Multiplexed and Miniaturized Protein Detectors with DNA-Modified Electrodes

描述(由申请人提供)：建议的研究是开发一种带有DNA修饰电极的小型化、多路复用的蛋白质检测器，以提供对DNA结合蛋白质的同步监测。这反过来将使探测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。通过DMA介导的电荷传输检测蛋白质提供了一个具有高结合特异性的纯电学检测平台，在快速、定量、廉价和可靠的疾病诊断方面显示出巨大的潜力。具体地说，这将涉及制造晶片阵列、组装电气硬件和编程计算机软件以实现多路复用操作。为了检测少量的蛋白质，晶片阵列将通过光刻和电子束光刻进行微型化。一旦多重作用的实验平台完成，就有可能分析蛋白质家族的位点特异性结合和结构。将研究甲基酶与不同碱基序列的DMA之间的位置选择性结合。将通过测试不同浓度的甲基酶来确定敏感度极限。结构变异将通过探测非特异性结合和比较与相同序列结合但甲基化不同位点的甲基酶来监测。随后，将确定具有不同结合基序的蛋白质对DMA介导的电荷传输的影响，包括螺旋-转弯-螺旋、锌指和BZIP蛋白。为了鉴定在DNA碱基对堆栈中引起很小干扰的蛋白质，甲基酶本身将被用于竞争分析。使用微型设备，将测试细胞水平的蛋白质水平，例如PAX5蛋白质，这是白血病和淋巴瘤的潜在特征。与公共卫生相关：建议的检测方法为检测包含疾病状态特征的DNA结合蛋白提供了一个平台，有可能对疾病进行快速、可量化、廉价和可靠的诊断。此外，这项研究将提供对DNA蛋白质相互作用的基本了解，这些相互作用可能导致疾病预防或治疗。

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.