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Sensors and Actuators B 238 (2017) 842–851

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

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

u-Ag bimetallic nanoparticles on reduced graphene oxideanosheets as peroxidase mimic for glucose and ascorbic acidetection

itashree Darabdhara a,b, Bhagyasmeeta Sharma a,b, Manash R. Das a,b,∗,abah Boukherroub c, Sabine Szunerits c

Advanced Materials Group, Material Sciences and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat 785006, Assam, IndiaAcademy of Scientific and Innovative Research, CSIR-NEIST Campus, IndiaInstitut d’Electronique, de Microélectronique et de Nanotechnologie (IEMN), UMR CNRS 8520, Université Lille1, Avenue Poincaré – CS 60069, 59652illeneuve d’Ascq, France

r t i c l e i n f o

rticle history:eceived 28 February 2016eceived in revised form 18 July 2016ccepted 20 July 2016vailable online 21 July 2016

eywords:

a b s t r a c t

Bimetallic nanoparticles (NPs) in several instances have resulted in improved catalytic properties whencompared to the monometallic analogues. This paper reports on a wet-chemistry approach for the synthe-sis of reduced graphene oxide (rGO) nanosheets decorated with bimetallic Cu-Ag NPs. The nanocompositepossesses the advantages of combining rGO and metallic NPs and exhibits excellent intrinsic per-oxidase like activity, which can be used to catalyse the reaction of peroxidase substrates such as3,3′,5,5′-tetramethylbenzidine (TMB) in presence of hydrogen peroxide (H2O2). A combination of the

educed graphene oxideopper-silver bimetallic nanoparticleseroxidaselucosescorbic acidensing

peroxidase-like activity of the Cu-Ag/rGO nanostructures with glucose oxidase (GluOx), allowed the con-struction of a sensitive and a selective colorimetric assay for glucose detection in blood serum with adetection limit of 3.8 �M. The monometallic analogues displayed a detection limit of 7.9 �M (Ag/rGO) and9.7 �M (Cu/rGO). In addition, the Cu-Ag/rGO nanostructures were also successfully applied for sensingascorbic acid with a detection limit of 3.6 �M.

© 2016 Elsevier B.V. All rights reserved.

. Introduction

The contemporary applications of reduced graphene oxiderGO) nanosheets loaded with metallic nanoparticles (NPs) inter-ect nowadays with a variety of disciplines, including theranostic,eterogeneous catalysis, sensing and biomimetics [1–3]. A promis-

ng application of the NPs-loaded rGO composites is their use asrtificial enzymes, notably as peroxidase mimetic catalysts [4–9].ike other natural enzymes, the catalytic activities of oxidore-uctases such as peroxidases are endowed with several benefits,

ncluding substrate specificity, selectivity and efficiency. However,roblems related to the sensitivity of the enzymatic activity to

nvironmental conditions such as pH, instability over time due tonzyme denaturation, as well as high costs associated with enzymereparation and purification limit their applications [10]. Since the

∗ Corresponding author at: Advanced Materials Group, Material Sciences andechnology Division, CSIR-North East Institute of Science and Technology, Jorhat85006, Assam, India.

E-mail address: mnshrdas@yahoo.com (M.R. Das).

ttp://dx.doi.org/10.1016/j.snb.2016.07.106925-4005/© 2016 Elsevier B.V. All rights reserved.

demonstration by Yan and co-workers that Fe3O4 magnetic parti-cles possess intrinsic peroxidase-like activity [11], numerous otherartificial structures have emerged as enzyme mimics [12], withNPs loaded rGO showing catalytic performances similar to or evenbetter than that of the natural enzyme [13].

Herein, we report on the synthesis of Cu-Ag bimetallic NPs dec-orated on rGO nanosheets with intrinsic peroxidase-like catalyticactivity towards the oxidation of 3,3′,5,5′-tetramethylbenzidine(TMB) in presence of hydrogen peroxide (H2O2). A large number ofgraphene based hybrid catalysts with peroxidase-like activity suchas gold [4,5,14], iron oxide [8,15] or platinum NPs [9] are reportedso far. On the contrary, this work takes into account the possiblesynergistic effects when rGO is loaded with two different metalcatalysts, Cu and Ag NPs. Both monometallic Cu NPs [16] and AgNPs [17] have shown excellent peroxidase-like properties [6,16].Bimetallic Cu-Ag NPs have resulted in improved performance incomparison to the monometallic analogues for instance in oxygen

reduction reaction (ORR) [18] and CO oxidation reaction [19]. Load-ing Cu-Ag bimetallic NPs onto rGO nanosheets is expected not onlyto improve the chemical and physical properties of Cu-Ag/rGO dueto the synergistic effects of all the three components, but also give

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ise to a new class of bimetallic graphene nanocomposite materialsith prospective catalytic applications. Moreover, the use of Cu-g/rGO nanocomposites as a peroxidase mimic towards detectionf glucose and ascorbic acid is novel and has not been reported yet.he Cu-Ag bimetallic NPs are low cost materials compared to otheroble bimetallic NPs. Indeed, by combining the peroxidase-likectivity of Cu-Ag/rGO with glucose oxidase (GluOx), a simple and aensitive colorimetric assay for analysis of glucose concentrationsn blood samples was developed. While the sensing of glucose levelsn real samples has gained remarkable attention over time [20–22],scorbic acid is another molecule of biological importance [16], theensing of which is also equally important. Ascorbic acid acts as

cofactor in several enzymatic reactions and plays an essentialole in metabolic reactions of both human and animals. It is a nat-rally occurring compound with antioxidant properties, essential

n cancer prevention and immunity development. Insufficiency ofscorbic acid is associated with severe symptoms of scurvy, cardio-ascular diseases, Parkinson’s disease and cancer. Thus, detectionf ascorbic acid is of great importance in clinical, pharmaceuticalnd food industry.

In this study, we report on the synthesis of Cu-Ag/rGO nanocom-osite and its evaluation as a peroxidase mimic in oxidation oferoxidase substrate TMB into blue coloured oxidized TMBDI.he Cu-Ag/rGO nanocomposite is found to follow Michaelisenten kinetics with good affinity towards TMB in comparison toonometallic Ag/rGO and Cu/rGO nanocomposites. Based on the

olour changing capability, a simple, low-cost and easy colorimetricetection technique is developed towards detection of both glucosend ascorbic acid.

. Materials and methods

.1. Materials

Graphite powder (<20 �m, Sigma-Aldrich, USA), sulfuric acidH2SO4, AR grade, Qualigens, India), hydrochloric acid (HCl, ARrade, Qualigens, India), hydrogen peroxide (H2O2 30%, Qualigens,ndia), potassium permanganate (KMnO4, >99%, Merck, India), cop-er (II) acetate monohydrate (Cu(CH3COO)2·H2O, Sigma-Aldrich,SA), silver nitrate (AgNO3, Qualigens, India), N-methyl-2-yrrolidone (NMP, Alfa Aesar, USA), oleylamine (>70%, Sigmaldrich, USA), trioctylphosphine (>90%, Sigma Aldrich, USA),,3′,5,5′-tetramethylbenzidine (TMB > 99%, Sigma Aldrich, USA),lucose oxidase (GluOx, Sigma Aldrich, USA), D-(+) glucose (>99.5%,igma Aldrich, USA), L-ascorbic acid (Sigma Aldrich, USA), uric acid≥99%, Sigma Aldrich, USA) and dopamine hydrochloride (Sigmaldrich, USA) were used as-received without any further purifica-

ion.

.2. Synthesis of Cu/rGO, Ag/rGO, Cu-Ag/rGO nanocomposites andu-Ag NPs

Graphene oxide (GO) was synthesized using a modified Hum-ers and Offemann’s procedure [23]. Typically, graphite powder

2 g) was oxidized in the presence of KMnO4 (6 g) and H2SO446 mL) followed by exfoliation of the formed graphite oxide in-methyl-2-pyrrolidone (NMP) solution under high power ultra-

onication (using SONICS Vibra cell ultrasonicator operating at arequency of 20 KHz ± 50 Hz) for 1 h to obtain a homogeneous sus-ension of GO. A GO suspension (1 mg mL−1) in NMP (20 mL) wassed to synthesize rGO modified with Cu-Ag bimetallic NPs. Typi-

ally, AgNO3 (17 mg) and Cu(CH3COO)2·H2O (18 mg) were added torioctylphosphine (0.2 mL) to which 5 mL of oleylamine was added.o the above reaction mixture GO in NMP was added followed byeating the reaction mixture at 60 ◦C for 10 min. The entire reac-

tuators B 238 (2017) 842–851 843

tion mixture was then heated to 200 ◦C for 2 h. The mixture wassubsequently cooled to room temperature followed by addition of20 mL ethanol to yield a black precipitate. The black precipitatewas then filtered and washed with a mixture of toluene:ethanol(1:3 v:v) three times and dried in an oven at 65 ◦C to obtain thedesired Cu-Ag/rGO bimetallic nanocomposite.

For the synthesis of Cu/rGO, Cu(CH3COO)2·H2O (18 mg) wasmixed with trioctylphosphine (0.2 mL) to which 5 mL of oleylaminewas added. GO in NMP was added to the above reaction mixture fol-lowed by heating at 60 ◦C for 10 min. The entire reaction mixturewas then heated to 200 ◦C for 2 h. The mixture was subsequentlycooled to room temperature followed by addition of 20 mL ethanolto yield a black precipitate which was filtered and washed witha mixture of toluene:ethanol (1:3 v:v) three times and dried in anoven at 65 ◦C to obtain the desired Cu/rGO nanocomposites.

Similarly, for Ag/rGO nanocomposites, AgNO3 (17 mg) wasmixed with trioctylphosphine (0.2 mL) to which 5 mL of oleylaminewas added. GO in NMP was added to the above reaction mixture fol-lowed by heating at 60 ◦C for 10 min. The entire reaction mixturewas then heated to 200 ◦C for 2 h. The mixture was subsequentlycooled to room temperature followed by addition of 20 mL ethanolto yield a black precipitate which was filtered and washed witha mixture of toluene:ethanol (1:3 v:v) three times and dried in anoven at 65 ◦C to obtain the desired Ag/rGO nanocomposite.

In a typical synthesis of Cu-Ag bimetallic NPs without rGO,AgNO3 (17 mg) and Cu(CH3COO)2·H2O (18 mg) were added to tri-octylphosphine (0.2 mL) followed by addition of 5 mL of oleylamine.The reaction mixture was stirred for 20 min followed by additionof 20 mL NMP. The entire reaction mixture was heated to 60 ◦C for10 min and then heated to 200 ◦C for 2 h, cooled to room temper-ature followed by addition of ethanol (20 mL). The solution wascentrifuged and then washed with a mixture of toluene:ethanol(1:3 v:v) three times and dried at 65 ◦C to obtain the Cu-Ag bimetal-lic NPs.

2.3. Characterization

Powder X-ray diffraction (XRD) patterns were recorded on aRigaku X-ray diffractometer (model: ULTIMA IV, Rigaku, Japan)with a scanning rate of 3◦ min−1 and 2� value ranging from 5 to100◦. The instrument uses Cu K� (� = 1.54056 Å) as the X-ray sourceand operates at a generator voltage of 40 kV and a generator currentof 40 mA.

The morphology and the crystal structure of the nanocom-posites were characterized by transmission electron microscopy(TEM) and high-resolution TEM (HRTEM) using a JEOL JEM 2100,transmission electron microscope (Japan) operated at an acceler-ating voltage of 200 kV. For analysis, dispersed colloidal solutionsof nanocomposites were prepared and dropped onto standardcarbon-coated copper grids which were then air dried at roomtemperature.

X-ray photoelectron spectroscopy (XPS) measurements wereperformed with an ESCALAB 220 XL spectrometer from Vac-uum Generators featuring a monochromatic Al K� X-ray source(1486.6 eV) and a spherical energy analyzer operated in the CAE(constant analyzer energy) mode (CAE = 100 eV for survey spectraand CAE = 40 eV for high-resolution spectra), using the electromag-netic lens mode. The angle between the incident X-rays and theanalyzer is 58◦. The detection angle of the photoelectrons is 30◦.

Thermal analysis (TGA) of the samples was carried out usinga Thermal Analyzer (Model: Netzsch STA 449F3). The temper-

ature calibration was done at a heating rate of 10 ◦C min−1

by analysing the TGA signal of the melting peak (Tm) ofthe pure substances: In (Tm = 431.8 K), Al (Tm = 959.2 K), Au(Tm = 1346.9 K), Sn (Tm = 508.4 K), and Zn (Tm = 700.8 K) using the

844 G. Darabdhara et al. / Sensors and Actuators B 238 (2017) 842–851

c), HR

sbma11m

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at

2

Hbotwtvv

sToatMrVTt

Fig. 1. Low resolution TEM images (a,b), size distribution histogram (

ame crucible (alumina) under similar conditions. Accordingly, aaseline correction was also performed in a nitrogen environ-ent at the same heating rate with an empty crucible. Then,

ll the TGA analyses were carried out by taking approximately0 mg of each sample in the crucible and heated from 20 to000 ◦C at a heating rate of 10 ◦C min−1 in a nitrogen environ-ent.

Diffuse reflectance infrared Fourier transform (DRIFT) spectraere recorded in transmittance mode at 4 cm−1 spectral resolution

sing an IR Affinity-1 FTIR spectrophotometer (Shimadzu, Japan)quipped with a DRS-8000 DRIFT accessory in the 400–4000 cm−1

ange.

The catalytic oxidation of TMB, colorimetric detection of glucosend ascorbic acid were all monitored using a UV–vis spectropho-ometer (Specord 200 Analytik Zena).

.4. Determination of peroxidase-like activity

To 2.5 mL of sodium acetate buffer (pH 3.8), TMB (0.5 mM) and2O2 (30%; 50 �L) were added and the reaction mixture was incu-ated at 35 ◦C for 30 min followed by monitoring the formation ofxidation product of TMB at 652 nm using UV–vis spectrophotome-ry. The effect of Cu-Ag/rGO concentration on the catalytic reactionas investigated over the range of 3–7 mg L−1. The effect of pH on

he catalytic reaction was evaluated by carrying out the reaction atarying pH from 2 to 10. Temperature effects were also studied byarying the temperature from 25 to 55 ◦C.

The enzyme kinetics of Cu-Ag/rGO was estimated using steadytate kinetics by varying the concentration of one substrate (eitherMB or H2O2) at a time. Typical Michaelis-Menten curves werebtained for Cu-Ag/rGO nanocomposites by varying both TMBnd H2O2 over a certain concentration range. The affinity ofhe enzyme towards the substrate was determined from the

ichaelis-Menten constant (Km) and the Lineweaver-Burk double( ) ( ) ( )

eciprocal plot: 1

v = KmVm

1[S] + 1

Vm , where v = initial velocity;

m = maximal reaction velocity and [S] = substrate concentration.he Michaelis constant Km is the substrate concentration at whichhe reaction rate is half of Vm.

TEM images (d,e), and SAED pattern (f) of Cu-Ag/rGO nanocomposite.

2.5. Detection of glucose and ascorbic acid

Glucose (0.001−3 mM; 0.1 mL) in phosphate buffer (10 mM,pH 7, 0.5 mL) was added to glucose oxidase (GluOx) (1 mg mL−1,0.1 mL) and the entire reaction mixture was incubated at 35 ◦C for30 min. TMB (20 mM, 0.3 mL), sodium acetate buffer (pH 3.8, 0.2 M,2.5 mL) and Cu-Ag/rGO (5 mg L−1; 0.040 mL) were added to the mix-ture and further incubated at 35 ◦C for 30 min and used for standardcurve measurements. The selectivity of the Cu-Ag/rGO catalyst forglucose was studied against glucose analogues (10 mM) such asmaltose, fructose and lactose.

Similar method was extended for the detection of glucose inreal samples, such as clinical blood samples and compared theresults with a standard hospital method. The real blood sampleswere first treated by centrifugation at 4000 rpm for 5 min. The col-lected supernatants were diluted ten times using 10 mM buffer (pH7) and used for further measurements. The absorbance at 652 nmwas then monitored using UV–vis spectrophotometry. The effi-ciency of glucose detection using Cu-Ag/rGO was compared to thatof monometallic Cu/rGO and Ag/rGO nanocomposites.

For ascorbic acid detection, H2O2 (30%, 0.5 mL) was added toascorbic acid solutions (0–3 mM) followed by addition of phos-phate buffer (10 mM, pH 3.8, 0.5 mL) and incubated for 20 min atroom temperature. TMB (20 mM, 0.3 mL), Cu-Ag/rGO (5 mg mL−1,0.040 mL) and sodium acetate buffer (pH 3.8; 0.2 M, 2.5 mL) wereadded to the mixture and incubated at 35 ◦C for 30 min. The absorp-tion spectrum of the reaction mixture was monitored at 652 nmusing UV–vis spectrophotometry. The selectivity of the Cu-Ag/rGOcatalyst for ascorbic acid (2 mM) was studied against dopamine(DA) and uric acid (UA).

3. Results and discussion

3.1. Formation and characterization of Cu-Ag/rGO, Cu/rGO,Ag/rGO nanocomposites and Cu-Ag NPs

The size, morphology and crystallinity of the nanocompositeswere determined using TEM and HRTEM imaging. RepresentativeTEM images of Cu-Ag/rGO are displayed in Fig. 1. The low resolu-

nd Actuators B 238 (2017) 842–851 845

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G. Darabdhara et al. / Sensors a

ion TEM images (Fig. 1a,b) show the formation of bimetallic Cu-AgPs on folded/wrinkled rGO nanosheets. The average particle sizef the polydisperse nanocomposite sample was determined to be6.4 ± 1.2 nm (Fig. 1c). Average particle size was obtained from 34articles which consisted of particles with very small size around0 nm as well as large particles of around 90 nm in diameter. Theata was obtained using Image J software. Also the particles seemo have agglomerated during the formation of bimetallic NPs. Thelloy nature of the Cu-Ag/rGO nanocomposite is confirmed by theppearance of clear lattice fringes in the HRTEM images (Fig. 1d,e)hat can be indexed to Cu(111) plane with a lattice fringe of.209 nm and to the Ag(111) plane with a lattice fringe of 0.236 nm,

ndicating random mixture of both metals in one particle. Further-ore, the selected area electron diffraction (SAED) pattern (Fig. 1f)

onfirms the polycrystalline nature of the Cu-Ag/rGO nanocompos-te. The (111), (220) and (222) planes for fcc Ag are obtained as wells the (111), (200) and (220) planes for fcc Cu.

For comparison, TEM images along with size distribution his-ograms, HRTEM and SAED patterns of monometallic Cu/rGO andg/rGO are displayed in Fig. S1 and S2 (see Supporting information,I). In the case of Cu/rGO, Cu NPs of 10.2 ± 0.5 nm in size, calculatedrom a population of 26 particles, are found to be distributed on rGOanosheets (Fig. S1a,b). The HRTEM image (Fig. S1c) clearly showshe lattice fringe of 0.209 nm of the (111) plane of fcc Cu. The crys-allinity of Cu/rGO is furthermore evidenced by the correspondingAED patterns (Fig. S1d). Similarly, Ag NPs of 22.7 ± 0.6 nm in size,alculated from a population of 23 particles, are observed on theGO nanosheets (Fig. S2 a,b). The HRTEM image (Fig. S2c) displays

lattice fringe of 0.236 nm, corresponding to the (111) plane of fccg. The crystalline nature of the Ag/rGO nanocomposite was furtherevealed by SAED patterns (Fig. S2d).

The successful formation of Cu-Ag bimetallic NPs, andonometallic Cu and Ag NPs along with reduction of GO to rGO

s confirmed from XRD analysis. As seen in Fig. 2, the XRD patternf Cu/rGO exhibits a diffraction at 2� = 42.9◦ corresponding to the111) plane of fcc Cu. In the case of Ag/rGO, diffraction peaks at� = 38.1◦, 44.3◦, 64.4◦, 77.4◦, 81.5◦, corresponding to (111), (200),220), (311) and (222) planes, respectively of fcc Ag are observed.owever, for Cu-Ag bimetallic NPs, diffractions of both Cu and Agre distinctly observable: 38.2◦ (111- fcc Ag), 43.4◦ (111- fcc Cu),4.4◦ (200- fcc Ag), 50.5◦ (200- fcc Cu), 64.6◦ (220- fcc Ag), 74.2◦

220- fcc Cu), 77.5◦ (311- fcc Ag) and 81.6◦ (222- fcc Ag). The addi-ional diffraction peak at 21.2◦ is indicative of the conversion of GO

o rGO nanosheets during NPs formation.

TGA analysis under N2 atmosphere indicates a major weight lossetween 300 and 500 ◦C and a slight loss above 600 ◦C (Fig. S3). Theeight loss around 300 ◦C is attributed to the removal of different

Fig. 3. FTIR spectra of Cu/rGO, Ag/rGO

Fig. 2. XRD patterns of Cu/rGO, Ag/rGO and Cu-Ag/rGO nanocomposites.

oxygen containing functional groups of GO like carboxyl, carbonyl,hydroxyl, and epoxy groups. The slight weight loss above 600 ◦Cresults from the loss of carbon skeleton of the rGO nanosheets.

FTIR analysis was used to examine the chemical composition ofthe materials (Fig. 3). The intensity of the stretching bands of oxy-gen functionalities like O H and C O are greatly reduced in thenanocomposites when compared to GO (Fig. S4), indicating suc-cessful formation of rGO. The bands at 1547, 1563 and 1540 cm−1

in the FTIR spectra of Cu/rGO, Ag/rGO and Cu-Ag/rGO correspond toN H bending modes, due to presence of some oleylamine remain-ing in the nanocomposites. Oleylamine acts both as a surfactant anda stabilizer, as well as a mild reducing agent during the formationof Cu/rGO, Ag/rGO and Cu-Ag/rGO nanocomposites.

XPS results also confirmed the formation of Cu-Ag/rGO. Fig. 4Adepicts the high resolution XPS C1s core level spectrum of Cu-Ag/rGO. It can be deconvoluted into three peaks at 284.3 eV (Csp2),285.0 eV (Csp3) and 286.5 eV (C O) with the Csp2 component beingdominant. The results suggest the reduction of GO during NPs depo-sition, in accordance with the results obtained using the variouscharacterisation techniques. The high resolution of the core levelof Cu2p reveals the presence of several peaks with binding energiesat 932.6, 934.4, 942.3, 952.5 and 954.9 eV (Fig. 4B). The peaks at932.6 and 952.5 eV are attributed to Cu2p3/2 and Cu2p1/2, respec-tively from metallic Cu0 or Cu+ (Cu2O). Cu0 cannot be distinguishedfrom Cu+ by XPS because of their spectral overlap [24,25]. However,

based on the literature data, the peak is most likely due to Cu0 (theISO standard Cu metal line is at 932.63 eV with a deviation set at±0.025 eV) [24]. The peaks at 934.4 and 954.9 eV are from Cu2+ dueto Cu2p3/2 and Cu2p1/2, respectively, arising from Cu(OH)2 rather

and Cu-Ag/rGO nanocomposites.

846 G. Darabdhara et al. / Sensors and Actuators B 238 (2017) 842–851

Fig. 4. High resolution XPS core level spectra of

FCs

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ig. 5. UV–vis spectra of 0.5 mM TMB in presence of (A) 50 mM H2O2, (B) 5 mg L−1

u-Ag/rGO, (C) both H2O2 and GO, and (D) both H2O2 and Cu-Ag/rGO. The insethows the corresponding photographs.

han CuO [4]. The presence of a shake-up satellite peak at 942.3 eV isharacteristic of materials having a d9 configuration in their groundtate, as in Cu2+. The overall Cu content is 0.6 at% (Table S1). Theigh resolution of the core level of Ag3d exhibits a characteristicoublet peak at 367 and 370 eV corresponding to Ag3d5/2 and Agd3/2, respectively (Fig. 4C). The band at 367 eV can be deconvoluted

nto two components: metallic Ag and AgO at binding energies of98.5 and 367.7 eV, respectively, suggesting that the surface of thearticles is mainly in the oxidized state. The overall Ag content is.9 at% (Table S1). The same is true for monometallic Cu/rGO andg/rGO particles with the atomic percentage of the metals listed inable S1.

Details of the formation and characterization of Cu-Ag bimetallicPs without GO support are illustrated in the SI. Fig. S5 and S6

epresent the XRD patterns and TEM images of Cu-Ag bimetallicPs without GO support.

.2. Intrinsic peroxidase like activity of Cu-Ag/rGOanocomposite and optimization of reaction conditions

The peroxidase-like activity of Cu-Ag/rGO was investigated by

tudying the catalytic oxidation reaction of the peroxidase sub-trate TMB, which in the presence of H2O2, produces a blue colourolution of 3,3′,5,5′-tetramethylbenzidine diimine (TMBDI) with anbsorbance maximum located at 652 nm (Fig. 5) [26]. As a control,

Cu-Ag/rGO: (A) C1s, (B) Cu2p and (C) Ag3d.

TMB oxidation catalysed by Cu-Ag/rGO was performed in absenceof H2O2. No colour change was observed, indicating that both H2O2and Cu-Ag/rGO nanocomposite are required for TMB oxidation. TheTMB oxidation reaction was also carried out in presence of GO andit was found that the activity of GO towards TMB was much lower incomparison to Cu-Ag/rGO nanocomposite (Fig. 5). Also, the activ-ity of free Cu-Ag NPs without GO support (Cu2O-Ag in our case)towards TMB oxidation reaction was found to be lower than thatof Cu-Ag/rGO (Fig. S7).

The dependence of the peroxidase-like activity of Cu-Ag/rGOcatalyst on various factors such as pH, temperature and amountof catalyst was investigated, which allowed the selection of theoptimum analytical conditions. The effect of pH on the activity ofCu-Ag/rGO catalyst is depicted in Fig. 6A. Maximal absorbance isobserved at pH 4, above which the catalytic activity of Cu-Ag/rGOsharply decreases. Maximal catalytic activity is recorded at 35 ◦C(Fig. 6B) at a concentration of 5 mg L−1 of Cu-Ag/rGO (Fig. 6C). Themaximum catalytic activity is thus obtained under the followingconditions: pH 4, 35 ◦C and 5 mg L−1 of Cu-Ag/rGO nanocomposite.

3.3. Kinetic analysis of Cu-Ag/rGO as peroxidase mimics anddetermination of Michaelis constant

The intrinsic peroxidase-like catalytic activity of Cu-Ag/rGOnanocomposite was investigated by initial rate method with TMBand H2O2 as substrates using steady state kinetics. Kinetic analy-sis was performed by changing the concentration of the substrates(TMB and H2O2), while keeping the concentration of other reagentsconstant. The values of absorbance were changed to their corre-sponding concentrations using the Beer Lambert’s law:

A = εTMBDI × c × L

Where, εTMBDI = 39000 M−1cm−1 at 652 nm [27]. The correspond-ing concentration terms were changed to velocity terms and typicalMichaelis Menten curves were obtained for TMB (Fig. 7A) and H2O2(Fig. 7B), as substrates in a certain concentration range. The catalyticparameters Km (Michaelis constant) and Vm (maximal velocity) ofthe enzyme mimics Cu-Ag/rGO were obtained by using LineweaverBurk double reciprocal plots (Fig. 7A, B insets). The intercept ofthe Lineweaver Burk double reciprocal plots gives the value ofVm and the slope provides the value of Km. For comparison, theMichaelis Menten curves and Lineweaver Burk double reciprocalplots for both TMB and H2O2 using the monometallic Ag/rGO and

Cu/rGO nanocomposites are exhibited in Fig. S8 and S9, respec-tively. The Km and Vm values for the three catalysts Cu-Ag/rGO,Ag/rGO, and Cu/rGO are listed in Table 1. The low Km values for thethree catalysts indicate high affinity towards both TMB and H2O2.

G. Darabdhara et al. / Sensors and Actuators B 238 (2017) 842–851 847

Fig. 6. UV–vis absorption spectra of TMB using Cu-Ag/rGO nanocomposite withvarying (A) pH, (B) temperature and (C) concentration of catalyst.

Table 1Michaelis Menten constant (Km) and maximum reaction rate (Vm) for Cu-Ag/rGO,Cu/rGO and Ag/rGO nanocomposites.

Catalysts Substance Km [mM] Vm [10−8 Ms−1]

Cu-Ag/rGO TMB 0.6340 4.2553Cu-Ag/rGO H2O2 8.6245 7.0175Ag/rGO TMB 0.8503 3.828Ag/rGO H2O2 20.928 6.2305Cu/rGO TMB 1.05 3.289Cu/rGO H2O2 26.332 5.385

Fig. 7. Steady-state kinetic assay and catalytic mechanism of Cu-Ag/rGO nanocom-posites. (A) Variation of TMB concentration at constant H2O2 concentration (50 mM).(B) Variation of H2O2 concentration at constant TMB concentration (0.5 mM). The

corresponding Lineweaver–Burk plots of the double reciprocal of Michaelis Mentenequation are shown in the inset.

The lowest Km value (0.6340) for TMB exhibited by Cu-Ag/rGOindicates its better substrate affinity in comparison to the corre-sponding monometallic analogues, 0.8503 for Ag/rGO and 1.05 forCu/rGO towards TMB. A comparative report of other enzyme mim-ics for both TMB and H2O2 are listed in Table S2. It can be seenthat, compared to other enzyme mimics, the Km value obtained forCu-Ag/rGO with TMB is much lower than for many other previouslyreported enzyme mimics (Table S2). Also, the Km value obtained forCu-Ag/rGO with H2O2 is very low, indicating that a lower H2O2 con-centration is required for TMB oxidation than many other enzymemimics.

The improved catalytic activity of Cu-Ag/rGO nanocompositeis most likely due to strong interaction of both metals and thesupport, rGO. The presence of highly delocalized � electrons onthe graphene’s basal plane improves electron transfer between therGO sheets and the Cu-Ag centres [28]. The rGO also provides alarge number of nucleation sites for growth of Cu-Ag and facilitateselectron transfer during the reaction process. Unlike rGO alone, Cu-Ag/rGO provides larger sites for TMB adsorption. Consequently, theamino group of TMB transfers its lone pair of electron to the Cu-Ag/rGO, resulting in increased electron density and mobility in thenanocomposite thus facilitating electron transfer from hybrids to

H2O2. As a result reduction of H2O2 to H2O occurs and TMB oxida-tion rate by H2O2 increases [29]. Also, the interaction between themetal centres and rGO increases the electron transfer from Cu-Ag

848 G. Darabdhara et al. / Sensors and Actuators B 238 (2017) 842–851

Fig. 8. Standard glucose concentration response curve under optimum conditionsand corresponding linear detection range (inset) using Cu-Ag/rGO nanocomposite.

Table 2Comparative table for the detection of glucose.

Catalyst Linear Range (mM) Limit of detection (�M) Reference

Ch-Ag NPs 0.005–0.002 0.099 [17]Ag NPs graphene 2–10 100 [28]Au NPs 0.02–5.7 8.2 [29]Au@Ag 0.05–20 25 [30]

ttC

3s

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das

ft

Fig. 9. UV–vis absorbance spectra of glucose and its analogues at 652 nm (A) andcorresponding bar diagrams (B) along with the corresponding photographs (Fig. 9Binset).

Cu NPs 0.001–0.1 0.68 [34]Cu-Ag/rGO 0.001–0.03 3.82 Present work

o rGO support thus reducing the activation energy of the reac-ion. These factors confer superior peroxidase-like activity to theu-Ag/rGO nanocomposite.

.4. Detection of glucose using Cu-Ag/rGO nanocomposite,electivity study and analysis of glucose in real blood samples

The colorimetric detection of glucose can be realized as H2O2 ishe main product of GluOx catalysed oxidation of glucose. At pH, GluOx can be denatured and hence detection of glucose wasealized in two steps. The first step involves catalytic oxidationf glucose to gluconic acid in pH 7 buffer solution by GluOx andimultaneous conversion of substrate oxygen to H2O2. The sec-nd step involves reduction of H2O2 by Cu-Ag/rGO in presence ofMB and formation of TMBDI, which is analytically determined asbove [30]. Fig. 8 exhibits a calibration curve for standard glucoseetection. Linearity between 1–30 �M glucose with a detection

imit of 3.82 �M are obtained. The efficiency of glucose detectionsing Cu-Ag/rGO catalyst was compared to that of monometal-

ic Ag/rGO and Cu/rGO. For Ag/rGO, a detection limit of 7.9 �Mith a linear range from 10–30 �M was recorded (Fig. S10A), while

detection limit of 9.7 �M with a linear range from 20–100 �Mas obtained for Cu/rGO (Fig. S10B). A comparative table illustrat-

ng the efficiency of our synthesized materials in comparison tother reported enzyme mimics is described in Table 2. The limitf detection of Cu-Ag/rGO towards glucose is better in comparisono reported AgNPs/graphene (100 �M) [31], AuNPs (8.2 �M) [32],u@Ag (25 �M) [33], etc.

The selectivity of the Cu-Ag/rGO catalyst for glucose was furtheremonstrated against glucose analogues such as maltose, fructosend lactose (Fig. 9A). From the bar diagram in Fig. 9B, the better

electivity of Cu-Ag/rGO towards glucose is confirmed.

We furthermore investigated the ability of Cu-Ag/rGO catalystor the detection of glucose in clinical blood samples. Fig. 10 depictshe UV–vis response of Cu-Ag/rGO to two serum samples. The

Fig. 10. UV–vis absorbance spectra obtained for blood serum samples. Inset showsthe colour change.

obtained result was compared with a standard hospital method

as shown in Table S3. It is found that our colorimetric detectionmethod using Cu-Ag/rGO catalyst can be successfully utilized foranalysing real samples with comparable results and good accuracy.

G. Darabdhara et al. / Sensors and Actuators B 238 (2017) 842–851 849

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synthesized Cu-Ag/rGO nanocomposite displayed good intrinsic

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ig. 11. Standard linear calibration plot for detection of ascorbic acid using Cu-g/rGO nanocomposite.

.5. Detection of ascorbic using Cu-Ag/rGO nanocomposite

We finally investigated the possibility for the detection of ascor-ic acid using Cu-Ag/rGO nanocomposite. When ascorbic acid isdded to a reaction mixture of H2O2 and TMB, the absorbance at52 nm decreases significantly and the colour changes from blueo colourless, indicating that addition of ascorbic acid prevents thexidation of TMB due to consumption of H2O2. Based on this obser-ation, we propose another colorimetric method for the detectionf ascorbic acid or vitamin C, which plays a significant role in manyiochemical processes. The standard ascorbic acid response curvender optimum conditions using Cu-Ag/rGO is displayed in Fig. 11.he linear range for ascorbic acid using Cu-Ag/rGO is 5–30 �Mith a limit of detection of 3.6 �M. The efficiency of ascorbic acid

etection using Cu-Ag/rGO was established by comparing it withonometallic Ag/rGO and Cu/rGO. For Ag/rGO, the linear ascorbic

cid detection range was 5–20 �M with a detection limit of 5.84 �MFig. S11A), while for Cu/rGO the linear range was 5–30 �M with aetection limit of 4.56 �M (Fig. S11B). Table 3 illustrates previouslyeported materials for ascorbic acid detection. As compared to otheranostructures, Cu-Ag/rGO nanocomposite exhibited a comparableetection limit and in addition provided an easy, simple and costffective method for ascorbic acid detection. This study also demon-trated better efficiency of Cu-Ag/rGO bimetallic nanocomposite inomparison to its monometallic analogues.

Finally, the selectivity of Cu-Ag/rGO for ascorbic acid sensingas investigated in the presence of uric acid (UA) and dopamine

DA), as ascorbic acid usually co-exists with UA and DA in humanlood. In the selectivity study, the response of the TMB-H2O2 sys-em using Cu-Ag/rGO catalyst was investigated against UA and DA2 mM) solutions. It was observed that only addition of ascorbiccid causes significant decrease in the absorbance of the peak at

52 nm and no such remarkable changes were seen upon additionf other interfering substances, UA and DA as shown in Fig. 12.he result clearly suggests the good selectivity of Cu-Ag/rGO for

able 3omparative table for the detection of ascorbic acid.

Catalyst/Method Linear Range (mM)

Cu NPs@C/Colorimetric 0.01–1

Au NPs-ssDNA/Colorimetric 0.001–0.015

Carbon nanotube/Electrochemistry 0.08–1.36

MIL-68/MIL-100/Colorimetric 0.03–0.485

MIL-53 (Fe)/Colorimetric 0.03–0.19

Cu-Ag/rGO 0.005–0.03

Fig. 12. UV–is absorbance spectra of ascorbic acid (AA) and its analogues dopamine(DA) and uric acid (UA) at 652 nm (A) and the corresponding bar diagrams (B) alongwith photographs (inset).

ascorbic acid detection. A similar selectivity investigation was alsocarried out by Wang et al. in which selectivity of ascorbic acid wasstudied against amino acids, carbohydrates and substances used asexcipients to accelerate drug absorption [16].

4. Conclusion

In conclusion, the synthesis of Cu-Ag bimetallic NPs dispersedon rGO nanosheets was successfully achieved using a solvother-mal reduction method. The obtained Cu-Ag NPs exhibited anaverage size of 36.4 ± 1.2 nm. Using a similar chemical route,monometallic Ag/rGO and Cu/rGO were also prepared. The as-

peroxidase-like activity and was successfully applied for the oxi-dation of peroxidase substrate TMB into blue coloured oxidizedTMB. The Cu-Ag/rGO nanocomposite was found to follow Michaelis

Limit of detection (�M) Reference

1.4 [16]0.3 [35]20 [36]6 [37]15 [38]3.6 Present work

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enten kinetics with good affinity towards TMB in comparison toonometallic Ag/rGO and Cu/rGO nanocomposites. Based on the

olour change capability, a simple, low-cost and easy colorimetricetection technique was developed towards detection of glucosend ascorbic acid. The results exhibited excellent linear detectionange and limit of detection for both glucose and ascorbic acid.esides, the efficiency of Cu-Ag/rGO nanocomposite in comparisono its monometallic counterparts was well established in this studyhich can be ascribed to the synergistic effect of the two metals in

imetallic NPs. This present study is anticipated to open up inno-ative directions towards the development of bimetallic-grapheneased nanocomposite for many other efficient biosensing and forotential applications in the area of biomedicine and biotechnol-gy.

cknowledgements

The authors are thankful to the Director, CSIR−NEIST, Jorhator his interest to carry out the work. The authors acknowledgedhe Department of Science and Technology, New Delhi for finan-ial support (DST No. INT/RUS/RFBR/P-193 and CSIR-NEIST Projecto. GPP-0301) GD gratefully acknowledges DST, New Delhi, India

or DST-INSPIRE Fellowship grant. The authors further acknowl-dge SAIF, NEHU, Shillong for the HRTEM facility and clinical centreSIR-NEIST, Jorhat for providing the blood serum sample.

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.2016.07.106.

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Biographies

Gitashree Darabdhara GD received her M.Sc degree in Chemistry in 2013 fromGauhati University, Guwahati, Assam, India. She is currently a research student inthe Advanced Materials Group, Material Sciences and Technology Division of CSIR-North East Institute of Science and Technology, Jorhat, India. Her research interestis in the field of synthesis of different bimetallic nanoparticles on graphene oxide

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Her current research interests are in the area of material science with emphasison the development of novel analytical platforms and interfaces for the study ofaffinity binding events and in the modification of nanostructures (diamond particles,magnetic particles, nanographene) for biomedical applications. She is co-author ofmore than 230 research publications, wrote several book chapters and has 6 patents.

G. Darabdhara et al. / Sensors a

anosheets and their application in various fields like photocatalysis, biosensing,ydrogen evolution reactions, organic catalytic transformations etc.

hagyasmeeta Sharma BS received her M.Sc degree in Chemistry in 2014 fromauhati University, Guwahati, Assam, India. Presently she is engaged as a Projectellow in the Advanced Materials Group, Material Sciences and Technology Divisionf CSIR-North East Institute of Science and Technology, Jorhat, India since 2015. She

s interested in photocatalytic degradation of harmful environmental contaminantssing different metal oxide based nanocomposites.

anash R. Das is, since 2008, a Research Scientist at Advanced Materials Group,aterial Sciences and Technology Division, CSIR-North East Institute of Science and

echnology (CSIR-NEIST), Jorhat, India. He is also an Assistant Professor in Academyouncil of Scientific and Innovative Research (AcSIR), India. He has carried out hishD research work at CSIR–NEIST, Jorhat and received his PhD degree from Dibru-arh University, Assam, India in 2007 in the field of Surface Chemistry. Between007 and 2008 he worked as a postdoctoral fellow with Dr. Rabah Boukherroub at

he Interdisciplinary Research Institute (IRI), France. He has published more than5 peer reviewed research articles and seven book chapters. His research interests

nclude functionalization of graphene, decoration of metal nanoparticles, bimetal-ic nanoparticles and metal oxide on the graphene sheets and their applications inrganic catalysis reaction, photocatalysis and water pollutant removal.

tuators B 238 (2017) 842–851 851

Rabah Boukherroub is a director of research at the CNRS and a group leader atthe Institute of Electronics, Microelectronics and Nanotechnology, University Lille1, France. He is Associate Editor for ACS Applied Materials & Interfaces. His researchinterests are in the area of synthesis of functional nanomaterials, surface chemistry,and photophysics of semiconductor/metal nanostructures with emphasis on biosen-sors, drug delivery, and development of new tools for studying molecular dynamicsin vivo. He is a co-author of more than 370 research publications and wrote 27 bookchapters in subjects related to nanotechnology, materials chemistry, and biosensors.He has 9 patents or patents pending.

Sabine Szunerits is since 2009 Professor in Chemistry at the University Lille 1, Franceand was nominated 2011 as member of the “Institut Universitaire de France” (IUF).