Preparation and optimization of superoxide microbiosensor

Analytica Chimica Acta 358 (1998) 27±33

Preparation and optimization of superoxide microbiosensor SÏtefan MesaÂrosÏa,*, ZÏaneta VanÏkovaÂa, Saul Grunfeldb, Adriana MesaÂrosÏovaÂc, Tadeusz Malinskib a

Department of Analytical Chemistry, Slovak Technical University, RadlinskeÂho 9, SK-812 37 Bratislava, Slovakia b Department of Chemistry, Oakland University, Rochester, MI 48309, USA c Department of Engineering Pedagogy, Slovak Technical University, Laurinska 14, SK-811 01 Bratislava, Slovakia Received 6 May 1997; received in revised form 8 October 1997; accepted 12 October 1997

Abstract An amperometric enzyme electrode for superoxide determination was prepared by anodic polymerization of pyrrole and concomitant incorporation of superoxide dismutase on a Pt wire in phosphate buffer solution. The amperometric response to superoxide was measured at a potential of 0.7 V to oxidize the hydrogen peroxide generated. The response time of biosensor was <5 s and a 15 nM detection limit was achieved. # 1998 Elsevier Science B.V. Keywords: Biosensors; Superoxide

1. Introduction Superoxide anion radical …Oÿ 2
† has attracted much attention in many ®elds of biology since it is realized that superoxide anion is produced in signi®cant quantities in various parts of biological systems, from microorganism to mammals [1]. However, in most cases, the biological signi®cance and mechanism of the generation of Oÿ 2
are not yet well understood. The measurement of the concentration of superoxide in a biological system is a challenging analytical problem. Most methods are indirect, obtaining quantitative information related to the amount of superoxide produced. These indirect methods for superoxide detection may provide misleading infor*Corresponding author. Tel.: +421 (7) 5325-313; fax: +421 (7) 393-198; e-mail: [email protected] 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00589-8

mation. The currently used instrumental techniques for superoxide measurements in biological samples are spectroscopic and electroanalytical in nature. Spectroscopic methods include chemiluminescence [2,3] and electron spin resonance spectroscopy [4]. Mostly used is a chemiluminescence method, based on the measurement of the intensity of the ¯uorescence radiation emitted after chemical oxidation of superoxide by lucigenin (bis-N-methylacridium nitrate) [5]. The detection limit is about 210ÿ8 mol/l. Electron spin resonance (also known as electron paramagnetic resonance) can be used to monitor molecules with an unpaired electron, including radicals such as Oÿ 2
. However, the detection limit is approximately 510ÿ6 mol/l. Electrochemical methods permit direct in situ measurement of superoxide in biological samples. Tanaka et al. [6,7] measured superoxide by direct oxidation on

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a carbon ®ber in a single cell. However, this experiment is not so valid under physiological conditions, where it is also possible to oxidize other species. Speci®city of determination can be found using an enzyme biosensor for the determination of superoxide. Enzymes named superoxide dismutase (SOD) [8] readily release O2 and H2O2 from biological systems of Oÿ 2
by a dismutation reaction. Several authors have used various types of immobilization of SOD for the determination of superoxide. McNeil et al. [9] immobilized covalently the cytochrome c or superoxide dismutase at a surface-modi®ed gold electrode and by passive adsorption to freshly platinized activatedcarbon electrodes. They measured superoxide production from human neutrophils. Methyl viologen was found by Ohsaka et al. [10] to ef®ciently mediate the electron transfer between polyethylene oxide-modi®ed superoxide dismutase and the electrode in dimethyl sulfoxide media. A new Clark-type electrode with immobilized SOD was used for detecting the superoxide. A detection limit of 5 mM was achieved [11]. Therefore, this enzyme (SOD) was used for the preparation of an enzymatic microelectrode. As can be seen from Fig. 1, the substrate (superoxide) penetrates from the tissue (from the bulk solution) into the polypyrrole ®lm, where it is dismuted by superoxide dismutase to hydrogen peroxide SOD

Oÿ 2
! H2 O2 The hydrogen peroxide generated is oxidized at the electrode surface: H2 O2 ! O2 ‡ 2H‡ ‡ 2eÿ

Fig. 1. General kinetic scheme for the polypyrrole superoxide dismutase (SOD) modified electrode.

Among all organic monomers that can be polymerized electrochemically, pyrrole was preferred for two main reasons. The oxidation of pyrrole occurs at low potentials and can be performed in an aqueous solvent without the formation of oxygen [12]. In addition, the polymer formed is conducting; it does not produce a passive ®lm during its preparation and consequently its thickness can be precisely controlled by the conditions of the electrolysis. The polymer can be used either in its conducting or insulating state. In the ®rst case, the electroactive product of the enzymatic reaction is oxidized at the ®lm surface and the electrons are transferred to the electrode through the conducting matrix [13,14]. In the second case, with the polymer in its insulating state, the mediator must diffuse into the inert ®lm and the electrochemical reaction occurs only at the electrode surface [15,16]. 2. Experimental 2.1. Chemicals Superoxide dismutase 3200 U mgÿ1 (E.C. 1.15.1.1.) from bovine liver and all other chemicals were from Sigma. The pyrrole solution was stored under nitrogen in the freezer to avoid any possibility of oxidation by air. All other solutions were prepared in 0.1 mol lÿ1 phosphate buffer aqueous solution (pH 7.4). Other reagents used were of analytical grade and supplied by Sigma. 2.2. Apparatus All the electrochemical experiments were carried out in a cell at 378C with the EG&G PAR Potentiostat/ Galvanostat M273A (Princeton, NJ) with custom data acquisition and control electrochemical software. The working electrode was a cylindrical platinum microelectrode (radius of 20 mm, length of 6 mm). A saturated calomel reference electrode (SCE) and Pt wire auxiliary electrode were used. The pH measurements were performed with a Metrohm pH meter. The superoxide dismutase activity was measured spectrophotometrically according to the Sigma protocol. This activity is de®ned as the amount of enzyme that inhibit the rate of reduction of cyto-

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chrome c by 50% in a coupled system with xanthine and xanthine oxidase at pH 7.8 at 258C in a reaction volume of 3.0 ml. 2.3. Enzyme electrode preparation The surface of the cylindrical Pt microelectrode was polished electrochemically in 1 M H2SO4 using cycling voltammetry from ÿ1.0 to ‡1.0 V vs. SCE, and was then rinsed with distilled water. To incorporate superoxide dismutase into the conducting polypyrrole ®lm, electropolymerization was conducted at 0.8 V vs. SCE using controlled potential coulometry at a Pt microelectrode from degassed phosphate buffer solution (pH 7.4) containing p-tosyl ions (1±10 mM) as supporting electrolyte, pyrrole (100±400 mM) as monomer and the enzyme SOD (ca. 50±500 U/ml). The deposition charge was monitored so as to determine the approximate ®lm thickness (based on the assumption that 45 mC/cm2 charge produces a ®lm of ca. 0.1 mm thickness) [17]. In order to obtain an inert stable non-conducting material, the polymer was electrochemically overoxidized after formation. This was performed by cycling the potential of the working electrode twice between ÿ0.3 and 1.2 V vs. SCE at a sweep rate of 5 mV sÿ1 in a deaerated phosphate buffer solution. The prepared enzyme electrode was washed with phosphate buffer solution (PBS) to rinse off the weakly bound enzyme from the electrode surface. The prepared enzyme electrode was stored in phosphate buffered saline (PBS, pH 7.4) at 48C when not in use. 2.4. Determination of superoxide The amperometric response of the enzyme electrode was obtained in a cell system using an SCE reference electrode and a platinum counter electrode. The cell system containing 4.00 ml of PBS with 0.002 U of xanthine oxidase was kept at 378C. A constant potential of 0.700 V (vs. SCE) was applied to the cell and the current was measured as a function of time. The potential supplied to the system had been predetermined by differential pulse voltammetry as being the diffusion-controlled region for hydrogen peroxide oxidation on the polypyrrole electrode. All the solutions were aerated by bubbling air for 15±20 min prior to use. The solution was

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kept under gentle stirring and after application of a potential to the working electrode, the background current was allowed to decay to a steady state. For the effects of the parameters, a single aliquot of 50 nM of xanthine was pipetted into the PBS solution with xanthine oxidase and the response was recorded. Combination of xanthine 50±400 nM and xanthine oxidase 0.002 U produced the superoxide anion in a manner dependent on the concentration of xanthine [3]. The reduction of oxygen by xanthine oxidase occurs via both univalent and divalent pathways [18]. The percent reduction to superoxide anion (univalent reduction) is dependent on pH. At pH 7.4, the yield of Oÿ 2
was 28% of the total xanthine present in the reaction [19]. We veri®ed this amount by chemiluminescence with lucigenin. For the linearity of the electrode response, successive additions of the required amount of xanthine were made with a micropipette from the stock solution and the current±time response was continuously recorded. The current increased after 3±5 s and became steady after about 10 s. 3. Results and discussion From the literature and our preliminary observations, the parameters that have an effect on the electrode response are ®lm thickness, concentration of monomer and supporting electrolyte, and enzyme loading. Film thickness seems to be the most important parameter as it determines whether the hydrogen peroxide oxidizes at the electrode surface or in the polymer backbone [20,21]. Therefore, the ®rst parameter to be investigated was chosen as the ®lm thickness while other parameters were kept constant (pyrrole: 250 mM; p-tosyl: 5 mM; and enzyme: 250 U/ml). Once the optimum value for the ®rst parameter was determined, the effect of the next parameter was studied at the optimum values of the already investigated parameters and at constant values of those not yet studied. The optimization was veri®ed also by the four-parameters Simplex method. After optimization of the above parameters that pertain to the sensor preparation, the effects of pH and the temperature of storage of the sensor were further evaluated.

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Fig. 2. Response of the enzyme electrode as a function of: (a) film thickness; (b) pyrrole concentration; (c) electrolyte (p-tosyl) concentration; (d) SOD concentration; to 56 nM superoxide in phosphate buffer (pH 7.4).

3.1. Effect of film thickness

3.2. Effect of the monomer (pyrrole) concentration

The effect of ®lm thickness on the amperometric response of the enzyme electrode is given in Fig. 2(a). A number of enzyme electrodes were prepared by varying the electrodeposition time during the electropolymerization for 0±1.0 mm ®lm thickness. From amperometric responses to superoxide of the prepared enzyme electrodes, the optimal ®lm thickness of the enzyme electrode was found to be 0.6 mm. The visual appearance of the electrode surface at this thickness revealed a homogeneously covered surface with a black polypyrrole ®lm.

Fig. 2(b) shows the effect of monomer (pyrrole) concentration in the electropolymerization solution on the amperometric response of the enzyme electrode. The optimal concentration of pyrrole was seen to be 250 mM. At a concentration of 100 mM pyrrole, very little current was passed and, therefore, an observable ®lm could not be obtained in a reasonable period. 3.3. Effect of electrolyte (p-tosyl) concentration The effect of electrolyte (p-tosyl) concentration was studied over the range of 1±10 mM p-tosyl. As

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depicted in Fig. 2(c), the enzyme electrode has a plateau response between 5±7 mM of p-tosyl concentrations. However, an optimal electrolyte concentration of 5 mM was chosen, as this level results in a slower growth of polymer ®lm and, hence, a more readily controllable ®lm thickness. 3.4. Effect of enzyme (SOD) concentration The amount of the enzyme (SOD) incorporated into the polypyrrole ®lm is reported to be proportional to its concentration in the electropolymerization solution. However, the response of the electrode does not seem to depend on the SOD level in the polymerization solution or on its presumably proportionately level incorporated into the polymer ®lm. The optimal enzyme (SOD) concentration in electropolymerization solution was found to be 200 to 350 U/ml, as depicted in Fig. 2(d). 3.5. Response to superoxide and calibration curve Fig. 3(a) shows the response to the addition of aliquots of stock xanthine solution. The response of the enzyme electrodes is rapid (3±5 s). By using the amperometric responses obtained in Fig. 3(a), a typical calibration curve for superoxide was obtained with the optimized enzyme electrode as indicated in Fig. 3(b). Observations were carried out on seven experiments. A linear relationship exist for the entire concentration range (rˆ0.9988). The existence of this linear relationship between the current and the concentration of superoxide is important for an accurate determination of superoxide in biological samples which lies within the narrow range of 50 to 200 nM. The detection limit calculated from the calibration curve (3-criterion) is 15 nM of superoxide. The amperometric determination of superoxide concentration was compared to the chemiluminescence method. A very good agreement between both methods of detection was found. 3.6. Effect of pH The effect of pH was investigated over the biologically relevant range. Fig. 4 shows the pH dependence of the steady-state amperometric response.

Fig. 3. (a) Response of the enzyme electrode to successive xanthine additions; (b) the calibration curve for superoxide.

However, it is important that the superoxide assay is based on the electrochemical oxidation of hydrogen peroxide which itself is a pH-dependent redox process [22]. As indicated in Fig. 4, optimal pH of the enzyme electrode was found to be 7.5, which is very close to the biological pH 7.4. 3.7. Specificity and stability of the enzyme electrode The potential applied to oxidize the hydrogen peroxide generated by the enzyme-catalyzed oxidation of glucose is suf®ciently anodic that several interfering substances could contribute to the oxidation current. The speci®city of the enzyme electrode was determined for various substances, such as ascorbic acid,

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The performance of the optimized polypyrrole± superoxide dismutase biosensor reveals some advantages:

Fig. 4. Effect of the pH on the response of the optimized enzyme electrode.

 This biosensor can be easily prepared in ca. 10 min (e.g. short co-immobilization period)  Response time of the sensor is <5 s (e.g. rapid substrate determination)  The amperometric response to superoxide of the biosensor is linear. Therefore, this biosensor can be easily applicable in biomedical analysis.  The biosensor response was unaffected by the presence of interfering substances. This shows that the biosensor can be used in the blood matrix.  The micro size of the sensor allows to use it for measuring in vivo and in a single cell, respectively.

References

Fig. 5. Effect of storage temperature on stability of the electrode.

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