The proposed project is continuation of a completed Phase I program entitled Real-Time Detection of Airborne Mycobacteria. ? ? The goal of Phase II efforts is to develop a multi-use automated portable system device for sampling and sensitive real-time detection of airborne pathogens, with special emphasis of Mycobacteria, and, most importantly, Mycobacterium tuberculosis. ? ? Experimental microorganisms will be aerosolized and a small-size (yet highly efficient) portable air sampler (MesoSystems Technology, Inc.) will be used with appropriate modifications for wet aerosol particle collection. Capture efficiency will be measured as a function of aerosol droplet size, microbe concentration, airborne residence time and relative humidity. The air sampler will be integrated with an automated immunoelectrochemical biosensor (developed and tested in the Phase I program), which will be capable of programmed sample acquisition (in the form of liquid aliquots), sample preconditioning (cleanup), and immunochemical labeling and quantitative detection. System calibration will be based on microscopic cell counting, colony counting, and when needed, quantitative polymerase chain reaction (PCR). ? ? The fluidic components of the biosensor will be designed as a sophisticated three-dimensional microfluidic cartridge, which carries out functions of rapid antibody binding (using chaotic advective mixer technology), removal of unbound label, and of an amplified highly sensitive enzyme-linked immunoassay. As an important practical feature, the reagents will be prepared in """"""""dry kits"""""""" suitable for room-temperature storage. The biosensor will selectively detect airborne pathogens within a 30-min timeframe post capture, at a sensitivity exceeding at least 100 times that of current bioagent strip tests; at a lower detection limit of 100-200 cells per sample. ? ? The biocapture/biosensor unit will be tested in aerosol chambers (during the -02 year) and hospital rooms (during the -03 year) with TB patients (at San Francisco General Hospital) for quantitative assessment of airborne Mycobacteria, and exposure assessment for clinical personnel. The modus operandi for the prototype design for applications in medical/clinical environment will be determined on the basis of results of the """"""""live"""""""" aerosol tests. ? ? Although the main body of proposed research and technology development will focus on airborne Mycobacteria, it will be demonstrated that the resulting biocapture/biodetection technology will be equally applicable for monitoring of other airborne bacteria (Streptococcus pyogenes), fungi (Aspergillus fumigatus), and airborne viruses. As such, proposed technology will deliver an essential tool for clinical, community-associated and environmental airborne pathogen monitoring. ? ? A product development/commercialization plan is proposed. ? ?
