Metagenomic studies suggest that only a small fraction of the viruses that exist in nature have been identified and studied. Characterization of unknown viral genomes is hindered by the many genomes populating any virus sample. A new method is reported that integrates drop‐based microfluidics and computational analysis to enable the purification of any single viral species from a complex mixed virus sample and the retrieval of complete genome sequences. By using this platform, the genome sequence of a 5243 bp dsDNA virus that was spiked into wastewater was retrieved with greater than 96 % sequence coverage and more than 99.8 % sequence identity. This method holds great potential for virus discovery since it allows enrichment and sequencing of previously undescribed viruses as well as known viruses.
Recombination is an important driver in the evolution of viruses and thus is key to understanding viral epidemics and improving strategies to prevent future outbreaks. Characterization of rare recombinant subpopulations remains technically challenging because of artifacts such as artificial recombinants, known as chimeras, and amplification bias. To overcome this, we have developed a high‐throughput microfluidic technique with a second verification step in order to amplify and sequence single recombinant viruses with high fidelity in picoliter drops. We obtained the first artifact‐free estimate of in vitro recombination rate between murine norovirus strains MNV‐1 and WU20 co‐infecting a cell (Prec=3.3×10−4±2×10−5) for a 1205 nt region. Our approach represents a time‐ and cost‐effective improvement over current methods, and can be adapted for genomic studies requiring artifact‐ and bias‐free selective amplification, such as microbial pathogens, or rare cancer cells.
Collagen is the main structural and load-bearing element of various connective tissues, where it forms the extracellular matrix that supports cells. It has long been known that collagenous tissues exhibit a highly nonlinear stress–strain relationship, although the origins of this nonlinearity remain unknown. Here, we show that the nonlinear stiffening of reconstituted type I collagen networks is controlled by the applied stress and that the network stiffness becomes surprisingly insensitive to network concentration. We demonstrate how a simple model for networks of elastic fibers can quantitatively account for the mechanics of reconstituted collagen networks. Our model points to the important role of normal stresses in determining the nonlinear shear elastic response, which can explain the approximate exponential relationship between stress and strain reported for collagenous tissues. This further suggests principles for the design of synthetic fiber networks with collagen-like properties, as well as a mechanism for the control of the mechanics of such networks.
Amorphous nanoparticles (a-NPs) have physicochemical properties distinctly different from those of the corresponding bulk crystals; for example, their solubility is much higher. However, many materials have a high propensity to crystallize and are difficult to formulate in an amorphous structure without stabilizers. We fabricated a microfluidic nebulator that can produce amorphous NPs from a wide range of materials, even including pure table salt (NaCl). By using supersonic air flow, the nebulator produces drops that are so small that they dry before crystal nuclei can form. The small size of the resulting spray-dried a-NPs limits the probability of crystal nucleation in any given particle during storage. The kinetic stability of the a-NPs—on the order of months—is advantageous for hydrophobic drug molecules.
Soft, solvent‐free poly(dimethylsiloxane) elastomers are fabricated by a one‐step process via crosslinking bottlebrush polymers. The bottlebrush architecture prevents the formation of entanglements, resulting in elastomers with precisely controllable low moduli from 1 to 100 kPa, below the lower limit of traditional elastomers; moreover, the solvent‐free nature enables their negligible adhesiveness compared to commercially available silicone products of similar stiffness.
We use droplet microfluidics to produce monodisperse elastomeric microbubbles consisting of gas encapsulated in a polydimethylsiloxane shell. These microbubbles withstand large, repeated deformations without rupture. We perform μN-scale compression tests on individual microbubbles and find their response to be highly dependent on the shell permeability; during deformation, the pressure inside impermeable microbubbles increases, resulting in an exponential increase in the applied force. Finite element models are used to interpret and extend these experimental results enabling the design and development of deformable microbubbles with a predictable mechanical response. Such microbubbles can be designed to repeatedly transit through the narrow constrictions found in a porous medium functioning as probes of the local pressure.
Knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale.
Recent development of liquid phase transmission electron microscopy (TEM) enables the study of specimens in wet ambient conditions within a liquid cell; however, direct structural observation of biological samples in their native solution using TEM is challenging since low-mass biomaterials embedded in a thick liquid layer of the host cell demonstrate low contrast. Furthermore, the integrity of delicate wet samples is easily compromised during typical sample preparation and TEM imaging. To overcome these limitations, we introduce a graphene liquid cell (GLC) using multilayer graphene sheets to reliably encapsulate and preserve biological samples in a liquid for TEM observation. We achieve nanometer scale spatial resolution with high contrast using low-dose TEM at room temperature, and we use the GLC to directly observe the structure of influenza viruses in their native buffer solution at room temperature. The GLC is further extended to investigate whole cells in wet conditions using TEM. We also demonstrate the potential of the GLC for correlative studies by TEM and fluorescence light microscopy imaging.
The importance of single-cell level data is increasingly appreciated, and significant advances in this direction have been made in recent years. Common to these technologies is the need to physically segregate individual cells into containers, such as wells or chambers of a micro-fluidics chip. High-throughput Single-Cell Labeling (Hi-SCL) in drops is a novel method that uses drop-based libraries of oligonucleotide barcodes to index individual cells in a population. The use of drops as containers, and a microfluidics platform to manipulate them en-masse, yields a highly scalable methodological framework. Once tagged, labeled molecules from different cells may be mixed without losing the cell-of-origin information. Here we demonstrate an application of the method for generating RNA-sequencing data for multiple individual cells within a population. Barcoded oligonucleotides are used to prime cDNA synthesis within drops. Barcoded cDNAs are then combined and subjected to second generation sequencing. The data are deconvoluted based on the barcodes, yielding single-cell mRNA expression data. In a proof-of-concept set of experiments we show that this method yields data comparable to other existing methods, but with unique potential for assaying very large numbers of cells.
The intracellular cytoskeleton is an active dynamic network of filaments and associated binding proteins that control key cellular properties, such as cell shape and mechanics. Due to the inherent complexity of the cell, reconstituted model systems have been successfully employed to gain an understanding of the fundamental physics governing cytoskeletal processes. Here, we review recent advances and key aspects of these reconstituted systems. We focus on the importance of assembly kinetics and dynamic arrest in determining network mechanics, and highlight novel emergent behavior occurring through interactions between cytoskeletal components in more complex networks incorporating multiple biopolymers and molecular motors.
In this study, a membrane-integrated glass capillary device for preparing small-sized water-in-oil-in-water (W/O/W) emulsion droplets is demonstrated. The concept of integrating microfluidics to prepare precise structure-controlled double emulsion droplets with the membrane emulsification technique provides a simple method for preparing small-sized and structure-controlled double emulsion droplets. The most important feature of the integrated device is the ability to decrease droplet size when the emulsion droplets generated at the capillary pass through the membrane. At the same time, most of the oil shell layer is stripped away and the resultant double emulsion droplets have thin shells. It is also demonstrated that the sizes of the resultant double emulsion droplets are greatly affected by both the double emulsion droplet flux through membranes and membrane pore size; when the flux is increased and membrane pore size is decreased, the generated W/O/W emulsion droplets are smaller than the original. In situ observation of the permeation behavior of the W/O/W emulsion droplets through membranes using a high-speed camera demonstrates (1) the stripping of the middle oil phase, (2) the division of the double emulsion droplets to generate two or more droplets with smaller size, and (3) the collapse of the double emulsion droplets. The first phenomenon results in a thinner oil shell, and the second division phenomenon produces double emulsion droplets that are smaller than the original.
Biocompatible, multifunctional, stimuli responsive, and high drug loading capacity are key factors for the new generation of drug delivery platforms. However, it is extremely challenging to create such a platform that inherits all these advanced properties in a single carrier. Herein, porous silicon nanoparticles (PSi NPs) and giant liposomes are assembled on a microfluidic chip as an advanced nano‐in‐micro platform (PSi NPs@giant liposomes), which can co‐load and co‐deliver hydrophilic and hydrophobic drugs combined with synthesized DNA nanostructures, short gold nanorods, and magnetic nanoparticles. The PSi NPs@giant liposomes with photothermal and magnetic responsiveness show good biocompatibility, high loading capacity, and controllable release. The hydrophilic thermal oxidized PSi NPs encapsulate hydrophobic therapeutics within the hydrophilic core of the giant liposomes, endowing high therapeutics loading capacity with tuneable ratio and controllable release. It is demonstrated that the DAO‐E A DNA nanostructures have synergism with drugs and importantly they contribute to the significant enhancement of cell death to doxorubicin‐resistant MCF‐7/DOX cells, overcoming the multidrug resistance in the cancer cells. Therefore, the PSi NPs@giant liposomes nano‐in‐micro platform hold great potential for a cocktail delivery of drugs and DNA nanostructures for effective cancer therapy, controllable drug release with tuneable therapeutics ratio, and both photothermal and magnetic dual responsiveness.
We propose a model for the dynamics of a probe embedded in a living cell, where both thermal fluctuations and nonequilibrium activity coexist. The model is based on a confining harmonic potential describing the elastic cytoskeletal matrix, which undergoes random active hops as a result of the nonequilibrium rearrangements within the cell. We describe the probe's statistics and we bring forth quantities affected by the nonequilibrium activity. We find an excellent agreement between the predictions of our model and experimental results for tracers inside living cells. Finally, we exploit our model to arrive at quantitative predictions for the parameters characterizing nonequilibrium activity, such as the typical time scale of the activity and the amplitude of the active fluctuations.
The actin cytoskeleton is a key element of cell structure and movement whose properties are determined by a host of accessory proteins. Actin cross-linking proteins create a connected network from individual actin filaments, and though the mechanical effects of cross-linker binding affinity on actin networks have been investigated in reconstituted systems, their impact on cellular forces is unknown. Here we show that the binding affinity of the actin cross-linker α-actinin 4 (ACTN4) in cells modulates cytoplasmic mobility, cellular movement, and traction forces. Using fluorescence recovery after photobleaching, we show that an ACTN4 mutation that causes human kidney disease roughly triples the wild-type binding affinity of ACTN4 to F-actin in cells, increasing the dissociation time from 29 ± 13 to 86 ± 29 s. This increased affinity creates a less dynamic cytoplasm, as demonstrated by reduced intracellular microsphere movement, and an approximate halving of cell speed. Surprisingly, these less motile cells generate larger forces. Using traction force microscopy, we show that increased binding affinity of ACTN4 increases the average contractile stress (from 1.8 ± 0.7 to 4.7 ± 0.5 kPa), and the average strain energy (0.4 ± 0.2 to 2.1 ± 0.4 pJ). We speculate that these changes may be explained by an increased solid-like nature of the cytoskeleton, where myosin activity is more partitioned into tension and less is dissipated through filament sliding. These findings demonstrate the impact of cross-linker point mutations on cell dynamics and forces, and suggest mechanisms by which such physical defects lead to human disease.
Stimuli‐responsive hydrogels are able to change their physical properties such as their elastic moduli in response to changes in their environment. If biocompatible polymers are used to prepare such materials and if living cells are encapsulated within these networks, their switchability allows the cell–matrix interactions to be investigated with unprecedented consistency. In this paper, thermo‐responsive macro‐ and microscopic hydrogels are presented based on azide‐functionalized copolymers of poly(N‐(2‐hydroxypropyl)‐methacrylamide) and poly(hydroxyethyl methacrylate) grafted with poly(N‐isopropylacrylamide) side chains. Crosslinking of these comb polymers is realized by bio‐orthogonal strain‐promoted azide–alkyne cycloaddition with cyclooctyne‐functionalized poly(ethylene glycol). The resulting hybrid hydrogels exhibit thermo‐tunable elasticity tailored by the polymer chain length and grafting density. This bio‐orthogonal polymer crosslinking strategy is combined with droplet‐based microfluidics to encapsulate living cells into stimuli‐responsive microgels, proving them to be a suitable platform for future systematic stem‐cell research.
Because of persistent economic pressure on cost reduction, inorganic fillers such as precipitated calcium carbonate (PCC) have become increasingly economically attractive in the papermaking process. The increase of filler level in paper can be achieved by adding it to pulp prior to the headbox, either as individual filler particles or as preaggregates, while maintaining paper strength and minimizing their negative impact. Consequently, the floc structure and strength of PCC aggregates was studied using flocculants and dry strength agents, using static light scattering/diffraction (SLS), real time fluorescent video imaging (RTFVI), image analysis, photometric dispersion analysis (PDA), and scanning electron microscopy (SEM). It was found that PEO/cofactor induced PCC aggregates were weaker at high shear and far more irreversible than those induced by the partially hydrolyzed polyvinyl formamide copolymerized with acrylic acid (PVFA/NaAA) or cationic starch. Flocs produced at low polymer dosages were smaller and weaker than those produced at higher dosages. The number of discrete PCC particles in aggregates was measured using real time fluorescent video imaging combined with image analysis. Finally, we speculate that when two scalenohedral crystal type PCC particles aggregate, there is a small effective surface area to bind them, mainly through classical bridging or charge neutralization flocculation. Moreover, additional polymer adsorption results in higher coverage of the external and internal surfaces and prevents further aggregation due to electrosteric repulsion.
It has long been the dream of biologists to map gene expression at the single-cell level. With such data one might track heterogeneous cell sub-populations, and infer regulatory relationships between genes and pathways. Recently, RNA sequencing has achieved single-cell resolution. What is limiting is an effective way to routinely isolate and process large numbers of individual cells for quantitative in-depth sequencing. We have developed a high-throughput droplet-microfluidic approach for barcoding the RNA from thousands of individual cells for subsequent analysis by next-generation sequencing. The method shows a surprisingly low noise profile and is readily adaptable to other sequencing-based assays. We analyzed mouse embryonic stem cells, revealing in detail the population structure and the heterogeneous onset of differentiation after leukemia inhibitory factor (LIF) withdrawal. The reproducibility of these high-throughput single-cell data allowed us to deconstruct cell populations and infer gene expression relationships.
Cells, the basic units of biological structure and function, vary broadly in type and state. Single-cell genomics can characterize cell identity and function, but limitations of ease and scale have prevented its broad application. Here we describe Drop-seq, a strategy for quickly profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets, associating a different barcode with each cell’s RNAs, and sequencing them all together. Drop-seq analyzes mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin. We analyzed transcriptomes from 44,808 mouse retinal cells and identified 39 transcriptionally distinct cell populations, creating a molecular atlas of gene expression for known retinal cell classes and novel candidate cell subtypes. Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at single-cell resolution.
Electrolyte gating of complex oxides enables investigation of electronic phase boundaries and collective response to strong electric fields. The origin of large conductance modulations and associated emergent properties in such field effect structures is a matter of intense study due to competing contributions from electrostatic (charge accumulation) and electrochemical (crystal chemistry changes) effects. Vanadium dioxide (VO2) is a prototypical correlated insulator that shows an insulator-to-metal transition at ∼67 °C and recent studies have noted a vast range of electronic effects in electric double-layer transistors (EDLT). In this study, we demonstrate that the response of electrolyte gated VO2 devices can be deterministically controlled by inserting a monolayer of graphene at the oxide–electrolyte interface. Several electrolytes as well as dopants (such as lithium ions and protons) were employed in EDL transistors to show that graphene serves as an inert barrier that successfully protects the oxide surface from chemical reactions. This monolayer interface has a striking effect on resistance modulation in the vanadium dioxide transistor channel up to several orders of magnitude and enables retention of the insulating phase. The studies allow new insights into the response of correlated insulators in EDLTs and inform design of correlated oxide–2D heterostructures for electronics and sensors.
Colloidal particles interacting via a long-range repulsion can, in contrast to hard-sphere systems, exhibit crystalline ordering at low volume fraction. Here we experimentally investigate the structure and properties of such “colloidal Wigner crystals.” We find a body-centered-cubic crystalline phase at volume fractions of ϕ≳15%, which exhibits large fluctuations of individual particles from their average positions. We determine the three independent crystalline elastic constants and find that these crystals are very compliant and highly anisotropic.