Fabrication of Substrate-Independent Protein Microarrays Using

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Langmuir 2009, 25, 1403-1411

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Fabrication of Substrate-Independent Protein Microarrays Using Polyelectrolyte Scaffolding Scott D. Spillman, Helen M. McEvoy, and Brian D. MacCraith* Optical Sensors Laboratory, School of Physical Sciences, National Centre for Sensor Research, Dublin City UniVersity, GlasneVin, Dublin 9, Ireland ReceiVed July 1, 2008. ReVised Manuscript ReceiVed September 26, 2008 While significant advances have been made in regard to protein microarray surface chemistries, the surface modification strategies developed are generally substrate-specific and cannot be interchanged between different materials and platforms. This current lack of substrate-independent surface modification strategies makes it difficult to compare or transfer fabrication methodologies between dissimilar substrates. To address this shortcoming, we have developed an interchangeable surface scaffold, which can be utilized to fabricate protein microarrays on a variety of materials with nearly identical results. The surface scaffold is deposited by alternate electrostatic assembly of the cationic and anionic polyelectrolytes poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate), respectively. Once assembled, the polyelectrolyte scaffold serves to mask the underlying surface properties of different materials and convert them to the surface properties of the scaffold itself. By obtaining common surface properties across different materials, it is possible to eliminate differences in protein surface density, spot diameter, and nonspecific binding of analytes on substrates as diverse as glass, gold, mica, silicon, and polymer. The concept of substrate-independent polyelectrolyte scaffolding described here provides researchers with a powerful new tool that can be utilized to compare, optimize, and transfer useful protein microarray surface chemistries across different materials and platforms.

Introduction Protein microarrays have become an important tool for the determination of protein function, proteome analysis, and basic biological research.1-6 The development of microarray technology has made it possible to simultaneously screen for thousands of compounds in an extremely high-throughput and parallel fashion while tremendously reducing the amount of reagents and analysis time. Despite these significant advantages of microarray technology, there are still many limitations that must be overcome before protein microarrays can reliably be used in practical and clinical applications.7-9 Limitations of particular importance in regard to protein microarray development are a lack of common assay, analysis, and fabrication methodologies.10-12 While the former can be overcome by the gradual adoption of common protein microarray assay and analysis methods, the development of common fabrication techniques presents a more challenging obstacle due to the use of different substrate materials. Depending on the method of detection, platform design, and specific application, it is often required to fabricate protein * Towhomcorrespondenceshouldbeaddressed.E-mail:[email protected]. (1) MacBeath, G.; Schreiber, S. L. Science 2000, 289(5485), 1760–1763. (2) 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(5537), 2101–2105. (3) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2003, 7(1), 55–63. (4) MacBeath, G. Nat. Genet. 2002, 32, 526–532. (5) Templin, M. F.; Stoll, D.; Schwenk, J. M.; Potz, O.; Kramer, S.; Joos, T. O. Proteomics 2003, 3(11), 2155–2166. (6) Borrebaeck, C. A. K. Immunol. Today 2000, 21(8), 379–382. (7) Templin, M. F.; Stoll, D.; Schrenk, M.; Traub, P. C.; Vohringer, C. F.; Joos, T. O. Trends Biotechnol. 2002, 20(4), 160–166. (8) Liotta, L. A.; Espina, V.; Mehta, A. I.; Calvert, V.; Rosenblatt, K.; Geho, D.; Munson, P. J.; Young, L.; Wulfkuhle, J.; Petricoin, E. F. Cancer Cell 2003, 3(4), 317–325. (9) Angenendt, P. Drug DiscoVery Today 2005, 10(7), 503–511. (10) Hultschig, C.; Kreutzberger, J.; Seitz, H.; Konthur, Z.; Bussow, K.; Lehrach, H. Curr. Opin. Chem. Biol. 2006, 10(1), 4–10. (11) Russo, G.; Zegar, C.; Giordano, A. Oncogene 2003, 22(42), 6497–6507. (12) Gulmann, C.; Sheehan, K. M.; Kay, E. W.; Liotta, L. A.; Petricoin, E. F. J. Pathol. 2006, 208(5), 595–606.

microarrays on various materials. Different substrate materials exhibit unique surface properties, which must be taken into consideration when developing suitable fabrication techniques for protein microarray applications. Consequently, numerous surface modification strategies have been developed for glass,13-15 metal,16-18 polymer,19-21 and other materials. While these strategies have made it possible to successfully fabricate protein microarrays on a variety of materials, the surface modification methods used are generally substrate-specific and produce different results on dissimilar materials. The dissimilar surface chemistries between materials result in different protein spot diameters and immobilization densities during the microarray fabrication. Furthermore, these dissimilar surface chemistries generate different levels of nonspecific binding (NSB) during subsequent stages of the protein assay. Together, these dissimilar results obtained on different materials significantly complicate the ability to compare and optimize protein microarray methodologies. To address these substrate-dependent limitations of protein microarray development, we explored the use of substrateindependent surface coatings as a means to obtain equivalent results on dissimilar materials. Previously, The´venet et al. (13) Angenendt, P.; Glokler, J.; Murphy, D.; Lehrach, H.; Cahill, D. J. Anal. Biochem. 2002, 309(2), 253–260. (14) Angenendt, P.; Glokler, J.; Sobek, J.; Lehrach, H.; Cahill, D. J. J. Chromatogr., A 2003, 1009(1-2), 97–104. (15) Kusnezow, W.; Jacob, A.; Walijew, A.; Diehl, F.; Hoheisel, J. D. Proteomics 2003, 3(3), 254–264. (16) Lee, Y.; Lee, E. K.; Cho, Y. W.; Matsui, T.; Kang, I. C.; Kim, T. S.; Han, M. H. Proteomics 2003, 3(12), 2289–2304. (17) Pavlickova, P.; Knappik, A.; Kambhampati, D.; Ortigao, F.; Hug, H. Biotechniques 2003, 34(1), 124–130. (18) Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20(3), 270–274. (19) Henry, A. C.; Tutt, T. J.; Galloway, M.; Davidson, Y. Y.; McWhorter, C. S.; Soper, S. A.; McCarley, R. L. Anal. Chem. 2000, 72(21), 5331–5337. (20) Rucker, V. C.; Havenstrite, K. L.; Simmons, B. A.; Sickafoose, S. M.; Herr, A. E.; Shediac, R. Langmuir 2005, 21(17), 7621–7625. (21) Nahar, P.; Wali, N. M.; Gandhi, R. P. Anal. Biochem. 2001, 294(2), 148–153.

10.1021/la8020723 CCC: $40.75  2009 American Chemical Society Published on Web 12/23/2008

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successfully used reactive polymer coatings as a substrateindependent method to generate DNA patterns on a variety of materials using the same protocol.22 Here, the use of polyelectrolyte multilayers (PEMs) was employed as a substrateindependent surface scaffold for protein microarray fabrication. PEMs are layer-by-layer (LbL) assembled thin films which can be deposited or patterned on a variety of materials.23-25 The use of PEMs has been shown as a simple and effective method to modify surfaces for a wide range of applications such as superhydrophobicity,26 nanoparticle modification,27 metal deposition,28 antireflective coatings,29 microfluidics,30 and antibody microarrays.31,32 Traditionally, the mechanism of formation of PEMs is electrostatic self-assembly. However, other forces such as hydrophobic interactions,33 hydrogen-bonding,34,35 and covalent linkage36,37 can be utilized to initiate multilayer buildup on a broad range of materials, making it feasible to exploit PE scaffolding as a substrate-independent method to fabricate protein microarrays with identical results on any material. The work presented here demonstrates that a PE scaffold composed of alternating layers of poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) can easily be assembled on a variety of different materials (organic, inorganic, metallic, and semiconductor) without the need for any surface pretreatments. Additionally, surface characterization revealed that the PE scaffold serves to mask a substrate’s underlying surface properties and convert them into the surface properties of the scaffold itself. The resultant PE scaffold provides a uniform surface that can be further utilized to fabricate protein microarrays on a variety of materials with nearly identical results. The ability to perform multianalyte protein assays on the PE scaffold was demonstrated by fabricating microarrays of the cardiac markers C-reactive protein (CRP) and myoglobin. Using a PAH-capped PE-scaffold-coated glass substrate, different concentrations of CRP and myoglobin protein were spotted onto the surface and then washed with poly(acrylic acid) (PAC). The surface was then blocked with bovine serum albumin (BSA) and the printed antigens were detected with their respective fluorescently labeled antibody, resulting in specific signal intensities with low NSB. This experiment was followed by a protein microarray assay carried out on multiple substrates using mouse IgG and anti-mouse IgG as a model antigen/antibody pair, respectively. Here, microarrays were prepared on PE-scaffoldcoated glass, gold, mica, silicon, and polymer substrates by spotting different concentrations of mouse IgG onto the surfaces and detecting with fluorescently labeled anti-mouse IgG. Microarrays fabricated using the PE-scaffold-coated materials (22) The´venet, S.; Chen, H. Y.; Lahann, J.; Stellacci, F. AdV. Mater. 2007, 19(24), 4333. (23) Decher, G. Science 1997, 277(5330), 1232–1237. (24) Bertrand, P.; Jonas, A.; Laschewsky, A.; Legras, R. Macromol. Rapid Commun. 2000, 21(7), 319–348. (25) Hammond, P. T. AdV. Mater. 2004, 16(15), 1271–1293. (26) Bravo, J.; Zhai, L.; Wu, Z.; Cohen, R. E.; Rubner, M. F. Langmuir 2007, 23(13), 7293–7298. (27) Caruso, F. AdV. Mater. 2001, 13(1), 11. (28) Hendricks, T. R.; Lee, I. Thin Solid Films 2006, 515(4), 2347–2352. (29) Hiller, J.; Mendelsohn, J. D.; Rubner, M. F. Nat. Mater. 2002, 1(1), 59–63. (30) Sui, Z. J.; Schlenoff, J. B. Langmuir 2003, 19(19), 7829–7831. (31) Zhou, X. C.; Zhou, J. Z. Proteomics 2006, 6(5), 1415–1426. (32) Dai, J. H.; Baker, G. L.; Bruening, M. L. Anal. Chem. 2006, 78(1), 135– 140. (33) Park, J.; Hammond, P. T. Macromolecules 2005, 38(25), 10542–10550. (34) Cho, J.; Caruso, F. Macromolecules 2003, 36(8), 2845–2851. (35) Lutkenhaus, J. L.; Hrabak, K. D.; McEnnis, K.; Hammond, P. T. J. Am. Chem. Soc. 2005, 127(49), 17228–17234. (36) Richert, L.; Boulmedais, F.; Lavalle, P.; Mutterer, J.; Ferreux, E.; Decher, G.; Schaaf, P.; Voegel, J. C.; Picart, C. Biomacromolecules 2004, 5(2), 284–294. (37) Such, G. K.; Quinn, J. F.; Quinn, A.; Tjipto, E.; Caruso, F. J. Am. Chem. Soc. 2006, 128(29), 9318–9319.

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produced nearly identical results in terms of antigen spot sizes, immobilization densities, and levels of NSB. The PE scaffold described here delivers a highly functional surface capable of assembling compounds designed to promote protein immobilization (PAH) and then assembling compounds designed to reduce NSB (PAC). The PE scaffold supplies a basic foundation that allows for additional improvement upon the results obtained here through utilization of the LbL self-assembly process further on the scaffold to incorporate compounds designed to further improve protein microarray performance, hence the term PE scaffolding. The use of substrate-independent PE scaffolding provides researchers with a method to easily compare, optimize, and transfer useful surface chemistries between different materials, platforms, and applications, making it possible to streamline protein microarray fabrication.

Experimental Section Materials. Glass microscope slides were obtained from SigmaAldrich (Dublin, Ireland); mica substrates from SPI Supplies (West Chester, PA, USA); silicon wafers from Silicon Inc. (Boise, ID); and Zeonor cycloolefin polymer slides from Åmic (Uppsala, Sweden). Gold slides were prepared by electron-beam evaporation of gold onto glass slides using an Edwards Auto 306 high vacuum system (West Sussex, U.K.). Poly-L-lysine (PLL) (Mw ) 150K-300K), PAH (Mw ) 70K), PSS (Mw ) 70K), and PAC (Mw ) 100K) were obtained from Sigma-Aldrich. Mouse IgG (IgG), anti-mouse IgG (whole molecule) antibody (anti-IgG), and BSA were obtained from Sigma-Aldrich. CRP was obtained from Meridian Life Science, Inc. (Saco, ME). Anti-CRP labeled with DY647 fluorescent dye was obtained from Exbio (Prague, Czech Republic). Myoglobin and antimyoglobin were obtained from Hytest Ltd. (Turku, Finland). Cy5 monoreactive dye pack and PD-10 columns were obtained from GE Healthcare (Buckinghamshire, U.K.). DY547 NHS-ester was obtained from Dyomics GmbH (Jena, Germany). All other chemicals and reagents were obtained from Sigma-Aldrich. Fluorescent Dye Conjugation. Anti-IgG was conjugated with Cy5 according to the manufacturer’s instructions. Briefly, 1 mL of 0.1 M sodium carbonate buffer at pH 9.3 was added to 1 mg of anti-IgG to obtain a final concentration of 1 mg/mL. The anti-IgG solution was then added to a vial of Cy5 monoreactive dye, inverted several times for mixing, and allowed to react for 30 min in the dark at room temperature. Following the reaction, the Cy5-labeled antiIgG was separated from unconjugated dye and buffer exchanged into 1× phosphate buffered saline (PBS) at pH 7.4 using a PD-10 column according to the manufacturer’s instructions. This labeling procedure was also used for anti-myoglobin. PAH was labeled with DY547 fluorescent dye by dissolving 200 mg of PAH in 8 mL of water, giving a final concentration of 50 mg/mL PAH. This solution was then added to 100 mg of DY547 and allowed to react for 2 h. Unconjugated dye was separated from the DY547-labeled PAH by dialysis in water. Substrate Preparation. The substrate materials used for IgG microarray fabrication in this study were glass, gold, mica, silicon, and polymer. Each material was used unmodified (native), PLLcoated, oxygen-plasma-treated (OPT) then PLL-coated, and PEscaffold-coated. All materials were initially cleaned by rinsing with distilled water, then ethanol, and dried with a gentle steam of air. The OPT substrates were prepared by exposing each material to an oxygen plasma using an Oxford Instruments PlasmaLab 80Plus (Abindon, Oxfordshire, UK) at 5 sccm O2, 40 mTorr pressure, and 50 W power for 2 min. OPT materials were used to improve PLL deposition by increasing the negative charge density on the surface, especially for the hydrophobic polymer material. PLL was deposited onto both native and OPT materials by incubating in 0.01% (w/v) PLL in water for 10 min. Following incubation in the PLL solution, the materials were washed with five exchanges of water and dried with air. The PE scaffold deposition process is illustrated in Figure 1. The materials were first incubated in 2 mg/mL PAH in water (pH 7.4)

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Figure 1. Stepwise assembly of PEMs. A suitable material is first washed with H2O (1) and then incubated in a positively charged PE (2). The material is then washed again (3) and incubated in a negatively charged PE (4). Steps 1-4 are then repeated (5) until the desired number of layers is achieved (6).

Figure 2. Layout of the dual-analyte microarrays spotted in this study. The spots are separated by 500 µm, and concentrations are shown in nM.

with 0.1 M sodium chloride (NaCl) for 10 min, followed by washing in five exchanges of water to remove any unbound PAH. The PAHcoated materials were then incubated in 2 mg/mL PSS in PBS (pH 7.4) for 10 min, followed by another wash in five exchanges of water. This process was repeated until a multilayer composed of 5.5 bilayers of PAH and PSS, or (PAH/PSS)5.5, was assembled, with PAH being the outermost layer. The PAH-capped scaffold provides an amine-rich surface primed for further LbL self-assembly, covalent activation, or direct adsorption of biomolecules. Dual-Analyte Protein Microarray Fabrication. The CRP and myoglobin antigens were each diluted to 5000 nM in water. These solutions were then used to prepare 1:2 serial dilutions in water. Using a Scienion AG sciFLEXARRAYER piezo-dispenser (Dortmund, Germany), individual 500 pL droplets of each sample were spotted in replicates of five onto glass substrates coated with the PAH-capped PE scaffold. The print layout of the dual-analyte assay is shown in Figure 2. After spotting the antigens onto each material, the microarrays were washed in two exchanges of PBST (PBS, 0.5% Tween 20) containing 2 mg/mL PAC (pH 7.4) for 5 min each to remove any unbound antigens from the surface. The addition of PAC into the wash solution was found to eliminate the appearance of “comet tails” caused by streaking and NSB of unbound protein during the wash step (see Results and Discussion). After washing, the microarrays were rinsed briefly with five exchanges of water to remove any unbound PAC. The washed microarrays were then blocked in PBST containing 2% BSA for 30 min, followed by further washing in two exchanges of PBST for 5 min each to remove any unbound BSA. The blocked microarrays were then briefly rinsed with water to remove excess salt and Tween 20 present in the PBST buffer and dried with air. Dual-analyte assays were performed by exposing the fabricated microarrays to 20 nM DY647-labeled anti-CRP and 0.8 nM Cy5labeled anti-myoglobin in PBS containing 2% BSA for 1 h to allow the binding event to occur between the antigens and their respective antibodies. Cy5-labeled IgG, not specific to either antigen, was used as a negative control. After the 1 h exposure to the fluorescently

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Figure 3. Layout of the IgG microarrays spotted in this study. IgG spots are separated by 500 µm, and concentrations are shown in nM.

labeled antibodies, the microarrays were washed twice in PBST for 5 min, then briefly rinsed with PBS to remove Tween 20, and finally dried with air. Protein Microarray Fabrication on Multiple Substrates. To compare the performance of microarrays fabricated on different substrates coated with the PE scaffold, simple direct assays were performed using IgG as an antigen. Here, spotting solutions were prepared as 1:2 serial dilutions of IgG in water with the highest concentration being 1250 nM. The print layout of the IgG microarrays is illustrated in Figure 3. After spotting IgG onto each coated substrate, the microarrays were washed and blocked in the same manner used for the dual-analyte assay. Assays were performed on each substrate by exposing the microarrays to 20 nM Cy5-labeled anti-IgG in PBS, and further processed in the same manner as the dual-analyte assays. Data Acquisition and Analysis. The PE scaffold thickness on glass and polymer was obtained using phase-shifting interferometry (PSI) with a Wyko NT1100 optical profiling system (Swavesey, Cambridge, U.K.). LbL buildup of the PE scaffold on glass and polymer was characterized using fluorescently labeled PAH and imaging the surfaces with a Genetic Microsystems 418 fluorescence scanner. The surface properties of glass, gold, mica, silicon, and polymer were monitored throughout the PE scaffold assembly process via contact angle measurements using a FTÅ200 dynamic contact angle analyzer. Microarray fluorescence images were analyzed by quantifying the pixel intensity of each spot using the image analysis software Scanalyze (http://rana.lbl.gov/EisenSoftware.htm). Mean signal intensities and the associated standard deviations were calculated from five replicate spots. All data generated were plotted with Microcal Origin.

Results and Discussion Characterization of the PE Scaffold. Initial characterization of the PE scaffold was performed on glass and polymer (Zeonor). Prior to multilayer assembly, polydimethylsiloxane (PDMS) was used to create a defined step in order to show the PE multilayer buildup on the glass and polymer substrates. A PE scaffold composed of PAH(PSS/PAH)5 was then assembled onto the substrates according to the procedure described in the Experimental Section. Following deposition of the PE scaffold onto glass and polymer, the PDMS was removed and the surfaces were examined. The images obtained from the PSI measurement are shown in Figure 4, where the blue regions represent the substrate surface and the green regions represent the PE scaffold surface. The spike at the step of the PE scaffold on the glass substrate was caused by the removal of PDMS. The measured PE scaffold thickness was similar for both substrates: 8.35 ( 0.73 nm on glass and 8.66 ( 2.33 nm on polymer. LbL buildup of the PE scaffold on glass and polymer was also characterized by measuring the fluorescence intensity increase

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Figure 6. Water contact angles measured throughout the PE scaffold deposition process. Solid lines represent native materials, and dotted lines represent OPT materials.

Figure 4. Images of the PE scaffold acquired via phase-shifting interferometry. The blue area represents the substrate surface, and the green area represents the PE scaffold surface. The PE scaffold thickness was measured to be 8.35 ( 0.73 nm on glass and 8.66 ( 2.33 nm on polymer.

Figure 5. Quantified signal intensities as a function of number of bilayers of PSS/DY547-labeled PAH on both glass and polymer.

using DY547-labeled PAH throughout the multilayer assembly process. First, a primer layer composed of PAH(PSS/PAH)2 was deposited onto untreated glass and polymer substrates to promote a more uniform adhesion of the DY547-labeled PAH. Following deposition of the primer layer, bilayers of PSS/DY547-labeled PAH were assembled. The fluorescence was measured over a 0.75 × 1.50 cm2 area on both the glass and polymer substrate following each DY547-labeled PAH layer deposited. The quantified fluorescence signals obtained from this analysis are shown in Figure 5. The quantified signal intensities plotted in Figure 5 show highly uniform deposition of DY547-labeled PAH on both glass and polymer, as indicated by the low standard deviation bars. The glass substrate also shows a linear increase in fluorescence signal, evidence that the thickness and density of each successive layer of DY547-labeled PAH deposited are equivalent throughout the assembly process. The polymer substrate initially shows a nonlinear increase in fluorescence signal but becomes linear after

the deposition of three PSS/DY547-labeled PAH bilayers. Additionally, the rate of fluorescence increase on polymer becomes nearly identical to that of glass after three bilayers of PSS/DY547-labeled PAH have been deposited. This is apparent from the equivalent slopes obtained for each material following the third bilayer, as shown in Figure 5. The equivalent rate of fluorescence signal increase on glass and polymer demonstrates that the DY547-labeled PAH assembles onto the surface of both materials at equal thicknesses and densities, indicating that the surface properties of the glass and polymer substrates become more alike as the PE scaffold is deposited. The ability to convert and obtain equivalent surface properties on different materials was further verified by monitoring the surface energies throughout PEM assembly on native and oxygenplasma-treated (OPT) glass, gold, mica, silicon, and polymer substrates using water contact angle measurements. Following deposition of each PE monolayer, the substrates were washed with five exchanges of water to remove any unbound PE from the surface and dried with a gentle stream of air. The water contact angle was then recorded for each material. This process was repeated for each successive PE layer deposited. Figure 6 shows the contact angles recorded throughout the PE scaffold assembly process. As the number of deposited PE layers increases, the contact angles of each surface gradually converge irrespective of the substrate, indicating that multilayer buildup occurs on all the materials tested. After the deposition of five bilayers, the contact angles of all materials are nearly identical, indicating that the surface properties of the underlying materials are completely masked and converted into the surface properties of the PE scaffold itself. At this point, the materials are ready for protein microarray fabrication and assays. Normalization of Intrinsic Background Fluorescence. Initially, the intrinsic background fluorescence of each material was recorded at an excitation wavelength of 635 nm using the microarray scanner at a maximum gain setting of 100%. At this gain setting, however, there is a large variation in intrinsic background fluorescence across the various materials, as shown in Figure 7. Additionally, materials with higher intrinsic background fluorescence resulted in saturated signal intensities at a gain setting of 100%, thereby preventing the ability to perform a simple background subtraction for accurate analysis across all materials. Therefore, prior to protein microarray assays, the intrinsic background fluorescence was normalized for each

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Figure 8. Microarray fluorescence images showing the effect of PAC in the wash solution. IgG was spotted onto PE-scaffold-coated glass and washed with either PBST or PBST with 2 mg/mL PAC. The fluorescence images show significant reduction of NSB of IgG caused by streaking when PAC is introduced into the wash solution.

Figure 7. Normalization of the intrinsic background fluorescence for each material. Each material was normalized relative to mica, which produced the lowest intrinsic background fluorescence. (A) Table showing the maximum and normalized gain settings used. (B) Quantified signal intensities of the intrinsic background fluorescence for each material at the maximum and normalized gain settings.

material to enable accurate analysis. Normalization was accomplished by manually adjusting the scanner gain in order to obtain equivalent intrinsic fluorescence signals for all materials. The gain for each material was adjusted relative to the mica substrate, which produced the lowest level of intrinsic background fluorescence at 100% gain. The normalized gain settings and the quantified intrinsic background fluorescence intensities for each material are shown in panels A and B, respectively, in Figure 7. Effect of PAC in the Wash Solution. PAC, a negative PE, is capable of electrostatic self-assembly onto positively charged surfaces such as PAH-coated substrates, and it has been shown to be effective at reducing NSB of proteins.32,38 Here, we investigated the effect of PAC in the microarray wash solution on the appearance of “comet tails” caused by streaking and NSB of the spotted IgG. IgG microarrays spotted onto PE-scaffoldcoated glass were washed with either PBST or PBST containing 2 mg/mL PAC, and direct assays were performed using Cy5labeled anti-IgG according to the procedure described in the Experimental Section. Figure 8 shows the fluorescence images acquired with the microarray scanner at the maximum gain setting of 100% to visualize the NSB. Microarrays washed in PBST alone show significant streaking caused by unbound IgG desorbing from the surface and then (38) Laib, S.; MacCraith, B. D. Anal. Chem. 2007, 79(16), 6264–6270.

readsorbing near the spot vicinity. The addition of PAC to the PBST wash solution, however, completely eliminated streaking of the IgG spots. Streaking of the IgG spots is highly reduced due to PAC rapidly adsorbing to the PAH surface, providing a barrier which significantly reduces the readsorption of unbound IgG onto the surface. Dual-Analyte Microarray Assays and Imaging. Dual-analyte assays were performed using CRP and myoglobin arrays printed onto PE-scaffold-coated glass slides as described in the Experimental Section. Printed microarrays were exposed to solutions containing 20 nM DY647-labeled anti-CRP, 0.8 nM Cy5-labeled anti-myoglobin, or a 1:1 mixture of both. Additionally, a blank solution containing no fluorescently labeled IgG and a solution containing 20 nM Cy5-labeled anti-IgG were used as controls. Following the assay, the microarrays were scanned. The acquired images from the dual-analyte assay are shown in Figure 9. These images were used to generate mean signal intensities and standard deviations of the dual-analyte assay. Dual-Analyte Assay Analysis. Three fluorescently labeled antibody solutions were used to evaluate the NSB and specificity of the dual-analyte assay. The fluorescence signal due to NSB for each detection solution was quantified on an area near the printed microarrays and compared to the signal obtained for the specific binding of fluorescently labeled anti-myoglobin to the 5 µM myoglobin spots. As shown in Figure 10, the fluorescence intensities obtained showed very low levels of NSB on the PEM scaffold surface for all the fluorescently labeled antibody detection solutions when compared to the microarray exposed to a blank sample containing no fluorescently labeled antibodies. Additionally, the fluorescence signal obtained on the 5000 nM myoglobin spots probed with 20 nM DY647-labeled anti-CRP and 0.8 nM Cy5-labeled anti-myoglobin is included in Figure 10 in order to shown the analyte signal intensity relative to the NSB signal intensity. The signal intensities obtained from the dual-analyte microarray exposed to both anti-myoglobin and anti-CRP are shown in Figure 11. DY647-labeled anti-CRP and Cy5-labeled anti-myoglobin showed only specific interactions, producing a signal only on arrays printed with their respective antigen. Additionally, both antigens produced concentration-dependent signal intensities, verifying the ability to fabricate multianalyte arrays with low NSB using the PE scaffold.

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Figure 10. Quantified background signal due to NSB of fluorescently labeled antibody detection solutions. The detection solutions contained either 20 nM DY647-labeled anti-CRP or 0.8 nM Cy5-labeled antimyoglobin. The fluorescence obtained on the 5000 nM myoglobin spots probed with both 20 nM DY647-labeled anti-CRP and 0.8 nM Cy5labeled anti-myoglobin is included in order to show the analyte signal intensity relative to the NSB signal intensity.

Figure 9. Dual-antigen protein microarray fluorescence images. Microarray fabrication and dual-analyte assays were performed as described in the Experimental Section using fluorescently labeled detection antibodies. The detection antibodies used for each printed microarray are shown to the left of each image.

Protein Microarray Assays and Imaging on Multiple Substrates. IgG assays were carried out on PE-scaffold-coated materials and compared against native, PLL-coated, and OPT PLL-coated materials. The substrate materials tested were glass, gold, mica, silicon, and polymer. Following microarray fabrication and assays as described in the Experimental Section, each substrate was scanned at both the maximum and normalized gain settings. The microarray images acquired are shown in Figure 12. These images were used to generate mean signal intensities and standard deviations of the IgG assay on multiple materials.

Figure 11. Quantified signal intensities obtained for the dual-analyte assay using CRP and myoglobin. The CRP and myoglobin array was exposed to a detection solution containing 20 nM DY647-labeled antiCRP and 0.8 nM Cy5-labeled anti-myoglobin.

Protein Spot Diameter Analysis. Spot diameters on each material were calculated from the IgG solutions spotted at a concentration of 1250 nM. Results of the spot diameter analysis are shown in Figure 13. The native, PLL-coated, and OPT PLLcoated materials produced very different spot diameters, ranging from 150 to 400 µm. Microarray spot diameters are influenced by surface hydrophobicity and spotting solution composition. Because the spotting solution composition was the same for each substrate, these differences in spot diameter can mainly be attributed to dissimilar surface energies between each material. The PE-scaffold-coated materials, on the other hand, produced nearly identical spot diameters of around 230 µm due to equivalent

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Figure 13. IgG spot diameter comparison on different materials with different surface treatments. Note that the spot diameters obtained on the PE scaffold are nearly identical for all materials.

native, PLL-coated, and OPT PLL-coated materials still show different microarray responses due to dissimilar surface properties, resulting in different spot diameters and immobilization densities. Although the PLL-coated materials underwent the same surface modification protocol, the underlying substrate still influences the surface properties, resulting in different assay responses on each material. The PE-scaffold-coated materials, shown in Figure 15D, however, produced nearly identical microarray responses. This result indicates that the IgG surface density and extent of NSB is the same on each substrate, which provides further indication of equivalent surface properties on each material following deposition of the PE scaffold.

Conclusions

Figure 12. Microarray fluorescence images on multiple substrates. Microarray fabrication and IgG assays were performed as described in the Experimental Section. (A) Images obtained by scanning each material at the maximum gain setting of 100%. (B) Images obtained by scanning at the normalized gain setting for each material. Note the equivalent signal intensities obtained on different materials using the PE scaffold in B.

surface energies, indicating that the surface properties of each material coated with the PE scaffold are the same. Assay Analysis on Multiple Substrates. The quantified signal intensities for each material obtained at the maximum gain setting are shown in Figure 14. As expected, the microarray assay responses are very different for all materials and surface modifications. These different responses are due to dissimilarities in either intrinsic background fluorescence, substrate surface properties, or both. Dissimilar surface properties between the materials result in different IgG spot diameters, IgG surface density, and NSB. Figure 15 shows the quantified signal intensities obtained at the normalized gain settings for each substrate material. Despite the normalization of the intrinsic background fluorescence, the

In this study, we have demonstrated the ability to obtain nearly identical protein microarray results on a wide range of materials using an interchangeable, substrate-independent PE scaffold composed of (PAH/PSS)5.5. The ability to perform multianalyte assays was demonstrated by printing microarrays of CRP and myoglobin onto PE-scaffold-coated glass and detecting the analytes simultaneously. Following that, assays were performed with multiple materials by spotting IgG directly onto different PE-scaffold-coated materials and performing direct assays using Cy5-labeled anti-IgG. Specifically, the materials examined were glass, gold, mica, silicon, and polymer. The PE scaffold served to convert the surface properties of each material into the surface properties of the scaffold itself, allowing for equal protein immobilization densities and levels of NSB. Additionally, the PE scaffold was found to easily assemble on each material without the need for any surface pretreatments. Microarrays with low NSB were fabricated by spotting IgG directly onto the PAH-capped PE scaffold. It should be noted additionally, however, that the PAH-capped PE scaffold provides a highly functional amine surface which allows for further development and improvement upon the results obtained here. This can be accomplished by exploiting the LbL self-assembly process further to incorporate other useful compounds which can increase surface receptor density (dendrimers, porous matrixes, covalent activation, etc.)39,40 or reduce NSB of target analytes (poly(ethylene glycol), copolymers, etc.).41,42 Furthermore, improvements developed on the PE scaffold using one (39) Benters, R.; Niemeyer, C. M.; Wohrle, D. ChemBioChem 2001, 2(9), 686–694. (40) Arenkov, P.; Kukhtin, A.; Gemmell, A.; Voloshchuk, S.; Chupeeva, V.; Mirzabekov, A. Anal. Biochem. 2000, 278(2), 123–131.

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Figure 14. Quantified signal intensities obtained for the IgG assays on multiple materials scanned at the maximum gain setting of 100%.

substrate material can be transferred to other materials of interest to obtain equivalent results. And finally, the concept of PE scaffolding can easily be extended to other application areas

Spillman et al.

Figure 15. Quantified signal intensities obtained for the IgG assays on multiple materials scanned at the normalized gain setting for each material. Note that the signal intensities on the PE scaffold are nearly identical for all materials.

which require surface modification and precise control over surface properties. Future work in this area will be devoted to

Substrate-Independent Protein Microarrays

expanding the application range of PE scaffolding to other areas which can benefit from interchangeable, substrate-independent surface modification techniques. (41) Kenausis, G. L.; Voros, J.; Elbert, D. L.; Huang, N. P.; Hofer, R.; RuizTaylor, L.; Textor, M.; Hubbell, J. A.; Spencer, N. D. J. Phys. Chem. B 2000, 104(14), 3298–3309. (42) Huang, N. P.; Michel, R.; Voros, J.; Textor, M.; Hofer, R.; Rossi, A.; Elbert, D. L.; Hubbell, J. A.; Spencer, N. D. Langmuir 2001, 17(2), 489–498.

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Acknowledgment. Special thanks go to Dr. Jimmy Bakker, Robert Copperwhite, and Michal Trnavsky for providing useful information and substrate materials essential for the completion of this study. This work was supported by the European Commission, Sixth Framework Programme, Information Society Technologies. CAREMAN (no. 017333). LA8020723