Publications by Year: 2016

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.

    Ofner, A. ; Moore, D. G. ; Rühs, P. A. ; Schwendimann, P. ; Eggersdorfer, M. ; Amstad, E. ; Weitz, D. A. ; Studart, A. R. High-Throughput Step Emulsification for the Production of Functional Materials Using a Glass Microfluidic Device. Macromolecular Chemistry and Physics 2016, 218, 1600472. Publisher's VersionAbstract

    High‐volume production of monodisperse droplets is of importance for industrial applications due to increased emulsion stability, precise control over droplet volumes, and the formation of periodic arranged structures. So far, parallelized microfluidic devices are limited by either their complicated channel geometry or by their chemically or thermally unstable embedding material. This study shows a scalable microfluidic step emulsification chip that enables production of monodisperse emulsions at a throughput of up to 25 mL h−1 in a glass device with 364 linearly parallelized droplet makers. The chemical and thermal stability of such a glass device allows for the preparation of a broad variety of functional particles and microdroplets by using any desired solvent together with nanoparticles, polymers, and hydrogels. Moreover, the microfluidic device can be stringently cleaned for nearly unlimited use and permits the alternating production of oil‐in‐water and water‐in‐oil emulsions. The combined high throughput, chemical and thermal stability offered by our device enables production of monodisperse functional materials for large‐scale applications.

    Maire, E. ; Redston, E. ; Persson Gulda, M. ; Weitz, D. A. ; Spaepen, F. Imaging grain boundary grooves in hard-sphere colloidal bicrystals. Phys. Rev. E 2016, 94, 042604. Publisher's VersionAbstract
    Colloidal particles were sedimented onto patterned glass slides to grow three-dimensional bicrystals with a controlled structure. Three types of symmetric tilt grain boundaries between close-packed face-centered-cubic crystals were produced: Σ5(100),Σ17(100), and Σ3(110). The structure of the crystals and their defects were visualized by confocal microscopy, and characterized by simple geometric measurements, including image difference, thresholding, and reprojection. This provided a quick and straightforward way to detect the regions in which the atoms are mobile. This atomic mobility was higher at the grain boundaries and close to the solid-liquid interface. This method was compared to the more conventional analysis based on the calculation of the local order parameter of the individual particles to identify the interface. This was used in turn to identify the presence of grooves at the grain-boundary–liquid triple junction for every type of grain boundary, except for the twin [Σ3(110)], for which no groove could be detected. Images of these grooves were processed, and the angle linking the grain boundary energy to the solid-liquid interfacial energy was measured. The resulting values of the grain boundary energy were compared to estimates based on the density deficit in the boundary.
    Zhang, L. ; Cai, L. - H. ; Lienemann, P. S. ; Rossow, T. ; Polenz, I. ; Vallmajo-Martin, Q. ; Ehrbar, M. ; Na, H. ; Mooney, D. J. ; Weitz, D. A. One-Step Microfluidic Fabrication of Polyelectrolyte Microcapsules in Aqueous Conditions for Protein Release. Angewandte Chemie 2016, 55, 13470-13474. Publisher's VersionAbstract

    We report a microfluidic approach for one‐step fabrication of polyelectrolyte microcapsules in aqueous conditions. Using two immiscible aqueous polymer solutions, we generate transient water‐in‐water‐in‐water double emulsion droplets and use them as templates to fabricate polyelectrolyte microcapsules. The capsule shell is formed by the complexation of oppositely charged polyelectrolytes at the immiscible interface. We find that attractive electrostatic interactions can significantly prolong the release of charged molecules. Moreover, we demonstrate the application of these microcapsules in encapsulation and release of proteins without impairing their biological activities. Our platform should benefit a wide range of applications that require encapsulation and sustained release of molecules in aqueous environments.

    Amstad, E. ; Chemama, M. ; Eggersdorfer, M. ; Arriaga, L. R. ; Brenner, M. P. ; Weitz, D. A. Robust scalable high throughput production of monodisperse drops. Lab on a Chip 2016, 16, 4163-4172. Publisher's VersionAbstract

    Monodisperse drops with diameters between 20 μm and 200 μm can be used to produce particles or capsules for many applications such as for cosmetics, food, and biotechnology. Drops composed of low viscosity fluids can be conveniently made using microfluidic devices. However, the throughput of microfluidic devices is limited and scale-up, achieved by increasing the number of devices run in parallel, can compromise the narrow drop-size distribution. In this paper, we present a microfluidic device, the millipede device, which forms drops through a static instability such that the fluid volume that is pinched off is the same every time a drop forms. As a result, the drops are highly monodisperse because their size is solely determined by the device geometry. This makes the operation of the device very robust. Therefore, the device can be scaled to a large number of nozzles operating simultaneously on the same chip; we demonstrate the operation of more than 500 nozzles on a single chip that produces up to 150 mL h−1 of highly monodisperse drops.

    Cowan, M. L. ; Page, J. H. ; Norisuye, T. ; Weitz, D. A. Dynamic sound scattering: Field fluctuation spectroscopy with singly scattered ultrasound in the near and far fields. The Journal of the Acoustical Society of America 2016, 140, 1992. Publisher's VersionAbstract

    Dynamic sound scattering (DSS) is a powerful acoustic technique for investigating the motion of particles or other inclusions inside an evolving medium. In DSS, this dynamic information is obtained by measuring the field autocorrelation function of the temporal fluctuations of singly scattered acoustic waves. The technique was initially introduced 15 years ago, but its technical aspects were not adequately discussed then. This paper addresses the need for a more complete account of the method by describing in detail two different implementations of this sound scattering technique, one of which is specifically adapted to a common experimental situation in ultrasonics. The technique is illustrated by the application of DSS to measure the mean square velocity fluctuations of particles in fluidized suspensions, as well as the dynamic velocity correlation length. By explaining the experimental and analytical methods involved in realizing the DSS technique in practice, the use of DSS will be facilitated for future studies of particulate suspension dynamics and particle properties over a wide range of particle sizes and concentrations, from millimeters down to nanometers, where the use of optical techniques is often limited by the opacity of the medium.

    Massenburg, S. S. ; Amstad, E. ; Weitz, D. A. Clogging in parallelized tapered microfluidic channels. Microfluidics and Nanofluidics 2016, 20, 1–5. Publisher's VersionAbstract

    Nearly all tubes and pores used to transport solids in fluids, such as arteries and filters, are subject to clogging. The length scales and geometries of these tubes are well defined. In spite of this knowledge, the collective clogging behavior of multiple tubes has not yet been connected to their shapes and sizes. We investigate the clogging behavior of ten parallel tubes, which we model with ten parallel tapered microchannels using poly(styrene) beads to induce clogging events. The clogging behavior depends on the channel geometry as well as the shear stress particles are subjected to. Although our microchannels model filters, our results can be applied to the clogging behavior of a broad range of applications such as the clogging in arteries, inkjets, or xylem in trees.

    Michaels, T. C. T. ; Dear, A. J. ; Kirkegaard, J. B. ; Saar, K. L. ; Weitz, D. A. ; Knowles, T. P. J. Fluctuations in the Kinetics of Linear Protein Self-Assembly. Physical Review Letters 2016, 116, 258103. Publisher's VersionAbstract

    Biological systems are characterized by compartmentalization from the subcellular to the tissue level, and thus reactions in small volumes are ubiquitous in living systems. Under such conditions, statistical number fluctuations, which are commonly negligible in bulk reactions, can become dominant and lead to stochastic behavior. We present here a stochastic model of protein filament formation in small volumes. We show that two principal regimes emerge for the system behavior, a small fluctuation regime close to bulk behavior and a large fluctuation regime characterized by single rare events. Our analysis shows that in both regimes the reaction lag-time scales inversely with the system volume, unlike in bulk. Finally, we use our stochastic model to connect data from small-volume microdroplet experiments of amyloid formation to bulk aggregation rates, and show that digital analysis of an ensemble of protein aggregation reactions taking place under microconfinement provides an accurate measure of the rate of primary nucleation of protein aggregates, a process that has been challenging to quantify from conventional bulk experiments.

      Amstad, E. ; Spaepen, F. A. ; Weitz, D. A. Stabilization of the Amorphous Structure of Spray-dried Drug Nanoparticles. The Journal of Physical Chemistry B 2016, 120, 9161–9165. Publisher's VersionAbstract

      The bioavailability of hydrophobic drugs strongly increases if they are formulated as amorphous materials because the solubility of the amorphous phase is much higher than that of the crystal. Moreover, the stability of these particles against crystallization during storage increases with decreasing particle size. Hence, it is advantageous to formulate poorly water soluble drugs as amorphous nanoparticles. The formulation of an amorphous structure is often difficult because many of these drugs have a high propensity to crystallize. This difficulty can be overcome if drugs are spray-dried using a microfluidic nebulator we recently developed. However, these nanoparticles agglomerate when they come in contact with each other, and this compromises the stability of their amorphous structure through crystallization. To improve their stability, we coat the nanoparticles with a sterically stabilizing polymer layer; this can be accomplished by co-spraying them with an excipient. However, this excipient must meet strict solubility limits, which severely limit the choice of polymers. Alternatively, the nanoparticles can be sterically stabilized by spraying them directly into a polymeric matrix; this enables a much wider choice of stabilizing polymers.

      Lee, H. ; Choi, C. - H. ; Abbaspourrad, A. ; Wesner, C. ; Caggioni, M. ; Zhu, T. ; Nawar, S. ; Weitz, D. A. Fluorocarbon Oil Reinforced Triple Emulsion Drops. Advanced Materials 2016, 28, 8425-8430. Publisher's VersionAbstract

      Fluorocarbon oil reinforced triple emulsion drops are prepared to encapsulate a broad range of polar and non‐polar cargoes in a single platform. In addition, it is demonstrated that the fluorocarbon oil within the emulsion drop acts as an effective diffusion barrier, as well as a non‐adhesive layer, enabling highly efficient encapsulation and retention of small molecules and active biomolecules in microcapsules.

      Mashaghi, S. ; Abbaspourrad, A. ; Weitz, D. A. ; van Oijen, A. M. Droplet microfluidics: A tool for biology, chemistry and nanotechnology. TrAC Trends in Analytical Chemistry 2016, 82, 118-125. Publisher's VersionAbstract
      The ability to perform laboratory operations on small scales using miniaturized devices provides numerous benefits, including reduced quantities of reagents and waste as well as increased portability and controllability of assays. These operations can involve reaction components in the solution phase and as a result, their miniaturization can be accomplished through microfluidic approaches. One such approach, droplet microfluidics, provides a high-throughput platform for a wide range of assays and approaches in chemistry, biology and nanotechnology. We highlight recent advances in the application of droplet microfluidics in chip-based technologies, such as single-cell analysis tools, small-scale cell cultures, in-droplet chemical synthesis, high-throughput drug screening, and nanodevice fabrication.
      Burger, J. A. ; Landau, D. A. ; Taylor-Weiner, A. ; Bozic, I. ; Zhang, H. ; Sarosiek, K. ; Wang, L. ; Stewart, C. ; Fan, J. ; Hoellenriegel, J. ; et al. Clonal evolution in patients with chronic lymphocytic leukaemia developing resistance to BTK inhibition. Nature communications 2016, 7 11589. Publisher's VersionAbstract

      Resistance to the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib has been attributed solely to mutations in BTK and related pathway molecules. Using whole-exome and deep-targeted sequencing, we dissect evolution of ibrutinib resistance in serial samples from five chronic lymphocytic leukaemia patients. In two patients, we detect BTK-C481S mutation or multiple PLCG2 mutations. The other three patients exhibit an expansion of clones harbouring del(8p) with additional driver mutations (EP300, MLL2 and EIF2A), with one patient developing trans-differentiation into CD19-negative histiocytic sarcoma. Using droplet-microfluidic technology and growth kinetic analyses, we demonstrate the presence of ibrutinib-resistant subclones and estimate subclone size before treatment initiation. Haploinsufficiency of TRAIL-R, a consequence of del(8p), results in TRAIL insensitivity, which may contribute to ibrutinib resistance. These findings demonstrate that the ibrutinib therapy favours selection and expansion of rare subclones already present before ibrutinib treatment, and provide insight into the heterogeneity of genetic changes associated with ibrutinib resistance.

      do Nascimento, D. F. ; Arriaga, L. R. ; Eggersdorfer, M. ; Ziblat, R. ; de Marques, M. F. V. ; Reynaud, F. ; Koehler, S. A. ; Weitz, D. A. Microfluidic Fabrication of Pluronic Vesicles with Controlled Permeability. Langmuir 2016, 32, 5350–5355. Publisher's VersionAbstract

      Block copolymers with a low hydrophilic-to-lipophilic balance form membranes that are highly permeable to hydrophilic molecules. Polymersomes with this type of membrane enable the controllable release of molecules without membrane rupture. However, these polymersomes are difficult to assemble because of their low hydrophobicity. Here, we report a microfluidic approach to the production of these polymersomes using double-emulsion drops with ultrathin shells as templates. The small thickness of the middle oil phase enables the attraction of the hydrophobic blocks of the polymers adsorbed at each of the oil/water interfaces of the double emulsions; this results in the dewetting of the oil from the surface of the innermost water drops of the double emulsions and the ultimate formation of the polymersome. This approach to polymersome fabrication enables control of the vesicle size and results in the efficient encapsulation of hydrophilic ingredients that can be released through the polymer membrane without membrane rupture. We apply our approach to the fabrication of Pluronic L121 vesicles and characterize the permeability of their membranes. Furthermore, we show that membrane permeability can be tuned by blending different Pluronic polymers. Our work thus describes a route to producing Pluronic vesicles that are useful for the controlled release of hydrophilic ingredients.

      Xu, Z. ; Huang, X. ; Wang, P. ; Wang, H. ; Weitz, D. A. Optimization and development of a universal flow-based microfluidic gradient generator. Microfluidics and Nanofluidics 2016, 20, 1–10. Publisher's VersionAbstract

      Generation of concentration gradients of reactive molecules is of fundamental importance for many applications including biology, pharmaceutical and chemical engineering. By numerically simulating the flow behaviour, we reveal the possible factors that cause significant error in the gradients generated by the conventional universal microfluidic gradient generator (MGG) device reported previously. Based on these computational analyses, we optimize the geometrical design of the conventional 2-inlet MGG devices and improve the accuracy of the generated gradients. Moreover, we innovatively propose a 3-inlet MGG design showing desirable accuracy and versatility on creating various gradient profiles using the one single device. We further demonstrate our numerical simulation by fabricating the MGG devices by soft lithography and experimentally produce concentration gradients of diverse power functions. In general, the current study substantially improves the performance of universal MGG devices, which can serve as powerful tools for widespread applications in biology and chemistry.

      Choi, C. - H. ; Wang, H. ; Lee, H. ; Kim, J. H. ; Zhang, L. ; Mao, A. ; Mooney, D. J. ; Weitz, D. A. One-step generation of cell-laden microgels using double emulsion drops with a sacrificial ultra-thin oil shell. Lab on a Chip 2016, 16, 1549–1555. Publisher's VersionAbstract

      Cell-laden microgels with highly uniform sizes have significant potential in tissue engineering and cell therapy due to their capability to provide a physiologically relevant three-dimensional (3D) microenvironment for living cells. In this work, we present a simple and efficient microfluidic approach to produce monodisperse cell-laden microgels through the use of double emulsion drops with an ultra-thin oil shell as the sacrificial template. Specifically, the thin oil shell in double emulsion spontaneously dewets upon polymerization of the innermost precursor drop and subsequent transfer into an aqueous solution, resulting in direct dispersion of microgels in the aqueous phase. Compared to conventional single emulsion-based techniques for cell encapsulation, this one-step approach prevents prolonged exposure of cells to the oil phase, leading to high-throughput cell encapsulation in microgels without compromising the cell viability. Moreover, this approach allows us to culture cells within a 3D microgel which mimics the extracellular matrix, thus enabling long-term cell functionality. This microfluidic technique represents a significant step forward in high-throughput cell microencapsulation technology and offers a potentially viable option to produce cell-laden microgels for widespread applications in tissue engineering and cell therapies.