Amperometric Sensors for Simultaneous Superoxide and Hydrogen

Feb 1, 1997 - Hwajeong Kim , Sung Soo Park , Jooyeok Seo , Chang-Sik Ha , Cheil Moon , and Youngkyoo Kim. ACS Applied Materials ..... A reliable and d...
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Anal. Chem. 1997, 69, 454-462

Amperometric Sensors for Simultaneous Superoxide and Hydrogen Peroxide Detection Vadim Lvovich† and Alexander Scheeline*

School of Chemical Sciences, University of Illinois, 600 South Mathews Street, Urbana, Illinois 61801

A two-channel sensor capable of almost instantaneous simultaneous detection of superoxide radical and hydrogen peroxide in the concentration range 10-7-10-4 M is very important for understanding of a number of rapid kinetics processes. A glassy carbon working microelectrode covered by an electrodeposited polypyrrole/horseradish peroxidase (PPy/HRP) membrane was employed as a H2O2 sensor. Another glassy carbon microelectrode covered by a composite membrane of an inside layer of PPy/HRP and an outside layer of superoxide dismutase was employed as a working electrode for superoxide detection. These two working electrodes with Pt counter and tungsten oxide (WO3) reference electrodes were contained in one 6 mm diameter Teflon cylinder. Simultaneous measurements were performed at a potential of -60 mV (vs WO3 reference, pH 5.1). Additional sensor characterization was performed for pH 5.1-9.0. Superoxide sensor behavior as a function of membrane deposition conditions and coating time is reported. Sensors’ mutual influence, selectivity, response times, linearity, stability, and sensitivity for hydrogen peroxide and superoxide are presented and discussed. A mathematical model of sensors’ responses is proposed, with model calculation corresponding to experiment within 10%. Detection of H2O2 and O2•- is very important for understanding the peroxidase/NADH oscillator,1-5 which is being studied in our laboratory.6-8 The Urbanalator model9-11 predicts spiking outbursts of hydrogen peroxide and superoxide resulting from various reactions in the peroxidase/NADH system. Theoretically predicted concentrations of H2O2 and O2•- peak in the range of * To whom correspondence should be addressed. E-mail: scheeline@ aries.scs.uiuc.edu. † E-mail: [email protected]. (1) Oppenheimer, N. J. In Pyridine Nucleotide Coenzymes; Dolphin, D., Avramovic, O., Poulson, R., Eds.; Wiley-Interscience: New York, 1987; Part A, Chapter 10. (2) Yamazaki, J.; Yokota, K.; Nakayama, R. In Oxidase and Related Redox Systems; King, T. E., Mason, H. S., Morrison, M., Eds.; Wiley-Interscience: New York, 1965; Vol. 1. (3) Fedkina, V. R.; Ataullakhanov, F. I.; Bronnikova, T. V. Theor. Exp. Chem. 1988, 24, 165-170. (4) Perahia, D.; Pullman, B.; Saran, A. In Structure and Conformation of Nucleotic Acids and Protein-Nucleic Acid Interactions; Sundaralingam, M., Rao, S. T., Eds.; University Park Press: Baltimore, MD, 1975; p 685. (5) Bielski, B. H.; Chan, P. C. J. Am. Chem. Soc. 1980, 102, 1713-1720. (6) Olson, D. L.; Scheeline, A. Anal. Chim. Acta 1993, 283, 703-743. (7) Olson, D. L.; Scheeline, A. J. Phys. Chem. 1995, 99, 1204-1211. (8) Olson, D. L.; Scheeline, A. J. Phys. Chem. 1995, 99, 1212-1217. (9) Olson, D. L. Ph.D. Thesis, University of Illinois, 1994. (10) Olson, D. L.; Williksen, E.; Scheeline, A. J. Am. Chem. Soc. 1995, 117, 2-15. (11) Bronnikova, T. V.; Fed’kina, V. R.; Olsen, L. F.; Schaffer, W. M. J. Phys. Chem. 1995, 99, 8431-8438.

454 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997

10-6-10-7 M. Therefore, selective, durable, and sensitive detectors with nanomole detection limits are needed to characterize superoxide and hydrogen peroxide concentration-time profiles. Nearly instantaneous (within 1 s after perturbation) sensor response, linear behavior for low substrate concentrations, long lifetime, and lack of interference with the chemistry of the peroxidase/NADH oscillator are also essential. Reactions of the peroxidase/NADH oscillator are typically carried out at pH 5.17.0, so both sensors have to perform throughout this pH range. The small reactor volume (∼5 mL) adds the requirement that the sensors be as compact as feasible. More broadly considered, the determination of hydrogen peroxide and superoxide is of practical importance in chemical, biological, clinical, environmental, and many other fields. However, conventional methods for the determination of superoxide and simultaneous detection of O2•- and H2O2 do not satisfy, at the same time, requirements of sensitivity, selectivity, reliability, response speed, low detection limit, and operational simplicity. Many enzyme electrodes have been reported as sensitive sensors for biologically important analytes, including hydrogen peroxide and superoxide.12-25 Among these, amperometric sensors based on electron transfer between an enzyme and the electrode are promising for fabricating sensitive and linearly responding devices. We developed such sensors, based on multilayer structures on glassy carbon microelectrode surfaces, with electrodeposited enzymes embedded in the polymer layers. The mechanism of these sensors is based on redox chemistry, leading to regeneration of native peroxidase by current supplied by the sensor electrode. A glassy carbon working microelectrode covered by an electrodeposited polypyrrole/horseradish peroxidase (PPy/HRP) membrane was employed as a H2O2 sensor. Another similarly prepared sensor with an additional adsorbed layer of superoxide dismutase (SOD) was employed as a working electrode for O2•- detection. The measurements were performed for constant potentials which (12) Kulis, J. J.; Samaluis, A. S. Electrokhimiya 1984, 20, 637-641. (13) Armstrong, F. A.; Lannon, A. M. J. Am. Chem. Soc. 1987, 109, 7211-7212. (14) Pan, S.; Arnold, M. A. Anal. Chim. Acta 1993, 283, 663-671. (15) Wollenberg, U.; Bogdanovskaya, V.; Bobrin, S.; Scheller, F.; Tarasevich, M. Anal. Lett. 1990, 23 (10), 1795-1808. (16) Belanger, D.; Nadreau, J.; Fortier, G. J. Electroanal. Chem. 1989, 274, 143155. (17) Csoregi, E.; Gorton, L.; Marko-Varga, G.; Tudos, A. J.; Kok, W. T. Anal. Chem. 1994, 66, 3604-3610. (18) Tatsuma, T.; Okawa, Y.; Watanabe, T. Anal. Chem. 1989, 61, 2352-2355. (19) Tatsuma, T.; Gondaira, M.; Watanabe, T. Anal. Chem. 1992, 64, 11831187. (20) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992, 64, 625-630. (21) Tatsuma, T.; Watanabe, T.; Tatsuma, S.; Watanabe, T. Anal. Chem. 1994, 66, 290-294. (22) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5970-5975. (23) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5976-5980. (24) Vreeke, M.; Maidan, R.; Heller, A. Anal. Chem. 1992, 64, 3084-3090. (25) Heller, A. J. Phys. Chem. 1992, 96, 3579-3587. S0003-2700(96)00626-9 CCC: $14.00

© 1997 American Chemical Society

were close to 0 V vs normal hydrogen electrode (NHE) to avoid reduction or oxidation of other components of the peroxidase/ NADH system under investigation. Membrane behavior dependence on fabrication and sensors’ storage conditions is demonstrated, with subsequent optimization of superoxide radical and hydrogen peroxide sensing. We also report sensors’ sensitivity, cross-talk, selectivity, response times, linearity, and stability for hydrogen peroxide and superoxide. To verify the validity of the data, a detailed mathematical model of the sensors’ responses to low substrate concentrations is proposed and compared with experimental results. EXPERIMENTAL SECTION Chemicals. A 30% solution of hydrogen peroxide (Fisher) was used to prepare 10 mM and 10 µM H2O2 stock solutions. After initial opening, H2O2 solutions were refrigerated at 4 °C. For generation of superoxide at pH 5.1-7.0, 99% xanthine (Sigma) and xanthine oxidase (Boehringer-Mannheim, GmbH, Germany) were used to prepare 1 mM xanthine (XA) and 2 µM xanthine oxidase (XOD) stock solutions. For characterization of superoxide electrodes at more alkalinic pHs, 10 µM KO2 (Aldrich) in potassium phosphate buffer (pH 7.0-9.0) was used as another O2•- stock solution. Uric acid (Sigma) was used to study its possible interference on superoxide sensor response. Superoxide dismutase (SOD, Boehringer-Mannheim, GmbH, Germany), polypyrrole (PPy, Aldrich), and horseradish peroxidase (HRP, Sigma) were used as received. A 0.1 M acetate buffer solution (pH 5.1-7.0) was prepared by combining the appropriate volumes of 0.1 M NaOAc and 0.1 M HOAc. A 0.1 M phosphate buffer (pH 7.0-9.0) was prepared by combining the appropriate volumes of 0.1 M Na2HPO4 and 0.1 M KH2PO4. Typical solution volumes were 5 mL. Buffer solutions were deoxygenated by nitrogen bubbling for 20 min, except for experiments using the XA/XOD reaction, which requires O2 to be present for superoxide production. Apparatus. Initial electrochemical measurements were made with a standard three-electrode potentiostat (Model 660 Electrochemical Workstation, CH Instruments Inc.) in a 50 mL glass beaker. For simultaneous detection of substrates with both superoxide and hydrogen peroxide sensors, we employed a locally built dual potentiostat interfaced to an ISA bus 486 computer with a CIO-DAS 1602/16 ADC board (Computer Boards Inc., Mansfield, MA). Data analysis was carried out with The Unscrambler Version 5.03 (CAMO Ltd., Trondheim, Norway), Quattro Pro Version 5.0 (Borland, Scotts Valley, CA), and Microsoft Excel (Microsoft Corp.). Initially potentials of the sensors were held at -60 and -50 mV vs WO3 reference electrode for O2•- and H2O2 detection, respectively (pH 5.1). When additional separate characterization of both sensors was performed at various pHs (5.1-9.0), potentials of the sensors were held at -230 and -220 mV vs Ag/AgCl reference electrode for O2•- and H2O2 detection, respectively. The choice of these potentials was based on the requirement to hold the potentials of both sensors close to 0 mV vs NHE to ensure their proper selectivity. However, when simultaneous superoxide and H2O2 detection with separate counter and reference electrodes for both sensors was carried out, severe cross-talk and electronic noise problems were encountered. Cyclic voltammograms in solutions containing superoxide and H2O2, recorded at superoxide and hydrogen peroxide sensors, respectively, demonstrated no significant dif-

ference in values of cathodic current corresponding to O2•- and H2O2 detection at -60 and -50 mV (vs WO3 reference electrode, pH 5.1), or at -230 and -220 mV (vs Ag/AgCl reference electrode, pH 5.1-9.0). Therefore, the final potentiostat setup for simultaneous superoxide and H2O2 detection was a fourelectrode design (with one reference, one counter, and two working electrodes), with potentials of both sensors held at -60 mV vs WO3 reference electrode for experiments at pH 5.1, and at -230 mV vs Ag/AgCl reference electrode for those at variable pH from 5.1 to 9.0. For all sensor performance experiments, current-time profiles were recorded to determine their sensitivity to hydrogen peroxide and superoxide and possible interferences by uric acid (a byproduct of the XA/XOD reaction) and the main components of the peroxidase/NADH system, namely NADH, NAD+, HRP, and methylene blue. Diffusion coefficients for H2O2 and superoxide were determined from Levich plots of limiting current vs square root of rotation rate26 on glassy carbon rotating disk (R ) 1.5 mm, Bioanalytical Systems Inc., West Lafayette, IN) using BAS-100 RDE-1 rotating disk module. An enzyme membrane, corresponding to the substrate under investigation, was deposited on the surface of the rotating disk electrode before experiments in H2O2 (pH 5.1) and superoxide solutions (pH 5.1 and 9.0) were performed. The methods for membrane deposition for the rotating electrode were the same as those for sensor microelectrodes, which are described below. Diffusion coefficients were determined as DH2O2 ) (2.34 ( 0.14) × 10-5 cm2 s-1 and DO2- ) (2.45 ( 0.12) × 10-5 cm2 s-1 for solutions containing 10-5 M H2O2 and 2.4 × 10-6 M superoxide, respectively. During all experiments, solution was stirred at a single constant rate with the magnetic stirrer from a Corning hot plate stirrer PC-351. Stirring efficiency was determined by detecting limiting current achieved by mixing with the magnetic stirrer and comparing that to limiting current at a rotating disk electrode. The limiting current value for all non-RDE experiments corresponded to that for 234 rpm for RDE of the same surface area. Corresponding diffusion layer thickness (d) was determined from the Levich equation26 as

d ) D1/3υ1/6/0.620ω1/2 ) 10-2/ω1/2 ≈ 5 × 10-3 cm

(1)

A chronocoulometry experiment26 was employed to obtain an approximate value for the heterogeneous electrode reaction rate constant, Kheterog, for HRP-mediated electron transfer: Kheterog

HRPox + 2e (electrode) 98 HRPred

(2)

Kheterog was determined as 0.176 ( 0.014 cm s-1, which compares well to a reported value of 0.133 ( 0.049 cm s-1.12 As will be seen later, only an approximate value was needed for this work, so this value was not refined with detailed experiments. Electrodes. Reference, Counter, and Working Electrodes. For all measurements at pH 5.1, a 0.8 mm diameter freshly polished tungsten rod was used as a reference electrode, with its potential -170 mV vs a Ag/AgCl reference electrode, as demonstrated by comparing cyclic voltammograms of buffer solution obtained using each of those reference electrodes under otherwise identical conditions. For variable pH from 5.1 to 9.0, a conventional Ag/ (26) Bard, A. J.; Faulkner, L. R. Electrochemical Methods; Wiley: New York, 1981; p 205.

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455

Figure 1. Two-channel superoxide/hydrogen peroxide sensing device.

AgCl reference electrode (Bioanalytical Systems) was employed. A 0.6 mm diameter platinum wire was used as counter electrode. The counter and reference electrodes and two working electrodes were contained in a 6 mm diameter Teflon cylinder (Figure 1). This setup showed far less electrical noise than an analogous six-electrode device, where separate counter and reference electrodes were employed for each working microelectrode. No electrical cross-talk between H2O2 and O2•- microsensors was detected. The procedure for preparation of glassy carbon and platinum electrodes was previously described.27 Glassy carbon rods with 1 mm diameter (Johnson Matthey Co., Ward Hill, MA) were sealed in a glass capillary tube (2 mm external diameter). Enzyme Immobilization. The hydrogen peroxide sensor was prepared by deposition of HRP on the glassy carbon microelectrode surface. Methods analogous to those employed here have been described previously.13-25 For adsorption of enzymes, glassy carbon was electrochemically activated by triplicate polarization to +2000 mV (100 s) and -1000 mV (200 s) in 0.1 M acetic buffer (pH 5.1, WO3 reference). This activated electrode was dipped into a 0.1 M pyrrole solution in 0.1 M acetate buffer (pH 5.1), where polypyrrole was electropolymerized and deposited for 100 s while the microelectrode was held at +1000 mV (WO3 reference). The microelectrode was then transferred into 2 mg/mL HRP solution in 0.06 M KCl, 0.1 M pyrrole in 0.1 M acetate buffer (pH 5.1), where potentiometric electrodeposition of membrane was continued at a constant current density of 0.5 mA/cm2 for 10 s. Typically, the microelectrode potential was from +900 to +1000 mV. After electrodeposition was completed, the microelectrode was conditioned in a 2 mg/mL HRP solution in 0.06 M KCl in 0.1 M acetate buffer (pH 5.1) for about 4 h. (27) Lvovich, V.; Scheeline, A. Arch. Biochem. Biophys. 1995, 320 (1), 1-13. (28) McNeil, C. J.; Smith, K. A.; Bellavite, P.; Bannister, J. V. Free Rad. Res. Commun. 1989, 7 (2), 89-96. (29) McNeil, C. J.; Greenough, K. R.; Weeks, P. A.; Self, C. H.; Cooper, J. M. Free Rad. Res. Commun. 1992, 17 (6), 399-406. (30) Cooper, J. M.; Greenough, K. R.; McNeil, C. J. Mol. Cryst. Liq. Cryst. Sci. Technol. 1993, 234, 409-414.

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The superoxide sensor was prepared by successive deposition of HRP/PPy and SOD on the glassy carbon microelectrode surface. The sequence and experimental conditions for glassy carbon electrochemical activation and HRP/PPY membrane deposition were identical to those for H2O2 sensor preparation. Following HRP/PPy deposition, the microelectrode was soaked at room temperature in 1 mL of 1 mM SOD in 0.1 M acetate buffer (pH 5.1) for 24 h, followed by air-drying. For detection of superoxide generated by XA/XOD reaction, the sensor was polarized at -60 mV (vs WO3 reference), and the rate of change of current as a function of enzymatically produced O2•- was measured. For experiments at more basic pHs when characterization of the sensor with KO2 was possible, the microsensor was polarized at -230 mV vs Ag/AgCl reference. Generation of Superoxide by Xanthine/Xanthine Oxidase. The kinetic model31-38 of superoxide generation resulting from interaction between xanthine (XA) and xanthine oxidase (XOD) is presented in Table 1. Superoxide was generated as an intermediate during the oxidation of xanthine by xanthine oxidase according to a two-stage mechanism (reactions 3a, 3b, and 4 in Table 1) proposed in refs 28-33. Superoxide is rapidly converted to H2O2 at step 6. The xanthine/xanthine oxidase reaction’s main path, which is independent of step 4, goes to the ultimate product H2O2 through an intermediate (reactions 5a and 5b in Table 1), with a Michaelis constant of ∼1.7 × 10-6 M. The detailed kinetic model was proposed to be pseudo-firstorder with respect to [XOD]. Rate constants can be expressed as k3 ) k3b [XOD]/(1/KEQ3 + [XOD]) and k5′ ) k5 [O2]/(1/KEQ5 + [O2]). It was reported that rate constant k4 is about 4 times higher than k5′, even if a correction for the following superoxide dismutation (6) is made.34,35 We experimentally recheck this value in the section titled Xanthine/Xanthine Oxidase Reaction Superoxide/Hydrogen Peroxide Production Ratio (below). A stock solution of 0.1 mM XA was prepared in 0.1 M acetate buffer and used as a background solution. After stable baseline response of the superoxide electrode had been obtained (∼2 min), XOD was injected in different amounts, to give final enzyme concentration over the range 0.01-5 µM. The rate of current production during the reduction of oxidized HRP was measured as a function of XOD concentration. Approximately 1 mM bulk oxygen concentration for non-deoxygenated solutions was determined by amperometry at a Pt electrode. RESULTS AND DISCUSSION Electron Transfer Path in Hydrogen Peroxide and Superoxide Sensors. Cyclic voltammograms were recorded for pure acetate buffer solution and solutions containing H2O2 and superoxide, respectively. At both electrodes, cathodic current appeared (31) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1968, 243, 5753-5760. (32) Fridovich, I.; Handler, P. J. Biol. Chem. 1958, 233, 1578-1580. (33) McCord, J. M.; McNeil, C. J.; Fridovich, I. In Superoxide and Superoxide Dismutases; Michelson, A. M., McCord, J. M., Fridovich, I., Eds.; Academic Press: New York, London, San Francisco, 1977; pp 11-17. (34) Olson, J. S.; Ballou, D. P.; Palmer, G.; Massey, V. J. Biol. Chem. 1974, 249 (14), 4350-4362. (35) Olson, J. S.; Ballou, D. P.; Palmer, G.; Massey, V. J. Biol. Chem. 1974, 249 (14), 4363-4382. (36) Bray, R. C. In The Enzymes; Boyer, P. D., Ed.; Academic Press: New York, 1984; pp 358-388. (37) Bommarius, A. S.; Hatton, T. A.; Wang, D. I. C. J. Am. Chem. Soc. 1995, 117, 4515-4523. (38) Behar, D.; Czapski, G.; Rabani, J.; Dorfman, L. M.; Schwarz, H. A. J. Phys. Chem. 1970, 74, 3209-3213.

Table 1. Rate and Equilibrium Constants for Reactions of Xanthine/Xanthine Oxidase Process eq no.

reaction

kf

KEQ3

3a

XA + XODox y\z XOD-XA

3b

XOD-XA 98 XODred + uric acid

k3b

KEQ (M-1)

ref

2 × 105 (pH 5)

31-37

885 min-1

34-37 34-37

k4

4

XODred + O2 9 8 O2•- + XODox HO

5a

XODred + O2 y\z XODred-O2

5b

XODred-O2 98 XODox + H2O2

6

2O2

2

2 × 103 (pH 6)

KEQ5

k5

205 s-1

k6

•-

34-37 34-37

2 × 107 M-1 s-1 (pH 5.1)

9 8 O2 + 2H2O + 4H

Figure 2. Sequence of electron transfer reactions for hydrogen peroxide reduction by HRP at surface-modified glassy carbon electrode. Redox potentials reported vs Ag/AgCl.

at potentials between 0 and -100 mV (vs WO3 reference). With electrodes prepared without enzymes, addition of substrate did not lead to any changes vs background. Since no mediators were present, this suggests that reduction current arose from direct electron transfer between the electrode and the enzyme membrane. The electron transfer path for the hydrogen peroxide sensor has been discussed12-21 and is illustrated in Figure 2. Superoxide generated by the interaction of xanthine and xanthine oxidase, or by injection of KO2 at basic pHs, was initially detected as illustrated in Figure 3. SOD present in the superoxide sensor membrane catalyzed disproportionation:38,39 SOD

2O2•- + 2H+ 98 O2 + H2O2

(6)

Sensed electrode current resulted from HRP-mediated reduction of enzymatically generated hydrogen peroxide or H2O2 from bulk solution which diffused through the SOD layer: HRP

H2O2 + 2H+ + 2e 98 2H2O

(7)

Superoxide Sensor Studies. Specificity of the superoxide sensor at pH 5.1 was investigated using injections of different volumes of 2 µM xanthine oxidase into the electrochemical cell, which initially contained 10 µM xanthine in acetate buffer solution. A linear relationship between the initial rate of change of cathodic current (A s-1) and the concentration of XOD from 0.01 to 10 µM was observed. Cathodic current increase was observed in less than 1 s after XOD injection, and limiting current was reached in 4-5 s. Typical calibration range was 5 × 10-8-1 × 10-6 M. Sensitivity, defined as the rate of change of current density per (39) Sawyer, D. T.; Valentine, J. S. Acc. Chem. Res. 1981, 14, 393-400.

Figure 3. Sequence of electron transfer reactions for superoxide dismutase and HRP-mediated superoxide reduction at glassy carbon electrode covered by a composite membrane. Redox potentials reported vs Ag/AgCl.

unit concentration of XOD, was calculated as 0.057 ( 0.003 A s-1 cm-2 M-1 from 45 experiments (henceforth noted as N ) 45). The superoxide microsensor response was also studied by another protocol. Superoxide was injected in the form of KO2 for more basic solutions (pH 7.0-9.0), where O2•- disproportionates more slowly. For those experiments, a steplike current response was observed, with the reduction current being linearly proportional to the amount of KO2 between 0.01 and 10 µM. The resulting sensitivity, defined as the current density per unit concentration of KO2, was determined as 0.114 ( 0.006 A cm-2 M-1 (N ) 32). The subsequent theoretical calculations, presented in Appendix 3 (eq A16), confirmed the validity of the determined sensitivity and its relation to those detected by measuring the sensor response to XA/XOD reaction. The studies of superoxide sensor response to injections of KO2 also showed that it responded to superoxide O2•- and not to hydroperoxyl radical or products and reactants of xanthine/xanthine oxidase reaction. The sensitivity of the superoxide sensor to H2O2 in acetate buffer was determined as 0.266 ( 0.008 A cm-2 M-1 (N ) 25). The response of the superoxide sensor to H2O2 can be explained by hydrogen peroxide diffusion past the SOD layer to the HRPloaded membrane. These results were verified by monitoring superoxide sensor response to joint injections of H2O2 and XOD into acetate buffer solution which also contained 0.1 mM of xanthine. The superoxide sensor sensitivity to XOD injections Analytical Chemistry, Vol. 69, No. 3, February 1, 1997

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Table 2. Theoretical and Experimental pH Dependence of Superoxide Sensor Response pH 5.1 5.5 5.9 6.4 7.0 0.72 0.83 0.89 0.96 0.99 [O2•-]/[O2•-] + [HO2•] Vmax 230 280 313 379 445 Vmax[O2•-]/[O2•-] + [HO2•] 166 232 279 364 441 current rate ratio for selected pH values

∆I/∆t theorb ∆I/∆t exptlc

∆I5.5/∆t ∆I5.1/∆t

∆I5.9/∆t ∆I5.5/∆t

∆I6.4/∆t ∆I5.9/∆t

∆I7.0/∆t ∆I6.4/∆t

1.40 1.46

1.20 1.25

1.31 1.27

1.21 1.18

a Obtained from K ) [H+] [O •-]/[HO •] ) 10-pH[O •-]/[HO •] a 2 2 2 2 for every pH value (row 1). b Obtained by dividing every following ••• Vmax[O2 ]/([O2 ] + [HO2 ]) value (row 4) by its previous value for every pH. c Experimental data.

was found to be 0.059 ( 0.003 A s-1 cm-2 M-1 (N ) 45). Additional superoxide sensor characterization was performed by monitoring its response to joint injections of H2O2 and KO2 at pH 9.0. The sensor sensitivity to H2O2 was found to be practically pH independent, while the sensitivity of 0.114 ( 0.006 A cm-2 M-1 for KO2 injections was reconfirmed. A series of experiments for pH 5.1-7.0 was conducted to determine differential response of the sensor to superoxide proper O2•- and its protonated form, hydroperoxyl radical HO2•.38-40 Hydroperoxyl radical has a pKa of 4.50-4.88,38-40 so HO2• may account for 25-35% of overall one-electron-reduced oxygen in solution at pH 5.1. Superoxide sensor response to XA/XODgenerated superoxide varied strongly with pH (Table 2). These changes could be explained due to increasing amount of O2•- in bulk solution, as well as by an increase of Vmax for the xanthine/ xanthine oxidase reaction.34 We compared theoretical pH dependence of the sensor response with averaged results from five sets of different O2•- detection experiments for pH between 5.1 and 7.0. For selected pHs, theoretical values of the sensor current rate ∆IpH/∆t were determined as being proportional to a product of Vmax34 and the ratio of [O2•-] to [O2•-] + [HO2•], determined by pKa. Results are presented at Table 2. Clearly the superoxide sensor responded to superoxide concentration [O2•-], and not to that of its acidic form, HO2•. In turn, the coefficient DO2- ) 2.45 × 10-5 cm2 s-1, determined in the RDE experiment described in the section titled Apparatus (above), is the diffusion coefficient for superoxide proper and not of HO2•. The superoxide microsensor response was unaffected by other components of the peroxidase/NADH system (methylene blue, HRP, NADH, and NAD+) or by products and reactants of the XA/ XOD reaction (uric acid and xanthine oxidase in the absence of xanthine or oxygen). Between experiments, the sensor was kept in buffer at room temperature, which was found to be more satisfactory than storage at 4 °C, as was suggested by several previous authors.28-30 Surprisingly, the working stability of our enzyme microelectrode preserved at room temperature turned out to be quite high, which made it possible to employ the same microelectrode for up to 10 days. Only about a 7% decrease in sensitivity was observed within a week, compared to a 30% decrease per 2 days for refrigerated (40) McCord, J. M.; Fridovich, I. J. Biol. Chem. 1969, 244 (22), 6049-6055.

458 Analytical Chemistry, Vol. 69, No. 3, February 1, 1997

Figure 4. Dependence of superoxide sensor sensitivity on time of SOD film deposition. Time of HRP film deposition was held constant for all experiments at the optimal value of 4 h. The sensor was characterized by superoxide produced by (solid bars) XA/XOD reaction (pH 5.1) and (hatched bars) KO2 (pH 9.0).

electrodes. Reasons for improved stability of microelectrodes at room temperature were not investigated. However, the loss of enzyme by desorption and additional denaturation at low temperatures was proposed in the literature as the reason for deterioration.28-30 Subsequent experiments with the two-channel sensing device for simultaneous detection, when H2O2 and XOD were injected into the cuvette containing XA (pH 5.1), or when potassium superoxide and hydrogen peroxide were injected in phosphate buffer solution (pH 9.0), did not show any mutual interference or electrical cross-talk. Response Properties of HRP/PPy/SOD Composite Membrane. Since the amount of enzyme on the electrode surface depends on the thickness of the membrane, the electrode response with thin enzyme membranes is expected to be limited by enzymatic reaction rate, and that of electrodes with thick films to be limited by mass transfer. Studies of the response of hydrogen peroxide sensor dependence on HRP/PPy film thickness were presented in a series of papers by Watanabe et al.18-21 We expanded their approach to a composite HPR/PPy/SOD membrane for the superoxide microelectrode sensor. The sensitivity vs amount of injected XOD (pH 5.1) and KO2 (pH 9.0) were separately examined for various HRP and SOD deposition times. The initial electrodeposited HRP/PPy corresponded to an electropolymerization charge of 5 mC cm-2.18,19,21 The overall thickness and composition of HRP and SOD films were controlled by varying the electrode soaking time in their respective solutions. Figure 4 shows the experimental dependence of current density vs time of electrode soaking in SOD for a constant optimal time of electrode soaking in HRP/PPy (4 h) for XOD/XA and KO2 characterizations. Figure 5 demonstrates the experimental dependence of current density vs soaking time in HRP/PPy for a constant optimal time of electrode soaking in SOD (24 h). These results corresponded well to a theoretically expected profile of sensitivity vs membrane thickness, with microelectrode sensor response increasing with time of enzyme immobilization until saturation at 4 h for the HRP-bearing film and 24 h for the SOD film. This is consistent with the view that response is

calibration was performed at pH 9.0. If data obtained for the experiments with KO2 at pH 9.0 are valid for more acidic solutions (as demonstrated above, for example, the hydrogen peroxide sensor response was pH independent), the final expression for superoxide sensor response is

IO2- sensor ) (0.114 ( 0.006)[O2•-] + (0.266 ( 0.008)[H2O2] (9)

Figure 5. Dependence of superoxide sensor sensitivity on time of HPR film deposition. Time of SOD film deposition was held constant for all experiments at the optimal value of 24 h. The sensor was characterized by superoxide produced by (solid bars) XA/XOD reaction (pH 5.1) and (hatched bars) KO2 (pH 9.0).

controlled by the enzymatic reaction at small deposition times while determined by mass transfer at long deposition times. Hydrogen Peroxide Sensor Studies. The specificity of the hydrogen peroxide sensor for H2O2 was investigated using injections of different volumes of substrate into the electrochemical cell, which initially contained only acetate buffer (pH 5.1) solution. Current step responses were observed in less than 1 s after injection, and steady-state current was reached in 1-2 s. Reduction current was proportional to [H2O2] between 0.02 and 600 µM. Sensitivity for hydrogen peroxide, defined as the change of current density per unit concentration H2O2, was calculated as 0.456 ( 0.014 A cm-2 M-1 (N ) 53). Theoretical calculations, presented in Appendix 2 (eq A10), also give expected sensitivity of this order. Between experiments, the sensor was kept in buffer at room temperature, as for the superoxide sensor. This also differs from conventional wisdom.14-17 Sensitivity decreased 5% per week, versus a 30% decrease every 2 days for refrigerated electrodes. Reasons for this behavior were not sought. It appeared that the presence of other components of the peroxidase/NADH systemsmethylene blue, HRP, NADH, and NAD+sdid not affect the electrode response. The hydrogen peroxide sensor was also introduced into xanthine/xanthine oxidase solution, which was not deoxygenated. The sensitivity of the hydrogen peroxide sensor to the XA/XOD reaction, which ultimately results in H2O2 generation in reactions 5a, 5b, and 6, was found to be 0.010 ( 0.0002 A cm-2 s-1 M-1. As indicated above, no mutual interference or cross-talk was observed between hydrogen peroxide and superoxide microsensors. These results will make it possible to employ both microsensors in the peroxidase/NADH oscillatory system with adequate signal differentiation. Overall hydrogen peroxide sensor response to H2O2 is

IH2O2 sensor ) (0.456 ( 0.014)[H2O2]

(8)

when current density is in A/cm2 and H2O2 concentration in M. The superoxide sensor responds, however, to both O2•- and H2O2. Because superoxide concentration is unstable at pH 5.1,

when current density is in A/cm2 and substrates’ concentrations in M. Employing eqs 8 and 9, we plan to detect, differentiate, and quantify bursts of H2O2 and O2•- resulting from various reactions in the peroxidase/NADH system. Xanthine/Xanthine Oxidase Reaction Superoxide/Hydrogen Peroxide Production Ratio. Sensitivity for hydrogen peroxide and superoxide sensors allowed us to verify XA/XOD reaction parameters (Table 1, refs 31-38). Since the sensitivities to both O2•- and H2O2 were independently measured, we could reverse the logic applied in calibration using XA/XOD to determine the branching ratio of O2•-/H2O2 production rate. The sensitivity of our hydrogen peroxide sensor to H2O2 generated in reactions 5a, 5b, and 6, was found to be 0.010 A cm-2 s-1 M-1, and that for the superoxide sensor was determined as 0.057 A s-1 cm-2 M-1. Sensitivity of the hydrogen peroxide sensor to pure H2O2 was measured as 0.456 A cm-2 M-1, and that for the superoxide sensor was measured as 0.266 A cm-2 M-1. Therefore, a contribution of H2O2 produced at stages 5a, 5b, and 6 of the XA/XOD process to overall sensitivity of the superoxide sensor can be determined as

0.010(0.266/0.456) ) 0.006 A cm-2 s-1 M-1 Sensitivity of superoxide sensor determined exclusively by O2•is 0.057 - 0.006 ) 0.051 A cm-2 s-1 M-1. A branching ratio of superoxide/hydrogen peroxide production in XA/XOD process can be calculated as 0.051/0.010 ) 5.1, slightly above the literature branching ratio.34,35 CONCLUSIONS A two-channel sensing device for simultaneous hydrogen peroxide and superoxide detection was fabricated and characterized. Dependence of composite membrane response on enzyme deposition time corresponded to a predicted behavior. Both microsensors demonstrated linear responses for substrate concentrations in the low micromole-high nanomole range and were able to selectively determine substrates at modest overpotentials without evident interference from the other components of the peroxidase/NADH system. We found that storing sensors at room temperature prolonged their effective lifetime up to 10 days. It became possible to achieve fast (in about 1 s), selective, and sensitive simultaneous detection of hydrogen peroxide and superoxide. Additional studies of superoxide sensor behavior showed that its response was determined by superoxide O2•- and not by hydroperoxyl radical or products and reactants of the xanthine/xanthine oxidase reaction. The mathematical analysis presented in the appendices was employed for validating performance of the microsensors. The proposed “linear region” model for low substrate concentrations Analytical Chemistry, Vol. 69, No. 3, February 1, 1997

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is presented in a series of first-order equations for steady-state kinetic conditions. In the process of theoretical calculation, we were able to verify a number of kinetics parameters, previously reported in the literature, and demonstrate the validity of our results. ACKNOWLEDGMENT The authors express their gratitude to E. Kirkor, G. Horras, and D. Bowlin for many helpful discussions. The dual potentiostat was assembled by Carl Reiner and the School of Chemical Sciences Electronic Service. The financial support of the National Science Foundation (Grant CHE-93-07547) and the University of Illinois is gratefully acknowledged. APPENDIX 1. MATHEMATICAL MODEL FOR SENSOR RESPONSE We constructed an overall model predicting the response of the hydrogen peroxide and superoxide sensors. Analysis of enzyme electrode kinetics is important for sensor design and optimization. Several such systematic studies in that field have been reported,41-48 including those related to multilayer kinetics models. However, many of these attempts46-48 were complicated because the enzyme molecules are distributed homogeneously in a relatively thick layer, in which the local concentration of substrate(s) and product(s), the local rate of enzymatic reaction, and the local velocities of mass transfer are not uniform, even in the steady state. In most of these cases, solution of differential equations is complex, if possible in closed form at all. In contrast, steady-state kinetics of a thin enzyme membrane electrode, where enzyme molecules are arranged on a plane, can be analyzed with a series of relatively simple differential equations. While developing the model, we assumed that (1) the electrode is an ideal plane; (2) active centers of HRP molecules are homogeneously distributed on a plane at a distance a from the electrode surface, and those of SOD at a distance b from the electrode surface, where 2a ) b; (3) both electrodes work in the so-called “linear region”, where substrate concentration is so low that the sensor response is proportional to the concentration [That assumption is true for specific conditions of low H2O2 and superoxide (10-6-10-7 M) concentrations generated in the peroxidase/NADH oscillatory system, which is the system motivating this work. Therefore, for the hydrogen peroxide sensor, H2O2 is completely consumed as a result of its reaction with HRP ([H2O2] ) 0 at X ) 0); for the superoxide sensor, O2•- is completely consumed as a result of its reaction with SOD ([O2•-] ) 0 at X ) a), and generated H2O2 is completely consumed as a result of its reaction with HRP ([H2O2] ) 0 at the electrode surface X ) 0)]; (4) diffusion layer thickness d is constant and is much larger than the enzyme membrane thickness; (5) solution is continuously stirred at a constant rate; (6) substrate concentrations in the bulk solution are constant; and (7) the substrate and enzymes carry the same number of charges n. (41) Bourdillon, C.; Bourgeois, J. P.; Thomas, D. J. Am. Chem. Soc. 1980, 102, 4231-4235. (42) Kulis, J. J. Enzyme Microb. Technol. 1981, 3, 344-352. (43) Schultz, A. R. Enzyme Kinetics; Cambridge University Press: New York, 1994. (44) Kuchel, P. W. In Kinetic Analysis of Multienzyme Systems; Welch, G. R., Ed.; Academic Press: New York, 1985; pp 327-334. (45) Bacha, S.; Bergel, A.; Comtat, M. Anal. Chem. 1995, 67, 1669-1678. (46) Mell, L. D.; Maloy, J. T. Anal. Chem. 1975, 47, 299-307. (47) Gough, D. A.; Leypoldt, J. K. Appl. Biochem. Bioeng. 1981, 3, 175-206. (48) Leypoldt, J. K.; Gough, D. A. Anal. Chem. 1984, 56, 2896-2904.

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Figure 6. H2O2 steady-state concentration profile simulation for a model enzyme-modified hydrogen peroxide sensor.

Figure 7. Superoxide and hydrogen peroxide steady-state concentration profile simulation for a model enzyme-modified superoxide sensor.

Figures 6 and 7 schematically illustrate the steady-state concentration profiles of H2O2, O2•-, and mediator enzymes in the vicinity of the electrode surface. Our ultimate goal was to rationalize the mechanism of enzyme electrode operation and compare experimental and theoretically predicted performance. APPENDIX 2. HYDROGEN PEROXIDE SENSOR MODEL We consider the kinetics where the HRP/H2O2 interaction determines the total enzymatic reaction rate. A sensor responds linearly to the substrate concentration for conditions of H2O2 mass transfer limitation, and this dependency is desirable for practical measurements. Enzyme is mostly unconsumed, and the rate of reaction in terms of flux J is given by

J ) K1Γ[H2O2]a

(A1)

where [H2O2]a is the substrate concentration at X ) a; K1 is the enzymatic rate constant, taken as KH2O2/HRP ≈ 1010 cm3 mol-1 s-1;7-11 and Γ is the total surface density of enzyme, taken as Γ ) 10-11 mol cm-2.20 The flux for substrate in the bulk [H2O2]bulk and for mediator HRP can be expressed also as

J ) DH2O2 ) DHRP

[H2O2]bulk - [H2O2]a d-a [HRP]a - [HRP]x)0 ) Kel[HRP]x)0 a

(A2)

where DH2O2 is the diffusion coefficient for H2O2 (2.34 × 10-5 cm2 s-1) obtained from rotating disk experiment; DHRP is the electron diffusion coefficient for HRP (assumed to be below 10-8 cm2 s-1 22,24); Kel is the heterogeneous electrode reaction rate constant, determined as Kheterog ) 0.176 cm s-1.

For the condition d . a and d - a ≈ d,

J ) DH2O2([H2O2]bulk - [H2O2]a)/d

(A3)

and, from (A1),

[H2O2]a ) J/K1Γ

(A4)

Therefore, the whole expression for the flux J can be rewritten as

J)

DH2O2([H2O2]bulk - J/K1Γ) [H2O2]bulk

)

Figure 8. Simulated diagram of typical response of superoxide sensor to XOD injection.

a (d/DH2O2) - (d K1Γ/DH2O2J)

(A5)

Or, simplifying (A5) for flux J,

J)

DH2O2[H2O2]bulk

[H2O2]bulk

d(1 + DH2O2/K1Γd)

)

(d/DH2O2) + (1/K1Γ)

(A6)

The output current density I can then be determined:

I ) nFchargesubstrateJ

(A7)

where charge is the enzyme/electrode charge transfer efficiency; substrate is the substrate supply efficiency, taken as ∼0.5;20 n is the number of transferred electrons (n ) 2); and F is the Faraday number. Therefore, current dependence on H2O2 concentration in the bulk is

IH2O2 )

2Fchargesubstrate[H2O2]bulk (d/DH2O2) + (1/K1Γ)

(A8)

We can determine enzyme/electrode charge transfer efficiency from the second part of eq A2 as

charge )

IH2O2 nFJ

)

Kel[HRP]x)0 d-a ) ≈1 J (DHRP/Kel) + d

(A9)

taking into consideration that DHRP/Kel , d, a , d. Average diffusion layer thickness was determined as d ) 5 × 10-3 cm (eq 1). Consequently, the final expression for the dependence of current vs bulk concentration of substrate for hydrogen peroxide electrode (A8) can be written as

IH2O2 [H2O2]bulk

2Fchargesubstrate )

(d/DH2O2) + (1/K1Γ)

(A10)

and is equal to about 4.31 × 108 nA cm-2 M-1, or 0.431 A cm-2 M-1, which is quite close to the experimental value of 0.457 A cm-2 M-1. APPENDIX 3. SUPEROXIDE SENSOR MODEL Similarly to the previous case with H2O2, for superoxide sensor performance we considered the kinetics where the enzyme/

substrate interaction determines the total enzymatic reaction rate. The sensor responds linearly to superoxide concentration for conditions of O2•- mass transfer limitation, and this dependency is desirable for practical measurements. Here we consider the case of superoxide sensor response to KO2 at pH 9.0 as the one practically related to the following O2•- detection. Moreover, the quantitative analysis for this case is more straightforward and simple than that for the case when superoxide is generated by the XA/XOD reaction (3-5). Response of the superoxide sensor while being characterized by XA/XOD is determined not only by the surface enzyme kinetics but also by the rate of increase of O2•- concentration in the bulk solution following XOD injection, and by the presence of H2O2, produced at the parallel stage (reaction 5b), as well as the result of the superoxide dismutation (reaction 6). Due to the presence of HRP/PPy film in the superoxide sensor composite membrane, it is necessary to propose that the sensor will respond not only to O2•- but also to H2O2 present in bulk solution from the sources different than XA/ XOD interaction. The difference between observed responses of the hydrogen peroxide sensor to H2O2 and superoxide sensor to KO2 on one side, and the superoxide sensor to XA/XOD on the other side, is demonstrated in Figure 8. While H2O2 and KO2 injections result in a nearly step current response, those of XOD lead initially to a current increase at the rate controlled by O2•production at stage 4 and, to a lesser extent, by H2O2 production in reaction 5b. After some initial period, characterized by the kinetics of superoxide production, O2•- dismutates in the bulk solution according to reaction 6. Solution at that point contains nearly exclusively H2O2 at a practically constant concentration, which results in a signal leveling-off, producing a nearly steadystate line. As H2O2 undergoes slow disproportionation in the solution by a mechanism similar to eq 7, a slow reduction current density decline is observed with time, as shown schematically in Figure 8. SOD film inclusion in a superoxide sensor composite membrane provides for an additional mass transfer limitation and slightly slows down the sensor response to substrate injections. In the superoxide sensor characterization experiments, a time delay on the order of 0.5 s has been noticed. We still kept the assumption of negligible consumption of enzyme while deriving equations of superoxide sensor response to O2•- and H2O2. The rate of O2•- reaction in terms of flux J is given by

JO2- ) K2Γ[O2•-]b

(A11)

where [O2•-]b is the substrate concentration at X ) b; K2 is the Analytical Chemistry, Vol. 69, No. 3, February 1, 1997

461

enzymatic rate constant, taken as KO2-/SOD ≈ 1012 cm3 mol-1 s-1;29 and Γ is the total surface density of enzyme, taken as Γ ) 10-11 mol cm-2.20 Equation A11 for detection of O2- bulk concentration [O2•-] bulk for the condition of d . b can be expressed also as

J ) JO2- ) DO2-([O2•-]bulk - [O2•-]b)/d

(A12)

where DO2- is the diffusion coefficient for O2•- (2.45 × 10-5 cm2 s-1) obtained from rotating disk experiment. The approach described above for the H2O2 sensor, namely eqs A3-A6, can be employed. Additional complications to O2•mass transfer through the SOD film and H2O2 through HRP film can be accounted for by inclusion of a second substrate supply efficiency parameter, substrate2 ≈ 0.5.20 The expression for current density for the superoxide sensor response IO2- can be written as

IO2- )

nFchargesubstrate1substrate2[O2•-]bulk (d/DO2-) + (1/K2Γ)

(A13)

or

IO2-

Fchargesubstrate1substrate2 )

[O2 ]bulk •-

(d/DO2-) + (1/K2Γ)

Analytical Chemistry, Vol. 69, No. 3, February 1, 1997

IO2[H2O2]bulk

2Fchargesubstrate1substrate2 )

(d/DH2O2) + (1/K1Γ)

(A15)

After performing the calculation, an expression for dependence of reduction current density vs H2O2 bulk concentration for the superoxide sensor was determined as being equal to about 2.25 × 108 nA cm-2 M-1, or 0.225 A cm-2 M-1, which is quite close to the experimental value of 0.266 A cm-2 M-1. And finally from eqs A14-A15, we will obtain for superoxide sensor response to O2•- and H2O2 injections

IO2 ) 0.118[O2•-]bulk + 0.225[H2O2]bulk

(A14)

After substituting previously determined values of n, F, enzyme/electrode charge transfer efficiency, substrate supply efficiency, diffusion coefficient for superoxide, average diffusion layer thickness, enzymatic rate constant, and total surface density of enzyme, an expression for dependence of reduction current

462

density increase vs superoxide bulk concentration was determined as being equal to about 1.18 × 108 nA cm-2 M-1, or 0.118 A cm-2 M-1. Derivation of an equation for detection of H2O2 by the superoxide sensor is analogous to that presented for eq A10. The additional complication to H2O2 mass transfer through SOD film can be accounted for by an inclusion of a second substrate supply efficiency parameter, substrate2 ≈ 0.5.20 The resulting equation will look like

(A16)

Received for review June 25, 1996. Accepted November 17, 1996.X AC9606261 X

Abstract published in Advance ACS Abstracts, January 1, 1997.