Radiation Monitoring Devices, Inc. (RMD) proposes to develop a field-portable instrument for the detection and identification of pathogenic viruses that are collected by an aerosol concentrator. It will incorporate modular, microfluidic platforms designed to capture and concentrate the sample for a rapid, two-stage analysis. The first employs pathogen capture on antibody-decorated, paramagnetic nanospheres and will provide rapid, preliminary warning based on the hydrodynamic properties of the nanoparticles when the targeted viruses are bound. This stage will monitor the relaxation of the magneto-optical birefringence of the nanoparticles and will trigger an alarm when the birefringence relaxation rate drops off dramatically. The methodology allows for continuous surveillance over virtually unlimited periods. The second stage will provide either confirmation or correction to the preliminary identification. It is based on the ability to interrogate the captured nucleic acid with a series of unique probes that change their fluorescence properties when they hybridize with specific nucleotide sequences of suspected viral genes. We will compare the speed and sensitivity of detecting these genes by fluorescence generated from molecular beacons, and by the temporal correlation of fluorescence issued from tagged probes. The detection method exhibiting the best performance will be incorporated into the sensor. In each case, Nucleic Acid Sequence Based Amplification (NASBA), an isothermal nucleotide amplification scheme, will be employed to raise the number of target template copies to detectable levels. The biosensor will integrate the two stages of sample transfer, amplification, and analysis in a microfluidic architecture that enables high throughput processing and parallel detection of multiple probes. Employing both proportional mode avalanche photodiode (APD) detectors and Geiger-mode muAPDs that are responsive to extremely low light levels, sensitivity to low virus titers will be possible. ? ? This proposal solicits funding for developing an instrument to detect and identify pathogenic viruses in a rapid, automated two-step analysis that can be used by emergency workers who are responding to a suspected release of airborne (waterborne) agents. The first step screens the sample to determine if there are any virus particles that possess a targeted group of exposed coat proteins. If the answer is affirmative, the virus is isolated and its nucleic acid is analyzed to determine if it carries pathogen genes. ? ? ?
