Biohazard Detection Using Metal-Enhanced Fluorescence

描述（由申请人提供）：该实验室的最新结果表明，荧光探针的光谱特性可以通过与金属颗粒的相互作用而有利地增强。我们建议使用这些相互作用，开发高灵敏度的检测生物恐怖主义相关的病原体。总体目标是证明对这些生物体特异性的核酸序列的单分子或低拷贝数检测。为了实现这些目标，我们建议： 具体目标1。开发并测试微阵列对所选Y的特异性。菌与 Y.假结核基因我们将从已知的鼠疫耶尔森氏菌基因组序列中选择基因。一些基因将是特异性的毒力质粒和其他检测两个Y。鼠疫菌和毒性较小的Y.假结核将针对来自耶尔森氏菌的其他种属和菌株的DNA确定特异性。 具体目标2。用于病原体特异性寡聚体的荧光团-金属颗粒检测方法的优化。我们将优化荧光团-金属表面的几何形状，以获得每个荧光团的最大光子数。这将包括：检查银岛膜和胶体以确定增强发射的最佳尺寸和形状;评估从荧光团到金属表面的最佳距离以获得最大发射;以及评估FRET用于DNA测定。具体目标1和2将同时进行。 具体目标3。确定金属表面增强检测Y的灵敏度和特异性。 鼠疫 将通过基于连续稀释的试验确定试验的灵敏度。将针对来自耶尔森氏菌的其他种属和菌株的DNA确定特异性。

DESCRIPTION (provided by applicant): 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.