POSS修饰的高生物相容性CdTe和CdSeTe荧光量子点的制备及其应用研究
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摘要
半导体荧光量子点相比于传统有机染料具有许多独特的光学特性,如尺寸可调的荧光发射波长、激发谱宽而连续、荧光发射谱窄而对称、荧光量子产率高、荧光寿命长、光稳定性好等,这些性质使得量子点作为一种新型的荧光探针和标记物,在化学分析及生物医学成像领域的应用日益广泛。Ⅱ-Ⅵ族半导体量子点是其中最具代表性的重要材料,有关其研究主要集中在水相制备具有高荧光效率的、光稳定性好、高生物相容性的量子点,同时通过对量子点进行适当的表面修饰,可以有效改善其表面性质并能保护量子点使其具有更好的光稳定性,同时抑制有毒Cd2+离子的泄露,降低其毒性。由于生物组织对近红外发光的量子点(650-900nm)光吸收和散射效应均最小,因此近红外发光可在生物组织中达到较大的穿透深度,并且可有效避免生物体自发荧光对外源荧光信号的干扰,提高细胞成像的质量。因此,合成高质量的近红外发光量子点具有重要的意义。
本论文的研究工作主要是以水相法合成多面体低聚倍半硅氧烷(POSS)修饰的CdTe量子点和近红外发射的CdSeTe三元合金量子点,研究了POSS-CdTe量子点作为荧光探针对金属Cu2+离子的分析检测,同时我们还系统研究了POSS修饰的CdTe和CdSeTe量子点的细胞毒性,并将其作为荧光探针初步应用于细胞标记。论文开展了以下工作：
1.采用水相合成方法,以8-巯丙基POSS (Octa-mercaptopropyl POSS, OM-POSS)和N-乙酰-L-半胱氨酸(NAC)作修饰剂合成了高质量的CdTe量子点,得到的量子点粒径分布均匀,分散性好,荧光量子产率最高可达95%。研究了前驱体溶液的pH值、NAC/Cd比例和Cd/Te比例对量子点的生长和光学性质的影响：经OM-POSS修饰后,量子点的生长速度变慢,这与多巯基的OM-POSS与Cd2+离子形成了更稳定的络合物有关。此外,我们制备的POSS-CdTe量子点还具有非常高的稳定性。
2.基于POSS-CdTe量子点的荧光淬灭效应,发展了一种探测金属Cu2+离子的新方法,并讨论了pH值、量子点浓度等条件对体系荧光强度和探测灵敏度的影响。结果表明,POSS-CdTe量子点作为荧光探针对Cu2+离子的检测具有低的检出限和高的灵敏度,并且有很好的选择性。在1×10-8mol L-1-1×10-6mol L-1浓度范围内POSS-CdTe量子点的荧光淬灭程度与Cu2+离子浓度存在很好的线性关系,回归方程为F0/F=0.68724+5.52783[Cu2+],探测限LOD为2.3×10-9mol L-1。我们还比较了CdTe量子点和POSS-CdTe量子点的探测灵敏度,通过变温实验确定了量子点静态淬灭的探测机理,并将POSS-CdTe量子点用于实际样品的检测分析。
3.系统研究了POSS-CdTe量子点对SiHa细胞和MC3T3-E1细胞的毒性效应和标记应用。结果表明,经OM-POSS修饰后,量子点的细胞毒性可有效降低,而目.由共聚焦荧光图像可以发现,POSS-CdTe量子点更容易通过脂质细胞膜而进入细胞,这与POSS-CdTe量子点具有的两亲性质有关。我们进一步讨论了量子点尺寸对SiHa细胞活性的影响,结果表明,随着量子点尺寸增加,其表现出的细胞毒性呈减小趋势。
4.利用水相法合成了8-氨丙基POSS (Octa-aminopropyl POSS, OA-POSS)修饰的近红外发光CdSeTe三元合金量子点,研究了量子点的能级结构和组分之间的光学弯曲效应,以及pH值、Cd/Te比例等条件对量子点生长的影响,并对其微观结构和光学性质进行了分析表征。合成的量子点粒径均匀,分散性好,发射峰位在681nm的POSS-CdSeTe量子点的绝对荧光量子产率为26.4%。利用SiHa细胞研究了POSS-CdSeTe量子点对细胞的毒性效应和标记作用,结果表明,经OA-POSS修饰后,CdSeTe量子点的细胞毒性大幅度降低；激光共聚焦图片表明POSS-CdSeTe量子点成功进入SiHa细胞实现了细胞标记。
Quantum dots (QDs) developed in recent years have attracted extensive attention since they offer substantial advantages over organic dyes, including size-tunable optical property, broad absorption, narrow emission, excellent photoluminescence efficiency, long fluorescence lifetime and high photochemical stability. These advantages make QDs an ideal fluorescent sensor for chemical or biological assays and real time, long term biomedical labeling. Ⅱ-Ⅵ group semiconductor QDs represent one of the most important types of nanomaterials. The aqueous synthesis route for QDs is relatively simpler, cheaper, less toxic and more environmentally friendly than the organometallic synthetic route, and the resulting QDs are water-soluble, also with high PLQYs, excellent photostability and biocompatibility, which make them convenient for the biological applications. QDs coated by capping ligands with their surrounding surface groups can obtain good biocompatibility since the release of Cd2+was effectively inhibited, and also retain their unique photophysical properties. The proper surface modification is needed to obtain desired functionality and improve photoluminescence response of QDs to some metal ions or molecules.
A new and exciting research avenue for QDs is their applications as optical probes for imaging of cells and tissue. As to biological optical imaging, the near-infrared (NIR)-emitting window is appealing because of the low tissue absorption and scattering effects in the emission range (650-900nm). This allows penetration of excitation and fluorescence photons deep into biological samples with reduced interaction and photodamage to the surrounding tissues, allowing fluorescence imaging depths on the order of centimeters. The near-infrared imaging is much prior to the visible light for biological applications.
In this paper, water soluble octa-mercaptopropyl POSS (OM-POSS) modified CdTe QDs have been successfully synthesized and employed as a novel fluorescent sensor for optical recognition of Cu2+in an aqueous medium. Their cytotoxicity and availability for cellular imaging in biomedical field were also studied. The near-infrared emitting CdSeTe QDs with octa-aminopropyl POSS (OA-POSS) capping were successfully synthesized and applied to SiHa cell imaging. The main contents are summarized as follows.
1. OM-POSS coated CdTe QDs with high fluorescence QYs were successfully synthesized in aqueous solution. TEM images reveal that these nanoparticles appear as spherical with excellent monodispersity. The relative photoluminescence quantum yield of the POSS-CdTe QDs was estimated to be95%using Rhodamine6G (PLQY95%) as reference. Temporal evolution of PL emitting wavelength and QYs of CdTe QDs growth under varied pH values, Cd/Te molar ratios and NAC/Cd molar ratios were studied. The growth rate decreases with the modification of OM-POSS due to its numerous mercapto groups. The POSS-CdTe QDs exhibit significantly good performance after two hours of UV exposure, indicating its excellent photostability.
2. The POSS-CdTe QDs have been employed as a novel fluorescent sensor for optical recognition of Cu2+in an aqueous medium. The fluorescence sensor for Cu2+is based on fluorescence quenching of the POSS-CdTe QDs. The selective quenching effect can be explained by the competitive binding of POSS capping layers between CdTe QDs and metal ions present in the solution. Fluorescence intensities of the POSS-CdTe QDs in PBS buffer solution at different pH values were studied. Stern-Volmer plots of different POSS-CdTe QDs concentrations were also investigated. Under optimum conditions, the detection limit for this sensing system is2.3×10-9mol L-1. The detection sensitivity for Cu2+is increased greatly with the capping of OM-POSS on CdTe QDs than the N AC-CdTe QDs. The Stern-Volmer plots at different temperatures show that POSS-CdTe QDs fluorescence was quenched by Cu2+through a static quenching mechanism. Furthermore, the practical utility of the POSS-CdTe QDs has been demonstrated by determination of trace Cu2+in water samples, obtaining satisfactory results with the standard addition method.
3. It is demonstrated that the POSS coating on CdTe core QDs confers it good bio compatibility, which is an important feature for cell labeling in the field of biomedicine. The SiHa cells and mouse preosteoblast cells MC3T3-E1were used to evaluate cytotoxicity of the as-prepared QDs. In comparison with CdTe QDs, the POSS-CdTe QDs show lower cytotoxicity under the same conditions. The OM-POSS coating can effectively inhibit the release of Cd2+and protect the QDs from aggregating caused by photobleaching. Moreover, large number of mercapto groups on OM-POSS can provide protection against QD-induced ROS stress and effectively prevent the decrease in cell viability. Confocal fluorescence images demonstrate that the QDs were internalized into the cells via endocytosis. The subcellular localization of POSS-CdTe QDs was found to depend upon QDs size. POSS-CdTe QDs are more easily taken up by cells than CdTe QDs due to its surface coating with amphiphilic moieties, which allows rapid intracellular uptake across the lipophilic cell membranes. The POSS-CdTe QDs allows rapid intracellular uptake, enabling the use of lower concentrations of QDs for an overall reduced toxicity.
4. The near-infrared emitting CdSeTe QDs with OA-POSS capping were successfully synthesized in aqueous solution. Temporal evolution of PL emitting wavelength and QYs of CdSeTe QDs growth under varied pH values, Cd/Te molar ratios and nonlinear composition effect were studied. TEM image reveals that these nanoparticles appear as spherical with excellent monodispersity. The absolute photoluminescence quantum yield of the POSS-CdTe QDs with681nm emitting wavelength was tested to be26.4%on Edinburgh FS-920fluorescence spectrometer. The SiHa cells were used to evaluate cytotoxicity of the as-prepared QDs. In comparison with CdSeTe QDs, the OA-POSS modified CdSeTe QDs show lower cytotoxicity under the same conditions. Confocal fluorescence images demonstrate that the near infrared emitting POSS-CdSeTe QDs were successfully internalized into the cells via endocytosis.
本论文的研究工作主要是以水相法合成多面体低聚倍半硅氧烷(POSS)修饰的CdTe量子点和近红外发射的CdSeTe三元合金量子点,研究了POSS-CdTe量子点作为荧光探针对金属Cu2+离子的分析检测,同时我们还系统研究了POSS修饰的CdTe和CdSeTe量子点的细胞毒性,并将其作为荧光探针初步应用于细胞标记。论文开展了以下工作：
1.采用水相合成方法,以8-巯丙基POSS (Octa-mercaptopropyl POSS, OM-POSS)和N-乙酰-L-半胱氨酸(NAC)作修饰剂合成了高质量的CdTe量子点,得到的量子点粒径分布均匀,分散性好,荧光量子产率最高可达95%。研究了前驱体溶液的pH值、NAC/Cd比例和Cd/Te比例对量子点的生长和光学性质的影响：经OM-POSS修饰后,量子点的生长速度变慢,这与多巯基的OM-POSS与Cd2+离子形成了更稳定的络合物有关。此外,我们制备的POSS-CdTe量子点还具有非常高的稳定性。
2.基于POSS-CdTe量子点的荧光淬灭效应,发展了一种探测金属Cu2+离子的新方法,并讨论了pH值、量子点浓度等条件对体系荧光强度和探测灵敏度的影响。结果表明,POSS-CdTe量子点作为荧光探针对Cu2+离子的检测具有低的检出限和高的灵敏度,并且有很好的选择性。在1×10-8mol L-1-1×10-6mol L-1浓度范围内POSS-CdTe量子点的荧光淬灭程度与Cu2+离子浓度存在很好的线性关系,回归方程为F0/F=0.68724+5.52783[Cu2+],探测限LOD为2.3×10-9mol L-1。我们还比较了CdTe量子点和POSS-CdTe量子点的探测灵敏度,通过变温实验确定了量子点静态淬灭的探测机理,并将POSS-CdTe量子点用于实际样品的检测分析。
3.系统研究了POSS-CdTe量子点对SiHa细胞和MC3T3-E1细胞的毒性效应和标记应用。结果表明,经OM-POSS修饰后,量子点的细胞毒性可有效降低,而目.由共聚焦荧光图像可以发现,POSS-CdTe量子点更容易通过脂质细胞膜而进入细胞,这与POSS-CdTe量子点具有的两亲性质有关。我们进一步讨论了量子点尺寸对SiHa细胞活性的影响,结果表明,随着量子点尺寸增加,其表现出的细胞毒性呈减小趋势。
4.利用水相法合成了8-氨丙基POSS (Octa-aminopropyl POSS, OA-POSS)修饰的近红外发光CdSeTe三元合金量子点,研究了量子点的能级结构和组分之间的光学弯曲效应,以及pH值、Cd/Te比例等条件对量子点生长的影响,并对其微观结构和光学性质进行了分析表征。合成的量子点粒径均匀,分散性好,发射峰位在681nm的POSS-CdSeTe量子点的绝对荧光量子产率为26.4%。利用SiHa细胞研究了POSS-CdSeTe量子点对细胞的毒性效应和标记作用,结果表明,经OA-POSS修饰后,CdSeTe量子点的细胞毒性大幅度降低；激光共聚焦图片表明POSS-CdSeTe量子点成功进入SiHa细胞实现了细胞标记。
Quantum dots (QDs) developed in recent years have attracted extensive attention since they offer substantial advantages over organic dyes, including size-tunable optical property, broad absorption, narrow emission, excellent photoluminescence efficiency, long fluorescence lifetime and high photochemical stability. These advantages make QDs an ideal fluorescent sensor for chemical or biological assays and real time, long term biomedical labeling. Ⅱ-Ⅵ group semiconductor QDs represent one of the most important types of nanomaterials. The aqueous synthesis route for QDs is relatively simpler, cheaper, less toxic and more environmentally friendly than the organometallic synthetic route, and the resulting QDs are water-soluble, also with high PLQYs, excellent photostability and biocompatibility, which make them convenient for the biological applications. QDs coated by capping ligands with their surrounding surface groups can obtain good biocompatibility since the release of Cd2+was effectively inhibited, and also retain their unique photophysical properties. The proper surface modification is needed to obtain desired functionality and improve photoluminescence response of QDs to some metal ions or molecules.
A new and exciting research avenue for QDs is their applications as optical probes for imaging of cells and tissue. As to biological optical imaging, the near-infrared (NIR)-emitting window is appealing because of the low tissue absorption and scattering effects in the emission range (650-900nm). This allows penetration of excitation and fluorescence photons deep into biological samples with reduced interaction and photodamage to the surrounding tissues, allowing fluorescence imaging depths on the order of centimeters. The near-infrared imaging is much prior to the visible light for biological applications.
In this paper, water soluble octa-mercaptopropyl POSS (OM-POSS) modified CdTe QDs have been successfully synthesized and employed as a novel fluorescent sensor for optical recognition of Cu2+in an aqueous medium. Their cytotoxicity and availability for cellular imaging in biomedical field were also studied. The near-infrared emitting CdSeTe QDs with octa-aminopropyl POSS (OA-POSS) capping were successfully synthesized and applied to SiHa cell imaging. The main contents are summarized as follows.
1. OM-POSS coated CdTe QDs with high fluorescence QYs were successfully synthesized in aqueous solution. TEM images reveal that these nanoparticles appear as spherical with excellent monodispersity. The relative photoluminescence quantum yield of the POSS-CdTe QDs was estimated to be95%using Rhodamine6G (PLQY95%) as reference. Temporal evolution of PL emitting wavelength and QYs of CdTe QDs growth under varied pH values, Cd/Te molar ratios and NAC/Cd molar ratios were studied. The growth rate decreases with the modification of OM-POSS due to its numerous mercapto groups. The POSS-CdTe QDs exhibit significantly good performance after two hours of UV exposure, indicating its excellent photostability.
2. The POSS-CdTe QDs have been employed as a novel fluorescent sensor for optical recognition of Cu2+in an aqueous medium. The fluorescence sensor for Cu2+is based on fluorescence quenching of the POSS-CdTe QDs. The selective quenching effect can be explained by the competitive binding of POSS capping layers between CdTe QDs and metal ions present in the solution. Fluorescence intensities of the POSS-CdTe QDs in PBS buffer solution at different pH values were studied. Stern-Volmer plots of different POSS-CdTe QDs concentrations were also investigated. Under optimum conditions, the detection limit for this sensing system is2.3×10-9mol L-1. The detection sensitivity for Cu2+is increased greatly with the capping of OM-POSS on CdTe QDs than the N AC-CdTe QDs. The Stern-Volmer plots at different temperatures show that POSS-CdTe QDs fluorescence was quenched by Cu2+through a static quenching mechanism. Furthermore, the practical utility of the POSS-CdTe QDs has been demonstrated by determination of trace Cu2+in water samples, obtaining satisfactory results with the standard addition method.
3. It is demonstrated that the POSS coating on CdTe core QDs confers it good bio compatibility, which is an important feature for cell labeling in the field of biomedicine. The SiHa cells and mouse preosteoblast cells MC3T3-E1were used to evaluate cytotoxicity of the as-prepared QDs. In comparison with CdTe QDs, the POSS-CdTe QDs show lower cytotoxicity under the same conditions. The OM-POSS coating can effectively inhibit the release of Cd2+and protect the QDs from aggregating caused by photobleaching. Moreover, large number of mercapto groups on OM-POSS can provide protection against QD-induced ROS stress and effectively prevent the decrease in cell viability. Confocal fluorescence images demonstrate that the QDs were internalized into the cells via endocytosis. The subcellular localization of POSS-CdTe QDs was found to depend upon QDs size. POSS-CdTe QDs are more easily taken up by cells than CdTe QDs due to its surface coating with amphiphilic moieties, which allows rapid intracellular uptake across the lipophilic cell membranes. The POSS-CdTe QDs allows rapid intracellular uptake, enabling the use of lower concentrations of QDs for an overall reduced toxicity.
4. The near-infrared emitting CdSeTe QDs with OA-POSS capping were successfully synthesized in aqueous solution. Temporal evolution of PL emitting wavelength and QYs of CdSeTe QDs growth under varied pH values, Cd/Te molar ratios and nonlinear composition effect were studied. TEM image reveals that these nanoparticles appear as spherical with excellent monodispersity. The absolute photoluminescence quantum yield of the POSS-CdTe QDs with681nm emitting wavelength was tested to be26.4%on Edinburgh FS-920fluorescence spectrometer. The SiHa cells were used to evaluate cytotoxicity of the as-prepared QDs. In comparison with CdSeTe QDs, the OA-POSS modified CdSeTe QDs show lower cytotoxicity under the same conditions. Confocal fluorescence images demonstrate that the near infrared emitting POSS-CdSeTe QDs were successfully internalized into the cells via endocytosis.
引文
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