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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


step.
For many decades, silver nanoparticles have been recognized To prevent the aggregation of formed metal nanoparticles, var-
as an antimicrobial agent [1] and used in many fields includ- ious types of stabilizers are used such as long-chain fatty acids
ing biomedical [2,3], food packaging [4,5], waste water treatment (stearic, palmitic, and lauric acids) [9], lauryl amine, poly(vinyl
[6], etc. The antimicrobial activities of silver nanoparticles were pyrrolidone) [16], poly(vinyl alcohol) [16], amphiphilic block
found to relate to their shapes and sizes [7]. Many attempts have copolymer PEA [15], soluble starch [17], heparin [18], chitosan
been made to synthesize the silver nanoparticles with controllable [8,11,12,19], etc.
shape, size, and size distribution. In most cases, the system for Chitosan, the second most naturally abundant polysaccharide,
fabricating silver nanoparticles consists of salt precursor, reduc- consists of glucosamine and N-acetyl glucosamine units liked
ing agent, and stabilizer. Silver nitrate (AgNO3 ) is usually applied together by ␤-1,4-glucosidic bonds. Due to its biodegradable, bio-
as the salt precursor. The reduction of silver ions (Ag+ ) to silver compatible, non-toxic and antimicrobial characteristics, chitosan
particles (Ag0 ) is conventionally achieved by a chemical method is utilized in many areas. For the preparation of silver nanoparti-
using reducing agents such as NaBH4 [8], formamide [9], dimethyl- cles, chitosan has been used either as a reducing agent [13] or as
formamide [9], triethanolamine [9], hydrazine [10], etc. Although a stabilizer [11–13]. Although the synthesis of silver nanoparticles
this method is simple and effective, the biological toxicity and the in chitosan–acetic acid solution using ␥-ray irradiation has been
environmental hazard of the residual reducing agent are problems. reported, the irradiation condition was under nitrogen atmosphere
Recently, reductions by other techniques such as ␥-ray irradiation [11,12,20].
[11,12], UV irradiation [13], microwave [14], and ultrasonic [15] The present research studies the fabrication of silver nanoparti-
have been alternative ways to overcome those problems and to 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-
∗ Corresponding author at: Department of Packaging Technology and Materials,
mon resonance (SPR) band and electron micrograph. The effects
Faculty of Agro-Industry, Kasesart University, 50 Paholyothin Road, Ladyao, Jatujak,
of ␥-ray dose (2.5–25.0 kGy), chitosan concentration (0.1 and
Bangkok 10900, Thailand. Tel.: +66 2 562 5097; fax: +66 2 562 5092. 0.5%, w/v) and silver nitrate content (0.02–0.10 mmole) on the
E-mail addresses: rangrong.y@ku.ac.th, yyrangrong@yahoo.com (R. Yoksan). 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) 296–302 297

tion, the antimicrobial activity of the silver nanoparticles is v/v). The mixtures were then cast onto acrylic plates and dried at 45 ◦ C overnight.
studied. 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-
2. Experimental methods 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,
2.1. Materials 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-
Chitosan (deacetylation degree of 0.95 and molecular weight of ∼700,000 Da) face were observed and the diameters of clear zones surrounding the film discs were
was purchased from the Seafresh Chitosan (Lab) Company Limited, Thailand. Silver measured.
nitrate and acetic acid were supplied by Merck, Germany. All chemicals were used
as received and without further purification. 3. Results and discussion
2.2. Instruments and equipment
Conventionally, silver nanoparticles are simply prepared by the
Gamma ray irradiation was carried out in a 60 Co Gammacell irradiator with a reduction of silver salts using chemical reducing agents. Although
dose rate of 12 kGy h−1 kindly provided by the Office of Atomic Energy for Peace, Min- the chemical reduction is effective, the toxicity of residual reduc-
istry of Science and Technology, Bangkok, Thailand. Ultraviolet and visible (UV–vis) ing agents and the environmental impact should be considered.
absorption spectra were recorded over a wavelength from 200 to 500 nm by a
Helios Gamma Thermo Spectronic spectrometer (England). Transmission electron
The present work, thus, focused on the ␥-ray irradiation–reduction
microscopy (TEM) photographs were taken at an accelerating voltage of 100.0 kV under simple conditions, i.e., air atmosphere, to prepare silver
by a Hitachi H-7650 (Hitachi High-Technology Corporation, Japan). Zeta potential nanoparticles. Silver nitrate was applied as a salt precursor, while
was determined at 20 ◦ C by a Malvern Zetasizer (model 3600, Malvern Instruments chitosan was used as a stabilizer. To study the factors affecting
Ltd., UK) equipped with a He–Ne laser operating at 4.0 mW and 633 nm with a fixed
formed particle size and number of particles, the ␥-ray dose, ini-
scattering angle of 90◦ .
tial AgNO3 content, and chitosan concentration were varied. The
2.3. Preparation of silver nanoparticles in chitosan solution using -ray amount of ␥-ray used was from 2.5 up to 25.0 kGy, initial AgNO3
irradiation 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
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
is non-toxic and has been approved by FDA, the silver nanoparticles
solutions were then centrifuged to eliminate traces of insoluble fractions. A fresh dispersing in chitosan solution do not need to be separated and/or
solution of 50 mM AgNO3 was prepared in 1% (v/v) aqueous acetic acid at room purified and may be directly used in antibacterial fields.
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
3.1. Formation of silver particles: physical appearance and
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 mechanism
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 In general, chitosan solution is transparent without color
same manner and used as a control.
(Fig. 1(a0 )). The solution turns pale yellow after ␥-ray irradi-
2.4. Study on antimicrobial activity of silver nanoparticles ation (Fig. 1(a)). The color intensity increased with increasing
␥-ray dose, especially in the case of 0.5% (w/v) chitosan solu-
Antimicrobial activity of silver nanoparticles was studied by an agar diffu- tion. This might involve the double-bond formation by the chain
sion method using polysaccharide as a film matrix. Silver particles incorporated in scission of polysaccharide as reported previously [21,22]. Here,
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
UV–vis spectrophotometry technique was also applied to deter-
solution. The content of silver nanoparticles was varied by changing concentration mine the structural changes of chitosan after ␥-ray irradiation. By
of the ␥-ray irradiated chitosan solutions containing AgNO3 (0 (control), 10 and 20%, comparison with unirradiated chitosan solution (Fig. 2(a)), the ␥-

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.
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 (6) [30,31]. Such clusters then gather together or absorb the neutral
(Fig. 2(b)–(g)). The absorption band that appeared at 260 nm Ag0 to form the silver particles [11,12,30,31].
might corresponds to the C O in COOH group, whereas the one
ioizing +
at 290 nm belonged to a terminal carbonyl structure formed at H2 O −→ OH • , e− •
aq , H , H2 O2 , H2 , H (1)
radiation
C1 or C4 after the main chain scission of chitosan and hydrogen
abstraction followed by the ring opening reaction [23–26]. The OH• (H• ) + R–H → R • (C1 –C6 ) + H2 O(H2 ) (2)
absorbance increased with increasing ␥-ray dose up to 20.0 kGy
(Fig. 2(b)–(f)) and chitosan concentration. However, at high ␥-ray R • (C1 , C4 ) → F1 • + F2 • (chainscission) (3)
dose, i.e., 25 kGy, the decomposition and/or conversion of carbonyl reduction
and carboxyl species might have taken place resulting in reduced Ag+ + e−
aq −→ Ag
0
(4)
absorbance (Fig. 2(g)).
Ag0 + Ag+ → Ag2 + (5)
The ␥-ray irradiated chitosan solutions containing AgNO3
exhibit more intense yellow color (Fig. 1(b)–(f)) than those without Ag2 + + Ag+ → Ag3 2+ (6)
the addition of AgNO3 (Fig. 1(a)) implying the formation of silver
particles [11,20]. This result was also confirmed by UV–vis spec- By considering the structure of chitosan in acidic solution, amino
trophotometry (see Section 3.2) and TEM techniques (see Section groups were protonated (–NH3 + ). Accordingly, the binding of silver
3.3). When the initial AgNO3 content and ␥-ray dose increased, clusters by chitosan may be achieved through the Ag–O bonds as
the solution color mostly changed from yellow to red-brown. In reported in previous studies [12,32,33], the ionic interaction formed
other words, the color intensity progressed as a function of AgNO3 between silver ion and carboxyl group on chitosan generated dur-
content and ␥-ray dose. For 0.1% (w/v) chitosan solution after ing ␥-ray irradiation (Ag+ . . .COO− ), or the combination thereof. The
25.0 kGy ␥-ray irradiation, the maximum color intensity (dark red- protonated amino groups (–NH3 + ) at the surface of formed silver
brown) appeared when AgNO3 content of 0.04–0.06 mmole was particles, thus, promoted the stability of the particles via static
used (Fig. 1(c) and (d), left). This might have resulted from either a repulsion. This speculation is supported by the result from zeta
large amount of silver particle formation or silver particle aggrega- potentiometer, e.g. a positively charged surface with the zeta poten-
tion [27]. tial of 40.41 ± 2.50 mV was observed for silver particles formed in
When the ␥-ray dose was in the range of 10.0–25.0 kGy, the color 25 kGy ␥-ray irradiated 0.5% (w/v) chitosan solution containing
of 0.1% (w/v) chitosan solutions containing AgNO3 was more intense 0.04 mmole AgNO3 . It should be pointed out that silver particles
than that of 0.5% (w/v) chitosan solutions. We speculate that the formed in 0.5% (w/v) chitosan solution were stable for more than 3
reduction of silver ions (Ag+ ) to silver particles (Ag0 ) was affected months, whereas those in 0.1% (w/v) chitosan solution were precip-
not only by ␥-ray dose, but also by weight ratio of AgNO3 to chitosan. itated out within a few weeks. This might be due to a larger number
The weight ratio of AgNO3 to chitosan in the case of 0.1% (w/v) chi- of chitosan chains enveloping the silver particle surface when 0.5%
tosan solution was fivefold higher than that of 0.5% (w/v) chitosan (w/v) chitosan solution was used.
solution. Herein, we suspect that ␥-ray irradiation induced both
reactions, which are the chain scission of chitosan and the reduction 3.2. Formation of silver particles: number of particles
of silver ions to silver particles. For the former, hydroxyl radicals and
hydrogen atoms, which are the intermediate products of water radi- UV–vis spectrophotometry technique was applied to confirm
olysis (Eq. (1)), react with polysaccharide molecules by hydrogen the formation of silver particles in the ␥-ray irradiated chitosan
abstraction resulting in the formation of macroradicals localized solution. Generally, ␥-ray irradiated chitosan solutions exhibit no
at various carbon atoms (C1 –C6 ) within the glucosamine unit (Eq. absorption peak at wavelengths longer than 350 nm (Fig. 2(b)–(g)),
(2)) [23,28]. Only radicals formed at C1 and C4 atoms can undergo while the ones containing AgNO3 show a peak at 401–409 nm corre-
rearrangement involving breakage of 1–4 glycosidic bonds (Eq. (3)) sponding to the surface plasmon resonance band of silver particles
and formation of C O species as discussed above. For the latter, the (Fig. 3) [34].
hydrated electron generated by water radiolysis can reduce Ag+ to An increase in absorbance and a slight shift of SPR band as a
Ag0 (Eq. (4)) [11,12,29]. The neutral atom, i.e., Ag0 , reacts with Ag+ function of ␥-ray dose and initial AgNO3 content can be observed
to form the relatively stabilized Ag clusters as shown in Eqs. (5) and 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 (see Section 3.3). However, the ␥-ray dose hardly affected the SPR
AgNO3 appear at the wavelength of 399–417 nm (Fig. 4). A red shift band.
of SPR band (to higher wavelength) was observed when the ini- The amount of silver particles formed related to the absorbance
tial AgNO3 content increased (Fig. 4). This indicated an increase in of ␥-ray irradiated chitosan solution containing AgNO3 [11,36]. For
particle size and/or the formation of silver aggregates [34,35]. It is low ␥-ray dose, i.e., 2.5 and 5.0 kGy, the absorbance was constant
speculated that the collision frequency of silver particles increased at 2–3 and 6–9 for 2.5 and 5.0 kGy, respectively, though AgNO3
significantly with increasing AgNO3 content resulting in silver par- content is varied (Fig. 5). In other words, AgNO3 content hardly
ticle aggregation. This speculation was also confirmed by TEM 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, 0.10 mmole for 25 kGy. This might be explained by the formation
the intensity increased up to 0.06 mmole AgNO3 content implying a rate of nuclei being more significant than the growth rate of sil-
larger number of silver particles formed for this condition. In addi- ver nanocrystal when the AgNO3 contents were less than those
tion, the largest number of silver particles formed was obtained specific values, e.g., 0.06 mmole for 10.0 and 15.0 kGy, 0.08 mmole
when AgNO3 content was 0.08 mmole for ␥-ray dose of 20 kGy and 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 3.3. Shape and size of silver particles
reduced number of silver particles formed in 25 kGy ␥-ray irradi-
ated chitosan solution might result from the particles’ aggregation Shape and size of silver particles formed in the ␥-ray
and/or the growth rate of silver nanocrystal over the formation rate irradiated chitosan solutions were observed by TEM. Most par-
of nuclei. In addition, the amount of silver particles formed in 0.1% ticles were spherical with an average diameter of 7–30 nm
(w/v) chitosan solutions was not significantly different from that (Figs. 6 and 7). The size of silver nanoparticles was dependent
in 0.5% (w/v) chitosan solutions when the same ␥-ray dose was on ␥-ray dose, initial AgNO3 content, and chitosan concentra-
applied. 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 4. Conclusion
Antimicrobial activity of polysaccharide-based film and silver nanoparticles incor-
porated polysaccharide-based film against E. coli and S. aureus.
The preparation of silver nanoparticles was carried out by ␥-
Concentration of 25 kGy ␥-ray Clear zone (cm)a ray irradiation under simple conditions, i.e., air atmosphere, using
irradiated 0.5% (w/v) chitosan solutions
chitosan as a stabilizer. The ␥-ray doses of 10–25 kGy were suffi-
containing 0.04 mmole AgNO3 (%, v/v
of polysaccharide aqueous solution)
cient to achieve maturely formed particles. The obtained particles
E. coli S. aureus 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
0 0.000 ± 0.000 0.000 ± 0.000
10 0.308 ± 0.038 0.079 ± 0.007
silver nitrate content. Chitosan–aqueous acetic acid solution with
20 0.354 ± 0.019 0.092 ± 0.036 the concentration of 0.5% (w/v) was preferred for fabrication of sta-
ble, monodispersed, and tiny silver nanoparticles compared to 0.1%
a
Reported as mean ± standard deviation (n = 3).
(w/v) chitosan solution. The obtained silver nanoparticles inhibited
the growth of the model Gram-positive and Gram-negative bacte-
Although, Long et al. revealed that silver nanoparticles could not ria. The results suggest that silver nanoparticles dispersed in chi-
be formed at the ␥-ray dose of less than 5 kGy [12], the present work tosan solution can be directly applied in antimicrobial fields, includ-
found a slightly different result in the formation of metal nanopar- ing antimicrobial food packaging and biomedical applications
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- Acknowledgements
ever, such a formation was incomplete as manifested in Fig. 6(a) and
(b). The nanoparticles were maturely formed when the dose was The authors thank the Thailand Research Fund (Grant No.
higher than 5.0 kGy. The size of particles increased with increasing MRG5080398) for the financial support. Appreciation is also
␥-ray dose, e.g., 20, 20, 25, and 30 nm for ␥-ray dose of 10.0, 15.0, expressed to the Office of Atomic Energy for Peace, Ministry of
20.0, and 25.0 kGy, respectively, when 0.1% (w/v) chitosan solution Science and Technology, Thailand for its kind allowance to use a
60 Co Gammacell irradiator and Hitachi Hi-Technologies Corpora-
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 tion, Japan for TEM observation. Sincere gratitude goes to Mr. Adrian
particles were 14, 14, 17, and 17, when ␥-ray dose was 10.0, 15.0, Hillman for his proof reading.
20.0, and 25.0 kGy, respectively (Fig. 6B(c)–(f)).
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