纳米组装体的可控制备及其在细胞成像分析中的应用研究
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摘要
研究纳米材料组装体对生化传感器构建和生物医学成像分析具有非常重要的意义。将金属纳米材料的表面等离子体共振特性或半导体纳米材料的荧光特性与碳纳米材料的生物相容性相结合,探索纳米组装体在生物成像和药物运载领域的应用具有独特的优势和发展潜力。本文研究了金属纳米颗粒形态、尺寸、等离子体耦合对表面等离子体共振特性的影响。在此基础上构建了金属纳米自组装体和金属纳米材料与碳纳米管的杂化组装体,并将组装方法拓展到了量子点与石墨烯的组装体制备,探索了组装体在生物成像和药物运载方面的用途。主要内容如下：
1.提出形态可控纳米金颗粒的一步合成法。研究发现,以盐酸四环素为还原剂,CTAB为包被试剂,通过调节CTAB与HAuCl4摩尔比,很容易制备孪晶片、单晶纳米片、孪晶十面体等三种形态晶体。进一步通过扫描电镜(SEM)、透射电镜(TEM)、高分辨透射电镜(HRTEM)、X射线衍射(XRD)、电子衍射(SAED)等表征手段对晶体结构进行了解析。通过UV-vis-NIR消光光谱、暗场散射成像及单颗粒散射光谱研究了形态、尺寸对其表面等离子体共振性质的影响。三种形态的纳米晶体都表现出优异的近红外吸收和暗场散射特性,可作为细胞成像探针用于癌症诊断和治疗。此外,本文还探讨了晶核形成及晶体生长机制,对今后纳米颗粒的合成及形态的控制具有理论参考意义。
2.金纳米棒的链型自组装和金纳米棒／纳米球的网状可控组装。以meso-四(4-磺基苯基)卟啉(TPPS)的H型聚集体为媒介,发现通过调节TPPS浓度可以调控组装体的粒子问距及其表面等离子体共振耦合。通过消光光谱、SEM等手段表征了自组装体的结构；通过ζ电位、动态光散射、共振散射光谱确证纳米组装体在溶液中形成。进一步通过有限差分时域(FDTD)计算发现,纳米颗粒表面等离子体共振峰红移程度随纳米颗粒间距呈双指数递减,基于此得到“等离子体标尺方程”。根据此方程计算组装体中粒子之间的距离,与SEM统计值吻合。进一步通过暗场散射成像和生物学方法探究金纳米棒的组装机理,证明卟啉聚集体吸附在金棒端头,是构成组装体的桥梁。
3.构建了以DNA为桥梁的金属纳米颗粒／碳纳米管异质组装体。实验结果与理论计算表明,单链DNA可以通过π-π共轭作用缠绕在碳纳米管表面,其π-π堆垛能力与碱基的电子结构密切相关。基于单链DNA与碳纳米管特殊相互作用,DNA修饰的金属纳米颗粒可吸附至碳纳米管表面从而构建金属纳米颗粒／碳纳米管异质组装体。金属纳米颗粒形态、电荷、组分可根据需要进行调节,修饰及组装过程对单组分的性质没有显著影响。组装后金属纳米颗粒的优异表面等离子体共振光学特性得到保持,而碳纳米管也保持其生物相容性。借助于暗场散射成像,发现组装体进入细胞的能力不仅与碳纳米管固有性质相关,还取决于金属纳米颗粒表面包被试剂。此外,结合金属纳米颗粒可见光区的散射特性和碳纳米管的近红外吸收性质,组装体可能同时应用于癌症的诊断和治疗。
4.以DNA为媒介构建了石墨烯／量子点异质组装体。研究发现,DNA通过共价键修饰在量子点表面,借助DNA在石墨烯表面的吸附,量子点与石墨烯进行组装。该组装体同时具备两种纳米材料的特点,如优异的荧光特性、良好的生物相容性。进一步研究表明,以石墨烯为药物载体,通过药物自身荧光可考察药物在细胞内的分布,借助量子点荧光可考察石墨烯内在化途径,利用该组装体不仅可以监测药物释放,还能观察载体在药物释放过程中的胞内分布情况。
上述研究内容探讨了金属表面等离子体共振特性与形态、组分、等离子体共振耦合等因素的关系,在此基础上通过DNA与碳纳米管的缠绕,构建了金属纳米颗粒与碳纳米管的组装体,利用DNA的吸附,该方法还可拓展至荧光量子点与石墨烯的组装,纳米组装体表现出优异的光学特性和良好的生物相容性,在生物成像和癌症治疗领域具有潜在的应用前景。
The investigations of nanoparticle assembly have promoted the development of nanocomposites in chemical and biological sensing, cell imaging and medical therapy. Combining the unique plasmon resonance properties of metal nanoparticles or fluorescence properties of semiconductor materials and biocompatibility of carbon nanomaterials, the nanocomposites demonstrate great potentials in biological imaging and cancer therapy. In this thesis, the dependence of surface plasmon resonance properties on particle morphology, size and plasmon coupling has been investigated, and then metal nanoparticle assembly as well as metal nanoparticle/carbon nanotube assembly have been facilely fabricated, which could be applied to the preparation of quantum dot/graphene nanocomposites. We have also investigated the potential applications of such nanocomposites in cell imaging and drug delivery. The main points are as follows:
1. A general one-step route to synthesize Au crystals with various morphologies has been proposed. Starting from HAuCl4 in an aqueous medium and by employing tetracycline hydrochloride as the reducing agent and cetyltrimethylammonium bromide (CTAB) as the capping agent, it is very easy to get twinned tabular crystals, single crystalline nanoplates, and multi-twinned decahedra by modulating the molar ratio of CTAB with HAuCl4. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), selected area electron diffraction (SAED) and high-resolution transmission electron microscopy (HRTEM) were employed to characterize the three types of symmetric morphology. UV-vis-NIR. extinction spectra, dark field light scattering imaging and single particle spectra were used to investigate the dependence of surface plasmon resonance scattering on crystal size and morphology. The unique optical properties of the obtained crystals, including NIR absorption and dark field light scattering demonstrate their potential applications in cancer cell diagnostics and photothermal therapy. Moreover, a tentative explanation for the growth mechanism of Au crystals with different morphologies has been made.
2. Linear chains of Au nanorods and bifurcated junctions of nanorods/nanospheres could be facilely fabricated via the cross-linking of H-type tetrakis(4-sulfonatophenyl)porphyrin (TPPS) aggregates in solutions. The tuning of the plasmon coupling between the Au nanocrystals has been demonstrated by varying the porphyrin concentration and thus the interparticle gap distances. The assembled nanocrystals were characterized by the extinction spectra and SEM images,ζ-potential measurements, dynamic light scattering analysis as well as resonance light scattering spectra confirmed the assembly was initiated in aqueous solutions other than solvent evaporation during the SEM specimen preparation. Further more, Finite-difference time-domain (FDTD) calculations showed that the redshift of the plasmon band exhibited a nearly exponential decay with increasing interparticle gap distances, giving rise to a "plasmon ruler equation". The gap distances determined according to this equation agreed well with the SEM observations. We also designed a biological procedure to investigate the interaction between Au nanocrystals and TPPS by the dark field light scattering technique, further confirming our view that TPPS preferentially bond to the end of Au nanorods to facilitate their end-to-end assembly.
3. A novel strategy to prepare metal nanoparticle/carbon nanotube (CNT) hybrids through DNA linkage has been proposed. Both experimental and theoretical results demonstrated that the adsorption ability of single-strand DNA (ssDNA) on carbon nanotubes is correlative with electronic structure of natural bases. Based on the special interaction between ssDNA and CNTs, DNA modified Au nanoparticles could deposite on CNTs throughπ-πconjugate interaction, and the morphology, size, charge as well as the composition of metal nanoparticles could be rationally modulated according to experimental requirements. The conjugation chemistry did not perturb the unique intrinsic properties of each component, such as the unique plasmon properties of metal nanoparticles, and the affinity of CNTs for cancer cells. We have further investigated the interaction between nanocomposites and cancer cells using dark field light scattering imaging, and the results demonstrated that the internalization of nanocomposites into cancer cells depends on not only intrinsic affinity of CNTs but also biocompatibility of capped agents on decorated metal nanoparticles. Furthermore, the enhanced absorption of CNTs in the NIR region and the strong scattering of AgNSs in the visible region enable AgNS/CNT hybrids to be efficient agents for both cell imaging and cancer therapy.
4. Quantum dots (QDs)/graphene hybrids have been facilely fabricated via DNA's cross-linking. It was found that QDs, which was first modified by DNA through colvalent bond, could be assembled on graphene surfaces throughπ-πstacking. The nanocomposites exhibited unique intrinsic properties of each component, such as high fluorescence intensity, good biocompatibility. By employing graphene as delivery vectors, we investigated the potential applications of the prepared nanocomposites in drug delivery, and it was found that the drug distribution could be visually observed by its own fluorescence. The internalization procedure of graphene could be monitored by QDs'fluorescence. In other words, the nanocomposites provided opportunities for monitoring both the drug delivery and vector distribution.
In conclusion, the dependence of surface plasmon resonance properties of metal nanoparticles on morphology, composition and plasmon coupling has been investigated in this thesis. Based on the interaction between ssDNA and CNTs, we proposed a universal method for fabrication of metal nanoparticle/carbon nanotube assembly via DNA's cross-linking. The method could be further applied to prepare QDs/graphene assembly. The nanocomposites exhibited unique optical properties as well as good biocompatibility, which demonstrated great potentials in biological imaging and cancer therapy.
1.提出形态可控纳米金颗粒的一步合成法。研究发现,以盐酸四环素为还原剂,CTAB为包被试剂,通过调节CTAB与HAuCl4摩尔比,很容易制备孪晶片、单晶纳米片、孪晶十面体等三种形态晶体。进一步通过扫描电镜(SEM)、透射电镜(TEM)、高分辨透射电镜(HRTEM)、X射线衍射(XRD)、电子衍射(SAED)等表征手段对晶体结构进行了解析。通过UV-vis-NIR消光光谱、暗场散射成像及单颗粒散射光谱研究了形态、尺寸对其表面等离子体共振性质的影响。三种形态的纳米晶体都表现出优异的近红外吸收和暗场散射特性,可作为细胞成像探针用于癌症诊断和治疗。此外,本文还探讨了晶核形成及晶体生长机制,对今后纳米颗粒的合成及形态的控制具有理论参考意义。
2.金纳米棒的链型自组装和金纳米棒／纳米球的网状可控组装。以meso-四(4-磺基苯基)卟啉(TPPS)的H型聚集体为媒介,发现通过调节TPPS浓度可以调控组装体的粒子问距及其表面等离子体共振耦合。通过消光光谱、SEM等手段表征了自组装体的结构；通过ζ电位、动态光散射、共振散射光谱确证纳米组装体在溶液中形成。进一步通过有限差分时域(FDTD)计算发现,纳米颗粒表面等离子体共振峰红移程度随纳米颗粒间距呈双指数递减,基于此得到“等离子体标尺方程”。根据此方程计算组装体中粒子之间的距离,与SEM统计值吻合。进一步通过暗场散射成像和生物学方法探究金纳米棒的组装机理,证明卟啉聚集体吸附在金棒端头,是构成组装体的桥梁。
3.构建了以DNA为桥梁的金属纳米颗粒／碳纳米管异质组装体。实验结果与理论计算表明,单链DNA可以通过π-π共轭作用缠绕在碳纳米管表面,其π-π堆垛能力与碱基的电子结构密切相关。基于单链DNA与碳纳米管特殊相互作用,DNA修饰的金属纳米颗粒可吸附至碳纳米管表面从而构建金属纳米颗粒／碳纳米管异质组装体。金属纳米颗粒形态、电荷、组分可根据需要进行调节,修饰及组装过程对单组分的性质没有显著影响。组装后金属纳米颗粒的优异表面等离子体共振光学特性得到保持,而碳纳米管也保持其生物相容性。借助于暗场散射成像,发现组装体进入细胞的能力不仅与碳纳米管固有性质相关,还取决于金属纳米颗粒表面包被试剂。此外,结合金属纳米颗粒可见光区的散射特性和碳纳米管的近红外吸收性质,组装体可能同时应用于癌症的诊断和治疗。
4.以DNA为媒介构建了石墨烯／量子点异质组装体。研究发现,DNA通过共价键修饰在量子点表面,借助DNA在石墨烯表面的吸附,量子点与石墨烯进行组装。该组装体同时具备两种纳米材料的特点,如优异的荧光特性、良好的生物相容性。进一步研究表明,以石墨烯为药物载体,通过药物自身荧光可考察药物在细胞内的分布,借助量子点荧光可考察石墨烯内在化途径,利用该组装体不仅可以监测药物释放,还能观察载体在药物释放过程中的胞内分布情况。
上述研究内容探讨了金属表面等离子体共振特性与形态、组分、等离子体共振耦合等因素的关系,在此基础上通过DNA与碳纳米管的缠绕,构建了金属纳米颗粒与碳纳米管的组装体,利用DNA的吸附,该方法还可拓展至荧光量子点与石墨烯的组装,纳米组装体表现出优异的光学特性和良好的生物相容性,在生物成像和癌症治疗领域具有潜在的应用前景。
The investigations of nanoparticle assembly have promoted the development of nanocomposites in chemical and biological sensing, cell imaging and medical therapy. Combining the unique plasmon resonance properties of metal nanoparticles or fluorescence properties of semiconductor materials and biocompatibility of carbon nanomaterials, the nanocomposites demonstrate great potentials in biological imaging and cancer therapy. In this thesis, the dependence of surface plasmon resonance properties on particle morphology, size and plasmon coupling has been investigated, and then metal nanoparticle assembly as well as metal nanoparticle/carbon nanotube assembly have been facilely fabricated, which could be applied to the preparation of quantum dot/graphene nanocomposites. We have also investigated the potential applications of such nanocomposites in cell imaging and drug delivery. The main points are as follows:
1. A general one-step route to synthesize Au crystals with various morphologies has been proposed. Starting from HAuCl4 in an aqueous medium and by employing tetracycline hydrochloride as the reducing agent and cetyltrimethylammonium bromide (CTAB) as the capping agent, it is very easy to get twinned tabular crystals, single crystalline nanoplates, and multi-twinned decahedra by modulating the molar ratio of CTAB with HAuCl4. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction (XRD), selected area electron diffraction (SAED) and high-resolution transmission electron microscopy (HRTEM) were employed to characterize the three types of symmetric morphology. UV-vis-NIR. extinction spectra, dark field light scattering imaging and single particle spectra were used to investigate the dependence of surface plasmon resonance scattering on crystal size and morphology. The unique optical properties of the obtained crystals, including NIR absorption and dark field light scattering demonstrate their potential applications in cancer cell diagnostics and photothermal therapy. Moreover, a tentative explanation for the growth mechanism of Au crystals with different morphologies has been made.
2. Linear chains of Au nanorods and bifurcated junctions of nanorods/nanospheres could be facilely fabricated via the cross-linking of H-type tetrakis(4-sulfonatophenyl)porphyrin (TPPS) aggregates in solutions. The tuning of the plasmon coupling between the Au nanocrystals has been demonstrated by varying the porphyrin concentration and thus the interparticle gap distances. The assembled nanocrystals were characterized by the extinction spectra and SEM images,ζ-potential measurements, dynamic light scattering analysis as well as resonance light scattering spectra confirmed the assembly was initiated in aqueous solutions other than solvent evaporation during the SEM specimen preparation. Further more, Finite-difference time-domain (FDTD) calculations showed that the redshift of the plasmon band exhibited a nearly exponential decay with increasing interparticle gap distances, giving rise to a "plasmon ruler equation". The gap distances determined according to this equation agreed well with the SEM observations. We also designed a biological procedure to investigate the interaction between Au nanocrystals and TPPS by the dark field light scattering technique, further confirming our view that TPPS preferentially bond to the end of Au nanorods to facilitate their end-to-end assembly.
3. A novel strategy to prepare metal nanoparticle/carbon nanotube (CNT) hybrids through DNA linkage has been proposed. Both experimental and theoretical results demonstrated that the adsorption ability of single-strand DNA (ssDNA) on carbon nanotubes is correlative with electronic structure of natural bases. Based on the special interaction between ssDNA and CNTs, DNA modified Au nanoparticles could deposite on CNTs throughπ-πconjugate interaction, and the morphology, size, charge as well as the composition of metal nanoparticles could be rationally modulated according to experimental requirements. The conjugation chemistry did not perturb the unique intrinsic properties of each component, such as the unique plasmon properties of metal nanoparticles, and the affinity of CNTs for cancer cells. We have further investigated the interaction between nanocomposites and cancer cells using dark field light scattering imaging, and the results demonstrated that the internalization of nanocomposites into cancer cells depends on not only intrinsic affinity of CNTs but also biocompatibility of capped agents on decorated metal nanoparticles. Furthermore, the enhanced absorption of CNTs in the NIR region and the strong scattering of AgNSs in the visible region enable AgNS/CNT hybrids to be efficient agents for both cell imaging and cancer therapy.
4. Quantum dots (QDs)/graphene hybrids have been facilely fabricated via DNA's cross-linking. It was found that QDs, which was first modified by DNA through colvalent bond, could be assembled on graphene surfaces throughπ-πstacking. The nanocomposites exhibited unique intrinsic properties of each component, such as high fluorescence intensity, good biocompatibility. By employing graphene as delivery vectors, we investigated the potential applications of the prepared nanocomposites in drug delivery, and it was found that the drug distribution could be visually observed by its own fluorescence. The internalization procedure of graphene could be monitored by QDs'fluorescence. In other words, the nanocomposites provided opportunities for monitoring both the drug delivery and vector distribution.
In conclusion, the dependence of surface plasmon resonance properties of metal nanoparticles on morphology, composition and plasmon coupling has been investigated in this thesis. Based on the interaction between ssDNA and CNTs, we proposed a universal method for fabrication of metal nanoparticle/carbon nanotube assembly via DNA's cross-linking. The method could be further applied to prepare QDs/graphene assembly. The nanocomposites exhibited unique optical properties as well as good biocompatibility, which demonstrated great potentials in biological imaging and cancer therapy.
引文
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