Plasmon-coupled Fluorescence Correlation Spectroscopy in Nanoholes

摘要 生物医学研究通常依赖于测量 大分子我们提出了原理验证实验来测试一种新的方法， 金属纳米结构与荧光相关光谱（FCS）的组合。我们 方法绕过了当前使用的方法的一些限制。银金属薄膜中的纳米孔 将被用来获得强度波动，这取决于旋转扩散，这是更多 比经典FCS中使用的平移扩散对分子量更敏感。此外，我们的 方法允许测量高分子量物质的长相关时间， 这对于典型的各向异性测量是困难的， 荧光团，通常在2至10 ns的时间尺度上。这些对较慢运动的测量将 可能是因为强度波动将是由于角度依赖的相互作用， 荧光团与纳米孔的内壁接触。 具体目标1。证明纳米孔中荧光团的强度波动可以被 用于测量荧光团的旋转扩散。这个目标是新颖的，因为旋转 在纳米孔中还没有报道扩散。以前的研究中使用的纳米孔 平移扩散进出观察体积。不同粘度的荧光团 将使用解来确定是否由于旋转扩散而发生强度波动。我们 将这种现象称为等离子体激元耦合荧光相关光谱（PC-FCS）。 具体目标2。使用PC-FCS测量宽谱带蛋白质的旋转相关时间 分子量范围。将检测PC-FCS，以检测模型之间的结合 蛋白质，如生物素化的人血清白蛋白（bt-HSA）和链霉亲和素（SA）。 冲击这个项目将有很高的影响，因为它扩展了荧光的使用 各向异性的高分子量分子或复合物，允许FCS测量在 微摩尔浓度，并且该方法可以扩展到高通量测定。 此外，PC-FCS的光学器件被简化，因为纳米孔，而不是物镜， 确定观察到的体积。PC-FCS具有用于高通量检测的潜力 因为最近报道的结构可以导致垂直于样品的发射 面

Abstract Biomedical research often depends on measurements of binding between macromolecules. We propose proof-of-principle experiments to test a new method using a combination of metallic nanostructures with fluorescence correlation spectroscopy (FCS). Our method bypasses some limitations of currently used methods. Nanoholes in silver metal films will be used to obtain intensity fluctuations which depend on rotational diffusion, which is more sensitive to molecular weight than translational diffusion used in classical FCS. Additionally, our method allows measurements of the long correlation times of high molecular weight species, which is difficult with typical anisotropy measurements that are limited by the lifetime of the fluorophores, typically on the 2 to 10 ns timescale. These measurements of slower motions will be possible because the intensity fluctuations will be due to angle-dependent interactions of fluorophores with the inner walls of the nanoholes. Specific Aim 1. Demonstrate that intensity fluctuations from fluorophores in nanoholes can be used to measure rotational diffusion of fluorophores. This Aim is novel because rotational diffusion has not yet been reported in nanoholes. Previous studies in nanoholes used translational diffusion into and out of the observed volume. Fluorophores in different viscosity solutions will be used to determine if intensity fluctuations occur due to rotational diffusion. We refer to this phenomenon as plasmon-coupled fluorescence correlation spectroscopy (PC-FCS). Specific Aim 2. Use PC-FCS to measure the rotational correlation times of proteins with a wide range of molecular weight. The PC-FCS will be tested for detection of binding between model proteins, such as biotinylated human serum albumin (bt-HSA) and streptavidin (SA). Impact. This project will have a high impact because it extends the use of fluorescence anisotropy to high molecular weight molecules or complexes, allows FCS measurements at micromolar concentrations, and the method can be extended to high-throughput assays. Additionally, the optics for PC-FCS is simplified because the nanoholes, and not the objective, determine the observed volume. PC-FCS has the potential for use in high-throughput assays because recently reported structures can result in emission perpendicular to the sample surface.