Electrochemical based DNA sensors

描述(由申请人提供)：我们建议开发一种基于DNA的电化学设备，该设备使用一种新的双电极策略用于DNA阵列的构图和检测。这一更新建议基于DNA介导的电化学，应该能够以高灵敏度检测核酸和DNA结合蛋白生物标记物，适合于定量诊断和研究。我们的双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、多路电催化传感。我们建议首先优化平台，包括采用基于管脚的图案。铜活化的点击化学将是形成电位相关阵列的主要手段。在检测方面，我们将优化电催化伙伴，并通过分析图案化平台上TATA结合蛋白与DNA的结合来评估检测极限(Attomoles)。微流体将被整合到该设备中。优化后，我们建议首先开发核酸检测平台，专门针对两个目标microRNA序列，miR-200c和let-7a。MicroRNAs在健康和癌症组织中有不同的表达，这使它们成为癌症早期检测和特征分析的理想靶点。我们将使用培养的大肠来监测表达水平的差异 有癌变和无癌变的细胞系。我们还建议将该传感器用于检测人类甲基酶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有关。我们将利用我们的“启动”甲基酶/限制性分析。我们将从不同表达DNMT1的细胞裂解物中定量DNMT1，然后测量组织样本。为了建立基于异常甲基化的新的早期诊断方法，将在不同癌症和甲基酶活性水平之间建立相关性。我们还将开发该平台，以筛选抑制甲基化的潜在疗法。接下来，我们将同时检测与疾病相关的miRNA表达和DNMT1水平。鉴于该设备检测的高灵敏度和重复性，我们还将探索OU生物标记物的单细胞检测。我们将探索我们的miRNA和甲基酶靶标，以比较整体平均结果和单细胞分布结果。结合我们实验室已经开发的蛋白质结合、RNA和DNA分析方法，以及新的阵列制作方法和双电极检测方案，我们提出了一种创新的方法，通过适用于系统生物学基础研究以及诊断和筛选的多路应用的坚固的传感器来检测多个生物标志物。

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.