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

Accepted Manuscript 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: 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 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, RI PT B.S.Hungund5 and K.Yogesh Kumar6* 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, SC Chikmagalore Dist., Karnataka, India-577 548 Centre for Incubation, Innovation, Research & Consultancy, Jyothy Institute of Technology, Bangalore560082, India MS R&D Centre, BMS College of Engineering Bangalore-560019, India Department of Biotechnology, KLE Technological University, Hubballi-580031, India 6* M AN U Department of Chemistry, School of Engineering and Technology, Jain University, Bangalore 562 112, India AC C EP TE D *Corresponding author/authors: Tel:(+91-8147673335) E-mail:yogeshkk3@gmail.com ACCEPTED MANUSCRIPT Abstract Graphene oxides embedded with copper oxide (GO@CuO) nanocomposite were successfully synthesized via hydrothermal method The nanoparticles were characterized by XRD, SEM, RI PT 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 SC 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 M AN U 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 TE D activities, which is due to the combining effect of GO and CuO Keywords: GO@CuO nanocomposite, Dopamine, Modified carbon paste electrode, Cyclic AC C EP voltammetry ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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 TE D 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 necessary EP development of a highly sensitive and selective electrode without an enzyme or noble metal is AC C 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 ACCEPTED MANUSCRIPT 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- RI PT 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 SC 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 M AN U 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 TE D 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 EP 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 AC C 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 ACCEPTED MANUSCRIPT 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 antimicrobial and anticancer activity are reported here SC Experimental RI PT biomolecules in the presence of different interfering analytes at biological pH and their 2.1 Materials M AN U 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 TE D 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 EP GO was prepared by utilizing a modified Hummers' method as follows [16] Briefly 15 g AC C of graphite powder was added into 250 mL of cooled sulfuric acid in an ice bath At that point, 25 g of KMnO4 and 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 and the temperature was raised to 80 °C after that 250 mL of doubly distilled water was gradually mixed at 80 oC for 30 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 ACCEPTED MANUSCRIPT ultrasonic treatment for 15 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 RI PT 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 SC 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 M AN U 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 TE D 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 EP 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 AC C 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 ACCEPTED MANUSCRIPT 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 RI PT an agate mortar to produce a homogenous paste and the carbon paste was packed into the cavity of electrode of 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 SC the tube MCPE was prepared by adding 2,4,6,8 and 10 mg NCS to above mentioned graphite 2.5 Electrochemical measurements M AN U powder and silicone oil mixture 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 TE D 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 mg of NCS EP 2.6 In vitro antimicrobial activity The in vitro antimicrobial activity of as synthesized NCS were evaluated against different AC C 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 °C for further use All the pure microbial strains obtained from National ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U 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 TE D 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 to 75 µg/mL The MIC value was determined as the lowest concentration of the NCS inhibiting the visual growth of the EP microorganism on the agar plate 2.8 In-vitro anticancer activity AC C 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 ACCEPTED MANUSCRIPT 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 RI PT 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 SC 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 M AN U incubated at 37 °C for about 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: ‒ Mean absorbance of toxicant × 100 Mean absorbance of ‒ve control TE D Cytotoxicity (%) = Cell viability (%) ꞊ 100 ‒ Cytotoxicity (%) EP Results and discussion 3.1 Growth Mechanism AC C 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 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 RI PT 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 SC to room temperature, and washed three times with DI water and ethanol At last, the compound 2.3 Characterization techniques M AN U was dried in an autoclave at 60 0C for 8h 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 TE D 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) EP 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 AC C 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 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 mg of RI PT GO@CuO NCS 2.5 Preparation of bare carbon paste electrode (BCPE) and modified carbon paste electrode (MCPE) SC 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 M AN U of electrode of 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 TE D 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 EP subtilis (NCIM 2999), Escherichia coli (NCIM 2574), Pseudomonas aeruginosa (NCIM 5029), Aspergilus flavus (NCIM 524) and Candida albicans (NCIM 3471) The microbial strains were AC C 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 °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 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 RI PT 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 experiment M AN U 2.7 Minimum inhibitory concentration (MIC) SC ≤0.05 was used to determine the significant differences between the results obtained in each 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 TE D from 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 EP 2.8.1 Cell culture The normal cells (Vero-ATCC® CCL-81™) and human cancer cells (HeLa-S3-ATCC ® AC C 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 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 nanomaterials RI PT 96 flat-bottom well plates, then cells were exposed to different concentration of prepared (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 SC cells were further incubated at 37 °C for about h in 5% CO2 atmosphere Thereafter, the formazan crystals were dissolved in 200 µl of DMSO and the absorbance was monitored in a Cytotoxicity (%) = ‒ M AN U colorimetric at 578 nm with reference filter as 630 nm The cytotoxicity effect was calculated as: Mean absorbance of toxicant Mean absorbance of ‒ve control × 100 2.8.3 Statistical analysis TE D Cell viability (%) ꞊ 100 ‒ Cytotoxicity (%) A statistical analyses values for all the experiments were expressed as a ± standard EP deviation The data were performed using Student t-test, where statistical significance was calculated for treated samples and untreated (as control) cells AC C 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 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 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 RI PT the CuO dominates the GO layer which is supported by SEM studies Fig 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 SC 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 M AN U 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 reveal that the product consists of a TE D 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 EP 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 AC C 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 ACCEPTED MANUSCRIPT nanosheets The EDX spectrum of the GO@CuO sample has been depicted in Fig 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 RI PT 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 SC 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 M AN U 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 TE D 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 EP 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 AC C 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 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 RI PT 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 M KCl respectively The electrochemical response of SC GO@CuO nanocomposites of an MCPE was studied by standard 1mM [K4Fe(CN)6] in M KCl as a supporting electrolyte with a scan rate 50 mVs-1 by the CV technique The corresponding M AN U peak potential differences ∆Ep=0.169105 V for the CPE/ GO@CuO NCS (b) are shown in Fig 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 TE D 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 EP NCS played an important role in improving the reversibility electrochemical performance of the CPE/ GO@CuO NCS AC C 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 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 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 RI PT 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 SC 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 and Fig 10.The oxidation peak potential M AN U (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 TE D 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 EP 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 AC C 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 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 RI PT 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) mL of DHI solution (40 mg/mL) were diluted to 25 mL of double distilled water SC 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 M AN U /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/ 3.7 Interference study TE D GO@CuONCS proposed efficiently used for the determination of DA in injections The influence of various foreign species as interfering compounds with the determination EP 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 AC C 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 ACCEPTED MANUSCRIPT unchanged after successive 20 cyclic voltammetric scans, confirms CPE/ GO@CuONCS has good stability 3.8 Antimicrobial activity RI PT 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 SC 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, M AN U Candida albicans) The results of the antibacterial activity of GO@CuO NCS are presented in Table 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 TE D 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 EP 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 AC C 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] 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 RI PT colorimetric assay The as obtained NCS was tested against different cells namely VeroATCC® 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 SC 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 M AN U 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 TE D 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 interaction AC C Conclusions EP However, more detailed studies required to understand the precise mechanism for cell 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 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 RI PT 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, SC 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 M AN U highly toxic for human cancer cells (HeLa-S3-ATCC ® CCL-2.2™ and (MDA-MB-231ATCC® HTB-26™) In summary, the new class of hybrid nanomaterials seemed to be highly beneficial especially for biomedical applications Acknowledgment TE D 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 AC C EP research work ACCEPTED MANUSCRIPT References AC C EP TE D M AN U SC RI PT Sharath Shankar S.; Kumara Swamy B E.; Chandrashekar B N.; J Mol Liq., 2012, 168, 80 Kiran Kumar S.R.; Mamatha G.P.; Muralidhara H.B.; Yogesh Kumar K.; Prashanth M.K.; Anal Bioanal Electrochem., 2015, 7, 175 Sathisha A.; Kumara Swamy B E.; Anal Bioanal Electrochem., 2015, 7, 12 Li-Dong Zhao; Bo-Ping Zhang; Jing-Feng Li Min Zhou; Wei-Shu Liu; Jing Liu.; J.Alloys Compd., 2008, 455, 259 Kima H.J.; Sohna H.J.; Kim S.; Yi S.N.; Ha D.H.; Sens Act B., 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Fig Cyclic voltammogram of 1mM [K4Fe(CN)6] in M KCl at BCPE andGO-CuO NCS /MCPE at scan rate 50 mVs-1 Fig Cyclic voltmmogram of 5×10−5M DA at different concentration of GO-CuO NCS in MCPE EP Fig 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 AC C 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 ACCEPTED MANUSCRIPT AC C EP TE D M AN U SC RI PT Fig 15 Cell viability (MTT) assay of NCS against different cell lines ...ACCEPTED MANUSCRIPT Highly efficient multipurpose graphene oxide embedded with copper oxide nanohybrid for electrochemical sensors and biomedical applications S.R Kiran Kumar1, G.P... Graphene and Graphene Oxide: Synthesis, Properties, and Applications Adv Mater 22 (2010) 3906 Sheng Chen, Junwu Zhu, Xiaodong Wu, Qiaofeng Han, Xin Wang, Graphene Oxide? ??MnO2 Nanocomposites for Supercapacitors... 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 TE D Graphene