Kinetic Separation of Amperometric Responses of Composite Redox

Chronoamperometry provides a sensitive method to elucidate the composition and properties of complex redox-active monolayers assembled onto Au ...
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3364

Langmuir 1997, 13, 3364-3373

Kinetic Separation of Amperometric Responses of Composite Redox-Active Monolayers Assembled onto Au Electrodes: Implications to the Monolayers’ Structure and Composition Eugenii Katz and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Received November 12, 1996X Chronoamperometry provides a sensitive method to elucidate the composition and properties of complex redox-active monolayers assembled onto Au electrodes. Chronoamperometry is applied to characterize a redox-active monolayer composed of a bisdiaminobenzoquinone dyad. The different distances of the quinone units in respect to the electrode yield different electron-transfer rate constants, k1 ) 25.3 s-1 and k2 ) 1.05 s-1, and the surface coverage of the two quinone units is estimated to be ΓQ1 ) 6.5 × 10-11 mol cm-2 and ΓQ2 ) 6.5 × 10-11 mol cm-2. The surface densities of the quinone units coincide nicely with those obtained by voltammetric analysis of the monolayer during the steps of its assembly. Similarly, a binary monolayer consisting of bipyridinium and anthraquinone redox-active units was characterized by chronoamperometry. The electron-transfer rate constant to the bipyridinium and anthraquinone components corresponds to k1 ) 503 s-1 and k2 ) 62 s-1, respectively, and the surface coverage of the redox-active groups is ΓV2+ ) 1.44 × 10-10 mol cm-2 and ΓQ ) 0.46 × 10-10 mol cm-2. Kinetic separation of the amperometric response of a redox-active monolayer formed by the coupling of N,N′-bis(7-carboxyheptyl)-4,4′-bipyridinium, V2+-(CO2H)2, to a cystamine-modified monolayer on an Au electrode, reveals the formation of two redoxactive groups within the monolayer assembly: one redox-active form consists of a single-point attachment of V2+-(CO2H)2 to the electrode, whereas the second form includes the bifunctional, two-point linkage of V2+-(CO2H)2 to the monolayer. The surface coverage of the two redox-active forms corresponds to Γ1 ) 1.11 × 10-10 and Γ2 ) 1.13 × 10-10 mol cm-2, respectively. The electron-transfer rate constants to the single-point-associated bipyridinium sites and to the two-point linkage bipyridinium units are k1 ) 508 s-1 and k2 ) 896 s-1, respectively. Chronoamperometric analysis of microperoxidase-11 assembled as a monolayer on an Au electrode reveals that the heme-substituted oligopeptide binds to the surface by two different modes. These include the covalent linkage of carboxylic functions associated with the porphyrin ligand or the peptide backbone to the primary cystamine monolayer assembled onto the Au electrode.

Functionalized monolayers1 assembled onto electrode surfaces are currently examined as sensor interfaces,2 optoelectronic devices,3,4 and active surfaces for patterning5 and chemical architecture6 of solid supports. The assembly of redox-active monolayers on electrode surfaces7 was recently employed for probing the electron-transfer theory,8 for the amperometric transduction of optical signals recorded by a photoactive/electroactive monolayer,9 and for the construction of supramolecular systems at the * Author to whom correspondence should be addressed. Fax: 972-2-6527715. Tel.: 972-2-6585272. E-mail: [email protected]. X Abstract published in Advance ACS Abstracts, May 15, 1997. (1) (a) Ulman, A. An Introduction to Ultrathin Organic Films: From Langmuir-Blodgett to Self-Assembly, Academic Press: Boston, 1991. (b) Ulman, A. Chem. Rev. 1996, 96, 1533-1554. (c) Finklea, H. O. In Electroanalytical Chemistry; Bard, A. J., Rubinstein, I., Eds.; Marcel Dekker: New York, 1996; Vol. 19, pp 109-335. (d) Xu, J.; Li, H.-L. J. Colloid Interface Sci. 1995, 176, 138-149. (2) (a) Willner, I.; Lapidot, N.; Riklin, A.; Kasher, R.; Zahavy, E.; Katz, E. J. Am. Chem. Soc. 1994, 116, 1428-1441. (b) Willner, I.; Riklin, A. Anal. Chem. 1994, 66, 1535-1539. (c) Katz, E.; Heleg-Shabtai, V.; Willner, B.; Willner, I.; Bu¨ckmann, A. F. Bioelectrochem. Bioenerg., in press. (d) Mandler, D.; Turyan, I. Electroanalysis 1996, 8, 207-213. (e) Schmidt, H.-L.; Schuhmann, W. Biosens. Bioelectron. 1996, 11, 127135. (f) Go¨pel, W.; Heiduschka, P. Biosens. Bioelectron. 1995, 10, 853883. (3) (a) Lion-Dagan, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 1994, 116, 7913-7914. (b) Katz, E.; Lion-Dagan, M.; Willner, I. J. Electroanal. Chem. 1995, 382, 25-31. (c) Lion-Dagan, M.; Katz, E.; Willner, I. J. Chem. Soc., Chem. Commun. 1994, 2741-2742. (d) Willner, I.; Willner, B. Adv. Mater. 1995, 7, 587-589. (e) Willner, I.; Lion-Dagan, M.; MarxTibbon, S.; Katz, E. J. Am. Chem. Soc. 1995, 117, 6581-6592. (f) Willner, I.; Lion-Dagan, M.; Katz, E. J. Chem. Soc., Chem. Commun. 1996, 623624. (g) Willner, I.; Willner, B. Bioelectrochem. Bioenerg., in press. (h) Katz, E.; Willner, B.; Willner, I. Biosens. Bioelectron., in press. (4) (a) Liu, Z.-F.; Hashimoto, K.; Fujishima, A. Nature 1990, 347, 658-660. (b) Morigaki, K.; Liu, Z.-F.; Hashimoto, K.; Fujishima, A. J. Phys. Chem. 1995, 99, 14771-14777. (c) Sekkat, Z.; Wood, J.; Geerts, Y.; Knoll, W. Langmuir 1995, 11, 2856-2859. (d) Sekkat, Z.; Wood, J.; Geerts, Y.; Knoll, W. Langmuir 1996, 12, 2976-2980.

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electrode surface.10 Two general strategies were employed in the assembly of redox-active monolayers on electrodes, including the direct immobilization of thiol-modified electroactive components,11 or the alternative stepwise assembly of a functionalized base monolayer followed by (5) (a) Willner, I.; Blonder, R. Thin Solid Films 1995, 266, 254-257. (b) Blonder, R.; Ben-Dov, I.; Dagan, A.; Willner, I. Biosens. Bioelectron., in press. (c) Abbott, N. L.; Folkers, J. P.; Whitesides, G. M. Science 1992, 257, 1380-1382. (d) Lopez, G. P.; Biebuyck, H. A.; Frisbie, C. D.; Whitesides, G. M. Science 1993, 260, 647-649. (e) Wilbur, J. L.; Kumar, A.; Kim, E.; Whitesides, G. M. Adv. Mater. 1994, 6, 600-604. (f) Kumar, A.; Biebuyck, H. A.; Whitesides, G. M. Langmuir 1994, 10, 1498-1511. (g) Gorman, C. B.; Biebuyck, H. A.; Whitesides, G. M. Chem. Mater. 1995, 7, 252-254. (h) Evans, S. D.; Flynn, T. M.; Ulman, A. Langmuir 1995, 11, 3811-3814. (i) Rozsnyai, L. F.; Wrighton, M. S. J. Am. Chem. Soc. 1994, 116, 5993-5994. (j) Rozsnyai, L. F.; Wrighton, M. S. Langmuir 1995, 11, 3913-3920. (k) Chan, K. C.; Kim, T.; Schoer, J. K.; Crooks, R. M. J. Am. Chem. Soc. 1995, 117, 5875-5876. (6) (a) Fuhrhop, J.-H.; Ko¨ning, J. Membranes and Molecular Assemblies: The Synkinetic Approach, Monographs in Supramolecular Chemistry; Stoddart, J. F., Ed.; Freie Universita¨t: Berlin, 1995; Chapter 6, pp 149-181. (b) Zhong, C.-J.; Porter, M. D. Anal. Chem. 1995, 67, 709A-715A. (c) Knoll, W. Curr. Opin. Colloid Interface Sci. 1996, 1, 137-143. (d) Bell, C. M.; Yang, H. C.; Mallouk, T. E. In Materials Chemistry; Interrante, L. V., Caspar, L. A., Ellis, A. B., Eds.; Advances in Chemistry Series 245; American Chemical Society, Washington, DC, 1995; Chapter 8, pp 211-230. (7) (a) Collard, D. M.; Fox, M. A. Langmuir 1991, 7, 1192-1197. (b) Rowe, G. K.; Creager, S. E. Langmuir 1991, 7, 2307-2312. (c) Uosaki, K.; Sato, Y.; Kita, H. Langmuir 1991, 7, 1510-1514. (d) Hickman, J. J.; Ofer, D.; Zou, C.; Wrighton, M. S.; Laibinis, P. E.; Whitesides, G. M. J. Am. Chem. Soc. 1991, 113, 1128-1132. (e) De Long, H. C.; Buttry, D. A. Langmuir 1990, 6, 1319-1322. (f) Lee, K. A. B. Langmuir 1990, 6, 709-712. (g) Chidsey, C. E. D.; Bertozzi, C. R.; Putvinski, T. M.; Muijsce, A. M. J. Am. Chem. Soc. 1990, 112, 4301-4306. (h) Akiyama, T.; Imahori, H.; Sakata, Y. Chem. Lett. 1994, 1447-1450. (i) Zak, J.; Yuan, H.; Ho, M.; Woo, L. K.; Porter, M. D. Langmuir 1993, 9, 27722774. (j) Zhang, L.; Lu, T.; Gokel, G. W.; Kaifer, A. E. Langmuir 1993, 9, 786-791. (k) Katz, E.; Schmidt, H.-L. J. Electroanal. Chem. 1994, 368, 87-94.

© 1997 American Chemical Society

Kinetic Separation of Amperometric Responses

Langmuir, Vol. 13, No. 13, 1997 3365

Scheme 1. Sequence of Reactions for the Assembly of a Quinone Dyad on an Au Electrode and Further Rigidification of the Monolayer with a Long-Alkyl Mercaptan

covalent attachment of the redox-active groups.12 Similarly, the organization of binary redox-active monolayer composites is accomplished by the coimmobilization of two different thiols,13 the stepwise, partial modification of a functionalized monolayer by two different electroactive (8) (a) Weber, K.; Creager, S. E. Anal. Chem. 1994, 66, 3164-3172. (b) Tender, L.; Carter, M. T.; Murray, R. W. Anal. Chem. 1994, 66, 3173-3181. (c) Finklea, H. O.; Hanshew, D. D. J. Am. Chem. Soc. 1992, 114, 3173-3181. (d) Guo, L. H.; Facci, J. S.; McLendon, G. J. Phys. Chem. 1995, 99, 8458-8461. (e) Chidsey, C. E. D. Science 1991, 251, 919-922. (f) Smalley, J. F.; Feldberg, S. W.; Chidsey, C. E. D.; Linford, M. R.; Newton, M. D.; Liu, Y.-P. J. Phys. Chem. 1995, 99, 13141-13149. (g) Richardson, J. N.; Peck, S. R.; Curtin, L. S.; Tender, L. M.; Terrill, R. H.; Carter, M. T.; Murray, R. W.; Rowe, G. K.; Creager, S. E. J. Phys. Chem. 1995, 99, 766-772. (h) Carter, M. T.; Rowe, G. K.; Richardson, J. N.; Tender, L. M.; Terrill, R. H.; Murray, R. W. J. Am. Chem. Soc. 1995, 117, 2896-2899. (i) Rowe, G. K.; Carter, M. T.; Richardson, J. N.; Murray, R. W. Langmuir 1995, 11, 1797-1806. (j) Katz, E.; Itzhak, N.; Willner, I. Langmuir 1993, 9, 1392-1396. (9) (a) Doron, A.; Katz, E.; Portnoy, M.; Willner, I. Angew. Chem., Int. Ed. Engl. 1996, 35, 1535-1537. (b) Doron, A.; Portnoy, M.; LionDagan, M.; Katz, E.; Willner, I. J. Am. Chem. Soc. 1996, 118, 89378944. (10) (a) Lu, T.; Zhang, L.; Gokel, G. W.; Kaifer, A. E. J. Am. Chem. Soc. 1993, 115, 2542-2543. (b) Zhang, L.; Godinez, L. A.; Lu, T.; Gokel, G. W.; Kaifer, A. E. Angew. Chem., Int. Ed. Engl. 1995, 34, 235-237. (c) Spinke, J.; Liley, M.; Guder, H.-J.; Angermaier, L.; Knoll, W. Langmuir 1993, 9, 1821-1825. (d) Rojas, M. T.; Koniger, R.; Stoddart, J. F.; Kaifer, A. E. J. Am. Chem. Soc. 1995, 117, 336-343. (e) Motesharei, K.; Myles, D. C. J. Am. Chem. Soc. 1994, 116, 7413-7414. (f) Rickert, J.; Weiss, T.; Kraas, W.; Jung, G.; Go¨pel, W. Biosens. Bioelectron. 1996, 11, 591-598. (g) Mittler, S.; Spinke, J.; Liley, M.; Nelles, G.; Weisser, M.; Back, R.; Wenz, G.; Knoll, W. Biosens. Bioelectron. 1995, 10, 903916. (h) Marx-Tibbon, S.; Ben-Dov, I.; Willner, I. J. Am. Chem. Soc. 1996, 118, 4717-4718. (i) Ranjit, K. T.; Marx-Tibbon, S.; Ben-Dov, I.; Willner, I. Angew Chem., Int. Ed. Engl. 1997, 36, 147-150. (j) Lahav, M.; Ranjit, K. T.; Katz, E.; Willner, I., Chem. Commun. 1997, 259-260. (11) (a) Katz, E.; Itzhak, N.; Willner, I. J. Electroanal. Chem. 1992, 336, 357-362. (b) Shimazu, K.; Yagi, I.; Sato, J.; Uosaki, K. Langmuir 1992, 8, 1385-1387. (12) (a) Duevel, R. V.; Corn, R. M. Anal. Chem. 1992, 64, 337-342. (b) Katz, E.; Solov’ev, A. A. J. Electroanal. Chem. 1990, 291, 171-186. (c) Katz, E. J. Electroanal. Chem. 1990, 291, 257-260. (d) Katz, E.; Schlereth, D. D.; Schmidt, H.-L. J. Electroanal. Chem. 1994, 367, 5970. (e) Katz, E.; Riklin, A.; Willner, I. J. Electroanal. Chem. 1993, 354, 129-144. (f) Schlereth, D. D.; Katz, E.; Schmidt, H.-L. Electroanalysis 1995, 7, 46-54. (13) (a) Rowe, G. K.; Creager, S. E. Langmuir 1994, 10, 1186-1192. (b) Creager, S. E.; Rowe, G. K. J. Electroanal. Chem. 1994, 370, 203211. (c) Yamada, S.; Kohrogi, H.; Matsuo, T. Chem. Lett. 1995, 639640.

substrates,14 or the sequential synthesis of electroactive dyads on a functionalized base monolayer.15 Voltammetric methods were used to characterize these monolayers, i.e., to find surface coverages, formal potentials, and interfacial electron-transfer rates. However, for complex monolayer composites, these methods are only applicable if the different redox components exhibit sufficiently different formal potentials that enable the separate analysis of the various voltammetric responses. Recently, kinetic separation of the electrical responses of composite redox-active monolayers exhibiting overlapping formal potentials, adsorbed onto mercury or Pt, was reported using chronoamperometry.16 This method assumes that different redox-active groups reveal different interfacial electron-transfer rates that allow the timedependent chronoamperometric analysis of the various electroactive species. Here we report on the characterization of composite electroactive thiol monolayers assembled onto Au electrodes by the kinetic separation of the amperometric responses of the redox components using chronoamperometry. We examine functionalized electrodes by simultaneous voltammetric and chronoamperometric experiments and reveal the possibility of determining the composition and properties of complex redox-active assemblies via kinetic resolution of their faradaic currents. We also demonstrate the application of the method to elucidate the composition and structure of redox-active monolayer assemblies, issues that cannot be resolved by single voltammetric studies. (14) (a) Katz. E.; Borovkov, V. V.; Evstigneeva, R. P. J. Electroanal. Chem. 1992, 326, 197-212. (b) Katz, E.; Lion-Dagan, M.; Willner, I. J. Electroanal. Chem. 1995, 382, 25-31. (15) (a) Katz, E.; Schmidt, H.-L. J. Electroanal. Chem. 1993, 360, 337-342. (b) Doron, A.; Katz, E.; Willner, I. Langmuir 1995, 11, 13131317. (c) Willner, I.; Heleg-Shabtai, V.; Blonder, R.; Katz, E.; Tao, G.; Bu¨ckmann, A. F.; Heller, A. J. Am. Chem. Soc. 1996, 118, 1032110322. (16) (a) Forster, R. J.; Faulkner, L. R. Anal. Chem. 1995, 67, 12321239. (b) Forster, R. J. Langmuir 1995, 11, 2247-2255. (c) Forster, R. J. Anal. Chem. 1996, 68, 3143-3150. (d) Forster, R. J. Analyst 1996, 121, 733-741.

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Scheme 2. Preparation of a Binary Monolayer Consisting of Anthraquinone-2-carboxylic Acid and N-Methyl-N′-(7-carboxyheptyl)-4,4′-bipyridinium Monolayer Redox-Active Components on an Au Electrode and Its Rigidification with a Long-Alkyl Mercaptan

Scheme 3. Assembly of N,N′-Bis(7-carboxyheptyl)-4,4′-bipyridinium as a Monolayer Consisting of Two Different Attachment Modes: Single- and Double-Point Coupling to a Cystamine-Modified Au Electrode

Experimental Section Materials. N-Methyl-N′-(7-carboxyheptyl)-4,4′-bipyridinium dichloride and N,N′-bis(7-carboxyheptyl)-4,4′-bipyridinium dichloride (mono- and dicarboxylic viologen derivatives: HO2C-V2+CH3 and HO2C-V2+-CO2H, respectively) were synthesized according to the published procedures.8j,2a All other materials, including microperoxidase-11, anthraquinone-2-carboxylic acid,

1,4-benzoquinone, 1,8-diaminooctane, and cystamine (bis(2aminoethyl)disulfide) were obtained commercially (Aldrich) and used as supplied. Ultrapure water from a Nanopure (Barnstead) source was used throughout this work. Electrode Modifications. An Au electrode (0.5 mm diameter Au wire of geometrical area ca. 0.2 cm2) was used for the modifications. Pretreatment of the electrode (to remove a

Kinetic Separation of Amperometric Responses Scheme 4. Assembly of the Microperoxidase-11 Monolayer on a Cystamine-Modified Au Electrode via Two Different Association Modes.

Langmuir, Vol. 13, No. 13, 1997 3367 potentials are reported with respect to this reference electrode. Argon bubbling was used to remove oxygen from the solutions in the electrochemical cell. RC cell time constants estimated as the fastest components in the current decay during chronoamperometric measurements were shorter than 400 ns. The interfacial kinetics were measured only at times greater than about 5-10 RC. Biexponential analysis of the current decays and smoothing of the semilogarithmic plots were performed using KaleidoGraph 3.0 software with a Macintosh LSIII computer. Fitting of the experimental decay curves was performed to the general relation A1 e-k1t + A2 e-k2t. For all fitting procedures, the correlation factor was R > 0.98.

Results and Discussion

previously adsorbed monolayer and to regenerate the bare surface) and its modification with a monolayer of cystamine were performed according to the published procedure.12b Amino groups of the adsorbed cystamine monolayer were used for covalent coupling of all redox-active materials used in this study. The carbodiimide coupling of the quinone12b,d and viologen8j carboxylic acid derivatives (cf. Schemes 2 and 3) as well as microperoxidase1117a (cf. Scheme 4) were performed in the presence of 1-ethyl3-(3-(dimethylamino)propyl)carbodiimide (EDC) in 0.05 M Hepes buffer, pH ) 7.2, according to published procedures. The multistep assemblage of 1,4-benzoquinone molecules as amino derivatives on the Au electrode, with different distances between the electrode surface and redox centers (cf. Scheme 1), was performed according to the recently developed procedure.15a Further incorporation of long-alkyl mercaptan into the viologen and quinone monolayers was recently described.7k,8j,9a,b Electrochemical Measurements. Electrochemical measurements (cyclic voltammetry and chronoamperometry) were performed using a potentiostat (EG&G VersaStat) connected to a personal computer. All the measurements were carried out at ambient temperature (22 ( 2 °C) in a three-compartment electrochemical cell consisting of the chemically modified electrode as a working electrode, a glassy carbon auxiliary electrode isolated by a glass frit, and a saturated calomel electrode connected to the working volume with a Luggin capillary. All (17) (a) Lo¨tzbeyer, T.; Schuhmann, W.; Katz, E.; Falter, J.; Schmidt, H.-L. J. Electroanal. Chem. 1994, 377, 291-294. (b) Moore, A. N. J.; Katz, E.; Willner, I. J. Electroanal. Chem. 1996, 417, 189-192.

A composite electroactive dyad monolayer consisting of two quinone units15a was assembled onto an electrode surface as outlined in Scheme 1. A primary cystamine monolayer was assembled onto the Au electrode as the base monolayer. Nucleophilic addition to benzoquinone yields the covalent attachment of the primary quinone to the monolayer. Subsequent reaction of the monolayer with 1,8-diaminooctane allows the coupling of the second quinone unit to the monolayer. The secondary quinone is further modified with butylamine to generate the diamino-substituted secondary quinone. Figure 1 shows the cyclic voltammograms recorded upon the stepwise construction of the dyad quinone monolayer. The benzoquinone attached to the primary amine monolayer revealed the redox wave at E° ) -0.17 V (pH ) 7.0) (curve a). Upon reaction with 1,8-diaminooctane, the original redox wave of the monoaminoquinone is depleted and a new wave characteristic of the diamino derivative is observed at E° ) -0.53 V (pH ) 7.0) (curve b). The negative shift in the reduction potential of the quinone component is consistent with the formation of a diaminosubstituted quinone where the electron-donating substituents shift the redox potential to negative values. Further reaction of the free amino function with benzoquinone results in the formation of the quinone dyad; here one of the quinones is monosubstituted by the amine function. This is evident by the appearance of two redox waves at E° ) -0.17 and -0.53 V, corresponding to the diamino- and monoamino-substituted quinones, respectively Figure 1 (curve c)). Further reaction of the dyad with butylamine results in the diamino-bisquinone dyad. This is supported by the formation of an overlapping redox wave at E° ) -0.53 V for the two quinone units (curve d). Note that the amperometric response of the wave at E° ) -0.53 V is substantially increased as compared to that of the diamino-monoquinone response (curve c), implying that the electrical signals of the two quinone units overlap. Quinone monolayers assembled onto Au surfaces form nondensely-packed monolayers. By treatment of the electrodes with a long alkanethiol, a mixed densely-packed monolayer can be generated.7k,8j,9a,b In the densely-packed monolayer, the redox-active dyad is rigidified in a confined alignment with respect to the electrode. Figure 1 (curve e) shows the redox wave of the resulting diaminobisquinone dyad after treatment with 1-octadecanethiol, C18SH. The redox wave of the dyad slightly decreases as compared to that of the primary nondense assembly, mainly because of the decrease in the capacitive current, and it appears at the same redox potential as the original nonrigidified dyad. By the integration of the area of the redox wave corresponding to the primary diaminoquinone component in the dyad (curve c), its surface coverage is calculated to be ΓQ1 ) 6.4 × 10-11 mol cm-2. Similarly, by coulometric analysis of the overlapping redox wave and subtraction of the charge associated with the reduction (or oxidation) of the primary quinone, the surface coverage

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Figure 2. (A) Current response of the Au electrode modified with bisamino-bisquinone dyad rigidified with C18SH following a potential step from -0.2 to -0.63 V. Inset: Current response of a nonelectroactive mixed monolayer of cystamine/C18SH assembled on an Au electrode following a potential step from -0.2 to -0.63 V. In all experiments the supporting electrolyte is 0.1 M phosphate buffer, pH ) 7.0. (B) Semilogarithmic plot for the current response. The time axis is referenced to the leading edge of the potential step.

resolved using chronoamperometry. For an ideal electrochemical reaction involving a surface-bound electroactive species, the faradaic current following a potential step that changes the redox composition of the monolayer decays with time according to eq 1,8e,16a-d where k is the apparent electron-transfer rate constant and Q is the total

iF(t) ) kQ exp(-kt)

Figure 1. Cyclic voltammograms of the Au electrode modified according to Scheme 1 at different steps of the assembly of the bisquinone dyad: (a) monoaminoquinone, (b) diaminoquinone, (c) mono- and diaminoquinone dyad, (d) bisamino-bisquinone dyad, and (e) bisamino-bisquinone dyad rigidified with C18SH. Background electrolyte: 0.1 M phosphate buffer, pH ) 7.0; potential scan rate, 100 mV s-1.

of the secondary diaminoquinone component in the monolayer is calculated to be ΓQ2 ) 6.4 × 10-11 mol cm-2. The interfacial electron-transfer rates can be analyzed using the Laviron theory18 on the basis of the peak-topeak separation of the redox waves of the electroactive species. However, the analysis of two overlapping waves by Laviron’s approach would only allow the elucidation of the slow electron-transfer rate. The electroactivity of the similar composite system, consisting of the diaminobisquinone dyad rigidified by C18SH chains, was kinetically (18) Laviron, E. J. Electroanal. Chem. 1979, 101, 19-28.

(1)

charge associated with electrolysis of the redox-active species. Figure 2A shows the current response of the diamino-bisquinone dyad upon the application of a potential step from -0.2 to -0.63 V. This potential step is ca. 100 mV more negative as compared to the quinone formal potential. The current response shown in Figure 2A cannot be analyzed in terms of a single-exponential decay. The semilogarithmic plot of the faradaic current decay as a function of time (Figure 2B), clearly indicates that the current decays biexponentially with two different rate constants. The inset in Figure 2A shows the chronoamperometric response of a nonelectroactive mixed monolayer of cystamine and C18SH assembled on an Au electrode. It is evident that the current associated with the background electrolyte decays within ca. 1 ms. Thus, the biexponential decay of the current can be attributed to only two different electron-transfer rate constants associated with the two redox species comprising the monolayer. The faradaic current decay shown in Figure 2 was fitted to a biexponential decay process (eq 2) The

iF(t) ) k1Q1 exp(-k1t) + k2Q2 exp(-k2t)

(2)

Kinetic Separation of Amperometric Responses

values k1 ) 25.3 s-1 and k2 ) 1.05 s-1 and the respective pre-exponential factors, A1 ) 63 µA and A2 ) 2.5 µA were derived. From the corresponding pre-exponential factors and the k1 and k2 values, the respective Q1 and Q2 values were derived. This corresponds to a surface coverage of the electrode of ΓQ1 ) 6.5 × 10-11 mol cm-2 and ΓQ2 ) 6.5 × 10-11 mol cm-2 of the primary and secondary quinones in the dyad monolayer assembly. Note that the surface coverages of the electroactive components in the dyad agree well with the respective surface densities elucidated by the voltammetric analysis. The kinetic resolution of the faradaic currents of the two electroactive species enabled also the characterization of the electron-transfer rates to the different quinone units. The electron-transfer rate to the primary quinone unit, close to the electrode surface, is k1 ) 25.3 s-1 whereas the electron-transfer rate to the secondary quinone component is k2 ) 1.05 s-1. An additional system to probe the applicability of the chronoamperometric method to elucidate the composition of complex electroactive monolayers, consisting of a binary mixed monolayer of two electroactive components, was examined. Scheme 2 outlines the assembly of the binary mixed monolayer. A primary cystamine monolayer was modified with a 1:1 mixture of N-methyl-N′-(8-octanic acid)-4,4′-bipyridinium, HO2C-V2+-CH3, and 2-carboxyanthraquinone, HO2C-Q, to yield the binary monolayer consisting of the bipyridinium and anthraquinone electroactive components. The resulting nondensely-packed monolayer was further rigidified with 1-tetradecanethiol, C14SH. The binary electroactive monolayer exhibits an important feature for the present study. While the electrochemistry of the bipyridinium component is pHindependent, the reduction potential of the quinone components is controlled by the pH of the electrolyte solution. Thus, by the appropriate tuning of the pH of the electrolyte solution, the redox waves of the two electroactive components could be either resolved or, alternatively, tuned to overlapping reduction-oxidation waves. This could allow us to analyze the binary monolayer by voltammetric and chronoamperometric means. Furthermore, kinetic resolution of the faradaic currents of the two electroactive components, under conditions where the waves of the two components overlap, would allow us to elucidate the composition of the binary monolayer and to compare it to the monolayer composition determined voltammetrically, under conditions where the redox waves of the two components are well separated. Figure 3 (curve a) shows the cyclic voltammogram of the rigidified binary mixed monolayer consisting of the bipyridinium-anthraquinone units at pH ) 5.3. The two redox waves at E° ) -0.59 and -0.38 V correspond to the redox processes of the bipyridinium and anthraquinone units, respectively. By coulometric analysis of the charge associated with the reduction (or oxidation) of the two components, the surface coverage of the electrode by the bipyridinium and anthraquinone units is calculated to be 1.45 × 10-10 and 0.47 × 10-10 mol cm-2, respectively. Figure 3 (curve b) shows the cyclic voltammogram of the binary monolayer at pH ) 8.8. A single redox wave of enhanced amperometric response is observed. At pH ) 8.8, the redox potential of the anthraquinone component is negatively shifted as compared to pH ) 5.3 and overlaps with the pH-independent redox wave of the bipyridinium unit. This gives rise to the enhanced amperometric response of the composite monolayer electrode. The electroactivity of the binary bipyridinium-anthraquinone mixed monolayer, under conditions where the redox waves of the two components overlap (pH ) 8.8), was kinetically resolved using chronoamperometry. Figure 4A shows the current response of the binary

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Figure 3. Cyclic voltammograms of the Au electrode modified with a binary monolayer consisting of anthraquinone-2-carboxylic acid and N-methyl-N′-(7-carboxyheptyl)-4,4′-bipyridinium redox components covalently linked to a cystamine monolayer and rigidified with C18SH. Background electrolyte: 0.1 M phosphate buffer titrated to (a) pH ) 5.3 and (b) pH ) 8.8; potential scan rate, 100 mV s-1.

Figure 4. (A) Current response of the Au electrode modified with the binary monolayer consisting of anthraquinone-2carboxylic acid and N-methyl-N′-(7-carboxyheptyl)-4,4′-bipyridinium rigidified with C14SH following a potential step from -0.3 to -0.69 V. Supporting electrolyte: 0.1 M phosphate buffer, pH ) 8.8. (B) Semilogarithmic plot for the current response. The time axis is referenced to the leading edge of the potential step.

monolayer upon the application of a potential step from -0.3 to -0.69 V. This potential step is ca. 100 mV more negative than the formal potential of the overlapping redox wave. The current response cannot be analyzed by a single-exponential decay. The semilogarithmic plot of the faradaic current as a function of time (Figure 4B) clearly implies a biexponential current decay. The experimental faradaic current response was fitted to a biexponential decay process (eq 2). Excellent fitting is obtained with

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Katz and Willner

Figure 6. Cyclic voltammograms of an Au electrode modified with N,N′-bis(7-carboxyheptyl)-4,4′-bipyridinium monolayer (a) before and (b) after monolayer rigidification with C14SH. Background electrolyte: 0.1 M phosphate buffer, pH ) 7.0; potential scan rate, 100 mV s-1.

Figure 5. (A) Current responses of the Au electrode modified with the binary monolayer consisting of anthraquinone-2carboxylic acid and N-methyl-N′-(7-carboxyheptyl)-4,4′-bipyridinium rigidified with C14SH following a potential step: (a) from -0.5 to -0.69 V and (b) from -0.3 to -0.49 V. Supporting electrolyte: 0.1 M phosphate buffer, pH ) 5.3. (B) Semilogarithmic plot for the current responses. The time axis is referenced to the leading edge of the potential steps.

two electron-transfer rate constants, k1 ) 503 s-1 and k2 ) 62 s-1, and the pre-exponential factors, A1 ) 1400 µA and A2 ) 110 µA, respectively. Thus, although the voltammetric redox responses of the two electroactive species overlap, the chronoamperometric experiments reveal the redox processes of the two electroactive components in the monolayer. To relate the electrontransfer rates to the specific redox components in the mixed monolayer, chronoamperometric experiments on the functionalized electrode were performed at pH ) 5.3, where the redox waves are well separated. Under these conditions, the faradaic currents of the anthraquinone component and of the bipyridinium units decay monoexponentially with time constants of 33 and 501 s-1, respectively (Figure 5). Thus, the fast electron-transfer rate, k1 ) 503 s-1, observed in the biexponential current decay at pH ) 8.8, is attributed to the interfacial electrontransfer reduction rate constant of the bipyridinium component, whereas the slow process, k2 ) 62 s-1 is attributed to the electron-transfer rate constant associated with the reduction of the anthraquinone component. It should be noted that the interfacial electron-transfer rate constant for the reduction of the anthraquinone units is ca. 2-fold slower at pH ) 5.3 as compared to that at pH ) 8.8. This is consistent with previous reports that addressed the slower electron-transfer rates of quinone monolayers at acidic pH values as compared to basic pH values.12b,d From the derived rate constants for the reduction of the two electroactive components in the binary monolayer (k1 and k2) and the respective pre-exponential factors (A1 and A2), the total charge associated with the reduction of the two redox components was calculated (eq 2). From these values the surface coverage of the binary

monolayer by the bipyridinium units (V2+) and the anthraquinone components (Q) was calculated to be ΓV2+ ) 1.44 × 10-10 mol cm-2 and ΓQ ) 0.46 × 10-10 mol cm-2, respectively. These values are in excellent agreement with the voltammetric-derived composition of the mixed monolayer. The two systems discussed to this point now were tailored in a way that enables the simultaneous analysis of the composition of complex redox-active monolayers by voltammetry and chronoamperometry. The results imply the validity of chronoamperometry in the kinetic separation of the amperometric responses of composite redoxactive monolayers and the elucidation of the monolayer composition. Nonetheless, the monolayers were artificially designed to allow the parallel voltammetric and chronoamperometric analyses. We will now turn to a more complex system where we demonstrate that only the chronoamperometric method enables the sensitive characterization of the structure and composition of a complex monolayer. Scheme 3 shows the method used to assemble the monolayer. A cystamine monolayer, assembled onto an Au electrode, is reacted with N,N′-bis(7-carboxyheptyl)4,4′-bipyridinium. The bifunctional electroactive bipyridinium substrate can bind to the base monolayer in two alternative modes: linkage of a single carboxylic acid residue to the monolayer or two-point binding of the two carboxylic acid sites to the base monolayer. We will point out that chronoamperometry is a sufficiently sensitive method to distinguish between the binding modes. The bipyridinium-modified Au electrode formed by covalent attachment of N,N′-bis(7-carboxyheptyl)-4,4′-bipyridinium, V2+-(CO2H)2, to the cystamine monolayer was further rigidified to a densely-packed monolayer by treatment of the electrode with C14SH. The resulting monolayer assembly reveals a reversible single redox wave at E° ) -0.59 V, (Figure 6). Figure 7A shows the faradaic current decay of a monolayer prepared by the interaction of the cystamine monolayer with V2+-(CO2H)2, 2 × 10-4 M, upon the application of a potential step from -0.3 to -0.69 V. This potential step is 100 mV more negative than the formal potential of the bipyridinium electroactive units. The current decrease with time does not fit a monoexponential decay but fits well with a biexponential decay process, k1 ) 500 s-1, k2 ) 900 s-1, and pre-exponential factors, A1 ) 1070 µA and A2 ) 1970 µA, respectively (Figure 7B). This result clearly indicates that two different electroactive populations of bipyridinium sites are present in the monolayer assembly. Chemical intuition suggests that the single-point attachment of the bipyridinium unit

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Figure 7. (A) Current response of the Au electrode modified with N,N′-bis(7-carboxyheptyl)-4,4′-bipyridinium monolayer rigidified with C14SH following a potential step from -0.3 to -0.69 V; modification was performed with 2 × 10-4 M of V2+(CO2H)2. Supporting electrolyte: 0.1 M phosphate buffer, pH ) 7.0. (B) Semilogarithmic plot for the current response. The time axis is referenced to the leading edge of the potential step.

Figure 8. (A) Current responses of the Au electrode modified with N,N′-bis(7-carboxyheptyl)-4,4′-bipyridinium monolayer rigidified with C14SH following a potential step from -0.3 to -0.69 V; modifications were performed with (a) 1 × 10-5 M and (b) 5 × 10-3 M of V2+-(CO2H)2. Supporting electrolyte: 0.1 M phosphate buffer, pH ) 7.0. (B) Semilogarithmic plot for the current responses. The time axis is referenced to the leading edge of the potential step.

to the cystamine monolayer will be favored upon modification of the base layer in the presence of high concentrations of V2+-(CO2H)2. The two-point modification of the monolayer will be favored in the presence of dilute solutions of V2+-(CO2H)2. Accordingly, the cystamine monolayer was modified in the presence of a concentrated solution of V2+-(CO2H)2, 5 × 10-3 M, or a diluted solution of V2+-(CO2H)2, 1 × 10-5 M. Figure 8A shows the chronoamperometric responses of the electrode prepared in the presence of a high concentration of the bipyridinium substrate (curve b) and a low concentration of V2+-(CO2H)2 (curve a). The two decay curves follow single-exponential kinetics (Figure 8B), indicating the formation of a single kind of electroactive species in each of the different modification procedures. The interfacial electron-transfer rate constant bipyridinium electroactive group, prepared at high concentrations of V2+-(CO2H)2, is k1 ) 508 s-1 (preexponential factor 2250 µA). The calculated electrontransfer rate constant to the bipyridinium units assembled onto the monolayer in the presence of a diluted solution of V2+-(CO2H)2 is k2 ) 896 s-1 (pre-exponential factor 4100 µA). Thus, for the monolayer prepared by modification of the cystamine monolayer with V2+-(CO2H)2, 2 × 10-4 M, where a biexponential decay of the faradaic current is observed, the slow electron-transfer rate constant, k1 ) 508 s-1 is attributed to the reduction of bipyridinium sites attached to the monolayer through a single-point linkage. The faster electron-transfer rate constant, k ) 896 s-1, is attributed to the reduction of bipyridinium groups associated with the monolayer via a two-point linkage.

Further support that the rapidly decaying faradaic current is associated with the redox process of the bipyridinium units linked to the monolayer via single-point attachment is obtained by chemical modification of the base cystamine monolayer with N-methyl-N′-(7-carboxyheptyl)-4,4′-bipyridinium, HO2C-V2+-CH3, and further rigidification of the monolayer with C14SH (cf. Scheme 2). In this system, only single-point linkage of the redox-active component can occur. Indeed, the resulting monolayer shows a singleexponential decay of the faradaic current, k ) 500 s-1, consistent with the decay constant of the faradaic current of the single-point linked V2+-(CO2H)2 to the monolayer assembly. From the respective pre-exponential factors and the derived k1 and k2 values, and using eq 1, the surface coverage of the electrodes by the bipyridinium components attached to the surface via single-point or two-point linkages were calculated to be 1.11 × 10-10 and 1.13 × 10-10 mol cm-2, respectively. The composition and structure of the bipyridinium monolayer formed upon reaction of the cystamine monolayer with V2+-(CO2H)2 can be found by analysis of the characteristic electron-transfer rate constants to bipyridinium sites linked to the base monolayer. Note that coulometric analysis of the voltammetric wave of the rigidified bipyridinium-modified electrode, (Figure 6 (curve b)), yields a surface coverage of the electrode corresponding to 2.3 × 10-10 mol cm-2. This corresponds to two types of bipyridinium units linked to the surface via single or double anchoring mode. Chronoamperometric measurements enable us to resolve the surface coverages of the two electroactive forms and their sum is in good agreement with the total surface coverage

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of the electroactive groups obtained by the voltammetric analysis. The method described to distinguish between one-point and two-point attachment of redox-active species to surfaces could be applied for the characterization of other redox species with multifunctional binding sites to surfaces.7i,j,19 In the different systems addressed to this point, the kinetic current decay curves fitted best to a biexponential function (easily realized upon plotting the ln I vs time). The fact that the current decay follows biexponential kinetics implies two significantly different electrontransfer rate constants to the respective two redox species associated with the monolayer. Each of these exponents coincides with the experimental chronoamperometric responses of monolayer electrodes that include one of the two redox species. Thus, the two rate constants derived by the biexponential analysis indeed correspond to the electron-transfer rates in each of the redox species associated with the monolayers. Nevertheless, it should be noted that each of these rate constants could represent an average of a sum of closely related values (i.e., redox species of slightly different orientation). Under such conditions, the average rate constants corresponding to each of the redox species differ significantly and still yield the biexponential current decay. It should be noted that the difference in the electrontransfer rate constants to the one-point- or two-pointattached bipyridinium redox-active units could originate from variable distances or orientations of the electroactive components. The two-point-linked bipyridinium component could be at a closer distance relative to the surface due to interchain H-bonds. Also, the horizontal configuration of the two-point-linked bipyridinium could enhance the electron transfer.19 We realize that chronoamperometric analysis of redoxactive groups is a sensitive method to detect variable modes of linkage of electroactive units to monolayer assemblies. Indeed, the method was employed to examine the binding modes of microperoxidase-11 to a cystamine monolayer assembled onto an Au electrode. Microperoxidase-11 is a heme-containing oligopeptide. It was previously immobilized as a monolayer onto Au electrodes, and its catalytic function for the electrocatalyzed reduction of H2O2 and organic peroxides was examined.17a,b Covalent attachment of microperoxidase-11 to a base cystamine monolayer could proceed via covalent linkage of the carboxylic acid residue of the oligopeptide or, alternatively, through coupling of the carboxylic acid groups of the heme ligand to the functionalized monolayer (Scheme 4). These two modes of binding might be sufficiently different so that the electroactivity of the heme site could be kinetically resolved by chronoamperometry. Figure 9 shows the cyclic voltammogram of the microperoxidase-11 monolayermodified electrode. A single reversible redox wave at E° ) -0.40 V is observed, and the two possible binding modes are indistinguishable. Figure 10A shows the faradaic current response of the modified monolayer upon the application of a potential step from 0 to -0.5 V. This potential step is ca. 100 mV more negative than the formal reduction potential of the microperoxidase-11 unit. The faradaic current decay does not follow a single-exponential decay, but fits well to a biexponential decay function (eq 2, k1 ) 16 s-1 and k2 ) 8.5 s-1 (pre-exponential factors, A1 ) 46 µA and A2 ) 27 µA)). The biexponential decay of the faradaic current is clearly evident from the semilogarithmic plot of the current as a function of time (Figure 10B). Thus, the chronoamperometric measurement clearly (19) Edwards, T. R. G.; Cunnane, V. J.; Parsons, R.; Gani, D. J. Chem. Soc., Chem. Commun. 1989, 1041-1043.

Katz and Willner

Figure 9. Cyclic voltammogram of an Au electrode modified with a monolayer of microperoxidase-11. Background electrolyte: 0.1 M phosphate buffer, pH ) 7.0; potential scan rate, 100 mV s-1.

Figure 10. (A) Current response of the Au electrode modified with a monolayer of microperoxidase-11 following a potential step from 0.0 to -0.5 V. Supporting electrolyte: 0.1 M phosphate buffer, pH ) 7.0. (B) Semilogarithmic plot for the current response. The time axis is referenced to the leading edge of the potential step.

reveals that covalent linkage of microperoxidase-11 to a cystamine monolayer leads to two distinct and different binding modes of the heme polypeptide to the monolayer, characterized by different electron-transfer rate constants. From the calculated electron-transfer rate constants k1 and k2, and the respective pre-exponential factors, the surface coverage of microperoxidase-11 populations linked to the surface by the two different anchoring modes, is calculated to be 1.50 × 10-10 and 1.65 × 10-10 mol cm-2. The sum of these surface densities coincides nicely with the total surface coverage of the heme electroactive sites obtained by coulometric analysis of the voltammetric wave (Figure 9, Γ ) 3.2 × 10-10 mol cm-2). It should be noted

Kinetic Separation of Amperometric Responses

that in the present system we are unable to address the specific rate constants to a defined mode of binding of microperoxidase to the monolayer. The chronoamperometric analysis of the system implies, however, that microperoxidase-11 is linked to the monolayer by two different binding modes and yields a composite redoxactive monolayer. Conclusions The present study has revealed the use of chronoamperometric measurements in the characterization of redox-active monolayers. It was demonstrated that kinetic resolution of the amperometric responses of composite redox-active monolayers provides a sensitive tool to elucidate the structure and composition of monolayer assemblies. Furthermore, we emphasized that chronoamperometric measurements enable us to resolve the

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structural characteristics of the complex monolayer assemblies that cannot be elucidated by simple voltammetric experiments. Voltammetric responses of redox-active monolayers are often insensitive to chemical processes on the monolayer assembly, i.e., formation of supramolecular complexes. We believe that the chronoamperometric method will be of general utility not only to elucidate the structure and composition of electroactive monolayers, but also to characterize chemical processes, i.e., formation of supramolecular complexes at the monolayer interface. Acknowledgment. This work was sponsored by the New Energy and Industrial Technology Development Organizatiion (NEDO)/Research Institute of Innovative Technology for the Earth (RITE), Japan. LA961095E