Anal. Chem. 1002, 64, 1254-1258
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Molecular Interface for an Activity Controlled Enzyme Electrode and Its Application for the Determination of Fructose Golam Faruque Khan, Eiry Kobatake, Hiroaki Shinohara, Yoshihito Ikariyama, and Masuo Aizawa* Department of Bioengineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 227, Japan
A PO0 enzyme (fructose dehydrogenase) Is electronlcally adsorbedInto a monolayer and then lmmoblllzedon a platlnum electrode surface by ekctrooxldatlve polymerlzatlon of polypyrrole. The conductlvepolymer matrhc works as an Interface that serves as an electron-shuttllng medlum between the enzyme and the electrode as well as the matrlx for enzyme Immoblllzatlon. The enzyme electrode demonstratesthat the PO0 enzyme Is rapidly turned over In the conductive materlal dependlng on the applled potentlal; Le., the actlvlty Is electrochemically controllable. I n other words, the enzyme In the conductlve thln membrane exhlblts a sharp Increase In catalytk actlvlty at the redox potentlal of the enzyme. On the other hand, less effklent electron transfer occurs at conventlonalelectrodeswithout polypyrrd.. Electrode propertks are reported when the electrode lo applld to the blosenrlng of o-fructose.
INTRODUCTION Direct electron transfer between the active-site prosthetic groups of enzymes and electrode materials is essential for the fabrication of the enzyme electrodes consisting of NADenzymes, FAD-enzymes, and PQQ-enzymes. Current extensive research makes us recognize that future developments are certain by improving reversible smooth electron transfer in bioelectronic devices. In the case of glucose oxidase, ferrocene and its derivatives are well-known to mediate electron exchange between the reduced form of the prosthetic FAD and electrode surfaces.lr2 Most recently, a significant number of systems have been developed that use synthetic redox couplesto shuttle electrons between the active prosthetic moiety and the electrode. Various sensors based on ferrocene derivatives,3-5 on quinone derivatives,&* and on electrodes comprising organic conducting materialslP”11 have recently been reported. In the case of dehydrogenase few effective approaches have been presented for the electron transfer between NADH and the transducer electrode mainly because of irreversible electrochemical regeneration of NADH. Recent innovative work
* To whom all correspondence should be addressed.
(1)Degani, Y.; Heller, A. J. Phys. Chem. 1987,91,1285-1289. (2)Degani, Y.; Heller, A. J. Am. Chem. SOC.1988,110,2615-2620. (3)Cass, A. E.G.; Davis, G.; Francis, D.; Hill, H. A. 0.;Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, A. P. F. Anal. Chem. 1984,56,667. (4)Lange, M. A.; Chambers, J. Q. Anal. Chim. Acta 1985,175,89-97. (5)Iwakura, C.; Kajiya,Y.;Yoneyama,H. J.Chem. Soc., Chem. Commun. 1988,1019-1020.(6)Ikeda, T.: Hamada, H.; Senda, M. Agric. B i d . Chem. 1986,50, 883-890. (7) Ikeda, T.; Shibata, T.; Senda, S. J.Electroanal. Chem. Interfacial Electrochem. 1989,261,351-362. (8)Kulys, J. J.; Cenas, N. K. Biochm. Biophys. Acta. 1983,744,57-63. (9)Kulys, J. J. Biosensors 1986,2,3-13. (10)Albery, W.J.; Bartlett, P. N.; Gaston, D. H. J.Electroanal. Chem. Interfacial Electrochem. 1985,194,223-235. (11)Mckenra, K.; Brajter, A. Anal. Chem. 1987,59,954-958. 0003-2700/92/0364-1254803.0010
on NAD regeneration has been the coupling of NAD with a mediator such as Meldola’s blue to make the coenzymecycling reproducible.12-14 However, these mediator-based biosensors suffer from inherent drawbacks: mediating species easily diffuse away from the electrode surface into the bulk solution and electron shuttling is inefficient. With these foregoing considerations in mind, several research groups have been pursuing new matrices where mediators can be stably retained and efficient electron shuttling can be assisted.IsJ6 The use of polypyrroleprovides a simplified procedure for electroimmobilization of enzymes on an electrode surface.”-19 In these studies the enzymecatalyzed oxidation of glucose was performed by the electrochemical oxidation of hydrogen peroxide either at the surface of the underlying electrode or at the conductive polymer matrix itself. However, in the clinical laboratory, an increasing trend toward whole blood testa requires the determination at less positive potentials where oxidizable ingredients such as ascorbate and uricate are hardly oxidized. We have demonstrated a new NAD-recycling system consisting of alcohol dehydrogenase, NAD, and Meldola’s blue, all of which are immobilized in a polypyrrole matrix. As the conductive polymer is shown to play the role of a molecular wire, electron shuttling between a base metal electrode and NAD proceeds smoothly with the aid of Meldola’s blue and the conductive wire. The molecular wiring was also effective for the reversible regeneration of glucose oxidase, a FADenzyme.20 The recent paper from this laboratory on PQQ (pyrroloquinolinelquinone) has extended the possibilities of using polypyrrole as a new interface for quinoproteins, since PQQ exhibits reversible electrochemistryat the polypyrrole-coated electrode.21 It is likely that the combination of polypyrrole and PQQ enzyme may be facilitated. We would like to emphasize that the conductive polymer plays dual roles, i.e., a medium of electron shuttering and a matrix for enzyme immobilization. In this report we present a novel approach for electron transfer between the prosthetic PQQ of fructose dehydrogenase and the base electrode through a polypyrroleinterface. (12)Gorton, L.;Torstenssen, A.; Joegfett, H.; Johnason, G. J. Electroanal. Chem. Interfacial Electrochem. 1984,161,103. (13)Lee, L. G.; Whitesides, G. M. Appl. Biochem. Biotechnol. 1987, 14,8414. (14)Bernofsky, C.; Swan, M. Anal. Biochem. 1973,53,452. (15)Hale, P. D.; Inagaki, T.; Karan, H. I.; Okamoto, Y.; Skotheim, T. A. J. Am. Chem. SOC. 1989,111,3482. (16)Hale, P. D.; Boguslavsky, L. I.; Inagaki, T.; Karan, H. I.; Lee, H. S.; Skotheim, T. A. Anal. Chem. 1991,63,677-682. (17)Iwakura, C.; Kajiya, Y.; Yoneyama, H. J.Chem. Soc., Chem. Commun. 1988,1010-1011: (18)Yabuki, S.:Shinohara, H.; Ikariyama, Y.; Aizawa, M. J. Electroanal. Chem. Interfacial Electrochem. 1990,277,179. (19)Aizawa, M.; Yabuki, S.; Shinohara, H. Biomolecular Interface. Molecular Electronics; Hong, F. T., Ed.; Plenum: New York, 1989;p 269. Chem. Com(20)Yabuki, S.;Shinohara, H.; Aizawa, M. J.Chem. SOC., mun. 1989,14,945. (21)Shinohara, H.; Khan, G. F.; Ikariyama, Y.; Aizawa, M. J. Electroanal. Chem. Interfacial Electrochem. 1991,304,75-84. 0 1992 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, lQQ2 1255
( b ) P P I FDH/ P I ( a ) FOH/ P I Flgurr 1. Schematlcrepresentation of electron transfer between PQQ of FDH and the electrode through the molecular Interface. Key: PP, Polypyrrole; FDH/R, FDKadsorbed Pt electrode;PP/FDH/Pt, PPcoated FDH/R electrode.
The concept of molecular interface is schematically presented in Figure 1. Although the PQQ moieties of FDH molecules in the vicinity of a transducer electrode can easily have an electron connection,22 it is a matter of difficulty to establish an electron transfer between the prosthetic groups of oppositely oriented FDH molecules and a transducer electrode (Figure la). Transfer of electrons between an enzyme and an electrode may be facilitated by introducing a kind of wiring which may assist the electron communication from the PQQ moiety to the electrode (Figure lb). In this study a monolayer FDH conductive polypyrrole with a thickness of 50-80 A is employed as a biomolecular interface in which an electron mediation is efficiently facilitated. In addition, we demonstrate that the activity of the membrane-bound FDH depends on applied potential and is controllable without losing ita activity in a certain potential range.
EXPERIMENTAL SECTION Chemicals. D-Fructose dehydrogenase (EC 1.1.99.11, grade 111,29.7 units/mg, MW 141 OOO) from Gluconobacter industrius, containing approximately 70% stabilizer (sugars, amino acids), was obtained from Toyobo Co. (Osaka), and was used without further purification. Pyrrole was purchased from Tokyo Kasei Co. (Tokyo) and was distilled before use. Other chemicals were guaranteed reagent grade. The supporting electrolyte was McIlvaine buffer of pH 4.5 prepared by mixing 0.1 M citric acid and 0.2 M disodium phosphate. Voltage-Assisted Adsorption of FDH on the Pt Electrode. Electrochemical adsorption of FDH on the Pt electrode surface was extensively studied in our previous paper.22 In this study, the optimum conditions clarified there for the monolayer FDH adsorption were employed; i.e., the enzyme (5 mg) was dissolved in 1mL of 0.1 M phosphate buffer of pH 6.0, which was followed by the voltage-misted adsorption by applying a positive potential of 0.5 V for 10min. A Pt plate having a surface of 1cm2was used as a working electrode. The FDH-adsorbed electrode was then thoroughly washed with distilled water and was kept in a buffer solution of pH 4.5 until further experiments. All the electrochemicalmeasurements were carried out by a conventional threeelectrode system. The electrode potential is referred to the Ag/ AgCl electrode. Polymerization of Pyrrole on t h e FDH-Adsorbed Electrode. Electrooxidative polymerization of pyrrole was performed on the FDH-adsorbed electrode in a solution containing 0.1 M pyrrole and 0.1 M KC1under anaerobic conditions at a potential of 0.7 V. The thickness of the polypyrrole membrane was controlled by a polymerization charge. After washing with distilled water, the PP/FDH/Pt electrode was kept in MaIlvaine buffer of pH 4.5 at 4 "C for several hours (8-20 h) until further experiments commenced. Measurement of Enzyme Activity. The enzymatic activity of the FDH/Pt and PP/FDH/Pt electrodes was determined by (22) Khan,G. F.; Shinohara, H.; Ikariyama, Y.; Aizawa, M. J. Electroanol. Chem. Interfacial Electrochem. 1991, 315, 263-274.
following the method in Toyobo Enzymes upon oxidation of D-fructose, the electron-acceptor ferricyanide is reduced to ferrocyanide, which forms Prussian blue with Fe2(S04)3.The appearance of Prussion blue was measured spectrophotometrically at 660 nm. One unit of FDH catalyzes the oxidation of 1 pmol of D-fructose (the formation of 2 pmol of Prussion blue) per min. Measurement of Electrochemical Activity. McIlvaine buffer solution of pH 4.5 was thoroughly deoxygenated by Nz bubbling for at least 10min. A three-electrode system consisting of a PP/FDH/Pt working electrode, a Pt-plate counter electrode, and a Ag/AgCl reference electrode was dipped in the deoxygenated buffer solution. A potential was applied,and the background current was allowed to decay to a steady-state value (5-15 min depending on applied potential). Then, fructose samples (in the same deoxygenated buffer solution) were injected into the gently stirred buffer solution. The resulting increase in anodic current was calculated at the response current which is directly proportional to the activity of the PP/FDH/Pt electrode. Apparatus. Differential pulse voltammetry was carried out by a Yanaco Polarographic P-1100 analyzer (Yanagimoto Co., Kyoto),and the results were recorded on a GraphtecX - Y recorder (WX 4421). The excitation waveform for the differential pulse voltammetry consisted of a small amplitude pulse (50 mV) superimposed upon a linear sweep (10 mV/s). The excitation signal with a frequency of 1Hz lasted for 50 ms, following which the sampling current was monitored at 30 ms. FDH adsorption and D-fructose detection were performed with a HA-301 potentiostat/galvanostat from Hokuto Denko Co. (Tokyo) and a YEW Type 3086 X-Y recorder from Graphtec Co. (Tokyo). Cyclic voltammetry was performed by the same instruments. All experiments were carried out in a conventional electrochemical cell consisting of a PP/FDH/Pt working electrode, Pt-plate auxiliary electrode, and a Ag/AgCl reference electrode. Except the fructose detection at constant potential, whichwas performed at 37 "C,all electrochemicalexperiments were conducted at room temperature (25 2 "C). The FDH activity was determined with a UV DEC-61OC thermostated spectrometer from Japan Spectroscopic Co. (Tokyo) with a Thermo Bath CTE-31 from Coolnics Co. (Tokyo).
*
RESULTS AND DISCUSSION Optimization of PP Membrane Thickness. Voltageassisted adsorption at 0.5 V for 5 min makes a monolayer-like coverage of FDH on the Pt electrode.22 However, in this present study, we continued adsorption for another 5 min to ensure 100% coverage of FDH on the electrode surface, because an uncovered Pt surface causes uneven electropolymerization on the surface. Almost all the FDH molecules on the FDH-adsorbed electrode surface seemed to demonstrate their activity because of the mild immobilization at less extreme potential and the easy diffusion of the substrate, although the employment of a polymer matrix to cover the enzyme layer seemed to affect the diffusion process of the substrate, causing the decrease in apparent enzyme activity. Therefore, it was very important to make the polymer membrane as thin as possible to minimize the effect of the membrane on substrate diffusion and to ensure the complete coverage of the enzyme layer. The effect of PP coating on the enzymatic activity of the PP/FDH/Pt electrode is shown in Figure 2 by changing the polymerization charge from zero to 20 mC. Four polypyrrole-interfaced electrodes were prepared at each polymerization charge, and the mean value was compared. The activity was determined by the optical method described previously. In general, the thickness of the polypyrrole membrane is directly proportional to the polymerization charge. Figure 2 shows that the apparent activity of FDH sharply dropped when the polym(23) Ameyama, A.; Shinagawa, E.; Mataushita, K.; Adachi, 0.J . Bacteriol. 1981, 145, 814. (24) Ameyama, A. Methods Enzymol. 1982,89, 20.
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ANALYTICAL CHEMISTRY, VOL. 64,NO. 11, JUNE 1, 1992
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Polymerization charge/ mC Effect of PP thickness on the enzymatic activity of the PPIFDHIR electrode. Activity was measured within 2-3 h after preparation. Figure 2.
1
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Potent i ai / V vs, AgIAgCI
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Figure 4.
Cyclicvoltammograms of the PP/FDH/Pt electrodeat ditferent
scan rates In Mcllvainebuffer of pH 4.5. Inset: a relationship between anodic peak current and scan rate. Scan rate (mV/s): (a) 500, (b) 400, (c)300, (d) 200, (e) 100. (CVs of the scan rates slower than 100 mV/s were not plotted here as these were recorded by upscaling the
current.)
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P o t e n t i a l I V v s , Agi AgCl Figure 3. Differential pulse voltammograms of FDH/Pt (- -) and PPI FDH/R (-) electrodes In McIlvaine buffer solution of pH 4.5. DPVs were obtained immediately after preparation of the FDH/R electrode and 20 after the preparation of the PP/FDH/Pt electrode. Conditions: pulse amplitude, 50 mV; pulse interval, 1000 ms;scan rate, 10 mV/s.
-
erization charge reached to 2-3-mC charge and then it fell gradually with the increase in polymerization charge. The P P membrane thickness prepared by passing a polymerization charge of 2-3 mC corresponds roughly to the monomolecular thickness of FDH. The thickness is estimated to be 50-80A,provided that the enzyme is spherical. In this study, a polymerization charge of 4 mC was passed to polymerize pyrrole, as it seems likely that this electricity is enough to cover the enzyme completely and to retain sufficient enzyme activity. Reversible Electron Transfer between FDH and t h e Electrode through the PP Interface. Differential pulse voltammetry of the FDH/Pt electrode was performed immediately after the enzyme adsorption because the enzyme on the Pt surface was easily desorbed from the surface.22 On the other hand, electrochemicalmeasurement of the PP/FDH/ P t electrode waa carried out after 20 h of storage in the buffer, since it took more than 6-8 h incubation to observe repeated electrochemicalresponse. After 6-8 h of storage the electrode demonstrated a stable response for a few days. The differential pulse voltammograms of the FDH/Pt and the PPIFDHI P t electrodes are shown in Figure 3. In both cases a pair of anodic and cathodic peaks were observed which were attributed to the electrochemical oxidation and reduction of the quinoprotein at redox potentials of 0.08 and 0.07 V (vs Ag/ AgCl), respectively. In our previous studyYz1 we found that the redox potential of PQQ in a solution of pH 4.5 is 0.06 V (vs Ag/AgCI) with the polypyrrole electrode. Therefore, the redox peaks of FDH are related directly to the electrochemical process of the prosthetic PQQ. The important difference between the electrochemicalactivities of these two electrodes
is as follows: by the employment of a conductiveP P interface, the redox potential of FDH shifted slightly (10 mV) to the redox potential of PQQ, which indicates more smooth and easier electrical shuttling between the prosthetic group of FDH and the electrode through the PP interface. In addition, the anodic and cathodic peak shapes and peak currents of the PP/FDH/Pt electrode were identical, which suggests revereibility of the electron-transfer process. One further point requires emphasis: the peak current of the PP/FDH/Pt electrode is about 8 times greater than that of the FDH/Pt electrode. The significant increase in redox peaks strongly supports our concept of the conductivebiomolecular interface illustrated in Figure 1. Due to the incorporation of FDH molecules in the *-conjugated conductive polymer on the surface of the electrode, the prosthetic PQQ electrically communicates with the base electrode through the molecular wiring, since the electron transfer between PQQ and the electrode was enhanced significantly in the presence of the PP film. These results clearly indicate that conductive PP works as an ideal molecular interface. Cyclic voltammograms of the FDH/Pt electrode were also obtained in a buffer solution of pH 4.5. However, as the background current was considerably larger than the far5daic currents, a pair of very small peaks were observed. CVs of the PP/FDH/Pt electrode at several scan rates are presented in Figure 4. A pair of symmetric and reversible anodic and cathodic peaks were obtained. Both peak currents increased with the increase of the scan rate. The peak height dependency on the scan rate is also insetted in Figure 4. The linear relationship between the peak current and the scan rate indicates that enough of the redox enzyme was immobilized in the P P interface electrode. The amount of FDH molecule immobilized on the surface was calculated to be 6.2 pg/cm2 from the gradient of the relationship by assuming that the electrochemical reaction proceeds in a two-electron-transfer process. This value was slightly higher than the actual amount of FDH adsorbed on the Pt electrode (6.0 pg/cm2).22These two values show some agreement. Therefore, this result also supports our concept that the FDH molecules interfaced to the electrode surface can reversibly and effectively transfer their electrons with the aid of molecular wiring. Potential-Controlled Activity of FDH in the Conductive Interface. Potential dependency of the biocatalytical activity of the PP/FDH/Pt electrode is shown in Figure 5.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992
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Potential/ V vs. AgIAgCI Flgurr 5. Dependency of the enzymatic activity of the PP/FDH/Pt electrode on applled potential. Potentials were applied to the same PP/FDH/R electrode. When the residualcurrent became steady state, fructose solution was injected to a final concentration of 5 mM, and the resultingIncrease In anodic current was measured as the response current.
The dependency was investigated by adding 5 mM fructose, and the resulting current response was compared. The applied potential-current response curve was divided into three parts. First, the potential range was less than the redox potential of FDH (0.07 V) where negligible response current was generated because in this potential range less oxidation of fructose occurred since very little FDH was in the oxidized form. Second, the potential range extended from the redox potential of FDH to the rest potential of the PP/FDH/Pt electrode (0.35 V), where a sharp increase of response current at around 0.1 V and then a gradual increase were observed. Third, the potential range was at a potential higher than the rest potential. The sharp increase in response current was observed up to 0.6 V. At a potential higher than this potential the response current fell sharply, probably due to the irreversible deactivation of FDH. The possible reasons for the deactivation a t higher potential may be (1)higher electrical field causes the conformational change of the FDH in such a way that the enzyme loses its prosthetic group, or it may change the structure of the enzyme into an inactive form, and (2) higher potential drastically changes the pH inside the membrane interface in a manner such that the enzyme loses ita activity. However, enzyme activity at the PP/FDH/Pt electrode can be controlled reversibly in the potential range from 0.1 to 0.6 V. The increase in anodic current upon the addition of fructose can be explained by considering the following three processes: (1)diffusion of substrate into the PP interface; (2) electron transfer from substrate to the enzyme upon the enzymatic oxidation of fructose (i.e., FDHPQQ is reduced to FDH-PQQH2); (3) reoxidation of reduced FDH to oxidized FDH-PQQ by releasing an electron to the electrode through the PP wiring. All of these three steps may be accelerated by the application of higher potential. However,we speculate that the last step is mainly responsible for the sharp increase in response current as described above. In such a case, the last step, Le., the electron-transferring step from FDH to the electrode, is the rate-limiting step. As the enzyme activity was reversibly controlled, the PP interface electrode was controllable as far as the applied potential was in the range from 0.1 to 0.6 V without any deterioration of the enzyme. Since PP has a positive charge in this potential
Flgure 6. Responses of (a) FDH/Pt and (b) PP/FDH/R electrodes upon the addiilon of o-fructose and glucose. The potentials of the electrodes were controlled at 0.4 V in a McIlvalne buffer of pH 4.5 at a controlledtemperature of 37 'C, and the solutionwas magnetlcally stlrred. When the background current became steady state (10-15 mln), each sample solution was injected and the resulting Increase of anodic current was measured as the response current.
range, the doped a-conjugated polymer works as a molecular wire which carries electrons between a biomolecule and an electrode. Application of the Reversible Electron-Transferring System to Biosensing of D-Fructose. As the potentials of the FDH/Pt and the PP/FDH/Pt electrodes are controlled more positive than 0.1 V, the FDH at the electrode surface can be retained in the oxidized form because the redox potential of FDH is less than 0.1 V (Figure 3). As long as the applied potential is higher than the redox potential, the oxidized form of FDH oxidizes D-fructose by transferring its two electrons to the electrode; thus a continuous flow of anodic current is observed. At a lower potential at around 0.1 V the residual current of both electrodes was found to be cathodic, as the rest potential in a solution of pH 4.5 was about 0.38 and 0.35 V for the FDH/Pt and the PP/FDH/Pt electrodes, respectively. To make the residual current as smallas possible and to observe the anodic current, the determination of Dfructose was performed a t 0.4 V. The response curves of these electrodes upon the addition of substrate are presented in Figure 6. To ascertain the selectivity of the enzyme electrode, we injected a series of saccharides. A typical example is shown in the figure. Satisfactory selectivity was obtained in the case of the PP/FDH/Pt electrode. The effectiveness of the PP film for the prevention of interfering substances was maintained as long as the electrode was covered with polypyrrole. However, the FDH/Pt electrode suffered from nonspecific response to saccharides such as glucose and galactose probably due to the simultaneous surface oxide formation-saccharide adsorption at the noble metal surface.25 The response time was as rapid as 2-3 s at the FDH/Pt and 3-5 s a t the PP/FDH/Pt electrode. The difference seems to be the reflection of the diffusion of the substrate in the interface. On the other hand, the response current of the PP/FDH/Pt electrode to D-fructose was about 4 times larger than that of the FDH/Pt electrode, which suggested effective electron shuttling in the interface. As mentioned earlier, the FDH/Pt electrode is unstable due to the desorption of the FDH molecules from the electrode surface. Therefore, it was not possible to use the same electrode repeatedly to draw a calibration curve for D-fructose. The calibration curve obtained by the PP/FDH/Pt electrode for D-fructose is presented in Figure 7. The response current ~~
~~
~
(25) Polta, J. A.; Johnson, D.C. Anal. Chem. 1985,57,1373.
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 11, JUNE 1, 1992 sensor decreased gradually after 1week, probably due to the inactivation of the immobilized enzyme and the deteorioration of the polypyrrole matrix. The stability of the PP/FDH/ P t electrode may be improved by handling in anaerobic conditions and storage in dry conditions.
CONCLUSION
0.0
0.2
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I
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was directly proportional to the D-fructose concentration in the concentration range from 10 pM to 10 mM. The slope of the linear curve was 90 nA/mM of D-fructose. The enzyme electrode was stable for over 1week with a slight increase on the third and fourth d a y . However, the output of the enzyme
Here, we have presented a novel approach for electron transfer between the prosthetic PQQ of fructose dehydrogenase and the base electrode through a polypyrrole interface. We demonstrated that the electrochemical activity of the membrane-bound enzyme is dependent on an applied potential and is controllable without the loss of enzyme activity in a wide potential range and activity is enhanced at an applied potential from 0.1 to 0.6 V because of the smooth and reversible electron transfer in the conductive interface. An application of this system in biosensors was described in the determination of D-fructose. The current response of the fructose sensor was found to be linear with the concentration from 10 pM to 10 mM. We believe that this study will open a new vista not only in electrochemical biosensors but also in the field of intelligent material applicable to activity-controllable bioreactors, biolelectrochemical fuel cells.
RECEIVED for review October 1, 1991. Accepted February 18, 1992. Registry No. PQQ, 72909-34-3; FDH,37250-85-4;polypyrrole, 30604-81-0; D-fructose, 57-48-7.
