Anal. Chem. 1994,66, 4337-4344
This Research Contribution is in Commemoration of the Life and Science of 1. M, Kolthoff (1894- 1993).
Electrocatalysis of NADH Oxidation with Electropolymerized Films of 3,4=Dihydroxybenzaldehyde F. Pariente,t E. Lorenzo,*lt and H. D. Abruiia*l# Departamento de Qulmica Anahtica y Analisis Instrumental, Universidad Autonoma de Madrid, Canto Blanco 28049, Madrid, Spain, and Department of Chemistry, Baker Laboratory, Cornel1 University, Ithaca, New York 14853-1301
The oxidation of 3,4-dihydroxybenzaldehyde(3,4-DHB) on glassy carbon electrodes gives rise to stable redoxactive electropolymerized f i s containing a quinone moiety. "he redox response of the f i s is that anticipated for a surface-immobilized redox couple, and the pH dependence of the redox activity of these films is 60 mV/ pH unit, which is very close to the anticipated Nernstian dependence of 59 mV/pH unit. We have characterized the dependence of the growth of these films on the solution concentration of 3,4-DHB, the potential for deposition, the time of deposition, and the pH. In addition, we have measured their permeability and apparent d f i s i o n coefficient (Dapp)of these films. These films exhibit potent and persistent electrocatalyticbehavior toward NADH oxidation. In a flow injection analysis determination, the limit of detection is estimated to be in the submicromolar regime. Because of their ubiquitous use as coenzymes by over 300 dehydrogenases, /?-nicotinamide adenine dinucleotide (NADH) and its oxidized form (NAD+) have been the subjects of numerous studies geared at understanding the factors that may control their redox activity. In particular, much effort has been devoted to identifying materials that may exhibit electrocatalytic oxidation of NADH. Of particular interest have been materials that may be attached to electrode surfaces'-* because of their potential utility in a wide range of sensor applications. The major difficulty arises as a result of the high overpotential that is typically encountered for NADH oxidation at bare electrode surface^.^ Although the reversible potential for NADH oxidation is estimated
' Universidad Aut6noma de Madrid. Come11 University. (1) Murray, R W. In Electroanalytical Chemistty; Bard, A. J., Ed.; Marcel Dekker: New York, 1984; Vol. 13,p 191. (2)Murray, R W. Annu. Rev. Mater. Sei. 1984,14,145. (3) Murray, R W. Accts. Chem. Res. 1980,13, 135. (4)Faulkner, L. R Chem. Eng. News 1982,60 (Feb 27),28. (5)Abruria H.D. Coord. Chem. Rev. 1988,86,135. (6)Abruiia, H. D. In Electroresponsive Molecular and Polymeric Systems; Skotheim, T. A. Ed.; Marcel Dekker: New York, 1988;p 92. (7)Fujihira, M. In Topics in Oiganic Chemistty; Fry, A. J., Britton, W. R, Eds.; Plenum: New York, 1986;p 255. (8)Gorton, L. J. Chem. Soc., Faraday Trans. 1 1986,82,1245. (9)Elving, P. J.; Schmakel, C. 0.;Santhanam, IC S. V. Crit. Rev. Anal. Chem. 1976,6,1.
0003-2700/94/0366-4337$04.50/0 0 1994 American Chemical Society
to be -0.32 vs NHE,Iothere is typically a very high overpotential, which can be as high as 1.0 V." A number of approaches have been employed in an effort to accelerate the kinetics of NADH oxidation. In one of the earlier attempts, Blaedel and JenkinsI2 employed an electrochemical pretreatment of carbon electrodes. They believed that the oxidative pretreatment produced surface hydroxyl, quinones, and other groups which were responsible for the improved performance. It is generally accepted that o-quinones can be quite active in the electrocatalytic oxidation of NADH, and as a result numerous derivatives incorporating such a group have been employed. For example, Kuwana and Tse were able to obtain electrodes with catalytic activity by covalent attachment of 1,Zhydroquinones to the surface of pyrolytic graphite.13 In addition, a number of investigators have employed graphite electrodes modified with adsorbed aromatics containing catechol functionalitie~.'~-'~ This approach was recently extended to the use of self-assembling monolayers on gold and platinum e1ectr0des.l~In addition, Miller and co-workers prepared polymeric materials derived from poly(methacrolyl chloride) and dopamine which, when adsorbed, also exhibited electrocatalytic activity toward NADH oxidation.I8 Porter et al. very recently reported that electrooxidation of amines on glassy carbon electrodes resulted in the formation of a C-N covalent bond, giving rise to electrodes modified with the amine-containing material. In this way they were able to immobilize dopamine (which has an o-quinone moiety) onto glassy carbon electrodes. Such modfied electrodes exhibited electrocatalytic activity toward NADH oxidation.lq (10) Clark, W. M. Oxidation Reduction Potentials of Organic Compounds; The Williams and Wilkins Co.: Baltimore, MD, 1960. (11) (a) Aizawa, M.; Coughlin, R W.; Charles, M. Biochim. Biophys. Acta 1975, 385,382. @) Leduc, P.; Thevenot, D. Bioelectrochem. Bioeng. 1974,1, 1. (12)Blaedel, W.J.; Jenkins, R A. Anal. Chem. 1976,48,1240. (13)Tse, D.C. S.; Kuwana, T. Anal. Chem. 1978,50,1315. (14)Jaegefeldt, H.;Torstensson, Gorton, L. G. 0.;Johansson, G. Anal. Chem. 1981,53,1979. (15)Ueda, C.; Tse, D. C. S.; Kuwana, T. Anal. Chem. 1982,54, 850. (16)Jaegefeldt, H.;Kuwana, T.; Johansson, G. /. Am. Chem. Soc. 1983, 105, 1805. (17)(a) Kunitake, M.;Akiyoshi, K; Kawatana, IC; Nakashima, N.; Manabe, 0. /. Electroanal. Chem. 1990,292,277.@) Lorenzo, E.;Pariente, F.; Sinchez, L.; Tirado, J.; Abruiia, H. D., manuscript in preparation. (18) (a) Degrand, C.; Miller, L. L.]. Am. Chem. SOC. 1980,102,5728. (b) Fukui, M.; Kitani, A; Degrand, C.; Miller, L. L.J Am. Chem. SOC.1982,104,28.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994 4337
In addition to quinone moieties, a number of dyes20-23have been employed. These are typically employed in carbon paste electrodes or as electropolymerized films. Moreover, Albery and co-workers have employed conducting salts derived from TCNQ for the catalytic oxidation of NADH.24,25 One of the major difficulties with virtually all of the approaches described above relates to the lifetime of the modified electrodes and in some cases the difficulty of modification. Thus, there is still a great deal of interest in the development of new or alternative approaches for the electrocatalytic oxidation of NADH, especially for materials immobilized on electrode surfaces. We have recently found that the electrooxidation of 3,4dihydroxybenzaldehyde on glassy carbon electrodes gives rise to stable redox-active electropolymerized films. These films exhibit very high and persistent electrocatalytic activity for the oxidation of NADH. We describe the preparation and characterization of such modified electrodes and their electrocatalytic activity for NADH oxidation. EXPERIMENTAL SECTION
Materials. 3,4-Dihydroxybenzaldhyde(3,4-DHB; 97%purity) from Aldrich Chemical Co. was recrystallized twice from water using activated charcoal. Methylviologen dichloride hydrate (Aldrich) was used as received. NADH (grade III) was obtained from Sigma Chemical Co. and was used as received. Tris and acetate buffers (0.10 M) containing 0.10 M NaN03 were used. 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. Teflonshrouded glassy carbon electrodes (area, 0.071 cmz) and platinum disk electrodes sealed in glass (area, 0.01 cm2) were used as working electrodes. A coiled platinum wire served as the auxiliary electrode. Except as described below for FIA measurements, all potentials are reported against a sodium saturated calomel electrode (SSCE) without regard for the liquid junction. A Pine Instruments rotating disk electrode system with a glassy carbon disk electrode (area, 0.23 cmz) was employed in rotating disk electrode experiments. Double potential step chronocoulometry experiments were carried out using a BAS 100 voltammetric analyzer with pulse widths from 5 to 100 ms. For the FIA measurements, a Metrohm 656 detector with a Metrohm EA-1096 flow-through wall-jet cell, equipped with a glassy carbon working electrode and a silver-silver chloride (3 M KC1) reference electrode, was used. The electrode was modified at a constant potential of +0.30 V for 3 min in 0.5 mM 3,4-DHB solution in pH 7 Tris/nitrate buffer. The potential of the modified electrode was held at f0.30 V (vs Ag/AgCl), and the currents were monitored with a Metrohm 641 VA potentiostat and recorded on a Liiseys L6512 Y-t recorder. The carrier stream (19) Deinhammer, R S.: Ho, M.: Anderegg, J. W.; Porter, M. D. Langmuir 1994, 10, 1306. (20) Torstensson, A:Gorton, L. J. Electroanal. Chem. 1981,130, 199. (21) Gorton, L.: Csoregi, E.; Dominguez, E.; Emneus, J.; Jonsson-Pettersson, G.; Marko-Varga, G.; Person, B. Anal. Chim. Acta 1991,250, 203. (22) Tanaka, K; Tokuda, K.; Ohsaka, T. J. Chem. SOC.,Chem. Commun. 1993, 1770. (23) Kulys, J.: Gleixner, G.; Schuhmann, W.; Schmidt, H.-L. Electroanalysis 1993, 5,201.
(24) Albery, W.J.; Bartlett, P. N. J. Chem. Soc., Chem. Commun. 1984,234. (25) Albery, W. J.; Bartlett, P. N.: C a s , A E. G. Phil. Trans. R. SOC.London, B 1987,316, 107.
4338 Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
was pumped by a Watson Marlow 202 U/1 peristaltic pump, and the sample was injected into the carrier stream by a six-way injection Rheodyne Type 50 valve.
Procedures. (a) Electrode Pretreatment and Activation. Prior to each experiment, glassy carbon electrodes were polished with 1 pm 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 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 3,4DHB was carried out as described below. SnOz electrodes (NESA glass from PPG Industries) were cleaned with methanol and acetone prior to use. (b) Electrode Modification with 3,4-DHB. Two different methods were used in the modification of the electrodes with 3,4DHB. In the first case, the activated electrode was placed in a solution containing 0.05-2.0 mM 3,4DHB in Tridnitrate buffer (PH 7.0 or 8.0) and the potential held at about +0.20 V (depending on pH, vide infra) for 3 min. In the second case, the activated electrode was modified by cycling the potential between -0.20 and +0.30 V (at 25 or 100 mv) in a 0.5 mM solution of 3,4DHB in Tris/nitrate buffer (PH 7.0 or 8.0). (c) FIAAnalysis. The carrier stream, Tris/nitrate buffer (PH 7.0), was pumped through the electrochemical cell at a flow rate of 0.7 mLmin-l. After a steady background current was obtained (typically 1 min), 75 pL of increasing concentrations of NADH were introduced, and the change in current at an applied potential of +0.30 V was recorded. RESULTS AND DISCUSSION 1. Electrochemical Response of 3,4-DHB in Solution and Its Electropolymerization. Cyclic voltammograms of 1mM 3,4 DHB (see Figure 1 for structure) in Tridnitrate buffer (PH 8.0) were carried out over the potential range of -0.30 to +OB0 V with glassy carbon electrodes that were either polished or polished and activated as described in the Experimental Section. On the first voltammetric scan at an activated electrode (Figure lA)(initial potential of -0.30 V), a sharp anodic peak is observed at a peak potential value of +0.25 V. No further anodic processes were observed up to potentials of +0.80 V. Upon scan reversal at f0.80 V, a small cathodic wave was observed at a peak potential value of +0.04 V. On further potential scanning, an additional anodic peak was observed at a peak potential value of +0.13 V. This wave could be due to hydroxylation of the immobilized material to the triol. Such behavior has been previously reported by Kuwana and co-workers.26 In addition, the initial sharp anodic peak was broadened significantly, and the peak potential value shifted to f0.42 V. Upon continuous scanning, this peak continued to broaden and shift. In addition, all of the voltammetric processes decreased in intensity with continued potential cycling (Figure lA). On the other hand, if the potential is cycled within the limits of -0.20 and +0.30 V, there is a continuous decrease in the amplitude of the initial sharp anodic peak (Figure 1B). Concomitant with this process is the emergence of a new reversible peak with a formal potential of +0.15 V and whose peak intensity increases with cycling. The shape of this new voltammetric feature is that of a surface-immobilized redox couple, suggesting (26) Gui, J. Y.; Hance, G. W.; Kuwana, T. J. Electroanal. Chem. 1991,309, 73.
I-
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h\
5p E vs SSCE
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I
1
l
1
l
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I
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l
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I
,
l
t0.80 +0.60 +0.40 +0.20
.
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1
0.0
1
I
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l
-0.20
EvsSSCE
i+-++H+0.20
1
l
-0.20
E vs SSCE
Figure t Cyclic voltammograms at 100 mV/s of 1 .O mM 3,4-DHB in Tridnitrate buffer (pH 7.0) at an activated glassy carbon electrode. Scan limits, -0.30 to +0.80V (A) and -0.20 to +0.30V (8). (C) Cyclic voltammogram of the modified electrode obtained in Figure 1B in Tris/ nitrate buffer solution (pH 7.0). Scan rate, 100 mV/s. Inset: structure of 3,4-DHB.
the formation of a redox-active polymeric (or oligomeric) film on the electrode surface. If the electrode is subsequently removed from the cell, rinsed with water, and placed in Tris/nitrate buffer (PH 7.0) containiig no 3,4DHB, a voltammetric peak typical of a surfaceimmobilized redox couple is observed with a formal potential value of f0.15 V (Figure 1C). Although there is some loss (typically about 10%) of material during the first 4-5 scans, further loss of the surface immobilized material takes place at a much slower rate as described below. On the other hand, if the potential is cycled to values more positive than f0.40 V, there is a very rapid and complete loss of the surfaceimmobilized material. Although this implies that care must be exercised in terms of what potentials are applied, it also provides for a very easy and convenient way to regenerate the electrode. Similar results, except with higher, more stable, and more reproducible coverages, are obtained if the electropolymerization is carried out at constant potential during a prescribed amount of time, with the potential being dependent on the pH of the electrolyte (vide infra). In general, best results were obtained when the applied potential was 50 mV beyond the peak for the oxidation of 3,4DHB. Although qualitatively similar results were obtained for electrodes that were only polished, consistently superior results were obtained with electrodes that had been polished and activated in base as described in the ExperimentaI Section. Thus, all further studies were carried out with activated glassy carbon electrodes. The electropolymerized films exhibited the behavior anticipated for a surfaceimmobilizedredox couple. As shown in Figure
0
100
200
300
400
500
600
v (mV/sec) Figure 2. Sweep rate dependence of the cyclic voltammetric response of a glassy carbon electrode modified with 3,4-DHB in Tris/ nitrate buffer (pH 7.0). Scan rates, 25(a); 50 (b); 75 (c); 100 (d); 200 (e); 300 (f); 400 (9); and 500 mV/s (h). Inset: plot of anodic peak current vs sweep rate.
2, the wave had the expected wave shape for a surfaceconfined redox center with a small (although not zero) AEp value. In addition, the current was directly proportional to the rate of potential sweep over the range of 25 to 500 mV/s, suggesting facile charge transfer kinetics (Figure 2). Experiments identical to those described above for glassy carbon electrodes were carried out on polished platinum electrodes (platinum electrodes were not activated, but rather prior to use, the potential was cycled in supporting electrolyte until a stable background response was obtained). However, in this case, no evidence of electropolymerization was apparent in the potential range used above. In an attempt to identify the species responsible for the observed polymerization reaction, the voltammetric response of benzaldehyde at an activated glassy carbon electrode was o b tained. Benzaldehyde exhibited a totally irreversible wave with a peak potential value of +1.80 V. However, there was no evidence of polymerization. In fact, when the electrode was removed from the benzaldehyde solution and placed in Trishitrate buffer (PH 7.01, there were no redox processes over the range of f0.50 to -0.20 V other than background currents. Although the 3,4DHB did not polymerize at a platinum electrode surface, we observed polymerization at a SnOz electrode, suggesting that the presence of oxygen containiig surface species is likely to play a role. In addition, the fact that activated electrodes gave rise to more stable deposits and higher coverages is also consistent with this. In the work by Porter et al. mentioned earlier,lg they found that, whereas deposition took place on glassy carbon electrodes, such was not the case on HOPG. This difference in reactivity Analytical Chemistry, Vol. 66, No. 23,December 1, 1994
4339
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(27) Hapiot, P.; Pinson, J.; Francesch, C.; Mhamdi, F.; Rolando, C.; Neb, P. J. Phys. Chem. 1994, 98, 2641. (28) Slabbert, N. P. Tetrahedron 1977, 33. 821.
4340 Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
0
10
20
Time (min.)
30
40
Figure 4. (Main panel) Variations in fractional coverage as a function of time of continuous scanning (scan rate, 100 mV/s) for a glassy carbon electrode modified with an electropolymerized film of 3,4-DHB. Deposition was carried out from 0.5 mM 3,4-DHB in Tris/ nitrate buffer (pH 7.0) at a fixed potential of +0.20 V for 3 (0)or 6 min (H). Insets: first- and second-order plots for the variation in fractional coverage as a function of time for electrodes modified for (A) 6 and (B) 3 min.
3. Mode of Deposition. 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 electropolymerization process, respectively. In order to determine the most effective method for electrode modification, comparisons were made in terms of the stability of the resulting films. This involved measuring the decay of the redox activity of the electropolymerized film. In the case where electropolymerization was at constant potential, the applied potential was f0.20 V, since this was determined to be the optimal value (vide infra). The electrodes were modified for 3 and 6 min. After modification, the electrodes were rinsed with water and placed in pH 7.0 Tridnitrate buffer, and the decay of the surface wave with time was determined. Whereas there was a significant decay in both cases, it was somewhat higher for electrodes that had been modified for 6 min relative to those modded for 3 min, as can be seen in the main panel of Figure 4. For both sets of experiments, plots of In coverage vs time as well as l/coverage vs time were constructed. As can be seen in insets A and B in Figure 4, better agreement (correlation) was obtained when the data were plotted as l/coverage vs time, suggesting that the decay process is second order. Similar experiments were carried out for electropolymerization effected by cycling of the potential. In this case, two different sweep rates were employed: 25 and 100 mV/s, respectively. As can be seen in the main panel of Figure 5, there was again a rather significant loss of electroactivity, with the effect being somewhat higher for electrodes where the sweep rate was 100 mV/s. As was done for the case of electrodes modified by holding the potential, plots of In coverage vs time as well as l/coverage vs time were constructed. As in the previous case, better fits were obtained for plots of l/coverage vs time, again suggesting that the decay process was second order. It should also be mentioned that after about 30 min of continuous cycling, the rate of decay slows down significantly for electrodes prepared by either method. In fact, the additional loss is less than about 5% for cycling of up to 8 continuous hours. This suggests that the initial decay observed in Figures 4 and 5 might
iin
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Figure 5. Variations in fractional coverage as a function of time of continuous scanning (scan rate, 100 mV/s) for a glassy carbon electrode modified with an electropolymerized film of 3,4-DHB. Deposition was carried out from 0.5 mM 3,4-DHB in Tridnitrate buffer (pH 7.0) cycling the potential between -0.2 and 0.3 V at 25 (0)or 100 mV/s (M).Insets: first- and second-order plots for the variation in fractional coverage as a function of time for electrodes modified at (A) 25 and (6) 100 mV/s.
be due to material that is weakly bound to the surface or to shortlength polymer chains that can desorb from the electrode surface with relative ease. The remaining material, however, is quite persistent, as mentioned above. We also studied the permeability and the apparent diffusion coefficient, D,,,, of films prepared by both methods. In the permeability studies, the limiting current, il for the electrochemical reaction of a solute (methyl viologen (MV) in this case) that partitions (with partition coefficient Pj and diffuses into a rotated disk electrode through a film barrier with diffusionconstant Ds,po~ (which is different from the value in solution) is described by eq 1:29
where d is the film thickness and o is in rad/s. The two terms on the right-hand side of eq 1 represent the rates of solute diffusion through the film and through the Levich depletion layer in solution, respectively. If diffusionthrough the film is slower than diffusion through the Levich layer, a plot of il vs (a Levich plot) will not be linear. However, a plot of i1-l vs o-lI2 (an inverse Levich plot) will be linear, and from the intercept, the value of (D,,,+‘/d) can be obtained. We have carried out such an analysis at both bare and 3,4 DHB-modfied electrodes (modified by cycling the potential as well as by electropolymerization at constant potential) using methyl viologen in solution. From values of the limiting current we constructed inverse Levich plots (Figure 6), and from their intercepts we calculated PDS,,,l/d values for MV permeation. We obtain values of 0.13 and 0.20 cm9-l for polymerization at constant potential and by potential cycling, respectively. From the slope of the inverse Levich plot, the diffusion coefficient for MV in ~~
~~
(29) Ikeda, T.;Schmehl, R; Denisevich, P.; Willman, K; Murray, R W. J. Am. Chem SOC. 1982,104, 2683.
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110I n Figure 6. Reciprocal Levich plots (lli, vs l/wl/*) for the reduction of methyl viologen (1 mM) in 0.10 M NaN03 at a bare glassy carbon electrode as well as at electrodes modified with 3,4-DHB at constant potential or by cycling the potential for 3 min. Inset: expanded region near the intercept.
solution was determined to be (8.7 f 0.1) x cmz s-l, in agreement with values obtained at an unmodified glassy carbon electrode as well as literature values.30 Thus, it is clear that permeation rates through electropolymerized films of 3,4DHB are rapid, so one would not anticipate kinetic limitations. In general, it was found that more stable and reproducible deposits were obtained when the electropolymerization was carried out at constant potential, and subsequent studies were carried out with electrodes modified in this fashion. Using double potential step chronocoulometry, we determined the apparent diffusion coefficients (Dapp) for electropolymerized films of 3,4DHB deposited at constant potential (+0.20 V, 0.5 mM concentration of 3,4DHB, pH 7) for 1,6,12, and 24 min. Values for both reduction and oxidation processes were determined. In all cases we found that the slopes of the resulting Anson plots were virtually identical, indicating the same value of D,,, (1.6 x 10-lo cm2/s) for all polymerization times as well as for both oxidation and reduction processes. 4. Properties of the ElectropolymerizedFilms Prepared at Constant Applied Potential as a Function of pH, Concentration of 3,4-DHB, and T i e of Deposition. The dependence of the properties of the electropolymerized films prepared at constant applied potential for 3 min was studied as a function of pH during deposition. In these studies the potential applied during electropolymerization was chosen to be 50 mV beyond the peak potential for the oxidation of 3,4DHB. (Recall that this process is pH dependent.) The results are presented in Table 1. In general, it was found that the best results were obtained at slightly acidic pH values, although variations in the pH range from 4.5 to 6.5 were not dramatic. However, for pH values above 7, a significant decrease in the surface coverage is observed which could be due to deprotonation.28 Nonetheless, in studying the electrocatalytic oxidation of NADH, it was found that a higher catalytic activity was obtained for electrodes prepared at pH 7-8 (vide infra). Thus, as a compromise, electrodes were m o d ~ e d at pH 7 and at a potential of +0.20 V. We also studied the effect of solution concentration of 3,4DHB on the properties of the resulting films. In these studies, the films were prepared at pH 7.0 at a constant applied potential of +0.20 V for 3 min. The effect was ascertained by plotting the surface (30)Steckhan, E.; Kuwana, T. Bey. Bunseges. Phys. Chem. 1974,78,253.
Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
4341
’
1
Table I. Variations in the Properties of Electropolymerized Films of 3,4 DHB as a Function of Deposition Potential and pH
3.5 4.5 5.5 6.5 7.0 8.0 9.0 10.2
+0.45 5.8 +0.40 6.6 f0.35 4.7 +0.25 4.5 $0.20 4.2 $0.17 4.1 +0.15 2.9 +0.10 2.9
5.7 6.2 4.5 4.3 4.1 4.0 3.1 2.7
+0.360 +0.305 +0.250 $0.170 +0.150 +0.120 +0.050 -0.030
+0.300 +0.255 +0.190 +0.100 +0.100 +0.080 +0.020 -0.060
60 50 60 70 40 40 30 30
f0.330 +0.280 +0.220 $0.135 +0.130
3.74 4.99 3.23 3.72 3.21 +0.100 2.53 +0.035 1.87 -0.045 1.70
3.83 4.98 3.47 3.90 3.06 2.67 2.20 1.70
Deposition potential vs SSCE. Anodic and cathodic peak currents. Anodic and cathodic peak potentials vs SSCE. Defined as Ep,aFp,c.e Formal potential defined as (Ep,a+ EP,d/2 vs SSCE. f Coverages in mol/cm2 x lolo.
P tO.50
+0.30
+0.10
-0.10
-0.30
E vs SSCE Figure 8. Voltammetric behavior at 10 mVls in Trislnitrate buffer (pH 8.0) of a glassy carbon electrode modified with 3,4-DHB in the absence (A) and the presence (8)of 1 .O mM NADH.
0.0
1.o
2.0
3.0
4.0
[3,4 DHBl (M) Figure 7. Dependence of fractional coverage on solution concentration of 3,4-DHB in Trislnitrate buffer (pH 7.0) for electrodes modified by electropolymerization at a fixed potential of +0.20 V for 3 min. The fractional coverage is defined as T/Tm,, where rm,= 3 x mollcm'. The data presented are the averages of three determinations.
coverage normalized to the solution concentration of 3,4DHB. As can be seen in Figure 7, there is a sharp rise in the normalized coverage with the solution concentration of 3,4DHB which levels off for solution concentrations above 0.50 mM. Finally, the effect of time of deposition was determined. In this case, the resulting coverage of material on the electrode surface was monitored as function of time of deposition using a solution concentration of 0.50 mM at a pH of 7.0 and a potential of +0.20 V. We observe that for deposition times below 60 s, the surface coverage increases with time. For deposition times over 120 s and up to 10 min, there is no further increase in the surface coverage. However, when deposition is carried out for extensive time periods (25 min), the measured coverage actually decreases. We believe that this might be due, at least in part, to transport limitations through an ostensibly thicker polymer film so that only a fraction of it is electrochemically active. Such behavior has been previously documented for other polymer films on With the intent of ascertaining this effect, a transparent Sn02 electrode was modified by cycling the potential between -0.20 and +0.30 V for 4 h. Afterward, the spectrum of the modified electrode was compared to that obtained prior to deposition. From an analysis of the interference peaks, we estimate the thickness of the film to be of the order of 1000A. However, from integration of the area under the voltammetric wave, a much lower coverage (about 5 x mol/cm2) was obtained, consistent with our earlier statement that only a very small fraction of the deposited polymer is electroactive. 4342 Analytical Chemistry, Val. 66, No. 23, December 1, 1994
Based on these observations, a deposition time of 3 min was chosen as optimal. From the results presented above, a solution concentration of 3,4DHB of 0.5 mM and a deposition time of 3 min at a constant potential of +0.20 V in pH 7 Tris/nitrate buffer were employed in subsequent studies. 5. 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 3,4DHB as a modifying agent since it has an &quinone moiety which, as mentioned earlier, has been identified as one that is capable of catalytically oxidizing NADH. In order to test for the potential electrocatalytic activity of these electropolymerized films, their cyclic voltammetric responses were obtained in the absence and presence of 1 mM NADH, and the data are presented in Figure 8. In the absence of NADH (Figure 8A), a well-behaved redox response for the polymer film on the electrode can be observed at a formal potential value of +0.18 V. Upon the addition of 1 mM NADH, there is a dramatic enhancement of the anodic current peak (Figure 8B), and, in addition, no current is observed in the return (cathodic) wave, consistent with a very strong electrocatalytic effect. Under identical conditions, NADH is oxidized at about +0.50 V at an unmodified but activated glassy carbon electrode. However, it should also be mentioned that, whereas for electrodes that are only activated, the oxidation peak potential for NADH progressively shifts to more positive values, in the case of the 3,4DHBmodified electrodes, the electrocatalytic response is quite persistent (vide infra). 6. Optimization of Electrocatalytic Response for the Oxidation of NADH. In order to optimize the electrocatalytic response of the modified electrodes for NADH oxidation, we determined the effect of potential and pH during deposition on the electrocatalytic oxidation of NADH at 1 mM concentration. As mentioned earlier, these two variables affect the surface coverage of the polymer on the electrode surface as well as the electrocatalytic oxidation of NADH.
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Figure 9 presents the results for the effects of the applied potential (for 3 min in pH 8.0 Trishitrate buffer) on the fractional coverage (rE/rm&l as well as on the catalytic current for NADH oxidation. As would be anticipated from the results presented earlier in Table 1,the highest coverage is obtained at an applied potential of +0.15 V. On the other hand, the catalytic current had a broad maximum over the range of +0.15 to +0.20 V. There was also a dramatic decrease both in the surface coverage and on the catalytic activity at potentials below +0.10 V and above +0.30 V, respectively. Thus, an applied potential of +0.20 V was deemed as most appropriate for maximizing the electrocatalytic response of the m o d ~ e delectrodes. These results are also consistent with those presented earlier in Table 1. The effects of pH during deposition on the fractional coverage and the catalytic activity are presented in Figure 10. As had been described earlier and as also seen in Figure 10, the highest coverages were obtained in slightly acidic media. The decrease in coverage at pH values above 7 could again be due to deprotonation.z8 The catalytic activity follows a similar trend, and this might reflect the fact that, at these slightly acidic pH values, there is more material on the electrode surface and thus a larger catalytic current. In order to account for this, the catalytic current was normalized by dividing by the peak current for the electropolymerized film in the absence of NADH. The results are presented in the inset to Figure 10, where it is apparent that the normalized catalytic activity is higher at higher pH. Based on
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NADH (mM) Figure 11. (A) Calibration curve for a 3,4-DHB-modified glassy carbon electrode in a FIA system in Trishitrate buffer solution (pH 8.0) at 0.70 mlmin-l. (6)Response in the lower concentration range to 1 .O x M). (C) Recorder tracings for a series of (3.2 x replicate injections of NADH at concentrations of (a) 3.2 x 10-6, (b) (c) 1.2 x and (d) 2.5 x M. 6.2 x
this observation, a pH value of 8.0 was chosen as representative of a compromise among the various dependencies. Based on these studies as well as on the results presented in Table 1, we chose a pH of 8.0, an applied potential of +0.20 V (for 3 min), and a solution concentration of 3,4-DHB of 0.5 mM as representing the best compromise for the modification of glassy carbon electrodes with electropolymerized films of 3,4DHB for their subsequent use in the electrocatalytic oxidation of NADH. 7. Calibration and Limit of NADH Detection of 3,4-DHBModitied Electrodes. The effect of increasing concentrations of NADH on the voltammetric response of the 3,4-DHB-modified electrodes was investigated. The response was linear from 5.0 x to 1.0 x M NADH. Hydrodynamic voltammetry under flow injection conditions,which possesses a much higher current sensitivity than cyclic voltammetry, was used to estimate the lower limit of detection for the electrocatalyticoxidation of NADH at a 3,CDHB-modified glassy carbon electrode. A calibration curve for NADH oxidation by continuous flow monitoring of the current at a fixed potential over the concentration range of 3.2 x to 6.0 x 10-3 M is shown in Figure I l k At higher concentrations, there appears to be a leveling off of the response, likely due to kinetic limitations. On the other hand, at the lower concentrations, a linear correlation is observed. For example, Figure 11B depicts the response over the range of concentrations of 3.2 x to 1.0 x 10-4 M. In all cases the response was rapid and reproducAnalytical Chemistry, Vol. 66, No. 23, December 1, 1994
4343
ible. Figure 11C shows the recorder tracings for a series of replicate injections of NADH at the lower concentrations from 3.2 x M (a) to 2.5 x M (d). From analysis of these data, we estimate that the limit of detection of NADH is of the order of 5.0 10-7 M. Although we have not carried out extensive stability studies, we typically employ the same electrode during the course of a day in numerous determinations such as a complete calibration (in replicate determinations). As mentioned earlier, after an initial loss of activity, the electrodes exhibit a very stable response during hours of continuous potential cycling. In addition, the electrode is quite stable at high NADH concentrations, conditions under which many other electrodes that exhibit electrocatalytic activity for NADH oxidation fail. For example, in replicate determinations of NADH at near saturation (1-5 mM), we observe less than 5% loss in activity. Although in principle a single electrode could be employed for long periods of time (days), due to the ease of preparation we typically modify an electrode and employ it in all determinations in the course of a day including calibration and other determinations. In addition, if a calibration curve is normalized to the surface coverage of the polymer, a universal calibration curve is obtained. Such an approach can be quite valuable, especially in sensor applications. It is likely that materials similar to 3,4-DHB will also undergo electropolymerization to give rise to redox-active polymer films that are also catalytically active in the oxidation of NADH. We are in the process of studying such materials, and the results of those investigations will be reported on elsewhere. CONCLUSIONS
The oxidation of 3,4 dihydroxy benzaldehyde (3,4DHB) on glassy carbon electrodes gives rise to stable redox-active elec-
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Analytical Chemistry, Vol. 66, No. 23, December 1, 1994
tropolymerized films. Film deposition by controlled potential gives consistently results superior to those obtained from films prepared by potential cycling. The permeability of the resulting films is such that rapid rates of transport are always obtained. The redox response of the films is that anticipated for a surfaceimmobilized redox couple, and the pH dependence of the redox activity of these films is 60 mV/pH unit. These films exhibit potent and persistent electrocatalytic behavior toward NADH oxidation. We have optimized the film growth conditions (in order to maximize the electrocatalytic activity) in terms of the solution concentration of 3,4-DHB, the potential for deposition, the time of deposition, and the pH. In a flow injection analysis determination, the limit of detection of NADH was estimated to be of the order of 5.0 x M. ACKNOWLEDGMENT
This work was supported by the National Science Foundation (DMR-9107116), a NATO Collaborative Research Grant (91-0047), and the DGICYT of Spain @IO 93-0660-CO4-02). We thank the reviewers for calling to our attention refs 19, 26, and 28.
ScientiFc Parentage ofthe Author. H. D. Abrufia, Ph.D. under R W. Murray, Ph.D. under R C. Bowers, Ph.D. under I. M. Kolthoff.
Received for review May 20, 1994. Accepted August 25,
1994.B e Abstract published in Advance ACS Abstracts, October 15, 1994.
