Electrocatalytic Oxidation of NADH at Glassy Carbon Electrodes

(B) Cyclic voltammogram at a sweep rate of 50 mV/s in phosphate buffer solution (pH 7.2) for a GC electrode modified with an electrodeposited film of ...
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Anal. Chem. 1996, 68, 3688-3696

Electrocatalytic Oxidation of NADH at Glassy Carbon Electrodes Modified with Transition Metal Complexes Containing 1,10-Phenanthroline-5,6-dione Ligands Q. Wu,† M. Maskus,† F. Pariente,‡ F. Tobalina,‡ V. M. Ferna´ndez,§ E. Lorenzo,‡ and H. D. Abrun˜a*,†

Baker Laboratory, Department of Chemistry, Cornell University, Ithaca, New York 14853-1301, Departamento de Quı´mica Analı´tica, Universidad Auto´ noma de Madrid, Canto Blanco 28049, Madrid, Spain, and Instituto de Cata´ lisis, CSIC, Madrid, Spain

The preparation and electrochemical characterization of glassy carbon electrodes modified with 1,10-phenanthroline-5,6-dione (phen-dione) complexes of transition metals including [M(phen-dione)3]2+ (M ) Fe, Ru, Co, Cr, Ni), [Re(phen-dione)(CO)3Cl], and [Ru(v-bpy)2(phen-dione)](PF6)2 (v-bpy is 4-vinyl-4′-methyl-2,2′-bipyridine) as well as their behavior as electrocatalysts toward the oxidation of NADH are described. The above-mentioned materials could be deposited by electrochemical deposition or electropolymerization. The resulting modified electrodes exhibited redox responses very similar to those of the complexes in solution, including the pH-dependent response ascribed to the pendant o-quinone groups. The deposited films also showed potent and persistent electrocatalytic activity toward NADH oxidation. In all cases, NADH oxidation took place at potentials around 0.0 V vs SSCE, which represents a dramatic diminution in the overpotential. In addition, interference effects due to ascorbate could be significantly decreased. Of all the materials tested, the [Fe(phen-dione)3]2+ complex was found to have the highest activity toward NADH oxidation. The kinetics of the catalytic reaction were characterized by using cyclic voltammetry and rotated disk electrode techniques, and a value of (6.2 ( 0.6) × 103 M-1 s-1 was obtained for electrodes modified with this complex. Moreover, this complex exhibited the best stability as determined from the time dependency of the decay of its redox activity. Using these observations as a point of departure, we have developed an ethanol biosensor based on the enzymatic activity of immobilized (on a nylon mesh) alcohol dehydrogenase, coupled with the electrocatalytic oxidation of NADH. Because over 300 dehydrogenases require nicotinamide coenzymes as cofactors, the electrocatalytic oxidation of β-nicotinamide adenine dinucleotide (NADH) has been of particular interest, especially since the direct oxidation of NADH (which at pH 7.0 should take place at about -0.55 V) at bare electrode surfaces takes place with high overpotentials.1 In addition, the development of such electrocatalysts could be coupled to biosensor design †

Cornell University. Universidad Auto´noma de Madrid. § CSIC. ‡

3688 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

for the determination of numerous species.2,3 The overpotentials for NADH oxidation at pH 7.0 are about +1.1 V for carbon4 and +1.3 V for platinum5 electrodes. In addition, the direct oxidation of NADH is often accompanied by electrode fouling and interfering background currents in real samples6-8 and sometimes leads to the formation of enzymatically inactive forms of NAD+.9 The high overpotential for the oxidation of NADH can be considerably reduced through the use of mediators, either in solution or immobilized on the electrode surface, which can undergo fast redox reactions with NADH. Much effort10-26 has been dedicated to identifying materials which could be employed as mediators. These include quinones,24 catechols,13,15 redox dyes,17,23 ferrocene derivatives,25 inorganic metal complexes,10,26 conducting organic salts,14 and conducting polymers.16 We have been examining means of lowering the overpotential of and enhancing the selectivity for NADH oxidation in homogeneous solution as well as with modified electrodes. We previously (1) Elving, P. J.; Schmakel, C. O.; Santhanam, K. S. V. Crit. Rev. Anal. Chem. 1976, 6, 1. (2) Blaedel, W. J.; Jenkins, R. A. Anal. Chem. 1976, 48, 1240. (3) Thomas, L. C.; Christian, D. Anal. Chim. Acta 1978, 78, 271. (4) Moiroux, J.; Elving, P. J. Anal. Chem. 1978, 50, 1056. (5) Jaegfeldt, H. J. Electroanal. Chem. 1980, 110, 295. (6) Coughlin, R. W.; Aizawa, M.; Alexander, B. F.; Charles, M. Biotechnol. Bioeng. 1975, 17, 515. (7) Schmakel, C. O.; Santhanam, K. S. V.; Elving, P. J. J. Am. Chem. Soc. 1975, 97, 5085. (8) Morroux, J.; Elving, P. J. J. Electroanal. Chem. 1979, 102, 93. (9) Bartalits, L.; Nagy, G.; Pungor, E. Anal. Lett. 1984, 17, 13. (10) Gorton, L. J. Chem. Soc., Faraday Trans. 1 1986, 82, 1245. (11) Cai, C. X.; Ju, H. X.; Chen, H. Y. J. Electroanal. Chem. 1995, 397, 185. (12) Essaadi, K.; Keita, B.; Nadjo, L. J. Electroanal. Chem. 1994, 367, 275. (13) Ueda, C.; Tse, D. C.; Kuwana, T. Anal. Chem. 1982, 54, 850. (14) Albery, W. J.; Bartlett, P. N. J. Chem. Soc., Chem. Commun. 1984, 234. (15) Jaegfeldt, H.; Kuwana, T.; Johansson, G. J. Am. Chem. Soc. 1983, 105, 1805. (16) Ohsaka, T.; Tanaka, K.; Tokuda, K. J. Chem. Soc., Chem. Commun. 1993, 222. (17) Schlereth, D. D.; Katz, E.; Schmidt, H.-L. Electroanalysis 1995, 7, 46. (18) Degrand, C.; Miller, L. L. J. Am. Chem. Soc. 1980, 102, 5728. (19) Deinhammer, R. S.; Ho, M.; Anderegg, J. W.; Porter, M. D. Langmuir 1994, 10, 1306. (20) Silber, A.; Bra¨uchle, C.; Hampp, N. J. Electroanal. Chem. 1995, 390, 83. (21) Pariente, F.; Lorenzo, E.; Abrun ˜a, H. D. Anal. Chem. 1994, 66, 4337. (22) Xu, F.; Li, H.; Cross, S. J.; Guarr, T. F. J. Electroanal. Chem. 1994, 368, 221. (23) Karyakin, A. A.; Karyakina, E. E.; Schubmann, W.; Schmidt, H. L. Electroanalysis 1994, 6, 821. (24) Miller, L. L.; Valentine, J. R. J Am. Chem. Soc. 1988, 110, 3982. (25) Matsue, T.; Suda, M.; Uchida, I,; Kato, T.; Akibi, U.; Osa, T. J. Electroanal. Chem. 1987, 234, 163. (26) Beley, M.; Colin, J.-P. J. Mol. Catal. 1993, 79, 133. S0003-2700(96)00395-2 CCC: $12.00

© 1996 American Chemical Society

reported the electrocatalysis of NADH oxidation using glassy carbon (GC) electrodes modified with electrodeposited films of 3,4-dihydroxybenzaldehyde (3,4-DHB), which exhibited very high and persistent electrocatalytic activity for the oxidation of NADH at low potentials.21 We also combined the electrocatalytic activity of GC electrodes modified with electrodeposited films of 3,4-DHB with the enzymatic activity of immobilized (on a nylon mesh) aldehyde dehydrogenase to develop an aldehyde biosensor.27 To further lower the overpotential for the oxidation of NADH, we have tested other potential mediators, and in the present case we have employed transition metal complexes of 1,10-phenanthroline-5,6-dione (phen-dione). This ligand has the ability to form stable complexes with a wide variety of metal ions. In addition, metal complexes of this ligand potentially allow for the variation and control of redox properties over a wide range. In a previous article, we reported the synthesis and the spectral and electrochemical characterization of phen-dione complexes of transition metals.28 Some of the materials were shown to be electrocatalytic toward the oxidation of NADH in solution and as electrodeposited films. We have recently found that electrodeposition of phen-dione complexes of transition metals as well as electropolymerization of [Ru(phen-dione)(v-bpy)2](PF6)2 on GC electrodes produces stable redox-active films which exhibit very high and persistent electrocatalytic activity for the oxidation of NADH at very low potentials. We describe here the preparation and characterization of such modified GC electrodes and their electrocatalytic behavior toward the oxidation of NADH. In addition, we demonstrate their utility in biosensor design using immobilized alcohol dehydrogenase for the preparation of an ethanol biosensor whose construction and characterization we also describe. EXPERIMENTAL SECTION Reagents. 1,10-Phenanthroline-5,6-dione (phen-dione) was obtained from G. F. Smith Co. [Re(phen-dione)(CO)3Cl], [Fe(phen-dione)3](PF6)2, [Co(phen-dione)3](PF6)2, [Cr(phen-dione)3](PF6)3, [Ni(phen-dione)3](PF6)2, [Ru(phen-dione)3](PF6)2, and [Ru(v-bpy)2(phen-dione)](PF6)2 (referred to in the text as the Re, Fe, Co, Cr, Ni, Ru, and Ru(v-bpy) complexes, respectively) were prepared as described previously.28-30 Electrochemical measurements were performed in water (purified by passage through a Milli-Q purification system) and acetonitrile (AN; Burdick and Jackson distilled in glass, dried over 4-Å molecular sieves). Tetra-n-butylammonium perchlorate (TBAP; G. F. Smith Co.) was recrystallized three times from ethyl acetate and dried under vacuum at 90 °C for 72 h. In aqueous media, potassium phosphate (Fisher) buffers were employed as supporting electrolyte, whereas for biosensor response, a Tris/HCl (Merck) buffer was employed. Alcohol dehydrogenase (ADH, EC 1.1.1.1) from baker’s yeast was obtained from Sigma Chemical Co. as a lyophilized powder containing 340 units of enzyme activity per milligram of protein. The preparation was stored below 0 °C. Under these conditions, no loss of enzymatic activity was observed for several months. Oxidized and reduced forms of nicotine adenine dinucleotide (NAD+ and NADH, grade III), glutaraldehyde (grade I, 50% aqueous solution), and bovine serum albumin (27) Pariente, F.; Lorenzo, E.; Tobalina, F.; Abrun ˜a, H. D. Anal. Chem. 1995, 67, 3936. (28) Goss, C. A.; Abrun ˜a, H. D. Inorg. Chem. 1985, 24, 4263. (29) Wrighton, M. S.; Morse, D. L. J. Am. Chem. Soc. 1974, 96, 998. (30) Breikss, A. I.; Abrun ˜a, H. D. J. Electroanal. Chem. 1986, 201, 347.

(BSA, fraction V, 96% purity) were obtained from Sigma Chemical Co. and used as received. Nylon mesh (Nytal) disks with 50- × 50-µm pores and 70-µm thickness were employed for enzyme immobilization. Ethanol, used as substrate of alcohol dehydrogenase, was a high-purity reagent obtained from Carlo Erba. All other reagents were of at least reagent grade quality and were used without further purification. Instrumentation. Electrochemical experiments were performed with an IBM EC-225 voltammetric analyzer or a BAS CV27 potentiostat. Data were recorded on a Soltec VP-64236 or Linseys X-Y recorder. Three-compartment electrochemical cells (separated by medium-porosity sintered glass disks) with the provision for addition of solutions were employed. All joints were standard-taper so that all compartments could be hermetically sealed with Teflon adapters. A glassy carbon (GC) electrode (geometric area, 0.071 cm2) was used as the working electrode. A coiled platinum wire was used as a counter electrode. For rotated disk electrode experiments, a Pine Instruments rotator and a RDE-3 bipotentiostat were employed. A GC electrode from Pine (geometric area, 0.283 cm2) was used as the working electrode. All potentials are referenced to a sodium saturated calomel electrode (SSCE) without regard for the liquid junction potential. Procedures. Electrode Activation and Modification. Prior to use, the GC electrodes were polished with 1-µm diamond paste (Buehler) and rinsed thoroughly with water and acetone. Before depositing the complexes, the polished electrode was activated by placing it in a 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 phosphate buffer (pH 7.0) for 5 min. The electrode was subsequently rinsed with water and modified with the desired complex by placing it in a 0.50 mM solution in phosphate buffer (pH 7.0) and holding the potential at about -0.20 V for 3 min. The electrode could also be modified by cycling the potential at 100 mV/s over the range from +0.50 to -0.30 V. The coated electrodes were rinsed thoroughly with water and placed in fresh buffer solution. In the case of the Ru(v-bpy) complex, with its polymerizable vinylbipyridine ligands, the polished electrode was modified by cycling the potential (at 100 mV/s) between +0.40 and -1.90 V for five or six cycles in a 1.0 mM solution of [Ru(phen-dione)(v-bpy)2](PF6)2 in AN (0.10 M TBAP) and then rinsing with AN and water.31 Surface coverages for the modified electrodes were determined in aqueous electrolyte (0.10 M phosphate buffer) solution, containing no dissolved complex, from the integrated charge under the anodic wave centered at around 0.0 V, assuming n ) 2 for the Re and Ru(v-bpy) complexes and n ) 6 for the Fe, Ru, Cr, Ni, and Co complexes. Although the surfaces of these electrodes are microscopically rough, it is difficult to estimate the roughness factor, so in all calculations we have employed the geometric area. Thus, the values reported represent upper limits. Typical coverage values were (1.3-2.0) × 10-10 mol cm-2 for electrodeposited films and (3.0-4.5) × 10-10 mol cm-2 for the electropolymerized films. Electrochemical Measurements of NADH. Prior to NADH measurements, the modified electrodes were placed in 4.0 mL of a thoroughly degassed buffer solution (oxygen can influence the linearity of the response), and the potential was cycled between (31) Abrun ˜a, H. D.; Denisevich, P.; Uman ˜a, M.; Meyer, T. J.; Murray, R. W. J. Am. Chem. Soc. 1981, 103, 1.

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-0.40 and +0.20 V at a rate of 20 mV/s for about 10 min. NADH measurements were performed by subsequently adding aliquots (10 and 20 µL) of NADH solutions (typically 0.025 or 0.0025 M) with a gas-tight syringe. The current due to the electrocatalytic oxidation of NADH was recorded after each addition. Enzyme Immobilization. ADH was dissolved in 0.10 M Tris/ HCl (pH 8.0) buffer containing 50% glycerol. As we have previously reported for aldehyde dehydrogenase, in the absence of glycerol a loss of enzymatic activity after immobilization was apparent.27 Nylon meshes were cut into 5.0-6.0-mm diameter disks, dipped in methanol, rinsed with water, and dried in air prior to use. For enzyme immobilization, the procedure employed followed closely the one previously described in the immobilization of aldehyde dehydrogenase.27 The following solutions were added to each disk: 2.0 µL of glutaraldehyde (2.5% v/v), 2.0 µL of BSA (10% w/v), and 1.5 µL (34 units) of ADH (1 mg in 15 µL of Tris buffer containing 50% (v/v) of glycerol). The mixture was carefully homogenized on the surface of the disk and allowed to dry at room temperature for 30 min. Prior to use, the unreacted carboxaldehyde groups in glutaraldehyde were inactivated by immersing the disks in 25.0 mL of 0.10 M phosphate buffer (pH 7.0) containing 0.10 M glycine for 15-30 min at room temperature. When not in use, the membranes were stored at -4 °C. Biosensor Preparation and Response. The biosensor was assembled by securing, with a holed cap, an enzyme-modified nylon mesh disk (prepared as described above) over a glassy carbon electrode previously modified with the iron complex. The assembled biosensor was placed in buffer solution for about 5 min prior to use to ensure solvent equilibration. The biosensor response was assayed in 0.1 M Tris/HCl (pH 8.0) buffer solution. For ethanol determinations, the biosensor was placed in 3.0 mL of buffer solution at an applied potential of 0.0 V. After the background current had decayed to a steady value, aliquots of a stock solution of ethanol in buffer were added. The solution was stirred for 30 s and allowed to stand for 2 min for equilibration. The steady-state current (achieved in less than 60 s) in the unstirred solution was subsequently recorded. RESULTS AND DISCUSSION Electrochemical Characterization of the Complexes in Solution and as Thin Films. The electrochemical behavior of phen-dione as well as its metal complexes has been previously described.28 In nonaqueous solvents such as AN, the complexes typically exhibit metal-based oxidations as well as ligand-based reductions. In the latter case, one can distinguish quinone-based redox processes (on the phen-dione) as well as additional ligand reductions at more negative potentials. For example, in AN (0.1 M TBAP), [Fe(phen-dione)3](PF6)2 exhibits one metal-based oxidation at +1.36 V and two ligand-based reductions at -0.18 and -0.92 V, respectively, which correspond to reductions of the quinone groups in the phen-dione ligands. It is of note that all three groups are reduced at the same potential, as ascertained from peak current values. As a second example, a reductive cyclic voltammogram of [Re(phen-dione)(CO)3Cl] in AN (0.10 M TBAP) exhibits three oneelectron redox processes at formal potential values of -0.13, -0.73, and -1.64 V, respectively (Figure 1A). The first two redox processes are ascribed to phen-dione-based reductions, where the potentials are shifted positively relative to those of the free ligand due to coordination to the rhenium center. The third redox process is assigned to a ligand-based reduction similar to the 3690 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

Figure 1. (A) Cyclic voltammogram at a sweep rate of 0.1 V/s for a GC electrode in AN (0.10 M TBAP) containing 1.0 mM [Re(phendione)(CO)3Cl]. (B) Cyclic voltammogram at a sweep rate of 50 mV/s in phosphate buffer solution (pH 7.2) for a GC electrode modified with an electrodeposited film of [Re(phen-dione)(CO)3Cl]. Table 1. Formal Potentials in Solution and for Immobilized Films of Transition Metal Complexes of Phen-dione and Peak Potential Value for the Electrocatalysis of NADH for Glassy Carbon Electrodes Modified with Films of the Same

complex

solutiona

depositeda

Ep for NADH electrocatalysisa

[Ru(phen-dione)3](PF6)2 [Cr(phen-dione)3](PF6)3 [Co(phen-dione)3](PF6)2 [Fe(phen-dione)3](PF6)2 [Ni(phen-dione)3](PF6)2 [Re(phen-dione)(CO)3Cl] [Ru(phen-dione)(v-bpy)2](PF6)2 ligand (phen-dione)

-0.04 -0.05 -0.12 -0.12 -0.05 -0.13b -0.13b -0.10

-0.04 -0.06 -0.08 -0.09 -0.05 -0.03 -0.03 -0.06

+0.05 -0.01 -0.02 -0.05 -0.02 0.0 0.0 -0.01

E0′ (V)

a

V vs SSCE. b In acetonitrile solution.

electrochemical response of [Re(dmbpy)(CO)3Cl] (dmbpy is 4,4′dimethyl-2,2′-bipyridine).30 In aqueous media, which is the focus of the present work, the complexes exhibited pH-dependent redox responses based on the quinones moieties. Values of the formal potentials for the different complexes, in aqueous solution and as deposited films, are presented in Table 1. As we previously described,28 some transition metal complexes of 1,10-phenanthroline-5,6-dione adsorb strongly to GC electrodes at negative potentials, and these modified electrodes retain their electroactivity in pure supporting electrolyte. Figure 1B shows a cyclic voltammogram for a GC electrode modified with an electrodeposited film of [Re(phen-dione)(CO)3Cl] in a phosphate buffer solution (pH 7.2). As can be ascertained, instead of two phen-dione-based one-electron reductions, as in AN, in aqueous media the adsorbed complex exhibits one reversible reduction, which is assigned to the two-electron/two-proton reduction of the quinone moiety to the corresponding hydroquinone. This reversible voltammetric wave, with the symmetrical shape anticipated for a surface-confined redox species, has a formal potential of -0.03 V at pH 7.2. In addition, the peak current was directly proportional to the scan rate, as predicted by theory. Average surface coverage values of (1.3-2.0) × 10-10 mol cm-2 were

Figure 2. Plot of surface coverage vs the applied potential during deposition for a GC electrode modified with [Fe(phen-dione)3](PF6)2 (obtained by holding the potential for 3 min in pH 7.2 phosphate buffer containing 0.4 mM of the complex). Inset: Plot of surface coverage as a function of the Fe complex concentration for a GC electrode modified at -0.20 V for 3 min in pH 7.2 phosphate buffer.

obtained (recall our discussion of surface coverages). The ∆Ep value was about 30 mV (at a sweep rate of 0.05 V/s) and increased at higher sweep rates, suggesting some kinetic limitations in the electron transfer. In a form analogous to the Re complex, all of the other complexes (except for Ru(v-bpy)) could also be electrodeposited onto GC electrodes by holding the potential at negative values in aqueous media. However, the specific coverage obtained was potential dependent. Figure 2 presents the dependence of coverage on applied potential for the Fe complex, and it can be seen that the deposition process is strongly dependent on the applied potential and that maximal coverages only were obtained when the potential was held negative of -0.20 V. The inset to Figure 2 shows the deposition isotherm for the [Fe(phen-dione)3](PF6)2 complex at a fixed potential of -0.20 V in phosphate buffer (pH 7.2), indicating that a saturation coverage is reached only at relatively high concentrations, of more than 0.5 mM, of the iron complex. As for the Re complex, the deposited Fe complex exhibited the voltammetric shape for a surface-confined redox couple, with the peak current being directly proportional to the scan rate. A qualitatively similar behavior was observed for all the complexes and the formal potentials of the resulting surfaceimmobilized complexes (at pH 7.2) are presented in Table 1, where it can be ascertained that, in all cases, the potentials are virtually identical to those in solution. In a way analogous to the process for the metal complexes described above, the free ligand itself (phen-dione) could also be deposited onto GC electrodes by holding the potential at about -0.30 V in an aqueous phosphate buffer solution of the ligand. As was the case for the metal complexes, the resulting modified electrode retained the redox activity of the phen-dione ligand, including the anticipated pH dependence of the formal potential. At pH 7.2, the value of the formal potential was found to be -0.06 V, a value that is very close to that of phen-dione in solution at the same pH (-0.10, Table 1). However, at pH values above 7, the modified electrodes were somewhat unstable, perhaps due to the types of reactions previously described by Evans and Griffith

Figure 3. Plot of the normalized coverage vs time of cycling over the potential range of +0.20 to -0.20 V at a sweep rate of 20 mV/s for GC electrodes modified with a film of (O) [Fe(phen-dione)3]2+ and (b) [Ru(phen-dione)(v-bpy)2]2+.

where the phen-dione undergoes a base-catalyzed decomposition to yield 4,5-diazafluorenone.32 As mentioned earlier, one of the objectives of the present work was the development of biosensors based on dehydrogenase enzymes. In such applications, the stability of the sensor is a particularly important aspect. We were thus interested in determining the stability of the modified electrodes. To ascertain stability, we continuously cycled the potential of the modified electrodes over the range of -0.20 to +0.20 V (which is the desired potential range for the determination of NADH and in biosensor applications; see below) and periodically recorded a voltammogram. From the charge under the voltammetric wave, we could determine the time dependence of the decay. To determine the general shape of the profiles, we present how the normalized coverage (Γt/Γ0; Γt, coverage at time t, Γ0, initial coverage) decreased as a function of time. Figure 3 (O) presents a plot of the normalized surface coverage (Γt/Γ0) vs time for continuous cycling between -0.20 and +0.20 V for an electrode modified with the Fe complex. As can be seen, the electrode is quite stable to cycling, with a loss of about 30% after 3 h of continuous cycling. It is also clear from the figure that most of the loss takes place during the first 20 min of cycling, suggesting that this likely represents material which is weakly associated with the surface. Afterward, the loss is greatly diminished. For this reason, electrodes were typically conditioned by cycling (at 20 mV/s) over the potential range of -0.20 to +0.20 V until a steady response was obtained. After that, the modified electrode exhibited a very stable response of the phen-dione moiety at around 0.0 V, allowing the reproducible determination of NADH. In an effort to further enhance the stability of the modified electrodes, we investigated the properties of the [Ru(v-bpy)2(phendione)]2+ complex, since it is well established that electropolymerized films of transition metal complexes of v-bpy are quite stable.31 This complex could be deposited onto GC electrodes by reductive electropolymerization of the vinyl groups of the v-bpy ligands, as has been previously described in numerous other cases.31 Consecutive cyclic voltammograms for a 1.0 mM solution of this complex in AN (0.10 M TBAP) depicting electropolymer(32) Evans, D. H.; Griffith, D. A. J. Electroanal. Chem. 1982, 134, 301.

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Figure 4. (A) Consecutive cyclic voltammograms at a sweep rate of 0.10 V/s, depicting electropolymerization for a GC electrode in contact with an AN (0.1 M TBAP) solution containing 1.0 mM [Ru(phen-dione)(v-bpy)2](PF6)2. (B) First cyclic voltammogram between +0.40 and -0.60 V in the same solution.

ization are shown in Figure 4A. The cyclic voltammetric response consists of two one-electron reductions at -0.10 and -0.40 V, corresponding to the phen-dione ligand, and two additional reductions at -1.50 and -1.75 V, associated with bipyridinelocalized reductions. Additionally, during the polymerization process, two new waves at -1.20 (cathodic) and -0.50 V (anodic) grow in, which we ascribe to so-called “charge trapping” as previously described.33 The voltammogram in Figure 4A has, at first, the rather peculiar feature of having the anodic wave at a potential negative of the cathodic counterpart. We believe that this is due to the overlap of other redox processes within the film (see above). This is confirmed by the fact that, if the sweep direction is reversed just after the second reduction, the anodic peak is now at potentials positive of the cathodic wave (Figure 4B). Average surface coverage values (for five or six consecutive polymerization cycles) of (3.0-4.5) × 10-10 mol cm-2 were obtained. As mentioned above, we were particularly interested in determining the stability of electrodes modified with this particular complex. The stability of the modified electrode was determined in the same way as described above for electrodes modified with the Fe complex, and data are presented in Figure 3 (b). As can be ascertained, the shape of the decay curve is qualitatively quite similar to that of the Fe complex, with most of the decay taking place during the first 20 min of cycling. In addition, it is also apparent that electrodes modified with the Fe complex are somewhat more stable than those modified with the Ru(v-bpy) complex. For this and other reasons (see below), and since electrode modification was easier with the Fe complex, biosensor studies were carried out with electrodes modified with the Fe complex (see below). As anticipated and previously shown for other metal complexes of phen-dione,28 the quinone-localized redox wave is pH-dependent in aqueous media. We also wanted to determine if such pH dependency was maintained for electrodes modified with electrodeposited or electropolymerized films of these materials. Thus, we obtained cyclic voltammograms over the pH range of 2.5-8 for electrodes modified with each of the complexes. From plots of the formal potentials vs pH, we obtained values of 57.5, 53, and 59 mV/pH unit for electrodes modified with the Re, Ru(v(33) Denisevich, P.; Willman, K. W.; Murray, R. W. J. Am. Chem. Soc. 1981, 103, 4727.

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Figure 5. Cyclic voltammograms in pH 7.2 phosphate buffer at a sweep rate of 20 mV/s for a glassy carbon electrode modified with a film of (A) [Re(phen-dione)(CO)3Cl], (B) [Ru(phen-dione)(v-bpy)2](PF6)2, and (C) [Fe(phen-dione)3](PF6)2, containing (a) 0.0, (b) 0.062, (c) 0.124, (d) 0.187, and (e) 0.248 mM NADH. (D-F) Plot of the corresponding catalytic currents vs NADH concentration.

bpy), and Fe complexes, respectively, which are quite close to the expected value of 59 mV/pH unit. Similar responses were obtained for the other complexes immobilized on the electrode surface. That the expected Nernstian response is maintained indicates that, during the deposition or polymerization process, the quinone groups remain largely unaffected. Electrocatalytic Oxidation of NADH. Metal complexes of 1,10-phenanthroline-5,6-dione have been previously shown to be active toward the catalytic oxidation of NADH,28 and their choice in the present study was based on this. Here we focus on results for [Fe(phen-dione)3](PF6)2, [Re(phen)(CO)3Cl], and [Ru(phendione)(v-bpy)2](PF6)2 immobilized as thin films on GC electrodes. Figure 5A-C shows cyclic voltammograms in pH 7.2 phosphate buffer for GC electrodes modified with films of the Re, Ru(v-bpy), and Fe complexes at coverages of 1.2-, 3.0-, and 1.5 × 10-10 mol cm-2, respectively, in the absence (a) and presence (b-e) of NADH. In the absence of NADH (a), the responses are those previously described for the modified electrodes and are ascribed to the reversible two-electron/two-proton oxidation/reduction of the hydroquinone/quinone group on the phen-dione ligands. Upon addition of NADH (b-e in Figure 5A-C), the cyclic voltammograms exhibit a dramatic enhancement of the anodic peak current with a decrease in the cathodic peak current. Both of these observations demonstrate a very strong catalytic effect in the oxidation of NADH. As can be seen in the voltammograms as well as in the corresponding calibration curves (Figure 5D-F), the anodic peak current increases linearly with NADH concentration. However, the current densities, at the same NADH concentration, for electrodes modified with different complexes are different, suggesting that the kinetics of NADH oxidation, mediated by the electrodeposited films, are likely rate limiting (see below).

Figure 6. Plot of the catalytic current vs NADH concentration over the range of 0-50 µM for GC electrodes modified with films of the Fe, Ru(v-bpy), and Re complexes.

Figure 5 shows that all three complexes exhibit very high activity for NADH oxidation at very low potentials, with peak potentials at values of 0.00 V for the Re and the Ru(v-bpy) and -0.05 V for the Fe complex in pH 7.2 phosphate buffer solutions. These values as well as those for the other complexes are presented in Table 1. As can be ascertained from the table, these represent dramatic potential shifts when compared to the NADH oxidation at a bare GC electrode which takes place around + 0.70 V.4 Figure 6 presents plots of the catalytic current vs NADH concentration over the range of 3-50 µM for GC electrodes modified with the Re, Fe, and Ru(v-bpy) complexes. The higher apparent sensitivity of the Ru(v-bpy) complex compared to the Re complex is due to the higher coverage of the Ru(v-bpy) complex as well as to other kinetic effects (see below). On the other hand, the higher sensitivity of the Fe complex is most likely due to the greater number of phen-dione ligands (three) on the complex and also to kinetic effects (see below). NADH Reaction Kinetics. Cyclic voltammetry and rotated disk electrode (RDE) techniques were employed to study the kinetics of the reaction between the various modified electrodes and NADH. In the first case, rate constants can be obtained according to the theory of Andrieux and Saveant34 by using the catalytic current at a given NADH concentration as a function of sweep rate. From such an approach, we calculated average rate constants, k, of 2.4 × 103, 5.6 × 103, and 4.4 × 103 M-1 s-1 for the Re, Fe, and Ru(v-bpy) complexes, respectively. It is worth noting that the Fe complex exhibits the fastest kinetics, likely due to the higher number of phen-dione ligands present. In addition, the Re complex exhibited the lowest rate. This might be due to the fact that this is a neutral molecule, so hydrophobic/hydrophilic as well as solvation effects may be playing a role. In the case of RDEs, the analysis is based on the dependence of the limiting current on the rate of rotation and the use of the Koutecky-Levich equation (1).

1 1 1 ) + ilim nFAkΓc0 0.62nFAν-1/6D

c0ω1/2

2/3 0

(1)

Figure 7 presents the results for a GC electrode modified with the Ru(v-bpy) complex. A plot of ilim vs ω1/2 at constant NADH (34) Andrieux, C. P.; Saveant, J. M. J. Electroanal. Chem. 1978, 93, 163.

Figure 7. Plot of ilim vs ω1/2 in pH 7.2 phosphate buffer in the presence of 31 µM NADH for a GC rotated disk electrode modified with a film of [Ru(phen-dione)(v-bpy)2](PF6)2. Inset: Koutecky-Levich plot of the same data set.

concentration (31 µM) exhibits linear (Levich) behavior at slow rates of rotation but levels off at higher rates, suggesting kinetic limitations. Rate constants can be obtained from the intercept of a plot of 1/ilim vs 1/ω1/2 (Koutecky-Levich plot). Such a plot of the experimental data is shown in the inset to Figure 7, where the anticipated linear behavior is observed. Qualitatively similar results were obtained in all cases. From the intercepts of the corresponding Koutecky-Levich plots, we calculated average values for k of 2.7 × 103, 6.8 × 103, and 4.3 × 103 M-1 s-1 for the Re, Fe, and Ru(v-bpy) complexes, respectively. It can be seen that the results obtained by RDE experiments and cyclic voltammetry are virtually identical and that the catalytic oxidation process of NADH at such modified electrodes is moderately fast. From studies of the dependence of the rate constant for NADH oxidation on its concentration for adsorbed phenoxazine mediators, Gorton10 has suggested that the process involves a charge transfer complex between NADH and the mediator in a mechanism akin to Michaelis-Menten kinetics. Although we have not explored this specific aspect in this study, it is plausible that a similar mechanism might be operative. To optimize the response of the modified electrodes toward NADH oxidation, we examined the effects of pH and coverage on the potential and peak current for NADH oxidation. As expected, with the pH-dependent shift of the quinone/hydroquinone redox wave as described above, the oxidation of NADH also shifts by about 59 mV/pH unit, again consistent with NADH being catalytically oxidized by the immobilized metal complexes. The peak current is also influenced by the pH, with a maximum value around pH 7.0. Finally, we also found that, with increasing coverage, the catalytic current increased and that the modified electrode itself was more stable when the coverage reached about one monolayer) [(1.5-2.5) × 10-10 mol cm-2]. Ascorbate Interference and Reproducibility. One of the main difficulties in the development of analytical applications of electrocatalysts for NADH oxidation is the interference of ascorbate, which is typically present in most biological media and whose oxidation tends to be catalyzed by the same type of systems that catalyze NADH oxidation. To mitigate such effects, electrodes are often coated with materials such as Nafion, which prevent anions (such as ascorbate) from reaching the electrode surface Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

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Figure 8. Cyclic voltammograms at a sweep rate of 10 mV/s for a GC electrode modified with a film of [Fe(phen-dione)3](PF6)2 in pH 7.2 phosphate buffer containing (a) buffer alone, (b) buffer plus 62 µM NADH, (c) buffer plus 62 µM ascorbate, and (d) buffer plus 62 µM NADH and 62 µM ascorbate. Inset: Calibration curve for NADH determination in the absence (b) and presence (3) of equal concentrations of ascorbate at -0.065 V for a GC electrode modified with a film of [Fe(phen-dione)3](PF6)2 in pH 7.2 phosphate buffer.

via Donnan exclusion. This, however, is often accompanied by a drastic diminution in response, especially since, at neutral pH, NADH is negatively charged and thus is also excluded. Because of the relatively low potentials at which the materials described here could catalyze the oxidation of NADH, we were interested in determining whether interference effects due to ascorbate could be decreased, and the results are presented in Figure 8 for an electrode modified with the Fe complex. Figure 8a shows the voltammetric response of the modified electrode in pH 7.2 phosphate buffer which was described earlier. Also shown are the voltammetric scans in the presence of 62 µM NADH (Figure 8b) and 62 µM ascorbate (Figure 8c), alone as well as when both NADH and ascorbate were both present at a concentration of 62 µM (Figure 8d), respectively. Comparing parts a and b of Figure 8, it can be seen that the peak for the catalytic oxidation of NADH is at a potential which is about 50 mV negative of that for ascorbate. This is a significant observation, since it suggests that NADH might be determined at these modified electrodes with significantly diminished interference due to the presence of ascorbate. Figure 8d shows the voltammetric response in the presence of both NADH and ascorabte at a concentration of 62 µM. The peak potential of this wave is intermediate between those for NADH and ascorbate alone, and the peak current value is about 20% higher than that for NADH alone. However, by measuring the current at about -0.065 V, where there is still a significant current due to NADH oxidation but where the current due to ascorabte oxidation is minimal, the interference due to ascorbate could be further diminished. The inset to Figure 8 shows calibration curves (obtained at an applied potential of -0.065 V) for NADH in the presence and absence of an equal concentration of ascorbate. As can be observed, although there is the anticipated diminution in sensitivity (relative to the determination at -0.05 V), the interference due to ascorbate is largely eliminated. One important goal for the development of any kind of sensor is to obtain reproducibility in the response. This is especially important in our case, since it is very difficult (if at all possible) to get the exact same surface coverage for each electrode and therefore would require a calibration curve for each electrode. 3694 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996

Figure 9. Plot of the coverage-normalized catalytic current vs NADH concentration for three GC electrodes modified under similar conditions with a film of [Re(phen-dione)(CO)3Cl] but with different surface coverages.

The fact that the surface coverage value of any electrode can be measured with great accuracy provides a means of circumventing the above-mentioned problem. By constructing a calibration curve normalized to the surface coverage (icat./Γ vs [NADH]), one obtains, in effect, a universal calibration curve so that only a single calibration is needed. To demonstrate this, we compared the results from three different electrodes modified with the Re complex and which were prepared under similar conditions, but with somewhat different surface coverages, and the results are presented in Figure 9. It can be seen that all the electrodes give virtually the same coverage-normalized calibration curve, consistent with the previous assertion. This is a very valuable aspect of this approach since, in essence, one can construct a universal calibration curve. Ethanol Biosensor. As mentioned earlier, one of the main objectives of these investigations was the development of biosensors based on dehydrogenases. To test for the potential utility in biosensors, of glassy carbon electrodes modified with phen-dione complexes of transition metals, we employed an electrode modified with the Fe complex, in conjunction with a nylon filter mesh modified with alcohol dehydrogenase (ADH), which in solution catalyzes the oxidation of short-chain alcohols to the corresponding aldehydes in the presence of NAD+ as a cofactor. In previous work,27 we have demonstrated that the use of a nylon mesh as support is superior to the direct immobilization of the enzyme over the electrode surface since the enzyme is preferentially bound onto the fibers of the mesh, without occlusion of the pores. Thus, transport to the electrode surface is not impeded. Moreover, in this assembly, the enzyme component is physically separated, thus allowing for its ready reuse with other electrodes. In the biosensor assembly described above, the immobilized ADH oxidizes ethanol to acetaldehyde in the presence of NAD+. This cofactor acts as an acceptor of electrons generated in the enzymatic reaction and is transformed to its reduced form, NADH. This, in turn, diffuses to the electrode, where ostensibly it is catalytically reoxidized back to NAD+ by the layer of the Fe complex. The modified electrode serves as a secondary acceptor of electrons able to regenerate the cofactor (NAD+) used in the enzymatic reaction, so the magnitude of this catalytic current can be employed as the analytical signal in the determination of the substrate (ethanol) concentration.

Figure 11. Steady-state current at 0.0 V for a [Fe(phen-dione)3]2+/ ADH biosensor (in Tris/0.1 M HCl buffer (pH 8.0)) containing 34 units of immobilized ADH at an ethanol concentration of 50 mM as a function of NAD+ concentration. Figure 10. Steady-state current at 0.0 V for a [Fe(phen-dione)3]2+/ ADH biosensor containing 34 units of immobilized ADH in the presence of increasing concentrations of ethanol in Tris/0.1 M HCl buffer (pH 8.0) and 10 mM NAD+. Inset: Expanded view of the region of 0-20 mM ethanol. Points are experimental data, and the lines are fits to the same.

That NADH is catalytically reoxidized back to NAD+ can be established by comparing the current for NADH oxidation at an electrode modified with the Fe complex alone with that for a biosensor in the presence of alcohol. The current density for a biosensor in the presence of NAD+ at a concentration of 50 µM is significantly higher than that for an electrode modified with the Fe complex in the presence of 50 µM NADH. This indicates that the NADH generated (by the enzymatic reaction), and which is subsequently oxidized to NAD+ (by the immobilized Fe complex), is enzymatically active and thus gives rise to the much higher currents observed. If the NADH were not oxidized to NAD+, such higher (catalytic) currents would not be observed. Moreover, if a biosensor is removed from a solution containing NAD+ and is placed in one containing ethanol but not NAD+ in solution, a catalytic current is still observed, again consistent with the above statements. In the development of the biosensor, we chose the Fe complex since, as mentioned earlier, it exhibited the fastest kinetics for the catalysis of NADH and the lowest potential in such a reaction (Table 1). In addition, the electrode modification could be easily and reproducibly controlled via the applied potential and solution concentration. Moreover, the response was more stable and reproducible than those obtained with the other complexes or the immobilized ligand by itself. For immobilized enzymes used in amperometric biosensors, the observed electrochemical response may be either mass transport limited or kinetically controlled. Mell and Malloy35 have suggested that, for an immobilized enzyme reaction that is kinetically controlled, the steady-state current, iss, is proportional to the initial rate of the enzymatic process. In this case, a plot of iss vs the substrate concentration, Cs, gives a typical MichaelisMenten-type response. In addition, a linear Lineweaver-Burk plot, 1/iss vs 1/Cs, will be diagnostic of kinetic control of the electrochemical response. Figure 10 shows the steady-state currents obtained at 0.0 V for increasing concentrations of ethanol employing a biosensor with 34 units of ADH immobilized on the (35) Mell, L. D.; Malloy, J. T. Anal. Chem. 1975, 47, 299.

nylon mesh. The iss vs Cs plot exhibits the typical MichaelisMenten shape, with a saturation response being reached for ethanol concentrations above 200 mM. In addition, the Lineweaver-Burk plot (not shown) is linear over the ethanol concentration range studied. These results suggest control by the enzymatic reaction, and the apparent Michaelis-Menten constant, Km′, was calculated to be 60 ( 5 mM. The ethanol biosensor exhibits good sensitivity (5 nA/mM, Figure 10 inset), and we estimate a limit of detection of about 1.0 × 10-4 M, which allows for determination of this analyte in whole blood. Moreover, the biosensor has a rapid response, reaching 90% of its steady-state value in less than 60 s. Our level of detection is of the same order as that reported by Park et al.36 but higher than that reported by Gorton et al.37 However, in this last case, the applied potential was +100 mV vs Ag/AgCl, which is significantly higher than that employed in our study and is a value where ascorbate interference would be significant. As would be anticipated, and as has been reported previously in numerous studies on dehydrogenase activity-based biosensors, the response will be dependent on the amount of active enzyme immobilized. To obtain a sensor with a long lifetime and to account for possible losses of enzymatic activity which would affect reproducibility, an enzyme loading level of 34 units was employed, since we had found that, for loadings above 25 units, a loadingindependent response was obtained (data not shown). In an analogous fashion, a concentration of NAD+ of 10 mM was employed in analytical determinations, again to ensure that the response was independent of its concentration. The value of the NAD+ concentration employed was significantly higher than that which we previously employed in the aldehyde biosensor.27 However, we have found that, at lower NAD+ concentrations, the biosensor response to increasing concentrations of alcohol was not linear nor as reproducible. Since at such high NAD+ concentrations there is the possibility of inhibition, we determined the response of the biosensor to an ethanol concentration of 50 mM as a function of the solution concentration of NAD+, and the results are presented in Figure 11. As can be seen, the biosensor response increases with increasing concentration of NAD+, reaches a maximum at about 6 mM, and exhibits a slight (10-15%) decrease at higher (36) Park, J.-K.; Yee, H.-J.; Kim, S.-T. Biosens. Bioelectron. 1995, 10, 587. (37) Domı´nguez, E.; Lan, H. L.; Okamoto, Y.; Hale, P. D.; Skotheim, T. A.; Gorton, L.; Hahn-Haegerdal, B. Biosens. Bioelectron. 1993, 8, 229.

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concentrations. This would suggest that there might be a slight inhibitory effect. However, the beneficial aspects of linearity and reproducibility were deemed to be more important than the slight decrease in response. Although we have not optimized the conditions for the operation of the ethanol biosensor, it is clear that the use of GC electrodes modified with films of transition metal complexes of phen-dione are very attractive in biosensor development. We are currently exploring the use of the materials described here in other biosensor applications, and the results of those investigations will be reported elsewhere.

catalytic response exhibits a linear dependency on the concentration of NADH in solution, with a limit of detection of about 1 µM. Moreover, the response was quite persistent and reproducible. Because of the very low potentials at which NADH was electrochemically oxidized, the interference effects of ascorbate could be largely minimized. The application of these materials to biosensor development has been demonstrated by preparing an ethanol biosensor based on the determination of NADH generated by the enzymatic activity (toward ethanol) of alcohol dehydrogenase immobilized on a nylon mesh membrane.

CONCLUSIONS Glassy carbon (GC) electrodes can be modified with electrodeposited or electropolymerized films of phen-dione complexes of transition metals including [Re(phen-dione)(CO)3Cl], [Fe(phendione)3](PF6)2, [Ru(phen-dione)3](PF6)2, [Cr(phen-dione)3](PF6)3, [Co(phen-dione)3](PF6)2, and [Ru(phen-dione)(v-bpy)2](PF6)2. As deposited films, the materials retain their redox activity, both metal- and ligand (quinone)-based responses, with the latter exhibiting the anticipated pH-dependent responses. GC electrodes modified with films of these materials exhibit very high activity toward the catalytic oxidation of NADH in solution at potentials as low as -0.05 V vs SSCE. This represents a dramatic diminution in the overpotential present at bare GC electrodes. The electro-

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.

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Received for review April 23, 1996. Accepted August 9, 1996.X AC960395Y X

Abstract published in Advance ACS Abstracts, September 15, 1996.