Bioprocess Biosyst Eng (2010) 33:159164 DOI 10.

1007/s00449-009-0373-2

ORIGINAL PAPER

Biological synthesis of platinum nanoparticles using Diopyros kaki leaf extract

Jae Yong Song Eun-Yeong Kwon Beom Soo Kim

Received: 10 June 2009 / Accepted: 5 August 2009 / Published online: 23 August 2009 Springer-Verlag 2009

Abstract The leaf extract of Diopyros kaki was used as a reducing agent in the ecofriendly extracellular synthesis of platinum nanoparticles from an aqueous H2PtCl66H2O solution. A greater than 90% conversion of platinum ions to nanoparticles was achieved with a reaction temperature of 95C and a leaf broth concentration of [10%. A variety of methods was used to characterize the platinum nanoparticles synthesized: inductively coupled plasma spectrometry, transmission electron microscopy, energydispersive X-ray spectroscopy, X-ray photoelectron spectroscopy, and Fourier-transform infrared spectroscopy (FTIR). The average particle size ranged from 2 to 12 nm depending on the reaction temperature and concentrations of the leaf broth and PtCl62-. FTIR analysis suggests that platinum nanoparticle synthesis using Diopyros kaki is not an enzyme-mediated process. This is the rst report of platinum nanoparticle synthesis using a plant extract. Keywords Biological synthesis Nanoparticles Platinum Plant extract Diopyros kaki

Introduction Nanoparticles of noble metals, such as gold, silver, and platinum, are widely used in products that come into direct contact with the human body, including shampoo, soap, detergent, shoes, cosmetics, and toothpaste, as well as medical and pharmaceutical applications. The well-known
J. Y. Song E.-Y. Kwon B. S. Kim (&) Department of Chemical Engineering, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea e-mail: bskim@chungbuk.ac.kr

platinum compound, cis-platin (cis-diaminedichloroplatinum), has been used as antitumor agent [1]. Platinum nanoparticles have been used in biomedical applications in combination with nanoparticles of other metals, in either alloy, core-shell, or bimetallic nanocluster form [1]. Yolkshell nanocrystals of FePt@CoS2 have been found to be more potent in killing HeLa cells as compared to cis-platin [2]. With such important applications, there is a growing need to develop processes for synthesizing nanoparticles that do not use toxic chemicals, and that are thus more likely to be environmentally friendly. Biological methods for nanoparticle synthesis using microorganisms, enzymes, and plants or plant extracts have been suggested as possible ecofriendly alternatives to chemical and physical methods [3, 4]. We recently demonstrated that silver and gold nanoparticles could be prepared using screened plant extracts [3, 57]. This process is rapid, with [90% conversion to silver and gold nanoparticles in only 11 and 3 min, respectively, in a reaction using Magnolia leaf broth at a temperature of 95C. Methods of nanoparticle synthesis that use plants have advantages over other types of biological methods, including an avoidance of the elaborate processes required to maintain cell cultures; they are also readily adapted for large-scale nanoparticle synthesis [8]. Gardea-Torresdey et al. [9, 10] have reported synthesizing gold and silver nanoparticles within live alfalfa plants from solid media. Extracellular nanoparticle synthesis using plant leaf extracts rather than whole plants would be more economical owing to easier downstream processing. Sastry and colleagues pioneered the use of plant extracts to synthesize nanoparticles [8, 1116] and have reported rates of synthesis comparable to those of chemical methods. While biological processes using microorganisms, plants, and plant extracts have been used to synthesize nanoparticles of

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gold and silver, there is relatively little knowledge concerning the biological synthesis of platinum nanoparticles. Biosorption of platinum by the sulfate-reducing bacterium Desulfovibrio desulfuricans has been reported [17]. It has also been found that resting cells of Shewanella algae reduced aqueous PtCl62- into elemental platinum within 60 min under room temperature and neutral pH conditions when lactate was provided as an electron donor [18]; transmission electron microscopy (TEM) images showed platinum nanoparticles approximately 5 nm in diameter within the periplasm of the algal cells. However, the use of plant extracts to synthesize platinum nanoparticles has not been reported as of yet. In this study, we synthesized platinum nanoparticles using screened leaf extracts of the Diopyros kaki plant. We also investigated how nanoparticle size was inuenced by reaction conditions, including temperature and the concentration of both leaf broth and PtCl62-. Fouriertransform infrared spectroscopy (FTIR) analysis was used to identify the biomolecules responsible for reducing platinum ions and stabilizing the platinum nanoparticles formed. To the best of our knowledge, this is the rst report of a plant extract being used to synthesize platinum nanoparticles.

spectroscopy (XPS; ESCALAB 210), and FTIR (Bomem MB100). Platinum concentrations and conversions were determined with inductively coupled plasma spectrometry (ICP; JY38Plus), and the average particle size ascertained from TEM micrographs.

Results and discussion Effects of temperature and reaction mixture composition The reduction of platinum ions to platinum nanoparticles when exposed to the plant leaf extract was tracked by monitoring changes in color with UVVis spectroscopy; absorbance at 477 nm was used to quantify platinum nanoparticle concentrations and conversion. There was a linear relationship between the platinum concentration values determined by ICP and those obtained by absorbance at 477 nm (data not shown). Figure 1 shows the time course of platinum nanoparticle synthesis with 20% Persimmon leaf broth at different reaction temperatures. The rate of platinum nanoparticle synthesis increased with increases in reaction temperature. At a reaction temperature of either 25 or 60C, \20% of platinum ions were converted to platinum nanoparticles. Increasing the reaction temperature to 95C improved the level of conversion to almost 100%. An increase in reaction rate with an increase in reaction temperature has been previously reported by Rai et al. for the synthesis of gold nanotriangles using lemongrass extract [14], and by our group for the synthesis of gold and silver nanoparticles using either Persimmon or Magnolia leaf broth [3, 5, 7]. In this study, we found that it took 150 min to achieve [90% conversion to platinum nanoparticles with a reaction temperature of 95C. This is much slower than the 3 and
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Materials and methods Persimmon (D. kaki) leaves were collected and left to dry for 2 days at room temperature. Leaf broth solution was prepared by boiling a mixture of 5 g of thoroughly washed and nely cut dried leaves and 100 mL of sterile distilled water in a 300-mL Erlenmeyer ask for 5 min. The solution was decanted and stored at 4C; it was used within a week of having been prepared. Our general method for reducing PtCl62- ions was to add 10 mL of leaf broth to 190 mL of 1 mM aqueous H2PtCl66H2O. The reaction was performed with reux at various temperatures, between 25 and 95C, to investigate the effects of temperature on platinum nanoparticle synthesis rate and size. The concentrations of H2PtCl66H2O solution and leaf broth were also varied, between 0.12 mM and 550% by volume, respectively. The resulting platinum nanoparticle solution was puried by repeated centrifugation at 15,000 rpm for 20 min, with the pellet produced by this process redispersed in deionized water. Ultravioletvisible (UVVis) spectra were recorded as a function of the reaction time on a UV-1650CP Shimadzu spectrophotometer operating with a resolution of 1 nm. Puried platinum nanoparticles were freeze-dried, and their structure and composition analyzed by high-resolution TEM (HR-TEM; JEOL-2010), energy-dispersive X-ray spectroscopy (EDS; Sigma), X-ray photoelectron

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Fig. 1 Effect of reaction temperature (2595C) on the time course of platinum nanoparticle synthesis using 20% D. kaki leaf broth and 1 mM PtCl62-

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11 min required for comparable levels of conversion when synthesizing gold and silver nanoparticles, respectively, with Magnolia leaf broth at the same temperature [3, 5, 7]. The relatively low rate of platinum nanoparticle synthesis is possibly due to a difculty in initially forming platinum nuclei, indicating that achieving close to 100% conversion to platinum nanoparticles requires longer reaction times and higher temperatures than those required for either gold or silver nanoparticles. The effect of Persimmon leaf broth concentration (550%) on platinum nanoparticle synthesis at 95C is shown in Fig. 2. The rate of synthesis increased with an increase in leaf broth concentration. With a leaf broth concentration of 5%, the level of conversion to nanoparticles achieved was only around 10%. Increasing the leaf broth concentration to more than 10% resulted in almost 100% conversion after 23 h. In synthesizing gold or silver nanoparticles, close to 100% conversion is achieved with a leaf broth concentration of 5% [3, 5, 7]. It is thus not only higher temperatures but also higher leaf broth concentrations that are required to achieve levels of platinum nanoparticle synthesis comparable to those for gold and silver. Figure 3 is a representative TEM image of the platinum nanoparticles obtained using Persimmon leaf broth. A mixture of spheres and plates was obtained, with the size range of 220 nm. HR-TEM images of the nanoparticles (Fig. 4) show lattice fringe distances of 0.227 and 0.199 nm that are consistent with interplanar spacings for metallic platinum (d111 = 0.2265 nm, d200 = 0.1962 nm), corresponding to the (111) and (200) pure platinum planes, respectively [19]. Characteristic platinum peaks on EDS and XPS spectra recordings (Fig. 5) are also consistent with the successful synthesis of platinum nanoparticles using Persimmon leaf broth.
Fig. 3 TEM images of platinum nanoparticles synthesized using 1 mM PtCl62- with (a) 10% D. kaki leaf broth at 95C and (b) 20% D. kaki leaf broth at 25C
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Fig. 2 Effect of D. kaki leaf broth concentration (550%) on the time course of platinum nanoparticle synthesis using 1 mM PtCl62- at 95C

We investigated the possibility of controlling the size of platinum nanoparticles synthesized via changes in the temperature and composition of the reaction mixture. Figure 6 summarizes the results of these investigations using Persimmon leaf broth. The average particle size decreased from 12 nm at 25C to 5 nm at 95C, a trend we previously observed with gold and silver nanoparticle synthesis using either Persimmon or Magnolia leaf broth. This effect of temperature on particle size may be explained in terms of an increased reaction rate at higher temperatures, and thus at which it is likely that most of the metal ions form nuclei so rapidly as to limit the extent to which secondary reduction processes on the surface of preformed nuclei can occur [3, 5, 7]. We also found that the

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162 Fig. 4 a HR-TEM image of platinum nanoparticles synthesized using 10% D. kaki leaf broth with 1 mM PtCl62at 95C. b An enlargement showing their crystalline platinum structure

Bioprocess Biosyst Eng (2010) 33:159164

Fig. 5 Characterization of platinum nanoparticles synthesized using 1 mM PtCl62- and 10% D. kaki leaf broth at 95C. a Spot prole EDS spectrum. b XPS spectrum

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4f Pt 74.15 70.89

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average platinum nanoparticle size decreased with an increase in leaf broth concentration, and increased with an increase in PtCl62- concentration. We previously found that large hexagonal or triangular gold nanoparticles were produced by reacting HAuCl4 with a low concentration of Magnolia leaf extract, but that increasing the broth concentration led to smaller spherical nanoparticles being

formed [7]. Ji et al. [20] have reported that the average size of gold nanocrystals formed by citrate reduction decreased as the ratio of sodium citrate to HAuCl4 precursor increased, and explained this in terms of a nucleationgrowth mechanism. The results of this study reveal a trend similar to that reported by Ji et al., with a greater concentration of platinum ions resulting in larger platinum

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Bioprocess Biosyst Eng (2010) 33:159164 Fig. 6 Effects of leaf broth concentration (with 1 mM PtCl62- at 95C), reaction temperature (with 20% leaf broth and 1 mM PtCl62-), and PtCl62- concentration (with 20% leaf broth at 95C) on the size of platinum nanoparticles
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nanoparticles being formed, provided the leaf broth (reducing agent) concentration and other reaction parameters remain the same. FTIR analysis of platinum nanoparticles FTIR analysis was used to characterize the synthesized platinum nanoparticles (Fig. 7). Intense bands were observed in the FTIR spectrum at 924, 1033, 1195, 1325, 1410, 1613, and 1692 cm-1, with these peaks assigned to alcohols, CN stretching vibration of aliphatic amines, phenolic groups, CN stretching vibration of aromatic amines, germinal methyls, C=C groups or aromatic rings, and carbonyl groups, respectively. There were no peaks in the amide I (1640 cm-1) or amide II (1540 cm-1) regions that are characteristic of proteins/enzymes responsible for the reduction of metal ions to nanoparticles by microorganisms such as fungi [21]. These results indicate that platinum nanoparticles synthesized with D. kaki extract are surrounded by some metabolites like terpenoids that have functional groups of amines, alcohols, ketones, aldehydes, and carboxylic acids. The mechanism by which nanoparticles form in biosynthesis procedures is not clear. The reduction necessary for the extracellular synthesis of gold nanoparticles using the fungus Fusarium oxysporum has been reported to occur via NADH-dependent reductase being released into solution [21]. It has also been suggested that nitroreductase enzymes may be involved in the synthesis of silver nanoparticles using culture supernatants of Enterobacteria [22]. Proteins in an extract of the unicellular green alga Chlorella vulgaris were thought to be involved in the biological synthesis of silver nanoplates using this organism [23]. Xie et al. [23] designed a simple bifunctional tripeptide (AspAsp-Tyr-OMe) that had a Tyr residue as a reduction source and two carboxyl groups in each of the Asp residues as shape-directors, and found that this produced a good yield

Fig. 7 FTIR spectrum of freeze-dried and puried platinum nanoparticles synthesized using 1 mM PtCl62- and 10% leaf broth at 95C

of silver nanoplates with low polydispersity. In the case of Neem leaf broth, it is believed that terpenoids are the surface-active molecules that stabilize nanoparticles, and that the reduction of metal ions is possibly also facilitated by these and/or sugars [8]. The terpenoids (isoprenoids) are a large and diverse class of naturally occurring lipids derived from 5-carbon isoprene units that can be assembled and modied in a variety of ways. These lipids can be found in all classes of living things and are the largest group of natural products. Therefore, many plant extracts can be used to synthesize metal nanoparticles owing to the existence of terpenoids and reducing sugars in them. The results of this study suggest that platinum nanoparticle synthesis using D. kaki is not an enzyme-mediated process because the rate of platinum nanoparticle synthesis is greatest at temperatures as high as 95C and there are no peaks associated with proteins/enzymes on FTIR analysis. It appears more likely that the reduction of platinum ions and stabilization of synthesized platinum nanoparticles is

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Bioprocess Biosyst Eng (2010) 33:159164 9. Gardea-Torresdey JL, Parsons JG, Gomez E, Peralta-Videa J, Troiani HE, Santiago P, Jose-Yacaman M (2002) Formation and growth of Au nanoparticles inside live alfalfa plants. Nano Lett 2:397401 10. Gardea-Torresdey JL, Gomez E, Peralta-Videa J, Parsons JG, Troiani HE, Santiago P, Jose-Yacaman M (2003) Alfalfa sprouts: a natural source for the synthesis of silver nanoparticles. Langmuir 19:13571361 11. Shankar SS, Ahmad A, Pasricha R, Sastry M (2003) Bioreduction of chloroaurate ions by geranium leaves and its endophytic fungus yields gold nanoparticles of different shapes. J Mater Chem 13:18221826 12. Shankar SS, Ahmad A, Sastry M (2003) Geranium leaf assisted biosynthesis of silver nanoparticles. Biotechnol Prog 19:1627 1631 13. Shankar SS, Rai A, Ankamwar B, Singh A, Ahmad A, Sastry M (2004) Biological synthesis of triangular gold nanoprisms. Nat Mater 3:482488 14. Rai A, Singh A, Ahmad A, Sastry M (2006) Role of halide ions and temperature on the morphology of biologically synthesized gold nanotriangles. Langmuir 22:736741 15. Rai A, Chaudhary M, Ahmad A, Bhargava S, Sastry M (2007) Synthesis of triangular Au core-Ag shell nanoparticles. Mater Res Bull 42:12121220 16. Chandran SP, Chaudhary M, Pasricha R, Ahmad A, Sastry M (2006) Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract. Biotechnol Prog 22:577583 17. Yong P, Rowson NA, Farr JPG, Harris IR, Macaskie LE (2002) Bioreduction and biocrystallization of palladium by Desulfovibrio desulfuricans NCIMB 8307. Biotechnol Bioeng 80:369379 18. Konishi Y, Ohno K, Saitoh N, Nomura T, Nagamine S, Hishida H, Takahashi Y, Uruga T (2007) Bioreductive deposition of platinum nanoparticles on the bacterium Shewanella algae. J Biotechnol 128:648653 19. JCPDS-ICDD International Center for Diffraction Data, JCPDS File No. 04-0802 (1999) 20. Ji XH, Song XN, Li J, Bai YB, Yang WS, Peng XG (2007) Size control of gold nanocrystals in citrate reduction: the third role of citrate. J Am Chem Soc 129:1393913948 21. Mukherjee P, Senapati S, Mandal D, Ahmad A, Khan MI, Kumar R, Sastri M (2002) Extracellular synthesis of gold nanoparticles by the fungus Fusarium oxysporum. ChemBioChem 5:461463 22. Shahverdi A, Minaeian S, Shahverdi HR, Jamalifar H, Nohi A-A (2007) Rapid synthesis of silver nanoparticles using culture supernatants of Enterobacteria: a novel biological approach. Proc Biochem 42:919923 23. Xie J, Lee JY, Wang DIC, Ting YP (2007) Silver nanoplates: from biological to biomimetic synthesis. ACSNano 1:429439

the responsibility of many functional groups, including amines, alcohols, ketones, aldehydes, and carboxylic acids, that are present in various metabolites such as terpenoids and reducing sugars. In conclusion, we have demonstrated in this study the rst ecofriendly use of a plant extract to synthesize platinum nanoparticles. An almost 100% conversion of platinum ions to platinum nanoparticles was achieved with a reaction temperature of 95C and a concentration of Persimmon leaf broth that was [10%. The average particle size was in the range of 212 nm and could be controlled via changes in reaction temperature and in the concentrations of leaf broth and PtCl62-.
Acknowledgments This research was nancially supported by the Ministry of Knowledge Economy (MKE) and Korea Institute for Advancement in Technology (KIAT) through the Workforce Development Program in Strategic Technology.

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