A new amperometric enzyme electrode for galactose determination

H. Gulcea,*, I . Atamana, A. Gulcea, A. Yıldızb

aDepartment of Chemistry, Suleyman Demirel University, Isparta, 32260, TurkeybDepartment of Chemistry, Hacettepe University, Beytepe, Ankara, 06532, Turkey

Received 17 May 2001; accepted 4 September 2001

Abstract

A new enzyme electrode for the determination of galactose was developed by immobilizing galactose oxidase in polyvinylferroceniummatrix coated on a Pt electrode surface. The amperometric response of the enzyme electrode was measured at a constant potential of�0.70Vvs SCE. The effects of galactose concentration and temperature on the response of the enzyme electrode were investigated. The responsetime was found to be 30–40 s and the upper limit of the linear working portion was found to be 40.0 mM galactose concentration. © 2002Elsevier Science Inc. All rights reserved.

1. Introduction

Determination of galactose is important in food andfermantation industries [1] and in clinical chemistry [2].Polarimetric, fluorometric, spectrophotometric and chro-matographic methods are generally used for the analysis ofgalactose. These methods are tiresome, costly and timeconsuming [3,4]. Following the improvements in enzymeimmobilization techniques fast, simple, reliable, sensitiveand inexpensive enzyme electrodes were developed for suchanalysis. Several amperometric sensors for the analysis ofgalactose using immobilized galactose oxidase have beenreported. This enzyme was either immobilized on the sur-faces of acetylcellulose or collagen membranes [5,6], po-rous nylon or nafion films [7,8] or sandwiched between amembrane and an electrode [4,9,10]. In another study theenzyme was immobilized in a polypyrolle matrix withoutthe use of any membrane [1].

The galactose response of the enzyme electrode wasmeasured either as an oxidation current of H2O2 which wasproduced as a result of the enzymatic reaction [1,8] or thepeak currents that belong to the osmium complex which wasused as an electron transfer mediator [11]. Clark oxygenelectrode can also be used to measure the electroreductioncurrent of oxygen when immobilized galactose oxidase wasused together with a second immobilized enzyme, catalase,

which generates oxygen from H2O2 enzymatically [12]. Theuse of gelatine immobilized galactose oxidase sandwichedbetween the two dialysis membranes was also reported [13].The construction and properties of a galactose biosensor,consisting of galactose oxidase and peroxidase co-immobi-lized by drop-coating on the surface of a graphite electrodewith adsorbed ferrocene as a mediator, were described byTkac et al. [14,15].

Gulce and coworkers [16,17] reported that a Pt elec-trode coated with a redox polymer, polyvinylferroceniumperchlorate (PVF�ClO4

�) catalyzed the electrooxidationand electroreduction of some organic species such asanthracenes in acetonitrile and the electrooxidation ofH2O2 in aqueous solution. Furthermore they showed thatthis matrix could be used as a preconcentration agent forthe analysis of some inorganic anions [18]. Anion ex-change properties of this redox polymer could also beused advantageously to develope an amperometric glu-cose sensor by immobilizing glucose oxidase [19] andsucrose sensor by coimmobilizing glucose oxidase andinvertase in this matrix at pH values above the isoelectricpoints of the respective enzymes [20].

By using the same strategy the development of an am-perometric galactose sensor using an immobilized galactoseoxidase in PVF� matrix is described in this work. Theamperometric response due to the electrooxidation of enzy-matically produced H2O2 was measured. The activity of theenzyme electrode was studied as a function of the substrateconcentration and working temperature.

* Corresponding author.E-mail address: handang@fef.sdu.edu.tr (H. Gu¨lce).

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2. Experimental

PVF�ClO4� modified Pt surface was prepared by elec-

trooxidizing polyvinylferrocene (PVF) at �0.70 V vs Ag/AgCl in a methylene chloride solution containing 0.10 Mtetrabutylammonium perchlorate (TBAP). PVF was pre-pared using a method of chemical polymerization [21] ofvinylferrocene (Alfa products). The electroprecipitation ofPVF�ClO4

� was carried out under nirogen atmosphere.The purification of methylene chloride was accomplishedaccording to the method proposed, by Perrin and Armarego[22]. TBAP was prepared by the reaction of tetrabutylam-monium hydroxide (40% aqueous solution) (Merck) withHClO4 (Merck), crystallized from an ethyl alcohol-watermixture (9:1) several times and kept under nitrogen atmo-sphere after vacuum drying at 120°C. The buffer solutionswere prepared using NaH2PO4 (AnalaR BDH) and NaOH(Merck). Galactose (Merck) solution was prepared in a0.075 M buffer solution of pH � 13. Galactose oxidasesolution was prepared by dissolving 64.0 mg of the enzyme(E.C. 1.1.3.9 Sigma G 7400) in 20 ml 0.010 M phosphatebuffer solution of pH � 13. The pH of the enzyme solutionwas kept at pH 13, which was well above the isoelectricpoint of GAO. The isoelectric point of the enzyme is 12.0[1]. This particular enzyme is quite stable at pH 13. Wehave used the same enzyme solution repeatedly and noticedno significant deactivation.

Enzyme was incorporated into the polymer matrix byimmersing PVF�ClO4

� coated Pt electrode in enzyme so-lution for 30 min according to the following ion exchangeprocess

PVF�ClO4�3 PVF�GAO� � ClO4

�

The enzyme is held electrostatically in the polymeric struc-ture. The enzyme electrode was then rinsed with the buffersolution of pH 13 to remove the excess enzyme which wasnot held electrostatically. The activity of the enzyme elec-trode was determined with a jacketed electrochemical cellwhich kept the solution at a desired temperature. Oxygenwas introduced into the solution in this cell at a constantflow rate to obtain an oxygen saturated solution. Oxygenflow was continued above the solution to keep it saturatedwith oxygen during the measurements.

Constant potential of �0.70 V vs SCE was applied to theenzyme electrode to measure the amperometric responsedue to the electrooxidation of H2O2 produced enzymati-cally. Steady state background current was first measured atthis potential with a blank buffer solution of pH 13. Afterthe steady state background current value was reached cer-tain volumes of galactose solution of known concentrationwere added and the currents for each added amount ofsubstrate were recorded.

A three electrode system was used as an electochemicalcell with separate compartments for the counter and refer-ence electrodes. SCE was used in aqueous solution as areference electrode. Ag/AgCl electrode immersed in 0.10 M

TBAP solution that contained saturated amount of AgClwas a reference electrodes in methylene chloride. Pt foilelectrode (A � 0,5 cm2) was a working electrode.

The electrochemical instrumentation consisted of PARModel 362 Potentiostat-Galvanostat. Current-voltagecurves were recorded on a model 16100-II Linseis recorder.

3. Results and discussion

During the preparation of PVF�ClO4� layer on the Pt

electrode, the amount of charge or the time of duration ofconstant potential electrolysis was controlled in order toobtain modified surfaces of different thicknesses. The ac-tivity of the enzyme electrode was found to increase withthe film thickness and remained constant after a certainvalue which corresponded to the constant potential electrol-ysis time of 5 minutes.

Galactose is oxidized in the presence of the enzymeaccording to:

Galactose � O2 3GAO

Galactohexodialdose � H2O2

At the applied potential of �0.70 V vs. SCE, H2O2 isoxidized to produce the amperometric response:

H2O23 O2 � 2H� � 2e�

Chemical oxidation of H2O2 by the redox polymer alsooccurs.

2 PVF� � H2O23 2 PVF � O2 � 2 H�

followed by the electroregeneration of the PVF� at thispotential,

PVF3 PVF� � e�

causing the measured current values to be much higher aswas the case in previous studies with glucose and sucrose[19,20]. Such catalytic property of the redox polymer resultsin an increase in the sensitivity of the enzyme electrode.

Fig. 1 shows the current-time responses of the enzymeelectrode for different amounts of added galactose. Theresponse is quite fast and the steady state current value wasreached within 30–40 s after the stirring was stopped fol-lowing each addition of the substrate. Chemical oxidation ofthe H2O2 by the redox polymer occurs within the polymerproducing catalytic current due to the electrooxidation ofPVF. The response time is comparable to the values re-ported in literature [1,8].

The steady state current values were used to construct thecalibration plot. The response reached a saturation valueafter 47.5 mM galactose concentration (Fig. 2). Each pointin Fig. 2 corresponds to the average of at least three mea-surements. Relative standart deviation values for successiveassays of galactose were calculated be �4%. The upperlimit of the linear working portion in the calibration plotwas found to be 40.0 mM galactose concentration. For

42 H. Gulce et al. / Enzyme and Microbial Technology 30 (2002) 41–44

comparison the linear concentration ranges were reported tobe between 10.0–50.0 mM by Schumacher et al. [13],0.01–5.0 mM by Miyata et al. [11] and 0–1.8 mM byCosnier et al. [1] and 0.25–4.25 mM by Ji et al. [8].

Apparent Michaelis-Menten constant (KMapp) for the im-mobilized enzyme was found to be 21.7 mM galactose fromthe Lineweaver-Burk plot. In order to compare this valuewith that obtained with free enzyme under same solutionconditions, the H2O2 generated from solutions of differentgalactose concentrations were measured electrochemically.The oxidation current of H2O2 was measured using un-coated Pt electrode of same geometric area by applying apotential step of 0.7 V vs. SCE (Fig. 3). Km for the free

enzyme system was also calculated and found to be 22.8mM galactose. Comparison of these Km values for theimmobilized and free systems indicate that no majorchanges occur in the structure of the enzyme as a result ofimmobilization and no diffusional limitations are caused bythe polymer.

The response of the enzyme electrode was tested atworking temperatures between 25°C and 45°C (Fig. 4). 60.0mM galactose solution was used in temperature studies. Asseen in Fig. 2 the electrode response was independent of theamount of the substrate around this concentration. The ac-tivity of the immobilized enzyme increased up to about40°C and started to decrease after this point due to thedenaturation of the enzyme at higher temperatures. Theactivation energy value obtained from this data were 39.1kJ/mole. The response of the free enzyme became maxi-mum at about 35°C (Fig. 4). The activation energy value forthe free enzyme was calculated to be 77.2 kJ/mole. Thecomparison of the two Ea values also confirm the fact thatthe immobilization of the enzyme causes no deformations inthe structure of the enzyme in immobilized state.

The operational stability was also investigated by record-

Fig. 1. Current-time responses obtained with the enzyme electrode fordifferent amounts of galactose solution additions (at 25°C in a 0.075 Mphosphate buffer of pH � 13).

Fig. 2. The change in the activity of the enzyme electrode with galactoseconcentration (at 25°C in a 0.075 M phosphate buffer of pH � 13).

Fig. 3. The change in the activity of the free enzyme with galactoseconcentration (at 25°C in a 0.075 M phosphate buffer of pH � 13).

Fig. 4. The effect of temperature on the activity of the enzyme electrodeand free enzyme (in 0.075 M phosphate buffer of pH � 13 with a galactoseconcentration of 60.0 mM).

43H. Gulce et al. / Enzyme and Microbial Technology 30 (2002) 41–44

ing over 25–30 assays in 3 days the current response toincreasing concentrations of galactose. Each series of mea-surements relative to each substrate was carried out in afresh electrolyte. Finally we found that the enzyme elec-trode activity was not changed significantly.

The novel galactose enzyme electrode whose character-istics described above seems to be simple to prepare, fast torespond, inexpensive and reasonably sensitive. Its higheractivity originates from the catalytic nature of the redoxpolymer in which the enzyme is immobilized. The responsetime and the linear working range of the electrode arecomparable to those already proposed in the literature. Theresponse of the electrode is in �A ranges which is higherthan nA ranges of the comparable amperometric sensorsproposed in literature.

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