From single-molecule sensing to extracellular vesicles in glioma cells under stress
This thesis describes the work I conducted in two different areas, namely biomolecular sensing and quantitative cell biology. The work in the former area was focused on the optimization of plasmonic metamaterials for sensing applications, and in the latter on the characterization of extracellular vesicles released by glioblastoma cells under stress conditions. If these two areas appear to be very far apart, it is because they are; however, they are not entirely disconnected. Understanding the biological function of extracellular vesicles depends on the information that can be obtained using bioanalytical sensors, which consequently relates to the information that can be gained from cellular experiments. Attempts were therefore made to review and exploit biophysical approaches that link the two areas, with focus on how detailed insights about the nature of these vesicles relate to their biological function. At its core, this thesis deals with: i) plasmonic biomolecular sensing, discussing noiserelated issues, single-molecule detection and a dual-wavelength method to extract more – and more accurate – information from ensemble measurements of complex macromolecular entities, including extracellular vesicles; ii) an introduction to glioblastomas, cellular models of hypoxic and oxidative stress, the role of extracellular vesicles in cellular communication in cancer, and an exploratory omics approach to understand how the biochemical composition of cells and vesicles changes across different cell lines in response to stress. As an encompassing bridge, a review on the techniques used to characterize extracellular vesicles also acts as an integrating element of this thesis and will help the reader understand different aspects of relevance with respect to how the physical and biological viewpoints intersect. The main findings of the work on biomolecular sensing consist in: i) the clarification of the interplay between inhomogeneous binding probability to a nanosensor (which depends on diffusion/geometry) and the inhomogeneous sensitivity distribution of the nanosensor itself as combined sources of detection uncertainty and ii) a method exploiting dual-wavelength plasmonic sensing to estimate the degree of deformation of adsorbed extracellular vesicles and hence more accurate determination of their mean size and bulk concentration. In the second area we find that iii) the proteome of vesicles mainly depends on cell type of origin and is greatly affected by stress, although in a heterogeneous way, with respect to the cells proteome; on the contrary, iv) the lipid composition of extracellular vesicles is very stable across cell types and stress.
single molecule sensing
surface plasmon resonance