肿瘤核酸标志物高灵敏SERS传感器的构建及应用研究

发布时间：2018-06-16 22:27

本文选题：表面增强拉曼散射 + 传感器　；参考：《南京邮电大学》2017年硕士论文

【摘要】：核酸的表达失控与肿瘤的发生发展密切相关,开展肿瘤相关的核酸标志物检测有望实现肿瘤的早期检测。然而在病变早期肿瘤核酸标志物含量极低,急需发展可靠的高灵敏检测新方法。SERS技术具有超高灵敏度、非破坏性、抗光漂白,谱峰尖锐适合多组分检测等优异的性能使其成为分子检测的重要手段。因此,发展并制备基于SERS的核酸标志物传感器及其多元联合检测技术,对于实现肿瘤的早期检测意义重大。针对这一需求,本论文基于SERS活性材料如银纳米棒阵列型基底、新型磁核枝杈状金壳纳米颗粒,设计并开发了多种灵敏度、特异性俱佳的SERS传感器,对血清中的痕量肺癌核酸标志物进行了快速、灵敏的联合检测,为SERS技术在肿瘤早期检测中的应用并建立了技术平台,主要研究内容分为以下3个方面:(1)基于阵列型固态SERS基底和分子信标发展了一种高灵敏多元mi RNA检测传感芯片。首先采用斜角沉积法蒸发镀膜技术制备了大面积均一的高性能银纳米棒(Ag NR)阵列型SERS基底,并在基底表面修饰了三种标记不同拉曼分子的DNA探针(分子信标),构建得到了mi RNA阵列型SERS传感器,通过表征识别目标mi RNA前后SERS强度的变化实现了对mi RNA的定性和定量检测。该阵列型传感器具有灵敏度和特异性高,均一性和稳定性好,可重复使用等优点,可同时检测血清中的三种肺癌标志物mi RNA-21/486/375,检测限分别达到393 a M、176 a M、144 a M。(2)基于磁核枝杈状金壳纳米颗粒结合四面体DNA探针,设计并构建了mi RNA全液相SERS传感器。首先基于种子生长法并引入定向生长控制剂Ag NO3,合成了一种磁核枝杈状金壳纳米颗粒;随后设计组装了四面体DNA探针,并将其修饰到磁核枝杈状金壳纳米颗粒表面构建得到了兼具SERS活性和超顺磁性的检测基底;然后通过在金纳米颗粒表面修饰单链DNA和拉曼活性分子DTNB构建了SERS探针;最后利用碱基互补配对形成“检测基底-mi RNA-SERS探针”三明治结构复合物,借助外加磁场分离检测液中的复合物并富集后进行SERS检测,实现了对于血清中肿瘤核酸标志物mi RNA-21的高灵敏、特异性检测,检测限达到623 a M。(3)发展了基于四面体DNA核酸探针的多元SERS传感器。针对性设计并组装了具有特异性识别功能的四面体DNA核酸探针,并将四面体DNA核酸探针有序组装到Ag NR阵列型基底表面构建了高灵敏SERS核酸检测基底;通过链置换反应,四面体DNA探针可同时识别两种不同的目标核酸,并捕获两种基于金纳米球构建的特异性SERS探针组装成金纳米球二聚体,形成SERS“热点”;借助SERS探针信号强度实现两种核酸分子的高灵敏检测。该传感器具有良好的特异性,线性检测范围宽(100 a M-1 n M),Target 1和2的检测限分别为50 a M和66 a M。
[Abstract]:The uncontrolled expression of nucleic acid is closely related to the occurrence and development of tumor. The detection of tumor-related nucleic acid markers is expected to realize the early detection of tumor. However, the content of nucleic acid markers in tumor is very low in the early stage of pathological changes, so it is urgent to develop a new method of high sensitivity, non-destructive and anti-photobleaching. The sharp spectrum peak is suitable for multicomponent detection and so on, which makes it an important method for molecular detection. Therefore, the development and preparation of SERS-based nucleic acid marker sensors and multivariate combined detection techniques are of great significance for the early detection of tumors. In order to meet this demand, SERS active materials such as silver nanorod array substrate and new magnetic core branched gold shell nanoparticles are used to design and develop a variety of SERS sensors with high sensitivity and specificity. The rapid and sensitive combined detection of trace lung cancer nucleic acid markers in serum was carried out, which provided a platform for the application of SERS technique in early detection of tumor. Based on the array solid state SERS substrate and molecular beacons, a highly sensitive and multivariate sensor chip for mi RNA detection is developed. Firstly, a large area uniform silver nanorod array SERS substrate was prepared by the oblique angle deposition method. Three kinds of DNA probes (molecular beacons) labeled with different Raman molecules were modified on the substrate surface, and the mi RNA array SERS sensor was constructed. The qualitative and quantitative detection of mi RNA was achieved by characterizing the changes of SERS intensity before and after recognition of target mi RNA. The array sensor has the advantages of high sensitivity and specificity, good uniformity and stability, and can be reused. Three lung cancer markers, mi RNA-21 / 486 / 375, with a detection limit of 393 a / M ~ (176) a / M ~ (-1) ~ (144) a 路m ~ (2), respectively, were detected simultaneously. Based on the magnetic core branched gold shell nanoparticles and tetrahedral DNA probe, a full-liquid phase SERS sensor was designed and constructed. Based on the seed growth method and the introduction of Agno _ 3, a magnetic core branched gold shell nanoparticles were synthesized, and the tetrahedron DNA probe was designed and assembled. The SERS probe was constructed on the surface of the branched gold shell nanoparticles with both SERS activity and superparamagnetism, and the SERS probe was constructed by modifying single strand DNA and Raman active molecule DTNB on the surface of gold nanoparticles. Finally, the sandwich structure complex of "detecting substrate-mi RNA-SERS probe" was formed by complementary pair of bases, and the complex in the solution was separated and enriched by external magnetic field, and then SERS was detected. A multivariate SERS sensor based on tetrahedral DNA probe was developed for the detection of tumor nucleic acid marker mi RNA-21 with a detection limit of 623 a M. A tetrahedral DNA probe with specific recognition function was designed and assembled, and the tetrahedron DNA probe was sequentially assembled onto the surface of Ag NR array substrate to construct a highly sensitive SERS nucleic acid detection substrate. Tetrahedral DNA probes can simultaneously recognize two different target nucleic acids, and capture two specific SERS probes based on gold nanospheres to form gold nanospheres dimer to form "hot spots" of SERS. The signal intensity of SERS probe is used to detect the two kinds of nucleic acid molecules with high sensitivity. The sensor has good specificity and the detection limits of the linear detection range are 50 mm and 66 mm, respectively.
【学位授予单位】：南京邮电大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212

【相似文献】