Microwave Assisted Synthesis of Ag-ZnO Particles and Their

Antibacterial Properties

BAZANT PAVEL1, 2

, KURITKA IVO1, 2

, MACHOVSKY MICHAL1, 2

, SEDLACEK TOMAS1, 2

PASTOREK MIROSLAV1, 2

1Centre of Polymer Systems, University Institute

Tomas Bata University in Zlín

Nad Ovcirnou 3685, 760 01 Zlín,

CZECH REPUBLIC 2 Polymer Centre, Faculty of Technology

Tomas Bata University in Zlín

Nam. T. G. Masaryka 275, 762 72 Zlín

CZECH REPUBLIC

bazant@uni.utb.cz http://cps.utb.cz

Abstract: Hybrid Ag-ZnO micro-structured nanoparticles were prepared by two different microwave techniques

from silver nitrate and zinc nitrate within 15 minutes. Crystalline structures of obtained Ag-ZnO powders were

characterized by X-ray diffractometer. Scanning electron microscopy was used for characterization of structure,

morphology, particle size Ag-ZnO filler. Elemental analysis of Ag-ZnO filler was made by Energy dispersive

X-ray analysis. Growth mechanism of particles was elucidated. The antibacterial activity was evaluated by

inhibition zone test; one of the materials performed well against Staphylococcus aureus, and Candida albicans.

Key-Words: microwave synthesis, hybrid filler, Ag-ZnO, antibacterial activity

1 Introduction Hybrid materials, especially metal-modified oxide

semiconductors, are of great interest amongst the

researchers and bring interesting possibilities of

property design, thus, they open new application

fields. Materials that combine silver and zinc oxide

(ZnO) have attracted attention because they were

successfully used in chemical and biological

sensors, electronics and photoelectronics devices

and have considerable bio-activity as well. [1-3].

Nowadays, various techniques are studied for

preparation of novel Ag-ZnO hybrid systems and

their antibacterial activity is tested [4-6]. The strong

antibacterial effects of both metallic Ag and Ag+

ions have been known for a long time [7, 8]. Zinc

oxide is another inorganic agent which, in form of

nanoparticles, exhibit strong antibacterial activity on

a broad spectrum of bacteria although its effect on

micro-organisms is not fully understood yet [9].

Preparation of such hybrid systems can be

significantly enhanced by the introduction of

microwave (MW) into synthesis process.

Microwave irradiation has attracted wide interest as

heating mechanisms in materials synthesis due to its

many advantages, including a very short reaction

time, and the ability to produce small inorganic

particles with narrow particle size distribution and

high purity [3, 10, 11]. In this paper, two different

facile microwave preparation methods of Ag-ZnO

nanostructured microparticles are presented using

hexamethylenetetramine (HMT) as precipitation and

reducing agent, with further aim to obtain new

antibacterial additives for prospective applications

in polymer medical devices.

2 Experimental 2.1 Material Materials silver nitrate AgNO3 (purum, ≥99.5%)

and zinc nitrate hexahydrate Zn(NO3)2·6H2O

(purum, >99%) were purchased from Penta (Prague,

Czech Republic). Hexamethylenetetramine (HMT)

C6H12N4 (purum, >99%, Fluka) was purchased from

Sigma-Aldrich (Prague, Czech Republic) and used

as precipitation and reduction agent and growth

modifier. Demineralised water was used in these

experiments.

2.2 Synthesis of Ag-ZnO For preparation of powders two different microwave

apparatures were used.

First material (sample 1) was prepared in the

microwave open vessel system MWG1K-10 (Radan,

Czech Republic, 800W, 2.45 GHz) based on

Mathematical Methods and Techniques in Engineering and Environmental Science

ISBN: 978-1-61804-046-6 341

modification of domestic oven by drilling a hole on

the ceiling for external cooler and equipped with

external MW power source that enables operate the

instrument in continuous mode. All the chemical

reagents were dissolved in demineralised water. The

solution of 0.015 mol Zn(NO3)2·6H2O was mixed

together with solution of 0.01 mol AgNO3. The total

amount of used water was 100 mL. The obtained

solution was placed into the microwave oven cavity

and heated for 2 minutes then a solution of 0.015

mol C6H12N4 in 50 mL water was added quickly

through the dropping system and microwave heating

continued for another 15 minutes. Obtained

suspension was cooled freely to room temperature.

For preparation of the second material (sample 2)

a commercial pressurized MW system Mars5 (CEM

Corporation) was used. Identical solutions were

prepared in smaller amounts, as they needed to be

mixed prior synthesis and filled in a Teflon reaction

vessel (XP-1500 Plus), sealed and heated in the

microwave oven. The total amount of used water

was 60 mL. The reaction solution in the vessel was

irradiated by MW at moderate power of 400 W to

ensure temperature regulation. The solution was

heated to 90°C for 15 minutes. When the reaction

was completed, the vessel was left to cool naturally

and then unsealed. A suspension was obtained.

Both suspensions were filtered, washed with

demineralised water and finally collected powders

were dried in the air at 37°C for over night.

2.3 Characterization The crystalline phase structure of obtained powders

was characterized by X-ray diffractometer

PANalytical X´Pert PRO (PANalytical, The

Netherlands) using Cu Kα1 radiation (λ = 0.1542

nm) operating at 40 kV and 30 mA with detector

PIXcel. Both materials were measured in

transmission mode with fixed setting and screen of

range angle 25-85° (2θ) and step 0.0263°. The phase

composition was evaluated by the software

PANalytical X'Pert High Score using normalized

RIR method. The RIR is the ratio between the

integrated intensities of the peak of interest and that

of a known standard [11].

The morphology of the products was investigated by

scanning electron microscope Vega II LMU

(Tescan, Czech Republic) with beam acceleration

voltage set at 10 kV, after coating with

gold/palladium by a high-resolution SEM sputter

coater SC 7640 (Quorum Technologies Ltd, UK).

SEM equipment includes Energy dispersive X-ray

analyser (Oxford INCA) used for elemental

analysis.

2.4 Testing antibacterial activity Antibacterial testing was performed against

Staphylococcus aureus CCM 4516, Escherichia coli

CCM 4517 and Candida albicans CCM 8215.

Amount of 0.1 g of obtained Ag-ZnO powder was

dispersed in 1 mL demineralised water and

homogenised in ultrasonic bath for 15 minutes.

Then 10 µL of suspension was applied on filter

paper with diameter 8 mm. Prepared specimens

were dried at the room temperature. Specimens

(diameter 8 mm) were put into nutrient agar which

was inoculated with bacteria (approximately 108

colony forming unit (CFU) per millilitre). The

bacteria used in this study were S. aureus (CCM

4517), E. coli and C. albicans (CCM 8215). After

24 h incubation at 37°C, the diameters of the

inhibition zones, which appeared on the surface,

were measured in five directions. Average values

were used for calculation of the inhibition zone area,

which was the measure of the antibacterial activity

of the studied samples. Each test was conducted

with six specimens. Blank samples without

suspension were prepared and tested in the same

way.

3 Results and discussion 3.1 Morphology and structure of prepared

Ag-ZnO particles SEM microphotographs are shown in Figure 1a and

1b. The images were taken by BSE detector that

allows distinguish composition of particles by

material (greyscale) contrast showing heavier

elements brighter. For sample 1 (a) it is apparent,

that the zinc oxide particles are hexagonal

microrods structures with the size up to 2 µm and

silver structures are aggregates of spherical particles

with the diameter up to 200 nm. In sample 2 (b) Ag-

ZnO particles of polyhedral shape with size up to 3

µm together with similar particle aggregates as in

sample 1. In left top corner of Figure 1b can be seen

a detail of one bigger particle revealing its

hexagonal symmetry. This kind of pyramid-like

particles can be considered as the final product of

the reaction, as similar growth was presented Huang

A.S. et al. (2010) who demonstrated grow of ZnO

hexagonal pyramids from hexagonal disks by

solvothermal reaction during 10 days. [16].

Figure 2 gives typical EDX spectra recorded for

the obtained powders. The EDX spectrum indicates

that samples 1 and 2 are composed of Ag, Zn, C and

O. Sample 1 contains 9.87 % (at.) of Zn and 32.36

% (at.) silver. Sample 2 contains 3.75 % (at.) of Zn

and 39.63 % (at.) of silver. Both materials contain

approximately 18% (at.) carbon and 40 % (at.)

Mathematical Methods and Techniques in Engineering and Environmental Science

ISBN: 978-1-61804-046-6 342

oxygen which can stem from adsorbed H2O, CH2O

(or its polymer, paraformaldehyde) and rests of

HMT. For possible sources of these components see

the discussion on growth mechanism below.

Figure 3 shows detailed microphotographs of

Ag-ZnO structures which were used for obtaining

precise composition of alone particles by point EDX

analysis. Structure “s” in image taken from sample 1

contains 43.58 % (at.) silver and 7.94 % (at.) of Zn.

Structure “z” has hexagonal structure composed

from 30.12 % (at.) of Zn and 6.99 % (at.) silver.

Note a hollow, calyx-like, particle in left corner of

the image. The big particle representing sample 2 is

composed from 10.69 % (at.) of ZnO and 52.91 %

(at.) silver. The rest is assigned to oxygen and

carbon. Therefore we assume that both materials are

not composed only from ZnO and silver particles,

but the particles are microsized aggregates with

some nanostructure involving also organic

compounds.

Fig. 3 Details of structures Ag-ZnO filler samples 1 and

samples 2. The picture indicates positions where point

EDX spectra were taken from.

Figure 4 presents results of the X-ray diffractometry

(XRD) of powders obtained by microwave

synthesis. Both samples exhibit the typical pattern

for the face-centered-cubic Ag metal consistent with

the reported values (JCPDS no. 01-087-0720).

Sample 1 contains hexagonal ZnO (JCPDS no. 01-

079-0207) also. Crystalline phase composition of

hybrid filler was estimated in sample 1 as 83 wt. %

2μμμμm

2.5 μμμμm

b

2μμμμm

2.5 μμμμm

2μμμμm2μμμμm

2.5 μμμμm2.5 μμμμm

b

Fig. 1 BSEM microphotographs of hybrid prepared Ag-

ZnO materials. Sample 1 (a), sample 2 (b).

X-ray photon energy/ keV X-ray photon energy/ keV

Fig. 2 EDX spectra of sample 1 (a), sample 2 (b)

Fig. 2 XRD patterns of the Ag/ZnO particles a) sample 1

and b) samples 2

a b

a

sample 2

s z

sample 1

b

a

1 µm

Mathematical Methods and Techniques in Engineering and Environmental Science

ISBN: 978-1-61804-046-6 343

of Ag and 17 wt. % of ZnO. Sample 2 was

characterized as 100 wt. % crystalline Ag. Overall

intensity of both observed spectra was relatively low

because X-ray diffraction patterns were measured in

transmitting mode; therefore diffraction lines

belonging to minor ZnO phase in sample 2 could be

hidden in background. However, these results seem

to be somewhat contradictory with respect to EDX

observation. It must be noted, that XRD allows to

analyse crystalline phase only, so it is reasonable to

expect, that at least certain portion of prepared

materials is amorphous, or the disorder introduced

by mutual doping of Ag and ZnO destroyed

periodicity of materials structure.

3.2 Mechanism of microwave assisted

synthesis Ag-ZnO powders were prepared by hydrothermal

microwave synthesis by two different microwave

devices, which involved the precipitation of ZnO

and the formation of Ag nanoparticles using HMT

as precipitation and reducing agent. During short

synthesis time were formed structures of Ag

containing ZnO with containing Ag nanoparticles.

Growth and morphology of ZnO and Ag particles

are dependent of many factors. Ashfold, M.N.R. et

al. 2007 describe the role of pH, zinc ion and HMT

concentration of the solution [13]. For controlled

growths of ZnO structure Baruah, S. and Dutta, J.,

2008 explain another parameters e.g. type of used

chemical (Zinc acetate, zinc nitrate and other),

reduction, precipitation agents or precursor and

surfactant (HMT, glucose, NH3, PEG, PVA, CTAB)

experimental parameters (temperature, time) and

describe basic role HMT [14]. This can be

summarized in the following equations:

(CH2)6N4 + 6H2O ↔ 6HCHO + 4NH3 (1)

NH3 +H2O ↔ NH4+ + OH

− (2)

2OH− + Zn

2+→ ZnO(s) + H2O (3)

Ye, X. Y. et al. 2008 refer that controlled growth

of ZnO structure can be influenced the presence Ag+

[4]. Zhang, Y.Y. and Mu, J. 2007 observed that the

morphology of ZnO varied from pillar-like to rod-

like with the increased concentration of Ag+. The

reactions involved in the formation of metallic silver

in basic conditions are believed to be as follows:

Ag+ + 2NH3 → [Ag(NH3)2]

+ (4)

HCHO + 2[Ag(NH3)2]+ + H2O →

HCOO- + 2Ag(s) + 3NH4

+ + NH3 (5)

The source of formaldehyde (reducing agent) is

decomposition of HMT at elevated temperature in

water solution as described in equation (1). The

addition of the silver nitrate changes the equilibrium

between zinc ions and ammonia due to the

coordination of silver ions by ammonia and

subsequently influences the morphology of ZnO and

efficiency of its growth. Increase the concentration

of HMT results in increase of the concentration of

ammonia in the reaction system, which will prevent

the influence of the silver ion on the equilibrium

between zinc ion and ammonia. [15]

The main difference between both synthesis

methods was the pressure in the reaction system. As

the sample 2 was prepared in sealed Teflon vessel it

is assumed that all chemical equilibria reactions

involving change in molarity were shifted to that

side where lower sum of stoichiometric indexes is.

Reactions (1) and (5) are shifted to the left side, the

reactions (3) and (4) shifts to the right side, reaction

(2) has the same number of moles on both sides.

Normally, elevated pressure would strongly enhance

reaction with solid phase formation, but it must not

be at the expenses of increase of other by-products

as in (5).

Observed composition of prepared materials, i.e.

higher content of Ag in sample 2 than in sample 1,

testifies for larger influence of silver nitrate addition

and competition for ammonia due to silver ions

complexation than the pressure effect on the

reaction system has, namely on HMT

decomposition (1) and ZnO formation (3), which

would prefer the growth of ZnO.

3.3 Antibacterial activity of Ag-ZnO filler Antibacterial activity of Ag-ZnO filler is shown in

Table 1. S. aureus represents gram positive bacteria

and seems to be sensitive towards sample 1,

whereas E. coli is a gram negative bacteria and did

not show inhibition zone. These two groups differ in

the structure of their cell walls since the cell walls of

gram positive bacteria contain more peptidoglycan

layers than gram negative bacteria. Gram positive

bacteria are more sensitive against ZnO than gram

negative. [17, 18] Sample 1 performed well also in

case of C. albicans. However, no effect of silver

present in sample 2 was observed on all tested

organisms. It can be expected that there is not

enough of free Ag+ ions available for diffusion

through agar plate to form the “halo” zone effect. To

complete the assessment of antibacterial properties

another more predicative test is needed which would

take into account contact inhibition effect of silver

surface on bacteria.

Mathematical Methods and Techniques in Engineering and Environmental Science

ISBN: 978-1-61804-046-6 344

Table 1 Antibacterial activity of Ag-ZnO filler

Inhibition zone (mm) Number

of sample E. coli

(CCM 4517)

S. aureus

(CCM 4516)

C. albicans

(CCM 8215)

Sample 1 8 14 9

Sample2 8 8 8

Blank 8 8 8

4 Conclusions 1. Two different microwave synthesis techniques

were successfully adapted for preparation of hybrid

Ag-ZnO. The methods are faster than any other

published previously in literature, moreover, they

are simple and do not require any template, catalyst,

or surfactants and allow control the morphology of

Ag-ZnO crystals from simple to complex.

2. As the main parameter influencing growth of

hybride particles is the complexation of silver ions

by ammonia as it can overbalance the effect of

pressure which would normally enhance

precipitation of ZnO.

3. The antibacterial activities of Ag-ZnO were tested

against E. coli, C. albicans and S. aureus. Powder

material prepared by microwave open vessel system

proved observable antibacterial activity against S.

aureus and C. albicans.

Acknowledgements This article was written with support of Operational

Program Research and Development for Innovations

co-funded by the European Regional Development

Fund (ERDF) and national budget of Czech

Republic, within the framework of project Centre of

Polymer Systems (reg. number:

CZ.1.05/2.1.00/03.0111).

This work was supported by the internal grant of

TBU in Zlín No. IGA/5/FT/11/D funded from the

resource of specific university research.

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