Thevetia peruviana-A Wild Natural Resource for the Green Synthesis of Gold Nanoparticles.
Advanced Materials Research Vol 1112 (2015) pp 71-75
© (2015) Trans Tech Publications, Switzerland
doi:10.4028/www.scientific.net/AMR.1112.71
Submitted: 2014-09-01
Revised: 2014-12-31
Accepted: 2015-01-06
Thevetia peruviana: A Wild Natural Resource for the Green Synthesis of
Gold Nanoparticles
N. Nyoman Rupiasih1, a, Avinash Aher2, b, Suresh Gosavi2, c
and P.B. Vidyasagar2, d
1
Department of Physics, Faculty of Sciences, Udayana University, Bali, Indonesia
2
a
Department of Physics, University of Pune, Ganeshkhind, Pune-411007, India
b
c
rupiasih69@yahoo.com, aviaherphysics@gmail.com, swg@physics.unipune.ac.in,
d
prof_pbv@yahoo.com
Keywords: Thevetia peruviana latex, aqueous extract, green chemistry, capping agent, gold
nanoparticles.
Abstract. The biosynthesis of nanostructures using plant extract (green chemistry) has emerged as an
ecofriendly and evergreen branch of nanoscience and nanotechnology for the development of
biomedical applications. In this study we report the use of aqueous extract of the latex of Thevetia
peruviana (TP) for the synthesis of gold nanoparticles (AuNPs). The reaction time was optimized for
maximum yield of AuNPs. The AuNPs formed were characterized using FTIR, EDS, TEM and XRD.
The formation of AuNPs was confirmed using SPR spectra observed at around 589 nm. XRD pattern
shows the FCC crystal structure of the nanoparticles, orresponding to (111), (200), (220) and (311)
faces of gold. TEM analysis revealed that the particles are spherical with size distributions ranging
from 41-50 nm. HRTEM image showed 2.35Å interplaner spacing. FTIR analysis confirmed that the
flavonoid and reducing sugar found in the extract were the main reductive and the protein was
capping components in the biosynthesis of AuNPs.
Introduction
Recently, biosynthesis of nanoparticles (NPs) has become as an emerging field and considered to be
promising alternative to the conventional synthetic methodologies due to its eco-friendly advantages.
Application of these principles has reduced the use of hazardous reagents and solvents, with improved
material efficiency and enhanced the design of products for various field applications [1]. In
biosynthesis methods one can use microorganisms or plants extracts for synthesis of metal NPs [2].
The reduction rate of metal ions using plant extracts or biomass has been found to be much faster as
compared to microorganisms [2]. Plant extract has become simple and viable alternatives, which
eliminates the elaborated process of maintaining cell cultures and storage of microorganisms. There
are many studied, which reports about synthesis metal NPs using plant extract or biomass, e.g.
Coriander alfalfa, Aloe vera, Avena sativa and lemongrass [3]. However there remains a big challenge
in a full understanding of the bio-protocol from a set of biosynthetic conditions such as choices of
plants and the description of the bio-compounds involved in the process could be tough. Therefore, it
requires a most universal explanation to address to those biosynthesis metal NPs. In this reason, it
would be interesting to study the nature of NPs formed using extracts from different parts of a plant.
Thevetia peruviana (TP) is an ever-green ornamental dicotyledonous plant that belongs to
Apocyanaceae family. It is commonly found in the tropics and sub-tropics regions. The plant contains
a number of phytoconstituents which reveals its uses for various therapeutic purposes such as
diabetes, liver toxicity, fungal infection, microbial infection, inflammation, pyrexia and relieves pain
[4]. The whole plant contains a milky juice (white latex) which is poisonous and vesicant. The
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans
Tech Publications, www.ttp.net. (ID: 125.162.145.94-23/04/15,06:11:37)
72
Advanced Materials Research and Production
poisonous as well as medicinal characteristics of TP would be interesting reasons to study about
synthesis gold nanoparticles (AuNPs) using latex extract from the green stems of the fruits of TP.
Experimental Detail
The protocol of preparation of aqueous latex extracts has been described earlier in our work [3].
For synthesis and characterization of AuNPs, following steps has been carried out. One milliliter of 2
mM AuCl4 solution was added into 20 mL of aqueous latex extract to make the final mixture solution
concentration 0.1 mM and it was kept at room temperature. After 6 h of reaction, the colour of the
mixture changed into light purple and continuously changed to dark purple.
The formation of AuNPs was monitored using JASCO V-670 UV-Vis spectrophotometer in the
range 200-800 nm, by periodical recording the spectra of reaction mixture within 0, 11, 24, 44, 71 and
96 hour reaction time. Elemental analysis was done using EDS (JEOL JSM 6360A). The crystal
structure of AuNPs was examined by XRD using BRUKER AXS D8 with Cu-Kα (λ = 1.54 Å), in the
range 20-80o. The morphology and size of AuNPs formed was investigated using SEM (JEOL JSM
6360A) and HRTEM images using TEM-TECNAI G2 20U-TWIN instrument. FTIR measurements
were done using JASCO (FT/IR-6100) spectrophotometer in the range 400-4000 nm.
Results and Discussion
From the study it is observed that, 0.1 mM concentration of AuCl4 solution is optimum
concentration and is used for further studies.
Fig. 1 shows the UV-Vis absorption spectra of AuNPs solutions at different time intervals. It
shows the progress of forming a strong surface plasmon resonance (SPR) band centered at 589 nm,
which is a characteristic of colloidal gold [5]. The spectra clearly showed an increase in the
absorbance intensity of the gold nanosuspension, which indicating an increased number of AuNPs
formed in the solution [5]. These also accompanied by darker purple colour of the AuNPs solution.
Aggregation was also observed. Fig. 1 also shows red shifted band from 609 nm to 681 nm which
occurred with increasing reaction time from 0 to 71 h and blue shifted band to 589 nm occurred after
96 h reaction. The UV-Vis absorption spectra in lower wavelength regions (200 - 400 nm) show the
absorption peaks at around 230 nm, 260 nm and 305 nm, suggesting the presence of protein in
solution and aromatic amino acids in these proteins to be responsible for absorbance. This observation
indicates the present of proteins in the solution might be playing an important role in the mechanism
of reduction of the metal ions which present in the solution to be metal.
1.4
h0
h11
h24
h44
h71
h96
Absorbance (a.u.)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
200
300
400
500
600
700
800
Wavelength (nm)
Fig 1. UV-Vis absorption spectra of AuNPs
synthesized using TP aqueous latex extract at
different time reactions: 0-96 h.
Fig 2. EDS spectrum of AuNPs
synthesized using TP aqueous
latex extract.
Advanced Materials Research Vol. 1112
73
Fig. 2 shows representative energy dispersive X-ray (EDS) spectrum of AuNPs formed. The EDS
analysis reveals a signal in the gold region and confirms the formation of AuNPs. There is also a
strong signal for Si, which due to Si (111) wafers that has used as substrate to prepare the sample.
Fig. 3 shows the FTIR spectrum of dried powder AuNPs formed after 96 h of reaction. The
spectrum showed two peaks related to OH/NH and C=O (carbonyl) groups, e.g. 3419 and 1640 cm-1
respectively. This finding was in agreement with studies reported in the literature [6, 7]. The FTIR
spectrum also shows a pair of bands 2928 and 2860 cm-1, which arising from C-H stretching of the
aldehyde group. The bands observed at 3250 cm-1 which corresponds to the stretching of primary
amines and for bending were observed at 1640 cm-1. Two bands observed at 1369 and 1221 cm-1 can
be assigned to the C–N stretching of aromatic and aliphatic amines, respectively [5]. The presence of
amide linkages suggests that there are some proteins in the reaction mixture. These proteins might be
play an important role in the stabilization of the nanoparticles formed [5].
Based on the major chemical components of aqueous extract of TP and from the FTIR data
analysis, it might be concluded that flavonoid and reducing sugar found in the aqueous extract were
the main reductive and protective components in the biosynthesis of AuNPs [1, 3, 5, 7].
100
Transmitance (%)
98
96
2860
2928
1730
1369
1418
1084
94
3250
92
623
1221
1640
90
3419
88
4000
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Fig 3. FTIR spectra of AuNPs synthesized aqueous latex extract.
4000
38.18 (111)
3600
3200
Counts
2800
2400
44.39 (200)
2000
64.57 (220)
1600
1200
77.58 (311)
800
400
20
30
40
50
2θ ( )
60
70
80
0
Fig 4. XRD patterns of AuNPs synthesized using TP using TP aqueous latex extract .
Fig. 4 shows the XRD pattern of AuNPs formed. This shows that AuNPs formed has crystalline
structure in accordance with the JCPDS data file no. 04-0784. This finding agreed with the crystal
structure of AuNPs which reported in the literatures [5]. A number of Bragg reflections with 2θ values
38.18o, 44.39o, 64.57o and 77.58o are observed which are indexed to (111), (200), (220) and (311)
74
Advanced Materials Research and Production
faces of gold respectively. This XRD pattern illustrates that AuNPs formed having FCC crystal
structure. The (200), (220) and (311) Bragg reflections are weak and considerably broadened relative
to (111) reflection. This interesting feature indicates that AuNPs is predominantly (111)-oriented.
Fig. 5 SEM image of AuNPs formed
10 kV × 30,000 resolutions.
Fig. 6a Representative TEM image of AuNPs at
synthesized using TP aqueous latex extract.
Fig. 5 shows SEM image of AuNPs. It clearly shows the formation of spherical particles. Further
insight into the morphology and size details was provided by TEM analysis, Fig. 6. Fig. 6a shows all
the nanoparticles to be well separated. The morphology was found spherical mostly and having
dominant size distributions at 41-50 nm as showed in Fig. 6b. Fig. 6c shows HRTEM image of
AuNPs with fringe spacing is 2.35Å, which is same as the value obtained from XRD analysis
(Bragg’s Law).
6b
Fig 6. b) Histogram of particle size distribution and c) HRTEM image of AuNPs formed.
Summary
The rapid synthesis of gold nanoparticles (AuNPs) using Thevetia peruviana (TP) aqueous latex
extract has been demonstrated. Present green synthesis showed that the environment friendly and
renewable latex of TP could be used as an effective reducing agent as well as capping for the synthesis
of AuNPs. This eco-friendly route for the synthesis is a challenging alternative to chemical synthesis
especially for biomedicine applications.
Advanced Materials Research Vol. 1112
75
Acknowledgments
The author thanks Udayana University, Bali, Indonesia for kind facility and also to Department of
Physics, University of Pune, India as the university based of research conducted.
References:
[1] Y. Zhou, W. Lin, J. Huang, W. Wang, Y. Gao, L. Lin, Q. Li, L. Lin and M. Du, Biosynthesis of
Gold Nanoparticles by Foliar Broths: Roles of Biocompounds and Other Attributes of the Extracts,
Nanoscale Res Lett. 5 (2010) 1351-1359.
[2] M. Mano Priya, B. Karunai Selvia, adn J.A. John Paul, Green Synthesis of Silver Nanoparticles
from the Leaf Extracts of Euphorbia Hirta and Nerium Indicum, Dig. J. Nanomater Bios . 6 (2) (2011)
869-877.
[3] N. Nyoman Rupiasih, A. Aher, S. Gosavi, and P.B. Vidyasagar, Green synthesis of silver
nanoparticles using latex extract of Thevetia peruviana: a novel approach towards poisonous plant
utilization, J. Phys: Conference Series. 423 (2013) 012032.
[4] S. Kishan, A. K. Kumar, M. Vimlesh, U. S. Mubeen, and S. Alok, A Review on: Thevetia
Peruviana, International Research Journal of Pharmacy. 3 (4) (2012) 74-77.
[5] R. Bhambure, M. Bule, N. Shaligram, M. Kamat, and R. Singhal, Extracellular Biosynthesis of
Gold Nanoparticles using Aspergillus niger - its Characterization and Stability, Chem. Eng. Technol.
32 (7) (2009) 1036–1041.
[6] M. Shakibaie, H. Forootanfar, K. Mollazadeh-Moghaddam, Z. Bagherzadeh, N. Nafissi-Varcheh,
A.R. Shahverdi, and M.A. Faramarzi, Green synthesis of gold nanoparticles by the marine microalga
Tetraselmis suecica, Biotechnol. Appl. Biochem. 57 (2) (2010) 71-75.
[7] M. Moshfegha, H. Forootanfara, B. B. Zarea, A.R. Shahverdia, G. Zarrinic, and M.A. Faramarzi,
Biological Synthesis of Au, Ag And Au-Ag Bimetallic Nanoparticles by Α-Amylase, Dig. J.
Nanomater Bios. 6 (3) (2011) 1419-1426.
© (2015) Trans Tech Publications, Switzerland
doi:10.4028/www.scientific.net/AMR.1112.71
Submitted: 2014-09-01
Revised: 2014-12-31
Accepted: 2015-01-06
Thevetia peruviana: A Wild Natural Resource for the Green Synthesis of
Gold Nanoparticles
N. Nyoman Rupiasih1, a, Avinash Aher2, b, Suresh Gosavi2, c
and P.B. Vidyasagar2, d
1
Department of Physics, Faculty of Sciences, Udayana University, Bali, Indonesia
2
a
Department of Physics, University of Pune, Ganeshkhind, Pune-411007, India
b
c
rupiasih69@yahoo.com, aviaherphysics@gmail.com, swg@physics.unipune.ac.in,
d
prof_pbv@yahoo.com
Keywords: Thevetia peruviana latex, aqueous extract, green chemistry, capping agent, gold
nanoparticles.
Abstract. The biosynthesis of nanostructures using plant extract (green chemistry) has emerged as an
ecofriendly and evergreen branch of nanoscience and nanotechnology for the development of
biomedical applications. In this study we report the use of aqueous extract of the latex of Thevetia
peruviana (TP) for the synthesis of gold nanoparticles (AuNPs). The reaction time was optimized for
maximum yield of AuNPs. The AuNPs formed were characterized using FTIR, EDS, TEM and XRD.
The formation of AuNPs was confirmed using SPR spectra observed at around 589 nm. XRD pattern
shows the FCC crystal structure of the nanoparticles, orresponding to (111), (200), (220) and (311)
faces of gold. TEM analysis revealed that the particles are spherical with size distributions ranging
from 41-50 nm. HRTEM image showed 2.35Å interplaner spacing. FTIR analysis confirmed that the
flavonoid and reducing sugar found in the extract were the main reductive and the protein was
capping components in the biosynthesis of AuNPs.
Introduction
Recently, biosynthesis of nanoparticles (NPs) has become as an emerging field and considered to be
promising alternative to the conventional synthetic methodologies due to its eco-friendly advantages.
Application of these principles has reduced the use of hazardous reagents and solvents, with improved
material efficiency and enhanced the design of products for various field applications [1]. In
biosynthesis methods one can use microorganisms or plants extracts for synthesis of metal NPs [2].
The reduction rate of metal ions using plant extracts or biomass has been found to be much faster as
compared to microorganisms [2]. Plant extract has become simple and viable alternatives, which
eliminates the elaborated process of maintaining cell cultures and storage of microorganisms. There
are many studied, which reports about synthesis metal NPs using plant extract or biomass, e.g.
Coriander alfalfa, Aloe vera, Avena sativa and lemongrass [3]. However there remains a big challenge
in a full understanding of the bio-protocol from a set of biosynthetic conditions such as choices of
plants and the description of the bio-compounds involved in the process could be tough. Therefore, it
requires a most universal explanation to address to those biosynthesis metal NPs. In this reason, it
would be interesting to study the nature of NPs formed using extracts from different parts of a plant.
Thevetia peruviana (TP) is an ever-green ornamental dicotyledonous plant that belongs to
Apocyanaceae family. It is commonly found in the tropics and sub-tropics regions. The plant contains
a number of phytoconstituents which reveals its uses for various therapeutic purposes such as
diabetes, liver toxicity, fungal infection, microbial infection, inflammation, pyrexia and relieves pain
[4]. The whole plant contains a milky juice (white latex) which is poisonous and vesicant. The
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of Trans
Tech Publications, www.ttp.net. (ID: 125.162.145.94-23/04/15,06:11:37)
72
Advanced Materials Research and Production
poisonous as well as medicinal characteristics of TP would be interesting reasons to study about
synthesis gold nanoparticles (AuNPs) using latex extract from the green stems of the fruits of TP.
Experimental Detail
The protocol of preparation of aqueous latex extracts has been described earlier in our work [3].
For synthesis and characterization of AuNPs, following steps has been carried out. One milliliter of 2
mM AuCl4 solution was added into 20 mL of aqueous latex extract to make the final mixture solution
concentration 0.1 mM and it was kept at room temperature. After 6 h of reaction, the colour of the
mixture changed into light purple and continuously changed to dark purple.
The formation of AuNPs was monitored using JASCO V-670 UV-Vis spectrophotometer in the
range 200-800 nm, by periodical recording the spectra of reaction mixture within 0, 11, 24, 44, 71 and
96 hour reaction time. Elemental analysis was done using EDS (JEOL JSM 6360A). The crystal
structure of AuNPs was examined by XRD using BRUKER AXS D8 with Cu-Kα (λ = 1.54 Å), in the
range 20-80o. The morphology and size of AuNPs formed was investigated using SEM (JEOL JSM
6360A) and HRTEM images using TEM-TECNAI G2 20U-TWIN instrument. FTIR measurements
were done using JASCO (FT/IR-6100) spectrophotometer in the range 400-4000 nm.
Results and Discussion
From the study it is observed that, 0.1 mM concentration of AuCl4 solution is optimum
concentration and is used for further studies.
Fig. 1 shows the UV-Vis absorption spectra of AuNPs solutions at different time intervals. It
shows the progress of forming a strong surface plasmon resonance (SPR) band centered at 589 nm,
which is a characteristic of colloidal gold [5]. The spectra clearly showed an increase in the
absorbance intensity of the gold nanosuspension, which indicating an increased number of AuNPs
formed in the solution [5]. These also accompanied by darker purple colour of the AuNPs solution.
Aggregation was also observed. Fig. 1 also shows red shifted band from 609 nm to 681 nm which
occurred with increasing reaction time from 0 to 71 h and blue shifted band to 589 nm occurred after
96 h reaction. The UV-Vis absorption spectra in lower wavelength regions (200 - 400 nm) show the
absorption peaks at around 230 nm, 260 nm and 305 nm, suggesting the presence of protein in
solution and aromatic amino acids in these proteins to be responsible for absorbance. This observation
indicates the present of proteins in the solution might be playing an important role in the mechanism
of reduction of the metal ions which present in the solution to be metal.
1.4
h0
h11
h24
h44
h71
h96
Absorbance (a.u.)
1.2
1.0
0.8
0.6
0.4
0.2
0.0
200
300
400
500
600
700
800
Wavelength (nm)
Fig 1. UV-Vis absorption spectra of AuNPs
synthesized using TP aqueous latex extract at
different time reactions: 0-96 h.
Fig 2. EDS spectrum of AuNPs
synthesized using TP aqueous
latex extract.
Advanced Materials Research Vol. 1112
73
Fig. 2 shows representative energy dispersive X-ray (EDS) spectrum of AuNPs formed. The EDS
analysis reveals a signal in the gold region and confirms the formation of AuNPs. There is also a
strong signal for Si, which due to Si (111) wafers that has used as substrate to prepare the sample.
Fig. 3 shows the FTIR spectrum of dried powder AuNPs formed after 96 h of reaction. The
spectrum showed two peaks related to OH/NH and C=O (carbonyl) groups, e.g. 3419 and 1640 cm-1
respectively. This finding was in agreement with studies reported in the literature [6, 7]. The FTIR
spectrum also shows a pair of bands 2928 and 2860 cm-1, which arising from C-H stretching of the
aldehyde group. The bands observed at 3250 cm-1 which corresponds to the stretching of primary
amines and for bending were observed at 1640 cm-1. Two bands observed at 1369 and 1221 cm-1 can
be assigned to the C–N stretching of aromatic and aliphatic amines, respectively [5]. The presence of
amide linkages suggests that there are some proteins in the reaction mixture. These proteins might be
play an important role in the stabilization of the nanoparticles formed [5].
Based on the major chemical components of aqueous extract of TP and from the FTIR data
analysis, it might be concluded that flavonoid and reducing sugar found in the aqueous extract were
the main reductive and protective components in the biosynthesis of AuNPs [1, 3, 5, 7].
100
Transmitance (%)
98
96
2860
2928
1730
1369
1418
1084
94
3250
92
623
1221
1640
90
3419
88
4000
3500
3000
2500
2000
1500
1000
500
-1
Wavenumber (cm )
Fig 3. FTIR spectra of AuNPs synthesized aqueous latex extract.
4000
38.18 (111)
3600
3200
Counts
2800
2400
44.39 (200)
2000
64.57 (220)
1600
1200
77.58 (311)
800
400
20
30
40
50
2θ ( )
60
70
80
0
Fig 4. XRD patterns of AuNPs synthesized using TP using TP aqueous latex extract .
Fig. 4 shows the XRD pattern of AuNPs formed. This shows that AuNPs formed has crystalline
structure in accordance with the JCPDS data file no. 04-0784. This finding agreed with the crystal
structure of AuNPs which reported in the literatures [5]. A number of Bragg reflections with 2θ values
38.18o, 44.39o, 64.57o and 77.58o are observed which are indexed to (111), (200), (220) and (311)
74
Advanced Materials Research and Production
faces of gold respectively. This XRD pattern illustrates that AuNPs formed having FCC crystal
structure. The (200), (220) and (311) Bragg reflections are weak and considerably broadened relative
to (111) reflection. This interesting feature indicates that AuNPs is predominantly (111)-oriented.
Fig. 5 SEM image of AuNPs formed
10 kV × 30,000 resolutions.
Fig. 6a Representative TEM image of AuNPs at
synthesized using TP aqueous latex extract.
Fig. 5 shows SEM image of AuNPs. It clearly shows the formation of spherical particles. Further
insight into the morphology and size details was provided by TEM analysis, Fig. 6. Fig. 6a shows all
the nanoparticles to be well separated. The morphology was found spherical mostly and having
dominant size distributions at 41-50 nm as showed in Fig. 6b. Fig. 6c shows HRTEM image of
AuNPs with fringe spacing is 2.35Å, which is same as the value obtained from XRD analysis
(Bragg’s Law).
6b
Fig 6. b) Histogram of particle size distribution and c) HRTEM image of AuNPs formed.
Summary
The rapid synthesis of gold nanoparticles (AuNPs) using Thevetia peruviana (TP) aqueous latex
extract has been demonstrated. Present green synthesis showed that the environment friendly and
renewable latex of TP could be used as an effective reducing agent as well as capping for the synthesis
of AuNPs. This eco-friendly route for the synthesis is a challenging alternative to chemical synthesis
especially for biomedicine applications.
Advanced Materials Research Vol. 1112
75
Acknowledgments
The author thanks Udayana University, Bali, Indonesia for kind facility and also to Department of
Physics, University of Pune, India as the university based of research conducted.
References:
[1] Y. Zhou, W. Lin, J. Huang, W. Wang, Y. Gao, L. Lin, Q. Li, L. Lin and M. Du, Biosynthesis of
Gold Nanoparticles by Foliar Broths: Roles of Biocompounds and Other Attributes of the Extracts,
Nanoscale Res Lett. 5 (2010) 1351-1359.
[2] M. Mano Priya, B. Karunai Selvia, adn J.A. John Paul, Green Synthesis of Silver Nanoparticles
from the Leaf Extracts of Euphorbia Hirta and Nerium Indicum, Dig. J. Nanomater Bios . 6 (2) (2011)
869-877.
[3] N. Nyoman Rupiasih, A. Aher, S. Gosavi, and P.B. Vidyasagar, Green synthesis of silver
nanoparticles using latex extract of Thevetia peruviana: a novel approach towards poisonous plant
utilization, J. Phys: Conference Series. 423 (2013) 012032.
[4] S. Kishan, A. K. Kumar, M. Vimlesh, U. S. Mubeen, and S. Alok, A Review on: Thevetia
Peruviana, International Research Journal of Pharmacy. 3 (4) (2012) 74-77.
[5] R. Bhambure, M. Bule, N. Shaligram, M. Kamat, and R. Singhal, Extracellular Biosynthesis of
Gold Nanoparticles using Aspergillus niger - its Characterization and Stability, Chem. Eng. Technol.
32 (7) (2009) 1036–1041.
[6] M. Shakibaie, H. Forootanfar, K. Mollazadeh-Moghaddam, Z. Bagherzadeh, N. Nafissi-Varcheh,
A.R. Shahverdi, and M.A. Faramarzi, Green synthesis of gold nanoparticles by the marine microalga
Tetraselmis suecica, Biotechnol. Appl. Biochem. 57 (2) (2010) 71-75.
[7] M. Moshfegha, H. Forootanfara, B. B. Zarea, A.R. Shahverdia, G. Zarrinic, and M.A. Faramarzi,
Biological Synthesis of Au, Ag And Au-Ag Bimetallic Nanoparticles by Α-Amylase, Dig. J.
Nanomater Bios. 6 (3) (2011) 1419-1426.