[59] Ultra-sensitive cholesterol biosensor based on low

Electrochemistry Communications 11 (2009) 118–121
Contents lists available at ScienceDirect
Electrochemistry Communications
journal homepage: www.elsevier.com/locate/elecom
Ultra-sensitive cholesterol biosensor based on low-temperature grown
ZnO nanoparticles
Ahmad Umar, M.M. Rahman, Mohammad Vaseem, Yoon-Bong Hahn *
School of Semiconductor and Chemical Engineering, BK21 Centre for Future Energy Materials and Devices and Nanomaterials Research Processing Centre, Chonbuk National University,
664-14, Duckjin Dong, 1 Ga Jeonju, Jeonju, Cholla Bukto 561-756, South Korea
a r t i c l e
i n f o
Article history:
Received 2 October 2008
Received in revised form 11 October 2008
Accepted 15 October 2008
Available online 30 October 2008
Keywords:
ZnO nanoparticles
Cholesterol oxidase
Cholesterol biosensor
a b s t r a c t
A high-sensitive cholesterol amperometric biosensor based on the immobilization of cholesterol oxidase
(ChOx) onto the ZnO nanoparticles has been fabricated which shows a very high and reproducible sensitivity of 23.7 lA mM1 cm2, detection limit (based on S/N ratio) 0.37 ± 0.02 nM, response time less
than 5 s, linear range from 1.0 to 500.0 nM and correlation coefficient of R = 0.9975. A relatively low value
of enzyme’s kinetic parameter (Michaelis–Menten constant) 4.7 mM has been obtained which indicates
the enhanced enzymatic affinity of ChOx to Cholesterol. To the best of our knowledge, this is the first
report in which such a very high-sensitivity and low detection limit has been achieved for the cholesterol
biosensor by using ZnO nanostructures modified electrodes.
Ó 2008 Elsevier B.V. All rights reserved.
1. Introduction
Cholesterol and its fatty acid esters are one of the main constituents for the human beings as they are the components of nerve
and brain cells [1] and are the precursors for other biological materials, such as bile acid and steroid hormones [2]. Due to high-rate of
clinical disorders, such as heart disease, coronary artery disease,
cerebral thrombosis, etc. (caused by the anomalous levels of cholesterol in blood), it is desirable to develop a reliable and sensitive
biosensor which can allow a convenient and rapid determination
of cholesterol [3,4]. The amperometric biosensor (which is based
on the proper immobilization of enzyme on suitable matrixes) offers a portable, cheap, and rapid method for the determination of
cholesterol [5–17]. Due to exotic properties, it is expected that biocompatible nanomaterials could be the promising matrixes for enzyme immobilization which can enhance the sensitivity and
selectivity of biosensors [5–10]. Among different nanomaterials,
the ZnO nanostructures possess a special place due to their own
merits such as high specific surface area, optical transparency,
bio-compatibility, non-toxicity, chemical and photochemical stability, ease of fabrication, high-electron communication features,
electrochemical activities, and so on. Even having versatile properties, the biosensor applications of ZnO nanostructures are very
rare. There are only two reports on ZnO nanostructures based cholesterol biosensor [11,12]. Cholesterol biosensor based on rf-sputtered ZnO nanoporous thin films exhibited a linear range of
* Corresponding author. Tel.: +82 63 270 2439; fax: +82 63 270 2306.
E-mail addresses: ahmadumar@chonbuk.ac.kr (A. Umar), ybhahn@chonbuk.ac.kr
(Y.-B. Hahn).
1388-2481/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.elecom.2008.10.046
25–400 mg dl1 and response time 15 s [11]. A cholesterol biosensor based on ZnO nanoparticles–chitosan composite films shows
the linear range of 5–300 mg dl1, detection limit of 5 mg dl1,
and sensitivity of 1.41 104 Amg dl1 [12]. In this paper, ultrasensitive cholesterol biosensors based on the immobilization of
ChOx onto the ZnO nanoparticles is presented showed a very high
and reproducible sensitivity of 23.7 lA mM1 cm2, detection limit
of 0.37 ± 0.02 nM and linear dynamic range from 1 to 500 nM. To
the best of our knowledge, this is the first report in which such a
very high-sensitivity and low detection limit has been achieved
for the cholesterol biosensor by using ZnO nanostructures modified electrodes.
2. Experimental details
In a typical synthesis process, aqueous solutions of 0.1 M zinc
acetate dihydrate (ZnAc) and 0.05 M HMTA (each 50.0 ml) were
mixed under continuous stirring and subsequently, stock aqueous
solution of 1.0 M LiOH was slowly added in this solution until the
solution pH became 10. The obtained solution was then refluxed at
90.0 °C for 3 h. White precipitates were obtained which were filtered off, washed thoroughly with deionized water and ethanol,
and dried at room temperature.
For the fabrication of cholesterol biosensors, ChOx was immobilized onto the ZnO nanoparticles, coated onto gold (Au) electrode
surface (3.0 mm2), by physical adsorption technique. The ZnO
nanoparticles surface was immobilized in a solution of ChOx
(1.0 mg/ml), prepared in phosphate buffer (PBS, pH 7.4) 150.0 mM
(0.9% NaCl) for 24 h. The electrode is kept overnight for ChOx
immobilization and subsequently washed with buffer solution,
A. Umar et al. / Electrochemistry Communications 11 (2009) 118–121
and dried in nitrogen environment. After drying the modified ChOx/
ZnO/Au electrode, a 10.0 ll Nafion solution was dropped onto the
electrode and dried for 24 h at 4.0 °C to form a film on the modified
electrode. When, not in use, the ZnO modified gold electrodes (i.e.,
Nafion/ChOx/ZnO/Au electrodes) were stored in PBS at 4.0 °C. The
electrochemical experiments were carried with a conventional
three-electrode configuration.
3. Results and discussion
Fig. 1a exhibits the typical FESEM image of the as-grown nanoparticles which shows that the grown products are synthesized in a
large-quantity with almost uniform sizes and shapes. The nanoparticles are almost spherical and triangular shaped while some of
them also possess hexagonal structures. The typical sizes of the
grown nanoparticles were 80 ± 20 nm. All the obtained peaks in
the X-ray diffraction pattern of as-grown nanoparticles are similar
to known wurtzite-structured hexagonal phase single crystalline
bulk ZnO (JCPDS Card No. 36–1451) confirming the synthesis of
119
pure ZnO nanoparticles (Fig. 1b). Even not shown here, the EDS
also confirmed that the grown nanoparticles are made with almost
1:1 stoichiometry of zinc and oxygen, respectively. The low-magnification TEM image (Fig. 1c) of as-grown ZnO nanoparticles revealed the full consistency with the FESEM observation (Fig. 1a)
in terms of density and dimensionality. The high-resolution TEM
(HRTEM) image shown in Fig. 1d gives the lattice fringe of about
0.26 nm, corresponding to the (0 0 0 2) fringes, clearly confirms
the single crystallinity and wurtzite hexagonal phase of the as-synthesized nanoparticles. The composition and quality of the product
was analyzed by the FTIR, in the range of 400–4000 cm1 (Fig. 1e).
The band at 434 cm1 is correlated to zinc oxide [13]. A broad band
in the range of 3350–3500 cm1 and at 1632 cm1 corresponds to
OAH stretching and bending modes of vibrations, respectively
[14]. The presence of other bands at 878, 1380, and 2359
cm1 are probably due to the carbonate moieties which generally
observed when FTIR samples are measured in air [13]. A sharp
band at 375 nm, from the room temperature UV–vis absorption
spectrum, was observed from the synthesized ZnO nanoparticles,
Fig. 1. Typical (a) FESEM image; (b) X-ray diffraction pattern; (c) low-magnification and (d) high-resolution TEM images; (e) FTIR spectrum and (f) UV–vis spectrum of the assynthesized ZnO nanoparticles grown at low temperature by aqueous solution process.
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A. Umar et al. / Electrochemistry Communications 11 (2009) 118–121
exhibiting a characteristic band for the wurtzite hexagonal pure
ZnO [14] (Fig. 1f).
Fig. 2a exhibits the schematic of the modification of gold electrode with ZnO nanoparticles, ChOx and Nafion for efficient detection of cholesterol. Fig. 2b shows a cyclic voltammetric (CV) sweep
curve for the ZnO-modified gold electrode (Nafion/ChOx/ZnO/Au)
without (dotted line) and with (solid line) 0.1 mM cholesterol in
0.1 M PBS buffer at pH 7.4 in the range of 0.3 to 0.7 V at scan rate
of 100 mV/s. The CV curve of Nafion/ChOx/ZnO/Au electrode with
cholesterol in PBS shows an increase in current from 0.01 to
0.68 V compared to PBS without cholesterol, confirming the electrochemical response of the Nafion/ChOx/ZnO/Au electrode in cholesterol. A peak at approximately +0.355 V has been observed from
the CV curve of Nafion/ChOx/ZnO/Au electrode in PBS with 0.1 mM
cholesterol which is because of the generation of H2O2 during the
oxidation of cholesterol by ChOx. Due to high surface area of ZnO
nanoparticles, the ChOx attached to the surfaces of nanoparticles
facilitates the faster direct electron transfer between the active
sites of immobilized ChOx and electrode surface which leads to a
sharper and well-defined peak. Therefore, cholesterol is efficiently
detected with the Nafion/ChOx/ZnO/Au electrodes. According to
the previous report, the electrochemical reaction for the detection
of cholesterol in presence of ChOx is proposed and the enzymatic
reaction is written as
ChOx
Cholesterol þ O2 ! Cholestenone þ H2 O2
The amperometry under stirred conditions has a much higher
current sensitivity than the cyclic voltammetry, hence the amperometric experiments have been performed under continuous stirring. A typical amperometric response of the Nafion/ChOx/ZnO/
Au electrode on a successive addition of cholesterol (from 1.0 to
700.0 nM) into continuously stirred 0.01 M PBS solution (pH 7.4)
at an applied potential of +0.355 V is shown in Fig. 2c. With successively increasing the concentration of cholesterol, the sensing current increases and 95% steady state response was achieved in less
than 5 s which confirms a good electro-catalytic and fast electron
exchange behavior of modified electrode. Fig. 2d exhibits the relation between the response current and cholesterol concentration
for the fabricated amperometric sensor which clearly shows that
the response current increases as the concentration of cholesterol
increases and saturated at high concentration of cholesterol which
suggests the saturation of active sites of the enzymes at those cholesterol levels. Under optimized conditions, the steady-state current showed a linear dynamic range of 1.0 to 500.0 nM (Fig. 2d).
The correlation coefficient (R) was estimated to be R = 0.9975 and
the sensitivity was found to be 23.7 lA mM1 cm2 from the fabricated biosensor. The detection limit, estimated based on signal to
noise ratio (S/N = 3), was found to be 0.37 ± 0.02 nM. To the best
of our knowledge, this is the first time such a very high-sensitivity
and low detection limit has been achieved for cholesterol biosensors by using ZnO nanostructures modified electrodes. The apparent Michaelis–Menten constant (Kmapp) which gives an indication
of the enzyme substrate kinetics can be calculated from the
Lineweaver–Burk equation 1/i = (Kmapp/imax) (1/C) + (1/imax), where
i is the current, imax is the maximum current measured under saturated substrate conditions, and C is the cholesterol concentration.
The Kmapp value was determined by the analysis of the slope and
intercept for the plot of the reciprocals of steady-state current vs.
cholesterol concentrations, i.e., the Lineweaver–Burk plot of 1/i
vs. 1/C (Fig. 2e). According to the Lineweaver–Burk plot, the Kmapp
is calculated to be 4. 7 mM. The lower Kmapp value can be attributed
to the favorable confirmation of the enzyme and an efficient ChOx
loading provided by the microenvironment of ZnO nanoparticles
surfaces. For comparison, the performances of the fabricated biosensor is compared with the previously reported cholesterol biosensors based on the utilization of different materials as the
working electrode (Table 1) and it was confirmed that the presented ZnO nanoparticles based cholesterol biosensor exhibited
an excellent and reproducible sensitivity [11–16]. It was examined
that the fabricated sensors did not show any significant decrease in
Fig. 2. (a) Schematic of the modification of gold electrode with ZnO nanoparticles, ChOx and Nafion for efficient detection of cholesterol; (b) cyclic voltammetric sweep curve
for the Nafion/ChOx/ZnO/Au electrode without cholesterol (dotted line) and with 0.1 mM cholesterol (solid line) in 0. 1 M PBS buffer (pH 7.4) in the range of 0.3 to 0.7 V at
scan rate of 100 mV/s; (c) amperometric response of the Nafion/ChOx/ZnO/Au electrode with successive addition of cholesterol into 0. 1 M PBS buffer solution (pH 7.4); (d)
calibration curve for cholesterol using Nafion/ChOx/ZnO/Au electrode; and (e) the plot of 1/Current vs. 1/Concentration exhibiting a linear relationship with the steady state
current and cholesterol concentration.
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A. Umar et al. / Electrochemistry Communications 11 (2009) 118–121
Table 1
Summary of the key performance parameters of cholesterol biosensor constructed based on ChOx-modified nanomaterials as working electrodes.
Electrode materials
Sensitivity (lA lM cm2)
Detection limit (lM)
KMapp (mM)
Linear range (lM)
Response time (s)
Ref.
ZnO nanoparticles
ZnO nanoporous thin films
ZnO nanoparticles + chitosan composite
Nanoporous CeO2 film
Polypyrrole films
Tertraethylortho-silicate
23.7
–
14.1
5.98
15.0
–
0.00037
–
0.125 103
–
–
0.500
4.7
2.1
0.223
2.06
9.8
21.2
0.001–0.5
(0.65–10.35) 106
(0.125–7.76) 106
(1.3–10.35) 106
(1.0–8.0) 103
(2.0–10.0) 103
5
15
15
15
–
50
Current work
11
12
9
15
16
the sensitivity for more than 50 days, while storing in an appropriate form when not in use. The long-term storage stability of the
sensor was tested for 50 days. The sensitivity retained 91.8% of initial sensitivity up to 50 days which gradually decreases afterwards
might be due to the loss of the catalytic activity.
4. Conclusions
In conclusion, an ultra-sensitive cholesterol biosensor has been
fabricated by modifying the gold electrode with well-crystallized
low-temperature grown ZnO nanoparticles. A high reproducible sensitivity of 23.7 lA mM1 cm2, response time less than 5 s and detection limit of 0.37 ± 0.02 nM was found from the fabricated biosensor.
A relatively low value of enzyme’s kinetic parameter (Michaelis–
Menten constant) 4.7 mM has been obtained which indicates the
enhanced enzymatic affinity to ChOx to Cholesterol. To the best of
our knowledge, this is the first time such a very high-sensitivity and
low detection limit has been achieved for cholesterol biosensors by
using ZnO nanostructures modified electrodes. Hence, one can concluded that due to the simple synthesis and electrode fabrication,
ultra-sensitivity, low detection limit, and fast response, the as-grown
well-crystallized ZnO nanoparticles opens a way for the fabrication of
highly efficient cholesterol biosensors.
Acknowledgements
This work was supported by the Korea Science and Engineering Foundation grant funded by Korea Government (MEST) (No.
R01-2006-000-11306-0). Author wish to thank Mr. T.S. Bae and
J.C. Lim, KBSI, Jeonju branch, and Mr. Kang, Centre for University
Facility for taking good quality SEM and TEM images,
respectively.
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