A 2D-CE System for Rapid and Quantitative Analysis of Clinical Protein Biomarkers

描述(申请人提供)：对于复杂的蛋白质混合物，如细胞裂解液或含有数千种蛋白质的血清，二维聚丙烯酰胺凝胶电泳法(2D-PAGE)是分离单个低丰度蛋白质用于鉴定和定量分析的标准方法。然而，这是一个繁琐和耗时的手动程序。可自动化的替代分离方法通常涉及不具备2D-PAGE分离能力或不容易用于定量分析的层析和质谱学程序。需要一个具有2D-PAGE分离效率、快速(不到几个小时)、易于自动化的平台来对临床样本进行定量分析。LCM Technologies目前正在开发一种类似于标准2D-PAGE方法的毛细管，在几厘米长的通道中对微流控芯片执行等电聚焦(IEF)，然后动员IEF片段注入毛细管阵列，用于激光诱导荧光检测(LIF)和MALDI-TOF质谱分析的SDS-CAE。一旦建成，该系统将完全自动化，总分离时间不到两个小时，预计分离效率将与2D-PAGE相当或更好。LiF检测将允许以四个数量级的动态范围定量测量分离的蛋白质。它还允许使用多个染料同时检测大小和等电点标记，以重复性地测量特定蛋白质。本论文的主要创新之处在于提出了一种高效、定量转移的高效毛细管阵列耦合芯片IEF的方案。该系统的特点将是整个阵列和逐个运行的效率，这是定量分析所必需的。此外，还将表征荧光检测灵敏度，以确定原始样品中的检测下限，预计单个蛋白质的检测下限将好于1 ng/mL。与公共健康相关的是，这项技术将被开发用于快速准确地测量血清和其他体液中的癌症和其他疾病标记物，这些体液具有早期诊断的能力。这可以用于患者癌症和其他疾病的常规筛查和监测，这将使早期治疗能够提高存活率。

DESCRIPTION (provided by applicant): For complex protein mixtures such as cell lysates or sera containing thousands of proteins, 2-D polyacrylamide gel-electrophoresis (2D-PAGE) is the standard method used to isolate individual low-abundance proteins for identification and quantitative analysis. However, it is a tedious and time-consuming manual procedure. Alternative separation methods that can be automated typically involve chromatographic and mass spectral procedures that do not have the separation capabilities of 2D-PAGE or are not easily used for quantitative analysis. A platform that has the separation efficiency of 2D-PAGE, is rapid (less than several hours), and easily automated is needed for quantitative analysis of clinical samples. LCM Technologies is currently developing a capillary analog to the standard 2D-PAGE method that performs iso-electric focusing (IEF) on a micro-fluidic chip in a channel a few centimeters long, after which IEF segments are mobilized for injection into a capillary array for SDS capillary array electrophoresis (SDS-CAE) with laser-induced fluorescence detection (LIF) and MALDI-TOF mass spectral analysis. Once completed, the system will be fully automated with a total separation time of less than two hours and is expected to have separation efficiency comparable to or better than 2D-PAGE. LIF detection will allow quantitative measurement of separated proteins with a dynamic range of four orders-of-magnitude. It also allows multiple dyes to be used for simultaneous detection of size and pI markers for reproducible measurement of specific proteins. The main innovation is a scheme to couple microchip IEF to the capillary array for SDS-CAE with high efficiency and with quantitative transfer. The system will be characterized for efficiency across the array and run-to-run, which is required for quantitative analysis. In addition, fluorescence detection sensitivity will be characterized to determine detection limits in the original sample, which are expected to be better than 1 ng/mL for individual proteins. The relevance to public health is that the technology will be developed for rapid and accurate measurement of cancer and other disease markers in serum and other bodily fluids that has the capability of early. This could be used for routine screening and monitoring for cancer and other diseases in patients, which will allow early treatment to increase survival rate.