ARTICLE pubs.acs.org/Langmuir
Rapid Synthesis of Stable and Functional Conjugates of DNA/Gold Nanoparticles Mediated by Tween 80 Shengmin Xu, Hang Yuan, An Xu, Jun Wang,* and Lijun Wu* Key Laboratory of Ion Beam Bioengineering, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, Anhui, P. R. China
bS Supporting Information ABSTRACT: Gold nanoparticles conjugated with DNA represent an attractive and alternative platform for broad applications in biosensors, medical diagnostic, and biological analysis. However, current methods to conjugate DNA to gold nanoparticles are time-consuming. In this study, we report a novel approach to rapidly conjugate DNA to gold nanoparticles (AuNPs) to form functional DNA/AuNPs in 23 h using Tween 80 as protective agent. With a fluorescence-based technique, we determine that the DNA density on the surface of AuNPs achieves about ∼60 strands per particles, which is comparable to the loading density in the current methods. Moreover, the DNA/AuNPs synthesized by our approach exhibit an excellent stability as a function of temperature, pH, and freezethaw cycle, and the functionality of DNA/AuNPs conjugates is also verified. The work presented here has important implications to develop the fast and reproducible synthesis of stable DNA-functionalized gold nanoparticles.
1. INTRODUCTION In the past decade, the development and study of DNAfunctionalized gold nanparticles (DNA/AuNPs) have attracted a great interest in the field of nanotechnology. Taking advantage of the strong AuS bond formed between the thiol-group and Au1 and the unique optoelectronic properties and excellent biocompatibility of AuNPs, DNA/AuNPs conjugates are applied widely in aspects of molecular diagnostics,2 bioanalytical systems,36 pollutant detections,79 gene regulation,10,11 and cancer labeling.12 The conjugation of DNA to AuNPs was first reported in 1996.13,14 This protocol to immobilize specific thiol-DNAs onto AuNPs surfaces has been widely accepted (referred to as the classic approach). However, a 2-day incubation process is needed to achieve a high DNA loading density in this approach. Considering the time-consuming nature of the classic approach, alternative methods that can shorten the time required while the quality of the DNA/AuNPs conjugates is preserved or improved are needed. One such method using vacuum centrifugation instead of a long aging step was reported by Brust et al.,15 and the labeling process was done in ∼20 h. Introduction of a polymeric layer (BSSP) to stabilize AuNPs prior to adding thiolDNA was developed by Alivisatos et al., which decreased the labeling time to ∼12 h.16 Ming et al. reported a strategy to rapid synthesis of DNA/AuNPs conjugates by adding mononucleotide.17 With dATP protection, the salt-dependent aggregation of AuNPs was brought under control and a high DNA loading density could be achieved in ∼3 h. The nonionic surfanctant polyoxyethylene (20) sorbitan monolaurate (Tween 20) has previously been shown to prevent AuNPs from aggregating through the physisorption of free r 2011 American Chemical Society
Tween molecules prior to the chemisorption of alkanethiols.18 With Tween 20 protection, a facile method for producing stable and functional peptide nucleic acid conjugated gold nanoparticles (PNA/AuNPs) has been developed.19 However, the conjugation process requires ∼24 h under gentle conditions. Apart from these examples, we herein present a simple and convenient method of rapidly synthesizing DNA/AuNPs conjugates in salt solution mediated by polysorbate 80 (Tween 80). This method takes advantage of the stabilizing effect of Tween 80 on AuNPs in a salt solution and a heating-facilitated ligand-exchange between Tween 80 and thiol-DNA. Prior to the addition of thiol-DNA, Tween 80 was added and adsorbed onto the AuNPs surfaces, which could effectively stabilize AuNPs in salt solutions by forming a protection layer on the surfaces of AuNPs. Under the protection of Tween 80, DNA/AuNPs conjugates could be rapidly formed in salt solution followed by a mild heatingfacilitated ligand-exchange between Tween 80 and thiol-DNA (Figure 1). Our results showed that the DNA/AuNPs conjugates could be completed within 23 h. The conjugates obtained by our method have similar or better quality in terms of DNA loading density and stability than the classic methods.
2. EXPERIMENTAL SECTION Materials. HAuCl4 3 4H2O, trisodium citrate, sodium acetate, sodium phosphate monobasic, sodium phosphate dibasic, and sodium Received: March 22, 2011 Revised: September 29, 2011 Published: September 30, 2011 13629
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Figure 1. Schematic representation of the principle of Tween 80-mediated conjugation of thiol-DNA to gold nanoparticles. carbonate were purchased from Sinopharm Chemical Inc. (Shanghai, China). DNA oligonucleotides, NP-40, Triton X-100, Tween 20, and Tween 80 were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). AuNPs 13 and 40 nm in size were synthesized following the procedures described in the literature.20,21,24 Buffers and Solutions. All buffer solutions were prepared to 10 mM concentration with 100 mM NaCl as previously reported:18 phosphoric acid/sodium phosphate monobasic (pH 3 and 4), acetic acid/sodium acetate (pH 5), sodium phosphate monobasic/sodium phosphate dibasic (pH 7), sodium phosphate dibasic (pH 8 and 9), and sodium carbonate (pH 11). Deionized (DI) water (electric resistivity = 18.2 MΩ cm) was purified using a Millipore filtration system. Exact pH values for buffer solutions were obtained using a FE20/EL20 pH meter (Mettler Toledo).
synthesized DNA (thiol-T10)/AuNPs conjugates were mixed together, and the final volume of mixture was diluted to 500 μL. After incubation overnight, the mixture was centrifuged at 12 000 rpm for 10 min, and the fluorescence signal of supernatants was measured.
Hybridization of Different Size of AuNPs Immobilized DNAs. Different-sized AuNPs labeled with cDNAs sequences were used to test the hybridization. Using thiol-T10 to functionalize AuNPs of 13 nm (thiol-T10/Au13) and thiol-A15 to AuNPs of 40 nm (thiol-A15/ Au40), two conjugates were mixed overnight in a solution of 0.15 M NaCl and 10 mM phosphate (pH 7.4) with a molar ratio of 1:5. The hybridization was observed by a JEM 2010 transmission electron microscope (TEM) operating at 200 kV (JEOL).
Rapid Synthesis of DNA/AuNPs Conjugates Mediated by Tween 80. Four microliters of 10% (v/v) Tween 80 in water and 1 mL
3. RESULTS AND DISCUSSION
of AuNPs were mixed and allowed to react for 30 min at room temperature. The mixture was then brought to 0.1 M PBS. Next, thiol-DNA [thiol-T10, 50 -d(TTT TTT TTT T)-C3-thiol-30 or thiolA15, 50 -d(AAA AAA AAA AAA AAA)-C3-thiol-30 ] was added to the solution of AuNPs to a final DNA concentration of 1 μM and allowed to age for 2.5 h at a constant temperature. All the temperature was controlled at 50 °C for Tween 80-mediated conjugation reactions, except for the temperature effect study. Unconjugated DNA was removed by centrifugation at 16 000g for 15 min and washed with 0.1 M PBS (10 mM phosphate, 0.1 M NaCl, 0.05% Tween 80, pH 7.4) three times. Final conjugates were redispersed in a solution of 0.15 M NaCl, 10 mM phosphate with 0.05% Tween 80 (pH 7.4). Measurement of DNA Loading on Gold Nanoparticles. The DNA loading density on AuNPs was determined by a fluorescencebased method using dithiothreitrol (DTT) as a disulfide reducing agent.22 To 500 μL FAM-A15/AuNPs centrifugation pellets was added 500 μL of 0.1 M DTT. After being incubated at 50 °C for 5 min and held at room temperature for 1 h to release the FAM-A15 from the AuNPs, the solutions were centrifuged at 12 000 rpm for 10 min, and the fluorescence signal of supernatants was measured with a fluorescence microplate reader (Molecular Devices Inc.). Characterization Methods and Stability Study. Dynamic light scattering measurements were performed using a Dynapro-99 (Wyatt Technology Corp.) operated in 173° backscatter mode with a laser wavelength of 633 nm. All UVvis spectra were recorded on a UV2550 spectrophotometer (SHIMADZH) using a 1 mL quartz cuvette. Absorbance and fluorescent intensity were measured on a microplate reader (Molecular Devices Inc.). The stability under various surfactants and temperatures were investigated without dilution by UVvis measurements. On the other hand, stability studies of AuNPs conjugates in various pHs and frozen condition were conducted with a dilution factor (f) of 3 by DLS and UVvis.
Salt and Temperature Effects on AuNPs Stabilization by Surfactants. Nonionic surfactants are often employed as block-
Measurement of Quenching Efficient Curve Using FAMA15. Twenty microliters of 1 μM FAM-A15 and different volumes of
ing agents in buffer to decrease nonspecific binding in Western blotting and systematic evolution of ligands by exponential enrichment (SELEX). Previous study showed that Tween 20 could be adsorbed onto the surface of AuNPs to shield the citrate ions and prevent the aggregation of gold nanoparticles.23 On the basis of this knowledge, Tseng and his co-workers developed a rapid method for detection of Hg2+ and Ag+23 and histidine24 using Tween 20-capped AuNPs. In the present study, we investigated the abilities of four different nonionic surfactants (NP-40, Triton X-100, Tween 20, and Tween 80) to stabilize the AuNPs. As seen from the UVvis spectra (Figure S1, Supporting Information), monodispersed AuNPs had a characteristic absorption peak at around 520 nm, while AuNPs in aggregated form gave a red-shift of the adsorption peak after addition of salt. But all four different surfactant-capped AuNPs remained monodispersed in 0.1 M PBS (0.1 M NaCl, 10 mM phosphate, pH 7.4), suggesting that these nonionic surfactants can stabilize AuNPs in a salt solution. Moreover, a shift to 523 nm was observed upon addition of surfactants, which was consistent with previous reports for Tween 20-coated AuNPs.18,19 We noted, however, that nanoparticles with surfactants also showed a slight increase in absorbance values at longer wavelengths (>600 nm). This was possibly due to changes in the dielectric properties of the layer around the gold nanoparticles and a small amount of aggregation induced by salt. We compared the stabilization effect of four nonionic surfactants by changing the salt concentration or temperature. In our studies, the salt effect on surfactant-capped AuNPs stabilization was monitored through the change of absorbance peak at 650 nm at different concentrations of NaCl.25 As shown in Figure 2A, a significant increase in absorbance at 650 nm appeared for the Triton X-100-capped AuNPs solutions at 0.1 M NaCl, while 13630
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Figure 2. Salt and temperature effects on surfactant-capped AuNPs. (A) Absorbance of four nonionic surfactant-capped AuNPs solutions at 650 nm under different salt concentrations. One milliliter of AuNPs was mixed with 4 μL of 10% (v/v) of different nonionic surfactant for 15 min at room temperature, respectively, and then the mixture was adjusted to different salt concentration with 5 M NaCl. (B) Absorbance ratio (A650/ A520) of AuNPs solutions under different temperature treatments. AuNPs were mixed with four different nonionic surfactants, respectively. The mixture was adjusted to 0.1 M PBS and incubated at different temperatures for 15 min.
those incubated with NP 40 or Tween 20 did not change much until the concentration of NaCl was increased to 0.3 M. It was noticeable that solutions of AuNPs incubated with Tween 80 could stay stabilized at NaCl concentration up to 1.0 M. The results suggested that Tween 80-capped AuNPs had a higher salt tolerance than the other three surfactant-capped AuNPs. However, the aggregated AuNPs did not return to the monodispersed state (data not shown), indicating that the salt-induced aggregation of four different nonionic surfactant-capped AuNPs was irreversible and was different from the phenomenon observed in the dNTP-protected AuNPs.25 The temperature dependency of surfactant-capped AuNPs stabilization was investigated by measuring the absorbance peak ratio between 650 and 520 nm, in order to quantify the stabilization of AuNPs.26 It was reported that enhancement of
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Figure 3. Efficiency of DNA loading on Tween 80-protected AuNPs as a function of temperature and incubation time. (A) Temperature effects on DNA loading study. A 500 μL portion of Tween 80-capped AuNPs was mixed with 5 μL of 200 μM FAM-A15 and the mixture was incubated for 2.5 h at different temperature. Afterward, the particles were washed three times and the DNA loading density was measured by a fluorescence-based method. The DNA/AuNPs conjugates obtained by the classic approach were used as control. The error bars represent standard deviation for three independent fluorescence measurements. (B) Time effects on DNA loading study. Tween 80-capped AuNPs were mixed with FAM-A15 and the mixture was incubated at 50 °C for different times, and then 500 μL of the mixture was used to study the DNA loading density by the fluorescent method.
reaction temperature could decrease the DNA loading time.27 This required that nonionic surfactant-capped AuNPs could also remain mondispered at higher temperature during synthesis of DNA/AuNPs conjugates in a salt solution. As shown in Figure 2B, Tween 80- and Tween 20-capped AuNPs maintained stabilization at 50 °C in 0.1 M PBS (0.1 M NaCl, 10 mM phosphate, pH 7.4), even when the temperature rose to 60 °C, while NP-40- and Triton X-100-capped AuNPs appeared aggregated. These results demonstrated that the abilities of different nonionic surfactants to stabilize AuNPs in higher temperature and higher salt concentration followed the same order: Tween 80 > Tween 20 > NP-40 > Triton X-100. As a result, Tween 80 13631
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Figure 4. Stability of Tween 80-capped DNA/AuNPs conjugates in different conditions. (A) UVvis spectra of Tween 80-capped DNA/AuNPs conjugates at a high temperature condition: solid line, Tween 80 capped-AuNPs was used as control without salt and heating; dashed line, Tween 80capped thiol-T10/AuNPs conjugates with 0.1 M PBS before heat treatment; dotted line, Tween 80-capped thiol-T10/AuNPs conjugates with 0.1 M PBS after 15 min heat treatment at 80 °C; dasheddotted line, Tween 80-capped AuNPs with 0.1 M PBS after 15 min heat treatment at 80 °C. (B) Stability as function of solution pH. The stability of conjugates was monitored by DLS (dilution factor f = 3) at different pH. The conjugates were suspended in different pH media and incubated for 2 h, and then the z-average size was measured by DLS. (C) Flocculation parameter versus pH of different conjugates after 2 h in different pH media. (D) Photographs of color changes of conjugates before and after a freezethaw cycle: a, bare AuNPs; b, Tween 80-capped AuNPs; c, Tween 80-capped thiol-T10/AuNPs; d, citrate-capped thiol-T10/AuNPs (top row, conjugates without freezing; bottom rows, conjugates thawed after freezing for 72 h at 20 °C).
was chosen as the protective agent during the synthesis of DNA/ AuNPs conjugates in the following study. DNA Loading Study of Tween 80-Mediated Conjugation. Figure 1 showed the basic principle of our method, and the detailed procedure for synthesis of the conjugates was described in the Experimental Section. As shown in Figure 2, physisorption of Tween 80 onto the gold nanoparticles prior to chemisorption of thiol-DNA prevented them from aggregating in salt solution at a higher temperature. Since the interaction of AuNPs surfaces with Tween 80 would be weaker than their interaction with thiol, the weakly adsorbed Tween 80 could be quickly substituted with thiol-DNA at a higher temperature condition. By assessing the effect of temperature on the Tween 80-mediated DNA/AuNPs conjugation process with fluorescence techniques,28 we demonstrated that the number of DNA loading on Tween 80-capped AuNPs
increased significantly as the temperature elevated from 20 to 60 °C but dropped at temperatures beyond that probably due to the AuNPs aggregation (Figure 3A). As seen from Figure 3A, the DNA loading density on AuNPs mediated by Tween 80 at optimal temperature can compare with the classic approach.28,29 Our experiment of temperature loading effect showed that the number of DNA strands calculated by fluorescence measurement increased significantly in the first 4 h at the optimized temperature and might achieve ∼90 strands when the incubation time was extended to 8 h (Figure 3B). The AuNPs surfaces were saturated and the DNA density cannot be further increased after 8 h. These results indicated that the conjugation of DNA to AuNPs was rapidly completed in salt solution followed by a temperature-facilitated ligand-exchange between Tween 80 and thiol-DNA. 13632
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Stability of DNA/AuNPs Conjugates Mediated by Tween 80 with Different Temptature, pH, and Storage Conditions.
The stability of DNA/AuNPs conjugates under various conditions is an important issue for their applications. In the present study, the temperature-dependent stability of the synthesized DNA/AuNPs conjugates mediated by Tween 80 was conducted at higher temperature (80 °C) by the measurement of surface plasmon resonance (SRP) using UVvis spectroscopy. Samples were incubated for 15 min at 80 °C before measurements were initiated. As shown in Figure 4A, Tween 80-capped DNA/ AuNPs conjugates were stable before and after the heat treatment (80 °C) in the 0.1 M PBS and there was only one peak located at 520 nm. Similar stability of citrate-capped DNA/ AuNPs conjugates obtained by the classic approach was observed (Figure S2, Supporting Information). In contrast, Tween 80capped AuNPs without DNA were observed with a significant increase in absorbance values at longer wavelengths (>600 nm), which indicated the aggregation of the conjugates. These results suggested that a much stronger covalent bond (AuS) was formed between thiol-DNA and AuNPs, which stabilized the conjugates even at a higher temperature. To determine the stability of DNA/AuNPs conjugates and Tween 80-capped AuNPs in different pHs, we diluted conjugates (dilution factor f = 3) into different pH buffer solutions ranging from 3 to 11 and then monitored their aggregation using dynamic light scattering (DLS) and UVvis absorbance. As shown in Figure 4B, Tween 80 dramatically enhanced AuNPs conjugates stability at a wide range of pH values from 3 to 11. The stabilization by Tween 80 might be explained by the formation of a physisorbed layer of surfactant chains that repel other colloidal particles. Citrate-capped DNA/AuNPs conjugates synthesized by the classic approach were more stable at a higher pH values (pH >7), but conjugates became unstable under highly acidic conditions (pH 600 nm). Based on these spectra, a “flocculation parameter” was introduced as semiquantitative of aggregation to evaluate the stability of conjugates, which was defined by Weisbecker30 and later modified by Mayya31 as the integral of the UVvis spectra between 600 and 800 nm. Similar results were obtained by “flocculation parameter” measurements (Figure 4C). This demonstrated that the net charge of DNA on the functionalized AuNPs depended strongly on pH, which caused the DNA/AuNPs conjugates aggregation at low pH values, and the surface modification using Tween 80 would increase the stability of DNA/AuNPs conjugates. The freezethaw stability of different AuNPs conjugates was also investigated in the present study. Samples were froze at 20 °C for 72 h and then thawed for measurements. It is known to all that bare AuNPs cannot be stored at 20 °C. This was also confirmed in our experiments. The AuNPs and citrate-capped DNA/AuNPs conjugates lost their characteristic red color and
Figure 5. (A) Hybridization titration curve of T10/AuNPs conjugates with complementary strand FAM-A15. Thiol-T10 was first immobilized on Tween 80-capped AuNPs, and FAM-A15 was used to hybridize with different volume of conjugates from 0 to 400 μL in a final volume of 500 μL. The concentration of FAM-A15 was kept at a constant concentration of 40 nM. (B) TEM images of hybridization between different sized DNA/AuNPs conjugates. Thiol-T10/Au13 and thiol-A15/Au40 conjugates were all synthesized through the Tween 80-mediated approach. Scale bar: 100 nm.
absorbance band after thawing (Figures 4D,a and D,d, and Figure S4a,d, Supporting Information), while the UVvis spectra of Tween 80-capped AuNPs had a significantly broader absorption increase from 600 to 800 nm (Figure S4b, Supporting Information), which was in good agreement with the observation of their colors (Figure 4D,b). However, Tween 80-capped DNA/ AuNPs conjugates exhibited more stability than the other three conjugates after thawing. The freezethaw stability was not surprising, since it has been proven that nanoparticles whose surface was modified in the presence of surfactant did not appear to undergo irreversible aggregation upon freezing or repeated drying and resuspension with mild sonication.18 Overall, our results suggested that Tween 80-capped DNA/ AuNPs conjugates exhibited excellent stability. This is attributed 13633
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Langmuir to the introduction of Tween 80 during the synthesis of DNA/ AuNPs conjugates. Validation of Functionality of Tween 80-Capped DNA/ AuNPs Conjugates. DNA hybridization experiments were conducted to verify the functionality of Tween 80-capped DNA/ AuNPs conjugates. On the basis of the property that AuNPs can quench the fluorescence of dyes, DNA-functionalized AuNPs are usually used in many hybridization-based biosensors.4,5 So in the present study, the quenching efficiency of conjugates was chosen to reflect the hybridization efficiency. As described in Figure 5A, the hybridization titration curve showed that 200 μL of DNA/ AuNPs conjugates was able to fully quench the fluorescence. It is known that poly A sequences exhibit high nonspecific binding on surfaces of AuNPs. But in our negative control group, 400 μL of Tween 80-capped AuNPs without immobolized thiol-T10 could not significantly quench the fluorescence of FAM-A15 after overnight incubation (the quenching efficiency was only 9.73%). This result demonstrated that Tween 80 occupied the surface of AuNPs so that DNA could not be absorbed onto AuNPs by nonspecific bindings through the other functional groups. This character of Tween 80 could enable the thiol-DNA to be immobilized onto the surface of AuNPs and maintained in a “standing-up” state, which is preferred for DNA hybridization on AuNPs surface. We further investigated the DNA hybridization by TEM. Complementary DNAs sequences were separately immobilized on the different sizes of AuNPs. We observed that the aggregates were formed between Tween 80-capped T10/Au13 and A15/Au40 by the TEM micrographs (Figure 5B). This indicated that the DNA immobilized on AuNPs still maintained the ability to hybridize with its complement. These results suggested that the DNA/AuNPs conjugates obtained by our approach are functionally stable.
4. CONCLUSIONS In this study, a new approach to rapidly synthesize DNAfunctionalized gold nanoparticles is established by using Tween 80 as protective agent. The DNA density on the surface of AuNPs is comparable to that obtained by the other current approaches, but the time required for the DNA labeling is significantly reduced to only several hours. The synthesized DNA/AuNPs exhibit an excellent stability and functionality, which could offer a wider range of application for DNA/AuNPs in nanobiotechnology. We envision that this method may offer an alternative for synthesis of AuNPs conjugates not only with DNA but also with other biomolecules. ’ ASSOCIATED CONTENT
bS
Supporting Information. UVvis spectra of AuNPs, Tween 80-capped AuNPs, and Tween 80 (or citrate)-capped DNA/AuNPs conjugates under different conditions. This material is available free of charge via the Internet at http://pubs.acs.org/.
’ AUTHOR INFORMATION Corresponding Author
*Phone: 86-551-5591602. Fax: 86-551-5595670. E-mail: ljw@ ipp.ac.cn (L.W.),
[email protected] (J.W.).
’ ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (grant no. 2007CB936602), National 863
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Research Program (2008AA062504), grants from the National Natural Science Foundation of China (20977093, 10935009 and 10225526), and Hundred Talents Program of the Chinese Academy of Sciences. We thank Dr. Zhiyuan Shen for his kind assistance and critical reading of the manuscript.
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