A Microfluidic Protein Separation Device Based on Dielectrophoresis

描述(申请人提供)：可靠和快速分离蛋白质是生物分析和生物医学研究的一个基本和具有挑战性的问题。传统的分离技术在极端情况下达到了极限，例如，当增加的样本复杂性要求在极小的浓度和巨大的背景下分析相关的疾病标志物时。对于时间关键的样品，即当需要快速回答时，例如具有暂时降解或成分变化的样品，或者当快速诊断是必要的时，例如在手术期间，进一步出现问题。这些限制对于较小的样本量和随之而来的低蛋白质量可能更加严重，例如在微创诊断或单细胞分析的情况下。后者对于了解特定的细胞途径和恶性进展尤其重要，否则这些路径和恶性进展将在整体测量中平均。另一个极其相关的例子代表了阿尔茨海默病的诊断，由于所涉及的多肽种类的暂时性及其在体液中的极低丰度，这一诊断特别具有挑战性。这一探索性的建议旨在开发在微流控平台上高效、快速和无凝胶地分离、纯化和预浓缩蛋白质的介电泳仪。该项目将一种新的原理应用于微流控系统中蛋白质的无胶分离。它利用蛋白质的介电(DEP)，作为可极化的物体，通过迁移运动响应不均匀的电场。由于极化率取决于各种参数，如形状、电荷、电荷密度、介电常数或变形性，因此它可以在很大范围内探测蛋白质的DEP响应。在芯片实验室平台上的定制微结构设计中，可以最佳地产生必要的非均匀电场梯度，从而激发蛋白质的介电泳动响应。对所选蛋白质的DEP行为的基础研究将深入了解必要的电驱动参数，并揭示更复杂的分离问题以及蛋白质和多肽的纯化和预浓缩的优化条件。这种新颖的设备能够在几分钟的时间尺度上分析蛋白质样本，并将样本量减少到Pl-Nl范围。特别是，我们开发了一种结合微流控免疫亲和和DEP的脑脊液中β-淀粉样蛋白(A2)寡聚体的分离检测方法。因此，这种新的DEP分离方法代表了对生物医学研究和护理点诊断学具有重要意义的发展。 公共卫生相关性(由申请人提供)：该项目的目标是设计和测试基于微流控平台上的介电泳法的快速、可靠和无凝胶的蛋白质分离和预浓缩设备。这些设备的发展极大地扩展了当前的分离技术，即用于低样品浓度、时间关键分析、小样本量和复杂样品。它将促进生物医学研究和最终的临床实验室实践，特别是快速和现场诊断点的护理。

DESCRIPTION (provided by applicant): Reliable and rapid separation of proteins is both a fundamental and challenging problem for bioanalytical and biomedical research. Conventional separation techniques reach their limits at extremes, for example, when increased sample complexity demands for the analysis of relevant disease markers in extremely small concentration and within a huge background. Problems further arise for time critical samples, i.e. when rapid answers are required, such as for samples with temporally degrading or altering composition or when rapid diagnosis is essential, such as during surgery. These limitations can be more drastic for smaller sample volumes and concomitantly low amount of proteins, such as in the case of minimal invasive diagnosis or single cell analysis. The latter is specifically important for understanding specific cellular pathways and malignant progressions, which would otherwise be averaged in the ensemble measurement. Another extremely relevant example represents the diagnosis of Alzheimer's Disease, which is particularly challenging due to the transient nature of the involved peptide species and their extremely low abundance in body fluids. This exploratory proposal aims to develop dielectrophoretic devices for the efficient, rapid and gel-free separation, purification and pre-concentration of proteins on microfluidic platforms. This project applies a new principle for the gel-free separation of proteins in microfluidic systems. It exploits dielectrophoresis (DEP) of proteins, which as polarizable objects respond to a non-uniform electric field with a migrational motion. As the polarizability depends on various parameters such as shape, charge, charge density, permittivity or deformability it thus allows probing the DEP response of proteins in a broad range. The dielectrophoretic response of proteins is provoked in tailored microstructure designs on a lab-on- a-chip platform in which the necessary inhomogeneous electric field gradient can be optimally generated. A fundamental investigation of the DEP behavior of selected proteins will give insight into the necessary electrical driving parameters and reveal optimized conditions for more complex separation problems as well as the purification and pre-concentration of proteins and peptides. This novel device is capable of analyzing protein samples in time scales of a few minutes and reduces sample volumes to the pL-nL range. In particular, we develop a combined microfluidic immunoaffinity and DEP based separation assay for beta-amyloid (A2) oligomers in cerebrospinal fluid. This novel DEP separation method thus represents a development of outstanding importance for biomedical research and point of care diagnostics. PUBLIC HEALTH RELEVANCE (provided by the applicant): The goal of this project is to design and test devices for fast, reliable and gel-free protein separation and pre-concentration based on dielectrophoresis on a microfluidic platform. The development of these devices extends current separation techniques at their limits, i.e. for low sample concentration, time critical analyzes, miniature sample amounts and complex samples. It will facilitate biomedical research and eventually clinical laboratory practice, in particular, rapid and on-site point of care diagnostics.