Liquid-liquid phase separation is a key phenomenon in the formation of membrane-less structures within the cell, appearing as liquid biomolecular condensates. Protein condensates are the most studied for their biological relevance, and their tendency to evolve, resulting in the formation of aggregates with a high level of order called amyloid. In this study, it is demonstrated that Human Insulin forms micrometric, round amyloid-like structures at room temperature within sub-microliter scale aqueous compartments. These distinctive particles feature a solid core enveloped by a fluid-like corona and form at the interface between the aqueous compartment and the glass coverslip upon which they are cast. Quantitative fluorescence microscopy is used to study in real-time the formation of amyloid-like superstructures. Their formation results driven by liquid-liquid phase separation process that arises from spatially heterogeneous distribution of nuclei at the glass-water interface. The proposed experimental setup allows modifying the surface-to-volume ratio of the aqueous compartments, which affects the aggregation rate and particle size, while also inducing fine alterations in the molecular structures of the final assemblies. These findings enhance the understanding of the factors governing amyloid structure formation, shedding light on the catalytic role of surfaces in this process.

The interactions and structural organization of water molecules play a crucial role in a wide range of physical, chemical, and biological processes. The ability of water to form hydrogen bonds (H-bonds) underpins its unique properties and enables it to respond dynamically to various environmental factors. These interactions at the molecular level may affect vital processes like protein folding, enzyme activity, and cellular organization. The presence of solutes and spatial constraints can alter the H-bonding network of water, and these effects are ubiquitous in the biological environment. In this study, we analyzed the fluorescence of 2-acetyl-6-(dimethylamino)naphthalene (ACDAN) fluorescence emission in water solutions containing kosmotropic and chaotropic salts and in agar hydrogels. Recently, this dye has proven invaluable in studying water network structure and dynamics, as its fluorescence signal changes based on the local dielectric environment, revealing variations in the dipolar relaxation of water. Our results show that ACDAN spectral response correlates with the degree of water ordering, providing important insights into solute–water interactions and water dynamics in free and confined environments.