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Electrochimica Acta 90 (2013) 203– 209

Contents lists available at SciVerse ScienceDirect

Electrochimica Acta

jou rn al hom epa ge: www.elsev ier .com/ locate /e lec tac ta

ydrophobic ionic liquid immoblizing cholesterol oxidase on thelectrodeposited Prussian blue on glassy carbon electrode for detectionf cholesterol

iuhui Liu ∗, Zhihan Nan, Yu Qiu, Lichun Zheng, Xiaoquan Lu ∗∗

ey Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070,hina

r t i c l e i n f o

rticle history:eceived 25 August 2012eceived in revised form5 November 2012ccepted 28 November 2012vailable online 5 December 2012

a b s t r a c t

A novel cholesterol biosensor was fabricated on hydrophobic ionic liquid (IL)/aqueous solution interface.The hydrophobic IL thin film played a signal amplification role because it not only enriched the cholesterolfrom the aqueous solution, but also immobilized matrix for cholesterol oxidase (ChOx). Prussian blue (PB)as advanced sensing materials was used as effective low-potential electron transfer mediation towardhydrogen peroxide (H2O2). The fabricated IL-ChOx/PB/Glassy carbon electrode (GCE) was characterized

eywords:onic Liquidholesterol oxidaseholesterolrussian blue

by electrochemical impedance spectroscopy (EIS) and cyclic voltammogram (CV), respectively. And itexhibited a linear response to cholesterol in the range of 0.01–0.40 mM with a detection limit of 4.4 �M.In addition, the kinetics behavior of cholesterol at IL-Chox/PB/GCE electrode was examined, and theelectrocatalytic mechanism was proposed as shown in first scheme. ChOx immobilized in hydrophobicIL thin film was used as the first electrocatalyst for the cholesterol into H2O2, and the PB film onto theGCE was used as the second electrocatalyst for the 2e− reduction of the produced H2O2 into H2O.

iosensor

. Introduction

The alarming rise in the rate of clinical disorders such as heartisease, hypertension, arteriosclerosis, coronary artery disease,erebral thrombosis, etc. due to abnormal levels of cholesteroln blood have stimulated public concern about the determina-ion of cholesterol level [1]. Meanwhile, the importance of theetermination of cholesterol has been reflected in recent years byn increase in the number of articles about the development ofholesterol biosensors [2]. In the fabrication of a cholesterol biosen-or, cholesterol oxidase (ChOx) is most commonly used as theiosensing element [3]. But the oxidation of H2O2 usually requires

high anodic potential (usually over +0.6 V, vs SCE) that maynduce simultaneous oxidation of other electrochemically activepecies presented in samples and lead to false positive signals [4].hus, it was expected that an additional electrocatalytic layer ofrussian blue (PB) placed on surface of electrode prior to Choxmmobilization would allow one to detect cholesterol at lower

otential (about +0.1 to −0.1 V) [5–7]. Many papers have demon-trated the PB-modified electrode exhibited high activity andelectivity in detection of hydrogen peroxide through its catalytic

∗ Corresponding author. Tel.: +86 0931 7970565; fax: +86 0931 7971323.∗∗ Corresponding author. Tel.: +86 0931 7971276; fax: +86 0931 7971323.

E-mail addresses: [email protected] (X. Liu), [email protected] (X. Lu).

013-4686/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.electacta.2012.11.119

© 2012 Elsevier Ltd. All rights reserved.

electroreduction [8–15]. However, the reference of PB enzymaticsensor for cholesterol detection was seldom reported, there are stillsome developmental challenges to be addressed. The main chal-lenge is developing suitable support matrix that provides betterenvironment for the efficient enzyme loading and maintaining ofthe enzymatic bioactivity so as to increase the sensitivity of thebiosensor [16–26].

Recently, the emerging ionic liquids (IL) material may offer greatopportunities to solve above problem because of the unique prop-erties of IL such as negligible vapor pressure, high thermal stabilityand viscosity, and good conductivity and solubility. Among these,the viscosity of the IL is an important consideration in electrochem-ical studies due to its strong effect on the rate of mass transportwithin solution [27]. In addition, it is also related to whether thedroplet of IL will hang up on the electrode surface. It was found thatonly few kind lipases could maintain their activity in ionic liquids of1-butyl-3-methylimidazolium bis[(trifluoromethyl)sulfonyl]imide([BMIM][NtF2]) [28,29]. Recent implementations suggested thatthe hydrophobic anion [NtF2]− can significantly improve the cat-alytic activity for H2O2 electrochemical oxidation/reduction andlower its overvoltage, which are beneficial in fabricating biosen-sors with high sensitivity and selectivity. Herein, the present paper

describes a cholesterol biosensor prepared by immobilization ofChOx in the hydrophobic ionic liquid 1-octyl-3-methylimidazoliumbis(trifluoromethylsulfonyl)imide([OMIM][NtF2]) on the top of PBmodified electrodes. Scheme 1 shows a multistep system for

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204 X. Liu et al. / Electrochimica A

St

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cheme 1. Schematic illustration of multi-step reactions between choles-erol/enzyme/electrode at the biosensor.

holesterol oxidation on the modified electrode. As shown in thecheme 1, the PB electrodeposited on the GCE is used as effec-ive low-potential electron transfer mediation toward H2O2, andhe hydrophobic IL plays the role of being the matrix immobiliz-ng ChOx and keeping the PB film stable. Our strategy is that ChOxmmobilized in hydrophobic IL thin film is used as the first elec-rocatalyst for the cholesterol into H2O2, and the PB film onto theCE serves as the second electrocatalyst for the 2e− reduction of

he produced H2O2 into H2O. Herein, there is the linear relationshipetween the concentration of the cholesterol and the value of theB redox peak current. Our results found that the immobilizationf enzyme with high activity in the hydrophobic ionic liquid matrixas made this technique to be a potential tool for developing newiosensors.

. Experimental

.1. Apparatus

Cyclic voltammetry experiments were performed on a CHI660Clectrochemical workstation (Austin, TX, USA) controlled by aicrocomputer with CHI660 software. A three-electrode systemas used, where a glass carbon electrode (GCE, 3.5 mm diameter)

r a ChOx-PB modified GCE served as the working electrode, a Ptire as the counter electrode and a SCE as reference electrode.

lectrochemical impedance (EI) experiments were performed on multi-channel electrochemical workstation (American Princetonnstruments Corporation).

.2. Reagents

The 1-octyl-3-methylimidazolium trifluoromethylsulfonate[OMIM][NtF2]) came from Shanghai Chengjie Chemistry Co. Ltd.Shanghai, China). Cholesterol oxidase (ChOx, EC 1.1.3. 6,15 U/mg)as purchased from Shanghai source leaf biological technology Co.

td. (Shanghai, China). Cholesterol (Tianjin Guangfu Fine Chemicalesearch Institute, Tianjin, China), Triton X-100 (Aladdin Chem-

stry Co. Ltd., Shanghai, China), isopropanol (Shanghai Zhongqinhemistry Co. Ltd., Shanghai, China), potassium ferricyanide and

cta 90 (2013) 203– 209

potassium chloride (Xi’an Chemical Reagent Factory, Xi’an, China)were of analytical reagent grades.

2.2.1. Preparation of cholesterol solutionIt is important to make the homogenous cholesterol solutions

because it is sparingly soluble in water. Non ionic surfactant Tri-ton X-100 and isopropanol were found to be effective solubilizingagents for cholesterol [30]. For preparing cholesterol stock solutionof 5 mmol/L, 0.04835 g cholesterol was dissolved in a 25 mL vol-umetric flask containing a mixture of 1 mL isopropanol and 1 mLTriton X-100 in a bath at 60 ◦C, and then was diluted with distilledwater. The cholesterol solution was stored at 4 ◦C.

2.2.2. Preparing of cholesterol oxidase-ionic liquid stock solutionThe stock cholesterol oxidase-ionic liquid solution was pre-

pared using a procedure by mixing 5 mg cholesterol oxidase (ChOx)with 200 �L 1-Octyl-3-methylimidazolium trifluoromethylsul-fonate (IL), The mixture was then ultrasonically treated for 30 minat room temperature until a stable solution was obtained. Thissolution was used throughout the experiment to prepare ChOx-ILmodified film.

2.3. Construction of IL-ChOx/PB/GCE electrode

The GCE was pretreated as following: mechanical polishing inmicrocloth pads with 0.05 �m alumina slurry, and then ultrasoni-cated in ethanol and doubly distilled water for 15 min. Finally theelectrode was washed with water, and dried at room temperature.Deposition of the PB was accomplished in a solution of 10 mmol/LK3[Fe(CN)6] +10 mmol/L FeCl3 + 0.1 mol/L KCl. A Prussian blue (PB)film was formed by applying a constant potential of 0.0 V to theGCE for 60 s, and then scanning 10 cycles at a sweep rate of 50 mV/sbetween 0.0 and 0.5 V. The PB modified electrode was electrochem-ically pretreated in 0.1 mol/L phosphate buffer containing 0.1 mol/LKCl (pH 6.8) by keeping at 0.0 V for 30 s and cycling between 0.5 and0.0 V for 10 cycles. The treaded electrode was washed with waterand dried under infrared lamp for 30 min. 3 �L of stock cholesteroloxidase-ionic liquid solution was cast onto the electrode surface. Allthe experiments were carried out at ambient temperature (about25 ◦C). The potentials recorded are vs. SCE.

3. Results and discussion

3.1. Monitoring the preparation of IL-ChOx/PB/GCE

3.1.1. The stability of the PB filmFrom the electrodeposited method [31]:

Fe4[Fe(CN)6]3 + 4K+ + 4e− ↔ K4Fe4[Fe(CN)6]3 (1)

we obtained the PB film as shown in Fig. S1 of SupportingInformation. A couple of well defined cathodic and anodic peakscorresponded to the reduction of PW and the oxidation of PB,respectively. The molecular structures of the compounds of PB andPW are shown in Scheme 2. In addition, Fig. 1A is the CV of the PBfilm scanned for 5 cycles in pH 6.8 phosphate buffer solution con-taining 0.1 M KCl. The peak currents of the Prussian blue obviouslydecreased with increase of scanning laps, illuminating that con-fined the PB film only on the GCE was unstable. But when thehydrophobic ionic liquid thin film cast onto the PB films, the peak

currents of the PB remained the same basically with the increase ofscanning laps as shown in Fig. 1B, indicating that the hydrophobicionic liquid film plays a crucial role of isolating and protecting PBfilm.

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X. Liu et al. / Electrochimica Acta 90 (2013) 203– 209 205

N)6]3)

3

smar

Fm

Scheme 2. (A) The molecular structure of the compound of PB (Fe4[Fe(C

.1.2. Electrochemical characterization of the IL-ChOx/PB/GCECVs of bare and modified GCE electrodes were obtained as

hown in Fig. 2 in pH 6.8PBS containing 0.1 M KCl in order to

onitor changes in their electrochemical behavior introduced

t different steps of electrode modification. No electrochemicalesponse was observed at the bare GCE in Fig. 2a. Whereas, when

ig. 1. CVs of the Prussian blue film modified GC electrode (A) and Prussian blue-ILodified GC electrode (B) in 0.1 M KCl pH 6.8 PBS for 5 cycles. Scan rate 50 mV/s.

. (B) The molecular structure of the compound of PW(K4 Fe4[Fe(CN)6]3).

the electrode was electrodeposited with PB film, CV of PB/GCE elec-trode showed a couple of redox peaks at about 0.3 V, correspondingto the reduction of Prussian white (PW) at 0.199 V and the oxida-tion of PB at 0.329 V, respectively, in Fig. 2b. After that, when theIL was cast onto the PB film, a quasireversible redox peak with thepeak-to-peak separation (�E) of 0.13 V was obtained as shown inFig. 2c. It is noted that both the cathodic and anodic peak currentsincreased sharply as compared to that in Fig. 2a and b, illuminatingthat IL could promote the electron transfer rate and enhance thereversibility of the redox of PB. Furthermore, Fig. 2d is the CV curveof ChOx immobilized in IL/PB/GCE, similar to that obtained fromIL/PB/GCE electrode except a little increase in the reduction peakcurrent.

Electrochemical impedance spectroscopy (EIS) can also provideinformation on the impedance changes of the electrode surfaceduring the modified process. In the electrochemical impedancespectroscopy, a semicircle portion observed at higher frequencieswould correspond to the electron-transfer-limited process, and alinear section characteristic of the lower frequency is attributableto a diffusion-limited electron transfer. The value of the electron-

transfer resistance (Ret) at the electrode surface can be estimateddirectly from the diameters of the high frequency semicircl, whichdepends on the dielectric and insulating features at the elec-trode/electrolyte interface. Using [Fe(CN)6]3−/4− redox couples as

Fig. 2. Cyclic voltammograms of 0.1 M KCl pH 6.8 PBS at (a) GCE, (b) PB/GCE, (c)IL/PB/GCE and (d) IL-ChOx/PB/GCE; scan rate 50 mV/s.

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206 X. Liu et al. / Electrochimica Acta 90 (2013) 203– 209

FIi

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3

3

puCrntar

C

O

P

P

twidH

Fig. 4. CVs obtained at the PB modified GCE (the red lines) in phosphate bufferwith 0.1 mol/L KCl and 1% Triton X-100 saturated with N2 (dashed line) or underambient air (solid line) and at the IL-ChOx/PB/GCE (the black lines) in 1 × 10−5 mol/L

ig. 3. Electrochemical impedance spectra obtained at (a) GCE, (b) PB/GCE, (c)L/PB/GCE, (d) IL-ChOx/PB/GCE in 5 mM K3Fe(CN)6/K4Fe(CN)6 (1:1) and 0.1 M KCln pH 6.8 PBS.

he electrochemical probe, the EIS plots of different modified elec-rodes are shown in Fig. 3, and the inset is the fits of equivalentircuit. The impedance spectra of the bare GCE (Fig. 3a) consistedf a small semicircle (Ret: 100 �) with an almost straight tail line,hich was the characteristic of a diffusion limiting step of the elec-

rochemical process. When the GCE electrode surface was coatedy PB film, the Ret value of Fig. 3b increased to about 450 �. This

mplies that the electrodepositing PB film generated an obstructiono the electron transfer of the electrochemical probe at electrodeurface. After the ionic liquid coated on the PB film, the diameter ofhe high frequency semicircle was significantly reduced with a Ret

alue of 250 � as shown in Fig. 3c, implying that ionic liquid maylay an important role in accelerating the electron transfer pro-ess. When the cholesterol oxidase-ionic liquid stock solution castnto the PB modified electrode, the diameter of the high frequencyemicircle significantly decreased to close to zero. These results areonsistent with that of CV experiments (Fig. 2).

.2. The investigation of effect factors on the IL-ChOx/PB/GCE

.2.1. The influence of the dissolved oxygen in cholesterol solutionThe cholesterol solution was deoxygenated by bubbling high-

urity N2 for at least 20 min and the experiments were undertakennder N2 atmosphere. As shown in Fig. 4, the black lines are theVs of the IL-ChOx/PB/GCE electrode in cholesterol solution satu-ated with N2 (dashed line) or under ambient air (solid line). It wasoted that the reduction peak current in the dashed line is smallerhan that of in the solid line. This may be ascribed to O2 influencend the possible reaction mechanism is proposed in the followingeactions:

hOx(FAD)+cholesterol→cholester-4-en-3-one+ChOx(FADH2)(2)

2 + ChOx(FADH2) → ChOx(FAD) + H2O2 (3)

W + 12

H2O2 → PB + H2O (4)

We−�PB + K+ (5)

As illustrated in Scheme 1, the cholesterol was first adsorbedo hydrophobic ionic liquid/aqueous solution interface and reacted

ith the cholesterol oxidase, which used as the first electrocatalyst

n reaction (2). Then the produced ChOx(FADH2) reacted with theissolved oxygen subsequently in reaction (3), and the produced2O2 would diffuse into the PB film coated on the GCE electrode.

cholesterol with 0.1 mol/L KCl and 1% Triton X-100 pH 6.8 PBS saturated with N2

(dashed line) or under ambient air (solid line). (For interpretation of the referencesto color in figure legend, the reader is referred to the web version of the article.)

It is very clearly to see in Scheme 2, Prussian white (PW) (K4Fe4[Fe(CN)6]3), oxidized form of Prussian blue (PB) (Fe4[Fe(CN)6]3)at the GCE electrode surface, used as the second electrocatalyst forreduction of H2O2 into H2O in reaction (4). Thus, when the exper-iment was undertaken under N2 atmosphere, the decrease of theO2 concentration induced the decrease of the produced H2O2, cor-responded to the smaller reduction peak current (dashed line inFig. 4, black).

To clarify this further, the CVs of PB film modified GC electrodewere performed in 0.1 mol/L KCl pH 6.8 PBS and 1% Triton X-100 asdepicted the red lines in Fig. 4. One can see that both the currentsand the potentials of the reduction peak show no change in sat-urated with N2 (dashed lines) or under ambient air (solid lines),which are different from the results obtained from the black lines.This indicated that dissolved oxygen in the solution has no influ-ence to the PB film modified electrode because the peaks currentin the red lines are ascribed to the reduction reaction of Prussianwhite (PW) occurred as shown in reaction (5) only.

3.2.2. Effect of buffer pHThe effect of buffer pH on the response for cholesterol was

investigated and results were shown in Fig. S2 of the SupportingInformation, in which the optimized pH of phosphate buffer was6.8. Normally, PB film is stable in acidic and neutral solution, butunstable in alkaline solution. Because ferric ions are known to behydrolyzed easily, and the hydroxyl ions (OH−) cannot be substi-tuted in their coordination sphere in course of PB crystallization.So it is beneficial to choose pH 6.8 buffer solution in the wholeexperiments.

3.3. Detecting cholesterol using the IL-ChOx/PB/GCE

Fig. 5 is CV curves of the IL-Chox/PB/GCE electrode at differ-ent concentrations of cholesterol. The linear relationship couldbe established between the peak currents and the concentrationsof cholesterol in the range of 10–400 �M. The sensitivity, cal-

culated from the slope of the plot (Fig. 5, Ipa(�A) = 0.15 + 0.04c(10−5 M)), was found to be 0.4 �A/�M. The detection limit (DL)for the constructed electrode, calculated from the expressionDL = (3 × SD)/sensitivity (where SD is the estimated standard

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X. Liu et al. / Electrochimica Acta 90 (2013) 203– 209 207

Table 1Comparison of different modified electrodes for cholesterol determination.

Electrode Line range Detection limit Reference

C/Fc-copolymer/HPR/ChOx 2–10 mM [36]CPE/hydroxymethylferrocene/HRP/ChOx 1 × 10−3–0.15 mM [37]Pt/nafion/cellulose/ChOx 5–100 mg/L [38]Pt/PPy/ChOx 0.025–0.3 mM 5.7 �M [39]W/ferrocyanide/ChOx 0.05–3 mM 0.01 mM [40]MWCNTs-AuNPs-CHIT-IL 0.5–5 mM [6]AuNPs 0.01–0.07 mM 10 mM [25]AuE/dithiol/AuNPs/MUA/ChOx 0.04–0.22 mM 34.6 �M [41]

dttisp

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wcstataKs[

Fc5Tr

ChOx-IL/PB 0.01–0.4 mM

eviation for the points used to construct the calibration curve andhe sensitivity, its slope), was found to be 4.4 �M. The results forhe determination of cholesterol using different methods are listedn Table 1, from where one can conclude that the proposed biosen-or has a higher sensitivity and a lower detection limit than thoserevious reported models.

The variation tendency of catalytic currents and the cholesteroloncentrations is shown in Fig. S3 of the Supporting Information.he reduction peak currents gradually increased with increas-ng concentrations of cholesterol. But when the concentrations ofholesterol came to 2 × 10−4 M, the increased trend became slow.he maximum catalytic current was obtained at the concentrationsf cholesterol about 4 × 10−4 M. The apparent Michaelis–Mentenonstant Km, which gives an indication of the enzyme-substrateinetics, can be obtained from the Lineweaver–Burk equation [32].

1Iss

= 1Imax

+ Km

Imax× 1

c(6)

here c is the concentration of cholesterol in solution, Iss is theatalytic reduction current (background current deducted) at theteady state when cholesterol concentration is at c, and Imax ishe maximum catalytic current. The Km value for the electrocat-lytic activity of IL-ChOx/PB/GCE to cholesterol was determinedo be 0.116 mM, implying that the present electrode exhibited

higher affinity for cholesterol. As well known, the smaller of

m, the higher catalytic ability of the cholesterol biosensor pos-esses. Compared with those reported in cholesterol biosensors33,34], the lower value of Km indicated that cholesterol oxidase

ig. 5. Cyclic voltammetric measurements with IL-ChOx/PB/GCE in various con-entrations of cholesterol pH 6.8 PBS solution saturated with air: 1 × 10−5, 3 × 10−5,

× 10−5, 7 × 10−5, 9 × 10−5, 1 × 10−4, 2 × 10−4, 4 × 10−4 mol/L from inner to outer.he inset is the calibration curve corresponding to amperometric responses. Scanate: 50 mV/s.

4.4 �M This paper

immobilized in hydrophobic ionic liquid film retained its bioactiv-ity and had a high biological efficiency to cholesterol.

Some possible interfering species, such as ascorbic acid,dopamine and uric acid were also investigated. CVs were takenin PBS containing 0.05 mM cholesterol and each interferences(0.05 mM). The results are shown in Fig. S4 of the SupportingInformation, where Ic+i and Ic are modified electrodes responseat 0.10 V for 0.05 mM cholesterol in the presence and absence ofeach interferences, respectively. We found that the value of Ic+i wasalmost equal to the value of Ic, indicating that AA, UA, and DA did notproduce significantly influence in the determination of cholesterol.So the biosensor we fabricated has high anti-interference ability.

The feasibility of the device for practical applications wascarried out by analyzing the 1% human serum. The cholesterolconcentration in the human serum sample was determined tobe 4.62 ± 0.04 mM (n = 3) by CV measurement, which is in therange of 2.85–5.98 mM, collected from the Chinese people’s serumcontaining the cholesterol published by Chinese PharmaceuticalAssociation.

3.4. Comparing the kinetics of cholesterol at IL-ChOx/PB/GCEelectrode with H2O2 at IL/PB/GCE electrode

Fig. 6A shows cyclic voltammograms of H2O2 at IL/PB/GCEelectrode with different scan rates (v). A pair of roughly symmet-ric anodic and cathodic peaks appeared with almost equal peakcurrents in the scan rate range from 0.01 to 0.30 V/s. The peak-to-peak separation also increased slightly with the scan rate. Inthese scan rates range, a good linear relationship was found for thepeak currents and scan rates, with the results as shown in Fig. 6C(Ipa(�A) = 8.938 + 394.9� (V/s) (r = 0.9925)) which was characteris-tic of quasi-reversible surface controlled electrochemical behavior.However, a linear behavior of the peak current versus the squareroot of scan rate was obtained when the scan rate was higher than0.10 V/s as shown in Fig. 6D, revealed a diffusion controlled mech-anism. Another similar experiment was performed for cholesterolat IL-ChOx/PB/GCE electrode. As shown in Fig. 6B and E, one cansee that there was always an ideal linear relationship for the peakcurrents and scan rates (Ipa(�A) = 5.50 + 59.9� (V/s) (r = 0.9999)) inthe scan rate range from 0.01 to 0.30 V/s which was the charac-teristic of adsorption controlled electrochemical behavior. Aboveexperiments demonstrated that cholesterol in the solution tendsto absorb to the aqueous solution and hydrophobic ionic liquidinterface, but H2O2 would like to stay in the aqueous solution.

With the increase of the scan rate, the peak-to-peak separation(�Ep) was also increased gradually as shown in Fig. 6A and B, so theelectrochemical parameters could be calculated using the method

developed by Laviron [35] as follows:

Epc = E0′ − RTln �

˛nF(7)

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208 X. Liu et al. / Electrochimica Acta 90 (2013) 203– 209

Fig. 6. (A) Cyclic voltammograms of 1.0 × 10−3 M H2O2 in pH 6.8 buffer solution with different scan rate at IL/PB/GCE. Scan rate: 0.30, 0.27, 0.24, 0.21, 0.18, 0.15, 0.12, 0.09,0.07, 0.05, 0.03, 0.01 V/s. Linear relationship of (C) Ip vs. v (v: 0.01–0.09 V/s) and (D) Ip vs. v1/2 (v: 0.12–0.30 V/s); (B) cyclic voltammograms of 1.0 × 10−5 M cholesterol in pH6 , 0.24,v

E

l

wfiptb

.8 buffer solution with different scan rate at IL-ChOx/PB/GCE. Scan rate: 0.30, 0.27 (v: 0.01–0.30 V/s).

pa = E0′ + RTln �

(1 − ˛)nF(8)

og ks = ̨ log(1 − ˛) + (1 − ˛) log ˛

− log(

RT

nF�

)− ˛(1 − ˛)nF�Ep

2.3RT(9)

here ̨ is the electron transfer coefficient, n is the electron trans-er number, ks is the apparent electron transfer rate constant, R

s the gas constant, T is the absolute temperature, and �Ep is theeak-to peak separation. So ̨ of H2O2 is calculated as 0.3550, andhe apparent electron-transfer rate constant (ks) is estimated toe 0.3408/s. Similarly, ̨ and the apparent electron-transfer rate

0.21, 0.18, 0.15, 0.12, 0.09, 0.07, 0.05, 0.03, 0.01 V/s. Linear relationship of (E) Ip vs.

constant (ks) of cholesterol are calculated as 0.1432 and 0.5871/s,respectively, revealing that the efficient enzyme loading and main-taining of the enzymatic bioactivity in ionic liquids of [BMIM][NtF2]would enhance the electrochemical reaction rate, and improve thesensitivity of the biosensor.

4. Conclusions

A novel scheme for the fabrication cholesterol biosensor wasdeveloped using electrodepositing PB film as electron-mediatorand hydrophobic ionic liquid as matrices for the assembly of inte-grated electrode. The hydrophobic ionic liquid film was found to

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nhance the current response of the fabricated bioelectrode androvide a biocompatible environment for the ChOx. ChOx immobi-

ized in hydrophobic IL thin film was used as the first electrocatalystor the cholesterol into H2O2, and the PB film onto the GC electrodeas used as the second electrocatalyst for the 2e− reduction of theroduced H2O2 into H2O. In addition, the fabricated bioelectrodeisplayed a series of excellent features such as good sensitivity,ide linear range, and low detection limit, which might pave aew and cost-effective way for biomonitoring of cholesterol both

n methodological study and in clinical laboratories.

cknowledgment

This work was supported by the National Natural Science Foun-ation of China (Nos. 21245004, 20875077)

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/j.lectacta.2012.11.119.

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