A Filtration-Based Protein Microarray Technique - American Chemical

technique can improve the overall reaction kinetic rate by 10-fold, yield a dynamic range of 4 decades, and enhance the assay sensitivity and specific...
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Anal. Chem. 2003, 75, 5345-5351

A Filtration-Based Protein Microarray Technique Yangqing Xu and Gang Bao*

Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia 30332

Protein microarrays are an emerging technology for studying protein expression profiling and protein functions. However, with the current design approaches, the overall performance of protein microarrays can be compromised by diffusion-limited kinetics. We developed a new protein microarray platform that utilizes a filtration assay with protein microarrays printed on protein-permeable nitrocellulose filter membranes. Compared with protein microarrays assayed with the conventional incubation-shaking method, this new approach overcomes the diffusion limit. We demonstrated that this novel technique can improve the overall reaction kinetic rate by 10-fold, yield a dynamic range of 4 decades, and enhance the assay sensitivity and specificity. Further, using multistacking protein chips, at least 14 chips can be probed simultaneously, with 22 400 different reactions in a single assay. The advantages of large fluorescent dyes, such as phycobilisome and quantum dots, can be better exploited using the filtration assay. The potential clinical applications of the filtration-based protein microarrays were demonstrated by detecting carcinoembryonic antigen in human plasma samples. Protein microarrays, or protein chips, are one of the most promising new technologies for a wide range of biomedical applications,1-4 including protein expression profiling for disease diagnosis;5-7 studies of protein-protein interactions8-10 and enzymatic activities;11,12 and the identification of protein-binding * Corresponding author. Phone: 404-385-0373. Fax: 404-894-4243. E-mail: [email protected]. (1) Macbeath G. Nat. Genet. 2002, 32 (Suppl), 526-532. (2) Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.; Vohringer, C. F.; Joos, T. O. Trends Biotechnol. 2002, 20, 160-166. (3) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2001, 5, 40-45. (4) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7, 55-63. (5) Joos, T. O.; Schrenk, M.; Hopfl, P.; Kroger, K.; Chowdhury, U.; Stoll, D.; Schorner, D.; Durr, M.; Herick, K.; Rupp, S.; Sohn, K.; Hammerle, H. Electrophoresis 2000, 21, 2641-2650. (6) Knezevic, V.; Leethanakul, C.; Bichsel, V. E.; Worth, J. M.; Prabhu, V. V.; Gutkind, J. S.; Liotta, L. A.; Munson, P. J.; Petricoin, E. F., 3rd; Krizman, D. B. Proteomics 2001, 1, 1271-1278. (7) Madoz-Gurpide, J.; Wang, H.; Misek, D. E.; Brichory, F.; Hanash, S. M. Proteomics 2001, 1, 1279-1287. (8) de Wildt, R. M.; Mundy, C. R.; Gorick, B. D.; Tomlinson I. M. Nat Biotechnol. 2000, 18, 989-994. (9) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-1763. (10) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293, 2101-2105. (11) Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat Biotechnol. 2002, 20, 270-274. 10.1021/ac034613g CCC: $25.00 Published on Web 09/05/2003

© 2003 American Chemical Society

molecules, such as peptides,13 phospolipids,10 and other small molecules.14,15 However, most of the current protein microarrays are formed on an impermeable solid surface, such as glass coverslides,9,16 and the assays are performed by incubation with shaking. As such, the reaction kinetics can be limited by the slow diffusion of analyte molecules toward the surface, where capture molecules are immobilized,5,17-20 resulting in slow binding kinetics and compromised assay sensitivity and dynamic range. The diffusion limit can become more severe when the concentration of a protein analyte is low. We developed a novel filtration-based protein microarray technique that can overcome the diffusion limit and enhance the overall performance of protein microarrays, including binding kinetics, detection limit, dynamic range, specificity, and throughput. As shown in Figure 1, in this approach, protein microarrays are printed on porous nitrocellulose filter membranes and are placed in a customized filtration apparatus for flow-through assays. Compared with modified glass/plastic surfaces and silanized silicon wafer, nitrocellulose membranes have higher proteinbinding capacity and are believed to be more biocompatible for immobilizing proteins.21 Instead of incubating and shaking with the microarray for binding reaction, the sample is filtered through the microarray-containing membrane chip using multiple cycles to facilitate the binding between analytes and their corresponding capture molecules (Figure 1A). To increase throughput, multiple protein chips can be stacked together and assayed simultaneously, as shown in Figure 1B. The array spots, 200-400 µm in diameter, are essentially three-dimensional as a result of the highly porous substrate membrane used (Figure 1C). This can significantly increase the protein-binding capacity. More importantly, as the analytes flow through the membrane pores, they react with the capture molecules that are immobilized on the porous surface (12) Zhu, H.; Klemic, J. F.; Chang, S.; Bertone, P.; Casamayor, A.; Klemic, K. G.; Smith, D.; Gerstein, M.; Reed, M. A.; Snyder, M. Nat. Genet. 2000, 26, 283-289. (13) Espejo, A.; Cote, J.; Bednarek, A.; Richard, S.; Bedford, M. T. Biochem. J. 2002, 367, 697-702. (14) Kuruvilla, F. G.; Shamji, A. F.; Sternson, S. M.; Hergenrother, P. J.; Schreiber, S, L. Nature 2002, 416, 653-657. (15) Winssinger, N.; Ficarro, S.; Schultz, P. G.; Harris, J. L. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11139-11144. (16) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2 2001, Research 4.1-4.13. (17) Karlsson, R.; Roos, H.; Fagerstam, L.; Persson, B. Methods; A companion to Methods in Enzymology 1994, 6, 99-110. (18) Stenberg, M.; Nygren, H. J. Immunol. Methods 1988, 113, 3-15. (19) Sapsford, K. E.; Liron, Z.; Shubin, Y.; Ligler, F. S. Anal. Chem. 2001, 73, 5518-5524. (20) Liu, R. H.; Lenigk, R.; Druyor-Sanchez, R. L.; Yang, J.; Grodzinski, P. Anal. Chem. 2003, 75, 1911-1917. (21) Tokinson, J. L.; Stillman, B. A. Front. Biosci. 2002, 7, c1-12.

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Figure 1. A schematic illustration of the filtration-based protein microarray technique. (A) The protein sample is filtered cyclically through a stack of protein chips shown in B. Each chip contains a microarray of 3D spots illustrated in C formed by capture molecules printed on a nitrocellulose membrane. (D) These capture molecules are immobilized on the surface of the microchannels of the membrane, increasing protein binding capacity and facilitating analyte binding during filtration.

(Figure 1D), and the depleted analytes near the spots are replenished more effectively by the continuous filtration flow. Therefore, the overall binding kinetics can be significantly increased. Unexpectedly, we found that the filtration-based protein microarrays not only improved sensitivity and extended the dynamic range, but also enhanced assay specificity. When the multistacking approach is used, we can simultaneously study more than 20 000 different reactions in a single assay. We also found that, by combining the filtration reaction with fluorescence labeling using large dyes, such as Phychobilisome and quantum dots, very high sensitivity and color-multiplexing was achieved in protein microarray assays. To demonstrate the clinical applications of the filtration-based microarray technique, a filtration-based microarray sandwich assay was performed to detect carcinoembryonic antigen (CEA) in human plasma samples. Although the filtration approach has been used in ELIFA22 and DNA microarray assays,23 it has not been applied to protein microarray studies. Since proteins in a sample cannot be amplified, the detection of low-concentration proteins using the conventional microarray approach is likely to sustain a more severe diffusion limit than in DNA detection, resulting in compromised performance. As demonstrated in the present work, the filtration-based protein chips promise a new platform for faster and more sensitive protein detection and analysis. MATERIAL AND METHODS Printing of Microarrays and Protein Conjugation. Four protein analytes were used as a model system for direct detection microarray assays; they were human serum albumin (HSA, Pierce), CEA (US Biological), mouse IgG (MIgG, Sigma), and NeutrAvidin (Pierce). The corresponding capture molecules were, respectively, mouse monoclonal anti-human serum albumin (AHSA, Biospacific), rabbit polyclonal anti-CEA (ACEA), goat anti-mouse IgG (GAM, Rockland) and protein G′ (Sigma), and biotinylated R-casein (Ca-biotin, with 1.5 biotin-DNP per casein). These reagents were chosen to represent a broad class of capture molecules, including monoclonal antibodies, polyclonal antibodies, nonantibody proteins, and small molecules.24 In microarray (22) Pinon, J. M.; Puygauthier-Toubs, D.; Lepan, H.; Boulant, J.; Marx-Chemila, C.; Dupont, H. Electrophoresis 1990, 11, 41-45. (23) Cheek, B. J.; Steel, A. B.; Torres, M. P.; Yu, Y. Y.; Yang, H. Anal. Chem. 2001, 73, 5777-5583. (24) Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.; Vohringer, C. F.; Joos, T. O. Drug Discovery Today 2002, 7, 815-22.

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sandwich assays for detecting CEA, a pair of mouse monoclonal anti-CEAs, ACEAh, with high affinity, and ACEAl, with low affinity were used (Biospacific). Spots of fluorescently labeled bovine serum albumin (BSA, from Sigma) were used as a standard for most of the microarray assays. The MicronSep nitrocellulose filter membrane (Osmonics) was used as the microarray substrate because of its ability to maintain protein conformation and activities and its high capacity for protein binding. It has a diameter of 13 mm (if not otherwise specified), a thickness of 140 µm, and an average pore size of 0.8 µm. The protein binding capacity of the filter is in the range of 80-120 µg/cm2. Prior to printing, the capture molecules were dialyzed into the spotting buffer that contained 0.05 M monobasic sodium phosphate and 0.05 M dibasic sodium phosphate, with pH 7.4. Printing of microarrays on both the nitrocellulose filter membrane and glass coverslide was performed using a noncontact Biochip ink-jet arrayer (Perkin-Elmer Bioscience). About 5-10 nL of capture molecule samples were dispensed at each spot, with an center-to-center distance of 300-600 µm, and the resulting spot diameter was in the range of 200-400 µm. The conjugation of proteins, including HSA, CEA, MIgG, NeutrAvidin, casein, and BSA with Alexa dyes (Molecular Probes) or biotin-DNP-SE (Molecular Probes) followed the protocol recommended by Molecular Probes. The conjugated protein samples were diluted in a reaction buffer containing 0.2% BSA, 10 mM Tris, 0.9% NaCl, and 0.05% Tween-20 (Pierce) unless otherwise indicated, with pH adjusted to 7.4. The cross-linking of NeutrAvidin to carboxylated quantum dots (kindly provided by Shuming Nie) was realized via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (Pierce). Binding Assays. The protein chip, that is, the microarraycontaining filter membrane, was first blocked for 15 min by 2% BSA in TBS buffer (Tris-buffered saline, containing 140 mM NaCl, 10 mM Tris-HCl, pH 7.4). A 100-200-µL portion of blocking reagent was cycled through the filter back and forth by using a customized filtration device. The flow rate was within the range of 0.05-0.5 cm/s, typically 0.4 cm/s, if not otherwise stated. A 100-200-µL portion of protein sample was then filtered through the chip in the same manner; the reaction time was typically 3045 min. For the multistacking assay, up to 14 chips were stacked together to allow simultaneous binding with 300-500 µL of sample. With some optimization of the device, only 15 µL of sample per cm2 of the chip area was needed. Two identical chips were used for each assay if not otherwise indicated. Following the binding reaction, chips were washed in the reaction buffer for 5-10 min to remove nonspecifically bound proteins. When a sandwich assay was performed, the buffer with fluorescently labeled detection antibodies was subsequently flown through the microarray for 15-30 min. Imaging and Data Analysis. Two instruments were used for imaging the microarrays, and the fluorescence intensities were quantified using the software provided by the instrument manufactures. The first instrument was a confocal-based array scanner (BioChip, Perkin-Elmer) that had a 30-µm depth of focus.25 It had high resolution (down to 5 µm) and high sensitivity (pmol per cm2), thus providing high quality images of microarrays. However, the thin focal plane was not ideal for quantitatively imaging the (25) Ramdas, L.; Zhang, W. B. Biophotonics Int. 2002, 9, 38-39.

3-dimensional spots. Alternatively, we used a FLA-3000 fluorescence imager (Fuji) that was able to more quantitatively measure the intensity of the spots. It is possible that the detection sensitivity of the filtration-based microarray technique can be further improved by using other nonconfocal array scanners with better imaging resolution and sensitivity.25,26 After quantification of fluorescence, the signal levels were determined by subtracting the backgrounds (defined by four to nine blank spots on the same chip) from the original intensity measurement. RESULTS AND DISCUSSION Accelerated Binding Kinetics. To evaluate the binding kinetics of filtration-based protein microarrays, we generated a microarray that contained five different capture molecules, including one monoclonal antibody (AHSA), two polyclonal antibodies (GAM and ACEA), one nonantibody protein (protein G′), and one small molecule conjugated to a protein carrier (Ca-biotin). Their corresponding analytes were HSA, CEA, MIgG (captured by both GAM and protein G′), and NeutrAvidin. This microarray represents a wide range of protein-binding molecules.2 Each capture molecule was prepared with two or three different concentrations, and the sample with each concentration was printed in triplicate. Assays were first performed using individual analyte species, and we found that the cross-reaction of each analyte to mismatched capture molecules was not detectable after 1 h of filtration assay. The microarrays were assayed with a sample containing all four analyte proteins labeled with Alexa647, with concentrations of 30 pM for HSA, CEA, and MIgG and 100 pM for NeutrAvidin. Two different assays were performed, both with identical microarrays on nitrocellulose filters. The first was the filtration assay described above. The second was an assay using incubation and shaking (shaking assay), in which the microarray-containing nitrocellulose filters were reacted with the analyte in a microplate well under shaking at 200 rpm with a sample volume of 200 µL. A comparison of fluorescence intensity resulting from filtration and shaking assays after 60 min of binding is shown in Figure 2A. When compared with the shaking assay, the fluorescence signal resulting from the filtration assay was much stronger for almost all spots, indicating that the filtration-based microarray assay could significantly accelerate the binding of different analytecapture molecule pairs, including antibodies, antigens, and small molecules. We found in a parallel study that this was also true for aptamers as capture molecules (data not shown). The degree of increase in binding kinetics was found to be different for different pairs of molecules because of their diverse intrinsic reaction rates and varied surface molar concentrations. The results in Figure 2A also demonstrated that the use of the filtration assay was more beneficial for spots with high surface concentration of capture molecules, which is often required to improve detection sensitivity when the analyte concentration is low, and to prevent premature saturation when the analyte is abundant. To further illustrate the accelerated binding reaction using filtration-based microarrays, the kinetics of filtration and shaking assays were compared in Figure 2B for AHSA-HSA, ACEA-CEA, and GAM-MIgG pairs with 1.0 mg/mL of capture molecules. The solid curves represent the fluorescence intensity of the spots as a function of time for the filtration assay, and dashed curves are (26) Zubritsky, E. Anal. Chem. 2000, 72, 761A-767A.

Figure 2. The comparison of binding kinetics of filtration-based and shaking-based microarrays. (A) Fluorescence intensity indicating the binding of five different analyte proteins to their corresponding capture molecules with two to three different concentrations after 60 min of reaction. The error bars represent the standard deviation of the signal intensity. (B) Binding kinetics of AHSA-HSA, ACEA-CEA, and GAM-MGG resulting from filtration and shaking assays for 60 min.

that for the shaking assay. Clearly, binding between analytes and capture molecules was much faster in the filtration assay than in the shaking assay. Using a nonlinear regression software (Prism 3.0), the apparent reaction rate constant in filtration assays was determined to be 1.6 ( 0.1 × 10-3, 4.39 ( 0.44 × 10-4, and 4.35 ( 0.38 × 10-4 s-1 for AHSA-HSA, ACEA-CEA, and GAM-MIgG respectively, whereas the kinetic rate constants for shaking assays could not be reliably determined. On the basis of a theoretical analysis (Xu and Bao, unpublished results), for a microarray assay that involves very low concentration of analytes and is not limited by transport, its apparent kinetic rate constant is determined by the intrinsic off-rate constant of the capture molecule-analyte pair, which is often in the range of ∼10-5 to 10-3 s-1. The apparent kinetic rate constants obtained from the experiment are within this range and, thus, indicate that the filtration-based assay is capable of completely eliminating the diffusion limit. On the basis of the intensity ratios, we found that the filtration assay was ∼10 times faster, as compared with the shaking assay. We also found that in some cases, it took 12 h for the shaking assay to reach the same signal intensity as that with 1 h of filtration (data not shown). Dynamic Ranges and Detection Limit. Our results indicated that the filtration-based protein microarray assay has broader Analytical Chemistry, Vol. 75, No. 20, October 15, 2003

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Figure 3. The dynamic range of filtration-based protein microarrays, shown as solid lines, in comparison with that of shaking-based microarrays, shown as dashed lines, for reaction times of 30 and 60 min.

dynamic ranges owing to its ability to detect low-concentration proteins within a short period. As illustrated in Figure 3, for a microarray with 1.0 mg/mL of ACEA as the capture molecule, the filtration assay had a linear response to CEA concentrations over 4 orders of magnitude after a 30-min reaction, whereas the shaking assay had a linear dynamic range over a little more than 2 orders of magnitude. The increase in the dynamic range of the assay was primarily due to the improved detection sensitivity of low-concentration proteins. Further, after 1 h of reaction, the dynamic range of the filtration assay was similar to that of 30 min; the dynamic range of the shaking assay was improved but still narrower than that of the filtration assay. This indicates that the filtration assay could reach the equilibrium state faster, reducing the experimental errors resulting from incomplete reaction. Other capture molecule/analyte pairs showed similar trends (data not shown). Using the filtration assay with the Fuji Imager, all five types of capture molecules could detect ∼50 pg/mL of directly labeled analytes within 30 min. It was also found that for certain samples diluted 5-10-fold prior to the filtration assay, the assay sensitivity was still higher than that of the incubation approach with the undiluted samples. The low detection limit of the filtration-based microarray assay is attributed to the efficient capture of analytes. We found that in certain assays, most analyte molecules were captured by the microarray, indicating that these assays are primarily mass-sensing instead of concentration-sensing. However, it is noteworthy that the signal-to-background ratio of a mass-sensing assay could be reduced by the relatively low fractional occupancy of specific analytes and by high nonspecific binding due to the large number of capture molecules. Therefore, the number of spots for a specific analyte molecule and the number of capture molecules at each spot should be optimized for the best assay sensitivity and specificity. In particular, it may be helpful to form small spots with a moderate number of capture molecules so that the surface concentration of the capture reagent is sufficient, yet the total number of capture molecules is not too high to deplete the analytes. Improved Specificity. We also found that the filtration-based approach can improve the specificity of protein microarray assays. 5348

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When the shaking assay lasted long enough (e.g., 12 h), the signal level could reach that of the filtration assay for 1 h; however, the cross-reactions of certain capture molecules with mismatched analytes were found to increase significantly. It was reported that cross-reactivity between proteins could reduce the specificity of microarray studies.1,27 When a microarray assay is diffusionlimited, cross-reactions can become more problematic, since both specific and nonspecific reactions will assume similar kinetics, which is determined by the diffusion rates of the analytes. For a microarray containing spots of AHSA, ACEA, GAM, and protein G′ with 1 mg/mL concentration, the binding of 30 pM MIgG to GAM and protein G′ in a 60-min filtration assay was highly specific, with essentially no detectable cross-reaction with AHSA and ACEA. The corresponding signal as a result of a 60-min shaking assay was much weaker, only ∼20% of the signal intensity of the filtration assay. After shaking overnight, the signal level of MIgG at GAM spots became comparable to that of the 60-min filtration assay; however, the nonspecific cross-reactions of MIgG to AHSA and ACEA were increased by more than 8-fold as compared with the filtration assay. This shows that filtration-assisted binding could improve the assay specificity by effectively accelerating the specific reaction. Therefore, the filtration-based microarray assays reflect more accurately the intrinsic specificity of the protein pairs, which is critical for both protein profiling and protein interaction studies. Multistacking Filtration Assay. The potential application of our technique for high-throughput studies was demonstrated using a novel multistacking filtration approach, that is, multiple chips are stacked together and filtered simultaneously with a sample. This approach also allows robust control of the reaction consistency of all the stacked chips, which is a unique feature that is different from all other flow-through techniques and protein microarrays. To determine the consistency of reaction through the stacked chips, eight identical microarrays were stacked together; each was formed with 1 mg/mL of AHSA, ACEA, GAM, and Ca-biotin spotted quadruplet on a nitrocellulose filter membrane. A total of 480 µL of the sample (a mixture of Alexa647-labeled HSA, CEA, MIgG, and NeutrAvidin, each with 30 pM concentration) was used in the assay, with a net filtration volume of 300 µL and a filtration rate of 0.5 cm/s. For comparison purposes, individual chips containing microarrays identical to that described above were assayed with 60 µL of the same sample under shaking at 200 rpm. The fluorescence intensities of the analytes on the eight microarray chips are displayed in Figure 4A, with the error bar on each column indicating the variation of signal level of the quadruplet spots on the same microarray. The average array-to-array variation (7-10%) is comparable to the spot-to-spot variations (5-15%) on each chip, and the fluorescence intensity was found to be independent of the chip location in the stack. No cross-contamination between adjacent chips was observed. This demonstrated that the multistacking assay can eliminate most of the chip-to-chip variation induced by both printing and reaction processes. In contrast, the signal resulting from the shaking assay using individual chips was not detectable after 30 min of reaction. We further demonstrated that using multistacking chips, a given analyte could be selectively detected by its corresponding capture molecule spotted on any of the stacked chips. Therefore, (27) Mitchell, P. Nat. Biotechnol. 2002, 20, 225-229.

Figure 4. Multistacking filtration assay. (A) Fluorescence intensity due to binding of a mixed sample through eight identical chips. The error bars indicate the on-chip variations of signals. (B) The selective detection of HSA-Alexa546 and CEA-Alexa547 by the multistacking filtration technique. Only rows 16-40 are shown. Left: the positive chip. Right: the third negative chip from the top. The Alexa488, Alexa546, and Alexa647 channels reflect the detection of MIgGAlexa488, HSA-Alexa546, and CEA-Alexa647, respectively. Rows 39 and 40 of the images in the Alexa647 channel are BSA-Alexa647 as a position marker. Although both the positive and negative chips captured MIgG-A488, only the positive chip detected HSA-Alexa546 and CEA-Alexa647.

many different capture molecules can be used in the multistacking microarrays to simultaneously detect their corresponding analytes. Two types of microarrays were used in this experiment, referred to as the positive and negative microarrays. Both were printed on nitrocellulose filters with 25-mm diameter. The positive chip contained a microarray that was formed by 40 × 40 spots. Rows 1-34 of the positive chip consisted of spots with 2.5 ng of GAM per spot. The left 20 spots and the right 20 spots of row 35 had 2.5 ng and 1.25 ng of ACEA per spot, respectively. In row 36, the left 20 and right 20 spots contained 2.5 ng and 1.25 ng of AHSA per spot, respectively. Rows 37 and 38 were formed by blank spots. Rows 39 and 40 were spots of BSA-Alexa647 as standard, with 1.25 pg and 2.5 pg per spot, respectively. The negative chip was

identical to the positive chip, except that rows 35 and 36 on the negative chip were blank spots. One positive chip was sandwiched by 13 negative chips, with 7 above and 6 blow. These 14 chips contained a total of 22 400 spots. The stacked chips were then assayed with a mixture of three analytes, including 200 ng/mL of MGG-Alexa488, 10 ng/mL of HSA-Alexa546, and 10 ng/mL of CEA-Alexa647, and the binding was monitored in Alexa488, Alexa546, and Alexa647 channels simultaneously. The total sample volume was 1.7 mL, or 30 µL of sample per cm2 of the chip area. As a control, single-microarray binding assays using filtration were also performed. Figure 4B shows the fluorescence images of rows 16-40 in the positive microarray and the third negative microarray from the top after 15 min of reaction. Images in the Alexa546 and Alexa647 channels indicated that anti-HSA and anti-CEA on the positive microarray selectively captured the HSA and CEA molecules in the sample, respectively, whereas the negative microarray did not show any signal. In the Alexa488 channel, only the captured MIgG-Alexa488 can be observed, and the GAM spots had similar intensities for both microarrays, confirming that both microarrays had equal reaction efficiency. The signal level of AHSA and ACEA spots was comparable to the results obtained in the individual microarray filtration assay (data not shown). Therefore, the specificity and binding kinetics of the multistacking filtration assay are similar to those of the single-chip filtration assay, but it has a much higher throughput and requires much less sample volume per chip. With smaller spots, a larger chip diameter and chip number in the stack, the assay throughput can be further improved. This feature enables quantitative proteomic studies in one assay. It may be advantageous to use the multistacking filtration approach in microarray assays with indirect detection, such as probing of captured analytes with secondary antibodies or other detection reagents, and the detection of posttranslational modifications (such as phosphorylation) on captured proteins. These assays typically involve two steps: the capturing of the target proteins from the sample (capture step) and the detection of the captured proteins or certain groups on them (detection step). The multistacking filtration assay can be used in the capture step to ensure a high reaction efficiency and uniformity throughout the multistacking chips. In the detection step, each individual chip, referred to as a subarray, can be probed with different groups of detection antibodies using single-chip assays. For subarrays that bind different groups of analytes, this approach can reduce crossreactions in sandwich assays28 as well as lower the detection reagent consumption. The multistacking filtration approach can also be combined with the detection step of an indirect assay. For example, to compare a patient sample with a control sample, one chip is needed for each sample. Using the multistacking approach in the detection step, the two chips can be probed together so that the experimental error and the consumption of detection reagents in the secondary detection can be reduced. Assays with Large Dye Conjugates. Directly labeling analytes with fluorescent dyes is currently the dominant detection method in microarray techniques.27 However, organic dyes are often less ideal because of their intensity, spectrum, and other fluorescence properties. Both phycobilisome and quantum dots (28) Schweitzer, B.; Roberts, S.; Grimwade, B.; Shao, W.; Wang, M.; Fu, Q.; Shu, Q.; Laroche, I.; Zhou, Z.; Tchernev, V. T.; Christiansen, J.; Velleca, M.; Kingsmore, S. F. Nat. Biotechnol. 2002, 20, 359-65.

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Figure 5. The comparison of fluorescence intensity resulting from filtration and shaking assays when streptavidin-PB1L and streptavidin-Alexa647 were reacted with arrays of BSA-biotin-DNP for 30 min.

are promising substitutes for the conventional organic dye molecules. Phycobilisomes are supramolecular clusters of highly fluorescent phycobiliproteins with a molecular weight of ∼15 MDa; they were reported to be 300 times brighter than FITC.29 The semiconductor nanocrystal quantum dots (QD) have high extinction coefficients and can facilitate color muliplexity.30 However, because of their large sizes, the binding reaction involving phycobilisome or QD conjugates can be severely limited by diffusion. We found that the filtration-based microarray can overcome this difficulty so that the advantages of these novel fluorescent reagents can be fully exploited. To demonstrate the detection of proteins directly conjugated with P1L and quantum dots, we printed biotinylated BSA (BSAbiotin-DNP) on nitrocellulose filters, which were subsequently assayed with streptavidin-phycobilisome conjugates SensiLight P1L (2.5 streptavidin molecules per phycobilisome, from Martek Bio, Inc.) or NeutrAvidin-QD585 conjugates (maximal emission at 585 nm), using both filtration and shaking assays. Alexa647labeled streptavidin (100 ng/mL) (Sigma) (1:1 labeled, its molar concentration is the same as that of streptavidin in 10 µg/mL P1L) was also assayed to represent the binding of organic-dye-labeled protein. The results shown in Figure 5 indicated that, with the filtration assay, the binding of the phycobilisome cluster was significantly improved. We found that the filtration assay with P1L conjugates had the highest sensitivity, followed by the P1L shaking assay, the streptavidin-Alexa647 filtration assay and the streptavidin-Alexa647 shaking assay. Since P1L is typically used in indirect detection assays as a secondary fluorescent reagent, we evaluated the assay performance by comparing the minimal amount of BSA-biotin-DNP that was detectable on the chip. We found that the P1L filtration assay can detect ∼2 pg/spot of BSAbiotin-DNP on the surface, which is ∼10 times more sensitive than the shaking assay using P1L and 500 times more sensitive than the shaking assay using Alexa647-labeled streptavidin. The filtration-based protein microarray also accelerated the binding of QD conjugates. For 1 ng BSA-biotin-DNP spots assayed with (29) Morseman, J. P.; Moss, M. W.; Zoha, S. J.; Allnutt, F. C. Biotechniques 1999, 26, 559-63. (30) Chan, W. C.; Nie, S. Science 1998, 281, 2016-2018.

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QD585-conjugated NeutrAvidin (∼500 ng/mL) for 30 min, the filtration assay gave a fluorescence signal 4 times higher than that of the shaking assay. Therefore, the filtration assay can facilitate the applications of QD to microarray studies with higher sensitivity and better color multiplexing. It was further demonstrated that by combining the filtration assay with the usage of P1L, picograms per milliliter of biotinylated CEA could be detected using an ACEA microarray and P1L conjugated with streptavidin. This sensitivity is almost comparable to the RCA assay,28,31 but the filtration assay is simpler and faster, and it consumes less secondary detection reagent. With a more suitable imaging instrument, the detection sensitivity can potentially be further improved. Filtration-Based Microarray Sandwich Assay for the Detection of CEA. To demonstrate the clinical applications of filtration-based microarrays, we performed a sandwich assay using human blood plasma, a highly heterogeneous sample. Compared to the direct assay using one antibody with direct labeling of the analyte, a sandwich immunoassay uses two antibodies, thus having higher specificity and avoiding the difficulty in labeling proteins quantitatively. We selected CEA in human plasma as the target molecule for detection, and HSA as an internal control. A smallscale microarray was created using four different antibodies (triplicate for each concentration of each antibody): ACEAh, with concentrations of 1.0 and 0.4 mg/mL; ACEAl, 1.0 mg/mL; polyclonal ACEA, 1.0 mg/mL; and AHSA, 1.0 mg/mL. The secondary detection antibodies for CEA and HSA were Alexa647conjugated ACEAl- and Alexa647-labeled monoclonal anti-HSA (Clone11, from Sigma), with concentrations of 1.0 and 1.5 µg/ mL, respectively. Using the filtration-based assay to detect purified CEA that was added in a plasma sample, this microarray could only detect 10 ng/mL of CEA in a total protein concentration of 10 µg/mL (equivalent to about 80 µg/mL of CEA in total blood), whereas the shaking assay failed to distinguish CEA-positive and CEA-negative samples. Using the sandwich assay, the complementary antibody pair formed by ACEAh and ACEAl detected 5 ng/mL of CEA that was added to a healthy individual’s plasma (with 10-fold dilution in TBS containing 2% BSA and 0.2% Tween20 before detection). The polyclonal ACEA spots showed slightly lower sensitivity. As expected, ACEAl as the capture molecule could not detect any CEA analytes because of its epitope competition with the detector. Figure 6A shows an example of images of ACEAh and AHSA spots after the assay. Following quantification of the fluorescence intensity, the signals of the ACEAh spots were normalized by that of AHSA spots, and the results are displayed in Figure 6B. It can be seen that adding 5 ng/mL of CEA induced a significant increase in the fluorescence signal. Estimated by an unpaired t-test, the results obtained from all the spots showed that the difference between the two samples was statistically significant. The shaking-based sandwich assay did not give good results, since the signals from both samples were statistically similar (data not shown). We have also successfully detected CEA in the plasma of pancreatic cancer patients by using the filtration-based microarray sandwich assay (data not shown). It should be noted, however, that the sensitivity of the assay could be compromised by nonspecific signals as a result of (31) Schweitzer, B.; Schweitzer, B.; Wiltshire, S.; Lambert, J.; O’Malley, S.; Kukanskis, K.; Zhu, Z.; Kingsmore, S. F.; Lizardi, P. M.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10113-10119.

that the fraction of specific binding at each spot was reduced by the sharing of limited CEA analytes among these spots. This could be mitigated by avoiding unnecessary spots and optimizing the amount of immobilized antibodies at each spot. In summary, we developed a novel protein microarray technique based on a filtration-assisted reaction. This new protein microarray platform is particularly attractive for protein expression profiling studies in which a sample contains low-abundance target proteins. In high-throughput studies of protein-protein interactions, the filtration-based microarrays can achieve better specificity and reflect more accurately the relative binding affinity than the current shaking-based approach. This new protein microarray approach can be easily extended to peptide microarrays, aptamer microarrays, and other small-molecular microarrays for disease detection and diagnosis, drug discovery, and other proteomic studies of biology and human health. ACKNOWLEDGMENT Figure 6. The detection of CEA in human plasma samples. (A) Top row: the results of CEA detection using plasma sample from a healthy individual. Bottom row: the results using the same plasma sample with 5 ng/mL of CEA added. The triplet spot groups were formed by, respectively, (i) 1.0 mg/mL of ACEAh, (ii) 0.4 mg/mL of ACEAh, and (iii) 0.4 mg/mL of AHSA. (B) Fluorescence intensity of spots formed by 1.0 mg/mL of ACEAh, normalized by the HSA signal intensity in filtration assay. Scale bar ) 500 µm.

the low intrinsic specificity of antibodies. Furthermore, since a total of 12 antibody spots were printed on each chip, it is possible

This work was supported in part by NSF (BES-0222211) and by a Seed Grant from the Institute for Bioengineering and Bioscience at Georgia Institute of Technology. We thank Shuming Nie for providing quantum dots and Lily Yang for providing human plasma samples for the CEA detection assay.

Received for review June 5, 2003. Accepted July 31, 2003. AC034613G

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