Atomic Force-FRET Microscope Using Quantum Dot for Cell Mechanobiology

描述(申请人提供)：心血管疾病是西方世界主要的死亡原因。这些疾病是多因素的，但研究揭示了机械因素的普遍作用。这项提议是一项多学科研究计划，旨在开发纳米级生物传感和成像技术，以了解细胞-细胞外基质的相互作用。将该技术应用于血管细胞的机械生物学，将在细胞生物学领域得到广泛应用。许多正常的和病理生理的血管过程依赖于细胞对机械力的反应，整合素起着核心作用。目前的技术还不能对机械力的整合素感觉的机械性质进行精确的定量分析。原子力显微镜(AFM)生物学应用的发展，特别是与纳米光学生物传感技术的结合，为机械生物学的研究提供了新的机遇。我们的策略将原子力显微镜纳米级尖端的空间分辨率和控制能力与纳米级量子点的独特光学特性相结合，形成了一种Forster共振能量转移(FRET)生物探针系统。该系统将确定引发细胞反应所需的力，检测细胞机械反应，并将它们与细胞膜上的整合素周转联系起来。在这种基于FRET的系统中，纳米级半导体量子点是一个很好的选择。其具体目标是：1.开发一种独特的具有光学活性和生物功能的纳米探针系统，用于集成原子力、多光学成像系统；以及2.通过开发上述基于纳米探针的系统，通过对血管平滑肌细胞外基质-整合素-细胞骨架轴的机械生物学的研究，展示该技术的实用性。这项建议中的合作建立在纳米级生物传感和成像技术的开发和使用方面现有的强有力的伙伴关系的基础上。公共卫生相关性：由于心血管疾病是主要的死亡原因，心血管研究在国内和国际上都很重要。我们建议开发一种纳米级的生物传感和成像技术，这将对了解心血管疾病有价值。拟议的纳米级成像和传感设备的开发也将为推进我们对细胞的分子理解提供一种灵活的新工具。

DESCRIPTION (provided by applicant): Cardiovascular diseases are a leading cause of death in the Western World. These diseases are multi- factorial, yet research reveals the pervasive role of mechanical factors. This proposal is a multidisciplinary research plan that aims to develop a nanoscale biosensing and imaging technology toward understanding cell-extracellular matrix interactions. Applied here to mechanobiology of vascular cells, the technology would be broadly applicable in the cell biology field. Many normal and pathophysiological vascular processes depend on cell responses to mechanical forces with integrins playing a central role. Current technologies have not permitted precise quantitative analysis of the mechanistic nature of integrin sensation of mechanical forces. Developments in biological applications of atomic force microscopy (AFM), particularly in combination with nanoscale optical biosensing technology, offer new opportunities for study of mechanobiology. Our strategy combines the spatial resolution and control capabilities of an AFM's nanoscale tip with the unique optical properties of nanoscale quantum dots to produce a Forster resonance energy transfer (FRET) bioprobe system. This system will determine the forces necessary to elicit cellular responses, detect cell mechanical responses and correlate them with integrin turnover at the cell membrane. Nanoscale semiconductor quantum dots present an excellent option in this FRET-based system. The specific aims are: 1. To develop a unique system consisting of optically active and biologically functionalized nano-probe for use in an integrated atomic force, multi-optical imaging system; and 2. To demonstrate the utility of this technology by investigation of the mechanobiology of the extracellular matrix- integrin-cytoskeletal axis in vascular smooth muscle cells through the development of the above nano-probe based system. The collaboration in this proposal is based on a strong existing partnership in the development and use of biosensing and imaging technologies at the nanoscale. Public Health Relevance: As cardiovascular disease is the leading cause of death, cardiovascular research is important nationally and internationally. We propose to develop a nanoscale biosensing and imaging technique that will be valuable for understanding cardiovascular disease. The development of the proposed nanoscale imaging and sensing device will also provide a flexible new tool for advancing our molecular understanding of the cell.