Immobilization of Amine-Modified Oligonucleotides on Aldehyde

Dec 9, 2004 - Dora Peelen and Lloyd M. Smith*. Department of Chemistry, 1101 ... imide functionality directly on an alkanethiol used in SAM formation...
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Immobilization of Amine-Modified Oligonucleotides on Aldehyde-Terminated Alkanethiol Monolayers on Gold Dora Peelen and Lloyd M. Smith* Department of Chemistry, 1101 University Avenue, University of Wisconsin, Madison, Wisconsin 53706-1396 Received July 21, 2004. In Final Form: October 11, 2004 Chemistry is described for the fabrication of DNA arrays on gold surfaces. Alkanethiols modified with terminal aldehyde groups are used to prepare a self-assembled monolayer (SAM). The aldehyde groups of the monolayer may be reacted with amine-modified oligonucleotides or other amine-bearing biomolecules to form a Schiff base, which may then be reduced to a stable secondary amine by treatment with sodium cyanoborohydride. The surface modifications and reactions are characterized by polarization modulation Fourier transform infrared reflection absorption spectroscopy (PM-FTIRRAS), and the accessibility, binding specificity, and stability of the DNA-modified surfaces are demonstrated in hybridization experiments.

Introduction Array technology has become an important tool for the parallel analysis of biomolecules.1-3 Examples include the development of DNA arrays for gene expression measurements,3 protein arrays for evaluation of protein-protein interactions,1,2 and peptide4,5 and carbohydrate arrays6,7 for the evaluation of ligand-receptor interactions and for screening enzymatic activities. A critical element in all such applications is a versatile and stable surface and surface attachment chemistry. We describe here such a chemistry and demonstrate its use in DNA array applications. A number of different types of surfaces have been employed for biomolecular array applications, including glass, gold, silicon, diamond, and glassy carbon.8-11 Attributes of gold surfaces include their ability to support the formation of SAMs, and compatability with surface plasmon resonance (SPR) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detection.12-14 Imaging SPR opens up the possibility of parallel detection without a need for labeling.14 Gold is * Corresponding author. E-mail: [email protected]. (1) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 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, 21012105. (3) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467-470. (4) Houseman, B. T.; Huh, J. H.; Kron, S. J.; Mrksich, M. Nat. Biotechnol. 2002, 20, 270-274. (5) Wegner, G. J.; Lee, H. J.; Corn, R. M. Anal. Chem. 2002, 74, 5161-5168. (6) Wang, D. N.; Liu, S. Y.; Trummer, B. J.; Deng, C.; Wang, A. L. Nat. Biotechnol. 2002, 20, 275-281. (7) Smith, E. A.; Thomas, W. D.; Kiessling, L. L.; Corn, R. M. J. Am. Chem. Soc. 2003, 125, 6140-6148. (8) Strother, T.; Hamers, R. J.; Smith, L. M. Nucleic Acids Res. 2000, 28, 3535-3541. (9) Strother, T.; Cai, W.; Zhao, X. S.; Hamers, R. J.; Smith, L. M. J. Am. Chem. Soc. 2000, 122, 1205-1209. (10) Strother, T.; Knickerbocker, T.; Russell, J. N.; Butler, J. E.; Smith, L. M.; Hamers, R. J. Langmuir 2002, 18, 968-971. (11) Yang, W.; Auciello, O.; Butler, J. E.; Cai, W.; Carlisle, J. A.; Gerbi, J. E.; Gruen, D. M.; Knickerbocker, T.; Lasseter, T. L.; Russell, J. N., Jr.; Smith, L. M.; Hamers, R. J. Nat. Mater. 2002, 1, 253-257. (12) Su, J.; Mrksich, M. Angew. Chem., Int. Ed. 2002, 41, 47154718. (13) Mouradian, S.; Nelson, C. M.; Smith, L. M. J. Am. Chem. Soc. 1996, 118, 8639-8645. (14) Brockman, J. M.; Nelson, B. P.; Corn, R. M. Annu. Rev. Phys. Chem. 2000, 51, 41-63.

also an electroactive substrate enabling electrochemical detection of biomolecules and modulation of surface properties by electrochemical means.15,16 A general approach for producing microarrays is to modify the biomolecule of interest with a functional group that allows covalent attachment to a reactive group on the surface. In the case of peptides and proteins, the reactive groups are often provided by the side chains of cysteine (thiol) and lysine (primary amine) residues. Amine and thiol modifications of synthetic oligonucleotides are also readily available. Many immobilization methodologies have taken advantage of the reactivity of thiol groups at neutral pH. Amine-terminated SAMs can be reacted with sulfosuccinimidyl 4-(N-maleimidomethyl cyclohexane)-1-carboxylate (SSMCC) to create a maleimide-terminated surface, which can in turn react with thiol-containing biomolecules.17-19 Mrksich created maleimide-terminated gold surfaces by creating the maleimide functionality directly on an alkanethiol used in SAM formation.20 A thiol-disulfide exchange reaction has been employed to couple thiol-containing biomolecules21,22 to a pyridyl disulfide-modified surface. Lam and co-workers prepared peptide arrays on glass slides by the reaction of N-terminal cysteine residues with immobilized aldehyde groups via thiazolidine ring formation.23 The disadvantage of using an immobilization chemistry involving the sulfhydryl group is that the sulfhydryl forms a disulfide bond in the presence of oxygen. The disulfide bond has to be reduced prior to immobilization, and the reducing agent (typically DTT or tris (2-carboxyethyl)phosphine (TCEP)) (15) Yousaf, M. N.; Mrksich, M. J. Am. Chem. Soc. 1999, 121, 42864287. (16) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (17) Brockman, J. M.; Frutos, A. G.; Corn, R. M. J. Am. Chem. Soc. 1999, 121, 8044-8051. (18) Oh, S. J.; Cho, S. J.; Kim, C. O.; Park, J. W. Langmuir 2002, 18, 1764-1769. (19) MacBeath, G.; Koehler, A. N.; Schreiber, S. L. J. Am. Chem. Soc. 1999, 121, 7967-7968. (20) Houseman, B. T.; Gawalt, E. S.; Mrksich, M. Langmuir 2003, 19, 1522-1531. (21) Smith, E. A.; Wanat, M. J.; Cheng, Y. F.; Barreira, S. V. P.; Frutos, A. G.; Corn, R. M. Langmuir 2001, 17, 2502-2507. (22) Rogers, Y. H.; Jiang-Baucom, P.; Huang, Z. J.; Bogdanov, V.; Anderson, S.; Boyce-Jacino, M. T. Anal. Biochem. 1999, 266, 23-30. (23) Falsey, J. R.; Renil, M.; Park, S.; Li, S. J.; Lam, K. S. Bioconjugate Chem. 2001, 12, 346-353.

10.1021/la048166r CCC: $30.25 © 2005 American Chemical Society Published on Web 12/09/2004

Immobilization of Amine-Modified Oligonucleotides

has to be separated from the solution of biomolecules.24 Purified thiol-modified probes have a limited lifetime and therefore may be stored only for a short time. These purification and storage issues become a problem when a complex array (e.g., hundreds to thousands of different probes immobilized) has to be constructed. Another approach is to use a Diels-Alder reaction between ligandcyclopentadiene conjugates and quinine groups immobilized on SAMs.4 Although this is an interesting and novel linking chemistry, it requires the biomolecule to be modified with cyclopentadiene, which is not a naturally occurring functional group (hence not naturally present in peptides and proteins) and is also not readily available for biomolecule modification at present. We present here a new surface attachment chemistry based upon the coupling of an amine group to aldehyde-modified gold surfaces. An amine can react with carboxylic acid-,25 aldehyde-,23,26,27 epoxy-,28 and isothiocyanate29,30-functionalized surfaces. Several papers have evaluated the performance of oligonucleotide arrays on aldehyde- or epoxy-modified glass substrates.31,32 Horton et al. reported the preparation of aldehyde-terminated SAMs on Au by exploiting the equilibrium between 2-hydroxypentamethylene sulfide (HPMS) and its open-chain aldehyde isomer.26 The aldehyde-terminated gold surface was reacted with alkylamines to form a Schiff base.26 However, the Schiff base linkage proved to be unstable in air as observed by the reduction in the intensity of the CdN (imine) band in the FTIR spectra.26 The Schiff base linkage is prone to hydrolysis: this instability can be eliminated by reducing the imine with NaBH4 or NaBH3CN to generate a stable secondary amine linkage. It was proposed that such surfaces may be useful for attaching biomolecules for sensor applications. We report here the synthesis and use of aldehyde alkanethiols to create aldehyde functionalities on gold surfaces via alkanethiol self-assembled monolayer formation. An 11-carbon alkane chain was employed, which has been shown to increase SAM stability and produce a more ordered monolayer than a shorter 5-carbon alkane chain.26 The aldehyde alkanethiol surfaces were characterized using PM-FTIRRAS. The utility of this surface attachment chemistry was demonstrated by fabricating and characterizing DNA arrays, and demonstrating their utility in DNA hybridization experiments. Experimental Section Materials and Reagents. Commercial gold slides (5 nm Cr, 100 nm Au) were purchased from Evaporated Metal Films Co. (Ithaca, NY). The following oligonucleotides were synthesized by the University of Wisconsin Biotechnology Center (reading in the 5′ f 3′ direction): oligonucleotide 1, FAM GCT TTT GCA (24) Shafer, D. E.; Inman, J. K.; Lees, A. Anal. Biochem. 2000, 282, 161-164. (25) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999, 71, 777-790. (26) Horton, R. C.; Herne, T. M.; Myles, D. C. J. Am. Chem. Soc. 1997, 119, 12980-12981. (27) Schena, M.; Shalon, D.; Heller, R.; Chai, A.; Brown, P. O.; Davis, R. W. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 10614-10619. (28) Lamture, J. B.; Beattie, K. L.; Burke, B. E.; Eggers, M. D.; Ehrlich, D. J.; Fowler, R.; Hollis, M. A.; Kosicki, B. B.; Reich, R. K.; Smith, S. R.; Varma, R. S.; Hogan, M. E. Nucleic Acids Res. 1994, 22, 2121-2125. (29) Guo, Z.; Guilfoyle, R. A.; Thiel, A. J.; Wang, R. F.; Smith, L. M. Nucleic Acids Res. 1994, 22, 5456-5465. (30) Charles, P. T.; Vora, G. J.; Andreadis, J. D.; Fortney, A. J.; Meador, C. E.; Dulcey, C. S.; Stenger, D. A. Langmuir 2003, 19, 15861591. (31) Lindroos, K.; Liljedahl, U.; Raitio, M.; Syvanen, A. C. Nucleic Acids Res. 2001, 29, e69. (32) Zammatteo, N.; Jeanmart, L.; Hamels, S.; Courtois, S.; Louette, P.; Hevesi, L.; Remacle, J. Anal. Biochem. 2000, 280, 143-150.

Langmuir, Vol. 21, No. 1, 2005 267 GGT CAT CGG-(spacer 18)10-NH2; oligonucleotide 2, FAM GCT TTT GCA GGT CAT CGG-(spacer 18)10-A; oligonucleotide 3, CTT CTG CAG GTC ATC GG-(spacer 18)10-NH2; oligonucleotide 4, CCG ATG ACC TGC AGA AGT-FAM; oligonucleotide 5, AAT GGT GGA AAT-T15-NH2; oligonucleotide 6, ATT TCC ACC ATTFAM; oligonucleotide 7, FAM GCT TTT GCA GGT CAT CGG(spacer 18)10-SH. All oligonucleotides were purified by reversephase HPLC with a binary gradient elution. The 3′-amine modifier was C3 phthalamide (Glen Research, Sterling, VA), and Spacer Phosphoramidite 18 (Glen Research) was used for the addition of the ethylene glycol spacer region. 11-Amino-1undecanethiol hydrochloride was purchased from Dojindo, absolute ethanol was from AAPER Alcohol and Chemical Co. (Shelbyville, KY), and SSMCC was from Pierce (Rockford, IL). Coupling of Amine-Modified Oligonucleotide to Aldehyde-Terminated Surfaces. Au-coated slides were placed in 1-10 mM di(10-decanal) disulfide (5) solution overnight. Each slide was rinsed well with 100 mL of ethanol and then with 100 mL of deionized (DI) water and dried under a stream of N2 gas. 3′-Amine-terminated oligonucleotide was diluted to ∼1 mM in 0.1 M NaHCO3 pH 10. A fresh solution of 50 mM NaBH3CN in 0.1 M NaHCO3 pH 10 was mixed with the oligonucleotide in a 1:1 volume ratio. Next, 0.4-0.5 µL of the (oligonucleotide + NaBH3CN) solution was spotted onto the amine reactive surface. Slides were allowed to react in a dry chamber overnight. They were then washed with DI water and incubated in 2×SSPE (0.3 M NaCl, 20 mM Na3PO4, 2 mM EDTA)/0.2% SDS (sodium dodecyl sulfate) for 1 h at 37 °C to remove excess unbound oligonucleotide. Coupling of Thiol Oligonucleotide to Maleimide-Terminated Surfaces. Au-coated slides were placed in 1-10 mM MUAM (11-amino-1-undecanethiol) solution overnight. Each slide was rinsed well with 100 mL of ethanol and then with 100 mL of DI water and dried under a stream of N2 gas. A fresh solution of SSMCC solution (0.4 mg/mL) was prepared in 0.1 M TEA pH 7 buffer. The slides were placed in a humidified chamber, and 500 µL of SSMCC solution/slide was uniformly dispersed onto the slides. The reaction was allowed to proceed for 15-20 min. The slide was then briefly rinsed with DI water and dried under a stream of N2 gas. 3′-Thiol-terminated oligonucleotide was diluted to ∼1 mM in 0.1 M TEA pH 7. Next, 0.4-0.5 µL of the oligonucleotide solution was spotted onto the thiol reactive surface for each spot. This produces spots of approximately 1 mm in diameter. Slides were allowed to react in a humid chamber overnight. They were then washed with DI water and incubated in 2×SSPE/0.2% SDS for 1 h at 37 °C to remove excess unbound oligonucleotide. Determination of Oligonucleotide Density on the Surface. A surface modified with single-stranded DNA as described above was incubated with 2 µM fluorescently labeled complement in 2×SSPE/0.2% SDS buffer for 30 min (in a total hybridization volume of 200 µL). The surface was rinsed with hybridization buffer (2 × 5 min). The chip was placed in 7 mL of 130 mM KOH/50 mM KCl solution for 5 min to elute the hybridized DNA from the surface. After removal of the chip, the concentration of the fluorescent complement remaining in the solution was determined as follows: calibration solutions in the range of 10-11 to 10-8 M were prepared by dissolving the fluorescent complements in 132 mM KOH/50 mM KCl. Using a fluorescence plate reader (BIOTEK, Flx 800, 100 µL/well), the fluorescence from the calibration solutions and the unknown samples was measured and the density of the hybridized DNA was calculated. Polarization Modulation FTIR Reflection Absorption Spectroscopy (PM-FTIRRAS). IR spectra in the 400-2000 cm-1 region were collected with a Mattson RS-1 spectrometer with real-time interferogram sampling electronics and optical layout (HgCdTe detector) as described previously.33,34 Spectra in the 2000-4000 cm-1 region were recorded using a Bruker Vector 22 spectrometer and InSb detector. Synthesis of Di(10-decanal) Disulfide (5). Di(10-decanal) disulfide (5) was synthesized from di(10,11-dihydroxyundecyl) disulfide (4) (step E in Figure 1). The synthesis of (4) was carried (33) Barner, B. J.; Green, M. J.; Saez, E. I.; Corn, R. M. Anal. Chem. 1991, 63, 55-60. (34) Green, M. J.; Barner, B. J.; Corn, R. M. Rev. Sci. Instrum. 1991, 62, 1426-1430.

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Peelen and Smith Di(10-decanal) Disulfide (5). This was adapted from the procedure by Marshall et al.36 To a stirred solution of 0.46 mmol (200 mg) of di(10,11-dihydroxyundecyl) disulfide 4 in 12.7 mL of dioxane and 5.1 mL of water was added 1 mmol (2.2 equiv, 214 mg) of NaIO4 in small portions. After 16 h, the reaction was poured into water and extracted with ethyl acetate and ether. The combined organic layers were washed with water and brine and dried over anhydrous MgSO4. The solvent was removed, and the material was taken in 10 mL of ether, washed again with water and brine, and dried over anhydrous MgSO4. Removal of solvent gave 155 mg of 5 as a yellow-white solid, 90% yield. 1H NMR (CDCl ) δ: 9.77 (t, J ) 1.8 Hz, 1H), 2.67 (t, J ) 7.4 Hz, 3 2H), 2.42 (td, J1 ) 7.2 Hz, J2 ) 1.8 Hz, 2H), 1.64 (m, 4H), 1.35 (m, 4H), 1.3-1.2 (m, 10H).

Results and Discussion Figure 1. Synthesis of di(10-decanal) disulfide 5. Reaction conditions: (A) OsO4(cat), tert-butyl hydroperoxide, Et4NOH, tert-butyl alcohol, 2000 cm-1 region (data not shown). The Fermi resonance between the aldehyde C-H stretch and the first overtone of the C-H bending vibration is not detected because it is below the detection limit, but the C-H stretching region absorbance bands at 2857 and 2926 cm-1 assigned respectively to the symmetric CH2 stretch and the asymmetric CH2 stretch were clearly visible. There is a broad band centered around 1120 cm-1 which could be assigned either to a C-O-C antisymmetric stretch in aliphatic ethers or to a CdS stretch in thiocarbonyl compounds (Figure 3A). Because this 1120 cm-1 band disappears after washing the surface with 10 mM HEPES pH 8.5 buffer overnight (Figure 3B), we attribute it to contaminating material physisorbed on the surface from the air or from the di(10-decanal) disulfide/ethanol solution. The reaction of methylamine with the aldehyde surface was monitored under various reaction conditions. Reaction of aldehyde with primary amine results in the formation of a Schiff base (Figure 2), whose vibrational signature at 1674 cm-1 (CdN stretch) has been reported previously.26 The Schiff base is water sensitive; that is, addition of water to the imine favors formation of the reactants, aldehyde and primary amine. Accordingly, when methylamine is reacted with the aldehyde surface in aqueous buffer (10 mM HEPES pH 8.5), both the imine CdN (at 1678 cm-1) and the carbonyl CdO (at 1732 cm-1) stretch are present in the spectrum (Figure 3C). On the other hand, if methylamine is reacted with the aldehyde surface in ethanol, that is, no water is present in the reaction environment, no carbonyl is left and only the imine peak is present in accordance with previous reports (Figure 3D).26 Although one can convert an aldehyde to a Schiff base quantitatively by removing water from the reaction environment, the resulting imine is not stable in air and can react with water vapor present in the air. In fact, Horton et al. observed the reduction of the CdN stretch band in the FTIR spectra to less than 20% of its original value over 3 days when the surface was exposed to ambient air.26 To solve the problem of instability, the imine can be reduced to a secondary amine using reducing agents NaBH3CN or NaBH4 (Figure 2).31,32,37,38 After reaction with (36) Marshall, J. A.; Flynn, K. E. J. Am. Chem. Soc. 1984, 106, 723730. (37) Dombi, K. L.; Griesang, N.; Richert, C. Synthesis 2002, 6, 816824.

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Figure 2. The reaction of primary amines with aldehyde functionalities on the gold surface.

Figure 3. PM-FTIRRAS spectra for aldehyde-terminated monolayers on gold. (A) Au slide immersed in 1 mM aldehyde/ ethanol solution; (B) aldehyde-terminated Au surface after incubation with 10 mM HEPES pH 8.5 buffer; (C) aldehydeterminated Au slide after reaction with 100 µM methylamine in 10 mM HEPES pH 8.5; (D) aldehyde-terminated Au slide after reaction with 100 µM methylamine in ethanol; and (E) aldehyde-terminated Au slide after reaction with 100 µM methylamine in 10 mM HEPES pH 8.5 in the presence of 50 mM NaBH3CN.

methylamine in the presence of NaBH3CN, we see the CdO stretch reduced in intensity and a new band appears at 1144 cm-1 which is assigned to the C-N stretch of secondary amines (Figure 3E). No residual imine peak at 1674 cm-1 is observed in Figure 3E, indicating that the imine bond has been quantitatively reduced to a secondary amine. Having characterized the reactivity of the surface to primary amines, we sought to immobilize oligonucleotides that had been modified to include a primary amine at the 3′-terminus. We were particularly interested in evaluating the possible reactivity of amines present in the nucleobases toward the aldehyde as well as the reactivity of the terminal primary amine at the 3′-terminus of the oligonucleotide. To evaluate this issue, the aldehyde surface was reacted with oligonucleotides with and without the 3′-amino modification (oligonucleotides 1 and 2). Figure 4A shows that the oligonucleotide with the 3′-amine modification reacted efficiently with the surface. In a separate experiment, the density of fluorescent oligonucleotides that hybridized to the surface was determined to be 5 × 1012 oligonucleotides/cm2 (8 pmol/cm2), which is (38) Kremsky, J. N.; Wooters, J. L.; Dougherty, J. P.; Meyers, R. E.; Collins, M.; Brown, E. L. Nucleic Acids Res. 1987, 15, 2891-2909.

the same as that observed for the maleimide/SH-DNA surface. On the other hand, oligonucleotide with no primary amine gave very low fluorescent intensity (0.2% of the signal from the sample with primary amine), indicating minimal nonspecific adsorption and the absence of reactivity with the nitrogens present in the nucleobases. We tested a number of reaction conditions such as buffer pH, solvent composition, and oligonucleotide and NaBH3CN concentration (Table 1). Published procedures for reacting aldehyde with amine-modified oligonucleotides on glass employ acidic reaction conditions (pH 5, 6.5).32,38 This is in accordance with the reaction mechanism, where the first step is the attack of the carbonyl oxygen by a proton which then facilitates nucleophilic attack on the carbonyl carbon by the primary amine. On the other hand, one can argue that basic conditions are necessary so that the amine is present in its free base form. We performed the coupling reaction over a wide range of pH values (pH 4, 5, 6.5, 7.3, 8.5, 10, 12) and found that the greatest fluorescence intensity (most oligonucleotide attached to the surface) occurred at pH 10, consistent with the latter argument (Table 1). We also investigated whether addition of NaBH3CN immediately or after imine formation results in better attachment. We found that adding the NaBH3CN immediately to the oligonucleotide coupling mixture results in a higher density of immobilized probe judged by the increase in fluorescence intensity. When using NaBH4 as a reducing agent, no attachment was achieved, possibly because NaBH4 is a stronger reducing agent than NaBH3CN and can reduce the aldehyde itself or the Au-S bond itself (AuSR + 1e- f Au(0) + RS-).39 Furthermore, we observed that increasing the NaBH3CN concentration over 50-500 mM decreases the amount of oligonucleotide immobilized, probably for the same reasons. We also tried to replace aqueous buffers with organic solvents to remove water from the reaction. We replaced the 0.1 M NaHCO3 pH 10 buffer with 20% or 50% dimethyl sulfoxide or 10% or 20% acetonitrile. The attachment of fluorescent oligonucleotide decreased as the amount of the organic solvent increased. The most successful strategy for efficient DNA coupling was to let the DNA solution dry out on the surface, which increased the measured fluorescence intensity by an order of magnitude (Table 1). One undesired consequence of allowing the DNA spots to dry out on the surface was that it gave rise to nonuniform surface coverage in some of the experiments performed.40 In conclusion, the best conditions for preparation of an oligonucleotide-modified aldehyde/Au surface are to incubate a 1 mM oligonucleotide solution (0.1 M NaHCO3 (39) Quinn, B. M.; Kontturi, K. J. Am. Chem. Soc. 2004, 126, 71687169. (40) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827-829.

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Figure 4. Reaction of the aldehyde surface with amine-modified oligonucleotides. Surfaces were prepared as described in the Experimental Section. (A) Oligonucleotide 1 (spots on the left, 3′ amine) and oligonucleotide 2 (spots on the right, no primary amine modification) were reacted with the aldehyde-terminated Au surface. (B) Oligonucleotide 3 (top spot) and oligonucleotide 5 (bottom spot) were reacted with the aldehyde-terminated Au surface, and oligonucleotide 4, complement to oligonucleotide 3, was added to the chip. (C) Surface shown in (B) was denatured to remove the oligonucleotide 4 complement. Oligonucleotide 3 (top spot) and oligonucleotide 5 (bottom spot) were reacted with the aldehyde-terminated Au surface, and oligonucleotide 6, complement to oligonucleotide 5, was added to the chip. (D) Histogram of fluorescence intensities from image A. (E) Histogram of fluorescence intensities from images B and C. Table 1. Relative Fluorescence Intensity Obtained from Aldehyde/Amine Attachment under Various Reaction Conditionsa conditions pH 4 pH 5 pH 6.5 pH 7.3 pH 8.5 pH 10 pH 12 ArrayIt (pH 8)b pH 10, 0% DMSO 20% DMSO 50% DMSO pH 10, NaBH3CN immediately day after NaBH4 pH 10, 0% ACN 10% 20% 50 mM NaBH3CN 100 500 humid conditions dry conditions

fluorescence intensity 7900 7900 4000 8700 11 000 28 000 13 000 700 20 000 4700 1200 20 000 4000 10 21 700 22 400 5500 40 000 26 000 8000 2000 35 000

a DMSO stands for dimethyl sulfoxide, and ACN stands for acetonitrile. b ArrayIt is a commercial buffer for microarray preparation available from Telechem (Sunnyvale, CA).

pH 10 buffer mixed with 50 mM NaBH3CN) with the surface in a dry chamber (as opposed to a humid chamber) overnight at room temperature. The procedure in the

Figure 5. Comparison of the stability of aldehyde/NH2-DNA and maleimide/SH-DNA surfaces. [ symbols denote the aldehyde/NH2-DNA surface, and 2 symbols denote the maleimide/ SH-DNA surface. Surfaces were prepared as described in the Experimental Section. The surface fluorescence intensity was measured periodically and divided by the fluorescence intensity of the freshly prepared sample to obtain the relative fluorescence intensity. Samples were kept in 2 × SSPE/0.2% SDS at room temperature between measurements.

Experimental Section follows these conditions, and the surfaces shown in Figures 4 and 5 were prepared in this manner. The surfaces were also characterized with respect to DNA hybridization (Figure 4B and C). Two oligonucleotides with different sequences (oligonucleotides 3 and 5)

Immobilization of Amine-Modified Oligonucleotides

were immobilized on the aldehyde-terminated Au surface as individual spots. The DNA-modified surface was covered with a solution of fluorescently labeled oligonucleotide (oligonucleotide 4) complementary to oligonucleotide 3 and allowed to hybridize. Figure 4B shows the image after hybridization and washing. Only the top spot containing oligonucleotide 3 fluoresced, indicating that oligonucleotide 4 selectively hybridized to its complementary probe on the surface. After denaturation with 8.3 M urea, the fluorescently labeled complement (oligonucleotide 6) to oligonucleotide 5 was added to the surface. After hybridization and washing, the fluorescence image revealed only the bottom spot where oligonucleotide 5 was immobilized (Figure 4C). These results demonstrate the accessibility of the surface-bound DNA to specific hybridization. The stability of the DNA-modified gold surface was evaluated by monitoring fluorescence from the covalently attached fluorescent oligonucleotides (Figure 5). Oligonucleotides were fluorescently labeled at the 5′-end and modified with an amino group on the 3′-end (oligonucleotide 1). After the covalent attachment of the oligonucleotide, the surface was stored overnight at room temperature in 1×SSPE/0.2% SDS. The fluorescence intensity of the surface was measured by fluorescence imaging, and

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then the surface was returned to the buffer. In parallel, a maleimide/thiol-DNA surface was prepared, and the same experiment was carried out. Figure 5 shows that over the course of 27 h no significant decrease in fluorescence intensity was observed for the aldehyde/ amine-DNA surface. A small (∼10%) decrease was observed for the maleimide/thiol-DNA surface. We concluded that the stability of the two surfaces is similar and they are stable under the hybridization conditions used here. Conclusions Aldehyde-modified alkanethiols were synthesized and used to create aldehyde-modified gold surfaces via alkanethiol self-assembled monolayer formation. The formation and reactivity of the aldehyde alkanethiol surfaces were characterized using PM-FTIRRAS. The fidelity and stability of the DNA-modified surface were demonstrated in DNA hybridization experiments. Acknowledgment. We would like to thank Professor R. Corn for the use of the PM-FTIRRAS instrument. This work was supported by NIH grant 5ROIEB00269 and NSF grant EIA0203892. LA048166R