基于FM NC-AFM方法对溶液中生物分子实现亚纳米级高分辨成像的机理研究

To obtain subnanometer resolution image of biomolecules (e.g. proteins) with atomic force microscopy (AFM) in solution under physiology conditions, is of great significance in analyzing the fine structures of these biomolecules and their functions. The frequency modulation non-contact AFM (FM NC-AFM) is the most promising technology to achieve this goal. However, obtaining subnanometer resolution FM NC-AFM image is still challenging. The improvement of the FM NC-AFM requires deep understanding of the imaging mechanism of high-resolution FM NC-AFM, of which the studies and quantitative analysis of the interaction between the probe and sample is critical. We aim to systematically evaluate the existing calculation methods of the interaction in FM NC-AFM and their application scope with our home-built FM NC-AFM, and to correct the methods if necessary. Then we study the interaction between the probe and simple biomolecule, streptavidin, with the corrected method and employ the Molecular Dynamic simulation to describe the process quantitatively. Finally, based on the contrast formation mechanism and expertise obtained in the study in the previous stage, we aim to obtain high resolution FM NC-AFM images on the biological important macromolecules (immunoglobulin IgM and perfringolysin O). Based on the detailed structural information revealed by this technique, we deepen the understanding of these biomolecules' functions.

在溶液中（生理条件）应用AFM方法获得亚纳米级分辨率，对于生物大分子（如蛋白质分子）的精细结构解析及其生物医学功能具有重要的意义。溶液中的FM NC-AFM技术是目前认为最有希望的方法，但目前亚纳米级分辨率还难以获得，深入研究和阐明FM NC-AFM成像机制是实现高分辨率的根本依据。其中，对于成像过程中力的研究和定量分析至关重要。我们将应用自行研制的FM NC-AFM仪器，系统地验证常用的成像力计算理论和其适用范围，并研究对其进行修正的必要性；然后依据通过验证（修正）的理论研究典型生物分子如链霉亲和素以及表面水层在振动探针下的行为，并计算生物分子所受到的力，利用分子动力学模拟方法阐释这一过程；进一步，利用根据成像机理研究获得的实验技术方法，对具有生物医学重要意义的生物大分子（如免疫球蛋白IgM和产气夹膜羧菌溶素PFO）进行高分辨成像，通过新的结构解析结合分子模型以深入阐述其生物功能。

本项目的总体目标为在溶液中应用FM NC-AFM技术获得生物分子亚纳米级分辨率结构信息。针对上述目标，本项目建立了表征原子力显微镜探针相关参数的方法以及研究探针振动频移与探针与样品相互作用力对应关系的实验体系，并对已有FM NC-AFM作用力理论进行分析验证，针对溶液状态下的应用，对已有公式进一步完善改进。并且，本项目还建立了FM NC-AFM成像条件对成像结果影响的研究体系，实现了对成像参数进行优化，从而获得了DNA折纸、双链DNA以及多肽GAV-9等典型生物样品的高分辨图像，展示了优化的FM NC-AFM对生物样品结构的亚纳米级解析能力。其中对多肽GAV-9的结构研究，将有助于神经退行性疾病中相关蛋白纤维化机制的研究。