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

建议：1402610 PI：Willets，KatherineTitle：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，数量和功能纳米生物传感器有可能彻底改变体外和体内诊断，因为它们允许在单个纳米级平台中靶向和信号传导疾病的生物标志物。 通常，纳米生物传感器基于纳米颗粒核心，例如20- 50 nm的金纳米颗粒，然后用分子（例如抗体）功能化，所述分子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，同时保留其功能，以最大限度地捕获和报告感兴趣的生物标志物。大量的努力已经投入到开发用于制备抗体功能化的金属纳米颗粒的新的合成策略中。然而，抗体和纳米颗粒核心的小尺寸使得难以测量单个抗体在纳米颗粒表面上的结合位置以及每个抗体是否保留其捕获目标的能力。 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间异质性和功能。该项目的成功完成将导致开发更好，更灵敏和更具体的纳米生物传感器。技术描述：在超分辨率单分子荧光成像中，荧光分子通过仔细控制其化学环境和激发激光的强度在发射和非发射状态之间切换。 通过一次仅允许单个分子发射，其衍射限制的发射可以拟合到模型函数，例如二维高斯，以定位具有优于10 nm的分辨率的发射器的位置。 在所提出的实验中，结合到金属纳米颗粒表面的抗体将用荧光分子标记，并且每个荧光发射体（以及因此每个抗体）的位置将使用上述超分辨率方法来确定。 靶抗原也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其靶抗原的能力，则将观察到不同荧光标签的位置之间的共定位，从而发出功能保留的信号。 通过在单分子水平上工作，将一次探测一个与单个纳米颗粒结合的单个抗体，从而可以完整地绘制整个纳米生物传感器群体的功能保留和异质性。

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.