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Fast Growth of ZnO Nanorods and Their Antimicrobial Properties

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Fast Growth of ZnO Nanorods and Their Antimicrobial Properties
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     Abstract —The present study reportsa simple, fast, and inexpensive sonochemistry method for ZnO nanorod synthesis. The ZnO nanorods were grown on glass substrates at room ambient. The successful growth of ZnO nanorods were determined by optical microscope and scanning electron microscope (SEM). The presence of ZnO crystal was investigated with Raman spectroscopy and energy dispersive X-ray spectroscopy (EDS). The antimicrobial properties of the ZnO nanorods against  Escherichia coli  (Gram-negative) and  Bacillus subtilis  (Gram-positive) were investigated using live and dead assays and agar flipping tests. Results show that ZnO nanorods present higher toxicity to  B. subtilis cells. The results suggest that sonochemistry can be utilized to grow ZnO nanorods on glass surfaces for antimicrobial purposes. BACKGROUND Zinc oxide (ZnO) is a group II-VI semiconductor with a wide range energy band (3.37eV) and it has a large exciton binding energy (60meV). Recently, researchers have produced ZnO nanostructures for various applications, such as gas, pH and temperature sensors as well as photo detectors [1, 2]. ZnO is stable at biological pH values and has also been found to be biocompatible and biodegradable [3]. ZnO presents high isoelectric point, which makes it suitable as an immobilization matrix [4]. Recent studies also showed that different ZnO nanostructures can exhibit antibacterial properties against various bacterial species, such as Staphylococcus epidermidis , Pseudomonas aeruginosa , Staphylococcus aureus , and  Escherichia coli [6, 7]. The antimicrobial properties of ZnO nanostructures is of extreme importance for biomedical applications, since the fourth leading cause of death are hospital acquired infections (i.e. nosocomial infections) with more than 2 million cases reported annually, and the majority of these nosocomial infections, about 60-70%, are associated with bacterial contamination of implanted medical devices [8]. The modification of implant surfaces has been suggested to be the most efficient strategy to combat infections of biomedical implants [5]. Sonochemistry is a promising method to grow ZnO nanostructures on various surfaces, since it is cost effective, straight forward and environmentally friendly. Here we report the sonochemical synthesis of ZnO nanorods and their antimicrobial properties against two types of bacteria,  E. coli  and  B. subtilis . CURRENT RESULTS ZnO nanorods sonochemically grown on the glass surface were investigated in this study. They were prepared ultrasonically on a glass substrate by first depositing a seed layer of ZnO on the surface via sonicating zinc acetate __________________________ 1 University of Houston, Department of Civil and Environmental Engineering, Houston, TX, USA. 2 Gediz University, Department of Electrical and Electronics Engineering, Menemen, Izmir, TURKEY. φ All authors contributed equally to this work. *Contacting Author: Debora F. Rodrigues is with the University of Houston, Department of Civil and Environmental Engineering; N136 Engineering Building 1, Houston, TX, 77204, USA (phone: 1- 713-743-1495; fax: 1- 713-743-4260; e-mail: dfrigirodrigues@uh.edu). dihydrate, C 4 H 6 Zn · 2H 2 O, solution. The seed solution contained 0.005 M solution of zinc acetate dihydrate in isopropyl alcohol. To seed the surface, the substrate was immersed in the solution and sonicated for 30 min at 50% of the maximum amplitude of the 400 W ultrasonic probe working at 20 kHz. Afterwards, ZnO nanorods were grown using an aqueous solution of 0.04 M zinc nitrate tetrahydrate, Zn(NO 3 ) 2 · 4H 2 O, in 0.04 M Hexamethylenetetramine (CH 2 ) 6 N 4 . The solutions were mixed and stirred with a magnetic stirrer at 750 rpm for 5 min. The substrate was then immersed in the solution and sonicated for 15 min at 50% of the maximum amplitude of the 20 kHz ultrasonic probe. After this step, solution was refreshed and sonicated for another 15 min. ZnO nanorods were characterized by scanning electron microscopy (SEM) (Fig. 1), Raman spectroscopy (Fig. 2), and energy dispersive X-ray spectroscopy (EDS) (Fig. 3). The Raman experiments were performed by a confocal Raman microstage with an excitation wavelength of 488 nm at room temperature. Raman peaks observed at 97and 457 cm -1  were assigned as E 2  (low) and E 2  (high) vibrational modes of ZnO crystal, respectively. Vibrational position of E 2  (low) mode agrees well with bulk ZnO crystal, however, that of E 2  (high) mode is 20 cm -1  greater than the bulk [9]. This shift might be attributed to a free dangling end of oxygen sublattice in nanorods. The remaining prominent Raman modes are distinct features of nanostructures. EDS result shows that zinc and oxygen are prominent in material and indicate that oxygen to zinc ratio is greater than the one which agrees well with the Raman results. The toxicity of the sonochemically synthesized ZnO nanorods were investigated against  Escherichia coli  MG 1655 and  Bacillus subtilis  102. Cells were grown in tryptic soy broth (TSB) and washed with phosphate buffered saline (PBS) solution to eliminate the culture media components. For the toxicity assays, nanorods and microbial cells were incubated together for 2 and 5 h.  LIVE/DEAD Baclight bacterial viability kit (Invitrogen) was used to distinguish metabolically active cells from injured and dead cells using fluorescence microscope. The toxicity was expressed as the percentage ratio of the total number of inactive cells to the total number of cells (Fig. 4). Moreover, the agar flipping tests measured the cell growth in the presence of the nanomaterials. Growth zones were measured after 24 and 48 h incubation to see the long term antimicrobial effect of ZnO (Fig. 5). We observed that the bacterial species presented different degrees of inactivation in the presence of ZnO nanorods. However, ZnO nanorods presented higher toxic effects against  B. subtilis . The results also show that the longer the cell exposure to the ZnO, the higher was the number of cells inactivated. Hence, our proposed technique is simple, fast and cost effective for the synthesis of ZnO nanorods compared to other methods, such as the conventional vapor phase method, which is sophisticated and requires high process temperatures. In sonochemical method, working at room ambient is a big advantage unlike other conventional growth methods which require extreme temperatures and vacuum conditions. Besides, sonochemical method tends to consume less time and energy for the production of ZnO nanorods; a continuous layer of ZnO nanorods can be synthesized within a short time Fast Growth of ZnO Nanorods and Their Antimicrobial Properties Tugba O. Okyay 1 φ , Rukayya K. Bala 2 φ , Ramazan Atalay 2 , Yavuz Bayam 2 φ ,  Member, IEEE, and Debora F. Rodrigues* φ     period for various surfaces and for different antimicrobial applications. R EFERENCES   [1]   O. H. Eugene, and S. H. Jeong "Sonochemical method for fabricating a high - performance ZnO nanorod sensor for CO gas detection",  J. of Korean Phy. Soc. ,Vol. 59, pp.8-9, 2011. [2]   A. Menzel, K. Subannajui, F. Guder, D. Moser, O. Paul, and M. Zacharias, "Multifunctional ZnO nanowire-based sensor",  Adv. Funct. Mater. , Vol.22, pp. 4342-4348, 2011. [3]   J. Zhou, N. S. Xu, and Z. L. Wang. "Dissolving behavior and stability of ZnO nanowires in biological fluids; A case study on biodegradability and biocompatibility of ZnO nanostructures",  Adv.  Mater. , Vol. 18, pp 2432, 2006. [4]   A. Fulati, S. M. Usman, M. H. Asif, N. H. Alvi, M. Willander, C. Birannmark, P. Stralfors, S. I. Borjesson, F. Elinder, and B. Danielsson, "An intracellular glucose biosensor based on nanoflakeZnO", Sensors and Actuators B , Vol. 150, pp 673-680, 2010. [5]   M. L. W. Knetsch and L. H. Koole, "New Strategies in the Development of Antimicrobial Coatings: The Example of Increasing Usage of Silver and Silver Nanoparticles", Polymers , Vol. 3, pp. 340-366, 2011. [6]   T. Jansson, J. Zachary, C. Salzler, T. D. Zaveri, S. mehta, N. V. Dolgova, B. H. Chu, F. Ren, and B. G. Keselowsky, "Antibacterial effects of Zinc oxide nanorodsurfaces",  Journal of Nanosci. and  Nanotech. , Vol. 12, pp 7132-7138, 2012. [7]   R. S. Subhasree, D. Selvakumar, and N. S. Kumar, "Hydrothermal mediated synthesis of ZnOnanorodsand their antibacterial properties",  Letters in Applied NanoBioScience , Vol. 1, pp. 2- 7, 2012. [8]   R. P. Wenzel, "Health care-associated infections: major issues in the early years of the 21st century", Clin. Infect. Dis., Vol. 45, pp. 85-88, 2007. [9]   C. Wang, Z. Chen, Y. He, L. Li, and D. Zhang, "Raman scattering of ZnO nanofilms prepared by the laser molecular beam epitaxy",  Materials Science-Poland  , Vol. 28, No. 1, 2010. Fig. 1.SEM image of sonochemically grown ZnO nanorods. Fig. 2.Raman spectrum of ZnO nanorods grown on glass substrate. Fig. 3.EDS spectrum of nanorods confirming glass surface coated with ZnO. E. coliB. subtilis  020406080100    T  o  x   i  c   i   t  y   (   %    d  e  a   d  c  e   l   l  s   )  Control-2h Control-5h ZnO (2h) ZnO (5h)  Fig. 4.Toxicity results of ZnO nanorods.  E. coli  and  B. subtilis  cells were incubated with ZnO coated glass substrates for 2 and 5 h. Controls have no nanomaterial. E. coli 24hE. coli 48hB. subtilis 24hB. subtilis 48h  0123      C  e   l   l  g  r  o  w   t   h  z  o  n  e  s   (  c  m   )  Control-2h Control-5h Control-24h Control-48h ZnO (2h) ZnO (5h) ZnO (24h) ZnO (48h) Fig. 5.Growth zones on agar plates.  E. coli  and  B. subtilis  cells were grown for 48 h and zone measurements were accomplished at 24h and 48h. Higher toxicity presents lower growth zone. 1 ɥ m
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