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One-pot synthesis of PVA-capped silver nanoparticles their characterization and biomedical

application

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2012 Adv. Nat. Sci: Nanosci. Nanotechnol. 3 015013

(http://iopscience.iop.org/2043-6262/3/1/015013)

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 015013 (7pp) doi:10.1088/2043-6262/3/1/015013

One-pot synthesis of PVA-capped silvernanoparticles their characterization andbiomedical applicationRupali S Patil1, Mangesh R Kokate1, Chitra L Jambhale2,Sambhaji M Pawar3, Sung H Han4 and Sanjay S Kolekar1

1 Department of Chemistry, Shivaji University, Kolhapur 416 004 (MS) India2 Department of Physics, Sangola College, Sangola 413307, (MS) India3 Department of Materials Science and Engineering, Chonnam National University, Gwangju 500-757,Korea4 Inorganic Nano Material Laboratory, Department of Chemistry, Hanyang University, Seoul 133-791,Korea

E-mail: kolekarss2003@yahoo.co.in

Received 21 October 2011Accepted for publication 6 January 2012Published 14 March 2012Online at stacks.iop.org/ANSN/3/015013

AbstractThe rapid one-pot synthesis of silver nanoparticles (SNPs) at room temperature by usinghydrazine hydrate as reducing agent and polyvinyl alcohol as stabilizing agent is reported. TheSNPs were characterized with UV-visible (UV-Vis) spectroscopy, x-ray diffraction (XRD),field emission scanning electron microscopy (FE-SEM), atomic force microscopy (AFM) andtransmission electron microscopy (TEM). The synthesized silver nanoparticle shows surfaceplasmon resonance at 410 nm. The XRD reveals face-centered cubic (FCC) structure of SNPs.FE-SEM, AFM and TEM show that nanoparticles have spherical morphology with diametersin the range of 10–60 nm. The antimicrobial activity of synthesized hybrid material againststrains of four different bacteria (Bacillus cereus, Escherichia coli, Staphylococus aureus,Proteus vulgaris), that are commonly found in hospitals has been studied. The results indicatethat such particles have potential applications in biotechnology and biomedical science.

Keywords: nanosilver, UV-Vis, AFM, TEM, antimicrobial activity

Classification numbers: 5.00, 5.08

1. Introduction

Nanotechnology has become one of the most interestingareas of scientific research in recent years, particularlyin the area of material research, including the synthesis,characterization and application of nanometer-sized metals,oxides, semiconductors and ceramics [1]. Nanoparticlesexhibit electronic and optical properties different fromthose displayed by their bulk-material counterparts. Theseproperties can further be tuned by altering the dimensionsand their mutual interactions [2–5]. Silver nanoparticles(SNPs) have been found to have applications in variousfields such as intercalation of materials for electricalbatteries [6], optical receptors [7], polarizing filters, catalystsin chemical reactions, bio-labelling [8], sensors [9], bioactive

materials [10], signal enhancers in SERS-based enzymeimmunoassay [11] and antimicrobial agents [12]. Theantimicrobial property of silver is being exploited in thebiomedical field [13]. The significant feature of silver isits broad spectrum antimicrobial property which is due tomicrobial colonization associated with biomaterial relatedinfections [14]. Silver nanoparticles in the form of silverpaste can be applied to electrodes due to its conductivity.Silver nanodispersion is used in conductive filler [15]. Forsynthesis of highly dispersed metallic nanoparticles, a widerange of reducing agents has been used [16, 17]. Thesenanoparticles are stabilized by a variety of different cappingagents such as thiols, amines and polymers (e.g. polyvinylpyrrolidone PVP) [18]. The synthesis of silver nanoparticlesby chemical route controls particle size, particle shape and

2043-6262/12/015013+07$33.00 1 © 2012 Vietnam Academy of Science & Technology

Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 015013 Rupali S Patil et al

morphology [19]. Polyvinyl alcohol (PVA) is a bio-friendlypolymer since it is water soluble and has extremely lowcytotoxicity. This allows a wide range of potential biomedicalapplications. It is used as a stabilizer due to its optical claritywhich enables investigation of nanoparticle formation. Also,introduction of nanosized silver into PVA matrix providesantibacterial activity, which is highly desired in textiles usedin medical clothing and household products [20].

Silver in colloidal state has germicidal properties and hasshown negligible toxicity even at high concentrations [21, 22].Silver nanoparticles are ideal for spectral interrogation ofvarious biological interactions [23, 24] and for use as tagsto indicate location and environment of a target of interest.Current research in bactericidal nanomaterial has opened anew era in pharmaceutical industries. Silver nanoparticles arethe metal of choice as they hold the promise to kill microbeseffectively [25]. Panacek et al have studied the antibacterialactivity of silver colloid with different size distribution and,according to them, 25 nm sized nanoparticles were mosteffective.

In this paper, we describe the synthesis of PVA-cappedSNPs in water. It has been shown that fine dispersion ofsilver nanoparticles can be achieved. UV-Vis absorptionspectra are used to monitor morphological evolution of silvernanoparticles.

2. Experimental

2.1. Materials

All chemicals, silver chloride, polyvinyl alcohol, hydrazinehydrate, ammonia, were of AR grade, purchased from SigmaAldrich and used without further purification. All solutionswere prepared in double distilled water.

The nutrient agar, type 1 agar and sodium chloride waspurchased from Hi-media. These agars were used to grow andmaintain the bacterial culture.

2.2. Synthesis of SNPs

In typical synthesis, 25 ml solution of 0.01 M silver chloridewas prepared in double distilled water and taken inErlenmeyer flask. The solution was maintained between pH8–9 by ammonia. To this solution, 20 ml, 2% polyvinylalcohol dissolved in distilled water was added with constantstirring. 1 ml 0.1 M solution of hydrazine hydrate was addeddrop wise. The reduction of silver chloride takes place toproduce SNPs colloid. The colloidal solution was analyzedby UV-Vis spectroscopy, Shimadzu UV-Vis near infrared(NIR) spectrophotometer (model-3600) by using distilledwater as [26]. The absorption spectra were recorded withradiation wavelength between 250 and 600 nm.

XRD pattern were measured using CuKα radiations(λ = 1.54 Å) on Bruker AXS D8. The microstructure ofthe prepared sample was mapped by FE-SEM (JSM-6160)at room temperature. The morphological characterizationof synthesized SNPs was studied by using AFM (AgilentTechnology) in contact mode. The size and morphology wasdetermined by TEM. TEM observations were performed onPhilips CM-200 operated at accelerating voltage 20–200 kVwith resolution 2.4 Å. Particle size of synthesized silver

nanoparticles was analyzed through dynamic light scattering.Light-scattering measurements were carried out at 90◦ ona photon correlation spectrometer (PCS)—Zetasizer 3000HAS equipped with a digital autocorrelation from MalvernInstruments UK.

2.3. Assays for minimum inhibitory concentration of SNPs

The synthesized SNPs were used in bactericidal studyagainst different bacterial strains. The bactericidal tests wereperformed with bacterial strains as bacillus cereus (B. cereus),Escherichia coli (E. coli), Staphylococcus aureus (S. aureus),Proteus vulgaris (P. vulgaris). Nutrient agar and type-I agarwere used as media to grow bacteria. The bacterial solutionwas prepared in 0.86% saline. The antibacterial activity ofsilver nanoparticle sample was assayed by following standardwell diffusion technique. The bacterial suspension was spreadon nutrient agar in Petri plate to create confluent lawnof bacterial growth. The wells of 6 mm were prepared byborer. The solutions of different SNP concentrations (5, 10and 15 µl) were poured into each well. The well withoutsilver nanoparticles was treated as control (water and PVA).These plates were incubated for 24 h at 35 ◦C. The lowestconcentration at which the Petri plate did not show any visiblegrowth after microscopic evaluations was considered asminimum inhibitory concentration (MIC). The susceptibilityof test organisms was determined after 24 h by measuringzone of inhibition around each well. In control no zone ofinhibition was observed. This proves that the antimicrobialactivity is due to silver nanoparticles. All antimicrobialparameters have been determined in triplicate.

3. Results and discussion

Reduction reaction of silver chloride in presence of PVA isshown below:

AgCl → Ag+ + Cl−,

Ag+ + Cl− + PVA + hydrazine hydrate

→ Ag0 (PVA-capped SNPS).

The UV-Vis spectroscopy was used to follow the reactionprocess and to characterize the optical properties of thenanoparticles. The as-prepared PVA-capped SNPs show asurface plasmon resonance (SPR) absorption peak at 410 nm(figure 1(a)). The synthesis of silver nanoparticles as afunction of the concentration of silver chloride and PVA andas a function of time was studied by UV-Vis spectroscopy(figures 1(b)). Figure 1(a) shows PVA-capped SNPs producedfrom solution. A peak centered at ∼410 nm is due tothe surface plasmon resonance of silver nanoparticles. Thisvalue is in good agreement with the literature reports onthe spectrum of silver nanoparticles in aqueous solutions.The position and shape of plasmon absorption of noblemetal nanocluster is strongly dependent on size, shape,dielectric medium surface absorbed species and surroundingmatrix [27, 28].

Figure 1(b) shows UV-Vis spectra of SNPs preparedwith different silver chloride concentrations. The SPR atapproximately 410 nm was consistent with SNPs. It can be

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 015013 Rupali S Patil et al

(a) (b)

(c) (d)

Figure 1. UV-Vis spectra of silver nanoparticles (a) showing SPR band, (b) synthesized with different silver chloride concentrations,concentration of PVA is 1%, (c) synthesis with different PVA concentrations, concentration of silver chloride is 0.01 M, (d) synthesis withdifferent reaction times, concentrations of silver chloride and PVA are 0.01 M and 1%, respectively.

seen that the intensity of plasmon maxima increases as afunction of increase in the silver chloride concentration from0.001 to 0.009 M, suggesting an increase in the concentrationof SNPs. It is important to note that although the silverchloride concentration is increased, the particle size does notincrease, possibly due to the capping action of PVA.

Figure 1(c) shows the influence of PVA concentrationon synthesis of SNPs. The SPR peak is slightly red shiftedby 5 nm. PVA plays an important role in the synthesis ofSNPs, by preventing them from aggregation. It is importantto note that PVA as a protective agent plays a decisive part incontrolling size distribution of SNPs. However, increasing theconcentration of PVA does not lead to the reduction of silvernanoparticle size.

Figure 1(d) shows UV-Vis spectra of SNPs as a functionof reaction time. From the absorption spectra it is clearthat, Ag+ ions are immediately reduced after the additionof hydrazine hydrate and hence there is not much effect onparticle size during the period from 10 min to 24 h. As weincrease time from 10 min to 24 h, the number of SNPs goeson increasing because hydrazine gradually reduces silver ionsto SNPs as time passes. Furthermore, to get closer insightsinto SNPs formation, some kinetic parameters of this process

30 40 50 60 70 8050

100

150

200

250

300

350

400

(31

1)(2

20

)

(20

0)

(11

1)

Inte

nsity

(A.U

)

2θ degree

Figure 2. XRD pattern of silver nanoparticles at 0.01 M of silverchloride.

should be evaluated. Effectively, from the formal kinetic pointof view, Ag+ reduction by hydrazine hydrate can be describedby the following equation

v = −d[Ag+]/dt = −kdt = k[Ag+]n,

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 015013 Rupali S Patil et al

(a) (b)

(c) (d)

Figure 3. FE-SEM image of SNPs with different silver chloride concentration (a) 0.005 M, (b) 0.007 M, (c) 0.009 M and (d) 0.01 M.

(a) (b)

Figure 4. Two-dimensional (a) and three-dimensional (b) AFM images of SNPs at 0.005 M silver chloride concentration.

where k is the rate constant, [Ag+] is the silver ionconcentration and n is the reaction order.

Figure 2 shows XRD pattern of SNPs deposited on glassplate. All prominent peaks at respective 2θ values are knownfor zero valent FCC silver, representing (111), (200), (220)and (311) crystal planes due to Braggs reflection at 2θ =

38.06◦, 44.24◦, 64.66◦ and 77.43◦ (JCPDS No 04-0783).XRD line broadening was used for the analysis of crystallitesize using Scherrer formula

D = 0.9λ/β cos θ.

The average particle size was found to be about 17 nm.The lattice constant calculated from this pattern is inagreement with the published results.

Figure 3 shows FE-SEM images of SNPs at differentconcentrations of silver chloride: 0.005, 0.007, 0.009 and0.01 M. The SNPs were deposited on glass substrate bydrop coating and used as the sample for FE-SEM analysis.The spherical nanoparticles uniformly distributed on glasssubstrate show average diameter of 10–50 nm. Particle sizeslightly increases with increasing concentration of silverchloride from 0.005 to 0.01 M.

The topography and morphology of SNPs were studiedby using AFM (figure 4). It was noticed that the SNPs wereregularly arranged on a glass slide obtained by drop coating.Particle size of SNPs was measured and found in the range10–50 nm. Figures 4(a) and (b) show two-dimensional andthree-dimensional AFM images of SNPs at 0.005 M silverchloride concentration.

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 015013 Rupali S Patil et al

(a) (b)

Figure 5. Two-dimensional (a) and three-dimensional (b) AFM images of SNPs at 0.01 M silver chloride concentration.

(a) (b)

(c) (d)

Figure 6. (a) TEM micrograph of PVA capped SNPs at 0.01 M concentration; (b) representative high resolution TEM image of PVA-cappedSNPs; (c) histogram showing particle size distribution of SNPs; (d) selected area electron diffraction (SAED) micrograph of SNPs.

Figures 5(a) and (b) show two-dimensional andthree-dimensional AFM images of SNPs at 0.01 M silverchloride concentration.

TEM image clearly demonstrates that the SNPswere spherical or pseudo-spherical (figures 6(a) and (b)),polydispersed, with more or less uniform size ranging from10 to 60 nm as dynamic light scattering using a Malvernzetasizer was used to determine the particle size distributionof the particles in solution. This is in agreement with the

TEM data as shown in the particle size distribution, illustratedby the histogram (figure 6(c)). The selected area electrondiffraction (SAED) micrograph of SNPs (figure 6(d)).shows FCC crystalline structure with indexed diffractingplanes [29].

The as-synthesized SNPs were studied for theiranti-bacterial activity (figure 7). The minimum inhibitoryconcentration (MIC) is the lowest concentration at which atested compound can respectively inhibit bacterium growth

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 015013 Rupali S Patil et al

(c) (d)

(a) (b)

Figure 7. Zone of inhibition (antimicrobial effect) by SNPs (a) on B. cereus; (b) on E. coli; (c) on S. aureus; (d) on P. vulgaris.

Table 1. The results of antibacterial activity with zone of inhibition.

No. Bacterial strains Zone of inhibition by SNPs

Control 5 µl 10 µl 15 µl

1 B. cereus 0 mm 10 mm 15 mm 20 mm2 E. coli 0 mm 10 mm 20 mm 25 mm3 S. aureus 0 mm 3 mm 5 mm 11 mm4 P. vulgaris 0 mm 5 mm 20 mm 20 mm

more than 99%. The MIC of SNPs and antibacterial study ofdifferent bacterial strains was studied by exposing bacterialstrains to SNPs. The different concentrations 5, 10 and15 µl were maintained, which shows a remarkable zone ofinhibition (figures 7(a)–(d)).

The various tested concentrations produce inhibitions of10, 15, 20 mm for B. cereus, 10, 20, 25 mm for E. coli, 3,5, 11 mm for S. aureus and 5, 20, 20 mm for P. vulgaris.This vast difference in zone of inhibition may be due to thesusceptibility of the organism used in the present study. Theabove results were obtained for 5, 10 and 15 ppm, respectively.The maximum antibacterial activity was recorded forE. coli and B. cereus. The zone of inhibition increases withincrease in concentration of SNPs. The control shows nozone of inhibition, which indicates that bactericidal activityis exclusively due to SNPs (figure 7, table 1). These resultscan be interpreted on the basis of the possible mechanisms,

all emphasizing the dependence of all antibacterial actionof SNPs on their size, dose, and morphology. Effectively,different mechanisms for bacterial action of silver NPs arethe following: (i) Ag+ ions are supposed to bind to sulfhydrylgroups, which leads to protein denaturation by the reductionof disulfide bonds; (ii) Ag+ can complex with electrondonor groups containing sulfur, oxygen, or nitrogen that arenormally present as thiols or phosphates on amino acidsand nucleic acids. Also, SNPs have been found to attach tothe surface of the cell membrane and disturb its function,penetrate into bacteria, and release Ag0; (iii) SNPs target thebacterial membrane, leading to a dissipation of the protonmotive force. Thus, a decrease in the NPs’ size can lead toan increase in the specific surface of a bactericidal specimen,inducing an increase in their ability to penetrate the cellmembrane, and thus improving antibacterial activity. It isalso reported that silver nanoparticles with size range of1–10 nm attach to the cell membrane and drastically disturbits proper function, like permeability and respiration. Further,upon penetration, more damage is caused by interacting withsulfur- and phosphorous-containing compounds like DNA.The higher antibacterial activity of E. coli and S. aureus isprobably driven by the difference in the structure of cell wallsbetween gram-negative and gram-positive bacteria. The cellwall of gram-negative bacteria consists of lipids, proteins andlipopolysaccharides (LPS) that ensure more effective defenseagainst biocides in comparison to gram-positive bacteria

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Adv. Nat. Sci.: Nanosci. Nanotechnol. 3 (2012) 015013 Rupali S Patil et al

where the cell wall does not contain outer membrane ofLPS [30, 31], thus leading to higher antibacterial activity ofPVA-capped SNPs.

4. Conclusion

We report a facile method for the synthesis of PVA-cappedSNPs. The absorption spectra confirm the presence of SPRat 410 nm, a characteristic of SNPs. The average particlesize is 10–60 nm. PVA can be applied as a cheap andenvironmentally friendly biomaterial for synthesis of SNPs.The PVA-capped SNPs demonstrate anti-bacterial activity.This activity was tested against B. cereus, E. coli, S.aureus, P. vulgaris bacteria and the results shows very goodantimicrobial activity. The silver nanoparticles produced inthis manner have potential applications in the biomedicalfields.

Acknowledgments

One of the authors (RSP) is grateful to Department ofChemistry, Shivaji University, Kolhapur and UniversityGrants Commission, New Delhi for SAP Fellowship. Weare also grateful to Department of Science and Technology,Government of India for DST-FIST programme facilities,Indian Institute of Technology, Bombay for TEM analysis.The authors gratefully acknowledge Department of MaterialsScience and Engineering, Chonnam National University,Gwangju, South Korea for characterization facilities.

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