ELECTROCHEMICAL DNA-BASED SENSORS

基于电化学 DNA 的传感器

• 批准号: 6363346
• 负责人: JACQUELINE K BARTON
• 金额: $ 24.66万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6363346
• 关键词: DNA binding protein; analytical method; bioengineering /biomedical engineering; biosensor device; biotherapeutic agent; electrochemistry; electrodes; electron transport; gene mutation; microarray technology; microelectrodes; molecular stacking; nucleic acid sequence; p53 gene /protein; phosphoester ligase; protein sequence

The proposed program has as its goal the design and application of DNA- based electrochemical sensors. Electrodes modified with DNA oligonucleotide duplexes and a non-covalently bound intercalator as redox probe will be constructed as sensors for single base mismatches. The approach is not based fundamentally on differential hybridization ir a molecular recognition event. Instead these DNA-based sensors rely upon DNA-mediated charge transport and its sensitivity to perturbations in DNA stacking. Preliminary results have established the individual elements necessary to construct a sensor for mutational analysis: 9i) different single base-mismatches can be detected electrochemically and the sequence composition of the film can be varied substantially; (ii0 single-stranded DNAs can be assayed with repeated cycling of hybridization and denaturation on the electrode; significantly, a high level of sensitivity and discrimination for single base mismatches are achieved by coupling of the DNA-mediated charge transported to an electrocatalytic cycle. It is proposed first that the scope and sensitivity associated with this design can be systematically examined, where oligonucleotide length, sequence, and intercalator probe are varied; additionally variations in linker length and the construction of mixed surfaces will be carried out. These studies will enable not only the development of mismatch probes but also of new electrochemical probes for DNA-binding proteins and small molecules. These DNA films will be utilized to develop electrochemical assays for DNA-binding proteins, for example base flipping enzymes, based upon their perturbations to DNA stacking. The DNA-modified electrodes will also be utilized to probe the redox chemistry of DNA repair proteins, such as photolyase and Mut Y, and chemotherapeutics, such as the enediynes and anthracycline antibiotics. Finally, DNA chips will be fabricated for the analysis first of mutational hot spots in the p53 gene and thereafter for the analysis of the entire p53 gene sequence. The proposed program therefore intends to exploit DNA-mediated charge transport for the development of a completely new family of DNA-based sensors.

拟议项目的目标是基于 DNA 的电化学传感器的设计和应用。用 DNA 寡核苷酸双链体和非共价结合嵌入剂作为氧化还原探针修饰的电极将被构建为单碱基错配的传感器。该方法并不是从根本上基于分子识别事件中的差异杂交。相反，这些基于 DNA 的传感器依赖于 DNA 介导的电荷传输及其对 DNA 堆积扰动的敏感性。初步结果已经确定了构建用于突变分析的传感器所需的各个元件：9i) 可以通过电化学方法检测不同的单碱基错配，并且可以显着改变膜的序列组成； （ii0 单链 DNA 可以通过电极上重复的杂交和变性循环进行测定；值得注意的是，通过将 DNA 介导的电荷传输到电催化循环耦合，可以实现对单碱基错配的高水平灵敏度和辨别力。首先建议可以系统地检查与此设计相关的范围和灵敏度，其中寡核苷酸 长度、序列和嵌入剂探针各不相同；另外，还将进行接头长度的变化和混合表面的构建。这些研究不仅可以开发错配探针，还可以开发用于 DNA 结合蛋白和小分子的新型电化学探针。这些 DNA 薄膜将用于开发 DNA 结合蛋白的电化学测定，例如碱基翻转 酶，基于它们对 DNA 堆积的扰动。 DNA 修饰电极还将用于探测 DNA 修复蛋白（例如光裂合酶和 Mut Y）以及化疗药物（例如烯二炔和蒽环类抗生素）的氧化还原化学。最后，将制造 DNA 芯片，首先分析 p53 基因中的突变热点，然后分析 整个p53基因序列的分析。因此，拟议的计划旨在利用 DNA 介导的电荷传输来开发全新的基于 DNA 的传感器系列。