Work

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

Year: 2026

Type: article

Abstract: 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.

Source: Journal of Molecular Liquids

DOI: 10.1016/j.molliq.2026.129378

Authors: Yevgeniya Karibjanova, Dimitri Radajewski, Sébastien Teychené, Isaac Rodriguez-Ruiz

Institutions: Centre National de la Recherche Scientifique, Université Toulouse III - Paul Sabatier, Laboratoire de Génie Chimique, Université de Toulouse, Laboratoire d'Analyse et d'Architecture des Systèmes

Language: English

Topic: Crystallization and Solubility Studies

Subfield: Materials Chemistry

Field: Materials Science

Domain: Physical Sciences

Open Access status: hybrid

Estimated APC paid: $3,480