Ultrasonics Sonochemistry 27 (2015) 466–473

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Ultrasonics Sonochemistry

journal homepage: www.elsevier .com/ locate/ul tson

Direct and indirect sonication affect differently the microstructureand the morphology of ZnO nanoparticles: Optical behaviorand its antibacterial activity

http://dx.doi.org/10.1016/j.ultsonch.2015.06.0161350-4177/� 2015 Elsevier B.V. All rights reserved.

⇑ Corresponding author at: Sonochemical Research Center, Environmental Chem-istry Research Center, Department of Chemistry, Faculty of Science, FerdowsiUniversity of Mashhad, 91779, Mashhad, Iran.

E-mail addresses: entezari@um.ac.ir, moh_entezari@yahoo.com (M.H. Entezari).

Zahra Sharifalhoseini a, Mohammad H. Entezari a,b,⇑, Razieh Jalal ca Sonochemical Research Center, Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, 91779, Mashhad, Iranb Environmental Chemistry Research Center, Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, 91779, Mashhad, Iranc Biochemical Research Center, Department of Chemistry, Faculty of Science, Ferdowsi University of Mashhad, 91779, Mashhad, Iran

a r t i c l e i n f o

Article history:Received 23 February 2015Received in revised form 7 June 2015Accepted 19 June 2015Available online 19 June 2015

Keywords:Green methodZnO NPsSono-synthesisOptical behavior

a b s t r a c t

In the present study, the sono-synthesis of ZnO nanoparticles (NPs) was performed by simple, low-cost,and the environmentally friendly method. The synthesis of zinc oxide as an antibacterial agent was per-formed by an ultrasonic bath (low intensity) for the indirect sonication and a horn system (high intensity)for the direct sonication. The samples synthesized by these two kinds of sonication were compared witheach other. Crystallographic structures and the morphologies of the resultant powders were determinedby X-ray diffraction (XRD) and transmission electron microscopy (TEM). The XRD patterns showed thatboth ZnO samples were crystallized in their pure phase. The TEM images confirmed that the morpholo-gies of the products were completely different from each other. Based on the obtained analysis, the prob-able growth mechanisms were proposed for crystallization of both samples. The antibacterial activity ofthe synthesized species was evaluated by the colony count method against Escherichia coli O157:H7.Moreover, the optical behavior of the samples was studied by UV–vis spectroscopy and the variationof the ZnO band gap was compared.

� 2015 Elsevier B.V. All rights reserved.

1. Introduction

Nanosized structures due to larger surface and higher efficiencythan bulk materials have been widely used in many industriesincluding materials science, optics, mechanics, plastics, medicine,and the textile industry [1]. ZnO is one of the most interestnanoparticle, which found as a safe and friendly material forhuman. This metal oxide has been developed in the dermatologicalapplications as an antibacterial agent and UV absorber in creams,lotions, and ointments [2]. Furthermore, ZnO as a very importantII–VI semiconductor with a wide band gap of 3.37 eV and an exci-ton binding energy of 60 meV has been widely used in a broadrange of the electronic, optical, and the piezoelectric applications[3–4].

Synthesis of the various ZnO nanostructures including nanotube[5], nanosheet [6], nanowire [7], and nanorod [8] has beenreported. Recently, the successful efforts have been made to make

the three-dimensional (3D) ZnO nanostructures due to theirpromising applications. For example, a scientific report confirmedthat the ZnO hierarchical microstructures show high photocat-alytic performance in comparison to the other nanoparticles,nanosheets and nanorods structures [9]. In addition, ZnOflower-like crystal, which used as the matrix enhances the enzymeimmobilization in biosensor for the detector of H2O2 [10].

Various methods such as chemical vapor deposition [11],hydrothermal [12–13], microwave-assisted method [14], and elec-trodeposition [15] have been successfully performed for ZnO syn-thesis. In the past decades, the sonochemical method as theefficient and powerful technique was developed for the synthesisof the wide range of the materials with controlled properties[16–17]. For this purpose, generally high intensity sonicatorsequipped with a horn have been developed while the bath systemsas the low power sonicators have been widely used for the cleaningapplications and has been less used in the synthesis processes. Inspite of this aspect, it should be mentioned that the use of thisapparatus provides the conditions to reach the product with thedifferent properties from those obtained under the high intensityirradiation or even under the conventional methods. Some

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Z. Sharifalhoseini et al. / Ultrasonics Sonochemistry 27 (2015) 466–473 467

researchers have conducted the experiments on the progress of thereaction in the ultrasonic bath [18–19].

The current paper deals with fabrication of the ZnO nanocrys-tals with the flower-like feature under the low intensity ultrasonicirradiation. The phase structure, morphology and the biologicalactivity of the product have been compared with the one sampleprepared under the high acoustic intensity. Furthermore, withregard to the morphological studies, probable mechanisms havebeen proposed for the growth processes of two sono-synthesizedsamples.

2. Experimental

2.1. Materials

The reagents including zinc acetate dihydrate, sodium hydrox-ide, glycerol, and ammonium citrate were purchased from Merckand used without further purification.

2.2. Preparation of Zn(OH)42�

An aqueous zinc acetate dihydrate solution(1.25 mM) was pre-pared by dissolving 5.5 g of Zn(Ac)2�2H2O, as a Zn2+ source, in the50 mL distilled water. Then, by the addition of 8 g of solid NaOHto this solution at 40 �C, a transparent solution saturated withZn(OH)42� was formed.

2.2.1. Direct method (method I)The resulting solution (final volume � 55 mL) was dropped to

the water-jacketed cell containing 100 mL of distilled water. Thetemperature of the water in the cell was adjusted at 65 �C. Theultrasonic irradiation was applied to the reaction medium by usingan ultrasonic apparatus equipped with a horn (20 kHz, model XL2020) for about 40 min. The obtained sample was named assample I.

2.2.2. Indirect method (method II)For this test, one Erlenmeyer containing 100 mL of distilled

water was placed in an ultrasonic bath (40 kHz, Branson8510E-DTE, internal dimensions: 49 cm � 29 cm � 16 cm). Thetemperature of the bath water was set at 65 �C and allowed toreach the thermal equilibrium between the bath and Erlenmeyerwater (for both methods, direct and indirect sonication, the tem-perature of the reaction containers was also checked by using anexternal thermometer during the reaction). Then the transparentcomplex of Zn(OH)42� (�55 mL) was added dropwise to theErlenmeyer. This product was named as sample II. The reactionconditions for the samples I and II are summarized in Table 1.

It is well known that the structure, particle size, purity and themorphology of the products are affected by the reaction conditions.Hence, to find the effect of the kind of ultrasonic irradiation on theproperties of the products, other parameters (including the startingmaterials and their concentration, temperature, and the reactiontime) were kept constant. Moreover, the addition of Zn(OH)42� asthe precursor to the reaction container from a burette was alsokept at a constant rate (�1 mL min�1).

Table 1Synthesis conditions for the samples I and II.

Sample Type of irradiation Ultrasonic status Ultrasonic parameters

Frequency (kHz) Inte

I Direct Horn 20 �3II Indirect Bath 40 �0

2.3. Preparation of nanofluids

The stability of the ZnO suspension during the antibacterialactivity tests is an important factor. Therefore, ZnO nanofluids(ZnO NFs) were prepared with the dispersion of ZnO NPs in glyc-erol as the base fluid and ammonium citrate as dispersant. Theweight ratio of dispersant to nanoparticles was kept at 1:1.

2.4. Antibacterial activity assay

2.4.1. Absorption methodTo estimate the antibacterial action of ZnO NPs and the fluid

components, including glycerol and ammonium citrate, absorptionmethod was applied. For this evaluation, sample I was chosen. TheZnO nanofluid was prepared as a stock solution at a concentrationof 5.6 mg mL�1 and was diluted twofold serially with tryptic soybroth (TSB) prior to each experiment. Escherichia coli (E. coli)O157:H7 (NTCC: 12900) was grown overnight with aeration inTSB at 37 �C. To prepare the logarithmic phase of bacteria in thebeginning state, 10 lL of inoculum was transferred into anErlenmeyer flask containing 10 mL TSB medium and was allowedto grow at 37 �C for 3–4 h on a reciprocal shaker (120 rpm). A celldensity was estimated by optical density measurement at 630 nm(OD630). The final concentration of 105 colony forming units (CFU)mL�1 was adjusted by dilution with TBS and then it was added intoZnO nanofluids with different concentrations (0.375, 0.750, and1.500 mg mL�1). The test samples were incubated at 37 �C on areciprocal shaker. For the absorption method which is based onthe turbidity measurement, after 24 h, the growth of the cultureswas monitored by measuring OD630 using an ELISA microplatereader (Biotek. Elx800). To exclude the influence of the base fluidand dispersant agent on the results, fluids containing glyceroland ammonium citrate without ZnO NPs were also cultured underthe same growth conditions. Bacterial suspension without ZnO NFsand culture medium (TSB) without microbial suspension were alsoconsidered as positive and negative controls, respectively.Composition of the fluids and ZnO NFs used in these experimentsare given in Table 2.

2.4.2. Colony count methodThe biological activity of the samples I and II were compared by

colony count method. Concentration of 0.750 mg mL�1 of ZnOnanofluids was chosen as the proper concentration based on theabsorption method results. For this test, a bacterial culture withconcentration of 105 CFU mL�1 was added into TSB medium con-taining ZnO nanofluids with concentration of 0.750 mg mL�1 andkept at 37 �C for 24 h on a reciprocal shaker. One hundred micro-liter of the culture was then diluted serially in TSB and inoculatedon tryptic soy agar (TSA) plates. After 24 h incubation at 37 �C, thenumbers of colonies were counted. To get access more accurateresults all measurements were performed in triplicate.

2.5. UV–visible absorption

UV–vis spectroscopy was used for the comparison of the opticalbehavior of the sono-synthesized samples I and II. The UV–visspectra were recorded by a Unico 2800 UV–vis spectrophotometer

Temperature (�C) Reagents

nsity (W cm�2) Zn2+ source O2� source

5.00 65 ± 5 Zn(CH3COO)2.2H2O NaOH.30 65 ± 5 Zn(CH3COO)2.2H2O NaOH

Table 2Composition of the fluids and ZnO NFs used in the absorption method.

Sample Concentration of ZnO NPs (mg mL�1) Concentration of Ammonium citrate (mg mL�1)

Fluidsa 0 0.3750 0.7500 1.500

ZnO nanofluidsa 0.375 0.3750.750 0.7501.500 1.500

a In all above suspensions glycerol was used as the base fluid.

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in the wavelength range of 200–800 nm. For these measurements,the ZnO powders were dispersed in the absolute ethanol in theultrasonic bath for 5 min at the ambient temperature.

3. Results and discussion

3.1. XRD analysis

The crystallographic structures for the ZnO powders weredetermined by X-ray diffraction. Recording of XRD spectra wasperformed by using of X-ray diffractometer (D8 Advance) withCu Ka radiation (k = 0.154 nm). Fig. 1(a) and (b) depict the XRDpatterns for the samples I and II, respectively.

Characteristic diffraction peaks of ZnO including (100), (002),(101), (102), (110), (103), (200), (112), (20 1), (004), and(202) were observed for two prepared samples. All the diffractionpeaks can be well indexed to the hexagonal phase ZnO reported inJCPDS card (No. 36-1451, a = b = 3.249 Å and c = 5.206 Å). The pres-ence of the high intensity peaks in Fig. 1(a) and (b) implied that theproducts were formed in the high crystalline structures. Moreover,these patterns confirm that the creation of the pure phases of ZnOoccurred under the different sonication conditions because of noextra peaks were detected in the XRD analysis. The observationof the similar peaks with different intensity in two patterns isattributed to the various crystallographic structures. On the otherword, the relative intensities of the diffraction peaks are implyingthat the preferred orientations occurred during the crystallization[20]. The (100)/(002) intensity ratio is reported 1.17 for the isotro-pic ZnO. This value is obtained from the XRD pattern collected forthe bulk sample. Any deviation from this value indicates a degreeof particle anisotropy. In fact, the small (100)/(002) ratio (intense(002) peak) suggests the crystal growth along c-axis and large(100)/(002) ratio indicate the shortening growth along the c-axis[21]. As can be seen in Fig. 1, the intensity of the (001) peak withrespect to the (002) peak of the samples I and II are different fromeach other. Based on our measurements, the values of this param-eter are 1.61 and 0.94 for the samples I and II, respectively.

Fig. 1. XRD patterns for the prepared ZnO powders; (a) the sample

Therefore, both of the samples show deviation from the isotropicstructure. As an interesting result, these values of the(110)/(103) ratio were repeated in the obtained XRD patterns.

3.2. FTIR spectroscopy

The mid-IR spectra were recorded by using Fourier transforminfrared spectrophotometer (FTIR, Bomem MB-154) in the rangeof 500–4000 cm�1. The mid-IR spectra are shown inFig. 2(a) and (b). As can be seen from these figures, the character-istic band of ZnO could be detected in two samples at about 500–530 cm�1. This sharp peak is ascribed to the Zn–O bond [22]. Sincethis peak is located near the end of the spectra, far-IR techniquewas employed to get more accurate data on this stretching vibra-tion mode. This technique by the scanning of the low wavenumberregion provided a better sight of the spectrum and clarified thepossible differences in the position of the ZnO peak for the samplesI and II. The far-IR spectra were collected in the region 300–600 cm�1 using a Nexus 870 spectrometer (Thermo Nicolet)equipped with a solid substrate beam splitter. The far-IR spectra(Fig. 2(a0) and (b0)) show clearly the differences in the peak positionfor the samples and confirm that the IR spectra are influenced byproperties of a sample such as the morphology and the particlesize. Based on the scientific reports, the pronounced peaks at430 cm�1, 555 cm�1and even at 670 cm�1 are assigned to thestretching vibrations of Zn–O bond [23]. Therefore, the presenceof three peaks (at 400 cm�1, 506 cm�1 and 545 cm�1) and twopeaks (at 425 cm�1and494 cm�1) observed in the far-IR spectraof the sample I and II respectively, could be assigned to the exis-tence of the Zn–O bonds with different strengths. It appears thatthe differences in the crystalline structure of the samples I and II,which were confirmed by the XRD patterns resulted in the slightshift in the position of the characteristic peaks.

In FTIR method, absorption of some unwanted molecules such asH2O and CO2 from the air on KBr pellet occurs during its preparationor in the recording of the spectrum. These molecules show thespecific infrared bands. The peak at 3200–3600 cm�1is attributedto the –OH bond and the peak at 1632 cm�1 is referred to bending

was synthesized by ultrasonic horn, and (b) by ultrasonic bath.

Fig. 2. FT-IR analysis for the prepared samples; (a & b) point to mid-IR, and (a0 & b0) are attributed to the far-IR spectra of the samples I and II, respectively.

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mode of H2O. Presence of CO2 is characterized by a weak peak at2400 cm�1, which is due to the asymmetric stretch –CO bond.

3.3. TEM images

Fig. 3(a) and (b) show the morphology of the samples I and II,respectively. The images showed that the direct sonication pro-duces the flower-like with the sharp part at the end of the branches

Fig. 3. TEM images of ZnO prepared under the different conditions; (a) flower-like of theunder low intensity irradiation.

(needle form) while under indirect sonication, particles crystal-lized in a star appearance with the leaf like parts. From the mor-phological studies, some points could be concluded as follows:

� The presence of the small particles and the ordered crystalswith the large size, which could be detected in TEM imagesimplying that the Ostwald ripening process, happened duringthe particle growth. According to this phenomenon, small crys-tals tend to dissolve and deposit on the surface of the larger

sample I synthesized under high intensity, and (b) star-like of sample II fabricated

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particles. Indeed, the formation of the larger crystals by reduc-tion of the external surface leads to approach the lower energylevels. Therefore, this process happens spontaneously.� The irradiation under direct and indirect ways promoted the

crystallization in two different paths. In addition to the inequal-ity of the frequency of these two systems, the different types ofthe ultrasonic wave propagation through the reaction medium,created the different structures.� The comparison of the crystallographic structures of the sam-

ples I and II with the conventional sample (prepared withoutthe ultrasound and the TEM images are shown in theSupplementary data, Fig. 1S) confirmed that the primary nucle-ation was deeply affected by the presence of the ultrasonic irra-diation. Applying of ultrasonic energy led to make the clusterform of ZnO while in the conventional procedure the isolatednanorod particles were formed. This phenomenon was assignedto the cavitation. As it is known, the strong streams, includingshockwaves and microjets produced during the cavitation pro-cess agitate the medium in the effective way and increase thepossibility of the particle joining. Based on our results thegrowth mechanisms were proposed for both samples.

3.3.1. Growth mechanismsFor crystallization of the flower-like structure (sample I), two

possibilities could be considered:

i. Joining of the nuclei and generation of the symmetrical formin the primary steps of the growth process.

ii. Agglomeration of the grown particles and formation of theordered structure in the next steps of the crystallization.

As it is known, many examples could be found in the naturewith the symmetric patterns in the macro and even in the microlevels. It has been scientifically proven that the existence of thesepatterns is due to the possessing of more stability in symmetricconfigurations.

Concerning to the tendency of having the symmetry and obser-vation of the flower-like structure with the equal length branches,the first path seems more possible to happen. By this assumption,

Fig. 4. Schematic view of the possible mechanism for the formation of the flower-like strof the hexagonal wurtzite ZnO.

the probable mechanism for crystallization of the flower-like struc-ture could be illustrated as follows:

At first, the hexagonal prisms were considered as the primarynuclei of ZnO. As it is known, a hexagonal prism includes twohexagonal ‘‘basal’’ faces and six rectangular ‘‘prism’’ faces. Thisstructure has two polar planes consist of (001) face(zinc-terminated plane) and (00 �1) face (oxygen-terminatedplane). The (001) face, which belongs to the symmetry group(C6) has higher symmetry and growth occurs along it (c-axis) [21].

It could be assumed that some primary nuclei (such as six oreight nuclei) were joined. The extension along the c-axis of eachnucleus and the formation of the pyramid after this step resultedin the final structure that is similar to the shape of the sample I.By the comparison of the morphology of the conventional sample(synthesized under the static sate) and the sample I (producedunder the high intensity irradiation), it can be concluded that theaggregation or fusion of the ZnO nuclei in the sample I was dueto the transferring of the acoustic energy to the reaction medium.The implosive collapse of the bubble generates localized hot spotsthrough adiabatic compression or shock waves. Fusion of thesenuclei could be illustrated by the high temperature (�5000 K)and high pressure (�20 MPa) produced in the hot spots. Fig. 4 rep-resents a possible growth path for this state in a schematic view.

Under the low intensity irradiation, crystallization progressoccurred in another path. The final structure of the sample IImay be shaped by the particle coalescence. The alignment of theparticles created the flake-like pieces. These blocks were orientedand formed the low-energy configuration in the star-like (Fig. 5).As it is well known, the combination of the small particles andtheir agglomeration during and even at the end of the crystalliza-tion is due to the inherent tendency for reduction of the fee energyof the particles. The oriented attachment process occurred undercontinuous sonication by the ultrasonic bath is in agreement withthe results reported in the literature [19].

3.4. UV–vis analysis

The room temperature UV–vis absorption spectra (Fig. 6) showan exciton band at 370 nm for both samples. These absorption

ucture (sample I). The hexagonal prism was considered as a simple model describing

Fig. 5. Schematic representation showing attachment of the flake like pieces to form the star-like structure.

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peaks present a blue shift compared to the bulk exciton absorptionof ZnO (380 nm). This shift is due to the size effect of the nanos-tructures [22]. The sample I shows marginally higher UV absorp-tion in comparison to the sample II. Moreover, absorption in thevisible range of wavelength could be observed for this sample.This optical behavior could be attributed to the existence of thedefect energy levels in this structure [24].

Fig. 6. Comparison of the UV–vis absorption spectra for samples I and II dispersedin ethanol.

Fig. 7. Diagram of the growth inhibitory percentage for ZnO nanofluids and fluids(glycerol and ammonium citrate) in three different concentrations (‘X’ indicatesthat no growth inhibition of E. coli O157:H7 was observed).

3.5. Biological activity

Evaluation of antibacterial activity was performed for the fluidsand ZnO NFs with three concentrations of 0.375, 0.750, and1.500 mg mL�1. The growth of the treated groups was comparedto the positive control and the percentage of growth inhibition incomparison with the positive control was determined usingEq. (1):

Growth inhibitory percentage ¼ 100� OD630 for the test sampleOD630 for the positive sample

ð1Þ

The results of these measurements are represented graphically inFig. 7.

As can be seen from the graph, ZnO NFs in low concentration(0.375 mg mL�1) shows the low antibacterial activity (about 16%)while in high concentration (1.5 mg mL�1), in addition to the ZnONFs, the fluid shows strong antibacterial action alone. This behav-ior is assigned to the presence of the high concentration of ammo-nium citrate, which has biological activity toward E. coli [25]. In themedium concentration (0.750 mg mL�1), the values of the growthinhibitory percentage were 75% and 20% for the ZnO NFs and fluidrespectively. Therefore, this concentration was chosen as a propervalue for the colony count method.

Comparison of the antimicrobial activity of the samples I and IIwas carried out by using of colony count method and the recoveryof E. coli on the TSA plate is shown in Fig. 8.

The colonies were counted and the results shown as a graph(Fig. 9). The values of the colonies grown on the plate are about18% and 22% respect to the positive control (set as %) for the sam-ples I and II, respectively (this value for the conventional samplewas evaluated 21%). The reported data for each specimen is anaverage of three measurements.

Fig. 8. Recovery of viable E. coli cells on TSA plates after 24 h incubation; (A) sample I, (B) sample II, (C) fluid (including glycerol and ammonium citrate), and (D) positivecontrol.

Fig. 9. Antibacterial activity of the samples I and II at the concentration of0.750 mg mL�1, and combination of glycerol (Gly.) and ammonium citrate (A.C.) onE. coli O157:H7 in tryptone soy agar. Data are shown with mean values and standarderrors of bacterial counts.

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Many researchers have been conducted on the evaluation of theantibacterial activity of ZnO nanoparticles against a broad spec-trum of microorganisms. In addition, different groups have workedto find the mechanism of the biocidal activity of this antibacterialagent. For instance, the results of a study carried out into the role ofsize scale of ZnO nano- and micro-particles on toxicity to bacteriaand osteoblast cancer cells indicated that the ZnO nanoparticlesshow antibacterial activity apparently greater than microparticles[26]. On the other hand, another study claimed that the ranges ofdifferently sized ZnO powders (101–104 nm) exhibited the similarantibacterial activity and this precluded discerning the effect ofsize on toxicity [27]. In fact, the effect of some factors, includingtesting methods and physicochemical properties of the particlessuch as size, crystal structure, defects, surface energy, surfacecharge, surface morphology, surface area, solubility, and evenagglomeration state on the antibacterial activity of the species isthe reason behind these observations [28]. In the case of its mech-anism, the biological activity of ZnO is mostly attributed to thegeneration of the reactive oxygen species (ROS) and the existenceof H2O2 in the presence of ZnO has been affirmed [29]. However,the exact mechanism of this generation has not yet been clarified

because of zinc oxide shows this property even under dark condi-tions. Release of zinc ion, membrane dysfunction, and nanoparticleinternalization are other paths considered for the biological actionof this metal oxide [30].

4. Conclusion

In summary, sono-synthesis of ZnO NPs was carried out viahigh-efficiency and green approach under direct and indirect son-ication. Characterization of the resulting samples showed that thepure phases of ZnO NPs were synthesized with the high crystallinestructures. The low and high intensity irradiation led to make theparticles with different morphologies. The optical property of thesamples were investigated. The preparation of the 3D structureof ZnO NPs with high purity and good antibacterial activity con-firmed that the bath sonicator could be more attended as a usefulvessel for the synthesis processes of the materials.

Acknowledgements

The support of Ferdowsi University of Mashhad (Research andTechnology) for this work (3/21049, 08/03/2012) is appreciated.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ultsonch.2015.06.016.

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Direct and indirect sonication affect differently the microstructure and the morphology of ZnO nanoparticles: Optical behavior and its antibacterial activity1 Introduction2 Experimental2.1 Materials2.2 Preparation of Zn(OH)42−2.2.1 Direct method (method I)2.2.2 Indirect method (method II)

2.3 Preparation of nanofluids2.4 Antibacterial activity assay2.4.1 Absorption method2.4.2 Colony count method

2.5 UV–visible absorption

3 Results and discussion3.1 XRD analysis3.2 FTIR spectroscopy3.3 TEM images3.3.1 Growth mechanisms

3.4 UV–vis analysis3.5 Biological activity

4 ConclusionAcknowledgementsAppendix A Supplementary dataReferences