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Alpha amylase assisted synthesis of TiO2 nanoparticles: Structural characterization and application as antibacterial agents
Accepted Manuscript Title: Alpha amylase assisted Synthesis of TiO2 Nanoparticles: Structural Characterization and Application as Antibacterial Agents Author: Razi Ahmad Mohd Mohsin Tokeer Ahmad Meryam Sardar PII: DOI: Reference:
S0304-3894(14)00754-7 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.08.073 HAZMAT 16264
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
28-6-2014 7-8-2014 26-8-2014
Please cite this article as: R. Ahmad, M. Mohsin, T. Ahmad, M. Sardar, Alpha amylase assisted Synthesis of TiO2 Nanoparticles: Structural Characterization and Application as Antibacterial Agents, Journal of Hazardous Materials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.08.073 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Green synthesis of TiO2 nanoparticles using an enzyme alpha amylase has been described.
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The morphology and shape depends upon the concentration of the alpha amylase enzyme.
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The biosynthesized nanoparticles show good bactericidal effect against both gram positive and gram negative bacteria.
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The bactericidal effect was further confirmed by Confocal microscopy and TEM.
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Alpha amylase assisted Synthesis of TiO2 Nanoparticles: Structural Characterization and Application as Antibacterial Agents Razi Ahmada, Mohd Mohsina, Tokeer Ahmadb and Meryam Sardara* Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India Department of Chemistry, Jamia Millia Islamia, New Delhi-110025, India
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*Corresponding author.
Dept. of Biosciences, Jamia Millia Islamia New Delhi-110025, India
E-mail address: [email protected] Tel: 011-26981717
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Abstract The enzyme alpha amylase was used as the sole reducing and capping agent for the synthesis
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of TiO2 nanoparticles. The biosynthesized nanoparticles were characterized by X-ray diffraction (XRD) and transmission electron microscopic (TEM) methods. The XRD data
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confirms the monophasic crystalline nature of the nanoparticles formed. TEM data shows that the morphology of nanoparticles depends upon the enzyme concentration used at the time of
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synthesis. The presence of alpha amylase on TiO2 nanoparticles was confirmed by FTIR. The nanoparticles were investigated for their antibacterial effect on Staphylococcus aureus and
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Escherichia coli. The minimum inhibitory concentration value of the TiO2 nanoparticles was found to be 62.50 µg/ml for both the bacterial strains. The inhibition was further confirmed
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using disc diffusion assay. It is evident from the zone of inhibition that TiO2 nanoparticles possess potent bactericidal activity. Further, growth curve study shows effect of inhibitory
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concentration of TiO2 nanoparticles against S. aureus and E. coli. Confocal microscopy and
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TEM investigation confirm that nanoparticles were disrupting the bacterial cell wall.
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Keywords: TiO2 nanoparticles; TEM; Minimum inhibitory concentration; Antibacterial effect
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1. Introduction In recent years the application of nanoparticles in various fields has expanded considerably. The synthesis of titanium dioxide (TiO2) nanoparticles and nanostructures have
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been a matter of interest due to their attractive material properties and applications in various fields like optical devices, sensors, photocatalysis, organic pollutants and antibacterial
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coatings[1-4]. TiO2 nanoparticles are considered to be amongst the best photocatalytic
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materials due to their long-term thermodynamic stability, strong oxidizing power, and relative non-toxicity [5, 6]. Presently there are chemical, physical and biological routes
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available for the synthesis of metal oxide nanoparticles [7, 8]. It is well known that many organisms can produce inorganic materials either intra or extracellular [9]. Biological
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synthesis of metal oxide nanoparticles is hallmarked by ambient experimental conditions of temperature, pH, and pressure [8, 10]. There is a need to develop environmentally safe
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protocols for the synthesis of TiO2 nanoparticles. So far, only few reports are available on the
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biosynthesis of TiO2 nanoparticles [8, 10-14]. Previously, nanoparticles has been synthesized
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by using enzymes silicatein [15] , lysozymes [11] and urease [13]. Recently, our group synthesized TiO2 nanoparticles using lactobacillus sp. [14]. We have also reported the synthesis of silver [16] and gold [17] nanoparticles using alpha amylase from Aspergillus oryzae. One of the major big challenges for researchers are facing is the occurrence of antibiotic resistant bacterial strains, hence there is the need to develop the new drugs [18]. Therefore, the finding of new antimicrobial agents with novel mechanisms of action is essential and extensively pursued in antibacterial drug discovery. In this paper, we describe the biosynthesis of TiO2 nanoparticles using hydrolytic enzyme alpha amylase from Aspergillus oryzae and their antibacterial effect against gram positive Staphylococcus aureus (S. aureus) and gram negative bacteria Escherichia coli (E. coli). 4
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2. Experimental
2.1. Materials and Methods Culture media Luria agar, Luria broth and Alpha amylase enzyme were purchased from
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Hi-media (India). TiO2 powder and TiO2 nanoparticles (size < 25nm) were purchased from
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Sigma Aldrich (USA). Bacterial strains Staphylococcus aureus (MTCC-3160) and Escherichia coli (MTCC-405) were procured from Microbial Type Culture Collection
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solvents used were of analytical and biotechnology grade.
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(MTCC), Institute of Microbial Technology (Chandigarh, India). All other chemicals and
2.2. Biosynthesis of TiO2 nanoparticles by alpha amylase
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Aqueous solution (25 ml) of 0.25 M TiO(OH)2 was added to 30 ml of alpha amylase enzyme (2 mg/ml dissolved in 20 mM sodium acetate buffer pH 4.5) and it was incubated at
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60 °C for 10 minutes. The solution was cooled and kept at 25°C with continuous shaking.
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After 24 h the mixture was centrifuged at 3000 g for 10 minutes and the nanoparticles of
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TiO2 obtained in the pellet were washed with distilled water for several times to remove the unbound enzyme. The washed and air dried nanoparticles were used for further characterization. In another set of experiment, enzyme concentration was increased to 15 mg/ml by keeping the other parameters constant.
2.3. XRD analysis
The XRD studies for the formation of metal oxide TiO2 nanoparticles was confirmed on Bruker D8 advance diffractometer
using Ni- filtered Cu-Kα X-rays of wavelength (λ)
=1.54056 Å over a wide range of Bragg angles (20°≤ 2θ ≤ 80°). The data was obtained at the
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scanning rate of 0.05º/s. The raw data obtained was subjected to the back ground correction and Kα2 reflections were stripped off using normal stripping procedure.
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2.4. Transmission electron microscope measurements of nanoparticles Transmission electron microscope measurements were performed on a JEOL, F2100
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instrument operated at an accelerating voltage at 200 kV for the size and morphological studies of the nanoparticles. The TEM specimens were prepared by adding a drop of the
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by a carbon coated copper grid, and dried in oven.
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dispersed sample in distilled water followed by sonication on a porous carbon film supported
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2.5. Fourier Transform infrared spectroscopy
Fourier transform infrared (FTIR) spectroscopy was recorded on a Bruker IR
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from 4000-650 cm-1.
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Spectrophotometer. The spectra of the washed and purified TiO2 nanoparticles were recorded
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2.6. Minimum inhibitory concentration (MIC) The microdilution method for estimation of MIC values was carried out to determine the
antimicrobial activity. The MIC values were determined on 96-well micro-dilution plates according to the protocols developed previously [19].
2.7. Agar diffusion assay
The antibacterial activity of TiO2 nanoparticles was evaluated against S. aureus and E. coli by agar diffusion method. Fresh cultures (0.2 ml) of S. aureus and E. coli were inoculated into 5 ml of sterile Luria broth separately, and incubated for 3–5 h to standardize the culture to McFarland standards (106 CFC/ml). Hundred µl of revived cultures from each 6
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was added on agar media and poured on three replicate plates for both cultures. Discs with the size of 6 mm were placed on the agar plates and 10 µl of TiO2 nanoparticles (4 mg/ml suspended in sterile water) loaded on one disc and on another disc 10 µl ampicillin (1 mg/ml)
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was loaded as standard. On separate discs 10 µl alpha amylase (15 mg/ml) and 10 µl TiO(OH)2 (4 mg/ml suspended in sterile water) were loaded as controls. The petriplates were
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incubated at 37 ºC for 18 h for bacterial growth.
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2.8. Growth curve study
S. aureus and E. coli were studied for their growth dynamics in the presence and absence
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of TiO2 nanoparticles. Briefly, 100 µl of overnight fresh bacterial culture (~106 CFU/ml) was
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inoculated in respective flasks containing 100 ml growth medium with and without TiO2 nanoparticles with varied concentration of nanoparticles from 15 to 250 µg/ml. The culture
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flasks were incubated at 37 ºC and optical density (OD600 nm) was recorded at the interval of
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2 h. The readings obtained were plotted and comparative studies were performed between
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control and treated culture with TiO2 nanoparticles.
2.9. Confocal scanning laser microscopy Confocal microscopy studies were performed on a confocal laser scanning microscope
Leica DMRE equipped with a confocal head TCS SPE (Leica, Wetzlar, Germany) and a 40X water immersion objective with a laser of 532 nm wavelength. S. aureus and E. coli cultures were labeled with propidium iodide (PI) dye. PI was used to determine the dead cells as PI specifically stains only dead bacteria. The TiO2 (125 µg/ml) nanoparticles were added to the overnight grown bacterial cultures and were incubated at 37 ºC for 30 minutes in the dark. 1 ml from both of these bacterial strain cultures were harvested by centrifuging at 2500 g for 5 min. The pellet was resuspended in 1 ml PBS buffer. Staining with PI was performed by 7
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adding 10 µl of PI to final concentration of 1 µg/ml. This solution was incubated for 3 h at 37 ºC. Likewise a control of both bacterial strains (not treated with TiO2 nanoparticles) was grown under similar conditions. A drop of the above prepared samples were then placed on a
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glass slide and mounted with the cover slip then cells were examined under a confocal scanning microscope.
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2.10. Transmission electron microscopy of bacterial cells
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Test compounds at MIC were added to the Bacterial cell S. aureus and E. coli
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suspensions in media (~1x106 c.f.u./ ml) and incubated for 16 h at 37 °C. All the cells were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 1 h at 20 °C [20]. Cells were
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washed with 0.1 M phosphate buffer (pH 7.2) and post-fixed by using 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h at 4 °C. For ultrastructure studies, samples were dehydrated
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with graded acetone, cleared with toluene and infiltrated with a toluene and araldite mixture
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at room temperature then finally in pure araldite at 50 °C and embedded in an polypropylene tube (1.5 ml) with pure araldite mixture at 60 °C. Samples were prepared using a sectioning
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ultramicrotome (Lecia EM UC6) and were observed using TEM [20].
3. Results and discussion
The “green synthesis” approach has been proven to be a better method due to their
slower kinetics, better manipulation, control over crystal growth and stabilization. Recent advancement in this area includes enzymatic method of synthesis suggesting the enzymes to be responsible for the nanoparticle formation. Biomacromolecules have been used in the past to synthesize metal oxides, including ZnO [21], Cu2O [22] and Ga2O3 [23]. Recently, Jayaseelan et al. [24] reported the synthesis of TiO2 nanoparticles using Aeromonas hydrophilla. The GC-MS analysis of the broth showed the major compound found in the 8
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Aeromonas hydrophilla is glycyl – proline and other compounds present in lesser amounts are uric acid, glycl-glumatic acid, and Leucyl-leucine and compounds containing –COOH and -C═O group. They concluded that glycil-L proline act as reducing agent for the synthesis
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of TiO2 nanoparticles and the water soluble carboxylic acid compounds acts as stabilizing agents [24]. In the present work we report the extracellular synthesis of TiO2 nanoparticles
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using an enzyme alpha amylase from a non pathogenic fungal strain Aspergillus oryzae. Alpha amylase from Aspergillus oryzae contains 478 amino acid residues out of which 21 Here it is assumed that the amino
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residues are proline [25], 12 of them are exposed [26].
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acid proline might have played an important role in the reduction of titanium hydroxide. Moreover, alpha amylase from Aspergillus oryzae is an acidic enzyme with optimum pH of
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4.5 and low isoelectric point (PI=4.2) [27, 28]. This enzyme has large percentage of carboxyl groups which might be responsible for the stabilization of TiO2 nanoparticles. Fig. 1
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represents the proposed mechanism for the synthesis of TiO2 nanoparticles with alpha
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amylase having proline residues. Use of enzyme for nanoparticles synthesis has advantage over other biological methods as they are available in pure form and their structure is also
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well known. The mechanism of biosynthesis can be easily understood using well characterized enzyme. So far lysozyme [11]
and urease [13] has been reported in the
literature for the synthesis of TiO2 nanoparticles, compared to these enzymes alpha amylase is a low cost enzyme.
The crystalline nature and phase purity of titania nanostructures were analyzed by X-ray
diffraction studies. Fig. 2 shows the XRD analysis of TiO2 nanoparticles synthesized with different concentrations of alpha amylase enzyme. Both diffraction patterns could be indexed to monophasic nanocrystalline structure of titanium dioxide and found to be in good agreement with the available literature reports (PCPDF No. #84-1285). The observed 9
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reflections in Fig. 2a and b corroborates to the pure nanocrystalline TiO2 with anatase structure. No peak corresponding to any impurity was present which indicates the high purity of the samples. Fig. 3 showing the TEM images of the TiO2 nanoparticles synthesized using
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different concentration of alpha amylase. The TEM analysis showed that the particle size and morphology depends upon the concentration of enzyme used during synthesis. With the
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alpha amylase concentration of 2 mg/ml, the grain size varies in the range of 30-70 nm with an average grain size of 50 nm (Fig. 3a), however the grain size decreases significantly to 25
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nm (Fig. 3b) by increasing the concentration of the enzyme upto 15 mg/ml. The morphology
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of the nanoparticles formed in the enzymatic synthesis also exhibits the strong dependence on enzyme concentration. The hexagonal along with spherical nanoparticles of TiO2 could be
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seen at 2 mg/ml concentration of enzyme as shown in Fig. 3a. It is important to note that as the enzyme concentration increased to 15 mg/ml, the nanorings were also observed in
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addition to nanoparticles (Fig. 3b). To arrive at the definite response of nanoring formation,
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the high resolution-TEM (HR-TEM) study has been carried out. HR-TEM image clearly showed the formation of nanoring (Fig. 3c). This is clear from Fig. 3b and c that the average
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ring outer and inner diameters are 55 nm and 33 nm respectively with the shell thickness of 13 nm. It has been reported that the nanoparticles of different sizes and shapes could be synthesized using biological methods [8, 14, 16, 29]. Thus, by controlling the enzyme concentration one can achieve the controlled shape and size of the nanoparticles. Further, research on the synthesis of TiO2 nanoparticles at different enzyme concentration is under investigation which will give some insight on this aspect. FTIR spectra of alpha amylase and biosynthesized TiO2 nanoparticles using amylase at two different concentrations were recorded. The FTIR of nanoparticles with different enzyme concentrations was found similar (Fig. 4a and b). The spectral bands of enzyme at 1625 cm-1 could be assigned to amide I and absorption signal at 1520 cm-1 indicate a characteristic amide II band [30, 31]. The 10
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characteristic amide I and amide II bands of alpha amylase was observed in the FTIR spectra (Fig. 4c). FTIR spectroscopy clearly indicating that alpha amylase is responsible for the synthesis of nanoparticles and in their stabilization.
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The antibacterial effect of enzyme assisted TiO2 nanoparticles was evaluated against gram positive and gram negative bacterial strains. Standard antibiotic ampicillin was used as
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a control. The MIC value for the TiO2 nanoparticles was found to be 62.5 µg/ml for both
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strain S. aureus and E. coli (Table 1). The MIC value of the biosynthesized nanoparticles was compared with the chemically synthesized nanoparticles (procured from sigma). The
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chemically synthesized nanoparticles show greater MIC values for both the strains as shown in Table 1. Further, the stability of the biosynthesized nanoparticles was also studied in terms
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of antibacterial effect. To check the stability of the enzyme assisted TiO2 nanoparticles, they were stored at 4°C for six months and used to calculate the MIC value against bacterial
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strains. The nanoparticles show good stability as no change in the MIC results was obtained.
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The Inhibition was further confirmed using disc diffusion assay as shown in Fig. 5. It is evident from the zone of inhibition that TiO2 nanoparticles possess potent bactericidal
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activity. The bactericidal effect of TiO2 nanoparticles has been attributed to the decomposition of bacterial outer membranes by reactive oxygen species (ROS), primarily hydroxyl radicals (OH), which leads to phospholipid peroxidation and ultimately cell death [32, 33]. It was proposed that nanomaterials that can physically attach to a cell can be bactericidal if they come in contact with cell [34]. The growth curve study of S. aureus and E. coli in the presence and absence of TiO2 nanoparticles showed the effect of inhibitory concentration of TiO2 nanoparticles against S. aureus and E. coli till 20 h of incubation (Fig. 6). In control cell lag phase ends upto 4-6 h after which log phase starts which ends upto 1618 h. However when cells were treated with TiO2 nanoparticles, then delay in the lag phase 11
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occurred. At MIC concentration lag phase ends upto 12 h. There occurred delay in lag phase on exposure to different concentration of TiO2 nanoparticles and thus a prominent decrease in growth of S. aureus and E. coli was observed. To prove the effect of TiO2 nanoparticles on
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the viability of S. aureus and E. coli strain, we performed confocal laser scanning microscopy (CLSM) in the presence of PI. Bacterial cells were grown and stained with PI as described
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above. PI penetrates only cells with severe membrane lesions; the entire bacterial cells appear red (Fig. 7). It should be noted that PI can only stain the cells in which the cell membrane is
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disrupted since it intercalates into the double-stranded nucleic acids [35]. Results presented
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here showing that PI penetrates the bacterial cell. S. aureus and E. coli were taken for the study. A control stained with PI without the TiO2 nanoparticles (Fig. 7a) and samples with
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the nanoparticles (Fig 7b and c) were tested for the invasiveness of the nanoparticles inside the cells. The confocal studies showed that control is not showing any fluorescence while the
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sample with TiO2 nanoparticles showing the good fluorescence proving the concept of
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invading the cells. Since the PI is binding to the double stranded DNA it is confirmed that the TiO2 nanoparticles damage the bacterial cell wall. The nanoparticles disrupt the bacterial cell
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walls whether it is gram positive or gram negative bacterial strain. TEM images of ultrathin sections of treated cells showing the damaged cell wall and cell membrane while in untreated cells the cell membrane structures are intact (Fig. 8a). Treated cells are showed the leakage of the intracellular contents (Fig. 8b). The focus of this study was to examine the antibacterial activity of the biologically synthesized nanoparticles and also the effect of their particle size. In this study size effect is not observed as seen by the MIC data, confocal and TEM studies.
4. Conclusion Here, the biosynthesis of TiO2 nanoparticles using the enzyme alpha amylase has been described. The present studies suggest that the proline residues present in the enzyme play an 12
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important role in the reduction of titanium hydroxide. The biosynthesized nanoparticles were exploited for their antibacterial activity. The disc diffusion and growth curve confirms their antibacterial effect and indicates that TiO2 nanoparticles can be considered as potent
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antibacterial compound. As the synthesis is eco-friendly, the antibacterial properties of these nanoparticles can be further explored in future on other bacterial strains, for their use in
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various industrial and medical applications.
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Acknowledgments
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First author RA acknowledge the Indian Council of Medical Research, Govt. of India,
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for providing the financial support in the form of Senior Research Fellowship.
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Legends to Figures Fig. 1. Possible mechanism for the synthesis of TiO2 nanoparticles. Fig. 2. X-ray diffraction pattern of TiO2 nanoparticles synthesized using alpha amylase (a)
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2mg/ml and (b) 15mg/ml.
Fig. 3. TEM images of TiO2 nanoparticles using (a) 2mg/ml and (b) 15mg/ml alpha amylase
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concentration (c) is the HRTEM image of the nanoring.
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Fig. 4. FTIR spectra of TiO2 nanoparticles: (a) alpha amylase. (b) Nanoparticles synthesized using 2mg/ml alpha amylase. (c). Nanoparticles synthesized using 15mg/ml alpha amylase.
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Fig. 5. Zone of Inhibition of TiO2 Nanoparticles against S. aureus and E.coli: (a) Ampicillin (b) TiO2 nanoparticles (Av. size 50 nm) (c) TiO2 nanoparticles (Av. size 25 nm) (d)
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TiO(OH)2 (e) alpha amylase.
Fig. 6. Growth curve study of bacterial strain S. aureus and E.coli: (a) S. aureus with
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different concentration of TiO2 nanoparticles (Av. size 50 nm) (b) S. aureus with different concentration of TiO2 nanoparticles (Av. size 25 nm) (c) E. coli with different concentration
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of nanoparticles (Av. size 50nm) (d) E. coli with different concentration of TiO2 nanoparticles (Av. size 25 nm). Each data point represents the mean ± standard deviation of
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three independent observations.
Fig. 7. Laser confocal images of S. aureus and E.coli: cells with membrane damage were stained with PI (red signals) (a) untreated control cells showing no fluorescence (b) cells treated with 125µg/ml of TiO2 nanoparticles (Av. size 50 nm) showing the red fluorescence (c) cells treated with 125µg/ml of TiO2 nanoparticles (Av. size 25 nm) showing the red fluorescence.
Fig. 8. TEM of S. aureus and E. coli: (a) untreated control cells (b) cells treated with 62.5µg/ml of TiO2 nanoparticles (Av. size 50 nm) (c) cells treated with 62.5µg/ml of TiO2 nanoparticles (Av. size 25 nm).
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Table 1 Zone of Inhibition (mm) and MIC (µg/ml) of alpha amylase and chemically synthesized TiO2 nanoparticles against S. aureus and E. coli. Particle size (Av. Size 25nm) MIC Inhibition (µg/ml)a Zone(mm) 62.5 16
E. coli (MTCC-405)
62.5
62.5
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200
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Particle size is not affecting the MIC of nanoparticles.
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200
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a
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Chemically synthesized NPS MIC (µg/ml)
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S. aureus (MTCC-3160)
Particle size (Av. Size 50nm) MIC Inhibition (µg/ml)a Zone(mm) 62.5 15
Bacterial strain
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S0304-3894(14)00754-7 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.08.073 HAZMAT 16264
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
28-6-2014 7-8-2014 26-8-2014
Please cite this article as: R. Ahmad, M. Mohsin, T. Ahmad, M. Sardar, Alpha amylase assisted Synthesis of TiO2 Nanoparticles: Structural Characterization and Application as Antibacterial Agents, Journal of Hazardous Materials (2014), http://dx.doi.org/10.1016/j.jhazmat.2014.08.073 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Highlights Green synthesis of TiO2 nanoparticles using an enzyme alpha amylase has been described.
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The morphology and shape depends upon the concentration of the alpha amylase enzyme.
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The biosynthesized nanoparticles show good bactericidal effect against both gram positive and gram negative bacteria.
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The bactericidal effect was further confirmed by Confocal microscopy and TEM.
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Alpha amylase assisted Synthesis of TiO2 Nanoparticles: Structural Characterization and Application as Antibacterial Agents Razi Ahmada, Mohd Mohsina, Tokeer Ahmadb and Meryam Sardara* Department of Biosciences, Jamia Millia Islamia, New Delhi-110025, India Department of Chemistry, Jamia Millia Islamia, New Delhi-110025, India
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*Corresponding author.
Dept. of Biosciences, Jamia Millia Islamia New Delhi-110025, India
E-mail address: [email protected] Tel: 011-26981717
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Abstract The enzyme alpha amylase was used as the sole reducing and capping agent for the synthesis
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of TiO2 nanoparticles. The biosynthesized nanoparticles were characterized by X-ray diffraction (XRD) and transmission electron microscopic (TEM) methods. The XRD data
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confirms the monophasic crystalline nature of the nanoparticles formed. TEM data shows that the morphology of nanoparticles depends upon the enzyme concentration used at the time of
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synthesis. The presence of alpha amylase on TiO2 nanoparticles was confirmed by FTIR. The nanoparticles were investigated for their antibacterial effect on Staphylococcus aureus and
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Escherichia coli. The minimum inhibitory concentration value of the TiO2 nanoparticles was found to be 62.50 µg/ml for both the bacterial strains. The inhibition was further confirmed
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using disc diffusion assay. It is evident from the zone of inhibition that TiO2 nanoparticles possess potent bactericidal activity. Further, growth curve study shows effect of inhibitory
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concentration of TiO2 nanoparticles against S. aureus and E. coli. Confocal microscopy and
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TEM investigation confirm that nanoparticles were disrupting the bacterial cell wall.
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Keywords: TiO2 nanoparticles; TEM; Minimum inhibitory concentration; Antibacterial effect
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1. Introduction In recent years the application of nanoparticles in various fields has expanded considerably. The synthesis of titanium dioxide (TiO2) nanoparticles and nanostructures have
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been a matter of interest due to their attractive material properties and applications in various fields like optical devices, sensors, photocatalysis, organic pollutants and antibacterial
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coatings[1-4]. TiO2 nanoparticles are considered to be amongst the best photocatalytic
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materials due to their long-term thermodynamic stability, strong oxidizing power, and relative non-toxicity [5, 6]. Presently there are chemical, physical and biological routes
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available for the synthesis of metal oxide nanoparticles [7, 8]. It is well known that many organisms can produce inorganic materials either intra or extracellular [9]. Biological
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synthesis of metal oxide nanoparticles is hallmarked by ambient experimental conditions of temperature, pH, and pressure [8, 10]. There is a need to develop environmentally safe
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protocols for the synthesis of TiO2 nanoparticles. So far, only few reports are available on the
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biosynthesis of TiO2 nanoparticles [8, 10-14]. Previously, nanoparticles has been synthesized
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by using enzymes silicatein [15] , lysozymes [11] and urease [13]. Recently, our group synthesized TiO2 nanoparticles using lactobacillus sp. [14]. We have also reported the synthesis of silver [16] and gold [17] nanoparticles using alpha amylase from Aspergillus oryzae. One of the major big challenges for researchers are facing is the occurrence of antibiotic resistant bacterial strains, hence there is the need to develop the new drugs [18]. Therefore, the finding of new antimicrobial agents with novel mechanisms of action is essential and extensively pursued in antibacterial drug discovery. In this paper, we describe the biosynthesis of TiO2 nanoparticles using hydrolytic enzyme alpha amylase from Aspergillus oryzae and their antibacterial effect against gram positive Staphylococcus aureus (S. aureus) and gram negative bacteria Escherichia coli (E. coli). 4
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2. Experimental
2.1. Materials and Methods Culture media Luria agar, Luria broth and Alpha amylase enzyme were purchased from
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Hi-media (India). TiO2 powder and TiO2 nanoparticles (size < 25nm) were purchased from
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Sigma Aldrich (USA). Bacterial strains Staphylococcus aureus (MTCC-3160) and Escherichia coli (MTCC-405) were procured from Microbial Type Culture Collection
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solvents used were of analytical and biotechnology grade.
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(MTCC), Institute of Microbial Technology (Chandigarh, India). All other chemicals and
2.2. Biosynthesis of TiO2 nanoparticles by alpha amylase
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Aqueous solution (25 ml) of 0.25 M TiO(OH)2 was added to 30 ml of alpha amylase enzyme (2 mg/ml dissolved in 20 mM sodium acetate buffer pH 4.5) and it was incubated at
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60 °C for 10 minutes. The solution was cooled and kept at 25°C with continuous shaking.
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After 24 h the mixture was centrifuged at 3000 g for 10 minutes and the nanoparticles of
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TiO2 obtained in the pellet were washed with distilled water for several times to remove the unbound enzyme. The washed and air dried nanoparticles were used for further characterization. In another set of experiment, enzyme concentration was increased to 15 mg/ml by keeping the other parameters constant.
2.3. XRD analysis
The XRD studies for the formation of metal oxide TiO2 nanoparticles was confirmed on Bruker D8 advance diffractometer
using Ni- filtered Cu-Kα X-rays of wavelength (λ)
=1.54056 Å over a wide range of Bragg angles (20°≤ 2θ ≤ 80°). The data was obtained at the
5
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scanning rate of 0.05º/s. The raw data obtained was subjected to the back ground correction and Kα2 reflections were stripped off using normal stripping procedure.
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2.4. Transmission electron microscope measurements of nanoparticles Transmission electron microscope measurements were performed on a JEOL, F2100
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instrument operated at an accelerating voltage at 200 kV for the size and morphological studies of the nanoparticles. The TEM specimens were prepared by adding a drop of the
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by a carbon coated copper grid, and dried in oven.
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dispersed sample in distilled water followed by sonication on a porous carbon film supported
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2.5. Fourier Transform infrared spectroscopy
Fourier transform infrared (FTIR) spectroscopy was recorded on a Bruker IR
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from 4000-650 cm-1.
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Spectrophotometer. The spectra of the washed and purified TiO2 nanoparticles were recorded
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2.6. Minimum inhibitory concentration (MIC) The microdilution method for estimation of MIC values was carried out to determine the
antimicrobial activity. The MIC values were determined on 96-well micro-dilution plates according to the protocols developed previously [19].
2.7. Agar diffusion assay
The antibacterial activity of TiO2 nanoparticles was evaluated against S. aureus and E. coli by agar diffusion method. Fresh cultures (0.2 ml) of S. aureus and E. coli were inoculated into 5 ml of sterile Luria broth separately, and incubated for 3–5 h to standardize the culture to McFarland standards (106 CFC/ml). Hundred µl of revived cultures from each 6
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was added on agar media and poured on three replicate plates for both cultures. Discs with the size of 6 mm were placed on the agar plates and 10 µl of TiO2 nanoparticles (4 mg/ml suspended in sterile water) loaded on one disc and on another disc 10 µl ampicillin (1 mg/ml)
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was loaded as standard. On separate discs 10 µl alpha amylase (15 mg/ml) and 10 µl TiO(OH)2 (4 mg/ml suspended in sterile water) were loaded as controls. The petriplates were
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incubated at 37 ºC for 18 h for bacterial growth.
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2.8. Growth curve study
S. aureus and E. coli were studied for their growth dynamics in the presence and absence
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of TiO2 nanoparticles. Briefly, 100 µl of overnight fresh bacterial culture (~106 CFU/ml) was
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inoculated in respective flasks containing 100 ml growth medium with and without TiO2 nanoparticles with varied concentration of nanoparticles from 15 to 250 µg/ml. The culture
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flasks were incubated at 37 ºC and optical density (OD600 nm) was recorded at the interval of
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2 h. The readings obtained were plotted and comparative studies were performed between
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control and treated culture with TiO2 nanoparticles.
2.9. Confocal scanning laser microscopy Confocal microscopy studies were performed on a confocal laser scanning microscope
Leica DMRE equipped with a confocal head TCS SPE (Leica, Wetzlar, Germany) and a 40X water immersion objective with a laser of 532 nm wavelength. S. aureus and E. coli cultures were labeled with propidium iodide (PI) dye. PI was used to determine the dead cells as PI specifically stains only dead bacteria. The TiO2 (125 µg/ml) nanoparticles were added to the overnight grown bacterial cultures and were incubated at 37 ºC for 30 minutes in the dark. 1 ml from both of these bacterial strain cultures were harvested by centrifuging at 2500 g for 5 min. The pellet was resuspended in 1 ml PBS buffer. Staining with PI was performed by 7
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adding 10 µl of PI to final concentration of 1 µg/ml. This solution was incubated for 3 h at 37 ºC. Likewise a control of both bacterial strains (not treated with TiO2 nanoparticles) was grown under similar conditions. A drop of the above prepared samples were then placed on a
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glass slide and mounted with the cover slip then cells were examined under a confocal scanning microscope.
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2.10. Transmission electron microscopy of bacterial cells
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Test compounds at MIC were added to the Bacterial cell S. aureus and E. coli
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suspensions in media (~1x106 c.f.u./ ml) and incubated for 16 h at 37 °C. All the cells were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer for 1 h at 20 °C [20]. Cells were
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washed with 0.1 M phosphate buffer (pH 7.2) and post-fixed by using 1% osmium tetroxide in 0.1 M phosphate buffer for 1 h at 4 °C. For ultrastructure studies, samples were dehydrated
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with graded acetone, cleared with toluene and infiltrated with a toluene and araldite mixture
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at room temperature then finally in pure araldite at 50 °C and embedded in an polypropylene tube (1.5 ml) with pure araldite mixture at 60 °C. Samples were prepared using a sectioning
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ultramicrotome (Lecia EM UC6) and were observed using TEM [20].
3. Results and discussion
The “green synthesis” approach has been proven to be a better method due to their
slower kinetics, better manipulation, control over crystal growth and stabilization. Recent advancement in this area includes enzymatic method of synthesis suggesting the enzymes to be responsible for the nanoparticle formation. Biomacromolecules have been used in the past to synthesize metal oxides, including ZnO [21], Cu2O [22] and Ga2O3 [23]. Recently, Jayaseelan et al. [24] reported the synthesis of TiO2 nanoparticles using Aeromonas hydrophilla. The GC-MS analysis of the broth showed the major compound found in the 8
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Aeromonas hydrophilla is glycyl – proline and other compounds present in lesser amounts are uric acid, glycl-glumatic acid, and Leucyl-leucine and compounds containing –COOH and -C═O group. They concluded that glycil-L proline act as reducing agent for the synthesis
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of TiO2 nanoparticles and the water soluble carboxylic acid compounds acts as stabilizing agents [24]. In the present work we report the extracellular synthesis of TiO2 nanoparticles
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using an enzyme alpha amylase from a non pathogenic fungal strain Aspergillus oryzae. Alpha amylase from Aspergillus oryzae contains 478 amino acid residues out of which 21 Here it is assumed that the amino
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residues are proline [25], 12 of them are exposed [26].
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acid proline might have played an important role in the reduction of titanium hydroxide. Moreover, alpha amylase from Aspergillus oryzae is an acidic enzyme with optimum pH of
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4.5 and low isoelectric point (PI=4.2) [27, 28]. This enzyme has large percentage of carboxyl groups which might be responsible for the stabilization of TiO2 nanoparticles. Fig. 1
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represents the proposed mechanism for the synthesis of TiO2 nanoparticles with alpha
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amylase having proline residues. Use of enzyme for nanoparticles synthesis has advantage over other biological methods as they are available in pure form and their structure is also
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well known. The mechanism of biosynthesis can be easily understood using well characterized enzyme. So far lysozyme [11]
and urease [13] has been reported in the
literature for the synthesis of TiO2 nanoparticles, compared to these enzymes alpha amylase is a low cost enzyme.
The crystalline nature and phase purity of titania nanostructures were analyzed by X-ray
diffraction studies. Fig. 2 shows the XRD analysis of TiO2 nanoparticles synthesized with different concentrations of alpha amylase enzyme. Both diffraction patterns could be indexed to monophasic nanocrystalline structure of titanium dioxide and found to be in good agreement with the available literature reports (PCPDF No. #84-1285). The observed 9
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reflections in Fig. 2a and b corroborates to the pure nanocrystalline TiO2 with anatase structure. No peak corresponding to any impurity was present which indicates the high purity of the samples. Fig. 3 showing the TEM images of the TiO2 nanoparticles synthesized using
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different concentration of alpha amylase. The TEM analysis showed that the particle size and morphology depends upon the concentration of enzyme used during synthesis. With the
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alpha amylase concentration of 2 mg/ml, the grain size varies in the range of 30-70 nm with an average grain size of 50 nm (Fig. 3a), however the grain size decreases significantly to 25
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nm (Fig. 3b) by increasing the concentration of the enzyme upto 15 mg/ml. The morphology
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of the nanoparticles formed in the enzymatic synthesis also exhibits the strong dependence on enzyme concentration. The hexagonal along with spherical nanoparticles of TiO2 could be
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seen at 2 mg/ml concentration of enzyme as shown in Fig. 3a. It is important to note that as the enzyme concentration increased to 15 mg/ml, the nanorings were also observed in
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addition to nanoparticles (Fig. 3b). To arrive at the definite response of nanoring formation,
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the high resolution-TEM (HR-TEM) study has been carried out. HR-TEM image clearly showed the formation of nanoring (Fig. 3c). This is clear from Fig. 3b and c that the average
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ring outer and inner diameters are 55 nm and 33 nm respectively with the shell thickness of 13 nm. It has been reported that the nanoparticles of different sizes and shapes could be synthesized using biological methods [8, 14, 16, 29]. Thus, by controlling the enzyme concentration one can achieve the controlled shape and size of the nanoparticles. Further, research on the synthesis of TiO2 nanoparticles at different enzyme concentration is under investigation which will give some insight on this aspect. FTIR spectra of alpha amylase and biosynthesized TiO2 nanoparticles using amylase at two different concentrations were recorded. The FTIR of nanoparticles with different enzyme concentrations was found similar (Fig. 4a and b). The spectral bands of enzyme at 1625 cm-1 could be assigned to amide I and absorption signal at 1520 cm-1 indicate a characteristic amide II band [30, 31]. The 10
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characteristic amide I and amide II bands of alpha amylase was observed in the FTIR spectra (Fig. 4c). FTIR spectroscopy clearly indicating that alpha amylase is responsible for the synthesis of nanoparticles and in their stabilization.
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The antibacterial effect of enzyme assisted TiO2 nanoparticles was evaluated against gram positive and gram negative bacterial strains. Standard antibiotic ampicillin was used as
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a control. The MIC value for the TiO2 nanoparticles was found to be 62.5 µg/ml for both
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strain S. aureus and E. coli (Table 1). The MIC value of the biosynthesized nanoparticles was compared with the chemically synthesized nanoparticles (procured from sigma). The
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chemically synthesized nanoparticles show greater MIC values for both the strains as shown in Table 1. Further, the stability of the biosynthesized nanoparticles was also studied in terms
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of antibacterial effect. To check the stability of the enzyme assisted TiO2 nanoparticles, they were stored at 4°C for six months and used to calculate the MIC value against bacterial
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strains. The nanoparticles show good stability as no change in the MIC results was obtained.
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The Inhibition was further confirmed using disc diffusion assay as shown in Fig. 5. It is evident from the zone of inhibition that TiO2 nanoparticles possess potent bactericidal
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activity. The bactericidal effect of TiO2 nanoparticles has been attributed to the decomposition of bacterial outer membranes by reactive oxygen species (ROS), primarily hydroxyl radicals (OH), which leads to phospholipid peroxidation and ultimately cell death [32, 33]. It was proposed that nanomaterials that can physically attach to a cell can be bactericidal if they come in contact with cell [34]. The growth curve study of S. aureus and E. coli in the presence and absence of TiO2 nanoparticles showed the effect of inhibitory concentration of TiO2 nanoparticles against S. aureus and E. coli till 20 h of incubation (Fig. 6). In control cell lag phase ends upto 4-6 h after which log phase starts which ends upto 1618 h. However when cells were treated with TiO2 nanoparticles, then delay in the lag phase 11
Page 11 of 29
occurred. At MIC concentration lag phase ends upto 12 h. There occurred delay in lag phase on exposure to different concentration of TiO2 nanoparticles and thus a prominent decrease in growth of S. aureus and E. coli was observed. To prove the effect of TiO2 nanoparticles on
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the viability of S. aureus and E. coli strain, we performed confocal laser scanning microscopy (CLSM) in the presence of PI. Bacterial cells were grown and stained with PI as described
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above. PI penetrates only cells with severe membrane lesions; the entire bacterial cells appear red (Fig. 7). It should be noted that PI can only stain the cells in which the cell membrane is
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disrupted since it intercalates into the double-stranded nucleic acids [35]. Results presented
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here showing that PI penetrates the bacterial cell. S. aureus and E. coli were taken for the study. A control stained with PI without the TiO2 nanoparticles (Fig. 7a) and samples with
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the nanoparticles (Fig 7b and c) were tested for the invasiveness of the nanoparticles inside the cells. The confocal studies showed that control is not showing any fluorescence while the
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sample with TiO2 nanoparticles showing the good fluorescence proving the concept of
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invading the cells. Since the PI is binding to the double stranded DNA it is confirmed that the TiO2 nanoparticles damage the bacterial cell wall. The nanoparticles disrupt the bacterial cell
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walls whether it is gram positive or gram negative bacterial strain. TEM images of ultrathin sections of treated cells showing the damaged cell wall and cell membrane while in untreated cells the cell membrane structures are intact (Fig. 8a). Treated cells are showed the leakage of the intracellular contents (Fig. 8b). The focus of this study was to examine the antibacterial activity of the biologically synthesized nanoparticles and also the effect of their particle size. In this study size effect is not observed as seen by the MIC data, confocal and TEM studies.
4. Conclusion Here, the biosynthesis of TiO2 nanoparticles using the enzyme alpha amylase has been described. The present studies suggest that the proline residues present in the enzyme play an 12
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important role in the reduction of titanium hydroxide. The biosynthesized nanoparticles were exploited for their antibacterial activity. The disc diffusion and growth curve confirms their antibacterial effect and indicates that TiO2 nanoparticles can be considered as potent
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antibacterial compound. As the synthesis is eco-friendly, the antibacterial properties of these nanoparticles can be further explored in future on other bacterial strains, for their use in
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various industrial and medical applications.
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Acknowledgments
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First author RA acknowledge the Indian Council of Medical Research, Govt. of India,
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for providing the financial support in the form of Senior Research Fellowship.
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TiO2, Journal of Photochemistry and Photobiology A: Chemistry, 181 (2006) 401-407. [34] H.A. Jeng, J. Swanson, Toxicity of metal oxide nanoparticles in mammalian cells,
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Journal of Environmental Science and Health Part A, 41 (2006) 2699-2711. [35] J.S. Miller, J.M. Quarles, Flow cytometric identification of microorganisms by dual
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te
d
staining with FITC and PI, Cytometry, 11 (1990) 667-675.
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Legends to Figures Fig. 1. Possible mechanism for the synthesis of TiO2 nanoparticles. Fig. 2. X-ray diffraction pattern of TiO2 nanoparticles synthesized using alpha amylase (a)
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2mg/ml and (b) 15mg/ml.
Fig. 3. TEM images of TiO2 nanoparticles using (a) 2mg/ml and (b) 15mg/ml alpha amylase
cr
concentration (c) is the HRTEM image of the nanoring.
us
Fig. 4. FTIR spectra of TiO2 nanoparticles: (a) alpha amylase. (b) Nanoparticles synthesized using 2mg/ml alpha amylase. (c). Nanoparticles synthesized using 15mg/ml alpha amylase.
an
Fig. 5. Zone of Inhibition of TiO2 Nanoparticles against S. aureus and E.coli: (a) Ampicillin (b) TiO2 nanoparticles (Av. size 50 nm) (c) TiO2 nanoparticles (Av. size 25 nm) (d)
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TiO(OH)2 (e) alpha amylase.
Fig. 6. Growth curve study of bacterial strain S. aureus and E.coli: (a) S. aureus with
d
different concentration of TiO2 nanoparticles (Av. size 50 nm) (b) S. aureus with different concentration of TiO2 nanoparticles (Av. size 25 nm) (c) E. coli with different concentration
te
of nanoparticles (Av. size 50nm) (d) E. coli with different concentration of TiO2 nanoparticles (Av. size 25 nm). Each data point represents the mean ± standard deviation of
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three independent observations.
Fig. 7. Laser confocal images of S. aureus and E.coli: cells with membrane damage were stained with PI (red signals) (a) untreated control cells showing no fluorescence (b) cells treated with 125µg/ml of TiO2 nanoparticles (Av. size 50 nm) showing the red fluorescence (c) cells treated with 125µg/ml of TiO2 nanoparticles (Av. size 25 nm) showing the red fluorescence.
Fig. 8. TEM of S. aureus and E. coli: (a) untreated control cells (b) cells treated with 62.5µg/ml of TiO2 nanoparticles (Av. size 50 nm) (c) cells treated with 62.5µg/ml of TiO2 nanoparticles (Av. size 25 nm).
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Table 1 Zone of Inhibition (mm) and MIC (µg/ml) of alpha amylase and chemically synthesized TiO2 nanoparticles against S. aureus and E. coli. Particle size (Av. Size 25nm) MIC Inhibition (µg/ml)a Zone(mm) 62.5 16
E. coli (MTCC-405)
62.5
62.5
us
200
te
d
M
an
Particle size is not affecting the MIC of nanoparticles.
13
200
Ac ce p
a
11
Chemically synthesized NPS MIC (µg/ml)
cr
S. aureus (MTCC-3160)
Particle size (Av. Size 50nm) MIC Inhibition (µg/ml)a Zone(mm) 62.5 15
Bacterial strain
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ip t cr us an M d te Ac ce p
Fig. 1
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ip t cr us an M d te Ac ce p Fig. 2
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d
te
Ac ce p us
an
M
cr
ip t
ip t cr us an
Ac ce p
te
d
M
Fig. 3
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Fig. 4
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Fig. 5
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d
te
Ac ce p us
an
M
cr
ip t
ip t cr us an M d te Ac ce p Fig. 6
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ip t cr us an M d te Ac ce p Fig. 7 28
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ip t cr us an M d te Ac ce p Fig. 8
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