Research Article Curious Case of Bactericidal Action...

Research ArticleCurious Case of Bactericidal Action of ZnO

Somnath Ghosh12 R Gowri Sankar2 and V Vandana2

1 Solid State and Structural Chemistry Unit (SSCU) Indian Institute of Science (IISc) Bangalore 560012 India2Department of Chemistry Central Research Laboratory Gandhi Institute of Technology and Management (GITAM) UniversityRushikonda Visakhapatnam Andhra Pradesh 530045 India

Correspondence should be addressed to Somnath Ghosh ghoshmnthgmailcom

Received 1 July 2014 Revised 19 September 2014 Accepted 3 October 2014 Published 17 November 2014

Academic Editor Irshad Hussain

Copyright copy 2014 Somnath Ghosh et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

ZnO nanoparticles (NPs) are well known for their bactericidal properties Various mechanisms are proposed for their bactericidalactivity An ambiguity still prevails to know which mechanism or property is mainly influencing the bactericidal activity of ZnONPsThe antibacterial properties of ZnO NPs were investigated against both Gram-positive and Gram-negative bacteria DifferentZnO samples with different degrees of surface oxygen vacancies were prepared from ZnO

2 The surface oxygen vacancy and

thereby reactive oxygen species (ROS) production in aqueous ZnO solution are quantified by photoluminescence (PL) and electronparamagnetic resonance (EPR) spin trapping experiments respectively Systematic experiments have been performed to validate aprecise antibacterial mechanism of ZnO particle

1 Introduction

The rapid development of nanotechnology emerges in adiverse range of nanomaterials and nanoproducts [1] Variousdesired targets have been accomplished in order to employmaterials in medicinal fields by maneuvering them at theiratomic size scale [2 3] through nanotechnology Unfortu-nately many such benevolent materials develop toxicity Astoxicities are selective to biological systems nanomaterialsare well exploited for antibacterial applications To combatbacterial infections metal and metal oxide NPs in variousforms are well studied [4ndash15]

The exposure of such NPs in the environment demandsa fundamental understanding about their mode and rangeof toxicity Their mode or modes of action towards bacteriaremained ambiguous

ZnO NPs have been extensively used as antibacterialagents for water purification [16ndash19] biofilm prevention [20ndash22] sunscreen lotion [23 24] wound dressing [25] and soforth Protection against intestinal bacterial infections bybulk ZnO was reported in late 1990s [26 27] though itsbactericidal activity against a broad spectrumof bacteria (egStaphylococcus aureus Escherichia coliBacillus subtilis Strep-tococcus agalactiae etc) was revealed little late when ZnO

NPswere exposed to bacterial solution [28ndash32] Although theantimicrobial properties of ZnONPs have been utilized to killbacteria in different issues their mechanistic pathways arestill imprecise The mechanisms of antibacterial property ofZnO NPs so far proposed are as follows (i) physical attack ofZnO NPs on the bacteria [18 33 34] (ZnO NPs can adhereto the bacterial cell wall surface and eventually pierce intocell leading to bacterial death by membrane disruption) (ii)oxidative stress generated by particles in solutions [35 36](hydroxyl radials are the result of interaction of ZnO NPswith aqueous solution which causes oxidation of bacterialmetabolic enzyme leading to bacterial death) and (iii) sol-ubility of NPs in aqueous solution [37 38] But none of themis solely responsible on a ldquoone fits allrdquo basis for the desiredactivity as reported in the literature The above-mentioneddifferent mechanisms play a role in the bactericidal activitybecause of size surface oxygen vacancy and active surfacearea of the ZnO particles A careful investigation is requiredto evaluate the influence of each such property towards thetoxicity of bacteria

The antibacterial properties of ZnO NPs were investi-gated against both Gram-positive and Gram-negative bacte-ria During bactericidal activity study various properties (egparticle size solubility surface area etc) of ZnO samples

Hindawi Publishing CorporationJournal of NanoscienceVolume 2014 Article ID 343467 8 pageshttpdxdoiorg1011552014343467

2 Journal of Nanoscience

were maintained unaltered when varying the other (egreactive oxygen species (ROS) production surface defect)through simple chemical route to correlate the antibacterialactivity of ZnO with such property Similarly when sol-ubility of ZnO samples was varied ROS production wasarrested by glutathione (GSH) in order to eliminate orestablish the possibility of oxidative stress mechanism fortheir bactericidal action These systematic studies state thatbactericidal efficiency of ZnO is little higher for Gram-positive Staphylococcus aureus ATCC 25923 compared toGram-negative Escherichia coli MTCC 1302 It has beenfound that as the surface area increases the solubility of thesame sized different ZnO samples in saline water increasesand also the bactericidal activity Interestingly when ROSproduction was stopped by employing GSH bactericidalactivity for each of the samples decreases a little Whenthe amount of ROS production was raised by creatingmore surface defects in ZnO samples maintaining the samesolubility the bactericidal activity increases with the rise inROS production Surprisingly for these samples when ROSproduction in aqueous solution was blocked bactericidalactivity decreases compared towhenROSproductionwas notarrested But level of antibacterial activity prevails similarlywithin those samples after ROS production was blocked Thewhole understanding of all the observations says that each ofthese individual physicochemical properties of ZnO samplescontributes individually towards the killing of bacteria andnoticeably the contribution byROSproduction due to surfacedefect in account of bactericidal activity is the leadingpart

2 Experimental Section

21 Materials Zinc acetate dihydrate (S D Fine-ChemPvt Ltd India) diethylene glycol (Merck India) hydro-gen peroxide (30 S D Fine-Chem Pvt Ltd India)2101584071015840-dichlorofluorescein diacetate (DCFH-DA) glutathione(GSH) 55-dimethyl-1-pyrroline-N-oxide (DMPO) (SigmaAldrich Chemical Co Inc Germany) and nutrient broth(Hi-Media Laboratories Ltd India) were used as such with-out further purification Escherichia coli MTCC 1302 andStaphylococcus aureus ATCC 25923 (MS Ramaiah HospitalBangalore) were used for the bacterial studies

22 Synthesis of ZnO Submicron Particles Zinc acetate dihy-drate (09855 gm) was added to 45mL of diethylene glycol(DEG) with vigorous stirring for 05 h at room temperatureThe mixture was refluxed at 180∘C for 1 h similar to theprocedure described by Ghosh et al [18] A milky whiteprecipitate appeared at the end of the reaction indicatingthe formation of ZnO The obtained milky precipitate wascentrifuged separated and washed with ethanol severaltimes by repeated sonication-centrifugation process Theprecipitate so obtained was dried under vacuum at 60∘C for6 h and characterized by powder X-ray diffraction

23 Synthesis of ZnO2 The pristine ZnO powder was stirred

with 30mL of aqueous KOH (1M) solution for 2 h at room

temperature and the solution was then washed with waterand 50mL of H

2O2(30) was added and stirring continued

for 24 h at 45∘C The precipitate was washed with water anddried under vacuum and characterized by powder X-raydiffraction thermogravimetric study

24 Synthesis of Various ZnO Samples from ZnO2 The

obtained ZnO2was heated at 230∘C in air and also at 300∘C

400∘C and 500∘C under H2(5 H

2and 95 Ar) atmo-

sphere (flow rate 10mLmin) for 3 h Heated samples weredesignated as ZnO-H ZnO-300 ZnO-400 and ZnO-500respectively and characterized by powder X-ray diffraction

25 DCFH-DA Test A fluorescence experiment was carriedout using a sensitive probe 2101584071015840-dichlorofluorescein diac-etate (DCFH-DA) for the detection of ROS by incubatingthe samples in 30mL of 005 times 10minus3M DCFH-DA solutionfor 2 h The colourless DCFH-DA solution changes to greenupon exposure with samples and imparts a fluorescence peakat sim525 nm (excited at 485 nm) which indicates the samplesproduced ROS in aqueous solution

26 Suppression of ROSProduction fromAqueous ZnObyGSHTreatment Glutathione (120574-glutamylcysteinylglycine GSH)a sulfhydryl (ndashSH) antioxidant an antitoxin is sued forthe suppression of ROS as produced from aqueous ZnOsolution Being water soluble and having facile electrondonating power GSH first arrests ROS (ie hydroxyl radicalOH∙) by reducing them Various aqueous ZnO samples wereincubated with excess GSH till the solution became colorlessand hence nonfluorescent (ie no peak at sim525 nm whenexcited at 485 nm)

27 Minimum Inhibition Concentration (MIC) The MIC isthe lowest concentration at which a material exhibits antimi-crobial activity and this was done by serial dilution tech-niques Sterile test tubes were taken separately with 99mL ofsaline water inoculated with corresponding microorganisms(fresh culture mid-log phase OD600 sim0045ndash0050) anddiluted up to 108 CFUmLminus1 Different concentrations (5ndash200120583gmLminus1) of various ZnO samples were added to individ-ual test tubes After 4 h incubation at 37∘C each 01mL of thissolution was taken and plated in sterile nutrient agar platesand the plates were incubated at 37∘C overnight and colonieswere counted to determine the MIC

3 Instrumentation and Characterization

31 Powder X-Ray Diffraction (XRD) Powder XRD patternsof the preparedZnOwere recorded onPhilipsXRDldquoXrdquoPERTPRO diffractometer using Cu-K120572 radiation (120582 = 15438 A) asX-ray source

32 Inductively Coupled Plasma-Optical Emission Spectropho-tometry (ICPOES) The amount of ZnO dissolved in knownvolume of saline water for different samples was estimatedusing Perkin-Elmer optima 2100 ICPOES at 120582 = 2138 nm

Journal of Nanoscience 3

30

40

50

60

70

80

90

100

110ZnO TGA

Wei

ght (

)

Hea

t flow

(mw

)

50 100 150 200 250 300 350 400 450

Temperature (∘C)

minus2

minus1

0

1

2

3

4

5

6

ZnO2 DTA

ZnO2 TGA

(a)

(210

)

(201

)

(200

)

(201

)(1

12)

(200

)(1

03)

(110

)

(102

)

(110

)

(002

)

Inte

nsity

(au

)

(222

)(3

11)

(220

)

(211

)(200

)

(100

)(1

11)

(112

)

(103

)

(110

)

(102

)

(110

)

(002

)(1

00)

ZnO-500

ZnO-400ZnO-300

ZnO

ZnO-H

ZnO2

10 20 30 40 50 60 70 80

2120579 (deg)

ZnO2 (JCPDS 13-311)

ZnO (JCPDS 36-1451)

(b)

Figure 1 (a) TGA (black) and DTA (blue) plot of ZnO2 (b) XRD of ZnO

2and different ZnO samples

Zn(CH3COO)2 middot2H2ODEG ZnO H2O2 ZnO2

Δ

230∘C

300∘C

400∘C

500∘C

ZnO-H

ZnO-300

ZnO-400

ZnO-500

H2 3h

H2 3h

H2 3h

Scheme 1 Flow chart of conversion of ZnO to ZnO2and again to different ZnO samples

33 BET Surface Area Surface area of different samples wasdetermined by NOVA surface area analyzer

34 Scanning ElectronMicroscopy (SEM) Themorphologicalstudies of the prepared ZnOwere carried out in field emissionscanning electron microscope (FE-SEM SIRION) A dropof well-dispersed particles in water was cast onto a piece ofsilicon wafer and air-dried A thin gold coating was appliedto avoid charging during scanning and a detailedmicroscopicstudy was carried out

35 Electron Paramagnetic Resonance Spectroscopy (EPR)Hydroxyl radicals were detected by EPR-spin trapping tech-nique using spin trapper 55-dimethyl-1-pyrroline-N-oxide(DMPO 002M) Aqueous suspensions of different ZnOsamples were drawn separately into quartz capillaries (oneend closed) along with DMPO The capillaries were placedin the EPR tube and spectra were recorded on a Bruker emxX-band EPR spectrometer

36 Photoluminescence (PL) Study PL spectra were recordedin Jobin Yvon FluoroLog 4 (Horiba) by exciting each equallyconcentrated ZnO sample solution (aq) at 370 nm

37 Thermogravimetric Study Thermogravimetric studieswere done in thermogravimetric system (Cahn TG131)

4 Results and Discussion

In order to elucidate proper mechanism of antibacterialactivity ZnO samples with different degrees of physico-chemical properties have been synthesized through a simpleroute as depicted in Scheme 1 Submicron size ZnO particleswere synthesized by polyol method The as-synthesized ZnOparticles were converted to ZnO

2by reacting with H

2O2and

heated in presence of H2gas at different temperatures

In thismethod various degrees of surface defects (oxygenvacancies) were obtained without much change in particlenature between the samples The thermogravimetric analysis(TGA) and differential thermal analysis (DTA) of ZnO

2show

a sharp exothermic peak (Figure 1(a)) indicating decomposi-tion of ZnO

2to ZnO atsim225∘C and the reactionswere carried

out above this temperatureXRD patterns were obtained for all the samples and are

depicted in Figure 1(b) All the reflections were assignedwith standard wurtzite structure of ZnO (JCPDS file number36-1451) while for ZnO

2the peaks are assigned with pure

cubic phase of ZnO2(JCPDS file number 13-311) SEM

pictures obtained for the samples along with ZnO2are

shown in Figure 2 It reveals that the pristine ZnO particlesare spherical and the size varies between 150 and 250 nm(Figure 2(a)) On treatment with H

2O2 few smaller sized

particles disintegrated (Figure 2(b)) and the larger particles(size 150ndash250 nm) remain intact even after heating at differenttemperatures (Figures 2(c) 2(d) 2(e) and 2(f))


(a) (b)

(c) (d)

(e) (f)

Figure 2 SEM micrographs of (a) ZnO (b) ZnO2 (c) ZnO-H (d) ZnO-300 (e) ZnO-400 and (f) ZnO-500 (all scale bars 500 nm)

The physicochemical properties of ZnO samples weretabulated along withMICs as shown in Table 1 To investigatethe mechanism of antibacterial activity MICs were deter-mined with and without treating ZnO samples with GSH

From pristine ZnO to ZnO-H the surface area andsolubility increase which may be attributed to the smallerfragmentation on treatment withH

2O2and heating but both

solubility and surface area remain unaltered for other samplesof ZnO-300 to ZnO-500 (Figure 3(a) and Table 1)

When bactericidal activity was evaluated without GSHtreatment MICs for ZnO to ZnO-500 decrease (Table 1and Figure 3(b)) The surface area (and hence solubility) ofZnO to ZnO-H increases but EPR peak-area due to ROSproduction does not differ much Bactericidal activity ofZnO NPs increases along with increase in solubility reportedearlier [37 38] Thus the decrease in MIC for ZnO to ZnO-H could be accounted by the solubility factor But the sameargument does not fit to account the MIC values of ZnO-300


0

20

40

60

80

100

5

6

7

8

9

10Without GSH (S aureus)

ZnO-500ZnO-400ZnO-300ZnO-HZnO

MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(a)

0

20

40

60

80

100



MIC

(120583g m

Lminus1 )

EPR

peak

area

(au

)

times106

(b)

50

60

70

80

90

100

110

120

130

140

5

6

7

8

9

10


With GSH (S aureus)

MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(c)

0

10

20

30

40

50

60

70

80

90 ZnO-500ZnO-400ZnO-300ZnO-HZnO

Wavelength (nm)450 500 550 600 650

120582exct = 370nm

PL in

tens

ity (a

u)

times103

(d)


Magnetic field (Gauss)3310 3320 3330 3340 3350 3360 3370

minus30

minus15

00

15

30

Inte

nsity

(au

)

times106

(e)

0

20

40

60

80

100


EPR

peak

area

(au

)

4

6

8

10

12

14

16

18

20

22

PL p

eak

area

(au

)

times102

(f)

Figure 3 Plots of (a) MICs (without GSH) versus solubility (b) MICs (without GSH) versus EPR peak area and (c) MICs (with GSH) versussolubility (d) PL spectra (e) EPR spectra and (f) correlation plot of EPR peak area versus PL peak area of different ZnO samples

to ZnO-500 Solubility (Table 1) of ZnO-300 to ZnO-500 insaline was the same but MIC value decreases or bactericidalactivity increases from ZnO-300 to ZnO-500 So not onlythe solubility of ZnO samples but also the other factors areresponsible for its bactericidal activity

Similarly when bactericidal activity was determined afterGSH (ie without ROS production) treatment with ZnOsamples MIC values (Table 1 Figure 3(c)) for ZnO-300 toZnO-500 were almost the same indicating oxidative stressdue to the fact that ROS is the governing factor as other


Table 1 Different physicochemical properties and minimum inhibition concentration (MIC) values

Materials Surface area(m2g)

Solubility (120583gmLminus1) EPR peak area(au times 106)

S aureus E coliMIC (120583gmLminus1) MIC (120583gmLminus1)

Without GSH With GSH Without GSH With GSHZnO 2381 plusmn 023 640 plusmn 052 0501 plusmn 023 10166 plusmn 288 13333 plusmn 288 12500 plusmn 500 16833 plusmn 288ZnO-H 3751 plusmn 016 763 plusmn 023 0489 plusmn 066 7666 plusmn 288 9666 plusmn 288 9666 plusmn 288 9166 plusmn 288ZnO-300 4184 plusmn 089 838 plusmn 018 0791 plusmn 053 2833 plusmn 288 7133 plusmn 709 4333 plusmn 288 8833 plusmn 288ZnO-400 4023 plusmn 057 858 plusmn 015 1573 plusmn 033 1166 plusmn 288 6666 plusmn 288 2666 plusmn 288 9833 plusmn 288ZnO-500 4139 plusmn 063 837 plusmn 013 2089 plusmn 052 833 plusmn 288 6166 plusmn 288 1333 plusmn 288 10166 plusmn 288

factors like solubility and particle size remain unchangedfor ZnO samples The mechanism by which ROS (ie OH∙)formed on the ZnO surface is not clear although someinvestigations on the nature of the ZnO surface such aschemisorbed OH species after interaction with water pro-duces ROS have been reported [39]

In addition a little is known about the nature of defectsites on a wet metal oxide surfaceThe formation of ROSmaybe explained by assuming the formation of hydroxyl radicalsby the reaction of water and (dissolved) oxygen over basicmetal oxides [40]

1

2O2larrrarr Os

2H2O + 2Os

minuslarrrarr 2OH∙ + 2OHs

minus

2OHsminuslarrrarr H

2O + VO +Os

2minus

Os + VO +Os2minuslarrrarr 2Os

minus

The net reaction is 12O2+H2Olarrrarr 2OH∙

(1)

whereVO refers to an oxygen vacancy and ldquosrdquo refers to surfacespecies

The higher oxygen vacancy the higher ROS productionThis is further shown in the PL spectra of the samples(Figure 3(d))The green emission between 440 and 700 nm isdue to surface oxygen vacancy The increase in PL peak area(Figure 3(e)) starting from ZnO to ZnO-500 indicates theincrease in oxygen vacancy [41] When such areas are plottedagainst the corresponding ZnO samples this shows almost alinear increase and the shape of the curve matches well withthe EPR peak area as shown in Figure 3(f) This supports theabove proposed mechanism for the ROS production throughoxygen vacancy of ZnO

5 Conclusions

The method adopted for the synthesis of ZnO and ZnO2is

very simple and effective Different physicochemical prop-erties of ZnO have been varied to validate their influencingnature towards their bactericidal action When oxidativestress has been nullified by arresting OH∙ (hydroxyl) radicalwith GSH MICs of ZnO samples (from ZnO to ZnO-H)decrease with the increase of their surface area (particle size

remains the same) and solubilityThe effect of oxidative stresstowards bactericidal action has been considered (ie withoutGSH treatment) and the MICs of ZnO samples decreasewith the increase of the surface defects or ROS production(from ZnO-300 to ZnO-500) Throughout the experimentsparticles sizes of ZnO samples were constant and the effectdue to physical attack (ie direct interactions between theparticles and bacteria) is the same and cannot be avoidedBactericidal properties of ZnO are due to the combination ofall depicted mechanisms It can be concluded that out of allmechanisms oxidative stress developed in bacteria throughROS production of ZnO samples is the most influencingfactor for its bactericidal activity

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Theauthors thankNanoCentre (IISc Bangalore) for electronmicroscope and XRD facilityThe authors are indebted to DrH N Vasan for his valuable scientific suggestions

References

[1] V Biju ldquoChemical modifications and bioconjugate reactions ofnanomaterials for sensing imaging drug delivery and therapyrdquoChemical Society Reviews vol 43 no 3 pp 744ndash764 2014

[2] K M L Taylor-Pashow J Della Rocca R C Huxford andW Lin ldquoHybrid nanomaterials for biomedical applicationsrdquoChemical Communications vol 46 no 32 pp 5832ndash5849 2010

[3] S Ghosh R Kaushik K Nagalakshmi et al ldquoAntimicrobialactivity of highly stable silver nanoparticles embedded in agar-agar matrix as a thin filmrdquo Carbohydrate Research vol 345 no15 pp 2220ndash2227 2010

[4] S Ghosh A Saraswathi S S Indi S L Hoti and H NVasan ldquoAgAgI coreshell structure in agarose matrix ashybrid synthesis characterization and antimicrobial activityrdquoLangmuir vol 28 no 22 pp 8550ndash8561 2012

[5] N Gao Y Chen and J Jiang ldquoAgFe2O3-GO nanocomposites

prepared by a phase transfer method with long-term antibacte-rial propertyrdquo ACS Applied Materials and Interfaces vol 5 no21 pp 11307ndash11314 2013


[6] P Bober J Liu K S Mikkonen et al ldquoBiocompositesof nanofibrillated cellulose polypyrrole and silver nanopar-ticles with electroconductive and antimicrobial propertiesrdquoBiomacromolecules vol 15 no 10 pp 3655ndash3663 2014

[7] R Kumar S Anandan K Hembram and T N Rao ldquoEfficientZnO-based visible-light-driven photocatalyst for antibacterialapplicationsrdquo ACS Applied Materials amp Interfaces vol 6 no 15pp 13138ndash13148 2014

[8] S H Hwang J Song Y Jung O Y Kweon H Song and J JangldquoElectrospun ZnOTiO

2composite nanofibers as a bactericidal

agentrdquoChemical Communications vol 47 no 32 pp 9164ndash91662011

[9] J Manna G Begum K P Kumar S Misra and R K RanaldquoEnabling antibacterial coating via bioinspired mineralizationof nanostructured ZnO on fabrics under mild conditionsrdquo ACSApplied Materials and Interfaces vol 5 no 10 pp 4457ndash44632013

[10] M Safarpour A Khataee and V Vatanpour ldquoPreparationof a Novel Polyvinylidene Fluoride (PVDF) ultrafiltrationmembrane modified with reduced graphene oxidetitaniumdioxide (TiO

2) nanocomposite with enhanced hydrophilicity

and antifouling propertiesrdquo Industrial amp Engineering ChemistryResearch vol 53 no 34 pp 13370ndash13382 2014

[11] I Perelshtein G Applerot N Perkas J Grinblat and AGedanken ldquoA one-step process for the antimicrobial finishingof textiles with crystalline TiO

2nanoparticlesrdquo Chemistry A

European Journal vol 18 no 15 pp 4575ndash4582 2012[12] J Xiong Z Li J Chen S Zhang L Wang and S Dou

ldquoFacile synthesis of highly efficient one-dimensional plasmonicphotocatalysts throughAgCu

2Ocorendashshell heteronanowiresrdquo

ACS Applied Materials amp Interfaces vol 6 no 18 pp 15716ndash15725 2014

[13] X Zhang T Zhang J Ng and D D Sun ldquoHigh-performancemultifunctional TiO

2nanowire ultrafiltration membrane with

a hierarchical layer structure for water treatmentrdquo AdvancedFunctional Materials vol 19 no 23 pp 3731ndash3736 2009

[14] X Wang H-F Wu Q Kuang R-B Huang Z-X Xie andL-S Zheng ldquoShape-dependent antibacterial activities of Ag

2O

polyhedral particlesrdquo Langmuir vol 26 no 4 pp 2774ndash27782010

[15] A Simon-Deckers S Loo M Mayne-LrsquoHermite et al ldquoSize-composition- and shape-dependent toxicological impact ofmetal oxide nanoparticles and carbon nanotubes toward bac-teriardquo Environmental Science and Technology vol 43 no 21 pp8423ndash8429 2009

[16] H Koga T Kitaoka and H Wariishi ldquoIn situ synthesis ofsilver nanoparticles on zinc oxide whiskers incorporated in apapermatrix for antibacterial applicationsrdquo Journal of MaterialsChemistry vol 19 no 15 pp 2135ndash2140 2009

[17] M Li S Pokhrel X Jin L Madler R Damoiseaux and EM V Hoek ldquoStability bioavailability and bacterial toxicityof Zno and iron-doped Zno nanoparticles in aquatic mediardquoEnvironmental Science and Technology vol 45 no 2 pp 755ndash761 2011

[18] S Ghosh V S Goudar K G Padmalekha S V Bhat S S IndiandHN Vasan ldquoZnOAg nanohybrid synthesis characteriza-tion synergistic antibacterial activity and its mechanismrdquo RSCAdvances vol 2 no 3 pp 930ndash940 2012

[19] Z Huang X Zheng D Yan et al ldquoToxicological effect of ZnOnanoparticles based on bacteriardquo Langmuir vol 24 no 8 pp4140ndash4144 2008

[20] G Applerot J Lellouche N Perkas Y Nitzan A Gedankenand E Banin ldquoZnO nanoparticle-coated surfaces inhibit bac-terial biofilm formation and increase antibiotic susceptibilityrdquoRSC Advances vol 2 no 6 pp 2314ndash2321 2012

[21] F Gladis A Eggert U Karsten and R Schumann ldquoPreventionof biofilm growth onman-made surfaces evaluation of antialgalactivity of two biocides and photocatalytic nanoparticlesrdquoBiofouling vol 26 no 1 pp 89ndash101 2010

[22] B M Geilich and T J Webster ldquoReduced adhesion of Staphy-lococcus aureus to ZnOPVC nanocompositesrdquo InternationalJournal of Nanomedicine vol 8 pp 1177ndash1184 2013

[23] J W Rasmussen E Martinez P Louka and D G WingettldquoZinc oxide nanoparticles for selective destruction of tumorcells and potential for drug delivery applicationsrdquo ExpertOpinion on Drug Delivery vol 7 no 9 pp 1063ndash1077 2010

[24] M J Osmond and M J McCall ldquoZinc oxide nanoparticlesin modern sunscreens an analysis of potential exposure andhazardrdquo Nanotoxicology vol 4 no 1 pp 15ndash41 2010

[25] P T Sudheesh Kumar V-K Lakshmanan T V Anilkumar etal ldquoFlexible and microporous chitosan hydrogelnano ZnOcomposite bandages for wound dressing in vitro and in vivoevaluationrdquo ACS Applied Materials and Interfaces vol 4 no 5pp 2618ndash2629 2012

[26] M Jensen-Waern L Melin R Lindberg A Johannisson LPetersson and P Wallgren ldquoDietary zinc oxide in weanedpigsmdasheffects on performance tissue concentrations morphol-ogy neutrophil functions and faecal microflorardquo Research inVeterinary Science vol 64 no 3 pp 225ndash231 1998

[27] S X Huang M McFall A C Cegielski and R N KirkwoodldquoEffect of dietary zinc supplementation on Escherichia coli sep-ticemia inweaned pigsrdquo Journal of SwineHealth and Productionvol 7 no 3 pp 109ndash111 1999

[28] R Brayner R Ferrari-Iliou N Brivois S Djediat M FBenedetti and F Fievet ldquoToxicological impact studies based onEscherichia coli bacteria in ultrafineZnOnanoparticles colloidalmediumrdquo Nano Letters vol 6 no 4 pp 866ndash870 2006

[29] L Zhang Y Jiang Y Ding M Povey and D York ldquoInves-tigation into the antibacterial behaviour of suspensions ofZnO nanoparticles (ZnO nanofluids)rdquo Journal of NanoparticleResearch vol 9 no 3 pp 479ndash489 2007

[30] X Li Y Xing Y Jiang Y Ding and W Li ldquoAntimicrobialactivities of ZnO powder-coated PVC film to inactivate foodpathogensrdquo International Journal of Food Science and Technol-ogy vol 44 no 11 pp 2161ndash2168 2009

[31] C Karunakaran V Rajeswari and P Gomathisankar ldquoAntibac-terial and photocatalytic activities of sonochemically preparedZnO and Ag-ZnOrdquo Journal of Alloys and Compounds vol 508no 2 pp 587ndash591 2010

[32] N Jones B Ray K T Ranjit and A C Manna ldquoAntibacterialactivity of ZnO nanoparticle suspensions on a broad spectrumof microorganismsrdquo FEMS Microbiology Letters vol 279 no 1pp 71ndash76 2008

[33] I Sondi and B Salopek-Sondi ldquoSilver nanoparticles as antimi-crobial agent a case study on E coli as a model for Gram-negative bacteriardquo Journal of Colloid and Interface Science vol275 no 1 pp 177ndash182 2004

[34] G Appierot A Lipovsky R Dror et al ldquoEnhanced antibac-terial actiwity of nanocrystalline ZnO due to increased ROS-mediated cell injuryrdquoAdvanced FunctionalMaterials vol 19 no6 pp 842ndash852 2009

[35] A Lipovsky Z Tzitrinovich H Friedmann G Applerot AGedanken and R Lubart ldquoEPR study of visible light-induced


ros generation by nanoparticles of ZnOrdquo Journal of PhysicalChemistry C vol 113 no 36 pp 15997ndash16001 2009

[36] L K Adams D Y Lyon and P J J Alvarez ldquoComparative eco-toxicity of nanoscale TiO

2 SiO2 and ZnO water suspensionsrdquo

Water Research vol 40 no 19 pp 3527ndash3532 2006[37] W Bai Z Zhang W Tian et al ldquoToxicity of zinc oxide

nanoparticles to zebrafish embryo a physicochemical study oftoxicity mechanismrdquo Journal of Nanoparticle Research vol 12no 5 pp 1645ndash1654 2010

[38] M Li L Zhu and D Lin ldquoToxicity of ZnO nanoparticlesto Escherichia coli mechanism and the influence of mediumcomponentsrdquo Environmental Science amp Technology vol 45 no5 pp 1977ndash1983 2011

[39] G Sengupta H S Ahluwalia S Banerjee and S P SenldquoChemisorption ofwater vapor on zinc oxiderdquo Journal of ColloidAnd Interface Science vol 69 no 2 pp 217ndash224 1979

[40] K B Hewett L C AndersonM P Rosynek and J H LunsfordldquoFormation of hydroxyl radicals from the reaction of waterand oxygen over basic metal oxidesrdquo Journal of the AmericanChemical Society vol 118 no 29 pp 6992ndash6997 1996

[41] K Vanheusden C H Seager W L Warren D R Tallant and JA Voigt ldquoCorrelation between photoluminescence and oxygenvacancies in ZnO phosphorsrdquo Applied Physics Letters vol 68no 3 pp 403ndash405 1996

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Nano

materials


Journal ofNanomaterials


were maintained unaltered when varying the other (egreactive oxygen species (ROS) production surface defect)through simple chemical route to correlate the antibacterialactivity of ZnO with such property Similarly when sol-ubility of ZnO samples was varied ROS production wasarrested by glutathione (GSH) in order to eliminate orestablish the possibility of oxidative stress mechanism fortheir bactericidal action These systematic studies state thatbactericidal efficiency of ZnO is little higher for Gram-positive Staphylococcus aureus ATCC 25923 compared toGram-negative Escherichia coli MTCC 1302 It has beenfound that as the surface area increases the solubility of thesame sized different ZnO samples in saline water increasesand also the bactericidal activity Interestingly when ROSproduction was stopped by employing GSH bactericidalactivity for each of the samples decreases a little Whenthe amount of ROS production was raised by creatingmore surface defects in ZnO samples maintaining the samesolubility the bactericidal activity increases with the rise inROS production Surprisingly for these samples when ROSproduction in aqueous solution was blocked bactericidalactivity decreases compared towhenROSproductionwas notarrested But level of antibacterial activity prevails similarlywithin those samples after ROS production was blocked Thewhole understanding of all the observations says that each ofthese individual physicochemical properties of ZnO samplescontributes individually towards the killing of bacteria andnoticeably the contribution byROSproduction due to surfacedefect in account of bactericidal activity is the leadingpart

2 Experimental Section

21 Materials Zinc acetate dihydrate (S D Fine-ChemPvt Ltd India) diethylene glycol (Merck India) hydro-gen peroxide (30 S D Fine-Chem Pvt Ltd India)2101584071015840-dichlorofluorescein diacetate (DCFH-DA) glutathione(GSH) 55-dimethyl-1-pyrroline-N-oxide (DMPO) (SigmaAldrich Chemical Co Inc Germany) and nutrient broth(Hi-Media Laboratories Ltd India) were used as such with-out further purification Escherichia coli MTCC 1302 andStaphylococcus aureus ATCC 25923 (MS Ramaiah HospitalBangalore) were used for the bacterial studies

22 Synthesis of ZnO Submicron Particles Zinc acetate dihy-drate (09855 gm) was added to 45mL of diethylene glycol(DEG) with vigorous stirring for 05 h at room temperatureThe mixture was refluxed at 180∘C for 1 h similar to theprocedure described by Ghosh et al [18] A milky whiteprecipitate appeared at the end of the reaction indicatingthe formation of ZnO The obtained milky precipitate wascentrifuged separated and washed with ethanol severaltimes by repeated sonication-centrifugation process Theprecipitate so obtained was dried under vacuum at 60∘C for6 h and characterized by powder X-ray diffraction

23 Synthesis of ZnO2 The pristine ZnO powder was stirred

with 30mL of aqueous KOH (1M) solution for 2 h at room

temperature and the solution was then washed with waterand 50mL of H

2O2(30) was added and stirring continued

for 24 h at 45∘C The precipitate was washed with water anddried under vacuum and characterized by powder X-raydiffraction thermogravimetric study

24 Synthesis of Various ZnO Samples from ZnO2 The

obtained ZnO2was heated at 230∘C in air and also at 300∘C

400∘C and 500∘C under H2(5 H

2and 95 Ar) atmo-

sphere (flow rate 10mLmin) for 3 h Heated samples weredesignated as ZnO-H ZnO-300 ZnO-400 and ZnO-500respectively and characterized by powder X-ray diffraction

25 DCFH-DA Test A fluorescence experiment was carriedout using a sensitive probe 2101584071015840-dichlorofluorescein diac-etate (DCFH-DA) for the detection of ROS by incubatingthe samples in 30mL of 005 times 10minus3M DCFH-DA solutionfor 2 h The colourless DCFH-DA solution changes to greenupon exposure with samples and imparts a fluorescence peakat sim525 nm (excited at 485 nm) which indicates the samplesproduced ROS in aqueous solution

26 Suppression of ROSProduction fromAqueous ZnObyGSHTreatment Glutathione (120574-glutamylcysteinylglycine GSH)a sulfhydryl (ndashSH) antioxidant an antitoxin is sued forthe suppression of ROS as produced from aqueous ZnOsolution Being water soluble and having facile electrondonating power GSH first arrests ROS (ie hydroxyl radicalOH∙) by reducing them Various aqueous ZnO samples wereincubated with excess GSH till the solution became colorlessand hence nonfluorescent (ie no peak at sim525 nm whenexcited at 485 nm)

27 Minimum Inhibition Concentration (MIC) The MIC isthe lowest concentration at which a material exhibits antimi-crobial activity and this was done by serial dilution tech-niques Sterile test tubes were taken separately with 99mL ofsaline water inoculated with corresponding microorganisms(fresh culture mid-log phase OD600 sim0045ndash0050) anddiluted up to 108 CFUmLminus1 Different concentrations (5ndash200120583gmLminus1) of various ZnO samples were added to individ-ual test tubes After 4 h incubation at 37∘C each 01mL of thissolution was taken and plated in sterile nutrient agar platesand the plates were incubated at 37∘C overnight and colonieswere counted to determine the MIC

3 Instrumentation and Characterization

31 Powder X-Ray Diffraction (XRD) Powder XRD patternsof the preparedZnOwere recorded onPhilipsXRDldquoXrdquoPERTPRO diffractometer using Cu-K120572 radiation (120582 = 15438 A) asX-ray source

32 Inductively Coupled Plasma-Optical Emission Spectropho-tometry (ICPOES) The amount of ZnO dissolved in knownvolume of saline water for different samples was estimatedusing Perkin-Elmer optima 2100 ICPOES at 120582 = 2138 nm


30

40

50

60

70

80

90

100

110ZnO TGA

Wei

ght (

)

Hea

t flow

(mw

)

50 100 150 200 250 300 350 400 450

Temperature (∘C)

minus2

minus1

0

1

2

3

4

5

6

ZnO2 DTA

ZnO2 TGA

(a)

(210

)

(201

)

(200

)

(201

)(1

12)

(200

)(1

03)

(110

)

(102

)

(110

)

(002

)

Inte

nsity

(au

)

(222

)(3

11)

(220

)

(211

)(200

)

(100

)(1

11)

(112

)

(103

)

(110

)

(102

)

(110

)

(002

)(1

00)

ZnO-500

ZnO-400ZnO-300

ZnO

ZnO-H

ZnO2

10 20 30 40 50 60 70 80

2120579 (deg)

ZnO2 (JCPDS 13-311)

ZnO (JCPDS 36-1451)

(b)




Δ

230∘C

300∘C

400∘C

500∘C

ZnO-H

ZnO-300

ZnO-400

ZnO-500

H2 3h

H2 3h

H2 3h









2by reacting with H

2O2and



2show












(a) (b)

(c) (d)

(e) (f)








0

20

40

60

80

100

5

6

7

8

9



MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(a)

0

20

40

60

80

100



MIC

(120583g m

Lminus1 )

EPR

peak

area

(au

)

times106

(b)

50

60

70

80

90

100

110

120

130

140

5

6

7

8

9

10


With GSH (S aureus)

MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(c)

0

10

20

30

40

50

60

70

80


Wavelength (nm)450 500 550 600 650

120582exct = 370nm

PL in

tens

ity (a

u)

times103

(d)



minus30

minus15

00

15

30

Inte

nsity

(au

)

times106

(e)

0

20

40

60

80

100


EPR

peak

area

(au

)

4

6

8

10

12

14

16

18

20

22

PL p

eak

area

(au

)

times102

(f)












1

2O2larrrarr Os

2H2O + 2Os


minus

2OHsminuslarrrarr H

2O + VO +Os

2minus


minus


(1)



5 Conclusions






Acknowledgments


References



























2O








































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




30

40

50

60

70

80

90

100

110ZnO TGA

Wei

ght (

)

Hea

t flow

(mw

)

50 100 150 200 250 300 350 400 450

Temperature (∘C)

minus2

minus1

0

1

2

3

4

5

6

ZnO2 DTA

ZnO2 TGA

(a)

(210

)

(201

)

(200

)

(201

)(1

12)

(200

)(1

03)

(110

)

(102

)

(110

)

(002

)

Inte

nsity

(au

)

(222

)(3

11)

(220

)

(211

)(200

)

(100

)(1

11)

(112

)

(103

)

(110

)

(102

)

(110

)

(002

)(1

00)

ZnO-500

ZnO-400ZnO-300

ZnO

ZnO-H

ZnO2

10 20 30 40 50 60 70 80

2120579 (deg)

ZnO2 (JCPDS 13-311)

ZnO (JCPDS 36-1451)

(b)




Δ

230∘C

300∘C

400∘C

500∘C

ZnO-H

ZnO-300

ZnO-400

ZnO-500

H2 3h

H2 3h

H2 3h









2by reacting with H

2O2and



2show












(a) (b)

(c) (d)

(e) (f)








0

20

40

60

80

100

5

6

7

8

9



MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(a)

0

20

40

60

80

100



MIC

(120583g m

Lminus1 )

EPR

peak

area

(au

)

times106

(b)

50

60

70

80

90

100

110

120

130

140

5

6

7

8

9

10


With GSH (S aureus)

MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(c)

0

10

20

30

40

50

60

70

80


Wavelength (nm)450 500 550 600 650

120582exct = 370nm

PL in

tens

ity (a

u)

times103

(d)



minus30

minus15

00

15

30

Inte

nsity

(au

)

times106

(e)

0

20

40

60

80

100


EPR

peak

area

(au

)

4

6

8

10

12

14

16

18

20

22

PL p

eak

area

(au

)

times102

(f)












1

2O2larrrarr Os

2H2O + 2Os


minus

2OHsminuslarrrarr H

2O + VO +Os

2minus


minus


(1)



5 Conclusions






Acknowledgments


References



























2O








































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) (b)

(c) (d)

(e) (f)








0

20

40

60

80

100

5

6

7

8

9



MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(a)

0

20

40

60

80

100



MIC

(120583g m

Lminus1 )

EPR

peak

area

(au

)

times106

(b)

50

60

70

80

90

100

110

120

130

140

5

6

7

8

9

10


With GSH (S aureus)

MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(c)

0

10

20

30

40

50

60

70

80


Wavelength (nm)450 500 550 600 650

120582exct = 370nm

PL in

tens

ity (a

u)

times103

(d)



minus30

minus15

00

15

30

Inte

nsity

(au

)

times106

(e)

0

20

40

60

80

100


EPR

peak

area

(au

)

4

6

8

10

12

14

16

18

20

22

PL p

eak

area

(au

)

times102

(f)












1

2O2larrrarr Os

2H2O + 2Os


minus

2OHsminuslarrrarr H

2O + VO +Os

2minus


minus


(1)



5 Conclusions






Acknowledgments


References



























2O








































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




0

20

40

60

80

100

5

6

7

8

9



MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(a)

0

20

40

60

80

100



MIC

(120583g m

Lminus1 )

EPR

peak

area

(au

)

times106

(b)

50

60

70

80

90

100

110

120

130

140

5

6

7

8

9

10


With GSH (S aureus)

MIC

(120583g m

Lminus1 )

Solu

bilit

y (120583

g mLminus

1 )

(c)

0

10

20

30

40

50

60

70

80


Wavelength (nm)450 500 550 600 650

120582exct = 370nm

PL in

tens

ity (a

u)

times103

(d)



minus30

minus15

00

15

30

Inte

nsity

(au

)

times106

(e)

0

20

40

60

80

100


EPR

peak

area

(au

)

4

6

8

10

12

14

16

18

20

22

PL p

eak

area

(au

)

times102

(f)












1

2O2larrrarr Os

2H2O + 2Os


minus

2OHsminuslarrrarr H

2O + VO +Os

2minus


minus


(1)



5 Conclusions






Acknowledgments


References



























2O








































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials











1

2O2larrrarr Os

2H2O + 2Os


minus

2OHsminuslarrrarr H

2O + VO +Os

2minus


minus


(1)



5 Conclusions






Acknowledgments


References



























2O








































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials























2O








































CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



Research Article Curious Case of Bactericidal Action...

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Transcript of Research Article Curious Case of Bactericidal Action...