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Immobilization Strategy and Characterization of Hydrogel-Based Thin Films for Interrogation of Ligand Binding with Staphylococcal Enterotoxin B (SEB) in a Protein Microarray Format Paul T. Charles,* Chris R. Taitt, Ellen R. Goldman, Jermain G. Rangasammy, and David A. Stenger U.S. Naval Research Laboratory, Center for Bio/Molecular Science & Engineering (Code 6910), 4555 Overlook Avenue SW, Washington, D.C. 20375 Received June 10, 2003
Introduction The progression of protein microarrays has given researchers a promising method to interrogate proteinprotein interactions, molecular expression profiles, and protein function.1-5 Microfabrication of protein microarrays on planar surfaces has been accomplished by a plethora of chemical modification strategies that range in diversity and complexity.6-16 However, concerns still remain regarding protein denaturation upon immobilization, the stringency of cross-linking conditions, and the hydration state of the protein when immobilized on a planar surface.2,15 In recent years, hydrogels or polyacrylamide gel pads have been used as a potential substrate for the development of protein microarrays.17-22 These three-dimensional * To whom correspondence should be addressed. E-mail: ptc@ cbmse.nrl.navy.mil. Tel: 202-404-6064. Fax: 202-404-8897. (1) MacBeath, G.; Schreiber, S. Science 2000, 289, 1760-1763. (2) Haab, B. B.; Dunham, M. J.; Brown, P. O. Gen. Biol. 2001, 2, 1-13. (3) MacBeath, G.; Koehler, A. N.; Schreiber, S. J. Am. Chem. Soc. 1999, 121, 7967-7968. (4) Lueking, A.; Horn, M.; Eickhoff, H.; Buessow, K.; Lehrach, H.; Walter, G. Anal. Biochem. 1999, 270, 103-111. (5) DeWildt, R. M. T.; Mundy, C. R.; Gorick, B. D.; Tomlinson, I. M. Nat. Biotechnol. 2000, 18, 989-994. (6) Anderson, N. L.; Matheson, A. D.; Steiner, S. Proteomic: Curr. Opin. Biotechnol. 2000, 11, 408-412. (7) Cahill, D. J. J. Immunol. Methods 2001, 250, 81-91. (8) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2001, 5, 40-45. (9) Emil, A. Q.; Cagney, G. Nat. Biotechnol. 2000, 18, 393-397. (10) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 4995, 767-773. (11) Mooney, J. F.; Hunt, A. J.; McIntosh, J. R.; Liberko, C. A.; Walba, D. M.; Rogers, C. T. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 1228712291. (12) Schena, M.; Shalon, D.; Heller, R.; Chai, A.; Brown, P. O.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10614. (13) Benters, R.; Niemeyer, C. M.; Wohrle, D. ChemBioChem 2001, 2, 686-694. (14) Fang, Y.; Frutos, A. G.; Lahiri, J. J. Am. Chem. Soc. 2002, 124, 2394. (15) Huang, R.-P. J. Immunol. Methods 2001, 255, 1-13. (16) Wang, C. C.; Huang, R.-P.; Sommer, M.; Lisoukov, H.; Huang, R.; Lin, Y.; Miller, T.; Burke, J. J. Proteome Res. 2002, 1, 337-343. (17) Stevens, P. W.; Wang, C. H.; Kelso, D. M. Anal. Chem. 2003, 75, 1141-1146. (18) Revzin, A.; Russell, R. J.; Yadavalli, V. K.; Koh, W.-G.; Deister, C.; Hile, D. D.; Mellott, M. B.; Pishko, M. V. Langmuir 2001, 17, 54405447. (19) Guschin, D.; Yershov, G.; Zaslavsky, A.; Gemmell, A.; Shick, V.; Proudnikov, D.; Arenkov, P.; Mirzabekov, A. Anal. Biochem. 1997, 250, 203-211. (20) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.; Mirzabekov, A. Anal. Biochem. 2000, 278, 123-131. (21) Parinov, S.; Barsky, V.; Yershov, G.; Kirillov, E.; Timofeev, E.; Belgovskiy; Mirzabekov, A. Nucleic Acid Res. 1996, 4, 2998-3004. (22) Angenendt, P.; Glokler, J.; Murphy, D.; Lehrach, H.; Cahill, D. J. Anal. Biochem. 2002, 309, 253-260.
10.1021/la0350195
(3-D) gel platforms offer a number of advantages: (1) an increased capacity for the immobilization of proteins compared to traditional glass surfaces, (2) an environment conducive for covalent attachment of proteins, (3) a stable support for ligand binding to occur, and (4) minimal background fluorescence. In particular, the 3-D gel mimics a solution-phase environment that provides free range of motion for the biomolecule, promoting increased interaction of the target ligand in contrast to the limited binding kinetics of a solid-liquid phase interaction evident on a glass surface. This paper describes the preparation and characterization of a 3-D hydrogel thin film that employed a novel chemical immobilization strategy for protein attachment in a microarray format. Three-dimensional protein microarrays were printed using a noncontact microdispensing system that eliminated contact with the hydrogel substrate and minimized the potential for cross-contamination. We used staphylococcal enterotoxin B (SEB) as the model protein and demonstrated antigenicity by a fluorescence-labeled anti-SEB antibody. We also demonstrated differences in ligand binding upon immobilization between the 3-D hydrogel and a two-dimensional (2-D) planar glass substrate chemically modified with identical cross-linking conditions. Experimental Section Materials. Acrylamide and bis-acrylamide monomers, potassium persulfate, and TEMED were purchased from Bio-Rad Laboratories (Hercules, CA). Silane coupling agents were received from Sigma-Aldrich-Fluka Chemical Corp. (Milwaukee, WI). The homobifunctional cross-linker, bis(sulfosuccinimidyl) suberate (BS3), was purchased from Pierce Chemical Co. (Rockford, IL). Ultrapure water (18 MΩ/cm) was obtained from a Milli-Q purification system (Millipore Corp., Bedford, MA). All other chemicals were of reagent grade quality or higher and were purchased from Sigma-Aldrich and Fisher Scientific, Inc. (Springfield, NJ). SEB antigen and the polyclonal sheep anti-SEB antibody were purchased from Toxin Technology, Inc. (Sarasota, FL). Cy5 mono-functional dye used for antibody labeling was purchased from Amersham Biosciences (Piscataway, NJ). Hydrogel Thin-Film Preparation/Homobifunctional Cross-Linking. Three-dimensional highly cross-linked polyacrylamide thin films were prepared on glass slides (2.5 cm × 7.5 cm) using the two monomers, acrylamide and bis-acrylamide. Two groups of acid-cleaned glass slides23 were silane treated with a 4% solution of 3-methacryloxypropyl trimethoxysilane (MTPTS) (group 1) in acidic-methanol and a 2% solution of dichlorodimethylsilane (DCDM) (group 2) dissolved in hexane following the protocol by Sanford et al.25 Utilizing the “flap technique”,24 a solution consisting of acrylamide (0.36 g, 5.1 mmol), bis-acrylamide (0.04 g, 0.26 mmol), potassium persulfate (2 mg, 7.4 µmol), and TEMED (3.0 µL, 20 µmol) dissolved in 1.0 mL of ultrapure H2O was deposited between the two silanetreated slides.25 Polymerization of the acrylamide mixture under nitrogen provided a 3-D hydrogel layer between the two silanetreated surfaces. After polymerization, the DCDM (hydrophobic)treated slide was separated from the MTPTS-treated slide revealing a thin film of highly cross-linked polyacrylamide possessing an amide moiety. Adherence of the polyacrylamide film to the MTPTS-treated surface was achieved with the vicinyl pendant group of the MTPTS surface in an inert (nitrogen) atmosphere during the free radical polymerization process. To control the thickness of the hydrogel, Teflon tape was used as (23) Chrisey, L. A.; Lee, G. U.; O’Ferrall, E. Nucleic Acid Res. 1996, 24, 3031-3039. (24) Radola, B. J. Electrophoresis 1980, 1, 43. (25) Sanford, M. S.; Charles, P. T.; Commisso, S. M.; Roberts, J. C.; Conrad, D. W. Chem. Mater. 1998, 10, 1510-1520.
This article not subject to U.S. Copyright. Published 2004 by the American Chemical Society Published on Web 12/03/2003
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Figure 1. Synthetic immobilization scheme using a 3-D hydrogel thin film cross-linked with bis(sulfosuccinimidyl) suberate (BS3) under acidic conditions (pH 6.0). Pendant NHSester reacts with the amide moiety within the internal network of the hydrogel film. The secondary NHS-ester group reacts with the primary amine on the protein, staphylococcal enterotoxin B (SEB), at neutral pH producing NHS as a side product for the formation of a stable bond between the protein and the hydrogel network. a spacer between the two silane-treated surfaces. Profilometry measurements were conducted for an estimated hydrogel thickness. The 3-D hydrogel film was allowed to incubate with the homobifunctional cross-linker, BS3, in 10 mM Na-phosphate buffer (pH 6.0) for 30 min. Slides were then rinsed with the phosphate buffer, allowed to air-dry, and then microarrayed with the SEB protein (Figure 1). Printing of SEB. Microarrayed elements of SEB were printed onto the BS3-modified 3-D hydrogel films and on the BS3-modified 2-D glass substrates using the noncontact piezotip dispensing Packard Biochip I Microarrayer (Meriden, CT). Each printed element was deposited in replicates of six with a print volume of 1.8 nL. Cy5-labeled SEB (0.1-100 µg/mL) was printed into the 3-D gel to serve as a standard curve for quantitative measurements. Microarrays were also printed with unlabeled SEB (0.03-100 µg/mL) for antigen-binding experiments with Cy5-labeled anti-SEB antibody. Cy5-Labeled Anti-SEB Antibody Binding Experiments. A series of experiments were performed that compared differences in ligand binding of an anti-SEB antibody to the immobilized SEB protein in the 3-D hydrogel film and the SEB immobilized on a 2-D glass surface. Two-dimensional glass surfaces were also functionalized with 3-aminopropylsilane, cross-linked with the homobifunctional cross-linker (BS3) with subsequent covalent attachment of the unlabeled-SEB biomolecule. Conditions for immobilization of SEB onto the 2-D substrates were optimized separately from the 3-D substrates. Unoccupied binding sites were blocked for 1 h with a 3% solution of bovine serum albumin (BSA)-casein prepared in PBS, pH 7.4. The patterned 2-D and 3-D surfaces were treated with Cy5-labeled anti-SEB antibody (15 µg/mL) prepared in PBS, pH 7.4, and allowed to incubate for 1 h at room temperature. All 3-D hydrogels and 2-D surfaces were post-washed with PBS-Tween 20 (0.1%) for 5 min to remove all noncovalently bound SEB. Fluorescence intensities were measured and recorded. Confocal Fluorescence Laser Scanning Microscopy. Confocal fluorescence laser scanning microscopy was conducted on both sets of 3-D microarrays using the Packard Biochip ScanArray Lite system (GSI Lumonics). Fluorescence intensities were measured with the QuantArray (GSI Lumonics) software package.
Results and Discussion A recent investigation using highly cross-linked 3-D hydrogels and a novel cross-linking strategy for the immobilization of SEB in a protein microarray format has been illustrated. Figure 2A illustrates the fluorescence signal measured from the printed microarray from immobilized Cy5-labeled SEB within the BS3-modified 3-D hydrogel. The mean spot diameter (six replicates per dilution) of the printed fluorescent protein was 419 ( 18 µm. The 3-D hydrogel thin films were transparent and provided reproducible films measured at a thickness
Figure 2. (A) Fluorescence microarray image depicting immobilized Cy5-labeled SEB in a 3-D hydrogel film (400 µm spot o.d.). The top row reflects six replicates of the immobilized SEB (100 µg/mL) with an 8-fold serial dilution shown in subsequent rows. The data represent means ( SD of six replicates. (B) Line plot of fluorescence intensity vs immobilized Cy5-SEB concentrations. The linear dynamic range was from 0.1 to 30 µg/ mL (r2 ) 0.997).
between 2 and 4 µm. As seen in Figure 2B, fluorescence signals showed a dose-dependent response as the concentration of SEB increased. Fluorescence signals reached saturated levels at immobilized Cy5-labeled SEB concentrations of 50 µg/mL (indicated by white; top row). Posttreatment of the 3-D microarrays with PBS-Tween 20 (0.01%) ensured the removal of all noncovalently bound Cy5-labeled SEB. Extrapolation of the experimental data with a linear plot (data not shown) produced a linear dynamic range from 0.1 to 30 µg/mL with a correlation of determination value (r2) calculated at 0.997. Calculated standard deviations (SDs) from the fluorescence intensity values at Cy5labeled SEB concentrations of >0.3 µg/mL were less than 5% with values as low as 1% at 100 µg/mL. Calculated SD values varied from 10% to 15% at SEB concentrations of 10 µg/mL. However, the fluorescence signals measured at immobilized SEB concentrations of
