J Biol Inorg Chem (2007) 12:527–534
DOI 10.1007/s00775-007-0208-z
ORIGINAL PAPER
Silver nanoparticles: partial oxidation and antibacterial activities
Chun-Nam Lok Æ Chi-Ming Ho Æ Rong Chen Æ Qing-Yu He Æ Wing-Yiu Yu Æ
Hongzhe Sun Æ Paul Kwong-Hang Tam Æ Jen-Fu Chiu Æ Chi-Ming Che
Received: 28 September 2006 / Accepted: 28 December 2006 / Published online: 16 February 2007

SBIC 2007
Abstract The physical and chemical properties of sil-
ver nanoparticles that are responsible for their antimi-
crobial activities have been studied with spherical silver
nanoparticles (average diameter approximately 9 nm)
synthesized by the borohydride reduction of Ag+ ions,
in relation to their sensitivity to oxidation, activities
towards silver-resistant bacteria, size-dependent activ-
ities, and dispersal in electrolytic solutions. Partially
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-007-0208-z) contains supplementary
material, which is available to authorized users.
Chun-Nam Lok and Chi-Ming Ho equally contributed to this
work.
C.-N. Lok
C.-M. Ho
R. Chen
Q.-Y. He
W.-Y. Yu

H. Sun
J.-F. Chiu (&)
C.-M. Che (&)
The Open Laboratory of Chemical Biology of the Institute
of Molecular Technology for Drug Discovery and Synthesis,
The University of Hong Kong,
Pokfulam Road, Hong Kong, China
e-mail: jfchiu@hkucc.hku.hk
C.-M. Che
e-mail: cmche@hku.hk
C.-M. Ho
R. Chen
Q.-Y. He
W.-Y. Yu
H. Sun

C.-M. Che
Department of Chemistry,
The University of Hong Kong,
Pokfulam Road, Hong Kong, China
C.-N. Lok
J.-F. Chiu
Department of Anatomy,
The University of Hong Kong,
Pokfulam Road, Hong Kong, China
P. K.-H. Tam
Department of Surgery, The University of Hong Kong,
Pokfulam Road, Hong Kong, China
(surface) oxidized silver nanoparticles have antibacte-
rial activities, but zero-valent nanoparticles do not. The
levels of chemisorbed Ag+ that form on the particle’s
surface, as revealed by changes in the surface plasmon
resonance absorption during oxidation and reduction,
correlate well with the observed antibacterial activities.
Silver nanoparticles, like Ag+ in the form of AgNO3
solution, are tolerated by the bacteria strains resistant
to Ag+. The antibacterial activities of silver nanoparti-
cles are related to their size, with the smaller particles
having higher activities on the basis of equivalent silver
mass content. The silver nanoparticles aggregate in
media with a high electrolyte content, resulting in a loss
of antibacterial activities. However, complexation with
albumin can stabilize the silver nanoparticles against
aggregation, leading to a retention of the antibacterial
activities. Taken together, the results show that the
antibacterial activities of silver nanoparticles are
dependent on chemisorbed Ag+, which is readily
formed owing to extreme sensitivity to oxygen. The
antibacterial activities of silver nanoparticles are
dependent on optimally displayed oxidized surfaces,
which are present in well-dispersed suspensions.
Keywords Silver nanoparticles
Silver ions

Antibacterial agents
Surface plasmon resonance
absorption
Oxidation
Abbreviations
BSA Bovine serum albumin
Hepes N-(2-Hydroxyethyl)piperazine-
N¢-ethanesulfonic acid
MIC Minimum inhibitory concentration
Nano-Ag Silver nanoparticles
SPR Surface plasmon resonance
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Introduction
The bioactivity of nano-sized metal particles and their
uses as molecular probes are research areas of growing
interest [1–2]. Owing to their large surface area and
high reactivity compared with the bulk solid, nano-
sized metal particles exhibit remarkable physical,
chemical, and biological properties. In particular, the
optical properties of silver nanoparticles (nano-Ag),
which vary according to their size and shape, as well as
their chemical environment, have been intensively
studied [3–6]. In addition, there is increasing interest in
utilization of nano-Ag as a special class of biocidal
agents, owing to the extraordinary antimicrobial
properties of silver [7–15]. For example, wound dress-
ings containing sputtered nanocrystalline silver mate-
rials are currently used in clinical practice to suppress
the microbial infection of burn wounds [7]. There are
also a number of silver composite materials that con-
tain nano-Ag as the active antimicrobial ingredient [8–
13, 33].
The antimicrobial activities of silver-containing
materials are often studied in terms of their Ag+ con-
tent. Ag+ has been reported to interact with cytoplas-
mic components and nucleic acids, to inhibit
respiratory chain enzymes, and to interfere with
membrane permeability (reviewed in [16]). Recently,
Dibrov and Häse [17] have demonstrated that low
concentrations of Ag+ induce a massive proton leakage
through the membrane of Vibrio cholerae, resulting in
a collapse of proton motive force. Holt and Bard [18]
examined the interaction of Ag+ with the respiratory
chain enzymes of Escherichia coli cells by electro-
chemical techniques, demonstrating that Ag+ inhibits
at a low potential point of the respiratory chain pos-
sibly the NADH dehydrogenase of complex I. Al-
though silver is a very potent and rapidly acting
antibacterial agent, silver-resistant bacteria exist [19].
Silver-resistant bacterial strains have been isolated
from silver mines, from clinics, as well as in the labo-
ratory by stepwise selection against increasing con-
centrations of silver [20–22]. The resistance is
associated with active efflux and the periplasmic
sequestration of Ag+ ions, reduction of Ag+ ions to a
colloidal form, and deficiency in outer-membrane po-
rins [20–22].
Several studies have investigated the interactions of
nano-Ag with bacteria. Electron microscopy studies by
Sondi and Salopek [23] and Morones et al. [24] re-
vealed that the majority of the nano-Ag were localized
on the membranes of treated E. coli cells. Dark-field
optical microscopy studies by Xu et al. [25] demon-
strated the association of nano-Ag to Pseudomonas
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J Biol Inorg Chem (2007) 12:527–534
aeruginosa when added at picomolar levels. Through
proteomic and biochemical studies, we have previously
demonstrated that the treatment of E. coli cells with
nanomolar concentrations of nano-Ag results in an
immediate dissipation of the proton motive force,
killing the cells within minutes [26]. Such a biological
mode of action is similar to that found for AgNO3
solution, suggesting that nano-Ag represent a special
physicochemical system which confers their antimi-
crobial activities via Ag+.
One fundamental aspect that remains to be estab-
lished concerns the identification of exactly which of
the physical and chemical properties of nano-Ag are
responsible for the effective manner in which they
deliver their antimicrobial activities. Henglein [5, 6]
has studied the oxidation of nano-Ag, and suggested
that nano-Ag are highly sensitive to oxygen, resulting
in formation of partially oxidized nano-Ag with
chemisorbed Ag+. In the present study, zero-valent
(reduced nano-Ag) and partially oxidized nano-Ag
(oxidized nano-Ag)1 were prepared with reference to
the study by Henglein [5] and the antibacterial activi-
ties were compared. The antibacterial activities in
relation to the particle size and the dispersal in elec-
trolytic solutions were also investigated. In addition,
the effects of nano-Ag on bacteria strains resistant to
Ag+ ions are reported.
Materials and methods
Zero-valent nano-Ag
Reduced nano-Ag were synthesized by the borohy-
dride reduction of AgNO3 in the presence of citrate as
a stabilizing agent [3, 26] under oxygen-free conditions.
Sodium citrate solution (0.7 mM, 100 mL) was boiled
for 1 h and cooled to room temperature under a stream
of nitrogen. AgNO3 (0.1 M, 100 lL) was added to the
citrate solution with vigorous stirring, and Ag+ reduc-
tion was initiated by the addition of 100 lL NaBH4
(5 mg/mL, 4 C). The solution became yellow imme-
diately and showed a surface plasmon resonance (SPR)
absorption peak at 375 nm and a shoulder at about
410 nm. The reduction was allowed to proceed under
nitrogen for 4 h, after which time no further spectral
changes were observed.
1
The term ‘‘oxidized nano-Ag’’ used throughout the text de-
notes the partially oxidized form of nano-Ag which possesses
chemisorbed Ag+ ions.
J Biol Inorg Chem (2007) 12:527–534
Partially oxidized nano-Ag
Reduced nano-Ag prepared as described in the previ-
ous section were transferred to a rubber-sealed test
tube and oxidized by bubbling with oxygen for 30 min.
The SPR absorption peak gradually shifted to a longer
wavelength, with a broadening of the band width and a
lowering of the maximum absorbance (Fig. 1b). The
oxidation reaction was complete when no further
spectral changes were observed. The solution finally
showed a SPR absorption at 397 nm. The redshift of
the absorption peak after exposure to oxygen is
attributed to the partial oxidation of nano-Ag and
indicates that they are highly sensitive to oxygen [5, 6].
Nanoparticles were examined using a Philips Tecnai
20 transmission electron microscope equipped with
energy-dispersive analysis of X-rays. The transmission
electron microscopy micrographs revealed that the
Fig. 1 Oxidation–reduction of silver nanoparticles (nano-Ag)
and their antibacterial activities. a The bacterial cell survival
(black bars) and ATP contents (gray bars) in the bacterial
culture treated with reduced nano-Ag (1), oxidized nano-Ag (2),
and the oxidized nano-Ag reduced by NaBH4 (3). The colony-
forming units and ATP content obtained from cells treated with
buffer control were approximately 1 · 108 cfu/mL and 4 nmol/
mg protein, respectively. These values are set at 100%. The data
shown are means and standard deviations of three independent
experiments. b The surface plasmon resonance (SPR) absorption
of reduced nano-Ag (solid line), oxidized nano-Ag (dotted line),
and oxidized nano-Ag reduced by NaBH4 (dashed line)
529
nano-Ag were spherical with an average diameter of
9.2 ± 2.8 nm. Determination of the particle concen-
trations was based on calculations using the density of
silver (10.5 g/cm3 at 27 C). For nanoparticles with an
average diameter of 9.2 nm, the physical attributes of
the nanoparticles can be summarized as follows:
10.8 lg/mL Ag is equivalent to 100 lM Ag or 4 nM
nanoparticles. The number of silver atoms in a 9.2 nm
nano-Ag is 2.4 · 105, also calculated using the density
of silver.
Large oxidized nano-Ag were first prepared by
reducing 500 mL of 100 lM AgNO3 with 10 mL of 1%
(w/v) sodium citrate solution under a nitrogen atmo-
sphere. The solution showed a SPR absorption peak
at 412 nm. After exposure to oxygen for 30 min, the
resulting solution showed a redshift of the SPR absorp-
tion peak to 415 nm, attributable to a partial oxidation of
nanoparticles (see the supplementary material). Spher-
ical particles of average diameter 62 ± 18 nm (examined
by transmission electron microscopy) were obtained
with this method.
Free silver content in oxidized nano-Ag solutions
The oxidized nano-Ag were allowed to pass through
YM-3 Centricon membranes (molecular weight cutoff
3,000; Millipore). After centrifugation, a clear filtrate
was obtained, while nano-Ag were retained on the
membrane. The silver content of the filtrate was
determined to be 0.1 lM by inductively coupled plas-
ma mass spectrometry (Agilent 7400a). This value is
equal to 0.1% of the total silver loaded. Importantly,
this filtrate did not exhibit significant antibacterial
activity (less than 10% decrease in colony-forming
units and ATP content). The efficiency of recovery of
the Centricon membranes was determined by filtering
10 ppb AgNO3 solution. The concentration of silver in
the filtrate was 9 ppb, corresponding to 90% recovery.
Bacteria strains
E. coli (K12 strain, MG1655) was obtained from the
E. coli Genetic Stock Center, Yale University (New
Haven, CT, USA). The silver-resistant strains J53
(pMG101) [21] and 116AgNO3R [22] were provided by
Simon Silver, Chicago, USA and Xian-Zhi Li, Ottawa,
Canada, respectively.
Antibacterial assays
For experiments on antibacterial activities of reduced
or oxidized nano-Ag (Fig. 1), E. coli cells at log
phase were washed and suspended in 50 mM sodium
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N-(2-hydroxyethyl)piperazine-N¢-ethanesulfonic acid
(Hepes) buffer (pH 7.0) containing 5 mM glucose to an
optical density at 650 nm (OD650) of 1. Then, the cells
were injected into the indicated preparations of nano-
Ag under nitrogen in test tubes sealed with a rubber
septum (final OD650 ~ 0.1). After 10 min, aliquots of
the treated cells were diluted appropriately with Luria–
Bertani medium containing 1 mM glutathione to dis-
solve the nanoparticles and quench the silver activity,
and were then spread on agar plates. Bacterial colonies
were counted after incubation overnight at 37 C.
Aliquots of the bacterial culture were also taken for
measurement of ATP and protein contents as previ-
ously described [26].
For experiments on the effects of nanoparticle size
on the antibacterial activities (Fig. 2), E. coli cells at
log phase were suspended in 50 mM sodium Hepes
buffer (pH 7.0) containing 5 mM glucose to OD650~0.1.
After treatment with nano-Ag, aliquots were taken and
diluted 100-fold with Luria–Bertani medium supple-
mented with 1 mM glutathione. The diluted bacteria
culture was allowed to grow at 37 C and OD650 was
measured.
Antibacterial assays were also performed on grow-
ing E. coli cells in culture (Fig. 3, Table 1). E. coli cells
were grown at 37 C with shaking to the early expo-
nential phase (OD650 ~ 0.1) in M9 defined medium
(47 mM Na2HPO4, 22 mM KH2PO4, 8.6 mM NaCl,
18 mM NH4Cl, 2 mM MgCl2, 0.1 mM CaCl2, supple-
mented with 0.4% glucose). Nano-Ag or silver nitrate
was added and growth was monitored by measuring
OD650. Nano-Ag aggregated in media with a high salt
content and lost their antibacterial activities. To sta-
bilize the nano-Ag in the M9 medium, bovine serum
albumin (BSA) was premixed with the nanoparticles
before adding them to the medium, and the final con-
centrations in the assays were typically 15 lM.
Fig. 2 Antimicrobial activities of oxidized nano-Ag with diam-
eters of 9.2 and 62 nm. Exponentially growing Escherichia coli
Results and discussion cells (OD650 ~ 0.1) were treated with oxidized nano-Ag of
average diameter 9.2 nm (a) or 62 nm (b) in 1 mL buffer
containing 50 mM N-(2-hydroxyethyl)piperazine-N¢-ethanesulf-
Oxidation–reduction of nano-Ag onic acid (Hepes), pH 7.0 and 5 mM glucose for 10 min at room
and their antibacterial activities temperature. Various concentrations of nano-Ag were prepared
by threefold serial dilution from a solution containing 108 lg/mL
Gibbard [27] documented that polished silver or Ag. Aliquots of treated cells were diluted by 100-fold with Luria–
Bertani medium containing 1 mM glutathione. The bacteria
molten silver cooled under hydrogen did not exhibit were allowed to grow at 37 C and OD650 was monitored
antibacterial activity; however, when molten silver continuously. c OD650 readings were taken at 15 h, by which time
was cooled in air, antibacterial activity was observed. the differences between the antibacterial activities of oxidized
Djokic and Burrell [28] showed that only silver films nano-Ag of the two sizes at various concentrations can be most
easily distinguished. Solid circles 9.2 nm, empty circles 62 nm
containing silver oxides (e.g., sputtered or electrooxi-
dized silver) showed antimicrobial activities. Fan and It is of fundamental concern to determine whether the
Bard [29] demonstrated that oxidized silver films can antibacterial activities of nano-Ag are similarly
be solubilized in the form of Ag(I) and Ag(0) species. dependent on their oxidation, considering that they

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Fig. 3 Oxidized nano-Ag aggregation in medium containing
high salt levels and the loss of antibacterial activities. a The SPR
absorption of oxidized nano-Ag in Hepes buffer (dashed line),
M9 medium (dotted line) and of those premixed with bovine
serum albumin (BSA) in M9 medium (solid line). b Shift of SPR
absorption maxima (Dkmax) of oxidized nano-Ag upon increasing
the concentration of BSA. c Antibacterial activities of oxidized
nano-Ag in M9 medium. E. coli cells were allowed to grow at
37 C to early exponential phase (OD650 ~ 0.1) and were treated
as indicated at the time marked with an arrow. OD650 of the
bacteria was monitored continuously. Empty squares untreated,
empty circles oxidized nano-Ag, solid circles oxidized nano-Ag
premixed with BSA, solid squares BSA alone
531
Table 1 Minimum inhibitory concentrations of silver nanopar-
ticles (nano-Ag) and AgNO3 against silver-sensitive and silver-
resistant Escherichia coli cells
Silver-sensitive Silver-resistant strains 116
strains 116S, J53 AgNO3R, J53 (pMG101)
AgNO3 3 lM >1 mM
Oxidized nano-Ag 2 nM >80 nMa
Minimum inhibitory concentration is defined as the minimum
concentration of AgNO3 or oxidized nano-Ag that completely
inhibits growth of the E. coli cells after 16-h incubation in M9
medium
a
The highest particle concentration of well-dispersed nano-Ag
that can be applied
are particularly sensitive to oxygen [5]. To this end,
reduced nano-Ag and oxidized nano-Ag were syn-
thesized and their antibacterial activities were mea-
sured in situ. Reduced nano-Ag were prepared by the
borohydride reduction of AgNO3 (100 lM) under a
nitrogen atmosphere, and particles with a diameter of
9.2 ± 2.8 nm were obtained. For the preparation of
partially oxidized nano-Ag, a portion of the reduced
nano-Ag was bubbled with oxygen for 30 min to
oxidize the nanoparticles. A buffer control was pre-
pared in the same way, with AgNO3 replaced by
NaNO3 in the reaction mixtures. All the preparations
were finally purged with nitrogen in sealed test tubes
to maintain the same experimental conditions. Con-
centrated suspensions of E. coli cells in sodium Hepes
buffer (pH 7.0) were injected into the nano-Ag
preparations, as well as the buffer control. After
10 min, aliquots were withdrawn and then diluted in
nutrient broth. The cell survivals were measured by
the colony-counting method. Figure 1a shows that
E. coli bacteria colony formation was not affected
by treatment of reduced nano-Ag. In contrast,
cells treated with oxidized nano-Ag showed marked
decreases in colony formation.
The rapid depletion of cellular ATP content within
E. coli cells is a characteristic of energy-uncoupling
effects, which is observed upon the addition of low
concentrations of silver [26]. Consistent with the cell
survival assays, the ATP levels within cells that had
been treated with reduced nano-Ag were similar to
that of the control (Fig. 1a). In contrast, ATP levels
in cells treated with oxidized nano-Ag were lowered
by more than 90%. Similar inhibition of cell survival
levels and depletion of ATP content were obtained
when cells were treated with oxidized nano-Ag pre-
pared under ambient conditions [26]. The antibacte-
rial activities of Ag+ in the form of AgNO3 solution
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532
were essentially unaffected by the presence or ab-
sence of oxygen, as under both sets of conditions,
there were more than 90% reductions in cell survival
and cellular ATP levels (see supplementary mate-
rial).
Measurement of SPR absorption of the nanoparti-
cles analogous to the studies performed by Henglein
[5] revealed a correlation between the SPR absorption
spectra obtained and the degree to which the nano-
particles had been oxidized (Fig. 1b). Reduced nano-
Ag exhibited a SPR absorption peaked at 375 nm.
Subsequent exposure to oxygen led to a rapid shift of
the absorption peak to 398 nm, with broadening of the
band width and lowering of the maximum absorption.
This indicated that nano-Ag were sensitive to oxygen.
This SPR absorption spectrum was similar to that of
nano-Ag prepared in ambient conditions. The changes
in the SPR absorption spectrum can be attributed to
the partial oxidation of the nano-Ag, with the forma-
tion of chemisorbed Ag+ on the particle surface [5, 6].
The relationship between the SPR absorption and the
silver concentration can be expressed as the following
equation [5]:
k = k0 (1 + [Agþ ]/[Ag])1=2 ;
where k0 and k are respective wavelengths of the
absorption maximum before and after oxidation, and
[Ag] and [Ag+] are the total Ag and chemisorbed Ag+
ion concentrations, respectively (assuming [Ag+] is a
small fraction of [Ag]). Thus, for the oxidized nano-Ag
preparation used in the aforementioned experiments,
where the total Ag concentration was 100 lM, the
chemisorbed Ag+ on the oxidized nano-Ag particles was
estimated to be 12 lM. The minimum inhibitory con-
centration (MIC) of AgNO3 under the aforementioned
assay conditions was determined to be 1 lM. Thus, the
levels of chemisorbed Ag+ ions appear to be sufficient to
mediate the antibacterial effects. It should be noted that
the levels of free Ag in all the aforementioned oxidized
nano-Ag preparations are relatively low (maximal
0.1 lM) as determined by centrifugal filtration methods.
Importantly, the resulting filtrates did not show signifi-
cant antibacterial activities. Ag+ is most likely tightly
adsorbed to the particle surface [5]. One possible mode
of delivery of Ag+ from oxidized nano-Ag may involve
the direct association of the oxidized nano-Ag with the
bacteria as demonstrated previously [23–25].
To further verify that the oxidation of nano-Ag is
required for the antibacterial activities, 2 equiv of
borohydride was added to the reduced nano-Ag un-
der a nitrogen atmosphere to reduce chemisorbed
Ag+. The reduction was confirmed by SPR absorption
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J Biol Inorg Chem (2007) 12:527–534
measurement, which showed that the band was
blueshifted to the same extent as for reduced nano-
Ag which had been prepared under nitrogen
(Fig. 1b). The antibacterial activities of the borohy-
dride-reduced nano-Ag were markedly diminished
(Fig. 1a). In addition, ascorbate, a moderate reduc-
tant, also abrogated the antibacterial activities of
nano-Ag, even under ambient conditions. Ascorbate
caused a blueshift of the SPR absorption peak, sug-
gesting that the nano-Ag were reduced (supplemen-
tary information).
Antimicrobial activities of oxidized nano-Ag
with diameters of 9.2 and 62 nm
To determine whether the antibacterial activities of
nano-Ag were affected by particle size, spherical nano-
Ag were synthesized by borohydride and citrate
reduction methods to yield particles with average
diameters of 9.2 and 62 nm, respectively (Fig. 2). On
the basis of equivalent total Ag mass contents, nano-
Ag of smaller size had much higher antibacterial
activities than those of larger size (Fig. 2a, b). Thus, the
62-nm nano-Ag exhibited the same activities as 9.2-nm
nano-Ag only when 9 times the total Ag was added
(Fig. 2c).
The relationship between the size of the nano-Ag
prepared by different synthetic routes (by sputtering of
bulk silver or reduction of Ag+) and their relative
antibacterial activities was investigated previously
[14, 24, 30]. Differences in the antibacterial activities of
nano-Ag of differing sizes can be interpreted from the
levels of chemisorbed Ag+ ions. The smaller the par-
ticles are, the larger the surface area to mass ratio, and
hence the higher the relative concentrations of chem-
isorbed Ag+. When the nano-Ag of the two sizes
exhibited equivalent antibacterial activities (e.g.,
12 lg/mL for 9.2-nm nano-Ag vs. 108 lg/mL for 62-nm
nano-Ag), the theoretical levels of the Ag atom dis-
played on the particle surface were also roughly
equivalent at the same order of magnitude (i.e.,
1.04 · 1016 vs. 1.44 · 1016 atoms; calculation derived
from the number of surface atoms of the spherical
particles, i.e., 6QN/q [5], where Q is the diameter of a
silver atom, i.e., 0.25 nm, q is the diameter of the
nanoparticles, N is the total number of silver atoms in
all the particles, volume 1 mL ). The size-dependent
antibacterial activities may also be explained by dif-
ferences in the levels of association of the nano-Ag to
the bacteria. In this regard, previous studies showed
that nano-Ag that present a direct association with the
bacteria preferentially have a diameter of approxi-
mately 1–10 nm [24].
J Biol Inorg Chem (2007) 12:527–534
Oxidized nano-Ag aggregation and the loss
of antibacterial activities
In addition to their sensitivity to oxidation, nano-Ag
have a propensity to aggregate in electrolytic solutions
[31, 32]. Although nano-Ag synthesized by borohy-
dride reduction (average diameter 9.2 nm) exhibited
antimicrobial activities in water or in buffered solu-
tions such as 50 mM sodium Hepes, the particles
aggregated in commonly used culture media and bio-
logical buffers which have high salt content (chloride
and phosphate). Figure 3a shows that when oxidized
nano-Ag were added to M9 bacterial culture medium,
the SPR absorption disappeared, with a broad increase
in the absorbance from 500 to 600 nm. Oxidized nano-
Ag had no inhibitory activity against the growth of
bacteria in this medium (Fig. 3c). However, when
oxidized nano-Ag were mixed with BSA and added to
the medium, aggregation was prevented and the SPR
absorption was preserved (Fig. 3a). Comparison of the
SPR absorption of oxidized nano-Ag in the presence of
BSA in M9 medium with that of oxidized nano-Ag in
Hepes buffer revealed that the SPR peak shifted
slightly from 397 to 404 nm, with no appreciable
change in the overall spectral shape. The SPR shift
reached a plateau at higher concentrations of BSA
(Fig. 3b), indicative of a saturable interaction. Impor-
tantly, oxidized nano-Ag premixed with BSA exhibited
significant antibacterial activities at nanomolar particle
concentrations (Fig. 3c), similar to their potency in
water or Hepes buffer. These data suggest that a good
dispersion of the nanoparticles is required for effective
antibacterial activities. Presumably, the aggregation of
oxidized nano-Ag leads to a decrease in the effective
surface of the nanoparticles, or the degree to which
they can associate with the cells. The physical nature of
the interaction between albumin and nano-Ag remains
to be investigated, but the albumin may be shielding
the charge interactions between the nanoparticles and
electrolytes.
Parallel experiments were performed to examine the
effects of albumin on the antibacterial activities of Ag+
ions in the form of a solution of AgNO3. In contrast to
the lack of activity found for oxidized nano-Ag in M9
medium, AgNO3 exerted antibacterial activities with a
MIC of 2.5 lM. The addition of albumin at low con-
centrations (15 lM), which restored the antibacterial
activities of nano-Ag, reproducibly increased the MIC
of AgNO3 to 5–10 lM. However, it should be men-
tioned that the antibacterial activities of both oxidized
nano-Ag and AgNO3 were both decreased when
albumin concentration was further increased. Thus,
while low concentration of BSA can stabilize the
533
nanoparticles without compromising the antibacterial
activities, excess BSA still appears to block the activi-
ties of Ag+ ions delivered from the nanoparticles.
Activities of oxidized nano-Ag towards
silver-resistant E. coli
Silver-resistant bacteria have been isolated both clini-
cally and experimentally [21, 22]. The silver-resistance
determinants involve various cellular mechanisms,
which lower the bioavailability of Ag+ [21, 22]. To
examine whether nano-Ag, representing an alternative
form of silver, could exert antibacterial activities
against silver-resistant strains, the 116 AgNO3R strain
(a laboratory strain isolated by stepwise selection
against increasing concentrations of AgNO3 [22]) and
strain J53 (pMG101) (which harbors a Salmonella
plasmid encoding silver-resistance determinants [21])
were tested. Both silver-resistant strains grew in M9
medium containing more than 1 mM AgNO3, whereas
the growth of sensitive parental strains was inhibited
with a MIC of 3 lM AgNO3 (Table 1). Albumin-sta-
bilized oxidized nano-Ag up to 80 nM (the highest
particle concentration of well-dispersed nano-Ag ap-
plied in the culture) were ineffective in inhibiting the
growth of the two silver-resistant strains. Thus, the
antibacterial silver activities delivered by oxidized
nano-Ag were similar to that of Ag+ in the form of
AgNO3 solution, and were tolerated by the silver-
resistant strains.
Conclusions
The antibacterial properties of nano-Ag synthesized by
borohydride reduction methods were tested in terms of
some of the physicochemical properties. The nano-Ag
are sensitive to oxygen and only partially oxidized
nano-Ag exhibit antibacterial activities. The formation
of Ag+ on the surface of the nanoparticles was revealed
by characteristic changes in SPR absorption during
oxidation and reduction. Thus, partially oxidized nano-
Ag may be carriers of chemisorbed Ag+ in quantities
that are sufficient to mediate antibacterial activities.
Nano-Ag, like Ag+ from AgNO3 solution, are tolerated
by the silver-resistant strains. The antibacterial activi-
ties of nano-Ag are related to their size, with the
smaller particles being more active when compared on
the basis of equivalent silver mass content. Analo-
gously to what is found for other metal nanoparticles,
nano-Ag aggregate in high salt, resulting in a loss of
antibacterial activities. The complexation of nano-Ag
with albumin can stabilize the nanoparticles, leading to
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a retention of their antibacterial activities. Taken to-
gether, the antibacterial activities of nano-Ag are
critically dependent on surface oxidation and optimal
particle dispersion.
Further studies examining the mechanism by which
nano-Ag deliver the Ag+ to the bacteria are warranted.
The levels of free Ag in the assay medium were low in
these experiments and did not significantly contribute
to the observed antibacterial activities. Possible
modes for the release of chemisorbed Ag+ species from
the nano-Ag may involve direct association between the
nanoparticles and the bacteria [23–25]. However, the
possibility for a higher degree of dissolution of nano-Ag
to free Ag+ in a more complex medium with sufficient
chelating properties cannot be excluded.
Acknowledgements We thank Simon Silver, Xian-Zhi Li, and
Keith Poole for providing the bacterial strains, and Rory Watt
for his help in editing the manuscript. This work was supported
by the Area of Excellence Scheme (AoE/P-10/01) established
under the University Grants Committee of the Hong Kong
Special Administrative Region, People’s Republic of China, the
Strategic Research Themes on Bionanotechnology, and the Re-
search Support Programs (to C.M.C., J.F.C., and C.N.L) and the
University of Hong Kong.
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