High-throughput on-chip platform for interrogation of mitochondrial membrane potential and apoptosis

 描述（由申请人提供）：在拟议的研究中，我们的目标是开发和验证技术，以高通量检测线粒体去极化（细胞凋亡的不归点）的抑制剂和诱导剂。由于细胞凋亡是肿瘤进展中关键的非调控途径，因此对其的研究和最终控制在肿瘤生物学中至关重要。我们中的一些人的初步研究已经显示了患者癌症无进展存活率与线粒体对毛地黄皂苷透化的患者原发性肿瘤细胞中促凋亡BH 3肽的响应（内膜的去极化，外膜的透化）之间的相关性。我们还表明，线粒体更容易被BH 3肽去极化的患者对化疗的反应更好。在概念验证工作中，我们展示了一种芯片上纳米流体技术，该技术能够捕获从细胞中分离的单个活线粒体， 组织，并使用这些个体线粒体的潜在敏感荧光探针来询问膜电位，以响应各种化学环境，包括电子传递链的底物和抑制剂，以及导致线粒体闪烁和去极化的钙挑战。此外，我们的初步工作表明， 在初步的研究中， 在脂质双层中使用纳米管电极。在拟议的研究中，我们将开发和验证这种技术，以感测线粒体和线粒体中单个离子通道的打开和关闭，研究它们的统计数据和时间，并开发一个平台，最终实现高通量测定线粒体电子传递链和膜去极化的电生理学在单个离子通道水平。该技术将在三个方面具有变革性：首先，它将验证一种用于研究细胞凋亡的定性新方法。细胞凋亡的失调被认为是癌症的6大标志之一，然而，目前还缺乏研究线粒体去极化的方法。第二，它将允许一个新的方式来研究电生理学在单离子通道水平。这将允许前所未有的时间，位置和线粒体膜闪烁和去极化的统计研究。最后，整体技术将使线粒体代谢的新研究成为可能。癌症代谢是去年添加到癌症的两个新的“标志”之一，这种仪器将使人们能够理解代谢通量的一个重要组成部分（即膜电位）。由于我们的高通量技术将能够从少量细胞（甚至是单个细胞）中检测数千个单个线粒体，因此它将使仪器的进步能够研究代谢，干细胞和癌症生物学之间的相互关系。

 DESCRIPTION (provided by applicant): In the proposed studies, we aim to develop and validate technology to allow for high-throughput assays of inhibitors and inducers of mitochondrial depolarization, the point of no return for apoptosis. As apoptosis is a key unregulated pathway in tumor progression, its study and ultimately control is of paramount important in cancer biology. Preliminary studies by some of us have shown a correlation between patient cancer progression-free survival rate and the response of mitochondria (depolarization of the inner membrane, permeabilization of the outer membrane) to pro-apoptotic BH3 peptides in digitonin permeabilized patient primary tumor cells. We also have shown that the patients whose mitochondria are more easily depolarized with BH3 peptides respond better to chemotherapy. In proof of concept work, we demonstrated an on-chip nanofluidic technology that was able to trap individual, living mitochondria isolated from cell and tissues, and to interrogate the membrane potential using potential sensitive fluorescence probes of these individual mitochondria in response various chemical environments, including substrates and inhibitors of the electron transport chain, as well as calcium challenges which resulted in mitochondrial flickering and depolarization. In addition, our initial work demonstrated in preliminary studies the electrical sensing of the opening and closing of individual ion channels in lipid bilayers using nanotube electrodes. In the proposed studies we will develop and validate this technology to sense the opening and closing of individual ion channels in mitoplasts and mitochondria, study their statistics and timing, and to develop a platform to enable, ultimately, high throughput assays of the electrophysiology of the mitochondrial electron transport chain and membrane depolarization at the single ion channel level. The technology will be transformative in three ways: First, it will validate a qualitatively new assay for study of apoptosis. Deregulation of apoptosis is well known as one of 6 "hallmarks" of cancer, however, methods to study mitochondrial depolarization have been lacking. Second, it will allow for a qualitatively new way to study electrophysiology at the single ion channel level. This will allow unprecedented studies of timing, location, and statistics of the mitochondrial membrane flickering and depolarization. Finally, the overall technology will enable new studies of mitochondrial metabolism. Cancer metabolism is one of the 2 new "hallmarks" added last year to cancer, and this instrumentation will enable the understanding of one important component of metabolic flux (namely, membrane potential). Because our high throughput technology will enable assays of thousands of individual mitochondria from small numbers of cells (even a single cell), it will enable an advance in instrumentation to study the interrelationships between metabolism, stem cells, and cancer biology.