Microfluidic Platform for Probing Ceramide Channels

描述（由申请人提供）：提出了一种微流体平台，该平台将有助于研究脂质通道形成和溶解中涉及的过程，从而更深入地了解分子跨细胞膜转运，并最终有助于我们了解生物通道及其与疾病过程的关系。该项目将利用我们团队的最新成果，包括首次在微流控离子通道芯片内使用单个膜结合生物离子通道作为电导传感元件进行单分子检测，并实时控制通道两侧的分析物浓度和缓冲液条件。在该提案中，概念验证微流体平台将被扩展以证明原位磷脂膜形成、用于增强膜稳定性的集成支持凝胶和微孔膜、自动化多路复用脂质通道测量以及膜处的薄膜热控制，以能够观察焓与膜形成、通道形成和动力学以及分析物/通道相互作用之间的关系。将进行一系列现有技术不可行或非常耗时的实验，重点是研究神经酰胺（一种与细胞凋亡有关的鞘脂）形成的通道。神经酰胺通道的结构和功能将使用微流控系统进行探测，控制因素被操纵，以研究它们对通道大小和稳定性的调节的影响。这一努力将导致一个新的和独特的电生理学平台，可应用于广泛的基础和应用研究的生物通道，连同实验结果，将扩大我们的理解一个重要的脂质通道系统参与程序性细胞死亡。公共卫生相关性：提出了一个独特的实验平台，将阐明参与inbiological膜通道的形成和溶解的过程，导致更深入地了解分子跨细胞膜的运输，并最终有助于我们的知识的生物通道及其与疾病过程的关系。

DESCRIPTION (provided by applicant): A microfluidic platform is proposed that will aid in investigating processes involved in lipid channel formation and dissolution, leading to a deeper understanding of molecular transport across cell membranes, and ultimately contribute to our knowledge of biological channels and their relationships to disease processes. The project will leverage recent results from our team, including the first demonstration of single-molecule detection within a microfluidic ion channel chip using a single membrane-bound biological ion channel as a conductometric sensing element, with real-time control of analyte concentration and buffer conditions on either side of the channel. In this proposal, the proof-of-concept microfluidic platform will be extended to demonstrate in situ phospholipid membrane formation, integrated supporting gels and microporous films for enhanced membrane stability, automated multiplexed lipid channel measurements, and thin film thermal control at the membranes to enable the observation of the relationships between enthalpy and membrane formation, channel formation and dynamics, and analyte/channel interactions. A range of experiments that are not feasible or extremely time-consuming with existing technology will be conducted, with a focus on investigating channels formed by ceramide, a sphingolipid implicated in apoptosis. The structure and function of ceramide channels will be probed using the microfluidic system, with controlling factors manipulated to investigate their impacts on the regulation of channel size and stability. This effort will result in a novel and unique electrophysiology platform which may be applied to a wide range of fundamental and applied investigations of biological channels, together with experimental results that will expand our understanding of an important lipid channel system involved in programmed cell death. PUBLIC HEALTH RELEVANCE: A unique experimental platform is proposed that will elucidate the processes involved inbiological membrane channel formation and dissolution, leading to a deeper understanding of molecular transport across cell membranes, and ultimately contribute to our knowledge of biological channels and their relationships to disease processes.