Microfluidics resolves non-classical lysozyme nucleation driven by attractive protein interactions

Protein crystalline nucleation pathways still remain a hot research topic after decades, due to the experimental challenges involved in its study and its vast implications in several applied and research fields. Here, we report a comprehensive investigation of lysozyme crystal nucleation, employing a high-throughput droplet-based microfluidic chip, developed ad-hoc for performing crystal nucleation kinetic measurements, and Dynamic Light Scattering (DLS) for local solution structure analysis. A temperature-controlled system with 500 independent nL-scale droplet microreactors enabled statistical analysis of nucleation events, and two distinct nucleation rates were identified, one attributed to prevalent heterogeneous nucleation (likely on impurities and/or denatured protein molecules), and the other to nucleation in the bulk of the solution. CNT-derived kinetic parameters for bulk nucleation aligned with previously reported values, yet deviated significantly from classical homogeneous predictions: the estimated density of active nucleation sites was found to be several orders of magnitude lower than the protein monomer concentration, strongly contradicting a homogeneous mechanism. Furthermore, when approaching the binodal, nucleation kinetics exhibited a marked deviation from the CNT trend, indicating a rupture in the nucleation behavior. DLS analysis further indicated that in supersaturated conditions, proteins do not remain monomeric but form dynamic locally densified regions. Results show that lysozyme nucleation proceeds via transient densifications driven by attractive intermolecular interactions, challenging the classical view of a homogeneous, monomer-dominated solution and underscoring the importance of mesoscopic solution structure. • High-throughput microfluidics resolves two distinct nucleation regimes • Bulk nucleation rates defy CNT predictions with extremely low density of nucleation sites • Bulk nucleation deviates from CNT trend near the LLPS boundary • DLS measurements reveal transient densifications in the solution • Nucleation proceeds via dynamic fluctuations driven by attractive protein interactions