Insight into the Mechanism of Antibacterial Activity of ZnO: Surface

Jul 29, 2015 - Sandeep K. Sehmi , Sacha Noimark , Sebastian D. Pike , Joseph C. Bear , William J. Peveler , Charlotte K. Williams , Milo S. P. Shaffer...
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Insight into the Mechanism of Antibacterial Activity of ZnO: Surface Defects Mediated Reactive Oxygen Species Even in the Dark V. Lakshmi Prasanna and Rajagopalan Vijayaraghavan* Centre for Excellence in Nano Materials, Materials Chemistry Division, School of Advanced Sciences, VIT University, 632 014 Vellore, India S Supporting Information *

ABSTRACT: A systematic and complete antibacterial study on well-designed and well-characterized microparticle (micro), nanoparticle (nano), and capped nano ZnO has been carried out in both dark and light conditions with the objective of arriving at the mechanism of the antibacterial activity of ZnO, particularly in the dark. The present systematic study has conclusively proved that reactive oxygen species (ROS) such as •OH, •O2−, and H2O2 are significantly produced from aqueous suspension of ZnO even in the dark and are mainly responsible for the activity in the dark up to 17%, rather than Zn2+ ion leaching as proposed earlier. This work further confirms that surface defects play a major role in the production of ROS both in the presence and absence of light. In the dark, superoxide (•O2−) radical mediated ROS generation through singly ionized oxygen vacancy is proposed for the first time, and it is confirmed by EPR and scavenger studies. ROS such as •O2−, H2O2, and •OH have been estimated by UV−visible spectroscopy using nitro blue tetrazolium (NBT), KMnO4 titrations, and fluorescence spectroscopy, respectively. These are correlated to the antibacterial activity of ZnO in the dark and light. The activity is found to be highest for nano ZnO and least for micro ZnO, with capped ZnO between the two, highlighting the important role of surface defects in generation of ROS. The surface charge density of ZnO in dark and light has been estimated for the first time to the best of our knowledge, and it can influence antibacterial activity. Our work proposes a new mechanism mediated by superoxide species, for antibacterial activity of ZnO especially in the dark.

1. INTRODUCTION Engineered nanoparticles (ENPs) due to their enhanced properties find potential applications in various fields such as optoelectronics, energy, sensors, drug delivery, and medical imaging.1−4 ENPs based on inorganic metal oxides are extensively investigated in the field of inorganic antibacterials recently and their activities are easily tunable by control over their particle size, morphology and crystal defects through appropriate synthesis methods.5,6 Metal oxides such as CuO, TiO2, ZnO, Al2O3, SiO2, Fe2O3, and CeO2 are exploited as antibacterials. Li et al. proved that TiO2 and ZnO are relatively efficient antibacterials compared to the others.7 ZnO is regarded as a generally regarded as safe material (GRASM) by the U.S Food and Drug Administration (21 CFR 182.8991).8 In addition to antibacterials, ZnO is proven to be promising materials for photocatalysts, photovoltaic cells, biochemical sensors, food packaging, and anticancer drugs.8−10 Due to its biocompatibility, safety, and activity against a broad spectrum of microorganisms, ZnO’s antibacterial activity has been well-investigated both in the presence (UV and visible) and absence of light.11−17 Reactive oxygen species (ROS) production and antibacterial activity of ZnO are found to be higher under UV light than in visible light.7 Nanoparticle (nano) ZnO has been proved be to more efficient © 2015 American Chemical Society

than microparticle (micro) ZnO due to more surface defects in the former and its high surface area which facilitates higher contact between nanomaterials and microbes.11,12 The mechanism of antibacterial activity of ZnO in the presence of light, studied by many researchers, is attributed to oxidative stress due to ROS,7,12,13 dissolution of zinc ions,14 and internalization of NPs leading to cell death.15 Among these, ROS is considered to be the dominant mechanism. ZnO, especially, nano ZnO, exhibits significant antibacterial activity in the dark also, although lower than that in the presence of light.17−19 The mechanism of antibacterial activity in the dark is mainly attributed to leaching of zinc ions from ZnO suspension which enter the cells damaging DNA and causing cell death.17−19 But Lipovsky et al. observed hydroxyl radical production in aqueous suspensions of nano ZnO even in the dark evidenced by ESR spectroscopy, but the mechanism of hydroxyl production was not discussed.20 Xu et al. also proved that H2O2 is generated form aqueous suspensions of ZnO even in the dark that can cause oxidative stress resulting in antibacterial activity, but the mechanism of generation of Received: December 24, 2014 Revised: July 29, 2015 Published: July 29, 2015 9155

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Langmuir H2O2 was not investigated.21 So understanding the origin of generation of ROS in ZnO in the dark is essential and in turn correlating its antibacterial activity in the dark. This may lead to design of novel antibacterial agents based on engineered oxides which may find potential applications as coatings in food packaging where light is absent. It is to be noted that nanostructures of ZnO may also influence the activity.19 Toward this end, we have synthesized and characterized micro ZnO), nano ZnO, and oxalic acid (OA) capped ZnO and studied systematically their antibacterial activity in the presence of ambient light and dark, also in the presence of a scavenger of ROS. Staphylococcus aureus (S. aureus), Gram-positive bacteria, is chosen as the model bacteria as it contains a very thick cell wall consisting of mucopeptides and lipoteichoic acids and also carotenoid compounds which assist in acquiring resistance to oxidative stress.13 Our results conclusively prove that ROS such as •OH, H2O2, •O2−, and •HO2 are produced even in the dark in defective nano ZnO measurable by simple methods and are mainly responsible for its antibacterial activity rather than Zn2+ ions leaching as proposed earlier.14,17,19 For the first time, we have proposed a mechanism involving superoxide species, especially in nano ZnO, operating to generate ROS in ZnO suspensions. These ROS are aided by surface defects of ZnO which are abundant in nanocrystalline form. We also provide evidence for the generation of ROS such as •OH, H2O2, and • O2−. Surface charge density of ZnO measured for the first time by acid base titrations is found to be different in light and dark which could affect the interaction between bacteria and nanoparticles.

resonance spectroscopy (EPR) was recorded using a BRUKER N500 EPR spectrometer. Antibacterial Activity of ZnO. Antibacterial activity of synthesized ZnO was studied against S. aureus (ATCC 25923). A 100 μL aliquot of S. aureus was added to sterilized Luria−Bertani (LB) broth and incubated overnight at 37 °C. The concentration of cells was estimated to be 3 × 1012 CFU/mL counting. To 0.1 g/L ZnO in LB broth, 100 μL of overnight culture was added and stirred on an orbital shaker. At different time intervals up to 2 h, 100 μL of broth was taken and serially diluted, plated on agar plates, and incubated at 37 °C overnight, and colonies were counted. Antibacterial activity was carried out in dark and ambient light. Zn2+ Dissolution from ZnO. Aqueous suspension of ZnO was stirred at room temperature. At regular intervals, 5 mL aliquots were drawn out and centrifuged at 8000 rpm and supernatant liquid was filtered with membrane (Anapore). Zn2+ ions leached out in the filtrate were estimated by AAS calibrated with standard Zn2+ solutions. Estimation of H2O2. H2O2 generated from aqueous ZnO suspensions was estimated by KMnO4 redox titrations.24 To aqueous suspensions of ZnO (0.1 g/L), 2 mL each of KMnO4 and H2SO4 at appropriate concentrations were added, kept under constant stirring at room temperature under ambient light and dark. At regular intervals, 5 mL aliquots were filtered through a membrane filter. H2O2 was estimated by a standard titration using a known amount of H2O2. Estimation of Hydroxyl Radical (•OH). Hydroxyl radicals were estimated using fluorescence spectroscopy. Terephthalic acid (TA) with hydroxyl radicals forms a 2-hydroxyl terephthalic acid complex which gives fluorescence, and its intensity is a direct measure of hydroxyl radical concentration.21 In a typical procedure, to aqueous suspensions of ZnO (0.1 g/L), 2 mM of TA was added and stirred under ambient light. At regular intervals, 3 mL aliquots were withdrawn and filtered through a membrane filter and fluorescence was measured at excitation wavelength of 312 nm. The intensity of emission at 425 nm is correlated to the hydroxyl radical concentration. The experiment was performed in the dark also. Estimation of Superoxide •O2−. Superoxide radicals from aqueous suspensions of ZnO were estimated by nitro blue tetrazolium (NBT).25 NBT shows a maximum absorbance at 259 nm, but with superoxide radicals, it will be converted to monoformazan and diformazan. The production of superoxide radicals was estimated by monitoring the degradation of NBT using UV−visible spectroscopy. This experiment was carried out both in ambient light and dark. Detection of OH• and •O2− by EPR. EPR spectroscopy was employed to detect ROS using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trap.20,26 DMPO traps OH• to form DMPO− OH which gives a quadrant signal. It also traps •O2− to form DMPO− OOH which is unstable and decomposes to DMPO−OH adduct. DMPO was purified in water with activated charcoal in the dark, and the solution was filtered and used for the experiment. To aqueous suspension of ZnO, DMPO was added and the EPR spectrum was recorded. The preceding experiments are performed both in light and dark. Surface Charge Density by Acid−Base Titration. In aqueous solutions of ZnO due to protonation/deprotonation of surface hydroxyl groups, charges are produced on the metal oxide surface. This charge developed on metal oxide can be measured as a function of pH by acid−base titrations.27,28 ZnO suspended in 50 mL of 0.1 M NaNO3 and 70 μL of 2.5 mM NaOH was added to the suspension, and excess NaOH was titrated with standardized HNO3.

2. EXPERIMENTAL SECTION Synthesis of Nanocrystalline ZnO and Oxalic Acid Capped ZnO. Zn(CH3COO)2·2H2O (1 M) and KOH (2 M) were dissolved in methanol separately. KOH solution was added slowly to zinc acetate, the mixture was refluxed at 50 °C under stirring for 1 h, and the precipitate was filtered and dried at 50 °C to obtain the products.22,23 OA capped ZnO was synthesized using OA as capping agent using the same procedure. Synthesis of Micrometer Sized ZnO. Zn(NO3)2·6H2O was dissolved in water, oven-dried at 100 °C, and then calcined at 300 °C for 1 h. Characterization. The phase purity and crystallite size of the synthesized products were analyzed by Bruker D8 Advance powder Xray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) with Cu Kα source. The morphology and particle size of the synthesized products were examined using transmission electron spectroscopy (TEM) and field emission electron microscopy (FE-SEM). TEM has been recorded employing a JEOL JEM 3010 electron microscope (JEOL Ltd.,Tokyo, Japan) using a copper grid in which is dispersed metal oxide suspension. FESEM was recorded using a 6701F instrument from JEOL. Room temperature photoluminescence spectrum (PL) was recorded using a Hitachi F-7000 fluorescence spectrophotometer with 150 W Xe lamp as excitation source. The slit widths at excitation and emission are 5 nm. X-ray photoelectron spectroscopic analysis was done using a K-Alpha instrument (XPS KAlpha surface analysis, Thermo Fisher Scientific, U.K.). Binding energy correction due to charging was made with respect to a C 1s (graphitic carbon) peak calibrated at 284.5 eV, and deconvolution of spectra was done using CASAXPS. UV−visible spectra were recorded at room temperature using a Jasco V 570 UV−vis spectrophotometer. Zinc ion concentration leached out from aqueous suspensions of ZnO was estimated using atomic absorption spectroscopy (AAS; Varian AA 240). FTIR spectra were recorded with a Shimadzu IR Affinity-1 spectrometer for capped ZnO products. X-band electron paramagnetic

σ0 =

F C T(Va − Vb) mSBET

(1)

where σ0 is the surface charge (C/m 2), F is the Faraday constant, C T is the concentration of titrant, m is the mass of the metal oxide, S is the surface area, and V a and V b are the titrant volumes added at a given pH for the suspension and for the blank titration (titration without metal oxide), respectively. The preceding experiment was performed for the aqueous suspensions of ZnO both in light and dark. 9156

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Figure 1. PL of (a) micro ZnO, (b) nano ZnO, (c) OA capped ZnO, and (d) O 1s XPS spectrum of nanocrystalline ZnO.

Figure 2. EPR of (a) nano ZnO in dark (black line), OA capped ZnO in dark (red line), and nano ZnO in light (blue line). (b) micro ZnO.

3. RESULTS AND DISCUSSION Characterization. Micro ZnO, nano ZnO, and oxalic acid capped nano ZnO synthesized have been confirmed to be single phase by XRD (Supporting Information Figure S1) with lattice parameters a ∼ 3.229 Å and c ∼ 5.225 Å. TEM (Figure S2a,b) and FESEM (Figure S3a,b) show that micro ZnO is of irregular shape with size distribution of length ranging from 300 to 550 nm and width from 175 to 200 nm and nano ZnO is nearly spherical particles with size ranging from 17 to 30 nm. OA capping was confirmed by FTIR spectroscopy (Figure S4). Parts a−c of Figure 1 give the photoluminescence spectra of ZnO. Micro ZnO (Figure 1a) shows only UV luminescence at 380 nm (3.26 eV) and negligible visible emission, whereas nano ZnO (Figure 1b) shows UV luminescence at 387 nm (3.20 eV)29 and broad visible luminescence at 506 nm (2.4 eV)30 due to oxygen vacancies. On capping, remarkable reduction in visible emission has been observed (Figure 1c) indicating that

capping blocks the vacancies. Figure 1d shows O 1s spectra of nano ZnO. XPS analysis shows that at O 1s the peak is asymmetric and can be deconvoluted into three peaks. The peak at 530.7 eV can be attributed to lattice oxygen, O2−, 532.1 eV corresponds to surface hydroxyl groups bound to oxygen vacancies or defects, and 533.4 eV corresponds to adsorbed water.21,31 Parts a and b of Figure 2 show X band EPR of nanocrystalline ZnO and capped ZnO in both light and dark. The signal at g = 2.002 corresponds to single ionized oxygen vacancies on the surface, whereas at g = 1.958 it corresponds to bulk species.32,33 Figure 2a confirms the presence of singly ionized oxygen vacancy even in the dark, and in the presence of visible light, enhancement in signals at g = 2.0002 is observed depicting increasing ionized oxygen species, whereas on capping the signal corresponding to g = 2.002 decreases confirming that singly ionized oxygen vacancy on the surface is less than nano ZnO (Figure 2a) . Micro ZnO (Figure 2b) 9157

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Figure 3. S. aureus inactivation by micro, nano, and capped nano ZnO: (a) dark; (b) visible.

Table 1. First Order Inactivation Rates and Antibacterial Efficiency of Different ZnO Products under Light and Dark dark composition nano ZnO OA capped ZnO micro ZnO

k (×10

−3

−1

min )

0.7 0.3 0.02

light

antibacterial efficacy (±2%) 17 9 nil

−3

k (×10

−1

min )

4.2 2.6 0.16

antibacterial efficacy (±2%) 80 48 3.8

Figure 4. Fluorescence spectra of hydroxyl terepthalic acid in aqueous suspensions of ZnO: (a) dark; (b) visible.

ZnO in the dark have mainly attributed it to Zn2+ ion dissolution or ZnO internalization14−18 In order to ascertain the effect of Zn2+ ions from ZnO, we estimated the concentrations of zinc ions leached out from aqueous suspensions of nano and micro ZnO of similar concentrations in LB broth, devoid of bacteria (stirred in dark and light for 2 h, pH = 8.5) through AAS, and the antibacterial activity of supernatant filtrate was estimated. The amounts of Zn2+ ions leached out from nano and micro ZnO after 2 h were found to be 10 and 2 ppm (Figure S5), respectively, indicating that nano ZnO leaches out more Zn2+ ions than micro ZnO.35 The antibacterial activity of supernatant filtrate (due to leached out Zn2+ ions from nano ZnO) in the dark and ambient light is found to be around 5% and negligible for micro ZnO. This study proves that zinc ion leaching plays only a minor role in antibacterial activity. Activity due to ZnO internalization should be insignificant as ZnO nano particles are negatively charged and hence cannot enter the negatively charged bacteria. ZnO seen within (Figure S6) the bacteria is due to ZnO particles embedded within bacteria after the cell membrane is damaged

shows a signal at g = 1.958 and no signal at g = 2.002 showing the absence of surface oxygen vacancies. Antibacterial Activity. Parts a and b of Figure 3 show the kinetics of antibacterial activity of micro, nano, and capped nano ZnO in the presence of dark and light, respectively, analyzed through the Chick−Watson model.34 The kinetics of inactivation was analyzed through the relation log(Nt /N0) = −kt

(2)

N0 is the initial S. aureus population, Ntis the population of S. aureus at time t, and k is the inactivation constant which is a direct measure of antibacterial activity. The results are tabulated in Table 1. In the light, nano ZnO exhibits 80% activity higher than capped ZnO (48%) but remarkably higher than micro ZnO (3.8%) (Table 1). The rate of inactivation of nano ZnO is much higher than that of micro ZnO (Table 1). In the dark, nano ZnO shows 17% efficacy, higher than micro and capped nano ZnO (Table 1), which we have investigated in detail, for the first time, as earlier reports on bactericidal effect of nano 9158

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documented.38−40 The electron−hole pair is generated from the semiconductor ZnO by excitation of electrons from the appropriate band levels through absorption of light (UV or visible) , and these electrons−holes produce ROS on interaction with water. ROS generation in light is given in Scheme 1. Surface oxygen vacancy may trap the photoexcited electrons, thus preventing recombination but increasing the production of ROS in nano ZnO.41 Capping reduces vacancies, causing relatively facile recombination, thus decreasing ROS. Micro ZnO devoid of surface defects generate the least amount of ROS. We propose the following mechanism for the production of ROS in the dark, especially in nano ZnO, involving superoxide species facilitated by surface defects (singly ionized oxygen vacancy) for the first time.

by ROS. It automatically leads us to conclude that there should be another dominant mechanism to operate in nano ZnO which exhibits bactericidal efficacy of 17% even in the dark, in addition to 5% activity attributed to Zn2+ leaching. To understand this better, we explored the possible generation of ROS mediated by defects in the dark in detail and hence studied the antibacterial activity of ZnO in the dark. Indeed, ROS release facilitated by defects was observed. If it were to exist, the activity of capped ZnO is expected to be less than that of uncapped nano ZnO in the dark also, due to a decrease in vacancy concentration in the latter and it was experimentally confirmed. We observed that the bactericidal efficacy is reduced to 9% in capped ZnO, a remarkable decrease from 17% in uncapped ZnO (Table 1). Micro ZnO showed negligible activity (Table 1) evidencing those effects of material parameters such as oxygen vacancy, particle size effect, and zinc ions leaching are absent in micro ZnO. Hence, for a given material, the preceding factors play a significant role in antibacterial activity both in light and dark. It is to be noted that S. aureus has transmembrane proteins that regulate the influx and out flux of zinc ions36,37 as well. Damage in the cell wall caused by ROS leading to the expulsion of cellular components was observed leading to cell death (Figure S6). In addition to S. aureus we have performed experiments on Escherichia coli (E. coli; ATCC 25922) and Pseudomonas aeruginosa (P. aeruginosa; ATCC 25619) also. In the dark, nano ZnO shows 25% activity against the former while the activity against the latter is 19%. Estimation of Reactive Oxygen Species. ROS responsible for antibacterial activity of nano ZnO, capped nano ZnO, and micro ZnO in both light and dark are estimated through fluorescence study, and in Figure 4a,b, we show the typical fluorescence spectra of nano ZnO in dark and visible light, respectively. Significant fluorescence intensity was observed within 5 min even in the dark (Figure 4a) depicting the spontaneous production of hydroxyl radical; the intensity also increases with time, indicting that more hydroxyl radicals are produced with time (Figure S7). As expected, hydroxyl radical production is much higher in light than in the dark. (Figure 4b). Table 2 lists the concentration of ROS produced from micro, nano, and capped ZnO in both visible light and the dark. In

Scheme 1: Mechanism of ROS Generation in Dark O2 + e− → •O2− •

dark composition

OH• (±0.01 ppm)

H2O2 (±0.2 ppm)

nano ZnO micro ZnO

0.95 N.D.

1.25 N.D.

8.6 0.9

14 0.64

a

−

(4)

•

HO2 + HO2 → H 2O2 •

−

(5) •

H 2O2 + O2 → O2 + OH + OH

−

(6)

EPR of nano ZnO in the dark (Figure 2a) shows the presence of singly ionized oxygen vacancy, i.e., oxygen vacancy with one electron. Oxygen from the atmosphere can react with an electron from the ZnO surface to form a superoxide radical (step 3) as reported in a sensor study42 Superoxide in water solvates to form hydroperoxyl radical43 (step 4) and hydroperoxyl radicals can recombine to form H2O2 (step 5). H2O2 may react with superoxide radical to form hydroxyl radical and hydroxyl ion44 (step 6). Singlet oxygen formation is not possible in the dark as a hole is required for its formation which will not be produced in the dark.31 Superoxide species, one of the ROS, responsible for antibacterial activity especially in the dark, has been investigated by NBT degradation study, spin trap ESR study using DMPO, and SOD scavenging study in order to elucidate its role in the antibacterial activity. Parts a and b of Figure 5 depict the degradation of NBT for nano ZnO in visible light and the dark, respectively. NBT undergoes high degradation in the presence of light as expected. Figure 5c shows degradation kinetics of NBT for nano ZnO, capped nano ZnO, and micro ZnO in the dark and visible light. It clearly reveals that, on capping, the degradation is decreased significantly both in dark and light. It confirms that capping decreases the concentration of superoxide radicals as capping blocks the oxygen vacancies which are primarily responsible for the production of •O2−, both in visible light and the dark. Micro ZnO shows the least degradation. The rates of degradation, k (min−1) , are tabulated in Table S1 which confirms that nano ZnO produces the highest amount of superoxide radicals compared to capped nano ZnO and micro ZnO in ambient light and dark. To confirm further superoxide radical generation from nano ZnO, EPR spin trapping coupled with DMPO was carried out in the dark.20 Figure 6I shows the characteristic DMPO−OH spin adduct in the case of nano ZnO, showing that ROS are generated even in the dark. Figure 6i,a shows the production of both hydroxyl and superoxide radical in the dark. To show only superoxide, a hydroxyl radical scavenging experiment was carried out in the presence of MeOH and Figure 6i,b shows the spectra correspond to superoxide radical only.

light

H2O2 (±0.2 ppm)

(3)

•

O2 + H 2O → HO2 + OH

•

Table 2. ROS from Different ZnO Products under Various Conditionsa OH• (±0.01 ppm)

−

N.D. = not detected.

visible light, micrometer sized ZnO releases the least amount of ROS, nano ZnO produces ROS (•OH), roughly three times that of capped ZnO and nine times that of micro ZnO. In the dark nano ZnO produces ROS (H2O2) two times that of capped ZnO because of increased surface area and more oxygen vacancies12 in the former. Generation of ROS in the dark is of significance in designing new oxide catalysts that can degrade dyes in the absence of light as well. Mechanism of Production of ROS in Light and Dark. The mechanism of generation of ROS in visible light is well9159

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Figure 5. UV−visible spectra of NBT in nano ZnO under (a) visible light and (b) dark, and (c) degradation kinetics of NBT in nano, capped, and micro ZnO in dark and visible.

Figure 6. (i) Hydroxyl and superoxide radical formation in dark (a) nano ZnO and (b) nano ZnO in the presence of MeOH. (ii) Inactivation of S. aureus by nano ZnO in the presence of SOD.

ROS will be produced, especially in nano ZnO in contrast to capped ZnO (in which defects are blocked) where superoxide contribution will be less. In the dark, micro ZnO shows no activity due to a lack of surface defects and very little Zn2+ ion leaching. We may conclude that defects mediate ROS through superoxide species in both dark (Scheme 1) and light (Scheme S1), but, in the latter, charge carrier excitation through absorption of light (visible and UV) can also occur, producing higher amounts ROS, resulting in higher antibacterial activity than in the dark. Surface Charge Density of ZnO in the Presence and Absence of Light. The pH of ZnO suspension in LB is 8.5. The surface charge densities of nano ZnO in light and dark at this pH are −1.7 and −2.8 C/m2, respectively. In aqueous suspension the surface charge density of metal oxide depends

To elucidate the role of superoxide radical in the mechanism of the production of ROS directly, the antibacterial activity of nano ZnO and capped nano ZnO was carried out in the dark in the presence of superoxide dismutase (SOD), a well-known scavenger for superoxide,45 and the plots are depicted in Figure 6ii. Antibacterial activity of nano ZnO is decreased significantly to 7% in the presence of SOD (Figure 6ii), in the dark, versus 17% in its absence, illustrating that ROS mediated through superoxide plays a major role in antibacterial activity in the dark confirming reactions 3−6 of Scheme 1. From Figure 3, if we subtract the antibacterial efficacy (5%) arising out of zinc ion from the activity (17%) of nano ZnO in the dark, we still need to account for 12% efficacy which should be due to ROS, solely resulting from superoxide ion mediated by surface defects. Under light, we may conclude that more 9160

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Langmuir on the surface hydroxyl groups.27 Lagström et al. correlated surface hydroxyl groups of metal oxide to surface charge density which decreases on protonation of species such as M−O−.46 In the presence of light, water molecules on interaction with generated holes form hydroxyl groups and protons. Lattice oxide ions take up the preceding protons forming a neutral hydroxyl group,47,48 resulting in a decrease of surface charge density. Hence, SCD of ZnO is lower in the presence of light than in the dark. There is a correlation between SCD and antibacterial activity.

(3) Su, S.; Wu, W.; Gao, J.; Lu, L.; Fan, C. Nanomaterials-Based Sensors for Applications in Environmental Monitoring. J. Mater. Chem. 2012, 22, 18101−18110. (4) Gao, J.; Gu, H.; Xu, B. Multifunctional Magnetic Nanoparticles: Design,Synthesis, and Biomedical Applications. Acc. Chem. Res. 2009, 42, 1097−1107. (5) Chen, D.; Wang, Z.; Ren, T.; Ding, H.; Yao, W.; Zong, R.; Zhu, Y. Influence of Defects on the Photocatalytic Activity of ZnO. J. Phys. Chem. C 2014, 118, 15300−15307. (6) Yin, H.; Casey, P. S.; McCall, M. J.; Fenech, M. Effects of Surface Chemistry on Cytotoxicity, Genotoxicity, and the Generation of Reactive Oxygen Species Induced by ZnO Nanoparticles. Langmuir 2010, 26, 15399−15408. (7) Li, Y.; Zhang, W.; Niu, J.; Chen, Y. Mechanism of Photogenerated Reactive Oxygen Species and Correlation with the Antibacterial Properties of Engineered Metal-Oxide Nanoparticles. ACS Nano 2012, 6, 5164−73. (8) Patra, P.; Mitra, S.; Debnath, N.; Goswami, A. Biochemical-, Biophysical-, and Microarray-Based Antifungal Evaluation of the Buffer-Mediated Synthesized Nano Zinc Oxide: An in Vivo and in Vitro Toxicity Study. Langmuir 2012, 28, 16966−16978. (9) Wang, Z. L. Zinc Oxide Nanostructures: Growth, Properties and Applications. J. Phys.: Condens. Matter 2004, 16, R829−R858. (10) Miyauchi, M.; Nakajima, A.; Watanabe, T.; Hashimoto, K. Photocatalysis and Photoinduced Hydrophilicity of Various Metal Oxide Thin Films. Chem. Mater. 2002, 14, 2812−2816. (11) Raghupathi, K. R.; Koodali, R. T.; Manna, A. C. Size-Dependent Bacterial Growth Inhibition and Mechanism of Antibacterial Activity of Zinc Oxide Nanoparticles. Langmuir 2011, 27, 4020−4028. (12) Padmavathy, N.; Vijayaraghavan, R. Enhanced bioactivity of ZnO Nanoparticles an antibacterial study. Sci. Technol. Adv. Mater. 2008, 9, 035004. (13) Applerot, G.; Lipovsky, A.; Dror, R.; Perkas, N.; Nitzan, Y.; Lubart, R.; Gedanken, A. Enhanced Antibacterial Activity of Nanocrystalline ZnO Due to Increased ROS-Mediated Cell Injury. Adv. Funct. Mater. 2009, 19, 842−852. (14) Li, M.; Zhu, L.; Lin, D. Toxicity of ZnO Nanoparticles to Escherichia coli: Mechanism and the Influence of Medium Components. Environ. Sci. Technol. 2011, 45, 1977−1983. (15) Brayner, R.; Ferrari-Iliou, R.; Brivois, N.; Djediat, S.; Benedetti, M. F.; Fievet, F. Toxicological Impact Studies Based on Escherichia coli Bacteria in Ultrafine ZnO Nanoparticles Colloidal Medium. Nano Lett. 2006, 6, 866−870. (16) Adams, L. K.; Lyon, D. Y.; Alvarez, P. J. J. Comparative Ecotoxicity of Nanoscale TiO2, SiO2, and ZnO Water Suspensions. Water Res. 2006, 40, 3527−3532. (17) Li, Y.; Niu, J.; Zhang, W.; Zhang, L.; Shang, E. Influence of Aqueous Media on the ROS-Mediated Toxicity of ZnO Nanoparticles toward Green Fluorescent Protein-Expressing Escherichia coli under UV-365 Irradiation. Langmuir 2014, 30, 2852−2862. (18) Jones, N.; Ray, B.; Ranjit, K. T.; Manna, A. C. Antibacterial Activity of ZnO Nanoparticle Suspensions on a Broad Spectrum of Microorganisms. FEMS Microbiol. Lett. 2008, 279, 71−76. (19) Sapkota, A.; Anceno, A. J.; Baruah, S.; Shipin, O. V.; Dutta, J. Zinc Oxide Nanorod Mediated Visible Light Photoinactivation of Model Microbes in Water. Nanotechnology 2011, 22, 215703. (20) Lipovsky, A.; Tzitrinovich, Z.; Friedmann, H.; Applerot, G.; Gedanken, A.; Lubart, R. EPR Study of Visible Light-Induced ROS Generation by Nanoparticles of ZnO. J. Phys. Chem. C 2009, 113, 15997−16001. (21) Xu, X.; Chen, D.; Yi, Z.; Jiang, M.; Wang, L.; Zhou, Z.; Fan; Wang, Y.; Hui, D. Antibacterial Mechanism Based on H2O2 Generation at Oxygen Vacancies in ZnO Crystals. Langmuir 2013, 29, 5573−5580. (22) Jayakumar, O. D.; Salunke, H. G.; Kadam, R. M.; Mohapatra, M.; Yaswant, G.; Kulshreshtha, S. K. Magnetism in Mn-doped ZnO Nanoparticles Prepared by a Co- Precipitation Method. Nanotechnology 2006, 17, 1278−1285.

4. CONCLUSION The mechanism of antibacterial activity of ZnO in the dark is attributed mainly to ROS originated through interaction of water/moisture with superoxide species which are facilitated by surface defects, for the first time. A correlation between oxygen vacancies and ROS is proved even in the dark. Experimental evidence for ROS generation even in the dark has been provided to prove the mechanism. This work demonstrates that engineered ZnO, with significant antibacterial efficacy is a potential antibacterial agent even in the dark and could find applications in food packaging. More importantly, nano ZnO coated surfaces could be expected to function effectively as antibacterial surfaces both in the presence and absence of light. The present study could be of interest not only to design new, potential antibacterial agents based on oxides but also to develop new oxide based catalysts that can degrade dyes in the absence of light also.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b02266. XRD, TEM, and FESEM of micro and nano ZnO, FTIR of oxalic acid capped nano ZnO, dissolution kinetics of Zn 2+ from aqueous suspensions of nano ZnO, FESEM and TEM of treated and untreated bacteria, kinetics of ROS production from nano and capped nano ZnO, and scheme of generation of ROS in visible light (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank VIT University for financial support and encouragement. V.L.P. thanks the UGC, Government of India, for a Rajiv Gandhi National Fellowship. R.V. dedicates this work to his mentor, Professor C. N. R. Rao FRS in honor of his 82nd birthday.

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