A Microchip-Based Multianalyte Assay System for the Assessment of

The development of a novel chip-based multianalyte detection system with a cardiac theme is reported. This work follows the initial reports of “elec...
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Anal. Chem. 2002, 74, 3030-3036

A Microchip-Based Multianalyte Assay System for the Assessment of Cardiac Risk Nick Christodoulides,† Maiyen Tran,† Pierre N. Floriano,† Marc Rodriguez,† Adrian Goodey,† Mehnaaz Ali,† Dean Neikirk,‡ and John T. McDevitt*,†,§,|

Department of Chemistry & Biochemistry, Department of Electrical & Computer Engineering, Center for Nanostructured Materials, and Texas Materials Institute, The University of Texas at Austin, Austin, Texas 78712

The development of a novel chip-based multianalyte detection system with a cardiac theme is reported. This work follows the initial reports of “electronic taste chips” whereby multiple solution-phase analytes such as acids, bases, metal cations, and biological cofactors were detected and quantitated. The newly fashioned “cardiac chip” exploits a geometry that allows for isolation and entrapment of single polymeric spheres in micromachined pits while providing to each bead the rapid introduction of a series of reagents/washes through microfluidic structures. The combination of these miniaturized components fosters the completion of complex assays with short analysis times using small sample volumes. Optical signals derived from single beads are used to complete immunological tests that yield outstanding assay characteristics. The power and utility of this new methodology is demonstrated here for the simultaneous detection of the cardiac risk factors, C-reactive protein and interleukin6, in human serum samples. This demonstration represents the first important step toward the development of a useful cardiac chip that targets numerous risk factors concurrently and one that can be customized readily for specific clinical settings. Coronary heart disease (CHD) is the leading cause of death in developed countries.1 In the United States alone, more than half a million people die each year from sudden cardiac arrest or from the consequences of chronic heart disease. The etiology of this common and often fatal disease is complex and multifactorial. Current screening and management strategies for the prevention of CHD target some of the plasma-based factors as predictors of cardiovascular risk.1-4 The typical analytical methods used for their measurement, however, are rather inefficient. These tests require long assay times, sophisticated instrumentation, and significant amounts of expensive reagents. Furthermore, these analytical * Corresponding author: (e-mail) [email protected]; (phone) 512471-0046, (fax) 512-232-7052. † Department of Chemistry & Biochemistry. ‡ Department of Electrical & Computer Engineering. § Center for Nanostructured Materials. | Texas Materials Institute. (1) Yu, H.; Rifai, N. Clin. Biochem. 2000, 33, 601-610. (2) Heller, R. F.; Chinn, S.; Pedoe, H. D. T.; Rose, G. Br. Med. J. 1984, 288, 1409-1411. (3) Wald, N. J.; Law, M.; Watt, H. C. Lancet 1994, 343, 15-79. (4) Kullo, I. F.; Gau, G. T.; Tajik, A. J. Mayo Clin. Proc. 2000, 75, 369-380.

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methods are limited to measuring just one analyte at a time. A more efficient, rapid, and inexpensive analytical system that targets numerous risk factors concurrently and one that can be customized for specific clinical settings would likely allow for a comprehensive and, thus, more reliable risk assessment for CHD. Therefore, the development of such a system could have a profound influence on the treatment and prevention of this important disease. Indeed, the areas of multianalyte immunoassays and immunosensors recently have become the focus of active research aiming for the use of smaller sample volumes, shorter assay times, simpler assay protocols, reduced cost per test, and larger information acquisition. Multianalyte array methods based on both dye labels and spatial resolution have been reported.5-10 Walt and coworkers exploited the former dye method using microbeads localized at the distal end of fiber bundles,5 while the latter approach was completed using spatial definition as applied by inkjet printing,6-7 micromachining,8 photolithography,9 and photodeposition.10 These array themes have yet to be employed specifically for the cardiac theme. As demonstrated previously, the newly fashioned electronic taste chip system has been shown to be suitable for the nearreal-time digital analysis of complex fluids.11-15 The central (5) Szurdoki, F.; Michael, K. L.; Walt, D. R. Anal. Biochem. 2001, 291, 219228. (6) Porakishvili, N.; Fordham, J. L. A.; Charrel, M.; Delves, P. J.; Lund, T.; Roitt, I. M. J. Immunol. Methods 2000, 234, 35-42. (7) Lemmo, A. V.; Fisher, J. T.; Geysen, H. M.; Rose, D. J. Anal. Chem. 1997, 69, 543-551. (8) Wu, Q. H.; Lee, K. M.; Liu, C. C. Sens, Actuators, B 1993, 13, 1-6. (9) Fodor, S. P. A.; Rava, R. P.; Huang, X. H. C.; Pease, A. C.; Holmes, C. P.; Adams, C. L. Nature 1993, 364, 555-556. (10) Ferguson, J. A.; Healey, B. G.; Bronk, K. S.; Barnard, S. M.; Walt, D. R. Anal. Chim. Acta 1997, 340, 123-131. (11) Goodey, A.; Lavigne, J. J.; Savoy, S. M.; Rodriguez, M. R.; Curey, T.; Tsao, A.; Simmons; G.; Yoo, S. J.; Youngsoo, S.; Anslyn, E. V.; Shear, J. B.; Neikirk, D. P.; McDevitt, J. T. J. Am. Chem. Soc. 2001, 123, 2559-2570. (12) Lavigne, J. J.; Savoy, S.; Clevenger, M. B.; Ritchie, J. E.; McDoniel, B.; Yoo, S. J.; Anslyn, E. V.; McDevitt, J. T.; Shear, J. B.; Neikirk, D. P. J. Am. Chem. Soc. 1998, 120, 6429-6430. (13) Savoy, S. M.; Lavigne, J. J.; Yoo, S.; Wright, J.; Rodriguez, M.; Goodey, A.; McDoniel, B.; McDevitt, J. T.; Anslyn, E. V.; Shear, J. B.; Ellington, A. E.; Neikirk, D. P. SPIE Conf. Chem. Microsens. Appl. 1999, 3539, 17-26. (14) Lavigne, J. J.; Metzger, A.; Niikura, K.; Cambell, L. A.; Savoy, S. M.; Yoo, S. J.; McDevitt, J. T.; Neikirk, D. P.; Shear, J. B.; Anslyn, E. V. Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3602, 220-231. (15) Curey, T. E.; Goodey, A.; Tsao, A.; Lavigne, J. J.; Sohn, Y.; McDevitt, J. T.; Anslyn, E. V.; Neikirk, D. P.; Shear, J. B. Anal. Biochem. 2001, 293 (2), 178-184. 10.1021/ac011150a CCC: $22.00

© 2002 American Chemical Society Published on Web 05/30/2002

components of this total analysis system are an extremely versatile silicon wafer platform that can be adapted to a number of customized applications and a video chip that serves to acquire optical data at the various microreactor sites. In this paper, we describe a novel, immunological application of this technology for the simultaneous detection of two serum cardiac risk factors, C-reactive protein (CRP) and interleukin-6 (IL-6). This report represents the initial application of a chip-based multianalyte detection system employed specifically for the cardiac theme. MATERIALS AND METHODS Reagents. The CRP-specific antibodies were purchased from Accurate Chemical, Scientific Corp. (Westbury, NY), Research Diagnostics, Inc. (Flanders, NJ), Biogenesis (Kingston, NH), Fitzgerald Industries Int., Inc. (Concord, MA), and Cortex Biochem (San Leandro, CA). Anti-Helicobacter pylori and antifibrinogen antibodies were obtained from Accurate Chemical and Scientific Corp., and IL-6-specific antibodies were obtained from Accurate Chemical, Scientific Corp., Pharmingen International (San Diego, CA), and Endogen (Woburn, MA). The CRP (Cortex Biochem) and IL-6 (Endogen) reagents were diluted in phosphatebuffered saline (PBS) or in serum diluent depleted of CRP by immunoaffinity chromatography (International Enzymes, Fallbrook, CA). Agarose microbeads were obtained from XC Corp. (Lowell, MA). Bead Derivitization. Analyte-specific and control antibodies were coupled to the microbeads by reductive amination. This coupling process is based on the principle that the aldehyde groups of “glyoxal” agarose beads react with ligands (such as antibodies) to form reversible Schiff base complexes that can be selectively reduced and stabilized as covalent linkages with sodium cyanoborohydride.16 Instrumentation. The components of the taste chip total analysis system were described previously.11-15 Briefly, individual microbeads were placed into chemically etched microcavities patterned in a square array on a silicon wafer chip. The beadloaded chip was packaged into a fluid flow cell that was held together by an aluminum casing. The flow cell assembly was positioned on a microscope stage that allowed for the microscopic observation of the beads from both transmission (bottom illumination) and epifluorescent (top illumination) perspectives. Solutions were introduced into the flow cell in an automated fashion using Amersham Pharmacia Biotech A¨ KTA fast protein liquid chromatography or high-pressure liquid chromatography systems controlled by Unicorn 3.0 software (Amersham Pharmacia Biotech). Beads within the sensor array were exposed to the serum samples and signaling reagents using fluid flow that was directed into the upper reservoir of the cell and forced down through the wells (containing the resin beads) to the lower reservoir and out through the drain. The flow cell was designed specifically to ensure that all introduced solution passed through the various wells of the array. The arrays were observed through the microscope optics, and images derived from the individual sensor beads were captured and analyzed using a charge-coupled device (CCD) in conjunction with Image Pro Plus 4.0 software (Media Cybernetics). (16) Borch, R. F.; Bernstein, M. D.; Durst, H. D. J. Am. Chem. Soc. 1971, 93, 2897-2904.

Assay Conditions. All assays were performed at room temperature under continuous fluid flow conditions. Prior to each assay, the flow cell and the inner walls of the capillary tubing used to introduce reagents to the flow cell were blocked with 3% bovine serum albumin (BSA) in PBS delivered at a flow rate of 0.2 mL/ min. Serum or 1% BSA/PBS solutions spiked with known amounts of CRP, IL-6, or both were delivered to the flow cell at a flow rate of 0.25 mL/min. These solutions were followed by the addition of detecting antibody delivered at 0.1 mL/min. In colorimetric assays utilizing horseradish peroxidase (HRP)-conjugated visualization antibodies, the presence of captured analyte was detected following injection of the chromogenic and precipitable HRP substrate 3-amino-9-ethylcarbazole (AEC) at 0.1 mL/min. In the bead regeneration experiments, the colorimetric precipitates were removed from the beads though their exposure to 80% methanol, followed by rinses with PBS solutions. The immunological components of the beads were regenerated with solution-phase exposure to 0.1 M glycine-HCl buffer (pH 2.5) and then reequilibrated with PBS. For the experiments involving the fluorescence signaling methodologies, the immunological components were regenerated using 10.0 M MgCl2 (pH 1.2) solution. Data Collection and Processing. The flow cell was maintained at a fixed position on the microscope stage throughout the duration of each immunoassay. Images of the array captured with the CCD before and after each run were subjected to analysis in which specific areas of interest (AOI) in the central region of each bead were selected and evaluated for their average red, green, and blue (RGB) intensities. Beads in the colorimetric CRP assay, which utilized the HRP/AEC-based detection system, developed a red-brown coloration that was quantified by measuring the average blue pixel intensity yielded by the CCD. This intensity, IBs, was subsequently converted to the “effective blue absorbance” value, AB, using Beer’s law (AB ) - log(IBs/IBo), where the reference blue intensity of the bead prior to the immunoassay, IBo, is used. Negative control beads (i.e., beads coated with H. pylori-specific antibody) allowed for the removal of nonspecific background signal through a simple baseline correction. At least five replicate beads for each concentration of CRP capturing antibody were included within the array from which the mean absorbance was calculated for the zero and CRP-containing serum samples. The limit of detection for a given type of bead (i.e., with a given CRP capturing antibody concentration) was defined at the 99.5% confidence level as the lowest CRP concentration yielding an average absorbance reading at least three standard deviations above the mean value recorded for the zero analyte. RESULTS AND DISCUSSION The taste chip total analytical system11-15 is adapted and expanded here for the development of bead-based immunoassays. Briefly, a liquid chromatography system is used to introduce solutions to a flow cell, which accommodates a silicon wafer platform with micromachined cavities arranged in 3 × 3, 3 × 4, 5 × 7, or 10 × 10 arrays (see Figure 1). The cavities are created with an anisotropic etch which yields pyramidal pits with transwafer openings.11 These openings allow for both fluid flow through and microscopic/optical analysis of the individual beads positioned within each of the cavities. Therefore, each cavity within the array serves as a miniaturized reaction vessel and analysis chamber, with selectivity determined by the specificity of the capturing Analytical Chemistry, Vol. 74, No. 13, July 1, 2002

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Figure 2. Demonstration of the various detection modes of the taste chip-based assay system. Agarose microbeads directly coated with a control-unlabeled (CTL)-, a horseradish peroxidase (HRP)-, a fluorescein isothiocyanate (FITC)-, and a colloidal gold (CG)conjugated antibody are shown. Beads coated with FITC-conjugated antibody are visualized directly by top (epi) illumination at the excitation wavelength maximum of the fluorophore (470 nm). Beads coated with the HRP- and CG-conjugated antibodies are visualized by bottom (transmission) illumination after exposure to AEC and silver enhancer solution, respectively. Control beads (CTL) shown here are also visualized by bottom illumination after exposure to AEC. Figure 1. Microchip-based immunosorbent assay system. (A) A scanning electron micrograph showing the microbeads positioned within the micromachined cavities localized on a silicon wafer chip. (B) A schematic illustration showing the bead array chip which is packaged into a fluid flow cell. The pyramidal pits extend through the entire thickness of the wafer, thereby creating square openings (∼100 µm × ∼100 µm) on the remote side of the chip. These openings both provide optical access to the bead element and serve as a drain for the reaction/analysis chambers. The package assembly is suitable for both fluorometric (based on epifluorescence) and colorimetric (based on transmission) analyses.

antibody coupled to the bead the cavity hosts. Consequently, the array in its current form has the potential capacity to capture and ultimately detect up to 100 different antibody/antigen analytes using a single drop of sample fluid (∼50 µL). Identification and quantitation of analytes occur via colorimetric and fluorescent optical signals (see Figure 2) acquired with a CCD allowing for the near-real-time digital analysis of complex fluids, including serum.11 Immunoassay Detection Modes. A series of detection/ visualization methods for the immunological adaptation of the taste chip analytical system are explored as shown in Figure 2. Here, colorimetric and fluorescent optical signals obtained from microspheres directly coated with the indicated immunoglobulins were evaluated. Most rapid analysis times are achieved using direct fluorescence detection methods. While dye precipitation and silver staining methods require slightly longer development times, these techniques yield excellent colorimetric signal capacities. Furthermore, these methodologies are compatible with simple optical components, making them more suitable for future use with simple hand-held reader units, such as the hand-held device utilizing the taste chip technology currently in development by 3032 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002

Constellation Technology Corp. (Largo, FL). Negative controls (i.e., beads possessing an unlabeled immunoglobulin) are examined also in the context of the various visualization strategies and are found to exhibit only minimal background activity in the colorimetric mode. Due to the excellent signal characteristics and simple colorimetric detection capacity, the dye precipitation method serves as the analysis method of choice for the majority of the studies discussed here. CRP Assay Description and Characteristics. As the first important target molecule for a cardiac theme chip, CRP is selected due to its recently recognized importance as an essential biomarker for cardiovascular risk.4 Likewise, CRP is a circulating acute-phase reactant that reflects active systemic inflammation. Inflammation contributes to the pathogenesis of atherosclerosis by destabilizing the fibrous cap of atherosclerotic plaque causing from time to time plaque rupture and increasing the risk of coronary thrombosis. Elevated plasma CRP levels are associated with the extent and severity of atherosclerosis and, consequently, with a higher risk for cardiovascular events.17-20 A schematic illustration showing relevant immunocomplex formation of the chip-based assay for the detection of CRP is provided in Figure 3A. The test sequence involves the successive addition of serum containing CRP and anti-CRP antibody conjugated to HRP and AEC to an array of beads coupled to a CRP(17) Ridker, P. M.; Buring, J. E.; Shih, J.; Matias, M.; Hennekens, C. H. Circulation 1998, 98, 731-733. (18) Ridker, P. M.; Glynn, R. J.; Hennekens, C. H. Circulation 1998, 97, 20072011. (19) Tracy, R. P.; Lemaitre, R. N.; Psaty, B. M.; Ives, D. G.; Evans, R. W. Arterioscler. Throm. Vasc. Biol. 1997, 17, 1121-1127. (20) Koenig, W.; Sund, M.; Frolich, M.; Fischer, H.-G.; Lowel, H.; Doring, A.; Hutchinson, W. L.; Pepys, M. B. Circulation 1999, 99, 237-242.

Figure 4. Detection limit and analytical range of the CRP-specific assay. Beads coupled to increasing concentrations of CRP-specific capture antibody (as labeled) are compared for their capacity to detect 0-10 000 ng/mL CRP in human serum. Shown here is the detection limit of the assay (utilizing the 6 mg/mL CRP antibody beads). The detection limit is defined as the minimum CRP concentration that yields a measurable mean signal above the value derived from the mean absorbance of 10 replicate beads plus three standard deviations from the mean of the zero analyte calibrator.

Figure 3. Application of the taste chip-based assay system for the detection of CRP. (A) The “sandwich” format immunoassay is based on the binding of human CRP (iii) from the fluid sample to two CRPspecific antibodies, one (ii) immobilized on the bead (i), and the other conjugated to HRP (iv). Results are evaluated based on the optical changes (blue color absorbance) in the individual sensing elements (beads) after addition of AEC. (B) Beads coupled to increasing concentrations of CRP-specific antibody (0-6 mg of antibody/mL of beads) are used for the assay of 1000 ng/mL CRP in human serum. (CTL beads indicated as having 0 mg of CRP-specific antibody/mL of beads are coated with rabbit anti-H. pylori-specific antibody at concentration of 3 mg of antibody/mL of beads.) (C) For the HRP/ AEC-based detection system used for the detection of CRP in (B), which develops a red-brown coloration at the beads, most changes in color intensity occur in the blue channel; provided here are the mean absorbance values derived from the blue color intensities of the sample beads after the assay, referenced to the same bead before the assay.11

specific antibody. The assay requires small amounts of sample and reagents (0.1-2.0 mL) and may be completed in less than 1 h. An attractive attribute associated with the taste chip approach is that each bead/well serves as its own microreactor and microanalysis chamber. This capacity is demonstrated from the data shown in Figure 3B. Here a series of 35 beads are simultaneously exposed to a serum sample containing 1,000 ng/ mL CRP. The 10 beads in the first two columns are coated with a high concentration of capture antibody (6 mg/mL), the next 10

beads/two columns with medium concentration (3 mg/mL), and the final 10 beads/two columns with a low level of capture probe (0.5 mg/mL). Also included in the array are five beads that serve as controls (coated with irrelevant rabbit anti-H. pylori immunoglobulin). With this arrangement, 10 identical trials are measured for each of the 3 loading levels, while 5 background levels are also determined. For 1000 ng/mL CRP, the average effective blue absorbance values are measured and shown in Figure 3C. As expected, the concentration of capture probe strongly influences the degree of blue color attenuation evaluated at each site.11 Moreover, a series of identical chips are prepared and exposed to different CRP levels to create concentration response curves for this type of array. The dose-dependent signals are collected as displayed in Figure 4. These results demonstrate the capacity of the taste chip system to selectively capture, detect, and quantitate CRP levels over clinically important ranges. The signals also reveal that the concentration of the capturing antibody on the bead determines the signal intensity for the assay for a given CRP concentration. Interestingly, this feature allows for an expansion of the analytical range and a reduction of the detection threshold of the assay. Indeed, beads coupled to the highest concentration of capturing antibody (i.e., 6 mg of antibody/mL of beads) yield the greatest range and lowest detection threshold. The CRP detection limit demonstrated here at 1 ng/mL is lower than those recently reported for automated CRP assay systems such as the Hitachi 911 (Iatron), BNII (Dade Behring), Hitachi 911 (Denka Seiken), Hitachi 917 (Wako), Immulite 2000 (Diagnostic Products Corp.), and Olympus AU640 (Olympus) with limits of detection at 5, 20, 30, 60, 20, and 80 ng/mL, respectively.21 (21) Roberts, W. L.; Moulton, L.; Law, T. C.; Farrow, G.; Cooper-Anderson, M.; Savory, J.; Rifai, N. Clin. Chem. 2001, 47, 418-425.

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Figure 5. Chip-based immunological assays demonstrating capacity for multiple uses. Beads coupled to 3 mg of antibody/mL of beads of either rabbit CRP-specific capture antibody (CRP) or an irrelevant rabbit anti-H. pylori-specific antibody (CTL) are tested for their capacity to detect 1000 ng/mL CRP in human serum in continuous repetitive runs. (A) In the upper panel, data are provided for a colorimetric method. Here each cycle involves the following: (i) injection of 1000 ng/mL CRP, (ii) addition of HRP-conjugated anti-CRP detecting antibody, (iii) addition of AEC, (iv) elution of signal with 80% methanol, (v) wash with PBS, (vi) regeneration with glycine-HCl buffer, and (vii) equilibration with PBS. Results shown here are for the mean blue absorbance values. (B) For the lower panel, data are provided for similar regeneration cycles completed with a fluorescence method. Here each cycle involves the following: (i) injection of 1000 ng/mL CRP, (ii) addition of FITC-conjugated anti-CRP detecting antibody, (iii) regeneration with MgCl2, and (iv) equilibration with PBS. The data provided here document the fluorescent intensity recorded at the various beads during the detection (assay) as well as from elution steps from each cycle. For brevity, only 5 of the 10 cycles are shown.

Furthermore, the taste chip array yields a useful analytical range (1-10 000 ng/mL without dilution) that exceeds the majority of the mature methods yet reported.21 Likewise, a large analytical range is a desirable and important feature for CRP assays in modern clinical settings.1 The precision of the CRP taste chip assay method is examined by testing a human serum sample spiked with 1000 ng/mL CRP in 35 replicates (i.e., one chip with 35 CRP-sensitized beads) in 10 independent assays over a period of two days. When results are evaluated by converting the signal from the blue channel into absorbance, the intra- and interassay precisions of the system are found to be 7.7 and 4.6% (coefficient of variance, CV), respectively. However, a significant improvement of the assay’s precision is achieved when the system is calibrated and the signal is expressed as a ratio of green to blue channels. Under these conditions, the taste chip system demonstrates excellent intra- and interassay precision at 3.0 and 1.3% CV, respectively, which is superior to the majority of the recently described automated CRP assay systems with CV between 4.3 and 6.8% (Dade Behring), 2.2-6.1% (Denka), 1.0-11% (Wako), 6.4-12% (Diagnostic Products Corp.), 1.1-3.4% (Iatron), and 3.2-44% (Olympus).21 Chip Assembly Regeneration Capacity. While many future applications of the cardiac theme chips will likely involve multiplexed analysis of patient samples whereby single-use, disposable 3034 Analytical Chemistry, Vol. 74, No. 13, July 1, 2002

cartridges are employed, the more rapid development of the relevant science and technology base will be facilitated by the identification of methods that can be used to recycle and reuse the arrays. Likewise, the well-established methods involving the reversible release of captured proteins and antibodies from immunoaffinity chromatographic supports would seem to suggest that similar methodologies may be adapted for the taste chip-based immunological assays. In this context, a series of potential elution buffers were tested. Solutions based on 0.1 M glycine-HCl buffer (pH 2.5) and MgCl2 (pH 1.2) were found to be efficient in releasing the captured CRP analyte and detecting antibody from the beads. The efficacy of the bead recycling method based on 0.1 M glycine-HCl treatment is shown in Figure 5A, where three successive repetitive CRP assays are achieved under these conditions of elution without a significant loss in sensitivity. Alternatively, when the assay is performed in fluorescence mode (Figure 5B), which utilizes a FITC-labeled CRP-detecting antibody, more than 20 repetitive CRP assays are achieved easily with the use of MgCl2 as the recycling agent. Multianalyte Detection Capacity. Perhaps the most powerful characteristic of the taste chip array is its capacity to detect multiple analytes simultaneously. The array multiplexing capacity is demonstrated here for the two cardiac risk factors, CRP and IL-6, as shown in Figure 6. The IL-6 analyte is the major initiator

Figure 6. Demonstration of the multianalyte detection capacity of the taste chip-based immunosorbent assay system. An array of beads sensitized as indicated in case A (upper left) is used in three independent tests for the assay of 1000 ng/mL CRP and IL-6 in PBS. (CTL refers to control beads coated with irrelevant immunoglobulin. The other beads are coated with antibodies specific for the protein indicated.) Cases shown in the left column (i) are CCD-captured images of the microarray prior to (case A) and after (cases B-D) each test; cases shown in the right column (ii) demonstrate the relative signal intensities of the beads, as determined by digital analysis of the corresponding image on the left. (Since the “before” images of all three tests are nearly identical, only one of them is shown here as way of an example.) In the first test (case B), CRP is selectively captured by the CRP-sensitized beads and detected with a rabbit anti-CRP/HRP-conjugated antibody. In the second test (case C), IL-6 is selectively captured by the IL-6-sensitized beads and detected with a rat anti-IL-6/HRP-conjugated antibody. In the third test (case D), both CRP and IL-6 are captured and detected concurrently by a mixture of their HRP-conjugated antibodies.

of the acute-phase response and induces the synthesis of CRP, as well as other acute-phase reactants.22-24 Given the role of IL-6 in CRP regulation, the combined use of IL-6 and CRP protein levels as indicators of inflammation may provide a better prediction of risk associated with inflammation than would use of either indicator alone.25 Prior to attempting simultaneous detection of (22) Lu, Z. Y.; Brailly, H.; Wijdenes, J.; Bataille, R.; Rossi, J. F.; Klein, B. Blood 1995, 86, 3123-3131. (23) Gauldie, J.; Richards, C.; Northemann, W.; Fey, G.; Baumann, H. Ann. N.Y. Acad. Sci. 1989, 557, 46-59. (24) Bataille, R.; Klein, B. Arthritis Rheum. 1992, 35, 982-983. (25) Harris, T. B.; Ferruci, L.; Tracy, R. P.; Corti, M. C.; Wacholder, S.; Ettinger, W. H.; Heimovitz, H.; Cohen, H. J.; Wallace, R. Am. J. Med. 1999, 106, 506-512.

both analytes, CRP and IL-6 are detected individually from a PBS sample containing 1000 ng/mL of each, Figure 6B and C, respectively. In separate experiments, requiring the same amount of time and reagents as the individual test, both analytes are detected concurrently from the same solution without an apparent loss in signal or a significant increase in nonspecific background level, Figure 6D. Similar results are obtained when a serum sample containing the same concentrations of CRP and IL-6 is tested (data not shown). These results demonstrate the capacity of the taste chip system to measure simultaneously both CRP and IL-6 without a compromise in sensitivity or specificity. We envision that a more complete definition of the risk for CHD will be achieved through an analysis of the interrelationship between epidemiology and serum biomarker concentrations using CRP and IL-6 as well as other novel risk factors such as soluble intercellular adhesion molecule-1, homocysteine, fibrinogen, and infectious agents such as H. pylori, Chlamydia pneumoniae, Herpesvirus hominis, and cytomegalovirus.4 The expansion of the molecular diversity and, thus, the increase in the utility of the cardiac theme chips is now in progress. It should be acknowledged that as the number of cardiac biomarkers represented within the array increases, the potential for interference and crosstalk also increases. Indeed, cases where nonspecific effects and interferences occur between the individual immunoassays have been documented already in our experiments (data not shown). However, this potential stumbling block has been surmounted successfully with the creation of multichamber flow cells and the associated fluid delivery system. Isolated-dedicated regions on the same chip can be utilized for the detection of compatible groupings of target molecules. Multiplexed cardiac marker analysis is executed more readily as is the selection of suitable capture and reporter agents using this “chip-on-a-chip” strategy. CONCLUSIONS From a careful inspection of the component features of the taste chip system along with those associated with the mature, macroscopic methods, some of the advantages of the taste chip approach become apparent. The important considerations here are as follows. First, current generation of the cardiac chips employ ∼280-µm-diameter beads that possess internal volumes of ∼10 nL and these spheres reside in containers (i.e., etched wells) with volumes of ∼30 nL. These low-volume elements, combined with fluidic channels capable of providing flow of 2 mL/ min with passage of reagents through “drains” at the bottom of each well, lead to a very efficient delivery of reagents and washes. In a typical experiment, >50 000 well-dead volumes are used to rinse away excess reagents as compared to the 2-3 washes for commonly exploited enzyme-linked immunosorbent assay (ELISA) methods. Significant reduction in the taste chip background signal is obtained with the fluidic channel and drain features. Second, in contrast to ELISA in which antigen-antibody interactions are built up from and limited by a single layer on the bottom surface of the well, the taste chip total analysis system benefits from the use of porous beads. This feature allows for the use of thicker layers of the capture agents. Moreover, the signal is localized in a confined volume, allowing for the production of larger signals. Third, fluids are transported rapidly into the analysis chamber using a pressure-driven flow or capillary forces. The active transport of reagents and small effective feature sizes of the Analytical Chemistry, Vol. 74, No. 13, July 1, 2002

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components used here allow for the more rapid delivery of the reagents and washes. Slow steps associated with diffusion of reagents over macroscopic distances, which is prevalent with the majority of the established methods, are minimized with the taste chip system. Finally, the ability to complete a full assay at each bead site allows for the simultaneous execution of multiple trials. This capacity provides more accurate results through signal averaging and allows for multiplexed testing to occur as demonstrated here for the initial cardiac theme chip. The taste chip adaptation of immunological assays described here has yielded a functional miniaturized platform that exhibits assay characteristics (analytical range, detection threshold, and coefficient of variance) superior, in many respects, to the mature macroscopic analogues. These characteristics, along with the capacity to complete multiplexed immunological assays, bode well for the future treatment and prevention of CHD using such chipbased sensor arrays. Adaptation of the taste chip methodology to

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other important multianalyte immunoassay systems for the areas of human health, veterinary sciences, environmental testing, drug monitoring, toxin detection, military, home land defense, and food/beverage processing is currently in progress. ACKNOWLEDGMENT We gratefully acknowledge Prof. Eric Anslyn, Prof. Jason Shear, and Prof. Andrew Ellington for helpful discussions. Funding for this project was provided by the National Institute of Health, the National Science Foundation (IGERT program), Labnetics Corp., the Beckman Foundation, the Army Research Office (MURI program), and the Welch Foundation.

Received for review November 5, 2001. Accepted February 5, 2002. AC011150A