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Colloids and Surfaces B: Biointerfaces 74 (2009) 328–335

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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iosynthesis, purification and characterization of silver nanoparticles usingscherichia coli

angiliyandi Gurunathana,b,∗, Kalimuthu Kalishwaralal a, Ramanathan Vaidyanathana,enkataraman Deepaka, Sureshbabu Ram Kumar Pandiana, Jeyaraj Muniyandia,ellaiah Hariharana, Soo Hyun Eomb

Division of Molecular and Cellular Biology, Department of Biotechnology, Kalasalingam University, Anand Nagar, Krishnankoil, 626190, Tamil Nadu, IndiaDepartment of Life Science, Cell Dynamics Research Centre, Gwangju Institute of Science and Technology, Gwangju, 500-712, South Korea

r t i c l e i n f o

rticle history:eceived 25 May 2009eceived in revised form 26 June 2009ccepted 31 July 2009vailable online 8 August 2009

eywords:iological synthesisurification. coliilver nanoparticles

a b s t r a c t

The application of nanoscale materials and structures, usually ranging from 1 to 100 nanometers (nm),is an emerging area of nanoscience and nanotechnology. Nanomaterials may provide solutions to tech-nological and environmental challenges in the areas of solar energy conversion, catalysis, medicine, andwater-treatment. The development of techniques for the controlled synthesis of nanoparticles of well-defined size, shape and composition, to be used in the biomedical field and areas such as optics andelectronics, has become a big challenge. Development of reliable and eco-friendly processes for synthesisof metallic nanoparticles is an important step in the field of application of nanotechnology. One of theoptions to achieve this objective is to use ‘natural factories’ such as biological systems. This study reportsthe optimal conditions for maximum synthesis of silver nanoparticles (AgNPs) through reduction of Ag+

ions by the culture supernatant of Escherichia coli. The synthesized silver nanoparticles were purified byusing sucrose density gradient centrifugation. The purified sample was further characterized by UV–vis

spectra, fluorescence spectroscopy and TEM. The purified solution yielded the maximum absorbancepeak at 420 nm and the TEM characterization showed a uniform distribution of nanoparticles, with anaverage size of 50 nm. X-ray diffraction (XRD) spectrum of the silver nanoparticles exhibited 2� valuescorresponding to the silver nanocrystal. The size-distribution of nanoparticles was determined usinga particle-size analyzer and the average particle size was found to be 50 nm. This study also demon-strates that particle size could be controlled by varying the parameters such as temperature, pH and concentration of AgNO3.

. Introduction

Nanoparticles are usually referred to as particles with a max-mum size of 100 nm. Nanoparticles exhibit completely new ormproved properties compared to larger particles of the bulk mate-ial and these novel properties are derived due to the variation inpecific characteristics such as size, distribution and morphologyf the particles. Nanoparticles present a higher surface area-to-olume ratio with decrease in the size of the particles. Specific

urface area is relevant for catalytic activity and other related prop-rties such as antimicrobial activity of AgNPs [1,2,3]. As the specificurface area of nanoparticles is increased, their biological effective-ess can also increase on the account of a rise in surface energy.

∗ Corresponding author at: Department of Biotechnology, Kalasalingam Univer-ity, Anand Nagar, Krishnankoil, 626190, Tamilnadu, India. Tel.: +91 4563 289042;ax: +91 4563 289322.

E-mail address: lvsangs@yahoo.com (S. Gurunathan).

927-7765/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2009.07.048

© 2009 Elsevier B.V. All rights reserved.

Nanoparticles have a wide range of applications, as in combat-ing microbes [4], biolabelling [5], and in the treatment of cancer [6].The antibacterial activity of silver species is known since ancienttimes [1,7] and it has been demonstrated that, at low concen-trations, silver is non-toxic to human cells [3]. It has also beenreported that Ag+ ions uncouple the respiratory chain from oxida-tive phosphorylation or collapse the proton-motive force acrossthe cytoplasmic membrane [8]. The interaction of Ag+ with bac-teria is directly related to the size and shape of the nanoparticles[3,9].

Size control during synthesis of particles is an important cri-terion in the arena of silver nanoparticle biosynthesis. Dependingon the size of the nanoparticles, their applications branch out.Although AgNPs are synthesized both intra- and extra-cellularly,

the latter method of biosynthesis of nanoparticles is highlyadvantageous because of ease of control over the environment,large-scale synthesis and easy downstream processing steps [10].Many organisms synthesize AgNPs extra-cellularly, among whichFusarium oxysporum [11], Bacillus licheniformis [12], Aspergillus

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umigatus [13] and Klebsiella pneumoniae, [14] have been reportedxtensively.

It is well known that the electronic and optical propertiesf metal nanoparticles are heavily size- and shape-dependent15]. Controlling the size, shape and surrounding media of metalanoparticles are important as many of their intrinsic proper-ies are determined by these parameters. Particular emphasis hasecently been placed on the control of shape, because, in many casest allows properties to be fine-tuned with a greater versatility thatives the particles a unique nature. It is only within the past decadehat it has become possible to control the shape of particles synthe-ized in solution, and numerous methods have been developed forhis. Recently, Song and Kim [16] reported the effects of reactiononditions (temperature, leaf broth concentration and concentra-ion of silver nitrate) on the synthesis-rate and particle-size ofgNPs. Previously silver nanoparticles were synthesized using theupernatant from various organisms [17–20]. However, there is noublished report so far involving novel approaches to synthesizeanoparticles of various sizes by controlling the temperature andH in E. coli. If biosynthesis of nanoparticles using micro-organisms

s to be a viable alternative to chemical methods currently in vogue,hen greater control over particle-size and polydispersity wouldeed to be established [21]. In this study the culture medium was

urnished with optimal environmental conditions that resulted inhigh yield of AgNPs and also made size controlled synthesis ofgNPs possible. The effect of reaction conditions, such as concen-

ration of silver nitrate (hereafter, AgNO3) temperature and pH onhe synthesis and particle-size reduction of AgNPs have also beennvestigated.

. Materials and methods

.1. Bacteria and chemicals

The E. coli strain used was DH5� (F-�80lacZ�M15�(lacZYA-rgF) U169 deoR recA1 endA1 hsdR17(rk−, mk+) phoA supE44 thi-gyrA96 relA1 �−) from Prof. Soo Hyun Eom (Department of life

cience, Gwangju Institute of Science and Technology). All otherhemicals were from Sigma (St Louis, MO) unless stated otherwise.

.2. Media and bacterial growth analysis

Nitrate media, Luria–Bertani (LB) media, and M9 minimal mediaere prepared and used as described [22]. E. coli cultures were first

rown aerobically at 37 ◦C in LB medium. The cells were harvestedy centrifugation, washed twice with phosphate-buffered salinepH 7.3), and re-suspended in the appropriate fresh medium, suchs M9 minimal medium, LB without NaCl or nitrate medium, toring the desired initial optical density (absorbance). Inoculatedultures were grown in a shaker (120 rpm) in Erlenmeyer flasksmedium volume/flask volume – 1/10) at 37 ◦C until they reachedhe stationary phase. Growth was monitored spectrophotomet-ically by periodically measuring the absorbance at 600 nm. Theacterium was routinely maintained on LB agar slants at 37 ◦C andreserved in glycerol stock solutions at −70 ◦C. Unless otherwisetated, three independent runs were made for all experiments.

.3. Synthesis of silver nanoparticles

Synthesis of AgNPs was carried out according to the methodescribed previously [12,17]. Briefly, bacteria were grown in a

00 mL Erlenmeyer flask that contained M9 minimal medium, LBroth without NaCl or nitrate medium. The flasks were incubatedor 21 h in a shaker set at 120 rpm and 37 ◦C. After the incuba-ion period, the culture was centrifuged at 10,000 rpm and theupernatant used for the synthesis of AgNPs. Three test tubes,

B: Biointerfaces 74 (2009) 328–335 329

the first containing AgNO3 (Sigma, USA, 99.9% pure) without thesupernatant, the second containing only the media and the thirdcontaining the supernatant and AgNO3 solution at a concentra-tion of 1 mM were incubated for 24 h. The absorption spectrumof the sample was recorded on a Shimadzu (model 9200) UV–visspectrophotometer operating at a resolution of 0.72 nm. The extra-cellular synthesis of AgNPs was monitored by visual inspection ofthe test-tubes for a change in the color of the culture mediumfrom a clear, light-yellow to brown, and by measurement of thepeak exhibited by AgNPs in the UV–vis spectra the synthesisof nanoparticles was confirmed. All samples for UV–vis spectrameasurement and particle-size distribution analysis were pre-pared by centrifuging an aliquot of culture supernatant (1.5 ml) at10,000 rpm for 10 min at 26 ◦C. The supernatant thus obtained wasa brown homogenous and clear suspension of AgNPs. All sampleswere diluted 10-fold for all experiments involving measurement ofUV–vis spectra.

2.4. Purification of nanoparticles

Briefly, bacteria were grown in a 1000 mL Erlenmeyer flask thatcontained 200 mL of nitrate medium. The flasks were incubated for21 h in an environmental shaker set at 120 rpm and 37 ◦C. Afterthe incubation period, the culture was centrifuged at 10,000 rpmand the supernatant used for the synthesis of AgNPs. 1 mM ofAgNO3 was mixed with 200 mL of cell filtrate in a 1000 mL Erlen-meyer flask. Bio-reduction was monitored by recording the UV–visabsorption spectra as a function of time of the reaction mixture.The particles were washed five times by centrifugation and re-dispersed in water to remove the remaining unconverted silverions. They were then transferred to a dialysis tube with a 12,000molecular weight cutoff. Nanoparticles were resuspended in 1 mLof HEPES buffer (20 mM, pH 7.4) supplemented with sucrose toreach a density of 2.5 g/ml and gradient was made according tomethod described earlier [23–25]. The solution was placed at thebottom of a centrifuge tube (13 mL). Twelve milliliters of a lineargradient of sucrose (0.25–1 M) density was layered on the nanopar-ticle suspension and subjected to ultracentrifugation (200,000 rpmat 4 ◦C for 16 h) by using an SW41 rotor (Beckman Instruments,Fullerton, CA, USA). Fractions (1 mL) were collected and purifiedsample was further characterized by UV–vis spectra and TEM.

2.5. Characterization of AgNPs

Characterization of the purified particles was carried out accord-ing to the method described previously [12,17]. Samples fortransmission electron microscopy (TEM) analysis were preparedon carbon-coated copper TEM grids. TEM measurements wereperformed on a JEOL model 1200EX instrument operated at anaccelerating voltage of 120 kV and later with an XDL 3000 pow-der X-ray diffractometer. Further characterization involved FourierTransform Infrared Spectroscopy (FTIR) analysis of the dried pow-der of AgNPs, by scanning it in the range 450–4000 cm−1 at aresolution of 4 cm−1. Finally, the size distribution of the nanopar-ticles was evaluated using DLS measurements conducted with aMalvern Zetasizer ZS compact scattering spectrometer (MalvernInstruments Ltd., Malvern, UK).

2.6. Particle-size distribution of AgNPs

Particle-size distribution analysis was carried out after treat-

ment of a 1 mM solution of AgNO3 with the culture supernatantof E. coli at room temperature for 24 h. The organism was grownin nitrate broth under incubation at 37 ◦C for 21 h. After the incu-bation period, the culture was centrifuged at 10,000 rpm and thesupernatant was used to reduce the AgNO3 solution. For the DLS

3 rfaces B: Biointerfaces 74 (2009) 328–335

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Fig. 1. Growth vs. Synthesis of silver nanoparticles monitored by UV–vis Spec-troscopy. (a) Cells were grown separately in LB and Nitrate media. Samples werewithdrawn at different time-points of growth and cells were centrifuged, washedwith distilled water and analyzed for growth at 600 nm. (filled diamond – Nitrate

30 S. Gurunathan et al. / Colloids and Su

easurements, the supernatant thus obtained was a clear brownomogenous suspension of AgNPs was diluted 10-fold for all exper-

ments involving measurement of DLS. The solutions were thenltered through syringe membrane filters with pores less than.4 �m, then centrifuged at 8000 rpm for 20 min.

.7. Effect of silver nitrate concentration on synthesis and particleizes

To obtain the optimum concentration of AgNO3 that yields theaximum synthesis of nanoparticles and particle-size distribution,gNO3, at concentrations ranging from 1 to 10 mM, was added to

he supernatant at pH 8.0 and temperature 30 ◦C.

.8. Effect of temperature and pH on nanoparticle synthesis andarticle sizes

To obtain optimum conditions for maximum synthesis ofanoparticles and particle-size distribution, the obtained the opti-um concentration of AgNO3 was added to the supernatant and

ncubated at various temperatures (20–90 ◦C) and pH conditions5–12). The pH of the incubation mixtures was adjusted using 1 MCl and 1 M NaOH solutions.

. Results and discussion

.1. Effect of media on silver nanoparticle synthesis

The strain DH5 � of E. coli was grown in different mediauch as M9 minimal medium, LB without NaCl (hereafter LB) anditrate medium. However, the cultures showed no increase in theirbsorbance values in M9 medium during the first 10 h of cultiva-ion. After a long lag-phase, growth proceeded normally as wasvidenced by the typical S-shaped growth curves of the cultureshough the final cell-densities of M9 cultures (data not shown) wereower than those for LB and nitrate media. In contrast to the M9

edium, the organism grew well in LB and nitrate media. How-ver, LB broth and nitrate medium showed the same amount ofiomass as is evident from the growth curve plotted between Timend A600 Fig. 1a. Although growth in both the media resulted in theame density of biomass, the synthesis of AgNPs was minimum inB but higher in nitrate broth. Nanoparticle synthesis was primarilyharacterized by the appearance of a brown color and a peak in the20 nm region of the spectrum. The intensity of the color increasedith increase in the incubation period.

Silver nanoparticle synthesis was also dependent on the growth-hase of the culture. Among the supernatant harvested fromarious growth phases, the culture supernatant obtained from thetationary phase resulted in the rapid synthesis of AgNPs whenompared with the other growth phases. This is apparent from thencreased absorbance values in the 420 nm region of the spectrum,.e. increased nanoparticle synthesis. These results corroborate withilver nanoparticle synthesis by B. licheniformis, where cultures inhe stationary phase showed the maximum synthesis of AgNPs12]. Even during cadmium sulfide nanoparticle biosynthesis, E.oli showed the maximum synthesis of nanoparticles in the sta-ionary phase when compared with other phases [26]. Moreover,he color of the supernatant changed from colorless to brown, withhe controls showing no change in color.

The spectra obtained for samples (taken every third hour) fromitrate broth showed maximum absorbance at 420 nm at all incu-

ation periods. The absorbance values increased with the age ofhe culture and the supernatant from the stationary phase showed

aximal synthesis of nanoparticles (Fig. 1b). In the case of LB brothhe A420 value (O.D420 nm – 0.42) was much lesser than that ofhe nitrate broth (O.D420 nm – 1.23), for the same culture ages and

medium; filled square – LB medium) (b) Samples were withdrawn at different time-points of growth, cells were sedimented, and the supernatant was incubated with1 × 10−3 M AgNO3 solution; AgNPs formation was followed by measurement ofabsorbance at 420 nm (filled diamond – Nitrate medium; filled square – LB medium).

incubation periods. Fig. 1b. shows the variation of A420 with incu-bation time for nanoparticle synthesis. These results show that therate of synthesis of silver nanoparticles at a given time is fasterin nitrate medium than in LB medium. This may be attributed topotassium nitrate that acts as an inducer for synthesis of nitratereductase/nitroreductase. Nitrate medium is a well known mediumfor the synthesis of the enzyme nitrate reductase [27]. The reportssuggest nitrate reductase to be the enzyme responsible for the syn-thesis of AgNPs [28]. The nitrate reductase enzyme is producedaerobically, barely induced by nitrate in the medium [29]. There-fore, further experiments for synthesis of were carried out only innitrate medium.

3.2. Characterization of synthesized silver nanoparticles

A study on extra-cellular biosynthesis of AgNPs by the culturesupernatant of E. coli was carried out in this work. Visual obser-vation of the culture supernatant incubated with AgNO3 showed acolor change from yellow to brown whereas no color change couldbe observed in culture supernatant without AgNO3 or media withAgNO3 solution alone (Fig. 2 inset). The appearance of a yellowish-brown color in AgNO3-treated culture supernatant suggested theformation of AgNPs [11,16,30]. The confirmation of the particle syn-thesis and stability of the AgNPs in colloidal solution was monitoredby UV–vis spectral analysis for which aliquots of the reaction mix-ture (after completion of the reaction) were withdrawn and usedfor UV–vis spectroscopy measurements. The primary characteri-zation of synthesized nanoparticles by UV–vis spectroscopy hasproven to be a very useful technique for the analysis of nanoparti-

cles [31]. In the UV–vis absorption spectrum, a strong, broad peak,located at about 420 nm, was observed for nanoparticles synthe-sized using the culture supernatant (Fig. 2). Observation of thispeak, assigned to a surface plasmon, is well documented for variousmetal nanoparticles with sizes ranging from 2 to 100 nm [30–32].

S. Gurunathan et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 328–335 331

Fig. 2. The absorption spectrum of silver nanoparticles synthesized by E. coli culturesupernatant. The absorption spectrum of silver nanoparticles exhibited a strongbroad peak at 420 nm and observation of such a band is assigned to surface plasmonresonance of the particles. The cells or samples were collected and were incubatedwith 1 × 10−3 M silver nitrate solution. After incubation period, the cultures werevo(c

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isualized in UV–vis spectra. Inset shows biosynthesis of silver nanoparticles – visualbservation. (A) AgNO3 solution without supernatant, after 24 h (no color change).B) Tube with medium and AgNO3, after 24 h (no color change) and (C) Tube with E.oli culture supernatant and AgNO3 solution, after 24 h (brown).

To explore the reduction process of AgNO3 by the culture super-atant of E. coli, FTIR measurements were carried out to identifyossible interactions between silver salts and protein molecules,hich could account for the reduction of Ag+ ions and stabiliza-

ion of AgNPs. The FTIR spectrum was recorded for the freeze-driedowder of AgNPs, formed after 24 h of incubation with the cul-ure supernatant of E. coli. The amide linkages between amino acidesidues in proteins give rise to the well-known signatures in thenfrared region of the electromagnetic spectrum. The bands seent 3500 and 3300 cm−1 were assigned to the stretching vibrationsf primary and secondary amines, respectively; while their cor-esponding bending vibrations were seen at 1641 and 1360 cm−1,espectively (data not shown). The overall observation confirms theresence of protein in samples of AgNPs. It has also been reportedarlier that proteins can bind to nanoparticles either through their

ree amine groups or cysteine residues [33]. Therefore, stabilizationf AgNPs by proteins is a clear possibility.

Further studies using X-ray diffraction were carried out to con-rm the crystalline nature of the particles, and the XRD patternbtained is shown in Fig. 3. The XRD pattern shows four intense

ig. 3. Representative XRD pattern of silver nanoparticles formed after reaction ofulture supernatant with silver nitrate (1 × 10−3 M) for 24 h. The XRD pattern showsour intense peaks in the whole spectrum of 2� values ranging from 20 to 80. Note 2�eak values of 39.01◦ , 46.48◦ , 64.69◦ and 77.62◦ , corresponding to 1 1 1, 2 0 0, 2 2 0,nd 3 1 1 planes, respectively, for silver.

Fig. 4. Particle-size distribution under unoptimized conditions. The particle-sizedistribution revealed that the particles ranging from 40–50 nm had the maximumintensity and thereafter the intensity was reduced. The average particle size wasfound to be 50 nm.

peaks in the whole spectrum of 2� values ranging from 20 to 80. Itis important to know the exact nature of the silver particles formedand this may be deduced from the XRD spectrum of the sample.A comparison of our XRD spectrum with the Standard confirmedthat the silver particles formed in our experiments were in the formof nanocrystals, as evidenced by the peaks at 2� values of 39.01◦,46.48◦, 64.69◦ and 77.62◦, corresponding to [111], [200], [220] and[311], respectively, for silver.

Fig. 4 shows that the particles range in size from 42.2 to 89.6 nmand possess an average size of 50 nm. Synthesis of AgNPs by treat-ing AgNO3 solution with the culture supernatant of K. pneumoniae(belonging to the family Enterobacteriaceae) has also been reported,in which the particles range in size from 28.2 to 122 nm and pos-sess an average size of 52.5 nm [14]. A study on synthesis of AgNPsusing Morganella sp., (belonging to the family Enterobacteriaceae)reported spherical nanoparticles of ∼20 nm size [17].

Transmission electron microscopy (TEM) was used to deter-mine the morphology and shape of nanoparticles. Purified silver

nanoparticles from extra-cellular culture supernatant using cen-trifugation was characterized by TEM. One representative exampleis shown in Fig. 5. In this example, TEM revealed the average sizeof particles as 50 nm. TEM images show that they are relatively

Fig. 5. TEM images obtained from purified fractions collected after sucrose densitygradient of silver nanoparticles synthesized using E. coli. After isolation and purifica-tion, nanoparticles from E. coli supernatant were examined by electron microscopy.Several fields were photographed and were used to determine the diameter ofnanoparticles. The range of observed diameters is 50 nm.

3 rfaces B: Biointerfaces 74 (2009) 328–335

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Fig. 6. Effect of various concentrations of silver nitrate on Synthesis (a) and Aver-age particle-size distribution (b). (a) UV–vis spectra were recorded after addition ofthe culture supernatant to AgNO3 solutions of different concentrations (1–8 mM).Intensity of the color formed was measured in terms of absorbance at 420 nm aftera period of 24 h. The organism was grown in nitrate broth under incubation at 37 ◦Cfor 21 h. After the incubation period, the culture was centrifuged at 10,000 × g and

32 S. Gurunathan et al. / Colloids and Su

niform in diameter and have spherical shape. The different frac-ions obtained on a continuous sucrose gradient were analyzed.he amount of nanoparticles in each gradient fraction was esti-ated by determining the intensity. The amount of nanoparticlesas maximum in fractions four corresponding to a peak density

.06 g/mL.All the above experiments were carried out with nitrate medium

f pH 8.0 (extra-cellular supernatant). The reaction temperatureas maintained at 30 ◦C and the final concentration of AgNO3 wasmM (unoptimized conditions). In order to further refine our meth-ds we brought about maximum and size controlled synthesis ofgNPs by providing optimum conditions of temperature, pH andoncentration of AgNO3.

.3. Effect of different parameters on the synthesis and sizeistribution of silver nanoparticles

.3.1. Concentration of silver nitrateThe possibility of controlling the reaction rate and particle size

as further investigated by changing the composition of the reac-ion mixture. In order to confirm, whether the concentration ofgNO3 plays an important role in the synthesis and size reduc-

ion of nanoparticles, different concentrations of AgNO3 were used.ig. 6a shows the effect of AgNO3 concentration on synthesis ofgNPs. The maximum synthesis of AgNPs occurred with respect

o Ag+ ion concentration in the range 1–10 mM. This was reflectedith an increase in the absorbance at 420 nm up to a concentra-

ion of 5 mM; however, the absorbance was found to decreaset higher concentrations of Ag+ ions. The control experimentsnvolving different concentrations of AgNO3 showed no synthesisf nanoparticles. Similar results were obtained for the synthesisgNPs using Morganella sp. [17]. The results clearly indicated that5 mM concentration of Ag+ ions was most appropriate for theaximum synthesis of AgNPs from the culture supernatant of E.

oli.Fig. 6b shows that the sizes of AgNPs decrease with increas-

ng concentrations of AgNO3. However, when the concentration ofgNO3 is more than 5 mM, the sizes of AgNPs were altered. The

ncrease in concentration of AgNO3 upto 5 mM resulted in sizeontrolled synthesis with the particle size being around 15 nm asevealed by the particle size analysis. This indicates that the size ofgNPs can be modulated by the concentration of AgNO3. The reason

or the decrease in particle size with increasing AgNO3 concentra-ion (1–5 mM) is not clear at this point. It is speculated that particleize and shape are dependent on various conditions, such as theulture supernatant, nanoparticle type, reaction temperature andeaction-mixture composition. This may also be because AgNO3orms a coat on growing particles, thereby preventing their aggre-ation and, thus, yielding particles of nanoscale size. This showshat silver ions, by their dispersive action, have a role in controllinghe growth of AgNPs.

.3.2. Reaction temperatureFig. 7a shows the effect of temperature on nanoparticle syn-

hesis. At an AgNO3 concentration of 5 mM and a pH of 8.0, it wasvident that increasing the temperature of the reaction, up to 60 ◦C,esults in an increase in the rate of synthesis of AgNPs. The enhancedate of synthesis of AgNPs might be the direct result of the effect ofemperature on a key enzyme present in the culture supernatantf E. coli. The results show that the optimum temperatures for cellrowth and silver accumulation are different. The rate of forma-

ion of AgNPs was related to the incubation temperature of theeaction mixture, with increased temperature levels allowing par-icle growth at a higher rate. At lower temperatures, bulk of AgNPsas formed only after 24 h of exposure to silver solution undernoptimized conditions.

the supernatant was used to reduce the AgNO3 solution. (b) Effect of concentrationof AgNO3 on size of AgNPs. Inset shows the representative particle-size distributionhistogram obtained using a particle analyzer.

Further, the effect of temperature on particle-size distribu-tion was also investigated. Fig. 7b shows that the sizes of AgNPsdecrease with increase in reaction temperature up to a maximumof 60 ◦C, beyond which they were found to increase. Increasingthe reaction temperature may enhance both the rate of adsorp-tion of AgNO3 and the viscosity of the coat-phase. This would, inturn, reduce the extent of aggregation of AgNPs, and lead to par-ticles of smaller size. Whereas the control experiment, i.e. AgNO3solution incubated at different temperature (20–90 ◦C) showed nosign of synthesis of nanoparticles. However, it appears that thegrowth of the AgNP nucleus is facilitated by temperatures above60 ◦C, resulting in larger size AgNPs. Besides, different time-courseswere observed for AgNPs production with different reaction tem-peratures, using the culture supernatant of E. coli: as the reactiontemperature increased, both synthesis rate and the final conversionalso increased (data not shown). Magnolia leaf broth demonstratedthat increase in reaction temperature increases the conversion as

well as synthesis rate of AgNPs and also mentioned that the aver-age particle size decreased from 50 nm at 25 ◦C to 16 nm at 95 ◦Cusing plant leaf extracts [16]. The reason for the decrease in particlesize with increase in temperature could be as follows: As the reac-

S. Gurunathan et al. / Colloids and Surfaces B: Biointerfaces 74 (2009) 328–335 333

Fig. 7. Effect of temperature on synthesis (a) and average particle-size distribution(b). (a) UV–vis spectra were recorded after treatment of 5 mM solutions of AgNO3

with the culture supernatant at different temperatures at pH 8.0. The intensity ofcolor formation was measured after a period of 5 h. The organism was grown innitrate broth under incubation at 37 ◦C for 21 h. After the incubation period, thecAta

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Fig. 8. Effect of pH on synthesis (a) and average particles size distribution (b). (a)UV–vis spectra recorded after the addition of culture supernatants with 5 mM AgNO3

at different pH at 60 ◦C. The intensity of color formation was measured after a periodof 5 h. The organism was cultivated in nitrate broth under incubation at 37 ◦C for21 h. After the incubation period, the culture was centrifuged at 10,000 × g and the

ulture was centrifuged at 10,000 × g and the supernatant was used to reduce thegNO3 solution. (b) Effect of various temperatures on size of AgNPs. Inset shows

he representative particle-size distribution histogram obtained using a particlenalyzer.

ion temperature increases, the reaction rate also increases, causingost silver ions to be consumed in the formation of nuclei and thus

topping the secondary reduction process on the surface of there-formed nuclei. The size is reduced initially due to the reduc-ion in aggregation of the growing nanoparticles. Increasing theemperature beyond a point aids the growth of the crystal aroundhe nucleus [34]. Therefore, by controlling the temperature of theynthetic environment the size of AgNPs can also be controlled.

.3.3. pH of the reaction mixture

In general, the reduction reaction of metallic ions is sensitive

o the pH of the solution as it may affect the morphology of theroduct via the formation of certain species as demonstrated [35].he present study involved a systematic analysis of pH-dependenthanges in the reaction mixture for synthesis of AgNPs. In this reac-

supernatant was used to reduce the AgNO3 solution. (b) Effect of various pH condi-tions on the size of silver nanoparticles. Inset shows the representative particle-sizedistribution histogram obtained using a particle analyzer.

tion, the concentration of AgNO3 was maintained at 5 mM and thereaction temperature at 60 ◦C. When pH was increased from 8.0 to12, maximum synthesis was observed at pH 10.0, with the synthe-sis time greatly reduced and the yield of AgNPs was significantlyenhanced, as evidenced by UV–vis spectroscopy (Fig. 8a). There-fore, the present study shows that the optimum pH for synthesisof AgNPs is 10.0. The control experiments i.e. AgNO3 solution incu-bated at different alkaline pH (8, 9, 10 & 11) showed no synthesisof nanoparticles. This is also in agreement with earlier reports thataddition of an alkaline ion is necessary to carry out the reductionreaction of metal ions [35]. In the absence of the hydroxide ion,the time taken for reduction of Ag+ ions was longer, indicating therequirement of OH− ions for the reduction reaction. The effect ofpH on nanoparticle synthesis has been explained previously in thecase of biorecduction of trivalent aurum [36] and the synthesis ofplatinum nanoparticles [37]. Similarly in our experiments, whenhydroxide ion was added, there was a rapid increase in silver con-version and the time taken was less than 30 min. This is mainlybecause the reducing power of the protein involved, which acts asthe reducing agent, is significantly increased under alkaline con-ditions. Increase or decrease of alkalinity can lead to aggregation

or distortion of Ag particles. It was also seen that increase in pHbeyond a value of 10.0 results in a fall in the absorbance at 420 nm.

Fig. 8b shows that the sizes of AgNPs reduce with increase in pHof the reaction mixture from 8.0 to 10. At pH below 10, the mean

334 S. Gurunathan et al. / Colloids and Surfaces

Fig. 9. TEM images obtained after synthesis of particles under optimal conditions.TmTr

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he optimal conditions contributing to the maximum synthesis of AgNPs was deter-ined. The synthesis was carried out using 5 mM AgNO3 at 60 ◦C and pH 8.0. The

EM image indicates the size controlled synthesis of particles with the particle sizeanging from 10 to 15 nm.

iameter of AgNPs decreased with increase in pH up to a value of0. It was also seen that the mean diameter of AgNPs increase whenhe pH of the reaction mixture was increased from 10 to 12. Thisndicates that nucleation resulting in AgNP synthesis was facilitatedt pH conditions lower than 10, while growth of the AgNP nucleusas favoured at pH conditions higher than 10.

The optimal conditions for synthesis were implemented andhe size controlled synthesis of particles was made possible. Fig. 9hows the image of the particles characterized using Transmissionlectron Microscopy and the particle size ranged from 10 to 15 nm.he small size of particles was obtained under optimal conditionsnd such small particles are attributed to have a large number ofpplications especially in the field of therapy and targeted drugelivery.

Currently, the mechanism of biological nanoparticle synthesiss not fully understood. With Neem leaf broth, it was reported thaterpenoids are believed to be the surface-active molecules stabi-izing the nanoparticles, and that, reaction of the metal ions isossibly facilitated by reducing sugars and/or terpenoids present

n the broth [38]. Recent results with Capsicum annuum L. extractndicate that the proteins which have amine groups play a reducingnd controlling role in the formation of AgNPs in the solution, andhat, the secondary structure of the proteins changes after reactionith Ag+ ions [39]. The phenomenon of a change in secondary struc-

ure of proteins was also reported during rapid synthesis of metallicanoparticles of silver by reduction of aqueous Ag+ ions using theulture supernatant of K. pneumoniae [14]. Therefore, more elabo-ate studies are required to elucidate the mechanism of biologicalanoparticle synthesis.

. Conclusion

We propose an environment-friendly method of synthesizinggNPs using the culture supernatant of E. coli. Since the low yieldnd particle size of AgNPs are drawbacks for practical applications,e provided optimum reaction conditions for the maximum syn-

hesis of AgNPs and reduction in particle size. For the synthesis of

gNPs, the medium was furnished with optimal conditions which

nclude concentration of AgNO3, reaction temperature and pH. Theedium contributing to the maximum synthesis was found to be

itrate medium; a concentration of 5 mM AgNO3, reaction temper-ture of 60 ◦C and pH 10.0 were found to be the optimal conditions

[[

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B: Biointerfaces 74 (2009) 328–335

for the maximum synthesis of AgNPs. Under these optimum con-ditions, only 30 min. was required for over 95% conversion usingthe culture supernatant of E. coli, which was faster than, or com-parable to, the synthesis rate of those particles obtained usingchemical methods. The average particle size could be controlledfrom 10–90 nm by varying the AgNO3 concentration, reaction tem-perature and pH. Under the optimized conditions, we also achievedsmaller-size AgNPs along with faster synthesis of particles whencompared to those under unoptimized conditions. The smaller-sizeof AgNPs has many positive attributes, such as good conductivity,chemical stability, catalytic and antibacterial activity, which wouldmake them suitable for many practical applications. To add to allthese, the extra-cellular synthesis of nanoparticles could be highlyadvantageous from the perspective of large-scale operations andeasy downstream processing. Our current research focuses on thebiological reduction mechanism of silver ions and effects of variousparticle sizes on vascular permeability using endothelial cells.

Acknowledgments

This work was supported by the Korea Research Foundation andthe Korean Federation of Science and Technology Societies grant,funded by Korea Government (MOEHRD, Basic Research Promo-tion Fund), for visiting professorship under the Brain Pool program.We are thankful to Dr. Sang Yong Jon for use of the particle ana-lyzer and Mr. Dongkyu Kim for helping us with the particle-sizeanalysis. The authors gratefully acknowledge the support of Dr.Pushpa Viswanathan, Professor, Cancer Institute (WIA), Chennai,who helped us in analyzing samples under the Transmission elec-tron microscope.

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