Synthesis and antibacterial activity of silver nanoparticles with different sizes

Abstract

Silver nanoparticles with different sizes (7, 29, and 89nm 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 ultraviolet–visible (UV–Vis) 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 29nm) and pseudospherical shape (89nm) 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.

Full text

BRIEF COMMUNICATION
Synthesis and antibacterial activity of silver nanoparticles
with different sizes
G. A. Martı
´nez-Castan
˜o
´nÆN. Nin
˜o-Martı
´nez Æ
F. Martı
´nez-Gutierrez ÆJ. R. Martı
´nez-Mendoza Æ
Facundo Ruiz
Received: 29 January 2008 / Accepted: 22 May 2008 / Published online: 2 July 2008
ÓSpringer 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 transmis-
sion electron microscopy (TEM), dynamic light
scattering (DLS), X-ray diffraction (XRD), and ultravi-
olet–visible (UV–Vis) absorption spectroscopy; the
antibacterial activity was assessed using the standard
microdilution method, determining the minimum inhib-
itory 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 nanopar-
ticles varies when their size diminishes.
Keywords Antibacterial-activity 
Ag-nanoparticles Synthesis Nanobiotechnology 
EHS
Abbreviations
TEM Transmission electron microscopy
DLS Dynamic light scattering
XRD X-ray diffraction
UV–Vis Ultraviolet–visible
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
et al. 2005; Pana
´c
ˇek et al. 2006). The antibacterial
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
G. A. Martı
´nez-Castan
˜o
´n(&)
Maestria en Ciencias Odontolo
´gicas, Facultad de
Estomatologı
´a, UASLP, Av. Manuel Nava 2,
Zona Universitaria, San Luis Potosi, SLP, Mexico
e-mail: mtzcastanon@fciencias.uaslp.mx
N. Nin
˜o-Martı
´nez
Instituto de Metalurgia, UASLP, Av. Sierra Leona
No. 550, Col. Lomas 2a. Seccio
´n, San Luis Potosi,
SLP, Mexico
N. Nin
˜o-Martı
´nez J. R. Martı
´nez-Mendoza F. Ruiz
Facultad de Ciencias, UASLP, A
´lvaro Obrego
´n 64,
C.P. 78000 San Luis Potosi, SLP, Mexico
F. Martı
´nez-Gutierrez
Facultad de Ciencias Quı
´micas, UASLP,
A
´lvaro Obrego
´n 64, C.P. 78000 San Luis Potosi,
SLP, Mexico
e-mail: fidel@uaslp.mx
123
J Nanopart Res (2008) 10:1343–1348
DOI 10.1007/s11051-008-9428-6

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 mem-
brane (Holt and Bard 2005). Silver nanoparticles
interactions with bacteria are dependent on the size
and shape of the nanoparticles (Pana
´c
ˇek et al. 2006;
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 (UV–Vis) absorption spectroscopy. An anti-
bacterial activity test (NCCLS M7-A4, 1997) was
conducted to observe differences in antibacterial
activity among the nanoparticles obtained.
Experimental section
Synthesis method
(a) 7-nm silver nanoparticles A total of 100 mL of
AgNO
3
0.001 M was placed in a 250-mL reaction
vessel. Under magnetic stirring, 10 mL of deion-
ized 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 solu-
tion of NaOH.
(b) 29-nm silver nanoparticles A total of 0.0169 g
of AgNO
3
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 NH
4
OH.
(c) 89-nm silver nanoparticles A total of 0.0169 g
of AgNO
3
was dissolved in 100 mL of deion-
ized water and this solution was placed in a
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
UV–Vis spectroscopy using a S2000-UV–Vis spec-
trometer 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 filtered Cu K
a
(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
The antimicrobial activity of the synthesized nano-
particles 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 2–128
times with 100 lL of Mueller–Hinton broth inocu-
lated with the tested bacteria at a concentration of
10
5
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
1344 J Nanopart Res (2008) 10:1343–1348
123

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, respec-
tively; 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 nanoparti-
cles did not have the spherical morphology; instead, a
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 1shows 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. 1a–c) 7-nm
silver nanoparticles present a peak centered at 7.2 nm
with 1.7 nm of width. Also 29-nm silver nanoparti-
cles present a peak centered at 29 nm with 9.5 nm of
width. Finally, 89-nm silver nanoparticles present 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
J Nanopart Res (2008) 10:1343–1348 1345
123

peak centered at 89 nm with 24 nm of width. These
results confirm a good stabilization of the nanopar-
ticles by gallic acid.
XRD analysis
Figure 2shows the diffraction pattern obtained for the
29-nm silver nanoparticles; this analysis was made to
confirm 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 confirms 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).
UV–Vis analysis
The absorption spectra of the silver nanoparticles are
presented in Fig. 3. All samples present the charac-
teristic surface plasmon of silver nanoparticles
(Martı
´nez-Castan
˜o
´n et al. 2005), 7-nm silver nano-
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.
1999; Manna et al. 2001;So
¨nnichsen et al. 2002). For
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 nanopar-
ticles 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 Kruskal–Wallis 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
30
Intensity (arb. units)
2(θ) (degress)
Ag29nm
40 50 60 70 80
Fig. 2 Diffraction pattern of the 29-nm silver nanoparticles
400
Absorption (arb. units)
Wavelength (nm)
Ag7nm
Ag29nm
Ag89nm
500 600 700 800
Fig. 3 UV–Vis spectra of the silver nanoparticles synthesized
in this work
1346 J Nanopart Res (2008) 10:1343–1348
123

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 micros-
copy. 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
significant difference between the MIC of 29 and
89-nm silver nanoparticles. For the 10-nm silver
nanoparticles, there is no significant difference of the
MIC against each bacteria. Here again the cellular
wall content plays an important role in these results.
Pana
´c
ˇek et al. (2006) reported the antibacterial
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
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 character-
ized using TEM, DLS, X-ray diffraction, and UV–Vis
absorption spectroscopy. The antibacterial activity of
the silver nanoparticles was analyzed and it was
found that it can be modified with the size of silver
nanoparticles. It decreases with an increase of the
particle size.
Acknowledgements This work was partially supported by
Fondo de Apoyo a la Investigacio
´n (FAI) of Universidad
Auto
´noma de San Luis Potosı
´(UASLP) and CONACYT-
61257. N. Nin
˜o-Martı
´nez would like to thank CONACYT for
the scholarship No. 185006.
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