highly efficient multipurpose graphene oxide embedded with...
TRANSCRIPT
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.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service toour customers we are providing this early version of the manuscript. The manuscript will undergocopyediting, typesetting, and review of the resulting proof before it is published in its final form. Pleasenote that during the production process errors may be discovered which could affect the content, and alllegal disclaimers that apply to the journal pertain.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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:[email protected].
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
1
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.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
2
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
3
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
4
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
5
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.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
6
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
7
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
8
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 ×
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
9
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
10
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
11
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
12
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
13
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
14
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
15
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.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
16
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.
References
1. Kiran Kumar S.R, Mamatha G.P, Muralidhara H.B, Yogesh Kumar K, Prashanth M.K,
Electrochemical Studies of Dopamine Using Titanium Dioxide Nanoparticle Modified
Carbon Paste Electrode, Anal. Bioanal. Electrochem. 7 (2015) 175.
2. Sathisha A, Kumara Swamy B. E, Simultaneous Determination of Dopamine, Serotonin
and Folic acid at Torasemide Modified Carbon Paste Electrode: A Cyclic Voltammetric
Study, Anal. Bioanal. Electrochem. 7 (2015) 12.
3. Mariammal R. N, Ramachandran K, Renganathanb B, Sastikumar D, High Sensitivity of
Porous Cu-Doped SnO2 Thin Films to Methanol. Sensors Actuators B. 169 (2012) 199.
4. Lu C. H, Bhattacharjee B, Chen S. Y, Synthesis of ZnS:Mn2+ and ZnS:Mn2+/ZnS core–
shell nanoparticles using poly(methyl methacrylate), J. Alloys Compd. 475 (2009) 116.
5. Kima H. J, Sohna H. J, Kim S, Yi S. N, Ha D. H, Electrochemical Detection of
Hydrazine Using a Copper oxide Nanoparticle Modified Glassy Carbon Electrode. Sens.
Act B.156 (2011) 990.
6. By Yanwu Zhu, ShanthiMurali, WeiweiCai, Xuesong Li, Ji Won Suk, Jeffrey R, Potts,
Rodney S. Ruoff, Graphene and Graphene Oxide: Synthesis, Properties, and
Applications. Adv. Mater. 22 (2010) 3906.
7. Sheng Chen, Junwu Zhu, Xiaodong Wu, Qiaofeng Han, Xin Wang, Graphene
Oxide−MnO2 Nanocomposites for Supercapacitors. ACS Nano. 4 (2010) 2822.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
17
8. Xianjun Zhu, Yanwu Zhu, ShanthiMurali, Meryl D, Stoller, Rodney S. Ruoff, Highly
Conductive and Porous Activated Reduced Graphene Oxide Films for High-Power
Supercapacitors. ACS Nano. 5 (2011) 3333.
9. Ye Cong, Mei Long, Zhengwei Cui, Xuanke Li, Zhijun Dong, Guanming Yuan, Jiang
Zhang, Anchoring a uniform TiO2 layer on graphene oxide sheets as an efficient visible
light photocatalyst. Applied Surface Science. 282 (2013) 400.
10. Yogesh Kumar K, Muralidhara H. B, Arthoba Nayaka Y, Magnificent adsorption
capacity of hierarchical mesoporous copper oxide nanoflakes towards mercury and cadmium
ions: Determination of analyte concentration by DPASV. Powder Technol, 258 (2014) 11.
11. Ameer A, Arham S. A, Oves M, Khan M. S, Adnan M, Antimicrobial activity of metal
oxide nanoparticles against Gram-positive and Gram-negative bacteria: a comparative study.
Int. J. Nanomed. 7 (2012) 3527.
12. Ratnika V, Seema B, Mulayam S. G, A Review: Biological Synthesis Of Silver And
Copper Nanoparticles. Nano Biomed. Eng. 4 (2012) 99.
13. Mohan R, Shanmugharaj A. M, Hun R. S, Advances and Applications Through Fungal
Nanobiotechnology. J. Biomed. Mater. Res. B. 96 (2011) 119.
14. Yong Qian, Fucheng Ye, Jianping Xu, Zhang-Gao Le, Synthesis of Cuprous Oxide
(Cu2O) Nanoparticles/Graphene Composite with an Excellent Electrocatalytic Activity
Towards Glucose. Int. J. Electrochem. Sci. 7 (2012) 10063.
15. Nurzulaikha R, Lim H. N, Harrison I, Lim S. S, Pandikumar A, Huang N. M, Lim S. P,
Thien G. S. H, Yusoff N, Ibrahim I, Graphene/SnO2 nanocomposite-modified electrode for
electrochemical detection of dopamine. Sens Biosensing Res. 5 (2015) 42.
16. Kakarla Raghava Reddy, Kwang-Pill Lee, Anantha Iyengar Gopalan, Min Seok Kim, Ali
Md Showkat, Young Chang Nho, Synthesis of metal (Fe or Pd)/alloy (Fe–Pd)-nanoparticles-
embedded multiwall carbon nanotube/sulfonated polyaniline composites by γ irradiation.
J. Polymer Sci., Part A: Polymer Chem. 44 (2006) 3355.
17. Hummers Jr W. S, Offeman R. E, Preparation of Graphitic Oxide. J Am Chem Soc. 80
(1958) 1339.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
18
18. Junwu Zhu, Guiyu Zeng, Fude Nie, Xiaoming Xu, Sheng Chen, Qiaofeng Han, Xin
Wang, Decorating graphene oxide with CuO nanoparticles in a water isopropanol system. Nanoscale. 2 (2010) 988.
19. Bradder P, Ling S. K, Wang S, Liu S, Dye Adsorption on Layered Graphite Oxide. J
Chem Eng Data. 56 (2010) 138.
20. Archana S, Yogesh Kumar K, Sharon Olivera, Jayanna B. K, Muralidhara H. B, Ananda
A, C. Vidyasagar C, Development of Multipurpose CuO–GO Nanocomposites for Heavy
Metals Adsorption and Super Capacitor Applications. Energy Environ. Focus. 5 (2016) 1.
21. Xu Y, Chen D, Jiao X, Fabrication of CuO pricky microspheres with tunable size by a
simple solution route. J Phys Chem B. 109 (2005) 13561.
22. Raoof J. B, Kiani A, Ojani R, Valiollahi R, Electrochemical Determination of Dopamine
Using Banana-MWCNTs Modified Carbon Paste Electrode. Anal Bioanal Electrochem. 3
(2011) 59.
23. Skeika T, Zuconelli C. R, Fujiwara S. T, Pessoa C. A, Preparation and Electrochemical
Characterization of a Carbon Ceramic Electrode Modified with Ferrocenecarboxylic Acid.
Sensors. 11 (2011) 1361.
24. Mohadesi A, Karimi M.A, Pourfarsi M, A New Negative Charged Self-Assembled
Monolayer for Selective Electroanalytical Determination of Dopamine in the Presence of
Ascorbic Acid. Int. J. Electrochem. Sci. 6 (2011) 309.
25. Zhu Z, Qu L. Guo Y, Zeng Y, Sun W, Huang X, Electrochemical detection of dopamine on a Ni/Al layered double hydroxide modified carbon ionic liquid electrode.Sens. Actuators B 151 (2010) 146.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
19
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.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
20
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)
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
21
Fig. 2.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
22
Fig. 3.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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)
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
25
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)
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
26
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
27
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)
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
28
Fig. 9
60 80 100 120 140 160 180 200
4
6
8
10
IpA
(10
-8A
)
Scan rate (mV/s)
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
29
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)
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
30
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
31
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
32
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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 ×
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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].
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
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.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
References 1. Sharath Shankar S.; Kumara Swamy B. E.; Chandrashekar B. N.; J. Mol. Liq., 2012, 168,
80. 2. Kiran Kumar S.R.; Mamatha G.P.; Muralidhara H.B.; Yogesh Kumar K.; Prashanth M.K.;
Anal. Bioanal. Electrochem., 2015, 7, 175. 3. Sathisha A.; Kumara Swamy B. E.; Anal. Bioanal. Electrochem., 2015, 7, 12. 4. Li-Dong Zhao; Bo-Ping Zhang; Jing-Feng Li Min Zhou; Wei-Shu Liu; Jing Liu.; J.Alloys
Compd., 2008, 455, 259. 5. Kima H.J.; Sohna H.J.; Kim S.; Yi S.N.; Ha D.H.; Sens. Act B., 2011, 156, 990. 6. Ramgir N.; Datta N.; Kaur M.; Kailasaganapathi S.; Debnath A. K.; Aswal D. K.; Gupta S.
K.; Colloids Surf. A Physicochem. Eng. Asp., 2013, 439, 101. 7. Mariammal R. N.; Ramachandran K.; Renganathanb B.; Sastikumar D.; Sensors Actuators
B., 2012, 169, 199. 8. Lu C. H.; Bhattacharjee B.; Chen S. Y.; J. Alloys Compd., 2009, 475, 116. 9. Kima H. J.; Sohna H. J.; Kim S.; Yi S. N.; Ha D. H.; Sens. Act B., 2011, 156, 990. 10. By Yanwu Zhu; ShanthiMurali; WeiweiCai; Xuesong Li; Ji Won Suk; Jeffrey R; Potts;
Rodney S. Ruoff ; Adv. Mater., 2010, 22, 3906. 11. Marcano D. C.; Kosynkin D. V.; Berlin J. M.; Sinitskii A.; Sun Z.; Slesarev A.; Alemany
L.B.; Lu W.; Tour J. M.; ACS Nano., 2010, 4, 4806. 12. Sheng Chen.; Junwu Zhu.; Xiaodong Wu.; Qiaofeng Han.; Xin Wang.; ACS Nano., 2010, 4,
2822. 13. Xianjun Zhu.; Yanwu Zhu; ShanthiMurali; Meryl D. Stoller.; Rodney S. Ruoff.; ACS Nano.,
2011, 5, 3333. 14. Ye Cong.; Mei Long.; Zhengwei Cui.; Xuanke Li.; Zhijun Dong.; Guanming Yuan.; Jiang
Zhang.; Applied Surface Science., 2013, 282, 400. 15. Liu R.; Kulp E .A.; Oba F.; Bohannan E.W.; Ernst F.; Switzer J. A.; Chem. Mat., 2005, 17,
725. 16. Zhou K.; Wang R.; Xu B.; Li Y.; Nanotechnol., 2006., 17, 3939. 17. Ameer A.; Arham S. A.; Oves M.; Khan M. S.; Adnan M.; Int. J. Nanomed., 2012, 7, 3527. 18. Ratnika V.; Seema B.; Mulayam S. G.; Nano Biomed. Eng., 2012, 4(2), 99. 19. Mohan R.; Shanmugharaj A. M.; Hun R. S.; J. Biomed. Mater. Res. B., 2011, 96(1), 119. 20. Ruparelia J. P.; Chatterjee A. K.; Duttagupta S. P.; Mukherji S.; Acta Biomaterialia., 2008, 4,
707. 21. Yong Qian.; Fucheng Ye.; Jianping Xu.; Zhang-Gao Le.; Int. J. Electrochem. Sci., 2012, 7,
10063. 22. Nurzulaikha R.; Lim H. N.; Harrison I.; Lim S. S.; Pandikumar A.; Huang N. M.; Lim S. P.;
Thien G. S. H.; Yusoff N.; Ibrahim I.; Sensing and Bio-Sensing Research., 2015, 5, 42. 23. Hummers Jr W. S.; Offeman R. E.; Journal of the American Chemical Society., 1958, 80,
1339. 24. Bradder P.; Ling S. K.; Wang S.; Liu S.; Journal of Chemical & Engineering Data., 2010,
56, 138. 25. Archana S.; Yogesh Kumar K.; Sharon Olivera.; Jayanna B. K.; Muralidhara H. B.; Ananda
A.; C. Vidyasagar C.; Energy and Environment Focus., 2016, 5, 1. 26. Dubal D.; Dhawale D.; Salunkhe R.; Jamdade V.; Lokhande C.; Journal of Alloys and
Compounds., 2010, 492, 26.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
27. Xu Y.; Chen D.; Jiao X.; The Journal of Physical Chemistry B., 2005, 109, 13561. 28. Gillespie S.H.; Medical Microbiology-Illustrated Butterworth Heinemann Ltd. United
Kingdom., 1994, 234. 29. Jones R. N.; Barry A. L.; Gaven T. L.; Washington J. A.; Lennette E. H.; Balows A.;
Shadomy W. H.; Manual of Clinical Microbiology; American Society for Microbiology, 4th edn. Washington DC., 1984, 972.
30. Tawale J. S.; Dey K.; Pasricha R.; Sood K. N.; Srivastava A. N.; Thin Solid Films., 2010, 519, 1244.
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.
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
Fig. 15 Cell viability (MTT) assay of NCS against different cell lines