Quantification of the Local Protein Content in Hydrogels Undergoing Swelling and Dissolution at Alkaline pH Using Fluorescence Microscopy

Wide-field fluorescence microscopy was used to quantify the evolution of the volumetric swelling ratio, Q, i.e., solids content, in a protein hydrogel undergoing swelling and dissolution. Heat-induced whey protein hydrogels labeled with Rhodamine B isothiocyanate (RITC) were used as a model system. Complications in the quantification of Q using fluorescence of proteins conjugated with RITC, arising from alkali destroying protein-dye interactions, were overcome using a reaction-diffusion numerical scheme. At pH 12-13, when the hydrogels dissolve readily, overlapping fluorescence intensity profiles were observed at different times, consistent with a system dissolving at a steady state. In stronger alkali (e.g., 1 M NaOH), when dissolution proceeds very slowly, we confirm that there is little swelling next to the gel boundary. These results present the first quantification of the solids distribution within protein hydrogels under reactive conditions.