Electroreduction of Hydrogen peroxide using Direct electrocatalysis of Cytochrome c on the a graphene-modified electrode

Introduction

The direct electron transfer (DET) of bio-molecules such as Cytochrome c (Cyt c) have been comprehensively studied for investigating their potential application in biofuels, enzyme mechanism and so on [1]. The water-soluble multi-functional hem-containing Cyt c at inner mitochondrial membrane [2] plays a significant role in the respiratory chain, acting as an interface for receiving electrons from the Cyt c reductase and transferring them to the Cyt c oxidase.

Cyt c with a spherical shape of 34°A in diameter (12 kDa) has a net positive charge of +5 to +6 at neutral pH (pI=10) [3]. Electrochemical studies have shown that proteins on a conventional bare electrode lead to substantial changes in their structure and function. Based on previous studies, DET between Cyt c and surface electrodes such as glassy carbon, Au, Hg, Ag and Pt is not possible. Most of the proteins are huge molecules and their redox center is embedded deeply in polypeptide chain structures, which hinder their direct electrochemical investigation. Furthermore, the proteins are being denatured on a bare electrode and poison the electrode surface [4]. Thus, modifying an electrode for preparing a biocompatible surface with a fast electron-transfer kinetic which prevents the denaturation of the protein is highly desirable [5, 6]. For this purpose, many innovative strategies have been proposed for modifying electrodes including self-assembled monolayers [7, 8], single-walled carbon nano tubes (SWNT) [9], multi-walled carbon nanotubes (MWNT) [10], nanomaterials [11, 12], and biomaterials modified electrodes [13]. These modifiers act as “tiny conducting wires” and increase the electron transfer rates between the protein redox center and the electrode.

Typically, graphene possesses a high surface area for immobilizing proteins [14-16], higher electron conductivity, and high mobility for transferring electrons [17], and strong irreversible adsorption [18].

Regarding the vital roles of hydrogen peroxide (H₂O₂) in pharmaceutics, food industry and other industries, and as a by-product of enzymatic reactions, its detection is a challenge [19].

In this study, a negative surface of the nafion/reduced graphene oxide (rGO)-modified glassy carbon electrode (GCE) acts as a suitable substrate for the immobilization of a positively charged protein. Cyt c shows a fast electron transfer kinetic on the graphene-modified electrode, stemming from electron transfer the mediating/accelerating effect of graphene. The immobilized Cyt c retains its electrocatalytic activity while immobilized on the electrode surface. Thus, an electrochemical hydrogen peroxide biosensor is constructed in which Cyt c catalyzes its reduction to water.

Experimental

Reagents

Extra pure graphite powder (particle size ≤50 μm) and equine heart Cytochrome c were purchased from Merck. The nafion solution in alcohol (0.5 wt.%) was purchased from Fluka. The experiments were conducted in the phosphate buffer solution (PBS, 0.1 M, pH 7.0).

Apparatus

The electrochemical signals were recorded using a µAutolabe type III potentiostat/galvanostat, controlled via aGPES4.9 software. A conventional electrochemical system with a working glassy carbon electrode, reference Ag|AgCl| 3 M KCl and counter electrode of Pt were used for recording the signals. In order to remove the O₂ from the solution, deaeration was applied using ultra-pureN₂ gas for 10 min.

Procedure

Synthesis of rGO

Modified Hummer’s method graphene oxide (GO) was prepared according to previous studies [20, 21]. To prepare rGO, 40 µL N₂H₄ was injected to stirred 1 mL GO (4 mg mL¹) and, subsequently, reacted at 80 °C for 24h. The color changed from brown to black, demonstrating the reduction of the oxygen group in GO.

Preparation of modified GC electrode

The bare glassy carbon electrode was cleaned with alumina slurries and water, then sonicated in ethanol, and finally exposed to 10µl of the dispersed rGO solution (1.0 mg/ml) with 100 µl of the nafion solution (0.08%) (Fig. 1). After that, the Cyt c-nafion-rGO/GC electrode was prepared by drop-casting the 1.0 mg/mL Cyt c solution (prepared PBS) for 24h at 4°C. In the same manner, a Cyt c-nafion/GC electrode was also prepared without using the rGO.

Results and discussion

DET of Cyt c on nafion/rGO GCE

To confirm the immobilization of Cyt c, the modification of the electrode was assessed using cyclic voltammograms (CVs). Fig. 2 shows CVs of the electrodes in PBS at a scan rate of 10 mV/s. Fig. 2a demonstrates the CV of a bare GC electrode, and Fig. 2b displays the CV of a nafion-rGO/GC electrode. As illustrated, no signal from both the Cyt c/GCE and nafion-rGO/GCE appeared. Notably, the nafion-rGO/GC electrode shows a high capacitive current resulting from the deposition of the graphene sheets. As shown in Fig. 2c, no characteristic redox peaks of Cyt c on a nafion/GC electrode was found, indicating that nafion alone either cannot adsorb Cyt c or fails to facilitate electron transfer to Cyt c due to its insulating properties (Fig. 2b). Interestingly, the defined redox peak currents on the nafion-rGO/Cyt c/GCE are attributed to wiring of graphene to the electroactive center of the immobilized Cyt c (Fig. 2d). In addition, it is revealed that graphene has a significant role in DET between Cyt c and the GCE. The Cyt c molecules are positively charged at pH 7.0 PBS. Therefore, hydrophobic and electrostatic interactions can contribute in immobilizing Cyt c on a graphene-modified electrode [22, 23].

The high stability of the nafion-rGO/Cyt c/GCE, with no significant redox peaks of Cyt c (30 cycles), demonstrated strong interactions between Cyt c and the graphene sheets. The immobilized protein shows an E_1/2 = -256 mV and peaks separation (∆E_p) of 86 mV at a scan rate of 20 mV s^-1 in PBS (pH 7.0), indicating a rapid and quasi-reversible electron-transfer process. This phenomenon is attributed to the heterogeneous dispersion of Cyt c on the electrode due to various kinetic/thermodynamic balances on uneven nafion-rGO/GCE [24]. Further, the scan rate effect was studied on the redox currents of Cyt c. The Linear increase in redox peak currents (cathodic and anodic) (Fig. 3) with the increment scan rate with correlation coefficients of 0.996 and 0.994, respectively, reveals an adsorption-controlled electrochemical process.

Based on the the Laviron’s equation [25]:

Where, n is the number of electrons transferred; I_p is the cathodic peak current; F is the Faraday constant; A is the electrode surface area; ν is the scan rate; Г is the surface coverage, and T is the temperature (K); R is the gas constant. The surface coverage of Cyt c on nafion-rGO/GC electrode was estimated as 1.8 pmol/cm^-2, close to values obtained for a monolayer coverage of Cyt c on an electrode surface [26]. Using the Laviron’s equation [25], the obtained heterogeneous electron transfer of K_s=1.3±0.09 s⁻¹ suggests a high kinetic electron transfer.

Electroreduction of H₂O₂

Based on previous studies, heme-proteins such as hemin, horseradish peroxidase, myoglobin, hemoglobin, could be used as a biocatalyst for the electroreduction of H₂O₂ [27]. For this reason, the electroactivity of Cyt c, immobilized on to the graphene-modified electrode, towards the reduction of H₂O₂ was studied.

The redox peak of nafion-rGO/Cyt c/GCE in the absence of H₂O₂ is observed (Fig. 4a). Upon the addition of 1.0 mM H₂O₂ to the PBS (Fig. 4b), the voltammetric signal of the relative electrode changed significantly. In the presence of H₂O₂, the reduction peak of Cyt c increased considerably while its oxidation peak decreased.

 The change in redox reaction of H₂O₂ is attributed to the electroactivity of Cyt c towards H₂O₂ (Scheme 1). Many research groups have investigated the mechanism of such an electrocatalytic reaction [28, 29], and the electrocatalytic process can be expressed as follow:         

Based on this mechanism, Fe³⁺-Cyt c is electrochemically reduced to Fe²⁺-Cyt c on the electrode surface (Eq. 1). H₂O₂ diffuses to the electrode surface where it can take an electron from electrochemically reduced Cyt c (Fe²⁺-Cyt c) (Eq. 2). In this process, Fe²⁺-Cyt c is oxidized back to its original oxidation state of Fe³⁺-Cyt c. The overall redox reaction taken place on the electrode surface is indicated in Eq. 3. The bimolecular electron transfer between H₂O₂ and Fe²⁺-Cyt c is rather a rapid process. Thus, in the presence of H₂O₂ the surface concentration of Fe³⁺-Cyt c increases while that of Fe²⁺-Cyt c decreases. This phenomenon results in increasing the reduction peak current of Fe³⁺-Cyt c with increasing H₂O₂ concentration (Fig. 5).

As shown in Fig. 5A and B, the CVs of Cyt c on a nafion-rGO/GCE in different concentrations of H₂O₂ in PBS (0.1M, pH 7) increased linearly in the range of 2.0 µM to 1.0 mM. As indicated above, the reduction peak current of Cyt c increases with the increase in the H₂O₂ concentration, thus providing a means for constructing an H₂O₂ sensor. The detection limit of the proposed sensor towards H₂O₂ was 0.4 µM at a signal-to-noise ratio of 3.

To investigate the selectivity of the proposed electrode, not only the 10-fold concentration of Cl^-, CO₃^2-, K⁺, Ca²⁺, Na⁺, and SO₄^2- does not interfere in the determination of H₂O₂, but also, no significant effect was observed in the presence of dopamine, glucose, and ascorbic acid.

Conclusion

In this study, successfully oriented immobilization of Cyt c as well as preserving its electroactivity on a nafion/rGO-modified electrode was reported. The electron-transfer rate constant of Cyt c was calculated, confirming a high kinetic electron transfer of Cyt c on the nafion/rGO-modified electrode. The high kinetic electron transfer of Cyt c on the modified electrode results from the excellent electron-transfer mediating effects of graphene. Cyt c showed an outstanding electrocatalytic activity towards the reduction of hydrogen peroxide. Thus, a hydrogen peroxide biosensor with an extended dynamic range and acceptable detection limit was constructed.

Acknowledgment

The authors express their gratitude to Shiraz University Research Council for the support of this work.

CONFLICT OF INTEREST

The authors declare no conflict of interest.