Nanofluidics Devices for Rapid Single Cell Analysis of Protein Expression

项目5：用于蛋白质表达的单细胞快速分析的纳米流体设备 癌症生物学中的一个关键问题源于无法进行生物化学和分子生物学 在单细胞水平上的表征。为了解决这个问题，我们建议开发一种护理点 微流控平台接受患者样本，并在30分钟内提供相关信息 向临床医生提供生化信息。微流控设备将包括样品处理和 微型流式细胞仪与基于纳米技术的生化分析联用研究基因 在已排序的单个细胞中的表达和功能。拟议的技术将针对复杂的患者衍生 外周血和骨髓等材料。初步工作将集中在 淋巴细胞中四个关键分子靶标的分类、计数和分子特征。 我们将开发基于纳米技术的分析，包括纳米孔随机传感器和荧光 分子生物传感器，用于在单细胞水平上测量蛋白质浓度和激酶活性。这个 纳米分析将询问FoxPS和磷酸化ERK的浓度以及 CDK4/6和CDC42？都是癌症生物学中已知的相关靶点。本文件中描述的方法 该提案将有几个即时的临床应用，包括列举和 T调节细胞的特征，一个重要的T细胞亚群，目前还不能 通过流式细胞术明确鉴定。在具体目标1-3中开发的技术 这一提议的实施将允许将流式细胞术的巨大好处纳入实时 临床决策，并将允许进行护理点测试。再加上纳米分析 在特定的AIMS 4-6中开发的能力，这些技术可以量身定做用于解决 肿瘤学中特别困难的临床问题，如确定靶向激酶的表达 循环中的恶性细胞(例如，急性心肌梗死中的Flt3)；识别循环中的实体瘤细胞(例如，在乳房中 癌症)；以及识别特征不佳的主要肿瘤细胞类型中的激酶相关信号事件 生物学兴趣(例如，造血干细胞)。

Project 5: Nanofluidics Devices for Rapid Single Cell Analysis of Protein Expression A critical problem in cancer biology stems from the inability to perform biochemical and molecular characterization at the single cell level. To address this issue, we propose to develop a point-of-care microfluidics platform that accepts "as collected" patient samples and within 30 min provides relevant biochemical information to the clinician. The microfluidics devices will include sample processing and a micro-flow cytometer coupled with nanotechnology-based biochemical assays to study gene expression and function in sorted single cells. The proposed technology will target complex patientderived materials such as peripheral blood and bone marrow. Initial efforts will concentrate on the sorting, enumeration and molecular characterization of four critical molecular targets in lymphocytes. We will develop nanotechnology based assays including nanopore stochastic sensors and fluorescent molecular biosensors to measure protein concentration and kinase activity at the single cell level. The nano-assays will interrogate FoxPS and phospho-ERK concentration as well as kinase activity of cdk4/6 and cdc42¿all targets of known relevance in cancer biology. The methods described in this proposal will have several immediate clinical applications including the enumeration and characterization of T-regulatory cells, an important T-cell subset that presently cannot be unambiguously identified by flow cytometric means. The technologies developed in specific aims 1-3 of this proposal will allow the tremendous benefits of flow-cytometry to be incorporated into real-time clinical decision making, and will allow for point-of-care testing. Coupled with the nano-analysis capabilities developed in specific aims 4-6, these technologies could be tailored for use in resolving particularly difficult clinical issues in oncology such as determining the expression of a targeted kinase in circulating malignant cells (e.g. Flt3 in AMI); identifying circulating solid tumor cells (e.g. in breast cancer); and identifying kinase-related signaling events in poorly characterized cell types of major biological interest (e.g. hematopoetic stem cells).