Life Sciences proposes the development of a novel pathogen detection system based on dye-doped nanoparticles (NPs) and supported by low cost, field portable instrumentation to facilitate the rapid detection of nucleic acid sequences present in low copy number; with or without amplification. Silica, dye-doped NPs to be used in this effort have been demonstrated to produce 10,000 to 100,000 fold increases in detectable signal from a single hybridization event with a DNA target when compared to commonly used fluorescent reporters. This high level of signal enhancement is expected to permit the use, with optimization, of an existing low cost, field portable fluorometer to support the application of the pathogen detection in products for rapid (10 minutes) low cost ($ 5-10 per test) for point-of collection diagnosis of infectious disease. The system may also be deployed for field detection and identification of environmental pathogens in food and water, to include agents that maybe employed as biological weapons. To achieve this goal there are two aims in this proposal. First, we will develop a novel DNA/RNA bioassay based on dye-doped NP. This assay will be performed on glass substrate and detected by a traditional spectrofluorometer. NP optimization will be completed and will emphasize smaller size, higher signal intensity, and ease of bioconjugation. Secondly, we intend to optimize an existing low-cost fluorometer such that it can be employed as a handheld gene detection analyzer. This part will be performed on magnetic beads and detected by the hand-held fluorometer. We will focus the immobilization of DNA on the surface of magnetic beads, optimization of the assay to detect DNA down to picomole range. More importantly, we will emphasize the combination of the hand-held fluorometer to the assay. Our project is an integrated one in which better NPs will be prepared, ultrasensitive optical detection systems will be optimized, NP bioconjugation and surface chemistry will be better understood and improved, new insights into NP's bioanalytical applications will be obtained, and portable protocols for ultrasensitive bioanalysis will be developed and utilized. ? ?
