Si

Ra

5b

c

a

ARR1A

KSNCIT

1

ai[fbsfiapufteR[h

FB

0d

Materials Chemistry and Physics 115 (2009) 296–302

Contents lists available at ScienceDirect

Materials Chemistry and Physics

journa l homepage: www.e lsev ier .com/ locate /matchemphys

ilver nanoparticles dispersing in chitosan solution: Preparation by �-rayrradiation and their antimicrobial activities

angrong Yoksana,b,∗, Suwabun Chirachanchaic

Department of Packaging Technology and Materials, Faculty of Agro-Industry, Kasesart University,0 Paholyothin Road, Ladyao, Jatujak, Bangkok 10900, ThailandDivision of Physico-Chemical Processing Technology, Faculty of Agro-Industry, Kasesart University, Bangkok 10900, ThailandThe Petroleum and Petrochemical College, Chulalongkorn University, Bangkok 10330, Thailand

r t i c l e i n f o

rticle history:eceived 25 September 2008eceived in revised form6 November 2008

a b s t r a c t

Silver nanoparticles were prepared by �-ray irradiation–reduction under simple conditions, i.e., airatmosphere, using chitosan as a stabilizer. The nanoparticles were spherical with an average size of7–30 nm as observed from TEM. The size decreased when chitosan concentration increased, while itincreased with increasing �-ray dose and initial silver nitrate content. The obtained silver nanoparti-

ccepted 1 December 2008

eywords:ilveranoparticlehitosan

rradiation

cles dispersed in a 0.5% (w/v) �-ray irradiated chitosan–aqueous acetic acid solution were stable for morethan 3 months without tendency to precipitate. The silver nanoparticles exhibited antimicrobial activitiesagainst Escherichia coli and Staphylococcus aureus. The results suggest that silver nanoparticles dispersedin chitosan solution can be directly applied in antimicrobial fields, including antimicrobial food packagingand biomedical applications.

© 2008 Elsevier B.V. All rights reserved.

ransmission electron microscopy (TEM)

. Introduction

For many decades, silver nanoparticles have been recognizeds an antimicrobial agent [1] and used in many fields includ-ng biomedical [2,3], food packaging [4,5], waste water treatment6], etc. The antimicrobial activities of silver nanoparticles wereound to relate to their shapes and sizes [7]. Many attempts haveeen made to synthesize the silver nanoparticles with controllablehape, size, and size distribution. In most cases, the system forabricating silver nanoparticles consists of salt precursor, reduc-ng agent, and stabilizer. Silver nitrate (AgNO3) is usually applieds the salt precursor. The reduction of silver ions (Ag+) to silverarticles (Ag0) is conventionally achieved by a chemical methodsing reducing agents such as NaBH4 [8], formamide [9], dimethyl-ormamide [9], triethanolamine [9], hydrazine [10], etc. Althoughhis method is simple and effective, the biological toxicity and the

nvironmental hazard of the residual reducing agent are problems.ecently, reductions by other techniques such as �-ray irradiation11,12], UV irradiation [13], microwave [14], and ultrasonic [15]ave been alternative ways to overcome those problems and to

∗ Corresponding author at: Department of Packaging Technology and Materials,aculty of Agro-Industry, Kasesart University, 50 Paholyothin Road, Ladyao, Jatujak,angkok 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).

254-0584/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.matchemphys.2008.12.001

eliminate the purification step or the removal of reducing agentstep.

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(vinylpyrrolidone) [16], poly(vinyl alcohol) [16], amphiphilic blockcopolymer 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 likedtogether by �-1,4-glucosidic bonds. Due to its biodegradable, bio-compatible, non-toxic and antimicrobial characteristics, chitosanis utilized in many areas. For the preparation of silver nanoparti-cles, chitosan has been used either as a reducing agent [13] or asa stabilizer [11–13]. Although the synthesis of silver nanoparticlesin chitosan–acetic acid solution using �-ray irradiation has beenreported, 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 irradiationunder 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 effectsof �-ray dose (2.5–25.0 kGy), chitosan concentration (0.1 and0.5%, w/v) and silver nitrate content (0.02–0.10 mmole) on theparticle size and particle number are also investigated. In addi-

Chem

ts

2

2

wna

2

diaHmbwLs

2i

ssst(wfr2s

2

sp(so

FcT

R. Yoksan, S. Chirachanchai / Materials

ion, the antimicrobial activity of the silver nanoparticles istudied.

. Experimental methods

.1. Materials

Chitosan (deacetylation degree of 0.95 and molecular weight of ∼700,000 Da)as purchased from the Seafresh Chitosan (Lab) Company Limited, Thailand. Silveritrate and acetic acid were supplied by Merck, Germany. All chemicals were useds received and without further purification.

.2. Instruments and equipment

Gamma ray irradiation was carried out in a 60Co Gammacell irradiator with aose rate of 12 kGy h−1 kindly provided by the Office of Atomic Energy for Peace, Min-

stry of Science and Technology, Bangkok, Thailand. Ultraviolet and visible (UV–vis)bsorption spectra were recorded over a wavelength from 200 to 500 nm by aelios Gamma Thermo Spectronic spectrometer (England). Transmission electronicroscopy (TEM) photographs were taken at an accelerating voltage of 100.0 kV

y a Hitachi H-7650 (Hitachi High-Technology Corporation, Japan). Zeta potentialas determined at 20 ◦C by a Malvern Zetasizer (model 3600, Malvern Instruments

td., UK) equipped with a He–Ne laser operating at 4.0 mW and 633 nm with a fixedcattering angle of 90◦ .

.3. Preparation of silver nanoparticles in chitosan solution using �-rayrradiation

Two concentrations of chitosan solutions (0.1 and 0.5%, w/v) were prepared bytirring chitosan in 1% (v/v) aqueous acetic acid at room temperature overnight. Theolutions were then centrifuged to eliminate traces of insoluble fractions. A fresholution of 50 mM AgNO3 was prepared in 1% (v/v) aqueous acetic acid at roomemperature. 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 mLith 1% (v/v) aqueous acetic acid. The mixture was stirred at room temperature

or 10 min and then �-ray irradiated in a 60Co Gammacell irradiator with the doseate of 12 kGy h−1. The dose of �-ray was varied as 0.0, 2.5, 5.0, 10.0, 15.0, 20.0, and5.0 kGy. Chitosan solution without the addition of AgNO3 was also prepared in theame manner and used as a control.

.4. Study on antimicrobial activity of silver nanoparticles

Antimicrobial activity of silver nanoparticles was studied by an agar diffu-ion method using polysaccharide as a film matrix. Silver particles incorporated inolysaccharide-based films were prepared by mixing 25 kGy �-ray irradiated 0.5%w/v) chitosan solution containing 0.04 mmole AgNO3 with polysaccharide aqueousolution. The content of silver nanoparticles was varied by changing concentrationf the �-ray irradiated chitosan solutions containing AgNO3 (0 (control), 10 and 20%,

ig. 1. Appearances of chitosan solutions (in 1% (v/v) aqueous acetic acid solution) with diontents: (a) 0.00 mmole, (b) 0.02 mmole, (c) 0.04 mmole, (d) 0.06 mmole, (e) 0.08 mmolehe first column (a0) is chitosan solution without the addition of AgNO3 and without �-ra

istry and Physics 115 (2009) 296–302 297

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 sterilizationin 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 seededwith 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). Theplates 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 weremeasured.

3. Results and discussion

Conventionally, silver nanoparticles are simply prepared by thereduction of silver salts using chemical reducing agents. Althoughthe 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–reductionunder simple conditions, i.e., air atmosphere, to prepare silvernanoparticles. Silver nitrate was applied as a salt precursor, whilechitosan was used as a stabilizer. To study the factors affectingformed particle size and number of particles, the �-ray dose, ini-tial AgNO3 content, and chitosan concentration were varied. Theamount of �-ray used was from 2.5 up to 25.0 kGy, initial AgNO3content 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 chitosanis non-toxic and has been approved by FDA, the silver nanoparticlesdispersing in chitosan solution do not need to be separated and/orpurified and may be directly used in antibacterial fields.

3.1. Formation of silver particles: physical appearance andmechanism

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 chainscission 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. Bycomparison with unirradiated chitosan solution (Fig. 2(a)), the �-

fferent concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v) containing different AgNO3

, and (f) 0.10 mmole, after �-ray irradiation with various doses from 2.5 to 25.0 kGy.y irradiation.

298 R. Yoksan, S. Chirachanchai / Materials Chemistry and Physics 115 (2009) 296–302

F cid soi 15.0 k

r(maCaa(daa

etpt3toc2bult

otrnTtsrohoaa(rahAt

ig. 2. UV–vis absorption spectra of chitosan solutions (in 1% (v/v) aqueous acetic arradiation with different doses: (a) 0.0 kGy, (b) 2.5 kGy, (c) 5.0 kGy, (d) 10.0 kGy, (e)

ay irradiated samples gave two new peaks at 260 and 290 nmFig. 2(b)–(g)). The absorption band that appeared at 260 nm

ight corresponds to the C O in COOH group, whereas the onet 290 nm belonged to a terminal carbonyl structure formed at1 or C4 after the main chain scission of chitosan and hydrogenbstraction followed by the ring opening reaction [23–26]. Thebsorbance increased with increasing �-ray dose up to 20.0 kGyFig. 2(b)–(f)) and chitosan concentration. However, at high �-rayose, i.e., 25 kGy, the decomposition and/or conversion of carbonylnd carboxyl species might have taken place resulting in reducedbsorbance (Fig. 2(g)).

The �-ray irradiated chitosan solutions containing AgNO3xhibit more intense yellow color (Fig. 1(b)–(f)) than those withouthe addition of AgNO3 (Fig. 1(a)) implying the formation of silverarticles [11,20]. This result was also confirmed by UV–vis spec-rophotometry (see Section 3.2) and TEM techniques (see Section.3). When the initial AgNO3 content and �-ray dose increased,he solution color mostly changed from yellow to red-brown. Inther words, the color intensity progressed as a function of AgNO3ontent and �-ray dose. For 0.1% (w/v) chitosan solution after5.0 kGy �-ray irradiation, the maximum color intensity (dark red-rown) appeared when AgNO3 content of 0.04–0.06 mmole wassed (Fig. 1(c) and (d), left). This might have resulted from either a

arge amount of silver particle formation or silver particle aggrega-ion [27].

When the �-ray dose was in the range of 10.0–25.0 kGy, the colorf 0.1% (w/v) chitosan solutions containing AgNO3 was more intensehan that of 0.5% (w/v) chitosan solutions. We speculate that theeduction of silver ions (Ag+) to silver particles (Ag0) was affectedot only by �-ray dose, but also by weight ratio of AgNO3 to chitosan.he weight ratio of AgNO3 to chitosan in the case of 0.1% (w/v) chi-osan solution was fivefold higher than that of 0.5% (w/v) chitosanolution. Herein, we suspect that �-ray irradiation induced botheactions, which are the chain scission of chitosan and the reductionf silver ions to silver particles. For the former, hydroxyl radicals andydrogen atoms, which are the intermediate products of water radi-lysis (Eq. (1)), react with polysaccharide molecules by hydrogenbstraction resulting in the formation of macroradicals localizedt various carbon atoms (C1–C6) within the glucosamine unit (Eq.2)) [23,28]. Only radicals formed at C1 and C4 atoms can undergo

earrangement involving breakage of 1–4 glycosidic bonds (Eq. (3))nd formation of C O species as discussed above. For the latter, theydrated electron generated by water radiolysis can reduce Ag+ tog0 (Eq. (4)) [11,12,29]. The neutral atom, i.e., Ag0, reacts with Ag+

o form the relatively stabilized Ag clusters as shown in Eqs. (5) and

lution) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), after �-rayGy, (f) 20.0 kGy, and (g) 25.0 kGy.

(6) [30,31]. Such clusters then gather together or absorb the neutralAg0 to form the silver particles [11,12,30,31].

H2Oioizing−→

radiationOH •, e−

aq, H•, H2O2, H2, H+ (1)

OH•(H•) + R–H → R•(C1–C6) + H2O(H2) (2)

R•(C1, C4) → F1• + F2

•(chainscission) (3)

Ag+ + e−aq

reduction−→ Ag0 (4)

Ag0 + Ag+ → Ag2+ (5)

Ag2+ + Ag+ → Ag3

2+ (6)

By considering the structure of chitosan in acidic solution, aminogroups were protonated (–NH3

+). Accordingly, the binding of silverclusters by chitosan may be achieved through the Ag–O bonds asreported in previous studies [12,32,33], the ionic interaction formedbetween silver ion and carboxyl group on chitosan generated dur-ing �-ray irradiation (Ag+. . .COO−), or the combination thereof. Theprotonated amino groups (–NH3

+) at the surface of formed silverparticles, thus, promoted the stability of the particles via staticrepulsion. This speculation is supported by the result from zetapotentiometer, e.g. a positively charged surface with the zeta poten-tial of 40.41 ± 2.50 mV was observed for silver particles formed in25 kGy �-ray irradiated 0.5% (w/v) chitosan solution containing0.04 mmole AgNO3. It should be pointed out that silver particlesformed in 0.5% (w/v) chitosan solution were stable for more than 3months, whereas those in 0.1% (w/v) chitosan solution were precip-itated out within a few weeks. This might be due to a larger numberof 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 confirmthe formation of silver particles in the �-ray irradiated chitosansolution. Generally, �-ray irradiated chitosan solutions exhibit noabsorption 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 afunction of �-ray dose and initial AgNO3 content can be observedin 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.

F d soluv ) 5.0 k

Aotpsst

Fc

ig. 4. Maximum wavelength of chitosan solutions (in 1% (v/v) aqueous acetic aciarious AgNO3 contents after �-ray irradiation with different doses: (�) 2.5 kGy, (©

The SPR bands of �-ray irradiated chitosan solutions containinggNO3 appear at the wavelength of 399–417 nm (Fig. 4). A red shiftf SPR band (to higher wavelength) was observed when the ini-

ial AgNO3 content increased (Fig. 4). This indicated an increase inarticle size and/or the formation of silver aggregates [34,35]. It ispeculated that the collision frequency of silver particles increasedignificantly with increasing AgNO3 content resulting in silver par-icle aggregation. This speculation was also confirmed by TEM

ig. 5. Absorbance at maximum wavelength of chitosan solutions (in 1% (v/v) aqueous acontaining various AgNO3 contents after �-ray irradiation with different doses: (�) 2.5 kG

tion) with different concentrations. (A) 0.1% (w/v) and (B) 0.5% (w/v), containingGy, (�) 10.0 kGy, (�) 15.0 kGy, (�) 20.0 kGy, and (�) 25.0 kGy.

(see Section 3.3). However, the �-ray dose hardly affected the SPRband.

The amount of silver particles formed related to the absorbance

of �-ray irradiated chitosan solution containing AgNO3 [11,36]. Forlow �-ray dose, i.e., 2.5 and 5.0 kGy, the absorbance was constantat 2–3 and 6–9 for 2.5 and 5.0 kGy, respectively, though AgNO3content is varied (Fig. 5). In other words, AgNO3 content hardlyaffected the absorbance or the number of particles formed at low

etic acid solution) with different concentrations; (A) 0.1% (w/v) and (B) 0.5% (w/v),y, (©) 5.0 kGy, (�) 10.0 kGy, (�) 15.0 kGy, (�) 20.0 kGy, and (�) 25.0 kGy.

3 Chem

�tltw

F(a

00 R. Yoksan, S. Chirachanchai / Materials

-ray dose. However, when the �-ray doses were 10.0 and 15.0 kGy,he intensity increased up to 0.06 mmole AgNO3 content implying aarger number of silver particles formed for this condition. In addi-ion, the largest number of silver particles formed was obtainedhen AgNO3 content was 0.08 mmole for �-ray dose of 20 kGy and

ig. 6. TEM micrographs at 100 kV (100,000×) of silver particles formed in chitosan solutiw/v) and (B) 0.5% (w/v), containing AgNO3 content of 0.10 mmole after �-ray irradiationnd (f) 25.0 kGy. Bar is 50 nm.

istry and Physics 115 (2009) 296–302

0.10 mmole for 25 kGy. This might be explained by the formationrate of nuclei being more significant than the growth rate of sil-ver nanocrystal when the AgNO3 contents were less than thosespecific values, e.g., 0.06 mmole for 10.0 and 15.0 kGy, 0.08 mmolefor 20.0 kGy, and 0.10 mmole for 25.0 kGy. The formation of silver

ons (in 1% (v/v) aqueous acetic acid solution) with different concentrations; (A) 0.1%with different doses: (a) 2.5 kGy, (b) 5.0 kGy, (c) 10.0 kGy, (d) 15.0 kGy, (e) 20.0 kGy

R. Yoksan, S. Chirachanchai / Materials Chemistry and Physics 115 (2009) 296–302 301

F soluti( 0.04 mw

praao(ia

Fb

ig. 7. TEM micrographs at 100 kV (100,000×) of silver particles formed in chitosanw/v) and (B) 0.5% (w/v), containing different AgNO3 contents: (a) 0.02 mmole, (b)ith the dose of 25.0 kGy. Bar is 50 nm.

articles increased with increasing �-ray dose up to 20 kGy. Theeduced number of silver particles formed in 25 kGy �-ray irradi-ted chitosan solution might result from the particles’ aggregationnd/or the growth rate of silver nanocrystal over the formation rate

f nuclei. In addition, the amount of silver particles formed in 0.1%w/v) chitosan solutions was not significantly different from thatn 0.5% (w/v) chitosan solutions when the same �-ray dose waspplied.

ig. 8. Photographs of antimicrobial test results against (A) E. coli and (B) S. aureus of (a) pased film.

ons (in 1% (v/v) aqueous acetic acid solution) with different concentrations. (A) 0.1%mole, (c) 0.06 mmole, (d) 0.08 mmole, and (e) 0.10 mmole, after �-ray irradiation

3.3. Shape and size of silver particles

Shape and size of silver particles formed in the �-rayirradiated 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 dependenton �-ray dose, initial AgNO3 content, and chitosan concentra-tion.

olysaccharide-based film and (b) silver nanoparticles incorporated polysaccharide-

302 R. Yoksan, S. Chirachanchai / Materials Chem

Table 1Antimicrobial activity of polysaccharide-based film and silver nanoparticles incor-porated polysaccharide-based film against E. coli and S. aureus.

Concentration of 25 kGy �-rayirradiated 0.5% (w/v) chitosan solutionscontaining 0.04 mmole AgNO3 (%, v/vof polysaccharide aqueous solution)

Clear zone (cm)a

E. coli S. aureus

0 0.000 ± 0.000 0.000 ± 0.000

2

bftae(h�2wtp2

bwcc0rs(0tL0fpcaio[

3

iwr2sgwciccTis(

[

[[

[

[[[

[

[

[[[[[[

10 0.308 ± 0.038 0.079 ± 0.0070 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 note formed at the �-ray dose of less than 5 kGy [12], the present workound a slightly different result in the formation of metal nanopar-icles at low �-ray dose, i.e., 2.5 and 5.0 kGy. This formation wasffirmable by the appearance of SPR band (see Section 3.2). How-ver, such a formation was incomplete as manifested in Fig. 6(a) andb). The nanoparticles were maturely formed when the dose wasigher 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, 15.0,0.0, and 25.0 kGy, respectively, when 0.1% (w/v) chitosan solutionas used (Fig. 6A(c)–(f)). For chitosan solution with the concentra-

ion of 0.5% (w/v), the result showed same trend. The diameters ofarticles were 14, 14, 17, and 17, when �-ray dose was 10.0, 15.0,0.0, and 25.0 kGy, respectively (Fig. 6B(c)–(f)).

The size of silver nanoparticles was more significantly affectedy the initial AgNO3 content than �-ray dose. The size increasedith increasing initial amount of AgNO3. For instance, silver parti-

les formed in 25 kGy �-ray irradiated 0.1% (w/v) chitosan solutionontaining different AgNO3 contents of 0.02, 0.04, 0.06, 0.08, and.10 mmole had average diameters of 9, 12, 17, 19, and 23 nm,espectively (Fig. 7A). In the same way, the average diameters ofilver particles formed in higher chitosan concentration, i.e., 0.5%w/v) chitosan solution containing AgNO3 contents of 0.02, 0.04,.06, 0.08, and 0.10 mmole were 7, 9, 11, 12, and 14 nm, respec-ively (Fig. 7B). This result corresponded to the one reported byong et al. [12] and Lei and Fan [15]. The nanoparticles formed in.1% (w/v) chitosan solution (Figs. 6A and 7A) were larger than thoseormed in 0.5% (w/v) chitosan solution (Figs. 6B and 7B). This sup-orted the above results (see Section 3.2). At high concentration ofationic polymer solution, the low collision, low aggregation and/orgglomeration, and strong electrostatic interactions were outstand-ng; as a result, tiny metal nanoparticles with monodispersity werebtained. This is in accordance with the result of Patakfalvi et al.16]

.4. Antimicrobial activity of silver nanoparticles

The antimicrobial activity of silver nanoparticles incorporatedn polysaccharide-based films against E. coli and S. aureus, which

ere applied as model Gram-negative and Gram-positive bacte-ia, respectively, was studied by an agar diffusion method. After4 h incubation at 37 ◦C, the contact areas of all films with agarurface turned transparent indicating the inhibition of bacteriarowth (Fig. 8). However, clear zones surrounding the film discsere observed only for the films containing silver nanoparti-

les (Fig. 8(b)). This implies that silver nanoparticles exhibitednhibitory activity against E. coli and S. aureus. The inhibitory effi-iency related to the size of the inhibition zone, i.e., the larger the

lear area around the film, the higher the inhibitory efficiency.he antimicrobial activity of the film increased with increas-ng silver nanoparticle content (Table 1). Silver nanoparticleshowed higher antimicrobial activity against E. coli than S. aureusTable 1).

[

[[

istry and Physics 115 (2009) 296–302

4. Conclusion

The preparation of silver nanoparticles was carried out by �-ray irradiation under simple conditions, i.e., air atmosphere, usingchitosan as a stabilizer. The �-ray doses of 10–25 kGy were suffi-cient to achieve maturely formed particles. The obtained particleswere spherical with an approximate size of 7–30 nm. The diame-ter of the particles increased as a function of �-ray dose and initialsilver nitrate content. Chitosan–aqueous acetic acid solution withthe 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 inhibitedthe 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 alsoexpressed to the Office of Atomic Energy for Peace, Ministry ofScience and Technology, Thailand for its kind allowance to use a60Co Gammacell irradiator and Hitachi Hi-Technologies Corpora-tion, Japan for TEM observation. Sincere gratitude goes to Mr. AdrianHillman for his proof reading.

References

[1] I. Sondi, B. Salopek-Sondi, J. Colloids Interf. Sci. 275 (2004) 177.[2] Q. Wu, H. Cao, Q. Luan, J. Zhang, Z. Wang, J.H. Warner, A.A.R. Watt, Inorg. Chem.

47 (2008) 5882.[3] L.S. Nair, C.T. Laurencin, J. Biomed. Nanotechnol. 3 (2007) 301.[4] Food casing based on cellulose hydrate with nanoparticles, United States Patent

20080145576.[5] M. Siegrist, M.-E. Cousin, H. Kastenholz, A. Wiek, Appetite 49 (2007) 459.[6] K. Dhermendra, J.B. Tiwari, P. Sen, World Appl. Sci. J. 3 (2008) 417.[7] H. Jiang, S. Manolache, A.C.L. Wong, F.S. Denes, J. Appl. Polym. Sci. 93 (2004)

1411.[8] H. Huang, Q. Yuan, X. Yang, Colloids Surf. B: Biointerf. 39 (2004) 31.[9] C.R.K. Rao, D.C. Trivedi, Synth. Met. 155 (2005) 324.

[10] W. Zhang, X. Qiao, J. Chen, H. Wang, J. Colloids Interf. Sci. 302 (2006) 370.[11] P. Chen, L. Song, Y. Liu, Y. Fang, Radiat. Phys. Chem. 76 (2007) 1165.12] D. Long, G. Wu, S. Chen, Radiat. Phys. Chem. 76 (2007) 1126.

[13] D.M. Cheng, X.D. Zhou, H.B. Xia, H.S.O. Chan, Chem. Mater. 17 (2005) 3578.[14] M. Tsuji, Y. Nishizawa, K. Matsumoto, N. Miyamae, T. Tsuji, X. Zhang, Colloids

Surf. A: Physicochem. Eng. Aspects 293 (2007) 185.[15] Z. Lei, Y. Fan, Mater. Lett. 60 (2006) 2256.[16] R. Patakfalvi, Z. Viranyi, I. Dekany, Colloids Polym. Sci. 283 (2004) 299.[17] N. Vigneshwaran, R.P. Nachane, R.H. Balasubramanya, P.V. Varadarajan, Carbo-

hydr. Res. 341 (2006) 2012.[18] H. Huang, X. Yang, Carbohydr. Res. 339 (2004) 2627.[19] D. Wei, W. Qian, Colloids Surf. B: Biointerf. 62 (2008) 136.20] X. Xu, Y. Yin, X. Ge, H. Wu, Z. Zhang, Mater. Lett. 37 (1998) 354.21] N. Nagasawa, H. Mitomo, F. Yoshii, T. Kume, Polym. Degrad. Stabil. 69 (2000)

279.22] W.S. Choi, K.J. Ahn, D.W. Lee, M.W. Byun, H.J. Park, Polym. Degrad. Stabil. 78

(2002) 533.23] P. Ulanski, J.M. Rosiak, Radiat. Phys. Chem. 39 (1992) 53.24] B. Kang, Y.-D. Dai, H.-Q. Zhang, D. Chen, Polym. Degrad. Stabil. 92 (2007) 359.25] R. Yoksan, M. Akashi, S. Biramontri, S. Chirachanchai, Biomacromolecules 2

(2001) 1038.26] R. Czechowska-Biskup, B. Rokita, S. Lotfy, P. Ulanski, J.M. Rosiak, Carbohydr.

Polym. 60 (2005) 175.27] J.J. Mock, M. Barbic, D.R. Smith, D.A. Schultz, S. Schultz, J. Chem. Phys. 116 (2002)

6755.28] R. Yoksan, M. Akashi, M. Miyata, S. Chirachanchai, Radiat. Res. 161 (2004) 471.29] Y. Zhu, Y. Qian, X. Li, M. Zhang, Chem. Commun. 12 (1997) 1081.30] E. Janata, A. Henglein, B.G. Ershov, J. Phys. Chem. 98 (1994) 10888.31] E. Janata, J. Phys. Chem. B 107 (2003) 7334.32] S.P. Chen, G.Z. Wu, H.Y. Zeng, Carbohydr. Polym. 60 (2005) 33.33] H.H. Huang, X.P. Ni, G.L. Loy, C.H. Chew, K.L. Tan, F.C. Loh, J.F. Deng, G.Q. Xu,

Langmuir 12 (1996) 909.34] A. Kumar, H. Joshi, R. Pasricha, A.B. Mandale, M. Sastry, J. Colloids Interf. Sci. 264

(2003) 396.35] J.P. Zhang, P. Chen, C.H. Sun, X.J. Hu, Appl. Catal. A: Gen. 266 (2004) 49.36] Z.Q. Zhang, R.C. Patel, R. Kothari, C.P. Johnson, S.E. Friberg, P.A. Aikens, J. Phys.

Chem. B 104 (2000) 1176.