Nanofluidics Devices for Rapid Single Cell Analysis of Protein Expression

用于蛋白质表达快速单细胞分析的纳米流体装置

• 批准号: 7934028
• 负责人: JOHN Michael RAMSEY
• 金额: $ 43.39万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7934028
• 关键词: Address; Bedside Testings; Binding; Biochemical; Biological; Biological Assay; Biosensor; Blood specimen; Bone Marrow; CD3 Antigens; Cancer Biology; Cancer Center; Cell Volumes; Cell physiology; Cell surface; Cells; Chemicals; Clinical; Complex; Core Facility; Coupled; Cytolysis; Decision Making; Devices; Electron Beam; Electronics; Electrophoresis; Event; Flow Cytometry; Gene Expression; Guanosine Triphosphate Phosphohydrolases; IL2RA gene; Ions; Kinetics; Leukocytes; Ligands; Liquid substance; Lymphocyte; Measures; Membrane; Methods; Microfluidic Microchips; Microfluidics; Molecular; Molecular Target; Nanotechnology; Operative Surgical Procedures; Optical Instrument; Patients; Phenotype; Phosphotransferases; Process; Proteins; Pump; Regulatory T-Lymphocyte; Sampling; Signal Transduction; Solid Neoplasm; Sorting - Cell Movement; Stem cells; T-Lymphocyte; T-Lymphocyte Subsets; Technology; Time; Trauma; base; cancer cell; cancer stem cell; cell type; chromophore; clinical application; combinatorial chemistry; instrument; interest; malignant breast neoplasm; nano; nanoassay; nanofluidic; nanopore; nanoscale; neoplastic cell; oncology; peripheral blood; point of care; pressure; protein expression; receptor; sensor; single cell analysis; single molecule; stem

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).

项目5：用于蛋白质表达的快速单细胞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 在单细胞水平上进行表征。为了解决这个问题，我们建议开发一个即时护理 微流体平台接受“采集的”患者样本，并在30分钟内提供相关的 生物化学信息提供给临床医生。微流体装置将包括样品处理和 一个微型流式细胞仪与基于纳米技术的生化分析相结合， 在分选的单细胞中的表达和功能。该技术将针对复杂的患者， 外周血和骨髓等材料。初期工作将集中于 淋巴细胞中四种关键分子靶标的分选、计数和分子表征。 我们将开发基于纳米技术的检测方法，包括纳米孔随机传感器和荧光检测。 分子生物传感器来测量单细胞水平的蛋白质浓度和激酶活性。的 纳米测定将询问FoxPS和磷酸化ERK浓度以及 cdk 4/6和cdc 42都是已知与癌症生物学相关的靶标。本文中描述的方法 该提案将有几个直接的临床应用，包括枚举和 T调节细胞的特征，一个重要的T细胞亚群，目前不能被 通过流式细胞术方法明确鉴定。在具体目标1-3中开发的技术 这一建议的实施将使流式细胞术的巨大好处被纳入实时 临床决策，并将允许即时检测。再加上纳米分析 在具体目标4-6中开发的能力，这些技术可以定制用于解决 肿瘤学中特别困难的临床问题，例如确定靶向激酶的表达 在循环恶性肿瘤细胞中（例如AMI中的Flt 3）;鉴定循环实体瘤细胞（例如乳腺癌中的 癌症）;以及在主要肿瘤的特征性细胞类型中鉴定激酶相关的信号传导事件。 生物学兴趣（例如造血干细胞）。