Electrochemical based DNA sensors

基于电化学的 DNA 传感器

• 批准号: 9024550
• 负责人: JACQUELINE K BARTON
• 金额: $ 36.76万
• 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 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9024550
• 关键词: Address; Affect; Basic Science; Binding Proteins; Biological Assay; Biological Markers; Buffers; Cancerous; Cell Line; Cells; Chemistry; Colorectal; Colorectal Cancer; Copper; Cytolysis; DNA; DNA Damage; DNA Methylation; DNA analysis; DNA-Binding Proteins; Detection; Development; Devices; Diagnosis; Diagnostic; Diagnostics Research; Disease; Early Diagnosis; Electrochemistry; Electrodes; Gene Expression Regulation; Genetic Transcription; Goals; Health; Hepatocyte; Human; Individual; Knock-out; Laboratories; Malignant Neoplasms; Measurement; Mediating; Methods; Methylation; Methyltransferase; MicroRNAs; Microfluidics; Monitor; Myeloid Leukemia; Nucleic Acids; Parents; Pattern; Principal Investigator; Proteins; RNA; RNA Interference; RNA analysis; RNA-Binding Proteins; Regulation; Reproducibility; Research; Scheme; Screening for cancer; Surface; System; Systems Biology; TATA-Box Binding Protein; Testing; Therapeutic; Tissue Sample; Tissues; Tritium; base; catalyst; differential expression; innovation; interest; new technology; novel diagnostics; nucleic acid detection; programs; protein biomarkers; research study; restriction enzyme; retinal rods; screening; sensor; targeted biomarker; tool; tumorigenic

DESCRIPTION (provided by applicant): We propose the development of a DNA-based electrochemical device using a new two-electrode strategy for DNA array patterning and detection. This renewal proposal is based on DNA-mediated electrochemistry and should allow the detection of nucleic acid and DNA-binding protein biomarkers with high sensitivity, suitable for quantitative diagnostics and research. Our 2-electrode platform provides a means to fabricate a DNA array on a single electrode, along with quantitative, multiplexed electrocatalytic sensing. We propose first to optimize the platform, including the incorporation of pin- based patterning. Click chemistry with copper activation will be the primary means for potential- dependent array formation. With respect to detection, we will optimize electrocatalysis partners and we will assess limits of detection (attomoles) through analysis of TATA-binding protein binding to DNA on the patterned platform. Microfluidics will be incorporated into the device. Once optimized, we propose developing the platform first for nucleic acid detection, specifically for two target microRNA sequences, miR-200c and let-7a. MicroRNAs are differentially expressed in healthy and cancerous tissues, which make them ideal targets for early cancer detection and profiling. We will monitor differences in expression levels using cultured colorectal cell lines with and without cancerous transformation. We also propose to test this sensor in detecting the human methylase DNMT1. DNA methylation modulates gene regulation and transcription, and both hyper and hypomethylation are associated with disease. We will take advantage of our "turn-on" methylase/restriction assay. We will quantify DNMT1 from cell lysates differing in expression of DNMT1, followed by measurements of tissue samples. Correlations will be drawn between different cancers and levels of methylase activity in order to establish a new early diagnostic based upon aberrant methylation. We will develop the platform also to screen potential therapeutics that inhibit methylation. Next we will move to simultaneous detection of disease-related miRNA expression and DNMT1 levels. Given the high sensitivity and reproducibility in detection with this device, we will also explore single cell detection of ou biomarkers. We will explore our miRNA and methylase targets to compare results between the bulk average and distribution among single cells. Combining assays for protein binding, RNA and DNA analysis already developed in our laboratory with new array fabrication methods and a two-electrode detection scheme, we propose an innovative approach to multiple biomarker detection through a robust sensor suitable for both basic research in systems biology as well as multiplexed applications for diagnosis and screening.

描述（由申请人提供）：我们建议使用新的两电极策略来开发基于DNA的电化学装置，以进行DNA阵列模式和检测。该更新建议基于DNA介导的电化学，应允许检测具有高灵敏度的核酸和DNA结合蛋白生物标志物，适合定量诊断和研究。我们的2电极平台提供了一种在单个电极上制造DNA阵列的方法，以及定量的多重电催化感应。我们首先建议优化平台，包括基于PIN的图案的合并。用铜激活点击化学将是潜在依赖阵列形成的主要手段。对于检测，我们将优化电催化伴侣，并通过分析塔塔结合蛋白与图案化平台上的DNA结合来评估检测限制（Attomoles）。微流体将纳入设备。一旦优化，我们就提出了首先开发用于核酸检测的平台，特别是针对两个目标microRNA序列miR-200c和let-7a。 microRNA在健康和癌性组织中差异表达，这使其成为早期癌症检测和分析的理想目标。我们将使用培养的结直肠监测表达水平的差异 具有和没有癌性转化的细胞系。我们还建议测试该传感器以检测人甲基酶DNMT1。 DNA甲基化调节基因调节和转录，并且超甲基化都与疾病有关。我们将利用我们的“转交”甲基酶/限制测定法。我们将对DNMT1表达不同的细胞裂解物进行量化DNMT1，然后对组织样品进行测量。将在不同的癌症和甲基酶活性水平之间提取相关性，以建立基于异常甲基化的新的早期诊断。我们将开发平台，以筛选抑制甲基化的潜在治疗剂。接下来，我们将同时检测与疾病相关的miRNA表达和DNMT1水平。鉴于该设备检测到高灵敏度和可重复性，我们还将探索OU生物标志物的单细胞检测。我们将探索我们的miRNA和甲基酶靶标，以比较单个细胞之间的总体平均值与分布之间的结果。结合蛋白质结合，RNA和DNA分析在我们的实验室已经通过新阵列制造方法和两种电极检测方案进行的结合，我们提出了一种创新的方法，通过适用于系统生物学的稳健传感器作为多种生物标志物检测，作为系统生物学的两种生物标志物检测以及用于诊断和筛查的多重应用。