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Sensors and Actuators B 160 (2011) 287– 294

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical

journa l h o mepage: www.elsev ier .com/ locate /snb

anocomposite film based on graphene oxide for high performance flexiblelucose biosensor

ian-Ding Qiua,b,∗, Jing Huanga, Ru-Ping Lianga

Institute for Advanced Study and Department of Chemistry, Nanchang University, Nanchang 330031, PR ChinaDepartment of Chemical Engineering, Pingxiang College, Pingxiang 337055, PR China

r t i c l e i n f o

rticle history:eceived 31 May 2011eceived in revised form 19 July 2011ccepted 24 July 2011vailable online 2 August 2011

eywords:raphene oxidelucose oxidasehitosan–ferroceneiosensor

a b s t r a c t

A homogeneous chitosan–ferrocene/graphene oxide/glucose oxidase (CS–Fc/GO/GOx) nanocompositefilm was successfully constructed as a novel platform for the fabrication of glucose biosensor. Themorphologies and electrochemistry of the nanocomposite film were investigated by using scanning elec-tron microscopy and electrochemical techniques including electrochemical impedance spectroscopy andcyclic voltammetry, respectively. Results demonstrated that the uniformly dispersed GO within the CSmatrix could significantly improve the stability of GO and make it exhibit a positive charge, which wasmore favorable for the further immobilization of biomolecules, such as GOx, with higher loading. Fur-ther attaching redox mediator ferrocene group (Fc) to CS could not only effectively prevent the leakageof Fc from the matrix and retain its electrochemical activity, but also improve the electrical conduc-tivity of CS and promote the electron-transfer between GOx and electrode. Biosensors based on this

CS–Fc/GO/GOx film had advantages of fast response, excellent reproducibility, high stability, and showeda linear response to glucose in the concentration range from 0.02 to 6.78 mM with a detection limitof 7.6 �M at a signal-to-noise ratio of 3 and exhibited a higher sensitivity of 10 �A mM−1 cm−2. Theproposed strategy based on CS–Fc/GO nanocomposite for the immobilization of enzymes can be of prac-tical relevance for the facile design of biosensors, as well as for the construction of new multifunctionalbioelectrochemical systems.

. Introduction

Electrochemical biosensors based on nanomaterials, such asarbon nanotubes [1–3], gold nanoparticles [4–6], metal oxides7,8], and semiconductors [9], have recently attracted considerablettention in the medicine and food quality control field. In partic-lar, because of the usefulness in diagnostic analysis of diabetes,lucose biosensors based on carbon nanotubes and metal nanopar-icles have been extensively studied in recent years [10–18].hough the suitability of functional graphene for ultracapacitor andnergy storage applications has been studied extensively [19,20],here are very few reports available that describe the suitabilityf graphene for electrochemical biosensing applications [21–26].nlike rolled structured CNTs and metal nanoparticles with dif-

erent sizes and shapes, graphene is a two-dimensional plenaryheet with open structure, hence both sides of graphene coulde utilized for supporting enzymes. Therefore, it is expected to

∗ Corresponding author at: Institute for Advanced Study and Department ofhemistry, Nanchang University, Nanchang 330031, PR China.el.: +86 791 3969518.

E-mail addresses: jdqiu@ncu.edu.cn, qiujianding@163.com (J.-D. Qiu).

925-4005/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2011.07.049

© 2011 Elsevier B.V. All rights reserved.

be a more promising enzymes carrier. However, the intrinsic vander Waals interactions between layers of graphene easily resultsin irreversible agglomeration or even restack to form graphite.This problem has been encountered in all previous efforts aimedat large-scale production of graphene through chemical conver-sion or thermal expansion/reduction [27–30]. The prevention ofaggregation is of particular importance for graphene because mostof their unique properties are only associated with individualsheets [31]. This limitation can be overcome by the attachment ofother molecules or polymers onto the graphene surfaces. Zhangand coworkers used deoxyribonucleic acid (DNA) for functional-izing graphene through �–� stacking interactions, the resultinghydrophilic DNA/graphene nanocomposite ideally improved thewater-solubility of graphene and thus prevented their aggrega-tion, meanwhile the obtained nanocomposite offered a promisingplatform for immobilizing horseradish peroxidase for the develop-ment of novel electrochemical biosensors [32]. Saha et al. found thatthe existence of polyacrylate on graphene and GO could assist thedispersion of both in different aqueous buffer solutions with dif-

ferent pHs [33]. Recently, it has been reported that CS could forma stable nanocomposite with GO through electrostatic attractionand hydrogen bonding [34]. Owing to such efficient combination,the resulting CS/GO nanocomposite is endowed with the excellent

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88 J.-D. Qiu et al. / Sensors and

roperties of the two independent components, such as the bio-ompatibility of CS and the outstanding electronic properties ofO. Furthermore, the existence of CS on GO not only assists theispersion of GO in aqueous solution but also makes it exhibit aositive charge, which is favorable for the further immobilizationf biomolecules through self-assembly.

As is well known, direct electron transfer between the activeite of the enzymes and the electrode surface is commonly forbid-en, due to the fact that this redox center is commonly buried inhe globular structure of the protein. For this reason, the use oflectron transfer mediators has been widely employed during theast few decades [35]. However, the operational simplicity desiredor biosensors is reduced by the use of these compounds in solu-ions. These drawbacks can be avoided by wiring the immobilizednzyme in conducting or redox polymers [36,37]. In particular, theodification of polymers with electron transfer mediators con-

titutes a simple, cheap and effective strategy for constructingeagentless amperometric biosensors [38,39]. On the other hand,nzymes are labile polypeptides that tend to be inactivated afterxposure to conditions commonly used for analytical determi-ations, and as a result, the stability and the sensitivity of thelectrode decay with time. To overcome this issue, the immobiliza-ion of enzymes in polysaccharide based supports has been widelymployed for the construction of biosensors [40,41]. Recently, wend other groups reported the synthesis of biocompatible and con-uctive ferrocene branched chitosan (CS–Fc) [42,43], which wassed as redox polymer for the construction of reagentless enzymeiosensors [44]. This novel redox active hybrid could not only effec-ively prevent the leakage of Fc from the matrix and improve thelectrical conductivity of CS but also show excellent biocompat-bility for enzyme immobilization without destructing its nativetructure [43,45].

In the present work, by taking the advantages of chitosan, GOnd ferrocene, we for the first time demonstrate the prepara-ion of a novel platform for the fabrication of enzyme electrodes,s illustrated in Scheme 1. As a common model enzyme, GOxs negatively charged in physiological media, it can be immo-ilized on CS/GO through a self-assembly method to form aulticomponent nanocomposite on a glassy carbon electrode. The

xperimental results demonstrated that such an electrochemi-al platform not only effectively prevented the leakage of bothnzyme and mediator, preserved the native structure of the immo-ilized enzyme, but also provided an attractive route to promotehe electron transfer between GOx and electrode. Meanwhile, the

s-prepared CS–Fc/GO/GOx electrode exhibited excellent analyti-al performance toward the quantification of glucose, with a wideinear range, excellent sensitivity, good reproducibility, and long-erm stability. Therefore, this kind of redox active, conductive and

Scheme 1. Schematic representation of the CS–Fc/GO/GOx modified electrode and th

tors B 160 (2011) 287– 294

biocompatible nanocomposite material offers a promising plat-form for the development of novel electrochemical biosensors andbiomedical devices.

2. Experimental

2.1. Reagents

Graphite flake (99.8%, 325 mesh) was provided by Alfa AesarChina Ltd. (China). Glucose oxidase (GOx, from Aspergillus niger,EC 1.1.3.4. 150,000 units g−1), �-d(+)-glucose, chitosan (CS, 85%deacetylation), sodium cyanoborohydride (NaCNBH3, 95%), andferrocenecarboxaldehyde (FcCHO, 98%) were purchased fromSigma–Aldrich Chemical (USA). All other chemicals were of ana-lytical grade and used without further purification. All solutionswere prepared using doubly distilled water.

2.2. Instruments

X-ray diffraction (XRD) patterns of the nanocomposites werecarried out using a Rigaku powder diffractometer equipped withCu K�1 radiation (� = 1.5406 A). UV–vis absorption spectra wererecorded with a UV-2450 spectrophotometer (Shimadzu). Scanningelectron microscopy (SEM) images were obtained by using a Quanta200 scanning electron microscope (FEI, USA). The accelerating volt-age was 20 kV. The Fourier-transform infrared (FTIR) spectra ofsamples in KBr pellets were recorded on a Nicolet 5700 FTIR spec-trometer (Nicolet, USA). Electrochemical experiments were carriedout on a PGSTAT30/FRA2 system (Autolab, The Netherlands). Athree-electrode system including an Ag/AgCl (saturated KCl) ref-erence electrode, a platinum wire as auxiliary electrode, and themodified electrode as the working electrode was employed. Theelectrolyte solutions were purged with N2 for at least 10 min toremove O2 and kept under N2 atmosphere during measurements.The electrochemical impedance spectroscopic (EIS) measurementswere performed at a bias potential of 210 mV in the presence of a5.0 mM K3[Fe(CN)6]/K4[Fe(CN)6] (1:1) mixture as a redox probe in0.1 M phosphate buffer (containing 0.1 M KCl, pH 6.98) by applyingan alternating current voltage with 5 mV amplitude in the fre-quency range from 0.01 Hz to 100 kHz.

2.3. Synthesis of graphene oxide

Graphene oxide (GO) was synthesized from graphite flake by a

modified Hummer’s method [46,47]. In brief, 0.5 g of graphite flake,0.5 g of NaNO3, and 23 mL of H2SO4 were stirred together in an icebath. While maintaining vigorous agitation, an amount of 3 g ofKMnO4 was slowly added, and the rate of addition was controlled

e mechanism of the oxidation of glucose, catalyzed by GOx and mediated by Fc.

J.-D. Qiu et al. / Sensors and Actuators B 160 (2011) 287– 294 289

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Fig. 1. (A) XRD patterns of (a) pristine graphite a

arefully to avoid a sudden increase in temperature. The mixtureas then maintained at 35 ◦C for about 1 h. Deionized water (40 mL)as gradually added, causing an increase in temperature to 90 ◦C.

inally, the mixture was further treated with 100 mL of deionizedater and 3 mL of 30% H2O2, turning the color of the solution fromark brown to yellow. GO was then obtained through filtering,ater washing and drying process.

.4. Preparation of ferrocene branched chitosan

CS–Fc was prepared according to the as-reported method [42].riefly, CS (75.0 mg) was dissolved in 0.1 M acetic acid solution15.0 mM). FcCHO (10.0 mg) was dissolved in methanol (10.0 mL)nd added to the CS solution. After the mixture was stirred at roomemperature for 2 h, NaCNBH3 (80.0 mg) was dropped and the reac-ion mixture was stirred for 24 h. The reaction was quenched byrecipitation with 5% NaOH and the yellow product was exhaus-ively washed with methanol and water. The product was dried inir and finally redispersed in 0.2 M acetate buffer (pH 5.0) usingonication.

.5. Sensor construction

Glassy carbon electrode (GCE, diameter of 3 mm) was carefullyolished with 1.0, 0.3, and 0.05 �m alumina slurry, respectively,nd rinsed thoroughly with doubly distilled water between eacholishing step. The electrode was successively sonicated in 1:1itric acid, acetone, and doubly distilled water, and then allowed tory under N2. Subsequently, 6 �L of the mixture consisting of CS–Fc0.5 mg mL−1), GO (0.03 mg mL−1), and GOx (4.0 mg mL−1) was castn the surface of well-polished GCE, and then dried in air at roomemperature. The obtained film was denoted as CS–Fc/GO/GOx. Foromparison, CS–Fc, CS–Fc/GO, and CS–Fc/GOx modified electrodesere also prepared using the same procedure. All the resultant elec-

rodes were stored at 4 ◦C when not in use. The fabrication of theS–Fc/GO/GOx modified electrode and the redox-enzyme catalyticycle that gives rise to the current in the presence of glucose ischematically shown in Scheme 1.

. Results and discussion

.1. Characterization of GO

The XRD patterns of the pristine graphite and GO were col-

ected (Fig. 1A). The diffraction peak at 2� value of 26.5◦ (Fig. 1A,urve a) in the XRD pattern of pristine graphite can be assignedo the (0 0 2) facet of the hexagonal crystalline graphite [48–50].ompared with the pristine graphite, disappearance of the peak at

GO. (B) SEM image and UV–vis spectrum of GO.

about 26.5◦ and appearance of the peak at 10.0◦ (Fig. 1A, curve b)reveal the successful oxidation of the starting graphite. SEM wasthen used to investigate the surface morphological characters ofthe as-synthesized GO (Fig. 1B). The isolated single-layer flakesdemonstrated that GO had been readily exfoliated into individualsheets in water by ultrasonic treatment. To further demonstratethe formation of GO, UV–vis spectroscopy was carried out (inset ofFig. 1B). The characteristic absorption peak of GO at 231 nm con-firmed that GO sheets were successfully synthesized, which wasconsistent with other studies [31].

3.2. Characterization of the CS–Fc/GO/GOx biocomposite film

Fig. 2A shows a flat and featureless morphology of CS–Fc/GOxfilm. When further entrapped GO into the CS matrix, as expected,the formed CS–Fc/GO/GOx membrane displayed a rough surfaceand many nanosheets uniformly dispersed throughout the hybridfilm without obvious aggregation (Fig. 2B), indicating that a bio-composite film composed of CS–Fc entrapped GO and GOx wasformed on the electrode surface. Fig. 2C and D represent the pho-tographs of GO and CS–Fc/GO, respectively, in PBS buffer solutionswith pH ranging from 5.0 to 10.0. It can be observed from Fig. 2C thatthe as-synthesized GO easily results in agglomeration in PBS solu-tions with different pHs, thus causing the color of solutions uneven(Fig. 2C, a–e) [33]. In contrast, the CS–Fc/GO nanocomposite couldbe readily dispersed in different PBS to form very homogenous darkbrown solutions (Fig. 2D, a–e) and no sediments were observedfor at least a month, which indicated that the nanocomposite washighly hydrophilic. In addition, the CS–Fc functionalized GO couldbe precipitated via high speed centrifuging and redispersed in freshwater or buffer solution at least 6 times. These observations sug-gested that GO was efficiently prevented from aggregations inPBS buffer solutions with different pHs by functionalizing withCS–Fc.

3.3. FTIR spectroscopic analysis of the CS–Fc/GO/GOx composite

FTIR experiments were carried out to investigate the formationof the CS–Fc/GO/GOx composite. Fig. 3 shows the FTIR spectra ofGO, CS–Fc, GO/CS–Fc, GOx, and GO/CS–Fc/GOx. In the spectrumof GO (Fig. 3a), the peak at 1734 and 1622 cm−1 are character-istics of the C O stretch of the carboxylic group on the GO anddeformations of the O–H bond in water, respectively. In the spec-trum of CS–Fc (Fig. 3b), there were two characteristic absorbance

bands centered at 1651 and 1596 cm−1, which corresponded tothe C O stretching vibration of –NHCO– and the N–H bendingof –NH2, respectively. Compared with pure CS–Fc and GO, bothpeaks at 1596 cm−1 related to –NH2 absorbance vibration and

290 J.-D. Qiu et al. / Sensors and Actuators B 160 (2011) 287– 294

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The semi-circle diameter in the impedance spectrum is equal to

ig. 2. Typical SEM images of (A) CS–Fc/GOx and (B) CS–Fc/GO/GOx. Photographs oH 6.0, (c) pH 7.4, (d) pH 9.0, (e) pH 10.0.

t 1734 cm−1 belonging to C O stretch of the carboxylic groupisappeared in the spectra of CS–Fc/GO nanocomposite. Moreover,he band corresponding to the C O characteristic stretching bandf the amide group shifted to a lower wavenumber (Fig. 3c). Theseould be ascribed to the synergistic effect of hydrogen bondingetween CS–Fc and the oxygenated groups in GO and electro-tatic interaction between polycationic CS and the negative chargen the surface of GO [33]. After CS–Fc/GO further interacted withOx, it could be seen that the FTIR spectrum of the CS–Fc/GO/GOxxhibited the amide I (1655 cm−1) and amide II (1585 cm−1)ands of the immobilized enzyme (Fig. 3d), which were essen-ially the same as those of the native GOx (Fig. 3e), demonstratedhe successful formation of CS–Fc/GO/GOx biocomposite and

he secondary structures of the immobilized enzyme were well

aintained.

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Wavenumber/ cm-1

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ig. 3. FTIR spectra of (a) GO, (b) CS–Fc, (c) GO/CS–Fc, (d) GO/CS–Fc/GOx, (e) GOx.

ure GO and (D) CS–Fc/GO in PBS buffer solutions with different pHs (a) pH 5.0, (b)

3.4. Electrochemical impedance spectroscopic characterization ofthe modified electrodes

The modified electrodes were monitored by EIS, which is aneffective method for probing the interfacial properties of theelectrodes [51] and often used for understanding chemical trans-formations and processes associated with the conductive supports[52–54]. The typical impedance spectrum (presented in the formof the Nyquist plot) includes a semicircle portion and a linearportion. The semicircle portion at higher frequencies correspondsto the electron-transfer limited process, and the linear portionat lower frequencies represents the diffusion-limited process.

the electron-transfer resistance, Ret, which controls the electron-transfer kinetics of the redox probe at the electrode interface. Fig. 4shows the EIS results of a bare GCE, CS–Fc/GCE, CS–Fc/GO/GCE, and

Fig. 4. The electrochemical impedance spectra of (a) bare GCE, (b) CS–Fc/GCE, (c)CS–Fc/GO/GCE, and (d) CS–Fc/GO/GOx/GCE. Inset: equivalent circuit used to modelimpedance data in the presence of the redox couples. Supporting electrolyte, 0.1 MPBS (pH 6.98) +0.1 M KCl +5.0 mM Fe(CN)6

3−/4− solution.

J.-D. Qiu et al. / Sensors and Actuators B 160 (2011) 287– 294 291

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ig. 5. CVs of CS–Fc/GO/GOx film electrode in 0.1 M PBS (pH 6.98, containing 0.1 MCl) at different scan rates (from inner to outer: 5, 10, 50, 100, 200, 300, 400, 500,00,700, 800, 900, 1000 mV s−1). Inset: plots of peak current vs.�1/2.

S–Fc/GO/GOx/GCE in the presence of redox probe Fe(CN)63−/4−. It

ould be seen that the bare electrode exhibited an almost straightine, which was the characteristic of a diffusion limiting step of thelectrochemical process (Fig. 4a). After coating a layer of CS–Fc, theet of the modified electrode obviously increased to 95 � (Fig. 4b),hich indicated the successful formation of CS–Fc film. When GOas incorporated into CS–Fc film, the Ret of the resulted CS–Fc/GOlm surprisingly decreased to 26 � (Fig. 4c), which was muchmaller than that of CS–Fc film, suggesting that the presence ofO on the CS–Fc modified electrode formed high electron conduc-

ion pathways between the electrode and electrolyte, and greatlymproved the electron transfer of the redox probe. After immobi-ization of GOx on CS–Fc/GO film, Ret increased to 182 � (Fig. 4d),he increase of electron-transfer resistance confirmed that the GOxas successfully assembled on the surface of GO, which resulted in

he hindered pathway of electron transfer. To obtain more detailednformation about the impedance of the modified electrode, a mod-fied Randles equivalent circuit (inset in Fig. 4) was chosen to fit the

easured results. The two components of the scheme, Rs and Zw,epresent the bulk properties of electrolyte solution and diffusionf the applied redox probe, respectively. The other two componentsf the circuit, Cdl and Ret, depend on the dielectric and insulatingeatures at the electrode/electrolyte interface.

.5. Electrochemical behavior of the modified electrode

Fig. 5 displays typical cyclic voltammograms (CVs) of theS–Fc/GO/GOx film electrode in 0.1 M PBS (pH 6.98) over the poten-ial range from −0.1 to 0.6 V at various scan rates. It is clear that aouple of well-defined redox peaks of the immobilized Fc groupre observed at scan rates ranging from 5 to 1000 mV s−1. Both theeduction and oxidation peak currents increased obviously withhe increasing scan rates, while the peak-to-peak separation (�E)as nearly independent of the scan rates, which indicated that the

lectron transfer of Fc in this composite film was relatively fast andeversible. Meanwhile, the CS–Fc/GO/GOx modified electrode wasound to be extremely stable during continuous potential cyclingetween −0.1 V and 0.6 V, indicated that the covalently bounded Fc

n CS chain could prevent mediator from leakage efficiently. Bothhe anodic and cathodic peak currents increased linearly with the

quare root of scan rate (�1/2) (inset in Fig. 5), confirming that theedox behavior of the electrodes was diffusion controlled, wherelectron transferred to and from the redox centers of the CS–Fcnvolved diffusion [55]. The linearity of ip against �1/2 plot also

Fig. 6. CVs of CS–Fc/GOx (a and c) and CS–Fc/GO/GOx (b and d) modified electrodesat 50 mV s−1 in 0.1 M PBS (pH 6.98, containing 0.1 M KCl) in the absence (a and b)and the presence (c and d) of 14 mM glucose.

suggested the electron-transfer model of Randles–Sevcik, whichassumes semi-infinite linear diffusion, the diffusion coefficient ofcharge transfer (D) was determined in accordance to the equationip = (2.69 × 105)n3/2ACD1/2�1/2 [55,56]. Where ip is the peak cur-rent, n is the number of electrons, A is the electrode area (in cm2),� is the scan rate (in V/s), D is the diffusion coefficient of chargetransfer, and C is the concentration of Fc redox centers in the film.The product D was calculated 6.7 × 10−8 cm−2 s−1, which is largerthan other redox polymers [57]. Electron-transfer under diffusioncontrol suggests that three-dimensional charge propagation in theCS–Fc/GO/GOx film is operative with this redox polymer, unlikethe acrylamide–ferrocene copolymer reported previously in whichthe redox charge decreased until a stable two-dimensional wiredenzyme was obtained in the monolayer level [58]. In the systemreported here, diffusion of the mediator and chain reptation are notpossible, thus charge propagate must through electron-hoppingbetween neighboring the conductive GO and polymer chain seg-ments containing ferrocene redox couples.

3.6. Electrochemical response of CS–Fc/GO/GOx film to glucose

To investigate the role of GO in biosensors, CVs of the CS–Fc/GOxand CS–Fc/GO/GOx modified electrodes in the absence and thepresence of glucose in 0.1 M PBS were studied (Fig. 6). In the absenceof glucose, the CS–Fc/GOx modified electrode exhibited a pair ofredox peaks of Fc (Fig. 6a). Further incorporation of GO in the film,the anodic peak and cathodic peak currents of the CS–Fc/GO/GOxmodified electrode increased considerably (Fig. 6b). Upon additionof glucose to the PBS solution, the anodic peak current increasednoticeably, while the cathodic peak current decreased, indicatingan obvious electrocatalytic oxidation of glucose at both CS–Fc/GOx(Fig. 6c) and CS–Fc/GO/GOx (Fig. 6d) modified electrodes. However,the electrocatalytic current on the CS–Fc/GO/GOx modified elec-trode was significantly improved, which was about 2.7 times higherthan that of the CS–Fc/GOx modified electrode. The improved per-formance of the CS–Fc/GO/GOx film could be attributed to thepresence of GO in the hybrid film. The nanoscale individual sheetsact as “molecular wires” to connect the active sites of GOx andelectron mediator Fc with the electrode, increasing the electrontransfer rate significantly. In addition, the high conductivity of GOis also responsible for the increased current. This typical enzyme-

dependent catalytic process shown in Scheme 1 can be furtherexpressed as follows [59]:

Glucose + GOx(FAD) → Gluconolactone + GOx(FADH2) (1)

292 J.-D. Qiu et al. / Sensors and Actuators B 160 (2011) 287– 294

Table 1Biosensor for glucose detection in comparison with literature.

Glucose Biosensor Detection limit (�M) KappM (mM) Linearity (mM) Reference

(GOD/GNP/CS)6/GOD/GNP/PAA/Pt 7.0 10.5 0.5–16 [60]GOD/graphite/Nafion 10 – Upto 6 [62]pH polymer/BSA/catalase/GOx 600 – 1–10 [63]GOD/MWNTs/CS–Fc 6.5 6.87 0.02–5.36 [64]GOD/mesocellular carbon foam/Nafion 70 – Upto 2 [65]

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here Fc and Fc+ represent reduced and oxidized forms of the fer-ocene, GOx (FAD) and GOx (FADH2) are the oxidized and reducedorms of glucose oxidase. In this process, the mediator takes thelace of oxygen in the native enzymatic reaction, and two elec-rons are transferred from glucose to the FAD of the enzyme. Theselectrons can then be transferred from FADH2 to the mediator,hich is then oxidized at the electrode surface producing a cur-

ent that is directly proportional to the concentration of glucose inolution.

.7. Amperometric determination of glucose

The current–time curve of the CS–Fc/GO/GOx film electrodepon successive additions of glucose at an applied potential of0.3 V clearly illustrated that the modified electrode could respondery rapidly to a change in the glucose concentration (Fig. 7), pro-ucing steady signals within only 5 s. Such a short response timeurther proves that the CS–Fc/GO material is a promising platformor the construction of biosensors. The response displayed a linearlucose concentration range from 0.02 to 6.78 mM with a detec-ion limit of 7.6 �M at a signal-to-noise ratio of 3. Based on threeimes the standard deviation of the slope, CS–Fc/GO/GOx electrodesxhibited an excellent sensitivity of 10 �A mM−1 cm−2 for glucoseetection, which was much higher than that of 7.86 �A mM−1 cm−2

or (GOD/GNP/CS)6/GOD/GNP/PAA/Pt [60]. The Michaelis–Mentenonstant (Kapp

M ), which is a reflection of both the enzymatic affin-

ty and the ratio of microscopic kinetic constant, was calculated toe 2.1 mM according to the Lineweaver–Burk equation [61]. Theurther comparison of CS–Fc/GO/GOx film electrode developed inhis study with other biosensors based on GOx is shown in Table 1.

600 900 1200 1500 1800 21000

1

2

3

4

5

6

0 2 4 6 8 100

2

4

6

0.5mM

Cur

rent

/μA

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0.02mM

0.04mM

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ig. 7. Amperometric response of CS–Fc/GO/GOx modified electrode at appliedotential of +0.3 V to successive addition of glucose in 0.1 M PBS (pH 6.98, containing.1 M KCl).

2.1 0.02–6.78 Current work

Results illustrate that the biosensor described in this work exhib-ited a lower detection limit and a wider linear range toward glucosedetection, thereby demonstrating the capability of the proposedbiosensor toward measuring the plasma glucose level (for the diag-nosis of diabetes).

3.8. Reproducibility and stability of the biosensor

The reproducibility of the CS–Fc/GO/GOx electrode was evalu-ated using five enzyme electrodes prepared at identified conditionson different days. A relative standard deviation (RSD) of the biosen-sor response to 1 mM glucose was 4.3%, indicating the satisfiedreproducibility of the resulting enzyme electrode for practicalapplication. The storage stability was investigated using the pre-pared electrode after having been stored in 0.1 M PBS (pH 6.98)at 4 ◦C for 1 month. No substantial decrease of the signal (>90%)was obtained, which may be arising from the biocompatibility ofthe CS–Fc/GO/GOx composite in maintaining the activity of theenzyme.

3.9. Interference study

Fig. 8 presents the selectivity testing results of theCS–Fc/GO/GOx modified electrode with successive additionsof ascorbic acid (AA), uric acid (UA), and glucose in 0.1 M pH 6.98PBS solution. Interestingly, the as-prepared biosensor gave outsignificant signals when adding 1 mM glucose for each time, whilethere were essentially negligible current responses of 0.1 mM AAand 0.02 mM UA at the normal physiological level. The results

obtained from several repetitions corroborated the fact that theCS–Fc/GO/GOx modified electrodes would give higher sensi-tivity and selectivity for glucose detection under physiologicalconditions.

700 800 900 1000 1100 1200

0.4

0.8

1.2

1.6

2.0Glucose

GlucoseAAUA

Cur

rent

/μA

t/s

UAAAGlucose

GlucoseGlucose

Fig. 8. Amperometric response of CS–Fc/GO/GOx modified electrode to successiveaddition of 0.02 mM UA, 0.1 mM AA, and 1 mM glucose into stirred 0.1 M PBS (pH6.98) containing 0.1 M KCl at a potential of +0.3 V.

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J.-D. Qiu et al. / Sensors and

. Conclusions

A novel ultrasensitive glucose biosensor by integratingS–Fc/GO nanocomposite as an ideal conductive platform forhe enzyme immobilization was designed through a simple self-ssembly method. The uniform GO dispersion within the CS matrixould significantly improve the stability of GO and make GO exhibit

positive charge, which was more favorable for the further immo-ilization of negative charged GOx without destructing its nativetructure and bioactivity. Further attaching redox mediator fer-ocene group to CS matrix could not only effectively prevent theeakage of Fc from the matrix and improve the electrical conduc-ivity of CS, but also show excellent biocompatibility for enzymemmobilization. The proposed CS–Fc/GO/GOx electrode performedood electrocatalytic oxidation for glucose with a broad linear-ty, good sensitivity, excellent reproducibility and storage stability.herefore, such a multicomponent platform, which integrates thedvantages of GO, ferrocene, chitosan, and GOx together, may haveromising potential for the fabrication of high performance flexibleiosensors or bioreactors.

cknowledgments

This work was supported by grants from the National Naturalcience Foundation of China (20865003, 20805023, 21065006) andhe Natural Science Foundation of Jiangxi Province (2010GZH0094).

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Biographies

Jian-Ding Qiu is a professor in Department of Chemistry, Nanchang University, PRChina. He received his PhD in analytical chemistry from Zhongshan University ofChina in 2004. He served as a postdoctoral research associate at the Nanjing Univer-sity (2004–2006) and Hokkaido University (2009–2010), respectively. His currentresearch interests include bioelectroanalysis, nanotechnology, and bioinformatics.

Jing Huang is a MS candidate in Department of Chemistry, Nanchang University.Her current research is the fabrication of a biosensor and its application in biological

fields.

Ru-Ping Liang received her PhD degree from Zhongshan University in 2004.Presently, she is a professor in Department of Chemistry, Nanchang University.Her main research interests include chemical modified electrode, microfluidics, andsurface modification.