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Antifungal activity of gold

nanoparticles prepared by
solvothermal method
Tokeer Ahmad, Irfan Hussain

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Materials Research Bulletin 48 (2013) 12–20

Contents lists available at SciVerse ScienceDirect

Materials Research Bulletin

journal homepage: www.elsevier.com/locate/matresbu

Antifungal activity of gold nanoparticles prepared by solvothermal method

Tokeer Ahmad a,*, Irshad A. Wani a, Irfan H. Lone a, Aparna Ganguly a, Nikhat Manzoor b,1, Aijaz Ahmad b,1,
Jahangeer Ahmed c, Ayed S. Al-Shihri d
a
Nanochemistry Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi 110025, India
b
Department of Biosciences, Jamia Millia Islamia, New Delhi 110025, India
c
Department of Chemistry, Michigan State University, East Lansing, MI 48824, USA
d
Department of Chemistry, Faculty of Science, King Khalid University, Abha 61413, P.O. Box 9004, Saudi Arabia

A R T I C L E I N F O A B S T R A C T

Article history: Gold nanoparticles have been successfully synthesized by solvothermal method using SnCl2 and NaBH4
Received 21 May 2012 as reducing agents. X-ray diffraction studies show highly crystalline and monophasic nature of the gold
Received in revised form 14 September 2012 nanoparticles with face centred cubic structure. The transmission electron microscopic studies show the
Accepted 29 September 2012
formation of nearly spherical gold nanoparticles of average size of 15 nm using SnCl2, however, NaBH4
Available online 8 October 2012
produced highly uniform, monodispersed and spherical gold nanoparticles of average grain size of 7 nm.
A high surface area of 329 m2/g for 7 nm and 269 m2/g for 15 nm gold nanoparticles was observed. UV–
Keywords:
vis studies assert the excitations over the visible region due to transverse and longitudinal surface
A. Metals
A. Nanostructures
plasmon modes. The gold nanoparticles exhibit excellent size dependant antifungal activity and greater
B. Chemical synthesis biocidal action against Candida isolates for 7 nm sized gold nanoparticles restricting the transmembrane
C. Electron microscopy H+ efflux of the Candida species than 15 nm sized gold nanoparticles.
D. Optical properties ß 2012 Elsevier Ltd. All rights reserved.

1. Introduction used in biological optical imaging and sensing applications [14–16]

Nanotechnology has experienced a rapid growth recently
because nanostructures exhibit physical and chemical properties
that are distinctly different from the bulk solid [1]. In particular,
metallic nanostructured materials have been the subject of intense
scientific research by virtue of their fundamental importance and
potential applications [2]. Among noble metals, gold nanoparticles
have been extensively investigated in the last 10 years because of
their potential applications in optics, electronics, and catalysis
[3,4]. The applications of the nanoparticles are mainly due to the
quantum size effect, which is the function of the number of free
electrons in the nanoparticles [5]. Few reports are also available
which show that the gold nanoparticles exhibit good antimicrobial
activity [6–8]. Their predominant antimicrobial activity can be
attributed to the strong cytotoxicity to various bacterial cells. They
can interact with the functional groups on the bacterial cell surface
to inactivate bacteria and destroy them [9]. Various methods have
been used to produce the nanoparticles, like chemical reduction
[10], photolytic reduction [11], radiolytic reduction [12] and metal
evaporation [13]. Gold and silver nanoparticles have also been
* Corresponding author. Tel.: +91 11 26981717x3261; fax: +91 11 26980229.
E-mail address: tahmad3@jmi.ac.in (T. Ahmad).
1
Contributed to the antifungal activity of gold nanoparticles.
due to the excellent elastic light scattering properties of metal
nanoparticles [14,17,18].
Nanoparticles could be characterized spectroscopically on the
basis of their sizes as their concentration depends on the method of
synthesis [19–22]. Various chemicals serve as reducing agents in
the process of nanoparticles production, such as sodium/potassi-
um borohydrate [23], hydrazine [24], salts of tartarate [25], sodium
citrate [26], ascorbic acid [27,28] and amino acids [29–31]. When
the nanoparticles are formed, they need to be stabilized for further
applications. Various reagents have also been reported to act as
stabilizing agent such as polyethylene glycol [32,33], polyvinyl
alcohol [34], polyvinyl pyrollidine [35,36], chitosane [37] and
various surfactants viz, sodium dodecyl sulphate [38–40], Triton-X
[41] etc. Numerous processes have been reported in literature for
the synthesis of gold nanoparticles, for example, citrate was used
as a reducing agent as well as a stabilizer for the synthesis of gold
nanoparticles [42,43]. Reverse microemulsion method [44,45],
polymeric matrix [46] and polyelectrolyte systems [47] have also
been used for the synthesis of gold nanoparticles. However, very
few reports are available in literature for the preparation of naked
gold nanoparticles with narrow size distribution, so that different
surface functionalities can be introduced for the applications in
nanoscale devices. In order to meet such requirements, we have
applied modified solvothermal method for the synthesis of silver
nanoparticles at a large scale [48]. Herein, we report the

0025-5408/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.materresbull.2012.09.069
 Author's personal copy
T. Ahmad et al. / Materials Research Bulletin 48 (2013) 12–20
solvothermal method for the synthesis of ultrafine gold nanopar-
ticles and studied their size, morphology, surface area and optical
properties by varying the reducing agents. The fungicidal activity
of the gold nanoparticles has also been investigated against the
various clinical strains of the fungus Candida. To the best of our
knowledge, there are no reports in literature for the antifungal
activity of gold nanoparticles against Candida and their mode of
action.
2. Materials and methods
2.1. Synthesis of gold nanoparticles
Gold nanoparticles have been successfully synthesized by the
solvothermal method using two different reducing agents. In the
first solvothermal reaction, 40 ml of 2.5  102 M aqueous
chloroauric acid, HAuCl4 (Spectrochem, Mumbai, India) was taken
in a round bottomed flask. To this flask 40 ml of 2.5  102 M
aqueous tin chloride, SnCl2 (Merck, India, 97%) was added slowly
with the help of burette. After the complete addition of the
reducing agent, the resulting mixture turned purple in colour. The
pH of the gold nanoparticle suspension was found to be 0.7. The
purple suspension was then refluxed at 60 8C for 2 h during which
there is no change in colour was observed. The pH of the gold
nanoparticles solution was recorded at an interval of 1 h which
shows that the pH was increased to 1.0 after 5 h as shown in Table
1. The pH of the gold nanoparticles suspension was also calculated
theoretically (see Eq. (1)) and was found to be 3. The observed
variation in the experimental and theoretical values of pH is
attributed to the fact that the tin chloride solution was prepared by
the addition of fuming HCl. The theoretical pH value after adding
the H+ ion concentration from HCl, comes out to be close to the
experimental value.
2HAuCl4 þ 3SnCl2 ! 2Au þ 3SnCl4 þ 2HCl
The solution was centrifuged at 7000 rpm for 20 min and the
precipitate was then washed thrice with double distilled water to
remove any water soluble impurity. The washed precipitate was
then dried in an oven at 60 8C. The flow chart representing the
sequence of experimental observations is shown in Fig. 1(a).
In the second set of reaction, tinchloride (SnCl2) was replaced by
the sodium borohydride (NaBH4) of the same molar concentration.
After the complete addition of the reducing agent, dark green
suspension was obtained. The pH of this suspension was recorded
and found to be 2.1. The suspension was then refluxed for 2 h
during which the colour turned to pale yellow and the pH of the
resulting gold nanoparticle suspension increased to 2.4. The pH of
the gold nanoparticles suspension was also calculated theoretical-
ly (see Eq. (2)) and was found to be 2.398. The experimental pH
value of 2.4 is in good agreement with the theoretically predicted
pH value of 2.398. The pH of the solution was also recorded with
time and the results are shown in Table 1. In general, both redox
Table 1
Variation of pH of gold nanoparticles suspension with time.
S. No. Reducing agent Time pH Color
1. SnCl2 1h 0.7 Purple
2h 0.8 Purple
3h 0.9 Purple
4h 1.0 Purple
5h 1.0 Purple
2. NaBH4 1h 2.1 Dark green
2h 2.3 Yellow
3h 2.4 Yellow
4h 2.4 Yellow
5h 2.4 Yellow
13
reactions (Eqs. (1) and (2)) led to increase the pH of the solution,
which may be due to the formation of HCl. Furthermore, the
constant pH value is attained in both the reactions with the
passage of time.
8HAuCl4 þ 3NaBH4 þ 12H2 O ! 8Au þ 3NaBðOHÞ4 þ 32HCl (2)
The pale yellow suspension was centrifuged at 7000 rpm for
20 min and the precipitate was washed thrice with double distilled
water. The washed precipitate was then dried in an oven at 60 8C.
The sequence of experimental observation is shown in Fig. 1(b).
2.2. Microorganisms and culture conditions
The Candida isolates were grown on yeast extract peptone
dextrose (YPD) medium containing 2% (w/v) glucose, 2% peptone
and 1% yeast extract (HiMedia, India). YPD plates containing 2.5%
Agar (HiMedia) were used to maintain the culture. Vanadate was
purchased from Aldrich Chemicals (Germany) whereas all inorganic
chemicals used in the antifungal assay were of analytical grade and
procured from E. Merck (India). Fuconazole and vanadate were
dissolved in water to make the desired concentrations.
2.3. Antifungal susceptibility test
The minimal inhibitory concentration (MIC) was defined as the
lowest concentration of the test gold nano-particles that causes
inhibition of visible growth (turbidity). MIC80 was determined in
vitro in liquid medium by the macrobroth dilution method as per
the guidelines of CLSI reference document M27-A3 [49] for fungi.
2.4. Disc diffusion assay
Strains were inoculated into liquid YPD medium and grown
(1) overnight at 37 8C. The cells were then pelletized and washed
a Aq. AuCl4- (2.5 x 10 -2) Aq. SnCl2 (2.5 x 10 -2)
Mixing
Purple suspension
Refluxing at 60 0C for two hours
Purple suspension
I. Centrifugation
II. Washing with double distilled water
Gold nanoparticles
b Aq. AuCl4- (2.5 x 10 -2) Aq. NaBH4 (2.5 x 10 -2)
Mixing
Dark green suspension
Refluxing at 60 0C for two hours
Pale yellow suspension
I. Centrifugation
II. Washing with double distilled water
Gold nanoparticles
Fig. 1. The flow chart for the synthesis of gold nanoparticles through solvothermal
method using (a) SnCl2 and (b) NaBH4 as the reducing agents.
 Author's personal copy
14 T. Ahmad et al. / Materials Research Bulletin 48 (2013) 12–20
thrice with distilled water. Approximately 105 cells/ml were
inoculated in molten agar media at 40 8C and poured into
100 mm diameter Petri plates. The test gold nanoparticles at the
concentrations of 4  MIC were pipetted onto 4 mm diameter filter
disk. 10 mg/mL of fluconazole was also applied on the discs to serve
as positive controls. The diameter of inhibition zone was recorded
in millimeters after 48 h and was compared with that of control.
The observed zone depicts fungicidal/static characteristic of test
nano-particles [50]. Values were shown in terms of mean  SD of
mean (SEM).
2.5. Proton efflux measurements
The proton pumping activity of Candida was determined by
monitoring the acidity of external medium by measuring the pH as
described earlier [51,52]. Briefly, mid-log phase cells harvested
from YEPD medium were washed twice with distilled water and
routinely 0.1 g cells were suspended in 5 mL solution containing
0.1 M KCl, 0.1 mM CaCl2 and distilled water. Suspension was kept
in a double-jacketed glass container with constant stirring. The
container was connected to a water circulator at 25 8C. Initial pH
was adjusted 7.0 using 0.01 M HCl/NaOH. 100 ml of 7 nm and
15 nm sized gold nanoparticles were added to achieve the desired
concentration of 4 mg/mL and 16 mg/mL, respectively in 5 mL. For
glucose stimulation experiments, 100 ml of glucose was added to
achieve a final concentration of 5 mM. H+ extrusion rate was
calculated from the volume of 0.01 M NaOH consumed. The
experiment was also performed with 5 mM vanadate-potent
inhibitor of the H+-ATPase and 5 mg/mL fluconazole which is an
antifungal drug and used very commonly.
2.6. Measurement of intracellular pH (pHi)
Intracellular pHi was measured as also done earlier [53] with
some modifications. Mid-log phase cells harvested from YEPD
medium were washed twice with distilled water and 0.1 g cells
were suspended in 5 mL solution containing 0.1 M KCl and 0.1 mM
CaCl2, total volume was made upto 5 mL with distilled water. To
study the effect of test gold nanoparticles, its desired concentra-
tions (MIC) were added to the suspension and pH was adjusted to
7.0 in each case. This was followed by an incubation process for
30 min at 37 8C with constant shaking at 200 rpm, pH was again
adjusted to 7.0. Nystatin (20 mM) dissolved in DMSO was added to
the suspension and incubated at 37 8C for 1 h. The value of external
pH at which nystatin permeabilization induced no further shift
was taken for calculation of intracellular pH. The change in pH of
suspension was followed on pH meter with constant stirring.
2.7. Instrumentation
The X-ray diffraction (XRD) patterns of as prepared gold
nanoparticles were carried out on Bruker D8 advance diffractom-
eter using Ni-filtered Cu-Ka radiation of wavelength
(l) = 1.54056 Å in the 2u range of 10–708 with the scanning rate
of 0.058/s. The raw data was subjected to background correction
and the Ka2 lines were removed using the normal stripping
procedure. The size and morphology of the gold nanoparticles
were recorded on FEI Technai G2 high resolution transmission
electron microscope (HRTEM) operating at an accelerating voltage
of 200 kV. The TEM specimen was prepared by dispersing the
nanoparticles in ethanol and then ultrasonicating it for half an
hour. A drop of the suspension was then put on a carbon coated
copper grid and then dried in air at a room temperature. The UV–
vis optical studies have been carried out on ocean-optics lambda
25 spectrophotometer in the wavelength range of 200–900 nm at
room temperature. The surface area studies of the nanoparticles
were obtained on BET surface area analyzer procured from
Quantachrome Instruments Limited (Model Nova 2000e, Make
Quantachrome, USA). In order to carry out the surface area
measurements, the gold nanoparticles was first degassed at
200 8C for 6 h at the degassing port to remove the contaminants
and gases adsorbed on the surface of gold nanoparticles. The
degassed samples were then analyzed and the surface area was
measured by using multi point BET method.
3. Results and discussion
3.1. X-ray diffraction studies
The powder X-ray diffraction patterns of the gold nanoparticles
prepared by the solvothermal method using tin chloride (SnCl2)
and sodium borohydride (NaBH4) as the reducing agents are
shown in Fig. 2(a) and (b) respectively. The observed reflections
belong to the plane families, [1 1 1], [2 0 0] and [2 2 0] respectively,
which corresponds to the formation of gold metal with face
centred cubic (FCC) structure (JCPDS no. – 030921). No diffraction
peak corresponding to any impurity could be seen indicating the
high purity of the gold in the sample. The pattern also shows the
broad peaks which could be possibly due to the small particle size
of nanocrystalline gold powder.
3.2. Transmission electron microscopic (TEM) studies
Transmission electron microscopic (TEM) image of the gold
nanoparticles prepared by the solvothermal method using
tinchloride (SnCl2) as a reducing agent is shown in Fig. 3(a). The
image shows the formation of polygonal gold nanoparticles of
nearly spherical geometry and the size ranges from 5 nm to 50 nm
with the average particle size of 15 nm. However, the high
resolution transmission electron microscopic (HRTEM) micro-
graph as shown in Fig. 3(b), have well defined lattice fringes which
indicates the highly crystalline nature of the gold nanoparticles.
The distance between the adjacent lattice fringes was calculated
and found to be 2.30 Å which matches approximately with the
interplaner distances of the [1 1 1] plane of the face centred cubic
gold. Fig. 4(a) shows the transmission electron microscopic (TEM)
image of the gold nanoparticles synthesized by using sodium
borohydride (NaBH4) as a reducing agent. As can be seen from the
111
b
200 220
10 20 30 40 50 60 70
a
10 20 30 40 50 60 70
Fig. 2. X-ray diffraction patterns of the gold nanoparticles using (a) SnCl2 and (b)
NaBH4 as the reducing agents.
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T. Ahmad et al. / Materials Research Bulletin 48 (2013) 12–20 15

Fig. 3. (a) TEM and (b) HRTEM images of the gold nanoparticles prepared by the
solvothermal method using SnCl2 as a reducing agent.

TEM image, the gold nanoparticles are highly uniform in size,

highly monodisperse and spherical in shape with the average grain
size of 7 nm. The histogram based on the particle size (Fig. 4b)
shows that the particle size varies in the range of 3–20 nm where
the maximum frequency of occurrence is centered at 7 nm. Fig. 4(c)
shows the high resolution transmission electron microscopic
(HRTEM) image of the gold nanocrystals. The image shows the
highly prominent fringe pattern indicating the highly crystalline
nature of the gold nanoparticles. The interplaner distance was also
calculated from the fringe pattern and found to be 1.44 Å which
corresponds with the interplaner distance of the [2 2 0] planes of
the gold nanocrystals. Thus HRTEM studies corroborate well with Fig. 4. (a) TEM micrograph, (b) particle size distribution plot and (c) HRTEM image
the X-ray diffraction studies. of the gold nanoparticles using NaBH4 as the reducing agent.

3.3. UV–vis spectroscopic studies

It is well-known that gold nanoparticles absorb light in the
visible and/or near-infrared region due to localized plasmon
resonance, which is light-induced collective oscillation of conduc-
tion electrons at the metal surface, and the magnitude of the
resonance wavelength depends on particle size, shape, and local
dielectric environment [54]. Fig. 5(a) shows the UV–vis absorption
spectrum of the gold nanoparticles prepared by tinchloride as a
reducing agent. As can be seen from the spectrum, the peak below
300 nm could be due to the charge transfer transition (from ligand
p to metal s*) of unreduced AuCl4 ions [55]. The absorption peak
at 590 nm represents the transverse surface plasmon resonance,
while the peak at the longer wavelength side (720 nm) may be
assigned to the longitudinal surface plasmon resonance of the
anisotropic gold nanoparticles. The frequency and intensity of the
longitudinal surface plasmon resonance (SPR) band depend on the
degree of aggregation and the orientation of the individual
particles within the aggregates [56]. The small hump at the short
wave length located around 420 nm could be due to the out of
plane surface plasmon resonance of the individual gold nanocrys-
tals. Fig. 5(b) shows the UV–vis absorption spectrum of the
solvothermally synthesized gold nanoparticles using sodium
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16 T. Ahmad et al. / Materials Research Bulletin 48 (2013) 12–20
Fig. 5. UV–vis absorption spectrum of the gold nanoparticle suspension synthesized
by the solvothermal method using (a) SnCl2 and (b) NaBH4.
borohydride (NaBH4) as the reducing agent. The absorption band
appears at 525 nm is characteristic of the surface plasmon
resonance peak of the spherical gold nanoparticles which is
normally observed between 520 nm and 530 nm [57,58]. In
addition to the SPR peak, a weak band of low intensity centered
at 730 nm can also be observed which may have an origin due to
the presence of gold nanoparticle couples in the gold nanoparticle
suspension. However, the peak at 275 nm can be assigned due to
the unreduced AuCl4 ions present in the reaction mixture [55].
The UV–vis reflectance spectra of gold nanoparticles prepared
by the solvothermal method using tin chloride and sodium
borohydride are shown in Fig. 6(a) and (b) respectively. A sharp
increase in reflectance can be observed around 500 nm which may
be attributed to the presence of surface plasmon resonance in gold
nanoparticles. Also a small fluctuation at 750 nm may be assigned
due to the longitudinal surface plasmon resonance or to the
Fig. 6. Reflectance spectra of gold nanoparticles using (a) SnCl2 and (b) NaBH4
respectively.
plasmon resonance of the nanoparticle couples formed in the solid
powder due to small amount of agglomeration.
3.4. BET surface area studies
The surface area properties of gold nanoparticles have been
measured using multipoint BET method at liquid nitrogen
temperature (78 K). Fig. 7(a) shows the adsorption isotherm of
the gold nanoparticles prepared by solvothermal method using
tinchloride (SnCl2) as a reducing agent. The adsorption isotherm
represents the unrestricted monolayer–multilayer adsorption [59]
and resembles to the type II adsorption isotherm. The BET plot of
the gold nanoparticles is shown in Fig. 8(a). The range of linearity is
restricted to a limited part of the isotherm which is not normally
outside the p/p8 range of 0.05–0.30. The surface area of the gold
nanopowder was found to be 269 m2/g. The nitrogen adsorption
isotherm of the gold nanoparticles prepared by the solvothermal
method using sodium borohydride (NaBH4) as a reducing agent is
shown in Fig. 7(b). The isotherm resembles to the type VI
adsorption isotherm and sharpness of the steps in this type of
isotherm depends on the system and the temperature at which the
multilayer adsorption occurs [59]. Fig. 8(b) shows the BET plot of
the gold nanoparticles. The surface area of the gold nanoparticles
was determined using the multipoint BET equation and found to be
329 m2/g. The surface area obtained is much higher than reported
so far up to the best of our knowledge [60]. Powder porosity and
pore size distribution of as prepared gold nanoparticles have been
carried out using the DA (Dubininz–Astakhov) model. Fig. 9(a) and
(b) show the DA plots of the gold nanoparticles prepared using tin
chloride and sodium borohydride as a reducing agent respectively.
A pore radius of 9.6 Å could be found using this method for gold
nanoparticles synthesized by using tinchlorode (Fig. 9a) as a
reducing agent, whereas for borohydride synthesized gold
nanoparticles, the pore radius was found to be 4.8 Å as shown
in Fig. 9(b). The much smaller pore radius of the borohydride
synthesized gold nanoparticles confirms the smaller grain size and
Fig. 7. (a) N2 adsorption isotherm of the gold nanoparticles using (a) SnCl2 and (b)
NaBH4 as the reducing agent.
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T. Ahmad et al. / Materials Research Bulletin 48 (2013) 12–20
Fig. 8. BET plot of the gold nanoparticles using (a) SnCl2 and (b) NaBH4 as the
reducing agent.
the close packing of the nanoparticles having very high specific
surface area than the tinchloride synthesized gold nanoparticles.
3.5. Antifungal activity
The antifungal effect of the as synthesized gold nanoparticles
was performed on the three clinical strains of Candida. The gold
nanoparticles were shown an excellent size dependant antifungal
activity.
3.6. Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) was defined as
the lowest concentration of the gold nanoparticles that causes 80%
decrease in absorbance (MIC80) compared with that of the control
(no test compound). The MIC80 values of 7 nm gold nanoparticles
against Candida albicans, Candida tropicalis and Candida glabrata
were found to be 4, 4 and 8 mg/mL respectively. Although, the
Fig. 9. DA plots of the gold nanoparticles using (a) SnCl2 and (b) NaBH4 as the
reducing agents.
17
MIC80 value for 15 nm gold nanparticles was found to be 16 mg/mL
in all the three strains of Candida.
3.7. Disc diffusion assay
The results summarized in Table 2 give the sensitivity assay,
using standard discs of 7 nm gold nanoparticles, 15 nm gold
nanparticles and fluconazole. All the three types of Candida isolates
showed high degree of sensitivity which is evident from the zone of
clearance in each case in Fig. 10(a). Index of sensitivity defined as
X Zone diameter ðmmÞ
¼ clearing ðmmÞ=mg
concentration ðmg=mLÞ
is maximum (4.2) for C. albicans when treated with 7 nm gold
nanoparticles and is minimum (1.1) for C. glabrata when treated
with 15 nm gold nanparticles. The discs impregnated with water
(negative control) showed no zone of inhibition and hence had no
effect on the strains tested in the present study.
3.8. Proton efflux measurements
Candida cells susceptible to the gold nanoparticles were
examined for the ability of pump intracellular protons to the
external medium (as measured by the alteration of pH of the
external medium) using the gold nanoparticles in the size range of
7–15 nm. Table 3 shows relative rates of H+-efflux by Candida
species in presence of 7 nm gold nanoparticles (4 mg/mL), 15 nm
gold nanparticles (16 mg/mL), vanadate (5 mM), and fluconazole
(5 mg/mL). Retentions of H+-efflux in C. albicans were found to be
19% and 23% for 7 and 15 nm gold nanoparticles, respectively. H+-
efflux rate was also decreased to 27% and 29% for 7 and 15 nm gold
nanparticles, respectively, in case of C. tropicalis. In case of C.
glabrata, H+-efflux again decreased to 57% and 51% when the cells
were treated with 7 and 15 nm gold nanoparticles, respectively.
Glucose (5 mM) stimulated H+-efflux in all the three strains by 3–5
folds. Glucose stimulated H+-efflux was also inhibited by 7 and
15 nm sized nanoparticles of gold. Glucose-stimulated H+-efflux
rates in C. albicans, C. tropicalis and C. glabrata isolates with respect
to control, were 39%, 47% and 59% in the presence of 7 nm gold
nanoparticles and 43%, 57% and 67% in presence of 15 nm gold
nanoparticles. In addition, the effect of vanadate and fluconazole
were also analyzed on the proton pumping activity. H+-extrusion
in every case was inhibited upto 91–100% following the addition of
5 mM vanadate – a well known inhibitor of H+-ATPase, whereas
fluconazole does not have any significant effect on the acidification
of the external medium.
3.9. Measurement of intracellular pH (pHi)
The internal pH of yeast cells is maintained between 6.0 and 7.5
by plasma membrane H+-ATPase activity. We tried to investigate
whether cells with normal H+-ATPase activity maintains the
constant internal pH as compared to cells with low H+-ATPase
activity. Fig. 10(b) shows changes in the pattern of pHi with control
and treated cells. Only yeast control cells with normal H+-ATPase
activity maintain the pHi (6.8), while the treated cells show
increase in internal acidification. The decrease in pHi was more in
cells that were exposed to 7 nm gold nanoparticles than 15 nm
gold nanoparticles.
3.10. Discussion on antifungal activity
Low MIC values of the nanoparticles obtained against various
clinically important Candida species encouraged us to study their
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18 T. Ahmad et al. / Materials Research Bulletin 48 (2013) 12–20

Table 2
In vitro sensitivity of 7 nm and 15 nm gold nanoparticles against Candida isolates as determined by disc diffusion assay. Sensitivity index is expressed as mean  SD and was
calculated as diameter of zone of inhibition (mm)/concentration of drug (mg/mL). Each isolate was tested in duplicate.

Candida isolates Sensitivity index (mm/mg)

7 nm gold nanoparticles 15 nm gold nanoparticles Fluconazole

Candida albicans ATCC 10261 4.2  1.14 3.8  0.98 3.2  0.74
Candida tropicalis ATCC 750 3.8  0.73 3.1  0.34 1.9  0.34
Candida glabrata ATCC 90030 2.1  0.51 1.1  0.12 1.0  0.08

mode of action. The effect of both the nanoparticles is spontaneous
and irreversible which suggests the cellular target(s) accessible to
these nanoparticles. Therefore, the effect of 7 nm and 15 nm gold
nanoparticles on H+ extrusion by plasma membrane H+-ATPase
was explored for various clinical Candida isolates. Candida isolates
showing susceptibility to the test gold nano-particles also showed
inhibition of H+-ATPase-mediated proton pumping, thus suggest-
ing that the two events are linked. Glucose induced acidification of
the external medium by yeast cells are a convenient measure of H+-
ATPase-mediated proton pumping [61]. The enzyme may exist in
different conformational states in the two situations. It was also
found that the fluconazole had no significant effect on H+-ATPase
where as vanadate completely inhibits H+-efflux. Thus, these
nanoparticles could interact directly with proteins in the regula-
tion of the proton gradient across the plasma membrane. One such
protein is H+-ATPase; an ATP-driven enzyme that transforms the
energy of ATP hydrolysis to electrochemical potential differences
of protons across diverse biological membranes via the primary
active transport of H+. In turn, the transmembrane electrochemical
potential of H+ is used to drive a variety of secondary active
transport systems via H+-dependent symporters, antiporters and
channel-mediated transport systems [62]. The interaction of gold
nanoparticles with such biologically active enzymes may alter
their normal conformations leading to the loss of the activity. Thus,
the fungal membrane is incapable to regulate the H+ transport
across the membrane leading the retardation of the cell growth,
which finally causes the cell death. The schematic diagram
showing the action of gold nanoparticles on the fungal cell is
shown in Fig. 11(a). The antifungal activity was found to be particle
size dependent. The extent of inhibition increased with decrease in
the particle size. This may be due to the fact that with a decrease in
the particle size, there is an increase in the surface area of the gold
nanoparticle, which enhances its interaction with the binding sites
of the plasma membrane proteins. Furthermore, it may be
assumed that the smaller gold nanoparticles may diffuse easily
through the cell membrane to the inside of the cell. Since gold (soft
acid) have greater tendency to react with the sulphur and
phosphorus containing soft bases. Thus gold nanoparticles may

Fig. 10. (a) Disk diffusion assay of Candida albicans ATCC 10261(A), Candida tropicalis ATCC 750 (B) and Candida glabrata ATCC 90030 (C). Cells were treated with 7 nm and
15 nm gold nanoparticles and fluconazole. Zone of inhibitions were measured after 48 h incubation of plates at 37 8C. (b) Intracellular pH in presence of test gold nano-
particles in Candida species. Mid logarithmic cells were incubated with MIC of 7 nm and 15 nm gold nanoparticles.
 Author's personal copy

T. Ahmad et al. / Materials Research Bulletin 48 (2013) 12–20 19

Table 3
Effect of 7 nm and 15 nm gold nanoparticles on the rate of H+-efflux by various Candida isolates at pH 7.0. Cells were suspended in 0.1 mM CaCl2 and 0.1 M KCl at 25 8C. Control
had an average (of 4 independent recordings) H+ efflux rate of 5.82  0.88 nmol/min/mg cells in standard isolates (1); 5.32  1.1 nmol/min/mg yeast cells in clinical isolates (1*)
and 4.96  0.64 nmol/min/mg yeast cells in resistant isolates (1**). Values in parentheses give the %-age inhibition of H+-efflux w.r.t. control.

Incubation with Range of relative H+-efflux rate (1011 moles min1 mg cells1)

C. albicans C. tropicalis C. glabrata

Control 1 1* 1**
7 nm gold nanoparticles (4 mg/mL) 1.13  0.17 (81) 1.39  0.18 (73) 2.53  0.91 (49)
15 nm gold nanoparticles (16 mg/mL) 1.89  0.23 (77) 1.51  0.32 (71) 2.84  0.32 (43)
Glucose (5 mM) 21.32  1.19 19.17  2.1 20.25  2.5
Glucose + 7 nm gold nanoparticles 8.21  1.94 (61) 9.10  2.1 (53) 11.89  1.78 (41)
Glucose + 15 nm gold nanoparticles 9.07  1.2 (57) 10.85  1.9 (43) 15.53  2.12 (33)
Vanadate (5 mM) 0  0 (100) 0  0 (100) 0.13  0.07 (97)
Fluconazole (5 mg/ml) 4.69  0.2 (19) 3.61  0.4 (31) 4.41  0.78 (11)

269 m2/g. However, uniform, monodisperse and spherical nano-

particles of gold of average size of 7 nm with higher surface area of
329 m2/g have been obtained using NaBH4. The reducing agent
NaBH4 is found to be more effective than SnCl2 because it produces
highly uniform and monodisperse gold nanoparticles with large
surface area. UV–vis spectrum of gold nanoparticles (obtained
from SnCl2) shows the presence of peaks at 590 nm and 720 nm,
which corroborates to transverse and longitudinal surface plasmon
resonance respectively and a hump at 420 nm is due to the out of
plane SPR of the gold nanoparticles. However, gold nanoparticles
using sodium borohydride shows band of high intensity at 525 nm
due to surface plasmon resonance (SPR) of spherical gold
nanoparticles. The gold nanoparticles show size dependent
antifungal activity against Candida and small sized nanoparticles
(7 nm) show a high fungicidal action than the larger ones. Thus, it
can be concluded that gold nanoparticles can be used as an
effective agent against human fungal pathogens. However,
exhaustive experimental trials are needed before using gold
nanoparticles as potential antifungal agent.

Acknowledgements

TA thanks to Department of Science and Technology (DST) for

the financial support (SR/FTP/CS-120/2006). The authors thank
Professor Ashok K. Ganguli for the use of HRTEM facility (funded by
DST, Nano Mission) at IIT Delhi.

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