Electrochemical based DNA sensors

描述（由申请人提供）：我们提出开发一种基于DNA的电化学装置，该装置使用新的双电极策略进行DNA阵列图案化和检测。该更新建议基于DNA介导的电化学，并且应该允许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，沿着定量的多路电催化传感。我们建议首先优化平台，包括结合针为基础的图案。点击化学与铜活化将是主要手段的潜在依赖性阵列的形成。关于检测，我们将优化电催化合作伙伴，我们将通过分析TATA结合蛋白与图案化平台上的DNA结合来评估检测限（阿托摩尔）。微流体将被纳入该器械。一旦优化，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-200 c和let-7a。microRNA在健康组织和癌组织中差异表达，这使它们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癌细胞来监测表达水平的差异， 具有和不具有癌性转化的细胞系。我们还建议测试这种传感器在检测人类甲基化酶DNMT 1。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“开启”甲基化酶/限制性分析。我们将从DNMT 1表达不同的细胞裂解物中定量DNMT 1，然后测量组织样品。将绘制不同癌症与甲基化酶活性水平之间的相关性，以建立基于异常甲基化的新的早期诊断。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，我们将同时检测疾病相关的miRNA表达和DNMT 1水平。鉴于该设备检测的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和甲基化酶靶点，以比较整体平均值和单细胞分布之间的结果。结合蛋白质结合，RNA和DNA分析已经在我们的实验室开发了新的阵列制造方法和双电极检测方案相结合的测定，我们提出了一种创新的方法，通过一个强大的传感器，适用于系统生物学的基础研究以及诊断和筛选的多重应用程序的多个生物标志物检测。

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.