金纳米颗粒比色分析新技术与肺癌基因目视诊断新方法研究

The method to synthesize DNA-modified gold nanoparticles that can be pulled onto the substrate surface via several connecting points has been studied, and the optical, electrochemical and aggregation behavior have been investigated. The strategy to amplify the signal based on electrochemical active probes pulled onto the surface via several connecting points (EPSP) has been developed, and the corresponding sensing interface has been characterized. The electrochemical sensing technology for the detection of p53 mutant gene has been fabricated, and high detection sensitivity and selectivity have been achieved. The applicability of this sensing system for the diagnosis of lung cancer has been primarily confirmed. These achievements indicate the accomplishment of the expected objectives of the study. .Twelve research papers have been published by the international academic journals, and the total number of impact factor is about 70. The cultivation of Master (5) and Ph.D. (3) has been accomplished on the basis of this research project, as well as other projects. Three scientific talents have been trained who had been awarded science foundation by Hunan Society of Chemistry and Chemical Industry, the Education Department in Hunan province and McMaster University. .On the basis of gold nanoparticles (GNP) aggregation-induced color change, this project is expected to develop a colorimetric detection method to screen the p53 mutation without any analytic instrument by utilizing a novel mechanism of GNP aggregation. This protocol for mutant gene diagnosis with an unaided eye offers the advantages of easy operation, simple detection, relative universality, exhibiting plausibly the sufficient flexibility to react to the actual situations of Chinese including an immense population base, a large floating population, a weak foundation, high incidence rate and high mortality rate. Moreover, this will provide a good opportunity to overcome the problems of clinical practice resulting from the high cost and complicated procedure of the diagnosis of lung cancer, contributing directly to the early diagnosis, timely medical treatment, accurate pathological classification, observation of the effect of therapy and prognosis. Based on the series of impressive studies, the project will present the solid supporting data and develop a promising method for the diagnosis and treatment of lung cancer, as well as for the regular screening and general survey of malignant tumors among High-Risk Groups.

研究了表面多位点牵引效应DNA衍生纳米金复合物合成路线与光学、电化学、团聚行为等性质，发展了电化学活性探针表面限定多位点牵引(EPSP)信号放大策略；探索了EPSP电化学传感界面构建路线；整合出一种灵敏度高、选择性好、适合于p53点突变准确识别的电化学传感技术，并初步运用于肺癌诊断。.发表论文12篇（总影响因子70左右），联合培养博士硕士研究生8名，培养省级、国际基金专项人才各3人次。.本项目根据纳米金（GNP）团聚发生颜色变化现象，拟通过利用GNP团聚新机制，发展一种无需任何检测仪器、容易操作、便于普及推广的p53基因目视识别技术，直接应对我国人口总数多、人口流动性大、经济不发达、肿瘤发病率与死亡率高的情况与目前肺癌临床诊断费用高、分析程序繁杂的问题，以期实现对肿瘤早期诊断、及时治疗、准确分期、疗效监测与预后评估等做出贡献，为肺癌诊治与高危人群定期筛查或普查提供可靠的数据支持与方法指导。

根据2016年2月世界卫生组织发布的《世界癌症报告》：世界范围内癌症发病率与死亡率呈持续上升趋势，发展中国家形势更加严峻。本项目重要研究内容就是解析金纳米颗粒GNP团聚的新机制，提出恶性肿瘤基因诊断目视比色检测新技术；并建立荧光与电化学等其它基因诊断技术，对比综合评价比色基因诊断的优势与应用前景。.本课题组发现了核酸两个末端巯基能够吸附同一金纳米颗粒表面的现象，提出末端吸盘附着核酸平躺于基底表面的组装行为，指出具有表观交联趋势的生物分子能够反转金纳米颗粒团聚行为的分子机制；利用核酸目标杂交诱导聚合链取代反应驱使双链核酸平躺组装，发展恶性肿瘤p53抑癌基因目视比色检测体系。.提出G-四股螺旋结构粘性末端匹配多维构筑物组装新技术，根据DNA-GNP能以桥连方式嵌入到多维核酸构物中，引起GNP团聚变色，研发了恶性肿瘤抑癌基因的目视比色检测体系。.此外，我们还设计了多功能一体化核酸探针、三茎二环探针、G-四股螺旋汇聚探针、核酸分子机器与核酸纳米线崩解式信号转换探针等；提出了支链递升RCA扩增技术与RCA产物支架聚合链取代双重扩增技术等。研发了其它目视比色核酸杂交检测策略、肿瘤基因高灵敏荧光分析平台与生物分子检测的电化学传感界面；发展了介稳态核酸纳米器件细胞内成像材料与核酸三角形卡子细胞原位成像方法。.所研发的肿瘤基因诊断体系，特别是目视比色分析体系，操作简易、检测时间短、成本低、适用性强，反应条件易于控制，且能同其它生物过程相融合，在恶性肿瘤的诊疗过程中具有明显的应用前景。所提出的细胞成像技术特异性好、灵敏度高，为肿瘤的识别定位提供了新思路。.本项目取得一系列的学术成果：发表SCI学术论文28篇, 总影响因子IF180左右；其中，IF>8.0，3篇；IF>7.0～8.0，13篇；中科院JCR一区期刊论文约20篇；负责人入选福建省闽江学者奖励计划、获批中央财政支持地方高校发展团队建设专项等。