Microfluidic Platform for Probing Ceramide Channels

描述(申请人提供)：提出了一个微流控平台，该平台将有助于研究涉及脂类通道形成和溶解的过程，从而加深对跨细胞膜的分子运输的了解，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，包括首次演示使用单个膜结合的生物离子通道作为电导传感元件在微流控离子通道芯片中进行单分子检测，并实时控制通道两侧的分析物浓度和缓冲条件。在这项提案中，概念验证微流控平台将扩展到展示原位磷脂膜形成、用于增强膜稳定性的集成支持凝胶和微孔膜、自动化多路脂通道测量以及膜上的薄膜热控制，从而能够观察焓和膜形成、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极其耗时的实验，重点是研究神经酰胺形成的通道，神经酰胺是一种与细胞凋亡有关的鞘磷脂。我们将利用微流控系统探索神经酰胺通道的结构和功能，并操纵控制因素来研究它们对通道大小和稳定性调节的影响。这一努力将产生一个新颖而独特的电生理学平台，它可能应用于对生物通道的广泛基础和应用研究，以及实验结果，将扩大我们对参与细胞程序性死亡的重要脂质通道系统的理解。公共卫生相关性：我们提出了一个独特的实验平台，该平台将阐明生物膜通道的形成和溶解所涉及的过程，从而加深对分子跨细胞膜运输的理解，并最终有助于我们了解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.