个性化医疗及针对细分人群进行个性化药物研发已成为癌症等重大疾病诊治的发展趋势。然而现有的生化实验技术手段存在分辨率低、功能单一、难以定量、离线检测等严重不足，亟需可在纳米尺度对细胞/分子多维生理特征信息进行原位检测的实验手段。随着纳米技术的发展，基于原子力显微镜(atomic force microscopy，AFM)的纳米操作机器人技术在纳米制造、加工、操作等领域取得了极大成功，然而其在细胞/分子领域的应用研究还很稀缺。本文以解决癌症靶向治疗中存在的不同患者疗效差异问题为目标，针对纳米操作机器人在细胞/分子尺度进行探测面临的挑战开展研究，发展基于纳米操作机器人的单细胞单分子生理特性可靠定量检测技术方法并将其应用于癌症临床治疗中，为推动纳米操作机器人在生物医药领域的应用并促进纳米-生物-信息的融合提供可行的思路。 针对纳米操作机器人难以对活体动物悬浮细胞进行探测的问题，提出了一种结合微柱/微坑机械夹持和多聚赖氨酸静电吸附的活体动物悬浮细胞固定方法，实现了溶液环境下在水平和垂直方向对单个动物悬浮活细胞的有效固定，并在此基础上对淋巴瘤细胞表面超微结构进行了精细成像。 针对生命科学领域免标记细胞生理特性检测技术的需求，建立了基于纳米操作机器人压痕技术的免标记单细胞机械特测量方法，基于此通过对不同类型细胞的机械特性进行测定，分析了癌细胞机械特性与侵袭能力之间的关系。 针对生命科学领域快速原位单分子生理特性检测技术的需求，发展了基于针尖化学修饰技术的分子力测量及细胞表面蛋白分布可视化表征方法，基于此分别对云母表面吸附的蛋白质分子和细胞表面的蛋白质分子的结合力进行了测定，并对其分布进行了可视化表征，实验结果证明了检测表征方法的准确性和可靠性。 将所建立的纳米操作机器人单细胞单分子生理特性检测表征方法应用于淋巴瘤的Rituximab靶向治疗中，揭示出Rituximab的三种作用机制中细胞超微形态、机械特性的变化和作用，同时发展了结合纳米操作机器人和荧光识别的活检样本细胞分子力及分布密度测试方法，基于此对多个淋巴瘤患者进行了测试并与通过实际疗效进行比较分析了分子间相互作用在Rituximab临床治疗中的作用。 本工作对研究从单细胞单分子尺度获取临床患者的病理信息，用以对药物进行评价并预测其临床疗效，进而辅助药物研发和医生制定用药方案，提供了一种新的途径，在生物学、医学、药学、机器人学方面都将有很大的应用前景。
Personalized medicine and personalized drug research for subpopulations has become the development trend of treating cancer and other major diseases. However, current biochemical experimental methods have many serious shortcomings (such as low resolution, single function, difficult to quantify, and off-line testing), and means that can in situ probe the cellular/molecular multiparameters at the nanoscale are urgently needed. With the development of nanotechnology, nanorobotics based on atomic force microscopy has achieved great success in the field of nano-fabrication, nano-machining and nano-manipulation, but its researches in the filed of cell and molecule are scarce. In this paper, in order to solve the problem of variable efficacies among different patients in the cancer targeted therapy, researches were performed directly for the challenges facing nanorobotics in probing the nanoscale cellular and molecular properties. By developing nanorobotic single-cell/single-molecule reliable quantitative detection methods and then applying them to the clinical cancer treatment, providing a viable idea for promoting the application of nano-robots in the field of biomedicine and boosting the fusion of nanotechnology, biology technology, and information technology. For addressing the problem that it is now difficult for nanorobots to probe the living mammalian suspended cells, a method combining micro-pillar/micro-well mechanical trapping and poly-L-lysine electrostatic adsorption was presented to immobilize mammalian suspended cells. Based on the presented method, single mammalian suspended cells were efficiently immobilized horizontally and vertically, and the ultra-structures on the surface of single lymphoma living cells were finely observed. For the needs of label-free probing cellular biophysical properties in life sciences, a method for label-free detecting the mechanical properties of single cells based on nanorobotic indentation technique was developed. With the developed method, the mechanical properties of different types of cells were quantified and the relationship between mechanical properties and invasive ability of cancer cells was analyzed. For the needs of in situ rapidly probing the molecular biophysical properties in life sciences, methods that can simultaneously measure the molecular binding forces and visualize the distribution of specific proteins on the cell surface were developed based on tip chemical modification. With the developed method, the binding forces of purified proteins on mica and native proteins on cells were measured and the distributions of proteins were also visualized. The experimental results demonstrated the accuracy and reproducibility of the developed methods. The eastablished nanorobotic single cell and single molecule biophysical properties were applied to the clinical Rituximab targeted therapy of lymphoma, revealing the changes of cellular ultra-microstructures and the role of cellular mechanical properties in the Rituximab’s three mechanisms. In addition, method that combined nanorobots and fluorescence recognition to measure the molecular forces and distribution density on cell from patient biopsy samples was developed. Based on the method, several lymphoma patients were tested and by comparing the results with the clinical data the roles of molecular interacionts in the clinical Rituximab therapy were analyzed. This work provides a new way to obtain the pathophysiological information of clinical patients at single cell and single molecule levels for predicting drug efficacies and ultimately assisting drug development and personalized medicine, having great application prospects in biology, medicine, pharmacy and robots.