amperometric enzyme electrode for organic peroxides determination

7/27/2019 Amperometric Enzyme Electrode for Organic Peroxides Determination

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Amperometric enzyme electrode for organic peroxides determinationprepared from horseradish peroxidase immobilized in

poly(vinylferrocenium) film

Mehtap Gundogan-Paul a, Serdar S. Celebi b, Haluk Ozyoruk c, Attila Yldz c,*a Ankara Nuclear Research and Training Center, 06100 Besevler, Ankara, Turkey

b Department of Chemical Engineering, Hacettepe University, 06532 Beytepe, Ankara, Turkeyc Department of Chemistry, Hacettepe University, 06532 Beytepe, Ankara, Turkey

Received 19 July 2001; received in revised form 28 March 2002; accepted 15 May 2002

Abstract

Organic peroxides, t-butyl hydroperoxide, 2-butanone peroxide, cumene hydroperoxide and t-butyl peracetate, were determined

by an amperometric enzyme electrode. The enzyme electrode was prepared through electrostatic immobilization of horseradish

peroxidase (HRP) in a polyvinylferrocenium (PVF) film. A PVF'ClO4( film was coated on a Pt foil at '/0.70 V by electrooxidation

of polyvinylferrocene in methylene chloride with 0.1 M tetrabutylammonium perchlorate (TBAP). The enzyme modified electrode

PVF'HRP( was prepared by anion-exchange in a solution of HRP( in 0.05 M phosphate buffer at pH 8.5. FTIR spectroscopy

was used to identify PVF, PVF'ClO4(, and PVF'HRP(. The immobilized amount of the enzyme in the film was determined by

UV spectroscopy. The effects of the polymeric film thickness, bulk enzyme concentration used in the immobilization treatment and

the temperature on the performance of enzyme electrode were investigated. The inhibitory effect of oxygen was also examined.

Linearities, lower detection limits, active life times and sensitivities of the electrode were determined for each peroxide. # 2002Elsevier Science B.V. All rights reserved.

Keywords: Amperometric enzyme electrode; Horseradish peroxidase; Organic peroxides; Poly(vinylferrocenium)

1. Introduction

Organic peroxides are increasingly used for bleaching

in the textile and paper industries and for epoxidation,

hydroxylation, oxidation, oxohalogenation and initia-

tion in polymerization processes in other industrial

sectors (Klenk et al., 1985). Monitoring peroxides in

these processes and during ozonation of drinking water,ozonation processes in air, in ambient aerosols, and in

clinical, food, pharmaceutical and environmental pur-

poses is desirable (Mulchandani et al., 1995; Glaze,

1987). This necessity has promoted work on biosensors

for peroxides. Such biosensors commonly constitute

peroxidases immobilized on conductive materials with

high specific areas and adsorption sites suitable for the

enzyme (Wang and Lin, 1989). High catalytic activity of

peroxidase with a broad range of substrates, activators

and inhibitors makes it attractive to use in amperometric

biosensors. Peroxidase biosensors for organic peroxides,

using both direct electron transfer and mediator-assisted

electron transfer from horseradish peroxidase (HRP)

have been reported in literature (Wollenberger et al.,

1990, 1991; Gorton et al., 1992; Jonsson and Gorton,1989; Toniolo et al., 1996; Ruzgas et al., 1996; Serge,

1999; Lin et al., 2000; Katja et al., 2000). Hexacyano-

ferrate (II), o-phenylenediamine, quinone, ferrocene and

its derivatives in dissolved forms have been used as

mediators. In general, the sensitivity for detection was

improved by a mediator compared with direct transfer

routes. Peroxidase is also coupled to oxidase type

enzymes to determine substrates such as glucose and

choline (Tatsuma et al., 1989; Garguilo et al., 1993).

We have recently developed an amperometric perox-

idase electrode for the determination of hydrogen

peroxide (Gundogan-Paul et al., 2001). The immobiliza-

* Corresponding author. Tel.: '/90-312-297-7964; fax: '/90-312-

299-2163

E-mail address: [email protected] (A. Yldz).

Biosensors and Bioelectronics 17 (2002) 875/881

www.elsevier.com/locate/bios

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

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tion of the enzyme was accomplished in a redox

polymer, polyvinylferrocenium (PVF') matrix. In this

polymer ferrocene centers are covalently bound to the

insoluble polymer skeleton. This way the leaching of the

sensor does not present a complication. The polymer

was deposited by the electrooxidation of the reduced

form polyvinylferrocene (PVF). The thickness of thepolymeric film can be controlled by the electrical charge

passed during electropolymerization. All of these unique

properties make this redox polymer ideal for the

construction of mediated amperometric biosensor.

PVF'E( modified electrodes were used for glucose

and sucrose determination (Gulce et al., 1995a,b). The

interaction between the negatively charged enzyme

species and the polymer matrix, which ensures that the

enzyme is immobilized in the polymeric matrix, is as

follows:

PVF'ClO(4 'E(0 PVF'E('ClO(4 (1)

The IR peaks in the spectrum of PVF'ClO4( that

appear at about 620 and 1100 cm(1 are due to the

ClO4( in the structure. When the polymer film immersed

in solution containing phosphate buffer, these peaks

decrease in intensity as a result of the uptake of

phosphate ions. The presence of phosphate ions, which

are held more strongly than perchlorate ions, also causes

the disapperance of the electroreduction/electrooxida-

tion peaks of ferrocenium/ferrocene centers (Gulce et

al., 1995c). This was interpreted as a result of the low

mobility of phosphate ions in comparison with that of

perchlorate ions.PVF' centers act as catalytic sites for H2O2 oxidation

according to the following reactions, giving rise to high

sensitivity of the electrode:

2PVF''H2O20 O2'2PVF'2H' (2)

PVF 0 PVF''e(

As found in earlier works (Gulce et al., 1995a), the

current values obtained with the PVF'ClO4( coated

electrodes for H2O2 oxidation were much higher than

those obtained with the uncoated Pt surface.

The colour of the PVF'ClO4( film is green and that

of the PVF film is yellow (Gulce et al., 1994). The greenfilm is soluble in dimethylformamide (DMF) and

insoluble in methylene chloride, whereas the yellow

film is soluble in methylene chloride and insoluble in

DMF. Immersion of the PVF'ClO4( film in an aqueous

solution containing 30 mM H2O2 for several hours

causes the colour of the film change from green to

yellow with concurrent gas evolution. The resulting film

is insoluble in DMF and soluble in methylene chloride.

This result provided independent evidence for the

involvement of H2O2 and for the mediation of H2O2oxidation by the ferrocene redox sites in the polymeric

film.

It is the aim of this study to use PVF' immobilized

peroxidase electrode to determine the organic peroxides.

2. Experimental

PVF was prepared by the chemical polymerization ofvinylferrocene (Alfa Products) (Aso et al., 1969). The

oxidized form of the polymer (PVF'ClO4() was elec-

troprecipitated on a Pt electrode by electrooxidizing the

polymer (PVF), at '/0.70 V versus AgjAgCl, in meth-ylene chloride 0.1 M tetrabutylammonium perchlorate

(TBAP). Electrochemical preparation of PVF'ClO4(

was performed in a dry oxygen-free nitrogen (BOS)

atmosphere. The electrical charge passed during the

electroprecipitation was measured to prepare polymer-

coated electrodes with varying thicknesses. For example,

0.0275 C and 0.96 cm2 electrode area corresponded to

about 2.97)/10(7

mol cm(2

of PVF'

ClO4(

. The drythickness of this film corresponded to approximately

72.5 mm or 7.24)/104 layers (Peerce and Bard, 1980).

Methylene chloride (BDH) was purified according to

previously published routines (Perrin and Armarego,

1980). TBAP was prepared by reacting tetrabutylam-

monium hydroxide (40% aqueous solution, Merck) with

HClO4 (BDH) and was recrystallized from a 9:1 mixture

of ethyl alcohol and water several times. It was kept

under a nitrogen atmosphere after vacuum drying at

120 8C.

HRP (E.C.1.11.1.7, 250 U mg(1, Sigma) was used as

received. All solutions were prepared from analytical-

grade chemicals and deionized water. t-Butyl hydroper-oxide, cumene hydroperoxide, t-butyl peracetate were

obtained from Merck and 2-butanone peroxide was

obtained from Aldrich. Stock t-butyl hydroperoxide

solutions were freshly diluted with 0.05 M phosphate

buffer at pH 8.5, 2-butanon peroxide and cumene

hydroperoxide solutions were freshly diluted with ethyl

alcohol, while tert-butyl peracetate solutions were

freshly diluted with acetone daily. The buffer solutions

were prepared by using Na2HPO4 and NaH2PO4(Merck). The enzyme electrode was prepared by immer-

sing the PVF'ClO4( coated Pt foil electrode in a

peroxidase solution (7.5 mg ml(1

). The polymer-coatedelectrodes were kept in the enzyme solution for 30 min

with stirring. The pH of the solution was kept at 8.5

which was above the isoelectric point (7.2) of the

enzyme. The enzyme exists an anion (E() under these

conditions, facilitating its interaction with the oxidized

polymer (PVF'). The amount of enzyme incorporated

in the modified electrode was determined by following

the decrease of the absorbance of the enzyme in solution

at 270 nm during the immobilization treatment. The

enzyme-attached electrode was rinsed with 0.05 M

phosphate buffer (pH 8.5) for 5 min to remove any

enzyme that was retained non-electrostatically on the

M. Gundogan-Paul et al. / Biosensors and Bioelectronics 17 (2002) 875/881876


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polymer surface. The amount of enzyme adsorbed onto

the glass surface that was lost by washing was measured

spectrophotometrically and found to be negligible.

For the determination of the steady-state background

current, a potential of'/0.70 V versus SCE was applied

to the enzyme electrode in the buffer solution. After the

steady-state background current was reached, knownamount of peroxide was added to the cell from fresh

stock peroxide solution and the solution was stirred for

5 s. The peroxide response of the electrode was

measured as the steady state current value obtained on

applying a constant potential of'/0.70 V versus SCE.

The enzyme electrode was kept in 0.05 M phosphate

buffer solution at pH 8.5 and '/4.0 8C when not in use.

Each point measured for each graph in this work was

repeated 3 times and no significant deviations have been

found. Tables 2 and 3 are produced by taking into

account these replicated experimental values. Further-

more, in some cases the measurements had to berepeated by another electrode fabricated at different

times and gave almost the same response under the same

conditions.

Electrochemical measurements were carried out in a

three-electrode cell. SCE was used as a reference

electrode for the analysis of organic peroxides. A Pt

foil electrode (area: 0.96 cm2) was used as a working

electrode. A Pt spiral electrode was used as a counter

electrode in all electrochemical measurements. The

reference electrode was AgAgCl during the polymer

oxidation in 0.1 M TBAP-methylene chloride system.

The electrochemical instrumentation was a PAR model

273 potentiostat/galvanostat. Current/time curves wererecorded on a Cole-Palmer 60648 X-t recorder. Cur-

rent/voltage curves were recorded on a BBC Goertz

Metrawatt SE 790 X-Y recorder. A Unicam Mattson

1000 FTIR spectrometer was used to obtain the IR

spectra. UV measurements were performed with a

Unicam UV/VIS spectrophotometer. Temperature of

the cell was kept constant by the recirculation of water

from the bath with a Grant LTD 66 Instrument.

3. Results and discussion

Organic peroxides for which the response of the

peroxidase electrode was measured are listed in Table

1. Only t-butyl hydroperoxide was found to be soluble in

0.05 M phosphate buffer solution at pH 8.5. For this

reason, the electrode responses for the other peroxides

were investigated in various mixtures of 0.05 M phos-

phate buffer solution of pH 8.5 and ethyl alcohol,

methyl alcohol and acetone. The media in which the

highest current response were obtained for each perox-

ide are given in Table 1.

The amount of the enzyme immobilized in the

polymeric matrix was determined using UV spectro-

photometric measurements at 270 nm and found to be

0.44 mg protein. The enzyme held was desorbed in a

0.025 M phosphate buffer solution at pH 4.0 which was

found to be the best medium for enzyme desorption.

This pH value is below the isoelectric point (7.2) of the

enzyme. The enzyme is positively charged and could bereleased from the polymeric matrix at this pH.

It was also found that the desorption process was

complete in 75 min in this medium. The optimum

immersion time during enzyme immobilization was

also determined spectrophotometrically. The length

time of immersion of the PVF'ClO4( coated electrode

into the 0.05 M phosphate buffer solution at pH 8.5 was

varied. The electrode was then transfered to the 0.025 M

phosphate buffer solution at pH 4.0, kept in this

solution for 75 min for the completion of the desorption

and the absorbance values at 270 nm in this solution was

measured. The optimum length of time of immersion forthe enzyme immobilization was determined to be 30

min.

3.1. The effect of the thickness of polymeric film

When the t-butyl hydroperoxide concentration was

kept constant at 0.5 mM the peak current values

increased with the polymer thickness up to a value

corresponding to the passage of a charge of 0.0275 C

during the electroprecipitation of the polymer, after

which it remained constant as seen in Fig. 1. This

behavior could be explained by the limited diffusion rateof t-butyl hydroperoxide from the bulk solution into the

inner regions of the polymer especially in thick films.

3.2. The effect of enzyme concentration

The effect of the enzyme concentration used in the

bulk solution during the immobilization of the enzyme

on the response of the electrode was also determined.

Using the optimum film thickness, the response of the

enzyme electrode that was prepared with bulk enzyme

concentration between 4.5 and 9.5 mg protein ml(1 to t-

butyl hydroperoxide (0.5 mM) is plotted in Fig. 2. As is

Table 1

Organic peroxides and the suitable media in which the measurements

were carried out

Peroxide Medium

t-butyl hydroperox-

ide

0.05 M phosphate buffer of pH 8.5

t-butyl peracetate 0.05 M phosphate buffer of pH 8.5

(60%)'acetone (40%)

2-butanone peroxide 0.05 M phosphate buffer of pH 8.5 (60%)'ethyl

alcohol (40%)

Cumene hydroper-

oxide

0.05 M phosphate buffer of pH 8.5 (60%)'ethyl

alcohol (40%)

M. Gundogan-Paul et al. / Biosensors and Bioelectronics 17 (2002) 875/881 877


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evident from the data, a substantial amount of current

values are measurable even when very low amounts of

enzyme used in solution during the immobilization

process. The optimum bulk enzyme concentration used

in immobilization process was found to be 7.5 mg

protein ml(1.

3.3. The effect of temperature

The response of the enzyme electrode was measured

at different temperatures varying from 15 to 25 8C for

organic peroxides. As seen in Fig. 3, the current valuesincreased up to a temperature of 20 8C for t-butyl

hydroperoxide. A decrease in the current value was

obtained after this temperature because of the possible

degradation of the peroxide in the medium used. The

maximum response of the enzyme electrode was also

measured at 20 8C for other three peroxides. The

calculated activation energies for the immobilized en-

zyme reaction systems are listed in Table 2.

3.4. The effect of oxygen

Fig. 4 shows the effect of dissolved oxygen on the

response of the enzyme electrode for t-butyl hydroper-

oxide. As was previously observed for hydrogen perox-

ide, the dissolved molecular oxygen has an inhibitory

effect on the response of this modified enzyme electrode

(Gundogan-Paul et al., 2001). When dissolved oxygenwas removed from the solution the activity of the

electrode increased substantially. The measured current

is due to the electrooxidative regeneration of PVF to

PVF'. Oxygen acts apparently as a chemical oxidant

for the conversion of some portion of PVF to PVF'

resulting in the lower measured currents. No current

response was detected when the amount of the dissolved

oxygen was increased to its saturation value.

3.5. The response of the electrode to substrate

concentration

Fig. 5 shows the current values measured for different

t-butyl hydroperoxide concentrations. The electrode

Fig. 1. The effect of film thickness on the current measured for the

solution of 0.5 mM t-butyl hydroperoxide in 0.05 M phosphate buffer

at pH 8.5 (the film thicknesses are expressed as the amount of charge

passed during the electroprecipitation of the polymer).

Fig. 2. The current response for the 0.5 mM t-butyl hydroperoxide

solution to the bulk enzyme concentration in pH 8.5, 0.05 M

phosphate buffer.

Fig. 3. The effect of temperature on the current measured for the 0.5

mM t-butyl hydroperoxide solution in pH 8.5, 0.05 M phosphate

buffer.

Table 2

Activation energies and apparent Michaelis/Menten constants with

corresponding regression coefficients for the organic peroxides studied

Peroxide Ea (kcal mol(1) KM (mM)

t-butyl hydroperoxide 17.8 (r00.968) 337 (r00.997)

t-butyl peracetate 19.8 (r00.985) 555 (r00.979)

2-butanone peroxide 14.5 (r00.999) 196 (r00.997)

Cumene hydroperoxide 15.2 (r00.999) 317 (r00.999)



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response varied linearly within a definite range of

substrate concentration. Table 3 lists the linear concen-

tration ranges determined for the four peroxides studied.

If the current values are compared to those in other

studies, it is seen that higher values are obtained. Theapparent KM values calculated using these data are listed

in Table 2. These are much smaller than the reported

values in literature (Popescu et al., 1996; Guo and Dong,

1997), indicating less structural changes of the enzyme in

immobilized state.

Amperometric responses of the PVF'E( coated,

PVF'ClO4( coated, and uncoated Pt electrodes for t-

butyl hydroperoxide are compared in Fig. 6. The current

values measured with PVF'E( coated Pt electrodewere highest than those measured with PVF'ClO4

(

coated, and uncoated Pt electrodes as expected. The

PVF'ClO4( electrode gave also a higher response than

uncoated Pt electrode.

2-Butanon peroxide behaved similarly as t-butyl

hydroperoxide on the three surfaces studied. No current

response was measured for cumene hydroperoxide on

uncoated Pt electrode. t-Butyl peracetate, on the other

hand gave no current response on uncoated and

PVF'ClO4( coated Pt surfaces.

The behaviour of the enzyme electrode can be

explained by the electrocatalytic effect of PVF'

filmcoated on Pt surface. The natural oxidized form of the

HRP is ferriperoxidase in which iron is at '/3 oxidation

state. The reaction between the enzyme and the sub-

strate generates the reduced form of the enzyme.

peroxide'ferriperoxidase (Eox)0 O2'2H'

'ferroperoxidase (Ered) (3)

The mediator PVF' serves to regenerate the oxidized

form of the enzyme (Gulce et al., 1995b).

Fig. 4. The effect of oxygen on the current measured for the 0.5 mM t-

butyl hydroperoxide solution (A) a deaerated solution; (B) an ambient

condition, in pH 8.5, 0.05 M phosphate buffer, at 20 8C.

Fig. 5. The current response of the enzyme electrode to the additions

ofvarious amounts of substrate in pH 8.5, 0.05 M phosphate buffer, at

20 8C.

Table 3

Linear working ranges with corresponding regression coefficients, sensitivities and active lifetimes of the enzyme electrode for different organic

peroxides

Peroxide Linear working range (mM) Sensitivity (A M(1 cm(2) Active lifetime (days)

t-butyl hydroperoxide 100/600 (r00.977) 0.002 40

2-butanone peroxide 25/400 (r00.999) 0.005 15

Cumene hydroperoxide 100/600 (r00.997) 0.003 15

t-butyl peracetate 100/400 (r00.993) 0.001 3

Fig. 6. The current measurements on (A) PVF'E( coated, (B)

PVF'ClO4( coated and (C) uncoated Pt electrodes with the different

amounts of substrate additions in pH 8.5, 0.05 M phosphate buffer, at20 8C.



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PVF''ferroperoxidase (Ered)0 PVF

'ferriperoxidase (Eox) (4)

Since the potential of the enzyme electrode was kept

constant at'/0.70 V versus SCE during the experiments

chemically generated PVF is oxidized to PVF' at this

potential.PVF 0 PVF''e (5)

The amperometric response of the PVF'ClO4( elec-

trode under nonenzymatic condition was also measur-

able as seen in Fig. 6. The reduced form of the polymer

is produced chemically;

peroxide'2PVF'0 O2'2PVF'2H' (6)

And the oxidized form of the polymer is regenerated

electrochemically at the applied potential of'/0.70 V.

PVF 0 PVF''e (7)

3.6. Stability of the enzyme electrode

The enzyme electrode which was prepared under

optimum conditions was tested at 20 8C with respect

to its stability for four peroxides. The lifetimes are

tabulated in Table 3. There is a noticeable decrease in

the response during the first 5 days for t-butyl hydro-

peroxide, 2-butanone peroxide, cumene hydroperoxide

owing to the desorption of the enzyme molecules, that

are weakly held by the surface. The activity remains

constant thereafter, indicating very good long-termstability of the electrode. The active life times were

determined as the time period during which a steady-

state response was obtained with the same electrode

under the same experimental conditions after the initial

desorption of the enzyme has been completed. The

activity for tert-butyl peracetate was measured only for

3 days, after which no current was measurable, indicat-

ing poor stability of this electrode for this substrate.

There are no literature stability studies for organic

peroxides for comparison.

4. Conclusions

It can be concluded that the biosensor developed in

this study responds to organic peroxides with a good

linear response region. The currents measured at sub-

strate concentration near saturated values were about

mA level. Table 3 compares the linear concentration

ranges and sensitivities obtained for four peroxides

studied with the peroxidase electrode. The electrode

tested for different organic peroxides appears to be

simple, fairly stable, sensitive, fast responding and of

low cost. The current responses and the Km values in this

work are comparable to the other peroxide based

electrode reported (Moore et al., 1996).

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amperometric enzyme electrode for organic peroxides determination

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