肽核酸共价功能化碳纳米管在甲基化DNA检测的应用

The project intends to develop a highly sensitive, highly selective, convenient, target label-free method for detecting the DNA methylation status of CpG sites of p16 gene promoter region in human hepatoblastoma HepG2 cancer cell. The system relies on highly selective single base extension reaction and significant optical amplification of peptide nucleic acid covalent functionalized carbon nanotubes(f-CNTs). When the target and probe pair are complementary at the methylation site, a higher fluorescence resonance energy transfer(FRET) efficiency occurs between f-CNTs and fluoresce in labeled dGTP(dGTP-Fl) is correlated to the incorporation of dGTP-Fl into the probe DNA. Because the probe is nearly not influenced by ionic strength, potentially interferences, pH, temperature, the developed method obtains low detection limit, high sensitivity, high FRET efficiency, and avoids tedious and repeated operations contrast to traditional methods. It is beneficial to the determination of trace methylated DNA in vivo or human metabolites materials, decrease the limited application range at the same time. These features make the system promising for future use for early cancer diagnosis. The method has theoretical significance and practical value.

本项目拟发展一种高灵敏度、高选择性、简便、免靶目标物标记定量检测原发性人肝母细胞瘤HepG2中p16基因启动子区CpG岛的DNA甲基化程度的方法。该检测体系基于甲基化敏感的单碱基延伸技术和肽核酸共价功能化碳纳米管（f-CNTs）探针的荧光信号转移放大作用。当靶向目标物与探针在DNA甲基化位点处完全互补时，f-CNTs与荧光标记dGTP-Fl之间发生高效率的荧光共振能量转移（FRET）。该方法因探针受离子强度、干扰物、pH、温度等多种因素影响较小，且具有检出限低、灵敏度高、FRET效率高等特点，可避免传统方法冗余繁琐的重复操作，有利于应用在复杂基质的生物体内痕量甲基化DNA及人体代谢物中痕量甲基化DNA的样品分析，解决应用范围受限的问题。这些特性有望应用于癌症的早期诊断。方法具有理论意义和实际价值。

在本项目研究中，我们选择具有荧光标记物与核酸双共价功能化的单壁碳纳米管荧光复合物作为探针，用于甲基化DNA的测定。详细考察了反应温度、介质酸度、浓度、反应时间等因素对碳纳米管功能化程度的影响，确定了最佳反应参数。通过透射电镜、红外光谱、荧光光谱、原子力显微镜、荧光显微镜等手段对荧光探针材料进行表征。同时，考察了分析过程中分析时间、pH值、离子强度等因素对探针荧光稳定性的影响。在样品的准备方面，我们尝试了人肝母癌细胞样品的培养及提取细胞裂解液相关操作处理。在甲基化DNA的样品前处理方面，进行了亚硫酸氢钠修饰，与聚合酶链反应程序的相关探索。有关研究工作已在学术刊物Mater. Lett.和大学化学上发表研究论文2篇，申请专利2项，其中1项已授权。还有2篇论文处于修改和审稿状态，完成了预期目标。