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Large-scale preparation of strawberry-like, AgNP-doped SiO2 microspheres using the electrospraying method
This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2011 Nanotechnology 22 305307 (http://iopscience.iop.org/0957-4484/22/30/305307) View the table of contents for this issue, or go to the journal homepage for more
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IOP PUBLISHING
NANOTECHNOLOGY
Nanotechnology 22 (2011) 305307 (10pp)
doi:10.1088/0957-4484/22/30/305307
Large-scale preparation of strawberry-like, AgNP-doped SiO2 microspheres using the electrospraying method Zhijun Ma1 , Huijiao Ji2 , Dezhi Tan1 , Guoping Dong3,4 , Yu Teng1 , Jiajia Zhou1 , Miaojia Guan1 , Jianrong Qiu1,3,4 and Ming Zhang2 1
State Key Laboratory of Silicon Materials, Zhejiang University, Hangzhou, Zhejiang 310027, People’s Republic of China 2 College of Life Science, Zhejiang University, Hangzhou 310058, People’s Republic of China 3 Key Laboratory of Specially Functional Materials of Ministry of Education and Institute of Optical Communication Materials, South China University of Technology, Guangzhou, Guangdong 510640, People’s Republic of China 4 Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, Qinghe Road 390, Jiading District, Shanghai 201800, People’s Republic of China E-mail:
[email protected] and
[email protected]
Received 23 March 2011, in final form 15 June 2011 Published 1 July 2011 Online at stacks.iop.org/Nano/22/305307 Abstract In this paper, we report on a novel strategy for the preparation of silver nanoparticle-doped SiO2 microspheres (Ag-SMSs) with an interesting strawberry-like morphology using a simple and efficient electrospraying method. SEM (scanning electron microscopy), TEM (transmission electron microscopy), XRD (x-ray diffraction), EDS (energy-dispersive spectroscopy) and UV–vis spectra (ultraviolet–visible spectra) were applied to investigate the morphology, structure, composition and optical properties of the hybrid microspheres, and E. coli (Escherichia coli) was used as a model microbe to evaluate their antibacterial ability. The results showed that the Ag-SMSs were environmentally stable and washing resistant. The Ag-SMSs exhibited effective inhibition against proliferation of E. coli, and their antibacterial ability could be well preserved for a long time. The environmental stability, washing resistance, efficient antibacterial ability and simple but productive preparation method endowed the Ag-SMSs with great potential for practical biomedical applications. (Some figures in this article are in colour only in the electronic version)
have been devoted to SiO2 as a matrix for stabilizing Ag NPs, due to its relative ease of preparation, hydrophilic nature, physical and chemical stability and good biocompatibility, etc. Diverse patterns of AgNP–SiO2 composites, including bulk, film, fiber and nano/microspheres, have been extensively studied [4, 9–11], wherein the nano/microspheres possess more widespread applications, such as cosmetic, pigment, SERS (surface-enhanced Raman scattering) substrate and antibacterial reagent, etc. Investigations on the preparation of AgNP–SiO2 nano/ microspheres have mainly focused on two types. In the first type, Ag NPs are first synthesized, then coated by a
1. Introduction The potent and broad-spectrum antibacterial effect of Ag nanoparticles (NPs) has been well established for a long time [1–3]. Recently, the use of Ag NPs as antibacterial reagent in diverse biomedical applications was revived, due to technical advances in the fabrication of Ag NPs [2]. However, for practical biomedical applications, Ag NPs usually have to be integrated with other materials that act as a stabilizing matrix in order to avoid undesired coagulation and/or oxidation, which would degrade or even remove their efficacy as antibacterial reagents [4–8]. Vast investigations 0957-4484/11/305307+10$33.00
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layer of SiO2 shell with tunable thickness through a modified St¨ober method [12–15]. These core–shell AgNP–SiO2 nano/microspheres can effectively circumvent the coagulation and/or oxidation problems, and thus are quite stable. In addition, the unique properties of the SiO2 shell also can endow the material with novel surface properties and new functionalities. However, for antibacterial application, their biocidal function is carried out through slow release of the silver ions [15]. As a result, the potent biocidal ability of the Ag NPs cannot be put into full play, as their biocidal function is mainly through direct contact with microorganisms [2]. In the second type, Ag NPs are grafted onto the SiO2 sphere through chemical coupling or physical adsorption [16–21]. In antibacterial applications, the Ag NPs can directly contact with the microorganisms, thus fierce and immediate sterilization can be achieved. Nevertheless, due to the relatively weak binding between the Ag NPs and the SiO2 sphere, this kind of material may release the Ag NPs relatively rapidly. As a result, it is hard to obtain a long-term antibacterial effect with this material. In practical biomedical applications, usually both fierce and immediate sterilization and long-term antibacterial effect are required. For example, in the treatment of wounds, immediate sterilization is demanded in the initial stage and relatively gentle but long-term antibacterial effect is required during the whole recovery stage to avoid further infections. Consequently, neither of the above-mentioned two kinds of AgNP–SiO2 nano/microspheres is ideal for practical antibacterial applications. A new strategy has to be proposed to design AgNP–SiO2 nano/microspheres with better performance. For the preparation of nano/microspheres, electrospraying is thought to be a versatile method. Compared with other methods, electrospraying is simpler, more efficient, more flexible and less demanding for the fabrication environment [22]. Some groups have also reported that its efficiency can be further improved through multi-nozzle or nozzle-less spraying techniques [23, 24]. In addition, with elaborate design of the capillary nozzle [25, 26] and proper compositional adjustment of the precursor sol [27, 28], this method also possesses high flexibility on morphology control over products. Core–shell structured particles or even products with complex morphology can be fabricated with the electrospraying technique. Here, we report on a novel design for the preparation of Ag NP-doped SiO2 microspheres (Ag-SMSs) as an antibacterial reagent using the electrospraying technique. Ag NPs are not only doped inside but also embedded on the surface of the SiO2 microspheres, making the samples appear like strawberries. These different loading styles of Ag NPs endow the sample with both fierce and immediate biocidal ability and long-term antibacterial effect.
3.8 wt%) was added drop wise. The solution was hydrolyzed in an oil bath pot under 80 ◦ C for 2 and a half hours to obtain spinnable SiO2 sol. x g AgNO3 (silver nitrate, >99.8 wt%) (x = 0.1, 0.5 and 0.7) was dissolved in 3 ml mixed solution of ethanol and acetonitrile (2 ml ethanol and 1 ml acetonitrile), and the obtained solution was mixed with the SiO2 to introduce the silver source. A home-made electrospraying setup with an aluminum plate as the collector was used for electrospraying. The applied voltage, feeding rate and collecting distance (distance from the tip of the capillary to the collector) were set to be 15 kV, 0.2 ml h−1 and 20 cm, respectively. The whole electrospraying process proceeded under room temperature (25 ◦ C) and room humidity (60%). After 2 h of collection, the samples were scraped off and dried at room temperature for 24 h, and then annealed in a reducing atmosphere (5% H2 , 95% N2 ) at 400 ◦ C for half an hour, in order to convert the ionized silver to Ag NPs. Samples prepared with the SiO2 sol containing x g AgNO3 were named as x Ag-SMSs. 2.2. Sample characterization SEM (scanning electron microscopy) and TEM (transmission electron microscopy) images were recorded with a Hitachi S-4800 scanning electron microscope and a CM200UT transmission electron microscope, respectively; XRD (xray diffraction) and EDS (energy-dispersive spectroscopy) patterns were tested using a Rigaku D/MAX 2550/PC polycrystalline x-ray diffractometer and an EMAX 350 energy dispersive spectrometer; UV–vis spectra were measured with a U-4100 spectrophotometer; the XPS (x-ray photoelectron spectrum) was tested with a Kratos AXIS Ultra DLDS Xray Photoelectron Spectroscope; statistical size distributions of the 0.5Ag-SMSs and its Ag NPs were collected with Nano Measure software according to the SEM and TEM images, respectively. To evaluate the antibacterial ability of the sample, inhibition zones against proliferation of E. coli were tested. Briefly, 100 μl E. coli culture suspension in logarithmic phase diluted to OD (optical density) 0.500 was added to the surface of a 10 cm glass dish containing 10 ml congealed agarose LB medium (composed of 10 g tryptone, 5 g yeast extract, and 5 g NaCl l−1 ). 0.1 g x Ag-SMSs (x = 0.1, 0.5, 0.7) were patterned into small discs with a diameter of 2 cm, then the small discs were immediately overlayed on the medium/bacterial surface. The dish was incubated at 37 ◦ C overnight. A bare sample (without loading of Ag NPs) was used as control (denoted as ‘con’). In order to evaluate the stability of the antibiotic effect of the Ag-SMSs, the inhibition zone of 0.5Ag-SMSs after being stored in air for two months was also investigated. To investigate the inhibition effect on the bacterial growth dynamic, E. coli strain DH5α culture suspension in logarithmic phase diluted to OD 0.500 was added to 5 ml liquid LB medium in glass tubes. Graded concentrations of 0.5Ag-SMSs were added into the tubes. The tubes were incubated in a 200 rpm shaker at 37 ◦ C. The absorbance at 600 nm wavelength was measured at selected time intervals with an Eppendorf Bio Photometer.
2. Experimental section 2.1. Sample preparation To prepare SiO2 sol, 30 ml TEOS (tetraethoxysilane, >98 wt%) was mixed with 30 ml EtOH (ethanol, >99.7 wt%) through magnetic stirring, then 4.5 ml HNO3 (nitric acid, 2
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Figure 2. Magnified SEM image of 0.5Ag-SMSs.
Figure 1. (a), (b) SEM and TEM images of 0.5Ag-SMSs; (c) HRTEM image of one single Ag NP that was embedded on the surface of a SiO2 microsphere.
Ag NPs on the SiO2 microsphere is beneficial for stabilization of the Ag NPs, which is vital for many applications. For example, when applied as antibacterial reagents, embedding of the Ag NPs will make the Ag-SMSs more washing resistant, thus providing a sustained antibacterial effect. Ordered onedirectional arranged crystal fringes can be observed on the whole Ag NP, indicative of a monocrystalline structure. The distance between adjacent lattice fringes was measured to be about 0.24 nm, corresponding to the (111) face of cubic silver crystal. Figure 2 is a magnified SEM image of 0.5Ag-SMSs. In this figure, the strawberry-like morphology of the sample can be observed more clearly. The Ag NPs of the Ag-SMSs are not only embedded on the surface, but also incorporated inside the SiO2 spheres. The decoration densities of Ag NPs on the surfaces of SiO2 spheres for individual Ag-SMSs, as a matter of fact, are very different from one another, as shown in figure 3(a). Some very small parts of the spheres are almost bare on their surfaces, with only very low decoration density of Ag NPs. Figures 3(b) and (c) show TEM images of two individual 0.5Ag-SMSs. By comparing these two images, we discover that the sphere in figure 3(b) has a lot of bulging-out Ag NPs along its rim, while the one in figure 3(c) almost has no such Ag NPs on its rim. However, one can see that the total Ag NP densities of the two spheres are similar, both are highly populated. So we assume that, besides the surface-embedded Ag NPs, there are a lot of Ag NPs inside the SiO2 spheres. The spheres in figures 3(b) and (c) just correspond to the white- and red-circled spheres in figure 3(a). We made a linear chemical scan across the 0.5AgSMS in figure 3(c) (the scanning route is shown by the green line in figure 3(c)), and found that the distribution content of silver increases with the thickness of the sphere, as shown in figure 3(d). This proves that most of the Ag NPs are doped inside the SiO2 spheres (otherwise, the distribution curve of the silver content would be parallel with the position axis). In order to further determine the chemical state of silver in the 0.5AgSMSs, we tested the sample’s XPS spectrum. Figure 4 shows the deconvoluted XPS spectrum of the Ag(3d) peaks for 0.5AgSMSs. The peaks at about 368.7 and 374.6 eV correspond to the Ag3d5/2 and Ag3d3/2 binding energies of the metallic Ag NPs. Besides the characteristic peaks of metallic silver, peaks at lower binding energies can also be observed, which should be ascribed to partial oxidation of the Ag NPs on the surfaces
The antibacterial efficiency of the Ag-SMSs was investigated using E. coli suspension. Gradient concentrations of 0.5Ag-SMSs were added when the initial bacteria concentration was set to be 1 × 108 cells ml−1 . The obtained mixtures were then spread uniformly in Petri dishes, and then the dishes were sealed and incubated at 37 ◦ C for two days. The surface spread-plate method was applied to measure the cell counts of the E. coli with viability. The cytotoxicity of the Ag-SMSs was investigated with BMSCs (bone mesenchymal stem cells) as the model human cells. Briefly, BMSCs were cultured in H-DMEM medium (Invitrogen, USA) with 15% fetal bovine serum (SiJiQing, HangZhou) at 37 ◦ C in a humidified atmosphere with 5% CO2 . The cells were seeded in 96-well plates at a density of 8 × 103 cells cm−2 and grown overnight, then incubated with fresh media containing gradient concentration of 0.5Ag-SMSs (from 20 to 200 μg cm−2 ). After four days of culture, 20 μl MTT solution (thiazolyl blue tetrazolium bromide, 10 mg ml−1 , Sigma-Aldrich,USA) was added to each well, then the plate was incubated at 37 ◦ C for 4 h. Finally, the cells were lysed using DMSO (Sigma, USA). A microplate reader (Bio-Rad 680, USA) was applied to monitor the absorbance of the supernatants at 570 nm. This experiment was repeated three times.
3. Results and discussion Figure 1(a) is an SEM image of 0.5Ag-SMSs. As shown, small dimples filled with Ag NPs (relatively white small particles) are distributed fully over the surface of the SiO2 microspheres, making the sample appear like strawberries. Figure 1(b) shows a TEM image of the 0.5Ag-SMSs. As can be seen, the exposed Ag NPs (the relatively dark particles) are actually partly embedded on the surface of the SiO2 microspheres. Figure 1(c) shows an HRTEM (high-resolution TEM) image of one single Ag NP on the surface of a SiO2 microsphere (the one white-circled in figure 1(b)). It can be seen clearly that half of the Ag NP is embedded rather than simply grafted onto the surface of the SiO2 microsphere. Such a loading style of 3
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Figure 3. (a) SEM image of 0.5Ag-SMSs; (b) TEM image of one single 0.5Ag-SMS with a lot of Ag NPs embedded on the surface, as white-circled in (a); (c) TEM image of one single 0.5 Ag-SMS almost without decoration of Ag NPs on the surface, as red-circled in (a); (d) distribution curve of silver content along the scan line as illustrated by the green line in (c).
randomly selected 0.5Ag-SMSs according to the TEM images. Figures 5(a) and (b) present a low-magnified SEM image and the size distribution of the 0.5Ag-SMSs, respectively. As shown, the size distribution of the sample has a wide range: most of the spheres have a diameter smaller than 1.6 μm, while some very small portion are larger than 2 μm. The inset in figure 5(b) shows the size distribution of the Ag NPs of 0.5AgSMSs. The size of the Ag NPs is still not so uniform, spanning from 12 to 32 nm. The average diameter of the 0.5Ag-SMSs and mean size of the Ag NPs were calculated to be 0.95 μm and 22.3 nm, respectively. During preparation of the precursor sol, we observed that the colorless SiO2 sol immediately became yellow after addition of the silver nitrate solution. The yellow color became darker and darker along with increase of the silver nitrate concentration, as shown in figure 6(a). By measuring the UV–vis spectra of the yellow Ag–SiO2 sol, the characteristic absorption peak of Ag NPs was detected, and the peak center shifted from 411 to 422 nm, along with increase of the silver nitrate concentration, as shown in figure 6(b). Consequently, it can be deduced that, at least, some of the Ag NPs were formed in the initial sol-preparation stage, and the peak redshift and intensity increase of the UV–vis spectra imply that both the mean size and quantity of the Ag NPs increased with the increase of silver nitrate concentration. The formation of the Ag NPs in the precursor SiO2 sol should be ascribed to the reduction of the silver ions by ethanol [32]. In the electrospraying process, Ag NPs together with the SiO2 sol are
Figure 4. XPS pattern of 0.5Ag-SMSs. The black line shows the experimental data and the green lines are the fitted results. The red curve is the stacking result of the green lines.
of the Ag-SMSs [29–31]. We estimated the molar ratios of metallic and ionized silver to be 34% and 66%, respectively. Considering that the analyzing depth for inorganic materials of the XPS technique is less than 4 nm, we can speculate that the oxidation of the Ag NPs on the sphere surface is trivial. We measured the diameter of 410 0.5Ag-SMSs according to the SEM image and the size of 200 Ag NPs on ten 4
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Figure 6. SiO2 sol with the addition of different concentrations of silver nitrate. (a) Digital images of SiO2 sol with the addition of different concentrations of silver nitrate ((a1) 0.1 g; (a2) 0.5 g; (a3) 0.7 g); (b) corresponding UV–vis spectra.
Figure 5. (a) SEM image of 0.5Ag-SMSs with low magnification; (b) statistical analysis of the size distribution of the 0.5Ag-SMSs (by investigating 410 pieces of spheres); the inset in (b) shows the size distribution of the Ag NPs on 0.5Ag-SMSs (by investigating in total 200 pieces of Ag NPs on ten randomly selected 0.5Ag-SMSs).
Figures 7(a1)–(a3) are TEM images of 0.1Ag-SMSs, 0.5Ag-SMSs, and 0.7Ag-SMSs, respectively. As can be seen, along with the increase of silver nitrate in the precursor SiO2 sol, both the population and size of the Ag NPs in the Ag-SMSs increased rapidly. According to the TEM images, the mean diameters of the Ag NPs for the three samples were measured to be about 6.63 nm, 22.3 nm and 25.8 nm, respectively.
ejected out. Some of the Ag NPs will be near the surface or emerge out of the SiO2 jet. Due to the surface tension, the SiO2 sol around the Ag NPs will become concave. After complete solidification of the jet, the concave state of the SiO2 sol around the Ag NPs is preserved, and small dimples filled with Ag NPs are formed, thus giving birth to the strawberry-like morphology of the Ag-SMSs.
Figure 7. (a1)–(c1) TEM images of 0.1Ag-SMSs, 0.5Ag-SMSs and 0.7Ag-SMSs; (a2)–(c2) XRD patterns of 0.1Ag-SMSs, 0.5Ag-SMSs and 0.7Ag-SMSs; (a3)–(c3) EDS patterns of 0.1Ag-SMSs, 0.5Ag-SMSs and 0.7Ag-SMSs.
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Figure 8. Environmental stability comparison of 0.5Ag-SMSs and Ag NPs. UV–vis spectra of: (a) freshly prepared Ag NPs (black line) and Ag NPs stored in the dark (red line) and in light conditions (blue line) for one week; (b) freshly prepared 0.5Ag-SMSs (black line) and 0.5Ag-SMSs stored in the dark (red line) and in light conditions (blue line) for two months.
Figure 9. Washing resistance comparison of 0.5Ag-SMSs and SiO2 microspheres grafted with Ag NPs. UV–vis spectra of (a) 0.5Ag-SMSs before (black line) and after (red line) ultrasonic washing; (b) SiO2 microspheres grafted with AgNPs before (black line) and after (red line) ultrasonic washing.
substantial decrease of the peak intensity, when either stored in the dark or in light conditions for one week. Here, the redshift of the peak should originate from aggregation of the Ag NPs, while the intensity decrease should be ascribed to their oxidation. Light played an important role in the oxidation or aggregation of the Ag NPs. Consequently, the spectral change of the Ag NPs stored in light conditions was more significant. Parallel investigation shows that the Ag-SMSs (the sample used here was 0.5Ag-SMSs) prepared in this investigation are quite stable, little change happened to their UV–vis spectra, no matter whether stored in the dark or in light conditions even for two months, as shown in figure 8(b). The high stability of the Ag-SMSs should be ascribed to the spatial distribution and impregnation of the Ag NPs, which anchors the Ag NPs, prevents their aggregation and reduces their oxidation, now the embedded-on-surface Ag NPs are partly buried and the doped-inside Ag NPs are totally incorporated in the SiO2 spheres. For SiO2 microspheres with Ag NPs loaded on the surface, being resistant to external force or influence, for example, washing or shaking, is very important for many applications, such as being applied as antibacterial reagents [34, 35].
Figures 7(a2), (b2) and (c2) are the corresponding XRD patterns. The peaks at about 38.3◦ , 44.3◦ , 64.5◦ and 77.5◦ are ascribed to the diffractions from the (111), (200), (220) and (311) faces of cubic silver nanocrystals [18], and their intensities increased sequentially, indicative of a quantitative increase of Ag NPs. Besides the diffraction peaks of metallic Ag NPs, no characteristic peaks from silver salts, such as Ag2 O or AgO, can be discerned, indicating that the silver of the Ag-SMSs mainly exists in the form of metallic Ag NPs. Figures 7(a3), (b3) and (c3) are EDS patterns of 0.1Ag-SMSs, 0.5Ag-SMSs, and 0.7Ag-SMSs. Characteristic peaks of silver can be discerned in the EDS patterns, and the contents of silver in the three samples were measured to be 0.1 wt%, 0.6 wt% and 0.8 wt%, respectively. Pure Ag NPs without stabilizing matrix easily become oxidized and/or aggregate in air, leading to degradation or even loss of their functionality, especially when applied as antibacterial reagents [7, 33]. Figure 8(a) shows the UV–vis spectra of pure Ag NPs dispersed in deionized water (these Ag NPs were obtained by centrifuging the precursor SiO2 sol with the addition of 0.5 g silver nitrate). The characteristic peak at 420 nm of Ag NPs moved to longer wavelength with 6
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Figure 10. (a), (b) SEM images of 0.5Ag-SMSs before and after fierce ultrasonic washing for 1 h; (c), (d) SEM images of SiO2 spheres with Ag NPs grafted on the surface before and after fierce ultrasonic washing for 10 min.
antibacterial effect of Ag-SMSs should be ascribed to Ag NPs. The above results have proved Ag-SMSs to be environmentally stable, thus their antibacterial efficacy is expected to be longlasting. It is shown that, after being stored for two months, the area of the inhibition zone of the 0.5Ag-SMSs did not decrease obviously (figure 12), indicating a long-lasting antibacterial effect of Ag-SMSs. A quantitative evaluation of the antibacterial effect was investigated by monitoring the growth curves of E. coli with treatment of gradient concentrations of 0.5Ag-SMSs. As in figure 13, the growth curves of E. coli showed a sensitive dose-dependent antibacterial effect of the sample. Along with the increase of the sample concentration, the lag phase of the growth curve was prolonged gradually, and was extended to longer than 12 h, when the sample concentration reached 200 ppm. When the concentration of 0.5Ag-SMSs reached more than 500 ppm, the growth of E. coli was absolutely inhibited in the whole incubation process, implying that the 0.5Ag-SMSs possess a long-lasting antibacterial ability. Figure 14 shows the antibacterial efficiency of the 0.5AgSMSs as a function of sample concentration. It is shown that the antibacterial efficiency increased rapidly with the increase of sample concentration. When the concentration of the 0.5AgSMSs reached 500 ppm, 100% antibacterial efficiency was obtained. According to the result of EDS, the content of Ag NPs in 0.5Ag-SMSs was about 0.6 wt%. Consequently, it can be estimated that the minimum concentration of Ag NPs for complete inhibition of E. coli was no more than 3 ppm. According to the loading styles of the Ag NPs on the AgSMSs and the parallel investigations from other groups, the antibacterial function of the sample should be realized in three ways. The first way is through direct contact of Ag NPs (which are embedded on the surface of the SiO2 microspheres) with the microbes. This is a fast and fierce biocidal process [2, 33].
As shown in figure 9(a), after fierce ultrasonic washing in deionized water for 1 h, the UV–vis spectrum of the 0.5AgSMSs only changed a little, the characteristic absorption peak at 420 nm shifted slightly to shorter wavelength with a tiny decrease of the peak intensity, which should be ascribed to the loss of a small number of relatively large Ag NPs on the surface. Compared with the 0.5Ag-SMSs, the control (SiO2 microspheres with Ag NPs merely grafted on the surface, its preparation method is the same as that in [17]) showed poor washing resistance. After only 10 min of ultrasonic washing, its UV–vis spectrum changed substantially with a drastic decrease of the peak intensity, due to a serious loss of Ag NPs, as shown in figure 9(b). SEM images of 0.5AgSMSs and the control before and after ultrasonic washing were also recorded to further exhibit the washing resistance of the sample. As shown in figure 10, the distribution density of the Ag NPs on the surfaces of the 0.5Ag-SMSs only decreased slightly. However, the loss of Ag NPs from the control was rather serious. By investigating 20 pieces of spheres, we found that the average numbers of Ag NPs on the surface per 0.5AgSMS were 178 and 163 for the cases before and after washing, and the corresponding numbers for the control were 166 and 58, respectively. Therefore, doping inside or embedding Ag NPs into the surface endowed the Ag-SMSs with great washing resistance. Here, the antibacterial effect of the Ag-SMSs was investigated to give an example of their practical applications. Figure 11 shows the inhibition zones of Ag-SMSs with different silver doses. Along with the increase of silver dose, the color of the samples darkened gradually, and the area of inhibition zones increased rapidly, indicating a silver-dosedependent antibacterial effect of the samples. In figure 11(a), the sample was pure SiO2 microspheres without Ag NPs, and no inhibition zone can be observed, confirming that the 7
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Figure 11. Inhibition zones of (a) bare SiO2 microspheres; (b)–(d) 0.1Ag-SMSs, 0.5Ag-SMSs and 0.7Ag-SMSs.
Figure 12. Inhibition zones of freshly prepared 0.5Ag-SMSs (left) and 0.5Ag-SMSs stored in the dark for two months (right).
The second way should be ascribed to the Ag NPs that are released from the inside of the SiO2 microspheres, due to gradual dissolving of the SiO2 microspheres. This is a relatively slow process [4, 36]. In the third way, a low concentration of silver ions can be released by the Ag NPs (both embedded on the surface and doped inside), due to the trivial but inevitable oxidation of the Ag NPs. This is an even slower and milder process [15, 37]. Such multiple antibacterial kinetics of the Ag-SMSs, as a matter of fact, is useful for some biomedical applications. For example, when used for treating trauma, the fast and fierce biocidal effect can cause immediate biocidal effect on the wounds, and the slow release of Ag NPs and Ag+ can provide sustained antibacterial effect, thus preventing infections during the whole regeneration process of the damaged skin. Figure 15 presents the result of cytotoxicity investigation of the Ag-SMSs. As shown, the 0.5Ag-SMSs had little
Figure 13. Bacteria growth curves with gradient concentrations of 0.5Ag-SMSs (0–1000 ppm).
influence on the proliferation and viability of BMSCs, when the concentration of the sample varied from 20 to 200 μg cm−2 (equivalent to 4000 to 40 000 ppm in the antibacterial efficiency test). It has been verified that the sample of 500 ppm concentration (equivalent to 0.5 μg cm−2 ) has 100% antibacterial efficiency. The viability of the cells was only slightly reduced, with no significance, even when the concentration of the 0.5Ag-SMSs reached up to 200 μg cm−2 . These results proved that the Ag-SMSs are biocompatible with human cells, and, at the same time, have effective antibiotic ability. 8
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Even after two months of storage, the antibacterial effect of the Ag-SMSs was still well preserved. The minimum inhibitory concentration of the Ag-SMSs was measured to be about 500 ppm, indicating that the antibacterial effect of the Ag-SMSs was very efficient. Considering their high environmental stability, washing resistance and simple but productive preparation method, Ag-SMSs may find great potential for practical biomedical applications.
Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Grant Nos 50872123 and 50802083), the National Basic Research Program of China (2006CB806000b), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT0651).
Figure 14. Antibacterial efficiency of gradient concentrations of 0.5Ag-SMSs (0–500 ppm).
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Figure 15. Cytotoxicity test of the Ag-SMSs. When the concentration of 0.5Ag-SMSs varied from 20 to 200 μg cm−2 , the sample showed no significant cytotoxicity to the proliferation and viability of the BMSCs.
4. Conclusion In summary, Ag NP-doped SiO2 microspheres (Ag-SMSs) were prepared using a simple and productive electrospraying method. Ag NPs were both doped inside and embedded on the surface of the SiO2 microspheres, making the AgSMSs appear like strawberries. Compared with pure Ag NPs or SiO2 microspheres with Ag NPs simply grafted on the surface, the Ag-SMSs are more environmentally stable and washing resistant, due to the unique loading style of the Ag NPs. In addition, the different loading styles of the Ag NPs also endowed the Ag-SMSs with a dual antibacterial effect (fast and fierce antibacterial effect from the Ag NPs on the surface and sustained antibacterial effect from the Ag NPs doped inside), which is beneficial for some biomedical applications. The mean size and the doping concentration of the Ag NPs were conveniently adjusted through variation of the silver nitrate concentration in the precursor SiO2 sol. Antibacterial investigation indicated that the Ag-SMSs had effective antibacterial ability against the proliferation of E. coli. 9
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