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One-step synthesis of biocompatible gold nanoparticles using gallic acid in the presence of poly-(N-vinyl-2-pyrrolidone)
Colloids and Surfaces A: Physicochem. Eng. Aspects 301 (2007) 73–79
One-step synthesis of biocompatible gold nanoparticles using gallic acid in the presence of poly-(N-vinyl-2-pyrrolidone) Wenxing Wang a , Qifan Chen a , Cha Jiang a , Dongzhi Yang a , Xingmin Liu b , Shukun Xu a,∗ b
a Department of Chemistry, Northeastern University, Shenyang, Liaoning 110004, China Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning 110016, China
Received 18 September 2006; received in revised form 30 November 2006; accepted 6 December 2006 Available online 15 December 2006
Abstract A one-step method for the synthesis of biocompatible gold nanoparticles at room temperature by reducing HAuCl4 with gallic acid in the presence of poly-(N-vinyl-2-pyrrolidone) (PVP) is presented. The effect of the molar ratio of gallic acid to gold (R) on the size and shape of gold nanoparticles and the characteristic of gold nanoparticles was investigated by UV–vis and infrared spectroscopy, transmission electron microscopy, and X-ray diffraction. For an R value of about 0.4, the prepared gold nanoparticles were the smallest in size and the most close to spherical in shape. At other molar ratios, particle sizes increased and various polyhedra were formed. A reaction mechanism for the reduction of HAuCl4 by gallic acid is proposed. The incorporation of PVP effectively protected the surface of gold nanoparticles and improved their stability. The PVP-protected gold nanoparticles were modified with 3 - and 5 -alkanethiol-capped 12-base oligonucleotides, respectively, to form two different nucleic acid probes. The probes were successfully used to complex a 24-base complementary polynucleotide target in a tail-to-tail fashion. © 2006 Elsevier B.V. All rights reserved. Keywords: Gold nanoparticles; Synthesis; Gallic acid; Poly-(N-vinyl-2-pyrrolidone) (PVP); DNA hybridization
1. Introduction In recent years, the synthesis and applications of gold nanoparticles have attracted the interest of many researchers, due to their unique physical and chemical properties and for potential bio-analytical applications. Since Frens [1] and Turkevitch et al. [2] initially introduced a sodium citrate reduction of HAuCl4 for the synthesis of stable gold nanoparticles, studies in this field have rapidly expanded. Many chemical and physical methods have been used to prepare a variety of gold nanoparticles; these methods include seed-mediated growth [3,4], use of reverse micelles [5], phase transfer reactions [6,7], thermolysis [8], radiolysis [9,10], photochemistry [11,12], and sonochemistry [13–15]. Among these, chemical methods are still the preferred method for the preparation of gold nanoparticles. The size and shape of gold nanoparticles are controlled by the
∗
Corresponding author. Tel.: +86 24 83681343; fax: +86 24 83681343. E-mail address: [email protected] (S. Xu).
0927-7757/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2006.12.037
ratio of HAuCl4 to the chemical reducing agent in the preparation with examples such as sodium citrate, borohydride, or other organic compounds. In addition, high molecular weight polymers [16–20], thiol derivatives [21–26] and other ligands [27,28], used as capping regents, are often used to control the particle size and shape, prevent aggregation, and improve function of the particle surface for application in bio-analytical methods. For instance, Mirkin and co-workers reported a “programmed assembly” strategy for utilizing nanoparticles aggregates modified with alkanethiol-capped single-stranded oligonucleotides and complementary linker oligonucleotides strands to analyze DNA [29–31]. In other work, particles have also been applied to the analysis of other biological substances such as lectin [32] and protein [33]. Gallic acid is a poly-phenolic compound that may be used as a reductant, which is obtained from the hydrolysis of natural plant poly-phenols. It has been used historically to yield blue ink as its reduction of iron chloride produces a blue precipitate. Here, we tried firstly to use it as reductant to prepare nanogold, and gold nanoparticles were facilely synthesized by
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reducing HAuCl4 with gallic acid at room temperature. PVP, as a polymeric stabilizer, has been frequently used to protect metal nanoparticles and improve the quality of particle [34,35]. Because of unique physical characteristics, polymer-protected gold nanoparticles are potentially used for a number of advanced functional applications, especially in the optical and photonic fields [34]. So far, it is not reported that PVP-protected gold nanoparticles are applied in bio-analytical field. PVP can be removed from the surface of gold nanoparticles by thiol derivatives according to a ligand-exchange process [36]. The quality of the prepared gold nanoparticles directly depends on thiol derivatives on the particle surface. In previous reactions, the used thiol derivatives were hydrophobic and therefore the prepared gold nanoparticles were soluble in aliphatic and aromatic hydrocarbon, chlorine solvents, and ethers, but insoluble in acetone, alcohol, and water. The insolubility in water made them difficult to be used in bio-analytical application. For solving the problem, in this work, alkanethiol-capped oligonucleotides soluble in water were utilized to remove the PVP on the gold surface. Furthermore, the alkanethiol-oligonucleotides-capped gold nanoparticles were applied in bio-analysis such as DNA hybridization. In this paper, a one-step method for the synthesis of biocompatible gold nanoparticles using gallic acid as the reducing agent in the presence of PVP at room temperature is reported. The effect of the molar ratio of gallic acid to gold (R) and the concentration of PVP on the size and shape of gold nanoparticles as well as the characteristic of gold nanoparticles were investigated. UV–vis and IR spectroscopy, transmission electron microscopy (TEM), and X-ray diffraction (XRD) were employed in the characterization of the prepared gold nanoparticles. The PVP-protected gold nanoparticles were used as a precursor material to prepare the thiol-modified single-strand oligonucleotides probes. The probes generated hybridization with complementary target single-strand oligonucleotides by a tail-to-tail fashion [30]. 2. Experimental 2.1. Materials All the reagents obtained commercially were of analytical grade and were used as received without further purification. All glassware used was cleaned in aqua regia (HCl:HNO3 , 3:1), rinsed thoroughly with triply distilled water, and oven dried prior to use. HAuCl4 ·3H2 O, gallic acid and poly-(N-vinyl-2-pyrrolidone) (PVP, Mw = 10000) were purchased from Shanghai Chemicals Company. All aqueous solutions were made using triply distilled water, and subsequently filtered through 0.22 m Millipore syringe filter. All DNA synthesis reagents were purchased from Shanghai Biological Engineering Company. The sequences of oligonucleotides used in this work were as follows: • 3 -(alkanethiol)oligonucleotide (S1): 5 -TAGGACGTACGC(CH2 )6 -SH-3 ;
• 5 -(alkanethiol)oligonucleotide (S2): 5 -SH-(CH2 )6 -TATCATCTAGTC-3 ; • complementary target oligonucleotide (S3): 5 -GCGTACGTCCTAGACTAGATGATA-3 . 2.2. Instrumental methods Gold nanoparticles were characterized by UV–vis spectroscopy, TEM, XRD and IR spectroscopy. UV–vis absorption spectra of gold nanoparticles were recorded using a Shimadzu UV-2550 UV–vis spectrophotometer using 1 cm quartz cuvette. TEM images were performed on a Philips EM 420 microscope operating at 120 kV. A typical sample for TEM was prepared by drying naturally a drop of solution containing gold nanoparticles at room temperature on a carbon-coated copper grid. XRD was recorded on a Panalytical PW 3040160 X-ray diffractometer using Cu K␣ radiation (λ = 0.1542 nm) operated at 50 kV and 100 mA. IR spectra were measured on a Perkin-Elmer Spectrum One FT-IR spectrometer. 2.3. Synthesis and characterization of gold nanoparticles 2.3.1. Method 1 Gold nanoparticles were synthesized by mixing HAuCl4 (0.3 mM, 80 ml) and gallic acid (varied concentration, 20 ml) solutions. That was, 20 ml of 0.25, 0.5, 1.25, 2.0 and 2.5 mM gallic acid was added rapidly to 80 ml of 0.3 mM HAuCl4 solution, respectively, under magnetic stirring in a water bath of room temperature. The color of solutions rapidly changed from colorless to different red depending on gallic acid concentration. The reactions were kept for 30 min. 2.3.2. Method 2 PVP was added to these solutions in Method 1 for improving the quality of prepared nanoparticles. Typically, 0, 0.01, 0.05, 0.1, 0.5, 2 and 4 ml of 1 mM PVP was added to the solution (the molar ratio of gallic acid to gold = 0.4) in Method 1, respectively. The volume of the reaction was 100 ml. The reactions were kept 30 min at room temperature. 2.4. Preparation of 3 - or 5 -(alkanethiol)oligonucleotidemodified gold nanoparticles Gold nanoparticles (about 12 nm diameter) synthesized in the presence of PVP by Method 2 were modified with 3 - or 5 alkanethiol-capped 12-base oligonucleotides (S1 or S2) to form two different gold nanoparticle probes [29]. Typically, 1.0 ml of gold nanoparticles aqueous solution with 3.6 M of S1 or S2 was shaken for 12 h and then incubated for 12 h at 45 ◦ C in a water bath. The solution was brought to 0.1 M NaCl, 10 mM phosphate buffer (pH 7) and was kept at 45 ◦ C for 40 h. To remove excess reagents, the solution was centrifuged at 14,000 rpm for 25 min, and the supernatant was replaced by 1.0 ml of the same buffer. After additional centrifugation under the same condition, the red oily precipitate was re-dispersed into 0.5 ml of 0.3 M NaCl 10 mM phosphate buffer (pH 7) to be used as gold nanoparticle probes.
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2.5. Hybridization of gold nanoparticle probes with complementary target oligonucleotide One hundred and fifty microlitres of each gold nanoparticle probe mixture prepared above was mixed with buffer (0.3 M NaCl, 10 mM phosphate, pH 7) containing 100 pmol complementary target oligonucleotide (S3). The solution was heated to 55 ◦ C for 5 min and allowed to cool to room temperature. The color of the solution changed from red to purple in minutes, and full precipitate occurred within approximately 1 h. The UV–vis spectrum of the hybridized solution was recorded. A TEM sample was prepared by dropping the hybridized solution onto a carbon-coated copper grid, and dried for 30 min under ambient conditions. The TEM sample was imaged using a Philips EM 420 transmission electron microscopy operating at 120 kV. 3. Results and discussion 3.1. Gold nanoparticles synthesized with gallic acid Initial synthesis focused on the formation of gold nanoparticles using gallic acid in the absence of PVP. The molar ratio of gallic acid to gold was defined R for abbreviation. When the R value was over 2.0, brown-red precipitates appeared in the reactor and resulted in a turbid solution. This phenomenon suggested that the prepared gold nanoparticles are too large to be stabilized in the solution. So the molar ratios of 0.2, 0.4, 1.0, 1.6 and 2.0 were chosen as illustrative examples. Fig. 1 shows the absorption spectra of gold nanoparticles synthesized by various R at room temperature in Method 1. The surface plasmon resonance band of gold nanoparticles varied with the change of R. Initially, the maximal plasmon absorption peaks of gold nanoparticles showed a blue shift and turned narrow in width with the increase of R, and then showed a red shift and broaden in width with the further increase of R. Only for the molar ratio of 0.4, the maximum plasmon absorption wavelength (λmax ) of gold nanoparticles was obtained for the least value (536 nm) without another absorption band in the longitudinal plasma resonance. It is interesting to note that, for
Fig. 1. Absorption spectra of gold nanoparticles synthesized with R of 0.2 (a), 0.4 (b), 1.0 (c), 1.6 (d) and 2.0 (e).
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the molar ratios of 1.0, 1.6 and 2.0, there was another absorption band appeared ranged 600–800 nm, which belongs to the longitudinal plasma resonance band of aggregates [22] or deviation from spherical geometry of gold nanoparticles [37]. Mie’s theory is often used to explain the deviation from spherical geometry of the particles, suggesting in the circumstance that the transverse and longitudinal dipole polarizability no longer produces equivalent resonance, leads to a broadening and red shift of longitudinal plasmon resonance as well as an appearance of transverse plasma resonance [38,39]. All the results indicated that the size and shape of gold nanoparticles were altered with R value, which was confirmed by TEM images further. Fig. 2 shows typical TEM images of gold nanoparticles synthesized by various R. With the increase of R, the average size of gold nanoparticles increased, but not significantly, meanwhile the shape of gold nanoparticles changed obviously. Gold nanoparticles obtained at the R of 0.2 were seen to be aggregated as shown in Fig. 2(A), resulting that the maximum absorption band shifted to longer wavelength than that at the molar ratio of 0.4. For the R of 0.4, the obtained gold nanoparticles exhibited better dispersibility; and near spherical in shape, as shown in Fig. 2(B). However, with the further increase of R, particle shape appeared various polygonal particles such as triangular, rod-like, hexagonal, spherical, etc. as shown in Fig. 2(C)–(E). These results were also in accordance with absorption spectra shown in Fig. 1. It indicated the occurrence of two plasmon resonance bands due to the increasing of the molar ratio mainly ascribed to the appearance of anisometric gold nanoparticles [37]. To investigate the structure of the obtained gold nanoparticles, the XRD pattern was measured. Fig. 3 shows the XRD pattern obtained from gold nanoparticles at R of 0.4. The spectrum included five peaks at 38.2◦ , 44.4◦ , 64.7◦ , 77.6◦ and 81.6◦ that can be assigned to the (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) planes, which have a good match with the standard diffraction pattern of JCPDS No. 089–3697. The XRD pattern indicated that gold nanoparticles were in the face-centered cubic (fcc) structure and crystal in nature. IR spectrum was also measured for the further investigation to identify the possible molecular response for efficient stabilization of gold nanoparticles synthesized with an R of 0.4. Gold nanoparticles were centrifuged at 14,000 rpm for 25 min, then the red precipitate was rinsed with water, re-centrifuged, the obtained precipitate was used for IR analysis. Fig. 4 shows the infrared spectra of gallic acid (A) and gold nanoparticles (B) obtained by gallic acid reduction of HAuCl4 . In Fig. 4(A), the strong and broad band between 3600–2500 cm−1 and the strong and narrow peak at 1702 cm−1 could be assigned to be stretching vibration of OH group and carbonyl group, which indicated that carboxyl group existed in the gallic acid. Three peaks observed at 1616, 1541, 1450 cm−1 are typical stretching vibrations of C C bonds in aromatic ring. There are several peaks in 1300–1000 cm−1 region that could be assigned to be the stretching vibration of C–O bond and bending vibration of O–H bond of gallic acid. Since phenolic compounds are easily oxidized to form quinones, it was speculated that the product of
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Fig. 2. TEM images of gold nanoparticles synthesized with R of 0.2 (A), 0.4 (B), 1.0 (C), 1.6 (D) and 2.0 (E).
gallic acid reduction of HAuCl4 might be a quinoid compound. In Fig. 4(B), the strong and broad band in the 3650–2700 cm−1 region was considered to be stretching vibration of OH group, which basically covered C–H bond stretching vibration at about 3100 cm−1 . Stretching vibration of C–O bond and bending vibration of O–H bond in 1300–1000 cm−1 region was still retained, but the intensity obviously decreased. In contrast with that of gallic acid shown in Fig. 4(A), the IR absorption spectrum of gold nanoparticles observed from Fig. 4(B) indicated that the stretching vibration peak of carbonyl group shifted from 1702 to 1640 cm−1 , and the stretching vibration of C C bond at 1616 and 1541 cm−1 was covered with a broad and middle intensity band at around 1640 cm−1 . The results indicated that quinoid compound with keto–enol system might be produced by gallic acid reduction of HAuCl4 and absorbed on the surface of gold nanoparticles. Usually, when molecule absorbs on the nano-scale metal island, surface-enhanced Raman and infrared spectra can be observed [40]. Electromagnetic field and chemical
Fig. 3. XRD pattern of gold nanoparticles synthesized with R of 0.4.
mechanisms have been proposed to explain this phenomenon. In this work, a chemical mechanism is more reasonable since significant conjugation exists within the quinoid compound with a keto–enol system. On the other hand, an inductive effect may also exist between the nucleophilic compound and the electron dense gold nanoparticles. These can be used to explain the significant shift of the stretching band of carbonyl group to lower wavenumber (from 1702 to 1640 cm−1 ). A proposed reaction scheme for the reduction of HAuCl4 by gallic acid is given below:
Fig. 4. IR spectra of gallic acid (A) and gold nanoparticles (B) synthesized with R of 0.4.
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Generally, with the increase of R, the particle size decreases, and the growth of particles have a tendency to form spheres [37]. In this synthesis, however, the increase of R led to the increase of the particle size and the formation of polygon gold nanoparticles. Probably this phenomenon is due to the instability of the quinoid compound in which keto–enol tautomerism usually exists. Compare to other reductive and capping agents, this compound is not strong enough to completely prevent the crystal growth after the nucleation stage. With the increase of gallic acid, more AuCl4 − was reduced, so the particle sizes increased. Even though not strong enough, gallic acid still partially prevents the aggregate and growth of the gold nanoparticles. Moreover, it may have special strong adsorption to some lattice planes of gold, which will change the growth rate of different planes and lead to the polygon shape of the particles.
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3.2. Effect of PVP concentration on the synthesis of gold nanoparticles
of PVP concentration, gold nanoparticles showed better monodispersive and uniformity. Experimental results showed that the stability of the gold nanoparticles prepared in the presence of PVP is very good. The stability of gold nanoparticles (PVP concentration of 0.02 mM) was confirmed by storing them in solution for 3 months without any precipitate observed and any change in absorption spectrum. These experimental results demonstrated that the use of PVP effectively protected gold nanoparticles and improved their quality. The presence of PVP made the particle size smaller and the particle shape closer to sphericity. It can be explained that PVP has a nucleating effect and therefore higher nucleation rates are obtained by increasing PVP concentrations, whereas the growth process of crystal nucleus is reduced [36]. In addition, PVP with long chain makes different lattice plane of nanoparticles grow at equal rate, which lead to the shape close to sphericity [42]. In the absence of PVP, the reduction products of HAuCl4, with
In general, for the synthesis of nano-scale metal clusters, molecules with long hydrocarbon chains absorbed on the surface of metal particles can decrease particle size and increased stability of the particles in solution [41]. The use of hydrophilic polymers with long chains as steric stabilizers is usually effective due to the intensive short-range steric repulsions that the polymers are able to produce [36]. Among the polymers, PVP is frequently used in many gel synthesis reactions as a good stabilizer, and was used here for improving the quality of the as prepared nanoparticles. Fig. 5 shows the UV–vis absorption spectra of gold nanoparticles obtained in the presence of PVP by Method 2 with an R of 0.4. The λmax of gold nanoparticles synthesized in the absence of PVP was 536 nm. With the increase of PVP concentration, the λmax of the obtained gold nanoparticles exhibited the blueshifts from 536 to 526 nm as shown in Fig. 5(A), meanwhile the absorbance intensity decreased gradually. The color of the solution changed from purple-red to deep red. The results indicated that the addition of PVP led to the decrease of particle size. When PVP concentration was over 0.001 mM, the λmax no longer exhibited blue-shift and almost fixed at 526 nm as shown in Fig. 5(B). At the same time, the absorbance increased with the increase of PVP concentration. To further investigate the effect of PVP on the size and shape of the gold nanoparticles, the microstructure of gold nanoparticles obtained in the presence of PVP with various concentrations was imaged by TEM. As shown in Fig. 6(A)–(C), the average size of gold nanoparticles was 23.3 ± 6.1, 16.1 ± 3.6 and 11.9 ± 1.6 nm for PVP concentration of 0.0001, 0.001 and 0.02 mM, respectively. The particle size decreased obviously with the increase of PVP concentration, and gold nanoparticles had an obvious tendency to be spherical in shape. It also can be seen from the figure, with the increase
Fig. 5. Absorption spectra of gold nanoparticles synthesized with different PVP concentration (R = 0.4).
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Fig. 6. TEM images of gold nanoparticles synthesized with PVP concentration of 0.0001 mM (A), 0.001 mM (B) and 0.02 mM (C) (R = 0.4).
shorter hydrocarbon chain, are not able to effectively control crystal growth, which lead to relatively large and polygonal gold nanoparticles. 3.3. Preparation and application in DNA hybridization of gold nanoparticles probes modified with 3 - and 5 -alkanethiol oligonucleotides Gold nanoparticles (about 12 nm diameter) synthesized in the presence of PVP by Method 2 were modified with 3 - or 5 -alkanethiol-capped 12-base oligonucleotides to form two different gold nanoparticles probes. The UV–vis spectra of unmodified gold nanoparticles in aqueous solution and gold nanoparticles modified with 3 - or 5 -alkanethiol 12-base oligonucleotides at 0.3 M NaCl are shown in Fig. 7(a)–(c), respectively. After modification, no shift in the surface plasmon resonance band was observed. However, the intensity of the plasmon band decreased apparently due to a decrease in particle concentration during the workup of the oligonucleotidemodified particles [30].
Fig. 7. Absorption spectra of (a) unmodified gold nanoparticles, (b) gold nanoparticles modified by 5 -(alkanethiol)-capped 12-mer ss-DNA, (c) gold nanoparticles modified by 3 -(alkanethiol)-capped 12-mer ss-DNA, and (d) the DNA-linked gold nanoparticles hybridized with complementary DNA.
The prepared probes were applied in the nanoparticlebased DNA hybridization detection system that two different oligonucleotides-modified probes (S1 and S2) would align in a tail-to-tail fashion onto a complementary target oligonucleotide strand (S3) [30]. When a buffer (0.3 M NaCl, 10 mM phosphate, pH 7) containing 100 pmol of complementary target oligonucleotide strand was mixed with the solution containing 150 l of each probe mixture, the solution color changed from red to purple in minutes. This color change can be attributed to the formation of large DNA-linked aggregates of gold nanoparticles, which lead to a red shift in the surface plasmon resonance from λmax = 526–585 nm as shown in Fig. 7(d) [29]. This was confirmed by a TEM image (as shown in Fig. 8). Visible is a large gold nanoparticle network structure, which consists of DNA-linked aggregates of gold probes and complementary target oligonucleotides, suggesting the presence of DNA hybridization. In the nanoparticle probes, thiol group (SH) connected with the oligonucleotides have strong affinity to the gold surface,
Fig. 8. TEM image of the DNA-linked gold nanoparticles after hybridization.
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due to a covalent interaction [21,43]. The physically absorbed nucleophile groups of PVP can be completely removed from the gold surface by thiol-modified compounds according to a ligand-exchange process [36]. Therefore, the PVP-protected gold nanoparticles prepared by gallic acid reduction of HAuCl4 are possible to be used as an excellent precursor material for the thiol-modified biological probes and further be applied in other bio-analysis. 4. Conclusions In summary, a one-step synthetic method for biocompatible gold nanoparticles using gallic acid as reductant in the presence of PVP at room temperature was demonstrated. The size and shape of gold nanoparticles could be tuned by altering R value. At the same time, fcc structure and crystal in nature of gold nanoparticles synthesized by gallic acid reduction of HAuCl4 were confirmed by UV–vis spectrometry, TEM and XRD pattern. It was known from IR spectroscopy that quinoid compounds with keto–enol structure were adsorbed on the surface of gold nanoparticles. Importantly, the addition of PVP could decrease gold particle size, improve the monodispersity and stability, as well as make it close to spherical in shape. The as prepared PVP-protected gold nanoparticles could be easily modified with thiol-capped oligonucleotides and then applied in DNA hybridization. Acknowledgements We are grateful for the support from Doctoral Research Found of Education Ministry of China (No. 20021045001) and National Natural Science Foundation of China (No. 20675011). References [1] G. Frens, Nat. Phys. Sci. 241 (1973) 20. [2] J. Turkevitch, P.C. Stevenson, J. Hillier, Discuss. Faraday Soc. 11 (1951) 55. [3] K.R. Brown, M.J. Natan, Langmuir 14 (1998) 726. [4] N.R. Jana, L. Gearheart, C.J. Murphy, Chem. Mater. 13 (2001) 2313. [5] C.L. Chiang, Colloid Interf. Sci. 230 (2000) 60. [6] K. Esumi, T. Hosoya, A. Suzuki, J. Colloid Interf. Sci. 229 (2000) 303. [7] H.-F. Zhu, Ch. Tao, S.-P. Zheng, J.-B. Li, Colloid Surf. A: Physicochem. Eng. Aspects 257/258 (2005) 411. [8] M. Yamamoto, M. Nakamoto, Chem. Lett. 32 (2003) 452. [9] A. Henglein, D. Meisel, Langmuir 14 (1998) 7392.
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One-step synthesis of biocompatible gold nanoparticles using gallic acid in the presence of poly-(N-vinyl-2-pyrrolidone) Wenxing Wang a , Qifan Chen a , Cha Jiang a , Dongzhi Yang a , Xingmin Liu b , Shukun Xu a,∗ b
a Department of Chemistry, Northeastern University, Shenyang, Liaoning 110004, China Shenyang National Laboratory for Materials Science, Institute of Metal Research, Chinese Academy of Sciences, Shenyang, Liaoning 110016, China
Received 18 September 2006; received in revised form 30 November 2006; accepted 6 December 2006 Available online 15 December 2006
Abstract A one-step method for the synthesis of biocompatible gold nanoparticles at room temperature by reducing HAuCl4 with gallic acid in the presence of poly-(N-vinyl-2-pyrrolidone) (PVP) is presented. The effect of the molar ratio of gallic acid to gold (R) on the size and shape of gold nanoparticles and the characteristic of gold nanoparticles was investigated by UV–vis and infrared spectroscopy, transmission electron microscopy, and X-ray diffraction. For an R value of about 0.4, the prepared gold nanoparticles were the smallest in size and the most close to spherical in shape. At other molar ratios, particle sizes increased and various polyhedra were formed. A reaction mechanism for the reduction of HAuCl4 by gallic acid is proposed. The incorporation of PVP effectively protected the surface of gold nanoparticles and improved their stability. The PVP-protected gold nanoparticles were modified with 3 - and 5 -alkanethiol-capped 12-base oligonucleotides, respectively, to form two different nucleic acid probes. The probes were successfully used to complex a 24-base complementary polynucleotide target in a tail-to-tail fashion. © 2006 Elsevier B.V. All rights reserved. Keywords: Gold nanoparticles; Synthesis; Gallic acid; Poly-(N-vinyl-2-pyrrolidone) (PVP); DNA hybridization
1. Introduction In recent years, the synthesis and applications of gold nanoparticles have attracted the interest of many researchers, due to their unique physical and chemical properties and for potential bio-analytical applications. Since Frens [1] and Turkevitch et al. [2] initially introduced a sodium citrate reduction of HAuCl4 for the synthesis of stable gold nanoparticles, studies in this field have rapidly expanded. Many chemical and physical methods have been used to prepare a variety of gold nanoparticles; these methods include seed-mediated growth [3,4], use of reverse micelles [5], phase transfer reactions [6,7], thermolysis [8], radiolysis [9,10], photochemistry [11,12], and sonochemistry [13–15]. Among these, chemical methods are still the preferred method for the preparation of gold nanoparticles. The size and shape of gold nanoparticles are controlled by the
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ratio of HAuCl4 to the chemical reducing agent in the preparation with examples such as sodium citrate, borohydride, or other organic compounds. In addition, high molecular weight polymers [16–20], thiol derivatives [21–26] and other ligands [27,28], used as capping regents, are often used to control the particle size and shape, prevent aggregation, and improve function of the particle surface for application in bio-analytical methods. For instance, Mirkin and co-workers reported a “programmed assembly” strategy for utilizing nanoparticles aggregates modified with alkanethiol-capped single-stranded oligonucleotides and complementary linker oligonucleotides strands to analyze DNA [29–31]. In other work, particles have also been applied to the analysis of other biological substances such as lectin [32] and protein [33]. Gallic acid is a poly-phenolic compound that may be used as a reductant, which is obtained from the hydrolysis of natural plant poly-phenols. It has been used historically to yield blue ink as its reduction of iron chloride produces a blue precipitate. Here, we tried firstly to use it as reductant to prepare nanogold, and gold nanoparticles were facilely synthesized by
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reducing HAuCl4 with gallic acid at room temperature. PVP, as a polymeric stabilizer, has been frequently used to protect metal nanoparticles and improve the quality of particle [34,35]. Because of unique physical characteristics, polymer-protected gold nanoparticles are potentially used for a number of advanced functional applications, especially in the optical and photonic fields [34]. So far, it is not reported that PVP-protected gold nanoparticles are applied in bio-analytical field. PVP can be removed from the surface of gold nanoparticles by thiol derivatives according to a ligand-exchange process [36]. The quality of the prepared gold nanoparticles directly depends on thiol derivatives on the particle surface. In previous reactions, the used thiol derivatives were hydrophobic and therefore the prepared gold nanoparticles were soluble in aliphatic and aromatic hydrocarbon, chlorine solvents, and ethers, but insoluble in acetone, alcohol, and water. The insolubility in water made them difficult to be used in bio-analytical application. For solving the problem, in this work, alkanethiol-capped oligonucleotides soluble in water were utilized to remove the PVP on the gold surface. Furthermore, the alkanethiol-oligonucleotides-capped gold nanoparticles were applied in bio-analysis such as DNA hybridization. In this paper, a one-step method for the synthesis of biocompatible gold nanoparticles using gallic acid as the reducing agent in the presence of PVP at room temperature is reported. The effect of the molar ratio of gallic acid to gold (R) and the concentration of PVP on the size and shape of gold nanoparticles as well as the characteristic of gold nanoparticles were investigated. UV–vis and IR spectroscopy, transmission electron microscopy (TEM), and X-ray diffraction (XRD) were employed in the characterization of the prepared gold nanoparticles. The PVP-protected gold nanoparticles were used as a precursor material to prepare the thiol-modified single-strand oligonucleotides probes. The probes generated hybridization with complementary target single-strand oligonucleotides by a tail-to-tail fashion [30]. 2. Experimental 2.1. Materials All the reagents obtained commercially were of analytical grade and were used as received without further purification. All glassware used was cleaned in aqua regia (HCl:HNO3 , 3:1), rinsed thoroughly with triply distilled water, and oven dried prior to use. HAuCl4 ·3H2 O, gallic acid and poly-(N-vinyl-2-pyrrolidone) (PVP, Mw = 10000) were purchased from Shanghai Chemicals Company. All aqueous solutions were made using triply distilled water, and subsequently filtered through 0.22 m Millipore syringe filter. All DNA synthesis reagents were purchased from Shanghai Biological Engineering Company. The sequences of oligonucleotides used in this work were as follows: • 3 -(alkanethiol)oligonucleotide (S1): 5 -TAGGACGTACGC(CH2 )6 -SH-3 ;
• 5 -(alkanethiol)oligonucleotide (S2): 5 -SH-(CH2 )6 -TATCATCTAGTC-3 ; • complementary target oligonucleotide (S3): 5 -GCGTACGTCCTAGACTAGATGATA-3 . 2.2. Instrumental methods Gold nanoparticles were characterized by UV–vis spectroscopy, TEM, XRD and IR spectroscopy. UV–vis absorption spectra of gold nanoparticles were recorded using a Shimadzu UV-2550 UV–vis spectrophotometer using 1 cm quartz cuvette. TEM images were performed on a Philips EM 420 microscope operating at 120 kV. A typical sample for TEM was prepared by drying naturally a drop of solution containing gold nanoparticles at room temperature on a carbon-coated copper grid. XRD was recorded on a Panalytical PW 3040160 X-ray diffractometer using Cu K␣ radiation (λ = 0.1542 nm) operated at 50 kV and 100 mA. IR spectra were measured on a Perkin-Elmer Spectrum One FT-IR spectrometer. 2.3. Synthesis and characterization of gold nanoparticles 2.3.1. Method 1 Gold nanoparticles were synthesized by mixing HAuCl4 (0.3 mM, 80 ml) and gallic acid (varied concentration, 20 ml) solutions. That was, 20 ml of 0.25, 0.5, 1.25, 2.0 and 2.5 mM gallic acid was added rapidly to 80 ml of 0.3 mM HAuCl4 solution, respectively, under magnetic stirring in a water bath of room temperature. The color of solutions rapidly changed from colorless to different red depending on gallic acid concentration. The reactions were kept for 30 min. 2.3.2. Method 2 PVP was added to these solutions in Method 1 for improving the quality of prepared nanoparticles. Typically, 0, 0.01, 0.05, 0.1, 0.5, 2 and 4 ml of 1 mM PVP was added to the solution (the molar ratio of gallic acid to gold = 0.4) in Method 1, respectively. The volume of the reaction was 100 ml. The reactions were kept 30 min at room temperature. 2.4. Preparation of 3 - or 5 -(alkanethiol)oligonucleotidemodified gold nanoparticles Gold nanoparticles (about 12 nm diameter) synthesized in the presence of PVP by Method 2 were modified with 3 - or 5 alkanethiol-capped 12-base oligonucleotides (S1 or S2) to form two different gold nanoparticle probes [29]. Typically, 1.0 ml of gold nanoparticles aqueous solution with 3.6 M of S1 or S2 was shaken for 12 h and then incubated for 12 h at 45 ◦ C in a water bath. The solution was brought to 0.1 M NaCl, 10 mM phosphate buffer (pH 7) and was kept at 45 ◦ C for 40 h. To remove excess reagents, the solution was centrifuged at 14,000 rpm for 25 min, and the supernatant was replaced by 1.0 ml of the same buffer. After additional centrifugation under the same condition, the red oily precipitate was re-dispersed into 0.5 ml of 0.3 M NaCl 10 mM phosphate buffer (pH 7) to be used as gold nanoparticle probes.
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2.5. Hybridization of gold nanoparticle probes with complementary target oligonucleotide One hundred and fifty microlitres of each gold nanoparticle probe mixture prepared above was mixed with buffer (0.3 M NaCl, 10 mM phosphate, pH 7) containing 100 pmol complementary target oligonucleotide (S3). The solution was heated to 55 ◦ C for 5 min and allowed to cool to room temperature. The color of the solution changed from red to purple in minutes, and full precipitate occurred within approximately 1 h. The UV–vis spectrum of the hybridized solution was recorded. A TEM sample was prepared by dropping the hybridized solution onto a carbon-coated copper grid, and dried for 30 min under ambient conditions. The TEM sample was imaged using a Philips EM 420 transmission electron microscopy operating at 120 kV. 3. Results and discussion 3.1. Gold nanoparticles synthesized with gallic acid Initial synthesis focused on the formation of gold nanoparticles using gallic acid in the absence of PVP. The molar ratio of gallic acid to gold was defined R for abbreviation. When the R value was over 2.0, brown-red precipitates appeared in the reactor and resulted in a turbid solution. This phenomenon suggested that the prepared gold nanoparticles are too large to be stabilized in the solution. So the molar ratios of 0.2, 0.4, 1.0, 1.6 and 2.0 were chosen as illustrative examples. Fig. 1 shows the absorption spectra of gold nanoparticles synthesized by various R at room temperature in Method 1. The surface plasmon resonance band of gold nanoparticles varied with the change of R. Initially, the maximal plasmon absorption peaks of gold nanoparticles showed a blue shift and turned narrow in width with the increase of R, and then showed a red shift and broaden in width with the further increase of R. Only for the molar ratio of 0.4, the maximum plasmon absorption wavelength (λmax ) of gold nanoparticles was obtained for the least value (536 nm) without another absorption band in the longitudinal plasma resonance. It is interesting to note that, for
Fig. 1. Absorption spectra of gold nanoparticles synthesized with R of 0.2 (a), 0.4 (b), 1.0 (c), 1.6 (d) and 2.0 (e).
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the molar ratios of 1.0, 1.6 and 2.0, there was another absorption band appeared ranged 600–800 nm, which belongs to the longitudinal plasma resonance band of aggregates [22] or deviation from spherical geometry of gold nanoparticles [37]. Mie’s theory is often used to explain the deviation from spherical geometry of the particles, suggesting in the circumstance that the transverse and longitudinal dipole polarizability no longer produces equivalent resonance, leads to a broadening and red shift of longitudinal plasmon resonance as well as an appearance of transverse plasma resonance [38,39]. All the results indicated that the size and shape of gold nanoparticles were altered with R value, which was confirmed by TEM images further. Fig. 2 shows typical TEM images of gold nanoparticles synthesized by various R. With the increase of R, the average size of gold nanoparticles increased, but not significantly, meanwhile the shape of gold nanoparticles changed obviously. Gold nanoparticles obtained at the R of 0.2 were seen to be aggregated as shown in Fig. 2(A), resulting that the maximum absorption band shifted to longer wavelength than that at the molar ratio of 0.4. For the R of 0.4, the obtained gold nanoparticles exhibited better dispersibility; and near spherical in shape, as shown in Fig. 2(B). However, with the further increase of R, particle shape appeared various polygonal particles such as triangular, rod-like, hexagonal, spherical, etc. as shown in Fig. 2(C)–(E). These results were also in accordance with absorption spectra shown in Fig. 1. It indicated the occurrence of two plasmon resonance bands due to the increasing of the molar ratio mainly ascribed to the appearance of anisometric gold nanoparticles [37]. To investigate the structure of the obtained gold nanoparticles, the XRD pattern was measured. Fig. 3 shows the XRD pattern obtained from gold nanoparticles at R of 0.4. The spectrum included five peaks at 38.2◦ , 44.4◦ , 64.7◦ , 77.6◦ and 81.6◦ that can be assigned to the (1 1 1), (2 0 0), (2 2 0), (3 1 1) and (2 2 2) planes, which have a good match with the standard diffraction pattern of JCPDS No. 089–3697. The XRD pattern indicated that gold nanoparticles were in the face-centered cubic (fcc) structure and crystal in nature. IR spectrum was also measured for the further investigation to identify the possible molecular response for efficient stabilization of gold nanoparticles synthesized with an R of 0.4. Gold nanoparticles were centrifuged at 14,000 rpm for 25 min, then the red precipitate was rinsed with water, re-centrifuged, the obtained precipitate was used for IR analysis. Fig. 4 shows the infrared spectra of gallic acid (A) and gold nanoparticles (B) obtained by gallic acid reduction of HAuCl4 . In Fig. 4(A), the strong and broad band between 3600–2500 cm−1 and the strong and narrow peak at 1702 cm−1 could be assigned to be stretching vibration of OH group and carbonyl group, which indicated that carboxyl group existed in the gallic acid. Three peaks observed at 1616, 1541, 1450 cm−1 are typical stretching vibrations of C C bonds in aromatic ring. There are several peaks in 1300–1000 cm−1 region that could be assigned to be the stretching vibration of C–O bond and bending vibration of O–H bond of gallic acid. Since phenolic compounds are easily oxidized to form quinones, it was speculated that the product of
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Fig. 2. TEM images of gold nanoparticles synthesized with R of 0.2 (A), 0.4 (B), 1.0 (C), 1.6 (D) and 2.0 (E).
gallic acid reduction of HAuCl4 might be a quinoid compound. In Fig. 4(B), the strong and broad band in the 3650–2700 cm−1 region was considered to be stretching vibration of OH group, which basically covered C–H bond stretching vibration at about 3100 cm−1 . Stretching vibration of C–O bond and bending vibration of O–H bond in 1300–1000 cm−1 region was still retained, but the intensity obviously decreased. In contrast with that of gallic acid shown in Fig. 4(A), the IR absorption spectrum of gold nanoparticles observed from Fig. 4(B) indicated that the stretching vibration peak of carbonyl group shifted from 1702 to 1640 cm−1 , and the stretching vibration of C C bond at 1616 and 1541 cm−1 was covered with a broad and middle intensity band at around 1640 cm−1 . The results indicated that quinoid compound with keto–enol system might be produced by gallic acid reduction of HAuCl4 and absorbed on the surface of gold nanoparticles. Usually, when molecule absorbs on the nano-scale metal island, surface-enhanced Raman and infrared spectra can be observed [40]. Electromagnetic field and chemical
Fig. 3. XRD pattern of gold nanoparticles synthesized with R of 0.4.
mechanisms have been proposed to explain this phenomenon. In this work, a chemical mechanism is more reasonable since significant conjugation exists within the quinoid compound with a keto–enol system. On the other hand, an inductive effect may also exist between the nucleophilic compound and the electron dense gold nanoparticles. These can be used to explain the significant shift of the stretching band of carbonyl group to lower wavenumber (from 1702 to 1640 cm−1 ). A proposed reaction scheme for the reduction of HAuCl4 by gallic acid is given below:
Fig. 4. IR spectra of gallic acid (A) and gold nanoparticles (B) synthesized with R of 0.4.
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Generally, with the increase of R, the particle size decreases, and the growth of particles have a tendency to form spheres [37]. In this synthesis, however, the increase of R led to the increase of the particle size and the formation of polygon gold nanoparticles. Probably this phenomenon is due to the instability of the quinoid compound in which keto–enol tautomerism usually exists. Compare to other reductive and capping agents, this compound is not strong enough to completely prevent the crystal growth after the nucleation stage. With the increase of gallic acid, more AuCl4 − was reduced, so the particle sizes increased. Even though not strong enough, gallic acid still partially prevents the aggregate and growth of the gold nanoparticles. Moreover, it may have special strong adsorption to some lattice planes of gold, which will change the growth rate of different planes and lead to the polygon shape of the particles.
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3.2. Effect of PVP concentration on the synthesis of gold nanoparticles
of PVP concentration, gold nanoparticles showed better monodispersive and uniformity. Experimental results showed that the stability of the gold nanoparticles prepared in the presence of PVP is very good. The stability of gold nanoparticles (PVP concentration of 0.02 mM) was confirmed by storing them in solution for 3 months without any precipitate observed and any change in absorption spectrum. These experimental results demonstrated that the use of PVP effectively protected gold nanoparticles and improved their quality. The presence of PVP made the particle size smaller and the particle shape closer to sphericity. It can be explained that PVP has a nucleating effect and therefore higher nucleation rates are obtained by increasing PVP concentrations, whereas the growth process of crystal nucleus is reduced [36]. In addition, PVP with long chain makes different lattice plane of nanoparticles grow at equal rate, which lead to the shape close to sphericity [42]. In the absence of PVP, the reduction products of HAuCl4, with
In general, for the synthesis of nano-scale metal clusters, molecules with long hydrocarbon chains absorbed on the surface of metal particles can decrease particle size and increased stability of the particles in solution [41]. The use of hydrophilic polymers with long chains as steric stabilizers is usually effective due to the intensive short-range steric repulsions that the polymers are able to produce [36]. Among the polymers, PVP is frequently used in many gel synthesis reactions as a good stabilizer, and was used here for improving the quality of the as prepared nanoparticles. Fig. 5 shows the UV–vis absorption spectra of gold nanoparticles obtained in the presence of PVP by Method 2 with an R of 0.4. The λmax of gold nanoparticles synthesized in the absence of PVP was 536 nm. With the increase of PVP concentration, the λmax of the obtained gold nanoparticles exhibited the blueshifts from 536 to 526 nm as shown in Fig. 5(A), meanwhile the absorbance intensity decreased gradually. The color of the solution changed from purple-red to deep red. The results indicated that the addition of PVP led to the decrease of particle size. When PVP concentration was over 0.001 mM, the λmax no longer exhibited blue-shift and almost fixed at 526 nm as shown in Fig. 5(B). At the same time, the absorbance increased with the increase of PVP concentration. To further investigate the effect of PVP on the size and shape of the gold nanoparticles, the microstructure of gold nanoparticles obtained in the presence of PVP with various concentrations was imaged by TEM. As shown in Fig. 6(A)–(C), the average size of gold nanoparticles was 23.3 ± 6.1, 16.1 ± 3.6 and 11.9 ± 1.6 nm for PVP concentration of 0.0001, 0.001 and 0.02 mM, respectively. The particle size decreased obviously with the increase of PVP concentration, and gold nanoparticles had an obvious tendency to be spherical in shape. It also can be seen from the figure, with the increase
Fig. 5. Absorption spectra of gold nanoparticles synthesized with different PVP concentration (R = 0.4).
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Fig. 6. TEM images of gold nanoparticles synthesized with PVP concentration of 0.0001 mM (A), 0.001 mM (B) and 0.02 mM (C) (R = 0.4).
shorter hydrocarbon chain, are not able to effectively control crystal growth, which lead to relatively large and polygonal gold nanoparticles. 3.3. Preparation and application in DNA hybridization of gold nanoparticles probes modified with 3 - and 5 -alkanethiol oligonucleotides Gold nanoparticles (about 12 nm diameter) synthesized in the presence of PVP by Method 2 were modified with 3 - or 5 -alkanethiol-capped 12-base oligonucleotides to form two different gold nanoparticles probes. The UV–vis spectra of unmodified gold nanoparticles in aqueous solution and gold nanoparticles modified with 3 - or 5 -alkanethiol 12-base oligonucleotides at 0.3 M NaCl are shown in Fig. 7(a)–(c), respectively. After modification, no shift in the surface plasmon resonance band was observed. However, the intensity of the plasmon band decreased apparently due to a decrease in particle concentration during the workup of the oligonucleotidemodified particles [30].
Fig. 7. Absorption spectra of (a) unmodified gold nanoparticles, (b) gold nanoparticles modified by 5 -(alkanethiol)-capped 12-mer ss-DNA, (c) gold nanoparticles modified by 3 -(alkanethiol)-capped 12-mer ss-DNA, and (d) the DNA-linked gold nanoparticles hybridized with complementary DNA.
The prepared probes were applied in the nanoparticlebased DNA hybridization detection system that two different oligonucleotides-modified probes (S1 and S2) would align in a tail-to-tail fashion onto a complementary target oligonucleotide strand (S3) [30]. When a buffer (0.3 M NaCl, 10 mM phosphate, pH 7) containing 100 pmol of complementary target oligonucleotide strand was mixed with the solution containing 150 l of each probe mixture, the solution color changed from red to purple in minutes. This color change can be attributed to the formation of large DNA-linked aggregates of gold nanoparticles, which lead to a red shift in the surface plasmon resonance from λmax = 526–585 nm as shown in Fig. 7(d) [29]. This was confirmed by a TEM image (as shown in Fig. 8). Visible is a large gold nanoparticle network structure, which consists of DNA-linked aggregates of gold probes and complementary target oligonucleotides, suggesting the presence of DNA hybridization. In the nanoparticle probes, thiol group (SH) connected with the oligonucleotides have strong affinity to the gold surface,
Fig. 8. TEM image of the DNA-linked gold nanoparticles after hybridization.
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due to a covalent interaction [21,43]. The physically absorbed nucleophile groups of PVP can be completely removed from the gold surface by thiol-modified compounds according to a ligand-exchange process [36]. Therefore, the PVP-protected gold nanoparticles prepared by gallic acid reduction of HAuCl4 are possible to be used as an excellent precursor material for the thiol-modified biological probes and further be applied in other bio-analysis. 4. Conclusions In summary, a one-step synthetic method for biocompatible gold nanoparticles using gallic acid as reductant in the presence of PVP at room temperature was demonstrated. The size and shape of gold nanoparticles could be tuned by altering R value. At the same time, fcc structure and crystal in nature of gold nanoparticles synthesized by gallic acid reduction of HAuCl4 were confirmed by UV–vis spectrometry, TEM and XRD pattern. It was known from IR spectroscopy that quinoid compounds with keto–enol structure were adsorbed on the surface of gold nanoparticles. Importantly, the addition of PVP could decrease gold particle size, improve the monodispersity and stability, as well as make it close to spherical in shape. The as prepared PVP-protected gold nanoparticles could be easily modified with thiol-capped oligonucleotides and then applied in DNA hybridization. Acknowledgements We are grateful for the support from Doctoral Research Found of Education Ministry of China (No. 20021045001) and National Natural Science Foundation of China (No. 20675011). References [1] G. Frens, Nat. Phys. Sci. 241 (1973) 20. [2] J. Turkevitch, P.C. Stevenson, J. Hillier, Discuss. Faraday Soc. 11 (1951) 55. [3] K.R. Brown, M.J. Natan, Langmuir 14 (1998) 726. [4] N.R. Jana, L. Gearheart, C.J. Murphy, Chem. Mater. 13 (2001) 2313. [5] C.L. Chiang, Colloid Interf. Sci. 230 (2000) 60. [6] K. Esumi, T. Hosoya, A. Suzuki, J. Colloid Interf. Sci. 229 (2000) 303. [7] H.-F. Zhu, Ch. Tao, S.-P. Zheng, J.-B. Li, Colloid Surf. A: Physicochem. Eng. Aspects 257/258 (2005) 411. [8] M. Yamamoto, M. Nakamoto, Chem. Lett. 32 (2003) 452. [9] A. Henglein, D. Meisel, Langmuir 14 (1998) 7392.
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