ETD

• biomolecular condensates
• Classical Nucleation Theory (CNT)
• Fused in Sarcoma (FUS)
• interfacial tension
• liquid-liquid phase separation (llps)
• microfluidics
• quantitative phase imaging (QPI)

The intracellular environment relies on membraneless biomolecular condensates formed via liquid-liquid phase separation (LLPS), a process crucial for cellular physiology that is also linked to neurodegenerative disorders like Amyotrophic Lateral Sclerosis (ALS). Understanding the thermodynamics and kinetics of LLPS is essential to deciphering condensate behaviour and involves measuring physical properties such as the interfacial tension γ. This thesis establishes an experimental framework to quantify γ from experimental studies of condensate nucleation carried out in droplet microfluidic platforms. The method was applied to three model compounds, namely poly(A) RNA, a designer peptide, and the ALS-associated protein Fused in Sarcoma (FUS). Label-free quantitative phase imaging (QPI) operating via Wavefront Angle Retrieval Process (WARP) validated early-stage condensates’ morphology, in particular their liquid-like character. PDMS microfluidic chips were fabricated via soft lithography to isolate monodisperse microdroplets, mitigating sample consumption and evaporation. Combinatorial screenings via droplet microfluidics and confocal microscopy enabled real-time tracking of phase boundaries across wide temperature ranges. Integrating Classical Nucleation Theory (CNT) with Flory-Huggins approximations allowed explicit calculation of interfacial tension from nucleation rates, and we foresee that this methodology will find widespread application for the characterization of biomolecular condensates in the future.