Moorthy Jaisree, Burgess Richard, Yethiraj Arun, Beebe David
Biomedical Engineering Department, University of Wisconsin, Madison, Wisconsin 53706, USA.
Anal Chem. 2007 Jul 15;79(14):5322-7. doi: 10.1021/ac070226l. Epub 2007 Jun 15.
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.
研究了微流控装置中的水凝胶柱作为反应环境，以表征蛋白质相互作用，目的是模拟生物环境的复杂性。水凝胶环境可以很容易地进行调节，以研究生物环境的特定特性。在本研究中，调节水凝胶孔径以模拟限制/拥挤对蛋白质相互作用的影响。通过光聚合在微流控通道内制备了不同交联比（4%和10%）的聚丙烯酰胺柱阵列。荧光标记的蛋白质（蛋白A（PA）和免疫球蛋白（IgG））通过扩散进入柱中，并使用荧光共振能量转移研究它们的相互作用。随着水凝胶孔径的减小，蛋白质之间的结合增强。拥挤增强结合相互作用的程度取决于蛋白质的固有特性；我们观察到，在水凝胶柱内，PA-山羊IgG的亲和力比PA-兔IgG的亲和力增加得更多。将可控纳米环境（水凝胶）与可控微环境（微通道）相结合，为研究蛋白质相互作用提供了增强的参数控制，这将有利于开发传感器、诊断以及在细胞和组织水平模拟生物环境。
