Synergistic Effect of Graphene Oxide and Different Valence of Cations

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Synergistic Effect of Graphene Oxide and Different Valence of Cations on Promoting Catalase Crystallization Jiayun Zou, Juntao Wu, Yunpeng Wang, Bo Zhang, Yao Wang, Fengqi Liu, Zhongqiang Yang, and Jerry Y.Y. Heng Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.9b00056 • Publication Date (Web): 21 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

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Crystal Growth & Design

Synergistic Effect of Graphene Oxide and Different Valence of Cations on Promoting Catalase Crystallization Jiayun Zoua,b, Juntao Wub, Yunpeng Wangb, Bo Zhangb, Yao Wangb, Fengqi Liua,*, Zhongqiang Yangb,*, Jerry Y. Y. Hengc,*

a. College of Chemistry, Jilin University, Changchun 130012, P. R. China

[email protected]

b. Key Laboratory of Organic Optoelectronics & Molecular Engineering of the Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China

[email protected]

ACS Paragon Plus Environment

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c. Surfaces and Particle Engineering Laboratory (SPEL), Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom [email protected]


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ABSTRACT: This study tests the effect of using the combination of graphene oxide (GO) with different valence cations as a heterogeneous nucleant on promoting catalase crystallization. By using GO and three types of salts with different valences, NaCl, MgCl2 and YCl3, the addition of GO with all three salts resulted in an increase in the percentage of crystal drops and a decrease in induction time. The experiment results further verified that there is a synergistic effect of GO and cations as the percentage of crystal drops was higher when GO with cations was added compared to control experiments where only GO or cations presented. It was also observed that the improvement in crystallization became more significant when cations with higher valence were utilized. It is believed that the enhancement in crystallization was due to the synergistic effect arising from the cation-π and electrostatic interactions between GO sheets and cations. These interactions subsequently contributed to the positively charged salt, which adsorbed and connected both negatively charged catalase molecules and the GO surfaces, increasing the local protein concentration and leading to crystallization. In addition, we compared LaCl3 and CeCl3 with YCl3 to verify the effect of the same valence salt on catalase crystallization and found that the higher the charge density, the more pronounced the


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promotion effect. This study provides a new protein crystallization methodology by exploiting GO with cations as heterogeneous nucleant to promote catalase crystallization, and brings about a new model for investigating protein crystallization mechanisms.

KEYWORDS:

synergistic effect

graphene oxide

cations

salt bridge

catalase crystallization

1. Introduction Protein crystallization is a powerful technique that brings advantages into protein engineering, drug design1,2 and the determination of protein structure.3 In this process, nucleation is one of the essential parts for controlling crystallization and includes homogeneous and heterogeneous nucleation.4,5 Compared with homogeneous nucleation which triggers nucleation at higher supersaturation levels, heterogeneous nucleation,6–9 considered as a surface or particle-assisted nucleation process,


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accelerates crystal growth at lower concentrations of proteins.10 This process is attributed to the surface or cavity interaction between macromolecules and nucleating agents.11 In 1988, minerals used as heterogeneous nuleants to promote protein crystallization was first reported.12 And along with further investigations on ‘universal’ nucleants, many substances, such as natural nucleants including small protein crystals,13 horse hair14, charged surfaces15 e.g., mica16,17 and other porous materials18,19 like bio-glass20 were utilized as heterogeneous nucleants to understand the role of interfacial properties in controlling heterogeneous nucleation. With the development of crystallization technology, some nanomaterials like 3D nanotemplates,21 carbon nanotubes,22 imprinted polymers23 and DNA origami24,25 were also applied in this research area due to their controllable structures and functional groups. Graphene oxide (GO), an unconventional soft material,26 has become a ‘star’ material in recent years. It can be treated and used as a two-dimensional (2D) polymer, highly anisotropic colloid,27 membrane liquid crystal28 or amphiphile due to its outstanding physical and chemical properties.29 This type of material has been applied for biosensors,30 drug delivery31 and the construction of various supramolecular


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architectures.32 Apart from these applications, GO has been used as a matrix to localize biological macromolecules on their surfaces33 because of its large surface area and functional groups. Some researches about using GO as a nucleant to promote nucleation of protein have been reported,34,35 and the best promotion of catalase crystallization with GO reached two-fold compared to the control in the absence of GO.34 However, a method with additional improvement in catalase crystallization experiment is always desired but still challenging. Herein, we propose that a synergistic effect of GO and cations can further promote crystallization of catalase. Scheme 1 shows that cations, serve as a salt bridge connecting protein molecules,36 are adsorbed on GO surfaces by cation-π37 and electrostatic interactions.38 In this case, protein molecules are attracted to the GO surfaces via the salt bridge,39 thereby increasing the local concentration of protein. This increment provides an environment to induce and accelerate the formation of thermodynamically stable aggregates, lower the free energy barrier and aid crystallization.14 In addition, cations with different valences may have an influence on protein crystallization in different degrees. To validate this assumption, we chose three types of salts with different valences: NaCl, MgCl2 and YCl3, and catalase was chosen as


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model protein in this study because of its important role in the immune system of living organisms.40 Findings from this work would allow the development in new methodologies and models for studying protein crystallization and their relevant applications.

Scheme 1. An illustration of the synergistic effect of GO and cations to promote protein crystallization.

2. Material and methods 2.1 Materials and methods Catalase as lyophilized powders (C40) and 2-methyl-1,3-propanediol (MPD) were

purchased from Sigma-Aldrich and utilized without any further purification. N(2hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) was purchased from Xinjing Biological Science Technologies Co. (Beijing, China). Polyethylene glycol 4000 (PEG-


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4000) was purchased from Alfa Aesar. Sodium chloride (NaCl), magnesium chloride (MgCl2), lanthanum chloride (LaCl3), cerium chloride (CeCl3) and yttrium chloride (YCl3) were purchased from Aladdin (Shanghai, China). Graphite powder (100 mesh) was purchased from Qingdao Huatai Lubricant Sealing S&T Co. Ltd., China. Hydrogen peroxide (30%), concentrated sulfuric acid (95%-98%), potassium permanganate, potassium hydroxide, sodium nitrate and hydrochloric acid (36%-38%) were all purchased from Beijing Chemical Reagent Co., China. Dialysis bags (molecule weight 8000-14000) were purchased from Lanyi Chemical Reagent Co., China. Filters of pore size 0.22 μm were purchased from Jinteng (Tianjin, China). Milli-Q water was used. All the reagents were of analytical grade or above.

2.2 Sample preparation Catalase was dissolved in 25 mM HEPES buffer solution (pH 7). All the solutions used for crystallization were filtered through filters of pore size 0.22 μm. The concentration of catalase was 5.0 mg/mL. GO was synthesized via the modified Hummers Method (Figure


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S1).41 Solutions of GO with different salts were prepared by mixing equal volume of 20 μg/mL GO and stated concentration of salts in protein precipitant solution.

2.3 Zeta potential measurements Zeta potential values of 10.0 μg/mL GO with 100.0 mM of different valence salt were measured by using Zetasizer Nano ZS system with irradiation from a standard 633 nm laser.

2.4 Protein crystallization experiment Protein crystallization experiments were conducted using conventional hanging-drop vapor diffusion technique. Crystallization drops were made by mixing 1.5 μL of protein solution and an equal volume of protein precipitant solutions in four conditions (blank without GO and salts, GO with NaCl, GO with MgCl2 and GO with YCl3) at different concentrations. The drops were equilibrated against 300 μL of the protein precipitant solution containing 25 mM HEPES, 5% w/v PEG-4000 and 5% v/v MPD in the reservoir well, as shown in Scheme 2. Concentration used for NaCl and MgCl2 were 0.1 mM, 1.0


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mM, 10.0 mM and 100.0 mM. However, for YCl3, a lower concentration range of 0.01 mM, 0.1 mM, 0.5 mM and 1.0 mM was chosen because serious precipitation was observed when catalase was introduced into the solution containing a higher concentration of YCl3 (≥ 10.0 mM). The crystallization plates were incubated at 4 °C for two weeks after the plates were sealed, and the crystal drops were observed daily by an optical microscope (Olympus SZM-T4). The sample size for each set of experiments was 144 drops. The effect of the additives that consist of GO and cations of different valences was evaluated by the percentage of crystal drops.

Scheme 2. An illustration of hanging drop vapor diffusion experiment setup.


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3. Results and Discussion 3.1 Zeta potential of the GO with different cations


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In this research, we aimed to study whether the combination of GO and cations have a


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synergistic effect on promoting protein crystallization. We have chosen the concentration


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of GO for all the experiments in this study to be 10.0 μg/mL, because mixing GO at low concentration with salt solutions performs better dispersity in water, which avoids serious

coagulation,42,43 and it supports clear images of droplets in the sight of the microscope. Three types of salts: NaCl, MgCl2 and YCl3 were selected to be combined with GO sheets based on their different valences. By mixing GO and cations, the π electrons from the GO surfaces are able to attract cations due to the cation-π interactions. Besides, the opposite charges between the functional groups (carboxyl and hydroxyl groups) on GO surfaces and cations may help to strengthen this attachment. To test this presumption, zeta potential experiment for GO combined with different salts at the same salt concentration of 100.0 mM was carried out.


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Figure 1. Zeta potential results of solutions of 10.0 μg/mL GO (grey bar) and GO combined with 100.0 mM NaCl (red bar), 100.0 mM MgCl2 (yellow bar) and 100.0 mM YCl3 (green bar) in catalase crystallization buffer.

Figure 1 presents the values of zeta potential for 4 types of mixtures in crystallization buffer. The zeta potential value for GO was about -7 mV, the negative charge can be attributed to the carboxyl and hydroxyl groups on GO surfaces. When higher valence of salts was added into the solution, the value moved towards positive region. The effect of NaCl, a monovalent salt, on the change of zeta potential of GO was minimum. With the increment of valence, the zeta potential value escalated. This data for GO+MgCl2 sample nearly arrived at 0 mV and the value of GO+YCl3 sample even reached 17 mV. The changes in zeta potential of GO was originated from the stronger combination between GO and cations due to the increase of valence,44,45 demonstrating that the cations were successfully adsorbed onto the GO surface by interactions containing cation-π and electrostatic interactions.


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3.2 Determination of optimal concentration of different salts

GO + NaCl

GO + MgCl2

GO + YCl3

a) 100 80 60 40 0 0.5

20

0. 01

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MgCl2 ] % [ s

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8 6 4 2

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an k

0 Bl

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Figure 2. a) Percentage of crystal drops of catalase using GO with different valence salt on the 14th day. (NaCl and MgCl2: 0.1 mM, 1.0 mM, 10.0 mM and 100.0 mM; YCl3: 0.01 mM, 0.1 mM 0.50 mM and 1.00 mM) b) The induction time for the first crystal to form. Concentration of NaCl MgCl2 was 10.0 mM and YCl3 was 0.5 mM. Note: Crystals


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appeared on the first day for all drops containing GO with YCl3, there was no deviation of the result.

A series of experiments were carried out to explore if the interactions between GO and cations of different valences have positive effect on promoting protein crystallization. Model protein catalase at a concentration of 5.0 mg/mL was chosen. Sets of hangingdrop vapor diffusion experiments were conducted to test the effect of different valence salts on catalase crystallization. Figure 2a presents the percentage of crystal drops by adding GO combined with different valences and concentrations of salts. For NaCl and MgCl2, concentrations used were 0.1 mM, 1.0 mM, 10.0 mM and 100.0 mM. However, for YCl3, a lower concentration range of 0.01 mM, 0.1 mM, 0.5 mM and 1.0 mM was chosen because serious precipitation was observed when catalse was introduced into the solution containing higher concentration of YCl3 (≥ 10.0 mM). Along x-axis of Figure 2a, the percentage of crystal drops increased with salt concentration when GO with NaCl was used as additive. From salt concentration of 0.1 mM to 10.0 mM, the percentage of crystal drops raised from 11 % to 22 %. However, when salt concentration was increased to


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100.0 mM, the effect on promoting crystallization dropped with only 6 % of crystal drops. Same trend was observed when GO and MgCl2 were used as additive. The highest crystallization ratio of this group was 90 % at 10.0 mM MgCl2. Similarly, the percentage of crystal drops declined to 38 % when the concentration of MgCl2 climbed to 100.0 mM. This phenomenon may be due to the protein molecules trapped by free cations in solution, thus the enrichment of proteins on GO surfaces were prevented. Nevertheless, GO combined with YCl3 achieved the peak value of 100 % at very low concentration of 0.5 mM, it did not decrease when the concentration was increased to 1.0 mM. Since 0.5 mM YCl3 has reached the threshold concentration for the best performance on promoting crystallization, and cations at low concentration in solution can not restrict protein molecules enrichment on GO surfaces. Above all, it is noted that GO combining with YCl3 had the best crystallizing performance, which indicates that the higher the valence, the better the synergistic effect. The reason behind this conclusion is that at the experiment condition of pH 7, the catalase is negatively charged due to its isoelectric point of 5.4, cations adsorbed on the GO sheet will further help to attract the catalase molecules by electrostatic interaction. With the increase in cation valence, the zeta potential value


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increases, causing an increment in the strength of cation-π and electrostatic interactions. As a result of this, the local protein concentration can be raised leading to crystallization effortlessly. Figure 2b shows the induction time of the catalase crystals when GO with different valence salts were added. Data were collected from the optimal crystallization concentration at 10.0 mM, 10.0 mM and 0.5 mM for NaCl, MgCl2 and YCl3 respectively as the first crystal was observed. The bar charts proved that by increasing the valence of the salts, the induction time can be significantly decreased from about 8 days to 1 day. This advantage arises from the stronger interaction between cations on GO surface and proteins in solution, and has potential applications for increasing the efficiency and reducing the time cost in pharmaceutical and biological applications.

3.3 The additional effect of GO on protein crystallization NaCl

MgCl2

YCl3

100 80 60 40 20 lt Sa

YCl3

lt Sa

NaCl

[

d l a t s y r c

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Figure 3. Percentage of crystal drops of catalase by adding GO, salts of different valences and GO combined with salts of different valences compared with blank (without GO and salt). All results were obtained on the 14th day.

A series of hanging-drop vapor diffusion crystallization experiments were set up to validate if the positive effect was from the combination of GO and salt but not from solely GO or salt. Each well contained 4 drops: blank (catalase precipitant without GO and salt), GO, salt and GO with salt. The results of these experiments are presented in Figure 3. The optimal concentrations for each salt chosen from the last experiment presented in Figure 2 were 10.0 mM for NaCl and MgCl2, 0.5 mM for YCl3. In the experiments of NaCl and MgCl2, the crystallization ratio for GO combined with salt was higher than that of salt or GO. For example, when 10.0 mM of MgCl2 was added as an additive, the crystallization ratio was only 28%. However, by addition of GO with MgCl2, the percentage of crystal drops sharply rose to 94%. In the case of YCl3, although the percentage of crystal drops was 100% for both GO+YCl3 and YCl3, obviously larger crystals were obtained in the former droplets (Figure S4). Larger crystal size benefits for the analysis of structure and


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the design of new types of medicine. These observations prove that there is indeed a synergistic effect when GO and cations are combined and used as a heterogeneous nucleant.

3.4 The effect of GO with same valence salts on protein crystallization From above experiments, we believe that trivalent salts have better promotion effect in catalase crystallization system comparing to mono- or di-valent salts. To further explore the effect of GO combined with salts at the same valence on catalase crystallization, we chose another two trivalent salts, LaCl3 and CeCl3. Figure 4 presents the percentage of crystal drops by adding GO combined with different salts at the same valence and concentration over a period of 14 days. It was obviously shown that GO with these three trivalent salts had the same influence on induction time (1 day) of catalase crystallization and the percentage of the crystal drops almost reached 100% in 6 days. This result attributes to the strong attraction between high valence cations on the GO surface and


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catalase in crystallization buffer. But a noticeable phenomenon that the proportion of the


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crystal drops in different salts conditions had an obvious distinction on the first day was observed. The percentage of crystal drops on the first day for LaCl3 was only 16% while this data for CeCl3 and YCl3 was 80% and 100%, respectively. In favor of exploring this phenomenon, we used zeta potential method to identify the electrical properties of different salt systems. As shown in Figure S5, the zeta potential value for GO+LaCl3 was 10 mV, followed by 15 mV for GO+CeCl3 and 17 mV for GO+YCl3. The lowest zeta potential value of GO with LaCl3 may be due to the lowest charge density because of its

100

Percentage of crystal drops [%]

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Blank GO + LaCl3 GO + CeCl3 GO + YCl3

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large atomic radius when the number of charges was the same as other cations. These observations prove that the promotion effect of catalase crystallization depends on the ionic charge intensity when salts with the same valence are selected.


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Figure 4. Percentage of crystal drops of catalase using GO with same valence salt over a period of 14 days. The concentration of salt was 1.0 mM. Four groups: Blank (gray), GO with LaCl3 (orange), GO with CeCl3 (blue), GO with YCl3 (green).


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A

B

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10.0 mM

C

100.0 mM

D

E

F

Blank

10.0 mM

100.0 mM

G

H

I

Blank

0.5 mM

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600 μm

3.5 Determination of morphology of catalase crystals Figure 5. Morphology of catalase crystals grew in (A, D, G) blanks without GO and salt, (B-C) GO with NaCl, (E-F) GO with MgCl2 and (H-I) GO with YCl3. The concentration of salts was indicated on the image.


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Another interesting observation was that different morphologies of catalase crystals appeared at different concentrations of salts, as shown in Figure 5. At the optimal concentration of different valence salts, the morphologies of crystals were almost the same in blade-like shape. However, when the salt concentrations further increased, the morphologies for different valence salts changed: crystals grew in addition of GO with 100.0 mM NaCl had tetragonal and hexagonal shapes, orthorhombic shape in the case of 100.0 mM MgCl2 and needle-like shape in the case of 1.0 mM YCl3. These changes may be originated from the specific interaction46,47 between the free cations and protein molecules in the solution.48

4. Conclusion In this paper, a new methodology which utilizes the synergistic effect of GO with cations to enhance protein crystallization has been reported. Notable improvements of crystallization effectiveness including increment in the percentage of crystal drops and decrement in induction time were observed. Besides that, these effects became more


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significant when employing cations with higher valence. Especially in the condition of GO with YCl3, the percentage of crystal drops even increased more than tenfold, and almost all droplets crystallized during the first day of the experiment. In addition, for different salts with trivalence, the promotion effect indicated that the higher the charge density, the higher promotion effect. It is believed that this phenomenon was originated from the synergistic effect of GO and cations which raised the local concentration of catalase. This synergistic effect may have outstanding potential in the system of other protein crystallization experiments. In future studies, GO sheets may be synthesized and functionalized more controllably to enhance the strength of the salt bridge, increasing the synergistic effect to promote crystallization of other proteins. With the development in the technology of 2D material, this methodology of the synergistic effect may be extended to other 2D materials as new heterogeneous additives to understand the process of biological macromolecules crystallization.

Supporting information:


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The characterization of GO (Figure S1), graphs of relevant experiments in the article with error bars (Figure S2, Figure S3), crystal morphology of catalase observed under an optical microscope (Figure S4) and zeta potential for GO with three trivalence salts (Figure S5).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (F.L.). *E-mail: [email protected] (Z.Y.). *E-mail: [email protected] (J.Y.Y.H.).

ORCID Zhongqiang Yang: 0000-0002-9399-4424

Notes The authors declare no competing financial interest.


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Acknowledgment This work was supported by the National Natural Science Foundation of China (21174053, 21474059). JYYH acknowledges the EPSRC (EP/N015916/1) for funding.

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For Table of Contents Use Only

Synergistic Effect of Graphene Oxide and Different Valence of Cations on Promoting Catalase Crystallization Jiayun Zoua,b, Juntao Wub, Yunpeng Wangb, Bo Zhangb, Yao Wangb, Fengqi Liua,*, Zhongqiang Yangb,*, Jerry Y. Y. Hengc,*

a. College of Chemistry, Jilin University, Changchun 130012, P. R. China

[email protected]

b. Key Laboratory of Organic Optoelectronics & Molecular Engineering of the Ministry of Education, Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China

[email protected]


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c. Surfaces and Particle Engineering Laboratory (SPEL), Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom

[email protected]


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Cations act as salt bridges to attract protein molecules onto graphene oxide surface to increase local protein concentration and promote protein crystallization.


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Synergistic Effect of Graphene Oxide and Different Valence of Cations

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