NANO LETTERS
Preparation of a Branched DNA Self-Assembled Monolayer toward Sensitive DNA Biosensors
2003 Vol. 3, No. 8 1083-1086
Fumio Nakamura,*,†,‡ Eisuke Ito,† Yosuke Sakao,§ Nobuo Ueno,§ Isaiah N. Gatuna,| Fumio S. Ohuchi,| and Masahiko Hara† Local Spatio-Temporal Functions Laboratory, Frontier Research System, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan, PRESTO, Japan Science and Technology (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, Faculty of Engineering, Chiba UniVersity, 1-33 Yayoicho, Inage-ku, Chiba 236-0022, Japan and Department of Materials Science and Engineering, Washington UniVersity, Box 352120, Seattle, Washington 98195 Received May 3, 2003; Revised Manuscript Received June 2, 2003
ABSTRACT To realize an efficient hybridization of oligonucleotides, a DNA self-assembled monolayer (SAM) composed of branched probe DNA containing single- and double-stranded portions was prepared on a gold substrate. Hybridization using the SAM was monitored in situ by surface plasmon resonance (SPR). By controlling the surface coverage of the probe DNA, hybridization efficiency was optimized. SPR results strongly indicate that all probe sites are accessible for hybridization under optimized conditions regardless of the position.
The efficient and reproducible hybridization of oligonucleotides with surface-immobilized DNA has become increasingly important in diagnostic studies because of its potential application to biosensing.1 It is, however, difficult to realize efficient hybridization because it is likewise difficult to immobilize single-stranded (ss) probe DNA on a surface while maintaining its activity. Because these difficulties in many cases arise from steric hindrance or the denaturation of probe portions on the substrates, hybridization depends on the immobilization methods of the probe DNA. To establish highly sensitive DNA biosensors, it is indispensable that the probe DNA should be designed so as not to be denatured on a solid substrate. Probe DNA molecules having multiple probe portions, such as branched probe DNA or a DNA dendrimer, are promising candidates for efficient hybridization.2 Highly sensitive DNA biosensors can be realized if the probe DNA can be immobilized, as self-assembled monolayers (SAMs), on a solid surface without causing the denaturation of the probe portions. SAMs3 have drawn considerable interest for their well-ordered thin-film applications, with thiol or disulfide derivatives spontaneously forming a closely packed * Corresponding author. E-mail:
[email protected]. † RIKEN. ‡ PRESTO. § Chiba University. | Washington University. 10.1021/nl0342794 CCC: $25.00 Published on Web 06/21/2003
© 2003 American Chemical Society
nanometer-level monolayer on a gold surface when the substrate is immersed in a thiol or disulfide derivative solution. If the branched probe DNA can form a monolayer and still maintain its activity, then highly efficient and reproducible hybridization can be realized using a DNASAM combination. In an effort to establish a novel DNA biosensor, we propose in this paper a novel DNA functionalized surface. In this method, a SAM containing branched probe DNA, consisting of double-stranded (ds) and singlestranded (ss) DNA portions, was prepared on a gold substrate, as shown in Scheme 1A. Chemical bonding of the thiol group with gold was induced to immobilize the probe DNA on gold substrates, and DNA SAMs were spontaneously formed on gold substrates.4 Surface plasmon resonance (SPR) was employed in detecting DNA hybridization on gold substrates. The target DNA was not in any way labeled. Scheme 1B shows a schematic illustration of the preparation of the branched probe DNA used here. Probe and target DNAs used in this study were purchased from Sawady Technology Co. (Tokyo, Japan). Their sequences are shown in Table 1 , where underlined sequences show probe portions. To prepare the branched probe DNA having three probe portions, a mixed aqueous solution of 80mer, 40mer, and thiolated 20mer was annealed up to 95 °C, leading to the double-stranded formation at the three portions. The mixed molar ratio of 80mer1, 40mer1, and 20merSH in the
Scheme 1. (A) Schematic Illustrations of Hybridization Using a SAM Containing Branched Probe DNA and (B) Formation of Branched Probe DNA Having Three Probe Portions
Table 1. Nomenclature and Sequences of DNA name
probe DNA
20merSH 80mer1
3′-ATCGTACGATTACGTACGTA-5′-C6-SH 5′-TGGAGAACTGATCGACACAGTTTTTAGAGGGGTCAAGAGGTTTTTAGAGGGGTCAAGAGGTAGCATGCTAATGCATGCAT-3′ 5′-TGGAGAACTGATCGACACAGTTTTTAGAGGGGTCAAGAGGTTTTTACACGCGACTACACGTAGCTAGCATGCTAATGCATGCAT-3′ 5′-CCTCTTGACCCCTCTTTTTTTGGAGAACTGATCGACACAG
80mer2
40mer1: 40mer2:
5′-CGTGTAGTCGCGTGTTTTTTAGCACATCAGTTGGTCTCTG-3′
40mer3:
5′-CCTCTTGACCCCTCTTTTTTTCGTGTAGTCAACCAGAGAC-3′
50mer
5′-TGGAGAACTGATCGACACAGTTTTTTTTTTTAGCATGCTAATGCATGCAT-3′ 5′-TAGCATGCTAATGCATGCAT-3′
20merC
solution was 1:2:1. The formation of the 80mer1/40mer1/ 20merSH complex (probe (I)) was confirmed by electrophoretic analysis. Probe (I) has three hybridization sites (20 bases), with “junk” spacers of five thymine bases to increase the mobility of the probe portions. To control the surface coverage of the probe DNA in the SAM, probe (I) and the 20merC/20merSH complex (mixture) were dissolved in a sterilized 20 mM MgCl2(OH)6 solution (MgCl2 solution) at various molar ratios (probe DNA (I)/mixture ) 100:0 SAM (100/0); 75:25 SAM (75/5); 50:50 SAM (50/50); 25:75 SAM (25/75); and 0:100 SAM (0/100)) to a final concentration of 2.7 µM. These SAMs were prepared by immersing a gold substrate into the aqueous MgCl2 solution containing the probe DNA for 15 h. The target DNA was dissolved in the aqueous MgCl2 solution to a final concentration of 6.5 µM. SPR measures the absorption process in real time that occurs on a solid surface through monitoring the reflection intensity as a function of time at a selected angle of incidence. Knowledge of the form of the resonance curve allowed this intensity to be interpreted as a shift in the angle of resonance. This simple experimental setup, called the Kretschmann configuration, was used in our SPR measurements.5 SPR measurement using a laser beam (HeNe, λ ) 632.8 nm, 5 mW) was carried out on the probe-modified gold substrate. The 50-nm-thick gold surface was vacuum deposited at 10-5 Torr onto a glass substrate (LaSFN9, n ) 1.85). The temperature of the SPR cuvette was controlled at 20 °C. The SAM prepared from probe (I) and/or mixture on a gold substrate was confirmed by SPR and X-ray photoemission spectroscopy (XPS). SPR reflectivity rose upon increasing the probe (I) fraction in the mixed solution (data not shown). The increments indicate that probe (I) molecules coexist with the mixture in the SAM and that the surface coverage of probe (I) increases with the molar fraction of probe (I) in the aqueous solution. 1084
name
target DNA
20comp1 20comp2 20comp3
5′-CTGTGTCGATCAGTTCTCCA-3′ 5′-GTCTCTGGTTGACTACACGA-3′ 5′-CAGAGACCAACTGATGTGCT-3′
name
probe DNA complex
probe (I) probe (II) probe (III) mixture
80mer1/40mer1/20merSH 80mer2/40mer2/40mer3/20merSH 50mer/20merSH 20merC/20merSH
XPS measurements were carried out with an ESCALAB 250 system (Thermo VG Scientific). A 150-W monochromatized Al KR line was used as an excitation source. The pass energy of the electron analyzer was set at 20 eV. The binding energy in the XPS spectra was calibrated with a Au 4f7/2 peak at 84.0 eV.6 In SAM (100/0), a peak in the S 2p XPS spectra was observed at approximately 162 eV, which can be assigned to sulfur bound to the gold substrate.7 The XPS result indicates that the probe DNA is adsorbed onto the gold substrate through chemical bonding via thiol and Au atoms. Figure 1A shows the adsorption kinetics of target DNA (20comp1) on the five kinds of SAMs. This adsorption behavior is observed by monitoring the reflected intensity at an appropriate angle (approximately 55°). The inset indicates the reflectivity change after the adsorption of the target DNA. In varying degrees, the 20comp1 molecule is adsorbed onto each of the SAMs prepared from the solution containing probe (I). This adsorption is due to the hybridization of the probe portion and target DNA. There is minimal adsorption of 20comp1 on SAM (0/100). Hybridization efficiency is optimized by SAM (50/50). The amount of hybridized 20comp1 on SAM (100/0) is less than that on SAM (75/25) or SAM (50/50) although the surface coverage of the probe DNA in SAM (100/0) should be higher than that in SAM (75/25) or SAM (50/50). The reason for the low efficiency of hybridization on SAM (100/0) might be steric hindrance, rendering the probe portions inaccessible to the solute target DNA. By introducing a certain average Nano Lett., Vol. 3, No. 8, 2003
Figure 2. Adsorption behavior of complementary DNA on a DNA SAM prepared from 1:1 probe (II)/mixture. “All mixture” means an equimolar mixture of 20comp1, 20comp2, and 20comp3.
Figure 1. (A) Adsorption behavior of target DNA on SAMs prepared from probe (I) and mixture mixed solutions: (a) SAM (100/0), (b) SAM (75/25), (c) SAM (50/50), (d) SAM (25/75), and (e) SAM (0/100). The inset shows the increments of the reflectivity after rinsing. (B) Comparisons with single-stranded system: (f) adsorption of 20merC on a 20merSH monolayer on a gold substrate; (g) adsorption of 20comp1 on a SAM prepared from an equimolar mixed solution of probe (III) and mixture. Arrows show rinsing points.
volume around the probes, the probe molecular motion is enhanced, and a larger site into which the target molecule can migrate and eventually hybridize is created. To confirm the superiority of the branched DNA to a nonbranched system, two experiments were performed. Figure 1B shows the comparative results. The hybridization of ss 20merSH and 20merC on a gold substrate was attempted by SPR. Curve f indicates the adsorption of 20merC on 20merSH preadsorbed monolayer. Little adsorption of 20merC was observed in the monolayer, but 20merSH formed a stable monolayer on a gold substrate. This result strongly suggests that the activity of the ss portion of 20merSH was so negligible that the hybridization of 20merC was not observed. As a second comparison, a complex of 50mer and 20merSH (probe (III)) composed of a ss portion and a ds portion was used for hybridization. The hybridization of 20comp1 with a DNA SAM prepared from an equimolar mixed solution of probe (III) and mixture is shown as curve g. This result shows that the hybridization signal of probe (III) is lower than that of probe (I). The hybridization signal of probe (I) is 1.74 times that of probe (III). If both probe (I) and probe (III) have the same hybridization efficiency, then the signal of probe (I) should be 3 times that of probe (III). This lower hybridization efficiency might be due to the surface coverage of probe (I). Because the molecular size Nano Lett., Vol. 3, No. 8, 2003
of probe (I) is larger than that of probe (III), the surface coverage of probe (I) might be less than that of probe (III) even when the SAMs were prepared from their equimolar mixtures.8 The conformation of the probe DNA raises the question of accessibility of the probe arms. It would appear that the two arms nestled at the top of the probe would have more superior performance, on the basis of their exposed location, than the third arm tucked away under the former two arms. To address this question, another probe DNA composed of 80mer2, 40mer2, 40mer3, and 20merSH (probe (II)) was used. Probe (II) has three probes with different base sequences. As such, they recognize three different complementary target DNAs. Figure 2 shows the hybridization of three kinds of target DNA (20comp2, 20comp3, and 20comp1, in the order added) on SAM (50/50) composed of probe (II) and mixture. Step-by-step hybridization is observed when these target DNAs are added one by one. The increments of the hybridization signal in these steps are 0.0169, 0.0135, and 0.0139, respectively. The first addition, which corresponds to the hybridization with the nearest portion from the surface, shows the maximum adsorption of target DNA, and there is a small difference in the level of adsorption between the second and third additions. This may be due to the distance from the surface. The intensity of a penetration wave, such as an evanescent wave or a plasmon wave, generated on the surface significantly decreases with the distance from the surface.9 Hence, the sensitivity of the hybridization signal may decrease in the cases of two probe portions that are farther from the surface. When the equimolar mixture of these three target DNAs was added, the efficiency of the hybridization was as much as that of the step-by-step hybridization. This result indicates that all probe portions are active on SAM (50/50) regardless of their positions in the probe DNA. We succeeded in the preparation of a SAM composed of branched probe DNA while maintaining the activity of probe portions and in the optimization of hybridization efficiency by controlling the surface coverage of the probe DNA. The advantage of this system is that a complex synthesis process for the preparation of the probe DNA is not required because the branched probe DNA can be prepared by annealing. The probe portions are still active even on the SAM regardless of their position. Furthermore, one probe DNA can recognize 1085
several kinds of target DNA using this system. This epochmaking method is promising and encouraging for highly sensitive DNA biosensors. Acknowledgment. F.N. gratefully acknowledges a Grantin-Aid for Encouragement of Young Scientists (no. 13740406) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Partial support from the U.S. Army Research Office DURINT program is also acknowledged. References (1) (a) Fodor, S. P. A.; Read, J. L.; Pirrung, M. C.; Stryer, L.; Lu, A. T.; Solas, D. Science 1991, 251, 767-773. (b) DeRisi, J. L.; Iyer, V. R.; Brown, P. O. Science 1997, 278, 680-686. (c) Landegren, U.; Nilsson, M.; Kwok, P. Y. Genome Res. 1998, 8, 769-776. (d) Thiel, A. J.; Frutos, A. G.; Jordan, C. E.; Corn, R. M.; Smith, L. M. Anal. Chem. 1997, 69, 4948-4956. (2) (a) Horn, T.; Uedea, M. S. Nucleic Acids Res. 1989, 17, 6959-6967. (b) Wang, J.; Jiang, M. J. Am. Chem. Soc. 1998, 120, 8281-8282. (3) (a) Ullman, A. Introduction to Ultra Organic Films: From Langmuir-Blodgett to Self-Assembly; Academic Press: Boston, 1991. (b) Nuzzo, R. G.; Allara, D. L. J. Am. Chem. Soc. 1983, 105, 4481-4483.
1086
(4) (a) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916-8920. (b) Levicky, R.; Herne, T. M.; Tarlov, M. J.; Satija, S. K. J. Am. Chem. Soc. 1998, 120, 9787-9792. (c) Peterson, A. W.; Heaton, R. J.; Georgiadis, R. J. Am. Chem. Soc. 2000, 122, 7837-7838. (d) Peterlinz, K.; Georgiadis, R. M.; Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 3401-3402. (e) Georgiadis, R. M.; Peterlinz, K.; Peterson, A. W. J. Am. Chem. Soc. 1997, 119, 3401-3402. (f) Peterson, A. W.; Wolf, L. K.; Georgiadis, R. M. J. Am. Chem. Soc. 2002, 124, 14601-14607. (5) Kretschmann, E.; Raether, H. Z. Naturforsch., A 1968, 23, 21352136. (6) Bird, R. J.; Swift, P. J. Electron Spectrosc. Relat. Phenom. 1980, 21, 227-240. (7) (a) Zubragel, Ch.; Deuper, C.; Schneider, F.; Neumann, M.; Grunze, M.; Schertel, A.; Woll, Ch. Chem. Phys. Lett. 1995, 238, 308-312. (b) Ishida, T.; Hara, M.; Kojima, I.; Tsuneda, S.; Nishida, N.; Sasabe, H.; Knoll, W. Langmuir 1998, 14, 2092-2096. (8) Weisser, M.; Kashammer, J.; Menges, B.; Matsumoto, J.; Nakamura, F.; Ijiro, K.; Shimomura, M.; Mittler, S. J. Am. Chem. Soc. 2000, 122, 87-95. (9) (a) Dickson, R. M.; Norris, D. J.; Tzeng, Y.; Moerner, W. E. Science 1996, 274, 966-969. (b) Xu, X.; Yeung, E. Science 1997, 275, 1106-1109. (c) Xu, X.; Yeung, E. Science 1998, 281, 1650-1653.
NL0342794
Nano Lett., Vol. 3, No. 8, 2003
