Collector Experiments with a Single Electrode: Introduction

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Generator/Collector Experiments with a Single Electrode: Introduction and Application to Exploring the Oxidation Mechanism of Serotonin Martin C. Henstridge, Gregory G. Wildgoose, and Richard G. Compton* Department of Chemistry, Physical and Theoretical Chemistry Laboratory, UniVersity of Oxford, South Parks Road, Oxford OX1 3QZ, United Kingdom ReceiVed: May 2, 2009; ReVised Manuscript ReceiVed: June 30, 2009

The facile chemical modification of a graphite electrode with pH sensitive redox active molecules results in the formation of a generator/collector electrode system using a single electrode. Using the oxidation of serotonin as a model system, we demonstrate that the single generator/collector electrode is capable of measuring changes in local pH immediately adjacent to the electrode surface, i.e., within the diffusion layer during the oxidation process. Comparison of experimental data with numerical simulations was used to ascertain that the serotonin oxidation mechanism in poorly buffered media initially involves the transfer of two electrons and only one proton in an electrochemical, chemical, electrochemical (ECE) mechanism. This approach compares favorably in terms of sensitivity to traditional double-electrode experiments such as the use of rotating ring-disk electrodes. 1. Introduction Many molecules undergo oxidation or reduction mechanisms involving electron transfer coupled with concomitant proton transfer, which may then lead to further homogeneous followup chemical reactions.1-3 Typically kinetic and/or mechanistic information concerning these reactions may be determined using a “generator” electrode, where the reaction is initiated, and then redox active intermediates are detected at a second “collector” electrode in close proximity to the generator electrode. Classical techniques include the use of hydrodynamic double-channel electrodes, rotating ring-disk electrodes, and wall-jet electrode systems,2,4-17 occasionally performed in combination with spectroelectrochemical techniques.5,10,18-20 However, such experiments are limited by the rate of mass transfer of reactive intermediates from the generator to the collector electrodes, with generator-collector separations usually on the millimeter scale. Recent advances in state-of-the-art fabrication have reduced the generator/collector gap to the micrometer/submicrometer scale such as the work by del Campo et al. on micro- and nanoringrecessed disk electrodes21 and Marken et al. who fabricated pairs of gold microdisk electrodes a mere 4 µm apart.22 Both of these latter approaches remove the need for hydrodynamic conditions and were applied under stagnant, diffusion only conditions. Attempts to probe reactive intermediates and/or changes in local pH within the diffusion layer adjacent to an electrode surface, during the reduction or oxidation of target molecules of interest, have been made by situating an ultramicroelectrode or even a nanometer-sized electrode close to the surface of the generator electrode where the redox reaction of interest is occurring, for example, using the methods of scanning electrochemical microscopy.23-26 The widespread use of any of the above approaches is limited by specialist equipment (bipotentiostats, spectrometers, XYZ piezoelectric controllers, etc) and the nontrivial fabrication of the very small electrodes that are required. In this report, we demonstrate proof-of-concept that by chemically attaching molecular pH probes onto the surface of * Corresponding author. E-mail: [email protected]. Telephone: +44 (0)1865 275413. Fax: +44 (0)1865 275410.

Figure 1. Schematic diagram comparing the operation of the nearzero gap generator/collector molecular pH probe configuration (above) versus the state-of-the-art microdisk arrangement (below) described by Marken et al.22

a carbon electrode, via a rapid and facile modification strategy, one can measure changes in local pH during a redox reaction, i.e., within the diffusion layer immediately adjacent to the sites of electroactivity. This is an example of an effectively zerogap generator/collector system made, conveniently, using a single macroelectrode (Figure 1). We further demonstrate how this approach can be used to elucidate mechanistic information (concerning the oxidation of the biologically relevant molecule, serotonin, as a model system) via simple one dimensional (1D) Digisim simulations as opposed to the complicated twodimensional (2D) and three-dimensional (3D) modeling required

10.1021/jp904083e CCC: $40.75  2009 American Chemical Society Published on Web 07/13/2009

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in the systems exemplified by del Campo21 and Marken,22 respectively. 2. Experimental Section All reagents were purchased from Aldrich (Gillingham, U.K.) and were used as received without further purification. All solutions were prepared using deionized water of resistivity not less than 18.2 MΩ cm at 298 K (Vivendi Water Systems, U.K.). Electrochemical measurements were recorded using a computer controlled µ Autolab potentiostat (EcoChemie, Utrecht, The Netherlands) with a standard threeelectrode configuration. An edge-plane pyrolytic graphite electrode (eppg, 5 mm diameter, Le Carbone, Sussex, U.K.) acted as the working electrode (see below). A platinum wire (99.99% GoodFellow, Cambridge, U.K.) counter electrode and a saturated calomel reference electrode (SCE, Radiometer, Copenhagen, Denmark) completed the cell assembly. The eppg electrode surface was renewed as necessary by successive polishing using 3.0 and 1.0 µm alumina slurry (Buehler) on soft lapping pads (Buehler). The electrode was thoroughly rinsed and sonicated briefly after each polishing stage to remove any adhered microparticles. All experiments were carried out in degassed solutions purged with nitrogen (BOC gases, Guildford, U.K.) at 20 ( 2 °C. Standard solutions of known pH were prepared as follows (all solutions contained in addition 0.1 M KCl): pH 1.0, 0.1 M HCl; pH 2.0, 0.01 M HCl; pH 4.6, 0.1 M acetic acid + 0.1 M sodium acetate; pH 6.6, 0.001 M Na2HPO4 + 0.001 M KH2PO4 (poorly buffered); pH 6.8, 0.025 M Na2HPO4 + 0.025 M KH2PO4 (well-buffered); pH 9.3, 0.05 M disodium tetraborate; pH 12.0, 0.01 M NaOH; pH 13.0, 0.1 M NaOH; pH 14.0, 1.0 M NaOH. 3. Results and Discussion 3.1. Electrode Modification and Characterization. In order to obtain good temporal resolution to demonstrate proof-ofconcept, it is necessary to use a molecular pH probe that has a redox potential close to that of our model system, serotonin. Therefore, our molecular pH probe of choice is the arylnitroso/ arylhydroxylamine redox system. To this end, an edge-plane pyrolytic graphite electrode (eppg) was chemically modified with 4-nitrophenyl groups according to a method developed previously (Supporting Information).27-32 Using cyclic voltammetry (CV), and upon scanning in a reductive direction (Figure 1 of the Supporting Information), we find the 4-nitrophenyl groups on the surface of the eppg electrode are irreversibly reduced to the corresponding arylhydroxylamine groups.27-32 This system was characterized using CV according to a standard literature method,30,31 during which voltammetry corresponding to that of an electrochemically quasireversible, surface-bound system involving the two-electron, two-proton arylnitroso/arylhydroxylamine redox couple was observed in agreement with the literature (Figure 2 and text of the Supporting Information).30,31 Because of the concomitant proton transfer being coupled to any electron transfer, the peak potential of this system is dependent on pH according to the Nernst equation,2,30,31 and the variation of the peak potential with bulk solution pH was recorded from pH 1 to 14 (Figure 3 of the Supporting Information). We envisage that the use of molecular pH probes will find application in a wide variety of biological systems, close to pH 7, and therefore, the arylnitroso/arylhydroxylamine molecular pH probe was examined further in a pH 6.8 phosphate buffer. In particular, the effect of the buffer capacity on the observed voltammetry of this redox system was examined using

Figure 2. Overlaid CVs of 1 mM serotonin in a 25 mM phosphate buffer (dashed line) and 1.0 mM phosphate buffer (solid line) recorded at a bare eppg electrode at a scan rate of 100 mV s-1.

Figure 3. Overlaid CVs recorded in a 1.0 mM phosphate buffer at 100 m Vs-1 of the arylnitroso/arylhydroxylamine-modified eppg electrode in the presence (solid line) and absence (dashed line) of 1.0 mM serotonin.

buffer solutions comprised of KHPO4 + Na2PO4 at equal concentrations ranging from 1.0 to 25 mM (all buffers also contained 0.1 M KCl). Even in a poorly buffered 1.0 mM electrolyte, the arylnitroso/arylhydroxylamine voltammetry was unchanged. However, in the absence of a buffer, i.e., in 0.1 M KCl alone, the corresponding voltammetry was poorly resolved. Therefore, to compare the effect of buffer capacity, we perfomed the following experiments in well-buffered 25 mM phosphate media and also in the weakly buffered 1.0 mM phosphate buffer. 3.2. Proof-of-Concept: Application of the Generator/Collector Single-Electrode to Elucidate the Mechanism of Serotonin Oxidation. In buffered media, the oxidation of serotonin, at a bare, unmodified eppg electrode is observed as a single oxidation wave (labeled I in Figure 2). Wrona et al. have speculated that this oxidation wave involves the removal of two electrons and two protons concomitantly.33-37 The intermediate species produced after this initial oxidation step can then react further to produce numerous other redox active products, giving rise to a second, small oxidation wave observed at higher potentials, labeled II in Figure 2.33-37 Upon reversing the scan direction, we observed several smaller reduction waves corresponding to the reduction of the products of the follow-up chemistry and may lead to fouling of the electrode upon repetitive scanning. However, in poorly buffered media such as a 1.0 mM phosphate buffer, also shown in Figure 2, the oxidation of serotonin is observed to be split into two distinct voltammetric peaks labeled Ia and Ib in Figures 2 and 3. One might, at first glance, suppose that each of these voltammetric peaks corresponds to two separate one-electron, one-proton oxidations of

Oxidation Mechanism of Serotonin

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TABLE 1: Experimental and Simulated Shifta in Reduction Peak Potential of the Arylnitroso Couple (Peak III′)b

SCHEME 1: Two Possible Reaction Mechanisms (ECE and ECEC)a

experimental simulated peak simulated peak scan rate peak shift (mV) shift (ECE) (mV) shift (ECEC) (mV) 50 100 250 500 100c 100d

+34 +30 +28 +24 +30 +4

+29 +29 +26 +25 +23 +2

+52 +47 +43 +40 +25 +2

a Mechanism for the shift is shown in parenthesis. b Values are for varying scan rates and with reversing the scan direction at various parts of the CV. c CV reversed between peaks Ia and Ib. d CV reversed at onset of peak Ia.

serotonin corresponding to an overall two-electron, two-proton oxidation as observed previously at higher buffer capacity. To explore whether this assumption is correct, and to demonstrate the proof-of-concept of the utility of using a molecular pH probe approach to this kind of mechanistic study, we next explored the oxidation of serotonin under identical conditions using the chemically modified eppg electrode with the arylhydroxylamine groups covalently attached to the electrode surface in well-buffered 25 mm and poorly buffered 1.0 mM phosphate media, at varying the scan rate from 50 to 500 mV s-1. Figure 3 shows the overlaid CVs (1st scans only to avoid any effects from the follow-up chemical products of serotonin oxidation fouling the electrode surface) of the modified electrode in the 1.0 mM phosphate buffer in the presence and absence of 1.0 mM serotonin. In either case, upon scanning initially in an oxidative direction, we observe no change in the oxidative peak potential of the arylnitroso/arylhydroxylamine couple (labeled III in Figure 3) as would be expected. However, after scanning to potentials just beyond the two oxidation waves of serotonin and subsequently reversing the scan direction, we observe that the reduction peak potential of the arylnitroso/arylhydroxylamine couple (labeled III′) has been shifted to more positive potentials compared to the situation where no serotonin is present in the solution. This indicates that the molecular pH probe, i.e., the reduction of the arylnitroso groups in this case, is sensing the reduction in the local pH adjacent to the electrode surface caused by the release of protons during the oxidation of the serotonin molecules. In the well-buffered media this shift was less pronounced, indicating that the buffer capacity of the solution adjacent to the electrode surface was sufficient to resist any measurable change in pH, and therefore, our attention now focuses exclusively on the poorly buffered case with the 1.0 mM buffer. Also note that the reduction of some redox active products of the serotonin oxidation follow-up chemistry gives rise to a reduction wave34 at slightly more negative potentials than that observed for the arylnitroso/arylhydroxylamine reduction wave, labeled IV in Figure 3. However, a comparison the voltammetry of serotonin at the bare eppg electrode allows us to identify, with some certainty, which of the reduction peaks corresponds to these products and which corresponds to the reduction of the arylnitroso groups. As peak IV occurs after peak III′ in the reductive scan, this will not perturb the local proton concentration experienced by the arylnitroso/arylhydroxylamine couple and can therefore be ignored. Table 1 details the experimentally determined shift in the potential of peak III′ and the corresponding change in the local pH at the electrode surface (estimated using the calibration plot in Figure 3 of the Supporting Information) at different points

a Mechanisms are considered for the initial oxidation of serotonin, together with buffering steps and optimal simulation parameters.

in the scan before and after oxidation of serotonin at the different scan rates studied. 3.2.1. Theory Wersus Experiment. In order to verify that the molecular pH probes are indeed sensing the change in the local pH upon oxidation of serotonin, we compared the experimentally determined change in local pH with that expected from Digisim simulations of the initial serotonin oxidation mechanism, including the effect of the concentration of the buffer component, using literature values of the diffusion coefficients of each species,34-37 including the diffusion coefficient of the proton, which can vary between 5 - 9.3 × 10-5 cm2 s-1 depending on ionic strength.38 However, using the values at either extreme of this range was found to have a negligibly small effect on the simulated local proton concentration, and so the typical literature value of 9.0 × 10-5 cm2s-1 was used.38 The pKa for the HPO4species and the kinetics of protonation of the PO4- ion were modeled using literature values.39 Simulations were performed for two cases: One case is where we assume the serotonin oxidation waves labeled Ia and Ib correspond to two separate one-electron, one-proton redox processes in an ECEC mechanism, i.e., where two protons are released upon oxidation of each serotonin molecule. The second is for the ECE case, where the removal of the second electron (peak Ib) does not involve coupled proton release, i.e., where the oxidation of serotonin involves the overall removal of twoelectrons but only one proton. The two mechanisms with optimized simulation parameters are given in Scheme 1. A more detailed discussion of the simulations and the optimization of constraining parameters are given in Figure 4 and text of the Supporting Information. In either case, we are only concerned with the initial oxidation mechanism of serotonin, and no attempt

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Henstridge et al. 4. Conclusions We have demonstrated proof-of-concept that by chemically attaching molecular pH probes onto the surface of a carbon electrode via a rapid and facile modification strategy one can measure changes in local pH during a redox reaction, i.e., within the diffusion layer immediately adjacent to the sites of electroactivity. This is an example of a single electrode generator/ collector system. The experimental application of this single generator/collector electrode system in combination with numerical simulations has enabled us to determine that the initial oxidation mechanism of serotonin in poorly buffered media close to pH 7 involves the transfer of two-electrons and only one proton in an ECE mechanism. No evidence was observed to suggest that the chemical modification of the electrode surface affected the oxidation of serotonin. The almost zero-gap spatial arrangement of this generator/ collector electrode system may allow its use in other pertinent mechanistic and/or kinetic studies, enabling one to determine information that was hitherto inaccessible using classical generator/collector electrochemical techniques. We note that the temporal resolution of this generator/collector electrode configuration is dependent on the choice of the molecular probe and difference in redox potentials between the sensing molecules on the electrode surface and molecule(s) of interest in the solution. However, this may be improved by the choice of molecular sensing probe attached to the electrode surface and also by simply varying the voltage scan rate. Either approach is likely more facile than the existing, classical approaches to improving the temporal resolution, which require complex micro- and nanofabrication processes to reduce the size of the generator/collector gap, fast-flow techniques (requiring specialist equipment), or both.

Figure 4. Simulated proton concentration profiles (ECE mechanism) adjacent to the electrode surface during the oxidation of 1.0 mM serotonin in a 1.0 mM phosphate buffer (pH 6.8) at a scan rate of 100 mV s-1 at the peak potential of (a) peak Ia and (b) peak III′.

to model any of the follow-up chemistry such as that detailed by Wrona et al.,34-37 has been made. The concentration profiles of all the species involved at the peak potential of the serotonin oxidation are given in Figure 5 of the Supporting Information, while panels a and b of Figures 4 show the proton concentration profile at the potential of peak Ib and at the experimentally observed peak potential for the reduction of the arylnitroso molecular pH probe, respectively. Values of the simulated surface proton concentration, for each scan rate studied, were then used to calculate the predicted local pH and associated expected shift in the arylnitroso reduction peak potential as detailed for the ECEC and ECE mechanisms in Table 1. It is immediately apparent from Table 1 that the ECEC mechanism is predicted by the simulation to increase the local proton concentration at the surface of the electrode by a significantly larger amount than is actually observed experimentally. In contrast, the ECE mechanism shows excellent agreement between theory and experiment. This would suggest that, contrary to what one might initially assume upon observing the voltammetry shown in Figure 2, the oxidation of serotonin in poorly buffered media around pH 7 does not involve two sequential one-electron, one-proton redox processes, but that the second oxidation wave involves the removal of an electron only, leaving the protonated oxidation product to undergo further homogeneous follow-up chemistry.

Acknowledgment. G.G.W. thanks St. John’s College, Oxford, for funding via a Junior Research Fellowship. Supporting Information Available: The procedure for the chemical modification of the eppg electrode with arylnitroso/ arylhydroxylamine groups and its subsequent voltammetric characterization and response to changes in solution pH as well as details of the simulation and optimization of parameters used in the modeling of the serotonin oxidation mechanism. This material is available free of charge via the Internet at http:// pubs.acs.org. References and Notes (1) Lund, H., Hammerich, O., Eds.; Organic Electrochemistry, 4th ed.; CRC Press: London, 2000. (2) Compton, R. G.; Banks, C. E. Understanding Voltammetry; World Scientific: Singapore, 2007. (3) Brett, C. M. A.; Brett, A. M. O. Electrochemistry: Principles, Methods, and Applications; Oxford University Press: Oxford, 1993. (4) Wang, J. Analytical Electrochemistry, 3rd ed.; Wiley VCH: Weinheim, 2006. (5) Albery, W. J.; Jones, C. C.; Mount, A. R. Compr. Chem. Kinet. 1989, 29, 129–48. (6) Brett, C. M. A.; Brett, A. M. C. F. O.; Compton, R. G.; Fisher, A. C.; Tyley, G. P. Electroanalysis 1991, 3, 631–6. (7) Compton, R. G.; Laing, M. E.; Mason, D.; Northing, R. J.; Unwin, P. R. Proc. Royal. Soc. London, A 1988, 418, 113–54. (8) Alden, J. A.; Compton, R. G. Anal. Chem. 2000, 72, 198A–203A. (9) Compton, R. G.; Fisher, A. C.; Tyley, G. P. J. Appl. Electrochem. 1991, 21, 295–300. (10) Cooper, J. A.; Compton, R. G. Electroanalysis 1998, 10, 1182– 1187. (11) Albery, W. J.; Calvo, E. J. J. Chem. Soc., Faraday Trans. 1983, 79, 2583–2596. (12) Albery, W. J.; Mount, A. R. J. Chem. Soc., Faraday Trans. 1989, 85, 1181–1188.

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