电化学阻抗免疫传感界面构建及其应用于多环芳烃的检测研究
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摘要
多环芳烃是一类具有致癌、致畸、致突变性的持久性有机污染物。1-芘丁酸是PAHs的衍生物,对人体和动植物均有毒性作用。因此,对PAHs及衍生物的快速检测具有重要意义。电化学免疫传感器作为一种有效的微量和痕量分析手段,具有灵敏度高、分析速度快、选择性强、仪器简单等优点,近年来被人们所广泛关注。文献调研表明,利用电化学阻抗免疫传感器来监测环境中的多环芳烃及其衍生物的研究较少,对小分子物质PAHs的电化学免疫分析有待进一步探索。
本文以1-芘丁酸为检测对象,采用不同的固定化技术将多环芳烃抗体有效固定在电极表面,构建灵敏和特异性的传感界面。以修饰的传感界面为敏感元件,根据循环伏安、交流阻抗等电化学方法表征免疫传感器的构建过程,通过电化学交流阻抗谱测定抗原抗体反应后传感器界面电子转移阻抗的变化来实现1-芘丁酸的定量分析,并据此探索了利用电化学阻抗免疫传感器进行多环芳烃快速检测的新方法。
采用电沉积纳米金膜物理吸附的固定化技术,将多环芳烃抗体固定在玻碳电极表面,应用电化学阻抗谱及循环伏安法测定修饰电极在电解液中的电化学行为。实验结果显示,随着样品1-芘丁酸浓度的增加,结合到纳米金修饰电极表面的抗原-抗体复合物也增加,使得氧化还原对在电极表面电子转移阻力变大,导致电极表面电子转移阻抗随之增加,这表明阻抗谱可以用于1-芘丁酸的定性检测分析。实验初步建立了电化学阻抗谱和免疫传感器联用的多环芳烃检测方法。
采用Nafion膜和金纳米粒子(nano-Au/Nafion)复合物作为电极材料,构建操作简单、高度灵敏、无标记型电化学阻抗免疫传感器,应用于1-芘丁酸的检测。在优化实验条件下,由于Nafion膜和金纳米粒子的协同作用和良好生物兼容性,电极表面的anti-PAHs抗体量极大提高。研究结果表明,nano-Au/Nafion复合电极对1-芘丁酸检测的灵敏度和稳定性比单一金纳米电极的性能更优异。nano-Au/Nafion免疫传感器的电子转移阻抗与1-芘丁酸的浓度对数成一定的线性关系,线性范围为0.1～150ng·mL-1,检测限为0.03 ng·ml-1。对电化学阻抗免疫传感器的选择性和准确性进行评价,均取得满意的结果。
利用巯基物质在金表面的自组装特性,在金电极表面形成稳定、有序、尾基为羧基的巯基乙酸(MAA)单分子层自组装膜,再利用偶联剂1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和N-羟基丁二酰亚胺(NHs)的活化作用形成活性酯中间体,anti-PAHs抗体与单分子层中的活性酯反应形成稳定的酰胺键,从而实现抗体在电极表面的有效固定。在Fe(CN)63-/Fe(CN)64-体系中以1-芘丁酸为检测对象,考察了传感器交流阻抗谱的电子转移阻抗在不同浓度1-芘丁酸中的响应变化,实现了对1-芘丁酸的定量分析。ΔRet与PBA的浓度对数在0.05-50 ng·mL-1范围内具有良好的线性关系,线性方程为ΔRet=3901.71gC+4135.3,相关系数R2=0.997,检测下限为0.05ng·mL-1。该免疫传感器在重现性和再生性方面也具有良好的表现。
Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants (POPs) widely present in the environment, many which are carcinogenic, teratogenesis and mutagenicity. 1-Pyrenebutyric acid (PBA) is one of PAHs derivatives and harmful for human being and other flora and fauna. Therefore, it is of necessary to develop a sensitive, rapid and cost-effective method for PAHs detection. As an efficient trace analysis method, electrochemical immunosensor possesses the merits of high sensitivity, quick analysis, strong selectivity and simple structure, etc. However, few researches have been concentrated on utilizing electrochemical impedance immunosensor to monitor PAHs and its derivatives up to now. Further investigations should be carried out for the electrochemical immunoassay for small molecules of PAHs.
1-Pyrenebutyric acid (PBA) was chosen as the examination object in this paper. Different immobilized technologies were adopted to fix the PAHs antibody on the electrode surface effectively, and then sensitive and specific sensing interfaces were constructed. The modified sensor interface is taken as the sensitive element. Then the construction process of the immunosensor is described by electrochemical means such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), etc. The quantitative analysis for PBA was realized according to the variation of the electron transfer impedance (Ret) after the immunoreaction between antigen and antibody on the sensing interface detected by EIS.
Glassy carbon is selected as the basal electrode. The anti-PAHs antibody was steadily immobilized at the nano-Au/GC electrode due to large specific surface area and satisfactory biocompatibility of gold nanoparticles. The electrochemical behavior of the modified electrode in the electrolyte is monitored according to EIS and CV. It is shown that the quantity of immunocomplex combined to the nanometer gold modified electrode surface would rise with the increase in the concentration of the PAHs sample. Therefore, the electronic transfer resistance on the electrode surface increases by the redox, which resulted in the increase in the electron transfer impedance on the electrode surface. It is proved that the impedance spectroscopy could be adopted in the qualitative detection and analysis for PBA. In addition, the EIS immunosensor for the detection of PAHs is set up preliminary during the experiment.
A simple, highly sensitive, and label-free electrochemical impedance spectroscopy (EIS) immunosensor was developed using Nafion and gold nanoparticles (nano-Au/Nafion) composites for the determination of PBA. Under the optimal conditions, the amount of immobilization of antibody was significantly improved on the nano-Au/Nafion electrode due to the synergistic effect and biocompatibility of Nafion film and gold nanoparticles composites. The results showed that the sensitivity and stability of nano-Au/Nafion composite electrode for PBA detection were much better than those of nano-Au modified glassy carbon electrode (nano-Au/GCE). The plot of increased electron transfer resistances (Rets) against the logarithm of PBA concentration is linear over the range from 0.1 to 150 ng-mL"1 with a detection limit of 0.03 ng·mL-1. The selectivity and accuracy of the proposed EIS immunosensor were evaluated with satisfactory results.
An electrochemical impedance spectroscopy (EIS) immunosensor for the detection of PAHs was developed by immobilizing antibody through the Au-S covalent bond. A pretreated gold electrode is first immersed in mercaptoacetic acid solution to form a self-assembled monolayer (SAM). Then anti-PAHs antibodies are immobilized to the SAM by means of covalenting with a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide(NHS). In the presence of Fe(CN)63-/Fe(CN)64- as a redox probe, the electrotransfer resistances (Rets) were directly detected by EIS after the immunosensor specific binding with PBA of different concentrations. A linear relationship between the increased Ret and the logarithmic value of PBA concentrations was found in the range of 0.05-50 ng·mL-1 with the detection limit of 0.05 ng·mL-1. The regeneration and repeating characteristics were also studied to be good.
本文以1-芘丁酸为检测对象,采用不同的固定化技术将多环芳烃抗体有效固定在电极表面,构建灵敏和特异性的传感界面。以修饰的传感界面为敏感元件,根据循环伏安、交流阻抗等电化学方法表征免疫传感器的构建过程,通过电化学交流阻抗谱测定抗原抗体反应后传感器界面电子转移阻抗的变化来实现1-芘丁酸的定量分析,并据此探索了利用电化学阻抗免疫传感器进行多环芳烃快速检测的新方法。
采用电沉积纳米金膜物理吸附的固定化技术,将多环芳烃抗体固定在玻碳电极表面,应用电化学阻抗谱及循环伏安法测定修饰电极在电解液中的电化学行为。实验结果显示,随着样品1-芘丁酸浓度的增加,结合到纳米金修饰电极表面的抗原-抗体复合物也增加,使得氧化还原对在电极表面电子转移阻力变大,导致电极表面电子转移阻抗随之增加,这表明阻抗谱可以用于1-芘丁酸的定性检测分析。实验初步建立了电化学阻抗谱和免疫传感器联用的多环芳烃检测方法。
采用Nafion膜和金纳米粒子(nano-Au/Nafion)复合物作为电极材料,构建操作简单、高度灵敏、无标记型电化学阻抗免疫传感器,应用于1-芘丁酸的检测。在优化实验条件下,由于Nafion膜和金纳米粒子的协同作用和良好生物兼容性,电极表面的anti-PAHs抗体量极大提高。研究结果表明,nano-Au/Nafion复合电极对1-芘丁酸检测的灵敏度和稳定性比单一金纳米电极的性能更优异。nano-Au/Nafion免疫传感器的电子转移阻抗与1-芘丁酸的浓度对数成一定的线性关系,线性范围为0.1～150ng·mL-1,检测限为0.03 ng·ml-1。对电化学阻抗免疫传感器的选择性和准确性进行评价,均取得满意的结果。
利用巯基物质在金表面的自组装特性,在金电极表面形成稳定、有序、尾基为羧基的巯基乙酸(MAA)单分子层自组装膜,再利用偶联剂1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐(EDC)和N-羟基丁二酰亚胺(NHs)的活化作用形成活性酯中间体,anti-PAHs抗体与单分子层中的活性酯反应形成稳定的酰胺键,从而实现抗体在电极表面的有效固定。在Fe(CN)63-/Fe(CN)64-体系中以1-芘丁酸为检测对象,考察了传感器交流阻抗谱的电子转移阻抗在不同浓度1-芘丁酸中的响应变化,实现了对1-芘丁酸的定量分析。ΔRet与PBA的浓度对数在0.05-50 ng·mL-1范围内具有良好的线性关系,线性方程为ΔRet=3901.71gC+4135.3,相关系数R2=0.997,检测下限为0.05ng·mL-1。该免疫传感器在重现性和再生性方面也具有良好的表现。
Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants (POPs) widely present in the environment, many which are carcinogenic, teratogenesis and mutagenicity. 1-Pyrenebutyric acid (PBA) is one of PAHs derivatives and harmful for human being and other flora and fauna. Therefore, it is of necessary to develop a sensitive, rapid and cost-effective method for PAHs detection. As an efficient trace analysis method, electrochemical immunosensor possesses the merits of high sensitivity, quick analysis, strong selectivity and simple structure, etc. However, few researches have been concentrated on utilizing electrochemical impedance immunosensor to monitor PAHs and its derivatives up to now. Further investigations should be carried out for the electrochemical immunoassay for small molecules of PAHs.
1-Pyrenebutyric acid (PBA) was chosen as the examination object in this paper. Different immobilized technologies were adopted to fix the PAHs antibody on the electrode surface effectively, and then sensitive and specific sensing interfaces were constructed. The modified sensor interface is taken as the sensitive element. Then the construction process of the immunosensor is described by electrochemical means such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), etc. The quantitative analysis for PBA was realized according to the variation of the electron transfer impedance (Ret) after the immunoreaction between antigen and antibody on the sensing interface detected by EIS.
Glassy carbon is selected as the basal electrode. The anti-PAHs antibody was steadily immobilized at the nano-Au/GC electrode due to large specific surface area and satisfactory biocompatibility of gold nanoparticles. The electrochemical behavior of the modified electrode in the electrolyte is monitored according to EIS and CV. It is shown that the quantity of immunocomplex combined to the nanometer gold modified electrode surface would rise with the increase in the concentration of the PAHs sample. Therefore, the electronic transfer resistance on the electrode surface increases by the redox, which resulted in the increase in the electron transfer impedance on the electrode surface. It is proved that the impedance spectroscopy could be adopted in the qualitative detection and analysis for PBA. In addition, the EIS immunosensor for the detection of PAHs is set up preliminary during the experiment.
A simple, highly sensitive, and label-free electrochemical impedance spectroscopy (EIS) immunosensor was developed using Nafion and gold nanoparticles (nano-Au/Nafion) composites for the determination of PBA. Under the optimal conditions, the amount of immobilization of antibody was significantly improved on the nano-Au/Nafion electrode due to the synergistic effect and biocompatibility of Nafion film and gold nanoparticles composites. The results showed that the sensitivity and stability of nano-Au/Nafion composite electrode for PBA detection were much better than those of nano-Au modified glassy carbon electrode (nano-Au/GCE). The plot of increased electron transfer resistances (Rets) against the logarithm of PBA concentration is linear over the range from 0.1 to 150 ng-mL"1 with a detection limit of 0.03 ng·mL-1. The selectivity and accuracy of the proposed EIS immunosensor were evaluated with satisfactory results.
An electrochemical impedance spectroscopy (EIS) immunosensor for the detection of PAHs was developed by immobilizing antibody through the Au-S covalent bond. A pretreated gold electrode is first immersed in mercaptoacetic acid solution to form a self-assembled monolayer (SAM). Then anti-PAHs antibodies are immobilized to the SAM by means of covalenting with a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide(NHS). In the presence of Fe(CN)63-/Fe(CN)64- as a redox probe, the electrotransfer resistances (Rets) were directly detected by EIS after the immunosensor specific binding with PBA of different concentrations. A linear relationship between the increased Ret and the logarithmic value of PBA concentrations was found in the range of 0.05-50 ng·mL-1 with the detection limit of 0.05 ng·mL-1. The regeneration and repeating characteristics were also studied to be good.
引文
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