Microfluidic Approaches for Investigating Aggregated Forms of Disease-Related Proteins

The self-assembly of soluble proteins is a common phenomenon observed in nature. It ranges from the formation of functional biomaterials, such as actin filaments and tubulin microtubules that form the cytoeskeleton, to the abnormal deposition of aberrant aggregates that lead to disease. There are nearly 50 diseases currently associated with protein misfolding and amyloid formation, among them several neurodegenerative disorders such as Alzheimer's, Parkinson's and prion diseases. Recent evidence indicates that the most toxic species are the low molecular weight oligomers formed during the onset of the aggregation process. These oligomeric species are commonly difficult to analyse due to their low abundance, transient nature and heterogeneity in their structure. In fact, prions, known to be associated with transmissible spongiform encephalopathies, have not yet been fully characterised as parts of the prion composition seem to lose infectivity through various \textit{in vitro} purification and isolation procedures. In nature, non-homogeneous solution conditions and molecular crowding are key factors as there might be multiple species associated with the disease. However, traditional biophysical techniques allow the study of biomolecules under ideal conditions, such as high concentrations and monodisperse size distribution. Furthermore, among the limitations of the traditional methods are the sensitivity as well as the sample preparation, which in some cases may require to chemically modify the molecule. In this thesis, I describe the development of a sensitive label-free method to enable the study of protein complexes and proteins under biologically relevant environment, such as brain homogenates, by combining immunochemistry with microfluidic strategies. In this thesis I describe the development of Immuno Diffusional Sizing (IDS), a method that combines diffusional sizing with the sensitivity and specificity of immunoassay detection. In Chapter 3, I explain the optimisation of the devices developed to size a range of particle sizes in heterogeneous mixtures, from small protein monomers to large aggregates. IDS is a label-free method capable of sizing proteins down to the nano- and picomolar concentrations, in complex solutions. The applications of this method to monomeric and aggregated forms of disease-related proteins are described in Chapter 4. IDS has been applied to study the Prion Protein (PrP) in its native and infectious forms. Furthermore, I studied of the proteinase resistant form (PrP\textsuperscript{res}) extracted from the brains of infected mice specimens. IDS was applied for studying disease-relevant species, such as intrinsically disordered proteins, e.g. $\alpha$-Synuclein (aSyn) and A$\beta$40, and their respective oligomers. Another advantage of this method is its applicability for studies of brain aSyn from transgenic overexpressing mice (OVX). Furthermore, I explore the potential of IDS for the study of post-translational modifications (PTMs) of specific proteins, such as the ubiquitination of aSyn \textit{in vitro}. The efforts performed during this work to achieve TR-FRET detection on-chip are described in Chapter 5. A comparison of two commercial immunoassays was performed in order to determine the suitability for its adaptation from a platereader to a microfluidic device. The design and adaptation of an optics platform for FRET detection on-chip is also discussed. My research on the mechanisms of the molecular chaperone $\alpha$B-Crystallin (aBC) is described in Chapter 6. I investigated the aggregation process of insulin in the presence of aBC. I have used a combination of bulk techniques to study the kinetics of the systems, as well as microfluidics to generate microdroplets that allow the direct observation of rare nucleation events leading to protein aggregation. Overall, in this work I describe the development of a novel microfluidic technique, its optimisation and application to multiple systems related to protein misfolding diseases, including prions and intrinsically disordered proteins. Purification of the sample is not necessary to study the target protein because the detection of the target protein after diffusional separation is made with a sandwich immunoassay, which allows to study disease-related proteins under native conditions in a label-free manner, which is usually difficult with traditional sizing techniques. The applications of this technology could be expanded to characterise other antigens in body fluids, like cerebrospinal fluid (CSF) of blood serum.CONACYT (Mexican National Council on Science and Technology) Cambridge Trus