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
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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
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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