Multiplexed and Miniaturized Protein Detectors with DNA-Modified Electrodes

描述（由申请人提供）：拟议的研究是开发一种带有dna修饰电极的小型化、多路复用蛋白质检测器，以同时监测dna结合蛋白。这将使探测DNA/蛋白质相互作用、诊断疾病状态和剖析DNA结合蛋白如何调节基因表达的新能力成为可能。dma介导的电荷输运蛋白检测提供了一种具有高结合特异性的纯电检测平台，在快速、可量化、廉价、可靠的疾病诊断方面具有巨大潜力。具体来说，这将涉及制造晶圆阵列，组装电气硬件和编程计算机软件，以实现多路操作。为了检测少量的蛋白质，晶圆阵列将通过光刻和电子束光刻实现小型化。一旦多重增殖的实验平台完成，就有可能分析蛋白质家族的位点特异性结合和结构。甲基化酶将被研究在不同碱基对序列之间的位点选择性结合。灵敏度限制将通过测试不同浓度的甲基化酶来确定。结构变化将通过探测非特异性结合和通过比较结合相同序列但甲基化不同位点的甲基化酶来监测。随后，我们将确定具有不同结合基序的蛋白对dma介导的电荷转运的影响，包括helix- turning -helix蛋白、锌指蛋白和Bzip蛋白。为了表征在DMA碱基对堆栈中引起小扰动的蛋白质，甲基化酶本身将用于竞争分析。有了小型化的设备，细胞水平的蛋白质水平将被测试，比如PAX5蛋白，它是白血病和淋巴瘤的潜在标志。公共卫生相关性：拟议的检测提供了一个检测含有疾病状态特征的dna结合蛋白的平台，具有快速、可量化、廉价和可靠的疾病诊断的潜力。此外，这项研究将提供对dna -蛋白质相互作用的基本理解，这可能导致疾病的预防或治疗。

DESCRIPTION (provided by applicant): The proposed research is to develop a miniaturized, multiplexed protein detector with DNA-modified electrodes to provide simultaneous monitoring of DNA-binding proteins. This will in turn enable new capabilities in probing DNA/protein interactions, diagnosing disease states, and dissecting how DNA-binding proteins regulate gene expression. Protein detection by DMA-mediated charge transport provides a purely electrical detection platform with high binding specificity and shows great potential for fast, quantifiable, inexpensive and reliable diagnosis of diseases. Specifically, this will involve fabricating wafer arrays, assembling electrical hardware and programming computer software to achieve multiplexed operation. In order to detect small quantities of proteins, wafer arrays will be miniaturized through photolithography and electron-beam lithography. Once the experimental platform for multiplexion is complete, it will be possible to assay the site-specific binding and structure of families of proteins. Methylases will be investigated for site-selective binding among DMA with various basepair sequences. Sensitivity limits will be established by testing for various concentrations of methylases. Structural variations will be monitored by probing for non-specific binding and by comparing methylases that bind to the same sequences but methylate different sites. Subsequently, the impact of proteins with different binding motifs on DMA-mediated charge transport will be determined, including helix-turn-helix, zinc finger and Bzip proteins. In order to characterize proteins that cause little perturbation in the DMA base-pair stack, the methylases themselves will be used for competition assays. With miniaturized devices, protein levels at cellular levels will be tested, such as the PAX5 protein, a potential signature of leukemias and lymphomas. PUBLIC HEALTH RELEVANCE: The proposed assay provides a platform for detecting DNA-binding proteins that contain signatures of disease states, having potential for fast, quantifiable, inexpensive and reliable diagnosis of diseases. In addition, this research will provide fundamental understanding of DNAprotein interactions that could lead to disease prevention or therapy.