Rapid highly sensitive general protein quantification through on-chip chemiluminescence.

Protein detection and quantification is a routinely performed procedure in research laboratories, predominantly executed either by spectroscopy-based measurements, such as NanoDrop, or by colorimetric assays. The detection limits of such assays, however, are limited to μ M concentrations. To establish an approach that achieves general protein detection at an enhanced sensitivity and without necessitating the requirement for signal amplification steps or a multicomponent detection system, here, we established a chemiluminescence-based protein detection assay. Our assay specifically targeted primary amines in proteins, which permitted characterization of any protein sample and, moreover, its latent nature eliminated the requirement for washing steps providing a simple route to implementation. Additionally, the use of a chemiluminescence-based readout ensured that the assay could be operated in an excitation source-free manner, which did not only permit an enhanced sensitivity due to a reduced background signal but also allowed for the use of a very simple optical setup comprising only an objective and a detection element. Using this assay, we demonstrated quantitative protein detection over a concentration range of five orders of magnitude and down to a high sensitivity of 10 pg mL - 1 , corresponding to pM concentrations. The capability of the platform presented here to achieve a high detection sensitivity without the requirement for a multistep operation or a multicomponent optical system sets the basis for a simple yet universal and sensitive protein detection strategy.Engineering and Physical Sciences Research Council Schmidt Science Fellows program in partnership with the Rhodes Trust European Research Council Newman Foundatio

Fluctuations in the Kinetics of Linear Protein Self-Assembly.

Biological systems are characterized by compartmentalization from the subcellular to the tissue level, and thus reactions in small volumes are ubiquitous in living systems. Under such conditions, statistical number fluctuations, which are commonly negligible in bulk reactions, can become dominant and lead to stochastic behavior. We present here a stochastic model of protein filament formation in small volumes. We show that two principal regimes emerge for the system behavior, a small fluctuation regime close to bulk behavior and a large fluctuation regime characterized by single rare events. Our analysis shows that in both regimes the reaction lag-time scales inversely with the system volume, unlike in bulk. Finally, we use our stochastic model to connect data from small-volume microdroplet experiments of amyloid formation to bulk aggregation rates, and show that digital analysis of an ensemble of protein aggregation reactions taking place under microconfinement provides an accurate measure of the rate of primary nucleation of protein aggregates, a process that has been challenging to quantify from conventional bulk experiments.We are grateful to St. John’s College, Cambridge (T. C. T. M., J. B. K.), the Schiff Foundation (A. J. D.), the EPSRC (K. L. S.), NSF Grant No. DMR-1310266 (D. A. W.), the Harvard MRSEC Grant No. DMR1420570 (D. A. W.), BBSRC (T. P. J. K.), ERC (T. C. T. M., T. P. J. K.), and Frances and Augustus Newman Foundation (T. P. J. K.) for financial support

Microfluidic approaches for probing amyloid assembly and behaviour.

The self-assembly of proteins into supramolecular structures and machinery underpins biological activity in living systems. Misassembled, misfolded and aggregated protein structures can, by contrast, have deleterious activity and such species are at the origin of a number of disease states ranging from cancer to neurodegenerative disorders. In particular, the formation of highly ordered protein aggregates, amyloid fibrils, from normally soluble peptides and proteins, is the common pathological hallmark of a range a group of over fifty protein misfolding disorders. Because of the critical role of the process in the aetiology of such disorders, as well as the quest to understand the basic principles of protein folding and misfolding, the amyloid phenomenon has become a central area of modern biomedical research. Advances in our knowledge of the physical properties of amyloid systems have, however, also highlighted the potential of amyloid structures in the context of materials science. In this review, we explore how microfluidic approaches can be used to study aspects of amyloid assembly and behaviour that are challenging to probe under bulk solution conditions. We discuss the use of volume confinement to probe very early events in the amyloid formation process. In addition, the well-defined fluid flow properties within channels with dimensions on the micron scale can be exploited to measure the physical properties of protein aggregates, such as their sizes and charges, to shed light on the physical and chemical parameters defining amyloid species. Moreover, the molecular species formed during aggregation reactions have physical dimensions spanning at least three orders of magnitude, and microfluidic techniques are well suited to work with analytes of such disparate dimensions. Furthermore, the flexibility of the design of microfluidic devices lends itself to adaptable experimental setups, including the study of protein self-assembly within living cells. Finally, we highlight the salient features of microfluidic experiments that facilitate probing complex biological systems, and discuss their use in the exploration of amyloids as a class of functional material

On-chip label-free protein analysis with downstream electrodes for direct removal of electrolysis products.

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

Real-Time Intrinsic Fluorescence Visualization and Sizing of Proteins and Protein Complexes in Microfluidic Devices.

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

Rapid Structural, Kinetic, and Immunochemical Analysis of Alpha-Synuclein Oligomers in Solution.

Oligomers comprised of misfolded proteins are implicated as neurotoxins in the pathogenesis of protein misfolding conditions such as Parkinson's and Alzheimer's diseases. Structural, biophysical, and biochemical characterization of these nanoscale protein assemblies is key to understanding their pathology and the design of therapeutic interventions, yet it is challenging due to their heterogeneous, transient nature and low relative abundance in complex mixtures. Here, we demonstrate separation of heterogeneous populations of oligomeric α-synuclein, a protein central to the pathology of Parkinson's disease, in solution using microfluidic free-flow electrophoresis. We characterize nanoscale structural heterogeneity of transient oligomers on a time scale of seconds, at least 2 orders of magnitude faster than conventional techniques. Furthermore, we utilize our platform to analyze oligomer ζ-potential and probe the immunochemistry of wild-type α-synuclein oligomers. Our findings contribute to an improved characterization of α-synuclein oligomers and demonstrate the application of microchip electrophoresis for the free-solution analysis of biological nanoparticle analytes

Rapid two-dimensional characterisation of proteins in solution

Abstract: 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

Quaternization of Vinyl/Alkynyl Pyridine enables ultrafast cysteine‐selective protein modification and charge modulation

© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.Quaternized vinyl- and alkynyl-pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine-tagged proteins at near-stoichiometric quantities. We have demonstrated that this method can effectively create a homogenous antibody-drug conjugate that features a precise drug-to-antibody ratio of 2, which was stable in human plasma and retained its specificity towards Her2+ cells. Finally, the developed warhead introduces a +1 charge to the overall net charge of the protein, which enabled us to show that the electrophoretic mobility of the protein may be tuned through the simple attachment of a quaternized vinyl pyridinium reagent at the cysteine residues. We anticipate the generalized use of quaternized vinyl- and alkynyl-pyridine reagents not only for bioconjugation, but also as warheads for covalent inhibition and as tools to profile cysteine reactivity.Funded under the EU Horizon 2020 Programme, Marie Skłodowska-Curie ITN GA No. 675007, the Royal Society (UF110046 and URF\R\180019 to G.J.L.B.), FCT Portugal (iFCT IF/00624/2015 to G.J.L.B. and PhD studentship SFRH/BD/115932/2016 to A.G.), Xunta de Galicia (Galician Plan of research, innovation and growth 2011–2015, ED481B 2014/086-0 and ED481B 2018/007 to M.J.M.), D.G.I. MINECO/FEDER (grants CTQ2015-70524-R and RYC-2013–14706 to G.J.-O. and C.D.N and CTQ2015-67727-R to F.C.), Universidad de la Rioja (FPI PhD studentship to I.C.), FAPESP (BEPE 2015/07509-1 and 2017/13168-8 to B.B.), and by an ERC StG (GA No. 676832).info:eu-repo/semantics/publishedVersio

Massively parallel C. elegans tracking provides multi-dimensional fingerprints for phenotypic discovery.

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