There is a need for novel analytical techniques to study the composition of single extracellular vesicles (EV). Such techniques are required to improve the understanding of heterogeneous EV populations, to allow identification of unique subpopulations, and to enable earlier and more sensitive disease detection. Because of the small size of EV and their low protein content, ultrahigh sensitivity technologies are required. Here, an immuno‐droplet digital polymerase chain reaction (iddPCR) amplification method is described that allows multiplexed single EV protein profiling. Antibody–DNA conjugates are used to label EV, followed by stochastic microfluidic incorporation of single EV into droplets. In situ PCR with fluorescent reporter probes converts and amplifies the barcode signal for subsequent read‐out by droplet imaging. In these proof‐of‐principle studies, it is shown that multiplex protein analysis is possible in single EV, opening the door for future analyses.
Monoclonal antibodies are powerful tools for scientific research and are the basis of numerous therapeutics. However, traditional approaches to generate monoclonal antibodies against a desired target, such as hybridoma-based techniques and display library methods, are laborious and suffer from fusion inefficiency and display bias, respectively. Here we present a platform, featuring droplet microfluidics and a bead-based binding assay, to rapidly identify and verify antigen-binding antibody sequences from primary cells. We used a defined mixture of hybridoma cells to characterize the system, sorting droplets at up to 100 Hz and isolating desired hybridoma cells, comprising 0.1% of the input, with a false positive rate of less than 1%. We then applied the system to once-frozen primary B-cells to isolate rare cells secreting target-binding antibody. We performed RT-PCR on individual sorted cells to recover the correctly paired heavy- and light-chain antibody sequences, and we used rapid cell-free protein synthesis to generate single-chain variable fragment-format (scFv) antibodies from fourteen of the sorted cells. Twelve of these showed antigen-specific binding by ELISA. Our platform facilitates screening animal B-cell repertoires within days at low cost, increasing both rate and range of discovering antigen-specific antibodies from living organisms. Further, these techniques can be adapted to isolate cells based on virtually any secreted product.
A hydrogel microcapsule with an intermediate thin oil layer is presented to achieve smart release of a broad range of cargoes triggered via diverse stimuli. A microfluidic technique is used to produce triple emulsion droplets with a thin oil layer that separates the innermost aqueous phase from the hydrogel prepolymer phase, which transforms into a hydrogel shell via photopolymerization. The intermediate oil layer within the hydrogel microcapsule acts as an effective diffusion barrier, allowing encapsulation of various small cargoes within a porous hydrogel shell until a stimulus is applied to destabilize the oil layer. It is demonstrated that diverse stimuli including chemical dissolution, mechanical stress, and osmotic pressure can be utilized to release the encapsulated cargo on‐demand. In addition, osmotic pressure and the hydrogel shell thickness can be independently tuned to control the onset time of release as well as the release behavior of multi‐cargo encapsulated hydrogel microcapsule. The release can be either simultaneous or selective.
Integration of ionic permselective medium (e.g., nanochannels, membranes) within microfluidic channels has been shown to enable on-chip desalination, sample purification, bioparticle sorting, and biomolecule concentration for enhanced detection sensitivity. However, the ion-permselective mediums are generally of fixed properties and cannot be dynamically tuned. Here we study a microfluidic device consisting of an array of individually addressable elastic membranes connected in series on top of a single microfluidic channel that can be deformed to locally reduce the channel cross-section into a nanochannel. Dynamic tunability of the ion-permselective medium, as well as controllability of its location and ionic permselectivity, introduces a new functionality to microfluidics-based lab-on-a-chip devices, for example, dynamic localization of preconcentrated biomolecule plugs at different sensing regions for multiplex detection. Moreover, the ability to simultaneously form a series of preconcentrated plugs at desired locations increases parallelization of the system and the trapping efficiency of target analytes.
Microcapsules with liquid cores and solid shells are attractive as dispersible protective micron-sized containers. Applications that rely on molecular mass transport often require a combination of size selectivity, high permeability, and mechanical stability. Capsule architectures that combine all these features represent a material property, design, and fabrication challenge. In this work, the design of an asymmetric microcapsule shell architecture is reported to achieve a good combination of the desired features. Poly(methyl methacrylate) phase-inverted microcapsules featuring an asymmetric graded macroporous shell covered with a dense skin separation layer are obtained from water-in-oil-in-water double emulsion drops that are phase-inverted in a water-based coagulation bath. The phase-inverted microcapsules exhibit good mechanical stability and allow for high permeability of its shell membrane with molecular size dependence.
Equilibrium interfaces were established between body-centered cubic (BCC) crystals and their liquid using charged colloidal particles in an electric bottle. By measuring a time series of interfacial positions and computing the average power spectrum, their interfacial stiffness was determined according to the capillary fluctuation method. For the (100) and the (114) interfaces, the stiffnesses were 0.15 and 0.18 kBT/σ2 (σ: particle diameter), respectively, and were isotropic in the plane of the interface. For comparison, similar charged colloids were used to create an interface between a face-centered cubic (FCC) crystal and its liquid. Its stiffness was significantly larger: 0.26 kBT/σ2. This result gives experimental support to the explanations offered for the preferential nucleation of BCC over FCC in metallic alloys.
Nanoparticles with diverse structures and unique properties have attracted increasing attention for their widespread applications. Co‐precipitation under rapid mixing is an effective method to obtained biocompatible nanoparticles and diverse particle carriers are achieved by controlled phase separation via interfacial tensions. In this Minireview, we summarize the underlying mechanism of co‐precipitation and show that rapid mixing is important to ensure co‐precipitation. In the binary polymer system, the particles can form four different morphologies, including occluded particle, core‐shell capsule, dimer particle, and heteroaggregate, and we demonstrate that the final morphology could be controlled by surface tensions through surfactant, polymer composition, molecular weight, and temperature. The applications of occluded particles, core‐shell capsules and dimer particles prepared by co‐precipitation or microfluidics upon the regulation of interfacial tensions are discussed in detail, and show great potential in the areas of functional materials, colloidal surfactants, drug delivery, nanomedicine, bio‐imaging, displays, and cargo encapsulation.
Understanding drug‐release kinetics is critical for the development of drug‐loaded nanoparticles. We developed a J‐aggregate‐based Förster‐resonance energy‐transfer (FRET) method to investigate the release of novel high‐drug‐loading (50 wt %) nanoparticles in comparison with low‐drug‐loading (0.5 wt %) nanoparticles. Single‐dye‐loaded nanoparticles form J‐aggregates because of the high dye‐loading (50 wt %), resulting in a large red‐shift (≈110 nm) in the fluorescence spectrum. Dual‐dye‐loaded nanoparticles with high dye‐loading using FRET pairs exhibited not only FRET but also a J‐aggregate red‐shift (116 nm). Using this J‐aggregate‐based FRET method, dye‐core–polymer‐shell nanoparticles showed two release processes intracellularly: the dissolution of the dye aggregates into dye molecules and the release of the dye molecules from the polymer shell. Also, the high‐dye‐loading nanoparticles (50 wt %) exhibited a slow release kinetics in serum and relatively quick release in cells, demonstrating their great potential in drug delivery.
Migratory dynamics of collective cells is central to the morphogenesis of biological tissues. The statistical distribution of cell velocities in 2D confluent monolayers is measured through large‐scale and long‐term experiments of various cell types lying on different substrates. A linear relation is discovered between the variability and the mean of cell speeds during the jamming process of confluent cell monolayers, suggesting time‐invariant distribution profile of cell velocities. It is further found that the probability density function of cell velocities obeys the non‐canonical q‐Gaussian statistics, regardless of cell types and substrate stiffness. It is the Tsallis entropy, instead of the classical Boltzmann–Gibbs entropy, that dictates the universal statistical laws of collective cell migration. The universal statistical law stems from cell–cell interactions, as demonstrated by the wound healing experiments. This previously unappreciated finding provides a linkage between cell‐level heterogeneity and tissue‐level ensembles in embryonic development and tumor growth.
Generally, one attempts to globally predict or interpret, from numerical simulations, the history of the effluents exiting porous media (i.e., the breakthrough curve), without a clear view of the detailed evolutions of deposition inside the medium. We developed a simple physical frame of description of the colloidal particle transport and adsorption, which allows to predict the main characteristics of transport and deposition in porous media from a set of directly measurable (macroscopic) physical parameters. More precisely, we show that the deposition distribution is basically a traveling wave propagating in the medium with a shape (frontal or extended) and velocity depending on the flow rate and the availability of particles with regards to the adsorption capacity. This in particular makes it possible to predict or interpret the breakthrough curve shape from a physical approach. We also show that additional effects may be included, such as a multiporosity leading to confinement effects (delayed deposition in less accessible regions). The validity of the model is checked from original direct visualizations by confocal microscopy of particle adsorption in time and space for nanoparticle suspensions flowing through a bead packing. This makes it possible to measure the evolution of the deposition profiles in time distinguishing the deposition in confined regions. The model appears to successfully predict the different trends: traveling wave, global deposition profile shape, profiles of deposition in confined regions.
Conformance control during waterflooding in an oil reservoir is utilized to redistribute water and increase the sweep efficiency and hence oil production. Using preformed gel particles can effectively redirect the flow by blocking the high-permeability zones and forcing water into low-permeability zones where the oil is trapped. However, the size of such gel particles can limit their applications deeper within the reservoir and can result in shear-induced degradation near the well bore. Here, we fabricate core–shell nanohydrogels with delayed swelling behavior; their volume increases by a factor of 200 after about 30 days in brine under reservoir conditions. We study their effect on the flow behavior in a three-dimensional porous medium micromodel consisting of randomly packed glass beads. Using confocal microscopy, we directly visualize the spatial variations of flow in the micromodel before and after nanohydrogel injection and swelling. The swollen nanohydrogels block some pores reducing the permeability of the micromodel and diverting the water into low-permeability regions. A core flood experiment further confirms that the nanohydrogels can significantly reduce the permeability of a reservoir sample and divert the fluid flow. Our results demonstrate that these core–shell nanohydrogels might be useful for flow control in porous media and can be used as a conformance control agent.
Droplet‐based single cell sequencing technologies, such as inDrop, Drop‐seq, and 10X Genomics, are catalyzing a revolution in the understanding of biology. Barcoding beads are key components for these technologies. What is limiting today are barcoding beads that are easy to fabricate, can efficiently deliver primers into drops, and thus achieve high detection efficiency. Here, this work reports an approach to fabricate dissolvable polyacrylamide beads, by crosslinking acrylamide with disulfide bridges that can be cleaved with dithiothreitol. The beads can be rapidly dissolved in drops and release DNA barcode primers. The dissolvable beads are easy to synthesize, and the primer cost for the beads is significantly lower than that for the previous barcoding beads. Furthermore, the dissolvable beads can be loaded into drops with >95% loading efficiency of a single bead per drop and the dissolution of beads does not influence reverse transcription or the polymerase chain reaction (PCR) in drops. Based on this approach, the dissolvable beads are used for single cell RNA and protein analysis.
Poor solubility often leads to low drug efficacy. Encapsulation of water‐insoluble drugs in polymeric nanoparticles offers a solution. However, low drug loading remains a critical challenge. Now, a simple and robust sequential nanoprecipitation technology is used to produce stable drug‐core polymer‐shell nanoparticles with high drug loading (up to 58.5 %) from a wide range of polymers and drugs. This technology is based on tuning the precipitation time of drugs and polymers using a solvent system comprising multiple organic solvents, which allows the formation of drug nanoparticles first followed by immediate precipitation of one or two polymers. This technology offers a new strategy to manufacture polymeric nanoparticles with high drug loading having good long‐term stability and programmed release and opens a unique opportunity for drug delivery applications.
Development of fast curing and easy modeling of colloidal photonic crystals is highly desirable for various applications. Here, a novel type of injectable photonic hydrogel (IPH) is proposed to achieve self‐healable structural color by integrating microfluidics‐derived photonic supraballs with supramolecular hydrogels. The supramolecular hydrogel is engineered via incorporating β‐cyclodextrin/poly(2‐hydroxypropyl acrylate‐co‐N‐vinylimidazole) (CD/poly(HPA‐co‐VI)) with methacrylated gelatin (GelMA), and serves as a scaffold for colloidal crystal arrays. The photonic supraballs derived from the microfluidics techniques, exhibit excellent compatibility with the hydrogel scaffolds, leading to enhanced assembly efficiency. By virtue of hydrogen bonds and host–guest interactions, a series of self‐healable photonic hydrogels (linear, planar, and spiral assemblies) can be facilely assembled. It is demonstrated that the spherical symmetry of the photonic supraballs endows them with identical optical responses independent of viewing angles. In addition, by taking the advantage of angle independent spectrum characteristics, the IPH presents beneficial effects in reflective cooling, which can achieve up to 17.4 °C in passive solar reflective cooling. The strategy represents an easy‐to‐perform platform for the construction of IPH, providing novel insights into macroscopic self‐assembly toward thermal management applications.
Immiscible displacement of fluids with large viscosity mismatch is inherently unstable due to viscous fingering, even in porous media where capillary forces dominate. Adding polymer to the displacing fluid reduces the viscosity mismatch and suppresses the viscous fingering instability thereby increasing the fluid displacement leading to extensive use in applications such as oil recovery. Surprisingly, however, an increase in displacement occurs even for very large viscosity mismatches. Moreover, significant additional displacement is observed when the polymer solution is followed by additional water flow. Thus, the fundamental physics of this phenomenon remains unclear. To understand this behavior, we use confocal microscopy to visualize the displacement of oil in a three-dimensional micromodel of a porous medium and simultaneously measure the local flow velocities of the displacing fluid. We find that the increased displacement results from a counterintuitive effect: polymer retention in the medium and the resultant local changes in flow. Typically retention is avoided since it reduces the permeability of the medium; instead, we find that large and heterogeneous local changes in flow lead to sufficiently large enough viscous forces at the interface of the immiscible fluids resulting in increased displacement.
Multiple sclerosis is a chronic inflammatory disease of the CNS1. Astrocytes contribute to the pathogenesis of multiple sclerosis2, but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC–seq), chromatin immunoprecipitation with sequencing (ChIP–seq), genome-wide analysis of DNA methylation and in vivo CRISPR–Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte–macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.
We generate droplets in a microfluidic device using a traveling surface acoustic wave (TSAW), and control droplet size by adjusting TSAW power and duration. We combine droplet production and fluorescence detection to selectively-encapsulate cells and beads; with this active method, the overwhelming majority of single particles or cells are encapsulated individually into droplets, contrasting the Poisson distribution of encapsulation number that governs traditional, passive microfluidic encapsulation. In addition, we lyse cells before selective encapsulation, and pico-inject new materials into existing droplets.
Glucose oxidase (GOx) is an important industrial enzyme that can be optimized for specific applications by mutagenesis and activity-based screening. To increase the efficiency of this approach, we have developed a new ultrahigh-throughput screening platform based on a microfluidic lab-on-chip device that allows the sorting of GOx mutants from a saturation mutagenesis library expressed on the surface of yeast cells. GOx activity was measured by monitoring the fluorescence of water microdroplets dispersed in perfluorinated oil. The signal was generated via a series of coupled enzyme reactions leading to the formation of fluorescein. Using this new method, we were able to enrich the yeast cell population by more than 35-fold for GOx mutants with higher than wild-type activity after two rounds of sorting, almost double the efficiency of our previously described flow cytometry platform. We identified and characterized novel GOx mutants, the most promising of which (M6) contained a combination of six point mutations that increased the catalytic constant kcat by 2.1-fold compared to wild-type GOx and by 1.4-fold compared to a parental GOx variant. The new microfluidic platform for GOx was therefore more sensitive than flow cytometry and supports comprehensive screens of gene libraries containing multiple mutations per gene.