Doan Tran T, Freeman Michael H, Schmidt Adrienne R, Nguyen Natalie D T, Leopold Michael C
Department of Chemistry, Gottwald Center for the Sciences, University of Richmond, USA.
J Vis Exp. 2011 Oct 4(56):3441. doi: 10.3791/3441.
Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM). In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (¹H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.
合成了用链烷硫醇配体保护的胶体金纳米颗粒，即单层保护金簇（MPC），随后将其纳入用作蛋白质单层电化学（PME）吸附平台的膜组件中。PME被用作研究氧化还原蛋白电化学性质的模型系统，通过将它们限制在修饰电极上的吸附平台上，该平台也作为电子转移（ET）反应的氧化还原伙伴。研究表明，与用链烷硫醇自组装单分子层（SAM）修饰的更传统系统相比，这种性质的金纳米颗粒膜组件提供了更均匀的蛋白质吸附环境，并促进了无距离依赖性的电子转移。在本文中，使用改进的布斯特反应合成了用己硫醇配体功能化的MPC，并用紫外可见（UV-Vis）光谱、透射电子显微镜（TEM）和质子（¹H）核磁共振（NMR）对其进行了表征。通过交替MPC层和二硫醇连接分子的“浸涂循环”方法，将MPC膜组装在SAM修饰的金电极界面上。通过测量系统双层充电电流的变化，以电化学方式跟踪金电极上的膜生长。在硅烷修饰的载玻片上组装的类似膜允许对膜生长进行光学监测，横截面TEM分析提供了估计的膜厚度。在膜组装过程中，对MPC配体保护以及颗粒间连接机制的操纵允许形成易于适应的网络膜，以与具有不同吸附机制的氧化还原蛋白界面。例如，铜绿假单胞菌天青蛋白（AZ）可以疏水吸附到己硫醇MPC的二硫醇连接膜上，而细胞色素c（cyt c）可以静电固定在羧酸修饰的MPC界面层上。在本报告中，我们仅关注AZ系统的膜方案。涉及蛋白质在MPC修饰的合成平台上吸附的研究可以进一步理解生物分子与人工材料之间的相互作用，从而有助于生物传感器方案、电子转移建模系统和合成生物相容性材料的开发。
