Synthesis of ZnO nanoparticles with chitosan as stabilizing agent and their antibacterial properties against Gram-positive and Gram-negative bacteria

Accepted Manuscript Synthesis of ZnO nanoparticles with chitosan as stabilizing agent and their antibacterial properties against Gram-positive and Gram-negative bacteria

Nurul Amira Ahmad Yusof, Norashikin Mat Zain, Norlin Pauzi PII: DOI: Reference:

S0141-8130(18)33853-4 https://doi.org/10.1016/j.ijbiomac.2018.11.228 BIOMAC 11105

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

28 July 2018 18 November 2018 25 November 2018

Please cite this article as: Nurul Amira Ahmad Yusof, Norashikin Mat Zain, Norlin Pauzi , Synthesis of ZnO nanoparticles with chitosan as stabilizing agent and their antibacterial properties against Gram-positive and Gram-negative bacteria. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.11.228

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ACCEPTED MANUSCRIPT Synthesis of ZnO nanoparticles with chitosan as stabilising agent and their antibacterial properties against Gram-

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positive and Gram-negative bacteria

1

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Nurul Amira Ahmad Yusof1, Norashikin Mat Zain1,*, Norlin Pauzi1

Faculty of Chemical & Natural Resources Engineering, Universiti Malaysia Pahang, 26300 Gambang,

SC

Pahang, Malaysia.

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Abstract

Antibacterial activity of zinc oxide (ZnO) nanoparticles have received significant interest, particularly by the

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implementation of nanotechnology to synthesize particles in nanometer region. ZnO nanoparticles were successfully synthesized through microwave heating by using chitosan as a stabilizing agent and characterized by UV-vis, FTIR, XRD and FESEM-EDX. The aim of the present study is to determine the antibacterial activity

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of ZnO nanoparticles against Gram-positive bacterium Staphylococcus aureus (S. aureus) and Gram-negative

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bacterium Escherichia coli (E. coli). The antibacterial effect of ZnO nanoparticles was investigated for the inhibition zone and inactivation of cell growth. The absorption of ZnO nanoparticles was found to be around 360nm. FTIR results showed the stretching mode of ZnO nanoparticles at 475 cm-1 of the absorption band. EDX

CE

results indicated that ZnO nanoparticles have been successfully formed with an atomic percentage of zinc and oxygen at 23.61 and 46.57% respectively. X-ray diffraction result was confirmed the single-phase formation of

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ZnO nanoparticles and the particle sizes were observed to be around 50 to 130nm. The results showed that ZnO nanoparticles have displayed inhibition zone of 16 and 13 mm against S. aureus and E. coli respectively. Gramnegative bacteria seemed to be more resistant to ZnO nanoparticles than Gram-positive bacteria.

Keywords: Zinc oxide, nanoparticle, microwave, chitosan, antibacterial

ACCEPTED MANUSCRIPT the properties of the prepared nanoparticles.

1. Introduction The

advantages

of

oxides

Therefore, it is necessary to use a stabilizing agent

nanoparticles as antimicrobial agents are their

in protecting them from any issues such as

greater

agglomeration

effectiveness

microbial

pathogens,

using

on less

inorganic

resistant toxicity

strains and

of heat

and

coalesence

because

the

nanoparticles are not thermodynamically stable in aqueous solutions (Rac-Rumijowska et al., 2017).

antibacterial agent due to its physiochemical

Due to its biodegradability, biocompatibility, and

properties and biocompatibility [2]. Plus, the

lack of toxicity, chitosan has been used in numerous

crystallite size and the nanoparticle shape have an

applications as an antibacterial coating [5,6]. The

effect on the antibacterial activity which smaller

combination

ZnO

antibacterial

nanoparticles was an efficient approach to produce

activity. Several reports have addressed the harmful

antibacterial materials with improved functional

higher

impact of nanomaterials on living cells, but

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properties.

chitosan

with

inorganic

Researchers

have

reported

on

antibacterial properties of chitosan incorporated

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relatively low concentrations of ZnO are nontoxic

of

SC

have

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nanoparticles

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resistance [1]. ZnO has biocidal action and strong

with Zn. The synergy effect has improved the

ZnO nanoparticles significantly inhibit the growth

antibacterial activity by strengthening of the

of a wide range of pathogenic bacteria under

positive charge on the amine group of chitosan

normal visible lighting conditions.

leading to easier complexation with anionic

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D

to eukaryotic cells [3]. Krishna et. al [4] stated that

components of cell surface [7,8].

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Microwave energy is precisely controllable and can In this study, ZnO nanoparticles have been

for warm-up and cool-down and microwave energy

synthesized by using microwave heating method.

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be turned on and off instantly, eliminating the need

is selectively absorbed by areas of greater moisture

The study was aimed to determine the antibacterial

resulting in more uniform temperature and moisture

activity of ZnO nanoparticles against Gram-positive

profiles, improved yields and enhanced product

(S. aureus) and Gram-negative bacteria (E. coli).

performance. 2. Experimental Although the microwave heating method is a relatively simple technique for synthesizing the

2.1

ZnO nanoparticles, there are some factors affecting

All chemicals used in this experiment were of

Material

ACCEPTED MANUSCRIPT analytical grade and they were used without further

UV/VIS Spectrophotometer, Hitachi, Berkshire,

purification. Zinc nitrate hexahydrate (Zn(NO3)2.

United Kingdom).

6H2O, 98%, Aldrich) and sodium hydroxide (NaOH, 98.9%, Bendosen) were used as the zinc cation

and

the

hydroxide

anion

2.3.2

Fourier

precursors,

transform

infrared

(FTIR)

analysis The

agent. Chitosan was employed as stabilizer and

wavenumber range of 400 cm-1 to 4000 cm-1 were

distilled water was used for preparation of

determined using FTIR (iD7 ATR Nicolet iS5

solutions.

Spectrometer, Thermo Fisher, USA).

absorption

spectra

within

the

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SC

2.2

FTIR

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respectively. Acetic acid was used as hydroxide

Preparation of ZnO nanoparticles

dissolving in 1.0% v/v of the acetic acid solution.

2.3.3

Nanosizer instrument analysis

The zeta potential of ZnO nanoparticles was

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After the chitosan was fully dissolved, 50mL of

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1.0% v/v of chitosan solution was prepared by

Zn(NO3)2. 6H2O and 50mL of NaOH solution were

measured using Zetasizer instrument (ZEM5002,

mixed with 5mL of chitosan solution. Following the

Malvern Instrument Ltd, Malvern, UK).

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reduction process of zinc ions, a milky solution was 2.3.4

mixture was then exposed to microwave radiation

XRD patterns recorded on a Philips PW 3050/10

at 600Watt for 6 minutes.

model. The samples were recorded on a Philips X-

2.3.1

CE

X-ray diffraction (XRD)

Pert MMP diffractometer. The diffractometer was

Characterization

controlled and operated by a PC computer with the

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2.3

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formed. Upon completing the reduction process, the

programs P Rofit and used a MoK (source with

UV-vis spectroscopy analysis

The optical properties of ZnO nanoparticles were

wavelength 0.70930 Å, operating with Mo-tube radiation at 50 kV and 40 mA).

studied by diluting 0.1mL of ZnO nanoparticles in 2.0mL of distilled water. The UV-vis absorption

2.3.5

Field

emission

scanning

electron

spectra of ZnO nanoparticles with wavelength

microscopy

range: 250nm to 800nm at room temperature was

dispersion X-ray (EDX) analysis

obtained using UV-vis spectrophotometer (U-1800

The morphology of an elemental composition of

(FESEM)

and

energy

ACCEPTED MANUSCRIPT ZnO nanoparticles was examined at 100K using

and stabilized by chitosan [10]. The synthesized

FESEM (JSM-7800F, JEOL, USA).

ZnO nanoparticles are presented in Figure 1.

2.4

Antibacterial assay

2.4.1

Zone of inhibition

Firstly, 50µL of 108 CFU/ml bacteria suspension

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was spread on tryptone soya agar (TSA) plate, and then 30µL the freshly prepared ZnO nanoparticles

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was loaded on sterile blank disc placed onto the

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surface of the agar. After overnight of incubation at 37°C, the diameter of zone of inhibition was

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measured [9].

Figure 1 ZnO nanoparticles suspension synthesized

Growth-reduction

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2.4.2

at 600 Watt for 6 minutes.

To examine bacterial growth, overnight cultures of

Figure 2 shows the effect of microwave radiation

fold into 100ml of tryptone soya broth in 250 ml

power on the UV absorption spectra of ZnO

flasks. ZnO nanoparticles were added to the

nanoparticles. All ZnO nanoparticles have shown a

respective flasks. Cultures were then grown for up

strong absorption peak at around 360 nm. The

to 13 h at 200 rpm, 37°C. Bacterial growth was

formation of ZnO agglomerates increases with

CE

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approximately 1 × 109 CFU/ml were diluted 100-

measured by optical density at 600 nm (OD600).

respect to radiation power. However, in the case of

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800W, the number of agglomerates is smaller as

3. Result and discussions

compared to that of 600W, which are consistent to

3.1

those reported by Barreto et al. [11] and Al-

The

Physical characteristics ZnO

nanoparticles

in

Gaashani et al. [12]. They found that the generation

accordance with the methods described above.

of agglomerates would cause the system to become

During the synthesis process of ZnO nanoparticles,

non-homogeneous. Also, the system was not well

the initially colorless solution will turn to the milky

dispersed as the one of 600W, thus decreasing the

solution.

intensity of ZnO nanoparticles.

This

has

were

indicated

prepared

that

the

ZnO

nanoparticles were successfully reduced by NaOH

As observed, when the sodium hydroxide was

ACCEPTED MANUSCRIPT nanoparticles and chitosan are presented in Figure

ZnO nanoparticles was formed. Exposure to

4. The FTIR spectra of chitosan reveal the main

microwave power radiation could dissolve the

absorption bands at 3352 cm-1, 1062 cm-1, and 1029

precipitate in supersaturated aqueous solution and

cm-1 which are associated with hydroxyl groups

form hydroxide ions (OH-) due to diffusion [12]. In

such as -OH, 3’-OH, and 5’-OH, respectively [15].

addition,

it

The bands at 1558 cm-1 and 1494 cm-1 are attributed

solubilized in dilute acetic acid as it reacted with H+

to strong carboxylate peaks [16]. The absorption

from the acid solution. This process would further

peaks at 2869 cm-1 and 2322cm-1 are attributed to

increase the solubility of chitosan in water. ZnO

asymmetric stretchings of CH2 and CH3 of chitosan

nanoparticles were formed by the electrostatic

polymer. The bands at 1646 cm-1 and 1581 cm-1

interaction of zinc nitrate and chitosan because the

correspond to the stretching vibration of C=O and

electron-rich oxygen atoms of polar hydroxyl and

the scissoring vibration of -NH2, respectively

after

ether groups of chitosan were likely to interact with

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CE

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D

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electropositive metal cations [13,14].

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protonated

SC

was

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chitosan

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mixed with zinc nitrate, the white precipitate of

Figure 2 Absorption spectra of ZnO nanoparticles synthesized at power radiation of 600 Watt.

[10,17].

For the ZnO nanoparticles synthesized with chitosan as a stabilizing agent, the absorption band has been split to 2915 cm-1 and 284 cm-1 from 2869 cm-1, signifying the vibrational modes of CH2 stretching in chitosan network. The band at 1555 cm-1 denotes N-H stretching [17]. The absorption band at 1382 cm-1 is attributed to the NH2 deformation stretching in chitosan backbone while bands at 1062 cm-1 and 1029 cm-1 denote C-N and C-O stretchings, respectively. For the FTIR spectrum of chitosan, the new peaks formed at 631 cm-1 and 475 cm-1 are due to the attachment of

3.2

FTIR

FTIR analysis was performed to identify the functional groups of ZnO nanoparticles and chitosan solution. The absorption spectra of ZnO

amide group and stretching mode of ZnO, respectively

[7,12,15].

Clearly,

these

results

indicate that chitosan, zinc nitrate and sodium hydroxide have been converted to a mixture of

ACCEPTED MANUSCRIPT chitosan and ZnO [18].

Table 1 Diffraction peaks of chitosan/ZnO nanoparticles

2θ (°)

Planes of

100

34.46

002

36.30

101

41.52

102

56.65

110

62.89

103

66.42

200

67.99

112

69.14

201

Figure 3 FTIR spectra of chitosan and ZnO

3.3

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nanoparticles

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31.82

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hexagonal

XRD

Figure 4 shows the XRD patterns of chitosan, ZnO

of

chitosan/ZnO

nanoparticles

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peaks

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nanoparticles, and chitosan/ZnO nanoparticles. All were

compared with the standard peaks of wurtzite ZnO (JCPDS card no. 36-1451) with high crystallinity

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resulting in excellent matching both samples. Furthermore, no characteristic peaks were observed

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for other added impurities. The typical peak of chitosan appeared at 20.59° while this peak became

Figure 4 XRD pattern of chitosan/ZnO

weak in the XRD pattern of chitosan/ZnO nanoparticles. Others diffraction peaks were listed and presented in Table 1.

nanoparticles The average crystalline sizes D (nm) of the ZnO nanoparticles was calculated using Scherrer’s formula.

ACCEPTED MANUSCRIPT θ = the Bragg angle

where; λ = the X-ray wavelength of Cu-Kα radiation source (1.5418 Å)

The average crystalline size is found to be 130 nm.

β = the full width at half maximum (FWHM)

FESEM-EDX

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3.4

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intensity of the diffraction peak located at 2θ

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The FESEM image of ZnO nanoparticles coated with platinum is presented in Figure 5. The ZnO

sizes are in the range from 50nm to 70nm. From the

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FESEM results, ZnO nanoparticles of smaller size

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nanoparticles are uniformly distributed and their

Figure 5 FESEM images of ZnO nanoparticles

can be produced by applying the microwave

Figure 6 shows the EDX spectrum of the

gradient within the particle, leading to the

synthesized ZnO nanoparticles. As seen, the

formation of small nanoparticles [10,19–21] due to

samples consist of zinc (Zn), carbon (C) and

the polarization of ZnO nuclei as highlighted by Al-

oxygen (O). The presence of platinum (Pt) is due to

Gaashani

ZnO

the coating applied before conducting the FESEM

nanoparticles are approximately spherical, which

analysis. The two primary elements in a ZnO

are consistent with those reported by Singh et al.

nanoparticle are zinc and oxygen (35.67% and

al.

[12].

The

AC

CE

et

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D

heating method. Radiation causes high-temperature

[20] and Anandhavelu et al. [18].

produced

17.22%, respectively). Meanwhile, the atomic percentages of zinc and oxygen are 23.61% and 46.57%, respectively. The EDX analysis indicates that ZnO nanoparticles have been successfully formed and they exhibit homogeneous dispersion within the chitosan matrix [17].

ACCEPTED MANUSCRIPT Table 2 Zone of inhibition of ZnO nanoparticles.

Bacterium

Diameter of inhibiton zone (mm)

S. aureus

16

E. coli

13

Time-kill study

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3.5.2

In this study, the time kill measurement was

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determined by the actual reduction in bacteria Figure 6 EDX spectrum of synthesized ZnO

growth curve at 13 h of the culturing period for S.

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nanoparticles

aureus and E. coli. bacteria. Data from the time–kill

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studies are shown in Figure 7 and Figure 8 which demonstrates the growth curve of S. aureus and E.

3.5

Evaluation of antibacterial activity

3.5.1

Zone of inhibition method

The

results

of

inhibition

zones

of

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coli. bacteria. S. aureus and E. coli. bacteria were in contact with 0.02ml of ZnO nanoparticles. The

ZnO

results exhibited that ZnO nanoparticles exhibit

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nanoparticles agaist S. aureus amd E. coli was

inhibitory effects on the growth as compared to

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presented in Table 2. The larger zone was observed bacteria culture without nanoparticles as a control.

at S. aureus culture which is 16mm compared to the These results demonstrated that ZnO nanoparticles

E. coli culture with 13mm of inhibition zone. Based showed inactivation effect against both types of

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on the results obtained, it can be suggested that Gram-negative bacteria are more resistance to ZnO

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nanoparticles compared to Gram-positive bacteria. This supports by Premanathan et. al (2011) [22] reports that ZnO nanoparticles showed a much stronger antibacterial effect on Gram-positive bacteria than on Gram-negative ones. Stoimenov et. al.[23] and Fu [24] stated that the possibilities of membrane damage are caused by electrostatic interaction between ZnO and cell surface.

bacteria.

ACCEPTED MANUSCRIPT works Figure 7 Effect of ZnO nanoparticles on the growth

ZnO

of S. aureus in TSB at 37°C

nanoparticles

were

successfully

been

synthesized by a microwave heating method. UVvis absorption analysis showed that the ZnO nanoparticles exhibited a peak at around 360 nm. The FTIR spectrum dispalyed the bond of ZnO

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nanoparticles at 475 cm-1. The nanoparticles are in spherical shaped with average size of 50 to 130nm.

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ZnO nanoparticles exhibited excellent antibacterial

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activity against Gram-positive and Gram-negative bacteria.

ZnO

nanoparticles

showed

better

Figure 8 Effect of ZnO nanoparticles on the growth

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of E. coli in TSB at 37°C

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antibacterial activity against S.aureus compared to

From Figure 8, Gram-negative bacteria is more

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resistance to the ZnO nanoparticles compared to the

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Gram-positive bacteria. This can be related to the cell wall of the membrane, as the Gram-positive bacteria have one cytoplasmic membrane with the

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multilayer of peptidoglycan polymer, and a thicker

E.coli.

Different process parameters can be studied for producing uniform size of the nanoparticles by varying the ratio of metal salts, pH and temperature during the synthesis process. The investigation also can be carried out by the use of natural material such as arabic gum as stabilising agent for producing better dispersion of ZnO nanoparticles.

cell wall [24]. Whereas Gram-negative bacteria

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wall is composed of two cell membranes, an outer membrane and a plasma membrane with a thin layer of peptidoglycan [25]. Besides according to Padmavathy et. Al (2008) [26], ZnO nanoparticles have an abrasive surface texture which influences the antibacterial mechanism, which in sequence destroys the bacterial membrane.

4. Conclusion and recommendation for future

Further studies should be done on the efficiency of ZnO nanoparticles against pathogenic bacterium and

fungi

such

as

Methicillin-resistant

Staphylococcus aureus (MRSA) and Trichophyton rubrum (T. rubrum) including the changes of morphological of the strains after in contact with ZnO nanoparticles. This study will contribute to the

ACCEPTED MANUSCRIPT understanding of inactivation effect of ZnO

activity

nanoparticles against pathogenic microorganisms.

Langmuir. 27 (2011) 4020–4028. [5]

Acknowledgment

A.

Gedanken,

PT

chitosan–ZnO-based

grant of RDU150333.

Chitosan

and

complex

nanoparticles: formation, characterization,

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