Materials Science and Engineering C 49 (2015) 408–415
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Synthesis of cerium oxide nanoparticles using Gloriosa superba L. leaf extract and their structural, optical and antibacterial properties Ayyakannu Arumugam a,⁎, Chandrasekaran Karthikeyan b, Abdulrahman Syedahamed Haja Hameed b, Kasi Gopinath a, Shanmugam Gowri a, Viswanathan Karthika a a b
Department of Nanoscience and Technology, Alagappa University, Karaikudi 630 004, Tamil Nadu, India PG and Research Department of Physics, Jamal Mohamed College, Tiruchirappalli 620 020, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 13 September 2014 Received in revised form 10 December 2014 Accepted 8 January 2015 Available online 10 January 2015 Keywords: Gloriosa superba Green synthesis Cerium oxide nanoparticles Raman spectroscopy X-ray photoelectron spectroscopy Antibacterial activity
a b s t r a c t CeO2 nanoparticles (NPs) were green synthesized using Gloriosa superba L. leaf extract. The synthesized nanoparticles retained the cubic structure, which was confirmed by X-ray diffraction studies. The oxidation states of the elements (C (1s), O (1s) and Ce (3d)) were confirmed by XPS studies. TEM images showed that the NPs possessed spherical shape and particle size of 5 nm. The Ce–O stretching bands were observed at 451 cm−1 and 457 cm−1 from the FT-IR and Raman spectra respectively. The band gap of the CeO2 NPs was estimated as 3.78 eV from the UV–visible spectrum. From the photoluminescence measurements, the broad emission composed of eight different bands were found. The antibacterial studies performed against a set of bacterial strains showed that Gram positive (G+) bacteria were relatively more susceptible to the NPs than Gram negative (G−) bacteria. The toxicological behavior of CeO2 NPs was found due to the synthesized NPs with uneven ridges and oxygen defects in CeO2 NPs. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Phytosynthesis of metal and metal oxide nanoparticles (NPs) is an emerging field of nanoscience and technology. Size, shape conjointly plays a vital role in physical, chemical, electrical and optical properties of nanomaterials. Cerium oxide (CeO2) is a semiconductor with wide band gap energy (3.19 eV) and large exciton binding energy. It is used in wide range of applications such as, catalyst, sensor, solid oxide fuel cells, sun screen cosmetics, bioimaging, biotransformation and antibacterial activity [1–7]. Generally, CeO2 NPs were synthesized by physical and chemical methods such as hydrothermal, flame spray pyrolysis, sonochemical, microwave, sol–gel, and co-precipitation [8–13]. However, most of the techniques are complex, time consuming, expensive and hazardous [14–23] (Table. 1). The green chemistry approaches to the development in phytosynthesis of metal and metal oxide NPs. This method offers a plenty of advantages such as cost-effectiveness, largescale commercial production and pharmaceutical applications. The CeO2 NPs was less toxic when compared to TiO2 and ZnO NPs in cell line activity [24]. Recently, the CeO2 NPs have been synthesized using honey, egg white and fungal extracellular compounds [18,25,26]. ⁎ Corresponding author at: Department of Nanoscience and Technology, Alagappa University, Karaikudi 630 004,Tamil Nadu, India. E-mail address: [email protected] (A. Arumugam).
http://dx.doi.org/10.1016/j.msec.2015.01.042 0928-4931/© 2015 Elsevier B.V. All rights reserved.
These biocomponents which act as capping and reducing agent render to produce a nanocrystalline nature of metal oxide NPs in different sizes and morphology. In this paper, the synthesis of CeO2 NPs using Gloriosa superba plant leaf extract and their characterization studies have been reported for the first time. G. superba L. belongs to Colchicaceae family. It is a perennial, greenish, climbing herb and native to South Africa. Its flower is a state flower of Tamil Nadu and national flower of Tamil Eelam [27]. Since 2000 B.C. it is being used as a traditional medicine by the tribes. Every part of the plant has been used in Siddha, Ayurveda and Unani system of medicine. G. superba is a tuberous plant with L- (or) V-shaped cylindrical tubers. The tuber powder has been effectively used against paralysis, rheumatism, snake bite, insect bites, against lice, intermittent fevers, wounds, anti-fertility, gonorrhea, leprosy, piles, debility, dyspepsia, flatulence, hemorrhoids, helminthiasis and inflammations [28]. It contains two major alkaloids namely colchicines and colchicosides. The seeds consist of colchicines, which are 2–5 times higher than in the tubers. Its leaf extract contains superbine, colchicine, gloriosine, gloriosol, phytosterils and stigmasterin [29]. In the present investigation, CeO2 NPs are synthesized by using G. superba leaf extract. We have studied the structural, optical and antibacterial properties of CeO2 NPs. To the best of our knowledge, this is the first report on the phytosynthesis of CeO2 NPs by using G. superba leaf extract and their characterization studies such as
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Table. 1 Synthesis of CeO2 nanoparticles by different methods. S. no
Reference article
Description
Number of steps involved in the process
Total processing time
1 2 3 4 5 6 7 8 9 10
B. Choughury and A. Choudhury [14] C. Hu et al. [15] Y. Huang et al. [16] A. Krishan et al. [17] S. Maensiri et al. [18] S. Maensiri et al. [19] S. Phoka et al. [20] S. Sathyamurthy [21] R. Suresh et al. [22] Y. Tao et al. [23]
Hydrolysis Composite hydroxide mediated Water-in-oil-micro-emulsion Thermolysis Egg white synthesis Plant extract Polymer complex Reverse micellar Precipitation Microwave
Single step Single step Two step Single step Single step Single step Single step Two step Single step Single step
5h 120 h 16 h 1h 12 h 12 h 3h 1h 20 h 5h
XRD, TEM, UV–visible, FT-IR, Micro-Raman, photoluminescence and antibacterial activity analyses.
was calcined at 400 °C for 2 h. Thus, CeO2 nanopowder was obtained. A schematic diagram for the formation of CeO2 NPs using G. superba leaf extract is shown in Fig. 1.
2. Materials and methods 2.3. Characterization of CeO2 NPs 2.1. Collection of plant material The G. superba leaves were collected from Endangered Medicinal Plants Conservation Centre, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India. Taxonomic identification was made by Dr. S. John Britto, The Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph's College, Tiruchirappalli, Tamil Nadu, India. The voucher specimen was numbered (KG-001) and preserved in the Department of Nanoscience and Technology, Alagappa University Karaikudi. 2.2. Synthesis of CeO2 NPs using G. superba leaf extract The 10 g of finely cut leaves was added with 100 mL of double distilled water and boiled at 50–60 °C for 5 min. The obtained extraction was filtered using Whatman No. 1 filter paper and the filtrate was collected in 250 mL Erlenmeyer flask and stored at room temperature for further usage. Thereafter, 3.72 g CeCl3 salt was added to 100 mL of G. superba leaf extract. This solution was stirred constantly at a temperature of 80 °C for 4–6 h. A white precipitate formed and then it became a yellowish brown in color on continuous stirring. Further the precipitate
The phytosynthesized CeO2 NP samples were subjected to XRD analysis. The XRD pattern was recorded using Cu Kα radiation (λ = 1.54060 Å) with nickel monochromator in the range of 2θ from 10° to 80°. The average crystallite size of the synthesized CeO2 NPs was calculated using Scherrer's formula [D = 0.9λ/βcos θ]. The XPS measurements were performed with XPS instrument (Carl Zeiss) equipment. The spectra were at a pressure using ultra high vacuum with Al Kα excitation at 250 W. The morphology of the synthesized CeO2 was examined using TEM. Samples for TEM analysis were prepared by drop coating the nanoparticle solutions on carbon-coated copper grids at room temperature. The excess nanoparticle solution was removed with filter paper. The copper grid was finally dried at room temperature and was subjected to TEM analysis by the instrument Tecnai F20 model operated at an accelerating voltage of 200 kV. Moreover, Fourier transform infra-red spectroscopy (FT-IR) analysis was carried out in the range of 400–4000 cm−1 (PerkinElmer). The Micro-Raman analysis of our samples was carried out using the instrument of Princeton Acton SP2500, CS spectrometer 0.5 Focal length triple grating monochromator excitation source Ar+ laser, 514.5 nm wavelength. UV–visible spectroscopy in the range of 200–850 nm used as Shimadzu spectrophotometer
Fig. 1. Formation of CeO2 NPs using G. superba leaf extract.
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(Model UV-1800) operating at a resolution of 1 nm. The 3 mg synthesized CeO2 NPs was disposed in 10 mL of double distilled water and this solution was sonicated for 15 min. After that, the liquid sample was carried out under UV–visible spectrophotometer. Photoluminescence measurement was carried out on a luminescence spectrophotometer (PerkinElmer LS-5513, PerkinElmer Instrument, USA) using xenon lamp as the excitation source at room temperature.
The antibacterial activity of the phytosynthesized CeO2 NPs were examined under two Gram positive (G+) (Staphylococcus aureus and Streptococcus pneumoniae) and five Gram negative (G −) bacteria (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Shigella dysenteriae) by disc diffusion method. These seven bacterial strains were grown in nutrient broth at 37 °C until the bacterial suspension reached 1.5 × 108 CFU/mL. Approximately 20 mL of molten nutrient agar was poured into the Petri dishes and cooled. All the bacterial suspension was swapped over the medium, the disc was loaded in three different concentrations 10, 50 and 100 mg of CeO2 NPs using sterile distilled water and they were placed over the medium using sterile forceps. The 100 μL of sterile distilled water was used as a negative control. The plates were then incubated for 24 h at 37 °C. The inhibition zone formed around each disc was measured. The experiment was performed for three times. 3. Results and discussion 3.1. X-ray diffraction analysis The X-ray diffraction peaks of CeO2 NPs synthesized using G. superba leaf extract are shown in Fig. 2. The XRD peaks are located at angles (2θ) of 28.51, 33.06 and 47.42 corresponding to (111), (200) and (220) planes of the CeO2 NPs. Similarly, other peaks found at angles (2θ) of 56.30, 59.09, 69.00, 76.57 and 78.99 are corresponding to (311), (222), (400), (331) and (420) planes of CeO2 NPs. The standard diffraction peaks show the face-center cubic phase of CeO2 NPs (JCPDS data card no: 34-0394). The lattice constant ‘a’ of CeO2 can be calculated by using the relation. h2 þ k2 þ l2 a2
Average crystalline size D ¼
0:9λ β cosθ
ð2Þ
where λ is the wavelength of X-ray used (1.54060 Å), β is the angular peak width at half maximum in radians and θ is Bragg's diffraction angle. The average crystalline size is estimated as 24 nm for CeO2 NPs.
2.4. Antibacterial activity of CeO2 NPs
1 ¼ d2
The average crystalline size of the samples is calculated after appropriated background corrections from X-ray line broadening of the diffraction peaks using Debye–Scherrer's formula.
! ð1Þ
3.2. X-ray photon spectroscopy XPS provides information on the oxidation state of each element in the sample as well as the composition of the surface functionalization of the CeO2. The XPS results show that the indexed peaks correspond to C (1s), O (1s), and Ce (3d) for CeO2 NPs. The C (1s) signals are most likely due to remaining trace amount of plant extract or simply due to absorption of organic contaminants during handling. The Ce (3d) signals are divided into eight signals, namely O1, O2, O3, O4, O5, O6, O7 and O8 in the Gaussian fitting shown in Fig. 3. Binding energy of CeO2 NP sample is having eight bands 882.26, 888.25, 896.04, 897.73, 900.24, 906.56, 916.34 and 921.33 eV. The main peaks (O7, O3 and O4) of Ce4 + 3d3/2 and Ce4 + 3d5/2 are shown at binding energies of (916.34 eV) and (896.04 & 897.73 eV) respectively. The O5 and O1 energy levels of Ce3+ 3d3/2 and Ce3 + 3d5/2 are located at 900.24 and 882.26 eV. Three additional satellite lines O6, O2 and O8 are shown at 906.56 eV on the Ce3 + 3d3/2 and (888.25 & 921.33 eV) on the Ce3 + 3d5/2, respectively. These spectra are found to be fully consistent with those reported previously [30–33]. Fig. 4 indicates oxygen O (1s) signals which are divided into three symmetrical signals namely G1, G2 and G3 in the Gaussian fitting. For CeO2 NPs, the lower energy level (G1) of O 4+ (1s) signal at 528.83 eV is attributed to O− 2 ions surrounded by Ce ions, which corresponds to the Ce–O bond in CeO2. The middle energy level (G2) of O (1s) at 530.86 eV can be ascribed to the O− 2 ions in the Ce–O bond where Ce is present in the 3+ state. Finally, higher energy level (G3) of O (1s) signals located at 532.47 eV is not related to the presence of either Ce3 + or Ce4 +. It has been suggested that the 532.47 eV peak is due to OH on the surface. 3.3. Transmission electron microscopy Fig. 5a–c shows HRTEM images of the as-prepared CeO2 NPs, calcined at a temperature of 400 °C, and the insets show the electron
The calculated value of ‘a’ is 5.416 Å for CeO2 NPs. The unit cell volume is calculated by the relation given by V = a3. The unit cell volume is found to be 158.867 Å3 for the CeO2 NPs.
Intensity (a.u.)
O1
Ce (3d) O1 O2 O3 O4 O5 O6 O7 O8 Peak sum
O8 O5
O2 O4 O6
O7
O3
880
890
900
910
Binding Energy (eV) Fig. 2. X-ray powder diffraction pattern of CeO2 NPs using G. superba leaf extract.
Fig. 3. XPS spectra of Ce (3d) for the CeO2 NPs.
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G1
411
O (1s) G1 G2 G3 Peak sum
G2 Intensity (a.u.)
G3
528
530
532
534
536
Binding Energy (eV) Fig. 4. XPS spectra of O (1s) for CeO2 NPs.
Fig. 6. FT-IR spectra of (a) G. superba plant extract, (b) as-prepared sample and (c) CeO2 NPs.
Fig. 5. (a–c) TEM images of CeO2 NPs and (d) selected area electron diffraction.
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mode of the Ce-8O vibrational unit and therefore it is very sensitive to any disorder in the oxygen sub-lattice resulting from thermal, doping, or grain size [35,36]. According to the Raman line broadening, the particle size of the CeO2 can be also estimated using the good correlation reported for nanocrystalline CeO2 powder specimens [37–39]. −1 ¼ 10 þ 124:7=DR Г cm
ð3Þ
where Г (cm−1) is the full-width at half-maximum of the Raman active mode peak and DR is the particle size of a CeO2 sample. By substituting Г (cm−1) = 457.24 cm−1 into Eq. (3) with the CeO2 particle size of 5 nm, this calculated particle size is found to be lower than that obtained from X-ray line broadening. 3.6. UV–visible spectroscopy studies Fig. 7. Raman spectrum of the CeO2 NPs calcined in air at 400 °C for 2 h.
diffraction patterns. The particle size is approximately 5 nm for spherical structure. The high crystallinity of the powder leads to its corresponding well-pronounced Debye–Scherrer diffraction rings in the selected area electron diffraction (SAED) pattern (Fig. 5d) that can be assigned to the reflections (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), (4 0 0), (3 3 1) and (4 2 0) of cubic CeO2. There are no additional rings in the SAED pattern stemming from any crystalline impurities.
Fig. 8a shows the UV–visible absorption spectrum of G. superba leaf extract assisted CeO2 nanoparticles. G. superba leaf extract consists of phytoconstituents acting as a capping and reducing agent. So, it has reduced the particle size. In this significant effect absorption peak shifted into UV–visible region [16,18,20,21]. The band gap can be determined by fitting the absorption data to the direct transition equation by extrapolating the linear portions of the curves to absorption equal to zero (the inset of Fig. 8b):
3.4. Fourier transform infra-red spectroscopy
1=2 αhυ ¼ A hυ−Eg
The FT-IR spectra of the prepared CeO2 NPs are shown in Fig. 6 using the KBr pellet method in the wave number range 400–4000 cm−1. The broad absorption in the frequency band 3750–3000 cm−1 is assigned to O–H stretching from residual alcohols, water and Ce–OH. The absorption peak is observed at 3475 cm−1 for CeO2 NPs. The CO2 peaks are observed at 2358 cm−1 and 1394 cm−1 for CeO2 NPs. These CO2 band may arise due to some trapped CO2 in air ambience. The band at 1647 cm−1 corresponds to the bending of H–O–H which is partly overlapping the O–C–O stretching band. The band due to the stretching frequency of Ce–O can be seen below 400 cm−1, indicating the formation of CeO2 [20]. In our case, Ce–O stretching is observed at 451 cm−1. Similarly, Goharshdi et al. [34] reported that the Ce–O stretching band appeared in 450 cm−1.
where α is the optical absorption coefficient, hυ is the photon energy, Eg is the direct band gap, and A is a constant. The estimated band gap of the CeO2 sample is to be 3.78 eV. It can be seen that the CeO2 sample shows an increase in Eg by a value exceeding 0.59, compared to the bulk CeO2 powders (Eg = 3.19 eV, determined by UV–visible spectroscopy) [40]. For the reason of increase in the optical band gap of CeO2. The quantum confinement effect is considered when the particle is down to a few nanometers [41]. Since the fundamental band gap is mainly deduced by quantum size effect when the particle size is less than or equal to 3 nm, the quantum size effect in the present CeO2 nanoparticles with particle size of 5 nm may be ruled out and the charge transition of Ce ion (Ce3 +–Ce4 +) may play an important role for the increase in the band gap of present CeO2 sample.
3.5. Micro-Raman spectroscopy
3.7. Photoluminescence studies
Fig. 7 shows a typical Raman spectrum of CeO2. In literature, Raman active mode peak has been observed at 458.9 cm−1 and 461. 04 cm−1 for CeO2 NPs [23,24]. From the present result, a sharp Raman active mode can be found at 457. 24 cm−1, which is attributed to a symmetrical stretching
Fig. 9 shows the photoluminescence spectra of the CeO2 NPs recorded with the excited wavelength of 290 nm. The PL emission is observed for CeO2 NP sample covering from the very short wavelength of 350 nm to long wavelength 500 nm. A good fit of eight peak Gaussian function is
Fig. 8. (a) UV–visible spectrum of the phytosynthesized CeO2 NPs. (b) Photon energy level of the phytosynthesized CeO2 NPs.
ð4Þ
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PL Intensity (a.u.)
G1
3.8. Antibacterial activity
CeO2
G4
G1 G2 G3 G4 G5 G6 G7 G8 Peak sum G8
G3 G2 G5 G6
G7
360
380
400
420
440
460
413
480
500
Wavelength (nm) Fig. 9. Photoluminescence emission spectra of CeO2 NPs using G. superba leaf extract.
obtained for CeO2 NPs. The PL spectra of the samples at the bottom are labeled as G1, G2, G3, G4, G5, G6, G7 and G8. The solid lines represent the linear combination of eight Gaussian peaks where G1 has the lowest and G8 has the highest wavelength. The emission spectra of the CeO2 NP sample are emphasized in eight peaks at 365, 378, 385, 395, 402, 421, 455 and 486 nm. These bands are four near band edge emissions, violet emission, two blue emissions and blue–green emission respectively. The NBE emissions G1, G2, G3 and G4 are located at UV region (365, 378, 385 and 395 nm) for CeO2 NPs. These NBE emissions are attributed to a band-to-band recombination process, possibly involving localized or free excitons [42]. The violet band G5 around 402 nm for the CeO2 sample originates from the defect states existing extensively between the Ce 4f state and O 2p valence band [43]. These defects possibly act as radiative recombination centers for electron initially excited from the valence band to the 4f band of the CeO2 [44]. The two blue emissions G6 and G7 at (421 and 455 nm) are related to the abundant defects such as dislocations, which is helpful for fast oxygen transportation. Ce 4f level with a width of 1 eV is localized at the forbidden gap, which lies at 3 eV over the valence band (O 2p). At room temperature, electron transition mainly occurs from defects level to O 2p level [45]. The blue–green emission G8 located at 486 nm is possibly due to surface defects in the CeO2 NPs, and the low intensity of the green emission may be due to the low density of oxygen vacancies.
In the present study, the NPs are studied extensively to explore their utility as a potential antibacterial agent. Several factors such as less toxicity and heat resistance are accountable for the use of NPs in the biological applications [46,47]. The antibacterial activity has been studied against (G +) and (G −) bacterial pathogens using three different concentrations of CeO2 samples (10, 50 and 100 mg of CeO2). Fig. 10 shows the size of the zone of inhibition and antibacterial activity formed around each CeO2 NPs loaded with test samples. The sample containing 100 mg of CeO2 shows most significant effect on zone of inhibition of 5.33 mm of S. aureus. The E. coli and S. dysenteriae exhibit a modulated effect on inhibition zone of 4.00 mm and 4.33 mm respectively. Then, P. aeruginosa, P. vulgaris, K. pneumonia and S. pneumoniae show similar inhibition zone of 4.67 mm for better activity as compared to that of the sample with 50 mg of CeO2. Recently, chemically synthesized CeO2 NPs showed a better antibacterial activity against E. coli by disc diffusion method. In addition, green synthesized CeO2 NPs were tested in antibacterial activity showed a significant effect of G + and G − bacteria by disc diffusion method [48,49]. The antibacterial efficiency of CeO2 NPs generally depends on their size, specific surface area, polar surface, morphology etc. Moreover, electrostatic attraction between negatively charged bacterial cells and positively charged nanoparticles is crucial for the activity of nanoparticles as bactericidal materials. This interaction not only inhibits the bacterial growth but also induces the reactive oxygen species (ROS) generation, which leads to cell death [50–52]. In particular, many previous studies have explored the photogeneration of ROS on the surfaces of metal-oxide NPs [53,54]. The general principle is that when NPs are illuminated by light with photoenergy greater than the band gap, the electrons (e−) of NPs are promoted across the band gap to the conduction band, which creates a hole (h+) in the valence band [53]. Electrons in the conduction band and holes in the valence band exhibit high reducing and oxidizing power respectively [54]. The electron can react with molecular oxygen to produce superoxide anion (O•− 2 ) through a reductive process [54]. The hole can abstract electrons from water and/or hydroxyl ions to generate hydroxyl radicals (•OH) through an oxidative process [54]. Singlet oxygen (1O2) is mostly produced indirectly from • aqueous reactions of O•− 2 [41]. OH is a strong and nonselective oxidant that can damage virtually all types of organic biomolecules, including carbohydrates, nucleic acids, lipids, proteins, DNA and amino acids [55]. 1O2 is the main mediator of photocytoxicity and can irreversibly damage the treated tissues [56], causing biomembrane oxidation and degradation [57]. Although O•− 2 is not a strong oxidant, as a precursor for •OH and 1O2, O•− 2 also has significant biological implications [58]. Although many previous studies have explored ROS generation by various
Fig. 10. Size of the zone of inhibition formed around each disc, loaded with test samples, indicating the antibacterial activity CeO2 NPs using G. superba leaf extract.
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Fig. 11. Diagrammatic representation of toxicity of CeO2 NPs against bacterial pathogens.
metal-oxide NPs, to the best of our knowledge, little research has examined the role of the electronic properties of metal-oxide NPs in ROS generation [52]. A deeper understanding in this aspect would allow us to interpret the underlying ROS generation mechanisms. Generally, nanoparticles with better photocatalytic activity have a large specific surface area and a small crystalline size which increase oxygen vacancies [59]. The antibacterial activity of CeO2 NPs can also be explained as follows. ROS include the most reactive hydroxyl radical (•OH), less toxic superoxide anion radical (−•O2), and singlet oxygen (1O2) with a weaker oxidizer, contributing to the major oxidative stress in biological systems [60]. ROS generation is closely associated with the efficiency of a photocatalyst, depending on the generation rate, rate of migration, and energy levels of the photoexcited electron-hole pairs. In the current study, such enhanced ROS yields may be connected with the electronic properties and microstructure (i.e., grain size, specific surface area, and pore, etc.) of the spherical CeO2 NPs. The electronic structure of metal-oxide NPs is characterized by the band gap (Eg), which is essentially the energy interval between the valence band (Ev) and the conduction band (Ec), each of which has a high density of states. Only the metal-oxide NPs with Eg higher than the
Fig. 12. Antibacterial activity of CeO2 NPs against G+ and G− bacterial strains. The data were obtained from three experiments and are expressed as the mean ± S.E. * indicates significant differences from the G+ve bacteria at P b 0.05, analyzed by two sample t-test.
incident photon energy (i.e., approximately 3.78 eV) can be photoexcited. Under UV excitation, electrons are promoted from the valence band to the conduction band, with the concomitant generation of a hole in the valence band. The photoexcited electrons and holes then react with an aqueous electron acceptor (i.e., molecular oxygen) and donor (i.e., water and hydroxyl ions) respectively to produce different types of ROS. The mechanism of light induced generation of ROS can be given as follows. − þ þ þ − − − CeO2 þ hυ→e þ h h þ H2 O→·OH þ H e þ O2 →·O2 ·O2 þ þ − · · þH →HO HO þ H þ e →H O 2
2
2
2
A higher ROS value generally comes from a photocatalyst with a larger surface area, appropriate crystal size, increase oxygen vacancies and the facilitation of diffusion and mass transportation of reactant molecules. From the XRD results, the crystalline size is found to be 24 nm. Smaller crystal size with a higher surface area leads to higher antibacterial activity [61]. From the photoluminescence results for the CeO2, the blue–green emission at 486 nm is due to the presence of oxygen vacancy and oxygen interstitial defect. Other possible mechanisms are involving the interaction of nanomaterials with the biological macromolecules. It is believed that microorganisms carry a negative charge while metal oxides carry a positive charge [62]. This creates an “electromagnetic” attraction between the microbe and treated surface. Once the contact is made, the microbe oxidizes and dies instantly. Since the CeO2 NPs interfere with the bacteria cell membrane and bind with mesosome, there is a perturbance in the mesosomal functions of cellular respiration, DNA replication, cell division and thereby the surface area of bacterial cell membrane is increased. These intracellular functional changes establish the oxidative stress induced by ROS generation due to the cell expiry. Considering the above facts, a schematic picture is represented in (Fig. 11). Generally, it is believed that nano-materials release ions, which react with the thiol groups (–SH) of the proteins present on the bactericidal cell surface [63]. Such proteins protrude through the bactericidal cell membrane, allowing the transport of nutrients through the cell wall. The nano-materials inactivate the proteins and decrease the membrane permeability, which eventually causes the cell death. The NPs with uneven surface texture due to rough edges and corners contribute to the mechanical damage of the cell membranes of E. coli [64]. From the TEM image, it is clear that the CeO2 NPs have uneven ridges at the outer surface which influence the antibacterial activity. The combination of various factors such as size and oxygen defects plays a critical role in the toxicological behavior of CeO2 NPs. From Fig. 12, it can be suggested that (G +) bacteria are relatively more
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susceptible to the NPs than (G−) bacteria. Comparing our results with the reported results regarding the sensitivity of (G +) and (G −) to the photogeneration of O− 2 in the case of ZnO and TiO2 [61,65], the same trend is also observed in the case of CeO2 NPs in such a way that (G+) is more sensitive to ROS than (G−). 4. Conclusion In summary, CeO2 NPs were successfully synthesized using aqueous leaf extract of G. superba. The XRD, Raman and SAED studies revealed the formation of CeO2 NPs. The TEM images clearly showed that CeO2 NPs possessed spherical shaped morphology with the average size of 5 nm. Smaller crystal sizes with a higher surface area lead to higher antibacterial activity. From the photoluminescence spectra of CeO2 NPs, the blue–green emission at 486 nm was observed due to the presence of oxygen vacancy and oxygen interstitial defects. As seen from the TEM image, the CeO2 NPs having uneven ridges at the outer surface influenced the antibacterial activity of the NPs, Thus, with the support of the photoluminescence and TEM results, the toxicological behavior of CeO2 NPs was found due to the size of the synthesized NPs with uneven ridges and oxygen defects in CeO2 NPs. Acknowledgments The authors gratefully thank School of Physics, Alagappa University for extending the XRD, and Micro-Raman and photoluminescence spectroscopy facilities. References [1] E. Bekyarova, P. Fornasiero, J. Kaspar, M. Graziani, Catal. Today 45 (1998) 179–183. [2] S.B. Khan, M. Faisal, M.M. Rahman, A. Jamal, Sci. Total Environ. 409 (2011) 2987–2992. [3] H. Yahiro, Y. Baba, K. Eguchi, A. Hiromichi, J. Electrochem. Soc. 135 (1998) 2077–2080. [4] S. Yabe, T. Sato, J. Solid State Chem. 171 (2003) 7–11. [5] S. Babu, J.H. Cho, J.M. Dowding, E. Heckert, C. Komanski, S. Soumen Das, J. Colon, C.H. Baker, M. Bass, W.T. Self, S. Seal, Chem. Commun. 46 (2010) 6915–6917. [6] P. Zhang, Y. Ma, Z. Zhang, X. He, J. Zhang, Z. Guo, R. Tai, Y. Zhao, Z. Chai, ACS Nano 11 (2012) 9943–9950. [7] A. Thill, O. Zeyons, O. Spalla, F. Chauvat, J. Rose, M. Auffan, A.M. Flank, Environ. Sci. Technol. 40 (2006) 6151–6156. [8] F. Zhang, S.W. Chan, J.E. Spanier, E. Apak, Q. Jin, R.D. Robinson, I.P. Herman, Appl. Phys. Lett. 80 (2002) 127–129. [9] J. Hu, Y. Li, X. Zhou, M. Cai, Mater. Lett. 61 (2007) 4989–4992. [10] H. Wang, J.J. Zhu, J.M. Zhu, X.H. Liao, S. Xu, T. Ding, H.Y. Chen, Phys. Chem. Chem. Phys. 4 (2002) 3794–3799. [11] X.H. Liao, J.M. Zhu, J.J. Zhu, J.Z. Xu, H.Y. Chen, Chem. Commun. (2001) 937–938. [12] F. Czerwinski, J.A. Szpunar, J. Sol-Gel Sci. Technol. 9 (1997) 103–114. [13] S.Y. Yao, Z.H. Xie, J. Mater. Process. Technol. 186 (2007) 54–59. [14] B. Choudhury, A. Choudhury, Mater. Chem. Phys. 131 (2012) 666–671. [15] C. Hu, Z. Zhang, H. Liu, P. Gao, Z.L. Wang, Nanotechnology 17 (2006) 5983–5987. [16] Y. Huang, Y. Cai, D. Qiao, H. Liu, Particuology 9 (2011) 170–173. [17] A. Krishnan, T.S. Sreeremya, E. Murray, S. Ghosh, J. Colloid Interface Sci. 389 (2013) 16–22. [18] S. Maensiri, C. Masingboon, P. Laokul, W. Jareonboon, V. Promarak, P.L. Anderson, S. Seraphin, Cryst. Growth Des. 7 (2007) 950–955. [19] S. Maensiri, S. Labuayai, P. Laokul, J. Klinkaewnarong, E. Swatsitang, Jpn. J. Appl. Phys. 53 (2014) 06JG14. [20] S. Phoka, P. Laokul, E. Swatsitang, S. Maensiri, Mater. Chem. Phys. 115 (2009) 424–428.
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Synthesis of cerium oxide nanoparticles using Gloriosa superba L. leaf extract and their structural, optical and antibacterial properties Ayyakannu Arumugam a,⁎, Chandrasekaran Karthikeyan b, Abdulrahman Syedahamed Haja Hameed b, Kasi Gopinath a, Shanmugam Gowri a, Viswanathan Karthika a a b
Department of Nanoscience and Technology, Alagappa University, Karaikudi 630 004, Tamil Nadu, India PG and Research Department of Physics, Jamal Mohamed College, Tiruchirappalli 620 020, Tamil Nadu, India
a r t i c l e
i n f o
Article history: Received 13 September 2014 Received in revised form 10 December 2014 Accepted 8 January 2015 Available online 10 January 2015 Keywords: Gloriosa superba Green synthesis Cerium oxide nanoparticles Raman spectroscopy X-ray photoelectron spectroscopy Antibacterial activity
a b s t r a c t CeO2 nanoparticles (NPs) were green synthesized using Gloriosa superba L. leaf extract. The synthesized nanoparticles retained the cubic structure, which was confirmed by X-ray diffraction studies. The oxidation states of the elements (C (1s), O (1s) and Ce (3d)) were confirmed by XPS studies. TEM images showed that the NPs possessed spherical shape and particle size of 5 nm. The Ce–O stretching bands were observed at 451 cm−1 and 457 cm−1 from the FT-IR and Raman spectra respectively. The band gap of the CeO2 NPs was estimated as 3.78 eV from the UV–visible spectrum. From the photoluminescence measurements, the broad emission composed of eight different bands were found. The antibacterial studies performed against a set of bacterial strains showed that Gram positive (G+) bacteria were relatively more susceptible to the NPs than Gram negative (G−) bacteria. The toxicological behavior of CeO2 NPs was found due to the synthesized NPs with uneven ridges and oxygen defects in CeO2 NPs. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Phytosynthesis of metal and metal oxide nanoparticles (NPs) is an emerging field of nanoscience and technology. Size, shape conjointly plays a vital role in physical, chemical, electrical and optical properties of nanomaterials. Cerium oxide (CeO2) is a semiconductor with wide band gap energy (3.19 eV) and large exciton binding energy. It is used in wide range of applications such as, catalyst, sensor, solid oxide fuel cells, sun screen cosmetics, bioimaging, biotransformation and antibacterial activity [1–7]. Generally, CeO2 NPs were synthesized by physical and chemical methods such as hydrothermal, flame spray pyrolysis, sonochemical, microwave, sol–gel, and co-precipitation [8–13]. However, most of the techniques are complex, time consuming, expensive and hazardous [14–23] (Table. 1). The green chemistry approaches to the development in phytosynthesis of metal and metal oxide NPs. This method offers a plenty of advantages such as cost-effectiveness, largescale commercial production and pharmaceutical applications. The CeO2 NPs was less toxic when compared to TiO2 and ZnO NPs in cell line activity [24]. Recently, the CeO2 NPs have been synthesized using honey, egg white and fungal extracellular compounds [18,25,26]. ⁎ Corresponding author at: Department of Nanoscience and Technology, Alagappa University, Karaikudi 630 004,Tamil Nadu, India. E-mail address: [email protected] (A. Arumugam).
http://dx.doi.org/10.1016/j.msec.2015.01.042 0928-4931/© 2015 Elsevier B.V. All rights reserved.
These biocomponents which act as capping and reducing agent render to produce a nanocrystalline nature of metal oxide NPs in different sizes and morphology. In this paper, the synthesis of CeO2 NPs using Gloriosa superba plant leaf extract and their characterization studies have been reported for the first time. G. superba L. belongs to Colchicaceae family. It is a perennial, greenish, climbing herb and native to South Africa. Its flower is a state flower of Tamil Nadu and national flower of Tamil Eelam [27]. Since 2000 B.C. it is being used as a traditional medicine by the tribes. Every part of the plant has been used in Siddha, Ayurveda and Unani system of medicine. G. superba is a tuberous plant with L- (or) V-shaped cylindrical tubers. The tuber powder has been effectively used against paralysis, rheumatism, snake bite, insect bites, against lice, intermittent fevers, wounds, anti-fertility, gonorrhea, leprosy, piles, debility, dyspepsia, flatulence, hemorrhoids, helminthiasis and inflammations [28]. It contains two major alkaloids namely colchicines and colchicosides. The seeds consist of colchicines, which are 2–5 times higher than in the tubers. Its leaf extract contains superbine, colchicine, gloriosine, gloriosol, phytosterils and stigmasterin [29]. In the present investigation, CeO2 NPs are synthesized by using G. superba leaf extract. We have studied the structural, optical and antibacterial properties of CeO2 NPs. To the best of our knowledge, this is the first report on the phytosynthesis of CeO2 NPs by using G. superba leaf extract and their characterization studies such as
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409
Table. 1 Synthesis of CeO2 nanoparticles by different methods. S. no
Reference article
Description
Number of steps involved in the process
Total processing time
1 2 3 4 5 6 7 8 9 10
B. Choughury and A. Choudhury [14] C. Hu et al. [15] Y. Huang et al. [16] A. Krishan et al. [17] S. Maensiri et al. [18] S. Maensiri et al. [19] S. Phoka et al. [20] S. Sathyamurthy [21] R. Suresh et al. [22] Y. Tao et al. [23]
Hydrolysis Composite hydroxide mediated Water-in-oil-micro-emulsion Thermolysis Egg white synthesis Plant extract Polymer complex Reverse micellar Precipitation Microwave
Single step Single step Two step Single step Single step Single step Single step Two step Single step Single step
5h 120 h 16 h 1h 12 h 12 h 3h 1h 20 h 5h
XRD, TEM, UV–visible, FT-IR, Micro-Raman, photoluminescence and antibacterial activity analyses.
was calcined at 400 °C for 2 h. Thus, CeO2 nanopowder was obtained. A schematic diagram for the formation of CeO2 NPs using G. superba leaf extract is shown in Fig. 1.
2. Materials and methods 2.3. Characterization of CeO2 NPs 2.1. Collection of plant material The G. superba leaves were collected from Endangered Medicinal Plants Conservation Centre, Science Campus, Alagappa University, Karaikudi, Tamil Nadu, India. Taxonomic identification was made by Dr. S. John Britto, The Rapinat Herbarium and Centre for Molecular Systematics, St. Joseph's College, Tiruchirappalli, Tamil Nadu, India. The voucher specimen was numbered (KG-001) and preserved in the Department of Nanoscience and Technology, Alagappa University Karaikudi. 2.2. Synthesis of CeO2 NPs using G. superba leaf extract The 10 g of finely cut leaves was added with 100 mL of double distilled water and boiled at 50–60 °C for 5 min. The obtained extraction was filtered using Whatman No. 1 filter paper and the filtrate was collected in 250 mL Erlenmeyer flask and stored at room temperature for further usage. Thereafter, 3.72 g CeCl3 salt was added to 100 mL of G. superba leaf extract. This solution was stirred constantly at a temperature of 80 °C for 4–6 h. A white precipitate formed and then it became a yellowish brown in color on continuous stirring. Further the precipitate
The phytosynthesized CeO2 NP samples were subjected to XRD analysis. The XRD pattern was recorded using Cu Kα radiation (λ = 1.54060 Å) with nickel monochromator in the range of 2θ from 10° to 80°. The average crystallite size of the synthesized CeO2 NPs was calculated using Scherrer's formula [D = 0.9λ/βcos θ]. The XPS measurements were performed with XPS instrument (Carl Zeiss) equipment. The spectra were at a pressure using ultra high vacuum with Al Kα excitation at 250 W. The morphology of the synthesized CeO2 was examined using TEM. Samples for TEM analysis were prepared by drop coating the nanoparticle solutions on carbon-coated copper grids at room temperature. The excess nanoparticle solution was removed with filter paper. The copper grid was finally dried at room temperature and was subjected to TEM analysis by the instrument Tecnai F20 model operated at an accelerating voltage of 200 kV. Moreover, Fourier transform infra-red spectroscopy (FT-IR) analysis was carried out in the range of 400–4000 cm−1 (PerkinElmer). The Micro-Raman analysis of our samples was carried out using the instrument of Princeton Acton SP2500, CS spectrometer 0.5 Focal length triple grating monochromator excitation source Ar+ laser, 514.5 nm wavelength. UV–visible spectroscopy in the range of 200–850 nm used as Shimadzu spectrophotometer
Fig. 1. Formation of CeO2 NPs using G. superba leaf extract.
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(Model UV-1800) operating at a resolution of 1 nm. The 3 mg synthesized CeO2 NPs was disposed in 10 mL of double distilled water and this solution was sonicated for 15 min. After that, the liquid sample was carried out under UV–visible spectrophotometer. Photoluminescence measurement was carried out on a luminescence spectrophotometer (PerkinElmer LS-5513, PerkinElmer Instrument, USA) using xenon lamp as the excitation source at room temperature.
The antibacterial activity of the phytosynthesized CeO2 NPs were examined under two Gram positive (G+) (Staphylococcus aureus and Streptococcus pneumoniae) and five Gram negative (G −) bacteria (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Shigella dysenteriae) by disc diffusion method. These seven bacterial strains were grown in nutrient broth at 37 °C until the bacterial suspension reached 1.5 × 108 CFU/mL. Approximately 20 mL of molten nutrient agar was poured into the Petri dishes and cooled. All the bacterial suspension was swapped over the medium, the disc was loaded in three different concentrations 10, 50 and 100 mg of CeO2 NPs using sterile distilled water and they were placed over the medium using sterile forceps. The 100 μL of sterile distilled water was used as a negative control. The plates were then incubated for 24 h at 37 °C. The inhibition zone formed around each disc was measured. The experiment was performed for three times. 3. Results and discussion 3.1. X-ray diffraction analysis The X-ray diffraction peaks of CeO2 NPs synthesized using G. superba leaf extract are shown in Fig. 2. The XRD peaks are located at angles (2θ) of 28.51, 33.06 and 47.42 corresponding to (111), (200) and (220) planes of the CeO2 NPs. Similarly, other peaks found at angles (2θ) of 56.30, 59.09, 69.00, 76.57 and 78.99 are corresponding to (311), (222), (400), (331) and (420) planes of CeO2 NPs. The standard diffraction peaks show the face-center cubic phase of CeO2 NPs (JCPDS data card no: 34-0394). The lattice constant ‘a’ of CeO2 can be calculated by using the relation. h2 þ k2 þ l2 a2
Average crystalline size D ¼
0:9λ β cosθ
ð2Þ
where λ is the wavelength of X-ray used (1.54060 Å), β is the angular peak width at half maximum in radians and θ is Bragg's diffraction angle. The average crystalline size is estimated as 24 nm for CeO2 NPs.
2.4. Antibacterial activity of CeO2 NPs
1 ¼ d2
The average crystalline size of the samples is calculated after appropriated background corrections from X-ray line broadening of the diffraction peaks using Debye–Scherrer's formula.
! ð1Þ
3.2. X-ray photon spectroscopy XPS provides information on the oxidation state of each element in the sample as well as the composition of the surface functionalization of the CeO2. The XPS results show that the indexed peaks correspond to C (1s), O (1s), and Ce (3d) for CeO2 NPs. The C (1s) signals are most likely due to remaining trace amount of plant extract or simply due to absorption of organic contaminants during handling. The Ce (3d) signals are divided into eight signals, namely O1, O2, O3, O4, O5, O6, O7 and O8 in the Gaussian fitting shown in Fig. 3. Binding energy of CeO2 NP sample is having eight bands 882.26, 888.25, 896.04, 897.73, 900.24, 906.56, 916.34 and 921.33 eV. The main peaks (O7, O3 and O4) of Ce4 + 3d3/2 and Ce4 + 3d5/2 are shown at binding energies of (916.34 eV) and (896.04 & 897.73 eV) respectively. The O5 and O1 energy levels of Ce3+ 3d3/2 and Ce3 + 3d5/2 are located at 900.24 and 882.26 eV. Three additional satellite lines O6, O2 and O8 are shown at 906.56 eV on the Ce3 + 3d3/2 and (888.25 & 921.33 eV) on the Ce3 + 3d5/2, respectively. These spectra are found to be fully consistent with those reported previously [30–33]. Fig. 4 indicates oxygen O (1s) signals which are divided into three symmetrical signals namely G1, G2 and G3 in the Gaussian fitting. For CeO2 NPs, the lower energy level (G1) of O 4+ (1s) signal at 528.83 eV is attributed to O− 2 ions surrounded by Ce ions, which corresponds to the Ce–O bond in CeO2. The middle energy level (G2) of O (1s) at 530.86 eV can be ascribed to the O− 2 ions in the Ce–O bond where Ce is present in the 3+ state. Finally, higher energy level (G3) of O (1s) signals located at 532.47 eV is not related to the presence of either Ce3 + or Ce4 +. It has been suggested that the 532.47 eV peak is due to OH on the surface. 3.3. Transmission electron microscopy Fig. 5a–c shows HRTEM images of the as-prepared CeO2 NPs, calcined at a temperature of 400 °C, and the insets show the electron
The calculated value of ‘a’ is 5.416 Å for CeO2 NPs. The unit cell volume is calculated by the relation given by V = a3. The unit cell volume is found to be 158.867 Å3 for the CeO2 NPs.
Intensity (a.u.)
O1
Ce (3d) O1 O2 O3 O4 O5 O6 O7 O8 Peak sum
O8 O5
O2 O4 O6
O7
O3
880
890
900
910
Binding Energy (eV) Fig. 2. X-ray powder diffraction pattern of CeO2 NPs using G. superba leaf extract.
Fig. 3. XPS spectra of Ce (3d) for the CeO2 NPs.
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G1
411
O (1s) G1 G2 G3 Peak sum
G2 Intensity (a.u.)
G3
528
530
532
534
536
Binding Energy (eV) Fig. 4. XPS spectra of O (1s) for CeO2 NPs.
Fig. 6. FT-IR spectra of (a) G. superba plant extract, (b) as-prepared sample and (c) CeO2 NPs.
Fig. 5. (a–c) TEM images of CeO2 NPs and (d) selected area electron diffraction.
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mode of the Ce-8O vibrational unit and therefore it is very sensitive to any disorder in the oxygen sub-lattice resulting from thermal, doping, or grain size [35,36]. According to the Raman line broadening, the particle size of the CeO2 can be also estimated using the good correlation reported for nanocrystalline CeO2 powder specimens [37–39]. −1 ¼ 10 þ 124:7=DR Г cm
ð3Þ
where Г (cm−1) is the full-width at half-maximum of the Raman active mode peak and DR is the particle size of a CeO2 sample. By substituting Г (cm−1) = 457.24 cm−1 into Eq. (3) with the CeO2 particle size of 5 nm, this calculated particle size is found to be lower than that obtained from X-ray line broadening. 3.6. UV–visible spectroscopy studies Fig. 7. Raman spectrum of the CeO2 NPs calcined in air at 400 °C for 2 h.
diffraction patterns. The particle size is approximately 5 nm for spherical structure. The high crystallinity of the powder leads to its corresponding well-pronounced Debye–Scherrer diffraction rings in the selected area electron diffraction (SAED) pattern (Fig. 5d) that can be assigned to the reflections (1 1 1), (2 0 0), (2 2 0), (3 1 1), (2 2 2), (4 0 0), (3 3 1) and (4 2 0) of cubic CeO2. There are no additional rings in the SAED pattern stemming from any crystalline impurities.
Fig. 8a shows the UV–visible absorption spectrum of G. superba leaf extract assisted CeO2 nanoparticles. G. superba leaf extract consists of phytoconstituents acting as a capping and reducing agent. So, it has reduced the particle size. In this significant effect absorption peak shifted into UV–visible region [16,18,20,21]. The band gap can be determined by fitting the absorption data to the direct transition equation by extrapolating the linear portions of the curves to absorption equal to zero (the inset of Fig. 8b):
3.4. Fourier transform infra-red spectroscopy
1=2 αhυ ¼ A hυ−Eg
The FT-IR spectra of the prepared CeO2 NPs are shown in Fig. 6 using the KBr pellet method in the wave number range 400–4000 cm−1. The broad absorption in the frequency band 3750–3000 cm−1 is assigned to O–H stretching from residual alcohols, water and Ce–OH. The absorption peak is observed at 3475 cm−1 for CeO2 NPs. The CO2 peaks are observed at 2358 cm−1 and 1394 cm−1 for CeO2 NPs. These CO2 band may arise due to some trapped CO2 in air ambience. The band at 1647 cm−1 corresponds to the bending of H–O–H which is partly overlapping the O–C–O stretching band. The band due to the stretching frequency of Ce–O can be seen below 400 cm−1, indicating the formation of CeO2 [20]. In our case, Ce–O stretching is observed at 451 cm−1. Similarly, Goharshdi et al. [34] reported that the Ce–O stretching band appeared in 450 cm−1.
where α is the optical absorption coefficient, hυ is the photon energy, Eg is the direct band gap, and A is a constant. The estimated band gap of the CeO2 sample is to be 3.78 eV. It can be seen that the CeO2 sample shows an increase in Eg by a value exceeding 0.59, compared to the bulk CeO2 powders (Eg = 3.19 eV, determined by UV–visible spectroscopy) [40]. For the reason of increase in the optical band gap of CeO2. The quantum confinement effect is considered when the particle is down to a few nanometers [41]. Since the fundamental band gap is mainly deduced by quantum size effect when the particle size is less than or equal to 3 nm, the quantum size effect in the present CeO2 nanoparticles with particle size of 5 nm may be ruled out and the charge transition of Ce ion (Ce3 +–Ce4 +) may play an important role for the increase in the band gap of present CeO2 sample.
3.5. Micro-Raman spectroscopy
3.7. Photoluminescence studies
Fig. 7 shows a typical Raman spectrum of CeO2. In literature, Raman active mode peak has been observed at 458.9 cm−1 and 461. 04 cm−1 for CeO2 NPs [23,24]. From the present result, a sharp Raman active mode can be found at 457. 24 cm−1, which is attributed to a symmetrical stretching
Fig. 9 shows the photoluminescence spectra of the CeO2 NPs recorded with the excited wavelength of 290 nm. The PL emission is observed for CeO2 NP sample covering from the very short wavelength of 350 nm to long wavelength 500 nm. A good fit of eight peak Gaussian function is
Fig. 8. (a) UV–visible spectrum of the phytosynthesized CeO2 NPs. (b) Photon energy level of the phytosynthesized CeO2 NPs.
ð4Þ
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PL Intensity (a.u.)
G1
3.8. Antibacterial activity
CeO2
G4
G1 G2 G3 G4 G5 G6 G7 G8 Peak sum G8
G3 G2 G5 G6
G7
360
380
400
420
440
460
413
480
500
Wavelength (nm) Fig. 9. Photoluminescence emission spectra of CeO2 NPs using G. superba leaf extract.
obtained for CeO2 NPs. The PL spectra of the samples at the bottom are labeled as G1, G2, G3, G4, G5, G6, G7 and G8. The solid lines represent the linear combination of eight Gaussian peaks where G1 has the lowest and G8 has the highest wavelength. The emission spectra of the CeO2 NP sample are emphasized in eight peaks at 365, 378, 385, 395, 402, 421, 455 and 486 nm. These bands are four near band edge emissions, violet emission, two blue emissions and blue–green emission respectively. The NBE emissions G1, G2, G3 and G4 are located at UV region (365, 378, 385 and 395 nm) for CeO2 NPs. These NBE emissions are attributed to a band-to-band recombination process, possibly involving localized or free excitons [42]. The violet band G5 around 402 nm for the CeO2 sample originates from the defect states existing extensively between the Ce 4f state and O 2p valence band [43]. These defects possibly act as radiative recombination centers for electron initially excited from the valence band to the 4f band of the CeO2 [44]. The two blue emissions G6 and G7 at (421 and 455 nm) are related to the abundant defects such as dislocations, which is helpful for fast oxygen transportation. Ce 4f level with a width of 1 eV is localized at the forbidden gap, which lies at 3 eV over the valence band (O 2p). At room temperature, electron transition mainly occurs from defects level to O 2p level [45]. The blue–green emission G8 located at 486 nm is possibly due to surface defects in the CeO2 NPs, and the low intensity of the green emission may be due to the low density of oxygen vacancies.
In the present study, the NPs are studied extensively to explore their utility as a potential antibacterial agent. Several factors such as less toxicity and heat resistance are accountable for the use of NPs in the biological applications [46,47]. The antibacterial activity has been studied against (G +) and (G −) bacterial pathogens using three different concentrations of CeO2 samples (10, 50 and 100 mg of CeO2). Fig. 10 shows the size of the zone of inhibition and antibacterial activity formed around each CeO2 NPs loaded with test samples. The sample containing 100 mg of CeO2 shows most significant effect on zone of inhibition of 5.33 mm of S. aureus. The E. coli and S. dysenteriae exhibit a modulated effect on inhibition zone of 4.00 mm and 4.33 mm respectively. Then, P. aeruginosa, P. vulgaris, K. pneumonia and S. pneumoniae show similar inhibition zone of 4.67 mm for better activity as compared to that of the sample with 50 mg of CeO2. Recently, chemically synthesized CeO2 NPs showed a better antibacterial activity against E. coli by disc diffusion method. In addition, green synthesized CeO2 NPs were tested in antibacterial activity showed a significant effect of G + and G − bacteria by disc diffusion method [48,49]. The antibacterial efficiency of CeO2 NPs generally depends on their size, specific surface area, polar surface, morphology etc. Moreover, electrostatic attraction between negatively charged bacterial cells and positively charged nanoparticles is crucial for the activity of nanoparticles as bactericidal materials. This interaction not only inhibits the bacterial growth but also induces the reactive oxygen species (ROS) generation, which leads to cell death [50–52]. In particular, many previous studies have explored the photogeneration of ROS on the surfaces of metal-oxide NPs [53,54]. The general principle is that when NPs are illuminated by light with photoenergy greater than the band gap, the electrons (e−) of NPs are promoted across the band gap to the conduction band, which creates a hole (h+) in the valence band [53]. Electrons in the conduction band and holes in the valence band exhibit high reducing and oxidizing power respectively [54]. The electron can react with molecular oxygen to produce superoxide anion (O•− 2 ) through a reductive process [54]. The hole can abstract electrons from water and/or hydroxyl ions to generate hydroxyl radicals (•OH) through an oxidative process [54]. Singlet oxygen (1O2) is mostly produced indirectly from • aqueous reactions of O•− 2 [41]. OH is a strong and nonselective oxidant that can damage virtually all types of organic biomolecules, including carbohydrates, nucleic acids, lipids, proteins, DNA and amino acids [55]. 1O2 is the main mediator of photocytoxicity and can irreversibly damage the treated tissues [56], causing biomembrane oxidation and degradation [57]. Although O•− 2 is not a strong oxidant, as a precursor for •OH and 1O2, O•− 2 also has significant biological implications [58]. Although many previous studies have explored ROS generation by various
Fig. 10. Size of the zone of inhibition formed around each disc, loaded with test samples, indicating the antibacterial activity CeO2 NPs using G. superba leaf extract.
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Fig. 11. Diagrammatic representation of toxicity of CeO2 NPs against bacterial pathogens.
metal-oxide NPs, to the best of our knowledge, little research has examined the role of the electronic properties of metal-oxide NPs in ROS generation [52]. A deeper understanding in this aspect would allow us to interpret the underlying ROS generation mechanisms. Generally, nanoparticles with better photocatalytic activity have a large specific surface area and a small crystalline size which increase oxygen vacancies [59]. The antibacterial activity of CeO2 NPs can also be explained as follows. ROS include the most reactive hydroxyl radical (•OH), less toxic superoxide anion radical (−•O2), and singlet oxygen (1O2) with a weaker oxidizer, contributing to the major oxidative stress in biological systems [60]. ROS generation is closely associated with the efficiency of a photocatalyst, depending on the generation rate, rate of migration, and energy levels of the photoexcited electron-hole pairs. In the current study, such enhanced ROS yields may be connected with the electronic properties and microstructure (i.e., grain size, specific surface area, and pore, etc.) of the spherical CeO2 NPs. The electronic structure of metal-oxide NPs is characterized by the band gap (Eg), which is essentially the energy interval between the valence band (Ev) and the conduction band (Ec), each of which has a high density of states. Only the metal-oxide NPs with Eg higher than the
Fig. 12. Antibacterial activity of CeO2 NPs against G+ and G− bacterial strains. The data were obtained from three experiments and are expressed as the mean ± S.E. * indicates significant differences from the G+ve bacteria at P b 0.05, analyzed by two sample t-test.
incident photon energy (i.e., approximately 3.78 eV) can be photoexcited. Under UV excitation, electrons are promoted from the valence band to the conduction band, with the concomitant generation of a hole in the valence band. The photoexcited electrons and holes then react with an aqueous electron acceptor (i.e., molecular oxygen) and donor (i.e., water and hydroxyl ions) respectively to produce different types of ROS. The mechanism of light induced generation of ROS can be given as follows. − þ þ þ − − − CeO2 þ hυ→e þ h h þ H2 O→·OH þ H e þ O2 →·O2 ·O2 þ þ − · · þH →HO HO þ H þ e →H O 2
2
2
2
A higher ROS value generally comes from a photocatalyst with a larger surface area, appropriate crystal size, increase oxygen vacancies and the facilitation of diffusion and mass transportation of reactant molecules. From the XRD results, the crystalline size is found to be 24 nm. Smaller crystal size with a higher surface area leads to higher antibacterial activity [61]. From the photoluminescence results for the CeO2, the blue–green emission at 486 nm is due to the presence of oxygen vacancy and oxygen interstitial defect. Other possible mechanisms are involving the interaction of nanomaterials with the biological macromolecules. It is believed that microorganisms carry a negative charge while metal oxides carry a positive charge [62]. This creates an “electromagnetic” attraction between the microbe and treated surface. Once the contact is made, the microbe oxidizes and dies instantly. Since the CeO2 NPs interfere with the bacteria cell membrane and bind with mesosome, there is a perturbance in the mesosomal functions of cellular respiration, DNA replication, cell division and thereby the surface area of bacterial cell membrane is increased. These intracellular functional changes establish the oxidative stress induced by ROS generation due to the cell expiry. Considering the above facts, a schematic picture is represented in (Fig. 11). Generally, it is believed that nano-materials release ions, which react with the thiol groups (–SH) of the proteins present on the bactericidal cell surface [63]. Such proteins protrude through the bactericidal cell membrane, allowing the transport of nutrients through the cell wall. The nano-materials inactivate the proteins and decrease the membrane permeability, which eventually causes the cell death. The NPs with uneven surface texture due to rough edges and corners contribute to the mechanical damage of the cell membranes of E. coli [64]. From the TEM image, it is clear that the CeO2 NPs have uneven ridges at the outer surface which influence the antibacterial activity. The combination of various factors such as size and oxygen defects plays a critical role in the toxicological behavior of CeO2 NPs. From Fig. 12, it can be suggested that (G +) bacteria are relatively more
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susceptible to the NPs than (G−) bacteria. Comparing our results with the reported results regarding the sensitivity of (G +) and (G −) to the photogeneration of O− 2 in the case of ZnO and TiO2 [61,65], the same trend is also observed in the case of CeO2 NPs in such a way that (G+) is more sensitive to ROS than (G−). 4. Conclusion In summary, CeO2 NPs were successfully synthesized using aqueous leaf extract of G. superba. The XRD, Raman and SAED studies revealed the formation of CeO2 NPs. The TEM images clearly showed that CeO2 NPs possessed spherical shaped morphology with the average size of 5 nm. Smaller crystal sizes with a higher surface area lead to higher antibacterial activity. From the photoluminescence spectra of CeO2 NPs, the blue–green emission at 486 nm was observed due to the presence of oxygen vacancy and oxygen interstitial defects. As seen from the TEM image, the CeO2 NPs having uneven ridges at the outer surface influenced the antibacterial activity of the NPs, Thus, with the support of the photoluminescence and TEM results, the toxicological behavior of CeO2 NPs was found due to the size of the synthesized NPs with uneven ridges and oxygen defects in CeO2 NPs. Acknowledgments The authors gratefully thank School of Physics, Alagappa University for extending the XRD, and Micro-Raman and photoluminescence spectroscopy facilities. References [1] E. Bekyarova, P. Fornasiero, J. Kaspar, M. Graziani, Catal. Today 45 (1998) 179–183. [2] S.B. Khan, M. Faisal, M.M. Rahman, A. Jamal, Sci. Total Environ. 409 (2011) 2987–2992. [3] H. Yahiro, Y. Baba, K. Eguchi, A. Hiromichi, J. Electrochem. Soc. 135 (1998) 2077–2080. [4] S. Yabe, T. Sato, J. Solid State Chem. 171 (2003) 7–11. [5] S. Babu, J.H. Cho, J.M. Dowding, E. Heckert, C. Komanski, S. Soumen Das, J. Colon, C.H. Baker, M. Bass, W.T. Self, S. Seal, Chem. Commun. 46 (2010) 6915–6917. [6] P. Zhang, Y. Ma, Z. Zhang, X. He, J. Zhang, Z. Guo, R. Tai, Y. Zhao, Z. Chai, ACS Nano 11 (2012) 9943–9950. [7] A. Thill, O. Zeyons, O. Spalla, F. Chauvat, J. Rose, M. Auffan, A.M. Flank, Environ. Sci. Technol. 40 (2006) 6151–6156. [8] F. Zhang, S.W. Chan, J.E. Spanier, E. Apak, Q. Jin, R.D. Robinson, I.P. Herman, Appl. Phys. Lett. 80 (2002) 127–129. [9] J. Hu, Y. Li, X. Zhou, M. Cai, Mater. Lett. 61 (2007) 4989–4992. [10] H. Wang, J.J. Zhu, J.M. Zhu, X.H. Liao, S. Xu, T. Ding, H.Y. Chen, Phys. Chem. Chem. Phys. 4 (2002) 3794–3799. [11] X.H. Liao, J.M. Zhu, J.J. Zhu, J.Z. Xu, H.Y. Chen, Chem. Commun. (2001) 937–938. [12] F. Czerwinski, J.A. Szpunar, J. Sol-Gel Sci. Technol. 9 (1997) 103–114. [13] S.Y. Yao, Z.H. Xie, J. Mater. Process. Technol. 186 (2007) 54–59. [14] B. Choudhury, A. Choudhury, Mater. Chem. Phys. 131 (2012) 666–671. [15] C. Hu, Z. Zhang, H. Liu, P. Gao, Z.L. Wang, Nanotechnology 17 (2006) 5983–5987. [16] Y. Huang, Y. Cai, D. Qiao, H. Liu, Particuology 9 (2011) 170–173. [17] A. Krishnan, T.S. Sreeremya, E. Murray, S. Ghosh, J. Colloid Interface Sci. 389 (2013) 16–22. [18] S. Maensiri, C. Masingboon, P. Laokul, W. Jareonboon, V. Promarak, P.L. Anderson, S. Seraphin, Cryst. Growth Des. 7 (2007) 950–955. [19] S. Maensiri, S. Labuayai, P. Laokul, J. Klinkaewnarong, E. Swatsitang, Jpn. J. Appl. Phys. 53 (2014) 06JG14. [20] S. Phoka, P. Laokul, E. Swatsitang, S. Maensiri, Mater. Chem. Phys. 115 (2009) 424–428.
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