Autonomous Microfluidic Sample Preparation System for Protein

Jun 26, 2007 - ... by wetted cyclone aerosol collector of ∼80% efficiency operating for 7 ... Zhenli Zhu , George C.-Y. Chan , Steven J. Ray , Xinro...
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Anal. Chem. 2007, 79, 5763-5770

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 system capable of continuously monitoring for airborne biothreat agents is necessary. At present, no system meets the requirements for size, speed, sensitivity, and selectivity 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 component modules, including aerosol collection via a commercially available collector, concentration, thermochemical lysis, size exclusion chromatography, fluorescent labeling, and chip gel electrophoresis were integrated together to create an autonomous collection/sample preparation/analysis system. The cycle time for sample preparation 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 collector 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. * Corresponding author. E-mail: [email protected]. Phone: 925-294-1287. Fax: 925-294-3020. † Currently at the University of California, Berkeley, Department of Mechanical Engineering. 10.1021/ac070567z CCC: $37.00 Published on Web 06/26/2007

© 2007 American Chemical Society

The primary fielded techniques for biothreat detection include particle size and native fluorescence analysis,1-3 immunoassaybased 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 intermediate speed (about 1 h4) and sensitivity (about 105 spores8) 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 organisms8) 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 (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.

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National Laboratory, has integrated immunoassay- and PCR-based techniques, resulting in low false positive rate for the detection of several common biothreat agents including airborne Bacillus spores.5 In the APDS protocol, immunoassays are run every hour and PCR analysis twice in 24 h or within about 5 h after an immunoassay trigger. The system sensitivity is approximately 49 agent-containing particles per liter of air (ACPLA) for Bacillus globigii spores.5 At present, no single biothreat detection system has been demonstrated to simultaneously meet the goals of specificity, sensitivity, and response time to warn and prevent the infection of military and civilian populations. Recently, microfluidic technologies for the processing and analysis of biological samples have been reported, showing great promise toward application in real-time, autonomous detection systems.9-17 For example, on-chip PCR technology promises reduced cycle times for a variety of reasons including enhanced heat transfer, smaller on-chip fluid volumes, and reduced transit times. Reduced cycle times and reagent quantities also lead to reduced costs.10,13,14 Recently, a device containing heaters, temperature sensors, and valves on a single chip was employed for the identification of bacterial cells.14 In addition to PCR-based systems, several reports have detailed the integration of multiple analytical processing modules into microfluidics-based systems for bioanalysis.9,11,17 Broyles et al. have integrated mechanical filtration, solid-phase extraction, and chip electrochromatography for the analysis of samples containing polycyclic aromatic hydrocarbons.17 Yin et al. have integrated a high-pressure liquid chromatography column, a sample enrichment column, and an electrospray tip to prepare peptide-containing samples for mass spectrometry.9 Skelley et al. have recently reported a system based on capillary electrophoresis, which includes all the necessary functionality for autonomous detection of amino acids in crushed rock samples.11 These examples motivate the application and further development of microfluidic technology for improved autonomous biochemical detection systems for aerosolized biothreats. In this work we present the coupling of a microfluidics-based automated sample preparation (ASP) system to our recently reported chip gel electrophoresis protein profiling (CGE-PP) system.18 We evaluate the potential of this coupled system to detect bacterial cells and spores with high speed and sensitivity. The CGE-PP system uses the microscale protein electrophoretic (9) Yin, H.; Killeen, K.; Brennen, R.; Sobek, D.; Werlich, M.; van de Goor, T. Anal. Chem. 2005, 77, 527-533. (10) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 158-162. (11) Skelley, A. M.; Scherer, J. R.; Aubrey, A. D.; Grover, W. H.; Ivester, R. H.; Ehrenfreund, P.; Grunthaner, F. J.; Bada, J. L.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 1041-1046. (12) McClain, M. A.; Culbertson, C. T.; Jacobson, S. C.; Allbritton, N. L.; Sims, C. E.; Ramsey, J. M. Anal. Chem. 2003, 75, 5646-5655. (13) Lagally, E. T.; Emrich, C. A.; Mathies, R. A. Lab Chip 2001, 1, 102-107. (14) Lagally, E. T.; Scherer, J. R.; Blazej, R. G.; Toriello, N. M.; Diep, B. A.; Ramchandani, M.; Sensabaugh, G. F.; Riley, L. W.; Mathies, R. A. Anal. Chem. 2004, 76, 3162-3170. (15) Hatch, A. V.; Herr, A. E.; Throckmorton, D. J.; Brennan, J. S.; Singh, A. K. Anal. Chem. 2006, 78, 4976-4984. (16) Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1997, 69, 3407-3412. (17) Broyles, B. S.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2003, 75, 27612767. (18) Pizzaro, S. A.; Lane, P.; Lane, T. W.; Cruz, E.; Haroldsen, B.; VanderNoot, V. A. Electrophoresis, in press.

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separation instrument, µChemLab (Sandia National Laboratories, California),19,20 combined with a specialized bacterial sample preparation protocol to create protein profiles in the 14-200 kDa range for agents including Escherichia coli cells and B. subtilis and B. anthracis (∆ Sterne) cells and endospores. While CGE-PP does not resolve individual proteins of bacterial species, it has been demonstrated to reliably discriminate between bacterial organisms within minutes.18 CGE-PP has the advantages of relative speed and sensitivity with cycle times of about 5 min and generally requiring lysate from less than 100 cells or spores in analysis volumes of about 1 nL. Additionally, CGE-PP can detect the presence of organisms even when the organism is not part of a predefined database or library, the unknown or unexpected threat agent problem. This is in contrast to immunoassay- or PCR-based methods, which will not generate a signal for an organism for which specific predefined antibodies or primer strands have not been developed and loaded into the system. For our integrated device, we constructed a series of microfluidic modules, each performing a step of the sample preparation protocol (concentration, thermochemical lysis,21 size exclusion chromatography (SEC),22 and in-capillary fluorescent labeling19). Capillary tubing connects these modules, and motorized pumps and valves control the flow between them, ultimately delivering a fully processed sample to the µChemLab instrument for analysis by CGE-PP. The combination of the CGE-PP and ASP systems required the unique integration of several diverse microfluidic subsystems that meet a demanding set of conditions including large temperature range (25-150 °C for certain components), large pressure range (1-300 psig), and exposure to various reagents (e.g., lysis chemicals and fluorescent dyes). This system represents an alternative for the detection of aerosolized bacterial cells and spores that offers the potential for improved detection speed while maintaining acceptable levels of sensitivity and selectivity. EXPERIMENTAL SECTION Reagents. Ovalbumin, R-lactalbumin, carbonic anhydrase, and bovine serum albumin were purchased from Sigma Chemical Co. (St. Louis, MO). Cholecystokinin flanking peptide (an 1100 Da peptide) was synthesized by Commonwealth Biotech (Richmond, VA). Acetonitrile, sodium borate and sodium dodecyl sulfate (SDS) were from Aldrich Chemical Co. (St. Louis, MO). Fluorescamine was purchased from Molecular Probes (Eugene, OR). BondBreaker TCEP (tris(2-carboxyethyl) phosphine hydrochloride) solution was purchased from Pierce Chemical Co. (Rockford, IL) at neutral pH and 0.5 M concentration. B. subtilis spores were purchased from Raven Labs (Omaha, NE). Radiation-killed Yersinia rohdei were provided by the Critical Reagents Program at U.S. Department of Defense (Aberdeen, MD) at an original concentration of about 5 × 108 cells/mL and observed to maintain (19) Fruetel, J. A.; Renzi, R. F.; VanderNoot, V. A.; Stamps, J.; Horn, B. A.; West, J. A.; Ferko, S.; Crocker, R.; Bailey, C. G.; Arnold, D.; Wiedenman, B.; Choi, W. Y.; Yee, D.; Shokair, I.; Hasselbrink, E.; Paul, P.; Rakestraw, D.; Padgen, D. Electrophoresis 2005, 26, 1144-1154. (20) Renzi, R. F.; Stamps, J.; Horn, B. A.; Ferko, S.; VanderNoot, V. A.; West, J. A.; Crocker, R.; Wiedenman, B.; Yee, D.; Fruetel, J. A. Anal. Chem. 2005, 77, 435-441. (21) West, J. A. A.; Hukari, K. W.; Renzi, R. F.; Patel, K. Proc. SPIE-Int. Soc. Opt. Eng. 2004, 5591, 56-63. DOI: 10.1117/12.578623. (22) Chirica, G.; Lachmann, J.; Chan, J. Anal. Chem. 2006, 78, 5362-5368.

Figure 1. Schematic representation of the automated sample preparation (ASP) system. Arrows clarify the flow direction where necessary. Blue lines and arrows represent the path taken by the incoming dilute sample from the aerosol collector. Red lines and arrows represent the path taken by the concentrated sample. Three-position valves are represented by three-port symbols. Fluidic connections between ports perpendicular to each other are made and broken as the valve actuates. Ports parallel to one another are never connected.

a spherical (intact) shape at the time of use. All reagents were used without further purification. Instrument. The ASP system was designed to prepare aqueous samples containing bacterial spores and cells in dilute concentration for proteomic signature detection and identification using Sandia’s µChemLab chip gel electrophoresis (CGE) instrument19,20 according to the previously published CGE-PP protocol.18 The samples used to test the system were intended to simulate those produced by a conventional wetted cyclone aerosol collector such as the SASS 2000+ (Research International, Monroe, WA). The ASP system accepted a relatively large volume (2 mL) aqueous sample containing dilute, intact microorganisms and delivered a small volume (5 µL) of concentrated (at least 10-100 nM), solubilized, fluorescently labeled proteins, which represent the proteome of the collected organisms. Generally, the ASP system is a series of microfluidic modules that perform specific consecutive processes on the collected aqueous samples. Individual modules were connected by fused-silica capillary (Polymicro Technologies, Inc., Phoenix, AZ). Miniature, motorized pumps and valves controlled by custom software written using LabVIEW (National Instruments, Austin, TX) accomplished fluid metering. Figure 1 is a schematic representation of the ASP system. Following the blue path, the dilute sample from the aerosol collector entered the system and was concentrated on a filter. Following the red path, the concentrated sample was flushed from the filter by lysis buffer (5 mM sodium borate, 1 wt % SDS, 50 mM Bond-Breaker TCEP solution) from the lysis buffer pump, which pushed the sample through a lyser, flow restrictor, filter, and into a 10 µL reservoir. Flow was redirected, and the sample within the reservoir was pushed through an SEC module by a pump containing CGE buffer (5 mM sodium borate, 5 mM SDS). Inside the SEC module, the lysis chemicals were separated from the solubilized proteins in the sample. Elution from the module was timed so that once the protein-containing fraction eluted from the module into a 5 µL reservoir, the flow from the CGE buffer pump was diverted to bypass the SEC module, leaving the lysis chemicals within the lysis module. Pushed by the CGE buffer pump, the sample passed a tee, where it was mixed with fluorescamine dye. The protein/dye mixture was then equilibrated for 90 s in a 10 µL reservoir, allowing labeling of the proteins and hydrolysis of unreacted dye to occur, prior to pressure injection into the on-chip sample reservoir of the µChemLab instrument. During sample analysis, the ASP system components were flushed with CGE buffer from the CGE pump, preparing the ASP system

for the next run. Figure 2 is a photograph of many of the components of the ASP system, such as pumps, valves, reservoirs, a lyser, and an SEC module, physically individual process modules. µChemLab System. The µChemLab System is a microfluidic, chip-based gel electrophoresis instrument, fully integrated onto a single microfabricated chip and packaged with miniaturized fluid reservoirs, electronics, and a laser-induced fluorescence detector. The system utilizes electrophoretic injections and has shown improved performance with respect to migration time reproducibility when constant current conditions are maintained during the separation run.19,20 Recently, µChemLab chips capable of online preconcentration have been developed and shown capable of amplifying signal magnitude by a factor of 5 using a chip design similar to that demonstrated by Foote et al.23 but without employing a separate silicate porous layer.24 Fluid Metering and Control. We have developed miniature stepper motor-driven pumps for accurate fluid control of microliter volumes. Pumps were built from stepper motor-driven linear actuators (Haydon Inc., Waterbury, CT), conventional glass syringes (Hamilton Inc., Reno, NV), and custom-built connectors and housings. Three different syringe models (250 µL, 1725; 100 µL, 1710; 50 µL, 1705) were used to vary pump capacity (120, 45, and 22 µL) and step volume (140, 66, and 33 nL), which are related proportionally. Flow rates ranging from 0.2 µL/min to well above 1 mL/min were possible. The maximum pressure produced by each syringe pump before motor stall decreased as the plunger area and perimeter (associated with seal friction) increased. Pumps built from the three syringe models above could produce approximately 150, 650, and 1500 psi, respectively. Miniaturized, low dead volume (22 nL), high-pressure (5000 psi) valves were custom built to satisfy the flow-switching requirements of the sample preparation system. The three-way and shut-off valves employed motorized rotating conical seats made from polyetheretherketone (PEEK). Automated actuations occurred in approximately 1 s. Custom in-house microfluidic interconnects including crosses, tees (polyetherimide), and capillary ferrules (PEEK) enabled low dead volume, space-efficient connections between sample processing modules. Concentration and Filtration. Concentration of the dilute aerosol collector sample was accomplished through mechanical (23) Foote, R. K. J.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2005, 77, 5763. (24) VanderNoot, V. A.; Weidenman, B.; Cruz, E. Sandia National Laboratories, unpublished results, 2005.

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Figure 2. Integrated components of the automated sample preparation (ASP) system including motorized pumps and three-way valves, pressure transducers, a thermochemical lyser, filters, an SEC module, check valves, and microfluidic interconnects such as crosses and tees.

filtration. A custom-built filter housing (PEEK plastic) housed a 0.2 µm polycarbonate track etched membrane filter (Nuclepore from Sterlitech Corp., Kent, WA) supported on both sides by fabric mesh sheets made from PEEK plastic with a 35 µm pore size (Peektex, Sefar Inc, Feibach, Switzerland). The total swept volume of the assembled filter was 3 µL. The filter housing was designed to minimize swept volume, while enabling high flow rates. During typical operation the filter was loaded at 1 mL/min and eluted at 13 µL/min. Assembled filters were cycled 100 times without clogging or significant loss of flow rate. During each cycle, 1 mL of diluted spore solution (1000 spores/µL) was passed through the filter in approximately 2 min. The filter was subsequently flushed in the opposite direction with 100 µL of the CGE buffer solution. Thermochemical Lysis. Filters were flushed with lysis buffer in the reverse direction to resuspend the solids collected on the concentration filter. This suspension was delivered to a heated lysis chamber made of PEEK plastic with an internal volume of 6 µL. Lysis temperatures ranged from 100 to 150 °C. Flow from the filter was approximately 13 µL/min, such that residence time in the lyser was approximately 30 s. A flow restriction capillary (50 µm i.d., 153 cm long) created over 300 psig of backpressure at this flow rate, adequate to prevent boiling. Size Exclusion Chromatography. Following lysis and passage through a 0.2 µm filter to remove unlysed species, the processed sample was injected into an SEC module (PEEK plastic) for the removal of TCEP.22 Sample loading occurred at a flow rate of 20 µL/min, and the sample was eluted at 30 µL/min. The internal volume of the module was approximately 90 µL. The stationary phase was separation media extracted directly from Micro BioSpin 6 columns (Bio-Rad Laboratories, Hercules, CA) and packed in the modules under vacuum.22 This stationary phase had a lower molecular weight exclusion limit of 6 kDa. Fluorescent Labeling and Injection. Labeling with the fluorogenic dye, fluorescamine, was performed as described by Fruetel, et al.19 Fluorescamine was stored at a concentration of 10 mM in dry acetonitrile. Mixing of dye stock solution and analyte proteins 5766

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occurred in a tee of 250 µm i.d. The mixing ratio was controlled by the relative flow rates of the sample and dye-containing pumps, which were approximately 8 to 1, respectively. The sample-dye mixture exited the tee in a capillary of 250 µm i.d.. After a 5 µL volume of sample was mixed with dye, the mixture was incubated 90 s in a capillary holding coil, prior to being pushed by the CGE pump into the µChemLab instrument for analysis. Spore Lysis Experiments. Experiments to determine the effectiveness of the thermochemical lysis methods used in this system were performed using a slightly modified lyser apparatus, similar to that used by West et al.21 Briefly, the lysis chamber consisted of a 6 cm section of 150 µm i.d. fused-silica capillary. This capillary was slipped inside a capillary of 536 µm i.d., which was wrapped with copper wire. The chamber was heated by passing a current through this wire. The temperature was measured by a thermocouple placed between the two capillaries. Following the heated section, a capillary with an i.d. of 25 µm was used to rate-limit the flow to achieve increased pressures. At a flow rate of about 2 µL/min, a 30 s residence time in the lyser and 200-300 psi were achieved, preventing boiling at temperatures up to 200 °C. Spore samples used in this device had a concentration of approximately 100 spores/nL in lysis buffer with or without TCEP, as explained in the Results and Discussion section. After passing through the lyser, samples were collected in microcentrifuge tubes. Any undissolved spore fragments remaining in the sample were sedimented at 5000g for 2 min. The supernatant was manually extracted and passed through a Micro Bio-Spin 6 SEC module according to the manufacturer’s protocol. The eluate from the module was manually labeled (by pipet injection of dye followed by vortex mixing) at a ratio of 10:1 with fluorescamine solution, prior to injection into the µChemLab instrument and subsequent CGE separation. SEC Reliability and Integration Experiments. The repeatability of the separation achieved by the SEC cartridge over 100 consecutive runs on the same cartridge was evaluated by monitoring the eluate over successive runs using a UVIS 200 UV detector (Linear Instruments, Reno, NV) at 210 nm, positioned at a fixed point downstream of the module. A solution of proteins extracted from B. subtilis spores and lysis buffer components was used to test the SEC modules. Briefly, the spore processing procedure began with concentrated spores (109 colony forming units/mL) stored in ethanol. These spores were diluted 1:1 with a buffer containing 5 mM sodium borate, 1 wt % SDS, and 50 mM TCEP. This mixture was boiled in a microcentrifuge tube at atmospheric pressure for 30 min. Following boiling, the sample was centrifuged at 5000g for 2 min. The supernatant was extracted and set aside for use in SEC testing. During continuous monitoring by the UV detector, a 5 µL volume of the supernatant mixture was loaded into the module at a flow rate of 20 µL/min and eluted at 30 µL/ min. The module was flushed with 250 µL of CGE buffer between successive runs. To test the integration of the SEC column, we assembled a portion of the ASP system components (all components to the right of the lyser in Figure 1), which included SEC, labeling, and injection functionality. A pump filled the 10 µL injection reservoir that preceded the SEC column with the protein sample containing lactalbumin, ovalbumin, and bovine serum albumin (10 µg/mL). When the reservoir had been flushed with several volumes of

sample to ensure that its contents were at their maximum concentration, valves were positioned so that the CGE pump loaded and eluted the SEC cartridge. The elution time was varied in order to sample different fractions of the eluate for analysis. After elution, valves were positioned such that flow bypassed the SEC cartridge, pushing the eluate fraction contained in the reservoir following the SEC module through the labeling module and into the µChemlab instrument. Dye-Labeling Efficiency Experiments. A mixture of protein standards consisting of cholecystokinin flanking peptide (1.1 kDa), lactalbumin (18.3 kDa), carbonic anhydrase (29 kDa), ovalbumin (45 kDa), and bovine serum albumin (66 kDa) was diluted in a solution of 5 mM sodium borate and 5 mM SDS (about 10 µg/ mL final protein concentration). This concentration remained constant in all experiments. Mixing experiments were conducted exactly as described in the Fluorescent Labeling and Injection section above, except that the diameter of the capillary exiting the mixing tee was varied (100, 150, 250 µm). Low-Concentration Detection Experiments. To establish the lower concentration detection limit of the fully integrated system (ASP coupled to CGE-PP) for B. subtilis spores, various concentrations of spores in water were prepared to simulate aerosolized particle concentrations of interest. Here the aerosol collector was assumed to collect 80% of the particles entering it. Reported efficiencies for conventional wetted cyclone aerosol collectors range from 80% to greater than 90% (Research International, Monroe, WA) for particles in the 2-10 µm range. For compatibility with the cycle times of the ASP system and the µChemlab instrument, we expect aerosol collection periods to last approximately 7 min. Therefore, to simulate collection of a sample containing about 16 ACPLA, we mixed an aqueous solution of spores at a concentration of 60 spores/µL, where we have assumed that each aerosolized particle (defined as 3 µm in diameter and chosen to be in the respirable range) contains 10 spores, collection capacity is 325 L/min, and the aqueous collection volume is 5 mL. It is important to note that the number of spores per particle is somewhat arbitrary in this exercise and may be expected to vary considerably with the method of particle production, the size of the particles produced, as well as the bacterial species involved. For significantly smaller particles one would expect fewer spores per particle down to approximately one spore per particle in the 1 µm size range. Sensitivity estimates can be modified accordingly to keep the absolute number of spores constant. Following collection, 1 mL of the collected solution was concentrated approximately 50 times (1 mL filtered, 20 µL eluted) on the filter, raising the concentration to 3000 spores/µL. The concentrated sample on the filter was eluted to the lyser. A 10 µL aliquot of the lysate was processed by the SEC column, which had a negligible effect on the concentration in the 5 µL collection fraction (see the Results and Discussion section). The collected fraction was mixed with fluorogenic dye and injected to the µChemlab system as described above. The µChemlab system was used with a microfluidic CGE chip with online preconcentration of approximately 5×.24 Therefore, the 1 nL injection volume processed in the CGE separation contains the lysate of approximately 15 spores, a small fraction out of the original aerosolized concentration of approximately 16 ACPLA.

RESULTS AND DISCUSSION We present a fully integrated and autonomous microfluidic system for the detection and identification of collected airborne bacterial spores and cells. Here, an ASP system, consisting of individual microfluidic process modules, prepares samples containing bacterial cells and spores for subsequent CGE-PP by the µChemLab instrument. A microfluidic approach was utilized in this work in order to reduce sample processing time through several well-known mechanisms that arise from the reduced dimensions of microfluidic systems; these include reduced process volumes, reduced thermal mass, and reduced mixing lengths. There were three primary challenges faced in automation of the sample preparation steps: development of process modules with suitable capacity, reliability, operational speed, and functional compatibility with a microfluidic, continuous flow platform; integration of process modules into a continuous, functional system; and testing of the integrated system to demonstrate performance and sensitivity. We present the development of modules for thermochemical spore lysis and protein labeling by mixing of interleaved plugs, the integration of a previously developed SEC module, and results from testing the fully integrated sample preparation system. In each case we took advantage of the flexibility and ease of design variation afforded by our “breadboard” construction, in which microcapillary tubes connected microfluidic process modules, and where flows were controlled by motorized pumps and valves. Thermochemical Spore Lysis. Bacterial spore coat is composed of a highly cross-linked matrix of proteins, which may be analyzed to help reveal the identity of the spore. These coat proteins resist lysis and solubilization.25 To solubilize the coat proteins, a reagent capable of reducing disulfide linkages was required. The use of reducing agents such as dithioerythreitol and β-mercaptoethanol for this purpose is widely reported. Typical protocols involve heating a mixture of spores, reducing agent, and detergent for tens of minutes to achieve lysis.25 In this work we adapted our previously reported CGE-PP protocol18 for use in a “flow-through” configuration at the microliter scale. We constructed a heated, microfluidic lyser and operated it with a backpressure adequate to prevent boiling at elevated temperatures. Figure 3A shows protein signatures obtained from the thermal and chemical treatment of samples containing B. subtilis spores. The number and size of peaks in the protein signature following treatment was used to access the effectiveness of lysis. All samples where passed through the lyser module at 150 °C (30 s residence time). Some samples were labeled and analyzed immediately without the addition of TCEP. To other samples, TCEP (50 mM final concentration) was added prior to a second, lower temperature (75 °C, 30 s) pass through the lyser to activate the TCEP. TCEP was removed from these latter samples using a standard benchtop protocol prior to labeling and analysis. Clearly, the B. subtilis protein signature recorded after lysis with 50 mM TCEP contains proteins not solubilized without the use of a reducing agent, illustrating that thermal treatment alone is not adequate to fully solubilize the spore proteome. In particular, the peaks occurring between 160 and 170 s, which are believed to contain the spore coat proteins, were significantly less prominent in the absence of the reducing agent. Additionally, we observed (25) Aronson, A. I.; Fitz-James, P. Bacteriol. Rev. 1976, 40, 360-402.

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Figure 4. Integration of the SEC module with system hardware. A moving time window was used to find the optimum collection fraction. The times refer to the total elution time, not including the loading, labeling, or injection times. The trace labeled “direct” resulted from injection of a manually labeled protein sample that did not pass through the SEC module into the µChemLab instrument. The sample used in these experiments contained lactalbumin (LAC), ovalbumin (OVA), and bovine serum albumin (BSA).

Figure 3. (A) Effect of TCEP on the signature achieved from lysis of B. subtilis spores. All samples were passed through the lyser module at 150 °C. Some samples were analyzed immediately (dotted line) without the addition of TCEP. To other samples, TCEP (50 mM final concentration) was added prior to a second, lower temperature (75 °C) pass through the lyser to activate the TCEP. TCEP was then removed from these samples using a standard benchtop protocol. (B) Comparison of lysis yields for (i) 30 s residence time in a microfluidic lyser at 150 °C and 300 psig, (ii) standard benchtop protocol (20 min at 100 °C in a microcentrifuge tube), and (iii) an unlysed sample for comparison. With the exception of spore lysis, all other processing steps (sample cleanup by SEC, labeling, injection) were done at the bench scale for all three samples for comparison purposes.

a greater yield from the automated microfluidic lysis process (30 s at 150 °C and 300 psi) than we did from the conventional benchtop lysis process (20 min at 100 °C and atmospheric pressure in a microcentrifuge tube) (Figure 3B). The increased yield and efficiency are likely due to the increase in temperature and the enhanced heat transfer (faster temperature rise) facilitated by the high surface area to volume ratio of the microfluidic lyser. Integration of SEC. Following lysis, the reducing agent, TCEP, must be removed prior to labeling the sample, because TCEP was observed to interfere with efficient fluorescent labeling of the proteins in the sample. Previously, the development of inline SEC modules suitable for the removal of TCEP from injected protein samples ranging in volume from 3 to 30 µL has been reported.22 Using SEC modules similar to those, we performed 100 consecutive runs on a given module and did not observe significant changes in the apparent concentrations and time 5768 Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

resolution of the protein and TCEP peaks as demonstrated by UV absorption monitored downstream of the module. Analysis of 20 runs revealed average elution times (full width at halfmaximum) of 14.7 ((1.7%) s and 37.7 ((3.6%) s for the protein and TCEP peaks, respectively. Prior to integrating the SEC module with the other system components, experimentation was required to determine the optimum fraction of eluate to collect for downstream processing. Figure 4 demonstrates the use of a 10 s moving collection window to determine the optimum collection fraction, which occurred ∼50-60 s after injection. Here the times listed correspond to the duration of the elution period, not including the time for loading, labeling, or injection. It is clear from examination of the figure that, when collecting the optimum eluate fraction, the resulting peaks are comparable to those resulting from analysis of an identical protein sample injected directly into the µChemLab instrument, without SEC treatment. Thus, no significant dilution of the optimum collected fraction occurs as it passes through the SEC module. Minimum dilution is possible because the analyzed portion of the eluate (5 µL) is less than the total volume loaded onto the SEC module (10 µL) and could be taken from the highest concentration portion of the eluting peak. Mixing of Interleaved Plugs. The final step in sample preparation prior to the injection of the processed sample into the µChemLab instrument is fluorescent labeling of the proteins contained in the sample. We sought an automated, capillary-based means of labeling the sample that had comparable efficiency to established manual protocols and could be easily integrated with the other automated process modules. The ASP system utilizes stepper motor-driven syringe pumps to push fluids through the system. When the flow from two such pumps, one containing the processed, unlabeled aqueous protein sample and the other containing the dye solution, met at a tee junction, the flow leaving the tee junction likely consisted of aqueous fluid interleaved axially along the exit capillary with dye solution.26 An interleaved pattern

Figure 5. Effect of exit capillary diameter on effectiveness of dye labeling. As labeled, the major peaks in the separation of the labeled sample are cholecystokinin flanking peptides (CCK), lactalbumin (LAC), carbonic anhydrase (CA), ovalbumin (OVA), bovine serum albumin (BSA), and a dimer of bovine serum albumin. Inset top right: close-up view showing increase in peak area with increased capillary i.d. Inset bottom left: schematic representation of the stepper-driven, plug mixing process. Inset top left: effect of capillary i.d. on labeling effectiveness.

is likely because the pumps expelled fluid in unsynchronized discrete steps, the rates of which were unequal (see the Experimental Section). In this application, the interleaved solution flowed from the tee into a reservoir, where it was held for approximately 1 min (as described above) and was then injected into the µChemlab fluid module. The geometry of the interleaved pattern likely depended on the i.d. of the capillary leaving the tee. That is, a larger diameter capillary would have resulted in axially shorter regions of the mixture fluid than did a capillary of smaller diameter. To maximize the number of protein labeling events, we sought to maximize the total amount of mass diffusion from the mixture regions to the aqueous regions. This diffusion correlates with the surface area of the boundary between regions, which is proportional to the square of the tube diameter. Based on these simplified arguments, we hypothesized that the effectiveness of mixing ought to increase with increasing capillary i.d. Figure 5 depicts the results of the labeling efficiency experiments conducted according to the protocol described in the Experimental Section. The peak areas increased as the capillary i.d. increased, and the peak areas obtained for a diameter of 250 µm were comparable to those obtained using vortex mixing in a microcentrifuge tube followed by direct manual injection into the µChemLab fluid module. The top left inset in Figure 5 shows the ratio of labeling effectiveness (average peak area) for each capillary diameter to turbulent mixing, averaged for four peaks: lactalbumin, carbonic anhydrase, ovalbumin, and bovine serum albumin dimer. This ratio increases with the capillary i.d. in agreement with our hypothesis. Integrated System Performance. The most distinctive aspect of this work is the integration of multiple component subsystems into a fully functional system capable of identifying collected airborne biological agents. The cycle time for the ASP system is (26) Okhonin, V.; Liu, X.; Krylov, S. N. Anal. Chem. 2005, 77, 5925-5929.

Figure 6. (A) Sensitivity of the system for detection of B. subtilis spores at different concentrations. (B) Comparison of protein signatures from B. subtilis spores and Y. rohdei cells.

approximately 5 min. When integrated with CGE, the combined cycle time is approximately 10 min. We performed two studies to characterize the performance of the coupled ASP, CGE-PP system, namely, determination of the system response to low concentrations of B. subtilis spores and comparison of the system responses to different biological agents. Figure 6A compares the coupled system response, including 5× on-chip preconcentration, for B. subtilis spores at simulated concentrations of approximately 16 and 160 ACPLA. Referring to the figure, the signature taken at 16 ACPLA displays many of the same features as the signature at 160 ACPLA, which is similar to signatures taken at higher concentrations and using bench top preparation (see Figure 3).18 Blank samples run immediately before the low-concentration samples yielded no significant signal, indicating that those signals observed in the 16 and 160 ACPLA tests were not the result of carryover from previous experiments (data not shown). Attempts to detect concentrations below 16 ACPLA did not yield recognizable signatures. Precise determination of the lower concentration sensitivity limit was not a goal of this initial work but is a subject of ongoing studies in our laboratory. The sensitivity of the coupled instrument is likely dependent upon the agent detected, the sample preparation Analytical Chemistry, Vol. 79, No. 15, August 1, 2007

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protocol, the parameters of the CGE analysis, and the algorithm used to interpret CGE spectra. Figure 6B compares the response of the coupled system for B. subtilis spores to the response for radiation-killed Y. rohdei cells. Here we note significant differences between the signatures for the two organisms including number of peaks, shape of molecular weight signature, and molecular weight range of signature, which could be used to differentiate between the two organisms. This result is similar to the findings of Pizarro et al., who differentiated between bacterial organisms based on their signatures from CGEPP analysis of Bacillus spores and E. coli cells.18 CONCLUSIONS This work demonstrates the successful integration of multiple microfluidic component modules, including collection using a commercial aerosol collector, concentration, thermochemical lysis, SEC (for removal of lysis agents), fluorescent labeling (of primary amines), and CGE, into a fully automated system for the detection of bacterial biothreat agents such as B. subtilis, a surrogate for B. anthracis. Biothreat agents may be identified according to the presence of specific protein profiles described previously.18 The “breadboard” approach used in this work, where capillary tubes connect microfluidic modules and motorized pumps and valves control flow between them, represents a powerful strategy for rapidly testing component compatibility and varying system design. An integrated system such as this, based on microfluidic modules, has to our knowledge not previously been applied to the problem of rapid detection of airborne bacterial biothreat agents and represents the development of an alternative to immunoassay- or PCR-based technologies for airborne biothreat detection. We are currently investigating the further integration of several of the ASP system components and streamlining of the overall system in order to take advantage of reduced system cost and reduced cycle time. (27) Lapizco-Encinas, B. H.; Davalos, R. V.; Simmons, B. A.; Cummings, E. B.; Fintschenko, Y. J. Microbiol. Methods 2005, 62, 317-326. (28) Lapizco-Encinas, B. H.; Simmons, B. A.; Cummings, E. B.; Fintschenko, Y. Electrophoresis 2004, 25, 1695-1704. (29) Lapizco-Encinas, B. H.; Simmons, B. A.; Cummings, E. B.; Fintschenko, Y. Anal. Chem. 2004, 76, 1571-1579.

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The primary contribution of this work lies in the integration of multiple microfluidic modules in an autonomous system and the design of those components such that integration is possible. In particular, this integration effort necessitated the development of process modules for rapid thermochemical lysis of spores as well as fluid mixing and labeling by the creation of a pattern of interleaved plugs. One potential disadvantage of using protein profiles will result from “dirty” or real-world samples. The very nature of the broad applicability of protein profiling suggests that the presence of multiple types of organisms in a sample will tend to complicate or mask the observed signature, severely limiting the ability to discriminate signatures. The authors acknowledge this as a very real challenge, and to mitigate this in the ultimate application, we envision incorporating a selective sorting technology known as insulator-based dielectrophoresis (iDEP), also under development at Sandia National Labs. iDEP, which exploits differences in behavior exhibited by different organisms in the presence of a nonuniform electric field, has been shown to be able to selectively separate and/or concentrate bacterial cells, spores, and viruses,27 and different species of bacterial cells,28 as well as to discriminate between live and dead cells of the same species.29 This phenomenon is attractive for differentiating biological particles prior to protein profiling because it can collect specific types of particles rapidly and reversibly. The combined approach of selective iDEP sorting with the rapid and widely applicable protein profiling should prove very effective. ACKNOWLEDGMENT This work was supported by the Department of Defense. Sandia is a multiprogram laboratory operated by Sandia Corporation, a Lockheed Martin Company, for the United States Department of Energy under Contract DEAC04-94AL85000.

Received for review March 21, 2007. Accepted May 9, 2007. AC070567Z