ORIGINAL PAPER

Biological synthesis of platinum nanoparticles using Diopyros kakileaf extract

Jae Yong Song Æ Eun-Yeong Kwon ÆBeom 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 H2PtCl6�6H2O

solution. A greater than 90% conversion of platinum ions

to nanoparticles was achieved with a reaction temperature

of 95�C 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, energy-

dispersive X-ray spectroscopy, X-ray photoelectron spec-

troscopy, and Fourier-transform infrared spectroscopy

(FTIR). The average particle size ranged from 2 to

12 nm depending on the reaction temperature and con-

centrations of the leaf broth and PtCl62-. FTIR analysis

suggests that platinum nanoparticle synthesis using Dio-

pyros kaki is not an enzyme-mediated process. This is the

first 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

platinum compound, cis-platin (cis-diaminedichloroplati-

num), 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]. Yolk-

shell 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 nano-

particles could be prepared using screened plant extracts

[3, 5–7]. 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 95�C. Methods of nanoparticle synthesis

that use plants have advantages over other types of bio-

logical 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 syn-

thesize nanoparticles [8, 11–16] 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

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

123

Bioprocess Biosyst Eng (2010) 33:159–164

DOI 10.1007/s00449-009-0373-2

gold and silver, there is relatively little knowledge con-

cerning 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 influenced

by reaction conditions, including temperature and the

concentration of both leaf broth and PtCl62-. Fourier-

transform 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 first report

of a plant extract being used to synthesize platinum

nanoparticles.

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 finely cut dried leaves and 100 mL of sterile distilled

water in a 300-mL Erlenmeyer flask for 5 min. The solu-

tion was decanted and stored at 4�C; 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

H2PtCl6�6H2O. The reaction was performed with reflux at

various temperatures, between 25 and 95�C, to investigate

the effects of temperature on platinum nanoparticle syn-

thesis rate and size. The concentrations of H2PtCl6�6H2O

solution and leaf broth were also varied, between 0.1–2 mM

and 5–50% by volume, respectively. The resulting plati-

num nanoparticle solution was purified by repeated

centrifugation at 15,000 rpm for 20 min, with the pellet

produced by this process redispersed in deionized water.

Ultraviolet–visible (UV–Vis) spectra were recorded as

a function of the reaction time on a UV-1650CP Shimadzu

spectrophotometer operating with a resolution of 1 nm.

Purified platinum nanoparticles were freeze-dried, and

their structure and composition analyzed by high-resolu-

tion TEM (HR-TEM; JEOL-2010), energy-dispersive

X-ray spectroscopy (EDS; Sigma), X-ray photoelectron

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 UV–Vis 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 absor-

bance 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 60�C, \20% of

platinum ions were converted to platinum nanoparticles.

Increasing the reaction temperature to 95�C improved the

level of conversion to almost 100%. An increase in reac-

tion 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 95�C. This is much slower than the 3 and

0 1 2 3 4 50

50

100

150

200

0

20

40

60

80

100C

onve

rsio

n (%

)

Pt

Con

cent

rati

on (

mg/

L)

Time (h)

25 °C

60 °C

95 °C

Fig. 1 Effect of reaction temperature (25–95�C) on the time course

of platinum nanoparticle synthesis using 20% D. kaki leaf broth and

1 mM PtCl62-

160 Bioprocess Biosyst Eng (2010) 33:159–164

123

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 difficulty 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

(5–50%) on platinum nanoparticle synthesis at 95�C 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 nanopar-

ticles achieved was only around 10%. Increasing the leaf

broth concentration to more than 10% resulted in almost

100% conversion after 2–3 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 concentra-

tions 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 2–20 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.

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 25�C to 5 nm at 95�C, 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

0 1 2 3 40

50

100

150

200

0

20

40

60

80

100

Pt

Con

cent

rati

on (

mg/

L)

Time (h)

Leaf broth 5% Leaf broth 10% Leaf broth 20% Leaf broth 50% C

onve

rsio

n (

%)

Fig. 2 Effect of D. kaki leaf broth concentration (5–50%) on the time

course of platinum nanoparticle synthesis using 1 mM PtCl62- at

95�C

Fig. 3 TEM images of platinum nanoparticles synthesized using

1 mM PtCl62- with (a) 10% D. kaki leaf broth at 95�C and (b) 20%

D. kaki leaf broth at 25�C

Bioprocess Biosyst Eng (2010) 33:159–164 161

123

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 nucleation-

growth mechanism. The results of this study reveal a trend

similar to that reported by Ji et al., with a greater con-

centration of platinum ions resulting in larger platinum

Fig. 4 a HR-TEM image of

platinum nanoparticles

synthesized using 10% D. kakileaf broth with 1 mM PtCl6

2-

at 95�C. b An enlargement

showing their crystalline

platinum structure

80 78 76 74 72 70 68

70.89

Binding Energy (eV)

4f Pt 74.15(B)

Fig. 5 Characterization of

platinum nanoparticles

synthesized using 1 mM

PtCl62- and 10% D. kaki leaf

broth at 95�C. a Spot profile

EDS spectrum. b XPS spectrum

162 Bioprocess Biosyst Eng (2010) 33:159–164

123

nanoparticles being formed, provided the leaf broth

(reducing agent) concentration and other reaction param-

eters 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, C–N stretching vibration of aliphatic amines,

phenolic groups, C–N 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 nanopar-

ticles by microorganisms such as fungi [21]. These

results indicate that platinum nanoparticles synthesized

with D. kaki extract are surrounded by some metabo-

lites like terpenoids that have functional groups of

amines, alcohols, ketones, aldehydes, and carboxylic

acids.

The mechanism by which nanoparticles form in bio-

synthesis 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 solu-

tion [21]. It has also been suggested that nitroreductase

enzymes may be involved in the synthesis of silver nano-

particles using culture supernatants of Enterobacteria [22].

Proteins in an extract of the unicellular green alga Chlo-

rella 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 (Asp-

Asp-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

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 modified 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 95�C 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

0

2

4

6

8

10

12

14

16

18

5 10 20 50 25 60 95 0.1 0.5 1 2

Leaf broth concentration (%)(°C)

PtCl6 ion concentration(mM)

Par

ticle

siz

e (n

m)

Reaction temperature

Fig. 6 Effects of leaf broth

concentration (with 1 mM

PtCl62- at 95�C), reaction

temperature (with 20% leaf

broth and 1 mM PtCl62-), and

PtCl62- concentration (with

20% leaf broth at 95�C) on the

size of platinum nanoparticles

Fig. 7 FTIR spectrum of freeze-dried and purified platinum nano-

particles synthesized using 1 mM PtCl62- and 10% leaf broth at 95�C

Bioprocess Biosyst Eng (2010) 33:159–164 163

123

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 first 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 95�C and a concentration of

Persimmon leaf broth that was[10%. The average particle

size was in the range of 2–12 nm and could be controlled

via changes in reaction temperature and in the concentra-

tions of leaf broth and PtCl62-.

Acknowledgments This research was financially 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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