Int. J. Adv. Res. Biol. Sci. (2016).

3(7): 75-84

International Journal of Advanced Research in Biological Sciences

ISSN: 2348-8069
www.ijarbs.com
Volume 3, Issue 7 - 2016
Research Article

SOI: http://s-o-i.org/1.15/ijarbs-2016-3-7-11

Biosynthesis and characterization of gold nanoparticles using leaves

extract of Piliostigma thonningii and their antimicrobial activity
P. Saranya and S. Premalatha*
PG & Research Department of Botany, Government Arts College (Autonomous), Coimbatore-641 018,
Tamil Nadu, India
Corresponding Author: Dr. S. Premalatha, Assistant Professor, Department of Botany, Government Arts
College, Coimbatore. E-mail: spr_latha@yahoo.com

Abstract
Synthesis and characterization of gold nano-particles using leaf extracts of Piliostigma thonningii and Estimation of antimicrobial
and antifungal activity. Method: Green approach has been utilized for the synthesis of gold nano-particles. Different aqueous
plant extracts has been prepared which was then utilized for the biosynthesis of gold nano-particles. Estimation for the synthesis
of nano-particles were done using UV-Visible spectroscopy and Fourier- Transform infrared spectroscopy. Antimicrobial and
antifungal activity of gold nano-particles prepared using aqueous extract was investigated using disc diffusion method. FTIR of
collected nano-particles gave an idea about the type of bio-molecules which helped in the reduction of auric and silver salts into
corresponding nano-particles. Antimicrobial and antifungal activity of gold nano-particles showed that the nano-particles have
better anti-microbial activity (kept as standard) when experiments were performed under similar conditions.

Keywords: gold nano-particles, Piliostigma thonningii, antimicrobial and antifungal activity, FTIR.

Introduction

Natural products once served humankind as the source (Pezzuto 1997). From 1983 to 1994 39% of the New
of all drugs, and higher plants provided most of these Approved Drugs were of natural origin, including
therapeutic Agents. Today, natural products (and their original natural products, products derived semi
derivatives and analogs) still represent over 50% of all synthetically from natural products, and synthetic
drugs in clinical use, with higher plant-derived natural products based on natural product models. (Cragg et
products representing ca. 25% of the total (Balandrin al, 1997). Further evidence of the importance of
et al, 1993). The World Health Organization estimates natural products is provided by the fact that almost
that 80% of the people in developing countries of the half of the world’s 25 best selling pharmaceuticals in
world rely on traditional medicine for their primary 1991 were either natural products or their derivatives
health care, and about 85% of traditional medicine (O’Neill and Lewis 1993). Conservative estimates
involves the use of plant extracts. This means that suggest that there are more than 250,000 species of
about 3.5 to 4 billion people in the world rely on higher plants existing on this planet, and only a very
plants as sources of drugs (Farnsworth 1988). In the small percentage of plants have been exhaustively
United States plant-derived drugs represent about 25% studied for their potential value as a source of drugs.
of the prescription drugs market, and in 1991 this Obviously natural products will continue to be
equated to a retail value of approximately $15.5 billion extremely important as sources of medicinal Agents.

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(7): 75-84
In addition to the natural products which have found unique properties like optoelectronic, magnetic, and
direct medicinal application as drug entities, many mechanical, which differs from bulk (Atul et al, 2010).
others can serve as chemical models or templates for Nanoparticles can be broadly grouped into two,
the design, synthesis, and semi synthesis of novel namely, organic nanoparticles which include carbon
substances for treating humankind’s diseases. nanoparticles (fullerenes) while, some of the
Although there are some new approaches to drug inorganic nanoparticles include magnetic
discovery, such as combinatorial chemistry and nanoparticles, noble metal nanoparticles (like gold
computer-based molecular modelling design, none of and gold) and semi- conductor nanoparticles (like
them can replaced the important role of natural titanium oxide and zinc oxide). There is a growing
products in drug discovery and development. interest in inorganic nanoparticles i.e. Of noble metal
nanoparticles (gold and silver) as they provide
The term “nanotechnology” describes the field of superior material properties with functional versatility.
developments in which size-dependent properties of Due to their size features and advantages over
materials in the nanometre regime play a dominant available chemical imaging drug Agents and drugs,
role, and where these properties can be used to inorganic particles have been examined as potential
generate new techniques and devices (Schmid et al, tools for medical imaging as well as for treating
2009). The materials can include nanoparticles with diseases. Inorganic nonmaterial have been widely
dimensions of less than 100nm as well as patterned used for cellular delivery due to their versatile
surfaces and more sophisticated assemblies. features like wide availability, rich functionality, good
Nanotechnology is an important field of modern compatibility, and capability of targeted drug delivery
research dealing with design, synthesis, and and controlled release of drugs (Xu et al, 2006). In the
manipulation of particles structure ranging from present study synthisis and characterization of gold
approximately 1-100 nm in one dimension. nano particles using leaf extract of Piliostigma
Nanotechnology is an interdisciplinary Field with thonningii and thier effects on microbes were reported.
contributions from physics, chemistry, biology, .
materials science, medicine and other disciplines.
Remarkable growth in this up-and-coming technology Materials and Methods
has opened novel fundamental and applied frontiers,
including the synthesis of nanoscale materials and Plant extract preparation
exploration or utilization of their exotic
physicochemical and optoelectronic properties. Fresh leaves of Piliostigma thonningii, (Family-
Fabaceae), were collected from in Tiruchirappalli
Nanotechnology is rapidly gaining importance in a district, Tamil nadu, and washed several times with
number of areas such as health care, cosmetics, food water to remove the dust particles and then Shade
and feed, environmental health, mechanics, optics, dried to remove the residual moisture and grinded to
biomedical sciences, chemical industries, electronics, form powder. Then plant extract was prepared by
space industries, drug-gene delivery, energy science, mixing 1% of plant extract with deionized water in a
optoelectronics, catalysis, reprography, single electron 250ml of (Borosil, India) conical flask. Then the
transistors, light emitters, nonlinear optical devices, solution was incubated for 30 min. and then subjected
and photo electrochemical applications (Colvin and to centrifuge for 30 min at room temperature with
Alivisatos 1994; Wang 1991). Nanomaterials are seen 5000 rpm. The supernatant was separated and filtered
as solution to many technological and environmental with (mm filter paper) filter paper with the help of
challenges in the field of solar energy conversion, vacuum filter. Then the solution was used for the
catalysis, medicine, and water treatment. In the reduction of gold ions (Au+) to gold nanoparticles
context of global efforts to reduce hazardous waste, (Auo).
the continuously increasing demand of nanomaterials
must be accompanied by green synthesis methods.
Nanotechnology is fundamentally changing the way in
which materials are synthesized and devices are
fabricated. Incorporation of nanoscale building blocks
into functional assemblies and further into
multifunctional devices can be achieved through a
“bottom-up approach”. Research on the synthesis of
nanosized material is of great interest because of their

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Plant leaf Powder form

Figure 1. Piliostigma thonningii Plant leaf and their Powder form

Synthesis of gold nanoparticles studied by Scanning Electron Microscope (JSM-6480

LV). After 24Hrs of the addition of HAuCl4 the SEM
For the synthesis of Gold nanoparticles, gold chloride slides were prepared by making a smear of the
prepared at the concentration of 10-3 M with pre- solutions on slides. A thin layer of platinum was
sterilized Milli Q water was used. A quantity of 1.5 ml coated to make the samples conductive. Then the
of each extract was mixed with 30 ml of 10-3 M of samples were characterized in the SEM at an
gold chloride for the synthesis of gold nanoparticles. accelerating voltage of 20 KV.
Gold chloride was taken in similar quantities of 1.5 ml
each without adding plant extracts to main respective DLS & Zeta potential analysis
controls. The saline bottles were tightly covered with
aluminium foil in order to avoid photo reduction of Dynamic light scattering (DLS) which is based on the
gold ions, incubated at room temperature under dark laser diffraction method with multiple scattering
condition and observations were recorded. techniques was employed to study the average particle
size of gold nanoparticles. The prepared sample was
Characterization of gold nanoparticles dispersed in deionized water followed by ultra-
sonication. Then solution was filtered and centrifuged
UV-vis analysis for 15 min. at 250C with 5000 rpm and the supernatant
was collected. The supernatant was diluted for 4 to 5
The optical property of AuNPs was determined by times and then the particles distribution in liquid was
UV-Vis spectrophotometer (Perkin-Elmer, Lamda 35, studied in a computer controlled particle size analyzer
Germany). After the addition of HAuCl4 to the plant (ZETA sizer Nanoseries,Malvern instrument Nano
extract, the spectra’s were taken in different time Zs).
intervals up to 24hrs between 450 nm to 540 nm. Then
the spectrum was taken after 24hrs of HAuCl4 Testing of antimicrobial activity
addition.
The test strains were: Aeromonas liquefaciens MTCC
FTIR analysis 2645 (B1), Enterococcus faecalis MTCC 439 (B2),
Klebsiella pneumonia NCIM 2883 (B3), Micrococcus
The chemical composition of the synthesized Gold luteus NCIM 2871 (B4), Salmonella typhimurium
nanoparticles was studied by using FTIR spectrometer NCIM 2501 (B5), Vibrio cholerae MTCC 3906 (B6),
(perkin-Elmer LS-55- Luminescence spectrometer). Candida albicans MTCC 1637 (F1), Cryptococcus sp.
The solutions were dried at 75o C and the dried MTCC 7076 (F2), Microsporum canis MTCC 3270
powders were characterized in the range 4000–400 (F3), Trichophyton rubrum MTCC 3272 (F4). The
cm-1 using KBr pellet method. cultures were obtained from MTCC, Chandigarh and
NCIM, Pune, India.
SEM analysis

The morphological features of synthesized gold

nanoparticles from P. thonningii plant extract were
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 Int. J. Adv. Res. Biol. Sci. (2016). 3(7): 75-84
Microbial strains were tested for antimicrobial light wave. The sharp bands of gold nanoparticles
sensitivity using the disc diffusion method (Bauer et al were observed around 540 nm in case of P. thonningii.
1966). This method was used to evaluate in vitro From different literatures it was found that the gold
antibacterial and antifungal activity of test sample nanoparticles show SPR peak at around 540 nm. From
against certain human pathogenic microorganisms on our studies we found the SPR peak for P. thonningii at
Muller Hinton agar (MHA) and potato dextrose agar 540 nm.
(PDA), respectively. A sterile cotton swab was used to
inoculate the standardized bacterial suspension on So we confirmed that P. thonningii leaf extract has
surface of agar plate. The 15 and 30 μL of test more potential to reduce Au ions into Au
solutions were poured in each disc (6 mm diameter), nanoparticles, which lead us for further research on
separately. One separate disc was used for control synthesis of gold nanoparticles from P. thonningii
study by taking sterile triple distilled water (without leaf extracts. The intensity of absorption peak
test sample). The plates were incubated at 37±1°C for increases with increasing time period. This
24–48 h (for bacteria) and 25 ±1°C for 48-72 h (for characteristic colour variation is due to the excitation
fungus). After incubation, the zone of inhibition was of the SPR in the metal nanoparticles. The reduction
measured with ruler/HiAntibiotic ZoneScale-C. The of the metal ions occurs fairly rapidly; more than 90%
assays were performed in triplicate and the average of reduction of Au+ ions is complete within 2 Hrs.
values are presented. Methicillin – 10mcg (for after addition of the metal ions to the plant extract.
bacteria) and Itraconazole – 10mcg (for fungus) was The metal particles were observed to be stable in
used as positive control. All the media, standard discs solution even 4 weeks after their synthesis. By
and HiAntibiotic ZoneScale-C were purchased from stability, we mean that there was no observable
Hi-Media (Mumbai, India). variation in the optical properties of the nanoparticles
solutions with time.
Results and Discussion
On the behalf of UV-vis data it was cleared that
Characterizations of AuNPs reduces metal ions. So the further characterizations
were carried out with P. thonningii (Figure. 2). The
UV-Vis spectrophotometer analysis UV-Vis absorption spectroscopy is one of the main
techniques followed to examine size and shape of the
Reduction of gold salt into gold nanoparticles during nanoparticles in the aqueous suspensions (Wiley et al.
exposure to plant extracts was observed as a result of 2006). Huang et al. (2007) reported formation of gold
the colour change. The colour change is due to the nanoparticles when constant aqueous HAuCl4 at 50
Surface Plasmon Resonance (SPR) phenomenon. The ml, 1 mM with 0.1 g biomass produced gold
metal nanoparticles have free electrons, which give the nanoparticles as indicated by sharp absorbance at
SPR absorption band, due to the combined vibration of around 540 nm in Cinnamomum camphora.
electrons of metal nanoparticles in resonance with

Figure 2. UV-VIS spectral analysis of Au nanoparticles

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(7): 75-84
FTIR analysis Therefore, the synthesized nanoparticles were
surrounded by proteins and metabolites having
FTIR measurements were carried out to identify the functional groups. From the analysis of FTIR studies
biomolecules for capping and efficient stabilization of we confirmed that the carbonyl groups from the amino
the metal nanoparticles synthesized. The FTIR acid residues and proteins has the stronger ability to
spectrum of gold nanoparticles wherein some bind metal indicating that the proteins could possibly
pronounced absorbance was recorded in the region from the metal nanoparticles (i.e.; capping of gold
between 4000 and 400 cmˉ¹. The FTIR spectra of nanoparticles) to prevent Agglomeration and thereby
aqueous P. thonningii extracts and AuNPs are shown stabilize the medium. This suggests that the biological
in Figure 3 and 3a respectively. P. thonningii extract molecules could possibly perform dual functions of
have shown in Figure. 3 the main peaks at around formation and stabilization of gold nanoparticles in the
3270, 2900, 1616, 1388, 766, 672 cm-1 whereas, the aqueous medium. Carbonyl groups proved that
gold nanoparticles in the presence of P. thonningii flavanones or terpenoids absorbed on the surface of
extract shows Figure. 3a the major peaks at 3434, metal nanoparticles.
2361, 2076, 1636, 1403.45, 1113.74 and 670.92 cm-1.
P. thonningii extract shows the peak at 3270 cm-1 can Flavanones or terpenoids could be adsorbed on the
be assigned to O-H stretch and peak at 2900 cm-1 surface of metal nanoparticles, possibly by interaction
corresponds to C-H stretch. The band at 1616 cm-1 is through carbonyl groups or π-electrons in the absence
assigned to C=O stretch. The band found at 1388 cm- of other strong ligating Agents in sufficient
1 can be assigned to C-O-C stretch. Another band at concentration. The presence of reducing sugars in the
766 cm-1 and 672 cm-1 assigned to C-H bend and C- solution could be responsible for the reduction of
H bond respectively. After synthesis the gold metal ions and formation of the corresponding metal
nanoparticles by the P. thonningii extracts have the nanoparticles. It is also possible that the terpenoids
broad peak at 3434 cm-1 assigned to O-H stretch and play a role in reduction of metal ions by oxidation of
also peak at 2361 cm-1 assigned to O-H stretch. The aldehydic groups in the molecules to carboxylic acids.
band at 2076 cm-1 and 1636 cm-1 are assigned to C-H These issues can be addressed once the various
stretch and C=O stretch respectively. The band found fractions of the plant extract are separated, identified
at 1403.45 cm-1 and 113.74 cm-1 are assigned to C-O- and individually assayed for reduction of the metal
C stretch. Another band found at 670.92 cm-1 ions. This rather elaborate study is currently
assigned to C-H bond. underway.
Table 1. Vibration modes of synthesized NP’s
Wave number ( cm ˉ ¹ ) Vibration modes
3434 O-H Stretch
2361 O-H Stretch
2076 C-H Stretch
1636 C=O Stretch
1403.45 C-O-C Stretch
1113.74 C-O-C Stretch
670.92 C-H bond

Figure 3. FTIR analysis of vibration modes and function groups of P. thonningii

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(7): 75-84

Figure 3a. FTIR analysis of vibration modes and function groups of AuNPs

SEM analysis prepared nanoparticles in the solution have sizes

several nano meters i.e. between 1-100 nm. The size
SEM provided further insight into the morphology and was more than the desired size as a result of the
size details of the gold nanoparticles. Comparison of proteins which were bound in the surface of the
experimental results showed that the diameters of nanoparticles (Figure 4).

Figure 4. SEM –microscopic view of P. thonningii reduced gold nano particles

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DLS analysis 100 nm range the percentage of nanoparticles present
is very less. The highest fraction of AuNPs present in
The particle size distribution (PSD) of synthesized the solution was of 73nm is very appropriate since it
gold nanoparticles, it was found that Au nanoparticles gives lowest average size of nanoparticles.
size were in the range of 80-120nm. However, beyond
Size Distribution by Intensity

10

8
Intensity (%)

0
0.1 1 10 100 1000 10000
Size (d.nm)
P. thonningii (Gold Nano particles); Z-Average (d.nm): 73.26
Record 3: MD-S 1
Figure 5. Dynamic Light Scattering of Particle Size Analyser of Au Nanoparticles

unstable. Therefore, the particles undergo

Zeta potential analysis Agglomeration/Aggregation to stabilize themselves.
So there were some potential charges on the surface of
The Figure 6 shows the zeta potential (ζ) is a measure the nanoparticles which makes them stable. These
of the electrostatic potential on the surface of the charge potential we got from this analysis. Zeta
nanoparticles and is related to the electrophoretic potential (Surface potential) has direct relation with
mobility and stability of the suspension of the stability of a form/structure as mentioned below
nanoparticles of the nanogold. The overall absorbance (Table 2; Figure 6).
of Zeta Potential revealed the energetically very

Figure 6. Zeta Potential Measurement of Au Nanoparticles

Zeta Potential (mV): from ±10 to ±30 = Incipient instability

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(7): 75-84
Table 2. A table showing the stability of the NPs according to the potential charge

Zeta potential [mV] Stability behaviour of the colloid

from 0 to ±5 Rapid coagulation or flocculation
from ±10 to ±30 Incipient instability
from ±30 to ±40 Moderate stability
from ±40 to ±60 Good stability
more than ±61 Excellent stability

Antibacterial and antifungal screening Selected microorganisms were showed significant

sensitivity against the biosynthesized nanoparticles.
Gold nanoparticles were tested in triplicates for The antimicrobial activity of test sample was
antimicrobial activity. The values were recorded and examined with various pathogenic microorganisms
averaged (Table 3 and Plate 1). P. thonningii has using the (measure the inhibition zone) Disc diffusion
tested and recorded the results for the gram-positive, test.
gram-negative bacteria and fungi. The gram-positive
were highly sensitive than gram-negative bacteria.

Table 3. Antimicrobial screening of AuNPs derived by P. thonningii leaves

Zone of inhibition (mm)

S.No Test Sample (15 & 30) µL / disc Diseases Route of
Microorganisms Transmission
15 30
Bacteria PC Remarks
µL µL
1 Aeromonas Wound Infections / Water / Food
liquefaciens 16 18 14 > PC Gastroenteritis
B1
2 Enterococcus Endocarditis / Bladder, Water / Food
fecalis Prostate, and Epididymal
15 17 8 > PC
B2 Infections / Nervous
system Infections
3 Micrococcus luteus Skin & Pulmonary Soil / Dust / Water /
B3 14 16 38 < PC infections / Septic shock / Airways / Food
Pneumonia endocarditis
4 Salmonella Typhoid Water / Food
typhimurium 15 18 0 > PC
B4
Fungi
5 Candida albicans Skin (Integument) Airways / Wound /
F1 Infections / Soil / Water
12 14 10 > PC
Gastrointestinal tract
Infection
6 Cryptococcus sp. Cryptococcal disease / Airways / Wound /
F2 12 13 9 > PC Bronchiectasis / Soil / Water
Endophthalmitis.
7 Microsporum canis Tinea capitis /Ringworm
Airways / Wound /
11 12 9 > PC
F3 Soil / Water
8 Trichophyton Tinea corporis / Tinea Airways / Wound /
rubrum 13 15 7 > PC cruris / Tinea pedis / Soil / Water
F4 Onychomycosis
PC - Positive Control (Using antibiotic disc; Bacteria – Methicillin (10mcg/disc) ;
Fungi – Itraconazole (10mcg/disc) Samples - 15 µL / disc & 30 µL / disc; > PC – greater than positive control; < PC
– less than positive control
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The results of the antimicrobial activities are noticed from B4. In fungi, this was effective against
summarized in Plates 1. In the present study, higher F4 whereas smaller effect was observed in F2.
(30 μL/disc) concentration of sample got greater
sensitivity than (15 μL/disc) lower concentration in all All the microbial strains depict higher sensitivity to
the tested microorganisms. In this study, all the the higher concentration (30 μL) and he concluded that
pathogens were fairly affected and nil effect was not the gold materials are an efficient alternative to
observed in the test samples. The gold nanoparticles antibiotics for the treatment. This nanoparticles release
not only interact at the surface of cell membrane, but gold ions in the bacterial cells, which enhance their
also enter inside the bacteria and cause damage of the bactericidal activity. There is no antimicrobial activity
cells by interacting with phosphorus/ sulfur containing in solution devoid of sample used as a vehicle control
DNA and its replication. In bacteria, the test sample (sterile triple distilled water), reflecting that
was most effective against B5 while smaller effect was antimicrobial activity was directly related to the
sample.
Plate 1. Antibacterial activity of biologically synthesized gold nanoparticles

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 Int. J. Adv. Res. Biol. Sci. (2016). 3(7): 75-84
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P. Saranya and S. Premalatha. (2016). Biosynthesis and characterization of gold nanoparticles using leaves
extract of Piliostigma thonningii and their antimicrobial activity. Int. J. Adv. Res. Biol. Sci. 3(7): 75-84.

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