Controlled synthesis of spin glass nickel oxide nanoparticles and evaluation of their potential antimicrobial activity: A cost effective and eco friendly approach

Article abstract of DOI:10.1039/c3ra42628a

Development of an easy sustainable synthetic pathway towards oxide nanomaterials (NMs) is a necessary challenge for nanotechnology research workers. Additionally, antimicrobial activity of oxide nanoparticles against multi drug resistance pathogenic bacteria motivates scientists to focus their research on oxide materials. We report here a cost effective, simple and eco-friendly pathway of synthesizing NiO nanoparticles (NPs). X-ray diffraction and energy dispersive X-ray study confirmed their crystallinity and composition. Field emission scanning electron microscope (FESEM) was employed to understand their surface architecture and the dimension of synthesized NiO NPs were found to be 20-30 nm from transmission electron microscope (TEM) study. The as synthesized NiO demonstrated typical spin glass behaviour which is one advantage of our synthetic procedure. Antimicrobial properties of NiO NPs were investigated using Gram negative and Gram positive bacteria and their bactericidal effects were determined from minimum inhibitory concentrations (MIC) and Minimum bactericidal concentrations (MBC). Haemolytic activity revealed the nontoxic nature of the NPs towards the blood proteins at MBC. TEM images of bacteria cells treated with NiO NPs showed irreversible damages to the cell wall leading to cell death. In light of our findings a possible mechanism of the antimicrobial effect of NiO NPs has been proposed.

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DOI: 10.1039/C3RA42628A
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ARTICLE TYPE
Controlled Synthesis of Spin Glass Nickel Oxide Nanoparticles and
Evaluation of their Potential Antimicrobial Activity: A Cost Effective
and Eco Friendly Approach
Soumyadipta Rakshit,a Srabanti Ghosh,b Sayantani Chall,a Soumya Sundar Mati,a S.P.Moulik,a,c
5
Subhash Chandra Bhattacharya,*a,c
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
Development of an easy sustainable synthetic pathway towards oxide nanomaterials (NMs) is a necessary
challenge for nanotechnology research workers. Additionally, antimicrobial activity of oxide
10 nanoparticles against multi drug resistance pathogenic bacteria motivates scientists to focus their research
on oxide materials. We report here a cost effective, simple and ecoꢀfriendly pathway of synthesizing NiO
nanoparticles (NPs). Xꢀray diffraction and energy dispersive Xꢀray study confirmed their crystallinity and
composition. Field emission scanning electron microscope (FESEM) was employed to understand their
surface architecture and the dimension of synthesized NiO NPs were found to be 20ꢀ30 nm from
15 transmission electron microscope (TEM) study. The as synthesized NiO demonstrated typical spin glass
behaviour which is on advantage of our synthetic procedure. Antimicrobial properties of NiO NPs were
investigated using gram negative and gram positive bacteria and their bactericidal effects were
determined from minimum inhibitory concentrations (MIC) and Minimum bactericidal concentrations
(MBC). Haemolytic activity revealed the nontoxic nature of the NPs towards the blood proteins at MBC.
20 TEM images of bacteria cells treated with NiO NPs showed irreversible damages to the cell wall leading
to cell death. In the light of our findings a possible mechanism of the antimicrobial effect of NiO NPs has
been proposed.
Introduction
There is an unceasing pursuance in material science and
25 chemistry to develop nanoscale materials having better
Typically Nickel oxalates (NiOX) are potential precursors to
produce NiO for their low cost, easy synthesis, good structure
stability and low decomposition temperature. Different
surfactants25,26, solꢀgel27,28 methods as a soft chemistry route were
performance with green approach due to environmental issues1ꢀ5
.
Since transitional metal oxides are key materials in catalysis,
microelectronics, optical and magnetic applications their use in 50 used for controlled synthesis of NiO NPs. Even though these
personal and commercial products is in a rush which necessitates
30 greater insight towards their ecological impact (as they have
intrinsic toxic property) on their release in environment6ꢀ9. Since
insoluble metal oxide nanomaterials are expected to be more
procedures have the ability to produce highly crystalline and
uniformly sized NPs, they are not without drawbacks since these
methods require expensive and toxic reagents and complicated
synthetic steps. Therefore, the development of a simple, versatile
environmentally persistent, investigations on environmental 55 and environment friendly method for the controlled synthesis of
perseverance, bioaccumulation and toxicity of these
35 nanomaterials help to assess potential risks and provide
metal oxide NPs is required, not only for its fascinating
utilization but also for the demand of time. Nevertheless, it has
been reported that cellulose fibers29, appoferritin30, mammalian
protein cages31, viral protein cages32 are effective materials for
information to industry to develop safer nanomaterials10,12
.
Owing to their specific functional characteristics and importance
in a wide variety of technical applications, NiO nanoparticles 60 bioꢀtemplated preparation of oxide NPs.
have been investigated extensively as catalyst13, electrode
Amid the diverse synthetic methodologies, we choose solꢀgel
40 material for lithium ion batteries and fuel cells14ꢀ16
in
electrochromic films17, electrochemical supercapacitors18ꢀ19
magnetic materials20 and gas sensors21. NiO is now also used for
,
technology since it is an impressive versatile approach for its
important applications in practice. Use of ecoꢀfriendly material in
this method makes it potential for different biomedical processes
,
recyclable protein separation22,23 and as biosensors24. Their 65 like enzyme, protein and antibody immobilization33ꢀ36, entrapping
increasing use in technology and biosystems motivated us to
45 undertake this work.
of different drugs in porous matrices for therapeutic application37,
selective coatings for optical and electrochemical biosensors38,39
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Bio gel derived nanoparticles show a very slow degree of release
From this perspective, we have focussed our work on whether our
of bioꢀactive agents40. Bioꢀgel templated synthesis route is an 60 synthesized NiO NPs can act as a promising antimicrobial agent
effective route towards metal oxide NPs. Though the major
problem of the gel mediated synthesis is to control the reaction
rates (which are generally too fast), resulting in formation of
precipitates with a high degree of structural disorder, the bioꢀgel
by determining Minimum Inhibitory Concentration (MIC) and
Minimum Bactericidal Concentration (MBC). We also
investigated the haemolytic activity for understanding the
influence of the nanomaterial on human health. In the light of our
5
composed of APSAꢀ80, Isopropyl myristate (IPM) and metal salt 65 findings we tried to propose
a possible mechanism of
solution is quite an exception to that. APSAꢀ80 is used in
agriculture as a dispersant for various fungicide, insecticides and
antimicrobial effect.
10 herbicides and as well as it is a very good spreader, activator for
herbicides for their ability to make water “wetter”. It has the
ability to enhance the activity of Limonine (a Citrus fruit extract)
to control Mealybugs, scale insects, white flies and aphides. It is
2. Marterials and Methods
2.1. Reagents and Materials
The precursor metal salts nickel (ΙΙ) nitrate hexahydrate
extensively used as spray adjuvant in agriculture and has merely 70 (Ni(NO3)2. 6H2O, 99.9%) and sodium oxalate (Na2C2O4,98%)
15 any antimicrobial effect41. Its formulation with IPM, another ecoꢀ
friendly material and water can be used as a more stabilised
vehicle for field application of pesticides with a better sustainable
environment effect and can be used as a green template for
were purchased from E. Merck. IPM (Isopropyl myristate, ≥
98%) and APSAꢀ80 were obtained from SRL (India) and
AMWAY (Mumbai, India) respectively. All the chemicals were
used as received. Acetone of analytical grade used in this work
nanoparticle synthesis42. In this present work, we have used a 75 was purchased from E.Merck and dried by standard procedure54.
20 green material APSAꢀ80 as a template to develop a controlled,
ecoꢀfriendly and costꢀeffective synthesis of NiO NPs. After
proper characterization we have discussed their interesting
magnetic property.
The water was treated with a MilliporeꢀQ water purification
system.
2.2. Synthesis of NiOX and NiO nanoparticles
All syntheses were performed under ambient atmospheric
80 condition. To prepare NiO nanoparticles we opted for a twoꢀstep
synthesis approach. In the first step, we prepared Nickel oxalate
by templating a bioꢀgel consisting of APSAꢀ80, IPM, Ni2+ and
C2O42ꢀ. In a typical synthetic procedure we prepared two different
solutions. The first one was composed of 20 ml APSAꢀ80, 10 ml
85 IPM, 8.8 ml Ni2+ and the other one of 20 ml APSAꢀ80, 10 ml
Antimicrobial agents are very important in water disinfection and
25 medicine since they can locally kill bacteria or slow down their
growth without being toxic to surrounding tissue. Although
chemically modified natural compounds are mostly applied
antibacterial agents, with their broad use and abuse such as
antibiotic therapy, horizontal gene transfer by conjugation,
30 transduction or transformation etc. but emergence of bacterial
resistance to these drugs are becoming a major problem these
days43,44. This has prompted the interest in inorganic metal oxides
which can provide an alternative strategy to treat bacterial
diseases.In this context, it is interesting to mention that metal
35 oxides are very easy to handle and are considered more
environment friendly as compared to many others currently used
as biocides. Gramꢀpositive Bacillus subtilis, Bacillus anthrax are
quite harmful towards human health since they can cause brain
inflammatory diseases such as meningitis and encephalitis and
2ꢀ
IPM, 8.8 ml C2O4 . The two solutions were mixed and stirred
vigorously to form gel. The resulted gel was put for 3 hours for
completion of formation of Nickel Oxalate. The gel comprised of
APSAꢀ80 and IPM was then dissolved by adding acetone in it.
90 After dissolving the gel the product (NiOX) was centrifuged and
washed for 6 times with acetone. The recovered NiOX was then
heated to 450°C at an increasing heat rate of 2°C. The optimal
annealing temperature was found from thermogravimetric
measurements to ensure complete conversion of precursor
95 oxalates to oxide. A synthesis scheme has been given in Fig S1.
40 they also produce allergic sensitivity45ꢀ47
.
Pseudomonas
aeruginosa is a heavy metal tolerant strain. It is mostly notorious
for causing lung infections or pneumonia in patients with
compromised immune systems. Patients who are hospitalized for
extended periods are frequently infected by this organism and can
45 cause malignant external otitis, endophthalmitis, endocarditis,
2.3. Screening for antimicrobial activity
Antibacterial activity of the NiO was determined by agarꢀ
diffusion assay as described previously55,56 against some of the
pathogenic Gramꢀpositive and Gramꢀnegative bacteria. Bacterial
100 strains were first grown in MuellerꢀHinton broth (MHB) under
shaking condition for 4 h at 37°C and after the incubation period
1 ml of culture was spread on MuellerꢀHinton agar (MHA). The
wells were made using sterile 6 mm cork borer in the inoculated
MHA plate. The wells were filled with 50 ꢁl (1mg/ml) of the
105 samples of NiO and blanks (phosphate buffer). Zone diameter
was measured after 24 h incubation at 37oC.
meningitis, pneumonia, and septicaemia48ꢀ50
.
However,
considering the significance of studying antimicrobial activity of
oxide nanomaterials, reports on NiO nanomaterials are quite rare.
YꢀW Baek et al. and Z.Wang et al. reported MIC and MBC
50 values using commercially available NiO NPs19,51,52. Haemolysis
is the destruction of red blood cells resulting in the release of
haemoglobin into the surrounding fluid and is a measure to
evaluate the toxicity of a material towards human blood. Thus,
haemolytic data of a particular nanoparticle is also very essential
55 for their possible usability in biomedical application. But
literature did not reveal any satisfying data concerning
haemolytic effect of the NiO NPs or even it did not discussed
how they affect the cell growth53.
2.4. Determination of Minimum inhibitory concentration
(MIC) and minimum bactericidal concentration (MBC)
The minimum inhibitory concentration (MIC) dictates the lowest
110 concentration at which an antiꢀmicrobial agent will inhibit the
growth of a microorganism57. Another related method is the
minimum bactericidal concentration (MBC). This indicates the
2
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lowest concentration at which 99.9% of bacteria are killed by the
2.0
100
80
compound under investigation. The MBC is the lowest
concentration of an antiꢀmicrobial agent that will restrict the
growth of the organism when subcultured into antibioticꢀfree
media. MIC and MBC of materials were determined using the
broth micro dilution58. An inoculum of the microorganism was
prepared from 24 h MHB cultures and suspensions were adjusted
with turbidity equivalent to that of a 0.5 McFarland standard.
Bacterial suspensions were further diluted to 1:10 in sterile MHB
TGA Curve
Exothermic
3580C
DTA Curve
1.6
19.1%
5
1.2
0.8
0.4
0.0
45.1%
60
10 to obtain a final inoculum of 5 × 105 CFU/ml. A 20 ꢁl aliquot of
the sample was added. Plates were covered and incubated for 24
h in ambient air at 37°C. After incubation, minimum inhibitory
concentrations (MIC) were read visually; all wells were plated to
nutrient agar and incubated. The minimal bactericidal
15 concentration (MBC) was defined as a 99.9% reduction in CFU
from the starting inoculum after 24 h incubation interval.
2180C
Endothermic
40
100 200 300 400 500 600 700 800
Temperature / oC
Figure 2: TGAꢀDTA plot of synthesized Nickel Oxalate
precursor.
2.5. Haemolytic activity
Haemolysis test was employed to determine cellular toxicity of
the NiO nanoparticles as previously described59. The 100%
20 haemolytic positive control was the buffer containing 1% Triton
Xꢀ100. Varying concentrations of NiO (0–1000 ꢂg/mL) were
then incubated with 1% human red blood cell suspension for 1 h
at 37°C. After 1 h incubation, the mixtures were iceꢀcooled and
centrifuged for 10 min at 1000 rpm at 4°C. The percent
25 haemolysis was determined by measuring the optical density of
the released haemoglobin in the supernatant at 540 nm. The
experiments were run in triplicate for each concentration of NiO
and were repeated three times.
heating rate of 10°C minꢀ1. Magnetic properties of Nickel Oxide
were measured by a SQUID magnetometer [MPMS XL7,
Quantum Design, USA]. The optical densities of released
45 haemoglobin were recorded with a Shimadzu UVꢀ1601 PC UVꢀ
Vis spectrophotometer using matched quartz cells of path length
1 cm. Surface charge of NiO NPs was measured by Malvern,
Zetasizer Nano ZS90.
3. Results and Discussions
50 3.1. X-Ray Diffraction Analysis
2.6. Measurement and Characterization
The overall crystallinity and phase composition of the
30 A field emission scanning electron microscope (FESEM, S4800,
Hitachi) equipped with an energyꢀdispersive Xꢀray spectrum was
applied to determine the surface morphology and chemical
composition of the asꢀprepared samples. Particle sizes, shapes,
and HRTEM characterization were carried out in a JEOL JEMꢀ
35 2100 with a tungsten filament at an accelerating voltage operated
at 200 kV. XꢀRay diffraction (XRD) spectra were taken in a
Bruker AXS Dꢀ8 Advance diffractometer at a scanning rate of
2°minꢀ1,using the Cu Kα radiation (λ = 1.5406 Å) at an
accelerating voltage and the applied current were 40 kV and 40
40 mA. Thermogravimetric analysis was carried out using a Perkinꢀ
Elmer Pyris instrument from room temperature to 600°C with a
1200
NiO
800
NiOX
400
0
20
60
80
40 2
θ
Figure 3: FESEM (a) and TEM (b) images of prepared Nickel
Oxalate. (c) Shows a single NiOX NP and (d), (e), (f) show
the elemental mapping of Ni, C, O respectively
Figure 1: Xꢀray Diffraction pattern of as synthesized NiOX and
NiO
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240°C corresponded to the exclusion of H2O associated with
an endothermic peak. The second weight loss at 240 ꢀ380°C
20 was ascribed to the decomposition of oxalate. The exothermic
peak associated with it clearly indicted loss of CO2 molecule.
Figure 4: FESEM images indicating gradual degradation of NiOX
NPs to NiO NPs at (a) 0 min (NiOX), (b) 60 minutes, (c) 120
minutes and (d) 180 minutes (NiO)
synthesized nanostructured NiOX and NiO were initially
determined by Xꢀray Powder Diffractometer (XRD) and are
shown in Figure 1. The XRD pattern of NiOX depicted in Figure
1 supported pure phase base centred orthorhombic NiOX (lattice
parameters a= 11.84, b= 5.345, c= 15.716, Z= 8 and space group
Cccm). The XRD pattern of NiO showed three major diffraction
peaks and was indexed as (111), (200) and (220) crystal planes
with a cubic structure which is in good agreement with literature
value (JCPDS 78ꢀ0423). High intensity and broad spectral width
10 in NiO XRD spectra affirmed their well grain size and detection
of no other peak related to impurities further confirmed their high
purity and crystallinity. Their compositions were further
confirmed by EDX spectrometry (Figure S2 a, b)
Figure 5: (a) TEM image of NiO obtained after calcination of
NiOX. (b) is its HRTEM image showing inter planar distance
2.08Å and 2.41Å for [200] and [111] crystal planes
respectively. (c) is the corresponding SAED pattern.
5
3.3. Electron Microscope Analysis (FESEM and TEM)
The morphology of Nickel Oxalate and Nickel Oxide
nanoparticles was determined by FESEM. Figure 3a showed the
25 typical FESEM images of Nickel Oxalate nanoparticles. The
dimensions of NiOX NPs were found to be 0.4ꢀ0.5 ꢂm and they
were free of surface cracks and interꢀstructure adherence. TEM
images of NiOX NPs also supported the observation.
In the elemental mapping Ni, C and O were found to be
30 homogenously distributed in the synthesized NiOX NPs (Figure
3d, e, f) and that of Ni and O in NiO (Figure S3).
3.2. Thermo Gravimetric Analysis
15 The TGA and DTA results of NiOX are presented in Figure 2.
Two distinct weight loss steps in the temperature range 35 ꢀ240°C
and 240 ꢀ380°C were observed. The first weight loss at 35–
For a substantial view of the growth mechanism of NiO NPs, the
time dependent evolution process was monitored. Figure 4
Figure 6: FC and ZFC magnetization curve for NiO (a) at an applied field 50 Oe. Inset shows ZFC and FC curves at the temperature region
below 30K, where there is a cusp at ~13K. Hysteresis loops at 10K, 50K and 300K (b) and their magnification (inset of b).
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50 3.5. Antimicrobial Effect of NiO
(a,b,c,d) represents the FESEM images of the products that were
obtained at different growth stages. The initial product Nickel
oxalate when subjected to heat at 450°C at a heating rate 2°C minꢀ
1, NiO nanoparticles (of smooth texture) were seen formed as
surface cracks after 1 hr. But after 3 hrs their surfaces were found
to be densely covered with small NiO NPs with dimension 20ꢀ30
nm. Their corresponding XRD patterns are also supported the
observation (Fig S4). The HRTEM image of synthesised NiO
The antimicrobial activity of NiO nanoparticles was investigated
against gram negative and gram positive bacteria using well
diffusion method. In an agar plate, NiO NPs were used as test
material and phosphate buffer as negative control. The inhibition
5
clearly showed the distribution of NiO NPs within
a
10 parallelogram of dimension 20ꢀ30 nm (Figure S5). The close
aggregation and packing of these NiO nanoparticles are shown in
Fig. S3. A highꢀresolution TEM image (Figure 5b) displays clear
lattices of NiO crystal, confirming the crystalline nature of NiO.
The corresponding SAED pattern (Figure c) recorded on one NiO
15 hollow microsphere suggests its polycrystalline nature. The
diffraction rings can be easily indexed to the (220), (111), and
(200) lattice planes of cubic NiO.
3.4. Magnetic Property
Figure 7: Determination of the effect of NiO nanoparticles on
gram negative and gram positive bacteria by agarꢀdiffusion
assay method. Pseudomonas aeruginosa and Bacillus subtilis
were spread on Agar plate. In each case, 1 represents Ni salt,
2, 3, 4 contains (8, 16, 32 ꢂg/well), 5 represent 50 ꢀl of water,
were added to the wells.
Magnetic properties of NiO were measured over a wide range of
20 temperature and magnetic field. Temperature dependent
magnetization curves (MꢀT) of synthesized NiO NPs were
recorded under Field Cooling (FC) and Zero Field Cooling (ZFC)
conditions from 10K to 300K at an applied field of 50 Oe. The
results are shown in Figure 6a. The main feature of the ZFC
25 curve was having two maxima: i) a broad maximum centred at
T1~150K and ii) a very sharp maximum was observed at a low
temperature T2~13K along with a bifurcation between FC and
ZFC curves around 220K, which corresponded to Néel transition
and also marked the largest nanoparticle present. Above this Néel
30 temperature, thermal motions destroyed the antiparallel
arrangement, and the nanomaterial then became paramagnetic60.
For MFC curve, magnetization increased monotonically with
decreasing temperature from 300 to 5 K. The spinꢀglassꢀlike
behavior indicated that a spinꢀfrozen transition might exist at
35 around 13 K61. The high temperature maximum is commonly
associated with mean blocking temperature denoting progressive
blocking of uncompensated magnetic moments of the particle
core62. As temperature was decreased the uncompensated surface
spins started to correlate and formed spin clusters at the particle
40 surface inducing a strong interꢀparticle interaction which led to a
sharp maximum at ~13 K63.
55 zone formed in the screening test indicating the antimicrobial
activity of NiO showed strong activity against both gram negative
and gram positive bacteria such as Pseudomonas aeruginosa and
Bacillus subtilis as evident from the clear zone of inhibition
around the well containing this solution (as shown in Table 1 and
60 Figure7). MIC (the lowest concentration at which no viable
bacterial cell is present) against Pseudomonas aeruginosa and
Bacillus subtilis was 8 ꢂg/mL whereas the MBC values were 16
ꢂg/mL and 32 ꢂg/mL repectively. For better understanding
antimicrobial effects of some other metal and metal oxide
65 nanoparticles against these two bacteria are given in Table ST1.
Furthermore, our study aimed to unravel the cellular events that
occurred upon exposure to NiO NPs with human blood cells in
order to utilize them as antimicrobial agent. Figure 8 illustrates a
100
Effec of NiO on human blood
80
60
To get a clear view of this interesting magnetic property
hysteresis loops were measured at different temperatures (10K,
50K, and 300K). At 10K it showed a fine hysteresis loop with a
45 remnant magnetization 5.6 emu molꢀ1 exhibiting typical
ferromagnetic behavior whereas for 50K and 300K the values
became small revealing a superparamagnetic character (Figure
6b). The high temperature paramagnetic behavior suggested
obliteration of longꢀrange antiferromagnetism ordering64.
40
Effect of NiO on human blood
20
Table 1: Minimum inhibitory concentration (MIC) and minimum
bactericidal concentration (MBC) of NiO against multiꢀdrugꢀ
resistant strains
0
1000
200 500
75 100
50
Positive control
[NiO](
ꢀ
g/mL)
Figure 8: A dose–response plot for the haemolysis of human red
blood cell in presence of NiO nanoparticles. In Haemolysis
assay, TritonꢀX used as a positive control and red blood cells
with different amount NiO nanoparticles.
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Figure 9: TEM images of P. Aeruginosa (a) control bacteria without nanoparticles treatment, treated with (b) MIC and (c) MBC of NiO
nanoparticles. (d) B. Subtilis before treatment treated with (e) MIC and (f) MBC of NiO nanoparticles
dose–response profile of haemolysis of the red blood cells in
presence of NiO. The haemolytic activity increased with
increasing concentration of NiO nanoparticles while at MBC of
NiO showed no haemolytic activity.
The haemolytic data suggested NiO has a strong affinity towards
haemoglobin protein at higher concentration than MBC. The
knowledge of the interaction of NiO with protein is crucial to
address the engineering of NiO materials towards
NiO for 2ꢀ3 h showed a significant change in their cell
morphology. The TEM Micrographs of control P. Aeruginosa
(Figure 9a) and B. Subtilis (Figure 9d) show relatively smooth
20 cell surface compared to the treated cells with typical characters
of rod shape. The TEM images revealed aggregation as well as
alteration of cell morphology (Figure 9 b, c and e, f) on exposure
to NiO nanoparticles.
5
The bacterial cells showed aberrant morphology with cracked and
biocompatibility. Thus at a suitable concentration NiO (100 25 ruptured cell. The transmission electron microscopic studies
10 ꢂg/mL) maintained the molecular structure of red blood cells
without any toxicity.
To get an insight into the effect of NiO on bacteria, the
morphologies of Gramꢀnegative (P. Aeruginosa) and Gramꢀ
revealed alteration of cellular morphology along with damage of
the membrane on treatment with NiO NPs. The interaction
between nanoparticle surface and the cell wall constituents might
have caused structural changes and damage to the cellular
positive bacteria (B. subtilis) were examined by taking electron 30 membranes.
15 microscopic images before and after treatment with NiO
nanoparticles at MIC and MBC. The incubation of bacteria with
Outer Membrane of Gramꢀnegative bacteria is composed of
lipopolysaccharide in addition to a thin peptidoglycan layer and
act as a primary permeability barrier for macromolecules and
hydrophobic drugs. But for Gramꢀpositive bacteria the structure
35 is rather simple as they have a membrane, which surrounds the
cell, and a cell wall primarily made up of peptidoglycan layer as
well as teichoic and lipoteichoic acids65. Still date the mechanism
of antimicrobial activity of NiO is not clear.
For both cases due to direct contact NiO NPs damaged the cell
40 membrane. The global charge of the bacterial cell at
physiological pH was negative due to the dissociation of excess
carboxylic groups at the cell surface66. Since NiO has a positive
surface charge and a ζ= 36.8 ± 0.9 mV (found from Zeta potential
measurement), they became electrostatically bound to the
Figure 10: Fluorescence micrograph of P. Aeruginosa before (a)
and after treating with NiO nanoparticles (b) in presence of
LIVE/DEAD® BacLight TM Bacterial Viability Kit
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Scheme 1: Schematic representation of possible mechanism of antimicrobial activity of NiO NPs
negative cell surface hindering the cell activity. Penetration of
NiO into the cell and its toxicity made it inactive and dead
bactericidal efficiency of the NiO nanoparticles.
followed by lysis. The above process has been depicted in
Scheme 1.
Conclusion
In short we developed a simple, controllable and environment
friendly bottom up pathway for synthesizing NiO nanoparticles
25 which were characterised by means of XRD, FESEM and TEM.
Magnetic study indicated their spin glass behaviour. The broad
spectrum of antimicrobial activity of these NiO nanoparticles
with low MIC values clearly indicated their efficiency towards
inhibiting the growth of gram positive and gram negative
30 bacteria. TEM pictures of the bacteria treated with NiO NPs
indicated that the antimicrobial action was governed via
membrane disruption and cell lysis and may be stable positive
surface charge played a vital role in it. Their low haemolytic
activity at bactericidal concentration further makes them useful
35 for biomedical application. For its easy preparation, low cost,
high abundance and excellent bactericidal effect, NiO has a
promising role for future antibacterial agent.
5
The antibacterial activity of the NiO nanoparticles was also
followed by fluorescence microscopy using LIVE/DEAD®
BacLightTM bacterial viability kit67 The kit contains two nucleic
acid binding stains, SYTO 9 and propidium iodide. These dyes
have different cell penetration properties along with distinct
10 spectral characteristics. SYTO 9 binds to the nucleic acid of both
living and dead cells and exhibits green fluorescence after
excitation with BP460–495 nm filter. Propidium iodide only
binds to the nucleic acid of dead cells and shows red
fluorescence. The fluorescence microscopic images of untreated
15 Gramꢀnegative, P. Aeruginosa and that of same bacterial cells
treated with 16 ꢁg/mL of NiO, respectively are shown in
Figure10. All the untreated cells exhibited green fluorescence
indicating the living status of bacteria. However, the observed red
fluorescence in the microscopic image (Figure 10b) of NiO
20 treated bacteria with propidium iodide further confirmed the
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Acknowledgement
Authors S.R and S.S.M acknowledge UGC for providing JRF and
SRF. S.G. gratefully acknowledges CSIR for financial support.
S.C., S.B and S.P.M greatly appreciate the funding from DST
(SR/S1/PCꢀ63/2009). SPM also thanks INSA for support. S.R
acknowledges Mr. I. Mukherjee for his help in preparing gel by
APSAꢀ80. Authors are grateful to Dr. K. K. Chattopadhyay, Mr.
N.S. Das and Mr. D. Sarkar of Jadavpur University for their kind
help in doing TEM and FESEM measurement. Help of Mr. Pulak
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15 sbjuchem@yahoo.com, scbhattacharyya@chemistry.jdvu.ac.in
b Centre for Advanced Materials, Indian Association for the Cultivation of
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NiOX and NiO Nanoparticles, Elemental Mapping of Ni and O in NiO,
Nickel Oxalate parallelograms and distribution of NiO NPs within the
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DOI: 10.1039/C3RA42628A
Controlled Synthesis of Spin Glass Nickel Oxide Nanoparticles
and Evaluation of their Potential Antimicrobial Activity:
A Cost-Effective and Eco-Friendly Approach
Soumyadipta Rakshit a, Srabanti Ghosh b, Sayantani Chall a, Soumya Sundar
ac
Mati a, S.P.Moulik ac, Subhash Chandra Bhattacharya
*
a Department of Chemistry, Jadavpur University, Kolkata -700032, India
b Centre for Advanced Materials, Indian Association for the Cultivation of
Science, Jadavpur, Kolkata-700032, India
c Centre for Surface Science, Department of Chemistry, Jadavpur University,
Kolkata -700032, India
*E-mail : sbjuchem@yahoo.com / scbhattacharyya@chemistry.jdvu.ac.in
*Phone No: 033 2414 6223; *Fax: 91(033) 24146584
Graphical Abstract

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Magnetic Property of NiO depicting Spin Glass behavior and their antimicrobial effect on gram-
positive and gram-negative bacteria