纳米粒子组装电化学生物传感器和电化学发光免疫分析法的研究
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摘要
生命科学、材料科学、环境科学的发展,对分析化学提出了越来越高的要求,同时也极大地促进了分析化学的发展。传统的分析方法往往需要烦琐费时的分离、复杂昂贵的仪器设备,分析速度慢、耗时长,且劳动强度大。建立高灵敏度、高选择性、简单、快速的分析方法和廉价的小型化分析装置是分析化学工作者的研究目标。我们长期的研究目的是建立快速、灵敏、可实现自动化的新型均相电化学发光免疫分析方法,研制具有实用价值的纳米组装电化学和电化学发光生物传感器,为临床免疫检测和基因检测提供性能优良的分析器件。
本论文研究工作旨在研究纳米粒子组装电极上电化学检测信号的增强作用,结合生物分子识别物质如酶、抗原/抗体和DNA的特异性,研制具有实用价值的纳米组装电化学生物传感器;利用多标记技术和磁性微粒的容易分离作用,结合生物分子识别物质—抗原/抗体的特异性,建立简单、快速、灵敏的电化学发光免疫分析新方法。本论文研究工作是在国家自然科学基金“新型功能纳米材料组装电化学发光生物亲合传感器的研究”(No.20375025)和“新型均相电化学发光免疫法的研究”(No.29975017)项目的资助下完成的。本论文研制了五种高灵敏度、高选择性、简单的碳纳米管组装电化学生物传感器;建立了两种简单、快速、灵敏的电化学发光免疫分析方法,并将其成功地用于过氧化氢、葡萄糖、酚类物质、IgG抗体、地高辛、碱基、DNA序列等物质的测定;还初步研究了DNA与电活性药物维生素B_6和氧氟沙星的相互作用,探讨了相互作用机理。
第一章 引言 详细介绍了电化学生物传感器和有关电化学分析的基本原理及电化学生物传感器的固定化技术,概述了电化学发光分析法的基本原理,着重评述了电化学生物传感器和电化学发光分析法的研究进展,还简要地介绍了超声电化学的基本原理和研究进展。
第二章 碳纳米管组装电化学生物催化传感器的研究 研制了碳纳米管组装基于血红蛋白直接电子转移的第三代过氧化氢生物传感器和碳纳米管组装预氧化电流型葡萄糖生物传感器,并将其用于实际样品眼药水中过氧化氢和血清中葡萄糖的测定。
第三章 电化学免疫传感器的研究 研究了碳纳米管组装电极上酚类物质的电化学行为,建立了同时测定对苯二酚和邻苯二酚异构体的电化学分析新方法;研制了碳纳米管组装电化学免疫传感器,建立了免疫竞争法检测IgG抗体的高灵敏度电化学分析新方法。
Current analytical chemistry trends the innovation. The development of bioscience, environmental science and new material science has made new research subjects on analytical chemistry. For instance, biologically active substances including DNA, proteins, drugs and environmental toxic substances were exclusively selected as research analytes. Analytical systems have been transferred from simple systems to complex systems. Analytical methods have trended forward biochemical methods involving enzymatic and immunochemical reactions. Considerable efforts have made during the last decades to improve conventional analytical methods in accuracy, sensitivity, selectivity, fastness and automation, and to develop new methodologies for industrial, environmental and biomedical processes. In recent years, highly selective and sensitive analytical methods such as immunoassay and biosensor have been received much attention. The development of a rapid, sensitive and selective method and the fabrication of a simple and cheap analytical device for the detection of biological molecular substances have been a long-standing goal.The aim of the present work is to develop novel electrogenerated chemiluminescence immunoassay and to design and fabricate electrochemical biosensors for the determination of biological molecule with sensitivity, selectivity and simplify. In this thesis, taking advantages of the unique properties of nanoparticles and the specificity of biological molecular recognition substances, such as enzyme, antigen/antibody, DNA, we have designed a series of electrochemical biosensors and developed a series of electrogenerated chemiluminescece immunoassay for the determination of hydrogen peroxide, glucose, phenol, IgG, digoxin, and DNA. A part of research work in this thesis is financially supported by the National Natural Science Foundation of China (Grant No. 20375025, No. 29975017).The major contents in this thesis are described as follows:In Chapter 1, general introduction to electrochemical biosensors, electrogenerated chemiluminescence (ECL) and sonoelectrochemistry including its principle and research development, and the purpose of this research work were presented.
In Chapter 2, two amperometric biosensors incorporating multiwall carbon nanotubes (MWNT) have been designed and applied to the determination of hydrogen peroxide in pharmaceutical injections of hydrogen peroxide and to the determination of glucose in human serum with satisfactory results, respectively.Amperometric third-generation hydrogen peroxide biosensor incorporating multiwall carbon nanotubes and hemoglobin An amperometric third-generation hydrogen peroxide biosensor was designed by immobilizing hemoglobin (Hb) on a glassy carbon electrode modified with multiwall carbon nanotubes (MWNT). The direct electron transfer of the Hb immobilized on the MWNT-modified electrode was observed. The formal potential of the immobilized Hb was -0.241 V vs. Ag/AgCl (3 mol/L NaCl) and the heterogeneous electron transfer rate constant was 0.58 s~(-1) in a 0.20 mol/L acetate buffer solution (pH 5.4). The immobilized Hb exhibited excellent electrocatalytic activity to reduce hydrogen peroxide in the absence and presence of oxygen, which facilitated designing an amperometric third-generation biosensor for hydrogen peroxide. In the presence of oxygen, the response to hydrogen peroxide of the designed biosensor at a potential of-0.35 V was linear in the concentration range from 6.0 ×10~(-6) mol/L to 6.0 ×10~(-3) mol/L, and the detection limit was 1.2×10~(-6) mol/L. The relative standard deviation was 2.4% for nine successive assays at 1.0×10~(-5) mol/L hydrogen peroxide. The designed biosensor was applied to the determination of hydrogen peroxide in pharmaceutical injections with satisfactory results.Pre-oxidative amperometric glucose biosensor incorporating multiwall carbon nanotubes and PbO_2 A pre-oxidative amperometric glucose biosensor incorporated with MWNT and PbO_2 was designed in order to make the biosensor possessing the interference-removing ability with simple structure and short response time and high sensitivity. The interferences coming from reduced substances, such as ascoribic acid and uric, were diminished by PbO_2. The oxidative current at the potential of + 0.40 V (vs. SCE) to the concentration of glucose was linear in the range from 0.5 to 20 mmol/L, and the detection limit of glucose was 0.1 mmol/L. The proposed biosensors have been applied to the determination of glucose in serum with satisfactory results.In Chapter 3, an electrochemical method for simultaneous determination of hydroquinone and catechol at a glassy carbon electrode modified with MWNT and an amperometric immunosensor for immunoglobulin G antibody (anti-IgG) based on immobilizing IgG antigen on a glassy carbon electrode modified with MWNT have
been developed.Simultaneous determination of hydroquinone and catechol at a glassy carbon electrode modified with multiwall carbon nanotubes A simply and selectively electrochemical method for simultaneous determination of hydroquinone and catechol had been developed at a glassy carbon electrode modified with MWNT. It was found that the oxidation peak separation of hydroquinone and catechol and the oxidation currents of hydroquinone and catechol greatly increased at MWNT modified electrode in a 0.20 mol/L acetate buffer solution (pH 4.5). The two corresponding well-defined oxidation peaks of hydroquinone in the presence of catechol at MWNT modified electrode occurred at 0.264 V and 0.162 V, respectively. Under the optimized condition, the oxidation peak current of hydroquinone was linear over a range from 1.0 ×10~(-6) mol/L to 1.0 ×10~(-4) mol/L hydroquinone in the presence of 1.0×10~(-4) mol/L catechol with a detection limit of 7.5×10~(-7) mol/L and the oxidation peak current of catechol was linear over a range from 6.0 ×10~(-7) mol/L to 1.0×10~(-4) mol/L catechol in the presence of 1.0 ×10~(-4) mol/L hydroquinone with a detection limit of 2.0 ×10~(-7) mol/L. The proposed method has been applied to simultaneous determination of hydroquinone and catechol in a water sample with simplicity and high selectivity.Amperometric immunosensor for IgG antibody based on immobilizing IgG antigen on a glassy carbon electrode modified with multiwall carbon nanotubes A highly sensitive amperometric immunosensor for anti-IgG based on immobilizing IgG antigen on a glassy carbon electrode modified with MWNT was designed. A MWNT monolayer formed on a glass carbon electrode was utilized as a sensing platform for the immobilization of IgG antigen and a competitive immunoreaction occurred while analyte anti-IgG and horseradish peroxidase (HRP)-labeled anti-IgG competed for the surface-immobilized IgG antigen binding sites. The catalytic reduction current at 0.030 V (vs. SCE), which is produced in the reaction of hydroquinone with hydrogen peroxide in the presence of HRP, was linear over a range from 0.30 to 10 μg/mL anti-IgG with a detection limit of 0.11 μg/mL.In Chapter 4, homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin and immunomagnetic electrogenerated chemiluminescence detection of digoxin based on Ru(bpy)_3~(2+) label have been developed.Homogeneous electrogenerated chemiluminescence immunoassay for the
determination of digoxin A novel homogeneous electrogenerated chemiluminescence immunoassay (ECLIA) for the determination of small hapten was developed. As a model system, digoxin was investigated while luminol served as luminescence label and BSA served as carrier protein. Digoxin was indirectly heavily labeled with luminol through BSA to form luminol-BSA-digoxin conjugate. The immunocomplex of luminol-BSA-digoxin conjugate with anti-digoxin antibody underwent less ECL reaction than luminol-BSA-digoxin conjugate after immunoreaction took place. Two immunoassay formats, directly homogeneous immunodetection for anti-digoxin antibody and competitive immunoassay for digoxin, were proposed to determine anti-digoxin antibody and digoxin, respectively. The anti-digoxin antibody concentration was determined in the range from 1/50000 to 1/2000 dilution. The ECL intensity vs. digoxin concentration was linear in the range from 5.0×10~(-10)g/mL to 3.0 ×10~(-8) g/mL. The detection limit was 2.8 ×10~(-10)g/mL. The relative standard derivation for 1.0 ×10~(-9)g/mL was 5.1%. The proposed method has been applied to assay digoxin in human control serum with satisfactory results.Immunomagnetic electrogenerated chemiluminescence detection of digoxin based on Ru(bpy)_3~(2+) label Immunomagnetic electrogenerated chemiluminescencedetection of digoxin based on Ru(bpy)_3~(2+) label was developed. Digoxin was covalently attached to magnetic beads and Ru(bpy)_3~(2+) was attached to anti-digoxin antibody. A competitive immunoreaction occurred between analyte digoxin and digoxin-labeled bead competing for the Ru(bpy)_3~(2+) labeled anti-digoxin antibody. After magnetic separation, the concentration of analyte digoxin, related to the concentration of the immunocomplex of digoxin and Ru(bpy)_3~(2+) labeled anti-digoxin conjugate, was determined by ECL produced by electrochemical oxidized Ru(bpy)_3~(2+) in the presence of TPA. The linear range for digoxin was 1.0 ×10~(-8) g/mL ~ 2.0 ×10~(-6) g/mL with a detection limit of 7.5×10~(-9)g/mL.In Chapter 5, MWNT paste electrode was fabricated in this chapter. At the MWNT paste electrode, electrochemical behaviors of bases of nuclueic acids and its analytical applications were investigated, and an electrochemical detection of DNA hybridization based on ss-DNA/ polypyrrole/MWNT paste electrode using ethidium bromide as a response indicator was developed. The interaction of vitamin B_6 /ofloxacin with DNA was also investigated.Electrochemical behaviors of bases of nuclueic acids at multiwall carbon
nanotube paste electrode and its analytical applications The electrochemical behaviors of five free bases of nucleic acids at a carbon paste electrode incorporated with MWNT were investigated by differential pulse voltammetry and cyclic voltammetry. It was found that in a 0.20 mol/L borate buffer solution (pH 11.0), the bases including guanine, adenine, thyrime, uracil and cytosine showed irreversible electrochemical behaviors and the corresponding oxidation peaks appeared at 0.556 V, 0.824 V, 0.952 V, 1.048 V and 1.160 V (vs. SCE), respectively. The oxidation peaks of bases were separated. A simple electrochemical method for the simultaneous determination of one purine or pyrimidine in the presence of related compounds (adenine in the presence of guanine, and guanine in the presence of cytosine) was developed using MWNT paste electrode. The proposed method has been applied to determination the value of (G+C)/(A+T) of HCl-digested DNA and other synthetic oligonucleotides with satisfactory results.Electrochemical detection of DNA hybridization based on ss-DNA/ polypyrrole/multiwall carbon nanotube paste electrode using EB as a response indicator A highly sensitive electrochemical method for the detection of DNA hybridization using a MWNT paste electrode and immobilizing nucleic acid probes within electropolymerized polypyrrole (PPy) was described. The detection approach relied on the oligonucleotide probes served as the counter anions during the growth of conducting PPy film on the MWNT paste electrodes. An electroactive intercalate, ethidium bromide, as an electrochemical hybridization indicator, was used to monitor the hybridization reaction by differential pulse voltammetry. The anodic peak current of EB after hybridization with the target ss-DNA was linearly related to the logarithmic value of the target ss-DNA concentration ranging from 5.0×10~(-10) to 1.0×10~(-8) mol/L. The detection limit was 1.0×10~(-10) mol/L.Electrochemical studies of the interaction between Vitamin B_6/ ofloxacin and DNA The interaction of vitamin B_6 /ofloxacin (OF) with DNA was investigated by electrochemical method. When DNA was added into vitamin B_6/OF solution, it was found that the oxidation current of vitamin B_6/OF decreased. The electrochemical behaviors in the absence and presence of DNA were investigated and the results showed that Vitamin B_6/OF and DNA formed an electrochemical inactive supermolecular complex.In conclusion, five electrochemical biosensors assembled by nanoparticles or
本论文研究工作旨在研究纳米粒子组装电极上电化学检测信号的增强作用,结合生物分子识别物质如酶、抗原/抗体和DNA的特异性,研制具有实用价值的纳米组装电化学生物传感器;利用多标记技术和磁性微粒的容易分离作用,结合生物分子识别物质—抗原/抗体的特异性,建立简单、快速、灵敏的电化学发光免疫分析新方法。本论文研究工作是在国家自然科学基金“新型功能纳米材料组装电化学发光生物亲合传感器的研究”(No.20375025)和“新型均相电化学发光免疫法的研究”(No.29975017)项目的资助下完成的。本论文研制了五种高灵敏度、高选择性、简单的碳纳米管组装电化学生物传感器;建立了两种简单、快速、灵敏的电化学发光免疫分析方法,并将其成功地用于过氧化氢、葡萄糖、酚类物质、IgG抗体、地高辛、碱基、DNA序列等物质的测定;还初步研究了DNA与电活性药物维生素B_6和氧氟沙星的相互作用,探讨了相互作用机理。
第一章 引言 详细介绍了电化学生物传感器和有关电化学分析的基本原理及电化学生物传感器的固定化技术,概述了电化学发光分析法的基本原理,着重评述了电化学生物传感器和电化学发光分析法的研究进展,还简要地介绍了超声电化学的基本原理和研究进展。
第二章 碳纳米管组装电化学生物催化传感器的研究 研制了碳纳米管组装基于血红蛋白直接电子转移的第三代过氧化氢生物传感器和碳纳米管组装预氧化电流型葡萄糖生物传感器,并将其用于实际样品眼药水中过氧化氢和血清中葡萄糖的测定。
第三章 电化学免疫传感器的研究 研究了碳纳米管组装电极上酚类物质的电化学行为,建立了同时测定对苯二酚和邻苯二酚异构体的电化学分析新方法;研制了碳纳米管组装电化学免疫传感器,建立了免疫竞争法检测IgG抗体的高灵敏度电化学分析新方法。
Current analytical chemistry trends the innovation. The development of bioscience, environmental science and new material science has made new research subjects on analytical chemistry. For instance, biologically active substances including DNA, proteins, drugs and environmental toxic substances were exclusively selected as research analytes. Analytical systems have been transferred from simple systems to complex systems. Analytical methods have trended forward biochemical methods involving enzymatic and immunochemical reactions. Considerable efforts have made during the last decades to improve conventional analytical methods in accuracy, sensitivity, selectivity, fastness and automation, and to develop new methodologies for industrial, environmental and biomedical processes. In recent years, highly selective and sensitive analytical methods such as immunoassay and biosensor have been received much attention. The development of a rapid, sensitive and selective method and the fabrication of a simple and cheap analytical device for the detection of biological molecular substances have been a long-standing goal.The aim of the present work is to develop novel electrogenerated chemiluminescence immunoassay and to design and fabricate electrochemical biosensors for the determination of biological molecule with sensitivity, selectivity and simplify. In this thesis, taking advantages of the unique properties of nanoparticles and the specificity of biological molecular recognition substances, such as enzyme, antigen/antibody, DNA, we have designed a series of electrochemical biosensors and developed a series of electrogenerated chemiluminescece immunoassay for the determination of hydrogen peroxide, glucose, phenol, IgG, digoxin, and DNA. A part of research work in this thesis is financially supported by the National Natural Science Foundation of China (Grant No. 20375025, No. 29975017).The major contents in this thesis are described as follows:In Chapter 1, general introduction to electrochemical biosensors, electrogenerated chemiluminescence (ECL) and sonoelectrochemistry including its principle and research development, and the purpose of this research work were presented.
In Chapter 2, two amperometric biosensors incorporating multiwall carbon nanotubes (MWNT) have been designed and applied to the determination of hydrogen peroxide in pharmaceutical injections of hydrogen peroxide and to the determination of glucose in human serum with satisfactory results, respectively.Amperometric third-generation hydrogen peroxide biosensor incorporating multiwall carbon nanotubes and hemoglobin An amperometric third-generation hydrogen peroxide biosensor was designed by immobilizing hemoglobin (Hb) on a glassy carbon electrode modified with multiwall carbon nanotubes (MWNT). The direct electron transfer of the Hb immobilized on the MWNT-modified electrode was observed. The formal potential of the immobilized Hb was -0.241 V vs. Ag/AgCl (3 mol/L NaCl) and the heterogeneous electron transfer rate constant was 0.58 s~(-1) in a 0.20 mol/L acetate buffer solution (pH 5.4). The immobilized Hb exhibited excellent electrocatalytic activity to reduce hydrogen peroxide in the absence and presence of oxygen, which facilitated designing an amperometric third-generation biosensor for hydrogen peroxide. In the presence of oxygen, the response to hydrogen peroxide of the designed biosensor at a potential of-0.35 V was linear in the concentration range from 6.0 ×10~(-6) mol/L to 6.0 ×10~(-3) mol/L, and the detection limit was 1.2×10~(-6) mol/L. The relative standard deviation was 2.4% for nine successive assays at 1.0×10~(-5) mol/L hydrogen peroxide. The designed biosensor was applied to the determination of hydrogen peroxide in pharmaceutical injections with satisfactory results.Pre-oxidative amperometric glucose biosensor incorporating multiwall carbon nanotubes and PbO_2 A pre-oxidative amperometric glucose biosensor incorporated with MWNT and PbO_2 was designed in order to make the biosensor possessing the interference-removing ability with simple structure and short response time and high sensitivity. The interferences coming from reduced substances, such as ascoribic acid and uric, were diminished by PbO_2. The oxidative current at the potential of + 0.40 V (vs. SCE) to the concentration of glucose was linear in the range from 0.5 to 20 mmol/L, and the detection limit of glucose was 0.1 mmol/L. The proposed biosensors have been applied to the determination of glucose in serum with satisfactory results.In Chapter 3, an electrochemical method for simultaneous determination of hydroquinone and catechol at a glassy carbon electrode modified with MWNT and an amperometric immunosensor for immunoglobulin G antibody (anti-IgG) based on immobilizing IgG antigen on a glassy carbon electrode modified with MWNT have
been developed.Simultaneous determination of hydroquinone and catechol at a glassy carbon electrode modified with multiwall carbon nanotubes A simply and selectively electrochemical method for simultaneous determination of hydroquinone and catechol had been developed at a glassy carbon electrode modified with MWNT. It was found that the oxidation peak separation of hydroquinone and catechol and the oxidation currents of hydroquinone and catechol greatly increased at MWNT modified electrode in a 0.20 mol/L acetate buffer solution (pH 4.5). The two corresponding well-defined oxidation peaks of hydroquinone in the presence of catechol at MWNT modified electrode occurred at 0.264 V and 0.162 V, respectively. Under the optimized condition, the oxidation peak current of hydroquinone was linear over a range from 1.0 ×10~(-6) mol/L to 1.0 ×10~(-4) mol/L hydroquinone in the presence of 1.0×10~(-4) mol/L catechol with a detection limit of 7.5×10~(-7) mol/L and the oxidation peak current of catechol was linear over a range from 6.0 ×10~(-7) mol/L to 1.0×10~(-4) mol/L catechol in the presence of 1.0 ×10~(-4) mol/L hydroquinone with a detection limit of 2.0 ×10~(-7) mol/L. The proposed method has been applied to simultaneous determination of hydroquinone and catechol in a water sample with simplicity and high selectivity.Amperometric immunosensor for IgG antibody based on immobilizing IgG antigen on a glassy carbon electrode modified with multiwall carbon nanotubes A highly sensitive amperometric immunosensor for anti-IgG based on immobilizing IgG antigen on a glassy carbon electrode modified with MWNT was designed. A MWNT monolayer formed on a glass carbon electrode was utilized as a sensing platform for the immobilization of IgG antigen and a competitive immunoreaction occurred while analyte anti-IgG and horseradish peroxidase (HRP)-labeled anti-IgG competed for the surface-immobilized IgG antigen binding sites. The catalytic reduction current at 0.030 V (vs. SCE), which is produced in the reaction of hydroquinone with hydrogen peroxide in the presence of HRP, was linear over a range from 0.30 to 10 μg/mL anti-IgG with a detection limit of 0.11 μg/mL.In Chapter 4, homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin and immunomagnetic electrogenerated chemiluminescence detection of digoxin based on Ru(bpy)_3~(2+) label have been developed.Homogeneous electrogenerated chemiluminescence immunoassay for the
determination of digoxin A novel homogeneous electrogenerated chemiluminescence immunoassay (ECLIA) for the determination of small hapten was developed. As a model system, digoxin was investigated while luminol served as luminescence label and BSA served as carrier protein. Digoxin was indirectly heavily labeled with luminol through BSA to form luminol-BSA-digoxin conjugate. The immunocomplex of luminol-BSA-digoxin conjugate with anti-digoxin antibody underwent less ECL reaction than luminol-BSA-digoxin conjugate after immunoreaction took place. Two immunoassay formats, directly homogeneous immunodetection for anti-digoxin antibody and competitive immunoassay for digoxin, were proposed to determine anti-digoxin antibody and digoxin, respectively. The anti-digoxin antibody concentration was determined in the range from 1/50000 to 1/2000 dilution. The ECL intensity vs. digoxin concentration was linear in the range from 5.0×10~(-10)g/mL to 3.0 ×10~(-8) g/mL. The detection limit was 2.8 ×10~(-10)g/mL. The relative standard derivation for 1.0 ×10~(-9)g/mL was 5.1%. The proposed method has been applied to assay digoxin in human control serum with satisfactory results.Immunomagnetic electrogenerated chemiluminescence detection of digoxin based on Ru(bpy)_3~(2+) label Immunomagnetic electrogenerated chemiluminescencedetection of digoxin based on Ru(bpy)_3~(2+) label was developed. Digoxin was covalently attached to magnetic beads and Ru(bpy)_3~(2+) was attached to anti-digoxin antibody. A competitive immunoreaction occurred between analyte digoxin and digoxin-labeled bead competing for the Ru(bpy)_3~(2+) labeled anti-digoxin antibody. After magnetic separation, the concentration of analyte digoxin, related to the concentration of the immunocomplex of digoxin and Ru(bpy)_3~(2+) labeled anti-digoxin conjugate, was determined by ECL produced by electrochemical oxidized Ru(bpy)_3~(2+) in the presence of TPA. The linear range for digoxin was 1.0 ×10~(-8) g/mL ~ 2.0 ×10~(-6) g/mL with a detection limit of 7.5×10~(-9)g/mL.In Chapter 5, MWNT paste electrode was fabricated in this chapter. At the MWNT paste electrode, electrochemical behaviors of bases of nuclueic acids and its analytical applications were investigated, and an electrochemical detection of DNA hybridization based on ss-DNA/ polypyrrole/MWNT paste electrode using ethidium bromide as a response indicator was developed. The interaction of vitamin B_6 /ofloxacin with DNA was also investigated.Electrochemical behaviors of bases of nuclueic acids at multiwall carbon
nanotube paste electrode and its analytical applications The electrochemical behaviors of five free bases of nucleic acids at a carbon paste electrode incorporated with MWNT were investigated by differential pulse voltammetry and cyclic voltammetry. It was found that in a 0.20 mol/L borate buffer solution (pH 11.0), the bases including guanine, adenine, thyrime, uracil and cytosine showed irreversible electrochemical behaviors and the corresponding oxidation peaks appeared at 0.556 V, 0.824 V, 0.952 V, 1.048 V and 1.160 V (vs. SCE), respectively. The oxidation peaks of bases were separated. A simple electrochemical method for the simultaneous determination of one purine or pyrimidine in the presence of related compounds (adenine in the presence of guanine, and guanine in the presence of cytosine) was developed using MWNT paste electrode. The proposed method has been applied to determination the value of (G+C)/(A+T) of HCl-digested DNA and other synthetic oligonucleotides with satisfactory results.Electrochemical detection of DNA hybridization based on ss-DNA/ polypyrrole/multiwall carbon nanotube paste electrode using EB as a response indicator A highly sensitive electrochemical method for the detection of DNA hybridization using a MWNT paste electrode and immobilizing nucleic acid probes within electropolymerized polypyrrole (PPy) was described. The detection approach relied on the oligonucleotide probes served as the counter anions during the growth of conducting PPy film on the MWNT paste electrodes. An electroactive intercalate, ethidium bromide, as an electrochemical hybridization indicator, was used to monitor the hybridization reaction by differential pulse voltammetry. The anodic peak current of EB after hybridization with the target ss-DNA was linearly related to the logarithmic value of the target ss-DNA concentration ranging from 5.0×10~(-10) to 1.0×10~(-8) mol/L. The detection limit was 1.0×10~(-10) mol/L.Electrochemical studies of the interaction between Vitamin B_6/ ofloxacin and DNA The interaction of vitamin B_6 /ofloxacin (OF) with DNA was investigated by electrochemical method. When DNA was added into vitamin B_6/OF solution, it was found that the oxidation current of vitamin B_6/OF decreased. The electrochemical behaviors in the absence and presence of DNA were investigated and the results showed that Vitamin B_6/OF and DNA formed an electrochemical inactive supermolecular complex.In conclusion, five electrochemical biosensors assembled by nanoparticles or
引文
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