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Gold Nanoparticles: Preparation, Characterization and Its Stability in Buffer

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 Nano Trends: A Journal of Nanotechnology and Its Applications
Volume 17, Issue 1, ISSN: 0973-418X

Gold Nanoparticles: Preparation, Characterization

and Its Stability in Buffer
Kiran Lata*, Arvind Kumar Jaiswal, Laxmana Naik, Rajan Sharma
Dairy Chemistry Division, National Dairy Research Institute, Karnal-132001, Haryana, India

Abstract
Among the colorimetric detection tactics, metallic nanoparticle based detection is gaining
more attention due to quickness and less resource requirement at the point of use. Unique
chemical inertness, shape, size and optical attributes of gold nanoparticles (GNPs) make them
ideal for various diagnostic tools. In this approach synthesized GNPs were stabilized by
citrate counter ions. These GNPs were characterized for their optical properties, shape, size,
zeta potential, and dynamic light scattering. Absorption maxima (λmax) was observed at
519.8 nm with the average particle size of 34.8 nm. Transmission electron microscopy also
revealed that the average size of GNPs were about 35 nm. Negative zeta potential (–43.2 mV)
indicates presence of negative charge at the surface of GNPs. To find the stability of GNPs in
buffer, two buffers (borate and phosphate) having different pH (6.5, 7.5 and 8.5) and same
molarity (0.1 M) were evaluated with a purpose to determine the suitable buffer required for
conjugation of GNPs to antibody for lateral flow assays.

Keywords: Antibody, citrate reduction, dynamic light scattering, gold nanoparticles, size
distribution, zeta potential

*Author for Correspondence E-mail: kiranlatahisar@gmail.com

INTRODUCTION range can also be used in fabrication of smart

In recent years, metal nanoparticles have
attracted an increasing attention in different
fields of sciences because of their easy
biofunctionalization, high surface area and
spectral properties [1]. Nanoparticles are
typically in the size range of 1–100 nm
(1 nm=10-9). Due to their small size they
comprise unusual physical, chemical and
optical properties [2] which makes them very
important for diagnostic, biological and
biomedical purposes [3–5]. Widely used
nanoparticles are, gold nanoparticles (AuNPs
or GNPs), quantum dots and super
paramagnetic nanoparticles. Among these
nanoparticles, GNPs are one of the most useful
nanoparticles in medical, chemical and
biological sciences [6, 7]. GNPs are employed
as anti-aging components for skin protection
[5], to treat wool or cotton fibres for a
permanent coloration of value textiles [8],
polymer/gold nanocomposites for novel
coatings and paintings [9], as catalysts [10],
labelling cancer cells, radiotherapy and light
based therapy for cancer [11, 12], scaffolds to
couple carbohydrates and protein [13], in gene
therapy [14]. The GNPs of 15–40 nm size
sensing devices such as lateral-flow devices,
lateral-flow immunoassay in biomedical
sciences as diagnostic tools [15].
GNPs exhibits high sensitivity for the
detection due to characteristic surface plasmon
resonance (SPR) absorption properties [16].
Size, shape, dielectric properties, and local
environmental conditions of the GNPs
strongly affect the resonance frequency [17].
Due to aggregation tendency the colour of
GNPs may range from red to purple, to blue
and almost black. In colloidal solutions, GNPs
are wine red in colour with the λmax at 520 nm
which can be visualized easily without any
additional development process [18]. As a
label GNPs is very stable in liquid or dried
form and is non-bleaching after staining on
membranes as well as they are widely
available in conjugated and unconjugated form
[19].
Nanoparticles can be synthesised by various
methods such as chemical reduction [20, 21],
photochemical using UV irradiation [22],
sonochemical [23], X-ray [24], laser ablation

©NSTC (2014) 1-10 © STM Journals 2014. All Rights Reserved Page 1
Gold Nanoparticles
of a metal gold plate in liquid [25, 26], seed-
mediated growth, sonoelectrochemical, etc.
[27]. The simplest and by far the most
commonly used preparation method for gold
nanoparticles is the aqueous reduction of gold
tetra-chloric acid through a “nucleation”
process by the reducing action of sodium
citrate [28]. Due to inexpensive reductant,
non-toxic water solvent, and low pollution by
reaction, the citrate reduction method is the
best option to fit the enlarging demand of
GNPs [29]. The main purpose of using
reducing agent is to convert one form of gold
into another form i.e. ionic form into metallic
form in a controlled condition. Some workers
have used sodium borohydride (NaBH4) as
reducing agent to prepare GNPs [30, 31].
GNPs have also been synthesized by eco-
friendly methods such as by biochemical
reduction [32]. On the other hand, by using
culture supernatant of Aspergillus niger
(fungi) for reduction of aqueous gold ions
[33]. Stable GNP with diameter 5–10 nm can
be synthesized in water at room temperature
by green photocatalytic method in less than
four minutes [34]. Most commonly used
methods for GNPs characterization includes
UV-Vis-NIR spectrophotometry, transmission
electron microscopy (TEM), scanning electron
microscopy (SEM) and high-resolution
transmission electron microscopy (HRTEM)
[35]. GNPs can also be characterized by zeta
potential and dynamic light scattering (DLS)
[36].
Rapidity and one step process in lateral-flow
immunoassay (LFIA) has recently generated
scientific and industrial interest for first-level
screening of food contaminants. Inertness
towards the functional activity of antibody or
antigen and other biomolecules, GNPs has
excellent compatibility with these molecules
even after immunisation [15]. Negative
surface charge of weekly bound citrate coating
on GNPs facilitates easy characterisation by
plasmon resonance (at 520 nm) and attraction
for positively charged antibody, which is a
basis for conjugation of GNPs with antibody.
This property can be used for the detection of
specific target analytes. As a result, surface
fictionalization of gold nanoparticles could
accelerate antibody-antigen reaction, which
further amplifies the signal in immunoassay
[37]. Gold nanoparticles (GNPs) of
©NSTC (2014) 1-10 © STM Journals 2014. All Rights Reserved
Lata et al.
approximately 40 nm mean diameter shows
good stability, better handling during
conjugation to antibodies and line detectability
[38]. Owing to these advantages, GNPs have
better suitability for lateral flow assay, which
is one-step on-site screening test for analyte
[39].
Considering above points, the objective of this
study was GNPs preparation, their
characterization, conjugation with antibody
and its stability in different buffer for
conjugation of GNPs to antibody for different
diagnostic assays.
MATERIALS AND METHODS
Materials
Gold chloride (tetrachloroauric acid trihydrate)
(HAuCl4.3H2O) and trisodium citrate
dihydrate were purchased from Sigma-Aldrich
(USA). All other chemicals were of analytical
grade. All synthetic preparations and
measurements were carried out in Millipore
water as having resistivity 18.2 MΩ/cm.
Cleaning of Glassware and Magnetic
Stirrer
All the glassware like beakers, pipettes,
condenser, double necked flasks (250 or
150 mL) and magnetic beads were first
washed with copious amount of normal tap
water. Then glassware and magnetic beads
were washed with liquid detergent. After
detergent washing, glassware were washed
with normal tap water. Then all materials were
dipped in aquaregia (3:1 mixture of
concentrated HCl and HNO3) for at least an
hour. Aqua regia should be prepared very
carefully wearing goggles and gloves in a
fume hood. It should be freshly prepared and
should never be stored in a closed vessel. The
capped aqua regia bottle may explode. After
aqua regia, glassware were washed with
copious amount of double distilled water and
then rinsed with Millipore water in sequence.
Gold Nanoparticles Synthesis
Gold nanoparticles were synthesized by citrate
reduction method originally introduced by
Turkevitch et al. [40] with slight modifications
described by Liu and Lu [28] in aqueous phase
using trisodium citrate and other agents for
reduction. Briefly, 98 mL of Millipore water
was added to a double necked flask (250 mL)
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 Nano Trends: A Journal of Nanotechnology and Its Applications
Volume 17, Issue 1, ISSN: 0973-418X
followed by 2 mL of 50 mM of gold chloride
solution (4 time diluted 200 mM stock gold
chloride solution). After putting magnetic bar
in the flask, glass condenser was connected to
central neck of the flask and stopper on the
side neck flask and whole assembly was
placed on the magnetic stirrer at 1000 rpm.
With continuous stirring gold chloride solution
was heated upto boiling. When refluxing was
started in gold chloride solution, stopper from
the side neck of the flask was removed
followed by quick addition of 10 mL of
38.8 mM trisodium citrate dihydrate with
simultaneous stirring and stopper placed back.
The boiling was further continued with
continuous stirring.
Fig. 1: Flow Diagram for Gold
Nanoparticles Synthesis.
Gradual change in the colour of the gold
chloride solution from pale yellow to deep red
©NSTC (2014) 1-10 © STM Journals 2014. All Rights Reserved
in one minute after addition of trisodium
citrate dihydrate solution indicates the
formation of colloidal GNPs. The mixture was
allowed to reflux for another 20 min. Heating
was turned off and the mixture was allowed to
cool upto 23–25ºC at room temperature with
continuous stirring. Procedure is shown in
Figure 1. Gold nanoparticles were collected in
a clean amber-coloured glass container
(washed with aquaregia) and stored at 4ºC.
Characterization of GNPs
The size, shape and other properties of
synthesized gold nanoparticles were
characterised by using UV-spectrophotometer
and dynamic light scattering (DLS). All the
analysis was performed in triplicates. The
absorption spectrum of prepared gold
nanoparticles was taken in double beam UV-
spectrophotometer (Shimadzu UV-2550;
Shimadzu, Kyoto, Japan) in the range of 400
to 700 nm using band width of 0.5 nm and
water as blank. Dynamic light scattering
(DLS) is a technique that is widely used to
characterize the size of various particles
including proteins, polymers, micelles,
carbohydrates, and nanoparticles [41]. It is a
rapid technique to determine the presence or
absence of aggregates in GNPs with less
sample volume requirement. It measures the
scattering of light by particles present in
solution. The size distribution and zeta
potential measurements of nanoparticles were
measured on DLS instrument Zetasizer
Nano ZS90 (Malvern instruments, UK) at
Aimil, New Delhi. Data was analysed in
automatic mode and measured values were
expressed as average value. The
morphological features of the particles were
studied with scanning electron microscopy as
described by Saha [42] and Yazid [43]. GNPs
were lyophilized and lyophilized sample
(GNPs) was placed on stub using double
adhesive tape and then coated with gold. They
were observed under Scanning electron
microscopic (SEM) using Carl Zeiss EVO 18
SEM at 850 x magnification. Transmission
Electron Microscopy (TEM) images of GNPs
were taken in Field Emission Transmission
Electron Microscopy JEOL JEM 2010F. The
prepared gold nanoparticle was taken (10 to
20 µL) in copper grid and air dried. Air dried
sample was used for observation in TEM.
Page 3
Gold Nanoparticles
Optimization of Buffer for Conjugation of
Antibodies to GNPs
To find out the suitable buffer and pH (6.5, 7.5
and 8.5) required for the stability of GNPs
during conjugation, two buffers (borate and
phosphate having different pH) were studied.
Colloidal gold solution (1 mL) was taken into
2 mL micro centrifuge tube and centrifuged at
13,200 rpm (15,973×g) for 10 min. The
supernatant was carefully removed by
pipetting and the resulting pellet re-suspended
in 1 mL of test buffer A or B. Change in
colour of GNP was noticed. After buffer
selection antibodies were conjugated with
GNPs by method described by Thobhani et al.
[44]. Obtained pellet containing the antibody-
gold conjugate were centrifuged and further
re-suspended in borate buffer (0.1 M, pH 7.5).
This conjugate was stored at 4°C until use and
further used for lateral flow strip.
RESULTS AND DISCUSSION
Synthesis of GNPs
GNPs were prepared in the lab using citrate
reduction method as suggested by Liu and Lu
[28]. The change in colour from pale yellow–
dark violet–cherry red after addition of
reducing agent citrate (Figure 2).
Fig. 2: Gold Nanoparticle (a) Before the
Addition of Reducing Agent (b) After Addition
of Reducing Agent.
©NSTC (2014) 1-10 © STM Journals 2014. All Rights Reserved
Lata et al.
The reducing agent should be added as rapidly
as possible in order to accomplish a nearly
mono dispersed solution. The shape, size, and
stability of the colloidal gold particles are
crucial factors for the accomplishment of the
lateral flow strip. The colloidal gold solution
with size range of 5 to 60 nm particle size are
stable for long duration in absence of any
special stabilizing agent [45]. The size as well
as the color of GNPs depends upon the amount
of sodium citrate used for reduction of gold
during GNPs preparation [46]. Beside this
temperature and the order of addition of
reagents also affects the size of GNPs [47].
Following synthesis reaction takes place
during the formation of GNPs [48].
6AuCl−
4 + C6 H8 O7 + 5H2 O
→ 6CO2 + 24Cl− + 6Au° + 18H+
Negatively charged gold ions released during
the reaction get surrounded and stabilized by
positively charged citrate ions and forms a
stern like layer. The GNPs were further
characterized for their size distribution and
other properties by different techniques.
Characterization of GNPs
Measurement of Plasmon Resonance
Plasmon resonance character of GNPs is
owing to strong absorption of electromagnetic
waves in the visible range, which causes
collective oscillations of the conduction
electrons of GNPs [49]. In present study
characteristic Plasmon resonance peak has
been observed at 519.8 nm with absorbance of
3.05 (Figure 3). Other workers have also
measured the characteristic Plasmon resonance
peak of prepared GNPs; used for lateral-flow
immunoassay, and other diagnostic tools [16,
28, 42, 44].
In general, it has been reported that absorption
maximum shifts to longer wavelengths with
increasing particle size [50, 19]. Further,
relative uniformity of the particles or the range
of the particles can be gauged by the width of
the absorption spectra; the sharper the band,
more the uniformity in the particles [19, 51].
However, literature indicates that this is not
always the case, and correlation between the
absorption maxima and size of the particles is
not good [16, 28, 44]. Heavy absorption of
visible light by GNPs at 520 nm results in
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 Nano Trends: A Journal of Nanotechnology and Its Applications
Volume 17, Issue 1, ISSN: 0973-418X

brilliant red colour, which also changes with
the size of GNPs [51].
Electron Microscopy
Synthesized GNPs were characterized by both
TEM and SEM for size distribution and
surface characteristics respectively. TEM
image (Figure 4) shows presence of small
spherical dark colored dot of colloidal gold in
mono dispersion state with the average
diameter of about 35 nm. Monodispersity of
colloidal gold might be due to presence of
negative charge of citrate ion on the surface of
GNPs.
Figure 5 shows scanning electron microscopy
(SEM) image of GNPs. Morphological
analysis of colloidal gold particles by SEM
revealed that the synthesized GNPs were
spherical in shape with smooth surface.

Fig. 3: Absorption Spectra (Plasmon Resonance) of Synthesized Gold Nanoparticles.

Fig. 4: Transmission Electron Microscopy Fig. 5: Scanning Electron Microscopy (SEM)

(TEM) of Colloidal Gold Nanoparticles at of Colloidal Gold Nanoparticles at
100 nm Scale. 5 nm Scale.

©NSTC (2014) 1-10 © STM Journals 2014. All Rights Reserved Page 5
Gold Nanoparticles
Measurement of Size Distribution and Zeta
Potential
GNPs synthesized by citrate reduction method
were analysed by DLS. Each sample was
analysed in triplicate. The average size of the
prepared GNPs was 34.8 nm (Figure 6). As the
intensity of the light scattered is proportional
to the six power of the particle size, larger
particles will give a greater signal than smaller
particles [44].
The size of the GNPs varies with the amount
of reducing agent. Jongjinakool et al. [36]
reported decrease in the particle size with
increase in the volume of NaBH4 ranging from
200 to 1,200 μL.
Fig.6: Size Distribution of Gold Nanoparticles
by Dynamic Light Scattering (DLS).
Zeta potential of the synthesized GNPs was
–43.2 mV (Figure 7) which indicates the
presence of negative charge at the surface
[52]. Other workers also synthesized GNP’s
by citrate reduction method with zeta potential
of –44.1 mV [53].
The particles with zeta potentials more than
+30 mV and more negative than –30 mV are
normally considered stable [53]. A novel one
step synthesis of water soluble Au and Ag
nanoparticles has been reported at room
temperature using a naturally occurring
bifunctional molecule, namely, gallic acid with
zeta potential –45 mV [54].
©NSTC (2014) 1-10 © STM Journals 2014. All Rights Reserved
Lata et al.
Fig. 7: Zeta Potential of Gold Nanoparticles.
Optimization of Conditions for Conjugation
of Antibodies to GNPs
Borate and phosphate buffers having different
pH (6.5, 7.5 and 8.5) and 0.1 M concentration
were evaluated with a purpose to determine
the suitable buffer required for conjugation of
GNPs to antibody. An aliquot of the prepared
GNPs was centrifuged and pelleted GNPs
were suspended in borate and phosphate
buffers and colour of the suspensions were
noted visually. The results indicated that GNPs
were unstable in phosphate buffer at all
pH (6.5, 7.5 and 8.5) as indicated by dark blue
(due to aggregation of GNPs). In case of
borate buffer, the blue color was observed
only at pH 8.5, while at pH 7.5 the color of the
suspension remained red. At pH 6.5, though
the suspension was red, but brightness was
less as compared to pH 7.5. Based on these
observations borate buffer at pH 7.5 was found
suitable for conjugation of antibodies with
GNPs (Figure 8). For the conjugation of
colloidal gold with proteins, electrostatic,
dative (sulphur bridge) and hydrophobic
interactions plays very important role.
Negative charge on the surface of citrate ion
coated GNPs shows attraction towards the
positive charge of amino acids of antibodies
[55]. However, in the conjugation studies
generally pH slightly above the isoelectric
point of ligand is selected as pH below the
isoelectric point causes ligand‐induced
flocculation [56, 44, 48]. This reduction in
electrostatic interaction is compensated by
other interactions playing part between GPs
and antibodies.
Page 6
 Nano Trends: A Journal of Nanotechnology and Its Applications
Volume 17, Issue 1, ISSN: 0973-418X
Fig. 8: Stability of Colloidal Gold Nanoparticles in Borate and Phosphate Buffer.
CONCLUSIONS
The multifunctional properties of GNPs offer
unique opportunities for their use in the
development of various diagnostic methods
and devices. Gold nanoparticles (GNPs)
prepared via citrate reduction method using
trisodium citrate show uniformity, fair
stability, and wider applicability for
immnunoassays.
A single plasmon resonance peak (519.8 nm)
of prepared GNPs was recorded in visible
spectra, indicating ideal labelling material for
lateral flow assay. Average size of prepared
GNPs was found to be 34.8 nm with zeta
potential of –43.2 mV confirming their
stability as nanoparticles with zeta potentials
more than +30 mV and more negative than
–30 mV are normally considered stable.
Borate buffer (0.1 M, pH 7.5) was found
suitable for GNPs conjugation with antibody.
These GNPs were suitable for use in lateral
flow strip with clear dark red line on
nitrocellulose membrane indicating presence
or absence of analyte in sample. Due to novel
applicability of metal nanoparticles in different
dimensions of sciences further research is
needed for other alternative methods for bulk
production of these multifunctional
nanoparticles in a cost effective approach.
ACKNOWLEDGEMENTS
The authors are very grateful to National Dairy
Research Institute, Karnal, Haryana, India.
This work was supported by Rajiv Gandhi
National Fellowship (RGNF) formulated and
©NSTC (2014) 1-10 © STM Journals 2014. All Rights Reserved
funded by Ministry of Social Justice and
Empowerment and Ministry of Tribal Affairs,
Government of India and authors sincerely
appreciate for the financial support.
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