American Journal of Analytical Chemistry, 2010, 2, 41-46
doi:10.4236/ajac.2010.12006 Published Online August 2010 (http://www.SciRP.org/journal/ajac)
Amperometric Determination of Serum Cholesterol with
Pencil Graphite Rod
Nidhi Chauhan, Jagriti Narang, Chandra S. Pundir*
Department of Biochemistry, Maharshi Dayanand University, Rohtak, India
Received March 14, 2010; revised July 14, 2010; accepted July 20, 2010
Abstract
A cholesterol oxidase from Streptomycin sp. was immobilized onto pencil graphite rod and employed for
amperometric determination of serum cholesterol. The method has the advantage over earlier amperometric
methods that it requires low potential to generate electrons from H2O2, which does not allow ionization of
serum substances. The optimum working conditions of amperometric determination were pH 6.8, 25˚C and
30 s. The current measured was in proportion to cholesterol concentration ranging from 1.29×10-3 to
10.33×10-3 M. Minimum detection limit of the method was 0.09 ×10-3 M. Mean analytical recovery of added
cholesterol (100 mg/dl and 200 mg/dl) in serum was 85.0% & 90.0% respectively. Within batch and between
batch coefficients of variations were 1.59% & 4.15% respectively. A good correlation (r = 0.99) was ob-
tained between serum cholesterol values by standard enzymic colorimetric method and the present method.
No interference by metabolites was observed in the method. The enzyme electrode was reused 200 times
over a period of 25 days, when stored at 4˚C.
Keywords: Cholesterol, Cholesterol Oxidase, Pencil Graphite, Enzyme Electrode, Serum, Amperometric
Biosensor
1. Introduction
Cholesterol an important steroid in human body plays a
vital role as a precursor to various hormones. Cholesterol
determination in blood is known to be clinically impor-
tant for diagnosis of various diseases like cardiac disor-
ders, atherosclerosis, nephritis, diabetes mellitus, myxo-
dema, obstructive jaundice and cerebral thrombosis [1].
Among various methods available for cholesterol deter-
mination, biosensors are comparatively simpler, rapid,
sensitive and specific [2-6]. Various amperometric cho-
lesterol biosensors have been reported, employing cho-
lesterol esterase, cholesterol oxidase and peroxidase im-
mobilized onto nylon mesh over a platinum electrode [7],
octyl-agarose gel activated with cyanogen bromide and
placed in a reactor [8], pyrrole membrane through elec-
tropolymerization and coupled with FIA for H2O2 analy-
sis [9], carbon paste electrode modified with hydroxy-
methyl ferrocene and hydrogen peroxide [10], poly (2-
hydroxyethyl methacrylate) (p(HEMA))/polypyrrole me-
mbrane [11], graphite-teflon composite matrix with in-
corporated potassium ferrocyanide [12], layer of silica
sol-gel matrix on the top of Prussian blue-modified
glassy carbon electrode [13], photosensitive polymer on
ultra-thin dialysis membrane [14], conducing polypyrrole
(PPY) films using electrochemical entrapment technique
[15], porous silicon [16], poly(vinylferrocenium) film
[17]. Due to high electrochemical reactivity, good me-
chanical rigidity, low cost and ease of modification, re-
newal and miniaturization, pencil graphite electrode
(PGE) received attention of workers for its use in work-
ing electrode of biosensor [18]. Furthermore, PGE has a
large active electrode surface area and therefore able to
detect low concentrations and/or volume of the analyte
[19]. The aim of this work was to develop a simple cho-
lesterol biosensor based on PGE bound cholesterol oxi-
dase. The electrode is better in the economic sense and a
small amount of material used; hence it seems to be a
better electrode than “high tech” electrodes described
previously [20].
2. Experimental
4-Amino-phenazone/4-aminoantipyrene, horseradish per-
oxidase from Sigma Aldrich, USA, Triton X-100, Cho-
lesterol, Cholesterol oxidase from Streptomycin sp. (500
units/10mg) from SRL, Mumbai. ‘HB’ lead pencil was
from local market. The kit of enzymic colorimetric

42 N. CHAUHAN ET AL.
method for cholesterol determination was from Erba
Transasia, Daman, India. All other chemicals were of
analytical reagent grade.
2.1. Assay of Free Cholesterol Oxidase
Assay of free cholesterol oxidase was carried out in a 15
ml test tube wrapped with black paper according to Al-
lain et al. (1974) with modification. The reaction mixture,
consisting of 1.8 ml sodium phosphate buffer (0.05 M,
pH 7.0) containing 0.4% Triton X-100, 0.1 ml of cho-
lesterol solution (10mM) and 0.1 ml of cholesterol oxi-
dase solution (13 Units) incubated for 5 min at 37˚C.
Color reagent (1.0 ml) was added and kept at 37˚C for 10
min to develop the colour. A520 was read and the content
of H2O2 was extrapolated from standard curve between
H2O2 concentration and A520. Color reagent consisted 50
mg 4-aminophenazone, 100 mg phenol and 1 mg horse-
radish peroxidase per 100 ml 0.4 M sodium phosphate
buffer (pH 7.0). It was stored in amber colored bottle at
4˚C & prepared fresh after one weak. One enzyme unit is
defined as the amount of enzyme required to generate 1.0
nmol of H2O2 per min per ml.
2.2. Immobilization of Enzyme onto Pencil
Graphite Rod
The wooden cover of a lead pencil was removed from its
both the ends with a sharp blade upto 2 cm height. The
one end of pencil graphite rod (0.15 diameter and 2 cm
long) was dipped into 60% HCl at room temperature for
24 h and then washed thoroughly with 0.05 M sodium
phosphate buffer (pH 7.4). It was dipped again into 70%
HNO3. After keeping it for 24h at room temperature, the
pencil rod was washed thoroughly with the same buffer
and then put into 0.2% enzyme solution. After keeping it
at 4˚C for 8h, the rod was taken off and washed thor-
oughly with the reaction buffer & tested for cholesterol
oxidase activity. The residual enzyme solution was tested
for activity and protein by Lowry method. The pencil
graphite rod containing immobilized enzyme acted as
working electrode (PGE).
2.3. Construction and Response Measurement of
Amperometric Cholesterol Biosensor
An amperometric cholesterol biosensor was constructed
by connecting pencil graphite electrode (PGE) as work-
ing electrode, silver/silver chloride (Ag/AgCl) as refer-
ence electrode and Cu wire as auxiliary electrode
through electrometer/high resistance meter (Keithley
6517A, Japan). To test the activity of this biosensor, the
electrode system was immersed into 1.8 ml 0.02 M so-
dium phosphate solution pH 7.0 and 0.2 ml of cholesterol
(12.9 mM) and polarized at a potential in the range 0-0.4
V versus Ag/AgCl. The current was maximum at 0.1 V.
Hence in the subsequent amperometric studies; the sen-
sor was polarized at 0.1 V to generate current. The elec-
trochemical reactions involved in response meas- ure-
ment are given in Figure 1.
2.4. Optimization of Cholesterol Biosensor
The optimal working conditions of cholesterol biosensor
were studied in terms of the current (mA) generated. To
study optimum pH, the pH of reaction buffer was varied
in the range pH 6.2 to pH 7.8 using the 0.02 M sodium
phosphate buffer. Similarly for optimum temperature, the
reaction mixture was incubated at temperature ranging
from 20 to 50˚C at an interval of 5˚C. Time course was
studied by incubating reaction mixture for different time
ranging from 5 to 40 s at an interval of 5 s. To study
effect of substrate concentration, the concentrations of
cholesterol was varied from 1.29 to 12.9 mM. Km (Micha-
elis Menten constant) and Imax (maximum current) were
calculated from L.B. plot.
2.5. Electrochemical Determination of
Cholesterol in Serum
Blood samples (1.0 ml each) from apparently healthy
male and female (10 each) and diseased persons (suffer-
ing from coronary heart diseases, hypertension and
atherosclerosis) were collected from local Pt BD Sharma
Post Graduate Institute of Medical Sciences, Rohtak, and
centrifuged at 5000 rpm for 5 min and their supernatant
(serum) was collected. Cholesterol content in serum was
determined by the present biosensor in the similar man-
ner as described for its response measurement, under its
optimal working conditions except that cholesterol was
replaced by serum. The current (mA) was measured and
the amount of cholesterol in serum extrapolated from
standard curve between cholesterol concentrations and
current (in mA) prepared under optimal working condi-
tions (Figure 2).
2.6. Evaluation of Cholesterol Biosensor
The biosensor was evaluated by studying analytical re-
Figure 1. Chemical reactions involved in generation of
electron in amperometric cholesterol biosensor based on
pencil graphite electrode (PGE) bound cholesterol oxidase
(ChOx).

N. CHAUHAN ET AL.
43
0
10
20
30
40
50
0100 200 300 400 500 600
Cholesterol (mg/dl)
Current (mA)/s
Figure 2. Standard curve of cholesterol by biosensor based
on pencil graphite rod bound cholesterol oxidase.
covery, precision and correlation. The effect of various
metals and metabolites found in blood, such as uric acid,
cholesterol, ascorbic acid, bilirubin, glucose, pyruate and
glutathione were studied at their physiological concen-
tration.
3. Results and Discussions
3.1. Immobilization of Cholesterol Oxidase onto
PG Rod and Construction of Amperometric
Cholesterol Biosensor
Commercial cholesterol oxidase from Streptomyces spe-
cies was immobilized on PGE through chemisorption
(Figure 3). The HCl treatment of PGE forms a mono-
layer which helps in electrostatic interaction of nega-
tively charged cholesterol oxidase at pH 7.0. Neverthe-
less treatment of graphite with HNO3 makes it highly
porous to provide the large surface area for adsorption or
chemical reaction [21]. The chemisorption is better than
physisorption for immobilization of enzyme which is
characterized by weak Van der Wall forces. A method is
described for construction of amperometric cholesterol
biosensor based on this PGE bound with cholesterol
oxidase. The biosensor showed optimum response at low
voltage i.e. 0.1 V had advantage that it does not allow the
ionization of number of serum substances which get ion-
ized at high voltage and interfere in current measurement
[9].
3.2. Optimization of Cholesterol Biosensor
The optimum response for pencil graphite electrode was
at pH 6.8 (Figure 4), which is comparable to earlier re-
ports pH 7.0 [8,9,15,24] and pH 7.5 [22].The PGE
showed optimum response at 40˚C (Figure 5), which is
higher than that of free enzyme in presence of free cho-
lesterol esterase and peroxidase (30˚C). The increase in
optimum temperature might be due to change in confor-
mation of enzyme after immobilization or due to steric
hindrance. PGE response was increased from 5 to 30 s
after which it became stable (Figure 6). Therefore in all
subsequent assays, the electrometer reading was recorded
at 30 s. A hyperbolic relationship was observed between
electrode response (current in mA/s) and cholesterol
concentration up to a final concentration of 12.9 × 10-3 M,
which is similar to earlier cholesterol biosensor , but
higher than 1 × 10-3 M to 8 × 10-3 M for cholesterol ester
[15], and 8 × 10-3 M for cholesterol [22]. Lineweaver-
Burk plot between the reciprocals of cholesterol concen-
tration and response of PGE working electrode was lin-
ear. Km (Michaelis constant) for cholesterol was 7.38 ×
10-3 M (Figure 7) which is lower than that for earlier
cholesterol biosensor (21.2 × 10-3 M) [23] and 19.6 ×
10-3 M [9]. This might be due to the hydrophobic forces
of pencil graphite, which facilitate the cholesterol bind-
ing with the graphite bound enzyme. Imax was 62.5 mA/s.
3.3. Evaluation of Cholesterol Biosensor
3.3.1. Linearity
A linear relationship was obtained between cholesterol
concentrations ranging from 1.29 × 10-3 M to 10.3 × 10-3
M and current (mA) measured (Figure 2).
Figure 3. Chemisorption immobilization of cholesterol oxi-
dase onto pencil graphite rod.
0
10
20
30
40
50
60
6.2 6.46.6 6.877.2 7.47.6 7.8
pH
Current (m A)/s
Figure 4. Effect of pH on the response of cholesterol bio-
sensor based on pencil graphite rod bound cholesterol oxi-
dase. Standard assay conditions were used except for the
pH that was varied from pH 6.2-7.8.

44 N. CHAUHAN ET AL.
0
10
20
30
40
20 25 30 35 40 45 50
Temperature (oC)
Current (mA)/s
Figure 5. Effect of incubation temperature on the response
of cholesterol biosensor based on pencil graphite rod bound
cholesterol oxidase. Standard assay conditions were used
except for the incubation temperature which was varied
from 15-45˚C.
0
5
10
15
20
25
0510 15 2025 30 3540 45
Time (s)
Current (mA)
Figure 6. Effect of time of incubation on the response of
cholesterol biosensor based on pencil graphite bound cho-
lesterol oxidase.
0
0.02
0.04
0.06
0.08
0.1
0.12
-0.01-0.00500.005 0.01 0.0150.02 0.025
1/[cholesterol] mg/dl
-1
___1___
mA/min
Figure 7. A Lineweaver-Burk plot for cholesterol biosensor
based on cholesterol oxidase immobilized onto pencil
graphite rod.
3.3.2. Minimum Detection Limit
The minimum detection limit of the present amperomet-
ric biosensor was 0.09×10-3 M, which is almost 3 times
lower than paleographic method employing soluble en-
zymes (0.32 × 10-3 M) [24], but higher than those meth-
ods employing silica gel bound enzyme (0.003 × 10-3 M)
[25] and amperometric cholesterol biosensor (0.064 ×
10-3 M) [9].
3.3.3. Analytical Recovery
In order to check the accuracy of the method, the ana-
lytical recovery of added cholesterol in the serum sam-
ples was determined. The mean analytical recovery of
added cholesterol (100 mg/dl and 200 mg/dl) in serum
was 85% (100 mg/dl) and 90% (200 mg/dl) (Table 1),
which is comparable with colorimetric method employ-
ing alkyl amine glass bound enzyme (95-102%) [26],
amperometric method (95-101% recovery) [7], enzymic
fluorometric method (103-104 %) for added cholesterol
concentration of 150 mg/dl and 50 mg/dl [26].
3.3.4. Precision
To check the reproducibility and reliability of the meth-
ods, the cholesterol content of the sample in one run
(Within batch) and after storage at -20˚C for one week
(Between batch) were determined. The results showed
that the cholesterol value of these determination agreed
with each other and within batch and between batch
coefficient of variation (CV) were 1.59% & 4.15 %
(Table 2), which is quite close to earlier reports such as
colorimetric, electrochemical method [26] employing
alkyl amine glass bound enzyme (1.6% for intrabatch
and 3.2% for interbatch), amperometric method using
silica gel bound enzyme (< 1.5% for all samples) [7] and
flow injection method employing controlled pore glass
bound cholesterol esterase and cholesterol oxidase
(within day < 1.0 and between day < 2.5%) [27], meas-
uring cholesterol after precipitation with phosphotugstic
acid/MgCl2 (within day 5.0 % and between day 8.2%)
[28] and amperometric detection of cholesterol (within
day 2%–between day 4%) [9]. The low coefficient of
variation values indicated the accuracy, reproducibility
and reliability of the method.
3.3.5. Accuracy
In order to know the accuracy of present method, the
level of cholesterol in 10 serum samples was determined
by standard enzymic colorimetric method with modifica-
tion and compared with those obtained by present
method. The serum cholesterol values obtained by stan-
dard enzymes colorimetric method (x) agreed with the
present biosensor (y) with a good correlation (r = 0.99)
(Figure 8).
3.3.6. Effect of metal ions and metal salts
The effect of some metal salts such as KCl, MgCl2, NaCl,
CaSO4, CuSO4, ZnSO4, CaCl2·2H2O and MgSO4·7H2O,

N. CHAUHAN ET AL.
45
Figure 8. Correlation between serum cholesterol value as
determined by enzo kit method employing free enzymes (x
axis) and present biosensor method (y-axis) based on pencil
graphite rod bound cholesterol oxidase.
Table 1. Analytical recovery of added cholesterol in serum
by biosensor based on pencil graphite electrode.
Cholesterol added
(mg/dl)
Cholesterol found
(mg/dl)
% Recov-
ery
Nil 150 -
100 235 85
200 330 90
Serum cholesterol was measured by cholesterol biosensor as described
in text. It was measured again after adding cholesterol into serum at
100 mg/dl & 200 mg/dl. % Recovery was calculated. Values are mean
of six serum samples.
Table 2. Precision measurement of serum cholesterol by a
cholesterol biosensor based on pencil graphite electrode
(PGE).
Total number of samples
(n = 6)
Mean Cholesterol
(mg/dl) % CV
6 (within assay) 54.25 1.59
6 (between assay)Ψ 54.2 4.15
Cholesterol was measured in six serum samples six times on the same
day (Within assay) and after one week storage at -20oC (Between as-
says) by cholesterol biosensor based on pencil graphite rod bound
cholesterol oxidase. % Coefficient of variation (CV) was calculated.
each at a final concentration of 1.0 mM was tested on the
response of the working electrode. Only Mg2+ caused
slight stimulation, while rest metals had practically no
effect.
3.3.7. Effect of Serum Metabolites
To study interference by serum metabolites glucose, uric
acid, ascorbic acid, acetone and bilirubin were added into
the reaction mixture at their normal physiological con-
centration before addition of cholesterol. The results
showed that there was practically no interference in
presence of these metabolites. Earlier uric acid and
ascorbic acid, at 0.6 V caused significant increase in the
value of current [29], which were attributed to the fact
that at high potentials for both uric acid & ascorbic acid
got oxidized contributing to oxidation current. Some
interference of endogenous electro reactive species like
uric acid, glucose had been reported when their concen-
tration was higher than their normal physiological con-
centrations [15].
3.4. Storage Stability and Reusability
The PG electrode lost 50% of its initial activity after its
regular use for 200 times over a period of 25 days, when
stored in 0.05 M sodium phosphate buffer, pH 7.0 at 4˚C.
4. Conclusions
A method is described for immobilization of cholesterol
oxidase onto pencil graphite (PG) rod and its use in con-
struction of a simple amperometric cholesterol biosensor.
The biosensor had an advantage that it worked at low
potential and thus had no interference by serum sub-
stances. The sensor was evaluated.
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