Flow lysometry and its biomedical applications

描述（由申请人提供）：流式细胞术的单细胞分析已经彻底改变了复杂细胞群的测量。只有有限的探针可用于测量单个细胞中的细胞质成分。虽然细胞成分如ATP可以在具有渗透性质膜的细胞悬浮液中灵敏地测量，但这些化合物在异质群体中单个细胞中的分布的测定是不可用的。我们提出了一种新的技术，流动溶解法，通过多学科的方法，结合细胞生物学和纳米技术开发的微机械工程，以测量单个细胞的细胞质成分。我们的初步数据表明，我们可以在流动通道中建立单个细胞的裂解，并通过类似于流式细胞术中的事件的化学发光脉冲检测ATP。传统流式细胞术与流式溶解法的同步将允许在异质细胞群中测量表面成分与细胞质成分。作为具体目标，我们建议：1。1 .研制一种流量测定装置；2 .与流式细胞术同步溶血测定；在异质细胞群上测试流溶法。与传统流式细胞仪接口的专用微流体通道流溶测定芯片的制造将允许同步细胞检测，细胞裂解和细胞质成分传感。我们将通过测量异质镰状红细胞中磷脂酰丝氨酸（PS）暴露细胞中的ATP水平来测试该技术的有效性。我们的研究将建立这种新技术来测量特定的单个细胞（亚群）中的细胞质成分。我们设想这项技术可以应用于各种类型的细胞，测量许多不同的细胞质成分。

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.