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Biosensors and Bioelectronics 17 (2002) 875 /881 www.elsevier.


Amperometric enzyme electrode for organic peroxides determination prepared from horseradish peroxidase immobilized in poly(vinylferrocenium) lm
zyo Mehtap Gu an-Paul a, Serdar S. C elebi b, Haluk O ru ndog k c, Attila Yldz c,*

Ankara Nuclear Research and Training Center, 06100 Bes evler, Ankara, Turkey Department of Chemical Engineering, Hacettepe University, 06532 Beytepe, Ankara, Turkey c 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 ( film was coated on a Pt foil at '/0.70 V by electrooxidation peroxidase (HRP) in a polyvinylferrocenium (PVF) film. A PVF ' ClO4 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. # 2002 Elsevier 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 initiation 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 purposes 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

* Corresponding author. Tel.: '/90-312-297-7964; fax: '/90-312299-2163 E-mail address: yildiz@hacettepe.edu.tr (A. Yldz).

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; Jo nsson and Gorton, 1989; Toniolo et al., 1996; Ruzgas et al., 1996; Serge, 1999; Lin et al., 2000; Katja et al., 2000). Hexacyanoferrate (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 peroxidase electrode for the determination of hydrogen peroxide (Gu an-Paul et al., 2001). The immobiliza ndog

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M. Gu an-Paul et al. / Biosensors and Bioelectronics 17 (2002) 875 /881 ndog

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 the polymeric 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 (Gu lce 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 ' ClO4

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 of vinylferrocene (Alfa Products) (Aso et al., 1969). The ( oxidized form of the polymer (PVF 'ClO4 ) was electroprecipitated on a Pt electrode by electrooxidizing the polymer (PVF), at '/0.70 V versus AgjAgCl, in methylene 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 polymercoated 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 dry thickness 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 tetrabutylammonium 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., 250 U mg (1, Sigma) was used as received. All solutions were prepared from analyticalgrade chemicals and deionized water. t-Butyl hydroperoxide, 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-coated electrodes 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


( The IR peaks in the spectrum of PVF 'ClO4 that (1 appear at about 620 and 1100 cm 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/electrooxidation peaks of ferrocenium/ferrocene centers (Gu lce 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' ' H2 O2 0 O2 ' 2PVF ' 2H' PVF 0 PVF ' e

' (


As found in earlier works (Gu lce 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 (Gu lce et al., 1994). The green film 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 H2O2 oxidation by the ferrocene redox sites in the polymeric film.

M. Gu an-Paul et al. / Biosensors and Bioelectronics 17 (2002) 875 /881 ndog


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, known amount 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. Furthermore, in some cases the measurements had to be repeated 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 were recorded on a Cole-Palmer 60648 X-t recorder. Current /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.

Table 1 Organic peroxides and the suitable media in which the measurements were carried out Peroxide t-butyl hydroperoxide t-butyl peracetate Medium 0.05 M phosphate buffer of pH 8.5

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- 0.05 M phosphate buffer of pH 8.5 (60%)'ethyl oxide alcohol (40%)

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 be released 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 for the enzyme immobilization was determined to be 30 min. 3.1. The effect of the thickness of polymeric lm 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 rate of 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 tbutyl hydroperoxide (0.5 mM) is plotted in Fig. 2. As is

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 phosphate 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 peroxide are given in Table 1. The amount of the enzyme immobilized in the polymeric matrix was determined using UV spectro-


M. Gu an-Paul et al. / Biosensors and Bioelectronics 17 (2002) 875 /881 ndog

Fig. 1. The effect of lm 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 lm thicknesses are expressed as the amount of charge passed during the electroprecipitation of the polymer).

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.

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 values increased 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 enzyme 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 hydroperoxide. As was previously observed for hydrogen peroxide, the dissolved molecular oxygen has an inhibitory effect on the response of this modified enzyme electrode (Gu ndogan-Paul et al., 2001). When dissolved oxygen was 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
Table 2 Activation energies and apparent Michaelis /Menten constants with corresponding regression coefcients for the organic peroxides studied Peroxide Ea (kcal mol( 1) t-butyl hydroperoxide 17.8 (r 0 0.968) t-butyl peracetate 19.8 (r 0 0.985) 2-butanone peroxide 14.5 (r 0 0.999) Cumene hydroperoxide 15.2 (r 0 0.999) KM (mM) 337 555 196 317 (r 0 0.997) (r 0 0.979) (r 0 0.997) (r 0 0.999)

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.

M. Gu an-Paul et al. / Biosensors and Bioelectronics 17 (2002) 875 /881 ndog


Fig. 4. The effect of oxygen on the current measured for the 0.5 mM tbutyl hydroperoxide solution (A) a deaerated solution; (B) an ambient condition, in pH 8.5, 0.05 M phosphate buffer, at 20 8C.

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 tbutyl hydroperoxide are compared in Fig. 6. The current values measured with PVF 'E( coated Pt electrode ( were 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 ' film coated 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 substrate 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 (Gu lce et al., 1995b).

Fig. 5. The current response of the enzyme electrode to the additions of various amounts of substrate in pH 8.5, 0.05 M phosphate buffer, at 20 8C.

response varied linearly within a definite range of substrate concentration. Table 3 lists the linear concentration 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. The apparent KM values calculated using these data are listed in Table 2. These are much smaller than the reported

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, at 20 8C.

Table 3 Linear working ranges with corresponding regression coefcients, sensitivities and active lifetimes of the enzyme electrode for different organic peroxides Peroxide t-butyl hydroperoxide 2-butanone peroxide Cumene hydroperoxide t-butyl peracetate Linear working range (mM) 100 /600 25 /400 100 /600 100 /400 (r 0 0.977) (r 0 0.999) (r 0 0.997) (r 0 0.993) Sensitivity (A M ( 1 cm ( 2) 0.002 0.005 0.003 0.001 Active lifetime (days) 40 15 15 3


M. Gu an-Paul et al. / Biosensors and Bioelectronics 17 (2002) 875 /881 ndog

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

work are comparable to the other peroxide based electrode reported (Moore et al., 1996).

Aso, C., Kunitake, T., Nakashima, T., 1969. Cationic polymerization and copolymerization of vinylferrocene. Makromol. Chem. 124, 232 /239. Garguilo, M.G., Huynh, N., Proctor, A., Michael, A.C., 1993. Amperometric sensors for peroxide, choline, and acetylcholine based on electron transfer between horseradish peroxidase and a redox polymer. Anal. Chem. 65, 523 /526. Glaze, H.W., 1987. Drinking-water treatment with ozone. Environ. Sci. Technol. 21 (3), 224 /230. Gorton, L., Petterson-Jo nsson, G., Cso regi, E., Johansson, K., Doninguez, E., Marko-Varga, G., 1992. Amperometric biosensors based on an apparent direct electron transfer between electrodes and immobilized peroxidases. Analyst 117, 1235 /1241. Guo, Y., Dong, Y., 1997. Organic phase enzyme electrodes based on organohydrogel. Anal. Chem. 69, 1904 /1908. zyo Gu ru lce, H., .O k, H., Yldz, A., 1994. Electrochemical reduction of anthracenes on poly (vinylferrocenium) coated Pt electrodes in acetonitrile. Ber. Bunsengen. Phys. Chem. 98, 228 /233. zyo Gu ru k, H., Yldz, A., 1995. Electrochemical response of lce, H., .O poly(vinylferrocenium)-coated Pt electrodes to some anions in aqueos media. Electroanalysis 7, 178 /183. zyo Gu elebi, S.S., Yldz, A., 1995. Amperometric ru k, H., C lce, H., .O enzyme electrode for aerobic glucose oxidase immobilized in poly(vinylferrocenium). J. Electroanal. Chem. 394, 63 /70. zyo Gu elebi, S.S., O ru lce, H., .C k, H., Yldz, A., 1995. Amperometric enzyme electrode for sucrose determination prepared from glucose oxidase and invertase co-immobilized in poly(vinyl ferrocenium). J. Electroanal. Chem. 397, 217 /223. zyo Gu an-Paul, M., .O elebi, S.S., Yldz, A., 2001. ru ndog k, H., C Amperometric enzyme electrode for hydrogen peroxide determination prepared with horseradish peroxidase immobilized in polyvinylferrocenium (PVF'/). Electroanalysis 14, 505 /511. Jo nsson, G., Gorton, L., 1989. An electrochemical sensor for hydrogen peroxide based on peroxidase adsorbed on a spectrograc graphite electrode. Electroanalysis 1, 465 /468. Katja, H., Marcus, M., Wolfgang, S., 2000. Electron-transfer mechanisms in amperometric biosensors. Fresenius J. Anal. Chem. 366, 560 /568. Klenk, H., Go tz, H.P., Siegmer, R., Mary, W., Degussa, A.G., 1985. Ullmanns Encyclopedia of Industrial Chemistry Vol. A.19, pp. 199 /201. Lin, X.Q., Chen, J., Chen, Z.H., 2000. Amperometric biosensor for hydrogen peroxide based on immobilization of horseradish peroxidase on methylene blue modied graphite electrode. Electroanalysis 12, 306 /310. Moore, A., Katz, E., Willner, I., 1996. Electrocatalytic reduction of organic peroxides in organic solvents by microperoxidase-11 immobilized as a monolayer on a gold electrode. J. Electroanal. Chem. 417, 189 /192. Mulchandani, A., Wang, C.L., Weetall, H., 1995. Amperometric detection of peroxides with poly(anilinomethylferrocene)-modied enzyme electrodes. Anal. Chem. 67, 94 /100. Peerce, P., Bard, A.J., 1980. Polymer lms on electrodes. Part II. Film structure and mechanism of electron transfer with electrodeposited poly(vinylferrocene). J. Electroanal. Chem. 112, 97 /115. Perrin, D.D., Armarego, W.L.F., 1980. Purication of Laboratory Chemicals. Pergamon Press, Oxford, p. 265. Popescu, I.C., Cso regi, E., Gorton, L., 1996. Peroxidase-modied carbon pasta microelectrode as amperometric FI-detector for peroxides in partial aqueous media. Electroanalysis 8, 1014 /1019.


( elecThe amperometric response of the PVF 'ClO4 trode under nonenzymatic condition was also measurable as seen in Fig. 6. The reduced form of the polymer is produced chemically;

peroxide ' 2PVF' 0 O2 ' 2PVF ' 2H'


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 hydroperoxide, 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-term stability of the electrode. The active life times were determined as the time period during which a steadystate 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, indicating 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 substrate 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

M. Gu an-Paul et al. / Biosensors and Bioelectronics 17 (2002) 875 /881 ndog Ruzgas, T., Cso regi, E., Emneus, J., Gorton, L., Marko-Varga, G., 1996. Peroxidase-modied electrodes: fundamentals and application. Anal. Chim. Acta 330, 123 /138. Serge, C., 1999. Biomolecule immobilization on electrode surfaces by entrapment or attachment to electrochemically polymerized lms. Biosens. Bioelectron. 14, 443 /456. Tatsuma, T., Okawa, Y., Watanabe, T., 1989. Enzyme monolayer-and bilayer-modied tin oxide electrodes for the determination of hydrogen peroxide and glucose. Anal. Chem. 61, 2352 /2355. Toniolo, R., Comisso, N., Bontempelli, G., Schiavon, G., 1996. Amperometric determination of peroxides by glassy carbon


electrodes modied with copper-phenanthroline complexes. Electroanalysis 8, 151 /157. Wang, J., Lin, M.S., 1989. Horseradish-root-modied carbon pasta bioelectrode. Electroanalysis 1, 43 /47. Wollenberger, U., Bogdanovskaya, V., Bobrin, S., Scheller, F., Tarasevich, M., 1990. Enzyme electrodes using bioelectrocatalytic reduction of hydrogen peroxide. Anal. Lett. 23 (10), 1795 /1808. zso Wollenberger, U., Wang, J., .O z, M., Gonzales-Romero, E., 1991. Bulk modied enzyme electrodes for reagentless detection of peroxides. Bioelectron. Bioenerg. 26, 287 /296.

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