Fabrication of gradient hydrogels using a thermophoretic approach in microfluidics.

The extracellular matrix presents spatially varying physical cues that can influence cell behavior in many processes. Physical gradients within hydrogels that mimic the heterogenous mechanical microenvironment are useful to study the impact of these cues on cellular responses. Therefore, simple and reliable techniques to create such gradient hydrogels are highly desirable. This work demonstrates the fabrication of stiffness gradient Gellan gum (GG) hydrogels by applying a temperature gradient across a microchannel containing hydrogel precursor solution. Thermophoretic migration of components within the precursor solution generates a concentration gradient that mirrors the temperature gradient profile, which translates into mechanical gradients upon crosslinking. Using this technique, GG hydrogels with stiffness gradients ranging from 20 to 90 kPa over 600µm are created, covering the elastic moduli typical of moderately hard to hard tissues. MC3T3 osteoblast cells are then cultured on these gradient substrates, which exhibit preferential migration and enhanced osteogenic potential toward the stiffest region on the gradient. Overall, the thermophoretic approach provides a non-toxic and effective method to create hydrogels with defined mechanical gradients at the micron scale suitable forin vitrobiological studies and potentially tissue engineering applications.

The liquid-to-solid transition of FUS is promoted by the condensate surface.

A wide range of macromolecules can undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed reversibly, and carry out essential functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics, and mechanics in time and space, that single-component FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases coexist simultaneously within the same condensate, resulting in highly inhomogeneous structures. Furthermore, our results show that this transition originates at the interface between the condensate and the dilute continuous phase, and once initiated, the gelation process propagates toward the center of the condensate. To probe such spatially inhomogeneous rheology during condensate aging, we use a combination of established micropipette aspiration experiments together with two optical techniques, spatial dynamic mapping and reflective confocal dynamic speckle microscopy. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a critical element in driving pathological protein aggregation.

Platelet accumulation in an endothelium-coated elastic vein valve model of deep vein thrombosis is mediated by GPIbα-VWF interaction.

Deep vein thrombosis is a life-threatening disease that takes millions of people's lives worldwide. Given both technical and ethical issues of using animals in research, it is necessary to develop an appropriate in vitro model that would recapitulate the conditions of venous thrombus development. We present here a novel microfluidics vein-on-a-chip with moving valve leaflets to mimic the hydrodynamics in a vein, and Human Umbilical Vein Endothelial Cell (HUVEC) monolayer. A pulsatile flow pattern, typical for veins, was used in the experiments. Unstimulated human platelets, reconstituted with the whole blood, accumulated at the luminal side of the leaflet tips proportionally to the leaflet flexibility. Platelet activation by thrombin induced robust platelet accrual at the leaflet tips. Inhibition of glycoprotein (GP) IIb-IIIa did not decrease but, paradoxically, slightly increased platelet accumulation. In contrast, blockade of the interaction between platelet GPIbα and A1 domain of von Willebrand factor completely abolished platelet deposition. Stimulation of the endothelium with histamine, a known secretagogue of Weibel-Palade bodies, promoted platelet accrual at the basal side of the leaflets, where human thrombi are usually observed. Thus, platelet deposition depends on the leaflet flexibility, and accumulation of activated platelets at the valve leaflets is mediated by GPIbα-VWF interaction.

The Effect of Bacteria on the Stability of Microfluidic-Generated Water-in-Oil Droplet.

Microencapsulation in emulsion droplets has great potential for various applications such as food which require formation of highly stable emulsions. Bacterial-emulsion interactions affect the physiological status of bacteria while bacterial cell characteristics such as surface-active properties and metabolic activity can affect emulsion stability. In this study, the viability and growth of two different bacterial species, Gram-negative Escherichia coli and Gram-positive Lactobacillus paracasei, encapsulated in water-in-oil (W/O) droplets or as planktonic cells, were monitored and their effect on droplet stability was determined. Microencapsulation of bacteria in W/O droplets with growth media or water was achieved by using a flow-focusing microfluidic device to ensure the production of highly monodispersed droplets. Stability of W/O droplets was monitored during 5 days of storage. Fluorescence microscopy was used to observe bacterial growth behaviour. Encapsulated cells showed different growth to planktonic cells. Encapsulated E. coli grew faster initially followed by a decline in viability while encapsulated L. paracasei showed a slow gradual growth throughout storage. The presence of bacteria increased droplet stability and a higher number of dead cells was found to provide better stability due to high affinity towards the interface. The stability of the droplets is also species dependent, with E. coli providing better stability as compared to Lactobacillus paracasei.

Combined Effect of Matrix Topography and Stiffness on Neutrophil Shape and Motility.

The crawling behavior of leukocytes is driven by the cell morphology transition, which is a direct manifestation of molecular motor machinery. The topographical anisotropy and mechanical stiffness of the substrates are the main physical cues that affect leukocytes' shape generation and migratory responses. However, their combined effects on the cell morphology and motility have been poorly understood, particularly for neutrophils, which are the fastest reacting leukocytes against infections and wounds. Here, spatiotemporally correlated physical parameters are shown, which determine the neutrophil shape change during migratory processes, in response to surface topography and elasticity. Guided crawling and shape generation of individual neutrophils, activated by a uniform concentration of a chemoattractant, are analyzed by adopting elasticity-tunable micropatterning and live cell imaging techniques. Whole cell-level image analysis is performed based on a planar geometric quantification of cell shape and motility. The findings show that the pattern anisotropy and elastic modulus of the substrate induce synergic effects on the shape anisotropy, deformability, and polarization/alignment of crawling neutrophils. How the morphology-motility relationship is affected by different surface microstructures and stiffness is demonstrated. These results imply that the neutrophil shape-motility correlations can be utilized for controlling the immune cell functions with predefined physical microenvironments.

Lab-on-a-Contact Lens Platforms Fabricated by Multi-Axis Femtosecond Laser Ablation.

Contact lens sensing platforms have drawn interest in the last decade for the possibility of providing a sterile, fully integrated ocular screening technology. However, designing scalable and rapid contact lens processing methods while keeping a high resolution is still an unsolved challenge. In this article, femtosecond laser writing is employed as a rapid and precise procedure to engrave microfluidic networks into commercial contact lenses. Functional microfluidic components such as flow valves, resistors, multi-inlet geometries, and splitters are produced using a bespoke seven-axis femtosecond laser system, yielding a resolution of 80 µm. The ablation process and the tear flow within microfluidic structures is evaluated both experimentally and computationally using finite element modeling. Flow velocity drops of the 8.3%, 20.8%, and 29% were observed in valves with enlargements of the 100%, 200%, and 300%, respectively. Resistors yielded flow rate drops of 20.8%, 33%, and 50% in the small, medium, and large configurations, respectively. Two applications were introduced, namely a tear volume sensor and a tear uric acid sensor (sensitivity 16 mg L-1 ), which are both painless alternatives to current methods and provide reduced contamination risks of tear samples.

Optimisation of bacterial release from a stable microfluidic-generated water-in-oil-in-water emulsion.

Application of droplet microfluidics for the encapsulation of bacteria in water-in-oil-in-water (W/O/W) emulsion allows for production of monodisperse droplets with controllable size. In this study the release of bacteria from W/O/W emulsion, the effect of the double emulsion structure on bacterial growth and metabolic activity, and the stability and mechanism of bacterial release were investigated. W/O/W emulsions were formed using a double flow-focusing junction microfluidic device under controlled pressure to produce droplets of approximately 100 µm in diameter containing an inner aqueous phase (W1) of about 40-50 µm in diameter. GFP-labelled Escherichia coli (E. coli-GFP) bacteria were encapsulated within the W1 droplets and the stability of emulsions was studied by monitoring droplet size and creaming behaviour. The double emulsions were stabilised using a hydrophilic (Tween 80) and a lipophilic surfactant (polyglycerol polyricinoleate) and were destabilised by altering the osmotic balance, adding NaCl either in the inner W1 phase (hypo-osmotic) or outer W2 phase (hyper-osmotic). The release of E. coli-GFP was monitored by plating on agar whereby the colony form unit (CFU) of the released bacteria was determined while fluorescent microscopy was employed to observe the mechanism of release from the droplets. The release of E. coli-GFP was significantly increased with higher concentrations of NaCl and lower amounts of Tween 80. Microscopic observation revealed a two-step mechanism for the release of bacteria: double W/O/W emulsion droplet splitting to release W1 droplets forming a secondary double emulsion followed by the collapse of W1 droplets to release E. coli-GFP into the continuous aqueous phase.

The role of valve stiffness in the insurgence of deep vein thrombosis.

Deep vein thrombosis is a life-threatening development of blood clots in deep veins. Immobility and blood flow stagnancy are typical risk factors indicating that fluid dynamics play an important role in the initiation of venous clots. However, the roles of physical parameters of the valves and flow conditions in deep vein thrombosis initiation have not been fully understood. Here, we describe a microfluidics in vitro method that enabled us to explore the role of valve elasticity using in situ fabrication and characterisation. In our experimental model the stiffness of each valve leaflet can be controlled independently, and various flow conditions were tested. The resulting complex flow patterns were detected using ghost particle velocimetry and linked to localised thrombus formation using whole blood and an aqueous suspension of polystyrene particles. In particular, valves with leaflets of similar stiffness had clot formation on the valve tips whereas valves with leaflets of different stiffness had clot formation in the valve pocket.

Integration of paper microfluidic sensors into contact lenses for tear fluid analysis.

In this article, using the integration of paper microfluidics within laser-inscribed commercial contact lenses, we demonstrate the multiplexed detection of clinically relevant analytes including hydrogen ions, proteins, glucose, nitrites and l-ascorbic acid, all sampled directly from model tears. In vitro measurements involved the optimization of colorimetric assays, with readouts collected, stored and analyzed using a bespoke Tears Diagnostics smartphone application prototype. We demonstrate the potential of the device to perform discrete measurements either for medical diagnosis or disease screening in the clinic or at the point-of-care (PoC), with future applications including monitoring of ocular infections, uveitis, diabetes, keratopathies and assessing oxidative stress.

Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition.

Membrane-less organelles resulting from liquid-liquid phase separation of biopolymers into intracellular condensates control essential biological functions, including messenger RNA processing, cell signalling and embryogenesis1-4. It has recently been discovered that several such protein condensates can undergo a further irreversible phase transition, forming solid nanoscale aggregates associated with neurodegenerative disease5-7. While the irreversible gelation of protein condensates is generally related to malfunction and disease, one case where the liquid-to-solid transition of protein condensates is functional, however, is that of silk spinning8,9. The formation of silk fibrils is largely driven by shear, yet it is not known what factors control the pathological gelation of functional condensates. Here we demonstrate that four proteins and one peptide system, with no function associated with fibre formation, have a strong propensity to undergo a liquid-to-solid transition when exposed to even low levels of mechanical shear once present in their liquid-liquid phase separated form. Using microfluidics to control the application of shear, we generated fibres from single-protein condensates and characterized their structural and material properties as a function of shear stress. Our results reveal generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. These observations suggest that shear can play an important role in the irreversible liquid-to-solid transition of protein condensates, shed light on the role of physical factors in driving this transition in protein aggregation-related diseases and open a new route towards artificial shear responsive biomaterials.

Microfluidic Templating of Spatially Inhomogeneous Protein Microgels.

3D scaffolds in the form of hydrogels and microgels have allowed for more native cell-culture systems to be developed relative to flat substrates. Native biological tissues are, however, usually spatially inhomogeneous and anisotropic, but regulating the spatial density of hydrogels at the microscale to mimic this inhomogeneity has been challenging to achieve. Moreover, the development of biocompatible synthesis approaches for protein-based microgels remains challenging, and typical gelation conditions include UV light, extreme pH, extreme temperature, or organic solvents, factors which can compromise the viability of cells. This study addresses these challenges by demonstrating an approach to fabricate protein microgels with controllable radial density through microfluidic mixing and physical and enzymatic crosslinking of gelatin precursor molecules. Microgels with a higher density in their cores and microgels with a higher density in their shells are demonstrated. The microgels have robust stability at 37 °C and different dissolution rates through enzymolysis, which can be further used for gradient scaffolds for 3D cell culture, enabling controlled degradability, and the release of biomolecules. The design principles of the microgels could also be exploited to generate other soft materials for applications ranging from novel protein-only micro reactors to soft robots.

Mass Transfer Accompanying Coalescence of Surfactant-Laden and Surfactant-Free Drop in a Microfluidic Channel.

The coalescence of two different drops, one surfactant-laden and the other surfactant-free, was studied under the condition of confined flow in a microchannel. The coalescence was accompanied by penetration of the surfactant-free drop into the surfactant-laden drop because of the difference in the capillary pressure and Marangoni flows causing a film of surfactant-laden liquid to spread over the surfactant-free drop. The penetration rate was dependent on the drop order, with considerably better penetration observed for the case when the surfactant-laden drop goes first. The penetration rate was found to increase with an increase of interfacial tension difference between the two drops, an increase of flow rate and drop confinement in the channel (for the case of the surfactant-laden drop going first), an increase of viscosity of the continuous phase, and a decrease of viscosity of the dispersed phase. Analysis of flow patterns inside the coalescing drops has shown that, unlike coalescence of identical drops, only two vortices are formed by asymmetrical coalescence, centered inside the surfactant-free drop. The vortices were accelerated by the flow of the continuous phase if the surfactant-laden drop preceded the surfactant-free one, increasing the rate of penetration; the opposite was observed if the drop order was reversed. The mixing patterns on a longer time scale were also dependent on the drop order, with better mixing being observed for the case when the surfactant-laden drop goes first.

Contact Lens Technology: From Fundamentals to Applications.

Contact lenses are ocular prosthetic devices used by over 150 million people worldwide. Primary applications of contact lenses include vision correction, therapeutics, and cosmetics. Contact lens materials have significantly evolved over time to minimize adverse effects associated with contact lens wearing, to maintain a regular corneal metabolism, and to preserve tear film stability. This article encompasses contact lens technology, including materials, chemical and physical properties, manufacturing processes, microbial contamination, and ocular complications. The function and the composition of the tear fluid are discussed to assess its potential as a diagnostic media. The regulatory standards of contact lens devices with regard to biocompatibility and contact lens market are presented. Future prospects in contact lens technology are evaluated, with particular interest given to theranostic applications for in situ continuous monitoring the ocular physiology.

Facile tuning of the mechanical properties of a biocompatible soft material.

Herein, we introduce a method to locally modify the mechanical properties of a soft, biocompatible material through the exploitation of the effects induced by the presence of a local temperature gradient. In our hypotheses, this induces a concentration gradient in an aqueous sodium alginate solution containing calcium carbonate particles confined within a microfluidic channel. The concentration gradient is then fixed by forming a stable calcium alginate hydrogel. The process responsible for the hydrogel formation is initiated by diffusing an acidic oil solution through a permeable membrane in a 2-layer microfluidic device, thus reducing the pH and freeing calcium ions. We characterize the gradient of mechanical properties using atomic force microscopy nanoindentation measurements for a variety of material compositions and thermal conditions. Significantly, our novel approach enables the creation of steep gradients in mechanical properties (typically between 10-100 kPa/mm) on small scales, which will be of significant use in a range of tissue engineering and cell mechanosensing studies.

Contact lenses for continuous corneal temperature monitoring.

Temperature variation is a ubiquitous medical sign to monitor ocular conditions including dry eye disease (DED), glaucoma, carotid artery stenosis, diabetic retinopathy, and vascular neuritis. The ability to measure OST in real time is desirable in point-of-care diagnostics. Here, we developed minimally invasive contact lens temperature sensors for continuous monitoring of the corneal temperature. The contact lens sensor consisted of a laser patterned commercial contact lens embedding temperature-sensitive Cholesteric Liquid Crystals (CLCs), which exhibited a fully reversible temperature-dependent color change in the visible spectrum. The contact lens allowed the corneal temperature to be mapped in four key areas, at distances of 0.0, 1.0, 3.0, and 5.0 mm from the pupil's edge. Liquid crystals exhibited a wavelength shift from 738 ± 4 nm to 474 ± 4 nm upon increasing the temperature from 29.0 °C to 40.0 °C, with a time responsivity of 490 ms and a negligible hysteresis. Readouts were performed using a smartphone, which output RGB triplets associated to temperature values. Contact lens sensors based on CLCs were fitted and tested on an ex vivo porcine eye and readouts were compared with infrared thermal measurements, resulting in an average difference of 0.3 °C.

Continuous Isotropic-Nematic Transition in Amyloid Fibril Suspensions Driven by Thermophoresis.

The isotropic and nematic (I + N) coexistence for rod-like colloids is a signature of the first-order thermodynamics nature of this phase transition. However, in the case of amyloid fibrils, the biphasic region is too small to be experimentally detected, due to their extremely high aspect ratio. Herein, we study the thermophoretic behaviour of fluorescently labelled ß-lactoglobulin amyloid fibrils by inducing a temperature gradient across a microfluidic channel. We discover that fibrils accumulate towards the hot side of the channel at the temperature range studied, thus presenting a negative Soret coefficient. By exploiting this thermophoretic behaviour, we show that it becomes possible to induce a continuous I-N transition with the I and N phases at the extremities of the channel, starting from an initially single N phase, by generating an appropriate concentration gradient along the width of the microchannel. Accordingly, we introduce a new methodology to control liquid crystal phase transitions in anisotropic colloidal suspensions. Because the induced order-order transitions are achieved under stationary conditions, this may have important implications in both applied colloidal science, such as in separation and fractionation of colloids, as well as in fundamental soft condensed matter, by widening the accessibility of target regions in the phase diagrams.

Controllable generation and encapsulation of alginate fibers using droplet-based microfluidics.

Herein we demonstrate the segmentation of alginate solution streams to generate alginate fibers of precisely controllable lengths between 200 and 1000 µm. Moreover, we demonstrate the subsequent encapsulation of the formed fibers within pL-volume microdroplets, produced within the same microfluidic device, in a direct manner. Finally, we show immediate and complete on-chip gelation of alginate fibers in a rapid and reproducible fashion.

Investigating the fluid dynamics of rapid processes within microfluidic devices using bright-field microscopy.

The widespread application of microfluidic devices in the biological and chemical sciences requires the implementation of complex designs and geometries, which in turn leads to atypical fluid dynamic phenomena. Accordingly, a complete understanding of fluid dynamics in such systems is key in the facile engineering of novel and efficient analytical tools. Herein, we present an accurate approach for studying the fluid dynamics of rapid processes within microfluidic devices using bright-field microscopy with white light illumination and a standard high-speed camera. Specifically, we combine Ghost Particle Velocimetry and the detection of moving objects in automated video surveillance to track submicron size tracing particles via cross correlation between the speckle patterns of successive images. The efficacy of the presented technique is demonstrated by measuring the flow field over a square pillar (80 µm × 80 µm) in a 200 µm wide microchannel at high volumetric flow rates. Experimental results are in excellent agreement with those obtained via computational fluid dynamics simulations. The method is subsequently used to study the dynamics of droplet generation at a flow focusing microfluidic geometry. A unique feature of the presented technique is the ability to perform velocimetry analysis of high-speed phenomena, which is not possible using micron-resolution particle image velocimetry (µPIV) approaches based on confocal or fluorescence microscopy.

Flow dependent performance of microfluidic microbial fuel cells.

The integration of Microbial Fuel Cells (MFCs) in a microfluidic geometry can significantly enhance the power density of these cells, which would have more active bacteria per unit volume. Moreover, microfluidic MFCs can be operated in a continuous mode as opposed to the traditional batch-fed mode. Here we investigate the effect of fluid flow on the performance of microfluidic MFCs. The growth and the structure of the bacterial biofilm depend to a large extent on the shear stress of the flow. We report the existence of a range of flow rates for which MFCs can achieve maximum voltage output. When operated under these optimal conditions, the power density of our microfluidic MFC is about 15 times that of a similar-size batch MFC. Furthermore, this optimum suggests a correlation between the behaviour of bacteria and fluid flow.

Unexpected trapping of particles at a T junction.

A common element in physiological flow networks, as well as most domestic and industrial piping systems, is a T junction that splits the flow into two nearly symmetric streams. It is reasonable to assume that any particles suspended in a fluid that enters the bifurcation will leave it with the fluid. Here we report experimental evidence and a theoretical description of a trapping mechanism for low-density particles in steady and pulsatile flows through T-shaped junctions. This mechanism induces accumulation of particles, which can form stable chains, or give rise to significant growth of bubbles due to coalescence. In particular, low-density material dispersed in the continuous phase fluid interacts with a vortical flow that develops at the T junction. As a result suspended particles can enter the vortices and, for a wide range of common flow conditions, the particles do not leave the bifurcation. Via 3D numerical simulations and a model of the two-phase flow we predict the location of particle accumulation, which is in excellent agreement with experimental data. We identify experimentally, as well as confirm by numerical simulations and a simple force balance, that there is a wide parameter space in which this phenomenon occurs. The trapping effect is expected to be important for the design of particle separation and fractionation devices, as well as used for better understanding of system failures in piping networks relevant to industry and physiology.