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Controlling DNA Orientation on Mixed ssDNA/OEG SAMs Christina Boozer, Shengfu Chen, and Shaoyi Jiang* Department of Chemical Engineering, UniVersity of Washington, Seattle, Washington 98195 ReceiVed October 28, 2005. In Final Form: February 3, 2006 We present and characterize a mixed self-assembled monolayer (SAM) consisting of single-stranded oligonucleotide (ssDNA)- and oligo(ethylene glycol) (OEG)-terminated thiols. The ssDNA/OEG SAMs are prepared by simultaneous coadsorption from a common thiol solution over a broad range of compositions. Electron spectroscopy for chemical analysis (ESCA) is used to measure the surface coverage of ssDNA, whereas surface plasmon resonance (SPR) sensor is used to measure the hybridization of complementary ssDNA and protein resistance. Through the complementary use of these techniques, we find that the composition of OEG in the assembly solution controls a key parameter: the surface coverage of ssDNA on the surface. There is evidence that it influences the orientation of the immobilized ssDNA probes. Lower OEG concentrations yield a surface with higher ssDNA coverage and less favorable orientation, whereas higher OEG concentrations produce a surface with lower DNA coverage and more favorable orientation. Competition between these two effects controls the hybridization efficiency of the ssDNA surface. Compared to ssDNA surfaces prepared with other diluent thiols, the use of OEG improves the protein resistance of the surface, making it more broadly applicable.
Introduction Surface-bound single-stranded oligonucleotide (ssDNA) probe arrays are a valuable tool with a broad assortment of biotechnology applications, including gene mapping,1,2 molecular diagnostics2, and biosensors.2-4 Regardless of the particular use, all DNA arrays are designed with a similar goal: the ssDNA probe sequences should be tethered to a solid support via a chemical linker group in a way that maximizes both the hybridization efficiency and specificity. Although commercial DNA arrays are most commonly prepared by microjet deposition5,6 or in situ photolithography,7,8 investigators have recently begun immobilizing ssDNA on gold surfaces via a thiol linkage, effectively forming an ssDNA self-assembled monolayer (SAM). Tarlov et al.9 have performed extensive studies on ssDNA probe surfaces prepared using a two-step sequential adsorption process. First, pure DNA thiols are immobilized on a clean gold surface, followed by backfilling with mercaptohexanol (MCH). Mercaptohexanol was chosen because it has been shown to resist nonspecific binding of noncomplementary target DNA, it is watersoluble, and its six-carbon chain is the same length as the C6 spacer group in HS-ssDNA. Additionally, it has been speculated that MCH helps reduce nonspecific binding of the DNA thiol to the gold surface, thereby improving the DNA probe orientation and hybridization efficiency. This approach has been extensively studied.9-19 Others have prepared DNA surfaces by the reverse * Corresponding author. E-mail:
[email protected]. (1) Lockhart, D. J.; Winzeler, E. A. Nature 2000, 405, 827-836. (2) Wang, J. Nucleic Acids Res. 2000, 28, 3011-3016. (3) Miller, M. M.; Sheehan, P. E.; Edelstein, R. L.; Tamanaha, C. R.; Zhong, L.; Bounnak, S.; Whitman, L. J.; Colton, R. J. J. Magn. Magn. Mater. 2001, 225, 138-144. (4) Boozer, C. L.; Ladd, J.; Chen, S.; Yu, Q.; Homola, J.; Jiang, S. Anal. Chem. 2004, 76, 6967-6972. (5) Marshall, A.; Hodgson, J. Nat. Biotechnol. 1998, 16, 27-31. (6) Heller, M. J. Annu. ReV. Biomed. Eng. 2002, 4, 129-153. (7) Pease, A. C.; Solas, D.; Sullivan, E. J.; Cronin, M. T.; Holmes, C. P.; Fodor, S. P. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 5022-5026. (8) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767-773. (9) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (10) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (11) Steel, A. B.; Herne, T. M.; Tarlov, M. J. Anal. Chem. 1998, 70, 0, 46704677.
approach: assembling a shorter-chain thiol first, followed by backfilling with ssDNA thiols.20 In this article, we present and characterize a mixed ssDNA and oligo(ethylene glycol) (OEG) SAM prepared by coadsorption from a single thiol solution. In DNA array applications such as biosensors, the surface can potentially interact with both DNA and proteins and therefore needs to be resistant to the nonspecific adsorption of proteins as well as noncomplementary DNA. Although mixed SAMs of DNA and MCH resist nonspecific binding of noncomplementary DNA strands, MCH is not sufficiently protein-resistant. Modification of surfaces by OEG is a common and effective means of rendering surfaces proteinresistant,21,22 and OEG-terminated thiols23-27 have been employed extensively for passivating gold surfaces. Coadsorption of the ssDNA and OEG thiols allows for a simple single-step assembly procedure where the surface density of DNA is controlled by (12) Steel, A. B.; Levicky, R. L.; Herne, T. M.; Tarlov, M. J. Biophys. J. 2000, 79, 975-981. (13) Petrovykh, D. Y.; Kimura-Suda, H.; Whitman, L. J.; Tarlov, M. J. J. Am. Chem. Soc. 2003, 125, 5219-5226. (14) Peterson, A. W.; Wolf, L. K.; Georgiadis, R. M. J. Am. Chem. Soc. 2002, 124, 14601-14607. (15) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. M. Nucleic Acids Res. 2001, 29, 5163-5168. (16) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. J. Am. Chem. Soc. 2000, 122, 7837-7838. (17) Peterlinz, K. A.; Georgiadis, R. M. J. Am. Chem. Soc. 1997, 119, 34013402. (18) Heaton, R. J.; Peterson, A. W.; Georgiadis, R. M. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 3701-3704. (19) Georgiadis, R.; Peterlinz, K. P.; Peterson, A. W. J. Am. Chem. Soc. 2000, 122, 3166-3173. (20) Satjapipat, M.; Sanedrin, R.; Zhou, F. M. Langmuir 2001, 17, 76377644. (21) Harris, J. M. Poly(ethylene glycol) Chemistry: Biotechnical And Biomedical Applications; Plenum Press: New York, 1992. (22) Harris, J. M., Ed; Zalipsky, S., Eds. Poly(ethylene glycol): Chemistry And Biological Applications; American Chemical Society: Washington, DC, 1997. (23) Prime, K. L.; Whitesides, G. M. J. Am. Chem. Soc. 1993, 115, 1071410721. (24) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164-1167. (25) Ostuni, E.; Yan, L.; Whitesides, G. M. Colloids Surf., B 1999, 15, 3-30. (26) Mrksich, M.; Whitesides, G. M. In Poly(ethylene glycol); Harris, J. M., Zalipsky, S., Eds.; American Chemical Society: Washington, DC, 1997; Vol. 680, pp 361-373. (27) Mrksich, M.; Sigal, G. B.; Whitesides, G. M. Langmuir 1995, 11, 43834385.
10.1021/la052908e CCC: $33.50 © 2006 American Chemical Society Published on Web 04/05/2006
Controlling DNA Orientation on Mixed SAMs
varying the composition of the SAM assembly solution. The result is a convenient method for preparing a mixed ssDNA/ OEG SAM that selectively hybridizes with complementary ssDNA yet resists nonspecific binding of both proteins and other DNA sequences. Materials and Methods Sequence Selection. All plain and modified (5′ thiolated) DNA sequences were custom synthesized by Synthegen. Two DNA probe sequences were used. The surface characterization was performed on surfaces prepared with sequence A (5′ TCC TGT GTG AAA TTG TTA TCC GCT 3′) whereas sequence B (5′ GTA ATC ATG GTC ATA GCT GTT 3′) was used for control experiments to test cross reactivity. Hybridization to the surface-bound probes was measured using the perfect complements to these sequences (denoted c-A or c-B). Additionally, a truncated version of c-A was formed by removing three bases from the 3′ end of the sequence. Where necessary, the full-length and truncated complements are distinguished using their respective lengths (c-A 24 and c-A 21). The sequences used in this work were chosen because they have been shown previously to have high hybridization efficiency.28 The OEGterminated thiols (HS(CH2)11(OCH2)4OH) were purchased from ProChimia (Poland). Substrate Preparation. Gold-coated silicon wafers were used as substrates for electron spectroscopy for chemical analysis (ESCA) measurements. Surface plasmon resonance (SPR) sensor substrates were prepared by coating clean BK-7 glass substrates with a 2 nm adhesion layer of chromium followed by a 50 nm layer of gold. Both the ESCA and SPR substrates were coated by electron beam evaporation at pressures below 1 × 10-6 Torr. Surface Functionalization. Prior to surface functionalization, all chips were rinsed with ethanol and water, blown dry with nitrogen, and cleaned by 20 min of UV/ozone treatment. After UV treatment, chips were rinsed again with water and ethanol and dried under a stream of N2. Chips were functionalized immediately following cleaning. Pure ssDNA or mixed ssDNA/OEG SAMs were formed by immersing clean Au chips in a 1.0 M KH2PO4 buffer solution of ssDNA and OEG thiols. The ssDNA thiol concentration was held constant at 100 nM for all experiments, whereas the OEG thiol concentration ranged from 0 to 100 µM. For the two-step assembly procedure, chips were immersed in 5 µM OEG thiol for a set amount of time and then transferred to a 100 nM DNA thiol solution. Following overnight assembly, the samples were rinsed thoroughly with water and dried with nitrogen. ESCA Measurements. ESCA measurements were performed on Surface Science Instruments X-Probe and S-Probe spectrometers. Both instruments are equipped with a monochromatized Al KR X-ray source and a hemispherical electron energy analyzer. Compositional survey and detail scans (P 2p, C 1s, N 1s, O 1s, and S 2p) were acquired using a pass energy of 150 eV. High-resolution spectra (C 1s and S 2p) were acquired for the DNA samples using a pass energy of 50 eV. For the high-resolution spectra, peak binding energies were referenced to the C 1s (C-C/C-H) peak at 285.0 eV. Three spots on two or more replicates of each DNA sample were analyzed. The compositional data are averages of the values determined at each spot. SPR Sensor Surfaces and Instrumentation. The SPR sensor setup has been described previously.29 Briefly, it is a custom-built instrument based on the Kretschmann configuration of the attenuated total reflection (ATR) method. The glass side of the gold-coated substrate is index matched to the prism coupler whereas the functionalized surface is mechanically pressed against an acrylic flow cell with a Mylar gasket. A polychromatic light beam is directed through the prism and the glass substrate and excites surface plasma waves at the metal-dielectric interface. The reflected light is analyzed with a spectrograph. (28) Niemeyer, C. M.; Sano, T.; Smith, C. L.; Cantor, C. R. Nucleic Acids Res. 1994, 22, 5530-5539. (29) Boozer, C. L.; Yu, Q. M.; Chen, S. F.; Lee, C. Y.; Homola, J.; Yee, S. S.; Jiang, S. Y. Sensors Actuators, B 2003, 90, 22-30.
Langmuir, Vol. 22, No. 10, 2006 4695 Table 1. Elemental Composition of a Pure ssDNA SAM as Measured by ESCA Composition of a Pure ssDNA SAM, Including the Substrate atom % experimental
C
N
O
P
S
Au
36.8
6.0
21.0
1.8
2.4
32.0
Composition of a Pure ssDNA SAM, Not Including the Substrate atom % experimental theoretical
atomic ratios
C
N
O
P
S
O/N
N/P
54.1 48.2
8.8 16.8
30.9 30.0
2.7 4.8
3.6 0.2
3.5 1.8
3.3 3.5
DNA and Protein Binding Experiments. All SPR experiments began with a buffer baseline (TE-NaCl: 10 mM tris-HCl with 1 mM EDTA, 1 M NaCl, pH 7.2) followed by the DNA or protein solution and a rinsing step. Complementary DNA (c-A or c-B) was used at 100 µM in TE-NaCl. The flow rate of all solutions was 50 µL/min.
Results and Discussion Surface Characterization by ESCA. The elemental composition of a pure ssDNA SAM (sequence A) was measured by electron spectroscopy for chemical analysis and is shown in Table 1. For comparison, the stoichiometric composition of this specific DNA thiol is also included. The presence of both N and P, from the nucleotide bases and backbone, respectively, is a strong indicator of DNA on the surface. Deviations in the measured composition from the stoichiometric composition are most likely due to contamination in the commercially purchased ssDNA thiol. It has been previously reported that the presence of these impurities will contribute additional C and S, thereby reducing the N and P concentrations.30 Note that the experimentally measured N/P ratio, which should not be affected by such contamination, is very close to the theoretical value. The surface coverage of DNA in the mixed ssDNA/OEG SAMs was calculated using the composition of a pure DNA SAM as a reference. A series of ssDNA/OEG SAMs were prepared with a range of OEG concentrations, and the elemental composition of each SAM was measured. Whereas C, O, and S are found in both DNA and OEG, N and P are elements unique to DNA. Because DNA contains more N atoms than P and hence produces a stronger signal, the percent of N in the pure DNA SAM was used as a reference for determining the DNA surface coverage as a function of solution composition. As shown in Figure 1, at very low DNA mole fractions (point a) the surface is dominated by OEG, and the DNA surface density is very low. As the DNA mole fraction in solution increases, the DNA surface coverage increases rapidly (point b) until a DNA mole fraction of about 0.2 is reached, where the surface coverage plateaus (points c and d). Note that the relationship between solution composition and surface composition is not linear: the ssDNA thiols show strong preferential adsorption over the OEG thiols. Sequence Specificity and Protein Resistance. Control experiments were performed to test the sequence specificity and protein resistance of the DNA/OEG SAM surface. To test the specificity of hybridization to the surface-bound ssDNA probes, the SPR response to both complementary and noncomplementary control oligonucleotide strands was measured. As shown in Figure 2, although the complement hybridized to the surface, there was no detectable binding of the noncomplementary control sequence. This is in agreement with previous results for DNA SAMs backfilled with MCH. (30) Lee, C. Y.; Canavan, H. E.; Gamble, L. J.; Castner, D. G. Langmuir 2005, 21, 5134-5141.
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Figure 1. Surface composition of mixed ssDNA/OEG SAMs as determined by ESCA. Figure 4. Complement hybridization to mixed ssDNA/OEG SAMs prepared by coadsorption.
Figure 2. Control experiment to test DNA specificity. Both c-A and c-B were flowed over a sequence A ssDNA/OEG SAM, but only the complementary DNA hybridized. The ssDNA/OEG surface was prepared from a solution with a DNA mole fraction of 0.02.
Figure 3. Comparison of protein resistance. BSA (1 mg/mL) was flowed over ssDNA SAMs prepared with MCH and OEG diluent thiols. The surface prepared with OEG is completely protein-resistant. The ssDNA/OEG surface was prepared from a solution with a DNA mole fraction of 0.02.
High concentration solutions of BSA were used to check the protein resistance of the DNA SAM surfaces. Figure 3 shows the SPR response of DNA SAMs with MCH and OEG backgrounds to a 1 mg/mL solution of BSA in TE-NaCl. Whereas
Figure 5. Complement hybridization to ssDNA/OEG SAMs prepared by the reverse two-step procedure (OEG thiols, followed by ssDNA).
the DNA/MCH SAM rapidly and irreversibly adsorbs large amounts of BSA, the DNA/OEG SAM is entirely resistant to the BSA. Complement Hybridization and DNA Orientation. Hybridization to ssDNA SAMs prepared with varying amounts of OEG thiol, and hence with varying probe densities, was measured by SPR and is shown in Figure 4. Complement hybridization exhibits a sharp maximum at a DNA mole fraction of approximately 0.02 (point b) and decreases rapidly at both higher and lower mole fractions. A comparison of Figure 4 with Figure 1 shows that the hybridization behavior is quite different from the DNA coverage that was measured by ESCA. First, the maximum hybridization was measured on a surface that contains only 60% of the maximum surface coverage (point b on each respective Figure). Additionally, ESCA suggests that all surfaces prepared with a DNA mole fraction greater than 0.2 should have approximately the same surface coverage, yet the SPR measurements indicate that complement hybridization decreases dramatically in this composition range (points c and d). We speculate that the observed variations in hybridization efficiency are due to the orientation of the ssDNA molecules. For maximum hybridization efficiency, the desired orientation of the DNA is perpendicular to the surface, with the ssDNA molecules interacting with the gold surface exclusively through
Controlling DNA Orientation on Mixed SAMs
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Scheme 1. Illustration of the Role of OEG in DNA Orientationa
a
The addition of OEG improves the orientation of the ssDNA probes, but too much OEG displaces the ssDNA.
Figure 6. Regeneration of a DNA SAM surface. NaOH was used to dehybridize the DNA duplex and recycle the probe surface five times. (a) SPR response and (b) direct comparison of successive sensorgrams. The ssDNA/OEG surface was prepared from a solution with a DNA mole fraction of 0.02.
their thiol groups. However, it has been shown that the DNA itself can nonspecifically physisorb onto the gold surface,9 resulting in a much less favorable orientation and lower hybridization efficiency. There is growing evidence that shortchain diluent thiols can help reduce this nonspecific binding and improve DNA orientation. For example, Tarlov et al. have developed a two-step assembly method where they adsorb thiolated ssDNA and then backfill with mercaptohexanol.9,10,12 Aqua et al.31 employ a reverse approach where they first block the surface with C18 thiol before assembling long dsDNA molecules. In both cases, the introduction of a second thiol component improves the hybridization efficiency of the immobilized DNA, presumably because of the more favorable orientation. We suggest that the OEG thiol plays a similar role in the orientation of the DNA in our mixed ssDNA/OEG SAMs. In this work, the ssDNA and OEG thiols are simultaneously coadsorbed from a common solution and are therefore competing for the exposed gold surface. As illustrated in Scheme 1, this competition controls both the coverage and the orientation of the DNA. At very high DNA mole fractions, there is not enough OEG to compete effectively with the nonspecific DNA interactions, and although the DNA coverage is high, the orientation is poor (point d in Scheme 1 and Figures 1 and 4). As the OEG concentration increases (decreasing DNA mole fraction), OEG competes with nonspecific DNA interactions more effectively, and the DNA orientation improves (points c). Eventually, OEG begins to dominate the surface, and the DNA coverage decreases (points b and a). It is this balance between DNA coverage and DNA orientation that determines the amount of complement hybrid(31) Aqua, T.; Naaman, R.; Daube, S. S. Langmuir 2003, 19, 10573-10580.
ization to the surface and produces the sharp maximum observed in Figure 4. Note that the SPR experiments performed in this work measure the absolute amount of DNA hybridization to a given surface, not the hybridization efficiency of the surface (e.g., surfaces a and c in Scheme 1). Although these two surfaces exhibit similar amounts of complement hybridization (Figure 4), surface c has a lower hybridization efficiency because most of the DNA strands are poorly oriented. As a test of this hypothesis, DNA/OEG SAMs were prepared by a two-step procedure where the OEG was assembled first, followed by the DNA. Clean chips were immersed in a 5 µM OEG solution for varying amounts of time, followed by a 100 nM DNA solution overnight. Complement hybridization to these chips is shown in Figure 5. At very short OEG assembly times, there is only a small amount of OEG on the surface, leaving most of the gold surface exposed. As a result, the DNA makes multiple contact points with the surface, and the orientation and hybridization are poor. In contrast, slightly longer exposure (∼10 min) to OEG thiols before assembly of the DNA thiols greatly enhances the complement hybridization. At this intermediate assembly time, an incomplete OEG monolayer is formed on the gold surface, with a limited number of gold regions exposed for DNA assembly. The DNA thiols can bind to the areas of exposed gold, but the presence of the OEG background reduces the likelihood of multiple binding events with the gold, thereby resulting in a more perpendicular DNA orientation. Longer OEG assembly times result in a more complete OEG monolayer, which inhibits DNA assembly and results in low hybridization. These results are consistent with those reported by Aqua et al.31 and provide additional evidence that the OEG thiol plays an important role in the orientation of the DNA.
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Surface Coverage. SPR measurements can be used to calculate how much ssDNA target hybridizes to the surface using the following equation
∂n ∂c
δλres ) SSΓ
where δλres is the observed wavelength shift, SS is the surface refractive index sensitivity, Γ is the surface coverage, and ∂n/∂c is the inverse slope coefficient relating refractive index to concentration.32 Using an SS value of ∼35 and a ∂n/∂c value for DNA of ∼0.14 cm3/g, the maximum SPR shift measured corresponds to a DNA duplex density of approximately 4 × 1012 duplexes/cm2. This is about 10% of the coverage of a full-packed theoretical DNA monolayer and is similar to coverages reported for other DNA SAMs.9,10,14,17 Note that this is an estimate of the amount of target ssDNA that hybridizes to the surface and not the total number of ssDNA probes on the surface and therefore represents the lower limit of ssDNA probe coverage. Surface Recycling. The DNA double helix can be dehybridized, or melted, by either chemical or thermal means. By dehybridizing the duplex, the DNA complement can be removed from the surface, thereby regenerating the ssDNA probe surface. In this work, we use 0.05 M NaOH to dehybridize the DNA duplex. Figure 6 shows a typical recycling experiment with five hybridization and melting cycles. As can be seen in the spectrograph, the baseline returns to zero after the dehybridization of the DNA duplex, indicating that all of the complement DNA has been removed from the surface. A comparison of the sequential hybridization curves shows that after five cycles the surface retains 85% of its original hybridization efficiency. Effect of Complement Length. It is interesting that the OEG used in this work contains four OEG groups and is considerably longer than both the MCH used by others and the C6 spacer group on the ssDNA thiols. On the basis of the reported length of our OEG,33 we expect it to block the lower three bases of the ssDNA probe and therefore reduce hybridization. To test this premise and improve hybridization, we compared the hybridization of a truncated complement (c-A 21) with the hybridization of the full-length complement (c-A 24). The length of the complement strand was reduced by three bases so that it would not interact with the three probe bases potentially blocked by the OEG thiol. As shown in Figure 7, hybridization of the truncated and standard complements was comparable, suggesting that the OEG did not significantly effect the DNA hybridization. Further (32) Ladd, J.; Boozer, C. L.; Chen, S.; Yu, Q.; Homola, J.; Jiang, S. Langmuir 2004, 20, 8090-8095. (33) Zheng, J.; Li, L. Y.; Chen, S. F.; Jiang, S. Y. Langmuir 2004, 20, 89318938.
Figure 7. Comparison of the hybridization of a truncated complement (c-A 21) with the hybridization of the full-length complement (c-A 24). The ssDNA/OEG surface was prepared from a solution with a DNA mole fraction of 0.02.
studies of the effect of the OEG chain length are needed to provide valuable insight.
Conclusions We present and characterize a mixed SAM consisting of ssDNA- and OEG-terminated thiols. Compared to the ssDNA SAMs presented in the literature, our DNA/OEG SAMs have two important features. First, we use OEG as the diluent thiol, which provides much better protein resistance than the commonly used mercaptohexanol. Second, we simultaneously coadsorb both thiols from a common solution, allowing for a convenient onestep assembly procedure. Results show that the OEG thiol composition plays an important role in the oligo surface coverage and may influence the orientation of the immobilized ssDNA probes. Whereas the presence of OEG helps the DNA probes stand up, high OEG compositions reduce the DNA coverage. Competition between these effects determines the capacity of the surface for complement hybridization. Acknowledgment. This work has been supported by the NSF (CTS-0528605) and FDA (FD-U-002250). We thank Lara Gamble and Dave Castner for their assistance with the ESCA experiments. All ESCA experiments were performed at the National ESCA and Surface Analysis Center for Biomedical Problems (NESAC/Bio, funded by NIH grant EB-002027 from the National Center for Research Resources). LA052908E
