基于无机纳米材料构建电流型甲胎蛋白免疫传感器的研究
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摘要
甲胎蛋白(a-fetoprotein, AFP)是肝癌细胞表达的高特异性蛋白质,很多肝癌病人(70～80%)在发病期间都有AFP基因高表达的特征。传统的医学理论认为AFP基因的高表达是肝癌发生过程中的一个伴随现象,是肝癌发生的特异性肿瘤标志物。新的研究结果表明细胞外的AFP能与其受体结合,介导生长信息的传递；细胞内的AFP不仅能抑制凋亡信号的转导,而且也能促进生长信号的传递,这是AFP具有抗凋亡和促进增殖功能的新发现,该研究结果赋予AFP新功能的认识和寻找到治疗肝癌的新靶点。因此,开发新型的AFP检测方法具有重要的理论意义和现实的应用价值。
电化学免疫传感器是将电分析技术与特异性免疫反应相结合,用于监测抗原-抗体反应的生物传感器。电流型免疫传感器是电化学传感器中技术最为成熟的一种。与其他电化学免疫传感技术相比这一方法无需分离、洗涤,可实现简便、快速免疫分析。本文利用无机纳米材料表面官能团密度大、吸附能力强、达到吸附平衡的时间短等诸多优点,结合金纳米粒子制备纳米复合材料并用于构建电流型甲胎蛋白免疫传感器。该免疫分析方法较传统测定AFP的方法,更简便、更快速。
本论文主要研究以下几个方面：
1.基于纳米金与牛血清白蛋白-二氧化钛复合物固定甲胎蛋白免疫传感器的研究
二氧化钛纳米粒子与常规材料相比具诸多优点,如：无毒,优良的电学性能,良好的热稳定性,不强的吸湿性,以及良好的成膜性等。本文将二氧化钛纳米粒子溶于氨基化溶剂牛血清白蛋白中制得牛血清白蛋白-二氧化钛纳米复合物。实验结果表明,利用该纳米复合物修饰金电极能够大量吸附生物亲和性好、比表面积大的纳米金颗粒,并通过纳米金成功固定甲胎蛋白抗体。利用此法研制的甲胎蛋白电流型免疫传感器对甲胎蛋白抗原有很好的电流响应,在最优实验条什下,其检测范围为0.01～80.0 ng/mL,检出限为0.003 ng/mL,实现了对甲胎蛋白抗原的定量分析。该免疫传感器制作简单、成本节约、环保、灵敏性高。
2.纳米金和磁性纳米复合物构建无试剂电流型免疫传感器的研究
四氧化三铁纳米粒子是一种典型的磁性纳米材料,具有易分离、易富集、易提取的优点,常用于制备生物感器。本文首先制备了具有易于磁性分离、硫堇(Thionine,Thi)包覆的四氧化三铁(Fe304)纳米复合物。通过静电吸附和共价键合作用,将Nafion、硫堇包覆的Fe3O4复合纳米粒子层层修饰到玻碳电极表面,最后利用纳米金吸附甲胎蛋白抗体(anti-AFP),从而制得了灵敏度高、稳定性好的无试剂电流型甲胎蛋白免疫传感器。实验通过透射电子显微镜(TEM)对该复合纳米粒子进行了表征,并用循环伏安法考察了电极的电化学特性。实验结果表明,Fe304/硫堇复合纳米粒子修饰的电极在实验过程中呈现出良好的氧化还原活性,省去了在测试底液中加入电活性物质的步骤。与类似方法相比,该传感器引入的四氧化三铁纳米粒子能显著降低检测下限。并且利用Fe304顺磁性,还能达到多次回收再生,既节约成本又环保。
3.基于碳纳米管/L-半胱氨酸/Fe3O4@ Au纳米复合材料的电流型甲胎蛋白免疫传感器的研究
将DMF(N，,N-二甲基甲酰胺)分散的多壁碳纳米管(MWNTs)修饰在金电极表面,再将修饰电极依次沉积纳米金、L-半胱氨酸,并通过半胱氨酸中的巯基吸附Fe3O4@Au纳米复合材料,最后固载甲胎蛋白抗体(anti-AFP),以牛血清白蛋白(BSA)封闭非特异性吸附位点,成功构建了高灵敏、稳定的新型电流型甲胎蛋白免疫传感器。实验通过扫描透射电子显微镜(TEM)对DMF-MWNTs和Fe3O4@Au复合纳米粒子进行了表征。与传统使用纳米金颗粒固定抗体的方法相比,Fe3O4@Au复合纳米粒子能显著增加甲胎蛋白抗体的吸附量,提高免疫传感器的灵敏度。同时,该传感器的制备方法不需要标记生物分子,也无需竞争或者夹心反应,只需在传感器表面进行常规的免疫反应,测量其电流响应信号即可。在优化的实验条件下,该免疫传感器对甲胎蛋白抗原的检测范围为0.1-150 ng/mL,检出限为0.03 ng/mL。该免疫传感器选择性好、稳定。
The expression of alpha-fetoprotein (a-fetoprotein, AFP) in hepatoma cells is a high specific protein. Many HCC patients (70～80%) in the incubation period have the characteristics of high AFP gene expression. The traditional medical theory holds that high expression of AFP gene is a process of liver cancer associated with the epiphenomenon, and is deemed as a liver cancer-specific tumor marker. It is reported that the AFP extracellular bind to its receptor and mediate the growth of information transfer. Cells within the AFP can not only inhibite the apoptosis signal transduction, but also promote the transmission of growth signals, which display new discoveries of anti-apoptosis and promoting proliferation for AFP, This study have endowed AFP new cognition of functionalization and new target for treating liver cancer.Therefore, the development of new detection methods for detection of AFP is of important theoretical and practical application value.
Electrochemical immunosensor is based on the combination of electroanalysis technology and specific immunoreaction. Among various electrochemical immunosensors, the amperometric immunosensor is the most sophisticated because it, avoids separation, washing, realizes easily, and achieves rapid immunoassay compared with other electrochemical immunoassay sensing technology. In our work, we successfully construct amperometric AFP immunosensor by utilizing inorganic nanomaterial for their advantages such as the adsorption ability, the surface with functional group density, short time to reach equilibrium.The prepared immunosensor exhibitmuch easier process and higher sensitivity compared with the traditional method for determination of AFP.
The main works and conclusions are as follows:
1. An Immunosensor for AFP Antigen Based on Gold Nanoparticles and Titanic Oxide-BSA Composite.
Titanic oxide nanoparticles own many advantages compared to conventional materials, such as, non-toxic, excellent electrical properties, good thermal stability, weak moisture absorption and good film-formation. A highly sensitive amperometric immunosensor for the determination of AFP is developed based on the composite of bovine serum albumin (BSA) and TiO2(BSA/TiO2),which is employed to link gold nanoparticles (nano-Au) for the immobilization of anti-AFP. The results show that the immunosensor presents highly sensitivity, good selectivity and a broad liner response from 0.01～80 ng/mL with a detection limit of 0.003 ng/mL.
2. Study on the self assembly reagentless amperometric immunosensor based on gold nanoparticles and magnetic nanocomposite.
Fe3O4 nanoparticles, a typical magnetic nano-materials, are proved to be easy to separate,be enriched,and extract the advantages of bio-sensors commonly prepared. A new reagentless amperometric immunosensor was successfully fabricated based on theα-fetoprotein antibody (anti-AFP) molecule coupled at Fe3O4@Thionine and gold nanoparticles. Firstly, magnetic nanoparticles and Fe3O4@Thionine Nanoparticles were prepared. Then, Fe3O4@Thionine nanoparticles was assembled on nafion modified glassy carbon electrode via electrostatic adsorption and covalent bonding. Finally, gold nanoparticles were absorbed on the electrode surface for the immobilization of anti-AFP. The proposed immunosensor possessed high sensitivity and good stability. Transmission electron microscopy (TEM) was used to investigate the nanoparticles composite and stepwise fabrication process of the immunosensor is characterized by the cyclic voltammetrys. The results show that, Fe3O4@Thionine modified immunosensor showed a good redox activity, avoiding the adding og electroactive materialsn. Compared with tranditional methods, the proposed nanomaterialsbased immunoassay displays the lower detection limit and the higher sensitivity.
3. An Immunosensor for AFP Antigen Based on MCNTS/L-Cysteine and Au@Fe3O4 Nanocomposite.
DMF dispersed MWNTS was firstly modified on electrode surface, which was in further eletrodeposited a nano-Au layer for immobilization of L-cysteine. Subsequently,Fe3O4@Au nanocompositeswere absorbed on the modified electrode surface by the SH-Au bond for increasing the specific surface area and further immobilizing anti-AFP. Finally, bovine serum albumin (BSA) was used to block the non-specific adsorption sites of the immunosensor to obtain a highly sensitive and stable immunosensor. Experiments by transmission electron microscopy (TEM) are carried out to characterize the prepared MWNTs and Fe3O4@ Au composite nanoparticles. Compared with the immunosensor based on the gold nanoparticles to immobilizethe antibodies, the proposed method can significantly increase the adsorption capacity of alpha-fetoprotein antibody to improve the sensitivity of immunosensor. Meanwhile, the sensor does not require the preparation of labeled biological molecules.avoid the competition or sandwich reaction and make the experiment easier. Under optimal conditions, the sensor has a good response to AFP. in the detection range of 0.1～150 ng/mL, and detection limit of 0.03 ng/mL.
电化学免疫传感器是将电分析技术与特异性免疫反应相结合,用于监测抗原-抗体反应的生物传感器。电流型免疫传感器是电化学传感器中技术最为成熟的一种。与其他电化学免疫传感技术相比这一方法无需分离、洗涤,可实现简便、快速免疫分析。本文利用无机纳米材料表面官能团密度大、吸附能力强、达到吸附平衡的时间短等诸多优点,结合金纳米粒子制备纳米复合材料并用于构建电流型甲胎蛋白免疫传感器。该免疫分析方法较传统测定AFP的方法,更简便、更快速。
本论文主要研究以下几个方面：
1.基于纳米金与牛血清白蛋白-二氧化钛复合物固定甲胎蛋白免疫传感器的研究
二氧化钛纳米粒子与常规材料相比具诸多优点,如：无毒,优良的电学性能,良好的热稳定性,不强的吸湿性,以及良好的成膜性等。本文将二氧化钛纳米粒子溶于氨基化溶剂牛血清白蛋白中制得牛血清白蛋白-二氧化钛纳米复合物。实验结果表明,利用该纳米复合物修饰金电极能够大量吸附生物亲和性好、比表面积大的纳米金颗粒,并通过纳米金成功固定甲胎蛋白抗体。利用此法研制的甲胎蛋白电流型免疫传感器对甲胎蛋白抗原有很好的电流响应,在最优实验条什下,其检测范围为0.01～80.0 ng/mL,检出限为0.003 ng/mL,实现了对甲胎蛋白抗原的定量分析。该免疫传感器制作简单、成本节约、环保、灵敏性高。
2.纳米金和磁性纳米复合物构建无试剂电流型免疫传感器的研究
四氧化三铁纳米粒子是一种典型的磁性纳米材料,具有易分离、易富集、易提取的优点,常用于制备生物感器。本文首先制备了具有易于磁性分离、硫堇(Thionine,Thi)包覆的四氧化三铁(Fe304)纳米复合物。通过静电吸附和共价键合作用,将Nafion、硫堇包覆的Fe3O4复合纳米粒子层层修饰到玻碳电极表面,最后利用纳米金吸附甲胎蛋白抗体(anti-AFP),从而制得了灵敏度高、稳定性好的无试剂电流型甲胎蛋白免疫传感器。实验通过透射电子显微镜(TEM)对该复合纳米粒子进行了表征,并用循环伏安法考察了电极的电化学特性。实验结果表明,Fe304/硫堇复合纳米粒子修饰的电极在实验过程中呈现出良好的氧化还原活性,省去了在测试底液中加入电活性物质的步骤。与类似方法相比,该传感器引入的四氧化三铁纳米粒子能显著降低检测下限。并且利用Fe304顺磁性,还能达到多次回收再生,既节约成本又环保。
3.基于碳纳米管/L-半胱氨酸/Fe3O4@ Au纳米复合材料的电流型甲胎蛋白免疫传感器的研究
将DMF(N，,N-二甲基甲酰胺)分散的多壁碳纳米管(MWNTs)修饰在金电极表面,再将修饰电极依次沉积纳米金、L-半胱氨酸,并通过半胱氨酸中的巯基吸附Fe3O4@Au纳米复合材料,最后固载甲胎蛋白抗体(anti-AFP),以牛血清白蛋白(BSA)封闭非特异性吸附位点,成功构建了高灵敏、稳定的新型电流型甲胎蛋白免疫传感器。实验通过扫描透射电子显微镜(TEM)对DMF-MWNTs和Fe3O4@Au复合纳米粒子进行了表征。与传统使用纳米金颗粒固定抗体的方法相比,Fe3O4@Au复合纳米粒子能显著增加甲胎蛋白抗体的吸附量,提高免疫传感器的灵敏度。同时,该传感器的制备方法不需要标记生物分子,也无需竞争或者夹心反应,只需在传感器表面进行常规的免疫反应,测量其电流响应信号即可。在优化的实验条件下,该免疫传感器对甲胎蛋白抗原的检测范围为0.1-150 ng/mL,检出限为0.03 ng/mL。该免疫传感器选择性好、稳定。
The expression of alpha-fetoprotein (a-fetoprotein, AFP) in hepatoma cells is a high specific protein. Many HCC patients (70～80%) in the incubation period have the characteristics of high AFP gene expression. The traditional medical theory holds that high expression of AFP gene is a process of liver cancer associated with the epiphenomenon, and is deemed as a liver cancer-specific tumor marker. It is reported that the AFP extracellular bind to its receptor and mediate the growth of information transfer. Cells within the AFP can not only inhibite the apoptosis signal transduction, but also promote the transmission of growth signals, which display new discoveries of anti-apoptosis and promoting proliferation for AFP, This study have endowed AFP new cognition of functionalization and new target for treating liver cancer.Therefore, the development of new detection methods for detection of AFP is of important theoretical and practical application value.
Electrochemical immunosensor is based on the combination of electroanalysis technology and specific immunoreaction. Among various electrochemical immunosensors, the amperometric immunosensor is the most sophisticated because it, avoids separation, washing, realizes easily, and achieves rapid immunoassay compared with other electrochemical immunoassay sensing technology. In our work, we successfully construct amperometric AFP immunosensor by utilizing inorganic nanomaterial for their advantages such as the adsorption ability, the surface with functional group density, short time to reach equilibrium.The prepared immunosensor exhibitmuch easier process and higher sensitivity compared with the traditional method for determination of AFP.
The main works and conclusions are as follows:
1. An Immunosensor for AFP Antigen Based on Gold Nanoparticles and Titanic Oxide-BSA Composite.
Titanic oxide nanoparticles own many advantages compared to conventional materials, such as, non-toxic, excellent electrical properties, good thermal stability, weak moisture absorption and good film-formation. A highly sensitive amperometric immunosensor for the determination of AFP is developed based on the composite of bovine serum albumin (BSA) and TiO2(BSA/TiO2),which is employed to link gold nanoparticles (nano-Au) for the immobilization of anti-AFP. The results show that the immunosensor presents highly sensitivity, good selectivity and a broad liner response from 0.01～80 ng/mL with a detection limit of 0.003 ng/mL.
2. Study on the self assembly reagentless amperometric immunosensor based on gold nanoparticles and magnetic nanocomposite.
Fe3O4 nanoparticles, a typical magnetic nano-materials, are proved to be easy to separate,be enriched,and extract the advantages of bio-sensors commonly prepared. A new reagentless amperometric immunosensor was successfully fabricated based on theα-fetoprotein antibody (anti-AFP) molecule coupled at Fe3O4@Thionine and gold nanoparticles. Firstly, magnetic nanoparticles and Fe3O4@Thionine Nanoparticles were prepared. Then, Fe3O4@Thionine nanoparticles was assembled on nafion modified glassy carbon electrode via electrostatic adsorption and covalent bonding. Finally, gold nanoparticles were absorbed on the electrode surface for the immobilization of anti-AFP. The proposed immunosensor possessed high sensitivity and good stability. Transmission electron microscopy (TEM) was used to investigate the nanoparticles composite and stepwise fabrication process of the immunosensor is characterized by the cyclic voltammetrys. The results show that, Fe3O4@Thionine modified immunosensor showed a good redox activity, avoiding the adding og electroactive materialsn. Compared with tranditional methods, the proposed nanomaterialsbased immunoassay displays the lower detection limit and the higher sensitivity.
3. An Immunosensor for AFP Antigen Based on MCNTS/L-Cysteine and Au@Fe3O4 Nanocomposite.
DMF dispersed MWNTS was firstly modified on electrode surface, which was in further eletrodeposited a nano-Au layer for immobilization of L-cysteine. Subsequently,Fe3O4@Au nanocompositeswere absorbed on the modified electrode surface by the SH-Au bond for increasing the specific surface area and further immobilizing anti-AFP. Finally, bovine serum albumin (BSA) was used to block the non-specific adsorption sites of the immunosensor to obtain a highly sensitive and stable immunosensor. Experiments by transmission electron microscopy (TEM) are carried out to characterize the prepared MWNTs and Fe3O4@ Au composite nanoparticles. Compared with the immunosensor based on the gold nanoparticles to immobilizethe antibodies, the proposed method can significantly increase the adsorption capacity of alpha-fetoprotein antibody to improve the sensitivity of immunosensor. Meanwhile, the sensor does not require the preparation of labeled biological molecules.avoid the competition or sandwich reaction and make the experiment easier. Under optimal conditions, the sensor has a good response to AFP. in the detection range of 0.1～150 ng/mL, and detection limit of 0.03 ng/mL.
引文
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