Research Article

Received: 22 March 2011, Revised: 27 April 2011, Accepted: 05 May 2011 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI: 10.1002/pat.2020

Polypyrrole/multiwalled carbon nanotubes‐

based biosensor for cholesterol estimation
K. Singha, Pratima R. Solankib, Tinku Basuc and B. D. Malhotrab*

The nanocomposite electrode comprising of polypyrrole (PPY) and carboxy functionalized multiwalled carbon nano-
tubes (MWCNT) has been electrochemically fabricated onto indium–tin–oxide (ITO) electrode using p‐toluene sul-
fonic acid (PTS). Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) have been immobilized onto this
PPY– MWCNT/ITO nanocomposite electrode using N‐ethyl‐N‐(3‐dimethylaminopropyl) carbodiimide and N‐hydroxy
succinimide chemistry for estimation of esterified cholesterol. The ChEt–ChOx/PPY–MWCNT/PTS/ITO bioelectrode
has been characterized using Fourier transform infrared spectroscopy, electrochemical techniques, and scanning
electron microscope. This ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode has a response time of about 9 s, lin-
earity of 4 × 10−4 to 6.5 × 10−3 M/l of cholesterol oleate concentration, Km of 0.02 mM, and thermal stability of upto
45°C. This electrode exhibits improved biosensing characteristics compared with other total cholesterol electrodes
reported in literature till date and can be used to estimate cholesterol in blood serum samples. Copyright © 2011
John Wiley & Sons, Ltd.

Keywords: polypyrrole; p‐toluene sulfonic acid; multiwalled carbon nanotubes; cholesterol oxidase; cholesterol esterase;
cholesterol oleate

INTRODUCTION electrode for the detection of reduced NADH at lower poten-

The cholesterol content in normal human blood plasma is
known to be about 130–260 mg/dl, of which two‐thirds is ester-
ified with fatty acids and one‐third is present as sterol.[1,2] A high
cholesterol level in human blood is related to arteriosclerosis, hy-
pertension, and myocardial infarction. The estimation of choles-
terol is thus very important in clinical diagnosis. Various
analytical methods including calorimetric, spectroscopic, and
electrochemical techniques have been utilized for cholesterol es-
timation in solutions.[3,4] However, most of these techniques are
expensive, non‐specific, and do not yield any satisfactory data. In
this context, enzyme‐based amperometric biosensors have re-
cently gained much attention as they provide easy operation, ac-
curacy, sensitivity, and selectivity for desired analyte (e.g.
cholesterol) estimation.[5–14]
Carbon nanotubes (CNT), because of their one‐dimensional
structure, exceptional optical, and electrical properties, are re-
ceiving much attention for biosensor applications.[15–17] Besides
this, they provide high surface area for higher enzyme loading
and a compatible microenvironment that helps enzyme to retain
its bioactivity.[18,19] However, poor solubility of CNT in most sol-
vents is currently a major barrier in developing desired CNT‐
based devices.[20,21] The existing challenge of solubilization of
CNT can perhaps be addressed through their covalent modifica-
tion[22] or non‐covalent functionalization.[23] In particular, “wrap-
ping” of CNT with polymeric chains may perhaps be useful in
improving their solubility without impairing their physical
properties.[24]
It has been reported that the composite of a conducting poly-
mer (CP) with CNT may perhaps provide a synergistic effect for
improved characteristics of biosensing devices.[25] Liu et al.
have used polyaniline (PANI)/poly(aminobenzene sulfonic acid)‐
modified single‐walled carbon nanotubes (PABS‐SWCNT) multilayer
tial.[26] They have found that PABS‐SWCNT inside a multilayer
electrode can be used to dope PANI effectively and shift its elec-
tro‐activity to the neutral pH environment. Singh et al. have
developed a DNA biosensor on the basis of CNT/PANI composite
for Neisseria gonorrhoeae detection by immobilizing 5′‐amino‐la-
beled N. gonorrhoeae probe using glutaraldehyde as a cross‐
linker.[27] Dhand et al. have fabricated the nanocomposite
electrode comprising of emeraldine salt and carboxyl group functio-
nalized multiwalled carbon nanotubes (MWCNT‐c) onto indium–tin–
oxide (ITO) for immobilization of cholesterol oxidase (ChOx) to
detect free cholesterol and have obtained a short response time
(10 s) and high sensitivity (6800 nA/mM).[28] Tuantranont et al. have
developed have cholesterol biosensor by using functionalized
CNT electrode in a polydimethylsiloxane/glass‐based flow
injection microfluidic chip and proposed a high‐speed real‐time
detection capability and very low sample consumption.[29]
* Correspondence to: B. D. Malhotra, Department of Science & Technology, Cen-
tre on Biomolecular Electronics, Biomedical Instrumentation Section, National
Physical Laboratory, Dr K. S. Krishnan Marg, New Delhi 110012, India.
E‐mail: bansi.malhotra@gmail.com
a K. Singh
Amity School of Engineering and Technology, Amity University, Noida, UP,
India
b P. R. Solanki, B. D. Malhotra
Department of Science & Technology, Centre on Biomolecular Electronics, Biomedical
Instrumentation Section, National Physical Laboratory, Dr K. S. Krishnan Marg,
New Delhi 110012, India
c T. Basu
Amity Institute of Nano Technology, Amity University, Noida 201 303, UP, India

Polym. Adv. Technol. (2011) Copyright © 2011 John Wiley & Sons, Ltd.

Among the various CPs, polypyrrole (PPY) is unique because of
its relatively facile synthesis, electrical conductivity, and biocom-
patibility. The CNT‐doped PPY exhibits a dramatically different
electronic architecture as compared with PPY prepared using
small anionic dopants.[30] Such differences reflect the conductiv-
ity of CNT dopant compared with the common insulating
dopants. It has been found that electron flow within the PPY–
CNT composite is apparently increased by the entrapped CNT
because of increased degree of delocalization and CNT bridg-
ing.[30] Brânzolâ et al. have reported glucose biosensor on the ba-
sis of SWCNTs‐PPY electrode. The biosensor exhibits a wide
linear response to glucose from 0.5 to 50 mM.[31] Fang et al. have
fabricated an electrochemical impedance‐based DNA biosensor
by using a nanocomposite material of PPY and MWNT.[25] Elec-
tropolymerization of nanocomposite of CNT‐doped pyrrole elec-
trode with electrochemically entrapped enzymes has proved to
be a simple and efficient method for constructing biosensors
with a high degree of selectivity.[19]
Attempts have recently been made to fabricate biosensors for es-
timation of total cholesterol.[32–44] Singh et al. have developed pho-
tometric cholesterol electrode on the basis of tetraethylorthosilicate
sol–gel films and found linearity of up to 12 mM/l.[33] They have
also utilized CPs for the total cholesterol determination.[34–36]
However, these biosensors have been found to have low sensi-
tivity (7.5 × 10−4 nA/mg/dl) and a high response time, like 240 s.
Li et al. have fabricated screen‐printed MWCNT polycarbonate
electrode to investigate total cholesterol and found that the
CNT‐modified biosensor offers a reliable calibration profile and
stable electrochemical properties.[1] Basu et al. have developed
optical cholesterol biosensor on the basis of polycarbonate
membrane for the determination of total cholesterol in food
samples.[37] Solanki et al. have immobilized cholesterol esterase
(ChEt) and ChOx via glutaraldehyde as a cross‐linker onto sol–
gel‐derived silica/chitosan/MWCNT‐based nanobiocomposite
electrode deposited onto ITO for the determination of esterified
cholesterol.[38] This bioelectrode shows a response time of 10 s,
sensitivity of 3.8 μA/mM, and shelf life of about 10 weeks for
the estimation of cholesterol oleate. The gold nanowire‐modified
microfluidic‐based amperometric total cholesterol bioelectrode has
linearity of 1 to 6 mM/l.[39] Arya et al. have reported self‐assembled
monolayer‐based cholesterol sensors and found linearity of 10 to
500 mg/dl.[40,41]
It is important to note that 70% of the total blood cholesterol
exists in ester form and 30% in free form. Most research work till
date is focused on the estimation of free cholesterol. In the clin-
ical diagnosis, it is necessary to monitor total cholesterol. In
present manuscript, we report results of the studies relating
to the immobilization of as ChEt and ChOx onto PPY–MWCNT/
p‐toluene sulfonic acid (PTS)/ITO nanocomposite electrode de-
posited onto ITO‐coated glass for the detection of total choles-
terol using amperometric technique. The novelty of this
investigation lies on its one‐step ease of fabrication, efficient
total cholesterol sensing characteristics, and precise application
of estimation of total cholesterol in real blood samples.
EXPERIMENTAL
Reagents
MWCNT (96%, 40–80 nm) were procured from Sigma‐Aldrich
(St. Louis, MO, USA). Cholesterol oleate (98%), ChEt (EC 3.1.1.13,
Pseudomonas species), with specific activity of 165 U/mg, and
wileyonlinelibrary.com/journal/pat Copyright © 2011 John Wiley & Sons, Ltd.
K. SINGH ET AL.
ChOx (EC 1.1.36, Pseudomonas fluorescens) with specific activity
of 24 U/mg were purchased from Sigma‐Aldrich. Horseradish
peroxidase (EC 1.11.1.7) with specific activity of 200 U/mg,
N‐hydroxysuccinimide (NHS), N‐ethyl‐N‐(3‐dimethylaminopropyl
carbodiimide) (EDC) were purchased from the Sigma‐Aldrich.
Pyrrole (PPY) (MW 67.09) (procured from Spectrochem, Pvt. Ltd.,
Mumbai, India) was distilled prior to polymerization. Di‐methyl
sulfoxide (DMSO) was obtained from Central Drug House, New
Delhi, India. All other chemicals were of analytical grade and
were used without further purification, and the solutions were
prepared in deionized water.
Cholesterol ester (cholesterol oleate) solution (400 mg/dl) was
first dissolved in 1% polidocanol (Brij) as a surfactant by heating
and gentle stirring resulting in clear and colorless suspension,
and the final volume was made by addition of 0.9% NaCl
solution.
Preparation of PPY/PTS/ITO and PPY–MWCNT/PTS/
ITO electrodes
MWCNT (2 mg) were functionalized with carboxylic acid groups
by refluxing in a 2:1 sulfuric acid/nitric acid mixture (20 ml) for
about 8 h, in accordance with the procedure reported in litera-
ture.[6] Subsequently, the pretreated MWCNT were washed with
distilled water, then with 0.1 M NaOH (to reach neutrality of
pH 7.0), filtered, and dried for about 1 h at 100°C. The desired
amount of the functionalized nanotubes (1 mg/ml) was dis-
persed in DMSO by sonication for about 15 min. Pyrrole mono-
mer solution was prepared by dissolving 0.1 M pyrrole in a
solution containing 10 ml of 0.1 M (p‐toluene sulfonic acid) and
30 µl of MWCNT dispersion in DMSO (1 mg/ml) via sonication
for 5–7 min until a homogeneous solution was obtained. The
PPY–MWCNT/PTS electrode was electrochemically deposited
onto the ITO electrode by using a cyclic voltammetric (CV) tech-
nique with a sweeping potential of −0.1 to 800 mV. PPY–
MWCNT/PTS/ITO electrodes were washed with deionized water
(Milli‐Q, Millipore Corporation, Billerica, MA, USA) followed by
phosphate buffer (50 mM, pH 7.0), in order to maintain the pH
over the electrode and is stored at 4°C. PPY/PTS/ITO electrode
was deposited onto ITO glass electrode by using pyrrole contain-
ing 10 ml of 0.1 M PTS solution via cyclic voltammetric technique
with sweeping potential of −0.1 to 700 mV.
Preparation of ChEt–ChOx/PPY–MWCNT/PTS/
ITO bioelectrode
ChEt and ChOx were covalently immobilized onto the electro-
chemically deposited nanocomposite electrode via covalent
bonding between the COOH group of PPY–MWCNT nanocompo-
site and NH2 of the enzymes (ChEt and ChOx) by using EDC as
the coupling agent and NHS as the activator.[42] For enzyme im-
mobilization, binding was achieved when 20 µl of freshly pre-
pared solution of ChEt (1 mg/ml) and ChOx (1 mg/ml) in the
ratio (1:1) with 0.4 M EDC and 0.1 M NHS are uniformly spread
on 1 cm2 of PPY–MWCNT electrode and kept in a humid cham-
ber for about 3 h. Thus fabricated nanobioelectrode (ChEt–
ChOx/PPY–MWCNT/ITO) was then washed with phosphate‐buff-
ered saline (PBS) solution (50 mM, 0.9% NaCl, pH 7.0) containing
0.05% Tween 20 (Sigma‐Aldrich) to remove any unbound en-
zyme and was stored at 4°C when not in use.
Polym. Adv. Technol. (2011)
NANOCOMPOSITE FOR CHOLESTEROL BIOSENSOR

Instrumentation the nanocomposite electrode, which is due to functionalized

MWCNT. In the FTIR spectrum of ChEt–ChOx/PPY–MWCNT/PTS/
The Fourier transform infrared (FTIR) studies of PPY/PTS/ITO, PPY–
ITO nanobioelectrode (curve c), ChOx and ChEt bindings are in-
MWCNT/PTS/ITO electrodes, and ChEt–ChOx/PPY–MWCNT/PTS/
dicated by the appearance of additional absorption bands at
ITO nanobioelectrodes were recorded on spectrophotometer
1524 and 1630 cm-1 assigned to the carbonyl stretch (amide I
BX‐1 (Waltham, MA, USA) in the frequency range, 400–4000/cm.
band) and ―N―H bending (amide II band), respectively.[28] A
Surface morphologies of PPY/PTS/ITO, PPY–MWCNT/PTS/ITO
broad band seen around 3200 cm-1 is attributed to amide bond
electrode and ChEt–ChOx/PPY–MWCNT/PTS/ITO bioelectrode
present in ChOx and ChEt (data not shown in Fig. 1).
were investigated by scanning electron microscope (LEO model;
Figure 2(a–c) shows scanning electron microscopy images
Carl Zeiss AG, Oberkochen, Germany). The electrochemical imped-
obtained for PPY/PTS/ITO and PPY–MWCNT/PTS/ITO electrodes.
ance spectroscopy, CV, and differential pulse voltammetry (DPV)
These micrographs clearly reveal PPY wrapping onto MWCNT
study measurements were conducted in phosphate buffer
through exohedral functionalization, which subsequently causes
(50 mM, pH 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3−/4− in a
increase in the diameter of MWCNT and the blurred edges of
three‐electrode cell consisting of Ag/AgCl as reference, platinum
MWCNT after the composite formation in the nanocomposite
(Pt) as counter electrode, and ITO as a working electrode
electrode (Fig 2(c)).[6] It can be seen (Fig. 2(b)) that MWCNT com-
(0.25 cm2) by using Autolab Potentiostat/Glavanostat Model
prising of small bundles and single tubes are uniformly distrib-
AUT83945 (PGSTAT302N, Metrohm Autolab B.V., Utrecht, The
uted on the surface. Such small bundles and single tubes
Netherlands).
(Fig. 2(c)) assembled homogeneously on the substrate may be
beneficial for the sensor performance. The surface morphology
RESULTS AND DISCUSSION of PPY–MWCNT/PTS/ITO nanobiocomposite electrode further
changes after the immobilization of ChOx and ChEt revealing im-
Characterization of PPY–MWCNT/PTS/ITO and ChEt–ChOx/ mobilization of enzymes (Fig. 2(d)). The covalently immobilized
PPY–MWCNT/PTS/ITO electrodes ChOx–ChEt is well dispersed on the electrode surface. Figure 2
(d) shows that the enzymes are uniformly spread over the nano-
The onset potential of electrochemical polymerization of
composite electrode. The nanocomposite matrix provides in-
MWCNT–PPY nanocomposite electrode on ITO is 800 mV,
creased surface area resulting in enhanced enzyme loading at
whereas for PPY, it is found to be 700 mV. It is observed that
the PPY–MWCNT/PTS/ITO bioelectrode surface.
magnitude of the current increases almost to three times for
the nanocomposite (data not shown) as compared with that of
the individual PPY. This indicates higher electrocatalytic behavior Electrochemical studies
in the nanocomposite electrode than PPY.
Impedance spectroscopy is a powerful tool for characterizing the
Figure 1 exhibits the FTIR spectra obtained for PPY/PTS/ITO
interfacial properties of modified electrodes. The value of elec-
(curve a), PPY–MWCNT/PTS/ITO electrode (curve b), and ChEt–
tron‐transfer resistance (RCT) depends on the dielectric and insu-
ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode (curve c). The FTIR
lating features at the electrode/electrolyte interface. Figure 3
spectra of PPY/PTS/ITO (curve a) and PPY–MWCNT/PTS/ITO
shows the Faradaic impedance spectra, presented as Nyquist
nanobioelectrode (curve b) show peaks at 791 and 830 cm-1 for
plots obtained from real (Z′) and imaginary (−Z″) in the frequency
ring formation and C―H bending. The presence of peaks at
range 0.01–105 Hz for PPY/PTS/ITO (curve a), PPY–MWCNT/PTS/
1010 and 1283 cm-1 is attributed to the C―H in‐plane bending.
ITO electrode (curve b), and ChEt–ChOx/PPY–MWCNT/PTS/ITO
The peak at 1710 cm-1 is attributed to ―C = O stretching vibra-
(curve c) electrode. This behavior originates mainly from the re-
tions, indicating the presence of carboxyl group (―COOH) in
sistance and capacitance of the cell, respectively.[43,44] The diam-
eter of the semicircle is related to the resistance of the charge
transfer reaction and the electronic resistance of the electrode.
70 The values of electron‐transfer resistance (RCT) derived from the
diameter of semicircle of impedance spectra are obtained as
c 2.09 × 103 Ω for PPY/PTS/ITO electrode (curve a), 4.31 × 102 Ω
Transmitance (T%)

1524
830 (curve b) for the PPY–MWCNT/PTS/ITO electrode, and 4.67 × 103 Ω
1630
for ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode, respectively.
60 Compared with the PPY/PTS/ITO electrode, RCT value (Fig. 3)
obtained for PPY–MWCNT/PTS/ITO nanobioelectrode (curve b)
1283
1541
decreases resulting in enhanced electron transfer to the elec-
1010 1710 b trode. This suggests that the presence of MWCNT enhances ionic
a transport in PPY resulting in the formation of complex leading to
50 792
improved charge transfer process. It can be seen that RCT
increases dramatically after the immobilization of ChEt and ChOx
onto PPY–MWCNT/PTS/ITO nanocomposite electrode arising be-
1000 1500 2000 2500
cause of insulating characteristics of ChEt and ChOx molecules to
Wavenumber (cm-1) the electron transport.
Figure 1. Fourier transform infrared spectra of (a) PPY/PTS/ITO elec-
Figure 4 shows DPV curves obtained for PPY/PTS/ITO (curve a),
trode, (b) PPY–MWCNT/PTS/ITO nanocomposite electrode, and (c) ChEt– PPY–MWCNT/PTS/ITO (curve b), and ChEt–ChOx/PPY–MWCNT/
ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode. PPY, polypyrrole; PTS, PTS/ITO electrodes (curve c). The DPV experiments are con-
p‐toluene sulfonic acid ITO, indium–tin–oxide; MWCNT, multiwalled car- ducted in PBS containing 5 mM [Fe(CN)6]3−/4− in the range of
bon nanotubes; ChEt, cholesterol esterase; ChOx, cholesterol oxidase. −0.4 to 1.0 V. The value of the maximum response current

Polym. Adv. Technol. (2011) Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pat
 K. SINGH ET AL.

Figure 2. Scanning electron micrographs of the electrodes (a) PPY/PTS/ITO, (b) PPY–MWCNT/PTS/ITO, (c) PPY–MWCNT/PTS/ITO (enlarged image), and
(d) ChEt–ChOx/PPY–MWCNT/PTS/ITO electrode. PPY, polypyrrole; PTS, p‐toluene sulfonic acid ITO, indium–tin–oxide; MWCNT, multiwalled carbon
nanotubes; ChEt, cholesterol esterase; ChOx, cholesterol oxidase.

obtained as 2.0 × 10−4 A for PPY (curve a) increases to
2.7 × 10−4 A on incorporation of MWCNT into PPY. This suggests
that the conducting nature of MWCNT results in increased
charge transport in PPY. This is attributed to the high electron
communication feature of PPY–MWCNT composite. The magni-
tude of current decreases to 9.1 × 10−5 A for ChEt–ChOx/
PPY–MWCNT/PTS/ITO bioelectrode, indicating slow redox
process at the nanobiocomposite due to insulating character-
istics of ChEt and ChOx revealing immobilization of ChEt and
ChOx on ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobiocomposite.
The single well‐defined oxidation peak for the ChEt–ChOx/
MWCNT–PPY/PTS/ITO bioelectrode is observed at 0.138 V.
Scheme 1 shows the various steps involved in the immobiliza-
tion of the enzymes. It appears that enzymes are linked through

c
b
2.5x10
400
a
2.0x10
300
Current (A)
Z ohms

a
1.5x10

200
1.0x10 c
100
5.0x10
b

0 0.0
0 200 400 600 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0
Z ohms Potential (V)

Figure 3. Impedance spectra of (a) PPY/PTS/ITO electrode, (b) PPY–
MWCNT/PTS/ITO electrode, (c) ChEt–ChOx/PPY–MWCNT/PTS/ITO elec-
trode. PPY, polypyrrole; PTS, p‐toluene sulfonic acid ITO, indium–tin–ox-
ide; MWCNT, multiwalled carbon nanotubes; ChEt, cholesterol esterase;
ChOx, cholesterol oxidase.
Figure 4. Differential pulse voltammetric spectra of (a) PPY/PTS/ITO
electrode, (b) MWCNT/PPY/PTS/ITO electrode, and (c) ChEt–ChOx/PPY–
MWCNT/PTS/ITO electrode. PPY, polypyrrole; PTS, p‐toluene sulfonic acid
ITO, indium–tin–oxide; MWCNT, multiwalled carbon nanotubes; ChEt,
cholesterol esterase; ChOx, cholesterol oxidase.

wileyonlinelibrary.com/journal/pat Copyright © 2011 John Wiley & Sons, Ltd. Polym. Adv. Technol. (2011)
NANOCOMPOSITE FOR CHOLESTEROL BIOSENSOR

ChOx
(Cholesterol Oxidase)

+ >
ChEt
(Cholesterol Esterase)

ITO PY (Pyrrole) (EDC +NHS)

(Indium
Tin +
Oxide) MWCNT (Multi
Electrode Walled Carbon
Nano Tube)

Scheme 1. Fabrication of ChET‐ChOx/PPY‐MWCNT/PTS/ITO electrode.

amide group with COOH group of the functionalized MWCNT via
carbodiimide chemistry and that PPY is attached with MWCNT
through exohedral functionalization via π conjugation.[28] It
seems that MWCNT perhaps act as a “wire,” connecting redox
sites of the enzyme and conducting polymeric PPY electrode
resulting in increased charge transfer rate.
Response studies of ChEt–ChOx/PPY–MWCNT/PTS/ITO
bioelectrode
Electrochemical studies
The DPV technique has been used for the determination of
cholesterol oleate concentration due to its better accuracy
and sensitivity. Inset in Fig. 5 shows that the anodic peak cur-
rent of DPV gradually increases with the increase in cholesterol
oleate concentration. Figure 5 shows the calibration plot of peak
current of DPV curve against concentration (4 × 10−4 to
6.5 × 10−3 M/l). The magnitude of the peak current increases lin-
early with increase in the cholesterol oleate concentration. In
the biochemical reaction, the positive charge on the PPY–
MWCNT/PTS/ITO nanobiocomposite accepts electrons gener-
ated during re‐oxidation of ChEt and ChOx prior to the evolution
of oxygen resulting in enhanced current response of the
nanobiocomposite matrix. The linear enhancement in the peak
current with increased concentration suggests that this nanobio-
composite provides favorable microenvironment to the enzyme
and that MWCNT provide enhanced electron transfer to the elec-
trode.[29] It has been found that the ChEt‐ChOx/PPY–MWCNT/ITO
bioelectrode in the above range for cholesterol follows the
relation: current (A) = 2.38 × 10−4 (A) + 3.06 × 10−4 (A/mM/l) × cho-
lesterol oleate concentration (mM), with standard deviation
8.3 × 10−6 A and regression coefficient (R) as 0.9943. The en-
hanced bioelectrocatalytic oxidation of cholesterol oleate by the
nanobiocomposite electrode is due to the effective charge that
facilitates electrical contact between the redox site of enzymes
and the electrode. All the experiments have been carried out in
triplicate, and the results reveal reproducibility of the system
within 3% error. The detection limit is found to be 0.04 mM/l. The
response time of the biosensor obtained by measuring the time
taken to reach the steady‐state current after applying a steady
voltage of 200 mV for 3 mM of cholesterol oleate solution in
pH 7.0 PBS buffer has been found to be 9 s.
The apparent Michaelis–Menten constant (Km), which gives an
indication of the enzyme–substrate kinetics, can be obtained
from the Lineweaver–Burke equation.[45]
1=I m ¼ 1=Imax þ Km =Imax C (1)

Here, Iss is the steady‐state current after the addition of sub-

f
strate, C is the bulk concentration of substrate, and Imax is the
4.0x10 e
maximum current measured under saturated substrate solution.
d
The value of the enzyme–substrate kinetics parameter (Michaelis–
3.0x10 c Mentenconstant, Km) reveals affinity of the enzyme for desired
b analyte. It is known that Km is dependent on both the nature of
Current (A)

a matrix and the method of immobilization of the enzymes that of-

2.0x10 ten exhibit conformational changes resulting in different values
of Km. Besides this, the value of Km for the bound enzyme can
1.0x10
be lower or higher than that of purified enzyme. We have
obtained the value of Km for the ChEt–ChOx/PPY–MWCNT/PTS/
ITO bioelectrode as 0.02 mM that is smaller than the reported
0.0 value.[29,35–42] The lower Km value indicates a high affinity for
-0.4 -0.2 0.0 0.2 0.4 0.6 cholesterol oleate attributed to the immobilization of ChOx and
Potential (V) ChEt onto PPY–MWCNT/ITO nanobiocomposite for faster bio-
chemical reaction. Furthermore, the bioelectrode in this range
Figure 5. Differential pulse voltammetric (DPV) spectra of ChEt–ChOx/
exhibits a high sensitivity of 30 μA/mM. Scheme 2 shows the
PPY–MWCNT/PTS/ITO electrode in different concentrations of cholesterol
oleate solutions: (a) 0.4, (b) 0.8, (c) 1.5, (d) 3.0, (e) 4.6, and (f) 6.1 mM/l. The
overall biochemical reaction involving [Fe(CN)6]3−/4− as a me-
inset exhibits plot of DPV peak current against the various concentration diating electrolyte and ChEt–ChOx with PPY–MWCNT/ITO
of cholesterol oleate. ChEt, cholesterol esterase; ChOx, cholesterol oxi- electrode.[28,37]
dase; PPY, polypyrrole; MWCNT, multiwalled carbon nanotubes; PTS, The ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode has
p‐toluene sulfonic acid ITO, indium–tin–oxide. been utilized for the estimation of total cholesterol in blood

Polym. Adv. Technol. (2011) Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pat
 K. SINGH ET AL.

Cholesterol Oleate

ChEt

ChOx(Oxi) [Fe(CN)6]3-

ELECTRODE
Fatty acid+ Cholesterol

MWCNT-PPY
nanocomposite e-

Cholest-4-ene-3-one [Fe(CN)6]4-
ChOx(Red)

Scheme 2. Proposed biochemical reaction on the nanobioelectrode.

serum samples. This nanobioelectrode is dipped into the reac-
tion mixture containing 100 µl blood serum in phosphate buffer
(50 mM, pH 7.0, 0.9% NaCl, 5 mM [Fe(CN)6]3−/4−), and DPV mea-
surements are taken using a three electrode system. The total
cholesterol of the blood serum has been calculated from the
DPV calibration curve (Fig. 5). These results are found to be
within 5% error of the values as obtained from clinical analyzer
from Chandra Pathological Lab, New Delhi, India (Table 1).
The performance of ChEt–ChOx/PPY–MWCNT/PTS/ITO bioelectrode
The response of ChEt–ChOx/PPY–MWCNT/PTS/ITO electrode has
been determined as a function of pH from 5.8 to 8.0 at room
temperature (25°C). The peak current increases with increase of
the pH, and the maximum value of the current obtained at
pH 7.0 indicates that nanobioelectrode is most active at pH 7.0.
At this pH, biomolecules retain their natural structures and do
not become denatured. Thus, all experiments have been con-
ducted at pH 7.0, and the nanobioelectrode is found to be stable
up to 40°C.
The effect of potential interferents at normal concentration
such as ascorbic acid (AA), uric acid (UA), glucose (Glu), lactic acid
(LA), sodium pyruvate (S.Pyr), and urea (Ur) present in the blood
samples on the response of ChEt–ChOx/PPY–MWCNT/PTS/ITO
bioelectrode has been investigated using CV studies in PBS con-
taining 5 mM [Fe(CN)6]3−/4−. The oxidation current is measured in
PBS containing equal amounts (1:1) of cholesterol oleate (3 mM)
and AA (05 mM), UA (0.1 mM), Glu (5 mM), LA (0.5 mM), S.Pyr
(5 mM), and Ur (1 mM) on ChEt–ChOx/PPY–MWCNT/PTS/ITO
bars show the variation of the anodic peak current
corresponding to the mixture of cholesterol oleate and interfer-
ents in a 1:1 ratio. The straight line parallel to the X‐axis shows
the observed current in the presence of desired interferents,
revealing a maximum of 3.7% interference. The percentage of
interference (% interference) has been calculated using various
interferents:
% Interference ¼ ½ðI cho –I int Þ= I cho   100 (2)
where ICho is the anodic peak current obtained with 3.7 mM cho-
lesterol oleate concentration, and Iinter is the anodic peak current
corresponding to the mixture of cholesterol and interferents in a
1:1 ratio. The negligible effect is observed with all the interfer-
ents (Fig. 6).
The storage stability of ChEt–ChOx/PPY–MWCNT/PTS/ITO bioelec-
trode has been determined by measuring the change in current re-
sponse at a regular interval of 1 week for about 2 months. This ChEt–
ChOx/PPY–MWCNT/PTS/ITO bioelectrode is stored at 4°C when not
in use. It is found that the bioelectrode exhibits a 98% response after
about 3 weeks. However, we have observed about a 15% decrease
in the amperometric current after about 9 weeks.
120
100
Current (micro amp)

bioelectrode using CV studies in PBS containing 5 mM [Fe(CN)

6]
3−/4−
. In Fig. 6, the first bar (Chol) shows the anodic peak cur- 80
rent obtained with 3.6 mM cholesterol oleate. The remaining
60

40
Table 1. Comparative results of blood serum analysis
20
Sl Total cholesterol content Total cholesterol content
No. (mg/dl) in blood serum (mg/dl) in blood serum
(from DPV results) (from clinical analyzer 0
cho AA Glu Ur LA S.Pyr UA
[Pathological Lab])
Interferents
1 257 249
Figure 6. Effect of various interferents on the response of ChEt–ChOx/
2 240 229
PPY–MWCNT/PTS/ITO bioelectrode AA, Glu, Ur, LA, S.Pyr, and UA. ChEt,
3 193 188 cholesterol esterase; ChOx, cholesterol oxidase; PPY, polypyrrole;
4 180 172 MWCNT, multiwalled carbon nanotubes; PTS, p‐toluene sulfonic acid
DPV, differential pulse voltammetry. ITO, indium–tin–oxide; AA, ascorbic acid; Glu, glucose; Ur, urea; LA, lac-
tic acid; S.Pyr, sodium pyruvate; UA, uric acid.

wileyonlinelibrary.com/journal/pat Copyright © 2011 John Wiley & Sons, Ltd. Polym. Adv. Technol. (2011)
NANOCOMPOSITE FOR CHOLESTEROL BIOSENSOR

Table 2. Characteristics of ChEt–ChOx/PPY–MWCNT/PTS/ ITO bioelectrode compared with other cholesterol electrodes reported
in the literature

Sl No. Components of biosensor Characteristics Reference

1 [Mat]: Laponite NP‐polypyrrolic [S]: 13.2 mA/M Singh et al.[32]
[E]: ChOx, ChEt
[MoI]: Physical entrapment
[M]: Ampero vs Ag/AgCl
2 [Mat]: Tetraethylorthosilicate (sol–gel) [L]: 0.184–12 mM/l Singh et al.[33]
[E]: ChEt–ChOx [RT]: 180 s
[Mol]: Covalent [SL]: 4 weeks
[M]: Photometric [S]: 5.4 × 10−5 Abs./mg/dl
3 [Mat]: PPY [L]: 1–8 mM/l Singh et al.[34]
[E]: ChOx, ChEt [SL]: 4 weeks
[Mol]: Adsorption
[M]: Ampero vs Ag/AgCl
4 [Mat]: Polyaniline [L]: 50–500 mg/dl Singh et al.[35]
[E]: ChEt–ChOx [RT]: 40 s
[Mol]: Covalent [SL]: 6 weeks
[M]: Ampero vs Ag/AgCl [S]: 7.4 × 10−4 nA
5 [Mat]: Polyaniline [L]: Up to 500 mg/dl Singh et al.[35]
[E]: ChEt, ChOx , HRP [Km]: 75 mg/dl
[Mol]: Covalent [RT]: 240 s
[M]: Ampero vs Ag/AgCl [SL]: 6 weeks
[S]: 0.042 μA
6 [Mat]: Polycarbonate [L]: 2–50 mg/dl Basu et al.[36]
[E]: ChOx, ChEt [SL]: 9 weeks
[Mol]: Covalent
[M]: Photometric
8 [Mat]: Au NW‐MEMS [L]: 0.15–7.68 mM Arya et al.[39]
[E]: ChOx, ChEt [Km]: 0.52 mM
[Mol]: Covalent
[M]: Ampero vs Ag/AgCl
9 [Mat]: N‐(2‐aminoethyl)‐3‐aminopropyltrimethoxysilane [L]: 10–500 mg/dl Salinas et al.[41]
[E]: HRP, ChOx, ChEt [Km]: 1.46 mM
[Mol]: Covalent [SL]: 10 weeks
[M]: Ampero vs Ag/AgCl [S]: 124 nA
10 [Mat]: Dithiobissuccinimidyl propionate [L]: 50–400 mg/dl Arya et al.[40]
[E]: ChOx, ChEt [Km]: 0.95 mM
[Mol]: Covalent [SL]: 7 weeks
[M]: Ampero vs Ag/AgCl
11 [Mat]: 3‐Aminopropyl modified [L]: 1.2 µl–1 mM/l Martin et al.[42]
Controlled‐pore glass (APCPG) [Km]: 2 mM
[E]: ChOx, ChEt, HRP [RT]: 120 s
[Mol]: Covalent [SL]: 3 weeks
[M]: Ampero vs Ag/AgCl
12 [Mat]: AT cut quartz crystal — Arya et al.[43]
[E]: ChOx, ChEt
[Mol]:
[M]: Impedance
13 [Mat]: CHIT–SiO2–MWCNT/ITO [L]: 0.15–7.68 mM Chen et al.[29]
[E]: ChEt, ChOx [Km]: 0.52 mM
[Mol]: Covalent
[M]: Ampero (DPV) vs Ag/AgCl
14 [Mat]: Polypyrrole/MWCNT nanocomposite [L]: 0.4 mM to 6.5 mM/l Present work
[E]: ChEt, ChOx [Km]: 0.02 mM
[Mol]: Covalent [RT]: 9 s
[M]: Ampero (DPV) vs Ag/AgCl [SL]: 9 weeks
Mat, matrix; E, enzyme; Mol, type of bonding; M, method of determination; L, linearity; RT, response time; SL, shelf life; S, sensitivity;
ChEt, cholesterol esterase; ChOx, cholesterol oxidase; PPY, polypyrrole; MWCNT, multiwalled carbon nanotubes; PTS, p‐toluene
sulfonic acid ITO, indium–tin–oxide; HRP, horseradish peroxidase; CHIT, chitosan; DPV, differential pulse voltammetry.

Polym. Adv. Technol. (2011) Copyright © 2011 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/pat

Table 2 shows characteristics of the ChEt–ChOx/PPY–MWCNT/
PTS/ITO bioelectrode including those reported in the litera-
ture.[29,32–43] It can be seen that this electrode exhibits higher
shelf life, low response time (9 s), and very low Km (0.02 mM/l)
value as compared with those of other cholesterol electrodes.
CONCLUSIONS
Functionalized MWCNT have been uniformly dispersed and cova-
lently linked with the ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobioe-
lectrode. The ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode
exhibits linearity of 4 × 10−4 to 6.5 × 10−3 M/l, high sensitivity
(30 μA/mM), low Km value of 0.02 mM, detection limit 0f
0.04 mM/l, and fast response time of 9 s by using CV. It has been
shown that this nanobiocomposite electrode can be used to es-
timate total cholesterol in blood serum samples. The unique fea-
tures of the ChEt–ChOx/PPY–MWCNT/PTS/ITO nanobioelectrode
lie in the ease of fabrication, minimum interference, very low Km
value, low response time, and its usefulness for blood serum
samples. It should be interesting to utilize this nanobioelectrode
for development of other biosensors for estimation of triglycer-
ides, lipo‐proteins and UA.
Acknowledgements
We thank Prof. R. C. Budhani, Director, NPL, India, and Dr Ashok
Kumar Chauhan (Founder President, Amity University Uttar Pradesh)
for providing the facilities. We also thank Dr (Mrs) Balvider Shukla,
Director General A.S.E.T, Dr R. P. Singh, Director, AINT, and AUUP
for interesting discussions. We acknowledge the financial assistance
received from the Department of Biotechnology, Govt. of India
(project no. BTPR 11123/MD/32/41/2008 DBT) for the financial
support. We thank Dr Chandra Pathological Lab at Mayur Vihar,
New Delhi, India, for providing the blood serum samples.
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