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Functionalized Surfaces of Mixed Alkanethiols on Gold as a Platform for Oligonucleotide Microarrays Michael Riepl,† Karin Enander,‡ and Bo Liedberg*,† Divisions of Sensor Science and Chemistry, Department of Physics and Measurement Technology, University of Linko¨ ping, SE-58183 Linko¨ ping, Sweden
Michael Scha¨ferling, Margit Kruschina, and Flavio Ortigao Thermo Hybaid Interactiva GmbH, Sedanstrasse 10, D-89077 Ulm, Germany Received November 28, 2001. In Final Form: June 3, 2002 Mixed self-assembled monolayers of biotinylated- and ethylene glycol-terminated long-chain alkanethiols were prepared on gold surfaces in an attempt to develop a reliable protocol for immobilization of streptavidin. A broad range of surface analytical techniques including ellipsometry, atomic force microscopy, and infrared, fluorescence, and X-ray photoelectron spectroscopy were used to characterize the SAMs before and after immobilization of streptavidin. The first part of the work was focused on finding the mixing conditions that lead to optimum binding capacity of streptavidin. Mixed SAMs prepared from loading solutions containing 75-95% of the biotinylated alkanethiol resulted in high immobilization levels of functional streptavidin. The thin layers of streptavidin subsequently can be used for the immobilization of a broad spectrum of biotinylated biomolecules (e.g. oligonucleotides, cDNA, peptides, proteins, antibodies, and carbohydrates) and provides therefore an excellent platform for the fabrication of chips/arrays for biosensor and screening applications. This is successfully demonstrated by monitoring the hybridization between a biotinylated 24-mer capturing oligonucleotide and a labeled target 89-mer DNA using a fluorescencebased DNA-microarray detection system. Moreover, the DNA-microarray experiments also revealed (i) good selectivity when comparing the response of the complementary oligonucleotide with that of a random 24-mer capturing oligonucleotide and (ii) low levels of nonspecific binding to the streptavidin surface.
Introduction Within the field of biotechnology there has been an increasing interest in surface modifications, with an emphasis on the development of sharp protocols for the immobilization of DNA, DNA fragments, proteins, lipids, or sugars. The realization of such surfaces for use in biosensors and microarrays complies with the demand of the pharmaceutical and biomedical industry for fast and parallel screening methods in their search for new drugs, genetic expressions, and polymorphisms. Since the works of Allara1-3 and Whitesides4,5 in the mid-1980s, the spontaneous adsorption of organosulfur compounds on gold is a widespread method for the preparation of self-assembled monolayers (SAMs). If the alkyl chain is long enough, these architectures are very stable and show a molecular orientation nearly perpendicular to the surface.2,6-8 The layers are densely packed and display excellent insulating properties, which, for example, can be examined with cyclic voltametry9 and * Corresponding author. E-mail:
[email protected]. Phone: +4613-281877. Fax: +46-13-288969. † Division of Sensor Science. ‡ Division of Chemistry. (1) Nuzzo R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 44814483. (2) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558-569. (3) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (4) Bain, C. D.; Whitesides, G. M. Science 1988, 240, 62-63. (5) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546-558. (6) Arnold, R.; Terfort, A.; Wo¨ll, C. Langmuir 2001, 17, 4980-4989. (7) Houssiau, L.; Graupe, M.; Colorado, R.; Kim, H. I.; Lee, T. R.; Perry, S. S.; Rabalais, J. W. J. Chem. Phys. 1998, 109, 9134-9147. (8) Buscher, C. T.; McBranch, D.; Li, D. Q. J. Am. Chem. Soc. 1996, 118, 2950-2953.
impedance measurements.10-12 Moreover, the alkyl chains can be terminated by reactive headgroups that are capable of supporting the immobilization of biomolecules via covalent chemical coupling, electrostatic physisorption or supramolecular interactions. Until now a large variety of self-assembled receptor-analyte systems have been successfully examined using optical,13-19 electrical,20-23 or mechanoacoustical24,25 techniques. The outcome of these (9) Finklea, H. O.; Avery, S.; Lynch, M.; Furtsch, T. Langmuir 1987, 3, 409-413. (10) Mirsky, V. M.; Riepl, M.; Wolfbeis, O. S. Biosens. Bioelectron. 1997, 12, 977-989. (11) Rickert, J.; Go¨pel, W.; Beck, W.; Jung, G.; Heiduschka, P. Biosens. Bioelectron. 1996, 11, 757-768. (12) Berggren, C.; Bjarnason, B.; Johansson, G. Electroanalysis 2001, 13, 173-180. (13) Knoll, W.; Zizlsperger, M.; Liebermann, T.; Arnold, S.; Badia, A.; Liley, M.; Piscevic, D.; Schmitt, F. J.; Spinke, J. Colloid Surf., A 2000, 161, 115-137. (14) Furuki, M.; Kameoka, J.; Craighead, H. G.; Isaacson, M. S. Sens. Actuators, B 2001, 79, 63-69. (15) Gaus, K.; Hall, E. A. H. J. Colloid Interface Sci. 1999, 217, 111118. (16) Svedhem, S.; Enander, K.; Karlsson, M.; Sjo¨bom, H.; Liedberg, B.; Lo¨fås, S.; Mårtensson, L. G.; Sjo¨strand, S. E.; Svensson, S.; Carlsson, U., Lundstro¨m, I. Anal. Biochem. 2001, 296, 188-196. (17) Vikinge, T. P.; Hansson, K. M.; Benesch, J.; Johansen, K.: Rånby, M.; Lindahl, T. L.; Liedberg, B.; Lundstro¨m, I.; Tengvall, P. J. Biomed. Opt. 2000, 5, 51-55. (18) Weiss, T.; Leipert, D.; Kaspar, M.; Jung, G.; Go¨pel, W. Adv. Mater. 1999, 11, 331-335. (19) Flink, S.; van Veggel, F. C. J. M.; Reinhoudt, D. N. Adv. Mater. 2000, 12, 1315-1328. (20) Riepl, M.; Mirsky, V. M.; Wolfbeis, O. S. Anal. Chim. Acta 1999, 392, 77-84. (21) Berggren, C.; Stålhandske, P.; Brundell, J.; Johansson, G. Electroanalysis 1999, 11, 156-160. (22) Qian, Z. B.; Shi, X. H.; Zhuang, J. H.; Kong, J. L.; Deng, J. Q. Bioelectrochem. Bioenerg. 1998, 46, 193-198. (23) Pandey, P. C.; Aston, R. W.; Weetall, H. H. Biosens. Bioelectron. 1995, 10, 669-674.
10.1021/la011732n CCC: $22.00 © 2002 American Chemical Society Published on Web 08/02/2002
Mixed Alkanethiols on Gold
analyses relies on the robustness of the layers and their ability to prevent the immobilized biomolecules from loosing their natural structure and therewith their functionality. For example, denaturation can be observed in the case of a direct contact between the biomolecule and the surface of a metallic or glass substrate. Oligo(ethylene glycol)- (OEG-) modified thiols are very interesting agents that can be used to form SAMs on noble metal surfaces. They are also well-known to hinder nonspecific adsorption of enzymes and proteins and appear therefore to be good candidates for biosensing applications.26-32 The structural properties of OEG monolayers as well as their functional properties in contact with various biofluids have been studied in detail by several groups.33-38 One promising approach for the preparation of a universal sensor platform is the use of streptavidin as a binding site for the immobilization of the recognition elements, utilizing the strong biotin-streptavidin interaction. Since there are protocols available for the biotinylation of a broad range of biomolecules, this sensor surface can be utilized in the analysis of a huge variety of analytes. Two distinctly different approaches can be used to generate a functional streptavidin monolayer: (1) coupling of biotin to a preordered SAM;39 (2) formation of a SAM of biotin-terminated alkanethiols.40 The biotinterminated thiols have to be mixed with nonfunctionalized alkanethioles to obtain optimal packing condition for streptavidin on the surface. In the following sections we describe the preparation of mixed monolayers, consisting of biotinylated alkanethiols and an ethylene glycolterminated alkanethiol. The latter helps to optimize the binding site density and reduces at the same time nonspecific binding to the surface.41 Ellipsometry as well as infrared reflection-absorption (RA), fluorescence (FL), and X-ray photoelectron spectroscopy (XPS) are the main techniques used to reveal the structural and mixing characteristics of the SAMs. We demonstrate also, using atomic force microscopy (AFM), that appropriate mixing leads to a high binding capacity of isolated streptavidin molecules. Finally, the streptavidin layers are used as a platform for the immobilization of a biotinylated capturing (24) Guilbault, G. G.; Jordan, J. M. CRC Crit. Rev. Anal. Chem. 1988, 19, 1-28. (25) Huang, E.; Zhou, F. M.; Deng, L. Langmuir 2000, 16, 32723280. (26) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (27) Harder, P.; Grunze, M.; Dahint, R.; Whiteside, G. M.; Laibinis, P. E. J. Phys. Chem. 1998, 102, 426-436. (28) Benesch, J.; Svedhem, S.; Svensson, S. C. T.; Valiokas, R.; Liedberg, B.; Tengvall, P. J. Biomater. Sci. 2001, 12, 581-597. (29) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777-790. (30) Zhu, B.; Eurell, T.; Gunawan, R.; Leckband, D. J. Biomed. Mater. Res. 2001, 56, 406-416. (31) Ostuni, E.; Chapman, R. G.; Holmlin, R. E.; Takayama, S.; Whitesides, G. M. Langmuir 2001, 17, 5605-5620. (32) Luk, Y. Y.; Kato, M.; Mrksich, M. Langmuir 2000, 16, 96049608. (33) Valiokas, R.; Svedhem, S.; Svensson, S. C. T.; Liedberg, B. Langmuir 1999, 15, 3390-3394. (34) Valiokas, R.; Svedhem, S.; O ¨ stblom, M.; Svensson, S. C. T.; Liedberg, B. J. Phys. Chem. B 2001, 105, 5459-5469. (35) Valiokas, R.; O ¨ stblom, M.; Svedhem, S.; Svensson, S. C. T.; Liedberg, B. J. Phys. Chem. B 2000, 104, 7565-7569. (36) Mrksich, M.; Grunwell, J. R.; Whitesides, G. M. J. Am. Chem. Soc. 1995, 117, 12009-12010. (37) Lang, H.; Duschl, C.; Vogel, H. Langmuir 1994, 10, 197-210. (38) Pertsin, A. J.; Grunze, M.; Kreuzer, H. J.; Wang, R. L. C. Phys. Chem. Chem. Phys. 2000, 2, 1729-1733. (39) Mecklenburg, M.; Danielsson, B.; Winquist, F. WO97/49989, 1997. (40) Knoll, W.; Schmitt, F.-J.; Klein, C. WO92/10757, 1992. (41) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 10714-10721.
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24-mer oligonucleotide that subsequently was hybridized with a fluorescent-labeled complementary target DNA. Experimental Section Materials. 16-Mercaptohexadecanoic acid (Aldrich) was recrystallized from hexane. Biotinyl-3,6-dioxaoctanediamine (KMF), 2-(2-aminoethoxy)ethanol (Aldrich), TSTU (Aldrich), diisopropylethylamine (DIPEA, Aldrich), and sodium methanolate (Merck) have been utilized for the synthesis of the ethylene glycolterminated and biotinylated alkanethiols. Absolute ethanol (SDS) and sterile water (Roth) was used without further purification. Streptavidin (Roche) and Cy5-streptavidin (Amersham Pharmacia) were utilized in TBS buffer (1.5 M NaCl/0.1 M Tris-HCl in steril water, Sigma). A 5′-biotinylated 24-mer oligonucleotide (HMb14, Thermo Hybaid) was immobilized as capture molecule, and another 5′-biotinylated 24-mer (EuB, Thermo Hybaid) for negative control (in 0.5× SSPE buffer, Sigma). A Cy5-labeled (5′-modified) 89-mer DNA (CRArg4, Thermo Hybaid) was used as target sequence for hybridization experiments. Preparation of Alkanethiol Monolayers. Gold substrates were prepared and cleaned according to previously described protocols.42,43 The freshly cleaned gold substrates were immersed in an ethanolic solution (99%, Kemetyl, Stockholm, Sweden) of the two alkanethiols at different molar ratios with an overall alkanethiol concentration of 100 µM for 16 h at room temperature. After adsorption the samples were rinsed with ethanol, ultrasonicated in ethanol for 5 min, and then rinsed again in ethanol to remove excess, loosely bound thiols. The samples were finally blown dry in a nitrogen stream and immediately analyzed. A deuterated thiol (HS(CD2)15CD3) was assembled on gold and used a reference sample for the collection of the background spectrum in the infrared RA experiments. Ellipsometry. The ellipsometric measurements were performed with an automatic Rudolph Research AutoEl III ellipsometer aligned at an angle of incidence of 70° with respect to the surface normal. The ellipsometer was equipped with a HeNe laser (632.8 nm) light source. An optical model based on isotropic optical constants for the organic layer (SAM and protein) Norg ) n + ik ) 1.50 + i0, with n ) refractive index and k ) extinction coefficient, was used for the evaluation of the thickness of the monolayers. The final film thickness was calculated by averaging the thickness values obtained at three different spots on at least three samples for each mixture. Infrared Measurements. The infrared reflection-absorption (RA) spectra were recorded at room temperature on a Bruker IFS 66 system equipped with a grazing angle (85°) infrared reflection accessory and a liquid nitrogen cooled MCT detector. The measurement chamber was continuously purged with nitrogen gas during the measurements. The acquisition time was around 10 min at a spectral resolution of 2 cm-1 using a three-term Blackman-Harris apodization function. X-ray Photoelectron Spectroscopy. The XPS measurements were performed on a Microlab 310-F spectrometer with a multichannel detection system. An Mg/Al twin anode was used as source operating at 300 W (15 kV, 20 mA). The base pressure for the system is 5 × 10-10 mbar. The data were taken at 60° incidence angle and 0° takeoff angle, with respect to the surface normal. For each sample a survey scan was performed, followed by narrow scans of the C 1s, O 1s, N 1s, and S 2p regions. The binding energies are lined up with respect to the Au 4f peak at 84.0 eV. Fluorescence Measurements. The chips have been read out with a XNA ScanPro20 microarray laser scanner (Thermo Hybaid). The Cy5-fluorescent dyes were excited with a 633 nm He-Ne laser. The resulting CCD camera images have been revised using the ScanPro20 array analysis software (Thermo Hybaid). Preparation of DNA Microarrays. XNA on Gold chips (Thermo Hybaid, 2 × 96 spot format) were used. The chip is prepared by depositing gold on a chromium-coated glass substrate and covering the gold surface with a Teflon layer via a mask to define the spots Φ ) 1.5 mm. The chips were coated with the (42) Bertilsson, L.; Liedberg, B. Langmuir 1993, 9, 141-149. (43) Enquist, I.; Lundstro¨m, I.; Liedberg, B. J. Phys. Chem. 1995, 99, 12257-12267.
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Figure 1. Schematic outline describing the synthesis of alkanethiols 1 and 2. mixed SAM as described above. The mixed monolayer was exposed to an aqueous streptavidin solution (200 µg/mL, TBS buffer) for 2 h. After being rinsed, the chip was spotted with a solution of the biotinylated oligonucleotide (in SSPE buffer, incubation time 2 h). Hybridization experiments were performed with Cy5-labeled DNA (hybridization time 2 h, SSPE buffer). After washing, the fluorescence intensity on the single spots was measured with a laser scanner. AFM. A NanoScope IIIa (Digital Instruments, Santa Barbara, CA) atomic force microscope (AFM) with the built-in Nanoscope IIIa software version 4.23r6 was used to produce high-resolution surface images. The surfacs were scanned in the tapping mode with a Pointprobe silicon tip (type: NCH-W) of radius
