Probing the location, number, and function of surface-bound antibodies on plasmonic nanoparticle biosensors using super-resolution fluorescence imaging

使用超分辨率荧光成像探测等离子体纳米颗粒生物传感器上表面结合抗体的位置、数量和功能

• 批准号: 1402610
• 负责人: Katherine Willets
• 金额: $ 30.1万
• 依托单位: University of Texas at Austin
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1402610&HistoricalAwards=false
• 关键词: Probing; location; number; function; surface

Proposal: 1402610PI: Willets, KatherineTitle: Probing the location, number, and function of surface-bound antibodies on plasmonic nanoparticle biosensors using super-resolution fluorescence imagingNanobiosensors have the potential to revolutionize both in vitro and in vivo diagnostics because they allow for targeting and signaling of biomarkers of disease in a single, nanoscale platform. Typically, nanobiosensors are based on a nanoparticle core, such as a 20- 50 nm gold nanoparticle, which is then functionalized with molecules, such as antibodies, that capture and report the presence of specific biomarkers. In the ideal case, the bound antibodies should cover the entire surface of each nanoparticle core while retaining their function, in order to maximize the chance of capturing and reporting on biomarkers of interest. A great deal of effort has gone into developing new synthetic strategies for preparing antibody-functionalized metal nanoparticles. However, the small size of both the antibodies and the nanoparticle core makes it incredibly difficult to measure where individual antibodies are bound on the nanoparticle surface and whether each retains its ability to capture its target. The proposed work will use super-resolution single molecule fluorescence imaging to measure the location, number, and function of antibodies bound to metal nanoparticles, in order to determine how different preparation strategies impact the particle-to-particle heterogeneity and function of this important class of nanobiosensors. Successful completion of this project will result in development of better, more sensitive and specific nanobiosensors.Technical Description: In super-resolution single molecule fluorescence imaging, fluorescent molecules are toggled between an emissive and non-emissive state via careful control of their chemical environment and the intensity of the excitation laser. By allowing only a single molecule to be emissive at a time, its diffraction-limited emission can be fit to a model function, such as a 2-dimensional Gaussian, to locate the position of the emitter with resolution better than 10 nm. In the proposed experiments, antibodies bound to the surface of metal nanoparticles will be labeled with fluorescent molecules, and the position of each fluorescent emitter (and thus each antibody) will be determined using the super-resolution approach described above. Target antigens will also be labeled with a different fluorescent dye. If surface-bound antibodies retain their ability to capture their target antigen, co-localization between the positions of the different fluorescent tags will be observed, signaling retention of function. By working at the single molecule level, individual antibodies bound to single nanoparticles will be probed one at a time, allowing a complete mapping of the function retention and heterogeneity across the nanobiosensor population.

提议：1402610PI：Willets，Katherine标题：使用超分辨率荧光成像探测血浆纳米颗粒生物传感器上表面结合抗体的位置、数量和功能纳米生物传感器具有革新体外和体内诊断的潜力，因为它们允许在单个纳米级平台上对疾病的生物标志物进行靶向和信号传递。通常，纳米生物传感器基于纳米颗粒核心，如20-50 nm的金纳米颗粒，然后用分子(如抗体)功能化，捕获并报告特定生物标志物的存在。在理想情况下，结合的抗体应该覆盖每个纳米颗粒核心的整个表面，同时保留它们的功能，以便最大限度地增加捕获和报告感兴趣的生物标志物的机会。为了制备抗体功能化的金属纳米颗粒，人们在开发新的合成策略方面做了大量的工作。然而，抗体和纳米颗粒核心的小尺寸使得测量单个抗体结合在纳米颗粒表面的位置以及每个抗体是否保持其捕获目标的能力变得极其困难。这项拟议的工作将使用超分辨率单分子荧光成像来测量与金属纳米颗粒结合的抗体的位置、数量和功能，以确定不同的制备策略如何影响这类重要的纳米生物传感器的颗粒到颗粒的异质性和功能。该项目的成功完成将导致开发出更好、更灵敏和更特异的纳米生物传感器。技术描述：在超分辨率单分子荧光成像中，通过仔细控制荧光分子的化学环境和激发激光的强度，荧光分子在发射和非发射状态之间切换。通过一次只允许一个分子发射，它的衍射限制发射可以符合一个模型函数，例如二维高斯，以定位分辨率优于10 nm的发射体的位置。在拟议的实验中，结合在金属纳米颗粒表面的抗体将被标记上荧光分子，并使用上述超分辨率方法确定每个荧光发射体(从而每个抗体)的位置。目标抗原也将被标记上不同的荧光染料。如果表面结合的抗体保持其捕获目标抗原的能力，将观察到不同荧光标记位置之间的共同定位，这表明功能保持不变。通过在单分子水平上的工作，结合到单个纳米颗粒上的单个抗体将被一次探测一个，从而实现整个纳米生物传感器群体的功能保留和异质性的完整映射。