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Contribution of Nanoscale Curvature to Number Density of Immobilized DNA on Gold Nanoparticles Atsushi Kira,†,‡ Hyonchol Kim,§ and Kenji Yasuda*,§,| Research & DeVelopment DiVision, ULVAC Inc., 2500 Hagizono, Chigasaki, Kanagawa 253-8543, Japan, Graduate School of Engineering, Yokohama National UniVersity, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan, Kanagawa Academy of Science and Technology, KSP East 310, 3-2-1, Sakado, Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan, and Department of Biomedical Information, DiVision of Biosystems, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental UniVersity, 2-3-10 Kanda-Surugadai, Chiyoda-ku, Tokyo 101-0062, Japan ReceiVed October 14, 2008. ReVised Manuscript ReceiVed December 18, 2008 We report the curvature size dependence of the density of attached single-stranded DNA (ssDNA) on the surface of gold nanoparticles. The densities of immobilized ssDNA on 10, 20, 30, and 50 nm gold nanoparticles were examined, and we found that the maximum density of the immobilized ssDNA on 10 nm particles was 13 times larger than that on 50 nm particles, which was still 10 times larger than that on flat gold surfaces. This result indicates the importance of curvature in the nanometer-scale attachment of ssDNAs to nanoparticles.
Introduction One of the challenges in the postgenome era is discovering key biomarker molecules at a single-cell level. These molecules determine the functions, conditions, and variations of each cell as a minimum unit of living systems. For this study, our goal was to establish a method to detect expressed biomarkers in single cells quantitatively and simultaneously. There are several ways to identify expressed critical biomarkers in cells using traditional detection methods, such as DNA microarray1-5 or in situ hybridization technologies;6-9 however, it is generally difficult to apply these methods for a comprehensive analysis of expressed molecules at a single-cell level without any amplification process such as the polymerase chain reaction. The fundamental difficulties of single-molecule-level counting using fluorescent dyes are its lower signal/noise ratio, the limitation of multicolor observation caused by the energy transfer of emitted light, and the excitation light of nearby different fluorescent dyes. * Corresponding author. E-mail:
[email protected]. Tel: +81 3 5280 8046. Fax: +81 3 5280 8049. † ULVAC Inc. ‡ Yokohama National University. § Kanagawa Academy of Science and Technology. | Tokyo Medical and Dental University. (1) Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O. Science 1995, 270, 467–470. (2) Lockhart, D. J.; Dong, H.; Byrne, M. C.; Follettie, M. T.; Gallo, M. V.; Chee, M. S.; Mittmann, M.; Wang, C.; Kobayashi, M.; Horton, H.; Brown, E. L. Nat. Biotechnol. 1996, 14, 1675–1680. (3) Cawley, S.; Bekiranov, S.; Ng, H. H.; Kapranov, P.; Sekinger, E. A.; Kampa, D.; Piccolboni, A.; Sementchenko, V.; Cheng, J.; Williams, A. J.; Wheeler, R.; Wong, B.; Drenkow, J.; Yamanaka, M.; Patel, S.; Brubaker, S.; Tammana, H.; Helt, G.; Struhl, K.; Gingeras, T. R. Cell 2004, 116, 499–509. (4) Bertone, P.; Stolc, V.; Royce, T. E.; Rozowsky, J. S.; Urban, A. E.; Zhu, X. W.; Rinn, J. L.; Tongprasit, W.; Samanta, M.; Weissman, S.; Gerstein, M.; Snyder, M. Science 2004, 306, 2242–2246. (5) Cheng, J.; Kapranov, P.; Drenkow, J.; Dike, S.; Brubaker, S.; Patel, S.; Long, J.; Stern, D.; Tammana, H.; Helt, G.; Sementchenko, V.; Piccolboni, A.; Bekiranov, S.; Bailey, D. K.; Ganesh, M.; Ghosh, S.; Bell, I.; Gerhard, D. S.; Gingeras, T. R. Science 2005, 308, 1149–1154. (6) Rudkin, G. T.; Stollar, B. D. Nature 1977, 265, 472–473. (7) Bauman, J. G. J.; Wiegant, J.; Borst, P.; Vanduijn, P. Exp. Cell Res. 1980, 128, 485–490. (8) Nederlof, P. M.; Robinson, D.; Abuknesha, R.; Wiegant, J.; Hopman, A. H. N.; Tanke, H. J.; Raap, A. K. Cytometry 1989, 10, 20–27. (9) Raap, A. K.; Vanderijke, F. M.; Dirks, R. W.; Sol, C. J.; Boom, R.; Vanderploeg, M. Exp. Cell Res. 1991, 197, 319–322.
To overcome these problems, we proposed a method in which different sizes of gold nanoparticles are used as nonquenchable probes to detect target biomarkers.10 The difference in size means the same as the difference in color of fluorescent dyes. Target biomarker molecules are distinguished by specific labeling with one size of gold nanoparticles on which single-stranded DNA (ssDNA) molecules are immobilized as probes. The target molecules are then quantified by counting the numbers and positions of the particular sizes of particles using a field-emission scanning electron microscope (FE-SEM). The nanoparticle probes have a high affinity for targets such as mRNA in comparison with free DNA probes; hence, they could be used as very sensitive biosensors.11,12 Although the use of gold nanoparticles as labels to detect target nucleic acids with the immobilization of probe DNAs onto particles was reported,13,14 the nanoparticle size dependence of the number of attached probe DNA molecules has not yet been studied. Thus, the relationship between the particle size and the number density of immobilized DNA was carefully studied in this work.
Materials and Methods The thiolated single-stranded DNA (to be denoted as SH-ssDNA hereafter), which is composed of 20 oligonucleotide bases with the sequence 5′-SH-(CH2)6-GCAACAAGTGAGCATCATTC-3′, was commercially obtained (Tsukuba Oligo Service, Co., Ltd., Ibaraki, Japan). Before immobilization of SH-ssDNA, concentrations of 10, 20, 30, and 50 nm gold nanoparticle solutions (in diameter, British BioCell International Inc., Cardiff, U.K.) were adjusted as 95, 12, 3, and 0.75 nM, respectively. The concentrations of both gold nanoparticles and SH-ssDNA were calculated from measurements of the value in absorbance at 520 nm for gold nanoparticles and absorbance at 280 nm for SH-ssDNA. SH-ssDNA was adjusted as 95, 12, 3, and 0.75 µM and mixed with 10, 20, 30, and 50 nm gold nanoparticle solutions, respectively, with a ratio of 1000 SH-ssDNA/ (10) Kim, H.; Kira, A.; Yasuda, K. Submitted for publication. (11) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757– 1760. (12) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503–1506. (13) Storhoff, J. J.; Elghanian, R.; Mirkin, C. A.; Letsinger, R. L. Langmuir 2002, 18, 6666–6670. (14) Gearheart, L. A.; Ploehn, H. J.; Murphy, C. J. J. Phys. Chem. B 2001, 105, 12609–12615.
10.1021/la803385x CCC: $40.75 2009 American Chemical Society Published on Web 01/08/2009
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nanoparticle. After incubation for 4 h, the mixture was diluted 10fold with 500 mM NaCl and 10 mM phosphate buffer (pH 7.4) and then incubated for 12 h. Excess reagents were removed by two rounds of centrifugation and resuspension. The amount of SH-ssDNA immobilized on the gold nanoparticles was quantified by comparing a 260 nm UV-vis spectra of the SH-ssDNA solution before the immobilization with that of the supernatant including nonreacted SH-ssDNA resulting from centrifugation using a U-3010 spectrometer (Hitachi High-Technologies, Ltd., Tokyo, Japan). The stability of the gold nanoparticle probes against various salt concentrations was evaluated by measuring the change in absorbance at a wavelength of 520 nm. Solutions (1 nM) of both 20 nm bare gold nanoparticles and those on which SH-ssDNA was immobilized were adjusted in a 10 mM phosphate buffer (pH 7.4) with 10 mM, 50 mM, 100 mM, 500 mM, or 1.0 M NaCl, and each UV-vis spectrum was measured.
Results and Discussion First, we evaluated the number density of SH-ssDNA immobilized on 10, 20, 30, and 50 nm gold nanoparticles. Typical UV-vis spectra of the SH-ssDNA solution before and after immobilization on gold nanoparticles are shown in Figure 1a. In this case, 2.05 µM SH-ssDNA was reacted with 1.2 nM 20 nm gold nanoparticles. As the absorbance of free ssDNA at 260 nm was decreased from 0.470 to 0.245, 981 nM () (0.470-0.245)/ 0.470 × 2.05 µM) was expected to react with the 1.2 nM gold nanoparticles. That is, 817 SH-ssDNA molecules would be immobilized onto a single gold nanoparticle. Nonthiolated ssDNA can also be immobilized on a gold surface,15 but this nonspecific adsorption is expected to require less bond energy than a sulfur-gold bond.16,17 The reaction mainly occurs because of the thiol group in SH-ssDNA. The thiol group also enables very sensitive detection of target DNA10-12 because of the group’s control of molecular direction. After the immobilization, the stability of the gold nanoparticle probes against various salt concentrations was checked. The UV-vis spectra of bare gold nanoparticles and that of SH-ssDNA-coated particles are shown in Figure 1b,c, respectively. As the salt concentration increased, the absorbance at 520 nm, which was derived from the surface plasmons of colloidal gold nanoparticles, decreased and shifted to a long-wavelength region of more than 520 nm (Figure 1b). This was because the bare particles aggregated. Usually, this aggregation is caused by the decrease in electrostatic repulsive force between particles according to the increase in ion concentration.18,19 In the dispersion state, floating bare colloids were softly covered with ionic molecules in solution and were kept in a stable dispersion state by their electrostatic repulsive force. In contrast, under a high salt concentration, the covered ions were canceled by the salt ions, and thus the particles aggregated because of the lack of long-distance repulsion. However, nanoparticles on which SH-ssDNAs were immobilized did not aggregate even in the solution with high salt concentration (Figure 1c). This might have been caused by the stable electrostatic repulsion of the negative charge generated by the phosphate groups of the immobilized SH-ssDNA. These results indicate that gold nanoparticle probes on which DNA molecules have been tightly and thickly immobilized can maintain a dispersed condition and thus can be stably used to label molecular markers even in a solution with high salt (15) Herne, T. M.; Tarlov, M. J. J. Am. Chem. Soc. 1997, 119, 8916–8920. (16) Nuzo, R. G.; Zegarski, B. R.; Dubois, L. H. J. Am. Chem. Soc. 1987, 109, 733–740. (17) Vargas, R.; Garza, J; Friesner, R. A.; Stern, H.; Hay, B. P.; Dixon, A. J. Phys. Chem. A 1995, 105, 4963–4968. (18) Shipway, A. N.; Lahav, M.; Gabai, R.; Willner, I. Langmuir 2000, 16, 8789–8795. (19) Li, D.; Hang, Y.; Li, J. J. Colloid Interface Sci. 2005, 283, 440–445.
Figure 1. Fabrication of stable gold nanoparticle probes on which SHssDNA was immobilized against high salt concentration. (a) Typical UV-vis spectra of gold nanoparticles before and after immobilization of SH-ssDNA. The salt concentration is 500 mM. (b, c) Change in UV-vis spectra of both bare gold nanoparticles (b) and SH-ssDNAimmobilized particles (c) against a gradual change in the salt concentration in the solution.
concentration, such as a physiological buffer. It should be noted that the absorbance of the 520 nm spectra of salt-added buffers was lower than that of the salt-free buffer and that the maximum absorption wavelength shifted from 523 to 530 nm (Figure 1c). This might have been influenced by the adsorption of particle surface SH-ssDNA. Next, we measured the particle size dependence of the number density of the immobilized SH-ssDNA on 10, 20, 30, and 50 nm particles under several different concentration conditions. The
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Figure 2. Immobilization of SH-ssDNA on various sizes of gold nanoparticles with various number ratios. The salt concentration is 500 mM.
Figure 3. Relationship between maximum number densities of SHssDNA immobilized on gold nanoparticles and diameters of particles. The salt concentration is 500 mM.
Table 1. Estimated Dissociation Constant (Kd), Total Number of Reaction Sites on a Particle (x0), and Estimated DNA/DNA Distance (d) for 10, 20, 30, and 50 nm Particles in a 500 mM Salt Concentration particle size (nm) 10 20 30 50
Kd (M)
x0 -5
1.00 × 10 1.98 × 10-6 3.76 × 10-7 5.06 × 10-8
d (nm)
1.01 × 10 1.58 × 103 1.51 × 103 2.05 × 103 3
1.3 2.2 3.3 4.7
relationship between the number density of immobilized SHssDNA on the particles and the concentration of applied free SH-ssDNA is shown in Figure 2. As can be seen, the increase in the concentration of free SH-ssDNA resulted in the increase in the immobilized number density. These relationships can be described as follows. Ka
X+Y T Z
(1)
where the total number of empty reaction sites on the gold nanoparticles is X, the number of SH-ssDNA molecules is Y, the total number of reaction sites at which SH-ssDNA was immobilized on particles is Z, and the association constant is Ka. The number of immobilized SH-ssDNA molecules on a single particle, z, would be
z)
{
}
1 (Kd/n + x0 + y0) - √(Kd/n + x0 + y0)2 - 4x0y0 (2) 2
where Kd is the dissociation constant, n is the number of particles, x0 is the total number of reaction sites on a particle, and y0 is the number ratio between SH-ssDNA and gold nanoparticles. The relationship between z and y0 in eq 2 is represented in Figure 2, and by fitting the results in Figure 2 to the equation, we can calculate both Kd and x0. Each Kd, x0, and estimated DNA/DNA distance d (calculated with x0 and the closely packed surface area of a particle) for the 10, 20, 30, and 50 nm particles is shown in Table 1. As can be seen, the value of Kd decreased as particle size increased. This was caused by the decrease in the potential barrier (due to the electrostatic repulsion of neighbor DNAs) around a particle surface as the surface becomes flatter (i.e., the number density of attached DNAs decreased). That indicates two facts: the shape of nanoparticles can contribute to the value of Kd, and the number density of attached ssDNAs was increased on the smaller particles, even when the value of Kd in smaller particles became larger. Kd is defined using the association
Figure 4. Number density of immobilized SH-ssDNA on 20 nm gold nanoparticles in various salt concentrations.
rate constant (k+) and dissociation rate constant (k-) (Kd ) k-/ k+). Because the thiol-gold interaction is high-energy, Kd decreased following the particle size increase, indicating that the association rate constant becomes large according to the change in the shape of the particles. x0 corresponds to the maximum number of reaction sites at which SH-ssDNA can be immobilized on a particle, so we can calculate the relationship between the maximum number density (calculated from x0 divided by the area of a single particle) and the diameters of the particles (Figure 3). As shown in Figure 3, the number density decreased according to the increase in the particle size. That is, the estimated number density of a 10 nm particle is 13 times larger than that of a 50 nm particle. Even that of a 50 nm particle is still 10 times larger than that of a flat gold surface (i.e., the number density of a flat gold substrate).20 The nanoscale structure, such as the curvature of the nanoparticles, contributes to the increase in the number density of SH-ssDNA.21,22 (20) Peterson, A. W.; Wolf, L. K.; Georgiadis, R. M. J. Am. Chem. Soc. 2002, 124, 14601–14607. (21) Hurst, S. J.; Lytton-Jean, A. K. R.; Mirkin, C. A. Anal. Chem. 2006, 78, 8313–8318. (22) Demer, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A.; Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000, 72, 5535–5541.
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This might be explained by the nanostructure possibly decreasing the electrostatic repulsion between the SH-ssDNA molecules attached to a particle surface. The number density of SH-ssDNA immobilized on 20 nm gold nanoparticles in 500 mM NaCl was 5 times greater than that in 0 M NaCl (Figure 4). This result indicates that the “electrostatic repulsion” resulting from the negative charges of phosphate groups on the backbone of the SH-ssDNA contributed to the density of the immobilized SH-ssDNA on gold surfaces. The possibility of a higher number density for smaller nanoparticles is helpful for the practical application of smaller nanoparticles as target probes. For example, without the contribution of nanoscale curvature to number density as described above, we expect that only about 400 DNA target probes could attach to the surface of a 10 nm nanoparticle using the number density of 20 nm nanoparticle (1580 DNAs maximum, x0). However, the contribution of nanoscale curvature can increase the number of probe DNAs to 1010 on a 10 nm nanoparticle, which has a 2.5 times higher density than that of a 20 nm particle.
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Summary The immobilization of SH-ssDNA on several sizes of gold nanoparticles was minutely studied, and the maximum number densities of immobilized SH-ssDNA were successfully estimated. The immobilizations were performed with a stepwise change in the buffer condition to prepare stable and reproducible nanoparticle probes. The number density decreased as the particle size increased. This could be explained by the electrostatic repulsion of SH-ssDNA on a nanoparticle surface. These results greatly help in preparing a set of stable gold nanoparticle probes with accurate control of number density. These probes can be used for the quantitative and reproducible detection of expressed biomarkers in or on a cell. Acknowledgment. We thank Dr. K. Okano for valuable discussions and experimental support. This work was supported by a Grant-in-Aid for JSPS Fellows and Grants-in-Aid for Science Research from The Ministry of Education, Culture, Sports and Technology of Japan and the Japan Science and Technology Agency (JST). LA803385X
