NSF Award
1264608
1264608<br/>Gryczynski, Ignacy <br/><br/>Technical Abtsract: The goal of this proposal is to develop a method for the measurement of intramolecular distances within a range of 100-200 Å, which is not currently achievable with conventional optical microscopy. The overall idea is to use a Förster Resonance Energy Transfer (FRET)with multiple acceptors. From our simulations and preliminary measurements we determined that the proposed multiple acceptor systems will provide energy transfer efficiencies that allow precise estimations of site-to-site distances of well over 100 Å. Using these systems, it was possible to reveal precise donor-to-horde of acceptors distances for systems with a DNA-avidin. We plan to apply both steady-state and time-resolved fluorescence techniques to further increase the accuracy of site-to-site distance measurements. Additionally, molecular/cellular Fluorescence Lifetime Imaging Microscopy (FLIM) and Fluorescence Correlation Spectroscopy(FCS) will be utilized. These methods are particularly useful in applications using extremely low concentrations of substrates and detected analytes. In order to predict and analyze the data for long FRET measurements, theoretical models will be developed. First, for a quick data analysis, a simple model will be established assuming a random distribution of acceptors on a sphere and a single donor. This will be followed by a Monte Carlo Simulations (MCS) based model to predict the intensity decays of the donor and acceptor for various numbers and distributions of interacting fluorophores. As a primary application of the long FRET strategy we propose developing an immunoassay for a celiac disease. This bioassay requires detection of site-to-site distances longer than 100 Å. A method allowing for easy, non invasive detection of this sickness has the potential to eliminate significant, unnecessary pain for a great many individuals.<br/><br/>General Abstract: The goal of this proposal is to develop more sensitive biosensors that work based on appearance of fluorescence upon binding of the target molecules. Such biosensors are often based on the fluorescence phenomenon where presence of two fluorophores cause quenching of signal and separation of these fluorophores causes for signal to come on. This proposal will develop a better fluorescence based system for biosensor development.
