Anal. Chem. 1997, 69, 1669-1673

An Enzymatic Clay-Modified Electrode for Aerobic Glucose Monitoring with Dopamine as Mediator Jyh-Myng Zen,* Chin-Wen Lo, and Ping-Jyh Chen

Department of Chemistry, National Chung-Hsing University, Taichung, Taiwan 402, ROC

Considering the unique adsorption and regeneration properties of dopamine, glucose oxidase, and nontronite, a glucose sensor was constructed by immobilizing glucose oxidase and nontronite clay coating on a glassy carbon electode with dopamine as mediator. The response of the glucose sensor was determined by measuring the cyclic voltammetric oxidation peak current values of dopamine under aerobic solution conditions. The effects of the amount of enzyme immobilized, the operating pH, and the common interferences on the response of the glucose sensor were studied. The detection limit was 7.4 µM (S/N ) 3), with a linear range extending to about 10 mM, giving a dynamic range of about 3 orders of magnitude for 0.8 mM dopamine. When stored in pH 7 phosphate buffer at 4 °C, the sensor shows almost no change in performance after operating for 45 days. Electrochemical sensors made of oxidase enzymes are mostly based on measuring current due to the electrochemical oxidation of the liberated hydrogen peroxide released by the reaction between oxidase enzyme and substrate in the presence of oxygen.1 Alternatively, many groups have sought electroactive species that can act as redox mediators and are able to regenerate the enzyme to its active form. Several previous studies showed that the redox mediators can be either in solution, immobilized onto the electrode surface, or incorporated into the structure of the enzyme or the electrode.1-8 Among these, it was demonstrated that the electrogenerated oxidized form of dopamine (DA) can react with glucose oxidase (GOx) in the presence of glucose to re-form GOx, as shown in Figure 1.8 Using the concept of hydrogen peroxide detection mentioned above, our previous study reported a novel glucose sensor (SWa1/MV-GS) constructed by immobilizing GOx between two nontronite clay coatings on a glassy carbon electrode (GCE) with methyl viologen (MV) as mediator.9 The sandwich configuration of the glucose sensor, as shown in Figure 2A, was proved to be very effective in the determination of glucose under aerobic conditions. As indicated in our previous study, part of the reason for the excellent glucose response using the SWa-1/MV-GS is the (1) Frew, J. F.; Hill, H. A. O. Anal. Chem. 1987, 59, 933A. (2) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1989, 111, 2357. (3) Coury, L. A.; Oliver, B. N.; Egekeze, J. O.; Sosnoff, C. S.; Brumfield, J. C.; Murray, R. W. Anal. Chem. 1990, 62, 452. (4) Bartlett, P. N.; Bradford, V. Q.; Whitaker, R. G. Talanta 1991, 38, 57. (5) Bartlett, P. N.; Daren, C. Analyst 1992, 117, 1287. (6) Bartlett, P. N.; Birkin, P. R. Anal. Chem. 1994, 66, 1552. (7) Garguilo, M. G.; Michael, A. C. Anal. Chem. 1994, 66, 2621. (8) Alfaro, I. M.; Pizarro, E. I.; Rodriguez, L.; Valdes, E. M. Bioelectrochem. Bioenerg. 1995, 38, 307. (9) Zen, J.-M.; Lo, C.-W. Anal. Chem. 1996, 68, 2635. S0003-2700(96)00830-X CCC: $14.00

© 1997 American Chemical Society

Figure 1. Proposed mechanism for the electrogenerated oxidized form of DA in the presence of glucose to re-form GOx and the initial electrochemical oxidation of DA.

Figure 2. Configurations for the (A) SWa-1/MV-GS and (B) SWa1/DA-GS.

strong adsorption of MV on the nontronite clay minerals.10 In reality, for MV (i.e., paraquat), the adsorption of bipyridylium cations by clay minerals is believed to be a major mechanism for its biological inactivation in the soil environment.11,12 Similarly, nontronite is also expected to be an excellent substrate for immobilizing DA to fabricate sensors by virtue of the amine group in DA. We report here another interesting nontronite-based glucose sensor based on the unique adsorption and regeneration properties of DA, GOx, and nontronite. The glucose sensor was constructed simply by coating GOx and nontronite clay onto GCE (10) Zen, J.-M.; Jeng, S.-H.; Chen, H.-J. J. Electroanal. Chem. 1996, 408, 157. (11) Theng, B. K. G. The Chemistry of Clay-Organic Reactions; Wiley: New York, 1974; Chapter 4. (12) Summers, L. A. The Bipyridinium Herbicides; Academic Press: New York, 1980; Chapter 6.

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with DA as mediator, as shown in Figure 2B. The DA centers present in the clay coatings can then act as redox mediators to regenerate the enzyme to its active form. In this paper, the preparation of the DA-incorporated, enzymatic clay-modifed glucose sensor, designated as SWa-1/DA-GS, and various factors influencing the determination of glucose are thoroughly investigated. Typical interferences encountered in the determination of glucose are discussed. Additionally, a comparison between the SWa-1/MV-GS and the SWa-1/DA-GS is also addressed. EXPERIMENTAL SECTION Chemicals and Reagents. Glucose oxidase (GOx, EC 1.1.3.4, 181 600 units/g, from Aspergillus niger), β-D-glucose, bovine serum albumin (BSA), and DA were bought from Sigma (St. Louis, MO). The interferents used, L-ascorbic acid, uric acid, cysteine, galactose, and oxalate, were also from Sigma. Glutaraldehyde (GA) was obtained from Merck. Standard clay minerals, kaolinite (KGa1), illite, bentonite, nontronite (SWa-1, ferruginous smectite), montmorillonite (SWy-1), and vermiculite (VTx-1), were brought from the Source Clay Minerals Repository (University of Missouri, Columbia, MO). All the other compounds (ACS-certified reagent grade) used in this work were prepared without further purification in doubly distilled, deionized water. Apparatus. Electrochemistry was performed on a Bioanalytical Systems (BAS, West Lafayette, IN) CV-50W electrochemical analyzer. A BAS Model VC-2 electrochemical cell was employed in these experiments. The three-electrode system consisted of a SWa-1/DA-GS, a Ag/AgCl reference electrode (Model RE-5, BAS), and a platinum wire auxiliary electrode. The supporting electrolyte was 0.1 M, pH 7 phosphate buffer solution in most cases. Electrode Preparation. The GCE (3 mm diameter, BAS) was polished with a polishing kit (BAS), and clay colloids were prepared in the sodium form, generally according to the procedures previously described.10 Enzymatic clay films were prepared by dropping 6 µL of a clay colloid (0.5 g/L) containing a suitable amount of GOx enzyme coating solution onto a clean GCE and drying under ambient conditions, usually ∼0.5-1 h. Uniform films could be cast reproducibly from clays. A typical GOx enzyme casting solution was prepared by dissolving 20 mg GOx, 15 mg of BSA, and 100 µL of 5% GA in sequence in 1 mL of 0.1 M, pH 7 phosphate buffer solution. General Procedure. All solutions were used directly for detection under open air at room temperature. The measurements can be done in two ways: First, a suitable amount of DA was added to the measuring system directly. Second, the steadystate background current of DA was measured first, and the enzyme electrode was then switched to a glucose solution for detection. Note that the measuring systems also contain the same concentration of DA to prevent leaching problem. Overall, as long as the steady state was reached, virtually the same results were obtained in either way. For convenience, the experiments were done in the first way. The glucose response of the electrode was measured as the oxidation peak current value from cyclic voltammograms (CVs) taken between -0.2 and +0.9 V vs Ag/AgCl at a scan rate of 50 mV/s. The glucose sensor was kept in 0.1 M, pH 7 phosphate buffer solution at 4 °C when not in use. RESULTS AND DISCUSSION Electrochemical Characterization of the SWa-1/DA-GS. In our previous study, a Nafion/clay-modified electrode was 1670 Analytical Chemistry, Vol. 69, No. 8, April 15, 1997

Table 1. Steady-State Oxidation Peak Currents Obtained from CVs for DAa Incorporated into the Six Different Clay-Modified Electrodes and Catalytic Oxidation Peak Currents of Glucoseb by Sensors Made of the Six Clays

GCE SWa-1 KGa-1 illite bentonite SWy-1 VTx-1

DA (ip/µA)

glucose (ip/µA)

-12.5 -17.4 -14.6 -14.5 -14.4 -14.1 -15.0

-32.7 -48.0 -42.9 -37.9 -37.9 -40.6 -38.0

a [DA] ) 0.8 mM in pH 7 phosphate buffer solution at a scan rate of 50 mV/s. b [DA] ) 0.8 mM and [glucose] ) 5 mM in pH 7 phosphate buffer solution at a scan rate of 50 mV/s.

Figure 3. Typical CVs for the SWa-1/DA-GS in 0.8 mM DA and 0.1 M, pH 7 phosphate buffer solution with (A) and without (B) the presence of 5 mM glucose under aerobic condition. Scan rate, 50 mV/s.

developed for the determination of paraquat (i.e., MV) by squarewave cathodic stripping voltammetry.13 Six representative clays were used to fabricate the paraquat sensor, and the clay that showed the best performance is nontronite. Part of the reason for the excellent response of the paraquat sensor is the strong adsorption of paraquat onto the nontronite clay minerals. We expect that a similar advantage should exist between nontronite and DA, and the electrochemical behavior of DA incorporated into the six different clay-coated GCEs was, therefore, first studied. The steady-state oxidation peak currents obtained from CVs for DA incorporated into the six different clay-modified electrodes are summarized in Table 1. As can be seen, all the clay-modified electrodes obtained a higher steady-state oxidation peak current than that with a bare GCE. Similar to the results obtained with the paraquat sensors,13 the SWa-1/GCE shows the highest steadystate oxidation current for DA among the clays tested. These results indicate good adsorption ability of DA onto the nontronite clay, which is, therefore, chosen for immobilizing DA to fabricate the glucose sensor. (13) Zen, J.-M.; Jeng, S.-H.; Chen, H.-J. Anal. Chem. 1996, 68, 498.

Figure 4. (A) Typical LSVs for the SWa-1/GCE in (a) 0.1 M, pH 7 phosphate buffer solution, (b) 0.8 mM catechol, and (c) 0.8 mM DA. (B) Typical LSVs for (a) SWa-1/catechol-GS and (b) SWa-1/DA-GS in the presence of 5 mM glucose under aerobic condition with 0.8 mM catechol and DA, respectively. (C) Typical CVs for the SWa-1/DA-GS in the presence of 5 mM glucose under (a) deaerated, (b) aerobic, and (c) oxygensaturated conditions with 0.8 mM DA. Scan rate, 50 mV/s.

The SWa-1/DA-GS is constructed by coating GOx and nontronite clay onto the GCE with DA as mediator. DA centers present in the clay coatings are expected to act as redox mediators to regenerate the enzyme to its active form. Catalytic oxidation of glucose by the proposed sensor was subsequently studied. Figure 3 shows the typical CVs for the SWa-1/DA-GS with (A) and without (B) the presence of glucose under aerobic condition. As can be seen, the oxidation peak current of DA increases in the presence of glucose, which suggests the proper function of the SWa-1/DA-GS. Meanwhile, the catalytic oxidations of glucose by sensors made of the six clays were studied for comparison, and the results obtained are also shown in Table 1. Again, the SWa-1/DA-GS shows the highest catalytic current and further verifies its appropriate operation. Finally, since the active form of GOx is regenerated through oxidation by the oxidized form of DA (i.e., quinone), as shown in Figure 1, the following experiments were designated to further elucidate the behavior of DA on the SWa-1 clay. Figure 4A shows the results obtained for both DA and catechol incorporated into the SWa-1/GCE under the same experimental conditions. As can be seen, the oxidation peak current for DA is much higher than that for catechol. Meanwhile, a similar advantage was also observed for the catalytic oxidation of glucose by the SWa-1/DAGS as for the SWa-1/catachol-GS, as shown in Figure 4B. These results suggest two important points. First, the adsorption ability onto the SWa-1 clay apparently has something to do with the amine group of DA. Second, in the presence of GOx/glucose, a large increase in the oxidation current is produced, which indicates that the reaction between the oxidized form of the DA (quinone) and the reduced form of the GOx predominates the oxidation reaction. Finally, as can be seen in Figure 4C, the active form of GOx can be regenerated either through oxidation by the oxidized form

of DA or simply by oxygen. The competition between DA and oxygen to regenerate the enzyme was clearly demonstrated by the fact that the higher the partial pressure of oxygen, the lower the catalytic oxidation peak of DA observed. In other words, an even lower detection limit can be achieved by deaeration, while it is more convenient to operate the glucose sensor under aerobic solution conditions. The results further confirmed the proper function of DA in regenerating the active form of GOx. Optimization of the SWa-1/DA-GS. The effect of experimental conditions, such as the amount of enzyme immobilized, the operating pH, and the mediator concentration, were investigated to optimize testing performance. Figure 5A shows the reult of the optimization of GOx loading in the SWa-1/DA-GS. The response increases with increasing enzyme concentration in the range of 0-5 mg/mL and starts to decrease when the enzyme concentration is higher than 5 mg/mL. An enzyme concentration of 5 mg/mL was, therefore, selected in subsequent study. Figure 5B shows the effect of solution pH on the CV response at the SWa-1/DA-GS. Similar to the phenomenon observed with the SWa-1/MV-GS,10 the best response for the SWa-1/DA-GS is at pH 7. A buffer solution of pH 7 was, therefore, used in subsequent study. Finally, the effect of DA concentration, ranging from 0.5 to 0.9 mM, on the electrode behavior for 5 mM glucose is illustrated in Figure 5C. The optimum mediator concentration was found to be around 0.8 mM for 5 mM glucose. When the mediator concentration was lower than 0.8 mM, the enzymatic reaction apparently was more than DA can mediate. On the other hand, at higher mediator concentration, the large background signal renders the measurement difficult. Analytical Characterizations of the SWa-1/DA-GS. Calibration data were obtained with the optimum experimental parameters mentioned above under aerobic conditions. Figure 6 presents CVs and a calibration plot for the glucose sensor with Analytical Chemistry, Vol. 69, No. 8, April 15, 1997

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Figure 5. Effects of (A) enzyme loading, (B) pH, and (C) DA concentration on the response to 5 mM glucose at the SWa-1/DA-GS. Data were obtained with a background subtraction of the signal without the addition of glucose.

Figure 6. CVs and calibration plot in 0.8 mM DA with (a) 0, (b) 2, (c) 4, (d) 6, (e) 8, and (f) 10 mM glucose at the SWa-1/DA-GS. Other conditions as in Figure 3.

glucose concentrations of 0 (a), 2 (b), 4 (c), 6 (d), 8 (e), and 10 mM (f), respectively. In all cases, a catalytic oxidation response of DA was observed. The observed oxidation peak currents were then used for the construction of the calibration plot. For 0.8 mM DA, the plot shows a very linear behavior with slope (µA/mM), intercept (µA), and correlation coefficient of 4.22, 13.61, and 0.9988, respectively. The detection limit was 7.4 µM (S/N ) 3), with a linear range extending to about 10 mM, giving a dynamic range of about 3 orders of magnitude. The sensitivity started to decrease when the concentration of glucose was higher than 10 mM. This concentration interval allows application of employed sensor in medical practice (for analyzing blood and serum with lower glucose concentration) as well as in the food industry (for analyzing products with higher glucose concentration). Figure 7 shows typical traces of the steady-state current-time response of the SWa-1/DA-GS and the SWa-1/MV-GS in the detection of 5 mM glucose under respective optimum conditions. As can be seen, the time required to reach 95% of the maximum response of the SWa-1/DA-GS (<10 s) is relatively faster than that of the the SWa-1/MV-GS (∼60 s). The results are expected, since the response time of the SWa-1/MV-GS relies on the detection of hydrogen peroxide produced by the enzymatic reaction. However, the sandwich configuration of the SWa-1/MVGS apparently can improve the sensitivity and selectivity but at the expense of response time. Overall, the response time of the SWa-1/DA-GS is largely improved due to a different mechanism, which has nothing to do with the generation and diffusion of the liberated hydrogen peroxide in the detection of glucose. 1672 Analytical Chemistry, Vol. 69, No. 8, April 15, 1997

Figure 7. Dynamic response to 5 mM glucose at (A) SWa-1/ DA-GS and (B) SWa-1/MV-GS. Data were obtained with CV every 10 s. Experimental conditions: (A) 0.8 mM DA in 0.1 M, pH 7 phosphate buffer solution with a scan rate of 50 mV/s; (B) 0.1 mM MV in 0.1 M, pH 7 phosphate buffer solution with a scan rate of 100 mV/s.

To characterize the reproducibility of the SWa-1/DA-GS, repetitive measurement cycles were carried out. After the voltammogram was recorded in 5 mM glucose and 0.8 mM DA solution, the electrode was then switched to a solution containing 0.8 mM DA to double-check the reproducibility of the glucose sensor before the next measurement. As shown in Figure 8A, the results of 10 successive measurements showed relative standard deviations of 1.2% and 3.2% for glucose and DA, respectively. Thus, the SWa-1/DA-GS shows a good, reproducible surface and can be used in repetitive measurements. When the enzyme electrode was stored in a 0.1 M, pH 7 phosphate buffer solution at 4 °C, the long-term stability was examined by measuring the response to various concentrations of glucose for 60 days. Note that the SWa-1/DA-GS was more frequently used in the first month, and sometimes it was continuously used for more than 10 h every day. In the second month, the electrode was used almost only for the purpose of the lifetime test and was operated for only several minutes. As can be seen in Figure 8B, the activity remains virtually constant during the first 45 days, indicating excellent long-term stability of the SWa1/DA-GS. The results of measurements during these 45 days showed a relative standard deviation of 1.2%. The result is not as good as that of the SWa-1/MV-GS, which can be stable for at least 60 days under similar experimental conditions. Obviously, the long-term stability of the GOx enzyme has something to do with the outer clay coating of the sandwich configuration of the SWa1/MV-GS.

Figure 8. (A) Ten repetitive measurement cycles at the SWa-1/DA-GS in (a) 5 mM glucose and 0.8 mM DA and (b) 0.8 mM DA. (B) Sensor stability over a 60 day period. Scan rate, 50 mV/s.

Finally, various substances were examined to determine their interference in the determination of 1 mM glucose with the SWa1/DA-GS under aerobic conditions. The reults showed that ascorbic acid (0.2 mM), uric acid (0.1 mM), oxalate (0.5 mM), galactose (0.5 mM), and cysteine (0.2 mM) do not cause any observable interference in the determination of glucose. CONCLUSION The SWa-1/DA-GS was proved to be very effective in the determination of glucose under aerobic conditions. The nontronite clay film shows good inherent chemical and physical stability of electrochemical response to DA. The method of the enzyme immobilization assures a great stability and anti-interference ability of the SWa-1/DA-GS. Notably, there is little interference from ascorbic acid and uric acid, which are common components of

biological fluids. The SWa-1/DA-GS is more rapid in comparison with the SWa-1/MV-GS, and it can be applied for the determination of glucose in a large concentration interval. ACKNOWLEDGMENT The authors gratefully acknowlege financial support from the National Science Council of the Republic of China under Grant NCS 86-2113-M-005-021.

Received for review August 14, 1996. Accepted January 20, 1997.X AC960830O X

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

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