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Tunable Stabilization of Gold Nanoparticles in Aqueous Solutions by Mononucleotides Wenting Zhao,† Thomas M. H. Lee,‡,§ Sharon S. Y. Leung,‡ and I-Ming Hsing*,†,‡ Bioengineering Graduate Program and Department of Chemical Engineering, The Hong Kong UniVersity of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR ReceiVed March 9, 2007. In Final Form: April 7, 2007 Gold nanoparticles are one of the popular nanomaterials, widely used in biosensor applications as well as nanostructure construction. An essential attribute of these gold nanoparticles (Au-nps) is their stabilization against salt-induced aggregation. In this work, utilization of deoxyribonucleotides (dNTPs) as a tunable surface-stabilization agent for Au-nps was investigated. It was found that surfaces of Au-nps are covered by a layer of dNTPs after an adequate incubation with dNTPs solutions. Electrostatic repulsion among dNTP-coated Au-nps could prevent aggregation of Au-nps at a high salt concentration. The strength of dNTP-based protection can be manipulated by changing preparation protocols (e.g., incubation temperature, ionic strength, and ratio of Au-nps to dNTPs). Four different types of dNTPs exhibit different binding affinity to Au-nps and thus various stabilization efficiency in the order of dATP > dCTP > dGTP ≈ dTTP. Moreover, this salt-induced aggregation can be reinitiated by the increase of solution temperature, which leads to a partial removal of the protective dNTP layer on Au-nps. The advantage of thermally tunable aggregation/dispersion of Au-nps mediated by dNTP adsorption offers a useful approach for the preparation of biomolecule (oligonucleotides and oligopeptides) modified nanoparticles in applications of bioassay and nanobiotechnology.
Introduction Gold nanoparticles (Au-nps) and oligonucleotide probemodified nanoparticles have been proven to be useful for the detection of nucleic acids.1-5 They are versatile materials for DNA nanostructure construction6-10 and offer interesting benefits to intracellular gene regulation.11 There have been many research investigations to study surface modification of Au-nps with an aim to provide stable biomolecule-functionalized nanoparticles for bioassay applications. Generally, a negatively charged citrate layer is electrostatically adsorbed on the Au-nps surface after the citrate reduction process. At a low ionic strength condition, Aunps are in a monodispersed state due to the mutual charge repulsion. However, at high ion strength scenarios, these citratestabilized Au-nps are vulnerable to aggregation as a result of charge neutralization. * Corresponding author. E-mail:
[email protected]. Telephone: (852) 23587131. Fax: (852) 3106-4857. † Bioengineering Graduate Program. ‡ Department of Chemical Engineering. § Current address: Department of Health Technology and Informatics, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong SAR. (1) Cao, Y. W. C.; Jin, R. C.; Mirkin, C. A. Science 2002, 297, 15361540. (2) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (3) Stoeva, S. I.; Lee, J. S.; Thaxton, C. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2006, 45, 3303-3306. (4) Storhoff, J. J.; Elghanian, R.; Mucic, R. C.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 1959-1964. (5) Weizmann, Y.; Patolsky, F.; Willner, I. Analyst 2001, 126, 1502-1504. (6) Claridge, S. A.; Goh, S. L.; Frechet, J. M. J.; Williams, S. C.; Micheel, C. M.; Alivisatos, A. P. Chem. Mater. 2005, 17, 1628-1635. (7) Mucic, R. C.; Storhoff, J. J.; Mirkin, C. A.; Letsinger, R. L. J. Am. Chem. Soc. 1998, 120, 12674-12675. (8) Pinto, Y. Y.; Le, J. D.; Seeman, N. C.; Musier-Forsyth, K.; Taton, T. A.; Kiehl, R. A. Nano Lett. 2005, 5, 2399-2402. (9) Zhang, J. P.; Liu, Y.; Ke, Y. G.; Yan, H. Nano Lett. 2006, 6, 248-251. (10) Zheng, J. W.; Constantinou, P. E.; Micheel, C.; Alivisatos, A. P.; Kiehl, R. A.; Seeman, N. C. Nano Lett. 2006, 6, 1502-1504. (11) Rosi, N. L.; Giljohann, D. A.; Thaxton, C. S.; Lytton-Jean, A. K. R.; Han, M. S.; Mirkin, C. A. Science 2006, 312, 1027-1030.
Monodispersed Au-nps can often be observed in oligonucleotide probe-linked Au-nps where the highly negatively charged phosphate group of alkanethiol-capped oligonucleotides selfassembled on Au-nps by Au-thiol bond offers resistance to saltinduced aggregation. A protocol developed by Mirkin and coworkers4 to form Au-nps and oligonucleotide conjugates was well-accepted by the community. In their approach, Au-nps and oligo probes were mixed at a low ionic strength condition, followed by an overnight incubation and a salt aging step. The whole procedure took about 2 days, because the chemisorption step in linking thiol oligo probes to citrate-protected Au-nps has to be carried out very slowly in a carefully controlled salt environment to avoid aggregation of Au-nps. An alternative strategy was reported by Alivisatos and co-workers where a polymeric layer was introduced to stabilize Au-nps before adding oligonucleotide probes.6,12 One drawback of their interesting method is the long polymer complexation step (more than 10 h), and also, the irreversible coating of polymers on Au-nps could hinder the diffusion and attachment of the probes to the particle surface. Considering the issues above, it would be of great value to develop new stabilization strategies for Au-nps that can quickly render the Au-nps stable against salt-induced aggregation and yet compatible to downstream processing. It should be noted that, in addition to terminal linkage of DNA and Au-nps through Au-S linkage, nonspecific binding of nucleotides to Au-nps through the nitrogen-containing bases13 or phosphate groups14,15 was also well-reported, and often this process was considered a disadvantage in many of the bioassay applications. The nonspecific and electrostatic interaction between Au-nps and nucleotides could result in an inadequate molecular conformation of oligonucleotides probe,16 which (12) Loweth, C. J.; Caldwell, W. B.; Peng, X. G.; Alivisatos, A. P.; Schultz, P. G. Angew. Chem., Int. Ed. 1999, 38, 1808-1812. (13) Demers, L. M.; Ostblom, M.; Zhang, H.; Jang, N. H.; Liedberg, B.; Mirkin, C. A. J. Am. Chem. Soc. 2002, 124, 11248-11249. (14) Li, H. X.; Rothberg, L. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1403614039. (15) Li, H. X.; Rothberg, L. J. J. Am. Chem. Soc. 2004, 126, 10958-10961.
10.1021/la7006843 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/23/2007
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compromises the hybridization performance17-19 or enzymatic reaction efficiency20 of oligo/Au-np conjugates. The idea of our approach was inspired by a recent work of Li and Rothberg where they took advantage of the preferential nonspecific binding of single-stranded oligonucleotides over double-stranded ones to the surface of Au-nps and devised a labelfree colorimetric DNA detection scheme.14,15 Fan and co-workers used a similar approach to detect conformational changes of potassium ion DNA aptamers.21 In this study, we attempt to develop a strategy to control the adsorptive coverage of mononucleotide (dNTPs) on Au-nps. When the Au-nps are well-covered by dNTPs, Au-nps will appear in a monodispersed state and be stabilized even at high salt concentrations, whereas the salt-induced aggregation of Au-nps could occur when the dNTP layers are removed from the surface by heating. Our results will demonstrate that the stabilization of Au-nps can be effectively tuned by manipulating coverage of dNTPs on Au-nps. dNTP-coated Au-nps display reversible aggregation/monodispersion by changing salt concentration. Irreversible salt-induced aggregation of dNTP-coated Au-nps can be triggered by gradually increasing solution temperature. Operation conditions and properties (e.g., different types of dNTPs, molar ratio of Au-nps to dNTPs, ionic strength, and incubation temperature) that would affect the binding strength and coverage of dNTPs on Au-nps are investigated. Adsorption kinetics of mononucleotides vs oligonucleotides to Au-nps is discussed. Transmission electron microscopy (TEM) and UVvis spectroscopy are employed to characterize the morphology and aggregation of Au-nps. Real-time fluorescent studies are conducted to monitor the thermal desorption process of the dNTPs from the Au-nps surfaces. Experimental Section Materials. Au-nps were purchased from Sigma (10 nm, approximately 0.01% HAuCl4). Four different mononucleotide solutions of dATP, dCTP, dGTP, and dTTP (10 mM) were ordered from Invitrogen, as well as dNTP mixture solution with an equal molar amount (10 mM each) of four different mononucleotides. Cy3dCTP was from GE Healthcare. Single-stranded DNA (ssDNA) was synthesized by Integrated DNA Technologies with the following sequence: 5′-GTA AAA CGA CGG CCA G -3′. Sodium chloride (NaCl, 99%), sodium phosphate monobasic monohydrate (NaH2PO4‚H2O, 99%), and disodium hydrogen phosphate (Na2HPO4, 99%) were provided by USB Corporation. Poly(L-lysine) was obtained from Sigma. Water was purified with NANOpure Diamond TOC Analytical Ultrapure Water System (Barnstead, U.S.A.) and autoclaved by ES-215/ES-315 Autoclaves (Tomy, Japan). UV-vis Spectroscopy. Au-nps were mixed with ssDNA or dNTPs in different molar ratios. After an incubation of 5-30 min, the mixture was adjusted to 10 mM sodium phosphate buffer (SPB) pH 7.0, with 0.1 M NaCl, unless otherwise stated. The samples for UV-vis spectroscopy were heated by an Eppendorf Mastercycler personal thermal cycler if needed and mixed vigorously by vortexing (VortexGenie 2) prior to the measurement. 1 mL plastic cuvettes (BRAGG&CO.) were used to hold the samples, and all UV-vis spectra were obtained using an Ultrospec 4300 pro UV/Visible Spectrophotometer (Amersham Biosciences) with 1 nm resolution (16) Parak, W. J.; Pellegrino, T.; Micheel, C. M.; Gerion, D.; Williams, S. C.; Alivisatos, A. P. Nano Lett. 2003, 3, 33-36. (17) Akamatsu, K.; Kimura, M.; Shibata, Y.; Nakano, S.; Miyoshi, D.; Nawafune, H.; Sugimoto, N. Nano Lett. 2006, 6, 491-495. (18) Maye, M. M.; Nykypanchuk, D.; van der Lelie, D.; Gang, O. J. Am. Chem. Soc. 2006, 128, 14020-14021. (19) Harris, N. C.; Kiang, C. H. Phys. ReV. Lett. 2005, 95, 046101-1046101-4. (20) Pena, S. R. N.; Raina, S.; Goodrich, G. P.; Fedoroff, N. V.; Keating, C. D. J. Am. Chem. Soc. 2002, 124, 7314-7323. (21) Wang, L. H.; Liu, X. F.; Hu, X. F.; Song, S. P.; Fan, C. H. Chem. Commun. 2006, 3780-3782.
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Figure 1. UV-vis spectra of Au-nps solutions mixed with and without nucleotide solutions. Dotted line: Au-nps only (3 nM). Solid line: Au-nps mixed with dNTPs mixture (containing an equal molar amount of four different mononucelotides) in a 1:1000 molar ratio for 5 min incubation at room temperature, followed by a salt concentration adjustment to 10 mM SPB (pH 7.0) with 0.1 M NaCl. Dashed line: Au-nps mixed with 16nts-ssDNA in a 1:1000 base molar ratio under the same incubation and salt adjustment as for the dNTPs (solid line) case. The insert images show the observed colors of the mixture solution of dNTPs/Au-nps (red) and ssDNA/Au-nps (purple). and scanning speed of 4626 nm/min. Spectra data were collected using the SWIFT II applications software. Transmission Electron Microscopy (TEM). Carbon-coated nonporous film supported by copper grid (SPI Supplies Inc., 400 mesh) was treated by 0.01% poly(L-lysine) prior to use. A 10 µL sample (prepared as in UV-vis spectroscopy study) was gently blotted with filter paper to obtain a thin layer on the grid. After overnight drying in air, the specimens were examined in a JEM 2010 transmission electron microscope (JEOL). Images were taken using a GATAN MSC 794 CCD Camera with the GATAN Digital Microscopy software package. Real-Time Fluorescent Study. Fluorescence signals of Cy3dCTP were recorded using Applied Biosystems 7300 Real-Time PCR System. 50 µL of the gold colloid solution was mixed with 1.92 µL of 25 µM Cy3-dCTP and 2.08 µL water. After standing for 30 min, 6 µL of 10 × SPB was added to bring the total volume to 60 µL. Then, the fluorescence signals of the Cy3-dCTP in the prepared samples were recorded every 1 min under certain temperature.
Results and Discussion Stabilization of Au-nps by Nonspecific Bindings: dNTPs vs Oligonucleotides. Nonspecific bindings of nucleotides with Au-nps surfaces through the functional groups (e.g., phosphate group and bases) are usually not desired, as these bindings could change the conformation of DNA probes (e.g., from standing-up to lying-down),16 subsequently influencing DNA hybridization efficiency on Au-nps. Nevertheless, the charged interaction between nucleotides (e.g., dNTPs or short oligonucleotides) and Au-nps, if properly controlled, can be used to shield away the salt-induced aggregation of citrate-reduced Au-nps, since they could introduce both charge repulsion and steric hindrance between particles. In this study, nucleotides of two different lengths (dNTPs containing an equal molar amount of four different mononucelotides and 16 nt ssDNA) were used to study the Aunps stabilization, and UV-vis spectroscopy was conducted to characterize the dispersity of Au-nps protected by nucleotides. Au-nps in monodispersed form gives rise to a characteristic absorption peak at around 520 nm, while a red-shift of the adsorption peak is expected for aggregated Au-nps. As can be seen from the UV-vis spectra (Figure 1), dNTPs can stabilize
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Au-nps, and dNTP-coated Au-nps stay monodispersed, as reflected by a sharp surface plasmon adsorption band at around 520 (red solution in the inset of Figure 1). On the other hand, a red shift of the adsorption band, accompanied by a decrease in absorbance at 520 nm and increase in absorbance at higher wavelengths, is seen for the ssDNA/Au-nps, suggesting the appearance of aggregated Au-nps (purple solution in the inset of Figure 1). A previous study by Mirkin et al. demonstrated that the binding of less-charged deoxynucleosides (dA, dC, and dG) to Au-nps leads to particle aggregation.22 However, in our case, the highly charged phosphate backbone of dNTPs sustains the electrostatic repulsion among dNTP-adsorbed Au-nps and thus stabilizes the particles. Our data also confirm that dNTPs provide better stabilization than ssDNA at the same incubation condition (Figure 1), suggesting that shorter mononucleotides should have a higher adsorption rate to Au-nps than longer ssDNA. This result is consistent with the study by Li and Rothberg.15 More dNTPs in Au-nps solutions should provide higher strength to guard against the salt-induced aggregation. Indeed, it was found from our experiments, which were repeated three times, that a Au-nps/dNTPs solution at molar ratio of 1:10 000 could resist aggregation in a high salt environment of 0.6 M NaCl in 10 mM SPB (pH 7.0), whereas a Au-nps/dNTPs solution at a molar ratio of 1:100 will aggregate at a salt concentration of 0.1 M NaCl in 10 mM SPB (pH 7.0). The stabilization effect of four different mononucleotides was also investigated in this study. Other investigations reported the binding affinity of less-charged bases (A, adenine; G, guanine; C, cytosine; T, thymine) and nucleosides (dA, dG, dC, dT) to gold surface.13,22,23 However, the binding interaction of highercharged mononucleotides (dATP, dGTP, dCTP, dTTP) on Aunps has not yet been investigated. In our experiments, the stabilization effect of different nucleotides on Au-nps was monitored separately by observing the absorbance peak change at 650 nm (a characteristic adsorption wavelength of aggregated Au-nps) at different salt concentrations. As shown in Figure 2A, there is a significant increase in absorbance for the Au-nps solutions incubated with dTTP and dGTP at 0.1 M NaCl condition, while those incubated with dATP or dCTP do not change much until the salt concentration is higher than 0.3 M NaCl. Similar results were obtained in repeated experiments. These results indicate that Au-nps could sustain a much higher salt concentration when protected by dATP or dCTP than by dGTP and dTTP. It should also be noted that the critical salt concentration to induce aggregation of dATP-protected Au-nps is higher than that of the dCTP protected one, suggesting that dATP is better than dCTP as a Au-nps protection agent. TEM images of different mononucleotide-protected Au-nps at 0.1 M NaCl are shown in Figure 2B. The results comply with the UV-vis data that Au-nps incubated with dATP and dCTP remain in dispersed form while those mixed with dTTP and dGTP undergo severe aggregation. From Figure 2, the abilities of different mononucleotides to stabilize Au-nps, in our experiments, follow the order of dATP > dCTP > dGTP ≈ dTTP. The stability effect of nucleotides should have a positive correlation to their binding affinity to Au-nps. Indeed, other than the G-base associated species, our results are largely consistent with the reported binding strength of bases and nucleosides on gold surfaces: G(dG) > A(dA) > C(dC) > T(dT).13,22,23 The inconsistency of ranking in the binding affinity for G(dG) and dGTP could be explained by the possible formation of G-quartet from dGTP in salt solution,24 which in (22) Storhoff, J. J.; Elghanian, R.; Mirkin, C. A.; Letsinger, R. L. Langmuir 2002, 18, 6666-6670. (23) Ostblom, M.; Liedberg, B.; Demers, L. M.; Mirkin, C. A. J. Phys. Chem. B 2005, 109, 15150-15160.
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Figure 2. (A) Absorbance of Au-nps solutions at 650 nm under different salt concentrations. Au-nps were mixed with dATP (9), dCTP (b), dGTP (2), or dTTP (1), respectively, in a 1:1000 molar ratio for 5 min room temperature incubation followed by a salt concentration adjustment to 10 mM SPB (pH 7.0) with 0.1 M NaCl. (B) TEM images of Au-nps/mononucleotides samples in A at 0.1 M NaCl salt condition. Top panel from left to right: dATP, dCTP; bottom panel from left to right: dTTP, dGTP. Scale bar: 100 nm.
turn reduces the free concentration of dGTP to adsorb on Au-nps despite the high affinity of G for Au-nps. Moreover, the higherdimensional configuration could also occur among the dGTP adsorbed on Au-nps, which results in a different form of Au-np aggregation. The phenomena that G-rich sequences would contribute to particle aggregation were reported elsewhere.21,25 Salt and Thermal Dependency of Au-nps Stabilization. Thermal properties of mononucleotide-stabilized Au-nps were evaluated by changing the salt concentration and the temperature of colloidal solutions at a fixed molar ratio of 1 Au-nps to 10 000 dNTP. As shown in Figure 3A, an obvious aggregation associated with a red shift of the maximum absorption band was observed when the salt concentration increased from 0.1 M NaCl in 10 mM SPB to 0.7 M NaCl in 10 mM SPB. But when the salt (24) Wong, A.; Wu, G. J. Am. Chem. Soc. 2003, 125, 13895-13905. (25) Li, Z.; Mirkin, C. A. J. Am. Chem. Soc. 2005, 127, 11568-11569.
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Figure 4. (A) Fluorescent signals of Au-nps mixed with Cy3-dCTP in buffer at different temperatures: 60 °C (9), 70 °C (b), 80 °C (2), or 90 °C (1). 50 µL of 10 nM Au-10 nm solution, 1.92 µL of 25 µM Cy3-dCTP, 2.08 µL water, and 6 µL 0.1 M PBS (0.1 M NaCl) were mixed together, and the final mixture is 0.8 µM Cy3-dCTP, 10 mM PBS, and 0.1 M NaCl. (B) A schematic representation of quenching and reactivation of fluorescence through the adsorption and desorption of Cy3-dCTP on Au-nps surface. Figure 3. (A) UV-vis spectra of Au-nps solutions undergo different treatments. Solid line: Au-nps (3 nM) mixed with dNTPs mixture (containing an equal molar amount of four different mononucelotides) in 1:10 000 molar ratio, in 10 mM SPB (pH 7.0) with 0.1 M NaCl. Dotted line: Same sample as in solid line but the buffer with 0.7 M NaCl. Dashed line: Same sample as in dotted line, but the salt concentration was further diluted from 0.7 M NaCl to 0.1 M NaCl by adding ddH2O while keeping the total volume unchanged. Dashdot line: Same sample as in solid line but after heating treatment at 80 °C for 15 min. (B) A schematic representation of dNTPs protected Au-nps undergo salt-induced aggregation and heating treatment.
concentration was diluted down to 0.1 M NaCl with the total volume unchanged, the aggregated particles returned to the monodispersed state and the characteristic peak at 520 nm reappears. The reproducibility of this phenomenon was investigated in three parallel experiments, which all showed similar changes in the UV-vis spectra. This result reveals that the aggregation of the dNTP-protected Au-nps under a high salt concentration is due to the neutralization of charge repulsion. Most importantly, the protection layer of mononucleotides on the Au-nps surface is stable enough to survive a high salt condition; thus, the salt-induced aggregation of the protected Au-nps in this case is reversible (Figure 3B, upper route). On the contrary, severe aggregation was observed when the temperature of the colloidal solution was increased to 80 °C, and the aggregation was irreversible (dash-dot line of Figure 3A). This result suggests that the protection layer formed by the mononucleotides is destroyed or weakened during the heating process (Figure 3B, lower branch). Other studies in temperature-programmed desorption (TPD) have reported the required desorption temperature of different bases from a planar gold film: adenine (210 ( 5 °C), guanine (220 ( 5 °C), cytosine (160 ( 5 °C), and thymine (100 ( 5 °C).23 Because of different underlying substrates (nano-
particles vs planar films) and the good heat transfer characteristics enhanced by Au-nps,26-28 the protective layer of mononucleotides adsorbed on Au-nps can be removed at a much lower temperature than that of the bases and nucleosides on the Au film. It was well-studied that Au-nps can quench fluorescence when the fluorophore is in close proximity to the particle surface.29,30 This unique property provides a good way to study the temperature dependency of binding of mononucleotides on Au-nps. As shown in the schematic diagram of Figure 4B, the relative signal intensity of fluorescence is an indication of adsorption (low fluorescence) or desorption (high fluorescence) of Cy3-dCTP on Au-nps. Figure 4A shows the changes in the fluorescence signals under different temperatures as a function of time. It is obvious that, the higher the temperature, the higher the fluorescence signal is, suggesting that more Cy3-dCTP molecules are being removed from the Au-nps surfaces. On the basis of the temperature dependency of dNTPs coverage on Au-nps, the stabilization of Au-nps protected by mononucleotides can be tuned by controlling the critical factors in this process including temperature, molar ratio of Au-nps/ dNTPs, and salt concentration, as well as the types of mononucleotides employed. To demonstrate the tuning effect, the dispersion vs aggregation of Au-nps solutions mixed with dATP at different molar ratios exposed to different salt concentrations and temperatures (50 and 80 °C) were carefully characterized by UV-vis spectroscopy (see Supporting Information). It was (26) Hu, M.; Hartland, G. V. J. Phys. Chem. B 2002, 106, 7029-7033. (27) Keblinski, P.; Phillpot, S. R.; Choi, S. U. S.; Eastman, J. A. Int. J. Heat Mass Transfer 2002, 45, 855-863. (28) Li, M.; Lin, Y. C.; Wu, C. C.; Liu, H. S. Nucleic Acids Res. 2005, 33, e184. (29) Dulkeith, E.; Ringler, M.; Klar, T. A.; Feldmann, J.; Javier, A. M.; Parak, W. J. Nano Lett. 2005, 5, 585-589. (30) Rupcich, N.; Chiuman, W.; Nutiu, R.; Mei, S.; Flora, K. K.; Li, Y. F.; Brennan, J. D. J. Am. Chem. Soc. 2006, 128, 780-790.
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found that the Au-nps form aggregates when the dATP concentration is low or when more salt is in the solution. If more dATP was added or the salt concentration was diluted, Au-nps could be protected more effectively from the aggregation. If the solution temperature was increased (e.g., from 50 to 80 °C), the desorption rate of dATP from Au-nps surfaces will increase and the surface coverage of dATP on Au-nps will be reduced, resulting in a higher percentage of bare Au-nps surface exposed to the salt solution. Once the bare surface coverage reaches a threshold value, Au-nps will aggregate. Our approach to tune the Au-np aggregation is to carefully balance the coverage of bare and nucleotide-protected surfaces on Au-nps through the portfolio of salt concentration, molar ratio of Au-nps/dNTPs, incubation temperature, and incubation time. The effect of these parameters can be seen from the UV-vis spectra in Figures S1 and S2 in the Supporting Information. The ability to control the state (aggregation vs mondispersion) and coverage (bare vs nucleotideprotected) of Au-nps demonstrated in this study will find many useful applications in bioassay and nanobiotechnology. For example, the linkage of various biomolecules (e.g., oligonucleotides and oligopeptides) on Au-nps could be established by the gradual removal of the nucleotide protection layer upon a different thermal/salt environment. Au-nps equipped with controllable aggregation properties and versatile modification by different biomolecules would be very useful nanomaterials in bioseparation and biodetection.
Conclusions We have demonstrated in this study that the nonspecific electrostatic adsorption of dNTPs to Au-nps can be used effectively to stabilize Au-nps in aqueous solutions. This electrostatic repulsion among Au-nps, as a result of the protective
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layer of dNTPs, prevents the aggregation of Au-nps even at a high salt concentration. Interestingly, this dNTP-protected stabilization could be weakened or destroyed by increasing the solution temperature. Reversible dNTP-coated Au-nps aggregation/monodispersion, which cannot be achieved in citrate-coated Au-nps, was demonstrated in this work by tailoring the salt concentrations. Different types of dNTPs were shown to exhibit different binding strengths to the Au-np surface. dATP and dCTP were proven to be better stabilization reagents than dGTP and dTTP. The approach demonstrated in this study by thermally tuning the stabilization of Au-nps provides an active route to manipulate the dispersion/aggregation of Au-nps upon specific requirements. This method could be readily applicable for a new bioconjugation method on the nanoparticle surface. Exemplary studies to synthesize Au-nps/oligonucleotide conjugates by thermally replacing dNTPs on Au-nps surfaces with thiol-capped ssDNA are ongoing. Acknowledgment. The authors thank the funding support from the Research Grants Council of the Hong Kong Special Administrative Region Government (Project No. HKUST 601305). Laboratory facilities provided by Bioengineering Laboratory and Materials Characterization & Preparation Facility of HKUST are also acknowledged. The authors thank Dr. XU Ying for her input to the manuscript. Supporting Information Available: UV-vis spectra of Aunps solution under different temperature, Au-nps/dATP molar ratio, and salt concentration to demonstrate the tuning effect on particles’ stabilization. This material is available free of charge via the Internet at http://pubs.acs.org. LA7006843
