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.

计划的目标是设计和应用基于DNA的电化学传感器。用DNA寡核苷酸双链修饰的电极和非共价结合的嵌入剂作为氧化还原探针将被构建为单碱基错配的传感器。这种方法从根本上不是基于分子识别事件的差异杂交。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堆积中的扰动的敏感性。初步结果已经确定了构建突变分析传感器所需的单个元件：9)可以用电化学方法检测到不同的单碱基错配，并且薄膜的序列组成可以有很大的变化；(I)可以通过重复杂交和电极上的变性来检测单链DNA；值得注意的是，通过耦合DNA介导的电荷传输到电催化循环，可以实现对单碱基错配的高水平灵敏度和区分度。首先提出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、序列和嵌入物探针是不同的；此外，将进行连接子长度和混合表面的构造的变化。这些研究不仅将使错配探针的开发成为可能，而且还将使新的DNA结合蛋白质和小分子的电化学探针的开发成为可能。这些DNA膜将被用于开发DNA结合蛋白的电化学分析，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索DNA修复蛋白的氧化还原化学，如光解酶和Mut Y，以及化疗药物，如烯二炔类和蒽环类抗生素。最后，将制造DNA芯片，首先分析p53基因的突变热点，然后分析整个p53基因序列。因此，拟议的计划打算利用DNA介导的电荷传输来开发一种全新的基于DNA的传感器家族。