Layer-by-Layer Construction of Enzyme Multilayers on an Electrode

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Anal. Chem. 1998, 70, 811-817

Technical Notes

Layer-by-Layer Construction of Enzyme Multilayers on an Electrode for the Preparation of Glucose and Lactate Sensors: Elimination of Ascorbate Interference by Means of an Ascorbate Oxidase Multilayer Jun-ichi Anzai,* Hiroki Takeshita, Yuka Kobayashi, Tetsuo Osa, and Tomonori Hoshi†

Faculty of Pharmaceutical Sciences, Tohoku University, Aramaki, Aoba-ku, Sendai 980-8578, Japan

A layer-by-layer structure of enzyme multilayers composed of glucose oxidase (GOx) or lactate oxidase (LOx) and ascorbate oxidase (AOx) was prepared on the surface of a platinum electrode. The amperometric response to glucose or lactate was studied in the presence of ascorbic acid as a possible interference. An alternating and repeated deposition of avidin and the biotin-labeled enzymes resulted in the layer-by-layer structure of GOx/ AOx and LOx/AOx multilayers. Optical and gravimetric measurements based on an ultraviolet-visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of monomolecular layers of the proteins. The GOx/AOx and LOx/AOx enzyme multilayers were useful to eliminate ascorbic acid interference in the glucose and lactate biosensors, because ascorbic acid can be converted to an electrochemically inert form, dehydroascorbic acid, before being oxidized directly on the Pt electrode. Thus, the GOx/AOx or LOx/AOx multilayer-modified biosensors can be used to determine the normal blood level of glucose (5 mM) and lactate (1 mM) in the presence of a physiological level of ascorbic acid (0.1 mM). The effects of the number of the AOx layers and geometry of the enzyme layers in the multilayer on the performance characteristics of the biosensors are discussed.

enzyme sensors, glucose sensors have been studied most extensively because of their usefulness in diagnostic analysis of diabetes. The glucose sensors usually detect an oxidation current originating from the electrolysis of hydrogen peroxide (H2O2), which, in turn, is produced enzymatically according to the following reaction:

The development of high-performance enzyme sensors has been a focal subject in analytical science and technology.1-3 Most enzyme sensors rely on the combination of an electrode and enzymes which catalyze reactions that consume or produce electrochemically active substances. Among the electrochemical

(4) Sasso, S. V.; Pierce, R. J.; Walla, R.; Yacynych, A. M. Anal. Chem. 1990, 62, 1111-1117. (5) Jung, S.-K.; Wilson, G. S. Anal. Chem. 1996, 68, 591-596. (6) Szentirmay, M. N.; Martin, C. R. Anal. Chem. 1984, 56, 1898-1902. (7) Wang, J.; Golden, T. Anal. Chem. 1989, 61, 1397-1400. (8) Yao, T. Anal. Chim. Acta 1983, 153, 175-180. (9) Nagy, G.; Rice, M. E.; Adams, R. N. Life Sci. 1982, 31, 2611-2616. (10) Wang, J.; Naser, N.; Ozsoz, M. Anal. Chim. Acta 1990, 234, 315-320. (11) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1988, 110, 2615-2620. (12) Liaudet, E.; Battaglini, F.; Calvo, E. J. J. Electroanal. Chem. 1990, 293, 55-68. (13) Nagata, R.; Clark, S. A.; Yokoyama, K.; Tamiya, E.; Karube, I. Anal. Chim. Acta 1995, 304, 157-164. (14) Kajiya, Y.; Sugai, H.; Iwakura, C.; Yoneyama, H. Anal. Chem. 1991, 63, 49-54. (15) Heller, A. J. Phys. Chem. 1992, 96, 3579-3587.

* To whom correspondence should be addressed. Tel: +81-22-217-6841. Fax: +81-22-217-6840. E-mail: [email protected]. † Current address: Abiko Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Abiko 270-11, Japan. (1) Janata, J.; Josowicz, M.; DeVaney, D. M. Anal.Chem. 1994, 66, 207R228R. (2) Byfield, M. P.; Abuknesha, R. A. Biosens. Bioelectron. 1994, 9, 373-400. (3) Reach, G.; Wilson, G. S. Anal. Chem. 1992, 64, 381A-386A. S0003-2700(97)00536-2 CCC: $15.00 Published on Web 01/21/1998

© 1998 American Chemical Society

GOx

glucose + O2 98 δ-gluconolactone + H2O2

(1)

(GOx ) glucose oxidase) H2O2 f O2 + 2H+ + 2e-

(2)

The amperometric detection of H2O2 is often accompanied by interference arising from electrooxidizable substances such as ascorbic acid and uric acid existing inherently in biological fluids. To address this problem, several methods have been proposed, including the use of polymeric membranes which can eliminate the interference by size exclusion4,5 or electrostatic repulsion6,7 and the catalytic8 or enzymatic decomposition9,10 of the interference. Recently, another strategy has been proposed to circumvent this problem, based on the use of electron mediators which transport electrons from a reduced form of GOx to the electrode.11-15 The electron mediators employed so far include ferrocene,11-13 hydroquinone,14 osmium complex,15 etc. The

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Figure 1. Layer-by-layer construction of enzyme layers on the electrode surface using avidin and biotin-labeled enzyme.

mediator-type glucose sensors can be operated at a low electrode potential, where the interferences are not oxidized directly, resulting in interference-free glucose sensors. Although these devices are potentially useful for the determination of glucose in biological fluids, the construction of these devices includes a somewhat complicated procedure due to the co-immobilization of the enzyme and electron mediator on the same electrode surface. Therefore, the H2O2 detection mode still remains the simplest and most common version of glucose sensors. In this context, we have recently reported that an ascorbic acid interference in the H2O2 detection-type glucose sensors can be eliminated by covering the GOx layer of the glucose sensors with a thin membrane of ascorbate oxidase (AOx).16 In the GOx/AOx multilayer-modified glucose sensors, ascorbic acid can be oxidized by AOx into dehydroascorbic acid, which is no longer electroactive, in the GOx/AOx multilayer before being electrooxidized directly on the electrode surface. Thus, it was possible to eliminate the interference arising from a physiological level of ascorbic acid in blood (∼0.1 mM). The GOx/AOx multilayermodified glucose sensors were fabricated on the basis of a layerby-layer deposition of the enzymes using avidin and biotin-labeled GOx and AOx, by taking advantage of the strong affinity of avidin for biotin.17-20 Alternating and repeated deposition of avidin and biotin-labeled enzymes produced a multilayer structure composed of monomolecular layers of protein, as schematically shown in Figure 1, because avidin contained four biotin-binding sites per molecule and the enzymes used were tagged with several residues of biotin. Based on this technique, the required type and amount of enzymes can be assembled arbitrarily in a thin layer on the electrode surface. The present paper describes the preparation of a layer-by-layer structure of enzyme multilayers composed of GOx or LOx and AOx on the surface of an electrode and their amperometric response to glucose or lactate in the presence of ascorbic acid as a possible interference. The possible use of the GOx/AOx and LOx/AOx multilayers for the elimination of ascorbic acid interference of the enzyme sensors is discussed in detail. EXPERIMENTAL SECTION Reagents. GOx (EC 1.1.3.4, from Aspergillus niger; Sigma, St. Louis, MO), LOx (EC 1.1.3.2, from Pediococcus species; Boehringer, Mannheim, Germany), and AOx (EC 1.10.3.3, from Cucurbita species; Oriental Yiest Co., Tokyo, Japan) were used (16) (17) (18) (19)

Chen, Q.; Anzai, J.; Osa, T. Denki Kagaku 1995, 63, 1141-1142. Green, N. M. Biochem. J. 1996, 101, 774-780. Wilchek, M.; Bayer, E. A. Anal. Biochem. 1988, 171, 1-32. Wilchek, M., Bayer, E. A., Eds. Method in Enzymology; Academic Press: San Diego, CA, 1990; Vol 184. (20) Leckband, D. E.; Schmitt, F.-J.; Israelachvili, J. N.; Knoll, W. Biochemistry 1994, 33, 4611-4624.

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as received. Avidin was obtained from Calzyme Lab. (San Luis Obispo, CA). Fluorescein-5-isothiocyanate (FITC)-conjugated avidin (nominally 5.5 FITC residues/molecule) was purchased from Molecular Probes (Eugene, OR). Sodium sulfosuccinimidyl6-(biotinamido)hexanoate (NHS-biotin) was obtained from Vector Laboratories (Burlingame, CA). Dulbecco’s phosphate-buffered saline (PBS, pH 7.4) was used to prepare enzyme and avidin solutions. The PBS was prepared by dissolving 8.0 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, and 1.15 g of Na2HPO4 in 1000 mL of distilled water. All other reagents were of the highest grade available and were used without further purification. Biotin-labeled enzymes were prepared according to the reported procedure:21 8.1 of mg GOx and 0.9 mg of NHS-biotin were dissolved in 0.1 M NaHCO3 (2 mL), and the solution was stirred for 3 h at 20 °C. The unreacted NHS-biotin was removed from the reaction mixture by centrifugal filtration. In a similar manner, LOx and AOx were reacted with ∼10 wt % of NHS-biotin to prepare biotin-labeled LOx and AOx. In these reactions, enzymes should be labeled with several residues of biotin per molecule because the enzymes were always reacted with an excess amount of NHSbiotin (the mole ratios of NHS-biotin/enzyme in the reaction were more than 10). Apparatus. Absorption spectra were measured using a Shimadzu UV-3100PC spectrophotometer. A quartz crystal microbalance (QCM) QCA917 system (Seiko EG & G) was employed for the gravimetric analysis of the deposition of AOx. A 9-MHz AT-cut quartz resonator coated with a thin platinum layer was used (effective surface area, 0.2 cm2). A potentiostat equipped with a function generator (Nikko Keisoku NPGF-2501A) was used for the electrochemical measurements. The working electrode used was a Teflon-supported Pt disk of 3.0 mm diameter. A Ag/ AgCl electrode with a liquid junction of 3.3 M KCl, saturated with AgCl, and a Pt wire were used as the reference and auxiliary electrodes, respectively. Spectrophotometric Measurement of AOx Multilayer. The surface of a quartz slide (5 cm × 1 cm × 0.5 mm) was first cleaned by treatment with a mixture of sulfuric acid and chromic acid. The clean surface of the quartz slide was modified with dichlorodimethylsilane (5% solution in toluene) for 24 h at room temperature and washed with toluene, acetone, and distilled water. By this treatment, the surface of the quartz slide should be hydrophobic. This silylated quartz slide was immersed in an FITC-avidin solution of 100 µg mL-1 for 1 h to deposit the first layer of FITC-avidin. After being rinsed with PBS for a few minutes, the quartz slide was immersed in a 100 µg mL-1 biotinlabeled AOx for 1 h and rinsed with PBS. This process provides both sides of the quartz slide with a FITC-avidin/biotin-labeled AOx layer. The deposition of FITC-avidin and biotin-labeled AOx was repeated, and the absorbance at 495 nm was recorded after every deposition. Gravimetric Measurement with QCM. The Pt surface of the quartz resonator was washed thoroughly with distilled water before use. The quartz resonator was mounted in a Teflon cell and immersed in the working buffer (PBS, 30 mL). After the resonance frequency had reached a steady-state value under gentle stirring, a concentrated stock solution of avidin or biotin-labeled enzyme was added to the buffer (final concentration, 100 µg mL-1). (21) Hoshi, T.; Anzai, J.; Osa, T. Anal. Chem. 1995, 67, 770-775.

The shift in the resonance frequency was monitored for 30-40 min at 20 °C. Preparation of Enzyme Sensors. The surface of the Pt disk electrode was polished thoroughly with alumina powder and rinsed with distilled water before use. The Pt electrode was immersed in an avidin solution (100 µg mL-1 PBS) for 20 min at room temperature to deposit the first avidin layer on the electrode surface and washed with PBS for 10 min to remove any weakly adsorbed avidin. The avidin-modified electrode was then immersed in a biotin-labeled enzyme solution (100 µg mL-1) for 20 min to immobilize the enzyme on the avidin-modified electrode through avidin/biotin complexation. To deposit the second layer of avidin and enzyme, the enzyme-modified electrode was treated similarly with the avidin and enzyme solutions. The same procedure was repeated to further deposit enzyme layers. The procedure for constructing the enzyme layer is shown schematically in Figure 1. Electrochemical Measurements of the Sensors. The electrochemical response of the enzyme sensors was measured with a conventional three-electrode system at 0.6 V vs Ag/AgCl. The GOx or LOx catalyzes the oxidation reaction of glucose or lactate, respectively, to produce H2O2, and the H2O2 can be oxidized at the Pt surface at this potential. A 0.1 M phosphate buffer (pH 6.8) was used for the electrochemical measurements. The apparent Michaelis-Menten constant (Kmapp) and maximum current (Imax) of the glucose sensor to glucose were determined using the following equation:

I ) ImaxC/(Kmapp + C)

(3)

where the current I is a measure of the rate of the enzymatic reaction, and C denotes the concentration of glucose. To verify the stability of the GOx/AOx and LOx/AOx multilayer sensors, the output current of the sensors to 1 mM glucose and 1 mM lactate, respectively, was measured once a day, and the sensors were stored in the buffer at 4 °C when not in use. RESULTS AND DISCUSSION Formation of Enzyme Multilayers. We have already reported that an enzyme multilayer can be constructed on the surfaces of a quartz plate and a Pt electrode by an alternate deposition of avidin and biotin-labeled enzymes.21-24 The enzyme multilayers thus prepared are characterized by a layer-by-layer structure composed of a monomolecular layer of avidin and enzyme (Figure 1), as characterized by UV spectrophotometry, QCM, and electrochemistry.22 Based on this technique, we have prepared enzyme sensors sensitive to glucose,21 lactate,23 alcohol,24 and acetyl choline.25 The enzyme loading of the sensors can be controlled arbitrarily by regulating the deposition number, resulting in stepwise and precise control of the size of the output current of the sensors thus prepared. These results prompted us to prepare bienzyme layers composed of monolayers of GOx or LOx (22) Anzai, J.; Kobayashi, Y.; Suzuki, Y.; Du, X.-Y.; Takeshita, H.; Chen, Q.; Hoshi, T.; Osa, T. Submitted. (23) Anzai, J.; Takeshita, H.; Hoshi, T.; Osa, T. Chem. Pharm. Bull. 1995, 43, 520-522. (24) Du, X.-Y.; Anzai, J.; Osa, T.; Motohashi, R. Electroanalysis 1996, 8, 813816. (25) Anzai, J.; Takeshita, H.; Chen, Q. Electroanalysis, in press.

Figure 2. Layer-by-layer deposition of FITC-avidin and biotinlabeled AOx (a) and native AOx (b) on a quartz slide, monitored by absorbance at 495 nm.

and AOx for the elimination of ascorbic acid interference in the glucose and lactate sensors. The preparation and characterization of GOx and LOx multilayer-modified sensors were reported previously.21-23 Prior to the construction of GOx/AOx and LOx/AOx bienzyme multilayermodified sensors, whether an AOx multilayer can be formed was examined by UV spectrophotometry. Using FITC-avidin and biotin-labeled AOx, an AOx multilayer was deposited on a quartz slide, and the absorbance at 495 nm, originating from the FITC moiety in avidin, was monitored after each deposition (Figure 2). The absorbance increased in proportion to the number of layers deposited, suggesting the formation of an AOx multilayer on the quartz slide. Using a molar extinction coefficient of 176 000 M-1 cm-1 at 495 nm for the FITC-avidin used and assuming that, upon each deposition, FITC-avidin forms a close-packed monomolecular layer, the density of FITC-avidin in each layer is calculated to be (6.3 ( 1.3) × 10-12 mol cm-2, depending on the orientation of the FITC-avidin molecule (the molecular dimensions of avidin are 6.0 nm × 5.5 nm × 4.0 nm).17-20 The experimental results in Figure 2 show that the density of the FITC-avidin in each layer is ∼7.0 × 10-12 mol cm-2. Therefore, these data suggest that FITC-avidin is immobilized in each layer as a nearly monomolecular layer. On the other hand, the increase in absorbance was negligibly small when native AOx containing no biotin residue was used in place of the biotin-labeled AOx. To verify the loading of AOx in the multilayer, a gravimetric measurement was carried out using QCM. For this purpose, the surface of a Pt-coated quartz resonator was first modified with a monolayer of avidin, and the change in resonance frequency (∆F) of the avidin-modified quartz resonator upon adsorption of biotinlabeled AOx was measured. The resonance frequency decreased upon addition of biotin-labeled AOx into the working buffer and reached a steady-state value in ∼4 min, showing that the biotinlabeled AOx was adsorbed onto the surface of the avidin-modified quartz resonator. The ∆F value was -80 Hz, which corresponds to 440 ng cm-2 of mass increase on the quartz resonator because the adsorption of 1 ng of substance induces a -0.91-Hz change in the 9-MHz QCM device used.26 If biotin-labeled AOx forms a close-packed monomolecular layer on the surface of the quartz resonator, the mass increase should be 520 ( 155 ng cm-2, depending on the orientation of the enzyme (the molecular (26) Muramatsu, H.; Suda, M.; Ataka, T.; Seki, A.; Tamiya, E.; Karube, I. Sens. Actuators 1990, A21pA23, 362-368.

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Figure 3. Calibration curves for the glucose sensors bearing GOx (10 layers) (O and b) and GOx/AOx (10 GOx layers plus 10 AOx outer layers) multilayers (4 and 2). O and 4, in the absence of ascorbic acid; b and 2, in the presence of 0.1 mM ascorbic acid.

dimensions of AOx are 6.5 nm × 5.3 nm × 9.8 m, and the molecular weight is ∼140 000).27 Consequently, the biotin-labeled AOx adsorbed on the quartz resonator is regarded as nearly a monomolecular layer. On the other hand, the ∆F value was less than -5 Hz when biotin-free native AOx was used instead of the biotin-labeled AOx, confirming the avidin-biotin interaction as a driving force of the multilayer formation. Thus, it is concluded that biotin-labeled AOx can be assembled into a layer-by-layer structure on the electrode surface through avidin-biotin complexation, as in the cases of GOx and LOx. GOx/AOx Multilayer-Modified Glucose Sensors. Avidin and biotin-labeled GOx were deposited alternately on a Pt electrode to fabricate a GOx multilayer-modified glucose sensor. In a similar manner, avidin and biotin-labeled AOx were deposited further on the surface of the GOx multilayer-modified sensor to prepare a GOx/AOx multilayer (10 GOx layers plus 10 AOx layers)-modified glucose sensor. The amperometric response of these glucose sensors to glucose was evaluated. Figure 3 illustrates calibration graphs of the responses of the sensors to glucose over the concentration range of 1 × 10-4-3 × 10-2 M in the presence and absence of a physiological level (0.1 mM) of ascorbic acid. In the absence of ascorbic acid, both sensors showed useful calibration responses to glucose in this concentration range, which covers both normal and diabetic blood levels of glucose (∼5-20 mM).3 It should be noted that coating of the AOx multilayer on the GOx layer of the sensor did not significantly reduce the output current of the sensor. About a 10% decrease in the output current was observed for the GOx/AOx multilayer sensor compared with that for the GOx multilayer sensor. These results suggest that the enzyme multilayers are so thin that they do not significantly influence the mass transfer of analyte and reaction products of the enzymatic reaction. The thickness of the GOx and AOx layers is calculated to be ∼10-15 nm per avidin/ enzyme unit layer, considering the molecular dimensions of avidin and enzymes.17,27-29 This view is further supported by the fact that the response time of the sensors remains virtually unchanged before and after coating of the AOx multilayer. Both sensors (27) Messerschmidt, A.; Ladenstein, R.; Huber, R.; Bolognesi, M.; Avigliano, L.; Petruzzelli, R.; Rossi, A.; Finazzi-Agro, A. J. Mol. Biol. 1992, 224, 179205. (28) Hecht, H. J.; Kalisz, H. M.; Hendle, J.; Schmid, R. D.; Schomburg, D. J. Mol. Biol. 1993, 229, 153-172. (29) Hecht, H. J.; Schomburg, D.; Kalisz, H.; Schmid, R. D. Biosens. Bioelectron. 1993, 8, 197-203.

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Figure 4. Response of the GOx/AOx multilayer sensor to 0.3 mM ascorbic acid in the presence and absence of dissolved oxygen.

responded rapidly to glucose and reached a steady-state current in ∼10 s or faster. Thus, it is clear that coating of the AOx multilayer does not have any undesirable influence on the response characteristics of the glucose sensor. A H2O2 detection-type glucose sensor often suffers from interference arising from the direct oxidation of redox-active substances such as ascorbic acid in blood sample. In fact, as illustrated in Figure 3, the calibration graph of the GOx multilayer (10 layers) sensor deviated significantly in the presence of 0.1 mM of ascorbic acid from that obtained in the absence of ascorbic acid. Consequently, the interference has to be eliminated in order for the glucose sensor to be used for accurate determination of glucose. We may be able to eliminate the interference originating from ascorbic acid using the GOx/AOx multilayer-modified glucose sensors because ascorbic acid can be oxidized into the redox-inactive form, dehydroascorbic acid, by AOx in the GOx/ AOx multilayer, according to the following reaction: AOx

ascorbic acid + 1/2O2 98 dehydroascorbic acid + H2O (4) To test this possibility, the electrochemical response of the GOx/ AOx multilayer-modified sensor to glucose and ascorbic acid was measured, and it was found that no interference was induced with the GOx/AOx multilayer sensor upon addition of a physiological level (0.1 mM) of ascorbic acid into a solution of 5 mM glucose. It is likely that ascorbic acid was oxidized to dehydroascorbic acid by AOx in the multilayer before being oxidized directly on the Pt surface. The calibration graph of the GOx/AOx multilayer sensor to glucose in the presence of 0.1 mM ascorbic acid was shown in Figure 3. The calibration graphs of the GOx/AOx multilayer sensor in the presence and absence of ascorbic acid are practically identical with each other (or deviation is 3% or less) over the glucose concentration range of ∼1-10 mM. Thus, the GOx/AOx multilayer sensor can be used for the determination of glucose in the samples containing a physiological level of ascorbic acid. To further ascertain that AOx retains its catalytic activity and oxidizes ascorbic acid in the multilayer, the amperometric response of the GOx/AOx multilayer sensor to ascorbic acid was measured in the presence and absence of dissolved oxygen (Figure 4). The GOx/AOx multilayer sensor exhibited ∼0.5 µA interfering current in response to 0.3 mM ascorbic acid in the absence of glucose, and the current was highly enhanced when dissolved oxygen was removed by introducing nitrogen (N2) gas through the sample solution. This is because the AOx-catalyzed

Table 1. Effect of the Number of AOx Outer Layers on the Response Characteristics of the Glucose Sensorsa no. of AOx layers

interference/%b

Imax/µA cm-2

Kmapp/mM

0 1 5 10

20 3 2 0

37 36 37 35

17.2 17.6 17.9 17.7

a GOx, 10 layers. b (Interfering current from 0.1 mM ascorbic acid)/ (response current to 5 mM glucose) × 100.

oxidation reaction (eq 4) is inhibited by lack of oxygen upon N2 bubbling, resulting in an enhanced effective concentration of ascorbic acid on the surface of the Pt electrode. Further bubbling with oxygen gas reversibly restored the original sensor current. These observations clearly show that AOx is catalytically active in the GOx/AOx multilayer and oxidizes ascorbic acid to eliminate the interference. Another plausible mechanism by which the interference was eliminated is that ascorbic acid may be excluded to some extent from the enzyme multilayer by electrostatic repulsion between the negative charges of ascorbate and AOx (both ascorbic acid and AOx are probably negatively charged at neutral pH due to their acidic nature; pKa of ascorbic acid ) 4.17, pI of AOx ) 6.0-7.8).30,31 In fact, these two mechanisms seem to contribute concurrently to the elimination of the interference. It is interesting to reveal the effect of the number of AOx layers (or the loading of AOx) on the response of the glucose sensors to glucose and ascorbic acid. We emphasize that the loading of AOx on the electrode can be optimized easily and precisely on the basis of the layer-by-layer deposition of avidin and biotinlabeled enzyme by simply regulating the number of depositions because known amounts of enzymes are deposited in each layer. For this purpose, the GOx multilayer (10 layers)-modified sensor was further modified with a monolayer, 5-layer, and 10-layer AOx. The response characteristics of these sensors are summarized in Table 1. About 20% of interfering current was observed for the GOx multilayer (10 layers)-modified glucose sensor when 0.1 mM ascorbic acid was added to the sample solution containing 5 mM glucose. On the other hand, the interference was suppressed to a negligibly low level, 3% or 2%, by coating the sensor with a monolayer or 5-layer AOx, respectively. This shows that, from a practical point of view, a monolayer AOx suffices for the selective determination of glucose level by this sensor. The Kmapp value of the sensors to glucose did not depend on the number of AOx layers. The Kmapp values are nearly consistent with literature results for glucose sensors in which GOx is immobilized on the basis of a conventional technique.32 The Imax of the sensors remained almost unchanged irrespective of the number of the AOx layers coated, though a slight decrease in the Imax value was observed in the 10-layer AOx-modified sensor. These data indicate that the reaction kinetics of GOx-catalyzed reaction in the GOx/ AOx multilayer are little affected by the AOx layers. Thus, the interference in the glucose sensors arising from a physiological level of ascorbic acid (0.1 mM) in a 5 mM glucose solution can be eliminated using the AOx multilayer. We also examined the (30) Pournagli-Azar, M. H.; Ojini, R. Talanta 1995, 42, 1839-1848. (31) Nakamura, T.; Makino, M.; Ogura, Y. J. Biochem. 1968, 64, 189-195. (32) Castner, J. F.; Wingard, L. B. Biochemistry 1984, 23, 2203-2210.

Table 2. Ascorbic Acid Interference with the GOx-AOx Multilayer (10 GOx Layers Plus 10 AOx Outer Layers)-Modified Glucose Sensor concn of glucose/ MM

concn of ascorbic acid/ mM

interference/ %a

5 5 5 5 5 1 3 10 30

0.01 0.03 0.1 0.3 1.0 0.1 0.1 0.1 0.1

0 0 0 -3b -40b 0 0 -3b -7b

a (Interfering current from ascorbic acid)/(response current to the glucose solution) × 100. b The negative value means that the response current to glucose was decreased.

effect of different concentrations of ascorbic acid (Table 2). For the sensor modified with 10 GOx layers and then 10 AOx outer layers, no interference was induced by ascorbic acid at a concentration lower than 0.2 mM. However, the output current of the sensor decreased slightly (∼3%) upon addition of 0.3 mM ascorbic acid. The interference was significant (∼40%) when the final concentration of ascorbic acid was 1.0 mM. Table 2 also summarizes the effect of 0.1 mM ascorbic acid on the calibration graph of the sensor to glucose. Ascorbic acid (0.1 mM) causes virtually no interference in 1 and 5 mM glucose solutions, while the response to glucose was decreased by 3% and 7% in the presence of 0.1 mM ascorbic acid in 10 and 30 mM glucose solutions, respectively. Note that the output current did not increase but decreased in the presence of a higher concentration of ascorbic acid. This is probably because the GOx-catalyzed oxidation reaction (eq 1) was prohibited in part due to the depletion of dissolved oxygen in the GOx multilayer, which, in turn, was caused by the consumption of oxygen through the oxidation reaction of ascorbic acid by AOx (eq 4) in the multilayer. The H2O2 concentration on the surface of the Pt electrode thus should be decreased, resulting in the reduced current. It has been reported that the response of glucose sensors significantly depends on the oxygen concentration in the medium, especially when the glucose concentration is higher than ∼10 mM.5 Another possible mechanism for the interference may relate to a homogeneous reaction of ascorbic acid with H2O2, by which the enzymatically generated H2O2 may be partly consumed.33 It is known that the homogeneous interference of ascorbic acid cannot be neglected in some bioanalytical systems involving H2O2 detection.34,35 In our case, the former mechanism seems to be predominant in view of the fact the ascorbic acid interference can be suppressed appreciably in an oxygen-saturated medium (data not shown). A different arrangement of GOx and AOx layers in the multilayer may modify the response characteristics of the sensors. For this reason, in addition to the 10 GOx layers plus 10 AOx outer layers sensor [i.e., (GOx)10(AOx)10 sensor], we prepared (33) Lowry, J. P.; O’Neill, R. D. Anal. Chem. 1992, 64, 456-459. (34) Matuszewski, W.; Trojanowicz, M. Electroanalysis 1990, 2, 147-153. (35) Inamdar, K.; Raghavan, K. G.; Pradham, D. S. Clin. Chem. 1991, 37, 864868.

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Table 3. Response Characteristics of the Lactate Sensors Modified with Different Types of Enzyme Multilayers

type of sensor (LOx)10 (LOx)10(AOx)n

Figure 5. Calibration curves for the lactate sensors bearing LOx (10 layers) (O and b) and LOx/AOx (10 LOx layers plus 10 AOx outer layers) multilayers (4 and 2). O and 4, in the absence of ascorbic acid; b and 2, in the presence of 0.1 mM ascorbic acid.

two different types of enzyme multilayer sensors, in which 10 AOx layers were deposited at first on the electrode surface, and then 10 GOx outer layers were added [(AOx)10(GOx)10 sensor], or single layers of avidin/GOx and avidin/AOx were deposited alternately up to 10 layers [(GOx/AOx)10 sensor]. The amperometric response of these sensors to glucose was measured in the absence and presence of ascorbic acid. The three different types of sensors gave almost identical calibration graphs for their responses to glucose in the absence of ascorbic acid, suggesting that the catalytic activity of GOx remains unchanged, regardless of the geometry of the enzyme layers. Although we measured very carefully the response of the sensors to glucose in the presence of 0.01, 0.1, 0.2, and 0.3 mM ascorbic acid, a clear difference could not be found among the sensors. The calibration graphs of the sensors were virtually identical with each other within the experimental error. For all sensors, the detectable concentration range of glucose was ∼1-10 mM in the presence of 0.1 mM ascorbic acid. LOx/AOx Multilayer-Modified Lactate Sensors. LOx/AOx multilayer-modified lactate sensors were prepared in analogy with the glucose sensors. Because the lactate sensor detects amperometrically H2O2 produced enzymatically according to the following reaction, the issue of ascorbic acid interference has to be addressed similarly to the glucose sensor case: LOx

lactic acid + O2 98 pyruvic acid + H2O2

(5)

For this purpose, a LOx multilayer (10 layers)-modified sensor was prepared using avidin and biotin-labeled LOx, and the surface of the LOx multilayer sensor was further coated with an AOx monolayer, a 5-layer AOx, or a 10-layer AOx outer layer. The four types of lactate sensors thus prepared exhibited almost the same calibration to lactic acid ranging from 10-6 to 3 × 10-3 M in the absence of ascorbic acid. Figure 5 illustrates typical calibration graphs of the LOx multilayer (10 LOx layers) sensor and LOx/ AOx multilayer (10 LOx plus 10 AOx layers) sensors. In the absence of ascorbic acid, the AOx-free lactate sensor showed a 14.8-µA output current in response to 1 mM lactic acid (a physiological level in normal blood), while the output current was reduced slightly (∼10%) by covering the sensor with the 5-layer or 10-layer AOx, the output being 13.0 µA for both sensors. These results suggest that the AOx outer layer practically does not 816 Analytical Chemistry, Vol. 70, No. 4, February 15, 1998

(AOx)n(LOx)10

(LOx/AOx)10

no. of AOx layers

interference/ %a

0 n)1 n)5 n ) 10 n ) 10 n ) 10 n ) 10 n)1 n)5 n ) 10 n ) 10 n ) 10 n ) 10 10 10 10 10

16 -1b -3b -7b

concn of ascorbic acid/mM

detection limits/ mMc

0.01 0.1 0.2

0.01-3.0 0.1-0.5 0.2-0.3

0.01 0.1 0.2

0.01-3.0 0.1-2.0 0.3-2.0

0.01 0.1 0.2

0.01-3.0 0.1-3.0 0.3-1.0

1 -3b -4b

-1b

a (Interfering current from 0.1 mM ascorbic acid)/(response current to 1 mM lactate) × 100. b The negative value means that the response current to lactate was decreased. c The lactate concentration where the deviation induced by ascorbic acid is within (5%.

disturb the diffusion of lactate into the LOx layer. The response time of the sensors was 10 s or faster, irrespective of the number of the AOx layer, confirming the smooth transport of lactate across the AOx multilayers. Figure 5 contains also the calibration graphs of the sensors in the presence of 0.1 mM ascorbic acid. The AOxfree lactate sensor suffered from a bias current induced by ascorbic acid, which originates from the direct oxidation of ascorbic acid at the Pt electrode, suggesting that the AOx-free lactate sensor cannot be used for the determination of lactate. The LOx/AOx multilayer sensor, on the other hand, exhibited a useful calibration graph in the lactate concentration higher than 0.1 mM, even in the presence of ascorbic acid, in which the output current was nearly comparable to that obtained in the absence of ascorbic acid. To assess the performance characteristics of the lactate sensors in more detail, we prepared different types of sensors by varying the arrangement of the LOx and AOx layers in the multilayer. Thus, (LOx)10(AOx)n, (AOx)n(LOx)10, and (LOx/AOx)10 type multilayers, where n ) 1, 5, and 10, were constructed on the electrode surface, and the amperometric responses of the sensors were measured in the presence of 0.01-0.2 mM ascorbic acid (Table 3). The AOx-free sensor exhibited a ∼16% bias current to 0.1 mM ascorbic acid. In contrast to the positive interference to the AOx-free sensor, the AOx-containing multilayer-modified sensors exhibited a negative interference in which the output current was reduced slightly in the presence of 0.1 mM ascorbic acid. The ascorbate interference was always less than (5%, except for the (LOx)10(AOx)10 sensor. This shows that these sensors can be used for the determination of physiological levels of lactate in the presence of 0.1 mM ascorbic acid. That the negative interference is more significant in the 10-AOx-layer sensors than in the AOx monolayer and the 5 AOx-layer sensors suggests the depletion of oxygen in the enzyme layer as a major origin of the interference, as in the case for the GOx/AOx multilayer sensors.

This view is compatible with the observation that the negative interference in the sensors can be reduced appreciably by saturating the working buffer with oxygen. Table 3 also summarizes the detection limits of the lactate sensors in the presence of 0.01, 0.1, and 0.2 mM ascorbic acid. The detection limits depended significantly on the concentration of ascorbic acid. The higher detection limits are considered to be determined mainly by oxygen consumption by AOx and partly by homogeneous interference,34,35 while the direct oxidation determines the lower detectable range. From the viewpoint of practical use of the sensors for determining normal levels of lactate, the (LOx/AOx)10 sensor seems more useful than the others in view of its lower interference and wider detectable range. CONCLUSIONS We have demonstrated that the GOx/AOx and LOx/AOx enzyme multilayers can be used to eliminate ascorbic acid interference in the glucose and lactate biosensors. The enzyme multilayers were prepared by alternating and repeated deposition of avidin and biotin-labeled enzymes on the electrode surface. Optical and gravimetric measurements revealed that the enzymes are assembled into a layer-by-layer structure composed of their monomolecular layers. The GOx/AOx and LOx/AOx enzyme multilayer sensors were useful for determining the normal level of glucose (5 mM) and lactate (1 mM) in the presence of a physiological level of ascorbic acid (0.1 mM). Unfortunately, the catalytic activity of AOx is inherently rather unstable and, as a result, the long-term stability of the sensors is not satisfactory. The activity of AOx in the multilayers decreased considerably in ∼10 days; thereafter, ascorbic acid interference was observed in

the glucose and lactate sensors. A possible problem in the GOx/ AOx and LOx/AOx multilayer sensors is that the output current is sometimes decreased in the presence of higher concentrations of substrate and ascorbic acid due to the depletion of dissolved oxygen in the multilayer (see Table 2). This problem should be addressed further for use of the sensors under oxygen-limited conditions (i.e., in vivo measurements). It should be emphasized that the layer-by-layer deposition technique used in the present study is quite useful in optimizing the loading of enzymes on the electrode surface and in regulating the geometry of the enzyme layers. Additionally, the present technique is well suited for the construction of a multienzyme system using two or more different types of enzymes, where the total activity of each enzyme and their deposition sequence can be regulated precisely and easily. It should be emphasized that the present technique makes it possible to design and construct enzyme multilayers precisely at the molecular level without loss of the catalytic activity. ACKNOWLEDGMENT The present work was supported in part by Grants-in-Aid (Nos. 08458282, 09237210, 09878206, and 09558110) from the Ministry of Education, Science, Sports and Culture of Japan and by the Suzuken Memorial Foundation.

Received for review May 27, 1997. Accepted December 4, 1997. AC970536B

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