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Enhancing the peroxidase-like activity of ficin via heme binding and colorimetric detection for uric acid
Talanta 185 (2018) 433–438
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Enhancing the peroxidase-like activity of ficin via heme binding and colorimetric detection for uric acid
T
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Yadi Pan, Yufang Yang, Yanjiao Pang, Ying Shi, Yijuan Long, Huzhi Zheng
The Key Laboratory of Luminescent and Real-time Analysis (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Heme-ficin complexes Peroxidase-like activity Uric acid detection
Ficin, a classical sulfhydryl protease, was found to possess intrinsic peroxidase-like activity. In this paper, we have put forward a novel strategy to improving the peroxidase-like activity of ficin through binding heme. Heme-ficin complexes were successfully obtained by simple one-step syntheticism. The results demonstrated that the catalytic activity and efficiency of heme-ficin complexes were about 1.7 times and 3 times higher than those of native ficin, respectively. Taking advantages of the high peroxidase-like activity, the heme-ficin complexes were used for colorimetric determination of uric acid with a low detection limit of 0.25 μM. Based on the excellent selectivity and sensitivity, we detected the concentration of uric acid in human serum successfully. On the basis of these findings, the heme-ficin complexes are promising for wide applications in various fields. Thus we not only optimized the peroxidase-like activity of the ficin, but also established a new strategy for development of artificial enzyme mimics by mimicking the architecture of the active site in horseradish peroxidase.
1. Introduction Artificial enzyme mimics have drawn considerable attentions in recent years, which are regarded as the alternatives for natural enzymes because of their excellent comprehensive properties. So far, more and more nanoparticle-based artificial enzyme mimics, such as metal-based nanoparticles [1–3], graphene-based materials [4,5], carbon nanodots [6–8], and metal-organic frameworks [9–11], have been designed and constructed. Unfortunately, some of these non-biological enzyme mimics, which often need complicated preparation procedures and modification process to avoid aggregation, have low biological compatibility and poor reproducibility in activity. Horseradish peroxidase (HRP) as a kind of nature biological enzyme, have been narrowed their industrial applications primarily for relatively high costs of preparation, complicated purification and meticulous storage environment [12,13]. Nevertheless, the intrinsic overwhelming superiorities of nature enzymes that we still can’t ignore, such as high-catalytic activity, good biological compatibility and low toxicity [14–17]. Development of artificial enzyme mimics based on the nature enzymes is highly desirable. Ficin was found to possess intrinsic peroxidase-like activity as classical cysteine protease that isolated from the latex of fig trees, which was firstly reported by our group in 2017 [17]. There is no denying that the catalytic efficiency of ficin is lower than HRP. However, compared with HRP, ficin could be used more extensively in analytic applications
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due to their outstanding robustness against harsh pH and temperature, higher affinity to H2O2 [17]. It could be mentioned that ficin is a simple enzyme while HRP is a conjugated enzyme containing the ferroprotoporphyrin cofactor in which iron plays a crucial role in the catalytic process [14,18–20]. In nature, the enzyme has evolved activity sites where the distribution of basic functional groups follows the precise spatial structure to activate the catalytic center effectively. Heme, a cofactor in HRP, is activated by H2O2 via the collaboration among a proximal His ligand, a distal His, and a distal Arg residue to form a high oxidation state intermediate compound I that accepts the electrons from the reducing substrate, which leading to a variety of basic biological functions [14]. A great number of efforts had been expended on incorporating heme into protein-like scaffolds in the looking for a similar active site promoting the creation of compound I [21–23]. Heme could bind His residues in amyloid-β to form a peroxidase mimic which plays an important role in the treatment of Alzheimer's disease(AD) [24–27], suggesting that heme also could bind His residues in ficin which contains about 200 amino acids to form complexes. In this paper, we have put forward a novel strategy to incorporate heme into ficin to obtain peroxidase-like complexes, in which ficin could offer a native protein microenvironment more than simple amino acid residue. To verify our hypothesis, we studied the UV–vis spectra, fluorescence spectroscopy and circular dichroism of the native ficin, heme and heme-incubated
Corresponding author. E-mail address: [email protected] (H. Zheng).
https://doi.org/10.1016/j.talanta.2018.04.005 Received 29 December 2017; Received in revised form 27 March 2018; Accepted 1 April 2018 Available online 03 April 2018 0039-9140/ © 2018 Elsevier B.V. All rights reserved.
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ficin to indicate the formation of heme-ficin complexes. Then we investigated the catalysis performance of heme-ficin complexes compared with native ficin and heme. And our results indicate heme-ficin complexes have enhanced peroxidase activity effectively. Uric acid (UA), an end product of purine metabolism, is an important biomarker of hyperuricemia in urine and serum can lead to several pathological symptoms such as gout, arthritis, neurological, renal, cardiovascular and kidney related disease. Hence, precise detection of UA concentration in urine and serum samples is a key of the early stage diagnosis and warning of these conditions. Colorimetric and fluorometric methods have always attracted considerable interest owing to its simple, cost-effective and routine analysis [28–32]. Based on the excellent peroxidase-like activity of heme-ficin complexes, we have applied the complexes to the colorimetric detection of uric acid with uricase. This system exhibited excellent selectivity and sensitivity for uric acid determination. And we detected the concentration of uric acid in human serum successfully, suggesting its great potential for biocatalysis and bioassays in the future.
DMPO/•OH spin adduct. The ESR spectra were obtained on a Bruker ESR 300 E with a microwave bridge (receiver gain, 1 × 105; modulation amplitude, 2 Gauss; microwave power, 10 mW; modulation frequency, 100 kHz). 2.5. Kinetic assays
2. Experiment section
Steady-state kinetic assays were carried out by monitoring the absorbance change at 652 nm at different reaction time. Experiments were carried out at 30 °C in 1.0 mL reaction buffer solution (20 mM PBS, pH = 5.0) contains 0.10 μg/mL heme-ficin complexes as catalyst in the presence of H2O2 and TMB by varying concentrations of TMB at a fixed concentration of H2O2 or vice versa. The Michaelis-Menten constant was calculated based on the Lineweaver-Burk plot: 1/v = (Km/Vmax)⋅ (1/[S] + 1/Km), where v is the initial velocity, Vmax is the maximal reaction velocity, and [S] is the concentration of substrate and Km is the Michaelis constant. The Michaelis constant is equivalent to the substrate concentration at which the rate of conversion is half of Vmax. Km indicates the affinity of the enzyme to the substrate: a lower Km value means a higher affinity.
2.1. Reagents
2.6. Detection of hydrogen peroxide and uric acid
Premium grade ficin (F4165, powder, molecular weight 23.8 kDa) were purchased from Sigma-Aldrich (America). Hemin (98%, which is referred to as heme in this paper) was obtained from Adamas (Shanghai, China). Uric acid and uricase were purchased from Yuanye Bio-Technology (Shanghai, China). NaCl, KCl, glucose, sucrose, urea, tryptophan, L-cysteine, ascorbic acid (AA) were purchased from Aladdin (Shanghai, China). Hydrogen peroxide (H2O2, 30%) was obtained from Chongqing Chuandong Chemical Co., Ltd. (Chongqing, China). The phosphate saline buffer (PBS) containing Na2HPO4 and NaH2PO4 was prepared and the pH was adjusted with H3PO4 or NaOH. All the commercial available regents were used without further purification. All solutions were prepared using ultrapure water (18.2 MΩ cm−1) from a Milli-Q automatic ultrapure water system.
H2O2 detection was carried out as follows: a) 1200 μL ultrapure water, 200 μL 0.20 M PBS (pH = 5.0), 200 μL of the 1.0 μg/mL hemeficin complexes, 200 μL of 0.80 mM TMB solution, and 200 μL of H2O2 with different concentrations were added into a 2.0 mL centrifuge tube and mixed together; b) the mixed solution was incubated for 210 min at 30 °C and then for standard curve measurement. Uric acid detection was performed as follows: a) 10 μL of 3.0 mg/mL uricase and 200 μL of uric acid of different concentrations in 0.02 M PBS (pH = 8.5) were incubated at 37 °C for 15 min; b) 200 μL of 1.0 μg/ mL heme-ficin complexes, 200 μL of 0.80 mM TMB and 1390 μL of 0.20 M PBS (pH = 5.0) were added to the above uric acid reaction solution; c) the mixed solution was incubated at 30 °C for 210 min and then for standard curve measurement.
2.2. Preparation of heme-ficin complexes
2.7. Uric acid Determination in Serum Samples
A stock solution of heme (40 mM) was prepared in 100 mM NaOH solution and stored at 4 °C in the dark, then was diluted to 2.0 mM in 50 mM PBS (pH = 7.0) before use. The stock solution of ficin (2.0 mg/ mL) was freshly prepared in 50 mM PBS (pH = 7.0). Then incubating 1 equiv of both heme and ficin solutions for 8 h at 4 °C in the dark. The molar ratio of heme to ficin was maintained at 25:1.
Serum samples were obtained from two healthy volunteers. The collected samples were first treated by ultrafiltration with 5000 Da at 1000 rpm for 30 min. Then, the filtrates were diluted 2 times using ultrapure water. The subsequent operations for uric acid detection in serum samples were the same as described above except the replacement of uric acid with serum samples. And the volunteers’ consent and approval from the Institutional Research Ethics Committee of Southwest University hospital were obtained for research purposes.
2.3. Peroxidase-like activity
3. Results and discussion
The peroxidase-like activity of heme-ficin complexes was measured using TMB as the chromogenic substrate in the presence of H2O2 following the increase in absorbance at 652 nm. In brief, 200 μL hemeficin complexes (0.50 μg/mL, which is the concentration of ficin presented in the complexes), 200 μL TMB (8.0 mM) and 200 μL H2O2 (8.0 mM) were orderly added into 200 μL PBS (pH = 5.0) and the final volume of the mixture was adjusted to 2.0 mL with ultrapure water. The mixed solution was then incubated in 30 °C water bath. After incubation for 210 min, the UV–vis absorption spectra of the solution were measured on a UV-2450 UV–vis spectrophotometer (Shimadzu, Japan).
3.1. Formation of heme-ficin complexes It has been well recognized that heme is an indispensable factor for some metabolism of organisms including oxygen transportation, catalysis and electron transfer [33]. Free heme, possessed low peroxidaselike activity, were available for binding His residues in amyloid-β (Aβ), which may hold a key towards development of the cure of AD [25]. Meanwhile, the catalytic rate of heme-Aβ complex was ~ 3 times faster than free heme [25]. In this study, we prepared heme-ficin complexes by mixing heme and ficin together, as shown in Scheme 1a. The main fabrication conditions we have optimized, which could be mainly influenced by pH and the molar ratio of heme to ficin that were investigated by measuring the corresponding peroxidase-like activities. As shown in Fig. S1a, at weakly acidic and neutral pH, we can obtain complexes with higher catalytic activity, and the optimal pH for fabricating complex was 7.0. The molar ratio of heme to ficin varied from 1
2.4. Electron spin resonance (ESR) Twenty μL ficin or heme-ficin complexes was added into 0.20 M PBS buffer (pH = 5.0), 20 μL of 15% H2O2, and 50 μL 100 mM 5,5-dimethyl1-pyrolin-N-oxide (DMPO) and proper amount of ultra-pure water into a plastic tube. The prepared sample solution was transferred to a quartz capillary tube and placed in the ESR cavity. DMPO was used to form the 434
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DMPO spin trap system, which is a powerful tool to verify the radicals involved in the reaction catalyzed by peroxidase mimics [35]. The generation rate of •OH radicals was confirmed by the characteristic ESR signal with a 1:2:2:1 quartet (Fig. 1b) [36]. Furthermore, the DMPO/ •OH adduct enhanced signal intensity is dependent on the concentration of heme-ficin complexes, indicating increase •OH radicals production by catalyzing the decomposition of H2O2. Moreover, the characteristic signal intensity produced by heme-ficin complexes was higher than that generated by same amount of ficin, which provides direct evidence that improved peroxidase-like activity of ficin via introducing heme. 3.3. Optimization of the catalytic reaction conditions Similar to the majority of peroxidases, the catalytic activity of heme-ficin complexes was influenced by pH, temperature, H2O2 concentration and incubation time. We measured the peroxidase-like activity of heme-ficin complexes while varying the pH from 1.0 to 8.0, the temperature from 20 °C to 50 °C, the H2O2 concentration from 0.10 to 4.0 mM, the incubation time from 30 min to 240 min (Fig. 2). Similar to ficin and other enzyme mimics, the acidic pH condition was suitable for H2O2-TMB chromogenic system, the optimal pH we selected was 5.0. In addition, at 20–30 °C, the relative catalytic activity was maintained about 95%, so we selected 30 °C as the optimal temperature for it can be accurately controlled, especially in the hot summer. The optimal concentration of H2O2 was 0.80 mM and the peroxidase-like activity of heme-ficin would be inhibited at higher H2O2 concentration according with the behavior of ficin and HRP [37]. As the reaction time increased, the absorbance value increased gradually. After 210 min incubation, the absorbance value reached a plateau, so we selected 210 min as the reaction time.
Scheme 1. (a) Scheme illustration of the synthesis of heme-ficin complexes. (b) Schematic illustration of uric acid detection in the presence of uricase and heme-ficin complexes catalyzed reaction.
to 50 was studied, we have discovered the satisfying ratio was 25 even superior to 50 (Fig. S1b). This may be ascribed to the binding sites of H2O2 or TMB have occupied by excess of free heme with lower peroxidase-like activities, thus blocked their combination with heme-ficin complexes. After getting rid of excess heme through ultrafiltration, the content of heme in the filtrate was determined by UV–vis spectra. We calculated the binding molar ratio of heme and ficin is about 17:1. Moreover, we selected 8 h as an adequate reaction time for incubation time. To verify the formation of heme-ficin complexes, we studied the UV–vis spectra, taking heme and ficin as the controls (Fig. S2a). It was discovered that heme had a Q band from 607 to 613 nm and two absorption peaks at about 250 nm and 384 nm, and ficin had an absorption peaks at 280 nm. However, heme-incubated ficin didn’t have any distinct absorption peak at 280 nm relative to native ficin and showed obvious increase at about 371 nm, yet the Q band was indistinct. Moreover, we measured fluorescence spectra with the same controls. It was noted that unbound ficin exhibited a high emission peak at 342 nm, while heme-incubated ficin showed almost no peak at the same location (Fig. S2b). It can be seen from the circular dichroism (CD) spectra (Fig. S3), both 260–340 nm and 200–260 nm UV region, there are some modification of distribution of the secondary structure elements and some loss of hydrophobic contacts (3D structure) upon complexes formation. These results amply indicate the successful formation of hemeficin complexes. Similar to heme-Aβ complexes, the formation of the complex could attribute to the connection of heme and His residues in ficin.
3.4. Steady-state kinetics Like ficin, the catalytic reaction of heme-ficin complexes follows the typical Michaelis-Menten model toward both substrates, H2O2 and TMB. By changing the concentration of H2O2 and TMB respectively, the data were fitted well to the Michaelis-Menten curves to acquire the enzyme kinetic parameters for the reaction (Fig. 3). We obtained the MichaelisMenten constant (Km) and maximum initial velocity (Vmax) from Lineweaver-Burk plot based on the equation 1/v = Km/Vmax·(1/[S] + 1/Km). Kcat/Km is often thought of as a measure of enzyme efficiency, it allows direct comparison of the effectiveness of enzyme toward different substrates or different enzymes with the same substrate. As listed in Table S1, the Kcat/Km of heme-ficin complexes was calculated to be 24.33 and 6.95 for TMB and H2O2 respectively. The catalytic efficiency of hemeficin complexes was approximately 3 times higher than ficin. In addition, the Km value of heme-ficin complexes towards TMB and H2O2 both are lower than that of ficin, which suggested a higher affinity of heme-ficin complexes to the substrates than that of ficin. There are two main factors could contribute to the higher peroxidase-like activity of heme-ficin complexes: (1) the lower Km value of heme-ficin complexes towards TMB and H2O2 ensure a higher affinity to the substrates, which could aggregate more substrates to approach to the active sites. (2) the synergistic effects between imidazole groups of His resides and the porphyrin of heme facilitate the location of H2O2 into the active site through H-bond interaction and the formation of initial compound I [18,38]. Furthermore, we measured initial velocity versus H2O2 concentration in a range of concentrations of TMB, or vice versa to obtain double reciprocal plots (Fig. 3). The slope of the lines is parallel according with the characteristic of a ping-pong mechanism [39].
3.2. The peroxidase-like activity of heme-ficin complexes A typical chromogenic system, H2O2-TMB was chosen to investigate the peroxidase-like activity of heme-ficin complexes in comparison to that of native ficin and heme. In the presence of H2O2, TMB was oxidized to blue-colored products (oxTMB) rapidly when peroxidase was added by following the increase in absorbance at 652 nm [34]. First, as shown in Fig. S4, the chromogenic reaction catalyzed by heme-ficin complexes showed an observably higher reaction velocity than the reaction catalyzed by ficin or heme. Furthermore, the catalytic activity of heme-ficin complexes was about 1.7 fold higher than those native ficin and increased by about 55% than the superposition peroxidase-like activity of heme and ficin (Fig. 1a). Thus the result is consistent with what we expected. ESR was applied to examine •OH radicals produced by the H2O2/
3.5. Detection of H2O2 and uric acid Considering the excellent biomimetic catalytic activity of heme-ficin complexes, we applied the complexes to H2O2 detection under the above optimum assay conditions. As shown in Fig. S5, the calibration 435
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Fig. 1. (a) The UV–Vis absorption spectra of different catalytic reaction systems: (1) hemeficin complexes + TMB + H2O2, (2) ficin + TMB + H2O2, (3) heme + TMB + H2O2, (4) TMB + H2O2 in a PBS solution (pH = 5.0) after incubation for 210 min at 30 °C. The concentrations were 0.050 μg/mL of native ficin or heme-ficin complexes, 0.050 μM heme, 0.80 mM for H2O2 and TMB. (b) Spin-trapping ESR spectra of •OH radicals in different systems.
Fig. 2. The peroxidase-like activity of hemeficin is dependent on pH (a), temperature (b), H2O2 concentration (c), and incubation time (d). Experiments were carried out using 0.10 μg/mL native ficin or heme-ficin complexes with 0.80 mM TMB and H2O2 as substrate, respectively. The pH of PBS was 5.0, and the temperature was 30 °C unless otherwise stated. Error bars represent the standard deviations of three independent experiments. ΔA = A − A0, where A and A0 is absorbance in the presence and absence of catalyst, respectively.
ascorbic acid (AA) as potential interferential substances in human serum. We found that as low as 60 µM uric acid caused a dramatic increase in absorbance, while other interferential substances presented negligible effect on the absorbance at 652 nm, even when their concentrations were 5 times higher than uric acid (Fig. 4c). In addition, we also investigate the effects of coexisting substances on the determination of uric acid. A relative error of less than ± 5% was considered to be acceptable. As shown in Fig. 4d, NaCl, KCl, glucose, sucrose, urea, tryptophan, L-cysteine, ascorbic acid (AA) have no significant effect on the detection of uric acid. These results demonstrated that our developed method not only had a low detection limit, but also an excellent selectivity for uric acid determination, which provide a possible to the application in the biomedical detection.
curve of the absorbance at 652 nm against H2O2 concentration was linear from 1.0 to 120 μM. The linear regression equation was A = 0.0085 C + 0.0387 (R2 = 0.9978), A represents the absorbance at 652 nm and C represents the concentration of H2O2. Uric acid, an important biological analyte, can be oxidized to produce H2O2 and allantoin in the presence of uricase. Hence, the above H2O2 response system made it possible to further develop a platform for uric acid detection (Scheme 1b). The blue color gradually darken with increased uric acid concentration, the naked eyes can observe obviously the color variation even the uric acid concentration was as low as 5.0 μM (Fig. 4a). The absorbance at 652 nm was linearly correlated to uric acid concentration from 1.0 to 120 μM with a detection limit of 0.25 μM which is comparative or even lower than some previous colorimetric methods listed in Table S2. The linear regression equation was A = 0.0099 C + 0.0169 (R2 = 0.9928), A represents the absorbance at 652 nm and C represents the concentration of uric acid (Fig. 4b). Furthermore, we evaluated the specifity of the proposed approach using NaCl, KCl, glucose, sucrose, urea, tryptophan, L-cysteine,
3.6. Uric acid determination in human blood samples Encouraged by the above results, we then determined the uric acid concentration in human serum to explore the practical usability of our 436
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Fig. 3. Steady-state kinetic assay and catalytic mechanism of heme-ficin complexes. The velocity (v) of the reaction was surveyed by using 0.10 μg/mL ficin presented in heme-ficin complexes in 1.0 mL of 0.20 M PBS solution at pH = 5.0 and 30 °C. The error bars express that the standard deviations derived from three repeated measurements. (a) The concentration of H2O2 was 0.30 mM and the concentration of TMB as a substrate was varied. (b) The concentration of TMB was 0.10 mM and the H2O2 concentration as a substrate was varied. (c) Double reciprocal plots of peroxidase-like activity of heme-ficin complexes with the concentration of H2O2 fixed and TMB varied, (d) the concentration of TMB fixed and H2O2 varied.
4. Conclusions
sensing system. We diluted 20 times of original serum samples to guarantee the uric acid content was in the range of our established standard curve. As listed in the Table 1, the concentrations of uric acid in human serum we measured were close to the values obtained through the routine clinical method in a local hospital. The results showed that our sensing methods possess high accuracy for uric acid determination in human system.
In summary, we have prepared the heme-ficin complexes through a simple and facile one-step synthesis. The formation of the complexes could attribute to the connection of heme and His residues in ficin. Compared with native ficin, the heme-ficin complexes have the enhanced peroxidase-like activity and catalytic efficiency. Based on these Fig. 4. (a) The absorption spectra corresponding to various concentrations of uric acid. Inset: color change with the corresponding concentration uric acid from 1.0 to 120 μM (from bottom to top: 1.0, 5.0, 10, 20, 40, 60, 80, 100, 120). (b) The linear regression to plots of the absorbance at 652 nm with the uric acid. (c) The selectivity analysis for uric acid detection by monitoring the relative absorbance change at 652 nm. The concentration of uric acid was 60 μM, while the concentrations of other interferential substances were 300 μM. (d) Effects of coexisting substances on the determination of uric acid. The concentration of uric acid, NaCl, KCl, glucose, sucrose, urea, tryptophan, L-cysteine, AA was 60 μM, 100 mM, 100 mM, 5 M, 5 M, 8 M, 500 μM, 10 μM, 10 μM.
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Table 1 Determination of UA in human serum samples. Sample
This method (µM)
RSD (n = 3; %)
Hospital result (µM)
t-test
1 2
225 ± 7.8 255 ± 12
3.4 4.7
229 251
0.89 0.58
t0.1,
2
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= 2.92 (P = 0.90).
findings, we have applied the complexes to the colorimetric detection of H2O2 and uric acid. The developed method exhibited a good sensitivity and high selectivity toward uric acid. More importantly, we had detected the concentration of uric acid in human serum successfully. Therefore, this good performance of the heme-ficin complexes made it possible to apply in various fields, such as materials science, biology, and medicine. In addition, this work provides an effective way to improve the peroxidase-like activity of ficin via mimicking the architecture of the active site in HRP. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (Nos. 21405124, 21175110) and the Fundamental Research Funds for the Central Universities (Nos. XDJK2013A022, XDJK2014C173, XDJK2017D052). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2018.04.005. References [1] Y. Chen, H. Cao, W. Shi, H. Liu, Y. Huang, Chem. Commun. 49 (2013) 5013. [2] L. Gao, et al., Nat. Nanotechnol. 2 (2007) 577. [3] W. He, H. Jia, X. Li, Y. Lei, J. Li, H. Zhao, L. Mi, L. Zhang, Z. Zheng, Nanoscale 4 (2012) 3501.
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Enhancing the peroxidase-like activity of ficin via heme binding and colorimetric detection for uric acid
T
⁎
Yadi Pan, Yufang Yang, Yanjiao Pang, Ying Shi, Yijuan Long, Huzhi Zheng
The Key Laboratory of Luminescent and Real-time Analysis (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Heme-ficin complexes Peroxidase-like activity Uric acid detection
Ficin, a classical sulfhydryl protease, was found to possess intrinsic peroxidase-like activity. In this paper, we have put forward a novel strategy to improving the peroxidase-like activity of ficin through binding heme. Heme-ficin complexes were successfully obtained by simple one-step syntheticism. The results demonstrated that the catalytic activity and efficiency of heme-ficin complexes were about 1.7 times and 3 times higher than those of native ficin, respectively. Taking advantages of the high peroxidase-like activity, the heme-ficin complexes were used for colorimetric determination of uric acid with a low detection limit of 0.25 μM. Based on the excellent selectivity and sensitivity, we detected the concentration of uric acid in human serum successfully. On the basis of these findings, the heme-ficin complexes are promising for wide applications in various fields. Thus we not only optimized the peroxidase-like activity of the ficin, but also established a new strategy for development of artificial enzyme mimics by mimicking the architecture of the active site in horseradish peroxidase.
1. Introduction Artificial enzyme mimics have drawn considerable attentions in recent years, which are regarded as the alternatives for natural enzymes because of their excellent comprehensive properties. So far, more and more nanoparticle-based artificial enzyme mimics, such as metal-based nanoparticles [1–3], graphene-based materials [4,5], carbon nanodots [6–8], and metal-organic frameworks [9–11], have been designed and constructed. Unfortunately, some of these non-biological enzyme mimics, which often need complicated preparation procedures and modification process to avoid aggregation, have low biological compatibility and poor reproducibility in activity. Horseradish peroxidase (HRP) as a kind of nature biological enzyme, have been narrowed their industrial applications primarily for relatively high costs of preparation, complicated purification and meticulous storage environment [12,13]. Nevertheless, the intrinsic overwhelming superiorities of nature enzymes that we still can’t ignore, such as high-catalytic activity, good biological compatibility and low toxicity [14–17]. Development of artificial enzyme mimics based on the nature enzymes is highly desirable. Ficin was found to possess intrinsic peroxidase-like activity as classical cysteine protease that isolated from the latex of fig trees, which was firstly reported by our group in 2017 [17]. There is no denying that the catalytic efficiency of ficin is lower than HRP. However, compared with HRP, ficin could be used more extensively in analytic applications
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due to their outstanding robustness against harsh pH and temperature, higher affinity to H2O2 [17]. It could be mentioned that ficin is a simple enzyme while HRP is a conjugated enzyme containing the ferroprotoporphyrin cofactor in which iron plays a crucial role in the catalytic process [14,18–20]. In nature, the enzyme has evolved activity sites where the distribution of basic functional groups follows the precise spatial structure to activate the catalytic center effectively. Heme, a cofactor in HRP, is activated by H2O2 via the collaboration among a proximal His ligand, a distal His, and a distal Arg residue to form a high oxidation state intermediate compound I that accepts the electrons from the reducing substrate, which leading to a variety of basic biological functions [14]. A great number of efforts had been expended on incorporating heme into protein-like scaffolds in the looking for a similar active site promoting the creation of compound I [21–23]. Heme could bind His residues in amyloid-β to form a peroxidase mimic which plays an important role in the treatment of Alzheimer's disease(AD) [24–27], suggesting that heme also could bind His residues in ficin which contains about 200 amino acids to form complexes. In this paper, we have put forward a novel strategy to incorporate heme into ficin to obtain peroxidase-like complexes, in which ficin could offer a native protein microenvironment more than simple amino acid residue. To verify our hypothesis, we studied the UV–vis spectra, fluorescence spectroscopy and circular dichroism of the native ficin, heme and heme-incubated
Corresponding author. E-mail address: [email protected] (H. Zheng).
https://doi.org/10.1016/j.talanta.2018.04.005 Received 29 December 2017; Received in revised form 27 March 2018; Accepted 1 April 2018 Available online 03 April 2018 0039-9140/ © 2018 Elsevier B.V. All rights reserved.
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ficin to indicate the formation of heme-ficin complexes. Then we investigated the catalysis performance of heme-ficin complexes compared with native ficin and heme. And our results indicate heme-ficin complexes have enhanced peroxidase activity effectively. Uric acid (UA), an end product of purine metabolism, is an important biomarker of hyperuricemia in urine and serum can lead to several pathological symptoms such as gout, arthritis, neurological, renal, cardiovascular and kidney related disease. Hence, precise detection of UA concentration in urine and serum samples is a key of the early stage diagnosis and warning of these conditions. Colorimetric and fluorometric methods have always attracted considerable interest owing to its simple, cost-effective and routine analysis [28–32]. Based on the excellent peroxidase-like activity of heme-ficin complexes, we have applied the complexes to the colorimetric detection of uric acid with uricase. This system exhibited excellent selectivity and sensitivity for uric acid determination. And we detected the concentration of uric acid in human serum successfully, suggesting its great potential for biocatalysis and bioassays in the future.
DMPO/•OH spin adduct. The ESR spectra were obtained on a Bruker ESR 300 E with a microwave bridge (receiver gain, 1 × 105; modulation amplitude, 2 Gauss; microwave power, 10 mW; modulation frequency, 100 kHz). 2.5. Kinetic assays
2. Experiment section
Steady-state kinetic assays were carried out by monitoring the absorbance change at 652 nm at different reaction time. Experiments were carried out at 30 °C in 1.0 mL reaction buffer solution (20 mM PBS, pH = 5.0) contains 0.10 μg/mL heme-ficin complexes as catalyst in the presence of H2O2 and TMB by varying concentrations of TMB at a fixed concentration of H2O2 or vice versa. The Michaelis-Menten constant was calculated based on the Lineweaver-Burk plot: 1/v = (Km/Vmax)⋅ (1/[S] + 1/Km), where v is the initial velocity, Vmax is the maximal reaction velocity, and [S] is the concentration of substrate and Km is the Michaelis constant. The Michaelis constant is equivalent to the substrate concentration at which the rate of conversion is half of Vmax. Km indicates the affinity of the enzyme to the substrate: a lower Km value means a higher affinity.
2.1. Reagents
2.6. Detection of hydrogen peroxide and uric acid
Premium grade ficin (F4165, powder, molecular weight 23.8 kDa) were purchased from Sigma-Aldrich (America). Hemin (98%, which is referred to as heme in this paper) was obtained from Adamas (Shanghai, China). Uric acid and uricase were purchased from Yuanye Bio-Technology (Shanghai, China). NaCl, KCl, glucose, sucrose, urea, tryptophan, L-cysteine, ascorbic acid (AA) were purchased from Aladdin (Shanghai, China). Hydrogen peroxide (H2O2, 30%) was obtained from Chongqing Chuandong Chemical Co., Ltd. (Chongqing, China). The phosphate saline buffer (PBS) containing Na2HPO4 and NaH2PO4 was prepared and the pH was adjusted with H3PO4 or NaOH. All the commercial available regents were used without further purification. All solutions were prepared using ultrapure water (18.2 MΩ cm−1) from a Milli-Q automatic ultrapure water system.
H2O2 detection was carried out as follows: a) 1200 μL ultrapure water, 200 μL 0.20 M PBS (pH = 5.0), 200 μL of the 1.0 μg/mL hemeficin complexes, 200 μL of 0.80 mM TMB solution, and 200 μL of H2O2 with different concentrations were added into a 2.0 mL centrifuge tube and mixed together; b) the mixed solution was incubated for 210 min at 30 °C and then for standard curve measurement. Uric acid detection was performed as follows: a) 10 μL of 3.0 mg/mL uricase and 200 μL of uric acid of different concentrations in 0.02 M PBS (pH = 8.5) were incubated at 37 °C for 15 min; b) 200 μL of 1.0 μg/ mL heme-ficin complexes, 200 μL of 0.80 mM TMB and 1390 μL of 0.20 M PBS (pH = 5.0) were added to the above uric acid reaction solution; c) the mixed solution was incubated at 30 °C for 210 min and then for standard curve measurement.
2.2. Preparation of heme-ficin complexes
2.7. Uric acid Determination in Serum Samples
A stock solution of heme (40 mM) was prepared in 100 mM NaOH solution and stored at 4 °C in the dark, then was diluted to 2.0 mM in 50 mM PBS (pH = 7.0) before use. The stock solution of ficin (2.0 mg/ mL) was freshly prepared in 50 mM PBS (pH = 7.0). Then incubating 1 equiv of both heme and ficin solutions for 8 h at 4 °C in the dark. The molar ratio of heme to ficin was maintained at 25:1.
Serum samples were obtained from two healthy volunteers. The collected samples were first treated by ultrafiltration with 5000 Da at 1000 rpm for 30 min. Then, the filtrates were diluted 2 times using ultrapure water. The subsequent operations for uric acid detection in serum samples were the same as described above except the replacement of uric acid with serum samples. And the volunteers’ consent and approval from the Institutional Research Ethics Committee of Southwest University hospital were obtained for research purposes.
2.3. Peroxidase-like activity
3. Results and discussion
The peroxidase-like activity of heme-ficin complexes was measured using TMB as the chromogenic substrate in the presence of H2O2 following the increase in absorbance at 652 nm. In brief, 200 μL hemeficin complexes (0.50 μg/mL, which is the concentration of ficin presented in the complexes), 200 μL TMB (8.0 mM) and 200 μL H2O2 (8.0 mM) were orderly added into 200 μL PBS (pH = 5.0) and the final volume of the mixture was adjusted to 2.0 mL with ultrapure water. The mixed solution was then incubated in 30 °C water bath. After incubation for 210 min, the UV–vis absorption spectra of the solution were measured on a UV-2450 UV–vis spectrophotometer (Shimadzu, Japan).
3.1. Formation of heme-ficin complexes It has been well recognized that heme is an indispensable factor for some metabolism of organisms including oxygen transportation, catalysis and electron transfer [33]. Free heme, possessed low peroxidaselike activity, were available for binding His residues in amyloid-β (Aβ), which may hold a key towards development of the cure of AD [25]. Meanwhile, the catalytic rate of heme-Aβ complex was ~ 3 times faster than free heme [25]. In this study, we prepared heme-ficin complexes by mixing heme and ficin together, as shown in Scheme 1a. The main fabrication conditions we have optimized, which could be mainly influenced by pH and the molar ratio of heme to ficin that were investigated by measuring the corresponding peroxidase-like activities. As shown in Fig. S1a, at weakly acidic and neutral pH, we can obtain complexes with higher catalytic activity, and the optimal pH for fabricating complex was 7.0. The molar ratio of heme to ficin varied from 1
2.4. Electron spin resonance (ESR) Twenty μL ficin or heme-ficin complexes was added into 0.20 M PBS buffer (pH = 5.0), 20 μL of 15% H2O2, and 50 μL 100 mM 5,5-dimethyl1-pyrolin-N-oxide (DMPO) and proper amount of ultra-pure water into a plastic tube. The prepared sample solution was transferred to a quartz capillary tube and placed in the ESR cavity. DMPO was used to form the 434
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DMPO spin trap system, which is a powerful tool to verify the radicals involved in the reaction catalyzed by peroxidase mimics [35]. The generation rate of •OH radicals was confirmed by the characteristic ESR signal with a 1:2:2:1 quartet (Fig. 1b) [36]. Furthermore, the DMPO/ •OH adduct enhanced signal intensity is dependent on the concentration of heme-ficin complexes, indicating increase •OH radicals production by catalyzing the decomposition of H2O2. Moreover, the characteristic signal intensity produced by heme-ficin complexes was higher than that generated by same amount of ficin, which provides direct evidence that improved peroxidase-like activity of ficin via introducing heme. 3.3. Optimization of the catalytic reaction conditions Similar to the majority of peroxidases, the catalytic activity of heme-ficin complexes was influenced by pH, temperature, H2O2 concentration and incubation time. We measured the peroxidase-like activity of heme-ficin complexes while varying the pH from 1.0 to 8.0, the temperature from 20 °C to 50 °C, the H2O2 concentration from 0.10 to 4.0 mM, the incubation time from 30 min to 240 min (Fig. 2). Similar to ficin and other enzyme mimics, the acidic pH condition was suitable for H2O2-TMB chromogenic system, the optimal pH we selected was 5.0. In addition, at 20–30 °C, the relative catalytic activity was maintained about 95%, so we selected 30 °C as the optimal temperature for it can be accurately controlled, especially in the hot summer. The optimal concentration of H2O2 was 0.80 mM and the peroxidase-like activity of heme-ficin would be inhibited at higher H2O2 concentration according with the behavior of ficin and HRP [37]. As the reaction time increased, the absorbance value increased gradually. After 210 min incubation, the absorbance value reached a plateau, so we selected 210 min as the reaction time.
Scheme 1. (a) Scheme illustration of the synthesis of heme-ficin complexes. (b) Schematic illustration of uric acid detection in the presence of uricase and heme-ficin complexes catalyzed reaction.
to 50 was studied, we have discovered the satisfying ratio was 25 even superior to 50 (Fig. S1b). This may be ascribed to the binding sites of H2O2 or TMB have occupied by excess of free heme with lower peroxidase-like activities, thus blocked their combination with heme-ficin complexes. After getting rid of excess heme through ultrafiltration, the content of heme in the filtrate was determined by UV–vis spectra. We calculated the binding molar ratio of heme and ficin is about 17:1. Moreover, we selected 8 h as an adequate reaction time for incubation time. To verify the formation of heme-ficin complexes, we studied the UV–vis spectra, taking heme and ficin as the controls (Fig. S2a). It was discovered that heme had a Q band from 607 to 613 nm and two absorption peaks at about 250 nm and 384 nm, and ficin had an absorption peaks at 280 nm. However, heme-incubated ficin didn’t have any distinct absorption peak at 280 nm relative to native ficin and showed obvious increase at about 371 nm, yet the Q band was indistinct. Moreover, we measured fluorescence spectra with the same controls. It was noted that unbound ficin exhibited a high emission peak at 342 nm, while heme-incubated ficin showed almost no peak at the same location (Fig. S2b). It can be seen from the circular dichroism (CD) spectra (Fig. S3), both 260–340 nm and 200–260 nm UV region, there are some modification of distribution of the secondary structure elements and some loss of hydrophobic contacts (3D structure) upon complexes formation. These results amply indicate the successful formation of hemeficin complexes. Similar to heme-Aβ complexes, the formation of the complex could attribute to the connection of heme and His residues in ficin.
3.4. Steady-state kinetics Like ficin, the catalytic reaction of heme-ficin complexes follows the typical Michaelis-Menten model toward both substrates, H2O2 and TMB. By changing the concentration of H2O2 and TMB respectively, the data were fitted well to the Michaelis-Menten curves to acquire the enzyme kinetic parameters for the reaction (Fig. 3). We obtained the MichaelisMenten constant (Km) and maximum initial velocity (Vmax) from Lineweaver-Burk plot based on the equation 1/v = Km/Vmax·(1/[S] + 1/Km). Kcat/Km is often thought of as a measure of enzyme efficiency, it allows direct comparison of the effectiveness of enzyme toward different substrates or different enzymes with the same substrate. As listed in Table S1, the Kcat/Km of heme-ficin complexes was calculated to be 24.33 and 6.95 for TMB and H2O2 respectively. The catalytic efficiency of hemeficin complexes was approximately 3 times higher than ficin. In addition, the Km value of heme-ficin complexes towards TMB and H2O2 both are lower than that of ficin, which suggested a higher affinity of heme-ficin complexes to the substrates than that of ficin. There are two main factors could contribute to the higher peroxidase-like activity of heme-ficin complexes: (1) the lower Km value of heme-ficin complexes towards TMB and H2O2 ensure a higher affinity to the substrates, which could aggregate more substrates to approach to the active sites. (2) the synergistic effects between imidazole groups of His resides and the porphyrin of heme facilitate the location of H2O2 into the active site through H-bond interaction and the formation of initial compound I [18,38]. Furthermore, we measured initial velocity versus H2O2 concentration in a range of concentrations of TMB, or vice versa to obtain double reciprocal plots (Fig. 3). The slope of the lines is parallel according with the characteristic of a ping-pong mechanism [39].
3.2. The peroxidase-like activity of heme-ficin complexes A typical chromogenic system, H2O2-TMB was chosen to investigate the peroxidase-like activity of heme-ficin complexes in comparison to that of native ficin and heme. In the presence of H2O2, TMB was oxidized to blue-colored products (oxTMB) rapidly when peroxidase was added by following the increase in absorbance at 652 nm [34]. First, as shown in Fig. S4, the chromogenic reaction catalyzed by heme-ficin complexes showed an observably higher reaction velocity than the reaction catalyzed by ficin or heme. Furthermore, the catalytic activity of heme-ficin complexes was about 1.7 fold higher than those native ficin and increased by about 55% than the superposition peroxidase-like activity of heme and ficin (Fig. 1a). Thus the result is consistent with what we expected. ESR was applied to examine •OH radicals produced by the H2O2/
3.5. Detection of H2O2 and uric acid Considering the excellent biomimetic catalytic activity of heme-ficin complexes, we applied the complexes to H2O2 detection under the above optimum assay conditions. As shown in Fig. S5, the calibration 435
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Fig. 1. (a) The UV–Vis absorption spectra of different catalytic reaction systems: (1) hemeficin complexes + TMB + H2O2, (2) ficin + TMB + H2O2, (3) heme + TMB + H2O2, (4) TMB + H2O2 in a PBS solution (pH = 5.0) after incubation for 210 min at 30 °C. The concentrations were 0.050 μg/mL of native ficin or heme-ficin complexes, 0.050 μM heme, 0.80 mM for H2O2 and TMB. (b) Spin-trapping ESR spectra of •OH radicals in different systems.
Fig. 2. The peroxidase-like activity of hemeficin is dependent on pH (a), temperature (b), H2O2 concentration (c), and incubation time (d). Experiments were carried out using 0.10 μg/mL native ficin or heme-ficin complexes with 0.80 mM TMB and H2O2 as substrate, respectively. The pH of PBS was 5.0, and the temperature was 30 °C unless otherwise stated. Error bars represent the standard deviations of three independent experiments. ΔA = A − A0, where A and A0 is absorbance in the presence and absence of catalyst, respectively.
ascorbic acid (AA) as potential interferential substances in human serum. We found that as low as 60 µM uric acid caused a dramatic increase in absorbance, while other interferential substances presented negligible effect on the absorbance at 652 nm, even when their concentrations were 5 times higher than uric acid (Fig. 4c). In addition, we also investigate the effects of coexisting substances on the determination of uric acid. A relative error of less than ± 5% was considered to be acceptable. As shown in Fig. 4d, NaCl, KCl, glucose, sucrose, urea, tryptophan, L-cysteine, ascorbic acid (AA) have no significant effect on the detection of uric acid. These results demonstrated that our developed method not only had a low detection limit, but also an excellent selectivity for uric acid determination, which provide a possible to the application in the biomedical detection.
curve of the absorbance at 652 nm against H2O2 concentration was linear from 1.0 to 120 μM. The linear regression equation was A = 0.0085 C + 0.0387 (R2 = 0.9978), A represents the absorbance at 652 nm and C represents the concentration of H2O2. Uric acid, an important biological analyte, can be oxidized to produce H2O2 and allantoin in the presence of uricase. Hence, the above H2O2 response system made it possible to further develop a platform for uric acid detection (Scheme 1b). The blue color gradually darken with increased uric acid concentration, the naked eyes can observe obviously the color variation even the uric acid concentration was as low as 5.0 μM (Fig. 4a). The absorbance at 652 nm was linearly correlated to uric acid concentration from 1.0 to 120 μM with a detection limit of 0.25 μM which is comparative or even lower than some previous colorimetric methods listed in Table S2. The linear regression equation was A = 0.0099 C + 0.0169 (R2 = 0.9928), A represents the absorbance at 652 nm and C represents the concentration of uric acid (Fig. 4b). Furthermore, we evaluated the specifity of the proposed approach using NaCl, KCl, glucose, sucrose, urea, tryptophan, L-cysteine,
3.6. Uric acid determination in human blood samples Encouraged by the above results, we then determined the uric acid concentration in human serum to explore the practical usability of our 436
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Fig. 3. Steady-state kinetic assay and catalytic mechanism of heme-ficin complexes. The velocity (v) of the reaction was surveyed by using 0.10 μg/mL ficin presented in heme-ficin complexes in 1.0 mL of 0.20 M PBS solution at pH = 5.0 and 30 °C. The error bars express that the standard deviations derived from three repeated measurements. (a) The concentration of H2O2 was 0.30 mM and the concentration of TMB as a substrate was varied. (b) The concentration of TMB was 0.10 mM and the H2O2 concentration as a substrate was varied. (c) Double reciprocal plots of peroxidase-like activity of heme-ficin complexes with the concentration of H2O2 fixed and TMB varied, (d) the concentration of TMB fixed and H2O2 varied.
4. Conclusions
sensing system. We diluted 20 times of original serum samples to guarantee the uric acid content was in the range of our established standard curve. As listed in the Table 1, the concentrations of uric acid in human serum we measured were close to the values obtained through the routine clinical method in a local hospital. The results showed that our sensing methods possess high accuracy for uric acid determination in human system.
In summary, we have prepared the heme-ficin complexes through a simple and facile one-step synthesis. The formation of the complexes could attribute to the connection of heme and His residues in ficin. Compared with native ficin, the heme-ficin complexes have the enhanced peroxidase-like activity and catalytic efficiency. Based on these Fig. 4. (a) The absorption spectra corresponding to various concentrations of uric acid. Inset: color change with the corresponding concentration uric acid from 1.0 to 120 μM (from bottom to top: 1.0, 5.0, 10, 20, 40, 60, 80, 100, 120). (b) The linear regression to plots of the absorbance at 652 nm with the uric acid. (c) The selectivity analysis for uric acid detection by monitoring the relative absorbance change at 652 nm. The concentration of uric acid was 60 μM, while the concentrations of other interferential substances were 300 μM. (d) Effects of coexisting substances on the determination of uric acid. The concentration of uric acid, NaCl, KCl, glucose, sucrose, urea, tryptophan, L-cysteine, AA was 60 μM, 100 mM, 100 mM, 5 M, 5 M, 8 M, 500 μM, 10 μM, 10 μM.
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Table 1 Determination of UA in human serum samples. Sample
This method (µM)
RSD (n = 3; %)
Hospital result (µM)
t-test
1 2
225 ± 7.8 255 ± 12
3.4 4.7
229 251
0.89 0.58
t0.1,
2
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= 2.92 (P = 0.90).
findings, we have applied the complexes to the colorimetric detection of H2O2 and uric acid. The developed method exhibited a good sensitivity and high selectivity toward uric acid. More importantly, we had detected the concentration of uric acid in human serum successfully. Therefore, this good performance of the heme-ficin complexes made it possible to apply in various fields, such as materials science, biology, and medicine. In addition, this work provides an effective way to improve the peroxidase-like activity of ficin via mimicking the architecture of the active site in HRP. Acknowledgements This research was financially supported by the National Natural Science Foundation of China (Nos. 21405124, 21175110) and the Fundamental Research Funds for the Central Universities (Nos. XDJK2013A022, XDJK2014C173, XDJK2017D052). Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.talanta.2018.04.005. References [1] Y. Chen, H. Cao, W. Shi, H. Liu, Y. Huang, Chem. Commun. 49 (2013) 5013. [2] L. Gao, et al., Nat. Nanotechnol. 2 (2007) 577. [3] W. He, H. Jia, X. Li, Y. Lei, J. Li, H. Zhao, L. Mi, L. Zhang, Z. Zheng, Nanoscale 4 (2012) 3501.
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