Accepted Manuscript

Study of structural, morphological, optical and biomedical properties of pH based

ZnO nanostructures

G. Magesh, G. Bhoopathi, A.P. Arun, E. Ranjith Kumar, Ch. Srinivas, S. Sathiyaraj

PII: S0749-6036(18)31964-5

DOI: 10.1016/j.spmi.2018.10.002

Reference: YSPMI 5914

To appear in: Superlattices and Microstructures

Received Date: 27 September 2018

Accepted Date: 01 October 2018

Please cite this article as: G. Magesh, G. Bhoopathi, A.P. Arun, E. Ranjith Kumar, Ch. Srinivas, S.
Sathiyaraj, Study of structural, morphological, optical and biomedical properties of pH based ZnO
nanostructures, Superlattices and Microstructures (2018), doi: 10.1016/j.spmi.2018.10.002

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 ACCEPTED MANUSCRIPT

Study of structural, morphological, optical and biomedical properties of pH based ZnO

nanostructures

G. Magesh1, G. Bhoopathi1*, A.P.Arun2, E. Ranjith Kumar3, Ch. Srinivas4, S. Sathiyaraj5

1 Department of Physics, PSG College of Arts and Science, Coimbatore – 641 014, India.

2 Department of Mechanical Engineering, Kumaraguru College of Technology, Coimbatore-

641035, India.

3Department of Physics, Dr. N.G. P. Institute of Technology, Coimbatore -643 048, India.

4Department of Physics, Sasi Institute of Technology & Engineering, Tadepalligudem 534101,

India.
5Department of Chemistry, Dr. N.G. P. Arts and Science College, Coimbatore -643 048, India.

Abstract

Nano ZnO has been synthesized at different pH values using chemical precipitation

technique. The X-ray diffraction pattern shows the hexagonal wurtzite phase of ZnO

nanoparticles. The crystallite size is found to decrease with increasing the pH value. From FE-

SEM analysis, it can understand with increasing the pH value from 8 to 12, the morphology of

ZnO nanoparticles changed from hexagonal faceted structure to nanospheroid structure. The

HRTEM images are in well accordance with the FE-SEM images. The average particle size of

nanospheroid morphology is 31.16 nm. The SAED pattern confirms the crystalline nature with

multifaceted growth of the samples. EDAX spectrum of ZnO nanoparticles at different pH values

confirms the formation of Zn and O. The absorption spectrum of ZnO nanoparticles shows that

the absorbance band is slightly shifted towards the lower wavelength with increasing the pH

value. The band gap energy values (by Tauc plot relation) of the ZnO nanoparticles are found in
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the range of 3.0 to 3.03eV. FTIR spectra revealed the presence of the characteristic stretching

vibrational band of Zn-O bonding at 445 – 418 cm-1 and this is shifted to lower frequency with

increasing the pH value from 8 to 12. PL spectra of ZnO nanoparticles at various pH values

exhibit a strong UV emission band and green emission band. The most intense green emission

band is obtained for ZnO nanoparticles at pH12. The synthesized nanoparticles show potential

antibacterial activity against Gram-positive (B. subtilis and S. aureus) and Gram-negative

bacteria (P. auregenia and K. Pneumonia). The cytotoxicity of ZnO nanoparticles at pH12 was

evaluated against Normal (L929) and Breast cancer cell line (MB231).

Key words: Nanoparticles; Chemical synthesis; Structural analysis; FE-SEM

1. INTRODUCTION

ZnO semiconductor material has drawn much interest in recent days due to their unique

application in various field of research such as gas sensors, highly functional photoelectron

devices, catalysts and effective devices like laser devices which are common nowadays [1]. Zinc

oxide (ZnO) has a large scope for various research groups as being wide band gap energy (3.37

eV) in a semiconductor with excellent electrical and optical properties. Zinc oxide nanostructures

have an appreciable advantage in the catalytic reaction process due to its large surface area and

high catalytic activity [2]. Several synthesis methods reported in the literature for the preparation

of ZnO nanostructures such as laser ablation [3], hydrothermal methods [4], chemical vapor

deposition [5], sol-gel method [6], thermal decomposition [7], electrochemical depositions [8]

and combustion method [9]. Nowadays, Zinc oxide nanoparticles were prepared by ultrasound

[10], co-precipitation [11], microwave assisted combustion method [12], anodisation [13], two-

step mechanochemical thermal synthesis [14] and electrophoretic deposition [15].

Nanostructured inorganic compound materials (ZnO) show potent antibacterial activity at low
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concentrations due to their high surface area to volume ratio and unique chemical and physical

properties [16]. The participation of metallic semiconductor ZnO nanoparticles (< 30 nm) has the

ability to enhance the natural biological properties in all the biological system [17-18]. ZnO is

genuinely one of the best bio friendly absorbers of UV radiation. Due to its photoactive

behavior, it generates superoxide and hydroxyl radicals (Reactive Oxygen Species; ROS) when

irradiated with UV in the presence of oxygen and water [19]. Literature review reveals that

several reports are available on the pH assisted synthesis of ZnO nanoparticles. Rayerfrancis et

al., synthesized ZnO nanostructures by hydrothermal method using Zinc Nitrate, HTMA and

HCL with NaOH as a pH regulator [20]. In another report, Mohammed Ali et al., synthesized

ZnO nanoparticles with different shape of varying the reactant concentration (NaOH) [21].

However, the extensive study of microstructural, optical and photoluminescence property of pH

assisted ZnO nanoparticles is still insufficient. In this manuscript, pH sensitive morphology of

the ZnO nanoparticles was synthesized by simple the chemical precipitation method. The main

objective of this chapter is to study the structural, morphological, optical and photoluminescence

properties of ZnO nanoparticles at different pH value with their antibacterial activity and

anticancer activity.

2. Experimental technique

2.1 Method of Synthesis

ZnO nanoparticles were prepared by simple chemical precipitation method. In a typical

preparation, 0.2 M of Zinc acetate dehydrate (Zn (CH3COO) 2.2H2O) is dissolved in 100 ml

distilled water and stirred for 30 minutes. Then 1 M of sodium hydroxide (NaOH) solution was

added to the above solution to attain pH-8. The resulting milky white colour colloidal solution

was stirred for another 1 hour. Then the wet white precipitate was filtered, washed and dried in

an oven to obtain ZnO nanoparticles. The same procedure was followed for pH-9, 10 and 12
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respectively. Finally, the dried white powder was ground well for further characterization. The

possible growth mechanism of ZnO is given below,

Zn (CH3COO)2.2H2O + NaOH    Zn (OH)2- + 2CH3COONa

AQUEOUSMEDIA

Zn (OH)2 + 2H2O 
 Zn (OH)2- + 2H+

2
Zn (OH) 4 TEMPERATUR
 E
 ZnO + H2O + 2OH-

3. Results and discussion

3.1 Structural analysis

The X-ray Diffraction analysis of the prepared samples with various pH values is shown

in Fig. (1). Sodium hydroxide (NaOH) is used as a pH regulator. The X-ray diffractograms

depicted the hexagonal wurtzite structure of ZnO nanoparticles belonging to space group:

p63mc. In XRD patterns of the chemically synthesized ZnO nanoparticles, the planes indexed

with miller indices (100), (002), (101), (102), (110), (103), (200), (112), (201), (004), (202),

(104) and (203) at 2θ angle 31.6 o, 34.4 o, 36.2 o, 47.5 o, 56.4 o, 62.75 o,66.1 o, 68.0 o, 69.02 o, 72.4o,

76.6o, 78.0 o and 86.1o in are well matched with diffraction pattern of JCPDS Card no. 89-

1397.The analysis of the prominent peaks reveals the information about the crystalline nature of

the prepared sample. Among the various diffraction peaks obtained, the peak which is indexed

with (101) plane is found to be the most prominent peak because it shows the preferential growth

orientation in the prepared sample as shown Fig. (1). Well-defined, sharp edged and high

intensity peaks proved the formation well crystalline nano ZnO. The absence of extra peaks

(impurity peaks) indicates the high phase purity of the prepared sample. The variation in the peak

intensities that depicted in the X-Ray diffractograms is presumed to be the effect of pH in

solubility. The highest intensity peak was observed at pH-8, due to adequate availability of OH-

ions in the formation of ZnO [22].

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The crystallite size of the pH mediated ZnO nanoparticles is estimated from the Debye-Scherer’s

equation (1),

k
D= (1)
 cos 

The crystallite size of ZnO nanoparticles decreases with the increase of pH value (8, 9, 10 and

12) is shown in Fig. (2). Furthermore, on comparison of pH from 8 to 12, the pH–12 provides a

suitable environment for the nucleation of sites and hence it attains the supersaturation state

where the crystallites are small [23].

The lattice parameters ‘a’ and ‘c’ are calculated from the equation (2),

1 4 h 2  hk  k 2 l 2
=  2 (2)
d hkl 2 3 a2 c

Where, dhkl interplanar spacing corresponding to the h, k, l lattice planes and a and c are the

lattice constants [24]. X-ray pattern reveals that the lattice parameter decreases with increase of

pH value. i.e., lattice contraction is shown in Fig. (3). The unit cell volume (v) and volume of a

crystallite (V) are evaluated from the relations (3) and (4), [24]

v = 0.866 a2c (3)

V = D3 (4)

The number of unit cells in the crystallite (Nu) is the ratio between the V and v which is

calculated from the relation (5), [25]

Nu = V/v (5)

The number of unit cells in the crystallite decreases with the increase in pH concentration, as

shown in Table 2. For pH-12, the number of unit cells in the crystallite is found to be 0.6198
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x106 which is 7 times lesser than pH-8. The effect of pH on the Zn-O bond lengths are calculated

from the relation, [26]

a2 1
L (  (  u) 2 c 2 ) (6)
3 2

Where a and c = Lattice Parameters, u = positional parameter

The positional parameter u calculated from the relation,

a2
u  0.25 (7)
3c 2

By increasing the pH value, the Zn-O bond length value decreases. The calculated structural

parameters are listed in Table (1). The reduction in crystallite size is due to the dissolution of

OH- ions. W-H method proposed a excellent method for the effect of size and strain on

broadening by the peak width as the function of diffraction angle [28]. By using Scherrer

formula and the volume-weighted average strain εstr = βhkl/4 tan θ results in the following Eq. (8),

k
 cos    4 tan  (8)
D cos 

Fig (4) exhibits the typical Williamson-Hall plot of ZnO nanoparticles by plotting 4sin (θ) as the

function of βcos (θ). From the straight line fit data, slope and the Y-intercept which gives the

microstrain induced crystallite size of the particles.

3.2 FESEM and EDAX analysis

Effect of pH on the morphology of the ZnO nanoparticles is shown in Fig. (5 (a-d)). The

morphology of the nanostructured ZnO nanoparticles drastically changes from hexagonal faceted

structure to nanospheroid structure. Fig. 5(a) shows the morphology of ZnO nanoparticles at

pH.8 with hexagonal faceted structure. Further, increasing the pH value to 9 (Fig. 5(b)) the
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morphology of ZnO transforms into nanosheets like structure and for pH.10 hexagonal faceted

nanorod like morphology was observed. The stable nanospheroid like morphology was observed

at pH.12. The reduced crystallite size and nanospheroid morphology enhance the surface to

volume ratio of nano ZnO, which easily penetrate through the bacterial cell membrane.

Therefore, ZnO nanoparticles at pH.12 are suitable for antibacterial and anticancer applications.

Energy dispersive analysis was carried out for the prepared sample in order to find the

composition as well as the purity of the ZnO nanoparticles. The elemental composition of the

nano ZnO at pH.12 were recorded at EDX spectrum in the binding energy range 0 to 20 KeV.

Fig. 6(a-d) shows the phase purity and existence of Zn and O also confirmed by EDAX analysis.

The occurrence of Cu signal may from the supporting filament copper grids used for FESEM

analysis.

3.3 HR-TEM analysis

Fig. 7(a-f) shows the typical HRTEM image of ZnO nanoparticles at various pH values.

The HRTEM images are well accordance with the FESEM images. The HRTEM micrograph of

ZnO nanoparticles at pH.12 with nano spheroid morphology is shown in Fig. 7 (d). Fig. 7 (e)

exhibits the average particle size distribution of nano spheroid morphology with an average

particle size 31.16 nm which is well matched with XRD data. The SAED pattern of the nano

ZnO portraits the polycrystalline nature of the ZnO nanoparticles with symmetrical orientations.

3.4 UV analysis

Fig. 8 (a-d) shows the optical UV-vis spectra of ZnO nanoparticles at various pH values.

The absorption edge of the nano ZnO depends on the band gap, surface roughness, oxygen

vacancy and impurity centres. The absorption spectra of ZnO nanoparticles show that the

absorption is decreased while increasing the pH value. The ZnO nanoparticles at various pH

value exhibits an absorption edge around 391-372 nm which might be due to the excitation of
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electrons from the valance band to the conduction band. The absorption edge shifts to lower

wavelength with the increase in pH value. The observed blue shift is owing to the formation of

smaller crystallite size. The direct absorption band gap energy of the ZnO nanoparticles at

various pH values obtained from the Tauc relation (9) [28].

h  B(h  E g ) n (9)

Where   Absorption co-efficient, h  Photon energy, Eg = Optical bandgap of the particles,

B = Material constant, n = ½ for a direct transition semiconductor material

Fig. 9 (a-d) shows the optical bandgap of ZnO nanoparticles evaluated by extrapolating the linear

portion of the curve (h ) 2 Vs (h ) .The estimated band gap energy of the ZnO nanoparticles at

pH.8 is 2.8 eV, for pH.9 it is 2.95 eV and the bandgap further increase to 3.0 eV and 3.03 eV for

pH 10 and 12 respectively. The energy gap of the ZnO nanoparticles increases with increasing

the pH value.

3.5 FTIR analysis

FTIR spectroscopy is engaged as an supplementary probe to confirm the existence of

functional groups of organic and in addition with other inorganic species. The synthesized ZnO

nanoparticles at different pH value were characterized by FTIR spectroscopic method in the

range of 4000-400 cm-1 as shown in the Fig. 10 (a-d).The broad band appeared at around 3440

cm-1 indicates the presence of stretching vibration band of a hydroxyl group (-OH). The peak

observed at 1546 cm-1 is assigned to the asymmetric stretching vibration band of C=O. The

symmetric stretching vibration band occurs at 1434 cm-1 which indicates the existence of the C-

O bond. The characteristic stretching vibrational band of Zn-O bond is occurred at 445 - 418 cm-
1 which shifts to lower wavelength with increasing the pH value. Therefore, the formation of

ZnO nanoparticles is confirmed by FT-IR spectrum [29].

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3.6 Photoluminescence spectrum

The PL spectrum of ZnO nanoparticles at an excitation wavelength 325 nm is shown in

Fig. 11 (a-d). The ZnO nanoparticles at various pH values exhibit two emission bands. The

strong UV absorption band observed at 387.5 nm, 405 nm, 407 nm and 409 nm for pH.8, 9, 10

and 12 respectively. The UV emission band is attributed to the radioactive recombination of the

free exciton-exciton collision process in the ZnO nanoparticles. Another emission band was

observed at green emission in the range of 530.5-547 nm which is due to the presence of

interstitial oxygen vacancy [30]. While increasing the pH value the intensity of the green

emission peak increases and shifted to higher wavelength. The most intense green emission band

is observed for ZnO nanoparticles at pH.12. The oxygen vacancy induced luminescence

properties of ZnO nanoparticles is suitable for biomedical applications.

3.7 Antibacterial activity

The antibacterial activity of ZnO nanoparticles at various pH values (8, 9, 10 and 12) was

determined against two gram-positive (B. subtilis and S. aureus) and two gram-negative (P.

aeruginosa and K. pneumoniae) bacteria. The gram positive and gram-negative bacterial

pathogens in the different plate were inoculated in the culture media. In each petridish four wells

per disc were placed with the help of sterile forceps. Then the synthesized ZnO nanoparticles at

different pH value were placed in the corresponding plates. After 24hrs the zone of inhibition

(mm) were measured around the samples with a ruler in mm. The samples were named as Z1 for

pH.8, Z2 for pH.9, Z3 for pH.10 and Z4 or pH.12. Fig (12) shows the effect of pH sensitive ZnO

nanoparticles on bactericidal activity against gram negative and gram-positive bacteria. The ZnO

nanoparticles exhibit potent antibacterial activity against the gram negative and gram-positive
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pathogenic bacteria which might be due to the electrostatic interface between the negatively

charged biosurfaces of the pathogens and positively charged (Zn2+) ZnO nanoparticles. Thus, the

attraction not only kills the bacteria, but also leads to the formation of ROS such as oxy and

hydroxyl radicals (OH-, O-, O2-, O2-) which causes cell death. The bactericidal efficacy increases

while increasing the pH value of ZnO nanoparticles as shown in Fig. (13).

On comparing the gram negative and gram-positive bacteria, the killing effect of gram

negative bacteria is higher than gram positive bacteria due to the variations in the cell structure

and thickness of the peptidoglycans in the cell wall [31]. The ZnO nanoparticles at pH.12 (Z4)

exhibit strong antibacterial activity against both gram negative and gram-positive bacteria. This

is because of large surface to volume ratio of the nanospheroid morphology of ZnO nanoparticles

with smaller crystallite size. The ZnO nanoparticles at pH.12 (Z4) shows highest antibacterial

activity against P. aeruginosa (17 mm) and K. pneumoniae (13 mm) and lower for S. aureus (13

mm) and B. subtilis (12 mm) respectively.The biocidal activity increases with decrease in

average particle size of ZnO nanoparticles. The influence of pH mediated ZnO nanoparticles

against the gram negative and gram-positive bacteria are given in the Table. (2).

3.8 Cytotoxic activity

The In vitro cytotoxicity of nano ZnO at pH.12was evaluated against MB-231 breast

cancer cell line and L929 fibro blast cell line at different concentrations. The cytotoxicity was

observed after 24hrs of incubation. Fig. 14 shows the cytotoxic activity of the ZnO Nanoparticles

at pH.12 against MB-231 breast cancer cell line. The maximum cell inhibition 80% was

observed at the concentration of 125μg/ml. Fig.15 shows the normal fibroblast cell line (L929)

treated with ZnO nanoparticles at pH.12 shows low toxicity 1% at 125μg/ml concentration. The

antiproliferative increases with increasing the concentration of the nanoparticles suspension (Fig.

16). Fig. 17 shows the dose response curves of the MB231 and L929cell lines. Therefore, the
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inhibitory concentration IC50 values of ZnO nanoparticles at pH.12 against MB231 breast cancer

cell was 80 μg/ml, although it was found to be significantly higher >100 μg/ml for normal cell

line. The IC50 value of MB-231 cell line is lesser than L929 cell line indicating the nano ZnO as

a dose dependent cytotoxic agent.

CONCLUSION

Nanosized ZnO at different pH values (8, 9, 10 and 12) were successfully synthesized by

chemical co precipitation method. The effects of pH on ZnO nanoparticles were also studied.

The XRD pattern revealed the hexagonal wurtzite structure. FESEM images exhibits hexagonal

faceted structure (pH.8), nanorod (pH.9), nanosheet (pH.10) and nanospheroid (pH.12)

morphology. The particle size distribution of the nanospheroid morphology was 31.16 nm. The

crystalline nature of the samples with symmetrical orientations was confirmed by SAED pattern.

EDX spectrum of ZnO nanoparticles confirms the formation of Zn and O. From UV analysis, the

absorbance band shifts to lower wavelength with increase in pH value. The occurrence of

functional groups and the characteristic vibrational band of Zn-O was confirmed from FTIR. PL

spectra of ZnO nanoparticles at different pH value described the shift in UV emission from 387.5

to 409 nm and a shift in green emission from 530.5-547 nm. In addition the prepared ZnO

nanoparticles at various pH value exhibits potent antibacterial activity against Gram-negative

and Gram-positive bacteria. The ZnO nanoparticles at pH.12 displayed dose dependent

cytotoxicity against MB-231 and L929 cell line.

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 Table.1. Structural Parameters of ZnO nanoparticles at different pH value

W-H plot Volume

Lattice Parameter Unit cell of Bond
Crystallite Atomic volume Crystallit Nu x 106 Length
pH value size packing (v) es (Zn–O)
(V/v)
(d) nm factor c/a
(Å3) V X 103 L (Å)
D (nm) Strain a (Å) c (Å) (nm)3

8 56.8 52.26 0.000482 3.141 5.225 1.663 44.64 183.25 4.105 1.936

9 43.65 43.57 0.000913 3.138 5.200 1.657 44.34 83.167 1.8757 1.931
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10 32.77 36.59 0.00166 3.135 5.181 1.653 44.98 35.513 0.8053 1.928

12 30 33.09 0.00224 3.123 5.157 1.651 43.56 27 0.6198 1.920

# Standard values: a=b=3.498 and c=5.2066 Å (JCPDS card no. (89-1397).

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Table.2. Antibacterial activity of ZnO Nanoparticles at different pH value

pH value Zone of Inhibition (mm)

P. aeruginosa K. pneumoniae S. aureus B. subtilis

8 12 11 9 10

9 15 11 10 13

10 16 11 12 12

12 17 13 13 12
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Fig. 1 XRD pattern of ZnO nanoparticles synthesized at different pH values

Fig. 2 Crystallite Size of ZnO nanoparticles against pH value

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Fig. 3 Lattice parameter “a” and “c” values of ZnO nanoparticles at different pH value

Fig. 4 Williamson-Hall (W–H) plot of ZnO nanoparticles at different pH values

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Fig. 5 FESEM images of ZnO nanoparticles at different pH values (a) pH.8, (b) pH.9 (c)
pH.10 and (d) pH.12

Fig. 6 EDX spectrum of ZnO nanoparticles

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Fig. 7 HRTEM images of ZnO nanoparticles at different pH values (a) pH.8, (b) pH.9
(c) pH.10 and (d) pH.12, (e) Particle size distribution histogram of nanospheroid
morphology of ZnO nanoparticles (f) SAED pattern of ZnO nanoparticles

Fig. 8 UV analysis of ZnO nanoparticles at different pH values (a) pH.8, (b) pH.9 (c)
pH.10 and (d) pH.12
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Fig. 9 Tauc plot of ZnO nanoparticles at different pH values (a) pH.8, (b) pH.9 (c)
pH.10 and (d) pH.12

Fig. 10 FTIR analysis of ZnO nanoparticles at different pH values (a) pH.8, (b) pH.9 (c)

pH.10 and (d) pH.12

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Fig. 11 PL spectrum of ZnO nanoparticles at different pH values (a) pH.8, (b) pH.9 (c)

pH.10 and (d) pH.12

Fig. 12 Antibacterial activity of ZnO nanoparticles at different pH value (pH.8, 9 10 and

12) against the gram negative and gram-positive bacteria
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Fig. 13 Graphical representation of Zone of inhibition

Fig. 14 Cytotoxic activity of ZnO Nanoparticles on L929 normal cell line at

different concentrations
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Fig. 15 Cytotoxic activity of ZnO Nanoparticles on MB-231 cell line at different

concentrations

Fig. 16 In vitro cytotoxic effect of ZnO particles (bar diagram) against MB-231 (Cancer)
and L929 (Normal) cell line
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Fig. 17 Percentage of viability vs concentration ZnO Nanoparticles against MB-231

(Cancer) and L929 (Normal) cell line
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 The crystallite size is found to decrease with increasing the pH value.

 The average particle size of nanospheroid morphology is 31.16 nm.
 The most intense green emission band is obtained for ZnO nanoparticles at pH12.