發(fā)布時(shí)間：2018-05-29 22:08

  本文選題：金納米星 + 表面增強(qiáng)拉曼散射　； 參考：《東南大學(xué)》2016年博士論文

【摘要】：拉曼光譜是一種無(wú)損光譜技術(shù),具有指紋識(shí)別的特征,能在分子水平獲得物質(zhì)的結(jié)構(gòu)信息。表面增強(qiáng)拉曼散射(SERS)主要是納米尺度的粗糙表面所具有的異常光學(xué)增強(qiáng)現(xiàn)象,它可以將吸附在材料表面分子的拉曼信號(hào)放大102-106倍,與常規(guī)拉曼散射相比具有更高的檢測(cè)靈敏度。SERS還具有穩(wěn)定、不易猝滅、不受生物樣品自身熒光及水的干擾等特點(diǎn),非常適于生物樣品的檢測(cè)。金納米星是一種由金屬核和多個(gè)枝角構(gòu)成的新型納米材料,不僅在近紅外區(qū)具有可調(diào)制的局域電磁場(chǎng),而且尖銳枝角的“熱點(diǎn)”(Hot Spots)極大地增強(qiáng)了周圍電磁場(chǎng)的強(qiáng)度。本論文基于金納米星構(gòu)筑了標(biāo)記SERS探針(SERS tags)和無(wú)標(biāo)記SERS探針(label-free SERS probes),開(kāi)展了細(xì)胞的SERS檢測(cè)研究。本論文具體開(kāi)展了如下工作：1.采用種子介導(dǎo)生長(zhǎng)法制備金納米星,通過(guò)調(diào)節(jié)反應(yīng)體系中氯金酸、對(duì)苯二酚以及金種子的比例,調(diào)控金納米星的枝角長(zhǎng)度和粒徑。利用掃描電子顯微鏡(SEM)、紫外-可見(jiàn)-近紅外(UV-vis-NIR)分光光度計(jì)和共聚焦拉曼光譜儀對(duì)金納米星的形貌、光學(xué)性質(zhì)和SERS效應(yīng)進(jìn)行了表征,優(yōu)化反應(yīng)條件,制得了形貌均一、單分散性好和SERS效應(yīng)強(qiáng)的金納米星。2.以尼羅蘭(NBA)和牛血清白蛋白(BSA)分別作為拉曼信號(hào)分子和保護(hù)層,制備了一種用于細(xì)胞成像的標(biāo)記SERS探針。聚集實(shí)驗(yàn)和CCK-8細(xì)胞毒性分析驗(yàn)證了標(biāo)記SERS探針具有很好穩(wěn)定性及生物相容性。將標(biāo)記SERS探針用于人肺腺癌細(xì)胞(A549)和人肺泡Ⅱ型上皮細(xì)胞(ATⅡ)的成像。結(jié)果表明標(biāo)記SERS探針主要分布在細(xì)胞質(zhì),少量進(jìn)入細(xì)胞核,負(fù)載標(biāo)記SERS探針的細(xì)胞產(chǎn)生的拉曼信號(hào)強(qiáng)度遠(yuǎn)高于未負(fù)載的細(xì)胞。通過(guò)細(xì)胞的暗場(chǎng)成像、TEM驗(yàn)證了SERS成像的結(jié)果。3. 以對(duì)巰基苯甲酸(4-MBA)作為拉曼信號(hào)分子和表皮生長(zhǎng)因子受體抗體(anti-EGFR)與金納米星之間的偶聯(lián)劑,構(gòu)筑了一種具有細(xì)胞膜靶向功能的標(biāo)記SERS探針。將標(biāo)記SERS探針用于人正常肺上皮細(xì)胞(BEAS-2B)和人肺癌細(xì)胞(A549、H1229)的成像研究。A549細(xì)胞和H1229細(xì)胞產(chǎn)生SERS信號(hào)的強(qiáng)度明顯強(qiáng)于BEAS-2B細(xì)胞,靶向到肺癌細(xì)胞上的SERS探針數(shù)量明顯多于正常細(xì)胞。暗場(chǎng)成像與X射線能譜(EDS)分析驗(yàn)證了anti-EGFR偶聯(lián)金納米星成功富集到肺癌細(xì)胞表面。4.將細(xì)胞膜穿透肽(TAT)連接到金納米星表面,構(gòu)筑了一種無(wú)標(biāo)記SERS探針用于區(qū)分肺干細(xì)胞(LR-MSCs)的分化。評(píng)價(jià)了無(wú)標(biāo)記SERS探針的穩(wěn)定性、生物相容性及探針與細(xì)胞的相互作用。測(cè)得了LR-MSCs定向分化為肺泡上皮細(xì)胞(ATⅡ)或間質(zhì)細(xì)胞(3T3)的平均SERS光譜。主成分分析(PCA)基于細(xì)胞SERS光譜很好地識(shí)別和區(qū)分了LR-MSCs的不同分化亞型。5.基于TAT功能化無(wú)標(biāo)記SERS探針,通過(guò)SERS表征了肺泡上皮細(xì)胞-間質(zhì)轉(zhuǎn)化(EMT)過(guò)程。免疫熒光和蛋白免疫印跡檢測(cè)了EMT過(guò)程中上皮標(biāo)志物和間質(zhì)標(biāo)志物的表達(dá),驗(yàn)證了博來(lái)霉素(BLM)能夠誘導(dǎo)ATⅡ細(xì)胞轉(zhuǎn)化為3T3細(xì)胞。檢測(cè)了EMT不同階段細(xì)胞的SERS光譜,在EMT過(guò)程中,許多細(xì)胞組分的特征峰發(fā)生了明顯的變化。PCA散點(diǎn)圖表明EMT不同階段的細(xì)胞被劃分在四個(gè)不同的區(qū)域中,不同類型的細(xì)胞區(qū)分明顯。
[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.
【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：O657.37;R318

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