基于空心金纳米花SERS探针对非小细胞肺癌中miRNA的检测研究

Previous studies found that MiRNA-196a-5p and miRNA-125a-5p were closely related to the development of lung cancer. The detection of their high sensitivity and specificity is of great significance in the early diagnosis and effective treatment of lung cancer. Surface-enhanced Raman scattering (SERS) is a highly sensitive technique that provides accurate information on the chemical structure of matter, and is well suitbale for the detection of miRNAs in body fluid samples. In contrast to other SERS tests, the hairpin-shaped SERS probe based on hollow gold nanoflowers just need a simple step matching hybridization. This method is simple and fast, with high sensitivity and low usage. In this project, we will prepare hollow gold nanoflowers with uniform morphology, excellent monodispersity and strong surface enhanced Raman scattering (SERS) effect. The hairpin-shaped SERS active probes based on hollow gold nanoflowers will be built for label and label-free detection of miRNA-196a-5p and miRNA-125a-5p, which present in cells, peripheral blood and sputum of patients with non-small cell lung cancer (NSCLC). In addition, we will study of the differences of these miRNAs expression level in different stages of NSCLC, and analyze the correlation between miRNAs and lung cancer progression. This study can develop methods of miRNA detection, and can be used for early diagnosis of lung cancer, even contribute to clinical testing and early intervention.

前期研究发现miRNA-196a-5p和miRNA-125a-5p与肺癌发生发展密切相关，它们的高灵敏和特异性的检测对肺癌早期诊断及有效治疗具有重要意义。表面增强拉曼散射（SERS）是一种高灵敏的检测技术，能够得到物质化学结构的准确信息，非常适于体液样本中miRNA的检测。相于其他SERS检测，基于空心金纳米花的发卡结构检测模型只要简单的一步配对杂化，具有简单快速、灵敏度高、用量少的特点。本项目拟基于空心金纳米花构建发夹型SERS活性探针，用于检测非小细胞肺癌（NSCLC）患者细胞、外周血和痰液中miRNA-196a-5p和miRNA-125a-5p的表达水平。研究在不同分期NSCLC患者外周血和痰液中两种miRNA表达水平的差异，并分析其与肺癌病程进展的相关性。本研究拓展miRNA检测方法，可应用于肺癌早期诊断，有助于临床检测及早期干预治疗。

miRNAs能够有效反映肿瘤的发生发展，它们的高灵敏和特异性的检测对肺癌早期诊断及有效治疗具有重要意义。表面增强拉曼散射（SERS）是一种高灵敏的检测技术，可以得到物质化学结构的准确信息，非常适于体液样本中miRNA的检测。相于其他检测模型，发卡结构检测模型只要简单的一步配对杂化，具有简单、快速、灵敏度高、用量少的特点。本项目基于空心金纳米花（HGNFs）构建了发夹型SERS活性探针，用于检测非小细胞肺癌（NSCLC）患者细胞、外周血和痰液中miR-196a-5p和miR-125a-5p的表达水平及长时程监测骨髓间充质干细胞（BMSCs）成骨分化过程中miR-31。.具体工作如下：.1.优化反应制备得到了形貌均一、单分散性好、SERS效应强的花状的中空金纳米花（GNCs）.2. 将HGNFs组装在ITO玻璃表面规模化获得大面积均匀性好且SERS活性高的GNCs固态基底（HGNFs@ITO），非标记SERS检测和区分了几种NSCLC相关miRNAs（miR-10a-5p、miR-125a-5p、miR-196a-5p）。 .3. 构建了一种胞内发夹DNA（hpDNA）功能化的空心金纳米花探针（hpDNA-HGNFs），实现了对正常肺上皮细胞和肺癌细胞中miR-125a-5p或miR-196a-5p的定量检测和成像。肺癌细胞产生SERS信号的强度明显强于正常肺上皮细胞，靶向到肺癌细胞上的SERS探针数量明显多于正常肺上皮细胞，qRT-PCR验证了SERS的准确性。.4. 在hpDNA-HGNFs制备的基础上，进一步通过SERS实现了长时程监测BMSCs成骨分化过程中miR-31的表达水平的动态变化。.5. 基于HGNFs@ITO基底和发卡结构检测模型相结合，通过简单的一步配对杂化，实现了快速、高灵敏、定量化、同时检测NSCLC患者和健康人外周血和痰液中miR-196a-5p和miR-125a-5p。相对于正常肺上皮细胞、健康人外周血和痰液，这两种miRNAs标志物在NSCLC细胞、NSCLC患者外周血和痰液中表达水平上调，证明它们与NSCLC发生发展的密切相关。