Selenium-functionalized Graphene Oxide that Can Modulate the

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Selenium-functionalized Graphene Oxide that Can Modulate the Balance of Reactive Oxygen Species Jiahao Xia, Feng Li, Shaobo Ji, and Huaping Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b05951 • Publication Date (Web): 06 Jun 2017 Downloaded from http://pubs.acs.org on June 13, 2017

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ACS Applied Materials & Interfaces

Selenium-functionalized Graphene Oxide that Can Modulate the Balance of Reactive Oxygen Species Jiahao Xia†, Feng Li†, Shaobo Ji†, Huaping Xu*† †

Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department

of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China Abstract

Graphene oxide (GO) is an important two-dimensional material since it is water soluble and can be functionalized to adapt to different applications. However, the current covalent functionalization methods usually require hash conditions, long duration and sometimes even multi-steps, while noncovalent functionalization is inevitably unstable, especially under physiological environment where competing species exist. Diselenide bond is a dynamic covalent bond and can respond to both redox condition and visible light irradiation in a sensitive manner. Thus, in this work by combining the stimuli response of diselenide bond and the oxidative/radical attackable nature of GO, we achieved the in situ covalent functionalization of GO simply by stirring GO with diselenide-containing molecules in aqueous solution. The covalent functionalization was proved by FT-IR, Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), Atomic Force Microscopy (AFM), Thermogravimetric Analysis (TGA), etc. and the functionalization mechanism was deduced to involve both redox reaction and radical addition reaction according to the XPS, Atomic Emission Spectroscopy (AES) and Raman spectroscopy. Moreover, we modified GO

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with a biocompatible diselenide-containing polymer (mPEGSe)2 and found selenium-functionalized GO could modulate the balance of reactive oxygen species (ROS). GOSe could decrease ROS level by accelerating the reduction of peroxides when the ROS concentration is high while boosting the ROS level by in situ generating ROS when its concentration is relatively low.

Keywords

Graphene oxide, selenium, reactive oxygen species, dynamic covalent bond, redox reaction

Introduction

With the rapid development of carbon materials, it is believed that we are entering the era of carbon. Graphene oxide (GO), without any doubt, is a crucial member of the carbon material family. GO is a typical two-dimensional material consisting of covalently bonded carbon sheet and a series of reactive oxygen-containing functional groups.1-3 These oxygen-containing groups not only render graphene oxide with dramatically enhanced solubility in aqueous solution, but also opportunities to be modified to adapt to different application environments.4-9 Chemically modified graphene oxide (CMGO) has been systematically studied and integrated into a range of research fields, including cancer treatment, drug delivery, catalysis, biosensors, quantum dots, etc.10-18 The surface modification of GO could be roughly divided into two types, i.e. covalent functionalization and noncovalent functionalization. Covalent functionalization is usually achieved by


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reacting with oxygen-containing functional groups.19-24 Noncovalent functionalization, in contrast, allows GO to bind with a series of species like polymers, drugs, metals, nanoparticles and graphene analogies via noncovalent interactions including hydrogen bonding, van der waals interactions, π-π interactions, etc.25-26 Noncovalent functionalization is inevitably unstable, especially under physiological environment where amounts of competing species exist. Meanwhile, covalent functionalization is stable enough, but usually requires hash conditions, long duration and sometimes even multi-steps. Hence, it is of vital importance to develop additional covalent functionalization method that can be performed under relatively mild and straightforward way. Selenium is an essential trace element in living organisms and possesses unique chemical features due to its atomic structure.27-28 Selenium atom has larger atomic radius and smaller electronegativity compared with sulfur atom, the bond energy of diselenide bond was much lower in contrast to that of disulfide bond, at 172 kJ mol−1 and 240 kJ mol−1 respectively.29 As a result, it has been proved that diselenide bond can respond to both redox condition and visible light irradiation in a highly sensitive manner.30-32 The diselenide bond could be cleaved to form seleninic acid or selenol under redox condition, and under visible light irradiation or under heating selenium radicals could be generated. Thus, a series of diselenide-containing polymer systems have recently been developed for use as promising drug delivery vehicles and self-healing materials.33-38



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Graphene Oxide has long been recognized to be rather oxidative and has been discovered to be capable to efficiently oxidize various alcohols and alkenes into aldehydes and ketones, respectively.39-40 Thus, it enlightened us that by taking the advantage of dual redox response and visible light response of diselenide bond, we may be able to in situ covalently functionalize GO in a simple and mild way via oxidation reaction and radical addition reaction. Moreover, selenium has proven to be capable of modulating the balance of reactive oxygen species (ROS). Specifically, selenium has long been integrated into the design of gluthathione peroxidase (GPx) mimics to catalyze the reduction of a variety of hydroperoxides (ROOH).41-43 Recent studies have also shown that selenium possesses anti-tumor effect since it can catalyze the generation of reactive oxygen species (ROS) in vitro.44-45 Therefore, we anticipate diselenide-containing PEG functionalized GO may be able to modulate the balance of reactive oxygen species (Scheme 1).

Scheme 1. Graphene oxide was in situ functionalized by diselenide-containing polyethylene glycol in a relatively mild and simple way. Experimental Section


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Materials. Selenium powder and sodium borohydride were purchased from Aladdin Chemical Reagent Co. Ltd. Poly (ethylene glycol) methyl ether (average Mn= 5000) was product from Sigma-Aldrich Co. LLC. 4-nitrothiophenol，Tosyl chloride, tetraethylene glycol and all deuterated solvents were purchased from J&K Chemical. Graphene oxide (GO) aqueous solution were purchased from Carmery Materials Technology Co. Ltd. Instruments. The

77

Se NMR and 1H NMR spectra were performed on a

BRUKER AVANCE III HD 400 (400 MHz) spectrometer. Gel permeation chromatography (GPC) measurements were recorded on the breeze system, Waters Corporation (Styragel® Columns) with polystyrene as standard and dimethyl formamide (DMF) as eluent. Electrospray ionization mass spectrometry (ESI-MS) spectrum was performed on a LTQ LC/MS apparatus. The FT-IR spectra were obtained from PERKIN ELMER SPECTRUM GX FT-IR SYSTEM. The samples were first mixed with potassium bromide and pressed into tablets. The Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) were performed on a ION-TOF GmbH TOF.SIMS 5. The X-ray Photoelectron Spectroscopy were measured on a ULVAC-PHI Quantro SXM. The samples were prepared by dropping on silica wafers and were then dried in vacuum to form thin films. The Raman Spectroscopy was obtained from HORIBA Evolution with the excitation wavelength at 532 nm. The Atomic Emission Spectroscopy was conducted on a VARIAN Vista-MPX. The X-ray Diffraction (XRD) was performed on a D8 ADVANCE Diffractometer. The Atomic Force Microscope images were recorded from a BRUKER Dimension Icon by



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tapping mode. The Thermogravimetric Analysis (TGA) was measured on a TA INSTRUMENTS Q5000IR. The UV-VIS Spectroscopy was measured by a HITACHI U-3010 spectrophotometer. The Confocal Microscopy was conducted on a Zeiss LSM710. Preparation of di-(tetraethylene glycol) diselenide. The synthetic route of di-( tetraethylene glycol) diselenide (HOEG4Se)2 were shown on Figure S1A. First 1.0017 g (0.0250 mol) NaOH, 3 mL deionized water, 22 mL (0.1274 mol) tetraethylene glycol and 3 mL THF were put into a 100 mL flask in sequence. Then 3.0040 g (0.0158 mL) Tosyl chloride were dissolved in 8 mL THF and dropwisely added into the flask within 30min. The reaction was performed on room temperature. After 6h 80 mL deionized water were poured into the flask to quench the reaction. The mixture was extracted by 150 mL CH2Cl2 in total for three times and washed with 900 mL saturated NaCl solution in total for six times. After drying by anhydrous sodium sulphite, the solvent was evaporated to obtain a colorless oil-like liquid (HOEG4OTs, 3.5041g). The crude product was directly used without further purification. Yield 63.9%. In the next step, Se powder 0.3800 g (0.0048 mol) and sodium borohydride 0.1701 g (0.0045 mol) were reacted with water (5 mL) for 30 minutes in nitrogen atmosphere to obtain disodium diselenide. THF solution of HOEG4OTs (1.5200 g, 0.0044 mol) was then added under nitrogen flow and reacted at 50 °C overnight. The product was purified by column chromatography using a 20:1 mixture of CH2Cl2 and methanol as eluent. At last 0.3436 g golden oil-like liquid was


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obtained. ESI-MS and NMR results are shown on Figure S1(B), (C), (D). Yield 30.7%. MS (ESI) (Figure S1B) Calc. (M+ = 514.06), obsvd. (M+NH4)+ = 532.09. 1H NMR (Figure S1C) (400MHz, CDCl3, 298 K) δ (ppm): 3.77 (4H, t, HOCH2), 3.70-3.60 (24H, m, CH2CH2O), 3.12 (4H, t, SeSeCH2), 2.45 (2H, s, HO).

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Se NMR

(Figure S1D) (CDCl3, 298 K) δ (ppm): 290 (2Se, s, CH2SeSeCH2). Preparation of di- (poly (ethylene glycol) methyl ether) diselenide (mPEGSe)2 (average Mn= 10000). The synthetic route of di- (poly (ethylene glycol) methyl ether) diselenide (mPEGSe)2 (average Mn= 10000) were shown on Figure S2A. 2.0000 g mPEG (average Mn= 5000) was previously dried by azeotropic distillation with toluene. Then 10 mL CH2Cl2 was added to dissolve mPEG and the bottle was quickly sealed with a rubber plug to prevent from moisture. 0.7626 g tosyl chloride was dissolved in 6.7 mL dry pyridine and carefully injected into the bottle. The reaction was performed on room temperature for 24h. After the reaction the solvents were removed and the obtained substance was re-dissolved in dry CH2Cl2. The product was purified by column chromatography using a 12:1 mixture of CH2Cl2 and methanol as eluent and precipitated in cold diethyl ether. Yield 82.1%. In the next step, Se powder 0.1580 g and sodium borohydride 0.0378 g were reacted with ethanol (5 mL) for 30 minutes in nitrogen atmosphere to obtain disodium diselenide. H2O solution of mPEGOTs (1.0000 g) was then added under nitrogen flow and reacted at 50 °C for 24h. The product was purified by column chromatography using a 10:1 mixture of CH2Cl2 and Methanol as eluent and precipitated in cold diethyl ether. At



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last 0.2841 g white powder was obtained. NMR and GPC results are shown on Figure S2(B), (C). Yield 28.4%. 1

H NMR (Figure S2B) (400MHz, CDCl3, 298 K) δ (ppm): 3.82-3.47 (m,

OCH2CH2), 3.38 (6H, s, CH3O), 3.11 (4H, t, SeSeCH2). GPC (Figure S2C) (DMF as the eluent, 303K) Average Mw = 10000 Da. Functionalization of Graphene Oxide. (HOEG4Se)2 was reacted with GO to form GO(HOEG4Se)2. In a typical procedure, 250 mg (HOEG4Se)2 was mixed with 5 mL GO solution (2 mg/mL) in a 25 mL Schlenk Flask. The solution was deoxygenated by three standard freeze–pump–thaw cycles and then reacted at 80 °C. After 24 h, the obtained solution was cooled and centrifuged at 15000 rpm for 60 min. The precipitate was collected and washed seven times by dichloromethane using a sonic probe (237.5 W) to remove the unreacted (HOEG4Se)2. The final product was obtained after vacuum drying. (mPEGSe)2 was reacted with GO to form GO(mPEGSe)2. The rest operations were the same as preparing GO(HOEG4Se)2. 50 mg (mPEGSe)2 was used to react with 5 mL GO solution (2 mg/mL).

Assay of Catalytic Activity. The catalytic activities of GO and GO(mPEGSe)2 to eliminate the H2O2 were determined by UV-Vis Spectrum. The decrease of the UV absorption at 317 nm due to the oxidation of the 4-nitrothiophenol (NTP) was regarded as the evaluation criterion of the catalytic ability. A typical test solution (400 µL) contained H2O2 (1500 µM), NTP (600 µM) and sample (0.05 mg/mL). The relatively hydrophobic substrate NTP was previously dissolved in methanol while the sample was dispersed in PBS (50 mM, pH = 7.0). Before testing, 500 µL NTP


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solution and sample solution were pre-mixed and put in a water bath to reach 37 °C. Then H2O2 solution was added to the mixture and the UV-Vis spectrum monitoring was immediately started. The initial rate was determined by the equation (1), where ε is the molar extinction coefficient of NTP (11780 M-1cm-1) and l is the thickness of the tested liquid layer (0.2 cm-1).

(1) ROS Assay. To prepare samples for confocal microscopy, MDA-MB-231 cells were seeded in confocal dishes with a density at 5 × 104 cells per dish. The cells were kept in culture for 24 h before substituting the medium with media containing samples at 10 µg/mL and 25 µg/mL, respectively. After 24 h, the media with samples were substituted with DCFH-DA (5 µg/mL) in DMEM. The cells were incubated for 30 min at 37 °C and were then washed with PBS for three times. Each dish was added with one milliliter of 4% paraformaldehyde to fix the cells. The cells were washed with PBS for three times before observing on a confocal microscope using an Ex/Em of 488/525 nm and a light field. Results and Discussion Functionalization of Graphene Oxide by diselenide-containing small molecule (HOEG4Se)2. First, to demonstrate the concept that diselenide bonds could react with GO, diselenide containing small molecules were experimented. A hydrophilic diselenide-containing molecule (HOEG4Se)2 was synthesized according to our previous report (Figure S1).46 Then (HOEG4Se)2 was dissolved in GO aqueous solution and stirred at 80 °C for 24 h. The unreacted diselenide-containing molecules



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were removed via consecutive washing by dichloromethane (see experimental sections for detailed information), the obtained material was named GO(HOEG4Se)2. Characterization of the GO(HOEG4Se)2 modified by (HOEG4Se)2. Fourier Transform Infrared Spectroscopy (FT-IR) was firstly conducted in order to verify that the diselenide-containing molecule has been successfully functionalized onto the surface of GO (Figure 1A).

The most obvious change before and after surface

functionalization was the appearance of the peak at 2924 cm-1 and 2853 cm-1, which could be ascribed to C-H stretches derived from (HOEG4Se)2 molecules grafted to GO. In order to rule out the effect of noncovalent functionalization, we used HOEG4OH without diselenide bond to mix with GO under the same condition and same washing process. From FT-IR result (Figure S3) the C-H stretches were neglectable, which implied that the relatively strong C-H signal of GO(HOEG4Se)2 was due to the covalent functionalization. We further used Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) to confirm the covalent functionalization (Figure 1B). After surface functionalization, a small peak at 79.92 appeared, which was in accordance with the molecular weight of one of the selenium’s isotope with highest relative abundance. The strong peak adjacent to selenium was sulfite ion derived from the GO preparation step. The rest of the isotope peaks of selenium could also be clearly found in the spectrum (Figure S4). The selenium signal in ToF-SIMS together with the FT-IR result indicated that (HOEG4Se)2 has indeed covalently grafted onto the GO surface.


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Figure 1. Evidence of covalent functionalization (HOEG4Se)2. A) FT-IR results of (HOEG4Se)2, GO and GO(HOEG4Se)2. The peak appeared at 2924 cm-1 and 2853 cm-1 after surface functionalization could be ascribed to C-H stretches derived from (HOEG4Se)2 molecules grafted to GO. B) ToF-SIMS result of GO and GO(HOEG4Se)2. The small peak at 79.9 represented one of the isotope of Selenium. Investigation of the functionalization mechanism. After confirming the successful functionalization, we then sought to investigate the mechanism of the reaction. XPS is a powerful tool to study the surface chemical compositions of GO and GO(HOEG4Se)2 (Figure 2). The XPS C 1s spectrum of GO showed roughly three separated peaks. The peak at 285.0 eV represents the C-C bonds, the peak at 286.9 eV represents the C-O bonds while the peak at 288.0 eV stands for C=O bonds. The relative large peak area of C-O compared with that of C-C implied a high percentage of oxygen functionality. After the reaction, however, the intensity of both C-O and C=O peaks declined significantly, which indicated the consumption of oxygen containing groups during the reaction. It is worth noting that from the FT-IR result we could also observe the relative decrease of the intensities of the C-O and C=O peaks, which was in accordance with the XPS result. Thus, as we anticipated, the GO was



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reduced during the reaction with (HOEG4Se)2. The XPS Se 3d spectrum of GO(HOEG4Se)2 showed two peaks, which indicated the existence of selenium elements in two different valences. This result implied the possibility of two types of grafting mechanism. Raman spectrum is a useful tool to detect the extent of the defects via the intensity ratio of D band and G band (i.e. ID/IG). From Figure S5 we can calculate the ID/IG before and after reaction were 0.91 and 0.98, respectively. The minor increase of the ID/IG indicated that the extent of defects rose slightly after reaction. Since heating and visible light irradiation can generate the selenium radicals in solution, it is probably due to the attack of the selenium radical that converted some of the sp2 carbon atoms into sp3 carbon atoms.


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Figure 2. XPS results of A) GO C 1s. B) GO(HOEG4Se)2 C 1s C) GO(HOEG4Se)2 Se 3d. The intensity decline of both C-O and C=O peaks in C 1s spectrum indicated the consumption of oxygen containing groups during the reaction. The two peaks in Se 3d spectrum of GO(HOEG4Se)2 indicated the existence of selenium elements in two different valences. We adjusted the concentration of (HOEG4Se)2 to investigate its effect on the grafting density. The concentration of selenium element was obtained from Atomic Emission Spectroscopy (AES) and the related grafting density was calculated. The grafting density increased from 1908 carbon atoms/chain to 359 carbon atoms/chain when the concentration of (HOEG4Se)2 was raised 100 folds (Table 1). The XPS C 1s spectra of GO(HOEG4Se)2 (1) and (2) (Figure S6) indicated that the extent of the reduction of GO declined with the decrease of the concentration of (HOEG4Se)2, which was in consistent with the AES results. Combining the XPS, FT-IR, Raman spectrum and AES results, we proposed two possible functionalization mechanisms. Some of the (HOEG4Se)2 molecules bound onto the GO surface were achieved by oxidation reaction with the reactive oxygen-containing groups, which leads to the reduction of the GO. Other (HOEG4Se)2 molecules were grafted onto the GO surface via selenium radicals attacking the carbon sheets. Table 1. Grafting density obtained from different (HOEG4Se)2 concentrations. Sample

Mass of

Concentration of

Grafting Density (carbon

(HOEG4Se)2 (mg)

Selenium (mg/mL)

atoms/ chain)



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GO(HOEG4Se)2 (1)

2.5

2.15

1908

GO(HOEG4Se)2 (2)

25

5.92

693

GO(HOEG4Se)2

250

11.42

359

Functionalization

of

Graphene

Oxide

by

diselenide-containing

macromolecule (mPEGSe)2. After confirming diselenide bond was applicable for GO functionalization, polymer system was further investigated. Polyethylene glycol (PEG), as a type of hydrophilic and biocompatible polymer, has been proven to render GO low cytotoxicity and high stability in physiological solutions.47-50 We therefore synthesized a diselenide-containing PEG named (mPEGSe)2 (average Mw = 10000 Da, methyl capped, Figure S2). The (mPEGSe)2 was then used to react with GO to obtain GO(mPEGSe)2 following the same procedure as preparing GO(HOEG4Se)2. Characterization of the GO(HOEG4Se)2 modified by (mPEGSe)2. Since the chemical structure of (mPEGSe)2 is quite close to (HOEG4Se)2, similar experimental results could be respected for some characterizations, though the relative concentration of diselenide bond were lower for (mPEGSe)2 compared with that of (HOEG4Se)2. From FT-IR results (Figure 3A) an apparent peak at around 2870 cm-1 can be observed which could be accounted for the C-H signals from PEG chains grafted on GO. When using mPEGOH without diselenide bond to replace (mPEGSe)2, no obvious peak could be spotted on the same wavenumber, which excluded the effect of noncovalent binding of PEG (Figure S7). Unlike pristine graphite, GO exhibited a


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single peak at 2θ = 11.48

o

in the X-Ray Diffraction (XRD) spectrum since the

decoration of oxygen-containing groups and intercalated water molecules expanded its interlayer distances. After grafted by (mPEGSe)2, the diffraction peak further shifted to 2θ = 9.47 o with the corresponding interlayer spacing increased from 7.70 Å to 9.33 Å (Figure 3B). This could be explained by the incorporation of PEG chains into the GO layer which further enlarged the layer distance. Atomic Force Microscopy (AFM) was utilized to characterize the surface morphology and the thickness of the materials (Figure 3C and 3D). The average thickness of GO was 1 nm with lateral dimensions at micrometer level, which indicated the single layer dispersion of GO. After functionalized by (mPEGSe)2, however, obvious protuberances could be spotted on the surface with the thickness of the GO sheets varies from 1nm to 3nm, which was probably due to the polymer wrapping on the GO surface.



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Figure 3. Evidence of covalent functionalization of (mPEGSe)2. A) FT-IR results of (mPEGSe)2, GO and GO(mPEGSe)2. The peak appeared at 2870 cm-1 after surface functionalization could be ascribed to C-H stretches derived from (mPEGSe)2 molecules grafted to GO B) XRD result of GO and GO(mPEGSe)2. C) AFM image of GO D) AFM image of GO(mPEGSe)2. Obvious protuberances could be spotted on the surface after functionalization.


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XPS (Figure S8) and Raman spectra (Figure 4A) also confirmed the covalent grafting of (mPEGSe)2 since the C-O/C-C ratio declined in XPS C 1s spectrum and the ID/IG ratio experienced a minor rise. We then employed the Thermogravimetric Analysis (TGA) to calculate the grafting density of the GO(mPEGSe)2 (Figure 4B). There were three major mass losses for GO(mPEGSe)2. The first weight loss before 200 °C referred to the loss of intercalated water molecules and the second one at around 240 °C should be ascribed to the loss of oxygen-containing functional groups. The third weight loss of GO(mPEGSe)2 around 270 °C to 400 °C was due to the decomposition of PEG chains which was in accordance with the decomposition process of (mPEGSe)2. In contrast, no obvious degradation of GO at this temperature window could be observed. It is worth noting that the thermal stability of GO material decreased a bit after functionalized by (mPEGSe)2, we deduced it was because the functionalization process caused more defects as implied by Raman spectra. The grafting density could therefore be calculated based on the third weight loss and the result was 2048 carbon atoms/chain. It was not surprising since diselenide bond concentration of (mPEGSe)2 was relatively low, and the chain size was much larger.



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Figure 4. A) Raman spectra of GO and GO(mPEGSe)2. ID/IG ratio experienced a minor rise after functionalization indicated the extent of defects rose slightly after reaction. B) TGA image of (mPEGSe)2, GO and GO(mPEGSe)2. The grafting density could be calculated to be 2048 carbon atoms/chain based on the third weight loss. After successful functionalization, we then sought to investigate the bioactivity of the GO(mPEGSe)2. Selenium has long been integrated into the design of gluthathione peroxidase (GPx) mimics, we wonder if the activity of selenium remained after connected to GO.41-43 GPx catalyzes the reduction of noxious hydroperoxides (ROOH) into nontoxic hydroxyl compounds (ROH) based on the glutathione (GSH) substrate. Hence, three ROOH with different structures and 4-nitrobenzenethiol were selected as the alternative of hydroperoxides and GSH, respectively, to evaluate the catalytic activity of GO and GO(mPEGSe)2 (Figure 5).51 The initial reduction rate of the control group for CuOOH was 4.97 µM/min. When GO was added, the initial reduction rate reached 10.06 µM/min, which was 2.0 times faster than that of control group. It was because the electron rich GO material could facilitate the electron transfer from thiol groups to CuOOH.52 When GO(mPEGSe)2 was used, however, a further increase was detected with initial reduction rate at 26.93 µM/min，which was 5.4 times faster than that of control group. Other two ROOH showed similar catalytic trends but the increase ratios were quite different. After adding GO(mPEGSe)2 as catalyst, the reduction rate of t-BuOOH increased by 3.5 times while the reduction rate of H2O2 only increased by 3.0 folds (Table S1). We anticipate it is because the catalyst favors organic substrates and the CuOOH could offer additional π-π


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interaction with GO catalyst due to the aromatic ring in the molecular structure. Our results indicated selenium-functionalized GO can accelerate the reduction of peroxides when the ROS concentration is high (1500 µM in this experiment).

Figure 5. Catalytic activity of GO and GO(mPEGSe)2 to accelerate the reduction of ROOH. Three different ROOH were selected in order to verify the universality of the catalytic activity and their structures were shown in the figure.

GO(mPEGSe)2 inducing reactive oxygen species in vitro. Recent studies have also revealed that selenium-containing compounds show potential applications in anti-cancer treatment as selenium can cause cytotoxicity by generating reactive oxygen species in vivo, where the biologically relevant concentration of ROS is 50-100 µM.44-45 Meanwhile, GO is also capable of generating ROS in vitro.53-55 We thus detected the ROS level via laser scanning confocal microscopy to verify if the ROS production could be boosted with the help of selenium. We used DCFH-DA which is one of the most commonly used fluorescent probes for detecting



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concentration level of intracellular reactive oxygen species (ROS). DCFH-DA itself has no fluorescence and can pass through the cell membrane freely. But once entering the cell, it will be hydrolyzed into DCFH by esterase. The DCFH can no longer pass out of the cell membrane and thus accumulates inside the cells. The intracellular ROS can oxidize the non-fluorescent DCFH to fluorescent DCF. So the green intensity of fluorescence is positively correlated to the level of ROS. It can be seen from Figure 6 that the GO only has marginal effect of inducing ROS，as the fluorescence intensity was rather low even at 25 µg/mL. But the GO(mPEGSe)2 at 25 µg/mL exhibited a strong fluorescence signal, which implied a great ability in inducing the ROS production. The result implied that selenium largely enhanced the capability of GO to produce ROS inside cells where the concentration of ROS is relatively low (50-100 µM).

Figure 6. Bio-functionality of GO(mPEGSe)2. Uptake of GO and GO(mPEGSe)2 at 10 µg/mL and 25 µg/mL by MDA-MB-231 cells after 24 h via laser scanning


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confocal microscopy. A strong fluorescence signal exhibited for GO(mPEGSe)2 indicated a great ability in inducing the ROS production.

Conclusion and outlook In this work, we have developed a relatively mild and straightforward method to covalently functionalize graphene oxide with diselenide-containing compounds. The functionalization mechanism was deduced to involve both redox reaction and radical addition reaction according to the XPS, FT-IR spectroscopy and Raman spectroscopy. Moreover, we found that the selenium-functionalized GO could modulate the balance of reactive oxygen species. Specifically, GOSe could decrease the ROS level by accelerating the reduction of peroxides when the ROS concentration is high while boosting the ROS level when the concentration is relatively low. We envision that this simple functionalization method could render GO new possibilities to be applicable to various situations.

Supporting Information The detailed synthetic routes and characterizations of diselenide compounds (HOEG4Se)2 and (mPEGSe)2, the FTIR, Raman spectra, XPS spectra and the Tof-SIMS results of GO(HOEG4Se)2 and GO(mPEGSe)2, the material’s initial reduction rates of the catalytic activities are available in the supporting information.

Author Information Corresponding Author *

E-mail: [email protected]



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Notes The authors declare no competing financial interest.

Acknowledgement This work was financially supported by the National Science Foundation for Distinguished Young Scholars (Grant 21425416), the National Basic Research Program of China (Grant 2013CB834502), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant 21421064), and the National Natural Science Foundation of China (Grant 91427301). The authors acknowledge Prof. Chun Li (Tsinghua University) for his suggestions about the characterizations of the material.

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Selenium-functionalized Graphene Oxide that Can Modulate the

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