In-vitro efficacy of different morphology zinc oxide nanopowders on Streptococcus sobrinus and Streptococcus mutans

Accepted Manuscript In-vitro efficacy of different morphology zinc oxide nanopowders on Streptococcus sobrinus and Streptococcus mutans

Siti Khadijah Mohd Bakhori, Shahrom Mahmud, Ling Chuo Ann, Amna Hassan Sirelkhatim, Habsah Hasan, Dasmawati Mohamad, Sam'an Malik Masudi, Azman Seeni, Rosliza Abd Rahman PII: DOI: Reference:

S0928-4931(16)32768-0 doi: 10.1016/j.msec.2017.04.085 MSC 7908

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

21 December 2016 13 April 2017 15 April 2017

Please cite this article as: Siti Khadijah Mohd Bakhori, Shahrom Mahmud, Ling Chuo Ann, Amna Hassan Sirelkhatim, Habsah Hasan, Dasmawati Mohamad, Sam'an Malik Masudi, Azman Seeni, Rosliza Abd Rahman , In-vitro efficacy of different morphology zinc oxide nanopowders on Streptococcus sobrinus and Streptococcus mutans. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Msc(2017), doi: 10.1016/j.msec.2017.04.085

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ACCEPTED MANUSCRIPT IN-VITRO EFFICACY OF DIFFERENT MORPHOLOGY ZINC OXIDE NANOPOWDERS ON STREPTOCOCCUS SOBRINUS AND STREPTOCOCCUS MUTANS Siti Khadijah Mohd Bakhori1,2*, Shahrom Mahmud1,2, Ling Chuo Ann1, Amna Hassan Sirelkhatim1, Habsah Hasan3, Dasmawati Mohamad4, Sam’an Malik Masudi5, Azman Seeni6, Rosliza Abd Rahman3 1

School of Physics, Universiti Sains Malaysia, 11800, USM, Penang, Malaysia

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Institute of Nano-optoelectronics Research & Technology (INOR), Universiti Sains Malaysia, 11800, USM, Penang, Malaysia

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Microbiology and Parasitology Laboratory, School of Medicine, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia Craniofacial Science Laboratory, School of Dentistry, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia 5 Faculty of Dentistry, Lincoln University College, No.2,Jalan Stadium, SS 7/15, Kelana Jaya, 47301 Petaling jaya, Selangor

Integrative Medicine Cluster, Advanced Medical & Dental Institute, Universiti Sains Malaysia, Bandar Putra Bertam, 13200 Kepala Batas, Pulau Pinang, Malaysia Corresponding author*: [email protected]

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Abstract ZnO with two different morphologies were used to study the inhibition of Streptococcus sobrinus and Streptococcus mutans which are closely associated with tooth cavity. Rod-like shaped ZnO-A and plate-like shaped ZnO-B were produced using a zinc boiling furnace. The nanopowders were characterized using energy filtered transmission electron microscopy (EFTEM), X-ray diffraction (XRD), photoluminescence (PL) spectroscopy, Raman spectroscopy and dynamic light scattering (DLS) to confirm the properties of the ZnO polycrystalline wurtzite structures. XRD results show that the calculated crystallite sizes of ZnO-A and ZnO-B were 36.6 and 39.4 nm, respectively, whereas DLS revealed particle size distributions of 21.82 nm (ZnO-A) and 52.21 nm (ZnO-B). PL spectra showed ion vacancy defects related to green and red luminescence for both ZnO particles. These defects evolved during the generation of reactive oxygen species which contributed to the antibacterial activity. Antibacterial activity was investigated using microdilution technique towards S. sobrinus and S. mutans at different nanopowder concentrations. Results showed that ZnO-A exhibited higher inhibition on both bacteria compared with ZnO-B. Moreover, S. mutans was more sensitive compared with S. sobrinus because of its higher inhibition rate. Keywords: ZnO nanopowder, in vitro, antibacterial activity, Streptococcus sobrinus, Streptococcus mutans

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1. Introduction

The wide band-gap ZnO is an attractive II–VI material for optical devices. This material offers various potential applications in short-wavelength optoelectronic devices, such as solar cells, thinfilm gas sensor, UV laser, varistor, and luminescent material [1]. ZnO has high exciton binding energy (60 meV) which assures more efficient exciton emission at room temperature [2]. Additionally, ZnO nanostructure also offers unique approaches for effective biological and medical applications. Studies have integrated these properties and are currently focused on incorporating nanostructures with biological properties. Nanostructured ZnO exhibits antibacterial activity that can inhibit bacterial growth and behaves as a bactericidal and bacteriostatic agent [3–5]. In addition, ZnO has been extensively used in dental applications such as for temporary fillers, root canal sealers, liner, bases, and pulp capping [6]. Considering that ZnO can alleviate pain and contains an antibacterial agent, ZnO-containing dental material is also better tolerated in tissue than other dental materials [7,8]. This compound also assists in the healing process [9]. 1

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In this study, the antibacterial activity of ZnO with two different morphologies (ZnO-A and ZnO-B nanopowders) against two bacteria commonly found in tooth cavities was investigated. The bacteria were Streptococcus sobrinus ATCC 33478 and Streptococcus mutans ATCC 35668. The American Type Culture collection (ATCC) is a standard reference microorganism. Thus, the ATCC should be used to avoid incorrect identification of microbes. The structural, optical, and morphological properties of both ZnO-A and ZnO-B were investigated to demonstrate the relevance of the physical properties and biological activity of ZnO nanopowder. Antibacterial activity was evaluated by the optical density measurement of bacterial suspension directly treated for 24 h with ZnO powder at specified concentrations. A previous study reported the antibacterial activity of dental materials with ZnO after three days [10]. They used biofilm inhibition studies with a biofilm model to test the ZnO composites with variant compositions of added chemicals [10]. By contrast, our study was focused on the independent treatment of bacteria using different morphologies of ZnO powders. Thus, no antibacterial activities from other chemicals added similar to those in composite ZnO dental materials were involved in the current study.

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2. Experimental 2.1. ZnO synthesis and suspension preparation

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A custom-made zinc boiling furnace pilot plan was implemented to produce two types of ZnO nanopowders, namely, ZnO-A and ZnO-B. The difference in the production of ZnO-A and ZnO-B was the crucible temperature during the melting process. Pure zinc block was heated in the crucible, and the vapor was captured and cooled using a piping line with an exhaust. Eventually, ZnO nanopowder was collected in the collecting bag. The crucible temperature for ZnO-A was over 1100 °C, while that of ZnO-B was approximately 980–1100 ° C. The French process was briefly described by Mahmud [11]. The nanopowders had different morphologies and size distributions. ZnO-A was equivalent to pharma grade used in cosmetics, medicine, and supplement product. By contrast, ZnO-B was equivalent to industrial grade use such as for rubber, paint, and coating. The nanopowders were suspended in liquid solution for antibacterial testing.

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Equal weights of ZnO-A and ZnO-B nanopowders (0.0586 g) were initially dispersed in 90 ml of deionized water. The mixture was vigorously sonicated for 30 min to avoid aggregation and deposition of particles. The resulting suspensions with a concentration of 0.1 M, which were considered as stock solutions, were then sterilized in an autoclave machine at 121 °C for 15 min and later diluted to a series of concentrations (1,1.5, and 2 mM) for bacterial susceptibility evaluation.

2.2. Characterizations of ZnO X-ray diffraction (XRD) was performed by PANanalytical X’pert Pro MRD (λ = 1.5405 Å) over the range of 20°–80° to determine phase purity and calculate the crystallite size of the nanopowders using Scherrer’s formula. Then, near-band edge (NBE) emission, deep level defects, and red luminescence were detected by photoluminescence (PL) spectroscopy at 300 K using Horiba Jobin Yvon HR800-UV system with 325 nm (HeCd) excitation laser sources over the range of 330– 1000 nm. Using the same system of Horiba Jobin Yvon HR800-UV, Raman scattering was used to determine the phonon modes of ZnO with Argon ion laser (514 nm) at room temperature. The morphology of ZnO nanopowder was revealed using energy filtered transmission electron

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microscopy (EFTEM) (Carl Zeiss Libra 120), in which the powder samples were dispersed in deionized water, spin-coated over a cleaned silicon wafer, and dried before analysis. Subsequently, field emission scanning electron microscopy (FESEM) was performed to depict the morphology of the microorganism using FEI Nova NanoSEM 450. Furthermore, dynamic light scattering (DLS) was performed using Malvern Zetasizer Nanoseries ZS to investigate the particle size distributions of ZnO-A and ZnO-B. ZnO powder (0.1 g) was suspended in 10 ml of deionized water and sonicated for 30 min before the measurement to ensure well dispersion in liquid. Measurements were performed thrice, and the average values were plotted. Broth dilution technique in 96-well plate was conducted for the antibacterial study with trypticase soy broth (TSB) as the growth medium for the microorganisms. Antibacterial activity was quantified by measuring the optical density at 600 nm using VersaMax microplate reader spectrophotometer after 24 h of incubation under anaerobic condition in an incubator containing 5% CO2.

2.3. Antibacterial testing

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Two Gram-positive bacteria, namely, S. sobrinus (ATCC 33478) and S. mutans (ATCC 35668), were used as the target microorganisms. Given that ZnO is the main component for zinc oxide eugenol (a temporary dental filler), ZnO was well-blended with eugenol resin bond and applied in tooth cavities. The bacteria selected were according to the most commonly found pathogenic bacteria in tooth decay and tooth caries [12–14]. Therefore, the effectiveness of ZnO as antibacterial agent against these cariogenic bacteria is relevant. All tools, consumables, and materials were sterilized in an autoclave before the experiments to eliminate unwanted contaminations. The bacteria were cultured using a streaking technique on blood agar and incubated for 48 h at 37 °C in an anaerobic incubator before treatment with ZnO nanopowders. After 48 h, the colonies formed were in optimum condition, and single colony from each S. sobrinus and S. mutans was inoculated into peptone water and adjusted to 0.5 McFarland (McF) using PhoenixSpec BD spectrophotometer, resulting in a suspension of 5×105 colonyforming units (CFU)/ml. The standardized 5×105 CFU/ml suspensions were then inoculated into 96-well plate and mixed with diluted 0.1 M ZnO stock solution suspended in TSB into 1, 1.5, and 2 mM. Concurrently, a quality control test was performed to ensure the reliability of the adjusted 0.5 McF by obtaining approximately 80 colonies on blood agar plate after 24 h of incubation. The optical density of the 96 well-plate was measured after 24 h using VersaMax microplate reader spectrophotometer at 600 nm at room temperature.

2.3.1 Reactive oxygen species (ROS) detection The ROS detection was performed in conjunction with antibacterial activity. ROS is the main component that causes the antibacterial effect [15,16]. ROS include superoxide anion (.O2−), hydrogen peroxide (H2O2), and hydroxyl radical (.OH) generated during the antibacterial process, but the cellular uptake and the specific species remain controversial. Therefore, the ROS detection probe H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) was used to study the ROS level in the cells. H2DCFDA is a non-fluorescent chemical oxidized by ROS to a highly fluorescent chemical DCF (2′,7′ diclorodihydrofluorescent). The fluorescence was detected by microplate reader spectroscopy BMG Omega Fluostar at λexcitation of 485 nm and λemission of 535 nm. The 48-hour old bacteria were exposed to H2DCFDA in TSB (10 µM) and incubated at 37 °C with 5% CO2 and under light protection. After 45 min, the probe was removed

ACCEPTED MANUSCRIPT from the bacteria by centrifugation and washed twice with phosphate buffer saline. The cleaned bacteria was returned in TSB and treated with ZnO-A and ZnO-B at 1, 1.5, and 2 mM. After incubation for 24 h at 37 °C and 5% CO2, the fluorescence was measured. The experiment was conducted in triplicate, and the value of ROS in percentage of fluorescence intensity was calculated with respect to the control.

2.3.2 Detection of Zn2+ ion released

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The release of Zn2+ ion was investigated using Perkin Elmer AAnalyst 400 Atomic Absorption spectroscopy (AAS). The calibration curve was obtained using Merck standard solution (1000 ppm). The standard dilution was diluted to 1, 2, 3, 4, and 5 ppm, and the absorption of dissolved Zn2+ was measured. Then, 5 mM ZnO stock solution was diluted into 1, 1.5, and 2 mM and incubated for 24 h at 37 °C (similar condition with antibacterial experiment). Then, the dissolved Zn2+ ions were detected using AAS. Distilled water was used in diluting ZnO, because distilled water was also the medium used for diluting ZnO in the antibacterial test. Moreover, the ZnO solution was incubated for 24 h prior to AAS measurement, because 24 h is similar to the antibacterial effect at 24 h. Thus, the dissolved Zn2+ ions were investigated at the selected concentrations.

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3. Results and Discussion 3.1. Structural properties

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XRD pattern was continuously scanned at 2θ=20°–80° for ZnO-A and ZnO-B and are depicted in Fig. 1. The detected peaks were (100), (002), (101), (102), (110), (103), (200), (112), and (201), indicating that both ZnO-A and ZnO-B were polycrystalline and had hexagonal structures. The sharpness of the (100), (002), and (101) peaks indicate the good crystallinity of ZnO. No evident diffraction from randomly oriented grains or impurity phases was observed from the XRD pattern. The average crystallite sizes of ZnO-A and ZnO-B were calculated using the full width at half maximum (FWHM) of (100), (002), and (101) peaks using Scherrer’s formula [17], as follows: D=

0.9 λ βcosθ

(1)

where λ is the wavelength of the Cukα radiation (0.154 nm), β is the amplitude of the FWHM, and θ is Bragg’s angle. The XRD pattern was determined thrice, and the average peaks of (100), (002), and (101) were used to calculate the crystallite size. The crystallite sizes for ZnO-A and ZnO-B were 36.6 nm and 39.40 nm, respectively (Table 1). The difference in crystallite size would affect the antibacterial response because of the surface-to-area ratio [3], which implies the total surface exposed to the bacteria for antibacterial reactions.

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Figure 1: XRD pattern of polycrystalline ZnO-A and ZnO-B.

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42.20

36.60

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Table 1: Crystallite size of ZnO-A and ZnO-B calculated using FWHM of (100), (002), and (101) diffraction peaks by Scherrer equation. Crystallite size (nm)

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3.2. Particle size distribution

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The particle size distributions of ZnO-A nad ZnO-B were investigated using DLS, and the results as shown in Fig. 2. The average particle size of 91.9% of the ZnO-A was 21.82 nm, while the rest was 501.2 nm. Meanwhile, the size of 85.7% of ZnO-B was 52.21 nm, while the rest was 520.3 nm. This result implies that ZnO-A had better homogeneous nanosize distribution compared with ZnO-B. This uniformity will induce more antibacterial activity because of the higher surfaceto-volume ratio [18].

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Figure 2: Particle size distribution of ZnO-A and ZnO-B

3.3. Morphology properties

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Figure 3 shows the EFTEM images and electron spectroscopy imaging (ESI) of oxygen and zinc for ZnO-A and ZnO-B. The EFTEM images in Figs. 3a and 3b revealed that ZnO-A and ZnO-B had nanorod and nanoplate structures, respectively. ESI were performed to map the distribution of oxygen and zinc on the specific surface of ZnO-A nanorod and ZnO-B nanoplate. ZnO-A nanorod contained more oxygen than zinc on its surfaces, compared with ZnO-B nanoplate because of the lower oxygen-to-zinc ratio of the latter. This characteristic plays a major role in the antibacterial response. Moreover, a histogram of the size distribution for ZnO-A and ZnO-B was plotted. ZnO-A particles were mostly found in the range size of 51–60 nm, while ZnO-B was at 71–80 nm. Table 2 shows a summary of the particle size data for ZnO-A and ZnO-B from EFTEM, DLS, and XRD. The discrepancies in the sizes measured were acceptable, because different approaches were performed to measure the sizes. The particle size measured by DLS (n=3, p<0.05) and EFTEM (n=100, p<0.05) shows significant different between ZnO-A and ZnO-B respectively.

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Figure 2: EFTEM images and mapping of zinc and oxygen element on (a) ZnO-A, (b) ZnO-B

Table 2: Particle size of ZnO-A and ZnO-B by using different equipment. The table shows particle size ± SD, (a p>0.05, b ,c p<0.05)

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Size by XRD, nm (n=3)

Rod like shape Plate like shape

36.6 ± 2.1 a 39.4 ± 1.8 a

Size by DLS, nm (n=3) 21.82 ± 0.9 b 52.21 ± 1.2 b

Size by EFTEM, nm (n=100) 51-60 ± 0.8 c 71-80 ± 1.7 c

ACCEPTED MANUSCRIPT 3.4. PL and Raman spectroscopy

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The PL spectra of ZnO-A and ZnO-B were recorded at room temperature over the range of 330– 1000 nm. ZnO-A and ZnO-B exhibited three PL bands in the area near UV, green, and red luminescence as shown in Fig. 3. The narrow and intense NBE peak at 371.34 nm for ZnO-A is attributed to the band-edge excitonic luminescence of ZnO [19]. The NBE peak for ZnO-B slightly red-shifted to a longer wavelength of 381.23 nm. Shim et al. [20] discussed that the red-shift might be due to a change in crystal structure. Furthermore, the difference in the intensities of NBE emissions between ZnO-A and ZnO-B also implies the degradation of crystalline structures. Tang et al. [21] showed that NBE is related to the microcrystalline structure, and this result is consistent with the XRD data, in which the average crystallite size of ZnO-A (36.60 nm) was smaller than that of ZnO-B (39.40 nm). Moreover, the intensity of NBE is an indicator of the quality of the nanostructure as suggested by Willander et al. [22]. FWHM values were calculated to evaluate the optical quality of the nanopowders. The FWHM values of the NBE peaks were calculated to be 137 and 131 meV for ZnO-A and ZnO-B, respectively. This result implies the better quality of ZnO-A compared with ZnO-B [23]. The broad and weak green emission bands at 514.3 and 527.09 nm for ZnO-A and ZnO-B, respectively, were detected (inset of Fig. 4). These bands originated from more than one deep level defect or deep band emission (DBE), and recent investigation has identified broad green luminescence band attributed to oxygen vacancies Voֿ [22–24]. The intensity of the DBE peak for ZnO-B was relatively very weak compared with that of ZnO-A. This characteristic can be attributed to the sufficient amount of oxygen diffused into the samples during synthesis and decrease in the concentration of oxygen vacancies, resulting in the suppression of the DBE peak [25]. Moreover, the red emissions at 752.39 and 762.44 nm are related to zinc vacancies or excess oxygen [22]. This red luminescence has been proposed to be caused by zinc vacancy complexes associated with excess oxygen [26]. This type of defect provides effective surfaces of ZnO to release ROS in antibacterial activity.

Figure 4: PL spectra of ZnO-A and ZnO-B at room temperature

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Figure 5 shows the room-temperature Raman spectra of ZnO-A and ZnO-B recorded at the laser power of 20 mW at 514 nm excitation wavelength of Argon ion laser. The Raman spectra were measured with scattering configuration . Under this configuration, the allowed phonon modes of the wurtzite structure ZnO that can be detected were A1(TO), E2(high), and E1(LO). Compared with the compilation of frequencies of Raman active phonon modes reported previously by Ashkenov et al. [27], the phonon modes for ZnO-A and ZnO-B are presented in Table 3. The peak at 383.8 and 380.5 cm−1 corresponds to A1(TO), 439.3 cm−1 corresponds to E2(high), and 590.0 and 586.8 cm−1 corresponds to E1(LO). All the allowed Raman phonon modes are clearly visible for both ZnO-A and ZnO-B samples, suggesting that the results are comparable to those reported results by Ashkenov et al. In addition, a second-order phonon 2E2 (M) was observed at 334.4 and 333.9 cm−1 for ZnO-A and ZnO-B, respectively [28,29]. Both ZnO-A and ZnO-B were assigned to the same E2 (high) phonon mode at 439.3 cm−1. In a previous study, changes in the frequency of the E2 (high) peak and FWHM were elucidated to be functions of the crystallite size of ZnO. As the crystallite size decreases, the E2(high) peak frequency decreases and FWHM increases [28]. The XRD data show that the crystallite sizes between ZnO-A and ZnO-B slightly differed. Hence, no significant changes could be observed in the frequency for Ram2an scattering. Nevertheless, the intensity of E2(high) phonon decreased as the crystallite size decreased relatively between ZnO-A and ZnO-B. This result indicates that E2(high) phonon exhibits size effect towards ZnO-A and ZnO-B [28].

Table 3: Phonon modes of Raman scattering for ZnO-A and ZnO-B at room temperature

A1(TO) 383.8 380.5

E2(high) 439.3 439.3

E1(LO) 590.0 586.8

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Figure 5: Raman spectra of ZnO-A and ZnO-B at room temperature.

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3.5. Evaluation of antibacterial properties and FESEM

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Figures 6 and 7 shows the FESEM images of S. sobrinus and S. mutans cells in the ZnO suspension, respectively. Both species were spherical shaped, nonmotile, and nonsporeforming Gram-positive bacteria. Moreover, these species grow in pairs. The species exhibited similar morphologies, because they belong to the same group of oral streptococci [30]. In addition, the EDS spectra showed no Zn in the untreated bacteria (Figs. 6d and 7d). Meanwhile, low-level Zn content was found on the surfaces of both strains (Figs. 6e, 6f, 7e, and 7f). This result demonstrates the existence of ZnO particle residuals on the bacteria surfaces treated at 2 mM.

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Figure 6: (a) S. Sobrinus untreated, (b) S. sobrinus treated with ZnO-A after 24 h at 2 mM, (c) S. sobrinus treated with ZnO-B after 24 h at 2 mM, (d-f) EDX spectra

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Figure 7: (a) S. mutans untreated, (b) S. mutans treated with ZnO-A after 24 h at 2 mM, (c) S. mutans treated with ZnO-B after 24 h at 2 mM, (d-f) EDX spectra

Figures 8a and b show the inhibition rates of ZnO-A and ZnO-B on S. sobrinus and S. mutans, respectively, after 24 h of incubation at 37 °C with the presence of 5% CO2. The inhibition rate after 24 h was calculated using the following formula [30]: (2)

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where 0% and 100% are the minimum and maximum values, respectively. As ZnO concentrations increased, the inhibition rates of S. sobrinus and S. mutans increased from 1 mM and 1.5 mM to 2 mM, and ZnO-A showed higher inhibition rate compared with ZnO-B at all concentrations. The inhibition rates of ZnO-A at 1, 1.5, and 2 mM for S. sobrinus increased gradually from 31.38%, 47.51%, and 82.32%, while those for ZnO-B were 22.35%, 31.38%, and 78.54%, respectively. By contrast, the inhibition rates for S. mutans at 1, 1.5, and 2 mM for ZnO-A were smaller at 70.91%, 80.15%, and 88.55%, whereas those for ZnO-B were 69.33%, 76.35%, and 86.33%, respectively. Both samples at 1, 1.5 and 2mM are significatly lower the bacteria growth (n= 3, p<0.05) except for 1 mM on S. sobrinus.

Figure 8: Percentage inhibition for ZnO-A and ZnO-B on (a) S. sobrinus and (b) S. mutans. Plots represent percentage inhibition ± SD (n=3, **p<0.05)

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The morphology of these nanopowders as depict by EFTEM images and ESI elemental mapping in Fig. 2 were considered when the inhibition rates of the nanopowders for both species were compared. ZnO-A consisted of rod-like shape that would affect the total surface area and particle sizes in addition to the higher O:Zn ratio on the specific particle surfaces compared with ZnO-B. Antibacterial activity increased with decreasing particle size, because hydrogen peroxide (H2O2) generated from ZnO surface is effective for inhibiting bacterial growth. Therefore, the smaller particles size will yield to a higher number of ZnO nanopowder particles per unit volume for H2O2 generation as shown by Yamamoto [18]. Furthermore, the small size of ZnO-A provided higher surface-to-area ratio compared with ZnO-B and increased the potential interaction between ZnO with building elements of the outer membrane. These characteristics led to structural changes and cell degradation [3]. These results are in close agreement with the XRD and DLS data, which showed the smaller crystallite sizes of ZnO-A compared with ZnO-B. Given the different morphology conditions, lower inhibition rate of ZnO-B compared with ZnO-A seems relevant for both microorganisms. Thus, larger surface-to-volume ratio could provide a more efficient mean for antibacterial activity [31]. Moreover, the release of H2O2 from the surface of ZnO, which cause damage to microorganism as described by Sunada et al. [32], is related to the defects in ZnO as revealed in the PL spectra. The positions of the green and red luminescence originating from the vacancies are in good agreement with the inhibition rate results in the antibacterial activity. This result indicates that the defects lead in releasing H2O2 to complete the antibacterial mechanism. Several mechanisms have been proposed for the antibacterial activity of nanomaterials. However, one of the mechanism responsible for the antibacterial activity of ZnO is the release of H2O2 [18]. The hole from the created electron–hole pairs (e−–h+) from the surface defects will split the H2O molecules into OH− and H+. O−2 transforms from dissolved oxygen molecules and generates (HO2 ) radicals after reacting with H+. Subsequently, the collision between HO2 and electrons will lead to HO2 production, resulting in H2O2 molecules after reacting with hydrogen ions. The generated H2O2 can penetrate the cell membrane and affect the bacteria system or kill the bacteria [33].

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3.5.1 ROS detection and Zn ion release In conjunction with the H2O2 released, the inhibition of bacteria could be attributed to other ROS that exist intacellularly within the cell, such as O−2and hydroxyl radical [15,34]. Therefore, the detection of ROS, including all type of oxygen species, was evaluated using H 2DCFDA probe to identify the percentage of total ROS in the cell that caused the inhibition or cell death. The H2DCFDA probe is a cell-permeant molecule and passively diffuses into the cell and trapped inside the cell. The H2DCFDA, which is a nonfluorescent chemical, is oxidized into DCF by ROS. The DCF is a fluorescent chemical that can be measured by fluorescence spectroscopy. Therefore, upon stimulation, the production of ROS will increase the fluorescence signal, which is dependent on concentration of ZnO. Figure 9 shows the percentages of fluorescence intensity detected for the control (untreated bacteria) and ZnO treated on S. sobrinus and S. mutans at 1, 1.5, and 2 mM. The control or untreated bacteria have 100% fluorescence intensity. This result indicates that ROS naturally occur within a healthy cell. In principle, ROS could be generated by exogenous factor or intracellularly

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from various sources involved in biochemical and physiological processes [35] and related to spontaneous hydrolysis, atmospheric oxidation, and/or light-induced oxidation [36,37,38]. Thus, the fluorescence intensity increased as the bacteria were treated with increasing ZnO concentration. This phenomenon is attributed to the ROS generated by ZnO. Higher concentration of ZnO will increase the ROS level and lead to higher fluorescence in the bacteria cells. One of the reasons is the higher electron–hole pairs in concentrated ZnO, leading to higher ROS generation. Furthermore, ZnO-A exhibited higher fluorescence intensity compared with ZnO-B. This phenomenon is related to enhanced oxygen level and smaller particle size of ZnO-A as shown in the elemental mapping by EFTEM in Fig. 3 and DLS spectra in Fig. 2. Both ZnO (1, 1.5, 2 mM) samples significantly generate ROS and lead to percentage of fluorescence.

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Figure 9: Percentage of fluorescence for control and ZnO treated at 1, 1.5 and 2 mM towards (a) S. sobrinus and (b) S. mutans. Plots represent average percentage of fluorescence ± SD (n=3, *p< 0.05)

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The release of Zn2+ ion has also been reported to contribute in antibacterial activity [39]. Table 4 shows the dissolved Zn2+ ion released in 1, 1.5, and 2 mM of ZnO-A and ZnO-B after 24 h. The dissolved Zn2+ ion was slightly higher in ZnO-A than in ZnO-B and dependent on ZnO concentration. This phenomenon may be attributed to the better solubity of ZnO-A because of its smaller particle size [40,41]. Moreover, the dissolved Zn2+ ion is involved in antibacterial activity [42,43]. The Zn2+ ion can penetrate into the bacteria cells and produce the toxic oxygen species. This oxygen species will damage the DNA and cell membrane or cell proteins, resulting in bacterial inhibition and/or bacterial death [44]. These results are in good agreement with the inhibition rates of S. sobrinus and S. mutans as shown in Fig. 8. At all concentrations, the Zn2+ ion dissolved is lower than the cytotoxic limits of 10 ppm as reported by Ferraris et. al [39]. Table 4: The release of Zn2+ ion from ZnO-A and ZnO-B after 24 h ZnO-A ZnO-B Concentration ppm ppm 1.0 mM 0.134 0.092 1.5 mM 0.143 0.104 2.0 mM 0.152 0.115

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In this study, although the cell wall of both microorganisms S.sobrinus (Fig 6b, 6c) and S.mutans (Fig 7b, 7c) did not rupture, the phenomena observed could have been caused by the interruption of protein synthesis and interference with DNA replication by the diffusion of oxygen species generated by the surfaces of ZnO nanoparticles into the cells. Four modes, namely, cell wall, protein synthesis (ribosomes), cytoplasmic membrane, and nucleic acid replication (DNA, mRNA), describe the target sites of antibacterial activity in the cells, and one or more of the target sites are probably involved (Fig. 10) [12,45,46]. If ribosomes are targeted, the oxygen species will interfere with the translocation, inhibiting protein synthesis that causes a drastic drop in bacterial growth. Moreover, interference in nucleic acid replication (DNA, mRNA) will suppress bacterial growth and increase the inhibition of bacterial growth [12] without damaging the cross-linking of the cell wall or disrupting the cell membrane.The FESEM images of bacteria as shown in Fig 6b-c and Fig 7b-c shows a good agreement with the cause mention above. Although there are no obvious rupture on bacteria’s cell wall, but the alteration and distortion on bacteria surface is believe to be originating from the membrane damage and effects from the generated ROS.

Conclusion

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Figure 10: Targeting sites at the cellular level in antibacterial activity; DNA and mRNA replication, protein synthesis (ribosomes), cell membrane and cell wall.

In summary, polycrystalline wurtzite structure of the obtained ZnO-A and ZnO-B nanostructures were confirmed to have good crystallinity with crystallite sizes of 36.6 and 39.4 nm, respectively, by XRD. Meanwhile, DLS revealed that the 91.9% of ZnO-A particles was 21.82 nm in size, and 85.7% of ZnO-B particles was 52.21 nm. PL measurement indicated the NBE peak and confirmed the existence of zinc vacancies or excess oxygen at the green and red emission peaks. These defects play a role in the antibacterial activity. ZnO-A exhibited higher inhibition rate compared with ZnOB at all concentrations for S. sobrinus and S. mutans. The contribution of zinc vacancy or excess oxygen in the antibacterial activity was apparently relevant for the antibacterial activity, in which the production of H2O2 and other oxygen species may damage or alter the cells. The EFTEM images supported the results shown by PL and inhibition rates for both ZnO-A and ZnO-B. The results show different morphologies between ZnO-A and ZnO-B, indicating different particle sizes and surface-to-area ratios. These characteristics lead to the effective antibacterial activity.

ACCEPTED MANUSCRIPT Acknowledgements The authors gratefully acknowledge support from Research University Individual Grant (RUI) Universiti Sains Malaysia [1001/PFIZIK/814174], [1001/PPSG/812160], Short Term Grant Universiti Sains Malaysia [304/PFIZIK/6312077], and Institute of Nano-optoelectronics Research & Technology (INOR), Universiti Sains Malaysia. Also Microbiology and Parasitology Laboratory, School of Medicine, and Craniofacial Science Laboratory, School of Dentistry, Health Campus, Universiti Sains Malaysia.

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This paper report the effect of different morphology of ZnO nanostructure as antibacterial agent towards Streptococcus sobrinus and Streptococcus mutans The nanostructure have been characterized for its optical, structure and morphology. Thus, relate these properties with antibacterial activity It shows that smaller particle size of ZnO revealed by EFTEM, XRD and DLS give higher inhibition on Streptococcus sobrinus and Streptococcus mutans. Moreover, defects of ion vacancy detected by photoluminescence spectroscopy influenced the antibacterial activity of ZnO.

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