• biomolecular condensates
• confocal microscopy
• droplet microfluidics
• kinetics
• liquid–liquid phase separation (LLPS)
• neurodegenerative diseases
• nucleation
• phase separation dynamics.

Biomolecular condensates are membraneless organelles that form through liquid-liquid phase separation (LLPS). They are increasingly recognized as key organizers of intracellular biochemical processes, signal transduction, and gene expression regulation, and have been implicated in a range of neurodegenerative diseases. While equilibrium thermodynamics defines the conditions under which phase separation occurs, it provides no insight into the dynamics of assembly. These dynamics are governed by kinetic parameters, including the rates at which condensates nucleate, grow, merge, and dissolve. Nucleation is the initial step of condensate formation, yet a systematic and quantitative investigation of this process has remained elusive.

Here, we introduce a comprehensive framework to probe the early stages of condensate formation by integrating Classical Nucleation Theory with experimental measurements using PolyA RNA, synthetic peptides, and proteins associated with neurodegenerative pathology. Leveraging droplet microfluidics, we generate monodisperse microdroplets that serve as controlled microenvironments, enabling precise modulation of local concentration, confinement, and temperature. Condensate formation in individual droplets is detected using confocal microscopy, allowing the extraction of nucleation rates. Our theoretical predictions are consistent with experimental data, providing a quantitative and mechanistic basis for understanding the early stages of LLPS-driven condensate formation. This work helps clarify the pivotal role of condensates in both physiological and pathological contexts.