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.

      Huang, X. ; Eggersdorfer, M. ; Wu, J. ; Zhao, C. - X. ; Xu, Z. ; Chen, D. ; Weitz, D. A. Collective generation of milliemulsions by step-emulsification. RSC Advances 2017, 7 14932–14938. Publisher's VersionAbstract

      Emulsification is a key step in many processes for the production and functionalization of dispersed liquid systems. Here, we report a versatile and robust device that generates monodisperse milliemulsions by step-emulsification. In contrast to the conventional design in which each channel is physically separated, we use a shallow plateau sandwiched between two parallel glass strips to connect all channels in a microcapillary film (MCF) before emerging in a deep reservoir. Because of the open plateau that connects different channels, the flow tips from neighboring channels may get immediately in contact with each other; this interaction may lead to the relative movement and deformation of the flow tips, to repulsion or even coalescence, enabling droplet generations from different channels to synchronize. By simply tuning the interaction, we achieve Janus droplets, drops of fluids mixed at different ratios and mixed drops of different compositions. The in situ generation of droplets with excellent control is essential for various applications.

      Ding, R. ; 丁睿骅, ; Ung, W. L. ; Heyman, J. A. ; Weitz, D. A. Sensitive and predictable separation of microfluidic droplets by size using in-line passive filter. Biomicrofluidics 2017, 11, 014114. Publisher's VersionAbstract
      Active manipulation of droplets is crucial in droplet microfluidics. However, droplet polydispersity decreases the accuracy of active manipulation. We develop a microfluidic “droplet filter” that accurately separates droplets by size. The droplet filter has a sharp size cutoff and is capable of distinguishing droplets differing in volume by 20%. A simple model explains the behavior of the droplets as they pass through the filter. We show application of the filter in improving dielectric sorting efficiency.
      Eggersdorfer, M. L. ; Zheng, W. ; Nawar, S. ; Mercandetti, C. ; Ofner, A. ; Leibacher, I. ; Koehler, S. ; Weitz, D. A. Tandem emulsification for high-throughput production of double emulsions. Lab on a Chip 2017, 17, 936–942. Publisher's VersionAbstract

      Core–shell double emulsions produced using microfluidic methods with controlled structural parameters exhibit great potential in a wide range of applications, but the low production rate of microfluidic methods hinders the exploitation of the capabilities of microfluidics to produce double emulsions with well-defined features. A major obstacle towards the scaled-up production of core–shell double emulsions is the difficulty of achieving robust spatially controlled wettability in integrated microfluidic devices. Here, we use tandem emulsification, a two-step process with microfluidic devices, to scale up the production. With this method, single emulsions are generated in a first device and are re-injected directly into a second device to form uniform double emulsions. We demonstrate the application of tandem emulsification for scalable core–shell emulsion production with both integrated flow focusing and millipede devices and obtain emulsions of which over 90% are single-core monodisperse double emulsion drops. With both mechanisms, the shell thickness can be controlled, so that shells as thin as 3 μm are obtained for emulsions 50 μm in radius.

      Xie, X. ; Zhang, W. ; Abbaspourrad, A. ; Ahn, J. ; Bader, A. ; Bose, S. ; Vegas, A. ; Lin, J. ; Tao, J. ; Hang, T. ; et al. Microfluidic Fabrication of Colloidal Nanomaterials-encapsulated Microcapsules for Biomolecular Sensing. Nano Letters 2017, 17, 2015-2020. Publisher's VersionAbstract

      Implantable sensors that detect biomarkers in vivo are critical for early disease diagnostics. Although many colloidal nanomaterials have been developed into optical sensors to detect biomolecules in vitro, their application in vivo as implantable sensors is hindered by potential migration or clearance from the implantation site. One potential solution is incorporating colloidal nanosensors in hydrogel scaffold prior to implantation. However, direct contact between the nanosensors and hydrogel matrix has the potential to disrupt sensor performance. Here, we develop a hollow-microcapsule-based sensing platform that protects colloidal nanosensors from direct contact with hydrogel matrix. Using microfluidics, colloidal nanosensors were encapsulated in polyethylene glycol microcapsules with liquid cores. The microcapsules selectively trap the nanosensors within the core while allowing free diffusion of smaller molecules such as glucose and heparin. Glucose-responsive quantum dots or gold nanorods or heparin-responsive gold nanorods were each encapsulated. Microcapsules loaded with these sensors showed responsive optical signals in the presence of target biomolecules (glucose or heparin). Furthermore, these microcapsules can be immobilized into biocompatible hydrogel as implantable devices for biomolecular sensing. This technique offers new opportunities to extend the utility of colloidal nanosensors from solution-based detection to implantable device-based detection.

      Liu, D. ; Zhang, H. ; Cito, S. ; Fan, J. ; Mäkilä, E. M. ; Salonen, J. J. ; Hirvonen, J. ; Sikanen, T. M. ; Weitz, D. A. ; Santos, H. A. Core/Shell Nanocomposites Produced by Superfast Sequential Microfluidic Nanoprecipitation. Nano Letters 2017, 17, 606-614. Publisher's VersionAbstract

      Although a number of techniques exist for generating structured organic nanocomposites, it is still challenging to fabricate them in a controllable, yet universal and scalable manner. In this work, a microfluidic platform, exploiting superfast (milliseconds) time intervals between sequential nanoprecipitation processes, has been developed for high-throughput production of structured core/shell nanocomposites. The extremely short time interval between the sequential nanoprecipitation processes, facilitated by the multiplexed microfluidic design, allows us to solve the instability issues of nanocomposite cores without using any stabilizers. Beyond high throughput production rate (∼700 g/day on a single device), the generated core/shell nanocomposites harness the inherent ultrahigh drug loading degree and enhanced payload dissolution kinetics of drug nanocrystals and the controlled drug release from polymer-based nanoparticles.

      Hati, A. G. ; Bassett, D. C. ; Ribe, J. M. ; Sikorski, P. ; Weitz, D. A. ; Stokke, B. T. Versatile, cell and chip friendly method to gel alginate in microfluidic devices. Lab on a Chip 2016, 16, 3718-3727. Publisher's VersionAbstract

      Alginate is used extensively in microfluidic devices to produce discrete beads or fibres at the microscale. Such structures may be used to encapsulate sensitive cargoes such as cells and biomolecules. On chip gelation of alginate represents a significant challenge since gelling kinetics or physicochemical conditions are not biocompatible. Here we present a new method that offers a hitherto unprecedented level of control over the gelling kinetics and pH applied to the encapsulation of a variety of cells in both bead and fibre geometries. This versatile approach proved straightforward to adjust to achieve appropriate solution conditions required for implementation in microfluidic devices and resulted in highly reliable device operation and very high viability of several different encapsulated cell types for prolonged periods. We believe this method offers a paradigm shift in alginate gelling technology for application in microfluidics.

      Keita, E. ; Kodger, T. E. ; Faure, P. ; Rodts, S. ; Weitz, D. A. ; Coussot, P. Water retention against drying with soft-particle suspensions in porous media. Physical Review E 2016, 94, 033104.Abstract

      Polymers suspended in granular packings have a significant impact on water retention, which is important for soil irrigation and the curing of building materials. Whereas the drying rate remains constant during a long period for pure water due to capillary flow providing liquid water to the evaporating surface, we show that it is not the case for a suspension made of soft polymeric particles called microgels: The drying rate decreases immediately and significantly. By measuring the spatial water saturation and concentration of suspended particles with magnetic resonance imaging, we can explain these original trends and model the process. In low-viscosity fluids, the accumulation of particles at the free surface induces a recession of the air-liquid interface. A simple model, assuming particle transport and accumulation below the sample free surface, is able to reproduce our observations without any fitting parameters. The high viscosity of the microgel suspension inhibits flow towards the free surface and a drying front appears. We show that water vapor diffusion over a defined and increasing length sets the drying rate. These results and model allow for better controlling the drying and water retention in granular porous materials.

        Yunker, P. J. ; Asahara, H. ; Hung, K. - C. ; Landry, C. ; Arriaga, L. R. ; Akartuna, I. ; Heyman, J. ; Chong, S. ; Weitz, D. A. One-pot system for synthesis, assembly, and display of functional single-span membrane proteins on oil–water interfaces. Proceedings of the National Academy of Sciences 2016, 113, 608–613. Publisher's VersionAbstract

        Single-span membrane proteins (ssMPs) represent approximately one-half of all membrane proteins and play important roles in cellular communications. However, like all membrane proteins, ssMPs are prone to misfolding and aggregation because of the hydrophobicity of transmembrane helices, making them difficult to study using common aqueous solution-based approaches. Detergents and membrane mimetics can solubilize membrane proteins but do not always result in proper folding and functionality. Here, we use cell-free protein synthesis in the presence of oil drops to create a one-pot system for the synthesis, assembly, and display of functional ssMPs. Our studies suggest that oil drops prevent aggregation of some in vitro-synthesized ssMPs by allowing these ssMPs to localize on oil surfaces. We speculate that oil drops may provide a hydrophobic interior for cotranslational insertion of the transmembrane helices and a fluidic surface for proper assembly and display of the ectodomains. These functionalized oil drop surfaces could mimic cell surfaces and allow ssMPs to interact with cell surface receptors under an environment closest to cell–cell communication. Using this approach, we showed that apoptosis-inducing human transmembrane proteins, FasL and TRAIL, synthesized and displayed on oil drops induce apoptosis of cultured tumor cells. In addition, we take advantage of hydrophobic interactions of transmembrane helices to manipulate the assembly of ssMPs and create artificial clusters on oil drop surfaces. Thus, by coupling protein synthesis with self-assembly at the water–oil interface, we create a platform that can use recombinant ssMPs to communicate with cells.

        Zhao, C. - X. ; Chen, D. ; Hui, Y. ; Weitz, D. A. ; Middelberg, A. P. J. Stable Ultrathin-Shell Double Emulsions for Controlled Release. ChemPhysChem 2016, 17, 1553–1556. Publisher's VersionAbstract

        Double emulsions are normally considered as metastable systems and this limit in stability restricts their applications. To enhance their stability, the outer shell can be converted into a mechanically strong layer, for example, a polymeric layer, thus allowing improved performance. This conversion can be problematic for food and drug applications, as a toxic solvent is needed to dissolve the polymer in the middle phase and a high temperature is required to remove the solvent. This process can also be highly complex, for example, involving UV initiation of polymeric monomer crosslinking. In this study, we report the formation of biocompatible, water-in-oil-in-water (W/O/W) double emulsions with an ultrathin layer of fish oil. We demonstrate their application for the encapsulation and controlled release of small hydrophilic molecules. Without a trigger, the double emulsions remained stable for months, and the release of small molecules was extremely slow. In contrast, rapid release was achieved by osmolarity shock, leading to complete release within 2 h. This work demonstrates the significant potential of double emulsions, and provides new insights into their stability and practical applications.

        Khavari, A. ; Nydén, M. ; Weitz, D. A. ; Ehrlicher, A. J. Composite alginate gels for tunable cellular microenvironment mechanics. Scientific reports 2016, 6 30854. Publisher's VersionAbstract

        The mechanics of the cellular microenvironment can be as critical as biochemistry in directing cell behavior. Many commonly utilized materials derived from extra-cellular-matrix create excellent scaffolds for cell growth, however, evaluating the relative mechanical and biochemical effects independently in 3D environments has been difficult in frequently used biopolymer matrices. Here we present 3D sodium alginate hydrogel microenvironments over a physiological range of stiffness (E = 1.85 to 5.29 kPa), with and without RGD binding sites or collagen fibers. We use confocal microscopy to measure the growth of multi-cellular aggregates (MCAs), of increasing metastatic potential in different elastic moduli of hydrogels, with and without binding factors. We find that the hydrogel stiffness regulates the growth and morphology of these cell clusters; MCAs grow larger and faster in the more rigid environments similar to cancerous breast tissue (E = 4–12 kPa) as compared to healthy tissue (E = 0.4–2 kpa). Adding binding factors from collagen and RGD peptides increases growth rates, and change maximum MCA sizes. These findings demonstrate the utility of these independently tunable mechanical/biochemistry gels, and that mechanical confinement in stiffer microenvironments may increase cell proliferation.

        Wang, X. ; Koehler, S. A. ; Wilking, J. N. ; Sinha, N. N. ; Cabeen, M. T. ; Srinivasan, S. ; Seminara, A. ; Rubinstein, S. ; Sun, Q. ; Brenner, M. P. ; et al. Probing phenotypic growth in expanding Bacillus subtilis biofilms. Applied microbiology and biotechnology 2016, 100, 4607–4615. Publisher's VersionAbstract

        We develop an optical imaging technique for spatially and temporally tracking biofilm growth and the distribution of the main phenotypes of a Bacillus subtilis strain with a triple-fluorescent reporter for motility, matrix production, and sporulation. We develop a calibration procedure for determining the biofilm thickness from the transmission images, which is based on Beer-Lambert’s law and involves cross-sectioning of biofilms. To obtain the phenotype distribution, we assume a linear relationship between the number of cells and their fluorescence and determine the best combination of calibration coefficients that matches the total number of cells for all three phenotypes and with the total number of cells from the transmission images. Based on this analysis, we resolve the composition of the biofilm in terms of motile, matrix-producing, sporulating cells and low-fluorescent materials which includes matrix and cells that are dead or have low fluorescent gene expression. We take advantage of the circular growth to make kymograph plots of all three phenotypes and the dominant phenotype in terms of radial distance and time. To visualize the nonlocal character of biofilm growth, we also make kymographs using the local colonization time. Our technique is suitable for real-time, noninvasive, quantitative studies of the growth and phenotype distribution of biofilms which are either exposed to different conditions such as biocides, nutrient depletion, dehydration, or waste accumulation.