Eggersdorfer, M. L. ; Seybold, H. ; Ofner, A. ; Weitz, D. A. ; Studart, A. R. Wetting controls of droplet formation in step emulsification. Proceedings of the National Academy of Sciences 2018, 115, 9479-9484. Publisher's VersionAbstract
The formation of droplets is ubiquitous in many natural and industrial processes and has reached an unprecedented level of control with the emergence of milli- and microfluidics. Although important insight into the mechanisms of droplet formation has been gained over the past decades, a sound understanding of the physics underlying this phenomenon and the effect of the fluid’s flow and wetting properties on the droplet size and production rate is still missing, especially for the widely applied method of step emulsification. In this work, we elucidate the physical controls of microdroplet formation in step emulsification by using the wetting of fluidic channels as a tunable parameter to explore a broad set of emulsification conditions. With the help of high-speed measurements, we unequivocally show that the final droplet pinch-off is triggered by a Rayleigh–Plateau-type instability. The droplet size, however, is not determined by the Rayleigh–Plateau breakup, but by the initial wetting regime, where the fluid’s contact angle plays a crucial role. We develop a physical theory for the wetting process, which closely describes our experimental measurements without invoking any free fit parameter. Our theory predicts the initiation of the Rayleigh–Plateau breakup and the transition from dripping to jetting as a function of the fluid’s contact angle. Additionally, the theory solves the conundrum why there is a minimal contact angle of α = 2π/3 = 120° for which droplets can form.
Rotem, A. ; Serohijos, A. W. R. ; Chang, C. B. ; Wolfe, J. T. ; Fischer, A. E. ; Mehoke, T. S. ; Zhang, H. ; Tao, Y. ; Ung, W. L. ; Choi, J. - M. ; et al. Evolution on the biophysical fitness landscape of an RNA virus. Mol. Biol. Evol. 2018, 35, 2390–2400. Publisher's VersionAbstract

Viral evolutionary pathways are determined by the fitness landscape, which maps viral genotype to fitness. However, a quantitative description of the landscape and the evolutionary forces on it remain elusive. Here, we apply a biophysical fitness model based on capsid folding stability and antibody binding affinity to predict the evolutionary pathway of norovirus escaping a neutralizing antibody. The model is validated by experimental evolution in bulk culture and in a drop-based microfluidics that propagates millions of independent small viral subpopulations. We demonstrate that along the axis of binding affinity, selection for escape variants and drift due to random mutations have the same direction, an atypical case in evolution. However, along folding stability, selection and drift are opposing forces whose balance is tuned by viral population size. Our results demonstrate that predictable epistatic tradeoffs between molecular traits of viral proteins shape viral evolution.

Qi, L. - Y. ; Shang, L. ; Chen, X. ; Ye, L. ; Zhang, W. ; Feng, P. ; Zou, W. ; Cao, N. ; Zhou, H. - H. ; Weitz, D. A. ; et al. A Versatile Strategy to Fabricate 3D Conductive Frameworks for Lithium Metal Anodes. Adv. Mater. Interfaces 2018, 5 1800807. Publisher's VersionAbstract
The suppression of lithium dendrite is critical to the realization of lithium metal batteries. 3D conductive framework, among different approaches, has shown very promising results in dendrite suppression. A novel cost‐effective and versatile dip‐coating method is presented here to make 3D conductive framework. Various substrates with different geometries are coated successfully with copper, including electrically insulating glass fiber (GF) or rice paper and conducting Ni foam. In particular, the as‐prepared copper coated GF shows promising results to serve as the lithium metal substrate by the electrochemical battery tests. The method significantly broadens the candidate materials database for 3D conductive framework to include all kinds of intrinsically insulating 3D substrates.
Werner, J. G. ; Deveney, B. T. ; Nawar, S. ; Weitz, D. A. Dynamic Microcapsules with Rapid and Reversible Permeability Switching. Adv. Funct. Mater. 2018, 28, 1803385. Publisher's VersionAbstract
Dynamic microcapsules are reported that exhibit shell membranes with fast and reversible changes in permeability in response to external stimuli. A hydrophobic anhydride monomer is employed in the thiol–ene polymerization as a disguised precursor for the acid‐containing shells; this enables the direct encapsulation of aqueous cargo in the liquid core using microfluidic fabrication of water‐in‐oil‐in‐water double emulsion drops. The poly(anhydride) shells hydrolyze in their aqueous environment without further chemical treatment, yielding cross‐linked poly(acid) microcapsules that exhibit trigger‐responsive and reversible property changes. The microcapsule shell can actively be switched numerous times between impermeable and permeable due to the exceptional mechanical properties of the thiol–ene network that prevent rupture or failure of the membrane, allowing it to withstand the mechanical stresses imposed on the capsule during the dynamic property changes. The permeability and molecular weight cutoff of the microcapsules can dynamically be controlled with triggers such as pH and ionic environment. The reversibly triggered changes in permeability of the shell exhibit a response time of seconds, enabling actively adjustable release profiles, as well as on‐demand capture, trapping, and release of cargo molecules with molecular selectivity and fast on‐off rates.
Montessori, A. ; Lauricella, M. ; Succi, S. ; Stolovicki, E. ; Weitz, D. Elucidating the mechanism of step emulsification. Phys. Rev. Fluids 2018, 3 072202. Publisher's VersionAbstract

Three-dimensional, time-dependent direct simulations of step emulsification microdevices highlight two essential mechanisms for droplet formation: first, the onset of an adverse pressure gradient driving a backflow of the continuous phase from the external reservoir to the microchannel, and second, the striction of the flowing jet which leads to its subsequent rupture. It is also shown that such a rupture is delayed and eventually suppressed by increasing the flow speed of the dispersed phase within the channel, due to the stabilizing effect of dynamic pressure. This suggests a new criterion for dripping-jetting transition, based on local values of the capillary and Weber numbers.

Werner, J. ; Nawar, S. ; Solovev, A. A. ; Weitz, D. A. Hydrogel Microcapsules with Dynamic pH-Responsive Properties from Methacrylic Anhydride. Macromolecules 2018, 51, 5798-5805. Publisher's VersionAbstract
Dynamic microcapsules are a highly sought-after class of encapsulant for advanced delivery applications with dynamically tunable release profiles, as actively manipulatable microreactors, or as selective microtraps for molecular separation and purification. Such dynamic microcapsules can only be realized with a nondestructive trigger-response mechanism that changes the permeability of the shell membrane reversibly, as found in hydrogels. However, the direct synthesis of a trigger-responsive hydrogel membrane around a water drop without the use of sacrificial templates remains elusive due to the incompatibility of the synthesis chemistry with aqueous emulsion processing. Here, we report on a facile approach to fabricate reversibly responsive hydrogel microcapsules utilizing reactive anhydride chemistry. Cross-linked and hydrophobic poly(methacrylic anhydride) microcapsules are obtained from microfluidic double emulsion drop templating that enables direct encapsulation of hydrophilic, water-suspended cargo within the aqueous core. Hydrolysis in aqueous environment yields microcapsules with a poly(acid) hydrogel shell that exhibit high mechanical and chemical stability for repeated cycling between its swollen and nonswollen states without rupture or fatigue. The permeability of the microcapsules is strongly dependent on the degree of swelling and hence can be actively and dynamically modified, enabling repeated capture, trap, and release of aqueous cargo over numerous cycles.
Li, W. ; Zhang, L. ; Ge, X. ; Xu, B. ; Zhang, W. ; Qu, L. ; Choi, C. - H. ; Xu, J. ; Zhang, A. ; Lee, H. ; et al. Microfluidic fabrication of microparticles for biomedical applications. Chem. Soc. Rev. 2018, 15, 5646-5683. Publisher's VersionAbstract
Droplet microfluidics offers exquisite control over the flows of multiple fluids in microscale, enabling fabrication of advanced microparticles with precisely tunable structures and compositions in a high throughput manner. The combination of these remarkable features with proper materials and fabrication methods has enabled high efficiency, direct encapsulation of actives in microparticles whose features and functionalities can be well controlled. These microparticles have great potential in a wide range of bio-related applications including drug delivery, cell-laden matrices, biosensors and even as artificial cells. In this review, we briefly summarize the materials, fabrication methods, and microparticle structures produced with droplet microfluidics. We also provide a comprehensive overview of their recent uses in biomedical applications. Finally, we discuss the existing challenges and perspectives to promote the future development of these engineered microparticles.
Zhang, H. ; Cui, N. ; Cai, Y. ; Lei, F. ; Weitz, D. A. Single-cell sequencing leads a new era of profiling transcriptomic landscape. Journal of Bio-X Research 2018, 1 2-6. Publisher's VersionAbstract
Understanding the complexity of biological systems requires a comprehensive analysis of their cell populations. Ideally, this should be done at the single cell level, because bulk analysis of the full population obscured many critical details due to artifacts introduced by averaging. However, this has been technically challenging due to the cumbersome procedure, low throughput, and high costs of performing analysis on a single-cell basis. Excitingly, technical improvements in single-cell RNA sequencing are making it economically practical to profile the transcriptomics of large populations of cells at the single-cell level, and have yielded numerous results that address important biological and medical questions. Further development of the technology and data analysis will significantly benefit the biomedical field by unraveling the function of individual cells in their microenvironments and modeling their transcriptional dynamics.
Liu, E. Y. ; Jung, S. ; Weitz, D. A. ; Yi, H. ; Choi, C. - H. High-throughput double emulsion-based microfluidic production of hydrogel microspheres with tunable chemical functionalities toward biomolecular conjugation. Lab Chip 2018, 18, 323–334. Publisher's VersionAbstract
Chemically functional hydrogel microspheres hold significant potential in a range of applications including biosensing, drug delivery, and tissue engineering due to their high degree of flexibility in imparting a range of functions. In this work, we present a simple, efficient, and high-throughput capillary microfluidic approach for controlled fabrication of monodisperse and chemically functional hydrogel microspheres via formation of double emulsion drops with an ultra-thin oil shell as a sacrificial template. This method utilizes spontaneous dewetting of the oil phase upon polymerization and transfer into aqueous solution, resulting in poly(ethylene glycol) (PEG)-based microspheres containing primary amines (chitosan, CS) or carboxylates (acrylic acid, AA) for chemical functionality. Simple fluorescent labelling of the as-prepared microspheres shows the presence of abundant, uniformly distributed and readily tunable functional groups throughout the microspheres. Furthermore, we show the utility of chitosan's primary amine as an efficient conjugation handle at physiological pH due to its low pKa by direct comparison with other primary amines. We also report the utility of these microspheres in biomolecular conjugation using model fluorescent proteins, R-phycoerythrin (R-PE) and green fluorescent protein (GFPuv), via tetrazine–trans-cyclooctene (Tz–TCO) ligation for CS-PEG microspheres and carbodiimide chemistry for AA-PEG microspheres, respectively. The results show rapid coupling of R-PE with the microspheres' functional groups with minimal non-specific adsorption. In-depth protein conjugation kinetics studies with our microspheres highlight the differences in reaction and diffusion of R-PE with CS-PEG and AA-PEG microspheres. Finally, we demonstrate orthogonal one-pot protein conjugation of R-PE and GFPuv with CS-PEG and AA-PEG microspheres via simple size-based encoding. Combined, these results represent a significant advancement in the rapid and reliable fabrication of monodisperse and chemically functional hydrogel microspheres with tunable properties.
Klepac, D. ; Kostkova, H. ; Petrova, S. ; Chytil, P. ; Etrych, T. ; Kereiche, S. ; Raska, I. ; Weitz, D. A. ; Filippov, S. K. Interaction of spin-labeled HPMA-based nanoparticles with human blood plasma proteins – the introduction of protein-corona-free polymer nanomedicine. Nanoscale 2018, 10, 6194–6204. Publisher's VersionAbstract
In this paper, we revised the current understanding of the protein corona that is created on the surface of nanoparticles in blood plasma after an intravenous injection. We have focused on nanoparticles that have a proven therapeutic outcome. These nanoparticles are based on two types of biocompatible amphiphilic copolymers based on N-(2-hydroxypropyl)methacrylamide (HPMA): a block copolymer, poly(ε-caprolactone) (PCL)-b-poly(HPMA), and a statistical HPMA copolymer bearing cholesterol moieties, which have been tested both in vitro and in vivo. We studied the interaction of nanoparticles with blood plasma and selected blood plasma proteins by electron paramagnetic resonance (EPR), isothermal titration calorimetry, dynamic light scattering, and cryo-transmission electron microscopy. The copolymers were labeled with TEMPO radicals at the end of hydrophobic PCL or along the hydrophilic HPMA chains to monitor changes in polymer chain dynamics caused by protein adsorption. By EPR and other methods, we were able to probe specific interactions between nanoparticles and blood proteins, specifically low- and high-density lipoproteins, immunoglobulin G, human serum albumin (HSA), and human plasma. It was found that individual proteins and plasma have very low binding affinity to nanoparticles. We observed no hard corona around HPMA-based nanoparticles; with the exception of HSA the proteins showed no detectable binding to the nanoparticles. Our study confirms that a classical “hard corona–soft corona” paradigm is not valid for all types of nanoparticles and each system has a unique protein corona that is determined by the nature of the NP material.
Anbari, A. ; Chien, H. - T. ; Datta, S. S. ; Deng, W. ; Weitz, D. A. ; Fan, J. Microfluidic Model Porous Media: Fabrication and Applications. Small 2018, 14, 1703575. Publisher's VersionAbstract
Complex fluid flow in porous media is ubiquitous in many natural and industrial processes. Direct visualization of the fluid structure and flow dynamics is critical for understanding and eventually manipulating these processes. However, the opacity of realistic porous media makes such visualization very challenging. Micromodels, microfluidic model porous media systems, have been developed to address this challenge. They provide a transparent interconnected porous network that enables the optical visualization of the complex fluid flow occurring inside at the pore scale. In this Review, the materials and fabrication methods to make micromodels, the main research activities that are conducted with micromodels and their applications in petroleum, geologic, and environmental engineering, as well as in the food and wood industries, are discussed. The potential applications of micromodels in other areas are also discussed and the key issues that should be addressed in the near future are proposed.
Choi, C. - H. ; Lee, H. ; Weitz, D. A. Rapid Patterning of PDMS Microfluidic Device Wettability Using Syringe-Vacuum-Induced Segmented Flow in Nonplanar Geometry. ACS Appl. Mater. Interfaces 2018, 10, 3170–3174. Publisher's VersionAbstract
We present a simple and rapid method to spatially pattern the surface wetting properties of PDMS microfluidic devices by layer-by-layer (LbL) deposition of polyelectrolytes using syringe-vacuum-induced segmented flow in nonplanar geometry. Our technique offers selective surface modification in microfluidic chips with multiple flow-focusing junctions, enabling production of monodisperse double- and triple-emulsion drops.
Wang, L. ; Xia, J. ; Li, J. ; Hagemann, T. L. ; Jones, J. R. ; Fraenkel, E. ; Weitz, D. A. ; Zhang, S. - C. ; Messing, A. ; Feany, M. B. Tissue and cellular rigidity and mechanosensitive signaling activation in Alexander disease. Nat. Commun. 2018, 9 1899. Publisher's VersionAbstract
Glial cells have increasingly been implicated as active participants in the pathogenesis of neurological diseases, but critical pathways and mechanisms controlling glial function and secondary non-cell autonomous neuronal injury remain incompletely defined. Here we use models of Alexander disease, a severe brain disorder caused by gain-of-function mutations in GFAP, to demonstrate that misregulation of GFAP leads to activation of a mechanosensitive signaling cascade characterized by activation of the Hippo pathway and consequent increased expression of A-type lamin. Importantly, we use genetics to verify a functional role for dysregulated mechanotransduction signaling in promoting behavioral abnormalities and non-cell autonomous neurodegeneration. Further, we take cell biological and biophysical approaches to suggest that brain tissue stiffness is increased in Alexander disease. Our findings implicate altered mechanotransduction signaling as a key pathological cascade driving neuronal dysfunction and neurodegeneration in Alexander disease, and possibly also in other brain disorders characterized by gliosis.
Zhang, L. ; Chen, K. ; Zhang, H. ; Pang, B. ; Choi, C. - H. ; Mao, A. S. ; Liao, H. ; Utech, S. ; Mooney, D. J. ; Wang, H. ; et al. Microfluidic Templated Multicompartment Microgels for 3D Encapsulation and Pairing of Single Cells. Small 2018, 14, 1-8. Publisher's VersionAbstract
Controlled encapsulation and pairing of single cells within a confined 3D matrix can enable the replication of the highly ordered cellular structure of human tissues. Microgels with independently controlled compartments that can encapsulate cells within separately confined hydrogel matrices would provide precise control over the route of pairing single cells. Here, a one‐step microfluidic method is presented to generate monodisperse multicompartment microgels that can be used as a 3D matrix to pair single cells in a highly biocompatible manner. A method is presented to induce microgels formation on chip, followed by direct extraction of the microgels from oil phase, thereby avoiding prolonged exposure of the microgels to the oil. It is further demonstrated that by entrapping stem cells with niche cells within separate but adjacent compartments of the microgels, it can create complex stem cell niche microenvironments in a controlled manner, which can serve as a useful tool for the study of cell–cell interactions. This microfluidic technique represents a significant step toward high‐throughput single cells encapsulation and pairing for the study of intercellular communications at single cell level, which is of significant importance for cell biology, stem cell therapy, and tissue engineering.
Atia, L. ; Bi, D. ; Sharma, Y. ; Mitchel, J. A. ; Gweon, B. ; Koehler, S. A. ; DeCamp, S. J. ; Lan, B. ; Kim, J. H. ; Hirsch, R. ; et al. Geometric constraints during epithelial jamming. Nature Physics 2018, 14, 613–620. Publisher's VersionAbstract
As an injury heals, an embryo develops or a carcinoma spreads, epithelial cells systematically change their shape. In each of these processes cell shape is studied extensively whereas variability of shape from cell to cell is regarded most often as biological noise. But where do cell shape and its variability come from? Here we report that cell shape and shape variability are mutually constrained through a relationship that is purely geometrical. That relationship is shown to govern processes as diverse as maturation of the pseudostratified bronchial epithelial layer cultured from non-asthmatic or asthmatic donors, and formation of the ventral furrow in the Drosophila embryo. Across these and other epithelial systems, shape variability collapses to a family of distributions that is common to all. That distribution, in turn, is accounted for by a mechanistic theory of cell–cell interaction, showing that cell shape becomes progressively less elongated and less variable as the layer becomes progressively more jammed. These findings suggest a connection between jamming and geometry that spans living organisms and inert jammed systems, and thus transcends system details. Although molecular events are needed for any complete theory of cell shape and cell packing, observations point to the hypothesis that jamming behaviour at larger scales of organization sets overriding geometric constraints.
Edery, Y. ; Berg, S. ; Weitz, D. Surfactant Variations in Porous Media Localize Capillary Instabilities during Haines Jumps. Physical Review Letters 2018, 120. Publisher's VersionAbstract
We use confocal microscopy to measure velocity and interfacial tension between a trapped wetting phase with a surfactant and a flowing, invading nonwetting phase in a porous medium. We relate interfacial tension variations at the fluid-fluid interface to surfactant concentration and show that these variations localize the destabilization of capillary forces and lead to rapid local invasion of the nonwetting fluid, resulting in a Haines jump. These spatial variations in surfactant concentration are caused by velocity variations at the fluid-fluid interfaces and lead to localization of the Haines jumps even in otherwise very uniform pore structure and pressure conditions. Our results provide new insight into the nature of Haines jumps, one of the most ubiquitous and important instabilities in flow in porous media.
Zhang, H. ; Zhu, Y. ; Qu, L. ; Wu, H. ; Kong, H. ; Yang, Z. ; Chen, D. ; Mäkilä, E. ; Salonen, J. ; Santos, H. A. ; et al. Gold nanorods conjugated porous silicon nanoparticles encapsulated in calcium alginate nano hydrogels using microemulsion templates. Nano Letters 2018, 18, 1448-1453. Publisher's VersionAbstract

Porous silicon nanoparticles (PSiNPs) and gold nanorods (AuNRs) can be used as biocompatible nanocarriers for delivery of therapeutics but undesired leakage makes them inefficient. By encapsulating the PSiNPs and AuNRs in a hydrogel shell, we create a biocompatible functional nanocarrier that enables sustained release of therapeutics. Here, we report the fabrication of AuNRs-conjugated PSi nanoparticles (AuNRsPSiNPs) through two-step chemical reaction for high-capacity loading of hydrophobic and hydrophilic therapeutics with photothermal property. Furthermore, using water-in-oil microemulsion templates, we encapsulate the AuNRsPSiNPs within a calcium alginate hydrogel nanoshell, creating a versatile biocompatible nanocarrier to codeliver therapeutics for biomedical applications. We find that the functionalized nanohydrogel effectively controls the release rate of the therapeutics while maintaining a high loading efficiency and tunable loading ratios. Notably, combinations of therapeutics coloaded in the functionalized nanohydrogels significantly enhance inhibition of multidrug resistance through synergism and promote faster cancer cell death when combined with photothermal therapy. Moreover, the AuNRs can mediate the conversion of near-infrared laser radiation into heat, increasing the release of therapeutics as well as thermally inducing cell damage to promote faster cancer cell death. Our AuNRsPSiNPs functionalized calcium alginate nanohydrogel holds great promise for photothermal combination therapy and other advanced biomedical applications.

Montessori, A. ; Lauricella, M. ; La Rocca, M. ; Succi, S. ; Stolovicki, E. ; Zibalt, R. ; Weitz, D. Regularized lattice Boltzmann multicomponent models for low capillary and Reynolds microfluidics flows. Computers & Fluids 2018, 167, 33-39. Publisher's VersionAbstract
We present a regularized version of the color gradient lattice Boltzmann (LB) scheme for the simulation of droplet formation in microfluidic devices of experimental relevance. The regularized version is shown to provide computationally efficient access to capillary number regimes relevant to droplet generation via microfluidic devices, such as flow-focusers and the more recent microfluidic step emulsifier devices.
Stolovicki, E. ; Ziblat, R. ; Weitz, D. A. Throughput enhancement of parallel step emulsifier devices by shear-free and efficient nozzle clearance. Lab on a Chip 2018, 18, 132-138. Publisher's VersionAbstract

Step emulsification is an attractive method for production of monodisperse drops. Its main advantage is the ability to parallelize many step emulsifier nozzles to achieve high production rates. However, step emulsification is sensitive to any obstructions at the nozzle exit. At high production rates, drops can accumulate at nozzle exits, disturb the formation of subsequent drops and impair monodispersity. As a result, parallelized step emulsifier devices typically do not work at maximum productivity. Here a design is introduced that parallelizes hundreds of step emulsifier nozzles, and effectively removes drops from the nozzle exits. The drop clearance is achieved by an open collecting channel, and is aided by buoyancy. Importantly, this clearance method avoids the use of a continuous phase flow for drop clearance and hence no shear is applied on the forming drops. The method works well for a wide range of drops, sizing from 30 to 1000 μm at production rates of 0.03 and 10 L per hour and achieved by 400 and 120 parallelized nozzles respectively.

Amstad, E. ; Weitz, D. A. Reply to the 'Comment on "Robust Scalable High Throughput Production of Monodisperse Drops. Lab on a Chip 2017, 17, 2332-2333. Publisher's VersionAbstract

This reply to the comment by Nakajima on our article that appeared in Lab on a Chip (E. Amstad, M. Chemama, M. Eggersdorfer, L. R. Arriaga, M. Brenner and D. A. Weitz, Lab Chip, 2016, 16, 4163–4172) highlights the differences between the microchannel step emulsification devices developed by the Nakajima group and the millipede device reported by us in Lab on a Chip.