Journal of Nanoparticle Research

, Volume 12, Issue 7, pp 2679–2687

A novel method to realize the transition from silver nanowires to nanoplates based on the degradation of DNA

Research Paper

DOI: 10.1007/s11051-010-0005-4

Cite this article as:
Li, C., Shen, Y., Xie, A. et al. J Nanopart Res (2010) 12: 2679. doi:10.1007/s11051-010-0005-4

Abstract

Silver “nano-necklaces” and nanoplates in DNA/Tris–EDTA (TE) solution are prepared using hydrothermal method. The nano-necklaces are composed of many spherical silver nanoparticles which are joined together by the DNA chain. Further the transition from silver nano-necklaces to triangular and hexagonal nanoplates is realized based on the degradation of DNA. Transmission electron microscopy, selected area electron diffraction, ultraviolet–visible spectroscopy, X-ray diffraction, and agarose gel electrophoresis are used to characterize silver nanoparticles and the change of DNA structure. The results show that TE acts as not only the buffer solution but also the reducing agent. DNA serves as templates to offer the nucleation sites and induce the growth of silver nanostructures. Hydrothermal process provides high temperature and pressure to activate the reducing property of TE and denature or degrade DNA molecules. The formation mechanism of silver nano-necklaces and nanoplates has also been studied.

Keywords

DNA Degradation Nanowire Nanoplate Hydrothermal synthesis Nanobiotechnology 

Introduction

As the genetic code carriers of almost all known living organisms, including all eukaryotes, bacteria, and many viruses, DNA play key roles in the storage, transmission, and expression of genetic information. Biologists and chemists show great interest in DNA research. Many biotechnology techniques, including recombinant DNA, anti-sense DNA and RNA, anti-gene technology, DNA vaccines, etc., have been developed.

Recently, DNA has entered nanoscience due to its unique structure: its minuscule size, with a diameter of about 2 nm, its short structural repeat (helical pitch) of about 3.4–3.6 nm, and its “stiffness,” with a persistence length (a measure of stiffness) of around 50 nm. It is considered as an ideal template for the preparation of metal nanostructure because DNA has high affinity for metal cations, which could be reduced to form metallic nanoparticles. The first report of DNA-templated nanomaterials comes from Braun et al. (1998). Till now the metal nanowires generated on DNA involve silver (Berti et al. 2005), gold (Mbindyo et al. 2001; Satti et al. 2007), platinum (Yang et al. 2006), palladium (Hatakeyama et al. 2008), copper (Monson and Woolley 2003; Becerril et al. 2004), cobalt (Gu et al. 2005), and nickel (Becerril et al. 2006), and carbon nanotube has been successfully localized on DNA molecules (Xin and Woolley 2003, 2005). Recently, Ivanisevic’s group (Kinsella and Ivanisevic 2005; Kinsella et al. 2007) has reported the preparation of magnetic nanowires templated by DNA. Many researchers also have used DNA as template to assemble nanomaterials with uniform morphology because of its sequence programmability, selective molecular recognition ability, and the relative rigidity of its double-helical form (Xiao et al. 2002; Becerril et al. 2005).

However, DNA may undergo damage by a number of endogenous and exogenous factors, such as heat, chemical reagents, and ultraviolet radiations (Scharer 2003). The consequence is the denaturation or degradation of DNA. Also the mismatch of DNA bases might occur. If not repaired, then these DNA lesions can initiate a cascade of biological effects at the cellular, organ, or individual level, leading to mutagenesis and carcinogenesis. Here, we introduced the unique property of DNA to nanoscience. The transition from silver nano-necklaces into nanoplates is realized based on the degradation of DNA, which has not been reported previously.

Experimental section

Materials

Calf thymus DNA (type I, sodium salt) was purchased from Sigma Corporation with product number D-1501. Silver nitrate, tris(hydroxymethyl)aminomethane (Tris), and disodium ethylenediamine tetraacetic acid (EDTA) were all received from Shanghai Reagent Co., China. The above reagents were of analytical purity and were used without further purification. Double distilled water was used in this experiment.

Preparation of silver nanostructures

DNA was dissolved in Tris–EDTA (TE) buffer (pH 7.4). The concentration of nucleotide bases in the DNA solution was determined based on the absorbance at 260 nm (ε = 6600 M−1 cm−1). In a typical experiment, 2 ml DNA (0.73 mM) and 14.6 ml AgNO3 (1 mM) were mixed to keep the molar ratio of silver ions to bases (s/b) at 10:1. The resulting solution was incubated for half an hour to complete the interaction between silver ions and DNA. Then the mixed solution was put into the 25-ml capacity stainless Teflon-lined autoclave, followed by the treatment at different temperatures (60, 70, 90, and 100 °C) for some time (1–3, and 5 h). Then, part of the solutions (4 ml) were taken out, centrifugated at 12,000 rpm, then washed with double distilled water five times, and finally dried under vacuum at 20 ± 2 °C for measurements.

Control experiments

Control experiment I: In order to determine the effect of s/b on the morphology and size of the silver nanoparticles, it was adjusted to 100:1, 1:1, and 10:0. Then, the solutions were treated at 70 and 90 °C for 1–3 and 5 h under hydrothermal conditions.

Control experiment II was done to demonstrate whether the reducing agent was TE or not. Here, DNA was dissolved into pure double distilled water instead of TE buffer. The s/b was 10:1. Then, the mixed solution was treated at 90 °C for 1–3 and 5 h, 16 h under hydrothermal conditions.

Control experiment III was to illustrate the indispensable effect of hydrothermal synthesis in the system. DNA was dissolved into TE buffer. S/b was kept at 10:1. The rest steps were the same as control experiment II except for replacing hydrothermal synthesis by direct heat in the open system.

Control experiment IV was to elucidate the changes of DNA structure during the hydrothermal process. DNA solution was divided into six parts with each volume of 4 ml. The first part was the original solution without any treatment. The next four parts were treated at 60 and 90 °C for 1 and 2 h, respectively, then placed in the ice bath immediately to lower the temperature avoiding the reassociation of DNA. Silver nitrate was added into the last part with s/b at 10:1 to confirm the interaction between DNA and silver ions.

Characterization

Samples were placed on the carbon-coated 400 mesh copper grids. Transmission electron microscopy (TEM) was performed on JEM model 100SX (Japan Electron Co.) at accelerating voltages of 80 kV. The pure DNA sample was stained by 1% uranyl acetate stock solution before TEM measurement. Selected area electron diffraction (SAED) measurements of the Ag nanoparticles were also carried out. Ultraviolet–visible (UV–vis) spectroscopic measurements were determined by a UV-3600 model UV–vis double beam spectrophotometer (Shimadzu Co. Japan) operating at a resolution of 2 nm. X-ray diffraction (XRD) was performed on a MAP18XAHF instrument, with the X-ray diffractometer using Cu Kα radiation (λ = 1.5 Å) at a scan rate of 0.05°2θ s−1 to determine the crystalline phase. The accelerating voltage and applied current were 35 kV and 25 mA, respectively (MAC Science, Japan). Agarose gel (1% w/v) electrophoresis was carried out under TBE buffer system at 50 V for 1 h on an electrophoretic instrument (Beijing Liuyi Co., China). Then the gel was visualized using a Bio-Rad Trans illuminator.

Results and discussion

Figure 1 shows the TEM images of pure DNA and silver nanoparticles obtained under various conditions. Figure 1a is the typical image of pure DNA. It assumes an elongated-coil conformation, consistent with the previous report (Zinchenko et al. 2005). The diameter of DNA chain is less than 10 nm, which is close to the theoretical value of the DNA diameter (2 nm), and the length of DNA chain reaches several microns. Figure 1b corresponds to the silver nanoparticles obtained from the reaction solution without DNA. It is clearly seen that the morphology of the particles is irregular, and the aggregation is obvious. In the presence of DNA, the s/b ratio highly influenced the morphology and size of the product. At very high s/b (100:1, Fig. 1c), only irregular particles are obtained.
Fig. 1

TEM images of the product. a Pure DNA. b 90 °C, 1 h, s/b = 10:0 (solution). c 90 °C, 1 h, s/b = 100:1. df 90 °C, s/b = 10:1; at 1, 3, and 5 h, respectively. The bars in e and f are 50 and 80 nm, respectively. gh 70 °C, s/b = 10:1; at 1, 3, and 5 h, respectively. Both the bars in the insets of h are 150 nm. The right-side inset in f is the SAED pattern of the product

Figure 1d–f shows images of the nanoparticles that were taken from the reaction solution after the reaction time of 1, 3, and 5 h at 90 °C with s/b at 10:1. Figure 1g–i corresponds to the product obtained at 70 °C. It can be clearly seen that DNA rings disappear, replaced by silver nano-necklaces and nanoplates. At the early stage of the reaction (1 h) silver nano-necklaces are formed (Fig. 1d, g), which are similar to the typical nanostructures generated along DNA chains (Becerril et al. 2004; Mertig et al. 2002). These nano-necklaces are composed of many spherical silver nanoparticles which are joined together by the DNA chain. The diameters of the silver nanoparticles in the nano-necklaces obtained at 90 and 70 °C are 15 and 70 nm, respectively. The length of the nano-necklaces reaches the micron scale. When the reaction time increased to 3 h, the silver nanoparticles exist in the shape of triangular and hexagonal nanoplates. The average edge lengths obtained at 90 and 70 °C are 50 ± 10 and 300 ± 100 nm, respectively (Fig. 1e, h). Increasing the reaction time to 5 h results in the formation of larger nanoparticles as shown in Fig. 1f, i, in which the average edge lengths of nanoplates are about 80 ± 10 nm (90 °C) and 500 ± 200 nm (70 °C), respectively. According to the sharp diffraction spots shown in SAED pattern (inset in Fig. 1f), the silver nanoplates are of single-crystal quality, and the plane could be indexed to the fcc silver.

Based on the above results three conclusions can be summarized. One is that with the increase of reaction time, the size of nanoparticles grows larger, and the transition from nano-necklaces to nanoplates is obtained. Another is that the nanoparticle size obtained at 70 °C is larger than that obtained at 90 °C. The last is the induced and regulated role of DNA on the formation of silver nanostructures. The detailed formation mechanism of silver nano-necklaces and nanoplates will be discussed in the next section.

Figure 1c shows the image of the silver nanostructures which are obtained at a high s/b (100:1) with the reaction time of 1 h at 90 °C. By comparison with Fig. 1d, the silver nanoparticles obtained are irregular and aggregate together. Here, the content of silver ions is so high that large numbers of ions could not be absorbed on the DNA chain, resulting in the formation of large metal particles by aggregation of silver clusters nucleated homogeneously. If s/b was adjusted to 1:1, few silver nanoparticles are formed even at the reaction time of 16 h. According to Gibbs free energy formula, the driving force for the formation of silver nanoparticles (ΔGv) is given by the equation as follows (Spanos and Koutsoukos 1998):
$$ {{\Updelta}}G_{\text{v}} = - RT_{\text{g}} { \ln }\,S_{\text{v}} $$
(1)
where R, Tg, and Sv are the gas constant, absolute temperature, and supersaturation, respectively. Based on the formula, lower supersaturation is unfavorable for the formation of silver nanostructures. Therefore, the low supersaturation might account for the few yield of silver nanoparticles.

In control experiments II and III, no phenomena were observed, indicating the role of reducing agent for TE and the importance of hydrothermal conditions in our system.

The reaction kinetics is monitored using UV–vis spectroscopy and the change of the solution color, which are shown in Fig. 2. Figure 2A shows the effect of reaction time on silver nanostructures. Before treating the mixed solution with hydrothermal conditions, only DNA absorption peak is observed at 266 nm (Fig. 2A-a). At the early stage of the reaction (1 h), a new absorption band centered at 398 nm appears (Fig. 2A-b), which is attributable to the surface plasmon resonance (SPR) of silver nanoparticle (Chen et al. 2008; Khanna et al. 2008), and the intensity of the absorption band of DNA exhibits an increase, possibly resulting from the denaturation of DNA. The shift of absorption band indicates that the microenvironment of the bases is perturbed. Increasing the reaction time results in the more intensive SPR peak (Fig. 2A-c, d, e) because of the formation of more silver nanoparticles, and the maximum wavelength displays red shift, from 430 to 433 nm and finally to 455 nm, indicating the increase of the particle size according to Mie theory (Mie 1908). The color of the solution (insets in the top right corner of the figures) becomes darker with reaction time. The change is due to the formation of more silver nanoparticles, which is consistent with that of SPR peak intensity. After aging for a month, the silver colloid obtained is also stable. However, in control experiment I without the addition of DNA, the products settle down on the bottom of the autoclave at the reaction time of 2 h. The regulated role of DNA on the formation of silver nanostructures can be also confirmed.
Fig. 2

UV–vis spectra and digital pictures of the solutions obtained under different conditions. A The time dependence of the variation of the absorption spectra of the silver colloidal solution obtained from the system with the ratio of s/b at 10:1 at 90 °C for different time. a 0 h, b 1 h, c 2 h, d 3 h, and e 5 h. B Recorded from the solutions treated with different temperatures with s/b at 10:1 for 5 h. a 60 °C, b 70 °C, c 90 °C, and d 100 °C

The effect of temperature on the shape and size of silver nanostructures is also investigated, as shown in Fig. 2b. As is well known, a high temperature will increase the collision rate of molecules, consequently accelerating chemical reaction, resulting in the higher yield of the product, which might be the reason for the increase of the SPR peak intensity along with the temperature. With the increase of the reaction temperature, the solution color becomes gradually opaque, relating well with the increase of the SPR peak intensity, meanwhile the SPR peak shifts toward longer wavelength. The detailed red shift is from 443 (60) to 469 nm (70 °C), finally to 475 nm (100 °C), indicating the formation of larger nanoparticles. However, an abnormal blue shift is presented from 70 to 90 °C. The same changes of the SPR peaks and solution color occur at the reaction time of 1 h.

In control experiments II and III, the resulting solutions experienced no changes on the color, and no sediments were observed on the bottom of the autoclave, which indicates that TE is the reducing agent and hydrothermal condition is very important in our system.

The XRD patterns of the products are displayed in Fig. 3. In Fig. 3a, the peaks are extremely weak, corresponding to the silver nanoparticles prepared without the addition of DNA. It illustrates that the formed particles are amorphous. Figure 3b is the XRD pattern of the sample obtained at the early stage of the reaction (1 h) with s/b at 10:1. The peak centered at the 2θ of 38.03° is assigned to the (111) plane of the cubic phase of silver. But the crystallinity is low according to the weak intensity of peak. While the reaction time increased to 3 h, the peaks become rather strong (Fig. 3c). These diffraction features appearing at about the 2θ of 37.99°, 44.11°, 64.45°, and 77.53°, correspond to the (111), (200), (220), and (311) planes of the cubic phase of silver, respectively. The strongest diffraction peak corresponds to the (111) faces, and its intensity is nearly six times (359:49) that of the (200) peak (the intensity ratio of (111) to (200) peaks is 100:40, PCPDF No. 040783). The similar phenomenon was also reported in a previous research (Chen and Carroll 2002), in which the samples obtained were all triangular nanoplates. The results indicate that the synthesized silver nanostructures are enriched in (111) faces. According to the above results, the regulated role of DNA on the formation of silver nanostructures can be concluded again.
Fig. 3

XRD patterns of silver nanoparticles synthesized under different conditions. (a) Corresponds to the sample obtained at 90 °C for 3 h with s/b at 10:0. (b) and (c) Correspond to the samples obtained at 90 °Cfor 1 and 3 h in the presence of DNA (s/b = 10:1)

In order to indicate the changes of DNA structure during the hydrothermal process, control experiments IV were performed. The resulting solutions were characterized by UV–vis spectrum and agarose gel electrophoresis, which are shown in Figs. 4 and 5. The little difference between Fig. 4a, b indicates that treating the solution at 60 °C for 1 h did not cause any changes on the structure of DNA. But when the temperature was increased to 90 °C, the structure might change a lot due to the great increase in the absorption band intensity (Fig. 4d). When the reaction time was 2 h, both treating at 60 and 90 °C induced the large changes of the structure observed from Fig. 4c, e. Compared with Fig. 4a, the absorption peak in Fig. 4f becomes more intensive and shifts toward longerwave, indicating the interaction between silver ions and DNA (Berti et al. 2005). Though the changes of structure could be estimated, the type of the changes (denaturation or degradation) is still unknown. Therefore, the same solutions were characterized by gel electrophoresis.
Fig. 4

UV–vis spectra of different DNA solutions obtained from control experiments IV. (a) DNA. (b) DNA, 60 °C, 1 h. (c) DNA, 60 °C, 2 h. (d) DNA, 90 °C, 1 h. (e) DNA, 90 °C, 2 h. (f) DNA + Ag+ (s/b = 10:1)

Fig. 5

Agarose gel electrophoresis of DNA. Lane 0 λ-DNA/EcoRI+HindIII marker. Lane 1 DNA without any manipulation. Lane 2 DNA + Ag+Lanes 3 and 4 treating pure DNA at 60 °C for 1 and 2 h, respectively. Lanes 5 and 6 treating pure DNA at 90 °C for 1 and 2 h, respectively

Figure 5 is an image of a 1% agarose gel electrophoresis of the different DNA solutions. Lane 0 corresponds to the DNA marker. For comparison, lane 1 shows the DNA that was kept in TE without any manipulations. Calf thymus DNA is of single form hence show a single band on electrophoresis. As shown in the figure the molecular mass of the DNA used might be close to 20 kilobase pairs, which is consistent with the value in previous report (Hossain and Huq 2002). When it was electrophoresed after adding the silver ions into the DNA solution (lane 2), no significant changes in intensity of the bands are observed. However, electrophoretic mobility of the band is found to decrease slightly. The decrease is believed due to the binding of silver ions to DNA, thus reducing its overall negative charge and increasing its molecular mass. After the pure DNA solution was treated at 60 °C for 1 h (lane 3), both the intensity and mobility were the same as the untreated DNA solution, indicating no changes on the DNA structure. Increasing the temperature to 90 °C results in an increase in the mobility of the band (lane 5), which is believed to be caused by the denaturation of double-stranded DNA (dsDNA). Because of the low molecular mass and more negative charge, the single-stranded DNA (ssDNA) migrates more quickly. No bands are observed in lanes 4 and 6 corresponding to the treatment for 2 h at 60 and 90 °C, respectively. There exist two possible explanations to the absence of DNA bands. One is that DNA molecules have been divided into many extremely short ones because of the cleavage of the phosphodiester bonds in the backbone under the high temperature and pressure in the system. Then the shorter DNA migrated so quickly that they passed through the gel and entered the buffer solution during the period for electrophoresis for about 1 h. The other is that the DNA molecules are decomposed into many small molecules, such as bases, sugars, etc. DNA could not be detected in the both cases. The two cases can support the degradation of DNA under our experimental conditions.

Amino groups possess weak reducing property, which could be used to reduce metal ions to prepare nanomaterials (Yamamoto et al. 2006). In our experiment, TE-containing amino groups serves as reducing agents under high temperature and pressure to reduce silver ions, resulting in the generation of metallic silver. Thus, the possible reaction in our experiments is shown as follows:
$$ {\text{AgNO}}_{3} \mathop{\longrightarrow}\limits_{{\text{Hydrothermal}}\;{\text{synthesis}}}^{{\text{Tris}}{\hbox{-}}{\text{EDTA}}}{\text{Ag}} \downarrow $$
(2)
As is well known, DNA is a linear polynucleotide chain, which exhibits double-stranded helical structure hybridized by two single strands, with a width of 2 nm and a length of 0.34 nm per nucleoside subunit. The joint of the two single strands is caused by the hydrogen bonds between A and T, C and G, and every subunit is connected with each other by phosphodiester bond. DNA is generally stable. However, when the temperature is increased to about 90 °C, DNA will be denatured. Subsequently the DNA will be decomposed into two single strands. It often takes only 1 min to complete the denaturation (Li et al. 2005). If the pressure reaches a very high value, then the long chain will be divided into many shorter ones because of the cleavage of the phosphodiester bond in the backbone. In addition, DNA has a high affinity for metal cations due to the two classes of binding sites: negatively charged phosphate groups and aromatic bases.
From the above discussion, we supposed the possible mechanism of the transition from silver nano-necklaces to nanoplates in DNA/TE solution. The schematic representation is shown in Fig. 6. After being added into the DNA solution, silver ions were absorbed on DNA chains via electrostatic forces. The molar ratio of s/b in our typical experiments is 10:1, guaranteeing saturation of all the possible DNA-binding sites (Berti et al. 2005). Subsequently, the denaturation of DNA and the redox reaction (2) might occur. The mixed solution may experience two different cases according to the different temperatures. When the mixture was treated with the temperature no <90 °C, DNA molecules would be immediately denatured and divided into two single strands. Then, with the increase of reaction time the redox reaction (2) takes place. If the reaction temperature is <90 °C, the redox reaction (2) might occur before the denaturation of DNA, resulting from that at the reaction time of 1 h the redox reaction (2) has taken place, while the denaturation has not occurred (Lane 3 in Fig. 5). Thus, the silver nanostructures obtained at no less than 90 °C might use the ssDNA as template, while the template used at <90 °C is the dsDNA. As shown in both Fig. 1d, g, there are also some discontinuous points in the nano-necklaces. As a result, parts of backbone were still bare in the solution. With the increase of reaction time, the pressure in the system was very high, then DNA might be degraded, resulting in the generation of shorter nano-necklaces. Then, more formed silver nanoparticles nucleated homogeneously on the surface of the silver nano-necklaces. Ultimately the triangular and hexagonal nanoplates were formed as seen in Fig. 1e, f, h, i.
Fig. 6

Schematic illustration of the formation of silver nanostructure

Two interesting phenomena must be noted here. One is that the size of silver nanostructures templated by ssDNA chain is smaller than that obtained by dsDNA. It is because that the ssDNA can provide more nucleation sites to combine silver ions owing to the weaker steric hindrance and more electrostatic adsorption sites. Then, the nucleation rate will be faster than the growth rate. Therefore, the size is smaller than that obtained by dsDNA. The other is the transition from nano-necklaces to nanoplates. According to the XRD analysis and previous publication (Germain et al. 2003), the (111) faces are the basal planes, while the (110) faces are the sidewalls of nanoplates as the terminated planes. Many metals with cubic structure have an equilibrium shape dominated by (111) faces and (110) faces indicating that these faces have the lowest energies. Here, after the generation of shorter nano-necklaces the higher surface energy faces, the (110) side faces, are prone to arrest the silver atoms, and the smaller plates could assemble together along the (110) boundary, forming the larger nanoplates which are rich in (111) faces. Then, the transition from nano-necklaces to nanoplates was completed.

Conclusions

We succeeded in bringing hydrothermal synthesis into DNA nanomaterials and realizing the transition from silver nano-necklaces to nanoplates based on the degradation of DNA. It might provide a novel method to prepare nanomaterials. A mechanism hypothesis was formulated. Further, the method can be extended to other metals. Thus, further studies have been currently undertaken in our lab.

Acknowledgments

This study is supported by the National Science Foundation of China (Grants 20871001, 20671001, and 20731001), the Research Foundation for the Doctoral Program of Higher Education of China (20070357002), the Important Project of Anhui provincial Education Department (Grant ZD2007004-1), the Key Laboratory of Environment-friendly Polymer Materials, and Functional Material of Inorganic Chemistry of Anhui Province.

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.School of Chemistry and Chemical EngineeringAnhui UniversityHefeiPeople’s Republic of China
  2. 2.State Key Laboratory of Coordination ChemistryNanjing UniversityNanjingPeople’s Republic of China

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