International Journal of Nano and Material Sciences, 2015, 4(1): 14-23

International Journal of Nano and Material Sciences ISSN: 2166-0182

Journal homepage:www.ModernScientificPress.com/Journals/ijnanos.aspx Florida, USA
Article
Phytosynthesis and Characterization of Gold Nanoparticles
using Callus of Jatropha curcas
Demissie A. G. * and Lele S.S.

Food Engineering and Technology Department, Institute of Chemical Technology (Deemed

University), Matunga, Mumbai, 400 019, India

* Author to whom correspondence should be addressed; Email: abebetse@yahoo.com,

abebe.demissie@amu.edu.et

Article history: Received 20 November 2014, Received in revised form 5 January 2015, Accepted 20
January 2015, Published 4 March January 2015.

Abstract: The present study reports a rapid plant-based biosynthesis of gold nanoparticles
using aqueous callus extract of Jatropha curcas L. The particle size analysis was carried
out using Zetasizer, SEM, TEM. The physicochemical properties were monitored using
UV–vis spectroscopic, IR, XRD and DSC. Formation gold nanoparticle was confirmed by
using UV–Vis spectrophotometer and absorbance peaks at 540nm were recorded. The gold
nanoparticle was found to be a negatively charged and sized between 2 to 50nm. The
morphology of the nanoparticle is of heterogeneous type in which the triangular is more
prominent at a neutral pH. The physicochemical study using DSC and XRD indicated
significant stability and crystalline nature of the nanoparticle. This phytosynthesis of gold
nanoparticles using plant tissue culture technology is a simple, cheap and eco-friendly than
other mechanical and physic-chemical approaches.

Keywords: Gold nanoparticle. Growth kinetics. In vitro selection .Jatropha curcas L.

Plant Growth Regulation, Suspension culture.

1. Introduction

Biosynthesis of important biomolecules through plant tissue culture has gained public attention
for the last couple of years. Of which callus, cell suspension and root/hairy root cultures proved to be

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Int. J. Nano & Matl. Sci. 2015, 4(1): 14-23 15

the alternate elements for production of these secondary metabolites. Shikonin (Curtin, 1984),
rosamiric acid (Karamet al. 2003), tropane alkaloid (Khanamet al. 2000), lithospermic acid B and
rosamiric acid (Morimoto et al. 1994), taxane, ginseng saponin and polysaccharide (Zhong and Wang,
1996) are some of the benefits of plant biotechnology.
However, so far there is no report on the synthesis of precious metal-based nanoparticle using
In vitro grown plant material. Nanoparticles of noble metals like gold, silver, and platinum have been
synthesized using a wide variety of methods such as bioreduction, hard template, and solution phase
syntheses (Canizal, et al., 2001). These nanoparticles have been mainly produced through chemical
synthesis schemes or a combination of various bio-chemical approaches. Neither chemicalnor bio-
chemical synthesis approaches are cost effective and eco-friendly, sometimes poses detrimental effects
to the living beings.
Various reports indicated that the rates of nanoparticle synthesis using intact plant extracts are
advantageous overother chemical methods (Shankaret al. 2004). Moreover, synthesis of such
nanoparticles using microorganisms or plants can be more environmentally friendly and
biocompatible. The biosynthesis of gold nanoparticles using microorganisms have been reported
elsewhere (Mandal, et al. 2006). Phytosynthesis of gold nanoparticles using plant leaf extracts has also
been reported (Gardea-Torresdey et al. 2002; Narayanan and Sakthivel, 2008; Song, et al. 2009).The
reports have shown that plant material based synthesis of nanoparticle is more stable and long lasting
than the microbial ones. However, there is no or limited report on the biosynthesis of nano-particle
through plant tissue culture.
The lack of complete reports on phytosynthesis of gold nanoparticle through plant cell tissue
culture technology, the remarkable importance of the gold nanoparticle and the significant increase in
the genotypic variations of Jatropha curcas interested us to pursue this study using in vitro grown
plant material. Keeping in view the applications, the biosynthesis route and role of the aforementioned
peptides for the synthesis of nanoparticles we carried out green phytosynthesis approach to synthesize
gold nanoparticle from Jatrophacurcas L. cell culture biomass. The size, shape and other
physicochemical properties of the nanoparticle was carried out using various spectroscopy and
microscopy facility like FTIR, DSC, XRD, TEM, SEM, Zetsizer and UV–vis spectroscopic studies.

2. Materials and Methods

2.1. Establishment of Aseptic Seedlings and Callus Initiation

Seeds of Jatropha curcas L. was washed in running tap water for 10 min to remove any
adherent particles and kept overnight immersed in distilled water (dH2O). The husk then carefully
removed from the kernel using scissor, gently washed and immersed back in dH2O containing a few

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Int. J. Nano & Matl. Sci. 2015, 4(1): 14-23 16

drops of levermed and Tween 20 for 5 min each. Thereafter, the seed was rinsed twice with dH2O and
ready for aseptic operation in a laminar hood. Therein, the seed was surface sterilized in 75% ethanol
for 3-5 min and 4% sodium hypochlorite for 18-20 min consecutively followed by three times dH2O
wash to remove all traces of sterilant. The sterilized seeds were transferred to petri dish and blotted on
sterile filter paper. The cotyledon leaf disc were excised and inoculated onto the respective growth
medium for germination. After 15-days the first emerging leaflets were excised from aseptic seedlings
and used as explants for callus induction and the culture were incubated in 16/8hrs photoperiods for
one month.
The callus initiated was harvested after 21 days of the culture time and taken for preparation of
gold nanoparticles. 10g of fresh callus tissue were minced using grinder and boiled in100ml of dH2O
for 3hrs. Thereafter, the extract was filtrated and centrifuged for further experiments. All the aqueous
solutions were prepared using millipore distilled deionized water. HAuCl3 (auric chloride) salt
analytical grade was purchased from Hi media.

2.2. Phytosynthesis Pattern of Gold Nanoparticles (AuNPs)

In a typical reaction procedure, 5ml of callus extract was added to 20ml of 1mM aqueous gold
solution, the mixture was heated at different temperature and pH as a function of time. The color
intensity was monitored using U-vis spectrophotometer at 450nm. Likewise, the effect of temperature,
pH, concentration of the metal solution and the crude extract were also carried out.

2.3. Morphological Characterization of AuNPs

The AuNP was characterized for their morphology and size using a Hitachi S-3400N scanning
electron microscope (SEM), transmission electron microscope (TEM) and Zetasizer. For SEM imaging
3-5mg dried AuNP sample was placed onto carbon tape mounted on an aluminum stub, and then the
images were taken at the desired magnification. For TEM analysis, around 20-50μL gold
nanosuspension sample was taken and dried thereafter kept on carbon-coated copper grids. Further
electrokinetic property and size distribution of the gold nanosuspensionwas also measured using
Zetasizer (NANO ZS, MALVERN, USA). Aliquot of the nanosuspension was sampled in cuvettes and
gold nanosuspension was then examined for equivalent charge and size distribution.

2.4. The Physicochemical Characterization of AuNPs

The physicochemical property of AuNP was carried out using Differential scanning calorimetry
(DSC, TA Instruments, model 2970) and FTIR. 4-6 mg of the sample was taken for analysis and sealed
into an aluminum pan and placed in the DSC cell under a nitrogen purge to determine the thermal
property. Dynamic scans were made on the samples at a heating rate of 5 ºC/min, from 30 to 300 ºC.

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Int. J. Nano & Matl. Sci. 2015, 4(1): 14-23 17

Thereafter, the sample was cooled to back to 30C0at a cooling rate of 5 ºC/min and the thermal
property of the AuNP was recorded. The possible functional group of the AuNP was determined using
FTIR. The measurements were carried out on a Perkin-Elmer Spectrum-One instrument in KBr pellets.
X-ray diffraction (XRD) measurement of the marine sponge reduced AuNP was carried out on films of
the respective solutions drop coated onto glass substrates on an Enraf Nonius CAD4-MV31 single
crystal X-ray diffractometer instrument operating at a voltage of 50 kV and a current of 40mA with Cu
K radiations.

2.5. Statistical Analysis

The particle size of nanoparticles obtained from the analysis was subjected to One-way
Analysis of Variance (ANOVA) to determine the significance of individual differences at p < 0.05
level. All the data analysis was done using Statgraphics.

3. Results and Discussion

3.1. Phytosynthesis Kinetics Pattern of AuNPs

The phytosynthesis kinetics profile of AuNPwas evaluated on the base of temperature, pH and
concentration of metal ion as a function of time as shown in fig 1. Various pH including 4, 6, 7, 8 and
10 were selected and monitored at room temperature (approximately 30 + 20C) in our study. As shows
in fig. 1, biosynthesis of the AuNP from the callus of Jatropha curcas as a function of time is pH and
temperature dependent. At pH 4 the synthesized AuNP was found to be formed in less than 5 min and
get precipitated thereafter. This might because the biomolecules involved at that particular pH get
denatured or losses its stability. pH=7 found to be the best for the biosynthesis of AuNP as well. From
our study we confirmed that the suitable pH range for the biosynthesis of the AuNP was alkaline. The
biosynthesis of the AuNP was even very fast (within 5-10min of the reaction time).

Fig. 1. Effect of pH on biosynthesis of AuNP as a function of time at room temperature.

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Int. J. Nano & Matl. Sci. 2015, 4(1): 14-23 18

3.2. Morphological Characterization of AuNPs

The morphological characterization of AuNP was carried out. Hence, the size of the AuNP
produced found to be between 2 to 50nm and their morphologically heterogeneous. The size and shape
of this kind of AuNP might be controlled maintaining optimum pH and temperature. The size and
morphological characteristics was determined using Zetasizer (Fig. 2),TEM ( Fig. 3.) and SEM(Fig.
4.).

A) B)

Fig.2. A) Zeta potential B) particle size measurement of the Au nanosuspension from Jatropha curcas

Fig. 3. TEM micrograph of AuNP by Jatropha curcas callus extract

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Int. J. Nano & Matl. Sci. 2015, 4(1): 14-23 19

Fig.4. SEM micrograph of AuNP by Jatropha curcas callus extract

3.3. The Physicochemical Characterization of AuNPs

UV-Vis spectroscopy was used to monitor the synthesis reaction of AuNP (Fig.5.). Infrared
spectroscopy was also used for chemical characterization of the AuNP (Fig. 6). The nature of the
biomolecules involved in the reduction and formation of gold nanoparticles was studied using FTIR
I. Uvisthe
analysis of AuNP formed. In addition DSC may be used to determine spectrum of GNP
thermal property of AuNP
(Fig. 7).

A B

 The pink c
and HAuCl4
@538nm
showed a str

 Characteri

 Synthesize
auric chlorid
volume of aq

Fig.5. UV–Vis absorption spectra of AuNP: A) AuNP formed by bio-reductionB) auric chloride
solution *A: Gold nanoparticle formed by reduction B: HAuCl4 solution

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Int. J. Nano & Matl. Sci. 2015, 4(1): 14-23 20

II. FTIR spectra of GNP

Fig. 6. FT–IR spectrum of AuNP formed using Jatropha curcascallus

*The strong absorption at 1733.89cm−1 is due car- bonyl stretching vibration of the acid
groups present in the extract. The bands at 1585.38 is the typical characteristic of amide band

Fig. 7. DSC spectrum of AuNP synthesized

Using FTIR, In order to determine the functional groups, the dried test sample was blended
with KBr to obtain a pellet. The strong IR absorption at 1733.89cm−1 is due —C═O stretching
vibration present in the amide linkages of the proteins. The band at 1585.38 is the typical characteristic
of amide band stretch vibrations present in the amide linkages of the proteins. The UV spectrum also
showed a strong peak at 540nm. This confirms further the phytosynthesis of stable gold nanoparticle.
Dried samples of 5-6mg were taken for DSC measurements. The AuNP indicated a
characteristic endotherm chemical condition. The melting temperature and the heat capacity were
found to be 139.14oC and -381.5mJ. The change in such physical parameter observed from the
nanoparticle also affirms its crystalline nature.
Biosynthesis of gold nanoparticle using microorganism and plant parts has been extensively
reviewed (Mohanpuria et al., 2008).Synthesis of AuNP from Coreander leaf has been reported
(Narayanan, and Sakthivel, 2008) and however production requires half a day to complete. On the

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Int. J. Nano & Matl. Sci. 2015, 4(1): 14-23 21

other hand in our study the phyto-reduction found to be completed within 10min. In addition the
phyto-synthesis of nano-particle in general gold in particular has not yet been reported using such plant
biotechnology approach.
Further studies were conducted using X-ray diffraction to confirm the crystalline nature of the
particles. The XRD pattern obtained is shown in Fig. 8. The XRD pattern shows four intense peaks in
the whole spectrum of 2θ values from 30 to 50. This helps to know the nature of the AuNP formed
using the phyto-extract. XRD spectra of pure crystalline gold structures was evidenced by the peaks at
2θ values of 38° and 44°, corresponding to 111 and 200 planes for gold, respectively. This also
confirmed the crystalline nature of AuNP. The mean particles diameter of AuNP was calculated from
the XRD pattern. The calculated average particles size of AuNP was found to be 18.15 nm. The
particle sizes obtained from XRD line broadening agreed well with that obtained from other
microscopy results like TEM and Zeta potential ( Fig. 2-4).

Fig. 8. XRD patterns of AuNP synthesized by treating J. curcas extract with HAuCl3 aqueous solution.

4. Conclusion

We reported phytosynthesis and characterization of AuNP using Jatropha curcas callus herein.
The phytosynthesis of AuNP using callus extract provides a simple, scalable and efficient route. These
results showed that the plant cell could be explored further for possible nanoparticle phytosynthesis.
The compound was characterized using spectrometric facilities such as SEM, TEM, Zetasizer, FTIR,
DSC, XRD and UV spectrometer. Various spectrometric and microscopic studies conformed that the
nanoparticle can successfully synthesized using plant cells. The AuNP formation was rapid at pH 7
and temperature 1000C. The result confirmed that the average size of AuNPis less than 50nm.

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Int. J. Nano & Matl. Sci. 2015, 4(1): 14-23 22

Skewness using particle sizer and TEM showed that the nanoparticles are significantly dispersed and
of reasonable size. In addition, the nanoparticle can be further exploited for its biotechnological
applications in medicine and agriculture.

Acknowledgment

The authors are grateful for The World Academy of Science (TWAS) Trieste, Italy and
Department of Biotechnology (DBT), India, for their financial support. This study is undertaken in the
Institute of Chemical Technology (DeemedUniversity), Food Engineering and Technology
Department.

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