Label-free, real-time detection of kinase activity in vitro and in single cells u

无标记、实时检测体外和单细胞中的激酶活性

• 批准号: 8079925
• 负责人: Laurie L. Parker
• 金额: $ 18.75万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8079925
• 关键词: ABL1 gene; Address; Adjuvant Therapy; Affect; Analysis of Variance; Antineoplastic Agents; Biological; Biological Models; Biology; Biopsy; Biosensor; Breast Cancer Model; Calibration; Cancer Etiology; Cancer Patient; Cells; Cessation of life; Clinic; Clinical; Complex; Detection; Development; Devices; Diagnosis; Disease; Drug resistance; Effectiveness; Environment; Future; Genetic; Goals; Hand; Hormones; In Vitro; Individual; Label; Laboratories; Lead; Left; Life; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mastectomy; Medicine; Methods; Microscopic; Microscopy; Monitor; Noise; Normal Cell; Oncogenic; Outcome; Pathologist; Pathway interactions; Patients; Peptides; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Physical environment; Pilot Projects; Population; Preclinical Drug Evaluation; Predictive Value; Radiation therapy; Raman Spectrum Analysis; Reporting; Reproducibility; Resistance; Resolution; Sampling; Scheme; Second Primary Cancers; Signal Transduction; Staging; Stem cells; Stimulus; Surface; System; Techniques; Technology; Therapeutic; Threonine; Time; Tissues; Toxic effect; Tyrosine; United States; Woman; Work; anticancer research; base; breast lumpectomy; c-abl Proto-Oncogenes; cancer cell; cancer stem cell; cellular imaging; design; drug discovery; experience; improved; in vitro Assay; in vitro activity; inhibitor/antagonist; instrumentation; malignant breast neoplasm; multidisciplinary; multiplex detection; mutant; nanoparticle; novel; overexpression; point of care; pressure; prevent; prognostic; response; single molecule; technology development; therapeutic target; tool; tumor; unpublished works

DESCRIPTION (provided by applicant): Our long-term goal is to develop a sensitive, multiplexed detection platform for real-time single-cell monitoring of prognostic kinase activity in tumor samples. The objective of this application is to develop the first steps towards a multiplex quantification of kinase activity in a breast cancer model system using surface-enhanced Raman spectroscopy (SERS) and peptide-functionalized nanoparticle (NP)-based biosensors. Recent unpublished work in our laboratory suggests that exogenously-added peptide substrates and SERS will allow for sensitive, direct monitoring of kinase activity in biological environments such as cells. In this multidisciplinary application, we combine the kinase biosensor expertise of Parker lab and the extensive SERS experience of Irudayaraj lab to develop a quantitative SERS platform to monitor the activity of Akt, Erk, Src, and c-Abl, kinases associated with drug resistance and clinical outcome of breast cancer patients. Towards demonstrating this proof-of-concept, we propose the following specific aims: Specific Aim 1. Standardize a SERS platform for the quantification of Akt, Erk, Src and Abl kinase activity. Specific Aim 2. Develop single cell mapping schemes to monitor differential kinase activity in response to various stimuli and inhibitors. Our approach represents a novel use of SERS microscopy and kinase substrate NP biosensor design. This technology is non-destructive (leaving cells viable and intact after analysis) and is adaptable to single molecule (and thus single cell) microscopy formats, providing exquisite spatial resolution and allowing us to monitor localized signaling in living cells. It is also tunable for different kinase substrates to allow simultaneous monitoring of more than one kinase activity, in other words 'multiplexing' the analysis, so we will be able to visualize kinases as complexes and systems rather than in isolation. This project has the potential to transform personalized medicine and therapy selection by facilitating single cell monitoring of therapeutic response. Using handheld SERS devices in the clinic, this technology could potentially benefit thousands of patients by giving pathologists (and thus clinicians) nearly 'real-time' mechanistic information about an individual's disease and therapeutic response. This work will also advance the field of signal transduction biology as a whole by enabling the analysis of multiple kinase activities (as opposed to just detecting the phosphorylation state of known endogenous substrates) in single cells in a microscopic platform. Upon demonstrating proof-of- concept with this pilot project, we will have the tools and experience in hand to undertake further technology and instrumentation development to facilitate real-time, live cell imaging using our technique, as well as implementation of a point-of-care, handheld SERS readout using commercially-available devices (goals for a future R33 application). Maturation of the detection platform to that stage would allow biologists for the first time ever to detect signal transduction in real-time in live cells without needing to develop and express e.g. a fluorescent genetic construct, potentially transforming cancer research and drug discovery. PUBLIC HEALTH RELEVANCE: This project has the potential to transform personalized medicine and therapy selection by facilitating single cell monitoring of cancer therapeutic response. Upon further development, this technology could benefit thousands of patients by giving pathologists (and thus clinicians) nearly 'real-time' mechanistic information about an individual's disease and therapeutic response.

描述（由申请人提供）：我们的长期目标是开发一种灵敏的多重检测平台，用于实时单细胞监测肿瘤样本中的预后激酶活性。该应用的目的是利用表面增强拉曼光谱 (SERS) 和基于肽功能化纳米粒子 (NP) 的生物传感器，迈出对乳腺癌模型系统中激酶活性进行多重定量的第一步。我们实验室最近未发表的工作表明，外源添加的肽底物和 SERS 将允许对细胞等生物环境中的激酶活性进行灵敏、直接的监测。在这个多学科应用中，我们结合了 Parker 实验室的激酶生物传感器专业知识和 Irudayaraj 实验室丰富的 SERS 经验，开发了一个定量 SERS 平台来监测 Akt、Erk、Src 和 c-Abl 的活性，这些激酶与耐药性和相关性相关。乳腺癌患者的临床结果。为了证明这一概念验证，我们提出以下具体目标： 具体目标 1. 标准化用于量化 Akt、Erk、Src 和 Abl 激酶活性的 SERS 平台。具体目标 2. 开发单细胞作图方案来监测响应各种刺激和抑制剂的差异激酶活性。 我们的方法代表了 SERS 显微镜和激酶底物 NP 生物传感器设计的新用途。该技术是非破坏性的（分析后使细胞保持活力和完整），并且适用于单分子（因此单细胞）显微镜格式，提供精致的空间分辨率，使我们能够监测活细胞中的局部信号传导。它还可以针对不同的激酶底物进行调节，以允许同时监测多种激酶活性，换句话说，“多重”分析，因此我们将能够将激酶可视化为复合物和系统，而不是孤立的。该项目有潜力通过促进治疗反应的单细胞监测来改变个性化医疗和治疗选择。在诊所中使用手持式 SERS 设备，该技术可以为病理学家（以及临床医生）提供有关个体疾病和治疗反应的近乎“实时”的机械信息，从而使数千名患者受益。这项工作还将通过在微观平台上分析单个细胞中的多种激酶活性（而不是仅仅检测已知内源底物的磷酸化状态）来推动整个信号转导生物学领域的发展。在通过该试点项目展示概念验证后，我们将拥有工具和经验来进行进一步的技术和仪器开发，以促进使用我们的技术进行实时活细胞成像，以及实施点分析- 使用市售设备的手持式 SERS 读数（未来 R33 应用的目标）。检测平台的成熟到该阶段将允许生物学家首次在活细胞中实时检测信号转导，而无需开发和表达例如信号转导。一种荧光基因结构，有可能改变癌症研究和药物发现。 公共健康相关性：该项目有潜力通过促进癌症治疗反应的单细胞监测来改变个性化医疗和治疗选择。经过进一步发展，这项技术可以为病理学家（以及临床医生）提供有关个体疾病和治疗反应的近乎“实时”的机械信息，从而使数千名患者受益。