Microfluidic based platform for characterization of protein interactions in hydrogel nanoenvironments. 
Hydrogel posts in microfluidic devices were investigated as reaction environments for characterizing protein interactions with the goal of mimicking the complexity of a biological environment.  The hydrogel environment can be easily tuned to study specific properties of the biological environment.  In this study, the hydrogel pore size was tuned to mimic the effect of confinement/crowding on protein interactions.  Arrays of polyacrylamide posts of different cross-link ratios (4 and 10%) were fabricated inside microfluidic channels via photopolymerization.  Fluorescence-labeled proteins (protein A (PA) and immunoglobulins (IgG)) were transported into the posts via diffusion, and their interaction was studied using FRET.  As the pore size of the hydrogel decreased, the binding between the proteins was enhanced.  The degree to which crowding enhances a binding interaction depends on the intrinsic properties of the proteins; we observed that, inside the hydrogel post, the PA-goat IgG affinity was increased more than PA-rabbit IgG affinity.  The integration of controlled nanoenvironments (hydrogels) with controlled microenvironments (microchannels) provides enhanced parametric control for studying protein interactions, which would be beneficial in developing sensors, in diagnostics, and for mimicking the biological environment at both the cell and the tissue level.