Colloids and Surfaces B: Biointerfaces 117 (2014) 233–239

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Amino acid-mediated synthesis of zinc oxide nanostructures and evaluation of their facet-dependent antimicrobial activity Meghana Ramani a , S. Ponnusamy a,∗ , C. Muthamizhchelvan a , Enrico Marsili b,c,∗∗ a Center for Materials Science and Nano Devices, Department of Physics, SRM University, Kattankulathur, Kancheepuram (D.t.), Chennai 603203, Tamil Nadu, India b School of Biotechnology, Dublin City University, Collins Avenue, Dublin 9, Dublin, Ireland c Singapore Centre on Environmental Life Sciences Engineering (SCELSE), Nanyang Technological University, 60 Nanyang Drive, 637551 Singapore, Singapore

a r t i c l e

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Article history: Received 21 August 2013 Received in revised form 9 January 2014 Accepted 9 February 2014 Available online 4 March 2014 Keywords: Zinc oxide Nanostructures Antimicrobial Reactive oxygen species

a b s t r a c t ZnO nanostructures (ZnO-NSs) of different morphologies are synthesized with the amino acids l-alanine, l-threonine, and l-glutamine as capping agents. X-ray diffraction (XRD) shows the formation of a crystalline wurtzite phase of ZnO-NSs. The surface modification of ZnO-NSs due to the capping agents is confirmed using Fourier transform infrared (FTIR) spectroscopy. Photoluminescence spectroscopy reveals that the concentration of surface defects correlates positively with the number of polar facets in ZnONSs. The antimicrobial activity of the ZnO-NSs has been tested against Escherichia coli and the common pathogens Staphylococcus aureus, Klebsiella pneumoniae, and Bacillus subtilis. Culture-based methods in rich medium show up to 90% growth inhibition, depending on the ZnO-NSs. Flow cytometry analyses indicate that the reactive oxygen species (ROS) generated by ZnO-NSs contribute mostly to the antibacterial activity. Control experiments in minimal medium show that amino acids and other reducing agents in Luria-Bertani (LB) medium quench ROS, thereby decreasing the antimicrobial activity of the ZnO-NSs. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Antibiotic-resistant pathogens like methicillin resistant Staphylococcus aureus (MRSA) [1] are major agents of nosocomial infections. These pathogens can survive common cleaning procedures that employ phenolic and cationic compounds [2]. Recent outbreaks of Enterococcus, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas and Enterobacter (ESKAPE) are responsible for two thirds of the healthcare associated infections in the period from 2004 to 2008 [3]. Antimicrobial nanostructures (NSs) comprise an efficient method to control infections in health care institutions. Inorganic metal-oxide NSs like MgO [4], Cu2 O [5], TiO2 [6], Ag2 O [7], ZnO [8,9] and their complexes [10] are preferred over organic antimicrobial agents due to their stability, robustness, and long shelf life.

∗ Corresponding author. Tel.: +91 44 27456255/27452818. ∗∗ Corresponding author at: School of Biotechnology, Dublin City University, Collins Avenue, Dublin 9, Dublin, Ireland. Tel.: +353 17008515; fax: +353 17005412. E-mail addresses: [email protected] (S. Ponnusamy), [email protected], [email protected] (E. Marsili). http://dx.doi.org/10.1016/j.colsurfb.2014.02.017 0927-7765/© 2014 Elsevier B.V. All rights reserved.

Metal-oxide NSs disrupt membrane activity, damage DNA, and oxidize microbial proteins, resulting in broad spectrum of antimicrobial activity. In this study, we focus on the low cost, easily synthesized ZnO. ZnO-NSs are effective against a broad range of Gram positive and Gram negative bacteria, making them an alternative to conventional antibiotics [8,9]. The antibacterial activity of ZnO-NSs depends on various factors such as particle size [8], shape, surface area, electronic states, surface charge, surface energy and roughness [8,11–13]. Smaller nanoparticles have large interfacial area and can easily penetrate bacterial membranes, increasing their antimicrobial effectiveness. Various ZnO-NSs having similar surface area but different shapes may have different active facets, which can lead to enhanced antimicrobial activity. Additionally, ZnO-NS shape can affect their internalization mechanism; it has been reported that one dimensional ZnO-NSs (i.e., rods and wires) penetrate the bacterial cell walls better than spherical ZnO-NSs [11]. Flower-shaped ZnO-NSs have shown higher biocidal activity against Escherichia coli and S. aureus, compared to rod-, and spherical-shaped ZnO-NSs [12]. Additionally, spherical ZnO particles of diameter 30 nm displayed higher antimicrobial effects against S. aureus and E. coli compared to hexagonal prisms of 1 ␮m in length and 100 nm in diameter, and ellipsoidal particles with 500–600 nm length and 100 nm diameter [13]. This was attributed

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to the large specific surface of the spherical particles. The toxicity of ZnO-NSs might also depend on their orientation, although published results are not conclusive. For example, crystallographic orientation was found to be inconsequential in one study [14], while randomly oriented nanorods indicated higher activity in another [15]. The antimicrobial mechanism of ZnO-NSs is not yet fully understood. Zn2+ ions in aqueous solution have been suggested to bind to the bacterial membrane and inhibit proliferation [16], glycolysis [17], transmembrane protein dislocation and acid tolerance [18], as well as increase the lag phase for suspended growth [19]. However, the toxicity of Zn2+ is very low in Luria-Bertani media, which is rich in complexing agents that decrease free Zn2+ ions concentration [19]. Zn is an essential element for bacterial cell metabolism and is not toxic at concentrations less than 100 ␮g mL−1 [20]. The toxicity of ZnO-NSs has been recently attributed to the production of reactive oxygen species (ROS). When the ZnO-NSs are exposed to UV or visible light, electron–hole pairs are produced. These split water molecules into OH− and H+ , which are in turn transformed into the ROS hydroxyl radicals (OH• ), superoxide anions (O2 − ) and hydrogen peroxide (H2 O2 ). H2 O2 can penetrate the cell membrane and disrupt the cellular organization, eventually leading to cell death [21,22]. Flow cytometry (FC) has been widely used to detect intracellular ROS and investigate the antimicrobial mechanism of ZnO-NSs [23]. The intracellular ROS are detected using a fluorescent probes such as propidium iodide [24] or dichlorofluorescein diacetate (DCFH-DA) [25]. Cellular internalization of the nanoparticles occurs by mechanical/electrostatic interactions between the nanoparticles and microbial cell walls [26]. Carboxyl, amide, phosphate, hydroxyl groups and carbohydrate-related moieties in bacterial cell walls provide adhesion sites for molecular-scale interactions with oxide nanoparticles [27–29]. The side-scattered (SSC) light in FC indicates intracellular density or granularity, which increases as nanoparticles are taken up by the cells. The uptake of ZnO and TiO2 nanoparticles in Salmonella typhimurium and E. coli was detected by FC and transmission electron microscopy (TEM) [33]. The number of polar facets in the NSs also contributes to antibacterial activity. The antibacterial activity of Ag2 O nanostructures increases as their surface area and number of the polar facets increases [7]. A similar trend was observed in Cu2 O nanostructures [5], where octahedral crystals bound by {1 1 1} polar facets have higher antibacterial activity than cubic crystals bound by non-polar {1 0 0} facets. Additionally, ZnO-NSs with large polar surfaces have a high concentration of oxygen vacancies, increasing the production of ROS, which in turn affects the photocatalytic performance of ZnO [30,31]. Based on this, a recent study [32] showed that ZnO morphologies with highly exposed (0 0 0 1)-Zn terminated polar facets possessed higher antibacterial activities. ZnO has been synthesized in various morphologies ranging from spheres to comb-like structures [33]. ZnO typically crystallizes in the hexagonal-wurtzite phase enclosed within the highly polar (0 0 0 1)-Zn and negatively charged (0 0 0 1)-O surfaces. Added surfactants (e.g., biopolymers [34], amino acids [35,36], and peptides [37]) decrease growth in a specific direction, thereby allowing a change in growth direction and hence imparting antimicrobial activity. Amino acids provide a strong surface bonding with the NSs due to their zwitterionic nature, which is defined as the promotion of charge change according to solution pH. Thus, NSs with various well-defined morphologies are expected to arise with the addition of amino acids. Here, we synthesize ZnO-NSs using the amino acids l-threonine, l-alanine, and l-glutamine as ligands. The hexagonal facet structures obtained show high antimicrobial activity against selected pathogens. Cellular uptake and surface defect analyses suggest that the antimicrobial toxicity is mainly due to the production of ROS, rather than to cellular internalization or generation of zinc ions,

as previously reported. We propose that the ROS generation at the polar facet/surface is the dominant mechanism for antimicrobial activity in metal oxide nanostructures. 2. Materials and methods 2.1. Materials Zinc acetate ((CH3 COO)2 Zn·2H2 O), sodium hydroxide (NaOH), l-glutamine, l-alanine, l-threonine, and phosphate buffer saline (PBS) were purchased from Merck. LB agar and LB broth were purchased from Himedia (Mumbai, India). E. coli MTCC 443, Bacillus subtilis MTCC 441, S. aureus MTCC 7443 and Klebsiella pneumoniae MTCC 3384 bacterial strains were purchase from microbial type culture collection (India). Dichlorofluoscein diacetate (DCFHDA) for flow cytometry was purchased from Sigma–Aldrich. All the chemicals were used as received. 2.2. Synthesis and characterization of ZnO nanostructures (ZnO-NSs) All samples were synthesized at room temperature. In a typical experiment, a 0.2 M solution of zinc acetate dihydrate ((CH3 COO)2 Zn·2H2 O) was prepared in de-ionized water and the required amino acid was added to the final concentration of 0.1 or 0.2 M. The pH was adjusted to 9 by adding concentrated NaOH. The reaction proceeded for 6 h under constant stirring, after which the precipitate was washed with deionized water and with 99% (v/v) ethanol, and then dried at room temperature for 4 h. The samples were labeled A1 and A2 for 0.1 M and 0.2 M solutions of l-alanine capped ZnO-NSs, T1 and T2 for 0.1 M and 0.2 M solutions of l-threonine capped ZnO-NSs, and G1 and G2 for 0.1 M and 0.2 M solutions of l-glutamine capped ZnO-NSs, respectively. The FTIR spectrum was recorded using infrared (IR) spectroscopy (Labindia, India) in the range of 400–4000 cm−1 by standard KBr pellet method. The morphological analyses were performed by field emission scanning electron microscopy (FESEM) (Quanta FEG, Netherlands) and TEM (Philips CM20, 200 kV). The crystal structure and quality was resolved using X’pert PRO (PANalytical, Netherlands) advanced X-ray diffractometer with Cu K␣ ˚ with 2 ranging between 20◦ and 80◦ radiation ( = 1.5406 A), at the scanning rate of 0.025◦ per second. The XPowder software package was used for background subtraction, K␣2 stripping and for implementing instrumental broadening correction and peak-profile fitting. Photoluminescence was carried out using the Fluorolog-3-11 spectrophotometer (Jobin Yvon, Horiba). The dissolved zinc ion concentration was measured by inductively coupled plasma mass spectrometry (ICP-MS) (Elan 6100; Perkin-Elmer SCIEX, USA). Cellular internalization of nanostructures and intracellular ROS were detected using BD FACSCalibur Flow Cytometry equipped with CellQuest software in a Mac Workstation. 2.3. Antibacterial activity of the ZnO-NSs We selected four strains: E. coli and the pathogens K. pneumoniae (Gram negative), S. aureus and B. subtilis (Gram positive). The antibacterial activity of the ZnO-NSs synthesized was tested at laboratory lighting conditions by: (a) Kirby–Bauer disc diffusion assay [38] and (b) growth curve in liquid medium spiked with ZnONSs. Mid-log phase cultures with OD600 0.5–0.6 grown in LB broth were swabbed on the LB agar plates for the Kirby–Bauer assay. Filter paper discs saturated with 0 (negative control), 5, 15, 25, 35, and 45 ␮g mL−1 of the ZnO-NSs dispersed in ultrapure water were placed on the plate. Plates were incubated at 37 ◦ C for 24 h and the diameters of the zone of inhibition (ZOI) were recorded. A disc saturated with 5 ␮g mL−1 ampicillin was used as positive control. In the

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liquid medium tests, single colonies from LB agar plates were grown overnight into 100 mL of LB, to OD600 of 1. Subsequently, 1 mL of the broth was inoculated in 100 mL of fresh LB medium supplemented with 0 (negative control), 5, 15, 25, 35, and 45 ␮g mL−1 of all the ZnO-NSs. All the flasks were then incubated in a rotary shaker at 150 rpm at 37 ◦ C. The growth was monitored at an interval of 1 h for 10 h by measuring OD600 . All experiments were performed in triplicate and the results are presented as mean ± squared deviation. The reduction in OD was calculated as follows: ODreduction (%) =

OD × 100 ODcontrol

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3. Results and discussion 3.1. Amino acids as capping agents for ZnO-NSs Amino acids serve as capping agents for the ZnO-NSs as they prevent NSs aggregation, thus determining their morphology and antimicrobial activity [35,36]. The FTIR spectra of all samples and their interpretation are shown in Figure S1. 3.2. Growth mechanism and morphology of ZnO-NSs

(1)

where ODcontrol is the OD measured in absence of ZnO-NSs. A toxicity index was calculated to determine the overall antibacterial activity for each sample across the four microorganisms tested. At each ZnO-NSs concentration (5, 15, 25, 35, and 45 ␮g mL−1 ) and for each strain, a number from 6 (most toxic) to 1 (least toxic) was assigned based on the OD reduction with respect to the control experiment. The sum over the four strains tested yielded the toxicity index. Two toxicity index values A and B were considered identical if |A − B| ≤ 2. 2.4. Determination of zinc ion concentration To determine the concentration of dissolved zinc ions, 1 mL of the aliquot was separated from the ZnO treated cell suspensions at the end of seventh hour. The samples were then centrifuged at 15,000 × g for 45 min and filtered using Whatman filter paper. The supernatant was collected and analyzed using ICP-MS.

2.5. Detection of nanoparticles uptake Log phase culture (OD600 = 0.6) of E. coli cell suspension was treated with 45 ␮g mL−1 of ZnO-NSs suspensions at 37 ◦ C. 1 mL aliquots of the sample were withdrawn at 60 min and 90 min each and washed thrice with PBS. The pellet was resuspended in 1 mL PBS and analyzed by flow cytometry (FACSCalibur, BD Biosciences). For each sample, 10,000 cells were acquired and analyzed with BD CellQuest Pro analysis software. The forward scatter (FSC) and side scatter (SSC) indicate the cell size and the intracellular density, respectively [39].

2.6. Detection of reactive oxygen species (ROS) Intracellular ROS in the bacterial cells were detected by observing a change in the fluorescence of the membrane-soluble dye DCFH-DA. However, the cleavage of its two ester bonds by intracellular esterase produces a polar, cell-membrane impermeable and non-fluorescent species (H2 DCF). Intra-cellular accumulation of H2 DCF and its oxidation by intracellular ROS yields highly fluorescent DCF, which can be monitored by detecting the increase in fluorescence at 530 nm when the sample is excited at 488 nm. The fluorescence observed is proportional to the concentration of ROS in cells. For the experiment, the E. coli culture was grown in LB media at 37 ◦ C with shaking (150 rpm) until the OD600 reached 0.6. This suspension was then treated with the 45 ␮g mL−1 of ZnONSs. 1 mL aliquots of the treated suspensions were withdrawn after 60 and 90 min of incubation and centrifuged at 3000 × g for 5 min. The pellet was washed twice with PBS and resuspended in 1 mL PBS containing 30 ␮g mL−1 of DCFH-DA at 37 ◦ C for 45 min in the dark with shaking (150 rpm). The sample was then pelleted by centrifugation at 3000 × g for 5 min, resuspended in 1 mL of PBS, and analyzed using flow cytometry.

ZnO crystallizes into a wurtzite phase having a hexagonal unit cell with a P6mc space group. The structure of ZnO is composed of tetrahedrally coordinated O2− and Zn2+ ions, stacked alternately along the c-axis in [0 0 1] direction. The oppositely charged ions produce positively charged (0 0 0 1)-Zn and negatively charged (0 0 0 1)-O surfaces. The side surfaces, normal to [1 1 0] or [1 0 0], carry a net negative charge. The difference in atomic arrangements of these faces results in an inherent asymmetry along the c-axis that leads to anisotropic growth of the ZnO crystallites. This anisotropic growth involves two simultaneous processes: axial growth along [0 0 1], resulting from deposition of positively charged ionic species like amines on the prismatic faces, and equatorial growth along [1 0 0] or [1 1 0] vectors, resulting from deposition of negatively charged species like halogen-containing ligands on the hexagonal facets. The final shape of the crystal is determined by variation in these facet-specific growth rates and can be controlled by changing the organic ligands and their concentration. Since amino acids are zwitterionic species, their net charge depends on the pH of the solution, and at pH 9 the three amino acids used here exist as anions with COO− being the dominant ions. Hence, axial growth is promoted by adsorption of COO− on the positively charged (0 0 0 1)-Zn terminated hexagonal facets. The FESEM image of samples A1 and A2 are shown in Fig. 1, indicating the formation of small nanoparticles (15 nm) and aggregates of nanoparticles (approx. 100 nm), respectively. l-Alanine is a non-polar amino acid bound to a methyl group. The non-polarity of l-alanine lowers the ionization rate, resulting in slow nucleation, disrupting the growth process and thus resulting in smaller nanoparticle formation. At higher concentrations, the excess COO− ions bind with NH2 of the neighboring molecule as evidenced by FTIR [Figure S1(A)(ii), curve a], causing cyclization and hence aggregation. TEM results suggest that the ZnO-NSs are embedded in a matrix of amino acids. l-Threonine is a polar amino acid with a hydroxyl side chain. At low concentration (0.1 M), the polarity promotes rapid nucleation and growth along the c-axis. Additionally, at the negatively charged prismatic facets of ZnO crystal lattice, the amide group is associated with the oxygen of ZnO via hydrogen bonding. However, the growth along the c-axis is terminated more effectively than along the prismatic facets due to the nature of the bonds, leading to the formation of hexagonal platelets of size 30–40 nm [Figure S2(i)]. At higher concentrations, the large number of COO− and NH+ ions, promote rapid nucleation and hence growth is suppressed, leading to the formation of small nanoparticles of size 15–20 nm. However, the presence of excess amino acid results in cross linking between the particles as in the case of A2, forming irregular thin sheet-like NSs. TEM analyses indicate that these sheets are formed by irregular aggregation of nanoparticles of diameter 20–30 nm [Figure S2(ii)]. l-Glutamine is a polar amino acid with an amine functional group. At low l-glutamine concentrations the negatively charged COO− suppresses the growth along the c-axis. However, the residual amine group and the amide group covalently bond to the ZnO by N H· · ·O onto the side surfaces and enhances the vertical growth, forming hexagonal tablets of diameter 30–35 nm [Figure S3(i)]. This leads to nucleation on the surface

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Fig. 1. FESEM and TEM (inset) of samples (i) A1 and (ii) A2, showing individual nanoparticles of 5–15 nm diameter and nanoaggregates, respectively.

of the hexagonal facet [Figure S3(i)] and a secondary growth process continues for a short time due to an insufficient quantity of ions. Increase in the concentration of l-glutamine provides more ionic groups for binding. These act as mineral bridges between two hexagonal platelets, further enhancing the growth along the c-axis, leading to the formation of twin-nanorods [Figure S3(ii)]. Formation of similar twin-structures has been reported previously for gelatin [35], glutamic acid and glycine capped nanostructures of ZnO [36]. 3.3. Structural analysis The crystalline phase of the products was identified as hexagonal wurtzite using XRD (Figure S4). The XRD pattern of all the samples shows high crystallinity and the crystallite sizes were estimated from Scherer’s equation [39]: k D= ˇ cos 

(2)

where  is the wavelength of Cu K␣ radiation, ˇ is the corrected half width of the diffracted peak (1 0 1),  is the diffraction angle and K = 0.9 is the Debye constant. The calculated crystallite size from XRD spectra are tabulated in Table S1. The (0 0 2) diffraction peak in the XRD of ZnO corresponds to growth along the c-axis. It is observed that the ratio (0 0 2)/(1 0 1) increases with increasing growth along the c-axis, as evidenced by TEM analyses. 3.4. Surface states analyses The presence of surface defects on the ZnO-NSs was determined by photoluminescence spectroscopy at room temperature (Figure S5). All samples exhibited two major peaks: a narrow band gap emission (350–380 nm) that is attributed to exciton recombination in the UV region [40], and a broad below-band gap blue-green emission, usually attributed to defects like oxygen vacancies and zinc interstitials [41,42]. The ratio of intensity of the band gap emission to the defect-level emission (Table 1) increases as the concentration of surface defects decreases. The samples can be arranged accordingly from lowest to highest concentration of surface defects as, G2 < A2 < G1 < A1 < T2 < T1. The intensity of defect level emission depends on the number of polar facets exposed to the surface, which is consistent with the high concentration of surface defects on the hexagonal platelets (T1) having a higher surface area of exposed (0 0 0 1)-Zn polar surface. The polar ZnO planes are stabilized by OH groups, which form oxygen vacancies by removing either OH− or H2 O groups from the surface [43,44]. As the particle aspect ratio increases, the area

of exposed polar facet decreases, thus decreasing the concentration of defects. Oxygen vacancies are the recombination centers of visible emissions and are mainly located on the surface; therefore, the samples with large specific surface (G2) show a high concentration of oxygen vacancies. Thinner hexagonal platelets are reported to possess higher concentration of oxygen vacancies [45,46]. The UV–vis spectra of these samples (Figure S6) show a broad absorption edge in the visible region (>400 nm). The spectra are red shifted from the bulk (370 nm) because of the defects on the surface that modifies the density of states of ZnO. 3.5. Toxicity of nano-ZnO to model pathogens in solid agar and liquid media The zone of inhibition (ZOI) increases with increasing concentration of ZnO-NSs (Figure S7). The toxicity sequence from highest to lowest was T1 > T2 > A1 > G1 > A2 > G2 across all microorganisms tested. The growth curves of B. subtilis in liquid medium spiked with the six ZnO-NSs are shown in Fig. 2; the other growth curves are included in Figure S8-S10. The average OD reduction with respect to the negative control (no ZnO-NSs added) for 5 ␮g mL−1 of ZnO-NSs was 62.7 ± 8.1% and 67.2 ± 8.7% for B. subtilis and S. aureus (Gram positive), respectively, while it was only 39.6 ± 4.6% and 49.7 ± 5.4% for K. pneumoniae and E. coli (Gram negative), respectively (Figure S11). At higher ZnO-NSs concentrations, the difference in antimicrobial activity across the four pathogens became less pronounced. The OD reduction at 45 ␮g mL−1 was approximately 80% for all four strains. It is likely that OD provides a reliable measure of antimicrobial activity at low ZnO-NSs concentration. However, at high concentration, the OD might be less reliable as it cannot discriminate between viable and dead cells. The samples T1, A1 and A2 were more toxic than the other nanostructures for all the microorganisms except E. coli. Sample G2 (nanorods) was the most toxic for E. coli. This difference in the

Table 1 The ratio of intensity of band gap emission to the band edge emission indicates the surface defect concentration variation of morphology of the nanostructures. The sample T1 had highest band edge emission and thus highest concentration of defects. Sample

Ratio IBG /IBE

A1 (nanoparticles) A2 (nanoparticle aggregates) T1 (nanoplatelets) T2 (nanosheets) G1 (nanodiscs) G2 (nanorods)

0.54 0.69 0.27 0.38 0.64 1.37

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Fig. 2. Growth curves of B. subtilis in the presence of different concentrations of sample: (a) A1, (b) A2, (c) T1, (d) T2, (e) G1 and (f) G2. Results are presented as mean ± SD (n = 3).

extent of toxicity could be attributed to the different membrane composition of the microorganisms tested [47,48]. Membrane disorganization in the Gram negative E. coli was previously observed following exposure to ZnO nanoparticles (80 ␮g mL−1 ) [49]. Experiments with E. coli lipid vesicles show that H2 O2 and other species produced by ZnO nanoparticles induce structural damage of the bacterial membrane [50]. However, very little mechanistic information is available for other microorganisms employed in this study. The IC50 for B. subtilis exposed to ZnO nanoparticles in water was 0.5 ␮g mL−1 , much lower than that reported here. This discrepancy is likely due to the medium used, very similar to river water, which does not quench the ROS produced by ZnO nanoparticles. The toxicity index was calculated across the four microorganisms as described previously in the material and methods section at a ZnO-NS concentration of 5 ␮g mL−1 . The sequence of decreasing toxicity was T2 = T1 > G1 > A1 > A2 > G2, similar to the sequence calculated by the surface defects data. This suggests that surface defects play a crucial role in microbial toxicity under our experimental conditions. The antimicrobial activity was significantly higher for Gram positive microorganisms than for Gram negative ones, suggesting that the cell membrane chemistry determines the resistance of the microorganism to ZnO-NSs.

3.6. Dissolution of Zn ions ICP-MS study did not detect soluble Zn2+ (data not shown), confirming that soluble Zn did not contribute to the antimicrobial activity in our setup. The absence of soluble Zn can be attributed to the surface functionalization of ZnO-NSs using amino acids. ZnO is known to form stable complexes with amino acids due to strong ionic bonding as confirmed by FTIR analyses. The LB media contains amino acids and peptides that can form complexes with zinc. Previous studies have shown that at concentrations G1 > T1 > A1 > T2 > A2. Almost 40% of the increase in granularity is observed for E. coli treated with 45 ␮g mL−1 of G2 for 90 min, a value higher than those reported previously for ZnO, 25% for 125 nm nanoparticles [29]. This could have resulted from the dimensional stability of ZnO-NSs due to the presence of biocompatible l-glutamine on their surface. The antimicrobial activity of the nanorods G2 and nanodiscs G1 is likely due to the interaction of the bacterial cell walls with the sharp prismatic edges of the ZnO-NSs. The microorganism suspension granularity increased moderately for the other four samples. As A1 and A2 are formed by the random aggregation of smaller nanoparticles, they cannot penetrate the bacterial membrane but rather accumulate on the cell membrane. The change in fluorescence with time of the DCFH-DA dye in the presence of various nanostructures at 45 ␮g mL−1 indicates the production of intracellular ROS (Fig. 4). The intensity of DCF increases with time for all the samples; nanoplatelets (T1) released the highest concentration of ROS. However, the cellular uptake of nanoplatelets was lower than the other ZnO-NSs, indicating that the prevalent mechanism of cell death is ROS-induced membrane damage. The intracellular ROS production observed by FC correlates well with the concentration of defects (Figure S5), indicating that polar facets enhance the antibacterial activities of the ZnO-NSs. The terminal polar (0 0 0 1) and (0 0 0 1) faces are highly active surfaces with a large concentration of defects in the form of oxygen vacancies [45,46]. The increased electron trapping due to higher defect sites lessens the electron–hole pair recombination rate to

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Fig. 3. Scatter analysis of increase in cellular granularity in E. coli after (A) 0 min, (B) 60 min and (C) 90 min of treatment with 45 ␮g mL−1 of sample G2. The overall analysis report is presented in (D) for all six microorganisms at a concentration of 45 ␮g mL−1 at the end of 60 min and 90 min incubation. Results are presented as mean ± SD (n = 3).

below the rate of electron transfer to adsorbed water molecules. If charge separation is maintained, the electron and hole may migrate to the surface where they participate in redox reactions with the adsorbed water molecules. Specifically, H+ can react with surface-bound H2 O or OH− to produce the hydroxyl radical and a superoxide radical anion (O2− ). Simultaneous reactions cause the generation of H2 O2 , which induces oxidative stress in living cells [22]. 3.8. ZnO-NSs toxicity in minimal medium Complex media like LB are known to reduce the antimicrobial toxicity of metal and other pollutants [51] in suspended cultures,

as LB components can complex toxic species and reduce their bioavailability. Minimal media provide a much simpler nutrient structure, enhance toxicity of organic and inorganic biocides, and thus are more suitable to study the microbial toxicity under controlled laboratory conditions [52]. To verify the role of LB in our study, we measure the toxicity of nanoplatelets (T1) in M9 against E. coli at the 45 ␮g mL−1 dose level, and measured a 98.5% OD reduction. The test was repeated at the same concentration of Zn2+ , and very little toxicity was observed. When cysteine, a known antioxidant, was added to the medium, the OD reduction decreased from 98.5% to 23.4% (Fig. 5). These results confirmed that the production and release of ROS in ZnO-NSs is the major toxicity mechanism for the ZnO-NSs synthesized in this study.

Fig. 4. Change in DCF intensity with time after treatment with all ZnO-NSs for (i) 60 min and (ii) 90 min.

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Fig. 5. The toxicity of sample T1 to E. coli in M9 minimal medium. Results are presented as mean ± SD (n = 3).

4. Conclusion Amino acids serve as shape-directing agents in the synthesis of ZnO-NSs. Photoluminescence of the ZnO-NSs surface shows high concentration of defects in NSs enclosed with polar facets. The antimicrobial activity of the ZnO-NSs synthesized in this study was higher for the Gram positive than for the Gram negative microorganisms. Overall, the ZnO-NSs enclosed by the highly polar (0 0 0 1) facets were most toxic. The correlation between antimicrobial toxicity and concentration of defects is consistent with the finding that the polar Zn-terminated plane is a source of oxygen vacancies. Flow cytometry experiments showed that internalization of the ZnO-NSs contributes to the overall toxicity. Minimal medium experiment showed that ZnO-NSs toxicity is mostly due to ROS production. We suggest that antimicrobial toxicity of ZnO-NSs should be carried out in minimal media to avoid ROS scavenging by reducing agents in rich media. Acknowledgements We acknowledge Nanotechnology Research Center for XRD and FESEM, College of Pharmacy for FTIR. We thank Dr K. Ilango, Dr M.R. Ganesh for flow cytometry analyses, and Dr S. Longford and Dr S. Guadarrama for reviewing the manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb. 2014.02.017. References [1] H. De Lencastre, D. Oliveira, A. Tomasz, Curr. Opin. Microbiol. 10 (2007) 428. [2] F. Daschner, A. Schuster, Am. J. Infect. Control 32 (2004) 224. [3] H.W. Boucher, G.L. Talbot, J.S. Bradley, J.E. Edwards, D. Gilbert, L.B. Rice, M. Scheld, B. Spellberg, J. Bartlett, Clin. Infect. Dis. 48 (2009) 1.

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Amino acid-mediated synthesis of zinc oxide nanostructures and evaluation of their facet-dependent antimicrobial activity.

ZnO nanostructures (ZnO-NSs) of different morphologies are synthesized with the amino acids L-alanine, L-threonine, and L-glutamine as capping agents...
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