Investigation of catecholamines (dopamine: DA, norepinephrine: NE and epinephrine EP) has been of importance, because of the crucial roles that these molecules play in neurological function and related diseases. Due to the similar chemical structures and oxidation potentials of these compounds, it has been a challenge to use simple electrochemical techniques to differentiate them in mixtures.¹ Redox cycling is known to have the capability to differentiate between species based on the propensity of the compounds to diffuse to and produce an electrochemical response at the neighboring electrode.² The oxidized species generated at an electrode (generator) can diffuse to neighboring electrodes held at a reducing potential (collector) that will yield the original species. This process can occur multiple times and can produce an amplified signal. If the oxidized form of the species is electrochemically inactive, selectivity can be achieved for the electroactive molecules at the collector. Chemical reactions that can happen during the transfer between generator and collector can also convert the oxidized species to an electroinactive one and prevent a signal at the collector electrode, as well. This can be the basis of separating and detecting species using redox cycling.

In their ECC’ mechanism, catecholamines undergo a two-electron oxidation (E) to yield the o-quinone.³ The o-quinone form can then go through an internal cyclization reaction (C) to yield the leucoaminochrome. The leucoaminochrome and o-quinone forms can react with each other (C’) to yield the original catecholamine and the aminochrome form. The internal cyclization reaction that is involved in the C part of the mechanism occurs at different rates for the different catecholamines. The apparent rate constants at pH=7.4 in phosphate buffer have been reported to be 0.13 ± 0.05 s^-1, 0.98 ± 0.52 s^-1 and 87 ± 10 s^-1 for DA, NE and EP, respectively.⁴ Due to the difference in these rate constants, the species can be distinguished by spatial distribution based on the survival of the o-quinone from the following chemistry. Step C in the mechanism can also affect the number of times the species can go back and forth between the electrodes and therefore the amplification of the signal, depending on the species.

The purpose of our research is to study the fundamental electrochemical redox cycling behavior of DA, NE and EP.  By understanding this behavior, it can then be determined whether their simultaneous detection in more complex samples will be feasible. Recent developments from our laboratory will be discussed. We are using microfabricated chips with individually-addressable microelectrode arrays to activate various combinations of electrodes in the redox cycling experiments. By activating different electrodes on the array, greater distances between the collector and generator electrodes can be achieved leading to longer transit times for the molecules to move between electrodes. This study will lead to the generation of a calibration map for different gaps, ratios of catecholamines, and concentrations. There is a greater decrease in collector signal with increasing gap width (beyond the loss due to diffusion) for species with greater rate constants for step C. Epinephrine does not exhibit a reduction signal when the gap is set at 4-µm.² Norepinephrine’s signal decreases as a function of increasing gap more sharply than dopamine’s. There is no observed collector signal for NE at the collector electrode, which is activated 28-µm away from the collector. The oxidation signal of the mixtures of catecholamines decreases even further due to the following chemistry producing the leucoaminochrome and increased concentrations of o-quinone and leucoaminochrome molecules.

References

1. Hawley, M. D.; Tatawawadi, S. V.; Piekarski, S.; Adams, R. N. Journal of the American Chemical Society 1967, 89, 447-450.
2. Hu, Mengjia; Fritsch, Ingrid. Analytical Chemistry 2015, 87, 2029−2032.
3. Ciolkowski, E. L.; Maness, K. M.; Cahill, P. S.; Wightman, R. M.; Evans, D. H.; Fosset, B.; Amatore, C. Analytical Chemistry 1994, 66, 3611-3617.
4. Ciolkowski, E. L.; Cooper, B. R.; Jankowski, J. A.; Jorgenson, J. W.; Wightman, R. M. Journal of the American Chemical Society 1992, 114, 2815-2821.