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）：本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。我们建议优化我们的DNA电化学突变分析，设计新的电化学传感器的核酸序列检测，开发和应用DNA修饰电极分析核酸和DNA-蛋白质结构及其相关的氧化还原化学。设计这些新的基于DNA的传感器的基础是长距离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，我们计划优化我们的电化学传感器用于单碱基突变，以从基因组DNA样品开始获得稳健、可重复和灵敏的检测。 在溶液中退火和组装测试样品后，将利用粘性端突出端将处理后的样品寻址到电化学芯片。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。为了构建一种灵敏的电化学传感器用于病原体检测，我们的基本DNA CT突变检测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电化学也将作为一种结构探针，用于监测与DNA碱基修饰相关的堆叠扰动，如DNA损伤或糖-磷酸骨架的改变。我们还建议应用DNA电化学探测DNA上蛋白质反应的动力学在真实的时间。我们将研究与DNA切割的限制性内切酶PvulI和胸腺嘧啶二聚体修复DNA光裂合酶的毫秒级时间尺度的结构扰动。使用DNA修饰的电极，DNA结合蛋白的氧化还原辅因子也将以其DNA结合形式被探测，天然产物化学治疗剂也将被还原活化以与DNA反应。因此，该计划旨在利用DNA介导的CT设计一个全新的基于DNA的传感器家族，用于基础探索和应用于新技术开发。