Autonomous Microfluidic Sample Preparation System for Protein Profile-Based Detection of Aerosolized Bacterial Cells and Spores Jeanne C. Stachowiak, † Erin E. Shugard, Bruce P. Mosier, Ronald F. Renzi, Pamela F. Caton, Scott M. Ferko, James L. Van de Vreugde, Daniel D. Yee, Brent L. Haroldsen, and Victoria A. VanderNoot* Sandia National Laboratories, Livermore, California For domestic and military security, an autonomous sys- tem capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectiv- ity to warn against and lead to the prevention of infection in field settings. We present a fully automated system for the detection of aerosolized bacterial biothreat agents such as Bacillus subtilis (surrogate for Bacillus anthracis) based on protein profiling by chip gel electrophoresis coupled with a microfluidic sample preparation system. Protein profiling has previously been demonstrated to differentiate between bacterial organisms. With the goal of reducing response time, multiple microfluidic compo- nent modules, including aerosol collection via a com- mercially available collector, concentration, thermochem- ical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample prepa- ration/analysis system. The cycle time for sample prepa- ration was approximately 5 min, while total cycle time, including chip gel electrophoresis, was approximately 10 min. Sensitivity of the coupled system for the detection of B. subtilis spores was 16 agent-containing particles per liter of air, based on samples that were prepared to simulate those collected by wetted cyclone aerosol col- lector of ∼80% efficiency operating for 7 min. Continuous monitoring of the air for particles containing biothreat agents such as bacterial spores is necessary for the security of civilian and military populations. Selectivity, sensitivity, detection time, autonomy, portability, power requirements, and reagent consumption must all be considered when determining the effectiveness of a monitoring system. Existing fielded systems address those considerations to varying degrees, but none has the selectivity to warn against, and lead to the prevention of, infection. In all cases, the time required for sample gathering, preparation, and analysis must be included in the total detection time. The primary fielded techniques for biothreat detection include particle size and native fluorescence analysis, 1-3 immunoassay- based techniques, 4 and polymerase chain reaction (PCR)-based techniques. 5, These technologies, which are covered in several recent reviews, 6-8 have been implemented to varying degrees in autonomous systems and meet some of the criteria listed above. Briefly, particle size and fluorescence analysis examines scattered and emitted light intensity from airborne particles to determine particle size and fluorescence, respectively. With single particle and 20 kHz capabilities, this technique has demonstrated superior performance compared to competing techniques in terms of sensitivity and speed, but it cannot reliably differentiate between various biological particles nor between biological and fluorescing nonbiological particles. 2 Immunoassay-based methods offer inter- mediate speed (about 1 h 4 ) and sensitivity (about 10 5 spores 8 ) but cannot detect pathogens for which antibodies are not available. Moreover, immunoassay-based techniques achieve their greatest specificity when two monoclonal antibodies are available for a sandwich assay against an identifying antigen on the agent surface, where detection is typically accomplished by flow cytometry. 4 PCR, which amplifies and detects specific target sequences of pathogen DNA, is extremely sensitive (10-100 organisms 8 ) but cannot detect pathogens for which specific sequence primers are not available. The time required for detection using instruments based on immunoassay or PCR depends on the details of the instrument design and the desired sensitivity, with longer times usually required for higher sensitivity. Notably, the autonomous pathogen detection system (APDS), developed at Lawrence Livermore * Corresponding author. E-mail: vavande@sandia.gov. Phone: 925-294-1287. Fax: 925-294-3020. † Currently at the University of California, Berkeley, Department of Mechanical Engineering. (1) Seaver, M.; Eversole, J. D.; Hardgrove, J. J.; Cary, W. K.; Roselle, D. C. Aerosol Sci. Technol. 1999, 30, 174-185. (2) Pinnick, R. G.; Hill, S. C.; Nachman, P.; Videen, G.; Chen, G.; Chang, R. K. Aerosol Sci. Technol. 1998, 28, 95-104. (3) Ho, J. Anal. Chim. Acta 2002, 457, 125-148. (4) Hindson, B. J.; Brown, S. B.; Marshall, G. D.; McBride, M. T.; Makarewicz, A. J.; Gutierrez, D. M.; Wolcott, D. K.; Metz, T. R.; Madabhushi, R. S.; Dzenitis, J. M.; Colston, B. W., Jr. Anal. Chem. 2004, 76, 3492-3497. (5) Hindson, B. J.; McBride, M. T.; Makarewicz, A. J.; Henderer, B. D.; Setlur, U. S.; Smith, S. M.; Gutierrez, D. M.; Metz, T. R.; Nasarabadi, S. L.; Venkateswaran, K. S.; Farrow, S. W.; Colston, B. W., Jr.; Dzenitis, J. M. Anal. Chem. 2005, 77, 284-289. (6) Gooding, J. J. Anal. Chim. Acta 2006, 559, 137-151. (7) Edwards, K. A.; Clancy, H. A.; Baeumner, A. J. Anal. Bioanal. Chem. 2006, 384, 73-84. (8) Lim, D. V.; Simpson, J. M.; Kearns, E. A.; Kramer, M. F. Clin. Microbiol. Rev. 2005, 18, 583-607. Anal. Chem. 2007, 79, 5763-5770 10.1021/ac070567z CCC: $37.00 © 2007 American Chemical Society Analytical Chemistry, Vol. 79, No. 15, August 1, 2007 5763 Published on Web 06/26/2007