as Substrates for DNA Oligonucleotide Microarrays - American

Dec 2, 2005 - Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, A-4020 Linz, Austria,. Biomaterials and Tissue Engineering, Eastman ...
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Langmuir 2006, 22, 277-285

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Glass Surfaces Grafted with High-Density Poly(ethylene glycol) as Substrates for DNA Oligonucleotide Microarrays Robert Schlapak,†,⊥ Patrick Pammer,†,⊥ David Armitage,‡ Rong Zhu,§ Peter Hinterdorfer,§ Matthias Vaupel,| Thomas Fru¨hwirth,† and Stefan Howorka*,†,# Center for Biomedical Nanotechnology, Upper Austrian Research GmbH, A-4020 Linz, Austria, Biomaterials and Tissue Engineering, Eastman Dental Institute, UniVersity College London, London WC1X 8LD, U.K., Institute for Biophysics, Johannes Kepler UniVersity Linz, A-4040 Linz, Austria, Nanofilm Technologie GmbH, D-37081 Go¨ttingen, Germany, and Department of Chemistry, UniVersity College London, London WC1H 0AJ, U.K. ReceiVed August 10, 2005. In Final Form: October 10, 2005 Surfaces carrying a dense layer of poly(ethylene glycol) (PEG) were prepared, characterized, and tested as substrates for DNA oligonucleotide microarrays. PEG bis(amine) with a molecular weight of 2000 was grafted onto silanized glass slides bearing aldehyde groups. After grafting, the terminal amino groups of the PEG layer were derivatized with the heterobifunctional cross-linker succinimidyl 4-[p-maleimidophenyl]butyrate to permit the immobilization of thiol-modified DNA oligonucleotides. The stepwise chemical modification was validated with X-ray photoelectron spectroscopy. Goniometry indicated that the PEG grafting procedure reduced surface inhomogeneities present after the silanization step, while atomic force microscopy and ellipsometry confirmed that the PEG layer was dense and monomolecular. Hybridization assays using DNA oligonucleotides and fluorescence imaging showed that PEG grafting improved the yield in hybridization 4-fold compared to non-PEGylated maleimide-derivatized surfaces. In addition, the PEG layer reduced the nonspecific adsorption of DNA by a factor of up to 13, demonstrating that surfaces with a dense PEG layer represent suitable substrates for DNA oligonucleotide microarrays.

Introduction DNA microarrays are important biomedical research tools owing their power and versatility to the ability to simultaneously and sequence specifically capture target DNA onto thousands of probe spots. The high degree of parallelism is key in the genomewide analysis of gene expression or SNP mapping for applications in diagnostics, toxicology, and pharmacology. DNA microarrays are usually fabricated by the photolithographic on-chip synthesis of oligonucleotides1 or by spotting DNA or oligonucleotides droplets onto solid supports.2 Irrespective of the chosen method of microarray fabrication, the chemically modified surfaces of the solid substrates play an essential part in the use and performance of DNA microarrays. Suitable surfaces have low intrinsic fluorescence, achieve high probe densities within the spots, obtain high and specific hybridization yields, resist the nonspecific adsorption of sample DNA, and show homogeneous surface characteristics within and between slides. The substrate most widely used for the fabrication of microarrays is silica or glass, and several surface modifications are available for the adsorption2 and covalent immobilization of DNA.3 For covalent tethering, the silanol groups of the substrate are usually first reacted with alkoxysilanes such as aminopropyl triethoxysilane or glycidoxypropyl trimethoxysilane to form a * To whom correspondence should be addressed. Department of Chemistry, University College London, Christopher Ingold Laboratories, 20 Gordon Street, London WC1H 0AJ, U.K. E-mail: [email protected]. † Upper Austrian Research GmbH. ‡ Eastman Dental Institute, University College London. § Johannes Kepler University Linz. | Nanofilm Technologie GmbH. # Department of Chemistry, University College London. ⊥ These authors contributed equally to this work. (1) Fodor, S. P.; Rava, R. P.; Huang, X. C.; Pease, A. C.; Holmes, C. P.; Adams, C. L. Nature 1993, 364, 555-556. (2) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467-470. (3) Schena, M. Microarray Analysis; John Wiley & Sons: Hoboken, NJ, 2002.

silane layer displaying amino, epoxide, or aldehyde groups. Chemically modified DNA is then commonly coupled to the surface via ester, ether, or imine linkages 4 or in the case of heterobifunctional cross-linkers5,6 via a thioether linkage. Despite its popularity, the conventional silanization of silica substrates remains challenging as the modification tends to produce irreproducibility in the macroscopic properties (e.g., wettability and contact angle hysteresis) of the deposited monolayers.7 The molecular reason for the problems is thought to originate from the complex chemistry of the multistep silanization reaction8,9 leading eventually to the formation of three-dimensional (3D) aggregates or multilayers instead of a pure monolayer.10 Strategies to address these problems have been developed and include the strict control of the silanization conditions7,11-13 or the building up of a 3D dendrimeric linker layer which can mask surface inhomogeneities and increase the number of functional groups for DNA immobilization.12 In this report, we explore an alternative surfacesa grafted layer of poly(ethylene glycol) (PEG)sas a substrate for DNA microarrays. Poly or oligo(ethylene glycol)-grafted surfaces are (4) Zammatteo, N.; Jeanmart, L.; Hamels, S.; Courtois, S.; Louette, P.; Hevesi, L.; Remacle, J. Anal. Biochem. 2000, 280, 143-150. (5) Chrisey, L. A.; Lee, G. U.; O’Ferrall, C. E. Nucleic Acids Res. 1996, 24, 3031-3039. (6) Oh, S. J.; Cho, S. J.; Kim, C. O.; Park, J. W. Langmuir 2002, 18, 17641769. (7) Brzoska, J. B.; Benazouz, I.; Rondelez, F. Langmuir 1994, 10, 43674373. (8) Legrange, J. D.; Markham, J. L.; Kurkjian, C. R. Langmuir 1993, 9, 17491753. (9) Zhao, X. L.; Kopelman, R. J Phys Chem B 1996, 100, 11014-11018. (10) Gray, D. E.; CaseGreen, S. C.; Fell, T. S.; Dobson, P. J.; Southern, E. M. Langmuir 1997, 13, 2833-2842. (11) Vallant, T.; Brunner, H.; Mayer, U.; Hoffmann, H.; Leitner, T.; Resch, R.; Friedbacher, G. J. Phys. Chem. B 1998, 102, 7190. (12) Benters, R.; Niemeyer, C. M.; Wohrle, D. ChemBioChem 2001, 2, 686694. (13) Lambert, A. G.; Neivandt, D. J.; McAloney, R. A.; Davies, P. B. Langmuir 2000, 16, 8377-8382.

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widely used as passivation layers to avoid nonspecific adsorption of proteins14-16 or as immobilization matrixes for peptide microarrays17 and biosensors.18-20 Reflecting the widespread use of PEGylated surfaces for these applications, many methods for the grafting of PEG have been developed, such as the coupling of epoxide-terminated PEG onto aminopropyl triethoxysilanederived layers,21 the reaction of the terminal hydroxyl group of PEG onto Cl2-activated silicon,22 the one-step grafting of methoxy poly(ethylene glycol) derivatives with terminal trimethoxysilane,23 methacryl onto silicate,24 or dihydroxyphenylalanine groups onto titanium oxide layers,25 and the chemisorption of the copolymer poly(L-lysine)-g-poly(ethylene glycol) on anionic surfaces of metal oxides.18,26 Despite the wealth of reports on PEG-covered surfaces, there are few reports on the use and performance of PEG-grafted substrates for DNA microarray applications.27 The shortage of reports stands in contrast to the potential advantages of PEG monolayers for DNA microarrays: (i) hydrophilic polymer chains can function as spacers to improve the recognition between target and probe and avoid sterical hindrance as in the case of linkerless DNA immobilization,28-31 (ii) PEG can help resist the nonspecific adsorption of DNA,32 and (iii) a dense PEG layer might also mask inhomogeneities of the underlying surface. Recently, Cha et al. reported on the preparation and use of PEG-grafted silicon as substrates for the immobilization of DNA oligonucleotides.27 In this study, the PEG layer was obtained by grafting PEG-carrying terminal hydroxyl groups onto Cl2-activated silicon displaying Cl-Si bonds. However, it is not clear, the extent to which the chemistry of this grafting procedure can be successfully applied to glass substrates, which are widely used in microarray applications.3 In addition, questions remain about the effect of the hydrophilic PEG layer on the hybridization yield and the reduction of nonspecific adsorption of DNA in comparison to surfaces without a PEG layer. Here, we report on the preparation of a PEG-grafted surface on glass slides and its characterization for DNA microarray applications in comparison to a PEG-less surface. Scheme 1 (14) McPherson, T.; Kidane, A.; Szleifer, I.; Park, K. Langmuir 1998, 14, 176-186. (15) Delamarche, E.; Donzel, C.; Kamounah, F. S.; Wolf, H.; Geissler, M.; Stutz, R.; Schmidt-Winkel, P.; Michel, B.; Mathieu, H. J.; Schaumburg, K. Langmuir 2003, 19, 8749-8758. (16) Papra, A.; Bernard, A.; Juncker, D.; Larsen, N. B.; Michel, B.; Delamarche, E. Langmuir 2001, 17, 4090-4095. (17) Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20, 270-274. (18) Ruiz-Taylor, L. A.; Martin, T. L.; Zaugg, F. G.; Witte, K.; Indermuhle, P.; Nock, S.; Wagner, P. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 852-857. (19) Huang, N. P.; Voros, J.; De Paul, S. M.; Textor, M.; Spencer, N. D. Langmuir 2002, 18, 220-230. (20) Stadler, B.; Falconnet, D.; Pfeiffer, I.; Hook, F.; Voros, J. Langmuir 2004, 20, 11348-11354. (21) Malmsten, M.; Emoto, K.; Van Alstine, J. M. J. Colloid Interface Sci. 1998, 202, 507-517. (22) Zhu, X. Y.; Jun, Y.; Staarup, D. R.; Major, R. C.; Danielson, S.; Boiadjiev, V.; Gladfelter, W. L.; Bunker, B. C.; Guo, A. Langmuir 2001, 17, 7798-7803. (23) Papra, A.; Gadegaard, N.; Larsen, N. B. Langmuir 2001, 17, 1457-1460. (24) Roosjen, A.; van der Mei, H. C.; Busscher, H. J.; Norde, W. Langmuir 2004, 20, 10949-10955. (25) Dalsin, J. L.; Lin, L. J.; Tosatti, S.; Voros, J.; Textor, M.; Messersmith, P. B. Langmuir 2005, 21, 640-646. (26) 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, 489-498. (27) Cha, T. W.; Boiadjiev, V.; Lozano, J.; Yang, H.; Zhu, X. Y. Anal. Biochem. 2002, 311, 27-32. (28) Shchepinov, M. S.; CaseGreen, S. C.; Southern, E. M. Nucleic Acids Res. 1997, 25, 1155-1161. (29) Southern, E.; Mir, K.; Shchepinov, M. Nat. Genet. 1999, 21, 5-9. (30) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 5163-5168. (31) Hong, B. J.; Oh, S. J.; Youn, T. O.; Kwon, S. H.; Park, J. W. Langmuir 2005, 21, 4257-4261. (32) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044-8051.

Schlapak et al.

illustrates the four-step procedure to obtain the PEG-grafted surface: (i) Glass slides are silanized with glycidoxypropyl trimethoxysilane, whose epoxide groups are (ii) hydrolyzed. The resulting diols are oxidized to yield an aldehyde-derivatized surface. (iii) Poly(ethylene glycol)-diamine is grafted onto the aldehyde surface using an improved method (see Experimental Section, Grafting of PEG). (iv) The terminal amino groups of the PEG layer are functionalized with the heterobifunctional cross-linker succinimidyl 4-[p-maleimidophenyl]butyrate (SMPB) to introduce maleimide groups for (v) the immobilization of thiol-modified oligonucleotides. For the purpose of comparison, a linkerless surface displaying maleimide groups is also prepared (Scheme 1, step vi). These surfaces are characterized by X-ray photoelectron spectroscopy, goniometry, ellipsometry, and atomic force microscopy (AFM). In addition, hybridization assays and fluorescence microscopy are used to assess the performance of the PEG-grafted surfaces in comparison to the PEG-less surface. Experimental Section Materials. Glass slides were bought from suppliers J. Melvin Freed (Sigma, Schnelldorf, Germany), Assistent (VWR, Darmstadt, Germany), and Carl Roth (Karlsruhe, Germany), which are being referred to as type 1, type 2, and type 3, respectively. OptiSlides of 1 mm thick BK7 glass substrate coated with a 80 nm Ta2O5 layer with refractive index 2.3 and a top 10 nm SiO2 layer were obtained from Nanofilm (Go¨ttingen, Germany) and were used for ellipsometric measurements. All slides had the format (75 × 25 × 1 mm) except Roth (50 × 24 × 0.15 mm). O,O′-Bis(2-aminoethyl) poly(ethylene glycol) (PEG-diamine) with a MW of 2000 was obtained from Rapp Polymere (Tu¨bingen, Germany). N-Succinimidyl 4-(4-maleimidophenyl)butyrate (SMPB) was purchased from Pierce (Rockford, IL). T4 polynucleotide kinase was obtained from New England Biolabs (Ipswich, MA), and [γ-32P]ATP was from GE Healthcare, Amersham Biosciences, U.K. Photoresist SU-8 2035 was from Microchem (Newton, MA), and 5 in. silicon wafers were from WaferNet Inc. (San Jose, CA). Poly(dimethyl siloxane) RTV 615 was obtained from GE Bayer Silicones (Leverkusen, Germany). DNA oligonucleotides were ordered from Integrated DNA Technologies (Coralville, IA) and VBC-Genomics (Vienna, Austria). All other chemicals were products from Sigma (Schnelldorf, Germany). Silanization of Substrates to Obtain Aldehyde Slides. By the use of a Teflon reactor, the glass slides were cleaned in 10:90, 50:50, and 90:10 methanol/CHCl3 for 15 min each in an ultrasonic bath followed by washing in deionized water. The slides were immersed in Piranha solution (30:70, 30% aqueous H2O2/H2SO4) for 30 min. (WARNING: Piranha solution reacts violently with organic material. It should not be combined with significant quantities of organic materials, and it should also not be stored for any length of time as there is a potential risk of explosive reactions.) The slides were dried in a stream of nitrogen after extensive washing with water. Following the cleaning procedure, slides were tested for low background fluorescence and small contact angles (