Reduced graphene oxide based on simultaneous detection of neurotransmitters

A R T I C L E I N F O Article history: Received: 2018-11-23 Revised: 2018-12-07 Accepted: 2018-12-25 Available online: 2019-01-06 Manuscript ID: PCBR-1811-1006


Introduction
The emerging field of carbon based allotropes such as graphene, multiwall and singlewall nanotubes exhibits important role in field of electronics, optoelectronics, electrochemical and biomedical applications due to its unique structure and remarkable electronic, optical, thermal, mechanical and electrical properties [1][2][3].One of the promising candidate among the allotropes of carbon is graphene is used for electrochemical sensing [4,5], since every atom in a graphene sheet is a surface atom, molecular interaction and thus electron transport through graphene can be highly sensitive to adsorbed molecules [6].Although various methods have been developed to prepare individual graphene sheets, such as chemical vapour deposition (CVD), micromechanical exfoliation of graphite and epitaxial growth on electrically insulating surfaces, chemical reduction of graphite oxide colloidal suspensions has been considered as an effective route to synthesize graphene sheets due to its simplicity, reliability, ability for large-scale production and cost effectiveness [7][8][9].Graphite oxide consists of a layered structure of 'graphene oxide (GO)' sheets bearing a strongly oxygenated, highly hydrophilic layered material that can be readily exfoliated in water to yield stable dispersions consisting mostly of single layer, which are referred to as graphene oxide [10][11][12][13][14].At present chemically converted graphene by the modified Hummers' method is the most widely used technique for preparing GO with excellent processability [15][16][17] which involves harsh oxidation of graphite to introduce abundant hydrophilic oxygenated functional groups.hormonal, renal and central nervous system [18].Uric Acid is primary end product of metabolic breakdown of purine.Any abnormal change of both the biomolecules leads to cause for several diseases, such schizophrenia, Alzheimer's disease and parkison's disease in case of dopamine abnormalities [19].On other hand, abnormal concentration levels of uric acid can cause gout, pneumonia, leukemia and hyperuricaemia [20][21][22].
Usually, dopamine coexists with uric acid in physiological samples which make major challenge in selectivity and sensitivity.Furthermore, their oxidation potentials are close to each other.To overcome these setbacks, we develop an rGO modified GC electrode for simultaneous electro-chemical detection of dopamine and uric acid.
Modified electrode shows well defined peak separation and low limit of detection for DA and UA with good stability and reproducibility.Particularly, these factors suggest that the proposed sensor could be applied for determination of DA and UA in real sample analysis.

Graphite oxide preparation (GO)
GO was synthesized from natural graphite powder by a modified Hummers method.Briefly, 2 g of natural graphite powder and 1 g of NaNO3 was added into a mixture of 70 ml of 98% concentrated sulfuric acid (Conc.H2SO4).The mixture was stirred in an ice bath for 50°C in an oven and the final product is collected and stored.

Preparation and modification of electrodes
Prior to modification, the bare Glassy carbon electrode GCE was polished carefully with 0.5µm alumina powder on polishing cloth and then rinsed ultrasonically in ethanol and water for 2 min, respectively.rGO was dispersed in deionized water (1mg/mL)by using ultrasonicator.Then, 10 μL of the prepared suspension was dropped onto the GCE surface by drop casting method and dried at room temperature for 1 hr to obtain the rGO modified GCE.

Characterization techniques
Fourier transform infrared (FTIR) spectra of the samples were recorded on a spectrophotometer (Perkin ElmerSpectrum RX I) using KBr as the mulling agent.
Ultraviolet-Visible (UV-Vis) spectra of the samples were collected on a UV-Vis spectrophotometer (Perkin-Elmer Lambda 35), and X-ray diffraction analysis (XRD) of powders were carried out on XPERT-PROX-ray diffractometer with monochromatic CuKα, radiation (λ=1.5406A°).Data were collected from 10° to 80° at a scan rate of 0.05° per 10.16 seconds.The morphologies of the samples were observed under scanning electron microscope (Carl Zeiss EVO 18).Cyclic voltammetry, electrochemical impedance spectroscopy and square wave voltammetry measurements were performed with a CHI 6005D workstation (CH Instruments,USA).A conventional three-electrode system was employed comprising a glassy carbon electrode (GCE, 0.07 cm 2 in diameter) coated with rGO as the working electrode, a platinum wire as the auxiliary electrode, and an Ag/AgCl as the reference electrode.corresponding to 0.8 nm which is very typical for GO [23].This is an evidence to show that graphite is converted to graphene oxide.An increase in interlayer spacing of graphene oxide could be attributed to the intercalation of the oxygen functional groups like hydroxyl, epoxy and carbonyl as well as water molecules between graphene layers during the process of oxidation [24].

Results and discussion
Also there is a very weak diffraction peak at 2 = 42.2°,which is believed due to the incomplete oxidation.After  conglomerates, in GO powder there may appear stable grain of size ranging even to single micrometers.and Na (13.87 %).

FTIR Analysis
Fig. 4 shows the FTIR spectrum of the reduced graphene oxide recorded in the range of 4000-400 cm -1 .
The band appeared at 3321 cm -1 corresponds to O-H stretching vibrations.The peak 2916 cm -1 corresponds to alkanes C-H stretching vibration.The peak 2141cm -1 corresponds to alkynes C -C stretching vibration.Hence, NaBH4 is found to be more effective in reducing oxygen functional groups.

UV-Vis Analysis
The reduction of GO into graphene is characterized by UV−Vis spectroscopy studies as shown in Fig. 5.The UV−vis spectrum of rGO shows an absorption peak at 270 nm which indicates the presence of few layer graphene.For few layer graphene, the absorption peak is expected to be from 230 nm to 270 nm during reduction and also suggesting that GO is reduced.suggests an absorption peak may be due to Na [26,27] and an n-π* transition at 350 nm of -C=O bonds [28,29].

Conclusion
We have reported a simple and convenient method for Dopamine (DA) and Uric Acid (UA) plays vital roles in physiological functions of mammalian.

3. 1
Structural characterization 3.1.1X-ray diffraction: XRD analysis is a non destructive technique to determine the phase formation, crystallinity of rGO sample as shown in the Fig. 1.Since XRD is not a perfect tool for identifying single-layer graphene, but it is very informative.The inset figure shows pristine graphite exhibits a basal reflection (002) peak at 2 = 25.5 o (d spacing = 0.349 nm) in the XRD pattern.After oxidation, graphite oxide (GO) shows a well-defined diffraction peak at 2θ=11 o with the interlayer spacing (d-spacing)

Fig. 1 .
Fig. 1.X-Ray diffraction patterns Graphite (Inset), Graphite Oxide and rGO.groups and restoration of carbon atoms in graphite network after the chemical reduction of NaBH4.Thus from the XRD results, it can be concluded that both epoxide and carboxylic functional groups were effectively reduced by sodium borohydride in line with the previous reports [25].

Fig. 2 Fig. 2 .
Fig. 2 shows typical results of the studies of reduced graphene oxide.Fig 2(a) represents the view of the sample at lower magnification which stands for examining the area of 800×800 μm 2 surface.Around the examined area, one can notice the presence of objects of sizes within 200 μm to 300 μm.Those objects consist of tiny particles, as can be proved by SEM studies results gathered on one of the particles.The above results are shown in Fig. 2(b) (25×25 μm 2 area, higher magnification).It is easy to notice that the examined particles consist of a number of smaller objects of 0.5 μm to few micrometers in size.The problem was caused by the factor responsible for difficulties connected with getting higher magnification were high susceptibility of nanoparticles to aggregate into larger conglomerates.It should be pointed here that, except for nanoparticles

Fig. 3 .
Fig. 3. shows a standard EDAX spectrum recorded to determine the elemental compositions of the sample.In the middle part of the presented spectrum one can clearly see three peaks located between 0.1kV and 1.2 kV.Those maxima are directly related to the GO characteristic lines K and L. The maximum located on the left part of the spectrum at 0.2 kV clearly comes from carbon.The hardly visible maximum located at 0.5 keV is connected with the oxygen characteristic line.The carbon and oxygen spots in the examined samples confirm the presence of stabilizers composed of alkyl chains.The spectra obtained during EDAX studies were used for carrying out the quantitative analysis.For that purpose, SEM Quant software and the ZAF procedure were applied.Quantitative analyses GO reveals an atomic percentage of C (57.68 %), O (28.45 %)

Fig. 6 Fig. 5 .
Fig. 6 shows CV behavior of bare and rGO modified GCE in presence of 1mM [Fe(CN)6] 3-/4-in 1.0 M KCl at a scan rate of 50 mVs -1 .CV values for bare and modified rGO are given in table1.Herein, we have noticed a pair of well-defined redox peak ∆Ep values of 73 mV.

Fig. 6 .
Fig. 6.Cyclic voltammetry analysis (a) bare GC, (b) rGO modified GCE.The Rct values of bare and rGO samples were 96.1 and 3919Ω Cm -2 respectively.It shows the electron transfer resistance of the electrode decreased after the modification.

Fig. 8
Fig. 8 shows the effect of varying scan rates on the cyclic voltammograms performed in 1mM [Fe(CN)6] 3-/4- precursor using GO modified electrode in 1.0 M KCl solution was studied over scan rate range of 10mV to 100 mV/s.Oxidation currents were observed to increase with scan rate due to heterogeneous kinetics.

Fig. 8 .
Fig. 8. Cyclic voltammetry of rGO modified GCE at different scan rates.Furthermore, the oxidation and reduction peak currents increased linearly with the square root of scan rate, while the cathodic peak potentials varied linearly with the logarithm of scan rate.

Fig. 9 .
Fig. 9.Linear fit plot of current against scan rate.

3. 5 . 3
Sensitivity of the modified electrode using SWV The sensitivity of the rGO a nanostructure towards the detection of DA & UA was calculated by performing the square wave voltammetry (SWV) at various concentrations.SWV is performed in the potential range -0.1V to 0.8V with 0.1 M phosphate buffer solution with pH 7.0.The DA and UA were injected from 500 nM to1mM into the PBS.In each step of addition of DA & UA, the corresponding current value is noted.It is evident from Fig. 10 (a) and (b), with an increase in the DA & UA concentration.We have witnessed an increase in the peak current.
synthesis of GO and converted into rGO via strong reducing reagent.The XRD results shows GO has 2θ at 11° with inter layer spacing equal to 0.8 nm and rGO shows a broad diffraction peak which implies the restoration sp 2 carbon network.The FT-IR analysis displays decreased intensity of characteristic peaks corresponds to oxygen functional groups which imply a successful reduction and formation of rGO phase.UV-vis spectrum of rGO exhibits maximum absorption peak at ~270 nm.SEM images demonstrate ultrathin and homogeneous graphene films.

Table 2 .
Comparison of different chemically modified electrodes for the determination of DA and UA with rGO/GCE.EDAX indicated the component of O elements in rGO films was about 57.68% elements of C and Na were also observed.The presence of oxygen-containing functional groups provided more opportunities for potential applications of rGO in many areas.These data will provide a reference to further study of graphene.Electrochemical behavior of rGO modified glassy carbon electrode investigated with reference to [Fe(CN)6] 3-/4-redox system reflect that the electrochemical behavior is controlled by the electron transfer.rGO/GCE displayed desirable properties including low detection limit, excellent stability, reproducibility, selectivity and sensitivity.Therefore, rGO/GCE would act as a promising sensor for a wide range of electrochemical sensing.