ARTICLE

Microbially Supported Synthesis of CatalyticallyActive Bimetallic Pd-Au Nanoparticles

Baharak Hosseinkhani,1,2,3 Lina Sveidal Sbjerg,2,3,4 Amelia-Elena Rotaru,2,3

Giti Emtiazi,1 Troels Skrydstrup,3,4 Rikke Louise Meyer2,3

1Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan, I.R. Iran2Department of Biological Sciences, Aarhus University, Ny Munkegade 114, 8000 Aarhus C,

Denmark; telephone:45 60202794; fax: 45 8942 2722; e-mail: rikke.meyer@biology.au.dk3Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus C, Denmark4Department of Chemistry, Aarhus University, Aarhus C, Denmark

Received 24 June 2011; accepted 3 August 2011

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.23293

ABSTRACT: Bimetallic nanoparticles are considered thenext generation of nanocatalysts with increased stabilityand catalytic activity. Bio-supported synthesis of monome-tallic nanoparticles has been proposed as an environmentallyfriendly alternative to the conventional chemical and physi-cal protocols. In this study we synthesize bimetallic bio-supported Pd-Au nanoparticles for the first time usingmicroorganisms as support material. The synthesis involvedtwo steps: (1) Formation of monometallic bio-supportedPd(0) and Au(0) nanoparticles on the surface of Cupriavidusnecator cells, and (2) formation of bimetallic bio-supportednanoparticles by reduction of either Au(III) or Pd(II) on tothe nanoparticles prepared in step one. Bio-supportedmonometallic Pd(0) or Au(0) nanoparticles were formedon the surface of C. necator by reduction of Pd(II) or Au(III)with formate. Addition of Au(III) or Pd(II) to the bio-supported particles resulted in increased particle size. UVVis spectrophotometry and HR-TEM analyses indicated thatthe previously monometallic nanoparticles had become fullyor partially covered by Au(0) or Pd(0), respectively. Fur-thermore, Energy Dispersive Spectrometry (EDS) and FastFourier Transformation (FFT) analyses confirmed that thenanoparticles indeed were bimetallic. The bimetallic nano-particles did not have a core-shell structure, but were super-ior to monometallic particles at reducing p-nitrophenol top-aminophenol. Hence, formation of microbially supportednanoparticles may be a cheap and environmentally friendlyapproach for production of bimetallic nanocatalysts.

Biotechnol. Bioeng. 2011;xxx: xxxxxx.

2011 Wiley Periodicals, Inc.KEYWORDS: bimetallic nanoparticles; gold; palladium;catalyst; microbial; bio-Pd

Introduction

Combination of two metals into single bimetallic nano-particles leads to higher physical stability and unique optical,magnetic, and catalytic properties compared to monome-tallic particles. Synthesis of bimetallic nanoparticles istherefore receiving much attention due to the many excitingapplications of such particles. The combination of aplatinum group metal with a noble metal is thought topromote stability and enhance the catalytic activity of theplatinum group metal (Akita et al., 2008; Ding et al., 2010;Huang et al., 2010). For example, bimetallic Pd-Au particleshave been applied as a catalyst for many different reactions,such as acetylene hydrogenation (Sarkany et al., 2002),trichloroethene hydrodechlorination (Nutt et al., 2005),hydrogen peroxide synthesis (Bernardotto et al., 2009), oxi-dation of benzyl alcohol (Marx and Baiker, 2009) oxidationof alcohol to aldehydes (Mertens et al., 2009), vinyl acetatesynthesis (Kumar et al., 2007), or removal of contaminantsin groundwater (Nutt et al., 2005).

Bimetallic nanoparticles have previously been preparedby chemical and physical methods (Mizukoshi et al., 1997;Patel et al., 2006). However, these methods are generallyexpensive, energy consuming, and environmentally hazard-ous (Korbekandi et al., 2009; Narayanan and Sakthivel,2010; Toshima and Yonezawa, 1998). Bio-supported syn-thesis of Pd nanoparticles was proposed as an environmen-tally friendly alternative to conventional protocols and hasbeen demonstrated on several microorganisms, such asDesulfovibrio desulfuricans, Shewanella oneidensis, Cupriavidusnecator, Paracoccus denitrificans, and Pseudomonas putida(Bunge et al., 2010; De Windt et al., 2005; Yong et al., 2002).Similarly, bio-supported deposition of Au nanoparticles hasalso been demonstrated on the surface of microorganismslike Cupriavidus metallidurans, Desulfovibrio desulfuricans,Esherichia coli, Rhodopseudomonas capsulata, and Shewanella

Correspondence to: R.L. Meyer

Contract grant sponsor: Danish Research Council for Technology and Production

Sciences

Contract grant number: 274-07-0254

2011 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2011 1

algae (Deplanche and Macaskie, 2008; He et al., 2007;Konishi et al., 2007; Reith et al., 2009). However, it has notbeen demonstrated that bimetallic nanoparticles couldbe obtained by similar processes on the surface ofmicroorganisms.

Only a few reports are available on biosynthesis of othertypes of bimetallic nanoparticles, such as Au-Ag(Govindaraju et al., 2008; Nair and Pradeep, 2002;Senapati et al., 2005; Shankar et al., 2004), Eu-Au(Ascencio et al., 2003), and Ti-Ni nanoparticles (Schabes-Retchkiman et al., 2006) synthesized with alfalfa as thereducing agent. In this work, we report on the synthesis ofbio-supported Pd(0) and Au(0) nanoparticles byCupriavidus necator H16, and demonstrate for the firsttime the synthesis of bio-supported bimetallic Au(0)-Pd(0)nanoparticles. The synthesis was performed by reductionwith formate at low pH, as we envision that nanoparticlesynthesis could be performed as a step in a metal recoveryprocess, where metals are leached at low pH prior tonanoparticle formation. The size and composition ofmonometallic and bimetallic nanoparticles was character-ized by UVVis spectrophotometry, transmission electronmicroscope (TEM), EDS, HR-TEM, and FFT analysis.Furthermore, the catalytic properties of bio-supportedmono- and bi-metallic nanoparticles were evaluated byreduction of p-nitrophenol.

Materials and Methods

Synthesis of Monometallic Bio-Supported Pd(0) andAu(0) Nanoparticles

Cupriavidus necatorH16 (DSM 428) was grown overnight inNutrient Broth (5 g L1 peptone, 3 g L1 meat extract, pH7.0) at 288C with mild shaking (120 rpm). Cells wereharvested by centrifugation (4,420g for 10min) in sterile50mL centrifuge tubes (TTP, Switzerland), washed threetimes with 20mMMOPS-NaOH (pH 7.0), and resuspendedin anaerobic 0.01M HNO3, (pH 2.1, degassed and purgedwith N2) to an optical density at 600 nm (OD600) of 1.3.

Four milliliter of the cell suspension was transferred tosterile N2-purged Hungate anaerobic culture tubes sealedwith butyl rubber stoppers and screw caps (GlasgeratebauOchs, Germany), and Pd(II) or Au(III) was added to a finalconcentration of 1mM from anaerobic stock solutionsof Na2PdCl4 or HAuCl4 (Sigma, Aldrich, St. Louis, MO).Formate was then added to a final concentration of 25mMfrom an anaerobic stock solution. The final volume of thecell suspension was 4.09mL after addition of Pd(II)/Au(III)and formate. The tubes were incubated for 24 h in the dark at308C with mild shaking (120 rpm) to allow reduction ofPd(II) or Au(III) to take place. The following controls wereincluded: (1) Pd(II)/Au(III)-free controls with cells andformate, (2) formate-free controls with cells and Pd(II)/Au(III), and (3) cell-free controls with formate and Pd(II)/Au(III). All samples and controls were prepared in triplicate.

Pd(II) reduction was previously shown to be 100% by thisprocedure (Bunge et al., 2010). The extent of Au(III)reduction was examined by measuring the concentration ofAu in the culture supernatant after the reduction step. Cellswere separated from the supernatant by centrifugation(4,420g for 10min) and Au(III) was measured in thesupernatant by the thiaminephloxine assay (Fujita et al.,1999). Au could not be detected in the supernatant at theend of the reduction step, and both Pd(II) and Au(III) wasthus reduced to Pd(0) and Au(0), which was associated withthe bacterial biomass.

Synthesis of Bimetallic Bio-Supported Nanoparticles

Synthesis of bimetallic particles involved two steps:Monometallic bio-supported nanoparticles were first pre-pared as described above. Then formate (25mM finalconcentration) was added again, and Au(III) (1mM finalconcentration) was added to tubes with monometallic Pd(0)particles, whereas Pd(II) (1mM final concentration) wasadded to tubes with monometallic Au(0) particles. All tubeswere then degassed with N2 for 5min, and incubated againfor 24 h in the dark at 308C with mild shaking (120 rpm).The resulting bimetallic particles are in the followingreferred to as bio-supported Pd(0)-Au(0) or Au(0)-Pd(0),nominating the order in which the two metals were added tothe bacterial biomass.

Characterization of Nanoparticles

UVVis absorbance spectra of monometallic Au(0) andbimetallic Au(0)-Pd(0) or Pd(0)-Au(0) nanoparticles wereobtained using a BioTeK UVVis spectrophotometer with ascan range of 300700 nm with an optical path length of1.0 cm (PowerWave XS2). The morphology and chemicalcomposition of the nanoparticles was evaluated using aPhilips CM20 TEM equipped with a LaB6 filamentoperating at 200 kV and a Gatan CCD camera (GatanInc., Pleasanton, CA). Energy dispersive spectra (EDS) werecollected and analyzed using the EDS Genesis software(Digital Micrograph, Gatan, Inc., Pleasanton, CA) mountedon the TEM.

Samples were prepared for TEM and EDS by fixation in4% glutaraldehyde for 10min followed by separation of cellsby centrifugation (5,000 rpm for 10min) and three washingsteps in MilliQ water. Finally, the cells were resuspended inMilliQ water. Formvar-coated copper grids were loadedwith 10mL of the suspension and air-dried before imaging.

TEM images were used to determine the size-distributionof the nanoparticles. The diameter of 1,000 randomlyselected nanoparticles from each sample was measured usingthe software ImageJ. Assuming a spherical structure, thesurface area of each particle was calculated. Particles weregrouped in intervals based on their diameter, and thecontribution of each group to the total particle surface areawas calculated. Surface area was chosen as the most relevant

2 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2011

parameter because the particle surface area is relevant fortheir catalytic properties. We did not attempt to estimate theconcentration of particles per volume sample, but the molarconcentration of the metals was 1mM each, since bothmetals were fully reduced during preparation of theparticles.

The bacterial biomass had to be removed in order tovisualize the nanoparticles by HR-TEM. The biomass waswashed three times with MOPS NaOH, once with acetone,dried at 60658C, and ground by pestle to give a dry blackpowder. This powder was then fixed in 4% glutaraldehyde asdescribed above. Fast fourier-transform (FFT) analyses ofHR- TEM images was carried out by using Gatan DigitalMicrograph software.

Catalysis of p-Nitrophenol Reduction by Mono andBimetallic Bio-Supported Nanoparticles

The catalytic activity of bio-supported mono- and bimetallicnanoparticles was evaluated by reduction of p-nitrophenol(Esumi et al., 2003). This assay was chosen for evaluating thecatalytic properties of Pd because Au does not catalyze thereaction, allowing us to address the catalytic properties of Pdspecifically.

The reaction was performed in tubes containing identicalmolar concentration of catalyst (1.25mM final concentra-tion). Suspensions with bio-supported nanoparticles con-tained 1mM Au, 1mM Pd, or 1mM Au 1mM Pd. Eachsuspension wasmixed thoroughly by shaking, and 24mL wastransferred with a Hamilton syringe from suspensions withmonometallic particles to a 12mL Exetainer containing9.5mL H2O (pH 13, pH adjusted with 5M NaOH) with230mM p-nitrophenol (final concentration). Only 12mLwere transferred from suspensions with bimetallic nano-particles in order to keep the molar concentration of metals(sum of Au Pd) similar in all samples. The solution was

then flushed with N2 for 4min and transferred to a secondExetainer containing 0.159mmol NaBH3 powder under N2atmosphere (ICN Biomedicals, Inc., Costa Mesa, CA). Thereaction started immediately when the NaBH3 wasdissolved. The reaction was run at RT and the Exetainerwas placed directly in the spectrophotometer (GEHealthcare, Uppsala, Sweden) where the optical density at400 nm was recorded every 10 s for 300 s. The p-nitrophenolreduction rates were calculated by linear regression of thedatapoints. All analyses were performed on triplicatesamples.

Results

Reduction of Au(III) and Pd(II) to MonometallicNanoparticles

The reduction of soluble Pd(II) to insoluble Pd(0) could beobserved visually by a color change of the solution fromamber (Fig. 1a) to black (Fig. 1b). The amber color and lackof precipitates in the formate-free controls (Fig. 1a) indicatethat Pd(II) was not reduced in the absence of formate. Thecolor shift indicating Pd(II) reduction was visible within 1 hin samples containing cells, whereas it took approximately24 h in cell-free controls. The Pd-free controls did notchange appearance after addition of formate (Fig. 1d).

The visual appearance of bio-supported Pd(0) was verydifferent from Pd(0) in cell-free suspensions (Fig. 1c). Bio-supported Pd(0) remained in suspension because it wasimmobilized on the surface of bacterial cells. The suspensionappeared as a black liquid, whereas Pd(0) formed largeaggregates at the bottom of the vial in the absence of cells(Fig. 1b,c). TEM revealed the presence electron densenanoparticles of 115 nm in diameter on the surface ofbacteria (Fig. 2a), which were not present on cells fromPd-free control samples (data not shown). EDS spectra

Figure 1. Palladium and gold solutions after 24 h incubation: (a) Cell-free control containing Pd(0) and formate, (b) bio-supported Pd(0), (c) formate-free control containingPd(II) and cells, (d) Pd(II)free control containing cell and formate, (e) cell-free control of Au(III), (f) bio-supported Au(0), (g) formate-free control containing Au(III) and cells,

(h) Au(III)- free control containing cell and formate, (i) bio-supported bimetallic Pd(0)/Au(0), (j) bio-supported bimetallic Au(0)/Pd(0).

Hosseinkhani et al.: Synthesis of Bimetallic Pd-Au Nanoparticles 3

Biotechnology and Bioengineering

confirmed that palladium was the constituent of thesenanoparticles (Fig. 3a).

The aqueous chloroaurate ions were reduced to metallicgold in the presence of bacteria and formate, resulting in avisible color shift from light yellow (Fig. 1e) to pink (Fig. 1f).There was no visible color change in formate-free (Fig. 1e),

cell-free (Fig. 1g), and Au-free controls (Fig. 1h). The UVVis spectra showed the appearance of an absorption peak at540550 nm within 46 h of adding formate to the samplescontaining Au(III) and cells, indicating the presence of goldnanoparticles (Fig. 4a). The intensity of this peak increasedover time, and no shift in the wavelength of the peak was

Figure 2. Transmission electron micrographs (a,d,g,j), high resolution TEM (b,e,h,k), and FFT analysis (c,f,i,l) of bio-supported Pd(0) nanoparticles (a,b,c) bio-supported Au(0)nanoparticles (d,e,f), bio-supported bimetallic Pd(0)-Au(0) nanoparticles (g,h,i) and Au(0)-Pd(0) nanoparticles (j,k,l). All particles were prepared by reduction with formate in 0.01M

HNO3, pH 2.1. Fast Fourier transform (FFT) analysis were performed on the areas squared in HR-TEM images.

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Figure 3. Energy dispersive spectra (EDS) of bio-supported Pd(0) nanoparticles (a), bio-supported Au(0) nanoparticles (b), bio-supported bimetallic Pd(0)-Au(0) nanoparticles(c), bio-supported bimetallic Au(0)-Pd(0) nanoparticles (d).

Abs

orba

nce

Wavelength (nm)

0

0,5

1

1,5

2

2,5

3

300 350 400 450 500 550 600 650 700

Bio-supported Au+ Pd (5 min)+ Pd (30 min)+ Pd (3 h)+ Pd (24 h)

0

0,5

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1,5

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300 350 400 450 500 550 600 650 700

Bio-supported Pd(0)+Au (5 min)+Au (30 min)+Au (3 h)+Au (24 h)

0

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2,5

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300 350 400 450 500 550 600 650 700

Pure cells+Au (2 h)+Au (4 h)+Au (6 h)+Au (9 h)+Au (12 h)+Au (24 h)

cba

Figure 4. UVVisible absorption spectra of monometallic and bimetallic nanoparticles at different time points after addition of Au(III) and formate to pure cells (a), addition ofAu(III) and formate to bio-supported Pd(0) (b), or addition of Pd(II) and formate to bio-supported Au(0) (c).

Hosseinkhani et al.: Synthesis of Bimetallic Pd-Au Nanoparticles 5

Biotechnology and Bioengineering

observed. TEM and EDS confirmed the presence of Auparticles of 257 nm in diameter on the cells (Figs. 2dand 3b). The bio-supported Au nanoparticles weresomewhat larger than the Pd nanoparticles (Fig. 5).

Synthesis of Bimetallic Bio-Supported Pd(0)-Au(0) andAu(0)-Pd(0) Nanoparticles

After addition of Au(III) and formate to bio-supportedPd(0), the suspension changed color from dark brown todeep purple (Fig. 1i), and the cells now containedsubstantially larger particles on the surface (Figs. 2gand 5). UVVis spectrophotometry showed the immediateappearance of an absorption peak around 540 nm afteraddition of Au(III) to the bio-supported Pd(0), whereas bio-supported Pd(0) alone had no adsorption peak in the 300700 nm range (Fig. 4b). Interestingly, the 540 nm absorptionpeak appeared already 5min after adding Au(III) to bio-supported Pd(0), whereas it took 6 h in samples with cellsnot containing bio-supported Pd(0) (Fig. 4a). The fasterAu(III) reduction in the presence of bio-supported Pd(0)could suggest that Pd(0) catalyzed Au(III) reduction.However, Au(III) was not reduced in cell-free samplescontaining Pd(0) (data not shown), so Pd(0) alone couldnot catalyze reduction of Au(III) by formate.

Addition of Pd(II) and formate to bio-supported Au(0)also resulted in formation of larger particles on the cellsurface (Figs. 2j and 5). The solution changed color frompink to dark brown within a few minutes (Fig. 1j), and the540 nm absorption peak from Au(0) nanoparticles de-creased in intensity over time (Fig. 4c).

EDS analysis showed that bio-supported Pd(0)-Au(0)and Au(0)-Pd(0) contained both metals on the cells(Fig. 3c and d), and HR-TEM confirmed that thenanoparticles were indeed bimetallic (Fig. 2b,e,h, and k).The second metal added to bio-supported monometallicnanoparticles grew pseudomorphically on the nanoparticles,and did not form core-shell structures. The atomic structure

of the lattice spacing in monometallic and bimetallicparticles observed by HR-TEM was analyzed by Fast FourierTransformation (FFT) (Fig. 2c,f,i, and l). The FFT profile ofmonometallic bio-supported Au(0) and Pd(0) particles wereanalyzed for comparison, and each of these metals containedone radial distance between two bright spots (Fig. 2c and f).The radial distance dA 2.24 nm corresponds to Pd(0)(Fig. 2c), whereas dA 2.35 nm corresponds to Au(0)(Fig. 2f). FFT analysis of nanoparticles from the Pd(0)-Au(0) and Au(0)-Pd(0) samples resulted in two differentradial distances, confirming the presence of palladium andgold together in one nanoparticle.

Catalysis of p-Nitrophenol Reduction

No reduction of p-nitrophenol occurred in the presence ofmonometallic Au(0) nanoparticles (Fig. 6), and the assaystherefore reflect the catalytic properties of Pd(0). Thereduction rate of p-nitrophenol to p-aminophenol wassubstantially higher (almost double) for bimetallic Pd(0)-Au(0) and Au(0)-Pd(0) nanoparticles compared to themonometallic Pd(0) nanoparticles (Fig. 6). It should benoted that the molar concentration of Pd was twice as highin the samples with monometallic particles compared tobimetallic particles. There was, however, no significantdifference in the catalytic properties of Pd(0)-Au(0) andAu(0)-Pd(0) bimetallic nanoparticles (Fig. 6).

Discussion

The cell surface of microorganisms has previously been usedto immobilize palladium and gold to synthesize bio-supported monometallic nanoparticles (Bunge et al., 2010;De Windt et al., 2005; Deplanche and Macaskie, 2008; Reith

Figure 5. Particle size distribution histogram of monometallic and bimetallicparticles determined by measuring the particle diameter of >1,000 particles in TEM

images.

Figure 6. Catalytic reduction of p-nitrophenol by monometallic and bimetallicbio-supported nanoparticles. Error barsS.D. (n 3).

6 Biotechnology and Bioengineering, Vol. xxx, No. xxx, 2011

et al., 2009; Yong et al., 2002). These results inspired us touse a similar approach to synthesize and immobilizebimetallic nanoparticles. We successfully synthesized bime-tallic Au(0)-Pd(0) particles on the surface of bacterial cells,using a two step protocol involving reductive synthesis ofimmobilized monometallic Pd(0) or Au(0) nanoparticleson the surface of C. necator cells, followed by bimetallicnanoparticle formation in a second step of Au(III) or Pd(II)reduction. Several different analyses (EDS, HR-TEM, andFFT analysis) confirmed the presence of bimetallic particleson the cells.

Bimetallic Au(0)-Pd(0) nanoparticles have been obtainedpreviously using stabilizing agents and chemical supportmaterials, such as C, Si, Al, or Ni (Davis and Boudart, 1994;Harada et al., 1993; Herzing et al., 2008; Huang et al., 2010;Toshima et al., 1992). However, all of the readily usedapproaches are lengthy and not environmentally friendly.Peptides engineered to bind multiple metals have been usedas a biomaterial for synthesis of bimetallic Au(0)-Pd(0)nanoparticles (Slocik and Naik, 2006). The use of suchpeptides is however costly for large-scale synthesis. Ourstudy demonstrates for the first time that bimetallic Au(0)-Pd(0) nanoparticles can be synthesized in short time usingbacterial cells as support material. These results may presenta new avenue for fast, clean, and cost effective preparation ofbimetallic catalysts.

A drawback of the approach presented here is that thebimetallic particles did not have a defined structure, as bothmetals agglomerated to form unorganized bimetallicparticles. Furthermore, we observed a mixture of bothmonometallic and bimetallic nanoparticles on microbialsurfaces. Our approach thus offered little control of thecomposition and structural arrangement of nanoparticles,which was probably a consequence of the complex nature ofthe cell surface used as support material. The size,composition, and structural organization of metal nano-particles are important to their catalytic properties. Previousstudies on Au(0)-Pd(0) bimetallic nanoparticles controlledthe bimetallic structure and size by using e.g., hightemperature, photoirradiation, mild reductants, or differentmolar ratios between Au(0)-Pd(0) (Herzing et al., 2008;Toshima, 2000). Despite the absence of a core-shellstructural arrangement, microbially supported bimetallicnanoparticles still proved superior to monometallic Pd(0)particles for catalysis of p-nitrophenol reduction to p-aminophenol. The increased catalytic activity is attributed togeometric or electronic effects by which Au is thought todraw electrons from Pd, which becomes depleted inelectrons and more prone to interaction with the catalyticreactants (Knecht et al., 2007; Scott et al., 2004; Slocik andNaik, 2006). In this study, we observed no difference incatalytic activity between Pd(0)-Au(0) and Au(0)-Pd(0)particles. It seems that the properties of the particles werenot affected by Pd being reduced onto Au(0) nanoparticlesor vice versa. Such similarities in catalytic properties arelikely the result of the synergistic effect of Au atoms on thecatalytic activity of Pd atoms. The incomplete core-shell

structure of the bimetallic nanoparticles allows in both cases(Au(0)-Pd(0) or Pd(0)/Au(0)) access of the reactant(p-nitrophenol) to Pd atoms, whereas a core-shell structuralarrangement would block access to Pd-atoms inside thePd(0)-Au(0) nanoparticles.

In conclusion, microbially supported synthesis ofnanoparticles could be an alternative method to immobilizeand stabilize Pd(0)-Au(0) and Au(0)-Pd(0) bimetallicnanoparticles in an environmentally friendly and costeffective manner, which could be suitable for industrial scaleapplications. The method could be only used to synthesizebimetallic nanoparticles with incomplete core-shell struc-ture, yet with better catalytic properties than theirmonometallic counterparts.

The authors would like to thank Jacques Chevallier for assistance with

EDX, HR-TEM, and FFT analysis. They gratefully acknowledge the

Danish Research Council for Technology and Production Sciences

(Grant no. 274-07-0254) for funding this work, and The University of

Isfahan for funding a travel stipend for B. Hosseinkhani.

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