High-throughput measurement of elastic moduli of microfibers by rope coiling.

There are many fields where it is of interest to measure the elastic moduli of tiny fragile fibers, such as filamentous bacteria, actin filaments, DNA, carbon nanotubes, and functional microfibers. The elastic modulus is typically deduced from a sophisticated tensile test under a microscope, but the throughput is low and limited by the time-consuming and skill-intensive sample loading/unloading. Here, we demonstrate a simple microfluidic method enabling the high-throughput measurement of the elastic moduli of microfibers by rope coiling using a localized compression, where sample loading/unloading are not needed between consecutive measurements. The rope coiling phenomenon occurs spontaneously when a microfiber flows from a small channel into a wide channel. The elastic modulus is determined by measuring either the buckling length or the coiling radius. The throughput of this method, currently 3,300 fibers per hour, is a thousand times higher than that of a tensile tester. We demonstrate the feasibility of the method by testing a nonuniform fiber with axially varying elastic modulus. We also demonstrate its capability for in situ inline measurement in a microfluidic production line. We envisage that high-throughput measurements may facilitate potential applications such as screening or sorting by mechanical properties and real-time control during production of microfibers.

Size Scaling of Condensates in Multicomponent Phase Separation.

Constant proportionalities between cells and their intracellular organelles have been widely observed in various types of cells, known as intracellular size scaling. However, the mechanism underlying the size scaling and its modulation by environmental factors in multicomponent systems remain poorly understood. Here, we study the size scaling of membrane-less condensates using microdroplet-encapsulated minimalistic condensates formed by droplet microfluidics and mean-field theory. We demonstrate that the size scaling of condensates is an inherent characteristic of liquid-liquid phase separation. This concept is supported by experiments showing the occurrence of size scaling phenomena in various condensate systems and a generic lever rule acquired from mean-field theory. Moreover, it is found that the condensate-to-microdroplet scaling ratio can be affected by the solute and salt concentrations, with good agreement between experiments and predictions by theory. Notably, we identify a noise buffering mechanism whereby condensates composed of large macromolecules effectively maintain constant volumes and counteract concentration fluctuations of small molecules. This mechanism is achieved through the dynamic rearrangement of small molecules in and out of membrane-free interfaces. Our work provides crucial insights into understanding mechanistic principles that govern the size of cells and intracellular organelles as well as associated biological functions.

Simple and complex coacervation in systems involving plant proteins.

Plant-based foods are gaining popularity as alternatives to meat and dairy products due to sustainability and health concerns. As a consequence, there is a renewed interest in the phase behaviour of plant proteins and of mixtures of plant proteins and polysaccharides, in particular in the cases where coacervation is found to occur, i.e., liquid-liquid phase separation (LLPS) into two phases, one of which is rich in biopolymers and one of which is poor in biopolymer. Here we review recent research into both simple and complex coacervation in systems involving plant proteins, and their applications in food- as well as other technologies, such as microencapsulation, microgel production, adhesives, biopolymer films, and more.

Three-Dimensionally Printed, Vertical Full-Color Display Pixels for Multiplexed Anticounterfeiting.

Information encryption strategies have become increasingly essential. Most of the fluorescent security patterns have been made with a lateral configuration of red, green, and blue subpixels, limiting the pixel density and security level. Here we report vertically stacked, luminescent heterojunction micropixels that construct high-resolution, multiplexed anticounterfeiting labels. This is enabled by meniscus-guided three-dimensional (3D) microprinting of red, green, and blue (RGB) dye-doped materials. High-precision vertical stacking of subpixel segments achieves full-color pixels without sacrificing lateral resolution, achieving a small pixel size of ∼µm and a high density of over 13,000 pixels per inch. Furthermore, a full-scale color synthesis for individual pixels is developed by modulating the lengths of the RGB subpixels. Taking advantage of these unique 3D structural designs, trichannel multiplexed anticounterfeiting Quick Response codes are successfully demonstrated. We expect that this work will advance data encryption technology while also providing a versatile manufacturing platform for diverse 3D display devices.

Tuning Material States and Functionalities of G-Quadruplex-Modulated RNA-Peptide Condensates.

RNA encodes sequence- and structure-dependent interactions to modulate the assembly and properties of biomolecular condensates. RNA G-quadruplexes (rG4s) formed by guanine-rich sequences can trigger the formation of liquid- or solid-like condensates that are involved in many aberrant phase transitions. However, exactly how rG4 motifs modulate different phase transitions and impart distinct material properties to condensates is unclear. Here, using RNA oligonucleotides and cationic peptides as model systems, we show that RNA-peptide condensates exhibit tunability in material properties over a wide spectrum via interactions arising from rG4 folding/unfolding kinetics. rG4-containing oligonucleotides formed strong pairwise attraction with peptides and tended to form solid-like condensates, while their less-structured non-G4 mutants formed liquid-like droplets. We find that the coupling between rG4 dissociation and RNA-peptide complex coacervation triggers solid-to-liquid transition of condensates prior to the complete unfolding of rG4s. This coupling points to a mechanism that material states of rG4-modulated condensates can be finely tuned from solid-like to liquid-like by the addition of less-structured RNA oligonucleotides, which have weak but dominant binding with peptides. We further show that the tunable material states of condensates can enhance RNA aptamer compartmentalization and RNA cleavage reactions. Our results suggest that condensates with complex properties can emerge from subtle changes in RNA oligonucleotides, contributing ways to treat dysfunctional condensates in diseases and insights into prebiotic compartmentalization.

Targeting unfolded protein response using albumin-encapsulated nanoparticles attenuates temozolomide resistance in glioblastoma.

BACKGROUND: Chemoresistant cancer cells frequently exhibit a state of chronically activated endoplasmic reticulum (ER) stress. Engaged with ER stress, the unfolded protein response (UPR) is an adaptive reaction initiated by the accumulation of misfolded proteins. Protein disulfide isomerase (PDI) is a molecular chaperone known to be highly expressed in glioblastomas with acquired resistance to temozolomide (TMZ). We investigate whether therapeutic targeting of PDI provides a rationale to overcome chemoresistance. METHODS: The activity of PDI was suppressed in glioblastoma cells using a small molecule inhibitor CCF642. Either single or combination treatment with TMZ was used. We prepared nanoformulation of CCF642 loaded in albumin as a drug carrier for orthotopic tumour model. RESULTS: Inhibition of PDI significantly enhances the cytotoxic effect of TMZ on glioblastoma cells. More importantly, inhibition of PDI is able to sensitise glioblastoma cells that are initially resistant to TMZ treatment. Nanoformulation of CCF642 is well-tolerated and effective in suppressing tumour growth. It activates cell death-triggering UPR beyond repair and induces ER perturbations through the downregulation of PERK signalling. Combination treatment of TMZ with CCF642 significantly reduces tumour growth compared with either modality alone. CONCLUSION: Our study demonstrates modulation of ER stress by targeting PDI as a promising therapeutic rationale to overcome chemoresistance.

Partitioning-Induced Isolation of Analyte and Analysis via Multiscaled Aqueous Two-Phase System.

Most fluorescence-based bioanalytical applications need labeling of analytes. Conventional labeling requires washing to remove the excess fluorescent labels and reduce the noise signals. These pretreatments are labor intensive and need specialized equipment, hindering portable applications in resource-limited areas. Herein, we use the aqueous two-phase system (ATPS) to realize the partitioning-induced isolation of labeled analytes from background signals without extra processing steps. ATPS is formed by mixing two polymers at sufficiently high concentrations. ATPS-based isolation is driven by intrinsic affinity differences between analytes and excess labels. To demonstrate the partitioning-induced isolation and analysis, fluorescein isothiocyanate (FITC) is selected as the interfering fluorophore, and a monoclonal antibody (IgG) is used as the analyte. To optimize ATPS compositions, different molecular weights and mass fractions of polyethylene glycol (PEG) and dextran and different phosphate-buffered saline (PBS) concentrations are investigated. Various operational scales of our approach are demonstrated, suggesting its compatibility with various bioanalytical applications. In centimeter-scale ATPS, the optimized distribution ratios of IgG and FITC are 91.682 and 0.998 using PEG 6000 Da and dextran 10,000 Da in 10 mM PBS. In millimeter-scale ATPS, the analyte is enriched to 6.067 fold using 15 wt % PEG 35,000 Da and 5 wt % dextran 500,000 Da in 10 mM PBS. In microscale ATPS, analyte dilutions are isolated into picoliter droplets, and the measured fluorescence intensities linearly correlated with the analyte concentrations (R2 = 0.982).

Flows of a nonequilibrated aqueous two-phase system in a microchannel.

Liquid-liquid phase separation is a rich and dynamic process, which recently has gained new interest, especially in biology and for material synthesis. In this work, we experimentally show that co-flow of a nonequilibrated aqueous two-phase system within a planar flow-focusing microfluidic device results in a three-dimensional flow, as the two nonequilibrated solutions move downstream along the length of the microchannel. After the system reaches steady-state, invasion fronts from the outer stream are formed along the top and bottom walls of the microfluidic device. The invasion fronts advance towards the center of the channel, until they merge. We first show by tuning the concentration of polymer species within the system that the formation of these fronts is due to liquid-liquid phase separation. Moreover, the rate of invasion from the outer stream increases with increasing polymer concentrations in the streams. We hypothesize the invasion front formation and growth is driven by Marangoni flow induced by the polymer concentration gradient along the width of the channel, as the system is undergoing phase separation. In addition, we show how at various downstream positions the system reaches its steady-state configuration once the two fluid streams flow side-by-side in the channel.

Hanging droplets from liquid surfaces.

Natural and man-made robotic systems use the interfacial tension between two fluids to support dense objects on liquid surfaces. Here, we show that coacervate-encased droplets of an aqueous polymer solution can be hung from the surface of a less dense aqueous polymer solution using surface tension. The forces acting on and the shapes of the hanging droplets can be controlled. Sacs with homogeneous and heterogeneous surfaces are hung from the surface and, by capillary forces, form well-ordered arrays. Locomotion and rotation can be achieved by embedding magnetic microparticles within the assemblies. Direct contact of the droplet with air enables in situ manipulation and compartmentalized cascading chemical reactions with selective transport. Applications including functional microreactors, motors, and biomimetic robots are evident.

One-Pot Self-Assembly of Dual-Color Domes Using Mono-Sized Silica Nanoparticles.

Spots with dual structural colors on the skin of some organisms in nature are of tremendous interest due to the unique function of their dye-free colors. However, imitation of them requires complicated manufacturing processes, expensive equipment, and multiple predesigned building blocks. In this work, a one-pot strategy based on the phase-separation-assisted nonuniform self-assembly of monosized silica nanoparticles is developed to construct domes with dual structural colors. In drying poly(ethylene glycol)-dextran-based (PEG-DEX) droplets, monosized nanoparticles distribute nonuniformly in two compartments due to the droplet inner flow and different nanoparticle compatibility with the two phases. The dome colors are derived from the self-assembled nanoparticles and are programmable by regulating the assembly conditions. The one-pot strategy enables the preparation of multicolor using only one type of building block. With the dual-color domes, encrypted patterns with a high volume of contents are designed, showing promising applications in information delivery.

Shape-Reconfigurable Ferrofluids.

Ferrofluids (FFs) can adapt their shape to a magnetic field. However, they cannot maintain their shape when the magnetic field is removed. Here, with a magneto-responsive and reconfigurable interfacial self-assembly (MRRIS) process, we show that FFs can be structured by a magnetic field and maintain their shape, like solids, after removing the magnetic field. The competing self-assembly of magnetic and nonmagnetic nanoparticles at the liquid interface endow FFs with both reconfigurability and structural stability. By manipulating the external magnetic field, we show that it is possible to "write" and "erase" the shape of the FFs remotely and repeatedly. To gain an in-depth understanding of the effect of MRRIS on the structure of FFs, we systematically study the shape variation of these liquids under both the static and dynamic magnetic fields. Our study provides a simple yet novel way of manipulating FFs and opens opportunities for the fabrication of all-liquid devices.

Synthetic Membraneless Droplets for Synaptic-Like Clustering of Lipid Vesicles.

In eukaryotic cells, the membraneless organelles (MLOs) formed via liquid-liquid phase separation (LLPS) are found to interact intimately with membranous organelles (MOs). One major mode is the clustering of MOs by MLOs, such as the formation of clusters of synaptic vesicles at nerve terminals mediated by the synapsin-rich MLOs. Aqueous droplets, including complex coacervates and aqueous two-phase systems, have been plausible MLO-mimics to emulate or elucidate biological processes. However, neither of them can cluster lipid vesicles (LVs) like MLOs. In this work, we develop a synthetic droplet assembled from a combination of two different interactions underlying the formation of these two droplets, namely, associative and segregative interactions, which we call segregative-associative (SA) droplets. The SA droplets cluster and disperse LVs recapitulating the key functional features of synapsin condensates, which can be attributed to the weak electrostatic interaction environment provided by SA droplets. This work suggests LLPS with combined segregative and associative interactions as a possible route for synaptic clustering of lipid vesicles and highlights SA droplets as plausible MLO-mimics and models for studying and mimicking related cellular dynamics.

Liquid Marbles under Electric Fields: New Capabilities for Non-wetting Droplet Manipulation and Beyond.

The study of liquid marbles (LMs) composed of stabilizing liquid droplets with solid particles in a gaseous environment has matured into an established area in surface and colloid science. The minimized "solid-liquid-air" triphase interface enables LMs to drastically reduce adhesion to a solid substrate, making them unique non-wetting droplets transportable with limited energy. The small volume, enclosed environment, and simple preparation render them suitable microreactors in industrial applications and processes such as cell culture, material synthesis, and blood coagulation. Extensive application contexts request precise and highly efficient manipulations of these non-wetting droplets. Many external fields, including magnetic, acoustic, photothermal, and pH, have emerged to prepare, deform, actuate, coalesce, mix, and disrupt these non-wetting droplets. Electric fields are rising among these external stimuli as an efficient source for manipulating the LMs with high controllability and a significant ability to contribute further to proposed applications. This Feature Article attempts to outline the recent developments related to LMs with the aid of electric fields. The effects of electric fields on the preparation and manipulation of LMs with intricate interfacial processes are discussed in detail. We highlight a wealth of novel electric field-involved LM-based applications and beyond while also envisaging the challenges, opportunities, and new directions for future development in this emerging research area.

Three-Dimensional Perovskite Nanopixels for Ultrahigh-Resolution Color Displays and Multilevel Anticounterfeiting.

Hybrid perovskites are emerging as a promising, high-performance luminescent material; however, the technological challenges associated with generating high-resolution, free-form perovskite structures remain unresolved, limiting innovation in optoelectronic devices. Here, we report nanoscale three-dimensional (3D) printing of colored perovskite pixels with programmed dimensions, placements, and emission characteristics. Notably, a meniscus comprising femtoliters of ink is used to guide a highly confined, out-of-plane crystallization process, which generates 3D red, green, and blue (RGB) perovskite nanopixels with ultrahigh integration density. We show that the 3D form of these nanopixels enhances their emission brightness without sacrificing their lateral resolution, thereby enabling the fabrication of high-resolution displays with improved brightness. Furthermore, 3D pixels can store and encode additional information into their vertical heights, providing multilevel security against counterfeiting. The proof-of-concept experiments demonstrate the potential of 3D printing to become a platform for the manufacture of smart, high-performance photonic devices without design restrictions.

The anti-scarring role of Lycium barbarum polysaccharide on cornea epithelial-stromal injury.

PURPOSE: Cornea epithelial-stromal scarring is related to the differentiation of fibroblasts into opaque myofibroblasts. Our study aims to assess the effectiveness of Lycium barbarum polysaccharide (LBP) solution as a pre-treatment in minimizing corneal scarring. METHODS: Human corneal fibroblasts were cultured in a three-dimensional collagen type I-based hydrogel in an eye-on-a-chip model. Fibroblasts were pre-treated with 2 mg/mL LBP for 24 h, followed by another 24-h incubation with 10 ng/mL transforming growth factor-beta 1 (TGF-ß1) to induce relevant physiological events after stromal injury. Intracellular pro-fibrotic proteins, extracellular matrix proteins, and pro-inflammatory cytokines that involved in fibrosis, were assessed using immunocytochemistry and enzyme-linked immunosorbent assays. RESULTS: Compared to the positive control TGF-ß1 group, LBP pre-treated cells had a significantly lower expression of alpha-smooth muscle actin, marker of myofibroblasts, vimentin (p < 0.05), and also extracellular matrix proteins both collagen type II and type III (p < 0.05) that can be found in scar tissues. Moreover, LBP pre-treated cells had a significantly lower secretion of pro-inflammatory cytokines interleukin-6 and interleukin-8 (p < 0.05). The cell-laden hydrogel contraction and stiffness showed no significant difference between LBP pre-treatment and control groups. Fibroblasts pretreated with LBP as well had reduced angiogenic factors expression and suppression of undesired proliferation (p < 0.05). CONCLUSION: Our results showed that LBP reduced both pro-fibrotic proteins and pro-inflammatory cytokines on corneal injury in vitro. We suggest that LBP, as a natural Traditional Chinese Medicine, may potentially be a novel topical pre-treatment option prior to corneal refractive surgeries with an improved prognosis.

Dynamic regimes of electrified liquid filaments.

We investigate the dynamics of an electrified liquid filament in a nozzle-to-substrate configuration with a close separation. The interplay between compressive viscous and electrostatic stresses dictates previously undocumented transitions between dynamic regimes of "jetting," "coiling," and "whipping." In particular, the onsets of both coiling and whipping instabilities are significantly influenced by the minimum radius along the liquid filament. Using a low-interfacial-tension system, we unravel the physics behind the transitions between jetting, coiling, and whipping of an electrified filament for a range of liquid properties and geometric parameters. Our results enrich the overall physical picture of the electrically forced jets, and provide insights for the emerging high-resolution instability-assisted printing of materials such as folded assemblies and scaffolds.

Emerging aqueous two-phase systems: from fundamentals of interfaces to biomedical applications.

Aqueous two-phase systems (ATPSs) have been recognized for their applications in extraction, separation, purification, and enrichment of (bio)molecules and cells. Recently, their unique ability to create aqueous-aqueous interfaces through phase separation and the characteristics of these interfaces have created new opportunities in biomedical applications. In this review, we summarize recent progress in understanding the dynamics at aqueous-aqueous interfaces, and in developing interface-assisted design of artificial cells and cyto-mimetic materials, fabrication of cyto- and bio-compatible microparticles, cell micropatterning, 3D bioprinting, and microfluidic separation of cells and biomolecules. We also discuss the challenges and perspectives to leverage the unique characteristics of ATPSs and their interfaces in broader applications.

Droplet and Microchamber-Based Digital Loop-Mediated Isothermal Amplification (dLAMP).

Digital loop-mediated isothermal amplification (dLAMP) refers to compartmentalizing nucleic acids and LAMP reagents into a large number of individual partitions, such as microchambers and droplets. This compartmentalization enables dLAMP to be an excellent platform to quantify the absolute number of the target nucleic acids. Owing to its low requirement for instrumentation complexity, high specificity, and strong tolerance to inhibitors in the nucleic acid samples, dLAMP has been recognized as a simple and accurate technique to quantify pathogenic nucleic acid. Herein, the general process of dLAMP techniques is summarized, the current dLAMP techniques are categorized, and a comprehensive discussion on different types of dLAMP techniques is presented. Also, the challenges of the current dLAMP are illustrated together with the possible strategies to address these challenges. In the end, the future directions of the dLAMP developments, including multitarget detection, multisample detection, and processing nucleic acid extraction are outlined. With recently significant advances in dLAMP, this technology has the potential to see more widespread use beyond the laboratory in the future.

On-Demand Droplet Collection for Capturing Single Cells.

Droplet-based microfluidic techniques are extensively used in efficient manipulation and genome-wide analysis of individual cells, probing the heterogeneity among populations of individuals. However, the extraction and isolation of single cells from individual droplets remains difficult due to the inevitable sample loss during processing. Herein, an automated system for accurate collection of defined numbers of droplets containing single cells is presented. Based on alternate sorting and dispensing in three branch channels, the droplet number can be precisely controlled down to single-droplet resolution. While encapsulating single cells and reserving one branch as a waste channel, sorting can be seamlessly integrated to enable on-demand collection of single cells. Combined with a lossless recovery strategy, this technique achieves capture and culture of individual cells with a harvest rate of over 95%. The on-demand droplet collection technique has great potential to realize quantitative processing and analysis of single cells for elucidating the role of cell-to-cell variations.

Steady and Unsteady Buckling of Viscous Capillary Jets and Liquid Bridges.

Steady buckling (coiling) of thin falling liquid jets is sensitive to surface tension, yet an understanding of these capillary effects lags far behind what is known about surface-tension-free coiling. In experiments with submillimetric jets and ultralow flow rates, we find that the critical dispensing height H_{c} for coiling decreases with increasing flow rate, a trend opposite to that found previously for inertia-free coiling. We resolve the apparent contradiction using nonlinear numerical simulations based on slender-jet theory which show that the trend reversal is due to the strong effect of surface tension in our experiments. We use our experiments to construct a regime diagram (coiling vs stagnation flow) in the space of capillary number Ca and jet slenderness ε and find that it agrees well with fully nonlinear numerical simulations. However, it differs substantially from the analogous regime diagram determined experimentally by Le Merrer, Quéré, and Clanet [Phys. Rev. Lett. 109, 064502 (2012)PRLTAO0031-900710.1103/PhysRevLett.109.064502] for the unsteady buckling of a compressed liquid bridge. Using linear stability analysis, we show that the differences between the two regime diagrams can be explained by a combination of shape nonuniformity and the influence of gravity.