Materials Science and Engineering B 152 (2008) 22–27

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Materials Science and Engineering B

journal homepage: www.elsevier.com/locate/mseb

Colloidal silver solutions with antimicrobial properties

A. Petica a,∗ , S. Gavriliu a , M. Lungu a , N. Buruntea a , C. Panzaru b
a
INCDIE ICPE-Advanced Research, Bucharest, Romania
b
Institute of Medicine and Pharmacy, Iassy, Romania

a r t i c l e i n f o a b s t r a c t

Keywords: Some colloidal silver solutions involving the electrochemical technique with “sacrificial anode method
Silver colloidal solutions and different stabilizers and co-stabilizers” have been prepared. A constant current pulse generator with
Stabilization with polymers stirrer at different working times has been used. To achieve stable colloidal silver solutions, a mix of
Antimicrobial
different tensioactive agents namely [poly (N-vinylpyrrolidone)], Na-naphthalene sulphonate, Na-lauryl
Antifungal
sulfate and Na-dodecyl sulphonate were tested. The effects of these various mixes of polymer and ionic
surfactants upon the Ag concentration and UV–vis spectra of silver nanoparticles were determined by
spectrophotometer techniques. The nanoparticles sizes have been analyzed through dynamic light scat-
tering technique and the silver nanoparticle morphology has been evidenced by transmission electron
microscopy (TEM). Micobiological analysis has been made by determining minimal inhibitorial con-
centration upon the following germs: Staphylococcus aureus (ATCC) (Gram-positive cocci), Pseudomonas
aeruginosa (ATTC), Escherichia coli (ATCC) and Acinetobacter spp. (Gram-negative coccobacillus). To evalu-
ate the antifungal effect, the antibiogram method involving various tests using a fungi mix of Aspergillus,
Penicillium and Trichoderma species has been used. The presented method allows obtaining of some
stable colloidal solutions containing up to 35 ppm of Ag with very good antimicrobial and antifungal
properties.
© 2008 Published by Elsevier B.V.

1. Introduction In addition, Ag+ from silver-based solution is a long lasting bio-

Noble metals nanoparticles synthesis attracts an increasing
interest due to their new and different characteristics as compared
with those of macroscopic phase, that allow attractive applications
in various fields, such as: medicine, biotechnology, optics, micro-
electronics, catalysis, information storage, energy conversion [1].
Silver nanoparticles have the properties of a high surface area, very
small size (<20 nm) and high dispersion. Moreover, colloidal silver
solutions (CSSs) have an increased interest due to their antimicro-
bial properties, with large applications, including pharmacology,
human and veterinary medicine, food industry, water purification
and so on. The interaction of metal nanoparticles with microor-
ganisms (from fungi to viruses, e.g. HIV) is an expanding field of
research [2]. It is believed that the mechanism of the antibacterial
effect of silver ions (Ag+ ) involves their absorption and accumula-
tion by bacterial cells and shrinkage of the cytoplasm membrane
or its detachment from the cell wall. As a result, DNA molecules
become condensed and loose their ability to replicate upon the
infiltration of Ag ions. The silver ions also interact with the S–H
bounds of the proteins, blocking and inactivating them [3].
∗ Corresponding author. Tel.: +40 213468297x129; fax: +40 213468299.
E-mail address: petica@icpe-ca.ro (A. Petica).
cide with high temperature stability and low volatility. The silver
nanoparticles may be used in form of colloidal sols or doping agents
for a lot of composite materials with polymer matrix.
The success of the introduction of the silver nanoparticles in
different forms in bioscience, healthcare and consumer goods is
well known [4,5]. Understanding the forces governing the col-
loidal stabilities [6] is an essential requirement for the preparation
of colloidal nanoparticles. In fact, stabilization of colloidal par-
ticles is involved in the preparation procedures. Particles can
be stabilized electrostatically and sterically. Steric stabilization
is accomplished by adsorbing polymers and/or surfactants onto
the surfaces of colloidal particles [7]. The capping by molecules,
which form chemical bonds with/or chemisorbs onto the par-
ticles provides an extremely useful method of stabilization, too
[8].
The paper presents some aspects upon the preparation and char-
acterization by an electrochemical approach and especially, the
antimicrobial effect of CSSs. Our original solution consists in using
as stabilizers of a mix of ionic and non-ionic biocompatible surfac-
tants, largely presented in reference [9].
The researches regarding to the obtaining of CSSs were focused
on the achieving of some silver nanoparticles with controllable
shape and grain size and certain surface chemistry, which in turn
determine the stability and functional properties. The obtained

0921-5107/$ – see front matter © 2008 Published by Elsevier B.V.

doi:10.1016/j.mseb.2008.06.021
 A. Petica et al. / Materials Science and Engineering B 152 (2008) 22–27 23

CSSs can be used in bio-medical applications like: bandages, 3. Results and discussion
dressings, cosmetics, consumer goods or as precursors for various
polymer matrices doping. 3.1. Role of stabilizers agents

To obtain stable CSSs, various mixes of stabilizer (polymer)

2. Experiments
CSSs were prepared by so-called “sacrificial anode method”,
which offers the advantage of a high purity of final formed solution,
especially for bio-medical applications. A constant current pulse
generator with a stirrer and two silver electrodes of Ag of 99.999%
purity were used.
The experiments were carried out using the following materials:
• deionized water with conductivity <1 ␮S and pH 6–6.5;
• poly [1-vinyl-2-pyrrolidone] (C6 H9 NO)n (PVP 10 with M = 10,000
and PVP 55 with M = 55,000 from Sigma–Aldrich), PVP 25 with
M = 25,000 from Merck;
• Na-lauryl sulfate, Na-naphthalene sulfonate and sodium dodecyl
benzenesulfonate from Sigma–Aldrich.
To achieve some stable CSSs, a mix of these different tensioac-
tive agents was tested. The Ag concentration of the obtained CSSs
was determined by quantitative analysis and UV–vis absorbance
spectra recording by a JASCO V 570 spectrophotometer. A cal-
ibration curve using standard colloidal silver nanoparticles in
the range of our measurements was recorded. The nanoparticles
sizes were analyzed by DLS (dynamic light scattering) tech-
nique using Zetasizer Nanoequipment. The silver nanoparticles
morphology and dispersability were evidenced by transmission
electron microscopy (TEM) measurements, using an electronic
microscope Philips CM 100. The specimens for TEM investigations
were prepared through deposition on 400 mesh grids with formvar
film.
To evaluate the antibacterial efficiency, the minimal inhibitorial
concentration (MIC) upon the S. aureus (ATCC) Gram-positive cocci,
P. aeruginosa (ATTC) and E. coli (ATCC) Gram-negative coccobacillus
was determined. According to the scheme of Ericcsson and Sherris
[10], in agar dilution technique was used.
To evaluate the antifungal effect, the antibiogram method was
used [11] with a fungi mix containing different mycotoxins (e.g.
aflatoxin). According to this method, Petri vessels with agarized
Czapek Dox medium were inoculated by spraying with a fungi
mix from the following species: Aspergillus, Penicillium and Tricho-
derma.
and co-stabilizer (surfactant) agents were used, such as: PVP
[poly(N-vinylpyrrolidone)] [2,3] with a molecular weight (M) of
10,000, 25,000 and 55,000, respectively Na-lauryl sulfate (Na-
LS), Na-naphtalene sulfonate (Na-NS) and SDBS (sodium dodecyl
benzenesulfonate). PVP exhibits excellent wetting properties [2]
and facilitates the formation of nanoparticles. Especially PVP 10
(M = 10,000), in the presence of a co-stabilizer, shows an inhibition
effect of nanoparticles agglomeration, assures a steric stability and
as a result, an increased Ag content in CSSs.
Co-stabilizers (surfactants) assure the wrapping and electro-
static stability of the silver nanoparticles in solutions. Using
concentrations of 3–20 g/l PVP and 0.25–1g/l Na-NS, Na-LS and
SDBS, stable colloidal nanosilver solutions were obtained with
concentrations up to 35 ppm, having a light yellow towards dark
orange color as a function of the total Ag particles content. Many
experiments were performed with various concentrations and elec-
troformation durations. The dependence of nano-Ag concentration
in colloidal solutions against PVP molecular weight, co-stabilizer
type and content and electroformation periods was largely pre-
sented in Reference [9].
Because this paper is focused on antibacterial and antifungal
characteristics of CSSs, the solutions with the best properties and
results have been presented.
According to our experiments [9], CSSs without any stabilizers
agents, have a low Ag content (up to 3.3 ppm Ag), regardless of the
processing time period. The addition of the PVP stabilizer facili-
tates an increase of Ag concentration (from 5.2 ppm for PVP 10 to
5.8 ppm for PVP 25 and to 6.76 ppm for PVP 55), but the increase of
PVP concentration over 6 g/l worsened the quality of colloidal solu-
tions, materialized by formation of grayish appearance suggesting
a growth of metallic particle size. To explain the reason for this
behavior it must take into account the kinetics of particle forma-
tion and growth. The formation of colloid particles involves two
phases: first, a nucleation takes place, followed by a growing pro-
cess of these nuclei. According to Patakfalvi et al. [12,13] the nuclei
may grow to primary particles by a diffusion mechanism and these
later aggregate to form secondary particles. The change in chemical
state of the system, e.g. greater concentrations of polymer, which
leads to an increasing of viscosity of electrosynthesis solution, hin-
ders the diffusion and decreases the electrostatic repulsion, thereby
promoting the particle agglomeration.

Table 1
Dependence of nano-Ag concentration in CSSs upon stabilizer mixture, working time and centrifugation

No. of CSS sample Composition Working time (h) Ag concentration (ppm) Ag concentration after centrifugation (ppm)

1 3 g/l PVP 10 + 0.25 g/l Na-NS 3 8.95 –

6 14.02 12.80

2 3 g/l PVP 10 + 0.25 g/l Na-LS 3 20.72 –

6 34.28 30.65

3 3 g/l PVP 10 + 0.25 g/l SDBS 3 13.50 –

6 19.80 5.47

4 3 g/l PVP 10 + 0.5 g/l Na-NS 3 17.50 –

6 34.57 31.80

5 3 g/l PVP 10 + 0.5 g/l Na-LS 3 17.70 –

6 28.08 26.40

6 3 g/l PVP 10 + 0.5 g/l SDBS 3 13.52 –

6 24.50 5.88
24 A. Petica et al. / Materials Science and Engineering B 152 (2008) 22–27

Fig. 2. Granulometric distribution of particles against their occupied volume for a

solution containing 28 ppm Ag formed in the presence of 3 g/l PVP 10 + 0.5 g/l Na-LS,
6 h.

Fig. 1. UV–vis spectra for silver nanoparticles obtained with different co-stabilizers
contents at a concentration of PVP 10 of 3 g/l.

Table 1 shows the dependence of nano-Ag concentration in

colloidal solutions with 3 g/l PVP 10 against three types of co-
stabilizers. In order to separate the agglomerate particles, the CSSs
obtained for 6 h working time were centrifuged for 30 min at a
speed of 6000 rotations/min.
As we can see from Table 1, the CSSs obtained in the presence of
SDBS have a high decrease of the concentration after centrifugation
because of the particle agglomeration formation, while the others
solutions have only a small decrease of concentration.
The protective mechanism of PVP in the electrochemical syn-
thesis of silver particles is generally proposed on the basis of its
structural features [13,14]. PVP has a structure of a polyvinyl skele-
ton with polar groups. The donated lone pairs of both nitrogen and
Fig. 3. Granulometric distribution of particles against their number for a solution
oxygen atoms in the polar groups of one PVP unit may occupy two
containing 34 ppm Ag formed in the presence of 3 g/l PVP 10 + 0.25 g/l Na-NS, 6 h.
sp orbitals of silver ions in the presence of PVP [15,16]. PVP has a
major role in steric stabilization upon CSSs.
The role of co-stabilizer type and content was proved to have a 10 and 30 nm. The stability of obtained colloidal solutions has been
significant influence on the nano-Ag particles content in the final determined by Zeta potential measurements that indicate if the
colloidal solution, regardless the electroformation time periods. particles from a liquid tend to associate and or/sediments. For the
Both addition of Na-NS, Na-LS and SDBS, lead to high contents of obtained CSSs, zeta potentials have not been affected either by
nano-Ag particles in the formed colloidal solutions that attain val- the consequent modification of particles’ sizes in time or by the
ues around 35 ppm for 6 h processing time. As it is exemplified in slight sedimentation tendency that appears during measurement
Table 1, for a solution of 3 g/l PVP 10, the addition of a co-stabilizer performing.
facilitates formation of concentrated colloidal solutions. Na-LS and According to Figs. 2–4, where are represented three successive
Na-NS have amphiphilic properties due to the C12 chain (lipophilic) measurements, the obtained solutions are stable and do not exhibit
attached to a sulfate or sulphonate group (hydrophilic). This bifunc- sedimentation tendency (materialized by peaks displacing to the
tionality in one molecule provides the basic properties useful in left) or agglomeration (peaks displacing to the right). This behav-
steric and electrostatic stabilization of silver nanoparticles [14].
The influence of presence and concentration of PVP 10 with
different co-stabilizers over the concentrations of silver particle
was evidenced through UV–vis spectroscopy. It should be men-
tioned that according to the literature [14], the absorption peak
at 400–420 nm may be assigned to Ag nanoparticles presence in
colloidal solution.
Fig. 1 presents the influence of the co-stabilizers upon the
absorbance recorded by UV–vis spectra. The maximum of absorp-
tion was evidenced in the case of the CSSs prepared in the presence
of 0.25 g/l Na-LS, in close agreement with the results presented in
Table 1.

3.2. Size, stability and morphology of CSSs

To analyze the nanoparticles size, their granulometric distribu-

Fig. 4. Granulometric distribution of particles against their occupied volume for a
tion has been recorded, expressed against the particles number solution containing 19.8 ppm Ag formed in the presence of 3 g/l PVP 10 + 0.25 g/l
and their occupied volume. The size of the particles is between SDBS, 6 h.
 A. Petica et al. / Materials Science and Engineering B 152 (2008) 22–27 25

Table 2
MIC of the tested CSSs

No. of CSS sample Composition and concentration of the CSSs CMI (ppm)

Stabilizers composition Ag conc. (ppm) Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa

4 3 g/l PVP 25 + 0.5 g/l Na-NS 31.80 31.80 7.95 7.95

5 3 g/l PVP 10 + 0.5 g/l Na-LS 26.40 6.60 1.65 1.65
6 3 g/l PVP 10 + 0.5 g/l SDBS 5.88 5.88 2.94 2.94

ior evidences again the role of the used stabilizers. Zeta potential results after 7 and 14 days of exposure have been evidenced for
distribution is a monomodal one, with values between −18 and solutions obtained in the presence of PVP with Na-LS, when inhi-
−35 mV that suggest the particles are entirely covered by stabilizer bition zone is more pronounced than in the case of solutions with
agents and thus the solution is stable, especially for solutions with Na-NS, and SDBS, respectively.
Na-LS.
In Fig. 2 it is observed that the three recordings, made
successively, are almost overlaid, that indicates a very good sta-
bility in time with a gentle tendency of agglomeration. Zeta
potential value was −30.5 mV, which also indicates a very good
stability.
From Fig. 3, it is observed a tendency of sedimentation (by shift-
ing to the left) and zeta potential value of −18.2 mV indicates a
poorer stability of the solution.
From Fig. 4, in agreement with the appearance of this solution,
there is a sedimentation process of a small percentage of agglom-
erate particles. Zeta potential value was −35.4 mV, which indicates
the presence of an electric charge on the particles surfaces, strong
enough, to hinder their agglomeration.
TEM micrographs show that colloidal solutions contain Ag
nanoparticles smaller than 20 nm, having a spherical shape, uni-
formly scattered (Fig. 5).

3.3. Antimicrobial effect

The obtained CSSs have been tested to evidence their antibac-

terial and antifungal properties.
The antibacterial activity was assayed against three frequently
bacterial species (E. coli, S. aureus and P. aeruginosa), by minimal
inhibitory concentration measurements using fractional diluting
(1; 1/2; 1/4; 1/8; 1/16). These bacteria offer by their morphological,
physiological and metabolic characteristics a useful diversity for
obtaining of some pertinent results. Table 2 presents the results of
these tests.
The samples No. 5 and No. 6 present the smallest values of
CMI for all tested bacteria. These results are in accordance with
the particles size showing by grain size distribution measurements
(Figs. 2–4) and TEM micrographs (Fig. 5), which evidence for the
samples No. 5 and No. 6 smaller mean diameters comparatively
with the sample No. 4.
We notice that, in the case of S. aureus, the CMI is higher than the
one for the other two bacteria. It is in agreement with the knowl-
edge regarding to its cell wall, which consist of a thick peptidoglycan
layer. Overall differences in pathogenicity of S. aureus are attributed
to genome islands encoding a lot of toxines [17]. This virulent and
opportunistic pathogen is resistant to most powerful antibiotics.
As a result, the increased pressure on researches to find alterna-
tive more effective ways of fighting against this strain represents a
challenge.
According to antibiogram method, the fungistatic properties
are expressed by the presence and magnitude of inhibition area
for mould growth around the filter paper padded with CSSs. The
used test fungi contained various mycotoxins as aflatoxin. Figs. 6–8
present photographic images of the inhibition area produced by the
three CSSs on a mould colony containing Aspergillus, Penicillium and Fig. 5. TEM micrographs of CSSs obtained in the presence of: 3 g/l PVP 10 + 0.5 g/l
Trichoderma, after 7 and 14 days of exposure. Generally, the best Na-NS (a), 3 g/l PVP 10 + 0.5 g/l Na-LS (b) and 3 g/l PVP 10 + 0.5 g/l SDBS (c).
26 A. Petica et al. / Materials Science and Engineering B 152 (2008) 22–27

Fig. 6. Photographic image of an inhibition zone produced by a silver colloidal solution with 28 ppm Ag (with a mix of 3 g/l PVP 25 + 0.5 g/l Na-LS, 6 h) against a mouldiness
cologne, containing Aspergillus, Penicillium, Trichoderma, after (a) 7 days and (b) 14 days.

Fig. 7. Photographic image of an inhibition zone produced by a silver colloidal solution with 15 ppm Ag (with a mix of 3 g/l PVP 25 + 0.25 g/l Na-NS, 6 h) against a mouldiness
cologne, containing Aspergillus, Penicillium, Trichoderma, after (a) 7 days and (b) 14 days.

Fig. 8. Photographic image of an inhibition zone produced by a silver colloidal solution with 20 ppm Ag (with a mix of 3 g/l PVP 10 + 0.25 g/l SDBS, 6 h) against a mouldiness
cologne, containing Aspergillus, Penicillium, Trichoderma, after (a) 7 days and (b) 14 days.

4. Conclusions Na-NS and SDBS facilitate a suitable dispersion of nanoparticles

and hinders their agglomeration. Zeta potential distribution is a
The used electrochemical method allows the formation of high monomodal one, with values up to −35 mV that suggest the par-
purity stable CSSs containing up to 35 ppm with colors varying from ticles are covered by stabilizer and thus, the solution is stable. For
yellow to dark-orange. PVP stabilizer with co-stabilizers as Na-LS, tested CSSs, MIC shows a very good efficiency against both Gram-
 A. Petica et al. / Materials Science and Engineering B 152 (2008) 22–27
positive and Gram-negative cocci. A good antimicrobial effect was
proved after 7–14 days of exposure at test fungi with no appearance
of mould in the area treated with CSSs. The results are promising
for the CSSs use in antimicrobial applications (antibacterial finishes
and disinfecting techniques for all types of textiles and leathers), for
food and health industry and, especially, for bio-medical applica-
tion.
Acknowledgements
This study is financially supported by the scientific
research project, which is in progress, CONTRACT CEEX No.
26/2005—NACOLAG, in the frame of the Romanian Excellence
Research Programme.
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