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Volume 1, December 2016, pages 13-20

Chemical synthesis and characterization of zinc nanoparticles and its antibacterial effect against pathogenic bacteria

Mansi Mishra1*, Nida Khan1, Sandeep Sirohi2

1*Department of Biotechnology, Meerut Institute of Engineering and Technology, Meerut, Uttar Pradesh, India
2Department of Biotechnology, Mewar University, Gangrar, Chittorgarh, Rajasthan, India


* Corresponding Author Email: maansi_mishra@yahoo.co.in | Tel: +919792148887

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Abstract

Metal nanoparticles have been intensively studied within the past decade. Nanosized materials have been an important subject  in basic and applied sciences. Zinc oxide nanoparticles have received considerable attention due to their unique antibacterial, antifungal, and UV filtering properties, high catalytic and photochemical activity. The objective of this work is to synthesize Zinc oxide nanoparticles using chemical method and characterize zinc oxide nanoparticles. Further antibacterial activity of Zinc oxide nanoparticles and antibiotics was analyzed against various pathogenic bacteria. It was found that thte minimum of zones of inhibition was found (0.2cm)   in P. aeruginosaS. typhimurium, M. luteus, and A. hydrophila. The maximum of zones of inhibition was observed (0.5 cm) in S. epidermis and E. aerogens. The observed results, indicates higher antibacterial activities in Tetracycline then Cholramphenicol> Penicillin and the antibacterial activities of antibiotics are slightly less against eight pathogenic bacterial species as compared to chemically synthesized zinc oxide nanoparticles.

Keywords

Nanoparticles, Zinc oxide, Antibacterial activity, Antibiotics,  Pathogenic bacteria,  Photochemical activity

Introduction

Nanotechnology is emerging as a rapidly growing field with its application in science and technology for the purpose of manufacturing new materials at the nanoscale level (Albrecht et al. 2006). Nanotechnology is understood as research and technology development at the atomic, molecular or macromolecular levels using a length scale of approximately 1-100 nm in any dimension including the ability to control or manipulate matter on an atomic scale. An important additional distinction should be made between nanostructured thin films or other fixed nanometer-scale objects (such as the circuits within computer microprocessors) and free nanoparticles (Tyagi et al. 2012).Ultrafine semiconductors particles are of great scientific interest as they are effectively a bridge
between bulk materials and atomic or molecular structures. They possess special properties such as a  large  surface to volume ratio, increased activity, special electronic properties and unexpected optical properties as they are small enough to confine their electrons and produce quantum effects. Among various semiconducting materials, zinc oxide (ZnO) is a distinctive electronic and photonic wurtzite n-type semiconductor with a wide direct band gap of 3.37 eV and a high exciton binding energy (60 meV) at room temperature (Wang 2004). The high exciton binding energy of ZnO would allow for excitonic transitions even at room temperature, which could mean high radiative recombination efficiency for spontaneous emission as well as a lower threshold voltage for laser emission microorganisms such as bacteria, actinomycetes, yeasts, and fungi continue to be researched and investigated in synthesis of metallic nanoparticles, the use of parts of whole plants for similar nanoparticle biosynthesis methodologies is an exciting possibility that is relatively unexplored and underexploited (Tyagi et al. 2012).Synthesis of AgNPs by reduction of aqueous Ag+ ions by the extract of onion, garlic, papaya and apple and their toxicity has been reported (Tyagi et al. 2016).ZnO nanoparticles are promising candidates for various applications, such as nanogenerators (Gao et al. 2005), gas sensors (Cheng et al. 2004), biosensors (Topoglidis et al. 2001), solar cells (Hames et al. 2010), varistors (Jun et al. 2002), photodetectors (Sharma et al. 2003), and photocatalysts (Kamat et al. 2002). ZnO is a mechanical actuators and piezoelectric sensors (Ko et al. 2003;  Zaouk et al. 2006). Zinc Oxide is widely used in a number of application like varistors (Rana et al. 2010) UV lasers, gas sensors, photoprinting, electrochemical nanodevice, sunscreen lotion cosmetics  and  medicated  creams (Ravichandrika et al. 2012) due to its several properties such as good  transparency,  high  electron mobility, strong room temperature luminescence. From the literature survey, it was found that various approaches for the preparation of ZnO nanopowders have been developed, namely, sol-gel, microemulsion, thermal decomposition of organic precursor, spray pyrolysis, electrodeposition, ultrasonic, microwave-assisted  techniques, chemical vapor deposition, and hydrothermal and precipitation methods (Singhai et al. 1997;  Rataboul et al. 2002; Okuyama & Wuled Lenggoro 2003; Wei & Chang 2008; Hu et al. 2004; Wu & Liu 2002; Zhai et al. 2008; Bitenc et al. 2008). Most of these techniques were not extensively used on a large scale, but chemical synthesis has been widely used due to its simplicity and is less expensive. In the present work, ZnO nanoparticles were synthesized by wet chemical method which was then characterized using UV-VIS spectroscopy and FTIR analysis. The antibacterial activity of synthesized ZnO nanoparticles was checked against various pathogenic bacteria and three antibiotics.

Materials and Methods

2.1. Sample collection
All chemicals used in this experiment were of the highest purity and obtained from Sigma and Merck. Zinc nitrate soluble starch and sodium hydroxide of analytical grade were used for chemical  synthesis  of  zinc  oxide nanoparticles. A solution of 0.1mol was prepared with 1% starch soluble starch and 300ml (0.2 Mol)  sodium  hydroxide  was prepared. For antibacterial activities against antibiotics  we  procured antibiotics    disc.
2.2. Collection  of pathogens
The toxic effects of zincoxide nanoparticles against eight pathogenic bacteria were used for and three antibiotics (Tetracycline, Chloramphenicol  and  Penicillin)  were  used.  Pseudomonas  aeroginosa  (MTCC-3160),  Bacillus cereus (MTCC-1305), Salmonella typhimurium (MTCC-1253), Micrococcus luteus (MTCC-1809), Staphylococcus epidermis (MTCC-3086), Aeromonas pneumonia (MTCC-3384), Aeromonashydrophila (MTCC-1739) and Enterobacter  aerogens  (MTCC-2823)  obtained from  IMTECH Chandigarh.
2.3. Chemical synthesis of zinc oxide nanoparticles  (CH-ZnONPs)
The ZnO nanoparticles was prepared by wet chemical method using zinc nitrate and sodium hydroxides  precursors and soluble starch as stabilizing agent. Soluble starch (0.5%) was dissolved in 500 ml of distilled water and treated in microwave oven for complete solubilization. Zinc nitrate, 14.874 g (0.1 Mol), was added in the above solution. Then the solution will keep under constant stirring at room temperature using magnetic stirrer for one hour. After complete dissolution of zinc nitrate, 300ml (0.2 Mol), of sodium hydroxide solution was added under constant stirring, drop by drop touching the walls of the vessel. The reaction was allowed to proceed for 2 hrs after complete addition of sodium hydroxide. After the completion of reaction, the solution was allowed to settle for overnight and then supernatant solution was discarded carefully. The remaining solution was centrifuged at 10,000 × g for 10 min and the supernatant was discarded. These produced nanoparticles were washed three times using distilled water. Washing was carried out to remove the byproducts and the excessive starch that were bound with the nanoparticles. After washing, the nanoparticles were dried at 80°C for overnight. During drying, complete conversion of Zn(OH)2 into ZnO takes place.
2.4. Characterization of CH-ZnONPs
The ZnO nanoparticle was characterized in a JASCO-V-530, UV-VIS spectrophotometer, to know the kinetic behavior of zinc oxide nanoparticles. The scanning range for the samples was  280-700 nm  at a scan speed of  400 nm/min. Base line correction of the spectrophotometer was carried out by using a blank reference. The UV- Vis spectra analysis of ZnONPs of all the samples was recorded.  The reduction of pure Zn+2   ions was monitored by measuring the UV-Vis spectrum of the reaction medium at 2 h after diluting a small aliquot of the sample into distilled  water. FTIR analysis was  also  done for the  same.
2.5. Antibacterial activity
Antibacterial activities of CH-ZnONPs were observed against eight pathogenic bacteria in two different sets of experminent.In first set of experiment antibacterial activities of CH-ZnONPs alone was studied while in next set of experiments antibacterial activities of CH-ZnONPs in combination with three antibiotics were studied. Antibacterial activities were determined, using the disc diffusion method. Approximately 20 mL of molten and cooled media (NA/SDA)  was poured in sterilized petri dishes. The  plates  were left overnight at room  temperature to check for    any contamination to  appear. The test  organisms were grown  in selected broth for  24  h. The  plates  containing    the test organism and CH-ZnONPs were incubated at 370C for 24 - 48 h. The plates were examined for evidence of zones of inhibition, which appear as a clear area around the disc. The diameter of such zones of inhibition was measured using a meter ruler and the mean value for each organism was recorded and expressed in centimeter.

Results and Discussions

In our study we did comparative analysis of antibacterial activity of chemical synthesized  zinc oxide nanoparticles   and commercially available  antibiotics  such  as  Tetracycline,  Chloramphenicol  and  Penicillin.Secondly we  wanted to investigate whether chemically synthesized zinc oxide can exhibit syngerstic effect with commercially available antibiotics to effectively target multidrug restistant bacteria? Our results demonstrates that the synerstic effect of chemically synthesized zinc oxide nanoparticles  and  antibiotics  results  in  the  maxium  antibacterial  activity  and can  even  target  multidrug  resistant bacteria.
3.1. Chemical  synthesis of ZnO nanoparticles
Zinc oxide nanoparticles have some excellent properties like exceptional mechanical strength and good antistatic, antibacterial and UV absorption properties. The zinc oxide nanoparticles were synthesized using wet chemical method (Figure 1).
3.2. Characterization of CH-ZnONPs
The UV-Visible (UV-Vis) spectrum of ZnONPs was recorded and is shown in the figure. Absorption spectrum of ZnONPs was in the range of 390- 420 nm. The data obtained from  UV-Visible  graphs  and  FTIR  annalysis  confirmed the synthesis of zinc oxide nanoparticles (Figure 2 &    3).
3.3. Antibacterial test for zinc oxide nanoparticles and antibiotics against pathogens
3.3.1. Antibacterial activity of  CH-ZnONPs
We investigated the antibacterial properties of CH-ZnONPs against the eight pathogenic bacteria (P.   aeroginosa,
B. cereus, S. typhimurium, M. luteus, S. epidermis, A. pneumonia, A. hydrophila and E. aerogens) on agar plates.     The observed results indicate that the high concentration (100%) of CH-ZnONPs leads to effective antibacterial activities against all pathogenic bacteria of CH-ZnONPs. The minimum of zones of inhibition was  observed  in  (0.2cm) in P. aeroginosa, S. typhimurium, M. luteus, and A. hydrophila and the maximum  of zones of  inhibition     (0.5 cm) was observed in in S. epidermis and E. aerogens observed (Figure 4 & 5).
3.3.2. Antibacterial  activity of  antibiotics  against pathogens
In the second set of experiments we  had checked  the antibacterial activities  of  eight pathogenic  bacteria against  three antibiotics e.g.  Penicillin (P), Tetracycline (T) and Cholramphenicol (CP)  on disc diffusion methods on NA   agar plate (Figure 6). The three bacterial species E.aerogens, P.aeroginosa and B. cereus were sensitive against Tetracyclin (T) drug and it exhibited 0.6cm, 0.5cm and 0.5 cm zones of inhibition respectively,  whereas  the  remaining two drugs Penicillin (P) and (CP) were inefficient against these three bacterial species. In case of P. aeroginosa, B. cereus and E.aerogens highest antibacterial activities was observed in drug Tetracyclin then Cholramphenicol followed by Penicillin.For S. typhimurium and M. luteus equal antibacterial activities were found  with drug Cholramphenicol and Tetracyclin while it was resistant to Penicillin. For A. pneumonia the highest antibacterial activities was observed in drug Tetracyclin  followed  by  Cholramphenicol  while  in  case  of  S.epidermis highest antibacterial activities was observed in drug Cholramphenicol followed by Tetracyclin. A. pneumonia and S.epidermis were resistant to Penicillin. In  case  of  A.  hydrophila  antibacterial  activity  was  observed only in Tetracyclin. On the other hands, we can say that these 4 bacterial species (M.  luteus,  S.  typhimurium, A. pneumonia and A. hydrophila) out of  8 bacterial  species  consired  in  our  study are  resistant  to these  drugs.
The observed results, indicates highest antibacterial activities in case of antibiotics was observed in Tetracyclin followed by Cholramphenicol and Penicillin. It was observed that antibacterial activities of antibiotics are slightly    less against  eight pathogenic  bacterial species  as  compared to CH-ZnONPs  (Figure 7 &   8).
3.3.3. Antibacterial activity of CH-ZnONPs + antibiotic complex
In this comparative study of ZnONPs and three drugs are combined, and observed results indicate that the higher concentration (100%) of AgNPs+ antibiotic complex are more efficient against pathogens(Figure11). On the other hands, we can say our results demonstrate that ZnONPs undergo a concentration-dependent interaction with the pathogenic bacterial species (Figure 9 & 10).

Conclusion

Zinc Oxide nanoparticles were synthesized using starch sodium hydroxide and zi c nitrate. The chemically synthesized zinc oxide nanoparticles were characterized using UV-Vis spectroscopy and FTIR spectroscopy. Microorganisms used for antibacterial activity are P. aeroginosa, B.  cereus,  S.  typhimurium,  M.  luteus,  S.  epidermis, A. pneumonia, A. hydrophila and E. aerogens. The three antibiotics were used  Chloramphenicol,  Tetracyclin and Penicillin for the antibacterial activity. The antibacterial activity performance of ZnO nanoparticles  was done by using disc diffusion method. From the above study it was assumed that the zone of inhibition was observed against different pathogenic bacteria for zinc  oxide nanoparticles and the antibiotics. But when antibiotic  disc was mixed zinc oxide nanooparticles zone of inhibition was increased which illustrates that the anitibacterial activity was increased when zincoxide nanoparticles  was  mixed  with  antibiotics  against  various  pathogenic  bacteria

Acknowledgement

Authors are thankful to the Department of Biotechnology, MIET, Meerut for providing infrastructure and financial support for the  project.

References

  1. Albrecht M, Evans C, Raston C.2006.Green chemistry and the health implications of nanoparticles. Green Chemistry. 8:417.
  2. Bitenc M, Marinšek M, Crnjak Orel Z.2008.Preparation and characterization of zinc hydroxide carbonate and porous zinc oxide particles. Journal of the European Ceramic Society. 28:2915-2921.
  3. Cheng X, Zhao H, Huo L, Gao S, Zhao J. 2004.ZnO nanoparticulate thin film: preparation, characterization and gas- sensing property. Sensors and Actuators B: Chemical. 102:248-252.
  4. Gao P, Ding Y, Mai W, Hughes W, Lao C, Wang Z.2005.Conversion of zinc oxide nanobelts into superlattice- structured nanohelices. science.  309:1700-1704.
  5. Hames Y, Alpaslan Z, Kösemen A, San S, Yerli Y.2010.Electrochemically grown ZnO nanorods for hybrid solar cell applications.  Solar  Energy. 84:426-431.
  6. Hu X, Zhu Y,Wang S.2004.Sonochemical and microwave-assisted synthesis of linked single-crystalline ZnO rods. Materials Chemistry and Physics. 88:421-426.
  7. Jun W, Changsheng X, Zikui B, Bailin Z, Kaijin H, Run W.2002.Preparation of ZnO-glass varistor from tetrapod  ZnOnanopowders. Materials Science and Engineering: B. 95:157-161.
  8. Kamat P, Huehn R, Nicolaescu R.2002. A “sense and shoot” approach for photocatalytic degradation of organic contaminants  in  water.  The  Journal of  Physical Chemistry B. 106:788-794.
  9. Ko S, Kim Y, Lee S, Choi S, Kim S.2003.Micromachined piezoelectric membrane acoustic device. Sensors and Actuators A: Physical. 103:130-134.
  10. Okuyama K , Wuled Lenggoro I.2003. Preparation of nanoparticles via spray route. Chemical Engineering Science. 58:537-547.
  11. Rana S, Singh P, Sharma A, Carbonar A, Dogra R. 2010.Synthesis and characterization of pure and doped ZnO  nanoparticles. Journal of Optoelectronics and Advanced Materials. 12:257 - 261.
  12. Rataboul F, Nayral C, Casanove M, Maisonnat A, Chaudret B.2002.Synthesis and characterization of monodisperse zinc and zinc oxide nanoparticles from the organometallic precursor [Zn(C6H11)2]. Journal of Organometallic Chemistry. 643-644:307-312.
  13. Ravichandrika K, Kiranmayi P, Ravikumar R.2012.Synthesis, characterization and antibacterial activity Of ZnO  nanoparticles. International Journal of Pharmacy and Pharmaceutical Sciences. 4:336-338.
  14. Sharma P, Sreenivas K, Rao K.2003.Analysis of ultraviolet photoconductivity in ZnO films prepared by unbalanced magnetron  sputtering. J  Appl  Phys. 93:3963.
  15. Singhai M, Chhabra V, Kang P, Shah D.1997.Synthesis of ZnO nanoparticles for varistor application using Zn- substituted aerosol to microemulsion. Materials Research Bulletin. 32:239-247.
  16. Topoglidis E, Cass A, O'Regan B, Durrant J.2001.Immobilisation and bioelectrochemistry of proteins on nanoporous  TiO2 and ZnO films. Journal of Electroanalytical Chemistry.   517:20-27.
  17. Tyagi  PK,  Shruti, Sarsar  V, Ahuja  A.2012.Synthesis of  metal Nanoparticles: A biological prospective for analysis.   Int.J. Pharm. Innov.4:48-60.
  18. Tyagi P, Mishra M, Khan N, Tyagi S, Sirohi S.2016.Toxicological study of silver nanoparticles on gut microbial community probiotic. Environmental Nanotechnology, Monitoring & Management.   5:36-43.
  19. Wang Z. 2004. Nanostructures of zinc oxide. Materials Today. 7:26-33.
  20. Wei Y, Chang P.2008.Characteristics of nano zinc oxide synthesized under ultrasonic condition. Journal of Physics and Chemistry of Solids. 69:688-692.
  21. Wu J, Liu S.2002.Low-temperature growth of well-aligned ZnO nanorods by chemical vapor deposition. Adv Mater. 14:215-218.
  22. Zaouk D, Zaatar Y, Asmar R, Jabbour J.2006.Piezoelectric zinc oxide by electrostatic spray pyrolysis. Microelectronics Journal. 37:1276-1279.
  23. Zhai H, Wu W, Lu F, Wang H, Wang C.2008. Effects of ammonia and cetyltrimethylammonium bromide (CTAB) on morphologies of ZnOnano- and micromaterials under solvothermal process. Materials Chemistry and Physics. 112:1024-1028.