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Synthesis, characterization and evaluation of copper

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based nanoparticles as agrochemicals against

Cite this: RSC Adv., 2013, 3, 21743
Phytophthora infestans†
K. Giannousi,a I. Avramidisb and C. Dendrinou-Samara*a

Copper based nanoparticles (Cu-based NPs) of different composition and sizes have been hydrothermally
synthesized by varying the reaction temperature and in the presence of biocompatible surfactants;
polyoxyethylene (20) sorbitan laurate, polyethylene glycol 1000 and polyethylene glycol 8000. Effective
control of the above synthetic parameters gave rise to Cu-based NPs of 11–55 nm. CuO, Cu2O
nanoparticles and Cu/Cu2O composite nanoparticles of nearly similar size and shape were selected
among the isolated samples and tested in the field on tomato (Lycopersicon esculentum) plants against
Phytophthora infestans. Their activity has been evaluated by assessing the fungal infection on the leaves
in comparison to the performance of the registered commercially used copper based products Kocide
Received 29th April 2013
Accepted 3rd September 2013
2000, Kocide Opti, Cuprofix disperss and Ridomil Gold Plus. The results showed that the prepared Cu-
based NPs can be applied in a lower formulated product and active ingredient rate and are more
DOI: 10.1039/c3ra42118j
effective than the trade agrochemicals. Moreover, assessments regarding phytotoxicity indicated that
www.rsc.org/advances they can be used without any deleterious effect on plants.

1 Introduction
The use of nanomaterials and nanotechnology has greatly
increased in the last decades, causing the incorporation of
innovative technologies in many elds of science spanning from
medicine to electronics.1,2 As far as biomedical applications
concerned, inorganic nanoparticles (NPs) are currently explored,
since their active participation in biological systems raises
expectations for achieving specic processes and selectivity.
Recently, in the eld of microbiology several metallic or oxide
NPs, such as Ag, Au, ZnO, NiO and TiO2, have been evaluated
in vitro as antimicrobial agents against a range of either Gram
negative or Gram positive bacteria, as Staphylococcus aureus and
Escherichia coli.1,3 Moreover, Pt, Au, Ag and Cu NPs have been
studied against the pathogenic fungi Candida albicans and Sta-
chybotrys chartarum and found to be more effective than the
corresponding bulk materials.4,5 The improved activity arising
from nano size effects, results in the appearance of new physi-
cochemical properties, different from their bulk counterparts or
from molecular compounds. The optimistic results that have
been obtained, caused lately the interest of evaluating inorganic
NPs in other elds of science like agriculture, where fungi and in
some cases bacteria, cause severe damages to many crops.6
a
Department of Chemistry, Aristotle University of Thessaloniki, 54124 Thessaloniki,
Greece. E-mail: samkat@chem.auth.gr
b
Agronomist, Field Trialist, Agrolab SA private sector, Thessaloniki, Greece
† Electronic supplementary information (ESI) available. See
10.1039/c3ra42118j
Copper has been used as a fungicide since 1882 in the area of
Bordeaux in France, where they observed that a mixture of copper
sulfate and calcium caused a dramatic reduction in the
percentage of grape plants from Plasmopara viticola fungi. This
mixture, known as Bordeaux mixture, is still used for preventing
damages in several crops, caused by many different types of
fungi.7 Since then, many copper based agrochemicals have been
produced and used in agriculture like copper oxychloride
[Cu(OH)2$CuCl2], copper hydroxide [Cu(OH)2] and cuprous oxide
(Cu2O). However, due to the low water solubility, the agrochem-
icals are applied in relatively large amounts in order to effectively
control the phytopathogenic fungi. When the spores of fungi
vegetate, dissolve them by causing the secretion of malic acid and
aminoacids.8,9 The biological activity of these products is based
on the free Cu2+ ions available for consumption.10 Copper toxicity
is attributed to the ability of copper ions to easily interconvert
between Cu(I)/Cu(II) by Fenton-like and Haber–Weiss reactions
that generate reactive oxygen species (ROS), which lead to lipid
peroxidation, protein oxidation and DNA damage.1,11 In conse-
quence, the limit between plant protection and phytotoxicity is
still a matter of discuss. One of the most important diseases that
copper based agrochemicals are able to control is late blight of
tomato, which is caused by the oomycete Phytophthora infestans
(P. infestans). The pathogen infects the new leaves of plants
belonging to the Solanacea family (like potato, tomato, eggplant,
pepper) and causes extended necrotic spots, which lead to general
necrosis of leaves and stems. Effective control of this disease is of
DOI: serious concern in Mediterranean countries, since their produc-
tion process is based on such yields.12

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However, the widespread use of conventional agrochemicals
causes severe environmental damage. A need exists for new
products having high biological activity and less copper in the
formulation. Inorganic NPs can be an approach in dissolving this
problem due to the increase in their surface to volume ratio.
Thus, the use of Cu-based NPs as nano-agrochemicals is chal-
lenging not only as a new research part but also as it is believed
that the applying dose of the formulated product can be
reduced.13 Moreover, it is a matter of concern which form of
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950 g mol1), polyethyleneglycol 8000 (Merck, M ¼ 8000 g mol1)
and polyoxyethylene (20) sorbitan laurate (Tween 20) (TCI, >97%,
M ¼ 1200 g mol1). All the reagents were of analytical grade and
were used without any further purication. Kocide 2000 35
(Ehymiadis agrochemicals, copper hydroxide, concentration in
metallic copper 35% w/w, formulation type: water dispersible
granules (WG)), Kocide Opti 30 (Dupont, copper hydroxide,
concentration in metallic copper 30% w/w, formulation type:
water dispersible granules (WG)), Cuprox disperss 20 (Alfa

copper is more suitable for agricultural applications, since in agrochemicals, Bordeaux mixture, calcium copper sulfate,
vitro antimicrobial studies of metallic copper (Cu), cuprous oxide concentration in copper 20% w/w, formulation type: water
(Cu2O) and cupric oxide (CuO) NPs reveal their effectiveness and dispersible granules (WG)) and Ridomil Gold Plus 42.5 (Syngenta,
specicity.14–17 Although the observed behavior is closely related copper oxychloride, concentration in metallic copper 40% w/w,
to the ability of NPs to release dissolved ions, relatively little are metalaxyl-M 2.5% w/w, formulation type: wettable powder (WP)).
known and thus it still remains under investigation the contri-
bution of the nanoparticle itself.1,17–20 Recently, Cu NPs have been
tested in laboratory conditions on Cucurbita pepo (zucchini),21,22 2.2 Preparation of Cu-based NPs
Phaseolus radiatus (mung bean),23 Triticum aestivum (wheat)23 All the synthetic routes followed the same procedure of hydro-
and Lactuca sativa (lettuce) seeds,6 while CuO NPs have been used thermal synthesis within a batch reactor (Parr Instrument
on Raphanus sativus (radish),24 Cucumis sativus (cucumber),24 Company, 5100 reactor). In total, three series of experiments
Triticum aestivum (wheat)25 and Lactuca sativa (lettuce) seeds.24 were carried out at 90, 120 and 160
C per each surfactant. The
The inuence on each kind of seeds, depends on concentration, surfactants that have been used were Tween 20, PEG 1000 and
composition, size and shape of NPs. In case of eld experiments, PEG 8000. Experimental results are summarized in Table 1.
it is necessary to evaluate not only the intrinsic characteristics of Reaction time (4 h), pressure (10 bar), the ratio of precursor to
each group of NPs but also the extrinsic conditions before their reducing agent (1 : 1) as well as precursor to surfactant ratio
application, as ora and fauna respond differently.6 (1 : 1) kept the same in all the experiments.
On that basis, the present study was designed to synthesize, In a typical synthesis (e.g. sample S1), 82 mmol (2 g)
characterize and evaluate in the eld the efficacy of Cu-based NPs Cu(NO3)2$3H2O were dissolved in 10 ml deionized water to
as agrochemical candidates against P. infestans in comparison obtain a blue solution. Then 82 mmol (0.414 g) N2H4$H2O were
with the registered and widely used conventional copper based added dropwise to the solution under vigorous stirring and the
agrochemicals Kocide 2000 35 WG, Kocide Opti 30 WG, Cuprox color changed from blue to yellow, indicating the reduction of
disperss 20 WG and Ridomil Gold Plus 42.5 WP. Thus, we Cu2+. Aerwards, a solution of 82 mmol (10 g) Tween 20 was
hydrothermally prepared Cu-based NPs of different composition added. The resulting mixture was stirred thoroughly and then
and sizes capped with biocompatible surfactants. In order to transferred into a 300 ml glass vessel, while the nal volume of
protect plant cells from increased NPs toxicity,24,25 the non ionic deionized water was lled up to 150 ml. The reactor was sealed
surfactants polyethylene glycol 1000 (PEG 1000), polyethylene and the solution was degassed for 5 min. By controlling the ow
glycol 8000 (PEG 8000) and polysorbate 20 (Tween 20) have been of the Ar into the vessel, the pressure was adjusted to 10 bar.
selected, while by monitoring the reaction temperature from 90 The temperature was adjusted to 90
C and maintained for 4 h.
to 160
C, Cu, Cu/Cu2O, Cu2O and CuO NPs have been isolated Finally, the reactor was cooled with water ow to room
and characterized. By passing through laboratory studies to real temperature and aer centrifugation at 1000 rpm, the super-
world applications and in proportion to our experimental natant liquids were discarded and a red-brown precipitate was
possibilities in the eld, three samples were selected to be tested
based on their size, shape and composition, considering that are
factors of key importance in many nano scale applications.26
Table 1 Summary of experimental results and main characteristics of samples
Cu2O, CuO and Cu/Cu2O NPs of similar sizes (11–14 nm) and
S1–S9. Emphasized samples have been selected for further structural and bio-
shapes have been tested in the eld for control of tomato late logical characterization
blight disease. Our ndings reveal that the newly prepared Cu-
based NPs are effective against P. infestans in lower copper weight Temperature Size
content and doses compared to the commercial products, indi- Surfactant Sample (
C) Phase (nm)
cating the possibility of future use as nano-agrochemicals. Tween 20 S1 90 Cu/Cu2O 14
S2 120 Cu/Cu2O 11
2 Experimental section S3 160 Cu2O 13
PEG 1000 S4 90 Cu/Cu2O 21
2.1 Materials S5 120 Cu2O 14
S6 160 CuO 14
Copper(II) nitrate trihydrate Cu(NO2)3$3H2O (Merck, $99.5%, M PEG 8000 S7 90 Cu 55
¼ 241.60 g mol1), hydrazine hydrate N2H4$H2O (Merck, about S8 120 Cu 52
100%, M ¼ 50.06 g mol1), polyethyleneglycol 1000 (Merck, M ¼ S9 160 Cu/Cu2O 44

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obtained and washed with ethanol, at least three times, to
remove the excess of ligands and the unreacted precursors.
2.3 NPs characterization
X-ray powder diffraction (XRD) measurements were performed
on a Philips PW 1820 diffractometer at a scanning rate of 0.050
per 3 sec, in the 2q range from 10–90
, with monochromatized Cu
Ka radiation (l ¼ 1.5406 nm). Conventional TEM images were
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minimum effective registered rate in terms of active ingredient
which refers to copper concentration; Kocide 2000 35 WG in 35 g
hl1, Kocide Opti 30 WG in 54 g hl1, Cuprox disperss 20 WG in
110 g hl1, Ridomil Gold Plus 42.5 WP in 224 g hl1. For the Cu-
based NPs active ingredients selected to be close to the lower
minimum of the reference products and in that manner for
sample S3 was 34 g hl1 and for sample S2 30 g hl1 while for
sample S6 we used half rate (15 g hl1) for comparison reasons.
One row of plants was kept as untreated control. The purpose of

obtained with a JEOL 100 CX microscope (TEM), operating at an

the untreated control was to check for the presence of adequate
acceleration voltage of 100 kV. For TEM observations we have
pest infestation and was not subjected to any of the plant
used suspensions of the NPs deposited onto copper grids. The
protection treatments under the study. The control arrangement
elemental composition of the samples was tested by inductively
was included into the experimental design.29
coupled plasma atomic emission spectroscopy (ICP-AES), Per-
The crop growth stage was between 69 and 89 according to
kin-Elmer Optima 3100XL. Thermogravimetric analysis (TGA)
BBCH crop growth stage, which is a decimal code system to
and Fourier transform infrared spectroscopy (FTIR) were used to
identify the phenological development stages of a plant, used in
examine the presence of the surfactant molecules on the surface
the agricultural industry.30 The air temperature was 29
C, the
of the nanocrystals. TG analysis (SETA-RAM SetSys-1200) was
air humidity 55% and the soil temperature in 10 cm depth was
carried out in the range from room temperature to 800
C at a
24.5
C. The method of application was foliar application with
heating rate of 10
C min1 under N2 atmosphere. Infrared
knapsack pressurized gas sprayer with lance (AZO type). The
spectra (280–4000 cm1) were recorded on a Nicolet FTIR 6700
application equipment was selected to simulate the commercial
spectrometer with samples prepared as KBr pellets. The micro-
practices in terms of spray pressure, droplet size and spray
Raman measurements were performed at room temperature
volume. The target application volume was 1000 liters per
using the 514.5 nm line of an Ar ion laser as the excitation source.
hectare and the application was performed before natural
The power was kept at 0.1 mW and a 100 mm lens was used. The
infection, when conditions were favorable for the disease. The
spectra were recorded using a micro-Raman Dilor XY triple
excess spray was sprayed into the waste plot, set up before the
monochromator coupled to a CCD detector. Finally, magnetic
initiation of the trial. In total, four assessments were made on
properties were measured using a superconducting quantum
the following dates: 11/06/12, 14/06/12, 18/06/12 and 21/06/12.
interference device (SQUID, Quantum Design MPMS-5) at 300 K.
Meanwhile, there were not observed any effects, positive or
negative, on other pests and non-target organisms (natural
2.4 Field experiments enemies, pollinators and/or adjacent crops) during this study.29
Field trial was conducted from June 11 to June 21 of 2012 in a
greenhouse in the village of Galatades, in the region of Central
Macedonia, Greece. The trial was set up under greenhouse 2.5 Statistical analysis
conditions in a growing area typical for commercial production of The experimental design was single replicate, non-randomized
the crop, in which infestation from the test organism has previ- plots and the layout of the plots was lengthwise. Two assess-
ously occurred.27 Typical symptoms of the disease like extended ment methods were used.29 The rst one was assessment on
necrotic spots on the young leaves were appeared.28 The experi- leaves, where 50 leaves were assessed on each plot to evaluate
ment was carried out under protected conditions (greenhouse) to the percentage of infection from P. infestans on each leaf. An
tomato (Lycopersicon esculentum) plants of Belladonna variety, average of these 50 leaves was used to estimate total percentage
transplanted on the 20th of April of 2012, with 0.4 m distance of
plants in the row and 0.8 m between rows. The plant height was in Table 2 Field experiment: treatment component, active ingredient, tested dose
average 1.5 m. Plot size was 3.2 m2 with 10 plants in one row. The in terms of active ingredient rate (g hl1) and in terms of formulated product rate
(g hl1)
trial was set up with a buffer zone of 5 m from the eld borders.
Growth conditions of the plants (e.g. age, height, form, blossom Active Formulated
density), cultural conditions (e.g. soil type, fertilization, tillage) Treatment ingredient product
and environmental conditions (temperature and humidity) were component Active ingredient rate (g hl1) rate (g hl1)
uniform for all plots and conform with the local horticultural
practice.29 Maintenance treatments did not have any activity on P. S3 Cu2O 34 43.87
S2 Cu/Cu2O 30 32.26
infestans.29 All Cu-based NPs treatments were destroyed at the end S6 CuO 15 27.78
of the trial by the trialist. The whole plants were uprooted and then Kocide Copper hydroxide 35 100
chopped up, dispersed and buried into the ground by ploughing. 2000 35 WG
The newly synthesized Cu-based NPs S2, S3 and S6 as well as Kocide Copper hydroxide 54 180
the reference products Kocide 2000 35 WG, Kocide Opti 30 WG, Opti 30 WG
Cuprox Calcium copper 110 550
Cuprox disperss 20 WG and Ridomil Gold Plus 42.5 WP have disperss 20 WG sulfate
been tested. In total, 7 treatments were applied and are presented Ridomil Copper oxychloride, 224 560
in Table 2. The standard reference products were applied in their Gold Plus 42.5 WP metalaxyl-M

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RSC Advances
of infection on each plot. The second type of assessment was the
percentage of phytotoxicity assessment. Each treated plot is
compared with an untreated plot for presence of phytotoxic
effects (stunting, chlorosis, bleaching) and % phytotoxicity is
estimated.29 Phytotoxicity assessment is done in accordance
with the EPPO Guideline no. 135 on Phytotoxicity Assessment.31
Results of assessment and statistical analyses have been
calculated with arcsine square root T(X) transformed response
for percentages. The raw data, arcsine square root transformed,
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were statistically analyzed using Analysis of Variance (ANOVA).32
Student–Newman–Keuls test was used for multiple compari-
sons of means. Efficacy was calculated using Abbot's formula.33
ARM statistical analysis tool was applied on this study.
3 Results and discussion
3.1 Synthesis and characterization of Cu-based NPs
In an attempt to evaluate the efficacy of Cu-based NPs for
potential use as agrochemicals, the hydrothermal synthesis in
the presence of hydrophilic surfactants was chosen, due to the
bio-application and the perquisite solubility of the products.
Water, as a solvent, makes the synthetic route eco-friendly
without producing hazardous waste and cost-effective.34,35
Surfactants/capping molecules are crucial especially in the case
of Cu-based NPs, as they do not act only as dispersant for
avoiding agglomeration and/or morphology control of the
samples,36–38 but also as a protective layer for preventing surface
oxidation, since Cu(I/II) exhibit exible redox behavior.39 In
addition, the antifungal activity is affected by the surface charge
of NPs, while the toxicity effect towards microbes is reduced
when charge is decreased.40 Thus, our study was focused on
three different surfactants, which are either polymers of
ethylene glycol (HO–CH2–CH2–OH) or natural mimetic poly-
sorbates, and are gaining increasing attention in the eld of
nanomedicine on account of being hydrophilic and non ionic.
Specically, we implemented Tween 20, which is a well-known
biomimetic polymer, and PEG of different molecular weights; a
relatively low molecular weight PEG 1000 and a higher one, PEG
8000. Concerning that there were not any previous synthetic
approaches on Cu-based NPs with the above capping agents, at
the rst step the synthetic conditions have been investigated.
Solution pH and temperature are the factors which affect the
mixed potential, and thus the most stable chemical species can
be changed by the pH and temperature.41,42 In the present study
we selected to vary only the temperature, as high temperatures
result in rapid nucleation and faster growth of the newly formed
nuclei, leading to the formation of small-sized NPs.43 The pH of
the reaction suspension is initially adjusted at 298 K by using
the same amount of reducing agent, hydrazine, in all the
experiments. In general, the mixed potential decreases with the
increase both in pH and temperature, while actual pH at reac-
tion temperatures differs from the adjusted value.44 Meanwhile,
earlier reports showed that the reducing agent affects the shape
of Cu-based NPs45,46 and that was also a matter of concern.
Thus, by differentiating the reaction temperature from 90 to
160
C for each surfactant under the same experimental
conditions samples S1–S9 were prepared. In all cases Cu nuclei
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are formed and depending on the experimental conditions
(reaction temperature, surfactant) they are oxidized further to
Cu2O or CuO. When Tween 20 was implemented, Cu/Cu2O
composite NPs were isolated at reaction temperatures of 90 and
120
C. However, when the reaction temperature was raised to
160
C, solely Cu2O NPs were formed. Similar behavior was
observed in the case of PEG 1000 for the temperatures of 90 and
120
C, while at 160
C solely CuO NPs were prepared. On the
other hand, the use of the more bulky PEG 8000 led to the
formation of Cu NPs at 90 and 120
C whereas at 160
C Cu/
Cu2O composite NPs have been isolated (Table 1), demon-
strating that the oxidation process depends on the molecular
weight of the polymer used and this is in accordance with
previous reports concerning lower and higher molecular weight
of poly(N-vinylpyrrolidone) (PVP).47
The phase composition and crystalline structure of all the
samples were determined via XRD measurements (Fig. S1 in the
ESI†). The peak positions of samples S1, S2, S4 and S9 consist of
two phases; one phase can be ascribed to face centered cubic
(fcc) copper (JCPDS card no. 04-0836) while the other phase
could be distinctly indexed to the cubic (Pn3m) crystal structure
of Cu2O (JCPDS card no. 05-0667). The patterns of samples S3
and S5 exhibit Bragg reections that correspond to the crystal
structure of Cu2O whereas the relative patterns of samples S7
and S8 correspond to fcc Cu. Finally, sample S6 exhibited the
monoclinic structure of CuO (ICDD, JCPDS card no. 80-1916).
However, it has to be denoted that copper is easily oxidized in
aqueous medium and thus further investigation by X-ray
photoelectron spectroscopy (XPS) needed for S7 and S8 samples
in order to testify if a CuO surface layer of different thickness
can exist.48 The average crystalline sizes of the NPs of all the
samples were determined by taking the full width at half-
maximum (FWHM) of the most intense peak and by using the
Scherrer equation (Table 1).
According to these data and in proportion to our experi-
mental possibilities in the eld, three samples were selected to
be tested based on their size and composition. The size of the
NPs is closely related to many essential properties, such as
surface property, solubility, chemical and biological reactivity.
NPs diffuse across the membrane and transport into cells
through ion channels and proteins, while in some cases
endocytosis occurs, depending on the size.49 The composition
is also a matter of concern as previously referred, since it is
linked to the exible Cu(I)/Cu(II) redox activity.11 Moreover, it is
stated that NPs are much more soluble than bulk materials
due to the larger surface areas, which allow them to interact
closer with solvent molecules and show faster dissolution.49
Taking into account the above factors, Cu2O (sample S3), CuO
(sample S6) NPs and Cu/Cu2O composite (sample S2) NPs with
relatively similar sizes 13, 14 nm and 11 nm respectively, were
depicted (Table 1). Metallic Cu NPs have been excluded at least
from the present study, as based on previous references found
to be more bioreactive compared to Cu2O and CuO NPs, since
were more effective against Escherichia coli bacteria and were
found to have antiviral activity.16,50 In that way, pure metallic
Cu NPs are expected to be toxic not only to fungi but also to
the plants.
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Further characterization of the selected samples achieved.
The weight content percentage (wt%) of copper of each sample
(S2, S3 and S6) has been measured by ICP elemental analysis as
the active ingredient of the commercial products refers to
copper concentration. Sample S6 is composed of relatively low
wt% Cu (54%) in comparison with samples S2 (93%) and S3
(77.5%). The differences in the wt% are attributed to the
different cell structure as the unit cell of CuO consists of four
copper and four oxygen atoms, whereas Cu2O of four copper
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and two oxygen atoms respectively.48
Lattice parameters were calculated by analyzing the XRD
patterns of the samples S2, S3 and S6 (Fig. 1) in detail and by
using MDI's Jade soware. As far as sample S6 concerned, the
corresponding values were found to be a ¼ 4.74 Å, b ¼ 3.42 Å,
c ¼ 5.13 Å, which are in good agreement with the standard
values reported by the JCPDS card no. 80-1916. The values of the
lattice parameters for samples S3 and Cu2O phase of the
composite sample S2 found: a ¼ 4.29 Å and 4.28 Å respectively
and are in accordance with the bulk lattice parameter of Cu2O
(4.27 Å). For the Cu phase of composite NPs (S2) the calculated
lattice parameter is a ¼ 3.62 Å, very close to the theoretical value
a ¼ 3.61 Å. These results reveal the high crystalline structure of
the samples. In addition, for the Cu/Cu2O composite NPs
(sample S2) the phase ratio was calculated by MDI's Jade so-
ware and found to be approximately 1 : 1 (Fig. 1 inset).
Fig. 1 XRD patterns of samples S2, S3 and S6. Vertical lines denote expected
diffraction peak positions for CuO (black and white triangles), Cu2O (full triangles)
and Cu (open triangles). Inset shows the phase ratio of sample S2.
Fig. 2 TEM images of samples S6 (a), S3 (b) and S2 (c).
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The chemical stability of the resulted NPs has been tested,
since Cu-based NPs tend to oxidize. For that reason, XRD
spectra of samples S2 and S3 were recorded aer 6 months and
the results clearly indicated that both samples are stable and no
sign of oxidation was observed even aer 6 months of storage
(Fig. S2 in the ESI†).
TEM micrographs are illustrated on Fig. 2 while the particle
size distributions are included in ESI (Fig. S3 in the ESI†).
Samples S3 and S2 isolated with the same surfactant (Tween 20)
display nearly spherical shape whereas sample S6 exhibits
truncated spherical shape due to the different surfactant (PEG
1000). Both surfactants can be considered as efficient capping
agents; they led to well dispersible NPs since no aggregation was
observed. In case of sample S2, Cu2O shell of approximately
1 nm thickness was formed, indicated by the dotted line and the
arrows. The Cu2O layer, showing light contrast, developed on
the particle surface while the particle interior showing a dark
contrast is composed of copper as found also elsewhere.51
Interestingly, we observed that Cu/Cu2O NPs exhibit also light
interior contrast in some spots in the center, which suggests the
formation of partial interior void, sacricing the copper core.
The hollowing process is closely related to the copper oxidation
as previously demonstrated.52
The presence of the surfactants has been testied by FT-IR
spectroscopy. FT-IR spectra of the samples (S6, S3 and S2) with
the corresponding patterns of PEG 1000 and Tween 20 are given
in Fig. 3(a) and (b) respectively for comparison reasons. In all
samples medium peaks at about 2920 cm1 and 2860 cm1 are
assigned to the asymmetric (nas) and symmetric (ns) stretching
vibrations of methylene (–CH2) groups, common in both
surfactant molecules. For sample S6 the broad peak around
1643 cm1 ascribed to the hydrogen bonds, formed from the
hydroxide groups while the C–O–C ether stretch band at 1106
cm1 is indicative of the presence of PEG 1000. For samples S3
and S2 two characteristic broad bands attributed to asymmetric
(nas) and symmetric (ns) stretching vibrations of laurate at 1628
and 1381 cm1 respectively are shown. The difference of
asymmetric (nas) and symmetric (ns) stretching vibrations [D ¼
nas(CO2)  ns(CO2)] found 247 cm1 indicating binding.53 For
sample S6 the peak located at 540 cm1 (Fig. 3(a)) can be
assigned to CuO metal–oxygen bond while for samples S3 and
S2 a peak at 625 cm1 is exhibited (Fig. 3(b)), which can be
assigned to the vibrations of Cu(I)–O bonds.54 Specically, in its
crystal lattice CuO is connected to four oxygen atoms in a square
planar geometry, whereas Cu2O is linked with two oxygen atoms
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Fig. 3 FTIR spectra of PEG 1000 and sample S6 (a) and FTIR spectra of Tween 20 and samples S2 and S3 (b).

in a linear coordination. Therefore, the Cu(II)–O bonds in CuO

are weaker than Cu(I)–O bonds in Cu2O and this will clearly
explain why the vibration of the Cu–O bond in CuO appears in
lower frequencies than those of Cu2O.54
Raman spectra of samples S2 and S3 were recorded to detect
if the samples contain a monolayer shell of CuO, too thin to be
detected by XRD and FT-IR spectra.48 Raman spectroscopy in
addition to FT-IR analysis provides a wide range of information
about the strength of interatomic and intermolecular bonds
within the NPs and can detect the presence of even thin oxide
layers. As shown in Fig. 4 there are four characteristic peaks of
crystalline Cu2O. The longitudinal optical phonon modes (LO)
G12 at 109 cm
1
and the G15
(1)
at approximately 150 cm1 are
inactive Raman modes while the most intense Raman signal is
the second-order overtone 2 G 1
12 (218 cm ). Additionally, a
broad peak with less intensity centered at approximately
630 cm1 is observed, which corresponds to the red-allowed
transverse phonon mode (TO) G (2) 55,56
15 . Thus, there is no Fig. 4 Raman spectra of samples S2 and S3.
evidence of any CuO layer in the samples, conrming the
presence of a pure phase of Cu2O NPs.
Fig. 5 shows the TGA curves of samples S2, S3 and S6 where decomposition of the laurate, while the last step to the
the total amount of weight losses are 21.2 wt%, 28.9 wt% and decomposition of the rest organic part. Thus, by implementing
25.7 wt%, respectively. Samples S2 and S3 reveal similar TG analysis the amount of surfactants coated on the surface was
thermal behavior due to the same surfactant (Tween 20); the quantitatively recorded and the thermal stability of the ligands
decomposition occurs in four steps between 25 and 450
C. In in the temperature range 0 to 200
C has been proved.
case of sample S6 the decomposition of PEG 1000 occurs in Field-dependent magnetization of the synthesized nano-
three steps. The rst and the second weight loss in all samples particles was measured at room temperature (300 K) and by
attribute to the water and CO2 decomposition respectively. In varying the magnetic eld up to 30 kOe (Fig. 6). The M–H data
case of samples S2 and S3 the third step in the temperature of sample S6 exhibit features of so magnetic material with
range of approximately 250 to 350
C can be attributed to the saturation magnetization 0.102 emu g1 and thereby CuO NPs

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Fig. 5 Thermogravimetric curves of Cu-based NPs S2, S3 and S6 showing the
relative weight loss attributed to the surfactant removal.
Fig. 6 Field dependent magnetization at 300 K for samples S2, S3 and S6.
were veried. The weak ferromagnetism may be attributed to
the exchange interactions between the uncompensated valence
states due to the oxygen vacancies at the surface of the NPs.57
Lack of magnetization in case of samples S2 and S3 is indicative
of their composition as expected.
3.2 Antifungal activity and phytotoxicity of Cu-based NPs
The late blight of tomato, as mentioned previously, is caused by
P. infestans and poses a well known threat to one of the major
agricultural products world-wide. The effective control of the
disease is of great economical importance. The efficacy of the
samples S2 (Cu/Cu2O composite NPs), S3 (Cu2O NPs) and S6
(CuO NPs) was evaluated in terms of antifungal activity against
P. infestans and crop safety on protected tomato plants and in
reference with registered agrochemicals. The criteria for the
selection of the referenced agrochemicals Kocide 2000 35 WG,
Kocide Opti 30 WG, Cuprox disperss 20 WG and Ridomil Gold
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Plus 42.5 WP were that are well-known trade products, widely
used in Greece, especially in the particular area. To our
knowledge, it is the rst time that such an in vivo test is reported
in the literature. For that reason, we chose four different agro-
chemicals as references that are already in use in the area,
which have different composition and rates of active ingredi-
ents and as a consequence are being sprayed on the plants in
distinctly different rates of formulated products. The aim of our
study was to test if the newly Cu-based NPs are more effective
against P. infestans compared to typical products. Thus, we
tested the registered products to their minimum effective
registered rate and the NPs were applied in even lower rates.
Two methods of assessment were used; the evaluation of the
percentage of infection on each leaf and in that way an average
of 50 leaves were assessed on each plot (Table 3) and the eval-
uation of phytotoxicity in which the presence of phytotoxic
effects as stunting, chlorosis, bleaching have been explored
(Table 4). The results depicted on Table 3 are also presented in
histograms (Fig. 7 and 8).
Treatment with CuO NPs (sample S6) resulted in signicant
protection from the fungal disease in comparison to the nega-
tive control and to the reference products, as proved to reduce
the percentage of infection from P. infestans on leaves (Fig. 7).
The efficacy of sample S6 was ranging from approximately 40%
three days aer application to 61.43% seven days aer appli-
cation (Fig. 8). Specically, during rst assessment CuO NPs
performed statistically better compared to Ridomil Gold Plus,
whereas the degree of efficacy did not differ signicantly in
comparison with the standard reference products Kocide Opti
30 WG and Cuprox disperss. The greatest level of disease
suppression on tomato leaves was observed seven days aer
application, while during last assessment sample S6 showed
higher reduction of disease severity than the standard reference
products Kocide 2000 35 WG and Kocide Opti 30 WG. Results
concerning sample S6 conrm that the applying dose of
formulated products can be reduced without affecting the effi-
cacy of the products, since CuO NPs were applied in the lowest
concentration of the formulated product (27.78 g hl1) and of
the active ingredient (15 g hl1) (Table 2).
Cu2O NPs (sample S3) exhibit a different pattern of plant
protection during the ten days of assessment. During rst and
last assessment greater disease suppression was observed in
comparison with all the applied products (Fig. 7). However,
seven days aer application, although the fungal disease was
signicantly suppressed in comparison with the untreated
plots, the values of the % infection on the leaves were not as low
as those of the other products. These results probably indicate a
different mechanism of action of Cu2O NPs. In terms of efficacy
sample S3 was the most efficient against P. infestans (73.53%) in
comparison to all products ten days aer application (Fig. 8).
The concentration of the formulated product (43.87 g hl1) and
the active ingredient (34 g hl1) still remain considerable lower
in comparison with all the tested registered products (Table 2).
The efficacy of Cu/Cu2O composite NPs ranged from
approximately 54.29% seven days aer application to 69.61%
ten days aer application (Fig. 8) indicating a more stable
antifungal activity than samples S3 and S6. During rst and last
RSC Adv., 2013, 3, 21743–21752 | 21749
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RSC Advances Paper

Table 3 Percentage of infection on 50 leaves per plot following application with samples S3, S2 and S6. Location: Galatades, Pella, Greece. Season: 2012. Means
followed by the same letter do not significantly differ (P ¼ 0.05, Student–Newman–Keuls)

11-Jun-12 14-Jun-12 18-Jun-12 21-Jun-12

Percentage of Percentage of Percentage of Percentage of

infection on infection on infection on infection on
Day of assessment 50 leaves per plot 50 leaves per plot 50 leaves per plot 50 leaves per plot

Student– Student– Student– Student–

Treatment Newman– Newman– Newman– Newman–
Published on 04 September 2013. Downloaded by Universidade de Brasilia on 08/05/2014 10:47:18.

component Mean% Keuls test Mean % Keuls test Efficacy % Mean % Keuls test Efficacy % Mean % Keuls test Efficacy %

Untreated plot 0.75 d 4.00 a 0.00 7.00 a 0.00 10.20 a 0.00

Sample S6 1.50 a 2.40 c 40.00 2.70 g 61.43 2.70 h 60.78
Sample S2 1.40 a 1.60 e 60.00 3.20 e 54.29 3.10 g 69.61
Sample S3 1.00 c 1.40 f 65.00 3.70 b 47.14 4.00 d 73.53
Kocide 2000 35 WG 1.20 b 1.70 d 57.50 3.40 c 51.43 4.30 b 57.84
Kocide Opti 30 WG 0.80 d 2.40 c 40.00 3.70 b 47.14 4.30 c 57.84
Cuprox disperss 0.90 cd 2.40 c 40.00 3.30 d 52.86 3.50 f 65.69
20 WG
Ridomil Gold Plus 1.20 b 3.00 b 25.00 2.60 f 62.86 3.60 e 64.71
42.5 WP

assessment, a trend of greater disease suppression was

observed in comparison with the reference products (Fig. 7).
Nonetheless, seven days aer application, Ridomil Gold Plus
proved to be the most effective antifungal agent.
In general, all the newly prepared Cu-based NPs found to be
effective while the applying dose of the products reduced
signicantly without affecting their efficacy. It has to be
mentioned that, although the samples were applied above the
dose of maximum effect, the copper weight content of the NPs
tested was not 100% and thus further investigation needed to
evaluate the maximum fungicidal activity. We observed that Cu-
based NPs diminish the percentage of infection as compared to
the untreated plots and proved to be more effective against
P. infestans than all the tested reference products Kocide 2000
35 WG, Kocide Opti 30 WG, Cuprox disperss and Ridomil Gold
Plus. Furthermore, Cu-based NPs exhibit differentiations on
their performance depending on the day of assessment, indi-
Fig. 7 % Infection on leaves of samples S2, S3 and S6 and of reference products
cating that their mechanism of action varies among NPs with
against P. infestans on protected tomato.
different composition in regard with the trade products. CuO

Table 4 Percentage of phytotoxicity in terms of necrotic and chlorotic spots

14-Jun-12 18-Jun-12 21-Jun-12

Day of assessment Percentage of phytotoxicity Percentage of phytotoxicity Percentage of phytotoxicity

Student–Newman– Student–Newman– Student–Newman–

Treatment component Mean % Keuls test Mean % Keuls test Mean % Keuls test

Untreated plot 0.00 b 0.00 a 0.00 a

Sample S6 0.00 b 0.00 a 0.00 a
Sample S2 3.75 a 1.25 a 0.00 a
Sample S3 2.50 ab 1.25 a 0.00 a
Kocide 2000 35 WG 0.00 b 0.00 a 0.00 a
Kocide Opti 30 WG 0.00 b 0.00 a 0.00 a
Cuprox disperss 20 WG 0.00 b 0.00 a 0.00 a
Ridomil Gold Plus 42.5 WP 0.00 b 0.00 a 0.00 a

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Fig. 8 % Efficacy of samples S2, S3 and S6 and of reference products against
P. infestans on protected tomato.
NPs (sample S6) found to be more effective seven days aer
application, while Cu2O NPs (sample S3) three and ten days aer
application. Regarding sample S2, which is Cu/Cu2O composite,
its performance was more stable among all days of assessment in
comparison with the other two samples. The dynamic nature of
NPs, in terms of stability, strength of aggregation and dissolu-
tion to ions, complicates the explanation of their observed bio-
reactivity and further investigation is needed.25
As far as phytotoxicity concerned, some symptoms were
observed in the plots treated with the samples S2 and S3, three
and seven days aer application, which disappeared ten days
aer application (Table 4). The phytotoxic symptoms were small
necrotic spots and some chlorotic spots on the leaves. However,
we did not observe any phytotoxic symptoms on fruits or
owers. Sample S2 exhibited the highest % phytotoxicity in
comparison with the rest of the samples. This behavior can be
attributed to the presence of the metallic core in those NPs
which can be considered more bioreactive than the oxides.16 As
a result in the present study the application of Cu/Cu2O
composite NPs to the plants approaches the limit between plant
protection and phytotoxicity.
4 Conclusions
Hydrothermal synthesis appears to be an easy preparation for a
variety of Cu-based NPs. Tailoring the synthetic conditions, by
means of temperature at 90, 120 and 160
C and by imple-
menting surfactants of relative low molecular weight (PEG 1000,
Tween 20) Cu/Cu2O, Cu2O, CuO NPs of small sizes (11–21 nm)
were formed. It is worth mentioning that Cu-based NPs of these
sizes are scarcely reported. On the other hand, the use of a
higher molecular weight surfactant, PEG 8000, protected effi-
ciently the metallic nuclei from any oxidation processes at 90
and 120
C and Cu NPs (50 nm) were isolated. However, when
the temperature was raised at 160
C Cu/Cu2O composite NPs
have been formed even in the case of PEG 8000. In conclusion,
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temperature and surfactants seem to selectively control the
composition of copper NPs.
Three different Cu-based NPs among the prepared, of similar
sizes (11–14 nm) and shapes (nearly spherical), Cu2O, CuO and
Cu/Cu2O respectively, were tested in the eld against P. infestans
on protected tomato. To our knowledge, this is the rst time
that such a study is reported in the literature. The results
illustrated that all the tested Cu-based NPs are more effective in
lower formulated product and active ingredient rate than the
four registered copper based agrochemicals. Beside the prom-
ising efficacy, concerning phytotoxicity Cu-based NPs did not
induce any permanent damage to the plants. Since the applied
amount of copper on the plants was minimized, further inves-
tigation is needed to nd out the minimum effective dose of
each sample. Nevertheless, a more detailed screening is
demanded to establish correlations between their characteris-
tics and nanosizes effects, keeping in mind that the limit
between plant protection and phytotoxicity is very close.
Acknowledgements
The authors C. Dendrinou-Samara and K. Giannousi are grate-
ful for the nancial support of the European Union (European
Social Fund – ESF) and Greek national funds through the
Operational Program “Education and Lifelong Learning” of the
National Strategic Reference Framework (NSRF) – Research
Funding Program: Thales. Investing in knowledge society
through the European Social Fund. All the authors would also
like to thank Dr Sakellari Despoina for her great assistance in
TEM analysis.
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