Electrochimica Acta 90 (2013) 203209

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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta

Hydrophobic ionic liquid immoblizing cholesterol oxidase on the

electrodeposited Prussian blue on glassy carbon electrode for detection
of cholesterol
Xiuhui Liu , Zhihan Nan, Yu Qiu, Lichun Zheng, Xiaoquan Lu
Key Laboratory of Bioelectrochemistry & Environmental Analysis of Gansu Province, College of Chemistry & Chemical Engineering, Northwest Normal University, Lanzhou 730070,
China

a r t i c l e

i n f o

Article history:
Received 25 August 2012
Received in revised form
25 November 2012
Accepted 28 November 2012
Available online 5 December 2012
Keywords:
Ionic Liquid
Cholesterol oxidase
Cholesterol
Prussian blue
Biosensor

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 lm played a signal amplication role because it not only enriched the cholesterol
from 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 toward
hydrogen peroxide (H2 O2 ). The fabricated IL-ChOx/PB/Glassy carbon electrode (GCE) was characterized
by electrochemical impedance spectroscopy (EIS) and cyclic voltammogram (CV), respectively. And it
exhibited a linear response to cholesterol in the range of 0.010.40 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 the
electrocatalytic mechanism was proposed as shown in rst scheme. ChOx immobilized in hydrophobic
IL thin lm was used as the rst electrocatalyst for the cholesterol into H2 O2 , and the PB lm onto the
GCE was used as the second electrocatalyst for the 2e reduction of the produced H2 O2 into H2 O.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
The alarming rise in the rate of clinical disorders such as heart
disease, hypertension, arteriosclerosis, coronary artery disease,
cerebral thrombosis, etc. due to abnormal levels of cholesterol
in blood have stimulated public concern about the determination of cholesterol level [1]. Meanwhile, the importance of the
determination of cholesterol has been reected in recent years by
an increase in the number of articles about the development of
cholesterol biosensors [2]. In the fabrication of a cholesterol biosensor, cholesterol oxidase (ChOx) is most commonly used as the
biosensing element [3]. But the oxidation of H2 O2 usually requires
a high anodic potential (usually over +0.6 V, vs SCE) that may
induce simultaneous oxidation of other electrochemically active
species presented in samples and lead to false positive signals [4].
Thus, it was expected that an additional electrocatalytic layer of
Prussian blue (PB) placed on surface of electrode prior to Chox
immobilization would allow one to detect cholesterol at lower
potential (about +0.1 to 0.1 V) [57]. Many papers have demonstrated the PB-modied electrode exhibited high activity and
selectivity 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: liuxh@nwnu.edu.cn (X. Liu), luxq@nwnu.edu.cn (X. Lu).
0013-4686/$ see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.electacta.2012.11.119

electroreduction [815]. However, the reference of PB enzymatic

sensor for cholesterol detection was seldom reported, there are still
some developmental challenges to be addressed. The main challenge is developing suitable support matrix that provides better
environment for the efcient enzyme loading and maintaining of
the enzymatic bioactivity so as to increase the sensitivity of the
biosensor [1626].
Recently, the emerging ionic liquids (IL) material may offer great
opportunities to solve above problem because of the unique properties of IL such as negligible vapor pressure, high thermal stability
and viscosity, and good conductivity and solubility. Among these,
the viscosity of the IL is an important consideration in electrochemical studies due to its strong effect on the rate of mass transport
within solution [27]. In addition, it is also related to whether the
droplet of IL will hang up on the electrode surface. It was found that
only few kind lipases could maintain their activity in ionic liquids of
1-butyl-3-methylimidazolium bis[(triuoromethyl)sulfonyl]imide
([BMIM][NtF2 ]) [28,29]. Recent implementations suggested that
the hydrophobic anion [NtF2 ] can signicantly improve the catalytic activity for H2 O2 electrochemical oxidation/reduction and
lower its overvoltage, which are benecial in fabricating biosensors with high sensitivity and selectivity. Herein, the present paper
describes a cholesterol biosensor prepared by immobilization of
ChOx in the hydrophobic ionic liquid 1-octyl-3-methylimidazolium
bis(triuoromethylsulfonyl)imide([OMIM][NtF2 ]) on the top of PB
modied electrodes. Scheme 1 shows a multistep system for

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X. Liu et al. / Electrochimica Acta 90 (2013) 203209

potassium chloride (Xian Chemical Reagent Factory, Xian, China)

were of analytical reagent grades.
2.2.1. Preparation of cholesterol solution
It is important to make the homogenous cholesterol solutions
because it is sparingly soluble in water. Non ionic surfactant Triton X-100 and isopropanol were found to be effective solubilizing
agents for cholesterol [30]. For preparing cholesterol stock solution
of 5 mmol/L, 0.04835 g cholesterol was dissolved in a 25 mL volumetric ask containing a mixture of 1 mL isopropanol and 1 mL
Triton X-100 in a bath at 60 C, and then was diluted with distilled
water. The cholesterol solution was stored at 4 C.
2.2.2. Preparing of cholesterol oxidase-ionic liquid stock solution
The stock cholesterol oxidase-ionic liquid solution was prepared using a procedure by mixing 5 mg cholesterol oxidase (ChOx)
with 200 L 1-Octyl-3-methylimidazolium triuoromethylsulfonate (IL), The mixture was then ultrasonically treated for 30 min
at room temperature until a stable solution was obtained. This
solution was used throughout the experiment to prepare ChOx-IL
modied lm.
Scheme 1. Schematic illustration of multi-step reactions between cholesterol/enzyme/electrode at the biosensor.

cholesterol oxidation on the modied electrode. As shown in the

Scheme 1, the PB electrodeposited on the GCE is used as effective low-potential electron transfer mediation toward H2 O2 , and
the hydrophobic IL plays the role of being the matrix immobilizing ChOx and keeping the PB lm stable. Our strategy is that ChOx
immobilized in hydrophobic IL thin lm is used as the rst electrocatalyst for the cholesterol into H2 O2 , and the PB lm onto the
GCE serves as the second electrocatalyst for the 2e reduction of
the produced H2 O2 into H2 O. Herein, there is the linear relationship
between the concentration of the cholesterol and the value of the
PB redox peak current. Our results found that the immobilization
of enzyme with high activity in the hydrophobic ionic liquid matrix
has made this technique to be a potential tool for developing new
biosensors.

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

The GCE was pretreated as following: mechanical polishing in
microcloth pads with 0.05 m alumina slurry, and then ultrasonicated in ethanol and doubly distilled water for 15 min. Finally the
electrode was washed with water, and dried at room temperature.
Deposition of the PB was accomplished in a solution of 10 mmol/L
K3 [Fe(CN)6 ] +10 mmol/L FeCl3 + 0.1 mol/L KCl. A Prussian blue (PB)
lm was formed by applying a constant potential of 0.0 V to the
GCE for 60 s, and then scanning 10 cycles at a sweep rate of 50 mV/s
between 0.0 and 0.5 V. The PB modied electrode was electrochemically pretreated in 0.1 mol/L phosphate buffer containing 0.1 mol/L
KCl (pH 6.8) by keeping at 0.0 V for 30 s and cycling between 0.5 and
0.0 V for 10 cycles. The treaded electrode was washed with water
and dried under infrared lamp for 30 min. 3 L of stock cholesterol
oxidase-ionic liquid solution was cast onto the electrode surface. All
the experiments were carried out at ambient temperature (about
25 C). The potentials recorded are vs. SCE.

2. Experimental
2.1. Apparatus
Cyclic voltammetry experiments were performed on a CHI660C
electrochemical workstation (Austin, TX, USA) controlled by a
microcomputer with CHI660 software. A three-electrode system
was used, where a glass carbon electrode (GCE, 3.5 mm diameter)
or a ChOx-PB modied GCE served as the working electrode, a Pt
wire as the counter electrode and a SCE as reference electrode.
Electrochemical impedance (EI) experiments were performed on
a multi-channel electrochemical workstation (American Princeton
Instruments Corporation).
2.2. Reagents
The 1-octyl-3-methylimidazolium triuoromethylsulfonate
([OMIM][NtF2 ]) came from Shanghai Chengjie Chemistry Co. Ltd.
(Shanghai, China). Cholesterol oxidase (ChOx, EC 1.1.3. 6,15 U/mg)
was purchased from Shanghai source leaf biological technology Co.
Ltd. (Shanghai, China). Cholesterol (Tianjin Guangfu Fine Chemical
Research Institute, Tianjin, China), Triton X-100 (Aladdin Chemistry Co. Ltd., Shanghai, China), isopropanol (Shanghai Zhongqin
Chemistry Co. Ltd., Shanghai, China), potassium ferricyanide and

3. Results and discussion

3.1. Monitoring the preparation of IL-ChOx/PB/GCE
3.1.1. The stability of the PB lm
From the electrodeposited method [31]:
Fe4 [Fe(CN)6 ]3 + 4K+ + 4e K4 Fe4 [Fe(CN)6 ]3

(1)

we obtained the PB lm as shown in Fig. S1 of Supporting

Information. A couple of well dened cathodic and anodic peaks
corresponded to the reduction of PW and the oxidation of PB,
respectively. The molecular structures of the compounds of PB and
PW are shown in Scheme 2. In addition, Fig. 1A is the CV of the PB
lm scanned for 5 cycles in pH 6.8 phosphate buffer solution containing 0.1 M KCl. The peak currents of the Prussian blue obviously
decreased with increase of scanning laps, illuminating that conned the PB lm only on the GCE was unstable. But when the
hydrophobic ionic liquid thin lm cast onto the PB lms, the peak
currents of the PB remained the same basically with the increase of
scanning laps as shown in Fig. 1B, indicating that the hydrophobic
ionic liquid lm plays a crucial role of isolating and protecting PB
lm.

X. Liu et al. / Electrochimica Acta 90 (2013) 203209

205

Scheme 2. (A) The molecular structure of the compound of PB (Fe4 [Fe(CN)6 ]3 ). (B) The molecular structure of the compound of PW(K4 Fe4 [Fe(CN)6 ]3 ).

3.1.2. Electrochemical characterization of the IL-ChOx/PB/GCE

CVs of bare and modied GCE electrodes were obtained as
shown in Fig. 2 in pH 6.8PBS containing 0.1 M KCl in order to
monitor changes in their electrochemical behavior introduced
at different steps of electrode modication. No electrochemical
response was observed at the bare GCE in Fig. 2a. Whereas, when

the electrode was electrodeposited with PB lm, CV of PB/GCE electrode showed a couple of redox peaks at about 0.3 V, corresponding
to the reduction of Prussian white (PW) at 0.199 V and the oxidation of PB at 0.329 V, respectively, in Fig. 2b. After that, when the
IL was cast onto the PB lm, a quasireversible redox peak with the
peak-to-peak separation (E) of 0.13 V was obtained as shown in
Fig. 2c. It is noted that both the cathodic and anodic peak currents
increased sharply as compared to that in Fig. 2a and b, illuminating
that IL could promote the electron transfer rate and enhance the
reversibility of the redox of PB. Furthermore, Fig. 2d is the CV curve
of ChOx immobilized in IL/PB/GCE, similar to that obtained from
IL/PB/GCE electrode except a little increase in the reduction peak
current.
Electrochemical impedance spectroscopy (EIS) can also provide
information on the impedance changes of the electrode surface
during the modied process. In the electrochemical impedance
spectroscopy, a semicircle portion observed at higher frequencies
would correspond to the electron-transfer-limited process, and a
linear section characteristic of the lower frequency is attributable
to a diffusion-limited electron transfer. The value of the electrontransfer resistance (Ret ) at the electrode surface can be estimated
directly from the diameters of the high frequency semicircl, which
depends on the dielectric and insulating features at the electrode/electrolyte interface. Using [Fe(CN)6 ]3/4 redox couples as

Fig. 1. CVs of the Prussian blue lm modied GC electrode (A) and Prussian blue-IL
modied GC electrode (B) in 0.1 M KCl pH 6.8 PBS for 5 cycles. Scan rate 50 mV/s.

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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X. Liu et al. / Electrochimica Acta 90 (2013) 203209

Fig. 3. Electrochemical impedance spectra obtained at (a) GCE, (b) PB/GCE, (c)
IL/PB/GCE, (d) IL-ChOx/PB/GCE in 5 mM K3 Fe(CN)6 /K4 Fe(CN)6 (1:1) and 0.1 M KCl
in pH 6.8 PBS.

the electrochemical probe, the EIS plots of different modied electrodes are shown in Fig. 3, and the inset is the ts of equivalent
circuit. The impedance spectra of the bare GCE (Fig. 3a) consisted
of a small semicircle (Ret : 100 ) with an almost straight tail line,
which was the characteristic of a diffusion limiting step of the electrochemical process. When the GCE electrode surface was coated
by PB lm, the Ret value of Fig. 3b increased to about 450 . This
implies that the electrodepositing PB lm generated an obstruction
to the electron transfer of the electrochemical probe at electrode
surface. After the ionic liquid coated on the PB lm, the diameter of
the high frequency semicircle was signicantly reduced with a Ret
value of 250  as shown in Fig. 3c, implying that ionic liquid may
play an important role in accelerating the electron transfer process. When the cholesterol oxidase-ionic liquid stock solution cast
onto the PB modied electrode, the diameter of the high frequency
semicircle signicantly decreased to close to zero. These results are
consistent with that of CV experiments (Fig. 2).
3.2. The investigation of effect factors on the IL-ChOx/PB/GCE
3.2.1. The inuence of the dissolved oxygen in cholesterol solution
The cholesterol solution was deoxygenated by bubbling highpurity N2 for at least 20 min and the experiments were undertaken
under N2 atmosphere. As shown in Fig. 4, the black lines are the
CVs of the IL-ChOx/PB/GCE electrode in cholesterol solution saturated with N2 (dashed line) or under ambient air (solid line). It was
noted that the reduction peak current in the dashed line is smaller
than that of in the solid line. This may be ascribed to O2 inuence
and the possible reaction mechanism is proposed in the following
reactions:

PW +
e

1
H2 O2 PB + H2 O
2

PWPB + K+

It is very clearly to see in Scheme 2, Prussian white (PW) (K4

Fe4 [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 for
reduction of H2 O2 into H2 O in reaction (4). Thus, when the experiment was undertaken under N2 atmosphere, the decrease of the
O2 concentration induced the decrease of the produced H2 O2 , corresponded to the smaller reduction peak current (dashed line in
Fig. 4, black).
To clarify this further, the CVs of PB lm modied GC electrode
were performed in 0.1 mol/L KCl pH 6.8 PBS and 1% Triton X-100 as
depicted the red lines in Fig. 4. One can see that both the currents
and the potentials of the reduction peak show no change in saturated 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 inuence to the PB lm modied electrode because the peaks current
in the red lines are ascribed to the reduction reaction of Prussian
white (PW) occurred as shown in reaction (5) only.

(3)

3.2.2. Effect of buffer pH

The effect of buffer pH on the response for cholesterol was
investigated and results were shown in Fig. S2 of the Supporting
Information, in which the optimized pH of phosphate buffer was
6.8. Normally, PB lm is stable in acidic and neutral solution, but
unstable in alkaline solution. Because ferric ions are known to be
hydrolyzed easily, and the hydroxyl ions (OH ) cannot be substituted in their coordination sphere in course of PB crystallization.
So it is benecial to choose pH 6.8 buffer solution in the whole
experiments.

(4)

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

ChOx(FAD)+cholesterolcholester-4-en-3-one+ChOx(FADH2 )(2)
O2 + ChOx(FADH2 ) ChOx(FAD) + H2 O2

Fig. 4. CVs obtained at the PB modied GCE (the red lines) in phosphate buffer
with 0.1 mol/L KCl and 1% Triton X-100 saturated with N2 (dashed line) or under
ambient air (solid line) and at the IL-ChOx/PB/GCE (the black lines) in 1 105 mol/L
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 references
to color in gure legend, the reader is referred to the web version of the article.)

(5)

As illustrated in Scheme 1, the cholesterol was rst adsorbed

to hydrophobic ionic liquid/aqueous solution interface and reacted
with the cholesterol oxidase, which used as the rst electrocatalyst
in reaction (2). Then the produced ChOx(FADH2 ) reacted with the
dissolved oxygen subsequently in reaction (3), and the produced
H2 O2 would diffuse into the PB lm coated on the GCE electrode.

Fig. 5 is CV curves of the IL-Chox/PB/GCE electrode at different concentrations of cholesterol. The linear relationship could
be established between the peak currents and the concentrations
of cholesterol in the range of 10400 M. The sensitivity, calculated from the slope of the plot (Fig. 5, Ipa (A) = 0.15 + 0.04c
(105 M)), was found to be 0.4 A/M. The detection limit (DL)
for the constructed electrode, calculated from the expression
DL = (3 SD)/sensitivity (where SD is the estimated standard

X. Liu et al. / Electrochimica Acta 90 (2013) 203209

207

Table 1
Comparison of different modied electrodes for cholesterol determination.
Electrode

Line range

C/Fc-copolymer/HPR/ChOx
CPE/hydroxymethylferrocene/HRP/ChOx
Pt/naon/cellulose/ChOx
Pt/PPy/ChOx
W/ferrocyanide/ChOx
MWCNTs-AuNPs-CHIT-IL
AuNPs
AuE/dithiol/AuNPs/MUA/ChOx
ChOx-IL/PB

210 mM
1 103 0.15 mM
5100 mg/L
0.0250.3 mM
0.053 mM
0.55 mM
0.010.07 mM
0.040.22 mM
0.010.4 mM

deviation for the points used to construct the calibration curve and
the sensitivity, its slope), was found to be 4.4 M. The results for
the determination of cholesterol using different methods are listed
in Table 1, from where one can conclude that the proposed biosensor has a higher sensitivity and a lower detection limit than those
previous reported models.
The variation tendency of catalytic currents and the cholesterol
concentrations is shown in Fig. S3 of the Supporting Information.
The reduction peak currents gradually increased with increasing concentrations of cholesterol. But when the concentrations of
cholesterol came to 2 104 M, the increased trend became slow.
The maximum catalytic current was obtained at the concentrations
of cholesterol about 4 104 M. The apparent MichaelisMenten
constant Km , which gives an indication of the enzyme-substrate
kinetics, can be obtained from the LineweaverBurk equation [32].
1
Km
1
1
=
+

Iss
Imax
Imax
c

(6)

where c is the concentration of cholesterol in solution, Iss is the

catalytic reduction current (background current deducted) at the
steady state when cholesterol concentration is at c, and Imax is
the maximum catalytic current. The Km value for the electrocatalytic activity of IL-ChOx/PB/GCE to cholesterol was determined
to be 0.116 mM, implying that the present electrode exhibited
a higher afnity for cholesterol. As well known, the smaller of
Km , the higher catalytic ability of the cholesterol biosensor possesses. Compared with those reported in cholesterol biosensors
[33,34], the lower value of Km indicated that cholesterol oxidase

Fig. 5. Cyclic voltammetric measurements with IL-ChOx/PB/GCE in various concentrations of cholesterol pH 6.8 PBS solution saturated with air: 1 105 , 3 105 ,
5 105 , 7 105 , 9 105 , 1 104 , 2 104 , 4 104 mol/L from inner to outer.
The inset is the calibration curve corresponding to amperometric responses. Scan
rate: 50 mV/s.

Detection limit

5.7 M
0.01 mM
10 mM
34.6 M
4.4 M

Reference
[36]
[37]
[38]
[39]
[40]
[6]
[25]
[41]
This paper

immobilized in hydrophobic ionic liquid lm retained its bioactivity and had a high biological efciency to cholesterol.
Some possible interfering species, such as ascorbic acid,
dopamine and uric acid were also investigated. CVs were taken
in PBS containing 0.05 mM cholesterol and each interferences
(0.05 mM). The results are shown in Fig. S4 of the Supporting
Information, where Ic+i and Ic are modied electrodes response
at 0.10 V for 0.05 mM cholesterol in the presence and absence of
each interferences, respectively. We found that the value of Ic+i was
almost equal to the value of Ic , indicating that AA, UA, and DA did not
produce signicantly inuence in the determination of cholesterol.
So the biosensor we fabricated has high anti-interference ability.
The feasibility of the device for practical applications was
carried out by analyzing the 1% human serum. The cholesterol
concentration in the human serum sample was determined to
be 4.62 0.04 mM (n = 3) by CV measurement, which is in the
range of 2.855.98 mM, collected from the Chinese peoples serum
containing the cholesterol published by Chinese Pharmaceutical
Association.

3.4. Comparing the kinetics of cholesterol at IL-ChOx/PB/GCE

electrode with H2 O2 at IL/PB/GCE electrode
Fig. 6A shows cyclic voltammograms of H2 O2 at IL/PB/GCE
electrode with different scan rates (v). A pair of roughly symmetric anodic and cathodic peaks appeared with almost equal peak
currents in the scan rate range from 0.01 to 0.30 V/s. The peakto-peak separation also increased slightly with the scan rate. In
these scan rates range, a good linear relationship was found for the
peak 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 characteristic of quasi-reversible surface controlled electrochemical behavior.
However, a linear behavior of the peak current versus the square
root of scan rate was obtained when the scan rate was higher than
0.10 V/s as shown in Fig. 6D, revealed a diffusion controlled mechanism. Another similar experiment was performed for cholesterol
at IL-ChOx/PB/GCE electrode. As shown in Fig. 6B and E, one can
see that there was always an ideal linear relationship for the peak
currents and scan rates (Ipa (A) = 5.50 + 59.9 (V/s) (r = 0.9999)) in
the scan rate range from 0.01 to 0.30 V/s which was the characteristic of adsorption controlled electrochemical behavior. Above
experiments demonstrated that cholesterol in the solution tends
to absorb to the aqueous solution and hydrophobic ionic liquid
interface, but H2 O2 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 the
electrochemical parameters could be calculated using the method
developed by Laviron [35] as follows:


Epc = E 0 RT

ln 
nF

(7)

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X. Liu et al. / Electrochimica Acta 90 (2013) 203209

Fig. 6. (A) Cyclic voltammograms of 1.0 103 M H2 O2 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: 0.010.09 V/s) and (D) Ip vs. v1/2 (v: 0.120.30 V/s); (B) cyclic voltammograms of 1.0 105 M cholesterol in pH
6.8 buffer solution with different scan rate at IL-ChOx/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 (E) Ip vs.
v (v: 0.010.30 V/s).

Epa = E 0 + RT

ln 
(1 )nF

(8)

log ks = log(1 ) + (1 ) log

log

 RT 
nF

(1 )nFEp
2.3RT

constant (ks) of cholesterol are calculated as 0.1432 and 0.5871/s,

respectively, revealing that the efcient enzyme loading and maintaining of the enzymatic bioactivity in ionic liquids of [BMIM][NtF2 ]
would enhance the electrochemical reaction rate, and improve the
sensitivity of the biosensor.

(9)

where is the electron transfer coefcient, n is the electron transfer number, ks is the apparent electron transfer rate constant, R
is the gas constant, T is the absolute temperature, and Ep is the
peak-to peak separation. So of H2 O2 is calculated as 0.3550, and
the apparent electron-transfer rate constant (ks) is estimated to
be 0.3408/s. Similarly, and the apparent electron-transfer rate

4. Conclusions
A novel scheme for the fabrication cholesterol biosensor was
developed using electrodepositing PB lm as electron-mediator
and hydrophobic ionic liquid as matrices for the assembly of integrated electrode. The hydrophobic ionic liquid lm was found to

X. Liu et al. / Electrochimica Acta 90 (2013) 203209

enhance the current response of the fabricated bioelectrode and

provide a biocompatible environment for the ChOx. ChOx immobilized in hydrophobic IL thin lm was used as the rst electrocatalyst
for the cholesterol into H2 O2 , and the PB lm onto the GC electrode
was used as the second electrocatalyst for the 2e reduction of the
produced H2 O2 into H2 O. In addition, the fabricated bioelectrode
displayed a series of excellent features such as good sensitivity,
wide linear range, and low detection limit, which might pave a
new and cost-effective way for biomonitoring of cholesterol both
in methodological study and in clinical laboratories.
Acknowledgment
This work was supported by the National Natural Science Foundation of China (Nos. 21245004, 20875077)
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
electacta.2012.11.119.
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