Anal. Chem. 1996, 68, 3135-3142
Electrodeposition of Redox-Active Films of Dihydroxybenzaldehydes and Related Analogs and Their Electrocatalytic Activity toward NADH Oxidation F. Pariente,† F. Tobalina,† M. Darder,† E. Lorenzo,† and H. D. Abrun˜a*,‡
Departamento de Quı´mica Analı´tica y Ana´ lisis Instrumental, Universidad Auto´ noma de Madrid, Canto Blanco 28049, Madrid, Spain, and Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301
Dihydroxybenzaldehyde (DHB) isomers can be oxidatively electrodeposited onto glassy carbon electrodes previously activated in base solution. We have studied the mechanism of such electrodeposition as well as the electrochemical features of films derived from 2,3-DHB, 3,4DHB, 2,5-DHB, and 2,4-DHB isomers as well as related analogs including 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenylacetic acid, and 4-nitrocatechol. The electrodeposition process and the electrochemical behavior of the modified electrodes were strongly dependent on pH as anticipated for quinone/hydroquinone functionalities. Glassy carbon electrodes modified with films derived from 2,3-DHB and 3,4-DHB exhibit catalytic activity in the electrooxidation of NADH. The catalytic currents are proportional to the concentration of NADH over the range 0.01-1.2 and 0.01-0.9 mM for films derived from 3,4DHB and 2,3-DHB, respectively. These modified electrodes can be used in the design of biosensors based on coupled dehydrogenase enzymatic activities. The electrochemical oxidation of NADH at low overpotentials is of particular interest in biosensor development because of its ubiquitous use as cofactor for over 300 dehydrogenases. Significant overpotentials, as large as 1.0 V, are often required to oxidize NADH at bare electrodes surfaces.1,2 At carbon electrodes, this high overpotential can be decreased by pretreatment although, in general, these surface-modified electrodes rapidly become deactivated.3 Oxidation of NADH at lower overpotentials can be achieved through a redox mediator, and a number of systems capable of this have been identified. It is generally accepted that o-quinones can be quite active in the electrocatalytic oxidation of NADH, and as a result, several surface-modified electrodes based on immobilization of o-quinone moieties on carbon electrodes have been reported.4-9 Immobilization methods applied to date include †
Universidad Auto´mona de Madrid. Cornell University. (1) Moiroux, J.; Elving, P. J. Anal. Chem. 1978, 50, 1056. (2) Jaegfeldt, H. J. Electroanal. Chem. 1980, 110, 295. (3) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1976, 48, 1240. (4) Huck, H.; Schmidt, H. L. Angew. Chem. 1981, 93, 421. (5) Jaegfeldt, H.; Torstensson, A.; Gorton, L.; Johansson, G. Anal. Chem. 1981, 53, 1979. (6) Tse, D. C. S.; Kuwana, T. Anal. Chem. 1978, 50, 1315. (7) Ueda, C.; Tse, D. C. S.; Kuwana, T. Anal. Chem. 1982, 54, 850. (8) Jaegfeldt, H.; Kuwana, T.; Johansson, G. J. Am. Chem. Soc. 1983, 105, 1805. ‡
S0003-2700(96)00280-6 CCC: $12.00
© 1996 American Chemical Society
adsorption onto graphite,4,5 covalent binding onto carbon surfaces,6,7 and the use of polymers derivatized with o-quinone moieties adsorbed onto carbon electrodes.8,9 Miller and co-workers prepared polymeric materials containing dopamine which exhibited electrocatalytic activity toward NADH oxidation.10 Porter et al. were able to immobilize dopamine onto glassy carbon electrodes previously modified with an aminecontaining material. Such modified electrodes exhibited electrocatalytic activity for the oxidation of NADH.11 More recently, modification of electrodes with compounds containing catechol functionalities has been extended to the use of self-assembling monolayers on gold and platinum electrodes.12 Other efforts to prepare modified electrodes that show electrocatalytic activity toward NADH oxidation include the use of redox mediators such as thionine derivatives13 and phenazine14 and phenoxazine15,16 derivatives adsorbed onto graphite or as electrodeposited films.17 One of the major difficulties with virtually all of the approaches described above relates to the stability of the modified electrode and, in some cases, the difficulty of modification. Thus, there continues to be a great deal of interest in the development of new materials for the electrocatalytic oxidation of NADH, especially those immobilized on electrode surfaces. We18 reported that the electrooxidation of 3,4-dihydroxybenzaldehyde (3,4-DHB) on glassy carbon electrodes gives rise to stable redox-active films which exhibited very high and persistent electrocatalytic activity for the oxidation of NADH, including enzymatically generated NADH. Combining this electrocatalytic activity with the enzymatic activity of aldehyde dehydrogenase immobilized on a nylon mesh, we developed an aldehyde biosen(9) Degrand, C.; Miller, L. L. J. Am. Chem. Soc. 1980, 102, 5728. (10) Fukui, M.; Kitani, A.; Degrand, C.; Miller, L. L. J. Am. Chem. Soc. 1982, 104, 28. (11) Deinhammer, R. S.; Ho, M.; Anderegg, J. W.; Porter, M. D. Langmuir 1994, 10, 1306. (12) (a) Kunitake, M.; Akiyoshi, K.; Kawatana, K.; Nkashima, N.; Manabe, O.; J. Electroanal. Chem. 1990, 292, 277. (b) Lorenzo, E.; Pariente, F.; Sa´nchez, L.; Tirado, J.; Abrun ˜a, H. D. Anal. Chim. Acta 1995, 309, 79. (13) Hajizadeh, K.; Tang, H. T.; Halsall, H. B.; Heinemann, W. R. Anal. Lett. 1991, 24, 1453. (14) Torstensson, A.; Gorton, L. J. Electroanal. Chem. 1981, 130, 199. (15) Gorton, L.; Torstensson, A.; Jaegfeldt, H.; Johansson, G. J. Electroanal. Chem. 1984, 161, 103. (16) Persson, B.; Gorton, L. J. Electroanal. Chem. 1990, 292, 115. (17) Persson, B.; Lan, H. L.; Gorton, L.; Okamoto, Y.; Hale, P. D.; Boguslavsky, L. I.; Skotheim, T. S. Biosens. Bioelectron. 1993, 8, 81. (18) Pariente, F.; Lorenzo, E.; Abrun ˜a, H. D. Anal. Chem. 1994, 66, 4337.
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sor.19 Such a biosensor showed great stability and sensitivity in analytical determinations. Wang and co-workers recently employed similarly modified electrodes in the development of a sensor for hydrazine and related materials.20 Taking as a point of departure the favorable characteristics of deposited films of 3,4-DHB for biosensor development, especially when coupled to dehydrogenase activity, we have extended our studies to the different isomers of 3,4-DHB as well as to other related compounds on glassy carbon electrodes and to their potential electrocatalytic activity toward the oxidation of NADH. Furthermore, we have carried out studies in order to elucidate the mechanism of electrodeposition and electron transfer kinetics of the deposited films. EXPERIMENTAL SECTION Materials. 2,3-, 3,4-, 2,4-, and 3,5-dihydroxybenzaldhydes (DHBs) (97% purity) from Aldrich Chemical Co. were recrystallized twice from water using activated charcoal. 4-Nitrocatechol and other DHB analogs were obtained from Fluka and used as received. NADH (grade III) was obtained from Sigma Chemical Co. (St. Louis, MO) and was used as received. DHB stock solutions (typically 25-50 mM) were prepared in water or absolute ethanol, and 0.10 M phosphate was used as buffer solution in all experiments. Water was purified with a Millipore Milli-Q system. All solutions were prepared just prior to use. Apparatus. Cyclic voltammetric studies were carried out with a BAS CV-27 potentiostat and a Linseys X-Y recorder. Teflon shrouded glassy carbon (GC) electrodes (area 0.071 cm2) were used as working electrodes. For ks measurements, an electrode whose area was 7.8 × 10-3 cm2 was employed. A coiled platinum wire served as the auxiliary electrode. All potentials are reported against a sodium-saturated calomel electrode (SSCE) without regards for the liquid junction. Fast cyclic voltammograms were obtained with a Princeton Applied Research Model 173 potentiostat coupled to a Model 175 universal programmer and recorded on a Nicolet Model 4094 digital oscilloscope. Procedures. a. Electrode Pretreatment and Activation. Prior to each experiment, GC electrodes were polished with 1 µm diamond paste (Buehler) and rinsed with water and acetone. In the cases where activated electrodes were employed, the polished electrodes were placed in 1.0 M NaOH solution and the potential was held at +1.20 V for 5 min followed by potential cycling from -0.20 to +1.0 V in buffer solution for 5 min. The electrode was rinsed with water, and the modification with the various DHB analogs was carried out as described below. b. Electrode Modification with DHB Isomers or Analogs. Two different methods were used in the modification of the GC electrodes with the DHBs and related analogs. In the first case, the activated electrode was modified by cycling the potential between -0.20 and +0.30 V (at 25 or 100 mV/s) in a 0.5-2.0 mM solution of the compound in phosphate buffer (pH 7.0 or 8.0). In the second case, the activated electrode was placed in a solution containing 0.5-2.0 mM of the compound in 0.1 M phosphate buffer (pH 7.0 or 8.0), and the potential was held at ∼+0.20 V (depending on pH) for 3 min. Surface coverages for the modified electrodes were determined by integration of the voltammetric wave and assuming an, n, value (19) Pariente, F.; Lorenzo, E.; Tobalina, F.; Abrun ˜a, H. D. Anal. Chem. 1995, 67, 3936. (20) Pamidi, P. V. A.; Wang, J. Electroanalysis 1996, 8, 244.
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of 2. Although the surfaces of the electrodes are microscopically rough, it is difficult to estimate the roughness factor. We have employed the geometric area of the electrodes in the determination of surface coverage. Thus, the values reported represent upper limits. For the determination of NADH, the potential of the modified electrode was stepped from -0.20 to +0.20 V and the steady state current measured after 30 s. RESULTS AND DISCUSSION Cyclic Voltammetry. Cyclic voltammetry of the DHB isomers (1.0 mM) in aqueous solution was carried out on base-activated GC electrodes in 0.10 M phosphate buffer (pH 7.0) at a scan rate of 100 mV/s from -0.20 V to 100 mV beyond the initial anodic wave and back to -0.20 V. The resulting voltammograms are presented in Figure 1A. On the first voltammetric scan a sharp, chemically irreversible anodic peak (a1) is observed for all isomers. A rapid followup chemical reaction takes place so that upon scan reversal a new reduction wave appeared (peak c2). On the second and subsequent scans, a new reversible and welldefined redox system (peaks c2 and a2) was apparent. The peak potentials obtained for the different DHB isomers are summarized in Table 1. The 2,4-DHB isomer did not show any electrochemical activity over the potential range from -0.2 to +1.0 V. In addition, upon continuous scanning, the initially sharp anodic peak (a1) broadened significantly, the peak potential (Epa1) was shifted slightly to more positive values and the peak current decreased. Concomitant with this behavior was an increase of the peak currents associated with the new reversible process (c2, a2). The shape of this new voltammetric feature was that of a surface-immobilized redox couple. If after several scans (typically five to eight) the electrode is removed from the cell, rinsed with water, and placed in fresh buffer solution containing no DHB isomer, a voltammetric response typical of a reversible redox couple confined to the electrode surface is observed for all isomers (Figure 1B) except 2,4-DHB, as mentioned above. The formal potentials and surface coverages obtained for each compound in 0.10 M phosphate buffer at pH 7.0 are also summarized in Table 1. Although there is some loss of material (typically ∼10%) during the first four to five scans, in supporting electrolyte alone, further loss of surface-immobilized material takes place at a much slower rate. We believe that the decrease in surface coverage is likely due to loss of material that is weakly associated with the surface. The electrodeposited films obtained from 2,5-DHB appeared to be more stable than those derived from 2,3-DHB or 3,4-DHB as would be anticipated for 1,4 vs 1,2-quinones.21 In addition, the films exhibited small (although not zero) ∆Ep values and the peak currents were directly proportional to the rate of potential sweep over the range of 5-500 mV/s, suggesting, in all cases, facile charge transfer kinetics over this range of sweep rates. However, for faster sweep rates, ∆Ep values increased significantly, as discussed below. The coverages, obtained from integration of the charge under the voltammetric wave, were significantly smaller for the 3,5-DHB isomer, which is the only one with a meta disposition of the hydroxyl groups. For the other isomers, the hydroxyl groups are in either ortho or para positions and these gave very similar and consistently higher coverages. However, in all cases the coverages were close to or less than one monolayer.18 This observation is consistent with a (21) Adams, R. N. Electrochemistry at Solid Electrodes; Marcel Dekker: New York, 1969.
Figure 1. (A) Cyclic voltammograms of the DHB isomers (1.0 mM) in 0.10 M phosphate buffer at pH 7.0; inset, structures of the various isomers. (B) Cyclic voltammograms, in 0.10 M phosphate buffer at pH 7.0, of the resulting modified electrodes with DHB isomers. In all voltammograms the starting potential was -0.20 V and the sweep rate was 0.1 V/s. Table 1 cyclic voltammetric peak potentials in solution (mV)
features of electrodeposited films
isomer
Epa1
Epa2
Epc1
E°'a
Γab
Γcb
2,5-DHB 2,3-DHB 3,4-DHB 3,5-DHB 2,4-DHB
+170 +240 +290 +640 none
+40 +160 +170 +270 none
0 +140 +120 +240 none
+20 +150 +120 +245
3.9 2.9 2.5 0.7
3.3 2.9 2.6 0.7
a Formal potential of electrodeposited redox couple (in mV). b Anodic and cathodic surface coverages (in mol cm-2 × 1010).
reaction involving functional groups present at the electrode surface, so that, at most, a monolayer of material would be deposited, as was observed in all cases. A voltammetric response similar to that described above was reported by Adams et al. for the oxidation of 1,4-dihydroxynaphthalene on carbon paste electrodes.22 In that case, the overall reaction resulted in the insertion of a hydroxyl group at the 4-position of the naphthalene ring. Such addition reactions have been also described for 2-substituted 1,4-hydroquinones when the 2- substituent is an electron-withdrawing group like COOH, CHO, (22) Papouchado, L.; Petrie, G.; Sharp, J. H.; Adams, R. N. J. Am. Chem. Soc. 1968, 90, 5620.
or NO2.23 After hydroquinone oxidation, a partial positive charge is generated at the 3-position of the ring, making it most susceptible to nucleophilic attack. In our case, the nucleophilic attack could be due to solvent molecules in competition with the active groups, like hydroxyl or carbonyl, present at the electrode surface, giving rise to an immobilization process via ether or ester bond formation (Scheme 1). Since the attacking group in such a reaction is electron-donating (nucleophile), the resulting products would, in general, be easier to oxidize than the starting DHB. From our earlier study on 3,4-DHB,18 we noted that the presence of oxygen-containing functionalities enhances the electrodeposition process. As we reported in that case, the surface coverage for GC electrodes that were activated was much larger, and the resulting films more stable, than those on electrodes that were only polished. In order to ascertain this possibility, the cyclic voltammetric behavior of different 4-substituted catechols (1.0 mM) in which the 4-substituent had a different electron-withdrawing group was investigated. Figure 2 shows the cyclic voltammograms of these materials as well as the electrochemistry of the electrodeposited films. On the first scan a sharp anodic peak, ascribed to oxidation of the hydroquinone, was observed for all derivatives. For both (23) Adams, R. N.; Hawley, D.; Feldberg, S. W. J. Phys. Chem. 1967, 71, 851.
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Figure 2. (Top) Consecutive cyclic voltammograms for 1.0 mM solutions of 3,4-DHB (A), 3,4-dihydroxybenzoic acid (B), 3,4-dihydroxyphenylacetic acid (C), and 4-nitrocatechol (D) in 0.10 M phosphate buffer (pH 7.0). (Bottom) Cyclic voltammograms obtained in 0.10 M phosphate buffer (pH 7.0) for the respective films derived after electrodeposition of 3,4-DHB and analogs on a GC electrode. In all cases the starting potential was -0.20 V and the sweep rate was 0.1 V/s.
Scheme 1
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3,4-DHB and 4-nitrocatechol the peak potential was ∼+0.30 V (Figure 2, traces A and D, respectively). When the 4-substituent was a carboxyl group, the oxidation was shifted to +0.20 V (Figure 2B). For 3,4-dihydroxyphenyl acetic acid (Figure 2C), the only species with no electron-withdrawing group in the 4-position, the oxidation was shifted to +0.05 V, consistent with the abovementioned arguments. On the subsequent cathodic scan, no reversible reduction of the o-quinone was obtained when the 4-substituent was carboxaldehyde or nitro, and a quasi-reversible reduction was obtained in the other two cases. In addition, only 3,4-DHB and 3,4dihydroxybenzoic acid showed evidence of electrodeposition. Cyclic voltammetry of the resulting modified electrodes in fresh buffer and in the absence of the compounds further corroborated the above assertions. Only 3,4-DHB showed evidence of an electrodeposited redox couple with a significant surface coverage (4.0 × 10-10 mol/cm2). The 3,4-dihydroxybenzoic acid film (Figure 2B) showed two reversible redox processes, but for both, the surface coverages were a factor of ∼10 smaller than for 3,4DHB films. These results can be rationalized by taking into account the stability of the different quinones resulting from the first oxidation step. For 4-nitrocatechol with the most electronwithdrawing substituent, the quinone would be anticipated to be quite unstable, resulting in rapid nucleophilic attack by the solvent.
Figure 3. (A) Consecutive cyclic voltammograms for 1.0 mM solutions of 2,3-DHB, 3,4-DHB, and 2,5-DHB in 0.10 M phosphate buffer at different pH. (B) Cyclic voltammograms for the electrodeposited films derived from 2,3-DHB, 3,4-DHB, and 2,5-DHB in 0.10 M phosphate buffer at the same pH as that used in the respective electrodeposition. In all cases the sweep rate was 0.1 V/s.
The quinone derived from 3,4-DHB is probably more stable and able to react only with the functional groups present at the electrode surface, resulting in stable and electrochemically wellbehaved films. The quinone from 3,4-dihydroxybenzoic acid would also be sufficiently stable, and in this case, the reaction could take place with different functional groups ostensibly present on the electrode surface giving rise to two reversible redox couples in the film. Finally, the quinone derived from 3,4-dihidroxyphenylacetic acid is very stable, but in this case, the 4-substituent has little if any electron-withdrawing effect, so the addition reaction is not favored. Effect of pH on the Electrodeposition of Films of DHB Isomers. Since the DHB isomers have quinone or hydroxyl functionalities, one would anticipate that the electrodeposition reaction and redox response of the modified electrodes would be pH dependent. In order to ascertain this, the electrochemical behavior of DHB isomers (1.0 mM) and the voltammetric response of the resulting modified electrodes were obtained in solutions of varying pH from 2 to 8. For each particular pH, the potential was scanned from -0.20 to 100 mV beyond the initial anodic wave, as indicated above, and after seven to eight scans the electrode was rinsed with water and transferred to a fresh buffer solution of the same pH but without dissolved isomer. Figure 3 shows the cyclic voltammograms of ortho and para isomers of DHB and the electrochemical response of the electrodeposited films obtained. In all cases electroactive films were obtained, but the resulting electrochemical behavior was very dependent on pH.
Figure 4. Dependence on pH of the first anodic wave peak potential for 1.0 mM solutions of 3,5-DHB ([), 3-4-DHB (b), 2,3-DHB (9), and 2,5-DHB (2) in 0.10 M phosphate buffer.
As anticipated, the peak potential for the first anodic wave was pH dependent, as can be seen in Figure 4, with slopes ranging from 60 to 64 mV/pH unit, for all isomers. These values are very close to the anticipated Nernstian value of 59 mV for a twoelectron, two-proton process. In addition, the position of the hydroxyl groups on the aromatic ring has a significant influence on the peak potential value. The para isomer (2,5-DHB) is easier to oxidize than the ortho (2,3-DHB, 3,4-DHB) and meta (3,5-DHB) isomers. Finally, 3,5-DHB shows a very high anodic peak potential Analytical Chemistry, Vol. 68, No. 18, September 15, 1996
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value when compared to the other isomers, as would be expected. On the basis of the above-mentioned results, we focused our attention on the study of the ortho and para isomers. At pH 2, the cyclic voltammetric responses of the modified electrodes showed a number of anodic and cathodic waves which, in general, were not too stable (30% of the deposited material was lost after 8-10 scans). Some of these waves were quite well defined and could be ascribed to reversible redox couples confined to the electrode surface and in some cases with ∆Ep values very close to zero as in films of 2,3-DHB. This high level of electrodeposition can be interpreted in terms of the enhanced stability of the oxidized forms at low pH values. In addition, the presence of a relatively high concentration of protons could minimize nucleophilic attack by the solvent, thereby favoring the reaction between the oxidized quinone and the functional groups present at the electrode surface. The differences in the electrochemical behavior for the different isomers could be due to the different positions of the hydroxyl groups. At pH 4, 2,3-DHB films exhibited two reversible and welldefined waves which were however, somewhat broad, and somewhat unstable. On the other hand, at this pH, 3,4-DHB films showed only one sharp and reversible response which was quite stable and with a surface coverage near to one monolayer. The significant differences obtained with the ortho isomers suggest, again, a significant role of the hydroxyl position in the electrodeposition process as well as in the stability of the resulting films. At pH 5, the electrochemical behavior of 2,5-DHB films exhibited only one reversible wave which was rather broad. In addition, the background currents were consistently large. At neutral pH (7.0), the films derived from the ortho isomers showed a similar electrochemical response with well-defined reversible waves and similar formal potentials. However, 3,4-DHB gave films with a higher surface coverage and greater stability than those derived from 2,3-DHB. In addition, 2,5-DHB films showed two reversible waves, the first with a formal potential of +0.020 V, with a high surface coverage and high stability, and a second with a formal potential of +0.30 V, with a low surface coverage and lower stability. These results at neutral pH suggest an increased nucleophilic attack by the solvent, so that only reaction with high-reactivity groups on the electrode surface is favored. Electrodeposition at pH values above 8.0 gave rise to films with very low surface coverages (data not show). In these cases the presence of higher concentrations of OH- (a strong nucleophile) in the medium enhances the reaction with the solvent and minimizes the reaction with the active functional groups on the electrode surface. Based on these results, all further studies were on films electrodeposited at neutral pH since they had high coverage and good stability. pH Dependence Studies of Electrodeposited Films of DHB Analogs. Since the redox response of the electrodeposited films is based on a quinone, one would anticipate that the response would be pH dependent. Thus the voltammetric response of electrodes modified with films derived from the DHB isomers was obtained in buffer solutions of varying pH from 2 to 9. As can be seen in Figure 5, the formal potentials of the different surface redox couples showed a linear dependence with pH, with a slope very close to the anticipated Nernstian value of 59 mV/pH unit for a two-proton, two-electron process. There is a slope change at pH 7.0 and 6.0 for 3,4-DHB and 3,5-DHB films, respectively, 3140 Analytical Chemistry, Vol. 68, No. 18, September 15, 1996
Figure 5. Dependence on pH of the formal potential for the electroactive films derived from DHB isomers in 0.10 M phosphate buffer.
which we ascribe to deprotonation of the deposited material. This is consistent with the fact that the first pKa for 3,4-DHB has been reported to be 7.21.24 In addition, the resulting slopes (35 and 38 mV/pH unit, respectively) are close to the anticipated value of 30 mV/pH unit. Similar to our previous report for films of 3,4-DHB,18 no detectable change in the surface coverage for the other isomers was observed over the pH range studied (2-9). Mode of Deposition and Film Stability. As described in the Experimental Section, two different deposition methods were employed and these involved cycling the potential or holding the potential constant during the electrodeposition process, respectively. In previous work, we carried out comparison studies for 3,4-DHB18 in terms of the stability of the resulting films. These studies involved measuring the decay of the redox activity of the electrodeposited films while continuously cycling the potential. In the case where electrodeposition was carried out at constant potential, the applied potential was +0.20 V, since as in the case of 3,4-DHB , this was determined to be the optimal value. In addition, electrodes were modified for 3 min. After modification, the electrodes were rinsed with water and placed in pH 7.0 phosphate buffer. The potential was cycled over the range of -0.20 to +0.30 V at 100 mV/s and the decay of the surface wave with time was determined. In our previous study on films derived from 3,4-DHB, there was some decay for both modification methods. However, it was somewhat higher for electrodes that had been modified by cycling the potential (25% loss after 10 min of continuous cycling) relative to those modified at a fixed potential (20% loss).18 Based on these previous results, modification at constant potential was used in all subsequent studies. Stability tests were carried out for electrodeposited films obtained from the different DHB isomers. Figure 6 presents plots of the fractional coverage decay as a function of time under continuous potential cycling at 100 mV/s between -0.20 and +0.30 V. Although a significant decay was observed during the initial minutes, afterward the rate of decay slowed significantly, (24) Slabbert, N. P. Tetrahedron 1977, 33, 821.
Figure 6. Fractional coverage dependence on time of continuous scanning for electrodeposited films derived from 2,5-DHB (9) and 3,4-DHB (b) in 0.10 M phosphate buffer at pH 7.0. Sweep rate was 0.10 V/s.
especially for films derived from 2,5-DHB whose electroactivity loss after 30 min was less than 20%. Ortho DHB isomers (2,3DHB, 3,4-DHB) exhibited virtually identical stability patterns (only 3,4-DHB is shown in the figure) with an electroactivity loss of 40% after 30 min of continuous cycling. After the initial loss of electroactivity, the additional loss was less than ∼5% for cycling of up to 8 continuous hours. Such behavior was exhibited by films of all isomers. These results suggest that the initial decay observed might be due to material that is weakly bound to the surface so that it can de displaced with relative ease. In addition, as mentioned above, films of 2,5-DHB were more stable than those derived from 2,3-DHB and 3,4-DHB. This is likely due to the fact that whereas 2,5-DHB is a p-quinone, both 2,3-DHB and 3,4-DHB are o-quinones and it is well established that the former are significantly more stable than the latter.21 Kinetics of Electron Transfer for Electrodeposited Films Prepared at Constant Potential. As mentioned earlier, the electrochemical responses of electrodeposited films from DHB isomers were those anticipated for surface-immobilized redox sites.25 The peak currents were proportional to the scan rate for sweep rates below 500 mV/s and the peak-to-peak potential separation (∆Ep) was small although not zero. In addition, the formal potential (E′) did not depend on the potential scan rate so that the transfer coefficient (R) was anticipated to be equal to 0.5. However, and as mentioned earlier for sweep rates above 500 mV/ s, ∆Ep values increased significantly, suggesting a limitation in the kinetics of charge transfer. Laviron derived general expressions for the linear potential sweep voltammetric response for the case of surface-confined electroactive species.26 From this theory it is possible to determine the standard rate constant (ks) for electron transfer as well as the transfer coefficient, R, by measuring the variations of the peak potentials with scan rate. We found that for scan rates above 1.0 V/s the values of ∆E (Epeak - E°′) were proportional to log v (Figure 7). Using such plots, values of ks and R were obtained for the electrodeposited films of the DHB isomers and the results are presented in Table 2. For scan rates above 10 V/s, significant peak distortions were observed particularly for electrodes modified (25) Murray, R. W. In Electroanalytical Chemistry; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13, p 191. (26) Laviron, E. J. Electroanal. Chem. 1979, 101, 19.
Figure 7. (A) Experimental variation of ∆E (∆E ) Ep - E°′) vs the logarithm of the sweep rate for a GC electrode modified with a 3,4DHB electrodeposited film in 0.10 M phosphate buffer (pH 7.0). The geometric area of the electrode was 7.8 × 10-3 cm2. Inset (B), magnification of the same plot for high sweep rates. Table 2. Formal Potentials, Transfer Coefficients, and Electron Transfer Rate Constants for Electrodeposited Films of DHB Isomers
a
isomer
E°′a
R
ks (s-1)
3,4-DHB 2,3-DHB 2,5-DHB 2,5-DHB
+120 +150 +20 +300
0.50 0.46 0.48 0.33
22 41 65 43
Formal potential of the electrodeposited redox couple (in mV).
with 2,3-DHB and 2,5-DHB. This could be due, at least in part, to the high double-layer capacitance present for these electrodeposited films. The results presented in Table 2 suggest a modest rate of electron transfer for electrodes modified with DHB and, in particular, for films derived from 2,5-DHB. Electrocatalysis on NADH Oxidation. One of the objectives of these investigations was the development of modified electrodes capable of the electrocatalytic oxidation of NADH. This, in part, was responsible for the choice of DHB isomers as modifying agents, since they have an o-quinone functionality which has been identified as one capable of catalytically oxidizing pyridine nucleotides. This capability can be strongly modified by the relative position of the hydroxyl groups on the aromatic ring. In order to test the potential electrocatalytic activity of electrodeposited films from DHB isomers, their cyclic voltammetric responses (v ) 10 mV/s) were obtained in pH 7.0 phosphate buffer in the absence and in the presence of 1.0 mM NADH, and the data are presented in Figure 8. In the absence of NADH, well-behaved redox responses for the polymer films on the electrode could be observed at the formal potential values presented in Table 2. Upon the addition of 1.0 mM NADH, there was a dramatic enhancement of the anodic peak current, and in addition, no current was observed in the return (cathodic) wave for electrodes modified with the o-quinone DHB isomers (2,3-DHB, 3,4-DHB). This behavior is consistent with a very strong electrocatalytic effect. That is, the films obtained from electrode position of ortho isomers exhibited potent electrocatalytic behavior toward NADH oxidation. On the other hand, a negligible electrocatalytic effect was observed for films obtained from the para isomer (2, 5-DHB), whose redox Analytical Chemistry, Vol. 68, No. 18, September 15, 1996
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Figure 9. Chronoamperometric calibration curve (Iss vs [NADH]) for a 2,3-DHB modified GC electrode (Γ ) 1.8 × 10-10 mol cm-2) in 0.10 M phosphate buffer (pH 8.0). The potential was stepped from -0.20 to +0.20 V, and the steady state current was measured after 30 s. Inset, linear range of the chronoamperometric response. Figure 8. Electrocatalytic oxidation of NADH on GC electrodes modified with electrodeposited films derived from dihydroxybenzaldehyde isomers. (Top) Cyclic voltammetry of electrodeposited films derived from ortho and para DHB isomers in 0.10 M phosphate (pH 7.0) in the absence of NADH. (Bottom) Cyclic voltammetry of the same films in the presence of 1.0 mM NADH. In all cases the sweep rate was 0.010 V/s.
response has the more negative formal potential. In this case, the oxidation of 2,5-DHB gives rise to very stable redox-active films with a relatively fast charge transfer rate constant but ineffective in the electrocatalytic oxidation of NADH. These observations are consistent with the fact that whereas o-quinones are quite active in the electrocatalytic oxidation of NADH, p-quinones are not. The magnitude of the catalytic currents was proportional to the solution concentration of NADH over the range of 0.02-0.8 mM for films derived from 2,3-DHB (Figure 9), which covers values of a great relevance in biosensor design and applications. In addition, for these films we estimated a detection limit of ∼10 µM. It is also apparent from Figure 9 that there is a leveling off in the response for NADH concentrations above 2 mM which we ascribe to kinetic limitations. This behavior is similar to that which we observed previously for films of 3,4-DHB and which we also ascribed to kinetic effects. In this context it is worth noting that the rate constant for the reaction of NADH at electrodes modified with films derived from 3,4-DHB was measured (by cyclic voltammetry and rotated disk electrode voltammetry) to be ∼2.7 × 103 M-1 s-1, which is a modest value.19 We are currently exploring the application of these and related materials to biosensor design, and the results of such investigations will be reported elsewhere. CONCLUSIONS Dihydroxybenzaldehyde isomers can be oxidatively electrodeposited onto GC electrodes previously activated in base. The
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mechanism of electrodeposition appears to involve oxidation of the parent compound to the corresponding quinone with a subsequent nucleophilic attack from active functional groups on the electrode surface. Under optimal conditions, the maximum surface coverage obtained is close to one monolayer for ortho and para DHB isomers. The electrodeposition process and the electrochemical behavior of the modified electrodes are strongly dependent on pH. At pH 7.0, the films derived from ortho and para DHB isomers show reversible electrochemical behavior with formal potentials around +0.15 V. These films are quite stable to continuous potential scanning, especially the films derived from 2,5-DHB. Glassy carbon electrodes modified with films derived from 2,3-DHB or 3,4-DHB are capable of catalyzing the electrochemical oxidation of NADH. The catalytic currents are proportional to the NADH concentration in solution. Modification of GC electrodes with ortho isomers of DHB could be employed in the design of biosensors based on dehydrogenase enzymatic activities. ACKNOWLEDGMENT This work was supported by the DGICYT of Spain through Grants BIO 93-0660C04-02 (E.L., F.P., F.T.) and PB92-0167 (H.D.A.), the National Science Foundation (DMR-9107116; H.D.A.), and a NATO Collaborative Research Grant (91-0047; H.D.A., E.L.). F.P. also acknowledges support by a NATO fellowship. M.D. was supported by a fellowship from the Universidad Auto´noma de Madrid.
Received for review March 21, 1996. Accepted June 20, 1996.X AC960280U X
Abstract published in Advance ACS Abstracts, August 1, 1996.
