基于树状分子的DNA电化学生物传感器研究
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摘要
脱氧核糖核酸(DNA)是重要的生命物质,其结构和功能的研究对遗传和变异有着重要意义,对DNA的检测分析是临床诊断的重要依据。常见的分析方法有色谱法、显微光度法、吸光光度法、荧光光度法、光散射技术、电化学分析法和传感器技术。其中DNA传感器又根据换能方法的不同分为电化学传感器、光化学传感器、压电传感器、光导纤维传感器等。DNA的电化学分析和电化学传感器的研究是近年来研究的热点,与荧光标记、发光标记等常见标记技术相比,具有选择性好、灵敏度高、测试费用低、易于微型化等优点,同时又不破坏测试体系,不受生物样品中混浊、溶血等情况干扰,所以能够在临床基因疾病检验、环境检测、法学鉴定等领域得到广泛的应用,逐渐成为分子生物学研究中直接进行DNA序列检测的方法之一。
树枝状大分子是上世纪中期开发的一类具有三维结构的合成高分子,其结构特点是高度支化,与传统线型聚合物相比,具有良好的溶解性和较低的黏度。其内部具有空腔,外部含有大量的功能基团,具有络合与分散各种金属微粒的潜在能力。作为一种新型分子,在表面活性剂、纳米复合材料等方面已展现出较好的应用前景。随着支化代数的增加,树枝状大分子的分子结构逐渐接近球形。与传统的表面活性剂相比,虽然分子结构不同,但是树枝状大分子的官能团也含有亲油基和亲水基,因此具有和传统表面活性剂相近的性质。同时,树枝状大分子的结构比传统高分子更明确,由于具有非结晶性、低黏度和较好的水溶性,并且末端可导入大量的反应性或功能性官能团,因此在生物医药,材料改性、工业催化及石油工业呈现出较好的应用性能。在纳米复合材料方面,树状大分子可以作为聚合模板,表面经修饰的PAMAM树状大分子还可以用来制备Cu—PAMAM、Ag—PAMAM、Au—PAMAM及CdS—PAMAM等树状金属纳米复合材料。树枝状大分子的特殊结构,即内部具有空腔,外部含有大量的功能基团,使其被广泛应用于DNA电化学传感器的制备。
本论文的工作致力于基于树状分子的DNA电化学生物传感器研究。主要包括以下三个方面:(1)PAMAM-NH_2(G4)、Ag/PAMAM-NH_2(G4)的光谱学研究;(2)羧基化多壁碳纳米管和PAMAM-NH_2(G2)增强的DNA电化学生物传感器;(3)基于DT4.5/Cu~(2+)标记DNA探针的DNA电化学传感器研究。本论文的新意在于将Ag/PAMAM-NH_2复合纳米粒子、羧基化多壁碳纳米管、PAMAM树状大分子和DT4.5/Cu~(2+)树枝状络合物应用到DNA电化学传感器中,并采用紫外-可见光谱、荧光光谱、红外光谱、原子力显微镜、差热-热重分析、X射线光电子能谱以及循环伏安、交流阻抗、差分脉冲伏安等电化学分析方法进行全方位表征。实验证明,制备的新型DNA电化学传感器灵敏度高、检测限低、稳定性和重现性好,且传感器能够再生。本论文主要内容分为以下几个部分:
一、PAMAM-NH_2(G4)、Ag/PAMAM-NH_2(G4)的光谱学研究
本文包括两部分内容:(一)通过原子力显微镜(AFM)成像,比较PAMAM-NH_2(G4)和PAMAM-NH_2(G2)结构形态的差异。研究四代以氨基为端基的聚酰胺胺树枝状化合物PAMAM-NH_2(G4)在不同条件下的荧光发射,讨论pH、陈化时间、浓度和生物分子dsDNA对其荧光强度的影响;(二)以PAMAM-NH_2(G4)为模版,制备Ag/PAMAM-NH_2(G4)树形纳米分子,讨论不同摩尔比(PAMAM-NH_2(G4):Ag+)的Ag+/PAMAM-NH_2(G4)溶液的滴定曲线图,从而定性地研究了不同摩尔比和pH值对Ag+与PAMAM-NH_2(G4)络合的影响,并用紫外-可见光谱,荧光光谱等测试手段对所制得的银树形纳米分子进行表征。Ag/PAMAM-NH_2(G4)树形纳米分子在DNA传感器中具有极其广阔的应用前景。
二、羧基化多壁碳纳米管和PAMAM-NH_2(G2)增强的DNA电化学生物传感器
本文介绍了一种新型的高灵敏度的DNA电化学传感器,即将羧基化的多壁碳纳米管和聚酰胺胺树枝状化合物应用到DNA电化学传感器中。我们在玻碳电极(GC)表面依次修饰羧基化的多壁碳纳米管(MWNT-COO-)和二代以氨基为端基的聚酰胺胺树枝状化合物( PAMAM-NH_2(G2) ),得到PAMAM/MWNT-COO-/GC电极。该修饰电极共价键合DNA探针序列,在不同浓度的互补序列溶液中经过杂交反应后,检测电极表面的阻抗变化,得到Rct—浓度曲线。采用CV、EIS、IR、XPS和TAG对层层修饰进行表征,同时讨论MWNT-COO-浓度及杂交时间的选择。该种传感器灵敏度高,线性范围宽,线性范围在5.0×10~(-10) M到5.0×10~(-14) M之间,最低检测限为1.034×10~(-14) M。实验证明,传感器稳定性佳、重现性好,同时具备良好的再生能力。另外,以柔红霉素为指示剂,检测DNA电化学探针对目标DNA的识别能力,柔红霉素响应信号明显。
三、基于DT4.5/Cu~(2+)标记DNA探针的DNA电化学传感器研究
利用DT4.5能与Cu~(2+)络合的特性,将大量Cu~(2+)嵌入DT4.5内部,首次将其标记DNA,制备DT4.5/Cu~(2+)标记的DNA电化学探针。本论文使用紫外吸收光谱表征DT4.5与Cu~(2+)络合的情况,研究了pH值对DT4.5与Cu~(2+)络合的影响和相关机理,并证明了DT4.5/Cu~(2+)标记的DNA电化学探针的成功制备。文章中还讨论和优化了包括修饰电极的制备、阳极溶出伏安法(ASV)检测Cu~(2+)的各种参数选择在内的各种实验条件,最终选择碳纳米管修饰电极、-0.4V作为最佳富集电位、300s作为还原富集时间。由于采用了灵敏度很高的阳极溶出伏安法并使用碳纳米管修饰电极进行测定,使传感器对特定序列DNA片段检测的灵敏度得到了极大的提高,检测下限达到1.0×10-12M,稳定性和重现性良好。
Deoxyribonucleic acid (DNA) is one of important materials to the life. Studies of its structure and function contribute to the research of genetic variation, and the detection and analysis of DNA is an important basis for clinical diagnosis. Common analytical methods include chromatography, micro-spectrophotometry, absorption spectrophotometry, fluorescence spectrophotometry, light scattering techniques, electrochemical analysis and sensor technology. DNA sensors, according to how the transducer works can be divided into different methods, such as electrochemical sensors, photochemical sensors, piezoelectric sensors, optical fiber sensors. Electrochemical DNA analysis and electrochemical sensor research are the hotspots in recent years. Compared with common markers such as the fluorescent, luminescent markers, its advantage is good selectivity, high sensitivity, testing low-cost, easy miniaturization, etc., without destroying the test system or disturbed by the turbidity and hemolytic interference from biological samples. It can be widely applied to clinical genetic diseases, environmental detection, and identification in the field of Law. DNA electrochemical sensors have gradually become direct DNA sequence detection methods in molecular biology research.
Dendrimers developed in the middle of last century are a kind of synthetic polymers of three-dimensional structure. Their structural characteristics are highly branched. As compared with traditional linear polymers, dendrimers have good solubility and low viscosity. With their internal cavity and external to contain a large number of functional groups, dendrimers can disperse or complex with various metal particles. As a new type of molecules in the surfactants, nano-composite materials have shown good application prospects. With an increase of their generation, the molecular structure of dendrimers is gradually close to spherical. With the traditional surfactants in comparison, although their molecular structure is different, the functional groups of the dendrimers also contain pro-oil-based and water-based, and therefore similar to traditional surfactants’nature. At the same time, the structure of dendrimers are much clearer than the traditional polymers, because of non-crystalline, low viscosity and good water-soluble, and the end to import a large number of reactive or functional groups, so dendrimers show better application performance in Biomedicine, Modification of Materials, Industrial Catalysis and the oil industry.The dendrimer can be used as a template polymerization in nanocomposites. After surface-modified, PAMAM dendrimer can also be used to prepare Cu-PAMAM, Ag-PAMAM, Au-PAMAM and CdS-PAMAM Dendrimer metal nanocomposites, etc. Because of the special structure of dendrimers, that is, with the internal cavity, external to contain a large number of functional groups, they are widely used in the preparation of DNA electrochemical sensor.
In this paper, the work is committed to developing DNA electrochemical biosensor based on dendrimers. This paper mainly includes the following three aspects: (1) The spectral analysis of PAMAM-NH_2 (G4), Ag/PAMAM-NH_2 (G4); (2) Carboxylated multi-wall carbon nanotubes and PAMAM-NH_2 (G2)-modified DNA electrochemical sensors; (3)Study of DNA electrochemical sensor based on DT4.5/Cu2+ -marked DNA probe. In this paper, the novelty lies in that we applied the composite nanoparticles Ag/PAMAM-NH_2, carboxylated multi-wall carbon nanotubes, PAMAM dendrimer and DT4.5/Cu2+ dendritic complexes to DNA electrochemical sensor, and got comprehensive characterization by ultraviolet - visible spectroscopy, fluorescence spectroscopy, infrared spectroscopy, atomic force microscopy, differential thermal - thermal gravimetric analysis, X-ray photoelectron spectroscopy and some electrochemical analysis methods such as cyclic voltammetry, AC impedance, differential pulse voltammetry, etc. Data show that this new type of DNA electrochemical sensor has high sensitivity and low detection line, good stability, good reproducibility and reproducibility. The main contents of this paper are divided into the following sections:
ⅠThe spectral analysis of PAMAM-NH_2 (G4), Ag/PAMAM-NH_2 (G4)
This article includes two parts: (1) Through the atomic force microscope (AFM) imaging, we compare PAMAM-NH_2 (G4) with PAMAM-NH_2 (G2) discovering morphology differences. A strong fluorescence emission of NH_2-terminated fourth-generation poly(amidoamine) dendrimers has been studied through changing pH, aging time, concentration of the solution of PAMAM-NH_2(G4)or adding biological moleculars dsDNA to this solution; (2) We take PAMAM as templates to create Ag/PAMAM-NH_2(?G4) dendrimer nanocomposites (DNC). We discuss titration curve of the solution of Ag+/PAMAM-NH_2 (G4). The results showed that different mole ratio of Ag+ to PAMAM and pH strongly influence complexation between Ag+ and PAMAM. The silver-DNCs were characterized by means of UV-visible absorption and fluorescence spectroscopy. Ag/PAMAM-NH_2 (G4) dendrimer nano moleculars in DNA sensors have an extremely wide application prospect.
ⅡCarboxylated multi-wall carbon nanotubes and PAMAM-NH_2 (G2)-modified DNA electrochemical sensors
In this paper, we introduce a new type of high-sensitivity DNA electrochemical sensor, that is, we applied carboxylated multi-wall carbon nanotubes and PAMAM dendrimer to DNA electrochemical sensor. Layer-by-layer films assembled by alternate adsorption of carboxylated multi-wall carbon nanotubes (MWNT-COO-) and NH_2-terminated second-generation poly(amidoamine) dendrimers(PAMAM-NH_2(G2)) onto a glassy carbon electrode were reported. After covalently adsorbed with PO4 probe sequence, PAMAM/MWNT-COO-/GC electrode was immersed in the solutions of complementary sequences of different concentrations, and then we can get Rct-concentration curve by EIS. Layer-by-layer films were characterized by CV, EIS, IR, XPS and TAG. At the same time, the concentration of MWNT-COO- and the timing of the hybridization were discussed. This sensor has demonstrated high sensitivity and wide linear range. Linear range is between 5.0×10~(-10) M to 5.0×10~(-14) M, and the minimum detection limit is 1.034×10~(-14) M. Experiments show that this sensor has good stability, good reproducibility, and at the same time, it has good regeneration capacity. In addition, we use daunomycin as indicator to test the recognition of DNA electrochemical probe on the target DNA. As a result, response signal is significant and satisfactory.
ⅢStudy of DNA electrochemical sensor based on DT4.5/Cu~(2+)-marked DNA probe
With the complexation between DT4.5 and Cu~(2+), it is the first time that we embed large amounts of Cu~(2+) into the inside of DT4.5, and then the complex is marked to ssDNA. Thus, we get DT4.5/Cu~(2+) labeled electrochemical DNA probe. In this paper, the complexation between DT4.5 and Cu~(2+) is charactered by UV absorption spectroscopy. At the same time, we study the impact of the pH value and mechanism of the complexation between DT4.5 and Cu~(2+), and prove the successful preparation of DT4.5/Cu~(2+) labeled DNA electrochemical probe. We also discuss and optimize a variety of experimental conditions, including the preparation of modified electrode and choosing the various parameters on the process of anodic stripping voltammetry (ASV) detection of Cu~(2+). As a result, we choose to use carbon nanotubes modified electrode, -0.4V as the optimal enrichment potential, and 300s as preconcentration time. As a result of using the highly-sensitive anodic stripping voltammetry and carbon nanotube modified electrode, the sensitivity of sensors on the specific sequence of DNA fragment detection has been greatly improved. The detection limit reachs 1.0×10~(-12)M. This sensor shows good stability and reproducibility.
树枝状大分子是上世纪中期开发的一类具有三维结构的合成高分子,其结构特点是高度支化,与传统线型聚合物相比,具有良好的溶解性和较低的黏度。其内部具有空腔,外部含有大量的功能基团,具有络合与分散各种金属微粒的潜在能力。作为一种新型分子,在表面活性剂、纳米复合材料等方面已展现出较好的应用前景。随着支化代数的增加,树枝状大分子的分子结构逐渐接近球形。与传统的表面活性剂相比,虽然分子结构不同,但是树枝状大分子的官能团也含有亲油基和亲水基,因此具有和传统表面活性剂相近的性质。同时,树枝状大分子的结构比传统高分子更明确,由于具有非结晶性、低黏度和较好的水溶性,并且末端可导入大量的反应性或功能性官能团,因此在生物医药,材料改性、工业催化及石油工业呈现出较好的应用性能。在纳米复合材料方面,树状大分子可以作为聚合模板,表面经修饰的PAMAM树状大分子还可以用来制备Cu—PAMAM、Ag—PAMAM、Au—PAMAM及CdS—PAMAM等树状金属纳米复合材料。树枝状大分子的特殊结构,即内部具有空腔,外部含有大量的功能基团,使其被广泛应用于DNA电化学传感器的制备。
本论文的工作致力于基于树状分子的DNA电化学生物传感器研究。主要包括以下三个方面:(1)PAMAM-NH_2(G4)、Ag/PAMAM-NH_2(G4)的光谱学研究;(2)羧基化多壁碳纳米管和PAMAM-NH_2(G2)增强的DNA电化学生物传感器;(3)基于DT4.5/Cu~(2+)标记DNA探针的DNA电化学传感器研究。本论文的新意在于将Ag/PAMAM-NH_2复合纳米粒子、羧基化多壁碳纳米管、PAMAM树状大分子和DT4.5/Cu~(2+)树枝状络合物应用到DNA电化学传感器中,并采用紫外-可见光谱、荧光光谱、红外光谱、原子力显微镜、差热-热重分析、X射线光电子能谱以及循环伏安、交流阻抗、差分脉冲伏安等电化学分析方法进行全方位表征。实验证明,制备的新型DNA电化学传感器灵敏度高、检测限低、稳定性和重现性好,且传感器能够再生。本论文主要内容分为以下几个部分:
一、PAMAM-NH_2(G4)、Ag/PAMAM-NH_2(G4)的光谱学研究
本文包括两部分内容:(一)通过原子力显微镜(AFM)成像,比较PAMAM-NH_2(G4)和PAMAM-NH_2(G2)结构形态的差异。研究四代以氨基为端基的聚酰胺胺树枝状化合物PAMAM-NH_2(G4)在不同条件下的荧光发射,讨论pH、陈化时间、浓度和生物分子dsDNA对其荧光强度的影响;(二)以PAMAM-NH_2(G4)为模版,制备Ag/PAMAM-NH_2(G4)树形纳米分子,讨论不同摩尔比(PAMAM-NH_2(G4):Ag+)的Ag+/PAMAM-NH_2(G4)溶液的滴定曲线图,从而定性地研究了不同摩尔比和pH值对Ag+与PAMAM-NH_2(G4)络合的影响,并用紫外-可见光谱,荧光光谱等测试手段对所制得的银树形纳米分子进行表征。Ag/PAMAM-NH_2(G4)树形纳米分子在DNA传感器中具有极其广阔的应用前景。
二、羧基化多壁碳纳米管和PAMAM-NH_2(G2)增强的DNA电化学生物传感器
本文介绍了一种新型的高灵敏度的DNA电化学传感器,即将羧基化的多壁碳纳米管和聚酰胺胺树枝状化合物应用到DNA电化学传感器中。我们在玻碳电极(GC)表面依次修饰羧基化的多壁碳纳米管(MWNT-COO-)和二代以氨基为端基的聚酰胺胺树枝状化合物( PAMAM-NH_2(G2) ),得到PAMAM/MWNT-COO-/GC电极。该修饰电极共价键合DNA探针序列,在不同浓度的互补序列溶液中经过杂交反应后,检测电极表面的阻抗变化,得到Rct—浓度曲线。采用CV、EIS、IR、XPS和TAG对层层修饰进行表征,同时讨论MWNT-COO-浓度及杂交时间的选择。该种传感器灵敏度高,线性范围宽,线性范围在5.0×10~(-10) M到5.0×10~(-14) M之间,最低检测限为1.034×10~(-14) M。实验证明,传感器稳定性佳、重现性好,同时具备良好的再生能力。另外,以柔红霉素为指示剂,检测DNA电化学探针对目标DNA的识别能力,柔红霉素响应信号明显。
三、基于DT4.5/Cu~(2+)标记DNA探针的DNA电化学传感器研究
利用DT4.5能与Cu~(2+)络合的特性,将大量Cu~(2+)嵌入DT4.5内部,首次将其标记DNA,制备DT4.5/Cu~(2+)标记的DNA电化学探针。本论文使用紫外吸收光谱表征DT4.5与Cu~(2+)络合的情况,研究了pH值对DT4.5与Cu~(2+)络合的影响和相关机理,并证明了DT4.5/Cu~(2+)标记的DNA电化学探针的成功制备。文章中还讨论和优化了包括修饰电极的制备、阳极溶出伏安法(ASV)检测Cu~(2+)的各种参数选择在内的各种实验条件,最终选择碳纳米管修饰电极、-0.4V作为最佳富集电位、300s作为还原富集时间。由于采用了灵敏度很高的阳极溶出伏安法并使用碳纳米管修饰电极进行测定,使传感器对特定序列DNA片段检测的灵敏度得到了极大的提高,检测下限达到1.0×10-12M,稳定性和重现性良好。
Deoxyribonucleic acid (DNA) is one of important materials to the life. Studies of its structure and function contribute to the research of genetic variation, and the detection and analysis of DNA is an important basis for clinical diagnosis. Common analytical methods include chromatography, micro-spectrophotometry, absorption spectrophotometry, fluorescence spectrophotometry, light scattering techniques, electrochemical analysis and sensor technology. DNA sensors, according to how the transducer works can be divided into different methods, such as electrochemical sensors, photochemical sensors, piezoelectric sensors, optical fiber sensors. Electrochemical DNA analysis and electrochemical sensor research are the hotspots in recent years. Compared with common markers such as the fluorescent, luminescent markers, its advantage is good selectivity, high sensitivity, testing low-cost, easy miniaturization, etc., without destroying the test system or disturbed by the turbidity and hemolytic interference from biological samples. It can be widely applied to clinical genetic diseases, environmental detection, and identification in the field of Law. DNA electrochemical sensors have gradually become direct DNA sequence detection methods in molecular biology research.
Dendrimers developed in the middle of last century are a kind of synthetic polymers of three-dimensional structure. Their structural characteristics are highly branched. As compared with traditional linear polymers, dendrimers have good solubility and low viscosity. With their internal cavity and external to contain a large number of functional groups, dendrimers can disperse or complex with various metal particles. As a new type of molecules in the surfactants, nano-composite materials have shown good application prospects. With an increase of their generation, the molecular structure of dendrimers is gradually close to spherical. With the traditional surfactants in comparison, although their molecular structure is different, the functional groups of the dendrimers also contain pro-oil-based and water-based, and therefore similar to traditional surfactants’nature. At the same time, the structure of dendrimers are much clearer than the traditional polymers, because of non-crystalline, low viscosity and good water-soluble, and the end to import a large number of reactive or functional groups, so dendrimers show better application performance in Biomedicine, Modification of Materials, Industrial Catalysis and the oil industry.The dendrimer can be used as a template polymerization in nanocomposites. After surface-modified, PAMAM dendrimer can also be used to prepare Cu-PAMAM, Ag-PAMAM, Au-PAMAM and CdS-PAMAM Dendrimer metal nanocomposites, etc. Because of the special structure of dendrimers, that is, with the internal cavity, external to contain a large number of functional groups, they are widely used in the preparation of DNA electrochemical sensor.
In this paper, the work is committed to developing DNA electrochemical biosensor based on dendrimers. This paper mainly includes the following three aspects: (1) The spectral analysis of PAMAM-NH_2 (G4), Ag/PAMAM-NH_2 (G4); (2) Carboxylated multi-wall carbon nanotubes and PAMAM-NH_2 (G2)-modified DNA electrochemical sensors; (3)Study of DNA electrochemical sensor based on DT4.5/Cu2+ -marked DNA probe. In this paper, the novelty lies in that we applied the composite nanoparticles Ag/PAMAM-NH_2, carboxylated multi-wall carbon nanotubes, PAMAM dendrimer and DT4.5/Cu2+ dendritic complexes to DNA electrochemical sensor, and got comprehensive characterization by ultraviolet - visible spectroscopy, fluorescence spectroscopy, infrared spectroscopy, atomic force microscopy, differential thermal - thermal gravimetric analysis, X-ray photoelectron spectroscopy and some electrochemical analysis methods such as cyclic voltammetry, AC impedance, differential pulse voltammetry, etc. Data show that this new type of DNA electrochemical sensor has high sensitivity and low detection line, good stability, good reproducibility and reproducibility. The main contents of this paper are divided into the following sections:
ⅠThe spectral analysis of PAMAM-NH_2 (G4), Ag/PAMAM-NH_2 (G4)
This article includes two parts: (1) Through the atomic force microscope (AFM) imaging, we compare PAMAM-NH_2 (G4) with PAMAM-NH_2 (G2) discovering morphology differences. A strong fluorescence emission of NH_2-terminated fourth-generation poly(amidoamine) dendrimers has been studied through changing pH, aging time, concentration of the solution of PAMAM-NH_2(G4)or adding biological moleculars dsDNA to this solution; (2) We take PAMAM as templates to create Ag/PAMAM-NH_2(?G4) dendrimer nanocomposites (DNC). We discuss titration curve of the solution of Ag+/PAMAM-NH_2 (G4). The results showed that different mole ratio of Ag+ to PAMAM and pH strongly influence complexation between Ag+ and PAMAM. The silver-DNCs were characterized by means of UV-visible absorption and fluorescence spectroscopy. Ag/PAMAM-NH_2 (G4) dendrimer nano moleculars in DNA sensors have an extremely wide application prospect.
ⅡCarboxylated multi-wall carbon nanotubes and PAMAM-NH_2 (G2)-modified DNA electrochemical sensors
In this paper, we introduce a new type of high-sensitivity DNA electrochemical sensor, that is, we applied carboxylated multi-wall carbon nanotubes and PAMAM dendrimer to DNA electrochemical sensor. Layer-by-layer films assembled by alternate adsorption of carboxylated multi-wall carbon nanotubes (MWNT-COO-) and NH_2-terminated second-generation poly(amidoamine) dendrimers(PAMAM-NH_2(G2)) onto a glassy carbon electrode were reported. After covalently adsorbed with PO4 probe sequence, PAMAM/MWNT-COO-/GC electrode was immersed in the solutions of complementary sequences of different concentrations, and then we can get Rct-concentration curve by EIS. Layer-by-layer films were characterized by CV, EIS, IR, XPS and TAG. At the same time, the concentration of MWNT-COO- and the timing of the hybridization were discussed. This sensor has demonstrated high sensitivity and wide linear range. Linear range is between 5.0×10~(-10) M to 5.0×10~(-14) M, and the minimum detection limit is 1.034×10~(-14) M. Experiments show that this sensor has good stability, good reproducibility, and at the same time, it has good regeneration capacity. In addition, we use daunomycin as indicator to test the recognition of DNA electrochemical probe on the target DNA. As a result, response signal is significant and satisfactory.
ⅢStudy of DNA electrochemical sensor based on DT4.5/Cu~(2+)-marked DNA probe
With the complexation between DT4.5 and Cu~(2+), it is the first time that we embed large amounts of Cu~(2+) into the inside of DT4.5, and then the complex is marked to ssDNA. Thus, we get DT4.5/Cu~(2+) labeled electrochemical DNA probe. In this paper, the complexation between DT4.5 and Cu~(2+) is charactered by UV absorption spectroscopy. At the same time, we study the impact of the pH value and mechanism of the complexation between DT4.5 and Cu~(2+), and prove the successful preparation of DT4.5/Cu~(2+) labeled DNA electrochemical probe. We also discuss and optimize a variety of experimental conditions, including the preparation of modified electrode and choosing the various parameters on the process of anodic stripping voltammetry (ASV) detection of Cu~(2+). As a result, we choose to use carbon nanotubes modified electrode, -0.4V as the optimal enrichment potential, and 300s as preconcentration time. As a result of using the highly-sensitive anodic stripping voltammetry and carbon nanotube modified electrode, the sensitivity of sensors on the specific sequence of DNA fragment detection has been greatly improved. The detection limit reachs 1.0×10~(-12)M. This sensor shows good stability and reproducibility.
引文
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