J Clust Sci DOI 10.1007/s10876-016-1048-1 ORIGINAL PAPER
Molybdenum Oxide Nanoparticles as Antimicrobial Agents Nitin Dighore1 • Sunita Jadhav1 • Priyanka Anandgaonker1 Suresh Gaikwad1 • Anjali Rajbhoj1
•
Received: 3 June 2016 Ó Springer Science+Business Media New York 2016
Abstract Molybdenum oxide nanoparticles were prepared by electrochemical reduction method using tetra ethyl ammonium bromide as structure directing agent in an organic medium tetrahydrofuran/acetonitrile in 4:1 ratio and at current density 14 mA/cm2. The synthesized molybdenum oxide nanoparticles were characterized by using ultra violet–visible spectroscopy, infra red spectroscopy, X-ray diffraction, scanning electron microscopy, energy dispersive spectroscopy and transmission electron microscope analysis techniques. The nanoparticles were tested for antibacterial activity against human pathogens like gram negative Escherichia coli, Salmonella typhi and gram positive Staphylococcus aureus, Bacillus subtilis strains, which showed excellent antibacterial properties. Keywords Electrochemical method Molybdenum oxide nanoparticles Human pathogens Antibacterial activity
Introduction Metal nanoparticles have been attracting intensive interest because of their outstanding properties and potential applications in the areas of catalysis [1, 2], magnetism [3], electronics [4], biological activity [5, 6] etc. Most of the metal nanoparticles show modified chemical and physical properties, which depend on the size of nanoparticles. In recent years, much effort has been devoted to the study of molybdenum oxide and related materials. Molybdenum oxide posses unique catalytic and electronic properties and have potential applications in chemical synthesis, petroleum refining, recording media and sensors [7–10]. They are also & Anjali Rajbhoj
[email protected] 1
Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad, Maharashtra 431004, India
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used in the optical device applications [11–14] and are promising material for photoelectrochemical energy production with high surface area having higher photo efficiencies [15, 16]. Molybdenum oxides are photochromic and electrochromic [17] materials, which change colour due to change in oxidation state by absorption of light [18, 19]. The molybdenum oxide nanoparticles show antimicrobial activity which can be attributed to their small size and high surface to volume ratio which allows them to attach closely with microbial membrane and the release of metal ion [20] in solution. Molybdenum oxide nanoparticles have been demonstrated to be useful and effective in bactericidal applications [21, 22] and therefore present a reasonable alternative for development of new bactericides. This study was designed to synthesize pure molybdenum oxide nanoparticles by electrochemical reduction method in which a controlled current is used throughout the electrolysis process. The tetra ethyl ammonium bromide (TEAB) salts were used which are highly soluble and dissociate in the solvent to play the role of electrolyte as well as capping agent. The bioevaluation assay of the inhibitory activity of synthesized molybdenum oxide on bacterial strain gram positive and gram negative were performed to establish the potential of these nanoparticles as antibacterial agents at different concentration and was also compared with standard amphicillin.
Materials and Methods Materials All chemicals (up to 98.99 % purity) were purchased from Aldrich and Rankem chemical suppliers and used as received. The sacrificial anode in the form of molybdenum sheet and platinum sheet as inert cathode having thickness 0.25 mm and purity 99.9 % were purchased from Alfa Asaer. Bacillus subtilis (ATCC No. 6633), Staphylococcus aureus (ATCC No. 25923), Escherichia coli (ATCC No. 25922) and Salmonella typhi (ATCC No. 23564) were provided by Govt. Institute of Science, Aurangabad 431004. Antibiotic amphicillin was used as standard and agar medium was used for screening the antibacterial activity. Synthesis of Molybdenum Oxide Nanoparticles The synthesis of molybdenum oxide nanoparticles by electrochemical reduction method is previously reported method [23–25]. In the initial experiment we have used a molybdenum metal sheet (1 9 1 cm) as anode and a platinum sheet (1 9 1 cm) as the cathode. These two electrodes were placed parallel to one another and were separated by 1.0 cm in 0.01 M solutions of TEAB prepared in ACN/THF (4:1) which also served as the supporting electrolyte. The electrolysis process was then carried out at current densities 14 mA/cm2. After completion of electrolysis process, the yellow coloured solution obtained was allowed to settle for 1 day. The agglomerated yellow solid molybdenum oxide nanoparticles were separated from
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the solution by decantation and washed three to four times with THF. The washed sample was then dried under vacuum desiccator and stored in air tight containers. Characterization of Molybdenum Oxide Nanoparticles The prepared molybdenum oxide nanoparticles were characterized by UV–Vis spectroscopy, FT-IR spectroscopy, XRD, SEM–EDS and TEM techniques. The UV–Vis spectrum recorded on Jasco-503 spectrophotometer with acetonitrile/ tetrahydrofuran as reference. The IR spectrum was recorded on FT-IR spectrophotometer [Jasco, FT-IR/4100 Japan] by using dry KBr as standard reference in the range of 400–4000 cm-1. The X-ray powder diffraction patterns of the molybdenum oxide nanoparticles were recorded on Bruker 8D advance X-ray diffractometer ˚ . To study the morphology of using CuKa radiation of wavelength = 1.54056 A molybdenum oxide nanoparticles the SEM analysis were carried out with JEOL; JSM- 6330 LA operated at 20.0 kV and 1.0000 nA. The presence and elemental composition in molybdenum oxide nanoparticles were examined using energy dispersive spectrophotometer (EDS). The TEM analysis was carried out with Philips model CM200 operated at 200 kV. Antibacterial Activity Experiment Preparation of Sub-culture Antibacterial activities were studied by the paper disc plate method [26]. A uniform suspension of test organism of 24 h old culture was prepared in test tube containing sterile saline solution. A sterile nutrient agar was then added in each of the petri dishes. The dishes were related to ensure the uniform mixing of the micro organism in the agar medium which was then allowed to solidify. The agar cups were prepared with sterile cork borer bearing suitable dimension. The solution of molybdenum oxide nanoparticles was tested aseptically into each cup. The ACN: THF was used a control of the solvent incubated at 37 °C for 24 h. The concentration of the molybdenum oxide nanoparticles in ACN: THF was 25 and 50 lg/ml-1. Amphicillin was used as a standard compound for comparison. After incubation the inhibitory zones around the agar cups were observed. The diameter of inhibition zones were measured in terms of mm. The antibacterial activity was found to depend on zone of inhibition in mm, if inhibition zone \7 mm the sample showed poorer antibacterial activity while [7 mm then sample would have better antibacterial activity. Keeping above mentioned points in view, the screening of the metal nanoparticles for antimicrobial activity has been performed.
Results and Discussion Figure 1 shows sample bottles (1–4) of the colloids of molybdenum oxide nanoparticles during the reaction at different intervals of time. During the electrolysis process, it has been observed that the color of the reaction mixture
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N. Dighore et al. Fig. 1 Molybdenum oxide colloidal nanoparticles
changed after 10 min from colorless to yellow and after 2 h the yellow molybdenum oxide nanoparticles were obtained because of yellow color Mo (VI) oxide composition. UV–Vis Spectroscopy The UV–Vis absorption spectrum recorded for molybdenum oxide nanoparticles is shown in Fig. 2 which exhibits maximum absorption at 640 nm for TEAB at 14 mA current density. The metal nanoparticles exhibit absorption bands or broad regions of absorption in the UV–Vis range due to the excitation of surface plasmon resonance (SPR) or interband transitions; these SPR are characteristic properties for the metallic nature of particles. The prominent peak observed at 640 nm in the visible wavelength is due to the absorption of surface Plasmon. A broad peak around 640 nm can be attributed to wide size distribution of particles formed in the 0 .8 0 .7
Absorbance (%)
0 .6 0 .5 0 .4 640nm
0 .3
(1 2 0 m in )
0 .2
(6 0 m in )
0 .1 0 .0 500
(1 80 m in )
600
700
800
Wavelength (nm) Fig. 2 UV–Vis spectrum for molybdenum nanoparticles capped with TEAB at 14 mA/cm2 current density
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Molybdenum Oxide Nanoparticles as Antimicrobial Agents
solution. The spectra were recorded after every 30 min reaction. In Fig. 2, the UV– Vis absorbance spectrum shows that a characteristic band at 640 nm the intensity of the characteristic band increased with time which indicated the formation of molybdenum oxide nanoparticles. The nanoparticles showed hardly any change in the absorption spectrum even after a month of ageing time indicating highly stable nature of particles. FT-IR Spectroscopy Figure 3 shows the IR spectrum of molybdenum nanoparticles at 14 mA/cm2 current density. The absorption of H2O on the sample takes place due to contact of sample with environment which gives rise to hydrogen bonding of all surface O–H groups. The broad band around 3424 cm-1 is the stretching mode of vibration O–H groups and 1633 cm-1 and can be assigned to the bending mode of O–H groups of absorbed water. The spectrum reveals peak at 2956 cm-1 which corresponds to the C–H stretching vibrations. The ammonium ion bending is seen at the corresponding absorbance band at 1462.74 cm-1. The frequency corresponding to 1593 cm-1 relates to the H–C–H bending vibrations. The C–N linkage in R4N? ion gives medium bands at 1056 cm-1 due to the C–N stretching vibrations. The spectrum also shows distinct bands at 1383 and 1215 cm-1, which can be attributed to the carbon dioxide and nitrate like impurities absorbed from atmosphere during the storage. The peaks at 496, 597, 669 and 848 cm-1 correspond to the mixed phase that contains molybdenum and molybdenum oxide [27]. X-Ray Diffraction In order to understand the phase symmetry of the prepared molybdenum oxide nanoparticles, a systematic study on the XRD was undertaken. Figure 4 shows XRD pattern for molybdenum oxide nanoparticles in which number of peaks are observed because of high crystallinity of molybdenum oxide nanoparticles. The lattice parameter was observed at a = 3.962, b = 13.85 c = 3.696 at alpha = 908,
Fig. 3 IR spectrum of molybdenum oxide nanoparticles
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(120)
500 400
0
(111)
100 20
30
(140)
200
(021)
300 (110)
Intensity (Count)
600
(150)
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40
50
2θ
60
70
80
Fig. 4 XRD spectra of molybdenum oxide nanoparticles
beta = 908 and gamma = 908. Sharp peaks were obtained corresponding to the planes (110), (120), (021), (111), (140) and (150) indicating the Orthorhombic structure [28] of molybdenum oxide nanoparticles which was found to be highly crystalline in nature. The diffraction is in good agreement with JCPDS Card No. 35-0609. Using the Debye–Sherrer method the average particle size was found to be 46 nm from peak broadening. This value is consistent with the TEM observations. Scanning Electron Microscopy with EDS The evaluated morphology of molybdenum oxide nanoparticles capped with TEAB studied by SEM is shown in Fig. 5a, b. The scanning electron microgram and the particle size distribution for the sample of molybdenum nanoparticles capped with TEAB at current density of 14 mA/cm2 is obtained for various resolutions. In Fig. 5a, b, SEM microstructures of derived molybdenum oxide nanoparticles reveal the presence of dense agglomerations. Figure 5a shows that these particles have irregular shape and their distribution is not uniform. This is probably due to the partial solubility of the surfactant in the solvent under the given experimental
Fig. 5 a, b SEM images and c EDS spectrum of molybdenum oxide nanoparticles
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Molybdenum Oxide Nanoparticles as Antimicrobial Agents
conditions. Figure 5a, b show the presence of porous nanoparticles that are agglomerated irregularly. Molybdenum nanoparticles synthesized with capping agent TEAB at current density 14 mA/cm-2 were analyzed qualitatively and quantitatively by EDS. Figure 5c shows the composition of atomic % of molybdenum and oxygen which was found to be 44.98 and 55.02 % respectively which clearly shows that formation of pure molybdenum oxide nanoparticles. Transmission Electron Microscopy TEM microscopic study has been done to evaluate particle shape and size distribution of molybdenum oxide nanoparticles as shown in Fig. 6a. TEM microstructure clearly indicated the decrease in particle size with increase in current density which resemble to the phenomenon observed in XRD pattern. Figure 6b shows a histogram of the molybdenum nanoparticles having average diameter (daverage) and standard deviation (r) to be 25.62 and 3.58 nm respectively. Antimicrobial Activity According to several studies, it is believed that the metal oxides carry the positive charge while the microorganisms carry negative charges; this cause’s electromagnetic attraction between microorganisms and the metal oxides which leads to oxidization causing pits or holes in the bacterial cell wall which could be associated with internalized particles, leading to increased permeability and cell death [29–31]. Molybdenum oxide nanoparticles due to their small size and high surface to volume ratio undergo a higher level of interaction with the bacterial cells surface than the larger particles, resulting in a good antibacterial activity. The antibacterial activity of molybdenum oxide nanoparticles was carried out by agar well diffusion method against two gram positive bacteria i.e. B. Subtilis, S. aureus and two gram negative bacteria i.e. S. Typhi and E. coli for two different concentrations of molybdenum oxide nanoparticles 50 and 100 ll. Amphicilin was
(a)
35
(b)
Particles Counts
30
Mean (d average )=25.62 Std. Dev(σ)=3.58
25 20 15 10 5 0
20
30
40
Diameter in nm
Fig. 6 a TEM image and b histogram of molybdenum oxide nanoparticles
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used as a standard for same the concentrations. From the results in Table 1 it can be seen that all samples have better antibacterial activity because their zone of inhibition is much [7 mm and the ACN/THF control did not show any antimicrobial activity against the tested bacterial strains. In addition, the antimicrobial activity is directly proportional to the molybdenum oxide nanoparticles concentration and 100 ll is the optimum concentration of molybdenum oxide nanoparticles for inhibiting growth of bacterial test organism. The molybdenum oxide nanoparticles proved to be very active on the tested Gram-positive strains; this differential sensitivity of Gram-negative and Grampositive bacteria towards nanoparticles depends upon their cell outer layer attribute and their interaction with the charged molybdenum oxide nanoparticles. The Grampositive bacteria have a relatively thick wall composed of many layers of peptidoglycan polymer and only one plasma membrane. The Gram-negative bacteria have only a thin layer of peptidoglycan and a more complex cell wall with two cell membranes, an outer membrane, and a plasma membrane. The addition of the outer membrane of the Gram-negative bacteria cells influences the permeability of many molecules. Under certain conditions, the Gram-negative bacteria are more resistant to many chemical agents than Gram-positive cells. In addition, the cell walls of Gram-negative bacteria are more prone to mechanical breakage because of the low amount of peptidoglycan [31]. Table 1 indicates the mean zone of inhibition which became prominent after treatment with nanoparticles suspension in care of both grams positive as well as gram negative bacteria. Interaction between nanoparticles and the cell wall of bacteria was congenial to the fact that growth of gram negative bacteria was more profoundly affected by molybdenum oxide nanoparticles than that of the gram positive bacteria. The maximum zone of inhibition was observed for molybdenum oxide nanoparticles against B. subtilis sp. Table 1 In vitro antibacterial screening of synthesized molybdenum oxide nanoparticles Human pathogenic bacteria
Mean zone of inhibition diameter ± SD (mm) Conc. (ll)
B. subtilis
S. aureus
50
14.33 ± 1.52a
E. coli
12.0 ± 2.0d
S. typhi
10.66 ± 1.52
Molybdenum oxide NPs
100
17.30 ± 0.57
15.66 ± 1.50
14.0 ± 1.0
Amphicillin
50
16.0 ± 1.0c
14.66 ± 0.57f
18.66 ± 1.15
100 ACN ? THF (4:1)
b
b
17.30 ± 1.52
e
c
16.0 ± 1.0
g
f
10.66 ± 0.57 13.0 ± 0.0
i
20.66 ± 1.52
h
13.33 ± 0.57j 16.33 ± 1.15
50
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
100
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
0.0 ± 0.0
F value
232.500
132.59
207.08
455.86
P value
\0.01**
\0.01**
\0.01**
\0.01**
Test applied—One way ANOVA with post hoc Tukey HSD test, P \ 0.05 statistically significant Post hoc: Values with same letter superscripted do not vary significantly SD standard deviation **
P value \0.01—The test is highly significant
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g
j
k
Molybdenum Oxide Nanoparticles as Antimicrobial Agents
(17.30 ± 0.57), S. aureus sp. (15.66 ± 1.50) at 100 ll concentration which were compared with standard amphicillin. Similar results obtained for B. subtilis sp. (17.30 ± 1.52), S. aureus sp. (16 ± 1.0) at 100 ll concentration. The antimicrobial data have been analyzed by data analysis tool [32]. One way ANOVA followed by the Tukey’ HSD test. The antimicrobial studies of molybdenum oxide nanoparticles against bacteria, the statistical analysis data was obtained in Table 1. The result obtained from Turkey’ HSD test showed that the P value corresponding to the F-statistic of ANOVA is \0.01 which strongly suggest that statistically significant different occur in antimicrobial screening.
Conclusions In summary, we have demonstrated the efficiency of electrochemical reduction method for the synthesis of molybdenum oxide nanoparticles. The TEAB salts used as ligands have played a significant role on controlling the particle size. The procedure offers several advantages including control of the particle size daverage 25.62 nm, excellent yield, operational simplicity and minimum environmental effects. Obtained values of zone of inhibition for gram positive and gram negative strains suggest that the prepared molybdenum oxide nanoparticles show excellent antibacterial activity and can be used as promising antibacterial agents in wide applications. The effect was dose dependent and was more pronounced against gram positive organisms than gram negative ones. Statistical analysis of antimicrobial screening resulted statistically significant. One way ANOVA Tukey post hoc test also strongly suggested that significant difference. Such efficient process provides new opportunities for the rapid screening of a wide range of synthesis of metal nanoparticles, either for the development of new drugs for the material scientist. Acknowledgments The Department of Chemistry acknowledges the financial assistance by UGC-SAPDRS scheme-1. One of the author NRD is thankful for financial assistance from University Scholar Fellowship, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad and Prof. Anjali Rajbhoj is thankful for financial assistance from Major Research project [F. No. 832/2010(SR)], University Grants Commission, New Delhi.
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