An enzymatic biosensor for hydrogen peroxide based on one-pot preparation of CeO₂-reduced graphene oxide nanocomposite

Abstract

The study describes cerium oxide-reduced graphene oxide (CeO₂-rGO) prepared by a facile one-pot hydrothermal approach and its assembly with horseradish peroxidase (HRP) for the detection of hydrogen peroxide (H₂O₂) at trace levels. The prepared nanocomposite was characterized by Fourier transform infrared spectroscopy, field-emission scanning electron microscopy, X-ray diffraction, and voltammetry. Furthermore, the direct electrochemistry of HRP/CeO₂-rGO composite has been studied. The immobilized enzyme retained its bioactivity and exhibited a pair of well-defined redox peaks, confirming the direct electron transfer (DET) of HRP with CeO₂-rGO composite modified electrode. A significant enzyme loading (4.270 × 10⁻¹⁰ mol cm⁻²) has been obtained on CeO₂-rGO composite as compared to the bare glassy carbon (GC), CeO₂, and rGO modified surfaces. This HRP/CeO₂-rGO film has been used for the sensitive detection of hydrogen peroxide by voltammetry and it exhibited a wide linear range of H₂O₂ from 0.1 to 500 μM with a detection limit of 0.021 μM. The apparent Michaelis–Menten constant (K^app_M) of HRP on the CeO₂-rGO composite was estimated as 0.011 mM. The combination of the direct electron transfer character of HRP and the promising feature of CeO₂-rGO composite favors the sensitive determination of H₂O₂ with improved sensitivity.

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1 Introduction

The direct electrochemistry of redox enzymes/proteins has been investigated with much interest for the development of a reagentless electrochemical biosensor.¹ These biosensors could avoid expensive redox mediators or electron shuttle molecules.² In addition, the direct electrochemistry of redox enzymes/proteins is well suited for electron transfer mechanistic studies in biological systems and greatly simplifies analytical device fabrication.^3,4 Reagentless hydrogen peroxide biosensors based on the direct electrochemistry of HRP play a leading role in monitoring a wide range of enzymatic reactions and also play an important role in biological processes, such as the metabolism of proteins, carbohydrates or in immune response, food industry, pharmaceutical and environmental protection. However, the heme catalytic center of HRP is deeply hindered within the insulated protein, which renders them inaccessible for the direct electron transfer (DET) with transducers. The use of a selected matrix that can promote the DET behavior of HRP and retention of the enzyme activity^5–8 can overcome these disadvantages.

Nano-sized materials have been identified as suitable immobilization matrixes for enzyme and protein bindings due to their high active surface areas and they also provide standard structures, as well as greater mechanical, thermal and chemical stability.⁹ A single-atom thick graphene with two dimensional sheet of sp² bonded carbon has been extensively used in electrochemistry due to the sheet like morphology with large surface area, small residual current, wide potential window, excellent chemical stability in various electrolytes and easily renewable surface. Furthermore, graphene has the most prospective application in synthesizing nanocomposites and application for biosensors.¹⁰

Recent research interest has focused on the preparation of nanocomposites, involving the combination of graphene and metal oxides. The anchoring and growing of metal oxide in the graphene sheets can improve mechanical and thermal properties as well as improve the electrical conductivity of graphene.^11,12 Among the various metal oxides, CeO₂ has been reported to exhibit important and interesting properties, such as electrical conductivity, chemical inertness, negligible swelling and good biocompatibility that can be used in electrochemistry, optics, catalyst and gas sensors.¹³ In addition, it is known to have a wide band gap (3.4 eV) with high isoelectric point (IEP, 9.0) and also excellent retention of biological activity for enzyme binding.^14,15 The CeO₂-graphene composite could combine the electrical characteristics of graphene and the catalytic properties of metal oxide. Many of these composites show enhanced electrocatalytic activity and catalytic applications as compared to those of pure CeO₂ or graphene.¹⁶ The strong electronic interaction between CeO₂ and graphene matrix could improve the electron transfer rate of enzymes. This is particularly important for studying the DET of enzymes.

Several methods have been applied for the preparation of metal oxide/graphene composite: microwave irradiation, pyrolysis, chemical reduction, thermal annealing and sonochemical.^17–21 Furthermore, these methods involve two steps, which involve the reduction of graphene sheets by either thermal annealing or the addition of toxic hydrazine. Hence, these methods evidently involve complicated experimental protocol with high energy consumption and expensive instrumentation techniques. Therefore, a facile materials preparation method and electrode modification protocol with improved DET of the enzyme is urgently required. Currently, several reports are available for H₂O₂ biosensors based on enzymatic reaction. However, they have generally used costly nanomaterials, such as MWCNT, Au and Ag and involve complex multiple steps for enzyme immobilization.^22–24 The main advantages of the newly fabricated H₂O₂ biosensor is that the CeO₂-rGO composite preparation (eco-friendly hydrothermal approach) and electrode modification (simple drop casting) procedure adopted in the present study are very simple as compared to previous reports.

In this study, we prepared CeO₂-rGO composite by a one-step hydrothermal approach. The CeO₂-rGO composite modified electrode provides a well-defined micro-environment for HRP immobilization and enhances the DET between enzyme and electrodes. Interestingly, the electron transfer ability of CeO₂-rGO composite was found to be higher than that of pure CeO₂ and rGO as revealed by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry measurements. Furthermore, HRP immobilized with CeO₂-rGO facilitates the good communication between electrode and HRP, and it accommodates a higher concentration of HRP on the electrode surface compared to that of CeO₂, rGO and bare GC electrode surfaces. These results demonstrate the application of the fabricated CeO₂-rGO based biosensors for electrochemical detection of H₂O₂. To the best of our knowledge, this is the first report on the preparation, direct horseradish peroxidase electrochemistry and electrocatalysis of HRP/CeO₂-rGO composite. This CeO₂-rGO composite material may provide new insight into the preparation of other enzymatic biosensors.

2 Experimental

2.1 Materials

Horseradish peroxidase (HRP) (EC 1.11.1.7, type V1-A, 250–330 units mg⁻¹, lyophilized powder from Armoracia rusticana) and graphite powder were procured from Sigma. Ce(NO₃)₃·6H₂O and H₂O₂ were purchased from Daejung Chemicals Ltd, South Korea. The phosphate buffer solutions with different pH values were prepared using Na₂HPO₄ and NaH₂PO₄. All the reagents were of analytical grade and used without further purification.

2.2 Instrumentation

The surface morphologies of the composite were characterized using a field emission-scanning electron microscope (FE-SEM) JEOL JSM-6700F. The phase purity and crystalline nature of the prepared materials were studied using the powder X-ray diffraction technique (XRD, Rigaku, Cu Kα radiation operating at 40 keV/40 mA). FT-IR spectra were recorded using a nicolet-6700 spectrometer from Thermo Scientific. Electrochemical measurements were performed in a conventional two compartment three electrode cell with a mirror polished 3 mm glassy carbon (GC) as the working electrode, Pt wire as the counter electrode and an Ag/AgCl (3 M KCl) as the reference electrode. The electrochemical measurements were carried out with an AUTOLAB (Model PGSTAT302N, Netherlands). Cyclic voltammograms were recorded between a potential window of −0.2 V and 0.6 V at a scan rate of 50 mV s⁻¹ in 0.1 M KCl solution containing 1.0 mM [Fe(CN)₆]^3−/4− redox couple. The electrochemical impedance spectroscopy (EIS) measurements were carried out by applying an ac potential of amplitude 10 mV over the dc potential of 250 mV in the frequency range of 100 kHz to 1 Hz. The impedance data are presented in the form of Nyquist plots. The value of the charge transfer resistance (R_ct) was determined using Zsimpwin software simulations.

2.3 Preparation of CeO₂-rGO composite

Graphene oxide (GO) was synthesized from natural graphite powders by a modified Hummers method as described in our earlier report.¹¹ The CeO₂-rGO nanocomposites were prepared by a hydrothermal method. Briefly, 50 mg graphene oxide was dispersed in 70 mL NaOH (15 M) solution and ultrasonicated for 30 min. Then, 0.1 g Ce(NO₃)₃·6H₂O was added into the solution. Furthermore, the mixture was sealed in a Teflon-lined stainless steel autoclave and maintained at 180 °C for 8 h and then cooled to room temperature gradually. Subsequently, the precipitation was filtered, washed with distilled water and ethanol several times, and dried at 80 °C for 6 h in a vacuum oven. For comparison, pure CeO₂ and reduced graphene oxide (rGO) were prepared using the same method.

2.4 Fabrication of the HRP/CeO₂-rGO biosensor

The HRP/CeO₂-rGO composite modified GC electrode was prepared as follows. First, the surface of the glassy carbon (GC) electrode, for each experiment, was mechanically polished with alumina suspensions (0.5 and 1.0 μM). Furthermore, the electrode was successively washed in ethanol and water for 2 minutes by ultrasonic method. The HRP/CeO₂-rGO modified electrodes were prepared by a simple drop casting method. Typically, 10 μL of CeO₂-rGO composite (0.5 mg mL⁻¹) suspension was mixed with 10 μL of HRP (5 mg mL⁻¹, 0.1 M PBS, pH 7.0) solution. Then, 10 μL of the mixed dispersion was cast onto the GC electrode and dried at room temperature; moreover, the HRP modified electrode was rinsed in 0.1 M PB solution (pH 7.0) to remove the unbound HRP. Subsequently 5 μL of 0.1 wt% Nafion solution was dropped over the HRP/CeO₂-rGO modified surfaces to form a tight membrane on the electrode surface (Scheme 1). In addition, HRP/CeO₂ and HRP/rGO modified GC electrodes were prepared by the abovementioned process for comparison. The modified electrodes were rinsed with PB solution and stored at 4 °C in a refrigerator when not in use.

Scheme 1 H₂O₂ detection scheme using HRP/CeO₂-rGO modified glassy carbon electrode.

3 Results and discussion

3.1 Characterization of the as-prepared Fe₂O₃/rGO composite

The microstructure and morphology of the GO, rGO, CeO₂-rGO composite and CeO₂ were characterized by FE-SEM. Fig. 1A–D shows the FE-SEM micrographs of the GO (A) and rGO (B), in which the GO and rGO appears corrugated into a wrinkle shape. Fig. 1C shows the CeO₂-rGO composite. It can be seen that the CeO₂ nanocubes were well decorated on the reduced graphene oxide sheets. These results clearly indicate the presence of strong interaction between rGO and CeO₂ nanocubes. Fig. 1D shows the CeO₂ nanocubes made under identical conditions without the GO. Fig. 1E and F shows the HR-TEM images of CeO₂-rGO and CeO_2. Comparison of Fig. 1E and F reveals that the average size of the CeO₂ nanocubes formed is ∼50–120 nm.

Fig. 1 Typical SEM images of (A) GO, (B) rGO, (C) CeO₂-rGO and (D) CeO₂; TEM images of (E) CeO₂-rGO and (F) CeO₂.

The XRD patterns of the CeO₂ and CeO₂-rGO are shown in Fig. 2. The XRD pattern of CeO₂ (Fig. 2a) shows the well-defined peaks that are centered at 28.5°, 33.0°, 47.5°, 56.4°, 59.1°, 69.5°, 76.5° and 79.0°, which correspond to the (111), (200), (220), (311), (222), (400), (331) and (420) plane reflections of CeO₂, respectively, as denoted by the International Center for Diffraction Data (JCPDS: 34-0394). All the diffraction peaks observed in the hybrid are related to the CeO₂ (as shown in Fig. 2b), confirming the CeO₂ has been grafted onto the rGO sheets.

Fig. 2 X-ray diffraction patterns of (a) CeO₂ and (b) CeO₂-rGO.

Fig. S1† depicts the FT-IR spectra of GO, rGO, CeO₂-rGO and CeO₂. In the FT-IR spectrum of GO (curve a), the peaks at 3435 and 1630 cm⁻¹ are attributed to O–H stretching and bending vibrations, respectively. All other oxygen functional groups are revealed by the peaks at 1730 cm⁻¹ (C=O stretching in COOH), 1058 cm⁻¹ (C–O stretching) and 1406 cm⁻¹ (C–O–C stretching in epoxy). In Fig. S1† (curves b and c), the intensity of oxygen functional groups are greatly suppressed and some peaks disappeared when compared to the GO peaks due to GO reduced into rGO during the hydrothermal process. Furthermore, the new vibration band appeared at 848 cm⁻¹ (metal–oxygen stretching vibration) for CeO₂-rGO hybrid (curve c). It is identical with CeO₂ (curve d). These data indicate that the CeO₂ was grafted onto the rGO sheets.

It is well known that the heme absorption provides a useful conformational probe for the investigation of heme proteins, and also the position of the Soret band gives information about the denaturation of heme proteins.²⁵ A film cast from HRP gave a heme band at 405 nm, which is similar to the Soret band at 403 nm for HRP in buffer solution.²⁶ The Soret band of the HRP-CeO₂-rGO film was also observed at 404 nm, which shows that not much shift was observed as compared to that of the HRP film alone (Fig. S2†). This result clearly demonstrated that the CeO₂-rGO film provides a good biocompatible environment for HRP attachment without any denaturation of enzyme, which is consistent with earlier reports.^27,28

3.2 Electrochemical behavior of the modified electrodes

The cyclic voltammogram (CV) curves of the bare GC (curve a), CeO₂ (curve b), CeO₂-rGO (curve C) and HRP-CeO₂-rGO (curve d) modified GC electrodes recorded in the presence of 1 mM [Fe(CN)₆]^3−/4− in 0.1 M KCl at a scan rate of 50 mV s⁻¹ are shown in Fig. 3A. Modification of the CeO₂-rGO (curve c) onto the GC electrode surface decreases the reversibility, while an apparent increase of the peak current of [Fe(CN)₆]^3−/4− (i_pa: 22 μA and ΔE_p (E_pa − E_pc): 73 mV) is noticed compared to the CeO₂ (i_pa: 17.2 μA and ΔE_p: 102 mV) and bare GC (i_pa: 19 μA and ΔE_p: 76 mV) modified electrodes. This could be attributed to the catalytic properties of CeO₂ being grafted over the rGO and also the restriction of the formation of face-to-face stacking in rGO sheets, which enhances the redox reaction of [Fe(CN)₆]^3−/4−. On the other hand, modification of the HRP/CeO₂-rGO (curve d) composite onto the GC electrode significantly reduced the redox peak current of [Fe(CN)₆]^3−/4− (i_pa: 12 μA and ΔE_p: 289 mV) compared to bare GC, CeO₂ and CeO₂-rGO composite modified GC electrodes due to the existence of a non-conducting amino acid protein chain of HRP molecules. It confirms the effective immobilization of HRP molecules into the CeO₂-rGO hybrid.

Fig. 3 (A) CV behavior of the modified GC electrodes in the presence of 1 mM [Fe(CN)₆]^3−/4− in 0.1 M KCl at a scan rate of 50 mV s⁻¹. (B) EIS behavior of the modified GC electrodes measured by impedance in the frequency region from 100 KHz to 0.1 Hz at DC potential of 200 mV and AC potential of ± 10 mV in the presence of 1 mM [Fe(CN)₆]^3−/4− in 0.1 M KCl solution. (C) Plot of peak current and charge transfer resistance against different modified GC electrodes. (D) Bode phase angle plot of the modified GC electrodes. Curve a: bare GC, curve b: CeO₂, curve c: CeO₂-rGO and curve d: HRP/CeO₂-rGO modified electrodes.

Electron transfer properties of the GC electrode after surface modification were characterized by electrochemical impedance spectroscopy (EIS). EIS is employed to investigate the interfacial properties of the modified electrode surface as it is a very sensitive technique to measure the intrinsic conductivity within the modified film. Fig. 3B shows Nyquist plots for bare GC (curve a), CeO₂ (curve b), CeO₂-rGO (curve c) and HRP-CeO₂-rGO (curve d) modified GC electrodes in 1 mM [Fe(CN)₆]^3−/4− as the redox probe in 0.1 M KCl solution. The value of charge transfer resistance (R_CT) was obtained by Randels equivalent circuit, R_s(Q_CPE(R_CTW)), where R_s is the solution resistance, R_CT is the charge transfer resistance, W is the Warburg impedance and Q_CPE is the constant phase element. Fig. 3B shows the EIS response of the bare and modified GC electrodes. This value of the charge transfer resistance (R_CT) for the bare GC (curve a), CeO₂ (curve b), CeO₂-rGO (curve c) and HRP-CeO₂-rGO (curve d) modified electrodes was estimated to be 157, 105, 878 and 9012 Ω, respectively. The R_CT value of CeO₂-rGO is lower than that of bare GC and CeO₂, which may due to the high surface area of the CeO₂-rGO hybrid. However, in the modification of the HRP/CeO₂-rGO electrode a much higher R_CT value is obtained compared to bare GC, CeO₂ and CeO₂-rGO. This R_CT value obtained for the HRP/CeO₂-rGO modified electrode is nearly 57, 10 and 85 fold higher when compared to bare GC, CeO₂ and CeO₂-rGO, respectively. The R_CT value increases after HRP modification could be well ascribed to hindering the diffusion of the redox probe by the non-conducting nature of HRP. The obtained R_CT values were in good agreement with the peak current values obtained from CV measurements (Fig. 3C). Further evidence was obtained from Bode phase–angle plot as shown in Fig. 3D. Bode plots give useful information about the behavior of electrodes depending upon the observed phase angles. When the phase angle is greater or equal to 90°, the electrode shows ideal capacitor characteristics, and when the phase angle is less than 90° the electrode does not behave like an ideal capacitor.²⁹ From Fig. 3D, the phase angle values of 22.7° (curve a), 46.2° (curve b), 15.3° (curve c) and 69.6° (curve d) were obtained for bare GC, CeO₂, CeO₂-rGO and HRP/CeO₂-rGO modified GC electrodes, respectively. Compared to those obtained at the bare GC and CeO₂ modified electrodes, a remarkably lower value was obtained at the CeO₂-rGO modified electrode, indicating that the electron transfer reaction of [Fe(CN)₆]^3−/4− was more facile at the CeO₂-rGO modified electrode. However, modification of the HRP/CeO₂-rGO (curve d) onto the GC electrode greatly enhances the phase angle value compared to bare GC, CeO₂ and CeO₂-rGO modified GC electrodes due to the activation of non-conducting HRP onto the CeO₂-rGO hybrid. These results of Nyquist and Bode phase–angle plot experiments were in good agreement with those of the CV experiment.

3.3 Electrochemical behavior of HRP/CeO₂-rGO modified electrodes

The cyclic voltammograms of HRP (curve a), HRP/rGO (curve b), HRP/CeO₂-rGO (curve c) and HRP/CeO₂ (curve d) modified GC electrodes in nitrogen saturated 0.1 M PBS (pH 7.0) are shown in Fig. 4A. Compared to those obtained at the HRP, HRP/rGO and HRP/CeO₂ modified GC electrodes, remarkably larger peak current was obtained at the HRP/CeO₂-rGO modified GC electrode. In addition, a pair of well-defined redox peaks at the formal potential of 365 ± 3 mV has been obtained, which is nearly similar to that of −377 ± 5 mV reported for HRP immobilized with a solid polymer matrix.³⁰ This reduction and oxidation process featured nearly symmetric redox peaks. It is evident that the electroactive center (heme Fe(III)/Fe(II)) of the HRP enzyme performed the redox reaction at the HRP/CeO₂-rGO modified electrode. The results suggest that the HRP enzyme entrapped in the CeO₂-rGO modified electrode retained the electrochemical activity. Furthermore, similar GC, rGO and CeO₂ modified electrodes with HRP gave smaller redox peak currents compared to that of the CeO₂-rGO modified GC electrode. According to Faraday's law, Q = nFAΓ*, where F is Faraday's constant, A is the geometrical surface area of the GC electrode, n is the number of electrons transferred and Q is the charge by integrating the cathodic peak of HRP at the CV.^31,32 The surface concentration of electroactive HRP (Γ*) at the HRP/CeO₂-rGO modified GC electrode was estimated to be 4.270 × 10⁻¹⁰ mol cm⁻² by assuming a one-electron transfer redox reaction. This value is higher than those of HRP at bare GC (1.611 × 10⁻¹⁰ mol cm⁻²), rGO (2.850 × 10⁻¹⁰ mol cm⁻²) and CeO₂ (2.280 × 10⁻¹⁰ mol cm⁻²). The higher peak current response was associated with the larger concentration of HRP present on its surface. These results revealed that a large amount of HRP accommodates onto CeO₂-rGO compared to the GC, rGO and CeO₂ modified surfaces. The two factors contributing to the increase in the redox peak current and high loading of HRP for the CeO₂-rGO modified surface are as follows: (i) in the CeO₂-rGO composite, restrict the rGO sheets aggregation by the attachment of CeO₂ nanocubes on both sides of the rGO sheets, which increases the surface-to-volume ratio of CeO₂-rGO hybrid as compared to that of rGO sheets alone and (ii) the high isoelectric point with good biocompatibility of CeO₂ nanocubes provides a well-defined microenvironment for HRP attachment, which enhances the direct electron transfer between HRP and electrode.

Fig. 4 (A) CVs of HRP on (a) bare, (b) rGO, (c) CeO₂-rGO and (d) CeO₂ in N₂ saturated 0.1 M PB solution (pH 7.0) at a scan rate of 100 mV s⁻¹ in the potential window between −0.7 and 0.0 V. (B) CVs of HRP/CeO₂-rGO modified GC electrodes at different scan rates. Curve a: 100 mV s⁻¹, curve b: 200 mV s⁻¹, curve c: 300 mV s⁻¹, curve d: 400 mV s⁻¹, curve e: 500 mV s⁻¹, curve f: 600 mV s⁻¹, curve g: 700 mV s⁻¹, curve h: 800 mV s⁻¹, curve i: 900 mV s⁻¹ and curve j: 1000 mV s⁻¹ in PB solution.

Fig. 4B shows the CVs of the HRP/CeO₂-rGO modified GC electrode with different scan rates (100–1000 mV s⁻¹) in N₂ saturated PB solution (pH 7.0). The redox process of the HRP/CeO₂-rGO modified electrode was a typical quasi-reversible electrochemical process relating an active substance attached to the electrode. The results suggest that all the electroactive HRP Fe(III) in the CeO₂-rGO modified electrode was reduced to HRP Fe(II) on the forward scan and then re-oxidized to Fe(III) on the reverse scan.³³ In addition, both the oxidation and reduction peak heights of the redox process were found to increase linearly with the scan rates from 100 to 1000 mV s⁻¹. The regression equation was deduced as I_pa (μA) = 0.006 (mV s⁻¹) + 0.371, r = 0.999; I_pc (μA) = −0.007 (mV s⁻¹) − 0.402, r = 0.999 (Fig. S3†). The characteristics of this particular redox reaction, involving the electroactive center of HRP, corresponded to those of other heme-containing proteins (myoglobin, hemoglobin, cytochrome P450 and HRP). These results indicated that the electron transfer between HRP enzyme and GC is a surface-controlled electrochemical process and the immobilized HRP enzyme on CeO₂-rGO is not denatured.

The influence of solution pH on the electrochemical behavior of HRP in CeO₂-rGO modified GC electrode has been examined by CV. The CV peaks of the HRP/CeO₂-rGO modified surface shifted negatively with increasing pH value (Fig. S4†). In addition, the results show that the electrode potential of the redox peaks and peak currents strongly depends on the pH of the solutions. All changes in CV peak potentials and peak currents with pH were reversible. The plot of formal potential (E⁰) versus pH was linear with a slope of about −53.9 mV per pH unit (inset in Fig. 5). The regression equation is derived as E⁰ (mV) = −53.9 pH + 18.2 with the regression coefficient of 0.996. The slope value was reasonably close to the theoretical value of −57.6 mV per pH, indicating that one proton participated in the electron-transfer process for neutralizing the charge change during redox reaction.³⁴ Moreover, the change in the solution pH did not affect the peak separation; hence, the diffusion of protons in the tri-helix scaffold of HRP/CeO₂-rGO was very fast. This is further evidence that the responses of the HRP/CeO₂-rGO biosensors are due to the redox reaction of the attached HRP on the electrode surface. In order to provide a good microenvironment of HRP enzyme, pH 7.0 was chosen in this study.

Fig. 5 (A) CVs of the HRP/CeO₂-rGO modified GC electrode in the absence (a) and presence (b) of 0.1 μM, (c) 100, (d) 200 μM and (e) 300 μM of H₂O₂ in 0.1 M PB solution (pH 7.0) at a scan rate of 100 mV s⁻¹. (B) Calibration plot of peak current vs. H₂O₂ concentration.

3.4 Electrocatalytic behavior of the HRP/CeO₂-rGO modified electrode

The HRP/CeO₂-rGO modified GC electrode showed good electrocatalytic activity towards H₂O₂ reduction. Fig. 5A shows the CVs of the HRP/CeO₂-rGO modified electrode in a solution containing different concentrations of hydrogen peroxide under the condition of nitrogen saturation. When H₂O₂ was added into PB solution (pH 7.0), there is a significant increase in the cathodic peak current (i_pc) at ∼ −0.40 V and a decrease in the anodic peak current (i_pc). Hence, the electrocatalytic reduction of H₂O₂ was due to the attached HRP in the CeO₂-rGO, which provides a fast direct electron transfer reaction between the heme group (Fe(III)/Fe(II)) of HRP and the electrode surface. The heme group of HRP reacts with H₂O₂ to form the first intermediate of compound I, which is the catalytic activity of H₂O₂. The electrocatalytic process of H₂O₂ by HRP can be derived as follows:³⁴

HRP (Fe³⁺) + H₂O₂ → compound I (Fe⁴⁺ = O) + H₂O (1)

Compound I (Fe⁴⁺ = O) + e + H⁺ → compound II (2)

Compound II + e + H⁺ → HRP (Fe³⁺) + H₂O (3)

Furthermore, a linear relationship between the catalytic reduction current and H₂O₂ concentration is obtained. The calibration curve for H₂O₂ detection is shown in Fig. 5B and a linear range from 0.1 to 500 μM with a correlation coefficient of 0.9991 was obtained. The detection limit was estimated to be 0.021 μM. The sensitivity of the HRP/CeO₂-rGO modified electrode was found to be 4650 nA mM⁻¹. When the H₂O₂ concentration was higher than 500 μM, the calibration curves tended to a plateau, which is a typical Michaelis–Menten kinetics behavior. The apparent Michaelis–Menten constant (K^app_M) was estimated to be 0.011 mM using the Lineweaver–Burk equation.³⁵ This value is much smaller than that reported for HRP entrapped in gold nanoparticle-SF of 1.22 mM,³⁶ the HRP immobilized onto the ZrO₂ nanoparticle of 8.01 mM (ref. 37) and HRP immobilized on gold-SPAN matrix of 2.21 mM.³⁸ The small K^app_M constant indicates that HRP immobilized into the CeO₂-rGO film retains its bio-activity and has a high affinity to H₂O₂. The analytical performance of the fabricated biosensor was very much comparable or better than the results reported for various H₂O₂ biosensors (ESI, Table S1†). The comparative data indicate the superiority of the present sensor over some reported H₂O₂ biosensors, and the possible reason being that the CeO₂-rGO modified surface possesses a good electron transfer property. In addition, the high surface-to-volume ratio of CeO₂-rGO can accommodate large amounts of enzyme, which might provide better communication between HRP and CeO₂-rGO electrode. Moreover, this CeO₂-rGO modified surface provides a friendly micro-environment to maintain HRP bioactivity.

3.5 The stability and reproducibility of the H₂O₂ biosensor

The storage stability of the HRP/CeO₂-rGO modified GCE was investigated. The peak current was measured using the same electrode, and it retained above 96.4% in 1 week and 94.2% in about 2 weeks of its initial response. To ascertain the reproducibility of the electrode fabrication, four different GC electrodes were modified with HRP/CeO₂-rGO. The redox peak current obtained in the measurements of three independent electrodes showed a relative standard deviation of 4.92%, confirming that the results are reproducible. In addition, the DET transfer of HRP/CeO₂-rGO modified GC electrode was stable. The CV responses of the HRP/CeO₂-rGO modified GC electrode in PB solution show no evident changes for 20 continuous cycles (Fig. S5†). The above results show that the fabricated HRP/CeO₂-rGO sensor is very much stable and reproducible.

4 Conclusion

We have demonstrated the synthesis of the CeO₂-rGO hybrid by an environment-friendly approach and have successively applied the immobilization of HRP to achieve the DET of HRP and also fabricated an electrochemical hydrogen peroxide biosensor. The electron transfer rate and the ability to retain the electroactivity of HRP are better than that of rGO and CeO₂ due to the synergistic effects from the combination between the two single components in the nanoscale. In addition, the fabricated HRP/CeO₂-rGO biosensors exhibit good affinity, fast response, wide linear range, lower detection limit, operational convenience, excellent stability and acceptable reproducibility. Therefore, our proposed biosensor offers an alternative method for the determination of H₂O₂ and has potential applications in the study of electrochemistry behaviors of enzymes as well as sensing applications.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (2013R1A2A2A01068926).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra12841a

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