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Living systems are characterized by their spatially highly inhomogeneous nature which is susceptible to modify fundamentally the behavior of biomolecular species, including the proteins that underpin biological functionality in cells. Spatial gradients in chemical potential are known to lead to strong transport effects for colloidal particles, but their effect on molecular scale species such as proteins has remained largely unexplored. Here, we improve on existing diffusiophoresis microfluidic technique to measure protein diffusiophoresis in real space. The measurement of proteins is made possible by two ameliorations. First, a label-free microscope is used to suppress label interference. Second, improvements in numerical methods are developed to meet the particular challenges posed by small molecules. We demonstrate that individual proteins can undergo strong diffusiophoretic motion in salt gradients in a manner which is sufficient to overcome diffusion and which leads to dramatic changes in their spatial organization on the scale of a cell. Moreover, we demonstrate that this phenomenon can be used to control the motion of proteins in microfluidic devices. These results open up a path towards a physical understanding of the role of gradients in living systems in the spatial organization of macromolecules and highlight novel routes towards protein sorting applications on device.

Parkinson’s disease (PD) is an increasingly prevalent and currently incurable neurodegenerative disorder linked to the accumulation of α-synuclein (αS) protein aggregates in the nervous system. While αS binding to membranes in its monomeric state is correlated to its physiological role, αS oligomerisation 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 αS (OαS) with lipid membranes and the factors that modulate such interactions. This is in large part due to experimental challenges underlying studies of OαS-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 OαS-membrane interactions. We find that OαS bind more strongly to highly curved, rather than flat, lipid membranes. By comparing the membrane-binding properties of OαS and monomeric αS (MαS), we further demonstrate that OαS bind to membranes with up to 150-fold higher affinity than their monomeric counterparts. Moreover, OαS compete with and displace bound MαS from the membrane surface, suggesting that disruption to the functional binding of MαS to membranes may provide an additional toxicity mechanism in PD. These findings present a unique binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD.Abstract Figure

Funder: EC | EC Seventh Framework Programm | FP7 Ideas: European Research Council (FP7-IDEAS-ERC - Specific Programme: "Ideas" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011199, Funder: EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council); doi: https://doi.org/10.13039/100010663, Molecular chaperones contribute to the maintenance of cellular protein homoeostasis through assisting de novo protein folding and preventing amyloid formation. Chaperones of the Hsp70 family can further disaggregate otherwise irreversible aggregate species such as α-synuclein fibrils, which accumulate in Parkinson’s disease. However, the mechanisms and kinetics of this key functionality are only partially understood. Here, we combine microfluidic measurements with chemical kinetics to study α-synuclein disaggregation. We show that Hsc70 together with its co-chaperones DnaJB1 and Apg2 can completely reverse α-synuclein aggregation back to its soluble monomeric state. This reaction proceeds through first-order kinetics where monomer units are removed directly from the fibril ends with little contribution from intermediate fibril fragmentation steps. These findings extend our mechanistic understanding of the role of chaperones in the suppression of amyloid proliferation and in aggregate clearance, and inform on possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies.

The ability to determine the identity of specific proteins is a critical challenge in many areas of cellular and molecular biology, and in medical diagnostics. Here, we present a macine learning aided microfluidic protein characterisation strategy that within a few minutes generates a three-dimensional fingerprint of a protein sample indicative of its amino acid composition and size and, thereby, creates a unique signature for the protein. By acquiring such multidimensional fingerprints for a set of ten proteins and using machine learning approaches to classify the fingerprints, we demonstrate that this strategy allows proteins to be classified at a high accuracy, even though classification using a single dimension is not possible. Moreover, we show that the acquired fingerprints correlate with the amino acid content of the samples, which makes it is possible to identify proteins directly from their sequence without requiring any prior knowledge about the fingerprints. These findings suggest that such a multidimensional profiling strategy can lead to the development of a novel method for protein identification in a microfluidic format., Protein classification and identification from their multidimensional fingerprints obtained on a microfluidic chip.

Caenorhabditis elegans is a valuable model organism in biomedical research that has led to major discoveries in the fields of neurodegeneration, cancer and aging. Because movement phenotypes are commonly used and represent strong indicators of C. elegans fitness, there is an increasing need to replace manual assessments of worm motility with automated measurements to increase throughput and minimize observer biases. Here, we provide a protocol for the implementation of the improved wide field-of-view nematode tracking platform (WF-NTP), which enables the simultaneous analysis of hundreds of worms with respect to multiple behavioral parameters. The protocol takes only a few hours to complete, excluding the time spent culturing C. elegans, and includes (i) experimental design and preparation of samples, (ii) data recording, (iii) software management with appropriate parameter choices and (iv) post-experimental data analysis. We compare the WF-NTP with other existing worm trackers, including those having high spatial resolution. The main benefits of WF-NTP relate to the high number of worms that can be assessed at the same time on a whole-plate basis and the number of phenotypes that can be screened for simultaneously.

The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation.

Microcapsules are a key class of microscale materials with applications in areas ranging from personal care to biomedicine, and with increasing potential to act as extracellular matrix (ECM) models of hollow organs, tissues, or biomolecular condensates. Such capsules are conventionally generated from non‐ECM materials including synthetic polymers. Here, robust microcapsules with controllable shell thickness from physically‐ and enzymatically‐crosslinked gelatin are fabricated, and a core–shell architecture is achieved by exploiting a liquid–liquid phase‐separated aqueous system in a one‐step microfluidic process. Microfluidic mechanical testing reveals that the mechanical robustness of thicker‐shell capsules could be controlled through modulation of the shell thickness. Furthermore, the microcapsules demonstrate environmentally‐responsive deformation, including buckling driven by osmosis and external mechanical forces. A sequential release of cargo species is obtained through the degradation of the capsules. Stability measurements show the capsules are stable at 37 °C for more than 2 weeks. Finally, through gel–sol transition, microgels function as precursors for the formation of all‐aqueous liquid–liquid phase‐separated systems that are two‐phase or multiphase. These smart capsules that can undergo phase transition are promising models of hollow biostructures, microscale drug carriers, and building blocks or compartments for active soft materials and robots.

Significance The formation of amyloid fibrils and toxic oligomeric species has been shown to be inhibited by their interactions with molecular chaperones, thus modulating monomer sequestration and toxicity in the context of neurodegenerative diseases. Understanding the physical and chemical properties underlying chaperone binding processes is essential to explore new therapeutic strategies to target toxic amyloid species. Here, we determine that the binding of the small heat-shock protein αB-crystallin to α-synculein fibrils, a protein which is related to the progression of Parkinson’s disease, is driven by entropic forces. By applying a microfluidic platform, we accurately quantified the thermodynamics and the kinetics of this intermolecular interaction in the condensed phase and hypothesize that αB-crystallin oligomers work as an entropic buffer system., Molecular chaperones are key components of the cellular proteostasis network whose role includes the suppression of the formation and proliferation of pathogenic aggregates associated with neurodegenerative diseases. The molecular principles that allow chaperones to recognize misfolded and aggregated proteins remain, however, incompletely understood. To address this challenge, here we probe the thermodynamics and kinetics of the interactions between chaperones and protein aggregates under native solution conditions using a microfluidic platform. We focus on the binding between amyloid fibrils of α-synuclein, associated with Parkinson’s disease, to the small heat-shock protein αB-crystallin, a chaperone widely involved in the cellular stress response. We find that αB-crystallin binds to α-synuclein fibrils with high nanomolar affinity and that the binding is driven by entropy rather than enthalpy. Measurements of the change in heat capacity indicate significant entropic gain originates from the disassembly of the oligomeric chaperones that function as an entropic buffer system. These results shed light on the functional roles of chaperone oligomerization and show that chaperones are stored as inactive complexes which are capable of releasing active subunits to target aberrant misfolded species.

Membrane proteins perform a vast range of vital biological functions and are the gatekeepers for exchange of information and matter between the intracellular and extracellular environment. However, membrane protein interactions can be challenging to characterise in a quantitative manner due to the low solubility and large size of the membrane protein complex with associated lipid or detergent molecules. Here, we show that measurements of the changes in charge and diffusivity on the micron scale allow for non-disruptive studies of membrane protein interactions in solution. The approach presented here uses measurements of key physical properties of membrane proteins and their ligands to characterise the binding equilibrium parameters. We demonstrate this approach for human aquaporins (AQPs), key membrane proteins in the regulation of water homeostasis in cells. We perform quantitative measurements to characterise the interactions between two full-length AQP isoforms and the regulatory protein, calmodulin (CaM), and show that CaM selectively binds AQP0. Through direct measurements of the diffusivity and mobility in an external electric field, the diffusion coefficients and electrophoretic mobilities are determined for the individual components and the resulting AQP0-CaM complex. Furthermore, we obtain directly the binding equilibrium parameters and effective charge of each component. These results open up a route towards the use of microfluidics as a general platform in protein science and open up new possibilities for the characterisation of membrane protein interactions in solution.

Circular dichroism spectroscopy has become a powerful tool to characterise proteins and other biomolecules. For heterogeneous samples such as those present for interacting proteins, typically only average spectroscopic features can be resolved. Here we overcome this limitation by using free-flow microfluidic size separation in-line with synchrotron radiation circular dichroism to resolve the secondary structure of each component of a model protein mixture containing monomers and fibrils. To enable this objective, we have integrated far-UV compatible measurement chambers into PDMS-based microfluidic devices. Two architectures are proposed so as to accommodate for a wide range of concentrations. The approach, which can be used in combination with other bulk measurement techniques, paves the way to the study of complex mixtures such as the ones associated with protein misfolding and aggregation diseases including Alzheimer's and Parkinson's diseases.

DNA circuits form the basis of programmable molecular systems capable of signal transduction and algorithmic computation. Some classes of molecular programs, such as catalyzed hairpin assembly, enable isothermal, enzyme-free signal amplification. However, current detection limits in DNA amplification circuits are modest, as sensitivity is inhibited by signal leakage resulting from noncatalyzed background reactions inherent to the noncovalent system. Here, we overcome this challenge by optimizing a catalyzed hairpin assembly for single-molecule sensing in a digital droplet assay. Furthermore, we demonstrate digital reporting of DNA computation at the single-molecule level by employing ddCHA as a signal transducer for simple DNA logic gates. By facilitating signal transduction of molecular computation at pM concentration, our approach can improve processing density by a factor of 104 relative to conventional DNA logic gates. More broadly, we believe that digital molecular computing will broaden the scope and efficacy of isothermal amplification circuits within DNA computing, biosensing, and signal amplification in general.

Molecular chaperones contribute to the maintenance of cellular protein homeostasis through a wide range of mechanisms, including the assistance of de novo protein folding, the rescue of misfolded proteins, and the prevention of amyloid formation. Chaperones of the Hsp70 family have a striking capability of disaggregating otherwise irreversible aggregate structures such as amyloid fibrils that accumulate during the development of neurodegenerative diseases. However, the mechanisms of this key emerging functionality remain largely unknown. Here, we bring together microfluidic measurements with kinetic analysis and show that that the Hsp70 protein heat chock complement Hsc70 together with its two co-chaperones DnaJB1 and the nucleotide exchange factor Apg2 is able to completely reverse the aggregation process of alpha-synuclein, associated with Parkinson’s disease, back to its soluble monomeric state. Moreover, we show that this reaction proceeds with first order kinetics in a process where monomer units are taken off directly from the fibril ends. Our results demonstrate that all components of the chaperone triad are essential for fibril disaggregation. Lastly, we quantify the interactions between the three chaperones as well as between the chaperones and the fibrils in solution, yielding both binding stoichiometries and dissociation constants. Crucially, we find that the stoichiometry of Hsc70 binding to fibrils suggests Hsc70 clustering at the fibril ends. Taken together, our results show that the mechanism of action of the Hsc70–DnaJB1–Apg2 chaperone system in disaggregating α-synuclein fibrils involves the removal of monomer units without any intermediate fragmentation steps. These findings are fundamental to our understanding of the suppression of amyloid proliferation early in life and the natural clearance mechanisms of fibrillar deposits in Parkinson’s disease, and inform on the possibilities and limitations of this strategy in the development of therapeutics against synucleinopathies and related neurodegenerative diseases.

Caenorhabditis elegans is a valuable model organism in biomedical research that has led to major discoveries in the fields of neurodegeneration, cancer and aging. Because movement phenotypes are commonly used and represent strong indicators of C. elegans fitness, there is an increasing need to replace manual assessments of worm motility with automated measurements to increase throughput and minimize observer biases. Here, we provide a protocol for the implementation of the improved wide field-of-view nematode tracking platform (WF-NTP), which enables the simultaneous analysis of hundreds of worms with respect to multiple behavioral parameters. The protocol takes only a few hours to complete, excluding the time spent culturing C. elegans, and includes (i) experimental design and preparation of samples, (ii) data recording, (iii) software management with appropriate parameter choices and (iv) post-experimental data analysis. We compare the WF-NTP with other existing worm trackers, including those having high spatial resolution. The main benefits of WF-NTP relate to the high number of worms that can be assessed at the same time on a whole-plate basis and the number of phenotypes that can be screened for simultaneously.

Nanofluidic devices have great potential for applications in areas ranging from renewable energy to human health. A crucial requirement for the successful operation of nanofluidic devices is the ability to interface them in a scalable manner with the outside world. Here, we demonstrate a hybrid two photon nanolithography approach interfaced with conventional mask whole-wafer UV-photolithography to generate master wafers for the fabrication of integrated micro and nanofluidic devices. Using this approach we demonstrate the fabrication of molds from SU-8 photoresist with nanofluidic features down to 230 nm lateral width and channel heights from micron to sub-100 nm. Scanning electron microscopy and atomic force microscopy were used to characterize the printing capabilities of the system and show the integration of nanofluidic channels into an existing microfluidic chip design. The functionality of the devices was demonstrated through super-resolution microscopy, allowing the observation of features below the diffraction limit of light produced using our approach. Single molecule localization of diffusing dye molecules verified the successful imprint of nanochannels and the spatial confinement of molecules to 200 nm across the nanochannel molded from the master wafer. This approach integrates readily with current microfluidic fabrication methods and allows the combination of microfluidic devices with locally two-photon-written nano-sized functionalities, enabling rapid nanofluidic device fabrication and enhancement of existing microfluidic device architectures with nanofluidic features., Nanoengineering: Flexible production of micro- and nanofluidic devices A laser-based manufacturing process can produce combined nanofluidic and microfluidic devices in a rapid and scalable manner. Nanofluidic devices allow for the observation of biological processes at the single-molecule level, but current fabrication methods are slow and costly. In this article, Tuomas Knowles and his team from the UK’s University of Cambridge used conventional light-blocking masks and laser technology to craft a light-sensitive material into devices with micron to nano-sized channels. The researchers used microscopy and single-molecule tracking of dyes to verify the functionality of the final imprinted device, which also demonstrates the integration of nanofluidic channels into existing microfluidics designs. The team says that further research may optimize their new platform, which they hope will offer a reliable and flexible pathway for nanofluidic device fabrication.

Optical detection has become a convenient and scalable approach to read out information from microfluidic systems. For the study of many key biomolecules, however, including peptides and proteins, which have low fluorescence emission efficiencies at visible wavelengths, this approach typically requires labeling of the species of interest with extrinsic fluorophores to enhance the optical signal obtained - a process which can be time-consuming, requires purification steps, and has the propensity to perturb the behavior of the systems under study due to interactions between the labels and the analyte molecules. As such, the exploitation of the intrinsic fluorescence of protein molecules in the UV range of the electromagnetic spectrum is an attractive path to allow the study of unlabeled proteins. However, direct visualization using 280 nm excitation in microfluidic devices has to date commonly required the use of coherent sources with frequency multipliers and devices fabricated out of materials that are incompatible with soft lithography techniques. Here, we have developed a simple, robust, and cost-effective 280 nm LED platform that allows real-time visualization of intrinsic fluorescence from both unlabeled proteins and protein complexes in polydimethylsiloxane microfluidic channels fabricated through soft lithography. Using this platform, we demonstrate intrinsic fluorescence visualization of proteins at nanomolar concentrations on chip and combine visualization with micron-scale diffusional sizing to measure the hydrodynamic radii of individual proteins and protein complexes under their native conditions in solution in a label-free manner.

Scanning probe microscopy provides a unique window into the morphology, mechanics, and structure of proteins and their complexes on the nanoscale. Such measurements require, however, deposition of samples onto substrates. This process can affect conformations and assembly states of the molecular species under investigation and can bias the molecular populations observed in heterogeneous samples through differential adsorption. Here, we show that these limitations can be overcome with a single-step microfluidic spray deposition platform. This method transfers biological solutions to substrates as microdroplets with subpicoliter volume, drying in milliseconds, a timescale that is shorter than typical diffusion times of proteins on liquid–solid interfaces, thus avoiding surface mass transport and change to the assembly state. Finally, the single-step deposition ensures the attachment of the full molecular content of the sample to the substrate, allowing quantitative measurements of different molecular populations within heterogeneous systems, including protein aggregates.

The current flowing through a Mott spin junction depends on the relative spin orientation of the two ferromagnetic layers comprising the “source” and “drain” sides of the junction. The resulting current asymmetry is detected as giant or tunnelling magnetoresistance depending on whether the two ferromagnets are separated by a metal or an insulator. Based on the fundamental principles of reciprocity for spin-dependent electron scattering, one can envisage a one-magnet-only spin junction in which the source is non-magnetic, and the spin information is encoded by the spin polarisation of the electrons that have crossed or are backscattered from the drain magnetic layer. The practical significance of using an unpolarised source is that the state of the magnetic layer can be modified without affecting the process of probing it. Whether this reciprocity is realised in the actual junctions is not yet known. Here, we demonstrate a nano-sized, one-magnet-only Mott spin junction by measuring the finite spin polarisation of the backscattered electrons. Based on this finding, we conclude that since the junction acts as a spin filter, the magnetic layer must experience a spin transfer that could become detectable in view of the high current densities achievable in this technology., Scientific Reports, 7 (1), ISSN:2045-2322

Summary Four expandable completion liners (ECLs) have been run in Algeria in two fields—these are the first fully compliant ECLs in Algeria, the first worldwide in gas wells, and the first worldwide in multilateral wells (from Weatherford database). This paper presents the first detailed benchmark study of ECL performance. All systems to date have been run in 8½-in. hole using 5½-in. base-pipe ECL compliantly expanded, and the installations went well. It has been possible to compare ECL performance data against a comprehensive surveillance data set for the two fields where data from existing openhole completions allow meaningful comparisons. The other completions consist largely of slotted liners and some barefoot completions. The tested production rates in each of the wells have been high relative to normal field trends; however, the predominant factor in this is the reservoir character. Pressure-buildup (PBU) analysis indicates that the second well has reduced Darcy skin, while it is considered unproved in the first well (more data over time are needed). There is, however, consistent evidence for a reduction in the rate-dependent proportion of total skin in both wells. This is supported by other studies and a consideration of basic principles. The reduction in the rate-dependent proportion of skin has given an increase in production rate of 5–20% as compared with the computed rate from a slotted liner. This difference assumes no borehole collapse, with analyses and discussion presented of the effect on well performance should borehole collapse occur. The joint venture (JV) is investigating the further use of this technology in smaller hole sizes and possibly in conjunction with fracture stimulation