Electrocatalysis of NADH Oxidation with an ... - ACS Publications

ACS2GO © 2019. ← → → ←. loading. To add this web app to the home screen open the browser option menu and tap on Add to homescreen...
1 downloads 0 Views 132KB Size
Download PDF
Anal. Chem. 2000, 72, 3203-3209

Electrocatalysis of NADH Oxidation with an Electropolymerized Film of 1,4-Bis(3,4-dihydroxyphenyl)-2,3-dimethylbutane Aleksander Ciszewski* and Grzegorz Milczarek

Institute of Chemistry and Applied Electrochemistry, Poznan University of Technology, 60-965 Poznan, Poland

The oxidation of 1,4-bis(3,4-dihydroxyphenyl)-2,3-dimethylbutane, known also as nordihydroguaiaretic acid, on a glassy carbon electrode anodically pretreated in KCl solution gives rise to a stabile redox-active polymer containing the o-quinone moiety. The redox response of the modified electrode is typical for a surface-immobilized species. The modifier thickness can be easy controlled by a number of potential cycles applied during electropolymerization, and a surface coverage up to 1.1 x 10-9 mol cm-2 can be achieved. The film exhibits catalytic activity toward NADH oxidation. Characteristic kinetic constants for the mediated oxidation of NADH were derived from rotating disk experiments performed in phosphate or Tris/ acetate buffers. The effects of film thickness, solution pH, and the presence of Mg2+ cation on the catalytic efficiency of the modified electrode were discussed and compared with literature data concerning related systems. Over 300 dehydrogenases are known that are dependent on the nicotinamide adenine dinucleotide coenzyme in its reduced (NADH) and oxidized (NAD+) forms, and a monitoring of enzymatically generated NADH is the basis of the development of biosensors specific to the particular substance characteristic for the dehydrogenase chosen. Hence, the detection of this coenzyme is important in both practical analysis and biochemical synthesis. Although the reversible potential of the NADH/NAD+ couple is estimated to be -0.32 V (vs NHE),1 the direct oxidation of NADH at bare electrodes is accompanied by very high overpotential, e.g., 1.1 V at carbon2 or 1.3 V at platinum.3 In the case of carbon electrodes, the overpotential can be reduced by a specific pretreatment4 which gives rise to different oxygen-containing surface groups promoting electron transfer, perhaps through a redox mediation mechanism. Unfortunately, this approach leads rather to a submonolayer modification, and the electrodes frequently suffer from fouling problems. A number of other approaches have been employed in an effort to accelerate the kinetics of NADH oxidation. Electrode modification with electroactive nickel hexacyanoferrate,5 tetrarutenated * Corresponding author: (fax) +48 (61) 6652571; (e-mail) [email protected]. (1) ) Clark, W. M. Oxidation Reduction Potentials of Organic Compounds; The Wiliams & Wiliams Co., Baltimore, MD, 1960. (2) Moiroux, J.; Elving, P. J. Anal. Chem. 1978, 50, 1056. (3) Jaegfeld, H. J. Electroanal. Chem. 1980, 110, 295. (4) Tse, D. C. S.; Kuwana, T. Anal. Chem. 1978, 50, 1315. 10.1021/ac991182m CCC: $19.00 Published on Web 06/06/2000

© 2000 American Chemical Society

complex of cobalt porphyrin,6 and different electroactive dyes7-10 can be mentioned. But, probably the most attention has been devoted to the modifiers containing o-hydroquinone groups, which are capable of effective mediating of NADH oxidation. In such a case, the oxidation potential can be reduced to the value approximately corresponding to the hydroquinone/quinone (QH2/ Q) redox transition. A variety of attachment schemes have been proposed to fabricate chemically modified electrodes possessing active ohydroquinones. Among the compounds studied, the common biomolecule dopamine (DA) is frequently used because of its quite active amine side chain, which can be utilized in the creation of covalent bonds. In the late 1970s, Tse and Kuwana4 demonstrated that DA and nearly related 3,4-dihydroxybenzylamine can be covalently attached to glassy carbon (GC) by amidization of carboxylic acid funtionalities of oxidized carbon. Covalent immobilization of DA can be also achieved by electrooxidation of DA on glassy carbon resulting in the formation of C-N bounds as reported by Deinhammer et al.11 In the case of gold electrodes, covalent modification was realized by reaction of a self-assembled cysteamine monolayer with DA12 in the presence of glutaradehyde as a linking agent. DA was also used to prepare methacrylateand styrene-based polymers with catechol side groups. The properties of these materials including their activity toward electrooxidation of NADH were extensively studied in the early 1980s by Miller’s group13-15 At the same time, another strategy of immobilization of catechol-based compounds had received considerable attention. It was based on the ability of condensed aromatics to be strongly adsorbed on graphite electrodes. Such (5) Cai, C.-X.; Ju, H.-X.; Chen, H.-Y. Anal. Chim. Acta 1995, 310, 145. (6) Angnes, L.; Azevedo, C. M. N.; Araki, K.; Toma, H. E. Anal. Chim. Acta 1996, 329, 91. (7) Gorton, L.; Torstensson, A.; Jaegfeld, H.; Johansson, G. J. Electroanal. Chem. 1984, 161, 103. (8) Schlereth, D. D.; Katz, E.; Smidt, H.-L. Electroanalysis 1994, 6, 725. (9) Karyakin, A. A.; Karyakina, E. E.; Schuhmann, W.; Smidt, H.-L.; Varfolomeyev, S. D. Electroanalysis 1994, 6, 821. (10) Zhou, D.-M.; Fang, H.-Q.; Chen, H.-Y.; Ju, H.-X.; Wang, Y. Anal. Chim. Acta 1996, 329, 41. (11) Deinhammer, R. S.; Ho, M.; Anderegg, J. W.; Porter, M. D. Langmuir 1994, 10, 1306. (12) Sun, J.-J.; Xu., J.-J.; Fang, H.-Q.; Chen, H.-Y. Bioelectrochem. Bioenerg. 1997, 44, 45. (13) Degrand, C.; Miller, L. L. J. Am. Chem. Soc. 1980, 102, 5728. (14) Fukui, M.; Kitani, A.; Degrand, C.; Miller, L. L. J. Am. Chem. Soc. 1982, 104, 28. (15) Lau, A. N. K.; Miller, L. L. J. Am. Chem. Soc. 1983, 105, 5271.

Analytical Chemistry, Vol. 72, No. 14, July 15, 2000 3203

compounds containing catechol functionalities were studied by Jaegfeldt and co-workers.16,17 An interesting example of incorporation of the o-hydroquinone moiety in the development of NADH-sensitive electrodes is modification of a Pt electrode by an appropriate derivative of pyridine using a self-assembling technique.18 Unfortunately, the modified electrode lost its activity when the pH value was higher than 7.0. This practically excludes the use of the proposed electrode in the construction of dehydrogenase-based biosensors since this class of enzymes usually requires a pH higher than 8.0. Recently, electropolymerization has also appeared to be useful in the modification of glassy carbon with catechol derivative-based electroactive films. Abruna et al.19-21 have found that such an approach is especially suited for o-dihydroxybenzaldehydes, which in their polymeric forms are highly active in the mediated oxidation of NADH. Using an electrochemical deposition technique, it is also possible to attach chlorogenic acid (another o-hydroquinone) to glassy carbon, as has been shown recently by Zare and Golabi.22 Electrocatalytic oxidation of NADH and the development of dyhydrogenase-based biosensors have been discussed in a number of research papers and reviews.23,24 In the work presented here, we report for the first time the electropolymerization of 1,4-bis(3,4-dihydroxyphenyl)-2,3-dimethylbutane, a compound known also as nordihydroguaiaretic acid (NDGA), and its activity toward NADH electrooxidation. To our very best knowledge, this is the first report on the application of a bis(o-hydroquinone) compound in the catalysis of NADH oxidation. EXPERIMENTAL SECTION Chemicals. 1,4-Bis(3,4-dihydroxyphenyl)-2,3-dimethylbutane was obtained from Lancaster Synthesis GmbH and was used as received. Aliquots of this compound were dissolved in a phosphate buffer solution (PBS; pH 7.4) and the solution was used for the electrode modification within 2 days. For the preparation of NDGA stock solutions in concentrations higher than 0.1 mM, a PBS/ acetonitrile (3/1, v/v) mixture was used instead of pure PBS in order to enhance monomer solubility. NADH (grade III, Sigma) was used to prepare standard NADH solutions in a Tris buffer (pH 8.3). Other chemicals were of analytical reagent grade and were used without purification. All solutions were prepared using triply distilled water and were deareated with prepurified nitrogen prior to use. Apparatus. Electrochemical experiments were carried out using a µAutolab electrochemical analyzer (EcoChemie) connected to a PC for control and data storage. A glassy carbon electrode (3 mm in diameter) was used as the substrate electrode. (16) Jaedfeldt, H.; Torstensson, A. B. C.; Gorton, L. G. O.; Johansson, G. Anal. Chem. 1981, 53, 1979. (17) Jaedfeldt, H.; Kuwana, T.; Johansson,G. J. Am. Chem. Soc. 1983, 105, 1805. (18) Lorenzo, E.; Sanchez, L.; Pariente, F.; Tirado, J.; Abruna, H. D. Anal. Chim. Acta 1995, 309, 79. (19) Pariente, F.; Lorenzo, E.; Abruna, H. D. Anal. Chem. 1994, 66, 4337. (20) Pariente, F.; Tobalina, F.; Darder, M.; Lorenzo, E.; Abruna, H. D. Anal. Chem. 1996, 68, 3135. (21) Pariente, F.; Tobalina, F.; Moreno, G.; Hernandez. L.; Lorenzo, E.; Abruna, H. D. Anal. Chem. 1997, 69, 4065. (22) Zare, H. R.; Golabi, S. M. J. Electroanal. Chem. 1999, 464, 14. (23) Gorton, L. Electroanalysis 1995, 7, 23. (24) Lobo, M. J.; Miranda, A. J.; Tunon, P. Electroanalysis 1997, 9, 191.

3204

Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

The counter electrode was a platinum wire. All potentials reported in this paper are referenced to an Ag/AgCl (saturated KCl) electrode without regard for the liquid junction potential. Electrode Pretreatment. Before its modification, the working surface was polished with 0.3-µm alumina slurry on a Buehler polishing cloth with distilled water as a lubricant, rinsed with triply distilled water, and sonicated in a water bath for 3 min. After sonicating, the electrode was placed in 1 M KCl and the electrode potential was cycled 30 times between 0.5 and 1.8 V (at 0.1 V s-1). The electrode was thoroughly rinsed with water, and the modification with NDGA was carried out as described below. Electrode Modification with Poly-NDGA. The anodically pretreated electrode was immersed in 50 µM solution of NDGA in phosphate buffer (pH 7.4) and modified by cycling its potential between -1.0 and 0.6 V for a number of cycles depending on the coverage to be achieved. The electrode was then thoroughly rinsed with water and was used in different experiments. Surface concentration of electrocatalytic sites (Γ in mol cm-2) was estimated by integrating the charge (Q) under the hydroquinone/quinone oxidation peak at low scan rate and using the common relation, Γ ) Q/nFA, where n, F, and A have their usual meaning. RESULTS AND DISCUSSION Electropolymerization of NDGA. Figure 1 A shows a continuous cyclic voltammogram (CV) of an activated electrode in a solution containing a micromolar concentration of monomeric NDGA. Since the first cycle a well-defined redox couple was observed with a formal potential of +135 mV vs Ag/AgCl. Upon continuous scanning, the peaks continued to increase. Such observation is typical for the deposition of a redox-active material on the electrode surface. Up to fifteenth scan, a nearly linear relationship is observed between the peak height and the number of scans applied. Then, the peak increments tend to decrease, and after 40 scans, the CV reaches a steady shape indicating no further growth of the film. If the electrode is subsequently removed from the cell, rinsed with water, and immersed in PBS containing no monomer species, the voltammetric peaks are still observed (Figure 1B), indicating successful modification. The separation between cathodic and anodic peaks is clearly dependent on surface coverage as shown in Figure 2. When the surface coverage is below 1 × 10-10 mol cm-2, which is characteristic of a submonolayer modification, a peak separation as low as 5 mV can be observed, indicating almost ideal behavior of the surface-attached redox molecules. The peak separation increases as the coverage increases, first very rapidly around 2 × 10-10 mol cm-2 and then logarithmically. As the film grows its value tends asymptotically probably to 60 mV, which is a value expected for solution species, but this was not confirmed, since the polymerization stops as the separation reaches 45 mV. The modification process was affected by the range of potential sweeps, monomer concentration, and electrode pretreatment as discussed below. First, we should mention the effect of the potential range on the electropolymerization process. As seen in Figure 1A, the lower limit of a potential sweep that we applied is fairly negative (-1000 mV) but it was not essential for the polymerization itself. The modification also takes place in a narrowed potential range; the

Figure 2. Dependence of the separation between cathodic and anodic peaks of the modifier as a function of its coverage during electrochemical polymerization of NDGA under conditions as in Figure 1.

Figure 1. Continuous cyclic voltammogram of 50 µM NDGA in PBS (pH 7.4) at a pretreated GC electrode (A) and cyclic voltammogram of poly-NDGA modified electrode in buffer containing no monomer species (B). Γ for (B), ∼2.5 × 10-10 mol cm-2. Scan rate, 100 mV s-1.

only requirement is that the potential window comprises the QH2/Q redox couple (for example, between 0 and +200 mV). But, the smaller the lower limit of the potential sweep, the higher are the peak increments between subsequent scans and the smaller is the peak separation between anodic and cathodic peaks. A possible explanation of this effect may be the reduction of some electroinactive compounds inhibiting polymerization, which are formed oxidatively apart from the redox-active matter. The upper limit of the cycling did not contribute to the polymerization rate within the range of +400 to +800 mV. Monomer concentration also has an important influence on the electropolymerization process. Generally, when the concentration was higher than 0.1 mM, during potential cycling the redox peaks not only did not grow but were suppressed on subsequent cycles and the resulting modified electrode suffered from significantly higher peak separation. Probably a less ordered structure is created at higher concentrations of the monomer species. Finally, we would like to mention the effect of electrode pretreatment on the electropolymerization of NDGA. A series of solutions which include NaOH, NaClO4, NaNO3, Na4B2O7, KCl,

and H2SO4 (all 1 M) were tested as the medium for the oxidative pretreatment of the working electrode. Our findings showed that the KCl solution was superior to the others. In such a case, surface loading as high as 1.1 x 10-9 mol cm-2 could be achieved in the polymerization step. It is noteworthy that pretreatment in NaOH solution proposed for the polymerization of 3,4-dihydroxybenzaldehyde (3,4-DHB)19 did not prove correct in the case studied. Surface loading for the electrodes pretreated in this manner did not differ from those obtained for polished electrodes and did not exceed 5 × 10-10 mol cm-2. However, some positive effect of such a pretreatment was manifested by smaller separation between redox peaks. It is also interesting to mention that the electrode pretreated in NaOH exhibits two redox systems in monomer solution during the few first scans with E°′ values of +135 and +190 mV, respectively. During continuous potential cycling, only the first couple grew. Referring to the mechanism proposed for 3,4-DHB,20 an electron-withdrawing group in the 1-position has some promoting effect on the attachment to the electrode surface. The alkyl chain linking two 3,4-dihydroxyphenyl groups in the compound studied does not have such properties. This suggests the differences in the polymerization mechanisms between these two groups of compounds. On the other hand, oxidative pretreatment in NaCl applied for graphite/epoxy electrodes25 had proved to enhance DA adsorption at the working surface and the reversibility of the oxidation reaction. Thus, we believe that in our case the adsorption proceeds the film formation. The polymerization is probably realized by intermolecular reactions between highly reactive o-quinones and the bifunctional nature of NDGA allows the formation of a highly cross-linked polymeric structure. That is just working hypothesis. Electrode pretreatment in NaNO3 appeared to inhibit the polymerization. Other solutions used in the pretreatment had a minor effect on the polymerization process. Voltammetric Characteristics of the Polymer-Modified Electrode. The voltammetric behavior of the polymer-modified electrode was investigated by monitoring the variation of the CV peak currents and positions of the QH2/Q couple as a function of (25) Falat, L.; Cheng, H.-Y. Anal. Chem. 1982, 54, 2111.

Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

3205

Figure 4. Cyclic voltammogram of the poly-NDGA-modified GC electrode in the absence (a) and presence (b) of 0.5 mM NADH. Electrolyte, as in Figure 1. Γ ∼1.5 × 10-10 mol cm-2. Scan rate, 10 mV s-1.

Figure 3. Dependence of anodic peak current of the QH2/Q couple of the modified electrode on scan rate (A) and the dependence of peak positions for anodic (circles) and cathodic (squares) peaks on the logarithm of scan rate (B). Open markers indicate data that were used to perform linear regression calculations. Equations of the fitting lines are given in the plot area.

scan rate. The results are shown in Figure 3. As seen, the peak current is directly proportional to the rate of a potential sweep over the range of 2-2500 mV s-1, suggesting very facile chargetransfer kinetics. At higher scan rates, kinetic effects in the electron transfer influence the linear relationship,26 but the leveling off is relatively low indicating a high value of the coefficient of a diffusion-like propagation of charge through the modifier at higher scan rates. We also studied the variation of peak positions on scan rate. Figure 3B shows the dependence of Ep on the logarithm of the scan rate for anodic and cathodic peaks. As seen, peak separation is almost constant over the range of 2-200 mV s-1 and does not exceed 30 mV (when Γ < 8 × 10-10); above it the separation grows, first exponentially and then linearly with log(v). As was proved by Laviron,26 if ∆Ep >200/n mV and Ep is linearly proportional to log(v), the standard rate constant ks and the charge-transfer coefficient R for the surface-immobilized redox species can be easy determined. In the case studied, the appropriate conditions were achieved over the range 4000-10 000 mV s-1. E° ′ was taken as the average of the cathodic and anodic peak potentials at the lowest scan rate applied (2 mV s-1), where no kinetic effects are apparent. Using the equations derived by the (26) Laviron, E.; Roulier, R. J. Electroanal. Chem. 1980, 115, 65.

3206

Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

author, we calculated ks and R values to be 43 s-1 and 0.44, respectively. The value of ks is higher that those for related systems reported so far.16,17,21,22 Mediated Electrooxidation of NADH. Figure 4. shows cyclic voltammograms of the poly-NDGA-modified electrode in buffer solution in the absence (a) and in the presence (b) of NADH, respectively. It can be seen that there is a great increase in the anodic peak of the QH2/Q couple in the presence of coenzyme. The reason is that electrochemically generated quinone is reduced back to hydroquinone by NADH species diffusing from the solution bulk to the electrode surface according to the equatiom k1

Q + NADH + H+ 98 QH2 + NAD+

(1)

For the same reason the cathodic peak of the QH2/Q couple is lowered. This example of an EC mechanism is that generally accepted for the mediated oxidation of NADH with 3,4-dihydroxyphenyl-containing modifiers.27 The catalytic effect can be seen directly when curve b is compared with the cyclic voltammogram of NADH oxidation at an unmodified GC electrode (not shown). The decrease in overvoltage is at least 450 mV. In the case studied, the catalytic peak current was proportional to the analyte concentration in the range 0.1-1.0 mM; above it a leveling off in the response is observed. The same shape of the current-concentration curve was observed for other modifiers8,12,20,28 and was ascribed to kinetic limitations. We also studied the electrode (27) Ueda, C.; Tse, D. C. S.; Kuwana, T. Anal. Chem. 1982, 54, 850. (28) Huan, Z.; Persson, B.; Gorton, L.; Sahni, S.; Skotheim, T.; Bartlett, P. Electroanalysis 1996, 8, 575.

Figure 5. Chronoamperogram of the catalytic response of the polyNDGA-modified GC electrode toward successive additions of NADH in 0.2 mM steps. Applied potential, +0.3 V. Arrows indicate moments of analyte addition. Electrolyte, as in Figure 1. Γ as in Figure 4. Inset: calibration curve.

response in an amperometric mode by monitoring the current at +0.3 V where the modifier exists in its fully oxidized state. The obtained current-time trace and corresponding calibration curve are shown in Figure 5. Again, a nonlinear relationship is observed. But it is important that there is no visible electrode deactivation during continuous oxidation of NADH on the modified electrode. This may be very valuable in biosensor development, which in most cases operates in an amperometric mode. Attempts were made to find further information about the kinetics of the electrocatalytic oxidation of NADH with electrodeposited poly-NDGA. We followed the diagnostic scheme based on rotating disk measurements recently proposed for polymeric 3,4-DHB.21 The model applied assumed that the reaction between the oxidized form of the mediator and NADH proceeds via an intermediate complex7,29 according to the expression k+1

k+2

NADH + MOX y\ z [NADH-M] 98 NAD+ + Mred (2) k -1

and the three characteristic constants can be lumped together giving the Michaelis-Menten (M-M) constant:

KM ) (k-1 + k+2)/k+1

(3)

The currents of NADH oxidation were measured at a fixed potential depending on pH, which was set 150 mV beyond the E°′ of the mediator, as a function of rotation rate and analyte concentration. Koutecky-Levich plots constructed (i-1 vs ω-1/2, not shown) were linear and nonzero intercepts allowed the determination of the rate constants for the chemical reaction between NADH and the mediator (k1). Next the plots of k1-1 vs CNADH were used to determine three characteristic kinetic constants, i.e., k1(Cd0), k+2, and KM.21 Since the mediator studied can form a multilayer structure, the diffusion of NADH through electrodeposited film can eventually affect the electrooxidation process. For this reason, independent experiments were per(29) Persson, B. J. Electroanal. Chem. 1990, 287, 61.

Figure 6. Dependence of the reciprocal of the k1 constant as a function of NADH concentration for three different coverages of the modifier.

Figure 7. Dependence of the constant k1(Cd0) on the coverage of the modifier. Inset shows the equation of fitting line.

formed using modified electrodes with different surface coverages. The theory mentioned above predicts that k1 values should be inversely related to the analyte concentration. Figure 6 shows the plot of the reciprocal of k1 against the concentration of NADH, for three representative surface coverages of the mediator. As seen, straight lines are obtained in each case, which confirms the formation of an intermediate complex. Inversion of the intercepts give asymptotic values of k1 anticipated for zero concentration of NADH. Such values calculated for a series of modified electrodes plotted as a function of modifier coverage are shown in Figure 7, and are also summarized in Table 1, among the corresponding values of k+2 and KM constants. Experimental data can be approximated with excellent correlation to the power function with an exponent of ∼-1. As it follows, the value k1 is inversely related to the surface coverage and thus k1(Cd0) f ∞ when Γ f 0. This suggests that the main limitations in the whole electrooxidation process may be those attributed to the mass transfer in the space of the modifier, i.e., analyte transportation to the active site (quinone moiety) of the catalyst and the diffusion of the oxidation products away to the bulk solution. This assertion may be supported by the fact that during electropolymerization probably only a small fraction of deposited material is redox active19 and a great deal of reacting species is consumed for the creation of an electroinactive polymeric framework which may impede mass transfer. As a consequence, an electrode with low coverage turns over a significantly larger number of NADH molecules than an Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

3207

Table 1. Comparison of Characteristic Kinetic Constants for Electrocatalytic Oxidation of NADH on the Poly-DHBand the Poly-NDGA-Modified Electrodes, as a Function of Electrolyte Type, Its pH, Film Thickness, and the Presence of Magnesium Cation electrode

solution buffer

pH

GC|poly-3,4-DHB

Tris/acetate

GC|poly-NDGA

phosphate

5.0 5.0 7.5 7.5 8.5 8.5 7.4 7.4 7.4 7.4 5.0 5.0 7.5 7.5 8.5 8.5

Tris/acetate

a

Γ x 1010 (mol cm-2)

CMg2+ (mM)

k1(CdO) × 10-3 (dm3 mol-1 s-1)

k+2 (s-1)

KM (mM)

1.3-2.0

0 20 0 20 0 20 0 0 0 0 0 20 0 20 0 20

14.8 13.8 2.8 9.8 1.3 10.2 6.7 3.4 1.2 0.6 20 32 3.3 14.1 0.85 9.7

1.00 1.10 0.54 0.59 0.31 0.61 0.85 0.30 0.15 0.07 1.10 2.3 0.35 0.7 0.2 0.58

0.06 0.08 0.19 0.06 0.20 0.06 0.13 0.09 0.12 0.14 0.06 0.07 0.11 0.05 0.24 0.06

0.8 1.9 4.7 8.0 1.5 1.9 1.9 1.9 1.8 1.5

ref 21

a

This work.

electrode with high coverage. So, an insulated single catalytic center could be capable, at least theoretically, of mediating the oxidation with huge speed. In practice, however, the maximal value of k1(Cd0) that could be achieved in the absence of transport limitation (Γ f 0) should be given by the equation

k1(Cd0,Γd0) ) ks/KM

(4)

k1(Cd0,Γd0) ) k+2/KM

(5)

because21

and the complex dissociation rate k+2 (eq 2) may be at most as high as the rate of the transformation QH2 f Q. Assuming a KM of 0.12 mM, since this constant is almost independent of Γ (see Table 1), a value of 3.6 × 105 M-1 s-1 can be evaluated for k1(Cd0,Γd0). Other effects on the catalytic efficiency we explored were the influence of solution pH and the presence of Mg2+ ions. These effects have been discussed in detail for poly-3,4-DHB,21 and it is interesting to compare them with respect to the modifier reported here. In these studies, we performed a series of experiments using electrodes with a coverage comparable with that of poly-3,4-DHB ((1.6 ( 0.2) × 10-10 mol cm-2) in three representative pHs (5.0, 7.5, 8.5) in the absence and presence of 20 mM magnesium chloride, respectively. Electrolytes applied in these experiments were always Tris/acetate buffers. In each case, the three kinetic constants were calculated according to the procedure mentioned above and are summarized in Table 1. As anticipated, similar dependencies of kinetic constants on solution pH are manifested; i.e., k1(Cd0) and k+2 decrease as pH increases, while the appositive tendency is seen for KM. However, catalytic oxidation of NADH on poly-NDGA seems to be somewhat more sensitive to pH than that on poly-3,4-DHB. The decrease in k1(Cd0) during increasing pH from 5.0 to 8.5 is 24 times for poly-NDGA and only 11 times 3208

Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

for poly-3,4-DHB. Another difference is the intensity of the catalytic effect of magnesium cation, attributed to the formation of a ternary complex: mediator-NADH-ion.21 In the case of poly-3,4-DHB, the effect is clearly seen only in neutral and basic solutions. The higher the pH, the stronger the enhancement of k1(Cd0). This also applies for poly-NDGA, but the effect is also seen at low pH. Over a 50% increase in k1(Cd0) and over 100% in k+2 is observed at pH 5.0 for poly-NDGA, while in the case of poly-3,4-DHB at the same pH, these constants remain almost unchanged with the addition of Mg2+. These differences can be attributed, at least in part, to the differences in the hydrophobicity between the two modifiers. It was reported that the stability of the intermediate complex is dependent on the polarity of the solvent and solution acidity.30 Hence, it is plausible that hydrophobicity/hydrophilicity of the electrode does also contribute to the complex formation. PolyNDGA is probably more hydrophobic than poly-3,4-DHB, due to the branched alkyl chain serving as a spacer between two catechol groups. Our working hypothesis is that the range of pH at which ternary intermadiate complex can form is shifted toward lower values as the hydrophobicity of the electrodic surfaces increases. Unfortunately, at this stage of investigation we have not other experimental data to support this assertion. Electrode Stability. It is well established that o-quinone mediators confined to the electrode surface are generally not very stabile and during continuous potential cycling or under potentiostatic conditions deactivation occurs. Although different methods have been applied for the modification of electrode surfaces such as covalent bonding, absorption, and electrodeposition, possible mechanisms to account for the loss of electroactivity are similar and may include the following: (i) desorptive loss of adsorbed15,16 or weakly bounded matter, also loss of low molecular weight oligomeric species in the case of electropolymerization; (ii) breakage of covalent bounds linking the modifier to the electrode surface or cross-linking the polymer; (iii) dimerization of a one-electron oxidation intermediate; (iv) hydroxylation reac(30) Gorton, L. J. Chem. Soc., Faraday Trans. 1 1986, 82, 1245.

Table 2. Deactivation Rate Constants for Electropolymerized NDGA as a Function of Applied Constant Potential

Figure 8. Change of fractional coverage of the poly-NDGA-modified GC electrode during continuous potential cycling between -0.2 and +0.6 V (at 100 mV s-1). Inset: second-order kinetic plot of deactivation. Γ°, 1.0 × 10-9 mol cm-2. Electrolyte, as in Figure 1.

tion in position 5 giving rise to a p-quinone compoundsobserved as a new redox couple appearing below the original one;27 (v) polymerization reactions between Q and Q or between Q and QH2 species producing electrochemically inactive products.17 Stability tests were carried out for electrodeposited film of NDGA under either potentiodynamic or potentiostatic conditions. In potentiodynamic mode, electrode potential was cycled continuously between -0.2 and +0.6 V (at 100 mV s-1) and the variation of peaks height and area was monitored. In potentiostatic mode, three independent experiments were carried out in which modified electrode was polarized either at -0.2, +0.14, or +0.3 V, respectively. Every 10 min the polarization was interrupted and a cyclic voltammogram was recorded, to determine actual surface coverage. Figure 8 shows the decay of the fractional coverage as a function of time under potentiodynamic conditions. As seen, 5 h of continuous cycling is required to deactivate the electrode 50%. Such stability is significantly better than that observed for polymeric 3,4-dihydroxybenzaldehyde, which was deactivated to a similar level during 35 min.20 It is worth mentioning that, after the first 100 min, during which likely monomeric and oligomeric weakly bonded material is loosed, the deactivation process obeys (31) Alfaro, I. M.; Pizarro, E. I.; Rodriguez, L.; Valdes, E. M. Bioelectrochem. Bioenerg. 1995, 38, 307. (32) Zen, J.-M.; Lo, C.-W.; Chen, P.-J. Anal. Chem. 1997, 69, 1669.

Eappl (mV)

kdeact × 10-4 (cm2 mol-1 s-1)

-200 135 300

0.40 3.08 1.92

second-order kinetics as confirmed by the corresponding kinetic plot (Figure 8, inset). The same dependence is observed under potentiostatic conditions. The slopes of kinetic plots were used to determine deactivation rate constants, which are given in Table 2. As follows from the data, the deactivation constant is the highest for the polarization potential close to E°′. At such a potential only half of the redox sites exist in oxidized form; so the very likely conclusion is that the predominant reaction responsible for the deactivation is that between Q and QH2 forms. At higher potential, deactivation is lower, making poly-NDGA potentialy useful in the development of amperometric sensors as was mentioned earlier (see also Figure 5). CONCLUSION Oxidative polymerization of 1,4-bis(3,4-dihydroxyphenyl)-2,3dimethylbutane on glassy carbon previously anodized in KCl solution gives rise to a very stable redox-active polymer. Film thickness can be easily controlled by a number of potential cycles applied in the polymerization step. The resulting film is permeable for NADH and exhibits potent and persistent electrocatalytic activity toward this species. These properties may be very valuable in the design of biosensors based on dehydrogenases. Athough we have no experimental evidence that the enzymes can be recycled by the modifier presented in this paper, many related systems, i.e., possessing 3,4-dihydroxybenzene groups, have proved to be effective in this reaction and we believe that our modifier will behave similarly. Additionally the mediation between electrode and FAD/FADH2 centers in oxidases recently reported for catecholamines31,32 also seems likely. Studies are in progress and the results will be published elsewhere. ACKNOWLEDGMENT The work was supported by Poznan University of Technology, Grant DS 31-339/98. Received for review October 13, 1999. Accepted April 11, 2000. AC991182M

Analytical Chemistry, Vol. 72, No. 14, July 15, 2000

3209