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: 1540926
• 负责人: Katherine Willets
• 金额: $ 25.11万
• 依托单位: Temple University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-16 至 2017-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1540926&HistoricalAwards=false
• 关键词: Probing; location; number; function; surface; 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.

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. 通常，纳米传感器基于纳米颗粒核，例如20-50 nm的金纳米颗粒，然后将其用抗体（例如抗体）功能化，从而捕获和报告特定生物标志物的存在。 在理想情况下，结合抗体应覆盖每个纳米颗粒芯的整个表面，同时保持其功能，以最大程度地捕获和报告感兴趣的生物标志物。开发了准备抗体功能化金属纳米颗粒的新合成策略的巨大努力。但是，抗体和纳米颗粒核的尺寸很小，因此难以测量单个抗体在纳米颗粒表面结合的地方变得非常困难，并且每个抗体是否保留其捕获其靶标的能力。 所提出的工作将使用超分辨率的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，以确定不同的制剂策略如何影响粒子到粒子之间的异质性和这一重要类别类别的纳米异种体的功能。该项目的成功完成将导致更好，更敏感和更特定的纳米传感器。技术描述：在超分辨率的单分子荧光成像中，通过仔细控制其化学环境和激发激励激励激发激光激素的强度，可以在发射和非发射状态之间进行荧光分子。 通过一次仅允许单个分子发射，其衍射限制的发射可以适合模型函数，例如二维高斯，以更好的分辨率位于发射器的位置，其分辨率优于10 nm。 在拟议的实验中，将用荧光分子标记与金属纳米颗粒表面结合的抗体，并使用上述超分辨率方法确定每个荧光发射极（以及每种抗体）的位置。 靶抗原还将用不同的荧光染料标记。 如果表面结合的抗体保留其捕获目标抗原的能力，则将观察到不同荧光标签位置之间的共定位，从而信号保留功能。 通过在单分子水平上工作，将一次探测与单个纳米颗粒结合的单个抗体，从而可以完整地映射整个纳米载体种群的功能保留和异质性。