Probing the microenvironment of polyacrylamide hydrogel matrix using turbidity and fluorescence recovery after photobleaching: One versus Two phases

The microstructure of polyacrylamide hydrogel matrices was probed by turbidity measurements, and by fluorescence recovery after photobleaching (FRAP) of fluorescently labeled bovine serum albumin (fBSA), as a function of polymer concentration and crosslink density. Turbidity increased with increasing polymer and/or crosslinker concentration. At low polymer and crosslinker concentrations, FRAP dynamics were well described by diffusion through a single polymer phase. However for higher concentrations of polymer and crosslinker, the diffusion model was inadequate, and a second mode, likely corresponding to release of fBSA from regions of relatively dense polymer, was observed. At low network concentrations, probe diffusivity (inversely related to FRAP relaxation time) decreased with increasing polymer and crosslinker concentrations. Following onset of the second mode, however, diffusivity increased with increasing monomer concentration, suggesting that the network through which fBSA diffused became looser. The optical and FRAP measurements, while showing qualitatively similar trends, were not directly correlated, and they likely reflected different modes of network phase separation.