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Sensors and Actuators B 152 (2011) 14–20

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l homepage: www.e lsev ier .com/ locate /snb

oly(brilliant cresyl blue) electrogenerated on single-walled carbon nanotubesodified electrode and its application in mediated biosensing system

ing Chena, Jia-Qi Xua, Shou-Nian Dingb, Dan Shana,∗, Huai-Guo Xuea, Serge Cosnierc, Michael Holzingerc

Key Laboratory of Environmental Materials & Environmental Engineering of Jiangsu Province, School of Chemistry & Chemical Engineering, Yangzhou University, 180# Si Wanging Road, Yangzhou, Jiangsu 225002, ChinaSchool of Chemistry & Chemical Engineering, Southeast University, Nanjing 211189, ChinaDépartment de Chimie Moléculaire UMR-5250, ICMG FR-2607, CNRS Université Joseph Fourier, BP-53, 38041 Grenoble, France

r t i c l e i n f o

rticle history:eceived 23 August 2010eceived in revised form7 September 2010ccepted 25 September 2010vailable online 1 November 2010

a b s t r a c t

Single-walled carbon nanotubes (SWCNTs) functionalized with carboxylic acid groups were cast toglassy carbon electrode (GCE) to construct a three-dimensional nano-micro structured scaffold. Bril-liant cresyl blue (BCB) was electropolymerized on the above-mentioned SWCNTs/GCE using continuouscycling between −0.7 and 0.9 V vs. SCE. PolyBCB yielded on SWCNTs/GCE exhibited the enhanced elec-trochemical redox behavior compared with that electrogenerated on bare GCE. The apparent surfacecoverage of PolyBCB obtained by SWCNTs/GCE was at least 10 times higher than that obtained by

eywords:lectropolymerizationingle-walled carbon nanotubesrilliant cresyl blueorseradish peroxidase

bare GCE, namely 4.8 × 10−9 and 3.6 × 10−10 mol cm−2. The cyclic voltammograms recorded by Poly-BCB/SWCNTs/GCE exhibited well-defined two peaks located at −0.25 V and −0.06 V, respectively, with asurface-controlled mechanism. In addition, morphologies of PolyBCB electrogenerated on GCE and SWC-NTS/GCE were characterized by atomic force microscopy. Finally, this proposed PolyBCB/SWCNTs/GCEwas used in the construction of the second-generation biosensors to hydrogen peroxide and glucose,with the enhanced analytical performance.

. Introduction

The development of in vitro electrochemical multi-enzymeiosensors in the last two decades has attracted great attentionsor bio-analysis in life. Mediator based enzyme biosensors are well-nown for its high sensitivity and low detection potential in theresence of an enhanced electron transfer redox promoter betweennzyme and electrode [1–3]. To facilitate the in vitro applicationf enzyme biosensors, both of enzyme and mediators should bemmobilized on the surface of electrode.

Dye molecules have been commonly used as mediators [4–6]nd numerous approaches and strategies have been used to co-mmobilize dye and enzyme molecules, for example, adsorption4], entrapment [7], and covalent linking. However, to developnhanced sensitive and long-term stable biosensors, there are still

everal challenges concerning the application of new dyes and themmobilization methods of dye molecules on the electrode surface,ecause low-molecular-weight, soluble mediating species sufferrom an inherent drawback that they can diffuse away easily from

∗ Corresponding author. Fax: +86 514 87975244.E-mail address: danshan@yzu.edu.cn (D. Shan).

925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2010.09.063

© 2010 Elsevier B.V. All rights reserved.

the electrode surface into the bulk solution when the mediated bio-electrode is used continuously. This would lead to significant signalloss and greatly affect the performance, especially life-time of thebiosensor.

Polymeric dye film can efficiently overcome above-mentionedproblems. Nevertheless, polymeric dye films directly generated onthe surface of bare electrode are generally impact and impose diffu-sion barriers, resulting in a slow and poor response of the sensors.

Due to good conductance, biocompatibility and high apparentsurface area of more than 1000 m2/g, single-walled carbon nan-otubes (SWCNTs), have been widely used to fabricate biosensors[8]. Furthermore, the SWCNTs exhibit a special sidewall curvatureand possess a �-conjugative structure with a highly hydrophobicsurface, which allow them to interact with some aromatic com-pounds such as anthracene derivatives [9], methylene blue (MB)[10], thionine [11–14] and nile blue A [15–17] through �–� elec-tronic and hydrophobic interactions.

In this work, SWCNTs functionalized with carboxylic acid

groups were used to for the construction of highly porous three-dimensional nano-micro structured scaffold. Positively chargedphenoxazine dye, brilliant cresyl blue (BCB), was used as tar-get dye in this work (Scheme 1). Due to the special interactionsbetween the functionalized SWCNTs and BCB, such as strong

M. Chen et al. / Sensors and Actuators B 152 (2011) 14–20 15

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Scheme 1. Possible mechanism of the BCB redox reaction, BC

lectrostatic interaction, �–� electronic, hydrophobic interactionsnd the synergistic effect, PolyBCB electrogenerated on the sur-ace of SWCNTs-modified GCE exhibited improved electrochemicalehavior with the increased and stable mediator concentration.urthermore, the above porous three-dimensional nano-microtructured scaffold (PolyBCB/SWCNTS/GCE) was firstly applied tommobilize enzymes to prepare the mediated biosensing system forhe detection of hydrogen peroxide and glucose by electric wiringf horseradish peroxidase (HRP) and bienzymatic system based onRP and glucose oxidase (GOD).

. Experimental

.1. Materials

Glucose oxidase (GOD) (EC 1.1.3.4, type II, 108 U mg−1) fromspergillus niger, horseradish peroxidase (HRP) (EC 1.11.1.7, type

I, 140 U mg−1) and brilliant cresyl blue (BCB) were purchased frommresco. Single-walled carbon nanotubes (SWCNTs), produced by

he HiPco® process (Purified, CNI grade, lot no. Po313), were pur-hased from Carbon Nanotechnologies, Inc. SWCNTs were oxidizednitially with 69% nitric acid (10.8 M) by stirring under reflux at10 ◦C for 3 h. After the reaction mixture was cooled down to roomemperature and the solid was settled out, the acidic supernatantas decanted off and the residue was diluted with distilled water.

his suspension was filtered over cellulose membrane (pore size.45 �m) and rinsed until the filtrate reaches a neutral pH value.he resulting solid was re-dispersed in water with ultra sound and

nsoluble particles were allowed to sediment. The obtained blackolution was again filtered over a cellulose membrane filter and thebtained oxidized SWCNTs were dried at 70 ◦C. Such chemicallyodified nanotubes can be dispersed in distilled water up to con-

entrations around 0.1 mg ml−1. All other chemical reagents were

merization and the possible structure of functional SWCNTs.

of analytical grade. Double-distilled water was used for aqueoussolution. Phosphate buffer solution was prepared by K2HPO4 andKH2PO4. H2O2 was freshly prepared before being used. Stock solu-tions of glucose were allowed to mutarotate at room temperature.

2.2. Measurements and apparatus

A CHI 660 electrochemical workstation (CHI Co., USA) wasused for cyclic voltammetry and amperometric technology. Allelectrochemical studies were performed with a conventional three-electrode system. A saturated calomel electrode (SCE) and a Pt foilelectrode were used as reference electrode and counter electrode,respectively. The working electrode was a glassy carbon electrode(GCE) (diameter 3 mm). Unless otherwise indicated, electrolyticsolutions were purged with highly purified nitrogen for at least20 min prior to the series of experiments. A nitrogen environmentwas then kept over solution in the cell to prevent the contact ofthe solution from oxygen. Atomic force microscopy (AFM) imageswere obtained with a multimode digital instrument (Vecco-DI).Electrochemical impedance spectra (EIS) measurements were con-duced using an Autolab/PGSTAT30 (Eco Chemie, Netherlands) witha three-electrode system. The EIS measurements were performedin 5 mM Fe(CN)6

3−/4− containing 0.1 M KNO3. The amplitude ofthe applied sine wave potential was 5 mV. The impedance mea-surements were recorded at a bias potential of 180 mV within thefrequency range of 0.05 Hz to 10 kHz.

2.3. Electrode modification

2.3.1. PolyBCB modified electrodeA GCE was polished carefully with 0.05 �m alumina particles

on silk followed by rinsing with the distilled water and dried in airbefore use. SWCNTs-modified GCE was obtained by casting 10 �lSWCNTs aqueous solution (0.1 mg ml−1) on the surface of GCE and

1 d Actuators B 152 (2011) 14–20

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ried in air. The electrochemical polymerization of BCB was per-ormed with bare GCE and SWCNTs/GCE, respectively, in a 0.1 MBS (pH 6.5) containing 2.5 mM BCB and 0.1 M KCl, using repeatedotential cycling between −0.7 and 0.9 V for 15 cycles at a scanate of 50 mV s−1. The above modified electrodes were denoted asolyBCB/GCE and PolyBCB/SWCNTs/GCE, respectively.

.3.2. Preparation of enzyme electrodesIn this work, calcium carbonate nanoparticles (Nano-CaCO3)

ere used as enzyme immobilization matrix. Thus, the protocolf preparation of enzyme electrode was followed by our previouseports [18–20]. The Nano-CaCO3 colloidal suspension (2 mg ml−1)as prepared by dispersing Nano-CaCO3 in deionized water with

tirring overnight. HRP or GOD was also dissolved in deionizedater with a concentration of 2 mg ml−1. The defined amount of

queous Nano-CaCO3/enzymes mixtures were spread on the sur-ace of as-prepared PolyBCB/GCE or PolyBCB/SWCNTs/GCE, leadingo an adherent Nano-CaCO3 film in which enzymes were entrapped.he resulting electrodes were placed in saturated glutaraldehydeapor at room temperature for 15 min in order to induce the chem-cal cross-linking of the entrapped enzyme molecules. Before use,he enzyme electrode was rinsed under stirring for 20 min withuffer solution to remove the enzyme not firmly immobilized.

. Results and discussion

.1. Electropolymerization of brilliant cresyl blue

The electropolymerization of BCB has been reported elsewheren the literature [21–23]. Fig. 1A and B shows the cyclic voltam-

ograms for the electropolymerization of BCB at bare GCE andWCNTs-modified GCE in a 0.1 M PBS (pH 6.5) containing 2.5 mMCB and 0.1 M KCl for 15 cycles, respectively. BCB species oxi-ized at potentials higher than +0.85 V. It has been proposed thathe amino group of BCB underwent oxidation to form a cationadical, followed by radical determination via carbon–nitrogenoupling reactions to form PolyBCB [23] (Scheme 1). Thus, in thisork, potentials were swept between −0.7 and 0.9 V with a scan

ate of 50 mV s−1. As shown in Fig. 1A, at about 0.87 V vs. SCEhe irreversible oxidation of monomer occurs, the more negativeotential at about −0.25 V corresponds to oxidation/reduction ofhe monomer [21] (Scheme 1). After the second cycles, there comesshoulder peak and this couple with formal potential around 0 V

ncreases in height with the continuous cycles. The latter couple isscribed to the polymer. After 15 cycles, there is a blue thin filmenerated on the surface of GCE.

SWCNTs exhibit sidewall curvature and possess a �–� conjuga-ive structure with a highly hydrophobic surface [24], interactedith BCB monomer by �–� stacking to form a 3D structure

omposite. In addition, the enrichment of BCB monomer can belso enhanced by the electrostatic interaction between positivelyharged BCB and negatively charged of SWCNTs. Thus, an obvi-usly different phenomenon can be obtained for the growth ofolyBCB on SWCNTS modified GCE: during the electropolymeriza-ion of BCB, the stronger and reversible signal of monomer redoxeaction (at about −0.25 V) decreases and reaches the steady stateith continuous cycling; simultaneous with the decrease of the

haracteristic redox peaks of the adsorbed species, a new pair ofell-shaped redox couple with formal potential of E0 ′ = −0.06 V in

he region of higher potential values and the intensity of this newouple increases with larger magnitude with the increasing cyclingFig. 1B).

.2. Topographic characterization using AFM

Three different modified electrodes: PolyBCB/GCE, SWC-Ts/GCE and PolyBCB/SWCNTS/GCE have been characterized using

Fig. 1. Growth of PolyBCB films on GCE (A) and SWCNTs modified GCE (B) duringelectrolysis of a PBS (0.1 M, pH 6.5) containing 2.5 mM BCB and 0.1 M KCl at a scanrate of 100 mV s−1.

AFM. From Fig. 2, it is significant that there are morphological dif-ferences between PolyBCB/GCE and PolyBCB/SWCNTs/GCE. The topview of structure in Fig. 2A on the surface of PolyBCB/GCE showsPolyBCB deposition is well uniform, smooth and compact. Fig. 2Bshows the AFM image of the surface for the SWCNTs-modified GCE.The presence of carbon nanotubes is obvious. The correspondingAFM image for SWCNTs-modified electrode after electropolymer-ization of BCB is shown in Fig. 2C. There was an increase in thediameter of the carbon nanotubes, indicating that PolyBCB moietieswere coated on the surface of the SWCNTs. Interestingly, there waslittle PolyBCB coated on the glassy carbon substrates. It suggestedthat BCB preferentially polymerized and coated on the surfaceof SWCNTs. The results indicated that SWCNTs were well dis-persed and embedded throughout the PolyBCB and interconnectedSWCNTs network formed on the electrode. This conductive CNTnetwork may establish electrical conduction pathways throughoutthe whole system, which is responsible for the electric conductivityand electrochemical sensing.

3.3. Electrochemical properties of PolyBCB film

3.3.1. Cyclic voltammetryThe PolyBCB film electrogenerated on the surface of GCE or

SWCNTs-modified GCE was investigated by cyclic voltammetry in0.1 M PBS (pH 6.5). The apparent surface coverage of PolyBCB on

M. Chen et al. / Sensors and Actuators B 152 (2011) 14–20 17

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Fig. 3. (A) Cyclic voltammograms of PolyBCB/SWCNTs/GCE and PolyBCB/GCE (InsetI) in 0.1 M PBS (pH 6.5) at different scan rates. The scan rates from inner to outerare 20 (a), 40 (b), 60 (c), 80 (d), 100 (e), 120 (f), 140 (g), 160 (h), 180 (i) and

Faradaic impedance spectroscopy (EIS) is an effective technique

ig. 2. AFM images of the surfaces of PolyBCB/GCE (A), SWCNTs/GCE (B) and Poly-CB/SWCNTs/GCE.

are GCE and SWCNTS-modified GCE obtained by 15 voltammetricycles were estimated coulometrically and was determined to bebout 3.6 × 10−10 and 4.8 × 10−9 mol cm−2, respectively. The corre-ponding PolyBCB coverage was about 10 times smaller than thatbtained on the SWCNTs-modified electrode. The presence of SWC-Ts offered significant increase in the surface area available for thelectropolymerization of BCB.

Cyclic voltammograms were recorded by PolyBCB/GCE andolyBCB/SWCNTs/GCE at different scan rates in 0.1 M PBS (pH 6.5).

n both cases the anodic and cathodic peak currents increase withhe increase of the scan rate from 20 to 200 mV s−1 (Fig. 3A and Inset). Compared with those obtained by PolyBCB/GCE, the CVs of Poly-CB/SWCNTs/GCE display well-defined two peaks shape at each

200 mV s−1 (j). Inset II: The plots of peak currents vs. scan rate for the CVs of Poly-BCB/SWCNTs/GCE. (B) Electrochemical impedance spectroscopy of bare GCE (a),PolyBCB/GCE (b) and PolyBCB/SWCNTs/GCE (c) in 0.1 M KNO3 solution containing5.0 mM Fe(CN)6

3−/Fe(CN)64− (1:1).

scan rate. The peak currents (IpaI, IpaII, IpcI and IpcII) vary linearly withthe scan rates, as shown in the Inset II of Fig. 3B. The linear regres-sion equation is IpaI(�A) = 233.5�(V s−1) + 4.7659, R2 = 0.9949;IpcI(�A) = −315.9�(V s−1) − 5.854, R2 = 0.9981; IpaII(�A) =285.6�(V s−1) + 3.9643, R2 = 0.9956; IpcII(�A) = −275.4�(V s−1) −3.0727, R2 = 0.9984, respectively. This indicates that the electrontransfer process for PolyBCB/SWCNTs/GCE is a surface-controlledmechanism in the range mentioned above.

3.3.2. Electrochemical impedance

to probe the features of surface-modified electrodes [25,26]. A mod-ified Randle’s equivalent circuit and the fitting measured spectrato the equivalent circuit are shown in Fig. 3B. The circuit, which isoften used to model interfacial phenomena, included the follow-

1 d Actuators B 152 (2011) 14–20

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Fig. 4. Typical steady-state responses: (a) Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCEand (b) Nano-CaCO3-HRP/PolyBCB/GCE on successive injection of H2O2 into 10 mlof stirring 0.1 M PBS (pH 6.5). Applied potential: −0.25 V. Inset A: Cyclic voltam-mograms of Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCE in PBS (0.1 M, pH 6.5) in theabsence (a) and in the presence of 0.12 mM (b), 0.16 mM (c), 0.2 mM (d) and

TC

8 M. Chen et al. / Sensors an

ng: Rs, the ohmic resistance of the electrolyte solution; Zw, thearburg impedance, resulting from the diffusion of ions from the

ulk electrolyte to the electrode interface; RCT, corresponding tohe interfacial electron-transfer resistance for the redox prober. Thearallel elements (CPE and Zw + RCT) of the equivalent circuit were

ntroduced since the total current through the working interfaceas the sum of respective contributions from the Faradaic pro-

ess and the double layer charging. The impedance spectra includesemicircle portion and a linear portion. The semicircle portion

t higher frequencies corresponds to the electron-transfer-limitedrocess, and the linear portion at lower frequencies representshe diffusion-limited process. The semicircle diameter equals thelectron-transfer resistance RCT. Thus, we can characterize elec-rochemical properties of the modified electrode by measuringhe diameter of semicircles in the spectrum. Faradaic impedance

easurements were performed in a 0.1 M PBS (pH 6.5) contain-ng 5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture. As shown inig. 3B, the Nyquist diameter of the GCE deposited with PolyBCBRCT ≈ 2450 �) is slight larger than that of bare GCE (RCT ≈ 2100 �).owever, it should be noted that PolyBCB/SWCNTS/GCE exhibitsnearly straight line that characteristic of diffusional limiting stepf the electrochemical process. It implies PolyBCB electrogeneratedn the SWCNTs modified GCE exhibits higher electrochemical prop-rties, in particularly with higher improved conductivity and fastlectron transfer behavior. Thus, PolyBCB/SWCNTs/GCE could findhe applications in the filed of electrochemistry.

.4. Application of PolyBCB/SWCNTs-modified GCE

.4.1. H2O2 biosensorImmobilization of HRP on the PolyBCB/GCE and Poly-

CB/SWCNTs/GCE was performed by the entrapment of the enzymeith a Nano-CaCO3 matrix followed by cross-linking step with

lutaraldehyde, as described previously [27,28]. This method isnown to be simple, versatile and efficient, leading to sensitive andtable biosensors. The electrochemical behavior of Nano-CaCO3-RP/PolyBCB/SWCNTs/GCE was studied by cyclic voltammetry

Inset A of Fig. 4). In the absence of H2O2, the enzyme electroderesents the cyclic voltammogram corresponding to the underly-

ng PolyBCB/SWCNTs (curve a). When H2O2 is added to the bufferolution, an increase in the reduction peak was observed with theecrease of the oxidation peak (Fig. 4A, curves b–e). Such phe-omenon unambiguously reveals an electrocatalytic reduction of2O2 by the Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCE as shown the

chematic illustration in Fig. 4B.The electrocatalytic reduction of hydrogen peroxide at

ano-CaCO3-HRP/PolyBCB/SWCNTs/GCE was also studied bymperometry, which is one of the most widely employedechniques for biosensor. Fig. 4 displays the amperomet-ic responses of different concentrations of H2O2 at Nano-aCO3-HRP/PolyBCB/SWCNTs/GCE (curve a) and Nano-CaCO3-

able 1omparison of the proposed mediated H2O2 biosensor with others based on HRP.

Electrode Mediator Eapp (V) L

Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCE PolyBCB −0.25 5HRP/TBa-MWCNT-GCE TB −0.4 ∼HRP/PolyBCB/GCE PolyBCB 0 ∼MWCNT/BCB/HRP/GCE BCB −0.32 5HRP/DNA-MBb/PAA/Au MB −0.2 ∼HRP/{TB/Au}n/Au TB −0.35 1Cs-CNTs-NBc-HRP NB −0.4 1GC/poly(GMA-co-VFc)d/HRP Poly(GMA-co-VFc) 0.35 2

a TB: toluidine blue.b MB: methylene blue.c NB: nile blue.d poly(GMA-co-VFc): poly(glycidyl methacrylate-co-vinylferrocene).

0.24 mM of H2O2 (e). Inset B: Schematic illustration of the proposed reaction mech-anism for the detection of H2O2 by Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCE. InsetC: Calibration curves of (a) Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCE and (b) Nano-CaCO3-HRP/PolyBCB/GCE to H2O2, in 0.1 M PBS (pH 6.5), Eapp = −0.25 V.

HRP/PolyBCB/GCE (curve b) with an applied potential of −0.25 Vvs. SCE in PBS (0.1 M, pH 6.5), respectively. As can be seen, thecurrent responses at Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCE aremuch higher than those at Nano-CaCO3-HRP/PolyBCB/GCE. Fromthe steady-state current response of H2O2 at the Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCE this proposed electrode achieved 95%of the steady-state current within 9 s, with a linear range of5 × 10−6 to 1.2 × 10−3 M (Inset C of Fig. 4). The detection limitwas 1.0 × 10−6 M based on S/N = 3. The Michaelis–Menten constant(Kapp

M ), which is a reflection of both the enzymatic affinity and theratio of microscopic kinetic constant, was calculated to be 0.17 mMaccording to the Lineweaver–Burk equation. The further compar-ison of Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCE developed in thisstudy with other H2O2 mediated biosensor based on HRP is sum-marized in Table 1.

3.4.2. Bienzymatic biosensor to glucoseSince the Nano-CaCO3-HRP/PolyBCB/SWCNTs/GCE may allow

the selective reduction of H2O2 to water in the presence ofoxygen, its use for fabrication of oxidase based biosensors has

been also investigated. For this purpose, GOD was chosen as arepresentative model of oxidase. The working principle of Nano-CaCO3-GOD-HRP/PolyBCB/SWCNTs/GCE to detection of glucose isdepicted in Fig. 5A. Glucose is transferred from bulk solution tothe Nano-CaCO3-GOD-HRP/PolyBCB/SWCNTs modified electrode

inear range (M) Detection limit (M) KappM (mM) Ref.

× 10−6 to 1.2 × 10−3 1.0 × 10−6 0.17 This work4.0 × 10−4 – 0.16 [8]6.5 × 10−5 4.2 × 10−6 – [18]× 10−7 to 1.8 × 10−5 1.0 × 10−7 1.5 [29]1.0 × 10−4 3.0 × 10−7 0.44 [30].5 × 10−7 to 8.6 × 10−3 7.0 × 10−8 – [31].0 × 10−6 to 2.4 × 10−4 1.2 × 10−7 – [32].0 × 10−3 to 3.0 × 10−2 2.6 × 10−6 1.14 [33]

M. Chen et al. / Sensors and Actuators B 152 (2011) 14–20 19

Fig. 5. (A) Schematic description of the proposed reaction mechanism for the amperometric detection of glucose at a Nano-CaCO3-GOD-HRP/PolyBCB/SWCNTs/GCE. (B) Cyclicv 6.5) inC PBSo ady-sti 0.125

tsTscStGctgmogHoaSctg(tr

oltammograms of Nano-CaCO3-GOD-HRP/PolyBCB/SWCNTs/GCE in 0.1 M PBS (pHalibration curve of Nano-CaCO3-GOD-HRP/PolyBCB/SWCNTs/GCE for glucose (0.1 Mf the proposed bienzymatic electrode on successive injection of glucose. (D) Stencrements: (a) 0.05 mM Urea, (b) 0.02 mM ascorbate, (c) 0.015 mM l-cysteine, (d)

hrough diffusion and GOD in the presence of the natural co-ubstrate O2, converts glucose to gluconic acid and O2 to H2O2.he H2O2 thus produced from the enzymatic reaction serves asubstrate for HRP and the oxidized state of HRP is in turn recy-led at the electrode surface. PolyBCB electrogenerated on theWCNTs modified GCE serves as an electron transducer. Thus,he amount of glucose is quantitatively estimated at Nano-CaCO3-OD-HRP/PolyBCB/SWCNTs modified electrode. Fig. 5B showsyclic voltammograms of the proposed bienzymatic electrode inhe absence and in the presence of glucose. In the presence of 2 mMlucose, the cyclic voltammogram displays a dramatic enhance-ent of the cathodic peak current and a concomitant decrease

f the anodic peak current (Fig. 5B curve b). This suggests thatlucose could be oxidized catalytically at the Nano-CaCO3-GOD-RP/PolyBCB/SWCNTs/GCE. GOD has the catalytic activity to thexidization of glucose; and HRP-PolyBCB has been used for the cat-lytic reduction of hydrogen peroxide at a low operating potential.ince GOD catalyzes the aerobic oxidation of glucose with con-omitant production of H2O2, the steady-state current response of

he bienzymatic Nano-CaCO3-GOD-HRP/PolyBCB/SWCNTs/GCE tolucose was investigated also by holding the biosensor at −0.25 VInset of Fig. 5C). The calibration plot for glucose shows thathe response current of the proposed biosensor is linear in theange from 2.5 × 10−6 to 5 × 10−3 M with a low detection limit

the absence (a) and in the presence (b) 2 mM glucose, at scan rate of 2 mV s−1. (C)with pH 6.5, at 25 ◦C, Eapp = −0.25 V). Inset shows the typical steady-state responsesate current–time responses of the proposed bienzymatic electrode to successive

mM glucose and (e) 0.125 mM glucose in 0.1 M PBS (pH 6.5) (Eapp = −0.25 V).

of 1 × 10−6 M based on signal/noise = 3 (Fig. 5C). At the appliedpotential of −0.25 V, interferences were evaluated by intercala-tion of three interferents: 0.05 mM urea, 0.02 mM ascorbate and0.015 mM l-cysteine. No appreciable signals were observed forthe successive injections of interferents into the electrolyte solu-tion (Fig. 5D). This non-significant interference effect is probablydue to the efficiency of the electrocatalytic process in the Nano-CaCO3-GOD-HRP/PolyBCB/SWCNTs sensing system, which permitsthe amperometric detection of glucose at −0.25 V vs. SCE.

4. Conclusion

In summary, the electrochemical behavior of the redox polymerbased on dye molecules can be feasibly enhanced by SWCNTs-modified electrode: (1) as nano-sized materials, SWCNTs canprovide higher electrochemically accessible surface area for theadsorption of dye molecules; (2) the advantageous interactionsbetween SWCNTs and dye molecules are favorable for the enrich-ment of dye molecules on the surface of SWCNTS-modified

electrode, in particular �–� stacking and the electrostatic inter-action; (3) the dye polymer film electropolymerized on theSWCNTs-modified electrode can provide even higher porous 3D-structure, which benefits the diffusion of the substrate; (4) dueto the high electronic conductivity of SWCNTs, the electron trans-

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er behavior of the redox polymer can be dramatically improved.hus, the enhanced redox polymer based on dye molecules throughntroducing of SWCNTs can be used as efficient mediator for theonstruction of second-generation biosensor with highly improvednalytical performance.

cknowledgements

The authors are grateful to the financial supports of Nationalatural Science Foundation of China (Grant Nos. 20773108 and0905011), University science research project of Jiangsu Province09KJB150015, BK2010396), Qing Lan Project of Jiangsu Province,he project sponsored for D. Shan by the Scientific Research Foun-ation for the returned overseas Chinese scholars, State Educationinistry, the Open Research Fund of State Key Laboratory of

ioelectronics, Southeast University and the Foundation of the Edu-ational Committee of Jiangsu Provincial Universities Excellencecience and Technology Invention Team in Yangzhou University.

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Biographies

Ming Chen is a researcher and lecturer in School of Chemistry & Chemical Engi-neering, Yangzhou University, PR China. He received his MS and Ph.D. in physicalchemistry from Yangzhou University, China in 2005 and 2008. His current fields ofinterest include supramolecule chemistry, electro-catalysis and interfacial electro-chemistry.

Jia-Qi Xu is currently studying MS at Yangzhou University.

Shou-Nian Ding is a researcher and lecturer in School of Chemistry and ChemicalEngineering, Southeast University, PR China. He received his MS in inorganic chem-istry from Anhui University, China in 2003, and his Ph.D. in analytical chemistry fromNanjing University, PR China in 2006. His current fields of interest include solar cell,biofuel cell, electrochemiluminescence, photoelectrochemistry, and microchip.

Dan Shan is a professor in College of Chemistry and Chemical Engineering, YangzhouUniversity, PR China. She received her MS in physical chemistry from YangzhouUniversity, China in 2001 and her Ph.D. in electrochemistry from Joseph Fourier Uni-versity of Grenoble (France) in 2004. Her current fields of interest include biosensor,interfacial electrochemistry and electroconductive polymer.

Huai-Guo Xue is presently employed as a professor in School of Chemistry & Chem-ical Engineering, Yangzhou University, PR China. He obtained Ph.D. from Instituteof Polymer Science and Engineering, Zhejiang University, PR China in 2002. Hisresearch interests include biosensor, polymer chemistry and electrochemistry.

Serge Cosnier is Research Director at CNRS and Head of the Biosystèmes Elec-trochimiques et Analytiques Laboratory at the Joseph Fourier University of Grenoble(France) where he began his research career in 1983. He received his doctoral degreein Chemistry from the Paul Sabatier University of Toulouse (1982) and Ph.D. from theJoseph Fourier University (1988). Since 1990, his main activity is focused on modifiedelectrodes and bioelectrochemistry. His research interests are in electrochemicalbiosensors, immunosensors and protein sensors, electrogenerated polymers and

biofuel cells.

Michael Holzinger is researcher at CNRS and works in the Department of Molec-ular Chemistry at the Joseph Fourier University of Grenoble (France) since 2006.He received Ph.D. degree from University Erlangen-Nürnberg (German) in 2002. Hismain research interest is focused on the development of biosensors and biofuel cellsbased on functionalized nanostructured carbon.