ORIGINAL PAPER

A novel nanocomposite matrix based on graphene oxideand ferrocene-branched organically modified sol–gel/chitosanfor biosensor application

Huaping Peng & Zhengjun Huang & Yanjie Zheng &

Wei Chen & Ailin Liu & Xinhua Lin

Received: 13 October 2013 /Revised: 21 January 2014 /Accepted: 28 January 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract A novel platform for the fabrication of a glucosebiosensor was successfully constructed by entrapping glucoseoxidase (GOD) in a ferrocene (Fc)-branched organically mod-ified silica material (ormosil)/chitosan (CS)/graphene oxide(GO) nanocomposite. The morphology, structure, and electro-chemistry of the nanocomposite were characterized by trans-mission electron microscopy, X-ray powder diffraction, UV–vis spectroscopy, Fourier transform infrared spectroscopy, andelectrochemical techniques. Results demonstrated that theproposed electrochemical platform not only provided an ex-cellent microenvironment to maintain the bioactivity of theimmobilized enzyme, but also effectively prevented the leak-age of both the enzyme and mediator from the matrix andretained the electrochemical activity of Fc. Furthermore, dis-persing GO within the Fc-branched ormosil/CS matrix couldsignificantly improve the stability of GO and make it exhibit apositive charge, which was more favorable for the furtherimmobilization of biomolecules, such as GOD, with higherloading. Moreover, it could also improve the conductivity ofthe matrix film and facilitate the electron shuttle between themediator and electrode. Under optimal conditions, the de-signed biosensor to glucose exhibited a wide and useful linearrange of 0.02 to 5.39 mM with a low detection limit of6.5 μM. The value of KM

app was 4.21 mM, indicating that thebiosensor possesses higher biological affinity to glucose. The

present approach could be used efficiently for the linkage ofother redox mediators and immobilize other biomolecules inthe process of fabricating novel biosensors.

Keywords Graphene oxide . Chitosan . Ferrocene-branchedorganically modified sol–gel . Glucose oxidase . Biosensor

Introduction

Immobilization of enzyme to electrode surface is a crucial stepfor the design, fabrication, and performance of the biosensor.The commonmethods for immobilizing enzymes on electrodesinclude assembling techniques [1], electrodeposition [2], cova-lent bonding or cross-linking [3, 4], adsorption [5], and sol–geltechnique [6]. Among them, sol–gel technology provides aunique means to prepare a three-dimensional network suitedfor the encapsulation of a variety of biomolecules [7–9].Considerable work has been done on immobilization of en-zymes in inorganic sol–gels using tetramethoxysilane (TMOS)and tetraethoxysilane (TEOS) as the precursors doped withother dopants [7]. Unfortunately, inorganic sol–gel matricesare usually fragile and film cracks during gelling to xerogel[10]. To overcome these problems, organically modified sol–gels (ormosils) have been developed [11], which added furtheradvantages [12], such as tailored surface property of the ma-trixes (e.g., hydrophilic, hydrophobic, and ionic), improvedflexibility of the film so that thicker and crack-free films canbe prepared, and being leak-free when the reagent is covalentlyattached to the silica framework. Furthermore, chitosan (CS), asa naturally regenerating polysaccharide, has intrinsic biode-gradability, nontoxicity, and good biocompatibility. Theseproperties make it a promising matrix for enzyme immobiliza-tion [13–15]. Moreover, its abundant source and low price aresuitable for commercial application. It has been reported thatthe hybrid of CS into TEOS-derived sol–gel matrix could not

Huaping Peng and Zhengjun Huang contributed equally to the presentstudy.

H. Peng :Y. Zheng :W. Chen :A. Liu :X. LinDepartment of Pharmaceutical Analysis, Faculty of Pharmacy, FujianMedical University, Fuzhou 350004, China

H. Peng : Z. Huang :Y. Zheng :W. Chen :A. Liu (*) :X. Lin (*)Nano Medical Technology Research Institute, Fujian MedicalUniversity, Fuzhou 350004, Chinae-mail: ailinliu@mail.fjmu.edu.cne-mail: xhl1963@sina.com

J Solid State ElectrochemDOI 10.1007/s10008-014-2415-1

only overcome the brittleness of the sol–gel matrix and improvethe long-term stability of the biosensor [16, 17], but also assistthe dispersion of graphene oxide (GO) in aqueous solution andmake the matrix exhibit a positive charge, which is morefavorable for the further immobilization of negatively chargedglucose oxidase (GOD) without destructing its native structureand bioactivity [18].

On the other hand, because the enzyme active sites aredeeply embedded inside the protein, the electron transfer effi-ciency of the redox enzyme is poor. For the electron transferand sensitive detection based on sol–gel technology, a mediatoris highly necessary for biosensors, which is generally added inthe detection solution [19]. The encapsulation of the mediator,such as ferrocene (Fc) derivatives in a siloxane homopolymer,for amperometric detection of glucose has been reported [11].However, leakage is a main problem in the entrapment ofmediator (low molecular weight) compounds in sol–gel matri-ces. This limitation can be resolved through direct cross-linkingof Fc derivatives with polymer, nanoparticles, and high molec-ular weight compounds before immobilization [15, 20, 21].However, Fc-conjugated sol–gel has been proposed to solvethis problem [22]. Herein, a novel Fc-conjugated ormosil wasp r e p a r e d b y l i n k i n g t h e a l d e h y d e g r o u p o fferrocenecarboxaldehyde (Fc-CHO) to the amino group of thesol–gel precursor 3-(aminopropyl)triethoxysilane (APTES) viaSchiff base reaction, and then TEOS and APTES–Fc were usedas the precursor for the sol–gel polymerization to modulatematerial properties. The functional TEOS–APTES–Fc compos-ite could effectively prevent the leakage of the mediator andshowed high stability.

Graphene is now considered as a “rising star” material andhas received much popularity because of its unique physicaland chemical properties and potential applications in manytechnological fields such as nanoelectronics, nanocomposites,batteries, sensors, and solar cells [23–25]. In recent years,graphene-based analytical systems are a very active area.Graphene not only could act as an advanced support with verylarge surface area for immobilizing different targets, but couldalso effectively promote the electron transfer between electrodeand analytes. Particularly, in some cases, graphene-based elec-trodes have shown superior performance in terms of electrocat-alytic activity and macroscopic scale conductivity than CNT-based ones [26, 27]. The incorporation of graphene into nano-composites is an efficient avenue for broadening and enhancingthe function and performance of other functionalnanomaterials. GO, a water-soluble derivative of graphene, isof great importance because of its unique characteristics such asexcellent dispersibility, good biocompatibility, and facile sur-face functionality. Compared with pristine graphene, the cova-lent oxygenated functional groups in GO can indeed give rise toremarkable structure defects. This is concomitant with someloss in electrical conductivity, which possibly limits the directapplication of GO in electrically active materials and devices.

However, in recent years, it has become popular to exploit GO-based materials for electrochemical biosensing. Due to itsfavorable electron mobility and unique surface properties, suchas one-atom thickness and high specific surface area, GO canaccommodate the active species and facilitate their electrontransfer at electrode surfaces [28]. Herein, we incorporatedGO into ormosil matrices to form a novel multifunctionalnanocomposite, which could facilitate the electron transferamong the immobilized enzyme, mediator, and electrode.

In view of the advantageous features of Fc-conjugatedormosil, chitosan, and graphene, this work prepared a new typeof reagentless glucose biosensor based on the multifunctionalFc-conjugated ormosil/CS/GO nanocomposite material, as illus-trated in Scheme 1. As a common model enzyme, GOD isnegatively charged in physiological media; it can beimmobilized in the Fc-conjugated ormosil/CS/GOnanocompos-ite through both self-assembly method and sol–gel technologyto form a multicomponent nanocomposite on the electrodesurface. Such an electrochemical platform not only effectivelyprevented the leakage of both enzyme and mediator, affordedhigh enzyme loadings, and maintained the high biological ac-tivity of the immobilized GOD, but also provided an attractiveroute to promote the electron transfer between the enzyme andelectrode and therefore enhance reaction efficiency. Thus, theproposed biosensor exhibited excellent analytical performancesuch as sensitivity, precision, accuracy, response time, stability,and reproducibility towards the quantification of glucose.

Materials and methods

Reagents

Graphite flakes (99.99 %, 325 mesh) were purchasedfrom Alfa Aesar. Ferrocenecarboxaldehyde (Fc-CHO,98 %), glucose oxidase (GOD, from Aspergillus niger,EC 1.1.3.4. 150,000 units g−1), β-D(+)-glucose,3-(aminopropyl)triethoxysilane (APTES), sodiumcyanoborohydride (NaCNBH3, 95 %), and chitosan (85 %deacetylation) were purchased from Sigma-Aldrich (St. Louis,USA) and used as received. Tetraethyl orthosilicate (TEOS,analytical reagent) was purchased from Beijing ChemicalCorporation and distilled before used. Phosphate bufferedsolutions (PBS, 0.1 M) with various pH values were preparedby varying the ratio of KH2PO4 to Na2HPO4. All otherchemicals were of analytical grade and used without furtherpurification. All the solutions were prepared using doublydistilled water.

Preparation of CS solution

A 0.50 wt% CS stock solution was prepared by dissolving CSflakes in a hot (80 °C) aqueous solution of 0.05 M HCl. The

J Solid State Electrochem

solution was cooled down to room temperature, and the pHwasadjusted to 5.0 using NaOH solution. After precipitates werefiltered, the CS solution was stored in a refrigerator (4 °C) whennot in use.

Preparation of Fc-branched ormosil

Solution I was prepared by dissolving 21.4 mg Fc-CHO inmethanol (1 mL), and then 50 μL of APTES was added dropby drop under stirring and agitated for 6 h at room tempera-ture; we called solution I as APTES–Fc. Solution II wasprepared by stirring TEOS (280 μL), water (24 μL), andHCl (62 μL, 0.01 M) magnetically at ambient temperaturefor 5 h to hydrolyze the monomer [8]. Then, solution II(6.5 μL) was mixed with the solution I, and the mixture wasstirred at room temperature. After 6 h, NaCNBH3 was addedand the reactionmixture was continuously stirred for 24 h, andthe products were collected by centrifugation and washedalternately with methanol and water. The Fc-branched ormosilsol–gel was prepared and called TEOS–APTES–Fc.

Preparation of GO

Initially, graphite oxide was synthesized from graphite flake by amodified Hummer's method [29, 30]. In brief, 0.5 g of graphiteflake, 0.5 g of NaNO3, and 23 mL of H2SO4 were stirredtogether in an ice bath. While maintaining vigorous agitation,an amount of 3 g of KMnO4 was slowly added, and the rate ofaddition was controlled carefully to avoid a sudden increase intemperature. The mixture was then maintained at 35 °C forabout 1 h. Deionized water (40 mL) was gradually added,causing an increase in temperature to 90 °C. Finally, the mixturewas further treated with 100mL of deionized water and 3mL of30%H2O2, turning the color of the solution from dark brown to

yellow. Then, the obtained graphite oxide was subsequentlysonicated to obtain GO sheets after being dispersed in water.

Sensor construction

Glassy carbon electrode (GCE, diameter of 3 mm) was care-fully polished with 1.0, 0.3, and 0.05 μm alumina slurry,respectively, and rinsed thoroughly with doubly distilled waterbetween each polishing step. The electrode was successivelysonicated in 1:1 nitric acid, acetone, and doubly distilled waterand then allowed to dry under N2. Subsequently, 40 μL0.5 wt% CS and 100 μL TEOS–APTES–Fc were mixed with20 μL GO sheets (0.1 mg mL−1) with ultrasonic agitation over5 min. Then, 5 μL of the resulting homogeneous solution wasplaced on the surface of the clean GCE and allowed to dry for24 h to form a TEOS–APTES–Fc/CS/GO composite film. Forthe preparation of enzyme-incorporated composite film, a dif-ferent amount of GOD (150,000 units g−1 in pH 7.0 PBS) wasadded to the mixture before casting. The obtained film wasdenoted as GOD/TEOS–APTES–Fc/CS/GO. After, the com-posite film modified electrode was washed thoroughly withwater and then stored in air at 4 °C until use. The fabricationof the GOD/TEOS–APTES–Fc/CS/GOmodified electrode andthe redox enzyme catalytic cycle that gives rise to the current inthe presence of glucose is schematically shown in Scheme 1.

Apparatus

An Autolab PGSTAT30 (Eco Chemie) electrochemical sys-tem driven by GPES 4.9 software was employed for all theelectrochemical techniques. A three-electrode system wasused, where Ag/AgCl (CH Instruments, Inc.) served asreference electrode, a platinum wire as the auxiliary electrode,and TEOS–APTES–Fc/CS, TEOS–APTES–Fc/CS/GO, or

Scheme 1 Schematic representation of the GOD/TEOS–APTES–Fc/CS/GO modified electrode and the mechanism of the oxidation of glucose,catalyzed by GOD and mediated by Fc

J Solid State Electrochem

GOD/TEOS–APTES–Fc/CS/GO composite modified GCEas the working electrode. The electrolyte solutions werepurged with highly pure N2 for at least 20 min to remove O2

and kept under N2 atmosphere during the measurements. Allthe electrochemical experiments were carried out at roomtemperature (25±1 °C). The morphology of the GO nanosheetwas examined using a transmission electron microscope(TEM, Hitach-600). UV–vis absorption spectra were acquiredwith a UV-2450 spectrophotometer (Shimadzu). Fouriertransform infrared (FTIR) spectra were recorded on aNicolet 5700 FTIR spectrometer (Nicolet). X-ray powderdiffraction (XRD) patterns of the products were recorded witha Rigaku/Max-3A diffractometer with Cu Kα radiation (λ=0.15418 nm).

Results and discussion

Characterization of GO

The XRD patterns of the pristine graphite and graphite oxidewere collected (Fig. 1a). The diffraction peak at 2θ value of26.5° (curve a) in the XRD pattern of pristine graphite couldbe assigned to the (002) facet of the hexagonal crystallinegraphite [31]. Compared with the pristine graphite, the disap-pearance of the peak at about 26.5° and appearance of thepeak at 10.0° (curve b) revealed the successful oxidation of thestarting graphite. The increased interlayer spacing for graphiteoxide could weaken the van der Waals interactions betweenlayers and make facile exfoliation via sonication possible.TEM was then used to investigate the morphologies of theas-synthesized GO (Fig. 1b). It is clear that the GO sheetsappeared transparent and entangled with each other, indicatingthat the GO with two-dimensional structure was successfullysynthesized by ultrasonic treatment. To further demonstratethe formation of GO, UV–vis spectroscopy was carried out(inset of Fig. 1b). The two absorption peaks at 230 and 300 nmare characteristic of GO, originating from the π–π* transitionof the C=C band and n–π* transition of the C=O band,respectively, which confirmed that GO sheets were success-fully synthesized [32].

FTIR characterization

Figure 2 shows the FTIR spectra of CS (a), GO (b), Fc-CHO(c), TEOS–APTES (d), and TEOS–APTES–Fc (e), respec-tively. The FTIR spectra of CS (curve a) shows two bandslocated at around 1,654 and 1,596 cm−1 which could beassigned to the amino I and amino II functional groups ofthe native CS, respectively. The spectrum for graphene oxideexhibited the presence of O–H (υO–H at 3,443 and1,392 cm−1), C=O (υC=O at 1,737 cm−1), C=C (υC=C at1,622 cm−1), and C–O (υC–O at 1,083 cm−1) (curve b), which

was in good agreement with previous work [33]. Comparedwith the FTIR of Fc-CHO (curve c), typical bands around1,127, 1,028, 780, and 465 cm−1 associated with the silicanetworks of ormosil sol–gel were observed on curves d and e.The strong absorption bands in the range 1,000–1,200 cm−1

were ascribed to asymmetric stretching vibration of the Si–O–Si bond of the silica component, and the deformation vibrationat 465 cm−1. The peak at 780 cm−1 was associated with SiCH3

rocking vibration, and the presence of the weak N–H bendingvibration at 694 cm−1. In addition to these peaks, the frequen-cies at 1,640 and 1,570 cm−1 could be assigned to the amino Iand amino II functional groups of the APTES, respectively.Meanwhile, the bands at 2,927 and 2,863 cm−1 were assignedto CH stretching of the CH2 group. It is worth noting that thecharacteristic peak of Fc-CHO at 1,680 cm−1 (v, C–O) disap-pears in the TEOS–APTES–Fc spectrum (curve e) because ofthe consumption of the aldehyde groups of Fc-CHO in the

Fig. 1 a XRD patterns of (a) pristine graphite and (b) graphite oxide. bTEM image and UV–vis spectrum of GO

J Solid State Electrochem

synthetic process. Simultaneously, compared to the TEOS–APETS spectrum (curve d), the TEOS–APTES–Fc spectrum(curve e) displays a new absorption band at about 483 cm−1

attributed to M-ring stretch and ring tilt of ferrocenyls [34]. Inaddition, another new absorption band at about 814 cm−1 inthe spectrum also indicated that the ferrocenyls exist in theTEOS–APTES–Fc conjugate [35]. These results confirmedthat the TEOS–APTES–Fc conjugate was successfullysynthesized.

Electrochemical behavior of modified electrodes

The electrochemical behavior of the composite modified elec-trode was studied using cyclic voltammograms (CVs). Asshown in Fig. 3a, no redox peak at the TEOS–APTES/CSmodified electrode was observed (curve a), while the TEOS–APTES–Fc/CS modified electrode exhibited a pair of well-defined redox peaks with 0.244 and 0.333 V with a formalpotential about 0.29 V (curve b), indicating that Fc wascovalently bonded in TEOS–APTES. As expected, after ad-dition of GO sheets to the composite film, the anodic peak andcathodic peak currents of the TEOS–APTES–Fc/CS/GOmodified electrode increased (curve c), which was consistentwith our previous result [18]. The reason could be due to thefact that doped GO greatly improved the conductivity of thematrix film and facilitated the electron shuttle between themediator and electrode. Furthermore, with a continuous cyclicscan, the voltammetric response of the TEOS–APTES–Fc/CS/GO composite electrode was stable, indicating that thecovalently bounded Fc in the APTES chain could preventthe mediator from leaking and retain its electrochemical ac-tivity efficiently. The effect of varying the scan rate on theperformance of the TEOS–APTES–Fc/CS/GO modified elec-trode was also studied (Fig. 3b). With an increasing scan rate,the CV peak currents of the TEOS–APTES–Fc/CS/GO mod-ified electrode increased in the scan rate range of 10–200 mV s−1. The inset of Fig. 3 shows that both the anodicand cathodic peak currents increased linearly with the squareroot of scan rate (ν1/2) (inset in Fig. 3b), confirming that theredox behavior of the electrodes was diffusion controlled,where electron transferred to and from the redox centers ofthe TEOS–APTES–Fc involved diffusion [18].

Electrochemical response of the biosensor to glucose

The ability of the biosensor in the electrocatalytic oxidation ofglucose was evaluated by CVs. Figure 4 shows the CVs of theGOD/TEOS–APTES–Fc/CS/GO modified electrode withoutand with 13.0 mM glucose in 0.1 M PBS. In the absence ofglucose, the modified electrode exhibited a pair of reversibleredox peaks of Fc (curve a). Upon the addition of glucose tothe PBS solution, the anodic peak current increased notice-ably, while the cathodic peak current decreased, indicating an

Fig. 2 FTIR spectra of the CS (a), GO (b), Fc-CHO (c), TEOS–APTES(d), and TEOS–APTES–Fc (e)

Fig. 3 a CVs of TEOS–APTES/CS (a), TEOS–APTES–Fc/CS (b), andTEOS–APTES–Fc/CS/GO (c) modified electrodes in 0.1 M pH 7.0 PBSat 100 mV s−1. b CVs of TEOS–APTES–Fc/CS/GO modified GCE in0.1 M pH 7.0 PBS at 10, 30, 50, 70, 100, 120, 150, 170, and 200 mV s−1

(from internal to external). Inset: plot of cathodic peak current and anodicpeak current vs. scan rate

J Solid State Electrochem

obvious electrocatalytic oxidation of glucose at the GOD/TEOS–APTES–Fc/CS/GO modified electrode (curve b).This typical enzyme-dependent catalytic process shown inScheme 1 can be expressed as follows [36]:

Glucoseþ GOD FADð Þ→Gluconolactoneþ GOD FADH2ð Þð1Þ

GOD FADH2ð Þ þ 2TEOS� APTES� Fcþ→GOD FADð Þþ2TEOS� APTES� Fcþ 2Hþ

ð2Þ

TEOS� APTES� Fc→TEOS� APTES� Fcþ þ e− ð3Þ

Optimization of measurement variables

The concentration of enzyme in the composite was an impor-tant factor affecting the amperometric response of the biosen-sor. As shown in Fig. 5a, with increasing of enzyme level onthe electrode surface up to 0.6 IU per electrode, the catalyticcurrent increased, and the response then reduced slowly due tothe increased protein concentration in the composite, whichmight be due to the fact that the increased protein concentra-tion decreased the conductivity of the composite film. Hence,0.6 IU GOD was selected for preparation of the glucosebiosensor.

The pH of the working solution may affect the life time,linear range, and detection limit of the biosensor as theimmobilized enzyme activity would be affected by the pH ofthe working solution and the higher or lower pH woulddestroy the microstructures of enzyme. The optimal pH re-ported for GOD is usually in the range of 6.5–7.5 [19, 37],

which varies with the immobilization method and microenvi-ronment around the enzyme. We explored the effect of pHbetween 5.6 and 9.2 (Fig. 5b); the present biosensor showed amaximum response at pH 7.0 PBS, which is in good agree-ment with the previous studies [14, 38].

Analytical performance of biosensor

Figure 6 shows a typical current–time response on the succes-sive addition of glucose under the optimized experimentalconditions. It was observed that the current value reached95 % steady-state responses within 5 s, indicating a fastresponse. Such a short response time further proved that theTEOS–APTES–Fc/CS/GO composite was a promising plat-form for the construction of biosensors. The amperometricresponse increased linearly to glucose concentration in therange from 0.02 to 5.39 mM with a detection limit of6.5 μM (S/N=3) (inset in Fig. 6). A substantially higher

Fig. 4 CVs of the GOD/TEOS–APTES–Fc/CS/GO film electrodes inthe absence (a) and the presence (b) of 13.0 mM glucose in PBS (pH 7.0)at 100 mV s−1

Fig. 5 a Influence of enzyme amount on electrode surface on ampero-metric response of biosensor in 0.1 M pH 7.0 PBS containing 5.0 mMglucose at +400 mV. b Effect of buffer pH on amperometric response of5.0 mM glucose at +400 mV

J Solid State Electrochem

sensitivity (19.5 μA×10−3 M−1 cm−2) was obtained with thisGOD/TEOS–APTES–Fc/CS/GO film as compared with otherglucose biosensors [16, 18, 34, 39]. At high glucose concen-tration, the calibration curve showed aMichaelis–Menten typeresponse. The apparent Michaelis–Menten constant (KM

app),which gives an indication of the enzyme-substrate kinetics,can be obtained from the Lineweaver–Burk equation [40].The KM

app value for this biosensor was calculated to be4.21 mM, which was much lower than those reported values,such as for the GOD in solution (14.4 mM) [41], for GODentrapped in sol–gel/chitosan composite (21 mM) [16], for theGOD immobilized in MWNTs/CS–Fc film (6.87 mM) [42],and the surface of nano-CaCO3 (21.4 mM) [43]. Low KM

app

value indicated that the enzyme immobilized in TEOS–APTES–Fc/CS/GO composite film retained its activity witha low diffusion barrier. The data in Table 1 revealed that thisbiosensor exhibited an excellent combination of the fast re-sponse time, high sensitivity, low detection limit, and lowKMapp. The excellent performance of the enzyme electrode

could be attributed to the high enzyme loadings due to thethree-dimensional porous structures of the composite film andthe biocompatible microenvironment around the enzyme.More importantly, the TEOS–APTES–Fc/CS/GO compositefilm effectively prevented the leakage of both enzyme andmediator. Meanwhile, the nanoscale individual GO sheetsacted as “molecular wires” to connect the active sites ofGOD and electron mediator Fc with the electrode, increasingthe electron transfer rate significantly [18].

Reproducibility and stability of biosensors

To prove the precision and practicability of the proposedmethod, the storage stability and reproducibility of the bio-sensor were also examined. The relative standard deviation(RSD) of the biosensor response to 1.0 mMglucose was 3.7%for 12 successive measurements. The RSD for five sensorsprepared using the same condition response to 5.0 mM glu-cose was 4.5 %. The biosensor was stored dry at 4 °C andmeasured at intervals of a week. After 1 week, the biosensorretained 90 % of its original sensitivity. After 1 month, thebiosensor still retained 83 % of its initial sensitivity. However,the biosensor without CS in the matrix only retained 82 and71 % of its original sensitivity after 1 week and 1 month,respectively. The excellent reproducibility and stability of thebiosensor can be attributed to the formation of the TEOS–APTES–Fc/CS/GO composite, which not only prevents leak-age of the mediator from the GOD/TEOS–APTES–Fc/CS/GO film structure efficiently, but alsomakes the matrix exhibita positive charge, which is more favorable for the furtherimmobilization of negatively charged GOD withoutdestructing its native structure and bioactivity.

Study on the interference

One of the most important analytical factors for an ampero-metric biosensor is the ability of the sensor to discriminate the

Fig. 6 Typical amperometric response of the biosensor to successiveaddition of glucose into stirred PBS at +400 mV. Inset: calibration curve

Table 1 Comparison of the performance of the present sensor and others reported in the literatures for glucose detection

Film Responsetime (s)

Linear range (mM) Detectionlimit (μM)

Sensitivity (μA×10−3 M−1 cm−2) KMapp (mM) References

GOD/ormosil/CS – 2.0–14 2.0 0.27 21 [16]

GOD/CS–Fc/GO <5 0.02–6.78 7.6 10 2.1 [18]

FMC–BSA/MWNTs/ormosil <15 0.06–8.0 20.0 – 6.6 [21]

CHIT–Fc/GOD <30 0.2–7.0 16 18 – [33]

(GOD/GNP/CS)6/GOD/GNP/PAA <8 0.5–16 7.0 – 10.5 [37]

GOD/MWNTs/CS–SiO2 sol–gel <5 0.001–14 1.0 – 14.4 [39]

GOD/CS–Fc/MWNTs <10 0.02–5.36 6.5 21.94 6.87 [40]

GOD/nano-CaCO3 <6 0.001–12 0.1 – 21.4 [41]

GOD/TEOS–APTES–Fc/CS/GO <5 0.02–5.39 6.5 19.5 4.21 This work

CS chitosan, FMC ferrocenemonocarboxylic acid, BSA bovine serum albumin, PAA poly(allylamine)

J Solid State Electrochem

interfering species having electroactivities similar to the targetanalyte. Ascorbic acid (AA) and uric acid (UA) are the mostcommon interfering electroactive species for the amperomet-ric detection of glucose. The presence of UA did not cause anyobservable interference, while the AA caused a few interfer-ences to the modified electrode, which can be avoided bycoating a selective Nafion film on the GOD/TEOS–APTES–Fc/CS/GO modified GCE [19].

Conclusions

In conclusion, a novel glucose biosensor by integratingTEOS–APTES–Fc/CS/GO nanocomposite as an ideal con-ductive platform for the enzyme immobilization was designedthrough both sol–gel technology and self-assembly method.The proposed GOD/TEOS–APTES–Fc/CS/GO compositefilm not only provided a very suitable environment for en-zyme entrapment and afforded high enzyme loadings, but alsoprevented the leakage of both enzyme and mediator.Furthermore, the GO in the composite film played an impor-tant role similar to a conducting wire, which made it easier forthe electron transfer to take place. The resulted reagentlessbiosensor had excellent analytical performance and stability.Therefore, such a multicomponent platform, which integratesthe advantages of GO, Fc-branched ormosil, and GOD togeth-er, can be extended to immobilize other mediators and bio-molecules and may have promising potential for the fabrica-tion of high-performance flexible biosensors or bioreactors.

Acknowledgments We gratefully acknowledge the financial support ofthe National High Technology and Development of China (863 Project:2012AA022604), the National Natural Science Foundation of China(21275028), the Research Fund for the Doctoral Program of HigherEducation of China (20123518110001), the Fujian Provincial ImportantScience and Technology Foundation (2011R1007-2), Sponsored byMed-ical Elite Cultivation Program of Fujian, P.R.C (2013–ZQN–JC–25), thescience and technology plan project of General Administration of QualitySupervision (2014IK060), the science and technology plan project ofFujian Entry-Exit Inspection and Quarantine Bureau (FK2012-01), Foun-dation of Fujian Provincial Department of Education (JA10126,JA11110, JA12130), and the Scientific Research Foundation for DoctoralProgram of Fujian Medical University (2011BS005).

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