Flow lysometry and its biomedical applications

描述(申请人提供)：用流式细胞术进行单细胞分析使复杂细胞群体的测量发生了革命性的变化。只有有限的探针可用于测量单个细胞中的胞浆成分。虽然在具有渗透性质膜的细胞悬浮液中可以灵敏地测量细胞成分，如三磷酸腺苷，但无法确定这些化合物在异质群体中单个细胞中的分布。我们提出了一种多学科的方法，结合细胞生物学和微机械工程开发的纳米技术，开发了一种新的技术-流动裂解法来测量单个细胞的胞浆成分。我们的初步数据表明，我们可以建立流动通道中单个细胞的裂解，并通过类似于流式细胞术中的事件的化学发光脉冲来检测ATP。传统的流式细胞术与流动裂解计量术的同步将允许测量异质细胞群体中的表面成分和胞浆成分。作为特定的目标，我们建议1.开发一种流动裂解测定仪，2.使流动裂解测定仪与流式细胞术同步，以及3.对异质细胞群体进行流动裂解测定法测试。与传统的流式细胞仪接口的专用微流控通道流动裂解芯片的制造将允许同步的细胞检测、细胞裂解和胞浆成分检测。我们将通过测量暴露在不同种类的镰刀状红细胞中的磷脂酰丝氨酸(PS)细胞中的ATP水平来测试这项技术的有效性。我们的研究将建立这项新技术来测量确定的单个细胞(亚群)中的胞浆成分。我们设想，这项技术可以应用于各种类型的细胞，测量许多不同的胞浆成分。

DESCRIPTION (provided by applicant): Single-cell analysis by flowcytometry has revolutionized the measurement of complex cell populations. Only limited probes are available for the measurement of cytosolic components in single cells. While cellular components such as ATP can be measured sensitively in a suspension of cells with permeabilized plasma membranes, the determination of the distribution of such compounds in individual cells in a heterogeneous population is not available. We propose the development a novel technique, flow-lysometry, by a multi- disciplinary approach, combining cell biology and nanotechnology developed by micromechanical engineering, to measure cytosolic components of single cells. Our preliminary data show that we can establish lysis of individual cells in a flow channel and detect ATP by a chemiluminescent pulse similar to an event in flowcytometry. The synchronization of conventional flowcytometry with flow-lysometry will allow measurement of surface components in conjunction with cytosolic components in a heterogeneous cell population. As specific aims we propose to 1. develop a device for flow-lysometry, 2. synchronize flow-lysometry with flowcytometry, and 3. test flow-lysometry on a heterogeneous cell population. Fabrication of dedicated micro fluidic channel flow-lysometry chips interfaced with a conventional flowcytometer will allow synchronized cell- detection, cell-lysis, and cytosolic component sensing. We will test the efficacy of this technology by measuring ATP levels in phosphatidylserine (PS) exposing cells in the heterogeneous population of sickle red blood cells. Our research will establish this novel technology to measure cytosolic components in defined, (subpopulations of) individual cells. We envision that this technique can be applied to various types of cells measuring many different cytosolic components.