Biosensors and Bioelectronics 64 (2015) 131–137

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Application of graphene–copper sulfide nanocomposite modified electrode for electrochemistry and electrocatalysis of hemoglobin Fan Shi, Weizhe Zheng, Wencheng Wang, Fei Hou, Bingxin Lei, Zhenfan Sun, Wei Sun n College of Chemistry and Chemical Engineering, Hainan Normal University, Haikou 571158, PR China

art ic l e i nf o

a b s t r a c t

Article history: Received 4 June 2014 Received in revised form 20 August 2014 Accepted 26 August 2014 Available online 28 August 2014

In this paper a graphene (GR) and copper sulfide (CuS) nanocomposite was synthesized by hydrothermal method and used for the electrode modification with a N-butylpyridinium hexafluorophosphate based carbon ionic liquid electrode (CILE) as the substrate electrode. Hemoglobin (Hb) was immobilized on the modified electrode to get a biocompatible sensing platform. UV–vis absorption spectroscopic results confirmed that Hb retained its native secondary structure in the composite. Direct electron transfer of Hb incorporated into the nanocomposite was investigated with a pair of well-defined redox waves appeared on cyclic voltammogram, indicating the realization of direct electrochemistry of Hb on the modified electrode. The results can be ascribed to the presence of GR–CuS nanocomposite on the electrode surface that facilitates the electron transfer rate between the electroactive center of Hb and the electrode. The Hb modified electrode showed excellent electrocatalytic activity to the reduction of trichloroacetic acid in the concentration range from 3.0 to 64.0 mmol L  1 with the detection limit of 0.20 mmol L  1 (3s). The fabricated biosensor displayed the advantages such as high sensitivity, good reproducibility and longterm stability. & 2014 Elsevier B.V. All rights reserved.

Keywords: Graphene Copper sulfide Hemoglobin Direct electrochemistry Electrocatalysis Carbon ionic liquid electrode

1. Introduction Investigation on direct electron transfer (DET) of the redox proteins has attracted much interest in recent years (Armstrong et al., 1988). However, the process is difficult to be realized due to the deeply embedment of the electroactive centers within the protein structures, and the partially denaturalization or the unfavorable orientation of proteins on the surface of electrode (Rusling, 1998; Armstrong and Wilson, 2000). Therefore different kinds of mediators have been used to facilitate the direct electron transfer of redox proteins with the underlying electrode. As a wellcharacterized redox protein with commercial availability and moderate cost, hemoglobin (Hb) has been extensively studied as the model for the DET of redox proteins. Hb is a heme protein that consists of four polypeptide chains with one heme group at each chain, which can store and transport oxygen in the red blood cells. Direct electrochemistry of Hb have been realized on different kinds of chemically modified electrodes such as biopolymers, surfactants, membranes and nanocomposites (Nadzhafova et al., 2007; Feng et al., 2007; Shi et al., 2007; Ma and Tian, 2010; Sun et al., 2012, 2013a, 2013b). n

Corresponding author. Tel./fax: þ 86 898 31381637. E-mail address: [email protected] (W. Sun).

http://dx.doi.org/10.1016/j.bios.2014.08.064 0956-5663/& 2014 Elsevier B.V. All rights reserved.

As a two-dimensional single-atom-thick carbon network, graphene (GR) has been the research focus due to its unique structure and physiochemical properties (Novoselov et al., 2004). GR has exhibited the specific characteristics including extremely electric conductivity, large specific surface area, unusual electronic structure, and upstanding thermal conductivity (Pumera, 2010). Electrochemical applications of GR and its related materials had been reviewed for their excellent electrochemical properties (Chen et al., 2010). Due to the easily aggregation of GR nanosheets by π–π stacking interaction and Van der waals, various GR composites have been investigated, which can prevent the aggregation of GR nanosheets and exhibit the synergistic effects (Singh et al., 2011). Huang et al. (2012b) reviewed the recent developments of GR based composites and their applications in different fields such as Li-ion batteries, supercapacitors, fuel cells and photovoltaic devices. Among the GR based composite, there are seldom reports about GR–CuS composite. CuS is an important p-type semiconductor with excellent optical, electronic, catalytic and other physiochemical properties, which has been extensively studied for various applications such as photocatalysis, solar cells, electrochemistry, catalyst, super ionic materials and Li-ion batteries (Chung and Sohn, 2002; Wang et al., 2003; Martinson et al., 2009; Zhuge et al., 2009; Basu et al., 2010). Various attempts have been proposed for the synthesis of CuS with different morphologies, such as flower, microsphere and nanotube (Wang et al., 2007; Li et al., 2007; Thongtem et al., 2009). Recently Goel et al. (2014)

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reviewed the synthesis and biomedical applications of CuS nanoparticles from sensors to theranostics. CuS has been proven to exhibit interesting properties including metal-like electrical conductivity, which may have potential application in electrochemical sensors (Mane and Lokhande, 2000; Zhang et al., 2008b). Liu and Xue (2011) prepared a CuS nanotube modified glassy carbon electrode (GCE) for the electrocatalytic oxidation of glucose. Zou et al. (2014) applied a CuS nanocrystal modified GCE for the electrochemical detection of hydroquinone. Yang et al. (2014) fabricated a CuS nanoflower modified GCE for the electrocatalytic sensing of hydrogen peroxide and glucose. Bo et al. (2010) applied a CuS nanoparticles decorated ordered mesoporous carbon modified GCE for the electrocatalytic detection of hydrogen peroxide. By combining GR with CuS to get a composite, GR–CuS nanocomposite has exhibited synergistic effects with intriguing properties. For example, Qian et al. (2014) prepared a CuS nanocrystal/ reduced graphene oxide (GO) composite by a one-pot solvothermal reaction and applied to the catalytic investigation on hydrogen peroxide. Park et al. (2013) prepared a CuS–GO/TiO2 composite for the high photonic effect and photocatalytic activity under visible light. Zhang et al. (2012) developed an environmental friendly strategy toward one-pot synthesis of CuS nanoparticles decorated reduced GO nanocomposite with enhanced photocatalytic performance. However, no references about the application of GR–CuS composite for the protein electrochemistry have been reported until now. The combination of GR with CuS nanomaterials can result in the synergetic effects of two components and exhibit the enhanced performance. GO with high surface area can act as a good matrix for the growth of CuS nanoparticles with excellent dispersion. Also the presence of CuS can prevent the aggregation of single-layer GR nanosheet. The GR–CuS nanocomposite with rough interface can provide sufficient spaces for the immobilization of redox proteins with close interconnection. The higher conductive GR and the excellent electrocatalytic activity of CuS can provide a favorable microenvironment for electrochemical communication, which is suitable for the electrochemical application with fast electron transfer. Therefore the GR–CuS nanocomposite is investigated to elucidate its potential application in direct electrochemistry of redox proteins. In the paper direct electrochemistry of Hb on GR–CuS nanocomposite modified electrode was realized and investigated in detail by using carbon ionic liquid electrode (CILE) as the substrate electrode. CILE is prepared by using ionic liquid (IL) as the modifier in the traditional carbon paste electrode (CPE). Due to the specific electrochemical characteristics of IL, such as higher ionic conductivity, wider electrochemical windows and lower background, CILE exhibits the advantage including good resistance towards electrode fouling, high electron transfer rate and the inherent catalytic activity (Sun et al., 2007a; Opallo and Lesniewski, 2011). The synergistic effects of CILE and GR–CuS nanocomposite were benefit for the realization of direct electrochemistry of Hb with a pair of well-defined redox peaks appeared. The as-prepared CTS/ Hb/GR–CuS/CILE demonstrated excellent electrocatalytic ability to the reduction of trichloroacetic acid (TCA) with wider dynamic range and lower detection limit. Therefore, the GR–CuS nanocomposite modified electrode can be served as a promising platform for the development of electrochemical biosensor with highefficiency.

2. Experimental 2.1. Reagents Bovine hemoglobin (Hb, MW. 64500, Sinopharm Chemical Reagent Co., China), 1-butylpyridinium hexafluorophosphate

(BPPF6, Lanzhou Greenchem ILS. LICP. CAS., China), graphite powder (particle size 30 μm, Shanghai Colloid Chem. Co., China), chitosan (CTS, Dalian Xindie Ltd. Co., China), trichloroacetic acid (TCA, Tianjin Kemiou Chemical Ltd. Co., China) and graphene oxide (GO, Taiyuan Tanmei Co., China) 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 All the electrochemical measurements were executed on a CHI 660D electrochemical workstation (Shanghai Chenhua Instrument, China). A conventional three-electrode system was used with an Hb modified electrode as the working electrode, a platinum wire as the auxiliary electrode and a saturated calomel electrode (SCE) as the reference electrode. FT-IR spectra and UV–vis absorption spectra were recorded on a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific Inc., USA) and a TU-1901 double beam UV–visible spectrophotometer (Beijing General Instrument Ltd. Co., China). Scanning electron microscopy (SEM) was recorded on a JSM-7100F scanning electron microscope (Japan Electron Company, Japan). Raman spectroscopy was performed using a Renishaw InVia Raman microspectrometer (Renishaw Plc., UK) using 514 nm lasers.

2.3. Synthesis of the GR–CuS nanocomposite 1.0 mL of GO suspension (1.0 mg mL  1) and 40.0 mL of ethylene glycol were mixed together, followed by stirring for 30 min. Then 0.001 mol of Cu(NO3)2 and 0.001 mol of thiourea were added into as-prepared GO solution with stirring. After being stirred for about 30 min, the reaction solution was transferred to a Teflonlined stainless-steel autoclave (50 mL capacity) and heated at 120 °C for 4.0 h in an electric oven. After the reaction, the autoclave was allowed to cool to room temperature. The final products were centrifuged, washed with deionized water and absolute ethanol, and then dried at 70 °C for 10 h in air to get the GR–CuS nanocomposite.

2.4. Preparation of the modified electrode CILE was fabricated according to a reported procedure (Sun et al., 2007b). In brief 3.0 g graphite powder and 1.0 g BPPF6 were mixed thoroughly in an agate mortar and the homogeneous paste was packed into a cavity of a glass tube (∅¼4.0 mm). A copper wire was inserted through the opposite end to establish an electrical contact. Prior to use the electrode was polished on a weighing paper to get a mirror-like surface. 6.0 μL of 0.05 mg mL  1 GR–CuS suspension was directly applied on the CILE surface and left to dry at room temperature. Then 8.0 μL of 15.0 mg mL  1 Hb was cast on the surface of GR–CuS/CILE and dried to get Hb/GR–CuS/CILE. Finally, 5.0 μL of 1.0 mg mL  1 CTS (in 1.0% HAC) solution was spread evenly onto the surface of Hb/GR–CuS/CILE to get the modified electrode as CTS/Hb/GR–CuS/ CILE. Other kinds of modified electrodes including CTS/Hb/CILE, CTS/GR–CuS/CILE, CTS/CILE etc. were prepared by the similar procedure and used for comparison.

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3. Results and discussion 3.1. Materials characterization The morphologies of materials used were examined by SEM with different magnification. As shown in Fig. 1A and B CuS exhibited as sphere with the increase of surface roughness and the average diameter was 3.5 μm. Fig. 1C exhibited the SEM image of GR nanosheets, which gave a typical wrinkled layered structure. The SEM image of GR–CuS nanocomposite showed that CuS microspheres were wrapped by GR sheet with both the morphologic structure of CuS spheres and the edge of GR clearly observed (Fig. 1D). So the presence of CuS microspheres increases the dispersion of GR nanosheets and the interface area of the nanocomposite. FT-IR spectra of GO and GR–CuS nanocomposite were recorded with the results shown in Fig. S1A. The characteristic peaks of GO appeared at 3442 cm  1 and 1398 cm  1 (OH), 1621 cm  1 (C ¼O), 1108 cm  1 and 933 cm  1 (C–O). As for GR–CuS nanocomposite, the peaks for the oxygen functional groups gradually decrease, which indicated that most oxygen functional groups in GO had been removed by ethylene glycol (Fan et al., 2012). Raman spectra of GO and GR–CuS nanocomposite were further recorded and shown in Fig. S1B. For the spectrum of GO, there were two strong and sharp peaks at 1357.51 cm  1 and 1593.50 cm  1, respectively, which was in agreement with the previous report (Chen et al., 2011). As for the GR–CuS nanocomposite, a strong and sharp peak appearing at 467.12 cm  1, which is in agreement with the previous report (He et al., 2013). The characteristic peaks at 1322.90 cm  1 and 1564.12 cm  1 in the Raman spectrum of the composites were attributed to the D and G bands from GR. So GO was reduced to GR with the recovery of the conductivity and the formation of sp2 domains The one-step growth mechanism of GR–CuS nanocomposite by a solvothermal method in ethylene glycol was illustrated in Fig. 1E. Accompanying the growth of CuS microspheres, GO was reduced and CuS microspheres grew between GR sheets directly at the same time. Finally, CuS microspheres were embedded in GR layers and almost enwrapped by GR sheets to get a uniform nanocomposite.

Fig. 1. SEM images of (A and B) CuS sphere, (C) GR and (D) GR–CuS composite and (E) the formation procedure of GR–CuS nanocomposite.

3.2. UV–vis spectroscopic results UV–vis absorption spectroscopy is an effective method to probe the structure 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. 2, the Hb molecules had a characteristic Soret absorption at 406.0 nm (curve a). While for the mixture solution of GR–CuS–Hb (curve b), the Soret band also appeared at 406.0 nm without changes, which indicated that Hb molecules kept its native structure in the mixture. The results proved the biocompatibility of GR–CuS with the protein and Hb retained the essential features of its native structure without any changes after mixed with in GR–CuS nanocomposite. 3.3. Electrochemical characteristics of the modified electrodes Electrochemical impedance spectroscopy (EIS) can provide interfacial information of the electrode during the modification process (Pan and Rothberg, 2005). The diameter of the semicircle usually equals to the electron transfer resistance (Ret), which controls the electron transfer kinetics of the redox probe at the electrode interface. EIS experiments were performed in a 0.1 mol L  1 KCl

Fig. 2. UV–vis absorption spectra of Hb (a) and CuS–GR–Hb (b) in water.

solution containing 1.0 mmol L  1 [Fe(CN)6]3 /4 with the frequencies swept from 105 to 10  1 Hz. Fig. 3A shows the EIS results of bare CILE (curve a), CTS/CILE (curve b), CTS/Hb/CILE (curve c) and CTS/ Hb/GR–CuS/CILE (curve d), respectively. The Ret value of CILE was got as 53.09 Ω (curve a), which was due to the presence of high conductive IL in the carbon paste. When a CTS layer was applied on the surface of CILE, the Ret value increased to 58.83 Ω (curve b), indicating that the presence of nonconductive CTS film on the electrode surface increased the interfacial resistances, then decreased the electron transfer rate. On CTS/Hb/CILE the Ret value was further increased to 102.99 Ω (curve c), which could be attributed to the presence of Hb on the electrode surface further hindered the electron transfer rate of [Fe(CN)6]3 /4 . While on CTS/ Hb/GR–CuS/CILE, the Ret value decreased to 13.64 Ω (curve d), indicating that the presence of good conductive GR–CuS nanocomposite in the film increased the electron transfer rate and decreased the resistance of the electrode surface. GR–CuS nanocomposite is a semiconductor with the advantages of high surface area, optical transparency, good biocompatibility and relatively good conductivity,

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Fig. 3. (A) EIS of different electrodes in a 5.0 mmol L  1 [Fe(CN)6]3  /4  and 0.1 mol L  1 KCl mixture solution with the frequencies ranging from 105 to 10  1 Hz. (B) Cyclic voltammograms of different electrodes in a 10.0 mmol L  1 [Fe(CN)6]3  /4  and 0.1 mol L  1 KCl mixture solution with scan rate as 100 mV s  1. Electrodes: (a) CILE, (b) CTS/ CILE, (c) CTS/Hb/CILE and (d) CTS/Hb/GR–CuS/CILE.

which exhibits excellent electrochemical conductivity on the electrode surface. Cyclic voltammograms of different electrodes were recorded in a 1.0 mmol L  1 [Fe(CN)6]3  /4  and 0.1 mol L  1 KCl mixture solution with the results shown in Fig. 3B. A pair of well-defined quasireversible redox peaks were observed on CILE (curve a), which was the typical properties of CILE. On CTS/CILE (curve b) the electrochemical response was weaken, indicating that the presence of nonconductive CTS film on the electrode surface hindered the electron transfer of [Fe(CN)6]3  /4  . On CTS/Hb/CILE (curve c) the redox peak currents were furthur decreased, indicating the presence of Hb on the electrode surface blocked the electron transfer. While on CTS/Hb/GR–CuS/CILE (curve d) the redox peak currents increased greatly, which could be attributed to the addition of GR– CuS nanocomposite that promoted the electron transfer rate of the redox probe. The cyclic voltammetric data were in good agreement with that of EIS results, indicating the successful preparation of the modified electrode. The effective electrode surface (A) was furthur calculated by the cyclic voltammetric method. By changing the scan rate and recording the redox peak currents, the effective surface area could be calculated by Randles–Servick equation (Bard and Fulkner, 2001), Ipc¼(2.69  105)n3/2AD1/2C*υ1/2, where Ipc is the reduction peak current (A), n is the electron transfer number, A is the effective surface area (cm2), D is the diffusion coefficient of K3[Fe(CN)6] in the solution (cm2/s), C* is the concentration of K3[Fe (CN)6] (mol L  1) and v is the scan rate (V/s). Based on this method, the A values of CILE and GR–CuS/CILE were calculated as 0.160 cm2 and 0.213 cm2. So the GR–CuS nanocomposite could greatly enhance the effective surface area of the modified electrode. 3.4. Direct electrochemical behavior of the Hb modified electrode Electrochemical behaviors of different modified electrodes were carefully investigated in pH 3.0 PBS by cyclic voltammetry with the results showed in Fig. 4. No electrochemical responses were observed at CTS/CILE (curve a) or CTS/GR–CuS/CILE (curve b), indicating no electrochemical reaction took place at the selected potential range. On CTS/Hb/CILE a pair of unsymmetric redox peaks appeared (curve c), indicating a slow direct electron transfer rate between Hb and CILE. Hb can exchange the electrons with CILE due to its high conductivity with biocompatible interface (Sun et al., 2007c). After the addition of GR–CuS nanocomposite in the Hb modified electrode, the redox peak currents increased gradually with the peak shape became more symmetry (curve d). At multi-scan cyclic voltammetry the redox peaks almost unchanged after continuous potential cycling. The results indicated that direct

Fig. 4. Cyclic voltammograms of (a) CTS/CILE, (b) CTS/GR–CuS/CILE, (c) CTS/Hb/CILE and (d) CTS/Hb/GR–CuS/CILE in pH 3.0 PBS with the scan rate as 100 mV s  1.

electron transfer rate of Hb was enhanced on GR–CuS/CILE with good stability. The presence of high conductive GR–CuS nanocomposite can establish a fast electron transfer path to facilitate the direct electrochemistry of Hb with the underlying electrode. GR– CuS nanocomposite on the electrode surface exhibits larger surface area with surface roughness increased. Also the GR–CuS nanocomposite combines the good conductivity and electrocatalytic ability of GR and CuS together, which is benefit for the acceleration of electron transfer between the active centers of Hb with electrode. From curve d the values of cathodic peak potential (Epc) and anodic peak potential (Epa) were got as at  0.302 V and  0.226 V, respectively, with the peak-to-peak separation (ΔEp) value as 76 mV. The formal peak potential (E0′), which is calculated from the midpoint of Epa and Epc, was estimated as  0.264 V (vs. SCE). The result was the typical characteristic of electroactive heme Fe(III)/Fe(II) redox couples. So the direct electron transfer of Hb was successfully realized on the GR–CuS nanocomposite modified electrode. The influence of scan rate (υ) on the electrochemical responses of the Hb modified electrode was further investigated with the results shown in Fig. 5A. At different scan rates a pair of welldefined quasi-reversible redox peaks appeared on cyclic voltammograms with almost equal height of redox peak currents. With the increase of scan rate the redox peak currents varied linearly with scan rate in the range from 0.1 to 0.7 V s  1, indicating

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Fig. 5. (A) Influence of scan rate on electrochemical responses of CTS/Hb/GR–CuS/CILE in pH 3.0 PBS with scan rates from a to m as 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700 mV s  1, respectively. (B) Linear relationship of cathodic (a) and anodic (b) peak current (Ip) versus scan rate (υ). (C) Linear relationship of Epa (a) and Epc (b) versus ln υ.

a typical surface-controlled electrochemical behavior. Two welldefined straight lines were got with the linear regression equations as Ipc (μA)¼ 80.25υ (V/s) þ4.70 (n ¼19, γ ¼0.998) and Ipa (μA) ¼  64.2υ (V/s)  4.16 (n ¼19, γ ¼0.998), respectively (as shown in Fig. 5B). By integration of the reduction or oxidation peak of cyclic voltammograms, the surface coverage (Γ*) can be estimated from the equation (Γ* ¼Q/nAF), where Q is the charge involved in the reaction, n is the number of electrons transferred, F is the Faraday constant, and A is the geometric area of electrode. Based on this equation the surface coverage (Γ*) of electroactive Hb was calculated as 7.9  10  10 mol cm  2, which was larger than that of monolayer coverage (1.89  10  11 mol cm  2) (Wang et al., 2005) and other kinds of Hb modified electrodes such as Hb–GR– CTS modified GCE (3.1  10  10 mol cm  2) (Xu et al., 2010) and Nafion/Hb/multi-walled carbon nanotubes/AuNPs modified GCE (6.87 0.3  10  10 mol cm  2) (Hong et al., 2013). While the total amounts of Hb cast on the electrode surface was calculated as 1.20  10  8 mol cm  2, so the fraction of electroactive proteins among the total proteins on CILE surface was 6.6%. The results suggested that multilayers of Hb in the composite film close to the electrode with suitable orientation could exchange electrons with electrode. With the increase of scan rate the redox peak potentials were also shifted gradually. As shown in Fig. 5C, the relationships of Ep with ln υ were constructed with two well-defined straight lines got as Epa (V)¼0.041 ln υ  0.10 (n ¼8, γ ¼ 0.997) and Epc (V) ¼ 0.02 ln υ 0.24 (n ¼8, γ ¼0.999). According to Laviron's equations (Laviron, 1979), the values of the electron transfer coefficient (α) and the heterogeneous electron transfer rate

Fig. 6. Cyclic voltammograms of CTS/GR–CuS/CILE with (a) 0 mmol L  1, (b) 10.0 mmol L  1 TCA and CTS/Hb/GR–CuS/CILE with 0, 1.0, 7.0, 12.0, 16.0, 25.0, 30.0, 38.0, 48.0, 56.0, 64.0 mmol L  1 TCA (curves c–m) in 0.1 mol L  1 pH 3.0 PBS at the scan rate of 100 mV s  1 (inset was the linear relationship of catalytic peak currents and TCA concentration).

constant (ks) were estimated as 0.36 and 1.58 s  1, respectively. The ks value of 1.58 s  1 has been obtained, which was larger than that of 0.64 s  1 on CTS/GR–LDH–Hb/CILE (Sun et al., 2013c), 0.65 s  1 on Nafion/GR–TiO2–Hb/CILE (Sun et al., 2013a), 0.83 s  1 on Hb–GNRs@SiO2/GCE (Zhang et al., 2008a), and 1.14 s  1 on Hb/ Chit-IL-Fc/GR/GCE (Huang et al., 2012a). This fast electron transfer rate indicated that GR–CuS nanocomposite could form a suitable microenvironment for Hb to undergo facile electron transfer reaction. The effect of buffer pH on cyclic voltammetric responses of the Hb modified electrode was further investigated in the pH range from 3.0 to 9.0 with a pair of stable and well-defined cyclic voltammetric curves appeared. With the increase of buffer pH the redox peak potential shifted to the negative direction, implying that protons were involved in the electrode process. A good linear regression relationship was got between the formal peak potential (E0′) and pH with the equation as E0′(mV)¼  56.6 pH  39.7 (n ¼7, γ ¼0.999). The slope value of  56.6 mV pH  1 was close to the theoretical value of  59.0 mV pH  1 at 20 °C for a single-proton coupled one-electron transfer process. So the electrochemical reaction could be expressed with the equation as: Hb heme Fe (III) þH þ þe2Hb heme Fe (II). Also the biggest readox peaks appeared at pH 3.0 buffer solution, which was used for the electrochemical investigation. 3.5. Electrocatalytic activity of the HB modified electrode Electrocatalytic activity towards TCA, which was an important target in the fields such as biochemistry and environmental analysis, was carefully investigated and the typical cyclic voltammograms were shown as Fig. 6. When different concentrations of TCA was added into the buffer solution, a great increase of the reduction peak was observed at  0.243 V with the simultaneously disappearance of the oxidation peak (curves c–m). With the further increase of the TCA concentration another new reduction peak appeared at  0.529 V. This peak could be attributed to the formation of a highly reduced form of Hb [Hb Fe(I)], which might dechlorinate di- and mono-chloroacetic acid after the dechlorination of TCA with Hb Fe(II) (Fan et al., 2000). While no redox peaks could be observed on CTS/GR–CuS/CILE for the electrocatalysis of TCA (curves a and b), indicating the presence of Hb in the composite film exhibited the electrocatalytic behavior. Thus, the electrocatalytic reaction may be deduced via the following equations, which involved the reduction of Hb Fe(III) to Hb Fe(II), the reduction of TCA with Hb Fe(II) on the electrode, the reduction of Hb Fe(II) to Hb Fe(I) on the electrode, the reduction of diand mono-chloroacetic acid with Hb Fe(II) and the reoxidation of Hb Fe(I).

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Hb Fe(III)þe-Hb Fe(II)

(1) þ

2Hb Fe(II) þ Cl3CCOOHþ H -2Hb Fe(III) þCl2CHCOOHþCl



Hb Fe(II)þe-Hb Fe(I)

(2) (3)

þ

2Hb Fe(I) þ Cl2CHCOOHþ H -2Hb Fe(II)þClCH2COOHþCl 2Hb Fe(I) þ ClCH2COOHþ H þ -2Hb Fe(II)þCH3COOHþ Cl 



(4)

retained its native structure and exhibited excellent electrocatalytic ability to the reduction of TCA with high sensitivity, wide linear range, low detection limit, long-term stability and good reproducibility. So the GR–CuS–Hb modified electrode has the potential application in constructing third-generation electrochemical biosensor based on mediator-free electrochemistry of the enzymes.

(5)

The catalytic reduction peak current had a good linear relationship with TCA concentration in the range from 1.0 mmol L  1 to 64.0 mmol L  1 with the linear regression equation as Ipc (μA)¼ 4.92C (mmol L  1)  7.28 (n ¼11, γ ¼0.998) and the detection limit as 0.20 mmol L  1 (3s). When the TCA concentration was more than 64.0 mmol L  1, the current reached a saturation value, which indicated a Michaelis–Menten kinetic for the electrocatalytic reaction of TCA. The apparent Michaelis–Menten constant (KMapp ) could be calculated from the electrochemical version of the Lineweaver–Burk equation: 1/Iss ¼(1/Imax) (1 þ KMapp /C), where Iss is the steady current after the addition of substrate, C is the bulk concentration of the substrate, and Imax is the maximum current measured under saturated substrate condition (Kamin et al., 1980). Then the KMapp value was calculated to be 6.3 mmol L  1, which was smaller than that of some previous reported values such as 47.0 mmol L  1 for Hb–agarose film (Wang et al., 2005), 14.52 mmol L  1 for Hb–CNT composite paste electrode (Zhai et al., 2009). The lower value of KMapp indicated that Hb immobilized on CTS/GR–CuS/CILE retained its bioactivity and had a high biological affinity to TCA.

Acknowledgments

3.6. Reproducibility and stability of CTS/HB/GR–CuS/CILE

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The reproducibility of CTS/Hb/GR–CuS/CILE for the determination of 10.0 mmol L  1 TCA was investigated by fabricating six bioelectrodes at the same procedure independently, which showed an acceptable reproducibility with a relative standard deviation (RSD) of 2.3%. The modified electrode was stored at 4 °C for a given period to examine the long-term storage stability when not in use. Every 5 days the voltammetric responses of CTS/Hb/ GR–CuS/CILE to 10.0 mmol L  1 TCA was examined. Up to 10 days the current response only decreased by 3% of the initial value. After a 30-day storage period, CTS/Hb/GR–CuS/CILE still retained 93% of initial current, which indicated that this bioelectrode had a good stability. So GR–CuS composite was a suitable matrix for the immobilization of Hb and retained its activity. The long lifetime of CTS/Hb/GR–CuS/CILE may be attributed to the good biocompatibility and stability of the composite used.

4. Conclusion A GR–CuS composite was synthesized by hydrothermal method and used for the realization of direct electrochemistry of Hb by cyclic voltammetry. On CTS/Hb/GR–CuS/CILE a pair of well-defined redox peaks appeared on cyclic voltammograms, which indicated that the direct electron transfer of Hb was realized. The result can be attributed to the high conductivity, big surface area and good biocompatibility of the GR–CuS nanocomposite present on the electrode surface. As for the direct electrochemistry of redox protein, the presence of nanomaterials on the electrode surface can establish the electron transfer tunnels for the electroactive center of Hb to exchange electrons with electrode. Also the biological activity of protein can be retained on the nanomaterials modified electrode. So different kinds of nanocomposites can be used for the realization of protein electrochemistry, which play synergistic roles for the accelerating the transfer of electrons and exhibit the advantages of each component. The immobilized Hb

We acknowledge the financial support from the National Natural Science Foundation of China (Nos. 21365010 and 51363008), the Natural Science Foundation of Hainan Province (213015), the Project of Hainan Applied Technology Research and Development (ZDXM2014098), the Undergraduate Training Programs for Innovation and Entrepreneurship of Hainan Province (2013116580) and the Foundation of Hainan Normal University.

Appendix A. Supplementary material Supplementary material associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014. 08.064.

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Application of graphene-copper sulfide nanocomposite modified electrode for electrochemistry and electrocatalysis of hemoglobin.

In this paper a graphene (GR) and copper sulfide (CuS) nanocomposite was synthesized by hydrothermal method and used for the electrode modification wi...
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