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Synthesis of antibacterial silver nanoparticles by g-irradiation

N. Sheikh a,, A. Akhavan a,b, M.Z. Kassaee b,

a Radiation Applications Research School, Nuclear Science and Technology Research Institute, Tehran, Iranb Chemistry Department, Tarbiat Modares University, Tehran, Iran

a r t i c l e i n f o

Article history:

Received 2 December 2008

Received in revised form

22 August 2009Accepted 18 September 2009Available online 2 October 2009

PACS:

07.85.m

61.46.Df

Keywords:

g-irradiation

Silver nanoparticles

Antibacterial

a b s t r a c t

Silver nanoparticles (Ag NPs) were synthesized by g-irradiation of silver ions in aqueous solutions

containing polyvinyl pyrrolidone (PVP). Increasing ofg-irradiation doses from 1 to 5 kGy enhanced the

concentration of Ag NPs, indicated by UVvis analysis. The XRD and the TEM measurements showed the

production of face-centered cubic (fcc) Ag NPs with a mean size of about 16 nm. The produced

nanoparticles were effectively stabilized by PVP through interactions, confirmed by the FT-IR. The

relatively higher antibacterial activities of Ag NPs, synthesized through g-irradiation method, against E.

coli indicate their potential for practical applications as biocidal materials.

& 2009 Elsevier B.V. All rights reserved.

1. Introduction

Silver nanoparticles (Ag NPs) have attracted considerableinterest for their unique optical properties [1], electrical con-

ductivities [2], oxidative catalysis [3], and antibacterial effects.

The antibacterial and antiviral actions of silver and silver-based

compounds have been thoroughly investigated since ancient

times [47]. Currently nano-sized silver particles are used to

control the bacterial growth in a variety of applications, including

medical devices, dental resin composites, and textile materials

[8,9]. The most common method for synthesis of Ag NPs is the

chemical reduction of silver ions in different stabilizers such as

polymers and surfactants [1014]. However, g-irradiation synth-

esis has been also employed as one of the most promising

methods to produce Ag NPs [1518] due to some important

advantages. As compared to conventional chemical/photochemi-

cal techniques, the radiochemical process can be performed toreduce Ag+ ions at the ambient temperature without using

excessive reducing agents or producing unwanted by-products

of the reductant. Moreover, reducing agent can be uniformly

distributed in the solution and Ag NPs are produced in highly pure

and stable form.

Considering the previous reports on the reduction of silver

ions to Ag NPs by g-irradiation, using PVP as a stabilizer

[1921], along with many articles on the antimicrobial activity

of Ag NPs [22,23], one may wonder about the novelty of this

work at the first glance. But the antibacterial activity ofcolloidal Ag NPs is influenced by their size [1012], shape

[13], and stability, which are in turn strongly dependent on the

preparation methods and the experimental conditions em-

ployed [14]. Hence, we found it worthwhile to synthesize Ag

NPs specifically through the g-irradiation in PVP and investigate

their antibacterial activity. To our knowledge, there is no such

report in the literature.

2. Experimental

2.1. Materials

Silver nitrate, PVP, and isopropyl alcohol were obtained from

Merck Chemicals Ltd. All chemicals were of analytical grade and

used without further purification. All aqueous solutions were

made using double distilled water, produced by a GFL company

water purification system.

2.2. Irradiation source

Irradiations were performed within a g-irradiation system

using 60Co source (Gammacell-220), at a dose rate of 18.6 Gy/min

calibrated by Fricke dosimeters.

ARTICLE IN PRESS

Contents lists available at ScienceDirect

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

Physica E

1386-9477/$- see front matter& 2009 Elsevier B.V. All rights reserved.

doi:10.1016/j.physe.2009.09.013

Corresponding authors. Fax: +98 2188221219 (N. Sheikh), + 98 2188006544

(M.Z. Kassaee).

E-mail addresses: [email protected] (N. Sheikh), [email protected]

(M.Z. Kassaee).

Physica E 42 (2009) 132135
http://-/?-http://www.elsevier.com/locate/physehttp://dx.doi.org/10.1016/j.physe.2009.09.013mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.physe.2009.09.013http://www.elsevier.com/locate/physehttp://-/?-


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2.3. Preparation and characterization of Ag NPs

In a typical experiment, PVP (0.1 g) was added to water (20 ml)

and stirred. After complete dissolution, 0.1 M silver nitrate (1 ml)

and isopropyl alcohol (0.2 ml) were added and stirred well. The

mixture was purged by N2 for 20 min, sealed and exposed to 1, 2,

and 5 kGy doses ofg-rays at room temperature. After irradiation,

the obtained colloidal solution was centrifuged at 17000 rpm at

101C for 30 min to remove the free PVP and excess silver ions. The

precipitated silver particles were washed with water, dried and

characterized by the UVvis spectroscopy, the TEM, the XRD, and

the FT-IR.

The UVvis spectra of Ag NPs were recorded using a Novaspec

III Biochrom Ltd., spectrophotometer from 330 to 800 nm where

distilled water was used as the blank. The TEM analyses were

performed using a ZEISS-EM 900 microscope operating at 120 kV.

For this purpose, the sample was prepared by drying a drop of the

silver colloidal solution on a TEM copper grid. The XRD pattern of

Ag NPs was recorded by a Holland Philips Xpert X-ray diffract-

ometer (CuKa). The FT-IR spectra of samples were obtained using

a Bruker IFS 45 spectrophotometer in the wave-number range of

4004000 cm1. The solid samples were grounded with KBr and

compressed into pellets.

2.4. Antimicrobial and bactericidal assays

To study the bactericidal effect of the prepared Ag NPs on

gram-negative bacteria, about 4.5104 colony forming units

(CFU) of E.coli ATCC 25922 were cultured on LB agar plates

supplemented with Ag NPs in concentrations ranging from 10 to

150mg/ml. Silver-free LB plates cultured under the same condi-

tions were used as the control. The plates were incubated at 37 1C

for 24 h, and then the numbers of colonies were counted. The

counts on 3 plates corresponding to each sample concentration

were averaged.

To examine the bacterial growth inhibition of Ag NPs in the

broth medium, aliquots of 10ml of E.coli cultured in nutrient brothwith a concentration of 4106 cells were transferred to sterile

cuvettes containing 50 and 150mg/ml concentration of Ag NPs in

LB broth. Cuvettes without inoculation of bacteria were used as

blanks, and silver-free cuvettes were used as the control. The

media were incubated at 37 1C and the optical density of each

medium was measured at 650 nm in different intervals.

3. Results and discussion

In our method, aqueous solutions are exposed to g-rays

creating hydrated electrons and primary radicals and molecules

as follows:

H2O e

aq;

H3O

;

H

;

OH

;

H2;

H2O2; . . .

1The solvated electrons and H atoms are strong reducing agents

so that in the following step they easily reduce silver ions down to

the zero-valent state:

Ag+ +eaq

- Ag0 (2)

Ag+ +H - Ag0+H+ (3)

In contrast, OHU radicals are able to oxidize the ions or the

atoms into a higher oxidation state, and thus to compensate the

reduction reactions (2) and (3). For this reason, the solution is

generally added with an OH radical scavenger like isopropyl

alcohol. The OH radical is capable of abstracting hydrogen from

the alcohol producing isopropyl radical, which acts as a reducing

agent to reduce silver ion [24].

(CH3)2CHOH+OH

- (CH3)2COH+H2O (4)

Ag+ +(CH3)2COH- Ag0+(CH3)2CO+H

+ (5)

Ag0+Ag + - Ag2+ (6)

Agn1+Ag+- Agn

+ (7)

Silver atoms formed by irradiation tend to coalesce into

oligomers (6), which progressively grow into larger clusters (7).

Here, the coalescence appears to be limited by the employed PVP

as the cluster stabilizer. The produced metallic clusters in the

early stage are stabilized by PVP through the steric hindrance or

the anchoring of the cluster by O or N atoms leading to the

formation of particles in nanometric scale [24].

To determine the influence ofg-irradiation doses on the Ag NPs

formation aqueous samples containing AgNO3, isopropyl alcohol

and PVP were exposed to 1, 2 and 5 kGy g-irradiation doses.

Depending on the absorbed doses, the reaction mixtures displayeda spectrum of yellow to brown colors. The UVvis spectra of the

samples show that after irradiation at a dose of 1 kGy, a low

intensity peak appears at 406 nm indicating the formation of Ag

NPs at a relatively low concentration (Fig. 1). On increasing the

irradiation dose from 1 to 5 kGy, the intensity of the absorption

band of Ag NPs increases significantly while their position does

not change noticeably (from 406 to 408 nm). These results suggest

formation of higher yields of Ag NPs at higher g-irradiation doses

[16]. The stability of Ag NPs stabilized with PVP was analyzed by

storing the samples at room temperature ($25 1C) for 90 days.

The absorbance at 406408 nm was monitored at an interval of

24h to check for agglomeration. No significant change in

absorbance was noticed during the storage, indicating a good

stability of Ag NPs.

The Ag NPs produced at 5 kGy (higher yield) g-irradiation dose

was characterized by different analytical techniques. The TEM

micrograph of the Ag NPs shows a relatively narrow size

distribution of the particles with uniform shape (Fig. 2). The

mean values of Ag particles diameter and the standard deviation,

estimated from their histogram, are 16.1 and 4.9 nm, respectively.

The XRD spectrum of the produced Ag NPs shows peaks at 37.8,

44.2, and 63.41, assigned to diffractions from the (111), (2 0 0),

0

0.5

1

1.5

2

2.5

300

Wavelength (nm)

Absorbance

400 500 600 700 800

5

210

Dose (kGy)

Fig. 1. UVvis absorption spectra of Ag NPs prepared at various g-irradiation

doses.

N. Sheikh et al. / Physica E 42 (2009) 132135 133


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and (2 2 0) planes of face-centered cubic (fcc) silver (JCPDS, 04-

0783), respectively (Fig. 3). The observed XRD peak broadenings of

Ag NPs are mostly due to the effects of nano-sized particles [25].

To probe the possibility of chemical interaction between PVP

molecules and Ag particles, FT-IR spectra of the pure PVP and the

PVP-stabilized Ag NPs were obtained (Fig. 4). Spectral comparison

indicates a red-shift of a peak from 1662 cm1 in the pure PVP to

1649 cm1 in the PVP containing Ag NPs. This band is attributed

to C=O stretching of PVP structure and its shift to lower energies

should be due to the chemical interaction between PVP molecules

and Ag NPs surface [26].

Antibacterial tests were performed against 4.5104CFU of

E.coli ATCC 25922 on LB agar plates containing differentconcentrations of Ag NPs ranging from 10 to 150mg/ml (Fig. 5).

The presence of these particles at a concentration of 10mg/ml

inhibits bacterial growth by 32%. By increasing the amount of Ag

NPs to 25 and 50mg/ml, the number of bacterial colonies, grown

on plates, is gradually reduced while the Ag NPs concentration

of 150mg/ml shows 99.5% inhibition of E.coli colonies growth

on the LB agar medium. Obviously, for all concentrations of

nanoparticles, the inhibition of bacterial growth depends on the

number of applied cells. As the number of cells is decreased from

4.5104 to 60 CFU, the inhibition of bacterial growth increases.

However, the 150 mg/ml concentration of Ag NPs completely

prevents bacterial growth for all concentrations of applied cells.

The dynamics of bacterial growth was also monitored in LB

medium supplemented with 4 106

E.coli cells and with 50

Fig. 2. The TEM micrograph and the corresponding size distribution histogram of

Ag NPs synthesized at 5 kGy.

Fig. 3. The XRD pattern of Ag NPs synthesized at 5 kGy.

Fig. 4. FT-IR spectra of (a) pure PVP and (b) PVP-stabilized Ag NPs synthesized at

5 kGy.

0

1

2

3

4

5

0

Concentration of silver nanoparticles (g/ml)

Num

berof

E.c

olico

lon

ies

(x104)

10 25 50 150

Fig. 5. Number ofE. coli colonies as a function of Ag NPs concentration in LB agar

plates. Upper right corner photograph shows LB plates containing 0 mg/ml (left)

and 150mg/ml (right) concentrations of Ag NPs.

0

0.1

0.2

0.3

1

Time (hours)

Op

tica

ldens

itya

t650nm 0 ug/ml

50 ug/ml

150 ug/ml

2 3 4 5 6 7

Fig. 6. Growth curves of E. coli in LB medium inoculated with 106 CFU of bacteria

in the presence of different concentrations of Ag NPs.

N. Sheikh et al. / Physica E 42 (2009) 132135134


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and 150mg/ml Ag NPs. These concentrations of nanoparticles

completely inhibited the growth of E.coli cells in first 7 h of curves

in the broth medium (Fig. 6). The inhibition of E.coli growth was

seen even at 24 h after the first inoculation (data not shown).

As a result, in comparison to antibacterial activity of Ag NPs

prepared by other chemical routes [22,23,27], these particles have

a relatively good biocidal effect depending on the concentration of

Ag NPs as well as on the CFU of the bacteria used in the

experiments.

4. Conclusion

PVP-stabilized Ag NPs were synthesized in a good yield by g-

irradiation of silver ions at 5 kGy as the applied appropriate dose.

The results of the X-ray diffraction, the TEM analysis and the

corresponding histogram showed a relatively narrow size dis-

tribution of face-centered cubic (fcc) Ag NPs. The FT-IR results

represented a chemical interaction between PVP molecules and

Ag NPs surface. The antibacterial activity of Ag NPs on gram-

negative bacteria is dependent on the concentration of Ag NPs.

These antibacterial Ag NPs, which can be prepared in a highly

stable state by a simple and clean method, may be suitable for the

formulation of new types of bactericidal materials.

Acknowledgement

The authors would like to thank R. Beteshobabrud, the head of

microbiological group, in Radiation Applications Research School

for performing the antibacterial tests.

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