14072
J. Phys. Chem. 1995,99, 14072-14077
Mechanism of a “Jumping Off’ Ferricenium in Glucose Oxidase-D-Glucose-Ferrocene Micellar Electrochemical Systems Alexander D. Ryabov,*,t,*Anton Amon>$ Raisa K. Gorbatova,’ Ekaterina S. Ryabova? and Boris B. Gnedenko” Division of Chemistry, G. V. Plekhanov Russian Economic Academy, Stremyanny per. 28, 113054, Moscow, Russia, Department of Chemistry, M. V. Lomonosov Moscow State University, 000958, Moscow, Russia, and Chemobyl Test Center Ltd., Rimsky-Korsakov St. 10, 127577, Moscow, Russia Received: April 4, 1995; In Final Form: June 10, 1 9 9 9
Incorporation of ferrocene (Fc), decamethylferrocene (DMFc), and n-dodecylferrocene (DDFc) into inner cavities of anionic, cationic, and nonionic micelles in aqueous solution tunes their observed redox potentials E112. The cyclic voltammetry study showed that anionic micelles of sodium dodecylsulfate (SDS) decrease while cationic and nonionic micelles of cetyltrimethylammonium bromide (CTAB) and Triton X- 100, in contrast, increase E112 of Fc. The effect of positively and negatively charged micelles on El12 in the case of DMFc was basically the same. Thus, solubilized ferrocene, but not its decamethyl and dodecyl analogs, couples electrochemically with glucose oxidase and provides a significant catalytic current in the presence of D-glucose. The rate constants for the oxidation of the reduced enzyme by the ferricenium ion are independent of the nature of the surfactant and, in the presence of 5% EtOH, fall in the range (4.3-5.7) x lo5 M-’ s-I. Based on this observation, a mechanism of the “jumping off’ ferricenium is presented and discussed. It is believed that Fc+ is captured by the enzyme in the rate-limiting step after its fast reversible dissociation from the micelle. The absence of such a coupling for n-dodecylferrocene in the Triton X-100 and CTAB micelles suggests that the “jump off’ is likely hampered by the hydrophobic side chain of this ferrocene derivative.
Introduction Micellar systems are customary in electrochemistry192and bi~chemistry.~-~ The interfacial field, which might be termed as micellar bioelectrochemistry,is a much less developed area,6v7 and this report is an attempt to fill this gap with some knowledge possibly useful for creating novel bioelectrochemical assemblies, designing water-insoluble electron-transfer mediators, and understanding mechanisms of bioelectrocatalysis in such rather complicated supramolecular systems. Here, we will describe properties of and provide some mechanistic insights into the well-known bioelectrochemical system glucose oxidase-Dglucose-ferrocene,8-10 the only difference of which compared to all previous reports is that intact ferrocene and its alkylated derivatives shown in Chart 1 are solubilized by neutral, negatively, and positively charged micelles of nonionic (Triton X-loo), anionic (SDS), and cationic (CTAB)” surfactants in aqueous solutions. Our current interests are associated with various aspects of organometallic biochemistry.10*12In this context, the present study had several objectives. It has been shown two decades ago that ferrocene solubilized by the nonionic surfactant, Tween 20, is an excellent mediator-titrant for cytochrome c and cytochrome c oxidase>a*cTherefore, micellar ferrocene systems, as well as other related assemblies incorporating poorly watersoluble electron transfer mediators, could be advantageous for coupling with glucose oxidase. A principal idea of the second objective is summarized in Scheme 1. Micelles of ionic surfactants are charged. The same is true for biomolecules and oxidoreductases in particular. It could thus be possible to tune the capability of one and the same mediator to couple with an G. V. Plekhanov Russian Economic Academy.
* Moscow State University.
9 IAESTE exchange student from the BOKU Universitat, Wien, Austria. II Chernobyl Test @
Center Ltd. Abstract published in Advance ACS Abstracts, August 15, 1995.
0022-3654/95/2099- 14072$09.00/0
CHART 1
Fc
DMFc
1-n-Bu-1’-(C0OH)Fc
DDFc
SCHEME 1
0- Fc** ^”o
oxidase by varying the micelle charge, since the charge of the water-soluble mediator is known to be crucial in determining the rate of the electron abstraction from reduced GO.13 It should also be noted that the values of pH around the isoelectric points (PI) of redox enzymes, likewise other proteins, are usually weakly acidic, neutral, or weakly basic. For example, the PI of GO equals 4.0.14 The net charge of GO can thus be adjusted by a simple pH change in the region of PI. If a redox mediator is comicellized with an ionic surfactant, the electrostatic effects could determine the rate of its interaction with the enzyme. We will, however, show that, because of a peculiar mechanism, the reactivity of solubilized ferrocene is insensitive to the micelle charge. 0 1995 American Chemical Society
Glucose Oxidase-D-Glucose-Ferrocene Systems
J. Phys. Chem., Vol. 99, No. 38, 1995 14073
Experimental Section Apparatus and Reagents. Electrochemical voltammetric measurements were made in a three-electrode cell with a pyrographite working electrode. Potentials are vs SCE throughout. Other details are described in our recent pub1i~ation.l~ Glucose oxidase @-D-glucose: oxygen oxidoreductase, EC 1.1.3.4)from Aspergillus niger was purchased from Serva and standardized as described elsewhereI6 using the extinction coefficient of 1.31 x lo4 M-' cm-' at 450 nm for the catalytically active FAD. Thus, the obtained concentration of the active enzyme was used for calculating the rate constants k3. Ferrocene and decamethylferrocene were obtained from Reakhim and Aldrich, respectively. 1-n-Butyl-1'-hydroxycarbonylferrocene and n-dodecylferrocene were kindly provided by Dr. M. D. Reshetova. SDS, CTAB, and Triton X-100 were Serva, Chemapol, and Aldrich reagents, respectively. Inorganic salts, mineral acids, and other chemicals were Reakhim reagents which in some cases were additionally purified according to standard procedure^.'^ Micellar solutions of ferrocenes in the presence of alcohols were prepared as follows. Ferrocene was first dissolved in EtOH, and this solution was then added to buffered (0.01 M phosphate for SDS and 0.1 M phosphate for CTAB and Triton X-100, pH 7.0) micellar solutions of SDS, CTAB, or Triton X-100. The concentration of Fc in the final mixture was 0.001 M, and the content of EtOH was 5%. DDFc was initially dissolved in MeOH. Its final concentration in Triton X-100 micellar solutions was 3.3 x M at [MeOH] = 5% (by volume). DMFc was fist dissolved in n - M H . Its final concentration in aqueous micellar solutions was 0.0001 M at [ n - M H ] = 10% (by volume). The SDS solutions of Fc were stable at 25 OC at least for 24 h, and no precipitate of ferrocenes was observed in the case of CTAB and Triton X-100 micelles. Alcohol-free micellar CTAB and Triton X-100 solutions of ferrocene were prepared by stirring overnight a weighted amount of Fc in the surfactant (0.05 M) buffered solution (0.1 M phosphate). SDS does not form stable solutions without EtOH even at a lower phosphate concentration.
Results Electrochemical Behavior of Fc in Micellar Systems. We share the views of Brajter-Toth that shifts in Eln due to partitioining can be useful for regulating reactivity.I8 Therefore, the electrochemical behavior of ferrocenes in micelles of different charge in the presence of 5% EtOH has been studied keeping in mind that changes in Ell2 may affect the interaction with GO. The representative data obtained in this work are in Figures l a and 2. The former shows the voltammetric trace of Fc incorporated into micelles of Triton X-100. Figure 2 indicates that the half-wave potential Eln, defined as a midpoint between the anodic and cathodic maxima, (1/2)(Epa EF), is a function of both the nature and the concentrations of Triton X-100, SDS, and CTAB in solution. The effect of surfactants on the behavior of redox-active species has been studied by several groups of researcher^.'^-^^ Our data are in general agreement with the results of other workers, although the experimental conditions were not always identical. In particular, we used pyrolytic graphite as an electrode exposed to water with its basal plane. The graphite seems to be very advantageous for electrochemical studies in micellar systems, since in this case "the surfactant assembly is thought to be more micellelike and di~ordered".~~ In accord with this, the current characteristics were not affected by the adsorption of surfactants on electrode surface. Only in the SDS case the anodic Fc maximum was sometimes followed by a smaller peak, probably of adsorptive origin. The plots of the limiting current i, against
+
I
I
I
I
I
0
.1
.2
.3
.4
I
1
.S
.6
EIV(vs SCE) Figure 1. Cyclic voltammograms of Fc (0.001 M) solubilized in Triton X-100 micelles (0.05 M) at pH 7.0 and 25 "C (a) in the absence and (b) in the presence of GO (0.945 x M) and D-glucose (0.1 M). Scan rate is 2 mV s-' and EtOH is 5%. E,,/mV
(vs SCE)
i
2oob>sDs, 150 0.00 0.05 0.10
,
0.15
,
j 0 20
[suMl/M
Figure 2. The half-wave redox potential of Fc vs SDS, CTAB, and Triton X-100 concentrations: scan rate 5 mV s-'; pH 7; EtOH 5%; 22 f 2 "C. the square root of the scan rate ( v ) are linear for all surfactants studied in the range 2-50 mV s-l, indicative of the absence of adsorption at the hydrophobic surface of the working electrode. This is also in accord with the report of Kamau et al., who showed that the CTAB micelles protect Fc from adsorption on pt
electrode^.^^
The peak separation (Epa- Epc)is only slightly dependent on surfactant concentration and the scan rate. In particular, it is 40 and 58 mV at [SDS]= 0.1 M at scan rates 2 and 40 mV s-', respectively. In the case of CTAB, the corresponding values are 52 and 67 mV. A tentative rationale for the peak separation being lower than 59 mV at low scan rates is that a micelle with incorporated ferrocenes might sometimes behave as a two-electron mediator under the conditions used. An attractive feature of these systems, which was discussed by other workers in more detai1,'9-25,27,28is the dependence of the observed redox potentials E I of~comicellized Fc on the nature and concentration of surfactant (Figure 2). The variation of El12 depends on the detergent charge. Anionic SDS reduces while cationic CTAB and nonionic Triton X-100 increase the observed redox potential. The values of E1n level off at high surfactant concentrations (Figure 2). To get a formalized feeling of the limiting values of E1/2,-, the data obtained mostly in the presence of spherical micelles31 were fitted to eq 1. E112,o + acE112.-
El/,
=
1 +ac
Ryabov et al.
14074 J. Phys. Chem., Vol. 99, No. 38, 1995
TABLE 1: Best Fit Values for EI/z,o, Euz,,, and a Calculated According to Equation 1 from the Data in Figure 2 surfactant .%,dmV E112,Jmv a/M-1 115 f 143 209 f 21 265 f 11 CTAB 30 f 39 205 f 5 173 f 12 SDS Triton X-100 229 f 2 362 f 7 6.4 f 0.8 Here, E1/2,0 is the redox potential in the absence of surfactant, c stands for the surfactant concentration, and a is a parameter. The best fit values for E112,0, E,/,,-, and a are summarized in Table 1. The largest difference in E112,- is caused by SDS and Triton X-100 micelles and is as high as 189 mV. The effect observed is comparable to that in the case of methylviologen solubilized in 70 mM SDS and CTAB, which was 40 and 170 mV for the first and the second reduction, re~pectively.~~ The results reported both here and in the literature reveal that the redox potentials are always increased in the presence of CTAB, while the effect of SDS is variable. As seen in Figure 2, SDS micelles decrease the potential of Fc. The same is observed for methyl~iologen,~~ while for the O~(3+/2+)'~ and related Co(3+/2+) complexes there is an increase in E112 as in the case of CTAB micelle^.^^^^^ A qualitative rationale for variation in E112 for Fc might readily be proposed, at least in the case of SDS and CTAB micelles. The oxidation is easier when a redox probe is incorporated into the anionic assembly, which has a stronger affinity to the anode compared to the cationic aggregate. Altematively, stabilization of the oxidized product in the Stem layer of the negatively charged SDS micelle can also contribute to the reduction of the redox potential. A quantitative rationalization of dependencies such as in Figure 2 is not an easy task. Attempts have previously been made,'9-24,28but a complete analysis, with limited assumptions, is still rather complicated. The most generally accepted, key equation which is commonly used for rationalization of the effect of surfactants or microemulsions on observed redox potentials is given by eq 2.'9921328
E,,, =
+ (RTl2nF) ln(DR/Do) + (RTInF) ln[Ko(KR
+ l)/KR(Ko + l)] (2)
Here, KRand KO are the partition coefficients between aqueous and micellar phases of the reduced and oxidized species, respectively, and DR and DO are the diffusion coefficients of the reduced and oxidized species, respectively. It is a common assumption in the ferrocene case, which is based on substantial aqueous solubility of Fc+, that KO > 1. Equation 2 simplifies to28
E,, =
+ (RT/2nF) ln(DR/Do) + (RT/nF) h[(KR
+ l)/KR] (3)
In principle, eq 3 accounts well for the increase in E112 by increasing the amount of surfactant,2ssince & becomes bigger at higher surfactant concentrations while DO remains constant. The situation is less obvious in the case of SDS (Figure 2). It appears that a positively charged ferricenium ion has an enhanced affinity toward negatively charged SDS micelles, and hence, the relative values of DRIDo and &/KO are different. Behavior of DMFc and DDFc in Micellar Systems. Decamethylferrocene is oxidized by 570 mV more cathodically than Fc in CHZC~,.~,Its self-exchange +IO rate constant in MeCN is also higher,33viz. 3.8 x lo7 vs 5.3 x lo6 M-' s-l for F c . ~DMFc ~ is more hydrophobic and thus deserves a comparative electrochemical and bioelectrochemical study in micellar solutions. The electrochemistry of DMFc was inves-
l
I
- .2
I
1
- .1
I
I
1
0
EIV(vs SCE) Figure 3. Decamethylferrocene (1 x M) in CTAB micelles (0.05 M) (a) without and (b) with GO and D-glucose (0.945 x M and 0.1 M, respectively). Parameters are 25 "C, 0.1 M phosphate, 10% n-PrOH, pH 7.15, and scan rate 5 mV s-l. tigated under similar conditions, but in the presence of n-PrOH which was used for solubilization. The final concentration of DMFc in solution was 1 x M at a total content of 10% n-PrOH by volume. In fact, this system is reminiscent of a microemulsion rather than a true micellar solution. A typical voltammogram of DMFc under such conditions is shown in Figure 3a. The effect of positively and negatively charged micelles on E112 was basically the same as that for Fc. SDS and CTAB micelles decreased and increased the values of El/,, and the limiting values were ca. -160 f 15 and -60 f 10 mV, respectively. The main dissimilarity was a higher peak separation, ca. 70 mV, which decreased with increasing [SDS]. Such an electrochemical behavior of DMFc contrasts to that in nonaqueous media were it is truly r e ~ e r s i b l e . ~ , , ~ ~ Dodecylferrocene was chosen for electrochemical studies in micellar systems on the following grounds. Obviously, its long hydrocarbon chain is an anchor that precludes KO > 1. In other words, DDFc will probably be bound to micelles in the oxidized form as well. It could still be a substrate of GO, since monosubstituted ferrocenes do usually couple electrochemically with the enzyme.8 DDFc is reasonably soluble in the Triton X-100 micelles. Its electrochemistry can be rationalized as reversible under these conditions. In particular, the peak separation was 55-60 mV with E112 at 294 mV. The peak current was a linear function of YI/,in the range 2-50 mV s-,. There was no true reversibility in CTAB micelles at [DDFc] = 5x M. Cyclic voltammograms of such solutions showed that E1/2 is close to 345 mV. The reduction peak was broader compared to the oxidation one. Correspondingly, the cathodic peak current was about 30% lower. Bioelectrochemistry in Micellar Systems. As seen in Figure lb, the ferrocene current in micellar solutions does strongly increase in the presence of GO and D-glucose. The "bioelectrochemical" voltammogram of Fc in Triton X-100 micelles matches such water-soluble ferrocene derivatives.8-'0 Similar voltammograms were observed in the presence of CTAB and SDS micelles. However, the catalytic current was not observed when micellar solutions of DMFc or DDFc were tested in the presence of GO and D-glucose (Figure 3). DMFc and DDFc are inappropriate for electrochemical coupling with GO. There was also no catalytic current in a micelle-free system when the solubility of DMFc was increased by the addition of a larger amount of the alcohol (30%) into the aqueous buffered solution. Thus, there is an interaction between two supramolecular associates, viz. GO and a Fc-containing micelle. And is it
Glucose Oxidase-D-Glucose-Ferrocene
Systems
J. Phys. Chem., Vol. 99,No. 38, 1995 14075
TABLE 2: Second-Order Rate Constants for Oxidation of
SDS
Reduced GO at 25 “C and pH 7.0 in the Presence of 5% EtOH ferrocene surfactant k3/M-’s-’ Fc CTAB (5.5 0.7) x 105 Fc SDS (5.7 f 0.2) x 105 Fc Triton X-100 (4.3 f 1.0) x 105 l-n-Bu-l’-(COOH)FC CTAB (PH7.15) (0.63 f 0.03) x 1CP FC CTAB (1.3 f 0.3) x lo5“ FC Triton X-100 (0.8 f 0.1) x 105“ Without EtOH.
10
*
‘ I
8
4.5xlg M 4 . 5 ~ 1 0M~ ~
6
SCHEME. 2
4
2
I
0.oo 0.01
1
0.02
I
I
I
I
I
0.03 0.04 0.05 0.06 0.07 0.08
/
([GO]rV)‘’* (MslV) 1’2 Figure 4. Plot for evaluation of the rate constants k3 as described in ref 36 by the example of Fc in the SDS micelles (0.05 M) at pH 7.0
and 25 “C. affected by the micelle charge? To provide an answer, the second-order rate constants for the oxidation of the reduced enzyme by ferricenium ions generated in the micellar solutions have been measured using the procedure proposed recently by Bourdillon et al.36 This seems to be a more general and justified routine for evaluation of rate constants as compared to the procedure used by Cass et aL8 and other ~ 0 r k e r s . l ~ The overall stoichiometry of the oxidation is given by eq 4. GO(red)
+ 2Fc’
-
GO(ox) 4-2Fc
+ 2H’
(4)
It is a combination of two successive one-electron steps associated with stepwise oxidation by Fc+ of reduced flavin adenine dinucleotide (FADH2) into its oxidized form FAD. According to Bourdillon et al.,36 the second-order rate constants for each step, Le., FADHFc+ FADH’ Fc (k4) and FAD’Fc+ FAD Fc (k3”), are close to each other (k3’ k3” = k3), and hence, one value of the rate constant k3 is obtained. Another assumption is imposed on the diffusion coefficient of a mediator which must be appreciably lower than that of GO.36 At f i s t glance, this does not perfectly hold since DRof Fc is practically the diffusion coefficient of a micelle as a whole.26 Fortunately, the value of DO is of primary importance, since this particular species interacts with reduced GO. As mentioned above, KO > 1, DO = 6.7 x cm2 s - ’ , ~ and ~ there is a possibility of using the approach of Bourdillon. A typical example for the evaluation of k3 is shown in Figure 4 where idiPois plotted against ([GO]/V)~’~. Here i, and i,” are the peak currents of Fc in the presence and in the absence of GO and D-glucose, respectively, and v is the scan rate. As seen, the slope is insensitive to the total concentration of Fc when the latter is around 4.5 x M. Therefore, the value of k3 can be calculated, since the concentration-independent slope equals 3.17 x (k3RT/F)It2. The procedure was carried out for Fc incorporated into cationic, anionic, and neutral micelles, and the results are summarized in Table 2. Surprisingly, the rate constants are practically independent of micelle charge. Since we found that DMFc and DDFc are unable to reoxidize GO(red), we tested 1-n-butyl-1’-hydroxycarbonylferrocene, a de-
+
-
+
+
-
+
rivative with intermediate bulkiness. The increase in current was not that impressive as compared to that of Fc, and correspondingly, the value of k3 is about 10 times lower (Table 2). It has been suggested by the referee that micellar systems containing alcohols might be treated as microemulsions rather than true micellar solutions. Therefore, we have also tested alcohol-free solutions of Fc in the presence of CTAB and Triton X-100. Basically, similar results as in the presence of 5% EtOH have been obtained. The rate constants k3 calculated as above from cyclic voltammograms obtained under these conditions are summarized in Table 2. It is seen that the k3 values are similar in Triton X-100 and CTAB micelles but lower than those in the presence of EtOH. The reason is that the alcohol activates the enzyme. This was directly confiied by comparing the VM/ KM values obtained by direct monitoring of the fading of the ferricenium dye Fc+PF6- 37 in the presence and the absence of EtOH. The ratio was found to be about 3 in the presence of 5% EtOH.
Discussion
This study has led to a few important and, probably, useful findings. First, some of poorly water-soluble species can electrochemically be coupled with GO in aqueous solution if solubilized by micelles. Second, the corresponding rate constants k3 are insensitive to micelle charge. The micellar solutions can thus be used for a systematic investigation of the bioelectrocatalysis of this type, but it is impossible to tune the reactivity of unsubstituted ferrocenes by varying the micelle surface charge. The latter seemed at first surprising since the observed values of El/2 are appreciably altered by differently charged surfactants and ferricenium has a diverse affinity toward miscellaneous micelles. What, then, makes the values of the k3 charge insensitive? A simple rationalization based on a concept of a “jumping off’ femcenium is summarized in Scheme 2. After the electrochemical oxidation of ferrocene, the femcenium ions formed are more hydrophilic than Fc itself. They are able to dissociate reversibly from micelles into the aqueous bulk and
14076 J. Phys. Chem., Vol. 99, No. 38, 1995
Ryabov et al.
micelles, the rate constants k3 for oxidation of reduced GO by do it more readily compared with the neutral species. In the ferricenium are charge-insensitive. Thus, a mechanism of a case of anionic SDS micelles, the cations could preferably be “jumping off’ femcenium was proposed to account for this localized in the anionic surface Stern layer, closer to the micelle phenomenon according to which Fc+ is trapped by GO(red) in exterior. In the CTAB case, lower amounts of Fc+ are to be a rate-limiting step after rapid and reversible dissociation of expected at the positively charged surface of the micelle. the femcenium ion from the micelle pseudophase. If a mediator However, the rate constants k3 are the same within the has a long hydrophobic tail that binds the former to micelles, experimental error for all the surfactants investigated in 5% the electrochemical coupling disappears since the “jump” is no EtOH, meaning that the enzymatic reaction between GO(red) longer possible. The approach reported here expands signifiand Fc+ is insensitive to the micelle charge. Consequently, the cantly the scope of mediators that can be tested in the GO enzyme must interact with Fc+ without the interference from bioelectrocatalysis in micellar systems. Activation of GO by micelles, Le., after dissociation. The reversible dissociation of ethanol might be an easy tool to increase the catalytic activity comicellized species is known to be fast.38 Hence, the rateof the enzyme. limiting step is the capture of dissociated, “jumped off’, ferricenium by GO(red). This model is supported by the following Acknowledgment. The research described in this publication observations. First, there is reasonable agreement between the was made possible in part by Grant No. ML2 000 from the values of rate constants k3 in alcohol-free media (Table 2) and International Science Foundation. We are grateful to the the values of (0.26-5.25) x 105 M-’ s-l (pH 7,25 “C) obtained Russian Foundation for Fundamental Research for financial by Cass et al. for a series of substituted ferrocenes in the micellesupport (Grant No. 03-09928a). We thank Miss Hanni Wilson free system using another procedure.8 Second, it is difficult to for experimental assistance. imagine a situation when the rate-limiting dissociation of a positively charged ferricenium ion is independent of the micelle References and Notes charge, especially if differently charged CTAB and SDS (1) Rusling, J. F. Acc. Chem. Res. 1991, 24, 75. micelles are compared. Third, the inability of DDFc to give (2) Rusling, J. F. Electroanal. Chem. 1994, 18, 1. catalytic current is additional evidence for the importance of (3) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Khmelnitsky, Yu. L.; Berezin, I. V. Eur. J. Eiochem. 1986, 155, 453. the jump. If tailored to micelle by the dodecyl radical, the (4) Khmelnitsky, Yu. L.; Kabanov, A. V.; Klyachko, N. L.; Levashov, ferrocene derivative couples no more with GO. A. V.; Martinek, K. Structure and Reactivity in Reverse Micelles; Pileni, There is a drop in k3 on going to 1-n-butyl-1’-hydroxycarM.-P., Ed.; Elsevier: Amsterdam, 1989; pp 230-261. (5) Luisi, P. L.; Giomini, M.; Pileni, M. P.; Robinson B. H. Eiochim. bonylferrocene (Table 2), while bulkier DMFc does not couple Eiophys. Acta 1988,947,209. Pileni, M. P. J. Phys. Chem. 1993,97,6961. with GO at all. The reason is believed to be steric in origin. A (6) (a) Fujihira, Y.; Kuwana, T. Eiochem. Biophys. Res. Commun. 1974, decrease in k3 for 1-n-butyl-1’-hydroxycarbonylferrocenemay 61,538. (b) Kuwana, T.; Heineman, W. R. Eioelecrrochem. Eioenerg. 1974, additionally be due to the carboxylic group which decreases 1, 389. (c) Yeh, P.; Kuwana, T. J. Electrochem. SOC. 1976, 123, 1334. (7) Kinnear, K. T.; Monbouquette, M. G. Eiotechnol. Eioeng. 1993, the affinity of the ferricenium ion to the enzyme binding site.36 42, 140. Rusling, J. F.; Nassar, A.-E. F. J. Am. Chem. SOC. 1993,115, 11891. Although the inability of the decamethylferricenium cation to (8) Cass, A. E. G.; Davis, D.; Francis, G. D.; Hill, H. 0. A.; Aston, oxidize reduced cytochrome c (Fe”) in an outer-sphere process W. J.; Higgins, I. J.; Plotkin, E. 0.;Scott, L. D. L.; Tumer, A. P. F. Anal. Chem. 1984, 56, 667. was attributed to tQe unfavorable (negative) driving force of (9) Heller, A. Arc. Chem. Res. 1990, 23, 128; J. Phys. Chem. 1992, the reaction in terms of it is still a downhill process in 96, 3579. the case of glucose oxidase (the FADH-EADH’ and FAD’-/ (10) Ryabov, A. D. Angew. Chem., Int. Ed. Engl. 1991,30, 931. FAD couples are at -330 and -520 mV, re~pectively~~). It (1 1) Abbreviations used in the text: Fc, ferrocene; DMFc, decamethylferrocene; DDFc, n-dodecylferrocene; SDS, sodium dodecyl sulfate; should also be noted that the value of E112 for DMFc in the CTAB, cetyltrimethylammonium bromide; GO, glucose oxidase; cmc, CTAB micelles is about -60 mV. It has been demonstrated critical micelle concentration. by the example of the osmium(I1) derivative [Os(4,4’-(NH2)2(12) Jaouen, G.; Vessibres, A.; Butler, I. S. Acc. Chem. Res. 1993,26, bpy)2(4,4’-Me~bpy)]Cl~that such a potential is quite sufficient 361. for achieving the rate constant k3 of 6.7 x lo6 M-’ s - ~ The . ~ ~ ~ (13) (a) Zakeeruddin, S. M.; Fraser, D. M.; Nazeeruddin, M.-K.; Gratzel, M. J. Electroanal. Chem. 1992, 337, 253. (b) Fraser, D. M.; Zakeeruddin, lack of correlation between the rate of oxidation of GO(red) by S. M.; Gratzel, M. J. Electroanal. Chem. 1993, 359, 125. ferricenium ions and the reaction driving force was previously (14) Hayashi, S.; Nakamura, S. Eiochim. Eiophys. Acta 1981,657,40. noticed.36 The evidence has recently been presented that GO(15) Gnedenko, B. B.; Ryabov, A. D. Anal. Chem. 1994, 66, 2240. (16) Weibel, M. K.; Bright, H. J. J. Eiol. Chem. 1971, 246, 2734. (red) has a high affinity toward the ferricenium ion.37 Hence, (17) Pemn, D. D.; Armarego, W. L. F.; Perrin, D. R. Purification of the oxidation of GO(red) has features typical of intramolecular Laboratory Chemicals; Pergamon Press: Oxford, New York, Toronto, electron transfer. Therefore, DMFc is likely to be too big for Sydney, Paris, Frankfurt, 1980. (18) Mackay, R. A.; Myers, S. A.; Bodalbhai, L.; Brajter-Toth, A. Anal. the corresponding binding site of GO. Our working hypothesis Chem. 1990.62, 1058. is based on the assumption that ferricenium might have the same (19) Ohsawa, Y.; Shimazaki, Y.; Aoyagui, S. J. Electroanal. Chem. binding site as D-glucose. The recent X-ray study of the enzyme 1980, 114, 235. from Aspergillus niger revealed that the glucose binding site is (20) Ohsawa, Y.; Aoyagui, S. J. Electroanal. Chem. 1982, 136, 353. (21) Georges, J.; Desmettre, S. Electrochim. Acta 1984, 29, 521. funnel-shaped with the cross section at the top being ap(22) Rusling, J. F.; Shi, C.-N.; Kumosiuski, T. F. Anal. Chem. 1988, proximately 10 x 10 A?o Larger ferrocenes may not fit the 60, 1260. binding center thus precluding the productive binding and (23) Kaifer, A. E.; Bard, A. J. J. Phys. Chem. 1985, 89, 4876. subsequent electron transfer. We are currently searching for (24) Davies, K.; Hussam, A. Langmuir 1993,9, 3270. (25) Davies, K.; Hussam, A.; Rector, B. R., Jr.; Owen, I. M.; King, P. supporting evidence for this hypothesis by probing the binding Inorg. Chem. 1994, 33, 1741. site of GO with a family of related femcenium ions.
Conclusion Poorly water-soluble redox mediators of glucose oxidase do electrochemically function if solubilized by micelles of cationic, anionic, and neutral surfactants. Although their observed redox potentials E I /are ~ subjected to variation by differently charged
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