Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 102 (2013) 15–23

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Spectrochimica Acta Part A: Molecular and

Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa

Green synthesis of gold nanoparticles using Citrus fruits (Citrus limon, Citrus
reticulata and Citrus sinensis) aqueous extract and its characterization
Mohanan V. Sujitha, Soundarapandian Kannan ⇑
Proteomics and Molecular Cell Physiology Lab, Department of Zoology, School of Life Sciences, Bharathiar University, Coimbatore 641 046, TN, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

" The biological synthesis of Au Nps

using citrus fruits juice extract as the
reducing and stabilizing agent.
" The size and shape of bio-
synthesized Au NPs depends on the
concentrations of the extract.
" The size of Au Nps varies in the order
of techniques DLS > TEM > XRD.

a r t i c l e i n f o a b s t r a c t

Article history: This study reports the biological synthesis of gold nanoparticles by the reduction of HAuCl4 by using
Received 23 July 2012 citrus fruits (Citrus limon, Citrus reticulata and Citrus sinensis) juice extract as the reducing and stabilizing
Received in revised form 28 August 2012 agent. A various shape and size of gold nanoparticles were formed when the ratio of the reactants were
Accepted 20 September 2012
altered with respect to 1.0 mM chloroauric acid solution. The gold nanoparticles obtained were charac-
Available online 28 September 2012
terized by UV–visible spectra, transmission electron microscopy (TEM) and X-ray diffraction (XRD).
TEM studies showed the particles to be of various shapes and sizes and particle size ranges from 15 to
Keywords:
80 nm. Selected-area electron diffraction (SAED) pattern confirmed fcc phase and crystallinity of the
Gold nanoparticles
Biological synthesis
particles. The X-ray diffraction analysis revealed the distinctive facets (1 1 1, 2 0 0, 2 2 0 and 2 2 2 planes)
Citrus fruits of gold nanoparticles. Dynamic light scattering (DLS) studies revealed that the average size for colloid
Surface plasmon resonance gp3 of C. limon, C. reticulata and C. sinensis are 32.2 nm, 43.4 nm and 56.7 nm respectively. The DLS graph
Transmission electron microscopy (TEM) showed that the particles size was larger and more polydispersed compared to the one observed by TEM
due to the fact that the measured size also includes the bio-organic compounds enveloping the core of the
Au NPs. Zeta potential value for gold nanoparticles obtained from colloid gp3 of C. limon, C. reticulata
and C. sinensis are 45.9, 37.9 and 31.4 respectively indicating the stability of the synthesized
nanoparticles. Herein we propose a novel, previously unexploited method for the biological syntheses
of polymorphic gold nanoparticles with potent biological applications.
Ó 2012 Elsevier B.V. All rights reserved.

Introduction and chemotherapy in recent period. Particularly for drug delivery,

Nanomaterials have achieved significant consideration due to
their potential applications in drug delivery, sensing, imaging
⇑ Corresponding author. Tel.: +91 422 2428310; fax: +91 422 2425706.
E-mail address: sk_protein@buc.edu.in (S. Kannan).
polymeric nanoparticles, dendrimers, liposomes and metal nano-
particles are being widely explored. Nanoparticles have a unique
optical and electronic property that greatly depends on the size
and shape of nanoparticles as an effect of quantum confinement
of electrons [1–3]. Metal is a poor catalyst in bulk form; however
nanometer-sized particles can exhibit excellent catalytic activity
owing to their relative high surface area-to-volume ratio and their

1386-1425/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.saa.2012.09.042
16 M.V. Sujitha, S. Kannan / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 102 (2013) 15–23
Fig. 1. UV–vis spectra of different colloidal gold solutions reduced with different quantity of Citrus fruit extract: (a) Citrus limon, (b) Citrus reticulata and (c) Citrus sinensis.
interface-dominated properties, which significantly differ from
those of the bulk material [4].
Gold nanoparticles have been considered important area of
research due to their unique and tunable surface plasmon reso-
nance (SPR) and have advantages in clinical applications over other
metallic particles in terms of biocompatibility and non-cytotoxic-
ity [5]. These can be easily synthesized and display high chemical
as well as thermal stability [6]. Many efforts have been made to
utilize AuNPs as multifunctional gene carriers owing to their excel-
lent biocompatibility, well-defined surface chemistry, and facile
molecular imaging using fluorescence resonance energy transfer
(FRET) [7–9]. Consequently, it is of significant importance to devel-
op new procedures for synthesis that are not only simple and fast,
but also environmentally benign.
Biosynthetic processes have received much attention as a viable
alternative for the development of metal nanoparticles where
plant extract is used for the synthesis of nanoparticles without
any chemical ingredients [10–14]. Recently there have been a
number of reports on the synthesis of Au nanoparticles of various
shapes using plants [15–18,12,19]. Plant mediated synthesis of
nanoparticles is more advantageous than the other biological pro-
cesses because it eliminates the elaborate process of maintaining
microbial cultures and can also be suitably scaled up for large scale
nanoparticle synthesis. Biological synthesis would have greater
commercial viability if the nanoparticles could be synthesized
more rapidly in the reaction vessels.
To the best of our knowledge, the use of naturally available
citrus fruit extract has not been investigated so far, for the biosyn-
theses of gold nanoparticles. Citrus limon, Citrus reticulata and Citrus
sinensis are a classical example of such abundantly available natu-
ral fruits. It is reported that Citrus fruit extract is composed of a
variety of phenolic acid, phenolic esters, flavonoids, triterpenoids,
thiamine and mangiferin. The rich source of citric acid and ascorbic
acid in the citrus fruit extract may possibly responsible for reduc-
tion of metal ions and efficient stabilization of synthesized nano-
particles. Since citrus fruits are a rich source of citric acid, C.
limon, C. reticulata and Cirtus sinensis were used as a bioreductant
in the present study for the synthesis of gold nanoparticles. The
gold nanoparticles were characterized using UV–visible spectrom-
etry, Transmission electron microscope (TEM) and X-ray diffrac-
tion (XRD) techniques.
Materials and methods
Materials
Hydrogen tetracholoroaureate (III) hydrate HAuCl4
3H2O
(99.9%) was procured from Sigma Aldrich. The citrus fruits (C. li-
mon, C. reticulata and C. sinensis) were purchased from Nilgiri
district.
Methods
Preparation of the extract
C. limon, C. reticulata and C. sinensis were squeezed to extract the
juice which was later strained through a fine pore nylon mesh. The
 M.V. Sujitha, S. Kannan / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 102 (2013) 15–23 17

juice obtained was centrifuged at 10,000 rpm for 10 min to remove
any undesired impurities. This juice was used for further experi-
ments. This solution was considered as 100% extract and other con-
centrations of the extract were prepared using this [20].
Synthesis of gold nanoparticles
All glasswares were cleaned with an aqua regia solution (1:3 -
nitric acid/hydrochloric acid) for complete removal of potential
artificial nucleation sites. Briefly, 1 mM of 50 mL solution of hydro-
gen tetrachloroaurate trihydrate (HAuCl4
3H2O) was brought to a
boil with vigorous stirring. To this solution, varying quantity
(1 mL, 2 mL, 3 mL, 4 mL, 5 mL and 6 mL) of C. limon, C. reticulata
and C. sinensis was added and the hot plate was turned off. A color
change was observed from colorless to purple to ruby red within
10 min. The colloidal solution was stirred for an additional
20 min, cooled at room temperature, transferred to an amber
bottle and stored at room temperature. AuNPs exhibit surface
plasmon resonance at 530–550 nm. The gold 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.
reduction of gold ions to the nanoparticle form was monitored
by measuring the UV–Visible spectra of the solutions after diluting
the sample with deionized water. The UV–visible spectra were re-
corded on a Shimadzu UV-1601 spectrophotometer with samples
in quartz cuvette operated at a resolution of 1 nm from 400 to
700 nm. Deionized water was used as blank. The spectra recorded
were then re-plotted using Origin 6.0 version.
Transmission electron microscopy. The shape and size of the parti-
cles were measured with high resolution transmission electron
microscopy (HRTEM) using JEOL 3010 equipped with selected area
electron diffraction pattern (SAED). For doing TEM measurement, a
drop of the synthesized colloidal sample is deposited on a carbon
coated copper grid followed by solvent evaporation under vacuum.
X-ray diffraction. Gold colloids were centrifuged (14,000 rpm,
25 °C) for 10 min, washed several times with distilled water, and
were then freeze dried and subjected to XRD analysis. The XRD
studies was performed with XPERTTVPVR-7130 (Philips), Cu Ka
radiation (k = 1.54 nm) in the 2h range of 30–80 operate data
voltage of 40 kV and a current of 30 mA.

Characterization Zeta potential measurement. The zeta potential of the synthesized

UV–Visible spectroscopy. Preliminary characterization of the gold nanoparticles was determined by means of zeta potential analyzer
nanoparticles was carried out using UV–Visible spectroscopy. The (90 Plus Particle Size Analyzer, Brookhaven Instruments

Fig. 2. (A) TEM image of Au colloids synthesized using Citrus limon: [a] (gp1) 1.0 mL, [b] (gp3) 3.0 mL and [c] (gp5) 5.0 mL and SAED pattern of Au colloids [a1] (gp1) 1.0 mL,
[b1] (gp3) 3.0 mL and [c1] (gp5) 5.0 mL. (B) TEM image of Au colloids synthesized using Citrus reticulata: [a] (gp1) 1.0 mL, [b] (gp3) 3.0 mL and [c] (gp5) 5.0 mL and SAED pattern
of Au colloids [a1] (gp1) 1.0 mL, [b1] (gp3) 3.0 mL and [c1] (gp5) 5.0 mL. (C) TEM image of Au colloids synthesized using Citrus sinensis: [a] (gp1) 1.0 mL, [b] (gp3) 3.0 mL and [c]
(gp5) 5.0 mL and SAED pattern of Au colloids [a1] (gp1) 1.0 mL, [b1] (gp3) 3.0 mL and [c1] (gp5) 5.0 mL.
18 M.V. Sujitha, S. Kannan / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 102 (2013) 15–23
Fig. 2. (continued)
Corporation, using Zeta Plus software). The measurement of zeta
potential is based on the direction and velocity of particles under
the influence of known electric field.
Particle size analysis. The particle size range of the nanoparticles
along with its polydispersity was determined using a particle size
analyzer (90 Plus Particle Size Analyzer, Brookhaven Instruments
Corporation). Particle size was arrived based on measuring the
time dependent fluctuation of scattering of laser light by the nano-
particles undergoing Brownian motion.
Results and discussion
Visual observations and UV–vis spectroscopy
UV–vis spectroscopy is one of the important techniques to
determine the formation and stability of metal nanoparticles in
aqueous solution. Colloidal solutions of Au nanoparticles show a
very intense color, which is absent in the bulk material as well as
for individual atoms. Their origin is attributed to the collective
oscillation of free conduction electrons induced by an interacting
electromagnetic field. These resonances are called surface plasmon
resonances [21]. Fig. 1a shows the UV–vis spectra of gold nanopar-
ticle formation at constant concentration of HAuCl4 (1 mM) with
varying quantity of C. limon fruit extract of (gp1) 1.0 mL, (gp2)
2.0 mL, (gp3) 3.0 mL, (gp4) 4.0 mL, (gp5) 5.0 mL and (gp6) 6.0 mL
aqueous medium. The gradual color change from pink to ruby
red to blue color was observed during reaction with varying quan-
tity of C. limon fruit extract which are characteristic of the Surface
Plasmon Resonance (SPR) of different size of gold nanoparticles in
solution. Gold nanoparticles are the aggregates of Au atoms at
nanoscale, which are red in color because of the absorption by their
surface plasmon oscillation that peaks at 520 nm [22]. Therefore,
the light scattering peak of gold nanoparticles at 556 nm can be
characterized as the Resonance Light Scattering (RLS) peak. When
gold nanoparticles aggregate and the interparticle distance in these
aggregates decreases to less than approximately the average parti-
cle diameter, the color of the aggregates turns blue, which results
in the shift of absorption band to longer wavelengths, because of
electric dipole–dipole interaction and coupling between the plas-
mons of neighboring particles in the formed aggregates [23].
Initially, the maximal plasmon absorption peaks of gold nano-
particles showed broaden in bandwidth of the peak with the de-
crease of C. limon fruit quantity [(gp1) 1.0 mL and (gp2) 2.0 mL],
followed by a blue shift and turned narrow in width with the in-
crease of C. limon fruit quantity [(gp3) 3.0 mL, (gp4) 4.0 mL and
(gp5) 5.0 mL] and then showed a red shift and broaden in width
with the further increase of C. limon fruit quantity [(gp6) 6.0 mL].
Only for (gp5) 5.0 mL, the maximum plasmon absorption
wavelength (kmax) of gold nanoparticles was obtained for the least
value (530 nm) without shifting to NIR (Near Infrared Region) re-
gion. The broad SPR at lower quantities of the extract is due to
the formation of large anisotropic particles. At lower quantities
of the extract, nanoparticle syntheses are not greatly favored due
to the absence of sufficient biomolecules responsible for capping
and efficient stabilization. The fairly sharp SPR band observed for
colloid (gp5) at 530 is indicative of spherical nanoparticles.
 M.V. Sujitha, S. Kannan / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 102 (2013) 15–23 19

Fig. 2. (continued)

Similar results were obtained for the gold nanoparticles biosyn-

thesized with C. reticulata and C. sinensis fruit extract. Fig. 1b shows
the UV–vis spectra of gold nanoparticle formation at constant con-
centration of HAuCl4 (1  10 3 M) with varying quantity of C. retic-
ulata fruit extract of (gp1) 1.0 mL, (gp2) 2.0 mL, (gp3) 3.0 mL, (gp4)
4.0 mL, (gp5) 5.0 mL and (gp6) 6.0 mL aqueous medium. Fig. 1c
shows the UV–vis spectra of gold nanoparticle formation at con-
stant concentration of HAuCl4 (1  10 3 M) with varying quantity
of C. sinensis fruit extract of (gp1) 1.0 mL, (gp2) 2.0 mL, (gp3) 3.0 mL,
(gp4) 4.0 mL, (gp5) 5.0 mL and (gp6) 6.0 mL aqueous medium. It is
interesting to note that in Fig. 1b, only for (gp5) 5.0 mL and (gp6)
6.0 mL the maximum plasmon absorption wavelength (kmax)
of gold nanoparticles was obtained for the least value (536 nm)
without another absorption band in the longitudinal plasmon
resonance.
Moreover, Absorption spectra of gold nanoparticles synthesized
with C. reticulata fruit extract of (gp1) 1.0 mL, (gp2) 2.0 mL, (gp3)
3.0 mL and (gp4) 4.0 mL showed another absorption band ranged
from 625 to 725 nm, which belongs to the longitudinal plasmon
resonance band of aggregates or deviation from spherical geometry
of gold nanoparticles [24]. Mie’s theory is often used to explain the
deviation from spherical geometry of the particles, suggesting in
the circumstance that the transverse and longitudinal dipole polar-
izability no longer produces equivalent resonance, leads to a
broadening and red shift of longitudinal plasmon resonance as well
as an appearance of transverse plasmon resonance [25,26]. For
Fig. 1c only for (gp6) 6.0 mL, the maximum plasmon absorption
wavelength (k max) of gold nanoparticles was obtained for the
least value (536 nm) without shifting to NIR region. Moreover
absorption spectra of gold nanoparticles synthesized with
C. sinensis fruit extract of (gp1) 1.0 mL, (gp3) 3.0 mL and (gp4)
4.0 mL and (gp5) 5.0 mL shifted to longer wave length region.
All the results indicated that the size and shape of gold nano-
particles were altered with C. limon, C. reticulata and C. sinensis fruit
extract quantity, which was further confirmed by TEM analysis.
The presence of nanoprism like particles again supports the
observed additional bands in the NIR region of the absorption spec-
trum indicating the formation of anisotropic particles. Haiss et al.
[27] reported the dependence of optical properties of spherical
gold nanoparticles on particle size and wavelength which was ana-
lyzed theoretically using multipole scattering theory, where the
complex refractive index of gold was corrected for the effect of a
reduced mean free path of the conduction electrons in small parti-
cles. Moreover the author showed both size and concentration of
gold nanoparticles can be determined directly from UV–vis spectra.
Transmission electron microscope (TEM) analysis
The results obtained are further corroborated by transmission
electron microscopic (TEM) observations, as shown in Fig. 2A–C.
From the figure it can be seen that the morphology consists of a
mixture of prism and spherical like particles. The presence of
nanoprism like particles again supports the observed additional
bands in the longer wave length region of the absorption spectrum
indicating the formation of anisotropic particles. The TEM observa-
tions clearly confirm that the Au NPs obtained at gp5 Fig. 2A[c], B[c]
and C[c] are smaller than those obtained at gp1 Fig. 2A[a], B[a] and
C[a] and gp3 Fig. 2A[b], B[b] and C[b]. The particle diameters ob-
served for various quantity of extract are as follows: 15 ± 20 nm
20 M.V. Sujitha, S. Kannan / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 102 (2013) 15–23
Fig. 3. X-ray diffraction pattern recorded from drop-coated films of the colloid gp3 deposited on glass substrates. The Bragg reflections are identified in the XRD pattern: (a)
Citrus limon, (b) Citrus reticulata and (c) Citrus sinensis.
at 5 mL, 17 ± 50 nm at 3 mL, and 18 ± 60 nm at 1 mL. With the
decrease of quantity of extract, the average size of gold nanoparti-
cles increased, but not significantly, meanwhile the shape of gold
nanoparticles changed obviously. Gold nanoparticles obtained at
the 1 mL seems to be aggregated as shown in (Fig. 2A[a], B[a]
and C[a], ensuing that the maximum absorption band shifted to
longer wavelength than that at 5 mL. For 5 mL, the obtained gold
nanoparticles exhibited improved dispersibility; and near spheri-
cal in shape, as shown in Fig. 2A[c], B[c] and C[c]. These results
were also in accordance with absorption spectra shown in
Fig. 1a–c. It indicated the occurrence of two plasmon resonance
bands due to the decrease of quantity of extract mainly attributed
to the appearance of anisometric gold nanoparticles. Sintering of
gold nanoparticles and their adherence to the nanotriangle in col-
loid is evident from Fig. 2A[a1], B[a1] and C[a1]. The blunt angled
nanotriangle is a result of the shrinking process arising from the
minimization of surface energy. The presence of large quantity of
extract causes strong interaction between protective biomolecules
and surfaces of nanoparticles preventing nascent gold nanocrystals
from sintering. With larger quantities of extract, the interaction is
increased, leading to size reduction of spherical nanoparticles.
Typical selected area electron diffraction (SAED) pattern
Fig. 2A[a1, b1 and c1], B[a1, b1 and c1] and C[a1, b1 and c1]) with
bright circular rings corresponding to the (1 1 1), (2 0 0), (2 2 0) and
(3 1 1) planes reveals that the particles are crystalline in nature. The
spots are indexed according to atomic planes from the gold face-
centered cubic (fcc) lattice. The hexagonal nature of the diffraction
spots is a clear indication that the Au nanoprisms are highly (1 1 1)
oriented with top normal to the electron beam.
X-ray diffraction (XRD) analysis
The crystalline nature of Au nanoparticles was further con-
firmed from X-ray diffraction (XRD) analysis. X-ray diffraction is
used to characterize crystallographic structure, grain size, and pre-
ferred orientation in polycrystalline or powder solid samples. This
is a preferred method of analysis for characterization of unknown
crystalline materials. Compounds are identified by comparing dif-
fraction data against a database of known materials. The results de-
picted in Fig. 3 shows the XRD pattern of gold nanoparticles
obtained from colloid gp3 of C. limon, C. reticulata and C. sinensis.
Gold nanoparticles synthesized from C. limon, showed Bragg Reflec-
tion peaks at 38.2°, 44.3°, 64.7° and 77.7° in the 2h range between
30° and 80° which can be indexed to the (1 1 1), (2 0 0), (2 2 0) and
(3 1 1) planes of face centered cubic (fcc) gold crystal respectively
which have a good match with the standard diffraction pattern
of JCPDS No. 04-0784, revealing that the synthesized gold nanopar-
ticles are composed of pure crystalline gold (Fig. 3a). The peak cor-
responding to (1 1 1) plane is more intense than the other planes,
suggesting that the (1 1 1) plane is in the predominant orientation.
 M.V. Sujitha, S. Kannan / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 102 (2013) 15–23
Fig. 4. Zeta potential analysis to determine the citrus fruits extract on NPS stability: (a) Citrus limon, (b) Citrus reticulata and (c) Citrus sinensis.
Similar results were reported by Ashok et al. [28], Shashi et al. [29],
Wang et al. [30] and Kasthuri et al. [31]. These authors biosynthe-
sized gold nanoparticles using Bananna peel extract, Tansy fruit ex-
tract, gallic acid and Apiin respectively.
In case of gold nanoparticles synthesized from C. reticulata, the
characteristic diffraction peaks were observed at 38.3°, 44.3°, 64.6°
and 76.9° in the 2h range 30–80° which can be indexed to the
(1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes of face centered cubic (fcc)
gold crystal respectively (Fig. 3b) and for C. sinensis, the character-
istic diffraction peaks were observed at 37.5°, 43.9°, 63.9° and 77.7°
in the 2h range 30–80° which can be indexed to the (1 1 1), (2 0 0),
(2 2 0) and (3 1 1) planes of face centered cubic (fcc) gold crystal
respectively (Fig. 3c). The biosynthesized gold nanoparticles pos-
sess a well defined atomic arrangement resembling the fcc struc-
ture occurring with bulk gold.
The peak positions explain about the translational symmetry
namely what is the size and shape of the unit cell whereas the peak
intensities give details about the electron density inside the unit
cell namely where the atoms are located. The peak widths and
shapes describe on deviations from a perfect crystal and make clear
about the crystallite size if it is less than roughly 100–200 nm. The
width of the (1 1 ) peak was employed to calculate the average
crystallite size using Scherrer equation. It is found that the average
21
size is 12 nm, 17 nm and 22 nm for gold nanoparticles ob-
tained from colloid gp3 of C. limon, C. reticulata and C. sinensis
respectively. The XRD pattern thus clearly shows that the Au nano-
particles formed by the reduction of AuCl4-ions by citrus fruit ex-
tract are crystalline in nature.
Effect of Citrus fruits extract on NPs stability
Zeta potential (ZP) values reveal information regarding the sur-
face charge and stability of the synthesized gold nanoparticles. At
different quantity of the citrus fruits extract, there was little vari-
ation in the zeta potential value of Au NPs. However, Au NPs dem-
onstrate lower ZP value at lower concentration of the extract,
whereas higher values were obtained at higher concentration of
the extract. In overall, the results of zeta potential value for gold
nanoparticles obtained from colloid gp3 of C. limon, C. reticulata
and C. sinensis are 45.9, 37.9 and 31.4 (Fig. 4a–c) respectively
indicating the stability of the synthesized nanoparticles. The rich
source of citric acid and ascorbic acid in the citrus fruits extract
may possibly responsible for reduction of metal ions and efficient
stabilization of synthesized nanoparticles.
The carbonyl groups from the citric acid and ascorbic acid may
have a stronger ability to bind metal ion, so that these compounds
22 M.V. Sujitha, S. Kannan / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 102 (2013) 15–23

Fig. 5. DLS measurement to determine the size distribution for colloid gp3 of (a) Citrus limon (b) Citrus reticulata and (c) Citrus sinensi.

could form a coat over the metal nanoparticle to prevent gathering also found same order of particle size observed by DLS, TEM and
of particles. XRD methods as DLS > TEM > XRD. Moreover, the large particle size
could also be due to the various forces of interaction in the solution
like van der Waals forces. The differences in the particle sizes ob-
DLS measurements
tained using the microscopic techniques were due to differences
in sample preparation and also due to the polydispersity of the
The DLS instrument is known to measure the shell thickness of
sample (as determined by DLS). Different techniques give different
a capping or stabilizing agent enveloping the metallic particles
size averages, depending on the instrument response to: particle
along with the actual size of the metallic core. The average size
numbers, volume, mass or optical property. But this is usually
of the Au NPs was also measured by DLS measurement for colloid
not the case for bio-based synthesis process which produces poly-
gp3 of C. limon, C. reticulata and C. sinensis. The size distribution vs.
disperse particles. Therefore, a great degree of caution needs to be
intensity graph has been shown in Fig. 5a–c. The average size for
taken when the particle sizes are measured by various techniques
colloid gp3 of C. limon, C. reticulata and C. sinensis were 32.2 nm,
for a bio-based process and need to be compared to determine
43.4 nm and 56.7 nm respectively. The particles size was larger
their reactivity for specific applications [31].
and more polydispersed compared to the TEM result. The large size
of particles observed by DLS is due to the fact that the measured
size also includes the bio-organic compounds enveloping the core Conclusion
of the Au NPs. Prathna et al. [32] found the particle size of silver
nanoparticles in the order of techniques DLS > AFM > TEM > XRD. In the present work we report a simple and novel method for
These authors stabilized Ag NPs by C. limon fruit extract. We have the biosynthesis of gold nanoparticles by the reduction of aqueous
 M.V. Sujitha, S. Kannan / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 102 (2013) 15–23
AuCl4 ions using Citrus fruits extract (C. limon, C. reticulata and C.
sinensis). The size and shape of gold nanoparticles could be chan-
ged by altering concentration of Citrus fruit extract which were
confirmed by UV–vis spectrometry and TEM analysis. The mor-
phology of the particles synthesized consists of a mixture of gold
nanoprisms and spheres. At lower concentrations of the extract,
different shapes of Au nanoparticles were observed, while at higher
concentrations almost spherical particles are observed. The XRD
study showed that the biosynthesized Au nanoparticles are crystal-
line in nature. The possible reducing agent is citric acid and cap-
ping material responsible for stabilization is proteins present in
citrus fruit extract. Finally, the overall result showed that C. limon
mediated biosynthesis of gold nanoparticle is more efficient fol-
lowed by C. reticulata and C. sinensis. These methods for gold nano-
particles biosynthesis do not use any chemicals and thus has the
potential for exploit in biomedical applications.
References
[1] D.D. Gutierrez, M. Surtchev, E. Eiser, C.J. Elsevier, Nano Lett. 6 (2006) 145–147.
[2] M. Everts, V. Saini, J.L. Leddon, R.J. Kok, M.S. Khalili, M.A. Preuss, C.L. Millican, G.
Perkins, J.M. Brown, H. Bagaria, D.E. Nikles, D.T. Johnson, V.P. Zharov, D.T.
Curiel, Nano Lett. 6 (2006) 587–591.
[3] B.G. Lohmeijer, U.S. Schubert, Angew. Chem. Int. Ed. 41 (2002) 3825–3829.
[4] S. Thangavel, R. Ramaraj, J. Phys. Chem. 112 (2008) 19825.
[5] E.E. Connor, J. Mwamuka, A. Gole, C.J. Murphy, M.D. Wyatt, Small 1 (2005)
325–327.
[6] T. Jennings, G. Strouse, Adv. Exp. Med. Biol. 620 (2007) 34.
[7] Yuhan Lee, Soo Hyeon Lee, Jee Seon Kim, Atsushi Maruyama, Xuesi Chen, Tae
Gwan Park, J. Control. Release. 155 (2011) 3–10.
[8] M.C. Daniel, D. Astruc, Chem. Rev. 104 (2004) 293–346.
[9] I.H. El-Sayed, X. Huang, M.A. El-Sayed, Nano Lett. 5 (2005) 829–834.
23
[10] V. Parashar, R. Parashar, B. Sharma, A.C. Pandey, Digest J. Nanomater. Biostruct.
4 (2009) 45–50.
[11] D. Philip, Spectrochim. Acta A. 73 (2009) 374–381.
[12] S.L. Smitha, D. Philip, K.G. Gopchandran, Spectrochim. Acta A. 74 (2009) 735–
739.
[13] J.Y. Song, Jang H-K, B.S. Kim, Process Biochem. 44 (2009) 1133–1138.
[14] K.N. Thakkar, S. S Mhatre, R.Y. Parikh, Nanomed. Nanotechnol. Biol. Med. 6
(2010) 257–262.
[15] S.P. Chandran, M. Chowdhary, R. Pasricha, A. Ahmad, M. Sastry, Biotechnol.
Prog. 22 (2006) 577–583.
[16] J. Huang, Q. Li, D. Sun, Y. Lu, Y. Su, X. Yang, H. Wang, Y. Wang, W. Shao, N. He, J.
Hong, C. Chen, Nanotechnology 18 (2007) 105104–105114.
[17] K.B. Narayanan, N. Sakthivel, Mater. Lett. 62 (2008) 4588–4590.
[18] P. Mohanpuria, N.K. Rana, S.K. Yadav, J. Nanopart. Res. 10 (2008) 507–517.
[19] D. Philip, Physica E 42 (2010) 1417–1424.
[20] T.C. Prathna, N. Chandrasekaran, Ashok M. Raichur, Amitava Mukherjee,
Colloid Surf. B: Biointerf. 82 (2011) 152–159.
[21] U. Kreibig, M. Vollmer, Optical Properties of Metal Clusters, Springer-Verlag,
Heidelberg, 1995.
[22] K.C. Grabar, R.G. Freeman, M.B. Hommer, M.J. Natan, Anal. Chem. 67 (1995)
735–743.
[23] U. Kreibig, L. Genzel, Surf. Sci. 156 (1985) 678700.
[24] C.S. Weisbecker, M.V. Merritt, G.M. Whitesides, Langmuir 12 (1996) 3763.
[25] G. Mie, Ann. Phys. 25 (1908) 377.
[26] J.A. Creighton, D.G. Eadon, J. Chem. Soc., Faraday Trans. 87 (1991) 3881.
[27] W. Haiss, N. Thanh, J. Aveyard, G. Fernig, Anal. Chem. 79 (2007)
4215–4221.
[28] B. Ashok, J. Bhagyashree, R.K. Ameeta, Z. Smita, Colloid Surf. B: Biointerf. 80
(2010) 45–50.
[29] P.D. Shashi, L. Manu, S. Mika, Process Biochem. 45 (2010) 1065–1071.
[30] W. Wang, Q. Chena, C. Jiang, D. Yang, X. Liu, S. Xua, Colloids Surf. A:
Physicochem, Eng. Aspects 301 (2007) 73–79.
[31] J. Kasthuri, S. Veerapandian, N. Rajendiran, Colloid Surf. B: Biointerf. 68 (2009)
55–60.
[32] T.C. Prathna, N. Chandrasekaran, M. Raichur, A. Mukherjee, Colloid Surf. B
Biointerf. 82 (2011) 152–159.