Bead-Assisted Displacement Immunoassay for Staphylococcal

Nov 2, 2006 - After a 30-min sample preparation step, the displacement assay was performed without user intervention and produced quantitative results...
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Anal. Chem. 2006, 78, 8412-8420

Bead-Assisted Displacement Immunoassay for Staphylococcal Enterotoxin B on a Microchip Amanda J. Haes,† Alex Terray,‡ and Greg E. Collins*

Chemistry Division, Naval Research Laboratory, 4555 Overlook Avenue, SW, Code 6112, Washington, DC 20375-5342

A microchip-based, displacement immunoassay for the sensitive laser-induced fluorescence detection of staphylococcal enterotoxin B is presented. The glass microchip device consists of a microchannel that contains a double weir structure for supporting antibody-functionalized microbeads. After a 30-min sample preparation step, the displacement assay was performed without user intervention and produced quantitative results in an additional 20 min. Linear detection responses were observed over 6 orders of magnitude and provided detection limits down to 1 fM (28.5 fg/mL). The surprisingly low detection limits are hypothesized to arise from field-based enrichment analogous to field-amplified stacking, chromatographic effects, and limited diffusion lengths in the microbead bed. The assay was challenged with bovine serum albumin, casein, and milk sample matrixes. This system has the potential to provide highly sensitive detection capabilities for target biomolecules. Analytical techniques that detect target proteins in a fast, sensitive, and selective manner without interferences from background materials are in high demand. For instance, detection platforms for toxins such as staphylococcal enterotoxin B (SEB), a 28.5-kDa protein, are important because the toxin can be used as a potential biowarfare agent and is a common source of foodborne illnesses.1 While its lethal dose is relatively high (LD50 ∼0.02 µg/kg), it can cause incapacitating effects at extremely low levels (effective dose, ED50 ∼0.4 ng/kg).2 For these reasons, methods that sensitively and specifically detect SEB at low concentrations are important for personal health and safety. Several techniques have been used to detect SEB. Some of the methods include (1) capillary electrophoresis (limit of detection (LOD) ∼85 ng/mL),3,4 (2) surface plasmon resonance (LOD ∼10 ng/mL in 10 min),5 (3) sandwich enzyme-linked immunosorbent assays (ELISA) “dipstick” (LOD ∼500 pg/mL in 1 h),6 (4) latex agglutination * To whom correspondence should be addressed. E-mail: greg.collins@ nrl.navy.mil. Phone: (202) 404-3337. Fax: (202) 404-8119. † Current address: Department of Chemistry, University of Iowa, Iowa City, IA 52242. ‡ Current address: SAIC, 1220 12th St., SE, Suite 140, Washington, DC 20003. (1) Walt, D. R.; Franz, D. R. Anal. Chem. 2000, 72, 738A-746A (2) Gill, D. M. Microbiol. Rev. 1982, 46, 86-94. (3) Lam, M. T.; Boulet, C. A.; Le, X. C. Anal. Chim. Acta 2002, 457, 21-28. (4) Lam, M. T.; Wan, Q. H.; Boulet, C. A.; Le, X. C. J. Chromatogr., A 1999, 853, 545-553. (5) Rasooly, A. J. Food Prot. 2001, 64, 37-43. (6) Morissette, C.; Goulet, J.; Lamoureux, G. Appl. Environ. Microbiol. 1991, 57, 836-842.

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(LOD ∼500 pg/mL in 24 h),7 (5) ELISA (LOD ∼200 pg/mL in 3 h),8 (6) fluorescence arrays (100 pg/mL),9,10 and (7) indirect double sandwich fluorogenic enzyme-linked immunoassay (LOD ∼0.1 fg/mL in 3 h).11 Since its introduction, immunoassays have become one of the most widely used bioanalytical methods.12,13 All immunoassays utilize antibodies as capture molecules for strong and specific binding to target antigens. Advances in the detection and analysis of this antibody-antigen interaction have driven the maturation of many chromatographic and spectrophotometric analytical systems. In practice, the usefulness of any immunoassay technique relies on several parameters, including the following: assay simplicity, convenience, and cost; total assay time; assay sensitivity and selectivity; and reagent stability. The success of an immunoassay is limited by (1) the binding constant between the antibody-antigen couple, (2) nonspecific binding, (3) diffusion distance between the antigen and antibody, (4) detector sensitivity, and (5) assay time. ELISA is one of the most sensitive immunoassay platforms.12,14,15 Using amplification, ELISA has been used to detect target biomolecules at attomolar concentrations in several hours.16,17 Recently, ELISA and other immunoassay techniques have been miniaturized onto microchip platforms.15,18-26 This combination of techniques is advantageous in that small sample volumes are (7) Di Pinto, A.; Forte, V. T.; Ciccarese, G.; Conversano, M. C.; Tantillo, G. M. J. Food Saf. 2004, 24, 231-238. (8) Pimbley; Patel. J. Appl. Microbiol. 1998, 84, 98S-109S. (9) Sapsford, K. E.; Taitt, C. R.; Loo, N.; Ligler, F. S. Appl. Environ. Microbiol. 2005, 71, 5590-5592. (10) Delehanty, J. B.; Ligler, F. S. Anal. Chem. 2002, 74, 5681-5687. (11) Bhatti, A. R.; Siddiqui, Y. M.; Micusan, V. V. J. Microbio. Methods 1994, 19, 179-187. (12) Diamandis, E. P., Christopoulos, T. K., Eds. Immunoassay; Academic Press: San Diego, CA, 1996. (13) Ekins, R. P. J. Pharm. Biomed. Anal. 1989, 7, 155-168. (14) Rossier, J. S.; Gokulrangan, G.; Girault, H. H.; Svojanovsky, S.; Wilson, G. S. Langmuir 2000, 16, 8489-8494. (15) Gao, Y.; Lin, F. Y. H.; Hu, G.; Sherman, P. M.; Li, D. Anal. Chim. Acta 2005, 543, 109-116. (16) Nam, J.-M.; Thaxton, C. S.; Mirkin, C. A. Science 2003, 301, 1884-1886. (17) Dijksma, M.; Kamp, B.; Hoogvliet, J. C.; Van, Bennekom, W. P. Anal. Chem. 2001, 73, 901-907. (18) Rubina, A. Y.; Dyukova, V. I.; Dementieva, E. I.; Stomakhin, A. A.; Nesmeyanov, V. A.; Grishin, E. V.; Zasedatelev, A. S. Anal. Biochem. 2005, 340, 317-329. (19) Herr, A. E.; Throckmorton, D. J.; Davenport, A. A.; Singh, A. K. Anal. Chem. 2005, 77, 585-590. (20) Phillips, K. S.; Cheng, Q. Anal. Chem. 2005, 77, 327-334. (21) Sato, K.; Yamanaka, M.; Hagino, T.; Tokeshi, M.; Kimura, H.; Kitamori, T. Lab Chip 2004, 4, 570-575. (22) Sato, K.; Yamanaka, M.; Takahashi, H.; Tokeshi, M.; Kimura, H.; Kitamori, T. Electrophoresis 2002, 23, 734-739. 10.1021/ac061057s CCC: $33.50

© 2006 American Chemical Society Published on Web 11/02/2006

required for analysis and assay times can be significantly minimized by the inherent reduction in diffusion distances between antigens and antibodies within the microchannels.14,27,28 Since the first microchip immunoassay experiments,26 detection sensitivity and analysis times have been further improved by immobilizing one of the reagents onto a solid phase.27,29-32 This approach is equivalent to macroscale immunoaffinity chromatography (IAC) techniques.33 In IAC, antibodies for a specific target are attached to a solid-phase support (i.e., sol-gels, beads, etc.) that is used for the capture and purification of the target biomolecule. Kitamori has used a bead-based immunoassay on a microfluidic device to detect subnanogram per milliliter concentrations of human interferon-γ in a 50-min total assay.21,34 Displacement immunoassays for the detection of hormones have been demonstrated on a microchip and were demonstrated to detect target analytes down to low-picomolar concentrations with under 10 min assay times.35 Design considerations such as flow rates, density of antibodies on the solid phase, and channel dimensions are important parameters for the miniaturization of ligand displacement immunoassays.12,32 Whether these assays occur in static or fluidic environments greatly influences the detection capabilities and efficiencies of the displacement response.28,36 It has been demonstrated that in a fixed channel dimension and fixed column porosity, as the flow rate is decreased, the signal response increases. This response increase has been attributed to increased residence time for antibody-antigen interactions in minimized diffusion distances. Herein, a displacement immunoassay for the detection of SEB is demonstrated on a glass microchip using laser-induced fluorescence detection and electrokinetically controlled fluidic delivery. A monoclonal antibody that is specific for SEB is covalently attached to silica beads. These beads are trapped on a microchip using narrowed pathways (weirs) on custom-made microchip devices. As a result of varying electric field strengths within the nonuniform channels, the sample undergoes an apparent fieldenhanced enrichment phenomenon. Additionally, given the limited diffusion lengths in this chromatographic microbead bed and the presaturation of the microbead bed with fluorescently labeled SEB, a more efficient interaction between antibody and antigen is achieved and chromatographic effects are maximized, respectively. Detection of low-femtomolar concentrations of SEB is demonstrated, and the assay is verified to provide sensitive detection in complex sample matrixes. (23) Guijt, R. M.; Baltussen, E.; van Dedem, G. W. K. Electrophoresis 2002, 23, 823-835. (24) Schmalzing, D.; Buonocore, S.; Piggee, C. Electrophoresis 2000, 21, 39193930. (25) Chiem, N. H.; Harrison, D. J. Electrophoresis 1998, 19, 3040-3044. (26) Chiem, N.; Harrison, D. J. Anal. Chem. 1997, 69, 373-378. (27) Rossier, J. S.; Girault, H. H. Lab Chip 2001, 1, 153-157. (28) Holt, D.; Rabbany, S. Y.; Kusterbeck, A. W.; Ligler, F. S. Rev. Anal. Chem. 1999, 18, 107-132. (29) Sato, K.; Tokeshi, M.; Kimura, H.; Kitamori, T. Anal. Chem. 2001, 73, 12131218. (30) Eteshola, E.; Leckband, D. Sens. Actuators, B 2001, 72, 129-133. (31) Sia, S. K.; Linder, V.; Parviz, B. A.; Siegel, A.; Whitesides, G. M. Angew. Chem., Int. Ed. 2004, 43, 498-502. (32) Ngo, T. T. Anal. Lett. 2005, 38, 1057-1069. (33) Hage, D. S. J. Chromatogr., B 1998, 715, 3-28. (34) Sato, K.; Kitamori, T. J. Nanosci. Nanotechnol. 2004, 4, 575-579. (35) Clarke, W.; Schiel, J. E.; Moser, A.; Hage, D. S. Anal. Chem. 2005, 77, 1859-1866. (36) Sheehan, P. E.; Whitman, L. J. Nano Lett. 2005, 5, 803-807.

MATERIALS AND METHODS Reagents and Chemicals. Bovine serum albumin (BSA), bleach, casein, dimethyl sulfoxide, hydrochloric acid, 2-morpholinoethanesulfonic acid (MES; luminescence grade), nitric acid, potassium phosphate monobasic, potassium phosphate dibasic, sodium azide, sodium bicarbonate, and sodium hydroxide were purchased from Sigma-Aldrich (St. Louis, MO). Microcons (regenerated cellulose, 30-kDa molecular weight cutoff (MWCO) filters) were purchased from Millipore (Billerica, MA). Water was purified to a resistivity of 18.2 MΩ cm-1 using Millipore cartridges. Sample Preparation. The run buffer and sample matrix buffer used were 100 mM MES, pH 7.1. The pH of the buffer was adjusted using 3 M NaOH. All solutions were filtered with 0.2µm nylon filters prior to use. Staphylococcal enterotoxin A (SEA) and staphylococcal enterotoxin B (SEB) were purchased from Toxin Technology, Inc. (Sarasota, FL). SEA and SEB were reconstituted in water to achieve a buffered solution of 10 mM PBS, pH 7.2. These solutions were stored at 4 °C until use. All dilutions were performed in a class II biosafety hood using 100 mM MES (pH 7.1). To simulate complex sample matrixes, a filtering/purification protocol was performed. In these cases, 150 µg/mL BSA in MES buffer, 150 µg/mL casein in MES buffer, and milk (Wholesome Farms, fat-free skim milk) were used. Identical samples were spiked with SEB to a final concentration of 10 pM. All samples were constantly mixed for 4 h and then stored at 4 °C for 3-7 days. Prior to analysis, the sample was vortexed briefly and 100 µL of the solution was added to a 30-kDa MWCO filter. The tube was centrifuged for 30 min at 8 rpm using an Eppendorf MiniSpin Plus centrifuge. The solution collected at the bottom of the tube was injected into a sample reservoir on the microfluidic chip. Identical filtering steps were performed with “uncontaminated” SEB samples. Approximately 10% of the sample is lost during this filtering process. Bead Functionalization. Uniform carboxyl-modified silica microspheres (diameter 5.06 µm, 10% (w/v)) were purchased from Bangs Laboratories, Inc. (Fisher, IN). A monoclonal antibody for SEB (mouse, Biodesign International, Saco, ME) was coupled to the bead surface according to literature protocol.37 The antibody was chosen because of its high selectivity for SEB. As per manufacturer’s analysis, this antibody showed no cross reactivity with other staphylococcal enterotoxins, ricin, and botulinum toxins. Briefly, 900 µL of 100 mM MES, 100 µL of a 10% bead solution, and 10 mg of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (Pierce, Rockford, IL) were mixed at room temperature for 15 min. The bead sample was centrifuged at 8 rpm for 15 min, and the sample was rinsed with buffer. Next, 388 µL of buffer and 12 µL of antibody (from 6.2 mg/mL stock) were added to the bead solution and allowed to react for 4 h at room temperature. The bead-antibody solution was then rinsed and resuspended in a quenching solution (2 µL of ethanoloamine, 100 µL of BSA (from 100 mg/mL stock), and 898 µL of MES buffer). After 30 min, the solution was washed twice and resuspended in a storage buffer (10 µL of BSA from 100 mg/mL stock, 50 µL of sodium azide from 20 mg/mL stock, and 940 µL of MES buffer). The resulting bead surface contained randomly oriented antibodies with a surface density of ∼1 × 104 antibodies/µm2.37 The bead solution was stored at 4 °C until use. (37) Bangs Laboratories, I. In TechNote 205, 2002.

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Fluorescently Labeled SEB (SEB*). SEB was modified with an Alexa Fluor 488 tetrafluorophenyl ester dye (Invitrogen, Carlsbad, CA) according to published protocols.38,39 Briefly, 5 mg of SEB was suspended in 500 µL of 100 mM sodium bicarbonate buffer at pH 9.0. Next, ∼1 mg of dye was added to the SEB solution. After 4 h, 50 µL of 1.5 M hydroxylamine pH 8.5 was added to the mixture to quench the coupling reaction. This solution was diluted to a final volume of 2.5 mL using 10 mM PBS, pH 7.4. To remove excess dye from the fluorescently labeled SEB (SEB*), PD-10 desalting columns made from Sephadex G-25M (Amersham Biosciences, Uppsala, Sweden) were used according to manufacturer protocol. Eluted fractions were collected, aliquoted, and stored at -20 °C until use. Microchip Fabrication. Using standard photolithography and wet etching techniques, microchips were fabricated from soda lime glass plates (thickness 0.06 in.) precoated with a layer of chrome and photoresist (Telic, Santa Monica, CA).40 The photomask was created with Adobe Illustrator and printed on a transparency at 20 000 dpi (Figure 1A, CAD/Art Services, Inc., Bandon, OR). The features were etched using a bath of buffered oxide etchant 6:1 (Transene Co., Inc., Danvers, MA) for short, 3-5-min intervals. Each exposure was followed by rinsing in deionized water and 1 M HCl. A KLA Tencor (P-15) Profilometer was used to measure weir depths to determine when the desired channel parameters had been achieved. Following etching, access holes were drilled at the end of the microchannels and the remaining photoresist and chrome layers were removed. Blank (cover) and etched plates were cleaned and hydrolyzed using a 30% Branson cleaning solution (Branson, Danbury, CT) and hydrolysis solution (1 part hydrogen peroxide, 2 parts ammonium hydroxide, and 2 parts water).40 The plates were rinsed with water and dried. Next, ∼1 mL of a 0.2-µm filtered 35 mM solution of KOH (Sigma Aldrich) was dispensed on an etched plate.41,42 A nonetched piece of soda lime was carefully brought into close contact such that no bubbles were apparent between the pieces. The devices were then placed in a heated hydraulic press (Carver Inc., Wabash, IN). A pressure of 1 ton was applied for 15 min at which point the heaters were turned on. When the temperature reached 250 °F, the pressure was increased to 4 tons and maintained for 45 min. Once cooled, the devices were removed, cleaned, placed in a high-temperature furnace (BF51800, Lindberg Blue), and heated to 550 °C. Reservoirs (glass tubing) were attached at the access holes using an epoxy (353ND-T; Epoxy Technology). Preparation of Microbead Column. Microfluidic chips were cleaned with 10-min cycles of 0.1 M HNO3, water, 0.1 M NaOH, water, and 100 mM MES buffer (pH 7.1) prior to use. Negative pressure was applied to the bead reservoir to facilitate chip rinsing. Beads were loaded into the microfluidic chip by adding 5 µL of the antibody-conjugated bead solution to 100 µL of MES buffer. Negative pressure was then applied to the buffer outlet reservoir to load the beads into the microfluidic channel. Although beads (38) Brinkley, M. Bioconjugate Chem. 1992, 3, 2-13. (39) Haugland, R. P. Methods Mol. Biol. 1995, 45, 205-221. (40) Ramsey, J. D.; Collins, G. E. Anal. Chem. 2005, 77, 6664-6670. (41) Elliffe, E. J.; Bogenstahl, J.; Deshpande, A.; Hough, J.; Killow, C.; Reid, S.; Robertson, D.; Rowan, S.; Ward, H.; Cagnoli, G. Classical Quantum Gravity 2005, 22. (42) Gwo, D.-H. Hydroxide-catalyzed bonding. U.S. Patent 6,548,176 B1, 2003.

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Figure 1. Double-weir structure fabricated in a soda lime glass microchip. (A) Mask design, scale bar equals 25 µm. (B) 3D illustration of the weir structure arbitrarily modified for visualization from profilometry data. The illustration shows the separation channel and side arm for bead loading. (Magnified region) Profilometry cross section of weir structure. The widest opening of the weir is ∼7.3 µm deep and ∼2.4 µm wide. (C) Schematic of the microfluidic chip for the displacement immunoassay. (1) Microchip design incorporates reservoirs for three different samples, SEB*, and bead loading/unloading. Sample 1, sample 2, sample 3, SEB*, and buffer reservoirs are ∼2.8 cm from buffer outlet. The dotted box designates the location of the bead bed area. (2) Bright-field image of the double-weir structure. Antibody-functionalized microbeads (diameter, 5.06 µm) are loaded into the bead bed by applying negative pressure at the buffer outlet reservoir. (3) Fluorescently labeled SEB (1 nM SEB*) binds to the antibody-functionalized microbeads.

were fully loaded in the bead bed within 15 s, the beads were allowed to equilibrate for ∼15 min prior to use. Following bead loading, all reservoirs were cleaned with sample buffer, and then ∼90 µL of the appropriate solution was added to each sample reservoir. Solutions were replenished after every third assay to limit the effects of buffer depletion.43 A single bead bed could be reused up to 15 times without a decrease in assay response (data not shown). When the microbeads began to lose their specificity to antigen, the reservoirs and channels were rinsed with a 1% bleach solution to decontaminate the microchip. Additionally, the beads were easily removed from the microchip by applying negative pressure at the bead reservoir in the presence of bleach. Microscopy. A Leica DMIL microscope (20× objective) was used for optical imaging experiments. A mercury arc lamp and blue filter block (470-490 nm) were used for fluorescence (43) McLaren, D. G.; Chen, D. D. Y. Electrophoresis 2003, 24, 2887-2895.

Table 1. Voltages Applied to Control SEB* Load, Rinse, and SEB Displacementa (kV vs Ground)

rinse SEB* load sample 1 sample 2 sample 3 a

buffer inlet

SEB*

sample 1

sample 2

sample 3

beads

buffer outlet

0.50 0.31 0.25 0.25 0.25

0.25 0.50 0.22 0.22 0.22

0.32 0.32 0.50 0.31 0.31

0.33 0.33 0.33 0.50 0.33

0.31 0.31 0.31 0.31 0.50

0.22 0.22 0.22 0.22 0.22

ground ground ground ground ground

Exact experimental details are included in the text.

excitation. A video camera (Astrovid StellaCam II) and MGI Videowave 4 software were used to capture the bright-field and fluorescence microscope images. Microfluidic Analysis. Microfluidic potentials were driven using an array of eight independently controlled high-voltage power supply channels (Microfluidics Tool Kit, Micralyne, Inc., Edmonton, Alberta, Canada). The power supplies were controlled using a manufacturer-provided Labview program. Table 1 summarizes the potentials used to control fluid flow in the microfluidic chip. Seven reservoirs were included on the chip. The sample reservoirs were filled as follows: (1) buffer to demonstrate a zero baseline response, (2) 10 pM SEB sample to verify that the displacement immunoassay was performing as expected, and (3) sample with varying composition. Alternatively, the three sample reservoirs were filled with varying sample concentrations and were analyzed in a random manner to assess the reproducibility of the assay. Laser-Induced Fluorescence Detection (LIF). LIF was used to monitor the fluorescence in the microfluidic channel during the displacement immunoassay. An air-cooled Ar-Kr laser (35KAP-431-208, Melles Griot, Carlsbad, CA) tuned to λex ) 488 nm (P ) 20 mW) was used in these experiments. The laser light was focused onto the microfluidic channel ∼150 µm downstream of the bead bed using a planoconvex lens with a 1000 mm focal length (Melles Griot). Fluorescence was collected using a 20× microscope objective (NA ) 0.4, Newport), spatially filtered with a 10000 µm pinhole (Melles Griot), and spectrally filtered with a band-pass filter centered at 515 ( 10 nm (Andover). The fluorescence from SEB* was monitored using a photomultiplier tube (PMT; Hamamtsu H7732-10). The PMT signal was amplified (SRS-SR570, Stanford Research Systems) and recorded using the same Labview program used to control the microfluidic channel potentials. All data were analyzed using GRAMS/AI (Version 7.02, Thermo-Galactic). RESULTS AND DISCUSSION Microchip Design for the Detection of SEB. Successful implementation of this displacement immunoassay on a lab-on-achip device required the containment of ∼5-µm beads within a microfluidic channel. This bead size was chosen to ensure that diffusion lengths in the bead bed were minimized, facilitating a fast and efficient assay response. By creating a dam or weir structure at both the top and bottom of the bead bed area, the microchip could be loaded, unloaded, and reloaded for repeated use with similar bead bed volumes and lengths. This is an important parameter for guaranteeing assay consistency from chip to chip. Several approaches, including polymer plugs,40,44 novel etching designs,21,45-48 and double etching designs,21,49 have been

used to create weir structures in glass microchips. Each of these techniques has distinct advantages and disadvantages. The weir design presented in this work offers the following advantages: (1) one-step etching process, (2) a glass structure for easy surface treatment and modification, (3) minimized weir length, (4) fixed bead bed length for reproducible studies, (5) ability to retain 4-min time period. These differences can be attributed to pH and ionic strength differences between milk and buffer.61 Given these differences, it is encouraging that the displacement band areas can still be used for calibration purposes. (61) In http://www.cfsan.fda.gov/˜comm/lacf-phs.html; U. S. Food and Drug Administration, 2003.

Similar Protein Interferences for the Displacement Immunoassay. While demonstrating that the displacement immunoassay can be used in complex (and real) sample matrixes, a final experiment testing the specificity of the system with proteins similar to SEB has been probed. Figure 4D displays preliminary results illustrating the specificity of the displacement immunoassay. In this case, equivalent masses of SEB and SEA were interrogated with the system. SEA is another member of the staphylococcal enterotoxin family, although exhibiting lower toxicity than SEB.62 When SEA is injected into the microchip, a small amount of displaced SEB* is detected (area, 3.8 AU). A control experiment with SEB is included for comparison. It is clear from this experiment that the displacement immunoassay presented here has a higher selectivity for SEB given the antibody’s specificity for SEB, blocking chemistry on the beads with BSA, and rinsing procedure. A small amount of SEB* runoff is detected in the control experiments. This undesired response could be limited by refining the rinsing procedures to include slightly harsher conditions; however, detection sensitivity might be sacrificed as a result of this change. CONCLUSIONS The results presented here systematically probe the effect of the miniaturization of an electrokinetically driven immunoassay on a microchip for the detection of SEB. This miniaturized immunoaffinity chromatographic microchip device utilizes the displacement of fluorescently labeled SEB from a bed of silica microbeads that have been functionalized with monoclonal SEB antibodies. The displacement immunoassay was performed without user intervention, revealing a quantitative response over 6 orders of magnitude in a 50-min total assay time (30-min sample preparation, 20-min analysis time), and a detection limit for SEB down to 1 fM (28.5 fg/mL) concentrations. This detection limit, which is several orders of magnitude more sensitive than standard immunoassays, arises from a combination of mechanisms. First, it is hypothesized that nonuniform channel dimensions in the microchip give rise to varying electric field strengths in the microchannel when a potential is applied. As a result, these structures induce a field-based sample enrichment event that concentrates the molecules. Second, elution band shape changes indicate that chromatographic effects are inducing sample enrichment. Third, because diffusion distances are minimized in the chromatographic bead bed, it is possible for SEB to displace fluorescently labeled SEB in ∼30 ms. Given the flow rate through the bead bed, this facilitates maximum displacement efficiency for the immunoassay. The assay was tested for specificity in complex sample matrixes. A small amount of nonspecific runoff is detected when the system is challenged by a 105-106 excess of bovine serum albumin and casein concentrations (in comparison to maximum SEB concentrations). However, when 10 pM SEB is incubated in these complex sample matrixes, the SEB response is ∼8 times greater than the nonspecific runoff response. SEB detection in milk was demonstrated to behave in a quantitative manner similar to the bovine serum albumin and casein studies. Further optimization of the rinsing procedure involved with the assay will improve the quantitative nature of the displacement (62) Balaban, N.; Rasooly, A. Int. J. Food Microbiol. 2000, 61, 1-10. (63) Martens, J. H. P. A.; Reijenga, J. C.; Ten, Thije, Boonkkamp, J. H. M.; Mattheij, R. M. M.; Everaerts, F. M. J. Chromatogr., A 1997, 772, 49-62.

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immunoassay. Further investigations into the mechanisms associated with sample enrichment will be pursued to help access new avenues for preconcentration of materials on microchip devices for more sensitive detection capabilities. ACKNOWLEDGMENT This publication was made possible by Grant AI056047 from the National Institute of Allergy and Infectious Diseases (NIAID). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the National

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Institutes of Health (NIH). This research was performed while A.J.H. held a National Research Council Research Associateship Award at the Naval Research Laboratory. The authors acknowledge Dr. Braden Giordano, Dr. Carl Newman, and Dr. Jeremy Ramsey for helpful discussions regarding the manuscript.

Received for review June 9, 2006. Accepted September 26, 2006. AC061057S