复合荧光二氧化硅纳米颗粒的制备及其在生物分析和光催化降解中的应用
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摘要
纳米材料是纳米技术的一个重要部分。由于在纳米尺度下物质中电子的量子力学性质和原子的相互作用将受到尺度大小的影响,因此纳米材料具有许多普通材料不可比拟的优良性能,也正因为如此,纳米材料的研制和应用一直是人们关注的焦点。复合纳米颗粒,尤其是核壳型复合纳米颗粒,克服了普通单组分纳米颗粒物质组成单一的不足,将不同物质所拥有的多种功能有机地结合在一起,显示出普通单组分纳米颗粒无可比拟的优越的理化性能,在生化检测、医学成像、生物物质分离等生物医学领域显示出了广阔的应用前景。本论文在对当前迅速发展的复合纳米颗粒进行简要综述的基础上,以几种复合纳米颗粒的制备、性能表征及其在生物医学领域中以及环境方面的应用为主线,主要开展了以下几个方面的研究工作:
1.使用异硫氰酸荧光素(FITC)与3-氨基丙基三甲氧基硅烷(APTMS)反应制得前驱体FITC-APTMS,然后采用油包水微乳液法,制备了FITC-APTMS前驱体掺杂的二氧化硅核壳型荧光纳米粒子。对其进行透射电镜,紫外可见光谱,荧光光谱及光稳定性实验与染料泄露实验等表征。由于这种纳米颗粒对pH敏感,在实验范围pH值3.6~ 9.7范围内,荧光强度与溶液酸度有良好的响应,此外,由于二氧化硅壳层是多孔网状立体结构,裸露的质子能穿透过去,对pH有良好的响应,而且该纳米颗粒能被单个小鼠神经干细胞吞噬,因此可望用做纳米pH传感器件,实现对单个细胞的pH实时监测。
2.以三联吡啶钌为内核材料,通过反相微乳液法合成了具有核壳结构的二氧化硅荧光纳米粒子Ru(bpy)32+/SiO2,利用透射电子显微镜、荧光光谱、紫外-可见光谱等手段进行表征,并进行了光稳定性、荧光分子泄露与纳米粒子表面氨基测定等实验,结果表明所合成的纳米粒子为规则的球形,大小均一,平均粒径为60±6 nm,有较高的荧光强度,呈单分散性,具有很好的光稳定性,在水溶液中不易发生染料泄露。通过硅烷化试剂APTMS与TEOS共水解与聚合作用,在纳米粒子的表面直接引入氨基活性基团,因而纳米粒子不需要进行表面修饰而直接标记生物分子。以该纳米粒子作为荧光探针标记链霉亲合素,建立了高灵敏度的小鼠IgG荧光免疫分析法。结果表明在实验值0.02 ng/mL ~350 ng/mL MIgG浓度范围内,荧光强度与MIgG浓度有良好的正相关性,最低检测限为10pg/mL。首次将此纳米粒子作为荧光探针应用于蛋白质微阵列芯片检测HIV p24抗原,结果显示荧光强度与p24浓度呈良好的正相关性,分析灵敏度为2.1ng/mL。同时我们还进行了纳米粒子的小鼠神经干细胞荧光成像实验。
3.以2 MeV 10mA GJ-2-Ⅱ电子加速器为辐射源,EDTA为稳定剂,采用电子束辐照法在水溶液中室温合成了CdSe量子点,然后采用油包水微乳液法,制备了表面带氨基的二氧化硅包覆CdSe量子点的核壳型荧光纳米粒子。经表征该纳米粒子粒径为200±8 nm,具有良好的荧光性能,其荧光强度及光稳定性均较CdSe量子点高。小鼠神经干细胞的荧光成像实验结果表明,该纳米粒子亦可应用于细胞成像并得到清晰的荧光成像图。
4.用电子束辐照法制备得的CdSe量子点,首次将其作为光催化剂用于光催化降解处理甲基橙进行了研究。对光催化的必要条件,CdSe量子点浓度,甲基橙浓度,催化剂种类以及CdSe量子点光催化反应的动力学进行了初步研究。研究表明,甲基橙浓度以及催化剂CdSe量子点的浓度对光催化效果有一定的影响,紫外灯照射和光催化剂CdSe量子点都是不可缺少的降解条件,在本实验条件下, CdSe量子点的光催化降解效果要略优于纳米TiO2,反应体系基本遵守一级反应动力学方程。
Compared with conventional single component nanoparticles,Composite nanoparticles, especially core-shell structure composite nanoparticles, have more unique chemical and physical properties due to the combination of binary or multi-component nanocomposites. In recent years, various core-shell composite nanoparticles have been synthesized and studied, and their applications in the fields of biology and medicine have gained increasing attention and shown broad prospects in medical imaging, molecular diagnosis, fluorescent immunoassay, biochip, biosensor, biomaterial separation, and so on. Aiming at this important research direction, this thesis mainly focused on the study of synthesis, characterization and biomedical application of several types of dye-doped core-shell silica composite nanoparticles.
First, a novel type of amino functionalized core-shell fluorescein isothiocyanate-doped silica nanoparticles was synthesized using a simple and effective approach of reverse microemulsion. Isothiocyanate coupled with a silane coupling agent, 3-aminopropyl-trimethoxysilane, was incorporated into silica sphere and the dye molecules were bound with silica sphere through hydrolysis and polymerization of tetraethoxysilane and 3-aminopropyl-trimethoxysilane. With the covalent binding between the dye molecules and silica sphere, the leakage of the dye was avoided. With amino groups on the surface, the nanoparticles can be directly conjugated with biological molecules with no need of complicated surface modification. The nanoparticles were spherical, monodisperse, uniform in size, pH sensitive, highly fluorescent and highly photostable. The pH response range was 3.6~9.7, respectively. More interestedly, the nanoparticles could be phagocytosed by murine neural stem cell and could be applied to detect pH value for single live cell.
Second, Ru(bpy)32+ doped silica fluorescent nanoparticles were prepared using the water-in-oil (W/O) microemulsion method. Characterizations by transmission electron microscopy, fluorescent spectra, UV-Vis absorption spectra, photostable experiments and dyeleaking experiments show that the nanoparticles were monodisperse, uniform in size with the diameter being 60±6 nm. Due to a lot of fluorescent dye molecules of Ru(bpy)32+ encapsulated in the silica matrix that also severed to protect Ru(bpy)32+ dye from photodamaging oxidation, the nanoparticles were extremetly bright, photostable and chemical stable. The nanoparticles utilized as fluorescent probe were conjugated with streptavidin and successfully applied in fluorescent immunoassay of mouse IgG and detection of HIV p24 with protein microarray. Results show that good positive correlations between fluorescence intensities and concentrations of mouse IgG or p24 were presented with 10 pg/mL and 2.1ng/mL of analytical sensitivities, respectively. The nanoparticles also could be effectively swallowed by mouse neural stem cells and highly resolute images were obtained.
Third, CdSe quantum dots was first prepared in aqueous solution at room temperature by a electron beam irradiation method using 2MeV 10mA GJ-2-II electronic accelerator as irradiation source and EDTA as stabilizer. Then a core-shell CdSe quantum dots-doped silica fluorescent nanoparticle were developed using the water-in-oil (W/O) microemulsion. It was found that the nanoparticles were spherical, monodisperse and uniform in size of 200±8 nm. Compared with pure CdSe quantum dots, the core-shell CdSe quantum dots-doped silica nanoparticle exhibited higher fluorescent intensity and higher photostability.
Finally, the synthesized CdSe quantum dots wer utilized as a photocatalyst for degradation of methyl orange (MO). The impact factors including the sorts of catalyst, concentrations of catalyst and MO, were studied. Moreover, the reaction kinetics of photocatalysis was also investigated. Results demonstrated that degradation efficiency was affected by the concentrations of MO and CdSe quantum dots. Interestedly, the degradation efficiency of CdSe quantum dots was better than TiO2 under optimal conditions in this study.
In conclusions, several different types of core-shell composite nanoparticles have been successfully synthesized. These dye or quantum dots doped composite silica nanoparticles exhibit excellent properties including photo,physics, and chemistry. They all can be applied in highly sensitive fluorescent immunoassay, sensor, cell imaging and biochip, and the CdSe quantum dots can be utilized as a photocatalyst for photocatalytic degradation.
1.使用异硫氰酸荧光素(FITC)与3-氨基丙基三甲氧基硅烷(APTMS)反应制得前驱体FITC-APTMS,然后采用油包水微乳液法,制备了FITC-APTMS前驱体掺杂的二氧化硅核壳型荧光纳米粒子。对其进行透射电镜,紫外可见光谱,荧光光谱及光稳定性实验与染料泄露实验等表征。由于这种纳米颗粒对pH敏感,在实验范围pH值3.6~ 9.7范围内,荧光强度与溶液酸度有良好的响应,此外,由于二氧化硅壳层是多孔网状立体结构,裸露的质子能穿透过去,对pH有良好的响应,而且该纳米颗粒能被单个小鼠神经干细胞吞噬,因此可望用做纳米pH传感器件,实现对单个细胞的pH实时监测。
2.以三联吡啶钌为内核材料,通过反相微乳液法合成了具有核壳结构的二氧化硅荧光纳米粒子Ru(bpy)32+/SiO2,利用透射电子显微镜、荧光光谱、紫外-可见光谱等手段进行表征,并进行了光稳定性、荧光分子泄露与纳米粒子表面氨基测定等实验,结果表明所合成的纳米粒子为规则的球形,大小均一,平均粒径为60±6 nm,有较高的荧光强度,呈单分散性,具有很好的光稳定性,在水溶液中不易发生染料泄露。通过硅烷化试剂APTMS与TEOS共水解与聚合作用,在纳米粒子的表面直接引入氨基活性基团,因而纳米粒子不需要进行表面修饰而直接标记生物分子。以该纳米粒子作为荧光探针标记链霉亲合素,建立了高灵敏度的小鼠IgG荧光免疫分析法。结果表明在实验值0.02 ng/mL ~350 ng/mL MIgG浓度范围内,荧光强度与MIgG浓度有良好的正相关性,最低检测限为10pg/mL。首次将此纳米粒子作为荧光探针应用于蛋白质微阵列芯片检测HIV p24抗原,结果显示荧光强度与p24浓度呈良好的正相关性,分析灵敏度为2.1ng/mL。同时我们还进行了纳米粒子的小鼠神经干细胞荧光成像实验。
3.以2 MeV 10mA GJ-2-Ⅱ电子加速器为辐射源,EDTA为稳定剂,采用电子束辐照法在水溶液中室温合成了CdSe量子点,然后采用油包水微乳液法,制备了表面带氨基的二氧化硅包覆CdSe量子点的核壳型荧光纳米粒子。经表征该纳米粒子粒径为200±8 nm,具有良好的荧光性能,其荧光强度及光稳定性均较CdSe量子点高。小鼠神经干细胞的荧光成像实验结果表明,该纳米粒子亦可应用于细胞成像并得到清晰的荧光成像图。
4.用电子束辐照法制备得的CdSe量子点,首次将其作为光催化剂用于光催化降解处理甲基橙进行了研究。对光催化的必要条件,CdSe量子点浓度,甲基橙浓度,催化剂种类以及CdSe量子点光催化反应的动力学进行了初步研究。研究表明,甲基橙浓度以及催化剂CdSe量子点的浓度对光催化效果有一定的影响,紫外灯照射和光催化剂CdSe量子点都是不可缺少的降解条件,在本实验条件下, CdSe量子点的光催化降解效果要略优于纳米TiO2,反应体系基本遵守一级反应动力学方程。
Compared with conventional single component nanoparticles,Composite nanoparticles, especially core-shell structure composite nanoparticles, have more unique chemical and physical properties due to the combination of binary or multi-component nanocomposites. In recent years, various core-shell composite nanoparticles have been synthesized and studied, and their applications in the fields of biology and medicine have gained increasing attention and shown broad prospects in medical imaging, molecular diagnosis, fluorescent immunoassay, biochip, biosensor, biomaterial separation, and so on. Aiming at this important research direction, this thesis mainly focused on the study of synthesis, characterization and biomedical application of several types of dye-doped core-shell silica composite nanoparticles.
First, a novel type of amino functionalized core-shell fluorescein isothiocyanate-doped silica nanoparticles was synthesized using a simple and effective approach of reverse microemulsion. Isothiocyanate coupled with a silane coupling agent, 3-aminopropyl-trimethoxysilane, was incorporated into silica sphere and the dye molecules were bound with silica sphere through hydrolysis and polymerization of tetraethoxysilane and 3-aminopropyl-trimethoxysilane. With the covalent binding between the dye molecules and silica sphere, the leakage of the dye was avoided. With amino groups on the surface, the nanoparticles can be directly conjugated with biological molecules with no need of complicated surface modification. The nanoparticles were spherical, monodisperse, uniform in size, pH sensitive, highly fluorescent and highly photostable. The pH response range was 3.6~9.7, respectively. More interestedly, the nanoparticles could be phagocytosed by murine neural stem cell and could be applied to detect pH value for single live cell.
Second, Ru(bpy)32+ doped silica fluorescent nanoparticles were prepared using the water-in-oil (W/O) microemulsion method. Characterizations by transmission electron microscopy, fluorescent spectra, UV-Vis absorption spectra, photostable experiments and dyeleaking experiments show that the nanoparticles were monodisperse, uniform in size with the diameter being 60±6 nm. Due to a lot of fluorescent dye molecules of Ru(bpy)32+ encapsulated in the silica matrix that also severed to protect Ru(bpy)32+ dye from photodamaging oxidation, the nanoparticles were extremetly bright, photostable and chemical stable. The nanoparticles utilized as fluorescent probe were conjugated with streptavidin and successfully applied in fluorescent immunoassay of mouse IgG and detection of HIV p24 with protein microarray. Results show that good positive correlations between fluorescence intensities and concentrations of mouse IgG or p24 were presented with 10 pg/mL and 2.1ng/mL of analytical sensitivities, respectively. The nanoparticles also could be effectively swallowed by mouse neural stem cells and highly resolute images were obtained.
Third, CdSe quantum dots was first prepared in aqueous solution at room temperature by a electron beam irradiation method using 2MeV 10mA GJ-2-II electronic accelerator as irradiation source and EDTA as stabilizer. Then a core-shell CdSe quantum dots-doped silica fluorescent nanoparticle were developed using the water-in-oil (W/O) microemulsion. It was found that the nanoparticles were spherical, monodisperse and uniform in size of 200±8 nm. Compared with pure CdSe quantum dots, the core-shell CdSe quantum dots-doped silica nanoparticle exhibited higher fluorescent intensity and higher photostability.
Finally, the synthesized CdSe quantum dots wer utilized as a photocatalyst for degradation of methyl orange (MO). The impact factors including the sorts of catalyst, concentrations of catalyst and MO, were studied. Moreover, the reaction kinetics of photocatalysis was also investigated. Results demonstrated that degradation efficiency was affected by the concentrations of MO and CdSe quantum dots. Interestedly, the degradation efficiency of CdSe quantum dots was better than TiO2 under optimal conditions in this study.
In conclusions, several different types of core-shell composite nanoparticles have been successfully synthesized. These dye or quantum dots doped composite silica nanoparticles exhibit excellent properties including photo,physics, and chemistry. They all can be applied in highly sensitive fluorescent immunoassay, sensor, cell imaging and biochip, and the CdSe quantum dots can be utilized as a photocatalyst for photocatalytic degradation.
引文
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