Biohazard Detection Using Metal-Enhanced Fluorescence

描述(申请人提供)：本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得到有利的增强。我们建议利用这些相互作用来开发与生物恐怖主义相关的病原体的高灵敏度检测。总的目标是展示对这种生物体特有的核酸序列的单分子或低拷贝数检测。为实现这些目标，我们建议： 具体目标1.建立和测试针对选定鼠疫杆菌和 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。一些基因将专用于毒力质粒，另一些基因将同时检测鼠疫耶尔森氏菌和毒力较低的假结核杆菌。这种特异性将根据耶尔森氏菌的其他物种和菌株的DNA来确定。 具体目标2.用于病原体特定寡聚体的荧光团-金属颗粒检测方法的优化。我们将优化荧光团-金属表面的几何结构，以获得每个荧光团的最大光子数。这将包括：检查银岛薄膜和胶体，以确定增强发射的最佳大小和形状；评估从荧光团到金属表面的最佳距离以实现最大发射；以及评估FRET用于DNA分析。具体目标1和2将同时进行。 具体目的3.确定金属表面增强试验对鼠疫杆菌的敏感性和特异性。 检测的灵敏度将通过基于系列稀释法的检测来确定。这种特异性将根据耶尔森氏菌的其他物种和菌株的DNA来确定。

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.