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Process Biochemistry 47 (2012) 992–998

Contents lists available at SciVerse ScienceDirect

Process Biochemistry

jo u rn al hom epa ge: www .e lsev ier .com/ locate /procbio

n amperometric H2O2 biosensor based on cytochrome c immobilized ontoickel oxide nanoparticles/carboxylated multiwalled carbonanotubes/polyaniline modified gold electrode

uman Lata, Bhawna Batra, Neelam Karwasra, Chandra S. Pundir ∗

epartment of Biochemistry, M.D. University, Rohtak 124001, Haryana, India

r t i c l e i n f o

rticle history:eceived 22 December 2011eceived in revised form 15 March 2012ccepted 27 March 2012vailable online 2 April 2012

eywords:

a b s t r a c t

Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalledcarbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) elec-trode. An amperometric H2O2 biosensor was constructed by connecting this modified Au electrode alongAg/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode wascharacterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning elec-tron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV)

2O2 biosensorytochrome cickel oxide nanoparticlesolyanilineultiwalled carbon nanotubes

studies of the electrode at different stages demonstrated that the modified Au electrode had enhancedelectrochemical oxidation of H2O2, which offered a number of attractive features to develop an ampero-metric biosensor based on split of H2O2. There was a good linear relationship between the current (mA)and H2O2 concentration in the range 3–700 �M. The sensor had a detection limit of 0.2 �M (S/N = 3) with ahigh sensitivity of 3.3 mA �M−1 cm−2. The sensor gave accurate and satisfactory results, when employed

2 in d

ruit juices for determination of H2O

. Introduction

The rapid and accurate determination of hydrogen peroxideH2O2) is very important, as it is not only the product of theeactions catalyzed by many highly selective oxidases, but alsomployed in various fields such as food, pharmaceutical and envi-onmental analysis [1–3]. H2O2, a powerful oxidizing agent, issually utilized as an antimicrobial agent in food and sterilizinggent on the foil lining of aseptic packages containing fruit juicesnd milk products. The higher concentrations of H2O2 are associ-ted with the diabetes, atherosclerosis and aging as it generatesree hydroxyl radicals, which cause oxidative damage of the tis-ue components such as lipids and proteins beside DNA [4,5]. Aumber of analytical methods are available for determination of2O2 such as titrimetry [2], colorimetry/spectrometry [3], chemi-

uminescence [6], high performance liquid chromatography (HPLC)nd idiometry [7]. Electrochemical biosensing method is one ofhe promising approaches, because of its simplicity, rapidity andigh sensitivity. In recent years, direct electrochemistry of met-

lloproteins and metalloenzymes has attracted the attention ofany scientists’, because of its potential application in the study

f redox and electron transfer properties of biomolecules and in

∗ Corresponding author. Tel.: +91 9416492413; fax: +91 126274640.E-mail address: pundircs@rediffmail.com (C.S. Pundir).

359-5113/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.procbio.2012.03.018

ifferent fruit juices.© 2012 Elsevier Ltd. All rights reserved.

fabricating mediator-free or the third generation biosensors [8].The proteins containing heme groups, such as hemoglobin, myo-globin and cytochrome c (Cyt c), possess peroxidase like catalyticactivity for reduction of H2O2, due to the electroactive center ofheme [9] and therefore employed for construction of H2O2 sensors[10,11].

Cyt c is a very basic redox heme protein, whose main func-tion is to deliver electrons from Cyt c reductase to Cyt c oxidase.However, electron transfer between heme and bare electrode isusually slow and the protein is irreversibly denatured. Thus, it isnecessary to search the way to develop a heme-modified elec-trode with well-behaved electrochemistry and good stability. Thenanoparticles are known to adsorb redox enzymes and proteins,without loss of their biological activity. In addition, the electrontransfer ability (direct electrochemistry) and biocatalytic activityof enzymes were increased, when they were adsorbed on nanoma-terials. In recent years, nickle oxide nanoparticles (NiO-NPs) hasattracted extensive interests due to its high surface to volume ratio,novel optical, electronic, magnetic, thermal, mechanical properties,quantum confinement effect and potential application as catalyst,in battery electrodes, gas sensors, electrochemic films and photo-electronic devices [12]. Carbon nanotubes (CNT) due to their unique

physical properties, including high electrical conductivity, supe-rior chemical and mechanical stability and large surface area, haveestablished themselves as a good material for immobilization ofenzymes, multiwalled carbon nanotubes (MWCNT) enhance the

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irect electron transfer between the enzyme’s active sites and thelectrode.

Among various conducting polymers, polyaniline (PANI) isnique, due to its relatively facile synthesis, conductivity and envi-onmental stability. PANI/CNT composites have been prepared bylectropolymerization of aniline or by in situ chemical polymeriza-ion [13].

We report herein the covalent immobilization of cytochrome cnto nickel oxide nanoparticles/carboxylated multiwalled carbonanotubes/polyaniline composite electrodeposited on Au electrode

or an amperometric determination of hydrogen peroxide.

. Materials and method

.1. Reagents and materials

Carboxylated multi-walled carbon nanotubes (cMWCNT) was obtained fromntelligent Materials Pvt. Ltd., Panchkula (Haryana) India. Nickle chloride was from

erck, Germany. Aniline and Cyt c were from SISCO Research Lab., Mumbai, India.ll other chemicals were of analytical reagent (AR) grade. Au electrode (2 cm × 1 cm)as purchased from local market.

.2. Apparatus

Potentiostat/galvanostat (Make: Autolab, model: AUT83785, manufactured byco Chemie) with a three electrode system composed of a Pt wire as an auxillary elec-rode, an Ag/AgCl electrode as reference electrode and Cyt c/NiONPs/MWCNT/PANI

odified Au electrode as a working electrode. Transmission electron microscopyTEM) and scanning electron microscopy (SEM) (Zeiss EV040). UV spectroscopyMake: Shimadzu, Model 160 A), transmission electron micrography (TEM) (JEOL100 F) X-ray diffractometer (XRD), (Make: 122 Rigaku, D/Max2550, Tokyo, Japan)ourier transform infra-red spectroscopy (FTIR) (Thermo Scientific, USA).

.3. Construction of Cyt c/NiO-NPs/cMWCNT/PANI modified Au electrode

.3.1. Preparation of NiO nanoparticlesNiO nanoparticles were synthesized by two step method [14]. Firstly 2.3 g NiCl2

n 10 ml double distilled water (DDW) and 1.5 g NaHCO3 in 10 ml DDW were dis-olved separately in glass beakers. The NiCl2 solution was stirred on a magnetictirrer for 15 min and then NaHCO3 solution was added to NiCl2 solution drop wisender constant stirring. After 15 min, the products were collected by centrifuga-ion and washed thoroughly with DDW and dried in air. The structural property ofiO-NPs was studied.

.3.2. Electrodeposition of NiO-NPs/cMWCNT/PANI onto Au electrodeThe surface of a Au electrode (2 cm × 1 cm) was polished manually by alumina

lurry (diameter 0.05 �m) with a polishing cloth, followed by thorough wash-ng with DDW, placed into ethanol, sonicated to remove adsorbed particles and

ashed finally with DDW. cMWCNT (1.0 mg) were suspended into 4 ml mixturef concentrated H2SO4 and HNO3 in 3:1 ratio and ultrasonicated for 6–8 h to getts finely dispersed black colored solution. Aniline (200 �l) was added to 25 ml ofN HCl and electropolymerized onto Au electrode through cyclic voltammetry bypplying ten successive polymerization cycles at −0.2 to 0.8 V. NiO-NPs suspen-ion (200 �l) and 1 ml of cMWCNT suspension (1:5 ratio) were added into 25 ml 1NCl to get the NiO-NPs/cMWCNT mixture. Finally, electrodeposition of NiO-NPs andMWCNT onto the Au electrode was carried out in an electrochemical cell systemy applying five polymerization cycles at −0.2 to 0.8 V. During the electrochemi-al polymerization, the surface of Au wire became green gradually, indicating theeposition of NiO-NPs/cMWCNT/PANI film on Au wire (Fig. 1A). The resulting NiO-Ps/cMWCNT/PANI modified Au electrode was washed thoroughly with distilledater to remove unbound matter and kept in a dry Petri-plate at 4 ◦C.

.3.3. Immobilization of cytochrome c onto NiO-NPs/cMWCNT/PANI modified Aulectrode

To prepare Cyt c solution, 100 mg of Cyt c (oxidized) was dissolved in 8 mlf 10 mM potassium phosphate buffer (pH 7.0). Cyt c (oxidized) was reduced bydding 3–5 mg of sodium ascorbate. The excess of ascorbate was removed by dia-yzing Cyt c solution against 10 mM of potassium phosphate buffer (pH 7.0) for 24 ht 4 ◦C with three changes of buffer. After dialysis, the volume of Cyt c solutionas brought to 10 ml with the same buffer. This Cyt c remains reduced for severalonths, if stored at 4 ◦C. Cyt c (reduced) solution (2 ml) was placed onto surface ofiO-NPs/cMWCNT/PANI modified Au electrode and kept overnight at 4 ◦C for immo-ilization. The resulting electrode (with immobilized Cyt c) was washed 3–4 times

ith a 50 mM potassium phosphate buffer (pH 7.0) to remove residual/unbound Cyt

. Protein concentration was calculated in wash out solution to calculate conjugationield. The resulting Cyt c/NiO-NPs/cMWCNT/PANI Au electrode was used as work-ng electrode and stored at 4 ◦C, until use. This working electrode was characterizedy SEM and FTIR at different stages of construction.

stry 47 (2012) 992–998 993

2.3.4. Cyclic voltammetric measurement and testing of H2O2 biosensorCyclic voltammogram (CV) of Cyt c/NiO-NPs/cMWCNT/PANI Au electrode was

recorded in potentiostat–galvanostat from −0.1 to 0.9 V vs. Ag/AgCl as referenceand Pt wire as counter electrode in a 25 ml of 10 mM potassium phosphate buffer(pH 7.0) containing 100 �l (100 �M) of H2O2. The maximum response (current inmA) was observed at 0.28 V (Fig. 1B) and hence subsequent studies were carried outat this voltage. The following electrochemical reactions occurred during responsemeasurement:

Chemical reaction : 2cyt c-Fe(II) + 2H+ + H2O2 → 2 cyt c-Fe(III)| + 2H2O

Electrochemical reduction : cyt c-Fe(III) + e− → cyt c-Fe(II)

2.4. Optimization of H2O2 biosensor

To optimize working conditions of the biosensor, effects of pH, incubationtemperature, time and substrate (H2O2) concentration were studied on biosensorresponse. To determine optimum pH, the pH was varied between pH 4.5 and 9.0at an interval of pH 0.5 using the following buffer, each at a final concentration of0.01 M: pH 4.5–7.5 potassium phosphate buffers and pH 8.0–9.0 Tris–HCl buffers.Similarly to determine optimum temperature the reaction mixture was incubatedat different temperature (20–50 ◦C) and time (1–20 s). The effect of H2O2 concentra-tion on biosensor response was determined by varying the concentration of H2O2

in the range 3–700 �M.

2.5. Application of H2O2 biosensor in fruit juices

The sensor was employed for quantitative determination of hydrogen perox-ide in commercially available juice of some fruits (green grapes, banana, papaya,mausmi, green apple) and sugarcane stem. Each juice was centrifuged at 8000 × gfor 5 min and supernatant was used for an amperometric analysis. The fruit juicewas analyzed for H2O2 as described for testing of biosensor under optimal condi-tions except that H2O2 solution was replaced by fruit juice. The H2O2 content in fruitjuices was calculated from standard curve between H2O2 concentration vs. currentin mA prepared under optimal assay conditions of Cyt c/NiO-NPs/cMWCNT/PANI Auelectrode (Fig. 1C).

2.6. Evaluation

The performance of this sensor was evaluated by studying its analytical recovery,detection limit, precision and correlation.

2.7. Interference study

It was carried out by measuring the sensor response before and after addition ofsome interferant (dopamine, ascorbic acid, l-cysteine, and glucose) into the 0.1 Mphosphate buffer containing 100 �M H2O2 at their physiological concentration. Thepercentage of current ratio before and after addition of interferrant was calculated.

2.8. Reusability and storage stability of Cyt c/NiO-NPs/cMWCNT/PANI/Auelectrode

The reusability and stability of the working electrode was studied by measuringits activity for six months at an interval of one week. The present electrode systemwas stored in dried condition at 4 ◦C when not in use.

3. Results and discussion

3.1. SEM studies of Au electrode during modification

The SEM images of the surfaces of the bare Au elec-trode, NiO-NPs/cMWCNTs/PANI/Au electrode and Cyt c/NiO-NPs/cMWCNT/PANI/Au electrode are shown in Fig. 2. The stepwisemodification of electrode could be seen clearly from these SEMimages. The SEM image of the bare Au electrode showed a smoothand featureless morphology Fig. 2(a). The NiO-NPs/cMWCNTs/PANIcomposite film showed net and porous structure, which provides

larger surface area Fig. 2(b). On immobilization of Cyt c, the globularstructures of Cyt c on the surface of NiO-NPs/cMWCNTs/PANI/Auwas seen due to the covalent interaction between Cyt c and modi-fied Au electrode Fig. 2(c).

994 S. Lata et al. / Process Biochemistry 47 (2012) 992–998

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ig. 1. (A) Cyclic voltammogram for electrochemical deposition of NiO-NPs/cMWCcan rate: 20 mV/s. (B) Cyclic voltammogram for 0.1 mM H2O2 solution in 0.01 M potn response of H2O2 biosensor based on Cyt c/NiO-NPs/cMWCNT/PANI Au electrod

.2. Characterization of NiO-NPs

The characterization of nanoparticles was carried out by UV andisible spectrophotometric TEM, XRD and FTIR spectra (Fig. 3A–D).

V spectrophotometric study revealed that strong absorbanceeak was positioned at around 400 nm (Fig. 3A). The typical TEM

mages of NiO nanoparticles showed the spherical shape of NiOanoparticles with a average diameter of 20 nm (Fig. 3B). The XRD

Fig. 2. SEM images of (a) bare Au electrode, (b) NiO-NPs/cMWCNT/PAN

NI composite film on Au wire (10 scans). Supporting electrolyte: 1N HCl solution; phosphate buffer (pH 7.0) with scan rate 20 mV/s. (C) Effect of H2O2 concentration

patterns of the prepared samples clearly showed the characteris-tics peaks of NiO appeared at 16.04◦, 27.66◦, 31.96◦, 33.28◦, 48.34◦,56.76◦, 75.56◦ (Fig. 3C). No characteristic peaks of impurities wereobserved, suggesting that high purity of the NiO-NPs. All peaks

were consistent with the peaks of NiO-NPs with high crystallinity.The FTIR spectrum of NiO-NPs is shown in Fig. 3(D). The significantpeaks at 1632.07 and 1350.69 cm−1 are assigned to the NiO stretch-ing vibration mode, which confirms the formation of NiO-NPs.

I Au electrode and (c) Cyt c/NiO-NPs/cMWCNT/PANI Au electrode.

S. Lata et al. / Process Biochemistry 47 (2012) 992–998 995

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ig. 3. (A) UV spectrum of NiO-NPs, (B) transmission electron microscopic (TEM) impectrum of NiO-NPs.

.3. Construction of an amperometric and non-enzymatic H2O2iosensor

A method is summarized in Fig. 4 for construction of a nonnzymic H2O2 biosensor based on immobilization of Cyt c oniO-NPs decorated cMWCNT and PANI composite film electrode-osited onto Au electrode. Firstly, aniline was electropolymerizedn the surface of Au electrode, as this method is easy and theayer thickness could be controlled. After that cMWCNT andiO-NPs were co-electrodeposited on the PANI coated Au elec-

rode. To construct a non enzymic H2O2 electrode, Cyt c wasmmobilized onto NiO-NPs/cMWCNT/PANI/Au electrode throughovalent coupling. Incubation of Cyt c solution with activatediO-NPs/cMWCNT/PANI/Au electrode leads to effective colli-

ion between COOH group of cMWCNT and NH2 groups onhe surface of the Cyt c to form an amide bond ( CO NH).he cyclic voltammograms of NiO-NPs/cMWCNT/PANI exhibitedigher currents than cMWCNT/PANI (Fig. 5) which indicate thatiO-NPs/cMWCNT/PANI composite film could provide a conduct-

ng path through the composite matrix for faster kinetics. Hence,he NiO-NPs, acting as electron transfer mediator help in enhancinghe sensor response and thus increase its sensitivity. These obser-ations also suggest that the NiO-NPs/cMWCNT/PANI compositelm provided a large surface area for immobilization of the Cyt c.

.4. FTIR spectra

Fig. 6 shows FTIR spectra obtained for Cyt c (curve A) PANI/Aulectrode (curve B), NiO-NPs/cMWCNT/PANI/Au electrode (curve) and Cyt c/NiO-NPs/cMWCNT/PANI/Au electrode (curve D). FTIRpectra of Cyt c alone showed the peaks of amide I and amide II at

650 cm−1 and 1500 cm−1 respectively (curve A). FTIR spectra oflectrodeposited PANI/Au composite showed bands at 1490 and601 cm−1 (curve B), which are caused by benzoid and quinoidibrations. The presence of peaks at 1135.53 and 3448 cm−1

of NiO-NPs and (C) X-ray diffraction (XRD) pattern of taken from NiO-NPs (D) FTIR

attributed to �-N+ Q and N H stretching vibrations of PANI.Fig. 5(b) shows the FTIR spectra of NiO-NPs/cMWCNT/PANI/Auelectrode, which showed several significant peaks. The significantpeaks in the region of 1632.07 cm−1 and 1350.69 cm−1 are assignedto Ni O stretching vibration mode. In FTIR spectrum of Cyt c/NiO-NPs/cMWCNT/PANI/Au bioelectrode, covalent binding of Cyt c isindicated by appearance of additional bands at 1631.70 cm−1 and1494.37 cm−1 assigned to the carbonyl stretch (amide I band) and

N H (amide II band) respectively (curve D). A comparison ofamide I and amide II peaks recorded for Cyt c alone and Cyt cimmobilized onto NiO-NPs decorated cMWCNT/PANI/Au electrode(Fig. 6D) shows that the latter peaks were broader than the formerpeaks. The broadening of amide I and II peaks after immobilizationof Cyt c on to modified cMWCNT shows the amide bond forma-tion between surface COOH groups of cMWCNT and surface NH2groups of Cyt c. Earlier we have also reported the covalent immobi-lization of laccase on AgNP decorated cMWCNT/PANI/Au electrode[15].

3.5. Responses measurements of non-enzymatic H2O2 biosensor

The sensor responses to H2O2 may be due to the partial (weak)oxidation of H2O2 molecules by carbon nanotubes. The calibrationcurve of the sensor response vs. the concentration of H2O2 showedthat the sensor’s response was proportional to the concentration ofH2O2. This relationship revealed that there was a charge transferinteraction between the carbon nanotubes and the H2O2 molecules.An amperometric response of an enzyme electrode was recordedtoward oxidation of H2O2 and the current response for the elec-trochemical oxidation of H2O2 was recorded for 100 �M of H2O2.There was a good linear relationship between current and H2O2

concentration in the range 3–700 �M. The detection limit calcu-lated as concentration that gave a signal equal to three times thestandard deviation of the blank signal, was 0.2 �M. For the purposeof comparison, electro oxidation of H2O2 was also performed at the

996 S. Lata et al. / Process Biochemistry 47 (2012) 992–998

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Fig. 6. FTIR spectra of Cyt c (A) PANI/Au (B) NiO-NPs/cMWCNT/PANI/Au (C) Cytc/NiO-NPs/cMWCNT/PANI Au electrode (D).

Fig. 4. Schematic representation of chemical reaction involved

are Au electrode. An oxidation wave was noticed around 0.28 V atodified electrode in comparison to the negligible at the bare Au

lectrode. Thus, it was clear that the modified electrodes have pro-ounced higher current response for the oxidation of 100 �M of2O2 than that at the bare Au electrode.

.6. Electrochemical impedance measurements

Fig. 7 shows electrochemical impedance spectra (EIS) of (i) Aulectrode (ii) NiO-NPs/cMWCNT/PANI/Au electrode and (iii) Cyt/NiO-NPs/cMWCNT/PANI/Au electrode, respectively. The chargeransfer process in Cyt c/NiO-NPs/cMWCNT/PANI/Au electrode wastudied by monitoring charge transfer resistance (RCT) at the elec-rode and electrolyte interface. The value of the electron transferesistance (semicircle diameter) (RCT) depends on the dielec-ric and insulating features at the electrode/electrolyte interface.he RCT values for bare Au electrode, NiO-NPs/cMWCNT/PANI Aulectrode, and Cyt c/NiO-NPs/cMWCNT/PANI Au electrodes were40 �, 110 � and 70 �, respectively. This decreased RCT value inresence of Cyt c revealed that the electron transfer via redox cou-le was enhanced by the presence of Cyt c on the electrode surface.

.7. Optimization of experimental conditions

The experimental conditions affecting the biosensor responseere studied in terms of effect of pH, incubation temperature,

ig. 5. Cyclic voltammogram for (a) cMWCNT/PANI/Au and (b) NiO-Ps/cMWCNT/PANI/Au.

Fig. 7. Impedance spectra of (i) bare Au electrode (ii) NiO-NPs/cMWCNTs/PANI/Auelectrode (iii) Cyt c/NiO-NPs/cMWCNT/PANI/Au electrode.

chemistry 47 (2012) 992–998 997

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Table 1Hydrogen peroxide level in different fruit samples by H2O2 biosensor based on Cytc/NiO-NPs/cMWCNT/PANI/Au electrode.

Type of juice sample H2O2 (�M) mean ± SEa

Grape juice 367 ± 0.04Sugarcane 408 ± 0.02Banana 150 ± 0.03Papaya 204 ± 0.02

S. Lata et al. / Process Bio

ime and substrate (H2O2) concentration. The optimum currentas obtained at pH 6.5, which is similar to that of sensor

ased on Meldola Blue, fumed-silica and horseradish peroxidasedsorbed into carbon paste (pH 6.5) [16] but comparable to thosenvolving self-deposited redox polyelectrolyte-oxidoreductasesrchitectures (pH 7.0) [17], direct electrochemistry of myoglobinmmobilized in poly-3-hydroxybutyrate film (pH 5.0) [18], Cyt

immobilized on a multi-walled carbon nanotubes modifiedlectrode (pH 7.0) [19], layer-by-layer assembly of horseradish per-xidase (HRP) and nile blue premixed with polyanion (pH 6.8) [20],rotein immobilized on zirconia nanoparticles enhanced graftedollagen matrix (pH 7.0) [21], and hemoglobin on multiwall carbonanotubes and gold colloidal nanoparticles (pH 6.0) [22] pyrrolelectropolymerized on screen-printed carbon paste electrodes (pH.0) [23], HRP entrapment in a polyacrylamide gel matrix (pH 6.0)24], hemoglobin immobilized on glassy carbon electrode modi-ed with Fe3O4/chitosan core–shell microspheres (pH 8.0) [25].he optimum temperature of sensor was 30 ◦C, which is com-arable to that reported for biosensor based on a multi-walledarbon nanotubes modified electrode [18–20], protein immobi-ized on zirconia nanoparticles enhanced grafted collagen matrix24] and hemoglobin immobilized on glassy carbon electrode mod-fied with Fe3O4/chitosan core–shell microsphere [25]. There was

hyperbolic relationship between sensor response and H2O2 con-entration in the range 3–900 �M, the response was constant after00 �M and the Km was 320 �M which is lower than 857 �M [19]nd 4900 �M [20].

.8. Evaluation of H2O2 biosensor

There was a linear relationship between H2O2 concentra-ion ranging from 3 to 700 �M and current (mA), which isomparable to the biosensors based on direct electrochemistryf myoglobin immobilized on to poly-3-hydroxybutyrate film1.0 × 10−7–4 × 10−4 M) [17], layer-by-layer assembly of HRP andile blue premixed with polyanion (0.20–7.03 mM) [20], protein

mmobilized on zirconia nanoparticles enhanced grafted col-agen matrix (1.0–85.0 �M) [21] and hemoglobin immobilizedn multiwall carbon nanotubes and gold colloidal nanoparti-les (2.1 × 10−7–3.0 × 10−3M) [22], screen-printed carbon pastelectrodes (0.1–2.0 mM) [23], hemoglobin immobilized on glassyarbon electrode modified with Fe3O4/chitosan core–shell micro-pheres (5.0 × 10−5–1.8 × 10−3 M and 1.8 × 10−3–6.8 × 10−3 M)25]. The detection limit of the biosensor was 0.20 �M, which isower than non-enzymatic hydrogen peroxide biosensor based onirect electrochemistry of cytochrome c on a multi-walled car-on nanotubes modified electrode (1.02 �M) [19] layer-by-layerssembly of HRP and nile blue premixed with polyanion (8.45 mM)20], protein immobilized on zirconia nanoparticles enhancedrafted collagen matrix (0.63 �M) [21], direct electrochemistryf hemoglobin immobilized on carbonized titania nanotubes0.92 �M) [26] hemoglobin immobilized on glassy carbon elec-rode modified with Fe3O4/chitosan core–shell microspheres4.0 × 10−6 M) [25], direct electrochemistry and electrocatalysis ofemoglobin in composite film based on ionic liquid and NiO micro-pheres with different morphologies (0.68 �M) [27].

In order to demonstrate the applicability of the present sensorn real sample analysis, 20 and 50 �M H2O2 solution were spikednto a fruit juice sample. The analytical recovery as measured byhe present sensor was 96.00% (n = 3) and 94.60% (n = 3), respec-ively. In order to check the repeatability and reproducibility of the

ethod, the H2O2 content in same juice sample was determined

ve times on a single day (within batch) and again after storaget −20 ◦C for one week (between batch). The results showed thateterminations were almost consistent and within and betweenatch coefficients of variation (CV) for juice sample determination

Mausami 250 ± 0.04Apple juice 350 ± 0.03

a SE = standard error.

were <5.1% and <5.34%, showing the good reproducibility and reli-ability of the method. To know the accuracy of present method,values of H2O2 in juice sample were determined by enzymic colori-metric method (x) and by the present method (y). The H2O2 valuesobtained by the present biosensor matched with those by stan-dard enzymic colorimetric method with a good correlation (r = 0.93,significant at 1% level).

3.9. Interference study

A well-defined current response was observed for H2O2(100 �M) at a potential of 0.28 V. The successive addition of rel-evant physiological concentrations of dopamine, ascorbic acid,l-cysteine, and glucose did not reveal obvious additional signalssuggesting their no interfering effect on sensor response.

3.10. Determination of H2O2 in fruit juices

A non enzymic an amperometric method was developed fordetermination of H2O2 in fruit juice using the present biosensor.The results are given in Table 1.

The working electrode lost 15% of its initial activity after its 100uses during the span of 2 months, when stored at 4 ◦C. This storagestability is higher than that of earlier electrodes [16].

4. Conclusion

A novel strategy is reported for developing a composite bioelectrode consisting of NiO-NPs/cMWCNT/PANI/Au, which showedremarkably enhanced sensitivity and selectivity for hydrogen per-oxide in the presence of interference by dopamine, ascorbic acid,l-cysteine, and glucose. The non-enzymatic biosensor showed rel-atively rapid response, broad linear range, low detection limit(0.2 �M), good reproducibility and long stability. The presentmethodology might be applied for a wide range of biosensors fordetection of metabolites.

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