金纳米星SERS探针的构筑及其在细胞检测中的应用研究

发布时间：2018-05-29 22:08

本文选题：金纳米星 + 表面增强拉曼散射　；参考：《东南大学》2016年博士论文

【摘要】：拉曼光谱是一种无损光谱技术,具有指纹识别的特征,能在分子水平获得物质的结构信息。表面增强拉曼散射(SERS)主要是纳米尺度的粗糙表面所具有的异常光学增强现象,它可以将吸附在材料表面分子的拉曼信号放大102-106倍,与常规拉曼散射相比具有更高的检测灵敏度。SERS还具有稳定、不易猝灭、不受生物样品自身荧光及水的干扰等特点,非常适于生物样品的检测。金纳米星是一种由金属核和多个枝角构成的新型纳米材料,不仅在近红外区具有可调制的局域电磁场,而且尖锐枝角的“热点”(Hot Spots)极大地增强了周围电磁场的强度。本论文基于金纳米星构筑了标记SERS探针(SERS tags)和无标记SERS探针(label-free SERS probes),开展了细胞的SERS检测研究。本论文具体开展了如下工作：1.采用种子介导生长法制备金纳米星,通过调节反应体系中氯金酸、对苯二酚以及金种子的比例,调控金纳米星的枝角长度和粒径。利用扫描电子显微镜(SEM)、紫外-可见-近红外(UV-vis-NIR)分光光度计和共聚焦拉曼光谱仪对金纳米星的形貌、光学性质和SERS效应进行了表征,优化反应条件,制得了形貌均一、单分散性好和SERS效应强的金纳米星。2.以尼罗兰(NBA)和牛血清白蛋白(BSA)分别作为拉曼信号分子和保护层,制备了一种用于细胞成像的标记SERS探针。聚集实验和CCK-8细胞毒性分析验证了标记SERS探针具有很好稳定性及生物相容性。将标记SERS探针用于人肺腺癌细胞(A549)和人肺泡Ⅱ型上皮细胞(ATⅡ)的成像。结果表明标记SERS探针主要分布在细胞质,少量进入细胞核,负载标记SERS探针的细胞产生的拉曼信号强度远高于未负载的细胞。通过细胞的暗场成像、TEM验证了SERS成像的结果。3. 以对巯基苯甲酸(4-MBA)作为拉曼信号分子和表皮生长因子受体抗体(anti-EGFR)与金纳米星之间的偶联剂,构筑了一种具有细胞膜靶向功能的标记SERS探针。将标记SERS探针用于人正常肺上皮细胞(BEAS-2B)和人肺癌细胞(A549、H1229)的成像研究。A549细胞和H1229细胞产生SERS信号的强度明显强于BEAS-2B细胞,靶向到肺癌细胞上的SERS探针数量明显多于正常细胞。暗场成像与X射线能谱(EDS)分析验证了anti-EGFR偶联金纳米星成功富集到肺癌细胞表面。4.将细胞膜穿透肽(TAT)连接到金纳米星表面,构筑了一种无标记SERS探针用于区分肺干细胞(LR-MSCs)的分化。评价了无标记SERS探针的稳定性、生物相容性及探针与细胞的相互作用。测得了LR-MSCs定向分化为肺泡上皮细胞(ATⅡ)或间质细胞(3T3)的平均SERS光谱。主成分分析(PCA)基于细胞SERS光谱很好地识别和区分了LR-MSCs的不同分化亚型。5.基于TAT功能化无标记SERS探针,通过SERS表征了肺泡上皮细胞-间质转化(EMT)过程。免疫荧光和蛋白免疫印迹检测了EMT过程中上皮标志物和间质标志物的表达,验证了博来霉素(BLM)能够诱导ATⅡ细胞转化为3T3细胞。检测了EMT不同阶段细胞的SERS光谱,在EMT过程中,许多细胞组分的特征峰发生了明显的变化。PCA散点图表明EMT不同阶段的细胞被划分在四个不同的区域中,不同类型的细胞区分明显。
[Abstract]:Raman spectroscopy is a nondestructive spectroscopy, which has the characteristics of fingerprint recognition and can obtain structural information at the molecular level. The surface enhanced Raman scattering (SERS) is mainly a phenomenon of abnormal optical enhancement in the rough surface of nanoscale. It can magnify the Raman signal adsorbed on the surface of the material by 102-106 times. The Raman scattering has a higher detection sensitivity than the.SERS, which is also stable, not easily quenched, not affected by the biological samples' self fluorescence and water interference. It is very suitable for the detection of biological samples. Gold nanoscale is a new type of nano material composed of metal nuclei and multiple dendrite, which not only has modulation local electromagnetic field in the near infrared region. The field, and the "hot spot" (Hot Spots) of the sharp corner (Hot Spots) greatly enhanced the intensity of the surrounding electromagnetic field. Based on the gold nanstars constructed the labeled SERS probe (SERS tags) and the unlabeled SERS probe (label-free SERS probes), the detection of SERS in the cell was carried out. The following work was carried out in this paper: 1. using seed mediate The length and particle size of gold nanstars are regulated by adjusting the proportion of chloroauric acid, hydroquinone and gold seeds in the reaction system. The morphology, optical properties and SERS effect of the gold nanstars are measured by scanning electron microscope (SEM), ultraviolet visible near infrared (UV-vis-NIR) spectrophotometer and confocal Raman spectrometer. A gold nanoscale star.2. with homogeneous morphology, good monodispersity and strong SERS effect was prepared, and a kind of labeled SERS probe used for cell imaging was prepared, respectively, and the aggregation experiment and CCK-8 cytotoxicity analysis verified the standard. The SERS probe has good stability and biocompatibility. The labeled SERS probe is applied to the imaging of human lung adenocarcinoma cell (A549) and human alveolar type II epithelial cells (AT II). The results show that the labeled SERS probe is mainly distributed in the cytoplasm, a little into the nucleus, and the intensity of the Raman signal produced by the cells loaded with SERS probe is far higher than that of the non negative. The cells carried by the cell. Through the dark field imaging of the cells, TEM verified the result of SERS imaging.3. to construct a tagged SERS probe with cell membrane targeting function with the coupling agent for mercapto benzoic acid (4-MBA) as a Raman signal molecule and the epidermal growth factor receptor antibody (anti-EGFR) and gold nanoscale. The labeled SERS probe was used to be used as a human positive. Imaging of normal lung epithelial cells (BEAS-2B) and human lung cancer cells (A549, H1229), the intensity of SERS signals produced by.A549 and H1229 cells is stronger than that of BEAS-2B cells. The number of SERS probes on target to lung cancer cells is more than that of normal cells. Dark field imaging and X ray energy spectrum (EDS) analysis verify the success of anti-EGFR coupling gold nanoscale. .4. was enriched on the surface of lung cancer cells to connect the cell membrane penetrating peptide (TAT) to the surface of the gold nanoscale. A unlabeled SERS probe was constructed to differentiate the differentiation of lung stem cells (LR-MSCs). The stability of the unlabeled SERS probe, the biocompatibility and the interaction between the probe and the cell were evaluated. The LR-MSCs directed differentiation into the alveolar epithelium was measured. The average SERS spectra of cell (AT II) or interstitial cells (3T3). Principal component analysis (PCA) based on cell SERS spectra well identified and distinguished the different differentiated subtypes of LR-MSCs.5. based on TAT functionalized unlabeled SERS probes and characterized the alveolar epithelial cell mesenchymal transition (EMT) process by SERS. Immunofluorescence and protein immunoblotting detected EMT. The expression of epithelial markers and interstitial markers in the course showed that BLM could induce AT II cells to be transformed into 3T3 cells. The SERS spectra of different stages of EMT were detected. In the EMT process, the characteristic peaks of many cell components were obviously changed by.PCA scatter plot, indicating that the cells in different stages of EMT were divided into four different stages. In the region, the differentiation of different types of cells is obvious.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：O657.37;R318

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