细胞色素C在SAM和离子液体修饰电极上的电化学行为研究
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摘要
构成生物体新陈代谢的几乎全部化学反应都是在活性蛋白质—酶的催化下进行的,生物体中最基本的运动便是电荷的运动,细胞色素C(Cytochrome C, Cyt.c)承担了呼吸链上重要的电子传递作用。在生物体内需要依靠其氧化还原酶的作用进行可逆的氧化还原反应,但在金属,如Hg、Pt、Au、Ag等电极上的电化学反应却是不可逆的,需要在一类称作电子传递促进剂(Promoter)或媒介体(Mediator)的存在下才能进行直接的电子传递。
本文主要应用分子自组装技术制备了2-氨基乙硫醇(2-aminoethanethiol,简称2AT)修饰的金电极,采用循环伏安(Cyclic voltammetry,CV),石英晶体微天平(Quartz crystal microbalance, QCM),拉曼光谱(Raman spectroscopy),电化学交流阻抗(Electrochemical impedance spectra, EIS)等方法对自组装膜进行了表征,并测定了Cyt.c在修饰电极上的循环伏安行为。实验结果证明,Cyt.c在2-氨基乙硫醇修饰电极上可进行直接的电子传递。结合电化学石英晶体微天平技术(EQCM)对修饰膜的研究,探讨了可能的电子传递反应机理。
此外,还利用层层组装技术将Cyt.c和2-氨基乙硫醇共同固定到金电极表面,制备了Cyt.c/2AT/Au修饰电极,测定了修饰电极在磷酸盐缓冲溶液(PBS)中的电化学行为,并与细胞色素C在2AT/Au修饰电极上的电化学行为进行了比较。结果表明,Cyt.c在2AT修饰的金电极上发生了明显的氧化还原反应;20mV/s的扫速下,其氧化还原峰电位分别为48mV和-7mV,峰峰电位差为55 mV,式量电位E0’约为20.5mV;而Cyt.c/2AT/Au修饰电极在磷酸盐缓冲溶液(PBS)中虽也出现一对明显的氧化还原电流峰,但其峰形不如前者。
离子液体(Ionic liquids)是在室温或稍高于室温的温度下呈液态的离子体系。它具有熔点低、蒸气压小、电化学窗口大、良好的溶解度、热稳定性好、粘度和密度大等特点,是一种绿色环保的溶剂。近年来,已在电化学、有机合成及分离分析等技术领域迅速得到应用。生物分子在离子液体中不会失活,是研究生物分子电化学行为的良好介质。
在参考相关文献资料的基础上,制备了咪唑类亲水性离子液体和疏水性离子液体﹑吡啶类亲水性离子液体和疏水性离子液体以及季胺盐离子液体,并利用核磁共振测试技术对制得的离子液体进行了表征。
将离子液体滴加到玻碳电极上制得修饰电极,测定了修饰电极在细胞色素C溶液中的循环伏安行为,并利用电化学交流阻抗谱技术对其电化学反应过程进行了原位测定,结合阻抗谱拟合的等效电路对离子液体促进细胞色素C电子传递的反应机理进行了探讨。
结果表明,无论是亲水性还是疏水性的咪唑类离子液体修饰的玻碳电极均能对Cyt.c的电子传递产生良好的促进作用,说明改变阴离子对离子液体修饰电极性能的影响不大;而细胞色素C在季胺盐修饰的玻碳电极上没有发生明显的电子传递反应,说明含季胺阳离子的离子液体不能对细胞色素C的电子传递反应产生良好的促进效果。
细胞色素C在吡啶疏水性离子液体修饰的玻碳电极上的CV图上也呈现出一对良好的氧化还原特征峰,说明吡啶类离子液体也可促进细胞色素C的电子传递。
Most chemical reactions during the metabolism are catalyzed by active protein-enzymes,and the basal movement in human body is charge movement during which Cytochrome c (Cyt.c) palys an very important role. Redox of Cyt.c in human body is reversible but irreversible on the metal electrodes such as Hg、Pt、Au、Ag and its direct electron transfer can only be completed with the help of promoter or mediator.
A gold electrode modified with 2-aminoethanethiol(2AT)self-assembled monolayer (SAM) has been fabricated. The characteristics of 2AT SAM have been investigated by cyclic votammetry(CV)、quartz crystal microbalance(QCM)、Raman spectroscopy、electrochemical impedance spectra(EIS). Results showed that 2AT SAM was an effective promoter for Cyt.c electron transfer.The 2AT assembles process and its possible promotion mechanism were discussed with the electrochemical quartz crystal microbalance (EQCM) and EIS measurements.
Cyt.c was assembled onto the Au electrode by using layer upon layer technic to form Cyt.c/2AT/Au modified electrode,the Cyt.c/2AT/Au electrode also showed a pair of redox peaks apparently in PBS solution but was not so good when compared to 2AT/Au electrode in Cyt.c solution.At the scan rate of 20mV/s reversible singals of Cyt.c on the 2AT/Au electrode was observed.It’s redox peak potential was 48mV and -7mV respectively. So the formal potential is 20.5mV. When in PBS solution, a pair of redox peaks was also found on the Cyt.c/2AT/Au electrode, but its’s not as well as 2AT/Au electrode in Cyt.c solution.
Room-temperature ionic liquids are compounds that only consist of ions and are liquids at or above room temperature.They have many advantages such as low melting point、lack of vapor pressure、broad electrochemical window、favorable solvent miscibility、thermal stability and high viscidity tolerance and density.It becomes a green solvent and is used widely in electrochemistry、organic synthesize、separate analyzing fields.Biologic molecules remain their activities in ionic liquids and it is a good medium for biologic molecules during their electrochemical behaviors.
We synthesized five ionic liquids according to references and the productions were proved by 1H-NMR.
Ionic liquids was dropped onto the GC electrode to form modified electrode.And the electrochemical behaviors of cytochrome c at the modified GC electrode were studied by CV,also EIS was used synchronously to detect the chemical reaction. According to the equivalent circuit experimental results was simulated by which we studied the electron transfer mechanism.
The results showed that both of the imidazole ionic liquid were good promoters for the direct electron transfer(DET) of Cyt.c which means that it affects little to the DET of Cyt.c when changing the anion of the ionic liquids;but no obvious electrochemical singals was found on the quaternary ammonium ionic liquid modified electrode which gave the information that quaternary ammonium cation was not a good promoter for DET of Cyt.c.
A pair of good electrochemical redox peaks were also found on the pyridine hexafluorophosphate ionic liquid modified GC electrode which told that pyridine cation was also a good DET promoter for Cyt.c.
本文主要应用分子自组装技术制备了2-氨基乙硫醇(2-aminoethanethiol,简称2AT)修饰的金电极,采用循环伏安(Cyclic voltammetry,CV),石英晶体微天平(Quartz crystal microbalance, QCM),拉曼光谱(Raman spectroscopy),电化学交流阻抗(Electrochemical impedance spectra, EIS)等方法对自组装膜进行了表征,并测定了Cyt.c在修饰电极上的循环伏安行为。实验结果证明,Cyt.c在2-氨基乙硫醇修饰电极上可进行直接的电子传递。结合电化学石英晶体微天平技术(EQCM)对修饰膜的研究,探讨了可能的电子传递反应机理。
此外,还利用层层组装技术将Cyt.c和2-氨基乙硫醇共同固定到金电极表面,制备了Cyt.c/2AT/Au修饰电极,测定了修饰电极在磷酸盐缓冲溶液(PBS)中的电化学行为,并与细胞色素C在2AT/Au修饰电极上的电化学行为进行了比较。结果表明,Cyt.c在2AT修饰的金电极上发生了明显的氧化还原反应;20mV/s的扫速下,其氧化还原峰电位分别为48mV和-7mV,峰峰电位差为55 mV,式量电位E0’约为20.5mV;而Cyt.c/2AT/Au修饰电极在磷酸盐缓冲溶液(PBS)中虽也出现一对明显的氧化还原电流峰,但其峰形不如前者。
离子液体(Ionic liquids)是在室温或稍高于室温的温度下呈液态的离子体系。它具有熔点低、蒸气压小、电化学窗口大、良好的溶解度、热稳定性好、粘度和密度大等特点,是一种绿色环保的溶剂。近年来,已在电化学、有机合成及分离分析等技术领域迅速得到应用。生物分子在离子液体中不会失活,是研究生物分子电化学行为的良好介质。
在参考相关文献资料的基础上,制备了咪唑类亲水性离子液体和疏水性离子液体﹑吡啶类亲水性离子液体和疏水性离子液体以及季胺盐离子液体,并利用核磁共振测试技术对制得的离子液体进行了表征。
将离子液体滴加到玻碳电极上制得修饰电极,测定了修饰电极在细胞色素C溶液中的循环伏安行为,并利用电化学交流阻抗谱技术对其电化学反应过程进行了原位测定,结合阻抗谱拟合的等效电路对离子液体促进细胞色素C电子传递的反应机理进行了探讨。
结果表明,无论是亲水性还是疏水性的咪唑类离子液体修饰的玻碳电极均能对Cyt.c的电子传递产生良好的促进作用,说明改变阴离子对离子液体修饰电极性能的影响不大;而细胞色素C在季胺盐修饰的玻碳电极上没有发生明显的电子传递反应,说明含季胺阳离子的离子液体不能对细胞色素C的电子传递反应产生良好的促进效果。
细胞色素C在吡啶疏水性离子液体修饰的玻碳电极上的CV图上也呈现出一对良好的氧化还原特征峰,说明吡啶类离子液体也可促进细胞色素C的电子传递。
Most chemical reactions during the metabolism are catalyzed by active protein-enzymes,and the basal movement in human body is charge movement during which Cytochrome c (Cyt.c) palys an very important role. Redox of Cyt.c in human body is reversible but irreversible on the metal electrodes such as Hg、Pt、Au、Ag and its direct electron transfer can only be completed with the help of promoter or mediator.
A gold electrode modified with 2-aminoethanethiol(2AT)self-assembled monolayer (SAM) has been fabricated. The characteristics of 2AT SAM have been investigated by cyclic votammetry(CV)、quartz crystal microbalance(QCM)、Raman spectroscopy、electrochemical impedance spectra(EIS). Results showed that 2AT SAM was an effective promoter for Cyt.c electron transfer.The 2AT assembles process and its possible promotion mechanism were discussed with the electrochemical quartz crystal microbalance (EQCM) and EIS measurements.
Cyt.c was assembled onto the Au electrode by using layer upon layer technic to form Cyt.c/2AT/Au modified electrode,the Cyt.c/2AT/Au electrode also showed a pair of redox peaks apparently in PBS solution but was not so good when compared to 2AT/Au electrode in Cyt.c solution.At the scan rate of 20mV/s reversible singals of Cyt.c on the 2AT/Au electrode was observed.It’s redox peak potential was 48mV and -7mV respectively. So the formal potential is 20.5mV. When in PBS solution, a pair of redox peaks was also found on the Cyt.c/2AT/Au electrode, but its’s not as well as 2AT/Au electrode in Cyt.c solution.
Room-temperature ionic liquids are compounds that only consist of ions and are liquids at or above room temperature.They have many advantages such as low melting point、lack of vapor pressure、broad electrochemical window、favorable solvent miscibility、thermal stability and high viscidity tolerance and density.It becomes a green solvent and is used widely in electrochemistry、organic synthesize、separate analyzing fields.Biologic molecules remain their activities in ionic liquids and it is a good medium for biologic molecules during their electrochemical behaviors.
We synthesized five ionic liquids according to references and the productions were proved by 1H-NMR.
Ionic liquids was dropped onto the GC electrode to form modified electrode.And the electrochemical behaviors of cytochrome c at the modified GC electrode were studied by CV,also EIS was used synchronously to detect the chemical reaction. According to the equivalent circuit experimental results was simulated by which we studied the electron transfer mechanism.
The results showed that both of the imidazole ionic liquid were good promoters for the direct electron transfer(DET) of Cyt.c which means that it affects little to the DET of Cyt.c when changing the anion of the ionic liquids;but no obvious electrochemical singals was found on the quaternary ammonium ionic liquid modified electrode which gave the information that quaternary ammonium cation was not a good promoter for DET of Cyt.c.
A pair of good electrochemical redox peaks were also found on the pyridine hexafluorophosphate ionic liquid modified GC electrode which told that pyridine cation was also a good DET promoter for Cyt.c.
引文
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