Two-Dimensional Electrophoresis Device for Biomarker Detection

描述(由申请人提供)：拟议研究的目标是开发用于药物发现和医疗诊断的蛋白质组学技术。具体地说，我们的目标是开发一种支持微流控的二维电泳仪，它有可能取代传统的二维凝胶电泳法(2DGE)。除了流行的蛋白质组学技术，如质谱仪(MS)和蛋白质微阵列，2DGE仍然是蛋白质组学研究中最有效的方法之一。2DGE的主要优势是能够将样品与对照进行比较，以识别疾病的生物标志物，但其主要限制是重复性差和过程耗时。 为了解决这些局限性，我们将开发一种2D电泳仪，它包括一个用于第一维(等电聚焦)的通道和多个用于第二维(聚丙烯酰胺凝胶电泳)的正交通道。为了引入不同的分离介质，将创建一系列微流控伪阀；一个流体网络允许蛋白质从第一维转移到第二维。由于分离电压较高，预计会有更快的电泳和更高的分离分辨率，这类似于DNA测序已经从平板凝胶电泳转变为毛细管阵列格式。此外，由于消除了凝胶翘曲，该设备将提供增强的2D地图的重复性，并由于消除了相邻凝胶通道中蛋白质之间的扩散和相互作用而提高了蛋白质鉴定的准确性。 具体目标包括：(1)设计和制造由微阀阵列和流体网络组成的2D电泳仪；(2)通过蛋白质分析演示该设备；(3)通过分析与创伤性脑损伤(TBI)相关的生物标志物来验证该设备。 这项研究的意义在于，2D电泳仪具有取代生物实验室中广泛使用的2DGE的潜力。其次，如果该平台成功实施，它很可能成为一个快速、一次性和可量化的诊断工具，帮助医生确定脑外伤的严重性，并指导实施适当的医疗治疗。此外，该平台可能有助于搜索生物标记物与各种其他疾病之间的联系，并利用已建立的生物标记物对特定疾病进行筛查/诊断。 公共卫生相关声明：拟议的研究是为了开发用于药物发现和医疗诊断的蛋白质组学技术。具体地说，我们将开发一种微流控的电泳仪，它有可能取代传统的二维平板凝胶电泳法。该设备将用于检测与创伤性脑损伤相关的生物标记物。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop proteomics technology for drug discovery and medical diagnostics. Specifically we aim to develop a microfluidics-enabled, two-dimensional electrophoresis device that has a potential to replace conventional two-dimensional gel electrophoresis (2DGE). In addition to popular proteomics techniques such as mass spectrometry (MS) and protein microarrays, 2DGE remains one of the most potent methods in proteomics studies. The key advantage of 2DGE is its ability to compare samples with controls for identification of biomarkers of diseases while its major limitations are poor reproducibility and time-consuming processes. To address the limitations, we will develop a 2D electrophoresis device consisting of one channel for the first dimension (isoelectric focusing) and a number of orthogonal channels for the second dimension (polyacrylamide gel electrophoresis). An array of microfluidic pseudovalves will be created for introducing different separation media; a fluidic network allows the transfer of proteins from the first to the second dimension. Faster electrophoresis and higher separation resolution are expected due to higher separation voltage, in an analogy to DNA sequencing that has shifted from slab gel electrophoresis to the capillary array format. In addition, the device will provide enhanced reproducibility of 2D maps due to elimination of gelwarping and improved accuracy in protein identification resulting from the elimination of the diffusion and interaction among proteins in adjacent gel lanes. The specific aims include: (1) designing and fabricating the 2D electrophoresis device consisting of an array of microvalves and the fluidic network; (2) demonstrating the device by protein analysis; and (3) validating the device by analyzing the biomarkers associated with traumatic brain injury (TBI). The significance of the research lies in the fact that the 2D electrophoresis device has the potential to replace 2DGE, which is widely used in biological laboratories. Secondly, if the platform is successfully implemented, it will likely become a rapid, disposable, and quantifiable diagnostic tool to help physicians determine the seriousness of TBI and to guide implementation of appropriate medical treatment. In addition, the platform will likely be useful in searching for the association between biomarkers and a variety of other diseases, and in screening/diagnosing a particular disease using the established biomarkers. Public Health Relevance Statement: The proposed research is to develop proteomics technology for drug discovery and medical diagnostics. Specifically we will develop a microfluidics-enabled electrophoresis device that has a potential to replace classical two-dimensional slab gel electrophoresis. The device will be used for detecting the biomarkers associated with traumatic brain injury.