A reliable microfluidic platform for the generation of stable and monodisperse multistage drug delivery systems is reported. A glass-capillary flow-focusing droplet generation device was used to encapsulate thermally hydrocarbonized porous silicon (PSi) microparticles into the aqueous cores of double emulsion drops, yielding the formation of a multistage PSi-lipid vesicle. This composite system enables a large loading capacity for hydrophobic drugs.
Many types of bacteria form colonies that grow into physically robust and strongly adhesiveaggregates known as biofilms. A distinguishing characteristic of bacterial biofilms is an extracellular polymeric substance (EPS) matrix that encases the cells and provides physical integrity to the colony. The EPS matrix consists of a large amount of polysaccharide, as well as protein filaments, DNA and degraded cellular materials. The genetic pathways that control the transformation of a colony into a biofilm have been widely studied, and yield a spatiotemporal heterogeneity in EPS production. Spatial gradients in metabolites parallel this heterogeneity in EPS, but nutrient concentration as an underlying physiological initiator of EPS production has not been explored. Here, we study the role of nutrient depletion in EPS production in Bacillus subtilis biofilms. By monitoring simultaneously biofilm size and matrix production, we find that EPS production increases at a critical colony thickness that depends on the initial amount of carbon sources in the medium. Through studies of individual cells in liquid culture we find that EPS production can be triggered at the single-cell level by reducing nutrient concentration. To connect the single-cell assays with conditions in the biofilm, we calculate carbon concentration with a model for the reaction and diffusion of nutrients in the biofilm. This model predicts the relationship between the initial concentration of carbon and the thickness of the colony at the point of internal nutrient deprivation.
Cell-generated mechanical forces play a critical role during tissue morphogenesis and organ formation in the embryo. Little is known about how these forces shape embryonic organs, mainly because it has not been possible to measure cellular forces within developing three-dimensional (3D) tissues in vivo. We present a method to quantify cell-generated mechanical stresses exerted locally within living embryonic tissues, using fluorescent, cell-sized oil microdroplets with defined mechanical properties and coated with adhesion receptor ligands. After a droplet is introduced between cells in a tissue, local stresses are determined from droplet shape deformations, measured using fluorescence microscopy and computerized image analysis. Using this method, we quantified the anisotropic stresses generated by mammary epithelial cells cultured within 3D aggregates, and we confirmed that these stresses (3.4 nN [mu]m-2) are dependent on myosin II activity and are more than twofold larger than stresses generated by cells of embryonic tooth mesenchyme, either within cultured aggregates or in developing whole mouse mandibles.
Coalescence of two kinds of pre-processed droplets is necessary to perform chemical and biological assays in droplet-based microfluidics. However, a robust technique to accomplish this does not exist. Here we present a microfluidic device to synchronize the reinjection of two different kinds of droplets and coalesce them, using hydrostatic pressure in conjunction with a conventional syringe pump. We use a device consisting of two opposing T-junctions for reinjecting two kinds of droplets and control the flows of the droplets by applying gravity-driven hydrostatic pressure. The hydrostatic-pressure operation facilitates balancing the droplet reinjection rates and allows us to synchronize the reinjection. Furthermore, we present a simple but robust module to coalesce two droplets that sequentially come into the module, regardless of their arrival times. These re-injection and coalescence techniques might be used in lab-on-chip applications requiring droplets with controlled numbers of solid materials, which can be made by coalescing two pre-processed droplets that are formed and sorted in devices.
We present a microfluidic device that enables high throughput production of relatively monodisperse emulsion drops while controlling the average size. The device consists of a two-dimensional array of regularly-spaced posts. Large drops of a highly polydisperse crude emulsion are input into the device and are successively split by the posts, ultimately yielding a finer emulsion consisting of smaller, and much more monodisperse drops. The size distribution of the resultant emulsion depends only weakly on the viscosities of the input fluids and allows fluids of very high viscosities to be used. The average size and polydispersity of the drops depend strongly on the device geometry enabling both control and optimization. We use this device to produce drops of a highly viscous monomer solution and subsequently solidify them into polymeric microparticles. The production rate of these devices is similar to that achieved by membrane emulsification techniques, yet the control over the drop size is superior; thus these post-array microfluidic devices are potentially useful for industrial applications.
Colloidal particles are microscopic solid particles suspended in a fluid. Colloids are small enough that thermal energy drives their dynamics and ensures equilibration with the suspending fluid; they are also large enough that their positions and motions can be measured precisely using optical methods, such as light scattering and laser-scanning confocal fluorescence microscopy. Colloidal suspensions are a powerful model system for the study of other phenomena in condensed matter physics, where the collective phase behavior of the solid particles mimics that of other condensed systems. We review three classes of interacting colloidal particles, crystals, glasses, and gels, each of which represents fascinating properties of colloidal particles as well as a model for more general types of materials and their behavior.
We demonstrate controlled growth of face-centered cubic (FCC), monodisperse hard-sphere colloidal crystals by centrifugation at up to 3000g onto FCC (100) templates. Such rapid deposition rates often result in an amorphous sediment. Surprisingly, however, growth onto (100) templates results only in single crystals with few or no extended defects. By contrast, deposition onto flat, (111), or (110) templates causes rapid disordering to an amorphous sediment if the dimensionless flux (particle volume fraction x Peclet number) exceeds a critical value. This crystalline-to-amorphous crossover results from the degeneracy of possible stacking positions for these orientations. No such degeneracy exists for growth onto (100). After growth, extended defects can nucleate and grow only if the crystal exceeds a critical thickness that depends on the lattice misfit with the template spacing. The experimental observations of the density of misfit dislocations are accounted for by the Frank-van der Merwe theory, adapted for the depth-dependent variation of lattice spacing and elastic constants that results from the gravitational pressure.
A hallmark of biopolymer networks is their sensitivity to stress, reflected by pronounced nonlinear elastic stiffening. Here, we demonstrate a distinct dynamical nonlinearity in biopolymer networks consisting of filamentous actin cross-linked by alpha-actinin-4. Applied stress delays the onset of relaxation and flow, markedly enhancing gelation and extending the regime of solidlike behavior to much lower frequencies. We show that this macroscopic network response can be accounted for at the single molecule level by the increased binding affinity of the cross-linker under load, characteristic of catch-bond-like behavior. DOI: 10.1103/PhysRevLett.110.018103
We show that when a non-wetting fluid drains a stratified porous medium at sufficiently small capillary numbers Ca, it flows only through the coarsest stratum of the medium; by contrast, above a threshold Ca, the non-wetting fluid is also forced laterally, into part of the adjacent, finer strata. The spatial extent of this partial invasion increases with Ca. We quantitatively understand this behavior by balancing the stratum-scale viscous pressure driving the flow with the capillary pressure required to invade individual pores. Because geological formations are frequently stratified, we anticipate that our results will be relevant to a number of important applications, including understanding oil migration, preventing groundwater contamination, and sub-surface CO2 storage. Copyright (C) EPLA, 2013
In nonpolar solvents, surfactants stabilize charge through the formation of reverse micelles; this enables the dissociation of charge from the surfaces of particles, thereby charge-stabilizing particle suspensions. We investigate the dynamics of such charged particles by directly visualizing their motion across a microfluidic channel in response to an external electric field. The presence of the reverse micelles has a significant effect on particle motion: in a constant field, the particles initially move, then slow down exponentially, and eventually stop. This is due to the accumulation of reverse micelles at the channel walls, which screens the applied field, leading to the subsequent decay of the internal electric field. The time constant of decay depends on the electrical conductivity of the particle suspension and the width of the channel; this behavior is modeled as an equivalent RC circuit.
Nanoparticle colloidosomes, whose release can be switched on and off in response to a temperature change, are fabricated. Unlike in other systems, the switchable release does not require the colloidosome shell to deform; it instead occurs due to the adsorption or desorption of a block copolymer, dissolved in the core, at the inner surface of the colloidosome shell, concomitantly blocking or unblocking the pores in the shell. The colloidosomes are prepared using double emulsion templates produced by microfluidics, and are thus highly monodisperse; moreover, they are mechanically stable and consist of biocompatible components, making them suitable for the encapsulation, delivery, and release of a broad range of active materials.
We introduce a novel type of droplet generator that produces droplets of a volume set by the geometry of the droplet generator and not by the flow rates of the liquids. The generator consists of a classic T-junction with a bypass channel. This bypass directs the continuous fluid around the forming droplets, so that they can fill the space between the inlet of the dispersed phase and the exit of the bypass without breaking. Once filled, the dispersed phase blocks the exit of the bypass and is squeezed by the continuous fluid and broken off from the junction. We demonstrate the fixed-volume droplet generator for (i) the formation of monodisperse droplets from a source of varying flow rates, (ii) the formation of monodisperse droplets containing a gradation of solute concentration, and (iii) the parallel production of monodisperse droplets. (C) 2013 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4801637]
We present a new type of microcapsule programmed with a tunable active release mechanism. The capsules are triggered by a plasticizing stimulus that induces a phase change transition of the polymeric membrane from a solid to a fluidized form; thereafter, the cargo is actively driven out of the capsule through a defect at the capsule wall with controllable release kinetics. Tuning the degree of membrane fluidity by tailoring the amount of plasticizing stimulus present allows us to obtain temporal variation of the release kinetics from a subsecond abrupt burst release to a slow sustained release of encapsulant over many minutes. Moreover, we demonstrate tuning of the collective capsule triggering response by adjusting stimulus content, polymer molecular weight, and capsule membrane thickness. For this model system, we use a microfluidic approach to fabricate polystyrene capsules triggered by a toluene stimulus. However, this active release approach is general and is applicable to diverse polymeric capsule systems; this versatility is demonstrated by extension of our trigger-release scheme to capsules fabricated from a rubberlike block copolymer. The utility of our technique further enhances the potential of these active release capsules for practical application.
Chromatin structure and dynamics control all aspects of DNA biology yet are poorly understood, especially at large length scales. We developed an approach, displacement correlation spectroscopy based on time-resolved image correlation analysis, to map chromatin dynamics simultaneously across the whole nucleus in cultured human cells. This method revealed that chromatin movement was coherent across large regions (4-5 mu m) for several seconds. Regions of coherent motion extended beyond the boundaries of single-chromosome territories, suggesting elastic coupling of motion over length scales much larger than those of genes. These large-scale, coupled motions were ATP dependent and unidirectional for several seconds, perhaps accounting for ATP-dependent directed movement of single genes. Perturbation of major nuclear ATPases such as DNA polymerase, RNA polymerase II, and topoisomerase II eliminated micron-scale coherence, while causing rapid, local movement to increase; i.e., local motions accelerated but became uncoupled from their neighbors. We observe similar trends in chromatin dynamics upon inducing a direct DNA damage; thus we hypothesize that this may be due to DNA damage responses that physically relax chromatin and block long-distance communication of forces.
Modern confocal microscopes enable high-precision measurement in three dimensions by collecting stacks of 2D (x-y) images that can be assembled digitally into a 3D image. It is difficult, however, to ensure position accuracy, particularly along the optical (z) axis where scanning is performed by a different physical mechanism than in x-y. We describe a simple device to calibrate simultaneously the x, y, and z pixel-to-micrometer conversion factors for a confocal microscope. By taking a known 2D pattern and positioning it at a precise angle with respect to the microscope axes, we created a 3D reference standard. The device is straightforward to construct and easy to use. (C) 2013 American Institute of Physics. [http://dx.doi.org/10.1063/1.4776672]
Simultaneous encapsulation of multiple active substances in a single carrier is essential for therapeutic applications of synergistic combinations of drugs. However, traditional carrier systems often lack efficient encapsulation and release of incorporated substances, particularly when combinations of drugs must be released in concentrations of a prescribed ratio. We present a novel biodegradable core shell carrier system fabricated in a one-step, solvent-free process on a microfluidic chip; a hydrophilic active (doxorubicin hydrochloride) is encapsulated in the aqueous core, while a hydrophobic active (paclitaxel) is encapsulated in the solid shell. Particle size and composition can be precisely controlled, and core and shell can be individually loaded with very high efficiency. Drug-loaded particles can be dried and stored as a powder. We demonstrate the efficacy of this system through the simultaneous encapsulation and controlled release of two synergistic anticancer drugs using two cancer-derived value for encapsulation of other active cell lines. This solvent-free platform technology is also of high potential ingredients and chemical reagents.
We report a microfluidic approach to produce monodisperse pH-responsive microcapsules with precisely controlled release behavior. The solid microcapsule shells are composed of a biocompatible pH-responsive polymer and robustly encapsulate an active material; however, when exposed to a trigger pH, the shells degrade and ultimately release the microcapsule contents. We control the trigger pH by using polymers that dissolve at different pH values. We independently control the time at which the microcapsule contents are released by carefully controlling the shell thickness. Moreover, we independently control the rate at which the encapsulated contents are released by making hybrid shells composed of a mixture of a pH-responsive polymer and varying proportions of another, solid, pH-unresponsive polymer. This enables us to achieve monodisperse microcapsules that robustly encapsulate an active material, only releasing it when exposed to a desired pH, after a prescribed time delay, and at a prescribed rate.
Droplet-based microfluidic techniques can form and process micrometer scale droplets at thousands per second. Each droplet can house an individual biochemical reaction, allowing millions of reactions to be performed in minutes with small amounts of total reagent. This versatile approach has been used for engineering enzymes, quantifying concentrations of DNA in solution, and screening protein crystallization conditions. Here, we use it to read the sequences of DNA molecules with a FRET-based assay. Using probes of different sequences, we interrogate a target DNA molecule for polymorphisms. With a larger probe set, additional polymorphisms can be interrogated as well as targets of arbitrary sequence.
We report a parallelized capillary microfluidic device for enhanced production rate of monodisperse polymersomes. This device consists of four independent capillary microfluidic devices, operated in parallel; each device produces monodisperse water-in-oil-in-water (W/O/W) double-emulsion drops through a single-step emulsification. During generation of the double-emulsion drops, the innermost water drop is formed first and it triggers a breakup of the middle oil phase over wide range of flow rates; this enables robust and stable formation of the double-emulsion drops in all drop makers of the parallelized device. Double-emulsion drops are transformed to polymersomes through a dewetting of the amphiphile-laden middle oil phase on the surface of the innermost water drop, followed by the subsequent separation of the oil drop. Therefore, we can make polymersomes with a production rate enhanced by a factor given by the number of drop makers in the parallelized device.