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Determination of reactive oxygen species from ZnO micro-nano structures with shape-dependent photocatalytic activity
Materials Research Bulletin 53 (2014) 246–250
Contents lists available at ScienceDirect
Materials Research Bulletin journal homepage: www.elsevier.com/locate/matresbu
Determination of reactive oxygen species from ZnO micro-nano structures with shape-dependent photocatalytic activity Weiwei He a , Hongxiao Zhao a , Huimin Jia a , Jun-Jie Yin b , Zhi Zheng a, * a Key Laboratory of Micro-Nano Materials for Energy Storage and Conversion of Henan Province, Institute of Surface Micro and Nano Materials, Xuchang University, Henan 461000, PR China b Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 September 2013 Received in revised form 28 January 2014 Accepted 20 February 2014 Available online 23 February 2014
ZnO micro/nano structures with different morphologies have been prepared by the changing solvents used during their synthesis by solvothermal reaction. Three typical shapes of ZnO structures including hexagonal, bell bottom like and multi-pod formed and were characterized by scanning electron microscopy and X-ray diffraction. Multi pod like ZnO structures exhibited the highest photocatalytic activity toward degradation of methyl orange. Using electron spin resonance spectroscopy coupled with spin trapping techniques, we demonstrate an effective way to identify precisely the generation of hydroxyl radicals, superoxide and singlet oxygen from the irradiated ZnO multi pod structures. The type of reactive oxygen species formed was predictable from the band gap structure of ZnO. These results indicate that the shape of micro-nano structures significantly affects the photocatalytic activity of ZnO, and demonstrate the value of electron spin resonance spectroscopy for characterizing the type of reactive oxygen species formed during photoexcitation of semiconductors. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Semiconductors Microstructure Electron spin resonance Catalytic properties
1. Introduction ZnO micro/nano structures have received a great deal of interest because of their current and potential importance in diverse applications [1], for example, in photocatalysis [2], optoelectronic nanodevices [3], gas sensors [4], and antimicrobial agents [5]. Owing to their superior ultraviolet light response, ZnO nanoparticles (NP) has also been often incorporated into sunscreens and cosmetics [6]. In the past couple of decades, many efforts have been made to improve the photocatalytic activity of ZnO. Synthesis of the ZnO micro-nano structures with various morphologies, including rod-like, tube-like, wire-like and flower-like structures [7–10], have been extensively studied to optimize photocatalytic activity. The photocatalytic activity of ZnO has been greatly enhanced by ion doping [11], dye-sensitization [12], and combining ZnO with metal [13,14] or graphene nanostructures [15,16] to form hybrid nanostructures. Understanding the mechanism of photocatalytic activity is essential for optimizing the performance of ZnO in photocatalysis and related applications (e.g., antibacterial activity). It is well established that reactive oxygen species (ROS) and photogenerated charge carriers are essential intermediates leading to the photocatalytic and antibacterial of semiconductors
* Corresponding author. Tel.: +86 374 2968783; fax: +86 374 2968783. E-mail address: [email protected] (Z. Zheng). http://dx.doi.org/10.1016/j.materresbull.2014.02.020 0025-5408/ ã 2014 Elsevier Ltd. All rights reserved.
[17–20]. ROS, including hydroxyl radicals, superoxide and singlet oxygen, are extremely reactive and can cause oxidative damage to surrounding molecules or species [21]. Singlet oxygen can cause oxidation and damage of biomembranes and tissues. Superoxide, though not as strong oxidant as hydroxyl radicals, is also biologically quite toxic and has been shown to denature enzymes, oxidize lipids and fragment DNA. Although a number of investigators have examined generation of ROS during photoexcitation of ZnO and other metal oxide NP, few studies have definitively identified and distinguished the type of ROS formed during photoexcitation of metal oxide NPs. In most available studies, ROS have been detected by indirect spectroscopic methods [22,23]. Unfortunately, definitive identification of ROS with these methods is difficult since ROS are short-lived and always produced simultaneously. Electron spin resonance spectroscopy (ESR) with spin trapping techniques is the most reliable and powerful tool for identification and quantification of short-lived free radicals and molecules with unpaired electrons [24]. We have employed ESR to investigate the generation of reactive oxygen species induced by Ag and Au NPs in biologically relevant systems [25,26]. In this paper, ZnO micro/ nano materials are considered because they are widely used in commercial products. Our objective is to use ESR with spin trapping technique to identify the type of ROS generated during irradiation of ZnO structures, and elucidate the mechanism for formation of ROS. Also, we developed a method to tune the
W. He et al. / Materials Research Bulletin 53 (2014) 246–250
morphology of ZnO micro-nano structures by changing solvents, and investigated their shape-dependent photocatalytic activity.
247
the effects of 10% DMSO, 1 U/ml SOD and 10 mM NaN3 were investigated as typical scavengers for hydroxyl radical, superoxide and singlet oxygen, respectively.
2. Experimental 3. Results and discussion 2.1. Materials and chemicals Zinc nitrate hexahydrate (Zn(NO3)26H2O), hexamethylenetetramine (HMTA), dimethylformamide (DMF), hexadecyl trimethyl ammonium bromide (CTAB), methyl orange, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMP), superoxide dismutase (SOD), NaN3, and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO). The spin-trap, 5-tertbutoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO), was purchased from Applied Bioanalytical Labs (Sarasota, FL). Milli-Q water (18 MV cm) was used for preparation of all solutions. 2.2. Synthesis of ZnO micro-nano structures In a typical synthesis of ZnO micro-nano structures, 2.5 mM zinc nitrate and 2.5 mM HMTA with molar ratio of 1:1 were dissolved in 15 mL of deionized water. Then the mixed solution was transferred into a 20 ml teflon-lined stainless steel autoclave. The autoclave was heated to 140 C for 12 h and then allowed to cool to room temperature. Similarly, dimethylformamide (DMF) and 10 mM hexadecyl trimethyl ammonium bromide (CTAB) were used to obtain different shaped ZnO structures. Finally, the white precipitates were centrifuged and washed with water and ethanol three times and dried under vacuum at room temperature. 2.3. Characterization The as-synthesized products were characterized by X-ray diffraction (Bruker D8 Advance diffractometer) with monochromatized Cu Ka radiation (l = 1.5418 Å). Scanning electron microscopy (Zeiss EVO LS-15) was used to further characterize the morphology of ZnO structures. A Micromeritics Gemini 2380 specific area analyzer was used to measure the BET surface areas of different shaped ZnO micro-nano structures by measuring nitrogen adsorption. The photocatalytic activities of different ZnO products were evaluated by measuring the degradation of methyl orange in an aqueous solution irradiated with 500 W Hg lamp at 365 nm. Typically, 20 mg ZnO photocatalyst was suspended in a 40 ml of aqueous solution of 20 mg/l methyl orange. The solution was continuously stirred for about 1 h to ensure the establishment of an adsorption–desorption equilibrium among the catalysts, methyl orange, and water before irradiation. The residual concentration of the organic dye was monitored by PerkinElmer Lambda 35 UV–vis Spectrometer.
Fig. 1 displays the typical SEM images of ZnO structures obtained hydrothermally in water, hexadecyl trimethyl ammonium bromide (CTAB) and dimethylformamide (DMF). As noted, three distinct shapes were formed. When synthesized in pure water, ZnO showed a bell bottom-like shape with average length 4–5 mm and was well dispersed (Fig. 1a). Hexagonal rod structures with average diameter of 0.5–1.0 mm and average length of 3–4 mm were obtained (Fig. 1b) when ZnO was synthesized in 10 mM CTAB. When DMF was used as solvent instead of water or CTAB, we observed a multi-pod structured ZnO having a smaller size (average diameter of 50 nm and average length 300–400 nm). Fig. 1d is a schematic illustration for the formation of ZnO structures with bell bottom-like, hexagonal rod and multi-pod shapes using the solvents water, CTAB and DMF, respectively. These results indicate that we can control the morphologies of ZnO micro-nano structures easily by changing the hydrothermal solvent. However, the mechanism for the effects of the solvent on the shape of ZnO requires further investigation. The purity and crystalline phase of the as-synthesized ZnO structures were characterized by X-ray diffraction (XRD) (Fig. 2). All the peaks can be clearly indexed to a hexagonal phase of wurtzite structure with lattice constants of a = 0.322 nm and c = 0.521 nm (JCPDS card No. 75–1526). The observed peaks could be assigned to diffraction from the (1 0 0), (1 0 1), (0 0 2), (11 0), (1 0 2) and (1 0 3) faces. No characteristic peaks from impurities are detected, indicating their excellent crystalline character. Among these three shapes of ZnO structures, bell bottom-like and hexagonal microrod have similar diffraction spectra (Fig. 2a and b), in which (1 0 0) plane shows the dominant diffraction signal, indicating that the growth is along the c-axis. Therefore, a rod-like shape formed. The XRD pattern from multi-pod ZnO nanostructures is quite different from the XRD patter of the other forms of ZnO synthesized. For the
2.4. Electron spin resonance spectroscopy All electron spin resonance measurements were carried out using a Bruker EMX electron spin resonance spectrometer (Billerica, MA) at ambient temperature. A light system consisting of a Xenon lamp coupled with a WG320 filter was used to generate light having wavelengths above 350 nm. Fifty microliter aliquots of control or sample solutions were put in quartz capillary tubes with internal diameters of 1 mm and sealed. The capillary tubes were inserted into the ESR cavity, and the spectra recorded at selected times. Other settings were as follows: 1 G field modulation, 100 G scan range, and 20 mW microwave power for detection of spin adducts using spin traps. In the detection of different reactive oxygen species, 25 mM BMPO was used as spin trap for hydroxyl radicals and superoxide, 2 mM 4-oxo-TEMP was chosen for detection of singlet oxygen. To confirm the identity of the ROS,
Fig. 1. SEM images of ZnO micro-nano structures prepared in (a) water, (b) 10 mM CTAB and (c) DMF with Zn2+/HMTA ratio of 1:1. Synthesis was done at 140 C for 12 h. (d) Schematic illustration for the formation of ZnO structures in different solvents.
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Fig. 2. XRD patterns of ZnO structures obtained in water (a), 10 mM CTAB (b) and DMF (c) corresponding to samples in Fig. 1.
multi-pod structure, the peak intensity of (1 0 1), (1 0 0) and (0 0 1) followed an order: (1 0 1) > (1 0 0) > (0 0 1), indicating crystal faces (1 0 1) and (1 0 0) had become dominant in the exposed facets of ZnO multi-pod. These results demonstrate that the solvent plays a critical role in crystal growth of ZnO in hydrothermal reactions. The photocatalytic activity of ZnO structures and the effects of the morphology on photocatalytic activity were examined by the degradation of methyl orange (MO) irradiated with a 365 nm Hg lamp (Fig. 3). MO itself is resistant to degradation in the absence of a photocatalyst. When ZnO structures were added to the MO solution, a considerable reduction in the absorbance of MO was detected for all three ZnO samples during irradiation with 365 nm light (Fig. 3a). Fig. 3b displays the evolution of the UV–vis Spectra for MO during irradiation in the presence of multi-pod ZnO. The ZnO structures with different shapes showed clear differences in their ability to photocatalytically degrade the MO (Fig. 3a). We observed the following order in photocatalytic activity: multipod > bell bottom > hexagonal microrod. After irradiation for 20 min, 10%, 31% and 44% MO were calculated to be degraded by hexagonal microrod, bell bottom like microrod and multi-pod ZnO,
respectively. ZnO multi-pod exhibited about four times higher catalytic activity than hexagonal microrod. These results demonstrate that the shape of ZnO structures plays a significant role in determining their photocatalytic activity. There are many factors that can affect the photocatalytic activity of a semiconductor, such as crystal structure, specific surface area, surface coating and chemical composition. In our work, these 3 ZnO structures have same crystalline phase and no additional surface chemical coating, therefore, the specific surface area and exposed crystal faces may be the main reasons for the observed differences in photocatalytic activity. The specific surface area was determined to be 44.8 1.2, 10.6 0.8 and 33.6 2.8 m2/g for bell bottom-like, hexagonal and multi-pod ZnO structures, respectively. These results are consistent with the observation that bell bottom and multi-pod ZnO showed higher catalytic activity than hexagonal rod, but cannot explain the order between multi-pod and bell bottom ZnO. Crystal face has been proposed to contribute to photocatalytic activity. As reported in [27], strongly face-dependent behavior was observed. The ZnO (1 0 1) surface showed the highest photocatalytic activity toward degradation of MO compared with (0 0 1) and (1 0 0) faces. The multi-pod structures have branches exposed with much larger (1 0 1) face than bottom bell ZnO, this may lead to the higher photocatalytic activity of multi-pod ZnO. Taken together, our results indicate that crystal face and specific surface area largely determined the observed shape-dependent photocatalytic activity of ZnO. To test the photocatalytic stability of ZnO, ZnO multi-pod, previously used as a photocatalyst, was collected by centrifugation and washed with water. The photocatalytic activity of this reclaimed ZnO was then examined. A slight decrease in photocatalytic activity was observed when reclaimed ZnO was used (Fig. 3c). It is well established that reactive oxygen species (ROS) play a central role in the photocatalytic activity of ZnO. Therefore, it is necessary to identify the ROS generated during photoexcitation of ZnO to understand the photocatalytic mechanism of ZnO. We developed a method based on ESR spectroscopy to definitively detect the ROS generated during irradiation of ZnO. In ESR, spin trapping is the most often used technique for detection of short lived free radicals and paramagnetic species. By appropriate selection of spin traps, it is possible to definitely identify ROS. In this work, BMPO was chosen as spin trap for capturing hydroxyl radicals and superoxide, and 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMP) was chosen for detection of singlet oxygen. We used multi-pod ZnO,
Fig. 3. (A) The photocatalytic activity toward degradation of methyl orange in the absence of any catalyst (control) and in the presence of 0.5 mg/ml ZnO structures with hexagonal-like (1#), bell bottom-like (2#) and multi-pod (3#) shapes; (B) Evolution of the absorption spectra over time for MO degradation photocatalyzed by multi-pod ZnO. The dashed navy line represents the original spectrum of MO; (C) Cycling curve for photocatalytic degradation of methyl orange using multi-pod ZnO.
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Fig. 4. Reactive oxygen species detection from irradiated ZnO multi-pod structures using BMPO (A) or 4-oxo-TEMP (B) as spin traps. ESR spectra were obtained from samples containing different spin probes (25 mM BMPO, or 2 mM 4-oxo-TEMP) and 0.1 mg/ml multi-pod like ZnO products before and after irradiating for 3 min with light having wavelengths above 350 nm. The control represents the sample containing spin probe alone under irradiation, or the sample containing spin probe and catalysts before exposure to light.
which had the highest photocatalytic activity among the synthesized forms of ZnO, to study the formation of ROS using ESR. Fig. 4 displays the ESR spectrum obtained using solutions containing BMPO or 4-oxo-TEMP and multi-pod ZnO before and during irradiation with light having wavelengths above 350 nm. For control samples containing only spin trap, or spin trap mixed with ZnO but without irradiation (Fig. 4), no ESR signal was observed in the cases of BMPO and 4-oxo-TEMP. Upon irradiation in the presence of ZnO, we observed a four-line spectrum with relative intensities of 1:2:2:1 and hyperfine splitting parameters of aN = 13.56, abH = 12.30, agH = 0.66 when BMPO was used as spin trap (Fig. 4A). This ESR spectrum is characteristic for the spin adduct between BMPO and the hydroxyl radical (BMPO/OH) [28]. This indicates that hydroxyl radical is generated by ZnO during irradiation. BMPO is also a spin trap frequently used for superoxide, but no characteristic ESR signal for the adduct BMPO/OOH appeared. This may be due to the spectra overlapping of BMPO/OH and BMPO/OOH. To elucidate the signal from BMPO, we investigated the effects of DMSO and superoxide dismutase (SOD) on the ESR signal. DMSO is the typically used scavenger for hydroxyl radical and SOD is a specific catalyst for the removal of superoxide. After addition of 10% DMSO, the ESR signal was significantly reduced. The remaining ESR signal can be assigned to a carbon center radical, associated with quenching of the hydroxyl radical by DMSO. These results further support the generation of OH. The addition of 1 U/ml SOD also partially suppressed the ESR signal, indicating the production of superoxide. These results demonstrate that both hydroxyl radical and superoxide are generated during irradiation of ZnO. 4-oxo-TEMP, a typical spin probe for singlet oxygen, has been widely used for detection of singlet oxygen [29]. In contrast with results obtained for the control in Fig. 4B, the characteristic ESR signal for the reaction between 4-oxo-TEMP and singlet oxygen was observed during the irradiation of ZnO. This observation directly verifies the production of singlet oxygen. Also, the addition of 10 mM NaN3, a singlet oxygen quencher, caused a substantial decrease in the observed ESR signal, which further supports that singlet oxygen is produced during photoexcitation of ZnO. It is noteworthy that SOD also a caused significant reduction in the ESR
signal when 4-oxo-TEMP was used (Fig. 4B). 4-oxo-TEMP is a specific probe to detect singlet oxygen. Superoxide and hydroxyl radicals cannot react with 4-oxo-TEMP to produce an ESR signal. In addition, SOD has no interaction with singlet oxygen. Therefore, these results indicate that superoxide must be involved in the generation of singlet oxygen. What determines which ROS are generated during photoexcitation of ZnO? When the semiconductors (e.g., ZnO) absorb light, electrons in the valence band are excited across the band gap to the conduction band with concomitant creation of holes in valence band. The electrons in conduction band and holes in valence band have strong reducing and oxidizing ability, respectively. Principally, the band gap structure determines the fate and power of the generated electrons/holes in semiconductor, and determines the type of ROS generated (Fig. 5). The redox potentials (E ) for H2O/OH, O2/O2 , and 1O2/O2 are 0.16, 2.32 and 1.88 V with respect to NHE at natural pH, respectively [30,31]. ZnO, with band gap (Eg = 3.2 eV), has a conduction band edge with redox potential of 0.31 V (Ec) and a valence band edge with redox potential of
Fig. 5. Proposed mechanisms for generation of ROS and photocatalytic reaction on the surface of a photoexcited ZnO particle.
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2.89 V (Ev) [32]. Comparing the Ec, Ev of ZnO and E of ROS, it is favorable thermodynamically to generate hydroxyl radical, superoxide and singlet oxygen from irradiated ZnO. The excited electrons in conduction band, having Ec ( 0.31 V) less than the E of O2/O2 , can drive the formation of superoxide. The holes in valence band (Ev = 2.89 V), enable photoexcited ZnO to oxidize H2O to form hydroxyl radicals. These predictions for superoxide and hydroxyl radical generation are consistent with our observations from ESR experiments. Although the holes in valence band from irradiated ZnO have the ability to directly generate singlet oxygen from oxygen, our ESR experimental results suggest that superoxide radicals play a major role in generating singlet oxygen. This mechanism is consistent with previous reports about the singlet oxygen formation as a result of the electron transfer reaction between superoxide and holes [29,33]. As we demonstrated using ESR with spin trapping technique, ROS including hydroxyl radicals, superoxide and singlet oxygen were produced during photoexcitation of ZnO multi-pod structures (Fig. 5). Because of their highly chemical reactivity, these ROS may contribute dominantly to the catalytic activity of ZnO micro-nano structures. 4. Conclusions In summary, micro/nano scale ZnO structures with different morphologies were prepared by changing solvents in solvothermal conditions. The ZnO micro-nano structures exhibited morphologydependent photocatalytic activity. Using ESR with spin trapping techniques, we detected separately and identified the ROS, including hydroxyl radicals, superoxide and singlet oxygen, generated during irradiation of ZnO. These reactive intermediates are proposed to be responsible for the photocatalytic behavior of ZnO micro-nano structures. The formation of ROS was determined by the band gap structures of ZnO, which also can predict the type of ROS generated in other semiconductors. These results may provide direct evidence and effective tools for understanding the photocatalytic mechanism of semiconductors. Acknowledgements This work was financially supported by National Natural Science Foundation of China (Grant No. 21303153, 61204009, 21273192) and Research Project of Basic and Advanced Technology of Henan Province (Grant No. 112300410106). This article is not an official US Food and Drug Administration (FDA) guidance or policy statement. No official support or endorsement by the US FDA is
intended or should be inferred. The authors appreciate Wayne G. Wamer from FDA for his scientific editing and discussions. References [1] A.B. Djuriši c, X.Y. Chen, Y.H. Leung, A.M.C. Ng, Journal of Materials Chemistry 22 (2012) 6526–6535. [2] C. Tian, Q. Zhang, A. Wu, M. Jiang, Z. Liang, B. Jiang, H. Fu, Chemical Communications 48 (2012) 2858–2860. [3] U. Ozgur, D. Hofstetter, H. Morkoc, Proceedings of the IEEE 98 (2010) 1255–1268. [4] S.T. Shishiyanu, T.S. Shishiyanu, O.I. Lupan, Sensors and Actuators B 107 (2005) 379–386. [5] Y.P. Xie, Y.P. He, P.L. Irwin, T. Jin, X. Shi, Applied and Environmental Microbiology 77 (2011) 2325–2331. [6] J.W. Wiechers, N. Musee, Journal of Biomedical Nanotechnology 6 (2010) 408–431. [7] Z. Wang, X. Qian, J. Yin, Z. Zhu, Langmuir 20 (2004) 3441. [8] Y. Lv, C. Li, L. Guo, Q. Wang, R. Wang, H. Xu, S. Yang, X. Ai, J. Zhang, Applied Physics Letters 87 (2005) 163103. [9] S. Bu, C. Cui, Q. Wang, L. Bai, Journal of Nanomaterials 2008 (2008) 610541. [10] M. Mo, J.C. Yu, L. Zhang, S.K.A. Li, Advanced Materials 17 (2005) 756–760. [11] X. Qiu, G. Li, X. Sun, L. Li, X. Fu, Nanotechnology 19 (2008) 215703. [12] S. Sarkar, A. Makhal, T. Bora, K. Lakhsman, A. Singha, J. Dutta, S.K. Pal, ACS Applied Materials & Interfaces 4 (2012) 7027–7035. [13] M.D.L.R. Peralta, U. Pal, R.S. Zeferino, ACS Applied Materials & Interfaces 4 (2012) 4807–4816. [14] N. Zhang, S. Liu, Y.J. Xu, Nanoscale 4 (2012) 2227–2238. [15] Z. Chen, N. Zhang, Y.J. Xu, CrystEngComm 15 (2013) 3022–3030. [16] Y. Zhang, Z. Chen, S. Liu, Y.J. Xu, Applied Catalysis B 140 (2013) 598–607. [17] Y. Zhang, Z.R. Tang, X. Fu, Y.J. Xu, ACS Nano 4 (2010) 7303–7314. [18] N. Zhang, Y. Zhang, Y.J. Xu, Nanoscale 4 (2012) 5792–5813. [19] M.R. Hoffmann, S.T. Martin, W. Choi, D.W. Bahnemann, Chemical Reviews 95 (1995) 69–96. [20] M.P. Brynildsen, J.A. Winkler, C.S. Spina, I.C. MacDonald, J.J. Collins, Nature Biotechnology 31 (2013) 160–165. [21] H.F. Lin, S.C. Liao, S.W. Hung, Journal of Photochemistry and Photobiology A: Chemistry 174 (2005) 82–87. [22] D. Guo, H. Bi, B. Liu, Q. Wu, D. Wang, Y. Cui, Toxicology in Vitro 27 (2013) 731–738. [23] M.K.K. Oo, Y. Yang, Y. Hu, M. Gomez, H. Du, H. Wang, ACS Nano 6 (2012) 1939–1947. [24] C.D. Jaeger, A.J. Brad, Journal of Physical Chemistry 83 (1979) 3146–3152. [25] W.W. He, Y.T. Zhou, W.G. Wamer, M.D. Boudreau, J.J. Yin, Biomaterials 33 (2012) 7547–7555. [26] W.W. He, Y.T. Zhou, W.G. Wamer, X. Hu, X. Wu, Z. Zheng, M.D. Boudreau, J.J. Yin, Biomaterials 34 (2013) 765–773. [27] N. Kislov, J. Lahiri, H. Verma, D.Y. Goswami, E. Stefanakos, M. Batzill, Langmuir 25 (2009) 3310. [28] H.T. Zhao, J. Joseph, H. Zhang, H. Karoui, B. Kalyanaraman, Free Radical Biology & Medicine 3 (2001) 599–606. [29] J.J. Yin, J. Liu, M. Ehrenshaft, J.E. Roberts, P.P. Fu, R.P. Mason, B. Zhao, Toxicology and Applied Pharmacology 263 (2012) 81–88. [30] P.M. Wood, Biochemical Journal 453 (1988) 287–289. [31] Y. Li, W. Zhang, J. Niu, Y. Chen, ACS Nano 6 (2012) 5164–5173. [32] Y. Xu, M.A.A. Schoonen, American Mineralogist 85 (2000) 543–556. [33] E.A. Mayeda, A.J. Bard, Journal of the American Chemical Society 95 (1973) 6223–6226.
Contents lists available at ScienceDirect
Materials Research Bulletin journal homepage: www.elsevier.com/locate/matresbu
Determination of reactive oxygen species from ZnO micro-nano structures with shape-dependent photocatalytic activity Weiwei He a , Hongxiao Zhao a , Huimin Jia a , Jun-Jie Yin b , Zhi Zheng a, * a Key Laboratory of Micro-Nano Materials for Energy Storage and Conversion of Henan Province, Institute of Surface Micro and Nano Materials, Xuchang University, Henan 461000, PR China b Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, College Park, MD 20740, USA
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 September 2013 Received in revised form 28 January 2014 Accepted 20 February 2014 Available online 23 February 2014
ZnO micro/nano structures with different morphologies have been prepared by the changing solvents used during their synthesis by solvothermal reaction. Three typical shapes of ZnO structures including hexagonal, bell bottom like and multi-pod formed and were characterized by scanning electron microscopy and X-ray diffraction. Multi pod like ZnO structures exhibited the highest photocatalytic activity toward degradation of methyl orange. Using electron spin resonance spectroscopy coupled with spin trapping techniques, we demonstrate an effective way to identify precisely the generation of hydroxyl radicals, superoxide and singlet oxygen from the irradiated ZnO multi pod structures. The type of reactive oxygen species formed was predictable from the band gap structure of ZnO. These results indicate that the shape of micro-nano structures significantly affects the photocatalytic activity of ZnO, and demonstrate the value of electron spin resonance spectroscopy for characterizing the type of reactive oxygen species formed during photoexcitation of semiconductors. ã 2014 Elsevier Ltd. All rights reserved.
Keywords: Semiconductors Microstructure Electron spin resonance Catalytic properties
1. Introduction ZnO micro/nano structures have received a great deal of interest because of their current and potential importance in diverse applications [1], for example, in photocatalysis [2], optoelectronic nanodevices [3], gas sensors [4], and antimicrobial agents [5]. Owing to their superior ultraviolet light response, ZnO nanoparticles (NP) has also been often incorporated into sunscreens and cosmetics [6]. In the past couple of decades, many efforts have been made to improve the photocatalytic activity of ZnO. Synthesis of the ZnO micro-nano structures with various morphologies, including rod-like, tube-like, wire-like and flower-like structures [7–10], have been extensively studied to optimize photocatalytic activity. The photocatalytic activity of ZnO has been greatly enhanced by ion doping [11], dye-sensitization [12], and combining ZnO with metal [13,14] or graphene nanostructures [15,16] to form hybrid nanostructures. Understanding the mechanism of photocatalytic activity is essential for optimizing the performance of ZnO in photocatalysis and related applications (e.g., antibacterial activity). It is well established that reactive oxygen species (ROS) and photogenerated charge carriers are essential intermediates leading to the photocatalytic and antibacterial of semiconductors
* Corresponding author. Tel.: +86 374 2968783; fax: +86 374 2968783. E-mail address: [email protected] (Z. Zheng). http://dx.doi.org/10.1016/j.materresbull.2014.02.020 0025-5408/ ã 2014 Elsevier Ltd. All rights reserved.
[17–20]. ROS, including hydroxyl radicals, superoxide and singlet oxygen, are extremely reactive and can cause oxidative damage to surrounding molecules or species [21]. Singlet oxygen can cause oxidation and damage of biomembranes and tissues. Superoxide, though not as strong oxidant as hydroxyl radicals, is also biologically quite toxic and has been shown to denature enzymes, oxidize lipids and fragment DNA. Although a number of investigators have examined generation of ROS during photoexcitation of ZnO and other metal oxide NP, few studies have definitively identified and distinguished the type of ROS formed during photoexcitation of metal oxide NPs. In most available studies, ROS have been detected by indirect spectroscopic methods [22,23]. Unfortunately, definitive identification of ROS with these methods is difficult since ROS are short-lived and always produced simultaneously. Electron spin resonance spectroscopy (ESR) with spin trapping techniques is the most reliable and powerful tool for identification and quantification of short-lived free radicals and molecules with unpaired electrons [24]. We have employed ESR to investigate the generation of reactive oxygen species induced by Ag and Au NPs in biologically relevant systems [25,26]. In this paper, ZnO micro/ nano materials are considered because they are widely used in commercial products. Our objective is to use ESR with spin trapping technique to identify the type of ROS generated during irradiation of ZnO structures, and elucidate the mechanism for formation of ROS. Also, we developed a method to tune the
W. He et al. / Materials Research Bulletin 53 (2014) 246–250
morphology of ZnO micro-nano structures by changing solvents, and investigated their shape-dependent photocatalytic activity.
247
the effects of 10% DMSO, 1 U/ml SOD and 10 mM NaN3 were investigated as typical scavengers for hydroxyl radical, superoxide and singlet oxygen, respectively.
2. Experimental 3. Results and discussion 2.1. Materials and chemicals Zinc nitrate hexahydrate (Zn(NO3)26H2O), hexamethylenetetramine (HMTA), dimethylformamide (DMF), hexadecyl trimethyl ammonium bromide (CTAB), methyl orange, 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMP), superoxide dismutase (SOD), NaN3, and dimethyl sulfoxide (DMSO) were purchased from Sigma–Aldrich (St. Louis, MO). The spin-trap, 5-tertbutoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO), was purchased from Applied Bioanalytical Labs (Sarasota, FL). Milli-Q water (18 MV cm) was used for preparation of all solutions. 2.2. Synthesis of ZnO micro-nano structures In a typical synthesis of ZnO micro-nano structures, 2.5 mM zinc nitrate and 2.5 mM HMTA with molar ratio of 1:1 were dissolved in 15 mL of deionized water. Then the mixed solution was transferred into a 20 ml teflon-lined stainless steel autoclave. The autoclave was heated to 140 C for 12 h and then allowed to cool to room temperature. Similarly, dimethylformamide (DMF) and 10 mM hexadecyl trimethyl ammonium bromide (CTAB) were used to obtain different shaped ZnO structures. Finally, the white precipitates were centrifuged and washed with water and ethanol three times and dried under vacuum at room temperature. 2.3. Characterization The as-synthesized products were characterized by X-ray diffraction (Bruker D8 Advance diffractometer) with monochromatized Cu Ka radiation (l = 1.5418 Å). Scanning electron microscopy (Zeiss EVO LS-15) was used to further characterize the morphology of ZnO structures. A Micromeritics Gemini 2380 specific area analyzer was used to measure the BET surface areas of different shaped ZnO micro-nano structures by measuring nitrogen adsorption. The photocatalytic activities of different ZnO products were evaluated by measuring the degradation of methyl orange in an aqueous solution irradiated with 500 W Hg lamp at 365 nm. Typically, 20 mg ZnO photocatalyst was suspended in a 40 ml of aqueous solution of 20 mg/l methyl orange. The solution was continuously stirred for about 1 h to ensure the establishment of an adsorption–desorption equilibrium among the catalysts, methyl orange, and water before irradiation. The residual concentration of the organic dye was monitored by PerkinElmer Lambda 35 UV–vis Spectrometer.
Fig. 1 displays the typical SEM images of ZnO structures obtained hydrothermally in water, hexadecyl trimethyl ammonium bromide (CTAB) and dimethylformamide (DMF). As noted, three distinct shapes were formed. When synthesized in pure water, ZnO showed a bell bottom-like shape with average length 4–5 mm and was well dispersed (Fig. 1a). Hexagonal rod structures with average diameter of 0.5–1.0 mm and average length of 3–4 mm were obtained (Fig. 1b) when ZnO was synthesized in 10 mM CTAB. When DMF was used as solvent instead of water or CTAB, we observed a multi-pod structured ZnO having a smaller size (average diameter of 50 nm and average length 300–400 nm). Fig. 1d is a schematic illustration for the formation of ZnO structures with bell bottom-like, hexagonal rod and multi-pod shapes using the solvents water, CTAB and DMF, respectively. These results indicate that we can control the morphologies of ZnO micro-nano structures easily by changing the hydrothermal solvent. However, the mechanism for the effects of the solvent on the shape of ZnO requires further investigation. The purity and crystalline phase of the as-synthesized ZnO structures were characterized by X-ray diffraction (XRD) (Fig. 2). All the peaks can be clearly indexed to a hexagonal phase of wurtzite structure with lattice constants of a = 0.322 nm and c = 0.521 nm (JCPDS card No. 75–1526). The observed peaks could be assigned to diffraction from the (1 0 0), (1 0 1), (0 0 2), (11 0), (1 0 2) and (1 0 3) faces. No characteristic peaks from impurities are detected, indicating their excellent crystalline character. Among these three shapes of ZnO structures, bell bottom-like and hexagonal microrod have similar diffraction spectra (Fig. 2a and b), in which (1 0 0) plane shows the dominant diffraction signal, indicating that the growth is along the c-axis. Therefore, a rod-like shape formed. The XRD pattern from multi-pod ZnO nanostructures is quite different from the XRD patter of the other forms of ZnO synthesized. For the
2.4. Electron spin resonance spectroscopy All electron spin resonance measurements were carried out using a Bruker EMX electron spin resonance spectrometer (Billerica, MA) at ambient temperature. A light system consisting of a Xenon lamp coupled with a WG320 filter was used to generate light having wavelengths above 350 nm. Fifty microliter aliquots of control or sample solutions were put in quartz capillary tubes with internal diameters of 1 mm and sealed. The capillary tubes were inserted into the ESR cavity, and the spectra recorded at selected times. Other settings were as follows: 1 G field modulation, 100 G scan range, and 20 mW microwave power for detection of spin adducts using spin traps. In the detection of different reactive oxygen species, 25 mM BMPO was used as spin trap for hydroxyl radicals and superoxide, 2 mM 4-oxo-TEMP was chosen for detection of singlet oxygen. To confirm the identity of the ROS,
Fig. 1. SEM images of ZnO micro-nano structures prepared in (a) water, (b) 10 mM CTAB and (c) DMF with Zn2+/HMTA ratio of 1:1. Synthesis was done at 140 C for 12 h. (d) Schematic illustration for the formation of ZnO structures in different solvents.
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Fig. 2. XRD patterns of ZnO structures obtained in water (a), 10 mM CTAB (b) and DMF (c) corresponding to samples in Fig. 1.
multi-pod structure, the peak intensity of (1 0 1), (1 0 0) and (0 0 1) followed an order: (1 0 1) > (1 0 0) > (0 0 1), indicating crystal faces (1 0 1) and (1 0 0) had become dominant in the exposed facets of ZnO multi-pod. These results demonstrate that the solvent plays a critical role in crystal growth of ZnO in hydrothermal reactions. The photocatalytic activity of ZnO structures and the effects of the morphology on photocatalytic activity were examined by the degradation of methyl orange (MO) irradiated with a 365 nm Hg lamp (Fig. 3). MO itself is resistant to degradation in the absence of a photocatalyst. When ZnO structures were added to the MO solution, a considerable reduction in the absorbance of MO was detected for all three ZnO samples during irradiation with 365 nm light (Fig. 3a). Fig. 3b displays the evolution of the UV–vis Spectra for MO during irradiation in the presence of multi-pod ZnO. The ZnO structures with different shapes showed clear differences in their ability to photocatalytically degrade the MO (Fig. 3a). We observed the following order in photocatalytic activity: multipod > bell bottom > hexagonal microrod. After irradiation for 20 min, 10%, 31% and 44% MO were calculated to be degraded by hexagonal microrod, bell bottom like microrod and multi-pod ZnO,
respectively. ZnO multi-pod exhibited about four times higher catalytic activity than hexagonal microrod. These results demonstrate that the shape of ZnO structures plays a significant role in determining their photocatalytic activity. There are many factors that can affect the photocatalytic activity of a semiconductor, such as crystal structure, specific surface area, surface coating and chemical composition. In our work, these 3 ZnO structures have same crystalline phase and no additional surface chemical coating, therefore, the specific surface area and exposed crystal faces may be the main reasons for the observed differences in photocatalytic activity. The specific surface area was determined to be 44.8 1.2, 10.6 0.8 and 33.6 2.8 m2/g for bell bottom-like, hexagonal and multi-pod ZnO structures, respectively. These results are consistent with the observation that bell bottom and multi-pod ZnO showed higher catalytic activity than hexagonal rod, but cannot explain the order between multi-pod and bell bottom ZnO. Crystal face has been proposed to contribute to photocatalytic activity. As reported in [27], strongly face-dependent behavior was observed. The ZnO (1 0 1) surface showed the highest photocatalytic activity toward degradation of MO compared with (0 0 1) and (1 0 0) faces. The multi-pod structures have branches exposed with much larger (1 0 1) face than bottom bell ZnO, this may lead to the higher photocatalytic activity of multi-pod ZnO. Taken together, our results indicate that crystal face and specific surface area largely determined the observed shape-dependent photocatalytic activity of ZnO. To test the photocatalytic stability of ZnO, ZnO multi-pod, previously used as a photocatalyst, was collected by centrifugation and washed with water. The photocatalytic activity of this reclaimed ZnO was then examined. A slight decrease in photocatalytic activity was observed when reclaimed ZnO was used (Fig. 3c). It is well established that reactive oxygen species (ROS) play a central role in the photocatalytic activity of ZnO. Therefore, it is necessary to identify the ROS generated during photoexcitation of ZnO to understand the photocatalytic mechanism of ZnO. We developed a method based on ESR spectroscopy to definitively detect the ROS generated during irradiation of ZnO. In ESR, spin trapping is the most often used technique for detection of short lived free radicals and paramagnetic species. By appropriate selection of spin traps, it is possible to definitely identify ROS. In this work, BMPO was chosen as spin trap for capturing hydroxyl radicals and superoxide, and 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy (4-oxo-TEMP) was chosen for detection of singlet oxygen. We used multi-pod ZnO,
Fig. 3. (A) The photocatalytic activity toward degradation of methyl orange in the absence of any catalyst (control) and in the presence of 0.5 mg/ml ZnO structures with hexagonal-like (1#), bell bottom-like (2#) and multi-pod (3#) shapes; (B) Evolution of the absorption spectra over time for MO degradation photocatalyzed by multi-pod ZnO. The dashed navy line represents the original spectrum of MO; (C) Cycling curve for photocatalytic degradation of methyl orange using multi-pod ZnO.
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Fig. 4. Reactive oxygen species detection from irradiated ZnO multi-pod structures using BMPO (A) or 4-oxo-TEMP (B) as spin traps. ESR spectra were obtained from samples containing different spin probes (25 mM BMPO, or 2 mM 4-oxo-TEMP) and 0.1 mg/ml multi-pod like ZnO products before and after irradiating for 3 min with light having wavelengths above 350 nm. The control represents the sample containing spin probe alone under irradiation, or the sample containing spin probe and catalysts before exposure to light.
which had the highest photocatalytic activity among the synthesized forms of ZnO, to study the formation of ROS using ESR. Fig. 4 displays the ESR spectrum obtained using solutions containing BMPO or 4-oxo-TEMP and multi-pod ZnO before and during irradiation with light having wavelengths above 350 nm. For control samples containing only spin trap, or spin trap mixed with ZnO but without irradiation (Fig. 4), no ESR signal was observed in the cases of BMPO and 4-oxo-TEMP. Upon irradiation in the presence of ZnO, we observed a four-line spectrum with relative intensities of 1:2:2:1 and hyperfine splitting parameters of aN = 13.56, abH = 12.30, agH = 0.66 when BMPO was used as spin trap (Fig. 4A). This ESR spectrum is characteristic for the spin adduct between BMPO and the hydroxyl radical (BMPO/OH) [28]. This indicates that hydroxyl radical is generated by ZnO during irradiation. BMPO is also a spin trap frequently used for superoxide, but no characteristic ESR signal for the adduct BMPO/OOH appeared. This may be due to the spectra overlapping of BMPO/OH and BMPO/OOH. To elucidate the signal from BMPO, we investigated the effects of DMSO and superoxide dismutase (SOD) on the ESR signal. DMSO is the typically used scavenger for hydroxyl radical and SOD is a specific catalyst for the removal of superoxide. After addition of 10% DMSO, the ESR signal was significantly reduced. The remaining ESR signal can be assigned to a carbon center radical, associated with quenching of the hydroxyl radical by DMSO. These results further support the generation of OH. The addition of 1 U/ml SOD also partially suppressed the ESR signal, indicating the production of superoxide. These results demonstrate that both hydroxyl radical and superoxide are generated during irradiation of ZnO. 4-oxo-TEMP, a typical spin probe for singlet oxygen, has been widely used for detection of singlet oxygen [29]. In contrast with results obtained for the control in Fig. 4B, the characteristic ESR signal for the reaction between 4-oxo-TEMP and singlet oxygen was observed during the irradiation of ZnO. This observation directly verifies the production of singlet oxygen. Also, the addition of 10 mM NaN3, a singlet oxygen quencher, caused a substantial decrease in the observed ESR signal, which further supports that singlet oxygen is produced during photoexcitation of ZnO. It is noteworthy that SOD also a caused significant reduction in the ESR
signal when 4-oxo-TEMP was used (Fig. 4B). 4-oxo-TEMP is a specific probe to detect singlet oxygen. Superoxide and hydroxyl radicals cannot react with 4-oxo-TEMP to produce an ESR signal. In addition, SOD has no interaction with singlet oxygen. Therefore, these results indicate that superoxide must be involved in the generation of singlet oxygen. What determines which ROS are generated during photoexcitation of ZnO? When the semiconductors (e.g., ZnO) absorb light, electrons in the valence band are excited across the band gap to the conduction band with concomitant creation of holes in valence band. The electrons in conduction band and holes in valence band have strong reducing and oxidizing ability, respectively. Principally, the band gap structure determines the fate and power of the generated electrons/holes in semiconductor, and determines the type of ROS generated (Fig. 5). The redox potentials (E ) for H2O/OH, O2/O2 , and 1O2/O2 are 0.16, 2.32 and 1.88 V with respect to NHE at natural pH, respectively [30,31]. ZnO, with band gap (Eg = 3.2 eV), has a conduction band edge with redox potential of 0.31 V (Ec) and a valence band edge with redox potential of
Fig. 5. Proposed mechanisms for generation of ROS and photocatalytic reaction on the surface of a photoexcited ZnO particle.
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2.89 V (Ev) [32]. Comparing the Ec, Ev of ZnO and E of ROS, it is favorable thermodynamically to generate hydroxyl radical, superoxide and singlet oxygen from irradiated ZnO. The excited electrons in conduction band, having Ec ( 0.31 V) less than the E of O2/O2 , can drive the formation of superoxide. The holes in valence band (Ev = 2.89 V), enable photoexcited ZnO to oxidize H2O to form hydroxyl radicals. These predictions for superoxide and hydroxyl radical generation are consistent with our observations from ESR experiments. Although the holes in valence band from irradiated ZnO have the ability to directly generate singlet oxygen from oxygen, our ESR experimental results suggest that superoxide radicals play a major role in generating singlet oxygen. This mechanism is consistent with previous reports about the singlet oxygen formation as a result of the electron transfer reaction between superoxide and holes [29,33]. As we demonstrated using ESR with spin trapping technique, ROS including hydroxyl radicals, superoxide and singlet oxygen were produced during photoexcitation of ZnO multi-pod structures (Fig. 5). Because of their highly chemical reactivity, these ROS may contribute dominantly to the catalytic activity of ZnO micro-nano structures. 4. Conclusions In summary, micro/nano scale ZnO structures with different morphologies were prepared by changing solvents in solvothermal conditions. The ZnO micro-nano structures exhibited morphologydependent photocatalytic activity. Using ESR with spin trapping techniques, we detected separately and identified the ROS, including hydroxyl radicals, superoxide and singlet oxygen, generated during irradiation of ZnO. These reactive intermediates are proposed to be responsible for the photocatalytic behavior of ZnO micro-nano structures. The formation of ROS was determined by the band gap structures of ZnO, which also can predict the type of ROS generated in other semiconductors. These results may provide direct evidence and effective tools for understanding the photocatalytic mechanism of semiconductors. Acknowledgements This work was financially supported by National Natural Science Foundation of China (Grant No. 21303153, 61204009, 21273192) and Research Project of Basic and Advanced Technology of Henan Province (Grant No. 112300410106). This article is not an official US Food and Drug Administration (FDA) guidance or policy statement. No official support or endorsement by the US FDA is
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