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Bioconjugate Chem. 2009, 20, 1218–1222
Rapid Synthesis of DNA-Functionalized Gold Nanoparticles in Salt Solution Using Mononucleotide-Mediated Conjugation Wenting Zhao,† Li Lin,† and I-Ming Hsing*,†,‡ Bioengineering Graduate Program and Department of Chemical and Biomolecular Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong SAR. Received February 20, 2009; Revised Manuscript Received April 8, 2009
DNA functionalized gold-nanoparticles (Au-nps) have been broadly used as labeling reagents in the development of molecular diagnostics as well as building blocks in nanotechnology. Conventional methods for the synthesis of DNA functionalized Au-nps require long incubation, typically overnight, and delicate control of the ionic strength to compensate for the charge repulsion between the nanoparticles surface and the DNA strands, which generally affect the stability of the nanoparticles and the DNA loading density. In this study, we present a novel mononucleotide-mediated conjugation approach to synthesize DNA-functionalized Au-nps within 4 h in a high ionic strength environment. Au-nps covered with a thermally tunable stabilization layer through mononucleotide adsorption were shown to readily conjugate with thiol-DNAs in 0.1 M NaCl solution upon heating. Monitoring this mononucleotide-mediated conjugation reaction through dynamic light scattering and UV-vis spectroscopy demonstrated the formation of stable DNA/Au-nps conjugates. The resulting conjugates, as characterized by fluorescence spectroscopy, are loaded by ∼80 strands per particle, comparable to the DNA loading density of current approaches. The general applicability of this approach was further verified in a nanoparticle-bound DNA hybridization test. Our results show that mononucleotide-mediated thermal conjugation is an attractive alternative that allows temperature-controlled and salt-enhanced functionalization of gold nanoparticles with DNAs in just a few hours.
INTRODUCTION DNA-functionalized gold nanoparticles (Au-nps) play an important role in biosensing and nanobiotechnology. A synergistic combination of the sequence-specific hybridization-based molecular recognition of DNA molecules and the size-dependent chemical, physical, and plasmonic properties of Au-nps has enabled DNA/Au-nps conjugates to find many critical applications in molecular diagnostics (1–5), nanofabrication (6–9), molecular nanoelectronics (10–12), cell imaging (13), and gene regulation (14). The use of DNA-functionalized gold nanoparticles is still in its infancy, and many more interesting approaches are expected. One of the key essentials to fully developing their application potentials is the fast and reproducible synthesis of stable DNA/Au-nps conjugates. On the basis of the chemistry of thiol self-assembly on gold, a widely accepted protocol to immobilize specific DNAs onto Au-nps surfaces was developed by Mirkin and co-workers (15). In their approach, a 2 day incubation process was needed to directly link thiol-terminated oligonucleotides to citrate-stabilized Au-nps (referred to as direct conjugation approach). Specifically, salt was added after an initial overnight incubation to neutralize the charge repulsion, and then, a 40 h aging step was performed to achieve a high DNA loading density, which is critical for the stabilization of the conjugates. Improvement on the direct conjugation approach was later reported by Brust and co-workers, who applied vacuum centrifugation instead of a long aging step to speed up the formation of stable conjugates, and the conjugation process was completed in ∼20 h (16). They suggested that the vacuum centrifugation step was essential to * Corresponding author. I-Ming Hsing, E-mail:
[email protected], Telephone: (852) 2358-7131, Fax: (852) 3106-4857. † Bioengineering Graduate Program. ‡ Department of Chemical and Biomolecular Engineering.
ensure a gradual and simultaneous rise of solution DNA concentration and ionic strength with a concomitant increase of DNA loading density on particles. Other than the direct conjugation method, Alivisatos and co-workers reported a strategy to minimize the salt effect on the particle stability by the complexation of Au-nps with bis(p-sulfonatophenyl)phenylphosphine dehydrate dipotassium salt (BSPP) prior to the immobilization of thiol-DNA molecules (referred to as BSPP coating approach) (17, 18). With BSPP protection, the saltdependent aggregation of Au-nps was controlled and the conjugation process could be done in ∼12 h. However, this process is suitable for the synthesis of mono or low DNA conjugated Au-nps, but not intended for preparing DNA/Aunps conjugates with a high DNA loading density. In this study, we report a new conjugation method enabling a rapid immobilization of high-density DNAs on stable Aunps in a salt environment, which to our knowledge has never been reported in the literature. The essence of our strategy relies on the fast and reversible binding of mononucleotides to Aunps surfaces. This binding was used to quickly stabilize Aunps in salt solution by forming a mononucleotide layer on particle surface, and the adsorption/desorption of this layer is also thermally tunable (19). As shown in Scheme 1, our conjugation process starts with a reversible adsorption of mononucleotides on the Au-nps for nanoparticle stabilization in salt solutions, followed by a temperature-facilitated ligandexchange reaction between the incoming thiol-DNA and the protecting mononucleotides on Au-nps surface. Our experimental characterization data will show that the synthesis of DNA/ Au-nps conjugates by our method could be achieved within several hours and the resulting conjugates have similar or better characteristic attributes (DNA loading density and stability) in comparison with those synthesized by conventional methods. The ability to modify Au-nps with specific DNAs in an efficient
10.1021/bc900080p CCC: $40.75 2009 American Chemical Society Published on Web 05/08/2009
Rapid Synthesis of DNA-Functionalized Au-NPs Scheme 1. Mononucleotide-Mediated Conjugation of Thiol-DNA to Gold Nanoparticles
and controlled manner opens up opportunities in molecular diagnostics and bionanotechnology.
EXPERIMENTAL PROCEDURES Materials. Thirteen nanometer Au-nps were prepared by reduction of tetrachloroaurate as reported previously (20, 21), while 20 nm Au-nps, hydrogen tetrachloroaurate trihydrate (HAuCl4 · 3H2O), sodium citrate, and poly(L-lysine) were purchased from Sigma. dATP (10 mM) was purchased from Invitrogen, and oligonucleotides were synthesized by Integrated DNA Technologies. Other salts were provided by USB Corporation. The water used in all experiments was purified with NANOpure Diamond TOC Analytical Ultrapure Water System (Barnstead, U.S.A.) and autoclaved by ES-215/ES-315 Autoclaves (Tomy, Japan) prior to use. Conjugation of DNA/Au-nps. The procedure for mononucleotide-mediated conjugation of DNA/Au-nps is illustrated in Scheme 1. Citrate-stabilized Au-nps were first incubated with dATP in a molar ratio (dATP/Au-nps) of 1000 for 15 min. The mixture was then brought to 10 mM sodium phosphate buffer (pH 8.0) and 0.1 M NaCl. After brief vortexing, thiol-DNA (thiol-T30, 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-C3-thiol-3′, 500 µM) was introduced in a molar ratio (thiolDNA to Au-nps) of 500, followed by heating (Eppendorf Mastercycler personal thermal cycler) at a constant temperature for 3 h. Except for the temperature effect study, all the heating steps were conducted at 60 °C. Afterward, the particles were washed three times in 10 mM sodium phosphate buffer (pH 8.0) with 0.1 M NaCl through centrifugation (13 200 rpm, 20 min) to remove excess reagents. Finally, the resulting conjugates were resuspended in 100 µL sodium phosphate buffer (10 mM, pH 8.0) with NaCl (0.3 M). For the direct conjugation and BSPP coating approaches, the procedure is the same as previously reported (15, 17, 18). Hybridization of Au-nps Linked DNAs. Hybridization test was performed using DNA/Au-nps conjugates of different sizes and labeled with DNAs of complementary sequences. Specifically, two conjugates were prepared using the mononucleotidemediated approach: Au-nps of 13 and 20 nm functionalized with respective cDNA sequences (thiol-T30, 5′-TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT-C3-thiol-3′, to Au-nps of 20 nm (thiol-T30/Au20) and thiol-A30, 5′-AAA AAA AAA AAA AAA AAA AAA AAA AAA AAA-C3-thiol-3′, to Au-nps of 13 nm (thiol-A30/Au13)). Two conjugates were mixed overnight at a molar ratio of 1:10 (thiol-T30/Au20:thiol-A30/Au13) in 10 mM sodium phosphate buffer with 0.3 M NaCl (pH 8.0). In the negative control, the thiol-A30 was substituted with a nonthiol-DNA (nonthiol-A24, 5′-AAA AAA AAA AAA AAA AAA AAA AAA-3′, forming nonthiol-A24/Au13). The resulting solutions of hybrids were imaged with TEM. UV-vis Spectroscopy. A 1 mL cuvette (Bragg and Co.) was used, and all spectra were obtained through Ultrospec 4300 pro UV-vis spectrophotometer (Amersham Biosciences). Dynamic Light Scattering (DLS). Measurements were performed using a ZetaPlus (Brookhaven Instruments Corpora-
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tion). Incident light was provided by a 35 mW solid state laser (660 nm). Scattered light was collected at a fixed angle of 90°. All samples were filtered through a 0.22 µm acetate membrane filter prior to measure. Five runs were performed per sample with 30 s per run. Fluorescence-Based Quantification of the DNA Loading Density on Au-nps. Centrifugation pellets of 300 µL Cy3-A20thiol/Au13 conjugates were incubated overnight with 50 µL mercaptoethanol (ME) (12 mM in 0.3 M PBS). The solution containing displaced Cy3-A20-thiol was separated from the Aunps by centrifugation. The fluorescence signal of each sample was measured using Applied Biosystems 7300 real-time PCR system. Standard curves were prepared to convert the fluorescence reading to molar concentration. Transmission Electron Microscopy (TEM). Specimens were prepared by deposition of a drop of colloidal solutions on 0.01% poly(L-lysine) pretreated carbon-nonporous film supported by copper grid (SPI Supplies Inc., 400 mesh). All the samples were examined in a JEM 2010 transmission electron microscope (JEOL) operated at 200 kV. Images were taken by GATAN MSC 794 CCD Camera and analyzed with GATAN Digital Microscopy software.
RESULTS Scheme 1 illustrates our strategy for the synthesis of DNA/ Au-nps conjugates. As shown, the process involves the protection of Au-nps from salt-induced aggregation by the mononucleotide adsorption and the thermally enhanced ligand exchange reaction between the surface-bound mononucleotides and the incoming DNA molecules. Deoxyadenosine triphosphate (dATP) was chosen among the four different mononucleotides, as in our previous study dATP-covered Au-nps were found to have a higher salt tolerance than the Au-nps protected by other mononucleotides (19). A detailed working procedure for conjugate synthesis is described in the Experimental Procedures. Critical characterization results of this conjugation approach are delineated in the following paragraphs. Conjugate Stability. It would be useful to understand the stability of the synthesized DNA/Au-nps conjugates, since the conjugates might also be vulnerable to the irreversible saltinduced aggregation at high temperature and high ionic strength as the mononucleotide-coated Au-nps do. From the measurement of surface plasmon resonance (SPR) using UV-vis spectroscopy, it is clear in Figure 1 that our DNA/Au-nps conjugates (thiol-T30/Au13) are stable at a salt solution (0.1 M NaCl) and exhibit a characteristic adsorption peak at 520 nm before and after the heat treatment at 80 °C. On the other hand, dATPcovered Au-nps without thiol-DNA attachments were found to suffer aggregation with an obvious color change from red to blue and a significant shift of the adsorptive peak to a higher wavelength (e.g., 650 nm), which is consistent with the previous report (19). The conjugate stability of the 20 nm Au-nps was also evaluated using UV-vis spectroscopy. Similar stability results (see Supporting Information) were observed. It is believed that the enhanced stability of the DNA/Au-nps conjugate comes from the ligand replacement of the initial dATP protection layer by thiol-DNA linkage, that stabilizes the conjugate even at high ionic strength and at elevated temperature. Other than a few-nanometer red shift caused by the newly formed thiol-DNA/Au linkage, the conjugate shows a similar SPR peak to that of the citrate Au-nps without salt. Effective Diameter of DNA/Au-nps Conjugates. In addition to assessing the dispersion of Au-nps, effectiveness of the dATPmediated conjugation method could also be reflected by measuring the “effective diameter” of DNA and nanoparticle conjugates using the dynamic light scattering (DLS) technique. The effective diameter of the DNA/Au-nps conjugates should
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Figure 1. UV-vis spectra of the solution mixture in the mononucleotide-mediated conjugation approach. Solid line (blue), Au-nps (13 nm) serving as control without dATP and salt; dashed line (yellow), thiolT30 and dATP protected Au-nps (thiol-T30/Au13) with 0.1 M NaCl before heat treatment; dotted line (purple), dATP protected Au-nps with 0.1 M NaCl after 3 h heat treatment at 80 °C; dash-dotted line (pink), thiol-T30 and dATP protected Au-nps (thiol-T30/Au13) with 0.1 M NaCl after 3 h heat treatment at 80 °C. The inset image shows the observed colors of the dATP protected Au-nps (right, blue) and the mixture of thiol-T30 and dATP protected Au-nps (left, red) after the heat treatment.
be significantly larger than that of the bare gold particles. Although the salt concentration might have influence on the effective diameter through its impact on the DNA inter/ intrastrand repulsion, the overall size of the conjugate is dominated by the surface loading density of thiol DNAs on Aunps (22). The higher the density, the larger is the size. In Figure 2,1 the effective diameters of our conjugates during the time course of conjugate synthesis was benchmarked with the ones synthesized by the conventional direct conjugation and BSPPbased methods. The effective diameter of the conjugates by the BSPP coating method was found to be the smallest among the three methods, and there is no significant size change of the conjugate during the synthesis. The limited size growth could be due to the low salt concentration and the hindered diffusion of DNAs during the synthesis. The conjugates prepared by our approach and the direct conjugation show very significant size increase (from 13 nm to ∼30 nm). However, the direct conjugation approach requires a much higher synthesis time, while the mononucleotide-mediated approach achieves a similar effective diameter in just 3 h. Temperature Effect on the Mononucleotide-Mediated Conjugation. The effect of temperature on the mononucleotidemediated conjugation process was evaluated using fluorescence techniques (23), by which the surface density of DNA per particle could be measured as a function of the conjugation temperature. As shown in Figure 3, the DNA loading densities on DNA/Au-nps synthesized by the three different approaches are compared. Straight lines (Figure 3A,C) were used here to represent the DNA surface density achieved by the conventional isothermal methods (direct conjugation approach and BSPP 1
The data were taken in the following operating time points: the first data point was taken at 0 h for the citrate-coated Au-nps for all the three methods. In the direct conjugation, the data points (A) at overnight incubation of thiol-DNA and Au-nps (18 h), salt concentration adjusting to 0.1 M NaCl (20 h) and after 40 h aging step (60 h) were taken. In BSPP method, the data points (B) at the formation of BSPP coating layer (18 h) and thiol-DNA incubation (20 and 60 h) were taken. In the mononucleotide-mediated approach, the data points (C) at the introduction of dATP and salts (0.5 h) and after thermal treatment (3 h) were taken.
Zhao et al.
Figure 2. Effective diameter of thiol-DNA/Au-nps conjugate during the time course of conjugate synthesis. Solid line (blue) with square mark (9), direct conjugation approach; dashed line (pink) with circle mark (•), BSPP coating approach; dash-dotted line (green) with triangle mark (2), mononucleotide-mediated approach. The starting size of bare Au-nps is 13 nm, and the error bars represent standard deviation for five independent measurements.
Figure 3. Temperature effect on DNA surface loading density. (A) dashed line (pink), direct conjugation approach; (B) solid line (blue) with circle mark (•), mononucleotide-mediated approach; (C) dash-dotted line (yellow), BSPP coating approach. The error bars represent standard deviation for three independent fluorescence measurements.
coating approach, respectively) for the convenience in comparison with the values achieved by mononucleotide-based approach under different temperatures. Our approach shows an increase of DNA density per particle (up to 80 oligo strands per particle) with temperature rising from 30 to 60 °C, and the density drops at higher temperatures, which indicates that the DNA loading on Au-nps could be optimized at an elevated temperature of ∼50 °C (Figure 3B). Validation of Hybridization by DNA/Au-np Conjugates. As the molecular orientation of immobilized DNAs may greatly affect the sensitivity in many hybridization-based biosensing applications (3, 24), hybridization experiments were therefore conducted to verify the functionality of immobilized DNAs. As shown by the TEM micrographs (Figure 4B), star-shaped aggregates were observed for the DNA/Au-nps conjugates with complementary thiol-DNA sequences (thiol-A30/Au13 and thiol-T30/Au20). However, in the negative control experiments, the hybridization between the thiol-T30/Au-20 conjugate and the 13 nm Au-with nonthiol-DNAs (nonthiol-A24/Au13) was not successful, and thus, no aggregates were found in Figure 4A. Misorientation of the nonthiol-DNA or the nonspecific
Rapid Synthesis of DNA-Functionalized Au-NPs
Figure 4. TEM images of DNA/Au-nps conjugates after hybridization. (A) thiol-T30/Au20 conjugates with nonthiol A24/Au13 particles; (B) thiol-T30/Au20 conjugates with thiol-A30/Au13 conjugates. All the conjugates were synthesized through mononucleotide-mediated conjugation approach. (Scale bar: 100 nm.) Schematic representations of scenarios (A) and (B) are placed next to the TEM micrographs.
adsorption of DNA on Au-nps could result in hybridization failure, as demonstrated in the negative control group. DNA/ Au-nps conjugates synthesized by the dATP-mediated approach retain DNA functionality and possess rich DNA strands per particle for hybridization-based medical diagnostic and biosensing applications.
DISCUSSION In the direct conjugation approach, careful control of the salt concentration is needed. Diffusion of thiol-linked DNA to citrate-covered Au-nps would require salt ions to reduce the repulsive force between the negatively charged incoming DNA molecules and the negatively charged citrate-capping on Aunps surface. However, too much salt could cause an irreversible aggregation of nanoparticles. Therefore, gradual addition of salts and long incubation are generally required in the direct conjugation approach in order to balance the need for DNA conjugation without causing particle aggregation. To address the long-standing issue related to salt tolerance, our approach utilizes the adsorbed mononucleotides as a mediating layer on Au-nps to perform the conjugation of thiol-DNA to citrate Aunps in salt solutions. The conjugates prepared by our approach and the direct conjugation show very significant size increase (from 13 nm to ∼30 nm). However, the direct conjugation approach requires a much higher synthesis time, while the mononucleotide-mediated approach achieves a similar effective diameter in just 3 h. The mediating component dATP plays an important role in shortening the synthesis time. Without the need for overnight incubation and a long aging step, the salt concentration in our approach can be adjusted to a higher value prior to the introduction of thiol-DNA, as the dATP-covered Au-nps could resist a much higher salt concentration without the salt-induced aggregation than that of the citrate-based counterpart. The controlled salt concentration and the temperature-monitored removal of mononucleotide from Au-nps facilitate the ligand exchange reaction between the incoming thiol-DNA and the outgoing dATP.
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Temperature plays an important role in the mononucleotidemediated conjugation. Although the removal rate of dATP is larger at higher temperatures (i.e., >60 °C), the re-exposed Aunp surfaces without dATP protection would suffer salt-induced aggregation and the bond of Au-thiol DNA might not survive at high-temperature (25). The increased loading density of thiolDNA on Au-nps due to the high dATP removal rate could be compromised by the unstable Au-thiol bonding at higher temperatures, which could result in the temperature dependence described earlier. In comparison with the DNA loading densities per Au-np particle in the direct conjugation method (∼76 oligo strands) and in the BSPP method (∼16 oligo strands) measured using the same procedure, we believe that the dATP-mediated approach, which demonstrates a loading density of 80 oligo strand, offers a synthesis strategy for rapid DNA/Au-nps conjugation with comparable quality of the established methods. In the direct conjugation method, other researchers reported loading density of ∼70 strands per 15 nm particle for a oligo DNA sample with poly dA spacer and 100 ( 30 DNA per 15 nm Au-np with the vacuum evaporation enhancement (16, 23, 26). Their results are consistent with ours (∼76 per 13 nm Au-np) for the direct conjugation approach. By balancing dATP displacement at a specific temperature and ion strength, stable DNA/Au-nps conjugates could be made within 4 h, which is much shorter than the conventional approaches. Moreover, dATP, a monomer unit of nucleic acids, shares similar nonthiol functional groups in DNA, and thus, dATP residues on Au-nps help to reduce the nonspecific DNA binding to the particle through the nonthiol entities. The reduction of nonspecific binding by dATPs contributes to the right molecular orientation of the immobilized DNAs for hybridization. Our study has demonstrated that a novel mononucleotidemediated conjugation approach enables the rapid DNA functionalization of Au-nps with DNA surface coverage comparable to that obtained by the direct conjugation method. The stable conjugates with about 80 DNA strands per particle can be formed in salt solution (0.1 M NaCl) at 60 °C without special equipment required, and the resulting conjugates are able to hybridize. We envision that this mononucleotide-mediated method will offer an attractive alternative for the preparation of DNA/Au-nps conjugates, and similar methodology can be readily applicable for the synthesis of nanoparticle conjugates with various biomolecules (e.g., DNA and peptides). The ability to modify Au-nps with biomolecules in a rapid and controlled manner would provide more opportunities in nanobiotechnology.
ACKNOWLEDGMENT The authors thank the funding support from the Research Grants Council of the Hong Kong Special Administrative Region Government (Project No.: HKUST 601305) and a University research grant (Project No: RPC07/08.EG21). Laboratory facilities provided by the Bioengineering Laboratory and Materials Characterization & Preparation Facility of HKUST are also acknowledged. Supporting Information Available: UV-vis spectra for the stability evaluation of 20 nm Au-nps during the mononucleotidemediated conjugation process. This material is available free of charge via the Internet at http://pubs.acs.org.
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