Materials Chemistry and Physics 115 (2009) 296–302

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Materials Chemistry and Physics

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

Silver nanoparticles dispersing in chitosan solution: Preparation by ␥-ray

irradiation and their antimicrobial activities
Rangrong Yoksan a,b,∗ , Suwabun Chirachanchai c
a
Department of Packaging Technology and Materials, Faculty of Agro-Industry, Kasesart University,
50 Paholyothin Road, Ladyao, Jatujak, Bangkok 10900, Thailand
b
Division of Physico-Chemical Processing Technology, Faculty of Agro-Industry, Kasesart University, Bangkok 10900, Thailand
c
The Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand

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

Article history: Silver nanoparticles were prepared by ␥-ray irradiation–reduction under simple conditions, i.e., air
Received 25 September 2008 atmosphere, using chitosan as a stabilizer. The nanoparticles were spherical with an average size of
Received in revised form 7–30 nm as observed from TEM. The size decreased when chitosan concentration increased, while it
16 November 2008
increased with increasing ␥-ray dose and initial silver nitrate content. The obtained silver nanoparti-
Accepted 1 December 2008
cles dispersed in a 0.5% (w/v) ␥-ray irradiated chitosan–aqueous acetic acid solution were stable for more
than 3 months without tendency to precipitate. The silver nanoparticles exhibited antimicrobial activities
Keywords:
against Escherichia coli and Staphylococcus aureus. The results suggest that silver nanoparticles dispersed
Silver
Nanoparticle
in chitosan solution can be directly applied in antimicrobial fields, including antimicrobial food packaging
Chitosan and biomedical applications.
Irradiation © 2008 Elsevier B.V. All rights reserved.
Transmission electron microscopy (TEM)

1. Introduction eliminate the purification step or the removal of reducing agent

For many decades, silver nanoparticles have been recognized
as an antimicrobial agent [1] and used in many fields includ-
ing biomedical [2,3], food packaging [4,5], waste water treatment
[6], etc. The antimicrobial activities of silver nanoparticles were
found to relate to their shapes and sizes [7]. Many attempts have
been made to synthesize the silver nanoparticles with controllable
shape, size, and size distribution. In most cases, the system for
fabricating silver nanoparticles consists of salt precursor, reduc-
ing agent, and stabilizer. Silver nitrate (AgNO3 ) is usually applied
as the salt precursor. The reduction of silver ions (Ag+ ) to silver
particles (Ag0 ) is conventionally achieved by a chemical method
using reducing agents such as NaBH4 [8], formamide [9], dimethyl-
formamide [9], triethanolamine [9], hydrazine [10], etc. Although
this method is simple and effective, the biological toxicity and the
environmental hazard of the residual reducing agent are problems.
Recently, reductions by other techniques such as ␥-ray irradiation
[11,12], UV irradiation [13], microwave [14], and ultrasonic [15]
have been alternative ways to overcome those problems and to
∗ Corresponding author at: Department of Packaging Technology and Materials,
Faculty of Agro-Industry, Kasesart University, 50 Paholyothin Road, Ladyao, Jatujak,
Bangkok 10900, Thailand. Tel.: +66 2 562 5097; fax: +66 2 562 5092.
E-mail addresses: rangrong.y@ku.ac.th, yyrangrong@yahoo.com (R. Yoksan).
step.
To prevent the aggregation of formed metal nanoparticles, var-
ious types of stabilizers are used such as long-chain fatty acids
(stearic, palmitic, and lauric acids) [9], lauryl amine, poly(vinyl
pyrrolidone) [16], poly(vinyl alcohol) [16], amphiphilic block
copolymer PEA [15], soluble starch [17], heparin [18], chitosan
[8,11,12,19], etc.
Chitosan, the second most naturally abundant polysaccharide,
consists of glucosamine and N-acetyl glucosamine units liked
together by ␤-1,4-glucosidic bonds. Due to its biodegradable, bio-
compatible, non-toxic and antimicrobial characteristics, chitosan
is utilized in many areas. For the preparation of silver nanoparti-
cles, chitosan has been used either as a reducing agent [13] or as
a stabilizer [11–13]. Although the synthesis of silver nanoparticles
in chitosan–acetic acid solution using ␥-ray irradiation has been
reported, the irradiation condition was under nitrogen atmosphere
[11,12,20].
The present research studies the fabrication of silver nanoparti-
cles in chitosan–aqueous acetic acid solution using ␥-ray irradiation
under simple conditions, i.e., air atmosphere. The formation of
silver nanoparticles is confirmed by the specific surface plas-
mon resonance (SPR) band and electron micrograph. The effects
of ␥-ray dose (2.5–25.0 kGy), chitosan concentration (0.1 and
0.5%, w/v) and silver nitrate content (0.02–0.10 mmole) on the
particle size and particle number are also investigated. In addi-

0254-0584/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.matchemphys.2008.12.001
 R. Yoksan, S. Chirachanchai / Materials Chemistry and Physics 115 (2009) 296–302
tion, the antimicrobial activity of the silver nanoparticles is
studied.
2. Experimental methods
2.1. Materials
Chitosan (deacetylation degree of 0.95 and molecular weight of ∼700,000 Da)
was purchased from the Seafresh Chitosan (Lab) Company Limited, Thailand. Silver
nitrate and acetic acid were supplied by Merck, Germany. All chemicals were used
as received and without further purification.
2.2. Instruments and equipment
Gamma ray irradiation was carried out in a 60 Co Gammacell irradiator with a
dose rate of 12 kGy h−1 kindly provided by the Office of Atomic Energy for Peace, Min-
istry of Science and Technology, Bangkok, Thailand. Ultraviolet and visible (UV–vis)
absorption spectra were recorded over a wavelength from 200 to 500 nm by a
Helios Gamma Thermo Spectronic spectrometer (England). Transmission electron
microscopy (TEM) photographs were taken at an accelerating voltage of 100.0 kV
by a Hitachi H-7650 (Hitachi High-Technology Corporation, Japan). Zeta potential
was determined at 20 ◦ C by a Malvern Zetasizer (model 3600, Malvern Instruments
Ltd., UK) equipped with a He–Ne laser operating at 4.0 mW and 633 nm with a fixed
scattering angle of 90◦ .
2.3. Preparation of silver nanoparticles in chitosan solution using -ray
irradiation
Two concentrations of chitosan solutions (0.1 and 0.5%, w/v) were prepared by
stirring chitosan in 1% (v/v) aqueous acetic acid at room temperature overnight. The
solutions were then centrifuged to eliminate traces of insoluble fractions. A fresh
solution of 50 mM AgNO3 was prepared in 1% (v/v) aqueous acetic acid at room
temperature. Chitosan solution (20 mL) was mixed with different amounts of AgNO3
(0.02, 0.04, 0.06, 0.08, and 0.10 mmole). The final volume was adjusted to be 22 mL
with 1% (v/v) aqueous acetic acid. The mixture was stirred at room temperature
for 10 min and then ␥-ray irradiated in a 60 Co Gammacell irradiator with the dose
rate of 12 kGy h−1 . The dose of ␥-ray was varied as 0.0, 2.5, 5.0, 10.0, 15.0, 20.0, and
25.0 kGy. Chitosan solution without the addition of AgNO3 was also prepared in the
same manner and used as a control.
2.4. Study on antimicrobial activity of silver nanoparticles
Antimicrobial activity of silver nanoparticles was studied by an agar diffu-
sion method using polysaccharide as a film matrix. Silver particles incorporated in
polysaccharide-based films were prepared by mixing 25 kGy ␥-ray irradiated 0.5%
(w/v) chitosan solution containing 0.04 mmole AgNO3 with polysaccharide aqueous
solution. The content of silver nanoparticles was varied by changing concentration
of the ␥-ray irradiated chitosan solutions containing AgNO3 (0 (control), 10 and 20%,
Fig. 1. Appearances of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v) containing different AgNO3
contents: (a) 0.00 mmole, (b) 0.02 mmole, (c) 0.04 mmole, (d) 0.06 mmole, (e) 0.08 mmole, and (f) 0.10 mmole, after ␥-ray irradiation with various doses from 2.5 to 25.0 kGy.
The first column (a0 ) is chitosan solution without the addition of AgNO3 and without ␥-ray irradiation.
297
v/v). The mixtures were then cast onto acrylic plates and dried at 45 ◦ C overnight.
The dried films were cut into discs of 28 mm diameter followed by sterilization
in an autoclave at 121 ◦ C for 15 min. The discs were placed on a surface of nutri-
ent agar (HiMedia Laboratories Pvt. Ltd., India), which had been previously seeded
with 20 ␮L of inoculum containing tested bacteria, i.e., Escherichia coli (ATCC 35218,
6.4 × 105 CFU mL−1 ) and Staphylococcus aureus (ATCC 6538, 7.4 × 108 CFU mL−1 ). The
plates were incubated at 37 ◦ C for 24 h. The contact areas of the films with agar sur-
face were observed and the diameters of clear zones surrounding the film discs were
measured.
3. Results and discussion
Conventionally, silver nanoparticles are simply prepared by the
reduction of silver salts using chemical reducing agents. Although
the chemical reduction is effective, the toxicity of residual reduc-
ing agents and the environmental impact should be considered.
The present work, thus, focused on the ␥-ray irradiation–reduction
under simple conditions, i.e., air atmosphere, to prepare silver
nanoparticles. Silver nitrate was applied as a salt precursor, while
chitosan was used as a stabilizer. To study the factors affecting
formed particle size and number of particles, the ␥-ray dose, ini-
tial AgNO3 content, and chitosan concentration were varied. The
amount of ␥-ray used was from 2.5 up to 25.0 kGy, initial AgNO3
content was in the range of 0.02–0.10 mmole, and chitosan concen-
trations were 0.1 and 0.5% (w/v). It should be noted that as chitosan
is non-toxic and has been approved by FDA, the silver nanoparticles
dispersing in chitosan solution do not need to be separated and/or
purified and may be directly used in antibacterial fields.
3.1. Formation of silver particles: physical appearance and
mechanism
In general, chitosan solution is transparent without color
(Fig. 1(a0 )). The solution turns pale yellow after ␥-ray irradi-
ation (Fig. 1(a)). The color intensity increased with increasing
␥-ray dose, especially in the case of 0.5% (w/v) chitosan solu-
tion. This might involve the double-bond formation by the chain
scission of polysaccharide as reported previously [21,22]. Here,
UV–vis spectrophotometry technique was also applied to deter-
mine the structural changes of chitosan after ␥-ray irradiation. By
comparison with unirradiated chitosan solution (Fig. 2(a)), the ␥-
298 R. Yoksan, S. Chirachanchai / Materials Chemistry and Physics 115 (2009) 296–302
Fig. 2. UV–vis absorption spectra of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), after ␥-ray
irradiation with different doses: (a) 0.0 kGy, (b) 2.5 kGy, (c) 5.0 kGy, (d) 10.0 kGy, (e) 15.0 kGy, (f) 20.0 kGy, and (g) 25.0 kGy.
ray irradiated samples gave two new peaks at 260 and 290 nm
(Fig. 2(b)–(g)). The absorption band that appeared at 260 nm
might corresponds to the C O in COOH group, whereas the one
at 290 nm belonged to a terminal carbonyl structure formed at
C1 or C4 after the main chain scission of chitosan and hydrogen
abstraction followed by the ring opening reaction [23–26]. The
absorbance increased with increasing ␥-ray dose up to 20.0 kGy
(Fig. 2(b)–(f)) and chitosan concentration. However, at high ␥-ray
dose, i.e., 25 kGy, the decomposition and/or conversion of carbonyl
and carboxyl species might have taken place resulting in reduced
absorbance (Fig. 2(g)).
The ␥-ray irradiated chitosan solutions containing AgNO3
exhibit more intense yellow color (Fig. 1(b)–(f)) than those without
the addition of AgNO3 (Fig. 1(a)) implying the formation of silver
particles [11,20]. This result was also confirmed by UV–vis spec-
trophotometry (see Section 3.2) and TEM techniques (see Section
3.3). When the initial AgNO3 content and ␥-ray dose increased,
the solution color mostly changed from yellow to red-brown. In
other words, the color intensity progressed as a function of AgNO3
content and ␥-ray dose. For 0.1% (w/v) chitosan solution after
25.0 kGy ␥-ray irradiation, the maximum color intensity (dark red-
brown) appeared when AgNO3 content of 0.04–0.06 mmole was
used (Fig. 1(c) and (d), left). This might have resulted from either a
large amount of silver particle formation or silver particle aggrega-
tion [27].
When the ␥-ray dose was in the range of 10.0–25.0 kGy, the color
of 0.1% (w/v) chitosan solutions containing AgNO3 was more intense
than that of 0.5% (w/v) chitosan solutions. We speculate that the
reduction of silver ions (Ag+ ) to silver particles (Ag0 ) was affected
not only by ␥-ray dose, but also by weight ratio of AgNO3 to chitosan.
The weight ratio of AgNO3 to chitosan in the case of 0.1% (w/v) chi-
tosan solution was fivefold higher than that of 0.5% (w/v) chitosan
solution. Herein, we suspect that ␥-ray irradiation induced both
reactions, which are the chain scission of chitosan and the reduction
of silver ions to silver particles. For the former, hydroxyl radicals and
hydrogen atoms, which are the intermediate products of water radi-
olysis (Eq. (1)), react with polysaccharide molecules by hydrogen
abstraction resulting in the formation of macroradicals localized
at various carbon atoms (C1 –C6 ) within the glucosamine unit (Eq.
(2)) [23,28]. Only radicals formed at C1 and C4 atoms can undergo
rearrangement involving breakage of 1–4 glycosidic bonds (Eq. (3))
and formation of C O species as discussed above. For the latter, the
hydrated electron generated by water radiolysis can reduce Ag+ to
Ag0 (Eq. (4)) [11,12,29]. The neutral atom, i.e., Ag0 , reacts with Ag+
to form the relatively stabilized Ag clusters as shown in Eqs. (5) and
(6) [30,31]. Such clusters then gather together or absorb the neutral
Ag0 to form the silver particles [11,12,30,31].
ioizing +
H2 O −→ OH • , e− •
aq , H , H2 O2 , H2 , H (1)
radiation
OH• (H• ) + R–H → R • (C1 –C6 ) + H2 O(H2 ) (2)
R • (C1 , C4 ) → F1 • + F2 • (chainscission) (3)
reduction
Ag+ + e−
aq −→ Ag
0
(4)
Ag0 + Ag+ → Ag2 + (5)
Ag2 + + Ag+ → Ag3 2+ (6)
By considering the structure of chitosan in acidic solution, amino
groups were protonated (–NH3 + ). Accordingly, the binding of silver
clusters by chitosan may be achieved through the Ag–O bonds as
reported in previous studies [12,32,33], the ionic interaction formed
between silver ion and carboxyl group on chitosan generated dur-
ing ␥-ray irradiation (Ag+ . . .COO− ), or the combination thereof. The
protonated amino groups (–NH3 + ) at the surface of formed silver
particles, thus, promoted the stability of the particles via static
repulsion. This speculation is supported by the result from zeta
potentiometer, e.g. a positively charged surface with the zeta poten-
tial of 40.41 ± 2.50 mV was observed for silver particles formed in
25 kGy ␥-ray irradiated 0.5% (w/v) chitosan solution containing
0.04 mmole AgNO3 . It should be pointed out that silver particles
formed in 0.5% (w/v) chitosan solution were stable for more than 3
months, whereas those in 0.1% (w/v) chitosan solution were precip-
itated out within a few weeks. This might be due to a larger number
of chitosan chains enveloping the silver particle surface when 0.5%
(w/v) chitosan solution was used.
3.2. Formation of silver particles: number of particles
UV–vis spectrophotometry technique was applied to confirm
the formation of silver particles in the ␥-ray irradiated chitosan
solution. Generally, ␥-ray irradiated chitosan solutions exhibit no
absorption peak at wavelengths longer than 350 nm (Fig. 2(b)–(g)),
while the ones containing AgNO3 show a peak at 401–409 nm corre-
sponding to the surface plasmon resonance band of silver particles
(Fig. 3) [34].
An increase in absorbance and a slight shift of SPR band as a
function of ␥-ray dose and initial AgNO3 content can be observed
in Fig. 3 and are summarized in Figs. 4 and 5.
 R. Yoksan, S. Chirachanchai / Materials Chemistry and Physics 115 (2009) 296–302 299

Fig. 3. UV–vis absorption spectra of 0.5% (w/v) chitosan solution (in 1% (v/v) aqueous acetic acid solution) containing different AgNO3 contents: (a) 0.02 mmole, (b) 0.04 mmole,
(c) 0.06 mmole, (d) 0.08 mmole, and (e) 0.10 mmole, after ␥-ray irradiation with different doses. (A) 20.0 kGy and (B) 25.0 kGy.

Fig. 4. Maximum wavelength of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), containing
various AgNO3 contents after ␥-ray irradiation with different doses: (䊉) 2.5 kGy, () 5.0 kGy, () 10.0 kGy, () 15.0 kGy, () 20.0 kGy, and () 25.0 kGy.

The SPR bands of ␥-ray irradiated chitosan solutions containing
AgNO3 appear at the wavelength of 399–417 nm (Fig. 4). A red shift
of SPR band (to higher wavelength) was observed when the ini-
tial AgNO3 content increased (Fig. 4). This indicated an increase in
particle size and/or the formation of silver aggregates [34,35]. It is
speculated that the collision frequency of silver particles increased
significantly with increasing AgNO3 content resulting in silver par-
ticle aggregation. This speculation was also confirmed by TEM
(see Section 3.3). However, the ␥-ray dose hardly affected the SPR
band.
The amount of silver particles formed related to the absorbance
of ␥-ray irradiated chitosan solution containing AgNO3 [11,36]. For
low ␥-ray dose, i.e., 2.5 and 5.0 kGy, the absorbance was constant
at 2–3 and 6–9 for 2.5 and 5.0 kGy, respectively, though AgNO3
content is varied (Fig. 5). In other words, AgNO3 content hardly
affected the absorbance or the number of particles formed at low

Fig. 5. Absorbance at maximum wavelength of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations; (A) 0.1% (w/v) and (B) 0.5% (w/v),
containing various AgNO3 contents after ␥-ray irradiation with different doses: (䊉) 2.5 kGy, () 5.0 kGy, () 10.0 kGy, () 15.0 kGy, () 20.0 kGy, and () 25.0 kGy.
300 R. Yoksan, S. Chirachanchai / Materials Chemistry and Physics 115 (2009) 296–302

␥-ray dose. However, when the ␥-ray doses were 10.0 and 15.0 kGy,
the intensity increased up to 0.06 mmole AgNO3 content implying a
larger number of silver particles formed for this condition. In addi-
tion, the largest number of silver particles formed was obtained
when AgNO3 content was 0.08 mmole for ␥-ray dose of 20 kGy and
0.10 mmole for 25 kGy. This might be explained by the formation
rate of nuclei being more significant than the growth rate of sil-
ver nanocrystal when the AgNO3 contents were less than those
specific values, e.g., 0.06 mmole for 10.0 and 15.0 kGy, 0.08 mmole
for 20.0 kGy, and 0.10 mmole for 25.0 kGy. The formation of silver

Fig. 6. TEM micrographs at 100 kV (100,000×) of silver particles formed in chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations; (A) 0.1%
(w/v) and (B) 0.5% (w/v), containing AgNO3 content of 0.10 mmole after ␥-ray irradiation with different doses: (a) 2.5 kGy, (b) 5.0 kGy, (c) 10.0 kGy, (d) 15.0 kGy, (e) 20.0 kGy
and (f) 25.0 kGy. Bar is 50 nm.
 R. Yoksan, S. Chirachanchai / Materials Chemistry and Physics 115 (2009) 296–302 301

Fig. 7. TEM micrographs at 100 kV (100,000×) of silver particles formed in chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1%
(w/v) and (B) 0.5% (w/v), containing different AgNO3 contents: (a) 0.02 mmole, (b) 0.04 mmole, (c) 0.06 mmole, (d) 0.08 mmole, and (e) 0.10 mmole, after ␥-ray irradiation
with the dose of 25.0 kGy. Bar is 50 nm.

particles increased with increasing ␥-ray dose up to 20 kGy. The
reduced number of silver particles formed in 25 kGy ␥-ray irradi-
ated chitosan solution might result from the particles’ aggregation
and/or the growth rate of silver nanocrystal over the formation rate
of nuclei. In addition, the amount of silver particles formed in 0.1%
(w/v) chitosan solutions was not significantly different from that
in 0.5% (w/v) chitosan solutions when the same ␥-ray dose was
applied.
3.3. Shape and size of silver particles
Shape and size of silver particles formed in the ␥-ray
irradiated chitosan solutions were observed by TEM. Most par-
ticles were spherical with an average diameter of 7–30 nm
(Figs. 6 and 7). The size of silver nanoparticles was dependent
on ␥-ray dose, initial AgNO3 content, and chitosan concentra-
tion.

Fig. 8. Photographs of antimicrobial test results against (A) E. coli and (B) S. aureus of (a) polysaccharide-based film and (b) silver nanoparticles incorporated polysaccharide-
based film.
302 R. Yoksan, S. Chirachanchai / Materials Chemistry and Physics 115 (2009) 296–302
Table 1
Antimicrobial activity of polysaccharide-based film and silver nanoparticles incor-
porated polysaccharide-based film against E. coli and S. aureus.
Concentration of 25 kGy ␥-ray Clear zone (cm)a
irradiated 0.5% (w/v) chitosan solutions
containing 0.04 mmole AgNO3 (%, v/v
of polysaccharide aqueous solution)
E. coli S. aureus
0 0.000 ± 0.000 0.000 ± 0.000
10 0.308 ± 0.038 0.079 ± 0.007
20 0.354 ± 0.019 0.092 ± 0.036
a
Reported as mean ± standard deviation (n = 3).
Although, Long et al. revealed that silver nanoparticles could not
be formed at the ␥-ray dose of less than 5 kGy [12], the present work
found a slightly different result in the formation of metal nanopar-
ticles at low ␥-ray dose, i.e., 2.5 and 5.0 kGy. This formation was
affirmable by the appearance of SPR band (see Section 3.2). How-
ever, such a formation was incomplete as manifested in Fig. 6(a) and
(b). The nanoparticles were maturely formed when the dose was
higher than 5.0 kGy. The size of particles increased with increasing
␥-ray dose, e.g., 20, 20, 25, and 30 nm for ␥-ray dose of 10.0, 15.0,
20.0, and 25.0 kGy, respectively, when 0.1% (w/v) chitosan solution
was used (Fig. 6A(c)–(f)). For chitosan solution with the concentra-
tion of 0.5% (w/v), the result showed same trend. The diameters of
particles were 14, 14, 17, and 17, when ␥-ray dose was 10.0, 15.0,
20.0, and 25.0 kGy, respectively (Fig. 6B(c)–(f)).
The size of silver nanoparticles was more significantly affected
by the initial AgNO3 content than ␥-ray dose. The size increased
with increasing initial amount of AgNO3 . For instance, silver parti-
cles formed in 25 kGy ␥-ray irradiated 0.1% (w/v) chitosan solution
containing different AgNO3 contents of 0.02, 0.04, 0.06, 0.08, and
0.10 mmole had average diameters of 9, 12, 17, 19, and 23 nm,
respectively (Fig. 7A). In the same way, the average diameters of
silver particles formed in higher chitosan concentration, i.e., 0.5%
(w/v) chitosan solution containing AgNO3 contents of 0.02, 0.04,
0.06, 0.08, and 0.10 mmole were 7, 9, 11, 12, and 14 nm, respec-
tively (Fig. 7B). This result corresponded to the one reported by
Long et al. [12] and Lei and Fan [15]. The nanoparticles formed in
0.1% (w/v) chitosan solution (Figs. 6A and 7A) were larger than those
formed in 0.5% (w/v) chitosan solution (Figs. 6B and 7B). This sup-
ported the above results (see Section 3.2). At high concentration of
cationic polymer solution, the low collision, low aggregation and/or
agglomeration, and strong electrostatic interactions were outstand-
ing; as a result, tiny metal nanoparticles with monodispersity were
obtained. This is in accordance with the result of Patakfalvi et al.
[16]
3.4. Antimicrobial activity of silver nanoparticles
The antimicrobial activity of silver nanoparticles incorporated
in polysaccharide-based films against E. coli and S. aureus, which
were applied as model Gram-negative and Gram-positive bacte-
ria, respectively, was studied by an agar diffusion method. After
24 h incubation at 37 ◦ C, the contact areas of all films with agar
surface turned transparent indicating the inhibition of bacteria
growth (Fig. 8). However, clear zones surrounding the film discs
were observed only for the films containing silver nanoparti-
cles (Fig. 8(b)). This implies that silver nanoparticles exhibited
inhibitory activity against E. coli and S. aureus. The inhibitory effi-
ciency related to the size of the inhibition zone, i.e., the larger the
clear area around the film, the higher the inhibitory efficiency.
The antimicrobial activity of the film increased with increas-
ing silver nanoparticle content (Table 1). Silver nanoparticles
showed higher antimicrobial activity against E. coli than S. aureus
(Table 1).
4. Conclusion
The preparation of silver nanoparticles was carried out by ␥-
ray irradiation under simple conditions, i.e., air atmosphere, using
chitosan as a stabilizer. The ␥-ray doses of 10–25 kGy were suffi-
cient to achieve maturely formed particles. The obtained particles
were spherical with an approximate size of 7–30 nm. The diame-
ter of the particles increased as a function of ␥-ray dose and initial
silver nitrate content. Chitosan–aqueous acetic acid solution with
the concentration of 0.5% (w/v) was preferred for fabrication of sta-
ble, monodispersed, and tiny silver nanoparticles compared to 0.1%
(w/v) chitosan solution. The obtained silver nanoparticles inhibited
the growth of the model Gram-positive and Gram-negative bacte-
ria. The results suggest that silver nanoparticles dispersed in chi-
tosan solution can be directly applied in antimicrobial fields, includ-
ing antimicrobial food packaging and biomedical applications
Acknowledgements
The authors thank the Thailand Research Fund (Grant No.
MRG5080398) for the financial support. Appreciation is also
expressed to the Office of Atomic Energy for Peace, Ministry of
Science and Technology, Thailand for its kind allowance to use a
60 Co Gammacell irradiator and Hitachi Hi-Technologies Corpora-
tion, Japan for TEM observation. Sincere gratitude goes to Mr. Adrian
Hillman for his proof reading.
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