In operando assessment of H₂O₂ production by a multicopper oxidase under direct electron transfer with a gold electrode

Abstract

A deeper understanding of the intricate redox processes at biotic/abiotic interfaces requires advanced analytical tools. We present the localized and real-time characterization of a multicopper oxidase adsorbed on a gold electrode using a dedicated microbiosensor, providing detailed and spatially resolved information. Enzymatic O₂ reduction is shown to proceed without detectable H₂O₂ accumulation. Instead, H₂O₂ originates from direct O₂ conversion at the gold surface.

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Multicopper oxidases (MCOs) are exceptional catalysts found in nature.^1–3 Coupling MCOs with electrodes enables mechanistic investigations and functional device development for relevant applications.^4–6 In particular, the use of enzymes from hyperthermophile organisms provides considerable advantages due to their robustness.⁷ The MCO from a hyperthermophilic archaeon Pyrobaculum aerophilum (McoP) shows outstanding stability across a wide pH range and high temperatures,⁸ with a half-life inactivation time of about 6 h at 80 °C.⁹ McoP-modified electrodes have confirmed remarkable long-term stabilities.^10,11 Effective direct electron transfer (DET) with McoP has been shown using carbon-based materials,⁸ particularly when integrating carbon nanotubes.^7,11–16 DET with Au electrodes has also been reported,^10,17 with the outstanding ability to sustain catalytic O₂ reduction activity even when immobilized on bare Au surfaces, which has been attributed to the compact and rigid structure of the hyperthermophilic enzyme.¹⁷ Other MCOs have shown DET on Au electrodes, as reported for ascorbate oxidase and laccase (LAC) when embedded into an anionic exchange resin film deposited over the electrode,^18,19 and LAC covalently attached to the electrode surface.²⁰ DET is also commonly observed using Au electrodes previously modified with thiol self-assembled monolayers, including examples for ascorbate oxidase,²¹ LAC and bilirubin oxidase (BOD),^22–28 and the copper efflux oxidase (CueO) from Escherichia coli.^29,30 Direct immobilization on flat bare Au electrodes is commonly associated with a fast inactivation caused by conformational changes,^31,32 leading to negligible bioelectrocatalytic activity for O₂ reduction, as reported for LAC,^28,32,33 or with activity sustained for only short periods, as in the case of BOD.^32,34 The use of Au nanostructures allowed active and relatively stable MCO-modified electrodes, as shown for BOD³⁵ and LAC^31,36,37 using Au nanoparticles, LAC-modified Au nanorods,³⁸ and nanoporous Au electrodes modified with BOD,^39–41 CueO,³⁹ and the MCO from Aquifex aeolicus (McoA).⁴² Remarkably, in addition to McoP, only hyperthermostable McoA has been shown to provide high and stable activity for O₂ reduction when immobilized on flat bare Au surfaces.⁴³

To date, several studies have shown that electrochemical O₂ reduction may lead to H₂O₂ as one of the reaction products when the enzyme is immobilized on Au surfaces.²⁸ However, H₂O₂ detection is commonly made at the end of an electrochemical experiment, taking a sample of the electrolyte solution and performing a colorimetric assay for H₂O₂ identification.^7,12,17,28 Advanced characterization methods are desired, allowing more reliable investigations. In contrast to post-hoc assays for H₂O₂ detection, we propose an enzymatic microbiosensor for localized H₂O₂ detection, allowing unprecedented in situ characterizations performed in real time, during the operation of the MCO-based bioelectrode under controlled conditions.

Polycrystalline Au electrodes modified with McoP from P. aerophilum have been selected, as it has been reported before that H₂O₂ is detected after bioelectrochemical O₂ reduction.^7,17 The Au electrodes were modified with McoP and characterized electrochemically at the optimal pH for bioelectrochemical conversion.⁴⁴ Voltammetric measurements confirmed the possibility of performing O₂ reduction with the enzyme under DET with the electrode surface (Fig. 1).

Fig. 1 Average voltammetric response for McoP on Au electrodes, the standard deviation between measurements is represented by the shaded region (n = 4). Responses for electrodes without enzyme are also shown (n = 2). Air-equilibrated buffer, pH 5.0. Scan rate: 10 mV s⁻¹.

When a clean electrode in the absence of enzyme was characterized, only minor catalytic responses for O₂ reduction were obtained in the same potential range (Fig. S1A), confirming that the enzyme catalyzed the reduction of O₂. Extending the range to more negative applied potentials revealed a significant O₂ reduction current, indicating that the Au electrode can effectively reduce O₂ at sufficiently negative applied potentials (Fig. S1B). This observation triggered the question of a possible contribution of H₂O₂ production when O₂ reduction is catalyzed directly by the Au electrode. O₂ reduction at polycrystalline Au surfaces in neutral aqueous media involves a two-electron process, causing H₂O₂ production.^45,46 In order to identify the contribution of the enzyme and the Au electrode for the production of H₂O₂, we developed a microbiosensor for H₂O₂ detection that could be used for the assessment of enzyme-modified electrodes under operando conditions, ensuring highly sensitive and selective measurements in contrast to conventionally used Pt microelectrodes. The microbiosensor consisted of a carbon paste microelectrode, which was modified with HRP embedded into poly(1-vinylimidazole-co-allylamine)-[Os(2,2′-bipyridine)₂Cl]Cl. In this way, the Os-complex modified redox polymer serves as both an immobilization matrix for the entrapment of HRP and a redox mediator for efficient electrochemical communication with the enzyme (Fig. 2).

Fig. 2 Schematic representation of the microbiosensor used as an electrochemical probe for the localized detection of H₂O₂ (HRP structure based on the PDB entry 1H5E⁴⁷). H₂O₂ is detected according to the electroenzymatic reduction pathway summarized on the right.

The microbiosensor was characterized electrochemically in the absence and presence of H₂O₂ (Fig. S2A). Cyclic voltammograms recorded in buffer solution showed the characteristic reversible response for the Os³⁺/Os²⁺ interconversion at the P–Os redox polymer, with a midpoint potential of about 420 mV vs. SHE. After the addition of H₂O₂, a well-defined cathodic catalytic wave was observed, confirming the enzymatic conversion of H₂O₂ and the associated transfer of electrons from the microelectrode to the enzyme, mediated by the P–Os redox polymer. Additional characterizations were performed using the microbiosensor at a constant applied potential and the current response was monitored with increasing H₂O₂ concentrations (Fig. S2B). The microbioelectrode allowed H₂O₂ detection in a wide concentration range, with a limit of detection of 0.4 µM, and a sensitivity of 3.03 ± 0.04 nA µM⁻¹ within a linear range of up to 200 µM H₂O₂ (see Fig. S2C). In comparison, a Pt microelectrode exhibits a sensitivity of 1.31 ± 0.02 nA µM⁻¹ (Fig. S2D), and requires a highly positive applied potential to follow H₂O₂ oxidation.

The microbiosensor was used as an electrochemical probe in a scanning electrochemical microscope (SECM). In this way, H₂O₂ production could be investigated in situ and under the operation of the McoP-modified electrode. The HRP-based microbiosensor was positioned over the investigated sample under a stationary configuration and at the applied potential required for H₂O₂ detection (i.e., 200 mV vs. SHE). The sample investigated consisted of a gold electrode modified with McoP. As summarized in Fig. 3, a chronoamperometric experiment was performed, applying different potentials to the investigated sample (sample potential). The current for the McoP-modified electrode (sample current) was recorded alongside the current obtained with the microbiosensor (tip current). The experiment started at 800 mV vs. SHE, where no catalytic reaction was observed (see Fig. 1 and Fig. S1), subsequently going gradually to more negative applied potentials where O₂ reduction can take place and finally coming back to the initially applied potentials. At the initial applied potentials (800 mV to 600 mV vs. SHE), no noticeable current was recorded for the sample, in agreement with the results obtained in the voltammetric characterization of McoP-modified electrodes (Fig. 1). At more negative applied potentials, increasing cathodic current responses were obtained with the investigated sample, which could be attributed to O₂ catalytic reduction. As the applied potential was later shifted to more positive values, the magnitude of the catalytic current decreased until it reached a negligible response, like that at the beginning of the experiment. When looking at the HRP microbiosensor current, only a background response was observed when the sample was polarized at applied potentials between 800 mV and 300 mV vs. SHE. Interestingly, significant sample currents were recorded at applied potentials between 500 mV and 300 mV vs. SHE, where the enzyme catalyzes O₂ reduction, in agreement with previous voltammetric experiments (see Fig. 1). In the same potential range, no response different from the background was observed for the microbiosensor, suggesting that, at least in this range, enzyme-catalyzed O₂ reduction proceeds without H₂O₂ production. At applied potentials of 200 mV vs. SHE and below, a considerable increase in the cathodic current was observed with the microbiosensor, indicating the collection of H₂O₂. The highest production rates were observed at more negative applied potentials, i.e., 100 mV and 0.0 mV vs. SHE, while H₂O₂ production ceased when more positive potentials were subsequently applied. These results correlate with the responses observed at an unmodified Au electrode (Fig. S1), showing notable activity for O₂ reduction at potentials equal to or below 200 mV vs. SHE and suggesting a prominent contribution of the Au substrate in the production of H₂O₂. Note that the response obtained with the microbiosensor corresponds to the local detection of H₂O₂ produced by the investigated sample. Under conditions favoring high production rates, the tip currents should be monitored preventing them from reaching values beyond the linear current response as a function of the H₂O₂ concentration (Fig. S2C), to avoid an incorrect interpretation of the results caused by enzyme saturation or inhibition.

Fig. 3 Analysis of the McoP-modified Au electrode (sample) using a H₂O₂ microbiosensor as a SECM probe. The sample was investigated at different applied potentials as indicated in the graph (sample potential), while simultaneously recording the current at the modified Au electrode (sample current) and the response of the microbiosensor (tip current). Air-equilibrated buffer. Tip-to-sample distance: 20 µm. During the analysis, the microbiosensor was polarized at 200 mV vs. SHE. The gray bars are a guide to the eye only, to help correlate the current responses with some of the applied potentials.

To assess whether the production of H₂O₂ originates from the enzyme-catalyzed reaction or from the Au electrode, a similar experiment as before was performed, this time with an unmodified Au electrode as the sample (Fig. 4). No noticeable sample current was observed until an applied potential of 200 mV vs. SHE was reached. H₂O₂ was detected with the SECM tip only after this potential was applied. At more negative applied potentials, increasing sample currents were recorded together with higher cathodic tip currents, indicating an increase in H₂O₂ production. When the sample potential was later shifted to more positive values, both the sample current and the microbiosensor response decreased. The obtained results indicated that H₂O₂ production is associated with O₂ reduction at the polycrystalline Au electrode. The lower cathodic sample currents obtained with the unmodified electrode (Fig. 4) in comparison with the McoP-modified electrode (Fig. 3) confirmed a mixed contribution to O₂ conversion with the latter at more negative applied potentials. Moreover, the higher intensity of the microbiosensor response for H₂O₂ detection with the bare Au electrode sample, in contrast to the McoP-modified electrode, could be explained by competition for O₂ conversion between the Au surface and the enzyme, effectively decreasing the amount of O₂ that can be converted directly at the electrode. In addition, the presence of the immobilized protein effectively blocks a part of the electrode surface, making it inaccessible for electrochemical O₂ reduction, leading to a decreased H₂O₂ production.

Fig. 4 Analysis of a bare Au electrode (sample) under the same conditions as indicated in the caption of Fig. 3.

An additional control measurement was conducted to confirm the previous observations, this time performing the analysis with an unmodified glassy carbon electrode as the sample (Fig. S3). In this case, negligible sample currents were observed for most of the applied potentials, with only small sample currents detected at 100 mV and 0.0 mV vs. SHE. This was expected, as the carbon surface has considerably lower catalytic activity for O₂ reduction in comparison with Au electrodes. As described before, sluggish kinetics for O₂ reduction are observed at glassy carbon electrodes, proceeding at high overpotentials. The reaction leads to a superoxide intermediate, which disproportionates to oxygen and peroxide, a process that is thermodynamically favorable.⁴⁸ Accordingly, H₂O₂ was detectable only at the two most negative applied potentials, verifying previous observations. The lower sample current and H₂O₂ collection responses for the carbon-based material in comparison with the Au electrode further confirmed that direct O₂ reduction at Au surfaces proceeds at substantial rates for H₂O₂ production at sufficiently negative applied potentials.

The use of the implemented microbiosensor stands as a more accurate analytical strategy, enabling in situ and real-time measurements, in contrast to previous investigations. The obtained results strongly suggest that H₂O₂ production at McoP-modified Au electrodes originates at the electrode substrate, without being produced by the enzyme during electrocatalytic O₂ reduction. This strategy opens new avenues for detailed and specific characterizations, which are envisioned to extend to other enzymes immobilized over different electrode surfaces.

This work was supported with funding from the Bundesministeriums für Forschung, Technologie und Raumfahrt (BMFTR), project CyFun [03SF0652A]; and FCT - Fundação para a Ciência e a Tecnologia, I.P., through MOSTMICRO-ITQB R&D Unit [UIDB/04612/2020, UIDP/04612/2020], and LS4FUTURE Associated Laboratory [LA/P/0087/2020]. F. C. acknowledges FCT for the researcher contract [2022.05842.CEECIND/CP1725/CT0001].

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d5cc03482e.

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