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Preparation of Self-Assembled Monolayers Containing Anthryl Groups toward Hybridization of Nucleotides Fumio Nakamura,*,†,‡ Keita Mitsui,† Masahiko Hara,† Stephan Kraemer,§ Silvia Mittler,§ and Wolfgang Knoll§,# Frontier Research System, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan, PRESTO, Japan Science and Technology (JST), Kawaguchi, Saitama, 335-0012, Japan, and Max-Planck Institute for Polymer Research, Ackermannweg, D-55128, Mainz, Germany Received December 3, 2002. In Final Form: March 11, 2003 To realize immobilization and hybridization of nucleotides, self-assembled monolayers (SAMs) containing anthryl groups which interact with nucleotides were prepared on a gold substrate. We used an unsymmetric disulfide containing an anthryl group (Anth-s-s-C8), which can form a SAM on a gold substrate, to immobilize nucleotides on the gold surface via the intercalation between DNA and anthryl groups. The SAM was characterized by time-of-flight mass spectroscopy. The interaction between polynucleotides and the SAM and hybridization were investigated by surface plasmon resonance. The surface coverage of anthryl groups on the SAM could be controlled by mixing Anth-s-s-C8 and 11-mercapto-1-undecanol in solution at various ratios. The efficient hybridization of polynucleotides could be realized by controlling 30% of Anth-s-s-C8 on the surface. This method can be applied to oligonucleotides, and the efficiency of hybridization is enhanced by introducing the double-stranded portion in the middle.
Introduction An efficient hybridization method and a highly sensitive method of detecting hybridization of nucleotides are required in genome research because the hybridization is utilized in DNA sensors, such as the DNA microarray chip.1-4 The hybridization of nucleotides, based on the molecular recognition of adenine-thymine and cytosineguanine via multiple hydrogen bonds, plays an important role in transferring genetic information in living systems. Today, a highly sensitive method of detecting the hybridization is required in the diagnosis of genetic diseases based on the detection of single nucleotide polymorphisms (SNPs).5-7 However, it is still difficult to immobilize singlestranded (ss) probe DNA on a solid surface while maintaining its activity or to realize efficient hybridization because of denaturation or steric hindrance of the ss parts on a solid surface. It is likewise difficult to realize reproducible hybridization using current methods because it is difficult to control the amount of immobilized probe DNA on a solid surface. To prepare a DNA-immobilized surface, it is essential to overcome such difficulties for the efficient hybridization of nucleotides, leading to highly sensitive biosensing and * Corresponding author. E-mail:
[email protected]. † Frontier Research System, RIKEN. ‡ PRESTO, Japan Science and Technology. § Max-Planck Institute for Polymer Research. # Also at the Department of Material Science and of Chemistry, The National University of Singapore, Lower Kent Ledge Road, 117534, Singapore. (2) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767-773. (3) DeRisi, J. L.; Iyer, V. R.; Brown, P. O. Science 1997, 278, 680686. (4) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467-470. (5) Lockhart, D. J.; Winzeler, E. A. Nature 2000, 405, 827-836. (6) The International SNP Map Working Group. Nature 2001, 409, 923-933. (7) Stoneking, M. Nature 2001, 409, 821-822. (8) Landegren, U.; Nilsson, M.; Kwok, P. Y. Genome Res. 1998, 8, 769-776.
bioengineering applications. Many researchers are attempting to immobilize DNA on a surface, while maintaining the activity of ss nucleotides. To immobilize ss nucleotides on a surface, some methods have been employed, such as electrostatic interactions,2,3 the avidinbiotin interaction,8-10 and the sulfur-Au interaction.11-17 In these methods, however, it is difficult to control the coverage of ss nucleotides on a surface or to realize reproducible hybridization. Although no modification is required to immobilize probe DNA on a surface in the electrostatic method, the efficiency of hybridization is not so high because of the tight interaction between probe DNA and the surface. To increase the efficiency of hybridization, a designed interaction between DNA and a surface is required, such as the van der Waals interaction. Although fluorescence imaging has been used in many cases to detect hybridization on the surface, the modification of the fluorescent probe for target DNA is strongly required.2,3,8 On the other hand, in the case of the surface plasmon resonance (SPR) method10,13-15 or the quartz crystal microbalance (QCM) method,9,16-18 no modification of target DNA for the detection of hybridization is required. (9) Kambhampati, D.; Jakob, T.; Robertson, J. W.; Cai, M.; Pemberton, J. E.; Knoll, W. Langmuir 2001, 17, 1169-1175. (10) Ijiro, H.; Ringsdorf, H.; Hirschfeld, E. B.; Hoffmann, S.; Schilken, U.; Strube, M. Langmuir 1998, 14, 2796-2800. (11) Jordan, C. E.; Frutos, A. G.; Thiel, A. J.; Corn, R. M. Anal. Chem. 1997, 69, 4939-4947. (12) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 89168920. (13) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (14) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. J. Am. Chem. Soc. 2000, 122, 7837-7838. (15) Peterlinz, K.; Georgiadis, R.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401-3402. (16) Georgiadis, R.; Peterlinz, K.; Peterson, A. W. J. Am. Chem. Soc. 1997, 119, 3401-3402. (17) Huang, E.; Satjapipat, M.; Han, S.; Zhou, F. Langmuir 2001, 17, 1215-1224. (18) Satjapipat, M.; Sanedrin, R.; Zhou, F. Langmuir 2001, 17, 76377644.
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Intercalation involves the incorporation of a planar chromophore, such as acridine orange or ethidium bromide, into stacked DNA base pairs via hydrophobic interaction.19,20 Attempts have been made to immobilize DNA on a two-dimensional surface by intercalation. Shimomura et al. succeeded in immobilizing DNA on a Langmuir-Blodgett monolayer using an amphiphilic intercalator, octadecyl acridine orange, at the air-water interface.21 Higashi and co-workers studied the interaction between DNA and an intercalator immobilized on selfassembled monolayers (SAMs) by QCM.22 Studies on the hybridization of nucleotides at the air-water interface using amphiphilic lipids have been reported.23 SAMs have attracted considerable attention for wellordered thin-film fabrication, by which thiol or disulfide derivatives can spontaneously form a closely packed monolayer on a gold surface when the substrate is immersed in a thiol or disulfide derivative solution.24-26 Using a SAM on a gold substrate, it is expected that polynucleotides can exist at the solid-liquid interface without denaturation. In this study, to establish a novel method of immobilizing DNA and a label-free method of detecting hybridization, we prepared a functionalized SAM on which DNA can be immobilized. To immobilize DNA on a SAM using an intercalator, we synthesized an unsymmetric disulfide having a terminal anthryl group and prepared a SAM containing the intercalator. The anthryl group is expected to act as an intercalator of DNA because of its planar structure.27 The interaction between DNA and the anthryl group in solution was well investigated and found to be based on intercalation because spectral shifts induced by intercalation were observed based on fluorescence measurement.28 The interaction between anthryl groups and DNA is weaker than the electrostatic interaction; therefore, if we can control the degree of interaction between anthryl groups and DNA on the surface, an efficient hybridization is expected in our method. The SAM was characterized by time-of-flight mass spectroscopy (TOFMS). The interaction between DNA and the SAM was investigated by SPR. The morphology of DNA on the SAM was observed by atomic force microscopy (AFM). We then attempted to hybridize ss nucleotides on the SAM containing anthryl groups. The adsorption behavior of ss polynucleotides onto the SAM and the hybridization processes were monitored in situ by SPR measurements. Finally, we applied this immobilization method to the hybridization of oligonucleotides on the SAM that was composed of ss and double-stranded (ds) portions. (19) Caruso, F.; Rodda, E.; Furlong, D. N.; Niikura, K.; Okahata, Y. Anal. Chem. 1997, 69, 2043-2049. (20) Shimomura, M.; Nakamura, F.; Ijiro, K.; Tanaka, M.; Taketsuna, T.; Nakamura, H.; Hasebe, K. J. Am. Chem. Soc. 1997, 119, 23412342. (21) Nakamura, F.; Shimomura, M.; Ijiro, K. Thin Solid Films 1998, 327-328, 603-605. (22) Ijiro, K.; Shimomura, M.; Tanaka, M.; Nakamura, H.; Hasebe, K. Thin Solid Films 1996, 284-285, 780-783. (23) Higashi, N.; Takahashi, M.; Niwa, M. Langmuir 1999, 15, 111115. (24) Sastry, M.; Ramakrishnan, V.; Pattarkine, M.; Gole, A.; Ganesh, K. N. Langmuir 2000, 16, 9142-9146. (25) Ullman, A. Introduction to Ultra Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: Boston, 1991. (26) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 44814483. (27) Nelles, G.; Schonherr, H.; Jaschke, M.; Wolf, H.; Schaub, M.; Kuther, J.; Tremel, W.; Bamberg, E.; Ringsdorf, H.; Butt, H.-J. Langmuir 1997, 14, 808-815. (28) Nakamura, F.; Mitsui, K.; Murase, T.; Kobayashi, K.; Hara, M.; Knoll, W.; Sasabe, H. Mol. Cryst. Liq. Cryst. 2000, 349, 219-222.
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Figure 1. Structure of thiol and disulfide derivatives. Table 1. Nomenclature and Sequences of DNA name
sequence
20T
Probe DNA 5′-GACACAGCTAGTCAAGAGGA-AAAAAAAAAAAAAAAAAAAA-GACACAGCTAGTCAAGAGGA-3′ 5′-TTTTTTTTTTTTTTTTTTTT-3′
20comp 20control
Target DNA 3′-CTGTGTCGATCAGTTCTCCT-5′ 3′-GACACAGCTAGTCAAGAGGA-5′
60mer
Materials and Methods Materials. Figure 1 shows the structural formulas of thiol and disulfide derivatives that can form a stable monolayer on a gold substrate. In our study, the unsymmetric disulfide containing anthryl groups (Anth-s-s-C8) was used to disperse anthryl groups on the surface because the anthryl groups easily aggregate when the thiol derivative containing anthryl groups is used.29 The anthryl group was attached to the terminal OH groups of the disulfide derivative by esterification in CH2Cl2 solution at room temperature. 1-Ethyl-3-(3′-(dimethylamino)-propyl) carbodiimide and 1-hydroxybenzotriazol were used as the condensing agent and the catalyst, respectively. The synthesis was reported previously.30 To control surface coverage of anthryl groups on the SAM, Anth-s-s-C8 and 11-mercapto-1-undecanol (HO-C11SH) were dissolved in chloroform at various ratios (Anth-s-sC8/HO-C11-SH ) 100:0, 99:1, 95:5, 50:50, 5:95, and 0:100; SAM (100/0), SAM (99/1), SAM (95/5), SAM (50/50), SAM (5/95), and SAM (0/100)), to a final concentration of 1 mM. SAMs were prepared by immersing a gold substrate into the chloroform solution for 24 h. DNAs used in this study were diluted with 10 mM tris(hydroxymethyl) aminomethane buffer solution containing 1 mM EDTA at pH 7.0 (TE buffer). Sonicating an aqueous cow thymus DNA (CT DNA, Sigma) solution produced the average number of base pairs of the DNA molecule, that is, 300-500 bp, as estimated by electrophoretic analysis. The polynucleotides, namely, polyadenylic acid (poly(A)), polyuridilic acid (poly(U)), polycytidilic acid (poly(C)), and polyguanylic acid (polyG)), purchased from Sigma, were dissolved in 0.2 M NaCl solution to a concentration of 0.05 g/L. Oligonucleotides, which were purchased from Sawady Technology Co., were dissolved in the TE buffer solution to a concentration of 6.5 µM. Table 1 shows the sequences of the probe and target nucleotides used in our experiments. The probe oligonucleotide containing both of the ss and ds portions was prepared by annealing 60mer and 20merT in their aqueous solutions at 95 °C, and the formation of double strands was confirmed by electrophoretic analysis. TOF-MS Measurements. TOF-MS measurements were performed in high vacuum at a pressure of 1 × 10-6 mbar. SAMs were prepared on 50-nm-thick gold films evaporated on glass slides that were covered first with a 2-nm-thick chromium film in order to increase mechanical stability. Details of the measurement were already reported previously.31,32 Atomic and molecular ions from the sample are released by spontaneous desorption, a secondary ion process by which the sample is not bombarded by (29) Kumar, C. V.; Asuncion, E. H. J. Am. Chem. Soc. 1993, 115, 8547-8553. (30) Fox, M. A.; Wooten, M. D. Langmuir 1997, 13, 7099-7105. (31) Nakamura, F.; Mitsui, K.; Hara, M. Mol. Cryst. Liq. Cryst. 2001, 370, 359-362. (32) Hangenhoff, B.; Benninghoven, A.; Spinke, J.; Liley, M.; Knoll, W. Langmuir 1993, 9, 1622-1624. (33) Weisser, M.; Kashammer, J.; Menges, B.; Matsumoto, J.; Nakamura, F.; Ijiro, K.; Shimomura, M.; Mittler, S. J. Am. Chem. Soc. 2000, 122, 87-95.
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Figure 3. TOF-MS spectra of SAMs prepared from HO-C11SH and Anth-s-s-C8 at various ratios.
Figure 2. (a) Schematic of the setup for SPR measurement. (b) Schematic reflectivity curves without and with a dielectric layer on a gold substrate, respectively, with different minima positions θ1 and θ2. (c) Schematic reflectivity versus time curve obtained by monitoring the reflected intensity at an angle θk. particles from an external source. Primary ions of adsorbates are field-desorbed from the edges of an acceleration grid located in front of the sample. These ions are accelerated toward the sample, gaining keV energies. They finally sputter secondary ions from the sample that are analyzed by a TOF mass spectrometer. Spectra of negative secondary electrons are recorded using the simultaneously emitted secondary electrons from the sample surface as trigger particles. An acceleration voltage of 9.5 kV was applied to the sample. The recording time of a spectrum was 20-40 min. Within one spectrum, mass peaks of interest were integrated and normalized by the number of start events with one or more corresponding stop events. This leads to a relative ion yield that allows comparison of intensities of equivalent peaks in different spectra. AFM Measurements. AFM measurement was performed using a NanoScope III (Digital Instruments, Santa Barbara, CA). The topology of DNA on the SAM was observed in ambient by AFM in the tapping mode. In AFM measurements, we used 100µm-long silicon nitride cantilevers (spring constant, 0.09 N/m) with integrated sharpened tips (Olympus, Tokyo, Japan). Images of SAMs were taken on the Au(111) that was prepared by evaporating gold onto freshly cleaved mica at a pressure of 10-7 mbar. After evaporation, the gold substrates were annealed at 300 °C under vacuum for 6 h and cooled to room temperature.33 SPR Measurements. The plasmon surface polaritons are excited at the metal/dielectric interface upon total internal reflection of a laser beam (HeNe, λ ) 632.8 nm, 5 mW) at the prism base. The simple experimental setup, called the Kretschmann configuration, was used in our SPR measurements, as shown in Figure 2.34 By varying the angles of incidence of the laser beam, we obtained a plot of reflected intensity as a function of the angle of incidence. The absorption process occurring at the solid-liquid interface can be monitored in real time, as shown in Figure 2, by selecting an appropriate angle of incidence θ (34) Noh, J.; Murase, T.; Nakajima, K.; Lee, H.; Hara, M. J. Phys. Chem. B 2000, 104, 7411-7416. (35) Weisser, M.; Nelles, G.; Wohlfart, P.; Wenz, G.; Neher, S. M. J. Phys. Chem. 1996, 100, 17893-17900.
Figure 4. Mole fraction x in the monolayer of two specific fragments of SAMs versus mole fraction of Anth-s-s-C8 in the self-assembly solution: HO-C11-S- (square); the sum of Anthand AnthCOO- (circle). (55-57°) and monitoring the reflected intensity as a function of time. Knowledge of the form of the resonance curve enabled interpretation of the reflected intensity as a shift of the angle of resonance. SPR measurement was carried out on the gold substrate that was prepared by evaporating gold onto glass (LaSFN9, n ) 1.85) under 10-5 Torr, the thickness of the gold being ∼50 nm. The temperature of the SPR cuvette was controlled at 20 °C. For rinsing, a pure buffer solution was used and the flow rate of sample injection and rising was controlled at 1.0 mL/min with a peristaltic pump.
Results and Discussion Characterization of SAMs on Gold Substrates. TOF-MS is a powerful tool for characterizing a thin film on an inorganic substrate. To control the amount of DNA adsorbed on a SAM, the surface coverage of anthryl groups on the SAM should be controlled. In this section, we monitored the surface coverage of anthryl groups on the SAM by TOF-MS. Figure 3 shows the TOF-MS spectra of monolayers prepared from HO-C11-SH and Anth-s-sC8 at various ratios. The peak attributed to anionic HOC11-SH (HO-C11-S-, m/z ) 203 u) is detected in spectrum a of the monolayer prepared from a HO-C11SH solution (SAM (0/100)). In spectrum b, fragments of anionic anthracene (Anth-, m/z ) 177 u) and anthracene carboxylic acid anion (AnthCOO-, m/z ) 221 u) are detected, while that of anionic Anth-s-s-C8 is not detected. Relative peak areas of mass numbers 204, 177, and 211 are plotted in Figure 4. Anthryl groups are hardly observed on SAM (0/100), SAM (5/95), and SAM (50/50). The intensities of the two peaks (Anth- and AnthCOO-) increase with increasing ratio of Anth-s-s-C8 in solution at more than 95% Anth-s-s-C8. On the other hand, the peak intensity of the HO-C11-S- fragment suddenly decreased at more than 95% Anth-s-s-C8 in solution. This
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Figure 5. Scanning SPR curves before and after immobilization of DNA: dashed line, before DNA injection; solid line, after DNA injection and rinsing.
Figure 6. SPR adsorption kinetics of CT DNA to SAMs prepared from HO-C11-SH and Anth-s-s-C8 at various ratios.
indicates that adsorption of HO-C11-SH to the gold substrate is preferred to that of Anth-s-s-C8. The poor adsorption of Anth-s-s-C8 to the substrate might be due to the steric hindrance because the mobility of the ester part in Anth-s-s-C8, which connects the anthryl group and the alkyl chain, prevents them from packing well. Assuming that the intensity of anthryl fragments reflects the surface coverage of the anthryl groups, the TOF-MS results show that the surface coverages of Anth-s-s-C8 are 0.06, 0.30, and 1.00 in the case of SAM (95/5), SAM (99/1), and SAM (100/0), respectively. Weisser et al. reported that adenine anion and adenine ester anion can be observed after cleavage of ester bonds in the case of a SAM prepared from adenine thiolated nucleobase.32 They showed that the adsorption of thiolated nucleobase to a gold substrate is less efficient than that of simple thiolated compounds, such as alkane thiol and OH-terminated alkane thiol. This means that the adsorption rate of thiolated bulky groups is much lower than that of normal chain thiol. Our finding of the behavior of Anth-s-s-C8 adsorption to the substrate is consistent with their results. Adsorption of DNA to the SAM. The adsorption processes of nucleotides can be monitored in situ by SPR. By varying the angles of incidence of the laser beam, we obtained many reflected intensities as a function of the angle of incidence. Figure 5 shows the reflectivity curves of a SAM on the gold substrate, which was prepared from pure Anth-s-s-C8 solution, before and after injection of an aqueous CT DNA solution. The curve shifted to the higher angle side after adding the DNA solution, indicating adsorption of DNA onto the SAM. By monitoring the reflectivity change at an appropriate angle, the progress of interaction occurring on the surface can be estimated in real time. Figure 6 shows the adsorption kinetics of SAMs prepared from various solutions upon injection of the CT DNA solution. The reflectivities of SAM (95/5), SAM (99/1), and SAM (100/0) increased after adding the
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Figure 7. AFM image of SAM (95/5) prepared on Au(111) after adsorption of CT DNA.
DNA solution, whereas there was little change in the case of SAM (0/100). This suggests that the anthryl groups of the SAM can interact with DNA at a solid-liquid interface. The amount of adsorption increases with the increment of surface coverage of anthryl groups on the surface. Since the anthryl groups interact with nucleic acid bases via the van der Waals force without any electrostatic interaction, the interaction is considered as an intercalation. As reported previously,22 a DNA multilayer might form on SAM (100/0) or SAM (99/1). To observe a monolayer of DNA on the SAM, surface coverage of anthryl groups should be controlled. Taking account of the average thickness of adsorbed DNA (0.3 nm) on SAM (95/5), twodimensional DNA structure can be observed on the SAM. Figure 7 shows an AFM image of SAM (95/5) taken in air after immersion in an aqueous DNA solution and rinsing with pure water. A network structure, which was not observed before the immersion, was observed here. The observed thickness of the network is 0.7-1.3 nm, well below the DNA diameter of 2.0 nm in solution but consistent with the reported height of DNA on substrates (between 0.1 and 1.5 nm) measured by AFM.35-37 This AFM image suggests that DNA can be immobilized on the SAM two-dimensionally using our method by controlling the surface coverage of the anthryl groups. These results obtained by SPR and AFM measurements indicate that the anthryl groups attached to the SAM can interact with DNA at the solid-liquid interface because the binding ability of the SAM for DNA increased after introducing anthryl groups to the SAM. Kinetics of Adsorption and Hybridization of ss Polynucleotides on the SAM. The anthryl groups on the SAM are also expected to interact with nucleobases of ss polynucleotides via the van der Waals interaction.38 The adsorption process of poly(A) and poly(U) occurring at the solid-liquid interface can be monitored in real time, as shown in Figure 8, by selecting an appropriate angle of incidence and monitoring the changes of the reflected intensity as a function of time. Poly(A) was adsorbed to SAM (100/0), while only a small amount of poly(U) was adsorbed to the poly(A)-preadsorbed SAM. On the other hand, poly(A) was also adsorbed to SAM (99/1) in the first step and poly(U), which was subsequently added, was adsorbed by the poly(A)-preadsorbed SAM (second step). The kinetic curves indicate that poly(A) interacts with (36) Lehnninger, L.; Nelson, D. L.; Cox, M. M. Principles of Biochemistry; Worth Publishers: New York, 1993. (37) Allison, D. P.; Bottomley, L. A.; Thundat, T.; Brown, G. M.; Woychik, R. P.; Schrick, J. J.; Jacobson, K. B.; Warmack, R. J. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 10129-10133. (38) Thundat, T.; Allison, D. P.; Warmack, R. J. Nucleic Acids Res. 1994, 22, 4224-4228. (39) Nakamura, F.; Hara, M. Mol. Cryst. Liq. Cryst. 2002, 377, 5760.
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Figure 8. SPR adsorption kinetics of poly(A) and poly(U) to (a) SAM (100/0), (b) SAM (99/1), and (c) SAM (0/100).
Figure 9. SPR adsorption kinetics to SAM (99/1): (a) adsorption to poly(A)-preadsorbed SAM; (b) adsorption to poly(U)preadsorbed SAM.
the anthryl groups on the SAM because poly(A) is not adsorbed to the SAM prepared from the pure HO-C11SH solution (SAM (0/100)). Assuming that the refractive index of a polynucleotide is 1.45, the increments of thickness in the first step and the second step were calculated to be 0.7 and 1.8 nm, respectively.39 This interaction between poly(A) and poly(U) was due to the formation of a ds polynucleotide through complementary base pairings on the SAM. In the case of SAM (99/1), the surface coverage of Anth-s-s-C8 was estimated to be 30% by TOF-MS, as shown previously. The reason efficient hybridization could not be realized on SAM (100/0) might be that poly(A) was immobilized tightly on the SAM such that poly(U) could not interact with poly(A) on the SAM. Figure 9a shows the kinetic curves of adsorption of poly(U) and poly(C) to the poly(A)-preadsorbed SAM. The curves suggest that poly(U) was adsorbed onto the SAM, while only a small amount of poly(C) was adsorbed. This selectivity strongly indicates that poly(A) on the SAM could recognize poly(U) at the solid-liquid interface through complementary base pairing. The adsorption behavior of (40) Wink, T.; Beer, J.; Hennink, W. E.; Bult, A.; van Bennekom, W. P. Anal. Chem. 1999, 71, 801-805.
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Figure 10. Schematic diagram showing immobilization and hybridization of oligonucleotides: (A) 60mer or (B) 60mer/20T was used as a probe DNA.
poly(A) or polyguanylic acid (poly(G)) to the poly(U)preadsorbed SAM is shown in Figure 9b. Similar to the adsorption behavior of poly(U) shown in Figure 9a, poly(A) was adsorbed onto the poly(U)-preadsorbed SAM. The thickness gradually increased following the addition of poly(G) although the change was not so significant. This adsorption of poly(G) onto the SAM might be due to the substitution of poly(G) for poly(U). Consequently, these SPR results strongly indicate that the formation of adenine-uracil base pairs can be realized on the SAM. These selective interactions strongly suggest that the hybridization of polynucleotides is realized on the SAM containing anthryl groups. For efficient hybridization of polynucleotides, SAM (99/1) is suitable because the SAM can immobilize ss polynucleotides without any denaturation. Although we cannot determine the degree of interaction between anthryl groups on SAM (99/1) and polynucleotides, the interaction is not so strong because the double-stranded formation with complementary polynucleotides breaks the interaction on the surface. If the interaction between a base and the SAM is too strong, a base cannot form a base pair. Hence, not all bases might be active on the surface. Hybridization of Oligonucleotides on the SAM. To utilize the SAM containing anthryl groups for the hybridization of oligonucleotides, DNA composed of ss and ds portions was used as a probe DNA, as shown in Figure 10. In 60mer, a ds portion composed of adenine-thymine base pairs is designed in the middle and two probe portions having the same sequence are connected in the terminal, as shown in Table 1. The ds portion composed of adeninethymine base pairs is expected to interact with the anthryl groups through intercalation. The formation of the ds portion after annealing was confirmed by electrophoretic analysis. The sequence of 20comp is complementary to that of the probe part of 60mer, while that of 20control is entirely noncomplementary. To apply this hybridization system to oligonucleotides, 60mer containing two probe portions in the terminal was used, as shown in Figure 10. Hybridization of target DNA (20comp) with the probe portion is expected to occur in both terminals because the two probe portions have the same sequence. Figure 11A shows the adsorption behavior of 60mer and 20comp added subsequently to SAM (99/1) as determined by SPR measurement (see Figure 10A). The 60mer was adsorbed to SAM (99/1) in the first step, and 20comp was adsorbed to the 60mer-preadsorbed SAM (second step). However, the intensity was decreased to half of the original after rinsing with buffer. This behavior indicates that physically adsorbed 20comp and/or hybridized 60mer/20comp was removed from the SAM by rinsing.
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Figure 13. SPR kinetics of adsorption of 20comp and 20control on SAM (99/1): solid line, diffusion-limited Langmuir fitting curve.
Figure 11. SPR kinetics of adsorption and hybridization on SAM (99/1) or bare gold: (A) 60mer on SAM (99/1); (B) 60mer/ 20T on (a) SAM (99/1) and (b) bare gold.
Figure 12. Summary of adsorption and desorption of probe DNA and target DNA on SAM (99/1) or bare gold: (i) before rinsing in the first step, (ii) after rinsing in the first step, (iii) before rinsing in the second step, and (iv) after rinsing in the second step.
To increase the stability of probe DNA on the SAM, another type of probe DNA having ds and ss portions in the molecule was used, as shown in Figure 10B. The intercalation of anthryl groups with the ds portion of the probe DNA was expected using this molecule. Figure 11B shows the behavior of the 60mer/20T complex or 20comp adsorption onto SAM (99/1), as detected by SPR. The 60mer/20T was adsorbed to SAM (99/1) in the first step, and 20comp added subsequently was adsorbed to the 60mer/20T-preadsorbed SAM (second step). Although probe DNA and 20comp were adsorbed to the Au bare substrate, the amount of 20comp adsorbed was much lower than that to the 60mer/20T- or 60mer-preadsorbed SAM (Figure 12). The poor adsorption of 20comp to the 60mer/ 20T-preadsorbed Au substrate indicates that most of the probe DNA is denatured on the Au bare substrate. Figure 12 summarizes the adsorption or desorption behavior of probe DNA and target DNA in both cases. In the first step, only a small amount of the 60mer/20T complex was
desorbed after rinsing, while 15% of the adsorbed 60mer molecules were removed from the SAM after rinsing, as shown in Figure 12. The rate of 60mer adsorption was higher than that of 60mer/20T adsorption although there was no significant difference in the amount of adsorbates between the two cases. The difference in rate implies that the physical adsorption of oligonucleotides is faster than the adsorption by intercalation; however, the intercalated DNA on the SAM is more stable than the physically adsorbed DNA. Although adsorbed probe DNA was stable on a Au bare substrate in the first step, the efficiency of hybridization was lower than that on SAM (99/1). Adsorption of 20comp and 20control to the 60mer/20Tpreadsorbed SAM is shown in Figure 13. Compared with 20comp, only a small amount of 20control was adsorbed to the SAM. This difference in adsorption behavior strongly indicates that the adsorption of 20comp to the SAM was due to hybridization. Although hybridization was also observed even when using 60mer as probe DNA, one-half of adsorbed 20comp was removed after rinsing. The difference in adsorption to the SAM and Au bare substrates is due to the activity of the DNA on the substrates. These results strongly indicate that the SAM containing anthryl groups can immobilize DNA while maintaining its activity on the surface. The analysis of hybridization by a diffusionlimited Langmuir adsorption model shows that the rate constant is 0.74 min-1/2 (5.8 h-1/2). This value is more than twice the previously reported value.14 In the case of 20comp, the reflectivity changes in the first and second steps (dRf and dRs) are 0.0242 and 0.0208, respectively. By taking into account the physical adsorption of 20control (dRs ) 0.004), dRs/dRf was calculated to be 0.67. This value seems larger than the expected value. Even when two target DNAs, 40 bases, hybridize with both probe portions of probe DNA, 80 bases, in the second step, dRs/ dRf should be below 0.5. The excess might be due to the orientation of double-stranded nucleotides on the substrate. Before hybridization, probe portions might be lying flat on the surface. On the other hand, base pairs might be perpendicular to the surface after the formation of the double strand. Although we did not estimate the exact efficiency of hybridization, reflectivity changes in the first and second steps suggest that most of the probe portions could hybridize with the target. Consequently, introduction of the ds portion supported a stable immobilization of oligonucleotides on the SAM, because attachment of ss portions to the SAM is not so tight such that they are easily removed from the SAM during rinsing with buffer.
SAMs Containing Anthryl Groups
Conclusions We prepared SAMs to immobilize ss and ds nucleotides while maintaining their activity through “delicate interaction” without any modification of the nucleotides. Moreover, we succeeded in hybridizing nucleotides using the SAM by controlling the surface coverage of the anthryl groups. The efficiency of hybridization was optimized using an appropriate amount of anthryl groups on the SAM, while efficient hybridization could not be observed in the case of the SAM prepared from pure Anth-s-s-C8 solution. The selectivity of adenine and uracil homopolynucleotides strongly indicates that they can recognize each other on the SAM through complementary adenine-uracil base pairing. In addition, the hybridization of oligonucleotides could be realized using this method, and the stability of probe DNA on the SAM was increased by introducing the ds portion, resulting in an efficient hybridization. The advantage of this method is that no modification of the polynucleotide itself is required in the hybridization process. By introduction of such functionalized SAMs, a polynucleotide monolayer can be realized without flattening or denaturation. In the case of SAM (99/1), the interaction between anthryl groups and polynucleotides, which is due to the van der Waals force, is moderate, so that the polynucleotides are still active and can interact
Langmuir, Vol. 19, No. 14, 2003 5829
with complementary polynucleotides on the SAM. A disadvantage of this method is that substitution of probe DNA and target DNA might occur during the hybridization process because the interaction between probe DNA and the SAM is not as strong as a chemical interaction. In a conventional DNA chip, an electrostatic interaction is used to immobilize cDNA prepared by polymerase chain reaction on a surface. Our method is also expected to immobilize many kinds of DNA, such as long DNA or ds DNA, on the surface via the delicate interaction and to realize an efficient hybridization. This label-free method can be applied to the construction of DNA chips, leading to easy and rapid sequencing of DNA and detection of SNPs. Acknowledgment. One of the authors (F.N.) acknowledges the support of the Grant-in-Aid for Encouragement of Young Scientists (No. 13740406) from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Special Postdoctoral Researchers Program of RIKEN. Part of this material was supported by the Science and Research Council, Singapore, under Grant Number MCE/TP/001.2, and was supported by an EU grant (QLRT-1999-31658, DNA-Track). LA020952D
