Accepted Manuscript

Structural, morphological, optical and biological properties of

pure ZnO and agar/zinc oxide nanocomposites

G. Magesh, G. Bhoopathi, N. Nithya, A.P. Arun, E. Ranjith

Kumar

PII: S0141-8130(18)31084-5
DOI: doi:10.1016/j.ijbiomac.2018.04.197
Reference: BIOMAC 9783
To appear in:
Received date: 6 March 2018
Revised date: 11 April 2018
Accepted date: 28 April 2018

Please cite this article as: G. Magesh, G. Bhoopathi, N. Nithya, A.P. Arun, E. Ranjith
Kumar , Structural, morphological, optical and biological properties of pure ZnO and agar/
zinc oxide nanocomposites. (2017), doi:10.1016/j.ijbiomac.2018.04.197

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Structural, Morphological, Optical and Biological properties of Pure ZnO

and Agar / Zinc Oxide nanocomposites

G. Magesh1, G. Bhoopathi1*, N.Nithya1, A.P.Arun2, E.RanjithKumar3

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

Tamilnadu, India.

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

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641035, Tamilnadu, India.

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3
Department of Physics, Dr. N.G. P. Institute of Technology, Coimbatore -643 048, India.
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Corresponding Author Email:bbijju03@gmail.com

Abstract:
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The present research work deals with the synthesis, characterization and antibacterial activity

of Pure ZnO and Agar/ZnO nanocomposites.The crystal structure of the samples has been
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investigated by XRD. TheXRDpatternof Pure ZnO and Agar/ZnO nanocomposites have been
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confirmsthatthe hexagonal wurtzite phase and Agar induced microstrain of the samples.The

morphology and elemental composition of the samples were characterized by FESEM with
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EDX, showed paddy shaped morphology for Agar/ZnO nanocomposites. Average particle

size has been determined by TEM. The optical properties of Pure ZnO and Agar/ZnO
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nanocomposites were characterized by UV, FTIR and PL.Oxygen vacancy induced

photoluminescence of ZnO are observed and its intensity is increasing linearly with the Agar

concentration.In The antibacterial activity of ZnO and Agar/ ZnO nanocomposites was

evaluated by disc diffusion method against Gram-positive (B.subtilis) and Gram-negative

(P.aeruginosa) bacteria. The cytotoxicity of Agar/ZnO nanocomposites was studied against

Normal (L929) and Breast cancer cell line (MB231). The result of this investigation shows
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that the Agar/ZnO nanocomposites deliver a dose dependent toxicity in normal and cancer

cell line.

Keywords: Bionanocomposites; polysaccharide; Antibacterial activity.

1. Introduction

Over the past decade, polysaccharide based bionanocomposites have a great research interest

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in enhancing the functional and mechanical properties of polymers [1-3]. As one of the most

often used biopolymer, Agar is a hydrophilic polysaccharide extracted from the marine algae

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that consists of a mixture of agarose and agaropectin. It is one of the most attractive material,

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because of its good gel strength, low cost, abundant in nature, non-toxic, eco-friendly and

recyclability [4]. The characteristics of the agar with a large array of functions and molecular
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diversity structure act as good stabilizing and reducing agent [5]. In-situ synthesis of
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semiconductor nanoparticles synthesized from their corresponding precursors in the presence

of a polymer matrix, prevent agglomeration and improve the size distribution of

nanocomposites is due to the strong interfacial interaction between organic and inorganic
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material [6-9]
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Among the semiconductor nanoparticles, the unique characteristics of biocompatible ZnO

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nanoparticles lend them attractive due to their potential application such as sensors,

antimicrobial agent, drug delivery system [10, 11]. Literature review reveals that, only
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limited reports are available in Agar/ZnO nanocomposites [12]. In this paper, a novel eco-

friendly Paddy shaped Agar/ZnO nanocomposite was synthesized by in-situ reduction

method. The influence of Agar concentration (0.5g, 1g and 3g) on optical, structural,

morphological and their biological properties were also discussed.

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2. Materials and Methods:

2.1 Chemicals:

Agar was purchased from Sigma Aldrich Co. Ltd. Acetic acid (Glacial 100%, Pro analysis),

Sodium Hydroxide and Zinc acetate dehydrate (Zn (CH3COO) 2.2H2O) were purchased

from Merck. All chemicals were used without further purification and freshly prepared

solutions were used in all experiments. The detailed sample preparation techniques are

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described below.

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2.2 Synthesis of Pure ZnO and Agar/ZnO nanocomposites:

Agar/ZnO nanocomposites were prepared by in-situ chemical precipitatation method. Firstly,

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0.5g, 1g, and 3g of Agar were dissolved in 100ml of preheated distilled with under stirring at

90oC for 30 mins until a clear solution obtained. Then Zinc acetate stock solution (0.2M of
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(Zn (CH3COO) 2. 2H2O) in 100ml distilled water) was added dropwise into the above Agar

solution with vigorous stirring. The solution turned to milky white gel and it was allowed to
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age for 24hrs. Agar/ZnO nanocomposites were obtained by centrifugation, washing,

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precipitation and drying in an oven for 60oC for 24 hrs. The same procedure is followed for

Pure ZnO nanoparticles without using Agar. The samples were designated as AZ0, AZ1, AZ2
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and AZ3 for Pure ZnO and Agar/ZnO nanocomposites with different concentrations (0.5g,
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1g, and 3g) of Agar.

2.3 Material Characterization:

The Agar /ZnO nanocomposites were characterized by XRD, FESEM with EDX, HRTEM,

UV, FTIR and PL. The phase purity of the samples was confirmed by X-ray Diffraction (X-

PERT -3, Philips PW3064/60) with Cukα1 (𝞴 = 1.54060 nm) and Cukα2 (𝞴 = 1.54443 nm)

radiation operating at 30mA and 45 kV. Data were collected over a 2𝞗 range of 10o to 90o.
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The structure morphology and composition of the samples were analysed by (SIGMA HV-

CARL ZEISS) FESEM with Bruker Quantax -200-Z10 EDX detector and HRTEM images

were taken in (JEOL – JEM -2010, JAPAN) with an accelerating voltage of 200kV with

SAED pattern were also performed. The optical properties of the samples were characterized

by UV spectrometer (JASCO-V-770 Spectroscopy) at wavelength range between 200 nm-900

nm. PL spectrum was performed with FP-3800 spectroflurometer. FTIR spectroscopic

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analysis was recorded using (Bruker Alpha FTIR spectrometer) at a wavenumber range of

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400 cm-1 – 4000 cm-1.

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2.4 Antibacterial Activity:

The antibacterial activity of Agar, Pure ZnO and various concentration of Agar/ZnO was
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performed against Gram-positive (B.subtilis and S.aureas) and Gram-negative (P.aeruginosa
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and K.neumonia) bacteria by disc diffusion method. The microorganisms were inoculated in

the fresh nutrient broth and incubated to get required turbidity. The nutrient agar plate was

swabbed with the test bacterial culture and various concentrations of sample (100μg) discs
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(discs are soaked overnight in nanoparticle suspension) was added into the well. After 24h
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incubation at 370 C, the antibacterial activity was measure based on the diameter of the zone

of clearance.
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2.5 Cytotoxicity of the samples:

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2.5.1 Cell line and Cell Culturing:

Breast cancer cell line (MB-231) and Normal Fibro Blast (L929) cell line were obtained from

National Centre of Cell Science (NCCS), Pune and Grown in Eagles Minimum Essential

Medium containing 10% Fetal Bovine Serum (FBS). The cells were cultured in a humidified

atmosphere with 5% Co2 at 37% in 96 well tissue plates.

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2.5.2 MTT Assay:

The Agar/ZnO Nanocomposites were evaluated for cytotoxic activity of MB-231 (cancer

cell) and L929 (Normal cell). The cells were plated in 96 well cell plates (1x10 5) cells/well

and left overnight at 37oC. The medium was then discarded and the cells were treated with

different concentrations of ZnO (25, 50, 75, 100, 125 μg/ml). Incubate the samples at 37o±

1oC for 18hrs and then MTT were added to all the wells and incubated for 4hrs. After

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incubation, DMSO were added in to the wells and read at 570 nm using photometer.

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Cytotoxicity and Cell viability of the samples were calculated using the formula given below.

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The concentrations required for 50% Inhibition of Viability (IC50) was determined

graphically.
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Cytotoxicity = [(Control – Treated)/Control] X 100

Cell Viability = (Treated/Control) X 100

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3. Result and discussion:

3.1 XRD analysis:

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Fig. 1(a) shows the XRD pattern of the (a) pure Agar, (b) AZ0, (c)AZ1, (d) AZ2 and (e) AZ3
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samples with miller indices. The diffractograms of pure Agar exhibits a broad band
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maximum at 19.23o which attributes to the polycrystalline nature of Agar molecular chain

[13]. All the obtained diffraction peaks at 2θ Positions of 36.1 o, 34.3 o, 31.7 o, 47.5 o, 56.5 o,
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62.73 o,66.3o, 67.8 o, 69.1 o, 72.4o and 76.6ocorresponding to (101), (002), (100), (102), (110),

(103), (200), (112), (201), (004) and (202) planes with hexagonal wurtzite of ZnO phase for

all samples and these are well matched with the JCPDS file no: 89-0510. As shown in Fig.1,

the XRD spectra of Agar/ZnO nanocomposites have less intense peak along with broadening

indicates that the ZnO nanoparticles was covered by the layer of Agar. The crystallite size of

AZ0, AZ1, AZ2 and AZ3 was determined using Debye-Scherrer equation (D=Kλ/β cos (θ)).
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The micro strain induced peak broadening of Agar/ZnO nanocomposites is determined by W-

H plot method (Eq (1)).

βhkl Cos(θ) = (Kλ/D) + (4ɛ sin(θ)) ------------(1)

Fig. 1(b) shows the W-H relation of the samples were plotted between x-axis (4 Sin(θ)) and

y-axis (β cos (θ)). From the linear fit data, the Y-intercept and slope represents the crystallite

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size and strain respectively. Table.1 summaries the influence of Agar concentration on

structural parameters calculated from the equation stated by Bindu and Saba et al [14,

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15].The microstarin increases with decrease in particle size. The calculated lattice constants

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‘a’ and ‘c’ are very close to the standard values [86-0511]. There is no significant change in
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lattice constants. The bond length of Agar /ZnO nanocomposites is 1.973, 1.975, 1.976 and

1.979 Å respectively. The reduction of ZnO nanoparticles in the Agar matrix may be the
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reason for changes in Zn-O bond length.

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3.2 FESEM and EDX Analysis:

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FESEM images of Pure ZnO and Agar/ZnO nanocomposites (AZ1, AZ2, and AZ3) are

shown in Fig. 2(a-d). The pure ZnO nanoparticles (AZ0) show the irregular hexagonal shape
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morphology. Moreover, the morphology of AZ1 and AZ2 were in Paddy like structure, but

AZ3 nanocomposites exhibit agglomerated spherical and paddy shape morphology. This may
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due to the large amount of Agar bound to the surface of ZnO and restrict the growth of the

particles. EDAX spectra Fig. 2(e and f) confirm the presence of Zinc and Oxygen in Pure

ZnO and Agar/ZnO nanocomposites which is in good agreement with XRD[16].

3.3 HRTEM and SAED Analysis:

Fig. 3(a-c) shows the TEM image of Pure ZnO (AZ0) and of Agar/ZnO nanocomposites

(AZ1 and AZ3) in the range of 100 nm. Fig.3 (a) shows hexagonal shaped ZnO nanoparticles
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is in the size range 40 nm. The structure changes from hexagonal to paddy like morphology

with an average diameter 31 nm for Agar/ZnO nanocomposites (AZ1) might be due to the

intermolecular interaction between ZnO nanoparticles and the hydroxyl group of Agar

matrix. For AZ3, agglomerated spherical and paddy morphology coated by the layer of agar

was observed with an average particle size at range 15.60 nm. Fig. 3(d-e) shows the measured

lattice fringes of AZ1, AZ2 and AZ3 corresponding to (101) plane of ZnO. Inset Fig.3(d-e),

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SAED pattern confirms the polycrystalline nature of the sample. From the TEM analysis, it is

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clear that the ZnO nanoparticles are well distributed and stabilized by Agar polymer matrix

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[17].

3.4 UV Visible Analysis (UV-Vis):

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Fig. 4(a-e) shows the UV-Vis spectra of Pure ZnO (AZ0) and Agar/ZnO nanocomposites.
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There was no absorption for Agar [18]. The AZ0 shows absorption band at 385 nm due to the

recombination energy of exciton. For Agar/ZnO nanocomposites (AZ1, AZ2 and AZ3), the

absorption spectra slightly shifts towards shorter wavelength from (385 to 364 nm). The
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energy band gap (Eg = hc/λ) of the samples were calculated as 3.2, 3.28, 3.33 and 3.4 eV
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respectively. The observed red shift in energy gap may be due the reduction of crystallite size

and strain [19].

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3.5 FTIR spectrum:

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Fig. 5(a-c) shows the FTIR spectrum of Agar, Pure ZnO (AZ0) and Agar/ZnO

nanocomposites (AZ3) respectively. The Pure ZnO exhibits the vibrational peak at 468 cm -1

which attributed to the stretching vibration mode of Zn-O bond. Fig. 5 (a) shows the

characteristic vibrational band of Agar at 3338 cm-1 (stretching O-H group), 2906 cm-1 (C-H

stretching of methane ring), 1653 cm-1 (stretching vibration of conjugated peptide bond

formation by amine (NH) and (CO) groups), 1377 cm-1 (ester sulphate) and the band at 1047

cm-1 and 935 cm-1 (3,6- anhydrogalactose bridges) [20]. IR spectrum of AZ3 has similar
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vibrational bands assigned to Agar with significant changes in intensity and broadening.

Although the Vibrational band decreases to the lower wavenumber indicates a strong bonding

between ZnO and Agar chains. In addition, the Zn-O stretching band also observed in

Agar/ZnO nanocomposites [21].

3.6 Photoluminescence Spectroscopic Characterization:

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The Photoluminescence spectrum exhibits two distinct bands of pure ZnO and Agar/ZnO

nanocomposites are shown in Fig. 6(a-d). First one in the blue - green region was observed at

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489 nm with dramatic changes in the peak position is due to the surface defects of ZnO and

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the second latter strong, intense peak in the greenish yellow emission band at around 546 nm

which may attribute to the presence of oxygen vacancies in the interstitial position. On
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varying the concentration of Agar, the band shifts (blue shift) towards smaller wavelength
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[22]. Fig. 6 inserts shows that the peak intensity increases with increasing the concentration

[23]. This indicates the transition effect of charge and higher energy between polymer chain

and ZnO [24].

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4. Biological properties:
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4.1 Antibacterial activity:

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Fig. 7(a and b) shows the antibacterial activity of the samples performed using disc diffusion
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method done by [25]. Agar did not show antibacterial activity, whereas ZnO and Agar/ ZnO

nanocomposites exhibit potent antibacterial activity against Gram-positive and Gram-

negative bacteria. In addition, it is clear that the bactericidal activity increases with decrease

in particle size, which might be due to the large surface to volume ratio. While increasing the

concentrations of Agar the zone of inhibition also increases (Fig.8).Enhanced bioactivity of

Agar/ZnO nanocomposites is due to the generation of H2O2 molecules on the surface of ZnO

which can penetrate through the cell membrane and inhibit the growth of bacteria [26].While
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comparing Gram-positive and Gram-negative bacteria, it has been frequently observed that

Agar/ZnO nanocomposites shows strong antibacterial activity towards Gram-negative than

Gram-positive which may attributed tothe different structure and thickness of peptide glycans

in the cell wall [27].Based on the experimental data (Table. 2) it was observed that, the

antibacterial activity of Agar/ZnO nanocomposites was greater than Agar and Pure ZnO

which may due to the synergistic biological properties of Agar and ZnO.

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4.2 Cytotoxicity Effect (MTT Assay) of Agar/ZnO nanocomposites:

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4.2.1 Normal Fibroblast Cell Line(L929):

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Fig. 9 shows images of cytotoxicity of the synthesized samples against L929 cell line. The

Agar/ZnO nanocomposites at concentrations of control, 25, 50, 75,100 and 125μg/ml showed
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0, 3, 8, 10, !3 and 15% low toxicity against L929 normal cell line.

4.2.2 Breast cancer cell line (MB-231):

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The antiproliferative effect of AZ3 (3g of Agar/ZnO nanocomposites) against MB-231 cancer

cell line is shown in Fig. 10. The Agar/ZnO nanocomposites at concentrations of control, 25,
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50, 75, 100 and 125μg/ml showed strong anticancer activity 0, 20, 32, 56, 69 and 82%
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respectively [28].
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From Fig. 11(a), increasing the concentration of the samples the cytotoxic rate also increases.
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The antiproliferative effect of the nanocomposites might be due to the electrostatic interaction

between negatively charged anionic phospholipids on cancer cells with positively charged
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surface of Agar stabilized ZnO. This might be due to the synergistic effect of bio functional

material agar and ZnO. Specifically, ZnO nanoparticles may generate ROS and react with

cell components leading to cell damage [29]. Cytotoxicity of the ZnO nanoparticles depends

upon the size of the particles [30].

Fig. 11(b) exhibits Inhibitory concentration values (IC50) values of MB-231 cancer cell line

and L929 normal cell line. The IC50of cell viability against MB-231 cancer cell line was
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observed at 66.79μg/ml.The IC50of cell viability against L929 cancer cell line was greater

than >100μg/ml. From the results, Agar/ZnO nanocomposites exhibits potential dose

dependent cytotoxicity against MB-231 (cancer cell line) than L929 (normal cell line).

Conclusion:

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Pure ZnO and Agar/ZnO nanocomposites with different concentrations (0.5g, 1g and 3g) of

Agar have been synthesized by in-situ chemical precipitatation method. The XRD pattern

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exhibits the hexagonal wurtzite structure with reduced crystallite size.The crystallite size

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decreased from 35 to 19 nm as an increase in Agar concentration. TEM reveals the average
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particle size of the irregular hexagonal shaped ZnO nanoparticles ~ 40 nm whereas for paddy

shaped Agar/ZnO nanocomposites ~20 nm. FTIR spectra confirm the intermolecular
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interaction between ZnO and Agar. Photoluminescence spectra of the samples displayed two

emission peak one at blue-green region and the other at yellow region. UV and PL spectra
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exhibit blue shift in wavelength which indicates the transition effect of higher energy and
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charge between polymer chain and ZnO.The prepared nanocomposites exhibit potent
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antibacterial activity against Gram-positive and Gram-negative bacteria. The Agar/ZnO

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nanocomposites displayed dose dependent cytotoxic effect against MB-231 and L929 cell

line.
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Table.1. Structural Parameters of Pure ZnO and Agar/ZnO Nanocomposites

Sample Crystallite W-H plot Lattice Strain c/a Dislocation Volume Bond
size parameter ratio density (Å)3 length
(nm) (Å) (δ) (Zn-
D Strain a c O)
(nm) L(Å)

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PA0 35.5 36.49 0.0012 3.144 5.185 0.0033 1.649 0.00079 44.39 1.932
PA1 29.59 29.01 0.001 3.135 5.166 0.0039 1.648 0.00114 43.94 1.926

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PA2 25.36 28.12 0.0006 3.129 5.161 0.0046 1.649 0.00156 43.76 1.923
PA3 19.73 18.48 0.0001 3.116 5.142 0.0059 1.650 0.00257 43.24 1.915

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#Standard values: a=b=3.249 and c=5.205 Å (JCPDS card no. 89-0511).
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Table.2. Antibacterial activity of Pure ZnO and Agar/ZnO Nanocomposites

Samples Zone of Inhibition (mm)
(g) B.subtilis P.aeruginosa
Agar (Control) - -
PA0 10 12
PA1 12 13

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PA2 13 17
PA3 16 18

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Caption of Figures

Fig. 1 (a) X-ray diffraction patterns and (b) W-H plot of (a) Agar (b) pure ZnO (AZ0) (c)
AZ1 (d) AZ2 and (e) AZ3

Fig. 2 (a-d) SEM images of (a) pure ZnO (AZ0) (b) AZ1 (c) AZ2 and (d) AZ3 and EDX
SPECTRUM (d-e) of Pure ZnO nanoparticles and Agar/ZnO nanoparticles

Fig. 3 HRTEM images (a- c) of Pure ZnO (AZ0) and Agar/ZnO nanoparticles (insert) particle

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size distribution histogram (d-e) Interplanar lattice fringes (insert) calculated Gatan
image of lattice fringes and SAED pattern

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Fig. 4 (a-e) UV Visible absorption spectra of (a) Agar (b) pure ZnO (AZ0) (c) AZ1 (d) AZ2

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and (e) AZ3

Fig. 5 (a-c) FTIR spectrum of (a) Agar (b) Pure ZnO (AZ0) (c) Agar/ZnO nanoparticles
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Fig. 6 (a-d) Photoluminescence spectra of (a) pure ZnO (AZ0) (b) AZ1 (c) AZ2 and (d) AZ3
(insert shows the PL peak intensity variation with the concentration of Agar)
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Fig. 7 (a & b) Antibacterial activity against Gram-positive and Gram-negative bacteria of (a)
Agar (b) pure ZnO (AZ0) (c) AZ1 (d) AZ2 and (e) AZ3
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Fig. 8 Graphical representation of Zone of inhibition (mm) of (a) pure ZnO (AZ0) (b) AZ1
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(c) AZ2 and (d) AZ3

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Fig. 9 Cytotoxic activity of Agar/ZnO Nanoparticles on L929 Normal cell line at different
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concentrations

Fig.10 Cytotoxic activity of Agar/ZnO Nanoparticles on MB-231 cell line at different

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concentrations

Fig. 11(a) In vitro cytotoxic effect of Agar/ZnO Nanoparticles (bar diagram) (b) Percentage
of viability vs concentration Agar/ZnO Nanoparticles against MB-231 (Cancer) and
L929 (Normal) cell line
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