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mutarotase-attached measuring cell
Biosensors & Bioelectronics Vol. 12. No. 9–10, pp. 1013–1020, 1997 1997 Elsevier Science Limited All rights reserved. Printed in Great Britain 0956–5663/97/$17.00 PII: S0956–5663(97)00057-2
Rapid determination of glucose and sucrose by an amperometric glucosesensing electrode combined with an invertase/mutarotase-attached measuring cell Fumio Mizutani* & Soichi Yabuki National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan (Received 26 February 1997; revised version received 1 May 1997; accepted 2 May 1997)
Abstract: Glucose and sucrose are simultaneously determined by the use of an enzyme sensor system consisting of a glucose-sensing electrode based on a lipid-modified glucose oxidase and a measuring cell that contains an invertase/mutarotase-coimmobilized layer. From the current response of the enzyme electrode after the addition of a glucose/sucrose mixture, the concentrations of the two kinds of sugars can be separately determined: the concentration of glucose (0·2 M–3 mM) is determined from the steady-state current increase obtained from 2 to 6 s after the addition of the mixture, and that of sucrose (10 M–6 mM), from the rate of current increase from 8 to 20 s after the addition. The relative standard deviations are 1·7% for glucose and 3·1% for sucrose (n = 10). The system can be applied to the rapid determination of glucose and sucrose in food samples. 1997 Elsevier Science Limited Keywords: enzyme electrode, glucose, sucrose
INTRODUCTION Of the various methods available for the determination of saccharides in industrial food laboratories, the most attractive are those based on amperometric enzyme electrodes (Bilitewski, 1994; Matsumoto et al., 1988). The combination of specific enzymes with sensitive electrochemical signal transduction yields enzyme electrodes which provide simple, rapid and accurate determination of the enzyme substrate (Mizutani & Asai, 1990a; Scheller & Schubert, 1992). In many food production processes, the simultaneous determination of glucose and sucrose is *To whom correspondence should be addressed. Rapid determination of glucose and sucrose
necessary for effective process control. Glucose and sucrose in mixtures have been determined successfully by using two different enzyme electrodes equipped with a measuring cell (Pfeiffer et al., 1980; Xu et al., 1989). For example, two enzyme electrodes, one with a layer of glucose oxidase (GOx) and the other with a layer containing GOx, invertase and mutarotase, are immersed in a testing buffer solution in the cell; the first electrode is used to measure glucose and the second, to measure the sum of glucose and sucrose. The simultaneous determination of glucose and sucrose has also been carried out by using multi-channel flow injection systems (Matsumoto et al., 1988; Masoom & Townshend, 1985). On the other hand, methods using a single
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enzyme electrode have been proposed for achieving the measurement with more simple and inexpensive instruments (Scheller & Karsten, 1983; Mizutani & Asai, 1990b). Scheller & Karsten (1983) have carried out a successive determination of glucose and sucrose. Glucose in a sample solution was determined from the change in a GOx-based electrode, then an invertase layer was immersed in the solution to produce glucose from sucrose; the concentration of sucrose was determined by measuring the rate of enzymatic glucose production, i.e. the rate of current change. However, this method is laborious and time-consuming: the invertase layer must be immersed in the test solution for every measurement, and more than 3 min are required to complete the measurement. We proposed a more convenient method which used a glucose-sensing electrode combined with a measuring cell that contained immobilized invertase. The concentration of glucose, C, in the measuring cell at t s after the addition of a sample is approximately expressed as (Mizutani & Asai, 1990b): C = CG(t ⬍ td)
(1)
C = CG + aCs(t − td) (t ⱖ td)
(2)
where CG and CS are the initial concentrations of glucose and sucrose, respectively, a is a constant and td a time lag for the production of glucose at the immobilized invertase. When the response time of the glucose-sensing electrode is very short (i.e. much shorter than td), the electrode can generate the current output proportional to C (see Eqs (1) and (2)). That is, the steady-state current response obtained at t ⬍ td would be proportional to CG and the rate of current change at t ⬎ td to CS; the determination of the two components can easily be carried out by monitoring the current response after the addition of the sample. However, in our previous work (Mizutani & Asai, 1990b), a relatively long response time for the glucose-sensing electrode used (100% response time, about 1 min) complicated the analysis of the current response for determining the two components and, of course, it took a long time (about 2 min) to complete the determination. Recently, we have prepared a glucose-sensing enzyme electrode with a 100% response time of less than 2 s by modifying a base electrode with a lipid-attached GOx (Mizutani et al., 1993). Such an enzyme electrode is considered to be useful for the rapid and simple determination of glucose and sucrose. It has been shown that glucose (0·2 M–3 mM) and sucrose (10 M–
6 mM) can be determined within 20 s. The method is applied to the determination of the two components in food samples.
EXPERIMENTAL Materials The enzymes used were GOx (EC 1.1.3.4, from Aspergillus sp., 150 U/mg, Toyobo Osaka), invertase (EC 3.2.1.6, from Candida utilis, 130 U/mg, Toyobo) and mutarotase (EC 5.1.1.3, from porcine kidney, 3300 U/mg, Sigma, St Louis, MO). A lipid, N-(␣-trimethylammonioacetyl)didodecylL-glutamate chloride [(C12)2gluN+Cl−], was obtained from Sogo Phermaceutical (Tokyo) and Nafion (5%(w/v) solution, 1100 equiv. wt) from Aldrich (Milwaukee, WI). Photo-crosslinkable poly(vinyl alcohol) bearing stilbazolium groups (PVA-SbQ (Ichimura, 1984); aqueous solution (9%(w/v)), pH 7; degree of polymerization, 1700; molar ratio of stilbazolium group, 1·3%) was a gift from Toyo Gosei Kogyo (Chiba). An F-kit (Boehringer, Indianapolis, IN) was used for the spectrophotometric measurement of glucose and sucrose. This kit uses an enzyme pair of hexokinase and glucose-6-phosphate dehydrogenase for determining glucose, and invertase in addition to the above enzyme pair for determining sucrose. All the other reagents were of analytical grade (Nacalai, Kyoto) and were used without further purification. Deionized, doubly distilled water was used throughout. Enzyme immobilization GOx modified with (C12)2gluN+ was prepared according to the previous literature (Mizutani et al., 1993; Okahata et al., 1989). A buffer solution (1 ml, 0·1 M potassium acetate buffer plus 0·2 M KCl, pH 6) containing 50 mg GOx was mixed with an aqueous dispersion (20 ml) of 200 mg of the lipid. The precipitate formed after the incubation of the mixture at 4°C for 24 h was lyophilized. A light yellow powder (about 120 mg) of a lipid-attached GOx was obtained. The lipid molecules are considered to form a monolayer on the surface of the enzyme molecule (Mizutani et al., (1993, 1997); Okahata et al., 1989); the monolayer would be formed based on the ionic bond between the cationic lipid and negatively charged enzyme molecule (Voet & Anderson, 1984) and also on the hydrophobic interaction between the lipid. A glassy carbon electrode (Bioanalytical Sys-
Fumio Mizutani & Soichi Yabuki
tems, West Lafayette, IN), 3 mm in diameter, was polished with a 0·05 m alumina slurry, rinsed with water, sonicated in water for 2 min and dried. Then the electrode was dipped into a benzene solution of the lipid-attached GOx (2%(w/v)) and the solvent was allowed to dry for 2 min at room temperature. Finally, a Nafion coating layer was formed by dip-coating the enzyme-modified electrode in 0·5%(w/v) solution, which was prepared by diluting the 5% solution as received with a mixture of 2-propanol (50%(v/v)) and water (Harrison et al., 1988), and the electrode was allowed to dry with the surface facing down, so as to form a pinhole-free Nafion overcoat (Gorton et al., 1990), for 3 h at room temperature. The thickness of the resulting enzyme/Nafion layer was 苲 1 m. A layer containing invertase and mutarotase was prepared by using PVA-SbQ as a support (Ichimura, 1984; Mizutani et al., 1985; Mizutani & Asai, 1990b). The salt content of the invertase (40 mg of the powder; salt content 苲 70%) was decreased by two 1 h dialyses against 200 ml water. Then the desalted invertase solution (about 1 ml) was mixed with mutarotase (5 mg) and the PVA-SbQ solution (200 mg). The desalting of invertase was necessary to form homogeneous enzyme/PVA-SbQ solution: the addition of the invertase as received into the PVA-SbQ solution caused salting-out of the polymer. The enzyme/polymer mixture was spread on a PTFE plate (to be 3 × 10 cm2) and dried for 16 h at 4°C. The dried film was carefully removed from the plate and put between two pieces of polyester mesh (100 mesh). The two pieces of mesh were sewn together, so that the thin invertase/mutarotase-containing film (thickness, ca 10 m) covered with polyester mesh could be manipulated easily. Then the enzyme film was irradiated for crosslinking of the polymer with a fluorescent lamp (100 W; distance between the light and film, about 10 cm) for 10 min on each side.
side-wall of the cell, as shown in Fig. 1. The solution in the cell was saturated with air and stirred with a magnetic bar. The temperature of the solution was kept at 30·0 ± 0·2°C. The potential of the enzyme electrode was usually set at 1·0 V versus Ag/AgCl. Glassy carbon is useful for the anodic detection of the hydrogen peroxide produced through an oxidase-catalyzed reaction, although a rather high potential is required (Gorton, 1985; Mizutani et al., (1993, 1995)) compared with the case of conventional noble metal electrodes. The spectrophotometric measurement of glucose and sucrose with the F-kit was performed by using a spectrophotometer (160A, Shimadzu, Kyoto).
RESULTS AND DISCUSSION Glucose concentration–time profiles in the measuring cell Fig. 2 shows the variations of glucose concentration in the invertase/mutarotase-attached cell with time after addition of sucrose (1, 2 and 4 mM) and a mixture of glucose (0·5 mM) and sucrose (2 mM). The glucose concentrations
Enzyme electrode system and measurement A potentiostat (HA-502, Hokuto Denko, Tokyo) was used in a three-electrode configuration for amperometric measurements: the enzyme electrode, an Ag/AgCl reference electrode (saturated with KCl, Bioanalytical Systems) and a platinum auxiliary electrode were immersed in 20 ml of a testing buffer solution (0·1 M potassium phosphate buffer, pH 7·0) in a cylindrical cell (3 cm in diameter). The invertase/mutarotase film covered with polyester mesh was attached to the
Fig. 1. Arrangement of the immobilized invertase/mutarotase in the measuring cell with the glucose-sensing enzyme electrode: (1) cylindrical glass cell; (2) invertase/mutarotase-coimmobilized layer; (3) enzyme electrode; (4) Ag/AgCl reference electrode; (5) platinum auxiliary electrode; (6) magnetic stirring bar; (7) potentiostat.
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C = CG + 1.70 × 10−3CS(t − 6)
(t
(4)
ⱖ 6 s, CS ⱕ 6 mM) From the measurement of the glucose-producing rate after the addition, in a relatively high concentration range (5–100 mM), of sucrose, the activity and Michaelis constant, Km, of invertase in the layer were determined to be 1·6 U/cm2 and 14 mM, respectively. The Km value of invertase in the crosslinked PVA-SbQ layer was close to that in solution (ca 15 mM, Hoshino & Momose, 1966), as in the cases of lactate oxidase (EC number, not assigned) and lactate dehydrogenase (EC 1.1.1.17) in the PVA-SbQ matrices (Mizutani et al., 1985). Electrode response
Fig. 2. Changes of glucose concentration in the measuring cell with time. The solutions tested initially contained (1) 1 mM sucrose, (2) 2 mM sucrose, (3) 4 mM sucrose and (4) 0·5 mM glucose and 2 mM sucrose.
shown in Fig. 2 were determined by using the F-kit. The glucose concentration increased linearly with time from 6 s (to at least 30 s) after the addition of sucrose. The glucose-producing rate was proportional to the initial sucrose concentration, CS, up to 6 mM. For enzyme layer systems, the time necessary to obtain a steadystate production rate after the addition of the enzyme substrate, td, is expressed as a function of the diffusion coefficient of the substrate in the layer, D, and the layer thickness, L (Mell & Maloy, 1975):
Curves 1–3 in Fig. 3 show the electrode response for 0·5 mM glucose, 2 mM sucrose, and a mixture of 0·5 mM glucose and 2 mM sucrose, respectively. After the addition of glucose, the electrode current increased immediately and reached another steady state within 2 s. Since the enzyme/Nafion layer is thin, the glucose added is expected to diffuse quickly so as to give a fast electrode response (Mizutani et al., 1993; Mell & Maloy, 1975). The current response was proportional to the glucose concentration up to
td ⱕ 1·5L2D−1 By introducing the diffusion coefficient of sucrose in the crosslinked PVA-SbQ layer, which was estimated as 2 × 10−7 cm2/s (Mizutani et al., 1985), and the thickness of the layer (10 m), the delay time is calculated to be 7·5 s. The calculated value agrees well with the experimental result. The glucose-producing rate from sucrose was independent of the initial glucose concentration in the samples (see, curves 2 and 4 in Fig. 2). Thus the glucose concentration, C, t s after the addition of a sample could be expressed as: C = CG (t ⬍ 6 s)
(3)
Fig. 3. Response/time curves of the enzyme electrode to (1) 0·5 mM glucose, (2) 2 mM sucrose and (3) 0·5 mM glucose and 2 mM sucrose.
Fumio Mizutani & Soichi Yabuki
3 mM (see Fig. 4). The detection limit was 0·2 M (signal-to-noise ratio, five). The relative standard deviation (rsd) for 10 successive measurements of 0·5 mM glucose was 1·7%. The current increased linearly with time from 8 s after the addition of sucrose (curve 2 in Fig. 3). The rate of current increase, which was recorded from 8 to 20 s after the addition of sucrose, was proportional to the concentration of sucrose up to 6 mM (see Fig. 5). The detection limit was 10 M (signal obtained 20 s after injecting sample to noise, 5). The rsd value for 10 successive measurement of 2 mM sucrose was 3·1%. The rsd value was compatible with the results reported previously for measuring the producing rate of enzyme substrates with amperometric sensors (Mizutani & Tsuda, 1982; Mizutani et al., 1983; Blum et al., 1983; Kihara et al., 1983; Wollenberger et al., 1989). Curve 3 in Fig. 3 shows the electrode response after the addition of a mixture of 0·5 mM glucose and 2 mM sucrose. The current response to the mixture was equal to the sum of the responses for glucose (curve 1 in Fig. 3) and sucrose (curve 2 in Fig. 3), as expected. This means that CG can be determined from the steady-state current increase obtained from 2 to 6 s after the addition of the mixture and CS, from the rate of current increase recorded from 8 to 20 s after the addition of the mixture. Fig. 4 shows the relationship between the steady-state current increase obtained from two to several seconds after the addition of glucose–
Fig. 5. Relationship between the rate of current increase obtained from 8 to 20 s after the sample addition and the sucrose concentration in the sample. The concentrations of coexisting glucose are: (䊊) 0 mM; (쎲) 2 mM; (䊐) 2·8 mM; and (䊏) 2·9 mM.
sucrose mixture and CG. As shown in Fig. 4, the steady-state current increase was dependent on the glucose concentration in the mixture but not on the concentration of coexisting sucrose at all for all the sucrose concentrations examined. Fig. 5 shows the relationship between the rate of current increase recorded from 8 to 20 s after injecting the sample and CS. The current increasing rate was independent of the CG when it was lower than 2·8 mM. As described above, the current response was linear against the glucose concentration up to 3 mM (Fig. 4). When the sum of the initial glucose concentration and the concentration of glucose produced through the cell-attached invertase reaction (at 20 s after the sample addition), C, are within 3 mM, the sucrose response is not affected by the coexisting glucose. On the other hand, when the initial glucose concentration in the sample is so high that C exceeds 3 mM, the current–time relationship for sucrose becomes non-linear which makes it difficult for determining CS. Determination of glucose and sucrose in food samples and stability of the electrode system
Fig. 4. Relationship between the steady-state current increase recorded from 2 to 6 s after the sample addition and the glucose concentration in the sample. The concentrations of coexisting sucrose are: (䊊) 0 mM; (쎲) 2 mM; (䊐) 4 mM; and (䊏) 6 mM.
The glucose-sensing electrode used Nafion, an anionic polymer, as the overcoat on the enzyme layer, which was effective for excluding anionic interferents (e.g. L-ascorbic acid) (Nagy et al., 1985; Harrison et al., 1988; Matsue et al., 1989;
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Dong et al., 1992). The ratio of response for Lascorbic acid to that for the same concentration of glucose was 0·9 (Mizutani et al., 1993). Further, the rate of current increase for sucrose ( ⱕ 6 mM) was not affected by the addition of L-ascorbic acid ( ⱕ 2 mM). The concentrations of L-ascorbic acid and glucose in fruit juices have been reported to be lower than 3 mM (Matsumoto et al., 1981) and 100–200 mM (Matsumoto et al., 1988; Mizutani et al., 1993), respectively. For measuring glucose in such a sample with the present glucose-sensing electrode, the level of electrochemical interference by L-ascorbic acid is expected to be within a few percent of the signal output. The addition of a mixture of glucose (1·5 mM) and L-ascorbic acid (0·03 mM) actually gave only 2% excess current compared with a pure glucose solution (1·5 mM). Hence the present electrode-based system is expected to be useful for the rapid and accurate determination of glucose and sucrose in beverages. Table 1 gives the results for the determination of glucose and sucrose in jams and fruit juices. Each jam was firstly dissolved in water to 10% (v/v), and 200 l of the solution was added to the buffer solution. Fruit juices (200 l) were used without any pretreatment. The results were compared to those obtained with the F-kit method. The agreement is excellent: the regression equations between the results obtained by the present method (x) and those by the F-kit method (y) were y = 0·985x + 0·00375 for glucose and y = 1·011x − 0·00233 for sucrose; the correlation coefficients were 0·9987 and 0·9964 for glucose and sucrose, respectively. Thus, the present enzyme electrode/enzyme-attached cell system has been proven to be useful for the simple and rapid assays of food samples. On the other hand, it would be difficult to neglect the
electrochemical interference for determining glucose in some kinds of vitamin C-enriched drinks by the present system. Hence, an enzyme electrode that gives a quick response with lower electrochemical interference response is still desired. The fabrication of such glucose-sensing electrodes is now in progress. The long-term stability of the enzyme electrode system was then examined: the current response for a mixture of 0·5 mM glucose and 2 mM sucrose was measured 10 times a day for 6 weeks. The average value of the steady-current response for glucose and that of the current increasing rate for sucrose in the 10 measurements did not decrease for 4 weeks.
CONCLUSION Rapid and precise measurements of glucose and sucrose were thus achieved by employing the simple system consisting of an enzyme electrode with rapid response to glucose and an invertase/mutarotase-attached measuring cell. The time required for the measurement was 30 s. The principle of the present method can be extended to other substrates by using different enzyme(s). For example, fructose and sucrose in food samples have been determined by the use of a fructose dehydrogenase-based electrode and an invertase-attached measuring cell.
ACKNOWLEDGEMENTS This work was partly supported by the Original Industry Technology R&D Promotion Program from the New Energy and Industrial Technology Development Organization (NEDO), Japan.
TABLE 1 Comparison of results obtained for glucose and sucrose in jams and fruit juices by different methods Sample
Glucose concentration (M) Proposed method
Jam (strawberry) Jam (orange) Juice (orange 1) Juice (orange 2) Juice (apple) Juice (grape fruit) Juice (mix 1) Juice (mix 2)
1·00 1·09 0·166 0·136 0·106 0·136 0·104 0·144
F-kit method 1·03 1·04 0·164 0·140 0·102 0·144 0·113 0·146
Sucrose concentration (M) Proposed method 0·327 0·319 0·098 0·141 0·094 0·061 0·119 0·049
F-kit method 0·326 0·303 0·096 0·138 0·094 0·061 0·117 0·048
Fumio Mizutani & Soichi Yabuki
REFERENCES Bilitewski, U. (1994). Enzyme electrode for food analysis. In Food Biosensor Analysis, ed. G. Wagner & G. G. Guilbault. Dekker, New York, pp. 31–61. Blum, L. J., Betterand, C. and Coulet, P. R. (1983) Amperometric sensor and immobilized enzyme electrode for the determination of enzymatic activities. Anal. Lett. 16B, 525–540. Dong, S., Wang, B. and Liu, B. (1992) Amperometric glucose sensor with ferrocene as an electron transfer mediator. Biosensors & Bioelectronics 7, 215–222. Gorton, L. (1985) A carbon electrode sputtered with palladium and gold for the amperometric detection of hydrogen peroxide. Anal. Chim. Acta 178, 247. Gorton, L., Karan, H. I., Hale, P. D., Inagaki, T., Okamoto, Y. and Skotheim, T. A. (1990) A glucose electrode based on carbon paste chemically modified with a ferrocene-containing siloxane polymer and glucose oxidase, coated with poly(estersulfonic acid) cation-exchanger. Anal. Chim. Acta 228, 23–30. Harrison, D. J., Turner, R. F. B. and Baltes, H. P. (1988) Characterization of perfluorosulfonic acid polymer coated enzyme electrodes and a miniaturized integrated potentiostat for glucose analysis in whole blood. Anal. Chem. 60, 2002–2007. Hoshino, J. and Momose, A. (1966) Comparative studies of the two forms of yeast invertase. J. Gen. Appl. Microbiol. 12, 163–177. Ichimura, K. (1984) A convenient photochemical method to immobilize enzymes. J. Polym. Sci., Polym. Chem. Ed. 22, 2917–2928. Kihara, K., Yasukawa, E. and Hirose, S. (1983) Sequential determination of glutamate-oxalacetate transaminase and glutamate-pyruvate transaminase activities in serum using an immobilized bienzymepoly(vinyl chloride) membrane electrode. Anal. Chem. 56, 1876–1880. Masoom, M. and Townshend, A. (1985) Simultaneous determination of sucrose and glucose in mixtures by flow injection analysis with immobilized enzymes. Anal. Chim. Acta 171, 185–194. Matsue, T., Aoki, A., Abe, T. & Uchida, I. (1989). Determination of catecholamines by an electrochemical flow-injection detector with indium-tin oxide microelectrode arrays coated with Nafion. Chem. Lett., 133–136. Matsumoto, K., Yamada, K. and Osajima, Y. (1981) Ascorbate electrode for determination of L-ascorbic acid in food. Anal. Chem. 53, 1974–1979. Matsumoto, K., Kamikado, H., Matsubara, H. and Osajima, Y. (1988) Simultaneous determination of glucose, fructose, and sucrose in mixtures by amperometric flow injection analysis with immobilized enzyme reactors. Anal. Chem. 60, 147–151. Mell, L. D. and Maloy, D. T. (1975) A model for the amperometric enzyme electrode obtained through digital simulation and applied to the immobilized glucose oxidase system. Anal. Chem. 47, 299–307.
Mizutani, F. and Tsuda, K. (1982) Amperometric determination of cholinesterase with use of an immobilized enzyme electrode. Anal. Chim. Acta 139, 359–362. Mizutani, F., Sasaki, K. and Shimura, Y. (1983) Sequential determination of L-lactate and lactate dehydrogenase with immobilized enzyme electrode. Anal. Chem. 55, 35–38. Mizutani, F., Yamanaka, T., Tanabe, Y. and Tsuda, K. (1985) An enzyme electrode for L-lactate with a chemically-amplified response. Anal. Chim. Acta 177, 153–166. Mizutani, F. & Asai, M. (1990a). Trends in enzyme sensors. In Bioinstrumentations, ed. D. L. Wise. Butterworths, Boston, pp. 314–353. Mizutani, F. and Asai, M. (1990b) Simultaneous determination of glucose and sucrose by a glucosesensing enzyme electrode combined with an invertase-attached cell. Anal. Chim. Acta 236, 245–250. Mizutani, F., Yabuki, S. and Katsura, T. (1993) Amperometric enzyme electrode with fast response to glucose using a layer of lipid-modified glucose oxidase and Nafion Anionic polymer. Anal. Chim. Acta 274, 201–207. Mizutani, F., Yabuki, S. and Hirata, Y. (1995) Amperometric L-lactate-sensing electrode based on a polyion complex layer containing lactate oxidase. Application to serum and milk samples. Anal. Chim. Acta 314, 233–239. Mizutani, F., Yabuki, S. & Sato, Y. (1997). Voltammetric enzyme sensor for urea using mercaptohydroquinone-modified gold electrode as the base transducer. Biosensors & Bioelectronics 12, 321– 328. Nagy, F., Gerhardt, G. A., Oke, A. F., Rice, M. E., Adams, R. N., Moore, R. B., Szentirmay, M. N. and Martin, C. R. (1985) Ion exchange and transport of neurotransmitters in Nafion films on conventional and microelectrode surfaces. J. Electroanal. Chem. 188, 85–94. Okahata, Y., Tsuruta, T., Ijiro, K. and Ariga, K. (1989) Preparations of Langmuir–Blodgett films of enzyme-lipid complexes: a glucose sensor membrane. Thin Solid Films 180, 65–72. Pfeiffer, P., Scheller, F., Janchen, M., Betermann, K. and Weise, H. (1980) Bienzyme electrodes for ATP, NAD+, starch and dissacharides based on a glucose sensor. Anal. Lett. B13, 1179–1200. Scheller, F. and Karsten, Ch. (1983) A combination of invertase reactor and glucose oxidase electrode for the successive determination of glucose and sucrose. Anal. Chim. Acta 155, 29–36. Scheller, F. & Schubert, F. (1992). Metabolism sensors. In Biosensors. Elsevier, Amsterdam, pp. 85–252. Voet, J. and Anderson, E. C. (1984) Electrostatic control of enzyme reactions: the mechanism of inhibition of glucose oxidase by putrescine. Arch. Biochem. Biophys. 233, 88–92. Wollenberger, U., Scheller, F. W., Boehmer, A., Passarge, M. and Mueller, H. -G. (1989) A specific
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enzyme electrode for L-glutamate—development and application. Biosensors 4, 381–391. Xu, Y., Guilbault, G. G. and Kuan, S. S. (1989)
Sucrose enzyme electrode. Anal. Chem. 61, 782– 784.
Rapid determination of glucose and sucrose by an amperometric glucosesensing electrode combined with an invertase/mutarotase-attached measuring cell Fumio Mizutani* & Soichi Yabuki National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan (Received 26 February 1997; revised version received 1 May 1997; accepted 2 May 1997)
Abstract: Glucose and sucrose are simultaneously determined by the use of an enzyme sensor system consisting of a glucose-sensing electrode based on a lipid-modified glucose oxidase and a measuring cell that contains an invertase/mutarotase-coimmobilized layer. From the current response of the enzyme electrode after the addition of a glucose/sucrose mixture, the concentrations of the two kinds of sugars can be separately determined: the concentration of glucose (0·2 M–3 mM) is determined from the steady-state current increase obtained from 2 to 6 s after the addition of the mixture, and that of sucrose (10 M–6 mM), from the rate of current increase from 8 to 20 s after the addition. The relative standard deviations are 1·7% for glucose and 3·1% for sucrose (n = 10). The system can be applied to the rapid determination of glucose and sucrose in food samples. 1997 Elsevier Science Limited Keywords: enzyme electrode, glucose, sucrose
INTRODUCTION Of the various methods available for the determination of saccharides in industrial food laboratories, the most attractive are those based on amperometric enzyme electrodes (Bilitewski, 1994; Matsumoto et al., 1988). The combination of specific enzymes with sensitive electrochemical signal transduction yields enzyme electrodes which provide simple, rapid and accurate determination of the enzyme substrate (Mizutani & Asai, 1990a; Scheller & Schubert, 1992). In many food production processes, the simultaneous determination of glucose and sucrose is *To whom correspondence should be addressed. Rapid determination of glucose and sucrose
necessary for effective process control. Glucose and sucrose in mixtures have been determined successfully by using two different enzyme electrodes equipped with a measuring cell (Pfeiffer et al., 1980; Xu et al., 1989). For example, two enzyme electrodes, one with a layer of glucose oxidase (GOx) and the other with a layer containing GOx, invertase and mutarotase, are immersed in a testing buffer solution in the cell; the first electrode is used to measure glucose and the second, to measure the sum of glucose and sucrose. The simultaneous determination of glucose and sucrose has also been carried out by using multi-channel flow injection systems (Matsumoto et al., 1988; Masoom & Townshend, 1985). On the other hand, methods using a single
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enzyme electrode have been proposed for achieving the measurement with more simple and inexpensive instruments (Scheller & Karsten, 1983; Mizutani & Asai, 1990b). Scheller & Karsten (1983) have carried out a successive determination of glucose and sucrose. Glucose in a sample solution was determined from the change in a GOx-based electrode, then an invertase layer was immersed in the solution to produce glucose from sucrose; the concentration of sucrose was determined by measuring the rate of enzymatic glucose production, i.e. the rate of current change. However, this method is laborious and time-consuming: the invertase layer must be immersed in the test solution for every measurement, and more than 3 min are required to complete the measurement. We proposed a more convenient method which used a glucose-sensing electrode combined with a measuring cell that contained immobilized invertase. The concentration of glucose, C, in the measuring cell at t s after the addition of a sample is approximately expressed as (Mizutani & Asai, 1990b): C = CG(t ⬍ td)
(1)
C = CG + aCs(t − td) (t ⱖ td)
(2)
where CG and CS are the initial concentrations of glucose and sucrose, respectively, a is a constant and td a time lag for the production of glucose at the immobilized invertase. When the response time of the glucose-sensing electrode is very short (i.e. much shorter than td), the electrode can generate the current output proportional to C (see Eqs (1) and (2)). That is, the steady-state current response obtained at t ⬍ td would be proportional to CG and the rate of current change at t ⬎ td to CS; the determination of the two components can easily be carried out by monitoring the current response after the addition of the sample. However, in our previous work (Mizutani & Asai, 1990b), a relatively long response time for the glucose-sensing electrode used (100% response time, about 1 min) complicated the analysis of the current response for determining the two components and, of course, it took a long time (about 2 min) to complete the determination. Recently, we have prepared a glucose-sensing enzyme electrode with a 100% response time of less than 2 s by modifying a base electrode with a lipid-attached GOx (Mizutani et al., 1993). Such an enzyme electrode is considered to be useful for the rapid and simple determination of glucose and sucrose. It has been shown that glucose (0·2 M–3 mM) and sucrose (10 M–
6 mM) can be determined within 20 s. The method is applied to the determination of the two components in food samples.
EXPERIMENTAL Materials The enzymes used were GOx (EC 1.1.3.4, from Aspergillus sp., 150 U/mg, Toyobo Osaka), invertase (EC 3.2.1.6, from Candida utilis, 130 U/mg, Toyobo) and mutarotase (EC 5.1.1.3, from porcine kidney, 3300 U/mg, Sigma, St Louis, MO). A lipid, N-(␣-trimethylammonioacetyl)didodecylL-glutamate chloride [(C12)2gluN+Cl−], was obtained from Sogo Phermaceutical (Tokyo) and Nafion (5%(w/v) solution, 1100 equiv. wt) from Aldrich (Milwaukee, WI). Photo-crosslinkable poly(vinyl alcohol) bearing stilbazolium groups (PVA-SbQ (Ichimura, 1984); aqueous solution (9%(w/v)), pH 7; degree of polymerization, 1700; molar ratio of stilbazolium group, 1·3%) was a gift from Toyo Gosei Kogyo (Chiba). An F-kit (Boehringer, Indianapolis, IN) was used for the spectrophotometric measurement of glucose and sucrose. This kit uses an enzyme pair of hexokinase and glucose-6-phosphate dehydrogenase for determining glucose, and invertase in addition to the above enzyme pair for determining sucrose. All the other reagents were of analytical grade (Nacalai, Kyoto) and were used without further purification. Deionized, doubly distilled water was used throughout. Enzyme immobilization GOx modified with (C12)2gluN+ was prepared according to the previous literature (Mizutani et al., 1993; Okahata et al., 1989). A buffer solution (1 ml, 0·1 M potassium acetate buffer plus 0·2 M KCl, pH 6) containing 50 mg GOx was mixed with an aqueous dispersion (20 ml) of 200 mg of the lipid. The precipitate formed after the incubation of the mixture at 4°C for 24 h was lyophilized. A light yellow powder (about 120 mg) of a lipid-attached GOx was obtained. The lipid molecules are considered to form a monolayer on the surface of the enzyme molecule (Mizutani et al., (1993, 1997); Okahata et al., 1989); the monolayer would be formed based on the ionic bond between the cationic lipid and negatively charged enzyme molecule (Voet & Anderson, 1984) and also on the hydrophobic interaction between the lipid. A glassy carbon electrode (Bioanalytical Sys-
Fumio Mizutani & Soichi Yabuki
tems, West Lafayette, IN), 3 mm in diameter, was polished with a 0·05 m alumina slurry, rinsed with water, sonicated in water for 2 min and dried. Then the electrode was dipped into a benzene solution of the lipid-attached GOx (2%(w/v)) and the solvent was allowed to dry for 2 min at room temperature. Finally, a Nafion coating layer was formed by dip-coating the enzyme-modified electrode in 0·5%(w/v) solution, which was prepared by diluting the 5% solution as received with a mixture of 2-propanol (50%(v/v)) and water (Harrison et al., 1988), and the electrode was allowed to dry with the surface facing down, so as to form a pinhole-free Nafion overcoat (Gorton et al., 1990), for 3 h at room temperature. The thickness of the resulting enzyme/Nafion layer was 苲 1 m. A layer containing invertase and mutarotase was prepared by using PVA-SbQ as a support (Ichimura, 1984; Mizutani et al., 1985; Mizutani & Asai, 1990b). The salt content of the invertase (40 mg of the powder; salt content 苲 70%) was decreased by two 1 h dialyses against 200 ml water. Then the desalted invertase solution (about 1 ml) was mixed with mutarotase (5 mg) and the PVA-SbQ solution (200 mg). The desalting of invertase was necessary to form homogeneous enzyme/PVA-SbQ solution: the addition of the invertase as received into the PVA-SbQ solution caused salting-out of the polymer. The enzyme/polymer mixture was spread on a PTFE plate (to be 3 × 10 cm2) and dried for 16 h at 4°C. The dried film was carefully removed from the plate and put between two pieces of polyester mesh (100 mesh). The two pieces of mesh were sewn together, so that the thin invertase/mutarotase-containing film (thickness, ca 10 m) covered with polyester mesh could be manipulated easily. Then the enzyme film was irradiated for crosslinking of the polymer with a fluorescent lamp (100 W; distance between the light and film, about 10 cm) for 10 min on each side.
side-wall of the cell, as shown in Fig. 1. The solution in the cell was saturated with air and stirred with a magnetic bar. The temperature of the solution was kept at 30·0 ± 0·2°C. The potential of the enzyme electrode was usually set at 1·0 V versus Ag/AgCl. Glassy carbon is useful for the anodic detection of the hydrogen peroxide produced through an oxidase-catalyzed reaction, although a rather high potential is required (Gorton, 1985; Mizutani et al., (1993, 1995)) compared with the case of conventional noble metal electrodes. The spectrophotometric measurement of glucose and sucrose with the F-kit was performed by using a spectrophotometer (160A, Shimadzu, Kyoto).
RESULTS AND DISCUSSION Glucose concentration–time profiles in the measuring cell Fig. 2 shows the variations of glucose concentration in the invertase/mutarotase-attached cell with time after addition of sucrose (1, 2 and 4 mM) and a mixture of glucose (0·5 mM) and sucrose (2 mM). The glucose concentrations
Enzyme electrode system and measurement A potentiostat (HA-502, Hokuto Denko, Tokyo) was used in a three-electrode configuration for amperometric measurements: the enzyme electrode, an Ag/AgCl reference electrode (saturated with KCl, Bioanalytical Systems) and a platinum auxiliary electrode were immersed in 20 ml of a testing buffer solution (0·1 M potassium phosphate buffer, pH 7·0) in a cylindrical cell (3 cm in diameter). The invertase/mutarotase film covered with polyester mesh was attached to the
Fig. 1. Arrangement of the immobilized invertase/mutarotase in the measuring cell with the glucose-sensing enzyme electrode: (1) cylindrical glass cell; (2) invertase/mutarotase-coimmobilized layer; (3) enzyme electrode; (4) Ag/AgCl reference electrode; (5) platinum auxiliary electrode; (6) magnetic stirring bar; (7) potentiostat.
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C = CG + 1.70 × 10−3CS(t − 6)
(t
(4)
ⱖ 6 s, CS ⱕ 6 mM) From the measurement of the glucose-producing rate after the addition, in a relatively high concentration range (5–100 mM), of sucrose, the activity and Michaelis constant, Km, of invertase in the layer were determined to be 1·6 U/cm2 and 14 mM, respectively. The Km value of invertase in the crosslinked PVA-SbQ layer was close to that in solution (ca 15 mM, Hoshino & Momose, 1966), as in the cases of lactate oxidase (EC number, not assigned) and lactate dehydrogenase (EC 1.1.1.17) in the PVA-SbQ matrices (Mizutani et al., 1985). Electrode response
Fig. 2. Changes of glucose concentration in the measuring cell with time. The solutions tested initially contained (1) 1 mM sucrose, (2) 2 mM sucrose, (3) 4 mM sucrose and (4) 0·5 mM glucose and 2 mM sucrose.
shown in Fig. 2 were determined by using the F-kit. The glucose concentration increased linearly with time from 6 s (to at least 30 s) after the addition of sucrose. The glucose-producing rate was proportional to the initial sucrose concentration, CS, up to 6 mM. For enzyme layer systems, the time necessary to obtain a steadystate production rate after the addition of the enzyme substrate, td, is expressed as a function of the diffusion coefficient of the substrate in the layer, D, and the layer thickness, L (Mell & Maloy, 1975):
Curves 1–3 in Fig. 3 show the electrode response for 0·5 mM glucose, 2 mM sucrose, and a mixture of 0·5 mM glucose and 2 mM sucrose, respectively. After the addition of glucose, the electrode current increased immediately and reached another steady state within 2 s. Since the enzyme/Nafion layer is thin, the glucose added is expected to diffuse quickly so as to give a fast electrode response (Mizutani et al., 1993; Mell & Maloy, 1975). The current response was proportional to the glucose concentration up to
td ⱕ 1·5L2D−1 By introducing the diffusion coefficient of sucrose in the crosslinked PVA-SbQ layer, which was estimated as 2 × 10−7 cm2/s (Mizutani et al., 1985), and the thickness of the layer (10 m), the delay time is calculated to be 7·5 s. The calculated value agrees well with the experimental result. The glucose-producing rate from sucrose was independent of the initial glucose concentration in the samples (see, curves 2 and 4 in Fig. 2). Thus the glucose concentration, C, t s after the addition of a sample could be expressed as: C = CG (t ⬍ 6 s)
(3)
Fig. 3. Response/time curves of the enzyme electrode to (1) 0·5 mM glucose, (2) 2 mM sucrose and (3) 0·5 mM glucose and 2 mM sucrose.
Fumio Mizutani & Soichi Yabuki
3 mM (see Fig. 4). The detection limit was 0·2 M (signal-to-noise ratio, five). The relative standard deviation (rsd) for 10 successive measurements of 0·5 mM glucose was 1·7%. The current increased linearly with time from 8 s after the addition of sucrose (curve 2 in Fig. 3). The rate of current increase, which was recorded from 8 to 20 s after the addition of sucrose, was proportional to the concentration of sucrose up to 6 mM (see Fig. 5). The detection limit was 10 M (signal obtained 20 s after injecting sample to noise, 5). The rsd value for 10 successive measurement of 2 mM sucrose was 3·1%. The rsd value was compatible with the results reported previously for measuring the producing rate of enzyme substrates with amperometric sensors (Mizutani & Tsuda, 1982; Mizutani et al., 1983; Blum et al., 1983; Kihara et al., 1983; Wollenberger et al., 1989). Curve 3 in Fig. 3 shows the electrode response after the addition of a mixture of 0·5 mM glucose and 2 mM sucrose. The current response to the mixture was equal to the sum of the responses for glucose (curve 1 in Fig. 3) and sucrose (curve 2 in Fig. 3), as expected. This means that CG can be determined from the steady-state current increase obtained from 2 to 6 s after the addition of the mixture and CS, from the rate of current increase recorded from 8 to 20 s after the addition of the mixture. Fig. 4 shows the relationship between the steady-state current increase obtained from two to several seconds after the addition of glucose–
Fig. 5. Relationship between the rate of current increase obtained from 8 to 20 s after the sample addition and the sucrose concentration in the sample. The concentrations of coexisting glucose are: (䊊) 0 mM; (쎲) 2 mM; (䊐) 2·8 mM; and (䊏) 2·9 mM.
sucrose mixture and CG. As shown in Fig. 4, the steady-state current increase was dependent on the glucose concentration in the mixture but not on the concentration of coexisting sucrose at all for all the sucrose concentrations examined. Fig. 5 shows the relationship between the rate of current increase recorded from 8 to 20 s after injecting the sample and CS. The current increasing rate was independent of the CG when it was lower than 2·8 mM. As described above, the current response was linear against the glucose concentration up to 3 mM (Fig. 4). When the sum of the initial glucose concentration and the concentration of glucose produced through the cell-attached invertase reaction (at 20 s after the sample addition), C, are within 3 mM, the sucrose response is not affected by the coexisting glucose. On the other hand, when the initial glucose concentration in the sample is so high that C exceeds 3 mM, the current–time relationship for sucrose becomes non-linear which makes it difficult for determining CS. Determination of glucose and sucrose in food samples and stability of the electrode system
Fig. 4. Relationship between the steady-state current increase recorded from 2 to 6 s after the sample addition and the glucose concentration in the sample. The concentrations of coexisting sucrose are: (䊊) 0 mM; (쎲) 2 mM; (䊐) 4 mM; and (䊏) 6 mM.
The glucose-sensing electrode used Nafion, an anionic polymer, as the overcoat on the enzyme layer, which was effective for excluding anionic interferents (e.g. L-ascorbic acid) (Nagy et al., 1985; Harrison et al., 1988; Matsue et al., 1989;
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Dong et al., 1992). The ratio of response for Lascorbic acid to that for the same concentration of glucose was 0·9 (Mizutani et al., 1993). Further, the rate of current increase for sucrose ( ⱕ 6 mM) was not affected by the addition of L-ascorbic acid ( ⱕ 2 mM). The concentrations of L-ascorbic acid and glucose in fruit juices have been reported to be lower than 3 mM (Matsumoto et al., 1981) and 100–200 mM (Matsumoto et al., 1988; Mizutani et al., 1993), respectively. For measuring glucose in such a sample with the present glucose-sensing electrode, the level of electrochemical interference by L-ascorbic acid is expected to be within a few percent of the signal output. The addition of a mixture of glucose (1·5 mM) and L-ascorbic acid (0·03 mM) actually gave only 2% excess current compared with a pure glucose solution (1·5 mM). Hence the present electrode-based system is expected to be useful for the rapid and accurate determination of glucose and sucrose in beverages. Table 1 gives the results for the determination of glucose and sucrose in jams and fruit juices. Each jam was firstly dissolved in water to 10% (v/v), and 200 l of the solution was added to the buffer solution. Fruit juices (200 l) were used without any pretreatment. The results were compared to those obtained with the F-kit method. The agreement is excellent: the regression equations between the results obtained by the present method (x) and those by the F-kit method (y) were y = 0·985x + 0·00375 for glucose and y = 1·011x − 0·00233 for sucrose; the correlation coefficients were 0·9987 and 0·9964 for glucose and sucrose, respectively. Thus, the present enzyme electrode/enzyme-attached cell system has been proven to be useful for the simple and rapid assays of food samples. On the other hand, it would be difficult to neglect the
electrochemical interference for determining glucose in some kinds of vitamin C-enriched drinks by the present system. Hence, an enzyme electrode that gives a quick response with lower electrochemical interference response is still desired. The fabrication of such glucose-sensing electrodes is now in progress. The long-term stability of the enzyme electrode system was then examined: the current response for a mixture of 0·5 mM glucose and 2 mM sucrose was measured 10 times a day for 6 weeks. The average value of the steady-current response for glucose and that of the current increasing rate for sucrose in the 10 measurements did not decrease for 4 weeks.
CONCLUSION Rapid and precise measurements of glucose and sucrose were thus achieved by employing the simple system consisting of an enzyme electrode with rapid response to glucose and an invertase/mutarotase-attached measuring cell. The time required for the measurement was 30 s. The principle of the present method can be extended to other substrates by using different enzyme(s). For example, fructose and sucrose in food samples have been determined by the use of a fructose dehydrogenase-based electrode and an invertase-attached measuring cell.
ACKNOWLEDGEMENTS This work was partly supported by the Original Industry Technology R&D Promotion Program from the New Energy and Industrial Technology Development Organization (NEDO), Japan.
TABLE 1 Comparison of results obtained for glucose and sucrose in jams and fruit juices by different methods Sample
Glucose concentration (M) Proposed method
Jam (strawberry) Jam (orange) Juice (orange 1) Juice (orange 2) Juice (apple) Juice (grape fruit) Juice (mix 1) Juice (mix 2)
1·00 1·09 0·166 0·136 0·106 0·136 0·104 0·144
F-kit method 1·03 1·04 0·164 0·140 0·102 0·144 0·113 0·146
Sucrose concentration (M) Proposed method 0·327 0·319 0·098 0·141 0·094 0·061 0·119 0·049
F-kit method 0·326 0·303 0·096 0·138 0·094 0·061 0·117 0·048
Fumio Mizutani & Soichi Yabuki
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