CN101198672B - 氧化硅基光致发光感应物及使用方法 - Google Patents

氧化硅基光致发光感应物及使用方法 Download PDF

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CN101198672B
CN101198672B CN2006800217500A CN200680021750A CN101198672B CN 101198672 B CN101198672 B CN 101198672B CN 2006800217500 A CN2006800217500 A CN 2006800217500A CN 200680021750 A CN200680021750 A CN 200680021750A CN 101198672 B CN101198672 B CN 101198672B
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安德鲁·A·伯恩斯
埃里克·赫茨
塔拉·C·泽戴科
乌尔里希·魏斯纳
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Abstract

本发明涉及包含氧化硅基的核及至少一种光致发光染料的感应颗粒。该氧化硅基的核可包含多个孔，而该至少一种光致发光染料可包括对其环境和分析物不敏感的参比染料以及对所述环境或分析物敏感的感应染料。该感应颗粒可用来在体内或体外感测生物或非生物系统中未知环境状态或分析物。

Description

氧化硅基光致发光感应物及使用方法
技术领域
本发明涉及氧化硅基颗粒，并且更具体地涉及含有适于用作感测剂(sensing agent)的光致发光物质的氧化硅基颗粒。
背景技术
近年来，基于光致发光成像的技术基本朝向高分辨率、实时、分子水平分析的方向发展，以直观化并获知生命及其过程。基于光致发光的分析的魅力源于其优异的对比度、高特异性以及快的响应时间。
尽管大多数光致发光染料被设计成对其环境不敏感，但各种具有特异亲和性并响应于化学分析物或环境刺激物的染料已经被合成出来。例如，已经将某些类型的光致发光染料游离地用于溶液中作为响应化学分析物或环境刺激物的感应物。
虽然敏感和不敏感的光致发光化合物通常都是游离地用于溶液中，但是在一些情况下，这些染料已被结合到感应平台(platform)如光学纤维、薄膜及膜上。感应平台的组分以及光致发光染料的特性，除了确定观察其感应能力的未知状态的类型外，还会影响染料的性能。
需要一种环境或分析物的感应物，其可以最优化光致发光染料的可变的性能并且在多种环境中感应未知状态。
发明内容
本发明涉及用于检测各种环境状况和分析物的改进的感应颗粒(sensor particle)。本发明还涉及在感测应用中使用这些颗粒的方法。
简要的说，本发明的感应颗粒包括核和至少一种光致发光染料。
在一些具体实施方式中，核是氧化硅基纳米颗粒，而至少一种光致发光染料包括参比染料和感应染料，其中至少之一可以与核共价连接。参比染料可以位于核内而感应染料可位于核的至少一部分表面上。可替换地，参比染料和感应染料可位于核表面或在核的外围。而且，氧化硅基颗粒可包括围绕核的氧化硅基的壳，其中参比染料位于核内并且氧化硅基的壳介于参比染料和感应染料之间。在其它具体实施方式中，参比染料和感应染料之一的每个感应颗粒包括在约1.0至1000.0个之间的染料分子，并且参比染料和感应染料响应于单波长或多波长激发源而发射光子。参比染料和感应染料响应适当激发的峰值发射波长通常被足够的间隙所分开，从而逐个地辨别它们的发射峰。
在其它具体实施方式中，核包括多个孔，其中至少一个孔具有在约0.1nm至约100.0nm之间的直径，并且更具体地在约2.0nm至约50.0nm之间。氧化硅基颗粒的直径可在约1.0nm至约250.0nm之间。
本发明的方法包括使用感应颗粒来感应未知的环境状况或分析物。
在一种具体实施方式中，该方法包括以下步骤：提供至少一种含有两种或多种光致发光染料的氧化硅基颗粒，将该至少一种氧化硅基颗粒引入到包括未知状态或分析物的环境中，将该至少一种氧化硅基颗粒暴露于激发光子，以使该两种或多种光致发光染料中的每一个发射光子，记录由该两种或多种光致发光染料发射的光子，以及从这些数据来确定未知的环境状态或分析物。该方法可以进一步包括在存在已知的环境状态或分析物的情况下用光子激发该两种或多种光致发光染料，并比较在已知的环境状态或分析物的存在下得到的该两种或多种光致发光染料的发射波。在这些情况下，在已知的环境状态或分析物存在下得到的发射可用来产生对照数据，其与已知的状态或分析物是相关的。因此，测定步骤可包括使用这些对照数据来确定未知状态或分析物。确定步骤还可以包括同时探寻(interrogation)其(即，多种感应染料)发射峰可去卷积(deconvolve)、以获得多个未知状态或分析物的状况的多种感应染料。
在另一个具体实施方式中，一种测定生物体中未知状态的方法包括以下步骤：提供含有能发射光子的感应染料的氧化硅基纳米颗粒，将该氧化硅基纳米颗粒插入到生物体中，用光子激发该感应染料，测定感应染料的光致发光发射，以及测定生物体的未知状态。感应染料可在红外或近红外波长处发射光子。氧化硅基纳米颗粒可放置在生物体的细胞、组织、血液或其它体液中。
附图说明
通过附图来描述要求保护的本发明的一些具体实施方式。应该理解，没有必要绘制的图形及对理解本发明没有必要的细节或其他难以理解的细节都被忽略了。当然，应该理解，本发明不必限制于这里所描述的特定具体实施方式。
图1A是本发明氧化硅基颗粒的一个具体实施方式的局部剖面透视图；
图1B是本发明氧化硅基颗粒的另一个具体实施方式的局部剖开的透视图；
图1C是本发明氧化硅基颗粒的又一个具体实施方式的局部剖开的透视图；
图1D是本发明氧化硅基颗粒的又一个具体实施方式的局部剖开的透视图。
图2A是图1A的氧化硅基颗粒的扫描电镜图像；
图2B是图1B的氧化硅基颗粒的扫描电镜图像；
图2C是图1C的氧化硅基颗粒的扫描电镜图像；
图3是在已知的pH下使用本发明的氧化硅基颗粒的光致发光强度对峰值波长的曲线图，以及
图4是校准曲线，其基于在图3中收集的数据，绘出了参比和感应染料与pH相关的峰值波长发射的比率。
具体实施方式
本发明的氧化硅基颗粒通常是纳米颗粒并包含氧化硅基的核以及两种或多种光致发光染料。如这里使用的，术语核是指中心或基本部分。用于形成氧化硅基基体(matrix)的氧化硅可具有通式R4-nSiXn，其中X可以是可水解基团，如乙氧基、甲氧基或2-甲氧基-乙氧基，R可以是有机基团(例如甲基或乙基)并且n是1至4的整数。两种或多种光致发光染料包括参比染料和感应染料。如这里使用的，术语参比染料是指这样的染料，其对环境和/或任何存在于其中的分析物基本不敏感或当在不同环境下暴露于激发光子时呈现出相同或基本相同光子发射。术语感应染料是指任何对其环境和/或分析物敏感的或有响应的，或在不同环境或分析物下暴露于光子时呈现出不同的光子发射的染料。在一些具体实施方式中，参比染料和感应染料可以与氧化硅基基质共价连接。在其它具体实施方式中，染料可以在无共价键的情况下通过物理捕获而限制在氧化硅基基体中或被吸附在基质的表面上。
在一种具体实施方式中，将参比和感应染料与氧化硅基基体结合在一起可增强染料的亮度并阻止光致褪色(即，染料保持明亮的时间延长)。在这些具体实施方式中，本发明的氧化硅基颗粒与溶液中的游离染料相比表现出增强的亮度水平，例如通过光致发光相关光谱测量的。
在另一个具体实施方式中，氧化硅基基体包括孔或缝隙，使得液体或分析物可以渗入到氧化硅基基体中，并且直接与位于其中的感应染料相互作用。以这种方式，感应能力可以增强。孔通常包括在约0.1nm至约100.0nm之间的直径。孔大小可表现出不同的分布，大小范围在2.0nm至约50nm及以上。
在另一个具体实施方式中，氧化硅基基体的使用可以用单激发波长激发两种或多种染料。对于具有可区分开的发射峰的参比和感应染料，改变每一颗粒中每种染料的分子数量可使通常需要多个激发波长的染料结合物响应于单激发波长。例如，如果参比染料为了发射比感应染料需要更长的激发波长，那么附加的参比染料分子可与氧化硅基基体结合来增强其相对吸收，使得这两种染料可测量地响应于相同的激发波长。氧化硅基基体每个颗粒可以结合约1.0个至约1000.0个染料分子，通常在2.0个至100.0个染料分子。
在又一个具体实施方式中，氧化硅基基体可包括多于一种的感应染料用于感应多于一种的环境状态或分析物。假定被引入的染料的发射波长是足够分开的从而可分辨不同的发射峰，约一种至约七种，并且更具体地约两种至五种不同的感应染料可与氧化硅基基体结合用于不同的感应应用。
本发明的氧化硅基颗粒也可引入添加物。可以出于各种目的在颗粒表面设置超顺磁性材料的核，如纳米尺寸的氧化铁或其它磁性合金或氧化物和/或一种或多种大量的官能团。例如，颗粒可与抗体或治疗剂结合以用于确定和治疗疾病的状态或症状。治疗剂可以被吸收到氧化硅基基体的孔中或涂在颗粒的表面上。
最终氧化硅基颗粒的表面可以用各种表面修饰剂，包括胺、抗体、微生物、病毒、酶、受体、半抗原、激素、或其组合加以功能化。氨基的使用，如氨丙基和甲基氨丙基基团，产生可吸引细胞表面负电荷的正表面电荷。烷基的使用，如甲基或十二烷基链，产生无电荷表面，使颗粒可存在于疏水环境中-如在细胞膜里。而且，可以用功能性的连接于氧化硅的基团与多种物质反应，包括聚合物例如：聚乙二醇(PEG)、交联剂以及其它表面修饰剂如羧酸、酯类或生物素，其可用于将其它部分(基团)连接到颗粒表面，包括肽、DNA、抗体以及抗生物素蛋白/链霉亲和素。表面修饰剂可包括围绕颗粒的层，其对某些分析物是选择性可渗透的。该层介于感应染料与环境之间。
用合适的表面改性剂功能化颗粒的表面使得颗粒可在多目标环境中使用。例如，疏水涂料的加入使颗粒可分散在疏水的环境里。按照这样的思路，合适的表面改性剂的选择可导致优先的相互作用，定义为特别需要的相互作用的可能性相对增大。实例包括使用至少一种对细胞表面抗原具有亲和性的抗体使颗粒表面功能化，和/或使用含有生物素和抗生物素蛋白/链霉亲和素的涂料，其优选在目标环境中与生物素相互作用。
如上所述，该两种或多种光致发光染料可包括参比染料和感应染料。参比染料和感应染料的发射峰值波长通常被足够的波长间隙分开从而在同一波长下激发时能单独地分辨它们的峰。
参比染料通常被螯合(sequester)在氧化硅基颗粒内部，一般在其核中。尽管参比染料对其环境和分析物是基本不敏感的，但是将参比染料螯合在颗粒内部可作为预防措施来阻止在确实存在敏感性时与目标环境或分析物的相互作用。合适的参比染料包括，但不限制于，如下：
参比染料名称    λex/λem(n  结合方法 发射
分子探针AlexaFluor 350     350/445  n-羟基琥珀酰亚胺酯(NHS酯) 近UV
分子探针Pacific Blue     416/451  n-羟基琥珀酰亚胺酯 可见光
分子探针AlexaFluor 488     488/520  n-羟基琥珀酰亚胺酯 可见光
分子探针AlexaFluor 532     530/555  n-羟基琥珀酰亚胺酯 可见光
罗丹明B异硫氰酸酯     540/573  异硫氰酸酯 可见光
四甲基罗丹明异硫氰酸酯     550/580  异硫氰酸酯 可见光
分子探针AlexaFluor 568     580/602  n-羟基琥珀酰亚胺酯 可见光
Dyomics DY 610     609/629  n-羟基琥珀酰亚胺酯 可见光
Dyomics DY 615     621/641  n-羟基琥珀酰亚胺酯 可见光
分子探针AlexaFluor 647     651/672  n-羟基琥珀酰亚胺酯 可见光
Dyomics DY 675     674/699  n-羟基琥珀酰亚胺酯 近IR
Dyomics DY 700     702/723  n-羟基琥珀酰亚胺酯 近IR
Dyomics DY 731     736/759  n-羟基琥珀酰亚胺酯 近IR
Dyomics DY 776     771/801  n-羟基琥珀酰亚胺酯 近IR
Sigama Aldrich NIR 797     795/835  异硫氰酸酯 近IR
Dyomics DY 485 XL     488/560  n-羟基琥珀酰亚胺酯 MegastokesT
Dyomics DY 510 XL     488/630  n-羟基琥珀酰亚胺酯 MegastokesT
所谓的MegastokesTM染料在这里定义为其激发和发射波长之间表现出的差值在约30.0nm到约200.0nm之间的染料。
感应染料一般放置在氧化硅基颗粒的表面上或附近，用来增强其与环境和/或任何分析物的相互作用。在这种结构中，感应染料更易于与需要研究的环境状态或分析物直接接触。感应染料的发射经受环境或分析物刺激。例如，依赖于环境的pH，pH感应染料基于感应染料电子状态的变化，通过光子的增加或减少，表现出变化的发射光谱。金属离子的存在通过淬灭(quenching)该感应染料而引起发射的减少。
下表是许多适合的多种感应染料以及其对环境或分析物敏感性的非详尽列表。
感应染料名称   λex/λem(nm) 敏感度 结合方法 发射
荧光异硫氰酸酯   485/500 pH(pKa＝6.4) 氨基结合 可见光
β-5-羧基荧光素-二-(5-羧甲氧基-2-硝基苄基)醚-丙氨酸-甲酰胺，琥珀酰亚胺酯   490/520 被笼住的荧光团+pH 氨基结合 可见光
氟-4碘乙酰胺   494/516 Ca2+ 巯基连接 可见光
5-羧基-2’，7’-二氯亚砜荧光素   500/530 pH(pKa＝6.4) 氨基结合 可见光
羧基Seminaphorhodafluor-1(SNARF-1)NHS酯   510-575/545-636 光谱移位pH(pKa＝7.7) 氨基结合 可见光
奥勒冈绿514羧酸，NHS酯   511/530 pH(pKa＝4.6) 氨基结合 可见光
赤藓红B异硫氰酸酯   530/555 氧气 氨基结合 可见光
Sigama Aldrich NIR667-NHS   667/685 氨基酸 氨基结合 近IR
荧光素   485/500 pH(pKa＝6.4) 葡聚糖连接 可见光
Fluo-4   488/530 Ca2+ 葡聚糖连接 可见光
钙绿   488/535 Ca2+ 葡聚糖连接 可见光
2’，7’-二-(2-羧乙基)-(5/6)-羧基荧光素   488/535 pH(pKa＝7.0) 葡聚糖连接 可见光
Seminapthorhodafluor-1(SNARF-1)   514/560-640 光谱移位pH(pKa＝7.5) 葡聚糖连接 近IR
4’，5’-二氯-2’，7’-二甲氧基荧光素   520/550 pH(pKa＝6.4) 葡聚糖连接 可见光
X-Rhod     575/620 Ca2+ 葡聚糖连接 可见光
PBFI(光谱移位)     338/557-507 K+ 物理捕获 MegaStokes
6-甲氧基-N-(3-磺丙基)喹啉     344/443 Cl 物理捕获 近UV
Zinquin Ethyl Ester     364/385 Zn2+ 物理捕获 近UV
二氨基萘     370/440 一氧化氮 物理捕获 可见光
钙黄     435/550 Ca2+ 物理捕获 可见光
4，5-二氨基荧光素     488/530 一氧化氮 物理捕获 可见光
4-氨基-5-甲基氨基-2’，7’-二氟荧光素     488/525 一氧化氮 物理捕获 可见光
钙绿     490/540 Ca2+ 物理捕获 可见光
镁绿     490/540 Mg2+ 物理捕获 可见光
二氢罗丹明     500/536 一氧化氮 物理捕获 可见光
钙橙     540/580 Ca2+ 物理捕获 可见光
corona红     547/576 Na+ 物理捕获 可见光
Rhodzin-3     550/575 Zn2+ 物理捕获 可见光
试卤灵(Resorufin)醚(苄基、乙基、甲基等)     560/580 酶活性+pH寿命 物理捕获 可见光
磺基罗丹明氨基乙基硫醇硼     561/581 硒 物理捕获 可见光
二吡咯甲烷(BODIPY 665-676)     665/676 活性氧 物理捕获 近IR
所谓被笼住的染料(caged dye)可作为感应染料。被笼住的染料是其光致发光状态被它们的环境活化的一类光致发光染料中。换句话说，束缚染料以非-光致发光状态进入其工作环境并被改变其染料化学结构的外部事件“打开”。被笼住的染料典型通常包括将特定基团共价连接至主要的共轭环状结构。当共价连接由于环境刺激物而断裂时活化就会发生。例如，试卤灵(7-羟基-phnoxazin-3-酮)是利用可断裂的醚连接的被笼住的染料家族的母体染料(parentdye)。
染料可以以多种设置引入到氧化硅基基体中。参照图1A、图1B、图1C和图1D，示出了本发明的氧化硅基颗粒的不同结构。如图1A所示，氧化硅基颗粒包括具有氧化硅基基体及参比染料的内核10、中间氧化硅基的壳12，以及含有感应染料的外壳14。在该具体实施方式中，中间氧化硅基的壳介于参比染料与感应染料之间。中间氧化硅基的壳可独自包括氧化硅基材料而没有光致发光染料的存在，或存在最小量的光致发光染料(如按重量计少于5％)。如图1B所示，氧化硅基颗粒包括含有氧化硅基基体及参比染料的内核10、和含有感应染料的外部壳14。如图1C所示，氧化硅基颗粒包括含有氧化硅基基体的内核10、含有参比染料的第一壳16、包围第一壳并含有感应染料的第二壳14。如图1D所示，氧化硅基颗粒包括具有氧化硅基基体的内核10、含有参比染料的第一壳16、包围第一壳并具有另一个氧化硅基基体的中间壳12，以及包围该中间壳并含有感应染料的外壳14。在这些具体实施方式中，核的直径可在约2.0nm至几十微米之间，并且通常在约25.0nm至200.0nm之间。第一壳和随后的壳的厚度可在约1.0nm至约400.0nm之间，并且通常在约10.0nm至约50.0nm之间。第一壳和随后的壳可以覆盖颗粒约10.0％至约100.0％的表面积。因此，颗粒的大小可在约10.0nm至几十微米之间，并且更具体地在约10.0nm至约1.0微米之间。
存在许多用来合成本发明的氧化硅基颗粒的方法。例如，可使用溶胶-凝胶法来产生颗粒。溶胶-凝胶化学使得氧化硅基颗粒的合成可在温和的条件下进行，如室温和不使用有害化学物质。可替换地，反胶束可用作反应器用于颗粒的合成。反胶束是由两亲的表面活性分子介导的，极性水相在疏水溶剂中的稳定混悬液。
St
Figure 2006800217500_0
ber溶胶-凝胶法一般可获得含有共价连接参比和感应染料的氧化硅基颗粒。通常，本发明的氧化硅基颗粒可使用St
Figure 2006800217500_1
ber溶胶-凝胶法而制备，步骤为：将活性参比染料与共反应性有机硅烷化合物混合来形成活性光致发光有机硅烷化合物，其可以被共缩合而形成核，随后通过将核颗粒与感应染料和另外的共反应性有机/烷氧基硅烷混合而将感应染料结合到核上。
然而，溶胶-凝胶法可根据颗粒所需的结构而改变。如上所述，本发明的氧化硅基颗粒可表现出各种构造，包括具有氧化硅基基体及参比染料的内核以及含有感应染料的外壳(图1A和图1B)，具有氧化硅基基体的内核、含有参比染料的第一壳及包围第一壳并含有感应染料的外壳(1C和图1D)。
图1A中的氧化硅基颗粒的制备包括核的合成、随后的氧化硅中间壳的生长以及富含感应染料的氧化硅壳的形成。核的合成一般是三个步骤。首先，在惰性气氛下，使硅烷化合物与参比染料以硅烷∶参比染料为约50∶1的摩尔比在脱气乙醇中结合，以形成光致发光硅烷化合物。然后，将该产物(参比染料结合物)在环境条件下加入到氨和水的无水乙醇溶液中，同时用力搅拌。接着，迅速地加入氧化硅前体，如四乙氧基硅烷，从而产生最终的反应溶液，其中参比染料结合物、氧化硅前体、氨及水的浓度是1.7×10-5M、0.05M、0.2M及0.855M。使该反应进行12-18小时，得到富含染料的硅酸盐核，直径为约5.0nm。在该溶液中，缓慢地加入另外的氧化硅前体来生长氧化硅壳，所加量为约5.55×10-6M/分钟，达到参比染料结合物、氧化硅前体、氨水及水分别为17×10-5M、1.055M、0.5M及0.855M的最终反应条件。
为了制备核/壳颗粒用于与感应染料结合，将颗粒对乙醇进行一次约24小时的渗析。然后稀释渗析溶液以避免颗粒在感应染料偶联反应过程中的团聚。使用以上用于参比染料的相同方法将感应染料与硅烷结合来形成感应染料结合物，但在这种情况中摩尔比低得多(即，硅烷与感应染料的摩尔比是2∶1)，以避免在功能化(functionalization)过程中颗粒的过度氨基化。这用于两个目的，即避免颗粒的团聚及有助于防止表面的氨基抑制感应染料的感应能力。此后，感应染料结合物与氧化硅前体在乙醇中以感应染料结合物∶氧化硅前体的摩尔比为约1.2×10-5M∶0.0125M下混合。将该混合物逐滴加入到核/壳颗粒的渗析溶液中。在溶液反应后，其被对(against)乙醇渗析，它可保持稳定至少数月而不发生团聚。在图2A的扫描电镜图中示出的最终颗粒的直径为约40.0nm。
通过溶胶-凝胶法制成的核的大小及颗粒的大小是可以改变的。导致图1B中示出的颗粒结构的合成如下。如上所述，将氧化硅和参比染料结合物在浓度是2.13×10-5M的参比染料结合物、0.2M的氧化硅前体、0.625M的氨及1.45M的水的乙醇溶液中共缩合(co-condense)，以获得直径为约55.0nm的单分散核颗粒。向该混合物中逐滴加入感应染料结合物及另外的氧化硅前体，以获得终反应条件：2.13×10-5M的参比染料结合物、2.13×10-5M的感应染料结合物、0.254M的氧化硅前体、0.625M的氨及1.45M的水。这些在核和含有感应染料的外壳之间缺少中间氧化硅壳的颗粒，被离心并在乙醇中及水中各重悬浮两次，以除去过多的试剂。如图2B所示，最终颗粒的大小为约60.0至80.0nm并具有相对大的富含参比染料的核及富含感应染料的壳。
图1C所示的氧化硅颗粒结构的制备对上述过程有微小修改。氧化硅前体、氨及水分别以0.17M、1.0M、2.5M的比例在环境条件下在绝对乙醇中通过用力搅拌而结合。在溶液反应后，通过离心收集反应产物，并通过用水和乙醇的超声处理进行重悬浮。接着，将含有氧化硅的种颗粒在具有水和氨的绝对无水乙醇中再悬浮，随后逐滴加入氧化硅前体、参比结合物及乙醇的预结合溶液，以获得终反应条件：参比结合物、氧化硅前体、氨及水分别是1.16M、0.027M、0.2M和2.0M。随后反应12-18小时，将颗粒离心并反复用无水乙醇和去离子水冲洗。使用在乙醇中、在惰性气体中以及有机硅烷与感应染料的摩尔比是50.0∶1.0的条件下合成出来的感应染料结合物，实施相似的合成方法以在这些颗粒上产生染料壳。如图2C所示的最终颗粒大小为约200.0nm并含有富含氧化硅的核，其被富含参比染料的第一壳包围，该第一壳被富含感应染料的第二壳包围。在图1D中的第四颗粒的结构中，参比和感应染料层之间的中间氧化硅壳是通过在第一壳上缩聚纯氧化硅前体而引入的，用来确保对包覆的参比染料的进一步保护。
为了制备本发明的氧化硅基颗粒(使用反胶束来引入缺少合适结合化学物质的染料)，微乳液合成方案如下。首先，使用St
Figure 2006800217500_2
ber溶胶-凝胶法合成具有参比染料的氧化硅基种籽颗粒。将这些种籽颗粒通过渗析或离心及清洗而从乙醇转移到含水环境中。足够的表面涂料所需的反应浓度根据可用的表面硅烷醇基团的数量(依赖于颗粒大小与浓度)来计算。将颗粒和任何葡聚糖连接物(dextran-bound)或其它亲水染料以水溶液形式加入到反相微乳液中，其中该反相微乳液是在环己烷中，在带电荷或不带电荷的两亲表面活性剂存在下，根据需要具有或不具有共-表面活性剂下形成的。加入氨的浓溶液以催化氧化硅前体的缩聚(condensation)，其被最后加入。一种合适的表面活性剂/共表面活性剂的结合是Triton X-100
Figure 2006800217500_3
和正己醇。
厚度为约2.0至约10.0nm的涂层可以在游离染料分子或葡聚糖连接的染料的存在下有效地附着在颗粒表面，该染料在形成氧化硅基体时被捕获，因此，封住感应染料分子并产生颗粒基感应物。换句话说，除了被共价连接到氧化硅基基体上，该光致发光染料一般被物理捕获或束缚在氧化硅基基体中。通过调节反应pH和氧化硅前体的浓度，可调整氧化硅基体的孔隙率，从而使分析物与捕获的染料分子相互作用。
在本发明的氧化硅基颗粒被合成后，它们可用于感测生物系统和非生物系统。多个感测方法，包括比例计量感测、光谱移动感测及寿命感测(life-time sensing)都是可能的。
环境状态以及某些分析物的存在和浓度，可以在生物和非生物系统中被探测。可以将氧化硅基颗粒引入到细胞、细胞膜、组织、原核系统(如大肠杆菌生物被膜)、溶液、反应和水体，如河和湖中。已存在多种将氧化硅基颗粒引入到细胞中的方法，包括基于商业可用的脂囊泡法、显微注射和特异性抗体摄取(在此过程中细胞表面受体介导颗粒的胞吞)。在吞噬细胞和一些内吞细胞中，摄取可以自发地发生。根据由颗粒表现的亮度水平，仅通过将单种本发明的氧化硅基颗粒引入到目标环境中，对于测定是有利的。然而，也可使用多种氧化硅基颗粒。
通过将多种类的分析物特异的感应物引入到未知环境中，可以实现多重检测。多重性允许同时探询其发射峰可被去卷积的多种光致发光物质从而获得多个分析物的状态。这种方法将非常适合于对细胞功能刺激(如新的治疗法)的影响的高通量分析。
氧化硅基颗粒也可作为感应物用于多种生物体中，在动物细胞或体液中，包括人细胞或体液，如血液或血清中。可以收集细胞或体液并在体外测试，或全身成像仪可刺激在体内的染料并测量发射波长。体外感应一般依赖于含有可见光谱的光致发光染料的感应物。在体内测试中，可将感应物局部注射到特定区域，或者用那些表现出与某些细胞表面受体优先相互作用的抗体将感应物功能化。用于体内测试的感应染料一般表现出红外或近红外发射波长。
处于被感应中的环境状态或分析物也可以是不同的。可以测定如pH水平和疏水的或亲水状态的环境状态。例如钾、磷酸根、钠、钙、铜、镁、铬、氯、氟及铁的离子，例如镉、锌、铅、硒、汞、镍的重金属以及生物分子物质，如维生素和氨基酸的存在及浓度也可以被检测。
而且，用本发明的氧化硅基颗粒进行体外和体内测试对于确定活的生物体中细胞的氧化还原状态是有利的。细胞的氧化还原状态反映其氧化-还原势能(potential)(即，细胞失去电子(氧化)的电位(potential)与其获得电子(还原)的电位(potential)的关系)。细胞所依赖的能量主要来自于通过氧化反应(从有机分子到氧气，在细胞内部产生总体的还原环境，其被谨慎地调节)所释放的能量。外界或内部对抗正常的氧化性/还原性的平衡-氧化还原应激-的攻击已经被视为对细胞不利事件的指示，尤其是肿瘤发生，并且在疾病或受伤状态如癌症和中风中是非常重要的。
通过各种感应方法可以研究前述未知的状态或分析物。
如本文使用的，比率计量感测(ratiometric sensing)是指基于为已知环境状态并相应于与已知环境状态建立的比率来测定未知的环境状态。在已知环境或分析物状态下测定两种或多种光致发光染料的主要发射峰强度。通过计算两种染料(感应染料/参比染料)的峰发射比率而进行校准；该比率与已知状态相对应。该校准形成用于测定未知状态的基础，其通过在未知状态下对含有相同参比和感应染料颗粒的激发和发射强度的测量。
基于光谱移动的测定是以相似的原理操作。通过使用校准曲线研究未知环境或分析物状态，其中该校准曲线是基于在已知状态下参比和感应染料被激发所表现出来的最大发射波长而建立起来的。然而，不是依赖峰值发射强度的比率外，参比和感应染料的最大峰值波长(λmax)之间的差值也被用作因变量。
基于寿命的感测测定两种或多种光致发光染料的激发态寿命，它们中的一种的寿命已知对环境是不敏感的，它们中的至少一种的寿命依赖于具体的环境状态或分析物。荧光寿命(τ)是光致发光染料花费在激发态的时间长度，从开始的光子吸收直到其发射；其最经常在10ps至数纳秒的范围内。通过测量荧光寿命可以为多种分析物浓度进行校准，为进一步的浓度测定建立基础。
为了为感测建立基线，首先将一个或多个颗粒引入到具有已知状态的环境中。然后颗粒被多光子光源中的一个或多个选择的波长激发，一般表现出的激发波长在约250.0nm至约800.0nm之间，这对颗粒中的参比和感应染料的吸收分布图是适当的。多光子激发利用激光(通常是Ti-蓝宝石)，其可以被调成很多种发射波长。长波长(700.0nm到＞1500.0nm)光子不易被组织或培养基吸收，但可能会在染料分子中以非常高的光子流量(即在光束的焦点)引发多光子激发。尽管参比染料一般表现出相对恒定的波长及发射强度，但感应染料却表现出与环境刺激物或某些分析物的存在或浓度相关的发射波长。
因此，在特定水平上激发每一种参比和感应染料后，就可以测量光子的发射。测量可使用荧光分光光度法或荧光显微镜法来实施。光致发光的寿命可使用脉冲激光源和栅极探测器测量，其测量作为时间随照射的函数的发射。光致发光发射被收集在石英或塑料比色皿中，作为与激发束成90度的发射波长的函数(通过在光电倍增管或雪崩光电二极管上的光子计数)。使用荧光显微镜法，采集单独的、含有经历不同已知状态的感应物的无细胞样品的图像。在测量参比和感应染料的光子发射后，发射比率(γ＝I感应/I参比)，绘出光谱移动或光致发光寿命对于已知环境状态的曲线以建立校准曲线。也就是说，在已知环境状态或分析物存在下产生的发射被用于产生对照数据，其与已知状态相关。例如，校准曲线示出对应于已知环境状态的比率范围(如，在一个具体实施方式中，在约0.5至约0.8之间的比率可对应于在6至7之间的pH)。
在产生对照数据后这些数据可以用于确定未知的环境状态或分析物的存在及浓度。在未知状态或分析物存在下记录颗粒的发射，并且通过将其与已知状态下建立的对照数据进行比较而测定未知状态。
本发明的氧化硅基颗粒可用于各种其它应用，如公开在共同拥有共同申请的题为“荧光氧化硅基纳米颗粒(Fluorescent SilicaBased Nanoparticles)”的美国申请序列第10/306,614号中的那些。将此处公开的所用应用通过引用并入本文。
实施例
以下的实施例用于说明的目的，并不限制本发明。这些实施例并不以任何方式限制本发明或其保护范围。
实施例1(pH感测)
合成含有参比染料和感应染料，基于图1C所示的结构的200.0nm的氧化硅基颗粒，用作比率计量感应物以检测环境的pH。
合成
在惰性气氛中，使3-氨基丙基-三乙氧基硅烷(APTS-SigmaAldrich Chem.Corp.)与参比染料四甲基罗丹明(TRITC)的异硫氰酸酯基团以APTS∶TRITC＝50∶1的摩尔比在乙醇(Pharmco 200 proof，脱气)中偶联以形成TRITC-结合物。将该TRITC-结合物在环境气氛下加入到氨(2.0M乙醇-Sigma Aldrich Chem.Corp.)和水的无水乙醇溶液中，然后其被迅速地加入氧化硅前体四乙氧基硅烷(tetraethoxysilane)(TEOS-99％ Sigma Aldrich Chem.Corp.)以获得[TRITC-结合物]、[TEOS]、[NH3]和[水]分别为1.7×10-5M、0.17M、1.0M和2.5M的最终反应溶液。该反应进行12-18小时，并且通过离心收集混浊的产物并使用水和乙醇超声处理而重悬浮。
以0.787mg/ml的浓度在具有合适的水和NH3浓度的无水乙醇中重悬浮这些颗粒，并逐滴(7.5×10-7MTEOS/min)加入TEOS、TRITC-结合物及乙醇的预-结合溶液，以达到[TRITC-结合物]、[TEOS]、[NH3]和[水]分别为1.16mM、0.27M、0.2M和2.0M的最终反应条件。然后反应12-18小时，离心这些颗粒并反复用无水乙醇和去离子水冲洗。
使用在惰性气氛下以4.25mM FITC及APTS∶FITC＝50∶1的摩尔比合成的FITC结合物，实施相似的合成方法以在这些颗粒上形成荧光(FITC)壳。然后，在相似条件下将该结合物加入到以上制备的TRITC结合混合物中，对于0.787mg/ml的颗粒密度，[FITC-结合物]、[TEOS]、[NH3]和[水]的最终浓度分别为0.4mM、0.029mM、0.2M和2.0M。颗粒大小为约200.0nm(图2C)并含有相对大的富含参比染料的核及富含感应染料的壳。
校准
颗粒从去离子水中以3∶20(v/v)稀释液(dilution)稀释到在pH5至pH9之间的磷酸钠缓冲溶液。然后激发这些颗粒，其通过引入540.0nm单光子光源来激发四甲基罗丹明，以及490nm来激发荧光素。发射波长通过荧光光光谱测定法测定(用一台光子技术国际荧光光谱测定仪)；最大峰在图3中示出。较大波长峰对应于荧光感应染料，而较小的峰对应于四甲基罗丹明参比染料。然后，绘制发射波长的最大峰比率(γ＝IFITC/ITRITC)对pH的曲线以给出图4所示的校准曲线。该校准曲线提供了对于每组测试颗粒在pH范围在5-9的线性校准。
感测
可以将以上合成的氧化硅基颗粒引入到未知pH的溶液中。该颗粒可以用在校准过程中使用的同样的单光子光源、以相同的激发波长激发。然后，可以计算对于发射波长的最大峰比率(γ＝IFITC/ITRITC)并与图4中的校准曲线对比。以这种方式，可以测定溶液的pH值。
本领域技术人员可在不背离本发明精神和范围的情况下进行这里所描述的变更、变形和其它实施方式。因此，本发明不以任何方式被以上例举性说明所限制。

Claims (18)

1.一种氧化硅基颗粒，包括氧化硅基的核及两种或多种光致发光染料，所述两种或多种光致发光染料含有至少一种参比染料和至少一种感应染料。
2.根据权利要求1所述的氧化硅基颗粒，其中，所述氧化硅基颗粒是纳米颗粒。
3.根据权利要求1所述的氧化硅基颗粒，其中，所述氧化硅基颗粒具有直径在0.1nm至100.0nm之间的孔。
4.根据权利要求1所述的氧化硅基颗粒，其中，所述参比染料和感应染料中至少其一共价连接至所述氧化硅基的核。
5.根据权利要求1所述的氧化硅基颗粒，其中，所述参比染料和感应染料中至少其一具有1.0个至100.0个之间的染料分子，并且所述参比染料和感应染料响应于在单预定波长发射的单激发源而发射光子。
6.根据权利要求1所述的氧化硅基颗粒，其中，所述参比染料设置在所述核内并且所述感应染料设置在所述氧化硅基颗粒的至少一部分表面上。
7.根据权利要求1所述的氧化硅基颗粒，其中，所述参比染料和感应染料设置在所述核的面上或在所述核的外围。
8.根据权利要求1所述的氧化硅基颗粒，进一步包括围绕所述核的氧化硅基的壳，其中，所述参比染料设置在所述核内并且所述氧化硅基的壳介于所述参比染料和所述感应染料之间。
9.根据权利要求1所述的氧化硅基颗粒，其中，所述参比染料和感应染料响应于在单个预定波长发射的单激发源而发射光子。
10.根据权利要求1所述的氧化硅基颗粒，其中，所述参比染料和感应染料响应光子暴露的峰值波长是被足够的间隙分开，以致能逐个辨别所述发射峰。
11.根据权利要求1所述的氧化硅基颗粒，其中，将所述参比染料或感应染料暴露于光子激发导致红外或近红外发射。
12.根据权利要求1所述的氧化硅基颗粒，其中，所述感应染料至少对离子的存在和浓度、化学物质或生物化学分析物中的一种有响应。
13.根据权利要求1所述的氧化硅基颗粒，进一步包括表面改性剂，所述表面改性剂显示出与目标分析物优先相互作用。
14.根据权利要求1所述的氧化硅基颗粒，进一步包括对疾病状态或损伤有响应的治疗剂。
15.根据权利要求1所述的氧化硅基颗粒，其中，所述感应染料对pH有响应。
16.一种氧化硅基光致发光纳米颗粒，包括两种或多种光致发光染料及多个孔，所述两种或多种光致发光染料包含至少一种参比染料和至少一种感应染料。
17.根据权利要求16所述的氧化硅基光致发光纳米颗粒，其中，至少一个所述孔的直径在0.1nm至100.0nm之间。
18.根据权利要求16所述的氧化硅基光致发光纳米颗粒，其中，所述纳米颗粒的直径在1.0nm至250.0nm之间。
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