Analyzer for 2D Multiplex Detection of Infectious Agents

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The goal of this grant is to develop a novel two-dimensional immuno-genetic analysis method and lab-on- a-chip apparatus that will be used for highly specific, sensitive, quantitative and rapid detection of pathogens. The immuno-genetic analyzer will be used for point-of-care diagnosis of infected individuals, for point-of- analysis identification of pathogens in food, water and other samples, and a version will be developed for high-throughput "clinical" screening. Our rationale is that in all these applications, it is critically important to have a method that is specific, that has very low false positives, that provides quantitation of the extent of contamination, and that provides genotypic information on the infection so that the nature of the threat as well as treatment are defined. These long-term goals will be approached by completing the following Specific Aims: (1) Reagents, methods and microfluidic networks will be developed for microfluidic capture/preconcentration chambers containing antibody decorated beads, monoliths or sol-gels that capture defined bacterial contaminants ( eg. from our development panel of food/water pathogens ). Samples will be pumped through these chambers and the bacteria will be separated, concentrated and purified via immunocapture. (2) These microfluidic capture methods will be extended to the segregation of mixed bacterial populations by using a series of chambers each targeted to a different class of pathogens. (3 and 4) Microfluidic methods will be developed at the breadboard level for integrating immunocapture chambers with microfabricated PCR amplification chambers and capillary electrophoresis (CE) analyzers to provide specificity, sensitivity and quantitation and most importantly genetic characterization of the infection. (5) The capture chambers, pumps, PCR chambers and CE analyzers will be monolithically integrated on a substrate as a part of a portable pathogen analyzer, and a series of instruments will be developed to improve performance and multiplex analysis. (6) The optimized portable systems will be used to perform analyses of more pathogenic bacteria, viruses and bacterial spores. (7) In addition, a high-throughput, parallel 96 sample version of the immuno-genetic analyzer will be developed.