荧光二氧化硅纳米粒子的合成、修饰及其在细胞生物学中的应用
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摘要
荧光二氧化硅纳米粒子具有独特的光学性质、易于进行表面修饰的特点,因此被广泛的应用于生物分析和生物检测领域,而磁性纳米粒子有磁力靶点和热力转化的性质,可以应用于肿瘤热疗,将二者性质相结合得到的二氧化硅磁性纳米粒子,具有相较磁性纳米粒子增强的化学稳定性、克服其易团聚的缺点,又可以实现对肿瘤细胞的双重靶向定位,或满足癌症的热化疗双重治疗。但是,目前仍甚少有研究针对二氧化硅磁性纳米粒子对细胞的生长状况及细胞表面结构和微结构的影响,因而,这就成为了本课题的一个研究重点。
目前,传统的痕量重金属离子的检测方法,所需仪器昂贵、离子选择性差、检测灵敏度低,同时,利用荧光纳米感应器对Hg2+等痕量重金属离子进行检测的工作中,荧光二氧化硅纳米粒子还是一个空白,因此,本课题的另一个研究重点旨在发现一种利用荧光二氧化硅纳米粒子对Hg2+的超灵敏快速检测方法,以期进一步将该方法应用于细胞内Hg2+含量测定。
(1)通过化学共沉淀法制备铁磁性纳米粒子(FeNPs),并以W／O反相微乳法制备包埋荧光染料三联吡啶钌配合物Ru(bpy)32+的二氧化硅纳米粒子(SiNPs)、二氧化硅磁性纳米粒子(Si/FeNPs)和氨基化二氧化硅纳米粒子(NH2NPs)。对制备FeNPs的后处理过程进行改进,使其更有利于进行二氧化硅壳层结构的包覆。本文通过改变反应物加样方式,解决了以往由于疏水性铁磁性纳米粒子制备条件苛刻、价格昂贵造成的实验费用高的问题。
(2)用TEM、XRD、UV-Vis、FTIR、荧光光谱以及磁滞回线测定等方法,对Si/FeNPs、SiNPs、NH2NPs、FeNPs进行了表征。Si/FeNPs、SiNPs、NH2NPs,均为球形实体,粒径均一,表面致密光滑,单分散性良好,并且三种纳米粒子均成功的包埋了Ru(bpy)32+,并且很好的保留了其光学性质；Si/FeNPs为非晶态物质,成功的包埋了FeNPs,具有超顺磁性,但其磁性能较FeNPs有所降低；FeNPs为类球形非晶态纳米粒子,单分散性良好,具有超顺磁性。
(3)研究了不同浓度的Si/FeNPs对肝癌细胞HepG2的生物效应,并以相同磁性能的FeNPs和相同硅含量的SiNPs作为参比。Si/FeNPs、SiNPs对HepG2细胞具有明显的促增殖作用,能够被通过包吞作用进入细胞,并未对细胞的表面形态和超微结构产生影响
(4)成功制备了富胸腺嘧啶核酸序列修饰的荧光二氧化硅纳米粒子核酸探针,利用胸腺嘧啶与Hg2+的特异性结合,可以实现对Hg2+的快速灵敏检测。为进一步检测细胞中的痕量Hg2+奠定了基础。
Due to its unique optical properties and ease of surface modifications, fluorescent silica-based nanoparticles are widely used in biological analysis and detection applications; on the other hand, magnetic nanoparticles can be used for hyperthermia treatment, taking advantage of their magnetic properties and energy transfer schema. Thus, silica coated magnetic nanoparticles, combining features of both nanoparticles, not only with improved chemical stability than magnetic nanoparticles, having less aggregation in liquid, but also can fulfill the goal of dual targeting or thermochemotherapy of cancer cells. But there are still limited research on their effects on cell growth, influences on surface structure and micro-structure of cancer cells, so this is one important topic of this study.
At present, the traditional detection method of trace heavy metal ions, has many defects, such as expensive equipments, low selectivity for ions and low sensitivity. Till now there is no fluorescent nano-sensors detection of Hg2+has been studied, therefore, to develop anultra-sensitive and rapid mercury-sensing method is another topic of this study, and next step is to apply this method to detect intracellular Hg
A series of research have been performed in this study: (1) Synthesized iron magnetic nanoparticles (FeNPs) by coprecipitation, and prepared silica nanoparticles(SiNPs), silica coated magnetic nanoparticles(Si/FeNPs), amino group modified silica nanoparticles (NH2NPs) with Ru(bpy)32+fluorescent by W/O microemulsion method. Improvements made on the post-process of FeNPs, making it more suitable for Si/FeNPs preparation. Changed the reactant addition methods and resolved the problems encountered in the past, such as harsh synthetic conditions, high cost of nanoparticles preparation.
(2) Si/FeNPs, SiNPs, NH2NPs, FeNPs were characterized by TEM, XRD, UV-Vis, FTIR, fluorescence spectroscopy and hysteresis analysis. Si/FeNPs, SiNPs, NH2NPs, were all spherical structure, had uniform particle size, single-well dispersed, and they were all successfully embedded with Ru(bpy)32-, retaining its optical properties; Si/FeNPs was superparamagnetic, amorphous material, and successfully embedded with FeNPs, although had relatively lower magnetic field than FeNPs; FeNPs amorphous nano-particles were spherical structure, single-well dispersed, and superparamagnetic.
(3) Studied the biological effects of Si/FeNPs with different concentrations on HepG2 hepatoma cells, using FeNPs with same magnetic property and SiNPs with same silicon content as reference. Si/FeNPs, SiNPs could promote proliferation of HepG2 cells, could enter cells by endocytosis and had no harmful effects on cell morphology and micro-structure.
(4) Successfully synthesized thymine-rich nucleic acid sequences modified silica-based fluorescent nanoparticles. As Hg2+can specific bind with thymidine, this nanoparticle-DNA sequence probe could be used to fast detect Hg2+ with high sensitivity. Further, this method could be applied to detection of intracellular Hg2+
目前,传统的痕量重金属离子的检测方法,所需仪器昂贵、离子选择性差、检测灵敏度低,同时,利用荧光纳米感应器对Hg2+等痕量重金属离子进行检测的工作中,荧光二氧化硅纳米粒子还是一个空白,因此,本课题的另一个研究重点旨在发现一种利用荧光二氧化硅纳米粒子对Hg2+的超灵敏快速检测方法,以期进一步将该方法应用于细胞内Hg2+含量测定。
(1)通过化学共沉淀法制备铁磁性纳米粒子(FeNPs),并以W／O反相微乳法制备包埋荧光染料三联吡啶钌配合物Ru(bpy)32+的二氧化硅纳米粒子(SiNPs)、二氧化硅磁性纳米粒子(Si/FeNPs)和氨基化二氧化硅纳米粒子(NH2NPs)。对制备FeNPs的后处理过程进行改进,使其更有利于进行二氧化硅壳层结构的包覆。本文通过改变反应物加样方式,解决了以往由于疏水性铁磁性纳米粒子制备条件苛刻、价格昂贵造成的实验费用高的问题。
(2)用TEM、XRD、UV-Vis、FTIR、荧光光谱以及磁滞回线测定等方法,对Si/FeNPs、SiNPs、NH2NPs、FeNPs进行了表征。Si/FeNPs、SiNPs、NH2NPs,均为球形实体,粒径均一,表面致密光滑,单分散性良好,并且三种纳米粒子均成功的包埋了Ru(bpy)32+,并且很好的保留了其光学性质；Si/FeNPs为非晶态物质,成功的包埋了FeNPs,具有超顺磁性,但其磁性能较FeNPs有所降低；FeNPs为类球形非晶态纳米粒子,单分散性良好,具有超顺磁性。
(3)研究了不同浓度的Si/FeNPs对肝癌细胞HepG2的生物效应,并以相同磁性能的FeNPs和相同硅含量的SiNPs作为参比。Si/FeNPs、SiNPs对HepG2细胞具有明显的促增殖作用,能够被通过包吞作用进入细胞,并未对细胞的表面形态和超微结构产生影响
(4)成功制备了富胸腺嘧啶核酸序列修饰的荧光二氧化硅纳米粒子核酸探针,利用胸腺嘧啶与Hg2+的特异性结合,可以实现对Hg2+的快速灵敏检测。为进一步检测细胞中的痕量Hg2+奠定了基础。
Due to its unique optical properties and ease of surface modifications, fluorescent silica-based nanoparticles are widely used in biological analysis and detection applications; on the other hand, magnetic nanoparticles can be used for hyperthermia treatment, taking advantage of their magnetic properties and energy transfer schema. Thus, silica coated magnetic nanoparticles, combining features of both nanoparticles, not only with improved chemical stability than magnetic nanoparticles, having less aggregation in liquid, but also can fulfill the goal of dual targeting or thermochemotherapy of cancer cells. But there are still limited research on their effects on cell growth, influences on surface structure and micro-structure of cancer cells, so this is one important topic of this study.
At present, the traditional detection method of trace heavy metal ions, has many defects, such as expensive equipments, low selectivity for ions and low sensitivity. Till now there is no fluorescent nano-sensors detection of Hg2+has been studied, therefore, to develop anultra-sensitive and rapid mercury-sensing method is another topic of this study, and next step is to apply this method to detect intracellular Hg
A series of research have been performed in this study: (1) Synthesized iron magnetic nanoparticles (FeNPs) by coprecipitation, and prepared silica nanoparticles(SiNPs), silica coated magnetic nanoparticles(Si/FeNPs), amino group modified silica nanoparticles (NH2NPs) with Ru(bpy)32+fluorescent by W/O microemulsion method. Improvements made on the post-process of FeNPs, making it more suitable for Si/FeNPs preparation. Changed the reactant addition methods and resolved the problems encountered in the past, such as harsh synthetic conditions, high cost of nanoparticles preparation.
(2) Si/FeNPs, SiNPs, NH2NPs, FeNPs were characterized by TEM, XRD, UV-Vis, FTIR, fluorescence spectroscopy and hysteresis analysis. Si/FeNPs, SiNPs, NH2NPs, were all spherical structure, had uniform particle size, single-well dispersed, and they were all successfully embedded with Ru(bpy)32-, retaining its optical properties; Si/FeNPs was superparamagnetic, amorphous material, and successfully embedded with FeNPs, although had relatively lower magnetic field than FeNPs; FeNPs amorphous nano-particles were spherical structure, single-well dispersed, and superparamagnetic.
(3) Studied the biological effects of Si/FeNPs with different concentrations on HepG2 hepatoma cells, using FeNPs with same magnetic property and SiNPs with same silicon content as reference. Si/FeNPs, SiNPs could promote proliferation of HepG2 cells, could enter cells by endocytosis and had no harmful effects on cell morphology and micro-structure.
(4) Successfully synthesized thymine-rich nucleic acid sequences modified silica-based fluorescent nanoparticles. As Hg2+can specific bind with thymidine, this nanoparticle-DNA sequence probe could be used to fast detect Hg2+ with high sensitivity. Further, this method could be applied to detection of intracellular Hg2+
引文
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