Amperometric fructose sensor based on direct bioelectrocatalysis

Biosensom& Bioektmnics 6 ( 199 1) 299-304

Amperometric fructose sensor based on direct bioelectrocatalysis Tokuji Ikeda, Fumio Matsushita & Mitsugi Senda Department

of Agricultural

Chemistry.

Faculty of Agriculture

University

(Received 4 May 1990; revised version received 7 September

of Kyoto. Sakyo-ku. Kyoto 606. Japan

1990: accepted 14 September

1990)

Abstract: Fructose dehydrogenase

(EC 1.1.99.I 1) from bacterial membranes was immobilized on a carbon paste electrode by covering the enzyme layer with a dialysis membrane. The fructose dehydrogenase-modified carbon paste electrode showed a current response to D-fructose without the addition of any external electron transfer mediators. The current response was independent of the oxygen concentration in the solution. Steady-state currents were obtained when measured at fixed electrode potentials. The dependence ofthe steady-state current on the potential, the pH of the solution and the temperature was studied. On the basis of this investigation. it was shown that the fructose dehydrogenasemodified carbon paste electrode could be used as an unmediated amperometric fructose sensor. D-Fructose in fruits was measured using the present electrode. A method of eliminating the effect of L-ascorbic acid is also described. Keywords: enzyme electrode. unmediated amperometric biosensor. L-ascorbic acid.

bioeiectrocatalysis,

D-fructose,

oxidation or reduction of the substrate in the absence of the mediators (Armstrong et al., 1988). We have found that carbon electrodes moditied with D-ghtconate dehydrogenase (GADH, EC 1.1.99.3) (Ikeda ec al., 1988~) and alcohol dehydrogenase (ADH, EC 1.1.99.8) (Fushimi, 1988) show a current response to their substrate attributable to direct bioelectrocatalysis. GADH and ADH are membrane-bound oxidoreductases containing FAD and heme c (GADH) or PQQ and heme c (ADH). We use here D-fructose dehydrogenase (EC 1.1.99.11). which is also a membrane-bound oxidoreductase containing PQQ and heme c as redox active sites, PQQ being considered to be the site to react with D-ftItCtOSe (Ameyama et al., 1981). A carbon paste electrode modified with this enzyme shows current response to D-frtXtOSe in the absence of external mediators. The use of the D-fIIKtOSe

INTRODUCTION Biocatalyst electrodes, i.e. modified electrodes carrying immobilized enzyme in which the electrode behaves as a substitute for a chemical electron acceptor or donor in the enzyme reaction, can oxidize or reduce the substrate of the enzyme bioelectrocatalytically. It has been shown (Tarasevich, 1985; Senda & Ikeda, 1986) that the bioelectrocatalytic oxidation or reduction can be achieved with the aid of small redox molecules serving as electron transfer mediators between the electrode and the enzyme immobilized on it. Electrodes carrying immobilized glucose oxidase with entrapped mediators could be used as a mediated amperometric enzyme electrode (Cass ef al., 1984; Ikeda et al., 1985). More recently, attention has been paid to the direct bioelectrocatalysis, i.e. the bioelectrocatalytic 299 Biosensom & Eioekctronh

09565663/91/$03.50 Q 1991 Elsevier Science Publishers Ltd. England. Printed in Great Britain

300 dehydrogenase-modified carbon paste electrode as an unmediated amperometric fructose sensor is described in this paper.

Tokuji Ikeda, Fumio Matsushita,Mitsugi Senda

potentials were measured against an AgJAgCI, saturated KC1 reference electrode. Preparation of electrodes

EXPERIMENTAL Reagents and apparatus D-Fructose dehydrogenase (FDH) preparation (D-fiuctose:(acceptor)5-oxidoreductase from Gluconobacter sp., Grade III, 297 units/mg, Lot 85710) was obtained from Toyobo Co. Stock solutions of FDH were prepared by dissolving 3 mg of the FDH preparation in 1 ml of MacIlvane buffer (pH 6.0) containing 0.1% Triton X-100. The FDH concentration of the stock solution was determined spectrophotometrically with reference to the spectrum given in the literature (Ameyama er al., 198 1). Ubiquinone, which might be contained in the FDH preparation, was determined by the method of Redfeam, 1967, and was not detected. The stock solutions were used within 2 weeks of their preparation, during which time the FDH activity of the stock solutions remained unchanged when stored at 5°C. Ascorbate oxidase (ASOD, L-ascorbate:oxygen oxidoreductase, EC 1.10.3.3, from cucumber, 133 units/mg, Lot 35003901) was purchased from Oriental Yeast Co. Paraffin liquid (for spectroscopy) and dialysis membranes (the thickness is 20pm in its dry state) were obtained from Merck Co. and Union Carbide Co., respectively. F-Kit for fructose measurements (product No. 139106) was purchased from Boehringer Mannheim Yamanouchi Co. The other chemicals used were of analytical reagent grade. A Yanagimoto Pl 100 polarographic analyzer was used to apply the potential to a threeelectrode system, and currents were measured with a Yokogawa 3025 x(t)-Y recorder. Electrochemical measurements The measurements were made at 25’C in an acetate buffer solution of pH 4.5 stirred at 500 rev/min with a magnetic stirrer unless otherwise stated. At this stirring rate, the concentration polarization of the substrate in the electrolyte solution was negligibly small. The electrolyte solution was not deaerated. All

Carbon paste electrodes were prepared in a manner similar to that described previously (Ikeda er al., 1985). In brief, graphite powder (5 g) was mixed with liquid paraffin (3 ml), and the mixture was packed into one end of a glass tube. The geometrical surface area of the electrode was 9.2 X 10:’ cm*. A 10 ~1 portion of the FDH stock solution, which contained 2.2 X lo-‘* mol FDH, was syringed onto the surface of the carbon paste electrode. The solvent was allowed to evaporate and then the electrode surface was covered with a dialysis membrane. The whole electrode was covered with a nylon net to give it physical strength. An electrode prepared in this way is referred to below as a film-FDH-CPE. When not in use, the film-FDH-CPE was stored at 5°C in MacIlvane buffer (pH 4.5) containing 0.1% Triton X-100.

RESULTS AND DISCUSSION Electrochemical response of the film-FDH-CPE to D-fructose (Fru) When a fixed potential was applied to the tilmFDH-CPE immersed in a pH4.5 MacIlvane buffer containing 0*10/oTriton X-100 and when Fru was added to the solution, the film-FDHCPE showed an anodic current response and a steady-state current was obtained about I min after the addition of Fru. Figure 1 shows the dependence of the steady-state current on the potential applied to the film-FDH-CPE. The steady-state current started to appear at 0 V and increased with increasing positive potential to approach a limiting current. In the following, we shall discuss the steady-state current I measured at 0.5 V. The dependence of I on the concentration cr,,, of Fru was studied with the film-FDH-CPE. I increased with increasing CF,.,, to approach saturation, as illustrated in Fig. 2. The result was analyzed as follows: 1 = In&(1

+ &I/+&

(1)

The two parameters K,,, and I,,,,, could be determined from the slope and the intercept of

Amperometric jktose

sensor based on direct bioelectrocatalysis

301

There was no significant difference in the K,,, values when measured using the Lx electrodes made with smaller amounts of immobilized FDH, namely 1.1 X lo-‘* mol and 2.2 X lOTi mol. The latter corresponds to 2.4 X lo-‘* mol/cm* and is of the order of monomolecular coverage for a protein of this size, of molecular weight 140 000. When FDH immobilized on the electrode was denatured by dipping the film-FDH-CPE into an acidic (pH 1.0) solution for 30 min, the film-FDH-CPE with the denatured FDH showed no such current response as that in Fig. 2. The results indicate that the current response observed with the tilmFDH-CPE in pH 4.5 buffer is due to the electroenzymic oxidation of D-fIUCtOSe. Since neither the electrolyte solution nor the film-FDH-CPE itself contains redox molecules functioning as an electron transfer mediator, the current response is attributed to the unmediated bioelectrocatalysis as observed with GADH (Ikeda ec al., 1988~) and ADH (Fushimi, 1988). Although the redox wave ascribable to the redox reaction of the immobilized FDH could not be observed in cyclic voltammetry, the site of the immobilized FDH which interacts directly with the electrode is assumed to be heme c, of which the redox potential may be considered to be about the same as that of GADH, -97 mV (Matsushita et al., 1982). Further studies in this direction are under way in our laboratory. In any event, the above results clearly show that the film-FDH-CPE shows a current response to Fru without the addition of any artificial electron transfer mediators to the solution. and

1

Cl4 uA I

/

0’

.o--

E I volt

vs.AglAgCl

Fig. 1. Dependence of the steady-state current I on the potential applied to thefilm-FDH-CPE. I was measured at 15°C in pH 4.5 Ma&vane buffer containing 0 1% Triton X-100 and 20 rnM Fru. The broken line shows the current obtained in the absence of Fru. 1.0

‘: 0.5

Film-FDH-CPE as an unmediated amperometric fructose sensor 0

I 10

I 20

30

cFnr/ mM Fig 2. Dependence of 1 at 05 Van CF,,,.1 was measured at 25°C in pH 4.5 MacIlvane bufler containing 0.1% Triton X-100. The inset shows the plot of cFm/I against cF,.,,.

versus cFm plot (Hanes-Woolf theCF,-&,,~~

plot in enzyme kinetics) given in the inset to Fig. 2. Experiments of the same type were performed using two other film-FDH-CPE devices prepared by the same procedure, and the K, and I,,,,, values of the electrodes tested were’determined as 8.0 f 1-OmM and I.4 f 0*4pA, respectively.

The dependence of Z on cF,,, was not linear over the whole range of cFrUstudied, 0.2-30 mM (Figs. 2 and 5) and the magnitude of Z was much smaller than that obtained (Ikeda et al., 1989) with the film-FDH-CPE in the presence of an electron transfer mediator such as p-benzoquinone. The results indicate that the dialysis membrane covering the FDH layer in the film-FDH-CPE has little effect on the magnitude of Z, i.e. Z is mainly controlled by the kinetics of the FDH reaction in the enzyme layer (Ikeda et al., 1988b). Thus, the magnitude of Z was sensitive to the pH of the solution, as seen in Fig. 3, in which two film-FDH-CPE devices were used: one for the

302

Tokuji Ikeda, Fumio Matsushita, Mirsugi Senda

o’201

IL: 0

cl

40

50

60

7.0

PH

Fig. 3. Dependence of I at .? rnht Fru on PH. Data denoted by 0 and 0 were obtained with two d@erentJilm-FDHCPE devices.

measurements at pH 5-3 and the other for pH 5-7. The maximum current response was obtained at around pH 45-5.0, close to the optimum pH of FDH, pH 4.0 (Ameyama et al., 1981). When the film-FDH-CPE devices were used for measurements at pH less than 3 or above 7, the electrodes became inactive owing to the denaturation of the enzyme. Accordingly, control of the solution pH is critical for the film-FDHCPE to be used as a sensor. The dependence of I on temperature was studied between 5°C and 30°C. I increased with increasing temperature in this range. The temperature coefficient of I was 2.3% at 25°C. The magnitude of I remained unchanged during the course of continuous measurements for 2 h at 25°C. At above 30°C however, Z decreased gradually; the higher the temperature, the greater the rate of decrease. In contrast, no decrease in I was observed during more than 24 h of continuous measurements at low temperatures such as 5°C. In the following, the characteristics of the tilmFDH-CPE are studied at pH 4.5 and at 25°C. Figure 4 shows the time dependence of the current response to Fru. The 90% level of the steady-state current was attained 50 s after the addition of Fru in the solution. This time dependence of the current response may be controlled by the permeation rate of the substrate in the dialysis membrane (Senda et al., 1986). The diffusional process in the membrane should also have an effect on the current response in the steady state, but the effect is not significant in our film-FDH-CPE, as mentioned above, the process of the enzymatic reaction being the step mainly controlling the magnitude of the steady-state

t 30s

Fig. 4. Time dependence of the current response of thefilmFDH-CPE. At the point indicated by the arrow, Fru was added to the solution to make a 02 rnMFru solution. The horizontal line shows the current zero level. 0.3 71

d /

0

0.2 /

a II

P 0.1 -

0

P P

1.0

2.0

‘Fru ’ mM

Fig. 5. Calibration graph for Ftu obtained with thejilmFDH-CPE,

current (Senda et al., 1986). Figure 5 shows the calibration graph for Fru in the range 0.2-2.0 mM. The reproducibility of the current response was examined using 2 mM Fru and the coefficient of variation was 1.9% (n = 10). Almost the same calibration graphs were obtained for the ftlmFDH-CPE devices with smaller amounts of FDH, as mentioned above, indicating that the diffusional resistance to the substrate in the enzyme layer is negligible in our film-FDH-CPE devices. Oxygen had no effect on the current response: deaeration by passing nitrogen into the solution caused no change of the current magnitude. No interference was observed on the additions of other sugars (2 mM), such as glucose, galactose, sucrose, lactose, maltose, xylose and arabinose. However, if the sample solutions contain small molecules directly oxidizable at the

Amperometricjiuctose

sensor based on direct bioelecttvcatalysis

carbon paste electrode, they may interfere with the measurement of Fru. In particular, when the film-FDH-CPE electrode is to be used for the measurement of Fru in foodstuffs, the interference from L-ascorbic acid (AsA) must be taken into account. Figure 6A shows the effect of AsA on I at 2 mM Fru, evidently showing a significant effect of AsA. Therefore, we examined a method for eliminating the interference from AsA using ASOD. ASOD catalyzes the oxidation of AsA by dioxygen: AsA + 02 +

dehydroascorbic

acid + Hz0 (2)

At the potential of +0*5 V where I is measured, both 02 and dehydroascorbic acid are electrochemically inactive. Therefore, if AsA is thoroughly consumed by the above reaction in the enzyme layer on an electrode, the electrode will show no current response to AsA. Figure 6B shows the current response to 2 mM Fru of the film-FDH-CPE which contains ASOD (1.2 units) together with FDH in the enzyme layer (tilmASOD/FDH-CPE), where pH of the solution was adjusted to 5.5, the optimum pH for ASOD. No

Fig. 6. E#& ofAsA on I at 2 mM Fru obtained with (A) the jilm-FDH-CPE and (B) the film-ASODLFDH-CPE.

303

interference was observed with the film-ASOD/ FDH-CPE up to 0.1 mM AsA as expected. Using the film-FDH-CPE and the tilmASOD/FDH-CPE, we measured the concentration of Fru in an apple and a lemon. The results are given in Table 1 together with the results obtained by the F-kit method. Both electrodes gave results agreeing well with that obtained by the F-kit method in the case of an apple. In the case of a lemon, however, the tilmFDH-CPE gave a rather high value compared with that by the F-kit method; the result obtained using the film-ASOD/FDH-CPE agreed well with that by the F-kit method. The lemon contained 1.72 mM AsA, corresponding to 2.9% of the concentration of Fru. This is a high enough concentration to affect the current response of the film-FDH-CPE but sufficiently low to allow the elimination of the effect by using the film-ASOD/ FDH-CPE (Fig. 6). The content of AsA in the apple was 7.6 X IO-* mM, which corresponds to 1.8 X 10-*0/o of the concentration of Fru. No interference is expected at this concentration level of AsA (Fig. 6A). In conclusion, the filmFDH-CPE can be used as a Fru sensor for the measurements of Fru in fruits so long as the AsA content is low, as in the case of an apple. When a considerable amount of AsA is present, the interference from AsA can be eliminated by using the film-ASOD/FDH-CPE. Finally, long-term stability was examined with the film-FDH-CPE; the-current fur 2 mM Fru was measured every 2 days at 25’C, and the electrode was stored when not in use at 5°C in pH 4.5 Macllvane buffer containing 0.1% Triton X-100, the solvent suitable for FDH (Ameyama ef al., 1981). Although the magnitude of the current response decreased gradually to 30% of the original value after 2 weeks, no appreciable decrease in current was observed during the course of continuous measurements for 2 h at 25°C even when the measurements were done

TABLE 1

Comparison of the present methods with the F-kit method Sample

Present methods Film-FDH-CPE (mM)

Apple Lemon n. number of runs.

422+4(n=3) 92.7 + 41 (n = 3)

F-kit method (mM)

Film-ASOD/FDH-CPE

(mM)

420 + 3(n = 3) 60.6 + 2.5 (n = 3)

420(n = 1) 58.9 (n = I)

304

after 10 days. The film-FDH-CPE could be used as a Fru sensor for at least 10 days.

ACKNOWLEDGMENT This work was supported in part by a Grant-in Aid for Scientific Research (No. 01560150) from the Ministry of Education, Science and Culture of Japan.

REFERENCES Ameyama, M., Shinagawa, E., Matsushita, K & Adachi, 0. (1981). o-Fructose dehydrogenase of Gluconobacter industrius: purification, characterization and application to enzymatic microdetermination of D-fructose. L Bacterial, 145(2), 814-23. Armstrong, F. A. Hill, H. A 0. &Walton, N. J. (1988). Direct electrochemistry of redox proteins. Act. Chem. Res., 21,407-13. Cass, A E. G., Davis, G.. Francis, G. D., Hill, H. A 0.. Aston. W. J., Higgins. 1. J.. Plotkin, E. V.. Scott, E. V. P. & Turner, A. P. F. (1984). Ferrocenemediated enzyme electrode for amperometric determination of glucose. Anal. Chem.. 56(4). 667-71. Fushimi, F. (1988). Bioelectrocatalysis at Dgluconate dehydrogenase-modified electrodes. Master’s Thesis, Kyoto University, 83-92. Ikeda, T., Hamada, H., Miki, K. & Senda, M. (1985). Glucose oxidase-immobilized benzoquinone-

Tokuji Ikeda, Fumio Matsushita. Mitsugi Senda

carbon paste electrode as a glucose sensor. Agric. Biol. Chem.. 49(2). 541-3. Ikeda, T., Fushimi, F., Miki, K. & Senda, M. (1988a). Direct bioelectrocatalysis at electrodes modified with D-gluconate dehydrogenase. Agric. Biol. Chem., 52(10), 2655-8. Ikeda, T., Miki, K & Senda, M. (1988b). Theory of catalytic current at the biocatalyst electrode with entrapped mediator. Anal. Sci. (Tokyo), 4, 133-8. Ikeda. T.. Fushimi, F., Miki, K & Senda, M. (1989). Amperometric biosensors based on quinoptotein-, flavoprotein-dehydrogenases: methods of steadystate current measurements and voltammetric measurements at higher sensitivity. Bunseki Kagaku (Tokyo), 38(1 l), 583-8. Matsushita, K. Shinagawa, E. & Ameyama, M. (1982). DGluconate dehydrogenase from bacteria, 2-keto-D-gluconate-yielding, membrane-bound. Methods Enzymology, 89, 187-93. Redfeam, E. R (1967). Isolation and determination of ubiquinone. Methods Enzymology, 10, 381-4. Senda, M. & Ikeda, T. (1986). Biopolymer-modified electrodes. In Koubunshi Hyoumen No Kiso To Ouyou (Polymer Surfaces, Fundamentals and Applications), Vol. 2. ed. Y. Ikada. Kagaku Dojin. Kyoto, pp. 201-26. Senda, M., Ikeda, T., Miki, K & Hiasa, H. (1986). Amperometric biosensors based on a biocatalyst electrode with entrapped mediator. Anal. Sci. (Tokyo), 2, 501-6. Tarasevich, M. R. (1985). Bioelectrocatalysis. In Comprehensive Treatise of Electrochemistry. Vol. 10. ed. S. Srinivasan. Y. A. Chizmadhev, J. GM. Bockris. B. E. Conway. & E. Yeager. Plenum. New York, pp. 23 I-96.