Advances in biophysical characterisation through micron scale flow engineering
Creators: name: Saar, Kadi Liis
Publisher: name: Apollo - University of Cambridge Repository
Year: 2019
Subjects: biophysical chemistry
protein biophysics
soft matter physics
protein self-assembly
microfluidics
microdroplets
microfabrication
soft-photolithography
micron scale flow engineering
micron scale separation approaches
electrophoresis
free flow electrophoresis
single cell proteomics
biophotovoltaics
biological solar cells
biophysics

description: 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.
description: Primary funding body: Engineering and Physical Sciences Research Council Additional funding: European Molecular Biology Organisation, NanoDTC