Accepted Manuscript

Highly efficient multipurpose graphene oxide embedded with copper oxide nanohybridfor electrochemical sensors and biomedical applications

S.R. Kiran Kumar, G.P. Mamatha, H.B. Muralidhara, M.S. Anantha, S. Yallappa, B.S.Hungund, K.Yogesh Kumar

PII: S2468-2179(17)30051-5

DOI: 10.1016/j.jsamd.2017.08.003

Reference: JSAMD 117

To appear in: Journal of Science: Advanced Materials and Devices

Received Date: 19 April 2017

Revised Date: 21 July 2017

Accepted Date: 9 August 2017

Please cite this article as: S.R.K. Kumar, G.P. Mamatha, H.B. Muralidhara, M.S. Anantha, S. Yallappa,B.S. Hungund, K.Y. Kumar, Highly efficient multipurpose graphene oxide embedded with copper oxidenanohybrid for electrochemical sensors and biomedical applications, Journal of Science: AdvancedMaterials and Devices (2017), doi: 10.1016/j.jsamd.2017.08.003.

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Highly efficient multipurpose graphene oxide embedded with copper oxide nanohybrid for electrochemical sensors and biomedical applications

S.R. Kiran Kumar1, G.P. Mamatha2*, H.B. Muralidhara3, M.S. Anantha1, S. Yallappa4,

B.S.Hungund5 and K.Yogesh Kumar6*

1Centre for Nanosciences, Department of Chemistry, K.S. Institute of Technology, Bangalore, 560 062,

India 2* Department of Pharmaceutical Chemistry, Kuvempu University, Post Graduate Centre, Kadur,

Chikmagalore Dist., Karnataka, India-577 548.

3 Centre for Incubation, Innovation, Research & Consultancy, Jyothy Institute of Technology, Bangalore-

560082, India 4 MS R&D Centre, BMS College of Engineering Bangalore-560019, India 5 Department of Biotechnology, KLE Technological University, Hubballi-580031, India 6* Department of Chemistry, School of Engineering and Technology, Jain University, Bangalore 562 112,

India

*Corresponding author/authors: Tel:(+91-8147673335) E-mail:yogeshkk3@gmail.com.

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Abstract Graphene oxides embedded with copper oxide (GO@CuO) nanocomposite were successfully

synthesized via hydrothermal method. The nanoparticles were characterized by XRD, SEM,

TEM and BET surface area analysis. The nanocomposite modified electrode is used for the

detection of dopamine and paracetamol using cyclic voltammetry with a scan rate of 50 mVs-1.

The voltammograms obtained during the oxidation studies revealed that as synthesized

GO@CuO nanocomposite sensor shows high catalytic activity in sensing. The oxidation peak

potential (Epa) of DA at BCPE and MCPE were observed at 0.1115 V and 0.1127 V

respectively. This electrode obtains good and satisfactory results in the determination of DA in a

commercial injection. Moreover, these NCS showed enhanced antimicrobial and anticancer

activities, which is due to the combining effect of GO and CuO.

Keywords: GO@CuO nanocomposite, Dopamine, Modified carbon paste electrode, Cyclic

voltammetry.

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1. Introduction Dopamine (DA) belongs to a catecholamine family, which plays an important role in the

functions of the central nervous system. In the brain, DA functions as a neurotransmitter and

shortage of DA, particularly the death of DA neurons in the nigrostriatal pathway, causes

Parkinson's disease [1]. Likewise, Paracetamol (PC) is a non-steroidal anti-inflammatory drug

that finds widespread application for its strong analgesic and antipyretic action. It is widely

applied for patients with a headache, backache, arthritis, migraines, neuralgia, menstrual cramps,

and postoperative pain; however, it does not show any harmful side effects [2]. Nevertheless, the

biomolecules of PC leads to hypersensitivity or overdose causes damage of the liver and kidney

which leads to hepatoxicity and nephrotoxicity.

Recently, many analytical methods have been employed for the determination of

biomolecules such as chemiluminescence, spectrophotometry, titrimetric and electrochemistry.

Among them, electrochemical sensors have attracted much attention due to their excellent

properties viz., low-cost, simplicity, high sensitivity and handing convenience [3]. Nevertheless,

the high cost of noble metal electrodes limits their usage in many applications. Hence, the

development of a highly sensitive and selective electrode without an enzyme or noble metal is

necessary.

In recent times, nanomaterials research has gained greater momentum owing to their

possession of thermo electric, optic, catalytic, mechanical properties. The surface coating of the

electrode with nanoparticles is an attractive approach for enhancing the scope of

electrochemically modified electrodes [4]. Graphene oxide (GO) stands out amongst the most

significant substituent of graphene and it is a trusted material for different innovative fields such

as optoelectronics, catalysis, nano-electronic compounds, gas sensors, super capacitors, and

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medical field [5]. Numerous composites comprising of graphene oxide and metal oxides viz.,

NiO, MnO2, CuO, Fe2O3, TiO2, ZnO, SnO2, In2O3 and Ce2O3 have been studied for many diverse

applications [6-8]. Similarly, CuO is an assured composite because of minimal effort, eco-

richness, non-poisonous quality, and effortless preparation in different states of nanosized

measurements [9]. Since ancient times, Cu and its oxides are known to apply for various

biomedical applications like wound healing ointments, dental work, food packaging, coating on

clinical equipment etc., due to its inherent antimicrobial and anticancer activity [10-12]. In order

to get an enhanced biological efficiency and also to meet some particular requirements, the

composite nanomaterials are in demand. In this way, GO can render the suitable platform to host

or functionalize with CuO nanoparticles [13]. The combination of GO and CuO could be a

productive integration of the properties of two components that can head to the novel series of

hybrid materials bearing new features. This type of hybridization of GO and CuO is known to

enhance the active sites including superior functioning and very good intrinsic properties. Thus,

in our quest for materials with enhanced biological activity (antimicrobial and anticancer

activity), we found these hybrid materials worth exploring. However, there are few studies on the

biological activity of carbon based materials hybridized with metal based nanoparticles (silver,

copper etc.) [14-15]. To the best of our knowledge, no studies exist concerning the biological

activity (antimicrobial and anticancer) of Graphene oxide embedded with copper oxide

(GO@CuO) nanocomposites (NCS). Thus, it is clinically necessary to identify new therapeutic

molecules that may significantly enhance biological efficacy. These aspects of nanomedicines

remain subjects of particular interest.

NCS was synthesized by adjusting the pH of the GO dispersion followed by mixing of

copper sulphate solution. The synthesized material was characterized by various analytical and

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spectroscopic techniques and then used to modify the carbon paste electrode. The

electrochemical effect of biomolecules on this GO@CuO modified electrode was studied. At the

same time we have used the novel NCS material for selective determination of different

biomolecules in the presence of different interfering analytes at biological pH and their

antimicrobial and anticancer activity are reported here.

2. Experimental

2.1. Materials

All chemicals were purchased from S.D. Fine-Chem Mumbai, India, until and unless stated

otherwise. Analytical Reagent (AR) grade chemicals without any purification were used in the

experiments. Silicone oil, Graphite powder, hydrogen peroxide (30 wt %), sodium nitrate (98%),

dopamine hydrochloride, sulphuric acid (98 wt%), sodium di-hydrogen orthophosphate

(NaH2PO4), potassium permanganate, copper(II) nitrate tri-hydrate, disodium hydrogen

phosphate (Na2HPO4) , sodium hydroxide and all of the stock solutions for the preparation of

composites were prepared by using double distilled water.

2.2. Synthesis of graphene oxide-copper oxide (GO–CuO) nanocomposite

GO was prepared by utilizing a modified Hummers' method as follows [16]. Briefly 15 g

of graphite powder was added into 250 mL of cooled sulfuric acid in an ice bath. At that point,

25 g of KMnO4 and 6 g of NaNO3 were added continuously with mixing and cooled so that the

temperature of the solution was kept at 15–20 °C. The solution was then mixed at 35 °C for 25

min and the temperature was raised to 80 °C after that 250 mL of doubly distilled water was

gradually mixed at 80 oC for 30 min. To prevent the oxidation, 50 mL of 30% H2O2 solution and

an extra 500 mL of deionized water added consecutively to decrease the effect of KMnO4.

Further, the sample was filtered, washed with 100 mL of deionized water and took after by

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ultrasonic treatment for 15 min. The precipitates was isolated by centrifugation and after that

dried in a vacuum stove at 50 °C for 18 h. NCS was prepared by fabrication of anchored CuO

nanoparticles on to GO. In the first stage, 20 ml of 0.2 mol/L NaOH solution was gradually

added into a 20 ml of 0.1 mol/L copper(II) nitrate trihydrate solution containing 0.005mol/L of

Triton X-100 with steady mixing. At that point, 65 ml of deionized (DI) water was added

gradually into the above solution with mixing to get Cu(OH)2. In the second step, a known

amount of GO (1:2) was diffused in 20 ml of DI water through ultrasonication. To this solution,

1.2 ml of Cu(OH)2 was added and the pH was adjusted to 10.0 by adding NaOH. The subsequent

dark solution was cooled normally to room temperature and washed three times with DI water

and ethanol. At last, the compound was dried in an autoclave at 60 °C for 8h.

2.3. Characterization techniques

The powder X-Ray diffraction (XRD) patterns of NCS were obtained by Bruker D2

Phaser X-Ray diffractometer equipped with graphite monochromatized Cu Kα radiation and a

Ni-filter. The structural morphology of NCS were observed by Field Emission Scanning Electron

Microscope (FESEM) (JEOL, JSM-840) operated at 15 kV and Transmission Electron

Microscope (TEM) (JEOL, JSM 1230) images were carried out by microscope at an accelerating

voltage of 200 kV. Thermo gravimetric analysis (TGA) was performed on TA instruments Q50.

Heating rate was maintained at 10 °C/min in an inert atmosphere. Fourier transform infrared

(FTIR) analysis was used to determine the surface functional groups (Bruker ATR) where the

spectra were recorded from 400 to 4000 cm-1. Moreover, the electrochemical experiments were

carried out in a three electrode cell system, which contained a bare carbon paste electrode

(BCPE), CPE/ GO@CuO nanocomposites (MCPE) as the working electrode.

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2.4. Preparation of bare carbon paste electrode (BCPE) and modified carbon paste

electrode (MCPE)

A BCPE was prepared by hand mixing of 80% graphite powder with 20% silicon oil in

an agate mortar to produce a homogenous paste and the carbon paste was packed into the cavity

of electrode of 4 mm in diameter. Then smoothed the surface of BCPE on a weighing paper and

the electrical contact was provided by a copper wire connected to the carbon paste in the end of

the tube. MCPE was prepared by adding 2,4,6,8 and 10 mg NCS to above mentioned graphite

powder and silicone oil mixture.

2.5. Electrochemical measurements

The electrochemical workstation (CHI 608E) was utilized to assess the electrochemical

properties of the NCS in 0.2M phosphate buffer (pH 7.2) as the electrolyte in a three-electrode

configuration utilizing cyclic voltammetry (CV). This contained three-electrode cell system, a

MCPE, as the working electrode an aqueous saturated calomel electrode (SCE) as the reference

electrode and Pt wire as the auxiliary electrode. The mass loading of the active material for each

modified carbon paste electrode was about 4 mg of NCS.

2.6. In vitro antimicrobial activity

The in vitro antimicrobial activity of as synthesized NCS were evaluated against different

human pathogens namely Staphylococcus aureus (NCIM 5021), Bacillus subtilis (NCIM 2999),

Escherichia coli (NCIM 2574), Pseudomonas aeruginosa (NCIM 5029), Aspergilus flavus

(NCIM 524) and Candida albicans (NCIM 3471). The microbial strains were cultured overnight

at 37 °C in nutrient broth and potato dextrose agar medium. The broth cultures were compared to

the turbidity with that of the standard 0.5 McFarland solution. All the Micro-organisms were

maintained at 4 °C for further use. All the pure microbial strains obtained from National

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Chemical Laboratory (NCL), Pune, India. The newly synthesized compounds were tested in vitro

using the agar disc diffusion method by taking streptomycin and fluconazole as standard drugs

for bacteria and fungi, respectively. The antimicrobial potentialities of the NCS were estimated

by pre-sterilized filter paper disks (6 mm in diameter) impregnated with NCS dissolved in 100

µg/mL was placed on the inoculated agar. The plates were incubated for about 24 h at 37 °C in

the case of bacteria and 48 h at 28 °C in the case of fungi. The zone of inhibition around the well

in each plate was measured in mm. The statistical analyses of the above results were performed

using IBM SPSS version 20 (2011). One way ANOVA (analysis of variance) at value p < 0.001

followed by Tukey’s Post Hoc test with p ≤0.05 was used to determine the significant differences

between the results obtained in each experiment.

2.7. Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration of the NCS was determined by dilution method.

The NCS was dissolved and diluted to give two-fold serial concentrations of the compounds was

employed to determine the MIC. In this method, NCS is made from 5 to 75 µg/mL. The MIC

value was determined as the lowest concentration of the NCS inhibiting the visual growth of the

microorganism on the agar plate.

2.8. In-vitro anticancer activity

2.8.1. Cell culture

The normal cells (Vero-ATCC® CCL-81™) and human cancer cells (HeLa-S3-ATCC ®

CCL-2.2™) and (MDA-MB-231-ATCC® HTB-26™) were maintained in Modified Eagles

Medium (MEM) supplemented with 10% FCS, 2% essential amino acids, 1% each of glutamine,

non-essential amino acids, vitamins and 100 U/ml Penicillin–Streptomycin. Cells were

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subcultured at 80–90% confluence and incubated at 37 °C in a humidified incubator supplied

with 5% CO2. The stock cells were maintained in 75 cm2 tissue culture flask.

2.8.2. Cell viability assay

The cytotoxicity effect of as obtained NCS was performed by 5-diphenyl-2H-tetrazolium

bromide (MTT) assay. Briefly, cultured cells (1 × 10‒6 cells/mL) were placed in 96 flat-bottom

well plates, then cells were exposed to different concentration of prepared nanomaterials (1–100

µg/mL) and incubated at 37 °C for about 24 h in 5% CO2 atmosphere. After 24 h incubation,

MTT (10 µl) was added to the incubated cancer cells. Then MTT added cells were further

incubated at 37 °C for about 4 h in 5% CO2 atmosphere. Thereafter, the formazan crystals were

dissolved in 200 µl of DMSO and the absorbance was monitored in a colorimetric at 578 nm

with reference filter as 630 nm. The cytotoxicity effect was calculated as:

Cell viability (%) ꞊ 100 ‒ Cytotoxicity (%)

3. Results and discussion

3.1. Growth Mechanism

Probable mechanism for the formation process of NCS is explained as follows: GO is a

layered material bearing oxygen-containing functional groups on their basal planes and edges;

these functional groups can act as anchor sites and consequently, make nanoparticles formed in

situ attach on the surfaces and edges of GO sheets. Accordingly, in the early stages, the positive

Cu2+ ions formed in the presence of solvent easily adsorb onto these negative GO sheets via the

electrostatic force. Large amount of nuclei were formed in a short time owing to the hydrolysis

Cytotoxicity (%) = 1 ‒ Mean absorbance of toxicant Mean absorbance of ‒ve control

100 ×

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reaction of Cu(NO3)2. The oxygen atoms of the crystal growth units then might form bonds with

the functional groups via intermolecular hydrogen bonds or coordination bonds, acting as anchor

sites for the crystallites to grow further [17].

3.2. Structural and morphological analysis

The phase composition and structures of NCS were examined by using X-ray powder

diffraction and the corresponding pattern is shown in Fig. 1c. The diffraction peaks observed at

2θ values of 32.50, 35.520, 38.780,46.30, 48.760, 53.760, 58.360, 61.760, 66.150 and 67.940

correspond to (110), (111), (200), (112), (202), (020), (021), (113), (311) and (220) planes

respectively, are similar to the characteristic diffractions of monoclinic phase of CuO (JCPDS

48-1548), where the (001) reflection peak of layered GO (Fig. 1b) has almost disappeared. The

previous work explains that the diffraction peak will not be prominent when GO is exfoliated. In

this composite the CuO dominates the GO layer which is supported by SEM studies [18-19].

Fig. 2 shows the surface morphology of NCS at different magnifications. A typical SEM

image shows non-uniform CuO nanoparticles with the sizes ranging from 100–200 nm. After

combination with GO to form a GO@CuO composite, CuO nanoparticles are decorated and

firmly anchored on the GO layers with a high density. GO may favor the hindrance of CuO from

agglomeration and enable their good distribution, whereas the CuO serves as a stabilizer to

separate GO sheets against aggregation. In addition, the GO@CuO was observed to have the

specific surface area 21.9 m2/g from Brunauer–Emmett–Teller (BET) examination and was

observed to be porous in nature [20].

The TEM images of NCS as shown in Fig. 3 reveal that the product consists of a large

quantity of CuO nanoparticles with sizes ranging from 100 to 200 nm. It can be seen that the GO

shows an ultrathin wrinkled paper-like structure and the CuO nanoparticles tend to aggregate like

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a needle with the size ranging from 100-200 nm. Based on the Energy-dispersive X-ray

spectroscopy (EDX) results, the atomic weight ratio of C, Cu and O is 5.03%, 71.25% and

23.72%, respectively. Thermogravimetric analysis (TGA) is also a significant analytical

technique. The decomposition behavior of the GO@CuO was studied by TGA. A large weight

loss can be observed at 250 °C, which is caused by the combustion of the carbon. After that

prominent loss in mass was not observed till 600 °C, but there was a sudden drop in the mass

around 640 °C.

In order to understand the nature of functional groups on their surface, FTIR

measurements were conducted. Fig. 4. shows FTIR spectra of GO@CuO. For GO, the peak at

3438 cm−1 corresponds to O-H stretching vibration. The vibration of C-OH was observed at

1262.21 cm−1. The peak 1634.9 cm−1 is attributed to C-C stretching vibration. The absorptions

peaks at 2856.29 and 2926.3 cm−1 are representing the symmetric and anti-symmetric stretching

vibrations of CH2. The absorption peaks at 1390.67 cm−1 and 1107 cm−1 are corresponding to the

stretching vibration of C-O of carboxylic acid and C-OH of alcohol, respectively. The

adsorptions at 506 and 622.83 cm−1 are the characteristic stretching vibrations of CuO bond in

monoclinic CuO [20].The other adsorption peaks may be due to OH bending vibrations of some

constitutional water incorporated in the CuO structure. From spectrum of the composite material,

characteristic peaks of both components can be seen. Thus, the FTIR results confirm the

anchoring of CuO nanoparticles on the surface of GO sheets.

3.3. Electrochemical response of [K4Fe(CN)6 ] at BCPE and MCPE

The MCPE was found to be stable, even after 20 cyclic voltammetric scans. The MCPE

is quite stable and prepared electrode could be used for more than 60 days if preserved in a

closed container. Relative standard deviation (RSD) calculated for anodic current and potential

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of 1mM [K4Fe(CN)6] in 1 M KCl respectively. The electrochemical response of GO@CuO

nanocomposites of an MCPE was studied by standard 1mM [K4Fe(CN)6] in 1 M KCl as a

supporting electrolyte with a scan rate 50 mVs-1 by the CV technique. The comparison of the

corresponding peak potential differences ∆Ep of different modified electrodes are given in

Table 1. At the BCPE the anodic peak potential (Epa) 0.1473 V peak currents significantly

increased at the MCPE with the anodic peak potential. Peak currents ipc and ipa of [K4Fe(CN)6]

at MCPE increased compared to those at the BCPE. Possibly a large pore volume of NCS

provides a large surface area leading to the enhancement in the peak current and these results

confirmed that the presence of NCS in the BCPE matrix improved the sensitivity by enhancing

electron transfer process. Therefore, NCS played an important role in improving the reversibility

electrochemical performance of the MCPE.

3.4. Effect of NCS MCPE for detection of Dopamine and Paracetamol

The effects of increasing the amount of modifier GO@CuO NCS in the carbon paste

matrix on the electrochemical behavior of PC and DA was also investigated (Fig. 5) in order to

optimize the conditions in a 0.2M phosphate buffer (pH 7.2) at a scan rate of 50 mV s-1. 4 mg

MCPE response to the maximum current as compared with the 2, 4, 6, 8 and 10 mg of NCS and

voltammograms of DA and PC in the same buffer solution were recorded separately. This

optimized concentration is maintained during further investigations of biomolecules.

3.5. Electrochemical response of DA and PC at BCPE and MCPE with NCS

The cyclic voltammograms obtained for the electrochemical responses of 5×10−5 M DA

and 1.0 ×10-6 M PC its voltammograms was recorded in 0.2 M phosphate buffer as the

supporting electrolyte at pH 7.2. Showed well-defined redox peaks at MCPE. The corresponding

peak potential differences ∆Ep=0.0802 V and ∆Ep=0.0998 V for the DA and PC at the MCPE

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are shown in Fig. 6 and Fig. 7. The oxidation peak potential (Epa) of DA at BCPE and MCPE

were observed at 0.1115 V and 0.1127 V respectively. PC peak currents significantly increased

at the MCPE with the Epa and Peak currents (Ipa) increased compared to those at BCPE

(Table 2). These results confirmed that the presence of NCS in CPE matrix improved the

sensitivity and the large pore volume NCS of provides a large specific area leading to the

enhancement in peak current.

3.6. Effect of scan rate on the peak current

The effect of a scan rate for DA and PC in a phosphate buffer solution at pH 7.2 was

studied by the CV at the MCPE. Fig. 8, show an increase in the redox peak current at a scan rate

of 0.05–0.200 V s−1 MCPE indicating that direct electron transfer in the modified electrode

surface of DA. The obtained graph for DA exhibited good linearity between the scan rate (v) and

the redox peak current (Fig. 9) for the MCPE with correlation coefficients of R2 = 0.99, which

indicates that the electron transfer reaction was diffusion-controlled process. The redox peak

current at a scan rate of 0.05–0.250 V s−1indicating that direct electron transfer in the MCPE

surface of PC and the graph obtained exhibited good linearity (Fig. 10) with correlation

coefficients of R2= 0.99, which indicates that the electron transfer reaction was adsorption-

controlled process.

3.7. Real sample analysis of Dopamine in dopamine hydrochloride injections

In order to verify the reliability of the method for the analysis of DA as a pharmaceutical

product the proposed MCPE was applied to the dopamine hydrochloride injection (DHI). 5 mL

of DHI solution (40 mg/mL) were diluted to 25 mL of double distilled water and then 0.2 mL of

this diluted solution was taken into 10 mL volumetric flask. The DHI solution in 0.2M phosphate

buffer solution of pH 7.2 at the BCPE and the MCPE were measured at a scan rate of 50 mV s−1

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by CV technique. The results confirmed that the proposed method could be effectively used for

the determination of DA in commercial samples and the MCPE proposed efficiently used for the

determination of DA in injections.

3.8. Interference study

The influence of various foreign species as interfering compounds with the determination

of DA, DHI solution and selectivity of the NCS sensor was investigated under the optimum

conditions 40 mg/mL at the 0.2M phosphate buffer solution of pH 7.2. Tolerance limit was

defined as the maximum concentration of interfering foreign species that caused an approximate

relative error of ±5% for the determination of neurotransmitter. Here we found that no significant

interference for the detection of DA was observed from the selected compounds such as KCl

5000 µM and CaCl2 4000 µM. These results indicate that the MCPE results confirmed here has a

high catalytic activity in sensing for DA analysis in the presence of other interfering substance.

Electrochemical response as the peaks remains unchanged after successive 20 cyclic

voltammetric scans, confirms MCPE has good stability.

3.9. Antimicrobial activity

The NCS was evaluated for antimicrobial activity by means of agar disc diffusion method

and minimum inhibitory concentration (MIC) was determined by dilution method. NCS

demonstrated in vitro antimicrobial activity against the four bacterial strains belonging to the

Gram-positive (S. aureus, Bacillus subtilis,) and Gram-negative (Escherichia coli, Pseudomonas

aeruginosa) and two strains of fungi namely Aspergilus flavus, Candida albicans). The results of

the antibacterial activity of NCS are presented in Table 3. The MIC is defined as the lowest

concentration of nanoparticles that inhibits the growth of a microorganism. NCS showed MIC at

28 and 31 µg/mL for E. coli and P. aeruginosa, respectively. According to MIC E. coli and

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P. aeruginosa exhibited the highest sensitivity toward NCS while B. subtilis, C. albicans and

A. flavus showed the least sensitivity among the tested microbes. The antimicrobial activity of

the tested NCS was compared to the positive control drugs, streptomycin and fluconazole. The

antibacterial properties of NCS are mainly attributed to adhesion with bacteria because of their

opposite electric charges resulting in a reduction at the bacterial cell wall. It was earlier reported

that the interaction between Gram-negative bacteria and NCS was stronger than that of Gram-

positive bacteria because of the difference in cell walls, cell structure, physiology, metabolism,

or degree of contact of organisms with nanoparticles. Gram-positive bacteria have thicker

peptidoglycan cell membranes compared to the Gram-negative bacteria and it is harder for NCS

to penetrate it, resulting in a low antibacterial response [21].

3.10. Cell viability assay

The biocompatibility of nanoparticles is an important issue in therapeutic applications.

Therefore the biocompatibility and cytotoxicity of NCS were evaluated by colorimetric assay.

The as obtained NCS was tested against different cell-lines namely Vero-ATCC® CCL-81™,

HeLa-S3-ATCC ® CCL-2.2™, and MDA-MB-231-ATCC® HTB-26™. The cell viability

results reveal that different cells treated with NCS exhibited dosage dependent and time-

dependent behavior. However, the as obtained NCS showed no obvious cytotoxic effect on

normal cells which indicates an excellent biocompatibility of prepared NCS. This lower

cytotoxicity of the NCS against normal cell line suggests its potential biological applications. For

instance the survivability of cells are found to be 78% for normal cells and 35% for cancer cells

at higher dose (100 µg/ml) of NCS, which is generally considered as high toxicity for cancer

cells. For all the cell lines with mentioned NCS concentration, the mean and standard error found

to be within acceptable limit. This statistical data indicates the repeatability and consistency of

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the experimentation. The biocompatibility for normal cells is perhaps due to the impact of

targeting agents. However, more detailed studies are required to understand the precise

mechanism for cell interaction.

4. Conclusions

In the present study, NCS was synthesized by modified hummers method followed by

hydrothermal treatment. The abundant porous architectures of NCS exhibited high selectivity

and good reproducibility of the voltammetric response, the prepared MCPE is considered to be

very useful in the construction of simple devices in the field of medicine for the diagnosis of

dopamine deficiency. The oxidation peak potential (Epa) of DA at BCPE and MCPE were

observed at 0.1115 V and 0.1127 V respectively. Electrochemical response as the peaks remains

unchanged after successive 20 cyclic voltammetric scans. Further, NCS hybrid nanomaterials

have shown very good biocide activity against tested microorganisms (S. aureus, B. subtilis, E.

coli, P. aeruginosa, A. flavus and C. albicans). In addition, NCS was found to be non-toxic for

normal cells (Vero-ATCC® CCL-81™), while highly toxic for human cancer cells (HeLa-S3-

ATCC ® CCL-2.2™ and (MDA-MB-231-ATCC® HTB-26™). In summary, the new class of

hybrid nanomaterials seemed to be highly beneficial especially for biomedical applications.

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Acknowledgment

The authors wish to thank Dr. B.E. Kumaraswamy, Department of Industrial Chemistry,

Kuvempu University, for his invaluable suggestions and moral support. The authors are also

thankful to K.S. Institute of Technology, Bangalore for providing the lab facility to carry out this

research work. Authors are thankful to Ms. Sangeetha Alwar for assisting us in improving

English language.

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Legends for Figure Fig. 1 XRD pattern of GO@CuO NCS. Fig. 2 FESEM images of GO@CuO NCS at different magnifications. Fig. 3 TEM images of GO@CuO NCS. Fig. 4 FTIR spectra of GO@CuO NCS. Fig. 5 Graph of current versus different concentration of GO@CuO NCS /MCPE in 0.2 M

phosphate buffer solution containing 5×10−5M DA. Fig. 6 Cyclic voltammogram of 5×10−5M DA in 0.2 M phosphate buffer solution at pH 7.2 using

bare CPE and GO@CuO NCS /MCPE at scan rate 50 mV s−1. Fig. 7 Cyclic voltmmogram of 1.0 ×10-6 M PC in 0.2 M phosphate buffer solution at pH 7.2

using bare CPE and GO@CuO NCS /MCPE at scan rate 50 mV s−1. Fig. 8 Cyclic voltmmogram of MCPE in 0.2 M phosphate buffer solution containing 5×10−5M

DA at different scan rates. Fig. 9 Graph shows the DA linear relationship between the anodic peak current and scan rate. Fig.10 Typical graph showing the PC linear relationship between the anodic peak current and

scan rate.

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Fig. 1.

10 20 30 40 50 60 70

Inte

nsi

ty (

a.u

.)

2θθθθ (degree)

CuO (a) GO (b) GO-CuO (c)

(a)(c)

(b)

(110)

(111)(200)

(112)

(202)

(020)(021)(113)(311)(002)

(001)

(101)

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Fig. 2.

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Fig. 3.

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23

Fig 4

500 1000 1500 2000 2500 3000 350050

55

60

65

70

75

80

Wavenumber (cm-1)

Tra

nsm

itta

nce

(%

)

506

622.83

808.7

1107

1262.21

1390.671634.9

2856.29

2926.3

3438O-H

C-OH

C-C-CH

2C-O

C-OH

-OH

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24

Fig.5

2 4 6 8 102.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

3.8

Ipa

(A)

Concentration (mg)

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Fig. 6

-0.2 0.0 0.2 0.4 0.6-8

-6

-4

-2

0

2

4

6

8

Cu

rren

t (1

0-8A

)

Potental (V)

(a) MCPE(b) BCPE

(a)

(b)

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Fig. 7

-0.2 0.0 0.2 0.4 0.6 0.8-6

-4

-2

0

2

4

(b)

(a)

Cu

rren

t (1

0-5A

)

Potential (V)

(a) BCPE(b) MCPE

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Fig. 8

-0.2 0.0 0.2 0.4 0.6

-12

-8

-4

0

4

8

12

16

50 mV/s 100 mV/s 150 mV/s 200 mV/s

Cu

rren

t (1

0-8A

)

Potential (V)

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Fig. 9

60 80 100 120 140 160 180 200

4

6

8

10

IpA

(10

-8A

)

Scan rate (mV/s)

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Fig. 10

50 100 150 200 250

1.40

1.42

1.44

1.46

1.48

1.50

1.52

1.54

1.56

Ipa

(10-1

A)

Scan rate (mV/s)

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Table 1 Antimicrobial activity of the GO@CuO NCS.

Tested In vitro activity zone of inhibition in mm (MIC in µg/ml)a compounds

Gram positive Gram negative Fungi

S. aureus B. subtilis E. coli P. aeruginosa C. albicans A. flavus

Ncs 13(50) 12(70) 13(28) 12(31) 11(75) 14(75)

Streptomycin 11.6(05) 10.4(05) 14.5(05) 13(05) Nt Nt

Flucanazole Nt Nt Nt Nt 16(05) 18(05)

a the values given are means of three experiments. Nt-denotes not tested

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Table 2: Comparison of the detection limits of different modified electrodes.

Electrode Detection limit (µM) Techniques Reference

Banana/MWCNTs/MCPE 2.09 DPV [22]

CCE/ferrocene carboxylic acid 0.45 SWV [23]

MEs/SAM-Au electrode 1.1 CV [24]

LDH/CILE 5 DPV [25]

GO-CuO / MCPE 0.5 CV Present work

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Table 3 Antimicrobial activity of the GO@CuO NCS.

Tested In vitro activity zone of inhibition in mm (MIC in µg/ml)a compounds

Gram positive Gram negative Fungi

S. aureus B. subtilis E. coli P. aeruginosa C. albicans A. flavus

Ncs 13(50) 12(70) 13(28) 12(31) 11(75) 14(75)

Streptomycin 11.6(05) 10.4(05) 14.5(05) 13(05) Nt Nt

Flucanazole Nt Nt Nt Nt 16(05) 18(05)

a the values given are means of three experiments. Nt-denotes not tested

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1. Introduction Dopamine (DA) belongs to a member of the catecholamine family, which plays an important

role in the functions of the central nervous system [1]. In the brain, DA functions as a

neurotransmitter and shortage of DA, particularly the death of DA neurons in the nigrostriatal

pathway, causes Parkinson's disease [2]. Likewise, Paracetamol (N-acetyl-p-aminophenol or 4-

acetamidophenol) is a non-steroidal anti-inflammatory drug that finds widespread application for

its strong analgesic and antipyretic action. It is widely applied for patients with a headache,

backache, arthritis, migraines, neuralgia, menstrual cramps, postoperative pain, however it does

not show any harmful side effects [3]. Nevertheless, the biomolecules of PC leads to

hypersensitivity or overdose causes damage of the liver and kidney this leads to hepatoxicity and

nephrotoxicity.

Recently, many analytical methods have been employed for the determination of

biomolecules such as chemiluminescence, spectrophotometry, titrimetry and electrochemistry

[4, 5]. Among them, electrochemical sensors are attracted much attention due to its excellent

properties viz., low-cost, simplicity, high sensitivity and handing convenience [6, 7].

Nevertheless, high cost of noble metal electrodes limits its usage in many applications. Hence,

the development of a highly sensitive and selective electrode without an enzyme or noble metal

is needed.

In recent times, nanomaterials research has gained greater momentum owing to their

possession of thermo electric, optic, catalytic, mechanical properties. The surface coating of the

electrode with nanoparticles is an attractive approach for enhancing the scope of

electrochemically modified electrodes [8, 9]. Graphene oxide (GO) is a standout amongst the

most significant substituent of grapheme and its trusted material for different innovative fields

such as optoelectronics, catalysis, nano-electronic compounds, gas sensors, super capacitors, and

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medical field [10,11]. Numerous composites comprising of graphene oxide and metal oxides

viz., NiO, MnO2, CuO, Fe2O3, TiO2, ZnO, SnO2, In2O3 and Ce2O3 have been studied for many

diversified applications [12-14]. Similarly, CuO is an assured composite because of minimal

effort, eco-richness, non-poisonous quality, and effortless preparation in different states of

nanosized measurements [15, 16]. Since ancient times, the Cu and its oxides are known to apply

for various biomedical applications like wound healing ointments, dental work, food packaging,

coating on clinical equipment etc., due to its inherent antimicrobial and anticancer activity [17-

19]. In order to get an enhanced biological efficiency and also to meet some particular

requirements, the composite nanomaterials are in demand. In this way, GO can render the

suitable platform to host or functionalize with CuO nanoparticles [20, 21]. The combination of

GO and CuO could be a productive integration of the properties of two components that can head

to the novel series of hybrid materials bearing new features. This type of hybridization of GO

and CuO is known to enhance the active sites including superior functioning and very good

intrinsic properties. Thus, in our quest for materials with enhanced biological activity

(antimicrobial and anticancer activity), we found that these hybrid materials are worth exploring.

However, there are few studies on the biological activity of carbon based materials (carbon

nanotubes) hybridized with metal based nanoparticles (silver, copper etc.) [22]. To the best of

our knowledge, there are no studies exist concerning the biological activity (antimicrobial and

anticancer) of GO@CuO nanocomposites. Thus, it is clinically necessary to identify possible

new therapeutic molecules that may significantly enhance biological efficacy. These aspects of

nanomedicines remain subjects of particular interest. Therefore, we have used the novel

GO@CuO composite material for selective determination of different biomolecules in the

presence of different interfering analytes at biological pH and their antimicrobial and anticancer

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activity were reported here. GO@CuO composite was synthesized by adjusting the pH of the GO

dispersion followed by mixing of copper sulphate solution. The synthesized material was

characterized by various analytical and spectroscopic techniques.

2. Experimental

2.1.Materials

All chemicals were of scientific grade, obtained from SD-Fine, Bangalore, India and used

as received without further purification. Silicone oil, Graphite powder, hydrogen peroxide

(30 wt %), sodium nitrate (98%), dopamine hydrochloride, sulphuric acid (98 wt%), sodium di-

hydrogen orthophosphate (NaH2PO4), potassium permanganate, copper(II) nitrate tri-hydrate,

disodium hydrogen phosphate (Na2HPO4) , sodium hydroxide and all of the stock solutions for

the preparation of composites were prepared by using double distilled water.

2.2. Synthesis of graphene oxide-copper oxide (GO–CuO) nanocomposite

Graphene oxide was prepared from characteristic graphite by utilizing a modified

Hummers' method as follows [23]. Briefly 15 g of graphite powder was added into 250 mL of

cooled sulfuric acid in an ice bath. At that point, 25 g of KMnO4 and 6 g of NaNO3 were added

continuously with mixing and cooled so that the temperature of the solution was kept up at 15–

20 °C. The solution was then mixed at 35 oC for 25 min and the temperature was raised to 80oC

after that 250 mL of doubly distilled water was gradually mixed at 80 oC for 30 min. To prevent

the oxidation, 50 mL of 30% H2O2 solution and an extra 500 mL of deionized water was added

consecutively to decrease the effect of KMnO4.Further, the sample was filtered , washed with

100 mL of deionized water and took after by ultrasonic treatment for 15 min. The precipitates

was isolated by centrifugation and after that dried in a vacuum stove at 50 °C for 18 h.

GO@@CuO nanocomposite was prepared by fabrication of anchored CuO nanoparticles on to

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GO. In the first stage, 20 ml of 0.2 mol/L NaOH solution was gradually added into a 20 ml of 0.1

mol/L copper(II) nitrate trihydrate solution containing 0.005mol/L of Triton X-100 with steady

mixing. At that point, 65 ml of deionized (DI) water was added gradually into the above solution

with mixing to get Cu(OH)2. In the second step, a known amount of GO (1:2) was diffused in 20

ml of DI water through ultrasonication. To this solution, 1.2 ml of Cu(OH)2 was added and the

pH was acclimated to 10.0 by adding NaOH. The subsequent dark solution was cooled normally

to room temperature, and washed three times with DI water and ethanol. At last, the compound

was dried in an autoclave at 60 0C for 8h.

2.3. Characterization techniques

The powder XRD patterns of NCS were obtained by Bruker D2 Phaser X-Ray

diffractometer equipped with graphite monochromatized Cu Kα radiation and a Ni-filter. The

structural morphology of NCS were observed by FESEM (JEOL, JSM-840) operated at 15 kV

and TEM (JEOL, JSM 1230) images were carried out by microscope at an accelerating voltage

of 200 kV. Thermo gravimetric analysis (TGA) was performed on TA instruments Q50. Heating

rate was maintained at 10 °C/min in an inert atmosphere. Fourier transform infrared (FTIR)

analysis was used to determine the surface functional groups using FTIR spectroscope (Bruker

ATR) where the spectra were recorded from 400 to 4000 cm-1. Moreover, the electrochemical

experiments were carried out in a three electrode cell system, which contained a bare carbon

paste electrode (BCPE), CPE/ GO@CuO nanocomposites, as the working electrode.

2.4. Electrochemical measurements

The electrochemical workstation (CHI 608E) was utilized to assess the electrochemical

properties of the NCS in 0.2M phosphate buffer (pH 7.2) as the electrolyte in a three-electrode

configuration utilizing cyclic voltammetry (CV). This contained three-electrode cell system, a

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CPE/ GO@CuO nanocomposites, as the working electrode an aqueous saturated calomel

electrode (SCE) as the reference electrode, and a Pt wire as the auxiliary electrode. The mass

loading of the active material for each modified carbon paste electrode was about 4 mg of

GO@CuO NCS.

2.5. Preparation of bare carbon paste electrode (BCPE) and modified carbon paste electrode

(MCPE)

A BCPE was prepared by hand mixing of 80% graphite powder with 20% silicon oil in

an agate mortar to produce a homogenous paste and the carbon paste was packed into the cavity

of electrode of 4 mm in diameter. Then smoothed the surface of BCPE on a weighing paper and

the electrical contact was provided by a copper wire connected to the carbon paste in the end of

the tube. MCPE was prepared by adding 2,4,6,8 and 10 mg GO@CuO nanocomposites to above

mentioned graphite powder and silicone oil mixture.

2.6. In vitro antimicrobial activity

The in vitro antimicrobial activity of as synthesized GO@CuO NCS were evaluated

against different human pathogens namely Staphylococcus aureus (NCIM 5021), Bacillus

subtilis (NCIM 2999), Escherichia coli (NCIM 2574), Pseudomonas aeruginosa (NCIM 5029),

Aspergilus flavus (NCIM 524) and Candida albicans (NCIM 3471). The microbial strains were

cultured overnight at 37 °C in nutrient broth and potato dextrose agar medium. The broth

cultures were compared to the turbidity with that of the standard 0.5 McFarland solution. All the

Micro-organisms were maintained at 4 °C for further use. All the pure microbial strains obtained

from National Chemical Laboratory (NCL), Pune, India. The newly synthesized compounds

were tested in vitro using the agar disc diffusion method by taking streptomycin and fluconazole

as standard drugs for bacteria and fungi, respectively. The antimicrobial potentialities of the

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GO@CuO NCS were estimated by pre-sterilized filter paper disks (6 mm in diameter)

impregnated with GO@CuO NCS dissolved in 100 µg/mL was placed on the inoculated agar.

The plates were incubated for about 24 h at 37 °C in the case of bacteria and 48 h at 28 °C in the

case of fungi. The zone of inhibition around the well in each plate was measured in mm. The

statistical analyses of the above results were performed using IBM SPSS version 20 (2011). One

way ANOVA (analysis of variance) at value p < 0.001 followed by Tukey’s Post Hoc test with p

≤0.05 was used to determine the significant differences between the results obtained in each

experiment.

2.7. Minimum inhibitory concentration (MIC)

The minimum inhibitory concentration of the GO@CuO NCS was determined by dilution

method. The GO@CuO NCS was dissolved and diluted to give two-fold serial concentrations of

the compounds was employed to determine the MIC. In this method, GO@CuO NCS is made

from 5 to 75 µg/mL. The MIC value was determined as the lowest concentration of the

GO@CuO NCS inhibiting the visual growth of the microorganism on the agar plate.

2.8. In-vitro anticancer activity

2.8.1. Cell culture

The normal cells (Vero-ATCC® CCL-81™) and human cancer cells (HeLa-S3-ATCC ®

CCL-2.2™ and (MDA-MB-231-ATCC® HTB-26™ were maintained in Modified Eagles

Medium (MEM) supplemented with 10% FCS, 2% essential amino acids, 1% each of glutamine,

non-essential amino acids, vitamins and 100 U/ml Penicillin–Streptomycin. Cells were

subcultured at 80–90% confluence and incubated at 37 °C in a humidified incubator supplied

with 5% CO2. The stock cells were maintained in 75 cm2 tissue culture flask.

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2.8.2. Cell viability assay

The cytotoxicity effect of as obtained NCS was performed by 5-diphenyl-2H-tetrazolium

bromide (MTT) assay [Lin et al 2014]. Briefly, cultured cells (1 × 10‒6 cells/mL) were placed in

96 flat-bottom well plates, then cells were exposed to different concentration of prepared

nanomaterials (1–100 µg/mL) and incubated at 37 °C for about 24 h in 5% CO2 atmosphere.

After 24 h incubation, MTT (10 µl) was added to the incubated cancer cells. Then MTT added

cells were further incubated at 37 °C for about 4 h in 5% CO2 atmosphere. Thereafter, the

formazan crystals were dissolved in 200 µl of DMSO and the absorbance was monitored in a

colorimetric at 578 nm with reference filter as 630 nm. The cytotoxicity effect was calculated as:

Cell viability (%) ꞊ 100 ‒ Cytotoxicity (%)

2.8.3. Statistical analysis

A statistical analyses values for all the experiments were expressed as a ± standard

deviation. The data were performed using Student t-test, where statistical significance was

calculated for treated samples and untreated (as control) cells.

3. Results and discussion

3.1. Structural and morphological analysis

The phase composition and structures of GO@CuO nanocomposites were examined by

using X-ray powder diffraction and the corresponding pattern is shown in Fig. 1. The diffraction

peaks observed at 2θ values of 35.520, 38.780, 48.760, 53.760, 58.360, 61.760, 66.150 and 67.940

correspond to (111), (111), (202), (020), (202), (113), (311) and (220) planes respectively, are

Cytotoxicity (%) = 1 ‒ Mean absorbance of toxicant Mean absorbance of ‒ve control

100 ×

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similar to the characteristic diffractions of monoclinic phase CuO (JCPDS 48-1548), where the

(001) reflection peak of layered GO has almost disappeared [24]. The previous work [25]

explains that the diffraction peak will not be prominent when GO is exfoliated. In this composite

the CuO dominates the GO layer which is supported by SEM studies.

Fig. 2 shows the surface morphology of GO@CuO at different magnifications. A typical

SEM image shows non-uniform CuO nanoparticles with the sizes ranging from 100–200 nm.

After combination with GO to form a GO@CuO composite, the CuO nanoparticles are decorated

and firmly anchored on the GO layers with a high density. GO may favor the hindrance of the

CuO from agglomeration and enable their good distribution, whereas the CuO serve as a

stabilizer to separate GO sheets against aggregation. In addition, the GO@CuO is observed to be

porous in nature, which will further help in the adsorption of heavy metal ions from waste water.

The TEM images of GO@CuO as shown in Fig. 3 reveal that the product consists of a

large quantity of CuO nanoparticles with sizes ranging from 100 to 200 nm. It can be seen that

the GO shows an ultrathin wrinkled paper-like structure and the CuO nanoparticles tend to

aggregate like a needle with the size ranging from 100-200 nm. As can be seen in Fig. 3a, CuO

nanoparticles were spread across the sheet with intimate contact. The corresponding HR-TEM

image (Fig. 3b) shows clear lattice fringes, which allows for the identification of crystallographic

spacing. The fringe spacing of ca.0.25 nm matches that of the (-111) crystallographic plane of

CuO. The selected area electron diffraction (SAED) pattern as shown in Fig. 3(c), is attributed to

(-111) and (111) and (202) diffraction of CuO respectively. Existence of the (-111) planes in

SAED characterization is also an evidence of the result which high resolution image (Fig. 3b) of

shown the corresponding lattice fringes. All these results are in agreement with the analysis of

XRD. EDX analysis was employed to determine the CuO nanoparticles on the surface of GO

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nanosheets. The EDX spectrum of the GO@CuO sample has been depicted in Fig. 4. As is seen,

C, O, and Cu are the only elements which were detected, revealing that the anchored particles on

GO sheets are composed of Cu and O. Based on the obtained results, the atomic weight ratio of

Cu and O is 71.25% and 23.72%, respectively. The inset figure shows the electron image of

GO@CuO, clearly indicating the anchoring of CuO on GO. The decomposition behavior of the

GO@CuO was studied by thermo gravimetric analysis (TGA) and the results are shown in Fig.

5. The weight loss below 110 °C is probably due to the evaporation of adsorbed moisture. A

large weight loss can be observed at 350 °C, which is caused by the combustion of the carbon.

Thereafter, no weight loss was obtained up to 1000 °C.

In order to understand the nature of functional groups on their surface, FTIR

measurements were conducted. Fig.6. shows FTIR spectra of GO@CuO. For GO, the peak at

3438 cm−1 corresponds to O-H stretching vibration. The vibration of C-OH was observed at

1262.21 cm−1. The peak 1634.9 cm−1 is attributed to C-C stretching vibration [26]. The

absorptions peaks at 2856.29 and 2926.3 cm−1 are representing the symmetric and anti-

symmetric stretching vibrations of CH2. The absorption peaks at 1390.67 cm−1 and 1107 cm−1

are corresponding to the stretching vibration of C-O of carboxylic acid and C-OH of alcohol,

respectively. The adsorptions at 506 and 622.83 cm−1 are the characteristic stretching vibrations

of CuO bond in monoclinic CuO [27].The other adsorption peaks may be due to OH bending

vibrations of some constitutional water incorporated in the CuO structure. From spectrum of the

composite material, characteristic peaks of both components can be seen. Thus, the FTIR results

confirm the anchoring of CuO nanoparticles on the surface of GO sheets.

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3.2. Electrochemical response of [K4Fe(CN)6 ] at BCPE and GO@CuO NCS/MCPE

The MCPE was found to be stable, even after 20 cyclic voltammetric scans. In the

present study, however the CPE/ GO@CuO nanocomposites were used only for a single scan.

The MCPE is quite stable and prepared electrode could be used for more than 60 days if

preserved in a closed container. Relative standard deviation (RSD) calculated for anodic current

and potential of 1mM [K4Fe(CN)6] in 1 M KCl respectively. The electrochemical response of

GO@CuO nanocomposites of an MCPE was studied by standard 1mM [K4Fe(CN)6] in 1 M KCl

as a supporting electrolyte with a scan rate 50 mVs-1 by the CV technique. The corresponding

peak potential differences ∆Ep=0.169105 V for the CPE/ GO@CuO NCS (b) are shown in Fig.

7. and at the BCPE the anodic peak potential (Epa) 0.1473 V peak currents significantly

increased at the MCPE with the anodic peak potential. Peak currents ipc and ipa of [K4Fe(CN)6]

at GO@CuO NCS/MCPE increased compared to those at the BCPE. Possibly a large pore

volume of CPE/ GO@CuO NCS provides a large surface area leading to the enhancement in the

peak current and these results confirmed that the presence of GO@CuO NCS in the BCPE

matrix improved the sensitivity by enhancing electron transfer process. Therefore, GO@CuO

NCS played an important role in improving the reversibility electrochemical performance of the

CPE/ GO@CuO NCS.

3.3. Effect of GO@CuO NCS MCPE for detection of Dopamine and Paracetamol

The effects of increasing the amount of modifier GO@CuO NCS in the carbon paste

matrix on the electrochemical behavior of PC and DA was also investigated (Fig. 8) in order to

optimize the conditions in a 0.2M phosphate buffer (pH 7.2) at a scan rate of 50 mV s-1. A 4 mg

GO@CuO /CPE response to the maximum current as compared with the 2, 6,8 and 10 mg of

GO@CuO NCS and voltammograms of DA and PC in the same buffer solution were recorded

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separately. This optimized concentration is maintained during further investigations of

biomolecules.

3.4. Electrochemical response of DA and PC at BCPE and MCPE with GO@CuO NCS

The cyclic voltammograms obtained for the electrochemical responses of 5×10−5 M DA

and 1.0 ×10-6 M PC its voltammograms was recorded in 0.2 M phosphate buffer as the

supporting electrolyte at pH 7.2. Showed well-defined redox peaks at GO@CuONCS/MCPE.

The corresponding peak potential differences ∆Ep=0.0802 V and ∆Ep=0.0998 V for the DA and

PC at the GO@CuONCS/MCPE are shown in Fig. 9 and Fig. 10.The oxidation peak potential

(Epa) of DA at BCPE and MCPE were observed at 0.1115 V and 0.1127 V respectively. PC peak

currents significantly increased at the GO@CuONCS/MCPE with the Epa and Peak currents

(Ipa) increased compared to those at BCPE. These results confirmed that the presence of

GO@CuO NCS in CPE matrix improved the sensitivity and the large pore volume GO@CuO

NCS of provides a large specific area leading to the enhancement in peak current.

3.5. Effect of scan rate on the peak current

The effect of a scan rate for DA and PC in a phosphate buffer solution at pH 7.2 was

studied by the CV at the GO@CuO NCS/MCPE. Fig. 11, show an increase in the redox peak

current at a scan rate of 0.05–0.200 V s−1GO@CuONCS /MCPE indicating that direct electron

transfer in the modified electrode surface of DA. The obtained graph for DA exhibited good

linearity between the scan rate (v) and the redox peak current (Fig. 12) for the GO@CuONCS

/MCPE with correlation coefficients of R2 = 0.99, which indicates that the electron transfer

reaction was diffusion-controlled process. The redox peak current at a scan rate of 0.05–0.250 V

s−1indicating that direct electron transfer in the GO@CuO NCS /MCPE surface of PC and the

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graph obtained exhibited good linearity (Fig. 13) with correlation coefficients of R2= 0.99, which

indicates that the electron transfer reaction was adsorption-controlled process.

3.6. Real sample analysis of Dopamine in dopamine hydrochloride injections

In order to verify the reliability of the method for the analysis of DA as a pharmaceutical

product the proposed GO@CuO /CPE was applied to the dopamine hydrochloride injection

(DHI). 5 mL of DHI solution (40 mg/mL) were diluted to 25 mL of double distilled water

and then 0.2 mL of this diluted solution was taken into 10 mL volumetric flask. The DHI

solution in 0.2M phosphate buffer solution of pH 7.2 at the BCPE and the GO@CuO NCS

/MCPE were measured at a scan rate of 50 mV s−1 by CV technique. The cyclic voltammograms

for the corresponding peak potential differences ∆Ep=0.0618 V for the DA at the

GO@CuONCS/MCPE are shown in Fig.14. The results confirmed that the proposed method

could be effectively used for the determination of DA in commercial samples and the CPE/

GO@CuONCS proposed efficiently used for the determination of DA in injections.

3.7. Interference study

The influence of various foreign species as interfering compounds with the determination

of DA, DHI solution and selectivity of the GO@CuO NCS sensor was investigated under the

optimum conditions 40 mg/mL at the 0.2M phosphate buffer solution of pH 7.2. Tolerance limit

was defined as the maximum concentration of interfering foreign species that caused an

approximate relative error of ±5% for the determination of neurotransmitter. Here we found that

no significant interference for the detection of DA was observed from the selected compounds

such as KCl 5000 µM and CaCl2 4000 µM. These results indicate that the GO@CuO

NCS/MCPE results confirmed here has a high catalytic activity in sensing for DA analysis in the

presence of other interfering substance. Electrochemical response as the peaks remains

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unchanged after successive 20 cyclic voltammetric scans, confirms CPE/ GO@CuONCS has

good stability.

3.8. Antimicrobial activity

The GO@CuO NCS was evaluated for antimicrobial activity by means of agar disc

diffusion method [28] and minimum inhibitory concentration (MIC) was determined by dilution

method [29].GO@CuO NCS demonstrated in vitro antimicrobial activity against the four

bacterial strains belonging to the Gram-positive (S. aureus, Bacillus subtilis,) and Gram-negative

(Escherichia coli, Pseudomonas aeruginosa) and two strains of fungi namely Aspergilus flavus,

Candida albicans). The results of the antibacterial activity of GO@CuO NCS are presented in

Table 1. The MIC is defined as the lowest concentration of nanoparticles that inhibits the growth

of a microorganism. GO@CuO NCS showed MIC at 28 and 31 µg/mL for E. coli and

P. aeruginosa, respectively. According to MIC E. coli and P. aeruginosa exhibited the highest

sensitivity toward GO@CuO NCS while B. subtilis, C. albicans and A. flavus showed the least

sensitivity among the tested microbes. The antimicrobial activity of the tested GO@CuO NCS

was compared to the positive control drugs, streptomycin and fluconazole. The antibacterial

properties of GO@CuO NCS are mainly attributed to adhesion with bacteria because of their

opposite electric charges resulting in a reduction at the bacterial cell wall. It was earlier reported

that the interaction between Gram-negative bacteria and GO@CuO NCS was stronger than that

of Gram-positive bacteria because of the difference in cell walls, cell structure, physiology,

metabolism, or degree of contact of organisms with nanoparticles. Gram-positive bacteria have

thicker peptidoglycan cell membranes compared to the Gram-negative bacteria and it is harder

for GO@CuO NCS to penetrate it, resulting in a low antibacterial response [30].

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3.9. Cell viability assay

The biocompatibility of nanoparticles is an important issue in pharmaceutical

applications. Therefore, to verify the biocompatibility and cytotoxicity of NCS was evaluated by

colorimetric assay. The as obtained NCS was tested against different cells namely Vero-

ATCC® CCL-81™, HeLa-S3-ATCC ® CCL-2.2™, and MDA-MB-231-ATCC® HTB-26™.

Fig. 15 shows the impact of NCS molecules on normal and cancer cells after incubated for 24 h

with different concentration, 25–250 µg/ml. The cell viability results reveal that different cells

treated with NCS exhibited dosage dependent and time-dependent behavior. However, the as

obtained NCS was no obvious cytotoxic effect on normal which indicates an excellent

biocompatibility of prepared NCS. This lower cytotoxicity of the NCS against normal cell line

fulfills the requirements of potential biological applications. Nevertheless, it is of worth to

explore the high cytotoxic effect of NCS when treated to cancer cells, as indicated in Fig. 15. For

instance the survivability of cells are found to be 78 % for normal cells and 35% for cancer cells

at higher dose (100 µg/ml) of NCS, which is generally considered as high toxicity for cancer

cells. The biocompatibility for normal cells perhaps due to the impact of targeting agents.

However, more detailed studies required to understand the precise mechanism for cell

interaction.

4. Conclusions

In the present study, GO@CuO NCs was synthesized by modified hummers method

followed by hydrothermal treatment. The abundant porous architectures of GO@CuO exhibited

high selectivity and good reproducibility of the voltammetric response, the prepared MCPE is

considered to be very useful in the construction of simple devices in the field of medicine for the

diagnosis of dopamine deficiency. Electrochemical behavior of the prepared nanocomposite was

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showing good result with its low cost, regeneration of the electrode surface and very easy

preparation of the MCPE. Notably, the composite material showed enhanced electro catalytic

behavior, attributing to the contributions of good electrical conductivity of GO@CuO NCS. Due

to the high stability, repeatability of the MCPE, it has the potential for the future development of

nanosensors for clinical research and electro-analytical chemistry. Further, GO@CuO hybrid

nanomaterials have shown very good biocide activity against tested microorganisms (S. aureus,

B. subtilis, E. coli, P. aeruginosa, A. flavus and C. albicans). In addition, GO@CuO hybrid

nanomaterial was found to be non-toxic for normal cells (Vero-ATCC® CCL-81™), while

highly toxic for human cancer cells (HeLa-S3-ATCC ® CCL-2.2™ and (MDA-MB-231-

ATCC® HTB-26™). In summary, the new class of hybrid nanomaterials seemed to be highly

beneficial especially for biomedical applications.

Acknowledgment

The authors wish to thank Dr. B.E. Kumaraswamy, Department of Industrial Chemistry,

Kuvempu University, for his invaluable suggestions and moral support. The authors are also

thankful to K.S. Institute of Technology, Bangalore for providing the lab facility to carry out this

research work.

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Legends for Figure Fig. 1 XRD pattern of GO@CuO NCS. Fig. 2 FESEM images of GO@CuO NCS at different magnifications. Fig. 3 TEM images of GO@CuO NCS. Fig. 4 EDX spectrum of the GO@CuO NCS. Fig. 5 Thermo gravimetric analysis of the GO@CuO NCS. Fig. 6 FTIR spectra of GO@CuO NCS. Fig. 7 Cyclic voltammogram of 1mM [K4Fe(CN)6] in 1 M KCl at BCPE andGO-CuO NCS /MCPE at scan rate 50 mVs-1. Fig. 8 Cyclic voltmmogram of 5×10−5M DA at different concentration of GO-CuO NCS in MCPE. Fig. 9 Cyclic voltammogram of 5×10−5M DA in 0.2 M phosphate buffer solution at pH 7.2 using bare CPE and GO@CuO NCS /MCPE at scan rate 50 mV s−1. Fig. 10 Cyclic voltmmogram of 1.0 ×10-6 M PC in 0.2 M phosphate buffer solution at pH 7.2 using bare CPE and GO@CuO NCS /MCPE at scan rate 50 mV s−1. Fig. 11 Cyclic voltmmogram of MCPE in 0.2 M phosphate buffer solution containing 5×10−5M DA at different scan rates. Fig. 12 Graph shows the DA linear relationship between the anodic peak current and scan rate. Fig.13 Typical graph showingthePC linear relationship between the anodic peak current and scan rate. Fig. 14 Cyclic voltammogram of bare CPE and GO@CuO NCS/MCPE in real samples (40 mg/ml DA in injection) using 0.2 M phosphate buffer solution at pH 7.2, at scan rate 50 mVs-1.

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Fig. 15 Cell viability (MTT) assay of NCS against different cell lines