拉曼散射技术在单细胞检测中的应用
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摘要
细胞是生命体的结构和生命活动的基本单位。早在1925年,生物学大师Wilson就提出：“一切生命的关键问题都要到细胞中去寻找。”生命体的生长发育是依靠细胞增殖、细胞分化与细胞凋亡来实现的。一切疾病的发病机制也要以细胞病变研究为基础。口腔鳞状细胞癌是一种高转移性口腔癌,其发病率占世界恶性肿瘤第六位,复发率高,患者生存率低,对其治疗至今仍缺乏十分有效的手段。肿瘤转移是口腔鳞癌导致患者死亡的重要原因。早期发现和阻断肿瘤的发生发展以及浸润转移是预防与治疗肿瘤的关键所在。传统的细胞生物学方法包括Western Blot,酶联免疫组化(ELISA)和免疫细胞化学(IC)等等,这些技术大多需要复杂的样品前处理,如细胞固定,成分提取和染色等。它们存在侵入性、耗时以及假阳／阴性结果等缺点,无法实时、原位地获得细胞内物质结构的具体信息。
拉曼光谱是基于光子激发振动而发生非弹性散射而建立起来的一种无损光谱技术,具有指纹识别特征,能从分子水平获得物质的结构及组成信息。而且,表面增强拉曼光谱(SERS)技术的发展极大地提高了检测分析的灵敏度,甚至能够实现单分子检测。与红外、荧光等其他光谱手段相比,它还具有不受水干扰、不易淬灭、可用红光激发、光谱带宽窄等优点,因此非常适合于生物体系,特别是在单个活细胞的研究。
基于以上,本论文展开了相关研究,其主要内容如下：
1、采用共聚焦显微拉曼对正常以及癌细胞／组织的生物组成进行研究
通过采集正常和鳞癌组织、正常和鳞癌细胞的拉曼光谱,对比研究正常和癌变组的光谱差异,进而追踪它们生物组成的变化,并尝试寻找其与病理学相符的致病机理,最后采用主成分分析法对光谱数据进行的归类,为拉曼光谱走进临床奠定基础。
2、一氧化氮刺激细胞过程的拉曼光谱研究
采用血管扩张药物硝普钠作为一氧化氮的供应源,通过调节培养液中硝普钠的浓度来控制一氧化氮的浓度,研究一氧化氮刺激癌细胞的作用过程。通过采集不同浓度一氧化氮刺激的细胞的拉曼光谱,研究细胞内各种成分含量和构型的变化情况,并且比较拉曼光谱与传统的流式细胞技术在研究细胞与药物作用上的差别。另外,在培养环境中一氧化氮浓度不变的情况下,研究一氧化氮刺激细胞作用的动力学过程。研究结果显示拉曼光谱是一种可靠、快速、无标记和灵敏的检测手段,有望用于检测新型药物的快速筛选。
3、膜靶向探针的制备及细胞成像的研究
首先制备稳定的金纳米粒子,然后通过静电吸附作用将膜抗体(表皮生长因子受体抗体,anti-Epidermal Growth Factor Receptor antibody, EGFR)与金纳米粒子连接,使SERS探针具备细胞膜靶向功能,然后将其与鳞癌细胞(TCA8113)共培养,金纳米粒子在EGFR抗体的帮助下靶向吸附于细胞膜的某些特异位点,采用拉曼成像技术对细胞进行表征,不仅获取了信号分子的SERS光谱,而且还得到细胞膜上蛋白质、脂类、氨基酸等生物分子的振动信息,该SERS探针有望用于药物进入细胞过程的监控以及细胞膜蛋白运输机制的研究。
4、核靶向探针的制备及细胞成像的研究
首先制备稳定的金纳米粒子,然后通过化学偶联法将核定位肽(SV-40Large T NLS peptide)与金纳米粒子连接,使SERS探针具备细胞核靶向功能,然后将其与鳞癌细胞共培养,金纳米粒子在NLS(?)太的帮助下靶向在细胞核的某些特异性位点上,采用拉曼成像技术此过程进行表征,光谱成像照片显示SERS探针具有高灵敏性和稳定性。另外,将“膜”与“核”靶向SERS探针与鳞癌细胞进行共培养,首次实现了单个活细胞内不同亚细胞结构的同时靶向成像,有望为细胞信号转导研究和抗癌药物机理的探讨提供一种多靶向成像技术。
Cell is the fundamental unite of life activity and structure in the living body. Early in1925, Wilson, the famous biologist, had proposed:"All of the key problems in the life have to resort to cell." Growth and development of living body rely on proliferation, differentiation and apoptosis of cell. Almost all diseases are caused by the pathologic change of cell. Oral squamous cell carcinoma (OSCC) is a kind of oral cancer with low survival rate, high metastatic and recurrence rate. OSCC ranks the sixth in the world cancer incidence. Up to now, no treatment has shown efficacy. Metastasis is an important reason which leads to the death of OSCC patient. Early detection and blocking of tumor genesis, progenesis, invasion and metastasis is critical for the prevention and treatment of OSCC. Conventional methods of cell biology include Western blot, enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (IC), etc. Most of the techniques require numerous preparation steps, such as cell fixation, extraction or staining. They have many disadvantages such as invasive, time consuming, as well as false positive/negative, and can not get specific structure information of biological composition in cells by real-time and in situ.
Raman spectroscopy, which is based on inelastic scattering of laser photons by molecular vibrations of biopolymers, is a quantitative and non-destructive optical technique that is capable of acquiring "fingerprint" information derived from the biochemical composition of cells at molecular leval. Moreover, the development of surface enhanced Raman spectroscopy (SERS) greatly improves the sensitivity of detection and analysis. Compared with infrared and fluorescent spectroscopy, Raman spectroscopy has advantages of low aqueous interference, unease to quench, can be excited with red laser, and narrow spectral band width, which make it very suitable for the research in biological system, especially for the study of single living cell.
Based on all of this, the paper set to study. The main contents are listed as follow:
1. Investaged normal and malignant tissues/cells by confocal Raman spectroscopy. After acquiring Raman spectroscopy of normal and malignant tissues/cells, spectral difference was obtained, from which the biological composition changes were seeked out, and finally principal component analysis (PCA) was employed to classify the Raman data into normal and malignant groups. The research has laid a foundation for the clinical application of Raman spectroscopy.
2. Monitored nitric oxide stimulus response of single cancer cell by Raman spectroscopy. Sodium nitroprusside (SNP) was chosed as a NO source. The amount of NO could be controlled by adjusting the SNP concentration in culture solution. TCA8113cells were firstly treated with the indicated concentration of SNP for certain time intervals in incubator, and then Raman spectroscopy was obtained from the cells. Content and structure changes of biological components in cells were observed after NO stimulas. Raman spectral analysis was compared with flow cytometric analysis in the study of NO stimlulus reponse. In addition, kinetic study was carried out to analyze the NO stimulus process. Research results indicated that Raman spectroscopy is a reliable, fast, label-free, and sensitive detection method, and it could potentially be used in rapid screening of novel drugs.
3. Synthesis of a novel membrane-targeted SERS nanoprobe and its application in cell imaging were presented. At first, highly stable gold nanoparticle with diameter of40nm was synthesized, and then it was linked with EGFR antibody by electrostatic adsorption. After incubating the SERS nanoprobe with TCA8113cells, gold nanoparticles were specifically targeted on the cell membrane with the aid of EGFR antibody. Raman mapping technique was employed to characterize the cells, and the results demonstrated the SERS nanoprobe is of high sensitivity and stability. It will be significant for the establishment of multi-targeted, highly sensitive, in situ and in vivo SERS nanoprobe.
4. Synthesis of a novel nuclear-targeted SERS nanoprobe and its application in cell imaging were presented. At first, highly stable gold nanoparticle with diameter of20nm was synthesized, and then it was conjugated with SV-40large T nuclear localization signal (NLS) peptide by chemical coupled reaction. After incubating the SERS nanoprobe with TCA8113cells, gold nanoparticles were specifically targeted on the cell nuclear with the aid of NLS peptide. Raman mapping technique was employed to characterize the nuclear-targeted process, and the results demonstrated the SERS nanoprobe is of high sensitivity and stability. In addition, simultaneously targeted detection of different subcellular structure was realized for the first time by incubating both the membrane-and the nuclear-targeted SERS nanoprobe with TCA8113cells. It will provide an effective analysis method for the research of cell signal transduction and anticancer mechanism of novel drugs.
拉曼光谱是基于光子激发振动而发生非弹性散射而建立起来的一种无损光谱技术,具有指纹识别特征,能从分子水平获得物质的结构及组成信息。而且,表面增强拉曼光谱(SERS)技术的发展极大地提高了检测分析的灵敏度,甚至能够实现单分子检测。与红外、荧光等其他光谱手段相比,它还具有不受水干扰、不易淬灭、可用红光激发、光谱带宽窄等优点,因此非常适合于生物体系,特别是在单个活细胞的研究。
基于以上,本论文展开了相关研究,其主要内容如下：
1、采用共聚焦显微拉曼对正常以及癌细胞／组织的生物组成进行研究
通过采集正常和鳞癌组织、正常和鳞癌细胞的拉曼光谱,对比研究正常和癌变组的光谱差异,进而追踪它们生物组成的变化,并尝试寻找其与病理学相符的致病机理,最后采用主成分分析法对光谱数据进行的归类,为拉曼光谱走进临床奠定基础。
2、一氧化氮刺激细胞过程的拉曼光谱研究
采用血管扩张药物硝普钠作为一氧化氮的供应源,通过调节培养液中硝普钠的浓度来控制一氧化氮的浓度,研究一氧化氮刺激癌细胞的作用过程。通过采集不同浓度一氧化氮刺激的细胞的拉曼光谱,研究细胞内各种成分含量和构型的变化情况,并且比较拉曼光谱与传统的流式细胞技术在研究细胞与药物作用上的差别。另外,在培养环境中一氧化氮浓度不变的情况下,研究一氧化氮刺激细胞作用的动力学过程。研究结果显示拉曼光谱是一种可靠、快速、无标记和灵敏的检测手段,有望用于检测新型药物的快速筛选。
3、膜靶向探针的制备及细胞成像的研究
首先制备稳定的金纳米粒子,然后通过静电吸附作用将膜抗体(表皮生长因子受体抗体,anti-Epidermal Growth Factor Receptor antibody, EGFR)与金纳米粒子连接,使SERS探针具备细胞膜靶向功能,然后将其与鳞癌细胞(TCA8113)共培养,金纳米粒子在EGFR抗体的帮助下靶向吸附于细胞膜的某些特异位点,采用拉曼成像技术对细胞进行表征,不仅获取了信号分子的SERS光谱,而且还得到细胞膜上蛋白质、脂类、氨基酸等生物分子的振动信息,该SERS探针有望用于药物进入细胞过程的监控以及细胞膜蛋白运输机制的研究。
4、核靶向探针的制备及细胞成像的研究
首先制备稳定的金纳米粒子,然后通过化学偶联法将核定位肽(SV-40Large T NLS peptide)与金纳米粒子连接,使SERS探针具备细胞核靶向功能,然后将其与鳞癌细胞共培养,金纳米粒子在NLS(?)太的帮助下靶向在细胞核的某些特异性位点上,采用拉曼成像技术此过程进行表征,光谱成像照片显示SERS探针具有高灵敏性和稳定性。另外,将“膜”与“核”靶向SERS探针与鳞癌细胞进行共培养,首次实现了单个活细胞内不同亚细胞结构的同时靶向成像,有望为细胞信号转导研究和抗癌药物机理的探讨提供一种多靶向成像技术。
Cell is the fundamental unite of life activity and structure in the living body. Early in1925, Wilson, the famous biologist, had proposed:"All of the key problems in the life have to resort to cell." Growth and development of living body rely on proliferation, differentiation and apoptosis of cell. Almost all diseases are caused by the pathologic change of cell. Oral squamous cell carcinoma (OSCC) is a kind of oral cancer with low survival rate, high metastatic and recurrence rate. OSCC ranks the sixth in the world cancer incidence. Up to now, no treatment has shown efficacy. Metastasis is an important reason which leads to the death of OSCC patient. Early detection and blocking of tumor genesis, progenesis, invasion and metastasis is critical for the prevention and treatment of OSCC. Conventional methods of cell biology include Western blot, enzyme-linked immunosorbent assay (ELISA), immunocytochemistry (IC), etc. Most of the techniques require numerous preparation steps, such as cell fixation, extraction or staining. They have many disadvantages such as invasive, time consuming, as well as false positive/negative, and can not get specific structure information of biological composition in cells by real-time and in situ.
Raman spectroscopy, which is based on inelastic scattering of laser photons by molecular vibrations of biopolymers, is a quantitative and non-destructive optical technique that is capable of acquiring "fingerprint" information derived from the biochemical composition of cells at molecular leval. Moreover, the development of surface enhanced Raman spectroscopy (SERS) greatly improves the sensitivity of detection and analysis. Compared with infrared and fluorescent spectroscopy, Raman spectroscopy has advantages of low aqueous interference, unease to quench, can be excited with red laser, and narrow spectral band width, which make it very suitable for the research in biological system, especially for the study of single living cell.
Based on all of this, the paper set to study. The main contents are listed as follow:
1. Investaged normal and malignant tissues/cells by confocal Raman spectroscopy. After acquiring Raman spectroscopy of normal and malignant tissues/cells, spectral difference was obtained, from which the biological composition changes were seeked out, and finally principal component analysis (PCA) was employed to classify the Raman data into normal and malignant groups. The research has laid a foundation for the clinical application of Raman spectroscopy.
2. Monitored nitric oxide stimulus response of single cancer cell by Raman spectroscopy. Sodium nitroprusside (SNP) was chosed as a NO source. The amount of NO could be controlled by adjusting the SNP concentration in culture solution. TCA8113cells were firstly treated with the indicated concentration of SNP for certain time intervals in incubator, and then Raman spectroscopy was obtained from the cells. Content and structure changes of biological components in cells were observed after NO stimulas. Raman spectral analysis was compared with flow cytometric analysis in the study of NO stimlulus reponse. In addition, kinetic study was carried out to analyze the NO stimulus process. Research results indicated that Raman spectroscopy is a reliable, fast, label-free, and sensitive detection method, and it could potentially be used in rapid screening of novel drugs.
3. Synthesis of a novel membrane-targeted SERS nanoprobe and its application in cell imaging were presented. At first, highly stable gold nanoparticle with diameter of40nm was synthesized, and then it was linked with EGFR antibody by electrostatic adsorption. After incubating the SERS nanoprobe with TCA8113cells, gold nanoparticles were specifically targeted on the cell membrane with the aid of EGFR antibody. Raman mapping technique was employed to characterize the cells, and the results demonstrated the SERS nanoprobe is of high sensitivity and stability. It will be significant for the establishment of multi-targeted, highly sensitive, in situ and in vivo SERS nanoprobe.
4. Synthesis of a novel nuclear-targeted SERS nanoprobe and its application in cell imaging were presented. At first, highly stable gold nanoparticle with diameter of20nm was synthesized, and then it was conjugated with SV-40large T nuclear localization signal (NLS) peptide by chemical coupled reaction. After incubating the SERS nanoprobe with TCA8113cells, gold nanoparticles were specifically targeted on the cell nuclear with the aid of NLS peptide. Raman mapping technique was employed to characterize the nuclear-targeted process, and the results demonstrated the SERS nanoprobe is of high sensitivity and stability. In addition, simultaneously targeted detection of different subcellular structure was realized for the first time by incubating both the membrane-and the nuclear-targeted SERS nanoprobe with TCA8113cells. It will provide an effective analysis method for the research of cell signal transduction and anticancer mechanism of novel drugs.
引文
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