Bioprocess Biosyst Eng (2009) 32:79–84
DOI 10.1007/s00449-008-0224-6
ORIGINAL PAPER
Rapid biological synthesis of silver nanoparticles
using plant leaf extracts
Jae Yong Song Æ Beom Soo Kim
Received: 19 January 2008 / Accepted: 15 April 2008 / Published online: 26 April 2008
Ó Springer-Verlag 2008
Abstract Five plant leaf extracts (Pine, Persimmon,
Ginkgo, Magnolia and Platanus) were used and compared
for their extracellular synthesis of metallic silver nanoparticles. Stable silver nanoparticles were formed by
treating aqueous solution of AgNO3 with the plant leaf
extracts as reducing agent of Ag+ to Ag0. UV-visible
spectroscopy was used to monitor the quantitative formation of silver nanoparticles. Magnolia leaf broth was the
best reducing agent in terms of synthesis rate and conversion to silver nanoparticles. Only 11 min was required for
more than 90% conversion at the reaction temperature of
95 °C using Magnolia leaf broth. The synthesized silver
nanoparticles were characterized with inductively coupled
plasma spectrometry (ICP), energy dispersive X-ray spectroscopy (EDS), scanning electron microscopy (SEM),
transmission electron microscopy (TEM), and particle
analyzer. The average particle size ranged from 15 to
500 nm. The particle size could be controlled by changing
the reaction temperature, leaf broth concentration and
AgNO3 concentration. This environmentally friendly
method of biological silver nanoparticles production provides rates of synthesis faster or comparable to those of
chemical methods and can potentially be used in various
human contacting areas such as cosmetics, foods and
medical applications.
Keywords Biological synthesis Nanoparticles Silver Plant extracts Size control
J. Y. Song B. S. Kim (&)
Department of Chemical Engineering,
Chungbuk National University, Cheongju,
Chungbuk 361-763, Republic of Korea
e-mail: [email protected]
Introduction
Nanoparticles usually referred as particles with a size up to
100 nm [1, 2]. Nanoparticles exhibit completely new or
improved properties based on specific characteristics such
as size, distribution and morphology, if compared with
larger particles of the bulk material they are made of.
Nanoparticles present a higher surface to volume ratio with
decreasing size of nanoparticles. Specific surface area is
relevant for catalytic reactivity and other related properties
such as antimicrobial activity in silver nanoparticles. As
specific surface area of nanoparticles is increased, their
biological effectiveness can increase due to the increase in
surface energy [1].
Silver has long been recognized as having an inhibitory
effect toward many bacterial strains and microorganisms
commonly present in medical and industrial processes [3].
The most widely used and known applications of silver and
silver nanoparticles are in the medical industry. These
include topical ointments and creams containing silver to
prevent infection of burns and open wounds [4]. Another
widely used applications are medical devices and implants
prepared with silver-impregnated polymers [5]. In addition,
silver-containing consumer products such as colloidal
silver gel and silver-embedded fabrics are now used in
sporting equipment.
Production of nanoparticles can be achieved through
different methods. Chemical approaches are the most
popular methods for the production of nanoparticles.
However, some chemical methods cannot avoid the use of
toxic chemicals in the synthesis protocol. Since noble
metal nanoparticles such as gold, silver and platinum
nanoparticles are widely applied to human contacting
areas, there is a growing need to develop environmentally
friendly processes of nanoparticles synthesis that do not use
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toxic chemicals. Biological methods of nanoparticles synthesis using microorganism [6–8], enzyme [9], and plant or
plant extract [10] have been suggested as possible ecofriendly alternatives to chemical and physical methods.
Using plant for nanoparticles synthesis can be advantageous over other biological processes by eliminating the
elaborate process of maintaining cell cultures [10]. It can
also be suitably scaled up for large-scale synthesis of
nanoparticles.
Shankar et al. [10] reported on the synthesis of pure
metallic nanoparticles of silver and gold by the reduction of
Ag+ and Au3+ ions using Neem (Azadirachta indica) leaf
broth. However, little has been carried out about engineering approaches such as rapid nanoparticles synthesis using
plant extracts and size control of the synthesized nanoparticles. The times required for more than 90% reduction of
Ag+ and Au3+ ions using Neem leaf broth were about 4 and
2 h, respectively. If biological synthesis of nanoparticles
can compete with chemical methods, there is a need to
achieve faster synthesis rates. In this study, we screened
several plant leaf extracts and compared their synthesis of
silver nanoparticles by monitoring the conversion using
UV-visible spectroscopy. We also investigated the effects
of reaction conditions such as reaction temperature, leaf
broth concentration and AgNO3 concentration on synthesis
rate and particle size of the silver nanoparticles.
Materials and methods
Five plant leaves were collected and dried for 2 days at
room temperature. They were Pine (Pinus desiflora), Persimmon (Diopyros kaki), Ginkgo (Ginko biloba), Magnolia
(Magnolia kobus) and Platanus (Platanus orientalis). The
plant leaf broth solution was prepared by taking 5 g of
thoroughly washed and finely cut leaves in a 300 mL
Erlenmeyer flask with 100 mL of sterile distilled water and
then boiling the mixture for 5 min before finally decanting
it. They were stored at 4 °C and used within a week.
Typically, 10 mL of leaf broth was added to 190 mL of
1 mM aqueous AgNO3 solution for reduction of Ag+ ions.
The effects of temperature on synthesis rate and particle
size of the prepared silver nanoparticles were studied by
carrying out the reaction in water bath at 25–95 °C with
reflux. The concentrations of AgNO3 solution and leaf
broth were also varied at 0.1–2 mM and 5–50% by volume,
respectively. The silver nanoparticle solution thus obtained
was purified by repeated centrifugation at 15,000 rpm for
20 min followed by redispersion of the pellet in deionized
water. UV-vis spectra were recorded as a function of
reaction time on a UV-1650CP Shimadzu spectrophotometer operated at resolution of 1 nm. After freeze drying of
the purified silver particles, the structure and composition
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Bioprocess Biosyst Eng (2009) 32:79–84
were analyzed by scanning electron microscopy (SEM,
Hitachi S-2500C), field emission transmission electron
microscopy (FE-TEM, Tecnai F30 S-Twin, FEI), energy
dispersive X-ray spectroscopy (EDS, Sigma), and X-ray
photoelectron spectroscopy (XPS, ESCALAB 210). Silver
concentrations and conversions were determined using
inductively coupled plasma spectrometry (ICP, JY38Plus).
Average particle size and distribution were measured using
particle analyzer (NICOMPTM 380 ZLS).
Results and discussion
Synthesis and characterization of silver nanoparticles
It is well known that silver nanoparticles exhibit yellowishbrown color in aqueous solution due to excitation of surface plasmon vibrations in silver nanoparticles [10].
Reduction of the silver ion to silver nanoparticles during
exposure to the plant leaf extracts could be followed by
color change and thus UV-vis spectroscopy. Figure 1a–c
show the UV-vis spectra recorded from the reaction medium as a function of reaction time using Persimmon,
Magnolia and Pine leaf broth, respectively. It is observed
that the maximum absorbance occurs at ca. 430 nm and
steadily increases in intensity as a function of reaction
time. The final absorption intensities at 430 nm were more
than 1.0 a.u. and increased up to 1.5 a.u. using Magnolia
leaf broth which was higher than about 0.5 a.u. reported
with Neem leaf broth by Shankar et al. [10], suggesting that
the conversion to silver nanoparticles may be higher using
the plant leaf extracts in this study. Since the peak wavelength did not shift during the reaction, we could
quantitatively monitor the concentrations of silver nanoparticles and thus conversion by measuring the absorbance
at 430 nm. The linear relationship was obtained between
the silver concentration determined by ICP and the absorbance at 430 nm.
Figure 2 shows the time courses of silver nanoparticles
production with different reaction temperatures obtained
with Persimmon leaf broth. As the reaction temperature
increased, both synthesis rate and final conversion to silver nanoparticles increased. The final conversion at 25 °C
was 60% and reached almost 100% at more than 55 °C.
Besides Persimmon leaf broth, we investigated the effect
of reaction temperature with Magnolia leaf broth and
obtained similar results. Rai et al. [11] also mentioned the
increase of reduction rate with increasing the reaction
temperature for the gold nanotriangles synthesized using
lemongrass extract. The average particle sizes with temperature are shown in the inset of Fig. 2. The particle size
decreased from 50 nm at 25 °C to 16 nm at 95 °C.
Regarding the reason of decrease in particle size with
Bioprocess Biosyst Eng (2009) 32:79–84
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55 oC
80
95 oC
100
80
60
60
40
20
80
60
40
40
20
0
20
20
0
0
5
10
Conversion (%)
25 oC
100
Particle Size (nm)
Ag Concentration (mg/L)
120
15
40
60
80
Temperature (oC)
20
100
0
25
Time (h)
Fig. 2 Time courses of silver nanoparticles formation obtained with
1 mM AgNO3 and 5% Diopyros kaki leaf broth with different
reaction temperature. Inset effect of reaction temperature on average
silver particle size
Fig. 1 UV-vis spectra recorded as a function of reaction time of
1 mM AgNO3 solution with plant leaf broth. a Diopyros kaki. b
Magnolia kobus. c Pinus densiflora. Insets respective plots of
absorbance at 430 nm as a function of time
temperature, we can hypothesize as follows. As the
reaction temperature increases, the reaction rate increases
and thus most silver ions are consumed in the formation
of nuclei, stopping the secondary reduction process on
the surface of the preformed nuclei. Similar trend was
observed with gold nanotriangles synthesized using lemongrass extract [11].
Figure 3a and b are SEM and TEM images, respectively, obtained with 5% Persimmon leaf broth and 1 mM
AgNO3 solution at 55 °C. It is shown that relatively
spherical nanoparticles are formed with average diameter
of 32 nm with some deviations. The silver nanoparticles
showed Gaussian distributions (the inset of Fig. 3a). The
coefficient of variance of the silver nanoparticles, defined
as the ratio of standard deviation to average diameter, was
0.672. Other values of the coefficient of variance were in
the range of 0.52–0.68. The inset of Fig. 3b shows the
selected area electron diffraction (SAED) pattern recorded
from the silver nanoparticles. The ring-like diffraction
pattern indicates that the particles are crystalline. The diffraction rings could be indexed on the basis of the fcc
structure of silver. Four rings arise due to reflections from
(111), (200), (220), and (311) lattice planes of fcc silver,
respectively. Similar SAED pattern was obtained with
silver nanoparticles synthesized using Aloe vera extract by
Chandran et al. [12]. EDS and XPS spectra recorded from
the silver nanoparticles are shown in Fig. 3c and d,
respectively. EDS profile shows strong silver signal along
with a weak oxygen and carbon peak, which may originate
from the biomolecules that are bound to the surface of the
silver nanoparticles. Together with TEM images, Shankar
et al. [10] reported that nanoparticles synthesized using
plant extracts are surrounded by a thin layer of some
capping organic material from plant leaf broth. Our TEM
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Bioprocess Biosyst Eng (2009) 32:79–84
Fig. 3 Characterization of silver nanoparticles formed with 1 mM
AgNO3 and 5% Diopyros kaki leaf broth at 55 °C. a SEM image.
Inset particle size distribution. b TEM image. Inset electron
diffraction pattern recorded from the particles shown in b with lattice
planes of fcc silver. c Spot profile EDS spectrum. d XPS spectrum
image in Fig. 3b also shows that the silver particles synthesized using plant extracts are surrounded by a thin layer
of some capping material and were stable in solution during 4 weeks after their synthesis possibly due to the
capping material on the surface of nanoparticles. XPS
spectrum shows characteristic silver peaks on the surface
of nanoparticles, suggesting that silver nanoparticles are
successfully synthesized using plant leaf broth in this
study.
In order to screen plant with high production capability
of silver nanoparticles, we compared several plant extracts
for their synthesis rate of silver nanoparticles. As shown in
Fig. 4, the synthesis rate was highest with Magnolia leaf
broth. Only 11 min was required for more than 90%
conversion at 95 °C. Although rapid synthesis of silver
nanoparticles within 5 min was recently reported using
culture supernatants of Enterobacteria [13], the silver
nanoparticles synthesized were unstable after 5 min. Using
plant extracts for nanoparticles synthesis is another
advantage over using bacteria because the nanoparticles are
stable for a long time.
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Control of reaction rate and particle size
We further investigated the possibility of controlling the
reaction rate and particle size by changing the composition
of the reaction mixture. Figure 5 shows the time courses of
silver nanoparticles formation with different Magnolia leaf
Bioprocess Biosyst Eng (2009) 32:79–84
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100
100
60
60
Diopyros leaf broth
Magnolia leaf broth
Ginkgo leaf broth
Platanus leaf broth
Pinus leaf broth
40
20
40
20
0
100
40
0.1 mM AgNO3
1 mM AgNO3
20
50
150
100
200
250
0
0
Fig. 4 Time courses of silver nanoparticles formation obtained with
1 mM AgNO3 and 5% various plant leaf extracts at 95 °C
broth concentrations at 5–50% and 1 mM AgNO3. The
reaction rate was highest at 20% leaf broth concentration,
but similar reaction rates were obtained with more than
10% leaf broth concentrations. The inset in Fig. 5 shows
that the average particle size increases with increasing the
leaf broth concentration. Sub-micro scale particles between
100 and 800 nm were obtained with high concentrations of
leaf broth more than 10%, suggesting that too many
reducing agents cause aggregation of the silver particles
synthesized possibly due to the interactions between capping molecules bound to the surface of particles and
secondary reduction process on the surface of the preformed nuclei.
Figure 6 shows the effect of AgNO3 concentration on
conversion and particle size with 5% Magnolia leaf broth.
The times required for more than 90% conversion were less
than 11 min with 0.1 and 1 mM AgNO3 concentration.
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Leaf Broth 5%
Leaf Broth 10%
Leaf Broth 20%
Leaf Broth 50%
Particle Size (nm)
80
60
40
20
800
5
10
60
600
40
400
200
0
0
80
Conversion (%)
100
100
20
0
10
15
20 30 40
Leaf Broth (%)
20
50
0
25
Time (min)
Fig. 5 Time courses of silver nanoparticles formation obtained with
1 mM AgNO3 and various concentrations of Magnolia kobus leaf
broth at 95 °C. Inset effect of leaf broth concentration on silver
particle size
20
40
80
60
40
20
0
2 mM AgNO3
300
Time (min)
Ag Concentration (mg/L)
60
0
0
0
80
Particle Size (nm)
80
80
Conversion (%)
100
Conversion (%)
Ag Concentration (mg/L)
120
60
0.0
0.5
1.0
1.5
2.0
AgNO3 Concentration (mM)
80
100
120
Time (min)
Fig. 6 Time courses of silver nanoparticles formation obtained with
5% Magnolia kobus leaf broth and various concentrations of AgNO3
at 95 °C. Inset effect of AgNO3 concentration on silver particle size
With 2 mM AgNO3, about 90 min was required for 90%
conversion, but the final conversion reached almost 100%.
The average particle size decreased with increasing the
AgNO3 concentration. The reason of decrease in particle
size with AgNO3 concentration is not clear at this point. It
is considered that particle size and shape are dependent on
various conditions such as plant type, nanoparticle type,
reaction temperature and composition.
Currently, the mechanism of biological nanoparticles
synthesis is not fully understood. For gold nanoparticles
synthesized extracellularly by the fungus Fusarium oxysporum, it was reported that the reduction occurs due to
NADH-dependent reductase released into the solution
[14]. With Neem leaf broth, it was reported that terpenoids are believed to be the surface active molecules
stabilizing the nanoparticles and reaction of the metal ions
is possibly facilitated by reducing sugars and/or terpenoids present in the Neem leaf broth [10]. Recent results
with Capsicum annuum L. extract indicated that the proteins which have amine groups played a reducing and
controlling role during the formation of silver nanoparticles in the solutions, and that the secondary structure of
the proteins changed after reaction with silver ions [15].
More elaborate studies are required to elucidate the
mechanism of biological nanoparticles synthesis. In conclusion, an environmentally friendly method using plant
extracts was proposed to synthesize silver nanoparticles.
Only 11 min was required for over 90% conversion by
using Magnolia leaf broth and increasing the reaction
temperature to 95 °C, which was faster or comparable to
the synthesis rate of chemical methods. The average
particle size could be controlled from 15 to 500 nm by
changing the reaction temperature, leaf broth concentration and AgNO3 concentration.
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