction or flow focus mechanisms. While both afford a high degree of control over drop formation, they are limited in maximum production rate by the jetting transition. Here, we introduce a new drop formation mechanism that is not limited by jetting, allowing much faster drop production.
We present a robust way to create multiple emulsions with controllable shell thicknesses that can vary over a wide range. We use a microfluidic device to create a coaxial jet of immiscible fluids; using a dripping instability, we break the jet into multiple emulsions. By controlling the thickness of each layer of the jet, we adjust the thicknesses of the shells of the multiple emulsions. The same method is also effective in creating monodisperse emulsions from fluids that cannot otherwise be controllably emulsified, such as, for example, viscoelastic fluids.
Monodisperse microscale drops formed with microfluidic devices are useful for encapsulating cells, microgel particles, or even additional drops. These techniques are thus useful for applications ranging from high-throughput biology to monodisperse particle and capsule synthesis, which require encapsulation of such objects. However, it is challenging to efficiently encapsulate the objects in all drops; often, the objects are encapsulated inefficiently, resulting in many improperly filled, unusable drops. Here, we describe a phenomenon that allows very efficient encapsulation. We use the inflow of the object to plug the drop maker nozzle; the continued injection of the outer phase pinches off a drop, thereby encapsulating the object; this yields precisely one object encapsulated per drop.
We present a simple method for creating monodisperse emulsions with microfluidic devices. Unlike conventional approaches that require bulky pumps, control computers, and expertise with device physics to operate devices, our method requires only the microfluidic device and a hand-operated syringe. The fluids needed for the emulsion are loaded into the device inlets, while the syringe is used to create a vacuum at the device outlet; this sucks the fluids through the channels, generating the drops. By controlling the hydrodynamic resistances of the channels using hydrodynamic resistors and valves, we are able to control the properties of the drops. This provides a simple and highly portable method for creating monodisperse emulsions. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3567093]