CN101387639A - 基于二氧化硅的荧光纳米颗粒 - Google Patents

基于二氧化硅的荧光纳米颗粒 Download PDF

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CN101387639A
CN101387639A CNA200810170588XA CN200810170588A CN101387639A CN 101387639 A CN101387639 A CN 101387639A CN A200810170588X A CNA200810170588X A CN A200810170588XA CN 200810170588 A CN200810170588 A CN 200810170588A CN 101387639 A CN101387639 A CN 101387639A
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U·威斯纳
H·奥
D·R·拉森
W·W·韦布
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Abstract

本发明总体涉及荧光纳米颗粒，更具体而言，涉及小于30nm的基于二氧化硅的荧光纳米颗粒，该荧光纳米颗粒具有共价连接的有机染料。本发明提供一种包括荧光团中心核和二氧化硅壳体的荧光单分散二氧化硅纳米颗粒，其中纳米颗粒的辐射性能取决于核的化学性质(组成)和二氧化硅壳体的存在。在本发明的一方面，核－壳结构使荧光量子效率得到了提高。本发明总体提供了对封装在大小为30nm及30nm以下的二氧化硅颗粒内的染料分子的光物理性能的控制。该控制通过改变二氧化硅的化学性质和以纳米尺度改变颗粒结构而实现，与游离染料相比导致亮度大大提高。

Description

基于二氧化硅的荧光纳米颗粒
本申请是2003年11月26日提交的200380109252.8号发明专利申请的分案申请。
本申请是申请日为2002年11月26日、序列号10/306614的共同未决非临时性美国专利申请的部分继续申请，其公开内容通过引用的方式纳入本申请。
技术领域
本发明总体涉及荧光纳米颗粒(nanoparticles)，更具体而言，涉及小于30nm的二氧化硅基荧光纳米颗粒，该荧光纳米颗粒与有机染料共价连接。
背景技术
作为生物技术和信息应用，例如生物成像、传感器技术、微阵列以及光计算的的指示剂和光子源，荧光纳米颗粒的应用前景无可限量。这些应用需要尺寸受控的单分散发光纳米颗粒，且所述纳米颗粒可以特异性地与生物大分子结合或者排布在高阶结构(higher-orderstructures)中。
近几年，出现了两种通用方法用以合成高荧光性、水溶性纳米颗粒，这些颗粒被用于一系列要求苛刻的生物和分析应用中。在第一种方法中，纳米颗粒材料本身是荧光性的(例如半导体纳米晶体或者金属纳米晶体)。在第二种方法中，荧光纳米颗粒(的荧光性)源于有机染料分子的掺混。考虑到有机染料分子的丰富多样性以及这些染料分子对它们所处环境的极度敏感性，后一种方法增加了开发多种具有精确受控荧光特性纳米颗粒的可能性。
基于等人的工作，已经合成了一系列不同大小、颜色和结构的、嵌有染料的二氧化硅纳米颗粒。在此前所有的光物理性质报告中，与游离染料相比，均观察到在二氧化硅颗粒内部共价结合的染料的(荧光)淬灭(be quenched)。然而，在聚(有机硅氧烷)微粒凝胶的情况下，可观察到荧光效率有轻微增加。其中，所述凝胶内部染料通过非共价键连接并通过扩散被负载。对于其他材料，例如聚苯乙烯微粒凝胶，包埋染料的量子效率(quantum effciency)等于或小于游离染料。荧光淬灭通常被归因为向二氧化硅基质的颗粒内能量转移或者非辐射衰减。这两种(能量转移)途径可能受颗粒内的染料所处环境影响，这意味着对颗粒内结构的精确控制可以缓解淬灭，或者，甚至引起荧光增强。
此外，仍然需要粒度小于30nm、与有机染料共价连接的高荧光性纳米颗粒。
发明内容
本发明涉及一类二氧化硅纳米颗粒，在所述纳米颗粒中，二氧化硅颗粒的内部结构对组份染料的辐射(radiative)性质具有明显的、受控的影响。
本发明提供一种荧光单分散二氧化硅纳米颗粒，所述纳米颗粒包括荧光团中心核(fluorophore center core)和二氧化硅壳体，其中，纳米颗粒的辐射性质取决于核的化学性质(组成)(composition)，以及二氧化硅壳体的存在。在本发明的一个方面，核-壳(core-shell)结构使荧光量子效率得以提高。本发明主要提供了染料分子的光物理性质的控制，所述染料分子被封装在粒度小于或等于30nm的二氧化硅颗粒内部。该控制通过改变二氧化硅化学性质(组成)以及纳米级的颗粒结构实现，从而，与游离染料相比，产生了显著的亮度提高。
在本发明的另一个方面，核-壳结构提供了对于纳米颗粒的光稳定性的控制能力。
本发明提供的量子效率提高源于以下两方面原因：辐射率的提高，以及非辐射率的降低，后者的影响在各结构之间是最易变的。非辐射率的变化与颗粒内部染料旋转迁移率(rotational mobility)的差相关性良好。
在本发明的一个实施方案中，荧光单分散纳米颗粒包括核-壳结构，其中，所述核包括被二氧化硅壳体包裹的密实核(compact core)。在本发明的另一个实施方案中，荧光单分散纳米颗粒包括核-壳结构，其中，所述核包括被二氧化硅壳包裹的扩充核(expanded core)。在本发明的又一个实施方案中，荧光单分散纳米颗粒包括核-壳结构，其中，所述核包括被二氧化硅壳体包裹的，且被染料稀疏包埋其中的均质颗粒(homogenous particle)。在本发明的再一个实施方案中，荧光单分散均质纳米颗粒不被二氧化硅壳体包裹。
本发明提供了一种制备具有密实核结构的荧光单分散纳米颗粒的方法，该方法通过将一种荧光化合物和一种有机硅烷化合物混合形成染料前体，混合得到的染料前体与水溶液形成密实荧光核，再将得到的密实核与二氧化硅前体混合，在密实核上形成二氧化硅壳体，从而制得本发明荧光单分散纳米颗粒。
本发明也提供了一种制备具有扩充核(expanded core)结构的荧光单分散纳米颗粒的方法，该方法通过将一种荧光化合物和一种有机硅烷化合物混合制成染料前体，将得到的染料前体与二氧化硅前体共缩合(co-condense)形成扩充荧光核，再将得到的扩充核与二氧化硅前体混合，在扩充核上形成二氧化硅壳体，从而制得荧光单分散纳米颗粒。
本发明还提供了一种制备荧光单分散均质纳米颗粒的方法，该方法通过将一种荧光化合物和一种有机硅烷化合物混合形成染料前体，并将得到的染料前体与二氧化硅前体共缩合制得均质荧光单分散纳米颗粒。
本发明的这些技术特征，以及其他技术特征，在本发明以下说明书中阐明。
附图说明
图1A-C示出了不同的二氧化硅纳米颗粒结构的示意图，这些示意图分指密实核-壳纳米颗粒(1A)、扩充核-壳纳米颗粒(1B)、以及均质纳米颗粒(1C)。密实核-壳结构和扩充核-壳结构的二氧化硅壳体含有二氧化硅，但不含有任何染料分子。均质纳米颗粒含有存在于基质中的二氧化硅和染料的混合物。
图2A-C示出了纳米颗粒和合成中间体的荧光相关光谱(fluorescence correlation spectroscopy)。
图2D-F示出了900nm处，激发值为1.2mW时密实核-壳结构、扩充核-壳结构以及均质结构合成阶段的亮度(计数(counts)/颗粒/秒)值。
图3示出了稳态光谱曲线。该曲线显示出通过核-壳结构所实现的量子效率提高。图3A-C示出了密实核-壳纳米颗粒的吸收光谱和荧光光谱(3A)，扩充核-壳纳米颗粒的吸收光谱和荧光光谱(3B)，以及均质纳米颗粒的吸收光谱和荧光光谱(3C)。吸光值以光密度单位计。荧光光谱相对使用的有机染料TRITC进行标度。图3D-F示出了密实核-壳纳米颗粒(3D)、扩充核-壳纳米颗粒(3E)和均质颗粒(3F)的吸收光谱与激发光谱的比较。
图4示出了随时间消减(time-resolved)的纳米颗粒的荧光(曲线)。
图4A示出了均质纳米颗粒、扩充核-壳纳米颗粒、密实核-壳纳米颗粒以及TRITC染料归一化的荧光衰减。图4B示出了扩充核-壳纳米颗粒、密实核-壳纳米颗粒、均质纳米颗粒以及TRITC染料的荧光各向异性(fluorescence anisotropy)。图4C示出了图4B所示的纳米颗粒曲线归一化后的荧光各向异性(曲线)。荧光寿命(τf)和旋转寿命(θ)的平均拟合值列于表III中。
具体实施方式
本发明荧光纳米颗粒包括，一个含有一种荧光硅烷化合物的核，和核上的二氧化硅壳体。所述纳米颗粒的核可以包括，例如一种活性荧光化合物与一种共反应性(co-reactive)有机硅烷化合物的反应产物，所述壳体可包括，例如可形成二氧化硅化合物的反应产物。该可形成二氧化硅化合物可产生，例如一层或多层二氧化硅，所述层数如1到20层，以及各种需要的壳体特征，例如壳层厚度、壳厚度与核厚度或直径的比、核的二氧化硅壳体表面覆盖率、二氧化硅壳体的孔隙度和承载能力，以及类似的特征(considerations)。
荧光单分散核-壳纳米颗粒的合成基于以下两步法。第一步，使有机染料分子异硫氰酸四甲基若丹明(tetramethylrhodamineisothiocynate TRITC)与二氧化硅前体结合，并缩合形成富含染料(dye-rich)的核。第二步，添加硅胶单体以在荧光核材料周围形成较密的二氧化硅网状物，以提供对光稳定性有害的溶剂相互作用的屏蔽作用。首先形成富含染料的核使得引入多种荧光团成为可能，所述荧光团覆盖整个紫外-可见吸收光谱和发射光谱谱段。
通过染料填筑(dyeplacement)合成不同核-壳结构，即，密实核、扩充核以及均质核-壳结构的过程中，使用的反应物的量始终保持相同，以产生精确的结构性能相关性。但导致形成核-壳结构的反应物反应顺序是变化的。因此，就对颗粒的光物理性质有重要影响的二氧化硅基质密度和有机部分(organic moiety)而言，各种结构中染料分子周围的化学环境是不同的。
为了合成密实核-壳纳米颗粒，将染料前体加入到装有适量氨、水和溶剂的反应容器中，反应过夜。染料前体可在无水条件下，通过使TRITC和3-氨基丙基三乙氧基硅烷以摩尔比1:50加成反应合成。富含染料的密实核合成完成后，随后加入原硅酸四乙酯(tetraethylorthosilcate TEOS)以生长包裹核的二氧化硅壳体。
通过使TEOS和前述染料前体共缩合并使混合物反应过夜，合成扩充核-壳纳米颗粒。扩充核合成完成后，加入额外的TEOS以生长包裹核的二氧化硅壳体。
通过同时共缩合所有反应物，即，染料前体和TEOS，并使混合物反应过夜，合成均质纳米颗粒。
图2A-C所示的FCS曲线和单指数型拟合曲线示出了各种结构在水溶液中的单分散条件(monodispersity requirment)。FCS曲线还提供了两个独立参数：扩散系数和绝对浓度。扩散系数与流体动力学半径直接相关，而绝对浓度使得颗粒亮度得以量化。
每种纳米颗粒以及它们各自的中间体的FCS曲线在图2A-C中示出。如图2A所示，密实核-壳结构的核的扩散系数(D＝0.098μm2/ms)大约是游离TRITC染料扩散系数(D＝0.21μm2/ms)的一半。完整的纳米颗粒大约大十倍，如图2A所示(D＝0.014μm2/ms)。在图2B中，游离TRITC染料、扩充核-壳结构和完整颗粒之间显示出了类似的关系。在扩充核-壳结构中，核的扩散系数为0.075μm2/ms，与之相对的游离染料TRITC的扩散系数是D＝0.21μm2/ms。对于均质纳米颗粒而言，没有中间体核结构，图2C示出了均质纳米颗粒相应于游离TRITC染料的扩散系数(D＝0.015μm2/ms)。
图2D-E示出了各种结构的亮度和合成中间体。对于使用核中间体进行的合成，尽管核中可能存在多种染料，可核(的亮度)总是比游离染料(的亮度)暗。但完整的纳米颗粒的亮度总是明显高于游离染料和/或核中间体。而且，亮度随结构不同而改变，尽管这些结构的颗粒大小相同且前体材料的绝对量相同。
本发明的荧光纳米颗粒包括一种有机染料TRITC，它在发光性质方面表现出提高量子效率的效果。当用初级能源照射本发明的荧光纳米颗粒时，发生相应于用于纳米颗粒的有机染料的带隙(band gap)的频率的能量次级发射。
每颗粒TRITC等价物(equivalent)的数目可通过FCS曲线和吸光度测定值的结合予以确定。其中，绝对浓度由FCS曲线确定，相关的吸光度针对溶液中的TRITC进行测量，如图3A-C所示。图3A-C中使用稀释溶液(大约50nM)，以便对一个样品进行FCS和吸光度两项内容的测量。每颗粒TRITC等价物的数目列于表II中，所述数目使用514.5nm处消光系数42,105M-1cm-1计算得到。类似地，如表II所示，所述纳米颗粒荧光相对强度和纳米颗粒吸收的相对强度产生相对于游离TRITC的量子效率提高。
对于密实核-壳和扩充核-壳纳米颗粒，每颗粒TRITC等价物的数目在8.6时不易区分。然而，相对于游离TRITC染料，扩充核-壳纳米颗粒显示了三倍的量子效率的提高，但密实核-壳仅显示了相对于游离TRITC染料的两倍的量子效率的提高，如图3A、B及表II所示。用均质纳米颗粒得到了最大的量子效率提高，其每颗粒TRITC等价物平均为2.3，如图3C和表II所示。
经纳米颗粒化学组成和结构对于光物理性质进行控制的能力，使得开发下一代荧光探针以及在溶液中难以进行的荧光性研究成为可能。
颗粒的荧光寿命表示在图4A中。每种颗粒的寿命，包括游离TRITC，都是多指数类型的，所述平均寿命值列于表III中。TRITC的寿命(τf＝2.1ns)与其在水中(测得)的文献值良好吻合。所述寿命依密实核-壳(1.8ns)到扩充核-壳(2.9ns)到均质颗粒(3.2ns)的顺序增加(图4A)。密实核-壳的寿命比游离染料小，尽管对该寿命的主要贡献来自快组分(fast component)(图4A)。仅由该寿命数据，可能认为染料在密实核-壳颗粒中淬灭，但稳态测量结果表明并非如此。荧光寿命和量子效率结合起来，使得可以对辐射率和非辐射率常数进行唯一确定，其以归一化和绝对形式(absolute form)列于表III中。在不同结构中，辐射率的提高是恒定的，其比游离TRITC大2.2倍。然而，在不同结构中，非辐射率可从密实核-壳纳米颗粒的较高非辐射率数值，变化至几乎为其三分之一的均质颗粒非辐射率值。
颗粒的旋转各向异性也显示为复杂的、多指数的行为(图4B、C)，目前，我们将本发明限定于平均旋转时间常数(表III，θ)。如期望的，颗粒内部染料的旋转被二氧化硅基质阻碍，导致其旋转时间比游离染料更长(图4B)。事实上，与香豆素对照品(请注意，此时也存在游离TRITC的一些残留的各向异性)相比，所述各向异性曲线在测量的时标(timescale)上不衰减到零。但无论如何，即使在纳米颗粒的紧密包裹的核内部，仍然存在显著的旋转迁移率(rotation mobility)，。如果旋转的时标太快，会导致30nm纳米的纳米颗粒无法完成整体的旋转，但太慢又会由于能量转移形成去极化。而且，曲线(r(θ))的振幅(amplitude)接近双光子各向异性(r(θ)＝0.57)的理论振幅，表明不存在通常与能量迁移相联系的的快速去极化。
因此，各向异性的衰减很可能是由于二氧化硅基质内染料分子的旋转受阻。每种结构的有标度的各向异性曲线如图4C所示。密实核-壳纳米颗粒具有最快、阻碍最小的旋转(图4C)，其次是扩充核-壳颗粒(图4C)，最后是均质纳米颗粒(图4C)。该旋转时间与非辐射率常数单调负相关(表III)，表明在二氧化硅纳米颗粒中受限的迁移率是造成非辐射衰减抑制的原因。
纳米颗粒合成原料
无水乙醇(Aldrich)、四氢呋喃(Aldrich)、氨水(Fluka，28％)、四乙氧基硅烷(Aldrich，98％)、3-氨基丙基三乙氧基硅烷(Aldrich，99％)、3-巯基丙基三乙氧基硅烷(Gelest，99％)、四甲基若丹明-5-(和-6-)-异硫氰酸酯*混合异构体*(TRITC)(MolecularProbes，88％)、Alexa 
Figure A200810170588D00101
488 C5Meleimide(Molecular Probes，97％)、Alexa 
Figure A200810170588D00102
488羧酸和琥珀酰亚胺酯(Molecular Probes，≥50％)。
核(荧光种子)纳米颗粒的通用制备方法
用量筒量取水、氨水和溶剂。在1L锥形瓶内进行荧光种子颗粒的合成，用
Figure A200810170588D00103
涂敷的磁性搅拌棒(stir bar)以大约600rpm的速度搅拌。向乙醇中加入去离子水和氨水溶液并搅拌。将溶于含425微摩尔APTS的乙醇或THF中的2mL活性染料前体加入反应容器中。根据所需结构，将得到的混合物在室温下搅拌1到12小时，搅拌时将反应容器用铝箔覆盖使曝光最小化，以获得荧光种子颗粒混合物。在氮气保护下将四氢呋喃(THF)和无水乙醇(EtOH)蒸出。然后，将有机染料从大约-20℃的存储温度移入室温，然后置于手套箱中。
核(荧光种子)颗粒上二氧化硅壳体的通用制备方法
二氧化硅壳包覆和生长步骤在上述荧光种子颗粒反应混合物中进行，向上述混合物中规则地加入溶剂，例如乙醇、甲醇或异丙醇，以防止在加入二氧化硅形成单体四乙氧基硅烷(TEOS)时溶液离子强度的剧烈变化。这一方法阻止了合成过程中的颗粒聚集，所述聚集会使颗粒尺寸分布变宽。
荧光纳米颗粒的表征
得到的荧光纳米颗粒的颗粒尺寸和颗粒尺寸分布通过电子显微镜(SEM)和荧光相干光谱(FCS)进行表征。
荧光相关光谱
所有FCS测量都使用基于双光子激发的、定制的FCS显微镜进行。仪器由脉宽为100fs、重复频率为80MHz的掺钛蓝宝石振荡器组成，所述振荡器由氩离子激光器(Spectra Physics，Palo Alto，CA)振动(pumped)。光束用与Zeiss Axiovert 35反向显微镜耦合的Bio-RadMRC600共焦扫描盒(Hercules，CA)定位。用Zeiss 63x C-Apochromat水浸物镜(N.A.＝1.2)聚焦激发光，并且收集发射也是通过相同的物镜。荧光用双色670DCLP从激发(光)中分离，并且通过HQ575/150发射滤光器到GaAsP光子计数PMT。得到的光电流与ALV 6010多τ自相关器数字自相关。
自相关函数G(τ)定义为：
G ( &tau; ) = < &PartialD; F ( 0 ) &PartialD; F ( &tau; ) > < F ( &tau; ) > 2 - - - ( 1 )
其中F(τ)是在延时τ时的体积(volume)获得的荧光值。括号表示系综平均，且δF(τ)＝F(τ)-<F(τ)>。拟合函数是用于单组分、三维扩散的标准函数：
G ( &tau; ) = 1 N { 1 + 4 D&tau; w xy 2 } - 1 { 1 + 4 D&tau; w z 2 } - 1 / 2 - - - ( 2 )
其中wxy与wz分别是双光子焦点体积(two-photo focal volume)横向和轴向尺寸；N是焦点体积内扩散种类(species)数；D是扩散系数。所有FCS测量在900nm激发波长下进行。在此波长时，确定横向尺寸为0.248微米，轴向尺寸为0.640微米。浓度标定由若丹明绿的标准样品确定为1.43nM/颗粒(即N＝1对应1.43nM)。
光漂白-对于光漂白实验，持续照亮在路径长度为3mm的石英比色管(Starna)内的30μL样品。激发为来自功率为5W、光点大小为1.5cm的氩激光器的514nm线激光(laser line)。样品制备为激发波长为514nm时具有相似的吸收度。
通过HQ605/90发射滤光器，于垂直于激发的角度收集荧光，并且通过双碱(bialkali)PMT(HC125-02，Hamamatsu)记录。
以下实施例仅用于说明目的而非限制本发明。
实施例1
表I  纳米颗粒和纳米颗粒核的流体动力学半径
 
纳米颗粒 核半径(nm)(SD)       核/壳半径(nm)(SD)         
密实核/壳 2.2(0.3) 15(1.2)
扩充核/壳均质      2.9(0.3)- 17(1.4)15(1.1)
在21℃时计算得到的流体动力学半径列于表I中。每种纳米颗粒曲线与单一扩散系数拟合，并且流体动力学尺寸在测量误差(r＝15,17nm)范围内相等。对于核中间体，扩充核(r＝2.9nm)稍大于密实核(r＝2.2nm)。
实施例2
表II纳米颗粒的亮度
 
样品 TRITC等价物 QE增加 亮度因子 计数/颗粒
TRITC 1.0 1.0 1.0 1.0
密实核/壳 8.6 2.0 18. 14.
扩充核/壳均质      8.72.3 3.13.3 27.07.5  22.06.0 
自相关的振幅提供了扩散种类(species)数，并且平均计数率是从光学定义(optically defined)焦点体积收集的光子的度量。由此数据，可以得到各扩散种类的每分子计数率，其为探针亮度的直接度量。
相对于游离染料的颗粒亮度的总提高值等于纳米颗粒内TRITC等价物的数目与染料的相对量子效率提高值，即表II所示亮度因子的乘积。然而，考虑到对TRITC等价物测定的不确定性，有必要进行附加的、独立的亮度度量以作比较，例如由FCS(表II，计数/颗粒)测得的每颗粒计数。亮度因子与由FCS(表II，比较最后两列)得到的独立亮度值相似，且在两种方法中，不同结构的亮度趋势相同。亮度因子始终高估用FCS测得的值，可能是由于染料等价物确定中的误差。
总的来说，量子效率的提高可能是由于辐射率(kr)的增加、非辐射率(knr)的减小或由于两种原因并存。这些因素的相对贡献与荧光寿命(τf)和量子效率(φ)以下列关系相关：
&tau; f = 1 k r + k nr - - - ( 1 )
&phi; = k r k r + k nr - - - ( 2 )
实施例3
表III  二氧化硅纳米颗粒的时间分辨参数
 
样品 <寿命>ns     φ krns-1 kr(归一化) knrns-1 knr(归一化) <θ>ns  
TRITC 2.1 0.15 0.072 1.0 0.41 1.0 0.21
密实核/壳 1.8 0.30 0.17 2.3 0.39 0.95 0.40
扩充核/壳 2.9 0.47 0.16 2.2 0.18 0.45 3.2
均质 3.2 0.50 0.16 2.2 0.16 0.39 6.7
所有前述出版物、专利和专利文献通过引用方式纳入本申请，如同通过引用单独纳入本申请一样。虽然通过引用多种具体和优选的实施方案和技术对本发明进行描述，但是，应该理解的是，可对其做多种变化和改进而仍处于本发明的主旨和范围之内。

Claims (13)

1.一种荧光单分散纳米颗粒，包括有机核和二氧化硅壳，其中核-壳的化学性质、组成及结构提供与游离染料和核相比增强的光物理性能。
2.权利要求1的荧光单分散纳米颗粒，其特征在于，所述增强的光物理性能包括增强的亮度、荧光量子效率、光稳定性或其组合。
3.权利要求1的荧光单分散纳米颗粒，其特征在于，所述核包括被二氧化硅壳体包裹的密实核。
4.权利要求1的荧光单分散纳米颗粒，其特征在于，所述核包括被二氧化硅壳体包裹的扩充核。
5.权利要求1的荧光单分散纳米颗粒，其特征在于，所述核包括被二氧化硅壳包裹的、染料稀疏地包埋其中的均质颗粒。
6.权利要求5的荧光单分散纳米颗粒，其特征在于，所述均质颗粒不被二氧化硅壳包裹。
7.权利要求1的荧光纳米颗粒，其特征在于，所述密实核具有小于2.5nm的半径。
8.权利要求1的荧光纳米颗粒，其特征在于，所述扩充核具有大于2.5nm的半径。
9.权利要求1的荧光纳米颗粒，其特征在于，所述核-壳化学性质、组成以及结构提供控制纳米颗粒的光稳定性的能力。
10.一种制备具有密实核结构的荧光单分散纳米颗粒的方法，包括：
将一种荧光化合物和一种有机硅烷化合物混合制得染料前体；
将得到的染料前体与水溶液混合制得密实荧光核；以及
将得到的密实核与二氧化硅前体混合在密实核上制得二氧化硅壳体，以提供荧光单分散纳米颗粒。
11.一种制备具有扩充核结构的荧光单分散纳米颗粒的方法，包括：
将一种荧光化合物和一种有机硅烷化合物混合制得染料前体；
将得到的染料前体与二氧化硅前体共缩合制得扩充荧光核；以及
将得到的扩充核与二氧化硅前体混合形成扩充核上的二氧化硅壳体，以提供荧光单分散纳米颗粒。
12.一种制备荧光单分散均质纳米颗粒的方法，包括：
将一种荧光化合物和一种有机硅烷化合物混合制得染料前体；以及，
将得到的染料前体与二氧化硅共缩合制得均质荧光单分散纳米颗粒。
13.权利要求12的方法，其特征在于，得到的均质纳米颗粒与二氧化硅共缩合在均质纳米颗粒上形成二氧化硅壳体，以提供荧光单分散纳米颗粒。
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