1. Art and Diseño

Galactomannan Mediated Green Synthesis of Highly Stable ZnO and CuO
Nanoparticles: Comparative effects of particle Size, pH and exposure time on
phyto-toxicity
Muhammad Amin1 *, Safyan Akram Khan2 , Mahmood Anwar1 , Manzar Sohail2 ,
Ahsanulhaq Qurashi2
1
Department of Chemistry, University of Sargodha, Sargodha-40100, Pakistan.
2
Center of Excellence in Nanotechnology Research Institute, King Fahd University of
Petroleum and Minerals, Dhahran 31261, Kingdom of Saudi Arabia
*Address for correspondence: Dr. Muhammad Amin, Department of Chemistry, University of
Sargodha-40100, Sargodha, Pakistan. E. mail: [email protected]
Abstract
This paper reports galactomannan mediated green synthesis of zinc and copper oxide
nanoparticles followed by their photo-toxicity evaluation.
The proposed green synthesis
involves the simultaneous use of galactomannan as reducing and stabilizing agent. The
synthesized zinc and copper oxide nanoparticles were structurally elucidated using SPR
spectroscopic, powder X-Ray diffraction spectroscopic, scanning electron microscopic,
transmission electron microscopic and atomic force microscopic measurements and had an
average size of 18 nm and 25 nm, respectively. Phyto-toxicity tests were performed in order to
assess the biological effect of nanoparticles on the seed germination and root length of
Abelmoschus esculentu seeds. Zinc oxide nanoparticles were found to be non -cytotoxic on the
seed germination and root elongation in the concentration range of 0.25 – 1.25 µg mL-1 .
However, at higher concentrations a retardation effect was noted. Copper oxide nanoparticles
showed deleterious effects on the seed germination and root elongation at all concentrations. The
likely mechanisms of the phytotoxic effects were also discussed. The phyto-toxicity related data
revealed that the tested Zinc oxide nanoparticles may play an important role towards improving
agriculture growth and productivity. Furthermore, the effect of nanoparticle size and exposure
time and pH of the media on the plant growth were optimized. The likely mechanisms of the
phytotoxic effects of nanoparticles were discussed.
Keywords:
Zinc
oxide
nanoparticles,
Copper
oxide
nanoparticles,
Galactomannan,
Hydrocolloids, Green synthesis, Phyto-toxicity.
1. Introduction.
Because of a wide band gap (3.3 eV) and high excitonic binding energy (60 MeV), Zinc
oxide (ZnO) NPs have recently been declared toxicologically safe by the U.S. Food and Drug
Administration (regulation code: 21CFR182.8991). Although, ZnO-NPs have demonstrated
potential antimicrobial activities against a wide range of gram-positive and gram- negative
bacteria yet they have limited therapeutic applications due to their instability and some structural
features [1]. Copper oxide (CuO) NPs have been explored as a possible substitute for noble
metals on account of their unique properties like potential role in catalysis [2], antimicrobial
agents [3] and semiconductors [4] etc. It has been reported that the unique properties exhibited
by metal NPs are very closely associated with their exposed crystallographic facets, sizes and
shapes [5]. Therefore, the preparation of highly stable, monodispersed and size-controlled NPs,
is a growing area of research in nanotechnology [6]. So far, the preparation of ZnO and CuO NPs
have been carried out through a number of methods involving radiation methods, microemulsion
techniques, electrode discharge and wet chemical reduction [6-8]. All these methods rely mostly
on chemicals and solvents which are highly undesirable because of their hazardous effects on the
environment [5, 9]. Furthermore, most of these techniques involve calcinations step to obtain the
final product [10]. The overall consequences of these techniques are high energy consumption
and heavier environmental pollution. Therefore, there is a need of developing cost effective and
environmental- friendly techniques as alternative to existing methods. In the light of growing
applications of ZnO and CuO NPs in various fields, the design and exploration of a facile and
environmentally benign strategy is highly desirable.
The Polysaccharide gums presents one of the most abundant naturally accruing raw materials
due to their sustainable, biodegradable, bio-safe characteristics [11] and versatile applications in
drug delivery [12], tissue engineering [13-14], biosensors and electronics [15]. Water soluble
natural gums, also known as hydrocolloids, consist of complex polysaccharides derived from
various sources including plant cell walls, tree exudates and seeds of the plants [11].
Galactomannan is a water soluble linear polysaccharide found in the endosperm of many plant
seeds, particularly the Leguminosae family, where they develop energy-reserve and hydration
functions [16]. On account of its specific physicochemical properties such as high molecular
weight, water solubility, non- ionic character and absence of toxicity, this polysaccharide finds
extensive applications in food, cosmetic and textile industry as stabilizer, emulsifier and gelling
agent [17-18]. Chemically, they are composed of a linear β (1-4) –mannane backbone attached to
a single D- galactopyranosyle residues via α-(1-6) linkages including side chains of galactose
remains [19]. Cyamopsis psoralioides L. (C. psoralioides) commonly known as ‘guar’ is a rich
and cheap source of galactomannan. It is commercially easily available in South Asian countries.
The galactomannan contents of the endosperm and seeds of guar range from 68-85%
respectively [20].
Some of the carbohydrates polymers have been used in the synthesis of nanoparticles (NPs)
and very recently it has been reported that highly stable silver and gold NPs were synthesized by
the use of arabinoxylan and glucoxylan as reducing and stabilizing agents [9, 21]. These studies
revealed the potential of carbohydrate polymers in the green synthesis of NPs without any use of
conventional chemicals or solvents. The NPs synthesized by carbohydrate polymers were found
to be exceptionally high stable as the polymeric nature of the carbohydrate polymers can cause
dispersion of particles into their matrix [9, 21]. Galactomannan has been used in the synthesis of
silver [22-23] and gold nanoparticles [24]. Most recently it has been used for the synthesis of
chitosan guar gum silver nanoparticles hybrid matrix having applications in immobilized
enzymes for fabrication of beta- glucan and glucose sensing photometric flow injection systems
[25].
Over the last decade, despite of nanomaterials fabrication advancements in various fields,
their uses in agriculture, especially for plant protection and production is an under-explored area
in the research community [26].
The objective of this study was to evaluate the potential of biocompatible carbohydrate
polymer, galactomannan, in synthesis of ZnO and CuO NPs for biomedical and agricultural
applications. To the best of our knowledge this is perhaps for the first time that we have
synthesized exceptionally highly stable ZnO and CuO NPs by the use of galactomannan
excluding the use of any conventional reducing and capping agent. It has been proposed that the
polymeric nature of the galactomannan will render high stability and provide a better opportunity
to the NPs to study the fluctuations in phytotoxicity studies under varying particle sizes, pH
conditions as demonstrated in this investigation.
2. Experime ntal
2.1 Materials
Zn (CH3 COO)2 .2H2O (E. Merck, Germany), Cu(CH3 COO)2 .H2 O (E. Merck, Germany)
sodium hydroxide, NaOH (E. Merck, Germany), and hydrochloric acid, HCl (E. Merck,
Germany) were of analytical grade and used without further purification. Ultra-pure produced
by Nanopure system was used throughout in this work. Cyamopsis tetragonolobus L. seeds were
purchased from a local market.
2.2 Isolation of galactomannan
C. tetragonolobus L. seeds were cleaned by washing with deionised water. Cleaned seeds
(100 g) were de-husked, milled and screened to obtain the ground endosperm, galactomannan, as
off- white powder. The %age yield of galactomannan was found to be 90. The powder thus
obtained was washed with ethanol, dried and dissolved in ultra-pure water (10 g/100 mL) and
stored as GN for further use.
2.3 Synthesis of ZnO-Nps
To zinc acetate dihydrate solution (10 mL, 1.0 mM) was added by drop wise, a solution of
GN (2.0 mL) and the reaction mixture was stirred constantly at 60 °C for 30 min. The reaction
mixture was filtered and the solid powder thus collected was dried in an oven at 120 °C for 3.0
hrs. Further experiments were performed by varying the reaction inputs in order to optimize the
effect of GN on the reduction of zinc acetate.
2.4 Synthesis of CuO-NPs
To a solution of copper acetate monohydrate (10 mL, 1.0 mM), GN (2.0 mL) was added drop
wise and the reaction mixture was stirred constantly at 75 °C for 45 min. The blue color of the
reaction mixture turned black indicating the formation of CuO NPs [27]. The reaction mixture
was filtered and the solid black powder thus collected was dried in an oven at 120 °C for 3.0 hrs.
The powder was washed with water 3-4 times and characterized. Further experiments were
performed by varying the reaction inputs in order to optimize the effect of GN on the reduction
of copper acetate. The black color of the solution was found to be stable for more than 2 years as
confirmed by SPR analysis.
2.4 Characterization of the samples
2.4.1. SPR spectroscopic measurements
UV-visible spectroscopic techniques were used to obtain the SPR spectrums of the NPs in the
range of 200–700
nm by using Pharmaspec UV-1700 (Shimadzu,
Tokyo, Japan)
spectrophotometer by setting GN and quartz cell as reference.
2.5. Powder X-Ray diffraction spectra
P-XRD spectra of the NPs were recorded on Bruker D8 Discover (Germany) diffractometer
using monochromatic Cu Kα radiation (λ= 1.5406 Å) operating at 40 kV and 30 mA in 10 – 80°
2θ range. Full width at half maxim (FWHM) of the most intense peak was determined and the
size of NPs was calculated by the use of Debye-Scherrer equation,
).
2.6. Scanning electron microscopy
SEM images of the selected NPs were obtained by using SEM S-3700N (Hitachi Japan)
without sputter coating because the NPs were self-conducting.
2.7. Transmission electron microscopy
An ultrasonically dispersed sample (one drop) of the solution of NPs was placed on a clean
carbon grid and TEM images were taken at room temperature by using JEM-1200EX (JEOL,
Japan) microscope at an accelerating voltage of 120 kV. By measuring the sizes of 140 different
particles, the average size was calculated by using Origin 7.5 software.
2.8. Atomic force microscopy
Scanning probe microscope SPM-9500 J3 (Shimadzu, Japan) was used to obtain the
atomic force microscopic (AFM) images from a 10.0 µm × 10.0 µm film of the samples
in contact mode under normal atmospheric conditions and a dust- free environment.
2.9. Phyto-toxicity study
Phyto-toxicity study of ZnO and CuO nanoparticles, having average size 18nm and 25nm
respectively, was performed on root length and percent seed germination of okra (Abelmoschus
esculentus) seeds by the use of plant agar method [28]. The seeds were sterilized in 5% sodium
hypochlorite solution and washed thoroughly with deionised water by several times. Petri dish
(83 mm × 17 mm) containing 25 mL of dual agar culture media (15 mL of 2.5% agar covered
with 10 mL of 1% agar) and ten different concentrations of NPs ,0.25, 0.5, 0.75, 0.1, 1.25, 1.5,
1.75, 2.0, 2.25 and 2.50 µg mL-1 were prepared. Petri dish without NPs containing only water
and with carbohydrate polymer (GN, 2.0 mg L-1 ) was sued as blank. Ten A. esculentus were
gently placed above the surface of agar media. The plates were placed in incubator at a
controlled temperature of 35 ± 1°C in dark. Seed germination and root length was measured and
compared with the blank for five days. In another experiment the same concentrations of the NPs
and control were used to determine the percent germination under similar conditions. The
germination was recorded for five days. These experiments were performed in triplicate and
data were statistically analyzed [29].
3. Results and discussion
3.1. Synthesis of nanoparticles
The ZnO-NPs and CuO-NPs were obtained by the reduction of zinc acetate and copper
acetate respectively with galactomannan isolated from C. tetragonolobus L. seeds, where the
galactomannan suspension, characterized to be a GN, served as reducing as well as capping
agent. The shape and size of the NPs was controlled by monitoring the SPR spectral absorptions
with respect to the amount of GN, stirring time, pH and temperature of the reaction media. As
shown in figure 1(a), ZnO-NPs exhibit a sharp peak at 327 nm and some broad peaks at 346,
350, 378 and 380 nm corresponding to the average size of 18, 30, 45, 38, 48 and 55 nm
respectively. On the other hand CuO-NPs exhibit SPR band at 549, 560, 570, 580 and 600 nm
with the average size of 25, 35, 42, 58 and 70 nm (Fig. 2b). The characteristic SPR band
appearing in at 346 nm and 549 nm reveal the formation of smallest ZnO-NPs and CuO-NPs
respectively (Nagajyothi et al., 2013). The reaction conditions optimized to obtain the smallest
ZnO-NPs in terms of amount of GN, pH of the reaction media, temperature and stirring time
were: amount of GM 6.0 mL/ 20 mL, zinc acetate dihydrate solution 10.0 mL/20 mL,
temperature 55°C and time 30 min. The optimum conditions to obtain polydisperse CuO-NPs
were:
amount of GN 8.0 mL/ 20 mL, copper acetate hydrate solution 10.0 mL/20 mL,
temperature 75°C and time 60 min. The SPR variations with respect to GN amount, pH,
temperature and reaction time is shown in Fig. 1. The SPR absorption bands were found to be
extremely sensitive to the GN amount because at higher GN concentration, aggregates of NPs
were formed perhaps due to increase in the viscosity of the reaction media as already reported for
the green synthesis of ZnO NPs by aloe barbadensis miller leaf extract [30]. It has generally been
recognized that SPR spectra of metal based NPs could be used to control their size and shape in
aqueous suspension [31].
3.2. Powder X-Ray diffraction spectra
The p-XRD spectrum were found to be characteristics of crystalline nature of the ZnO NPs
as shown is Fig. 2 a. The peaks appeared in the spectrum were compared to the data JCPDS
(Card Number 36-1451) and were characterized to be (100), (002), (101), (102), (110), (103), (2
00), (112), (201), (004) and (202) planes of ZnO in the wurtzite structure. The powder XRD
pattern (Fig. 2b) peaks appeared in the P-XRD spectrum consists of (2Ѳ) = 32.25º(111),
34.33°(002), 39.90° (– 111), 46.53º (– 202), 52.10º (020), 58.14° (202), 61.9º (– 113), 67.2° (022)
and 68.49° (220) corresponds to different planes of monoclinic phase of CuO. The
nanocrystallite of CuO was characterized to be of typical monoclinic structure space group C2/c
after comparing with JCPDS file no. 89-5896. Some additional peaks found in the pXRD spectra
of ZnO and CuO NPs were characterized to be due to the crystallization of organic material
belongs to polymer matrix of GN. This is in agreements to the previous study of glucoxylan
mediated green synthesis of gold and silver NPs [21]. The average size of the ZnO and CuO NPs
determined from the full width at half maximum of the most intense peak by applying DebyeScherrer equation was found to be 18 nm and 25 nm respectively.
3.3 AFM, SEM and TEM analysis
The surface morphology of the carbohydrate polymer matrix having incorporated ZnO and
CuO NPs is shown in AFM images (Fig. 3a, 3b). The roughness of the surface is due to the
dispersion of the NPs in the GN matrix. Typical SEM and TEM images, the TEM micrographs
depicting the effect of GN concentration on the size and shape of ZnO NPs and the size
distribution histogram of the ZnO NPs under optimized conditions are shown in Fig. 4 (a, b ,c
and d). It is clear from the Fig. 4b, that the NPs are monodispersed and of spherical shapes. The
particle size distribution was calculated by analyzing the TEM image and average particle size
was calculated from the size distribution histogram. From these measurements the average size
of ZnO NPs was found to be 18 nm.
The TEM image of CuO NPs (Fig. 5a) showed some quasi-spherical nanosized particles
incorporated in a polymer matrix. The selected area electron diffraction pattern (Fig. 5c)
confirmed the crystalline nature of the CuONPs. The circular rings appearing in SAED pattern
were assigned to (100), (102), (101), (110), (103) and (112) crystalline planes. The average size
of the nanocrystalite calculated from TEM measurements was found to be 25 nm. The results are
in consistent to a previous study [31]. The average sizes of ZnO NPs and CuO-NPs calculated
from TEM measurements match well with the sizes calculated from XRD and SPR calculations.
This investigation demonstrated that GN can act as an efficient template for the synthesis of ZnO
and CuO without calcination by acting itself a reducing and dispersing agent just like
arbinoxylan and glucoxylan. The mechanism of nanoparticle formation was supposed to be the
hydrolysis of polysaccharides into monosaccharide which in turn exist most probably into
aldehyde forms [32]. These aldehyde groups cause reduction of metal ions and the polymeric
nature of the reducing agents (GN) causes dispersion of the NPs. Therefore, this study
consolidates our previous findings [9] that carbohydrate polymers can act as an efficient
reducing agent and capping agent in nanosize reduction of metals. The NPs in this study were
found to be stable for a period of more than two years (at the time of writing of this ma nuscript)
and exhibited unchanged SPR (UV- vis not shown). Therefore, the NPs prepared by this method
may be stored for a long period of time and be utilized for biomedical, engineering and other
applications without being contaminated by the conventional reducing agents. The size range of
the ZnO-NPs obtained by our method falls in the range, which is suitable for being used as
antimicrobial agents against drug resistant bacteria [33]. ZnO NPs having size in the range of 2530 nm can efficiently enter the cell wall, produce reactive oxygen species and finally can cause
cell destruction of bacteria [21].
3.4. Phyto-toxicity study of nanoparticles
The dose response curve of the ZnO NPs and CuO NPs on the seed germination (%age) and
root length tests performed on okra seeds are shown in Figs. 6a and 6b respectively. It is clear
from these figures that both of the NPs exhibited different responses to seed germination and
root lengths. In the case of ZnO NPs (Fig. 5a) it was observed that the seed germination
increases with the increase in NPs concentration up to1.25 µg mL-1 . However, at higher
concentrations of the NPs the %age germination was found to be declined. The maximum and
the minimum %age germination was found to be 95.4% (p value 0.054) and 25% (p value 0.052)
at 1.25 µg mL-1 and 2.50 µg mL-1 respectively. A comparatively higher germination than the
control and comparable with that of the polymer control was observed (data not shown), which
may perhaps be due to the presence of highly hydrophilic material, GN, in the samples. In the
root length test, ZnO NPs exhibited an increasing trend at lower concentrations (0.25 to 1.00 µg
mL-1 ) whereas as higher concentrations an inhibitory effect was observed (ig. 6b. The reduction
in root growth at higher dose maybe attributed to a number of factors including the toxicity of
ZnO NPs above a certain level [34]. This phenomenon may perhaps be due to the sterilizing
effect of the ZnO NPs at lower concentrations which stimulates the growth whereas at higher
concentrations a toxic effect retards the plant growth. A significant inhibitory effect on the seed
germination and root length was observed in the case of CuO NPs (Fig. 6b ) even at lower
concentrations. The main factors contributing to this trend can be physical attachment of
nanoparticles on a rough seed surface, electrostatic attraction and hydrophobic interactions
between seeds and NP agglomerates [34].
3.5 The effect of ZnO nanoparticle size and exposure time on germination
On account of the interesting fluctuations in phyto-toxicities exhibited by ZnO NPs, further
experiments were performed in order to evaluate the effects of particle size (10-60 nm) and
exposure time (12-72 hrs) on the germination of A. esculentus seeds. The results of this study are
shown in Fig. 7a. It is clear from the ‘germination vs size’ curve that by increasing the size of
NPs germination (%age) was decreased. The best %age germination investigated in this study
was found to be 90 at a size of 10 nm, followed by 75, 45, 30, 10 and 5 at 15, 25, 35, 50 and 60
nm size respectively. The reduction in germination observed at increased size may be attributed
to the growth medium pH-altering behavior of the of ZnO NPs [35]. In order to investigate the
physicochemistry of NPs under increased exposure to A. esculentus seeds, the experiments were
performed for 12 nm size of ZnO NPs and the germination (%age) was calculated after 12, 24,
36, 48, 60 and 72 hrs time. The results are shown in Fig. 7 a by ‘Germination vs Time’ curve. It
is clear from this curve that %age germination increases with time, reaches to a maximum 90%
after 36 hrs and then decreases gradually. This behavior may perhaps be attributed to the
sterilizing effects of ZnO NPs thereby boosting the plant growth initially followed by retarding
effects at further exposure [21].
3.7 Effect of pH on the root length
In order to understand the role of pH on nanoparticle phytotoxicity, plants were allowed to grow
at different pH values (1-8) by the use of ZnO NPs of different sizes (10 nm to 65 nm). The pH
of the agar media was adjusted by the use of 0.1 N HCl and 0.1 NaOH solution as appropriate
(other conditions being the same as were described in experimental section). The root length
(mm) were measured after 5 days. The results of this study are shown in Fig. 7b. The plant
growth was found to be increasing from lower to higher values of pH followed by a decreasing
trend at very high pH values. Therefore, the better growth was observed to be in the pH range of
3.5 to 4.5. This may perhaps be due to the fact that an acidic and more basic pH may limit the
availability of nutrients and result in growth deficiencies. Therefore, the plant growth was
reduced by the more basic medium relative to the control [35]. Hence, A. esculentus plants was
found to grow optimally at pH 4.5 where the necessary nutrients are more biologically available
for uptake. Same growth was also observed for the particles of all size range at a given pH value
indicating the stability of the nanoparticles under the fluctuation of pH. Some previous
investigators have noted that alkaline stress (pH ≥ 8.0) in Arabidopsis can reduce root growth
and
begin
de-polymerization
of
microfilaments
[36].
3.4 Conclusion
In conclusion, the present investigation explored biocompatible carbohydrate polymer,
galactomannan, as a template for the synthesis of nanoparticles having exceptionally high
stability without any calcinations. The synthesis process is based upon the principles of green
and sustainable chemistry and eliminates the use of conventional reducing and capping agents.
Different concentrations of the polymer can efficiently be optimized to synthesize the
nanoparticles of the desired size. On the basis of optimized size of the nanoparticle, pH of the
media and exposure time on phyto- toxicity studies it is revealed
that the engineered metal
oxide nanoparticles may hold significant potential applications in agriculture and gardening, as
they may selectively inhibit unwanted plants and weeds. Additionally, the nanoparticles of size ~
12 nm have the potential to release essential metal elements necessary for plant growth as
observed in the case of zinc oxide nanoparticles at low concentrations in this investigation.
Conflict of Interests
This authors declare that there is no conflict of interests regarding the p ublication of this
paper.
Figure 1. Optimization curves in terms of SPR spectra: (a) SPR curves vs. amount of GN for (a)
ZnO NPs and (b) CuO NPs, (c) SPR curves vs. temperature and (d) SPR curves vs. pH
(a)
(b)
(c)
(d)
Figure 2. P-XRD spectra of: (a) ZnO NPs (b) CuO NPs
(a)
(b)
Figure 3. AFM image of GN film: (a) with ZnO NPs (b) with CuO NPs
(a)
(b)
Figure 4. Size and shape characterization of ZnO NPs: (a) typical SEM image, (b) typical TEM
image at 6.0 mL GN, (c) size distribution and (d) TEM images at various GN amounts: (a) 2.0
mL, (b) .0 mL, (c) 8.0 mL and (d) 10 mL
(a)
(b)
(c)
(d)
Figure 5. (a) TEM image of CuO NPs dispersed in polymer matrix, (b) Size distribution and (c)
SAED of CuO NPs
(a)
(b)
(c)
Figure. 6. Phyto-toxicity of NPs against A. esculentus: (a) Effect on seed germination and (b)
Effect on root length
(a)
(b)
Figure. 7(a) Effect of ZnONPs size and exposure time on seed germination of A. esculentus
seeds
Germination vs Time
70
80
70
60
50
40
30
20
10
0
Particle Size (nm)
60
50
40
30
20
10
0
0
Exposure Time (h)
Germination vs Size
10 20 30 40 50 60 70 80 90 100
Germination (%)
Figure. 7(a) Effect of pH of ZnO-nanoparticle of various sizes on root length of A.
esculentus
seeds
6
10 nm
15 nm
18 nm
25 nm
50 nm
65 nm
Root length (mm)
5
4
3
2
1
0
0
2
pH
4
6
8
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