Study on antimicrobial activity of undoped and Mn

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Scholars research library Archives of Applied Science Research, 2011, 3 (6):173-179

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Study on antimicrobial activity of undoped and Mn doped ZnO nanoparticles synthesized by microwave irradiation Bindiya H. Sonia #, M. P. Deshpandea *, Sandip V. Bhatta , Sunil H. Chakia and Haresh Kaheriab a

Department of Physics, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India B. R. D. school of Biosciences, Sardar Patel University, Vallabh Vidyanagar, India ______________________________________________________________________________ b

ABSTRACT Nanotechnology is an emerging interdisciplinary subject that has been booming in many areas during the recent decade. Looking to this aspect, we synthesized undoped and Mn doped ZnO nanoparticles by chemical method using microwave irradiation for 20 sec at 720 watt. Energy dispersive analysis of X-rays (EDAX) confirmed that the synthesized nanoparticles do not contain any foreign elements in them. The particle size, shape and morphology were examined by Transmission electron microscopy (TEM). The potential toxicity of nanosized ZnO and Mn doped ZnO were investigated using Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Serratia marceseus and Proteus vulgaris bacteria as test organism. The result showed that nanoparticles dispersed in methanol medium enhanced the antibacterial activity. Keywords: ZnO nanoparticles, particle size, microwave irradiation, antimicrobial activity. ______________________________________________________________________________ INTRODUCTION In recent years, ZnO being a wide band-gap semiconductor (3.3eV) has received increased attention regarding potential electronic applications due to its unique optical, electrical and chemical properties. ZnO nanostructures are currently being widely investigated because of their great potential for electronic, photonic and spintronics applications and also for use in biosensors [1–2], bioimaging [3-4], drug delivery [5] and other such biological applications. The availability of a wide range of nanostructures makes ZnO an ideal material for nanoscale optoelectronics and piezoelectric nanogenerators as well as in biotechnology [6]. The utilization of microwave irradiation in the preparation of nanoparticles is reported in recent years [7]. Compared to the conventional methods, the microwave synthesis has the advantages of producing small particle size metal oxide with high purity owing to short reaction time. It is found that this method is fast, mild, energy efficient and environment friendly route to produce ZnO nanoparticles. Surface area and surface defect play an important role in photocatalyst activities of metal oxides. The reason is that doping of metal oxide and/or transition metals [like Mn] increase the surface defects [8]. In addition, it affects the optical and electronic properties and can presumably shift the optical 173 Scholars research library

Bindiya H. Soni et al

Arch. Appl. Sci. Res., 2011, 3 (6):173-179 ______________________________________________________________________________

absorption towards the visible region [9]. Inorganic materials such as metal and metal oxide have attracted lots of attention over the past decade due to their ability to withstand harsh process conditions. Of the inorganic materials, metal oxides such as TiO2, ZnO, MgO and CaO are of particular interest as they are not stable under harsh process conditions but also generally regarded as safe material to human beings and animals [10]. ZnO demonstrates significant growth inhibition of a broad spectrum of bacteria [11-12]. The suggested mechanism for the antibacterial activity of ZnO is based mainly on catalysis of formation of reactive oxygen species (ROS) from water and oxygen [13-17] that disrupt the integrity of the bacterial membrane, although additional mechanisms are also been suggested [18-24]. In the present work, we are reporting the synthesis of undoped and Mn doped (Mn content: 5 mol%, 10 mol% and 15 mol%) ZnO nanoparticles by microwave irradiation and their study on antimicrobial activity. MATERIALS AND METHODS 2.1 Synthesis of undoped and Mn doped ZnO nanoparticles To prepare undoped ZnO nanoparticles, following steps were undertaken: 0.2M of Zinc acetate dehydrate and 0.4M Sodium hydroxide in 25ml distilled water were prepared and mixed together aided by magnetic stirrer for 30 minutes. During this, 0.12gm PolyVinylPyrolidone (PVP) in 25 ml distilled water was added dropwise to the above mixture and then followed by microwave irradiation for 20 sec at 720 Watt. The precipitates which get formed are separated from the solution by filtration, washed several times with deionized water and absolute ethanol, and then dried in oven at 600C for 24 hrs to obtain ZnO nanoparticles. For Mn doped ZnO nanoparticles (with Mn content: 5 mol%, 10 mol% and 15 mol %), 45 mM of Zinc acetate dihydrate and X mM (X= 2.4, 4.4 and 6.4) of Manganese acetate tetrahydrate in 200 ml of deionized water were mixed together and stirred at room temperature followed by microwave treatment for 20 sec at 720 Watt. 140 mM of Potassium hydroxide in 200 ml deionized water was prepared and added drop by drop to the above solution. The precipitates which get formed were separated from the solution by filtration, washed several times with distilled water and absolute ethanol, and then dried in oven at 600C for 24 hrs to obtain Mn doped ZnO nanoparticles. 2.2 Characterization Energy dispersive analysis of X-rays (EDAX) attached to Philips EM 400 electron microscope is used for determining composition of undoped and Mn doped ZnO nanoparticles. Transmission electron microscopy (TEM) patterns taken were used to study the size, shape and surface morphology of the synthesized nanoparticles. 2.3 Bacterial cultures and evaluation of antibacterial activities 2.3.1 Master solution To make dispersed undoped and Mn doped ZnO nanoparticles for the antibacterial test, we took a preset amount of dry undoped and Mn doped ZnO nanoparticles mixed with distilled water in a glass beaker separately with the aid of magnetic stirrer. Once the particles were dispersed in water the beaker was placed in an ultrasonicator. The reason for the use of sonication was to breakdown the agglomerate. After 30 minutes of sonication the so called dispersed undoped and Mn doped ZnO nanoparticles were produced which had concentration of 5mg/ml. Similar procedure was followed to obtain disperse undoped and Mn doped ZnO nanoparticles in methanol medium.

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Arch. Appl. Sci. Res., 2011, 3 (6):173-179 ______________________________________________________________________________ 2.3.2 Culture and Inoculums Standard bacterial cultures Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, Serratia marceseus, Proteus vulgaris were procured as from B. R. D school of Biosciences, S. P. University, Gujarat. The bacterial cultures were streaked to check the purity and a single colony was inoculated in 5ml Luria Bertani broth, incubated overnight at 300C, 150 rpm. We determined the optical densities of the actively growing cultures by measuring absorbance at 600nm.

2.3.3 Agar diffusion method The anti-bacterial/microbial activity was studied using gel diffusion method [6]. 100 l of actively growing target culture was affixed with 5ml of 1% top agar vortexed and pour onto the agar plate, allowed to solidify. The solidified plate was bored to form 5 wells of 4mm diameter using cork borer. The wells were then inoculated with 100 l aliquots of 5mg/ml dispersed undoped and Mn doped ZnO nanoparticles. The nanoparticles were allowed to diffuse into the agar followed by incubating the plates at 300C for 48 hours. Upon incubation the zone of clearance around the wells were measured and evaluated with respect to water/methanol media respectively. RESULTS AND DISCUSSION

Figure 1 EDAX Spectra of (a) undoped (b) 5 mol% Mn (c) 10 mol% Mn (d) 15 mol% Mn doped ZnO nanoparticles respectively



Arch. Appl. Sci. Res., 2011, 3 (6):173-179 ______________________________________________________________________________

3.1 Energy Dispersive Analysis of X-rays (EDAX) Figure 1 (a), (b), (c) and (d) shows the EDAX spectra of undoped and Mn doped (with Mn content: 5 mol%, 10 mol% and 15 mol %) ZnO nanoparticles and clearly reflects the absentia of other impurities and contaminants in the samples respectively. Table 1 gives the weight percentage observed from the EDAX spectra and those calculated for each element present in the sample. Table 1 Composition of ZnO and Mn doped ZnO nanoparticles Dopant concentration (mol %) Calculated 0 Observed Calculated 5 Observed Calculated 10 Observed Calculated 15 Observed

Zn (wt %) 80.33 81.07 76.81 77.15 73.24 80.90 69.62 78.84

O (wt %) 19.66 18.93 19.78 19.50 19.91 14.53 20.04 17.27

Mn (wt %) --------3.39 3.35 6.83 4.56 10.32 3.89

3.2 Transmission Electron Microscopy (TEM) TEM image and selected area electron diffraction pattern of undoped and Mn doped (with Mn content: 5 mol%, 10 mol% and 15 mol %) ZnO nanoparticles are shown in Figure 2 (a), (b), (c) and (d) respectively. The selected area electron diffraction pattern (SAED) indicates (inset) that the synthesized nanoparticles are polycrystalline in nature. The diffracting planes are indexed as (2 0 2), (1 1 2), (1 0 3), (1 0 0), (1 1 0), (1 0 2) and (1 0 1) reflections which are corresponding to the hexagonal phase of ZnO. A statistical analysis reveals that the particle size ranges from 3554nm, 14-27nm, 12-22nm and 6-10nm for undoped and Mn doped (with Mn content: 5 mol%, 10 mol% and 15 mol%) respectively. The particle size decreases with the increase in Mn concentration. After recording the diffraction pattern, we measured the distances used in the following equation derived from the Bragg equation to calculate the d spacing. =

…………….. (1)

In this expression, R is the distance from the central bright spot to corresponding rings, L is the camera length between specimen and photographic film and λ is the wavelength of the electron based on the accelerating voltage: 200 kV = 0.02736 Å. The lattice parameters for hexagonal undoped and Mn doped ZnO nanoparticles were estimated from the equation: =

+

…………….. (2)

where a and c are the lattice parameters and h, k and l are the Miller indices and is the interplanar spacing for the plane (h k l). The lattice parameters a and c calculated from TEM using equation (2) are found to be 3.23 Å and 5.18 Å respectively which are in the good resemblance with the lattice parameters values calculated from standard JCPDS (Joint Committee for Powder Diffraction Standard) file no.1314-13-2.



Arch. Appl. Sci. Res., 2011, 3 (6):173-179 ______________________________________________________________________________

Figure 2 TEM image and selected area electron diffraction pattern (SAED) of (a) undoped (b) 5 mol% Mn (c) 10 mol% Mn (d) 15 mol% Mn doped ZnO nanoparticles respectively

Figure 3 Evaluation of antibacterial action for Bacillus subtilis with samples dispersed in water (b) methanol



Arch. Appl. Sci. Res., 2011, 3 (6):173-179 ______________________________________________________________________________ 3.3 Evaluation of antibacterial properties Figure 3(a) and (b) shows agar diffusion test of undoped and Mn doped ZnO nanoparticles dispersed in water and methanol media respectively, against Bacillus subtilis with inhibition zone around the cavity. In Figure 3(a) number 2 indicates undoped ZnO nanoparticles and number 3, 4 and 5 indicates the 15 mol%, 10 mol% and 5 mol% Mn doped ZnO nanoparticles dispersed in water medium respectively. Similarly in Figure 3(b), number 6,7and 8 indicates 10 mol%, 15 mol% and 5 mol% Mn doped and 9 indicates undoped ZnO nanoparticles dispersed in methanol medium respectively. Table 2 Antibacterial assessment by agar diffusion method

Symbols indicated in figure Sample details Test organism Staphylococcus aureus Escherichia coli Bacillus subtilis Pseudomonas aeruginosa Serratia marceseus Proteus vulgaris

2 3 4 5 6 7 8 9 Undoped 15mol% 10mol% 5mol% 10mol% 15mol% 5mol% Undoped Sample dispersed in water medium Sample dispersed in methanol medium Diameter of the clear inhibition zone (mm) 5 2 2 4 1.5 5 6 3.5 nil nil nil nil 3 2.5 4.5 2.5 6.5 2.5 nil nil 7.5 2.5 9 5 1.5 nil nil nil 4.5 1 4 2.5 3 1 nil nil 3.5 3 3.5 1 3 1 0.5 1 3.5 5 4 4.5

The results of the quantitative antibacterial assessment by agar diffusion is shown in table 2 and it is observed that the size of the inhibition zone is higher for all samples dispersed in methanol medium than the water medium and hence we can say that the antibacterial activity by the test microorganism was higher for the samples dispersed in methanol. No antibacterial activity was found against the Escherichia coli for the samples dispersed in water medium. The Bacillus subtilis exhibits the strongest antibacterial activity in both media compared to the other test microorganisms and Escherichia coli show the weakest antibacterial activity. The reason for the difference in the antibacterial activity for different test microorganisms may be due to the difference in structure and thickness of the membrane cell wall. The antibacterial activity depends on the surface area and concentration, while the crystalline structure and particle shape have little effect. Water and methanol media don’t play any role in the zone exhibition. The presence of an inhibition zone clearly indicates that the mechanism of the biocidal action of ZnO involves disrupting the membrane. Doping of the ZnO nanoparticles did not result in appreciable enhancement of the activity. CONCLUSION In this present work, undoped and Mn doped nanoparticles were prepared by microwave irradiation. EDAX spectra showed the purity of the samples with no foreign contaminants and from TEM image, we concluded that the particle size decreases with the increase in Mn concentration. Selected area electron diffraction pattern (SAED) showed the ring pattern which confirms the polycrystalline nature of the nanoparticles and reflecting planes are corresponding to the wurtzite structure of the nanoparticles. Bioactivity of undoped and Mn doped ZnO nanoparticles were studied by antimicrobial activity of suspension with various media using a standard microbial method. The enhanced activity of undoped and Mn doped (with Mn content: 5 mol%, 10 mol% and 15 mol%) ZnO nanoparticles dispersed in methanol medium are attributed to the higher surface area to volume ratio due to higher dispersion compared to water medium. 178 Scholars research library


Arch. Appl. Sci. Res., 2011, 3 (6):173-179 ______________________________________________________________________________ Acknowledgements All the authors are thankful to the Sophisticated Instrumentation Centre for Applied Research & Testing (SICART), Vallabh Vidyanagar, Gujarat, INDIA for carrying out EDAX and TEM of our samples. The authors are also grateful to Miss Anjali Bose, research student, B. R. D. school of BioSciences, S. P. University, Vallabh Vidyanagar for antibacterial analysis of these samples. REFERENCES

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