Electrochemical DNA-based Sensors

基于电化学 DNA 的传感器

• 批准号: 6868151
• 负责人: JACQUELINE K BARTON
• 金额: $ 32.66万
• 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 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6868151
• 关键词: DNA; DNA binding protein; analytical method; bioengineering /biomedical engineering; carbon; communicable disease diagnosis; electrochemistry; electrodes; electron transport; gene mutation; intermolecular interaction; microarray technology; microelectrodes; molecular stacking; nucleic acid quantitation /detection; nucleic acid sequence; nucleic acid structure; oxidation reduction reaction; p53 gene /protein; protein structure

DESCRIPTION (provided by applicant): The goal of this renewal proposal continues to be the development of novel, sensitive electrochemical sensors for DNA diagnosis. We propose to optimize our DNA electrochemistry assay for mutational analysis, to design novel electrochemical sensors for nucleic acid sequence detection, to develop and apply DNA modified electrodes to analyze nucleic acid and DNA-protein structures and their associated redox chemistry. Fundamental to the design of these new DNA-based sensors are long-range DNA-mediated charge transport (CT) chemistry and its sensitivity to perturbations in DNA structure and base pair stacking. Specifically we plan to optimize our electrochemical sensor for single base mutations to obtain robust, reproducible and sensitive detection starting from genomic DNA samples. After annealing and assembling test samples in solution, processed samples will be addressed to the electrochemical chip utilizing sticky-end overhangs for addressing. The length of DNA targets will be varied, multiplexed addressing will be explored, and different biochemical methods for the preparation of test samples will be optimized. Graphite will also be probed as an alternate electrode surface. With the aim of constructing a sensitive electrochemical sensor for pathogen detection, our basic DNA CT strategy for mutation detection will be modified and expanded to allow the direct detection of nucleic acid sequences without biochemical amplification. DNA CT electrochemistry will also be applied as a structural probe in monitoring perturbations in stacking associated with modifications to the DNA bases, as found in DNA lesions, or alterations in the sugar-phosphate backbone. We also propose to apply DNA electrochemistry to probe the kinetics of protein reactions on DNA in real time. We will examine on the millisecond timescale structural perturbations associated with DNA cleavage by the restriction enzyme PvulI and thymine dimer repair by DNA photolyase. Using DNA-modified electrodes, redox cofactors of DNA-binding proteins will also be probed in their DNA-bound form, as will natural product chemotherapeutics which are reductively activated for reaction with DNA. The proposed program therefore intends to exploit DNA-mediated CT in the design of a completely new family of DNA-based sensors forboth fundamental exploration and applied to new technology development.

描述（由申请人提供）：该更新提案的目标仍然是开发用于 DNA 诊断的新型、灵敏的电化学传感器。我们建议优化用于突变分析的 DNA 电化学测定，设计用于核酸序列检测的新型电化学传感器，开发和应用 DNA 修饰电极来分析核酸和 DNA 蛋白质结构及其相关的氧化还原化学。这些新型 DNA 传感器设计的基础是长程 DNA 介导的电荷传输 (CT) 化学及其对 DNA 结构和碱基对堆积扰动的敏感性。 具体来说，我们计划针对单碱基突变优化我们的电化学传感器，以便从基因组 DNA 样本开始获得稳健、可重复且灵敏的检测。 在溶液中退火和组装测试样品后，处理后的样品将利用粘端突出物寻址到电化学芯片。 DNA靶标的长度将有所不同，将探索多重寻址，并将优化用于制备测试样品的不同生化方法。石墨也将作为替代电极表面进行探测。为了构建用于病原体检测的灵敏电化学传感器，我们用于突变检测的基本 DNA CT 策略将被修改和扩展，以允许直接检测核酸序列而无需生化扩增。 DNA CT 电化学还可用作结构探针，用于监测与 DNA 碱基修饰相关的堆积扰动，如 DNA 损伤或糖磷酸骨架的变化。我们还建议应用 DNA 电化学来实时探测 DNA 上蛋白质反应的动力学。我们将在毫秒时间尺度上检查与限制性内切酶 PvulI 进行的 DNA 切割和 DNA 光裂合酶进行的胸腺嘧啶二聚体修复相关的结构扰动。使用 DNA 修饰电极，DNA 结合蛋白的氧化还原辅助因子也将以其 DNA 结合形式进行探测，天然产物化疗药物也会被还原激活以与 DNA 发生反应。因此，拟议的计划旨在利用 DNA 介导的 CT 设计全新的基于 DNA 的传感器系列，以进行基础探索并应用于新技术开发。