CN107121549A - 一种快速的检测癌胚抗原的比色分析方法 - Google Patents
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Abstract
本发明涉及医学中肿瘤标志物检测领域,具体是利用标记物构建比色免疫传感器,实现对癌胚抗原的检测。一种快速的检测癌胚抗原的比色分析方法,制备Ag3PO4/Ag纳米复合材料并将其修饰癌胚抗原抗体,制备Fe3O4/Ag纳米复合材料并将其修饰癌胚抗原抗体,将Fe3O4@Ag‑Ab1纳米球分散液依次孵育不同浓度的癌胚抗原CEA 30分钟,然后用水清洗2‑5次,再用Ab2‑Ag3PO4/Ag纳米球分散液孵育30分钟,构建成免疫传感器,加入ABS缓冲液和四甲基联苯胺TMB溶液,通过紫外分光光度计检测TMB的紫外吸收来定量CEA的浓度。
Description
技术领域
本发明涉及医学中肿瘤标志物检测领域,具体是利用标记物构建比色免疫传感器,实现对癌胚抗原的检测。
背景技术
肿瘤标记物是肿瘤细胞产生的,含量远高于正常细胞的特异性物质(W. Chen, R.Zheng, P. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X. Yu, J. He, Ca.Cancer J. Clin. 66 (2016) 115–132.)。癌胚抗原是目前公认的一种肿瘤标志物,它的临床检测在评估病变范围、预测疗效、监测复发等方面具有较高参考价值,检测 CEA 有助于提高肿瘤患者的诊断率,监测肿瘤早期转移,并可为患者根治性手术后是否进行辅助化疗、疗效判断、复发转移等具有一定的指导意义,对患者的病情预后判断有着至关重要的临床意义。基于抗原-抗体特异性反应的免疫分析法是检测CEA最为常用的方法,最近几年,多种多样的免疫分析技术被开发利用,如光电免疫分析,电化学免疫分析,ELISA,表面加强拉曼散射免疫分析,比色免疫分析(A. Hlaváček, Z. Farka, M. Hübner, V. Horňáková, D.Němeček, R. Niessner, P. Skládal, D. Knopp, H. Gorris, Anal. Chem. 88 (2016)6011–6017;Z. Yang, Y. Cao, J. Li, M. Lu, Z. Jiang, X. Hu, ACS Appl. Mater.Interfaces 8 (2016) 12031–12038;N. Zhang, Y. Ruan, Z. Ma, W. Zhao, J. Xu, H.Chen, Biosens. Bioelectron. 85 (2016) 294–299;X. Cai, S. Weng, R. Guo, L.Lin, W. Chen, Z. Zheng, Z. Huang, X. Lin, Biosens. Bioelectron. 81 (2016)173–180.)等等。其中比色分析方法是一种灵敏度和准确度均很高的分析方法,只需裸眼观察就可进行半定量分析,结合紫外可见分光光度计能实现微量甚至痕量检测。由于其价格低廉、设备小型化、操作简便等优点,比色分析方法被广泛应用于环境、医学、食品检测等领域,已成为检测抗生素的有力方法。
传统的比色分析方法往往需要生物酶和催化底物的参与,但是生物酶的结构容易发生变化、在生物体内含量很低、储存条件比较苛刻等因素大大限制了其实际应用。由于纳米材料模拟酶对酸、碱、温度具有较好的稳定性且催化活性较高,已成为生命分析化学等相关领域的研究热点之一。纳米材料模拟酶在比色传感、生物传感、降解环境污染物、电化学传感等方面已显示出诱人的应用前景(Dutta, S., Ray, C., Mallick, S., Sarkar, S.,Sahoo, R., Negishi, Y., Pal, T., 2015. J. Phys. Chem. C. 119, 23790-23800.Qin, W., Su, L., Yang, C., Ma, Y., Zhang, H., Chen, X., 2014. J. Agric. Food.Chem. 62, 5827-5834. Zhao, H., Dong, Y., Jiang, P., Wang, G., Zhang, J.,2015. ACS Appl. Mater. Interfaces. 7, 6451-6461.)。从实际应用的角度考虑,探寻具有高催化活性的、稳定的、可重复利用的模拟酶纳米材料尤为重要。
尽管如此,模拟酶纳米材料在比色分析方法中的应用也需要过氧化氢的参与,为了减小添加底物带来的误差,近几年,一些光催化纳米材料也被发现具有氧化TMB显色的活性,且不需要过氧化氢的参与,如石墨烯,TiO2, AgI等(Wang, G., Xu, X., Qiu, L.,Dong, Y., Li, Z., Zhang, C., 2014a. ACS Appl. Mater. Interfaces. 6, 6434−6442. Wang, G., Xu, X., Wu, X., Cao, G., Dong, Y., Li, Z. J., 2014b. J. Phys.Chem. C. 118, 28109-28117. Wang, G., Jin, L., Dong, Y., Wu, X., Li, Z., 2015.Biosens. Bioelectron. 64, 523-529.)。本文选择制备Ag3PO4/Ag复合纳米材料,并用其作为标记物构建比色免疫传感器,用于CEA的检测。纳米材料的制备方法简单、快速、成本低,以此构建的比色免疫传感器具有较好的选择性、稳定性和重现性。
发明内容
本发明所要解决的技术问题是:如何提供一种快速检测癌胚抗原的比色免疫传感分析方法,实现对癌胚抗原的检测,并具有较低的检测限,且能用于实际血清样品的检测。
本发明所采用的技术方案是:一种快速的检测癌胚抗原的比色分析方法,按照如下的步骤进行
步骤一、制备Ag3PO4/Ag纳米复合材料并将其修饰癌胚抗原抗体,将硝酸银和乙二醇溶液混合均匀形成第一溶液,将Na2HPO4·12H2O 和乙二醇溶液混合均匀形成第二溶液,将第一溶液和第二溶液混合均匀后搅拌1-3小时形成第三溶液,第三溶液在160°C 油浴条件下搅拌30分钟,通过离心和清洗,得到Ag3PO4/Ag 纳米球分散液,将CEA抗体加入到Ag3PO4/Ag纳米球分散液中,在4 °C下孵育8-12小时,磁性分离并清洗得到抗体标记的Ag3PO4/Ag 结合物即Ab2-Ag3PO4/Ag纳米球分散液;
步骤二、制备Fe3O4/Ag纳米复合材料并将其修饰癌胚抗原抗体,将四氧化三铁和多巴胺加入到二次水中混合均匀,再将硝酸银加入到该混合溶液中,搅拌5-7小时,通过磁性分离得到沉淀物,经清洗后得到Fe3O4@Ag 纳米球分散液,将CEA抗体加入到Fe3O4@Ag 纳米球分散液中,在4 °C下孵育8-12小时,磁性分离并清洗得到抗体标记的Fe3O4@Ag 结合物即Fe3O4@Ag-Ab1纳米球分散液;
步骤三、将Fe3O4@Ag-Ab1纳米球分散液依次孵育不同浓度的癌胚抗原CEA 30分钟,然后用水清洗2-5次,再用Ab2-Ag3PO4/Ag纳米球分散液孵育30分钟,构建成免疫传感器,加入ABS缓冲液和四甲基联苯胺TMB溶液,通过紫外分光光度计检测TMB的紫外吸收来定量CEA的浓度。
作为一种优选方式:步骤一中,第一溶液中硝酸银的量为1-1.8a毫克,乙二醇的量为20-36a毫升,浓度为0.1毫摩尔/升;第二溶液中Na2HPO4·12H2O的量为20-36a毫升,质量分数为0.5 %,乙二醇的量为20-36a毫升,浓度为0.1毫摩尔/升;CEA抗体的量为20-36a毫升,浓度为0.1毫摩尔/升;步骤二中四氧化三铁的量为1-1.8a毫克;多巴胺的量为15-27a毫克;二次水的量为8-10a毫升;硝酸银的量为11-20a毫克;CEA抗体的量为500-900a微升,浓度为1毫克/毫升;a为正整数。
作为一种优选方式:步骤三中,依次孵育不同浓度的癌胚抗原CEA 30分钟是指依次在浓度为2.5纳克/毫升、2纳克/毫升、1.5纳克/毫升、1纳克/毫升、0.8纳克/毫升、0.5纳克/毫升、0.2纳克/毫升的癌胚抗原CEA中在室温下孵育30 分钟。
作为一种优选方式:步骤三中,Fe3O4@Ag-Ab1的量为1-1.8b毫升,Ab2-Ag3PO4/Ag的量为1-1.8b毫升;ABS缓冲液的量为2-3.6b毫升,pH为3;TMB的量为100-180b微升,浓度为5毫摩尔/升,b为正整数。
作为一种优选方式:通过紫外分光光度计检测TMB的紫外吸收来定量CEA的浓度是指,根据CEA浓度与吸光度成线性相关性建立对应的线性方程为y=0.297 x + 0.176,x是CEA的浓度,单位是纳克/毫升,y是检测的吸光度。
本发明的有益效果是:本发明方法利用Ag3PO4/Ag氧化TMB显色,构建比色免疫传感器对癌胚抗原进行检测。此比色免疫分析方法具有较好的灵敏度,并且检测速度快,准确率高,检测癌胚抗原有较好的专一性。
附图说明
图1是Ag3PO4/Ag复合物透射电子显微镜(TEM)图;
图2是Fe3O4@Ag复合物透射电子显微镜(TEM)图;
图3是比色免疫分析方法的构建示意图;
图4是癌胚抗原的浓度与TMB吸光度的线性图。
具体实施方式
一种快速的检测癌胚抗原的比色分析方法,按照如下的步骤进行
步骤一、配置第一溶液:将1-1.8毫克硝酸银和20-36毫升乙二醇溶液混合均匀,配置第二溶液,将20-36毫升浓度为0.1毫摩尔/升的Na2HPO4·12H2O 和20-36毫升乙二醇溶液混合均匀,将第一溶液和第二溶液混合均匀搅拌2小时形成第三溶液,第三溶液在160°C 油浴条件下搅拌30分钟,通过离心和清洗,得到Ag3PO4/Ag 纳米球分散液,其透射电镜如图1所示。将500-900微升,浓度为1毫克/毫升的CEA抗体加入到该Ag3PO4/Ag 纳米球分散液中,在4 °C下孵育过夜,最后磁性分离清洗得到抗体标记的Ag3PO4/Ag 结合物(记为Ab2-Ag3PO4/Ag)。
步骤二、将1-1.8毫克四氧化三铁和15-27毫克多巴胺加入到8-10毫升的二次水中混合均匀,再将11-20毫克硝酸银加入到该混合溶液中,搅拌6个小时,通过磁性分离得到沉淀物,经清洗后得到Fe3O4@Ag 纳米球,其透射电镜图如图2所示。进一步,将500-900微升,浓度为1毫克/毫升的CEA抗体加入到该Fe3O4@Ag 纳米球分散液中,在4 °C下孵育过夜,最后磁性分离清洗得到抗体标记的Fe3O4@Ag 结合物(记为Fe3O4@Ag-Ab1)。
利用Ag3PO4/Ag和Fe3O4/Ag复合物构建比色免疫分析方法检测癌胚抗原的方法,示意图如图3所示,将1-1.8a毫升Fe3O4@Ag-Ab1依次孵育2.5纳克/毫升、2纳克/毫升、1.5纳克/毫升、1纳克/毫升、0.8纳克/毫升、0.5纳克/毫升、0.2纳克/毫升的癌胚抗原(CEA)30分钟,然后用二次水清洗2-5次,进一步孵育1-1.8毫升Ab2-Ag3PO4/Ag 30分钟,构建成免疫传感器,随后加入2-3.6毫升,pH为3的ABS缓冲液和100-180a微升,浓度为5毫摩尔/升四甲基联苯胺(TMB)溶液,通过紫外分光光度计检测TMB的紫外吸收来定量CEA的浓度。
通过紫外分光光度计检测TMB的紫外吸收来定量CEA的浓度是指,将发生反应后的TMB溶液在200-800波长范围内进行紫外光谱扫描,当CEA的浓度在0纳克/毫升时,观察到空白缓冲液的紫外吸收峰为0.210,当CEA的浓度在0.2纳克/毫升时,得到的紫外吸收峰开始大于0.210,CEA的浓度检测范围是2.5纳克/毫升到0.2纳克/毫升,如图4,在此范围内,CEA的浓度与紫外吸收成线性相关性,其线性方程为y = 0.297 x + 0.176其中,x是CEA的浓度,单位是纳克/毫升,y是紫外吸光度。其最低检测限为0.03纳克/毫升(信噪比为3),与其它检测方法相比,构建的比色免疫分析方法具有较低的检测限和较宽的检测范围。
CEA浓度与吸光度的对应关系如下表所示:
实际样品分析
用构建的比色免疫分析方法检测血清中的癌胚抗原,检测结果与商业化的酶联免疫分析法检测结果相对比,两组结果相对比,通过t验证法计算,得到的t值都小于tcrit(4.30),证明该免疫分析方法可以用来检测实际血清样品中的癌胚抗原的浓度。
比色方法结果和酶联免疫分析法结果如下表所示:
专一性分析
将制得的比色免疫传感器分别在空白缓冲溶液、10纳克/毫升的不同干扰物质(Ca2+,Mg2+, Zn2+,Fe2+,H2O2,葡萄糖)的缓冲溶液中孵育30分钟后,用二次水充分洗涤,然后检测,构建的比色传感器与上述六种干扰物质作用后,测得的吸光度值与空白组比较(0.210)相差不大(< 2%)。相反,当构建的比色传感器与1纳克/毫升的CEA作用时,吸光度值变化显著,为0.513。说明基于Ag3PO4/Ag复合物构建的比色免疫分析方法对检测CEA有较好的专一性。
Claims (5)
1.一种快速的检测癌胚抗原的比色分析方法,其特征在于:按照如下的步骤进行
步骤一、制备Ag3PO4/Ag纳米复合材料并将其修饰癌胚抗原抗体,将硝酸银和乙二醇溶液混合均匀形成第一溶液,将Na2HPO4·12H2O 和乙二醇溶液混合均匀形成第二溶液,将第一溶液和第二溶液混合均匀后搅拌1-3小时形成第三溶液,第三溶液在160°C 油浴条件下搅拌30分钟,通过离心和清洗,得到Ag3PO4/Ag 纳米球分散液,将CEA抗体加入到Ag3PO4/Ag纳米球分散液中,在4 °C下孵育8-12小时,磁性分离并清洗得到抗体标记的Ag3PO4/Ag 结合物即Ab2-Ag3PO4/Ag纳米球分散液;
步骤二、制备Fe3O4/Ag纳米复合材料并将其修饰癌胚抗原抗体,将四氧化三铁和多巴胺加入到二次水中混合均匀,再将硝酸银加入到该混合溶液中,搅拌5-7小时,通过磁性分离得到沉淀物,经清洗后得到Fe3O4@Ag 纳米球分散液,将CEA抗体加入到Fe3O4@Ag 纳米球分散液中,在4 °C下孵育8-12小时,磁性分离并清洗得到抗体标记的Fe3O4@Ag 结合物即Fe3O4@Ag-Ab1纳米球分散液;
步骤三、将Fe3O4@Ag-Ab1纳米球分散液依次孵育不同浓度的癌胚抗原CEA 30分钟,然后用水清洗2-5次,再用Ab2-Ag3PO4/Ag纳米球分散液孵育30分钟,构建成免疫传感器,加入ABS缓冲液和四甲基联苯胺TMB溶液,通过紫外分光光度计检测TMB的紫外吸收来定量CEA的浓度。
2.根据权利要求1所述的一种快速的检测癌胚抗原的比色分析方法,其特征在于:步骤一中,第一溶液中硝酸银的量为1-1.8a毫克,乙二醇的量为20-36a毫升,浓度为0.1毫摩尔/升;第二溶液中Na2HPO4·12H2O的量为20-36a毫升,质量分数为0.5 %,乙二醇的量为20-36a毫升,浓度为0.1毫摩尔/升;CEA抗体的量为20-36a毫升,浓度为0.1毫摩尔/升;步骤二中四氧化三铁的量为1-1.8a毫克;多巴胺的量为15-27a毫克;二次水的量为8-10a毫升;硝酸银的量为11-20a毫克;CEA抗体的量为500-900a微升,浓度为1毫克/毫升;a为正整数。
3.根据权利要求1所述的一种快速的检测癌胚抗原的比色分析方法,其特征在于:步骤三中,依次孵育不同浓度的癌胚抗原CEA 30分钟是指依次在浓度为2.5纳克/毫升、2纳克/毫升、1.5纳克/毫升、1纳克/毫升、0.8纳克/毫升、0.5纳克/毫升、0.2纳克/毫升的癌胚抗原CEA中在室温下孵育30 分钟。
4.根据权利要求1所述的一种快速的检测癌胚抗原的比色分析方法,其特征在于:步骤三中,Fe3O4@Ag-Ab1的量为1-1.8b毫升,Ab2-Ag3PO4/Ag的量为1-1.8b毫升;ABS缓冲液的量为2-3.6b毫升,pH为3;TMB的量为100-180b微升,浓度为5毫摩尔/升,b为正整数。
5.根据权利要求1所述的一种快速的检测癌胚抗原的比色分析方法,其特征在于:通过紫外分光光度计检测TMB的紫外吸收来定量CEA的浓度是指,根据CEA浓度与吸光度成线性相关性建立对应的线性方程为y=0.297 x + 0.176,x是CEA的浓度,单位是纳克/毫升,y是检测的吸光度。
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CN109342420A (zh) * | 2018-12-07 | 2019-02-15 | 上海工程技术大学 | Fe3O4@C一维纳米线的应用 |
CN109459569A (zh) * | 2018-07-28 | 2019-03-12 | 南昌大学 | 一种免血样肿瘤早期监测的方法 |
CN110133266A (zh) * | 2019-05-22 | 2019-08-16 | 太原理工大学 | 癌胚抗体和聚苯胺@金的结合物制备及其构建光热免疫传感器检测癌胚抗原的方法 |
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CN110133266A (zh) * | 2019-05-22 | 2019-08-16 | 太原理工大学 | 癌胚抗体和聚苯胺@金的结合物制备及其构建光热免疫传感器检测癌胚抗原的方法 |
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