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 i s 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.
Biomedical research and clinical testing is strongly dependent on measurements of binding reactions between biomolecules in homogeneous bioassays. There are many examples, such as immunoassays to detect disease markers and drug development. Currently used spectroscopic methods have llimitations regarding the molecular weight of the target molecules which restricts these methods to smaller biomolecues. We propose to develop a new concept which is based on fluorescence and nanotechnology. We will construct nanoscale structures that alter the intensities of fluorescent molecues based on their orientation relative to a metallic nanostructure. Our method will reveal binding by the non-contact interactions of fluorphores attached to biomolecules with the metal surfaces in nanoholes. Our approach is novel because it uses a new and sensitive method to detect rotational motions which depend on the size of biomolecules, and thus on association reactions, but can be used with very high molecular weight biomolecular complexes. The concept will eliminate the need for complex and expensive optics, can be readily extended to multi-analyte and/or high-throughput testing and thus provide a new measurement technology for research and clinical testing.
