a new amperometric carbon paste enzyme electrode for ethanol determination

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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

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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

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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

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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

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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.


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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.

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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–


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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).


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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).


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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).


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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).


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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.


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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.


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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.


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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).


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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


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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.

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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


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a new amperometric carbon paste enzyme electrode for ethanol determination

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