ELECTROCHEMICAL DNA-BASED SENSORS

基于电化学 DNA 的传感器

• 批准号: 6636423
• 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/6636423
• 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.

该计划的目标是基于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的介导子作为氧化还原探针修饰的电极将被构造为单个碱基不匹配的传感器。该方法从根本上基于差异化杂交IR分子识别事件。相反，这些基于DNA的传感器依赖于DNA介导的电荷转运及其对DNA堆叠中扰动的敏感性。初步结果已经确定了构造一个传感器进行突变分析所需的个体元素：9i）可以从电化学上检测到不同的单个基本不匹配，并且膜的序列组成可以大大变化； （II0单链DNA可以通过反复的杂交和杂交和变性的循环进行测定；显着地，通过耦合将DNA介导的电荷耦合到与电启动循环相关的元素相关的DNA介导的电荷来实现单个基础不匹配的高敏感性和歧视。长度，序列和互化探针是不同的，并且将进行混合表面的构造，这些研究将不仅能够开发不匹配探针，还可以对新的电化学探针进行新的DNA结合蛋白和小分子的示例。基于酶对DNA堆叠的扰动。因此，对整个p53基因序列进行分析。