基于碳纳米管及离子液体增敏效应的电化学生物传感器：研制、表征与应用
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摘要
电化学传感器采用电极作为换能元件,具有便携、成本低、灵敏度高、稳定性良好等优点,被分析化学工作者寄予厚望。电化学生物传感器由生物材料作为敏感元件,以电势、电导(电阻)或电流为特征检测信号,具有高度选择性,是快速、直接获取复杂体系组成信息的理想分析工具。以纳米结构材料为媒介体,通过对生物分子或电极进行修饰,设计新颖的、功能化的纳米仿生界面进行生物界面与电极之间的信息转换的研究已遍布整个生物电化学研究领域并值得人们进一步付出更多的努力。
碳纳米管(CNTs)具有比表面积大、电荷传递能力强、吸附性好、生物相容性好和催化能力强等优良特性。室温离子液体(RTILs)不但可用作溶剂,又可作为支持电解质,具有电化学窗口宽、能促进电子传递、高离子导电性和良好的生物相容性等特点。特别是,RTILs与CNTs的结合有利于它们电分析化学优异特性的进一步发挥,为新型电化学传感器和电化学生物传感器的制备展现了美好前景。
本论文所作研究工作的重要目的是高灵敏度、高实用性电化学传感器和电化学生物传感器的制备与应用。其中提高常用电极的电分析化学性能是实验的基础目标。为此,CNTs被用作修饰电极的材料或者被用来制备阵列电极。结合RTILs的电分析化学特性,论文还构建了一种可用于超痕量检测的三明治模式。在以上基础上,对多种重要分子进行了电化学研究与检测。
全文包括十个章节。
第一章为文献综述,对研究背景和选题依据进行综述。
第二章至第六章的主要内容为脱氧核糖核酸(DNA)在高敏电极上的电化学研究。DNA是生物体内遗传信息的携带者、基因表达的物质基础,在生物的生长、发育和繁殖过程中起着十分重要的作用。对DNA的研究是生命科学研究中的一个极其重要的方面。就DNA检测与分析而言,电化学方法拥有许多方面的优势,比如高灵敏度、低检测限、快反应速度、低费用、微样品需求量等。基于高度灵敏的修饰电极或CNTs阵列电极,DNA及相关分子的伏安行为得到了研究。在此基础上,实验通过两种途径对源于转基因生物的特殊序列DNA片段进行了检测。
第二章:预处理过的多壁碳纳米管(MWNTs)修饰玻碳电极的制备(涂膜法)与表征和脱氧鸟苷三磷酸(dGTP)在修饰电极上的电化学行为及检测。在修饰电极上dGTP的氧化峰电位较之在裸玻碳电极上负移0.108 V;峰电流较之在裸玻碳电极上具有显著地增加。电子转移系数α为0.50。电极反应标准速率常数k's为0.16 s-1。
第三章:构建聚合酶链式反应(PCR)与电化学伏安技术相结合的检测模式用于特殊序列基因研究。以铁氰化钾和亚甲基蓝(MB)为电化学探针,表征结果表明羧基化短单壁碳纳米管(S-SWNTs)修饰玻碳电极具有非常优良的电分析化学性能。以之为换能器微分脉冲伏安(DPV)检测PCR反应前后混合液中游离dGTP的浓度变化,据结果得到PCR扩增反应是否成功的信息,从而推测出模板DNA中目标基因的存在与否,建立一种低费用、快捷的转基因生物鉴定模式并应用于转基因生物实际样品,所得结果与用凝胶电泳法测定所得结果一致。
第四章:S-SWNTs与疏水性室温离子液体(RTIL)——1-丁基-3-甲基咪唑六氟磷酸盐( BMIMPF6 )研成胶,修饰在玻碳电极上制备修饰电极S-SWNT&RTIL/GCE。以铁氰化钾、抗坏血酸(AA)和MB为电化学探针表征结果表明,该修饰电极具有优异的电催化性能和富集效应。单链DNA(ssDNA)在其上具有灵敏的伏安响应,于0.532 V和0.808 V处分别出现鸟嘌呤碱基和腺嘌呤碱基的氧化峰。鸟嘌呤碱基和腺嘌呤碱基在S-SWNT&RTIL/GCE上的电极反应标准速率常数k's分别为1.84×10~(-2) s~(-1)和3.69×10~(-2) s~(-1)。
第五章:把S-SWNTs与BMIMPF6混合起来制备一种新型糊电极(S-SWNT/IL PE)。与以石蜡为粘合剂制备的S-SWNT糊电极(S-SWNT/oil PE)相比,该新型电极对多种电化学探针都具有更好的电催化活性和富集效应。ssDNA在其上具有非常灵敏的催化氧化伏安响应。利用鸟嘌呤碱基的DPV信号,寡核苷酸的浓度检测限可达9.9 pmol/L。该电极可以准确指示出一定浓度范围内寡核苷酸中鸟嘌呤碱基或腺嘌呤碱基的个数。
第六章:在活化剂1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)存在下,以乙二胺为链接剂,羧基化的S-SWNTs被垂直组装在玻碳电极表面,构建成阵列修饰电极(SWNTE)。基于ssDNA与SWNTs的特殊相互作用机理,探针ssDNA被非共价固定在SWNTE上。鸟嘌呤碱基和腺嘌呤碱基都具有灵敏的微分脉冲伏安响应。与互补ssDNA杂交后所形成的dsDNA在负电位等因素的帮助下离开SWNTE,造成鸟嘌呤碱基和腺嘌呤碱基的氧化峰电流降低。据此检测特殊序列基因片段。通过超声洗涤,该免指示剂杂交传感器表面可以很方便地更新,可快捷、灵活地运用于不同靶基因的检测。
第七章到第十章内容为高灵敏度CNTs修饰电极及三明治检测法在其它一些与环境和生命相关的重要分子的研究中的应用。
第七章:以具有宽电化学窗口的疏水性RTIL—BMIMPF6为膜材料,将待检测的目标物质封在S-SWCNT/GCE的表面,实验建立了一种三明治结构的电化学伏安检测模式。该模式具有高的灵敏度、精密度和稳定性,对于样品量较少的痕量检测尤其具有现实意义。将之应用于AA和多巴胺(DA)的检测,检测限分别可低达400 fmol和80 fmol,且可以用于在大量AA存在的情况下选择性测定DA。
第八章:研究了鸟嘌呤在S-SWNT/GCE上发生的电催化氧化特性,根据其氧化电位对支持电解质溶液的pH值具有灵敏的响应,制备了以鸟嘌呤为指示剂的固体电位型pH传感器。该传感器制备简单、使用方便,在优化各种影响因子后,于pH 2.0 - 12.0范围内具有宽的线性响应。酸碱滴定终点具有较明显的突跃。
第九章:运用三明治模式把富勒烯C60和富勒烯C60纳米管(FNTs)分别封在S-SWCNT/GCE的表面,考察了常温下该结构在B-R缓冲溶液中的伏安行为,并对富勒烯C60和FNTs的电极过程从机理上进行了推测。此外,基于DNA和FNTs的相互作用,实验制备了在水溶液中分散性较好的FNTs,将之修饰在GCE表面,进一步研究了FNTs的电极过程机理。该修饰电极在碱性支持电解质中还原后能够明显增大DA和AA的氧化峰电位间距,使两峰能够完全分开,从而可以在共存时进行选择性检测。
第十章:研究了AA在MWNT/GCE上的电催化氧化行为。在pH 4.0的B-R缓冲溶液中首次得到AA两个分离良好的氧化峰。根据AA分子与表面羧基化的MWNTs相结合后的电子传递性质对AA的两个氧化峰进行了初步解释。应用MWNT/GCE对AA以微分脉冲伏安法测定得到了很低的检测限。
Electrochemical sensors are centered great expectations for their obvious advantages, such as portability, low-cost, sensitivity and stability. Among them, electrochemical biosensors, which employ electrode as transducer and biomaterials as sensing organ, are ideal devices for directly and selectively getting information from a complex system by monitoring the signal of potential, conductance or current. It is an attractive strategy to develop novel biosensors via the modification of electrode and biomolecules. Especially, the nanostructure materials can be exploited to increase the conversion of information between the target substance and electrode in the investigation of bioelectrochemistry and related fields. So far there are still great opportunities for us to carry on further research.
Carbon nanotubes (CNTs) exhibit many extraordinarily attractive physical and chemical properties, such as the large surface area, the good ability to promote electro-transfer reactions, remarkable catalysis towards biomolecules, good biocompatibility, accumulating many kinds of molecules and etc. Room temperature ionic liquids (RTILs) can be used as electrolyte as well as solvent. They also have attracted considerable attention due to their unique physical and chemical properties, such as high thermal and chemical stability, high conductivity, the ability to dissolve a wide range of organic and inorganic molecules, wide electrochemical potential window, ability to facilitate direct electron-transfer reactions, good biocompatibility and etc. Since the introduction, CNTs and RTILs have received enormous attention in the fields of electrochemistry and analytical electrochemistry. Moreover, it’s attractive that the combined application of them has more promising prospects in many fields of electroanalytical chemistry, for example, the fabrication of electrochemical sensors and electrochemical biosensors.
The goal of the research work is the achievement of sensitive and practical electrochemical sensors and electrochemical biosensors. To fulfill it, CNTs are used to modify electrodes or to be assembled as array electrode to increase the electroanalytical properties of common electrode. Based on the preparation of the sensitive transducer, a sandwich-type mode, which is competent for trace amount detection, is established by employing RTILs as membrane material.
The paper consists of ten chapters in all.
The first chapter is the introduction of the subject. The background of the research is reviewed and the direction of my work is stated.
DNA is the carriers of hereditary information and the basic substance of gene expression, and plays key roles in the growth, development and breeding of lives. So, it’s an important aspect to discover the information about DNA molecule. Electrochemical protocols exhibit many advantages in the assay of DNA, such as high sensitivity, low detection limit, low cost, fast response, and so on. From Chapter Two to Chapter Six, the contents are focused on the investigation of DNA and related molecules at the sensitive modified electrode. Combining polymerase chain reaction (PCR) technology or using label-free hybridization biosensor, we established two modes to detect sequence-specific DNA related to genetically modified organisms (GMOs) successfully with voltammetric methods.
In Chapter Two, the treated multi-walled carbon nanotubes (MWNTs) are coated onto the glassy carbon electrode (GCE) to fabricate a modified electrode (MWNT/GCE). At the MWNT/GCE the electrochemical behaviors of 2′-dexyguanosine 5′-triphosphate trisodiums salt (dGTP), which is one of the four reactants in PCR, are investigated. In 0.2 mol/L B-R buffer solution the oxidative peak potential (Epa) of dGTP at the MWNT/GCE shifted 0.108 V negatively in contrast to that at the GCE. And the oxidative peak current (ipa) increased dramatically in contrast to that at the GCE. The electron-transfer coefficientαand the heterogeneous electron transfer rate constant k's are 0.50 and 0.16 s-1, respectively.
Chapter Three centers the establishment and application of a fast and cost-effective protocol for detecting sequence-specific DNA by combining PCR and electrochemical technologies. Characterizing with cyclic voltammetry by using potassium ferricyanide and methylene blue (MB) as probes, we find that the short single-walled carbon nanotubes (S-SWNTs) modified glassy carbon electrode (S-SWNT/GCE) has attractive electroanalytical properties. A dramatic enhancement of the ipa and a visible decrease of overpotential towards dGTP can be realized at it. The ipa of the free dGTP decreases remarkably after a successful PCR amplification owing to the participation of the free dGTP as one of reactive substances for the PCR products, namely dsDNA. Based upon this response change of the free dGTP before and after incorporation in PCR, the target gene in the DNA template is present or not can be deduced. Thus, the GMOs sample, which provides template DNA, can be detected easily. And the result is in good accordance with that obtained with gel electrophoresis.
In Chapter Four, a gel-like paste is made by mixing 1:1(w/w) S-SWNTs and a kind of RTIL of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6). The composite film is modified onto the GCE to prepare the S-SWNT&RTIL/GCE. The modified electrode shows attractive electrocatalytic ability and can enhance the current response of electro-active molecules by employing potassium ferricyanide, AA and MB as probes. ssDNA has a sensitive voltammetric response at the S-SWNT&RTIL/GCE and the Epas of guanine base and adenine base are 0.532 V and 0.808 V, respectively. The heterogeneous electron transfer rate constants k's for the two bases are 1.84×10~(-2) s~(-1) and 3.69×10~(-2) s~(-1), respectively.
The content of Chapter Five consists of the preparation, characterization and application of a novel kind of paste electrode (S-SWNT&RTIL PE), which is fabricated with S-SWNTs mixed RTIL of BMIMPF6. Its electrochemical behavior is investigated by voltammetry and electrochemical impedance spectroscopy (EIS) in comparison with the paste electrode using mineral oil as a binder. Results highlight the advantages of it: not only higher conductivity, but also lower potential separation (?Ep), higher peak current (ip) and better reversibility towards a number of molecules. Based on the current response of guanine bases, the S-SWNT&RTIL PE can be used to detect ssDNA sensitively with a detection limit of 9.9 pmol/L. And the number of guanine bases and adenine bases contents in per mol oligonucleotides can be monitored according to the current response within a rather wide range.
In Chapter Six, carboxylic group-functionalized S-SWNTs are assembled vertically on the GCE using ethylenediamine as linking agent to fabricate an aligned electrode (SWNTE) in the presence of EDC and NHS. ssDNA wrap around the SWNTs to form ssDNA-wrapped-SWNTE structures based on the interaction between ssDNA and SWNT. Sensitive differential pulse voltammetric (DPV) responses are obtained at the ssDNA-wrapped-SWNTE owing to the electrooxidation of guanine bases and adenine bases. dsDNA is formed when ssDNA on the ssDNA-wrapped-SWNTE is hybridized with complementary ssDNA (cDNA). The dsDNA is removed from the SWNTs through preconditioning and rinsing processes. Consequentially, the DPV current response of guanine bases decreased. Based on this mechanism, a label-free and readily reusable electrochemical DNA hybridization biosensor is designed by directly monitoring the current change of guanine bases. The used SWNTE can be renewed easily via ultrasonically rinsing. Thus, the biosensor can be switched to detect different target DNAs easily.
From Chapter Seven to Chapter Ten, the contents consist of the establishment of sandwich mode for trace amount detection and the application of the sensitive electrode in some other important molecules.
In consideration with the advantages of SWNTs and RTIL in detecting target molecules (TMs), a novel strategy of sandwich-type electrode is established with TMs confined by RTIL between the S-SWNT/GCE and RTIL membrane. This strategy shows high sensitivity, good precision and good stability. It is of especial importance towards trace amount detection. It can be used for electrochemical detection of AA and dopamine (DA) with the detection limits of 400 fmol and 80 fmol, respectively. The selective detection of DA in the presence of high amount of AA can be performed, too.
In Chapter Eight, the electrochemical properties of guanine at the S-SWCNT/GCE are studied. The Epa of guanine responds sensitively to the pH of buffer solution. Based on this, the S-SWCNT/GCE/guanine system is used to prepare a novel potentiometric pH sensor. The sensor shows linear response to pH values in the range of 2.0 - 12.0 with a slope of 0.0497 V/pH. And there is a remarkable potential jump on the potentiometric titration curve.
In B-R buffer solution, the voltammetric responses of fullerene C60 and fullerene C60 nanotubes (FNTs) are investigated with sandwich mode at room temperature. The mechanism of the electrode processes are presumed for the first time. Moreover, with the aid of DNA, FNTs are dispersed into aqueous solution, followed by being immobilized onto the GCE surface. And the mechanism of the electrode process of FNTs is characterized further. After undergoing a reductive reaction in NaOH solution, the modified electrode can be used to selectively detect DA in the coexistence of AA owing to the perfect separation of their electrooxidative peaks.
In the last Chapter, the catalytic electrooxidation of AA at the MWNT/GCE is investigated. AA has two well-separated oxiditive peaks in B-R buffer solution of pH 4.0 and the reaction mechanism is illustrated according to the molecular structure of AA and the carboxylic acid group-functionalized MWNTs. A rather low detection limit of AA is obtained with DPV measurement using MWNT/GCE as working electrode.
碳纳米管(CNTs)具有比表面积大、电荷传递能力强、吸附性好、生物相容性好和催化能力强等优良特性。室温离子液体(RTILs)不但可用作溶剂,又可作为支持电解质,具有电化学窗口宽、能促进电子传递、高离子导电性和良好的生物相容性等特点。特别是,RTILs与CNTs的结合有利于它们电分析化学优异特性的进一步发挥,为新型电化学传感器和电化学生物传感器的制备展现了美好前景。
本论文所作研究工作的重要目的是高灵敏度、高实用性电化学传感器和电化学生物传感器的制备与应用。其中提高常用电极的电分析化学性能是实验的基础目标。为此,CNTs被用作修饰电极的材料或者被用来制备阵列电极。结合RTILs的电分析化学特性,论文还构建了一种可用于超痕量检测的三明治模式。在以上基础上,对多种重要分子进行了电化学研究与检测。
全文包括十个章节。
第一章为文献综述,对研究背景和选题依据进行综述。
第二章至第六章的主要内容为脱氧核糖核酸(DNA)在高敏电极上的电化学研究。DNA是生物体内遗传信息的携带者、基因表达的物质基础,在生物的生长、发育和繁殖过程中起着十分重要的作用。对DNA的研究是生命科学研究中的一个极其重要的方面。就DNA检测与分析而言,电化学方法拥有许多方面的优势,比如高灵敏度、低检测限、快反应速度、低费用、微样品需求量等。基于高度灵敏的修饰电极或CNTs阵列电极,DNA及相关分子的伏安行为得到了研究。在此基础上,实验通过两种途径对源于转基因生物的特殊序列DNA片段进行了检测。
第二章:预处理过的多壁碳纳米管(MWNTs)修饰玻碳电极的制备(涂膜法)与表征和脱氧鸟苷三磷酸(dGTP)在修饰电极上的电化学行为及检测。在修饰电极上dGTP的氧化峰电位较之在裸玻碳电极上负移0.108 V;峰电流较之在裸玻碳电极上具有显著地增加。电子转移系数α为0.50。电极反应标准速率常数k's为0.16 s-1。
第三章:构建聚合酶链式反应(PCR)与电化学伏安技术相结合的检测模式用于特殊序列基因研究。以铁氰化钾和亚甲基蓝(MB)为电化学探针,表征结果表明羧基化短单壁碳纳米管(S-SWNTs)修饰玻碳电极具有非常优良的电分析化学性能。以之为换能器微分脉冲伏安(DPV)检测PCR反应前后混合液中游离dGTP的浓度变化,据结果得到PCR扩增反应是否成功的信息,从而推测出模板DNA中目标基因的存在与否,建立一种低费用、快捷的转基因生物鉴定模式并应用于转基因生物实际样品,所得结果与用凝胶电泳法测定所得结果一致。
第四章:S-SWNTs与疏水性室温离子液体(RTIL)——1-丁基-3-甲基咪唑六氟磷酸盐( BMIMPF6 )研成胶,修饰在玻碳电极上制备修饰电极S-SWNT&RTIL/GCE。以铁氰化钾、抗坏血酸(AA)和MB为电化学探针表征结果表明,该修饰电极具有优异的电催化性能和富集效应。单链DNA(ssDNA)在其上具有灵敏的伏安响应,于0.532 V和0.808 V处分别出现鸟嘌呤碱基和腺嘌呤碱基的氧化峰。鸟嘌呤碱基和腺嘌呤碱基在S-SWNT&RTIL/GCE上的电极反应标准速率常数k's分别为1.84×10~(-2) s~(-1)和3.69×10~(-2) s~(-1)。
第五章:把S-SWNTs与BMIMPF6混合起来制备一种新型糊电极(S-SWNT/IL PE)。与以石蜡为粘合剂制备的S-SWNT糊电极(S-SWNT/oil PE)相比,该新型电极对多种电化学探针都具有更好的电催化活性和富集效应。ssDNA在其上具有非常灵敏的催化氧化伏安响应。利用鸟嘌呤碱基的DPV信号,寡核苷酸的浓度检测限可达9.9 pmol/L。该电极可以准确指示出一定浓度范围内寡核苷酸中鸟嘌呤碱基或腺嘌呤碱基的个数。
第六章:在活化剂1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)存在下,以乙二胺为链接剂,羧基化的S-SWNTs被垂直组装在玻碳电极表面,构建成阵列修饰电极(SWNTE)。基于ssDNA与SWNTs的特殊相互作用机理,探针ssDNA被非共价固定在SWNTE上。鸟嘌呤碱基和腺嘌呤碱基都具有灵敏的微分脉冲伏安响应。与互补ssDNA杂交后所形成的dsDNA在负电位等因素的帮助下离开SWNTE,造成鸟嘌呤碱基和腺嘌呤碱基的氧化峰电流降低。据此检测特殊序列基因片段。通过超声洗涤,该免指示剂杂交传感器表面可以很方便地更新,可快捷、灵活地运用于不同靶基因的检测。
第七章到第十章内容为高灵敏度CNTs修饰电极及三明治检测法在其它一些与环境和生命相关的重要分子的研究中的应用。
第七章:以具有宽电化学窗口的疏水性RTIL—BMIMPF6为膜材料,将待检测的目标物质封在S-SWCNT/GCE的表面,实验建立了一种三明治结构的电化学伏安检测模式。该模式具有高的灵敏度、精密度和稳定性,对于样品量较少的痕量检测尤其具有现实意义。将之应用于AA和多巴胺(DA)的检测,检测限分别可低达400 fmol和80 fmol,且可以用于在大量AA存在的情况下选择性测定DA。
第八章:研究了鸟嘌呤在S-SWNT/GCE上发生的电催化氧化特性,根据其氧化电位对支持电解质溶液的pH值具有灵敏的响应,制备了以鸟嘌呤为指示剂的固体电位型pH传感器。该传感器制备简单、使用方便,在优化各种影响因子后,于pH 2.0 - 12.0范围内具有宽的线性响应。酸碱滴定终点具有较明显的突跃。
第九章:运用三明治模式把富勒烯C60和富勒烯C60纳米管(FNTs)分别封在S-SWCNT/GCE的表面,考察了常温下该结构在B-R缓冲溶液中的伏安行为,并对富勒烯C60和FNTs的电极过程从机理上进行了推测。此外,基于DNA和FNTs的相互作用,实验制备了在水溶液中分散性较好的FNTs,将之修饰在GCE表面,进一步研究了FNTs的电极过程机理。该修饰电极在碱性支持电解质中还原后能够明显增大DA和AA的氧化峰电位间距,使两峰能够完全分开,从而可以在共存时进行选择性检测。
第十章:研究了AA在MWNT/GCE上的电催化氧化行为。在pH 4.0的B-R缓冲溶液中首次得到AA两个分离良好的氧化峰。根据AA分子与表面羧基化的MWNTs相结合后的电子传递性质对AA的两个氧化峰进行了初步解释。应用MWNT/GCE对AA以微分脉冲伏安法测定得到了很低的检测限。
Electrochemical sensors are centered great expectations for their obvious advantages, such as portability, low-cost, sensitivity and stability. Among them, electrochemical biosensors, which employ electrode as transducer and biomaterials as sensing organ, are ideal devices for directly and selectively getting information from a complex system by monitoring the signal of potential, conductance or current. It is an attractive strategy to develop novel biosensors via the modification of electrode and biomolecules. Especially, the nanostructure materials can be exploited to increase the conversion of information between the target substance and electrode in the investigation of bioelectrochemistry and related fields. So far there are still great opportunities for us to carry on further research.
Carbon nanotubes (CNTs) exhibit many extraordinarily attractive physical and chemical properties, such as the large surface area, the good ability to promote electro-transfer reactions, remarkable catalysis towards biomolecules, good biocompatibility, accumulating many kinds of molecules and etc. Room temperature ionic liquids (RTILs) can be used as electrolyte as well as solvent. They also have attracted considerable attention due to their unique physical and chemical properties, such as high thermal and chemical stability, high conductivity, the ability to dissolve a wide range of organic and inorganic molecules, wide electrochemical potential window, ability to facilitate direct electron-transfer reactions, good biocompatibility and etc. Since the introduction, CNTs and RTILs have received enormous attention in the fields of electrochemistry and analytical electrochemistry. Moreover, it’s attractive that the combined application of them has more promising prospects in many fields of electroanalytical chemistry, for example, the fabrication of electrochemical sensors and electrochemical biosensors.
The goal of the research work is the achievement of sensitive and practical electrochemical sensors and electrochemical biosensors. To fulfill it, CNTs are used to modify electrodes or to be assembled as array electrode to increase the electroanalytical properties of common electrode. Based on the preparation of the sensitive transducer, a sandwich-type mode, which is competent for trace amount detection, is established by employing RTILs as membrane material.
The paper consists of ten chapters in all.
The first chapter is the introduction of the subject. The background of the research is reviewed and the direction of my work is stated.
DNA is the carriers of hereditary information and the basic substance of gene expression, and plays key roles in the growth, development and breeding of lives. So, it’s an important aspect to discover the information about DNA molecule. Electrochemical protocols exhibit many advantages in the assay of DNA, such as high sensitivity, low detection limit, low cost, fast response, and so on. From Chapter Two to Chapter Six, the contents are focused on the investigation of DNA and related molecules at the sensitive modified electrode. Combining polymerase chain reaction (PCR) technology or using label-free hybridization biosensor, we established two modes to detect sequence-specific DNA related to genetically modified organisms (GMOs) successfully with voltammetric methods.
In Chapter Two, the treated multi-walled carbon nanotubes (MWNTs) are coated onto the glassy carbon electrode (GCE) to fabricate a modified electrode (MWNT/GCE). At the MWNT/GCE the electrochemical behaviors of 2′-dexyguanosine 5′-triphosphate trisodiums salt (dGTP), which is one of the four reactants in PCR, are investigated. In 0.2 mol/L B-R buffer solution the oxidative peak potential (Epa) of dGTP at the MWNT/GCE shifted 0.108 V negatively in contrast to that at the GCE. And the oxidative peak current (ipa) increased dramatically in contrast to that at the GCE. The electron-transfer coefficientαand the heterogeneous electron transfer rate constant k's are 0.50 and 0.16 s-1, respectively.
Chapter Three centers the establishment and application of a fast and cost-effective protocol for detecting sequence-specific DNA by combining PCR and electrochemical technologies. Characterizing with cyclic voltammetry by using potassium ferricyanide and methylene blue (MB) as probes, we find that the short single-walled carbon nanotubes (S-SWNTs) modified glassy carbon electrode (S-SWNT/GCE) has attractive electroanalytical properties. A dramatic enhancement of the ipa and a visible decrease of overpotential towards dGTP can be realized at it. The ipa of the free dGTP decreases remarkably after a successful PCR amplification owing to the participation of the free dGTP as one of reactive substances for the PCR products, namely dsDNA. Based upon this response change of the free dGTP before and after incorporation in PCR, the target gene in the DNA template is present or not can be deduced. Thus, the GMOs sample, which provides template DNA, can be detected easily. And the result is in good accordance with that obtained with gel electrophoresis.
In Chapter Four, a gel-like paste is made by mixing 1:1(w/w) S-SWNTs and a kind of RTIL of 1-butyl-3-methylimidazolium hexafluorophosphate (BMIMPF6). The composite film is modified onto the GCE to prepare the S-SWNT&RTIL/GCE. The modified electrode shows attractive electrocatalytic ability and can enhance the current response of electro-active molecules by employing potassium ferricyanide, AA and MB as probes. ssDNA has a sensitive voltammetric response at the S-SWNT&RTIL/GCE and the Epas of guanine base and adenine base are 0.532 V and 0.808 V, respectively. The heterogeneous electron transfer rate constants k's for the two bases are 1.84×10~(-2) s~(-1) and 3.69×10~(-2) s~(-1), respectively.
The content of Chapter Five consists of the preparation, characterization and application of a novel kind of paste electrode (S-SWNT&RTIL PE), which is fabricated with S-SWNTs mixed RTIL of BMIMPF6. Its electrochemical behavior is investigated by voltammetry and electrochemical impedance spectroscopy (EIS) in comparison with the paste electrode using mineral oil as a binder. Results highlight the advantages of it: not only higher conductivity, but also lower potential separation (?Ep), higher peak current (ip) and better reversibility towards a number of molecules. Based on the current response of guanine bases, the S-SWNT&RTIL PE can be used to detect ssDNA sensitively with a detection limit of 9.9 pmol/L. And the number of guanine bases and adenine bases contents in per mol oligonucleotides can be monitored according to the current response within a rather wide range.
In Chapter Six, carboxylic group-functionalized S-SWNTs are assembled vertically on the GCE using ethylenediamine as linking agent to fabricate an aligned electrode (SWNTE) in the presence of EDC and NHS. ssDNA wrap around the SWNTs to form ssDNA-wrapped-SWNTE structures based on the interaction between ssDNA and SWNT. Sensitive differential pulse voltammetric (DPV) responses are obtained at the ssDNA-wrapped-SWNTE owing to the electrooxidation of guanine bases and adenine bases. dsDNA is formed when ssDNA on the ssDNA-wrapped-SWNTE is hybridized with complementary ssDNA (cDNA). The dsDNA is removed from the SWNTs through preconditioning and rinsing processes. Consequentially, the DPV current response of guanine bases decreased. Based on this mechanism, a label-free and readily reusable electrochemical DNA hybridization biosensor is designed by directly monitoring the current change of guanine bases. The used SWNTE can be renewed easily via ultrasonically rinsing. Thus, the biosensor can be switched to detect different target DNAs easily.
From Chapter Seven to Chapter Ten, the contents consist of the establishment of sandwich mode for trace amount detection and the application of the sensitive electrode in some other important molecules.
In consideration with the advantages of SWNTs and RTIL in detecting target molecules (TMs), a novel strategy of sandwich-type electrode is established with TMs confined by RTIL between the S-SWNT/GCE and RTIL membrane. This strategy shows high sensitivity, good precision and good stability. It is of especial importance towards trace amount detection. It can be used for electrochemical detection of AA and dopamine (DA) with the detection limits of 400 fmol and 80 fmol, respectively. The selective detection of DA in the presence of high amount of AA can be performed, too.
In Chapter Eight, the electrochemical properties of guanine at the S-SWCNT/GCE are studied. The Epa of guanine responds sensitively to the pH of buffer solution. Based on this, the S-SWCNT/GCE/guanine system is used to prepare a novel potentiometric pH sensor. The sensor shows linear response to pH values in the range of 2.0 - 12.0 with a slope of 0.0497 V/pH. And there is a remarkable potential jump on the potentiometric titration curve.
In B-R buffer solution, the voltammetric responses of fullerene C60 and fullerene C60 nanotubes (FNTs) are investigated with sandwich mode at room temperature. The mechanism of the electrode processes are presumed for the first time. Moreover, with the aid of DNA, FNTs are dispersed into aqueous solution, followed by being immobilized onto the GCE surface. And the mechanism of the electrode process of FNTs is characterized further. After undergoing a reductive reaction in NaOH solution, the modified electrode can be used to selectively detect DA in the coexistence of AA owing to the perfect separation of their electrooxidative peaks.
In the last Chapter, the catalytic electrooxidation of AA at the MWNT/GCE is investigated. AA has two well-separated oxiditive peaks in B-R buffer solution of pH 4.0 and the reaction mechanism is illustrated according to the molecular structure of AA and the carboxylic acid group-functionalized MWNTs. A rather low detection limit of AA is obtained with DPV measurement using MWNT/GCE as working electrode.
引文
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