Peroxidase-modified carbon paste microelectrode as amperometric FI-detector for peroxides in partial aqueous media

1014

Peroxidase-Modified Carbon Paste Microelectrode as Amperometric FI-Detector for Peroxides in Partial Aqueous Media Iond C. Popescu,

Elisuheth Csfirrgi,f

a n d Lo Gorton*

'

Department of Physical Chemistry. University Babes -Bolyai, ROM-3400 Cluj-Napoca, Romania +

+ Department of Analytical Chemistry, University of Lund, P.O. Box 124, S-221 00 Lund. Sweden

Received: December 27, 1995 Final version: March 4, 1996 Abstract Biosensor characteristics of peroxidase-lactitol-modified carbon paste microclectrodes have been evaluated in aqueous and partial aqueous media (H20-100/ov./v. CH;OH and H20-10% v./v. CH3CN), for hydrogen peroxidc and 2-butanone peroxide amperometric detcction, using a flow injection system. The activity of the investigated peroxidases (horseradish and fungal peroxidase from A r t l z r o n ~ ~ wramosus) s was higher for hydrogen peroxide than for 2-butanone peroxide, irrcspective of the solvent composition. For both peroxidases, the microelectrodes' sensitivity. estimated from the slopes of the linear domains as well as calculated from the Eddie-Mofstee plots decreased in the sequence: H 2 0 > HZO-lO% v.jv. CHqCN > H20-10% v./v. CHIOH. It is suggested that this behavior was due to the difference between the polarity and other physical properties, such as kinematic viscosity and diclcctric constant of the nonaqueous solvents. Keywords: Peroxidases, Hydrogen peroxide. Carbon paste electrodes, Amperumctric, Organic-phase biosensor

1. Introduction Peroxidases are among the most investigated oxidoreductases both in enzymatic catalysis and in electrochemical systems [I]. Several groups reported on the direct electroil transfer between horseradish peroxidase (HRP) and different bare electrode surfaces, such as, carbon black [2], pyrolytic graphite [3-61, carbon paste [7-91, and organic metals [lo, 111. It was also demonstrated that the immobilized H R P has high enough activity and stability, allowing the construction of various biosensors for amperometric detection of hydrogen peroxide and organic peroxides. Such electrodes have been shown to perform in organic solvents [ 12- 1 71. and/or at microscale level [14, 18-22]. In this context, taking advantage of the convenience offered by carbon paste for electrode miniaturization [23] and for enzyme immobilization [9], including the inherent advantage of bulk modification, this article reports a study of peroxidasemodified micro-carbon paste electrodes. A previously optimized peroxidase- and lactitol-modified carbon paste design [24] was used for electrode construction. The biosensors were used for hydrogen peroxide and 2-butanone peroxide detection in a flow injection (FI) system using a potential of -0.05 V (vs.Ag/AgCl). Performance characteristics are presented with regard to the nature of the immobilized enzymes, i.e., horseradish peroxidase vs. fungal peroxidase, and the nature of the analysis media i.e., aqueous V.S. partial aqueous media (H,O-with either 10% v./v. CH3CN or CH,OH), and compared to those obtained for surface modified peroxidase-based carbon fiber electrodes. Both types were used for the detection of organic peroxides, including paramenthane-, and di-isopropyl-benzol hydroperoxide reported for the first time to be detectable with this type of biosensors.

2. Experimental 2.1. Materials Two horseradish peroxidases, one (HRP-S) from Sigma, St.Louis, MO, USA, (type VI, cat. no. P-8375,288 U mg-') and one (HRP-B) from Boehringer Mannheim, Mannheim, Elertroanrr!,?ris 1996, 8. No. I I

Germany (90% homogeneous concerning isoenzyme C, cat. no. 814393, 1000 U mg-I); arid two fungal peroxidascs from Arthromyces rumo.sz~.s, one (ARP-S) from Sigma (cat. no. P4794, 270 U ing ') and one (ARP-J) from Suntory Ltd, Tokyo, Japan, (lot no. 90051 I , 250 U mg I ) , were used for electrode construction. H 2 0 2 (30% sol) from Merck, Darmstadt, Germany (cat. no. 72091, 2-butanone peroxide (2BP 30% w solution in dimethyl phthalate, cat. no. 24,401-51, and cumol hydroperoxide (CHP, cat. no. 24,750-2) from Aldrich, Steinheim, Germany, were used as received. Paramenthane hydroperoxide (PMHP) and di-isopropyl-benzol hydroperoxide (DIPBHP) were donated by Prof. Klaus Unger (Department of Analytical and Inorganical Chemistry, University of Mainz, Germany). Graphite powder (GP) was from Heraeus, Karlsruhe, Germany (cat. no. 00641) and paraffin oil from Fluka, Buchs, Switzerland (cat. no. 76235). Lactitol (LA) was a generous gift from Dr. T. D . Gibson (Department of Biochemistry and Molecular Biology, University of Leeds, UK). All other chemicals were of analytical grade (Merck) and were used without further purification. Solutions were prepared with water purified in a Milli-Q system from Millipore, Bedford, MA, USA, except for the stock solutions of organic peroxides (0.1 M), which were prepared with HPLC-grade acetonitrile; further dilutions were made daily with Millipore water and the diluted solutions were used within 3 h.

2.2. Electrode Preaparation The graphite powder was pretreated by heating at 700 "C for 145s in a Muffle furnance (Carbolite, Sheffield, UK, mod. LMF2/P-EIP) and then cooled to 25°C in a desiccator [8]. All peroxidases were immobilized only by absorption [24] as follows; 400 pL of 0.1 M phosphate buffer (pH 8) containing 2 mg of the above mentioned peroxidases and l 0 m g of LA were added to 100mg of GP and the mixture was allowed to react at 4 "C for 2 h, then dried under reduced pressure for 4.5 h. The carbon paste electrodes (CPE) were prepared following a previous protocol [25], by manually mixing 4 0 p L of paraffin oil to the enzyme-lactitol-modified composite to form a homogeneous paste. Next, a l 0 c m length PTFE tubing (1. D. 0.5mm) was filled with the modified carbon paste to form an approximative 3-4 mm high active layer (Fig. 1A). Electrical

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Fig. 1. Scheme of the peroxidase-modifiedcarbon paste microelectrode (A) and the flow-through aniperometric cell (B) contact was made by inserting a silver wire into the pressed paste from the opposite side of the working electrode surface. Finally, the pCPE was gently rubbed on fine paper tissue to produce a flat electrode surface. The inicroelectrodes were stored in a dry state at 4 ”C until use.

2.3. Instrumentation The pCPE was mounted in a two electrode quasi-flow-through amperometric cell [26] (Fig. lB), connected to a potentiostat BAS4B (Bioanalytical Systems, West Lafayette, IL, USA). An Ag/AgCl (0.01 M NaCI) electrode served both as a quasireference and a counter electrode. The output of the potentiostat was displayed on a strip chart recorder (Kipp & Zonen, Delft, Thc Netherlands, Model BDI 11). The cell was coniiectcd to a single line FI system consisting of pulse-free microdialysis pump (CMA, Stockholm, Sweden, mod.lOO) and a manually operated injection valve (Valco, model CI4W, Switzerland) with an injection volume o f 60 nL. The flow carrier consisted of a n aqueous solution of 0.01 M NaCI-0.1 M phosphate buffer at pH 7, or containing as previously specified, 10% v./v. CH,CN or CH30H. A flow rate of l 0 p L min was used. The dispersion factor ( D ) of the flow system, determined as the ratio between the steady-state and peak currents [27], was evaluated from measurements made with 0.1 m M K,Fe(CN), and bare carbon paste microelectrodes at -0.05 V V.Y. (Ag/AgCI, 0.01 M NaCI). For the flow rate used, D was found equal to 35. I . Each FI result is reported as the average of three measurements.

’

3. Results and Discussion H 2 0 2 is an important substrate being produced in many enzymatic reaction, hence its determination is of vital importance. Albeit, hundreds of new configurations have been produced, only one based on “wired” H R P was recently marketed by BAS (see catalogue). Moreover, these electrodes are often usedpo be used as post column electrochemical detectors, where the liquid phase is frequently organic o r partially organic. Since, ultra microelectrodes have been shown to display higher sensitivities than similarly built macroscaled ones [28], our aim was to study the behavior of some peroxide microelectrodes in aqueous and partially aqueous environment. We chose though, micro-sized electrodes, since their constructation did not require use of any complicated instrumentation.

Previous investigations carried out on the influence of various commercially available GPs and some additives on the behavior of horseradish peroxidase modified carbon pastc electrodes, showed that the presence of LA resulted in improved H 2 0 2 detection, the beneficial properties being independent of the chosen G P and enzymc immobilization conditions [24]. I t w a s also noted that electrodes based on G P with the lowcst background current displayed the lowcst detection limit for H202. Previous experiments carried out with various peroxidases [2 I], and alcohol oxidases [29], showed that the biosensoicharacteristics were influenced not only by the source of the used enzyme, but also differed using the same cnzyme produced by different manufacturers. Taking into consideration the above mentioned premises. pCPE with a working area of about 0.2 mm’ were constructed incorporating different peroxidases. Bioscnsor characteristics of the obtained microelectrodes were compared for H 2 0 2in aqueous and partial aqueous media (H20--10% v./v. CH3CN and H20-I0% v./v. CH30H), and for 2-butanone peroxide (2BP) detection only in H20- lo‘% v./v. C H 3 0 H . 2BP was chosen as a substrate because of its hydrophobic nature. Other experimental conditions, flow rate and injected volumc, were deliberately chosen in order to simulate the use of microelectrodes as a dctcctor in a microliter per minute flow injection system or chromatographic system. Calibration plots obtained for H 2 0 2 detection with clcctrodes based on the four different peroxidase modified CPEs and three different flow carriers, are shown in Figs. 2A-D. It can be concluded, that in spite of the obtained low sensitivity (all values in the pA range), the pCPEs displayed a well-defined Michaelis-Menten behavior (see Table I). I t can bc also noted, that examining the influence of the flow carrier composition on the biosensor sensitivity, the four investigated different types of peroxidase modified electrodes show two different patterns. For HRP-based microelectrodes (Figs. 2A and B) the responses decreased in the following order: H2O > H20-10% v./v. C H 3 0 H > H20-10Yo v./v. CH3CN, while for the ARP-based ones (Figs. 2C and D), the sequence was different: H 2 0 > H 2 0 10% v./v. CH3CN > H20-10% v./v. CH3OH. The FI calibration plots for 2BP obtained for the four types of pCPEs and H20-10% v./v. CH,OH as the flow carrier (Fig. 3), showed a much smaller efficiency than that observed for H2Oz. The same behavior was recently reported for peroxidase-modified carbon fiber microelectrodes [21]. This behavior might be induced by a specific effect of methanol o n the peroxidase activity, as also seen from the comparison of the reported sensitivities in CH3CN and in C H 3 0 H for Eleefrouncilysi~s1996, 8. No. I I

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HRP-modified carbon-fiber microelectrodes [ 141, showing less sensitivity in the latter media. A summarized view giving the overall microbiosensor characteristics, the linear calibration parameters and kinetic parameters obtained from electrochemical Eadie-Hofstee plots are presented in Table 1. Based on the sensitivities for H L 0 2 detection, in the limits of the experimental errors and irrespective of the solvent composition, an activity sequence for the investigated peroxidases could be established as follows: ARP-S > HRP-S > ARP-J > HRP-B. For ;!BP detection the peroxidase activity decreased in a different sequence: HRPS > HRP-B > ARP-J > ARP-S. In the same context, it is worth mentioning that the pCPEs sensitivities for H 2 0 2 detection, obtained from the slopes of the linear domains, and those calculated as the ratio of I,,,,, vs. K"Mpp, showed the same general trend when the flow carrier composition was changed. Thus, irrespective of the used peroxidase, the H202sensitivities decreased in the sequence: H 2 0 > H20-10% v./v. CH3CN > H20-10% v./v. CH30H. This finding is in accordance with recently reported results showing higher responses to H 2 0 2for a HRP-modified biosensor in CH3CN than in C H 3 0 H Electroanalysis 1996, 8, No. 1 1

[ 171, and higher sensitivity for 2BP detection in CH3CN than in C H 3 0 H when a HRP-modified Pt electrode was used [16, 301. This behavior is belived to be due to the higher polarity of methanol which may lead to the denaturation of the enzyme and some specific interactions with the active center of the peroxidase [31, 321. However, other physical properties of the solvents, such as kinematic viscosity and dielectric constants were also shown to influence the diffusion and partition coefficients of the analytes and the electrostatic forces around the enzyme's charge and polar active site, respectively, and thus the sensitivity of an organic phase biosensor [30, 331. Also, it is worth mentioning that increased solvent polarity resulted in an enhanced inhibition of H R P activity in nonaqueous solvents [33]. On the other hand, the use of coupled enzyme based biosensors in organic media could be advantageous since increased solvent polarity was shown to enhance the H 2 0 2 production in reactions between glucose oxidase [34], o r alcohol oxidase [35] with their specific substrate . Concerning the influence of the flow carrier composition on the KZp value for H 2 0 2as substrate, it is interesting to note that again, a general pattern could be established irrespective to the

Peroxides in Partial Aqueous Media

1017

Table 1. Linear calibration and electrochemical Eadie-Hofstee plot parameters for pCPEs incorporating different peroxidases; FI parameters as in Figures 2 and 3 . Linear domain

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0.9962

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0.8 f O.l[a]

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0.982 1

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0.9960

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0.9 fO.l[a]

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0.9896

0.9 fo.l[d]

1.6 f0. I[a]

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0.9866

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0.3fO.l[a]

0.9948

0.7 fO.l[a]

0.6 f0. I[a]

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0.9964

2.4 f0.4[a]

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0.1 fO.OI[a]

0.9962

3.2 f 1.8[a]

0.5 f0.2[a]

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125-600 7

0.9765

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0.2iOl[a]

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0.9990

1.7 f0.3[a]

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used peroxidase: Fip/H2O> e$/H20-10% v./v. CH3OH > f l ~ p / H 2 0 - 1 0 % v./v. CH3CN. The higher Michaelis-Menten constant obtained in the presence of methanol might be due to the lower dielectric constant displayed by methanol, favoring the substrate binding at the enzyme active center. The kinetic parameters and substrate sensitivities of the pCPEs differed, however, from those displayed by carbon fiber microelectrodes (pCFE) [ 211. Table 2 presents comparative values, where each entry represents the mean of 3 equally made electrodes and the sensitivity of the pCFEs is obtained by calculating the minimum and maximum surface area of an electrode made of 50 fibers with 1.5mm length and 7 p m ID, assuming that the enzyme layer covers either only the top (minimum) or the whole surface (maximum) of the pCFEs. Other organic peroxides than 2BP were also studied, their molecular weight increasing as follows; CHP < 2BP < P M H P < DIPBHP. The sensitivity pattern for the pCPEs was H2Oz > 2BP > C H P > P M H P > DIPBHP, while CFEs displayed decreasing sensitivities in order PMPH M DIPBHP N C H P > 2BP > H202, their structures being shown in Figure 4. The organic nature of the pCPEs is believed to influence the partition coeffiecient of the studied organic

peroxides, and hence the sensitivity of the electrode. However, it has been previously shown for other enzymes that when immobilized in the organic phase of carbon paste their selectivity pattern may change drastically compared when in an aqueous environment [25, 36, 371. Generally pCFEs displayed lower detection limits (calculated as twice the signal-to-noise ratio), higher K g P , and broader linear dynamic range for H 2 0 2detection than pCPEs. However, pCPEs are much easier to build, handle and miniaturize. They could be used as amperometric detectors for various peroxides even in partial aqueous media, when coupled after a suitable separation technique. Figure 5 shows F1 peaks obtained for H202,2BP, and C H P in buffer and for H 2 0 2and C H P in buffer with 10% v./v. C H 3 0 H using HRP-S based electrodes. Other biosensor characteristics such as reproducibility (less than 10% for equally made electrodes from the same batch), operational (2% decrease of FI signal/4 h [24]) and storage (no loss of sensitivity for HRP-LA/2 months, and no loss of sensitivity for ARP-LA pastes/3 weeks) stability, can allow the use of such pCPEs for practical applications in food or medical industry. More detailed studies in this direction were beyond the scope of this work. Elrctroanuly.ris 1996, 8, No. I1

I.C.Popescu et al.

1018

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Table 2 Comparison between electi ode charactrristics obtained for ~ L C P -and p C F electrodes for hydrogen peroxide and 2-butanone hydroperoxide The detection limit wab Lalculated as twice the signalto-noise ratio, without taking the dispeisioii factor of the used cell into account ~.

Electinde type/ sub rtrulc CFE-HRP-S/H?O2 CFE-ARP-J/H,O> CFE-ARP-J/ZBP CPE-HRP-S/H?O? CPE-ARP-J/H>02 CPE-ARP-J/2BP

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Fig. 5. F I peaks obtained for HzOz (A), 2BP (B) and CH (C) in 0.1 M phosphate buffer, and in buffer with 10% v./v. CH30H at pH 7.0. Other characteristics as in Figure 2.

4. Conclusions Peroxidase-modified carbon paste microelectrodes were constructed, and characterized with respect to the used peroxidases and composition of the flow carrier. These electrodes displayed a well featured Michaelis-Menten behavior for H202and 2BP. Taking into account the dispersion factor of the flow system, the lowest detection limits (calculated as twice the signal-to-noise ratio) obtained with these electrodes (pCPE-HRP-S) were approximatively 0.1 p M for H202 and 0.3 pM for 2-butanone peroxide. The organic nature of the carbon paste caused a change in the selectivity pattern of the biosensors, the obtained sensitivities for H202and some organic peroxides were different from those obtained with similarly built carbon fiber based microsensors. The developed microelectrodes could be used as amperonietric detectors in various microflow systems operated in partial aqueous media, for on-line monitoring of various peroxides.

5. Acknowledgements

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The authors thank the Swedish Natural Science Research Council (NFR) and Swedish Technical Research Council (TFR) for financial support, Suntory, Japan, Dr. T.D. Gibson (Department of Biochemistry and Molecular Biology, University of Leeds, UK) and Prof. K. Unger (Department of Analytical and Inorganical Chemistry, University of Mainz, Germany) for their generous gift of Arthromyces rumosus, lactitol, and organic peroxides, respectively. The Swedish Institute is gratefully acknowledged by I.C. Popescu for financial help.

6. References CHI

I HT -

0-

CH,

CH3

I f -0 0 - H

4

en-,

Fig. 4. Formulae of the studied peroxides; cumol hydroperoxide (I), 2butanone hydroperoxide (2), paramenthane hydroperoxide ( 3 ) and di-isopropyl-benzol hydroperoxide (4). Elec.tr.ounalysis1996, 8. No. 1 1

[ I ] H.B. Dunford, Peroriduse in C'hemisrr),and Biology 11, CRC Press. New York 1991, p. 2. [2] A. Yaropolov, V. Malovik, S.D. Varfolomeev, I.V. Berezin, Dokl. Akud. Nuuk SSSR 1979, 249, 1399. [3] G. Jonsson, L. Gorton, Electrouncilysi~1989, I , 465. [4] U. Wollenberger, V. Bogdanovskaya, S. Bobrin, F. Scheller, M.R. Tarasevitch, Anal. Lett. 1990, 23, 1795. [5] L. Gorton. E. Csoregi, E. Dominguez, J. Emneus, G. Jonsson-Petterson, G. Marko-Varga B. Person, Anal. Chim. Actu 1991, 250, 203.

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Electroanalysis 1996, 8, No. 1 I