CN112098649A - 一种检测肝癌患者血清生物标志物的信号控释纳米材料及其制备方法 - Google Patents
一种检测肝癌患者血清生物标志物的信号控释纳米材料及其制备方法 Download PDFInfo
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Abstract
本发明涉及一种检测肝癌患者血清生物标志物的信号控释纳米材料及其制备方法。甲胎蛋白AFP与多种肿瘤的发生、发展密切相关，临床上主要作为肝癌的血清标志物，用于原发性肝癌的诊断和疗效监测。因此，甲胎蛋白的高灵敏检测对于肝癌患者的早期诊断与治疗具有重要意义。本发明提出了一种新型的基于甲胎蛋白与其一抗和二抗的无酶免疫检测的信号控释纳米材料及其制备方法，并将其用于人血清中甲胎蛋白的检测，不但克服了传统方法依赖过氧化物酶产生信号及信号放大的困难，而且显著提高了检测灵敏度、简化了检测过程、降低了成本、拓宽了应用领域，解决了酶联免疫检测技术复杂、价格居高、灵敏度不足等难题，在肿瘤早期诊断及治疗等领域具有广阔的应用前景，为无酶免疫检测提供高灵敏度、高特异性的新方法。
Description
技术领域
本发明涉及肝癌患者血清生物标志物的检测，具体涉及一种检测肿瘤患者血清生物标志物AFP的方法，属于光电化学生物传感分析及生物医学临床诊断领域。
背景技术
甲胎蛋白(AFP)是一种糖蛋白，是重要的肿瘤标志物。正常情况下，这种蛋白主要来自胚胎的肝细胞，胎儿出生约两周后甲胎蛋白从血液中消失，因此正常人血清中甲胎蛋白的水平低于20ng/mL，而在肝癌患者血清中显著升高，成为肝癌的特异性标志物。除此之外，甲胎蛋白还与多种肿瘤的发生、发展密切相关，睾丸癌、卵巢肿瘤、恶性畸胎瘤、胰腺癌、胃癌、肠癌、肺癌等多种肿瘤中均可表现出较高浓度，可作为多种肿瘤的阳性检测指标。临床上主要作为肝癌的血清标志物，用于原发性肝癌的诊断和疗效监测。因此，AFP的灵敏检测对于肝癌患者的早期临床诊断和治疗具有重要意义。
到目前为止，报道了多种检测AFP的方法，例如酶联免疫测定法(ELISA)，免疫放射测定法(IRMA)、酶标电泳法、电化学免疫传感等检测方法，由于步骤繁琐、耗时、成本高且缺乏灵敏度和选择性而限制了这些方法的实际应用。荧光检测技术因其具有显著的优点，包括高灵敏度，优异的选择性，良好的重现性和稳定性，快速的响应，简单的仪器和低成本等优点，使其得到了广泛研究并被开发应用。因此，为了克服传统技术的缺点和不足，迫切需要开发出既简单、灵敏又特异、准确的荧光检测方法用于满足生物医疗领域对肝癌等多种恶性肿瘤的早期诊断及早期治疗的需求。
本发明根据抗原AFP与其一抗和二抗之间能够发生特异性结合反应形成免疫夹心结构的特性，利用聚多巴胺(Polydopamine,PDA)和空心纳米金构建的信号控释纳米材料，实现了对AFP简单、高效地检测。迄今为止，利用聚多巴胺与空心纳米金构建基于AFP与其一抗和二抗的无酶免疫检测的信号控释纳米材料及其制备方法和应用还未见文献报道。
发明内容
为了克服现有技术的不足，针对利用PDA与空心纳米金构建基于AFP与其一抗和二抗的无酶免疫检测的信号控释纳米材料及其制备方法和应用的文献还未见报道，因此，本发明的第一目的：提出一种新型的利用PDA与空心纳米金构建基于AFP与其一抗和二抗的无酶免疫检测的信号控释纳米材料，具体地说，本发明利用多巴胺在特定条件下发生氧化自聚，在空心纳米金表面形成PDA，使纳米材料被含有大量官能团的PDA包裹，再通过迈克尔加成反应与胺端检测抗体共价连接形成二抗复合物，最终获得用于检测AFP的信号控释纳米材料。为了实现对AFP高灵敏的荧光检测，本发明在空心纳米金内部载带大量荧光分子，通过氧化自聚合反应在空心纳米金表面生成具有非凡表面活性的PDA“纳米衣”，形成荧光纳米胶囊。当目标抗原AFP出现时，被固定于孔板内的捕获抗体捕获并与检测抗体构建的二抗复合物结合从而形成免疫夹心结构。将pH调至2.0，二抗复合物中纳米胶囊表面的聚合物PDA发生降解，导致内部的荧光分子RhB得到释放，产生荧光信号，通过检测增强的荧光信号实现对AFP的检测。本发明的显著优点是，克服了传统方法依赖过氧化物酶产生信号及信号放大的困难，最大程度地提高了检测灵敏度、简化了检测过程、降低了检测成本、拓宽了应用领域，解决了酶链免疫检测技术复杂、价格居高、灵敏度不足等难题，在肿瘤早期诊断及治疗等领域具有潜在的应用价值；本发明的第二目的：提出一种新型的利用PDA与空心纳米金构建基于AFP与其一抗和二抗的无酶免疫检测的信号控释纳米材料的制备方法；本发明的第三目的：提供一种新型的利用PDA与空心纳米金构建基于AFP与其一抗和二抗的无酶免疫检测的信号控释纳米材料用于检测AFP的方法。
本发明是通过以下技术方案实现发明目的的。本发明提出了一种新型的利用PDA与空心纳米金构建基于AFP与其一抗和二抗的无酶免疫检测的信号控释纳米材料，具体地说，本发明利用多巴胺在特定条件下发生氧化自聚，在空心纳米金表面形成PDA，使纳米材料被含有大量官能团的PDA包裹，再通过迈克尔加成反应与胺端检测抗体共价连接形成二抗复合物，最终获得用于检测AFP的信号控释纳米材料。当目标抗原AFP出现时，被固定于孔板内的捕获抗体捕获并与检测抗体构建的二抗复合物结合从而形成免疫夹心结构。将pH调至2.0，二抗复合物中纳米胶囊表面的聚合物PDA发生降解，导致内部的荧光分子RhB得到释放，产生荧光信号，通过检测增强的荧光信号实现对AFP的检测。
利用PDA构建信号控释纳米材料的优点在于该物质是一种具有多种优良理化性质的黏合性聚合物，在光学性质、粘附性质、生物相容性等方面性能优异，被广泛用于各种材料的功能化。本发明在空心纳米金表面通过氧化自聚合反应生成的PDA，因其自身的黏附性将空心纳米金包裹，进而形成“纳米衣”，使原本带负电的纳米金负电增强，强静电排斥作用使PDA包裹的纳米金表现出极强的稳定性，不但有效抑制了荧光分子的释放，而且使形成的纳米胶囊具有PDA的特性，除了-OH、-NH2官能团以外还含有活泼的双键，可与多个基团发生化学反应，如氨基(-NH2)、巯基(-SH)等。因此，本发明将氨基修饰的AFP二抗分子共价连接于纳米胶囊表面，有如在PDA“纳米衣”上安装了天线，可以探测到抗原AFP并与其结合。通过这种方式成功构建了基于抗原-抗体免疫反应的信号控释纳米材料，无需加入传统方法采用的辣根过氧化物酶等任何生物酶，就可以实现对AFP的高灵敏检测。
本发明基于抗原-抗体无酶免疫检测机理，不但实现了无酶条件下对于AFP的特异性识别和响应，释放出大量的信号分子从而获得显著增强的荧光信号，而且由于信号控释纳米材料的特性，当形成免疫夹心结构后，通过控制pH值降解PDA纳米衣，使纳米胶囊内大量的荧光分子被释放，产生增强的荧光信号。由于采用了信号控释纳米材料，即便只有微量的AFP，也能够通过特异性的免疫反应形成夹心结构，而每个夹心结构仅通过简单的降解，就可释放出纳米胶囊内大量的信号分子，因而获得信号的放大。在该体系中，荧光信号的增强放大不需要外加生物酶进行催化，也不需要外加传统方法采用的辣根过氧化物酶与底物反应产生信号，而仅通过控制pH值降解免疫夹心结构中的纳米衣，就能释放出大量的信号分子，从而获得超高的检测灵敏度，为AFP的高灵敏检测提供了特异、高效的信号放大检测技术。
一种本发明提出的新型的利用PDA与空心纳米金构建基于AFP与其一抗和二抗的无酶免疫检测的信号控释纳米材料的制备方法，包括如下步骤：
(1)在1mL空心纳米金悬浮液中加入5μL浓度为1.0×10-5mol/L的RhB溶液，37℃摇床反应12h；
(2)离心分离，除去上清液，溶解在1mL 10mM的PBS缓冲液中；
(3)在1mL0.05 mg/mL的多巴胺Tris-HCl溶液中加入上步已制备好的溶液，室温反应6h；
(4)离心分离，加入100μL浓度为1mg/mL的二抗悬浮液，室温孵育30min，置于4℃冰箱过夜；
(5)离心收集沉淀并用PBS溶液洗涤后获得信号控释纳米材料，将其分散于400μL含1.0％BSA和0.1％NaN3的PBS溶液中。
本发明的有益效果：本发明提出的信号控释纳米材料，即便只有微量的靶标AFP，也能够通过特异性的免疫反应形成夹心结构，而每个夹心结构仅通过简单的降解，就可释放出大量的信号分子，使检测信号被显著放大，因而可获得高灵敏的检测。
本发明的显著优点：荧光信号的增强放大不需要任何生物酶进行催化，也不需要外加传统方法采用的辣根过氧化物酶与底物反应产生信号，而仅通过控制pH值的简单方式，即可降解免疫夹心结构中的纳米衣，导致大量的信号分子被释放出来，从而获得超高的检测灵敏度，为AFP的高灵敏检测提供了特异、高效的信号放大检测技术。结果表明，本发明不但克服了传统方法依赖过氧化物酶产生信号及信号放大的困难，而且最大程度地提高了检测灵敏度、简化了检测过程、降低了检测成本、节约了试剂、拓宽了应用领域，解决了酶联免疫检测技术复杂、价格居高、灵敏度不足等难题，为肿瘤早期诊断及治疗提供无酶免疫检测新技术和新方法。
本发明提出的基于无酶免疫检测AFP的信号控释纳米材料具有高效、灵敏、易制备、稳定、生物相容性好、适用范围广等优异性能，也不会受到干扰物质如IgG、CEA和BSA的影响，表现出优异的特异性和选择性。
实验结果表明，采用本发明提出的信号控释纳米材料检测AFP，不但显示了远高于文献值的灵敏度和优异的选择性，所获检测限低至0.047pg/mL(S/N＝3)，而且，还获得了更宽的线性检测范围0.0001-25ng/mL，能够很好地满足人血清常量及微量样品的高灵敏检测，其线性方程和相关系数分别为F/F0＝1.52+1.20×CAFP(ng/mL)和0.9931，F为荧光强度，F0为荧光空白值。可以预见，本发明提出的基于无酶免疫检测AFP的信号控释纳米材料及其制备方法和应用在肿瘤的早期诊断及治疗等领域具有广阔的应用前景，为无酶免疫检测提供更灵敏和特异的新方法。
附图说明
图1.信号控释纳米材料对AFP的选择性分析，mix为IgG、CEA、BSA与AFP的混合物。
具体实施方式
以下是本发明涉及的具体实施例，对本发明的技术方案做进一步描述，但是本发明的保护范围并不限于这些实施例。凡是不背离本发明构思的改变或等同替代均包括在本发明的保护范围之内。
下面通过实施例具体说明本发明，但本发明不受下述实施例的限定。
实验仪器：THZ-82A气浴恒温振荡器(金坛市医疗器械厂)；DF-101S集热式恒温加热磁力搅拌器(天津市工兴电器厂)；TDL-40B 4000转/分离心机(上海安亭科学仪器厂)；TGL-16B 10000转/分离心机(上海安亭科学仪器厂)；F-4600荧光分光光度计(日立，日本)。
实验试剂：盐酸多巴胺(Dopamine hydrochloride，探索平台)；罗丹明B(上海阿拉丁生化科技股份有限公司)；乙烯吡咯烷酮K-30(PVP，中国国药化学公司)；AFP抗原(青岛云山生物科技有限公司)；AFP捕获抗体(Ab1，青岛云山生物科技有限公司)；AFP检测抗体(Ab2，青岛云山生物科技有限公司)；人甲胎蛋白(AFP)检测试剂盒(HumanAFP ELISAKit)；pH＝7.4、10mM的PBS缓冲溶液；pH＝8.5、50mM的Tris-HCl缓冲溶液；肝癌患者筛查的血清样品收集于青岛市第八人民医院；二次水等，所用试剂均为分析纯。
实施例1：
一种制备本发明提出的新型的利用PDA与空心纳米金构建基于AFP与其一抗和二抗的无酶免疫检测的信号控释纳米材料的制备方法，包括如下步骤：
(1)在1mL空心纳米金悬浮液中加入5μL浓度为1.0×10-5mol/L的RhB溶液，37℃摇床反应12h；
(2)离心分离，除去上清液，溶解在1mL 10mM的PBS缓冲液中；
(3)在1mL 0.05mg/mL的多巴胺Tris-HCl溶液中加入上步已制备好的溶液，室温反应6h；
(4)离心分离，加入100μL浓度为1mg/mL的二抗悬浮液，室温孵育30min，置于4℃冰箱过夜；
(5)离心收集沉淀并用PBS溶液洗涤后获得信号控释纳米材料，将其分散于400μL含1.0％BSA和0.1％NaN3的PBS溶液中；
所述的空心纳米金按文献方法获得(J.Y.Chen,M.X.Yang,Y.N.Xia,et.al.Adv.Funct.Mater.,2010,20:3684-3694)。
实施例2:
一种采用本发明提出的利用PDA与空心纳米金构建基于AFP与其一抗和二抗的无酶免疫检测的信号控释纳米材料用于检测人血清样品中AFP的方法，包括如下步骤：
(1)在包被一抗的孔板内，室温下用封闭缓冲液温育2h，洗涤孔板；
(2)加入50μL从医院获得的人血清样品的稀释液，37℃孵育1h；
(3)洗涤孔板，加入300μL实施例1中制备的信号控释纳米材料溶液，37℃孵育1h；
(4)洗涤孔板，用0.1M的HCl将pH调至2.0，放置4h后，取上清液进行荧光检测，激发波长553nm，狭缝宽度为5.0nm。
其中，检测样品采用青岛市第八人民医院获取的3组人血清样品按照1：10倍稀释后进行检测并与酶联免疫试剂盒检测结果进行对照，实验结果表明，本发明方法测得人血清样品中AFP的浓度分别为0.72、1.98和4.99ng/mL，与试剂盒的检测结果相符。由此可见，本实验所采用的无酶免疫信号放大检测方法能够高效、特异、灵敏地用于人血清实际样品的检测，不但克服了传统方法依赖过氧化物酶产生信号及信号放大的困难，而且最大程度地提高了检测灵敏度、简化了检测过程、降低了检测成本、节约了试剂、拓宽了应用领域，解决了酶联免疫检测技术复杂、价格居高、灵敏度不足等难题，在肿瘤早期诊断及治疗等领域具有广阔的应用前景，为无酶免疫检测提供更灵敏和特异的新方法。
Claims (7)
1.一种用于检测肿瘤标志物AFP的信号控释纳米材料，其特征在于：该材料基于AFP与其一抗和二抗的无酶免疫检测策略，由纳米载体、信号分子、纳米衣和检测抗体构建而成。
2.一种如权利要求1所述的用于检测肿瘤标志物AFP的信号控释纳米材料，其特征在于：所述的信号控释纳米材料用于检测肝癌患者血清中的AFP。
3.一种如权利要求1所述的用于检测肿瘤标志物AFP的信号控释纳米材料，其特征在于：所述的纳米载体是空心纳米金。
4.一种如权利要求1所述的用于检测肿瘤标志物AFP的信号控释纳米材料，其特征在于：所述的纳米衣是利用多巴胺在特定条件下发生氧化自聚，在空心纳米金表面形成聚多巴胺外壳。
5.一种如权利要求1所述的用于检测肿瘤标志物AFP的信号控释纳米材料，其特征在于：所述的检测抗体是氨基修饰的，通过加成反应将其与所述的纳米载体表面的纳米衣共价连接形成二抗复合物，最终获得用于检测AFP的信号控释纳米材料。
6.一种如权利要求1所述的用于检测肿瘤标志物AFP的信号控释纳米材料，其特征在于：所述的信号分子是罗丹明B。
7.一种如权利要求1所述的用于检测肿瘤标志物AFP的信号控释纳米材料的制备方法，其特征在于步骤如下：
(1)在空心纳米金悬浮液中加入5μL浓度为1.0×10-5mol/L的RhB溶液，37℃摇床反应12h；
(2)离心分离，除去上清液，溶解在1mL PBS缓冲溶液中；
(3)在1mL 0.05mg/mL的多巴胺的Tris-HCl溶液中加入上步已制备好的溶液，室温反应6h；
(4)离心分离，加入100μL二抗悬浮液，室温孵育30min，置于4℃冰箱过夜；
(5)离心收集沉淀并用PBS溶液洗涤后获得信号控释纳米材料，将其分散于400μL含1.0％BSA和0.1％NaN3的PBS溶液中。
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