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Role of oxygen vacancies on photo-catalytic activities of green synthesized ceria nanoparticles in Cydonia oblonga miller seeds extract and evaluation of its cytotoxicity effects
Journal Pre-proof Role of oxygen vacancies on photo-catalytic activities of green synthesized ceria nanoparticles in Cydonia oblonga miller seeds extract and evaluation of its cytotoxicity effects Behrouz Elahi, Mahdi Mirzaee, Majid Darroudi, Reza Kazemi Oskuee, Kayvan Sadri, Leila Gholami PII:
S0925-8388(19)33799-5
DOI:
https://doi.org/10.1016/j.jallcom.2019.152553
Reference:
JALCOM 152553
To appear in:
Journal of Alloys and Compounds
Received Date: 30 July 2019 Revised Date:
20 September 2019
Accepted Date: 3 October 2019
Please cite this article as: B. Elahi, M. Mirzaee, M. Darroudi, R.K. Oskuee, K. Sadri, L. Gholami, Role of oxygen vacancies on photo-catalytic activities of green synthesized ceria nanoparticles in Cydonia oblonga miller seeds extract and evaluation of its cytotoxicity effects, Journal of Alloys and Compounds (2019), doi: https://doi.org/10.1016/j.jallcom.2019.152553. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Role of oxygen vacancies on photo-catalytic activities of green synthesized Ceria nanoparticles in Cydonia oblonga miller seeds extract and evaluation of its cytotoxicity effects
Behrouz Elahia, Mahdi Mirzaeea, *, Majid Darroudib,c, **, Reza Kazemi Oskueec, Kayvan Sadrib, Leila Gholamid a
Faculty of Chemistry, Shahrood University of Technology, Shahrood, Iran
b
Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
c
Department of Medical Biotechnology and Nanotechnology, School of Medicine, Mashhad University of Medical
Sciences, Mashhad, Iran d
Nanotechnology research center, Mashhad medical university of science, Mashhad, Iran
*,**
*
Corresponding authors
M. Mirzaee
Faculty of Chemistry, Shahrood University of Technology, Shahrood, Iran E-mail: [email protected], Tel.: +98-23-32395441 & Fax: +98-23-32395441. **
M. Darroudi
Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran E-mail: [email protected], Tel.: +98-513-8002286 & Fax: +98-513-8002287
Abstract Ceria nanoparticles (CN) were produced via green route in Cydonia oblonga miller (Com) seeds extract as capping and stabilizing agent. They were characterized by a variety of physicochemical methods such as XRD, FT-IR, UV-Vis, FESEM, TGA/DTA, and photoluminescence (PL). Crystalline size of CN was increased with ascending the calcination temperature while band gap energy of them was descending from 2.4 to 3.1 eV. Cell viability was determined by MTT assay and results showed that CN had no significance toxicity on A549 cell line. Also, antioxidant effect of CN on the same cell line was performed via 2´,7´–dichlorofluorescin diacetate (DCFDA). All concentration of CN specially 15.6 µg/ml could neutralize the oxygen reactive species (ROS). Photo-catalytic study was performed to evaluate dyes degradation ability of CN under UV-A irradiation. Results illustrated that samples calcined in lower temperature showed better photo-catalytic activity. Also, PL study showed that the photoluminescence emission intensity of samples was diminished with increasing the calcination temperature. In conclusion, enhance the photo-catalytic activity and photoluminescence emission was related to presence of oxygen defects in CN structure which were increased in lower calcination temperature and responsible for improving the optical properties of CN. Keywords: Ceria nanoparticles, Plant extract, Cytotoxicity effect, Dye degradation, Oxygen vacancies.
1. Introduction Ceria is a direct semiconductor with band gap about 3.19 eV which crystallize in a face-centered cubic (fcc) system in the Fm3m space group [1, 2]. Cerium atoms can take two oxidation numbers +3 and +4 (most stable) in ceria structure [3]. Electron transfer between Ce3+ and Ce4+ leads to several oxygen vacancies in ceria lattice [3]. Nano-crystallite ceria showed higher concentration of oxygen vacancies in comparison to bulk ceria which these defects could be in the surface or inside of its lattice. These defects play a key role in variety of ceria applications such as catalyst [4], photo-catalyst [5], oxygen sensor [6], optical devices [7], and ultraviolet absorber [8]. Also, ceria has shown antioxidant properties due to these oxygen vacancies which develops its using in biological studies [9]. The band energy of these oxygen vacancies is lower than ceria band gap of 3.19 eV. Consequently, the oxygen vacancies of ceria could expand its absorption edge to visible region and makes it a potential candidate for electron trapping [10]. The concentration of oxygen vacancies in ceria were increased when it was heated in a poor O2 atmosphere and caused to produce some oxygen deficient phases of ceria (CeO2-x , 0 ≤ x ≤ 0.5) [11-13]. It means that, synthesis conditions can effectively controls the amount of oxygen vacancies as well as optical properties of ceria. In photo-catalytic processes, the electron-hole pairs are responsible for degradation of dye molecules by producing energized holes (h+), hydroxyl (•OH), and superoxide anion (O2•-) radicals. These species in aqueous media can oxidize/reduce the organic dye molecules [14-16]. As mentioned above, oxygen vacancies could effectively trapped the electrons and therefore the amount of energized holes (h+) would be enhanced by increasing the oxygen vacancies [5, 10]. The CN has been synthesized by various chemical-physical methods such as co-precipitation [17], hydrothermal [18], sol-gel [19], microwave [20], and solvo-thermal [21]. Among them, an eco-friendly green synthesis method
has been highlighted because in this process biological media such as plant extracts, bacteria, fungus, algae and yeasts were used which all of them are bio-degradable and do not produced toxic compounds [22, 23]. Plant extracts contain organic compounds which could act as capping agents. These compounds in Com seed extract are including phenolic compounds, organic acids and free amino acids [24]. These are electron donor substituents which could effectively avoided particle aggregation. Here, CN was produced via a green route using Com seed extract in different calcination temperatures for investigating the effect of temperature on the concentration of oxygen vacancies. In addition, the influence of oxygen vacancies on the photo-catalytic activity of CN was perused in degradation of RhB dye as an organic pollutant in wastewaters.
2. Materials and Method 2.1. Materials and reagents Cerium nitrate hexa-hydrate (99%) was purchased from Merck, Germany. Com seeds were bought from a grocery market in Mashhad, Iran. Phosphate Buffered Saline (PBS), Trypsin and 3-(4,5–dimethyl-2-thiazolyl)-2,5–diphenyl-2H-tetrazolium bromide (MTT), and Dimethyl sulfoxide (DMSO) were bought from Sigma (USA). To assess cytotoxicity effect of CN on A549 cell line, cells (bought from Pasteur institute of Iran, Tehran, Iran) had been cultured in streptomycin (100 mg/ml), penicillin (100 µg/ml), and DMEM (Dulbecco’s modified Eagle’s medium) along with 10% FBS (Fetal bovine serum). To evaluate reactive oxygen species (ROS), (DCFDA) was bought from Sigma-Aldrich. In all processes, doubled distilled water was engaged. 2.2. Com seed extract preparation
10.0 g of Com seeds were washed by distilled water for removing dust. After that, they were appended to 100 ml of distilled water and mixture was stirred for 4 h at 60 ˚C. Then, the extract was separated from the mixture by filtration and stored in 4 ˚C. 2.3. Synthesis method of CN 30 ml of prepared Com seeds extract was mixed with 50 ml of distilled water and then 20 ml of an aqueous solution containing 5.0 g cerium nitrate hexa-hydrate was drop-wisely added to it. This solution was heated and stirred at 80 ˚C for 6 h. Next, due to remove water and attain gel, the solution was put into an oven (60 ˚C). Achieved yellow gel was divided to three portions and they were calcined for 2 h at 400, 500, and 600 ˚C with rate of 3 ˚C/min due to eliminate organic composites and produce yellow powders of CN. 2.4. Characterization Green synthesized CN samples were characterized by several laboratory device including D8Advance Bruker equipped with Cu Kα1 radiation (λ=0.15406 nm), Rayleigh-WQF-510A spectrometer, Cecil-CE9500 spectrophotometer, TESCAN MIRA3, Bahr STA 503, and Shimadzu spectrofluorometer for PXRD, FTIR, UV-Vis, FESEM, TGA/DTA, and PL analysis, respectively. Also, Epoch micro-plate spectrophotometer from BioTek and PerkinElmer multiplate reader were employed to read absorbance of the MTT and fluorescence emission of 2´, 7´ – dichlorofluorescin (DCF) dye. 2.5. Photo-catalytic processes The photo-catalytic experiments were accomplished using UV-A light with an intensity of 4.4 W/m2 provided by an 11 W fluorescence lamp with maximum wavelength at 365 nm. Irradiation without any filtration was directly glowed into the reaction mixture under air atmosphere at room
temperature. In all processes 100 mg of CN was added into 50 ml solution (4 ppm) of RhB in a 250 ml beaker and mixture was stirred vigorously. Before starting the irradiation, mixtures were stirred in the dark for 60 min due to complete absorption/desorption of dye on the CN surface. Also, all reactions were repeated three times due to investigate reproducibility. The absorbance of dye was recorded by UV–Vis spectrophotometer during the processes. 2.6. Determination of Cytotoxicity effects The cytotoxic effect of CN on cell viability of A549 cell line was assessed by MTT assay [25]. A549 cells were developed in DMEM medium supplemented with 10% FBS and 1% antibiotic. Cells were seeded on 96 well plate with density of 104 cell per well for 24h. After that, different concentrations of dispersed CN in distilled water were exposed to cells and another incubation time for 24h. Next, optimized amount of MTT solution (5 mg/ml) were added to each well and cells were incubated for 4h at 37˚C. Then, media was removed and formazan crystals were dissolved in 100 µl of fresh DMSO. The optical absorbance of each well was measured at 570 and 630 nm with a micro-plate reader. Due to certify the repeatability, each experiment was frequented triplicate and viability was expressed as a relative percentage mean ± SD versus to the untreated control cells. For the ROS assay, seeded A549 cells were treated by CN similar to MTT assay and were incubated at the same incubation time. Afterward, the medium was eliminated and each well was washed with 100 µl PBS. Next step, DCFDA with specific concentration of 10 mM was added to wells and after 24h of incubation and then fluorescence emission of DCF was read using a multi micro-plate reader.
3. Results and Discussions 3.1. Synthesis and characterization of CN CN was produced in Com seeds extract as capping and stabilizing agent through green route.
TGA/DTA analysis was performed to determine calcination temperature and evaluate thermal stability of product in range of 25-700 ˚C with rate of 3 ˚C per minute under air environment (Fig. 1). TGA curve shows three stages of weight loss which all of them are accompanied with endothermic peaks in DTA diagram. The first stage with 20% weight loss below 110 ˚C which attended by two endothermic peaks in the DTA curve, is related to the evaporation of water which physically absorbed on the pores and surface of CN [26]. Other two weight losses up to 45% in the range of 160 to 250 ˚C could be related to the removal of remaining organic compositions of Com seeds extract [27, 28]. These process cause to alteration of CeOY (Y=H, R) to pure ceria crystals at about 300 ˚C [29, 30]. Total weight loss in TGA curve is about 65%; also, there is no significance weight loss at higher temperatures which confirmed thermal stability of the product. In accordance with TGA/DTA and to ensure complete ignition of residual organic compounds in the product and investigate the effect of calcination temperature on the crystallite size and optical properties of CN, jelly samples were calcined at 400, 500 or 600 ˚C with rate of 3 ˚C per minute in an electric furnace and yellow powders were obtained. Fig. 2a illustrated UV-Vis spectra of CN colloidal solution. Maximum absorption for all samples was in range of 315 to 345 nm which is corresponded to charge transfer from O2P to Ce4f orbital [5, 31]. Band gap energy of CN samples was computed by Eq. 1 [32], ℎ
=
ℎ −
(1)
wherein, hν is the photon energy, α is the absorption coefficient, B is a constant, and n is 2 for semiconductor with direct transitions like ceria [33]. Band gap energy can be obtained from the junction spot of
ℎ
. ℎ plot slope on the X-axis (Fig. 2b). The λmax along with band gap
energy (Eg) are presented in Tab. 1. As it shown, CN-400 has lowest λmax because in direct
semiconductor decrease in particle size cause a blue shift in λmax [34, 35]; moreover, CN-400 has sharper peak than other which is related to the more uniform distribution of its particle size. FTIR spectra of green synthesized samples are showed in Fig. 3. All samples showed a sharp band about 400 cm-1 and weak band about 1050 cm-1 that are corresponded to stretching vibration of Ce─O and O─Ce─O bonds, separately [36]. Bands about 3400 cm-1 could be corresponded to OH group of adsorbed water on the CN surface. Weak bands about 1300 to 1500 cm-1 could be related to stretching vibration of C─H and C═O bonds of organic molecules, respectively, which are maybe remained from Com seeds extract [37]. XRD patterns of CN-400, 500, and 600 are illustrated in Fig. 4. All patterns were matched with single phase Cerianite (JCPDS No. 34-0394). Full width at half maximum (FWHM) was decreased and also peaks were sharpen with increasing the calcination temperature due to increasing the size and crystallinity of particles. Crystallite size of particles were calculated by Scherrer equation with using FWHM of (111), (200), (220), (311), (400), and (331) crystallographic planes [32]. Average crystallite size of particles was presented in Tab. 1. FESEM images were shown agglomerated shapeless particles for all samples (Fig. 5). As illustrated in Fig. 5, the size of CN-400 particle is smaller than CN-500 and CN-600; also increasing the calcination temperature resulted to the particle agglomeration. EDX analysis confirmed the attendance of O and Ce elements on the surface of all samples (Fig. 5). FESEM showed that CN-400 has smallest particle size with more uniform morphology among these samples which approved the outcomes of XRD and UV-Vis analysis. Fig. 6a demonstrates the PL spectra of dispersed CN-400, CN-500, and CN-600 in distilled water with an excitation wavelength of 200 nm. The bands in range of 420 to 520 nm are corresponded to the surface structural defects of CeO2 including oxygen vacancies [38, 39]. The
blue-green emission band at 470 nm had the maximum intensity among others which are blue bands at 440 and 450 nm, blue-green bands at 485 and 495 nm, and green band at 515 nm. Moreover, intensity of bands in CN-400 was greater than others which could be depended on morphology of particles, excitation wavelength, and density of oxygen vacancies [5, 38, 40]. Structural defects including surface oxygen vacancies were decreased by increasing the calcination temperature in presence of air and cause to reduce the intensity of PL peaks [10, 41]; therefore, surface defect density of CN-400 was higher than others. 3.2. Effect of oxygen vacancies on the photo-catalytic degradation strength The photo-catalytic experiments were done to evaluate the influence of calcination temperature on the photo-catalytic strength in dye degradation. 50 ml of the RhB colored solution (8 ppm) was degraded by 100 mg of CN-400, CN-500, and CN-600, respectively, which they were dispersed in RhB solution under UV-A irradiation with intensity of 4.4 W/m2. The amount of catalyst was determined according to optimum achieved from our recent study [32]. The solution was stirred vigorously and during process, absorption peak of RhB solution was monitored by UV-Vis spectrophotometer. All reactions were repeated in dark with the above conditions to ensure the rule of UV irradiation on degradation process. The results of Fig. 6b showed that CN400 could degrade approximately 92% of RhB in lower time in comparison to CN-500 and CN600. Moreover, to investigate the reproducibility of dye degradation results, all reactions were repeated three times (Fig. 6b). The results showed that photo-degradation of RhB dye in presence of CN under UV-A light was repeatable with accuracy more that 94%.
As a result,
increasing the calcination temperature had reverse influence on the photo-catalytic strength of the CN samples and this is in accordance with more surface oxygen vacancies in lower calcination temperature as mentioned above. Because, surface vacant oxygen sites can catch the
conducting electron released and decrease recombination of electron-hole pairs. Therefore, energized holes reacted with dye molecules and oxidized them faster [5, 42]. If oxygen defects have absorption edges in visible region, it causes to produce more electron-hole sites which should improve its photo-catalytic effect. But in this study, CN has not showed any photocatalytic effect in visible region, therefore it should be acted as an electron trapper which increased the energized holes. Meanwhile, according to the photo-degradation mechanism of RhB in the presence of CN, the holes (h+) had more effective role than •OH and O2•‾ [32]. The kinetic of photo-degradation process was obtained from Eq. 2 [43], −
=
(2)
Wherein, C and C0 are the dye concentration at time ‘t’ and ‘t=0’, separately, and k is the rate constant which it can be calculate from the plot of lnC/C0 versus irradiation time (Fig. 6c). This plots showed straight line which it confirmed the photo-degradation by CN follows a pseudo first-order reaction rate. The CN-400 has better activity with rate constant of 0.012 min-1 in comparison to CN-500 (0.007 min-1) and CN-600 (0.006 min-1). The percentage of photodegradation after 3 h for CN-400, CN-500, and CN-600 were demonstrated in Fig. 6d which among them, CN-400 showed the best efficiency (92%). Fig. 7 showed the photo-catalytic mechanism of RhB dye degradation in the presence of CN under UV-A irradiation. 3.3. Cytotoxicity study Based on above results, CN-400 had smallest particle size with better optical properties therefore it was selected for cytotoxic study. MTT assay was executed to evaluate in-vitro cell viability of A549 cell line in presence of different concentrations of CN-400 (1 to 500 µg/ml). Treated cells were incubated for 24 h, and then absorption of MTT was read by micro-plate
spectrophotometer. Fig. 8a showed the percentage of cell viability versus various concentrations of CN-400. The viability was further than 80% for density up to 250 µg/ml while it was 62% for 500 µg/ml. All concentrations of CN-400 were indicated cell viability more than 50%, therefore green synthesized CN-400 showed no significance toxicity on A549 cells. The ROS scavenger influence of CN-400 on the A549 cell line was evaluated by DCFDA assay. Cells were treated by CN-400 as it was done for MTT assay and the emission of DCF dye was recorded at 530 nm by a micro-plate reader. Fig. 8b illustrated the percentage of ROS scavenging versus different concentrations of CN-400. All concentration of CN-400 could neutralize ROS; moreover, 15.6 µg/ml of CN-400 had best antioxidant influence and could defuse about 76% of ROS formed by cellular metabolism. The antioxidant properties of CeO2 is due to electron transfer from Ce3+ to Ce4+ which it can reduce ROS [44]. The result of MTT and ROS assays confirmed that CN is a proper candidate for biological studies such as cell imaging or drug/gene delivery which were reported for CN [45, 46].
4. Conclusion The CN were produced via green route by using Com seeds extract as capping agent. Agglomerated uniform particles were obtained which their size were increased by enhancing the calcination temperature. According to the PL analysis, CN-400 showed higher emission intensity and therefore it may contained more oxygen vacancies in its structure. In addition, this low temperature calcined sample showed higher photo-catalytic activity in degradation of RhB under UV-A irradiation. Green synthesized CN-400 showed no significance toxicity on the A549 cell line; moreover, it showed worthy antioxidant effect which confirmed CN could be suitable candidate for biological studies.
Acknowledgements The authors gratefully acknowledge Shahrood University of Technology and Mashhad university of Medical Sciences for the technical and financial support of this research.
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Legends Fig. 1. The thermal gravimetric and differential thermal curves of gel sample Fig. 2. a) UV-Vis spectra of dispersed CN in distilled water, b) The graph of (αhν)2 vs. hν for the calculation the band gap energy of samples Fig. 3. The FTIR spectra of the achieved samples Fig. 4. The XRD patterns of calcined samples in 400, 500, and 600 ˚C Fig. 5. The FESEM images and the EDX spectrum of the samples Fig. 6. a) PL spectrums of samples, b) The comparison of dye degradation ability between CN400, CN-500, and CN-600 respect to time along with variations of RhB absorbance spectra as function of UV-A light irradiation time, c) The plot of ln C/C0 vs. irradiation time for CN400, CN-500, and CN-600, d) The photo-degradation efficiencies of all samples after 180 min. Fig. 7. Photo-catalyst mechanism of RhB dye degradation in presence of CN under UV-A light irradiation Fig. 8. a) A549 cell viability in attendance of CN-400, b) The ability of CN-400 in scavenging ROS Tab. 1. Temperature of calcination, photo-catalytic degradation rate constant, optical properties, and particle size of the CN samples
Fig.1. M. Darroudi et al., 2019
Fig.2. M. Darroudi et al., 2019
Fig.3. M. Darroudi et al., 2019
Fig.4. M. Darroudi et al., 2019
Fig.5. M. Darroudi et al., 2019
Fig. 6. M. Darroudi et al., 2019
Fig.7. M. Darroudi et al., 2019
Fig.8. M. Darroudi et al., 2019
Tab. 1. M. Darroudi et al., 2019 Sample
Calcination temperature (˚C)
CN-400
400
Degradation rate constant (min-1) a 0.012
CN-500
500
CN-600
600
λmax (nm)
Eg (eV) b
Crystallite size (nm) c
315
3.1
9.0
0.007
323
3.0
9.5
0.006
342
2.4
11.8
a
Computed from the plot of ln C/C0 vs. time
b
Computed according to Eq. 1 from UV-Vis spectra
c
Computed corresponding to the Scherrer equation
Research Highlights ►Green
synthesis of Ceria nanoparticles was carried out in Cydonia oblonga miller
seeds extract as capping agent. ►Ceria nanoparticles calcined in lower temperature had more oxygen vacancies in their structure and shown higher intensity in photoluminescence emission
►The more amounts of oxygen
vacancies caused to enhance the photo-catalytic strength in dyes degradation ► Ceria nanoparticles has not shown toxic effect on cell viability and could remarkably scavenge the ROS produced by cell metabolism.
S0925-8388(19)33799-5
DOI:
https://doi.org/10.1016/j.jallcom.2019.152553
Reference:
JALCOM 152553
To appear in:
Journal of Alloys and Compounds
Received Date: 30 July 2019 Revised Date:
20 September 2019
Accepted Date: 3 October 2019
Please cite this article as: B. Elahi, M. Mirzaee, M. Darroudi, R.K. Oskuee, K. Sadri, L. Gholami, Role of oxygen vacancies on photo-catalytic activities of green synthesized ceria nanoparticles in Cydonia oblonga miller seeds extract and evaluation of its cytotoxicity effects, Journal of Alloys and Compounds (2019), doi: https://doi.org/10.1016/j.jallcom.2019.152553. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Role of oxygen vacancies on photo-catalytic activities of green synthesized Ceria nanoparticles in Cydonia oblonga miller seeds extract and evaluation of its cytotoxicity effects
Behrouz Elahia, Mahdi Mirzaeea, *, Majid Darroudib,c, **, Reza Kazemi Oskueec, Kayvan Sadrib, Leila Gholamid a
Faculty of Chemistry, Shahrood University of Technology, Shahrood, Iran
b
Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
c
Department of Medical Biotechnology and Nanotechnology, School of Medicine, Mashhad University of Medical
Sciences, Mashhad, Iran d
Nanotechnology research center, Mashhad medical university of science, Mashhad, Iran
*,**
*
Corresponding authors
M. Mirzaee
Faculty of Chemistry, Shahrood University of Technology, Shahrood, Iran E-mail: [email protected], Tel.: +98-23-32395441 & Fax: +98-23-32395441. **
M. Darroudi
Nuclear Medicine Research Center, Mashhad University of Medical Sciences, Mashhad, Iran E-mail: [email protected], Tel.: +98-513-8002286 & Fax: +98-513-8002287
Abstract Ceria nanoparticles (CN) were produced via green route in Cydonia oblonga miller (Com) seeds extract as capping and stabilizing agent. They were characterized by a variety of physicochemical methods such as XRD, FT-IR, UV-Vis, FESEM, TGA/DTA, and photoluminescence (PL). Crystalline size of CN was increased with ascending the calcination temperature while band gap energy of them was descending from 2.4 to 3.1 eV. Cell viability was determined by MTT assay and results showed that CN had no significance toxicity on A549 cell line. Also, antioxidant effect of CN on the same cell line was performed via 2´,7´–dichlorofluorescin diacetate (DCFDA). All concentration of CN specially 15.6 µg/ml could neutralize the oxygen reactive species (ROS). Photo-catalytic study was performed to evaluate dyes degradation ability of CN under UV-A irradiation. Results illustrated that samples calcined in lower temperature showed better photo-catalytic activity. Also, PL study showed that the photoluminescence emission intensity of samples was diminished with increasing the calcination temperature. In conclusion, enhance the photo-catalytic activity and photoluminescence emission was related to presence of oxygen defects in CN structure which were increased in lower calcination temperature and responsible for improving the optical properties of CN. Keywords: Ceria nanoparticles, Plant extract, Cytotoxicity effect, Dye degradation, Oxygen vacancies.
1. Introduction Ceria is a direct semiconductor with band gap about 3.19 eV which crystallize in a face-centered cubic (fcc) system in the Fm3m space group [1, 2]. Cerium atoms can take two oxidation numbers +3 and +4 (most stable) in ceria structure [3]. Electron transfer between Ce3+ and Ce4+ leads to several oxygen vacancies in ceria lattice [3]. Nano-crystallite ceria showed higher concentration of oxygen vacancies in comparison to bulk ceria which these defects could be in the surface or inside of its lattice. These defects play a key role in variety of ceria applications such as catalyst [4], photo-catalyst [5], oxygen sensor [6], optical devices [7], and ultraviolet absorber [8]. Also, ceria has shown antioxidant properties due to these oxygen vacancies which develops its using in biological studies [9]. The band energy of these oxygen vacancies is lower than ceria band gap of 3.19 eV. Consequently, the oxygen vacancies of ceria could expand its absorption edge to visible region and makes it a potential candidate for electron trapping [10]. The concentration of oxygen vacancies in ceria were increased when it was heated in a poor O2 atmosphere and caused to produce some oxygen deficient phases of ceria (CeO2-x , 0 ≤ x ≤ 0.5) [11-13]. It means that, synthesis conditions can effectively controls the amount of oxygen vacancies as well as optical properties of ceria. In photo-catalytic processes, the electron-hole pairs are responsible for degradation of dye molecules by producing energized holes (h+), hydroxyl (•OH), and superoxide anion (O2•-) radicals. These species in aqueous media can oxidize/reduce the organic dye molecules [14-16]. As mentioned above, oxygen vacancies could effectively trapped the electrons and therefore the amount of energized holes (h+) would be enhanced by increasing the oxygen vacancies [5, 10]. The CN has been synthesized by various chemical-physical methods such as co-precipitation [17], hydrothermal [18], sol-gel [19], microwave [20], and solvo-thermal [21]. Among them, an eco-friendly green synthesis method
has been highlighted because in this process biological media such as plant extracts, bacteria, fungus, algae and yeasts were used which all of them are bio-degradable and do not produced toxic compounds [22, 23]. Plant extracts contain organic compounds which could act as capping agents. These compounds in Com seed extract are including phenolic compounds, organic acids and free amino acids [24]. These are electron donor substituents which could effectively avoided particle aggregation. Here, CN was produced via a green route using Com seed extract in different calcination temperatures for investigating the effect of temperature on the concentration of oxygen vacancies. In addition, the influence of oxygen vacancies on the photo-catalytic activity of CN was perused in degradation of RhB dye as an organic pollutant in wastewaters.
2. Materials and Method 2.1. Materials and reagents Cerium nitrate hexa-hydrate (99%) was purchased from Merck, Germany. Com seeds were bought from a grocery market in Mashhad, Iran. Phosphate Buffered Saline (PBS), Trypsin and 3-(4,5–dimethyl-2-thiazolyl)-2,5–diphenyl-2H-tetrazolium bromide (MTT), and Dimethyl sulfoxide (DMSO) were bought from Sigma (USA). To assess cytotoxicity effect of CN on A549 cell line, cells (bought from Pasteur institute of Iran, Tehran, Iran) had been cultured in streptomycin (100 mg/ml), penicillin (100 µg/ml), and DMEM (Dulbecco’s modified Eagle’s medium) along with 10% FBS (Fetal bovine serum). To evaluate reactive oxygen species (ROS), (DCFDA) was bought from Sigma-Aldrich. In all processes, doubled distilled water was engaged. 2.2. Com seed extract preparation
10.0 g of Com seeds were washed by distilled water for removing dust. After that, they were appended to 100 ml of distilled water and mixture was stirred for 4 h at 60 ˚C. Then, the extract was separated from the mixture by filtration and stored in 4 ˚C. 2.3. Synthesis method of CN 30 ml of prepared Com seeds extract was mixed with 50 ml of distilled water and then 20 ml of an aqueous solution containing 5.0 g cerium nitrate hexa-hydrate was drop-wisely added to it. This solution was heated and stirred at 80 ˚C for 6 h. Next, due to remove water and attain gel, the solution was put into an oven (60 ˚C). Achieved yellow gel was divided to three portions and they were calcined for 2 h at 400, 500, and 600 ˚C with rate of 3 ˚C/min due to eliminate organic composites and produce yellow powders of CN. 2.4. Characterization Green synthesized CN samples were characterized by several laboratory device including D8Advance Bruker equipped with Cu Kα1 radiation (λ=0.15406 nm), Rayleigh-WQF-510A spectrometer, Cecil-CE9500 spectrophotometer, TESCAN MIRA3, Bahr STA 503, and Shimadzu spectrofluorometer for PXRD, FTIR, UV-Vis, FESEM, TGA/DTA, and PL analysis, respectively. Also, Epoch micro-plate spectrophotometer from BioTek and PerkinElmer multiplate reader were employed to read absorbance of the MTT and fluorescence emission of 2´, 7´ – dichlorofluorescin (DCF) dye. 2.5. Photo-catalytic processes The photo-catalytic experiments were accomplished using UV-A light with an intensity of 4.4 W/m2 provided by an 11 W fluorescence lamp with maximum wavelength at 365 nm. Irradiation without any filtration was directly glowed into the reaction mixture under air atmosphere at room
temperature. In all processes 100 mg of CN was added into 50 ml solution (4 ppm) of RhB in a 250 ml beaker and mixture was stirred vigorously. Before starting the irradiation, mixtures were stirred in the dark for 60 min due to complete absorption/desorption of dye on the CN surface. Also, all reactions were repeated three times due to investigate reproducibility. The absorbance of dye was recorded by UV–Vis spectrophotometer during the processes. 2.6. Determination of Cytotoxicity effects The cytotoxic effect of CN on cell viability of A549 cell line was assessed by MTT assay [25]. A549 cells were developed in DMEM medium supplemented with 10% FBS and 1% antibiotic. Cells were seeded on 96 well plate with density of 104 cell per well for 24h. After that, different concentrations of dispersed CN in distilled water were exposed to cells and another incubation time for 24h. Next, optimized amount of MTT solution (5 mg/ml) were added to each well and cells were incubated for 4h at 37˚C. Then, media was removed and formazan crystals were dissolved in 100 µl of fresh DMSO. The optical absorbance of each well was measured at 570 and 630 nm with a micro-plate reader. Due to certify the repeatability, each experiment was frequented triplicate and viability was expressed as a relative percentage mean ± SD versus to the untreated control cells. For the ROS assay, seeded A549 cells were treated by CN similar to MTT assay and were incubated at the same incubation time. Afterward, the medium was eliminated and each well was washed with 100 µl PBS. Next step, DCFDA with specific concentration of 10 mM was added to wells and after 24h of incubation and then fluorescence emission of DCF was read using a multi micro-plate reader.
3. Results and Discussions 3.1. Synthesis and characterization of CN CN was produced in Com seeds extract as capping and stabilizing agent through green route.
TGA/DTA analysis was performed to determine calcination temperature and evaluate thermal stability of product in range of 25-700 ˚C with rate of 3 ˚C per minute under air environment (Fig. 1). TGA curve shows three stages of weight loss which all of them are accompanied with endothermic peaks in DTA diagram. The first stage with 20% weight loss below 110 ˚C which attended by two endothermic peaks in the DTA curve, is related to the evaporation of water which physically absorbed on the pores and surface of CN [26]. Other two weight losses up to 45% in the range of 160 to 250 ˚C could be related to the removal of remaining organic compositions of Com seeds extract [27, 28]. These process cause to alteration of CeOY (Y=H, R) to pure ceria crystals at about 300 ˚C [29, 30]. Total weight loss in TGA curve is about 65%; also, there is no significance weight loss at higher temperatures which confirmed thermal stability of the product. In accordance with TGA/DTA and to ensure complete ignition of residual organic compounds in the product and investigate the effect of calcination temperature on the crystallite size and optical properties of CN, jelly samples were calcined at 400, 500 or 600 ˚C with rate of 3 ˚C per minute in an electric furnace and yellow powders were obtained. Fig. 2a illustrated UV-Vis spectra of CN colloidal solution. Maximum absorption for all samples was in range of 315 to 345 nm which is corresponded to charge transfer from O2P to Ce4f orbital [5, 31]. Band gap energy of CN samples was computed by Eq. 1 [32], ℎ
=
ℎ −
(1)
wherein, hν is the photon energy, α is the absorption coefficient, B is a constant, and n is 2 for semiconductor with direct transitions like ceria [33]. Band gap energy can be obtained from the junction spot of
ℎ
. ℎ plot slope on the X-axis (Fig. 2b). The λmax along with band gap
energy (Eg) are presented in Tab. 1. As it shown, CN-400 has lowest λmax because in direct
semiconductor decrease in particle size cause a blue shift in λmax [34, 35]; moreover, CN-400 has sharper peak than other which is related to the more uniform distribution of its particle size. FTIR spectra of green synthesized samples are showed in Fig. 3. All samples showed a sharp band about 400 cm-1 and weak band about 1050 cm-1 that are corresponded to stretching vibration of Ce─O and O─Ce─O bonds, separately [36]. Bands about 3400 cm-1 could be corresponded to OH group of adsorbed water on the CN surface. Weak bands about 1300 to 1500 cm-1 could be related to stretching vibration of C─H and C═O bonds of organic molecules, respectively, which are maybe remained from Com seeds extract [37]. XRD patterns of CN-400, 500, and 600 are illustrated in Fig. 4. All patterns were matched with single phase Cerianite (JCPDS No. 34-0394). Full width at half maximum (FWHM) was decreased and also peaks were sharpen with increasing the calcination temperature due to increasing the size and crystallinity of particles. Crystallite size of particles were calculated by Scherrer equation with using FWHM of (111), (200), (220), (311), (400), and (331) crystallographic planes [32]. Average crystallite size of particles was presented in Tab. 1. FESEM images were shown agglomerated shapeless particles for all samples (Fig. 5). As illustrated in Fig. 5, the size of CN-400 particle is smaller than CN-500 and CN-600; also increasing the calcination temperature resulted to the particle agglomeration. EDX analysis confirmed the attendance of O and Ce elements on the surface of all samples (Fig. 5). FESEM showed that CN-400 has smallest particle size with more uniform morphology among these samples which approved the outcomes of XRD and UV-Vis analysis. Fig. 6a demonstrates the PL spectra of dispersed CN-400, CN-500, and CN-600 in distilled water with an excitation wavelength of 200 nm. The bands in range of 420 to 520 nm are corresponded to the surface structural defects of CeO2 including oxygen vacancies [38, 39]. The
blue-green emission band at 470 nm had the maximum intensity among others which are blue bands at 440 and 450 nm, blue-green bands at 485 and 495 nm, and green band at 515 nm. Moreover, intensity of bands in CN-400 was greater than others which could be depended on morphology of particles, excitation wavelength, and density of oxygen vacancies [5, 38, 40]. Structural defects including surface oxygen vacancies were decreased by increasing the calcination temperature in presence of air and cause to reduce the intensity of PL peaks [10, 41]; therefore, surface defect density of CN-400 was higher than others. 3.2. Effect of oxygen vacancies on the photo-catalytic degradation strength The photo-catalytic experiments were done to evaluate the influence of calcination temperature on the photo-catalytic strength in dye degradation. 50 ml of the RhB colored solution (8 ppm) was degraded by 100 mg of CN-400, CN-500, and CN-600, respectively, which they were dispersed in RhB solution under UV-A irradiation with intensity of 4.4 W/m2. The amount of catalyst was determined according to optimum achieved from our recent study [32]. The solution was stirred vigorously and during process, absorption peak of RhB solution was monitored by UV-Vis spectrophotometer. All reactions were repeated in dark with the above conditions to ensure the rule of UV irradiation on degradation process. The results of Fig. 6b showed that CN400 could degrade approximately 92% of RhB in lower time in comparison to CN-500 and CN600. Moreover, to investigate the reproducibility of dye degradation results, all reactions were repeated three times (Fig. 6b). The results showed that photo-degradation of RhB dye in presence of CN under UV-A light was repeatable with accuracy more that 94%.
As a result,
increasing the calcination temperature had reverse influence on the photo-catalytic strength of the CN samples and this is in accordance with more surface oxygen vacancies in lower calcination temperature as mentioned above. Because, surface vacant oxygen sites can catch the
conducting electron released and decrease recombination of electron-hole pairs. Therefore, energized holes reacted with dye molecules and oxidized them faster [5, 42]. If oxygen defects have absorption edges in visible region, it causes to produce more electron-hole sites which should improve its photo-catalytic effect. But in this study, CN has not showed any photocatalytic effect in visible region, therefore it should be acted as an electron trapper which increased the energized holes. Meanwhile, according to the photo-degradation mechanism of RhB in the presence of CN, the holes (h+) had more effective role than •OH and O2•‾ [32]. The kinetic of photo-degradation process was obtained from Eq. 2 [43], −
=
(2)
Wherein, C and C0 are the dye concentration at time ‘t’ and ‘t=0’, separately, and k is the rate constant which it can be calculate from the plot of lnC/C0 versus irradiation time (Fig. 6c). This plots showed straight line which it confirmed the photo-degradation by CN follows a pseudo first-order reaction rate. The CN-400 has better activity with rate constant of 0.012 min-1 in comparison to CN-500 (0.007 min-1) and CN-600 (0.006 min-1). The percentage of photodegradation after 3 h for CN-400, CN-500, and CN-600 were demonstrated in Fig. 6d which among them, CN-400 showed the best efficiency (92%). Fig. 7 showed the photo-catalytic mechanism of RhB dye degradation in the presence of CN under UV-A irradiation. 3.3. Cytotoxicity study Based on above results, CN-400 had smallest particle size with better optical properties therefore it was selected for cytotoxic study. MTT assay was executed to evaluate in-vitro cell viability of A549 cell line in presence of different concentrations of CN-400 (1 to 500 µg/ml). Treated cells were incubated for 24 h, and then absorption of MTT was read by micro-plate
spectrophotometer. Fig. 8a showed the percentage of cell viability versus various concentrations of CN-400. The viability was further than 80% for density up to 250 µg/ml while it was 62% for 500 µg/ml. All concentrations of CN-400 were indicated cell viability more than 50%, therefore green synthesized CN-400 showed no significance toxicity on A549 cells. The ROS scavenger influence of CN-400 on the A549 cell line was evaluated by DCFDA assay. Cells were treated by CN-400 as it was done for MTT assay and the emission of DCF dye was recorded at 530 nm by a micro-plate reader. Fig. 8b illustrated the percentage of ROS scavenging versus different concentrations of CN-400. All concentration of CN-400 could neutralize ROS; moreover, 15.6 µg/ml of CN-400 had best antioxidant influence and could defuse about 76% of ROS formed by cellular metabolism. The antioxidant properties of CeO2 is due to electron transfer from Ce3+ to Ce4+ which it can reduce ROS [44]. The result of MTT and ROS assays confirmed that CN is a proper candidate for biological studies such as cell imaging or drug/gene delivery which were reported for CN [45, 46].
4. Conclusion The CN were produced via green route by using Com seeds extract as capping agent. Agglomerated uniform particles were obtained which their size were increased by enhancing the calcination temperature. According to the PL analysis, CN-400 showed higher emission intensity and therefore it may contained more oxygen vacancies in its structure. In addition, this low temperature calcined sample showed higher photo-catalytic activity in degradation of RhB under UV-A irradiation. Green synthesized CN-400 showed no significance toxicity on the A549 cell line; moreover, it showed worthy antioxidant effect which confirmed CN could be suitable candidate for biological studies.
Acknowledgements The authors gratefully acknowledge Shahrood University of Technology and Mashhad university of Medical Sciences for the technical and financial support of this research.
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Legends Fig. 1. The thermal gravimetric and differential thermal curves of gel sample Fig. 2. a) UV-Vis spectra of dispersed CN in distilled water, b) The graph of (αhν)2 vs. hν for the calculation the band gap energy of samples Fig. 3. The FTIR spectra of the achieved samples Fig. 4. The XRD patterns of calcined samples in 400, 500, and 600 ˚C Fig. 5. The FESEM images and the EDX spectrum of the samples Fig. 6. a) PL spectrums of samples, b) The comparison of dye degradation ability between CN400, CN-500, and CN-600 respect to time along with variations of RhB absorbance spectra as function of UV-A light irradiation time, c) The plot of ln C/C0 vs. irradiation time for CN400, CN-500, and CN-600, d) The photo-degradation efficiencies of all samples after 180 min. Fig. 7. Photo-catalyst mechanism of RhB dye degradation in presence of CN under UV-A light irradiation Fig. 8. a) A549 cell viability in attendance of CN-400, b) The ability of CN-400 in scavenging ROS Tab. 1. Temperature of calcination, photo-catalytic degradation rate constant, optical properties, and particle size of the CN samples
Fig.1. M. Darroudi et al., 2019
Fig.2. M. Darroudi et al., 2019
Fig.3. M. Darroudi et al., 2019
Fig.4. M. Darroudi et al., 2019
Fig.5. M. Darroudi et al., 2019
Fig. 6. M. Darroudi et al., 2019
Fig.7. M. Darroudi et al., 2019
Fig.8. M. Darroudi et al., 2019
Tab. 1. M. Darroudi et al., 2019 Sample
Calcination temperature (˚C)
CN-400
400
Degradation rate constant (min-1) a 0.012
CN-500
500
CN-600
600
λmax (nm)
Eg (eV) b
Crystallite size (nm) c
315
3.1
9.0
0.007
323
3.0
9.5
0.006
342
2.4
11.8
a
Computed from the plot of ln C/C0 vs. time
b
Computed according to Eq. 1 from UV-Vis spectra
c
Computed corresponding to the Scherrer equation
Research Highlights ►Green
synthesis of Ceria nanoparticles was carried out in Cydonia oblonga miller
seeds extract as capping agent. ►Ceria nanoparticles calcined in lower temperature had more oxygen vacancies in their structure and shown higher intensity in photoluminescence emission
►The more amounts of oxygen
vacancies caused to enhance the photo-catalytic strength in dyes degradation ► Ceria nanoparticles has not shown toxic effect on cell viability and could remarkably scavenge the ROS produced by cell metabolism.