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小型化的，多路复用的蛋白质检测器与DNA修饰的电极，以提供DNA结合蛋白质的同时监测。这将反过来使新的能力，在探测DNA/蛋白质相互作用，诊断疾病状态，并剖析如何DNA结合蛋白调节基因表达。通过DMA介导的电荷传输进行的蛋白质检测提供了具有高结合特异性的纯电学检测平台，并且显示出快速、可量化、廉价和可靠的疾病诊断的巨大潜力。具体来说，这将涉及制造晶片阵列，组装电子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，将通过光刻和电子束光刻使晶片阵列小型化。一旦完成了多重抗体的实验平台，就有可能测定蛋白质家族的位点特异性结合和结构。甲基化酶将被调查之间的DMA与各种碱基对序列的位点选择性结合。将通过检测不同浓度的甲基化酶来确定灵敏度限度。通过探测非特异性结合和比较结合相同序列但甲基化不同位点的甲基化酶来监测结构变异。随后，将确定具有不同结合基序的蛋白质对DMA介导的电荷转运的影响，包括螺旋-转角-螺旋、锌指和Bzip蛋白。为了表征在DMA碱基对堆叠中引起小扰动的蛋白质，甲基化酶本身将用于竞争测定。使用小型化设备，将测试细胞水平的蛋白质水平，例如PAX 5蛋白，这是白血病和淋巴瘤的潜在特征。公共卫生相关性：所提出的测定提供了用于检测包含疾病状态特征的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蛋白相互作用的基本理解，从而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.