Anal. Chem. 1995, 67,1684-1690
Preparation and Amperometric Glucose Sensitivity of Covalently Bound Glucose Oxidase to (2-Aminoethy1)ferroceneon an Au Electrode Susumu Kuwabata, Tsuyoshi Okamoto, Yoshio Kajiya, and Hiroshi Yoneyama*
Deparfment of Applied Chemistty, Faculty of Engineering, Osaka Univemity, Yamada-oka 2-1, Suita, Osaka 565, Japan
A multilayer film consisting of glucose oxidase (Gox)and (2-aminoethy1)ferrocene (2-AF) as an electron mediator has been prepared on an Au electrode with the use of glutaraldehyde as a cross-linkingagent. The amount of GOx and 2-AF deposited on the electrode is varied by changing the concentrationof 2-AFrelative to that of GOx in the preparation bath, and the highest deposition is obtained at an optimum concentration of 2-AF. Interestingly, the molar ratio of 2-AF to GOx in the film is -10, independent of relative compositions of 2-AF to GOx in the preparation bath. The prepared electrodeexhibitsfast amperometric responses to glucose, its sensitivitybeing almost proportional to the amount of the immobilized GOx. Kinetic studies employhgvoltammew and rotating disk electrode measurements suggest that the readion rate is controlled by glucose oxidation by GOx and/or the electronmediation of 2-AFrather than difhisionof glucose in the film. Amperometric glucose sensors based on glucose oxidase (GOx) have been the focus of intense interest for the past two and their development has reached the point where they are now commercially available. The ultimate goal would be the development of a solid-state chip upon which the GOx and an appropriate electron mediator are immobilized. Thus, many of the most recent efforts to refine this technology have focused on the reduction of size of the sensor device and improvements in both sensitivity and response time."-8 In order to achieve both high sensitivity and fast response time, the choice of the mediator species and immobilization of both GOx and the electron mediator are certainly important. However, it is most critical that the immobilization of GOx does not hinder its catalytic activity. It has recently been demonstrated that GOx can easily be incorporated into conducting polymer filmsg such as p ~ l y p y r r o l e , poly(N-methylpyrrole) ~~-~~ ,'6J7 polyaniline,1-8-20 and polyindoleZ1by electrochemical polymerization in the presence (1) Mell, L. D.; Maloy, J. T. Anal. Chem. 1975,47,299. (2) Shu, F. R.; Wilson, G. S. Anal. Chem. 1976,48, 1679. (3) Murray, R W. Acc. Chem. Res. 1980,13,135. (4)Ianniello, R; Yacynych, A. M. Anal. Chem. 1981,53,2090. (5) Cass, A. E. G.; Davis, G.; Francis, G. D.; Hill, H. A. 0.;Aston, W. J.; Higgins, I. J.; Plotkin, E. V.; Scott, L. D. L.; Turner, P. F. Anal. Chem. 1984,54,667. (6) Degani, Y.; Heller, A. J. A m . Chem. SOC. 1989,111, 2357. (7) Schuhmann, W.; Ohara, T. J.; Schmidt, H.-L.; Heller, A J .Am. Chem. SOC. 1991,113,1394. (8) Hale, P. D.; Boguslavsky, L. I.; Inagaki, T.; &an, H. I.; Lee, H. S.; Skotheim, T.A. Anal. Chem. 1991,63,677. (9) Bartlet, P. N.; Cooper, J. M. J. Electroanal. Chem. 1993,1. (10) Umarla, M.; Waller, J. Anal. Chem. 1986,58,2979. 1259. (11) Foulds, N. C.; Lowe, C. R. J. Chem. Soc., Faraday Trans. 1 1986,82, 1684 Analytical Chemistty, Vol. 67,No. 10, May 15, 7995
of GOx. In our previous w 0 r k , ~ 2 -we ~ ~demonstrated that polypyrrole films containing both GOx and an electron mediator such as ferro~enecarboxylate~~~~~ or hydroq~inonesulfonate~4 possess reasonable amperometric sensitivities to glucose. It is most significant that these composite films function as sensors in the absence of any electron mediator species in the sample solution. However, the sensitivity of these film systems was lower than predicted based on the amount of GOx and mediator known to be present in the film.24 It was concluded that the sensitivity and response were limited by analyte diffusion in the polymer layer. Other workers have employed self-assembled monolayers to immobilize m o m and multilayers of such species as malic glutathione r e d u c t a ~ e ? ~and ,~~ In a recent comm~nication,~~ we reported our attempts to immobilize GOx and a mediator species of (2-aminoethy1)ferrocene(2-AI9 to a selfassembled monolayer of Caminothiophenol on an Au electrode using glutaraldehyde as a cross-linking agent. Our immobilization method produced a multilayer of GOx whose sensitivity exceeded that of monolayer Later, it was found that the multilayer film of GOx and 2-AF can be adsorbed quite strongly on the electrode substrate even in the absence of Caminothiophe no1 monolayer. This finding led us to investigate the adsorption behavior and electrochemical properties of the multilayer, with specific emphasis on its sensitivity to glucose. As will be shown below, the multilayer flm exhibits a high amperometric sensitivity to solution phase glucose. We will discuss the marked improve(12) Foulds, N. C.; Lowe, C. R Anal. Chem. 1988,60, 2473. (13) Tamiya, E.; Karube, I.; Hattori, S.; Suzuki, M.; Yokama, K Sens. Actuators 1989,18,297. (14) Matsue, T.; Kasai, N.; N a n " , M.; Nishizawa, M.; Yamada, H.; Uchida, I.J. Electroanal. Chem. 1991,300,111. (15) Janda, P.; Weber, J.J. Electroanal. Chem. 1991,300,119. (16) Barlett, P. N.; Whitaker, R G. J. Electroanal. Chem. 1987,224,27. (17) Bartlett, P. N.; Whitaker, R G. J. Electroanal. Chem. 1987,224,37. 359. (18) Bartlett, P. N.; Whitaker, R G. Biosenson 1987/1988,3, (19) Shinohara, H.; Chiba, T.; Aizawa, M. Sens. Actuators 1988,13,79. (20) Cooper, J. C.; Hall, E. A H. Biosensors 1992,7,473. (21) Pandey, P. C. J. Chem. Soc., Faraday Trans. 1988,84,2259. (22) Iwakura, C.; Kajiya, Y.;Yoneyama, H.J. Chem. Soc., Chem. Commun. 1988, 1019. (23) Kajiya, Y.; Iwakura, C.; Yoneyama, H. MRS Int. Meet. Ado. Mater. 1989, 12,249. (24) Kajiya, Y.; Sugai, H.; Iwakura, C.; Yoneyama, H. Anal. Chem. 1991,63,49. (25) Willner, I.; Riklin, A Anal. Chem. 1994,66,1535. (26) Katz, E.; Riklin, A; Willner, I. J. Electroanal. Chem. 1993,354,129. (27) Willner, I.; Lapidot, N.; Riklin, A; Kasher, R; Zahavy, E.; Katz, E. J. Am. Chem. SOC.1994,116,1428. (28) Tatsuma, T.; Okawa. Y.; Watanabe, T. Anal. Chem. 1989,61,2352. (29) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992,64,625. (30) Tatsuma, T.; Watanabe, T. Anal. Chem. 1992,64,630. (31) Willner, I.; Riklin, A; Shoham, B.; Rivenzon, D.; Katz, E.Adu. Mater. 1993, 5, 912. (32) Kajiya, Y.; Okamoto, T.; Yoneyama, H. Chem. Left. 1993,2107. 0003-2700/95/0367-1684$9.00/0 0 1995 American Chemical Society
ment over conducting polymer systems in terms of the open structure of the multilayer film of GOx and 2-AF. EXPERIMENTAL SECTION Glucose oxidase (EC 1.1.3.4) obtained from Aspergillus niger (Type VII) was commercially available from Sigma. Horseradish peroxidase (EC 1.11.1.7) was purchased from Wako Pure Chemicals. Water was purified by double distillation of deionized water. Diethyl ether used for preparation of (2-ammonioethy1)ferrocene iodide was twice distilled under dry Nz prior to use. (2-Ammonioethy1)ferrocene iodide was synthesized according to the l i t e r a t u ~ - e , ~as~follows. -~~ (Nflfl-Triethy1ammonio)methyll ferrocene iodide was prepared by quaternization of [(NJV-dimethy1amino)methyllferrocene using CH3I in methan01.3~A 0.2 mol sample of the obtained compound was dissolved in 0.8 dm3 of aqueous solution containing 1 mol dm-3 potassium cyanide, and the resulting solution was boiled until ferrocylacetonitrile solid was pre~ipitated.~~ Then, ferrocylacetonitrile was dissolved in dried diethyl ether to give 0.35 mol dm-3, and the resulting solution was added drop by drop to 0.5 mol dm-3 LiAlH4 suspended in diethyl ether to give (2-aminoethy1)ferrocene precipitate, which was then dissolved in acetonitrile and quaternized by adding hydroiodic acid (57%HI in water) to give a final product of (2-ammonioethy1)ferroceneiodide. Careful control of the addition of the ferrocylacetonitrile solution is required to prevent vigorous reaction.34 AU other chemicals were of reagent grade and used without further purification. All electrochemical measurementswere carried out using a phosphate buffer (PH 7.0) prepared by mixing 0.1 mol dm-3 NaHzP04 solution and 0.1 mol dm-3 NaOH, after being bubbled with Nz gas for more than 1h. An Au plate having an exposed area of 0.27 or 0.13 cm2 was used as an electrode substrate except for rotating disk electrode measurements, where an Au disk electrode (6 mm diameter) was used. Prior to measurements, the electrode surface was successively polished with 1.0 and 0.3 pm alumina, followed by ultrasonication in distilled water for 1 h. A platinum foil having a 5 cm2 area and a saturated calomel electrode (SCE) served as counter electrode and reference electrode, respectively. Immobilization of glucose oxidase and (2-aminoethy1)ferrocene to the Au electrodewas accomplished by immersing the electrode in phosphate buffer (PH 7.0) containing 1 g dm-3 GOx, 5 mmol dm3(2-ammonioethy1)ferrocene iodide, and 5.6 wt % (-455 mmol dm-3) glutaraldehyde for 2 h except where noted. White precipitates appeared and increased gradually with time, showing that random polymerization of GOx took place in the bath. The Au electrode kept its mirror-finished surface as if nothing was deposited on the electrode. It was, however, found that a very thin layer of GOx was immobilized on the electrode surface, as will be mentioned below. The resulting electrode was rinsed with phosphate buffer to remove weakly adsorbed species. This electrode will be denoted here as GOx/AF/Au. Amperometric responses of the prepared electrodesto glucose were measured by addition of aliquots of a glucose stock solution into phosphate buffer in which the electrode was polarized at given potentials, and the resulting oxidation currents were monitored. The measurements were carried out at 30 "C with use of a potentiostat (Nikko Keisoku NPOT-2501), a potential sweeper (33) Lindsay, J. K.; Hauser, C. RJ. Org. Chem. 1957,22,355. 653. (34) Lednicer, D.;Lindsay, J. IC; Hauser, C. R J. Ow. Chem. 1958,23, (35) Hauser, C. R.; Lindsay, J. K.; Lednicer, D. J. Org. Chem. 1958,23, 358.
Wikko Keisoku N P S W , and a polyrecorder (Yokogawa LR4120). Except where noted, the measurements were carried out without agitation of the solution. Detailed procedures were given in our previous paper.24 Rotating disk electrode measurements were carried out using a Nikko Model W E - 1 ring-disk electrode measurement system. The amount of GOx immobilized on the electrode surface was estimated by measuring the fluorescence intensity of flavin adenine dinucleotide (FAD), which is known to be present as the redox centers in 2 mol/for mol of GOx molecules. Since the fluorescenceintensity of FAD bound to apo-GOx is it was desired to get measurements of FAD on its release from GOx. For this purpose, the GOx/AF/Au electrodes were soaked in 5 mL of 8 mol dm-3 urea solution under agitation for 2 days under a NZ atmosphere in the dark. The fluorescence intensity of the resulting solution was measured at 525 nm with excitation at 370 nm, and the amount of the immobilized GOx was evaluated using a calibration curve between the concentration of FAD and the fluorescence intensity, which was obtained by dissolving commercially available FAD into 8 mol dm-3 urea solution for concentrations smaller than 1.0 x mol dm-3. The 2 day soaking of the electrode in the urea solution seemed enough to release FAD from GOx as already r e p ~ r t e d ?because ~ . ~ no change in the fluorescence intensity was observed for more than 2 days, as long as the solution was stored in the dark under Nz atmosphere. The enzyme activity of the immobilized GOx was evaluated by determining the amount of indamine dye produced by the following reaction:39
+ 0, + H,O GOx gluconolactone + H,O, + MBTH + DEA indamine dye + H,O
glucose
(1)
H,O,
(2)
where MBTH is 3-methyl-2-benzothiazolinonehydrazone, DEA is Nfldiethylaniline, and POX is horseradish peroxidase. The measurement bath was prepared by mixing three kinds of solutions. The fist solution was 2 mL of 0.1 mol dm3acetic acid solution containing 0.27 mmol dm-3 disodium ethylenediaminetetraacetate, 2.5 mmol d r r 3 MBTH, and 10 mmol dm-3 DEA. The second was 0.5 mL of 0.1 mol dm-3 acetic acid solution containing 4 units of POX. The last was 0.5 mL of 0.1 mol dm-3 acetic acid solution containing 0.12 mol dm-3 glucose. After each solution was bubbled with air for 30 min, all solutions were mixed together, followed by being kept at 25 "C under agitation. It has been confirmed that before addition of GOx no appreciable change occurred in the absorption spectrum of the mixed solution for more than 2 h. The GOx/AF/Au electrode was immersed in the solution for 5 min, and then 1.0 mL of 0.5 mol dnr3HCl solution was added to stop the reaction. The amount of indamine dye produced was determined from absorbance of the resulting solution at 590 nm using the molar extinction coefficient of 29.05 cm2pmol-l obtained under the assay condition. RESULTS AND DISCUSSION Immobilization of GOx and 2-AF on Au Electrode. The GOx/AF/Au electrode prepared was dried in a vacuum for 5 h (36) Tsuge, H.; Mitsuda, H. J. Vitaminol. 1971,17, 24. (37) Swoboda, B. E. P. Biochem. Biophys. Acta 1969,175, 365. (38) Swoboda, B. E. P. Biochem. Biophys. Acta 1969,175, 380. (39) Toyobo Enzyme Catalog, 1988 p 220.
Analytical Chemistry, Vol. 67, No. 10, May 15,1995
1685
Au
glutaraldehyde
2-AF
Schematic illustration of the hypothetical structure of a multilayer of GOx and 2-AF deposited on the Au electrode.
Figure 2.
t
- 4
-6 0
0.2
0.4
0.6 - 3
E vs. SCE ( V )
1686 Analytical Chemistry, Vol. 67,No. 70,May 75, 7995
-5
E
P
'0
Figure 1. Cyclic voltammogram of GOx/AF/Au electrode taken in deaerated phosphate buffer (pH 7.0) at dHdt = 10 mV s-l. The electrode was prepared by immersing the Au electrode for 2 h in phosphate buffer (pH 7.0) containing 1 g dm-3 glucose oxidase, 5 mmol d m 3 2-AF, and 5.6 wt % glutaraldehyde.
and observed by a scanning electron microscope (SEM; Hitachi S800).It was found that a film layer having a thickness of 0.2 f 0.01 pm was deposited on the Au electrode. The amount of GOx immobilized in the prepared film was evaluated to be 4.26 x lo-" mol cm-? The enzyme activity of GOx in the film was 64 mU cm-2, which was more than 30 times as large as that of a polypyrrole film containing GOx and hydroquinonesulfonate prepared with the deposition charge of 50 mC cm+, although the amount of GOx immobilized was larger in the latter case (2.8 x mol cm-2)?4 Referring to the principle of the enzyme activity measurement, the diffusion of oxygen and/or glucose should influence the activity value determined. The higher enzyme activity obtained at the GOx/AF/Au electrode suggests, therefore, that the GOx layer prepared in this study had a porous structure allowing easy diffusion of both oxygen and glucose in it as compared to the GOx-incorporated polypyrrole film. Figure 1 shows a cyclic voltammogram of the GOx/AF/Au electrode taken at 10 mV ssl in 0.1 mol dm-3 phosphate buffer @H 7). Well-defined oxidation and reduction waves appeared at around 0.3 V vs SCE, indicating the achievement of immobilization of 2-AF in the iilm. The peak potentials of the anodic and cathodic waves were 0.312 and 0.281 V vs SCE, respectively, from which the redox potential (E,,) of 0.297 V vs SCE was determined. This value was 0.130 V more positive than the original redox potential of 2-AF dissolved in the same electrolyte solution, suggesting that the amino group of 2-AF in the film made Schiff base bonding with an electron-withdrawingaldehyde group of glutaraldehyde. The integration of the anodic current wave shown by a hatched area in Figure 1gives the amount of the immobilized 2-AF of 4.37 x mol cm-2 that was -10 times as large as that of the immobilized GOx. Figure 2 is proposed as a structure model of the GOx/AF/Au electrode. In our previous study,32we used an Au electrode coated with a self-assembled monolayer of 4-ami-
N
7
- 2
I
2
L
-1
O l 0
I
I
1
I
1
I
20
40
60
80
100
120
'0 140
Immersion time / min Amount of GOx (0)and 2-AF ( 0 )immobilized on the Au electrode by immersing the electrode in phosphate buffer (pH 7.0) containing 1 g dm-3 glucose oxidase, 5 mmol d w 3 2-AF, and 5.6 wt YO glutaraldehyde as a function of the immersion time. Figure 3.
nothiophenol to bind covalently the GOx layer on the electrode surface by bridging GOx and amino group of the monolayer with glutaraldehyde. However, it was found that the GOx layer was strongly immobilized on the Au electrode in the absence of the self-assembledmonolayer if (2-aminoethy1)ferrocene and glutaraldehyde were present. Since GOx itself has low adsorbability to the Au substrate,4O a three-dimensional network created by reactions of GOx, 2-AF, and glutaraldehyde seems favored for high adhesion of the GOx layer to the electrode. Figure 3 shows the effect of immersion time on the amount of immobilized GOx and 2-AF in the prepared film. The effect of the concentration of 2-AF dissolved in the preparation bath is given in Figure 4. It is seen from these figures that the molar ratio of the immobilized 2-AF to GOx was -10 for all the electrodes prepared, suggesting that -10 amino groups of amino acid residues in one GOx molecule are involved in the immobilization of 2-AF. As Figure 3 shows, the amount of both GOx and 2-AF increased with increasing the immersion time up to 1h, beyond which a saturation tendency appeared. When the immersion chosen was long enough, the concentration of 2-AF influenced the immobilization of both GOx and 2-AF, and the maximum amount was achieved at 5 mmol dm-3, as shown in Figure 4. Even when no 2-AF was dissolved in the bath, the immobilization of (40) Szucs, A; Hichens, G. 0.;Bockris, J. 0 ' M . j . Electrochem. Soc. 1989,136, 3748.
0 0
N
E
0
s
-h
I
i' 0
2
4
6
8
0
0.05
0.1
0.15
(dE/dt) I V si
10
Concentration of 2-AF / mmol dm'3 Figure 4. Effects of concentration of 2-AF dissolved in the preparation baths on the amount of GOx (0)and 2-AF (0)immobilized on the Au electrode. Preparation bath: phosphate buffer (pH 7.0) containing 1 g dm-3 glucose oxidase, 5.6 wi % glutaraldehyde, and 2-AF of various concentrations given in the figure.
GOx took place, but its amount was as small as 5.5 x mol cm-2, suggesting that the presence of 2-AF in the deposition bath is essential to grow the GOx/AF film. Amiio groups of GOx and glutaraldehyde may react with each other even in the absence of 2-AF, but judging from the finding that the successive growth of GOx film did not occur in that case, glutaraldehyde may be involved in making bridges between neighboring amino groups presented in one GOx molecule rather than between those of different GOx molecules. It is well-known that glutaraldehyde dissolved in aqueous solutions is polymerized to give several kinds of polymers, though all of the generated polymers are not yet identi.tied.41-43 The polymerization of glutaraldehydeoccurs in such a way as to leave a very low concentration of the which must be involved in the reaction to give the three-dimensional network. As shown in Figure 3, the reaction to make the network occurs slowly due to the low concentration of glutaraldehydemonomers, taking -1 h to complete the reaction. During the course of the reaction, some glutaraldehyde monomers react with 2-AF to give N-4-formylbutylidene2-ferrocylethylamine, which is denoted here as 2-AF-boundglutaraldehyde. The produced 2-AF-boundglutaraldehyde can react with amino groups of GOx, giving 2-AF-bound GOx. Unbound glutaraldehyde also reacts with amino groups of GOx. However, the bridging between one amino group and another one of the same GOx molecule with glutaraldehydemust be hindered by the presence of the 2-AF bound to GOx, the degree being dependent on its surface concentration. If an unbound amino group or glutaraldehyde-bound amino group of GOx is surrounded by the bound 2-AF, it is used to bond to another GOx with the assistance of glutaraldehyde. Then the chance of binding two GOx molecules becomes great with increasing concentration of 2-AF in the preparation bath, but if the concentration of 2-AF exceeds an optimum value, a large fraction of the amino groups
of COX is occupied by the bound %AF,and the amino groups available for making bonds with other GOx molecules become scarce, resulting in a decrease in the amount of GOx and 2-AF immobilized in the film, as shown in Figure 4. According to the study on covalently binding of ferrocenecarboxylate to the amino group of GOx," the number of amino groups available for chemical mod~cationof one GOx molecule without denaturing of the enzyme is 13. If this number is valid for making bonds with glutaraldehydes,-3 amino groups of one GOx molecle are utilized for making bonds to other GOx molecules, as judged from the finding that the molar ratio of the immobilized 2-AF to GOx was -10 as mentioned above. ElectrochemicalProperties of WAF'/Au in the Absence of Glucose. As already shown in Figure 1,the cyclic voltammogram of the GOx/AF/Au electrode taken in phosphate buffer in the absence of glucose showed definite anodic and cathodic waves due to the redox reaction of the immobilized 2-AF. If the same measurements were made at a potential sweep rate (dE/dt) smaller than 0.05 V s-l, the separation of anodic and cathodic peak potentials became less than 59 mV, and anoidic peak current (Ip)increased linearly with increasing the potential sweep rate (dEldt), as shown in Figure 5a, indicating that the immobilized 2-AF exhibited electrochemical responses characteristic of the redox species adsorbed on the ele~trode.4~ In a range where dE/ dt is higher than 0.05 V s-l, however, the peak separation became
(41) Richards, F. M.; Knowles, J. R J. Mol. Biol. 1968,37, 231. (42) Wold, F. In Method in Enzymology, Vol. Enzyme Sfnrcture;Hirs, C. H. W., Timasheff, S. N., eds.; Academic Press: New York, 1972; p 623. (43) Monsan, P.; Puzo, G.; Mazarguil, H. Biochim. Biophys. Actu 1975,229,384,
(44) Badia, A; Carlini, R; Fernandez, A; Battaglini, F.; Mikkelsen, S. R; English, A M. J. Am. Chem. SOC.1993, 115, 7053. (45) Bard, A J.; Faulkner, L. R Electrochemical Methods; John Wiley & Sons Inc.: New York. 1980.
0
0.2 0.4 0.6 0.8 (dEldt)"/ Vi'* S i R
1
Figure 5. Anodic peak currents of cyclic voltammograms of the GOx/AF/Au electrode taken in deaerated phosphate buffer (pH 7.0) as a function of potential scanning rates (a) and their square roots (b).
Analytical Chemistry, Vol. 67,No. IO, May 15, 1995
1687
I LV
loo 100
Y
80
6 -
. 9
60
a0
6
-
\
Y
I
60
c C
-
f
0 40
40
20
20
0
0 0-
0.2
0.4
E / V vs. SCE 0
0.4
0.2
Figure 7. Steady-state /-E curves of the GOx/AF/Au electrode taken in deaerated phosphate buffer (pH 7.0) containing 100 (a), 10 (b), and 1 (c) mmol dm-3 glucose.
0.6
E vs. SCE I V
Figure 6. Cyclic voltammograms of the GOx/AF/Au electrode taken in deaerated phosphate buffer (pH 7) in the absence (a) and presence (b) of 30 mmol dm-3 glucose. dHdt = 10 mV s-l.
-60 mV, and Zp was in proportion to (dE/dt)’I2 as shown in Figure 5b, suggesting that the redox reaction of 2-AF changed its mode into the diffusioncontrolled reaction. The conversion of 2-AF to its oxidized form (2-AFY) accompanies two kinds of diffusion processes: one concerns the diffusion of electrons that must occur by hopping between 2-AF and 2-AF+ molecules fixed in the film, and the other that of electrolyte anions (HzPOd-) which must be incorporated into the film layer to compensate the generated positive charges. It seems unlikely that the latter process controls the redox reaction of 2-AF in this case because the film layer has the open structure, as already discussed. This anticipation was further conlimed by the rotating disk electrode measurements, as will be shown below. It is speculated that the electron hopping between 2-AF and 2-AJ? molecules determines the rate of the redox reaction of the GOx/ AF/Au at a potential sweep rate higher than 0.05 V The slope of the Zp vs (dE/dt)1/2 plots shown in Figure 5b is 3.75 x A V-1/2 s1/2 cm-2 from which DappllzC~ of 1.39 x mol cm-2 s1/2 is obtained by using15
Zp= (2.69 x 105)n3/2D,,,”2[2-AF](dE/dt)’/2
(3)
where n is the number of electrons involved in the reaction, Dapp is the diffusion coefficient, and Cm is the concentration of 2-AF in the film layer. If the film thickness of 0.2 pm obtained by the SEM observation was used, Cm was estimated to be 2.2 x mol ~ m -which ~ , gave Dapp value of 4.1 x cm2ss1. However, since it is likely that the GOx layer was swollen in the electrolyte solution, the actual Dappvalue might be a little larger than that estimated here. Electrochemical Oxidation of Glucose on GWAF/Au. Figure 6 shows cyclic voltammograms of the GOx/AF/Au electrode taken in phosphate buffer (PH7) in the absence (curve a) and presence (curve b) of 30 mmol dm-3 glucose. When glucose was dissolved in the electrolyte solution, a remarkable 1688 Analytical Chemistry, Vol. 67,No.
IO, May 15, 1995
increase in the oxidation currents was observed at potentials positive of 0.15 V vs SCE, indicating that 2-AF molecules immobilized in the film worked very well as an electron mediator between the electrode substrate and the redox center of GOx (FAD).When the GOx/AF/Au electrode was polarized at 0.35 V vs SCE and then a glucose stock solution was injected into the solution under agitation, the oxidation current reached its maximum for only few seconds, followed by a gentle decrease to a steady-statevalue. The steady-statecurrents were stable, and even if the agitation of the electrolyte solution were stopped, no change in the current value was seen. The fast reponse of the electrode is indebted to the electrode structure and makes a marked contrast to that of the GOx-incorporated polypyrrole film where a gradual increase in the oxidation currents was observed after injection of glucose.22 Figure 7 shows dependencies of the steady-state currents on applied potentials obtained in electrolyte solutions containing 100, 10, and 1mmol dm-3 glucose. Each current value was measured after applying the potential for 1 min, which was long enough to obtain steady-state currents. As the figure shows, the current value becomes constant at potentials positive of 0.32 V vs SCE. It is noticed that the half-wave potential ( E I ~ of z ) the obtained I-E curves was more negative than the redox potential of 2-AF in the film (0.287 V vs SCE). Calvo et al. reported the theoretical treatments of a polarization curve for electrochemical oxidation of glucose at an electrode that has immobilized GOx and an electron mediator on its surface.46According to their theory, E112 must coincide with E, of the electron mediator if the immobilized layer is so thin as to develop no significant concentrationgradient of all species in the layer, being in disagreement with the results obtained at the GOx/AF/Au electrode. It is suggested then that diffusional processes in the multilayer of GOx affect the kinetics of electrochemical oxidation of glucose at the GOx/AF/Au electrode. If this were the case, the diffusion of electrons by hopping between 2-AF and 2-AF+ would have a principal effect on the reaction kinetics because the porous structure of the GOx/ AF layer allows fast diffusion of glucose as well as electrolyte (46)Calvo, E.J.; Danilowicz, C.; Diaz, L.J. Chem. SOC.,Faraday Trans. 1993, 89,377.
0
20
40
80
60
100
120
140
Immersiontime I min
2ov 0 8 0
100
I
1
I
I
I
5
10
15
20
25
30
Concentration / mmol dme3 80
Figure 9. Calibration curves of the GOx/AF/Au electrode for amperometric detection of glucose at 0.35 V vs SCE taken in phosphate buffer (pH 7) saturated with NP (a) and air (b).
20
0 0
2
4
6
8
1
0
1
2
Concentrationof 2-AF/ mmol dm’ Figure 8. (a) Effects of the immersion time for preparation of the GOx/AF/Au electrode on the current response of the resulting electrode to 30 mmol dm-3 glucose. (b) As in (a) but for the concentration of 2-AF in preparation bath. The applied potential for glucose detection was 0.35 V vs SCE.
anions in it compared with the above electron hopping, as w ill be discussed in the rotating disk electrode measurements. As shown in Figures 3 and 4, the amount of the immobilized GOx and 2-AF was changed by changing the soaking time and the concentration of 2-AF dissolved in the preparation bath. Figure 8 shows the effect of these variables on the sensitivity obtained at 0.35 V vs SCE. It is apparent by comparing Figure 8 with Figures 3 and 4 that the sensitivity correlates well to the amount of GOx and 2-AF immobilized, suggesting that the oxidation of glucose by GOx was the ratedetermining step when the polarization potential was positive enough for the oxidation reaction to proceed rapidly. Figure 9 shows the steady-state currents obtained at 0.35 V vs SCE as a function of the glucose concentration for electrolyte solutions saturated with Nz (curve a) and air (curve b). In both cases, the response currents increased with increasing concentration of glucose up to 25 mmol dm-3, though the relation was not linear. When air is present in the solution, both 2-AF and hydrogen peroxide must be competitively produced in the regeneration of the redox center of GOx, but since hydrogen peroxide is not oxidized at the measurement potential chosen, the response currents in the presence of dissolved 02 are small compared with those in its absence. As noticed from this figure, the percentage of suppression of the currents became smaller with increasing glucose concentration, and above 10 mmol dm-3,which gave response currents greater than 60 f i cm-2, the suppression eventually became constant and -15 pA cm-2 suppression occurred on introducing 02, though the current response itself
showed an increasing tendency with increasing glucose concentration. The neutral phosphate buffer saturated with air contains -0.3 mmol dm-3 which is not large enough to give the maximum reaction rate of glucose oxidation since this value is less than the Michaelis constant of -0.5 m o l dm-3 obtained for the reduction of 02 to HzO2 by dissolved G O X . ~If~the concentration of glucose is increased under such the conditions, the supply of 02 should become the ratedetermining step of the glucose oxidation catalyzed by GOx. Certainly the results shown in Figure 9 suggest that the rate of glucose oxidation with 02 at the GOxl AFlAu electrode seems to be controlled by the 02 supply if the concentration of glucose is greater than 10 mmol d m 3 ,because a constant suppression by O2is seen for this concentration range. However, since the reaction rate determined by the 02 supply is lower than the rate of the regeneration reaction of GOx with the immobilized 2-AF+, a relatively small suppression of the response currents appears on introducing air into the electrolyte solution. If glucose oxidation by GOx is the ratedetermining step at the GOx/AF/Au electrode, the Michaelis-Menten equation is applicable to its steady-state response curve. Usually, the EadieHofstee form given by eq 4 is where Kmapp is the a p
z =,I
- K,”P(Z/CJ
(4)
parent Michaelis constant, I,, is the maximum current, and C is the concentration of glucose in the solution. Plots of the oxidation currents obtained under NZF i e 9a) as a function of I/[glucosel give a good linear relationship, from which Kmapp = 5.54 mmol dm-3 and I- = 102 ,LAcmW2are determined. The stability of the GOx/AF/Au electrode was tested by intermittent measurements of the glucose sensitivity at 0.35 V vs SCE in phosphate buffer containing 30 mmol dm-3 glucose at 30 “C after being taken from storage in glucosefree phosphate buffer (PH 7) at 4 “C. The measurements were made twice a day. A gradual decrease in the current response of -8% was observed (47) Gibson, Q. H.; Swoboda, B. E.; Massey,V.J.Bid. Chem. 1964,239, 3927. (48) Gregg, B. A; Heller, A. J. Phys. Chem. 1991,95,5976. (49) Gregg, B. A; Heller, A J. Phys. Chem. 1991,95, 5970. (50) Kamin, R A.; Wilson, G. S. Anal. Chem. 1980,52,1198.
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for the initial 8 days, but further storage for 20 days caused no change in the sensitivity. Rotating Disk Electrode Measurements. The steady-state oxidation current of 82.5 pA cm-2 was obtained by polarizing a rotating GOx/AF/Au disk electrode at 0.35 Vvs SCE in phosphate buffer containing 40 mmol dm-3 glucose. This value was completely independent of the rotating rate from 0 to 4000 rpm, indicating that the diffusion of glucose in the electrolyte solution has no influence on its electrochemicaloxidation. S i a r behavior has been reported by Gregg and Heller for an electrode coated with poly(viny1pyridine) in which (bpy)2OsC1 and GOx were imm~bilized.~~ If 5 mmol dm-3 anthraquinone-2-sulfonate (AQS) were used in place of glucose, the Levich plots shown in Figure 10 were obtained for the rotating bare Au and GOx/AF/Au electrodes. In these cases, since the electrode was polarized at 0.1 V vs SCE, which is positive enough to oxidize AQS but not to oxidize the immobilized 2-AF, the electron mediation of 2-AF to AQS is not expected. Then, AQS must diffuse in the GOx/AF film toward the Au electrode substrate to be oxidized. As Figure 10 shows, the oxidation currents increased with increasing rotating rate and the magnitude of the currents was much larger than that of glucose oxidation. The Coutecky-Levich plots (Z-l12 vs w 1 1 2 ) of the obtained results gave a linear relationship, and the diffusion coefficients of 5.30 x and 5.01 x cm2ssl were obtained at the bare Au and the GOx/AF/Au electrodes, respectively. Judging from very little d ~ e r e n c eof the diffusion coefficients between the two kinds of electrodes, the GOx/AF layer must be porous enough to allow easy diffusion of AQS molecules in the film layer. The same thing must be valid for glucose because the maximum molecular size of glucose (-0.5 nm) is smaller than that of AQS (-0.8 nm). Furthermore, it is noteworthy that the diffusioncoefficient of AQS obtained at the GOx/AF/Au electrode is 4 orders of magnitude larger than the Dappobtained for the redox reaction of the immobilized 2-AF. This finding supports the idea that the electron hopping between immobilized 2-AF molecules rather than diffusion of electrolyte anions of HzP04(-0.4 nm) determined kinetics of the redox reactions of 2-AF.
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o"'/ rpm"' Figure 10. Levich plots obtained from rotating disk electrode measurements of bare Au (a) and GOx/AF/Au (b) electrodes taken in deaerated phosphate buffer (pH 7.0) containing 5 mmol d m 3 hydroquinonesulfonate. The electrodes were polarized at 0.1 V vs SCE.
ACKNOWLEWMENT This research was supported by Grant-in-Aid for Scientific Research on Priority Area, 05235229 from the Ministry of Education, Science and Culture. The authors are indebted to Assistant Professor Colby k Foss Department of Chemistry, Georgetown University) for his help in preparation of this paper. Received for review September 23, 1994. Accepted March 2, 1995.@ AC940945X @
Abstract published in Advance ACS Absfructs, April 15, 1995.
