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

描述（由申请人提供）：对于复杂的蛋白质混合物，如细胞裂解物或含有数千种蛋白质的血清，2-D聚丙烯酰胺凝胶电泳（2D-PAGE）是用于分离单个低丰度蛋白质以进行鉴定和定量分析的标准方法。然而，这是一个繁琐且耗时的手动过程。可自动化的替代分离方法通常涉及不具有2D-PAGE分离能力或不容易用于定量分析的色谱和质谱程序。临床样品的定量分析需要一个具有2D-PAGE分离效率、快速（少于几小时）且易于自动化的平台。LCM Technologies目前正在开发一种与标准2D-PAGE方法类似的毛细管，该方法在几厘米长的通道中的微流控芯片上进行等电聚焦（IEF），之后IEF片段被移动以注入毛细管阵列中，用于SDS毛细管阵列电泳（SDS-CAE），并进行激光诱导荧光检测（LIF）和MALDI-TOF质谱分析。完成后，该系统将完全自动化，总分离时间不到两小时，预计分离效率与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。它还允许使用多种染料同时检测大小和pI标记物，以重复测量特定蛋白质。主要创新点是将微芯片IEF耦合到SDS-CAE的毛细管阵列，具有高效率和定量转移。将对系统在整个阵列和运行间的效率进行表征，这是定量分析所需的。此外，将表征荧光检测灵敏度，以确定原始样品中的检测限，预计单个蛋白质的检测限优于1 ng/mL。与公共卫生的相关性在于，该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志物，该技术具有早期诊断的能力。这可以用于常规筛查和监测患者的癌症和其他疾病，这将允许早期治疗以提高存活率。

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.