Plasmon-coupled Fluorescence Correlation Spectroscopy in Nanoholes

摘要 生物医学研究通常依赖于测量两者之间的结合 大分子。我们提出了原则证明实验来测试一种新方法，该方法使用 金属纳米结构与荧光相关光谱(FCS)的结合。我们的 方法绕过了当前使用的方法的某些限制。银金属薄膜中的纳米孔 将被用来获得依赖于旋转扩散的强度波动，这比 与经典FCS中使用的平移扩散相比，它对分子量更敏感。此外，我们的 该方法允许测量高分子量物种的长相关时间， 这对于典型的各向异性测量来说是困难的，这些测量受到 荧光团，通常在2到10 ns的时间尺度上。这些对较慢运动的测量将 这是可能的，因为强度波动将是由于 荧光团与纳米孔的内壁。 具体目标1.证明纳米孔中荧光团的强度波动可以是 用于测量荧光团的旋转扩散。这个目标是新颖的，因为旋转 纳米孔中的扩散还没有报道。先前在纳米孔中使用的研究 平移扩散进入和流出观测体积。不同粘度的荧光团 解决方案将被用来确定强度波动是否由于旋转扩散而发生。我们 将这种现象称为等离子体耦合荧光相关光谱(PC-FCS)。 具体目的2.用PC-FCS测量蛋白质的转动相关时间 分子量范围。将测试PC-FCS以检测模型之间的绑定 蛋白质，如生物素化人血清白蛋白(bt-HSA)和链霉亲和素(SA)。 冲击力。这个项目将产生很大的影响，因为它扩大了荧光的使用 对高分子量分子或络合物的各向异性，使FCS测量在 该方法可扩展到高通量分析中。 此外，PC-FCS的光学系统也得到了简化，因为纳米孔，而不是物镜， 确定观察到的体积。PC-FCS具有用于高通量分析的潜力 因为最近报道的结构可以导致垂直于样品的发射 浮出水面。

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.