Electrochemical based DNA sensors

基于电化学的 DNA 传感器

基本信息

批准号：
9220832
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
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-03-01 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9220832
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 Hepatocyte Human Hybrids Individual Knock-out Laboratories Malignant Neoplasms Measurement Mediating Methods Methylation Methyltransferase MicroRNAs Microfluidics Monitor Myeloid Leukemia Nucleic Acids Parents Pattern Premalignant Principal Investigator Proteins RNA RNA Interference RNA analysis Regulation Reproducibility Research Scheme Screening for cancer Surface System Systems Biology TATA-Box Binding Protein Testing Therapeutic Tissue Sample Tissues Tritium Tumorigenicity base cancer diagnosis catalyst differential expression experimental study innovation interest new technology novel diagnostics nucleic acid detection programs protein biomarkers public health relevance restriction enzyme retinal rods screening sensor targeted biomarker tool tumorigenic

项目摘要

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）：我们建议开发一种基于 DNA 的电化学装置，使用新的两电极策略进行 DNA 阵列图案化和检测。这项更新提案基于 DNA 介导的电化学，应该能够以高灵敏度检测核酸和 DNA 结合蛋白生物标志物，适用于定量诊断和研究。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、多重电催化传感。我们建议首先优化平台，包括结合基于引脚的图案。铜活化的点击化学将是电位依赖性阵列形成的主要手段。在检测方面，我们将优化电催化合作伙伴，并通过在图案化平台上分析 TATA 结合蛋白与 DNA 的结合来评估检测限（阿摩尔）。微流体技术将被整合到该设备中。优化后，我们建议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 在健康组织和癌组织中表达存在差异，这使其成为早期癌症检测和分析的理想靶标。我们将使用培养的结直肠细胞监测表达水平的差异有或没有癌变的细胞系。我们还建议测试该传感器检测人类甲基化酶 DNMT1。 DNA 甲基化调节基因调控和转录，高甲基化和低甲基化都与疾病相关。我们将利用我们的“开启”甲基化酶/限制性测定。我们将从 DNMT1 表达不同的细胞裂解物中定量 DNMT1，然后测量组织样本。将绘制不同癌症和甲基化酶活性水平之间的相关性，以便根据异常甲基化建立新的早期诊断。我们还将开发该平台来筛选抑制甲基化的潜在疗法。接下来我们将转向同时检测疾病相关的 miRNA 表达和 DNMT1 水平。鉴于该设备检测的高灵敏度和重现性，我们还将探索生物标志物的单细胞检测。我们将探索 miRNA 和甲基化酶靶标，以比较总体平均值和单细胞分布之间的结果。将我们实验室已经开发的蛋白质结合、RNA 和 DNA 分析测定与新的阵列制造方法和双电极检测方案相结合，我们提出了一种创新方法，通过适用于系统生物学基础研究和双电极检测方案的强大传感器来检测多种生物标志物。以及用于诊断和筛查的多重应用。