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

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品中的预后激酶活性。本申请的目的是开发的第一步，在乳腺癌模型系统中使用表面增强的拉曼光谱（Sers）和肽功能化的纳米粒子（NP）为基础的生物传感器的激酶活性的多重定量。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将允许敏感，直接监测生物环境中的激酶活性，如细胞。在这个多学科的应用中，我们联合收割机结合帕克实验室的激酶生物传感器专业知识和Irudayaraj实验室的丰富Sers经验，开发了一个定量Sers平台，以监测Akt，Erk，Src和c-Abl的活性，这些激酶与乳腺癌患者的耐药性和临床结局相关。为了证明这一概念验证，我们提出了以下具体目标：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。开发单细胞绘图方案，以监测不同的激酶活性对各种刺激和抑制剂的反应。 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。这项技术是非破坏性的（分析后细胞保持活力和完整），并适用于单分子（因此是单细胞）显微镜形式，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。它还可以针对不同的激酶底物进行调节，以允许同时监测一种以上的激酶活性，换句话说，“多路复用”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。该项目有可能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。在临床中使用手持式Sers设备，这项技术可以通过为病理学家（以及临床医生）提供有关个人疾病和治疗反应的几乎“实时”的机械信息，从而使数千名患者受益。这项工作也将推进信号转导生物学领域作为一个整体，使多个激酶活性的分析（而不是只检测已知的内源性底物的磷酸化状态）在单细胞在显微镜平台。在展示了这个试点项目的概念验证后，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，以及使用商用设备实现即时护理，手持Sers读数（未来R33应用的目标）。检测平台的成熟将使生物学家首次能够在活细胞中实时检测信号转导，而无需开发和表达荧光基因构建体，从而可能改变癌症研究和药物发现。

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 Recent unpublished work in our laboratory suggests that exogenously-added peptide substrates and SERS will allow for sensitive, direct monitoring of kinase activity in biological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.