Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (2014) xxx–xxx
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Green synthesis of ZnO nanoparticles using Solanum nigrum leaf extract and their antibacterial activity M. Ramesh a,⇑, M. Anbuvannan b, G. Viruthagiri b a b
Department of Physics, Physics Wing (DDE), Annamalai University, Annamalai Nagar 608002, Tamil Nadu, India Department of Physics, Annamalai University, Annamalai Nagar 608002, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
We synthesized ZnO nanoparticles
with wurtzite hexagonal structure using green synthesis. Method is simple, novel, nontoxic and cost effective and environment friendly. Antibacterial activity of ZnO greenly synthesized by Solanum nigrum.
a r t i c l e
i n f o
Article history: Received 30 July 2014 Received in revised form 19 September 2014 Accepted 24 September 2014 Available online xxxx Keywords: Green synthesis ZnO NPs Solanum nigrum Antibacterial activity
Zinc nitrate
a b s t r a c t In the present investigation, we have described the green biosynthesis of ZnO nanoparticles (NPs) by using Solanum nigrum as capping agent. The functionalization of ZnO particles through S. nigrum leaf extract mediated bioreduction of ZnO was investigated through UV–Vis DRS, photoluminescence (PL), X-ray diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy, Field Emission Scanning Electron Microscopy (FE-SEM), Transmission Electron Microscopy (TEM), thermal gravimetric–differential thermal analysis (TG–DTA), X-ray photoelectron spectroscopy (XPS) and antibacterial activities. UV–Vis-DRS studies revealed that the indirect band gap 3.38 eV and photoluminescence study reveals the blue emission at 402, 447, 469 and 483 nm and the green emission at 529 nm respectively. In addition, the synthesized NPs are wurtzite hexagonal structure with an average grain size lies between 20 and 30 nm were found from XRD analysis. Further, FT-IR spectra revealed the functional groups and the presence of protein as the stabilizing agent for surrounding the ZnO NPs. The diameter of the NPs in the range of 20–30 nm was found from FE-SEM study. TEM analysis was investigated the ZnO NPs as a quasi-spherical in shape and their diameter at around 29.79 nm. Finally, the current study has clearly demonstrated that the particle size variations and surface area to volume ratios of ZnO NPs are responsible for significant higher antibacterial activities. Further, the present investigation suggests that ZnO NPs has the potential applications for various medical and industrial fields so, that the investigation is so useful and helpful to the scientific communities. Ó 2014 Published by Elsevier B.V.
⇑ Corresponding author. Tel.: +91 9976994926. E-mail address: [email protected] (M. Ramesh). http://dx.doi.org/10.1016/j.saa.2014.09.105 1386-1425/Ó 2014 Published by Elsevier B.V.
Please cite this article in press as: M. Ramesh et al., Green synthesis of ZnO nanoparticles using Solanum nigrum leaf extract and their antibacterial activity, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), http://dx.doi.org/10.1016/j.saa.2014.09.105
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Introduction
Preparation of zinc oxide NPs
The field of nanotechnology is one of the most active researches in modern material science. Nanotechnology is emerging as a rapid growing field with its applications in science and technology for the purpose of manufacturing new materials at the nanoscale level. Nano-materials are called a wonder of modern medicine. It is stated that antibiotics kill perhaps a half dozen different diseasecausing organisms but nano-materials can kill some 650 cells [1]. Previously reported that, metal NPs have various functions that are not observed in bulk phase [2,3] and have been studied extensively because of their exclusive catalytic, optical, electronic, magnetic and antimicrobial [4,5] wound healing and anti-inflammatory properties [6]. The different types of NPs are used in different applications, among them; ZnO NPs have a wide range of applications like cosmetics and sunscreen lotions because of their efficient UV-A and UV-B efficient absorption properties without scattering visible light [7]. ZnO NPs have also used as the antimicrobial properties [8,9] and agriculture and anticancer therapy [10,11]. The production of ZnO NPs increased day by day and also increased the accidental exposure to humans and animals. Biological methods for NPs synthesis using microorganisms, enzymes, and plants or plant extracts have been suggested as possible eco-friendly alternatives to chemical and physical methods. Thus, there is a need for a new simple and eco-friendly green synthesis processes, those avoids the use of toxic chemicals and high energy inputs. Recently, a variety of research work has been carried out to investigate the bioreduction of various metal ions (e.g. Ag, Au, Pd, and Pt) into metal NPs [12–15]. The bioreduction process exploits the reduction capability of various biochemicals in natural and renewable materials such as microbes [16–18] and plant extracts [19,20] to reduce metal ions into zero-valance metal NPs. Green synthesis of ZnO has also been reported for antibacterial properties [21–23]. Solanum nigrum is a medicinal plant belonging to the family Solanaceae. Its common names are Makoi and blacknight shade. S. nigrum has been used traditionally to treat various ailments such as pain, inflammation fever and enteric diseases. It possesses many activities like anti-tumorigenic, anti-oxidant, anti-inflammatory, diuretic, anti-pyretic agent, and anti-bacterial respectively. It is also used against sexually transmitted diseases. S. nigrum possesses numerous compounds that are responsible for the bioreduction. Its active components are glycoalkaloids, glycoproteins, and polysaccharides, polyphenolic compounds such as gallic acid, catechin, protocatechuic acid (PCA), caffeic acid, epicatechin, rutin, and naringenin [24]. Hence, the present study was aimed to rapidly green synthesize of ZnO NPs using aqueous leaves extract of S. nigrum, to investigate the biomolecules responsible for synthesis of ZnO NPs and finally to evaluate its antibacterial activities.
For the synthesis of NPs, 50 mL of S. nigrum leaves extract was taken and boiled at 60–80 °C by using a stirrer-heater. Then, 5 g of zinc nitrate was added to the solution as the temperatures reached at 60 °C. This mixture was then boiled until it converted to a deep yellow coloured suspension. This paste was then collected in a ceramic crucible and heated in an air heated furnace at 400 °C for 2 h. A light white coloured powder was obtained and this powder was carefully collected and sent for different characterizations. The material was powered using a mortar and pestle so, that got a fine powder, which is easy for further characterizations.
Materials and methods Preparation of the leaf extract S. nigrum plant leaves were collected from Polur, Thiruvannamalai district, Tamil Nadu, India. The leaves were washed several times with distilled water to removes the dust particles. The extract was prepared by placing 20 g of washed dried fine cut leaves in 250 mL glass beaker along with 100 mL of sterile distilled water. The mixture was then boiled for 20 min until the colour of the aqueous solution changes from watery to light yellow. The extract was cooled at room temperature and filtered by Whatman filter paper. Then, the extract was stored in a room temperature in order to use for further characterizations.
Characterization of zinc oxide NPs The UV–Vis reflectance spectra (UV–Vis DRS) were carried out with a measurements UV140404B in the wavelength range of 200–850 nm in reflectance mode. Photoluminescence (PL) spectra were recorded using FLUOROLOG – FL3-11 fluorescence spectrometer. The crystalline structure of the samples was analyzed by using (PAN analytical X’PERT PRO model X-ray diffractometer), on the instrument operating at a voltage of 50 kV and a current of 30 mA. Fourier Transform Infrared spectra were recorded under identical conditions in the 400–4000 cm1 region using Fourier Transform Infrared Spectrometer (SHIMADZHU). The morphology and size distribution were characterized using FE-SEM (JEOL JSM 6701-F) and TEM measurement in a TECHNAI10-Philphs instrument. Antibacterial assay Synthesized compound are tested for inhibition against the human pathogenic bacteria. Microbial assay are carried out by Kelman et al. disc diffusion method [25]. All the bacterial strains were enriched in nutrient broth at 37 °C for 18–24 h. After that they were streaked over the surface of Muller Hinton Agar (MHA) by using sterile cotton swabs. Then 20 ll of the extract was pipetted on a 6 mm sterile paper disc, the solvent was allowed to evaporate and a disc was placed on the surface of the plate, the plates were incubated for 24 h at 37 °C. Control disc were placed with solvent and standard to assess the effect of solvent and standard on pathogens. Inhibited bacterial and fungal growths were observed as clear halos (zones) around the disc. Antimicrobial activity was measured as diameter of zone of inhibition excluding the paper disc diameter. Inhibition zone was observed after 24 h. Results and discussion Optical studies Fig. 1a shows the optical absorption spectrum of ZnO NPs synthesized by using S. nigrum extract. The sample has a clear and strongly observed absorption peak below at 400 nm. The band gap energy (Eg) of ZnO was obtained from the wavelength value corresponding to the intersection point of the vertical and horizontal part of the spectrum, using the equations:
Eg ¼
hc 1240 eV; Eg ¼ eV k k
ð1Þ
The band gap energy corresponds to the absorption limit and can be roughly evaluated by the above relation. Where, Eg is the band gap energy (eV), h is the Planck’s constant (6.626 1034 J s), C is the light velocity (3 108 m/s) and k is the wavelength (nm). From Fig. 1a the absorption edge are positioned at 358 nm which, corresponding the band gap value of 3.46 eV which indicates red shift in the UV region.
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ZnO
70
450000
Intensity (CPS)
50
%R
- ZnO
400000
60
40 30
447
350000
529 250000 200000 150000
10
100000 50000 200
300
400
500
600
700
800
900
Wavelength (nm)
Wavelength (nm)
Indirect band gap energy (Kubelka–Munk plot) The reflectance spectra were analyzed using the Kubelka–Munk relation Eq. (2). To convert the reflectance data into a Kubelka– Munk function (equivalent to the absorption coefficient) F(R), the following relation was used.
ð1 RÞ2 2R
ð2Þ
where R is the reflectance value. Bandgap energy of the sample were estimated from the variation of the Kubelka–Munk function with photon energy. Fig. 1b shows the Kubelka–Munk plots for the ZnO NPs. It is used to determine their band gap energy associated with their indirect transitions. The ZnO exhibits indirect Eg of 3.38 eV.
Fig. 2. Photoluminescence spectrum of ZnO NPs synthesized using Solanum nigrum leaf extract.
1000
101
100
600
002 400
102
200
110 103
112 201 004202
0 10
Photoluminescence (PL) analysis Photoluminescence (PL) studies were performed to emphasize its emission properties as shown in Fig. 2. The photoluminescence of ZnO sample suggested five emission bands, including three blue bands at 402, 447,469 and at 483 nm, probable green band at 529 nm have been observed from the prepared ZnO NPs sample. The blue band at 417, 440 and 462 nm may be in correlation with the defect structures in ZnO crystal. The green bands at 520 nm and shoulder red band at 675 nm may be correlated to a transition between the oxygen vacancy and interstitial oxygen [26,27].
ZnO
800
Intensity (arb.units)
Fig. 1a. UV-DRS spectrum of ZnO NPs synthesized using Solanum nigrum leaf extract.
FðRÞ ¼
483
300000
20
0
469
402
20
30
40
50
60
70
80
2 Theta (Deg) Fig. 3. XRD spectrum of ZnO NPs synthesized using Solanum nigrum leaf extract.
Table 1 The structure and geometric parameters of ZnO NPs. 2h (°)
FWHM (b)
d-spacing (Å)
Cos h
Crystallite size (nm)
31.29 33.95 36.04 47.05 56.09 62.38 65.90 67.45 68.60 72.03 76.48
0.20 0.20 0.23 0.25 0.36 0.36 0.4 0.36 0.30 0.7 0.4
2.85 2.63 2.50 2.21 1.63 1.48 1.41 1.38 1.36 1.31 1.24
0.9629 0.9564 0.9509 0.9981 0.8825 0.8554 0.8391 0.8317 0.8260 0.8088 0.7854
41.38 41.76 36.48 34.83 25.11 25.91 23.74 26.66 32.16 14.07 25.67
Average
29.79
XRD analysis
Fig. 1b. Plot of indirect band gap energy for ZnO NPs.
Structure and phase purity of ZnO NPs are shown in Fig. 3. From the diffractogram of XRD are very well matched with the hexagonal phase (wurtzite structure) by comparison with the data from JCPDS card No. 89-1397 and no indication of a secondary phase or impurity peaks were obtained. The strong and narrow diffraction peaks indicate that the product has good crystalline structure. The sharp intense diffraction peaks appearing at about 2h of 31.29°, 33.95°, 36.04°, 47.05°, 56.09°, 62.38°, 65.90°, 67.45°, 68.60°, 72.03°,
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FT-IR analysis
%T
Leaf Extract
4000
ZnO
3500
3000
2500
2000
1500
1000
500
Wavenumber (cm-1) Fig. 4. FT-IR spectra of leaf extract and ZnO NPs synthesized using Solanum nigrum leaf extract.
and 76.48° corresponding with those from (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4) and (2 0 2) orientations, respectively. The average grain size of the sample was calculated using the Scherer’s Eq. (3):
D¼
Kk Å bCosh
ð3Þ
where D is the average crystallite size in Å, K is the shape factor (0.9), k is the wavelength of X-ray (1.5406 Å) Cu Ka radiation, h is the Bragg angle, and b is the corrected line broadening of the NPs. The average crystallite size of the ZnO NPs is about 29.79 nm. Estimation of the crystallite sizes using this relation are depicted in Table 1.
Possible biomolecules responsible for the reduction of ZnO and capping agent of bioreduced ZnO NPs through particular bond vibration peaks identified from defined wave numbers in FT-IR technique. The FT-IR spectra of control leaf extract (before reaction without Zn (NO3)2 and synthesized ZnO (after reaction with Zn (NO3)2 are shown in Fig. 4. The absorption bands at 3429, 2926, 2349, 1635, 1404, 1261, 1031, 605, 540, and 470 cm1 in S. nigrum leaf extract wound be shifted into 3425, 2924, 1627, 1382, 1126, 1030, 833, and 445 cm1 in synthesized ZnO NPs sample. The peak found at around 1450–1500 cm1 showed the stretching of N–H vibration. Whereas the stretching of ZnO NPs were found around 400–800 cm1. The FT-IR spectra showed the presence of bonds due to O–H stretching (around 3429 cm1) and aldehydic C–H stretching (around 2924 cm1) [28]. The peak at around 1240– 1261 cm1 present in both the cases signified amide III band of the random coil of protein [29]. Peak at 1404 cm1 may be assigned to the symmetric stretching of the carboxyl side groups in the amino acid residues of the protein molecules [30]. The band at 1029 cm1 corresponds to C–N stretching vibration of amine [31]. The bands incidence at 1126, 1030 and 605 cm1 illustrates the chemical bonding, crystal structure and relative intensities of the IR bands of the carbonate [32,33]. Therefore the synthesized ZnO NPs were surrounded by proteins and metabolites such as terpenoids having functional groups. From the analysis of FTIR studies, we confirmed that the carbonyl groups from the amino acid residues and proteins has the stronger ability to bind metal ions, indicating that the proteins could possibly from the metal NPs (i.e., capping of silver NPs) to prevent agglomeration and
Fig. 5. FE-SEM image and EDX spectrum of ZnO NPs synthesized using Solanum nigrum leaf extract.
Fig. 6. TEM image ZnO NPs synthesized using Solanum nigrum leaf extract.
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ZnO NPs and these absorption peaks were due to the surface plasmon resonance of zinc oxide NPs. The origin of these elements lies in the biological components; mostly align along with ZnO NPs [34].
4 100
DTA (mW/mg)
3
95
TG %
90 2 85 1
80 75
TEM analysis The size and morphology of ZnO nanoparticles analyzed by TEM and SAED pattern obtained from TEM studies are depicted in Fig. 6a and b. This image reveals that most of the ZnO NPs are quasi-spherical and their diameter is about 29.79 nm. The SAED pattern revealed that the diffraction rings of the synthesized ZnO exhibited Debye–Scherrer rings assigned (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4) and (2 0 2) respectively, lattice planes of the face centered cubic (fcc) ZnO, indicating that the biogenic NPs seen in the TEM images are nano-crystalline in nature as shown in Fig. 6b. The particle size determined from TEM analysis is in good agreement with that of the XRD analysis.
0
70 0
200
400
600
800
1000
o Temperature ( C)
Fig. 7. TG–DTA curves of the ZnO NPs synthesized using Solanum nigrum leaf extract.
TG–DTA analysis thereby stabilize the medium. This suggests that the biological molecules could possibly perform dual functions of formation and stabilization of ZnO NPs in the aqueous medium.
Fig. 7 shows the TG–DTA measurement results of the ZnO nanoparticles. The DTA analysis of Zn (NO3)26H2O demonstrated several endothermic effects at 50 °C due to loss of residual water. The thermal dehydration at 165 °C and 318 °C, respectively, which corresponds to the dehydration and hydrolysis to basic zinc nitrate. The endothermic peak with maximum at 391 °C corresponded to the decomposition of the basic zinc nitrate to zinc oxide [35].
FE-SEM analysis The FE-SEM image of prepared ZnO NPs was presented in Fig. 5a. The diameter of the cluster ZnO NPs found to be in the range of 20–30 nm and is nearly spherical in shape with rough surface. Fig. 5b. EDX spectrum shows the high values of zinc (62%) and oxygen (29%) respectively. These results confirmed the present of zinc as a majority label compared to oxygen in precursor material. The EDX analysis displays the optical absorption peaks of
XPS analysis In the O 1s profile is asymmetric and can be fitted to one symmetrical peak (a locating at 530.08 eV, respectively), indicating one peak of O species in the sample. The peaks a should be Survey
1.20E+06 Zn2p
Counts / s
1.00E+06 8.00E+05 O1s
6.00E+05 4.00E+05 2.00E+05
K2p
0.00E+00 1200
1000
800
600
400
200
0
Binding Energy (eV) Zn2p Scan
O1s Scan
2.00E+05
8.00E+04
Element ZnO
1.40E+05 1.20E+05 1.00E+05 8.00E+04
Counts / s
7.00E+04
1.60E+05
Counts / s
Metal oxide
1.80E+05
6.00E+04 5.00E+04 4.00E+04 3.00E+04
6.00E+04 4.00E+04 1050
1040
1030
Binding Energy (eV)
1020
2.00E+04 544 542 540 538 536 534 532 530 528 526
Binding Energy (eV)
Fig. 8. XPS spectra of ZnO NPs synthesized using Solanum nigrum leaf extract.
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Fig. 9. Antibacterial activity of ZnO NPs synthesized using Solanum nigrum leaf extract.
associated with the lattice oxygen (OL) of ZnO Nps and chemisorbed oxygen (OH) caused by the surface hydroxyl respectively [36]. The Zn 2p core level spectrum is presented in Fig. 8. Two strong peaks centred at 1021.08 and 1044.18 eV, corresponding to the Zn 2p3/2 and Zn 2p1/2, are clearly observed. These present value are in agreement with the binding energies of Zn2+ ion [36]. However, it is hard to identify whether the Zn XPS signal comes from the ZnO Nps. Antibacterial activity The antibacterial activity of ZnO NPs was investigated both Gram positive (Staphylococcus aureus) and Gram negative (Salmonella paratyphi, Vibrio cholerae, Escherichia coli) bacteria by zone inhibition methods. The results of zone inhibition method as depicted in Fig. 9. The diameter of inhibition zones (mm) around each well with ZnO NPs solution are represented as shown in Fig. 9. Antibacterial activity of prepared ZnO nanoparticles was evolved against S. aureus, E. coli, S. paratyphi, and V. cholerae. The ZnO NPs synthesized by S. nigrum extracts are found the highest antimicrobial activity against S. paratyphi (17 mm) than compared to standard tablet, and the lesser antimicrobial activity of ZnO NPs was found against S. aureus (18 mm), V. cholerae (11 mm), and E. coli (7 mm) than compared to a standard tablet. The positive control (Leaf extract) was showed activity against all the microbial strains tested the maximum activity against V. cholerae, and S. aureus (6 mm and 6 mm). The antibacterial activity of ZnO NPs depends on the size, the smaller the size, the better the antibacterial activity [37]. We supposed that the ZnO NPs formed in this experiment might have good antibacterial activities. Conclusion ZnO NPs synthesized were successfully using a S. nigrum leaf extract mediated bioreduction process. The structure, morphology
and size (dimension) of prepared ZnO NPs were examined by FT-IR, XRD, FE-SEM and TEM analysis. UV–Vis-DRS studies confirmed the indirect band gap 3.38 eV and photoluminescence was found the blue band at 402, 447, 469 and 483 nm. The average grain size lies between 20–30 nm were found from XRD study as well as FT-IR spectra revealed the functional groups of stretching bands for ZnO NPs were found around 800–400 cm1. The diameter of the NPs and the quasi-spherical in shape and their diameter around 29.79 nm were confirmed by TEM study. The endothermic peak with maximum at 391 °C corresponded to the decomposition of the basic zinc nitrate to zinc oxide was confirmed by TG–DTA study. Two strong peaks centred at 1021.08 and 1044.18 eV, corresponding to the Zn 2p3/2 and Zn 2p1/2, were confirmed by XPS study. The synthesized NPs was studied for antibacterial activities against the microorganisms both Gram positive (S. aureus) and Gram negative (S. paratyphi, V. cholerae, E. coli) bacteria. Finally, the present study is so helpful and useful to the scientific community for using the Zno NPs as the potent applications to the antidiabetic and antihyperlipidemic activities. In addition, it is inexpensive, stable and nontoxic, safe and eco-friendly without side effects of human beings. Therefore, in the present research was designed to prepared eco-friendly, stable and nontoxic ZnO nanoparticles from the S. nigrum leaf extract. Acknowledgements The authors are thankful and grateful to Dr. S. Barathan, Professor and Head, Department of Physics, Annamalai University, Tamilnadu–608 002, for providing all necessary facilities to carry out the present work successfully. References [1] I.O. Sosa, C. Noguez, R.G. Barrera, J. Phys. Chem. B 107 (2003) 6269–6975. [2] Y.G. Sun, B. Mayers, T. Herricks, Y.N. Xia, Nano Lett. 3 (2003) 955–960.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Green synthesis of ZnO nanoparticles using Solanum nigrum leaf extract and their antibacterial activity M. Ramesh a,⇑, M. Anbuvannan b, G. Viruthagiri b a b
Department of Physics, Physics Wing (DDE), Annamalai University, Annamalai Nagar 608002, Tamil Nadu, India Department of Physics, Annamalai University, Annamalai Nagar 608002, Tamil Nadu, India
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
We synthesized ZnO nanoparticles
with wurtzite hexagonal structure using green synthesis. Method is simple, novel, nontoxic and cost effective and environment friendly. Antibacterial activity of ZnO greenly synthesized by Solanum nigrum.
a r t i c l e
i n f o
Article history: Received 30 July 2014 Received in revised form 19 September 2014 Accepted 24 September 2014 Available online xxxx Keywords: Green synthesis ZnO NPs Solanum nigrum Antibacterial activity
Zinc nitrate
a b s t r a c t In the present investigation, we have described the green biosynthesis of ZnO nanoparticles (NPs) by using Solanum nigrum as capping agent. The functionalization of ZnO particles through S. nigrum leaf extract mediated bioreduction of ZnO was investigated through UV–Vis DRS, photoluminescence (PL), X-ray diffraction (XRD), Fourier Transform Infrared (FTIR) spectroscopy, Field Emission Scanning Electron Microscopy (FE-SEM), Transmission Electron Microscopy (TEM), thermal gravimetric–differential thermal analysis (TG–DTA), X-ray photoelectron spectroscopy (XPS) and antibacterial activities. UV–Vis-DRS studies revealed that the indirect band gap 3.38 eV and photoluminescence study reveals the blue emission at 402, 447, 469 and 483 nm and the green emission at 529 nm respectively. In addition, the synthesized NPs are wurtzite hexagonal structure with an average grain size lies between 20 and 30 nm were found from XRD analysis. Further, FT-IR spectra revealed the functional groups and the presence of protein as the stabilizing agent for surrounding the ZnO NPs. The diameter of the NPs in the range of 20–30 nm was found from FE-SEM study. TEM analysis was investigated the ZnO NPs as a quasi-spherical in shape and their diameter at around 29.79 nm. Finally, the current study has clearly demonstrated that the particle size variations and surface area to volume ratios of ZnO NPs are responsible for significant higher antibacterial activities. Further, the present investigation suggests that ZnO NPs has the potential applications for various medical and industrial fields so, that the investigation is so useful and helpful to the scientific communities. Ó 2014 Published by Elsevier B.V.
⇑ Corresponding author. Tel.: +91 9976994926. E-mail address: [email protected] (M. Ramesh). http://dx.doi.org/10.1016/j.saa.2014.09.105 1386-1425/Ó 2014 Published by Elsevier B.V.
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Introduction
Preparation of zinc oxide NPs
The field of nanotechnology is one of the most active researches in modern material science. Nanotechnology is emerging as a rapid growing field with its applications in science and technology for the purpose of manufacturing new materials at the nanoscale level. Nano-materials are called a wonder of modern medicine. It is stated that antibiotics kill perhaps a half dozen different diseasecausing organisms but nano-materials can kill some 650 cells [1]. Previously reported that, metal NPs have various functions that are not observed in bulk phase [2,3] and have been studied extensively because of their exclusive catalytic, optical, electronic, magnetic and antimicrobial [4,5] wound healing and anti-inflammatory properties [6]. The different types of NPs are used in different applications, among them; ZnO NPs have a wide range of applications like cosmetics and sunscreen lotions because of their efficient UV-A and UV-B efficient absorption properties without scattering visible light [7]. ZnO NPs have also used as the antimicrobial properties [8,9] and agriculture and anticancer therapy [10,11]. The production of ZnO NPs increased day by day and also increased the accidental exposure to humans and animals. Biological methods for NPs synthesis using microorganisms, enzymes, and plants or plant extracts have been suggested as possible eco-friendly alternatives to chemical and physical methods. Thus, there is a need for a new simple and eco-friendly green synthesis processes, those avoids the use of toxic chemicals and high energy inputs. Recently, a variety of research work has been carried out to investigate the bioreduction of various metal ions (e.g. Ag, Au, Pd, and Pt) into metal NPs [12–15]. The bioreduction process exploits the reduction capability of various biochemicals in natural and renewable materials such as microbes [16–18] and plant extracts [19,20] to reduce metal ions into zero-valance metal NPs. Green synthesis of ZnO has also been reported for antibacterial properties [21–23]. Solanum nigrum is a medicinal plant belonging to the family Solanaceae. Its common names are Makoi and blacknight shade. S. nigrum has been used traditionally to treat various ailments such as pain, inflammation fever and enteric diseases. It possesses many activities like anti-tumorigenic, anti-oxidant, anti-inflammatory, diuretic, anti-pyretic agent, and anti-bacterial respectively. It is also used against sexually transmitted diseases. S. nigrum possesses numerous compounds that are responsible for the bioreduction. Its active components are glycoalkaloids, glycoproteins, and polysaccharides, polyphenolic compounds such as gallic acid, catechin, protocatechuic acid (PCA), caffeic acid, epicatechin, rutin, and naringenin [24]. Hence, the present study was aimed to rapidly green synthesize of ZnO NPs using aqueous leaves extract of S. nigrum, to investigate the biomolecules responsible for synthesis of ZnO NPs and finally to evaluate its antibacterial activities.
For the synthesis of NPs, 50 mL of S. nigrum leaves extract was taken and boiled at 60–80 °C by using a stirrer-heater. Then, 5 g of zinc nitrate was added to the solution as the temperatures reached at 60 °C. This mixture was then boiled until it converted to a deep yellow coloured suspension. This paste was then collected in a ceramic crucible and heated in an air heated furnace at 400 °C for 2 h. A light white coloured powder was obtained and this powder was carefully collected and sent for different characterizations. The material was powered using a mortar and pestle so, that got a fine powder, which is easy for further characterizations.
Materials and methods Preparation of the leaf extract S. nigrum plant leaves were collected from Polur, Thiruvannamalai district, Tamil Nadu, India. The leaves were washed several times with distilled water to removes the dust particles. The extract was prepared by placing 20 g of washed dried fine cut leaves in 250 mL glass beaker along with 100 mL of sterile distilled water. The mixture was then boiled for 20 min until the colour of the aqueous solution changes from watery to light yellow. The extract was cooled at room temperature and filtered by Whatman filter paper. Then, the extract was stored in a room temperature in order to use for further characterizations.
Characterization of zinc oxide NPs The UV–Vis reflectance spectra (UV–Vis DRS) were carried out with a measurements UV140404B in the wavelength range of 200–850 nm in reflectance mode. Photoluminescence (PL) spectra were recorded using FLUOROLOG – FL3-11 fluorescence spectrometer. The crystalline structure of the samples was analyzed by using (PAN analytical X’PERT PRO model X-ray diffractometer), on the instrument operating at a voltage of 50 kV and a current of 30 mA. Fourier Transform Infrared spectra were recorded under identical conditions in the 400–4000 cm1 region using Fourier Transform Infrared Spectrometer (SHIMADZHU). The morphology and size distribution were characterized using FE-SEM (JEOL JSM 6701-F) and TEM measurement in a TECHNAI10-Philphs instrument. Antibacterial assay Synthesized compound are tested for inhibition against the human pathogenic bacteria. Microbial assay are carried out by Kelman et al. disc diffusion method [25]. All the bacterial strains were enriched in nutrient broth at 37 °C for 18–24 h. After that they were streaked over the surface of Muller Hinton Agar (MHA) by using sterile cotton swabs. Then 20 ll of the extract was pipetted on a 6 mm sterile paper disc, the solvent was allowed to evaporate and a disc was placed on the surface of the plate, the plates were incubated for 24 h at 37 °C. Control disc were placed with solvent and standard to assess the effect of solvent and standard on pathogens. Inhibited bacterial and fungal growths were observed as clear halos (zones) around the disc. Antimicrobial activity was measured as diameter of zone of inhibition excluding the paper disc diameter. Inhibition zone was observed after 24 h. Results and discussion Optical studies Fig. 1a shows the optical absorption spectrum of ZnO NPs synthesized by using S. nigrum extract. The sample has a clear and strongly observed absorption peak below at 400 nm. The band gap energy (Eg) of ZnO was obtained from the wavelength value corresponding to the intersection point of the vertical and horizontal part of the spectrum, using the equations:
Eg ¼
hc 1240 eV; Eg ¼ eV k k
ð1Þ
The band gap energy corresponds to the absorption limit and can be roughly evaluated by the above relation. Where, Eg is the band gap energy (eV), h is the Planck’s constant (6.626 1034 J s), C is the light velocity (3 108 m/s) and k is the wavelength (nm). From Fig. 1a the absorption edge are positioned at 358 nm which, corresponding the band gap value of 3.46 eV which indicates red shift in the UV region.
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ZnO
70
450000
Intensity (CPS)
50
%R
- ZnO
400000
60
40 30
447
350000
529 250000 200000 150000
10
100000 50000 200
300
400
500
600
700
800
900
Wavelength (nm)
Wavelength (nm)
Indirect band gap energy (Kubelka–Munk plot) The reflectance spectra were analyzed using the Kubelka–Munk relation Eq. (2). To convert the reflectance data into a Kubelka– Munk function (equivalent to the absorption coefficient) F(R), the following relation was used.
ð1 RÞ2 2R
ð2Þ
where R is the reflectance value. Bandgap energy of the sample were estimated from the variation of the Kubelka–Munk function with photon energy. Fig. 1b shows the Kubelka–Munk plots for the ZnO NPs. It is used to determine their band gap energy associated with their indirect transitions. The ZnO exhibits indirect Eg of 3.38 eV.
Fig. 2. Photoluminescence spectrum of ZnO NPs synthesized using Solanum nigrum leaf extract.
1000
101
100
600
002 400
102
200
110 103
112 201 004202
0 10
Photoluminescence (PL) analysis Photoluminescence (PL) studies were performed to emphasize its emission properties as shown in Fig. 2. The photoluminescence of ZnO sample suggested five emission bands, including three blue bands at 402, 447,469 and at 483 nm, probable green band at 529 nm have been observed from the prepared ZnO NPs sample. The blue band at 417, 440 and 462 nm may be in correlation with the defect structures in ZnO crystal. The green bands at 520 nm and shoulder red band at 675 nm may be correlated to a transition between the oxygen vacancy and interstitial oxygen [26,27].
ZnO
800
Intensity (arb.units)
Fig. 1a. UV-DRS spectrum of ZnO NPs synthesized using Solanum nigrum leaf extract.
FðRÞ ¼
483
300000
20
0
469
402
20
30
40
50
60
70
80
2 Theta (Deg) Fig. 3. XRD spectrum of ZnO NPs synthesized using Solanum nigrum leaf extract.
Table 1 The structure and geometric parameters of ZnO NPs. 2h (°)
FWHM (b)
d-spacing (Å)
Cos h
Crystallite size (nm)
31.29 33.95 36.04 47.05 56.09 62.38 65.90 67.45 68.60 72.03 76.48
0.20 0.20 0.23 0.25 0.36 0.36 0.4 0.36 0.30 0.7 0.4
2.85 2.63 2.50 2.21 1.63 1.48 1.41 1.38 1.36 1.31 1.24
0.9629 0.9564 0.9509 0.9981 0.8825 0.8554 0.8391 0.8317 0.8260 0.8088 0.7854
41.38 41.76 36.48 34.83 25.11 25.91 23.74 26.66 32.16 14.07 25.67
Average
29.79
XRD analysis
Fig. 1b. Plot of indirect band gap energy for ZnO NPs.
Structure and phase purity of ZnO NPs are shown in Fig. 3. From the diffractogram of XRD are very well matched with the hexagonal phase (wurtzite structure) by comparison with the data from JCPDS card No. 89-1397 and no indication of a secondary phase or impurity peaks were obtained. The strong and narrow diffraction peaks indicate that the product has good crystalline structure. The sharp intense diffraction peaks appearing at about 2h of 31.29°, 33.95°, 36.04°, 47.05°, 56.09°, 62.38°, 65.90°, 67.45°, 68.60°, 72.03°,
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FT-IR analysis
%T
Leaf Extract
4000
ZnO
3500
3000
2500
2000
1500
1000
500
Wavenumber (cm-1) Fig. 4. FT-IR spectra of leaf extract and ZnO NPs synthesized using Solanum nigrum leaf extract.
and 76.48° corresponding with those from (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4) and (2 0 2) orientations, respectively. The average grain size of the sample was calculated using the Scherer’s Eq. (3):
D¼
Kk Å bCosh
ð3Þ
where D is the average crystallite size in Å, K is the shape factor (0.9), k is the wavelength of X-ray (1.5406 Å) Cu Ka radiation, h is the Bragg angle, and b is the corrected line broadening of the NPs. The average crystallite size of the ZnO NPs is about 29.79 nm. Estimation of the crystallite sizes using this relation are depicted in Table 1.
Possible biomolecules responsible for the reduction of ZnO and capping agent of bioreduced ZnO NPs through particular bond vibration peaks identified from defined wave numbers in FT-IR technique. The FT-IR spectra of control leaf extract (before reaction without Zn (NO3)2 and synthesized ZnO (after reaction with Zn (NO3)2 are shown in Fig. 4. The absorption bands at 3429, 2926, 2349, 1635, 1404, 1261, 1031, 605, 540, and 470 cm1 in S. nigrum leaf extract wound be shifted into 3425, 2924, 1627, 1382, 1126, 1030, 833, and 445 cm1 in synthesized ZnO NPs sample. The peak found at around 1450–1500 cm1 showed the stretching of N–H vibration. Whereas the stretching of ZnO NPs were found around 400–800 cm1. The FT-IR spectra showed the presence of bonds due to O–H stretching (around 3429 cm1) and aldehydic C–H stretching (around 2924 cm1) [28]. The peak at around 1240– 1261 cm1 present in both the cases signified amide III band of the random coil of protein [29]. Peak at 1404 cm1 may be assigned to the symmetric stretching of the carboxyl side groups in the amino acid residues of the protein molecules [30]. The band at 1029 cm1 corresponds to C–N stretching vibration of amine [31]. The bands incidence at 1126, 1030 and 605 cm1 illustrates the chemical bonding, crystal structure and relative intensities of the IR bands of the carbonate [32,33]. Therefore the synthesized ZnO NPs were surrounded by proteins and metabolites such as terpenoids having functional groups. From the analysis of FTIR studies, we confirmed that the carbonyl groups from the amino acid residues and proteins has the stronger ability to bind metal ions, indicating that the proteins could possibly from the metal NPs (i.e., capping of silver NPs) to prevent agglomeration and
Fig. 5. FE-SEM image and EDX spectrum of ZnO NPs synthesized using Solanum nigrum leaf extract.
Fig. 6. TEM image ZnO NPs synthesized using Solanum nigrum leaf extract.
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ZnO NPs and these absorption peaks were due to the surface plasmon resonance of zinc oxide NPs. The origin of these elements lies in the biological components; mostly align along with ZnO NPs [34].
4 100
DTA (mW/mg)
3
95
TG %
90 2 85 1
80 75
TEM analysis The size and morphology of ZnO nanoparticles analyzed by TEM and SAED pattern obtained from TEM studies are depicted in Fig. 6a and b. This image reveals that most of the ZnO NPs are quasi-spherical and their diameter is about 29.79 nm. The SAED pattern revealed that the diffraction rings of the synthesized ZnO exhibited Debye–Scherrer rings assigned (1 0 0), (0 0 2), (1 0 1), (1 0 2), (1 1 0), (1 0 3), (2 0 0), (1 1 2), (2 0 1), (0 0 4) and (2 0 2) respectively, lattice planes of the face centered cubic (fcc) ZnO, indicating that the biogenic NPs seen in the TEM images are nano-crystalline in nature as shown in Fig. 6b. The particle size determined from TEM analysis is in good agreement with that of the XRD analysis.
0
70 0
200
400
600
800
1000
o Temperature ( C)
Fig. 7. TG–DTA curves of the ZnO NPs synthesized using Solanum nigrum leaf extract.
TG–DTA analysis thereby stabilize the medium. This suggests that the biological molecules could possibly perform dual functions of formation and stabilization of ZnO NPs in the aqueous medium.
Fig. 7 shows the TG–DTA measurement results of the ZnO nanoparticles. The DTA analysis of Zn (NO3)26H2O demonstrated several endothermic effects at 50 °C due to loss of residual water. The thermal dehydration at 165 °C and 318 °C, respectively, which corresponds to the dehydration and hydrolysis to basic zinc nitrate. The endothermic peak with maximum at 391 °C corresponded to the decomposition of the basic zinc nitrate to zinc oxide [35].
FE-SEM analysis The FE-SEM image of prepared ZnO NPs was presented in Fig. 5a. The diameter of the cluster ZnO NPs found to be in the range of 20–30 nm and is nearly spherical in shape with rough surface. Fig. 5b. EDX spectrum shows the high values of zinc (62%) and oxygen (29%) respectively. These results confirmed the present of zinc as a majority label compared to oxygen in precursor material. The EDX analysis displays the optical absorption peaks of
XPS analysis In the O 1s profile is asymmetric and can be fitted to one symmetrical peak (a locating at 530.08 eV, respectively), indicating one peak of O species in the sample. The peaks a should be Survey
1.20E+06 Zn2p
Counts / s
1.00E+06 8.00E+05 O1s
6.00E+05 4.00E+05 2.00E+05
K2p
0.00E+00 1200
1000
800
600
400
200
0
Binding Energy (eV) Zn2p Scan
O1s Scan
2.00E+05
8.00E+04
Element ZnO
1.40E+05 1.20E+05 1.00E+05 8.00E+04
Counts / s
7.00E+04
1.60E+05
Counts / s
Metal oxide
1.80E+05
6.00E+04 5.00E+04 4.00E+04 3.00E+04
6.00E+04 4.00E+04 1050
1040
1030
Binding Energy (eV)
1020
2.00E+04 544 542 540 538 536 534 532 530 528 526
Binding Energy (eV)
Fig. 8. XPS spectra of ZnO NPs synthesized using Solanum nigrum leaf extract.
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Fig. 9. Antibacterial activity of ZnO NPs synthesized using Solanum nigrum leaf extract.
associated with the lattice oxygen (OL) of ZnO Nps and chemisorbed oxygen (OH) caused by the surface hydroxyl respectively [36]. The Zn 2p core level spectrum is presented in Fig. 8. Two strong peaks centred at 1021.08 and 1044.18 eV, corresponding to the Zn 2p3/2 and Zn 2p1/2, are clearly observed. These present value are in agreement with the binding energies of Zn2+ ion [36]. However, it is hard to identify whether the Zn XPS signal comes from the ZnO Nps. Antibacterial activity The antibacterial activity of ZnO NPs was investigated both Gram positive (Staphylococcus aureus) and Gram negative (Salmonella paratyphi, Vibrio cholerae, Escherichia coli) bacteria by zone inhibition methods. The results of zone inhibition method as depicted in Fig. 9. The diameter of inhibition zones (mm) around each well with ZnO NPs solution are represented as shown in Fig. 9. Antibacterial activity of prepared ZnO nanoparticles was evolved against S. aureus, E. coli, S. paratyphi, and V. cholerae. The ZnO NPs synthesized by S. nigrum extracts are found the highest antimicrobial activity against S. paratyphi (17 mm) than compared to standard tablet, and the lesser antimicrobial activity of ZnO NPs was found against S. aureus (18 mm), V. cholerae (11 mm), and E. coli (7 mm) than compared to a standard tablet. The positive control (Leaf extract) was showed activity against all the microbial strains tested the maximum activity against V. cholerae, and S. aureus (6 mm and 6 mm). The antibacterial activity of ZnO NPs depends on the size, the smaller the size, the better the antibacterial activity [37]. We supposed that the ZnO NPs formed in this experiment might have good antibacterial activities. Conclusion ZnO NPs synthesized were successfully using a S. nigrum leaf extract mediated bioreduction process. The structure, morphology
and size (dimension) of prepared ZnO NPs were examined by FT-IR, XRD, FE-SEM and TEM analysis. UV–Vis-DRS studies confirmed the indirect band gap 3.38 eV and photoluminescence was found the blue band at 402, 447, 469 and 483 nm. The average grain size lies between 20–30 nm were found from XRD study as well as FT-IR spectra revealed the functional groups of stretching bands for ZnO NPs were found around 800–400 cm1. The diameter of the NPs and the quasi-spherical in shape and their diameter around 29.79 nm were confirmed by TEM study. The endothermic peak with maximum at 391 °C corresponded to the decomposition of the basic zinc nitrate to zinc oxide was confirmed by TG–DTA study. Two strong peaks centred at 1021.08 and 1044.18 eV, corresponding to the Zn 2p3/2 and Zn 2p1/2, were confirmed by XPS study. The synthesized NPs was studied for antibacterial activities against the microorganisms both Gram positive (S. aureus) and Gram negative (S. paratyphi, V. cholerae, E. coli) bacteria. Finally, the present study is so helpful and useful to the scientific community for using the Zno NPs as the potent applications to the antidiabetic and antihyperlipidemic activities. In addition, it is inexpensive, stable and nontoxic, safe and eco-friendly without side effects of human beings. Therefore, in the present research was designed to prepared eco-friendly, stable and nontoxic ZnO nanoparticles from the S. nigrum leaf extract. Acknowledgements The authors are thankful and grateful to Dr. S. Barathan, Professor and Head, Department of Physics, Annamalai University, Tamilnadu–608 002, for providing all necessary facilities to carry out the present work successfully. References [1] I.O. Sosa, C. Noguez, R.G. Barrera, J. Phys. Chem. B 107 (2003) 6269–6975. [2] Y.G. Sun, B. Mayers, T. Herricks, Y.N. Xia, Nano Lett. 3 (2003) 955–960.
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