Colloids and Surfaces B: Biointerfaces 101 (2013) 162–170

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Colloids and Surfaces B: Biointerfaces

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Size and shape dependant antifungal activity of gold nanoparticles:

A case study of Candida
Irshad A. Wani, Tokeer Ahmad ∗
Nanochemistry Laboratory, Department of Chemistry, Jamia Millia Islamia, New Delhi 110025, India

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

Article history: A simple and economical sonochemical approach was employed for the synthesis of gold nanoparticles.
Received 26 April 2012 The effect of the reducing agents has been studied on the particle size, morphology and properties at
Accepted 7 June 2012 the same ultrasonic frequency under ambient conditions. Gold nanodiscs of average diameter of 25 nm
Available online 16 June 2012
were obtained using tinchloride (SnCl2 ) as a reducing agent, while sodium borohydride (NaBH4 ) produced
polyhedral structures of the average size of 30 nm. The time evolution of the UV–visible absorption spectra
Keywords:
of the gold nanostructures shows the origin of peaks due to higher order quadrupolar modes apart from
Nanoparticles
the peaks of the in plane and out plane dipolar surface plasmon modes. Surface area studies reveal the
Sonochemical synthesis
Electron microscopy
much higher surface area of the gold nanodiscs (179.5 m2 /g), than the gold nanoparticles (150.5 m2 /g)
Surface area prepared by the sodium borohydride as the reducing agent. The gold nanoparticles exhibit excellent
Antifungal activity antifungal activity against the fungus, Candida. We investigated the effect of the gold nanoparticles on
the H+ -ATPase mediated H+ pumping by various Candida species. Gold nanodiscs displayed the stronger
fungicidal activity compared to the gold polyhedral nanoparticles. The two types of gold nanoparticles
inhibit H+ -ATPase activity at their respective MIC values.
© 2012 Elsevier B.V. All rights reserved.

1. Introduction expansion/compression cycles or grow over one (sometimes two or

Gold nanostructures have been the focus of intense research
due to their fascinating optical, electronic, and chemical properties
as well as promising applications in nanoelectronics, biomedicine,
sensing, and catalysis [1,2]. The size and shape of gold nanos-
tructures significantly affects the intrinsic properties and relevant
applications, thus the great efforts have been taken to the con-
trolled synthesis of gold nanostructures in the past few years [3].
In particular, a variety of wet chemical methods have been devel-
oped to fabricate gold nanoparticles with various shapes such as
rods [3], wires [4], plates [5,6], prisms [7], cubes [8], polyhedra [9]
and branched particles [10]. Sonochemical method has received a
great attention of researchers to produce metallic nanostructures
in the past decade. This route involves the use of acoustic waves
with frequencies of more than 20 kHz which causes the structural
changes and accelerates the chemical reactions [11]. The initializa-
tion of the sonochemical reactions in aqueous solution is caused
by the acoustic cavitation. The evolution of cavitation bubbles fol-
lows the sound field in a liquid during the cycles of compression
and expansion and is stimulated by time-varying pressure. Bub-
bles either oscillate around their equilibrium position over several
∗ Corresponding author. Tel.: +91 11 26981717x3261; fax: +91 11 26980229.
E-mail address: tahmad3@jmi.ac.in (T. Ahmad).
three) acoustic cycles to double their initial size and finally collapse
violently. This short-time cavitational collapse produces hot spots
inside the medium with transient temperatures of about 5000 K
and the pressures can reach over 1800 atm [12,13]. If a medium is
heterogeneous and has metallic particles in it, tremendous heat-
ing/cooling rates of bubbles and transient high temperature inside
hot spots cause the dispersion of particles, reduction in size, acti-
vation, and changes of the crystalline structure [14].
The effect of the ultrasound frequency on rate of reduction
of Au(III) and the synthesis of gold nanoparticles was studied
by Okitsu et al. [15]. Sonochemical formation of gold nanopar-
ticles with a narrow size distribution in nonionic polyethylene,
glycol monostearate (PEG40-MS), polyoxyethylenesorbitan mono-
laurate, anionic SDS, or water-soluble polyvinylpyrrolidone (PVP)
under an ultrasound wave of 200-kHz frequency are also reported
[16,17]. Single-crystalline Au nanobelts have been sonochemi-
cally synthesized in presence of ␣-d-glucose and incorporated into
the functional electronic, optoelectronic, and sensing miniatur-
ized devices [18]. Single-crystal Au nanoprisms with triangular or
hexagonal shape, 30–40 nm planar dimension, and 6–10 nm thick-
ness in an ethylene glycol solution in the presence of PVP were also
synthesized by a simple sonochemical method [19].
It is well known that inorganic nanoparticles can act as antibac-
terial and antifungal agents, and thus have the ability to interact
with microorganisms [20–24]. Few reports are also available which

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http://dx.doi.org/10.1016/j.colsurfb.2012.06.005
 I.A. Wani, T. Ahmad / Colloids and Surfaces B: Biointerfaces 101 (2013) 162–170
shows the gold nanoparticles exhibiting good antimicrobial activity
[25–27]. 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 [28]. In this paper, we report the sono-
chemical method for the synthesis of anisotropic gold nanoparticles
and studied their size and shape dependent fungicidal activity
against various strains of Candida. To the best of our knowledge
this is the first report showing the antifungal activity of gold
nanoparticles against the fungus Candida. The gold nanoparticles
were characterized by Powder X-ray diffraction (XRD), Transmis-
sion electron microscopy (TEM), UV–visible spectroscopy and BET
surface area studies.
2. Experimental
2.1. Materials and Methods
All the chemicals listed are of analytical grade and were used
without any further purification. Chloroauric acid (HAuCl4 ) and
Sodium borohydride (NaBH4 ) were purchased from Spectrochem
(Mumbai, India). Tin chloride (SnCl2 ) was obtained from Merck
(India, 99%). All of the solutions were prepared in double distilled
water.
The gold nanoparticles were synthesized by an ultrasonic
method using two different reducing agents. In the first reaction,
20 ml of 2.5 × 10−2 M aqueous chloroauric solution (HAuCl4 ) was
taken in a conical flask fitted inside a sonicating bath operating at
20 kHz frequency with the power intensity of 2.5 W/cm2 . To this
aqueous gold salt solution, 20 ml of 2.5 × 10−2 aqueous tin chloride
(SnCl2 ) was added dropwise with the help of a buret. The resulting
mixture turned light brown in color. The light brown suspension
was subjected to ultrasonic waves for 3 h during which the reac-
tion mixture turned purple in color. On further sonication for 7 h
the color changed from light violet to deep violet and finally to deep
brown. The sonication was stopped at this stage and the reaction
mixture was centrifuged at 8000 rpm for 20 min. The precipitate
was washed thrice with double distilled water and then dried in an
oven at 60 ◦ C.
The second reaction was performed under the same environ-
mental conditions, except that aqueous tin chloride (SnCl2 ) was
replaced by the aqueous sodium borohydride (NaBH4 ) solution of
the same molar concentration. The reaction mixture obtained ini-
tially was then subjected to ultrasonic waves for 8 h during which
the color changed from light brown to yellowish brown. Upon son-
ication for next 2 h the precipitation occurred and the precipitate
settled down as a mud at the bottom of the flask. The precipitate
was centrifuged and washed with double distilled water and then
dried in an oven at 60 ◦ C.
2.2. Microorganisms and culture conditions
The strains used in this study are listed in Table 1. All of
the strains 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)
in addition were used to maintain the culture. Vanadate was pur-
chased from Aldrich chemicals (Germany), whereas all inorganic
chemicals used in the antifungal essay were of analytical grade
and procured from Merck (India). Fuconazole and vanadate were
dissolved in water to make to desired concentrations.
2.3. Antifungal susceptibility test
Microtiter assay: Cells were grown for 48 h at 30 ◦ C to obtain
single colonies, which were resuspended in a 0.9% normal saline
163
Table 1
Isolates used in the present study.
Classification of isolates Type of isolates
Standard (n = 3)
ATCC 90028 C. albicans
ATCC 750 C. tropicalis
ATCC 90030 C. glabrata
Clinical (n = 8)
HIV patients (n = 3) C. krusei, C. albicans, C. glabrata
Cutaneous (n = 2) C. tropicalis, C. krusei
Burn patients (n = 2) C. glabrata, C. tropicalis
Candidemia (n = 1) C. tropicalis
solution to give an optical density at 600 nm (OD600 ) of 0.1. The
cells were then diluted 100-fold in YNB medium containing 2%
glucose and the respective auxotrophic supplements. The diluted
cell suspensions were added to the wells of round-bottomed 96-
well microtiter plates (100 ␮l/well) containing equal volumes of
medium (100 ␮l/well) and different concentrations of the gold
nano-particles [29,30]. A drug-free control was also included. The
plates were incubated at 37 ◦ C for 24 h. The MIC test end point was
evaluated both visually and by observing the OD595 in a microplate
reader (Bio-Rad, iMark, US) and is defined as the lowest compound
concentration that gave ≥80% inhibition of growth compared to the
controls.
2.4. Growth curve studies
Before the tests were performed, all the Candida species were
sub-cultured at least twice and grown for 24 h at 37 ◦ C. For growth
studies, 106 cells (optical density A595 = 0.1) of all the isolates
were grown aerobically in 50 ml media on automated shaker at
37 ◦ C with agitation of 200 rpm. Test gold nanoparticles with final
concentrations of 0 × MIC (control), 0.5 × MIC, and 1 × MIC for
standard and clinical isolates were added to the cultures. At a pre-
determined time (after 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 h)
aliquots were taken and growth was followed turbidometrically at
595 nm using Lab Med Spectrophotometer (USA). Optical density
was recorded for each concentration against time in hours.
2.5. Proton efflux measurements
The proton pumping activity of Candida was determined by
monitoring acidification of external medium by measuring the pH
as previously described [31,32]. Briefly, mid-log phase cells har-
vested from YEPD medium were washed twice with distilled water
and routinely 0.1 g cells were suspended in 5 ml solution contain-
ing 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 ◦ C. Initial
pH was adjusted 7.0 using 0.01 M HCl/NaOH. 100 ␮l of 25 nm gold
nanodiscs and 30 nm polyhedral gold nanoparticles were added
to achieve the desired concentration of 16 ␮g/ml and 32 ␮g/ml,
respectively, in 5 ml. For glucose stimulation experiments 100 ␮l
of glucose was added to achieve a final concentration of 5 mM. H+
extrusion rate was calculated from the volume of 0.01 N NaOH con-
sumed. The experiment was also performed with 5 mM vanadate
– a potent inhibitor of the H+ -ATPase and 5 ␮g/ml fluconazole –
antifungal drugs commonly used.
2.6. Measurement of intracellular pH (pHi)
Intracellular pHi was measured as done earlier [33] with slight
modifications. Mid-log phase cells harvested from YEPD medium
were washed twice with distilled water and 0.1 g cells were sus-
pended in 5 ml solution containing 0.1 M KCl and 0.1 mM CaCl2 ,
164 I.A. Wani, T. Ahmad / Colloids and Surfaces B: Biointerfaces 101 (2013) 162–170

total volume was made upto 5 ml with distilled water. To study 111
the effect of test gold nano-particles, its desired concentrations
(MIC) were added to the suspension and pH was adjusted to 7.0
in each case. Following incubation for 30 min at 37 ◦ C with con- a
stant shaking at 200 rpm, pH was again adjusted to 7.0. Nystatin
200
(20 ␮M) dissolved in DMSO was added to the suspension and incu- 220
bated at 37 ◦ C 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. b

2.7. Instrumentation

The powder X-ray diffraction of the gold nanoparticles was

recorded on Bruker D8 advance diffractometer using Ni-filtered
10 20 30 40 50 60 70
Cu-K␣ X-rays of wavelength (␭) = 1.54056 Å in the 2Â range of
2-Theta
10◦ –70◦ with the scanning rate of 0.05◦ /s. The raw data was sub-
jected to background correction and the K␣2 lines were stripped Fig. 1. X-ray diffraction patterns of the gold nanoparticles using (a) SnCl2 and (b)
off using stripping procedure. The size and morphology of the gold NaBH4 as the reducing agents.
nanoparticles have been recorded on FEI Technai G2 TEM operating
at an accelerating voltage of 200KV. The TEM grid was prepared
by adding a small quantity of the sample into 10 ml ethanol and samples were placed in an oven to dry at ambient temperature
then ultrasonicated for half an hour to ensure proper dispersion before examining. The UV–visible optical studies were recorded
of the sample into the ethanol. A drop of this dispersed sample on ocean-optics lambda 25 spectrophotometer in the wavelength
was then placed on a TEM grid coated with the carbon film. The range of 200–900 nm at room temperature. The surface area

Fig. 2. (a) TEM image and (b) HRTEM image of the gold nanoparticles using tinchloride as a reducing agent. The inset of Fig. 2(a) and (b) shows high magnification TEM
image and the particle size distribution plot of the gold nanodiscs respectively. (c) TEM image and (d) HRTEM image of the gold nanocrystals using sodium borohydride as
the reducing agent.
 I.A. Wani, T. Ahmad / Colloids and Surfaces B: Biointerfaces 101 (2013) 162–170 165

studies of the samples were obtained by B.E.T. surface area analyzer

procured from Quantachrome Instruments Limited (Model Nova
2000e, Make Quantachrome, USA). The samples were first degassed
at 200 ◦ C for 6 h to remove the contaminants such as water and
other adsorbed gases. The degassed samples were then subjected
to analysis and the surface area was recorded by using the multiple
point B.E.T. method.

3. Results and discussion

3.1. Powder X-ray diffraction studies

Fig. 1(a) and (b) shows the powder X-ray diffraction (XRD) pat-
terns of gold nanoparticles prepared by the sonochemical reduction
of Au(III) ions using tin chloride (SnCl2 ) and sodium borohydride
(NaBH4 ) as the reducing agents respectively. The diffraction peaks
in both the patterns have been indexed as [1 1 1], [2 0 0] and [2 2 0]
to the lattice planes of the face centered cubic gold with the cor-
responding 2Â values of 38.184, 44.392 and 64.476 respectively
(JCPDS file no: 04-0784). The X-ray diffraction patterns reveal the
monophasic nature and high order of crystallinity in ultrafine gold
nanoparticles.

3.2. TEM studies

The TEM image of ultrasonically synthesized gold nanocrystals

using tinchloride as the reducing agent is shown in Fig. 2(a). The
TEM images show the formation of nanodiscs of gold which are cir-
cular in shape with smooth and well defined boundaries. The visible
feature of the TEM images of the gold nanodiscs is depicted by the
presence of the fringes of variable contrasts (inset of Fig. 2(a)). The
origin of these contrast variation may be explained due to the pres-
ence of dislocation networks on [1 1 1] plane, because the surface
layer presents the lattice parameters slightly different from those of
the bulk material [34]. The crystal lattice restructures to accommo-
date the difference between the parameters of the surface layer and
those of the inner crystal, thus leading to such dislocations [35]. The
origin of these contrast fringes has also been explained on the basis
of mass thickness contrast mechanism [36]. The high resolution
transmission electron microscopic (HRTEM) image of the gold nan- Fig. 3. Time evolution absorption spectra of gold nanoparticle suspension using (a)
odisc is shown in Fig. 2(b). The image shows the well defined lattice tinchloride and (b) sodium borohydride as the reducing agents. Insets in Fig. 3(a)
fringes which indicate that the sample is highly crystalline. The dis- and (b) shows the aliquots of the reaction mixtures confirming the presence of gold
tance between the adjacent fringes have been calculated and found nanodiscs and polyhedral gold nanoparticles respectively.

to be ∼2 Å which matches approximately with the interplaner plan-

ner distance between the [2 0 0] planes of the face centered cubic
gold. The particle size histogram in the inset of Fig. 2(b) shows
that the size of these nanodiscs varies in the range of 10–55 nm
with the average disc size of 25 nm and the maximum frequency of
occurrence is located at ∼23 nm.
Fig. 2(c) shows the TEM image of the ultrasonically synthe-
sized gold nanoparticles using sodium borohydride as a reducing
agent. The particles show low size and shape uniformity. The micro-
graph shows the size of the polyhedral gold nanocrystals ranging
from 20 nm to 40 nm with varying edge lengths having average
size of 30 nm. The HRTEM image of gold nanoparticles is shown in
Fig. 2(d). The image shows the well defined fringe pattern indicat-
ing the crystalline nature of the gold nanoparticles. The interplaner
spacing was calculated from the fringe pattern and was found to
be 1.41 Å which matches approximately with the interplaner dis-
tances between the [2 2 0] planes of the face centered cubic gold
nanoparticles.
3.3. UV–visible spectroscopic studies
The progress of the reaction and structural features of gold
nanoparticles were established by the UV–visible spectroscopy. The
inset of Fig. 3(a) and (b) shows the aliquots of the reaction mixtures
taken at the completion of the reaction. The deep brown and bright
yellow colors indicate the presence of gold nanoparticles in solu-
tion. Fig. 3(a) shows the time evolution of absorption spectra of the
gold nanoparticle suspension prepared using tincloride (SnCl2 ) as a
reductant. A peak located below 300 nm is a unique fingerprint for
AuCl4 − and directly originates from ␲ to ␴* charge transfer transi-
tion of the unreduced AuCl4 − ions present in the solution [37,38].
The formation of gold nanoparticles was evident after 15 min of the
reaction when an absorption spectrum revealed an in-plane dipole
plasmon resonance band around 530 nm. The intensity of this band
enhanced gradually with time due to increase in the density of the
number of particles in the colloidal solution. After sonicating the
reaction mixture for 5 h a new set of peaks of increasing intensity
arise at 360 nm and 330 nm corresponds to higher order plasmon
modes of the gold nanodiscs which are often called quadrupolar
modes. A band of weak intensity appears around 450 nm after 10 h
of sonication which represent the out of plane dipole plasmon res-
onance of the gold nanodiscs. The oscillation of absorption band is
visible in the range of 300–600 nm which may be attributed to the
166 I.A. Wani, T. Ahmad / Colloids and Surfaces B: Biointerfaces 101 (2013) 162–170
Fig. 4. N2 adsorption isotherm of the gold nanoparticles using (a) SnCl2 and (b) NaBH4 as the reducing agent (c) and (d) gives the B.E.T. plots of the gold nanoparticles using
SnCl2 and NaBH4 as the reducing agent respectively.
shape transition in gold nanostructures [14] as shown in the TEM
image.
Time evolution of absorption spectra of sonochemically syn-
thesized gold nanoparticle suspension using sodium borohydride
is shown in Fig. 3(b). The spectra give two absorption bands,
one of moderate intensity located around 525 nm and another
of weak intensity appeared around 770 nm can be assigned to
the SPR absorption bands of gold nanoparticles for two differ-
ent size distributions [39] which corroborates to the TEM studies.
After sonication for 5 h, the absorption band located around
525 nm shows a slight blue shift of few nanometers which may
arise from the electromagnetic coupling of the particle assem-
blies or aggregates [40]. In addition, a small peg located at
380 nm can be assigned to the out of plane SPR of the gold
nanoparticles.
3.4. B.E.T. surface area studies
The N2 adsorption isotherm of the samples was measured by
Brunauer–Emmett–Teller (B.E.T.) method with the help of B.E.T.
surface area analyzer. B.E.T method is the most widely used pro-
cedure for the determination of the surface area of solid materials
in spite of the oversimplification of the model on which the the-
ory is based [41]. Fig. 4(a) shows the adsorption isotherm of the
gold nanopowder prepared by using Tin chloride (SnCl2 ) as a
reducing agent. The isotherm resembles to the type VI adsorption
isotherm according to the BDDT (Brunauer, Deming, Deming and
Teller) type of classification [42]. The sharpness of the isotherm
depends on the system as well as temperature and represents
the multilayer adsorption on the uniform surfaces [43]. Fig. 4(c)
shows the B.E.T. plot of the as synthesized gold nanoparticles
which gives the specific surface area of 179.5 m2 /g. Fig. 4(b) shows
the nitrogen adsorption isotherm of the gold nanoparticles syn-
thesized by the sonochemical method using sodium borohydride
(NaBH4 ) as a reducing agent. It can be seen that the curve resem-
bles the type II adsorption isotherm. This type of adsorption
isotherm represents unrestricted monolayer–multilayer adsorp-
tion [43]. The BET plot of the gold nanoparticles is shown
in Fig. 4(d). The range of linearity is restricted to a limited
part of the isotherm - usually not outside the P/P◦ range of
0.05–0.30. The surface area of the gold nanoparticles was found to
be 150.5 m2 /g.
Powder porosity of the as synthesized gold nanoparticles was
determined with the help of DA (Dubinin–Astakhov) and BJH
(Barrett–Joyner–Halenda) models. For ultrasonically synthesized
gold nanodiscs using tinchloride as a reducing agent, the pore
radius varies in the range of 9.6 Å in DA plot as shown in Fig. 5(a)
up to 25 Å in BJH plot as shown in Fig. 5(b). Whereas pore radius
ranges from 9.6 Å to 38 Å for gold nanoparticles synthesized by
using sodium borohydride (NaBH4 ), as shown in Fig. 5(c) and (d)
respectively. Thus, gold nanodiscs possess narrow pore size distri-
bution than the gold nano polyhedral synthesized by using sodium
borohydride as a reducing agent. Thus the narrow pore size distri-
bution of the gold nanodiscs confirms the smaller grain size and
 I.A. Wani, T. Ahmad / Colloids and Surfaces B: Biointerfaces 101 (2013) 162–170
Fig. 5. (a) DA and (b) BJH plots of gold nanoparticles using SnCl2 as a reducing agent. (c) DA and (d) BJH plots of gold nanoparticles using NaBH4 as a reducing agent.
the close packing having high specific surface area than the boro-
hydride synthesized gold nanopolyhedrals.
3.5. Antifungal activity of gold nanoparticles
3.5.1. Minimum inhibitory concentration
The minimum inhibitory concentration (MIC) was defined as
the lowest concentration of the nano-particles that causes 80%
decrease in absorbance (MIC80 ) compared with that of the con-
trol (no test compound). The MIC80 values of 25 nm gold nanodiscs
against both the standard lab strain as well as clinical Candida
isolates ranges between 16 and 32 ␮g/ml while as for 30 nm poly-
hedral gold nanocrystals, MIC80 values ranges between 32 and
128 ␮g/ml. In vitro studies have shown that the test gold nano-
particles significantly inhibit both standard as well as clinical
Candida isolates.
3.5.2. Growth studies
In case of growth curve studies, the effect of increasing concen-
trations of the test gold nanoparticles on the growth of test Candida
species has been studied. Fig. 6(A) and (B) depict the growth curves
of lab standard Candida albicans ATCC 90028 and clinical strain
Candida tropicalis 105 in the presence of the test gold nanoparti-
cles at MIC and sub-MIC concentrations. The absorbance obtained
for the growth control (only organism) showed that the Candida
reached the stationary growth phase after 14–16 h showing a nor-
mal growth pattern. The curve depicts a lag phase in the initial
phase of growth, active log phase and stationary phase. All the
species were found to be susceptible to the test gold nanoparti-
cles. At sub-MIC value (0.5 × MIC) of the test gold nano-particles,
the Candida did not show alterations in their growth curves to
distinct extent, except that of the extension of the lag phase by
2 to 4 h; while as more than 80% inhibition was observed at
167
MIC values of all the three test compounds by both the tested
microorganisms.
3.6. Proton efflux measurements
H+ -efflux is an immediate event associated with H+ -ATPase
activity. Proton-pumping ability of fungi mediated by the H+ -
ATPase at the expense of energy is crucial for the regulation of
internal pH and growth regulation of fungal cell. Fungal cells
depleted of carbon source when exposed to glucose, rapidly acid-
ify medium to generate proton motive force for nutrient uptake.
Candida cells susceptible to the gold nano-particles were exam-
ined for the ability to pump intracellular protons to the external
medium (as measured by the alteration of pH of the external
medium) in the presence of 25 nm gold nanodiscs and 30 nm poly-
hedral gold nanoparticles. Table 2 gives relative rates of H+ -efflux
by Candida species in presence of 25 nm gold nanodiscs (16 ␮g/ml),
30 nm polyhedral gold nanoparticles (64 ␮g/ml), vanadate (5 mM),
and fluconazole (5 ␮g/ml). Retention of H+ -efflux in standard lab
strains was 43% and 47% when treated with 25 nm gold nanodiscs
and 30 nm polyhedral gold nanoparticles, respectively. H+ -efflux
rate was also decreased to 45% and 51% respectively, for 25 nm
gold nanodiscs and 30 nm polyhedral gold nanoparticles in case
of clinical isolates. Glucose (5 mM) stimulated H+ -efflux in both
the types of isolates by 3–5-folds. Glucose stimulated H+ -efflux
was also inhibited by 25 nm gold nanodiscs and 30 nm polyhedral
gold nanoparticles. Glucose-stimulated H+ -efflux rates in standard
and clinical Candida isolates with respect to control, were 49% and
61% in the presence of 25 nm gold nanodiscs and 53% and 59%
in presence of 30 nm polyhedral 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
by 91–100% following addition of 5 mM vanadate – a well known
168 I.A. Wani, T. Ahmad / Colloids and Surfaces B: Biointerfaces 101 (2013) 162–170

Fig. 6. Representative dose dependent growth curve of Standard C. albicans 90028 (A) and clinical lab strain C. tropicalis 750 (B). The cells were grown with 1/2 × MIC of
30 nm gold nanocrystals (b), 1/2 × MIC of 25 nm gold nanodiscs (c), MIC of 30 nm gold nanocrystals (d) and MIC of 25 nm gold nanodiscs (e). (a) Represents untreated cells
(control). (C) Intracellular pH in presence of 25 nm gold nanodiscs and 30 nm gold nanocrystals at the respective MIC values. (D) Schematic diagram showing the action of
gold nanoparticles on the fungal cell.

Table 2 inhibitor of H+ -ATPase, whereas fluconazole does not have any sig-
Effect of 25 nm gold nanodiscs and 30 nm gold nanocrystals on the rate of H+ -efflux
nificant effect on the acidification of the external medium.
by standard lab Candida and clinical isolates at pH 7.0. Cells were suspended in
0.1 mM CaCl2 and 0.1 M KCl at 25 ◦ C. Glucose starved control had an average (of
4 independent recordings) proton efflux rate of 4.9 ± 1.1 nmoles min−1 mg−1 cells 3.7. Measurement of intracellular pH (pHi)
in standard isolates (1); 4.5 ± 0.9 nmoles min−1 mg−1 yeast cells in clinical iso-
lates (1*). Control in presence of glucose had an average H+ -efflux rate Intracellular pH controlled by the H+ pump is supposed to play a
of 21.7 ± 1.7 nmoles−1 min−1 mg−1 yeast cells in clinical isolates (1␣ ) and
18.3 ± 1.4 nmoles−1 min−1 mg−1 yeast cells in clinical isolates (1␤ ). Values in paren-
crucial role in normal growth of the yeast cells. Many cellular trans-
theses give the %-age inhibition of H+ -efflux with respect to control. port processes are regulated by the internal pH controlled by the
H+ cycle. Normally internal pH of yeast cells is regulated between
Incubation with Range of relative H+ -efflux rate
6.0 and 7.5 by plasma membrane H+ -ATPase activity. In this find-
(×10−11 moles min−1 mg cells)
ing, we tried to investigate the change in the internal pH associated
Standard isolates Clinical isolates with the H+ -ATPase in treated cells compared to the control cells
Control 1 1* with normal H+ -ATPase activity. Fig. 6(C) shows changes in the pat-
Gold nanodiscs (25 nm) 2.1 ± 0.3 (57) 2.1 ± 0.4 (53) tern of pHi with control and treated cells. Only yeast control cells
(16 ␮g/ml)
with normal H+ -ATPase activity maintain the pHi (6.5), while the
Gold nanocrystals 2.2 ± 0.3 (55) 2.3 ± 0.4 (49)
(30 nm) (64 ␮g/ml) treated cells show increase in internal acidification. The decrease in
Glucose (5 mM) 1␣ 1␤ pHi was more in cells exposed to 25 nm gold nanodiscs (5.8) than
Glucose + Au nanodiscs 10.6 ± 1.1(51) 9.7 ± 1.6 (47) 30 nm polyhedral gold nanoparticles (6).
(25 nm)
Glucose + Au 13.2 ± 0.5 (39) 10.8 ± 0.4 (41)
Nanocrystals (30 nm)
3.8. Discussion on antifungal activity of gold nanoparticles
Vanadate (5 mM) 0±0 (100) 0.4 ± 0.05 (91)
Fluconazole (5 ␮g/ml) 3.9 ± 0.5 (19) 3.8 ± 0.4 (15) Low MIC values of the gold nano-particles obtained against vari-
ous clinically important Candida species intrigued us to study their
 I.A. Wani, T. Ahmad / Colloids and Surfaces B: Biointerfaces 101 (2013) 162–170
mode of activity. The effect of the gold nano-particles is sponta-
neous and irreversible suggesting that there may be cellular sites
prone to the attack by these gold nanoparticles. Hence we explored
the effect of 25 nm gold nanodiscs and 30 nm polyhedral gold
nanoparticles on H+ extrusion by plasma membrane H+ – of vari-
ous standard and clinical Candida isolates. The gold nanoparticle
susceptible Candida isolates exhibited inhibition of H+ -ATPase-
mediated proton pumping suggesting that the two events are
linked. The H+ -ATPase-mediated proton pumping activity can be
conveniently measured by the glucose induced acidification of the
extracellular medium by the yeast cells [44]. The fluconazole had
no significant effect on H+ -ATPase while as vanadate completely
inhibits H+ -efflux. The result suggests that the interaction of any
compound with the plasma membrane is insufficient to affect the
function of the enzyme. It is thus possible that the gold nano-
particles may be directly interacting with the protein, which serves
as the primary reason for their antifungal activity. Regulation of
pHi is fundamental prerequisite for growth of Candida and activa-
tion of plasma membrane ATPase is involved in maintenance of pHi
[33]. We therefore studied the role of plasma membrane ATPase
activation in the regulation of pHi, in control as well as treated
cells. The pHi was near neutrality in absence of test gold nanopar-
ticles and then declined to 5.8 and 6.0 in presence of 25 nm gold
nanodiscs and 30 nm polyhedral gold nanoparticles, at concentra-
tions inhibitory to H+ -efflux and growth of Candida. Thus, the gold
nanoparticles may be supposed to act both at the membrane and
cytoplasmic level. On the formal level, based on the proton efflux
measurements, it can be assumed that the test gold nanoparticles
may be interacting directly with the transmembrane proteins espe-
cially the proteins responsible for maintaining the transmembrane
electrochemical potential gradient. One such protein of our study
is H+ -ATPase; an ATP-driven enzyme that transforms the energy
of ATP hydrolysis to electrochemical potential differences of pro-
tons across diverse biological membranes via the primary active
transport of H+ . The proton gradients are used to drive secondary
transport processes, essential for the growth of the fungal cell [45].
The interaction of gold nanoparticles with such biologically active
enzymes may alter their normal conformation leading to the loss
of the activity. Thus the normal proton pump activity is disturbed,
due to which the fungal cell is unable to pick up the nutrients
from the surroundings, leading to the retardation of growth and
finally the cell dies. The schematic diagram showing the action of
the gold nanoparticles on the Candidal cell is shown in Fig. 6(D). The
results also show that the 25 nm gold nanodiscs inhibit the fungal
growth to the larger extent than the 30 nm sized gold nanocrystals.
This may be due to the fact that 25 nm gold nanodiscs are having
higher surface area (179.5 m2 /g) than the 30 nm gold nanocrystals
(150.5 m2 /g). Hence, the increase in surface area may result in the
greater enhancement of interaction of the gold nanodisc with the
binding sites of the plasma membrane proteins. Further it may be
assumed that there may be some morphology specific interaction
between the gold nanoparticles and the plasma membrane proteins
that resulted in the higher antifungal activity of the gold nanodiscs.
It may also be suggested that the smaller gold nanoparticles might
have diffused easily through the cell membrane to the inside of the
cell. Since gold being a soft acid might have interacted strongly with
the soft bases like sulphur containing proteins in the membrane or
phosphorus containing bases in the DNA, thus retarding their nor-
mal functions like synthesis, repair and replication leading to the
cell death [46].
4. Conclusion
The gold nanodiscs and polyhedral structures have been suc-
cessfully synthesized by sonochemical reduction of aqueous gold
169
salt solution without using any stabilizer. TEM images show that
tinchloride (SnCl2 ) has proved to be more efficient reducing agent
in producing gold nanodiscs of controlled morphology with aver-
age disc size of 25 nm. However, sodium borohydride reduction
produced mixture of nanocrystals of different morphologies con-
taining cubes, hexagons and other polyhedral structures with
average size of 30 nm. Time evolution of the UV–visible absorption
spectra of the gold nanodiscs shows in-plane dipolar surface plas-
mon resonance (SPR) peak located at 530 nm. While borohydride
stabilized gold nanocrystals exhibit a set of peaks located around
525 nm and 770 nm due to two size distributions and a hump at
380 nm owing to the out of plane dipolar SPR of these nanocrys-
tals. BET surface area studies reveal that nanodiscs synthesized by
using tinchloride (SnCl2 ) shows type VI adsorption isotherm with
the specific surface area of 179.5 m2 /g while borohydride synthe-
sized gold nanocrystals show type II isotherm and have less surface
area of 150.5 m2 /g than the nanodiscs. The gold nanoparticles show
excellent size and shape dependant antifungal activity against Can-
dida isolates. The smaller sized gold nanodiscs proved to be more
fungicidal than large sized gold polyhedral nanocrystals. The pos-
sible mechanism of the fungicidal action of the gold nanoparticles
was also worked out which involves the inhibition of H+ -ATPase
leading to intracellular acidification and cell death. Thus, the cur-
rent work can act as a bridge between materials chemistry and
biotechnology to design more effective antifungal agents against
human fungal pathogens.
Acknowledgments
TA thanks to Department of Science and Technology (DST) for
the financial support (SR/FTP/CS-120/2006). The authors thank Pro-
fessor Ashok K. Ganguli for the use of HRTEM facility (funded by
DST, Nano Mission) at IIT Delhi. The authors also thank Mr. Aijaz
Ahmad and Dr. Nikhat Manzoor, Department of Biosciences, Jamia
Millia Islamia, New Delhi for the experiments and discussion for
the Antifungal studies.
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