A Microfluidic Protein Separation Device Based on Dielectrophoresis

描述（由申请人提供）：蛋白质的可靠和快速分离是生物分析和生物医学研究的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，当增加的样品复杂性需要在极小的浓度和巨大的背景下分析相关疾病标记物时。对于时间关键型样品，问题进一步出现，即当需要快速回答时，例如对于具有暂时降解或改变成分的样品，或当必须进行快速诊断时，例如在手术期间。对于较小的样本量和伴随的低蛋白质量，例如在微创诊断或单细胞分析的情况下，这些限制可能更加严重。后者对于理解特定的细胞途径和恶性进展特别重要，否则它们将在集合测量中被平均。另一个非常相关的例子是阿尔茨海默病的诊断，由于所涉及的肽种类的短暂性和它们在体液中的极低丰度，这尤其具有挑战性。本探索性提案旨在开发介电装置，用于在微流控平台上高效、快速和无凝胶的蛋白质分离、纯化和预浓缩。本项目为微流体系统中蛋白质的无凝胶分离应用了一种新的原理。它利用蛋白质的介电电泳（DEP），作为可极化的物体，对具有迁移运动的非均匀电场作出反应。由于极化率取决于各种参数，如形状、电荷、电荷密度、介电常数或可变形性，因此可以在很宽的范围内探测蛋白质的DEP响应。在芯片上的实验室平台上，蛋白质的介电泳响应是在定制的微观结构设计中引起的，其中必要的不均匀电场梯度可以最佳地产生。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肽的纯化和预浓缩的优化条件。这种新型装置能够在几分钟的时间尺度内分析蛋白质样品，并将样品体积减少到pL-nL范围。特别是，我们开发了一种基于微流体免疫亲和和DEP的脑脊液中β -淀粉样蛋白（A2）低聚物分离试验。因此，这种新颖的DEP分离方法代表了生物医学研究和护理点诊断的重要发展。

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.