Advances in biophysical characterisation through micron scale flow engineering

Proteins are the chief actor molecules of cells central to the majority of biochemical and biophysical processes that sustain life. Interactions between proteins and other biomolecules are crucial to a faultless execution of biological function, yet it has remained challenging to analyse these biomolecular interactions with current protein science tools - they commonly rely on non-physiological conditions for performing analysis, thereby compromising the ability to analyse biological interactions. This thesis describes the development and applications of platforms that facilitate rapid analysis of heterogeneous systems of proteins and protein interactions directly in solution, under fully native conditions. I achieved this objective by fabricating micron scale structures, where, in contrast to macroscale systems, chaotic mixing of fluids and the molecules therein was suppressed. In this manner, I was able to dispense with the support structures that prevent mixing in conventional protein analysis platforms and decrease analysis times by orders of magnitude, from hours to seconds. The first part of the thesis was centred around the use of micron scale strategies for probing proteins, protein interactions and protein self-assembly in vitro. First, I demonstrated a platform for performing automated high-throughput measurements on protein self-assembly in a label-free environment. I proceeded by addressing two challenges at the core of creating micron scale separation platforms - the integration of strong and stable electric fields with micron scale channels and the enhancing of the resolution limit of such separation systems. Finally, I devised and demonstrated devices for combined biomolecular separation and analysis, which allowed me to size mixtures of proteins at an unprecedented resolution and gain multidimensional data on biomolecular systems. The second part focussed on probing protein behaviour inside cells. I first described a strategy for detecting intracellular proteins in individual cells in a high throughput manner, offering a substantially advanced multiplexing capability in comparison to existing approaches for analysing intracellular proteins. I then focussed on a specific application of cellular biophysics and measured electrical outputs of cells. This work led to record high power outputs for systems that use biological matter for converting sunlight into electricity. To my knowledge, this was also the first demonstration of a biological solar cell equipped with energy storing capacity, the lack of which had been viewed as one of the most notable limitations of current solar cells.Primary funding body: Engineering and Physical Sciences Research Council Additional funding: European Molecular Biology Organisation, NanoDT

Enhancing the resolution of micro free flow electrophoresis through spatially controlled sample injection

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 minimising 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 by spatially restricting the sample injection and collection to the regions where the gradients in the velocity distribution of the carrier medium are the smallest, this broadening effect can be substantially suppressed and hence the resolution of microscale free flow electrophoresis devices increased. To demonstrate this concept we fabricated microfluidic free flow electrophoresis devices with spatially restricted injection nozzles implemented via the use of multilayer soft-photolithography and further integrated quartz based observation areas for fluorescent detection and imaging. With these devices we demonstrated a five fold reduction in the beam broadening extent compared to conventional free flow electrophoresis approaches with non-restricted 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 more complex mixtures

Automated Ex Situ Assays of Amyloid Formation on a Microfluidic Platform.

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.Financial support from the Frances and Augustus Newman Foundation, the BBSRC, the EPSRC, the ERC and the Swiss National Science Foundation is gratefully acknowledged.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.bpj.2015.11.352

Supplementary Information from Mechanism of biosurfactant adsorption to oil/water interfaces from millisecond scale tensiometry measurements

Many biological molecules are by their nature amphiphilic and have the ability to act as surfactants, stabilizing interfaces between aqueous and immiscible oil phases. In this paper, we explore the adsorption kinetics of surfactin, a naturally occurring cyclic lipopeptide, at hexadecane/water interfaces and compare and contrast its adsorption behaviour with that of synthetic alkyl benzene sulfonate isomers, through direct measurements of changes in interfacial tension upon surfactant adsorption. We access millisecond time resolution in kinetic measurements by making use of droplet microfluidics to probe the interfacial tension of hexadecane droplets dispersed in a continuous water phase through monitoring their deformation when the droplets are exposed to shear flows in a microfluidic channel with regular corrugations. Our results reveal that surfactin rapidly adsorbs to the interface, thus the interfacial tension equilibrates within 300 ms, while the synthetic surfactants used undergo adsorption processes at an approximately one order of magnitude longer time scale. The approach presented may provide opportunities for understanding and modulating the adsorption mechanism of amphiphiles on a variety of interfaces in the context of life sciences and industrial applications

α-Synuclein Oligomers Displace Monomeric α-Synuclein from Lipid Membranes.

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 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 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 binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD

α-Synuclein Oligomers Displace Monomeric α-Synuclein from Lipid Membranes.

Publication status: PublishedParkinson'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 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 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 binding mechanism of oligomers to model membranes, which can potentially be targeted to inhibit the progression of PD