Elsevier

Electrochimica Acta

Volume 147, 20 November 2014, Pages 401-407
Electrochimica Acta

Pyrolyzed Photoresist Carbon Electrodes in Aprotic Solvent: Bilirubin Electrochemistry and Interaction with Electrogenerated Superoxide

https://doi.org/10.1016/j.electacta.2014.09.046Get rights and content

Highlights

  • Pyrrolized photoresist carbon electrodes (PPCEs) are characterized and applied in dimethylsulfoxide (DMSO).

  • PPCEs present a wider accessible potential window and a smaller capacitance with respect to glassy carbon electrodes.

  • The mechanism of the electrochemical oxidation and reduction of bilirubin is studied in detail.

  • Bilirubin is an efficient scavenger for the superoxide radical electrogenerated in DMSO.

Abstract

Pyrolyzed photoresist carbon electrodes (PPCEs) are fabricated by the photopatterning of a negative tone epoxy-based photoresist, SU-8, through optimized standard UV photolithography. The electrochemical characteristics of PPCEs are investigated in dimethyl sulfoxide (DMSO), observing a wider accessible potential window and a smaller capacitance with respect to glassy carbon electrodes. PPCEs are used to study the cyclic voltammetric behavior of bilirubin (BR) in DMSO. Detailed information is obtained on the multiple steps involved both in the electrochemical oxidation and reduction of BR. Interesting points concerning the electrochemical oxidation of BR to biliverdin are clarified, identifying the formation of an intermediate whose fate depends on the time scale of the electrochemical experiment.

PPCEs are also used to electrogenerate the superoxide anion O2−• in DMSO for studying possible reactions between BR and O2−•. The results obtained demonstrate that BR is an efficient superoxide scavenger and that a concentration 2 mM of BR is high enough to consume all the O2−• generated by oxygen reduction at the PPCE/DMSO interface.

Introduction

Carbon electrodes possess many advantages including low fabrication cost, wide accessible potential window, chemical and electrochemical stability [1] so that, in many cases, these electrodes are superior with respect to noble metal electrodes [2]. Among common carbon electrode materials, such as pyrolytic graphite, carbon fibers or carbon paste [3], [4], [5], [6], glassy carbon (GC) is the most widely used [7], [8]. Glassy carbon is impermeable to gases and liquids, has a very small porosity and can be polished to a mirror-like finish an almost infinitive number of times. Moreover, it is very resistant both to high oxidizing and reducing potentials thereby allowing one to perform electrochemistry in a 4–5 V wide potential window [9], when suitable electrolytes are used.

Recently, a new procedure to fabricate carbon electrodes based on the controlled pyrolysis of polymeric photoresist has been introduced, obtaining so-called pyrolyzed photoresist carbon electrodes (PPCEs) [10]. This is a very promising approach since, by using simple UV photo-lithography it is possible to pattern high-performance carbon electrodes with complex geometries, such as interdigitated arrays of microelectrodes [11], [12]. The optimization of the pyrolysis conditions allows one to obtain PPCEs in which the carbon is mostly amorphous GC [13]. Studies performed in aqueous media demonstrated that the electrochemical behavior of PPCEs compares with that of classical electrodes prepared from bulk glassy carbon rods, with the advantage of being cheaper and with the possibility to customize their design [13]. In this paper we report, for the first time, the application of PPCEs for voltammetric measurements in an aprotic solvent, namely dimethyl sulfoxide (DMSO), used here as medium to dissolve bilirubin (BR) and to study its electrochemical behavior together with its reactivity towards electrogenerated superoxide radical anions.

Bilirubin (BR) is the yellow-orange bile pigment found in blood, mostly bound to the plasma protein albumin. It is a linear tetrapyrrole (see scheme 1), insoluble in water at neutral pH, but very soluble in organic solvents. BR is an important serum biomarker used in clinical medicine for assessing hemolysis, hepatic function and cardiovascular risk [14]. This molecule originates from the degradation of the heme moiety in hemoglobin, other hemoproteins, such as cytochromes, catalase, peroxidase and tryptophan pyrrolase, and free heme [15]. In human body BR is present mainly as conjugated and unconjugated BR; the first forms a complex with gluconic acid, which makes it water soluble. Because of the important role played by this redox compound in several physiological processes and diseases [16], BR has been the subject of several previous electrochemical studies performed both in aqueous [17], [18], [19], [20], [21] and non aqueous media [22], [23], [24], [25], [26], [27], [28], [29]. The results of these studies indicate that the electrochemical oxidation of BR is a multistep process in which, depending on the applied potential, BR is electrochemically oxidized to biliverdin (BV), purpurin or choletelin (see Scheme 1) [22].

Because of the poor water solubility of unconjugated BR, the use of non aqueous media is to be preferred for gathering fundamental information on its electrochemical behavior. This notwithstanding, even for measurements performed in aprotic media like DMF [22], [23], [24], [25], [26], [27] and DMSO [28], [29], in the literature there is no agreement on the oxidation mechanisms or even on the number of electrons involved in the oxidation steps, since different authors report different results. For instance, concerning the first oxidation process, coulometric data support a two-electrons oxidation [28], [29]. However, voltammetric data gathered at the corresponding oxidation peak do not agree with a two-electron process; for instance, |Ep - Ep/2| values are significantly larger than the 28.5 mV value expected for a reversible two-electron oxidation [30]. Moreover, Ribo et al. [26] reported voltammograms in which the first oxidation peak of bilirubin presented the same peak current as the one-electron oxidation of similar molecules. Similarly, for the BR reduction, it is not clear which peaks are ascribed to the direct reduction of BR and which are related to the electroactivity of BV electrogenerated in the anodic portion of the cyclic voltammetric experiments. These controversial results prompted us to undertake the present study with the goal of studying the electrochemical performance of PPCEs in DMSO, and then revisiting the electrochemical behavior of BR in this aprotic solvent. The findings enabled us to use the PPCE for the electrochemical generation of O2−• (by reduction of dissolved oxygen) and to study the reactivity of BR towards this radical as a proof of the radical scavenging capabilities of BR.

Section snippets

Electrochemical apparatus

All voltammetric measurements were carried out at room temperature (22 ± 1 °C) with a CHI1222A potentiostat controlled via a personal computer with its own software. A three-electrode single-compartment cell made of dark glass and equipped with a PPCE (geometric area = 0.031 cm2) or a glassy carbon working electrode (GCE; geometric area = 0.071 cm2), a platinum counter electrode and a platinum pseudo - reference electrode (Pt-pseudo). All potential values are finally referred to the E1/2 of the

Characterization of PPCE in DMSO

Fig. 1 reports the blank CVs recorded in 0.1 M supporting electrolyte (TBABF4) at 100 mV s−1 with a PPCE (full line) and a conventional GCE (dashed line) in the absence of dissolved oxygen. The current values in these voltammograms are normalized with respect to the geometrical area of the electrodes and reported as current densities (J). The comparison shows a similar cathodic limit for the accessible potential window on both electrode materials. On the anodic side, a significantly higher

Conclusions

The results obtained in this study indicate that PPCEs can be successfully employed for performing reliable voltammetric measurements in aprotic solvents. The comparison of the electrochemical behaviour of PPCE vs. GCE puts is evidence some significant differences and advantages for the former. First of all, PPCEs present a very small double-layer charging current with a capacitance which is less than half that of GCEs. This is mainly a consequence of the very smooth surface of the PPCE, as

Acknowledgments

This work was supported financially by the Cross-Border Cooperation Italy-Slovenia Program 2007-2013 - Strategic Project TRANS2CARE and, partially, by MIUR (Rome), project PRIN 2010AXENJ8, and Veneto Region. We thank Ermelinda Fioriniello for preliminary measurements with GCE and Dr. Veronica Vascotto for the AFM measurements.

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      In the case of hemin, the FeIII-heme-FeII-heme couple (CHE/AHE) appears as a reversible voltammetric signature at a midpeak potential of −0.24 V accompanied by the corresponding FeII-heme-FeI-heme couple at −1.26 V and an apparently irreversible oxidation process at +1.15 V, attributable to the oxidation of the porphyrin motif [12]. The electrochemistry of bilirubin is more complicated consisting, in the region of positive potentials, of an oxidation to a radical cation followed by dimerization (ABR) and a two-electron, two-proton subsequent oxidation (ARR), as recently described by Ugo et al. [59]. In DMSO, the oxidation of bilirubin occurs at a potential more positive than the oxidation of hemin so that, as indicated by repetitive voltammetry (see Fig. 7a and b), there is no generation of interfering voltammetric signals during electrochemical turnovers.

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