Parkinson's disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of alpha-synuclein (alpha S) protein aggregates in the nervous system. While alpha S binding to membranes in its monomeric state is correlated to its physiological role, alpha S oligomerization and subsequent aberrant interactions with lipid bilayers have emerged as key steps in PD-associated neurotoxicity. However, little is known of the mechanisms that govern the interactions of oligomeric alpha S (O alpha S) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of O alpha S-membrane interactions due to their dynamic and transient nature. Here, we address this challenge by using a suite of microfluidics-based assays that enable in-solution quantification of O alpha S-membrane interactions. We find that O alpha S bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of O alpha S and monomeric alpha S (M alpha S), we further demonstrate that O alpha S bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, O alpha S compete with and displace bound M alpha S from the membrane surface, suggesting that disruption to the functional binding of M alpha S to membranes may provide an additional toxicity mechanism in PD. These findings present a binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.

The biological function of proteins is dictated by the formation of supra-molecular complexes that act as the basic machinery of the cell. As such, measuring the properties of protein species in heterogeneous mixtures is of key importance for understanding the molecular basis of biological function. Here, we describe the combination of analytical microfluidic tools with liquid chromatography for multidimensional characterisation of biomolecules in complex mixtures in the solution phase. Following chromatographic separation, a small fraction of the flow-through is distributed to multiple microfluidic devices for analysis. The microfluidic device developed here allows the simultaneous determination of the hydrodynamic radius, electrophoretic mobility, effective molecular charge and isoelectric point of isolated protein species. We demonstrate the operation principle of this approach with a mixture of three unlabelled model proteins varying in size and charge. We further extend the analytical potential of the presented approach by analysing a mixture of interacting streptavidin with biotinylated BSA and fluorophores, which form a mixture of stable complexes with diverse biophysical properties and stoichiometries. The presented microfluidic device positioned in-line with liquid chromatography presents an advanced tool for characterising multidimensional physical properties of proteins in biological samples to further understand the assembly/disassembly mechanism of proteins and the nature of complex mixtures.

Microfluidic platforms provide an excellent basis for working with heterogeneous samples and separating biomolecular components at high throughput, with high recovery rates and by using only very small sample volumes. To date, several micron scale platforms with preparative capabilities have been demonstrated. Here we describe and demonstrate a microfluidic device that brings preparative and analytical operations together onto a single chip and thereby allows the acquisition of multidimensional information. We achieve this objective by using a free-flow electrophoretic separation approach that directs fractions of sample into an on-chip analysis unit, where the fractions are characterised through a microfluidic diffusional sizing process. This combined approach therefore allows simultaneously quantifying the sizes and the charges of components in heterogenous mixtures. We illustrate the power of the platform by describing the size distribution of a mixture comprising components which are close in size and cannot be identified as individual components using state-of-the-art solution sizing techniques on their own. Furthermore, we show that the platform can be used for two-dimensional fingerprinting of heterogeneous protein mixtures within tens of seconds, opening up a possibility to obtain multiparameter data on biomolecular systems on a minute timescale.

Spray atomisation is a highly versatile materials processing method that has found applications in the pharmaceutical, food, chemical, electronic and automotive industries. However, common commercial spray nozzle systems offer little processing flexibility and usually require large amount of liquid. Microfluidics-based spray nozzles, that have a small dead volume and that can incorporate mixing, filtering or dilution functions have the potential to open up interesting opportunities for advanced materials processing as well as analytical chemistry and biology. In this study, we propose a flexible 3 dimensional microfluidic spray nozzle fabricated using soft-lithography. The nozzle, whose performances are comparable with commercial systems, generates ultrafine, sub-picolitre droplets and enables the deposition of homogeneous layers over large areas. The droplets dry so quickly that no coalescence is observed for flow rates up to 500 mu L/h when the spray is positioned 8 cm from the substrate. The amount deposited can be controlled by varying the flow rate and deposition time.

Free flow electrophoresis is a versatile technique for the continuous separation of mixtures with both preparative and analytical applications. Microscale versions of free flow electrophoresis are particularly attractive strategies because of their fast separation times, ability to work with small sample volumes, and large surface area to volume ratios facilitating rapid heat transfer, thus minimizing the detrimental effects of Joule heating even at high voltages. The resolution of microscale free flow electrophoresis, however, is limited by the broadening of the analyte beam in the microfluidic channel, an effect that becomes especially pronounced when the analyte is deflected significantly away from its original position. Here, we describe and demonstrate how restricting spatially the sample injection and collection to the regions where the gradients in the velocity distribution of the carrier medium are the smallest allows this broadening effect to be substantially suppressed and hence the resolution of microscale free flow electrophoresis devices to be increased. To demonstrate this concept, we fabricated microfluidic free flow electrophoresis devices with spatially restricted injection nozzles implemented through the use of multilayer soft-photolithography and further integrated quartz based observation areas for fluorescent detection and imaging. With these devices, we demonstrated a 5-fold reduction in the extent of beam broadening relative to conventional free flow electrophoresis approaches with nonrestricted sample introduction. The manifold enhancement in the achievable resolution of microscale free flow electrophoresis devices opens up the possibility of rapid separation and analysis of complex mixtures.

Biophotovoltaic devices (BPVs), which use photosynthetic organisms as active materials to harvest light, have a range of attractive features relative to synthetic and non-biological photovoltaics, including their environmentally friendly nature and ability to self-repair. However, efficiencies of BPVs are currently lower than those of synthetic analogues. Here, we demonstrate BPVs delivering anodic power densities of over 0.5 W m−2, a value five times that for previously described BPVs. We achieved this through the use of cyanobacterial mutants with increased electron export characteristics together with a microscale flow-based design that allowed independent optimization of the charging and power delivery processes, as well as membrane-free operation by exploiting laminar flow to separate the catholyte and anolyte streams. These results suggest that miniaturization of active elements and flow control for decoupled operation and independent optimization of the core processes involved in BPV design are effective strategies for enhancing power output and thus the potential of BPVs as viable systems for sustainable energy generation. Biological photovoltaic devices (BPVs) use photosynthetic microorganisms to generate electricity, but their efficiency is low. Here the authors report power densities of over 0.5 W per m2 for a flow-based BPV system, by decoupling the charging and the power delivery units.

Many molecular chaperones exist as oligomeric complexes in their functional states, yet the physical determinants underlying such self-assembly behavior, as well as the role of oligomerization in the activity of molecular chaperones in inhibiting protein aggregation, have proven to be difficult to define. Here, we demonstrate direct measurements under native conditions of the changes in the average oligomer populations of a chaperone system as a function of concentration and time and thus determine the thermodynamic and kinetic parameters governing the self-assembly process. We access this self-assembly behavior in real time under native-like conditions by monitoring the changes in the micrometer-scale diffusion of the different complexes in time and space using a microfluidic platform. Using this approach, we find that the oligomerization mechanism of the Hsp70 subdomain occurs in a cooperative manner and involves structural constraints that limit the size of the species formed beyond the limits imposed by mass balance. These results illustrate the ability of microfluidic methods to probe polydisperse protein self-assembly in real time in solution and to shed light on the nature and dynamics of oligomerization processes.

Elucidation of the fundamental interactions of proteins with biological membranes under native conditions is crucial for understanding the molecular basis of their biological function and malfunction. Notably, the large surface to volume ratio of living cells provides a molecular landscape for significant interactions of cellular components with membranes, thereby potentially modulating their function. However, such interactions can be challenging to probe using conventional biophysical methods due to the heterogeneity of the species and processes involved. Here, we use direct measurements of micron scale molecular diffusivity to detect and quantify the interactions of α-synuclein, associated with the etiology of Parkinson's disease, with negatively charged lipid vesicles. We further demonstrate that this microfluidic approach enables the characterization of size distributions of different binary mixtures of vesicles, which are not readily accessible using conventional light scattering techniques. Finally, the size distributions of the two α-synuclein conformations, free α-synuclein and membrane-bound α-synuclein, were resolved under varying lipid:protein ratios, thus, allowing the determination of the dissociation constant and the binding stoichiometry associated with this protein-lipid system. The microfluidic diffusional sizing platform allows these measurements to be performed on a time scale of minutes using microlitre volumes, thus, establishing the basis for an approach for the study of molecular interactions of heterogeneous systems under native conditions.

The ability to apply highly controlled electric fields within microfluidic devices is valuable as a basis for preparative and analytical processes. A challenge encountered in the context of such approaches in conductive media, including aqueous buffers, is the generation of electrolysis products at the electrode/liquid interface which can lead to contamination, perturb fluid flows and generally interfere with the measurement process. Here, we address this challenge by designing a single layer microfluidic device architecture where the electric potential is applied outside and downstream of the microfluidic device while the field is propagated back to the chip via the use of a co-flowing highly conductive electrolyte solution that forms a stable interface at the separation region of the device. The co-flowing electrolyte ensures that all the generated electrolysis products, including Joule heat and gaseous products, are flowed away from the chip without coming into contact with the analytes while the single layer fabrication process where all the structures are defined lithographically allows producing the devices in a simple yet highly reproducible manner. We demonstrate that by allowing stable and effective application of electric fields in excess of 100 V cm-1, the described platform provides the basis for rapid separation of heterogeneous mixtures of proteins and protein complexes directly in their native buffers as well as for the simultaneous quantification of their charge states. We illustrate this by probing the interactions in a mixture of an amyloid forming protein, amyloid-β, and a molecular chaperone, Brichos, known to inhibit the process of amyloid formation. The availability of a platform for applying stable electric fields and its compatibility with single-layer soft-lithography processes opens up the possibility of separating and analysing a wide range of molecules on chip, including those with similar electrophoretic mobilities.

The release of nanoscale structures from microcapsules, triggered by changes in the capsule in response to external stimuli, has significant potential for active component delivery. Here, we describe an orthogonal strategy for controlling molecular species' release across oil/water interfaces by modulating their intrinsic self-assembly state. We show that although the soluble peptide Boc-FF can be stably encapsulated for days, its self-assembly into nanostructures triggers jet-like release within seconds. Moreover, we exploit this self-assembly-mediated release to deliver other molecular species that are transported as cargo. These results demonstrate the role of self-assembly in modulating the transport of peptides across interfaces.

The sensitive detection of proteins is a key objective in many areas of biomolecular science, ranging from biophysics to diagnostics. However, sensing in complex biological fluids is hindered by nonspecific interactions with off-target species. Here, we describe and demonstrate an assay that utilizes both the chemical and physical properties of the target species to achieve high selectivity in a manner not possible by chemical complementarity alone, in complex media. We achieve this objective through a combinatorial strategy, by simultaneously exploiting free-flow electrophoresis for target selection, on the basis of electrophoretic mobility, and conventional affinity-based selection. In addition, we demonstrate amplification of the resultant signal by a catalytic DNA nano circuit. This approach brings together the inherent solution-phase advantages of microfluidic sample handling with isothermal, enzyme-free signal amplification. With this method, no surface immobilization or washing steps are required, and our assay is well suited to monoepitopic targets, presenting advantages over conventional ELISA techniques.

Background: The nematode worm C. elegans is a model organism widely used for studies of genetics and of human disease. The health and fitness of the worms can be quantified in different ways, such as by measuring their bending frequency, speed or lifespan. Manual assays, however, are time consuming and limited in their scope providing a strong motivation for automation. New method: We describe the development and application of an advanced machine vision system for characterising the behaviour of C. elegans, the Wide Field-of-View Nematode Tracking Platform (WF-NTP), which enables massively parallel data acquisition and automated multi-parameter behavioural profiling of thousands of worms simultaneously. Results: We screened more than a million worms from several established models of neurodegenerative disorders and characterised the effects of potential therapeutic molecules for Alzheimer's and Parkinson's diseases. By using very large numbers of animals we show that the sensitivity and reproducibility of behavioural assays is very greatly increased. The results reveal the ability of this platform to detect even subtle phenotypes. Comparison with existing methods: The WF-NTP method has substantially greater capacity compared to current automated platforms that typically either focus on characterising single worms at high resolution or tracking the properties of populations of less than 50 animals. Conclusions: The WF-NTP extends significantly the power of existing automated platforms by combining enhanced optical imaging techniques with an advanced software platform. We anticipate that this approach will further extend the scope and utility of C. elegans as a model organism. (C) 2018 Published by Elsevier B.V.

With the push to reduce in vivo approaches, the demand for microphysiological models that recapitulate the in vivo settings in vitro is dramatically increasing. Here, we present an extracellular matrix-integrated microfluidic chip with a rounded microvessel of ~100 µm in diameter. Our system displays favorable characteristics for broad user adaptation: simplified procedure for vessel creation, minimised use of reagents and cells, and the ability to couple live-cell imaging and image analysis to study dynamics of cell-microenvironment interactions in 3D. Using this platform, the dynamic process of single breast cancer cells (LM2-4175) exiting the vessel lumen into the surrounding extracellular matrix was tracked. Here, we show that the presence of endothelial lining significantly reduced the cancer exit events over the 15-hour imaging period: there were either no cancer cells exiting, or the fraction of spontaneous exits was positively correlated with the number of cancer cells in proximity to the endothelial barrier. The capability to map the z-position of individual cancer cells within a 3D vessel lumen enabled us to observe cancer cell transmigration ‘hot spot’ dynamically. We also suggest the variations in the microvessel qualities may lead to the two distinct types of cancer transmigration behaviour. Our findings provide a tractable in vitro model applicable to other areas of microvascular research.

Naturally spun silks generate fibres with unique properties, including strength, elasticity and biocompatibility. Here we describe a microfluidics-based strategy to spin liquid native silk, obtained directly from the silk gland of Bombyx mori silkworms, into micron-scale capsules with controllable geometry and variable levels of intermolecular β-sheet content in their protein shells. We demonstrate that such micrococoons can store internally the otherwise highly unstable liquid native silk for several months and without apparent effect on its functionality. We further demonstrate that these native silk micrococoons enable the effective encapsulation, storage and release of other aggregation-prone proteins, such as functional antibodies. These results show that native silk micrococoons are capable of preserving the full activity of sensitive cargo proteins that can aggregate and lose function under conditions of bulk storage, and thus represent an attractive class of materials for the storage and release of active biomolecules.

The isoelectric point (pI) of a protein is a key characteristic that influences its overall electrostatic behaviour. The majority of conventional methods for the determination of the isoelectric point of a molecule rely on the use of spatial gradients in pH, although significant practical challenges are associated with such techniques, notably the difficulty in generating a stable and well controlled pH gradient. Here, we introduce a gradient-free approach, exploiting a microfluidic platform which allows us to perform rapid pH change on chip and probe the electrophoretic mobility of species in a controlled field. In particular, in this approach, the pH of the electrolyte solution is modulated in time rather than in space, as in the case for conventional determinations of the isoelectric point. To demonstrate the general approachability of this platform, we have measured the isoelectric points of representative set of seven proteins, bovine serum albumin, β-lactoglobulin, ribonuclease A, ovalbumin, human transferrin, ubiquitin and myoglobin in microlitre sample volumes. The ability to conduct measurements in free solution thus provides the basis for the rapid determination of isoelectric points of proteins under a wide variety of solution conditions and in small volumes.

Conventional approaches for simulating steady-state distributions of dilute particles under diffusive and advective transport involve solving the diffusion and advection equations in at least two dimensions. Here, we present an alternative computational strategy by combining a particle-based rather than a field-based approach with the initialisation of particles in proportion to their flux. This method allows accurate prediction of the steady state and is applicable even at intermediate and high Peclet numbers (Pe > 1) where traditional particle-based Monte-Carlo methods starting from randomly initialised particle distributions fail. We demonstrate that generating a flux of particles according to a predetermined density and velocity distribution at a single fixed time and initial location allows for accurate simulation of mass transport under flow. Specifically, upon initialisation in proportion to their flux, these particles are propagated individually and detected by summing up their Monte-Carlo trajectories in predefined detection regions. We demonstrate quantitative agreement of the predicted concentration profiles with the results of experiments performed with fluorescent particles in microfluidic channels under continuous flow. This approach is computationally advantageous and readily allows non-trivial initial distributions to be considered. In particular, this method is highly suitable for simulating advective and diffusive transport in microfluidic devices, for instance in the context of diffusive sizing.

The measurement of molecular interactions with pathological protein aggregates, including amyloid fibrils, is of central importance in the context of the development of diagnostic and therapeutic strategies against protein misfolding disorders. Probing such interactions by conventional methods can, however, be challenging because of the supramolecular nature of protein aggregates, their heterogeneity, and their often dynamic nature. Here we demonstrate that direct measurement of diffusion on a microfluidic platform enables the determination of affinity and kinetics data for ligand binding to amyloid fibrils in solution. This method yields rapid binding information from only microlitres of sample, and is therefore a powerful technique for identifying and characterising molecular species with potential therapeutic or diagnostic application.

Protein nanofibrils were first discovered in the context of misfolding and neurodegenerative diseases but have recently been found in naturally occurring functional materials including algal adhesives, bacterial coatings, and even mammalian melanosomes. These physiologically beneficial roles have led to the exploration of their use as the basis for artificial protein-based functional materials for a range of applications as bioscaffolds and carrier agents. In this work, we fabricate core-shell protein microgels stabilized by protein fibrillation with hierarchical structuring on scales ranging from a few nanometers to tens of microns. With the aid of droplet microfluidics, we exploit fibrillar protein self-assembly together with the aqueous phase separation of a polysaccharide and polyethylene glycol to control the internal structure of the microgels on the micro- and nanoscales. We further elucidate the local composition, morphology, and structural characteristics of the microgels and demonstrate a potential application of core-shell protein microgels for controlling the storage and sequential release of small drug-like molecules. The controlled self-assembly of protein nanofibrils into hierarchical structures can be used in this manner to generate a class of nanomaterials with a range of potential functions and applications.

Increasingly prevalent neurodegenerative diseases are associated with the formation of nanoscale amyloid aggregates from normally soluble peptides and proteins. A widely used strategy for following the aggregation process and defining its kinetics involves the use of extrinsic dyes that undergo a spectral shift when bound to β-sheet-rich aggregates. An attractive route to carry out such studies is to perform ex situ assays, where the dye molecules are not present in the reaction mixture, but instead are only introduced into aliquots taken from the reaction at regular time intervals to avoid the possibility that the dye molecules interfere with the aggregation process. However, such ex situ measurements are time-consuming to perform, require large sample volumes, and do not provide for real-time observation of aggregation phenomena. To overcome these limitations, here we have designed and fabricated microfluidic devices that offer continuous and automated real-time ex situ tracking of the protein aggregation process. This device allows us to improve the time resolution of ex situ aggregation assays relative to conventional assays by more than one order of magnitude. The availability of an automated system for tracking the progress of protein aggregation reactions without the presence of marker molecules in the reaction mixtures opens up the possibility of routine noninvasive study of protein aggregation phenomena.