Alkanethiolate Bilayer Immobilizing

Anal. Chem. 1999, 71, 4278-4283

A Biomimetic Phospholipid/Alkanethiolate Bilayer Immobilizing Uricase and an Electron Mediator on an Au Electrode for Amperometric Determination of Uric Acid 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

A biomimetic bilayer membrane immobilizing uricase (urate oxidase; EC 1.7.3.3) (UOx) and a redox agent of 1-methoxy-5-methylphenazinium (MMP) was fabricated on an Au electrode substrate with use of the Au substrate coated with a self-assembled monolayer of n-octanethiolate (OT/Au) and L-r-phosphatidylcholine β-oleoyl-γpalmitoyl (PCOP). The preparation was carried out by successively immersing an Au electrode substrate in an ethanol solution of OT, an MMP aqueous solution, and a suspension of proteoliposome formed by PCOP containing UOx and MMP. The prepared electrode exhibited such fast steady amperometric responses to uric acid as to allow its determination within 20 s after injecting uric acid, indicating that UOx-catalyzed electrochemical oxidation of uric acid was accomplished with assistance of electron mediation by MMP between UOx and the Au substrate. An increase in the response currents with increasing concentration of uric acid was obtained in a concentration range of uric acid found in healthy human blood. Any interference in the current response that is caused by direct anodic oxidation of uric acid or ascorbic acid was not observed at the prepared sensor electrode because the densely packed bilayer effectively blocked the diffusion of these substrates toward the Au surface, making it possible to determine amperometrically uric acid at the electrode with high precision. Fabrication of a self-organized interface on a solid support has been of great interest.1-7 For example, self-assembled monolayers (SAMs) of alkanethiolate on an Au substrate were extensively investigated and various functions were given to the SAMs by * Corresponding author: (Fax) +81-6(6879)7374; (e-mail) [email protected]. eng.osaka-u.ac.jp. (1) Ulman, A. An Introduction to Ultrathin Organic Films: From LangmuirBlodgett to Self-Assembly; Academic Press: Boston and Tokyo, 1991. (2) Varner, J. E. Self-Assembling Architecture; Alan. R. Liss: New York, 1988. (3) Sauvage, J.-P.; Hosseini, M. W. Templating, Self-Assembly, and SelfOrganization. In Comprehensive Supramolecular Chemistry; Pergamon: New York, 1996; Vol. 9. (4) Riste, T., Sherrington, D., Eds. Physics of Biomaterials: Fluctuations, Selfassembly and Evolution; NATO ASI Series E, Applied Sciences, Vol. 322; Kluwer Academic: Dordrecht, 1996. (5) Gompper, G., Schick, M., Eds. Phase Transitions and Critical Phenomena; Self-Assembling Amphiphilic Systems Vol. 16; Academic Press: London and Tokyo, 1994.

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introducing functional molecules.8-17 Willner et al. covalently attached enzymes and/or biological cofactors to a short alkanethiolate SAM on an Au electrode, resulting in successful development of amperometric sensors.18-22 The ordered interface of biochemical functionality was also constructed by utilizing the self-organized fusion of a phospholipid vesicle to the alkanethiolate SAM on an Au substrate as reported by Plant et al.23-25 and Ding et al.26 The resulting phospholipid/alkanethiolate bilayer supported on an Au substrate was studied as a model of a biomembrane, and it has recently been reported that so-called membrane proteins that are fixed in biomembranes in vivo can be incorporated in the artificial bilayers.27-32 Uricase (urate oxidase, UOx), catalyzing oxidation (6) Lindman, B., Rosenholm, J. B., Stenius, P., Eds. Surfactants and Macromolecules: Self-Assembly at Interfaces and in Bulk; Progress in Colloid & Polymer Science, Vol. 82; Springer-Verlag: New York, 1990. (7) Lindman, B., Ninham, B. W., Eds. The Colloid Science of Lipids: New Paradigms for Self-Assembly in Science and Technology; Progress in Colloid & Polymer Science, Vol. 108; Springer-Verlag: New York, 1998. (8) Ulman, A., Ed. Self-Assembled Monolayers of Thiols; Thin films; Vol. 24; Academic Press: San Diego, 1998. (9) Finklea, H. O., Ed Electrochemistry of Organized Monolayers of Thiols and Related Molecules on Electrodes; Electroanalytical Chemistry: A Series of Advances, Vol. 19; Marcel Dekker: New York, 1996. (10) Bard, A. J.; Abrun ˜a, H. D.; Chidsey, C. E.; Faulkner, L. R.; Feldberg, S. W.; Itaya, K.; Majda, M.; Melroy, O.; Murray, R. W.; Porter, M. D.; Soriaga, M. P.; White, H. S. J. Phys. Chem. 1993, 97, 7147-73. (11) Uosaki, K.; Kondo, T.; Zhang, X.-Q.; Yanagida, M. J. Am. Chem. Soc. 1997, 119, 8367-8. (12) Bilewicz, R.; Majda, M. J. Am. Chem. Soc. 1991, 113, 5464-6. (13) Chailapakul, O.; Crooks, R. M. Langmuir 1993, 9, 884-8. (14) Turyan, I.; Mandler, D. Anal. Chem. 1994, 66, 58-63. (15) Rojas, M. T.; Ko¨niger, R.; Stoddart, J. F.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336-43. (16) VanVelzen, E. U. T.; Engbersen, J. F. J.; Reinhoudt, D. N. J. Am. Chem. Soc. 1994, 116, 3597-8. (17) Hickman, J. J.; Ofer, D.; Laibinis, P. E.; Whitesides, G. M.; Wrighton, M. S. Science 1991, 252, 688-91. (18) Willner, I.; Katz, E.; Riklin, A.; Kasher, R. J. Am. Chem. Soc. 1992, 114, 10965-6. (19) Willner, I.; Riklin, A.; Shoham, B.; Rivenzon, D.; Katz, E. Adv. Mater. 1993, 5, 912-5. (20) Willner, I.; Katz, E.; Willner, B. Electroanalysis 1997, 9, 965-77. (21) Riklin, A.; Willner, I. Anal. Chem. 1995, 67, 4118-26. (22) Bardea, A.; Katz, E.; Bu ¨ ckmann, A. F.; Willner, I. J. Am. Chem. Soc. 1997, 119, 9114-9. (23) Plant, A. L. Langmuir 1993, 9, 2764-7. (24) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126-33. (25) Meuse, C. W.; Niaura, G.; Lewis, M. L.; Plant, A. L. Langmuir 1998, 14, 1604-11. (26) Ding, L.; Li, J.; Dong, S.; Wang, E. J. Electroanal. Chem. 1996, 416, 10512. 10.1021/ac981371p CCC: $18.00

© 1999 American Chemical Society Published on Web 08/12/1999

Scheme 1. Preparation of the Au Electrode Coated with the Bilayer of OT and PCOP Incorporating UOx and MMP

of uric acid to allantoin in the presence of O2, is a key enzyme in the purine degradation pathway in living bodies.33-35 Interestingly, recent studies revealed that UOx is a peroxisomal globular protein enclosing a tunnel,35 which possesses a characteristic property as a transporter of uric acid across a biomembrane, implying that UOx is a membrane protein.36-39 The present study is conducted to mimic the functions of UOx in the biomembrane by embedding UOx in an artificial bilayer membrane. In our recent paper, electrochemical oxidation of uric acid catalyzed by UOx was achieved by mediating electrons between UOx and an electrode substrate with use of an artificial redox electron mediator, e.g.,1-methoxy-5-methylphenazinium (MMP).40,41 Furthermore, we have developed a reagentless amperometric uric acid sensor by immobilizing UOx on an Au electrode surface together with a redox polymer mediator of an MMP analogue.41 (27) Cullison, J. K.; Hawkridge, F. M.; Nakashima, N.; Yoshikawa, S. Langmuir 1994, 10, 877-82. (28) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580-3. (29) Pierrat, O.; Lechat, N.; Bourdillon, C.; Laval, J.-M. Langmuir 1997, 13, 4112-8. (30) Naumann, R.; Jonczyk, A.; Wolf, H.; Liley, M.; Ringsdorf, H.; Knoll, W. Angew. Chem., Int. Ed. Engl. 1995, 34, 2056-8. (31) Naumann, R.; Jonczyk, A.; Hampel, C.; Ringsdorf, H.; Knoll, W.; Bumjes, N.; Gra¨ber, P. Bioelectrochem. Bioenerg. 1997, 42, 241-7. (32) Li, J.; Ding, L.; Wang, E.; Dong, S. J. Electroanal. Chem. 1996, 414, 1721. (33) Baker, B. F. Free Radical Biol. Med. 1993, 14, 615-31. (34) Kahn, K.; Tipton, P. A. Biochemistry 1998, 37, 11651-9. (35) Colloc’h, N.; El Hajji, M.; Bachet, B.; L’Hermite, G.; Schiltz, M.; Prange´, T.; Castro, B.; Mornon, J.-P. Nature Struct. Biol. 1997, 4, 947-52. (36) Damsz, B.; Dannenhoffer, J. M.; Bell, J. A.; Webb, M. A. Plant Cell Physiol. 1994, 35, 979-82. (37) Pordy, W. T.; Lipkowitz, M. S.; Abramson, R. G. Am. J. Physiol. 1987, 253, F702-11. (38) Abramson, R. G.; King, V. F.; Reif, M. C.; Leal-Pint, E.; Baruch, S. B. Am. J. Physiol. 1982, 242, F158-70. (39) Wu, X., Lee, C. C.; Muzny, D. M.; Caskey, C. T. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9412-6. (40) Kuwabata, S.; Nakaminami, T.; Ito, S.; Yoneyama, H. Sens. Actuators B 1998, 52, 72-7. (41) Nakaminami, T.; Ito, S.; Kuwabata, S.; Yoneyama, H. Anal. Chem 1999, 71, 1928-34.

The use of an artificial electron mediator having less positive redox potential than the natural mediator of O2/H2O2 should be desirable in the amperometric determination of uric acid by utilizing the selectivity of UOx, because direct anodic oxidation of uric acid itself and blood components such as ascorbic acid at the electrode can be suppressed. More perfect suppression of the oxidation of the current-interfering compounds in blood may be attained by coating a UOx-immobilized electrode with an external permselective membrane against the interfering compounds, as already demonstrated in amperometric detection of glucose.42-46 In the present study, we developed a novel technology for fabrication of an amperometric uric acid sensor allowing a highly selective determination of uric acid without any serious interference; an Au electrode coated with a biomimetic bilayer immobilizing UOx and the above-mentioned MMP as an electron-transfer mediator was prepared as schematically illustrated in Scheme 1. Although there are several reports on the application of the supported artificial biomembrane to the fabrication of the amperometric biosensors,29,47,48 no paper deals with the detection selectivity and the immobilization of both electron mediator and membrane enzyme molecules in the bilayer. It will be demonstrated in this paper that the prepared electrode is valid for the selective, rapid, and regentless determination of uric acid, which is clinically important for diagnosing gout caused by crystallization of uric acid accumulated in blood.49-53 EXPERIMENTAL SECTION Uricase (urate oxidase; EC 1.7.3.3) (UOx) from Candida sp. and L-R-phosphatidylcholine β-oleoyl-γ-palmitoyl (PCOP) were (42) Lobel, E.; Rishpon, J. Anal. Chem. 1981, 53, 51-3. (43) Bindra, D. S.; Wilson, G. S. Anal. Chem. 1989, 61, 2565-70. (44) Harrison, D. J.; Turner, R. F. B.; Baltes, H. P. Anal. Chem. 1988, 60, 20027. (45) Maidan, R.; Heller, A. Anal. Chem. 1992, 64, 2889-96. (46) Ikeda, T.; Katasho, I.; Senda, M. Anal. Sci. 1985, 1, 455-7. (47) Tien, H. T.; Wurster, S. H.; Ottova, A. L. Bioelectrochem. Bioenerg. 1997, 42, 77-94. (48) Tominaga, M.; Kusano, S.; Nakashima, N. Bioelectrochem. Bioenerg. 1997, 42, 59-62.

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commercially available from Wako Pure Chemicals and used without further purification. All other chemicals used were of analytical grade and obtained from Wako Pure Chemicals except for 1-methoxy-5-methylphenazinium sulfate (Dojindo Laboratories). All aqueous solutions were prepared using twice-distilled water. PCOP liposome (i.e., lipid vesicle) was prepared as follows.23-26,54 PCOP (10 mmol dm-3) dissolved in CHCl3 was put in a roundbottom flask, and then the solvent was evaporated under reduced pressure overnight using a rotary evaporator (Tokyo Rikakikai, EYELA) to deposit PCOP thin films on the inner surface of the flask. The resulting PCOP films were dissolved in 2-propanol to give 40 mmol dm-3 PCOP, and 50 mm3 of this solution was added to 1 cm3 of 20 mmol dm-3 Tris-HCl buffer containing 150 mmol dm-3 NaCl (pH 7.3), followed by agitating vigorously for 10 min to give a suspension of PCOP liposomes. UOx (0.83 µmol dm-3) and 10 mmol dm-3 MMP were dissolved in the suspension of PCOP liposomes, followed by agitating vigorously for 10 min to incorporate both UOx and MMP in the liposome.37,38 An Au disk plate (0.88-cm diameter) was used as an electrode substrate. The Au disk was 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,9,55 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! Formation of a bilayer of PCOP and n-octanethiol (OT) on the Au electrode substrate and fixation of UOx and MMP in the bilayer were accomplished by procedures as shown in Scheme 1. The Au electrode was immersed in an ethanol solution containing 1 mmol dm-3 OT for 4 h to prepare the Au electrode coated SAM of OT (OT/Au), followed by immersing the OT/Au electrode in 10 mmol dm-3 MMP aqueous solution for 1 h to incorporate MMP molecules in the OT-SAM. After each immersion step, the resulting electrode was rinsed well with ethanol and water, respectively. The resulting electrode was immersed in the liposome suspension described above for 12 h to give the Au electrode coated with the bilayer of OT and PCOP in which UOx and MMP were fixed. In this paper, the electrode obtained in this way will be denoted as the PCOP/UOx/MMP/OT/Au. It is thought that the immobilized UOx molecules in the PCOP layer of the bilayer prepared must extrude into the solution as shown in Scheme 1 because UOx is a homotetramic globular protein of 50 × 50 Å, whereas the thickness of the PCOP layer was estimated to be ∼30 Å at most by assuming that alkyl chains of PCOP molecules stood perpendicularly to the Au substrate.23,27 The enzymatic activity of UOx immobilized in the bilayer was examined by chemical oxidation of uric acid with use of the PCOP/UOx/MMP/ OT/Au as a catalyst and O2 as an oxidizing agent.56 For this purpose, 2 cm3 of air-saturated 0.1 mol dm-3 borate buffer (pH 8.5) at 25 °C was put in a quartz cell having a light path length of (49) Fox, I. H. Metabolism 1981, 30, 616-34. (50) Ullman, B.; Wormsted, M. A.; Cohen, M. B.; Martin, D. W., Jr. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 5127-31. (51) Yamanaka, H.; Togashi, R.; Hakoda, M.; Terai, C.; Kashiwazaki, S.; Dan, T.; Kamatani, N. Adv. Exp. Med. Biol. 1998, 431, 13-8. (52) Liang, M. H.; Fries, J. F. Ann. Intern. Med. 1978, 88, 666-70. (53) Simkin, P. A. Ann. Intern. Med. 1979, 90, 812-6. (54) Batzri, S.; Korn, E. D. Biochim. Biophys. Acta 1973, 298, 1015-9. (55) Hayes, W. A.; Shannon, C. Langmuir 1996, 12, 3688-94.

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Figure 1. Cyclic voltammograms at (a) the bare Au and (b) the OT/Au electrodes taken in N2-saturated 0.1 mol dm-3 borate buffer (pH 8.5) containing 1 mmol dm-3 uric acid.

1 cm and then the PCOP/UOx/MMP/OT/Au was immersed in it. An aliquot of 20 mmol dm-3 uric acid dissolved in borate buffer was added to the quartz cell to give a concentration of 0.1 mmol dm-3, and absorption spectra were measured between 200 and 350 nm using a diode array spectrophotometer (Hewlett-Packard, 8452A). The time course of decrease in absorbance at 290 nm that is due to uric acid was monitored for 5 min. Using a molar absorptivity of uric acid of 1.22 × 104 dm3 mol-1 cm-1 at that wavelength, the enzymatic activity of UOx on the electrode was determined. The rate of consumption of 1 µmol of uric acid min-1 cm2 of electrode area was defined here as 1 unit cm-2 of the enzymatic activity of the electrode. Amperometric responses of the PCOP/UOx/MMP/OT/Au to uric acid were measured at 0 V vs a saturated calomel electrode (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 electrode, respectively. In this paper, potentials referred to SCE will be cited for all cases. The cell was placed in an incubator controlled at 30 ( 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 after polarizing the electrode, an aliquot of 20 mmol dm-3 uric acid dissolved in the borate buffer was added to the electrolyte solution to give a desired concentration, followed by magnetically agitating the solution for 5 s. Then, the time course of the oxidation currents in quiescent solution was monitored on an electric polyrecorder (Toa Denpa, EPR-151A). Cyclic voltammetry was performed using an electrochemical analyzer (Bioanalytical Systems, BAS-100B/W) connected to a Gateway 2000 computer. The potential scan rate used was 50 mV s-1 for all cases, and other measurement conditions will be described elsewhere in the present paper. RESULTS AND DISCUSSION Diffusion of Uric Acid and Electron Mediators to an Au Electrode Substrate Coated with OT-SAM. Figure 1 shows cyclic voltammograms at bare Au and OT-SAM-coated Au (OT/ Au) electrodes taken in 0.1 mol dm-3 borate buffer (pH 8.5) (56) Dilena, B. A.; Peake, M. J.; Pardue, H. L.; Skoug, J. W. Clin. Chem. 1986, 32, 486-91.

Figure 2. Cyclic voltammograms at (a, b) the bare Au, and (c, d) the OT/Au taken in N2-saturated 0.1 mol dm-3 borate buffer (pH 8.5) containing 0.5 mmol dm-3 (a, c) ferrocenecarboxylate and (b, d) MMP.

containing 1 mmol dm-3 uric acid. As shown by voltammogram a, the bare Au electrode gave significant anodic currents at potentials positive of ∼0.2 V, indicating that electrochemical oxidation of uric acid occurred directly at the bare Au electrode. However, if the Au electrode was covered with an OT-SAM, anodic currents were largely suppressed as shown by voltammogram b, evidencing that diffusion of uric acid toward the Au electrode substrate is blocked by the densely packed OT-SAM. Several redox compounds, which were found to work as electron mediators for UOx in our previous studies,40,41 were tested to investigate their abilities to penetrate into the OT-SAM on the Au substrate. Figure 2 shows cyclic voltammograms at the OT/ Au and the bare Au electrodes taken in a borate buffer either containing 0.5 mmol dm-3 ferrocenecarboxylate or MMP. Ferrocenecarboxylate gave reversible voltammetric waves at the bare Au electrode, but at the OT/Au, the magnitude of redox waves was decreased and its shape was deformed, suggesting that the alkyl chains of OT-SAM hindered the electron exchanges between ferrocenecarboxylate and the Au substrate. If [Fe(CN)6]4-/3- was used as a redox agent, no redox wave appeared at the OT/Au, though not shown here, indicating that the OT-SAM blocked the redox reaction completely. In the case of the redox reaction of MMP, the magnitude of currents was decreased a little by covering the Au electrode surface with OT-SAM, but no shift in the current peak potentials appeared. Since MMP is a highly planar molecule, as illustrated in Scheme 1, it might be able to penetrate deeply into the OT layer and undergo the redox reaction at the Au substrate. If the OT/Au electrode was immersed in 10 mmol dm-3 MMP aqueous solution for 1 h, the resulting electrode exhibited redox activity of MMP in 0.1 mol dm-3 borate buffer (pH 8.5), even if the electrode was carefully washed with water, indicating that MMP was incorporated in the OT layer. However, the redox peak currents gradually decreased and disappeared if the electrode was immersed in the borate buffer for 1.5 h, suggesting that MMP was physically adsorbed in the OT layer. Immobilization of MMP and UOx Molecules in a Bilayer of PCOP and OT. Figure 3 shows cyclic voltammograms of PCOP/UOx/MMP/OT/Au taken in borate buffer (pH 8.5) when prepared and after soaking in the measurement solution for 4 h. With a 4-h soaking, current values were a little decreased, but further soaking did not cause any significant decrease, suggesting that the PCOP adlayer effectively works as a “cap” which prevents the leaching of MMP molecules from the OT and/or PCOP layer.

Figure 3. Cyclic voltammograms at the PCOP/UOx/MMP/OT/Au electrode (a) as prepared and (b) immersed in the electrolyte solution for 4 h. Electrolyte solution, N2-saturated 0.1 mol dm-3 borate buffer (pH 8.5).

The integration of the anodic wave of voltammogram b allowed the determination of the surface concentration of MMP in the bilayer to be 3.9 × 10-11 mol cm-2. The achievements of immobilization of UOx in the bilayer were also confirmed by enzymatic activity tests conducted as follows; immersion of the PCOP/UOx/MMP/OT/Au in air-saturated borate buffer containing 0.1 mmol dm-3 uric acid for 5 min resulted in chemical oxidation of 6.57 µmol of uric acid, from which an enzymatic activity of 4.87 units cm-2 was evaluated. The obtained enzymatic activity of the electrode was not less than half the activity (8.01 units cm-2) obtained at our previously studied electrode, which was modified with cross-linked UOx multilayers and an electron mediator of polymerized MMP analogue.41 Considering that the amount of enzyme immobilized on the electrode used in the present study was so small as to give less than a monolayer and that the electrode was prepared with the use of UOx having the same original activity (unit mol-1) as that used in the previous study, it may be said that the immobilization technique used in the present study did not give significant loss of original activities of UOx molecules. Amperometric Responses of the PCOP/UOx/MMP/OT/ Au to Uric Acid. The current responses of the PCOP/UOx/ MMP/OT/Au to uric acid were examined at 0 V, which was positive enough to oxidize reduced state of MMP in the bilayer (see Figure 3). Figure 4A shows the time course of changes in oxidation currents caused by adding uric acid to the electrolyte solution. A rapid increase in oxidation currents followed by the appearance of steady currents was obtained by addition of 0.1 mmol dm-3 uric acid. Another addition of 0.1 mmol dm-3 uric acid gave a further increase in the anodic currents. The time required to obtain steady currents was within 20 s after each injection of uric acid. If the bilayer-coated Au electrode that did not contain MMP and/or UOx was used, no oxidation currents of uric acid were obtained. The possibilities of direct anodic oxidation of uric acid and MMP-catalyzed chemical oxidation of uric acid at the PCOP/UOx/MMP/OT/Au are also ruled out because the OT layer suppressed the direct anodic oxidation of uric acid as indicated from voltammogram b in Figure 1, and furthermore, MMP had no catalytic activity for oxidation of uric acid, as reported in our previous paper.41 It is believed that the following reactions definitely occurred at the PCOP/UOx/MMP/ OT/Au electrode. Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

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Figure 5. Dependence of the steady response currents at the PCOP/UOx/MMP/OT/Au on the concentration of uric acid in N2saturated 0.1 mol dm-3 borate buffer (pH 8.5). Electrode potential, 0 V vs SCE. Shaded region given in the figure shows the range of the uric acid concentration contained in healthy human blood.

Figure 4. (A) Time course of currents at the PCOP/UOx/MMP/OT/ Au obtained by stepwise additions of 0.1 mmol dm-3 uric acid. (B) Time course of currents (a) at the bare Au electrode obtained by addition of 0.1 mmol dm-3 ascorbic acid and (b) at the PCOP/UOx/ MMP/OT/Au obtained by alternative stepwise additions of 0.1 mmol dm-3 ascorbic acid and 0.1 mmol dm-3 uric acid. Note that ascorbic acid was added in the beginning of the measurement also in the case of (b). (A, B) Electrolyte solution, N2-saturated 0.1 mol dm-3 borate buffer (pH 8.5); electrode potential, 0 V vs SCE. The times when uric acid and ascorbic acid were added to the solution are designated by arrows marked of “U” and “A”, respectively. UOx

uric acid + MMP 98 product + MMPred electrode

MMPred 98 MMP + 2e-

(1) (2)

where MMPred denotes the reduced form of MMP. Since UOx is reported to be a big enzyme enclosing a tunnel of 12 Å in diameter,35 as described in the introduction, diffusion of uric acid from the solution bulk to the active sites of immobilized UOx molecule would occur through the tunnel. This view is supported by the finding that the bilayer-coated Au electrode without UOx and MMP did not show any activity for oxidation of uric acid due to complete blocking its diffusion; by covering the OT/Au with the PCOP layer, voltammogram b shown in Figure 1 was changed to that giving no currents even at the applied potential of 0.5 V. Considering that the catalytic oxidation of uric acid was accomplished with assistance of the electron acceptor of MMP, followed by mediation of the electron transfer from UOx to Au substrate, the redox couple of MMP/MMPred seems to possess a high mobility in the bilayer. Ascorbic acid is known to interfere with the amperometric determination of a human blood component. Indeed, large oxidation currents of ascorbic acid were observed at a bare Au electrode polarized at 0 V vs SCE as shown by curve a in Figure 4B. On the contrary, as shown by curve b in the figure, the PCOP/ 4282 Analytical Chemistry, Vol. 71, No. 19, October 1, 1999

UOx/MMP/OT/Au electrode did not show any current response to ascorbic acid, indicating that the densely packed bilayer of PCOP and OT blocked completely the approach of ascorbic acid to the Au electrode substrate. If uric acid was successively added to the electrolyte solution, an increase in anodic currents was observed at the PCOP/UOx/MMP/OT/Au. Further addition of ascorbic acid did not disturb such current responses of the electrode to uric acid, and the magnitude of the steady currents obtained in each addition of uric acid was identical to that obtained in the presence of the same concentration of uric acid only (see Figure 4A), indicating that the PCOP/UOx/MMP/OT/Au worked as a highly selective amperometric uric acid sensor. If the obtained steady currents for uric acid were plotted as a function of the concentration of uric acid added, the results shown in Figure 5 were obtained. The magnitude of the steady currents increased with increasing uric acid concentration for a concentration range that covers the concentration contained in the blood of healthy human beings (0.15-0.4 mmol dm-3 51-53,57,58). Lineweaver-Burk analysis according to the following equation allows the determination of the kinetic parameters of the sensor responses.21,59-64

1/ir ) (1 + KMapp/CS)/Imax

(3)

where ir is the obtained steady currents and CS is the concentration of uric acid. KMapp and Imax are the apparent Michaelis constant and the maximum current response of the PCOP/UOx/MMP/ OT/Au, respectively. A good linear relation was obtained by plots (57) Motonaka, J.; Miyata, K.; Faulkner, L. R. Anal. Lett. 1994, 27, 1-13. (58) Harper, H. A. Review of Physiological Chemistry, 17th Asian ed.; Lange Medical: San Fransisco, 1979; Chapter 14. (59) Segel, I. H. Enzyme Kinetics; Wiley-Interscience: New York, 1993; Chapters 2 and 9-I. (60) Nakaminami, T.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1997, 69, 236772. (61) Nakaminami, T.; Ito, S.; Kuwabata, S.; Yoneyama, H. Anal. Chem. 1999, 71, 1068-76. (62) Kuwabata, S.; Okamoto, T.; Kajiya, Y.; Yoneyama, H. Anal. Chem. 1995, 67, 1684-90. (63) Foulds, N. C.; Lowe, C. R. Anal. Chem. 1988, 60, 2473-8. (64) Liaudet, E.; Battaglini, F.; Calvo, E. J. J. Electroanal. Chem. 1990, 293, 5568.

of 1/ir as a function of 1/CS, from which KMapp ) 1.32 mmol dm-3 and Imax ) 0.74 µA cm-2 were obtained. If KM for the chemical oxidation of uric acid was estimated for dissolved native UOx, about one-tenth of the KMapp value of the electrode was obtained, suggesting that electrochemical oxidation of uric acid at the PCOP/UOx/MMP/OT/Au might be controlled by the diffusion of uric acid.65-67 If it were the case, it may be said that most of the active sites of UOx molecules are buried in the PCOP layer rather than extruded to the solution. Imax at the PCOP/UOx/ MMP/OT/Au is comparable to that obtained at the electrode previously studied (∼0.8 µA cm-2) which was modified with a multilayer of cross-linked UOx and a mediator of the polymerized MMP analogue.41 However, it should be emphasized again that the electrode prepared in the present study contained a much smaller amount of UOx and therefore possessed a smaller enzymatic activity as compared to the previous one. Considering that Imax is usually apt to increase with increasing concentration and/or activity of UOx,65,68 it seems likely that the efficiency of electron mediation by MMP in the PCOP/UOx/MMP/OT/Au was much higher than that of the electrode coated with the UOx multilayer; i.e., the number of UOx substantially involved in the detection reaction was much larger than that obtained in the previous electrode. Changes in the sensitivity that may be caused by repeated measurements were examined for the PCOP/UOx/MMP/OT/ Au electrode. During the interruption of measurements, the electrode was stored in uric acid-free 0.1 mol dm-3 borate buffer (pH 8.5) at 4 °C in the dark for 30 min. Essentially the same current responses to 0.2 mmol dm-3 uric acid (0.95 ( 0.01 µA cm-2) were obtained for the initial four measurements (total storage time, ∼2 h), but after that the currents gradually decreased by ∼3% for every measurement. As already discussed for the results shown in Figure 3, MMP molecules encapsulated in the bilayer seemed to be dissolved out to the buffer solution by soaking for the first 4 h after preparation of the PCOP/UOx/ (65) Calvo, E.; Danilowicz, C.; Diaz, L. J. Chem. Soc., Faraday Trans. 1 1993, 89, 377-84. (66) Sundaram, P. V.; Tweedale, A.; Laidler, K. J. Can. J. Chem. 1970, 48, 1498504. (67) Horvath, C.; Engasser, J.-M. Biotechnol. Bioeng. 1974, 16, 909-23. (68) Bartlett, P. N.; Pratt, K. F. E. J. Electroanal. Chem. 1997, 397, 61-78.

MMP/OT/Au. Since the current response to uric acid was stable for the first 2 h, the decrease in the current response observed after 2 h is not necessarily attributed to leaching of MMP. Presumably, thermal deactivation and/or desorption of UOx would be responsible for the loss of amperometric activities of the electrode with time. Though a bilayer consisting of PCOP and OT-SAM on an Au electrode without UOx and MMP is reported to be stable at least for several days,23 bulky UOx molecules might leach gradually out of the bilayer at the electrode prepared in the present study, leading to partial collapse of the ordered bilayer structure. CONCLUSION In the present study, a novel technology for fabrication of a biosensor was successfully developed; the self-organized PCOP/ OT bilayer served not only as a tool for the immobilization of both an enzyme and an electron mediator on an electrode substrate but also as a membrane for perfect elimination of the interference signal which could be caused by compounds other than analyte. The PCOP/UOx/MMP/OT/Au electrode prepared by utilizing the present methodology was useful to achieve rapid amperometric determinations of uric acid with high selectivity. However, further investigations are needed to improve the stability of the electrode. Since the leaching of the UOx molecules from the bilayer on the electrode was suggested, it might be useful to bind PCOP molecules on the electrode with each other to solve this problem; tightly bound PCOP molecules would tightly fix UOx in the bilayer of PCOP/OT. Studies toward this direction are under way and will be reported in future. ACKNOWLEDGMENT This work was partially supported by Asahi Glass Foundation and by a Grant-in-Aid for Scientific Research (103555305) and that on the Priority Area of “Electrochemistry of Ordered Interfaces” (09237104) from the Ministry of Education, Science, Sports and Culture. Received for review December 11, 1998. Accepted June 25, 1999. AC981371P

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