Single-Molecule Protein Analysis by Centrifugal Droplet Immuno-PCR with Magnetic Nanoparticles.

Detecting proteins in ultralow concentrations in complex media is important for many applications but often relies on complicated techniques. Herein, a single-molecule protein analyzer with the potential for high-throughput applications is reported. Gold-coated magnetic nanoparticles with DNA-labeled antibodies were used for target recognition and separation. The immunocomplex was loaded into microdroplets generated with centrifugation. Immuno-PCR amplification of the DNA enabled the quantification of proteins at the level of single molecules. As an example, ultrasensitive detection of α-synuclein, a biomarker for neurodegenerative diseases, is achieved. The limit of detection was determined to be ∼50 aM in buffer and ∼170 aM in serum. The method exhibited high specificity and could be used to analyze post-translational modifications such as protein phosphorylation. This study will inspire wider studies on single-molecule protein detection, especially in disease diagnostics, biomarker discovery, and drug development.

Monodisperse Microshell Structured Gelatin Microparticles for Temporary Chemoembolization.

Embolization is a nonsurgical, minimally invasive procedure that deliberately blocks a blood vessel. Although several embolic particles have been commercialized, their much wider applications have been hampered owing mainly to particle size variation and uncontrollable degradation kinetics. Herein we introduce a microfluidic approach to fabricate highly monodisperse gelatin microparticles (GMPs) with a microshell structure. For this purpose, we fabricate uniform gelatin emulsion precursors using a microfluidic technique and consecutively cross-link them by inbound diffusion of glutaraldehyde from the oil continuous phase to the suspending gelatin precursor droplets. A model micromechanic study, carried out in an artificial blood vessel, demonstrates that the extraordinary degradation kinetics of the GMPs, which stems from the microshell structure, enables controlled rupturing while exhibiting drug release under temporary chemoembolic conditions.

Compression and Reswelling of Microgel Particles after an Osmotic Shock.

We use dedicated microfluidic devices to expose soft hydrogel particles to a rapid change in the externally applied osmotic pressure and observe a surprising, nonmonotonic response: After an initial rapid compression, the particle slowly reswells to approximately its original size. We theoretically account for this behavior, enabling us to extract important material properties from a single microfluidic experiment, including the compressive modulus, the gel permeability, and the diffusivity of the osmolyte inside the gel. We expect our approach to be relevant to applications such as controlled release, chromatography, and responsive materials.

Long-range repulsion of colloids driven by ion exchange and diffusiophoresis.

Interactions between surfaces and particles in aqueous suspension are usually limited to distances smaller than 1 µm. However, in a range of studies from different disciplines, repulsion of particles has been observed over distances of up to hundreds of micrometers, in the absence of any additional external fields. Although a range of hypotheses have been suggested to account for such behavior, the physical mechanisms responsible for the phenomenon still remain unclear. To identify and isolate these mechanisms, we perform detailed experiments on a well-defined experimental system, using a setup that minimizes the effects of gravity and convection. Our experiments clearly indicate that the observed long-range repulsion is driven by a combination of ion exchange, ion diffusion, and diffusiophoresis. We develop a simple model that accounts for our data; this description is expected to be directly applicable to a wide range of systems exhibiting similar long-range forces.

Convection associated with exclusion zone formation in colloidal suspensions.

The long-range repulsion of colloids from various interfaces has been observed in a wide range of studies from different research disciplines. This so-called exclusion zone (EZ) formation occurs near surfaces such as hydrogels, polymers, or biological tissues. It was recently shown that the underlying physical mechanism leading to this long-range repulsion is a combination of ion-exchange at the interface, diffusion of ions, and diffusiophoresis of colloids in the resulting ion concentration gradients. In this paper, we show that the same ion concentration gradients that lead to exclusion zone formation also imply that diffusioosmosis near the walls of the sample cell must occur. This should lead to convective flow patterns that are directly associated with exclusion zone formation. We use multi-particle tracking to study the dynamics of particles during exclusion zone formation in detail, confirming that indeed two pronounced vortex-like convection rolls occur near the cell walls. These dramatic flow patterns persist for more than 4 hours, with the typical velocity decreasing as a function of time. We find that the flow velocity depends strongly on the surface properties of the sample cell walls, consistent with diffusioosmosis being the main physical mechanism that governs these convective flows.

Soft colloids make strong glasses.

Glass formation in colloidal suspensions has many of the hallmarks of glass formation in molecular materials. For hard-sphere colloids, which interact only as a result of excluded volume, phase behaviour is controlled by volume fraction, phi; an increase in phi drives the system towards its glassy state, analogously to a decrease in temperature, T, in molecular systems. When phi increases above phi* approximately 0.53, the viscosity starts to increase significantly, and the system eventually moves out of equilibrium at the glass transition, phi(g) approximately 0.58, where particle crowding greatly restricts structural relaxation. The large particle size makes it possible to study both structure and dynamics with light scattering and imaging; colloidal suspensions have therefore provided considerable insight into the glass transition. However, hard-sphere colloidal suspensions do not exhibit the same diversity of behaviour as molecular glasses. This is highlighted by the wide variation in behaviour observed for the viscosity or structural relaxation time, tau(alpha), when the glassy state is approached in supercooled molecular liquids. This variation is characterized by the unifying concept of fragility, which has spurred the search for a 'universal' description of dynamic arrest in glass-forming liquids. For 'fragile' liquids, tau(alpha) is highly sensitive to changes in T, whereas non-fragile, or 'strong', liquids show a much lower T sensitivity. In contrast, hard-sphere colloidal suspensions are restricted to fragile behaviour, as determined by their phi dependence, ultimately limiting their utility in the study of the glass transition. Here we show that deformable colloidal particles, when studied through their concentration dependence at fixed temperature, do exhibit the same variation in fragility as that observed in the T dependence of molecular liquids at fixed volume. Their fragility is dictated by elastic properties on the scale of individual colloidal particles. Furthermore, we find an equivalent effect in molecular systems, where elasticity directly reflects fragility. Colloidal suspensions may thus provide new insight into glass formation in molecular systems.

Tough stimuli-responsive supramolecular hydrogels with hydrogen-bonding network junctions.

Hydrogels were prepared with physical cross-links comprising 2-ureido-4[1H]-pyrimidinone (UPy) hydrogen-bonding units within the backbone of segmented amphiphilic macromolecules having hydrophilic poly(ethylene glycol) (PEG). The bulk materials adopt nanoscopic physical cross-links composed of UPy-UPy dimers embedded in segregated hydrophobic domains dispersed within the PEG matrix as comfirmed by cryo-electron microscopy. The amphiphilic network was swollen with high weight fractions of water (w(H2O) ≈ 0.8) owing to the high PEG weight fraction within the pristine polymers (w(PEG) ≈ 0.9). Two different PEG chain lengths were investigated and illustrate the corresponding consequences of cross-link density on mechanical properties. The resulting hydrogels exhibited high strength and resilience upon deformation, consistent with a microphase separated network, in which the UPy-UPy interactions were adequately shielded within hydrophobic nanoscale pockets that maintain the network despite extensive water content. The cumulative result is a series of tough hydrogels with tunable mechanical properties and tractable synthetic preparation and processing. Furthermore, the melting transition of PEG in the dry polymer was shown to be an effective stimulus for shape memory behavior.

Capillary micromechanics for core-shell particles.

In this work, we have developed a facile, economical microfluidic approach as well as a simple model description to measure and predict the mechanical properties of composite core-shell microparticles made from materials with dramatically different elastic properties. By forcing the particles through a tapered capillary and analyzing their deformation, the shear and compressive moduli can be measured in one single experiment. We have also formulated theoretical models that accurately capture the moduli of the microparticles in both the elastic and the non-linear deformation regimes. Our results show how the moduli of these core-shell structures depend on the material composition of the core-shell microparticles, as well as on their microstructures. The proposed technique and the understanding enabled by it also provide valuable insights into the mechanical behavior of analogous biomaterials, such as liposomes and cells.

Topochemical polymerization in self-assembled rodlike micelles of bisurea bolaamphiphiles.

Rod-like micelles, formed from bolaamphiphiles with oligo(ethylene oxide) hydrophilic outer segments and a hydrophobic segment with diacetylene flanked by two urea moieties, were covalently fixated by topochemical photopolymerization to high degrees of polymerization by optimizing the hydrophobic core and the hydrophilic periphery of the bolaamphiphiles. Analysis of the polymerized product with dynamic light scattering in chloroform showed degrees of polymerization of approximately 250. Cryo-TEMof bolaamphiphiles before and after UV irradiation showed that the morphology of the rods was conserved upon topochemical polymerization.

Mesoscale characterization of supramolecular transient networks using SAXS and rheology.

Hydrogels and, in particular, supramolecular hydrogels show promising properties for application in regenerative medicine because of their ability to adapt to the natural environment these materials are brought into. However, only few studies focus on the structure-property relationships in supramolecular hydrogels. Here, we study in detail both the structure and the mechanical properties of such a network, composed of poly(ethylene glycol), end-functionalized with ureido-pyrimidinone fourfold hydrogen bonding units. This network is responsive to triggers such as concentration, temperature and pH. To obtain more insight into the sol-gel transition of the system, both rheology and small-angle X-ray scattering (SAXS) are used. We show that the sol-gel transitions based on these three triggers, as measured by rheology, coincide with the appearance of a structural feature in SAXS. We attribute this feature to the presence of hydrophobic domains where cross-links are formed. These results provide more insight into the mechanism of network formation in these materials, which can be exploited for tailoring their behavior for biomedical applications, where one of the triggers discussed might be used.

Interplay of electrokinetic effects in nonpolar solvents for electronic paper displays.

HYPOTHESIS: Electronic paper displays rely on electrokinetic effects in nonpolar solvents to drive the displacement of colloidal particles within a fluidic cell. While Electrophoresis (EP) is a well-established and frequently employed phenomenon, electro-osmosis (EO), which drives fluid flow along charged solid surfaces, has not been studied as extensively. We hypothesize that by exploiting the interplay between these effects, an enhanced particle transport can be achieved. EXPERIMENTS: In this study, we experimentally investigate the combined effects of EP and EO for colloidal particles in non-polar solvents, driven by an electric field. We use astigmatism micro-particle tracking velocimetry (A-µPTV) to measure the motion of charged particles within model fluidic cells. Using a simple approach that relies on basic fluid flow properties we extract the contributions due to EP and EO, finding that EO contributes significantly to particle transport. The validity of our approach is confirmed by measurements on particles with different magnitudes of charge, and by comparison to numerical simulations. FINDINGS: We find that EO flows can play a dominant role in the transport of particles in electrokinetic display devices. This can be exploited to speed up particle transport, potentially yielding displays with significantly faster switching times.

Single Hydrogel Particle Mechanics and Dynamics Studied by Combining Capillary Micromechanics with Osmotic Compression.

Hydrogels can exhibit a remarkably complex response to external stimuli and show rich mechanical behavior. Previous studies of the mechanics of hydrogel particles have generally focused on their static, rather than dynamic, response, as traditional methods for measuring single particle response at the microscopic scale cannot readily measure time-dependent mechanics. Here, we study both the static and the time-dependent response of a single batch of polyacrylamide (PAAm) particles by combining direct contact forces, applied by using Capillary Micromechanics, a method where particles are deformed in a tapered capillary, and osmotic forces are applied by a high molecular weight dextran solution. We found higher values of the static compressive and shear elastic moduli for particles exposed to dextran, as compared to water (KDex≈63 kPa vs. Kwater≈36 kPa, and GDex≈16 kPa vs. Gwater≈7 kPa), which we accounted for, theoretically, as being the result of the increased internal polymer concentration. For the dynamic response, we observed surprising behavior, not readily explained by poroelastic theories. The particles exposed to dextran solutions deformed more slowly under applied external forces than did those suspended in water (τDex≈90 s vs. τwater≈15 s). The theoretical expectation was the opposite. However, we could account for this behaviour by considering the diffusion of dextran molecules in the surrounding solution, which we found to dominate the compression dynamics of our hydrogel particles suspended in dextran solutions.

Magnetically actuated glaucoma drainage device for regulating intraocular pressure after implantation.

The key risk factor for glaucoma is increased intraocular pressure (IOP). Glaucoma drainage devices implanted in the eye can reduce IOP and thus stop disease progression. However, most devices currently used in clinical practice are passive and do not allow for postsurgical IOP control, which may result in serious complications such as hypotony (i.e., excessively low IOP). To enable noninvasive IOP control, we demonstrate a novel, miniature glaucoma implant that will enable the repeated adjustment of the hydrodynamic resistance after implantation. This is achieved by integrating a magnetic microvalve containing a micropencil-shaped plug that is moved using an external magnet, thereby opening or closing fluidic channels. The microplug is made from biocompatible poly(styrene-block-isobutylene-block-styrene) (SIBS) containing iron microparticles. The complete implant consists of an SIBS drainage tube and a housing element containing the microvalve and fabricated with hot embossing using femtosecond laser-machined glass molds. Using in vitro and ex vivo microfluidic experiments, we demonstrate that when the microvalve is closed, it can provide sufficient hydrodynamic resistance to overcome hypotony. Valve function is repeatable and stable over time. Due to its small size, our implant is a promising, safe, easy-to-implant, minimally invasive glaucoma surgery device.

Injectable hydrogels from segmented PEG-bisurea copolymers.

We describe the preparation of an injectable, biocompatible, and elastic segmented copolymer hydrogel for biomedical applications, with segmented hydrophobic bisurea hard segments and hydrophilic PEG segments. The segmented copolymers were obtained by the step growth polymerization of amino-terminated PEG and aliphatic diisocyanate. Due to their capacity for multiple hydrogen bonding within the hydrophobic segments, these copolymers can form highly stable gels in water at low concentrations. Moreover, the gels show shear thinning by a factor of 40 at large strain, which allows injection through narrow gauge needles. Hydrogel moduli are highly tunable via the physical cross-link density and the length of the hydrophilic segments. In particular, the mechanical properties can be optimized to match the properties of biological host tissues such as muscle tissue and the extracellular matrix.

An integrated system for automated measurement of airborne pollen based on electrostatic enrichment and image analysis with machine vision.

Here we describe an automated and compact pollen detection system that integrates enrichment, in-situ detection and self-cleaning modules. The system can achieve continuous capture and enrichment of pollen grains in air samples by electrostatic adsorption. The captured pollen grains are imaged with a digital camera, and an automated image analysis based on machine vision is performed, which enables a quantification of the number of pollen particles as well as a preliminary classification into two types of pollen grains. In order to optimize and evaluate the system performance, we developed a testing approach that utilizes an airflow containing a precisely metered amount of pollen particles surrounded by a sheath flow to achieve the generation and lossless transmission of standard gas samples. We studied various factors affecting the pollen capture efficiency, including the applied voltage, air flow rate and humidity. Under optimized conditions, the system was successfully used in the measurement of airborne pollen particles within a wide range of concentrations, spanning 3 orders of magnitude.

A model for designing intraocular pressure-regulating glaucoma implants.

Glaucoma is a group of eye conditions that damage the optic nerve, the health of which is vital for vision. The key risk factor for the development and progression of this disease is increased intraocular pressure (IOP). Implantable glaucoma drainage devices have been developed to divert aqueous humor from the glaucomatous eye as a means of reducing IOP. The artificial drainage pathway created by these devices drives the fluid into a filtering bleb. The long-term success of filtration surgery is dictated by the proper functioning of the bleb and overlying Tenon's and conjunctival tissue. To better understand the influence of the health condition of these tissues on IOP, we have developed a mathematical model of fluid production in the eye, its removal from the anterior chamber by a particular glaucoma implant-the PRESERFLO® MicroShunt-, drainage into the bleb and absorption by the subconjunctival vasculature. The mathematical model was numerically solved by commercial FEM package COMSOL. Our numerical results of IOP for different postoperative conditions are consistent with the available evidence on IOP outcomes after the implantation of this device. To obtain insight into the adjustments in the implant's hydrodynamic resistance that are required for IOP control when hypotony or bleb scarring due to tissue fibrosis take place, we have simulated the flow through a microshunt with an adjustable lumen diameter. Our findings show that increasing the hydrodynamic resistance of the microshunt by reducing the lumen diameter, can effectively help to prevent hypotony. However, decreasing the hydrodynamic resistance of the implant will not sufficiently decrease the IOP to acceptable levels when the bleb is encapsulated due to tissue fibrosis. Therefore, to effectively reduce IOP, the adjustable glaucoma implant should be combined with a means of reducing fibrosis. The results reported herein may provide guidelines to support the design of future glaucoma implants with adjustable hydrodynamic resistances.

Biophysical properties of normal and diseased renal glomeruli.

The mechanical properties of tissues and cells including renal glomeruli are important determinants of their differentiated state, function, and responses to injury but are not well characterized or understood. Understanding glomerular mechanics is important for understanding renal diseases attributable to abnormal expression or assembly of structural proteins and abnormal hemodynamics. We use atomic force microscopy (AFM) and a new technique, capillary micromechanics, to measure the elastic properties of rat glomeruli. The Young's modulus of glomeruli was 2,500 Pa, and it was reduced to 1,100 Pa by cytochalasin and latunculin, and to 1,400 Pa by blebbistatin. Cytochalasin or latrunculin reduced the F/G actin ratios of glomeruli but did not disrupt their architecture. To assess glomerular biomechanics in disease, we measured the Young's moduli of glomeruli from two mouse models of primary glomerular disease, Col4a3(-/-) mice (Alport model) and Tg26(HIV/nl) mice (HIV-associated nephropathy model), at stages where glomerular injury was minimal by histopathology. Col4a3(-/-) mice express abnormal glomerular basement membrane proteins, and Tg26(HIV/nl) mouse podocytes have multiple abnormalities in morphology, adhesion, and cytoskeletal structure. In both models, the Young's modulus of the glomeruli was reduced by 30%. We find that glomeruli have specific and quantifiable biomechanical properties that are dependent on the state of the actin cytoskeleton and nonmuscle myosins. These properties may be altered early in disease and represent an important early component of disease. This increased deformability of glomeruli could directly contribute to disease by permitting increased distension with hemodynamic force or represent a mechanically inhospitable environment for glomerular cells.

Conventional glaucoma implants and the new MIGS devices: a comprehensive review of current options and future directions.

Glaucoma is a progressive optic neuropathy that is the second leading cause of preventable blindness worldwide, after cataract formation. A rise in the intraocular pressure (IOP) is considered to be a major risk factor for glaucoma and is associated with an abnormal increase of resistance to aqueous humour outflow from the anterior chamber. Glaucoma drainage devices have been developed to provide an alternative pathway through which aqueous humour can effectively exit the anterior chamber, thereby reducing IOP. These devices include the traditional aqueous shunts with tube-plate design, as well as more recent implants, such as the trabeculectomy-modifying EX-PRESS® implant and the new minimally invasive glaucoma surgery (MIGS) devices. In this review, we will describe each implant in detail, focusing on their efficacy in reducing IOP and safety profile. Additionally, a critical and evidence-based comparison between these implants will be provided. Finally, we will propose potential developments that may help to improve the performance of current devices.

Compression and swelling of hydrogels in polymer solutions: A dominant-mode model.

The swelling and compression of hydrogels in polymer solutions can be understood by considering hydrogel-osmolyte-solvent interactions which determine the osmotic pressure difference between the inside and the outside of a hydrogel particle and the changes in effective solvent quality for the hydrogel network. Using the theory of poroelasticity, we find the exact solution to hydrogel dynamics in a dilute polymer solution, which quantifies the effect of diffusion and partitioning of osmolyte and the related solvent quality change to the volumetric changes of the hydrogel network. By making a dominant-mode assumption, we propose a model for the swelling and compression dynamics of (spherical) hydrogels in concentrated polymer solutions. Osmolyte diffusion induces a biexponential response in the size of the hydrogel radius, whereas osmolyte partitioning and solvent quality effects induce monoexponential responses. Comparison of the dominant-mode model to experiments provides reasonable values for the compressive bulk modulus of a hydrogel particle, the permeability of the hydrogel network, and the diffusion constant of osmolyte molecules inside the hydrogel network. Our model shows that hydrogel-osmolyte interactions can be described in a conceptually simple manner, while still capturing the rich (de)swelling behaviors observed in experiments. We expect our approach to provide a roadmap for further research into and applications of hydrogel dynamics induced by, for example, changes in the temperature and the pH.