a new amperometric carbon paste enzyme electrode for ethanol determination
TRANSCRIPT
This article was downloaded by: [University of Waterloo]On: 27 November 2014, At: 06:07Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office:Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Analytical LettersPublication details, including instructions for authors and subscriptioninformation:http://www.tandfonline.com/loi/lanl20
A New Amperometric Carbon Paste EnzymeElectrode for Ethanol DeterminationDerya Koyuncu a , Pınar E. Erden b , Şule Pekyardımcı b & Esma Kılıç b
a Department of Biotechnology , Institute of Biotechnology, AnkaraUniversity , Ankara, Türkiyeb Faculty of Science, Department of Chemistry , Ankara University , Ankara,TürkiyePublished online: 18 Aug 2007.
To cite this article: Derya Koyuncu , Pınar E. Erden , Şule Pekyardımcı & Esma Kılıç (2007) A NewAmperometric Carbon Paste Enzyme Electrode for Ethanol Determination, Analytical Letters, 40:10,1904-1922, DOI: 10.1080/00032710701384691
To link to this article: http://dx.doi.org/10.1080/00032710701384691
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”)contained in the publications on our platform. However, Taylor & Francis, our agents, and ourlicensors make no representations or warranties whatsoever as to the accuracy, completeness, orsuitability for any purpose of the Content. Any opinions and views expressed in this publicationare the opinions and views of the authors, and are not the views of or endorsed by Taylor &Francis. The accuracy of the Content should not be relied upon and should be independentlyverified with primary sources of information. Taylor and Francis shall not be liable for anylosses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilitieswhatsoever or howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any substantialor systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, ordistribution in any form to anyone is expressly forbidden. Terms & Conditions of access and usecan be found at http://www.tandfonline.com/page/terms-and-conditions
ENZYME ELECTRODE
A New Amperometric Carbon PasteEnzyme Electrode for Ethanol
Determination
Derya Koyuncu
Department of Biotechnology, Institute of Biotechnology, Ankara
University, Ankara, Turkiye
Pınar E. Erden, Sule Pekyardımcı, and Esma Kılıc
Faculty of Science, Department of Chemistry, Ankara University,
Ankara, Turkiye
Abstract: In this study, a new amperometric carbon paste enzyme electrode for
determination of ethanol was developed. The carbon paste was prepared by mixing
alcohol dehydrogenase, its coenzyme nicotinamide adenine dinucleotide (oxidized
form, NADþ), poly(vinylferrocene) (PVF) that was used as a mediator, graphite
powder and paraffin oil, then the paste was placed into cavity of a glass electrode
body. Determination of ethanol was performed by oxidation of nicotinamide adenine
dinucleotide (reduced form, NADH) generated enzymatically at þ0.7 V. The effects
of enzyme, coenzyme and PVF amounts; pH; buffer concentration and temperature
were investigated. The linear working range of the enzyme electrode was
4.0 � 1024–4.5 � 1023 M, determination limit was 3.9 � 1024 M and response
time was 50 s. The optimum pH, buffer concentration, temperature, and amounts of
enzyme, NADþ and PVF for enzyme electrode were found to be 8.5, 0.10 M, 378C,
2.0, 6.0, and 12.0 mg, respectively. The storage stability of enzyme electrode
Received 22 December 2006; accepted 15 March 2007
We gratefully acknowledge the financial support of T.R. Prime Ministry State
Planning Organization (Project No: 98-K-120830), Ankara University, Biotechnology
Institute (Project No: 155) and a scholarship by Scientific and Technical Research
Council of Turkey for P. E. ERDEN.
Address correspondence to D. Koyuncu, Department of Biotechnology, Institute
of Biotechnology, Ankara University, Ankara, Turkiye. E-mail: derya_koyuncu@
yahoo.com
Analytical Letters, 40: 1904–1922, 2007
Copyright # Taylor & Francis Group, LLC
ISSN 0003-2719 print/1532-236X online
DOI: 10.1080/00032710701384691
1904
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
at þ48C was 7 days. Enzyme electrode was used for determination of ethanol in two
different wine samples and results were in good agreement with those obtained by
gas chromatography.
Keywords: Alcohol dehydrogenase, amperometry, enzyme electrode, carbon paste,
ethanol, poly(vinylferrocene)
INTRODUCTION
Rapid, sensitive, and accurate determination of ethanol is very important in
different fields such as biotechnology, medicine, food industry, and clinical
analysis. For ethanol determination, various instrumental methods such as
gas chromatography (Clarkson et al. 1995), liquid chromatography coupled
with absorbance or fluorescence detection (Vitrac et al. 2002), spectropho-
tometry (Lazaro et al. 1986; Segundo and Rangel 2002) or enzymatic
test-kits have been reported. However, these methods are laborious,
expensive, time-consuming, and complex to perform.
An alternative method for ethanol determination is the use of electroche-
mical enzyme electrodes that allow direct, rapid and inexpensive measurement
of the ethanol in the samples. Several amperometric enzyme electrodes for
ethanol based on alcohol oxidase (AOD) (Verduyn et al. 1983; Patel et al.
2001; Gulce et al. 2002; Shkotova et al. 2006) and alcohol dehydrogenase
(ADH) (Kitagawa and Kitabatake 1989; Sim, 1990; Miyamoto et al. 1991;
Wang et al. 1993; Persson et al. 1993; Dominguez et al. 1993; Park et al.
1995; Boujtita et al. 1996; Castanon et al. 1997; Leca and Marty 1997; Cai
et al. 1997; Park et al. 1999; Serban and El Murr 2004) have been
proposed. Amperometric ethanol biosensors are based on the measurement
of amperometically detectable products such as hydrogen peroxide (H2O2)
and b-nicotinamide adenine dinucleotide reduced form (NADH). For
ethanol determination, alcohol oxidase-peroxidase coupled enzyme system
(Vijayakumar et al. 1996), microbial biosensors (Reshetilov et al. 2001;
Tkac et al. 2002; Akyılmaz and Dinckaya 2005) and plant tissue (Akyılmaz
and Dinckaya 2000) have also been described.
Instead of AOD biosensors, ADH biosensors have some advantages for
ethanol monitoring, such as it is not oxygen dependent and more selective
to ethanol. In ADH based amperometric ethanol biosensors, ADH depends
on the coenzyme NADþ for enzymatic activity. Ethanol is converted to acet-
aldehyde by the ADH enzyme in the presence of NADþ and the reduced
NADH can be detected amperometrically, according to the following
reactions (Santos et al. 2003):
CH3 CH2 OH þ NADþ �!ADH
CH3CHO þ NADH þ Hþ
NADH �! NADþ þ Hþ þ 2e–
Amperometric Carbon Paste Enzyme Electrode 1905
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
The most important drawback of the ADH biosensors is the high overpo-
tentials that the electrochemical oxidation of NADH occurs (Castanon et al.
1997; Park et al. 1999; Patel et al. 2001; Barlett et al. 2002). At these high
potentials, other electroactive species present in the sample are also
oxidized and interfere in the analysis and the intermediates produced during
the oxidation reaction may foul the electrode (Elving et al. 1982; Barlett
et al. 2002). To decrease the high overpotential and the number of interfer-
ences, the use of different organic and inorganic mediators have been
reported for electrochemical oxidation of NADH (Kitagawa and Kitabatake
1989; Kubiak and Wang 1989; Chi and Dong 1994; Leca and Marty 1997;
Katrlik et al. 1998; Serban and El Murr 2004; Yao et al. 2000; Prieto-
Simon and Fabregas 2004). In addition, several studies using conducting
polymer films in the construction of enzyme electrodes based on the electro-
catalytic NADH oxidation to facilitate the incorporation of mediators have
been reported (Persson et al. 1993; Castanon et al. 1997). Another approach
is the use of polymers with mediating functions that facilitate the electron
transfer between NADH and electrode surface (Xu et al. 1994).
Unlike the classical dehydrogenase biosensors, the incorporation of the
biocatalysts into carbon paste matrix is an interesting approach for ethanol
biosensors. A number of ethanol biosensors based on the incorporation of
the enzymes, coenzymes, and mediators into carbon paste matrix have been
reported. These reports provide an effective way for overcoming the
drawback of ADH biosensors (Kubiak and Wang 1989; Wang et al. 1993;
Persson et al. 1993; Dominguez et al. 1993; Chi and Dong 1994; Castanon
et al. 1997; Yao et al. 2000).
In this work, we developed an ethanol carbon paste enzyme electrode by
the incorporation of ADH, the coenzyme NADþ, and a new redox mediator
poly(vinylferrocene) within a carbon paste matrix. We also investigated that
(a) the parameters that influence the electrode performance, (b) its analytical
characteristics, (c) operational and storage stability of the electrode, and (d)
the use of the biosensor for ethanol determination in real samples. Although
the working principles of the enzyme electrode constructed are based upon
the same reactions, there is no study in the literature where PVF was used
as a mediator for the ethanol carbon paste enzyme electrodes.
EXPERIMENTAL
Equipment and Reagents
The electrochemical studies were carried out using BAS 100 B/W electroche-
mical analyzer using a three-electrode cell. The working electrode was a
modified carbon paste electrode. The counter and the reference electrodes
were a Pt wire (MW 1034) and Ag/AgCl (MF 2052) electrode, respectively.
The pH values of the buffer solutions were measured with ORION Model
D. Koyuncu et al.1906
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
720A pH/ion meter. Temperature control was achieved with Grant LTD GG
thermostat. Gas chromatography measurements were performed by GC 8000
Top CE Instruments Gas Chromatography Instrument.
Alcohol dehydrogenase (E.C.1.1.1.1 from Saccharomyces cerevisia)
NADþ, NADH, and dialysis membrane were purchased from Sigma,
sodium monohydrogenphosphate, sodium dihydrogenphosphate, ethanol,
propan-1-ol, butan-1-ol, propan-2-ol, butan-2-ol were supplied from Riedel-
de Haen. Sodium hydroxide, methanol, and n-amyl alcohol were obtained
from Merck. Vinylferrocene was from Aldrich. Phosphoric acid was from
PRS Panreal and benzene was from Fluka. The standard ethanol solutions
were prepared every day freshly and stored at þ48C. PVF was prepared by
the chemical polymerization of vinylferrocene (Smith et al. 1976).
Preparation of Carbon Paste, PVF Modified Carbon Paste, and
Carbon Paste Enzyme Electrodes
Glass tubes with an inner diameter of 3 mm, outer diameter of 5 mm, and
length of 12 cm were used as working electrode body. A spectroscopic
graphite rod was placed in the glass tube leaving about 5 mm in the tube
bottom empty to be filled with carbon paste and modified carbon paste. Elec-
trical contact was made by the insertion of a copper wire in the graphite rod.
The unmodified carbon paste was prepared by hand-mixing 43.0 mg of
graphite powder with 15 ml paraffin oil until a uniform paste was obtained.
PVF modified carbon paste was prepared by mixing 5.0 mg PVF, 38.0 mg
graphite powder and 15 ml paraffin oil. The paste was then placed into the
bottom of the working electrode body and the electrode surface was
polished with a weight paper to have a smooth surface.
The carbon paste for enzyme electrode was prepared by mixing the
different amounts of ADH enzyme, coenzyme NADþ, mediator PVF,
graphite powder, and paraffin oil until a uniform paste was obtained.
Table 1 shows the different carbon paste compositions used for electrode prep-
aration. Electrodes were stored in refrigerator at þ48C when not in use.
Amperometric Measurements
All amperometric measurements were performed in phosphate buffer solutions
(0.10 M; pH 8.5) with constant stirring. First of all we investigated the electro-
chemical oxidation of NADH at unmodified carbon paste electrodes. 5.0 ml
phosphate buffer solution was added to the cell. After application of þ0.7 V
potential, the background current was allowed to decay constant value. Then
an aliquot of NADH stock solution was added to the cell and stirred for
4 min and the cell was purged with argon for one minute prior to each measure-
ment. The response of the electrode against NADH was measured after 50 s.
Amperometric Carbon Paste Enzyme Electrode 1907
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
Table 1. Carbon paste compositions used for electrode preparation and comparison of the working ranges, sensitivities and regression coefficients of
different enzyme electrodes
Enzyme electrode no.
Carbon paste compositions (mg) Electrode characteristics
ADH NADþ PVF Graphite powder
Linear working
range (mM)
Sensitivity
(mA/mM)
Regression
coefficient (R2)
1 1.0 6.0 12.0 24.0 1.4–3.6 0.30 0.8923
2 1.5 6.0 12.0 23.5 0.7–2.7 0.51 0.9595
3 2.0 6.0 12.0 23.0 0.4–4.5 0.72 0.9936
4 2.5 6.0 12.0 22.5 0.7–2.8 0.38 0.9886
5 2.0 2.0 12.0 27.0 — — —
6 2.0 4.0 12.0 25.0 0.7–3.4 0.23 0.7682
7 2.0 8.0 12.0 21.0 1.1–3.4 0.22 0.9367
8 2.0 6.0 6.0 29.0 — — —
9 2.0 6.0 7.0 28.0 0.4–3.0 0.32 0.9830
10 2.0 6,0 9.0 26.0 0.4–1.7 0.42 0.9851
11 2.0 6.0 14.0 21.0 0.7–3.4 0.05 0.9850
D.Koyuncu
etal.
1908
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
The current values against NADH concentrations were plotted in order to
determine whether the electrode was sensitive to NADH or not. The same
experiment was performed for modified carbon paste electrode.
In order to determine whether the carbon paste enzyme electrode was
sensitive to ethanol, 5.0 ml buffer solution was added to the cell. The
solution was purged with argon for 5 minutes and after stabilization of the
background current at þ0.7 V, the aliquots of ethanol stock solution was
added to the cell successively. The responses of the enzyme electrode
against ethanol were measured at þ0.7 V vs. Ag/AgCl after 5 min constant
stirring and calibration curve was plotted.
For real sample measurements, the electrode was covered with dialysis
membrane to prevent the interferences. The carbon paste electrode was
placed in the cell containing 5.0 ml phosphate buffer and after the stabilization
of background current, 250 ml sample (dilution of the samples for biosensor
measurements was 1:100) was added and stirred for 5 min than the response
of the electrode against ethanol was determined. After the current was again
reached a steady-state value, standard addition procedure was used to
determine the ethanol. The standard addition procedure was performed by
subsequent addition of 100 ml 0.010 M standard ethanol solution.
RESULTS AND DISCUSSION
In this study, we prepared the carbon paste-based ethanol enzyme electrode
by using of redox polymer poly(vinylferrocene) as a new mediator. The
parameters affecting the performance of the enzyme electrode, optimum
working conditions of the biosensor and the real sample measurements were
investigated and discussed below.
NADH Responses of Carbon Paste and Modified Carbon Paste
Electrodes
Figure 1 shows the current values against NADH concentration obtained with
carbon paste, and PVFþ modified carbon paste electrodes. It is clear from the
figure that the sensitivity of the PVFþ modified electrode is higher than that of
carbon paste electrode. According to the below reactions, NADH is electroox-
idized at þ0.7 V vs Ag/AgCl on carbon paste electrode (Chi and Dong 1994);
PVFþ catalyzes the electrooxidation of NADH; PVF is also electrooxidized
at þ0.7 V potential and the oxidized polymer, PVFþ, is
re-formed (Gulce et al. 1995):
NADH�!NADþ þ 2e– þ Hþ
NADH þ 2PVFþ�!NADþ þ Hþ þ 2PVF
PVF�!PVFþ þ e–
Amperometric Carbon Paste Enzyme Electrode 1909
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
This catalytic process provides an increase in the sensitivity of the PVFþ
modified carbon paste electrodes.
Carbon Paste Compositions of the Prepared Electrodes
Various enzyme electrodes were prepared according to the carbon paste com-
positions given in Table 1. The ethanol responses of these electrodes were
investigated and the sensitivities and working ranges are also shown in the
table. It is clear from the table that enzyme electrode 3 showed the best per-
formance and we decided to use the enzyme electrode with this composition
for our following measurements.
The Effect of Enzyme, PVF, and NAD1 Amounts
The effect of the enzyme amount used in carbon paste matrix on the electrode
response was determined. By keeping the amount of NADþ and PVF constant
and varying the ADH amount, the responses of the enzyme electrodes
(Electrode no 1–4 in Table 1) were measured at four different enzyme
amounts and plotted their calibration graphs. The sensitivities obtained from
calibration graphs were recorded against enzyme amount (Fig. 2). The
Figure 1. Current difference-concentration curves obtained for the electrooxidation
of NADH at carbon paste and PVFþ modified carbon paste electrodes: O: Carbon
paste electrode, B: PVFþ modified carbon paste electrode (0.10 M pH 8.5 phosphate
buffer, 378C).
D. Koyuncu et al.1910
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
maximum sensitivity was recorded with the electrode containing 2.0 mg
enzyme. However, the linearity and the working ranges of the calibration
graphs recorded for electrodes with 1.0, 1.5, and 2.5 mg enzyme were not sat-
isfactory. The equation of the calibration graph recorded with 2.0 mg enzyme
(Electrode 3) is y ¼ 0.72 � 2 0.31 and R2 ¼ 0.9936. Chi and Dong (1994)
reported wide linear working ranges with nominal sensitivities for low
enzyme amounts and narrow working ranges for high enzyme amounts.
The effect of the NADþ was determined by changing the NADþ amount
as 2.0, 4.0, 6.0, 8.0 mg and optimum NADþ amount was found as 6.0 mg
(Fig. 3). As NADþ amount increases the slow reaction between ethanol and
NADþ favours the production of NADH and thus an increase in sensitivity
is expected. This case was observed until 6.0 mg after this value, its sensitivity
was decreased. This can be caused by the direct interactions between PVF,
PVFþ, and NADþ in the carbon paste matrix. These results were found to
be in good compliance with the similar studies in literature. Castanon et al.
(1997) reported that the ethanol response increased substantially with increas-
ing NADþ content between 2 and 10% (w/w) after which at 12% (w/w), it
decreased slightly. As reported by Santos et al. (2003) NADþ percentages
varied from %1.5 to %17 and sensitivity increases sharply until %5, for
higher NADþ percentages the sensitivity decreases.
The influence of PVF amount on the enzyme electrode response was also
studied. Carbon paste electrodes (Electrode No: 3, 8–11) were prepared in the
compositions given in Table 1 and the sensitivities, linear working ranges and
the linearity of calibration curves obtained for each of them were compared.
Figure 2. The effect of enzyme amount on the sensitivity of ethanol electrode
(0.10 M pH 8.5 phosphate buffer, 378C).
Amperometric Carbon Paste Enzyme Electrode 1911
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
The highest sensitivity and working range was obtained with the carbon paste
electrode prepared with 12.0 mg PVF. The sensitivity and linear working
range of this electrode was found to be 0.72 and 4.0 � 1024 2 4.5 � 1023 M
respectively. For lower PVF amounts, the electrochemical oxidation of NADH
was not achieved. The effective oxidation of NADH occurs with two electron
transfers. However, one-electron oxidants such as ferrocenium ions give low
rates of oxidation of NADH (Barlett et al. 2002). For higher PVF amounts, the
rate of PVFþ formation from PVF and PVF from PVFþ decreases and time
consumed for constant background current is higher (Figure 4).
In this study the carbon paste enzyme electrode 3 prepared with 2.0 mg
ADH, 6.0 mg NADþ, 12.0 mg PVF, and 23.0 mg graphite powder showed
the best performance and thus all following electrode characteristics and
operating conditions were determined with this electrode. The detection
limit of this electrode was found to be 3.9 � 1024 M, and the linear
working range was 4.0 � 1024– 4.5 � 1023 M (Fig. 5).
These results show that the carbon paste enzyme electrode prepared by
using PVFþ as a new mediator can readily catalyze the oxidation of NADH
that is generated from the reaction of NADþ and ethanol catalyzed by ADH
as schematized in Fig. 6.
Response Time
The amperometric response time of the carbon paste enzyme electrode to
ethanol was determined at two different ethanol concentrations. The current
Figure 3. The effect of NADþ amount on the sensitivity of ethanol electrode (0.10 M
pH 8.5 phosphate buffer, 378C).
D. Koyuncu et al.1912
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
differences for 1.2 � 1023 M and 1.5 � 1023 M ethanol against time were
plotted. The response time can be taken as 3 min where the currents are
approximately constant. Since the response curves are parallel to each
other, the measurements can be taken before 3 min provided that they are
Figure 4. The effect of PVF amount on the sensitivity of ethanol electrode (0.10 M
pH 8.5 phosphate buffer, 378C).
Figure 5. Ethanol response of the modified carbon paste enzyme electrode (Elec-
trode 3) (0.10 M pH 8.5 phosphate buffer, 378C).
Amperometric Carbon Paste Enzyme Electrode 1913
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
made exactly at the same time. The response time was expected as 50 s and all
the parameters were investigated basing upon the measurements taken after
50 s. This response time is quite fast and highly suitable for biosensor
response. There are many studies in the literature for ethanol enzyme electro-
des with longer (Kitagawa and Kitabatake 1989; Sim, 1990; Sprules et al.
1996; Katrlik et al. 1998; Akyılmaz and Dinckaya 2003; Akyılmaz and
Dinckaya 2005) and shorter (Kubiak and Wang 1989; Wang et al. 1993;
Boujtita et al. 1996; Castanon et al 1997; Tkac et al. 2002) response times
than that of our proposed electrode.
The Effect of pH
The buffers at various pH values were tested to investigate the effect of pH.
The pH of the buffers was varied from 5.0 to 9.0. The measurements were
performed at a constant ethanol concentration of 1.2 � 1023 M. Figure 7
shows that the maximum response was obtained at pH 8.5. Therefore, pH
8.5 was selected as the optimum pH and all following measurements were
performed at this pH. This value is in good agreement with the data
reported by Castanon et al. (1997). For some carbon paste ethanol enzyme
electrodes, different pH values than 8.5 were also reported in the literature;
pH 8.8 (Chi and Dong 1994; Katrlik et al. 1998), pH 8.0 (Wang et al.
1993), pH 7.8 (Santos et al. 2003) and pH 7.5 (Dominguez et al. 1993; Yao
et al. 2000). This was attributed to the fact that the mediator used, enzyme
supply, immobilization method and electrode preparation procedures were
different. The decrease in the responses of the enzyme electrode at pH
values below and above 8.5 can be attributed to the change of the enzyme con-
formations and thus decrease of the enzyme activities.
The Effect of Temperature
Temperature has a great effect on enzyme activity and it is important to inves-
tigate the temperature dependence of the response of the enzyme electrode
(Telefoncu, 1999). The temperature influence on the response of the
modified carbon paste enzyme electrode was tested between 20 and 508C at
Figure 6. The response mechanism of the purposed ethanol biosensor.
D. Koyuncu et al.1914
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
pH 8.5 using constant ethanol concentration of 1.2 � 1023 M (Fig. 8). The
electrode response increases with temperature up to 508C and increases
suddenly afterwards. The sudden increase in the responses after 508C is
thought to be caused by enzyme oxidation at the working potential of
þ0.7 V. We performed the ethanol measurements at 378C because of the
Figure 7. The effect of the solution pH on the response of the electrode in 0.10 M
phosphate buffer solution at 378C in 1.2 � 1023 M ethanol.
Figure 8. The effect of temperature on the response of modified carbon paste enzyme
electrode in 0.10 M pH 8.5 phosphate buffer and 1.2 � 1023 M ethanol.
Amperometric Carbon Paste Enzyme Electrode 1915
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
fact that the linearity of the calibration curves plotted at 378C was better than
the linearity of the calibration curves plotted at 258C.
The Effect of Phosphate Concentration
The amperometric response of the modified carbon paste enzyme electrode was
determined at five different phosphate concentrations of the phosphate buffer
used in the study at a pH value of 8.5 with a constant ethanol concentration
of 1.2 � 1023 M. The response of the enzyme electrode was plotted against
the phosphate concentration (Fig. 9). The best response was obtained in a
buffer solution with phosphate concentration of 0.10 M. This result was
found to be in good compliance with the similar studies in literature
(Dominguez et al. 1993; Chi and Dong 1994; Lobo et al. 1996; Castanon
et al. 1997; Yao et al. 2000; Santos et al. 2003). Above or below this concen-
tration, the response was found to show a significant decrease. It can be
concluded that the buffering capacity of the solution was decreased at lower
phosphate concentration resulting in the change of the buffer pH. In addition
to this, the complex formed between phosphate ions and NADH makes the
oxidation of NADH easier (Rover et al. 1998). At higher phosphate concen-
tration, on the other hand, the decrease in the response can be attributed to
the decrease in the efficiency of the enzymatic reaction, cause of the increase
in the interaction of PVFþ with phosphate ions (Gulce et al. 1995).
Figure 9. The effect of the phosphate concentration on the response of the modified
carbon paste enzyme electrode in pH 8.5 phosphate buffer; 378C and 1.2 � 1023 M
ethanol.
D. Koyuncu et al.1916
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
The Reproducibility of the Modified Carbon Paste Enzyme Electrode
The reproducibility of the modified carbon paste enzyme electrode was also
investigated. Three calibration curves were plotted by the use of the same
electrode in one day (Fig. 10). The relative standard deviation of the sensi-
tivities (the slopes of the curves) was found to be 2.7%. This result
indicates that the reproducibility of the enzyme electrode was highly satisfac-
tory and electrode can be used for many analyses.
Storage Stability
The response of the enzyme electrode prepared under optimum conditions was
measured for a period of 20 days at constant ethanol concentration. There was
no significant change in the electrode responses between first and fourth days,
but the current values decreased sharply after seventh day. It can be calculated
that the electrode lost 28% of its initial sensitivity after 6 days and 64% after
10 days. After 17 days, the electrode lost almost all its initial sensitivity. This
shows that the enzyme electrode can be used for one week.
Response of Electrode to Other Alcohols
To investigate the response of the proposed ethanol biosensor to other alcohols
(propan-1-ol, propan-2-ol, butan-1-ol, butan-2-ol, amyl alcohol, and methanol),
0.010 M alcohol solutions were prepared and calibration curves for each alcohol
Figure 10. The reproducibility of the modified carbon paste enzyme electrode
(0.10 M pH 8.5 phosphate buffer, 378C).
Amperometric Carbon Paste Enzyme Electrode 1917
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
were plotted. The sensitivities obtained from these calibration curves were
compared with the ethanol response of the electrode. The relative response of
the biosensor to alcohols was found to decrease in the following order; ethanol .
propan-1-ol . butan-1-ol . propan-2-ol . methanol . butan-2-ol . amyl
alcohol (Fig. 11). The yeast ADH can readily oxidize primary alcohols except
methanol and slowly oxidize secondary alcohols. This is in good agreement
with the biosensor response to propan-1-ol, butan-1-ol, propan-2-ol and butan-
2-ol. However, the response of the biosensor to methanol is not coherent with
the biospecificity of the enzyme. This is probably caused by the differences in
the conformation between the immobilized enzyme and solution-phase
enzyme (Chi and Dong 1994). Chi and Dong (1994) reported that the
following trend in sensitivity was observed: ethanol . propan-1-ol . propan-
2-ol . butan-1-ol . amyl alcohol . butan-2-ol . methanol. The enzyme
electrode described by Castanon et al. (1997) readily responds to primary
alcohols but no response was observed for methanol.
Figure 11. Relative responses of the biosensor to other alcohols. (1: ethanol; 2: propan-
1-ol; 3: butan-1-ol; 4: propan-2-ol; 5: methanol; 6: butan-2-ol; 7: amyl alcohol).
Table 2. Comparison of ethanol content (% v/v) in wine samples using the proposed
ethanol biosensor and gas chromatography
Ethanol, % (v/v)
Biosensor GC Labeled value
Wine 1 13.71 + 0.16 13.35 + 0.07 13
Wine 2 11.90 + 0.35 11.96 + 0.08 12
D. Koyuncu et al.1918
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
Determination of Ethanol in Wine Samples
The proposed carbon paste ethanol biosensor was used to determine the
ethanol in two wine samples as explained in amperometric measurements
sections. For the evaluation of the new ethanol biosensor, the measurement
results were compared to those obtained by the gas chromatography. In
Table 2, the results obtained for the two samples containing ethanol by
using our new enzyme electrode are presented together those obtained from
the gas chromatography. Results are in good agreement and show that the
new enzyme electrode can be used for ethanol determination in wines.
CONCLUSION
It can be concluded that the presented redox polymer, PVFþ is a suitable
mediator to construct new type of amperometric ethanol biosensor based on
modified carbon paste for ethanol determination.
As a result, the determination of ethanol in wine samples can be also
successfully carried out using PVF modified carbon paste ethanol biosensor
prepared in this study.
The properties and the optimum working conditions of the carbon paste
enzyme electrode are summarized in Table 3.
REFERENCES
Akyılmaz, E. and Dinckaya, E. 2005. An amperometric microbial biosensor develop-ment based on Candida tropicalis yeast cells for sensitive determination of ethanol.Biosens. Bioelectron., 20: 1263–1269.
Akyılmaz, E. and Dinckaya, E. 2003. Development of a catalase based biosensor foralcohol determination in beer samples. Talanta, 61: 113–118.
Table 3. The properties and the optimum working conditions of the PVF modified
carbon paste ethanol biosensor
Parameters Optimum conditions and characteristics
Response time 50 seconds
Temperature 378CpH 8.5
Phosphate concentration 0.10 M
Working range 4.0 � 1024–4.5 � 1023 M
Detection limit 3.9 � 1024 M
Reproducibility The standard deviation computed from the
sensitivities of calibration curves is 2.7%
Storage stabilization About one week
Amperometric Carbon Paste Enzyme Electrode 1919
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
Akyılmaz, E. and Dinckaya, E. 2000. A mushroom (Agaricus Bisporus) tissue hom-
ogenate based alcohol oxidase electrode for alcohol determination in serum.
Talanta, 53: 505–509.
Barlett, P.N., Simon, E., and Toh, C.S. 2002. Modified electrodes for NADH oxidation
and dehydrogenase-based biosensors. Bioelectrochem., 56: 117–122.
Boujtita, M., Chapleau, M., and Murr, N.E. 1996. Biosensors for analysis of ethanol infood: Effect of pasting liquid. Anal. Chim. Acta, 319: 91–96.
Cai, C.X., Xue, K.H., Zhou, Y.M., and Yang, H. 1997. Amperometric biosensor for
ethanol based on immobilization of alcohol dehydrogenase on a nickel hexacyano-
ferrate modified microbial gold electrode. Talanta, 44: 339–347.
Castanon, M.J.L., Ordieres, A.J.M., and Blanco, P.T. 1997. Amperometric detection of
ethanol with poly-(o-phenylenediamine)-modified enzyme electrodes. Biosens.
Bioelectron., 12: 511–520.
Chi, O. and Dong, S. 1994. Electrocatalytic oxidation of reduced nicotinamide
coenzymes at methylene green-modified electrodes and fabrication of amperometricalcohol biosensors. Anal. Chim. Acta, 285: 125–133.
Clarkson, S.P., Onnrod, I.H.L., and Sharpe, F.R. 1995. Determination of ethanol in beer
by direct injection gas chromatography. A comparison of six identical systems.
J. Institu. of Brewing, 101: 191–193.
Dominguez, E., Lan, H.L., Okamoto, Y., Hale, P.D., Skotheim, T.A., Gorton, L., and
Hahn-Hagerdal, B. 1993. Reagentless chemically modified carbon paste electrode
based on a phenothiazine polymer derivative and yeast alcohol dehydrogenase for
the analysis of ethanol. Biosens. Bioelectron., 8: 229–237.
Elving, P.J., Bresnahan, W.T., Moiroux, J., and Samec, Z. 1982. NAD/NADH asa model redox system: Mechanism, mediation, modification by the environment.
Bioelectrochem. Bioenerg., 9: 365–378.
Gulce, H., Gulce, A., Kavanoz, M., Coskun, H., and Yıldız, A. 2002. A new ampeo-
metric enzyme electrode for alcohol determination. Biosens Bioelectron., 17:
517–521.
Gulce, H., Ozyoruk, H., Celebi, S.S., and Yıldız, A. 1995. Amperometric enzyme
electrode for aerobic glucose monitoring prepared by glucose oxidase immobilized
in poly(vinilferrocenium). J. Electroanal. Chem., 394: 63–70.
Kitagawa, Y. and Kitabatake, K. 1989. Alcohol sensor based on membrane-boundalcohol dehydrogenase. Anal. Chim. Acta, 218: 61–68.
Katrlik, J., Svorc, J., Stred’ansky, M., and Miertus, S. 1998. Composite alcohol biosen-
sors based on solid binding matrix. Biosens. Bioelectron., 13: 181–191.
Kubiak, W.W. and Wang, J. 1989. Yeast-based carbon paste bioelectrode for ethanol.
Anal. Chim. Acta, 221: 43–51.
Lazaro, F., Luque de Castro, M.D., and Valcarcel, M. 1986. Individual and simul-
taneous enzymatic determination of ethanol and acetaldehyde in wines by flow
injection analysis. Anal. Chim. Acta, 185: 57–64.
Leca, B. and Marty, J.L. 1997. Reuseable ethanol sensor based on a NADþ-dependent
dehydrogenase without coenzyme addition. Anal. Chim. Acta, 340: 143–148.Lobo, M.J., Miranda, A.J., Lopez-Fonesca, J.M., and Yunon, P. 1996. Electrocatalytic
detection of nicotinamide coenzymes by poly(o-aminophenol)- and poly(o-phenyle-
nediamine)-modified carbon paste electrodes. Anal. Chim. Acta, 325: 33–42.
Miyamoto, S., Murakami, T., Saito, A., and Kimura, J. 1991. Development of an
amperometric alcohol sensor based on immobilized alcohol dehydrogenase and
entrapped NADþ. Biosens. Bioelectron., 6: 563–567.
Park, J.K., Yee, H.J., and Kim, S.T. 1995. Amperometric biosensor for determination
of ethanol vapor. Biosens. Bioelectron., 10: 587–594.
D. Koyuncu et al.1920
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
Park, J.K., Yee, H.J., Lee, K.S., Lee, W.Y., Shin, M.C., Kim, T.H., and Kim, S.R. 1999.
Determination of breath alcohol using a differential–type amperometric biosensor
based on alcohol dehydrogenase. Anal. Chim. Acta, 390: 83–91.
Patel, N.G., Meier, S., Cammann, K., and Chemnitius, G.C. 2001. Screen-printed
biosensors using different alcohol oxidases. Sens. Act. B, 75: 101–110.
Persson, B., Lan, H.L., Gorton, L., Okamoto, Y., Hale, P.D., Boguslavsky, L.I., andSkotheim, T. 1993. Amperometric biosensors based on electrocatalytic regeneration
of NADþ at redox polymer-modified electrodes. Biosens. Bioelectron., 8: 81–88.
Prieto-Simon, B. and Fabregas, E. 2004. Comparative study of electron mediators used
in the electrochemical oxidation of NADH. Biosens. Bioelectron., 19: 1131–1138.
Reshetilov, A.N., Trotsenko, J.A., Morozova, N.O., Iliasov, P.V., and Ashin, V.V.
2001. Characteristic of gluconobacter oxydans B-1280 and Pichia methanolica
MN4 cell based biosensors for detection of ethanol. Proc. Biochem., 36: 1015–1020.
Rover, L., Jr., Fernandes, J.C.B., Neto, G.O., Kubota, L.T., Katekawa, E., and
Serrano, S.H.P. 1998. Study of NADH stability using Ultraviolet–Visible spectro-photometric analysis and factorial design. Anal. Biochem., 260: 50–55.
Santos, A.S., Freire, R.S., and Kubota, L.T. 2003. Highly stable amperometric
biosensor for ethanol based on Meldola’s blue adsorbed on silica gel modified
with niobium oxide. J. Electroanal. Chem., 547: 135–142.
Segundo, M.A. and Rangel, A.O.S.S. 2002. Sequential injection flow system with
improved sample throughput: Determination of glycerol and ethanol in wines.
Anal. Chim. Acta, 458: 131–138.
Serban, S. and El Murr, N. 2004. Synergetic effect for NADH oxidation of ferrocene
and zeolite in modified carbon paste electrodes. New approach for dehydrogenasebased biosensors. Biosens. Bioelectron., 20: 161–166.
Shkotova, L.V., Soldatkin, A.P., Gonchar, M.V., Schuhmann, W., and Dzyadevych, S.V.
2006. Amperometric biosensor for ethanol detection based on alcohol oxidase
immobilised within electrochemically deposited Resydrol film. Mat. Sci. Eng. C,
26: 411–414.
Sim, K.W. 1990. Development of a sensor for ethanol. Biosens. Bioelectron., 5:
311–325.
Smith, W.T., Kuder, J., and Wychick, E. 1976. Voltammetric behavior of poly(vinyl-
ferrocene). J. Polym. Sci., 14: 2433–2448.Sprules, S.D., Hartley, I.C., Wedge, R., Hart, J.P., and Pittson, R. 1996. A disposable
reagentless sceern-printed amperometric biosensor for the measurement of alcohol
beverages. Anal. Chim. Acta, 329: 215–221.
Telefoncu, A. 1999. Biyosensorler; (Biyokimya Lisansustu Yaz Okulu, Haziran 20–26
Ege Universitesi, Kusadası), pp. 136–137.
Tkac, J., Vostiar, I., Gemeiner, P., and Sturdik, E. 2002. Monitoring of ethanol during
fermentation using a microbial biosensor with enhanced selectivity. Bioelectro-
chem., 56: 127–129.
Verduyn, C., Van Dijken, J.P., and Scheffers, W.A. 1983. A simple, sensitive, and
accurate alcohol electrode. Biotechnol. Bioeng., 25: 1049–1055.Vijayakumar, A.R., Csoregi, E., Heller, A., and Gorton, L. 1996. Alcohol biosensors
based on coupled oxidase-peroxidase systems. Anal. Chim. Acta, 327: 223–234.
Vitrac, X., Monti, J., Vercauteren, J., and Merillon, J. 2002. Direct liquid chromato-
graphic analysis of resveratrol derivatives and flavanonols in wines with absorbance
and fluorescence detection. Anal. Chim. Acta, 458: 103–110.
Wang, J., Romera, E.G., and Reviejo, A.J. 1993. Improved alcohol biosensor based on
ruthenium-dispersed carbon paste enzyme electrodes. J. Electroanal Chem., 353:
113–120.
Amperometric Carbon Paste Enzyme Electrode 1921
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14
Xu, F., Li, H., Cross, S.J., and Guarr, T.F. 1994. Electrocatalytic oxidation of NADH atpoly(metallophthalocyanine)-modified electrodes. J. Electroanal. Chem., 368:221–225.
Yao, Q., Tabuki, S., and Mizutani, F. 2000. Preparation of a carbon paste/alcoholdehydrogenase electrode using polyethylene glycol-modified enzyme andoil-soluble mediator. Sens. Act. B, 65: 147–149.
D. Koyuncu et al.1922
Dow
nloa
ded
by [
Uni
vers
ity o
f W
ater
loo]
at 0
6:07
27
Nov
embe
r 20
14