We propose to develop a new approach to measuring biomolecule association reactions. This method will be based on diffusion-enhanced FRET (DE-FRET). With nanosecond decay time fluorophores diffusion has essentially no effect on the extent of FRET. We will overcome this limitation by using lanthanide oxide nanoparticles (LNP) which have surface-bound silver particles. The presence of multiple lanthanide ions per particle and Ag particles will compensate for the typical low extinction and intensity of lanthanides, and also circumvent the need for sensitizing chelators. We will use the LNPs as donors and acceptor-labeled detection molecules. Binding of the detection molecules to a target will alter the acceptor diffusion coefficient, the LNPs intensity and decay times. The DE-FRET data will reveal translational diffusion coefficients. Similar information can be obtained by FCS, but FCS requires careful control of the focal volume and can only be used at nanomolar concentrations. In contrast our method will require no special optics, will work with any volume sample and will be sensitive to binding constants in the micromolar range, which is ideal for binding to receptors and drug discovery. We will demonstrate the usefulness of our approach using two types of binding reactions. The first will be self-association of a protein melittin to form tetramer. Measurements of protein- protein association reactions are important for proteomics. The second will be a clinical testing assay of digoxin in serum. This application is representative of large class of small molecule assays of drugs and substances of abuse. In this revised proposal we included an important biomedical application, screening of antibodies against HIV pseudo-virons or gp120. This project will have a high impact because the method can be immediately used with some commercial plate readers and easily added to most existing plate readers.
Measurement of binding between biomolecules is a central topic of medical diagnostics and biological research. Many of these methods are complex, such as PCR and Elisa assays, and requiring multiple handling steps. Some desirable methods such as fluorescence correlation spectroscopy (FCS) are difficult to use because of complex optics and accessible concentration range. We propose to develop a new class of binding assays which provides information similar to FCS, but without the constraints of FCS on precise optics and concentration. This approach will be implemented with a stage scanning microscope with modified electronics and software. We will use the effects of molecular diffusion on fluorescence resonance energy transfer (FRET) using novel lanthanide oxide nanoparticles with noble metals to increase their emission intensities. The decay times of these particles will reveal the diffusion coefficient of molecules in the solution. Binding of molecules like antibodies to target analytes will be detected by changes in diffusion coefficients. We will test our method with binding assays which are representative of those used in biological research. These systems will include a protein-protein association reaction and detection of a clinical analyte digoxin which requires close control of its concentrations in blood. Our method will also be used to screen potential neutralizing antibodies against HIV.