Electrochemistry of myoglobin on graphene–SnO₂ nanocomposite modified electrode and its electrocatalysis

2.1 Reagents

1-Butylpyridinium hexafluorophosphate (BPPF₆, ⩾99%, Lanzhou Greenchem. ILS. LICP. CAS., China), horseheart myoglobin (Mb, Sigma, MW = 17,800), graphite powder (average particle size 30 μm, Shanghai Colloid Chemical Plant, China) and Nafion (5% ethanol solution, Sigma) were used as received. 0.1 mol L⁻¹ phosphate buffer solutions (PBS) with various pH values were used as the supporting electrolyte. All the other chemicals used were of analytical reagent grade and doubly distilled water was used in the experiments.

2.2 Apparatus

A CHI 750B electrochemical workstation (Shanghai CH Instrument, China) was used for all the electrochemical measurements. A conventional three-electrode system was used with an Mb-modified electrode as the working electrode, a platinum wire as the auxiliary electrode and a saturated calomel electrode (SCE) as the reference electrode. Ultraviolet–visible (UV–Vis) absorption spectra and Fourier transform infrared (FT-IR) spectra were recorded on a Cary 50 probe spectrophotometer (Varian Company, Australia) and a Tensor 27 FT-IR spectrophotometer (Bruker Company, Germany), respectively. Transmission electron microscopy (TEM) was performed on an FEI Tecnai G2 F20 transmission electron microscope (FEI Company, USA) and scanning electron microscopy (SEM) was recorded on a JSM-7100F scanning electron microscope (JEOL Inc., Japan).

2.3 Preparation of the GR–SnO₂ nanocomposite

GR was synthesized by a solvothermal method reported in our previous work (Zhu et al., 2013). Briefly, 3.0 mL of tetrachloromethane was reacted with 4.0 g of potassium in an autoclave at 200 °C for 15 h. When the autoclave cooled down to room temperature after the reaction, the product was transferred to a beaker, and washed subsequently by HCl solution, de-ionized water, acetone, and de-ionized water, until the pH value of the solution reached to 7. Then the product was dried at 100 °C for 12 h.

For the synthesis of GR-supported SnO₂ nanoparticles (Dong et al., 2012), 192 mg of SnCl₂·2H₂O and 331 μL of HCl (38%) were dispersed in 60 mL of deionized water. Then 30 mg of GR was added into the solution, and the mixed solution was sonicated for 5 min and stirred for 30 min. Finally, the mixed solution was filtered, washed and dried in an oven at 70 °C for 12 h.

2.4 Preparation of the modified electrodes

Based on the reported procedure (Sun et al., 2007), CILE was fabricated by mixing 1.6 g of graphite powder and 0.8 g of BPPF₆ thoroughly in a mortar, which was filled into one end of a glass tube (Ф = 4.2 mm) with a copper wire inserted through the opposite end to establish an electrical contact. Prior to use a mirror-like surface was obtained by polishing the electrode on a weighing paper.

A mixture solution containing 12.0 mg mL⁻¹ Mb and 0.5 mg mL⁻¹ GR–SnO₂ nanocomposite was prepared and mixed homogeneously to obtain a suspension solution, and then 7.0 μL of the mixture was pipetted onto the surface of CILE and dried to get the modified electrode. Finally, 3.0 μL of 0.5% Nafion ethanol solution was cast on the electrode surface and dried to get a uniform film modified electrode, which was denoted as Nafion/Mb–GR–SnO₂/CILE and kept in refrigerator at 4 °C.

2.5 Procedure

Electrochemical measurements were carried out in a 10 mL electrochemical cell containing 0.1 mol L⁻¹ PBS as the supporting electrolyte, which was purged with highly purified nitrogen for 30 min prior to the experiments and maintained in a nitrogen atmosphere during the experiments. UV–Vis absorption spectroscopic experiments were performed using a mixture solution containing certain concentrations of Mb and GR–SnO₂ nanocomposite, which was scanned in the wavelength range from 300 to 600 nm. The Mb–GR–SnO₂ film was assembled on a glass slide and air-dried to generate the composite, which was stripped off and tableted with KBr powder for the FT-IR experiments. For TEM characterizations, GR–SnO₂ nanocomposite was dispersed in an ethanol solution and ultrasonicated for 5 min. Several drops of suspension were then transferred onto a holey carbon TEM grid.

3.1 Characteristics of GR–SnO₂ nanocomposite

The synthesis of GR–SnO₂ nanocomposite was similar to our former reported procedure (Dong et al., 2012). The morphology and structure of GR-supported SnO₂ nanoparticles were examined on an FEI Tecnai G2 F20 transmission electron microscope. Due to their low contrast under electron beam and small size of SnO₂ nanoparticles, it is challenging to show their distribution on GR surface at low magnifications. As given in Fig. 1A, GR sheet wrinkled together to reduce surface energy, and the dimension of GR sheets was smaller than 100 nm. High resolution TEM (HRTEM) image of GR–SnO₂ was presented in Fig. 1B. It was observed that SnO₂ nanoparticles with a dimension of 2–4 nm were uniformly formed on GR sheets. The tetragonal (1 1 0) and (1 0 1) planes with lattice spacing of 0.34 and 0.26 nm were marked in Fig. 1B, respectively. HRTEM characterizations reveal the formation and distribution of SnO₂ nanoparticles on the surface of GR. The resulted GR–SnO₂ nanocomposite still remains graphene-like nanosheet with large surface area, which is beneficial for its further interaction with Mb molecules. SEM image of GR–SnO₂ nanocomposite on the electrode surface was recorded and shown in Fig. 1C. It can be seen that the flake like GR sheets were present on the electrode surface, which was interconnected and associated with each other to give a porous structure. Therefore the effective surface area was increased greatly on the GR–SnO₂ modified electrode.

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Figure 1 (A) TEM and (B) HRTEM images of GR–SnO₂ nanocomposite, (C) SEM image of GR–SnO₂/CILE.

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3.2 Spectroscopic results

UV–Vis absorption spectroscopy is an effective method to probe the structural change of heme proteins. The location of the Soret absorption band from the four iron heme groups of proteins can provide structural information about possible denaturation of heme proteins, especially the possible denaturation or the conformational change in the heme group region (George and Hanania, 1953). As shown in Fig. 2A, the Soret band of Mb dissolved in water appeared at 410.2 nm (curve a), while the similar Soret band appeared at 410.6 nm for a Mb–GR–SnO₂ mixture suspension solution in pH 5.0 PBS (curve b). The similar values indicated that Mb was not denatured after mixing with GR–SnO₂ nanocomposite.

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Figure 2 (A) UV–Vis absorption spectra of Mb in water (a) and Mb–GR–SnO₂ suspension in PBS (b); (B) FT-IR spectra of Mb (a) and Mb–GR–SnO₂ (b).

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FT-IR spectroscopy is usually used to provide detailed information on the secondary structure of polypeptide chains and to detect conformational changes of proteins. It is well-known that the amide I band at 1700–1600 cm⁻¹ is caused by C⚌O stretching vibrations of the peptide linkage and the amide II band at 1600–1500 cm⁻¹ results from a combination of N⚌H in-plane bending and C⚌N stretching of the peptide groups. If Mb molecule is denatured, the intensity and shape of the amide I and II bands would diminish or disappear (Song et al., 1992). From Fig. 2B, it can be seen that the amide I and II bands of native state of Mb appeared at 1656.44 cm⁻¹ and 1535.54 cm⁻¹ (Fig. 2Ba), and that of Mb in GR–SnO₂ film located at 1655.13 cm⁻¹ and 1541.56 cm⁻¹ (Fig. 2Bb). The similar positions of amide band suggested that Mb molecules still retained their native states after mixing with GR–SnO₂ nanocomposite.

3.3 EIS of the modified electrodes

EIS is a useful tool to investigate the impedance information of the electrode surface during the modification process. Fig. 3 presented the Nyquist diagrams of different electrodes in a 10.0 mmol L⁻¹ [Fe(CN)₆]^3−/4− solution containing 0.1 mol L⁻¹ KCl across the frequency range from 10⁴ to 0.1 Hz. The semicircle diameter observed at higher frequency range equals to electron transfer resistance (R_et), which can imply the electron transfer kinetics of the redox probe at the electrode. As for CILE the R_et value was around 43.65 Ω (curve c), which can be ascribed to the presence of high conductive IL in the carbon paste. After the addition of GR–SnO₂ nanocomposite onto the CILE surface, the R_et value of GR–SnO₂/CILE decreased to 20.58 Ω (curve d). The result indicated that GR–SnO₂ nanocomposite exhibited good conductivity and decreased the interfacial resistance. On Nafion/GR–SnO₂/CILE the R_et value was increased to 75.89 Ω (curve b), which was due to the presence of Nafion film on the electrode surface that hindered the electron transfer of [Fe(CN)₆]^3−/4−. After Mb was immobilized in the film, the R_et value was further increased to 93.26 Ω (curve a), which indicated that the successful immobilization of Mb on the electrode surface further hindered the electron transfer and increased the resistance.

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Figure 3 EIS for (a) Nafion/Mb–GR–SnO₂/CILE, (b) Nafion/GR–SnO₂/CILE, (c) CILE, and (d) GR–SnO₂/CILE in the solution of 10.0 mmol L⁻¹ [Fe(CN)₆]^3−/4− and 0.1 mol L⁻¹ KCl with the frequencies swept from 10⁴ to 0.1 Hz.

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3.4 Cyclic voltammetric behavior of the Mb modified electrode

Cyclic voltammograms of different modified electrodes were recorded in a 0.1 mol L⁻¹ PBS (pH 5.0) at a scan rate of 100 mV s⁻¹. As shown in Fig. 4, no redox peaks appeared on CILE (curve a) and Nafion/GR–SnO₂/CILE (curve b) but with the increase of background current. At Nafion/Mb/CILE a pair of unsymmetrical redox peaks were observed with a cathodic peak (E_pc) at −0.303 V and an anodic peak (E_pa) at −0.226 V (curve c). CILE has been proven to be an excellent working electrode with many advantages, which can be used for the realization of direct electron transfer of redox proteins (Sun et al., 2007). On Nafion/Mb–GR–SnO₂/CILE a pair of well-defined and nearly symmetric redox peaks were observed with the redox peak currents increased obviously (curve d). The cathodic and anodic peak potentials were located at −0.318 V (E_pc) and −0.236 V (E_pa), respectively. The formal peak potential (E^0′), which was defined as the average value of E_pc and E_pa, was got as −0.272 V with a peak-to-peak separation (ΔE_p) of 82 mV. So the direct electron transfer of Mb was accelerated on the GR–SnO₂ modified CILE. The GR–SnO₂ nanocomposite exhibited as nanosheets with SnO₂ nanoparticles dispersed on the surface of GR, which showed synergistic effects, including the high conductivity and large surface area of GR and the biocompatibility of SnO₂ nanoparticles. Therefore direct electrochemistry of Mb was accelerated on the GR–SnO₂ modified electrode with a pair of well-defined redox peaks appeared.

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Figure 4 Cyclic voltammograms of (a) CILE, (b) Nafion/GR–SnO₂/CILE, (c) Nafion/Mb/CILE, and (d) Nafion/Mb–GR–SnO₂/CILE in pH 5.0 PBS with the scan rate as 100 mV s⁻¹.

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3.5 Electrochemical behaviors

Cyclic voltammograms of Nafion/Mb–GR–SnO₂/CILE in 0.1 mol L⁻¹ PBS at different scan rates were further investigated from 30 to 500 mV s⁻¹ with the results shown in Fig. 5A. It can be seen that a pair of well-defined quasi-reversible redox peaks appeared at different scan rates with almost equal value of peak currents. The relationship of the electrochemical data with scan rate was further calculated. The linear regression equations of redox peak currents with scan rate were calculated as I_pc(μA) = 227.33υ (V s⁻¹) + 9.52 (n = 12, γ = 0.996) and I_pa(μA) = −232.14υ (V s⁻¹) −2.46 (n = 12, γ = 0.996), respectively (as shown in Fig. 5B), which indicated an adsorption-controlled electrochemical process. Based on the equation of Γ^∗ = Q/nAF (Bard and Faulkner, 1980), the surface concentration (Γ^∗) of electroactive Mb was calculated to be 1.3 × 10⁻⁹ mol cm⁻². The total amount of Mb cast on the electrode surface was 4.72 × 10⁻⁹ mol cm⁻², therefore 27.5% of the Mb molecules on the electrode surface participated in the electrochemical reaction. So the presence of GR–SnO₂ nanocomposite exhibited a larger surface area that was efficient for Mb immobilization, which could provide a specific conductive interface for multiple layers of Mb near the electrode surface to transfer electrons with the basal electrode.

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Figure 5 (A) Influence of scan rate on electrochemical responses of Nafion/Mb–GR–SnO₂/CILE in pH 5.0 PBS with scan rates from (a) to (l) as 30, 50, 70, 100, 150, 200, 250, 300, 350, 400, 450, 500 mV s⁻¹, respectively. (B) Linear relationship of redox peak current (Ip) versus scan rate (υ). (C) Linear relationship of the redox potential versus ln υ.

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From Fig. 5A it can be seen that with the increase of scan rate the redox peak potential shifted slightly with ΔE_p value increased gradually. Two straight lines were obtained with the equations of E_pc(V) = −0.026 ln υ −0.37 (γ = 0.995) and E_pa(V) = 0.28 ln υ −0.19 (γ = 0.997), respectively. According to the Laviron’s equation (Laviron, 1974; Laviron, 1979), the values of the electron transfer coefficient (α) and the heterogeneous electron transfer rate constant (k_s) were calculated as 0.519 and 1.034 s⁻¹, respectively. It is well known that k_s value reflects the local microenvironment of protein immobilized on the electrode. This k_s value is higher than the values of Mb when immobilized on C₆₀-multiwalled CNT based electrode (0.39 s⁻¹, Zhang et al., 2006), Ag–CNTs modified GCE (0.41 s⁻¹, Liu and Hu, 2009), NiO nanoparticle modified GCE (0.34 s⁻¹, Moghaddam et al., 2008), Co nanoparticle modified CILE (0.588 s⁻¹, Sun et al., 2009) and GO–IL modified CILE (0.584 s⁻¹, Sun et al., 2013), indicating the electron transfer of Mb was facilitated on GR–SnO₂ nanocomposite modified electrode.

In most cases the redox behavior of protein is significantly dependent on the solution pH, therefore the influence of buffer pH from 2.0 to 8.0 on cyclic voltammetric behaviors of Nafion/Mb–GR–SnO₂/CILE was investigated in 0.1 mol L⁻¹ PBS with the results shown in Fig. 6. Nearly reversible voltammograms with stable and well-defined redox peaks were observed and the maximum redox peak current was occurred at pH 5.0, which was selected for the electrochemical investigation. Furthermore, negative shifts in both cathodic and anodic peak potentials were observed with increasing pH value, indicating that protons took part in the electrode reaction. The formal peak potential (E^0′) had a linear relationship with the buffer pH and the regression equation was calculated as E^0′(mV) = −48.1 pH +13.5 (γ = 0.998). The slope of −48.1 mV pH⁻¹ was smaller than the theoretical value of −59.0 mV pH⁻¹ for a one-electron one-proton reaction between the electrode and Mb. The reason might be the influence of the protonation states of transligands to the heme iron and amino acids around the heme or the protonation of the water molecule coordinated with the central iron. So the electrochemical reaction can be expressed with the equation as: Mb Fe(III) + H⁺ + e → Mb Fe(II), which involved a single protonation accompanied with one electron transfer of Mb Fe(III) to electrode.

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Figure 6 Cyclic voltammograms of Nafion/Mb–GR–SnO₂/CILE in different pH PBS (from a to g as 2, 3, 4, 5, 6, 7, 8) with the scan rate of 100 mV s⁻¹.

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3.6 Electrocatalytic behaviors of the immobilized Mb

The Mb immobilized on the electrode surface exhibited excellent electrocatalytic ability to the reduction of NaNO₂. Fig. 7A shows the cyclic voltammograms of Nafion/Mb–GR–SnO₂/CILE in PBS containing different concentrations of NaNO₂ at a fixed scan rate of 100 mV s⁻¹. Upon the addition of NaNO₂, a new irreversible reduction peak appeared at −0.812 V, which was attributed to the reduction of NO₂⁻ by Mb molecules on the electrode. While on Nafion/GR–SnO₂/CILE the direct electrochemical reduction of nitrite required a large potential with the reduction peak potential appeared −0.998 V (Fig. 7B curves i and j) without the appearance of the oxidation peak. The overpotential of electroreduction of nitrite was lower for 0.186 V. Therefore the presence of Mb molecules on the electrode exhibited the catalytic behaviors with the decrease of the activation energy for the reaction and the increase of the reduction peak current. Based on the references (Yomathan et al., 1992; Shan et al., 2005), the reaction process can be proposed as follows:

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Figure 7 Cyclic voltammograms of (A) Nafion/Mb–SnO₂–GR/CILE in 0.1 mol L⁻¹ pH 5.0 PBS with different concentrations of NaNO₂ (From a to h: 0, 2.0, 5.0, 7.0, 9.0, 14.0, 18.0, 20.0 μmol L⁻¹) at the scan rate of 100 mV s⁻¹. Inset is linear relationship of catalytic reduction peak currents versus the NaNO₂ concentration; (B) Nafion/SnO₂–GR/CILE in 0.1 mol L⁻¹ pH 5.0 PBS with 0 and 2.50 mmol L⁻¹ NaNO₂ (From i to j) at the scan rate of 100 mV s⁻¹.

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This reaction mechanism indicates that nitrite combines with a proton to get HNO₂ firstly_, which is disproportionated to NO and NO₃⁻ subsequently. Then Mb Fe(II) formed on the electrode surface reacts with NO to get the ferrous nitrosyl complex of Mb Fe(II)—NO, which can directly reduce on the electrode surface to release Mb Fe(II) again with a catalytic cycle formed.

The reduction peak current increased along with NaNO₂ concentration in the range from 0.2 to 350.0 μmol L⁻¹ with the linear regression equation got as I_ss(μA) = 8.901 + 29.77C (μmol L⁻¹) and the detection limit of 0.483 μmol L⁻¹ (S/N = 3). When the concentration of NaNO₂ was more than 400.0 μmol L⁻¹, a current plateau appeared, which was the typical Michaelis–Menten process. For a thin film of immobilized proteins, the maximum current measured under the saturated substrate conditions (I_max) and the apparent Michaelis–Menten constant (K_M^app), which give an indication of the enzyme-substrate kinetics, can be obtained from the Lineweaver–Burk equation (Kamin and Willson, 1980): $$\frac{1}{I_{\mathit{SS}}}=\frac{1}{I_{\text{max}}}+\frac{K_{\mathit{app}}^M}{I_{\text{max}}C}$$ where C is the bulk concentration of the substrate. Based on the above equation the K_M^app value for this NaNO₂ biosensor was calculated to be 3.98 μmol L⁻¹. A comparison of the proposed bioelectrode for the electrocatalytic detection of NaNO₂ with other redox protein modified electrodes was summarized and listed in Table 1. It can be seen that this electrode showed excellent electrochemical behaviors with wider linear range and lower detection limit, indicated the specific characteristic of GR–SnO₂ nanocomposite for the efficiency electron transfer of Mb molecules with electrode.

3.7 Stability and reproducibility of Nafion/Mb–GR–SnO₂/CILE

The stability of Nafion/Mb–GR–SnO₂/CILE was also investigated. Firstly the modified electrode was evaluated by examining the cyclic voltammetric peak currents after continuous scanning for 50 cycles. There was nearly no decrease of the voltammetric response and 99.1% of the initial current response was retained. After the modified electrode was stored in a refrigerator at 4 °C for two weeks, 5.5% decrease of the peak current appeared. After a 30-day storage, Nafion/Mb–GR–SnO₂/CILE still retained 91.2% of its initial current response. The relatively good stability of the Mb electrode could be attributed to the biocompatibility of the GR–SnO₂ nanomaterial in the film. Six Mb modified electrodes prepared by the same procedure gave the relative standard deviation (RSD) of 3.2% for the determination of 10.0 μmol L⁻¹ NaNO₂ solution, which indicated the modified electrode had good repeatability.

3.8 Sample determination

To evaluate the possible application of the proposed electrode for the real samples detection, different water samples including tap water and lake water were tested, which were diluted with pH 5.0 PBS and detected by the recommended procedure. All the samples were further measured by the standard addition method to calculate the recovery with the results summarized in Table 2. It can be seen that the recovery of this method was in the range from 99.3% to 103.1%, which was satisfied for the routine analysis.