p53肿瘤抑制基因突变状态的电化学识别及应用

The p53 tumor suppressor gene is a well known transcription factor of cell regulation in human genome that plays a central role in inhibiting the growth of tumor cell. The mutation status of p53 tumor suppressor gene is closely related to the formation and progression of tumor cell. Therefore, the development of new molecular approaches for the detection of the mutation status of p53 tumor suppressor gene could provide a powerful tool for early cancer diagnosis as well as insight into fundamental mechanisms of epigenetic regulation of genetic information. Herein, the electrochemical recognition of the mutation status of p53 tumor suppressor gene and its medical applications are proposed. Based on the electrochemical techniques, several strategies for effective recognition of the mutation status of p53 tumor suppressor gene are presented, such as the electrochemical investigation of the mutation status of p53 tumor suppressor gene by direct electrocatalytic oxidation, the label-free electrochemical identification of the mutation status of p53 tumor suppressor gene according to bisulfite conversion analysis, the development of charge transport channel method for gene mutation detection using a hairpin DNA probe, the real time tracking of the dynamic mutation process of p53 tumor suppressor gene based on surface plasmon resonance (SPR) technique in combination with electrochemical biosensor, and the simultaneous detection of multiple mutational hotspots in the sequence of p53 tumor suppressor gene using microarray chips. Finally, the mutation statuses of p53 tumor suppressor gene from tumor cells are recognized according to the presented methods, which may make the proposed project promising for potential applications in early cancer prediction. The investigation of this project will definitely enrich new clinical diagnosis technique, which is of great significance for the diagnosis and treatment of cancer and the research of epigenetic evaluation.

p53肿瘤抑制基因的突变状态与肿瘤细胞的形成和发展密切相关。本项目拟开展p53肿瘤抑制基因突变状态的电化学识别与应用研究。针对不同序列的p53肿瘤抑制基因片段，建立电化学检测方法，主要包括：构建合适的电化学界面，并将其应用于基因突变状态的直接电催化氧化研究；以亚硫酸盐转化分析为基础，建立基因突变状态的免标记识别新方法；基于双链DNA的电荷传递路径，建立p53肿瘤抑制基因突变的电化学通道检测法；根据表面等离子共振与电化学技术联用的双重信号，实时跟踪基因突变的动态变化过程；制备操作简便、快速灵敏的微阵列芯片，发展p53肿瘤抑制基因序列中多个"突变热点"的同时检测技术。并根据p53肿瘤抑制基因的突变状态，考察肿瘤细胞的形成和发展过程，为疾病诊断和药物筛选提供依据。本课题的开展必将丰富临床检验新技术，对癌症的早期诊断和治疗，以及遗传学的机理研究具有十分重要的理论意义和实用价值。

基因突变是一种重要的生物学现象，p53肿瘤抑制基因的突变状态与癌症的形成过程密切相关。本项目以电化学方法为研究工具，以功能化的电极界面构建为基础，研究了p53肿瘤抑制基因突变状态的电化学检测新方法，并探究了其潜在的应用价值。主要研究内容包括：构建了多种适宜的电化学界面，例如石墨烯纳米墙界面、功能化的单分子层界面、双酶协同催化界面、碳纳米网界面、贵金属合金纳米材料界面、聚合物导向下的碳纳米管微电极界面等；建立了基因突变状态的直接电催化氧化研究法；实现了p53肿瘤抑制基因序列中158密码子四种突变状态的电化学检测；建立了以亚硫酸盐转化分析为基础的基因突变检测法；实现了p53肿瘤抑制基因序列中两个甲基化突变位点的同时检测；构建了GOD/HRP双酶协同催化体系，并探究了其催化机理和传感应用；制备了纸基传感设备，实现了多个目标的电化学测定。本项目所构建的电化学界面具有电位窗口宽、电极活性高、背景电流小、抗污染能力强等优点，可应用于DNA碱基序列的信号导出。基于界面构建，实现了各种DNA碱基的信号识别和同时测定。与文献的分析方法相比，本项目所建立的检测方法具有较快的分析速度，较低的检出限和较高的准确度。这些工作的开展对癌症的早期诊断和遗传学的机理研究具有重要的意义。本项目所取得的研究成果先后发表于Chem. Commun.，Biosens. Bioelectron.，Electrochem. Commun.，J. Electroanal. Chem.，Sensor Actuat. B-Chem.，J. Electrochem. Soc.等学术期刊。这些论文已经被Proc. Natl. Acad. Sci. U.S.A.，Trends in Analytical Chemistry，Chem. Commun.，Biosens. Bioelectron.，Electrochem. Commun.等杂志多次引用，并得到了肯定的评价。