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Synthesis and antibacterial activity of silver nanoparticles against gram-positive and gram-negative bacteria
POTENTIAL CLINICAL RELEVANCE Nanomedicine: Nanotechnology, Biology, and Medicine 8 (2012) 37 – 45
Review Article
nanomedjournal.com
Synthesis and antibacterial activity of silver nanoparticles against gram-positive and gram-negative bacteria Maribel Guzman, PhDa,⁎, Jean Dille, PhDb , Stéphane Godet, PhDb a Pontificia Universidad Católica del Perú, Engineering Department, Lima, Peru Université Libre de Bruxelles, Matters and Materials Department, Brussels, Belgium Received 11 November 2009; accepted 4 May 2011
b
Abstract Synthesis of nanosized particles with antibacterial properties is of great interest in the development of new pharmaceutical products. Silver nanoparticles (Ag NPs) are known to have inhibitory and bactericidal effects. In this article we present the synthesis of Ag NPs prepared by chemical reduction from aqueous solutions of silver nitrate, containing a mixture of hydrazine hydrate and sodium citrate as reductants and sodium dodecyl sulfate as a stabilizer. The results of the characterization of the Ag NPs show agglomerates of grains with a narrow size distribution (from 40 to 60 nm), whereas the radii of the individual particles are between 10 and 20 nm. Finally, the antibacterial activity was measured by the Kirby-Bauer method. The results showed reasonable bactericidal activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The standard dilution micromethod, determining the minimum inhibitory concentration leading to inhibition of bacterial growth, is still under way. Preliminary results have been obtained. From the Clinical Editor: In this paper the synthesis of Ag NPs via chemical reduction from aqueous solutions is discussed. Reasonable bactericidal activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus was demonstrated. © 2012 Elsevier Inc. All rights reserved. Key words: Antibacterial activity; Chemical reduction; Silver nanoparticles; Surface plasmon; UV-vis absorption spectrum
In recent years there has been growing interest in the preparation and study of silver nanoparticles (Ag NPs), because those nanoparticles have been found to exhibit interesting antibacterial activities.1,2 Production of nanosized metallic silver particles with different morphologies and sizes using different routes has been reported.3-11 Along with those methods, the simple process involving a reduction of silver salts has already been well developed.12,13 This synthetic method involves reduction of an ionic salt in an appropriate medium in the presence of surfactant using various reducing agents,14 such as sodium borohydride15 hydrazine hydrate,16 sodium citrate,17 and ascorbic acid.18 Contrary to the bactericidal effects of ionic silver, the antimicrobial activity of colloidal silver particles is influenced This research work has been supported by The Engineering Department of Pontificia Universidad Católica del Peru (grants DAI-E034 and 53831. R702). The aim of this research work was completely academic. The grants DAI-E034 and 53831.R702 supporting this research work were offered by the Research Academic Office of M.G.'s institution; with the aim of developing research at the University, without any commercial association, within the past 5 years until now. There are no patent licensing arrangements. ⁎Corresponding author: Pontificia Universidad Católica del Perú, Engineering Department, Av. Universitaria 1801, Lima-32, Peru. E-mail address: [email protected] (M. Guzman).
by the dimensions of the particles: the smaller the particles, the greater the antimicrobial effect.19 The use of Ag NPs as an antibacterial agent is relatively new. Antibacterial activity of the Ag NPs can be exploited for disinfection in wastewater treatment plants, to prevent bacterial colonization and eliminate microorganisms on medical and silicone rubber gaskets to protect and transport food and textile fabrics among others.20 Antimicrobial susceptibility tests are classified into different methods, based on the applied principle. They include: diffusion (Kirby-Bauer and Stokes), dilution (minimum inhibitory concentration, MIC), and diffusion and dilution (E-test method). The Kirby-Bauer and Stokes methods are usually used for antimicrobial susceptibility testing, and the former is recommended by the Clinical and Laboratory Standards Institute (CLSI). This method is well documented, and standard zones of inhibition have been determined for susceptible and resistant values.21 The antibacterial characteristics of Ag NPs produced have been demonstrated by directly exposing bacteria to colloidal silver particles solution.22 The exact antibacterial action of Ag NPs is not completely understood. There are reports in the literature that show that electrostatic attraction between negatively charged bacterial cells and positively charged nanoparticles is crucial for the activity of nanoparticles as bactericidal materials.23,24 Several possible
1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.05.007 Please cite this article as: M. Guzman, J. Dille, S. Godet, Synthesis and antibacterial activity of silver nanoparticles against gram-positive and gramnegative bacteria. Nanomedicine: NBM 2012;8:37-45, doi:10.1016/j.nano.2011.05.007
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mechanisms have been proposed that involve the interaction of silver with biological macromolecules25 such as enzymes and DNA through an electron-release mechanism.26 It is believed that DNA loses its replication ability and cellular proteins are inactivated upon Ag+ treatment.27 In addition, it was also shown that Ag+ binds to functional groups of proteins, resulting in protein denaturation. In this work, Ag NPs were prepared from aqueous silver nitrate (AgNO3) solution using a mixture of hydrazine hydrate and citrate of sodium as reductant and sodium dodecyl sulfate (SDS) as a stabilizer.28 The antibacterial activity of these Ag NPs against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus was tested with the Kirby-Bauer diffusion and MIC dilution methods. Results show that Ag NPs were effective against these pathogens.
Methods Materials AgNO3, hydrazine hydrate, sodium citrate, and SDS were purchased from Merck Peruana (Lima, Peru). All chemicals were used as received. Double-distilled deionized water was used. Characterization techniques Ultraviolet-visible (UV-vis) spectroscopy was performed in a Spectrophotometer (Perkin-Elmer Lambda 2 Spectrophotometer; Bremen, Germany) in a range of 300-700 nm. The studies of size, morphology, and composition of the nanoparticles were performed by means of transmission electron microscopy (TEM), energy-dispersive x-ray analysis (EDX), and highenergy electron diffraction (HEED) using a transmission electron microscope operating at 200 kV (Philips CM20-Ultra Twin microscope; Eindhoven, The Netherlands). Histograms of size distribution were calculated from the TEM images by measuring the diameters of at least 50 particles. Samples for TEM studies were prepared by placing drops of the Ag NP solutions on carbon-coated TEM copper grids. Preparation of Ag NPs For the synthesis of Ag NPs, solutions of AgNO3 (1.0 mM to 6.0 mM), hydrazine hydrate (2.0 mM to 12 mM), sodium citrate (1.0 mM to 2.0 mM), and 8 wt% SDS were used. After the reaction, we filtered the colloids containing nanoparticles. The nanoparticles remained in the paper (filter), while the solution containing the remaining silver ions was received in a special flask and sent for analysis by atomic absorption spectroscopy (AAS) in an atomic absorption spectrometer (Varian SpectrAA 220 Atomic Absorption Spectrometer; Victoria, Australia). The yields of the samples were calculated by determining the final concentration of Ag+ by AAS in the remaining solution. To remove excess silver ions, the silver colloids were washed with deionized water under a nitrogen stream. The nitrogen stream was used to avoid the oxidation of Ag NPs obtained. The use of deionized water avoids the presence of chloride ions that can precipitate the silver ions as silver chloride (AgCl). All solutions from the rinsing of the nanoparticles were also
Table 1 Silver concentration in the initial solution and in the last rinsing solution Sample
Initial silver concentration (mg/L)
Silver concentration in the final rinsing solution (mg/L)
SHC-1 SHC-2 SHS-1 SHCS-1
116.28 112.72 621.33 595.44
1.05 0.89 0.66 1.19
collected in a special flask and analyzed by AAS; the number of rinsings was determined by the concentration of silver in the water solutions (Table 1). A dried nanopowder of silver was obtained by freeze-drying. To carry out all characterization methods and interaction of the Ag NPs with bacteria, the silver nanopowder in the freeze-drying cuvette was again suspended in deionized water. The suspension was homogenized with an ultrasonic cleaning container (Fisher Bioblock Scientific, Suwanee, Georgia). Antibacterial assays The antimicrobial susceptibility of Ag NPs was evaluated using the disk diffusion or Kirby-Bauer methods.21 Zones of inhibition were measured after 24 hours of incubation at 35°C. The comparative stability of disks containing oxacillin and ciprofloxacin was prepared. Disposable plates inoculated with the tested gram-positive and gram-negative bacteria at a concentration of 105 to 106 colony-forming units/mL were used for the tests. Ag NPs sols were inoculated in the disposable plates containing gram-positive and gram-negative bacteria. Zones of inhibition were measured after 24 hours of incubation at 35°C. The standard dilution micromethod, determining the MIC leading to inhibition of bacterial growth, is still under way. However, preliminary results have been obtained. Antimicrobial activities of the synthesized silver colloidal sols were assessed using the standard dilution micromethod, determining the MIC leading to inhibition of bacterial growth.21 The silver sols were diluted two to eight times with 100 μL of Mueller-Hinton broth and inoculated with the tested bacteria at a concentration of 105 to 106 colony-forming units/mL. The MIC was read after 24 hours of incubation at 37°C. The MIC was determined as the lowest concentration that inhibited the visible growth of the bacterium. The silver sols were used in the form in which they had been prepared. Therefore, control bactericidal tests of solutions containing all the reaction components with the exception of AgNO3 and reducing agents were performed.
Results and discussion Ag NPs were synthesized according to the method described in the previous section. First, the effects in the particle size were investigated in two cases: when a second reducing agent is added in the synthesis, and when the concentration of reducing agent is increased. Finally, the antibacterial activity of all silver colloids prepared was examined.
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Figure 1. UV-vis absorption spectrum of the Ag NPs prepared from 1.1 mM AgNO3 solution (sample SHS-1).
Table 1 shows the initial silver concentration (before the reducing agents were added) and the concentration of silver ion in the last rinsing solution. We know that silver ions are soluble in deionized water. In accordance with these results, we can consider that all remaining silver ions were removed. Effect of nature of reducing agent Ag NPs using hydrazine and a mixture of hydrazine-sodium citrate, both in presence of SDS as stabilizing agent, were obtained at room temperature (20°–25°C). Normally the synthesis with AgNO3 and sodium citrate is possible only at 80°C in 24 hours. The silver colloids obtained show different colors, pale brown when only hydrazine was used (sample SHS-1) and pale yellow when sodium citrate as a second reducing agent was used (sample SHSC-1). UV-vis spectroscopy is a valuable tool for structural characterization of Ag NPs. It is well known that the optical absorption spectra of metal nanoparticles are dominated by surface plasmon resonances (SPRs) that shift to longer wavelengths with increasing particle size.29 Also, it is well recognized that the absorbance of Ag NPs depends mainly upon size and shape.30 In general, the number of SPR peaks decreases as the symmetry of the nanoparticle increases.31 The UV-vis absorption spectra of samples SHS-1 and SHSC1 are presented in Figures 1 and 2, respectively. Figure 1 shows a surface plasmon absorption band with a maximum at 418 nm, indicating the presence of spherical or roughly spherical Ag NPs.32 The position and shape of the plasmon absorption depends on the particles' size and shape, and
the dielectric constant of the surrounding medium. It can be observed that the surface plasmon absorption maximum, initially at 418 nm, is shifted to a lower wavelength (Figure 2) when sodium citrate is added as reducing agent. The shape of the second UV-vis spectrum is also of interest; it shows a smooth shoulder near 540 nm. The surface plasmon absorptions at 418 nm and 412 nm are consistent with other reports.17,18 The morphology and size of the silver colloids were investigated by TEM analysis. TEM micrographs of samples SHS-1 and SHSC-1 are presented in Figures 3 and 4, respectively. The TEM micrograph in Figure 3 shows agglomerates of small grains and some dispersed nanoparticles. The size of the particles ranges from 8 to 50 nm, with a mean diameter of 24 nm and standard deviation of 6 nm, as illustrated on the histogram on the right-hand side of Figure 3. The TEM micrograph in Figure 4 shows the presence of well-dispersed particles that are more or less spherical, as well as faceted particles. The distribution of size of this sample (Figure 4, right) shows a bimodal distribution of the particle sizes, the first cluster ranging from 15 to 30 nm and the second ranging from 32 to 48 nm, with a mean diameter of 30 nm and standard deviation of 7 nm. In comparison with particle sizes obtained using only hydrazine hydrate (Figure 3, right), the mean diameter does not increase significantly. In consequence, the shift of the surface plasmon absorption maximum from 418 nm (Figure 1) to 412 nm (Figure 2) could be due to a difference in particle shape. That was consistent with the report of Schultz and co-workers,32 in which there was a correlation of the absorption spectra of individual Ag NPs with their size (40-120 nm) and shape (spheres,
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Figure 2. UV–vis absorption spectrum of Ag NPs prepared using a mixture of hydrazine and citrate of sodium as a reducing agent (sample SHCS-1).
Figure 3. TEM image of spherical Ag NPs (left) and their particle size distributions (right). Ag NPs prepared from 1.1 mM AgNO3 solution (sample SHS-1).
decahedrons, triangular truncated pyramids, and platelets) as determined by TEM. They found that spherical and roughly spherical nanoparticles, decahedral or pentagonal nanoparticles, and triangular truncated pyramids and platelets absorb in the blue, green, and red parts of the spectrum, respectively. Table 2 shows the yield of those reactions. For both samples the yield was higher than 80%. It can be observed that the yield of the reaction does not increase significantly when the sodium citrate is added.
The elemental analysis of the Ag NPs was performed using the EDX method in the transmission electron microscope. Figure 5 shows the EDX spectrum of sample SHS-1. All the K and L emission peaks for silver are observed; similar results have been reported by Shahverdi et al1 and Lim et al.33 The CKα, CuKα and CuKβ peaks are also detected. These carbon and copper peaks are due to the transmission electron microscope holding grid. No other obvious peak attributable to impurity is detected. This result indicates that the as-synthesized product is
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Figure 4. TEM image of spherical and faceted Ag NPs (left) and particle size distributions of Ag NPs prepared using a mixture of hydrazine and citrate of sodium as a reducing agent (sample SHSC-1).
Table 2 Surface plasmon absorption maxima of silver nanoparticles prepared with and without sodium citrate Sample
Mean diameter (nm)
λ (nm)
Yield(%)
SHS-1 SHSC-1
24 ± 6 30 ± 7
418 412
82.49 88.56
6.0 mM AgNO3 + SDS.
composed of high-purity Ag NPs. A similar EDX spectrum was obtained for sample SHSC-1. The crystallographic properties such as structure of the silver colloids were determined by HEED analysis. The corresponding HEED pattern of sample SHSC-1 is shown on the right-hand illustration in Figure 6. When the electron diffraction is carried out on a limited number of crystals, one can only observe some spots of diffraction distributed in concentric circles. The ring patterns of sample SHSC-1 with plane distances of 2.36 Å, 2.04 Å, 1.45 Å, 1.23 Å, and 0.94 Å are consistent with the plane families {111}, {200}, {220}, {311}, and {331} of pure facecentered cubic (FCC) silver structure (JCPDS, File No. 4-0787; Powder Diffraction File, International Center for Diffraction Data, Newtown Square, Pennsylvania). A similar HEED spectrum was obtained for sample SHS-1. In both cases the formation of FCC Ag NPs is confirmed. Those results were consistent with the reports of Chen and Gao,12 Mandal et al28 and Hasell et al.34 Effect of the concentration of reducing agent Ag NPs using a mixture of both hydrazine hydrate and sodium citrate at different concentrations were prepared. In both cases, SDS was not used. The pale-red silver colloids obtained with concentration of 1.0 mM (sample SHC-1) and 2.0 mM (sample SHC-2) of sodium citrate were characterized by UV-vis and TEM analysis. The UV-vis spectra of samples SHC-1 and SHC-2 are presented in Figure 7, A and B, in that order. The absorptions of SPR are situated at 406 nm and 405 nm for sample SHC-1 and
sample SHC-2, respectively. This is evidence that the surface plasmon absorption does not change appreciably when the concentration of sodium citrate is doubled. The shapes of the spectra are similar. The values of surface plasmon absorption are consistent with other reports.12,17 In comparison with Figures 1 and 2, shifts of the surface plasmon absorption maximum from 418 nm to 406 nm and from 412 nm to 406 nm are observed. Those shifts can be attributed to the difference in particle sizes of the silver colloids.32 The TEM micrographs of samples SHS-1 and SHSC-1 are presented in Figure 8, A and B, respectively. The TEM results indicate that the nanoparticles consist of agglomerates of small grains with mean diameters between 9 ± 2 and 14 ± 5 nm. However, some particles with diameters larger than 60 nm were formed as a result of aggregation during preparation of the TEM holding grid. Figure 8, A shows agglomerates of small grains and some dispersed nanoparticles that are more or less spherical. Figure 8, B shows spherical small particles, whereas the larger particles have an almost square shape. The particle size histograms of both samples are presented in Figure 9. With a 1.0 mM solution of sodium citrate, the size of the particles ranges from 7 to 20 nm with a mean diameter of 9 ± 2 nm (Figure 9, A). When the concentration is doubled, the size distribution becomes bimodal. Figure 9, B shows that the particles of silver range in size from 3 to 6 nm and from 21 to 35 nm, with mean diameter of 14 nm ± 5 nm. The particle size distributions become narrower and the average particle sizes decrease when the second agent reduction is used. In comparison with particle sizes obtained using only hydrazine (Figure 3) and a mixture of hydrazine‑sodium citrate, the mean diameter decreases significantly. This can be attributed to the double role of sodium citrate as reducing and dispersing agent.17,35 In addition, when the concentration of sodium citrate is doubled, the yield of the reaction is increased by approximately 17% (Table 3). The particle size data, the UV-vis surface plasmon absorption maxima, and the yield are given in Table 3. According to Panacek et al20 the size of Ag NPs can be controlled by using different concentrations of reducing agents and silver ions undergoing reduction. In addition, when SDS was used the mean diameter was increased (samples SHS-1 and SHCS-1).
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Figure 5. EDX spectrum of Ag NPs prepared from 1.1 mM AgNO3 solution (sample SHS-1).
Figure 6. TEM (left) and HEED (right) images of sample SHCS-1 (metal ion concentration and citrate of sodium solution 6.0 mM and 2.0 mM, respectively).
Antibacterial activity Finally, the antimicrobial susceptibility of Ag NPs synthesized was investigated. The Kirby-Bauer diffusion method was used as antimicrobial susceptibility testing method. For antibacterial function testing, S. aureus and S. aureus MRSA (both gram-positive), as well as E. coli and P. aeruginosa (both gramnegative) were used as the bacilli. The average of particle sizes used for the antibacterial test was 9 ± 2 nm, 14 ± 5 nm, 24 ± 6 nm, and 30 ± 7 nm. The presence of nanoparticles at a certain level inhibited bacterial growth by more than 90%. The diameter of inhibition zones (in millimeters) measured is shown in Table 4. The clearzone diameter of the bacterial inhibition zone was correlated to antibiotic activity (oxacillin and ciprofloxacin) for gram-positive and Gram-negative bacteria, respectively. Table 4 shows that clear-zone diameter increased as the concentration of Ag NPs increased, because of the bactericidal activity of Ago. These data are consistent with previously reported studies.36 Antimicrobial activities of the synthesized silver colloidal sols were assessed using the standard dilution micromethod.
Table 5 summarizes the MICs of the silver particles samples tested against gram-positive and gram-negative bacteria. Smaller Ag NPs synthesized using sodium citrate showed a considerable antibacterial activity (14 ppm and 29 ppm). This phenomenon is related to the size of colloidal silver particles. Thus, the 9 ± 2 nm and 14 ± 5 nm silver particles synthesized showed the highest activity against gram-positive and gramnegative bacteria. The lowest antimicrobial effect (216 ppm) obtained with sample (SHCS-1) can be attributed to larger mean diameter (30 ± 8 nm) or a contrary effect of the SDS dispersant, which can be retained on the surface of Ag NPs after the synthesis. Silver particles obtained using only hydrazine (sample SHS-1) show the lowest antimicrobial effect (259 ppm) for E. coli and S. aureus. Nevertheless, this sample shows the highest activity (7 ppm) against P. aeruginosa bacteria. This can be attributed to an effect of the shape of nanoparticles. Similar results have been reported previously by Pal and co-workers.2 The results of antibacterial activities against E. coli, P. aeruginosa, and S. aureus around 14.38 ppm, 6.74 ppm, and 14.38 ppm are close to those reported earlier by Song et al37 and Sondi and Salopek-Sondi,24 around 1 ppm and 10 ppm for gram-negative and gram-positive bacteria, respectively. The mechanism of the bactericidal effect of silver colloid particles against bacteria is not very well known. It is possible that Ag NPs act similarly to the antimicrobial agents used for the treatment of bacterial infections. Those agents show four different mechanisms of action: (1) interference with cell wall synthesis, (2) inhibition of protein synthesis, (3) interference with nucleic acid synthesis, and (4) inhibition of a metabolic pathway.38 Three different mechanisms of action of Ag NPs have been proposed by Feng et al27: First, Ag NPs attach to the surface of the cell membrane and disturb its power functions, such as permeability and respiration.39 Plasmolysis (cytoplasm separated from bacterial cell wall) in P. aeruginosa and the inhibition of bacterial cell wall synthesis in S. aureus bacteria were reported by Song et al.37 It is reasonable to state that the binding of the particles to the bacteria depends on the interaction of the surface area available. Smaller particles having a larger surface area available for interaction will have a stronger bactericidal effect
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Figure 7. UV-vis absorption spectra of samples SHC-1 and SHC-2, respectively. Ag NPs prepared using hydrazine hydrate (2.0 mM) and citrate of sodium solution with a concentrate ranging from 1.0 mM (A) to 2.0 mM (B).
Figure 8. TEM image of spherical Ag NPs obtained with 1.0 mM (A) and 2.0 mM (B) of citrate of sodium solution (samples SHC-1 and SHC-2, respectively).
than will larger particles.40 This is moderately in accordance with the results shown in Table 4. However, an antibacterial effect of the sample SHS-1 can be attributed to the nanoparticle shape. Second, Ag NPs are able to penetrate the bacteria and cause further damage, possibly by interacting with sulfur- and phosphorus-containing compounds such as DNA.41 Raffi and co-workers42 reported TEM images of Ag NPs in the membranes of the bacteria as well as in the interior. In addition, it is believed that Ag NPs after penetration into the bacteria have inactivated
Figure 9. Particle size distributions of Ag NPs (prepared from 1.1 mM AgNO3 solution) and citrate of sodium solution with a concentration ranging from 1.0 mM (A) to 2.0 mM (B).
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Table 3 Mean diameter and surface plasmon absorption maxima of silver nanoparticles obtained at different sodium citrate concentrations Sample
Mean diameter (nm)
λ (nm)
Yield (%)
SHC-1 SHC-2
9±2 14 ± 5
406 405
65.77 82.51
1.1 mM AgNO3.
Table 5 Minimum inhibitory concentrations of silver nanoparticles Bacteria
Escherichia coli CCM 3954 Pseudomonas aeruginosa CCM 3955 Staphylococcus aureus CCM 3953 Staphylococcus aureus MRSA
Minimum inhibitory concentrations (μg/mL) SHC-1 SHC-2 SHS-1
SHCS-1
14.38 14.38 14.38 14.38
215.74 215.74 215.74 215.74
28.77 28.77 28.77 28.77
258.89 6.74 258.89 258.89
Table 4 Zone of inhibition (mm) of silver nanoparticles sols against test strains Bacteria
SHC-1
Escherichia coli CCM 3954 9 ± 1.0 Pseudomonas aeruginosa 9 ± 0.5 CCM 3955 Staphylococcus aureus CCM 3953 12 ± 0.4 Staphylococcus aureus MRSA 11 ± 0.6
SHC-2
SHS-1
SHCS-1
10 ± 0.5 32 ± 0.7 26 ± 0.5 11 ± 0.3 29 ± 1.0 19 ± 0.6 11 ± 0.6 34 ± 0.5 31 ± 1.0 11 ± 1.0 40 ± 0.3 29 ± 0.5
their enzymes, generating hydrogen peroxide and causing bacterial cell death.42,43 In addition, it is believed that the high affinity of silver for sulfur or phosphorus is the key element of its antibacterial property, in that sulfur and phosphorus are found in abundance throughout the cell membrane. Ag NPs react with sulfur-containing proteins inside or outside the cell membrane, which in turn affects cell viability.23 Third, Ag NPs release silver ions, which make an additional contribution to the bactericidal effect.27 In fact, Morones et al44 showed that Ag NPs (where silver is present in the Ag0 form) also contain micromolar concentrations of Ag+, and they have shown that Ag+ and Ag0 both contribute to the antibacterial activity. The mechanism of inhibition by silver ions on microorganisms is partially known. It is believed that DNA loses its replication ability and cellular proteins become inactivated on silver ion treatment.45,46 Higher concentrations of Ag+ ions have been shown to interact with cytoplasmic components and nucleic acids.27,36 The antibacterial effect of Ag NPs determined in this study was found to be similar to that described in the earlier reports.40 The particle size has an effect on microbes; the effect increased with smaller particle size. In our case we have a distribution of particle sizes with a mean diameter ranging from 9 nm to 30 nm. From the results in Tables 2, 3, and 5 we can say that there is a relationship between the size of the nanoparticles and the bacterial activity. For instance, as the nanoparticle size decreases the MIC decreases for all bacteria. In other words, we need a smaller dose of silver suspension nanoparticles to inhibit the growth of bacteria. However, there is an anomalous behavior of sample SHS-1 with average diameter of 24 nm, having a stronger bactericidal activity with an MIC of only 7 μg/mL. This behavior can be attributed to an additional effect of nanoparticle shape, but this relationship has not yet been determined.
Summary Ag NPs with mean diameters of 9 ± 2 nm, 14 ± 5 nm, 24 ± 6 nm, and 30 ± 7 nm were synthesized using hydrazine hydrate and
sodium citrate as reducing agent at room temperature. The nanoparticles were characterized by UV-vis spectroscopy, EDX, and TEM. UV-vis spectra show the characteristic plasmon absorption peak for the Ag NPs between 405 and 418 nm. The EDX of the nanoparticles dispersion confirmed the presence of elemental silver signal. No elemental impurity was detected. HEED confirmed the formation of FCC Ag NPs. Additionally, the antibacterial activity of the nanopartículate dispersion was measured by the Kirby-Bauer method. The results of this study clearly demonstrated that the colloidal Ag NPs inhibited the growth and multiplication of the tested bacteria, including highly multidrug-resistant bacteria such as methicillinresistant S. aureus, S. aureus, E. coli, and P. aeruginosa. A strong antibacterial activity was observed at very low total concentrations of silver (below 7 ppm). Acknowledgment M. Guzman wants to thank the Matter and Materials Department of Université Libre de Bruxelles of Belgium, which provided facilities for use of their transmission electron microscope.
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27. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO. A mechanistic study of the antibacterial effect of silver ions on E. coli and Staphylococcus aureus. J Biomed Mater Res 2000;52:662-8. 28. Mandal S, Karumugam S, Pasricha R, Sastry M. Silver nanoparticles of variable morphology synthesized in aqueous foams as novel templates. Bull Mater Sci 2005;28:503-10. 29. Brause R, Moeltgen H, Kleinermanns K. Characterization of laser ablated and chemically reduced silver colloids in aqueous solution by UV/VIS spectroscopy and STM/SEM microscopy. Appl Phys B: Lasers Opt 2002;75:711-6. 30. Kerker M. The optics of colloidal silver: something old and something new. J Colloid Interface Sci 1985;105:297-314. 31. Sosa IO, Noguez C, Barrera RG. Optical properties of metal nanoparticles with arbitrary shapes. J Phys Chem B 2003;107:6269-75. 32. Mock JJ, Barbic M, Smith DR, Schultz DA, Schultz S. Shape effects in plasmon resonance of individual colloidal silver nanoparticles. J Chem Phys 2002;116:6755-60. 33. Lim PY, Liu RS, She PL, Hung CF, Shih HC. Synthesis of Ag nanospheres particles in ethylene glycol by electrochemical-assisted polyol process. Chem Phys Lett 2006;420:304-8. 34. Hasell T, Yang J, Wang W, Brown PD, Howdle SM. A facile synthetic route to aqueous dispersions of silver nanoparticles. Mater Lett 2007;61: 4906-10. 35. Yin H, Yamamoto T, Wada Y, Yanagida S. Large-scale and sizecontrolled synthesis of silver nanoparticles under microwave irradiation. Mater Chem Phys 2004;83:66-70. 36. Kim JS. Antibacterial activity of Ag+ ion-containing silver nanoparticles prepared using the alcohol reduction method. J Indust Eng Chem 2007; 13:718-22. 37. Song HY, Ko KK, Oh IH, Lee BT. Fabrication of silver nanoparticles and their antimicrobial mechanisms. Eur Cells Mater 2006;11:58-9. 38. Tenover FC. Mechanisms of antimicrobial resistance in bacteria. Am J Med 2006;119:3-10. 39. Murray RGE, Steed P, Elson HE. The location of the mucopeptide in sections of the cell wall of Escherichia coli and other gram-negative bacteria. Can J Microbiol 1965;11:547-60. 40. Shrivastava S, Bera T, Roy A, Singh G, Ramachandrarao P, Dash D. Characterization of enhanced antibacterial effects of novel silver nanoparticles. Nanotechnology 2007;18:225103-12. 41. Gibbins B, Warner L. The role of antimicrobial silver nanotechnology. Med Device Diagnostic Indust Mag 2005;1:1-2. 42. Raffi M, Hussain F, Bhatti TM, Akhter JI, Hameed A, Hasan MM. Antibacterial characterization of silver nanoparticles against E. coli ATCC-15224. J Mater Sci Technol 2008;24:192-6. 43. Shockman GD, Barrett JF. Structure, function, and assembly of cell walls of gram-positive bacteria. Annu Rev Microbiol 1983;37:501-27. 44. Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Tapia J, et al. The bactericidal effect of silver nanoparticles. Nanotechnology 2005;16:2346-53. 45. Kumar A, Kumar-Vemula P, Ajayan PM, John G. Silver-nanoparticleembedded antimicrobial paints based on vegetable oil. Nat Mater 2008; 7:236-41. 46. Gupta P, Bajpai M, Bajpai SK. Investigation of antibacterial properties of silver nanoparticle-loaded poly (acrylamide-co-itaconic acid)-grafted cotton fabric. J Cotton Sci 2008;12:280-6.
Review Article
nanomedjournal.com
Synthesis and antibacterial activity of silver nanoparticles against gram-positive and gram-negative bacteria Maribel Guzman, PhDa,⁎, Jean Dille, PhDb , Stéphane Godet, PhDb a Pontificia Universidad Católica del Perú, Engineering Department, Lima, Peru Université Libre de Bruxelles, Matters and Materials Department, Brussels, Belgium Received 11 November 2009; accepted 4 May 2011
b
Abstract Synthesis of nanosized particles with antibacterial properties is of great interest in the development of new pharmaceutical products. Silver nanoparticles (Ag NPs) are known to have inhibitory and bactericidal effects. In this article we present the synthesis of Ag NPs prepared by chemical reduction from aqueous solutions of silver nitrate, containing a mixture of hydrazine hydrate and sodium citrate as reductants and sodium dodecyl sulfate as a stabilizer. The results of the characterization of the Ag NPs show agglomerates of grains with a narrow size distribution (from 40 to 60 nm), whereas the radii of the individual particles are between 10 and 20 nm. Finally, the antibacterial activity was measured by the Kirby-Bauer method. The results showed reasonable bactericidal activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The standard dilution micromethod, determining the minimum inhibitory concentration leading to inhibition of bacterial growth, is still under way. Preliminary results have been obtained. From the Clinical Editor: In this paper the synthesis of Ag NPs via chemical reduction from aqueous solutions is discussed. Reasonable bactericidal activity against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus was demonstrated. © 2012 Elsevier Inc. All rights reserved. Key words: Antibacterial activity; Chemical reduction; Silver nanoparticles; Surface plasmon; UV-vis absorption spectrum
In recent years there has been growing interest in the preparation and study of silver nanoparticles (Ag NPs), because those nanoparticles have been found to exhibit interesting antibacterial activities.1,2 Production of nanosized metallic silver particles with different morphologies and sizes using different routes has been reported.3-11 Along with those methods, the simple process involving a reduction of silver salts has already been well developed.12,13 This synthetic method involves reduction of an ionic salt in an appropriate medium in the presence of surfactant using various reducing agents,14 such as sodium borohydride15 hydrazine hydrate,16 sodium citrate,17 and ascorbic acid.18 Contrary to the bactericidal effects of ionic silver, the antimicrobial activity of colloidal silver particles is influenced This research work has been supported by The Engineering Department of Pontificia Universidad Católica del Peru (grants DAI-E034 and 53831. R702). The aim of this research work was completely academic. The grants DAI-E034 and 53831.R702 supporting this research work were offered by the Research Academic Office of M.G.'s institution; with the aim of developing research at the University, without any commercial association, within the past 5 years until now. There are no patent licensing arrangements. ⁎Corresponding author: Pontificia Universidad Católica del Perú, Engineering Department, Av. Universitaria 1801, Lima-32, Peru. E-mail address: [email protected] (M. Guzman).
by the dimensions of the particles: the smaller the particles, the greater the antimicrobial effect.19 The use of Ag NPs as an antibacterial agent is relatively new. Antibacterial activity of the Ag NPs can be exploited for disinfection in wastewater treatment plants, to prevent bacterial colonization and eliminate microorganisms on medical and silicone rubber gaskets to protect and transport food and textile fabrics among others.20 Antimicrobial susceptibility tests are classified into different methods, based on the applied principle. They include: diffusion (Kirby-Bauer and Stokes), dilution (minimum inhibitory concentration, MIC), and diffusion and dilution (E-test method). The Kirby-Bauer and Stokes methods are usually used for antimicrobial susceptibility testing, and the former is recommended by the Clinical and Laboratory Standards Institute (CLSI). This method is well documented, and standard zones of inhibition have been determined for susceptible and resistant values.21 The antibacterial characteristics of Ag NPs produced have been demonstrated by directly exposing bacteria to colloidal silver particles solution.22 The exact antibacterial action of Ag NPs is not completely understood. There are reports in the literature that show that electrostatic attraction between negatively charged bacterial cells and positively charged nanoparticles is crucial for the activity of nanoparticles as bactericidal materials.23,24 Several possible
1549-9634/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nano.2011.05.007 Please cite this article as: M. Guzman, J. Dille, S. Godet, Synthesis and antibacterial activity of silver nanoparticles against gram-positive and gramnegative bacteria. Nanomedicine: NBM 2012;8:37-45, doi:10.1016/j.nano.2011.05.007
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mechanisms have been proposed that involve the interaction of silver with biological macromolecules25 such as enzymes and DNA through an electron-release mechanism.26 It is believed that DNA loses its replication ability and cellular proteins are inactivated upon Ag+ treatment.27 In addition, it was also shown that Ag+ binds to functional groups of proteins, resulting in protein denaturation. In this work, Ag NPs were prepared from aqueous silver nitrate (AgNO3) solution using a mixture of hydrazine hydrate and citrate of sodium as reductant and sodium dodecyl sulfate (SDS) as a stabilizer.28 The antibacterial activity of these Ag NPs against Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus was tested with the Kirby-Bauer diffusion and MIC dilution methods. Results show that Ag NPs were effective against these pathogens.
Methods Materials AgNO3, hydrazine hydrate, sodium citrate, and SDS were purchased from Merck Peruana (Lima, Peru). All chemicals were used as received. Double-distilled deionized water was used. Characterization techniques Ultraviolet-visible (UV-vis) spectroscopy was performed in a Spectrophotometer (Perkin-Elmer Lambda 2 Spectrophotometer; Bremen, Germany) in a range of 300-700 nm. The studies of size, morphology, and composition of the nanoparticles were performed by means of transmission electron microscopy (TEM), energy-dispersive x-ray analysis (EDX), and highenergy electron diffraction (HEED) using a transmission electron microscope operating at 200 kV (Philips CM20-Ultra Twin microscope; Eindhoven, The Netherlands). Histograms of size distribution were calculated from the TEM images by measuring the diameters of at least 50 particles. Samples for TEM studies were prepared by placing drops of the Ag NP solutions on carbon-coated TEM copper grids. Preparation of Ag NPs For the synthesis of Ag NPs, solutions of AgNO3 (1.0 mM to 6.0 mM), hydrazine hydrate (2.0 mM to 12 mM), sodium citrate (1.0 mM to 2.0 mM), and 8 wt% SDS were used. After the reaction, we filtered the colloids containing nanoparticles. The nanoparticles remained in the paper (filter), while the solution containing the remaining silver ions was received in a special flask and sent for analysis by atomic absorption spectroscopy (AAS) in an atomic absorption spectrometer (Varian SpectrAA 220 Atomic Absorption Spectrometer; Victoria, Australia). The yields of the samples were calculated by determining the final concentration of Ag+ by AAS in the remaining solution. To remove excess silver ions, the silver colloids were washed with deionized water under a nitrogen stream. The nitrogen stream was used to avoid the oxidation of Ag NPs obtained. The use of deionized water avoids the presence of chloride ions that can precipitate the silver ions as silver chloride (AgCl). All solutions from the rinsing of the nanoparticles were also
Table 1 Silver concentration in the initial solution and in the last rinsing solution Sample
Initial silver concentration (mg/L)
Silver concentration in the final rinsing solution (mg/L)
SHC-1 SHC-2 SHS-1 SHCS-1
116.28 112.72 621.33 595.44
1.05 0.89 0.66 1.19
collected in a special flask and analyzed by AAS; the number of rinsings was determined by the concentration of silver in the water solutions (Table 1). A dried nanopowder of silver was obtained by freeze-drying. To carry out all characterization methods and interaction of the Ag NPs with bacteria, the silver nanopowder in the freeze-drying cuvette was again suspended in deionized water. The suspension was homogenized with an ultrasonic cleaning container (Fisher Bioblock Scientific, Suwanee, Georgia). Antibacterial assays The antimicrobial susceptibility of Ag NPs was evaluated using the disk diffusion or Kirby-Bauer methods.21 Zones of inhibition were measured after 24 hours of incubation at 35°C. The comparative stability of disks containing oxacillin and ciprofloxacin was prepared. Disposable plates inoculated with the tested gram-positive and gram-negative bacteria at a concentration of 105 to 106 colony-forming units/mL were used for the tests. Ag NPs sols were inoculated in the disposable plates containing gram-positive and gram-negative bacteria. Zones of inhibition were measured after 24 hours of incubation at 35°C. The standard dilution micromethod, determining the MIC leading to inhibition of bacterial growth, is still under way. However, preliminary results have been obtained. Antimicrobial activities of the synthesized silver colloidal sols were assessed using the standard dilution micromethod, determining the MIC leading to inhibition of bacterial growth.21 The silver sols were diluted two to eight times with 100 μL of Mueller-Hinton broth and inoculated with the tested bacteria at a concentration of 105 to 106 colony-forming units/mL. The MIC was read after 24 hours of incubation at 37°C. The MIC was determined as the lowest concentration that inhibited the visible growth of the bacterium. The silver sols were used in the form in which they had been prepared. Therefore, control bactericidal tests of solutions containing all the reaction components with the exception of AgNO3 and reducing agents were performed.
Results and discussion Ag NPs were synthesized according to the method described in the previous section. First, the effects in the particle size were investigated in two cases: when a second reducing agent is added in the synthesis, and when the concentration of reducing agent is increased. Finally, the antibacterial activity of all silver colloids prepared was examined.
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39
Figure 1. UV-vis absorption spectrum of the Ag NPs prepared from 1.1 mM AgNO3 solution (sample SHS-1).
Table 1 shows the initial silver concentration (before the reducing agents were added) and the concentration of silver ion in the last rinsing solution. We know that silver ions are soluble in deionized water. In accordance with these results, we can consider that all remaining silver ions were removed. Effect of nature of reducing agent Ag NPs using hydrazine and a mixture of hydrazine-sodium citrate, both in presence of SDS as stabilizing agent, were obtained at room temperature (20°–25°C). Normally the synthesis with AgNO3 and sodium citrate is possible only at 80°C in 24 hours. The silver colloids obtained show different colors, pale brown when only hydrazine was used (sample SHS-1) and pale yellow when sodium citrate as a second reducing agent was used (sample SHSC-1). UV-vis spectroscopy is a valuable tool for structural characterization of Ag NPs. It is well known that the optical absorption spectra of metal nanoparticles are dominated by surface plasmon resonances (SPRs) that shift to longer wavelengths with increasing particle size.29 Also, it is well recognized that the absorbance of Ag NPs depends mainly upon size and shape.30 In general, the number of SPR peaks decreases as the symmetry of the nanoparticle increases.31 The UV-vis absorption spectra of samples SHS-1 and SHSC1 are presented in Figures 1 and 2, respectively. Figure 1 shows a surface plasmon absorption band with a maximum at 418 nm, indicating the presence of spherical or roughly spherical Ag NPs.32 The position and shape of the plasmon absorption depends on the particles' size and shape, and
the dielectric constant of the surrounding medium. It can be observed that the surface plasmon absorption maximum, initially at 418 nm, is shifted to a lower wavelength (Figure 2) when sodium citrate is added as reducing agent. The shape of the second UV-vis spectrum is also of interest; it shows a smooth shoulder near 540 nm. The surface plasmon absorptions at 418 nm and 412 nm are consistent with other reports.17,18 The morphology and size of the silver colloids were investigated by TEM analysis. TEM micrographs of samples SHS-1 and SHSC-1 are presented in Figures 3 and 4, respectively. The TEM micrograph in Figure 3 shows agglomerates of small grains and some dispersed nanoparticles. The size of the particles ranges from 8 to 50 nm, with a mean diameter of 24 nm and standard deviation of 6 nm, as illustrated on the histogram on the right-hand side of Figure 3. The TEM micrograph in Figure 4 shows the presence of well-dispersed particles that are more or less spherical, as well as faceted particles. The distribution of size of this sample (Figure 4, right) shows a bimodal distribution of the particle sizes, the first cluster ranging from 15 to 30 nm and the second ranging from 32 to 48 nm, with a mean diameter of 30 nm and standard deviation of 7 nm. In comparison with particle sizes obtained using only hydrazine hydrate (Figure 3, right), the mean diameter does not increase significantly. In consequence, the shift of the surface plasmon absorption maximum from 418 nm (Figure 1) to 412 nm (Figure 2) could be due to a difference in particle shape. That was consistent with the report of Schultz and co-workers,32 in which there was a correlation of the absorption spectra of individual Ag NPs with their size (40-120 nm) and shape (spheres,
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Figure 2. UV–vis absorption spectrum of Ag NPs prepared using a mixture of hydrazine and citrate of sodium as a reducing agent (sample SHCS-1).
Figure 3. TEM image of spherical Ag NPs (left) and their particle size distributions (right). Ag NPs prepared from 1.1 mM AgNO3 solution (sample SHS-1).
decahedrons, triangular truncated pyramids, and platelets) as determined by TEM. They found that spherical and roughly spherical nanoparticles, decahedral or pentagonal nanoparticles, and triangular truncated pyramids and platelets absorb in the blue, green, and red parts of the spectrum, respectively. Table 2 shows the yield of those reactions. For both samples the yield was higher than 80%. It can be observed that the yield of the reaction does not increase significantly when the sodium citrate is added.
The elemental analysis of the Ag NPs was performed using the EDX method in the transmission electron microscope. Figure 5 shows the EDX spectrum of sample SHS-1. All the K and L emission peaks for silver are observed; similar results have been reported by Shahverdi et al1 and Lim et al.33 The CKα, CuKα and CuKβ peaks are also detected. These carbon and copper peaks are due to the transmission electron microscope holding grid. No other obvious peak attributable to impurity is detected. This result indicates that the as-synthesized product is
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Figure 4. TEM image of spherical and faceted Ag NPs (left) and particle size distributions of Ag NPs prepared using a mixture of hydrazine and citrate of sodium as a reducing agent (sample SHSC-1).
Table 2 Surface plasmon absorption maxima of silver nanoparticles prepared with and without sodium citrate Sample
Mean diameter (nm)
λ (nm)
Yield(%)
SHS-1 SHSC-1
24 ± 6 30 ± 7
418 412
82.49 88.56
6.0 mM AgNO3 + SDS.
composed of high-purity Ag NPs. A similar EDX spectrum was obtained for sample SHSC-1. The crystallographic properties such as structure of the silver colloids were determined by HEED analysis. The corresponding HEED pattern of sample SHSC-1 is shown on the right-hand illustration in Figure 6. When the electron diffraction is carried out on a limited number of crystals, one can only observe some spots of diffraction distributed in concentric circles. The ring patterns of sample SHSC-1 with plane distances of 2.36 Å, 2.04 Å, 1.45 Å, 1.23 Å, and 0.94 Å are consistent with the plane families {111}, {200}, {220}, {311}, and {331} of pure facecentered cubic (FCC) silver structure (JCPDS, File No. 4-0787; Powder Diffraction File, International Center for Diffraction Data, Newtown Square, Pennsylvania). A similar HEED spectrum was obtained for sample SHS-1. In both cases the formation of FCC Ag NPs is confirmed. Those results were consistent with the reports of Chen and Gao,12 Mandal et al28 and Hasell et al.34 Effect of the concentration of reducing agent Ag NPs using a mixture of both hydrazine hydrate and sodium citrate at different concentrations were prepared. In both cases, SDS was not used. The pale-red silver colloids obtained with concentration of 1.0 mM (sample SHC-1) and 2.0 mM (sample SHC-2) of sodium citrate were characterized by UV-vis and TEM analysis. The UV-vis spectra of samples SHC-1 and SHC-2 are presented in Figure 7, A and B, in that order. The absorptions of SPR are situated at 406 nm and 405 nm for sample SHC-1 and
sample SHC-2, respectively. This is evidence that the surface plasmon absorption does not change appreciably when the concentration of sodium citrate is doubled. The shapes of the spectra are similar. The values of surface plasmon absorption are consistent with other reports.12,17 In comparison with Figures 1 and 2, shifts of the surface plasmon absorption maximum from 418 nm to 406 nm and from 412 nm to 406 nm are observed. Those shifts can be attributed to the difference in particle sizes of the silver colloids.32 The TEM micrographs of samples SHS-1 and SHSC-1 are presented in Figure 8, A and B, respectively. The TEM results indicate that the nanoparticles consist of agglomerates of small grains with mean diameters between 9 ± 2 and 14 ± 5 nm. However, some particles with diameters larger than 60 nm were formed as a result of aggregation during preparation of the TEM holding grid. Figure 8, A shows agglomerates of small grains and some dispersed nanoparticles that are more or less spherical. Figure 8, B shows spherical small particles, whereas the larger particles have an almost square shape. The particle size histograms of both samples are presented in Figure 9. With a 1.0 mM solution of sodium citrate, the size of the particles ranges from 7 to 20 nm with a mean diameter of 9 ± 2 nm (Figure 9, A). When the concentration is doubled, the size distribution becomes bimodal. Figure 9, B shows that the particles of silver range in size from 3 to 6 nm and from 21 to 35 nm, with mean diameter of 14 nm ± 5 nm. The particle size distributions become narrower and the average particle sizes decrease when the second agent reduction is used. In comparison with particle sizes obtained using only hydrazine (Figure 3) and a mixture of hydrazine‑sodium citrate, the mean diameter decreases significantly. This can be attributed to the double role of sodium citrate as reducing and dispersing agent.17,35 In addition, when the concentration of sodium citrate is doubled, the yield of the reaction is increased by approximately 17% (Table 3). The particle size data, the UV-vis surface plasmon absorption maxima, and the yield are given in Table 3. According to Panacek et al20 the size of Ag NPs can be controlled by using different concentrations of reducing agents and silver ions undergoing reduction. In addition, when SDS was used the mean diameter was increased (samples SHS-1 and SHCS-1).
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Figure 5. EDX spectrum of Ag NPs prepared from 1.1 mM AgNO3 solution (sample SHS-1).
Figure 6. TEM (left) and HEED (right) images of sample SHCS-1 (metal ion concentration and citrate of sodium solution 6.0 mM and 2.0 mM, respectively).
Antibacterial activity Finally, the antimicrobial susceptibility of Ag NPs synthesized was investigated. The Kirby-Bauer diffusion method was used as antimicrobial susceptibility testing method. For antibacterial function testing, S. aureus and S. aureus MRSA (both gram-positive), as well as E. coli and P. aeruginosa (both gramnegative) were used as the bacilli. The average of particle sizes used for the antibacterial test was 9 ± 2 nm, 14 ± 5 nm, 24 ± 6 nm, and 30 ± 7 nm. The presence of nanoparticles at a certain level inhibited bacterial growth by more than 90%. The diameter of inhibition zones (in millimeters) measured is shown in Table 4. The clearzone diameter of the bacterial inhibition zone was correlated to antibiotic activity (oxacillin and ciprofloxacin) for gram-positive and Gram-negative bacteria, respectively. Table 4 shows that clear-zone diameter increased as the concentration of Ag NPs increased, because of the bactericidal activity of Ago. These data are consistent with previously reported studies.36 Antimicrobial activities of the synthesized silver colloidal sols were assessed using the standard dilution micromethod.
Table 5 summarizes the MICs of the silver particles samples tested against gram-positive and gram-negative bacteria. Smaller Ag NPs synthesized using sodium citrate showed a considerable antibacterial activity (14 ppm and 29 ppm). This phenomenon is related to the size of colloidal silver particles. Thus, the 9 ± 2 nm and 14 ± 5 nm silver particles synthesized showed the highest activity against gram-positive and gramnegative bacteria. The lowest antimicrobial effect (216 ppm) obtained with sample (SHCS-1) can be attributed to larger mean diameter (30 ± 8 nm) or a contrary effect of the SDS dispersant, which can be retained on the surface of Ag NPs after the synthesis. Silver particles obtained using only hydrazine (sample SHS-1) show the lowest antimicrobial effect (259 ppm) for E. coli and S. aureus. Nevertheless, this sample shows the highest activity (7 ppm) against P. aeruginosa bacteria. This can be attributed to an effect of the shape of nanoparticles. Similar results have been reported previously by Pal and co-workers.2 The results of antibacterial activities against E. coli, P. aeruginosa, and S. aureus around 14.38 ppm, 6.74 ppm, and 14.38 ppm are close to those reported earlier by Song et al37 and Sondi and Salopek-Sondi,24 around 1 ppm and 10 ppm for gram-negative and gram-positive bacteria, respectively. The mechanism of the bactericidal effect of silver colloid particles against bacteria is not very well known. It is possible that Ag NPs act similarly to the antimicrobial agents used for the treatment of bacterial infections. Those agents show four different mechanisms of action: (1) interference with cell wall synthesis, (2) inhibition of protein synthesis, (3) interference with nucleic acid synthesis, and (4) inhibition of a metabolic pathway.38 Three different mechanisms of action of Ag NPs have been proposed by Feng et al27: First, Ag NPs attach to the surface of the cell membrane and disturb its power functions, such as permeability and respiration.39 Plasmolysis (cytoplasm separated from bacterial cell wall) in P. aeruginosa and the inhibition of bacterial cell wall synthesis in S. aureus bacteria were reported by Song et al.37 It is reasonable to state that the binding of the particles to the bacteria depends on the interaction of the surface area available. Smaller particles having a larger surface area available for interaction will have a stronger bactericidal effect
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Figure 7. UV-vis absorption spectra of samples SHC-1 and SHC-2, respectively. Ag NPs prepared using hydrazine hydrate (2.0 mM) and citrate of sodium solution with a concentrate ranging from 1.0 mM (A) to 2.0 mM (B).
Figure 8. TEM image of spherical Ag NPs obtained with 1.0 mM (A) and 2.0 mM (B) of citrate of sodium solution (samples SHC-1 and SHC-2, respectively).
than will larger particles.40 This is moderately in accordance with the results shown in Table 4. However, an antibacterial effect of the sample SHS-1 can be attributed to the nanoparticle shape. Second, Ag NPs are able to penetrate the bacteria and cause further damage, possibly by interacting with sulfur- and phosphorus-containing compounds such as DNA.41 Raffi and co-workers42 reported TEM images of Ag NPs in the membranes of the bacteria as well as in the interior. In addition, it is believed that Ag NPs after penetration into the bacteria have inactivated
Figure 9. Particle size distributions of Ag NPs (prepared from 1.1 mM AgNO3 solution) and citrate of sodium solution with a concentration ranging from 1.0 mM (A) to 2.0 mM (B).
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Table 3 Mean diameter and surface plasmon absorption maxima of silver nanoparticles obtained at different sodium citrate concentrations Sample
Mean diameter (nm)
λ (nm)
Yield (%)
SHC-1 SHC-2
9±2 14 ± 5
406 405
65.77 82.51
1.1 mM AgNO3.
Table 5 Minimum inhibitory concentrations of silver nanoparticles Bacteria
Escherichia coli CCM 3954 Pseudomonas aeruginosa CCM 3955 Staphylococcus aureus CCM 3953 Staphylococcus aureus MRSA
Minimum inhibitory concentrations (μg/mL) SHC-1 SHC-2 SHS-1
SHCS-1
14.38 14.38 14.38 14.38
215.74 215.74 215.74 215.74
28.77 28.77 28.77 28.77
258.89 6.74 258.89 258.89
Table 4 Zone of inhibition (mm) of silver nanoparticles sols against test strains Bacteria
SHC-1
Escherichia coli CCM 3954 9 ± 1.0 Pseudomonas aeruginosa 9 ± 0.5 CCM 3955 Staphylococcus aureus CCM 3953 12 ± 0.4 Staphylococcus aureus MRSA 11 ± 0.6
SHC-2
SHS-1
SHCS-1
10 ± 0.5 32 ± 0.7 26 ± 0.5 11 ± 0.3 29 ± 1.0 19 ± 0.6 11 ± 0.6 34 ± 0.5 31 ± 1.0 11 ± 1.0 40 ± 0.3 29 ± 0.5
their enzymes, generating hydrogen peroxide and causing bacterial cell death.42,43 In addition, it is believed that the high affinity of silver for sulfur or phosphorus is the key element of its antibacterial property, in that sulfur and phosphorus are found in abundance throughout the cell membrane. Ag NPs react with sulfur-containing proteins inside or outside the cell membrane, which in turn affects cell viability.23 Third, Ag NPs release silver ions, which make an additional contribution to the bactericidal effect.27 In fact, Morones et al44 showed that Ag NPs (where silver is present in the Ag0 form) also contain micromolar concentrations of Ag+, and they have shown that Ag+ and Ag0 both contribute to the antibacterial activity. The mechanism of inhibition by silver ions on microorganisms is partially known. It is believed that DNA loses its replication ability and cellular proteins become inactivated on silver ion treatment.45,46 Higher concentrations of Ag+ ions have been shown to interact with cytoplasmic components and nucleic acids.27,36 The antibacterial effect of Ag NPs determined in this study was found to be similar to that described in the earlier reports.40 The particle size has an effect on microbes; the effect increased with smaller particle size. In our case we have a distribution of particle sizes with a mean diameter ranging from 9 nm to 30 nm. From the results in Tables 2, 3, and 5 we can say that there is a relationship between the size of the nanoparticles and the bacterial activity. For instance, as the nanoparticle size decreases the MIC decreases for all bacteria. In other words, we need a smaller dose of silver suspension nanoparticles to inhibit the growth of bacteria. However, there is an anomalous behavior of sample SHS-1 with average diameter of 24 nm, having a stronger bactericidal activity with an MIC of only 7 μg/mL. This behavior can be attributed to an additional effect of nanoparticle shape, but this relationship has not yet been determined.
Summary Ag NPs with mean diameters of 9 ± 2 nm, 14 ± 5 nm, 24 ± 6 nm, and 30 ± 7 nm were synthesized using hydrazine hydrate and
sodium citrate as reducing agent at room temperature. The nanoparticles were characterized by UV-vis spectroscopy, EDX, and TEM. UV-vis spectra show the characteristic plasmon absorption peak for the Ag NPs between 405 and 418 nm. The EDX of the nanoparticles dispersion confirmed the presence of elemental silver signal. No elemental impurity was detected. HEED confirmed the formation of FCC Ag NPs. Additionally, the antibacterial activity of the nanopartículate dispersion was measured by the Kirby-Bauer method. The results of this study clearly demonstrated that the colloidal Ag NPs inhibited the growth and multiplication of the tested bacteria, including highly multidrug-resistant bacteria such as methicillinresistant S. aureus, S. aureus, E. coli, and P. aeruginosa. A strong antibacterial activity was observed at very low total concentrations of silver (below 7 ppm). Acknowledgment M. Guzman wants to thank the Matter and Materials Department of Université Libre de Bruxelles of Belgium, which provided facilities for use of their transmission electron microscope.
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