CN106290898B - 一种金‑上转换纳米粒子三聚体的制备方法及其应用 - Google Patents

一种金‑上转换纳米粒子三聚体的制备方法及其应用 Download PDF

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CN106290898B
CN106290898B CN201610613233.8A CN201610613233A CN106290898B CN 106290898 B CN106290898 B CN 106290898B CN 201610613233 A CN201610613233 A CN 201610613233A CN 106290898 B CN106290898 B CN 106290898B
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徐丽广
瞿爱华
胥传来
匡华
刘丽强
吴晓玲
宋珊珊
胡拥明
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Abstract

一种金‑上转换纳米粒子三聚体的制备方法及其应用，属于分析化学技术领域。本发明主要包括20nm粒径的金纳米粒子的合成、金纳米粒子上Mucin‑1适配体的修饰、上转换纳米粒子上AFP适配体的修饰、金纳米粒子上AFP和Mucin‑1适配体部分互补序列的修饰、金‑上转换纳米粒子三聚体的组装、荧光和拉曼检测；在外加AFP后，荧光会增强；而在外加Mucin‑1后，拉曼信号会减弱，从而建立荧光信号与AFP浓度、拉曼信号与Mucin‑1浓度的标准曲线。本发明提供了一种用金‑上转换纳米粒子三聚体对癌症标志物的多重超灵敏检测方法，与传统检测方法相比成本低，灵敏度高，方便快捷，具有很好的实际应用前景。

Description

一种金-上转换纳米粒子三聚体的制备方法及其应用
技术领域
本发明涉及一种金-上转换纳米粒子三聚体的制备方法及其应用，属于分析化学技术领域。
背景技术
甲胎蛋白（α-fetoprotein，AFP），是一种致癌糖蛋白，是诊断原发性肝癌的特异性肿瘤标志物，具有确立诊断、早期诊断、鉴别诊断的作用。大量的临床发现，70%-95% 的原发性肝癌患者的AFP 升高。粘蛋白-1（Mucin-1）发生在许多腺癌，包括胰腺癌、肺癌、乳腺癌、卵巢癌、结肠癌和其它组织。粘蛋白也过度表达在肺疾病，如哮喘、支气管炎、慢性阻塞性肺病或囊性纤维化。Mucin-1正诊断标记为恶性肿瘤和其他疾病过程中，通常会过度或错误表达。
传统检测癌症标志物的方法有：酶联免疫法、电化学和基于仪器的液质联用、高效液相色谱等。酶联免疫法检测肿瘤标志物时，因蛋白质容易热变性，所以很难检测蛋白类肿瘤标志物，另外酶联免疫法的灵敏度较低，而且耗力耗时；电化学对样品的基质要求较高，很难实施；而用仪器检测时成本很高，并且对操作人员的操作技能有很高的要求。
所以，本发明提出了一种新型的、简便的、灵敏的检测方法，把纳米材料作为一种新型探针，进行AFP和Mucin-1的超灵敏检测。
通过 DNA 杂交将20nm粒径的金纳米粒子和20nm粒径的上转换纳米粒子组装成三聚体结构。目标物AFP和其适配体部分互补序列修饰的上转换纳米粒子竞争结合，荧光信号增强；而目标物Mucin-1和其适配体部分互补序列修饰的金纳米粒子竞争结合，拉曼信号减弱。将荧光信号与目标物AFP的浓度建立标准曲线，拉曼信号与目标物Mucin-1的浓度建立标准曲线，以此来定量检测癌症标志物甲胎蛋白及粘蛋白-1。
发明内容
本发明的目的是提供一种金-上转换纳米粒子三聚体的制备方法及其应用，其提供的新型的、简便的、灵敏的检测方法，把纳米材料作为一种新型探针，进行AFP和Mucin-1的超灵敏检测。
本发明的技术方案，一种金-上转换纳米粒子三聚体的制备方法，具体包括金纳米粒子的合成，上转换纳米粒子的提供，金-上转换纳米粒子三聚体的组装及结构表征，荧光、拉曼信号测试，用金-上转换纳米粒子三聚体检测AFP和Mucin-1。具体步骤如下：
（1）金纳米粒子的合成：进行20nm 粒径金纳米粒子的合成；48.75mL超纯水在搅拌下加入1.25mL 4g/L的氯金酸，使其沸腾，沸腾状态保持2-3min，加入1.2mL 10mg/mL的柠檬酸钠水溶液，观察颜色变化，待颜色不变后冷却至室温，放入冰箱中待用：
（2）上转换纳米粒子的提供：由北京万德高科技发展有限公司购买；
（3）金-上转换纳米粒子三聚体的组装：取两管50mL步骤（1）制备的已浓缩好的10nM 20nm粒径金纳米粒子，分别与巯基修饰的Mucin-1的适配体及互补序列进行偶联，金纳米粒子︰巯基修饰的Mucin-1的适配体/互补序列的摩尔比为1︰3；再将50mL上转换纳米粒子与巯基修饰的AFP的适配体偶联，其中上转换纳米粒子︰巯基修饰的AFP摩尔比为1︰5；三管试剂均在室温下过夜反应，13000r/min 离心15min，弃上清，然后用50mL的TBE缓冲液分散沉淀，分别得到Au-Mucin-1适配体、Au-互补序列及UCNP- AFP适配体，待用；
将50mL Au-Mucin-1适配体、50mL Au-互补序列和50mL UCNP- AFP适配体杂交反应12h，得到组装产物金-上转换纳米粒子三聚体Au-Au-UCNP；离心15min，弃上清，后用100mL的超纯水分散，进行TEM、荧光和拉曼表征。
Mucin-1适配体：5’-GCAGTTGATC CTTTGGATAC CCTGG-SH-3’；
互补序列：5’-GATCAACTGC ACAGCACCAC AGACC-SH-3’；
AFP适配体：5’-SH-GGCAGGAAGA CAAACAGGAC CGGGTTGTGT GGGGTTTTAAGAGCGTCGCC TGTGTGTGGT CTGTGGTGCT GT-3’。
金-上转换纳米粒子三聚体的应用，用于癌症标志物的多重超灵敏检测，具体采用金-上转换纳米粒子三聚体检测甲胎蛋白AFP和粘蛋白-1Mucin-1。
（4）AFP的检测：取100mL的金-上转换纳米粒子三聚体Au-Au-UCNP，加入1mL不同浓度的甲胎蛋白（AFP），使其终浓度在0-100aM（具体为 0、1、2、5、10、20、50、100 aM），反应10min，进行荧光测试，绘制标准曲线。
（5） Mucin-1的检测：取100mL的金-上转换纳米粒子三聚体Au-Au-UCNP，加入1mL不同浓度的粘蛋白-1（Mucin-1），使其终浓度在0-10fM（具体为 0、0.01、0.05、0.1、0.5、1、5、10 fM），反应 10min，进行拉曼测试，绘制标准曲线。
（6）样品测试：从无锡市第二人民医院获取三名病人的血清样品，稀释后取1mL，加入100mL Au-Au-UCNP组装体，进行荧光和拉曼测试，根据标准曲线得出样品所含AFP及Mucin-1的浓度，并与标准浓度进行比较。
本发明的有益效果：本发明基于荧光和拉曼作为检测信号，可以分别检测两种癌症标志物甲胎蛋白AFP及粘蛋白-1Mucin-1。与传统的检测手段相比，具有灵敏度高、检测限低、方便、快捷的优点，有非常好的应用前景。
附图说明
图1金-上转换纳米粒子三聚体Au-Au-UCNP组装结构的 TEM 图。
图 2 不同浓度AFP下金-上转换纳米粒子三聚体的荧光信号。
图 3 基于金-上转换纳米粒子三聚体组装结构进行AFP检测标准曲线。
图 4 不同浓度Mucin-1下金-上转换纳米粒子三聚体的拉曼信号。
图 5 基于金-上转换纳米粒子三聚体组装结构进行Mucin-1检测标准曲线。
具体实施方式
以下实施例中的上转换纳米粒子购自北京万德高科技发展有限公司。
实施例 1 基于金-上转换纳米粒子三聚体的癌症标志物的多重超灵敏检测方法。
（1）金纳米粒子的合成：将48.75mL超纯水在搅拌下加入1.25mL 4g/L的氯金酸，使其沸腾，沸腾状态保持2-3min，加入1.2mL 10mg/mL的柠檬酸钠水溶液，观察颜色变化，待颜色不变后冷却至室温，放入冰箱中待用。
（2）上转换纳米粒子的提供：由北京万德高科技发展有限公司购买。
（3）金-上转换纳米粒子三聚体的组装：取两管50mL已浓缩好的20nm粒径金纳米粒子，分别与巯基修饰的Mucin-1的适配体及互补序列进行偶联，偶联摩尔比为1︰3；再将50mL上转换纳米粒子与巯基修饰的AFP的适配体偶联，偶联摩尔比为1︰5，三管在室温下过夜反应，13000r/min 离心15min，弃上清，然后用50 mL的TBE缓冲液分散沉淀，待用；将50 mLAu-Mucin-1适配体、50 mL Au-互补序列、50 mL UCNP- AFP适配体杂交反应12h，组装产物为金-上转换纳米粒子三聚体Au-Au-UCNP，离心15min，弃上清，后用100mL的超纯水分散，进行TEM、荧光和拉曼表征。
（4）AFP的检测：取100mL的金-上转换纳米粒子三聚体，加入1 mL 不同浓度的AFP溶液，使其终浓度范围分别为 0、1、2、5、10、20、50、100 aM，反应 10min，进行荧光测试。最低检测限达0.059aM。
（4） Mucin-1的检测：取100mL的金-上转换纳米粒子三聚体，加入1 mL不同浓度的Mucin-1溶液，使其终浓度范围分别为0、0.01、0.05、0.1、0.5、1、5、10 fM，反应 10min，进行拉曼测试。最低检测限达4.1aM。

Claims (5)

1.一种金-上转换纳米粒子三聚体的制备方法，其特征在于包括金纳米粒子的合成，上转换纳米粒子的提供，金-上转换纳米粒子三聚体的组装及结构表征，具体步骤如下：
（1）金纳米粒子的合成：进行20nm 粒径金纳米粒子的合成；48.75mL超纯水在搅拌下加入1.25mL 4g/L的氯金酸，使其沸腾，沸腾状态保持2-3min，加入1.2mL 10mg/mL的柠檬酸钠水溶液，观察颜色变化，待颜色不变后冷却至室温，放入冰箱中待用；
（2）上转换纳米粒子的提供：由北京万德高科技发展有限公司购买；
（3）金-上转换纳米粒子三聚体的组装：取两管50mL步骤（1）制备的已浓缩好的 10nM20nm粒径金纳米粒子，分别与巯基修饰的Mucin-1的适配体及互补序列进行偶联，金纳米粒子︰巯基修饰的Mucin-1的适配体的摩尔比为1︰3，金纳米粒子︰互补序列的摩尔比也为1︰3；再将50mL上转换纳米粒子与巯基修饰的AFP的适配体偶联，其中上转换纳米粒子︰巯基修饰的AFP摩尔比为1︰5；三管试剂均在室温下过夜反应，13000r/min 离心15min，弃上清，然后用50mL的TBE缓冲液分散沉淀，分别得到Au-Mucin-1适配体、Au-互补序列及UCNP- AFP适配体，待用；
将50mL Au-Mucin-1适配体、50mL Au-互补序列和50mL UCNP- AFP适配体杂交反应12h，得到组装产物金-上转换纳米粒子三聚体Au-Au-UCNP；离心15min，弃上清，后用100mL的超纯水分散，进行TEM、荧光和拉曼表征。
2.根据权利要求1所述金-上转换纳米粒子三聚体的制备方法，其特征在于：
Mucin-1适配体：5’-GCAGTTGATC CTTTGGATAC CCTGG-SH-3’；
互补序列：5’-GATCAACTGC ACAGCACCAC AGACC-SH-3’ ；
AFP适配体：5’-SH-GGCAGGAAGA CAAACAGGAC CGGGTTGTGT GGGGTTTTAA GAGCGTCGCCTGTGTGTGGT CTGTGGTGCT GT-3’。
3.用权利要求1方法制备的金-上转换纳米粒子三聚体的应用，其特征在于：用于癌症标志物的多重超灵敏检测，具体采用金-上转换纳米粒子三聚体检测甲胎蛋白AFP和粘蛋白-1Mucin-1。
4.根据权利要求3所述金-上转换纳米粒子三聚体的应用，其特征在于具体步骤如下：
（1）AFP的检测：取100mL的金-上转换纳米粒子三聚体Au-Au-UCNP，加入1mL不同浓度的甲胎蛋白AFP，使其终浓度在0-100aM，反应10min，进行荧光测试，绘制标准曲线；
（2）Mucin-1的检测：取100mL的金-上转换纳米粒子三聚体Au-Au-UCNP，加入1mL不同浓度的粘蛋白-1Mucin-1，使其终浓度在0-10fM，反应 10min，进行拉曼测试，绘制标准曲线；
（3）样品测试：取血清样品，稀释后取1mL，加入100mL Au-Au-UCNP组装体，进行荧光和拉曼测试，根据标准曲线得出样品所含AFP及Mucin-1的浓度，并与标准浓度进行比较。
5.根据权利要求4所述金-上转换纳米粒子三聚体的应用，其特征在于所述不同浓度的甲胎蛋白AFP具体为0、1、2、5、10、20、50、100aM；不同浓度的粘蛋白-1Mucin-1具体为0、0.01、0.05、0.1、0.5、1、5、10fM。
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