Electrochemically Triggered Reaction of a Surface-Confined Reagent

Langmuir 1999, 15, 127-134

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Electrochemically Triggered Reaction of a Surface-Confined Reagent: Mechanistic and EQCM Characterization of Redox-Active Self-Assembling Monolayers Derived from 5,5′-Dithiobis(2-nitrobenzoic acid) and Related Materials Elena Casero,† Margarita Darder,† Kazutake Takada,‡ He´ctor D. Abrun˜a,‡ Fe´lix Pariente,† and Encarnacio´n Lorenzo*,† Departamento de Quı´mica Analı´tica y Ana´ lisis Instrumental, Universidad Auto´ noma de Madrid, Cantoblanco 28049, Madrid, Spain, and Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301 Received June 4, 1998. In Final Form: October 13, 1998 The adsorption processes and electrochemical behavior of DTNB (5, 5’-dithiobis(2-nitrobenzoic acid) adsorbed onto gold electrodes have been investigated in aqueous phosphate buffer/0.1 M NaNO3 electrolyte solutions using cyclic voltammetry in conjunction with the electrochemical quartz crystal microbalance. DTNB adsorbs onto gold electrode surfaces, and upon potential cycling past -0.55V, is transformed into the hydroxylamine which exhibits a well-behaved pH-dependent redox couple centered at -0.04 V at pH 7.0. To elucidate the mechanism of this electrochemically triggered surface-confined transformation, nine different compounds with structures similar to that of DTNB have also been studied. From these studies, it appears that the nitrogroup is responsible for the redox behavior and a mechanism for the overall reaction is proposed. The pH dependence of the redox response has been investigated and the kinetics of electron transfer evaluated. In addition, the EQCM technique has been employed to follow the deposition process in real time as well as the characteristics of the charge transfer associated with the surfaceconfined redox-active couple. The electrocatalytic activity of such modified electrodes toward the oxidation of NADH has also been explored.

Introduction There continues to be a great deal of interest in the use and applications of self-assembled monolayers (SAMs),1 especially those containing redox-active groups.2 Because of their strong chemisorptive bonds, particular emphasis has been placed on the adsorption of sulfur-containing materials such as thiols,3 sulfides,4 and disulfides5 on gold and silver surfaces, since in numerous cases they form highly ordered monolayers. Moreover, because of the strength of this interaction, these materials can be adsorbed onto such surfaces even in the presence of other functional groups, thus allowing for the preparation of surfaces modified with a wide array of functional groups.6 The use of such highly organized and structured interfaces can, in principle, provide the means to control † ‡

Universidad Auto´noma de Madrid. Cornell University.

(1) (a) Swalen, J. D.; Allara, D. L.; Andrade, J. D.; Chandross, E. A.; Garoff, S.; Isreaelachvili, J.; McCarthy, T. J.; Murray, R.; Pease, R. F.; Rabolt, J. F.; Wynne, K. J.; Yu, H. Langmuir 1987, 3, 932-950. (b) Ulman, A. An Introduction to Ultrathin Organic Films from LangmuirBlodgett to Self-Assembly; Academic Press: San Diego, CA, 1991. (2) Finklea, H. O. Electroanal. Chem. 1996, 19, 109-335. (3) (a) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 112, 558-569. (b) Bain, C. D.; Evall, J.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7155-7164. (c) Walczak, M. M.; Chung, C.; Stole, S. M.; Widrig, C. A.; Porter, M. D. J. Am. Chem. Soc. 1991, 113, 23702378. (4) (a) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546-558. (b) Rubinstein, I.; Steinberg, S.; Tor, Y.; Shanzer, A.; Sagiv, J. Nature 1988, 332, 426-429. (c) Katz, E.; Borovkov, V. V.; Evstigneeva, R. P. J. Electroanal. Chem. 1992, 326, 197-212. (5) (a) Hickman, J. J.; Ofer, D.; Zou, C.; Wrighton, M. S.; Laibinis, P. E.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 1128-1132. (b) Bain, C. D.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1989, 5, 723-727. (c) Katz, E. J. Electroanal. Chem. 1990, 291, 257-260. (6) Nuzzo, R. G.; Fusco, F. A.; Allara, D. L. J. Am. Chem. Soc. 1987, 109, 2358-2368.

the chemical and physical properties of interfaces for a variety of heterogeneous processes, including catalysis, corrosion, lubrication, and adhesion. Numerous methods have been used for the characterization of the packing, structure, and composition of SAMs, including Fourier transform infrared spectroscopy,7 Raman spectroscopy, UV-vis spectroscopy, X-ray photoelectron spectroscopy,8 surface X-ray scattering,9 and others. Although these are very powerful techniques, they generally encounter difficulties with monitoring the adsorption processes in real time. However, if the self-assembling molecules have redox-active sites, the adsorption process can be monitored in real time using electrochemical techniques such as cyclic voltammetry (CV). The quartz crystal microbalance (QCM) technique has been employed as a highly sensitive detector for measuring in situ changes in mass with nanogram resolution.10 It is a powerful technique for monitoring the self-assembly process and, in fact, has been employed to study the adsorption kinetics of thiols and thiol-substituted viologens onto gold surfaces.11 For self-assembling molecules containing redox centers, the adsorption process can be studied by combining (7) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559-3568. (8) Folkers, J. P.; Laibinis, P. E.; Whitesides, G. M. Langmuir 1995, 8, 1330-1341. (9) Fenter, P.; Eisenberger, P.; Li, J.; Camillone, N.; Bernasek, S.; Scoles, G.; Ramanarayanan, T. A.; Liang, K. S. Langmuir 1991, 7, 20132016. (10) Ward, M. D.; Buttry, D. A. Science 1990, 249, 1000-1007. (11) (a) Karpovich, D. S.; Blanchard, G. J. Langmuir 1994, 10, 33153322. (b) Frubo¨se, C.; Doblhofer, K. J. Chem. Soc., Faraday Trans. 1995, 13, 1949-1953. (c) De Long, H. C.; Buttry, D. A. Langmuir 1990, 6, 1319-1322. (d) De Long, H. C.; Buttry, D. A. Langmuir 1992, 8, 2491-2496.

10.1021/la9806552 CCC: $18.00 © 1999 American Chemical Society Published on Web 12/12/1998

128 Langmuir, Vol. 15, No. 1, 1999

Figure 1. Structure of DTNB as well as of some of the other materials employed in these studies.

electrochemical and QCM techniques into what is generally termed the electrochemical quartz crystal microbalance (EQCM). In this case, one can determine not only the coverage of the adsorbed redox-active species but also obtain information about the solvation (and its changes) of the adsorbed monolayer. The surface coverage is obtained from the electrochemical measurement while changes in mass are obtained from the series resonant frequency changes during the adsorption process. Moreover, since the adsorbed molecules show an electrochemical response, interfacial mass transport caused by redox reactions can be measured using the EQCM technique. In this paper we report on the preparation and characterization of redox-active self-assembled monolayers derived from 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) adsorbed onto a gold electrode in phosphate buffer using in situ EQCM and CV techniques. This is an especially interesting system since reduction of the adsorbed DTNB results in the formation of the corresponding hydroxylamine which exhibits reversible pH-dependent redox chemistry. Thus, it represents the novel case of deliberately triggering a surface reaction by electrochemical means. From EQCM studies we have been able to follow the deposition process in real time and have demonstrated that during the self-assembly as well as during the redox process of the DTNB there are no significant changes in the viscoelastic properties of the monolayer, as determined by admittance measurements of the quartz crystal resonator, on the basis of the electrical equivalent circuit. Thus, changes in the resonant frequency of the EQCM can be used to calculate mass changes directly. We have also carried out studies in order to elucidate the mechanism of the electrochemically triggered transformation and the nature of the redox process. With these purposes in mind, we have also investigated the electrochemical behavior of gold electrodes modified with materials that are closely related to DTNB. In addition, we have observed that the self-assembled monolayer derived from 2,2′-dithiobis(5-nitropyridine) (DTNPy) and adsorbed onto a gold electrode exhibits electrocatalytic activity in the oxidation of NADH. Experimental Section Materials. All reagents used to prepare the self-assembled monolayers on gold electrodes were obtained from Aldrich, were of, at least, 95% purity, and used as received. 5,5′-dithiobis(2nitrobenzoic acid), 6,6′-dithiodinicotinic acid, 2,2′-dithiobis(5nitropyridine), and cystamine were stored at room temperature. Thioctic acid, 2,2′-dithioethanol, 2-mercaptopyridine, 4-mercaptopyridine, and aldrithiol were stored at 4 °C. Figure 1 presents the structures of the materials studied and employed in the preparation of SAM’s. β-Nicotinamide adenine dinucleotide (NADH) was obtained from Sigma and used as received. All solutions were prepared immediately prior to use. Sodium phosphate and sodium nitrate were used in the preparation of electrolyte and buffer solutions. Water was purified with a Millipore Milli-Q system.

Casero et al. Apparatus and Procedures. Voltammetric Measurements. CV studies were carried out with a BAS CV-27 potentiostat connected to a BAS X-Y recorder or with an Autolab/PGSTAT10 potentiostat from Eco-Chemie. The electrochemical experiments were carried out in three-compartment electrochemical cells with standard taper joints so that all three compartments could be hermetically sealed with Teflon adapters. Gold-disk electrodes sealed in soft glass were used as working electrodes. A largearea coiled platinum wire served as the auxiliary electrode, and all potentials are reported against a sodium-saturated calomel electrode (SSCE) without regard for the liquid junction potential. Surface coverages were determined electrochemically by integrating the charge under the CV wave obtained at 100 mV/s. Values were typically (10%. Prepurified nitrogen gas was used to deaerate all solutions before use and flowed over the solutions during experiments. All measurements were carried out at room temperature. EQCM Measurements. AT-cut quartz crystals (5 MHz) with a 25 mm diameter and with Au electrodes deposited over a Ti adhesion layer (Maxtek Co.) were used for EQCM measurements. An asymmetric-keyhole-electrode arrangement was used, in which the circular electrodes’ geometrical areas were 1.370 cm2 (front side) and 0.317 cm2 (backside). The electrode surfaces were overtone polished. Prior to use, the quartz crystals were cleaned by immersion in piranha solution H2SO4/H2O2 (Caution! Use extreme care when using piranha solution.) They were subsequently rinsed with water and acetone and dried in air. The quartz-crystal resonator was set in a probe (TPS-550, Maxtek) made of Teflon in which the oscillator circuit was included, and the quartz crystal was held vertically. For monitoring the selfassembly process, the probe was immersed in 60 mL of phosphate buffer solution (pH 7.0) which was thermostated at 25.0 ( 0.1 °C by a water-jacketed beaker (which served as the cell). For EQCM measurements, the probe was connected to a waterjacketed conventional three-chamber electrochemical cell by a homemade Teflon joint. The cell was thermostated at 25.0 ( 0.1 °C with a thermostated bath (digital temperature controller 9101, Fisher Scientific, USA). In short-time (time required for a CV) experiments the stability of the oscillator was on the order of ( 0.1 Hz. Nitrogen gas, passed through hydrocarbon and oxygen traps, was used to degas the solutions before use and flowed over the solutions during experiments. One of the electrodes of the quartz-crystal resonator, in contact with the solution, was also used as the working electrode. The potential of the working electrode was controlled with a potentiostat (CV-27, BAS). A sodium chloride saturated calomel electrode (SSCE) and a coiled Pt wire were used as reference and counter electrodes, respectively. The frequency response measured with a plating monitor (PM-740, Maxtek) and the current measured with the potentiostat were simultaneously recorded by a personal computer which was interfaced to the above instruments using Lab-View. The admittance of the quartz-crystal resonator was measured near its resonant frequency by an impedance analyzer (HP4194A, Hewlett-Packard) equipped with a test lead (HP16048A). A probe similar to the one used in the EQCM measurements, but which did not include an oscillator circuit inside, was used to accomplish a direct connection of the quartz-crystal resonator to the impedance analyzer. Electrode Conditioning and Monolayer Preparation. Polycrystalline gold disk electrodes were polished with 1.0 µm diamond paste (Buehler), rinsed in water, and sonicated for 10 min in distilled water. Afterward, they were immersed in HNO3 at 30 °C for 10 min and rinsed in distilled water. The electrodes were then dipped in a 0.1 M NaOH solution, and the potential was held at -1.5 V for 1 min. The electrodes were subsequently activated by holding the potential at +2.0 V for 5 s in a 0.1 M H2SO4 solution and then at -0.35 V for 10 s in the same solution, followed by potential cycling from -0.2 to +1.5 V at 5 V/s for 2 min. Finally the CV characteristic of a clean polycrystalline gold surface was recorded12 in 0.1 M H2SO4. By integration of the cathodic peak associated with the reduction of the gold oxide, the microscopic area of the electrode was determined. The (12) Sabatani, E.; Rubinstein, I.; Maoz, R.; Sagiv, J. J. Electroanal. Chem. 1987, 219, 365-371.

Redox-Active Self-Assembling Monolayers

Figure 2. Cyclic voltammograms at 100 mV s-1 of an Au electrode modified with a DTNB monolayer in 0.1 M phosphate buffer (pH 7) in the potential range from +0.1 to -0.8 V.

Langmuir, Vol. 15, No. 1, 1999 129

Figure 3. Cyclic voltammograms at 100 mV s-1 of an Au electrode modified with a DTNB monolayer in 0.1 M phosphate buffer (pH 7) in the potential range from +0.1 to -0.6 V.

electrode was rinsed with water and used immediately in the preparation of the monolayer. Organic monolayers were prepared by immersion (for 3 h at room temperature) of an activated gold electrode in ca. 10 mM ethanolic solution of the material to be deposited. The electrode was subsequently rinsed with copious volumes of ethanol and water. Electrodes were employed immediately after preparation.

Results and Discussion 1. Electrochemical Response of DTNB-SAMs. CV of gold electrodes modified with DTNB (and related materials) were carried out in 0.1 M phosphate buffer (pH 7.0) at 100 mV/s over the potential range of +0.10 to -0.80 V. It should be noted that in all of these studies the thiol modifier is not present in solution. The results for DTNB are presented in Figure 2. On the first cathodic scan (solid line) two well-resolved, chemically irreversible processes are observed at peak potential values of -0.55 and -0.75 V. Upon scan reversal at -0.80 V, an anodic wave with a peak potential centered at -0.03 V is observed. On the second (dashed line) and subsequent potential scans, an additional cathodic peak was observed at -0.05 V which is the cathodic counterpart to the anodic wave at -0.03 V. This appears to indicate that the first cathodic sweep gives rise to a reversible redox couple with a formal potential of -0.04 V. In addition, the peak currents of the cathodic waves centered at -0.55 and -0.75 V decreased significantly on the second and on subsequent scans. Focusing our attention on the reversible redox couple centered at -0.04 V, cyclic voltammograms of gold electrodes modified with DTNB were carried out over the potential range of -0.25 to +0.25 V (starting at -0.25 V) and compared with those obtained for an unmodified gold electrode. No Faradaic processes were observed over this potential range for either the bare or the DTNB-modified gold electrode. However, in the latter case there is a significant decrease in the capacitative current as would be anticipated for an electrode covered with a low-dielectric layer. To ascertain whether the first or the second of the cathodic processes present on the initial scan gave rise to the subsequent generation of the reversible redox couple centered at -0.04 V, voltammograms were carried out in which the negative potential limit was progressively increased. When the onset of the first wave was reached (ca. -0.4, see Figure 3, solid line), there was the appear-

Figure 4. Surface coverage obtained from the cyclic voltammetric wave centered at -0.04 V, as a function of the triggering potential.

ance of the reversible couple centered at -0.04 V. The generation of this new wave increased as the negative potential limit increased and reached a maximum value at about -0.55 V. Sweeping the potential to more negative values did not result in the generation of additional material, and in fact, the stability decreased. Figure 4 presents a plot of the surface coverage Γ of the redox couple at -0.04 V as a function of the trigger potential where the above-mentioned behavior is clearly evident. On a second cathodic sweep (Figure 3, dashed line) the peak current at -0.55 V was greatly attenuated, suggesting that the reaction consumed virtually all of the adsorbed DTNB. In addition, after repeated cycling to -0.55 V, the amplitude of the wave at -0.04 V increased slightly, and the wave at -0.55 V eventually disappeared. The effect of the time of triggering was also studied. In this case, the surface coverage of the resulting reversible redox response was monitored as function of time using pH 7.0 phosphate buffer and at an applied potential of -0.55 V. We observe that for times up to 5 s the surface coverage increased with time, but then decreased. We believe that this might be a result of partial desorption of the deposited material (vide infra).

130 Langmuir, Vol. 15, No. 1, 1999

Casero et al. Scheme 1

by Kemula and co-workers,13 by Lindbeck and Freund,14 and most recently by Rubinstein15 who carried out studies at gold-electrode surfaces.

(S-Ar-NO2)2 + 8H+ + 8e- f (S-Ar-NHOH)2 + 2H2O (1)

Figure 5. Cyclic voltammograms of a DTNB-modified (by applying -0.55 V for 5 s) Au electrode in pH 7.0 phosphate buffer over the potential range of -0.25 to +0.20 V at 100 mV s-1.

On the basis of these observations, we adopted a standard protocol for the generation of the redox process at -0.04 V. This was that following the adsorption of the DTNB onto the electrode, the potential was held at -0.55 V for 3 s which resulted in the maximal amplitude of the wave at -0.04 V. The voltammetric behavior observed for the wave centered at -0.04 V (Figure 5) is that anticipated for a reversible, surface-confined redox couple with a small (although not zero) ∆Ep value. The peak current was directly proportional to the rate of potential sweep over the range of 25-500 mV/s, suggesting facile chargetransfer kinetics within this range of sweep rates. However, for sweep rates above 500 mV/s the ∆Ep values increased significantly, suggesting kinetic limitations. To ascertain the nature of the reaction leading to the generation of the reversible surface redox couple, gold electrodes were modified with molecules closely related to DTNB and their electrochemical response was studied by CV in 0.1 M phosphate buffer (pH 7.0) following the protocol employed for DTNB. That is, the potential was initially held at -0.55 V for 5 s and then scanned over the range of +0.20 to -0.25 V. Under these conditions, neither aldrithiol nor 6,6′-dithionicotinic acid (see Figure 1 for their structures) exhibited an electrochemical response. On the other hand, DTNPy (2,2′-dithiobis(5-nitropyridine)) exhibited a reversible electrochemical response similar to that observed for DTNB. These results suggest that the reduction of the nitro group (at -0.55 V) is likely responsible for the generation of the reversible redox couple centered at -0.04 V. On the basis of these results, we propose that for electrodes modified with DTNB, when the potential is held at -0.55 V in pH 7.0 phosphate buffer, an irreversible four-electron, four-proton electrochemical reduction of each of the nitro groups of DTNB with the corresponding arylhydroxylamine takes place according to the mechanism presented in Scheme 1A and eq 1 (where Ar represents an aromatic system) previously proposed (13) Kemula, W.; Krygowski, T. M. In Encyclopedia of Electrochemistry of the Elements. Organic Section; Bard, A. J., Lund, H., Eds.; Dekker: New York, 1979; Vol. 23, Chapter 2, p 77. (14) Lindbeck, M. R.; Freund, H. Anal. Chim. Acta 1966, 35, 74-84. (15) Rubinstein, I. J. Electroanal. Chem. 1985, 183, 379-386. (16) (a) Miller, I. R.; Teva, J. J. Electroanal. Chem. 1972, 36, 157166. (b) Stankovich, M. T.; Bard, A. J. J. Electroanal. Chem. 1977, 75, 487-505.

A similar reaction sequence is proposed for electrodes modified with DTNPy. The second redox process (with a peak potential value of -0.75 V) is ascribed to the reduction of the disulfide to the corresponding thiol which is ostensibly less strongly adsorbed. Such reactivity is well established for disulfide/ thiol couples with the cystine/cysteine system having received the most attention as a result of its biological relevance.16 It should also be mentioned that in basic media and when the potential scan was extended to even more negative values, an additional, irreversible wave centered at about -1.15 V was observed (data not shown). We believe that this corresponds to reductive desorption of the monolayer as has been described previously by Porter et al. (and others) for alkane thiols.17 These last two redox processes result in partial or total loss of material from the electrode surface as stated above. The reversible redox couple centered at -0.04 V is thus ascribed to

(S-Ar-NO)2 + 4H+ + 4e- a (S-Ar-NHOH)2 (2) as depicted in Scheme 1B. The surface coverage, calculated by integration of the charge under the voltammetric wave, was found to be 3.1 × 10-11 mol cm-2. Although specific values of the electrochemically determined surface coverage exhibited some variations, they were typically in the range of (3-4) × 10-11 mol cm-2. Comparing the charge passed during the first cycle to about -0.60 V (Figure 2) and associated with the wave with a peak potential value of -0.55 V, it is about twice the value anticipated from the ratio of the stoichiometries of reactions 1 and 2, suggesting that only about 50% of the charge passed gives rise to the reversible redox couple. The difference in charge is likely due to the generation of nonredox-active secondary products, as is often found in these types of transformations, as well as to some potential desorption. 2. Effect of pH, Electrolyte, and Oxygen. According to eq 1, the reduction of adsorbed DTNB should be pH dependent. In fact, the peak potential for the first reduction wave shifted with pH at a rate of 59 mV/pH unit as predicted by eq 1. In all cases, the hydroxylamine product was generated, although triggering at lower pH values decreased the desorptive losses previously discussed since the surface reaction occurred at less negative potentials. Similarly, eq 2 predicts that the formal potential of the reversible wave associated with the arylhydroxylamine/ (17) Widrig, C. A.; Chung, C.; Porter, M. D. J. Electroanal. Chem. 1991, 310, 335-359.

Redox-Active Self-Assembling Monolayers

Langmuir, Vol. 15, No. 1, 1999 131

Figure 6. pH dependence of the formal potential E°′ for a DTNB-modified (by applying -0.55 V for 5 s) Au electrode. The inset shows the peak currents obtained by cyclic voltammetry in 0.1 M phosphate buffer at different pH values: (a) 1.57, (b) 2.24, (c) 3.48, (d) 5.59, (e) 6.98, (f) 7.98, (g) 9.19, (h) 10.02, (i) 11.06, and (j) 12.03.

nitroso couple should also be pH dependent. To ascertain this, the voltammetric response of modified gold electrodes was obtained in solutions of varying pH from a pH of 1.3 to 12.0. As can be seen in Figure 6, the formal potential of the redox couple was pH dependent, with a slope of 62 mV per pH unit which is very close to the anticipated Nernstian value of 59 mV for electrochemical processes involving the same number of protons and electrons and which is also in accord with eq 2. In addition, no significant variations in the slope were observed, suggesting that the aryl hydroxylamine groups remain protonated over the entire pH range studied. In addition, the voltammetric response and the coverage remained virtually constant, indicating that the redox process is stable over the entire range of pH (Figure 6, inset). Finally, the effects of the supporting electrolyte and of pH on the cyclic voltammetric responses of modified gold electrodes were studied. Experiments were carried out in phosphate buffer and in sodium nitrate solutions at different pH values. The responses obtained in both electrolytes and at low (pH ) 1.3) or neutral pH were virtually identical, indicating that the supporting electrolyte and pH have no effect on the redox response. In addition, measurements could also be carried out in the presence of oxygen, since its presence does not modify the redox response. 3. Charge-Transfer Kinetics. As mentioned earlier, the electrochemical response of the wave at -0.04 V was that anticipated for a surface-confined redox couple. The peak currents were proportional to the scan rate for sweep rates below 500 mV/s, and the peak potential separation ∆Ep was small although not zero, as expected for an ideal Nernstian reaction.18 Laviron has derived general expressions for the linear potential sweep voltammetric response for the case of surface-confined electroactive species.19 (18) Bard, A. J.; Faulkner, L. R. Electrochemical Methods. Fundamentals and Applications; Wiley & Sons: New York, 1980; p 522. (19) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28.

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 variation in the peak potential with scan rate. Figure 7 presents plots of ∆E (defined as Epeak - E°′) vs log v where it can be seen that ∆E values are independent of log v at low scan rates and proportional to log v for scan rates above 1.0 V/s. From such plots, values of ks and R were obtained for gold electrodes modified with DTNB and DTNPy monolayers, and the results are presented in Table 1. From Table 1 it appears that the couple derived from DTNB exhibits significantly slower kinetics, with ks ) 450 s-1, than that derived from DTNPy, with ks ) 600 s-1. Although we are not certain as to the origin of this effect, we speculate that it might be due, at least in part, to the negative charges present in the former but not in the latter. 4. Electrochemical Quartz Crystal Microbalance (EQCM) Studies. a. Adsorption of DTNB. The EQCM technique allows measurement of mass changes at surfaces by changes in the resonant frequency of the quartz crystal. Thus, decreases in mass correspond to increases in frequency and vice versa. The frequency and mass changes are related by the Sauerbrey equation20

∆f ) -Cf∆m

(3)

where ∆f is the change in frequency (Hz), ∆m is the mass changes (ng cm-2), and Cf (0.0566 Hz cm2 ng-1) is a proportionality constant for the 5.0 MHz crystals used in this study. To study the mass changes associated with the adsorption of DTNB onto a gold electrode, a QCM probe containing a gold resonator was immersed in a thermostated solution (0.1 M phosphate, pH 7.0) as described in the Experimental Section. After the temperature and frequency had stabilized, an aliquot of a 20 mM ethanolic stock solution of DTNB was added so that (20) Sauerbrey, G. Z. Phys. 1959, 155, 206-222.

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Casero et al.

Figure 7. Experimental variation of ∆E (∆E ) Ep - E°′) versus the logarithm of the sweep rate for a Au electrode modified with 2,2′-dithiobis(5-nitropyridine) in 0.1 M phosphate buffer (pH 7). The geometric area of the electrode was 0.03 cm2. Inset shows the same plot for the higher sweep rates. Table 1. Formal Potentials, Transfer Coefficients and Electron Transfer Rate Constants for Gold Electrodes Modified with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) and 5,5′-dithiobis (2-nitropyridine) (DTNPy) at pH 7.0 DTNB DTNPy

E°′ (V)

R

ks (s-1)

-0.041 -0.074

0.46 0.49

450 600

the final concentration was 0.5 mM. The solution was stirred for a few seconds, and the frequency was subsequently recorded. Figure 8 shows the frequency changes as a function of time. As can be seen, the frequency decreases gradually during the first 5 min and then a steady state is obtained. The final decrease in frequency was 12 Hz. From this change in frequency and using eq 3, the mass deposited on the electrode surface was estimated. The value obtained was 214 ng which, if due only to adsorbed DTNB, would correspond to about 5.4 × 10-10 mol cm-2. This value is larger than that obtained from electrochemical experiments described earlier. This apparent discrepancy would suggest that not only is the DTNB adsorbed, but also waters of hydration and possibly electrolyte ions. The shape of the frequency-time profile can be employed in order to study the kinetics of adsorption. The process can be controlled by either transport (diffusion) or by kinetics (activation controlled), which predict time dependencies that are t1/2 and exp (t), respectively. The fit to the data (Figure 8, solid line) is well-described by an exponential decay, indicating that the process is kinetically controlled. From the fit to the data a rate constant of the order of 0.015 s-1 was obtained. Attempts to fit the data to a t1/2 dependence yielded consistently poor results (data not shown). Although, as mentioned above, the fit of the data to an exponential function was quite good, one should also note that adsorption of the DTNB molecules is accompanied by the

Figure 8. Time dependence of the frequency changes of a quartz-crystal resonator in a 0.5 mM DTNB phosphate buffer solution at 25.0 ( 0.1 °C. Solid line represents data fitted to a first-order kinetic equation.

incorporation of solvent molecules and implicit in the analysis is the fact that the time dependence of the frequency change is controlled by the adsorption of the DTNB and not the solvent. b. Redox process. The EQCM could also be used to follow the generation of the arylhydroxylamine from the adsorbed DTNB. Figure 9 shows current/time and frequency/time plots for a gold electrode modified with DTNB following a potential step from -0.25 to -0.55 V, a 5 s holding period, and stepping back to -0.25 V. Following the initial step, the current decayed smoothly, and upon stepping back to

Redox-Active Self-Assembling Monolayers

Figure 9. (A) Current/time and (B) frequency/time plots for a quartz-crystal resonator modified with DTNB in 0.1 M (pH 7) phosphate buffer upon stepping the potential from -0.25 V to -0.55 V (5 s) and back to -0.25 V.

-0.25, the reverse current (and charge) were significantly smaller for the reasons previously discussed. (See previous discussion associated with Figure 3.) The frequency/time plot exhibited an overall frequency increase of 3.5 Hz which represents a mass loss of about 62.3 ng. Assuming that the surface reaction involves conversion of the nitro groups to the corresponding hydroxylamines, this would indicate an overall loss in mass of the deposited material and thus a corresponding frequency increase. However, the observed frequency increase of 3.5 Hz is significantly higher than that calculated if the only process involved was the one mentioned above. Thus, there must also be other reactions involved. These could be loss of solvating water molecules and/or partial desorption of the deposited material. At this time it is difficult to establish the relative contributions of these two processes. To study the mass transfer during the redox reaction of the hydroxylamine/nitroso process, changes in the frequency of the quartz-crystal resonator were monitored while scanning the applied potential between -0.25 and +0.20 V versus SSCE in a 0.1 M phosphate buffer at pH 7. (These measurements were carried out just after the adsorption of the DTNB was completed and a potential of -0.55 V was applied, as mentioned in the previous section.) Figure 10 presents (A) the cyclic voltammogram and (B) the frequency responses as a function of applied potential in a 0.1 M phosphate buffer (pH 7.0) solution for a gold electrode modified as described above. As mentioned previously, since the frequency of the quartz-crystal resonator is sensitive not only to mass but also to the rigidity, roughness, and solvophilicity of the film surface, it is important to establish the main factors giving rise to the frequency changes during the redox reaction of the adsorbed layer on the quartz-crystal

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Figure 10. (A) Cyclic voltammogram and (B) frequencypotential curve of the DTNB-modified (by applying -0.55 V for 3 s) quartz-crystal resonator in 0.1 M phosphate buffer (pH 7). Sweep potential rate: 50 mV s-1.

resonator. Admittance measurements of the quartz crystal resonators were carried out, and no differences were observed in the resistance parameter (Ω ) 334) between the reduced (-0.25 V) and oxidized (+0.20 V) states. This result demonstrates that the main factor giving rise to the frequency changes is a change in the mass of the deposited layer. Therefore, changes in mass can be calculated from the changes in the frequency of the quartzcrystal resonator using eq 3. As can be seen in Figure 10, the oxidation reaction causes an increase in the frequency, corresponding to a decrease in the mass of the adsorbed film, while the reduction reaction causes a decrease in the frequency, corresponding to an increase in the mass of the film. Given that the redox reaction involves a nitroso-to-hydroxylamine (-NO/NHOH) conversion, the difference in mass would be 2 amu which, taking into account the surface coverage, should give rise to a frequency change of only 0.003 Hz, which is well below our resolution. However, as can be ascertained from Figure 10, the overall frequency change was 0.4 Hz. We thus consider these frequency changes as arising from changes in the degree of solvation of the nitroso and hydroxylamine groups with the latter being significantly more solvated than the former, as a result of enhanced ability of the hydroxylamine group to form multiple hydrogen bonds. From the measured frequency changes, we estimate that about 6.5 water molecules are exchanged per proton. 5. Electrocatalysis of NADH Oxidation. One of the applications of SAMs is the development of modified electrodes capable of the electrocatalytic oxidation of (21) See: (a) Gorton, L J. Chem. Soc., Farady Trans. 1986, 82, 12451258. (b) Persson, B.; Gorton, L. J. Electroanal. Chem.. 1990, 292, 115138. (c) Lorenzo, E.; Pariente, F.; Herna´ndez, L.; Wu, Q.; Maskus, M.; Tobalina, F.; Darder, M.; Abrun˜a, H. D. Biosens. Bioelectron. 1998, 13, 319-332, and references therein.

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value, the uncatalyzed reaction which typically takes place at about +0.70 to +1.0 V (depending on electrode material). In addition, it suggests that the nitroso/hydroxylamine couple might find general use in the electrocatalytic oxidation of NADH and related materials as well as in biosensor applications. Moreover, this approach might prove general for the generation-on-demand of surface-immobilized reactants by electrochemically triggered reactions. Conclusions

Figure 11. CV of the Au electrode modified with 2,2′-dithiobis(5-nitropyridine) in 0.1 M phosphate buffer (pH 7) (a) in the absence of NADH, (b) in the presence of 1.0 mM NADH. Sweep potential rate: 10 mV s-1.

molecules of biological interest such as NADH.21 To test for the potential electrocatalytic activity of these materials, the cyclic voltammetric responses of modified electrodes were obtained in the absence of and in the presence of NADH. Figure 11 shows the cyclic voltammetric response (at 10 mV/s) in phosphate buffer (pH 7) of a gold electrode modified with a layer derived from adsorbed DTNPy in the absence and in the presence of 1.0 mM NADH. In the absence of NADH (Figure 11a), a well-behaved redox response for the adsorbed nitroso/hydroxylamine couple can be observed at a formal potential value of -0.06 V. Upon the addition of 1.0 mM NADH, there is an enhancement of the anodic current peak (Figure 11b), and, in addition, there is a decrease in the return (cathodic) wave. This behavior is consistent with a strong electrocatalytic effect. It is also of note that the potential at which this electrocatalytic reaction takes place is well below, in

DTNB adsorbs onto gold electrode surfaces and, upon potential cycling past -0.55 V, is transformed into the hydroxylamine which exhibits a well-behaved pH-dependent redox couple centered at -0.04 V at pH 7.0 and with a moderately fast charge-transfer rate constant (450 s-1). This electrochemically triggered transformation, ascribed to reduction of the nitro group to the corresponding hydroxylamine, has been studied by electrochemical and EQCM techniques. Adsorption of DTNB is activation controlled, and its reduction generates the hydroxylamine product with a coulometric yield that is estimated to be on the order of 50%. The resulting redox-active layer exhibits a well-behaved redox response that is stable over a very broad pH range (pH 1.5-12). Studies of the reactivity of analogues of DTNB and related materials are consistent with the proposed mechanism. From EQCM studies, it appears that the redox response of the immobilized layer is accompanied by significant changes in solvation. Electrodes modified with a redox-active layer derived from DTNPy catalyze the electrooxidation of NADH. Acknowledgment. This work was supported by the DGICYT of Spain through the Grant BIO96-1016-C02-02 (E.L., F.P.), the Comunidad Auto´noma de Madrid (06M/ 044/96), and the Office of Naval Research. E.C. and M.D. also acknowledge support by the Comunidad Auto´noma de Madrid. LA9806552