Recent results from this laboratory have shown that the spectral properties of fluorescent probes can be favorably enhanced by interactions with metallic particles. We propose to use these interactions to develop high sensitivity detection of bioterrorism-related pathogens. The overall goal is to demonstrate single molecule or low copy number detection of nucleic acid sequences specific for such organisms. To accomplish these goals we propose:
Specific Aim 1. Develop and test specificity of microarrays for selected Y. pestis and Y. pseudotuberculosis genes. We will select genes from the known genomic sequence of Yersinia pestis. Some genes will be specific to the virulence plasmids and others to detect both Y. pestis and the less virulent Y. pseudotuberculosis. The specificity will be determined against DNA from other species and strains of Yersinia.
Specific Aim 2. Optimization of the fluorophore-metallic particle detection methodology for use with pathogen-specific oligomers. We will optimize the fluorophore-metallic surface geometries to obtain the maximal number of photons per fluorophore. This will include: examination of silver island films and colloids to determine the optimal size and shape for enhanced emission; evaluation of the optimal distance from the fluorophore to the metallic surface for maximal emission; and evaluation of the use of FRET for DNA assays.
Specific Aims 1 and 2 will proceed concurrently.
Specific Aim 3. Determine the sensitivity and specificity of the metallic-surface enhanced assays for Y. pestis. The sensitivity of the assays will be determined by assays based on serial dilutions. The specificity will be determined against DNA from other species and strains of Yersinia.
| Lakowicz, Joseph R (2005) Radiative decay engineering 5: metal-enhanced fluorescence and plasmon emission. Anal Biochem 337:171-94 |
| Lakowicz, Joseph R; Geddes, Chris D; Gryczynski, Ignacy et al. (2004) Advances in surface-enhanced fluorescence. J Fluoresc 14:425-41 |
| Malicka, Joanna; Gryczynski, Ignacy; Gryczynski, Zygmunt et al. (2004) Use of surface plasmon-coupled emission to measure DNA hybridization. J Biomol Screen 9:208-15 |
| Lukomska, Joanna; Malicka, Joanna; Gryczynski, Ignacy et al. (2004) Fluorescence enhancements on silver colloid coated surfaces. J Fluoresc 14:417-23 |
| Matveeva, Evgenia; Malicka, Joanna; Gryczynski, Ignacy et al. (2004) Multi-wavelength immunoassays using surface plasmon-coupled emission. Biochem Biophys Res Commun 313:721-6 |
| Lakowicz, Joseph R; Malicka, Joanna; Huang, Jun et al. (2004) Ultrabright fluorescein-labeled antibodies near silver metallic surfaces. Biopolymers 74:467-75 |
| Lakowicz, Joseph R (2004) Radiative decay engineering 3. Surface plasmon-coupled directional emission. Anal Biochem 324:153-69 |
| Gryczynski, Ignacy; Malicka, Joanna; Gryczynski, Zygmunt et al. (2004) Radiative decay engineering 4. Experimental studies of surface plasmon-coupled directional emission. Anal Biochem 324:170-82 |
| Gryczynski, Zygmunt; Gryczynski, Ignacy; Matveeva, Evgenia et al. (2004) Surface-plasmon-coupled emission: new technology for studying molecular processes. Methods Cell Biol 75:73-104 |
| Matveeva, Evgenia; Gryczynski, Zygmunt; Gryczynski, Ignacy et al. (2004) Immunoassays based on directional surface plasmon-coupled emission. J Immunol Methods 286:133-40 |
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