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

基本信息

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
项目类别：
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point of care pressure prevent prognostic response single molecule technology development therapeutic target tool tumor unpublished works

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

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项目摘要

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个敏感的多重检测平台，用于对肿瘤样品中预后激酶活性的实时单细胞监测。该应用的目的是使用表面增强的拉曼光谱（SER）和肽官能化的纳米颗粒（NP）的生物传感器来开发在乳腺癌模型系统中对激酶活性多重定量的第一步。我们实验室中最近未发表的工作表明，外源性肽底物和SERS将允许在生物学环境（例如细胞）中对激酶活性进行敏感，直接监测。在此多学科应用中，我们将帕克实验室的激酶生物传感器专业知识和Irudayaraj Lab的广泛SERS经验相结合，以开发一个定量的SERS平台，以监测与耐药性和乳腺癌患者耐药性和抗药性和临床抗药性和乳腺癌患者临床相关的AKT，ERK，SRC和C-ABL的活性。为了证明这一概念验证，我们提出了以下特定目的：特定目标1。标准化一个SERS平台，以量化AKT，ERK，SRC和ABL激酶活性。具体目标2。开发单细胞图方案，以响应各种刺激和抑制剂来监测差异激酶活性。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颖使用。该技术是无损的（分析后使细胞可行且完整）适应单分子（以及单细胞）显微镜格式，提供了精美的空间分辨率，并允许我们监视活细胞中的局部信号传导。对于不同的激酶底物，允许同时监视多个激酶活性，换句话说，“多路复用”分析也可以调整，因此我们将能够将激酶视为复合物和系统而不是隔离。该项目有可能通过促进治疗反应的单细胞监测来改变个性化的医学和治疗选择。使用诊所中的手持式SERS设备，该技术可能会通过给予病理学家（以及临床医生）几乎“实时”机械性信息和治疗反应来使数千名患者受益。这项工作还将通过在微观平台中的单个细胞中对多种激酶活性进行分析（而不是仅检测已知内源性底物的磷酸化状态）来推进整体信号转导生物学领域。通过该试点项目证明了概念证明，我们将拥有手头的工具和经验，以实现进一步的技术和仪器开发，以促进使用我们的技术实时，实时细胞成像，并实施使用良心的手持式SERS读数（未来R33应用程序的目标）。检测平台到该阶段的成熟将使生物学家有史以来首次检测活细胞中实时的信号转导，而无需开发和表达例如荧光遗传构建体，可能改变癌症研究和药物发现。公共卫生相关性：该项目有可能通过促进癌症治疗反应的单细胞监测来改变个性化的医学和治疗选择。在进一步开发后，这项技术可以通过给病理学家（以及临床医生）几乎“实时”机械性信息和治疗反应来使数千名患者受益。