Time-Revolved Monitoring of β-Lactoglobulin Assembly and Aggregation in Microdroplets

Protein aggregation strongly depends on the local environment, yet investigating it in compartmentalized, high-surface-area volumes remains challenging. Here, we present an optically controlled platform for inducing and monitoring protein aggregation in microliter droplets. Local pH changes are generated in situ via photoconversion of 2-nitrobenzaldehyde, enabling noninvasive environmental control without mechanical mixing or direct perturbation of the sample. By combining fluorescein-based pH measurements with Raster Image Correlation Spectroscopy (RICS), pH variations and protein diffusion were monitored. As a model system, we investigated β-lactoglobulin, whose association state strongly depends on pH. Light-induced acidification progressively shifted the protein toward its isoelectric region, resulting in a measurable decrease in the diffusion coefficient consistent with the formation of larger supramolecular species. Furthermore, modulation of electrostatic interactions through NaCl addition revealed the sensitivity of the system to environmental factors governing the balance between electrostatic repulsion and short-range attractive interactions. Under these conditions, the platform enabled the observation of aggregation behaviors that were significantly less pronounced in corresponding bulk conditions. Overall, this droplet-based approach allows for the precise manipulation of local chemistry and quantitative analysis of biomolecular self-assembly, making it readily extendable to various noninvasive, time-resolved monitoring applications.