Ung, W. L. ; Mutafopulos, K. ; Spink, P. ; Rambach, R. W. ; Franke, T. ; Weitz, D. A. Enhanced Surface Acoustic Wave Cell Sorting by 3D Microfluidic-Chip Design. Lab on a Chip 2017, 17, 4059-4069. Publisher's VersionAbstract
We demonstrate an acoustic wave driven microfluidic cell sorter that combines advantages of multilayer device fabrication with planar surface acoustic wave excitation. We harness the strong vertical component of the refracted acoustic wave to enhance cell actuation by using an asymmetric flow field to increase cell deflection. Precise control of the 3-dimensional flow is realized by topographical structures implemented on the top of the microchannel. We experimentally quantify the effect of the structure dimensions and acoustic parameter. The design attains cell sorting rates and purities approaching those of state of the art fluorescence-activated cell sorters with all the advantages of microfluidic cell sorting.
Chaudhuri, M. ; Allahyarov, E. ; Löwen, H. ; Egelhaaf, S. U. ; Weitz, D. A. Triple Junction at the Triple Point Resolved on the Individual Particle Level. Phys. Rev. Lett. 2017, 119, 128001. Publisher's VersionAbstract

At the triple point of a repulsive screened Coulomb system, a fcc crystal, a bcc crystal, and a fluid phase coexist. At their intersection, these three phases form a liquid groove, the triple junction. Using confocal microscopy, we resolve the triple junction on a single-particle level in a model system of charged PMMA colloids in a nonpolar solvent. The groove is found to be extremely deep and the incommensurate solid-solid interface to be very broad. Thermal fluctuations hence appear to dominate the solid-solid interface. This indicates a very low interfacial energy. The fcc-bcc interfacial energy is quantitatively determined based on Young’s equation and, indeed, it is only about 1.3 times higher than the fcc-fluid interfacial energy close to the triple point.

    Zhao, C. - X. ; Chen, D. ; Hui, Y. ; Weitz, D. A. ; Middelberg, A. P. J. Controlled Generation of Ultrathin-Shell Double Emulsions and Studies on Their Stability. ChemPhysChem 2017, 18, 1393–1399. Publisher's VersionAbstract
    Double emulsions with a hierarchical core-shell structure have great potential in various applications, but their broad use is limited by their instability. To improve stability, water-in-oil-in-water (W/O/W) emulsions with an ultrathin oil layer of several hundred nanometres were produced by using a microcapillary device. The effects of various parameters on the generation of ultrathin-shell double emulsions and their droplet size were investigated, including the proper combinations of inner, middle and outer phases, flow rates and surfactants. The surfactant in the middle oil phase was found to be critical for the formation of the ultrathin-shell double emulsions. Furthermore, the stability of these double emulsions can be notably improved by increasing the concentration of the surfactant, and they can be stable for months. This opens up new opportunities for their future applications in cosmetics, foods and pharmaceuticals.
    Beroz, F. ; Jawerth, L. M. ; Münster, S. ; Weitz, D. A. ; Broedersz, C. P. ; Wingreen, N. S. Physical limits to biomechanical sensing in disordered fibre networks. Nature communications 2017, 8 16096. Publisher's VersionAbstract

    Cells actively probe and respond to the stiffness of their surroundings. Since mechanosensory cells in connective tissue are surrounded by a disordered network of biopolymers, their in vivo mechanical environment can be extremely heterogeneous. Here we investigate how this heterogeneity impacts mechanosensing by modelling the cell as an idealized local stiffness sensor inside a disordered fibre network. For all types of networks we study, including experimentally-imaged collagen and fibrin architectures, we find that measurements applied at different points yield a strikingly broad range of local stiffnesses, spanning roughly two decades. We verify via simulations and scaling arguments that this broad range of local stiffnesses is a generic property of disordered fibre networks. Finally, we show that to obtain optimal, reliable estimates of global tissue stiffness, a cell must adjust its size, shape, and position to integrate multiple stiffness measurements over extended regions of space.

    Kong, L. ; Amstad, E. ; Hai, M. ; Ke, X. ; Chen, D. ; Zhao, C. - X. ; Weitz, D. A. Biocompatible microcapsules with a water core templated from single emulsions. Chinese Chemical Letters 2017, 28, 1897-1900. Publisher's VersionAbstract

    Biocompatible microcapsules with a water core are widely used to encapsulate hydrophilic actives. Here, a facile method to fabricate monodisperse biocompatible microcapsules with a water core in large quantity is reported. Microfluidic technology is utilized to emulsify the inner aqueous phase containing the shell polymer into monodisperse drops in the outer oil phase. As the cosolvent in the inner aqueous phase diffuses into the outer oil phase, the solubility of the shell polymer decreases, which eventually precipitates. Since the shell polymer, shellac, contains both hydrophilic and hydrophobic groups, it tends to wet both the inner aqueous phase and the outer oil phase, thus forming a solid shell at the periphery of the drop. We show that the diffusion rate of hydrophilic molecules encapsulated in the water core decreases as their molecular weight increases and the property of the microcapsules could further be modified by polyelectrolyte multilayer coating. These microcapsules are fabricated using FDA-approved polymer and non-toxic solvents and are of great use in drugs, cosmetics and foods.

    Fan, J. ; Kim, S. - H. ; Chen, Z. ; Zhou, S. ; Amstad, E. ; Lin, T. ; Weitz, D. A. Creation of Faceted Polyhedral Microgels from Compressed Emulsions. Small 2017, 13, 1701256. Publisher's VersionAbstract
    Compressed monodisperse emulsions in confined space exhibit highly ordered structures. The influence of the volume fraction and the confinement geometry on the organized structures is investigated and the mechanism by which structural transition occurs is studied. Based on the understanding of ordering behavior of compressed emulsions, a simple and high‐throughput method to fabricate monodisperse polyhedral microgels using the emulsions as the template is developed. By controlling the geometry of the confined spaces, a variety of shapes such as hexagonal prism, Fejes Toth honeycomb prism, truncated octahedron, pyritohedron, and truncated hexagonal trapezohedron are implemented. Moreover, the edge sharpness of each shape is controllable by adjusting the drop volume fraction. This design principle can be readily extended to other shapes and materials, and therefore provides a useful means to create polyhedral microparticles for both fundamental study and practical applications.
    Zhang, W. ; Abbaspourrad, A. ; Chen, D. ; Campbell, E. ; Zhao, H. ; Li, Y. ; Li, Q. ; Weitz, D. A. Osmotic Pressure Triggered Rapid Release of Encapsulated Enzymes with Enhanced Activity. Advanced Functional Materials 2017, 27, 1700975. Publisher's VersionAbstract
    In this study, a single‐step microfluidic approach is reported for encapsulation of enzymes within microcapsules with ultrathin polymeric shell for controlled release triggered by an osmotic shock. Using a glass capillary microfluidic device, monodisperse water‐in‐oil‐in‐water double emulsion droplets are fabricated with enzymes in the core and an ultrathin middle oil layer that solidifies to produce a consolidated inert polymeric shell with a thickness of a few tens to hundreds of nanometers. Through careful design of microcapsule membranes, a high percentage of cargo release, over 90%, is achieved, which is triggered by osmotic shock when using poly(methyl methacrylate) as the shell material. Moreover, it is demonstrated that compared to free enzymes, the encapsulated enzyme activity is maintained well for as long as 47 days at room temperature. This study not only extends industrial applications of enzymes, but also offers new opportunities for encapsulation of a wide range of sensitive molecules and biomolecules that can be controllably released upon applying osmotic shock.
    Amstad, E. ; Chen, X. ; Eggersdorfer, M. ; Cohen, N. ; Kodger, T. E. ; Ren, C. L. ; Weitz, D. A. Parallelization of microfluidic flow-focusing devices. Physical Review E 2017, 95, 043105. Publisher's VersionAbstract

    Microfluidic flow-focusing devices offer excellent control over fluid flow, enabling formation of drops with a narrow size distribution. However, the throughput of microfluidic flow-focusing devices is limited and scale-up through operation of multiple drop makers in parallel often compromises the robustness of their operation. We demonstrate that parallelization is facilitated if the outer phase is injected from the direction opposite to that of the inner phase, because the fluid injection flow rate, where the drop formation transitions from the squeezing into the dripping regime, is shifted towards higher values.

      Kodger, T. E. ; Lu, P. J. ; Wiseman, G. R. ; Weitz, D. A. Stable, fluorescent PMMA particles for long-term observation of slow colloidal dynamics. Langmuir 2017, 33, 6382–6389. Publisher's VersionAbstract

      Suspensions of solid micron-scale colloidal particles in liquid solvents are a foundational model system used to explore a wide range of phase transitions, including crystallization, gelation, spinodal decomposition, and the glass transition. One of the most commonly used systems for these investigations is the fluorescent spherical particles of polymethylmethacrylate (PMMA) suspended in a mixture of nonpolar solvents that match the density and the refractive index of the particles to minimize sedimentation and scattering. However, the particles can swell in these solvents, changing their size and density, and may leak the fluorescent dye over days to weeks; this constrains the exploration of slow and kinetically limited processes, such as near-boundary phase separation or the glass transition. In this paper, we produce PMMA colloidal particles that employ polymerizable and photostable cyanine-based fluorescent monomers spanning the range of visible wavelengths and a polymeric stabilizer prepared from polydimethylsiloxane, PDMS-graft-PMMA. Using microcalorimetry, we characterize the thermodynamics of an accelerated equilibration process for these dispersions in the buoyancy- and refractive-index-matching solvents. We use confocal differential dynamic microscopy to demonstrate that they behave as hard spheres. The suspended particles are stable for months to years, maintaining fixed particle size and density, and do not leak dye. Thus, these particles enable longer term experiments than may have been possible earlier; we demonstrate this by observing spinodal decomposition in a mixture of these particles with a depletant polymer in the microgravity environment of the International Space Station. Using fluorescence microscopy, we observe coarsening over several months and measure the growth of the characteristic length scale to be a fraction of a picometer per second; this rate is among the slowest observed in a phase-separating system. Our protocols should facilitate the synthesis of a variety of particles.

      Haliburton, J. R. ; Kim, S. C. ; Clark, I. C. ; Sperling, R. A. ; Weitz, D. A. ; Abate, A. R. Efficient extraction of oil from droplet microfluidic emulsions. Biomicrofluidics 2017, 11, 034111. Publisher's VersionAbstract

      Droplet microfluidic techniques can perform large numbers of single molecule and cell reactions but often require controlled, periodic flow to merge, split, and sort droplets. Here, we describe a simple method to convert aperiodic flows into periodic ones. Using an oil extraction module, we efficiently remove oil from emulsions to readjust the droplet volume fraction, velocity, and packing, producing periodic flows. The extractor acts as a universal adaptor to connect microfluidic modules that do not operate under identical flow conditions, such as droplet generators, incubators, and merger devices.

      Prakadan, S. M. ; Shalek, A. K. ; Weitz, D. A. Scaling by shrinking: empowering single-cell'omics' with microfluidic devices. Nature Reviews Genetics 2017, 18, 345–361. Publisher's VersionAbstract

      Recent advances in cellular profiling have demonstrated substantial heterogeneity in the behaviour of cells once deemed 'identical', challenging fundamental notions of cell 'type' and 'state'. Not surprisingly, these findings have elicited substantial interest in deeply characterizing the diversity, interrelationships and plasticity among cellular phenotypes. To explore these questions, experimental platforms are needed that can extensively and controllably profile many individual cells. Here, microfluidic structures — whether valve-, droplet- or nanowell-based — have an important role because they can facilitate easy capture and processing of single cells and their components, reducing labour and costs relative to conventional plate-based methods while also improving consistency. In this article, we review the current state-of-the-art methodologies with respect to microfluidics for mammalian single-cell 'omics' and discuss challenges and future opportunities.

      Qin, Y. ; Hu, Y. ; Koehler, S. A. ; Cai, L. ; Wen, J. ; Tan, X. ; Xu, W. L. ; Sheng, Q. ; Hou, X. ; Xue, J. ; et al. Ultrafast Nanofiltration through Large-Area Single-layered Graphene Membranes. ACS Applied Materials & Interfaces 2017, 9 9239–9244. Publisher's VersionAbstract

      Perforated single-layered graphene has demonstrated selectivity and flux that is orders of magnitude greater than state-of-the-art polymer membranes. However, only individual graphene sheets with sizes up to tens of micrometers have been successfully fabricated for pressurized permeation studies. Scaling-up and reinforcement of these atomic membranes with minimum cracks and pinholes remains a major hurdle for practical applications. We develop a large-area in situ, phase-inversion casting technique to create 63 cm2 high-quality single-layered perforated graphene membranes for ultrafast nanofiltration that can operate at pressures up to 50 bar. This result demonstrates the feasibility of our technique for creating robust large-area, high quality, single-layered graphene and its potential use as a pressurized nanofiltration membrane.

      He, Y. ; Battat, S. ; Fan, J. ; Abbaspourrad, A. ; Weitz, D. A. Preparation of microparticles through co-flowing of partially miscible liquids. Chemical Engineering Journal 2017, 320, 144-150. Publisher's VersionAbstract

      Monodisperse microparticles find applications in a wide range of areas. Generation of such particles of several microns in size at a large scale is a difficult task. This paper reports a proof of concept using a microfluidic device that coaxially flows partially miscible liquids for the production of microparticles. The approach makes use of both the physical forces of the process and the chemical properties of the liquid systems that allows for careful control of the mixing and droplet formation processes. Initial results show that, with this approach, particles with a reasonably narrow size distribution can be produced and liquid miscibility can be used as an additional avenue to manipulate the mean particle size and morphology.

        Eggersdorfer, M. L. ; Koren, V. ; Stolovicki, E. ; Amstad, E. ; Weitz, D. A. Rapid Production of Submicron Drug Substance Particles by Supersonic Spray Drying. Crystal Growth & Design 2017, 17, 2046–2053. Publisher's VersionAbstract

        Many newly developed active pharmaceutical ingredients (APIs) are poorly soluble in water and thus have a dissolution-limited bioavailability. The bioavailability of Biopharmaceutical Classification System (BCS) class II APIs increases if they dissolve faster; this can be achieved by increasing their surface-to-volume ratio, for example, through formulation as submicron particles. In this paper, we develop a supersonic spray dryer that enables rapid synthesis of submicron-sized APIs at room temperature. Dispersing gas is accelerated to supersonic velocities in the divergent portion of a de Laval nozzle. The API solution is directly injected in the divergent portion and fully nebulized by impinging high velocity gas and pressure gradients across shocks at the exit of the nozzle. In such a device, we produce crystalline danazol particles with a Sauter mean diameter as small as 188 nm at a production rate up to 200 mg/h. The smallest particles with the narrowest size distributions are formed in overexpanded flows with a shock front close to the nozzle exit. Moreover, we demonstrate the scalability up to 1500 mg/h by increasing the danazol solution concentration; in this case, the Sauter mean diameter of the spray-dried particles increases to 772 nm.

        Yissachar, N. ; Zhou, Y. ; Ung, L. ; Lai, N. Y. ; Mohan, J. F. ; Ehrlicher, A. ; Weitz, D. A. ; Kasper, D. L. ; Chiu, I. M. ; Mathis, D. ; et al. An Intestinal Organ Culture System Uncovers a Role for the Nervous System in Microbe-Immune Crosstalk. Cell 2017, 168, 1135–1148. Publisher's VersionAbstract

        Investigation of host-environment interactions in the gut would benefit from a culture system that maintained tissue architecture yet allowed tight experimental control. We devised a microfabricated organ culture system that viably preserves the normal multicellular composition of the mouse intestine, with luminal flow to control perturbations (e.g., microbes, drugs). It enables studying short-term responses of diverse gut components (immune, neuronal, etc.). We focused on the early response to bacteria that induce either Th17 or RORg+ T-regulatory (Treg) cells in vivo. Transcriptional responses partially reproduced in vivo signatures, but these microbes elicited diametrically opposite changes in expression of a neuronal-specific gene set, notably nociceptive neuropeptides. We demonstrated activation of sensory neurons by microbes, correlating with RORg+ Treg induction. Colonic RORg+ Treg frequencies increased in mice lacking TAC1 neuropeptide precursor and decreased in capsaicin-diet fed mice. Thus, differential engagement of the enteric nervous system may partake in bifurcating pro- or anti-inflammatory responses to microbes.

        Qu, X. ; Zhang, H. ; Chen, H. ; Aldalbahi, A. ; Li, L. ; Tian, Y. ; Weitz, D. A. ; Pei, H. Convection Driven Pull-Down Assays in Nanoliter Droplets using Scaffolded Aptamers. Analytical Chemistry 2017, 89, 3468–3473. Publisher's VersionAbstract

        One of the great challenges in cellular studies is to develop a rapid and biocompatible analytical tool for single-cell analysis. We report a rapid, DNA nanostructure-supported aptamer pull-down (DNaPull) assay under convective flux in a glass capillary for analyzing the contents of droplets with nano- or picoliter volumes. We have demonstrated that the scaffolded aptamer can greatly improve the efficiency of target molecules’ pull down. The convective flux allows complete reaction in <5 min, which is an 18-fold improvement compared to purely diffusive flux (traditional model of the stationary case). This established DNaPull assay can serve as a rapid and sensitive analytical platform for analyzing a variety of bioactive molecules, including small molecules [ATP, limit of detecton (LOD) of 1 μM], a drug (cocaine, LOD of 1 μM), and a biomarker (thrombin, LOD of 0.1 nM). Significantly, the designed microfluidic device compartmentalizes live cells into nanoliter-sized droplets to present single-cell samples. As a proof of concept, we demonstrated that cellular molecules (ATP) from a discrete number of HNE1 cells (zero to five cells) lysed inside nanoliter-sized droplets can be analyzed using our DNaPull assay, in which the intracellular ATP level was estimated to be ∼3.4 mM. Given the rapid assay feature and single-cell sample analysis ability, we believe that our analytical platform of convection-driven DNaPull in a glass capillary can provide a new paradigm in biosensor design and will be valuable for single-cell analysis.

        Sprakel, J. ; Zaccone, A. ; Spaepen, F. ; Schall, P. ; Weitz, D. A. Direct Observation of Entropic Stabilization of bcc Crystals Near Melting. Physical Review Letters 2017, 118, 088003. Publisher's VersionAbstract

        Crystals with low latent heat are predicted to melt from an entropically stabilized body-centered cubic symmetry. At this weakly first-order transition, strongly correlated fluctuations are expected to emerge, which could change the nature of the transition. Here we show how large fluctuations stabilize bcc crystals formed from charged colloids, giving rise to strongly power-law correlated heterogeneous dynamics. Moreover, we find that significant nonaffine particle displacements lead to a vanishing of the nonaffine shear modulus at the transition. We interpret these observations by reformulating the Born-Huang theory to account for nonaffinity, illustrating a scenario of ordered solids reaching a state where classical lattice dynamics fail.

          Amato, D. V. ; Lee, H. ; Werner, J. G. ; Weitz, D. A. ; Patton, D. L. Functional Microcapsules via Thiol- Ene Photopolymerization in Droplet-Based Microfluidics. ACS applied materials & interfaces 2017, 9 3288–3293. Publisher's VersionAbstract

          Thiol–ene chemistry was exploited in droplet-based microfluidics to fabricate advanced microcapsules with tunable encapsulation, degradation, and thermal properties. In addition, by utilizing the thiol–ene photopolymerization with tunable cross-link density, we demonstrate the importance of monomer conversion on the retention of omniphilic cargo in double emulsion templated microcapsules. Furthermore, we highlight the rapid cure kinetics afforded by thiol–ene chemistry in a continuous flow photopatterning device for hemispherical microparticle production.

          Kalinich, M. ; Bhan, I. ; Kwan, T. T. ; Miyamoto, D. T. ; Javaid, S. ; LiCausi, J. A. ; Milner, J. D. ; Hong, X. ; Goyal, L. ; Sil, S. ; et al. An RNA-based signature enables high specificity detection of circulating tumor cells in hepatocellular carcinoma. Proceedings of the National Academy of Sciences 2017, 114, 1123-1128. Publisher's VersionAbstract

          Circulating tumor cells (CTCs) are shed into the bloodstream by invasive cancers, but the difficulty inherent in identifying these rare cells by microscopy has precluded their routine use in monitoring or screening for cancer. We recently described a high-throughput microfluidic CTC-iChip, which efficiently depletes hematopoietic cells from blood specimens and enriches for CTCs with well-preserved RNA. Application of RNA-based digital PCR to detect CTC-derived signatures may thus enable highly accurate tissue lineage-based cancer detection in blood specimens. As proof of principle, we examined hepatocellular carcinoma (HCC), a cancer that is derived from liver cells bearing a unique gene expression profile. After identifying a digital signature of 10 liver-specific transcripts, we used a cross-validated logistic regression model to identify the presence of HCC-derived CTCs in nine of 16 (56%) untreated patients with HCC versus one of 31 (3%) patients with nonmalignant liver disease at risk for developing HCC (P < 0.0001). Positive CTC scores declined in treated patients: Nine of 32 (28%) patients receiving therapy and only one of 15 (7%) patients who had undergone curative-intent ablation, surgery, or liver transplantation were positive. RNA-based digital CTC scoring was not correlated with the standard HCC serum protein marker alpha fetoprotein (P = 0.57). Modeling the sequential use of these two orthogonal markers for liver cancer screening in patients with high-risk cirrhosis generates positive and negative predictive values of 80% and 86%, respectively. Thus, digital RNA quantitation constitutes a sensitive and specific CTC readout, enabling high-throughput clinical applications, such as noninvasive screening for HCC in populations where viral hepatitis and cirrhosis are prevalent.

          Amstad, E. ; Spaepen, F. ; Brenner, M. P. ; Weitz, D. A. The microfluidic nebulator: production of sub-micrometer sized airborne drops. Lab on a Chip 2017, 17, 1475-1480. Publisher's VersionAbstract

          Many powders employed in the food and pharmaceutical industries are produced through spray drying because it is a cost efficient process that offers control over the particle size. However, most commercially available spray-driers cannot produce drops with diameters below 1 μm, limiting the size of spray-dried particles to values above 300 nm. We recently developed a microfluidic spray-drier that can form much smaller drops than commercially available spray-driers. This is achieved through a two-step process: first, the microfluidic spray-drier operates in the dripping regime to form 100 μm diameter primary drops in air and, second, subjects them to high shear stresses due to supersonic flow of air to break them into many much smaller secondary drops. In this paper, we describe the two essential steps required to form sub-μm diameter airborne drops inside microfluidic channels. We investigate the influence of the device geometry on the ability to operate the microfluidic spray-drier in the dripping regime. Moreover, we describe how these primary drops are nebulized into many secondary drops that are much smaller than the smallest dimension of the spray-drier channels.