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

Therefore, we have used the novel GO@CuO composite material for selective determination of different biomolecules in the presence of different interfering analytes at bi[r]

Highly efficient multipurpose graphene oxide embedded with copper oxide nanohybrid

for 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 oxide

nanohybrid for electrochemical sensors and biomedical applications, Journal of Science: Advanced

Materials 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 to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highly efficient multipurpose graphene oxide embedded with copper oxide nanohybrid for

electrochemical sensors and biomedical applications S.R Kiran Kumar 1 , G.P Mamatha 2* , H.B Muralidhara 3 , M.S Anantha 1 , S Yallappa 4 , B.S.Hungund 5 and K.Yogesh Kumar 6*

1

Centre 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,

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

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

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

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

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

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

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 µ g/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

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

×

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 67.940correspond 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

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 [K 4 Fe(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

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

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

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

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

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 µ g/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

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 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

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

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

(110)

(111) (200)

(112)

(202)

(002) (001)

(101)

1390.67

1634.9

2856.29 2926.3

C-OH

-OH