Abstract
A high-performance modified electrode was developed to sense hydrogen peroxide, based on immobilization of Cytochrome c onto a glassy carbon electrode modified with polyaniline and carboxylated multi-walled carbon nanotubes nanocomposite (Cyt c/PAN/cMWCNTs/GC electrode). Using Fourier transform infrared spectroscopy and electrochemical impedance spectroscopy, the hybrid matrix properties of the Cyt c/PAN/cMWCNTs/GC electrode were characterized. The surface morphology of the Cyt c/PAN/cMWCNTs/GC electrode was examined by scanning electron microscopy. The electrochemical properties of the designed electrode were investigated by cyclic voltammetry and amperometry. A couple of well-defined redox peaks were obtained and the formal potential, electron transfer rate constant, average surface coverage and α value were calculated to be -0.35 V, 3.01 s-1, 3.1 × 10-9 mol cm-2 and 0.37, respectively. The modified electrode was highly sensitive for the quantification of hydrogen peroxide with a sensitivity of 97.6 μA mM-1, a detection limit of 0.2 μmol L-1, and a linear range of 2-600 μmol L-1. According to the obtained results, the proposed hybrid material could facilitate the direct electron-transfer between the electrode surface and the redox center of Cyt c, expecting to find applications in the next-generation biosensors.
Introduction
Hydrogen peroxide (H2O2) has been widely employed to sterilize and bleach in food, aquaculture, pharmaceutical, textile, dye industries, diagnostic detection and cancer therapy [1–7]. In addition, H2O2 is an intermediate product in many of enzymatic and bi-enzymatic biosensors for analyte detection, such as glucose, oxalate, xanthine, and creatinine [8–12]. Sometimes, it is produced by ionizing radiation in the cell, which could damage DNA and cause the development and progression of diseases such as heart disease, neurodegeneration, cancer, and diabetes [13–15].
Biosensors have been used in the variety of fields (e.g. agricultural science, food industry, pharmacology, medical diagnostics, and environmental monitoring) for biological detection, signal processing, and electronics applications [16]. The most important step in the biosensor manufacturing processes is the preparation of a conductive platform to guarantee the structural stability of the immobilized biological components [17]. In order to quantify and qualify the protein immobilization and electron transmission processes, in recent years, conductive polymer-based nanocomposites have received more attention in biosensors structure [18–24].
Due to their extraordinary properties such as large surface area, high electrical conductivity and superior mechanical behavior of carbon nanotubes (CNTs), they were frequently used in the fabrication of the biosensors [25–29]. The improved functionalized CNTs such as large fiber carboxylated multi-walled carbon nanotubes (cMWCNTs) with high porous structure have been recommended for high protein loading in a variety of immobilization applications [30]. The hydrophobic nature of CNTs has been reduced and controlled using the conductive polymers [31], ionic liquids [32], ionic polymers [33, 34], and surfactants [35, 36], as well as surface functionalization with hydrophilic groups [37].
Among the conductive polymers, due to its high conductivity, environmental stability, facile synthesis, easy handling and low-cost monomer, polyaniline (PAN) has been drawn considerable interest for using in the extensive electrical applications such as fuel cells [38], super capacitors [39, 40], rechargeable batteries [41, 42], sensors and biosensors [31, 43]. Because of strong interaction between the graphite structure of CNTs and the aromatic ring of PAN, the PAN/CNTs nanocomposites show a favorable charge transfer properties [44–46].
In this work, Cyt c was immobilized on PAN/cMWCNTs-modified glassy carbon (GC) electrode during a simple procedure. The electrochemical behaviors of the fabricated electrode were studied by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). This bio-electrode showed the ability of direct electron transfer between Cyt c and electrode surface for applying in electrochemical sensing of H2O2.
Experiment
Materials
Horse heart cytochrome c (Cyt c) was obtained from Sigma Aldrich and MWCNTs (diameter: 30-50 nm; length 20 µm) were purchased from Times nano Co. (Chengdu, China) with 95% purity. Aniline monomer was obtained from Sigma; it was purified by simple distillation twice and held in 4 ºC until it was used in electropolymerization. The stock phosphate buffer solution (PBS; 0.05 mol L-1, pH 7.0) was prepared from potassium dihydrogen phosphate and dipotassium hydrogen phosphate, both from Merck, in double-distilled water. The stock solution of 8 mg mL-1 Cyt c was prepared with 0.02 mol L-1 PBS at pH 7.0. H2O2 solution (30%) was bought from Carlo Erba Co. Potassium chloride, N, N’-dimethyl formamide (DMF), β-D-(+)-glucose, L-cysteine and L-tyrosine were prepared from Merck, ascorbic acid and uric acid were purchased from Acros Organics. Other used chemicals and reagents were of analytical grade.
Apparatus and measuring methods
Electrochemical experiments were performed using an electrochemical analysis system (SAMA500 potentiostat/galvanostat, Isfahan, Iran) and an electrochemical cell, which was equipped with a platinum auxiliary electrode, an Ag/AgCl (3 mol L-1 KCl) reference electrode and a working Cyt c/PAN/cMWCNTs-modified GC electrode (Azar Electrode, Uromia, Iran). Before CV measurements, the PBS was deoxygenated by bubbling pure nitrogen for 10 min and a nitrogen atmosphere was kept over the solutions to protect the solution from oxygen diffusion during measurements. The EIS studies were performed using an Autolab PGSTAT 30 electrochemical analyzer (Eco Chemie, Netherlands) in 10 mmol L-1 K3[Fe(CN)6]/K4[Fe(CN)6] solution containing 0.1 mol L-1 KCl in the frequency range 0.1-105 Hz with an AC voltage amplitude 0.05 V and a bias potential of 0.2 V. The amperometric responses of modified electrode to H2O2 addition were evaluated by a potentiostat/galvanostat (EG&G, Model 273A, USA) equipped with a rotating disk electrode (RDE) device (PerkinElmer, USA) and “Power Suite” software package. The scanning electron microscopy (SEM) images of working electrode surface were taken by electronic microscopy (KYKY, Model EM3200, China). An ultrasonic bath with a digital timer and a temperature control (Euronda, Montecchio Precalcino, Italy) was used for acid treatment and dispersion of MWCNTs. The washing process of treated MWCNTs was carried out by a suction filtration system through mixed cellulose ester membrane filter with a pore size of 0.45 μm.
Preparation of Cyt c/PAN/cMWCNTs/GC electrode
10 mg of MWCNTs were treated with 10 mL of 35% HNO3 at 40 ºC for 6 h by ultrasonic vibration, which was periodically relaxed for at least 10 min after each 20 min agitation. Functionalized MWCNTs (cMWCNTs) were washed with double-distilled water several times to reach the pH of the distilled water and then were placed under the infrared lamp until dried to obtain a black powder of cMWCNTs. One mg of cMWCNTs was mixed with 1 mL of N, N’-dimethyl formamide (DMF), and then was dispersed under ultrasonic agitation until a homogenous black suspension was obtained.
Before the preparation of the modified electrode, the surface of glassy carbon (GC) electrode was thoroughly polished with 5.0 and 0.5 μm alumina slurry, respectively. The GC electrode was sonicated in a mixture of ethanol/water for 5 min and then 2 µL of cMWCNTs suspension in DMF was cast on the sonicated GC electrode surface. After drying cMWCNTs/GC electrode, it was dipped in the electrochemical cell containing 4 mL H2SO4 (0.25 mol L-1) and aniline monomer (0.025 mol L-1). Electropolymerization was carried out in the potential range from -0.1 to +0.9 V (vs. Ag/AgCl (3 mol L-1 KCl)) at a scan rate of 0.02 V s-1, and it was repeated for 6 cycles. When PAN was electropolymerized, the surface of modified cMWCNTs/GC (PAN/ cMWCNTs/GC) electrode appeared to be dark green that was attributed to synthesize the emeraldine salt form of PAN [31]. The surface of PAN/cMWCNTs/GC electrode was gently washed several times using double-distilled water to remove non-reacted aniline monomers. The dissolved Cyt c (8 mg mL-1) in the PBS was dropped onto the surface of PAN/cMWCNTs/GC electrode and dried at room temperature. The Cyt c/PAN/cMWCNTs/GC electrode was washed to take away non-immobilized Cyt c and it was stored in the refrigerator (4 ºC) until use.
Results and discussion
FTIR analysis of Cyt c/PAN/cMWCNTs nanocomposite film
The FTIR spectrum of the cMWCNTs, PAN/cMWCNTs nanocomposite, and Cyt c/PAN/cMWCNTs nanocomposite are shown in Fig. 1 from 4000 to 400 cm-l wave number. According to Fig. 1a, in the FTIR spectrum of the cMWCNTs, the wide peak at 3435 cm-1 is attributed to the hydroxyl groups [47, 48] and the bending vibration at 1718 cm-1 is assigned to the -C=O stretching vibrations [49–51]. After the PAN electropolymerization as shown in Fig. 1b, the peak at 564 cm-1 is attributed to the existence of sulfate anions as a dopant in the polymerized PAN [52]. The intensity peak at 3428 cm-1 was increased, indicating -N-H stretching, due to the addition of PAN [51]. The presence of immobilized Cyt c on the PAN/cMWCNTs/GC electrode surface as shown in Fig. 1c was confirmed by the stretching vibration peak at 1498 cm-1, which is a band of amid II after casting protein solution on PAN/cMWCNTs/GC electrode [53]. Indeed, as shown in Fig. 1b, the FTIR spectrum of this nanocomposite reveals weak peaks. The polymer chains of PAN are constrained to grow around cMWCNTs during electropolymerization, decreasing the intensity of vibration mode peaks by the presence of cMWCNTs in nanocomposite due to the polymer chain motion restriction in the nanocomposite matrix [52].
Surface morphology of the modified electrode
SEM was applied to observe the surface morphology of the modified electrode. The SEM images of modified electrodes with cMWCNTs and PAN/cMWCNTs nanocomposite layer (Fig. 2A and 2B) have shown that the structure of nanocomposite platform is more compact than cMWCNTs. The presented surface area of cMWCNTs is covered by a layer of PAN that is similar to snowflakes. The observed morphology of PAN/cMWCNTs nanocomposite layer is in agreement with earlier reported works [12, 46]. According to Fig. 1B, the porous platform has enough pores to place biomolecules onto the surface of the nanocomposite modified electrode. The SEM of Cyt c/PAN/cMWCNTs/GC electrode surface as shown in Fig. 2C, in comparison with the PAN/cMWCNTs/GC electrode surface (Fig. 2B), indicates that most of the pores in the PAN/cMWCNTs nano composite layer have been filled and the diameter of cMWCNTs coated with PAN has been increased, which could be explained by Cyt c location on the fabricated platform.
EIS characterization of Cyt c/PAN/cMWCNTs/GC electrode
The electrochemical interfacial properties of the modified electrodes were investigated by EIS studies in 10 mmol L-1 of K3Fe(CN)6/K4Fe(CN)6 and 0.1 mol L-1 KCl solution in a frequency range between 0.1 and 1 × 105 Hz. The Nyquist plots of variously modified electrodes are represented in Fig. 3. As shown in this figure, the Nyquist plots show a single semicircle at high frequencies followed by a 45° straight line at low frequencies due to the charge-transfer and diffusion-controlled processes, respectively [54, 55]. The charge-transfer resistance (Rct), indicating by the semicircle diameter of the plot, was estimated to be 3 Ω for PAN/cMWCNTs/GC and 5 Ω for Cyt c/PAN/cMWCNTs/GC electrodes. According to the obtained results as presented in Fig. 3, Rct of Cyt c/PAN/cMWCNTs/GC electrode shows an increase with respect to that of PAN/cMWCNTs/GC electrode [56].
Electrochemical behavior of Cyt c/PAN/cMWCNTs modified GC electrode
The electrochemical behavior of the fabricated electrodes was studied by cyclic voltammetry (CV). The recorded cyclic voltammograms (CVs) in Fig. 4 (curve a) demonstrated that there is no electron transfer between Cyt c and the bare GC electrode. However, as shown in Fig. 4 (curve c), obvious redox peaks were observed for Cyt c immobilization on the PAN/cMWCNTs/GC electrode, while immobilized Cyt c on the cMWCNTs/GC electrode showed small hump peaks (curve b). The protonated emeraldine salt formula is a conductive form of PAN, which is hydrophilic [57, 58], resulting in the peak intensity enhancement due to an increase in the immobilized amount of Cyt c caused by the more surface hydrophilicity of nanocomposite platform.
In Fig. 5, the CVs of bare GC (curve a), cMWCNTs/GC (curve b) and PAN/cMWCNTs/GC (curve c) electrodes were presented without any peaks between -0.1 to -0.6 V at a scan rate of 0.1 V s-1, whereas the CVs of Cyt c/PAN/cMWCNTs/GC electrode showed well-defined redox peaks (curve d). The results indicate that the PAN/cMWCNTs nanocomposite is a relevant platform for the immobilization of Cyt c as well as a very good promoter for the electron transfer between Cyt c and the electrode. The cathodic and anodic peak potentials were -0.377 and -0.323 V, respectively, and the calculated formal potential (E0´) from the average of redox peak potentials was -0.35 V. The peak separation (ΔEP) was obtained to be 0.054 V, indicating the rapid quasi-reversible electron transfer of the heme group of Cyt c. The ΔEP value of Cyt c/PAN/cMWCNTs/GC electrode obtained in this work is comparable to other modified electrodes, such as Cyt c/GNPs/PANS/GC electrode, 0.075 V [59] and Cyt c/AuNP/ITO, 0.07 V [60].
The scan rate dependence of cathodic and anodic peak potentials was shown by CVs of the Cyt c/PAN/PPY/cMWCNTs/GC electrode between -0.1 to -0.6 V at different scan rates from 0.02 to 2.0 V s-1 (Fig. 6A). According to the obtained results, with the increase of scan rate, the cathodic peak potentials were shifted to more negative values and the anodic peak potentials were shifted to more positive values, which directed to increase in the ΔEp values. The effect of potential scan rate on the cathodic and anodic peak currents was studied and reported in Fig. 6B, showing that the cathodic and anodic peak currents increase linearly in the scan rate of 0.02-2.0 V s-1, indicating the stability of immobilized Cyt c and a surface-controlled process for electron transfer [32, 61]. The electron transfer rate constant, ks, was calculated by the Laviron theory for ΔEp>0.2 V [62]:
log〖k_s =α〗 log(1-α)+(1-α) logα-log(□(RT/nFν))-α(1-α) □((nF∆E_p)/2.3RT) (1)
where n, F, υ, R and T are the electron transfer number, the Faraday constant, potential scan rate, the universal gas constant and absolute temperature, respectively. The charge transfer coefficient, α value was calculated to be 0.37 using the plot of cathodic peak potential (Ep) versus the logarithm of scan rate (log υ) by Laviron’s Equation [62]:
E_pc=E^0+RT/(α_c nF) ln(α_c/m) (2)
where m=((RTk_s ))⁄((Fυ) ). The calculated as of the immobilized Cyt c on PAN/cMWCNTs/GC electrode (3.01 s-1) is higher than that of the immobilized Cyt c on poly-3-methylthiophene/MWCNTs/GC electrode (0.49 s-1) [63], sodium dodecyl sulfate/polyacrylamide/GC electrode (1.56 s-1) [55], SiO2/Cyt c/SiO2/boron-doped diamond film (1.39 s-1) [64] and ionic liquid/cMWCNTs/GC electrode (1.24 s-1) [65]. These results indicate that the PAN/cMWCNTs/GC electrode can act as a relevant promoter for facilitating electron transfer between the electrode and redox-active site of Cyt c.
According to equation of I_p=(n^2 F^2 νAΓ)⁄4RT [66], the surface coverage (Γ) was calculated to be 3.1 × 10-9 mol cm-2, where A is the surface area of electrode and IP is cathodic peak current, that the slope of IP versus υ was used to calculate Γ (Fig. 6D). The surface coverage of Cyt c/cMWCNTs/GC electrode was reported elsewhere as 3.8 × 10-10 mol cm-2 [65] and 4.1 × 10-10 mol cm-2 [67].
Stability of modified electrode
The operational stability of the Cyt c-modified electrode was examined by continuous cyclic voltammetry. Cyt c can be closely adsorbed on the modified electrode. Therefore, after 100 cycles at the scan rate of 0.1 V s-1, no noticeable change in the CVs of Cyt c was observed (Fig. 7). To explore the storage stability of Cyt c/PAN/cMWCNTs/GC electrode, it was stored in PBS at 4 °C and the CVs of Cyt c was measured for 4 consecutive weeks. The current response of the Cyt c/PAN/cMWCNTs/GC electrode retained approximately 93% of the initial response after three measurements in 5 days, and it maintained about 89% of its initial current response after measurements in the 4-week period. This result exhibits that the composite film shows a reasonable stability for 4 weeks.
Electrocatalytic behavior of Cyt c/PAN/cMWCNTs/GC electrode
The electrocatalytic reduction of H2O2 at the Cyt c/PAN/cMWCNTs/GC electrode was shown in Fig. 8. The CVs of the modified electrode was recorded before and after addition of 100 μL H2O2 (10 mmol L-1) to 10 mL PBS. The cathodic peak currents increase with increasing H2O2 concentration, indicating the electrocatalytic ability of the modified electrode to H2O2 reduction. The obtained results indicate the decrease of oxidative peak current together with the increase of the reductive peak current of the Cyt c/PAN/cMWCNTs/GC electrode. The electrocatalytic reduction of H2O2 can be explained as in the following form [60, 61]:
Heme group reduction: [Cyt c(FeIII)]+ e− → [Cyt c(FeII)] (4)
Heme group oxidation: 2[Cyt c(FeII)] + 2H+ + H2O2 → 2[Cyt c(FeIII)]+ 2H2O (5)
The overall reaction: H2O2 + 2H+ +2e− → 2H2O (6)
Amperometric detection of H2O2
To evaluate the ability of Cyt c/PAN/cMWCNTs/GC electrode for amperometric detection of H2O2, the current-time curve was obtained by successive addition of different volumes of H2O2 (5 mmol L-1) to a continuously stirred 5 mL PBS solution at the applied potential of -0.4 V versus Ag/AgCl. The results are presented in Fig. 9 and the corresponding calibration curve of amperogram for the Cyt c/PAN/cMWCNTs/GC electrode is plotted in the inset of Fig. 9. The amperometric response time of the modified electrode to H2O2 was 7 s. This time is an acceptable response performance to H2O2. The linear range of Cyt c/PAN/cMWCNTs/GC electrode was 2-600 μmol L-1 of H2O2, which is covered substantial levels of H2O2 in the human organs such as kidney, vascular endothelial, circulating blood cells, urinary tract and bladder [68]. Levels of H2O2 at or below about 20-50 μmol L-1 gave the impression to have limited cytotoxicity to many cell types [68]. The sensitivity of modified electrode was obtained using the slope of the linear regression equation of calibration curve in the inset of Fig. 9, I/µA = 0.0976 [H2O2]/(µmol L-1) + 7.1754, which that was equal to 97.6 μA mM-1. The concentration of H2O2 as the detection limit (DL) was estimated to be 0.2 μmol L-1 at the signal-to-noise ratio of 3. The obtained linear range and DL of the modified Cyt c/PAN/cMWCNTs/GC electrode were compared with those of some Cyt c-based electrodes introduced in other works, and the results were presented in Table 1 [63, 69-80].
Influence of interferences
The effects of five potential interfering substances, including glucose, ascorbic acid, uric acid, cysteine, and tyrosine were studied to confirm the selectivity of the proposed electrode. For this purpose, a determined amount of each species was added to the 5 mL reaction mixture, while the final concentration of each sample after a successive addition was 100 μmol L-1, and the amperometric response of the modified Cyt c/PAN/cMWCNTs/GC electrode was recorded. As shown in Fig. 10, according to the experimental results, no significant responses were observed for 100 μmol L-1 glucose, 100 μmol L-1 ascorbic acid, 100 μmol L-1 uric acid, 100 μmol L-1 cysteine and 100 μmol L-1 tyrosine, while a repeatable current response was observed for 100 μmol L-1 H2O2. These results revealed that the proposed Cyt c/PAN/cMWCNTs/GC electrode exhibits high selectivity toward H2O2 which makes it suitable for use in different practical applications.
Conclusion
In this work, Cyt c was immobilized on a platform, which has been designed by a nanocomposite containing PAN and cMWCNTs on the surface of GC electrode. The properties of Cyt c/PAN/MWCNTs/GC electrode, such as electron transfer rate, coverage surface area, charge transfer resistance and response time were investigated by electrochemical CV evaluations and EIS studies. The experimental results showed that large amounts of Cyt c were successfully immobilized on PAN/MWCNTs/GC electrode and the modified electrode could transfer electrons with quasi-reversible and well-defined redox peaks. The electroactivity behavior of prepared Cyt c/PAN/cMWCNTs/GC electrode was studied by CV measurements and the amperometric detection of H2O2. The proposed platform in this work can be applied to fabricate third generation biosensor for sensing H2O2 in practical applications.