Journal of Colloid and Interface Science 431 (2014) 255–263

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Journal of Colloid and Interface Science

www.elsevier .com/locate / jc is

Electrochemically active biofilm assisted synthesis of Ag@CeO2

nanocomposites for antimicrobial activity, photocatalysis andphotoelectrodes

http://dx.doi.org/10.1016/j.jcis.2014.06.0260021-9797/� 2014 Elsevier Inc. All rights reserved.

⇑ Corresponding author. Fax: +82 53 810 4631.E-mail address: mhcho@ynu.ac.kr (M.H. Cho).

Mohammad Mansoob Khan, Sajid Ali Ansari, Jin-Hyung Lee, M. Omaish Ansari, Jintae Lee, Moo Hwan Cho ⇑School of Chemical Engineering, Yeungnam University, Gyeongsan-si, Gyeongbuk 712-749, South Korea

a r t i c l e i n f o

Article history:Received 28 March 2014Accepted 12 June 2014Available online 21 June 2014

Keywords:NanocompositesAg@CeO2

CeO2

Biogenic synthesisElectrochemically active biofilmVisible light activeAntimicrobial activityPhotocatalysisPhotoelectrodePhotoelectrochemical studies

a b s t r a c t

Ag@CeO2 nanocomposites were synthesized by a biogenic and green approach using electrochemicallyactive biofilms (EABs) as a reducing tool. The as-synthesized Ag@CeO2 nanocomposites were character-ized and used in antimicrobial, visible light photocatalytic and photoelectrode studies. The Ag@CeO2

nanocomposites showed effective and efficient bactericidal activities and survival test against Escherichiacoli O157:H7, and Pseudomonas aeruginosa. The as-synthesized Ag@CeO2 nanocomposites also exhibitedenhanced visible light photocatalytic degradation of 4-nitrophenol and methylene blue than pure CeO2. Aphotocatalytic investigation showed that the Ag@CeO2 nanocomposites possessed excellent visible lightphotocatalytic activities compared to pure CeO2. Electrochemical impedance spectroscopy and photocur-rent measurements showed that the as-synthesized Ag@CeO2 nanocomposites exhibited excellent andenhanced responses to visible light irradiation. These results suggest that the AgNPs anchored at CeO2

induced visible light photoactivity by decreasing the recombination of photogenerated electrons andholes, and extending the response of pure CeO2 to visible light. Overall, as-synthesized Ag@CeO2 nano-composites are smart materials that can be used for a range of applications, such as antimicrobial activity,visible light photocatalysis and photoelectrode.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

The demand for efficient and smart materials that can be usedfor multipurpose applications, such as visible light photocatalysis,photoelectrode and medicine is increasing. Metal oxide nanostruc-tures, such as CeO2, serving as supporting materials, have attractedincreasing interest in catalysis because of their unique structuralproperties, such as high surface area, uniform pore distributionand rigid framework [1–6]. Li et al. [7] recently designed a uniquemesoporous multicomponent nanocomposite catalyst using ther-mally-stable noble-metal and oxide nanoparticles. Sophisticatedand supported heterogeneous catalysts have also been producedby controlling the size and shape of the active metal species, andby selecting and changing the composition of the support [8].

Fine-tuning of the interfacial interactions between the activemetal species and supports are the most important factors throughwhich high catalytic performance can be achieved. Because of themany intrinsic advantages of heterogeneous catalysts over homo-geneous ones, such as their durability at high temperatures and

reusability, the fine-tuning of metal–metal oxide interactions inheterogeneous catalysts is an emerging and challenging objective[9]. Interfacial interactions in noble metal-supported catalystsdetermine the catalytic processes of many fundamental chemicalreactions [8,10]. To strengthen the interfacial interactions betweenthe noble metals and support, supported catalysts prepared byimpregnation or combustion methods need to undergo high-temperature sintering (500 �C) for activation [11]. During the sin-tering process, unavoidable aggregation of the noble metals andoxide supports occurs, which can lead to a significant decrease incatalytic activity. Such problems can be avoided by anchoring verysmall noble metal nanoparticles to the support under mild condi-tions, which might give rise to high catalytic performance becauseof the strengthened interfacial interactions and high surface area.Unfortunately, most methods generally used are complicated,and the sizes of the metal nanoparticles cannot be controlled easilyto increase the interfacial interactions for enhanced catalytic per-formance [11,12]. Some of the studies have deposited the smallmetal nanoparticles on the surface of a support but those thatdid involve complicated steps or harmful materials [13–16].

The present study extended the biogenic protocol to synthesizeAg@CeO2 nanocomposites using electrochemically active biofilms

256 M.M. Khan et al. / Journal of Colloid and Interface Science 431 (2014) 255–263

(EABs), which is novel, green and does not involve toxic chemicals[17–21]. The main aim of this study is to enhance the visible lightresponse of P-CeO2 by anchoring the silver nanoparticles (AgNPs).AgNPs were anchored to CeO2, which is quite small than theconventional noble metal-supported structures [12,14,22]. Theinterfacial interactions between AgNPs and CeO2 nanoparticlescould be adjusted conveniently in a simple manner. The synergisticeffects of AgNPs and CeO2 would become enhanced, yieldingincrease in the visible light catalytic activity and stability of theas-synthesized Ag@CeO2 nanocomposites. The as-synthesizedAg@CeO2 nanocomposites were assessed for their antimicrobialactivity, visible light photocatalysis and photoelectrodes, andshowed an excellent response in each application. The significanceof these findings is twofold: First, by supporting nanosized AgNPs,it is possible to induce a visible light response to CeO2, whichotherwise is inactive under visible light irradiation. Second,anchoring the small size AgNPs on CeO2 keeps them dispersedand prevents aggregation, which helps retain and maximize theirantimicrobial and photo activities.

2. Experimental section

2.1. Materials

Cerium oxide (nano-CeO2), silver nitrate (AgNO3, 99%) andmethylene blue (MB) were purchased from Sigma–Aldrich.4-Nitrophenol (4-NP), sodium acetate, sodium phosphate, sodiumsulfate and sodium chloride were obtained from Duksan PureChemicals Co. Ltd. South Korea. Ethyl cellulose and a-terpineol weresupplied by KANTO Chemical Co., Japan, and fluorine-doped trans-parent conducting oxide glass (FTO; F-doped SnO2 glass; 7 X/sq)was acquired from Pilkington, USA. All other chemicals wereof analytical grade and used as received. The bacterial culture med-ium was purchased from Becton Dickinson and Company (NJ, USA).All solutions were prepared with deionized (DI) water obtainedfrom a PURE ROUP 30 water purification system.

2.2. Methods

The diffuse absorbance/reflectance UV–Vis spectra (DRS) of thepowder Ag@CeO2 nanocomposites and pure CeO2 (P-CeO2) sampleswere obtained using an ultraviolet–visible–near infrared (UV–VIS–NIR) double beam spectrophotometer (VARIAN, Cary 5000, USA)equipped with a diffuse reflectance accessory. A given amount ofthe Ag@CeO2 nanocomposites and P-CeO2 powder was presseduniformly in the sample holder, which was then placed at the inte-grating sphere for the absorbance/reflectance measurements.X-ray diffraction (XRD, PANalytical, X’Pert-PRO MPD) was per-formed using Cu Ka radiation (k = 0.15405 nm). The microstructurewas examined by field emission transmission electron microscopy(FE-TEM, Tecnai G2 F20, FEI, USA) operating at an accelerating volt-age of 200 kV. Selected-area electron diffraction (SAED) and a highangle annular dark field (HAADF) were carried out by TEM. Quan-titative analysis was performed by energy dispersive spectrometry(EDS) attached to the TEM. X-ray photoelectron spectroscopy (XPS,ESCALAB 250 XPS System, Thermo Fisher Scientific U.K.) wasconducted using the following X-ray source: monochromated AlKa, hm = 1486.6 eV, X-ray energy: 15 kV, 150 W and spot size:500 lm, take-off angle: 90�, pass energy: 20 eV, BE resolution:0.6 eV (calibrated by Ag 3d5/2) at Korea Basic Science Institute,(KBSI) South Korea. XPS fitting was done using ‘‘AVANTAGE’’ soft-ware by Shirley subtraction and shape of the peaks used for thedeconvolution was Gaussian–Lorentzian shapes. Sensitivity factorused for Ce 3d5 and Ag 3d5 was 10.66 and 30.5 respectively.

The photocatalytic degradation and photoelectrochemicalexperiments (Electrochemical impedance spectroscopy (EIS) andlinear scan voltammetry (LSV)) were conducted using a 400 Wlamp (3 M, USA). The EIS and LSV measurements were taken usinga potentiostat (Versa STAT 3, Princeton Research, USA) using astandard three-electrode system. Ag/AgCl (saturated with KCl), aPt gauge and FTO glass coated with 1 mM and 3 mM of the as-synthesized Ag@CeO2 nanocomposites, and P-CeO2 samples wereused as the reference, counter and working photoelectrodes,respectively. The experiments were performed in a 0.2 M Na2SO4

solution, as the electrolyte, at room temperature. The projectionarea of each photoelectrode was 1 cm2. The experimental condi-tions for EIS were 0.0 V and the frequency ranged from 1 to104 Hz. The working electrodes for the photoelectrochemical mea-surements were prepared as follows. One hundred milligrams ofeach sample was suspended thoroughly by adding ethyl celluloseas a binder and a-terpineol as a solvent for the paste. The pastewas later coated on a fluorine-doped tin oxide glass electrode usingthe doctor-blade method.

2.3. Development of electrochemically active biofilms

The EABs on carbon paper were prepared using the procedurereported elsewhere [17–21]. Briefly, 0.2 g sodium acetate wasadded as a substrate to a 200 mL of mineral salt medium [23]. Thiswas followed by the addition of 10 mL of anaerobic sludge (Biogasplant in Paju, Republic of South Korea) and sparging with N2 gas for5 min to create an anaerobic environment. Finally, carbon paper(2.5 cm � 4.5 cm) was dipped into the mixture and the bottlewas sealed. All media, including the bacterial inoculum, werechanged every two days under strict anaerobic conditions. The pro-cess was repeated for two weeks. The EABs formed on the carbonpaper were confirmed using a microbial fuel cell by obtaining theappropriate voltage. The living EABs formed on the carbon paperspecimens were used to synthesize the Ag@CeO2 nanocomposites.

2.4. Electrochemically active biofilm mediated synthesis of Ag@CeO2

nanocomposites

Two sets of 200 mL, 5 mM CeO2 aqueous suspensions wereprepared. Subsequently, 0.2 g sodium acetate was added to eachsuspension as an electron donor. Either a 1 mM or a 3 mM AgNO3

aqueous solution was added to the above reaction mixtures. Thereaction mixtures were sparged with N2 gas for 5 min to maintainthe anaerobic environment. The EAB was hung in each reactionbottle, and the systems were sealed and left for magnetic stirringat 30 �C. The reaction mixture was stirred for a further �8 h tocomplete the reaction. In each case, the initial white color beganchanging to a light brown color within 30 min. Finally, grayish-brown and blackish-brown precipitates were obtained in the1 mM and 3 mM AgNO3 cases, respectively. The reaction mixtureswere centrifuged, and the powdered Ag@CeO2 nanocompositeswere isolated for further characterization and applications.The obtained supernatant was tested using an UV–vis spectropho-tometer to determine the quantity of AgNPs remaining in thesupernatant.

Two controlled syntheses were performed to confirm the role ofthe EAB and sodium acetate. Two 5 mM CeO2 aqueous suspensions(200 mL) were prepared. In the first controlled synthesis, an aque-ous solution containing a 0.2 g sodium acetate and 1 mM AgNO3

was added. In the second controlled synthesis, only a 1 mM AgNO3

aqueous solution was added. Both reaction mixtures were spargedwith N2 gas for 5 min to maintain the anaerobic conditions. TheEAB was hung in the second controlled synthesis only. Both sys-tems were sealed and stirred with a magnetic stirrer at 30 �C. No

Scheme 1. Biogenic synthesis of the Ag@CeO2 nanocomposites using an electro-chemically active biofilm.

M.M. Khan et al. / Journal of Colloid and Interface Science 431 (2014) 255–263 257

changes were observed, even after 48 h. This confirmed that boththe EAB and sodium acetate are essential to complete the reaction.

2.5. Antimicrobial activity of the Ag@CeO2 nanocomposites

The antimicrobial activity test was performed using the dropassay method to test the antibacterial activities of the P-CeO2,and the as-synthesized 1 mM and 3 mM Ag@CeO2 nanocompositesunder normal laboratory condition. Two pathogenic bacteria,Escherichia coli O157:H7 (ATCC43895) [24], and Pseudomonasaeruginosa PAO1 [25] of the sequenced strains were used. Allexperiments were conducted at 37 �C and Luria–Bertani (LB) med-ium [26] was used to culture E. coli, and P. aeruginosa. Two patho-genic bacterial strains were initially streaked from �80 �C glycerolstock on an LB plate, and a fresh single colony was then inoculatedin LB (25 mL) in 250 mL flasks and cultured at 37 �C and 250 rpm.Overnight cultures were re-inoculated in LB medium at a 1:100dilution. 0.1 mL of the overnight culture was spread on a LB agarplate and incubated at 37 �C for 24 h with 5 lL of P-CeO2, andthe as-synthesized 1 mM and 3 mM Ag@CeO2 nanocomposites,which were dispersed in water. The final concentrations of 1 mg/mL were used to detect the bactericidal effect. The plates wereincubated at 37 �C for 18 h and the antibacterial activities of thesamples were evaluated by measuring a clear zone, where theinhibition of bacterial growth was observed. All experiments wereperformed using at least two independent cultures.

Survival test was performed on two pathogenic bacteria such asE. coli O157:H7 (ATCC43895) and P. aeruginosa PAO1 againstP-CeO2, 1 mM Ag@CeO2, and 3 mM Ag@CeO2. The overnight grownseed culture of pathogenic bacteria were inoculated in 2 mL LB andincubated for 4 h until the OD600 reached �1.0. The stock solutionsof each chemical were prepared in water. The cultured cells (1 mL)of each bacterium were separately mixed with P-CeO2, 1 mMAg@CeO2 and 3 mM Ag@CeO2 by vortexing at final concentrationof 3 mg/mL. The mixed cells were incubated at 37 �C for 1 h undershaking condition (250 rpm). After incubation, cells were seriallydiluted in phosphate buffer solution (PBS) and spread plated onLB-agar plates. These plates were incubated at 37 �C for 24 h. Totalnumber of bacterial colonies appeared on plates were counted. Theexperiments were performed using two independent cultures.

2.6. Photocatalytic degradation of 4-NP and MB by Ag@CeO2

nanocomposites under visible light irradiation

The photocatalytic activities of the as-synthesized 1 mM and3 mM Ag@CeO2 nanocomposites and P-CeO2 samples were testedfor the degradation of 4-NP and MB under visible light irradiation.The photocatalytic activities of the 1 mM and 3 mM Ag@CeO2

nanocomposites and P-CeO2 were measured by the photodecom-position of 4-NP and MB at a concentration of 5 mg/L and 10 mg/L, respectively. The rate of 4-NP and MB degradation was calcu-lated using the decrease in the absorbance of the degradedsolutions [13,27]. For photodecomposition, 2 mg of each photocat-alyst was suspended in a 20 mL of aqueous solutions of 4-NP andMB. Each solution was sonicated for 5 min in the dark. The solu-tions were stirred in the dark for 30 min to complete the adsorp-tion and desorption equilibrium of the specific substrate on theAg@CeO2 nanocomposites and P-CeO2. Visible light irradiation ofthe solutions was performed using a 400 W lamp (k > 400 nm).The two sets of experiments were observed for 5 h and 6 h in the4-NP and MB degradation experiment, respectively. The rate ofdye degradation was monitored by taking 1.7 mL of the samplesfrom each set every 1 h, centrifuging to remove the catalyst andrecording the UV–vis spectrum. Each experiment was performedin triplicate to ensure that the photocatalytic activities of theAg@CeO2 nanocomposites and P-CeO2.

For the control experiment, P-CeO2 was taken as the referencephotocatalyst. Normally, the photocatalytic activity of any newmaterial was compared with that of P-CeO2 as a standard. The sec-ond control experiment was performed without a catalyst (i.e.P-CeO2 and Ag@CeO2) to check the degradation of 4-NP and MBunder visible light irradiation under similar conditions. To checkthe adsorption of dyes on the catalyst third control experimentwas performed in the dark condition under similar conditions.

2.7. Ag@CeO2 nanocomposites as photoelectrodes for EIS andphotocurrent

The photoelectrode response of the as-synthesized 1 mM and3 mM Ag@CeO2 nanocomposites and P-CeO2 samples were exam-ined by photoelectrochemical experiments, such as EIS and LSV,which were carried out under ambient conditions in the darkand under visible light irradiation in a 50 mL, 0.2 M Na2SO4 aque-ous solution at room temperature. For each electrode, EIS was firstperformed in dark conditions and later under visible light irradia-tion (k > 400 nm) at 0.0 V and with a frequency ranging from 1 to104 Hz. The photocurrent response was obtained by LSV in darkand under visible light irradiation at a scan rate of 50 mV/s overthe potential range of �1.0 to 1.0 V.

2.8. Stability and reusability of the Ag@CeO2 nanocomposites

A preliminary test for stability was performed by suspendingthe Ag@CeO2 nanocomposites in water and sonicating them for1 h. The level of AgNPs leaching in the solution was examined byUV–vis spectrophotometry to confirm the stability of the Ag@CeO2

nanocomposites and their possible use as a catalyst. The reusabilityof the Ag@CeO2 nanocomposite was tested after centrifuging thecatalyst from the 4-NP and MB solution. The isolated catalyst fromfirst and second run were washed with DI water, dried in an ovenat 90 �C, and reused for a second and third run to check their cat-alytic activity with a 4-NP and MB solution under the sameconditions.

3. Results and discussion

3.1. Mechanism of biogenic synthesis of Ag@CeO2 nanocomposites

The EABs are well known in microbial fuel cells and thebio-inspired synthesis of nanomaterials [17–21,27–30]. EAB bio-logically decomposes the carbon source (sodium acetate) as shownby the equation CH3COO� + 4H2O + EABs ? 8e� + 9H+ + 2HCO3

�

[29,30].This study extended this biogenic and green approach to the

synthesis of Ag@CeO2 nanocomposites for antimicrobial, photoca-talysis and photoelectrode studies. Scheme 1 shows the proposedsynthesis of the Ag@CeO2 nanocomposite in the presence of EABand sodium acetate (carbon source) in water at 30 �C. The electronsproduced by EAB, after biologically decomposing the acetate areconsumed in the simultaneous reduction of Ag+ to Ag0 and the

(a)

0.30 nm(111)

5 nm

2 nm

(b)

258 M.M. Khan et al. / Journal of Colloid and Interface Science 431 (2014) 255–263

anchoring or adsorption of AgNPs at the CeO2 surface[15,20,21,28,29]. Supernatant analysis by UV–vis spectrophotome-try did not show any absorbance in the 350–500 nm range(Fig. S1). This confirms that all the AgNPs had adsorbed oranchored at the CeO2 surface. The product obtained in this synthe-sis is quite pure and free of impurities because the biofilm was sup-ported on carbon paper. Moreover, the control experiments clearlyshowed the role of EAB and sodium acetate in the synthesis. Theproposed approach is quite novel, and does not involve any harm-ful chemicals.

3.2. Characterization of the Ag@CeO2 nanocomposites

3.2.1. UV–vis spectroscopyFig. 1 shows the UV–vis absorption spectra of P-CeO2, and the

1 mM and 3 mM Ag@CeO2 nanocomposites. Fig. S2 shows their dif-fuse reflectance spectra and color of the samples. The UV–visabsorption spectrum of P-CeO2 showed a strong absorption peakat �316 nm with no other absorption peaks in the visible region.In contrast, the 1 mM and 3 mM Ag@CeO2 nanocomposites showedbroad absorption in the visible region due to the surface plasmonresonance (SPR) effect of the AgNPs (Fig. 1). The surface plasmonabsorption provided the evidence that AgNPs exist in the nano-composites. Significant absorption was observed in the visibleregion, �400–500 nm, because of the anchoring of AgNPs to thesurface of the CeO2 nanoparticles. In case of 1 mM and 3 mMAg@CeO2 nanocomposites the absorption in the visible regionshowed a red shift, and the absorption increased with increasingAg content. In comparison to P-CeO2 the absorption for Ag@CeO2

nanocomposites is red shifted whereas after increase in the con-centration of Ag content from 1 mM to 3 mM the absorption is blueshifted. The red shift is attributed to the visible light absorption ofAg@CeO2 nanocomposites whereas the blue shift in the absorptionof the 3 mM Ag@CeO2 could be attributed to the decrease in theparticles size of AgNPs at the surface of CeO2. The SPR absorptionband of the AgNPs can help to improve the absorption in visiblelight, and enhance the absorption intensity of the Ag@CeO2 nano-composites [22]. The maximum absorption and shifts in the visibleregion could be because of the interaction between AgNPs andCeO2 nanoparticles and the difference in their work function[22,31].

3.2.2. Transmission electron microscopyFig. S3 presents a TEM image of the P-CeO2 nanoparticles, which

are in the range of 20–25 nm. Fig. 2 presents HR-TEM images and

200 300 400 500 600 700 800

Ag plasmon absorption

Wavelength (nm)

Abs

orba

nce

(a.u

.)

P-CeO2

1 mM Ag@CeO2

3 mM Ag@CeO2

Fig. 1. UV–vis diffuse absorbance spectra of P-CeO2, and the 1 mM and 3 mMAg@CeO2 nanocomposites.

SAED patterns of P-CeO2, and the 1 mM and 3 mM Ag@CeO2 nano-composites. The dark spots in Fig. 2(b and c) revealed the presenceof AgNPs on the surface of CeO2, which are in the range of 3–7 nm.The overall sizes of the Ag@CeO2 nanocomposites were in therange of 25–30 nm. The SAED patterns (Fig. 2 insets) of the 1 mMand 3 mM Ag@CeO2 nanocomposites clearly showed well-resolvedlattice fringes and diffraction cycles, indicating the highly crystal-line nature of the Ag@CeO2 nanocomposites, which is in goodagreement with DRS (Fig. 1) and XRD (Fig. 3). Figs. S4 and S5 show

(c)

0.30 nm(111)

5 nm

Fig. 2. HRTEM images of (a) P-CeO2, (b) 1 mM Ag@CeO2 nanocomposite, and (c)3 mM Ag@CeO2 nanocomposite. The inset shows the respective SAED pattern.

M.M. Khan et al. / Journal of Colloid and Interface Science 431 (2014) 255–263 259

the HAADF image and EDX spectrum of the 1 mM Ag@CeO2 nano-composite, respectively. Figs. S6 and S7 show a HAADF image andEDX spectrum of the 3 mM Ag@CeO2 nanocomposites, respec-tively, corresponding to the O (K), Ag (K) and Ce (K) lines. TheEDX elemental spectra are also supported by the elemental compo-sition, as listed in Tables S1 and S2 for the 1 mM and 3 mMAg@CeO2 nanocomposites, respectively. This further confirms thepresence of AgNPs at the CeO2 surface. EDX revealed the 3 mMAg@CeO2 nanocomposite to have a higher silver content than the1 mM Ag@CeO2 nanocomposite, which is in agreement with DRSand XRD results.

3.2.3. X-ray diffractionThe phase and structure of the as-synthesized samples were

examined by XRD. Fig. 3 shows the XRD patterns, which confirmsthe formation of metallic AgNPs at the surface of CeO2 as well asthe overall formation of Ag@CeO2 nanocomposites and their crys-tallinity. The prominent XRD peaks confirmed that the main bodywas CeO2. The XRD peaks at 28.54�, 33.07�, 47.48�, 56.33�, 59.08�,69.4�, 76.69� and 79.06� 2h were assigned to the (111), (200),(220), (311), (222), (400), (331), and (420) planes, respectively,of the face centered cubic fluorite structure of CeO2 (JCPDS File No.81-0792). When the Ag concentration was lower i.e. 1 mM, onlythe face centered cubic fluorite structure of CeO2 was observedin the XRD pattern. On the other hand, when the Ag concentrationwas increased to 3 mM, the Ag crystalline phase began to appearwith a face-centered cubic phase structure. In the 3 mM Ag@CeO2

nanocomposite, the (111), (200), (220), and (311) peaks for face-centered cubic (fcc) crystalline silver were detected at 38.15�,44.21�, and 64.38� 2h, respectively (JCPDS File No. 89-3722) [22].The peak at 38.15� 2h is the characteristic peak for Ag metal, whichconfirmed the deposition of Ag on the CeO2 surface. The intensityof the silver peaks increased with increasing amount of AgNPsfrom 1 mM to 3 mM, which confirmed the anchoring of AgNPs tothe CeO2 surface. This is in accordance with the UV–vis spectrum(Fig. 1).

The mean crystallite size of P-CeO2, and the 1 mM and 3 mMAg@CeO2 nanocomposites were calculated using the Scherrer’s for-mula, D = jk/bcosh, where j is the shape factor and has a typicalvalue of �0.9, k is the wavelength (Cu Ka = 0.15405 nm), b is thefull width at half maximum of the most intense peak (in radians)and h is the peak position [32]. Using this equation, the calculatedcrystallite sizes of P-CeO2, and the 1 mM and 3 mM Ag@CeO2 nano-composites were 25.79 nm, 26.09 nm and 26.78 nm, respectively.The likely reason for the increase of overall size of the Ag@CeO2

nanocomposites could be the anchoring of AgNPs to the CeO2

21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78

21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78

21 24 27 30 33 36 39 42 45 48 51 54 57 60 63 66 69 72 75 78

Inte

nsity

(a.u

.)

2 Theta (degree)

P-CeO2

(200

)

∗∗

1 mM Ag@CeO2

(420

) (3

31)

(400

)

(222

) (3

11)

(220

)

(220

)

(111

)

∗

(200

)

(111

)

3 mM Ag@CeO2

Fig. 3. XRD patterns of P-CeO2, and the 1 mM and 3 mM Ag@CeO2 nanocomposites.The peaks marked with (⁄) represent the Ag signals.

surface [21,32]. In the case of the 1 mM and 3 mM Ag@CeO2 nano-composites, sharpening of the diffraction peaks and a slight shift tolower h values (Fig. 3) were observed. Since anchoring of AgNPs atCeO2 may cause some stress on the CeO2 crystals [32]. Therefore,caused stress seems responsible for minor shift in the XRD peaktowards lower angle which may leads to the expansion of the crys-tal lattice [32]. In the present case too, stress appears to be respon-sible for the peak shift [21,32].

3.2.4. XPS analysisThe surface chemical composition and chemical states of

P-CeO2 and Ag@CeO2 were examined by XPS. Fig. S8(a) presentsthe XP survey spectra of the P-CeO2 and Ag@CeO2, nanocompos-ites, showing that the material contains only Ce, Ag, O and C withno other impurity elements. The carbon peak in Fig. S8(b)(C 1s = 284.8 eV) was assigned to residual carbon from the sampleand hydrocarbons from the XPS instrument, whereas Fig. S8(c) pre-sents the O 1s spectra of P-CeO2 and Ag@CeO2, in which the peak at�531.39 eV was assigned to the lattice oxygen present in CeO2,whereas the peaks at 531.94 eV could be assigned to surfacehydroxyl groups within the of CeO2 matrix [10]. The presence ofsurface hydroxyl groups facilitates the trapping of photoinducedelectrons and holes, thereby enhancing the photocatalytic degra-dation process [5,21].

The XP spectra of Ce 3d, and Ag 3d (Fig. 4a, and b) showed thatthe binding energies of these electrons in the case of Ag@CeO2

were slightly different (Tables S3–S5) from those of P-CeO2, sug-gesting a strong interaction between the Ag and CeO2 nanoparti-cles [10]. Fig. 4(a) shows the Ce 3d spectra with specific peaks at898.94 eV for 3d5/2 and 916.43 eV for 3d3/2, indicating the oxida-tion state of Ce4+ in the form of CeO2 in the materials [10].Fig. 4(b) shows the core level Ag 3d spectra of the AgNPs inAg@CeO2 nanocomposites, showing two distinct individual peaksfor Ag 3d5/2 and Ag 3d3/2 at 367.5 eV and 373.6 eV, respectively,with �6 eV splitting between the two peaks, which is evidence ofthe reduction of Ag ions by EAB to produce metallic silver[10,33,34]. This is attributed to the complete reduction of Ag+ toAg0 [34]. Moreover, the binding energy (BE) of Ag 3d5/2 for theAg@CeO2 shifted to a lower binding energy (Tables S3–S5)compared to the corresponding value of the synthesized puremetallic Ag (the BE of Ag0 is approximately 368.2 eV). This is attrib-uted to the anchoring of AgNPs to the CeO2 surface [28,35]. There-fore, a new Fermi energy level in Ag@CeO2 is formed, indicating astrong interaction between Ag and the CeO2 nanoparticles [35].Once the AgNPs are deposited on the surface of the metal oxides,electron transfer occurs from Ag to the CeO2 because the workfunction of silver (4.2 eV) is smaller than that of CeO2 (4.7 eV)[22,31,33,35,36,46]. The atomic % (Fig. 4b) of Ag 3d5/2 found inthe 1 mM and 3 mM Ag@CeO2 nanocomposites was 0.36, and0.54 respectively which is in agreement with the EDX.

4. Applications of Ag@CeO2 nanocomposites

4.1. Antimicrobial activities of the Ag@CeO2 nanocomposites

Silver has extensive antibacterial activity and relatively highsafety [37–41]. When silver is supported on an inorganic carrierand is released slowly from the surface, it acts as an inorganicdisinfectant with superior safety, durability and heat resistance[40–42]. Therefore, an antibacterial test against enterohemorrhag-ic E. coli O157:H7, and multi-drug resistant P. aeruginosa PAO1 wasperformed to compare the bacterial colonies on agar plates in thepresence of P-CeO2, and the as-synthesized 1 mM and 3 mMAg@CeO2 nanocomposites. The growth of the bacteria in thepresence of the 1 mM and 3 mM Ag@CeO2 nanocomposites was

924 918 912 906 900 894 888 882 876

924 918 912 906 900 894 888 882 876

924 918 912 906 900 894 888 882 876

Binding Energy (eV)

(a) Ce 3d

P-CeO2

3 mM Ag@CeO2

1 mM Ag@CeO2

Inte

nsity

(a.u

.)

378 376 374 372 370 368 366 364 362

378 376 374 372 370 368 366 364 362

Inte

nsity

(a.u

.)

Atomic % Ag 3d5 = 0.36

Atomic % Ag 3d5 = 0.54

(b) Ag 3d 3 mM Ag@CeO2

1 mM Ag@CeO2

Binding Energy (eV)

Fig. 4. Fitted XP spectra of P-CeO2, 1 mM and 3 mM Ag@CeO2 nanocomposites for (a) Ce 3d peaks, and (b) Ag 3d peaks.

Table 1Inhibition zone by the P-CeO2, as-synthesized 1 mM and 3 mM Ag@CeO2 nanocom-posites against E. coli, and P. aeruginosa.

E. coli O157:H7 (mm) P. aeruginosa (mm)

P-CeO2 0.0 0.01 mM Ag@CeO2 0.5 1.53 mM Ag@CeO2 1.0 2.0

260 M.M. Khan et al. / Journal of Colloid and Interface Science 431 (2014) 255–263

inhibited (Fig. 5(I)), suggesting that the 1 mM and 3 mM Ag@CeO2

nanocomposites have good antibacterial properties. Table 1 liststhe zone of inhibition (Fig. 5(I)) shown by the as-synthesized1 mM Ag@CeO2 nanocomposites and 3 mM Ag@CeO2 nanocom-posites against E. coli, and P. aeruginosa. The as-synthesized1 mM and 3 mM Ag@CeO2 nanocomposites showed better bacteri-cidal activity than P-CeO2. In addition, the 3 mM Ag@CeO2 nano-composites showed much better bacterial inhibition than the1 mM Ag@CeO2 nanocomposites, P-CeO2 and other studies [43].This suggests that the antibacterial properties of the Ag@CeO2

nanocomposites increase with increasing AgNPs concentration.The survival rate of E. coli O157:H7 and P. aeruginosa against

P-CeO2 and 1 mM and 3 mM Ag@CeO2 is shown in Fig. 5(II).P-CeO2 did not show bactericidal effect on the survival of E. coliO157:H7 and P. aeruginosa PAO1, whereas 1 mM Ag@CeO2 and3 mM Ag@CeO2 reduced the survival of these pathogenic bacteriadramatically. The bactericidal effect of both 1 mM Ag@CeO2 and3 mM Ag@CeO2 seems working on two tested pathogenic bacteriawell with 1 h exposure. These results indicate that the synthesizednanocomposites show high antibacterial activity as compared tothe P-CeO2 and pervious reports [44,45].

The enhanced antibacterial activity might be due to Ag ionseluted from the Ag@CeO2 nanocomposites, which could beabsorbed on the surface of the bacterial cells, damaging the cellmembrane and solidifying the protein structure. This will lead toa distortion of the bacterial cell shape, which gives rise to the leak-age of intracellular constituents, resulting in the inhibition anddeath of the bacteria [42,45,46].

4.2. Photocatalytic activities of the Ag@CeO2 nanocomposites

The photocatalytic activities of P-CeO2, and the 1 mM and 3 mMAg@CeO2 photocatalyst for the degradation of 4-NP and MB under

Surv

ival

rate

(%)

1

10

100

1000 E. co

None CeO

(II)

(a) (b)

P-CeO2

1 mMAg@CeO2

3 mMAg@CeO2

(I)

Fig. 5. (I) Antimicrobial activities for (a) E. coli O157:H7, and (b) P. aeruginosa, and (II) s3 mM Ag@CeO2 nanocomposites.

visible light (k > 400 nm) irradiation were examined [10,12]. 4-NPis a colorless aromatic compound, whereas MB is a colored dye.Both the 1 mM and 3 mM Ag@CeO2 photocatalysts showed betterdegradation than P-CeO2 under visible light irradiation. The degra-dation was estimated from the decrease in the absorption intensityof 4-NP and MB at fixed wavelengths, kmax = 400 and 665 nm,respectively, during the course of the visible light photocatalyticdegradation reaction. The degradation was calculated from C/C0,where C0 is the initial concentration and C is the concentrationafter photo-irradiation (Fig. 6). Fig. S10 shows that there is almostno adsorption of 4-NP and MB on the photocatalyst in the darkcondition.

The Ag@CeO2 photocatalyst showed better catalytic activitythan the P-CeO2. The likely reasons for the enhanced photocatalyticactivities compared to P-CeO2 are the surface modification byAgNPs in the 1 mM and 3 mM Ag@CeO2 photocatalyst [21]. Theexplanation for the variation in the photocatalytic activities ofP-CeO2, and the 1 mM and 3 mM Ag@CeO2 photocatalyst is alsosupported by DRS (Figs. S2 and 1). These results suggest that thevisible light photocatalytic activities of the 1 mM and 3 mMAg@CeO2 photocatalyst were improved considerably. On the otherhand, the 3 mM Ag@CeO2 photocatalyst showed the maximumphotocatalytic activities to degrade 4-NP and MB. This suggests thatin the 3 mM Ag@CeO2 photocatalyst, the increased amount of

li O157:H7

2 1 mM 3 mMAgCeO2 AgCeO2

(a)

Surv

ival

rate

(%)

0.01

0.1

1

10

100

1000 P. aeruginosa

None CeO2 1 mM 3 mMAgCeO2 AgCeO2

(b)

urvival rate for (a) E. coli O157:H7, (b) P. aeruginosa against the P-CeO2, 1 mM and

0 1 2 3 4 5

0.2

0.4

0.6

0.8

1.0 (a)

Time (hours)

C/C

0

P-CeO2

1 mM Ag@CeO2 3 mM Ag@CeO2

0 1 2 3 4 5 60.0

0.2

0.4

0.6

0.8

1.0

(b)

Time (hours)

C/C

0

P-CeO2

1 mM Ag@CeO2

3 mM Ag@CeO2

Fig. 6. Visible light photocatalytic degradation of (a) 4-NP, and (b) MB by the P-CeO2, 1 mM and 3 mM Ag@CeO2 photocatalyst.

M.M. Khan et al. / Journal of Colloid and Interface Science 431 (2014) 255–263 261

AgNPs induced the visible light photocatalytic degradation of 4-NPand MB by decreasing the recombination of the photogeneratedelectrons and holes as well as extending the response of P-CeO2

to visible light [21,31,46,47]. The photocatalytic activities shownby these nanocomposites were better than those reported for othernanomaterials [2,13,21,48]. Fig. 7 shows the proposed mechanismfor the visible light activities of the Ag@CeO2 photocatalysts whichis supported by other reports [8].

When two materials with different work functions such as Ag(Wf = 4.2 eV) and CeO2 (Wf = 4.7 eV) were in contact with eachother, Schottky barrier will be formed [35,36]. As shown in Fig. 7the Fermi energy level of Ag (Fm) is higher than that of CeO2 (Fs)because of the larger work function of CeO2. This leads the transferof electrons from Fermi level of Ag (Fm) to Fermi level of CeO2 (Fs),until the two levels attain equilibrium and form the new Fermienergy level (Fms). The Fermi levels of Ag and CeO2 were adjustedto the same value during the formation of the composite and hav-ing more free electrons above the new Fermi level (Fms). When vis-ible light is irradiated, the equilibrated Fermi level (Fms) electronsare excited to CeO2 conduction band by surface plasmon resonancephenomenon [35,36].

In general, photogenerated electrons can react with oxygenmolecules adsorbed on the surface of the Ag@CeO2 photocatalystto yield free radicals such as �O2

� and HO�. The presence of extrahydroxyl species delays the recombination through hole trappingat AgNPs which helps to retards the recombination of photogener-ated electron hole pairs [21,35]. Therefore, the Ag@CeO2 photocat-alyst exhibits superior visible light photocatalytic activity toP-CeO2. In addition, the photocatalytic activity has a direct correla-tion with the rate of formation of reactive free radicals, i.e. the fas-ter formation of free radicals leads to higher photocatalytic activityof the catalyst. These results suggest that the AgNPs at the surfaceof CeO2 helps decrease the recombination of photogenerated

Ag

CB

VB CeO2

•OH

H2Oē ē ēē ē ē ēē

Fs

FmFms

h+HO-

•OH

Mineralized products

4-NP or MB

Wf = 4.2 eV

Wf = 4.7 eV

Eg = 3.2 eV

EvEv

•O2-

Fig. 7. Proposed mechanism for the photocatalytic degradation of 4-NP and MB bythe Ag@CeO2 photocatalyst under visible light irradiation.

electrons and holes, increase the rate of formation of �O2� and HO�

radicals, and facilitate the degradation of 4-NP and MB [21,35].The first control experiment suggests that Ag@CeO2 nanocom-

posites have much better visible light photocatalytic activities thanP-CeO2. The second control experiment showed that without thecatalysts (i.e. P-CeO2 and Ag@CeO2) no degradation of 4-NP andMB occurred under visible light irradiation (Fig. S9). The third con-trol experiment showed that there is no adsorption of dyes on thecatalyst in the dark condition (Fig. S10).

4.3. Stability and reusability of the Ag@CeO2 nanocomposite

The stability of the Ag@CeO2 nanocomposite was evaluated bysonicating the aqueous suspension of Ag@CeO2 nanocompositesfor one hour. The solution was later analyzed for any leached silverusing an UV–vis spectrophotometer (Fig. S11). The analysis did notshow any absorbance for silver. This confirms that the Ag@CeO2

nanocomposite was quite stable. The reusability of the Ag@CeO2

nanocomposite was tested by centrifuging the catalyst from thedye solutions, washing with DI water and drying in an air ovenat 90 �C. The reused catalyst showed almost same response for sec-ond and third run to that of the fresh catalyst (Fig. 6) which high-lights the reusability of the Ag@CeO2 nanocomposites.

4.4. Ag@CeO2 nanocomposites as photoelectrodes

The photoelectrochemical activity of the nanomaterials can bedetermined by both the light-harvesting capacity and the separa-tion of electron–hole pairs [48]. EIS was used to examine the elec-trochemical properties of the photoelectrodes in the dark andunder visible light irradiation. EIS was performed on P-CeO2, andthe 1 mM and 3 mM Ag@CeO2 photoelectrodes to examine thecharge transfer resistance and separation efficiency between thephotogenerated electrons and holes because the charge separationefficiency of the photogenerated electrons and holes is a criticalfactor for the photoactivity of photoelectrodes [49,50]. Fig. 8(a)shows the typical EIS Nyquist plots of P-CeO2, and the 1 mM and3 mM Ag@CeO2 photoelectrodes in the dark and under visible lightirradiation. For all photoelectrodes, the resistance under light irra-diation was much lower than that in the dark because of the highercharge carrier densities induced by photo-excitation. The arcradius of the EIS spectra reflects the interface layer resistance atthe surface of the electrode [50]. A smaller arc radius indicateshigher charge transfer efficiency [49]. The arc radii of the 1 mMand 3 mM Ag@CeO2 photoelectrodes were smaller than that ofP-CeO2 in the dark and under visible light irradiation. This suggests

0 30 60 90 120 150 1800

30

60

90

120

150 (a)Z'

'(Ohm

)

Z'(Ohm)

P-CeO2DarkP-CeO2Light1 mM Ag@CeO2Dark1 mM Ag@CeO2Light3 mM Ag@CeO2Dark3 mM Ag@CeO2Light

-1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0

0.00

0.01

0.02

0.03

0.04

0.05

0.06(b)

Phot

ocur

rent

(mA

)

Potential (V vs Ag/AgCl)

P-CeO2 DarkP-CeO2 Light1mM Ag@CeO2 Dark1mM Ag@CeO2 Light3mM Ag@CeO2 Dark3mM Ag@CeO2 Light

Fig. 8. (a) EIS Nyquist plots, and (b) LSV curves for the photocurrent response of the P-CeO2, 1 mM and 3 mM Ag@CeO2 photoelectrodes in dark and under visible lightirradiation.

262 M.M. Khan et al. / Journal of Colloid and Interface Science 431 (2014) 255–263

that the 1 mM and 3 mM Ag@CeO2 photoelectrodes have lowerresistance than P-CeO2, which can accelerate the interfacialcharge-transfer process. The arc radii of the 3 mM Ag@CeO2 photo-electrode were smaller than those of the 1 mM Ag@CeO2 photo-electrodes in the dark and under visible light irradiation,respectively. The 3 mM Ag@CeO2 photoelectrode had the lowestresistance in both the dark and under visible light irradiation,confirming that the 3 mM Ag@CeO2 photoelectrodes could betterfacilitate electron transfer from the photoelectrode to the electro-lyte. This can explain the best photo-response and stability. Thissuggests that the 3 mM Ag@CeO2 photoelectrodes have the lowestcharge transfer resistance and are most suitable for photocatalyticapplications [49–51]. These observations confirm that the 3 mMAg@CeO2 photoelectrodes can be used effectively as a photocata-lyst and as a photoelectrode material.

To obtain the photocurrent response of P-CeO2, and the 1 mMand 3 mM Ag@CeO2 photoelectrodes (Fig. 8(b)), LSV over thepotential range of �1.0 to 1.0 V were recorded in the dark andunder visible light irradiation [52–54]. All the samples showed adrastic increase in the photocurrent at an onset potential of�0.30 V vs. Ag/AgCl, and the photocurrent increases linearly untila potential of �1.0 V vs. Ag/AgCl. Under visible light irradiation,the 1 mM and 3 mM Ag@CeO2 exhibited an enhanced photocurrentresponse compared to P-CeO2 but the highest photocurrent wasrecorded for 3 mM Ag@CeO2 under visible light irradiation. Theenhanced photocurrent for the 1 mM and 3 mM Ag@CeO2 can beattributed to the improvement of visible light harvesting, whichis due to efficient electron transfer and greater surface areaachieved by the anchoring of AgNPs at the CeO2 surface [49,52].Under visible light irradiation, photocurrent shown by the 1 mMand 3 mM Ag@CeO2 electrodes were approximately 3.1 and 4.5times higher, respectively, than those of the P-CeO2 electrode,which suggests that the separation efficiency of photo-inducedelectrons and holes was improved by an electronic interactionbetween AgNPs and CeO2.

5. Conclusions

This study proposed a biogenic approach to synthesize Ag@CeO2

nanocomposites, in which nanosized Ag could (i) enhance the anti-bacterial activity, (ii) extend the response of the nanocomposite tovisible light, and (iii) improve the rapid transfer of electrons andinhibit charge recombination. The antimicrobial experimentsshowed that the 1 mM and 3 mM Ag@CeO2 nanocomposites haveconsiderable bactericidal activity due to the synergistic effect

between the AgNPs and CeO2 nanoparticles. The visible light photo-catalysis and photoelectrochemical experiments (EIS and LSV) con-firmed that the Ag@CeO2 nanocomposites could be a very smartmaterial that can be used for a range of applications. This paperreports an approach to synthesize novel and smart materials formultiple uses.

Acknowledgment

This study was supported by Basic Science Research Programthrough the National Research Foundation of Korea (NRF) fundedby the Ministry of Education, Science and Technology (Grant No:2012R1A1A4A01005951).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jcis.2014.06.026.

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