【作者】 王锋；

【导师】 王民权； 樊先平；

【作者基本信息】 浙江大学 ， 材料学， 2006， 博士

【摘要】 几十年来，发光标记材料的研究与应用极大地促进了生物化学、临床医学及疫病防治与控制等生物学研究的进步与发展。目前广泛使用的有机染料和量子点（Quantum Dots，QDs）等生物标记材料因存在发射光谱宽、光热稳定性差、间歇性发光（闪烁，Blinking）和细胞毒性等诸多缺陷，有很大的应用局限。近年来，人们发现镧系掺杂发光纳米微粒兼具有毒性低、化学稳定性高、发光强度高而稳定（无闪烁）、斯托克斯位移（Stokes shift）大等综合优异性能，可望成为一种极具发展前景的新型发光生物标记材料，因而对其产生了浓厚的研究兴趣。本文综述了发光生物标记材料的研究现状，系统介绍了镧系离子的光谱理论及镧系掺杂发光纳米微粒的研究背景，概括和评述了近年来镧系掺杂发光纳米微粒的合成和表面修饰所取得的进展和面临的问题，并对其今后的研究方向进行了总结和展望。在此基础上，我们针对生物标记对发光材料的要求，研究建立了一系列低温湿化学合成方法和原位表面化学修饰技术，成功制备了多种具有不同光学性质的镧系掺杂氟化物发光纳米微粒。应用X射线衍射（XRD）、透射电镜（TEM）、光致发光谱（PL）、傅立叶变换红外光谱（FT-IR）、热分析（DTA-TG）以及Eu³⁺离子荧光探针和细胞毒性检测等手段研究了合成条件和掺杂离子浓度等对镧系掺杂发光纳米微粒的晶体结构、形貌和尺寸、表面化学性质、掺杂离子的固溶度和掺杂格位以及发光性能的影响和控制规律，取得了一系列重要的结论和创新性成果，为镧系掺杂纳米微粒在生物标记领域应用的推广和普及打下了坚实的基础。以湿化学法制备镧系掺杂发光纳米微粒时，通常都需要在起始反应溶液中加入其它试剂来控制微粒的生长，从而得到纳米级的微粒。本文中，我们基于溶度积原理，研究建立了一种简便易行的在纯乙醇溶液中进行的化学共沉淀法，在不使用任何外加试剂的情况下，分别制备了粒径为15nm、10nm和30nm左右的CaF₂：Eu³⁺、LaF₃：Eu³⁺和GdF₃：Eu³⁺纳米微粒。所得产物均为纯单相晶体，其晶型分别为立方、六方和斜方。通过改变起始原料的配比，在纳米微粒中掺入了不同浓度的Eu³⁺离子。研究表明，在所考察的掺杂浓度范围内(CaF₂、LaF₃和GdF₃纳米微粒中Eu³⁺离子的最高掺杂浓度分别为30mol％、60mol％和40mol％)，增加Eu³⁺离子掺杂浓度不会引起新结晶相的产生，三种纳米微粒的发光强度随Eu³⁺离子掺杂浓度增大均表现出了浓度猝灭现象，其猝灭浓度分别为～15mol％、～30mol％和～20mol％。大多数生物实验都要求标记材料亲水，获得亲水性纳米微粒的常用方法是在样品制备过程中加入一些具有亲水基团的有机配体，使这些配体结合在产物表面从而赋予其亲水性。本文中，我们基于水分子与稀土离子的配位作用，研究建立了一种非常简单的在水溶液中进行的化学共沉淀法，在不使用任何外加有机配体的情况下制备了粒径30nm以下的亲水性LaF₃：Ln³⁺（Ln=Eu，Ce-Tb，Nd）纳米微粒。所得产物具有纯六方相晶体结构，能稳定存在于水溶液中并产生较强的发光（量子效率为16％）。掺杂不同的镧系离子的样品能够产生从可见到近红区域的发光，适用于多种不同的生物标记，特别是掺杂Nd³⁺离子的纳米微粒，其激发位置和主要发射峰都位于近红外区域，非常适用于超灵敏的生物检测和生物呈像。生物标记（特别是活细胞和活体标记）实验中通常还要求纳米微粒表面具有化学官能团并且生物相容（即没有细胞毒性），获得这些属性的常规方法是在纳米微粒制备好之后再对其进行表面修饰。本文中，我们基于表面包覆剂分子结构上的氨基与稀土离子间的配位作用，研究建立了一种镧系掺杂发光纳米微粒的原位表面修饰方法，通过单步反应分别制备了壳聚糖包覆的CTS／LaF₃：Eu³⁺和聚乙烯亚胺包覆的PEI╱NaYF₄：Yb³⁺，Er³⁺等一系列纳米微粒。其中CTS／LaF₃：Eu³⁺纳米微粒具有纯六方相晶体结构，粒径为25nm左右；PEI／NaYF₄：Yb³⁺，Er³⁺纳米微粒为六方相和立方相混合的晶体，粒径约为50nm。研究表明，两种表面包覆膜对人结肠腺癌HT-29细胞系均无细胞毒性，也不会影响纳米微粒的发光光谱性质和发光量子效率。经表面修饰的纳米微粒在中性水溶液中可稳定存在5天以上，能较好地满足活细胞和活体标记的需要。常规的发光标记技术通常以紫外光或短波长的可见光作为激发源，这些激发光因能量较高，除能引起生物体的自发荧光，还会对细胞和生物组织产生光损伤。为了克服这些缺陷，我们开展了镧系掺杂上转换发光纳米微粒的研究。上转换发光材料能够在近红外光的激发下产生可见发光，可以有效解决常规激发方式引起的一系列问题。我们应用本文建立的纯乙醇溶液中进行的化学共沉淀法，制备了共掺yb³⁺_Er³⁺的LaF₃上转换发光纳米微粒，研究了热处理对其结构、形貌和上转换发光性能的影响规律，发现随着热处理温度升高，纳米微粒的晶体结构保持不变、粒径和上转换发光强度则均有显著增加，经过400℃以上温度的热处理后，样品表现出了肉眼可见的明亮上转换发光。但高温热处理会引起纳米微粒的长大、胶体属性的丧失以及表面化学官能团的破坏，因此不是制备上转换发光生物标记纳米微粒的最佳途径。为了制备出具有较好胶体属性和较强上转换发光的纳米微粒，我们选用了NaYF₄这种更有利于实现上转换的材料作为基质，采用本文建立的镧系掺杂发光纳米微粒的原位表面修饰方法，制备了PEI／NaYF₄：Yb³⁺，Er³⁺上转换发光纳米微粒，研究了合成工艺对其晶型和形貌的影响规律，发现延长反应时间有助于NaYF₄晶体由四方相向六方相转变，从而有利于提高其上转换发光的强度。延长水热处理时间不会引起产物的形貌和大小的改变。研究确认比较适宜的反应时间是24h，研究了该条件下制备的纳米微粒在水溶液中的上转换发光行为，发现它们在600mW近红外二极管激光器的激发下能够产生明亮的上转换发光，非常适用于上转换生物检测和活体生物呈像。在生物标记中，往往需要同时标记体系中的多个不同组分，这些实验需要采用多种具有不同颜色的发光标记材料，并且这些标记物应能被单一波长的激发源同时激发，以简化实验设备、提高信噪比。针对这一要求，我们采用本文建立的镧系掺杂发光纳米微粒的原位表面修饰方法，制备了共掺同一敏化剂(Ce³⁺)和不同激活剂(Tb³⁺，Eu³⁺，Sm³⁺，Oy³⁺)的一系列PEI／NaGdF₄纳米微粒。产物具有六方相晶体结构，形貌为棒状（棒直径约20nm-30nm，棒长约40nm-60nm）。研究发现，当Ce³⁺离子受到激发后，能够通过基质晶格中Gd³⁺离子的辅助作用有效地将所吸收的能量传递给镧系激活离子，从而引发后者的特征发光。正是由于这一光学特性，在单一波长（254nm）紫外光的激发下，掺杂不同镧系激活离子的PEI／NaGdF₄纳米微粒的水溶液产生了不同颜色的明亮发光，表明它们在多色生物标记实验中具有很大的应用潜力。更多还原

【Abstract】 The use of luminescent labeling agents has greatly assisted the studies in the field of biology. Conventional luminescent labeling agents such as organic dyes and quantum dots （QDs） have several limitations caused by their intrinsic properties such as, for example, broad emission profiles, poor photochemical stability, intermittent on/off emission （blinking）, and cytotoxicity etc. Recently, considering their attractive optical and chemical features such as low toxicity, large effective Stokes shifts, as well as high resistance to photobleaching, blinking, and photochemical degradation, lanthanide-doped luminescent nanoparticles were proposed to be a promising new class of luminescent labeling agents, which have the potential and ability to overcome a number of problems associated with the commonly used luminescent labels.This thesis gives an overview of the recent advances on luminescent materials for biological labeling, systemically introduces the luminescence theories of lanthanide ions and backgrounds of lanthanide-doped luminescent nanoparticles, summarizes the recent achievements on syntheses and surface modifications of lanthanide-doped luminescent nanoparticles, and puts forward the challenges they faced and the future directions of them in the area of biological labeling. On the above bases, we developed a series of mild wet chemical routes and in situ surface modification methods, and synthesized several kinds of lanthanide-doped luminescent fluoride nanoparticles accordingly. X-ray diffraction （XRD）, transmission electronic microscope （TEM）, photoluminescence spectroscopy （PL）, fourier transform infrared spectroscopy （FT-IR）, thermal analysis （DTA-TG）, and MTT assay were used to study the crystal structure, size and morphology, surface property, and luminescence property of the nanoparticles. A series of important conclusions and innovative results with practical significance were obtained.In wet chemical routes to produce lanthanide-doped luminescent nanoparticles, organic ligands were usually required to control the particle growth in order to get products in the nanometer scale. In this thesis, we developed a very simplecoprecipitation method carried out in absolute ethanol to synthesize lanthanide-doped luminescent nanoparticles without the use of any ligands. A series of nanoparticles include CaF₂:Eu³⁺ （-15 nm）, LaF₃:Eu³⁺ （-10 nm）, and GdF₃:Eu³⁺ （-30 nm） were synthesized using this method. The products consist of well crystallized pure cubic, hexagonal, and orthorhombic phases, respectively. By varying the ratios of the start materials, nanoparticles doped with different concentrations of Eu³⁺ ions were synthesized. In the doping concentration range studied (the maximum doping concentrations of Eu³⁺ ions for CaF₂, LaF₃, and GdF₃ nanoparticles are 30 mol%, 60 mol%, and 40 mol% respectively), no second phase were found for all the three samples at high doping concentrations of Eu³⁺ ions and they all showed a concentration quenching with increasing the Eu³⁺ ions doping concentration （the quenching concentrations are -15 mol%, -30 mol%, and -20 mol%,for CaF₂, LaF₃, and GdF₃ respectively）.Since most biological studies are situated in an aqueous environment, nanoparticles with hydrophilic surfaces are usually required. Conventional method for making hydrophilic nanoparticles is to use organic ligands with hydrophilic groups when synthesizing the nanoparticles. In this thesis, we developed a simple method to synthesize hydrophilic lanthanide-doped luminescent LaF₃ nanoparticles directly in aqueous solution without using any ligands. The nanoparticles have a nearly spherical shape with average size of below 30 nm and consist of well crystallized pure hexagonal phase. The nanopartcles are stable in aqueous solutions and show strong luminescence （quantum yield 16 %）. LaF₃ nanoparticles doped with different lanthanide ions or ion pair (Eu³⁺, Ce³⁺-Tb³⁺, and Nd³⁺) were synthesized, which emit in the visible （VIS） and near-infrared （NIR） spectral regions, and can be used for different biological applications.For certain biological labeling, especially for in vivo cell and animal labeling, the nanoparticles should be biocompatible （free of cytotoxicity） and contain functional groups for further attachment of biomolecules. Conventional method for obtaining such nanoparticles is via post surface modification. In this thesis, we developed an in-situ surface modification method to prepare biocompatible lanthanide-doped luminescent nanoparticles with functional chemical groups in a one-pot synthesis process. Chitosan capped CTS/LaF₃:Eu³⁺ nanoparticles and polyethylenimine capped PEI/NaYF₄:Yb³⁺,Er³⁺ nanoparticles were synthesized using this method. The CTS/LaF₃:Eu³⁺ nanoparticles have an average size of about 25 nm and consist of well crystallized pure hexagonalphase. The PEI/NaYF₄:Yb³⁺,Er³⁺ nanoparticles have an average size of about 50 nm and consist of well crystallized cubic and hexagonal mixed phases. It is revealed that both CTS and PEI coatings are biocompatible to human colon HT-29 cells and have no impacts on the luminescence property and quantum yield of the nanoparticles. The as-prepared nanoparticles stay stable in aqueous solutions for more than 5 days and thus have good potentials for in vivo cell and animal labeling.Conventional luminescent labeling technologies are most based on the use of ultraviolet （UV） or short VIS irradiation. A major problem associated with these systems in biological applications is the autofluorescence from and photo-damage to the biological specimens. A possible way to circumvent this problem is labeling with lanthanide-doped up-conversion nanoparticles, which show VIS emissions under NIR irradiation. Using the above described coprecipitation method carried out in absolute ethanol, we prepared Yb³⁺ and Er³⁺ codoped LaF₃ nanoparticles and investigated the influence of synthesis temperatures on the size, morphology, and up-conversion luminescence intensity of the nanoparticles. It is found that elevating the synthesis temperatures will cause significant increase in the particle size and up-conversion luminescence intensity of the nanoparticles. Annealing at above 400 °C is necessary to obtain products with practical up-conversion luminescence. However, heat treatment at high temperatures will cause the nanoparticles to lose colloidal properties and functional chemical groups, and thus is not the optimal way to produce up-conversion luminescent biolabels.In order to prepare colloidal lanthanide-doped nanoparticles with intense up-conversion luminescence, we replace the host matrix with NaYF₄, which is the most efficient host material for performing up-conversion known to date. We synthesized the PEI/NaYF₄:Yb³⁺,Er³⁺ nanoparticles using the in situ surface modification method described above and investigated the influence of reaction time on the properties of the resulting products. It is revealed that prolonging the reaction time will cause phase transfer from cubic to hexagonal of the products and thus promote their up-conversion luminescence intensity. The size and morphology of the nanoparticles show no obvious changes with varying the reaction time. The optimum reaction time is found to be 24 h and the nanoparticles synthesized under this condition show strong up-conversion luminescence in aqueous solutions when excited with a 600 mW diode laser at 980 nm, and thus have good potentials for use as labels in up-conversion detection and in vivo animal imaging.For multiplexing labeling, which is of especial interest in biological field currently, nanoparticles of different emission colors should be readily available and the whole group of labels can be excited at a single wavelength. To cater for these applications, we prepared the PEI/NaGdF₄:Ce³⁺,Ln³⁺ （Ln = Tb, Eu, Sm, and Dy） nanoparticles using the in situ surface modification method described above. The nanoparticles have a rod shape （20-30 nm in diameter and 40-60 nm in length） and consist of well crystallized hexagonal phase. It is found that after excitation into the Ce³⁺ ions, the excitation energy can be transferred to the luminescent centers via the Gd³⁺ sublattice followed by emission from the these luminescent centers. As such, the PEI/NaGdF₄ nanoparticles activated with different lanthanide ions show bright luminescence of different colors under single wavelength excitation at 254 nm, and thus have good potentials for multiplexing labeling.更多还原