Residence-Time-Dependent Evolution of the Protein Matrix Modulates Nucleation and Pattern Formation in Drying Protein Droplets

The formation of internal patterns in drying protein droplets is commonly interpreted as a consequence of evaporation-driven hydrodynamic transport. Here, we show that the residence time of the HSA–NaCl system before the hygrometric transition systematically modulates the morphology of the final dried deposit. By varying the residence time before the hygrometric transition (tr), that is, the time for which the droplet is kept at high relative humidity, we controlled the duration of the pre-transition evolution of the protein matrix before accelerated drying. Short residence times produced heterogeneous aggregates, whereas prolonged residence favored coherent dendritic deposits with higher inter-sample reproducibility. These results are consistent with the interpretation that time-dependent structural evolution of the gel matrix constrains the accessible nucleation pathways and thereby influences the final deposit geometry. Multiscale morphological analysis based on texture descriptors, directional coherence, and radial features, combined with principal component analysis (PCA) and Gaussian mixture modeling, revealed residence-time-dependent changes in morphological organization, together with a progressive reduction in the number of recurrent morphotypes at prolonged residence times. Atomic force microscopy showed a parallel change in surface relief, from dispersed micro-islands and isolated peaks at short residence times to recurrent topographic motifs after prolonged residence. Nucleation dynamics changed from spatially distributed competitive growth at short residence times to highly localized and recurrent nucleation from a reduced number of reproducible sites at long residence times. Time-resolved lateral profiles showed similar early thinning trajectories under high relative humidity, while also revealing that solvent loss occurs during the residence stage: the hygrometric transition was applied to a still-curved droplet at short residence time and to a macroscopically flattened matrix at prolonged residence times. Together, these results indicate that the residence time, tr, before the hygrometric transition modulates the nucleation regime and final architecture of protein deposits.