J Mater Sci: Mater Med DOI 10.1007/s10856-014-5165-9
Synthesis, characterization and bactericidal activity of silica/silver core–shell nanoparticles Pooja Devi • Supriya Deepak Patil • P. Jeevanandam • Naveen K. Navani M. L. Singla
•
Received: 3 September 2013 / Accepted: 27 January 2014 Ó Springer Science+Business Media New York 2014
Abstract Silica/silver core–shell nanoparticles (NPs) were synthesized by coating silver NPs on silica core particles (size *300 ± 10 nm) via electro less reduction method. The core–shell NPs were characterized for their structural, morphological, compositional and optical behavior using X-ray diffraction, scanning electron microscopy, energy dispersive X-ray analysis and UV– Visible spectroscopy, respectively. The size (16–35 nm) and loaded amount of silver NPs on the silica core were found to be dependent upon reaction time and activation method of silica. The bactericidal activity of the NPs was tested by broth micro dilution method against both Bacillus subtilis (gram positive) and Escherichia coli ATCC25922 (gram negative) bacterium. The bactericidal activity of silica/silver core–shell NPS is more against E. coli ATCC25922, when compared to B. subtilis. The minimal inhibitory concentration of the core–shell NPs ranged from 7.8 to 250 lg/mL and is found to be dependent upon the amount of silver on silica, the core. These results suggest that silica/silver core–shell NPs can be utilized as a strong substitutional candidate to control pathogenic bacterium, which are otherwise resistant to antibiotics, making them applicable in diverse medical devices.
P. Devi M. L. Singla (&) Academy of Scientific and Innovative Research, Central Scientific Instruments Organisation, Sector 30 C, Chandigarh 160030, India e-mail:
[email protected] S. D. Patil P. Jeevanandam (&) N. K. Navani Indian Institute of Technology Roorkee, Roorkee 247667, Uttarakhand, India e-mail:
[email protected] 1 Introduction Among colloids of metallic nanoparticles (NPs), silver NPs stand out as one of the most intensively investigated systems owing to their sharp plasmon band in visible region and very high extinction coefficient [1]. Furthermore, they possess unique properties such as excellent electrical conductivity, catalytic activity, nonlinear optical behavior and antimicrobial effects, making them potential candidate for applications in different areas such as sensors, textiles, electronics, catalysis, food, and healthcare [2, 3]. Silver salts have been known for their antimicrobial effects with a history dating back to antiquity [4] and are being used as a control agent in dental work, catheters, textiles, washing machines, water purifiers, burn wounds, etc. [5, 6]. However, silver NPs are reported to have better antimicrobial activity in comparison to silver salt and other metallic and semiconducting NPs [7–9]. The antimicrobial effect of silver is due to silver ions, which interact with disulfide or –SH groups of enzymes, causing structural changes that lead to disruption of metabolic processes followed by cell death [10]. In the case of NPs, due to increased surface area and associated increased potential for the release of silver, they may accumulate in the bacterial cytoplasmic membrane, causing a significant increase in permeability and cell death [8, 11]. The bactericidal activity of silver NPs is dependent on the size, shape and the amount of silver NPs [12, 13]. To further improve their antibacterial efficiency, dispersibility in other matrices like polymers, textiles, synthetic fibres and other thin film materials etc., hybrids of silver NPs with other materials are investigated extensively to cut down the application cost. For example, Egger et al. [14] have shown that Ag NPs dispersed in amorphous silica produced using an industrial flame spray pyrolysis process exhibit effective
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antimicrobial properties; however, the size of matrix used was very large *1,000 nm. Akhavan et al. [15] studied the bactericidal effect of Ag NPs immobilized on the surface of SiO2 thin films. Mahltig et al. [16] have reported the antibacterial activity of Ag and silica coated Ag sols impregnated into textiles. They have also shown that the coating of crystalline silver with silica improves the adhesion of particles in textile fabric. Additionally, Jasiorski et al. [17] have investigated the antimicrobial activity of textiles doped with silver in different forms. They have reported that the textiles only doped with SiO2/Ag spheres are bacteriostatically active in comparison to silver salt and silver NPs. Also, composite of Ag2S with silica has been demonstrated to show antifungal activity by Fateixa and co-workers [18]. Silica particles, used as a carrier as well as core for antimicrobial agents, are known to have high surface area, which enables loading of large amounts of antimicrobial materials and slow release of the bactericidal agents over an extended time period. In addition, silica tends to provide good stability against coagulation due to a very low value of Hamaker constant, which defines the Vander Waals forces of attraction among the particles and the medium [19]. To the best of authors’ knowledge, up to now silica core particles coated with silver, independently, have not been investigated systematically for their bactericidal behavior. Motivated by this need, we have synthesized silver coated silica NPs by electro-less reduction method and performed a systematic series of experiments to test their bactericidal behavior with respect to different loading amount of silver. The silica/silver core–shell NPs were probed using X-ray diffraction (XRD), infrared and UV– visible spectroscopy and thereafter their antibacterial activity was tested against Bacillus subtilis (gram positive) and Escherichia coli ATCC25922 (gram negative) by broth micro dilution method.
2 Materials and methods All the chemicals were used as received, without further purification: silver nitrate (SISCO Research Laboratories Pvt. Ltd.), tetraethylorthosilicate (98 %, Sigma Aldrich), NaOH, Ammonia solution (25 %, RFCL Limited), SnCl2, Sn powder (HIMEDIAÒ), formaldehyde (S D Fine Chem. Limited), HCl (THOMAS BAKER Chem. Pvt. Ltd.), ethanol (Jigansu Hauxi International Trade Co. Ltd.) and Mueller–Hinton Broth (Merck, Germany). All glassware used for the solution synthesis were first rinsed with chromic acid solution, then thoroughly with Millipore deionized water, and dried in an oven before use. Antibacterial activity was tested in 96 well plates (Genaxy, India).
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Table 1 Surface activation methods for four different samples set Sample
Activation method
s1
No activation
s2
2 % NaOH
s3
Calcination at 450 °C
s4
Calcination at 750 °C
A Hitachi FE-SEM (S-4300) equipped with an X-ray analyzer was employed for morphological and elemental studies. Powder XRD patterns of the samples were recorded at room temperature using Bruker powder X-ray diffractometer operated at 30 kV (Cu Ka radiation, 40 mA, ˚ ). FT-IR analysis was done using Varian FTk = 1.5406 A IR spectrophotometer and optical absorption studies were performed using Hitachi UV–visible spectrophotometer at CSIR-CSIO. 2.1 Synthesis of silica/silver core–shell NPs Silica spheres, which acted as the core, were synthesized by hydrolysis and condensation of TEOS in an alkaline medium. In a typical experiment, TEOS (3.6 mL) was added to ammonium hydroxide (11.9 mL) containing ethanol (88 mL) solution and stirred for 15 h. Various reaction conditions such as precursor concentration, type of solvent, reaction time and temperature were controlled to vary the size of resultant silica NPs, as reported in our previous work [20]. In order to coat Ag NPs on these silica spheres, a simple electro-less reduction method is used [21]. Initially, four different samples set namely s1, s2, s3 and s4 were prepared by varying the activation method for silica spheres as described in Table 1. These activated particles were then added to a mixture (50 mL) containing SnCl2 (0.053 mol/L), HCl (0.01 mol/L), and Sn powder (20 mg). The colloids were later rinsed, collected and then incubated into ammoniacal silver solution (50 mL) for varying period of time (2–4 h). The above solution was rinsed again with de-ionized water and added into a mixture of formaldehyde (0.025 M), silver nitrate (0.05 M) and ethanol. The mixture was stirred under N2 environment for 24 h. 2.2 Antimicrobial activity of silica/silver core–shell NPs The antibacterial activity of silica/silver core–shell NPs was studied by broth micro dilution method against a gram negative and gram positive representative namely E. coli and B. subtilis, respectively. The NPs were added in a series of dilutions in Mueller–Hinton (MH) broth to 96-well plates to make a final volume of 100 lL. Then, 100 lL of MH broth containing approximately 105 cfu/mL
J Mater Sci: Mater Med Table 2 Crystallite size calculation for the silver NPs coated on silica spheres activated by different methods
Sample
s1
s2
Activation method
No activation
2 % NaOH
Incubation time (h)
FWHM (b) (°)
(Rad)
3
0.2,487
4.336 9 10-3
34.20
2
0.51
8.90 9 10-3
16.6
3
0.369
6.445 9 10-3
23.01
4
0.337
5.8,817 9 10-3
25.20
-3
32.97
s3
Calcination at 450 °C
3
0.2,576
4.4,959 9 10
s4
Calcination at 750 °C
3
0.3,374
5.88 9 10-3
of bacterial cells in their log phase was added to these dilutions. To prepare cells, E. coli and B. subtilis were grown overnight in 10 mL of MH broth. Overnight cultures in MH broth were subcultured (1 % inoculum) in fresh medium and incubated further till optical density at 600 nm (OD600) reached 0.5–0.7. This culture was then diluted and standardized by OD600 readings to approximately 105 cfu/ mL and then 100 lL of this diluted culture was added to each well of the 96-well plate to make a final volume of 200 lL. Silica particles alone were taken as negative control and chloramphenicol as positive control for bacterial inhibition. The plates were incubated at 37 °C in a shaking incubator with 100 rpm shaking to prevent the NPs from settling down at the bottom of the wells. Optical density at 600 nm was recorded before and after incubation using Spectramax Plus Plate Reader (Molecular Devices, USA) at IIT Roorkee. MIC was deduced as the lowest concentration of NPs at which the growth of the tested bacteria was inhibited completely after 12 h of incubation at 37 °C. The MIC for each bacterium was determined in triplicates.
3 Results and discussion Figure 1a shows the XRD patterns of silica and silica/silver NPs, while Fig. 1b corresponds to the XRD spectra of different samples set namely s1, s2, s3 and s4 (Fig. 1b). The presence of single broad diffraction peak at 2h = 25° for silica NPs reveals the amorphous nature of the silica, while the appearance of three distinct diffraction peaks for silica/silver at 2h values of 38.1°, 44.3°, and 64.4° which correspond to (111), (200) and (220) crystalline planes, respectively of the cubic Ag (JCPDS No. 4-0783), confirms the coating of silver NPs onto silica core surface. Furthermore, powder diffraction patterns of different samples set shown in Fig. 1b, also confirmed the presence of silver for all activation methods used. However, the crystallite size of coated silver is observed to be dependent upon the surface activation method and incubation time in ammoniacal silver salt solution for growth step, as summarized in
Crystallite size (nm)
25.18
Table 2. The mean crystallite size of silver NPs was calculated by Debye–Scherrer equation using the FWHM of the (111) reflections and is found in the range of 16.0–35 nm. Figure 2 is associated with the scanning electron microscopy (SEM) images of synthesized silica particles at lower (6,0009) (Fig. 2a) and higher (15,0009) (Fig. 2b) magnification. The images show that the particles are spherical and highly monodispersed in nature with a size range *300–350 nm. These particles were later calcined at 450 and 750 °C in an electric furnace. When calcined at 450 °C, no significant change in morphology of silica spheres was observed. However, when calcined at 750 °C, agglomeration of particles was observed, which could be seen from their SEM images [ESI]. This can be explained by the fact that calcination at higher temperature leads to the removal of most of the –OH groups present on the surface of silica [22]. Figure 3 shows the SEM images and respective EDXA analysis of these four different samples set (s1–s4). It is evident from SEM images that the morphology of the particles is not altered even after coating with silver NPs and the particles retain their spherical morphology. Point and area EDXA analysis of samples further confirms the coating of silver NPs on silica core. However, it can be observed from EDXA analysis of all samples set, that they have varying amount (wt%) of loaded silver on the silica core as shown in Table 3, which can be attributed to the variation in the surface activation method of silica core particles. This is because the activation method affects the number of activated –OH groups present on the surface of silica spheres. When, activation is done with 2 % NaOH, it dissolves the uppermost layer of silica spheres and exposes more number of functional –OH groups in comparison to no activation approach. Additionally, activation of silica surface by heating at high temperature (*750 °C) reduces the number of –OH groups on surface, and hence the silver deposition as summarized in Table 3. Also, the influence of incubation time, during growth step, on the silver weight percent is deduced. With increase in time, the silver weight percent as well as the size of silver NPs increases, which
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J Mater Sci: Mater Med Fig. 1 Powder diffraction spectra of a silica and silica/ silver NPs and b sample sets s1, s2, s3, and s4
Fig. 2 SEM images of silica NPs at a lower magnification (96,000), b higher magnification (915,000)
can be explained by the fact that with increase in deposition time, the size of seed particles on silica spheres increases, which further grow to form a uniform layer of silver NPs around the silica spheres. Figure 4 presents the effect of surface activation method (Fig. 4a) and incubation time of activated spheres in ammoniacal silver salt solution during growth step (Fig. 4b), onto optical behavior of silica/silver core–shell NPs. Activation method affects the number of surface –OH groups and hence the silver loading on the silica spheres. Silica NPs themselves don’t show surface plasmon resonance phenomenon, because the dielectric function of the silica has no imaginary part and is almost constant in the wavelength range (300–800 nm). In addition silica is transparent in UV–Visible range due to its high band gap. On the other hand, silver NPs coated silica spheres show a surface plasmon peak at about *400 nm, which is actually the characteristic band of silver NPs and is dependent upon size, shape, dielectric constant, and aggregation level of particles [23]. It can be seen from Fig. 4a that the extinction band has broadened significantly for samples set s3 and s4, and has almost covered the wavelength range from 320 to 800 nm. The dipole–dipole interactions between the neighboring silver particles and the Mie scattering of the silver shell as a whole may serve as the main reason for the large band broadening and shift. The ratio of silica core
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radius to silver shell thickness also affects the band position to a great extent. This ratio directly depends on the activation methods. Several other factors, such as NPs size, shape, and aggregation state, may be responsible for observed features of band width, position, etc. Furthermore, it can be seen from Fig. 4 that with increase in deposition time, the intensity of Ag LSPR band is increased, which can be assigned to the increased wt% of loaded silver on silica. 3.1 Antibacterial study of silica/silver core–shell NPs The antibacterial activity of silver NPs coated silica spheres was studied against both gram positive, B. subtilis, and gram negative bacteria, E. coli ATCC25922. The initial culture containing *105 bacterial cells/mL was incubated with different dilutions of the silver NPs coated silica spheres, whereas silica spheres were used as negative control and chloramphenicol as a positive control. The results observed for different samples are presented in Table 4. It can be revealed from Table 4 that silver NPs coated silica spheres kill both gram positive and gram negative bacteria, however, the amount of particles required to kill gram positive bacteria was more than that for gram negative bacteria, which can be due to the difference in their wall characteristics, since the thick
J Mater Sci: Mater Med Fig. 3 SEM micrographs and corresponding EDXA analysis of silver coated silica NPs samples set s1, s2, s3, and s4
Table 3 Variation of silver weight percent with variation in the activation method and the deposition time Sample
Activation method
s1
No activation
Incubation time (h)
Amount of Ag present (wt%)
3
14–17
2
8–13
3
12–16
s2
2 % NaOH
4
18–20
s3
Calcination at 450 °C
3
10–13
s4
Calcination at 750 °C
3
4–6
peptidoglycan layer of the cell wall of gram positive bacteria resist the penetration of the NPs inside the cytoplasm [24]. The antibacterial effect is associated with the interaction of NPs with the bacterial surfaces. When silica spheres alone were used as the negative control, no effect
on microbial growth was observed even after 24 h of incubation. This is because silica doesn’t possess any bactericidal activity and is reported to be biocompatible in many studies [25]. Furthermore, for the different samples set namely, s1, s2, s3 and s4, the minimum amount of silica/silver core–shell NPs, i.e. minimal inhibitory concentration (MIC), required for complete inhibition of bacterial growth is observed to be dependent upon the weight percent of silver present on the silica core surface. As evidenced from the Table 4, MIC for sample s4 is highest (250 lg/mL, E. coli ATCC25922) because of low weight percent of silver NPs on silica spheres and that of sample s2 (4 h) is the lowest (7.8 lg/mL, E. coli ATCC25922), having highest wt% of silver. In addition to the wt% of silver on silica, the size of silver seems to play an important role in bacterial killing. Sample s2 (3 h), having silver NPs size *23 nm, is observed to show better result than sample s1 (crystallite size *34 nm) irrespective of loading amount (*12–17 %), which is in agreement with reported results [13]. This has been attributed to the high
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J Mater Sci: Mater Med Fig. 4 UV–Visible spectra of silica and silica/silver core–shell NPs; a effect of activation method and b incubation time on optical spectra of silica/silver NPs
Table 4 Minimal inhibitory concentration (MIC) of silver NPs coated silica sphere samples against E. coli ATCC25922 and Bacillus subtilis Samples detail
s1
Minimal inhibitory concentration (lg/mL) E. coli ATCC25922
Bacillus subtilis
62.5
125
s2 2h
15.625
31.25
3h
31.25
62.25
4h
7.8
15.625
15.625 250
31.25 500
s3 s4
surface to volume ratio of small size NPs and increase potential for slow release of surface atoms/ions. The actual mechanism of inhibition of growth by silver NPs is not very clear but the inhibitory action of silver NPs is based on the controlled release of Ag. In addition to the increased surface area and associated increased potential for the release of Ag, when dispersed in liquid suspensions, silver NPs may accumulate in the bacterial cytoplasmic membrane. Sondi and co-workers [26] have shown formation of pits on bacterial membrane surfaces, when treated with silver NPs, which results in significant increase in cell permeability, leaving the bacterial cells incapable of properly regulating transport through the plasma membrane and, finally causing cell death. Recently, it has been suggested that the antimicrobial mechanism of silver NPs may also be related to membrane damage due to free radicals that are derived from the surface of the NPs. This bactericidal activity also appears to be dependent on the size and shape of the silver NPs [12, 13]. However, further study is underway for understanding the core mechanism behind bactericidal effect of silica/silver core–shell NPs. This study is an attempt to bridge a gap between bactericidal behavior of silver NPs alone and silica/silver nanocomposite in textiles. It may help in better understanding of
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required parameters for dispersing these particles in other matrix like polymers, textiles, resins, etc. for antibacterial application like in medical devices, bandages, etc.
4 Conclusion In conclusion, silica/silver core–shell NPs were synthesized by electro-less reduction method and studied for their antibacterial activity against both gram positive and gram negative bacteria. The surface activation method of silica core particles is observed to affect the final loading amount (wt%) and size of silver NPs coated on the silica core. Incubation time is also an important factor to control loading value and associated bactericidal behavior. A sample with highest amount of silver is observed to show the lowest value of MIC (7.8 lg/mL) against E. coli ATCC25922. The synthesized NPs exhibit inhibitory effects for both gram positive and gram negative bacteria and hence hold potential in various antibacterial applications. Acknowledgments Authors are thankful to both CSIR-CSIO and IIT Roorkee for infrastructural and experimental facilities.
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