Development of an Automated Sample Preparation Module for

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Anal. Chem. 2004, 76, 3492-3497

Development of an Automated Sample Preparation Module for Environmental Monitoring of Biowarfare Agents Benjamin J. Hindson,† Steve B. Brown,† Graham D. Marshall,‡ Mary T. McBride,† Anthony J. Makarewicz,† Dora M. Gutierrez,† Duane K. Wolcott,‡ Thomas R. Metz,† Ramakrishna S. Madabhushi,† John M. Dzenitis,† and Billy W. Colston, Jr.*,†

Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94551, and Global FIA, Inc., P.O. Box 480, Fox Island, Washington 98333

An automated sample preparation module, based upon sequential injection analysis (SIA), has been developed for use within an autonomous pathogen detection system. The SIA system interfaced aerosol sampling with multiplexed microsphere immunoassay-flow cytometric detection. Metering and sequestering of microspheres using SIA was found to be reproducible and reliable, over 24-h periods of autonomous operation. Four inbuilt immunoassay controls showed excellent immunoassay and system stability over five days of unattended continuous operation. Titration curves for two biological warfare agents, Bacillus anthracis and Yersinia pestis, obtained using the automated SIA procedure were shown to be similar to those generated using a manual microtiter plate procedure. Early detection is essential for reducing the impact of a biological warfare attack on the civilian population. Toward this end, we have begun development of an environmental sensory network capable of alerting authorities of an aerosolized biowarfare agent release in the public domain. The current gold standard for this type of monitoring was demonstrated at the 2002 U.S. Winter Olympics in Salt Lake City and consists of dry, filter-based aerosol collection at a number of distributed sites with centralized, manual analysis at a bioanalytical field laboratory.1 This monitoring scheme offers a multitiered, nucleic acid-based detection approach resulting in extremely low false positive rates. Many deployment scenarios, however, require a more cost-effective and rapidresponse environmental monitoring network. Based on these requirements, we have developed an autonomous sensory device for detection of aerosolized pathogens. The autonomous pathogen detection system (APDS) is a field-deployable unit composed of an aerosol sampler, a sample processing unit, and a detector. The aerosol sampler2 selectively concentrates airborne particles within the respirable size range (1-10 µm) into the liquid phase (∼4 mL) via a wetted-wall cyclone (Research International, Monroe, WA). The collected liquid is then screened for target pathogens * Corresponding author. E-mail: [email protected]. † Lawrence Livermore National Laboratory. ‡ Global FIA, Inc. (1) Davidson, J. W.; Imbro, D. R. UCRL-JC-149311-EXT-ABS. (2) Masquelier, D. LLNL File No. IL-11041.

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using a microsphere array immunoassay with flow cytometric detection. Recently, we have demonstrated the capability of the APDS to detect live aerosolized biowarfare agents.3 Two design specifications, autonomy and the ability to manipulate microspheres, presented certain challenges to the selection, development, and integration of the sample-processing platform for the APDS. Common bioanalytical platforms are designed for high-throughput applications, where bioassays are performed manually or by sophisticated robotic systems.4,5 In contrast to these systems, the APDS uses a simple, robust, sequential injection analysis (SIA) module that is extremely flexible and designed to process single, serial samples in a continuous fashion. SIA, first reported by Ruzicka and Marshall,6 is a fluid manipulation platform with proven capability to perform complex analyses; for an excellent review of this technique and its applications, see Lenehan et al.7 Core SIA hardware components include a bidirectional pump, a holding coil, a multiposition selection valve, and a computer. Additional components such as sample collectors, reagent reservoirs, and detectors are interfaced with the system via a multiposition selection valve. Ruzicka8,9 and others8,10-17 have demonstrated that SIA is capable of manipulating small particles (less than 200 µm in diameter) for various applications, including immunoassay10 and nucleic acid analy(3) McBride, M. T.; Masquelier, D.; Hindson, B. J.; Makarewicz, A. J.; Brown, S.; Burris, K.; Metz, T.; Langlois, R. G.; Tsang, K. W.; Bryan, R.; Anderson, D. A.; Venkateswaran, K. S.; Milanovich, F. P.; Colston, B. W., Jr. Anal. Chem. 2003, 75, 5293-5299. (4) Wheeler, M. J. Ann. Clin. Biochem. 2001, 38, 217-229. (5) Hallborn, J.; Carlsson, R. BioTechniques 2002, 33, S30-S37. (6) Ruzicka, J.; Marshall, G. D. Anal. Chim. Acta 1990, 237, 329-343. (7) Lenehan, C. E.; Barnett, N. W.; Lewis, S. W. Analyst 2002, 127, 997-1020. (8) Ruzicka, J.; Scampavia, L. Anal. Chem. 1999, 71, 257A-263A. (9) Ruzicka, J. Analyst 2000, 125, 1053-1060. (10) Willumsen, B.; Christian, G. D.; Ruzicka, J. Anal. Chem. 1997, 69, 34823489. (11) Egorov, O.; O’Hara, M. J.; Grate, J. W.; Ruzicka, J. Anal. Chem. 1999, 71, 345-352. (12) Hodder, P. S.; Ruzicka, J. Anal. Chem. 1999, 71, 1160-1166. (13) Hodder, P. S.; Beeson, C.; Ruzicka, J. Anal. Chem. 2000, 72, 3109-3115. (14) Lahdesmaki, I.; Scampavia, L. D.; Beeson, C.; Ruzicka, J. Anal. Chem. 1999, 71, 5248-5252. (15) Bruckner-Lea, C. J.; Stottlemyre, M. S.; Holman, D. A.; Grate, J. W.; Brockman, F. J.; Chandler, D. P. Anal. Chem. 2000, 72, 4135-4141. (16) Chandler, D. P.; Brockman, F. J.; Holman, D. A.; Grate, J. W.; BrucknerLea, C. J. Trac, Trends Anal. Chem. 2000, 19, 314-321. (17) Wang, J.; Hansen, E. H. J. Anal. At. Spectrom. 2001, 16, 1349-1355. 10.1021/ac035365r CCC: $27.50

© 2004 American Chemical Society Published on Web 04/24/2004

Figure 1. Schematic diagram of the SIA manifold used to perform automated microsphere immunoassays within an autonomous pathogen detection system.

sis.15,16 Several devices, including a jet ring cell,10,12-14 a porous frit,11 a rotating rod,15,18 a leaky piston,9 microfabricated weirs,19-22 and a tube restriction,17 have also been described previously to withhold the solid phase (particles, beads, microspheres) in a fixed location to which fluids (sample, reagents) are then delivered. Here we describe an autonomous sample-processing platform based upon SIA for use within an environmental monitor for aerosolized pathogens. The SIA instrumentation and procedures presented have enabled complete automation of a multistep microsphere immunoassay procedure. We developed a continuously stirred reservoir used to maintain microspheres in suspension and a reusable coaxial membrane sequestering cell in which immunoassays were performed. The sequestering cell described herein, enabled capture, repeated washing, and recovery of microspheres to realize optimal immunoassay performance. Performance data are presented for microsphere metering and sequestering during autonomous operation of the APDS over 24 h. The SIA immunoassay methodology was challenged with two biowarfare agents, Bacillus anthracis and Yersinia pestis, and the titration curves generated on the fluidics platform were compared to standard curves obtained using a manual benchtop microtiter plate procedure. We also incorporated four immunoassay controls into the microsphere array that were used as indicators of both assay and system performance during unattended operation of the APDS. EXPERIMENTAL SECTION Sequential Injection Analysis. The SIA system was a FloPro4P (Global FIA, Fox Island, WA) fitted with a syringe pump (1 mL, Cavro, Sunnyvale, CA) and two multiposition selection Cheminert valves (10 and 14 port, VICI, Houston, TX). PFA tubing (0.8-mm i.d., 1.6-mm o.d., Cole-Parmer, Vernon Hills, IL) was used throughout the SIA manifold. Clean tubing cuts were obtained with a rotating blade (Upchurch Scientific, www.upchurch.com). A schematic diagram of the SIA manifold is shown in Figure 1. Flangeless 1/4-28 and 10-32 nuts and ferrules (VICI) provided tubing connections throughout the manifold. Microsphere Reservoir. The microsphere reservoir was machined from poly(methyl methacrylate) with an internal volume (18) Grate, J. W.; Bruckner-Lea, C. J.; Jarrell, A. E.; Chandler, D. P. Anal. Chim. Acta 2003, 478, 85-98. (19) Sato, K.; Tokeshi, M.; Kimura, H.; Kitamori, T. Anal. Chem. 2001, 73, 12131218. (20) Seong, G. H.; Zhan, W.; Crooks, R. M. Anal. Chem. 2002, 74, 3372-3377. (21) Ekstrom, S.; Malmstrom, J.; Wallman, L.; Lofgren, M.; Nilsson, J.; Laurell, T.; Marko-Varga, G. Proteomics 2002, 2, 413-421. (22) Bergkvist, J.; Ekstrom, S.; Wallman, L.; Lofgren, M.; Marko-Varga, G.; Nilsson, J.; Laurell, T. Proteomics 2002, 2, 422-429.

and maximum fill volume of 21 and 16 mL, respectively. The stirrer shaft and paddle were stainless steel, the paddle pitch was 30°, and this assembly was coupled to a stirrer motor (Instech Laboratories, Plymouth Meeting, PA) driven by a power supply (model 1060, Instech). Microspheres were maintained in suspension by continuous stirring at 400 rpm. Rotation speed of the shaft was measured with an Evolution noncontact laser tachometer (Monarch Instrument, Amherst, NH). The exterior of the reservoir was painted black to prevent photobleaching of the microspheres. Microsphere Sequestering Cell. The coaxial membrane sequestering cell (Global FIA, Inc., Fox Island, WA) was composed of porous polypropylene with a mean internal diameter, wall thickness, pore size, and internal volume of 600 µm, 200 µm, 0.2 µm, and 26 ( 5 µL, respectively. The membrane was mounted within a shell that enabled fluid connections to selection valves to be made. Prior to use, the membrane was treated with an aqueous solution of the fluorosurfactant Zonyl FSN (0.05% v/v, Dupont) to improve wetting. Sample Cup. A cup (internal volume of 200 µL), also machined from poly(methyl methacrylate), provided the interface between the fluidics manifold and the sample probe of the flow cytometer. Inlet and outlet tubing were positioned at the side and bottom of the cup, respectively, and were fixed in place with epoxy glue. Flow Cytometer. A Luminex 100 (Luminex Corp., Austin, TX) flow cytometer fitted with a sheath fluid delivery system was used. Sample aspiration volume, injection rate, and analysis time were 50 µL, 60 µL/min, and 60 s, respectively. System Control, Data Collection, and Analysis. The aerosol collector, SIA, and Luminex 100 were controlled via serial cables by a computer using a graphical user interface written in our laboratory with Labview Version 6.1 (National Instruments, Austin, TX). Communication between the Labview graphical user interface and the Luminex 100 was achieved using the software program Luminex LXR Library Version 1.1.7.1 (Luminex Corp.). Data output from the Luminex 100 was processed using the software program FCS Express Version 1.0 (De Novo Software, Thornhill, ON, Canada). Reagents. Phosphate-buffered saline (PBS) solution (50 mM sodium dihydrogen orthophosphate, 150 mM sodium chloride, pH 7.4) was prepared from the salts. The SIA used PBS-T (PBS, Tween 20 (0.02% v/v)) as the carrier solution that propelled zones of fluid throughout the manifold and served as a bioassaycompatible reaction medium. Stock microspheres and reagents were stored in PBS-TBN (PBS-T, bovine serum albumin (BSA, 1% m/v), sodium azide (0.01% m/v)). Reagent grade BSA, sodium Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

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azide, and Tween-20 were obtained from Sigma (St. Louis, MO). BupH citrate-MOPS buffer pack No. 28386 was purchased from Pierce Chemicals (Rockford, IL). Buffer solutions were filtered (0.2-µm cellulose acetate) prior to use. Immunoassay Reagents. Protein-G purified capture and biotinylated detector antibodies were obtained from Tetracore (Gaithersburg, MD). N-hydroxysulfosuccinimide and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide were from Pierce. Chicken immuno-γ-globulin (ChIgG) and biotinylated rabbit-anti ChIgG were purchased from Jackson Immunochemicals (West Grove, PA). The detector antibody solution contained either the biotinrabbit-anti B. anthracis (3 µg/mL) or biotin-mouse-anti Y. pestis (3 µg/mL) together with biotin-rabbit-anti ChIgG (0.18 µg/mL). The fluorescent reporter, streptavidin phycoerythrin (2.4 µg/mL) was from Caltag Laboratories (Burlingame, CA). Lc-biotin-BSA was purchased from Pierce Chemicals (Rockford, IL). Stock solutions of certified killed B. anthracis (Sterne strain) and Y. pestis (India strain) were from Dugway Proving Grounds (Dugway, UT) and were diluted with PBS as required. Preparation of Microsphere-Protein Conjugates. Proteins were covalently coupled to carboxylated microspheres (Luminex Corp.) in accordance with the manufacturer’s protocol. A mixed suspension containing 10 classes of protein-labeled microspheres was prepared from a combination of individual stock suspensions followed by dilution with PBS-T. Benchtop Microtiter Plate Microsphere Assay Procedure. In prewetted wells of a 96-well microtiter plate (MultiScreen-BV, Millipore, MA), aqueous samples (100 µL) and microsphere suspension (60 µL) were incubated for 20 min. The plate was vacuum aspirated and the wells washed twice with PBS-T (100 µL). The microspheres were resuspended in PBS-T (100 µL) and incubated with biotinylated detector antibody solution (60 µL) for 15 min. The plate was vacuum aspirated and washed, and the microspheres were resuspended in PBS-T (100 µL). The wells were incubated with streptavidin phycoerythrin (60 µL) for 5 min. The plate was vacuum aspirated and washed. The microspheres were resuspended in PBS-T (100 µL), transferred from the wells of the microtiter plate to microtubes, and then analyzed using the Luminex 100. Safety Considerations. Personal protective equipment (gloves, lab coat, goggles) was worn. All consumables (filtration plates, pipet tips, tubes, etc.) and aqueous waste were collected in biohazard bags and autoclaved. Instrumentation and benchtops were disinfected with a sodium hypochlorite solution (1% m/v) after use. RESULTS AND DISCUSSION SIA Requirements. Within the APDS, the SIA platform acted as the central interface for fluid movement between several components (Figure 1). An APDS cycle consisted of four steps. First, the aerosol sampler collected and concentrated particles from large volumes of air (up to 3000 L/min) into small volumes of water (∼4 mL) via a wetted wall cyclone. Second, the SIA took an aliquot of this sample and performed a three-step Luminex microsphere immunoassay. Third, the SIA delivered the processed microspheres to a Luminex 100 flow cytometer for analysis. Finally, the remaining sample (∼3.9 mL) was archived to a fraction collector for confirmatory testing. The cycle time of the APDS is 3494

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∼2 h; however, as sample collection and processing occur concurrently, an immunoassay result is posted every hour. The principal challenge associated with automation of the immunoassay procedure was the requirement for precise, reproducible manipulation of microspheres. In a typical assay, a minimum quota of 100 microspheres of a given classification should be counted by the Luminex 100 to acquire reliable statistics. Therefore, we identified both microsphere metering and microsphere sequestering as two essential SIA operations. Although referred to as SIA in this paper, Marshall et al.23 proposed the term “zone fluidics” for this type of fluid manipulation. Zone fluidics is seen to go beyond SIA to achieve complex samplehandling procedures such as those described in this paper. Detection Overview. Luminex microspheres (5.6 µm) are embedded with precise ratios of red and near-infrared fluorescent dyes. Within the Luminex 100, microspheres are hydrodynamically focused and flow single file through a narrow detection channel (200 µm) where they are interrogated by classification (635 nm) and reporter (532 nm) lasers. Light scattering measures each particle’s size; those outside the range of single microspheres are rejected. Each microsphere is then assigned to a distinct spectral region using the fluorescence intensities measured at two wavelengths (658, 712 nm). Fluorescence (575 ( 15 nm) from reporter molecules, bound to each microsphere during an assay, is also measured and related to analyte concentration. As 100 distinct microsphere classes are available, highly multiplexed assays are potentially accessible with this array. The APDS immunoassay employed a typical sandwich format (Figure 2) where antigen-specific capture antibodies covalently bound to microspheres are incubated with antigen. Bound antigen is detected using biotinylated antibodies subsequently labeled with the fluorescent reporter, streptavidin phycoerythrin (SAPE). SAPE is a large protein with a strong affinity for biotin and a high quantum yield, making this a rapid, sensitive labeling scheme. A two-step procedure using prelabeled detector antibodies was considered less desirable as the nonspecific coupling of fluorophore can hinder antigen binding, reducing assay sensitivity. Microsphere Metering. Luminex microspheres are denser than water; therefore, a mechanism to keep them in suspension was required. We fabricated a reservoir that held up to 16 mL of the microsphere suspension and connected to the multiposition valve of the SIA. The microsphere suspension was stirred continuously with a rotating impellor powered by a small electric motor, capable of rotation speeds from 100 to 7000 rpm. Aliquots of microsphere suspension were withdrawn from the reservoir into the holding coil and then delivered to other locations within the system. Bracketing a liquid plug with zones of air (10 µL) effectively minimized dispersion within the manifold tubing; this approach was used to transport microspheres between components (e.g., microsphere reservoir, sequestering cell, Luminex 100). The suspension used for all experiments was composed of 10 microsphere classes, where each class was present at approximately equal concentrations (12 microspheres/µL). Injection of the stock microsphere suspension into the Luminex 100 yielded ∼6000 counts in a 60-s analysis cycle. (23) Marshall, G.; Wolcott, D.; Olson, D. Anal. Chim. Acta 2003, 499, 29-40.

Figure 2. Schematic depiction of the three-step immunoassay procedure. (1) A microsphere labeled with an antigen-specific capture antibody was incubated (20 min) with the antigen, washed to remove unbound material, then (2) incubated (15 min) with detector antibody, washed, then (3) incubated (5 min) with the reporter fluorophore, streptavidin phycoerythrin, washed, and finally sent to the Luminex 100 for analysis. The two key operations required for the automated SIA immunoassay system were microsphere metering and sequestering.

Figure 3. Microsphere metering performance of a continuously stirred reservoir coupled to the automated SIA and Luminex 100 flow cytometer. Error bars indicate one standard deviation of the mean (n ) 3). The concentration of the suspension was ∼100 microspheres/ µL. The frequency of microsphere metering was 1/h and matched that of the immunoassay.

The performance of the continuously stirred reservoir was evaluated over 24 h. A simple SIA protocol was used whereby every hour, an aliquot (60 µL) of the microsphere suspension was aspirated into the holding coil and then dispensed to the Luminex for analysis. The results of the metering experiments (Figure 3) indicated that continuously stirring the suspension at 400 rpm maintained the microsphere concentration over 24 h. The relative standard deviation of the microsphere counts for each metering experiment was less than 3%, demonstrating the excellent reproducibility of our SIA platform. Microsphere Sequestering. Our initial experiments on the use of SIA for automated immunoassays were performed using a

“no-wash” procedure (i.e., microspheres and assay reagents were added sequentially to a single reaction vessel and excess reagents were not removed between additions). This methodology suffered from elevated background fluorescence signals caused by nonspecific binding of assay components (sample, detector antibodies, streptavidin phycoerythrin) to the microspheres, which in turn decreased the immunoassay sensitivity. For benchtop methodologies, nonspecific binding may be reduced through the use of filter bottom microtiter plates, which enable wash steps to be included between incubations. To capture, wash, and recover microspheres using SIA, a suitable sequestering device was needed. Various bead-trapping (sequestering) device designs have been reported in the literature, but only a few are suitable for capturing microspheres as small as Luminex’s (5.6 µm).18 To address this need, we fabricated a coaxial membrane sequestering cell, which served as a flow-through immunoassay reaction vessel. The cell comprised a length of porous polypropylene tubular membrane with a mean pore size of 0.2 µm held within an outer shell, to which fluid connections were made. A schematic depiction of the device and the fluidics sequences used to deliver, sequester, and recover microspheres is shown in Figure 4. Prior to each analysis, the membrane and its supporting shell were primed with the immunoassay buffer, PBS-T. A small zone of air (10 µL) was then positioned in the tubing above the membrane. Microspheres, sample, reagents, and wash solutions were delivered to the cell without using bracketing air bubbles, since additional air zones within the cell resulted in nonuniform staining of the microspheres. During the immunoassay, a total volume of 520 µL, including sample (100 µL), biotinylated antibody (60 µL), SAPE (60 µL), and wash (300 µL) solutions, was pumped across the membrane on which the microspheres were trapped. Therefore, after the incubation steps were complete, the microspheres were resuspended by back-flushing the membrane with a small volume (48 µL) of buffer at a high flow rate (300 µL/s). The zone of microsphere suspension was then recovered from the cell into the holding coil. The prepositioned air bubble was critical to the operation of the device as it minimized dispersion and subsequent dilution of the microsphere zone during the back-flush and recovery sequences. The processed sample, in a volume of 100 µL, was then delivered to the Luminex 100 for analysis. Microsphere recovery was identified as an important performance criterion for the coaxial membrane cell, to count a minimum quota of microspheres per assay. Microsphere recovery data were obtained using two coaxial membrane cells (Figure 5). Experiments were conducted using the APDS over three consecutive days, representing 72 individual immunoassays per cell. Recoveries were calculated using the following formula: microsphere recovery (%) ) (number of microsphere counts in an aliquot of suspension recovered from the cell after an immunoassay/number of microsphere counts in an aliquot of suspension taken from the microsphere reservoir) × 1.4 (dilution factor) × 100). A recovery of 100% represents ∼3600 microsphere counts. Each data point in Figure 5 represents the mean recovery obtained at a particular time during three consecutive 24-h experiments. Given that the mean microsphere recoveries for cells 1 and 2 were 75 and 77%, respectively, our minimum quota of 1000 total microsphere counts per assay (i.e., 100 per class) was always exceeded. The relative standard deviations of same-day microAnalytical Chemistry, Vol. 76, No. 13, July 1, 2004

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Figure 4. Schematic illustration of the microsphere sequestering and recovery sequence used to perform immunoassays within a coaxial membrane cell. The cell was primed with buffer solution, then (A) an air bubble was positioned in the tubing above the membrane, (B) microspheres were delivered, and (C) sample, reagents, and wash solutions were pumped into the cell where incubation with the microspheres occurred. Next (D), the microspheres were resuspended by back-flushing the membrane with buffer solution, then (E) drawn back into the holding coil aided by the trailing air bubble, and pumped to the Luminex 100 for analysis.

Figure 5. Microsphere recovery versus time for two coaxial membrane cells that were each fitted to an automated SIA system coupled to a Luminex 100 flow cytometer that was running a threestep sandwich immunoassay protocol. Error bars indicate one standard deviation of the mean (n ) 3). Assays were conducted continuously; a sample was analyzed every hour.

sphere recovery for the two coaxial membrane cells ranged from 4.6 up to 7.0%. As the coaxial membrane cell was intended to serve as a reusable reaction vessel, the accumulation of microspheres and proteins at the membrane surface could limit the useful lifetime of this device within the APDS. During our initial experiments, we noted that when the membrane was not reconditioned between assays, the microsphere recoveries decreased quickly as a function of the number of assays performed in the cell (data not shown). As both the membrane (polypropylene) and the Luminex microspheres (polystyrene) were hydrophobic, protein adsorption was most likely responsible for this behavior. Scanning electron micrographs of a used membrane revealed microspheres adsorbed onto the surface. We also noted some selective retention of certain microsphere types by a used cell. To minimize protein adsorption and microsphere retention, the membrane was treated with sodium hypochlorite solution (60 µL, 1.2% m/v) after each assay. Residual bleach in the cell was detrimental to subsequent assays as it oxidized both the protein coatings and fluorescent classification dyes of the microspheres. Following the bleach treatment with a buffer rinse (60 µL, morpholinepropanesulfonic acid 100 mM, sodium citrate 600 mM, pH 7.5) effectively reacted with and removed residual bleach from the cell. Using this reconditioning 3496 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

protocol, which took only 2 min to complete, microsphere carryover between assays was less than 1% (n ) 11). This simple sequestering cell enabled us to perform the microsphere and fluid manipulations demanded by the immunoassay. The coaxial membrane cell was able to accommodate the small-diameter Luminex microspheres. The robust nature of this device makes it an attractive candidate for use in the APDS as its performance was dependent entirely upon a series of fluid manipulation sequences performed by the SIA and not on mechanically driven components or additional filtration media.18 Furthermore, the device was relatively inexpensive and could be readily replaced as required. SIA Luminex Immunoassay. The SIA immunoassay methodology was designed to mimic the benchtop microtiter plate procedure developed previously in our laboratory.24 (In the benchtop protocol, beads are mixed with antigen and incubated 20 min, followed by incubations with detector antibody (15 min) and reporter (5 min).) A multistep protocol used to drive the SIA system was constructed from simple sequences, representing basic fluid manipulations. Sample and reagent zones were pumped to the cell in 1-µL increments at a flow rate of 1 µL/s. The delay time between pumping increments determined the incubation times. The SIA system was challenged with two biowarfare agents. B. anthracis is a Gram-positive bacterium that forms spores approximately 1-3 µm in size and causes the disease, anthrax.25 Plague results from infection with Y. pestis, a Gram-negative rodshaped bacterium between 0.5 and 0.8 µm in width and 1-3 µm in length. Titration curves obtained using SIA exhibit dynamic ranges and limits of detection that are comparable to results obtained on the benchtop (Figure 6). Median fluorescent intensities obtained using the SIA system were, on average, 1.6 times higher than those achieved using the microtiter plate procedure for both B. anthracis and Y. pestis. This may have resulted from (24) McBride, M. T.; Gammon, S.; Pitesky, M.; O’Brien, T. W.; Smith, T.; Aldrich, J.; Langlois, R. G.; Colston, B.; Venkateswaran, K. S. Anal. Chem. 2003, 75, 1924-1930. (25) Inglesby, T. V.; Henderson, D. A.; Bartlett, J. G.; Ascher, M. S.; Eitzen, E.; Friedlander, A. M.; Hauer, J.; McDade, J.; Osterholm, M. T.; O’Toole, T.; Parker, G.; Perl, T. M.; Russell, P. K.; Tonat, K. J. Am. Med. Assoc. 1999, 281, 1735-1745.

also contained biotin-rabbit-anti ChIgG. Finally, the fluorescent control microsphere was labeled with biotinylated BSA that confirmed the correct addition and incubation of the reporter, streptavidin phycoerythrin. The performance of the immunoassay controls was evaluated during autonomous operation of the APDS over five consecutive days within our laboratory, representing 135 individual immunoassays. The relative standard deviation of the median fluorescence intensity values for the negative, instrument, antibody, and fluorescent controls were 13, 3, 3, and 5%, respectively. The reproducibility of the four assay controls was excellent. Controls in autonomous sensors such as the APDS are critical. For instance, their behavior can be incorporated into algorithms for setting alarm thresholds or they can be used to perform instrument diagnostics from remote locations, to monitor instrument and assay performance or schedule maintenance cycles. The APDS has been deployed in environments that have high concentrations of airborne particles, including subways. As the Luminex 100 uses both size and fluorescence intensity ratios for microsphere classification, interferences from other particles present in the sample are minimal. We have evaluated the performance of our immunoassay platform using many different environmental aerosol samples, the results of which will be published in a separate report. Figure 6. Comparison of the titration curves obtained for two biological warfare agents, B. anthracis and Y. pestis, obtained using either an automated SIA system or a manual benchtop microtiter plate procedure. Error bars indicate one standard deviation of the mean (n ) 3).

the smaller volume (27 µL) of the coaxial membrane cell compared to the microtiter plate well (200 µL), resulting in more favorable reaction kinetics due to better mixing. For the coaxial membrane cell, both sample and reagent solutions were pumped to the cell in 1-µL increments that were equally spaced over the duration of the incubation period. This constant delivery of fresh reagent to the microspheres during the incubation steps may have increased the likelihood of antigen-antibody binding occurring. This is in contrast to the microtiter plate format where mixing during incubation steps was dominated by diffusion. Inbuilt Immunoassay Controls. The Luminex array enabled controls to be built into the assay by simply adding appropriately labeled microspheres to the suspension. Other immunoassay methodologies, such as enzyme linked immunosorbent assays, require control experiments to be run separately from the sample. The microsphere suspension was composed of 10 classes. Six were used for antigen capture and four were employed as controls. The negative control microsphere was coated with BSA to monitor for nonspecific binding. The instrument control microsphere was labeled with the fluorescent protein R-phycoerythrin, which indicated the correct function of the Luminex 100 detection components, particularly the YAG laser employed for excitation of streptavidin phycoerythrin. The antibody control microsphere was labeled with ChIgG and indicated correct addition and incubation of the detector antibody, as the latter reagent solution

CONCLUSION The inherent simplicity and robustness of SIA make it an excellent choice for tackling the fluid-handling challenges presented by an autonomous environmental monitoring device such as the APDS. Using SIA, we have successfully demonstrated the integration and automation of sample collection, processing, and detection. The coupling of SIA to microsphere array immunoassay and flow cytometry yielded a powerful detection strategy without additional hardware complexities. The system is suitably robust and capable of generating high-quality analytical data for remote environmental sensing applications. Recently, we have also demonstrated the capability of the APDS to detect live aerosolized biowarfare agents.3 We are currently working on the development of additional SIA-compatible components, for cell lyses, DNA extraction, purification, amplification, and hybridization, to realize orthogonal detection of target nucleic acid sequences. It is envisaged that the integration of these components to the APDS may be done via the multiposition valve of the SIA system, using a “plug and play” type approach. ACKNOWLEDGMENT This work was performed under the auspices of the U.S Department of Energy by the University of California, Lawrence Livermore National Laboratory under Contract W-7405-ENG-48, with funding from the DOE-CBNP Program.

Received for review November 18, 2003. Accepted March 19, 2004. AC035365R

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