静电力驱动非接触调频模式原子力显微镜的研制与生物膜的高分辨成像

Achieving a spatial resolution down to (sub)nanometer scale on biological macromolecules will be great helpfule for understanding the structure-function relationship at a molecular level. Atomic force microscopy (AFM) is one of the most powerful and versatile tools to resolve the biological structure under physiological liquid conditions. However, the limitation of conventional contact and tapping modes in the nature of contact between the tip and sample as well as its low sensitivity is not able to achieve nm to sub-nm resolution on macromolecules. Hence, the ultimate goal of this project is to construct a new AFM with low noise and high sensitivity, based on our recently developed non-contact frequency modulation AFM (NC FM-AFM), combing with electrostatic excitation method, to achieve nm to sub-nm resolution on individual macromolecules. This new instrument will be used to detect the two dimensional lattice structure of Bacteriorhodopsin (BR) and headgroups of lipid molecules in purple membranes. This will provide basic fundamental information to enable an understanding of the interactions between BR proteins and lipids as well as their functions in the photo cycle process. Further, we will use electrostatic excitation FM NC-AFM to analyze the influence of ions on purple membranes, to elucidate their binding sites, then provide the theoretical basis and data supports to reveal the molecular mechanisms of photochemical transformation.

在纳米/亚纳米尺度上获得生物分子的高分辨结构将有助于在分子层次理解其结构与功能关系。原子力显微镜（AFM）是能在溶液生理条件下解析生物结构的最有效手段之一，但常规AFM模式（轻敲、接触模式）由于探针和样品的接触及低灵敏度，阻碍了对生物分子的纳米/亚纳米级高分辨成像。本项目将基于自主研制的非接触调频模式AFM，结合静电力直接驱动悬臂振动的方法，发展一种新型的低噪声、高灵敏度AFM，提高AFM对单个分散生物分子高分辨率成像能力，以期获得其纳米/亚纳米级结构信息。作为典型应用，我们将利用该系统解析紫膜上BR蛋白分子结构及磷脂分子头部结构，研究它们的相互作用关系及在光循环中所起的作用；进一步高分辨成像研究金属离子对紫膜产生的影响，从而推测出金属离子在紫膜上的结合位点，为揭示光化学转化的分子机制奠定理论基础和数据支持。

在纳米/亚纳米尺度上获得生物分子的高分辨结构将有助于在分子层次理解其结构与功能关系。原子力显微镜（AFM）是能在溶液生理条件下解析生物结构的最有效手段之一，但常规AFM模式（轻敲、接触模式）由于探针和样品的接触及低灵敏度，阻碍了对生物分子的纳米/亚纳米级高分辨成像。本项目自主研制了非接触调频模式AFM结合静电力直接驱动悬臂振动的方法，发展了一种新型的低噪声、高灵敏度AFM 。该系统在空气中本底噪声密度约为18fm/√Hz；在溶液中本底噪声密度约为23fm/√Hz; 反馈信号的频率偏移噪声小于10Hz; 同时本项目消除了商业上利用压电陶瓷驱动悬臂振动时由于液体槽和周围水溶液的耦合作用产生的杂峰，提高了品质因子Q。在此基础上，使用新发展的装置，本项目成功地获得了DNA折纸、双链DNA、神经退行性疾病相关蛋白GAV9的高分辨结构。结果表明本项目改进后的仪器对软的生物样品具有纳米/亚纳米级分辨率的解析能力，进而为研究生物大分子精细结构与相关生物功能的关系提供新的、精确定量的手段。