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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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