Anal. Chem. 1999, 71, 1928-1934
Uricase-Catalyzed Oxidation of Uric Acid Using an Artificial Electron Acceptor and Fabrication of Amperometric Uric Acid Sensors with Use of a Redox Ladder Polymer Takahiro Nakaminami, Shin-ichiro Ito, Susumu Kuwabata,* and Hiroshi Yoneyama*
Department of Applied Chemistry, Faculty of Engineering, Osaka University,Yamada-oka 2-1, Suita, Osaka 565-0871, Japan
Electrochemical oxidation of uric acid catalyzed by uricase (uric acid oxidase, UOx; EC 1.7.3.3) was studied using several redox compounds including 5-methylphenazinium (MP) and 1-methoxy-5-methylphenazinium (MMP) as electron acceptors for UOx, which does not contain any redox cofactor. It was found that MP and MMP were useful to mediate electrons from UOx to an electrode in the enzymatic oxidation of uric acid. A novel redox polymer, poly(N-methyl-o-phenylenediamine) (poly-MPD), containing the MP units was also found to possess the mediation ability for UOx, and poly-MPD was immobilized together with UOx onto an electrode substrate covered with a self-assembled monolayer of 2-aminoethanethiolate with use of glutaraldehyde as a binding agent. The resulting electrode (poly-MPD/UOx/Au) exhibited amperometric responses to uric acid with very fast response of ∼30 s, allowing reagentless amperometric determination in a concentration range covering that in the blood of a healthy human being. Kinetic parameters of the apparent Michaelis constant and the maximum current response obtained at the poly-MPD/UOx/Au suggested that electrochemical oxidation of uric acid was controlled by diffusion of uric acid into the enzyme film and that the redox polymer worked well in mediating between active sites of UOx molecules and the electrode substrate. It is important to determine the concentration of uric acid dissolved in human urine and/or blood to diagnose diseases caused by disorder of purine biosynthesis and/or purine catabolism, such as gout, hyperuricemia, and Lesch-Nyhan syndrome.1-5 Several determination methods have been developed using uricase (uric acid oxidase, UOx; EC 1.7.3.3) which catalyzes in vivo oxidation of uric acid as given by eq 1. Addition of UOx to samples UOx
uric acid + O2 98 allantoin + CO2 + H2O2
(1)
in the presence of dissolved O2 causes a decrease in the concentration of uric acid. By determining changes in absorbance * Corresponding author: (fax) +81-6(6879)7373; (e-mail) yoneyama@ ap.chem.eng.osaka-u.ac.jp. (1) Fox, I. H. Metabolism 1981, 30, 616-34.
1928 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
due to uric acid at a given reaction time, the original concentration of uric acid is determined.6,7 Several electrochemical measurements have also been reported as the substitute. Potentiometric determination of CO2 produced by reaction 18 and amperometric determination of consumption rate of O29-12 may be useful, but the results are largely influenced by solution pH and original concentration of dissolved O2. Amperometric determination of H2O2 is also useful,13-15 but the potential (>0.4 V vs a saturated calomel electrode (SCE)) at which anodic oxidation of H2O2 takes place is positive enough to cause direct oxidation at the electrode of uric acid and other components such as ascorbate and acetoaminophen dissolved in human fluid, resulting in inaccuracy of the determinations.14,15 To solve this problem, the use of horseradish peroxidase (HRP) together with UOx has been proposed for enzymatic reduction of H2O2, and then the amperometric determination was achieved at less positive potentials of 0.5-0.15 V vs SCE than that causing oxidation of H2O2.16,17 In our previous paper, it was reported that [Fe(CN)6]3- was found to work as an electron acceptor for UOx in place of O2 in the reaction given by eq 1 and that the concentration of uric acid can be determined by a convenient way of oxidizing electrochemically the produced [Fe(CN)6]4- at a UOx-immobilized Au electrode polarized at 0.1 V vs SCE.18 In this paper, we will show that several (2) Ullman, B.; Wormsted, M. A.; Cohen, M. B.; Martin, D. W., Jr. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 5127-31. (3) Yamanaka, H.; Togashi, R.; Hakoda, M.; Terai, C.; Kashiwazaki, S.; Dan, T.; Kamatani, N. Adv. Exp. Med. Biol. 1998, 431, 13-8. (4) Liang, M. H.; Fries, J. F. Ann. Intern. Med. 1978, 88, 666-70. (5) Simkin, P. A. Ann. Intern. Med. 1979, 90, 812-6. (6) Dilena, B. A.; Peake, M. J.; Pardue, H. L.; Skoug, J. W. Clin. Chem. 1986, 32, 486-91. (7) Feichtmeir, T. V.; Wrenn, H. T. Am. J. Clin. Pathol. 1955, 25, 833-9. (8) Kawashima, T.; Rechnitz, G. A. Anal. Chim. Acta 1976, 83, 9-15. (9) Nanjo, M.; Guilbault, G. G. Anal. Chem. 1974, 46, 1769-72. (10) Janchen, M.; Walzel, G.; Neef, B.; Wolf, B.; Scheller, F.; Kuhn, M.; Pfeiffer, D.; Sojka, W.; Jaross, W. Biomed. Biochim. Acta 1983, 42, 1055-9. (11) Uchiyama, S.; Shimizu, H.; Hasebe, Y. Anal. Chem. 1991, 66, 1873-6. (12) Uchiyama, S.; Suzuki, S.; Sato, T. Electroanalysis 1990, 2, 559-61. (13) Markas, A.; Gilmartin, T.; Hart, J. P. Analyst 1994, 119, 833-40. (14) Shaolin, M.; Jinqing, K.; Jianbing, Z. J. Electroanal. Chem. 1992, 334, 12132. (15) Motonaka, J.; Miyata, K.; Faulkner, L. R. Anal. Lett. 1994, 27, 1-13. (16) Tatsuma, T.; Watanabe, T. Anal. Chim. Acta 1991, 242, 85-9. (17) Miland, E.; Ordieres, A. J. M.; Blanco, P. T.; Smyth, M. R.; Fa´ga´in, C. O Ä. Talanta 1996, 43, 785-96. (18) Kuwabata, S.; Nakaminami, T.; Ito, S.; Yoneyama, H. Sens. Actuators B 1998, 52, 72-7. 10.1021/ac981168u CCC: $18.00
© 1999 American Chemical Society Published on Web 04/13/1999
Chart 1. Chemical Structure of (a) MP and (b) MMP
other redox species including 5-methylphenazinium (MP) and 1-methoxy-5-methylphenazinium (MMP), whose chemical structures are shown in Chart 1, also serve as useful electron acceptors for UOx. The most convenient amperometric uric acid sensors would be an electrode on which electron mediator molecules are immobilized together with UOx. Searches for improved electron mediators for UOx have extended to the use of a novel redox polymer of poly(N-methyl-o-phenylenediamine) (poly-MPD) containing MP units in its polymer structure, which was synthesized by methylation of chemically prepared poly(o-phenylenediamine) (poly-PD). The poly-PD, which can also be prepared by electrochemical polymerization of o-phenylenediamine, was already utilized as a permselective membrane in glucose sensors of high selectivity.19-24 In this study, it will be shown that the poly-MPD works well as a promising electron mediator for UOx and reagentless uric acid sensors have been successfully prepared by immobilizing this redox polymer onto cross-linked UOx films on an Au substrate. EXPERIMENTAL SECTION UOx from Candida sp. was commercially available from Wako Pure Chemicals and used without further purification. Poly-PD was synthesized according to literature25 with the following partial modifications. Ammonium peroxosulfate (1 mol dm-3) as an oxidizing agent dissolved in dimethyl sulfoxide (DMSO) was added drop by drop to give a final concentration of 170 mmol dm-3 to an agitated 50 cm3 of DMSO containing 50 mmol dm-3 o-phenylenediamine monomer. The colorless solution gradually turned red and a black precipitate of poly-PD appeared. After the solution was stirred for 12 h, the resulting suspension was filtered, and the precipitate was washed several times with distilled water and acetone to remove unreacted monomer and low-molecularweight products. Poly-MPD was obtained by N-methylation of polyPD. Ethanol (50 cm3) containing 2 mmol of monomer units of poly-PD was mixed with 2 mmol of methyl iodide and refluxed at 60 °C for 24 h. Then, the solvent and unreacted methyl iodide were evaporated under reduced pressure. All other chemicals used were of analytical grade and obtained from Wako Pure Chemicals except for thionin (3,7-diaminophenothiazinium) (Aldrich), cystamine (Aldrich), 1-methoxy-5-methylphenazinium sulfate (Dojindo (19) Sasso, S. V.; Pierce, R. J.; Walla, R.; Yacynych, A. M. Anal. Chem. 1990, 62, 1111-7. (20) Malietesta, C.; Palmisano, F.; Torsi, L.; Zambonin, P. G. Anal. Chem. 1990, 62, 2735-40. (21) Centonze, D.; Palmisano, F.; Torsi, L.; Zambonin, P. G. Electroanalysis 1994, 6, 423-9. (22) Lowry, J. P.; O’Neill, R. D. Electroanalysis 1994, 6, 369-79. (23) Trojanowicz, M.; Geschke, O.; Krawczynski vel Krawczyk, T.; Cammann, K. Sens. Actuators B 1995, 28, 191-9. (24) Wang, J.; Chen, L.; Liu, J.; Lu, F. Electroanalysis 1996, 8, 1127-30. (25) Premasiri, A. H.; Euler, W. B. Macromol. Chem. Phys. 1995, 196, 3655-66.
Laboratories), and anthraquinone-1-sulfonate (Tokyo Chemical Industries). All aqueous solutions were prepared using twicedistilled water. The ability of redox agents as an electron acceptor for UOxcatalyzed oxidation of uric acid was tested by measuring the time course of changes in their absorbance caused by reduction of the oxidized form of the redox agent. The reaction was initiated by adding 0.5 mmol dm-3 uric acid to N2-saturated 0.1 mmol dm-3 borate buffer (pH 8.5) containing the oxidized form of the redox agent of desired concentration and 8.3 µmol dm-3 UOx at 30 °C. A quartz cell having a light path length of 1 or 0.2 cm was used, and absorption spectra were measured between 300 and 800 nm using a diode array spectrophotometer (Hewlett-Packard, 8452A). A Pt disk plate (0.88 cm diameter) was used as an electrode substrate for electrochemical oxidation of uric acid, and an Au disk plate of the same area was used as the electrode substrate for immobilization of UOx and/or a redox polymer. The electrode substrates were polished successively with alumina slurries of 1.0 and 0.3 µm, subjected to ultrasonication in deionized water for 30 min, soaked overnight in a piranha solution,26 and then mounted in a Teflon electrode holder which restricted the exposed area to 0.27 cm2. Note: Piranha solution is a strong oxidant and must be used with extreme caution! A vapor-deposited Au on a glass plate having an area of 17.5 cm2 was also used as the Au substrate for observations of the cross section of the prepared enzyme films with a scanning electron microscope (SEM; Hitachi, S-800). Immobilization of UOx and poly-MPD onto the electrode substrate was accomplished as follows. The Au electrode was immersed in 1 mmol dm-3 cystamine aqueous solution for 8 h to deposit a self-assembled monolayer of 2-aminoethanethiolate, followed by casting 30 mm3 () 120 mm3 per 1 cm2 of electrode area) of 20 mmol dm-3 phosphate buffer (pH 7.0) containing 8.3 µmol dm-3 UOx, 1.8 g dm-3 poly-MPD, 1 wt % (∼80 mmol dm-3) glutaraldehyde (GA), and 10 vol % ethanol, and then the electrode was kept in a glass vessel filled with dry N2 at 35 ( 1 °C for 6 h to allow the binding of amino residues of UOx molecules with the amino groups of 2-aminoethanethiolate on the Au substrate via glutaraldehyde. When electrodes having different amounts of poly-MPD were prepared, the concentration of poly-MPD in the cast solution was varied without changing the concentrations of the other components. The resulting electrodes were rinsed well with phosphate buffer (pH 7.0) to remove weakly adsorbed species. When only UOx was desired to be immobilized onto the gold electrode, the same procedure was employed but the phosphate buffer solution used did not contain poly-MPD and ethanol. In this paper, the electrodes modified with both UOx and poly-MPD and those with UOx alone will be denoted as polyMPD/UOx/Au and UOx/Au, respectively. The enzymatic activity of UOx immobilized on the Au substrate was determined by measuring the time course of a decrease in the concentration of uric acid in the presence of dissolved O2.6 For this purpose, 2 cm3 of air-saturated 0.1 mol dm-3 borate buffer (pH 8.5) was put in a quartz cell having a light path length of 1 cm and then the UOx/Au or the poly-MPD/UOx/Au was immersed in it. An aliquot of 20 mmol dm-3 uric acid dissolved in the borate buffer was added to the quartz cell as to give a concentration of 0.1 mmol dm-3, and absorbance of the solution (26) Hayes, W. A.; Shannon, C. Langmuir 1996, 12, 3688-94.
Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
1929
time after addition of uric acid, and the spectrum approached that of reduced MMP, which is shown by a broken line in Figure 1. Since the injection of uric acid into the solution in the absence of UOx did not cause any changes in the absorption spectrum, the results given in Figure 1 should have resulted from such electrontransfer events that uric acid molecules were oxidized by MMP with catalysis of UOx. UOx
MMP + uric acid 98 reduced MMP + products (2)
Figure 1. Changes in absorption spectra of N2-saturated 0.1 mol dm-3 borate buffer (pH 8.5) containing 0.25 mmol dm-3 MMP and 0.83 µmol dm-3 UOx caused by addition of 0.5 mmol dm-3 uric acid. The time given is that elapsed after addition of uric acid. Broken line shows the spectrum of MMP reduced by 0.2 mmol dm-3 sodium hydrosulfite in the same solution composition. Inset shows time course of the concentration of MMP obtained from the absorbance peak at 510 nm.
at 290 nm which is due to uric acid, was monitored. Using a molar absorptivity of uric acid of 1.22 × 104 dm3 mol-1 cm-1 the rate of consumption of 1 µmol of uric acid per min () 1 unit) per cm2 of electrode area was determined. Amperometric responses at the poly-MPD/UOx/Au to uric acid were measured at 0 V vs SCE using a potentiostat (Bioanalytical Systems, BS-1) in 0.1 mol dm-3 borate buffer (pH 8.5). A one-compartment electrochemical cell was used and it was equipped with a platinum foil of 2 cm2 and an SCE as a counter and a reference electrodes, respectively. In this paper, potentials referred to SCE will be cited for all cases. The cell was placed in an incubator controlled at 45 ( 1 °C during the course of measurements. The electrolyte solution was deoxygenated by bubbling N2 for 20 min prior to the measurements. When constant background currents were obtained, an aliquot of 20 mmol dm-3 uric acid dissolved in the borate buffer was added to the electrolyte solution to give the desired concentration, followed by agitation of the electrolyte solution for 5 s. The time course of the oxidation currents in quiescent solution was monitored on an electric polyrecorder (Toa Denpa, EPR-151A). Steady currents obtained are termed here as the current response (ir) and the time required to give the steady currents after the addition of uric acid as the response time (tr). In the measurements of amperometric responses at the UOx/Au, the same procedure was employed except that the electrolyte solution containing 1 mmol dm-3 redox agent in its oxidized state was used. Cyclic voltammetry was performed using an electrochemical analyzer (Bioanalytical Systems, BAS100B/W) connected to a Gateway 2000 computer. RESULTS AND DISCUSSION Artificial Electron Acceptor for UOx-Catalyzed Uric Acid Oxidation. Figure 1 shows changes in absorption spectra caused by addition of uric acid of MMP dissolved in N2-saturated borate buffer containing UOx. As shown in this figure, absorbance peaks of MMP, which appear at around 380 and 510 nm, decreased with 1930 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
The inset of the figure shows the time course of changes in the concentration of MMP, which was obtained from the absorbance peak at 510 nm. The initial rate of the enzymatic oxidation of uric acid was determined to be 46.4 µmol dm-3 min-1, and the rate became gradually low with time, approaching the reaction equilibrium. If the reduced MMP is directly oxidized by electrochemical means, the uric acid concentration can easily be determined from the oxidation currents, as will be shown later. Unlike flavine adenine dinucleotide (FAD)-containing redox enzymes such as glucose oxidase and cholesterol oxidase, UOx does not have any redox cofactor.27,28 Accordingly, the electron transfer from UOx to oxidized MMP cannot be explained on the basis of redox reactions of UOx. The following discussion have been presented for the case of using O2 as an electron acceptor for UOx. UOx-catalyzed oxidation of uric acid may proceed with an ordered mechanism where uric acid and O2 are bound to UOx to give a trimolecular transitory complex which is dissociated to give the products.27 An important reaction step in that case is an activation step of uric acid molecule at UOx prior to transferring electrons to O2.28 Considering that MMP can work as an electron acceptor like dissolved O2, the same mechanism may be valid for the case of MMP, too. UOx-Catalyzed Electrochemical Oxidation of Uric Acid with Use of an Electron Mediator. Electrochemical detections of uric acid using UOx and an electron mediator were carried out using three kinds of electrolyte solutions: borate buffer solution (pH 8.5) containing UOx and one of redox agents listed in Table 1, borate buffer containing only the redox agent, and unadulterated borate buffer solution. The results obtained for using p-benzoquinonesulfonate (BQS) as the redox agent are shown in Figure 2. A Pt working electrode used was polarized at 0.3 V which was positive enough to oxidize the reduced BQS, i.e., p-hydrobenzoquinonesulfonate (H2BQS). As shown by curve a in the figure, if a borate buffer containing BQS and UOx was used as an electrolyte solution, anodic currents increased with time after adding uric acid to the solution and reached a steady value of 20.4 µA cm-2. The results then seem to suggest that BQS works as an electron mediator between UOx molecules and the Pt electrode, but anodic currents were also obtained with the use of the electrolyte solution in the absence of UOx, as shown by curve b in the figure, though the magnitude of the steady currents was almost half of that shown by curve a. If the borate buffer containing neither UOx nor BQS was used, much smaller oxidation currents, which are related to direct oxidation of uric acid at the Pt electrode, appeared as shown by curve c. The oxidation currents obtained in the presence of BQS only were (27) Kahn, K.; Tipton, P. A. Biochemistry 1997, 36, 4731-8. (28) Kahn, K.; Serfozo, P.; Tipton, P. A. J. Am. Chem. Soc. 1997, 119, 5435-42.
Table 1. Direct and UOx-Catalyzed Electrochemical Oxidation of Uric Acida redox compound f
FC BQS ferricyanide MP MMP thionin AQS g
redox potentialb/ V vs SCE
electrode potential/ V vs SCE
I1c/µA cm-2
I2d/µA cm-2
I3e/µA cm-2
0.28 0.20 0.15 -0.18 -0.21 -0.34 -0.52
0.38 0.30 0.10 -0.08 -0.11 -0.10 -0.10
13.1 20.4 8.93 0.175 0.165 0 0
5.57 11.7 0.130 0 0 0 0
5.57 2.55 0.130 0 0 0 0
rUOx/% 57.5 42.5 98.5 100 100
a Performed under experimental conditions similar to those listed in Figure 2 except that the redox compound and electrode potential listed were used. b Estimated as an intermediate potential between anodic and cathodic peak potentials in the cyclic voltammogram at Pt electrode taken in N2-saturated 0.1 mol dm-3 borate buffer (pH 8.5) containing 1 mmol dm-3 redox compound listed. c Steady-state oxidation currents of uric acid obtained in the presence of UOx and the redox agent listed. d Steady-state oxidation currents of uric acid obtained in the presence of the redox agent only. e Steady-state currents for direct oxidation of uric acid in the absence of UOx and the redox compound. f Ferrocenecarboxylate. g Anthraquinone-1-sulfonate.
Figure 2. Time course of changes in oxidation currents at Pt electrode after addition of 0.1 mmol dm-3 uric acid to N2-saturated 0.1 mol dm-3 borate buffer (pH 8.5) containing (a) 0.83 µmol dm-3 UOx and 1.0 mmol dm-3 BQS, (b) 1.0 mmol dm-3 BQS, and (c) none of these compounds. Electrode potential, 0.3 V vs SCE.
much greater than those obtained by the direct oxidation of uric acid, indicating that chemical oxidation of uric acid by BQS as an oxidizing agent took place to give H2BQS, which was oxidized at the Pt electrode. Since the electron mediator has to pick up electrons from uric acid bound to UOx selectively without any serious interferences, the selectivity (rUOx) was evaluated for the redox agents listed in Table 1 with the simple definition as given by eq 3, where I1 are
rUOx (%) ) 100(I1 - I2)/I1
(3)
currents obtained in the presence of both UOx and a redox agent and I2 are those obtained in the presence of the redox agent only. According to this definition, BQS gave an rUOx of 42.5%. Similar tests were carried out for several redox agents other than BQS, and the results are summarized in Table 1. The redox agents of ferrocenecarboxylate (FC) and ferricyanide did not give any noticeable difference between I2 and direct oxidation currents I3,
indicating that they do not possess any ability for oxidation of uric acid without UOx. As shown in the table, if redox agents having redox potentials more negative than BQS were used, direct oxidation of uric acid was suppressed, as shown for ferricyanide, MP, and MMP. From the viewpoint of selective detection of uric acid, MP and MMP are preferable, because only the UOxcatalyzed uric acid oxidation currents are seen. Redox agents having even more negative redox potentials than MP and MMP gave no current response to uric acid, suggesting that the redox potential of uric acid complexed with UOx is approximately between -0.21 and -0.34 V. Since the one-electron redox potential of free uric acid is reported to be 0.02 V,29 uric acid is said to be activated at UOx by ∼0.2 V for its oxidation. It took more than 12 min to obtain steady-state currents after addition of uric acid to an electrolyte solution containing dissolved UOx and MMP. The time course of changes in oxidation currents in that case was quite consistent with changes in the concentration of MMP caused by the enzyme reaction as shown in the inset of Figure 1. It is then concluded that the current response was determined by an equilibrium concentration of MMP in its reduced state in the solution bulk, and the response time by the time required to attain the equilibrium, as already discussed for the electrochemical oxidation of cholesterol catalyzed by cholesterol oxidase.30 It was shown in our previous paper that the concentration of uric acid can be determined using UOx-immobilized Au electrodes in the presence of ferricyanide as a dissolved electron mediator.18 The immobilization of UOx on the electrode was made by binding amino residues of UOx molecules with amino groups of a selfassembled monolayer of 2-aminoethanethiolate on the Au electrode substrate with the assistance of glutaraldehyde (UOx/Au; see Experimental Section). In the present study, attempts were made to prepare UOx/Au electrodes using the same technique. The resulting electrode was found to have an enzymatic activity of 9.68 units cm-2. When 0.1 mmol dm-3 uric acid was injected in a borate buffer containing 1 mmol dm-3 MMP, oxidation currents appeared at 0 V, indicating that MMP worked as the electron mediator for the immobilized UOx molecules. A steady current response of 0.072 µA cm-2 was obtained and the response time was a little shorter than 1 min, which was much shorter than that obtained when dissolved UOx was used (12 min). The following detection scheme seems valid for the appearance of the fast response observed. When UOx-immobilized gold electrode is used, reduction of MMP takes place in the thin UOx film on the gold electrode, and the resulting reduced MMP is then anodically oxidized at the electrode substrate before it is diffused away into the solution bulk. The response time must be determined by the time needed for uric acid in the whole enzyme film to diffuse to the immobilized UOx.31 Electrochemical Properties of Poly-PD and Poly-MPD and Their Abilities as Electron Acceptors for UOx. If MMP or MP molecules are immobilized on UOx-covered electrode substrate, the resulting electrode will work as an amperometric uric acid sensor which does not require any reagents in the (29) Jovanovic, S. V.; Simic, M. G. J. Phys. Chem. 1986, 90, 974-8. (30) Nakaminami, T.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1997, 69, 236772. (31) Nakaminami, T.; Ito, S.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1999, 71, 1068-76.
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Chart 2. Chemical Structure of (a) Poly-PD and (b) Poly-MPD
measurements. However, it is not easy to immobilize covalently MMP and MP molecules onto the electrode surface, because they do not have any functional groups available for covalent attachments.32-38 Also physical incorporation of MMP and MP molecules into a cross-linked UOx network on the electrode substrate is of little significance because of their high solubility in water.39-43 So we searched for a redox polymer44-49 that can work as a mediator for UOx when entrapped in the immobilized UOx layer formed on an electrode substrate. The redox polymer poly-PD, obtained by oxidative polymerization of o-phenylenediamine, possesses a ladderlike structure in which phenazine units are organized as shown by (a) of Chart 2.50,51 It was reported that the poly-PD exhibits an electrical conductivity of ∼10-4 S cm-1 due to its π-conjugation.52 The polymer also shows redox activities with a redox potential of (32) Kuwabata, S.; Okamoto, T.; Kajiya, Y.; Yoneyama, H. Anal. Chem. 1995, 67, 1684-90. (33) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J. Am. Chem. Soc. 1992, 114, 10965-6. (34) Willner, I.; Riklin, A.; Shoham, B.; Rivenzon, D.; Katz, E. Adv. Mater. 1993, 5, 912-5. (35) Willner, I.; Katz, E.; Willner, B. Electroanalysis 1997, 9, 965-77. (36) Bardea, A.; Katz, E.; Bu ¨ ckmann, A. F.; Willner, I. J. Am. Chem. Soc. 1997, 119, 9114-9. (37) Foulds, N. C.; Lowe, C. R. Anal. Chem. 1988, 60, 2473-8. (38) Degani, Y.; Heller, A. J. Phys. Chem. 1987, 91, 1285-9. (39) Iwakura, C.; Kajiya, Y.; Yoneyama, H. J. Chem. Soc., Chem. Commun. 1988, 1019-20. (40) Kajiya, Y.; Sugai, H.; Iwakura, C.; Yoneyama, H. Anal. Chem. 1991, 63, 49-54. (41) Kajiya, Y.; Tsuda, R.; Yoneyama, H. J. Electroanal. Chem. 1991, 301, 15564. (42) Kajiya, Y.; Matsumoto, H.; Yoneyama, H. J. Electroanal. Chem. 1991, 319, 185-94. (43) Bartlett, P. N.; Ali, Z.; Eastwick-Field, V. J. Chem. Soc., Faraday Trans. 1992, 88, 2677-83. (44) Calvo, E. J.; Danilowicz, C.; Diaz, L. J. Chem. Soc., Faraday Trans. 1993, 89, 377-84. (45) Calvo, E. J.; Danilowicz, C.; Diaz, L. J. Electroanal. Chem. 1994, 369, 27982. (46) Calvo, E. J.; Etchenique, R.; Danilowicz, C.; Diaz, L. Anal. Chem. 1996, 68, 4186-93. (47) Hodak, J.; Etchenique, R.; Calvo, E. J.; Shinghal, K.; Bartlett, P. N. Langmuir 1997, 13, 2708-16. (48) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1989, 111, 2357-8. (49) Gregg, B.; Heller, A. J. Phys. Chem. 1991, 95, 5976-80. (50) Wu, L.-L.; Luo, J.; Lin, Z.-H. J. Electroanal. Chem. 1996, 417, 53-8. (51) Wu, L.-L.; Luo, J.; Lin, Z.-H. J. Electroanal. Chem. 1997, 440, 173-82. (52) Chiba, K.; Ohsaka, T.; Ohnuki, Y.; Oyama, N. J. Electroanal. Chem. 1987, 219, 117-24.
1932 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
Figure 3. Cyclic voltammograms of Au electrode coated with (a) poly-PD (poly-PD/Au) and (b) poly-MPD (poly-MPD/Au) taken in N2saturated 0.1 mol dm-3 borate buffer (pH 8.5). dE/dt ) 50 mV s-1. The electrodes were prepared by casting 10 mm3 of ethanol containing 6 g dm-3 of each polymer onto Au substrates, followed by drying at 35 ( 1 °C for 5 h.
∼-0.65 V at pH 8.5, which seems to be from the phenazine moiety, whose redox potential is ∼-0.6 V.52 Since MP, which works as the electron acceptor for UOx as shown in Table 1, is derived by N-methylation of phenazine, attempts were made to quaternize the nitrogen atom of poly-PD with a methyl group. The prepared poly-MPD is believed to have the chemical structure as given by (b) of Chart 2. Cyclic voltammograms of Au electrodes coated with poly-PD (poly-PD/Au) and poly-MPD (poly-MPD/ Au) in borate buffer (pH 8.5) are shown in Figure 3. Both electrodes were prepared by casting 10 mm3 of ethanol containing 6 g dm-3 of one of the polymers onto the Au electrode substrate, followed by drying at 35 ( 1 °C for 5 h. The voltammograms as shown in this figure were not significantly changed by repeating cycles for at least 1 h. Both the poly-PD/Au and the poly-MPD/ Au exhibited redox activities due to the deposited polymers. From an average of peak potentials of anodic and cathodic waves, the redox potentials of poly-PD and poly-MPD were evaluated to be ∼-0.67 and ∼-0.27 V, respectively. The more positive redox potential of poly-MPD results from the presence of an electrondonating methyl group at the nitrogen atom, although the N-methylation seems to have occurred in different degrees in the polymer, as suggested from the appearance of broad redox waves. If the pH of an electrolyte solution was changed, the redox potentials of poly-PD and poly-MPD were changed at a rate of ∼-0.07 V pH-1, indicating that protonation and deprotonation reactions were involved in the redox reactions of both polymers. Judging from the reported results that similar shifts in the redox potential were observed for phenazine and MP molecules,52,53 the protonation and deprotonation seem to a occur at methyl groupfree nitrogen atom in the redox reaction of poly-PD and poly-MPD. If 0.1 mmol dm-3 uric acid was added to 0.1 mol dm-3 borate buffer (pH 8.5) in the presence of dissolved UOx under polarization of the poly-MPD/Au at 0 V, a current response of 80 nA cm-2 was obtained with a response time of ∼30 min. Since no oxidation currents were obtained in the absence of UOx, the current (53) Komura, T.; Funahashi, Y.; Yamaguchi, T.; Takahashi, K. J. Electroanal. Chem. 1998, 446, 113-23.
Figure 4. Time course of currents at the poly-MPD/UOx/Au obtained by stepwise addition of 0.1 mmol dm-3 uric acid in N2-saturated 0.1 mol dm-3 borate buffer (pH 8.5). Electrode potential, 0 V vs SCE.
response must arise from oxidation of the poly-MPD which picks up electrons from the UOx-uric acid complex produced in the solution bulk. The poly-PD/Au electrode, on the contrary, did not exhibit any current responses, indicating that poly-PD has a too negative redox potential of -0.60 V to capture electrons from the UOx-uric acid complex, the results being consistent with those listed in Table 1. Amperometric Response of Poly-MPD/UOx/Au. Both poly-MPD and UOx were immobilized on the Au substrate with the assistance of a self-assembled monolayer of 2-aminoethanthiolate and a cross-linking agent of GA. The prepared electrode is denoted as poly-MPD/UOx/Au. SEM observations of the cross section of the resulting electrode revealed that an enzyme film whose thickness was 1.1 ( 0.02 µm was adhered on the Au substrate. The film was durable against vigorous agitation of the solution in which the electrode was immersed. However, if the enzyme film was prepared on a naked Au substrate, it was easily exfoliated from the electrode substrate, suggesting the importance of covalent bond formation between the Au substrate and the enzyme film of UOx via the self-assembled monolayer. The enzymatic activity of UOx of the poly-MPD/UOx/Au electrode was 8.01 units cm-2 as determined by consumption rates of uric acid in the presence of oxygen as an electron acceptor, and the electrode showed amperometric sensitivities to uric acid, as shown in Figure 4. The addition of 0.1 mmol dm-3 uric acid under polarization of the electrode at 0 V gave a significant increase in anodic currents, and steady currents, ir, of 0.066 µA cm-2 were obtained within ∼30 s. If another 0.1 mmol dm-3 uric acid was added (total concentration, 0.2 mmol dm-3), currents increased further and reached again another steady value. The magnitude of ir obtained at 0.1 mmol dm-3 uric acid was comparable to that obtained at the UOx/Au in the presence of dissolved MMP, where 0.072 µA cm-2 was obtained. The enzymatic activities of polyMPD/UOx/Au (8.01 units cm-2) and UOx/Au (9.68 units cm-2) were comparable, too, suggesting that the immobilized poly-MPD in the poly-MPD/UOx/Au possesses the electron mediation
Figure 5. Dependence of the current response (ir) at the poly-MPD/ UOx/Au electrode on the concentration of uric acid in N2-saturated 0.1 mol dm-3 borate buffer (pH 8.5) obtained for the poly-MPD/UOx/ Au prepared by using cast solution containing poly-MPD of (0) 1.2, (4) 1.5, (b) 1.8, (O) 2.4, (2) 3.3, and (9) 4.2 g dm-3 (see Experimental Section). Electrode potential; 0 V vs SCE.
ability, the efficiency of which is as high as that of MMP dissolved in the solution. Figure 5 shows ir obtained at the poly-MPD/UOx/Au containing different amounts of poly-MPD as a function of the uric acid concentration. For all cases, the ir values increased with increase in the uric acid concentration, indicating that the poly-MPD/UOx/ Au worked as a reagentless amperometric uric acid sensor. The concentration range shown in Figure 5 covers that of uric acid in healthy human blood (0.15-0.40 mmol dm-3).3-5,15,54 As recognized in this figure, amperometric response to uric acid was dependent on the amount of poly-MPD, and the highest sensitivity of the electrode was obtained at an intermediate amount of polyMPD of 1.8 mg dm-3. To discuss the presence of the optimum amount of poly-MPD, kinetic parameters for the poly-MPD/UOx/ Au electrode were obtained. Plots of the reciprocals of ir as a function of CS-1 (Lineweaver-Burk plots) allowed us to determine the apparent Michaelis constant for uric acid (KMapp) and the maximum current response (Imax).37,55-58
ir-1 ) (1 + KMappCS-1)Imax-1
(4)
where CS is the concentration of uric acid. The electrodes prepared by using various amounts of poly-MPD satisfied the relation given by this equation. Figure 6 shows the obtained KMapp and Imax as functions of the amount of poly-MPD in the casting solution. The obtained KMapp were much larger than the Michaelis constant (KM) of dissolved native UOx, which ranges from 10-2 to 10-1 mmol dm-3 and showed a tendency to increase with an increase in the (54) Harper, H. A. Review of Physiological Chemistry, 17th Asian ed.; Lange Medical: San Francisco, 1979; Chapter 14. (55) Segel, I. H. Enzyme Kinetics; Wiley-Interscience: New York, 1993; Chapters 2 and 9-I. (56) Foulds, N. C.; Lowe, C. R. J. Chem. Soc., Faraday Trans. 1 1986, 82, 125964. (57) Riklin, A.; Willner, I. Anal. Chem. 1995, 67, 4118-26. (58) Liaudet, E.; Battaglini, F.; Calvo, E. J. J. Electroanal. Chem. 1990, 293, 5568.
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Figure 6. Maximum current response (Imax) and apparent Michaelis constant (KMapp) of the poly-MPD/UOx/Au as a function of amount of poly-MPD contained in the cast solution used for preparation of polyMPD/UOx/Au. The values of Imax and KMapp were obtained from Lineweaver-Burk plots for the results shown in Figure 5. The curves are aids to the eye.
amount of poly-MPD. It is thought that permeation of uric acid into the film of the poly-MPD/UOx/Au influences the kinetics in the electrochemical oxidation of uric acid at this electrode;44,59,60 the permeation is reduced as the amount of poly-MPD increases and the film becomes dense. As shown in the figure, Imax also increased with increasing amount of poly-MPD up to ∼1.8 mg dm-3, above which a gradual depletion of Imax was observed. Imax is the current response obtained at an excess of the analyte and is usually larger for a higher concentration of immobilized enzyme.44-46,61 However, since the same amount of UOx was immobilized on all poly-MPD/UOx/Au electrodes prepared in the present study, Imax values might have been determined by another cause. It was reported that Imax obtained at the glucose oxidaseimmobilized electrodes for glucose detection varied with the thickness of the enzyme film, even if the same amount of the enzyme was immobilized on the electrode.62-64 According to the theoretical predictions made by Hall et al.63-65 and some other groups,66,67 the sensor response should increase with an increase in the thickness of the enzyme layer up to an optimum thickness, beyond which it decreases, because in such thick films the analyte is entirely consumed before reaching the electrode substrate and enzymatic reaction occurs only near the film/solution interface. Such prediction seems valid for the results shown in Figure 6, since the film thickness would be increased with an increase in the amount of poly-MPD in the film. However, for the poly-MPD/ (59) Sundaram, P. V.; Tweedale, A.; Laidler, K. J. Can. J. Chem. 1970, 48, 1498504. (60) Horvath, C.; Engasser, J.-M. Biotechnol. Bioeng. 1974, 16, 909-23. (61) Bartlett, P. N.; Pratt, K. F. E. J. Electroanal. Chem. 1997, 397, 61-78. (62) Bartlett, P. N.; Whitaker, R. G. J. Electroanal. Chem. 1987, 224, 37-48. (63) Gooding, J. J.; Hall, E. A. H. Electroanalysis 1996, 8, 407-13. (64) Gooding, J. J.; Hall, E. A. H. J. Electroanal. Chem. 1996, 417, 25-33. (65) Martens, N.; Hall, E. A. H. Anal. Chem. 1994, 66, 2763-70. (66) Bartlett, P. N.; Whitaker, R. G. J. Electroanal. Chem. 1987, 224, 27-35. (67) Sorochinskii, V. V.; Kurganov, B. I. Biosens. Bioelectron. 1994, 9, 481-9.
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UOx/Au prepared in the present study, the film should also become dense with an increase in the immobilized amount of polyMPD, as indicated by the increase in KMapp. Presumably, a small fraction of the loaded polymer would be accommodated into free spaces which are present between the immobilized bulky UOx enzymes of a molecular mass of 130 kDa. Because the theoretical prediction is made for a model enzyme electrode with use of dissolved electron mediator in solution and under the assumption that the diffusion and partition coefficients of reaction substrates are constant, the results shown in the figure might not completely be explained by the prediction. An alternative speculation is that an increase of poly-MPD causes an increase in the number of UOx molecules adjacent to the poly-MPD, resulting in the increase of Imax up to the optimum amount of poly-MPD. Beyond the optimum amount of poly-MPD, however, the film should become dense and the diffusion of uric acid through the film would be retarded to suppress the Imax value obtained at the electrode. Both of these effects, i.e., increase in thickness and density of the film, seems to be related to the observed variation in the current response depending on the amount of loaded poly-MPD. CONCLUSIONS In the present study, UOx-catalyzed oxidation of uric acid with use of an artificial electron acceptor was achieved for the first time to our best knowledge. Since the electron acceptor, e.g., MP and MMP, has the redox potential as negative as ∼-0.2 V vs SCE (at pH 8.5), the electrochemical oxidation of uric acid catalyzed by UOx was accomplished in potentials where direct anodic oxidation of uric acid was not simultaneously induced. These findings lead us to attempt to fabricate an amperometric uric acid sensor, and a redox ladder polymer of poly-MPD was found to be suitable for an electron mediator, which can be immobilized on an Au electrode substrate. However, in order for this novel polymer mediator to be applied to other biosensor systems, more detailed investigations of its electrical and electrochemical properties have to be made. The amperometric uric acid sensor (polyMPD/UOx/Au) which does not require the use of special reagents in test solutions was successfully constructed in the present study, but further characterizations of the poly-MPD/ UOx/Au are also needed to obtain information on the stability of the electrodes and influences of the possible interfering compounds which are present in blood or urine such as O2 and ascorbate. For example, the working potential of this sensor of 0 V vs SCE may still cause oxidation of ascorbate more or less. The investigations concerning these subjects are now under way and will be reported in future. ACKNOWLEDGMENT The present work was supported by the Asahi Glass Foundation and by Grant-in-Aids for Scientific Research (10555305) from the Ministry of Education, Science, Culture, and Sports, Japan. Received for review October 27, 1998. Accepted February 26, 1999. AC981168U
