【作者】 李敏；

【导师】 吴琪琳；

【作者基本信息】 东华大学 ， 纳米纤维及杂化材料， 2017， 硕士

【摘要】 铜离子(Cu²⁺),人体中第三丰富的过渡金属离子,在生物系统中起着至关重要的作用。作为众多酶的辅酶因子,铜离子的含量过低或过高,都会对人体的生物系统造成巨大影响。目前对于Cu²⁺检测的方法很多,其中荧光检测技术因具有较高的灵敏度,易操作,光谱干扰少,吸收波段宽等优点而受到广泛关注。但是大部分荧光探针主要使用有机溶剂,在水中的溶解性差,限制了探针在环境及其生物领域中的应用。碳量子点（Carbon Quantum Dots,CQDs）是一种发现于2004年的新型荧光材料,因其突出的荧光性能、良好的光学稳定性以及表面易修饰性等特点,尤其是其优越的生物相容性与低毒性,被认为是可以替代金属半导体荧光染料的有力替选材料之一。但是与无机量子点和有机染料相比,碳量子点的荧光产率较低。因此制备一种具有较高荧光产率,良好荧光性能以及生物相容性的碳量子点意义重大。基于以上所述,论文主要内容如下:1、以废弃山竹壳为原料,通过一步碳化的方法获得含碳量75%以上,具有相对完整碳网结构的碳化山竹壳。山竹壳材料结构的完善,有助于制备CQDs效率的提高。2、以碳化山竹壳为碳源,采用混酸氧化切割的方法,成功制备具有蓝绿色荧光的山竹壳基碳量子点,荧光产率为5.1%,该碳量子点平均尺寸2.7nm,在高分辨率透射电镜下可以观察到明显的晶格条纹,晶面间距为0.246 nm,对应类石墨的（001）晶面。红外图谱分析证明其表面富含羟基羧基,水溶性良好,易于修饰钝化。3、在山竹壳基碳量子点CQDs的基础上,以聚乙烯亚胺BPEI作为修饰剂,通过一步水热的方法对CQDs进行修饰,使其表面富含可与Cu²⁺发生络合反应的氨基。经过优化制备条件,BPEI-CQDs水溶性良好,粒径分布在3⁹ nm范围内,平均粒径为4.9 nm。红外光谱证明BPEI成功在CQDs表面修饰,与修饰前CQDs相比,BPEI-CQDs荧光性能得到改善,荧光产率从5.1%提升到24.2%。同时荧光发射峰发生蓝移,紫外灯照射下可发出明亮蓝色荧光。通过测试BPEI-CQDs水溶液中对不同金属离子以及不同浓度Cu²⁺的荧光响应情况,可知Cu²⁺可以使BPEI-CQDs发生荧光猝灭,从而可以对Cu²⁺进行特异性识别。同时BPEI-CQDs的荧光猝灭程度与Cu²⁺浓度线性相关,线性范围为5⁶0μmol/L,检测限为0.69μmol/L,满足人体细胞内Cu²⁺含量（10-4¹0-6 mol/L）的检测要求,为其应用于细胞内Cu²⁺检测提供理论基础。4、将制备的BPEI-CQDs应用在小鼠成纤维细胞L929的细胞成像以及细胞内铜离子的检测上。MTT结果表明BPEI-CQDs具有低毒性以及良好的生物相容性,BPEI-CQDs可以通过细胞膜,停留于细胞质中。改变激发波长,细胞荧光成像颜色发生变化,对应其波长依赖的性质。在外源Cu²⁺的作用下,细胞内荧光发生明显猝灭,为实现细胞内Cu²⁺的检测提供建议。更多还原

【Abstract】 Copper ions(Cu²⁺)which is the third most abundant transition metal ion in the human body play a crucial role in biological systems.As the cofactor of many enzymes,the content of copper ion will have a huge impact on biological systems.At present,there are many methods for the detection of Cu²⁺,and the fluorescence detection technology has attracted much attention because of its high sensitivity,easy operation,less spectral interference and wide absorption band.However,most of the fluorescent probes use organic solvents mostly and the poor solubility in water,it will limit the probe in the environment and its biological applications.Carbon Quantum Dots（CQDs）,a novel fluorescent material discovered in 2004,are characterized by outstanding fluorescence,good optical stability and easy surface modification,especially their excellent biocompatibility and low toxicity.It is considered to be a powerful alternative to metalsemiconductor fluorescent dye material.However,compared to inorganic quantum dots and organic dyes,the fluorescence quantum yield of carbon dots is lower.Therefore,it is of great significance to prepare a carbon quantum dot with high fluorescence quantum yield,good fluorescence property and biocompatibility.Based on the above,the main contents of this paper are as follows:1)By using the mangosteen shells as the raw material,the carbonized mangosteen shells with carbon content of more than 75% and relatively complete carbon network structure was obtained by one-step carbonization method.The perfection of structure of mangosteen shells is helpful for the preparation efficiency of CQDs.2)The water-soluble CQDs with blue green fluorescence were prepared by carbonic acid-catalyzed oxidative cleavage from carbonized mangosteen shells.The average size of the carbon quantum dots was 2.7 nm,and the lattice spacing was 0.246 nm,corresponding to the（001）plane of graphite.FTIR spectra analysis showed that the surface of CQDs is rich in hydroxyl and carboxyl groups,which means the good water solubility and easy to modification.3)The CQDs were modified by one-step hydrothermal method on the basis of the mangosteen shells based quantum dots,and the surface of the CQDs was enriched with amino groups whichcould react with Cu²⁺.FTIR spectra analysis showed that BPEI was successfully modified on the CQDs’ surface.After optimization,the fluorescence intensity of BPEI-CQDs could be greatly enhanced,and the fluorescence quantum yield was improved from 3.7% to 21.6%. Besides,the fluorescence emission peak moved to blue shift,which could emit bright blue light under the ultraviolet light.Through the fluorescence response for BPEI-CQDs to different ions and Cu²⁺ ions with different concertation,the fluorescence of BPEI-CQDs was quenched,so that BPEI-CQDs can show specific recognition to Cu²⁺.The degree of fluorescence quenching linear range of BPEI-CQDs is 5⁶0 μmol/L and the detection limit can reach to 0.69 μmol / L, which can meet the requirements of the detection of Cu²⁺ in the human body（10-4 10-6 mol / L）.4)The as prepared BPEI-CQDs were applied to the imaging of mouse fibroblast L929 cells and intracellular copper ion detection.MTT test results showed that BPEI-CQDs exhibited low toxicity and good cell membrane permeability.BPEI-CQDs could pass through the cell membrane and stay in the cytoplasm.The colour of fluorescence imaging in L929 cells was changed when the excitation wavelength changed,which corresponding to that the emission wavelength of the BPEI-CQDs depends on the excitation wavelength.Under the action of exogenous Cu²⁺,the intracellular fluorescence was quenched,which provided suggestion for the detection of intracellular Cu²⁺.更多还原