Plasmon-coupled Fluorescence Correlation Spectroscopy in Nanoholes

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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.