Hong Mi-Young, Lee Dohoon, Kim Hak-Sung
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Yusung-ku, Taejon.
Anal Chem. 2005 Nov 15;77(22):7326-34. doi: 10.1021/ac051045r.
The interaction of streptavidin (SA) with a biotinylated surface has been of great interest in the development of an interfacial layer for protein immobilization based on self-assembled monolayers (SAMs) and polymeric layers. Here, we demonstrate the unique characteristics of protein-ligand interactions on dendrimer monolayers based on kinetic and equilibrium binding analyses. With amine-ended poly(amidoamine) dendrimers from the first (G1) to fourth (G4) generation, the formation of even, compact dendrimer monolayers on gold was confirmed using FT-IR spectroscopy and ellipsometry. For the SA-biotin interaction, quantitative analysis of bound SA using surface plasmon resonance showed that the saturation binding level of SA was fairly higher in all dendrimer layers when compared to other tested systems of 11-mercaptoundecylamine SAMs and a poly(L-lysine) layer. Kinetic studies revealed that the initial binding rate of SA up to the saturation level was 2-fold higher in all dendrimer layers than in the SAMs regardless of the surface density of functionalized biotin. Concurrently, the dendrimer layers led to much higher values of sticking probability, which is defined as the probability that the SA molecule adsorbs upon collision with a biotinylated surface, at a fixed SA coverage, and prolonged the significant levels around the maximum probability with increasing SA coverage. Plots of the saturation coverage of SA versus the SA concentration in solution showed that SA binding onto the biotinylated G1 and G3 layers fit to a Langmuir isotherm model. Taken together, faster binding of SA and highly ordered packing of the molecules seems to be achieved through typical properties of the dendrimer monolayers such as surface distribution of functionalized biotin, surface corrugation, and flexibility of highly branched larger dendrimers, which provides a guideline for the construction and analysis of an interfacial layer in biosensing applications.
链霉亲和素（SA）与生物素化表面的相互作用在基于自组装单分子层（SAMs）和聚合物层的蛋白质固定界面层的开发中备受关注。在此，我们基于动力学和平衡结合分析证明了树枝状聚合物单分子层上蛋白质-配体相互作用的独特特性。使用第一代（G1）到第四代（G4）的胺端基聚（酰胺胺）树枝状聚合物，通过傅里叶变换红外光谱（FT-IR）和椭偏仪证实了在金表面形成了均匀、致密的树枝状聚合物单分子层。对于SA-生物素相互作用，使用表面等离子体共振对结合的SA进行定量分析表明，与其他测试系统（11-巯基十一胺SAMs和聚（L-赖氨酸）层）相比，在所有树枝状聚合物层中SA的饱和结合水平都相当高。动力学研究表明，无论功能化生物素的表面密度如何，在所有树枝状聚合物层中SA达到饱和水平的初始结合速率都比SAMs高2倍。同时，树枝状聚合物层导致在固定的SA覆盖率下具有更高的粘附概率值，粘附概率定义为SA分子与生物素化表面碰撞时吸附的概率，并随着SA覆盖率的增加延长了最大概率附近的显著水平。SA的饱和覆盖率与溶液中SA浓度的关系图表明，SA与生物素化的G1和G3层的结合符合朗缪尔等温线模型。综上所述，SA的更快结合和分子的高度有序堆积似乎是通过树枝状聚合物单分子层的典型特性实现的，如功能化生物素的表面分布、表面波纹以及高度分支的较大树枝状聚合物的柔韧性，这为生物传感应用中界面层的构建和分析提供了指导。
