ACTA FACULTATIS PHARMACEUTICAE UNIVERSITATIS COMENIANAE

Tomus LII 2005

INTERFERENCE-FREE AMPEROMETRIC BIOSENSOR

FOR DIRECT CHOLESTEROL DETERMINATION
Katrlk, J. Valach, M. Jantoov, L.
Department of Pharmaceutical Analysis and Nuclear Pharmacy,
Faculty of Pharmacy, Comenius University, Bratislava

Amperometric biosensors enable rapid analyses with a minimum sample pretreatment. In this
work interference-free biosensor for direct amperometric determination of cholesterol is presented.
The biosensor system consists of a planar gold electrode modified with immobilised enzyme
peroxidase, insoluble mediator, layers of acetate cellulose and enzyme cholesterol oxidase (ChOx)
in a buffer solution. Both layers of acetate cellulose and low applied potential vs. reference
electrode lead to exclusion of interferents as ascorbate, urea, lactate, glucose, nitrite and nitrate on
biosensor response, so direct cholesterol determination without need of sample pretreatment is
enabled. The cholesterol can be analysed in range of concentrations of 1.1 40 mol/l (linear
range) in the buffer solution, what means concentrations range of 0.55 20 mmol/l in the sample
at dilution ratio 1:500. The base biosensor transducer (without ChOx in solution) can be also used
for the rapid hydrogen peroxide determination in range of concentrations of 1 250 mol/l in the
buffer solution. The base biosensor transducer also showed a good storage stability, after 50 days
of the storage about 77 % of the original sensitivity was obtained.
Key words: biosensor enzyme electrode amperometric electrochemistry electrode cholesterol cholesterol oxidase hydrogen peroxide

INTRODUCTION
Many studies demonstrate that increased concentrations of cholesterol are related to
cardiovascular diseases [1-3]. High cholesterol levels in blood signalise danger of either
formation or occurrence of hypertension, hyperthyroidism, anaemia and coronary artery
diseases. The monitoring of blood cholesterol concentrations is important both in
prevention and therapy, particularly in the case of risk people and persons treated with
hypolipidemics. Cholesterol determination is also significant for the investigation of
relation between cholesterol blood level and causation and prevalence of cardiovascular
diseases, because this research is still in progress. For all this purposes is necessary to
employ rapid and accurate analytical technique to determine cholesterol concentrations.
116

Prevalent techniques are (i) well known Lieberman-Burchard reaction involved

colorimetric determination of cholesterol in chloroform after precipitation of proteins
and extraction of cholesterol, (ii) colorimetry of coloured dyes produced during
enzymatic reaction of 4-aminoantipyrine and phenol with hydrogen peroxide (hydrogen
peroxide is produced by oxidation of cholesterol with an enzyme cholesterol oxidase
(ChOx)), catalysed with an enzyme horseradish peroxidase (HRP) [4], and (iii)
separation methods, mostly HPLC [5]. However, these methods need either
preseparation of serum and plasma to avoid optical interferences (colorimetry), either
pretreatment of the sample (HPLC), or a lot of time and reagents (Lieberman-Burchard
reaction).
Biosensors are analytical devices consisted of two components: a bioreceptor and a
transducer. The bioreceptor is a biomolecule that recognises the target analyte whereas
the transducer converts the recognition event into a measurable signal. The simplicity
and the high speed of measurement are the main advantages of biosensors. Among
biosensors electrochemical biosensors have the most important position, especially
amperometric ones.
Amperometric biosensors are also used for appropriate determination of cholesterol,
what means both rapidity and no need of sample pretreatment. ChOx contains flavin
adenine dinucleotide (FAD) as the active redox centre. The amperometric detection of
cholesterol can be based on enzymatic reaction employing ChOx and this reaction is
followed by electrochemical oxidation of the mediator (M1) on the electrode surface
(Eqs. 1, 2).

ChOx

cholest-4-enone + M2red

cholesterol + M1ox
M1red

electrode

M1ox + e

cholesterol + O2

ChOx

cholest-4-enone + H2O2

(1)
(2)
(3)

The oxygen is a physiological mediator for ChOx (Eq. 3). In the case of its
utilisation, it can be electrochemically measured either speed of oxidation of produced
H2O2 (increase of concentration) or speed of reduction of consumed O2 (decrease of
concentration). However, both types of electrochemical detection of H2O2 and O2 have
some disadvantages discussed in the amperometric determination of glucose using
biosensors [6,7]. The another way is to replace O2 by some redox compounds artificial
mediators. There were used different compounds as mediators e.g. phenothiazine dyes as
thionin [8], derivates of polynaphthalene [9] and Os-complex [10].
Produced H2O2 in the case of utilisation of oxygen can reacts in the presence of the
enzyme HRP with the mediator (M2) in its reduced form (Eq. 4), and oxidised mediator
can be electrochemically reduced on the electrode surface (Eq. 5). This concept was used
also in this work. Although it suffers from influence by changes of the concentration of
dissolved O2, this is not crucial fact. It provides a good sensitivity, a possibility of
formation of polymeric layer to exclude interferences and enables to apply low potential
to eliminate interferences.
117

H2O2 + 2H+ + M2red

M2ox + e

electrode

HRP

M2ox + 2H2O

M2red

(4)
(5)

The fundamental problems in whole blood analyses are interferences caused by

compounds such as hemoglobin, proteins, ascorbic acid, residues of drugs etc. In
biosensor systems the interferences are suppressed by layer or multilayer fabrication
[11]. This method enables analyses without sample purification or pretreatment. The
biosensor device for total cholesterol and its subfractions determination based on feeder
strip where the sample is transferred by capillary action was developed [12]. There can
be named also optical biosensors based on above mentioned colorimetry where the
system of layers enables to eliminate the need of sample preseparation and can be used
direct for whole blood samples [13]. The optical cholesterol biosensors are also on
market supplied as test strips by Roche. Amperometric cholesterol biosensor was
developed and commercialised by Medisense.
The total content of cholesterol in blood is sum of free cholesterol and its esters of
fatty acids. To determine this total content by a biosensor, the cholesterol biosensor can
be moreover modified with enzyme cholesterol esterase [8] which hydrolyses cholesterol
esters to free cholesterol and fatty acids. The released cholesterol is then consecutively
determined by cholesterol biosensor with naturally free cholesterol as a sum of total
cholesterol.
In this paper, results of the development of cholesterol amperometric interference-free biosensors based on ChOx and HRP, cellulose acetate membrane and insoluble
mediator are described.

EXPERIMENTAL
Cholesterol oxidase (ChOx) (EC 1.1.3.6) from Pseudomonas fluorescens with
activity 5.2 U/mg and horseradish peroxidase (HRP) with activity 116 U/mg
commercially available from Sigma. All enzymes were used without further purification.
Nafion and cellulose acetate were used for protective layer forming. Ferrocenyl
methanol (Fluka) was used as mediator. Cholesterol were purchased from Sigma and
hydrogen peroxide from MikroChem Bratislava and they were used for preparation of
standard solutions. Reagents used for enzyme immobilisation were cellulose acetate
(Sigma), glutaraldehyde (Sigma), Triton X (Fluka), Nafion (Fluka), poly(carbamoyl)
sulfonate (PCS) (SensLab Leipzig) and polyethyleneglycol diglycidylether (PEG-DGE)
PEG 400 (Polysciences). Other chemicals were of analytical grade and commercially
available. For preparation of aqueous solutions deionized water was used.
The cholesterol as a hydrophobic compound is practically insoluble in pure water.
For this fact there was used Triton X up to 4% w/w. In prepared standard solutions the
concentration of cholesterol was in range of millimoles.
The gold planar electrodes (active surface diameter of 1.6 mm) were purchased from
Institute of Informatics, SAS, Bratislava. These electrodes were used for a biosensor

118

preparation. Active surface of the electrode was rinsed with ethanol. Appropriate volume
of solution of HRP containing 2.3 U of enzyme was applied and spread over the active
surface. After drying at room temperature the bottom of the electrode with active surface
was immersed into solution containing mediator (0.4% w/w) and cellulose acetate (2%
w/w) in acetone to fix both enzyme onto electrode surface and mediator inside polymeric
layer. After drying the next layer containing only cellulose acetate (2% w/w) in acetone
was applied. In some experiments another layer containing ChOx was also applied.
However, the surface composition of the biosensor could be changed in dependence on
actual experiment. Prepared biosensors were stored dry at 4 C.
The electrochemical detection of cholesterol was carried out using 0.1 mol/l
phosphate buffer pH 7.0 (5 ml) containing 0.8 U ChOx. All experiments were carried out
at room temperature in a vessel equipped with a magnetic stirring. Two-electrode
system, prepared biosensor and saturated calomel reference electrode (SCE) (ED
Turnov) was used connected with potentiostat PST-3 (FEI STU Bratislava) and recorder
TZ 4620 (Laboratorn pstroje Praha). The applied potential was of -30 mV vs. SCE.
Current-time response curves were recorded. The height of the recorded wave (current
increase) corresponding to the concentration of analyte was evaluated. The portions of
analyte solutions were added with micropipette.

RESULTS AND DISCUSSION

For the preparation of cholesterol biosensor there has been chosen system containing
enzymes ChOx and HRP and insoluble mediator onto planar electrode. This composition
can secure good sensitivity of the biosensor and possibility of formation of polymeric
layer to exclude interferences. The mediator ferrocenyl methanol was used because of its
good electrochemical properties. Its electrochemical reduction at potentials about 0 V vs.
SCE helps to reduce interferences and also is water-insoluble. As a suitable potential for
its reduction onto gold planar electrode was found potential of -30 mV. Measurement at
this potential ensures the elimination of majority of electrochemical interferences.
It is important to achieve sufficient sensitivity of the ChOx-free biosensor (only with
HRP and mediator) to hydrogen peroxide to obtain a good performance of complete
cholesterol sensor. This sensitivity depends on activity of HRP, amount of mediator and
thickness of polymeric layer. In the case of high loading of mediator, after addition of
H2O2 standard rapid increase of current (for about 1 second) was observed following by
slightly slower decrease of current. One minute after addition of H2O2 the current
achieved its prior value. The created membrane of cellulose acetate contains likely so
high amount of mediator that this amount can not be incorporated in and the mediator
leaks into solution. After addition of H2O2 this superfluous mediator catalyses
decomposition of hydrogen peroxide in the solution. These experiments were carried out
with sensor modified by HRP and solution containing mediator (2% w/w) and cellulose
acetate (2% w/w) in acetone. The sensors were immersed into solution of mediator and
cellulose acetate in acetone 1, 2, or 3 times, to load different amount of mediator and
polymer, and the results were practically the same for all prepared sensors.
In the case of sensors modified by HRP and solution containing only 0.4% w/w of
mediator (the amount of cellulose acetate was 2% w/w) the decomposition of hydrogen

119

peroxide was not already observed. To secure no leakage of mediator into solution this
sensor was immersed into solution of cellulose acetate (2% w/w) in acetone to form
second, mediator-free protective layer. Though the sensitivity of this biosensor was
lower, after its reuse the decrease of sensitivity was only small, in comparison with one-layer sensor (Fig. 1). Also, the shape of calibration curve of one-layer biosensor
indicates leakage of the mediator. One protective layer is suitable to protect leakage of
the mediator. The increase of number of protective layers led to increase of response
time.
400

i [nA]

300

200

100

0
0

100

200

300

400

c (H2O2) [mol/l]
Fig. 1. Dependences of the current changes on hydrogen peroxide concentration
of the biosensor modified with HRP and: () layer of mediator-cellulose acetate, ()
second measurement with previous one-layer electrode, () layer of mediator-cellulose acetate and another layer of cellulose acetate and (
) second measurement
with previous two-layer electrode. Conditions of measurements: 0.1 mol/l pH 7.0
potassium phosphate buffer, working potential of -30 mV vs. SCE

The response of two-layer biosensor sensitive to hydrogen peroxide with different

HRP loading (0.46, 0.93, 2.32 and 4.64 U) was measured. The sensitivity of biosensor
with the highest loading of enzyme (HRP 4.64 U) was the highest on the other hand the
signal was very noised, so for further experiments electrode with amount of HRP of 2.3
U was chosen. The response of this biosensor had a good sensitivity, an adequate linear
range (up to 250 mol/l of H2O2) and the signal was of low noise. This sensor can be
also used for hydrogen peroxide detection.
The immobilization of ChOx on surface of bioelectrode sensitive to hydrogen
peroxide was carried out by different methods. These methods included (i) simple
physical absorption, (ii) immobilization under cellulose acetate membrane, (iii)
immobilization under Nafion membrane, (iv) reticulation with glutaraldehyde, (v)
entrapment by a poly(carbamoyl) sulfonate (PCS) hydrogel [14] and (vi) covalent
binding with PEG-DGE [15]. However, not a single one was successful. The reasons
were weak adhesion of enzyme to the surface (i), denaturation of enzyme during
chemical reaction with binding agent (iv, vi), too small membrane pores of diameter for
cholesterol (ii) and probably hydrophobic nature of cholesterol that did not enable to

120

pass molecules of cholesterol through created hydrophilic membranes (iii, v). This was
a reason for using of dissolved ChOx in solution.
The found optimum amount of ChOx was 0.83 U in 5 ml of buffer solution. The
increase of enzyme amount did not increase the biosensor sensitivity, and the decrease
led to decrease of sensitivity. The calibration curve of such biosensor is shown on Fig. 2.
It can be seen that the biosensor response is linear up to 40 mol/l of cholesterol.
70
60

i [nA]

50
40
30
20
10
0
0

50

100

150

200

250

300

350

400

c (cholesterol) [mol/l]
Fig. 2. Dependence of the current changes on concentration measured
with biosensor. Conditions of measurement: 0.1 mol/l pH 7.0 potassium phosphate
buffer containing 0.83 U ChOx in 5 ml of buffer solution, working potential
of -30 mV vs. SCE

The determination limit of the biosensor was 1.1 mol/l of cholesterol in a buffer
solution. This value is sufficient for determination of cholesterol in biological samples.
In the case of sample dilution during sample addition by factor of 500 (10 l of sample
into 5 ml of buffer solution), the determinable concentration of cholesterol in sample is
0.55 mmol/l.
The tested interferents were ascorbate, urea, lactate, glucose, nitrite and nitrate. The
concentrations of these interferents were three times higher than their limit blood
concentrations are. The sample dilution was again 500 times during measurement. No
response to any interferent was observed. This is caused by using of low potential of
-30 mV vs. SCE (removal of electrochemical interferences) and by applying of layers of
acetate cellulose (retardation or stopping of diffusion of large and middle large
molecules, while hydrogen peroxide can diffuse through the membrane).
The model samples of cholesterol were prepared with concentration of 1 to
10 mmol/l. The model samples contains cholesterol, 10 mmol/l of urea, 3 mmol/l of
lactate, 0.5 mmol/l of ascorbate, 5 mmol/l of glucose and 0.3 mmol/l of nitrite. During
measurement, 10 l of sample was added into 5 ml of buffer solution. The response was
evaluated by method of standard addition. The results are shown in Table 1.

121

Table 1. Analyses of model samples by amperometric cholesterol biosensor

c1,s [mmol/l]
1.050
1.275
2.550
5.100
7.650
10.200
c1,s
c1,m
c2,s
c2,m
er

c1,m [
mol/l]
2.10
2.55
5.10
10.2
15.3
20.4

c2,s [mmol/l]
0.997
1.230
2.500
5.386
7.707
10.155

c2,m [
mol/l]
1.994
2.461
5.001
10.772
15.415
20.310

er [%]
-5.04
-3.50
-1.95
5.61
0.75
0.44

concentration of cholesterol in model sample

concentration of cholesterol in measured buffer after sample addition (dilution 1:500)
concentration of cholesterol in model sample determined by cholesterol biosensor
concentration of cholesterol in measured buffer after sample addition (dilution 1:500)
determined by amperometric cholesterol biosensor
relative error

The operational stability of the biosensor was tested by repeated measurements of

20 mol/l solution of cholesterol. Each one was carried out in a new buffer solution
containing ChOx. The sensitivity of the biosensor decreased to about 74 % of its initial
value after 15 measurements. For evaluation of the storage stability, six sensors were
stored dry at 4 C. The sensitivity was checked once in ten days, whilst each
measurement was carried out with a new biosensor. After 50 days of the storage about
77 % of the original sensitivity was obtained.
In both cases, the decrease of the sensitivity of approximately 75 % still enables use
of biosensor for cholesterol determination.

CONCLUSIONS
Interference-free amperometric biosensors for cholesterol determination have been
described. The developed biosensor system was based on planar gold electrode modified
with immobilised enzyme HRP, mediator, layers of acetate cellulose and enzyme ChOx
in buffer solution. This device enables direct cholesterol determination of cholesterol in
samples with concentrations of physiological level without need of sample pretreatment.
The base transducer (without ChOx in solution) can be also used for rapid hydrogen
peroxide determination.
This concept can be transformed to strip biosensor system with simple argentochloride reference electrode of small diameter that could be used for direct amperometric
cholesterol determination in a drop of sample. This research is still in progress.
Acknowledgement: The present work was supported by the Scientific Grant Agency of the
Ministry of Education of the Slovak Republic (VEGA Grant No. 1/1196/04) and by the Grant of
Comenius University Bratislava (Grant No. UK/126/2004).

REFERENCES
1. BITTMAN, R.: Cholesterol: Its Functions and Metabolism in Biology and Medicine. New
York, Plenum Press 1997.

122

2. BOWMAN, T.S. SESSO, H.D. MA, J. KURTH, T. KASE, C.S. STAMPFER, M.J.
GAZIANO, J.M.: Cholesterol and the risk of ischemic stroke. Stroke 34, 2003, p. 2930-2934.
3. RIFAI, N. MA, J. SACKS, F.M. RIDKER, P.M. HERNANDEZ, W.J.L.
STAMPFER, M.J. MARCOVINA, S.M.: Apolipoprotein(a) size and lipoprotein(a)
concentration and future risk of angina pectoris with evidence of severe coronary
atherosclerosis in men: The physicians' health study. Clin. Chem. 50, 2004, p. 1364-1371.
4. NANJEE, M.N. MILLER, N.E.: Sequential microenzymatic assay of cholesterol,
triglycerides, and phospholipids in a single aliquot. Clin. Chem. 42, 1996, p. 915-926.
5. HIROWATARI, Y. YOSHIDA, H. KUROSAWA, H. DOUMITU, K. TADA, N.:
Measurement of cholesterol of major serum lipoprotein classes by anion-exchange HPLC with
perchlorate ion-containing eluent. J. Lipid Res. 44, 2003, p. 1404-1412.
6. CASS, A.E.G. DAVIS, G. FRANCIS, G.D. HILL, H.A.O. ASTON, W.J. HIGGINS,
I.J. PLOTKIN, E.V. SCOTT, L.D.L. TURNER, A.P.F.: Ferrocene-mediated enzyme
electrode for amperometric determination of glucose. Anal. Chem. 56, 1984, p. 667-671.
7. KATRLIK, J. BRANDSTETER, R. SVORC, J. ROSENBERG, M. MIERTUS, S.:
Mediator type of glucose microbial biosensor based on Aspergillus niger. Anal. Chim. Acta
356, 1997, p. 217-224.
8. NAKAMINAMI, T. ITO, S. KUWABATA, S. YONEYAMA, H.: Amperometric
determination of total cholesterol at gold electrodes covalently modified with cholesterol
oxidase and cholesterol esterase with use of thionin as an electron mediator. Anal. Chem. 71,
1999, p. 1068-1076.
9. VIDAL, J.C. ESPUELAS, J. GARCIA-RUIZ, E. CASTILLO, J.R.: A polymeric bilayer
configuration for a cholesterol amperometric biosensor based on the combination of
overoxidized polypyrrole and a polynaphthalene derivative. Anal. Lett. 35, 2002, p. 837-853.
10. ZHANG, C.X. GAO, Q. AIZAWA, M.: Flow injection analytical system for glucose with
screen-printed enzyme biosensor incorporating Os-complex mediator. Anal. Chim. Acta 426,
2001, p. 33-41.
11. GOBI, K.V. MIZUTANI, F.: Layer-by-layer construction of an active multilayer enzyme
electrode applicable for direct amperometric determination of cholesterol. Sensor. Actuat. B
Chem. 2001, p. 272-277.
12. FOSTER, R. CASSIDY, J. O'DONOGHUE, E.: Electrochemical diagnostic strip device
for total cholesterol and its subfractions. Electroanal. 12, 2000, p. 716-721.
13. RAM, M.K. BERTONCELLO, P. DING, H. PADDEU, S. NICOLINI, C.: Cholesterol
biosensors prepared by layer-by-layer technique. Biosens. Bioelectron. 16, 2001, p. 849-856.
14. GRNDING, B.: Application of polycarbamoylsulfonate(PCS) for fabrication of enzyme
sensors. Landbauforsch. Vlk. SH 241, 2002, p. 63-70.
15. OHARA, T.J. RAJAGOPALAN, R. HELLER, A.: Glucose electrodes based on cross-linked [Os(bpy)2Cl]+/2+ complexed poly(1-vinylimidazole)films. Anal. Chem. 65, 1993,
p. 3512-3517.

Registered: March 24, 2005

Accepted: May 17, 2005

Ing. Jaroslav Katrlk, PhD.

Faculty of Pharmacy
Comenius University
Odbojrov 10
832 32 Bratislava
Slovak Republic
katrlik@fpharm.uniba.sk

123

AMPEROMETRICK BIOSENZOR PRE PRIAME STANOVENIE

CHOLESTEROLU BEZ VPLYVU INTERFERENCI
Katrlk, J. Valach, M. Jantoov, L.
Katedra farmaceutickej analzy a nuklernej farmcie, Farmaceutick fakulta,
Univerzita Komenskho, Bratislava
Amperometrick biosenzory umouj rchle analzy, ktor nevyaduj prakticky iadnu
predpravu vzorky. V tejto prci je predstaven amperometrick biosenzor na priame stanovenie
cholesterolu bez vplyvu interferenci. Opsan biosenzorov systm sa sklad z plochej zlatej
elektrdy pokrytej vrstvou imobilizovanho enzmu peroxidzy, z nerozpustnho meditora,
s vrstvami acettu celulzy a enzmu cholesteroloxidzy (ChOx) v meranom roztoku. Vrstvy acettu celulzy spolu s nzkou hodnotou vkladanho potencilu zabezpeuj potlaenie vplyvu
interferentov (askorbt, moovina, laktt, glukza, dusitany, dusinany) na odozvu elektrdy.
Tmto je umonen priame stanovenie cholesterolu bez potreby predpravy vzorky. Cholesterol
me by analyzovan v koncentrcich 1,1 40 mol/l (linerny rozsah) v meranom roztoku, o
zodpoved koncentrcim 0,55 20 mmol/l vo vzorke riedenej v pomere 1:500.
Zkladn prevodnk biosenzora (bez ChOx v roztoku) me by pouit na rchle stanovenie
koncentrci peroxidu vodka v rozsahu 1 250 mol/l v meranom roztoku. Tento zkladn prevodnk biosenzora m tie dobr skladovaciu stabilitu. Po 50 doch skladovania klesla jeho
citlivos na 77 % pvodnej citlivosti.

Acta Facult. Pharm. Univ. Comenianae 52, 2005, p. 116-124.

124