A new amperometric enzyme electrode for alcohol determination

H. Gulce a,*, A. Gulce a, M. Kavanoz a, H. Coskun a, A. Yıldız b

a Department of Chemistry, Suleyman Demirel University, Isparta 32260, Turkeyb Department of Chemistry, Hacettepe University, Beytepe, Ankara 06532, Turkey

Received 25 May 2001; received in revised form 23 October 2001; accepted 20 December 2001

Abstract

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium

matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H2O2

was measured at a constant potential of �/0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and

temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 8C. The steady-state

current of this enzyme electrode was reproducible within 9/5.0% of the relative error. The sensitivity of the enzyme electrode

decreased in the following order: methanol�/ethanol�/n -butanol�/benzyl alcohol. The linear response was observed up to 3.7 mM

for methanol, 3.0 mM for ethanol, 6.2 mM for n -butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis�/Menten

constant (KMapp) value and the activation energy, Ea, of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/

mol for methanol, respectively. # 2002 Elsevier Science B.V. All rights reserved.

Keywords: Amperometric; Enzyme electrode; Alcohol determination

1. Introduction

Quantitative determination of alcohols is important infood, fermentation and wine industries and in clinical

chemistry. The classical methods such as refractometry,

chromatography and steam distillation which require

expensive equipment and are time consuming are being

replaced by inexpensive, rapid and reliable methods

based on immobilized enzyme electrodes. The ampero-

metric response of these electrodes is either due to the

electrooxidation of H2O2 generated from the analyte inthe presence of the enzyme, alcohol oxidase (AOx),

R � CH2OH�O2 0AOx

R � CHO�H2O2

or the electrooxidation of the reduced form (NADH) of

the coenzyme b-nicotinamide adenine dinucleotide

(NAD�) at the applied potentials when alcohol dehy-

drogenase (ADH) is used as an enzyme.

CH3CH2OH�NAD� 0ADH

CH3CHO�NADH�H�

NADH 0 NAD��2e�H�

AOx catalyzes other alcohols as well and is thereforenot selective. ADH is selective for ethanol but the

electrode is unstable (Rebelo et al., 1994). The enzymes

immobilized in various matrixes catalyze one of the

above reactions generating the electroactive species.

Several amperometric enzyme electrodes using AOx

(Mason, 1983; Lubrano et al., 1991) and ADH/NAD�

system (Wangsa and Danielson, 1991; Lobo et al., 1996;

Castanon et al., 1997; Gotoh and Karube, 1994; Zhaoand Buck, 1991) were reported. Microbial alcohol

sensor using the yeast candida vini which was immobi-

lized in the pores of acetylcellulose filter was also

reported (Mascini et al., 1989). Bienzyme electrode

which works with immobilized alcohol oxidase�/catalase

system measures the electroreduction current of O2

molecule that is generated from H2O2 by the catalytic

action of catalase (Verduyn et al., 1983).

CH3CH2OH�O2 0AOx

CH3CHO�H2O2

H2O2 0catalase

O2�2H��2e

O2�4H��4e 0 2H2O

Alcohol oxidase�/peroxidase system which requires an* Corresponding author. Fax: �90-246-237-1106.

E-mail address: handang@fef.sdu.edu.tr (H. Gulce).

Biosensors and Bioelectronics 17 (2002) 517�/521

www.elsevier.com/locate/bios

0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 9 5 6 - 5 6 6 3 ( 0 2 ) 0 0 0 0 8 - 8

addition electron transfer mediator such as 1,1-di-

methylferrocene (DMFc) for the reduction of H2O2

measures the current due to the reduction of the

oxidized form of the mediator (Kulys and Schmid,1991).

CH3CH2OH�O2 0AOx

CH3CHO�H2O2

2DMFc�H2O2�2H� 0peroxidase

2DMFc��2H2O

DMFc��/e0/DMFc

The use of a redox polymer, polyvinylferroceniumperchlorate (PVF�ClO4

�) as an immobilization matrix

allowed the development of novel amperometric sensors

for glucose, galactose and sucrose and lactose. It is

known that the reduced form of this redox polymer film,

PVF, is a homogeneous compact film whereas the

oxidized form, PVF�, is inhomogeneous film (Inzelt

and Bacskai, 1992). The pores or pinholes exist in PVF�

film through which dissolved reactants could diffuse tothe underlying metal surface (Peerce and Bard, 1980). It

was already reported in previous reports related to the

enzymatic sensors for glucose (Gulce et al., 1995a),

galactose (Gulce et al., 2002a), sucrose (Gulce et al.,

1995b) and lactose (Gulce et al., 2002b) that the

covalently bonded ferrocenium centers (PVF�) of this

redox polymer act as chemical oxidants for H2O2

oxidation and the reduced forms of the redox couple(PVF) get oxidized at the applied potential. This

catalytic property of the polymer can be used as a basis

for the development of a rapidly responding sensitive

alcohol sensor.

This work describes a new amperometric enzyme

electrode in polyvinylferrocenium perchlorate

(PVF�ClO4�) matrix. Electrooxidation current of the

enzymatically produced H2O2 was measured and theeffects of enzyme concentration, buffer concentration,

the amount of the free enzyme in solution during

immobilization, pH, temperature and long term stability

were determined.

2. Experimental

PVF�ClO4� modified Pt surface was prepared by

electrooxidizing polyvinylferrocene (PVF) at �/0.70 Vversus Ag/AgCl in a methylene chloride solution con-

taining 0.1 M tetrabutylammonium percholarate

(TBAP). PVF was prepared using a method of chemical

polymerization (Smith et al., 1976) of vinylferrocene

(Alfa products). The electroprecipitation of PVF�ClO4�

was carried out under nitrogen atmosphere. Methylene

chloride was purificated before use. For this aim,

methylene chloride was shaken with portions of conc.H2SO4 until the acid layer remained colorless. It was

washed with water, aqueous 5% Na2CO3, and then

water again. Then it was predried with CaCl2 and

distillated from P2O5 (Perrin and Armorego, 1980).

TBAP was prepared by the reaction of tetrabutylam-

monium hydroxide (40% aqueous solution) (Merck)

with HClO4 (Merck), crystallized from an ethylalcohol�/water mixture (9:1) several times and kept

under nitrogen atmosphere after vacuum drying at 120

8C. The buffer solutions were prepared using NaH2PO4

(AnalaR BDH) and NaOH (Merck). Alcohol (Merck)solutions were prepared in a 0.10 M phosphate buffersolution of pH 8.0. Alcohol oxidase (E.C. 1.1.3.13Sigma A6941) solution was prepared in 0.01 M phos-phate buffer solution of pH 8.0. At pH values above 5the enzyme is in the form of anion and can be ion-exchanged and electrostatically held by the polymer.The isoelectric point of the enzyme is between pH 4.0and pH 5.0 (Mizutani et al., 1997). Enzyme wasincorporated into the polymer matrix by immersingPVF�ClO4

� coated Pt electrode in enzyme solution for30 min according to the ion exchange process

PVF�ClO�4 �AOx�0 PVF�AOx��ClO�

4

The enzyme is held electrostatically in the polymeric

structure. The enzyme electrode was then rinsed with the

buffer solution of working pH to remove the excess

enzyme which was not held electrostatically. We know

quite well that enzyme molecules are immobilized

through the ion exchange process. In a previous study,

the amount of enzyme incorporated in the modified

electrode was determined by following the decrease ofthe absorbance of the enzyme solution at 277 nm during

the surface immobilization procedure. The amount of

enzyme adsorbed onto the glass surface that is lost by

washing was measured spectrophotometrically and

found to be negligible. Furthermore, the amount of

the enzyme immobilized within the polymer could then

be determined using the same absorption peak after

desorbing the enzyme in a solution that has a pH valueless than the isoelectric point of the corresponding

enzyme (Gulce et al., 1995a). The activity of the enzyme

electrode was determined with a jacketed electrochemi-

cal cell which kept the solution at a desired temperature.

Oxygen was introduced into the solution in this cell at a

constant flow rate to obtain an oxygen saturated

solution. Oxygen flow was continued above the solution

to keep it saturated with oxygen during the measure-ments.

Constant potential of �/0.70 V versus SCE was

applied to the enzyme electrode to measure the ampero-

metric response due to the electrooxidation of H2O2

produced enzymatically. Steady state background cur-

rent was first measured at this potential with a blank

buffer solution of working pH. After the steady state

background current value was reached, certain volumesof alcohol solution of known concentration were added

and the currents for each added amount of substrate

were recorded.

H. Gulce et al. / Biosensors and Bioelectronics 17 (2002) 517�/521518

A three electrode system was used as an electroche-

mical cell with separate compartments for the counter

and reference electrodes. SCE was used in aqueous

solution, Ag/AgCl electrode immersed in 0.10 M TBAPsolution that contained saturated amount of AgCl was

used in methylene chloride as reference electrodes. Pt

foil electrode (A�/0.5 cm2) was a working electrode.

The electrochemical instrumentation consisted of

PAR Model 362 Potentiostat�/Galvanostat. Current�/

time curves were recorded on a model 16100-II Linseis

recorder.

3. Results and discussion

When a constant potential of �/0.70 V versus SCE

was applied to the enzyme electrode and alcohol was

added to the electrochemical cell while stirring, a sharp

current response was obtained. No response was ob-

tained when the polymer coated Pt electrode did not

contain any immobilized enzyme. Fig. 1 shows the

responses of the enzyme electrode to methanol. Theresponse reached a steady state value within 30�/50 s.

Steady state current values were used to construct the

calibration plots.

The amount of the enzyme present in solution during

the immobilization process effects the current response

value as seen in Fig. 2. The response of the electrode did

not change significantly after about 6.4 units/ml solu-

tion. The following measurements were therefore taken

with an enzyme electrode prepared with a solution that

contained 10.24 units/ml.

An increase in the buffer concentration caused some

decrease in the response of the enzyme electrode after

about 0.10 M buffer concentration. The concentration

of buffer solution was kept at 0.10 M for all measure-

ments.

The pH dependence of the response of the enzyme

electrode was determined using a buffer solution con-

taining concentrated methanol solution (9.0 mM). The

current values increased up to a pH value of 8.0 and

decreased thereafter (Fig. 3). For each point in Fig. 3 a

new electrode was prepared in order to eliminate the

errors that might arise from re-use of the enzyme loaded

polymer surface. The data points are the averages of

three measurements. The pH of the solution was kept at

8.0 for all measurements. The maximum current values

obtained for the free enzyme using a bare Pt electrode

under the same conditions were with a solution of pH

8.0 and at 80 8C as well as seen in Fig. 3.The effect of temperature on the activity of the

enzyme electrode was determined at pH 8.0. The

temperature was increased from 17 to 90 8C. The

temperature at which the enzyme electrode yielded a

Fig. 1. The response of the alcohol oxidase electrode to methanol

additions (0.10 M buffer concentration, pH 8.0, 30 8C).

Fig. 2. The changes in Imax values of methanol with the enzyme

concentration in solution during immobilization (0.10 M buffer

concentration, pH 8.0, 30 8C).

Fig. 3. The effects of pH on the responses of the enzyme electrode and

free enzyme for methanol (0.10 M buffer concentration, 9.0 mM

methanol concentration, 30 8C).

H. Gulce et al. / Biosensors and Bioelectronics 17 (2002) 517�/521 519

maximum current was found to be 80 8C (Fig. 4a). The

activation energy of this immobilized enzyme system,

Ea, was found to be 38.07 kJ/mol from Arrhenius

plot. The response of the bare Pt electrode was similar

to that of the immobilized enzyme electrode at a

temperature range between 30 and 908C (Fig. 4b). The

response was found to reach a maximum at 808C as seen

in Fig. 4b.

Fig. 5 shows the changes in the response of the

enzyme electrode as a function of the concentration of

various alcohols. Each point in Fig. 5 is the average of at

lest three measurements and relative standard deviation

values were calculated to be less than 5%. The sensitivity

of the enzyme electrode decreased in the following

order: methanol�/ethanol�/n -butanol�/benzyl alco-

hol. The upper limit of the linear working portion in

the calibration plot was found to be 3.7 mM for

methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol,

and 5.2 mM for benzyl alcohol. The apparent

Michaelis�/Menten constant (KMapp) value calculated

from the Lineweaver�/Burk plot data for methanol was

5.78 mM.

The Ea and KM values for the free enzyme system for

methanol were calculated to be 32.40 kJ/mol and 15.57

mM, respectively. The comparison of these values with

those of immobilized enzyme system gives evidence for

the fact that no substantial structural changes and

diffusional limitations occur in the immobilized state.

The electrode which was prepared optimum condi-

tions was tested at 30 8C with respect to its stability

using 9.0 mM methanol solution. A total of 132

measurements were made in 36 days. The electrode

stability was found to be satisfactory for a few days and

decreased considerably for the following periods as seen

in Fig. 6.The effects of several possible interferants such as

cholesterol, sucrose, D-glucose, D-fructose, D-galactose,

lactose, L-tyrosine and ascorbic acid were also investi-

gated. The most important interference was caused by

trozin and ascorbic acid which produced current values

of 1.5 and 2.4 mA when they are present in 5 mM

concentration in 0.10 M buffer solution of pH 8.0 at 30

8C.The alcohol sensor whose characteristics are outlined

above showed better value for the upper limit in the

linear portion of the calibration plot when compared to

the reported values in the literature. The response time

of the electrode was comparable to the published values

(Rebelo et al., 1994; Mizutani et al., 1997; Mascini et al.,

1989; Verduyn et al., 1983). The redox polymer, PVF�,

acts as a suitable immobilizing medium for alcohol

Fig. 4. (a) The changes in the activity of the enzyme electrode for

methanol with temperature. (b) The changes in the activity of free

enzyme for methanol with temperature (0.10 M buffer concentration,

9.0 mM methanol concentration, pH 8.0).

Fig. 5. The changes in the activity of the enzyme electrode for various

alcohols as a function of substrate concentration (0.10 M buffer

concentration, pH 8.0, 30 8C).

Fig. 6. Long-term stability of the enzyme electrode (0.10 M buffer

concentration, 9.0 mM methanol concentration, pH 8.0).

H. Gulce et al. / Biosensors and Bioelectronics 17 (2002) 517�/521520

oxidase and catalyzes the H2O2 oxidation that was

produced enzymatically.

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