Ccc

Va

b

c

a

ARRAA

KCHCCG

1

ftaiahcnc

odrs

I

h0

Sensors and Actuators B 240 (2017) 338–348

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

jo u r nal homep age: www.elsev ier .com/ locate /snb

ytocompatible peroxidase mimic CuO:graphene nanosphereomposite as colorimetric dual sensor for hydrogen peroxide andholesterol with its logic gate implementation

inay Sharma a, Shaikh M. Mobin a,b,c,∗

Centre for Biosciences and Bio-Medical Engineering, Indian Institute of Technology Indore, Simrol, Indore, 453552, IndiaDiscipline of Chemistry, Indian Institute of Technology Indore, Simrol, Indore- 453552, IndiaMetallurgical Engineering and Material Science, Indian Institute of Technology Indore, Simrol, Indore- 453552, India

r t i c l e i n f o

rticle history:eceived 3 June 2016eceived in revised form 26 August 2016ccepted 29 August 2016vailable online 31 August 2016

eywords:

a b s t r a c t

Faster and easy detection of cholesterol still remains a challenge. Thus, by introducing colorimetric sen-sor for detection of cholesterol may lead to the fabrication of a ready to use sensing strip. Our presentwork demonstrates the use of a cytocompatible CuO:Graphene nanosphere (CuO:GNS) composite as aperoxidase mimic for detection of H2O2 and free cholesterol. The synthesized CuO:GNS composite wasinvestigated systematically for structural, morphological and functional aspects. The proposed methodol-ogy involves detection of H2O2 produced during oxidation of free cholesterol in the presence of cholesterol

holesterolydrogen peroxideolorimetricopper oxideraphene

oxidase. The nanocomposite sensor has shown excellent detection sensitivity for cholesterol and hasdemonstrated a linear response in the range of 0.1 mM –0.8 mM with LOD as low as 78 �M. This nanocom-posite sensor also detected a very low concentration of H2O2 (0.01–0.1 mM) with LOD of 6.88 �M. AnAND logic gate system based on CuO:GNS and Cholesterol input was also proposed. The CuO:GNS wasfound to have better cytocompatibility than standalone CuO.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

Cholesterol plays an important role in a range of physiologicalunctions in the body such as maintaining cell membranes, produc-ion of bile acids, a precursor in several biochemical pathways ands a construction unit in the hormonal system [1]. The abnormal-ty in cholesterol concentration can lead to various diseases suchs coronary heart disease, arteriosclerosis, myocardial infarction,ypertension etc. [2,3]. The routinely practised quantification ofholesterol concentration has been a vital need for clinical diag-osis [4]. Hence, there is a considerable interest in making newholesterol biosensor [5–7].

Nanomaterials are getting vital attention as enzyme mimicswing to their unique properties, high stability under abrasive con-itions, low cost, ease of synthesis and their utilization in a wide

ange of applications [8]. Although, natural enzymes have very highpecific activity but their extraction, purification, expenses, storage

∗ Corresponding author at: Discipline of Chemistry, Indian Institute of Technologyndore, Simrol, Indore- 453552, India.

E-mail address: xray@iiti.ac.in (S.M. Mobin).

ttp://dx.doi.org/10.1016/j.snb.2016.08.169925-4005/© 2016 Elsevier B.V. All rights reserved.

and inability to sustain harsh conditions, limits their applications[9].

Metal and metal oxide nanoparticles are shown to have enzymemimic activity [10,11] and has gained much attention for theirapplications towards biological systems [12,13] and sensors [14].The CuO nanoparticles are being studied extensively for enzymemimic [15], sensing [16] or catalytic properties [17–19]. Recently,various enzyme mimic inorganic nano-hybrid combinations andnanocomposites have been explored due to their enhanced prop-erties compared to their solo counterparts [9,20–22].

Graphene quantum dots (GQD) are the graphene sheets withplanner size less than 100 nm and due to edge effect and quantumconfinement, the GQD’s are reported to have excellent lumines-cence, remarkable mechanical properties, good chemical resistanceand excellent biocompatibility [23–25] but enzymatic activity ofgraphene is relatively less explored [26]. Graphene has gained sig-nificant attention in the area of chemical sensing due to its carbonbase and 2D structure [27]. Graphene is expected to be an idealsubstrate for anchoring of metallic and metal oxide nanoparticles

for enhanced performance [28]. The large surface area and perfecttwo-dimensional carbon structure of graphene results in a highercatalytic response of its composites [29]. The catalytic activity ofgraphene can be tuned by tuning its shape, size and thickness [30].

and A

Td

afspOem

ganoaheag

oairo

dsasonttl

2

2

patRwrfiftip1gaomSw

2

p

V. Sharma, S.M. Mobin / Sensors

he sensing response of nanoparticles is also shown to have a shapeependency [31].

The majority of work reported in biosensor area is based onmperometric detection of analytes [32], but these methods suf-er from manufacturing issues, narrow range of diagnosis andometimes the detected concentrations fall considerably below thehysiological range which requires quite high sample dilution [33].n the other hand, the colorimetric sensors have the advantage offficiency, convenience and no requirement of sophisticated instru-ents [34].

The sensing behaviour of CuO is extensively reported towardslucose, lactate, hydrogen peroxide, volatile organic compoundsnd gases [17,18,35–39], but its composite with grapheneanosphere is not yet explored. The toxicity associated with metalxide nanoparticles limits their applicability and cytocompatiblelternatives are being sought. CuO nanoparticles are reported toave genotoxicity and can cause DNA damage. In view of this,fforts are being made to reduce the toxicity of CuO [40]. Therere various reports available for toxic impacts of CuO [41] but itsraphene composite is rarely studied [42].

The area of molecular logic gates is growing rapidly. The devel-pment of molecular logic gates having chemical signals as inputnd optical signals as measurable output are gaining significantnterest recently. The conventional silicon based electronics iseaching its limits due to physical constraints and hence the devel-pment of new molecular logic gates is need of the hour [43].

Herein, we report a simple, cytocompatible and highly efficientual sensor for colorimetric detection of H2O2 and cholesterol. Theynthesized CuO:GNS nanocomposite not only mimic peroxidasectivity but also give enhanced sensing response as compared totandalone CuO nanoparticles with reduced toxicity. To the bestf our knowledge, this is the first report on the use of CuO:GNSanocomposite as a dual sensor for cholesterol and H2O2 detec-ion. The proposed methodology has shown excellent selectivityowards cholesterol. The methodology is also utilised as molecularogic gate to implement AND logic function.

. Experimental

.1. Materials and instrumentation

Copper acetate monohydrate, Sodium hydroxide, Hydrogeneroxide and Phenol were purchased from Merck. 4-amino-ntipyrine (4-AAP), 3,3′,5,5′-tetramethylbenzidine (TMB), choles-erol and cholesterol oxidase were purchased from sigma aldrich.hodamine-123 and 2,7-dichlorofluorescin diacetate (DCFH-DA)as purchased from sigma-aldrich. All chemicals were used as

eceived without further purification. The water used was puri-ed by a Sartorius Milli-Q system. Synergy H1 microplate reader

rom BioTek was used for absorption measurements. IR spec-ra [4000–400 cm−1] were recorded with a Bio-Rad FTS 3000MXnstrument on KBr pellets. Spectrophotometric measurement waserformed on a Varian UV–vis spectrophotometer (model: Carry00) using a quartz cuvette with a path length of 1 cm. Thermo-ravimetric analyses were performed on Metler Toledo thermalnalysis system. The measurements were done at a heating ratef 5 ◦C/min from 25 ◦C to 1000 ◦C under flowing nitrogen environ-ent. Powder X-ray diffraction studies were carried out on Rigaku

mart Lab X-ray diffractometer using CuK� radiation (1.54 Å). TEMas done using Philips CM 200 Transmission Electron Microscope.

.2. Synthesis of CuO nanoparticles

The synthesis of Copper oxide nanoparticles was based on a sim-le chemical co-precipitation method reported earlier [44]. Briefly,

ctuators B 240 (2017) 338–348 339

copper acetate monohydrate (0.02 M) was dissolved in water and500 �L glacial acetic acid was added to the solution. The mixturewas stirred, heated and refluxed for two hours. NaOH aqueous solu-tion was added to above reaction. The final concentration of NaOHwas 0.05 M. The addition of NaOH resulted in quick precipitationand color of the solution turned black. The mixture was centrifugedat 7000 rpm for 30 min and washed thrice with absolute ethanoland dried in the air. The synthesized CuO nanoparticles were fairlysoluble in water.

2.3. Synthesis of graphene nanosphere (GNS)

GNS was synthesized using ultrasonication assisted method inwhich graphite powder was mixed with 20 mL of H2SO4:HNO3(3:1) solution and sonicated for 3 h, during sonication the tempera-ture was raised from RT to 60 ◦C. Further, the solution was heated at90 ◦C and stirred vigorously. After 90 min the reaction was stoppedand the solution was left to cool down. The pH of the solution wasadjusted using NaOH. The supernatant was dialysed using 3.5 kDadialysis tubes for 4 days. Finally, a yellow coloured solution wasobtained. The effect of H2SO4:HNO3 on the interlayer spacing ofgraphite was reported earlier [45].

The catalytic response of graphene is size and shape dependent[30]. The accurate control of the size of graphene is still a challenge.The size of graphene nanosphere can possibly be altered by differ-ent sonication/mechanical condition, varying etching time, heatingrate and time [46–49].

2.4. Preparation of CuO:GNS nanocomposite

A dark brown coloured solution of water soluble CuO nanopar-ticle (5 mL) was mixed with the yellow coloured solution of GNS(5 mL) and stirred vigorously and sonicated for 15 min. The mix-ture was further heated for 30 min left to cool down. Within 30 minCuO:GNS nanocomposite settles down. The supernatant was dis-carded and the composite was air dried.

2.5. Determination of peroxidase activity

A micro-assay was used to investigate the catalytic behaviour. Astock solution of TMB (5 mM) was made in absolute ethanol. Stocksolutions of CuO and CuO:GNS (0.2 mg/mL) were made in water.In a typical experiment 76 �L of stock TMB (final concentration750 �M) was added to 320 �L PBS (pH − 7.4) and incubated for2 min. To this 84 �L of H2O2 was added. The above solution wasdivided into two wells of 96 well plate (240 �L each). 10 �L of CuOand CuO:GNS solution was added to well 1 and 2 respectively. Cor-responding control was kept without adding H2O2. The plate wasread after 5 min using a microplate reader and respective controlwas reduced.

2.6. H2O2 detection using CuO:GNS nanocomposite

The stock solutions of Phenol (6 mM) and 4-AAP (12 mM) wasprepared in 20 mM PBS. CuO:GNS stock solution of 0.2 mg/mL andH2O2 solution of various concentrations were prepared. In a typicalexperiment, a multiwell plate was used where each well was sup-plied with 70 �L of Phenol and 30 �L of 4-AAP along with 100 �Lof 20 mM PBS and 5 �L of CuO:GNS. H2O2 ranging from 0.01 mM to

0.1 mM in steps of 0.01 mM was added to each well in such a waythat the final volume was maintained at 250 �L. The multiwell platewas incubated at 37 ◦C for 40 min and was read using a microplatereader.

340 V. Sharma, S.M. Mobin / Sensors and Actuators B 240 (2017) 338–348

of sy

2

Cpdtito4tm

2

[i1mo

3

no(

Scheme 1. Schematic representation

.7. Cholesterol sensing

The stock solutions of Cholesterol (in absolute ethanol) andholesterol Oxidase (ChOx) (0.5 mg/mL in 20 mM PBS) were pre-ared. The stocks of Phenol, 4-AAP and CuO:GNS prepared for H2O2etection were used as it is. The experimental assay was designedo be of 250 �L hence multiwell plate was used. In a typical exper-ment different concentration of cholesterol ranging from 0.1 mMo 1.0 mM was incubated with 5 �L of ChOx at 37 ◦C. The dilutionf cholesterol was performed in 20 mM PBS. 70 �L Phenol, 30 �L-AAP and 5 �L of CuO:GNS was added to above incubation mix-ure. The solution was further incubated at RT and transferred to a

ultiwell plate which was read using a microplate reader.

.8. Cytotoxicity studies

The toxicity of CuO and CuO:GNS was analysed using MTT assay50], ROS generation and mitochondrial membrane potential stud-es [51]. For MTT assay the dose of CuO and CuO:GNS (0.1 �g/mL to00 �g/mL) was given for a period of 24 h. The ROS generation anditochondrial membrane potential were studied at a concentration

f 50 �g/mL of CuO and CuO:GNS.

. Results and discussion

A freshly prepared Copper oxide (CuO) and Grapheneanosphere (GNS) was taken as a precursor for the synthesisf (CuO:GNS) composite by using ultrasonic assisted methodScheme 1). The CuO:GNS composite was employed as a colori-

nthesis of CuO:GNS nanocomposite.

metric sensor for detection of H2O2 and Cholesterol. Further, thecytotoxicity and ‘AND’ logic implementation was studied.

3.1. Characterization of CuO:GNS nanocomposite

The structural, optical and thermal properties of CuO, GNSand CuO:GNS was investigated using Powder X-ray diffrac-tion (PXRD), Transmission Electron Microscopy (TEM), FourierTransform Infrared Spectroscopy(FT-IR), UV–vis spectroscopy andThermogravimetric analysis.

The structural characterisation was performed using PXRD pat-terns of nanoparticles and nanocomposite as shown in Fig. 1A.The XRD pattern of CuO revealed high phase purity and crystallinenature with characteristic peak broadening of nanomaterials. Thepeaks were well indexed as a monoclinic crystal structure (JCPDSCard no. − 80–1916) and the average crystallite size determinedusing the Debye-Scherer equation for the most intense peak (111)was found to be 8.5 nm. The PXRD pattern of GNS shows a charac-teristic peak at 2� = 24.37◦ (d spacing = 3.65 Å) corresponding to 002plane of graphene. The PXRD pattern of CuO:GNS composite shows,peaks corresponding to CuO at 2� = 38.58◦, 35.57◦ and 48.56◦ alongwith a broad peak at 2� = 24.9◦ which corresponds to GNS. The restof the CuO peaks, in the composite, are of low intensity whichprevails that the interaction resulted in a decreased crystallinity.It indicates a good interfacial interaction between the compositecomponents.

Fig. 1B depicts the FT-IR spectra of CuO, GNS and CuO:GNS. The

frequency modes obtained at 532 and 595 cm−1 are due Cu(II)-Obond vibrations. The peak around 2922 cm−1 indicates the presenceof COO group of acetic acid. The peak around 1630 in GNS andCuO:GNS composite can be assigned to C C stretching mode of

V. Sharma, S.M. Mobin / Sensors and Actuators B 240 (2017) 338–348 341

NS, Cu

gb

pnwTtoGccr

F2

Fig. 1. Characterisation of G

raphene. The strong broad peak near 3440 cm−1 is due to O Hond stretching of the adsorbed water molecule.

The morphology of synthesized CuO, GNS and CuO:GNS com-osite were examined by TEM (Fig. 1C). The CuO particles wereearly spherical in shape with an average diameter of approx. 8 nmhich is in accordance with the crystallite size estimated by PXRD.

he GNS were also found to be spherical with an average diame-er of approx. 60 nm. Interestingly, the GNS were attached to eachther resulting in a random network like structure (Fig. 1C). TheNS was seemed to be converted in a sheet-like morphology in theomposite as evident by TEM micrograph, whereas the CuO parti-les retained their size and shape in composite and appeared to beeinforced in the graphene sheet.

The UV–vis spectra of CuO, GNS and CuO:GNS are depicted inig. 1D. The CuO dispersed in water shows absorption peak at75 nm. The band gap of CuO was determined to be 2.91 eV (Fig.

O and CuO:GNS composite.

S1), that is larger than the reported band gap of bulk CuO (1.2 eV).The increase in band gap can be attributed to the quantum confine-ment effect. The GNS has given a long absorption edge which wasalso present in CuO:GNS.

The TGA of CuO, GNS and CuO:GNS are depicted in Fig. 1E. Thetotal weight loss up to 1000 ◦C was 17.5% and 25.8% in case of CuOand GNS respectively, whereas 11.2% weight loss was observed inthe case of CuO:GNS indicating increased thermal stability of thecomposite due to positive synergistic effect.

3.2. Peroxidase mimic activity

The peroxidase mimic activity of CuO nanoparticles andCuO:GNS composite was determined using the catalytic oxidationof 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of hydro-gen peroxide based on the following reaction [52] (1)

3 and Actuators B 240 (2017) 338–348

(1)

acatata

3

o

42 V. Sharma, S.M. Mobin / Sensors

It is to be noted that both CuO and CuO:GNS composite can act as catalyst for the oxidation of peroxidase substrate TMB which wasonfirmed by a sudden color change. The solution containing TMBnd H2O2 undergoes a color change from transparent to blue byhe addition of CuO or CuO:GNS which shows an absorption peakt 652 nm. The catalytic activity of CuO:GNS composite is foundo be 60% higher than that of CuO nanoparticles as estimated bybsorbance at 652 nm (Fig. 2).

.3. H2O2 and cholesterol detection

The detection of Hydrogen peroxide is of significant importancewing to its presence as an intermediate in several processes. A

Fig. 2. Absorption spectra of TMB catalytic

Fig. 3. A) Absorbance change with increasing concentration of H2O2 B) L

colorimetric process in which phenol is coupled with 4-amino-antipyrine (4-AAP) in the presence of hydrogen peroxide and aperoxidase enzyme, yields a red coloured product 1, which wasreported by Trinder (reaction 2) [53]. A similar colorimetric processwas explored for H2O2 and Cholesterol detection using peroxidasemimic CuO:GNS composite.

ally oxidized by CuO and CuO:GNS.

inear correlation between concentration of H2O2 and absorbance.

and Actuators B 240 (2017) 338–348 343

(

Hifia

V. Sharma, S.M. Mobin / Sensors

2)It can be seen from Fig. 3A that the increasing concentration of

2O2 in the presence of CuO:GNS results in increasing absorbancentensity due to higher production of a red coloured product con-rming the role of CuO:GNS as a peroxidase. A linear relation inbsorbance and concentration of H2O2 is found to be in the range

Scheme 2. Schematic illustration of colorim

Fig. 4. A) Absorbance change with increasing concentration of Cholesterol B) L

0.01 mM to 0.1 mM (Fig. 3B). The detection limit of the method iscalculated to be 6.88 �M. The ESI–MS (m/z) of production of 1 is

given in Fig. S2.

etric Cholesterol detection process.

inear correlation between concentration of Cholesterol and absorbance.

344 V. Sharma, S.M. Mobin / Sensors and A

Fig. 5. Time dependent absorption change at 490 nm of differentrA+

r

C

tCp

uSctmrtT

L

wr

da

eaction systems. A) 4-AAP + Phenol B) 4-AAP + Phenol + Cholesterol C) 4-AP + Phenol + Cholesterol + ChOx D) 4-AAP + Phenol + Cholesterol + ChOx

CuO:GNS E) Cholesterol + ChOx + CuO:GNS.

Cholesterol oxidase is an enzyme that catalyses the chemicaleaction

holesterol + O2 → Choles − 4 − en − 3 − one + H2O2

Hydrogen peroxide is the main product of oxidation of choles-erol by cholesterol oxidase, hence the H2O2 detection usinguO:GNS composite can be coupled with this cholesterol oxidationrocess to determine the presence of cholesterol (Scheme 2).

The H2O2 released during cholesterol oxidation was detectedsing a reaction between 4-AAP and phenol as depicted in thecheme 2. It was observed that the increasing concentration ofholesterol results in increasing absorption intensity at 490 nm andhe same red coloured product formation (Fig. 4A). The proposed

ethodology gave a linear relation for cholesterol detection in theange of 0.1 mM-0.8 mM as depicted in Fig. 4B. The limit of detec-ion (LOD) of the process for cholesterol was found to be 78 �M.he LOD is calculated using the following equation [54]

OD = 3.3(�/S) (1)

here � and S are standard error and slope of calibration curve

espectively.

Fig. 5 represents the time-dependent absorption changes inifferent reaction mixtures which show a continuous increase inbsorbance at 490 nm as the reaction proceeds in the presence of

Fig. 6. Selectivity and Repeatab

ctuators B 240 (2017) 338–348

CuO:GNS composite. However, no obvious change is observed with-out CuO:GNS composite, which reveals that the sensing behaviouris solely due to CuO:GNS.

3.4. Specificity and repeatability of the sensor

The specificity of the proposed methodology towards choles-terol was tested using its relative activity with other interferingspecies such as urea, uric acid, ascorbic acid, glucose and sucrose.The absorbance intensity for 1 mM of all the interfering species isplotted against absorbance intensity for 0.1 mM cholesterol. As evi-dent by Fig. 6(a), the sensor is highly selective towards cholesteroldetection without being affected by other species. To determine theconsistency of the results obtained by the sensor, the experimentwas repeated 6 times using the same set of samples made in sameconditions. The measurements were performed in triplicates andthe mean was calculated (Fig. 6(b)).

3.5. Stability of the sensor

The sensor has been tested for its activity at different temper-atures. The relative activity was tested in the temperature range(−20 ◦C–90 ◦C). The sensor is found to be stable at a wide rangeof temperature with maximum activity at around 40 ◦C (Fig. S3).The robustness of the CuO:GNS composite makes it a good alterna-tive for other peroxidase where the environmental factor such ashigh temperature is a bottleneck for their application under harshconditions.

On comparison with the recently reported probes, our CuO:GNSbased dual sensor has demonstrated better performance in termsof the limit of detection (Table 1). Moreover, the sensor also has theadvantage of facile synthetic approach.

3.6. Implementation of the logic system

An aqueous solution of 4-AAP, phenol and cholesterol oxidasewith input cholesterol and CuO:GNS can be used to construct anabsorbance based logic system. For AND logic implementation, asolution of working concentrations of Phenol, 4-AAP, and ChOx inPBS was used [Input (0,0)]. The addition of CuO:GNS to this solu-tion was used as [Input (1,0)]. The addition of cholesterol to [Input(0,0)] was treated as [Input (0,1)]. The addition of both CuO:GNS andcholesterol to (0,0) were treated as [Input (1,1)]. The inputs were

ility in sensor response.

allowed to react at RT and output was measured on a microplatereader.

Fig. 7(a) represents the truth table followed by the logic system.Fig. 7(b) represents the absorbance intensity at a different input

V. Sharma, S.M. Mobin / Sensors and Actuators B 240 (2017) 338–348 345

Table 1Performance comparison of different sensors for detection of Cholesterol.

Material Linear Range LOD Method Ref

Au Nanocomposite 0.5–450 mg dL−1 0.235 mg dL−1 Electrochemical [55]ChOx/CS–GR/GCE 5–1000 mmol L−1 0.715 mM Electrochemical [36]AgNPs/GCE 3.9–773.4 mg dL−1 0.99 mg dL−1 Electrochemical [56]Chitosan capped CdS 0.64–12.9 mM 0.47 mM Electrochemical [57]CNT/Gold 0.18–11 mM 0.02 mM Electrochemical [58]ZnO@ZnS 0.4–3.0 mM 0.02 mM Amperometric [06]CuO:GNS composite 0.1–0.8 mM 78 �M Colorimetric This Work

GCE: glassy carbon electrode, CS: chitosan, CS–GR: chitosan–graphene.

sorban

s

3

CM

v(tto

tT(wdc

Fig. 7. Logic gate implementation (a) Truth table of AND logic (b) Ab

ignal. The corresponding logic symbol is shown in Fig. 7(c).

.7. Cytocompatibility of the CuO:GNS sensor

In order to investigate the biocompatibility of synthesizeduO:GNS, its cytotoxicity was compared to standalone CuO usingTT assay and ROS generation.

As exhibited in Fig. 8, CuO:GNS shows significantly better celliability than standalone CuO. A very low concentration of CuO∼40 �g/mL) cause cell death to more than 50% of cells while morehan 90% cells are live at this concentration of CuO:GNS. Morehan 50% cells are found live at a high concentration 100 �g/mLf CuO:GNS.

The reason of reduced toxicity of CuO:GNS was studied usinghe level of ROS generation and mitochondrial membrane potential.he breast cancer cell line MCF-7 treated with CuO and CuO:GNS

50 �g/mL) was incubated with 20 �M DCFH-DA. DCFH-DA reactsith ROS inside the cell and forms a green fluorescent compound

ichlorofluorescein (DCF) [59], which is imaged under a fluores-ence microscope. As illustrated in Fig. 9, the relative proportion

ce at 490 nm at different input signals (c) Logic symbol of AND gate.

of cells having ROS induction is considerably less in the case ofCuO:GNS than CuO. In the case of CuO treatment, almost all cellsare showing green fluorescence indicating high ROS levels, whilethe relative proportion of fluorescent cells as compare to non-fluorescent is less in the case of CuO:GNS treatment indicatingfewer levels of ROS (Fig. 10).

Further, the mitochondrial membrane potential, which isknown to reduce during apoptosis was studied using the inten-sity of Rhodamine-123 in CuO and CuO:GNS treated DU145 andMCF-7 cells. The intensity of red fluorescence was reduced in CuOexposed cells as compared to CuO:GNS exposed cells indicating areduction in mitochondrial membrane potential. High fluorescenceintensity in CuO:GNS treated cells indicates non-apoptosis whileCuO is causing apoptotic cell death.

4. Conclusions

In summary, a simple and cost-effective methodology forthe fabrication of a cytocompatible CuO:GNS nanocomposite isreported. The composite was systematically characterized and

346 V. Sharma, S.M. Mobin / Sensors and Actuators B 240 (2017) 338–348

Fig. 8. The dose-dependent toxicity against skin melanoma (A375) and prostate cancer (DU145) cell lines.

ress g

iiTco

Fig. 9. Comparative oxidative st

nvestigated for its peroxidase mimic activity. Further, the compos-

te was employed as a colorimetric sensor for H2O2 and cholesterol.he sensor has shown excellent sensitivity and selectivity towardsholesterol with LOD values of 78 �M and towards H2O2 with LODf 6.88 �M. The sensor has found to be robust and stable at varied

eneration by CuO and CuO:GNS.

temperature. An AND logic gate was proposed using cholesterol

and CuO:GNS as inputs. The composite CuO:GNS was found morebiocompatible than CuO.

V. Sharma, S.M. Mobin / Sensors and Actuators B 240 (2017) 338–348 347

sessm

A

tAwfr

A

t

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

Fig. 10. Mitochondrial membrane potential as

cknowledgements

We sincerely acknowledge Sophisticated Instrumentation Cen-re (SIC), IIT Indore for providing the characterization facility.uthors would like to thank SAIF, IIT Bombay for TEM facility. SMMould like to acknowledge CSIR, New Delhi, India and IIT Indore for

unding. VS would like to thank UGC, Govt. of India for providingesearch fellowship.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.snb.2016.08.169.

eferences

[1] M. Orth, S. Bellosta, Cholesterol: its regulation and role in central nervoussystem disorders, Cholesterol (2012) e292598, http://dx.doi.org/10.1155/2012/292598.

[2] R. Ji, L. Wang, G. Wang, X. Zhang, Synthesize thickness copper (I) sulfidenanoplates on copper rod and it’s application as nonenzymatic cholesterolsensor, Electrochim. Acta 130 (2014) 239–244.

[3] Y. Tong, H. Li, H. Guan, J. Zhao, S. Majeed, S. Anjum, F. Liang, G. Xu,Electrochemical cholesterol sensor based on carbon nanotube@molecularlyimprinted polymer modified ceramic carbon electrode, Biosens. Bioelectron.47 (2013) 553–558.

[4] R. Ahmad, N. Tripathy, S.H. Kim, A. Umar, A. Al-Hajry, Y.-B. Hahn, Highperformance cholesterol sensor based on ZnO nanotubes grown on Si/Agelectrodes, Electrochem. Commun. 38 (2014) 4–7.

[5] N. Ruecha, R. Rangkupan, N. Rodthongkum, O. Chailapakul, Novel paper-basedcholesterol biosensor using graphene/polyvinylpyrrolidone/polyanilinenanocomposite, Biosens. Bioelectron. 52 (2014) 13–19.

[6] A.K. Giri, C. Charan, S.C. Ghosh, V.K. Shahi, A.B. Panda, Phase and compositionselective superior cholesterol sensing performance of ZnO@ZnSnano-heterostructure and ZnS nanotubes, Sens. Actuators B: Chem. 229(2016) 14–24.

[7] J. Jaime, G. Rangel, A. Munoz-Bonilla, A. Mayoral, P. Herrasti, Magnetite as aplatform material in the detection of glucose, ethanol and cholesterol, Sens.Actuators B: Chem. 238 (2017) 693–701.

[8] H. Wei, E. Wang, Nanomaterials with enzyme-like characteristics

(nanozymes): next-generation artificial enzymes, Chem. Soc. Rev. 42 (2013)6060–6093.

[9] A. Hayat, W. Haider, Y. Raza, J.L. Marty, Colorimetric cholesterol sensor basedon peroxidase like activity of zinc oxide nanoparticles incorporated carbonnanotubes, Talanta 143 (2015) 157–161.

[

ent following exposure to CuO and CuO:GNS.

10] C.-L. Hsu, C.-W. Lien, C.-W. Wang, S.G. Harroun, C.-C. Huang, H.-T. Chang,Immobilization of aptamer-modified gold nanoparticles on BiOCl nanosheets:tunable peroxidase-like activity by protein recognition, Biosens. Bioelectron.75 (2016) 181–187.

11] Y. Liu, G. Zhu, J. Yang, A. Yuan, X. Shen, Peroxidase-like catalytic activity ofAg3PO4 nanocrystals prepared by a colloidal route, PLoS One 9 (2014)e109158.

12] P.R. Solanki, A. Kaushik, V.V. Agrawal, B.D. Malhotra, Nanostructured metaloxide-based biosensors, NPG Asia Mater. 3 (2011) 17–24.

13] V. Sharma, A. Mohammad, V. Mishra, A. Chaudhary, K. Kapoor, S.M. Mobin,Fabrication of innovative ZnO nanoflowers showing drastic biological activity,New J. Chem. 40 (2016) 2145–2155.

14] A. Liu, Towards development of chemosensors and biosensors withmetal-oxide-based nanowires or nanotubes, Biosens. Bioelectron. 24 (2008)167–177.

15] M. Saraf, K. Natarajan, S.M. Mobin, Non-enzymatic amperometric sensing ofglucose by employing sucrose templated microspheres of copper oxide (CuO),Dalton Trans. 45 (2016) 5833–5840.

16] S. Steinhauer, A. Chapelle, P. Menini, M. Sowwan, Local CuO nanowire growthon microhotplates: In situ electrical measurements and gas sensingapplication, ACS Sens. (2016), http://dx.doi.org/10.1021/acssensors.6b00042.

17] P. Gao, D. Liu, Facile synthesis of copper oxide nanostructures and theirapplication in non-enzymatic hydrogen peroxide sensing, Sens. Actuators B:Chem. 208 (2015) 346–354.

18] A.-L. Hu, Y.-H. Liu, H.-H. Deng, G.-L. Hong, A.-L. Liu, X.-H. Lin, X.-H. Xia, W.Chen, Fluorescent hydrogen peroxide sensor based on cupric oxidenanoparticles and its application for glucose and l-lactate detection, Biosens.Bioelectron. 61 (2014) 374–378.

19] H. Ma, Y. Li, Y. Wang, L. Hu, Y. Zhang, D. Fan, T. Yan, Q. Wei, Cubic Cu2Onanoframes with a unique edge-truncated structure and a goodelectrocatalytic activity for immunosensor application, Biosens. Bioelectron.78 (2016) 167–173.

20] Y. Dong, H. Zhang, Z.U. Rahman, L. Su, X. Chen, J. Hu, X. Chen, Grapheneoxide–Fe3O4 magnetic nanocomposites with peroxidase-like activity forcolorimetric detection of glucose, Nanoscale 4 (2012) 3969.

21] M.M. Khan, S.A. Ansari, M.E. Khan, M.O. Ansari, B.-K. Min, M.H. Cho, Visiblelight-induced enhanced photoelectrochemical and photocatalytic studies ofgold decorated SnO2 nanostructures, New J. Chem. 39 (2015) 2758–2766.

22] C. Balamurugan, S. Arunkumar, D.-W. Lee, Hierarchical 3D nanostructure ofGdInO3 and reduced-graphene-decorated GdInO3 nanocomposite for COsensing applications, Sens. Actuators B: Chem. 234 (2016) 155–166.

23] W. Liu, H. Yang, C. Ma, Y. Ding, S. Ge, J. Yu, M. Yan, Graphene—palladiumnanowires based electrochemical sensor using ZnFe2O4—graphene quantumdots as an effective peroxidase mimic, Anal. Chim. Acta 852 (2014) 181–188.

24] M.E. Khan, M.M. Khan, M.H. Cho, Green synthesis, photocatalytic andphotoelectrochemical performance of an Au-graphene nanocomposite, RSC

Adv. 5 (2015) 26897–26904.

25] A. Rengaraj, Y. Haldorai, C.H. Kwak, S. Ahn, K.-J. Jeon, S.H. Park, Y.-K. Han, Y.S.Huh, Electrodeposition of flower-like nickel oxide on CVD-grown graphene to

3 and A

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

tant Professor in Discipline of Chemistry. Apart from his X-ray crystallographic work,he had develop his own research group working in wide area of research includingSingle-Crystal-to-Single-Crystal(SCSC) Transformation, Organometallic complexes,Metal-ion and anion sensing, Metal nano-oxide materials as catalyst in organictransformation, cell imaging and molecular docking.

48 V. Sharma, S.M. Mobin / Sensors

develop an electrochemical non-enzymatic biosensor, J. Mater. Chem. B 3(2015) 6301–6309.

26] X. Yang, C. Zhao, E. Ju, J. Ren, X. Qu, Contrasting modulation of enzyme activityexhibited by graphene oxide and reduced graphene, Chem. Commun. 49(2013) 8611–8613.

27] E.C. Nallon, V.P. Schnee, C. Bright, M.P. Polcha, Q. Li, Chemical discriminationwith an unmodified graphene chemical sensor, ACS Sens. 1 (2016) 26–31.

28] M.E. Khan, M.M. Khan, M.H. Cho, Biogenic synthesis of a Ag-graphenenanocomposite with efficient photocatalytic degradation, electricalconductivity and photoelectrochemical performance, New J. Chem. 39 (2015)8121–8129, http://dx.doi.org/10.1039/c5nj01320h.

29] M.E. Khan, M.M. Khan, M.H. Cho, Fabrication of WO3 nanorods on graphenenanosheets for improved visible light-induced photocapacitive andphotocatalytic performance, RSC Adv. 6 (2016) 20824–20833.

30] J. Benson, Q. Xu, P. Wang, Y. Shen, L. Sun, T. Wang, M. Li, P. Papakonstantinou,Tuning the catalytic activity of graphene nanosheets for oxygen reductionreaction via size and thickness reduction, ACS Appl. Mater. Interfaces 6 (2014)19726–19736.

31] M.M. Khan, S.A. Ansari, J. Lee, M.H. Cho, Novel Ag@TiO2 nanocompositesynthesized by electrochemically active biofilm for nonenzymatic hydrogenperoxide sensor, Mater. Sci. Eng. C 33 (2013) 4692–4699.

32] U. Saxena, A.B. Das, Nanomaterials towards fabrication of cholesterolbiosensors: key roles and design approaches, Biosens. Bioelectron. 75 (2016)196–205.

33] R. Ahmad, N. Tripathy, Y.-B. Hahn, High-performance cholesterol sensorbased on the solution-gated field effect transistor fabricated with ZnOnanorods, Biosens. Bioelectron. 45 (2013) 281–286.

34] H. Zhang, Y. Xia, Ratiometry, wavelength, and intensity: triple signal readoutfor colorimetric sensing of mercury ions by plasmonic cu2-xSe nanoparticles,ACS Sens. (2016).

35] W. Chen, J. Chen, Y.-B. Feng, L. Hong, Q.-Y. Chen, L.-F. Wu, X.-H. Lin, X.-H. Xia,Peroxidase-like activity of water-soluble cupric oxide nanoparticles and itsanalytical application for detection of hydrogen peroxide and glucose, Analyst137 (2012) 1706–1712.

36] Z. Li, Y. Chen, Y. Xin, Z. Zhang, Sensitive electrochemical nonenzymaticglucose sensing based on anodized CuO nanowires on three-dimensionalporous copper foam, Sci. Rep. 5 (2015) 16115.

37] F. Wang, H. Li, C. Yuan, Y. Sun, F. Chang, H. Deng, L. Xie, H. Li, High sensitivegas sensor based on CuO nanoparticles Synthetized by sol-gel method, RSCAdv. (2016), http://dx.doi.org/10.1039/C6RA13876D.

38] H. Yan, X. Tian, J. Sun, F. Ma, Enhanced sensing properties of CuO nanosheetsfor volatile organic compounds detection, J. Mater. Sci. Mater. Electron. 26(2014) 280–287.

39] J. Zhang, J. Ma, S. Zhang, W. Wang, Z. Chen, A highly sensitive nonenzymaticglucose sensor based on CuO nanoparticles decorated carbon spheres, Sens.Actuators B: Chem. 211 (2015) 385–391.

40] X. Zheng, Y. Su, Y. Chen, R. Wan, M. Li, H. Huang, X. Li, Carbon nanotubes affectthe toxicity of CuO nanoparticles to denitrification in marine sediments byaltering cellular internalization of nanoparticle, Sci. Rep. 6 (2016) 27748.

41] A. Wongrakpanich, I.A. Mudunkotuwa, S.M. Geary, A.S. Morris, K.A. Mapuskar,D.R. Spitz, V.H. Grassian, A.K. Salem, Size-dependent cytotoxicity of copperoxide nanoparticles in lung epithelial cells, Environ. Sci. Nano 3 (2016)365–374.

42] T. Alizadeh, S. Mirzagholipur, A Nafion-free non-enzymatic amperometricglucose sensor based on copper oxide nanoparticles—graphenenanocomposite, Sens. Actuators B: Chem 198 (2014) 438–447.

43] Madhuprasad, M.P. Bhat, H.-Y. Jung, D. Losic, M.D. Kurkuri, Anion sensors aslogic gates: a close encounter? Chem.—Eur. J. 22 (2016) 6148–6178.

44] J. Zhu, D. Li, H. Chen, X. Yang, L. Lu, X. Wang, Highly dispersed CuOnanoparticles prepared by a novel quick-precipitation method, Mater. Lett. 58(2004) 3324–3327.

45] S. Sheshmani, M.A. Fashapoyeh, Suitable chemical methods for preparation ofgraphene oxide, graphene and surface functionalized graphene nanosheets,

Acta Chim. Slov. 60 (2013) 813–825.

46] S.D. Oh, J. Kim, D.H. Lee, J.H. Kim, C.W. Jang, S. Kim, S.-H. Choi, Structural andoptical characteristics of graphene quantum dots size-controlled andwell-aligned on a large scale by polystyrene-nanosphere lithography, J. Phys.Appl. Phys. 49 (2016) 25308.

ctuators B 240 (2017) 338–348

47] K.-Y. Shin, S. Lee, S. Hong, J. Jang, Graphene size control via amechanochemical method and electroresponsive properties, ACS Appl. Mater.Interfaces 6 (2014) 5531–5537.

48] J. Zheng, H. Liu, B. Wu, Y. Guo, T. Wu, G. Yu, Y. Liu, D. Zhu, Production ofgraphene nanospheres by annealing of graphene oxide in solution, Nano Res.4 (2011) 705.

49] Q. Shao, J. Tang, Y. Lin, F. Zhang, J. Yuan, H. Zhang, N. Shinya, L.-C. Qin,Synthesis and characterization of graphene hollow spheres for application insupercapacitors, J. Mater. Chem. A 1 (2013) 15423–15428.

50] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Evaluation ofa tetrazolium-based semiautomated colorimetric assay: assessment ofchemosensitivity testing, Cancer Res. 47 (1987) 936–942.

51] M.A. Siddiqui, H.A. Alhadlaq, J. Ahmad, A.A. Al-Khedhairy, J. Musarrat, M.Ahamed, Copper oxide nanoparticles induced mitochondria mediatedapoptosis in human hepatocarcinoma cells, PLoS One 8 (2013) e69534.

52] P.D. Josephy, T. Eling, R.P. Mason, The horseradish peroxidase-catalyzedoxidation of 3,5,3′ ,5′-tetramethylbenzidine. Free radical and charge-transfercomplex intermediates, J. Biol. Chem. 257 (1982) 3669–3675.

53] P. Trinder, Determination of glucose in blood using glucose oxidase with analternative oxygen acceptor, Ann Clin. Biochem. Int. J. Biochem. Lab. Med. 6(1969) 24–27.

54] V. Sharma, A.K. Saini, S.M. Mobin, Multicolour fluorescent carbonnanoparticle probes for live cell imaging and dual palladium and mercurysensors, J. Mater. Chem. B 4 (2016) 2466–2476.

55] L. Xu, M. Zhang, Y. Hou, W. Huang, C. Yao, Q. Wu, An Au nanocomposite basedbiosensor for determination of cholesterol, Anal. Methods 7 (2015)3480–3485.

56] S. Nantaphol, O. Chailapakul, W. Siangproh, Sensitive and selectiveelectrochemical sensor using silver nanoparticles modified glassy carbonelectrode for determination of cholesterol in bovine serum, Sens. Actuators B:Chem. Part A 207 (2015) 193–198.

57] H. Dhyani, M.A. Ali, S.P. Pal, S. Srivastava, P.R. Solanki, B.D. Malhotra, P. Sen,Mediator-free biosensor using chitosan capped CdS quantum dots fordetection of total cholesterol, RSC Adv. 5 (2015) 45928–45934.

58] X. Cai, X. Gao, L. Wang, Q. Wu, X. Lin, A layer-by-layer assembled and carbonnanotubes/gold nanoparticles-based bienzyme biosensor for cholesteroldetection, Sens. Actuators B: Chem. 181 (2013) 575–583.

59] M.A. Siddiqui, M.P. Kashyap, V. Kumar, A.A. Al-Khedhairy, J. Musarrat, A.B.Pant, Protective potential of trans-resveratrol against 4-hydroxynonenalinduced damage in PC12 cells, Toxicol. In Vitro 24 (2010) 1592–1598.

Biographies

Vinay Sharma Vinay Sharma is a research scholar at Indian Institute of TechnolgyIndore. His research work is devoted to the development of biosensors based onnanomaterials.

Dr. Shaikh M. Mobin Dr. Shaikh completed his Bachelor’s and Master’s from WilsonCollege, University of Mumbai with major in Chemistry. Further, he completed Ph.D.from University of Mumbai in Chemistry. He had worked as X-ray Crystallographerat National Single Crystal X-ray Diffraction Facility, IIT Bombay. Dr. Shaikh co-authormore than 250 publications in last 15 years of research experience. So far he hadsolved and refined over 6000 crystal structures. In 2012, he joined IIT Indore as Assis-