Amperometric Glucose-Sensing Electrodes with the Use of Modified

Chapter 4

Amperometric Glucose-Sensing Electrodes with the Use of Modified Enzymes Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 8, 2014 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0556.ch004

F. Mizutani, S. Yabuki, and T. Katsura National Institute of Bioscience and Human Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan

Two kinds of modified glucose oxidases (GOD's), a polyethylene glycol-modified GOD and a lipid-modified GOD were prepared and used in the construction of enzyme electrodes. The polymer- modified GOD and a mediator (ferrocene) were incorporated into carbon paste (CP) to prepare a ferrocene-mediated glucose-sensing electrode. The polymer-modified enzyme exhibited higher activity than native enzyme in the CP matrix, owing to the enhanced affinity toward the hydrophobic matrix resulting from the enzyme modification. The higher enzyme activity resulted in an enhanced electrode response to glucose. Another glucose-sensing electrode was prepared by using the water-insoluble, lipid-modified GOD: the modified enzyme was immobilized on the surface of a glassy carbon (GC) electrode with a thin Nafion coating. The lipid-modified GOD-based electrode showed high performance characteristics such as rapid response, high sensitivity and superior stability. The modification of enzyme by attaching ions or molecules is a suitable way for providing it with useful functions. For example, the use of modifiers such as polyethylene glycol (PEG) (7,2) and synthetic lipids (3) enhances the affinity for hydrophobic environments. PEG-modified enzymes are soluble and active in various organic solvents as well as aqueous solutions (1,2). Lipid-modified enzymes are insoluble in aqueous solutions, but show catalytic activities in both aqueous and organic media (3). These unique properties lead us to apply PEG- and lipid-modified enzymes in the construction of enzyme electrodes. In this article, we describe the preparation and use of amperometric electrodes for sensing the analytically significant substrate glucose based on a PEG-modified glucose oxidase (GOD) (4,5) and on a lipid-modified GOD (6). The PEG-modified GOD has been incorporated into a carbon paste (CP) electrode: the modified enzyme exhibits higher activity in a hydrophobic CP matrix than hydrophilic, native GOD (710). The lipid-modified GOD has been immobilized on a glassy carbon (GC) electrode with a thin Nafion overcoat. The water-insoluble modified enzyme is far more stable between the electrode surface and the polymer layer than native GOD (77).

0097-6156/94/0556-0041$08.00/0 © 1994 American Chemical Society

In Diagnostic Biosensor Polymers; Usmani, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

42

DIAGNOSTIC BIOSENSOR POLYMERS

Downloaded by UNIV OF CALIFORNIA SAN FRANCISCO on December 8, 2014 | http://pubs.acs.org Publication Date: May 5, 1994 | doi: 10.1021/bk-1994-0556.ch004

Experimental Reagents. The enzymes used were GOD (E.C. 1.1.3.4, from Aspergillus sp., Grade II, Toyobo) and peroxidase (E.C. 1.11.1.7, from horseradish, Grade I-C., Toyobo). Methoxypolyeâiylene glycol activated with cyanuric chloride (activated PEG; Mwt., 5,000) was obtained from Sigma Chemical, a lipid, iV-(atrimemylammomoacetyl)didodecyl-L-glutamate chloride, from Sogo Pharmaceutical, and Nafion [5% (w/v) solution, 1,100 equiv. wt.], from Aldrich Chemical. F-kit (Boehringer Mannheim) was used for the spectrophotometric measurement of glucose. The kit uses the enzyme pair hexokinase/glucose-6-phosphate dehydrogenase. Other reagents were of analytical reagent grade. Deionized doubly distilled water was used throughout. Modified GOD's. GOD modified with PEG was prepared as follows. The GOD, as received (native GOD, 20 mg), and activated PEG (200 mg) were dissolved into 3 ml of 0.1 M borate-KOH buffer (pH 10). The solution was then incubated at 37°C for 2 h, after which the reaction between GOD and activated PEG was stopped by adding acetic acid to the solution to pH 6. Unattached PEG was removed in an ultrafiltration cell (Millipore) using a dialysis membrane (PTTK membrane, Millipore; cut off molecular weight, 30,000) and 0.05 M (NH4)HC03 as a dialyzing solution. Finally, a light yellow-colored powder of PEG-modified GOD (ca. 30 mg) was obtained by lyophilization. GOD modified with the lipid was prepared according to the procedure of Okahata et al. (5). A buffer solution (5 ml, 0.02 M potassium acetate buffer plus 0.1 M KC1, pH 6) containing 25 mg GOD was mixed with an aqueous dispersion (50 ml) of 100 mg of the lipid. The precipitate formed after incubation of the mixture at 4°C for 24 h was lyophilized. A light yellow powder (ca. 70 mg) was obtained. The GOD content in these modified enzymes were determined by measuring the adsorption by flavin adenine dinucleotide in solutions of the lyophilized products (the solvents used were water and benzene for the PEG- and lipid-modified GOD, respectively) at 450 nm. The enzyme activities of the native and modified GOD's were measured by using a peroxid^se/phenoy4-ammoantipyrine chromogenic system. The solution (or dispersed medium in the case of the lipid-modified GOD) was 0.1 M potassium phosphate buffer (pH 7, 25°C). Enzyme Electrode Systems. Two kinds of glucose-sensing CP electrodes, CPE I and II, were prepared by using the PEG-modified and native GOD, respectively. The modified or native GOD, Ι,Γ-dimethylferrocene, and carbon paste (CP-O; Bioanalytical Systems), 1:1:8 by weight, were thoroughly mixed together. A portion of the mixture was placed in a hole (3 mm diameter, 4 mm diameter) at the end of the electrode body (model 11-2010, Bioanalytical Systems). Similarly two kinds of glucose-sensing GC electrodes, G C E I and Π were prepared by using the lipid-modified and native GOD, respectively. The GC disk electrodes used (diameter, 3 mm; model 11-2012, Bioanalytical Systems) were first activated according to the procedure of Wang and Tuzhi (72). After activation, a drop of benzene solution containing the lipid-modified GOD or of an aqueous solution (pH 7) containing native GOD was placed on the GC electrode surface, and each solvent was allowed to evaporate at room temperature. The surface density of each GOD on GC electrode was 0.7 mg cm"2. Finally, a Nafion membrane coating was made by dip-coating each electrode in 0.5% (w/v) Nafion solution, which was prepared by diluting the 5% solution as received with a mixture of 2-propanol [50% (v/v)] and water (13), and the electrode was allowed to dry with the surface facing down at room temperature for 1 h. The thickness of each GOD/Nafion layer was several microns. A potentiostat (HA-502, Hokuto Denko) was used in a three-electrode configuration for amperometric measurements. The enzyme electrode thus prepared,



4. MIZUTANI ET AL.

Amperometric Glucose-Sensing Electrodes

43

an Ag/AgCl reference electrode, and a platinum auxiliary electrode were immersed in 10 ml of a test solution of 0.1 M potassium phosphate buffer solution (pH 7) in a cylindrical cell. The measurements were carried under argon atmosphere with CPE I and II and in air with G C E I and II. The solution was stirred with a magnetic bar, and the temperature of the solution was kept at 25°C. Further, a G C electrode coated with a lipid-modified GOD/Nafion layer was incorporated into an electrochemical flow detection system (Bioanalytical Systems) and the response to glucose was recorded. A flow-cell electrode (Model 11-1000, Bioanalytical Systems; electrode area, 0.14 cm^) was used as the base electrode, and a lipid-modified GOD layer and a Nafion overcoat were similarly prepared by the procedure described above. The sample volume used was 10 μΐ and the flow rate was 1 ml min"l. Glucose-Sensing Electrode based on PEG-Modified Enzyme Properties of PEG-modified GOD. The GOD content in the lyophilized powder of PEG-modified enzyme was determined to be 50%. The PEG-modified GOD was soluble in organic solvents, such as benzene and hexane, as well as in aqueous media. The GOD activities of the modified- and native enzymes were 15 and 105 m U mg"l, respectively. This indicates that the enzyme activity is considerably reduced by the modification process. On the other hand, when the PEG-modified GOD was doped into CP, it exhibited a much higher activity than the native enzyme. The GOD activities on the surfaces of CPE I and II were ca. 0.1 and 0.02 U cm"2, respectively. The higher activity of modified GOD in the CP matrix is attributable to its enhanced affinity for the hydrophobic matrix, which can be proven by the solubility of the modified enzyme in organic solvents such as hexane. The PEG-modified GOD is highly dispersed in the CP matrix, and the modifier protects GOD from denaturation by the oil contained in CP. Glucose Response of Enzyme Electrode. The increase in the GOD activity in CP by modifying the enzyme with PEG resulted in enhanced response toward glucose on CPE I, compared to CPE II. Figure 1 shows the current responses to 5 m M glucose on CPE I and II. The electrode potential, 0.4 V vs. Ag/AgCl, was sufficient for the oxidation of Ι,Γ-dimethylferrocene to ferricinium ion, and hence for obtaining the ferrocene-mediated current response for glucose (14): Glucose + GODox -> gluconolactone + GODred GODred + 2FcR+ GODox + 2FcR + 2H+ 2FcR-> 2FcR+ + 2e" (at the electrode) In this scheme, GODox and GODred represent the oxidized and reduced forms of GOD, respectively, and FcR /FcR, the ferricinium ion/ferrocene couple. As shown in Figure 1, the glucose response on CPE I was about five times as large as that on CPE II. CPE I gave a linear current response up to 10 mM, and a significant increase in the current was still observed with glucose concentrations between 10-50 mM. The detection limit was 0.1 m M (signal-to-noise ratio = 5). The relative standard deviation for ten successive measurements of 5 m M glucose on CPE I was ca. 2%. The effect of storage (in the test solution at 4°C) of CPE I and II were then examined. On each electrode, the response to glucose gradually decreased and become ca. 60% of the initial value after 2 weeks. Long-term stability was not improved by modification of the enzyme. The decrease in the electrode response was caused by leaching of the (modified or native) enzyme out of the CP matrix, since the solution used for storing CPE I or II showed significant GOD activity and the current response was not reduced when the electrode surface was covered with a dialysis membrane (Viscase Seals). +


44



Glucose-Sensing Electrode based on Lipid-Modified Enzyme Properties of Lipid-Modified GOD. The G O D content in the lyophilized product of lipid-modified GOD was calculated to be 33%. The modified enzyme was soluble in nonpolar organic solvents, such as benzene and chloroform. The GOD activity of the modified enzyme was 38 U mg"^-solid, which corresponds to 115 U mg"l-GOD. This shows that the enzyme activity does not decrease during the modification process and that the enzyme substrates, glucose and oxygen permeate easily into the lipid layer on the GOD molecule to reach active site. After immersing GCE I and Π in the test solution for 1 h, the GOD activities on the electrode surfaces were measured. GCE I exhibited a GOD activity of ca. 0.2 U cm~2, whereas GCE II exhibited an activity lower than 1 m U cm"2. In the case of CPE II, the native GOD placed on the electrode surface easily leaked through the Nafion overcoat, and only a small amount of the enzyme remained immobilized. In contrast, the water-insoluble, lipid- modified GOD was stable between the electrode surface and the polymer overcoat which permitted the much higher enzyme activity. Glucose Response of Enzyme Electrode. Figure 2 shows current-time curves for GCE I and II. The potential of each electrode was set at 0.9 V vs. Ag/AgCl. The current on each electrode increased immediately after the addition of glucose and reached a steady state within a few seconds. As the Nafion layer is thin, the added glucose is expected to diffuse quickly through the layer, and is available to be oxidized by the GOD reaction: GOD Glucose + O 2 —*· gluconolactone + H 2 O 2

The hydrogen peroxide produced near the electrode surface is immediately oxidized to give an anodic current response. As shown in Figure 2, the glucose response on GCE I was far larger than that on G C E Π. The much higher activity on the electrode surface with G C E I is responsible for the larger glucose response. The relative standard deviation for ten successive measurements of 0.2 m M glucose on GCE I was 1.3 %. GCE I gave a linear current response up to 3 m M , and a significant increase in the current was observed with glucose concentration in the range of 3-10 mM. The detection limit was as low as 0.2 u M (signal-to-noise ratio, 5). The effect of storage (in the test solution at 4°C) of CPE I was then examined for 3 weeks: the response to glucose did not decrease during this period. Flow Injection Measurement of Glucose. The G C E coated with a lipidmodified GOD/Nafion layer thus showed high performance characteristics such as rapid response, high sensitivity, and high stability. These characteristics are particularly promising for the use of the electrode in flow injection measuring systems. Therefore we examined the analytical potential of the modified GOD/Nafion-coated GC electrode by measuring glucose in beverages. Figure 3 shows typical responses by the electrode. The peak current was proportional to the glucose concentration up to 10 m M , under the present exrjerimental conditions, and the detection limit was 10 μΜ. Table I gives the results for the determination of glucose in beverages. Each sample was diluted with 0.1 M potassium phosphate buffer (pH 7) by a factor of 50 before use. The results were compared with those given by the F-kit method. The agreement was excellent; the regression equation between the results obtained by the present electrode method (x) and those by the F-kit method (y) was y = 0.993x + 0.572 for the seven samples given in Table I. The long-term stability of the electrode was examined by measuring 10 m M glucose 30 times a day each day for 12 weeks. The average value of the electrode response for the 30 measurements did not decrease for 10 weeks. Such an accurate and stable measuring system is suitable for the use in a flow arrangement. In Diagnostic Biosensor Polymers; Usmani, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


4. MIZUTANI ET AL·

Amperometric Glucose-Sensing Electrodes

0

30 Time/s

60

Figure 1. Response/time curves for (A) CPE I and (Β) Π to 5 m M glucose.

1-5 < M-0

Ξ0-5 t

10 20 Time/s

30

Figure 2. Response/time curves for (A) G C E I and (Β) II obtained on increasing the glucose concentration in 0.5 m M steps.

Time

Figure 3. Recorded calibration and sample peaks for determination of glucose in beverages. 1, 2, 5 and 10, glucose concentrations of standard solutions given in m M ; A and B , sample solutions (samples 1 [orange juice] and 2 [cola] in Table I, respectively). Each of the beverage was diluted by a factor of 50 with 0.1 M phosphate buffer [pH 7]). In Diagnostic Biosensor Polymers; Usmani, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.

45

46


Table I. Comparison of results obtained for glucose in beverages by different methods Sample no.

Glucose concentration (mM)


proposed method (x) 1 2 3 4 5 6 7

138 279 138 250 291 142 146

F-kit method (y) 141 276 137 253 288 141 142

Conclusion Modification of GOD with PEG was effective for enhancing the affinity of the enzyme for a hydrophobic CP matrix. A water-insoluble, lipid-modified GOD could be immobilized on the GC electrode surface far more tightly than the native enzyme. These modifiers produced high performance characteristics for GOD-based electrodes. We have prepared and tested other modified enzymes. These include a cholinesensing CP electrode based on a PEG-modified choline oxidase, which exhibits a wide dynamic range (75), and a G C electrode, using a lipid-modified lactate oxidase (16), which shows a fast response to L-lactate. Literature Cited (1)

Inada, Y.; Nishikawa, H.; Takahashi, K.; Yoshimoto, T.; Saha, A. R.; Saito, Y. Biochem. Biophys. Res. Commun. 1984, 122, 845-850. (2) Takahashi, K.; Ajima, Α.; Yoshimoto, T.; Okada. M.; Matsushima, M.; Tamura, Y.; Inada, Y. J. Org. Chem. 1985, 50, 3414-3415. (3) Okahata, Y.; Tsuruta, T.; Ijiro, K.; Ariga, K. Thin Solid Films, 1989, 180, 6572. (4) Mizutani, F.; Yabuki, S.; Katsura, T. Bull. Chem. Soc. Jpn. 1991, 64, 28492851. (5) Yabuki, S.; Mizutani, F.; Katsura, T. Biosens. Bioelectron., 1992, 7, 695-700. (6) Mizutani, F.; Yabuki, S.; Katsura, T. Anal. Chim. Acta, 1993, 274, 201- 207. (7) Matsuszewski, W.; Trojanowicz, M.; Analyst, 1988, 113, 735-738. (8) Gorton, L.; Karan, H. I.; Hale, P. D.; Inagaki, T.; Okamoto, Y.; Skotheim, T. A. Anal. Chim. Acta, 1990, 228, 23-30. (9) Wang, J.; Wu, L.-H.; Lu, Ζ.; Li, R.; Sanchez, J. Anal. Chim. Acta, 1990, 228, 251-257. (10) Amine, Α.; Kauffmann, J.-M.; Patriarche, G. J. Talanta, 1991, 38, 107- 110. (11) Wang, J.; Leech, D.; Ozsoz, M.; Martines, S.; Smyth, M. R. Anal. Chim. Acta, 1991, 245, 139-143. (12) Wang, J.; Tuzhi, P. Anal. Chem. 1986, 58, 1787-1790. (13) Harrison, D. J.; Turner, R. F. B.; Baltes, H. P. Anal. Chem. 1988, 60, 20022007. (14) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. O.; Aston, W. J.; Higgins, I. J.; Protokin, Ε. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984, 56, 667-671. (15) Yabuki, S.; Mizutani, F.; Katsura, T., unpublished work. (16) Mizutani, F.; Yabuki, S.; Katsura, T., Denki Kagaku, in press. RECEIVED September 14, 1993


Amperometric Glucose-Sensing Electrodes with the Use of Modified

Recommend Documents