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Effective Enhancement of Fluorescence Detection Efficiency in Protein Microarray Assays: Application of a Highly Fluorinated Organosilane as the Blocking Agent on the Background Surface by a Facile Vapor-Phase Deposition Process Hsin-Yi Hsieh,† Pen-Cheng Wang,‡ Chun-Lung Wu,† Chi-Wen Huang,‡ Ching-Chang Chieng,†,‡ and Fan-Gang Tseng*,†,‡,§ Institute of NanoEngineering and MicroSystems (NEMS) and Department of Engineering and System Science, National Tsing Hua University, Hsinchu 30013, Taiwan R.O.C., and Division of Mechanics, Research Center for Applied Sciences, Academia Sinica, Taipei 11574, Taiwan R.O.C. Protein microarrays are emerging as an important enabling technology for the simultaneous investigation of complicated interactions among thousands of proteins. The solution-based blocking protocols commonly used in protein microarray assays often cause cross-contamination among probes and diminution of protein binding efficiency because of the spreading of blocking solution and the obstruction formed by the blocking molecules. In this paper, an alternative blocking process for protein microarray assays is proposed to obtain better performance by employing a vapor-phase deposition method to form self-assembled surface coatings using a highly fluorinated organosilane as the blocking agent on the background surfaces. Compared to conventional solutionbased blocking processes, our experimental results showed that this vapor-phase process could shorten the blocking time from hours to less than 10 min, enhance the binding efficiency by up to 6 times, reduce the background noise by up to 16 times, and improve the S/N ratio by up to 64 times. This facile blocking process is compatible with current microarray assays using silica-based substrates and can be performed on many types of silane-modified surfaces. Enzyme-linked immunosorbent assays (ELISA) for quantification of antibodies or antigens were developed by Peter Perlmann and Eva Engvall in 1971.1 Different from conventional ELISA performed in a 96-well plate or a microfluidics-based microchip,2 protein microarrays are capable of simultaneously and dynamically analyzing tens to thousands of proteins.3-5 As protein expression * To whom correspondence should be addressed. E-mail: fangang@ ess.nthu.edu.tw. Phone: 886-3-5715131-34270. Fax: 886-3-5720724. † Institute of NanoEngineering and MicroSystems (NEMS), National Tsing Hua University. ‡ Department of Engineering and System Science, National Tsing Hua University. § Academia Sinica. (1) Engvall, E.; Perlmann, P. Immunochemistry 1971, 8, 871–874. (2) Mahmoudian, L.; Kaji, N.; Tokeshi, M.; Nilsson, M.; Baba, Y. Anal. Chem. 2008, 80, 2483–2490.
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profiles explicitly reveal the functions and biological properties of various cells or tissues, the data collected from protein microarrays could provide more direct and functional information in high-throughput ways for protein-network investigation, therapy guidance, prognostics in cancer, drug discovery, and entire genome screening.6-8 In spite of being a powerful tool, protein microarrays are more complicated than DNA microarrays,9 and face many challenges, such as the difficulties of maintaining a proper conformation, function, and orientation of proteins when they are immobilized on surfaces,10 steric problems, or nonspecific protein adsorption originating from wet blocking protocols that interfere with the specific binding between target proteins and recognition proteins,10,11 or the presence of cross-contamination and background noise which diminish signal-to-noise (S/N) ratio, and so on. To fabricate protein microarrays with high stability, robust protein microarray platforms based on glass slides,12-14 matrix slides,12,15 or nanowells12,16 have been developed. Glass slides are relatively inexpensive substrates and compatible with the detection (3) Haab, B.; Dunham, M.; Brown, P. Genome Biol. 2001, 2, research0004.10004.13. (4) Kusnezow, W.; Jacob, A.; Walijew, A.; Diehl, F.; Hoheisel, J. D. Proteomics 2003, 3, 254–264. (5) Fall, B. I.; Eberlein-Konig, B.; Behrendt, H.; Niessner, R.; Ring, J.; Weller, M. G. Anal. Chem. 2003, 75, 556–562. (6) Poetz, O.; Schwenk, J. M.; Kramer, S.; Stoll, D.; Templin, M. F.; Joos, T. O. Mech. Ageing Dev. 2005, 126, 161–170. (7) Gulmann, C.; Sheehan, K. M.; Kay, E. W.; Liotta, L. A.; Petricoin, E. F. J. Pathol. 2006, 208, 595–606. (8) Kingsmore, S. F. Nat. Rev. Drug Discov. 2006, 5, 310–321. (9) Trau, D.; Lee, T. M. H.; Lao, A. I. K.; Lenigk, R.; Hsing, I.-M.; Ip, N. Y.; Carles, M. C.; Sucher, N. J. Anal. Chem. 2002, 74, 3168–3173. (10) Peluso, P.; Wilson, D. S.; Do, D.; Tran, H.; Venkatasubbaiah, M.; Quincy, D.; Heidecker, B.; Poindexter, K.; Tolani, N.; Phelan, M.; Witte, K.; Jung, L. S.; Wagner, P.; Nock, S. Anal. Biochem. 2003, 312, 113–124. (11) Eteshola, E.; Leckband, D. Sens. Actuators, B 2001, 72, 129–133. (12) Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2001, 5, 40–45. (13) Poetz, O.; Ostendorp, R.; Brocks, B.; Schwenk, J. M.; Stoll, D.; Joos, T. O.; Templin, M. F. Proteomics 2005, 5, 2402–2411. (14) Angenendt, P.; Glo ¨kler, J.; Sobek, J.; Lehrach, H.; Cahill, D. J. J. Chromatogr. A 2003, 1009, 97–104. (15) Kersten, B.; Possling, A.; Blaesing, F.; Mirgorodskaya, E.; Gobom, J.; Seitz, H. Anal. Biochem. 2004, 331, 303–313. (16) Angenendt, P.; Nyarsik, L.; Szaflarski, W.; Glo ¨kler, J.; Nierhaus, K. H.; Lehrach, H.; Cahill, D. J.; Lueking, A. Anal. Chem. 2004, 76, 1844–1849. 10.1021/ac900552v CCC: $40.75 2009 American Chemical Society Published on Web 08/25/2009
equipment of DNA microarrays, but the possibility for crosscontamination during blocking or washing processes is the main disadvantage. On the other hand, matrix slides and nanowells have low contamination for protein spots separated by walls. However, the cost is higher because of photolithography fabrication. As regard to protein immobilization interfaces, the employable materials with reasonable binding ability and stable properties include agarose gels,17 dextran-based hydrogels,18 porous polyacrylamide hydrogels,19 hydrophilic polymers,20 chemically reactive surfaces,21,22 or affinity surfaces.23 For protein microarrays applied to more general medical applications, substrates compatible with proteins, low cost, easy preparation, low cross-contamination, and capability for correct diagnosis are desired. As a result, self-assembled monolayers (SAMs) or self-assembled coatings have been developed with their easy preparation, low cost, and high acceptance for protein immobilization by providing reactive sites for covalent bonding or positively charged surfaces for electrostatic adsorption.4,22 3-Aminopropyltrimethoxysilane (APTS) is an organosilane commonly used to form self-assembled coatings on silica-based substrates for protein immobilization.4,22 Although APTS is commonly used to modify silica-based substrates for protein immobilization, it is also well-known that unblocked surfaces based on self-assembled APTS coatings tend to cause serious nonspecific binding of target proteins, which can often result in background surfaces with high-noise signals and cross-contamination among probes to interfere with high-throughput protein microarray assays. To resolve the non-specific binding issues, blocking solutions containing non-fat/low-fat milk (MILK),4,13,14 bovine serum albumin (BSA),4,13,24 or horse serum (HS)24 can be used to block the background surfaces of protein microarrays to improve assay performance. However, those solution-based procedures are still not ideal because they take a long time (usually more than hours) to perform. Besides, cross-contamination among probes during blocking/washing processes can not be effectively prevented, especially for amine-rich surfaces.25,26 Moreover, comet-tailing and/or smearing of protein spots frequently occur because of (i) high spotting concentrations, (ii) vigorous shaking in the blocking solution, (iii) rough application of the coverslip before conjugation, and (iv) inappropriate blocking agents.25,26 Furthermore, the relatively large sizes of the proteinbased blocking agents used in wet blocking processes can cause obstruction and/or steric hindrance to interfere with specific protein binding assays.11 (17) Afanassiev, V.; Hanemann, V.; Wolf, S. Nucleic Acids Res. 2000, 28, e66. (18) Piehler, J.; Brecht, A.; Hehl, K.; Gauglitz, G. Colloids Surf., B 1999, 13, 325–336. (19) Brueggemeier, S. B.; Kron, S. J.; Palecek, S. P. Anal. Biochem. 2004, 329, 180–189. (20) Kim, J.-K.; Shin, D.-S.; Chung, W.-J.; Jang, K.-H.; Lee, K.-N.; Kim, Y.-K.; Lee, Y.-S. Colloids Surf., B 2004, 33, 67–75. (21) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760–1763. (22) Angenendt, P.; Glokler, J.; Murphy, D.; Lehrach, H.; Cahill, D. J. Anal. Biochem. 2002, 309, 253–260. (23) Schmid, E. L.; Keller, T. A.; Dienes, Z.; Vogel, H. Anal. Chem. 1997, 69, 1979–1985. (24) Ruotsalainen, V.; Ljungberg, P.; Wartiovaara, J.; Lenkkeri, U.; Kestila¨, M.; Jalanko, H.; Holmberg, C.; Tryggvason, K. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7962–7967. (25) Taylor, S.; Smith, S.; Windle, B.; Guiseppi-Elie, A. Nucleic Acids Res. 2003, 31, e87. (26) Dolan, P. L.; Wu, Y.; Ista, L. K.; Metzenberg, R. L.; Nelson, M. A.; Lopez, G. P. Nucleic Acids Res. 2001, 29, e107.
To avoid the undesired implications caused by conventional wet blocking protocols, it is necessary to develop an alternative protocol for high-throughput protein microarray assays. In this study, a facile vapor-phase deposition method using a highly fluorinated organosilane, 1H,1H,2H,2H-perfluorooctyltrichlorosilane (FOTS), as the blocking agent to treat the background surfaces of protein microarrays for effective enhancement of fluorescence detection efficiency is described. Although it has been shown that FOTS is useful for anti-stiction application,27-30 it is not considered by first intuition that FOTS can be an appropriate component for integration in protein microarrays because of the high surface hydrophobicity of the self-assembled coating formed by this highly fluorinated organosilane. Nonetheless, by employing the facile vapor-phase deposition method described in this paper, it was successfully demonstrated by our experiments that FOTS could be exploited as an effective blocking agent to enhance the fluorescence detection efficiency in protein microarray assays involving multistep chemical and/or biochemical wet incubation processes. Unlike the conventional solutionbased blocking processes, our vapor-phase blocking process can effectively prevent tailing/smearing of protein spots to eliminate probe cross-contamination, and minimize steric hindrance that interferes with the conjugation of target proteins and recognition proteins to improve specific protein binding efficiency. Above all, the whole vapor-phase blocking process can be completed in less than 30 min, including reaction time and air-extraction time. EXPERIMENTAL SECTION Materials. Kimble glass slides (Kimble Chase Life Science and Research Products LLC, U.S.A.) and Superior glass slides (Paul Marienfeld GmbH & Co. KG, Germany) were used as substrates to fabricate our protein arrays. To minimize the interference of substrate roughness with surface characterization and/or to facilitate reflective optical measurements, silicon wafers (one-side polished; with 1 µm thermally grown silicon dioxide) were used for Atomic Force Microscopy (AFM), FourierTransform Infrared Spectroscopy (FTIR), and X-ray Photoelectron Spectroscopy (XPS) experiments. APTS (3-aminopropyltrimethoxysilane; H2N-(CH2)3-Si(OCH3)3, catalogue number: 281778), FOTS (1H,1H,2H,2H-perfluorooctyltrichlorosilane; CF3-(CF2)5CH2CH2-SiCl3, catalogue number: 448931) and TWEEN 20 (catalogue number: P1379) were purchased from Sigma-Aldrich. Mouse IgG (catalogue number: PP54), Cy3-conjugated rabbit antimouse IgG (ex/em 550/570 nm, catalogue number: AP160C) and Cy5-conjugated goat anti-rabbit IgG (ex/em 650/680 nm, catalogue number: AP132S) were purchased from Chemicon (Millipore). Surface Silanization. The silica-based substrates (i.e., the glass slides and silicon wafers described in the Materials section) were first cleaned in freshly prepared piranha solution (7:1 v/v mixture of 96% sulfuric acid and 30% hydrogen peroxide) at 90 °C for 10 min or 10% NaOH(aq) at room temperature for 1 h. After (27) Jung, G.-Y.; Li, Z.; Wu, W.; Chen, Y.; Olynick, D. L.; Wang, S.-Y.; Tong, W. M.; Williams, R. S. Langmuir 2005, 21, 1158–1161. (28) Wu, C.-W.; Shen, Y.-K.; Chuang, S.-Y.; Wei, C. S. Sens. Actuators, A 2007, 139, 145–151. (29) Knieling, T.; Lang, W.; Benecke, W. Sens. Actuators, B 2007, 126, 13–17. (30) Zhou, W.; Zhang, J.; Liu, Y.; Li, X.; Niu, X.; Song, Z.; Min, G.; Wan, Y.; Shi, L.; Feng, S. Appl. Surf. Sci. 2008, 255, 2885–2889.
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Figure 1. General protein array preparation procedure used in this study is schematically illustrated in (a) to (f). Among them, steps (c1) to (f1) are for conventional wet blocking, and steps (c2) to (f2) are for vapor-phase blocking. The house-built vapor deposition apparatus is shown in (g). Vapor-phase FOTS molecules undergoing hydrolysis (with the release of HCl) near or on the APTS surface are shown in (h). FOTS molecules bound with APTS coating or silicon dioxide surface via the Si-O-Si bonds are shown in (i).
the above piranha solution or NaOH solution treatment, the substrates were rinsed with copious amounts of deionized water and dried by a compressed-air stream. To perform the APTS silanization treatment, the cleaned substrates were immersed in a 0.5% APTS solution (v/v in 99.5% absolute anhydrous ethanol) for 65 min at room temperature. After the APTS treatment, the slides were rinsed with copious amounts of deionized water and dried by a compressed-air stream. 7910
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To perform the FOTS silanization treatment, a house-built apparatus for vapor-phase deposition of FOTS was used (Figure 1g). Briefly, the substrates were first fixed on the sample holders in a house-built plastic box (130 mm L × 90 mm W × 70 mm H) with two tubings. The box was then placed in the vacuum chamber of the vapor-phase deposition apparatus, with one tubing connected to the gas inlet of the vacuum chamber, and the other tubing connected to a mechanical pump via the gas outlet of the
vacuum chamber (Figure 1g). When the box containing the substrates for deposition was appropriately loaded into the vacuum chamber, the pressure of the vacuum chamber was pumped down to
