Real-Time Quantification of Methanol in Plants Using a Hybrid Alcohol

An amperometric biosensor immobilizing two enzymes and an electron mediator in an identical plane has been fabricated by the self-assembly technique f...
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Anal. Chem. 2004, 76, 1500-1506

Real-Time Quantification of Methanol in Plants Using a Hybrid Alcohol Oxidase-Peroxidase Biosensor Tomohisa Hasunuma,† Susumu Kuwabata,*,‡ Ei-ichiro Fukusaki,† and Akio Kobayashi*,†

Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan, and Department of Materials Chemistry, Graduate School of Engineering, Osaka University, 2-1, Yamadaoka, Suita, Osaka 565-0871, Japan

An amperometric biosensor immobilizing two enzymes and an electron mediator in an identical plane has been fabricated by the self-assembly technique for determination of methanol in crude plant samples. A self-assembled mixed monolayer of 4,4′-dithiodibutyric acid covalently attached two enzymes (Hansenula sp. alcohol oxidase and horseradish peroxidase) and 11-ferrocenyl-1-undecanethiol as an electron mediator on an Au electrode is exploited to produce a two-dimensional reaction matrix. The composition of the two enzymes and electron mediator molecules was optimized for detection of methanol in 0.1 M sodium phosphate buffer (pH 6.0). We successfully quantified methanol in low-purity tobacco (Nicotiana tabacum) plant extracts with the biosensor, which showed sensitivity comparable to that of gas chromatography/ mass spectrometry. The redox-relay biosensor is quite simple and stable due to its covalent attachment to the Au surface, making it possible to downsize the construction. We fabricated a miniature methanol biosensor that fitted a well of a 96-well micro assay plate available for high-throughput assay. The biosensor is advantageous for the sensitive, continuous, and convenient determination of methanol. Since the new importance of effects of methanol on plant physiology has been recently elucidated,1-4 methods allowing facile and rapid measurements of slight amounts of methanol have become more significant. The measurement system would require the capability to distinguish methanol from other contaminants generated in plant tissues. As such, the means of measurement , spectrophotometry,5 fluorometry,6,7 and gas chromatography (GC), * To whom correspondence should be addressed. Phone and Fax: +81-66879-7372. E-mail: [email protected]. † Department of Biotechnology. ‡ Department of Materials Chemistry. (1) Nonomura, A. M.; Benson, A. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9794-9798. (2) Shiraishi, T.; Fukusaki, E.; Miyake, C.; Yokota, A.; Kobayashi, A. J. Biosci. Bioeng. 2000, 89, 564-568. (3) Fall, R.; Benson, A. A. Trends Plant Sci. 1996, 1, 296-301. (4) Kobayashi, A.; Fukusaki, E.; Isogai, A.: U.S. Patent 6465396, 2002. (5) Mangos, T. J.; Haas, M. J. J. Agric. Food Chem. 1996, 44, 2977-2981. (6) Nemecek-Marshall, M.; MacDonald, R. C.; Franzen, J. J.; Wojciechowski, C. L.; Fall, R. Plant Physiol. 1995, 108, 1359-1368. (7) Wojciechowski, C. L.; Fall, R. Anal. Biochem. 1996, 237, 103-108.

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have been utilized so far, but each has both advantages and disadvantages. Spectrophotometric assay allows continuous measurements but has lower sensitivity that is around 10 -3 mol dm-3. In a fluorometric assay, methanol is oxidized to formaldehyde by alcohol oxidase (EC 1.1.3.13; AOD), followed by reaction with Fluoral-P (4-amino-3-penten-2-one) to produce DDDP (3,5-diacethyl-2,6-dihydrolutidine),8 then fluorescence from DDDP at 510 nm with excitation at 412 nm is used as a signal for continuous quantification of methanol with ∼30 times higher sensitivity than the spectrophotometric measurement.7 However, this method is susceptible to fluorescent backgrounds, leading sometimes to difficulties in precise evaluation of methanol in crude plant extracts. The GC method with a detector of mass spectrometry (GC/MS) has an advantage in both sensitivity and selective detection (the detection limit is ∼10-5 mol dm-3), but it is discontinuous and often complicated. Herein, we would like to demonstrate the usefulness of an electrochemical method by developing a convenient biosensor. Several biosensors have been developed using AOD and alcohol dehydrogenase (EC 1.1.1.1, ADH) as an alcohol recognition site.9-13 AOD catalyzes oxidation of short-chain aliphatic alcohols, including methanol, whereas ADH possesses low selectivity for catalytic oxidation of both aromatic and aliphatic alcohols.14 In fabrication of alcohol sensor using AOD, electrochemical detection of produced hydrogen peroxide is essential. The simplest way is direct oxidation at an electrode surface, but detection by electrochemical reduction employing peroxidase (EC 1.11.1.7; POD) and an appropriate mediator is currently recognized to be a novel technique.15 Although direct electron exchanges between peroxidase and an electrode can be conducted if the enzyme is adsorbed on a gold electrode is reported,16 the electrontransfer rate for the native horseradish peroxidase is much lower (8) Compton, B. J.; Purdy, W. C. Anal. Chim. Acta 1980, 119, 349-357. (9) Guilbault, G. G.; Danielsson, B.; Mandenius, C. F.; Mosbach, K. Anal. Chem. 1983, 55, 1582-1585. (10) Guilbault, G. G.; Lubrano, G. J. Anal. Chim. Acta 1974, 69, 189-194. (11) Miyamoto, S.; Murakami, T.; Saito, A.; Kimura, J. Biosens. Bioelectron. 1991, 6, 563-567. (12) Wang, J.; Chen, Q.; Pedrero, M.; Pingarro´n, J. M. Anal. Chim. Acta 1995, 300, 111-116. (13) Lubrano, G. J.; Faridnia, M. H.; Palleschi, G.; Guilbault, G. G. Anal. Biochem. 1991, 198, 97-103. (14) Barman, T. E. Enzyme handbook; Springer-Verlag: New York, 1969. (15) Pen ˜a, N.; Ta´rrega, R.; Reviejo, A. J.; Pingarro´n, J. M. Anal. Lett. 2002, 35, 1931-1944. 10.1021/ac035309q CCC: $27.50

© 2004 American Chemical Society Published on Web 02/04/2004

Figure 1. Schematic representation of an electronic relay on the AOD/POD/11-FUT/Au electrode. Inset shows illustration of three kinds of functional alkanethiol: 6-ferrocenyl-1-hexanethiol (6-FHT), 11-ferrocenyl-1-undecanethiol (11-FUT), and 3-carboxy-1-propanethiol (3-CPT).

than a recombinant one; therefore, electron mediators are effective in detecting hydrogen peroxide. When a ferrocene derivative (Fc) is used as an electron mediator, biosensing by combining the oxidase-catalyzed reaction and reduction of H2O2 with POD gives cathodic currents with the following reactions

Sub + O2 f Pro + H2O2

(1)

PODred + H2O2 f PODox + H2O

(2)

PODox + 2Fc + 2H+ f PODred + 2Fc+ + H2O

(3)

2Fc+ + 2e- f 2Fc

(4)

where Sub and Pro, PODred, PODox are, respectively, substrate, product, and the reductive and oxidative forms of POD. Reductive detection is advantageous from viewpoints of the sensing selectivity because there are several species which are easily oxidized, including ascorbic acid in tissues. Usefulness of the reaction system was first reported for glucose sensing with the use of glucose oxidase (GOD).17,18 Its applicability to alcohol sensing has been also confirmed by using AOD, POD, and appropriate electron mediators.15,19-25 To fabricate practical alcohol sensors, a graphite electrode,19 carbon paste electrode20,21 and carbon-epoxy resin electrode 15,22 were usually used, and attempts were made to immobilize enzymes and electron mediators. A self-assembly technique has been very widely used to construct ordered molecular layers possessing various functions. (16) Presnova, G.; Grigorenko, V.; Egorov, A.; Ruzgas, T.; Lindgren, A.; Gorton, L.; Bo ¨rchers, T. Faraday Discuss. 2000, 116. (17) Frew, J. E.; Hill, H. A. O. Anal. Chem. 1987, 59, 933A. (18) Tatsuma, T.; Okawa, Y.; Watanabe, T. Anal. Chem. 1989, 61, 2352-2355. (19) Kulys, J.; Schmid, R. D. Biosens. Bioelectron. 1991, 6, 43-48. (20) Vijayakumar, A. R.; Cso ¨regi, E.; Heller, A.; Gorton, L. Anal. Chim. Acta 1996, 327, 223-234. (21) Gorton, L.; Marko-Varga, G.; Persson, B.; Huan, Z.; Linde´n, H.; Burestedt, E.; Ghobadi, H.; Smolander, M.; Sahni, S.; Skotheim, T. Adv. Mol. Cell Biol. 1996, 15B, 421-450. (22) Ohara, T. J.; Vreek, M. S.; Battaglini, F.; Heller, A. Electroanalysis 1993, 5, 825-831. (23) Buttler, T.; Gorton, L.; Jarskog, H.; Marko-Varga, G. Biotechnol. Bioeng. 1994, 44, 322-328. (24) Marko-Varga, G.; Johansson, K.; Gorton, L. J. Chromatogr. 1994, 660, 153167. (25) Gu ¨ lce, H.; Gu ¨ lce, A.; Kavanoz, M.; Coskun, H.; Yildiz, A. Biosens. Bioelectron. 2002, 17, 517-522.

In particular, self-assembled monolayers (SAMs) of alkanethiols on metal substrates have been extensively investigated as a method to fabricate functional electrodes.26-33 For example, Willner et al. have attached covalently several kinds of enzymes or biological cofactors to a short alkanethiol SAM on gold electrodes, resulting in the successful development of amperometric sensors.34-37 Efficient electrical communication between redox enzymes and electrodes has also been achieved by the reconstitution of apoenzymes on electron-relay-cofactor monolayerfunctionalized electrodes.38,39 In the present study, we fabricated a biosensor containing three components, AOD, POD, and a ferrocenyl group, as an electron mediator, all immobilized onto a Au electrode surface by a selfassembly technique (Figure 1). The unique feature of the architecture is a self-assembled “mixed” monolayer, dithiodibutyric acid and ferrocenylalkanethiol. The carboxyl groups of the former are used for anchoring two enzymes (AOD and POD), and the latter is used as an electron relay molecule. Use of a ferrocenylalkanethiol SAM as an electron relay has been attempted for fabrication of a glucose sensor with GOD immobilized on the surface of SAM.40 To our knowledge, however, fabrication of a (26) Troughton, E. B.; Bain, C. D.; Whitesides, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter, M. D. Langmuir 1988, 4. (27) Turyan, I.; Mandler, D. Anal. Chem. 1994, 66, 58-63. (28) Bard, A. J.; Abrun ˜a, H. D.; Chidesy, C. E.; Faulkner, L. R.; Feldberg, S. W.; Itaya, K.; Majda, M.; Merloy, O.; Murray, R. W.; Porter, M. D.; Soriaga, M.; White, H. S. J. Phys. Chem. 1993, 97, 7. (29) Uosaki, K.; Kondo, T.; Zhang, X.-Q.; Yanagida, M. J. Am. Chem. Soc. 1997, 119, 8367-8368. (30) Bilewicz, R.; Majda, M. J. Am. Chem. Soc. 1991, 113, 5464-5466. (31) Rojas, M. T.; Ko ¨niger, R.; Stoddart, J. F.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336-343. (32) van Velzen, E. U. T.; Engbersen, J. F. J.; Reinhoudt, D. N. J. Am. Chem. Soc. 1994, 116, 3597-3598. (33) Hickman, J. J.; Ofer, D.; Laibinis, P. E.; Whitesides, G. M.; Wrighton, M. S. Science 1991, 252, 688-691. (34) Willner, I.; Katz, E.; Rilkin, A.; Kasher, R. J. Am. Chem. Soc. 1992, 114, 10965-10966. (35) Patolsky, F.; Zayats, M.; Katz, E.; Willner, I. Anal. Chem. 1999, 71, 31713180. (36) Willner, I.; Rilkin, A.; Shoham, B.; Rivenzon, D.; Katz, E. Adv. Mater. 1993, 13, 912-915. (37) Alfonta, L.; Katz, E.; Willner, I. Anal. Chem. 2000, 72, 927-935. (38) Zayats, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 2002, 124, 14724-14735. (39) Willner, I.; Heleg-Shabtai, V.; Blonder, R.; Katz, E.; Tao, G.; Bu ¨ ckmann, A. F.; Heller, A. J. Am. Chem. Soc. 1996, 118, 10321-10322. (40) Rubin, S.; Chow, J. T.; Ferraris, J. P.; Zawodzinski, T. A. Langmuir 1996, 12, 363-370.

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Figure 2. Schematic drawing of the microsensor on a 96-well polypropyrene assay plate. The 2-electrode system was composed with AOD/POD/11-FUT/Au sheet as the working electrode (WE) and coiled Ag/AgCl wire as the counter electrode (CE), respectively.

sensor electrode, on which mediator-SAM and two enzymes are independently and covalently attached in an identical plane, has not yet been reported. It was, therefore, an important subject in this work to elucidate the optimal composition of three species for obtaining high sensitivity through the reaction system, including H2O2 species dissolved in solution. The preparation of functional electrodes by a self-assembly technique has a great advantage in that all steps, including monolayer formation and the attachment of functional molecules, can be performed by only repeatedly immersing the electrode surface. This allows desired molecules to be layered onto electrodes having any kind of shape with any sizes. By utilizing this advantage, we successfully fabricated a miniature methanol sensor, which fitted into a well of an ordinary 96-well micro assay plate. As will be shown, such a sensing method made it possible to perform real-time quantification of methanol in crude plant extracts with accuracy comparable to GC/MS. EXPERIMENTAL SECTION Reagents. Alcohol oxidase (AOD) from Hansenula sp. (EC 1.1.3.13, 7.7 units/mg), peroxidase (POD) type VI from horseradish (EC 1.11.1.7, 250 units/mg), pectin methylesterase (PME) from orange peel (EC 3.1.1.11, 700 units/mg), and citrus pectin (59% esterified) were purchased from Sigma Chemical (St. Louis, MO). 4,4′-Dithiodibutyric acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 6-ferrocenyl-1-hexanethiol (6-FHT), and 11-ferrocenyl-1-uncedanethiol (11-FUT) were obtained from Dojindo Laboratories(Kumamoto, Japan). N-Hydroxysuccinimide was obtained from Nacalai Tesque (Kyoto, Japan). Hydrogen peroxide, ethanol, 1-propanol, 2-propanol, 1-butanol, and benzyl alcohol were obtained from Wako Pure Chemicals (Osaka, Japan). Methanol used as a standard was purified by distillation. Water was purified with a Millipore Milli-Q-System. All other reagents were of analytical grade and were used as received. Methanol-d3 was obtained from Kanto Chemicals (Tokyo, Japan) Preparation of Enzyme Electrode. A Au disk electrode of 3.0 mm φ (BAS, Tokyo, Japan) was polished with alumina slurries of 1.0 and 0.3 µm, subjected to ultrasonication in distilled water for 10 min, and rinsed thoroughly with distilled water. A Au sheet (0.2 mm thick, Nilaco, Tokyo, Japan) was cut to 6 mm × 18 mm, bent into a hoop (Figure 2), and used as an electrode substrate for enzyme immobilization. The Au sheet was cleaned with piranha 1502

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solution41 and rinsed with water. Note: Piranha solution is a strong oxidant and must be used with extreme caution! Both the Au electrode and the Au sheet were immersed in ethanol containing 0.1 µM of ferrocenylalkanethiol compound and 10 µM dithiodibutyric acid for 30 min to deposit a mixed self-assembled monolayer, followed by rinsing with ethanol, activating with 16 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 13 mM N-hydroxysuccinimide in 90% dimethyl sulfoxide for 3 min and rinsing well with distilled water. The resulting electrodes were immersed in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.12 mg/mL AOD and 0.08 mg/mL POD for 30 min at 4 °C, followed by rinsing with 0.1 M sodium phosphate buffer (pH7.0) to remove weakly adsorbed species. The enzymemodified electrode was stored at 4 °C until use and is denoted here as AOD/POD/6-FHT/Au or AOD/POD/11-FUT/Au. Electrochemical Measurements. Amperometric responses of the enzyme-modified electrode to substrate (methanol or H2O2) were measured by polarizing the electrode at 200 mV vs Ag/AgCl with a Bioanalytical Systems BAS-100B/W in 0.1 M sodium phosphate buffer (pH 6.0) at 25 °C unless otherwise stated. A onecompartment electrochemical cell equipped with a platinum foil counter electrode and an Ag/AgCl reference electrode was used. When constant background currents were obtained, substrate dissolved in water was added to the electrolyte solution so as to give the desired concentration, followed by agitation for 5 s with a magnetic stirrer. After use, the composite electrode was rinsed in 0.1 M sodium phosphate buffer (pH 7.0) before being stored at 4 °C. In the amperometric measurements with the two-electrode system, one well (φ 6.4 mm) of a polypropylene 96-well assay plate (Corning, Catalogue No. 3364) was used as an electrolysis cell containing enzyme-modified gold sheet and coiled Ag/AgCl wire as working and counter electrodes, respectively (Figure 2). In this case, an aliquot of 5 mM methanol was used as a standard solution, and agitation was performed for 5 s with a micro plate mixer, NS-P (Iuchi, Osaka, Japan). Preparation of Plant Extracts. Leaf samples of tobacco (Nicotiana tabacum L. cv. Petit Habana SR1) plants were frozen in liquid nitrogen and ground to a fine powder. The homogenized samples were stirred for 2 h at room temperature in 0.1 M sodium phosphate buffer (pH 7.0) and then centrifuged at 15000g for 20 min. The supernatant was retained as crude plant extracts. The extract samples were stored at -20 °C until use. Methanol Calibration with GC/MS. Concentration of methanol in the tobacco extracts was measured with methanol-d3 as an internal standard. Plant samples containing 4 mM methanol-d3 were subjected to GC/MS analysis, which was carried out using a QP-5000 (Shimadzu, Kyoto, Japan), fitted with a CP-PoraBOND Q fused-silica PLOT column (25 m × 0.25-mm i.d., df ) 3 mm film thickness; Varian, Inc.) in the splitless mode, and the splitter was subsequently opened. The column head pressure was 50 kPa, the injector port temperature was 250 °C, and the initial column temperature was 80 °C. This temperature was maintained for 2 min and then increased to 180 °C at a constant rate of 30 °C /min. Mass spectrometry was performed using a QP-5000 mass selective detector (Shimadzu). The electron multiplier was set at 1.8 kV. The peak ratio of the ion abundance between m/z 32 (generated (41) Hayes, W. A.; Shannon, C. Langmuir 1996, 12, 3688-3694.

Figure 3. Comparison of the current response of the different mixed monolayer. Preparation of self-assembled monolayer was carried out in ethanol containing 10 µM dithiodibutyric acid and 0.005-8 µM ferrocenylalkanethiol. Activated carboxyl groups were used to immobilize peroxidase. Current response to 0.4 mM H2O2 was measured in 0.1 M sodium phosphate (pH 7.0). Operating potential, 200 mV.

from methanol) and m/z 35 (from methanol-d3) was determined by selective ion detection. Determination of PME Activity. After adding orange peel PME into 0.1 M sodium phosphate buffer (pH 7.0) containing 50 mg/mL pectin, amperometric responses of the AOD/POD/11FUT/Au electrode were measured with a three-electrode system as described above. RESULTS AND DISCUSSION Preparation of Mixed Monolayer. An electrode on which ferrocenylalkanethiol and POD were immobilized was prepared to test performance of ferrocenyl groups in SAM as an electron mediator. Two kinds of ferrocenylalkanethiols, 6-ferrocenyl-1hexanethiol (6-FHT) and 11-ferrocenyl-1-undecanethiol (11-FUT), were used to examine the influence of the methylene chain length on electronic communication between an enzyme and an electrode. The immersion bath for preparing the mixed monolayer was an ethanolic solution containing 10 µM 4,4′-dithiodibutyric acid and either 6-FHT or 11-FUT. The concentration of the ferrocenylalkanethiols was varied to evaluate the optimal molar ratio between the electron mediator and the enzyme anchor molecules. The carboxyl groups derived from dithiodibutyric acid were activated with water-soluble carbodiimide and N-hydroxysuccinimide to immobilize peroxidase via amido bonds. The current responses were then taken by addition of hydrogen peroxide to 0.1 M sodium phosphate electrolyte buffer (pH 7.0). The measurements conducted under different electrode potentials revealed that the same reduction currents were obtained when the electrode potentials more negative than 250 mV vs Ag/AgCl were chosen. Figure 3 shows plots of current responses as a function of concentration of 6-FHT or 11-FUT in the immersion bath. Significant currents exhibiting largest values at around 0.1 µM were observed for both electrodes, indicating that both 6-FHT and 11-FUT worked definitely as an electron mediator for POD and their amounts in SAM influenced the current responses, as expected. However, contrary to our expectation, two ferrocenylalkanethiols having different chain lengths gave similar magnitude of response currents. As already shown in Figure 1, the sizes of 6-FHT and 11-FUT are ∼11 and ∼17 Å, respectively, whereas the POD molecule has an ellipsoid shape (∼10 × 50 Å). Since 3-carboxy-1-propanethiol (3-CPT) derived from 4,4′-dithiodibutyric

acid has an ∼6 Å length, ferrocenyl groups of both 6-FHT and 11-FUT should reach to the POD bound to the terminals of 3-CPT. If electron exchanges between ferrocenyl groups and electrode substrate were considered, 6-FHT would be favorable. However, ferrocenyl groups of 11-FUT should have a higher capability to approach the active center, which is buried in the POD molecule. Such a desired situation for ferrocenyl groups of both thiols might provide comparable effects on the response currents. Furthermore, it seems likely that the solvent and double layer effects on the redox reaction of ferrocenyl-SAM observed, especially for 6-FHT, were small in the present cases.42 If these were true, the optimal length would exist between 6-FHT and 11-FUT. Since the two electrodes prepared exhibited sufficient sensitivities, nevertheless, as shown in Figure 3, we chose an immersion bath containing 0.1 µM 11-FUT and 10 µM dithiodibutyric acid for preparing the mixed monolayer in further works. Immobilization of Alcohol Oxidase and Peroxidase on the Electrode Surface. The enzymes used in this study were commercial AOD from Hansenula sp. and POD from Horseradish, which have been well-characterized.43,44 The active form of AOD is an octamer, and each subunit has a molecular weight of ∼74 000. As the molecular model shown in Figure 1, the architecture of AOD is deduced to be cubic with every octameric subunit making the same contact with four neighbors in one plane.45 POD is expected to form an ellipsoid structure having a weight of ∼40 000.46 After activating the carboxylates in the mixed monolayer as described above, the electrode was immersed in mixed AOD/POD solution for 30 min. Surface plasmon resonance (SPR) analysis revealed that immobilization of both AOD and POD to the activated electrode occurred at a relatively fast rate, and 30 min of the immobilization time was long enough to obtain the electrode coated completely with these enzymes. A total amount of AOD and POD in the immersion bath was fixed at 0.2 mg/mL, whereas the weight ratio of AOD to the total amount of the two enzymes was varied from 0 to 1. Current responses to 4 mM methanol are given in Table 1. Apparently, the electrodes possessing both AOD and POD gave current responses, whereas lack of one enzyme decreased currents to null, evidencing that the successive reactions 1-4 given in the introductory section took place certainly on the prepared electrodes. The current responses exhibited a peak at around 0.6 (w/w) of the AOD ratio, which corresponded to a 0.67 weight ratio of POD to AOD. Considering molecular weight of the AOD octamer (∼590 000), the number of POD in the bath was deduced to be ∼9.9 times as large as that of the AOD octamer. If it is assumed that the POD/AOD ratio on the resulting electrode is roughly equal to that in the immersion bath, an arrangement that an AOD octamer is surrounded with POD molecules can be speculated as the two-dimensional structure of the electrode surface. It seems likely that such an arrangement is favorable for the effective capture of H2O2 released from AOD by POD. For subsequent works, the AOD ratio to total amount was fixed to be 0.6 in the immersion bath. (42) Creager, S. E.; Rowe, G. K. J. Electroanal. Chem. 1997, 420, 291-299. (43) van der Klei, I. J.; Bystrykh, L. V.; Harder, W. Methods Enzymol. 1990, 188, 420-427. (44) Smith, A. T.; Santama, N.; Dacey, S.; Edwards, M.; Bray, R. C.; Thorneley, R. N.; Burke, J. F. J. Biol. Chem. 1990, 265, 13335-13343. (45) Vonck, J.; van Bruggen, E. F. Biochim. Biophys. Acta 1990, 1038, 74-79. (46) Gajhede, M.; Schuller, D. J.; Henriksen, A.; Smith, A. T.; Poulos, T. L. Nat. Struct. Biol. 1997, 4, 1032-1038.

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Table 1. Comparison of the Current Responses of the Different Modification with Enzymea ratio of AOD/(AOD + POD)

change in current [nA]

0 0.4 0.5 0.6 0.7 0.8 0.95 1

0 -5.84 -4.09 -12.47 -9.21 -2.13 -1.18 0

a After the self-assembled mixed monolayer was preparated with 10 µM dithiodibutyric acid and 0.1 µM 11-FUT, the electrode was immersed in 0.2 mg/mL total protein containing different weight ratio of AOD/(AOD + POD). Current response for 4 mM methanol was measured in 0.1 M sodium phosphate (pH 7.0). Operating potential, 200 mV vs Ag/AgCl.

Figure 6. Calibration curve of AOD/POD/11-FUT/Au electrode for methanol in 0.1 M sodium phosphate buffer (pH 6.0) at 30 °C. Operating potential, 200 mV vs Ag/AgCl. Table 2. Substrate Selectivity of Biosensora substrate

relative response (%)

methanol ethanol 1-propanol 2-propanol 1-butanol benzyl alcohol

100.0 8.9 3.4