Surface spectroscopy and microscopy studies of functional molecular films at electrified interfaces

本研究计划的总体目标是研究沉积在金属电极上的磷脂双层膜在金属-溶液界面的结构和反应活性。这些膜的功能将由多肽和蛋白质的存在来决定。我们将构建模拟革兰氏阴性细菌外膜(OM)的不对称双层，并将研究大型抗菌肽与构成细菌外膜外叶的脂多糖(LPS)的相互作用。我们的目标是了解多肽如何与内毒素结合，以及它们是如何导致膜破裂的。我们将研究电场如何影响不对称双层膜的稳定性，其中顶叶由脂多糖组成，下叶由磷脂组成，以及抗生素的掺入。我们将使用朗缪尔-谢弗技术组装不对称双层膜。我们将使用电化学阻抗谱(EIS)来提供有关离子传输的膜屏障属性(电阻率)的信息。扫描探针显微镜，如扫描隧道显微镜(STM)和原子力显微镜(AFM)将被用来提供脂多糖(LPS)单分子层和不对称双层的分子水平图像。原位光子偏振调制傅里叶变换红外反射吸收光谱(PM-IRRAS)、表面增强红外光谱(SEIRAS)和表面增强拉曼光谱(SERS)将被用来研究多肽与双层之间的构象、取向、相互作用和穿透。光谱技术是互补的；PM-IRRAS将提供含有和不含有多肽的双层的稳态性质的信息。SEIRAS将被用于研究抗生素多肽引起的结构变化以及它们在双层中的插入或运输的动力学。表面增强拉曼光谱在多糖的研究中具有独特的潜力。它的光谱范围扩展到很低的波数，它的信号不受水存在引起的光谱干扰的影响。我们将使用几个平台来获得高质量的多糖SERS光谱，例如壳层分离的金纳米颗粒、纯金纳米颗粒阵列和金纳米空穴阵列。在另一个主题中，我们将应用光谱工具来研究没有和有金属中心的二氧化碳还原酶(如甲酸脱氢酶，FDH)与导电载体的键合机理，以及它们将二氧化碳转化为有用化学物质的性能。我们的任务将是应用光谱方法来提供有关与导电载体形成的键的性质的信息，以及随着其氧化还原中心氧化还原状态的变化而发生的酶的结构变化。

The general objective of this research program is to study the structure and reactivity of phospholipid bilayers deposited onto a metal electrode at the metal-solution interface. The functionality of these films will be determined by the presence of peptides and proteins. We will build asymmetric bilayer that mimics outer membrane (OM) of a gram-negative bacterium and will investigate interactions of the large antibacterial peptides with lipopolysaccharides (LPS) that constitute the outer leaflet of the outer bacteria membrane. Our goal is to learn how the peptides bound to the LPS and how they cause a poration of the membrane. We will investigate how the electric field affects the stability of the asymmetric bilayers in which top leaflet consisting of LPS and the bottom leaflet of phospholipids and incorporation of antibiotics into such membrane. We will use Langmuir-Schaeffer technique to assemble the asymmetric bilayer. We will employ electrochemical impedance spectroscopy (EIS) to provide information about the membrane barrier properties for the transport of ions (resistivity). The scanning probe microscopies such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM) will be used to provide molecular level images of the monolayer of lipopolysaccharides (LPS) and the asymmetric bilayer. In situ photon polarization modulation Fourier transform infrared reflection absorption spectroscopy (PM-IRRAS), surface enhanced infrared (SEIRAS) and surface enhanced Raman spectroscopy (SERS) will be used to investigate conformation, orientation, interactions, and penetration of the peptides with and through the bilayer. The spectroscopic techniques are complementary; PM-IRRAS will provide information about steady state properties of the bilayer with and without the peptides. SEIRAS will be used to investigate kinetics of the structural changes induced by antibiotic peptides and their insertion or transport across the bilayer. SERS has unique potential for studies of polysaccharides. Its spectral range is extended down to very low wavenumbers and its signal is not affected by the spectral interferences caused by the presence of water. We will employ several platforms to obtain high quality SERS spectra of polysaccharides such as shell isolated gold nanoparticles, array of pure gold nanoparticles and array of gold nanocavities. In another theme we will apply spectroscopic tools to investigate the mechanism of bonding of CO2 reducing enzymes such as formate dehydrogenase, (FDH) without and with metal centers to conductive supports and their performance to convert CO2 to useful chemicals.  Our task will be to apply spectroscopic methods to provide information about the nature of the bond formed with conductive support, and structural changes of the enzyme accompanying changes of the redox state of its redox center.