Assessment of antioxidant activity of pure graphene oxide (GO) and composite V2O5/GO using DPPH radical and H2O2 scavenging assays

In this research, the Co precipitation method was utilized to synthesize a nanocomposite of vanadium oxide (V2O5) and graphene oxide (GO). Pure GO was synthesized by the modified hammers method. Using a 1:2 ratio of GO and V2O5 and heating in the oven at 70 °C leading to the formation of V2O5/GO nanocomposite. By grafting of GO on V2O5 surface, a high range of graphene oxidation in V2O5/GO allowed for better reduction with V2O5 metal-oxide. The scanning electron microscope (SEM) images and X-ray diffraction (XRD) spectra provide evidence of the distinct phase of graphene oxide formation. The antioxidant activity of V2O5/GO nanocomposite was conducted in two vitro assays, focusing on neutralization of 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and H2O2 radicals. The uniqueness of the nanocomposite was evident from its concentration-dependent antioxidant activities. Interestingly, the V2O5/GO samples has showed 60% antioxidant performance in the H2O2 assays compared to its constituent. Nanocomposite of V2O5/GO was synthesized by grafting of GO on V2O5 surface. The oxidation of graphene within the V2O5/GO system facilitated improved reduction when combined with V2O5 metal oxide. The distinctiveness of the nanocomposite became apparent through its antioxidant activities, which were dependent on its concentration. It is noteworthy that the V2O5/GO samples exhibited a 60% higher antioxidant performance in the H2O2 tests compared to their individual components. Nanocomposite of V2O5/GO was synthesized by grafting of GO on V2O5 surface. The oxidation of graphene within the V2O5/GO system facilitated improved reduction when combined with V2O5 metal oxide. The distinctiveness of the nanocomposite became apparent through its antioxidant activities, which were dependent on its concentration. It is noteworthy that the V2O5/GO samples exhibited a 60% higher antioxidant performance in the H2O2 tests compared to their individual components.

Graphene • Co-precipitation method • Assays Highlights • Nanocomposite of V 2 O 5 /GO was synthesized by grafting of GO on V 2 O 5 surface.

•
The oxidation of graphene within the V 2 O 5 /GO system facilitated improved reduction when combined with V 2 O 5 metal oxide.

•
The distinctiveness of the nanocomposite became apparent through its antioxidant activities, which were dependent on its concentration.

Introduction
The development of carbon nanomaterials in nanoscience over the past few decades has created a significant new area of inquiry for all carbon allotropes [1].Because of its unique different properties which include electrical, electronic, and mechanical graphene, a two-dimensional carbonaceous material with atomic thickness, has garnered a great deal of attention in the scientific community [2,3].Biosensors, bioimaging, and drug delivery are just a few of the medical applications that have drawn increased attention to graphene-based materials [4,5].Rapid growth in the number of publications on graphene and related materials has coincided with the development of methods for broadscale synthesis of graphene.Graphene is typically synthesized by de-scaling graphene oxide (GO) in an acid medium.By mixing slurry of graphite with fuming nitric acid and a trace amount of potassium chlorate, Brodie was the first to demonstrate in 1859 that GO could be synthesized in this way.In 1898, Staudenmaier modified this procedure by adding chlorate to a mixture of sulfuric acid and fume of nitric acid over time.Using KMnO 4 and NaNO 3 in a sulfuric acid (H 2 SO 4 ), Hummers modified the GO synthesis protocol in 1958 [6].As an alternative to Hummers' method, the Tour process has been proposed in recent years [7].This process aims to reduce the amount of toxic gas produced during synthesis while maintaining precise control over the reaction parameters.Graphene's unique properties inspire scientists to synthesize metallic nanoparticle-graphene nanocomposites for improving sensor and biosensor performance [8,9].
Graphene's with high surface area, magnificent conductivity, functionalization ease, and fabrication make it an exceptional platform for making valuable nanomaterials.In addition, GO can be used as a 2D matrix that is decorated with chemical elements or metal oxides, such as in the production of metal oxide-reduced graphene oxide [10].The photocatalytic activity of graphene nanocomposite of Au and Ag was enhanced by loading graphene sheets with gold nanoparticles (AuNPs) or silver nanoparticles (AgNPs).Adsorption and photocatalysis applications have benefited greatly from the synergistic effects of AuNPs' surface plasmonic resonance or the hydrophilic nature of the Ag-graphene nanocomposite and the specific electronics effect of graphene [11].Superior electrochemical performance and photocatalytic degradation of organic dyes distinguish graphene nanosheets decorated with vanadium oxide (V 2 O 5 ) nanoparticles as a cutting-edge material [12,13].In these cases, V 2 O 5 can act as a photocatalyst, while graphene can be used as an electron-acceptor/transport material to aid in the transport of photo-generated electrons; prevent recombination of electron, and hole [12].Decorating the surface of graphene with V 2 O 5 has been shown to significantly boost its antibacterial and antioxidant properties [14][15][16].In metal oxide, Vanadium can be applied to potential the rapeutic because of anti-diabetic properties [17][18][19].Vanadium pentoxide application has been increasing due to their chemical and physical properties.It shows many applications in medical science and biology [20].Nanocomposite of vanadium oxide exhibit anti-cancer activity in the cell of breast cancer [21].The ultrasound assisted V 2 O 5 nanoparticles are appropriate material for bio-medical and waste-water treatment [22].
Vanadium pentoxide has various properties including less toxicity, narrower bandgap and electrochemical activity [23].Vanadium pentoxide goes through a transitional phase of semi-conductor to metal phase near 257 °C [24].Orthorhombic unit cell of crystalline V 2 O 5 with parameters a = 11.510Å, b = 3.563 Å, c = 4.369 Å [25].V 2 O 5 nanoparticles have been prepared that exhibit cancer of melanoma of cancer in mice by using a technique of antiangiogenesis [25].We greatly feel that in the coming period, V 2 O 5 NPs likely used as a potential therapeutic agent for cancer treatment further to melanoma [21].The production of oxidative stress is one of the few hypothesized mechanisms of vanadium toxicity.It includes kinase activity and interference with protein phosphates and DNA repair obstruction [26][27][28].The genotoxicity linked to the stress of oxidation is based on the oxidation process of vanadium (V)reduction, which produces reactive oxygen species like hydroxyl-radicals [29].Antioxidants have the ability to inactivation of highly reactive molecules like ROS, which produces throughout various biochemicalprocessincells [30], making the potential block age and coadjutants in the treatment of chronic diseases linked to DNA damage and oxidation stress [31].Ascorbic acid and alpha to copherol are two antioxidants that are turnout to prove and to diminish the medicine clastogenicity and microbicide in experimental animals [32][33][34].Cameron and Pauling highlighted the therapeutic characteristics of AA, claiming that high dose of AA (>10 g/ day) might heal and bock cancer cells by boosting collective tissues formation [35].Scientist now believes that Ascorbic acid helps the body ROS eliminate starting age cancers by neutralizing reactive oxygen species before they affect DNA and trigger tumor maturation [36][37][38].Ascorbic acid may also operate as a pro-oxidant, assisting the body ROS in destroying cancer [39,40].
TOH has also been found to beneficent reducing the effects of a variety of Geno-toxic chemicals [41,42].Because it efficiently decrease the oxidation stress and control lipid per oxidation and ROS toxic effects in biological systems, -TOH is in biological sciences and in functionally, it is the most significant and vigorous antioxidant of all the vitamin E is-forms in humans in terms of biological availability and biological activity [43,44].Dietary -TOH supplementation has been shown to be relatively efficient in extinguish induced carcinogen malignancies in mice since the1960s, similar to AA [45].In this research, the optimum graphite-to-KMnO 4 ratio was determined for GO synthesis using hydrogen peroxide and a modification of the Hummers' method.V 2 O 5 -decorated graphene oxide was produced when GO was decorated with V 2 O 5 /GO [46].This study provided further evidence that Zn nanoparticles can be used to attractive effect on the surface of GO sheets, with the quality of the decoration depending on the degree of oxidation.We utilized X-ray diffraction (XRD), Scanning Electron Microscopy and Raman spectroscopy to gain insight into the molecular composition and atomic arrangement of GO and V 2 O 5 /GO composites.These techniques allow for a semi quantitative assessment of the reduction and decoration of GO and V 2 O 5 /GO.The antioxidant activity of GO and V 2 O 5 /GO nanocomposites was evaluated using DPPH radical and hydrogen peroxide scavenging assays, respectively.
2 Experimental section

Materials required
Vanadium pentoxide (V 2 O 5 , 98%), graphite flake, sulfuric acid, potassium permanganate, hydrogen peroxide, hydrochloric acid, and nitric acid were purchased from sigma Aldrich and used without further purification.

Synthesis of GO
Hummer's procedure was used to synthesize graphene oxide.Graphite powder (3.0 g) were sonicated for 40 min with 23 mL of hydrogen peroxide and 46 mL of hydro sulfuric acid [7].At 5 °C, 9 g of KMnO 4 was added gradually.At 8 °C and with continuous stirring for 1 h, 200 mL of deionized water (DI) were added drop by drop, followed by 30 mL of hydrogen peroxide (H 2 O 2 ).With the addition of H 2 O 2 , a remarkable color change was seen, confirming the synthesis of GO [47].

Synthesis of V 2 O 5 /GO nanocomposite
In practical, Co precipitation was used to create the V 2 O 5 / GO nanocomposite [48][49][50].V 2 O 5 was dispersed in acetic acid and ethylene glycol followed by sonication for 1 h.In the next step, GO was added to the V 2 O 5 solution, followed by constant stirring for 2 h at 85 °C Table 1.The final mixture was centrifuged for 2 h.By centrifuging the final mixture at 3500 rpm for 15 min, the precipitates were baked for 24 h at 70 °C to dry them out.

V 2 O 5 /GO nanocomposite antioxidant activity
Samples were evaluated for their antioxidant potential by measuring their ability to scavenge the stable DPPH free radical [51].To sum up, a 1.9 methanol solution of DDPH was mixed with 0.1 ml of variously concentrated methanol solutions of each compound (0.004%).The Shimadzu UV-1280 spectrometer was used to calculate the absorbance at 517 nm.The ascorbic acid was used as a control.There were two separate tests conducted.Equation ( 1) was used to determine the DPPH radical-scavenging capacity.

Scavenging ability
The control absorbance (A c ) is subtracted from the sample absorbance (A s ).The method was used to calculate the scavenging efficiency of the samples against H 2 O 2 .Fresh H 2 O 2 (40 mM) was made in a phosphate buffer solution (pH = 7.4) containing 0.05 M KH 2 PO 4 -K 2 HPO 4 [52].A total volume of 1.4 mL of phosphate buffer and 0.6 mL of 40 mM H 2 O 2 was combined with the compounds at varying concentrations.Using a Shimadzu UV-1280 spectrophotometer, the H 2 O 2 concentration was measured after 10 min at 230 nm.The vitamin C ascorbic acid served as the reference standard.

X-ray diffraction (XRD)
The crystal structure and the phase purity of our nanomaterial were analyzed by utilizing XRD on the-axis values ranges with 2θ.The XRD result supported the crystalline nature and purity of the V 2 O 5 /GO nanocomposites.In Fig. 1a, the highest peak of graphene  oxide is observed at 10.7°, corresponding to the (001) plane.Notably, the d-spacing value for graphite was measured to be 3.3 Å. Upon oxidation of GO, the d spacing increased to 7.6 Å indicating successful oxidation with only minor alterations in the structure.In Fig. 1b, the XRD measurement of Vanadium pentoxide, reflection peaks were noticed at 2θ values with (001), (101), and (002) diffraction planes.Most of the intense XRD peaks corresponded to the orthorhombic V 2 O 5 phase, consistent with the standard JCPDS data (card No: 01-070-8747).Additionally, the presence of some structural peaks suggests different oxidation state of vanadium oxide.The peak observed at a 2θ angle of 27.70°indicates the existence of the metastable monoclinic (β) phase of VO 2 (according to JCPDS card No. 82-0661).Interestingly, it is evident that the oxide layers developed on Vanadium metal consist of a combination of oxide phases.
The XRD of nanocomposite as shown in Fig. 1c.Based on XRD pattern, the GO/V 2 O 5 nanocomposites validate their purity, as there were no impurities or characteristic peaks observed apart from those corresponding to V 2 O 5 /GO nanocomposites.Moreover, the XRD pattern indicates that the synthesized V 2 O 5 /GO nanocomposites were composed of a single phase, and the diffraction peaks were well-defined and distinct.The diffraction peaks appearing at 2θ are 15.4°, 20.5°, 26.2°, 31.1°whichrepresents (200), (010), (100), (400), (020), (211), (222) planes respectively, confirming orthorhombic structure of the nanocomposite.A crystallinity of the material is calculated by using equation (2) [53].
The observed crystallinity is 82% and the highest peak obtained at 20.5°with 010 planes.The advantage of using co-precipitation method is that it removes all the functional groups, results a successful adornment of graphene oxide on the surface of vanadium pentoxide particles and the suppression peak of graphene oxide in the composition of nanocomposite [17].
The average particle size was calculated by employing Debye Scherer Eq. ( 3) as indicated in Table 2.
The calculated values of lattice parameters (A-C) and unit cell volume are given in Table 2.The data matched well with the values in the JCPDS data reported in literature [54]

Scanning electron microscopy
The SEM analysis of the GO, V 2 O 5 and V 2 O 5 /GO nanocomposite was conducted using a scanning electron microscope (SEM, LEO-0430) at room temperature.The sample was directly mounted on a sample stub and coated with a layer of gold film (~200 nm) under reduced pressure (0.133 Pa) before the investigation.SEM image of GO is displayed in Fig. 2a.This image serve as evidence, demonstrating that the process of graphite oxidation leads to a reduction in the number of layers, a decrease in crystallinity, and the occurrence of amorphization [36].In Fig. 2b, the SEM images display hexagonal structures for V 2 O 5 .The particles have uniform particle size and have spherical morphology.In Fig. 2c, the SEM images of the V 2 O 5 /GO nanocomposite reveal the presence of overlapping and nonhomogeneous grafting of GO on the surface of V 2 O 5 .The results indicate that the agglomerated structure is formed due to the dense coverage [55,56].

Raman spectroscopy
Raman spectroscopy is extensively employed for assessing various properties of nanocomposite structures.The Raman spectrum of GO which includes the D and G peaks as depicted in Fig. 3a.The D peak at 1350 cm −1 result from defects in the graphene sheets, while the G peak at 1600 cm −1 results from bond stretching of sp 2 hybridized carbons [56,57].In Fig. 3b, with an excitation wavelength of 514 nm, the Renishaw Laser Confocal Raman Microscope was used to record the Raman spectra for this study.The layered structure of V 2 O 5 , indicates good crystallinity.For V 2 O 5 /GO, the ID/IG proportion improves from 1.01 to 1.09, which is indicative of GO being removed.Due to the necessity of using a powerful laser, a complete lack of peaks is observed in relation to the vibrational modes of V 2 O 5 [57].Overall, the conclusion demonstrates that the mass proportion in Hummers' approach is a considerable factor of graphene oxide in both reduction and metal oxide decoration.Our study keeps its primary focus on the GO matrix, where we seek to optimize GO oxidation by increasing the amount of oxidant agent used.Specifically, the higher the oxidation

Energy dispersive X-ray analysis
Energy-dispersive X-ray analysis is a method employed to quantify nanoparticles using scanning electron microscopy (SEM).In this approach, the nanoparticles undergo analysis through activation using an energy-dispersive X-ray spectrophotometer (EDS), a component commonly found in modern and advanced SEM.The isolated individual nanoparticles are placed onto a compatible substrate that doesn't disrupt the process of nanoparticle characterization.Based on the EDS spectrum of V 2 O 5 , no peak of carbon distinctly supports the suppression of GO in the formation of nanocomposite with V 2 O 5 .The results confirm formation of single phase V 2 O 5 /GO nanocomposite as shown in the Fig. 4. The inset table in EDS spectrum shows the ratios of vanadium and oxygen according to their atomic weight and ratios.The data confirming a dominant peak of oxygen can be seen in the table [60].
As noted in the DPPH radical experiments, the capacity of the tested samples to scavenge H 2 O 2 showed a correlation with the dosage used (Fig. 6).The H 2 O 2 scavenging activity of different samples and ascorbic acid at 350 μg/mL was in the order: Ascorbic acid (75%) > V 2 O 5 /GO1:3 (60%) > GO1:4(53%) > V 2 O 5 /GO1:2(47%) > V 2 O 5 /GO1:4(43%) > GO1:2(38%) > GO1:3(17%).Based on the obtained results, the nanocomposite has showed concentration-dependent antioxidant activities.The H 2 O 2 molecule is relatively unreactive, but the hydroxyl radicals it can generate within a cell can be harmful.As a result, getting rid of H 2 O 2 is crucial for safeguarding the biological system and, in particular, food components [14].The antioxidant activity pattern can depend on the structure of graphene oxide or few-layer graphene.
Our results align with prior research that highlights the capacity of graphene-based materials (including graphene, rGO, and GO) to counteract free radicals, scavenge oxygen, or break down peroxides due to their antioxidative properties [61,64].Our results are compatible with past study that shown the effectiveness of graphene-based materials [65].
After a second adduct is formed, the radical is destroyed through electron transfer, hydrogen donation from functional groups, chelation of transition metal ions, or inhibition of Fenton-based radical generation [64,66,67].This is based on the hypothesis that radical adduct formation at sp 2 carbon sites delocalizes spin across the conjugated graphene backbone.Graphene oxide outperformed V 2 O 5 -decorated GO in the DPPH radical scavenging assay, while the reverse was true in the H 2 O 2 antioxidant assays.Compared to GO, the data suggest that V 2 O 5 /GO has greater antioxidant activity perhaps as a result of synergistic effects or the interaction between V 2 O 5 nanoparticles and GO [68,69].

Conclusion
Results confirms subsequent characterization of the synthesized V 2 O 5 /GO nanocomposite using XRD and Raman spectroscopy provided unequivocal evidence that GO was effectively reduced and the nanocomposite was successfully synthesized.The presence of characteristic peaks corresponding to the hexagonal wurtzite structure of V 2 O 5 confirmed the successful synthesis of the nanocomposite.Moreover, the PL results revealed the presence of oxygenated functional groups, such as hydroxyl, carboxyl, and epoxy groups, on GO, and demonstrated the variation in their intensity following GO reduction.Further structural characterizations substantiated that the most effective reduction and decoration of graphene oxide by vanadium oxide occurred in the highly oxidized samples remarkably, the prepared nanocomposites exhibited notable antioxidant properties.The V 2 O 5 /GO nanocomposites demonstrate dose-dependent electron or hydrogen atom donation, allowing them to effectively react with free radicals.This was evident from the results of the DPPH radical and H 2 O 2 scavenging assays, where the nanocomposites outperformed GO in scavenging H 2 O 2 .These compelling results underscore the importance of conducting further research on both pristine and metal oxide-decorated graphene oxide.The ultimate objective is to obtain high-quality matrices of both types to assess their biological viability.However, additional investigations are necessary to determine the optimal degree of decoration and antioxidant capacity to fully harness their potential.

Fig. 6
Fig. 6 H 2 O 2 scavenging capability of GO and V 2 O 5 /GO nanocomposites.The numbers displayed above the final columns in the bar graph indicate the maximum level of antioxidant activity achieved by each sample at a concentration of 350 µg

Table 1
The XRD analysis was used to determine the hkl values of V 2 O 5 /GO

Table 2
Average size values observed for various compounds from X-ray diffraction