纳米功能材料的可控制备、表面修饰及其荧光标记和细胞毒性的研究
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摘要
随着纳米技术和纳米材料的迅速发展,量子点、磁性Fe3O4、纳米金、氧化铈、碳纳米管和石墨烯等纳米功能材料在生物医学领域的应用越来越广泛。其中量子点和氧化铈的应用尤为引人关注。论文瞄准这一研究方向,在对当前迅速发展的量子点和纳米氧化铈进行大量文献调研的基础上,以电泳分析技术,MTT法及抗氧化性测试作为主要分析手段,开展了量子点和纳米氧化铈的可控制备、表面修饰及其荧光标记和细胞毒性的研究工作,论文具体章节的内容介绍如下：
第一章针对量子点荧光标记物高灵敏、原位、实时、动态的前沿和热点,概述了量子点荧光标记物在细胞和组织成像、靶向药物、DNA分子检测方面的研究进展；针对纳米氧化铈独特的储放氧功能和催化/再生的特性,扼要的介绍了纳米氧化铈在自由基清除、抗氧化性、肿瘤标记和靶向药物等生物医学领域的研究热点和进展。基于这两种纳米功能材料在荧光标记和抗氧化性方面的应用,提出了本论文的工作内容和创新点。
第二章在高温油相、N2保护的条件下,依据“奥斯瓦尔特”熟化原理,利用“反向注入法”合成尺寸可控的CdSe量子点,并在其表面包覆了能级较宽的ZnS,合成了核壳型结构的CdSe@ZnS量子点。为了提高CdSe@ZnS量子点的生物兼容性,我们在其表面修饰了两亲性的高分子材料,将脂溶性量子点转换为水溶性量子点,为量子点进一步的功能组装奠定基础。
第三章以量子点标记DNA分子为主要研究内容,针对寡核苷酸探针需特异性设计、传统的有机荧光小分子和生物素不稳定和传统的放射性同位素标记有辐射的问题,尝试采用PCR技术获得的DNA与量子点进行荧光标记,以期获得一种廉价、便捷的DNA分子荧光标记的方法。
第四章以制备超细单分散、水溶性纳米氧化铈的制备为主要研究内容,利用X-射线粉末衍射(XRD)、透射电子显微镜(TEM)、X-光电子能谱(XPS)、傅里叶红外变换(FT-IR)、紫外-可见光(UV-vis)和Zeta电位等表征方法,深入细致地分析纳米氧化铈的物相组成、形貌、大小、价态、表面官能团和电荷性质。确立一种行之有效的超细单分散、水溶性纳米氧化铈的合成及表面修饰的方法,为下一步体外细胞测试奠定了基础。
第五章以超细单分散纳米氧化铈对人体胃癌细胞(BGC-803)的细胞毒性和抗氧化性测试为研究主线,利用MTT, GSH, SOD, FCA, SEM和TEM等检测方法,分析BGC-803细胞与超细单分散纳米氧化铈作用后的细胞形态,抗氧化性能力和细胞摄入情况,探讨纳米氧化铈的化学组成和价态、尺寸和形貌、表面官能团和电荷性质对BGC-803细胞的细胞毒性和抗氧化性的影响规律,为研究纳米氧化铈在抗氧化药物方面的应用提供实验数据,并为内蒙古地区开展轻稀土氧化铈在生物医学领域的应用提供实验和理论依据。
With the rapid development of nanotechnology and wide application of nanomaterials, quantum dots (QDs), magnetic Fe3O4, nano gold, cerium oxide, carbon nanotubes grapheme and so on functional nanomaterials are already widely applied in the biomedical field. Among these functional nanomaterial, quantum dots and cerium oxide are particularly concerned. Just aiming at this important research direction, based on a survey of a large number of documents, by means of electrophoresis analysis techniques, MTT method and anti-oxidation test (SOD and GSH), the controllable synthesis, surface modification of functional nanomaterial (quantum dots and cerium oxide) and their fluorescence labeling and cell cytotoxicity study are carried out. Content of specific sections of the paper are described below.
In the first chapter, for the frontier and hot of quantum dots fluorescent labels at highly sensitive, in situ, real-time, dynamic, the paper summarizes the research progress of quantum dot fluorescent labels in the cell and tissue imaging, targeted drug, DNA molecular testing aspects. Considering the nanocerium oxide with the unique feature at storing and releasing oxygen, catalytic/regenerative properties, the article sums up the research progress of nanocerium oxide in the biomedical field at radical scavenging, antioxidant activity, tumor markers and targeted drugs. Based on these two functional nanomaterials in fluorescent markers and antioxidant aspects of the application, the research concent and innovation also is come up with.
In the second chapter, based on "Oswalt" ripening principle, size-controlled CdSe quantum dots were synthesized by use of "reverse injection method" in the high temperature of the oil phase and N2protection conditions. Then core and shell CdSe@Zns QDs were prepared through coating the wide level nanomaterials ZnS on the surface of CdSe quantum dots. In order to enhance biocompatibility of CdSe@Zns QDs, a kind of two dear of polymer materials was modified on the surface of CdSe@Zns QDs, which make the fat-soluble quantum dots conver to water-soluble quantum dots. The obtained QDs with excellent solubility could be further functional assembled.
In the third chapter, DNA molecules labeled with quantum dots is the main content. Because the oligonucleotide molecule probe needs special design, its cost is high. While conventional organic fluorescent small molecule and biotin are instability, at the same time, conventional radioisotope labeling has radiation. In order to obtain an inexpensive and convenient method of fluorescently labeled DNA molecules, we try to label DNA molecules obtained by PCR technology by means of quantum dot.
In the fourth chapter, controllable synthesis and surface modification of nanoceria are the main content. Nanoceria's phase composition, morphology, size, valence, and the charge properties of surface functional groups were analyzed in great detail by X-ray powder diffraction (XRD), Transmission electron microscopy (TEM), X-photoelectron spectroscopy (XPS), Fourier transform microscopy infrared spectrometer (FT-IR), UV-vis absorption (UV-vis) and Zeta potential technologies. In this part of work, it corfirms an effective method, which could prepare ultrafine monodisperse and soluble nanoceria modified with functional group. Furthermore these nanoceria particles are completely in conformity with the requirements of biomedical field and established the foundation for the next test in vitro.
In the fifth chapter, the cell cytotoxicity and antioxidant property of ultrafine monodisperse nanoceria are the main content. Cell morphology, antioxidant property and cell intake are analyzed by use of Scanning electron microscope (SEM), Transmission electron microscopy (TEM), MTT assay, GSH, SOD and Flow Cytometric assay (FCA) after the BGC-803cells and ultrafine monodispersed nano ceria interact. In this work, we explored the relationship between the soluble property, chemical valence, surface functional groups of nanoceria and the cytotoxicity and antioxidant properties of BGC-803cells, which could be benefit for determining the law between the chemical composition and valence state, size and morphology, surface charge and surface coating, agglomeration state of cerium oxide nanoparticles and cytotoxicity of BGC-803cells, revealing the incentive of oxidative stress in BGC-803cells. This work could provide experimental data for the study of cerium oxide nanoparticles in antioxidant drugs and could help to carry out light rare earth cerium oxide's application of the biomedical field in Inner Mongolia region.
第一章针对量子点荧光标记物高灵敏、原位、实时、动态的前沿和热点,概述了量子点荧光标记物在细胞和组织成像、靶向药物、DNA分子检测方面的研究进展；针对纳米氧化铈独特的储放氧功能和催化/再生的特性,扼要的介绍了纳米氧化铈在自由基清除、抗氧化性、肿瘤标记和靶向药物等生物医学领域的研究热点和进展。基于这两种纳米功能材料在荧光标记和抗氧化性方面的应用,提出了本论文的工作内容和创新点。
第二章在高温油相、N2保护的条件下,依据“奥斯瓦尔特”熟化原理,利用“反向注入法”合成尺寸可控的CdSe量子点,并在其表面包覆了能级较宽的ZnS,合成了核壳型结构的CdSe@ZnS量子点。为了提高CdSe@ZnS量子点的生物兼容性,我们在其表面修饰了两亲性的高分子材料,将脂溶性量子点转换为水溶性量子点,为量子点进一步的功能组装奠定基础。
第三章以量子点标记DNA分子为主要研究内容,针对寡核苷酸探针需特异性设计、传统的有机荧光小分子和生物素不稳定和传统的放射性同位素标记有辐射的问题,尝试采用PCR技术获得的DNA与量子点进行荧光标记,以期获得一种廉价、便捷的DNA分子荧光标记的方法。
第四章以制备超细单分散、水溶性纳米氧化铈的制备为主要研究内容,利用X-射线粉末衍射(XRD)、透射电子显微镜(TEM)、X-光电子能谱(XPS)、傅里叶红外变换(FT-IR)、紫外-可见光(UV-vis)和Zeta电位等表征方法,深入细致地分析纳米氧化铈的物相组成、形貌、大小、价态、表面官能团和电荷性质。确立一种行之有效的超细单分散、水溶性纳米氧化铈的合成及表面修饰的方法,为下一步体外细胞测试奠定了基础。
第五章以超细单分散纳米氧化铈对人体胃癌细胞(BGC-803)的细胞毒性和抗氧化性测试为研究主线,利用MTT, GSH, SOD, FCA, SEM和TEM等检测方法,分析BGC-803细胞与超细单分散纳米氧化铈作用后的细胞形态,抗氧化性能力和细胞摄入情况,探讨纳米氧化铈的化学组成和价态、尺寸和形貌、表面官能团和电荷性质对BGC-803细胞的细胞毒性和抗氧化性的影响规律,为研究纳米氧化铈在抗氧化药物方面的应用提供实验数据,并为内蒙古地区开展轻稀土氧化铈在生物医学领域的应用提供实验和理论依据。
With the rapid development of nanotechnology and wide application of nanomaterials, quantum dots (QDs), magnetic Fe3O4, nano gold, cerium oxide, carbon nanotubes grapheme and so on functional nanomaterials are already widely applied in the biomedical field. Among these functional nanomaterial, quantum dots and cerium oxide are particularly concerned. Just aiming at this important research direction, based on a survey of a large number of documents, by means of electrophoresis analysis techniques, MTT method and anti-oxidation test (SOD and GSH), the controllable synthesis, surface modification of functional nanomaterial (quantum dots and cerium oxide) and their fluorescence labeling and cell cytotoxicity study are carried out. Content of specific sections of the paper are described below.
In the first chapter, for the frontier and hot of quantum dots fluorescent labels at highly sensitive, in situ, real-time, dynamic, the paper summarizes the research progress of quantum dot fluorescent labels in the cell and tissue imaging, targeted drug, DNA molecular testing aspects. Considering the nanocerium oxide with the unique feature at storing and releasing oxygen, catalytic/regenerative properties, the article sums up the research progress of nanocerium oxide in the biomedical field at radical scavenging, antioxidant activity, tumor markers and targeted drugs. Based on these two functional nanomaterials in fluorescent markers and antioxidant aspects of the application, the research concent and innovation also is come up with.
In the second chapter, based on "Oswalt" ripening principle, size-controlled CdSe quantum dots were synthesized by use of "reverse injection method" in the high temperature of the oil phase and N2protection conditions. Then core and shell CdSe@Zns QDs were prepared through coating the wide level nanomaterials ZnS on the surface of CdSe quantum dots. In order to enhance biocompatibility of CdSe@Zns QDs, a kind of two dear of polymer materials was modified on the surface of CdSe@Zns QDs, which make the fat-soluble quantum dots conver to water-soluble quantum dots. The obtained QDs with excellent solubility could be further functional assembled.
In the third chapter, DNA molecules labeled with quantum dots is the main content. Because the oligonucleotide molecule probe needs special design, its cost is high. While conventional organic fluorescent small molecule and biotin are instability, at the same time, conventional radioisotope labeling has radiation. In order to obtain an inexpensive and convenient method of fluorescently labeled DNA molecules, we try to label DNA molecules obtained by PCR technology by means of quantum dot.
In the fourth chapter, controllable synthesis and surface modification of nanoceria are the main content. Nanoceria's phase composition, morphology, size, valence, and the charge properties of surface functional groups were analyzed in great detail by X-ray powder diffraction (XRD), Transmission electron microscopy (TEM), X-photoelectron spectroscopy (XPS), Fourier transform microscopy infrared spectrometer (FT-IR), UV-vis absorption (UV-vis) and Zeta potential technologies. In this part of work, it corfirms an effective method, which could prepare ultrafine monodisperse and soluble nanoceria modified with functional group. Furthermore these nanoceria particles are completely in conformity with the requirements of biomedical field and established the foundation for the next test in vitro.
In the fifth chapter, the cell cytotoxicity and antioxidant property of ultrafine monodisperse nanoceria are the main content. Cell morphology, antioxidant property and cell intake are analyzed by use of Scanning electron microscope (SEM), Transmission electron microscopy (TEM), MTT assay, GSH, SOD and Flow Cytometric assay (FCA) after the BGC-803cells and ultrafine monodispersed nano ceria interact. In this work, we explored the relationship between the soluble property, chemical valence, surface functional groups of nanoceria and the cytotoxicity and antioxidant properties of BGC-803cells, which could be benefit for determining the law between the chemical composition and valence state, size and morphology, surface charge and surface coating, agglomeration state of cerium oxide nanoparticles and cytotoxicity of BGC-803cells, revealing the incentive of oxidative stress in BGC-803cells. This work could provide experimental data for the study of cerium oxide nanoparticles in antioxidant drugs and could help to carry out light rare earth cerium oxide's application of the biomedical field in Inner Mongolia region.
引文
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