基于荧光光谱的血浆中亚甲蓝含量测定和二氧化硅强荧光纳米粒子的制备及应用
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摘要
本文第一部分利用β-环糊精对亚甲蓝荧光信号的增敏作用，建立了直接检测血浆中微量亚甲蓝含量的荧光分光光度法。亚甲蓝的线性范围为0.089-3.57μmol/L（r=0.9988），RSD为6.1%，回收率在95.9-108.3%之间。该方法简便、准确，不需要分离浓缩等预处理，可以直接用于血浆中微量亚甲蓝的含量测定。
本文第二部分就二氧化硅强荧光纳米粒子的制备及应用作了相关研究。
采用水相法，在不同的时间点终止反应，获得了发射出绿色、黄色、橙色和红色荧光的CdTe量子点。其中，绿光、黄光和橙光CdTe量子点容易得到，并且荧光较强，但是要想制备出红光CdTe量子点，反应时间较长，随之荧光将变弱，发射峰将展宽。因此，本文在橙光CdTe量子点的基础上，采用包裹CdS壳的方法合成了发射波长为615nm的荧光较强的红色CdTe/CdS核壳型量子点。CdTe/CdS量子点的发射峰明显红移，可以缩短制备红光量子点的时间。基于静电自组装的方式合成了阳离子聚电解质聚二烯丙基二甲基氯化铵（PDADMAC）保护的CdTe量子点，即CdTe/PDADMAC纳米复合物。该纳米复合物为条带状形态，不影响CdTe量子点的荧光性质，并且能够保护CdTe量子点表面的配体，从而改善CdTe量子点的荧光稳定性。
以CdTe/PDADMAC纳米复合物为核，通过反相微乳法合成了CdTe/PDADMAC@SiO_2。该核壳型二氧化硅荧光纳米粒子为球形，大约为25nm，粒径分布均一，分散性较好。与CdTe@SiO_2相比，CdTe/PDADMAC@SiO_2具有更强的荧光，荧光稳定性明显改善。通过优化反应条件，得到剩余荧光为76.4%的CdTe/PDADMAC@SiO_2。而且，CdTe/PDADMAC@SiO_2显示出良好的生物兼容性，可以用于细胞成像。
将常用的反相微乳体系放大5倍，通过调节各组分的加入量，重现了放大之前制备CdTe/PDADMAC@SiO_2的结果，解决了以往一次反应合成量较少的问题。同时制备了荧光较强、稳定性较好的能够发出红色荧光的CdTe/CdS/PDADMAC@SiO_2。CdTe/CdS/PDADMAC@SiO_2与发射出绿色荧光的CdTe1/PDADMAC@SiO_2的荧光光谱之间没有重叠，彼此不存在干扰，可以同时作为标记物用于定量分析。
成功构建了Ag@SiO_2复合纳米粒子，其中Ag核为50nm，二氧化硅壳厚10nm，Ag@SiO_2复合纳米粒子结合了Ag纳米粒子的表面等离子体共振性能和二氧化硅的优良性质，是表面等离子体共振增强荧光的常用平台。正电荷的罗丹明6G与具有负电荷表面的Ag@SiO_2复合纳米粒子可以通过静电相互作用结合形成Ag@SiO_2/Rh6G复合体。过氧化氢不影响罗丹明6G的荧光，而且能够透过硅壳将Ag核降解。由于Ag纳米粒子的表面等离子体共振增强荧光效应，相对没有Ag核的SiO_2/Rh6G，Ag@SiO_2/Rh6G复合体的荧光增强了1倍。采用类似的方法，将Ag@SiO_2/PDADMAC复合纳米粒子与IgG-FITC结合，结果Ag@SiO_2/PDADMAC/IgG-FITC复合体的荧光增强了0.7倍。
In the first part of the paper, the fluorescence spectrophotometry has beenestablished to determine directly the trace amount of methylene blue in the plasmabased on the sensitizing effect of β-cyclodextrin on the fluorescence signal of methyleneblue. The linear range of methylene blue was0.089-3.57μmol/L (r=0.9988), RSD was6.1%, and the recovery rate was95.9-108.3%. The method was simple and accurate,which could be used to determine the trace amount of methylene blue in the plasmawithout pretreatments, such as separation and enrichment.
In the second part, this paper has studied the preparation of strong fluorescencesilica nanoparticles and their applications
The CdTe quantum dots (QDs) emitting green, yellow, orange and red fluorescencewas obtained in aqueous solution by adjusting the reaction time. The green, yellow andorange CdTe QDs with strong fluorescence could be got easily. But it need long time toprepare red CdTe QDs. At the same time, its fluorescence would become weak and theemission peak would broaden. Therefore, the red CdTe/CdS core-shell QDs with strongfluorescence was synthesized by epitaxial growth the nuclear layer of CdS on thesurface of the orange CdTe QDs. The emission peak of CdTe/CdS QDs was red-shiftedrapidly to615nm and the reaction time was reduced. By self-assembling of CdTe QDsand poly-diallyldimethylammonium chloride (PDADMAC) through electrostaticattraction, the polyelectrolyte-protected CdTe QDs (that is, QDs/PDADMACnanocomposites) was prepared. The QDs/PDADMAC nanocomposites showedstrip-like shape. Because the fluorescence properties of CdTe QDs were not affected andPDADMAC could effectively prevent mercaptoacetic acid ligands dropping out fromthe surface of QDs, the nanocomposites had more significant fluorescence stability than the bare CdTe QDs.
Based on CdTe/PDADMAC nanocomposites, the core-shell silica fluorescencenanoparticles (CdTe/PDADMAC@SiO_2) was prepared via a water-in-oil reversemicroemulsion method. All of the particles were almost spherical and there was auniform distribution of the particle size with the average diameter about25nm. Ascompared with the CdTe@SiO_2, the CdTe/PDADMAC@SiO_2had much strongerfluorescence, and their fluorescence stability could be obviously improved. Theremaining fluorescence of CdTe/PDADMAC@SiO_2was up to76.4%by optimizingreaction conditions. The cytotoxicity and staining effect of CdTe@SiO_2andCdTe/PDADMAC@SiO_2nanoparticles were studied using A549lung cancer cell as amodel cell. The results showed that the cytotoxicity of CdTe/PDADMAC@SiO_2waslitter and the staining effect was better than those of CdTe@SiO_2.
In order to solve the problem of litter yield, the common reverse microemulsionsystem was magnified five times and the results before amplification were reproducedby regulating the amount of each component. At the same time, we also synthesized redCdTe/CdS/PDADMAC@SiO_2with strong fluorescence and good stability.Thefluorescence spectra of CdTe/PDADMAC@SiO_2and CdTe/CdS/PDADMAC@SiO_2didnot overlap. Since their fluorescence spectra had no interference, they could be used asmarkers simultaneously for quantitative analysis.
Ag@SiO_2composite nanoparticles were constructed successfully, of which Agcore was50nm and the thickness of silica shell was10nm. The composite nanoparticlescombined the surface plasmon resonance property of Ag nanoparticles and the perfectqualities of the silica, which was the common platform of metal enhancementfluorescence. Ag@SiO_2/Rh6G complex was formed by combining the positivelycharged rhodamine6G with Ag@SiO_2composite nanoparticles with the negativelycharged surface in the help of electrostatic interaction. Hydrogen peroxide did not affectthe fluorescence of rhodamine6G, and could degrade Ag core through the silica shell.Compared with the SiO_2/Rh6G without Ag core, the fluorescence intensity ofAg@SiO_2/Rh6G complex was enhanced1-fold due to the surface plasmon resonancefluorescence enhancement effect of Ag nanoparticles. Similarly, the fluorescenceintensity of IgG-FITC was enhanced0.7-fold after being assembed on the surface of Ag@SiO_2/PDADMAC composite nanoparticles.
本文第二部分就二氧化硅强荧光纳米粒子的制备及应用作了相关研究。
采用水相法，在不同的时间点终止反应，获得了发射出绿色、黄色、橙色和红色荧光的CdTe量子点。其中，绿光、黄光和橙光CdTe量子点容易得到，并且荧光较强，但是要想制备出红光CdTe量子点，反应时间较长，随之荧光将变弱，发射峰将展宽。因此，本文在橙光CdTe量子点的基础上，采用包裹CdS壳的方法合成了发射波长为615nm的荧光较强的红色CdTe/CdS核壳型量子点。CdTe/CdS量子点的发射峰明显红移，可以缩短制备红光量子点的时间。基于静电自组装的方式合成了阳离子聚电解质聚二烯丙基二甲基氯化铵（PDADMAC）保护的CdTe量子点，即CdTe/PDADMAC纳米复合物。该纳米复合物为条带状形态，不影响CdTe量子点的荧光性质，并且能够保护CdTe量子点表面的配体，从而改善CdTe量子点的荧光稳定性。
以CdTe/PDADMAC纳米复合物为核，通过反相微乳法合成了CdTe/PDADMAC@SiO_2。该核壳型二氧化硅荧光纳米粒子为球形，大约为25nm，粒径分布均一，分散性较好。与CdTe@SiO_2相比，CdTe/PDADMAC@SiO_2具有更强的荧光，荧光稳定性明显改善。通过优化反应条件，得到剩余荧光为76.4%的CdTe/PDADMAC@SiO_2。而且，CdTe/PDADMAC@SiO_2显示出良好的生物兼容性，可以用于细胞成像。
将常用的反相微乳体系放大5倍，通过调节各组分的加入量，重现了放大之前制备CdTe/PDADMAC@SiO_2的结果，解决了以往一次反应合成量较少的问题。同时制备了荧光较强、稳定性较好的能够发出红色荧光的CdTe/CdS/PDADMAC@SiO_2。CdTe/CdS/PDADMAC@SiO_2与发射出绿色荧光的CdTe1/PDADMAC@SiO_2的荧光光谱之间没有重叠，彼此不存在干扰，可以同时作为标记物用于定量分析。
成功构建了Ag@SiO_2复合纳米粒子，其中Ag核为50nm，二氧化硅壳厚10nm，Ag@SiO_2复合纳米粒子结合了Ag纳米粒子的表面等离子体共振性能和二氧化硅的优良性质，是表面等离子体共振增强荧光的常用平台。正电荷的罗丹明6G与具有负电荷表面的Ag@SiO_2复合纳米粒子可以通过静电相互作用结合形成Ag@SiO_2/Rh6G复合体。过氧化氢不影响罗丹明6G的荧光，而且能够透过硅壳将Ag核降解。由于Ag纳米粒子的表面等离子体共振增强荧光效应，相对没有Ag核的SiO_2/Rh6G，Ag@SiO_2/Rh6G复合体的荧光增强了1倍。采用类似的方法，将Ag@SiO_2/PDADMAC复合纳米粒子与IgG-FITC结合，结果Ag@SiO_2/PDADMAC/IgG-FITC复合体的荧光增强了0.7倍。
In the first part of the paper, the fluorescence spectrophotometry has beenestablished to determine directly the trace amount of methylene blue in the plasmabased on the sensitizing effect of β-cyclodextrin on the fluorescence signal of methyleneblue. The linear range of methylene blue was0.089-3.57μmol/L (r=0.9988), RSD was6.1%, and the recovery rate was95.9-108.3%. The method was simple and accurate,which could be used to determine the trace amount of methylene blue in the plasmawithout pretreatments, such as separation and enrichment.
In the second part, this paper has studied the preparation of strong fluorescencesilica nanoparticles and their applications
The CdTe quantum dots (QDs) emitting green, yellow, orange and red fluorescencewas obtained in aqueous solution by adjusting the reaction time. The green, yellow andorange CdTe QDs with strong fluorescence could be got easily. But it need long time toprepare red CdTe QDs. At the same time, its fluorescence would become weak and theemission peak would broaden. Therefore, the red CdTe/CdS core-shell QDs with strongfluorescence was synthesized by epitaxial growth the nuclear layer of CdS on thesurface of the orange CdTe QDs. The emission peak of CdTe/CdS QDs was red-shiftedrapidly to615nm and the reaction time was reduced. By self-assembling of CdTe QDsand poly-diallyldimethylammonium chloride (PDADMAC) through electrostaticattraction, the polyelectrolyte-protected CdTe QDs (that is, QDs/PDADMACnanocomposites) was prepared. The QDs/PDADMAC nanocomposites showedstrip-like shape. Because the fluorescence properties of CdTe QDs were not affected andPDADMAC could effectively prevent mercaptoacetic acid ligands dropping out fromthe surface of QDs, the nanocomposites had more significant fluorescence stability than the bare CdTe QDs.
Based on CdTe/PDADMAC nanocomposites, the core-shell silica fluorescencenanoparticles (CdTe/PDADMAC@SiO_2) was prepared via a water-in-oil reversemicroemulsion method. All of the particles were almost spherical and there was auniform distribution of the particle size with the average diameter about25nm. Ascompared with the CdTe@SiO_2, the CdTe/PDADMAC@SiO_2had much strongerfluorescence, and their fluorescence stability could be obviously improved. Theremaining fluorescence of CdTe/PDADMAC@SiO_2was up to76.4%by optimizingreaction conditions. The cytotoxicity and staining effect of CdTe@SiO_2andCdTe/PDADMAC@SiO_2nanoparticles were studied using A549lung cancer cell as amodel cell. The results showed that the cytotoxicity of CdTe/PDADMAC@SiO_2waslitter and the staining effect was better than those of CdTe@SiO_2.
In order to solve the problem of litter yield, the common reverse microemulsionsystem was magnified five times and the results before amplification were reproducedby regulating the amount of each component. At the same time, we also synthesized redCdTe/CdS/PDADMAC@SiO_2with strong fluorescence and good stability.Thefluorescence spectra of CdTe/PDADMAC@SiO_2and CdTe/CdS/PDADMAC@SiO_2didnot overlap. Since their fluorescence spectra had no interference, they could be used asmarkers simultaneously for quantitative analysis.
Ag@SiO_2composite nanoparticles were constructed successfully, of which Agcore was50nm and the thickness of silica shell was10nm. The composite nanoparticlescombined the surface plasmon resonance property of Ag nanoparticles and the perfectqualities of the silica, which was the common platform of metal enhancementfluorescence. Ag@SiO_2/Rh6G complex was formed by combining the positivelycharged rhodamine6G with Ag@SiO_2composite nanoparticles with the negativelycharged surface in the help of electrostatic interaction. Hydrogen peroxide did not affectthe fluorescence of rhodamine6G, and could degrade Ag core through the silica shell.Compared with the SiO_2/Rh6G without Ag core, the fluorescence intensity ofAg@SiO_2/Rh6G complex was enhanced1-fold due to the surface plasmon resonancefluorescence enhancement effect of Ag nanoparticles. Similarly, the fluorescenceintensity of IgG-FITC was enhanced0.7-fold after being assembed on the surface of Ag@SiO_2/PDADMAC composite nanoparticles.
引文
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