Electrochemical DNA-based Sensors

基于电化学 DNA 的传感器

• 批准号: 7027671
• 负责人: JACQUELINE K BARTON
• 金额: $ 31.9万
• 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/7027671
• 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)：这项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电化学分析方法，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。设计这些基于DNA的新型传感器的基础是远程DNA介导的电荷传输(CT)化学及其对DNA结构和碱基对堆积的扰动的敏感性。具体地说，我们计划针对单碱基突变优化我们的电化学传感器，以获得从基因组DNA样本开始的强大、可重复和灵敏的检测。在溶液中对测试样品进行热处理和组装后，处理后的样品将利用粘端悬臂寻址到电化学芯片上进行寻址。DNA靶标的长度将是不同的，将探索多路寻址，并将优化不同的生化方法来制备测试样品。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不需要生化放大。DNA CT电化学还将作为结构探针应用于监测与DNA碱基修饰相关的堆积扰动，如在DNA损伤中发现的，或糖-磷酸骨架的变化。我们还建议应用DNA电化学来实时探测蛋白质在DNA上的反应动力学。我们将研究与限制酶Pual I切割DNA和DNA光解酶修复胸腺嘧啶二聚体相关的毫秒时间尺度结构扰动。利用DNA修饰的电极，DNA结合蛋白的氧化还原辅因子也将以DNA结合的形式进行探索，天然产物化疗药物也将被还原激活以与DNA反应。因此，拟议的计划打算利用DNA介导的CT来设计一种全新的基于DNA的传感器家族，既用于基础探索，又应用于新技术开发。