J Nanopart Res (2008) 10:13431348 DOI 10.

1007/s11051-008-9428-6

BRIEF COMMUNICATION

Synthesis and antibacterial activity of silver nanoparticles with different sizes

n N. Nin nez-Castan nez o o-Mart G. A. Mart nez-Gutierrez J. R. Mart nez-Mendoza F. Mart Facundo Ruiz

Received: 29 January 2008 / Accepted: 22 May 2008 / Published online: 2 July 2008 Springer Science+Business Media B.V. 2008

Abstract Silver nanoparticles with different sizes (7, 29, and 89 nm mean values) were synthesized using gallic acid in an aqueous chemical reduction method. The nanoparticles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), and ultravioletvisible (UVVis) absorption spectroscopy; the antibacterial activity was assessed using the standard microdilution method, determining the minimum inhibitory concentration (MIC) according to the National Committee for Clinical Laboratory Standards. From the microscopies studies (TEM) we observed that silver nanoparticles have spherical (7 and 29 nm) and

pseudospherical shape (89 nm) with a narrow size distribution. The sizes of the silver nanoparticles were controlled by varying some experimental conditions. It was found that the antibacterial activity of the nanoparticles varies when their size diminishes. Keywords Antibacterial-activity Ag-nanoparticles Synthesis Nanobiotechnology EHS

nez-Castan n (& ) o G. A. Mart gicas, Facultad de Maestria en Ciencias Odontolo a, UASLP, Av. Manuel Nava 2, Estomatolog Zona Universitaria, San Luis Potosi, SLP, Mexico e-mail: mtzcastanon@fciencias.uaslp.mx nez o-Mart N. Nin Instituto de Metalurgia, UASLP, Av. Sierra Leona n, San Luis Potosi, No. 550, Col. Lomas 2a. Seccio SLP, Mexico nez J. R. Mart nez-Mendoza F. Ruiz o-Mart N. Nin lvaro Obrego n 64, Facultad de Ciencias, UASLP, A C.P. 78000 San Luis Potosi, SLP, Mexico nez-Gutierrez F. Mart micas, UASLP, Facultad de Ciencias Qu lvaro Obrego n 64, C.P. 78000 San Luis Potosi, A SLP, Mexico e-mail: del@uaslp.mx

Abbreviations TEM Transmission electron microscopy DLS Dynamic light scattering XRD X-ray diffraction UVVis Ultravioletvisible MIC Minimum inhibitory concentration Introduction Due to the increasing bacterial resistance to classic antibiotics, the investigations on the antibacterial activity of silver nanoparticles have increased (Li c ek et al. 2006). The antibacterial et al. 2005; Pana activity of silver species has been well known since ancient times (Holt and Bard 2005; Shrivastava et al. 2007) and it has been demonstrated that, in low concentrations, silver is non toxic to human cells (Zhang et al. 2003; Pal et al. 2007). The actual bactericide mechanism of silver nanoparticles is not

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well known. Some researchers support the idea that silver species release Ag+ ions and they interact with the thiol groups in bacteria proteins, affecting the replication of DNA (Marini et al. 2007). It has also been reported that Ag+ ions uncouple the respiratory chain from oxidative phosphorylation or collapse the proton-motive force across the cytoplasmic membrane (Holt and Bard 2005). Silver nanoparticles interactions with bacteria are dependent on the size c ek et al. 2006; and shape of the nanoparticles (Pana Morones et al. 2005; Pal et al. 2007). In this work, a method to synthesize silver nanoparticles using gallic acid was developed; with this method, we are able to obtain silver nanoparticles with different sizes by just controlling some reaction parameters. The obtained nanoparticles were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), X-ray diffraction (XRD), and ultraviolet visible (UVVis) absorption spectroscopy. An antibacterial activity test (NCCLS M7-A4, 1997) was conducted to observe differences in antibacterial activity among the nanoparticles obtained.

250-mL reaction vessel inside a UV light reactor. Under magnetic stirring, 0.01 g of gallic acid in 10 mL of deionized water was added to the Ag+ solution and the mixture was irradiated with UV light (254 nm, 15 W) for 30 min. Then, the solution was heated for 30 min at 80 C. Characterization The produced nanoparticles were characterized by UVVis spectroscopy using a S2000-UVVis spectrometer from OceanOptics Inc. DLS analysis was performed in a Malvern Zetasizer Nano ZS. TEM analysis was performed on a JEOL JEM-1230 at an accelerating voltage of 100 kV. X-ray diffraction patterns were recorded with a GBC-Difftech MMA ) diffractometer. The nickel ltered Cu Ka (k = 1.54 A radiation was used at 34.2 mA and 35 kV. All the characterization analyses except X-ray diffraction were made using the obtained aqueous dispersions of silver nanoparticles. Antibacterial test

Experimental section Synthesis method 7-nm silver nanoparticles A total of 100 mL of AgNO3 0.001 M was placed in a 250-mL reaction vessel. Under magnetic stirring, 10 mL of deionized water containing 0.01 g of gallic acid was added to the Ag+ solution. After the addition of gallic acid, the pH value of the solution was immediately adjusted to 11 using a 1.0 M solution of NaOH. (b) 29-nm silver nanoparticles A total of 0.0169 g of AgNO3 was dissolved in 100 mL of deionized water and this solution was placed in a 250-mL reaction vessel. A total of 0.01 g of gallic acid was dissolved in 10 mL of deionized water and under magnetic stirring were added to the Ag+ solution. After the addition of gallic acid, the pH value of the solution was immediately adjusted to 10 using a 7.7 M solution of NH4OH. (c) 89-nm silver nanoparticles A total of 0.0169 g of AgNO3 was dissolved in 100 mL of deionized water and this solution was placed in a (a)

The antimicrobial activity of the synthesized nanoparticles was tested using the standard microdilution method, which determines the minimum inhibitory concentration (MIC) leading to the inhibition of bacterial growth (NCCLS M7-A4, 1997). Disposable microtitration plates were used for the tests. The composites in dispersion form were diluted 2128 times with 100 lL of MuellerHinton broth inoculated with the tested bacteria at a concentration of 105 CFU/mL. The MIC was read after 24 h of incubation at 37 C as the MIC of the tested substance that inhibited the growth of the bacterial strain. The dispersions were used in the form in which they had been prepared. Therefore, control bactericidal tests of solutions were performed containing all the reaction components.

Results and discussion Synthesis In this work, gallic acid was used as a reducing and stabilizing agent, the oxidation reaction of phenol

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groups in gallic acid was responsible for the reduction of silver ions, and the produced quinoid compound with a ketoenol-system could be adsorbed on the surface of silver nanoparticles accounting for their stabilization (Wang et al. 2007). In the preparation of 7- and 29 nm silver nanoparticles the reduction reaction was carried out at pH 11 and 10, respectively; at this pH value it is expected that phenol groups are ionized so that the reduction reaction is very fast and the particles obtained are spherical (Fig. 1a, b). When 89-nm silver nanoparticles were prepared, the pH value was not raised, and the ionization of phenol groups was achieved using UV light. This photoionization lead us to a slower reduction reaction and the obtained silver nanoparticles did not have the spherical morphology; instead, a
Fig. 1 TEM images and DLS analysis (insets) of the silver nanoparticles synthesized in this work (a) 7-, (b) 29- and (c) 89-nm silver nanoparticles

polygonal morphology was achieved. The aging process using temperature promotes the growth and narrowing of the size distribution of these particles (Fig. 1c). TEM and DLS analysis Figure 1 shows the TEM images and the results of DLS analysis. The nanoparticles prepared (7, 29, and 89 nm) have a narrow size distribution and present spherical (7 and 29 nm) and pseudospherical shape (89 nm). In DLS analyses (insets in Fig. 1ac) 7-nm silver nanoparticles present a peak centered at 7.2 nm with 1.7 nm of width. Also 29-nm silver nanoparticles present a peak centered at 29 nm with 9.5 nm of width. Finally, 89-nm silver nanoparticles present a

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XRD analysis Figure 2 shows the diffraction pattern obtained for the 29-nm silver nanoparticles; this analysis was made to conrm the identity of the products. The diffractogram shows peaks corresponding to elemental silver (JCPS 04-0783). Rietveld analysis (Lutterotti et al. 1999) made using this diffraction pattern conrms the results obtained by DLS analysis, the nanoparticles have a mean diameter around 25 nm. The 7- and 89-nm silver nanoparticles present similar results (12 and 92 nm, respectively). UVVis analysis The absorption spectra of the silver nanoparticles are presented in Fig. 3. All samples present the characteristic surface plasmon of silver nanoparticles nez-Castan n et al. 2005), 7-nm silver nanoo (Mart particles present a narrow band with a maximum at 410 nm, 29-nm silver nanoparticles also have a narrow band, which presents a maximum at 425 nm, and 89-nm silver nanoparticles present a wider band with a maximum at 490 nm. It is reported that the absorption spectrum of spherical silver nanoparticles present a maximum between 420 and 450 nm with a blue or red shift when particle size diminishes or increases, respectively (Pal et al. 2007; Jana et al.

Absorption (arb. units)

peak centered at 89 nm with 24 nm of width. These results conrm a good stabilization of the nanoparticles by gallic acid.

Ag7nm Ag29nm Ag89nm

400

500

600

700

800

Wavelength (nm)

Fig. 3 UVVis spectra of the silver nanoparticles synthesized in this work

nnichsen et al. 2002). For 1999; Manna et al. 2001; So this reason, 7-nm silver nanoparticles present a plasmon, which is blue shifted with respect to that of 29-nm silver nanoparticles. The width of each plasmon is related to the size distribution of the nanoparticles. For irregular particles (non spherical), two or more plasmon bands are expected depending on the symmetry of the particles (Pal et al. 2007). For the 89-nm silver nanoparticles, this could be the reason why the width of its plasmon does not correspond with the narrow size distribution found in DLS analysis. The faceted 89-nm silver nanoparticles raise multiples bands, which combine and form a wider band. Antibacterial results MIC values were obtained for the synthesized nanoparticles tested against E. coli (Gram negative bacteria, ATCC 25922) and S. aureus (Gram positive bacteria, ATCC 25923). The results are presented as average values in Table 1 (the KruskalWallis test was applied). From Table 1, we can see that the 7-nm silver nanoparticles present the best antibacterial against E. coli and S. aureus. Because of their size, 7-nm silver nanoparticles can easily reach the nuclear content of bacteria and they present the greatest surface area; therefore the contact with bacteria is the greatest (Lok et al. 2006). This could be the reason why they present the best antibacterial activity. For solid systems, some authors argue that Ag+ ions

Ag29nm
Intensity (arb. units)

30

40

50

60

70

80

2() (degress)

Fig. 2 Diffraction pattern of the 29-nm silver nanoparticles

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J Nanopart Res (2008) 10:13431348 Table 1 Minimum inhibition concentrations of Ag nanoparticles Sample Minimum inhibition concentration (lg/mL) Bacteria E. coli 7-nm silver nanoparticles 29-nm silver nanoparticles 89-nm silver nanoparticles Gallic acid 6.25 13.02 11.79 a S. aureus 7.5 16.67 33.71 a

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6.75 lg/mL for E. coli and S. aureus, respectively), although our particles are bigger; this difference could be due to the strains used: we used ATCC strains while they used strains from the Collection of Samples of the Masaryk University, Brno, Czech Republic. Conclusions Silver nanoparticles with different sizes (7, 29, and 89 nm) were synthesized using gallic acid in an aqueous chemical reduction method and characterized using TEM, DLS, X-ray diffraction, and UVVis absorption spectroscopy. The antibacterial activity of the silver nanoparticles was analyzed and it was found that it can be modied with the size of silver nanoparticles. It decreases with an increase of the particle size.
Acknowledgements This work was partially supported by n (FAI) of Universidad Fondo de Apoyo a la Investigacio noma de San Luis Potos (UASLP) and CONACYTAuto nez would like to thank CONACYT for o-Mart 61257. N. Nin the scholarship No. 185006.

a No antibacterial activity was found with the concentrations used in this work

released from the surface of Ag nanoparticles are responsible for their antibacterial activity (Morones et al. 2005; Lee et al. 2005; MacKeen et al. 1987; Li et al. 2006; Jeong et al. 2005); for aqueous systems (as the system tested here), the results found by Lok et al. (2006) show that the antibacterial activity of Ag+ ions is low at the concentrations levels reached by releasing, and the presence of nanoparticles is vital, which reinforces the idea that the greatest the surface area the greatest the antibacterial activity (Jeong et al. 2005; Thiel et al. 2007). In order to demonstrate that the silver nanoparticles reported here have a direct contact with bacteria, our group is developing a methodology to image them by atomic force microscopy. Atomic force microscopy enables the direct observation of bacteria without an alteration of the cellular content. This work is still in progress. The MIC of all samples is lower when tested against E. coli than when tested against S. aureus. These results can be explained on the basis of the differences on the cellular wall of each strain; the cellular wall for gram-positive strains is wider than the cellular wall for gram-negative strains (Thiel et al. 2007). These results agree with those presented by Kim et al. (2007). For E. coli, there is no signicant difference between the MIC of 29 and 89-nm silver nanoparticles. For the 10-nm silver nanoparticles, there is no signicant difference of the MIC against each bacteria. Here again the cellular wall content plays an important role in these results. c ek et al. (2006) reported the antibacterial Pana activity of 25 nm silver nanoparticles using the same method reported here, but their results, compared with our 29-nm silver nanoparticles results, are slightly different. They report a lower MIC (3.38 and

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