蛋白质在金刚石和玻碳表面的共价修饰及电化学生物传感
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摘要
作为电化学生物传感器的研究中应用最广泛的固定化生物功能试剂，生物大分子蛋白质或酶参与生命体系中的新陈代谢等许多生理过程，这些过程中都要经历电子转移的过程，因此采用电化学方法来研究蛋白质或酶的电子传递过程有着特殊的优越性。对蛋白质分子的直接电化学的研究有助于获得蛋白质的热力学和动力学性质，为揭示生物氧化还原过程奠定了基础，对开发新型生物传感器、生物燃料电池等生物电子器件具有十分重要的理论及应用指导意义。然而，由于生物大分子的分子体积比较庞大，生物活性中心包埋于大分子内部不易暴露，从而造成使蛋白质或酶等生物分子在电极上的直接电化学反应难以实现，从而使蛋白质的电化学研究存在着诸多困难。另一方面，近几年来，碳电极在电化学生物传感器研究中应用受到了广泛关注。碳电极自身有着高于传统材料的诸多优点，如：良好的生物兼容性，稳定的形态，较好的化学和电化学稳定性，良好的机械加工性能以及较低的背景电流等；而且由于碳晶体独特的特点，使得对其表面易于进行化学修饰；也使得具有p键共轭结构的有机分子或配合物更容易被修饰到碳电极的表面，为该电极上的化学修饰提供了更多的途径。而特别值得关注的是，近二、三年来，对具有独特性能的金刚石膜电极(Boron-Doped Diamond Film Electrode，BDD)在生物电分析领域中的研究已经成为人们关注的焦点和研究的重要领域之一。然而，由于金刚石膜本身独特的化学及电化学惰性使得在其表面的共价修饰具有一定的难度，而这将金刚石膜作为电化学生物传感器基底材料的研究工作在国际上刚刚兴起。
本论文的研究工作采用芳香分子重氮盐的电化学还原反应修饰方法，对比了玻碳(Glassy Carbon Electrode，GC)和金刚石电极的修饰情况，实验结果表明，共价键合在电极上的芳香分子膜对探针分子Fe(CN)_6~(3-／4-)的电子传递反应具有明显的阻碍效应，表现为芳香分子共价修饰的GC(或BDD)电极的阻抗R_(ct)值比未修饰的GC电极(或BDD)电极的R_(ct)值明显增大。其中与GC电极以及芳香分子共价修饰的GC电极相比，BDD电极以及芳香分子共价修饰的BDD电极均表现出更大的R_(ct)值，说明Fe(CN)_6~(3-／4-)电子传递反应在BDD电极以及芳香分子共价修饰的BDD电极上较为缓慢。
As a most widely-used biofunctional reagent for immobilization in the field of electrochemical biosensor, biomacromolecules, such as protein, enzyme and et al, are involved in metabolism and other important physiological processes which are characteristics of electron transfer. So there must be advantage for electrochemistry method to be adopted for the investigation of the electron-transfer processes of protein and enzyme. Moreover, the investigation of the direct electrochemistry of protein can help us comprehend the dynamic and thermodynamic properties of protein, establish a solid foundation to explore the biological oxidation and reduction processes, and be vitally significant for the theoretical and applicational research of new-generation biosensors, biological fuel batteries and et al. However, due to the huge dimension of bio-macromolecules' structure, their bio-active centre is usually buried deeply inside the bio-macromolecules, which must cause difficulty in the realization of the direct electrochemistry of the bio-macromolecules, such as protein or enzyme. On the other hand, carbon electrode has been broadly adopted as a kind of substrate material for the investigation of electrochemical biosensors. There are many good properties of carbon electrodes, such as perfect biological affinity, stable morphology, high chemical and electrochemical stability, good machining property, low background current and et al. Besides, due to carbon crystal's unique property and carbon materials' conjugate structure of p bond, the surface modification of carbon material can be achieved more easily. And especially, a lot of attentions have been paid for the investigation of Boron-Doped Diamond Film Electrode (BDD) which is a kind of new material with many perfect and unique properties. Despite the unique chemical and electrochemical inertness of BDD causes difficulty in its surface modification, the investigation of BDD for developing biosensors has just been carried out.
In this paper, the surface pre-modification of Glassy Carbon Electrode (GC) and BDD electrode has been studied with a method named the electrochemical reduction of aromatic diazonium salts. The results show that the film monolayer of aromatic molecules covalently modified at the electrodes has an apparent blocking effect on the electron-transfer process of the redox system, Fe(CN)_6~(3-/4-) It was found that comparing with the R_(ct) value of GC or BDD electrode, the R_(ct) values of GC or BDD electrode covalently modified with aromatic molecules increased apparently. More than that, BDD electrode's R_(ct) value was found to be more than GC electrode's R_(ct) value, and the R_(ct) value of aromatic molecules-modified BDD electrode more than the one of aromatic molecules-modified GC electrode. So it indicates that the electron-transfer process of Fe(CN)_6~(3-/4-) at BDD electrode or aromatic molecules-modified BDD electrode is correspondingly slower than at GC electrode or aromatic molecules-modified GC electrode.
With the characterization results obtained from the electrodes modified under different conditions of electrochemical reduction of aromatic molecules, the R_(ct)
本论文的研究工作采用芳香分子重氮盐的电化学还原反应修饰方法，对比了玻碳(Glassy Carbon Electrode，GC)和金刚石电极的修饰情况，实验结果表明，共价键合在电极上的芳香分子膜对探针分子Fe(CN)_6~(3-／4-)的电子传递反应具有明显的阻碍效应，表现为芳香分子共价修饰的GC(或BDD)电极的阻抗R_(ct)值比未修饰的GC电极(或BDD)电极的R_(ct)值明显增大。其中与GC电极以及芳香分子共价修饰的GC电极相比，BDD电极以及芳香分子共价修饰的BDD电极均表现出更大的R_(ct)值，说明Fe(CN)_6~(3-／4-)电子传递反应在BDD电极以及芳香分子共价修饰的BDD电极上较为缓慢。
As a most widely-used biofunctional reagent for immobilization in the field of electrochemical biosensor, biomacromolecules, such as protein, enzyme and et al, are involved in metabolism and other important physiological processes which are characteristics of electron transfer. So there must be advantage for electrochemistry method to be adopted for the investigation of the electron-transfer processes of protein and enzyme. Moreover, the investigation of the direct electrochemistry of protein can help us comprehend the dynamic and thermodynamic properties of protein, establish a solid foundation to explore the biological oxidation and reduction processes, and be vitally significant for the theoretical and applicational research of new-generation biosensors, biological fuel batteries and et al. However, due to the huge dimension of bio-macromolecules' structure, their bio-active centre is usually buried deeply inside the bio-macromolecules, which must cause difficulty in the realization of the direct electrochemistry of the bio-macromolecules, such as protein or enzyme. On the other hand, carbon electrode has been broadly adopted as a kind of substrate material for the investigation of electrochemical biosensors. There are many good properties of carbon electrodes, such as perfect biological affinity, stable morphology, high chemical and electrochemical stability, good machining property, low background current and et al. Besides, due to carbon crystal's unique property and carbon materials' conjugate structure of p bond, the surface modification of carbon material can be achieved more easily. And especially, a lot of attentions have been paid for the investigation of Boron-Doped Diamond Film Electrode (BDD) which is a kind of new material with many perfect and unique properties. Despite the unique chemical and electrochemical inertness of BDD causes difficulty in its surface modification, the investigation of BDD for developing biosensors has just been carried out.
In this paper, the surface pre-modification of Glassy Carbon Electrode (GC) and BDD electrode has been studied with a method named the electrochemical reduction of aromatic diazonium salts. The results show that the film monolayer of aromatic molecules covalently modified at the electrodes has an apparent blocking effect on the electron-transfer process of the redox system, Fe(CN)_6~(3-/4-) It was found that comparing with the R_(ct) value of GC or BDD electrode, the R_(ct) values of GC or BDD electrode covalently modified with aromatic molecules increased apparently. More than that, BDD electrode's R_(ct) value was found to be more than GC electrode's R_(ct) value, and the R_(ct) value of aromatic molecules-modified BDD electrode more than the one of aromatic molecules-modified GC electrode. So it indicates that the electron-transfer process of Fe(CN)_6~(3-/4-) at BDD electrode or aromatic molecules-modified BDD electrode is correspondingly slower than at GC electrode or aromatic molecules-modified GC electrode.
With the characterization results obtained from the electrodes modified under different conditions of electrochemical reduction of aromatic molecules, the R_(ct)
引文
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